Wilson Disease [1st Edition] 9780444636270, 9780444636256

Table of contents :
Content:
Series PagePage ii
CopyrightPage iv
Handbook of Clinical Neurology 3rd SeriesPages v-vi
ForewordPage viiMichael J. Aminoff, François Boller, Dick F. Swaab
PrefacePage ixAnna Członkowska, Michael L. Schilsky
ContributorsPages xi-xii
Chapter 1 - History of Wilson disease: a personal accountPages 1-5John M. Walshe
Chapter 2 - Epidemiology and introduction to the clinical presentation of Wilson diseasePages 7-17Christine Lo, Oliver Bandmann
Chapter 3 - The genetics of Wilson diseasePages 19-34Irene J. Chang, Si Houn Hahn
Chapter 4 - Genetic and environmental modifiers of Wilson diseasePages 35-41Valentina Medici, Karl-Heinz Weiss
Chapter 5 - Pathogenesis of Wilson diseasePages 43-55Ivo Florin Scheiber, Radan Brůha, Petr Dušek
Chapter 6 - Animal models of Wilson diseasePages 57-70Valentina Medici, Dominik Huster
Chapter 7 - Wilson disease – liver pathologyPages 71-75Maciej Pronicki
Chapter 8 - Wilson disease: brain pathologyPages 77-89Aurélia Poujois, Jacqueline Mikol, France Woimant
Chapter 9 - Hepatic features of Wilson diseasePages 91-99Salih Boga, Aftab Ala, Michael L. Schilsky
Chapter 10 - Wilson disease: neurologic featuresPages 101-119Anna Członkowska, Tomasz Litwin, Grzegorz Chabik
Chapter 11 - Cognitive and psychiatric symptoms in Wilson diseasePages 121-140Paula Zimbrean, Joanna Seniów
Chapter 12 - Wilson disease in childrenPages 141-156Eve A. Roberts, Piotr Socha
Chapter 13 - Other organ involvement and clinical aspects of Wilson diseasePages 157-169Karolina Dzieżyc, Tomasz Litwin, Anna Członkowska
Chapter 14 - Diagnosis of Wilson diseasePages 171-180Peter Ferenci
Chapter 15 - Wilson disease – currently used anticopper therapyPages 181-191Anna Członkowska, Tomasz Litwin
Chapter 16 - Liver transplantation for Wilson diseasePages 193-204Ahsan Ahmad, Euriko Torrazza-Perez, Michael L. Schilsky
Chapter 17 - Wilson disease: symptomatic liver therapyPages 205-209Jan Pfeiffenberger, Karl-Heinz Weiss, Wolfgang Stremmel
Chapter 18 - Symptomatic treatment of neurologic symptoms in Wilson diseasePages 211-223Tomasz Litwin, Petr Dušek, Anna Członkowska
Chapter 19 - Novel perspectives on Wilson disease treatmentPages 225-230Christian Rupp, Wolfgang Stremmel, Karl-Heinz Weiss
Chapter 20 - Patient support groups in the management of Wilson diseasePages 231-240Mary L. Graper, Michael L. Schilsky
IndexPages 241-248

Citation preview

HANDBOOK OF CLINICAL NEUROLOGY Series Editors

MICHAEL J. AMINOFF, FRANÇOIS BOLLER, AND DICK F. SWAAB VOLUME 142

ELSEVIER Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States © 2017 Elsevier B.V. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. With respect to any drug or pharmaceutical products identified, readers are advised to check the most current information provided (i) on procedures featured or (ii) by the manufacturer of each product to be administered, to verify the recommended dose or formula, the method and duration of administration, and contraindications. It is the responsibility of practitioners, relying on their own experience and knowledge of their patients, to make diagnoses, to determine dosages and the best treatment for each individual patient, and to take all appropriate safety precautions. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-444-63625-6 For information on all Elsevier publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Mara Conner Editorial Project Manager: Kristi Anderson Production Project Manager: Sujatha Thirugnana Sambandam Cover Designer: Alan Studholme Typeset by SPi Global, India

Handbook of Clinical Neurology 3rd Series Available titles Vol. 79, The human hypothalamus: basic and clinical aspects, Part I, D.F. Swaab, ed. ISBN 9780444513571 Vol. 80, The human hypothalamus: basic and clinical aspects, Part II, D.F. Swaab, ed. ISBN 9780444514905 Vol. 81, Pain, F. Cervero and T.S. Jensen, eds. ISBN 9780444519016 Vol. 82, Motor neurone disorders and related diseases, A.A. Eisen and P.J. Shaw, eds. ISBN 9780444518941 Vol. 83, Parkinson’s disease and related disorders, Part I, W.C. Koller and E. Melamed, eds. ISBN 9780444519009 Vol. 84, Parkinson’s disease and related disorders, Part II, W.C. Koller and E. Melamed, eds. ISBN 9780444528933 Vol. 85, HIV/AIDS and the nervous system, P. Portegies and J. Berger, eds. ISBN 9780444520104 Vol. 86, Myopathies, F.L. Mastaglia and D. Hilton Jones, eds. ISBN 9780444518996 Vol. 87, Malformations of the nervous system, H.B. Sarnat and P. Curatolo, eds. ISBN 9780444518965 Vol. 88, Neuropsychology and behavioural neurology, G. Goldenberg and B.C. Miller, eds. ISBN 9780444518972 Vol. 89, Dementias, C. Duyckaerts and I. Litvan, eds. ISBN 9780444518989 Vol. 90, Disorders of consciousness, G.B. Young and E.F.M. Wijdicks, eds. ISBN 9780444518958 Vol. 91, Neuromuscular junction disorders, A.G. Engel, ed. ISBN 9780444520081 Vol. 92, Stroke – Part I: Basic and epidemiological aspects, M. Fisher, ed. ISBN 9780444520036 Vol. 93, Stroke – Part II: Clinical manifestations and pathogenesis, M. Fisher, ed. ISBN 9780444520043 Vol. 94, Stroke – Part III: Investigations and management, M. Fisher, ed. ISBN 9780444520050 Vol. 95, History of neurology, S. Finger, F. Boller and K.L. Tyler, eds. ISBN 9780444520081 Vol. 96, Bacterial infections of the central nervous system, K.L. Roos and A.R. Tunkel, eds. ISBN 9780444520159 Vol. 97, Headache, G. Nappi and M.A. Moskowitz, eds. ISBN 9780444521392 Vol. 98, Sleep disorders Part I, P. Montagna and S. Chokroverty, eds. ISBN 9780444520067 Vol. 99, Sleep disorders Part II, P. Montagna and S. Chokroverty, eds. ISBN 9780444520074 Vol. 100, Hyperkinetic movement disorders, W.J. Weiner and E. Tolosa, eds. ISBN 9780444520142 Vol. 101, Muscular dystrophies, A. Amato and R.C. Griggs, eds. ISBN 9780080450315 Vol. 102, Neuro-ophthalmology, C. Kennard and R.J. Leigh, eds. ISBN 9780444529039 Vol. 103, Ataxic disorders, S.H. Subramony and A. Durr, eds. ISBN 9780444518927 Vol. 104, Neuro-oncology Part I, W. Grisold and R. Sofietti, eds. ISBN 9780444521385 Vol. 105, Neuro-oncology Part II, W. Grisold and R. Sofietti, eds. ISBN 9780444535023 Vol. 106, Neurobiology of psychiatric disorders, T. Schlaepfer and C.B. Nemeroff, eds. ISBN 9780444520029 Vol. 107, Epilepsy Part I, H. Stefan and W.H. Theodore, eds. ISBN 9780444528988 Vol. 108, Epilepsy Part II, H. Stefan and W.H. Theodore, eds. ISBN 9780444528995 Vol. 109, Spinal cord injury, J. Verhaagen and J.W. McDonald III, eds. ISBN 9780444521378 Vol. 110, Neurological rehabilitation, M. Barnes and D.C. Good, eds. ISBN 9780444529015 Vol. 111, Pediatric neurology Part I, O. Dulac, M. Lassonde and H.B. Sarnat, eds. ISBN 9780444528919 Vol. 112, Pediatric neurology Part II, O. Dulac, M. Lassonde and H.B. Sarnat, eds. ISBN 9780444529107 Vol. 113, Pediatric neurology Part III, O. Dulac, M. Lassonde and H.B. Sarnat, eds. ISBN 9780444595652 Vol. 114, Neuroparasitology and tropical neurology, H.H. Garcia, H.B. Tanowitz and O.H. Del Brutto, eds. ISBN 9780444534903 Vol. 115, Peripheral nerve disorders, G. Said and C. Krarup, eds. ISBN 9780444529022 Vol. 116, Brain stimulation, A.M. Lozano and M. Hallett, eds. ISBN 9780444534972 Vol. 117, Autonomic nervous system, R.M. Buijs and D.F. Swaab, eds. ISBN 9780444534910 Vol. 118, Ethical and legal issues in neurology, J.L. Bernat and H.R. Beresford, eds. ISBN 9780444535016 Vol. 119, Neurologic aspects of systemic disease Part I, J. Biller and J.M. Ferro, eds. ISBN 9780702040863 Vol. 120, Neurologic aspects of systemic disease Part II, J. Biller and J.M. Ferro, eds. ISBN 9780702040870 Vol. 121, Neurologic aspects of systemic disease Part III, J. Biller and J.M. Ferro, eds. ISBN 9780702040887 Vol. 122, Multiple sclerosis and related disorders, D.S. Goodin, ed. ISBN 9780444520012 Vol. 123, Neurovirology, A.C. Tselis and J. Booss, eds. ISBN 9780444534880

vi

AVAILABLE TITLES (Continued)

Vol. 124, Clinical neuroendocrinology, E. Fliers, M. Korbonits and J.A. Romijn, eds. ISBN 9780444596024 Vol. 125, Alcohol and the nervous system, E.V. Sullivan and A. Pfefferbaum, eds. ISBN 9780444626196 Vol. 126, Diabetes and the nervous system, D.W. Zochodne and R.A. Malik, eds. ISBN 9780444534804 Vol. 127, Traumatic brain injury Part I, J.H. Grafman and A.M. Salazar, eds. ISBN 9780444528926 Vol. 128, Traumatic brain injury Part II, J.H. Grafman and A.M. Salazar, eds. ISBN 9780444635211 Vol. 129, The human auditory system: Fundamental organization and clinical disorders, G.G. Celesia and G. Hickok, eds. ISBN 9780444626301 Vol. 130, Neurology of sexual and bladder disorders, D.B. Vodušek and F. Boller, eds. ISBN 9780444632470 Vol. 131, Occupational neurology, M. Lotti and M.L. Bleecker, eds. ISBN 9780444626271 Vol. 132, Neurocutaneous syndromes, M.P. Islam and E.S. Roach, eds. ISBN 9780444627025 Vol. 133, Autoimmune neurology, S.J. Pittock and A. Vincent, eds. ISBN 9780444634320 Vol. 134, Gliomas, M.S. Berger and M. Weller, eds. ISBN 9780128029978 Vol. 135, Neuroimaging Part I, J.C. Masdeu and R.G. González, eds. ISBN 9780444534859 Vol. 136, Neuroimaging Part II, J.C. Masdeu and R.G. González, eds. ISBN 9780444534866 Vol. 137, Neuro-otology, J.M. Furman and T. Lempert, eds. ISBN 9780444634375 Vol. 138, Neuroepidemiology, C. Rosano, M.A. Ikram and M. Ganguli, eds. ISBN 9780128029732 Vol. 139, Functional neurologic disorders, M. Hallett, J. Stone and A. Carson, eds. ISBN 9780128017722 Vol. 140, Critical care neurology Part I, E.F.M. Wijdicks and A.H. Kramer, eds. ISBN 9780444636003 Vol. 141, Critical care neurology Part II, E.F.M. Wijdicks and A.H. Kramer, eds. ISBN 9780444635990 All volumes in the 3rd Series of the Handbook of Clinical Neurology are published electronically, on Science Direct: http://www.sciencedirect.com/science/handbooks/00729752.

Foreword

It is a pleasure to present this volume of the Handbook of Clinical Neurology (HCN), which is dedicated to Wilson disease. There are plenty of eponyms in medicine and particularly in neurology, but very few HCN volumes include a person’s name in their title and this is only the third in the present series, after Parkinson and Huntington. Kinnier Wilson (1878–1937) fully deserves this honor. In 1912, he published in Brain what had been his MD thesis: “Progressive lenticular degeneration: a familial nervous system disease associated with cirrhosis of the liver.” As is almost always the case, similar conditions associating liver and brain disease had been described previously, going as far back as Morgagni and, more recently, by Westphal and Strumpell. However, the Wilson monograph included the main clinical and pathologic features of the disease, and it opened the way to a new chapter in the history of neurology, namely that of extrapyramidal system disorders, a term Wilson introduced. The volume opens with a chapter describing in detail the history of the disease written by one of its protagonists, John M. Walshe, who in 1956 proposed the use of penicillamine, one of the first disease-modifying drugs in neurology. The volume includes chapters dealing with the epidemiology and genetics of the disease. It focuses on the two main organs affected, the brain and the liver, with detailed descriptions of the pathology and pathogenesis of the disease, including animal models, as well as its diagnosis and the various available forms of management and treatment. Novel perspectives are discussed in a chapter that introduces a new chelating agent, tetrathiomolybdate, as well as nonchelating drugs currently under clinical investigation. As is appropriate for such a rare yet highly complex hereditary disease, the volume concludes with a chapter on the groups that advocate for and support patients with Wilson disease. We have been fortunate to have as volume editors two distinguished scholars and clinicians, Anna Członkowska, Professor at the Second Department of Neurology, Institute of Psychiatry and Neurology in Warsaw, and Michael L. Schilsky, Associate Professor of Medicine and Surgery and Medical Director of Adult Liver Transplantation at the Yale-New Haven Transplantation Center. Both have been on the forefront of research on Wilson disease for many years. They have assembled a truly international group of authors with acknowledged expertise and together they have produced this authoritative, comprehensive, and up-to-date volume. Its availability electronically on Elsevier’s Science Direct site as well as in print format should ensure its ready accessibility and facilitate searches for specific information. We are grateful to them and to all the contributors for their efforts in creating such an invaluable resource. As series editors we read and commented on each of the chapters with great interest. We are therefore confident that both clinicians and researchers in many different medical disciplines will find much in this volume to appeal to them. And last, but not least, we thank Elsevier, our publisher – and in particular Michael Parkinson in Scotland, and Mara Conner and Kristi Anderson in San Diego – for their unfailing and expert assistance in the development and production of this volume. Michael J. Aminoff Franc¸ois Boller Dick F. Swaab

Preface

There is almost no other treatable disorder with as wide a clinical spectrum for presentation as Wilson disease. From child to septuagenarian at diagnosis, patients may be asymptomatic or present with advanced cirrhosis or liver failure, with a movement disorder ranging from mild tremor or bradykinesia to severe dystonia, or with an affective disorder or psychosis. Although this is a volume in a series focused on neurologic and neuropsychiatric disorders, it was inescapable that we cross between disciplines and consider the full spectrum of the disease, the underlying hepatic disease that accompanies the disorder, the diverse neurologic presentations, and the fascinating history of the discovery of its basis and treatment. Recent advances in molecular genetics testing allow easier and faster diagnosis of Wilson disease, for which treatment may prevent, stop, or even reverse tissue injury. Also introduced more recently are imaging techniques that help estimate the stage of the disease at the time of diagnosis and which are helpful for monitoring the progress of treatment. With a better understanding of the disease pathogenesis, there are new opportunities for safer and even more effective therapy. The aim of this text was to provide a thorough examination of Wilson disease in an easy-to-digest format. To accomplish this, we engaged a group of international experts from Europe and North America and asked for their help in covering a wide range of relevant topics important for all caregivers of patients with Wilson disease, whether pediatric or adult physicians, neurologists, psychiatrists, or hepatologists. The text begins with a historic perspective, and then provides overviews of disease pathogenesis and its underlying genetic basis, the effects of the disease on both the central nervous system and liver, its diagnosis, and the range of clinical disease and treatments. Unusually, we have included the viewpoint of a lay patient organization on what is important from a patient and advocacy perspective. We thank all our contributors for their time and help in putting together this volume. A number of revisions were necessary to make the handbook more cohesive, and we appreciate their patience and forbearance, which made this possible. We would like particularly to acknowledge John M. Walshe for his historic perspective and his contributions to the field, and Mary L. Graper for her advocacy for all patients with Wilson disease. We especially thank all of our coworkers, with whom we have worked over the years to assist hundreds of patients, broaden our personal experience, and share it with others. We are also grateful for the cooperation of our patients and their families who put their trust in us and from whom we continue to learn. Anna Członkowska and Michael L. Schilsky

Contributors

A. Ahmad Section of Transplantation and Immunology, Department of Surgery, Yale University School of Medicine, New Haven, CT, USA

First Faculty of Medicine and General University Hospital in Prague, Prague, Czech Republic and Institute of Neuroradiology, University Medicine Goettingen, Goettingen, Germany

A. Ala Department of Clinical and Experimental Medicine, Faculty of Health and Medical Sciences, University of Surrey and Department of Gastroenterology and Hepatology, Royal Surrey County Hospital NHS Foundation Trust, Guildford, Surrey, UK

K. Dziezyc Second Department of Neurology, Institute of Psychiatry and Neurology, Warsaw, Poland

O. Bandmann Sheffield Institute for Translational Neuroscience (SITraN), University of Sheffield, Sheffield, UK S. Boga Department of Gastroenterology, Sisli Etfal Education and Research Hospital, Istanbul, Turkey R. Brůha Fourth Department of Internal Medicine, Charles University in Prague, First Faculty of Medicine and General University Hospital in Prague, Prague, Czech Republic G. Chabik Second Department of Neurology, Institute of Psychiatry and Neurology, Warsaw, Poland



P. Ferenci Department of Internal Medicine 3, Gastroenterology and Hepatology, Medical University of Vienna, Vienna, Austria M.L. Graper Wilson Disease Association, Milwaukee, WI, USA S.H. Hahn Division of Genetic Medicine, Department of Pediatrics, University of Washington School of Medicine, Seattle Children’s Hospital, Seattle, WA, USA D. Huster Department of Gastroenterology and Oncology, Deaconess Hospital Leipzig, Leipzig, Germany T. Litwin Second Department of Neurology, Institute of Psychiatry and Neurology, Warsaw, Poland

I.J. Chang Division of Medical Genetics, Department of Medicine, University of Washington School of Medicine, Seattle, WA, USA

C. Lo Sheffield Institute for Translational Neuroscience (SITraN), University of Sheffield, Sheffield, UK

A. Członkowska Second Department of Neurology, Institute of Psychiatry and Neurology and Department of Experimental and Clinical Pharmacology, Medical University of Warsaw, Poland

V. Medici Division of Gastroenterology and Hepatology, Department of Internal Medicine, University of California Davis, Sacramento, CA, USA

P. Dušek Department of Neurology and Center of Clinical Neuroscience, Charles University in Prague,

J. Mikol Pathology Department, Paris Diderot University, Paris, France

xii

CONTRIBUTORS

J. Pfeiffenberger Department of Gastroenterology and Hepatology, University Hospital of Heidelberg, Heidelberg, Germany

P. Socha Departments of Gastroenterology, Hepatology, Nutritional Disorders and Pediatrics, The Children’s Memorial Health Institute, Warsaw, Poland

A. Poujois French National Reference Centre for Wilson Disease, Neurology Department, Lariboisière Hospital, Paris, France

W. Stremmel Department of Gastroenterology and Hepatology, University Hospital of Heidelberg, Heidelberg, Germany

M. Pronicki Department of Pathology, The Children’s Memorial Health Institute, Warsaw, Poland

E. Torrazza-Perez Division of Gastroenterology and Hepatology, Department of Internal Medicine, University of New Mexico, Albuquerque, USA

E.A. Roberts Departments of Paediatrics, Medicine and Pharmacology and Toxicology, University of Toronto, Toronto, Canada C. Rupp Department of Internal Medicine, University Hospital Heidelberg, Heidelberg, Germany I.F. Scheiber Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic M.L. Schilsky Section of Digestive Diseases and Transplantation and Immunology, Department of Medicine and Surgery, Yale University School of Medicine, New Haven, CT, USA J. Seniów Second Department of Neurology, Institute of Psychiatry and Neurology, Warsaw, Poland

J.M. Walshe Formerly of Addenbrookes Hospital, Cambridge and the Middlesex Hospital, London, UK K.-H. Weiss Department of Gastroenterology and Hepatology, University Hospital of Heidelberg, Heidelberg, Germany F. Woimant French National Reference Centre for Wilson Disease, Neurology Department, Lariboisière Hospital, Paris, France P. Zimbrean Departments of Psychiatry and Surgery (Transplant), Yale University, New Haven, CT, USA

Handbook of Clinical Neurology, Vol. 142 (3rd series) Wilson Disease A. Członkowska and M.L. Schilsky, Editors http://dx.doi.org/10.1016/B978-0-444-63625-6.00001-X © 2017 Elsevier B.V. All rights reserved

Chapter 1

History of Wilson disease: a personal account JOHN M. WALSHE* Formerly of Addenbrookes Hospital, Cambridge and the Middlesex Hospital, London, UK

Abstract This chapter focuses on the historic aspects of the development of much of our current knowledge of the diagnosis and treatment of Wilson disease. Included are descriptions of the clinical signs of neurologic and hepatic disease, the natural history of disease progression, studies of disease pathogenesis and a unique perspective on the development of diagnostic testing and pharmacological therapy.

“Begin at the beginning,” the King said very gravely, “then go on until the end, then stop.” Alice’s Adventures Through the Looking Glass, Lewis Carroll This admirable advice for any story teller is honored more often in the breach than the observance. The difficulty remains, where is the beginning and does the story have any definitive end? With the history of disease the probability is that there is, in all likelihood, no end. Knowledge and theories will continue to develop almost indefinitely. Well then, is there a beginning? Probably, but exactly where that is, is not always obvious. In this case, it might seem self-evident that the story begins with Wilson’s original article in Brain in 1912, when he described four cases of what he called “progressive lenticular degeneration: a familial nervous disease associated with cirrhosis of the liver.” (Fig. 1.1) In addition he found four cases in the literature which fitted his description, one of which was dated back to 1890. Furthermore, there is a case report in Frerichs’ book of 1860 that was almost certainly a case of Wilson disease. Indeed, going back even further, Morgagni, in 1761, described several cases of liver disease associated with nervous symptoms which may possibly have been cases of Wilson disease. It is interesting that one of the defining physical signs of Wilson disease, the Kayser–Fleischer corneal rings, had been described 10 years before the disease to which

they later became associated (Kayser, 1902). It is surprising, in view of the very detailed nature of his descriptions of his patients, that Wilson did not observe the corneal rings and indeed he refused to admit of their relationship to his disease for 10 years after his original publication (Wilson, 1922). These rings were for many years thought to pathognomonic of Wilson disease but it is now known that they can be found in other forms of liver disease, such as primary biliary cirrhosis and chronic cholestasis (Fleming et al., 1975). Only 1 year after Wilson’s original publication in Brain, the Austrian pathologist, Rumpel, reported finding an excess of copper in the liver of a patient dying of Wilson disease, an observation whose importance was completely missed (Rumpel, 1913). He also suggested that there was excess of silver in the eyes. Another important observation which passed unnoticed was by Bramwell, whose house physician Wilson had once been (Bramwell, 1916). Bramwell described a family in which four siblings died, between the ages of 9 and 14 years, of rapid-onset liver failure and suggested that this might be related to Wilson disease. It would be interesting to see if any descendants of this family still exist so that they could be tested to see if they carry, in single dose, the mutation for Wilson disease. The next important addition to knowledge came when Hall (1921) showed that this disease was inherited in a recessive mode, later confirmed in more detail by Bearn (1960), who studied 30 families he saw in New York

*Correspondence to: J.M. Walshe, MD, ScD, FRCP, 58 High Street Hemingford Grey, Huntingdon PE 28 9BN, UK. E-mail: [email protected]

2

J.M. WALSHE

Fig. 1.1. Dr. John Walshe (right) with Dr. James Kinner Wilson, son of neurologist Samuel Alexander Kinner Wilson, whose 1912 publication on hepatolenticular degeneration was being celebrated at the Centennial Symposium on Wilson’s disease in London, October 5–6, 2012.

whose ancestry could be traced back to Eastern Europe or Southern Italy. It must be noted that in Wilson’s original publication of only 4 patients, he described almost all of the signs and symptoms which we now know are characteristic of this disease (Wilson, 1912). He carried out his own pathologic studies and even traveled to Switzerland to collect the brain of a patient dying of his disease. Wilson did not believe that the liver pathology was a significant factor in the natural history of the disease, although one of his own patients actually died of bleeding esophageal varices. Over the next 30 years there were only minor advances in the understanding of the disease, whose course remained remorseless and invariably fatal. In 1922 Siemerling and Oloff described the association of sunflower cataracts with Kayser–Fleischer rings and noted the similarity to lesions caused by intraocular damage with intraocular copper fragments. Vogt (1929) and Haurowitz (1930) reported finding excess copper in the brain and liver of Wilson disease patients – an observation which was not followed up. In 1945 Sir Rudolph Peters and his team in Oxford were able to report their earlier work in the development of the antiarsenical drug, dimercaprol (British anti-Lewisite, BAL). This was developed during the Second World War to combat anticipated attack by Hitler with the arsenical gas Lewisite. The importance of this became apparent in 1948 when Cumings showed that Wilson disease was indeed due to accumulation of excess copper in the brain and liver of all patients dying of this disease and postulated that treatment with BAL might halt the progress of the illness (Cumings, 1951). By coincidence, at the same time, Mandelbrote et al. (1948) reported their findings on the effect of BAL on copper excretion on a wide spectrum of patients with neurologic diseases; one of these patients had Wilson disease and in this particular patient there was a great increase in the

copper excreted in the urine. This important observation was followed up in 1951 by Cumings himself in London and by Denny Brown and Porter (1951) in Boston. Both teams showed that courses of BAL resulted in significant improvement in their patients’ neurologic symptoms. However it soon became apparent that patients needed repeated courses of the drug and subsequent courses were less effective than the initial one. In addition the injections were painful and associated with a wide variety of toxic reactions. This was clearly not an ideal treatment. The picture changed when, in 1956, I reported that penicillamine promoted a much larger excretion of copper in the urine than either BAL or the other medical chelating agent ethylenediamine tetraacetic acid (EDTA) (Walshe, 1956, 1960). Penicillamine is a breakdown product of penicillin and is excreted in the urine of all patients treated with penicillin, an observation I had made some years earlier when studying changes in amino acid metabolism in patients with liver damage (Walshe, 1956). This idea came to me when working in the Liver Unit under Dr. Charles Davidson, at the Boston City Hospital. Having seen one of Professor Denny Brown’s Wilson disease patients who was not prospering on treatment with BAL, it occurred to me that penicillamine, with its –SH and –NH3 groups, might have the right chemical structure to chelate copper and promote its excretion in the urine. Dr. Davidson was able to obtain for me 2 grams of penicillamine from Professor Sheehan at the Massachusetts Institute of Technology. Having taken 1 gram myself to prove its safety, I administered the second gram to Professor Denny Brown’s patient and recorded the very satisfactory cupresis so induced. With this simple procedure the start of penicillamine therapy was launched. In those far-off days there were no ethical committees to be approached for permission; life was a lot easier when trying out new ideas. By 1960 I was able to publish the first account of a patient with Wilson disease improving significantly as a result of this new therapy (Walshe, 1960). This was confirmed by similar studies by Scheinberg and Sternlieb, also in 1960. Whilst these important advances in the treatment of this disease were progressing, so also was our understanding of its pathogenesis. In 1948 Holmberg and Laurell described the finding of a copper-carrying protein in the plasma which they named ceruloplasmin. In 1952, working independently in New York, both Bearn and Kunkel and Scheinberg and Gitlin showed that patients with Wilson disease all had low or absent concentrations of this protein (Bearn and Kunkel,1952; Scheinberg and Gitlin, 1952). Shortly after this Cartwright and his team (1954) in Salt Lake City published an important article on copper metabolism in

HISTORY OF WILSON DISEASE: A PERSONAL ACCOUNT Wilson disease, an article which inspired my own interest in this malady, in which they discussed the various therapies then available and their shortcomings. Despite these real advances Uzman, working in Denny Brown’s department in Boston, believed that Wilson disease was due to an abnormality of peptide metabolism and that copper deposition was a side-effect and not the directly causative etiology of the disease (Uzman et al., 1956). This hypothesis was later firmly disproved by Asator et al. (1976) using a technique more sophisticated than that available to Uzman. Although Uzman was wrong on this point, he was able to confirm Bramwell’s hypothesis that Wilson disease could present as liver disease in children before the onset of neurologic signs (Bramwell, 1916; Uzman et al., 1956; Chalmers et al., 1957). The clinical picture of the disease was widened by Bearn, who showed that there were abnormalities of renal function and skeletal lesions in many patients (Bearn, 1957). He and Kunkel also introduced the idea of using radiolabeled copper, the short half-isotope, to study copper metabolism in Wilson disease patients (Bearn and Kunkel, 1954). About the same time the role of ceruloplasmin in the pathogenesis was hotly debated and Uzman and Hood (1952) rightly pointed out that the concentrations of ceruloplasmin found in Wilson disease patients was quite variable and frequently overlapped that found in symptomless carriers of the Wilson disease gene in single dose. In 1959 Cumings published a book, Heavy Metals and the Brain, in which he tried to cover the entire literature of Wilson disease to date and this remains an invaluable historical source of information for those interested in the history of the disease, as also does Scheinberg and Sternlieb’s (1984) monograph. Beginning in the 1960s I realized the potential of using radiocopper in the investigation of Wilson disease, working first with Osborn, and later with Potter (Osborn and Walshe, 1965, 1967; Walshe and Potter, 1977). Together we devised a method of determining the uptake of copper by the liver and its further distribution throughout the body (Osborn and Walshe, 1967). It was thus possible to show that in the presymptomatic stages of the disease the liver had a high affinity for the metal and 90% of the injected dose was present in the liver within 24 hours. As the disease progressed the liver uptake of radiocopper became less and less effective and the isotope could be found distributed through many other tissues. However treatment with chelating agents effectively decoppered the liver and restored its ability to sequester the metal. But in no patient was it possible to show, as in normal individuals, a peak in the lower abdomen which represented copper excreted via the bile in to the intestine (Walshe and Potter, 1977). Later it was possible to show, in patients and controls, who had

3

undergone cholecystectomy for gallstones, that the difference in copper excretion in the bile was of an order of magnitude between normal individuals and patients (Gibbs and Walshe, 1980). Furthermore, by studying the difference in urine copper concentration in pigment stones the mean figure for copper in patients was 50 mg/g compared with the figure of 1072.5 mg/g in controls (Walshe, 1998). The first description of hemolysis and a serious complication of Wilson disease was first described by Professor Sherlock and her team at the Royal Free Hospital in London (Sherlock et al., 1968), whilst Roche-Sicot and Benhamou (1977) described the first case of massive hepatic necrosis and hemolysis, thus proposing the idea of acute fatal Wilson disease. Recently I showed that hemolysis was to be found in 6.9% of 321 patients and that the hemolysis was extravascular, the age of onset was 12.6 years, and there was a female-to-male ratio of 2:1 (Walshe, 2013). Again, in the 1960s, in addition to our increased understanding of the pathogenesis of Wilson disease was an increased range of therapies which favorably influenced the prognosis. In 1961 Schouwink reported that zinc salts could block the uptake of copper from the intestine and thereby induce, although very slowly, a negative copper balance and this was later taken up enthusiastically by Hoogenraad et al. (1979) in the Netherlands and by Brewer et al. (1981) in the United States. In an attempt to overcome the toxic side-effects of penicillamine, which were becoming apparent by the late 1960s (Walshe, 1968), with the invaluable help of Dr. Hal Dixon, I proposed the use of triethylene tetramine 2HCl as an alternative chelating agent and this proved to be an effective therapy and remarkably free of sideeffects (Walshe, 1982). The next advance was the introduction of liver transplantation by Starzl and his team in 1971 (DuBois et al., 1971). This approach actually cured the Wilson disease so patients no longer needed chelation treatment, but they did require lifelong immunosuppression. The final advance came with the use of ammonium tetrathiomolybdate (Walshe, 1986). I first took this myself for a week to test its safety before giving it to a patient who had proved intolerant to penicillamine and trientine and had found the side-effects of zinc salts intolerable. Within a year it had effectively decoppered her liver and changed the histology from a fatty liver with cellular infiltrates to normal (Walshe, 1986). It is of interest that Bickel et al. (1957), had tried the use of molybdate in the 1950s, but they had not used the tetrathio salt and their preparation had no anticopper properties so the possibilities of this form of therapy were set back some 30 years. They had relied on the observation that sheep

4

J.M. WALSHE

fed on pasture contaminated with molybdenum developed copper deficiency but failed to realize that in the rumen of the sheep the MoO4 was converted, by reducing conditions, to MoS4, an action which does not take place in the human gut. The most recent, and perhaps the most important, breakthrough came in 1993 when three separate groups (Bull et al., 1993; Tanzi et al., 1993; Yamaguchi et al., 1993) reported in Nature Genetics that they had identified the Wilson disease gene as a P-type APTase, 140-kDa copper-transporting enzyme located on chromosome 13q14. There have been no further important advances since then, except for the identification of more than 500 mutations of this gene. As most patients are compound heterozygotes the number of permutations and combinations is enormous and will make phenotype–genotype correlations almost impossible. Having brought the story up to date the question remains, what of the future? It would seem that, as far as natural history and biochemistry and genetics are concerned, the disease is worked out. However therapy is far from ideal. The initial therapy with BAL still has a place for severely dystonic patients but it has too many side-effects and its administration is something of an ordeal for the patient. Penicillamine is effective in many cases but also has too many side-effects, mostly immunogenic in nature and, in a small percentage of patients, can induce a sudden deterioration which is, unfortunately, unpredictable (Walshe, 2011). Trientine has far fewer side-effects and is a very useful alternative (Walshe, 1982) but, like all forms of treatment, is sometimes associated with initial worsening of symptoms, which is always a worrying state of affairs. Tetrathiomolybdate is yet another alternative but is not yet in the pharmacopeia so is not readily available. Zinc salts are effective in blocking copper absorption from the gut but can only produce a very slow negative copper balance and are probably more useful for maintenance than initial therapy. Many zinc salts are gastric irritants and are probably contraindicated in patients with prominent esophageal varices. Finally there is liver transplantation, in effect a cure, but which condemns the patient to long-term immunosuppression with all its possible complications. Its use suggests a failure of either diagnosis or initial treatment. One day it may be possible to offer gene replacement but this is not yet even on the horizon. It remains to be seen what the future holds for patients with Wilson disease.

REFERENCES Asator AM, Milne MD, Walshe JM (1976). The urinary excretion of peptides and hydroxyproline in Wilson’s disease. Clin Sci Mol Med 51: 369–378.

Bearn AG (1957). Wilson’s disease. An inborn error of metabolism with multiple manifestations. Am J Med 22: 747–764. Bearn AG (1960). A genetical analysis of 30 families with Wilson’s disease (hepatolenticular degeneration). Ann Hum Genet 24: 33–43. Bearn AG, Kunkel HG (1952). Biochemical abnormalities in Wilson’s disease. J Clin Invest 31: 616. Bearn AG, Kunkel HG (1954). Localisation of 64Cu in serum fractions folloqwing oral administration in Wilson’s disease. Proc Soc Expl Biol 85: 44–48. Bickel H, Neale FC, Hall G (1957). A clinical and biochemical study of hepatolenticular degeneration (Wilson’s disease). QJM 26: 527–558. Bramwell B (1916). Familial cirrhosis of the liver; four cases of acute fatal cirrhosis in the same family, the patients being respectively nineteen, fourteen and fourteen years of age; suggested relationship to Wilson’s progressive degeneration of the lenticular nucleus. Edin Med J 17: 90–99. Brewer GJ, Prasad AS, Cossack ZT et al. (1981). Treatment of Wilson’s disease with oral zinc. Clin Res 29: 758. Bull PC, Thomas GR, Romanes JN et al. (1993). The Wilson’s disease gene is a putative copper transporting P-type ATP-ase similar to the Menkes gene. Nat Genet 5: 327–337. Cartwright GE, Hodges RE, Gubler CJ et al. (1954). Studies on copper metabolism. Hepatolenticular degeneration. J Clin Invest 33: 1487–1501. Chalmers TC, Iber FL, Uzman LL (1957). Hepatolenticular degeneration (Wilson’s disease) as a form of idiopathic cirrhosis. N Engl J Med 256: 235–242. Cumings JN (1951). The effect of BAL in hepatodegeneration. Brain 74: 10–22. Cumings JN (1959). Heavy metals and the brain, Blackwell Scientific Publications, Oxford. Denny Brown D, Porter H (1951). The effect of BAL (2.3dimercapto propanol) on hepatolenticular degeneration (Wilson’s disease). N Engl J Med 245: 917–925. DuBois RS, Rodgerson DO, Martineau G et al. (1971). Orthopic liver transplantation for Wilson’s disease. Lancet 1: 505–508. Fleming CR, Dickson ER, Hellenhorst ER et al. (1975). Pigmented corneal rings in a patient with biliary cirrhosis. Gastroenterology 69: 220–225. Frerichs FT (1860). In: C Murchison (Ed.), Clinical treatise on diseases of the liver, Vol. 1. New Sydenham Society, London. Gibbs K, Walshe JM (1980). Biliary excretion of copper in Wilson’s disease. Lancet 2: 538–539. Hall HC (1921). La degenerescence hepatolenticulare. Maladie de Wilson-pseudosclerose, Mason, Paris. Haurowitz F (1930). Uber eine anamolides Kupferstoffwechsels. Hoppe-Seyl Z 190: 72–74. Holmberg CG, Laurell CB (1948). Investigations on serum copper II. Isolation of the copper containing protein and a description of properties. Acta Chem Scand 85: 550–556. Hoogenraad TU, Koevoet R, du Ruyter Korvet EG (1979). Oral zinc sulphate as long term treatment in Wilson’s disease (hepatolenticular degeneration). Eur Neurol 18: 205–211.

HISTORY OF WILSON DISEASE: A PERSONAL ACCOUNT Kayser B (1902). Uber einen Fall von angeborener Grunlicherverfarbung der Cornea. Kiln Mbl Augenheilk 40: 22–25. Mandelbrote BM, Stanier MW, Thompson RHS et al. (1948). Studies on copper metabolism in demyelinating diseases. Brain 71: 212–228. Morgagni GB (1761). De sedibus et causis morborum, Trans Alexander B, London. Osborn SB, Walshe JM (1965). Studies with radiocopper (64Cu) in Wilson’s disease; the dynamics of copper transport. Clin Sci 29: 575–581. Osborn SB, Walshe JM (1967). Studies with radioactive copper (64Cu, 67Cu) in relation to the natural history of Wilson’s disease. Lancet 1: 346–350. Peters RA, Stocken LA, Thompson RHS (1945). BAL. Nature 156: 616. Roche-Sicot J, Benhamou JN (1977). Acute intravascular harmolysis and acute liver failure associated as a first manifestation of Wilson’s disease. Ann Intern Med 86: 301–303. Rumpel A (1913). Ueber das Wesen und die Bedeutung der Leberveranderungen und der Pigmentierungen bei den damit verbundenen Fallen von pseudosclerose (WestphalStrumpell). Deutche Zeitschrift Nervenheilkde 49: 54–73. Scheinberg IH, Gitlin D (1952). Deficiency of ceruloplasmin in patients with hepatolenticular degeneration (Wilson’s disease). Science 116: 484–485. Scheinberg IH, Sternlieb I (1960). Long term management of hepatolenticular degeneration (Wilson’s disease). Am J Med 29: 316–333. Scheinberg IH, Sternlieb I (1984). Wilson’s disease, Saunders, Philadelphia. Schouwink G (1961). De hepatocerebrale degeneratie, met een onderzoek nar zinkstofwisseling. In: University of Amsterdam, Thesis, MD. Sherlock S, McIntyre N, Clink HM et al. (1968). Haemolytic anaemia in Wilson’s disease. In: S Bergsma, IH Scheinberg, I Sternlieb (Eds.), Wilson’s disease. Birth defects, original article series, Vol. IV. National Foundation - March of Dimes, New York, pp. 99–102. Siemerling E, Oloff H (1922). Pseudosclerose (Westphal– Strumpell) mit Cornealring (KayserFleischer) und doppelseitger Scheinkatarakt dienurbie Seitlicherbeuchtung sichtbar ist und die, der nach Verletzung durch kupfersplitterentstehenden Katarakt ahnlich ist. Klin Wschr 1: 1087–1089.

5

Tanzi R, Petrukhin K, Chernov I et al. (1993). The Wilson’s disease gene is a copper transporting ATPase with homology to the Menkes gene. Nat Genet 5: 44–50. Uzman LL, Hood B (1952). The familial nature of the amino-aciduria of Wilson’s disease. Am J Med Sci 223: 392–400. Uzman LL, Iber FL, Chalmers TC et al. (1956). The mechanism of copper deposition in the liver in hepatolenticular degeneration (Wilson’s disease). Am J Med Sci 231: 511–518. Vogt A (1929). Kupfer und siber aufgespeichert in Auge, leber Milz und Nieren als Symptom der Peudoskerlose. Klin Mbl Augenheilk 83: 417–419. Walshe JM (1956). Penicillamine, a new oral therapy for Wilson’s disease. Am J Med 21: 487–495. Walshe JM (1960). The treatment of Wilson’s disease with penicillamine. Lancet 1: 188–192. Walshe JM (1968). Toxic reactions to penicillamine in patients with Wilson’s disease. Postgrad Med J 6–8. Suppl. Walshe JM (1982). Triethylene tetramine dihydrochloride: a new chelating agent for copper. In: FE Karch (Ed.), Orphan Drugs, Marcel Dekker, New York. Walshe JM (1986). Tetrathiomolybdate (MoS4) as an anti copper agent in man. In: IH Scheinberg, JM Walshe (Eds.), Orphan Diseases and Orphan Drugs. Manchester University Press, Manchester. Walshe JM (1998). Wilson disease: gall stone copper following liver transplantation. Ann Clin Biochem 35: 681–682. Walshe JM (2011). Penicillamine neurotoxicity: a hypothesis. ISRN Neurology. Article ID 464572. Walshe JM (2013). The acute haemolytic syndrome in Wilson disease – a review of 22 patients. Q J Med 106: 1003–1008. Walshe JM, Potter G (1977). The pattern of the whole body distribution of radioactive copper (67Cu, 64Cu) in Wilson’s disease and various control groups. Q J Med 46: 445–462. Wilson SAK (1912). Progressive lenticular degeneration; a familial nervous disease associated with cirrhosis of the liver. Brain 34: 295–507. Wilson SAK (1922). La maladie de Wilson. In: P Marie (Ed.), Question Neurologique d’Actualitie. Mason, Paris. Yamaguchi Y, Heiny M, Gitlib JD (1993). Isolation and characterisation of human liver cDNA as a candidate gene for Wilson’s disease. Biochem Biophys Res Commun 197: 271–277.

Handbook of Clinical Neurology, Vol. 142 (3rd series) Wilson Disease A. Członkowska and M.L. Schilsky, Editors http://dx.doi.org/10.1016/B978-0-444-63625-6.00002-1 © 2017 Elsevier B.V. All rights reserved

Chapter 2

Epidemiology and introduction to the clinical presentation of Wilson disease CHRISTINE LO AND OLIVER BANDMANN* Sheffield Institute for Translational Neuroscience (SITraN), University of Sheffield, Sheffield, UK

Abstract Our understanding of the epidemiology of Wilson disease has steadily grown since Sternlieb and Scheinberg’s first prevalence estimate of 5 per million individuals in 1968. Increasingly sophisticated genetic techniques have led to revised genetic prevalence estimates of 142 per million. Various population isolates exist where the prevalence of Wilson disease is higher still, the highest being 885 per million from within the mountainous region of Rucar in Romania. In Sardinia, where the prevalence of Wilson disease has been calculated at 370 per million births, six mutations account for around 85% of Wilson disease chromosomes identified. Significant variation in the patterns of presentation may however exist, even between individuals carrying the same mutations. At either extremes of presentation are an 8-month-old infant with abnormal liver function tests and individuals diagnosed in their eighth decade of life. Three main patterns of presentation have been recognized – hepatic, neurologic, and psychiatric – prompting their presentation to a diverse range of specialists. Deviations in the family history from the anticipated autosomal-recessive mode of inheritance, with apparent “pseudodominance” and mechanisms of inheritance that include uniparental isodisomy (the inheritance of both chromosomal copies from a single parent), may all further cloud the diagnosis. It can therefore take the efforts of an astute clinician with a high clinical index of suspicion to clinch the diagnosis of this eminently treatable condition.

INTRODUCTION Sternlieb and Scheinberg (1968) first estimated the prevalence of Wilson disease in 1968 to be 5 per million individuals. Subsequent work by Bachmann et al. (1979a, b) at the Leipzig Centre for Wilson’s Disease in East Germany between 1949 and 1977 calculated a birth prevalence of Wilson disease of 29 per million. Saito (1981) reported a similar birth prevalence of 33 per million in 1981. With this expanded data set and taking into account mortality figures from the USA pertaining to the number of deaths from Wilson disease, which they assumed to be half the true number, Scheinberg and Sternlieb (1984) revised their worldwide prevalence estimate to 30 per million with a heterozygote carrier frequency of 1 in 90, whilst acknowledging the existence

of population isolates in which a higher prevalence was likely. Park and coworkers (1991), concerned by the potential for a significant number of undiagnosed individuals with Wilson disease, sought to determine the prevalence of Wilson disease in Scotland. Examining computerized hospital statistical records, death certificates, and the results of a postal questionnaire sent to relevant clinicians, they identified 21 patients with Wilson disease alive at the point of analysis in a population of 5 090 700 individuals, equating to a prevalence of 4 per million. The authors re-examined the data presented by Bachmann et al. (1979a, b) from East Germany and calculated a revised prevalence rate of 4.6 per million, which was comparable with their own figure (Park et al., 1991). This rate was still higher than that of

*Correspondence to: Oliver Bandmann, SITraN, 385a Glossop Road, Sheffield S10 2HQ, UK. Tel: +44-114-2222262, E-mail: [email protected]

8

C. LO AND O. BANDMANN

2.7 per million reported by Przuntek and Hoffmann (1987) from West Germany, notwithstanding their less than ideal response rate of 45%. Reilly et al. (1993) adopted a methodologic approach similar to that taken by Park et al. in order to determine the prevalence of Wilson disease in the Republic of Ireland. Twenty-six cases of Wilson disease over a 19-year period were identified, 5 of which had died of an illness consistent with the disease before its formal diagnosis. An adjusted birth incidence rate of 17 per million was calculated for the period of 1950–1969, corresponding to a gene frequency of 0.41% and heterozygote incidence of 0.82%. To allow for the maximal degree of anticipated consanguinity, the gene frequency and heterozygote incidence estimates were revised to 0.36% and 0.72% respectively, providing minimum disease estimates. In this chapter we will provide an overview of clinical genetic and biochemical studies to date and examine their roles in further delineating the epidemiology of Wilson disease. We will explore factors which may help to explain the disparity between the number of individuals predicted to be affected on the basis of screening studies and those diagnosed clinically. Finally, we will review the clinical course of Wilson disease and the diverse ways in which it may present.

BIOCHEMICAL SCREENING FOR THE STUDY OF PREVALENCE Many of the early epidemiologic studies are thought to have underestimated the prevalence of Wilson disease, not least due to their reliance on symptomatic patients having received the correct diagnosis. Considerable interest lies in the early diagnosis of presymptomatic individuals with Wilson disease so that the early instigation of treatment may prevent future complications. The UK NHS newborn blood spot screening program is already utilized to screen for six inherited metabolic diseases. A number of pilot studies have investigated the possibility of mass screening for Wilson disease. Serum copper is typically reduced in Wilson disease. However, its utility as a target agent for dried blood spot screening is limited by its environmental abundance and presence in unpredictable amounts in dried filter paper itself (Hahn, 2014). Instead an enzyme-linked immunosorbent assay using a monoclonal antibody specify to holoceruloplasmin (ceruloplasmin bound with copper) has been developed to measure ceruloplasmin in dried blood spots (Ohura et al., 1999; Yamaguchi et al., 1999; Hahn et al., 2002; Kroll et al., 2006). Screening of 1045 anonymous newborn screening dried blood spots failed to identify any cases of Wilson disease (Kroll et al., 2006). The results of screening a total of 126 810 newborns by Yamaguchi et al. (1999)

were similarly disappointing. They achieved greater success when blood samples from a slightly older population of 24 165 children aged 6 months to 9 years old were used to measure ceruloplasmin with a combination of oxidase activity, particle-coated fluorescence immunoassay, and specific monoclonal antibody. Three patients with Wilson disease were identified, the youngest patient being 8 months old, equating to a prevalence of 124 per million. The authors suggested that, considered in combination with existing screening programs, serum ceruloplasmin level stability and ease of blood collection meant that the age of 3 years old was the most opportune time to undertake any population-wide ceruloplasmin-based mass screening in children (Yamaguchi et al., 1999). In apparent support of their supposition, a presymptomatic 32-month-old boy was the sole positive case identified through a pilot study in which ceruloplasmin levels in dried blood spots were assessed in 3667 asymptomatic Korean children aged between 3 months and 15 years old (Hahn et al., 2002). A similar study, with a 2.6-fold higher prevalence of 717 per million, screened the dried blood spots of 2789 children, identifying 2 aged 30 and 39 months old, with markedly low ceruloplasmin levels (0.24 and 0.40 mg/dL compared to an average of 12.4 mg/dL). The diagnoses were later confirmed genetically by the identification of compound heterozygous A803T/2871delC and R778L/G1035V mutations (Ohura et al., 1999). Noninvasive techniques to assess the amount of urinary holoceruloplasmin protein have also been developed. Screening of urine from 48 819 children led to the identification of 36 children with very low urinary and serum holoceruloplasmin levels, 2 of whom additionally demonstrated low serum copper and increased urinary copper excretion. Both children were otherwise asymptomatic with no signs on physical examination. Yet on further genetic testing they were found to be compound heterozygotes for mutations recognized in Japanese patients with Wilson disease (Owada et al., 2002). The calculated prevalence of 41 per million was less than half that derived from another Japanese study, where a further 11 362 children underwent measurement of urinary ceruloplasmin concentration. An immunologic latex agglutination assay kit was applied to an automated analyzer and results were shown to correlate closely with urinary holoceruloplasmin level measurements. Three successive screening stages led to the identification of 9 children as positive for low urinary ceruloplasmin levels from an initial 668 positive subjects. A diagnosis of Wilson disease was excluded in 8 children in the context of normal biochemical data. Ultimately, in the absence of symptoms or clinical signs, genetic confirmation was necessary to diagnose 1 child, aged 3 years old, as a compound heterozygote (Nakayama et al., 2008).

EPIDEMIOLOGY AND INTRODUCTION TO THE CLINICAL PRESENTATION OF WILSON DISEASE

MODERN GENETICS STUDIES As techniques in molecular genetics have developed, attempts have been made to better characterize the underlying ATP7B mutations in patients with Wilson disease from different populations. In 1999 Curtis et al. studied Wilson disease mutations in Britain in 52 patients by screening parts of the ATP7B gene using single-strand conformational polymorphism (SSCP) and subsequent sequencing. At the time SSCP screening of three exons was predicted to identify around 60% of mutations. Indeed, complete or partial genetic characterization was achieved in 37 individuals, with 4 patients having a homozygous ATP7B mutation, 18 being compound heterozygotes, and 15 in whom a mutation in only one allele was detected (Curtis et al., 1999). An earlier study had similarly managed to characterize around 60% of disease alleles (Nanji et al., 1997). A significant improvement in the overall mutation detection frequency to 98% was reported in a subsequent study from the UK by Coffey et al. in 2013. Genetic confirmation was achieved in 177 out of 181 patients with Wilson disease diagnosed on the basis of clinical and biochemical findings. Of the remaining 4 patients, 2 were found to have a single ATP7B mutation whilst, in the others, no mutations were identified (Coffey et al., 2013). The improved detection rate was largely due to improved mutation techniques as well as the analysis of the entire ATP7B coding region. Though population estimates vary, in Europe the most common mutation observed is the H1069Q missense mutation (Shah et al., 1997; Stapelbroek et al., 2004; Gomes and Dedoussis, 2015). Ivanova-Somlenskya et al. (1999) described a cohort of 40 unrelated patients with Wilson disease from Slavonic families from the European part of Russia to have H1069Q mutations, representing 48.7% of Wilson disease chromosomes. Tarnacka et al. in 2000 described 148 Polish patients with Wilson disease from 95 families. The H1069Q mutation was found in 57% of chromosomes studied appearing in the homozygous and heterozygous states in 39.9% and 30.4% of cases respectively. Though they attempted genotype–phenotype correlation, no relationship was found either with symptomatology or age of onset. In contrast, a study by Shah et al. (1997) reported a younger age of onset in individuals heterozygous for the H1069Q mutation at 15.4 years of age compared to 20 in homozygotes. Through their use of the Hardy–Weinberg equilibrium, they estimated that the ratio of H1069Q heterozygotes to homozygotes was in the order of 3.26. In a study of 70 symptomatic Dutch patients from 59 unrelated families with predominant hepatic presentation, the H1069Q mutation was reported to account for 33% of alleles, with 23% of patients homozygous for

9

the mutation compared to 20% being heterozygous. Homozygotes were reported to more often present with neurologic symptoms, though this did not reach statistical significance. However, compared to patients without the H1069Q mutation, homozygotes and heterozygotes were 3.5 and 2.13 times more likely to present neurologically (Stapelbroek et al., 2004). Mirroring studies by Shah et al. (1997) and Gromadzka et al. (2006), patients heterozygote for the H1069Q mutation were found to present at a slightly earlier age than homozygotes but presented at a later age than patients harboring non-H1069Q mutations (Stapelbroek et al., 2004). A greater disparity in the existence of heterozygous and homozygous H1069Q mutations was found in Romanian patients, where an allelic frequency of 38.1% was reported, accounting for 34.2% of mutations in the heterozygous and 21.1% in the homozygous state (Iacob et al., 2012). In a German cohort described by Merle et al. in 2010, patients with homozygous H1069Q mutations occurred at an even greater frequency, being identified in 50.9% of patients with a further 25.4% of patients being compound heterozygotes with one H1069Q mutant allele. Though the H1069Q mutation is considered to be the most common mutation in Eastern and Northern European populations, this is not necessarily the case worldwide. Indeed, in a Japanese study by Nanji et al. in 1997, out of 21 unrelated families the H1069Q mutation was not detected in a single case. Instead, the R778L mutation occurred at the greatest frequency, accounting for 12% of the identified Wilson disease mutations. The R778L mutation has also been reported in Taiwanese families with Wilson disease (Chuang et al., 1996), as well as in Korean patients (Kim et al., 1998) and in the Chinese Han population, in whom the allele frequency is considerably higher, at 45.6% (Liu et al., 2004). A Spanish study identified the M645R missense mutation as the most frequent mutation; its detection in the heterozygous state was reported in 22 out of 40 unrelated patients with Wilson disease (Margarit et al., 2005). In Costa Rica another missense mutation, the N1270S mutation, also observed in Sicilian and continental Italian and Turkish populations, was found to represent 61% of all mutations (Tanzi et al., 1993; Figus et al., 1995; Shah et al., 1997). Whilst interpatient variability may in part be explained by differences in the underlying genotype, studies have examined rates of intrafamilial concordance between individuals sharing similar genetic characteristics. In a study involving 73 index cases from 73 unrelated families and 95 of their siblings, with an overall H1069Q allelic frequency of 77%, an 86% rate of concordance of presenting symptoms was noted among individuals first presenting with hepatic symptoms. A lower

10

C. LO AND O. BANDMANN

concordance rate of 66% was noted between index cases presenting primarily with neurologic symptoms and their symptomatic siblings (Chabik et al., 2014). In a survey of Wilson disease in Iceland over a 40-year period, 8 patients, from two kindreds, were diagnosed with Wilson disease. All patients were found to be homozygous for the same 2010del7 mutation, presumably inherited from a shared common progenitor. Despite their common genotype and primarily neurologic symptomatology, differences in their presentation were noted, with 3 out of the 8 manifesting with psychiatric symptoms (Thomas et al., 1995). Differing phenotypes were also reported by Wang et al. (2011) in two siblings, both compound heterozygous for the I1148T missense mutation and the S105Stop mutation. Whilst neuropsychiatric symptoms were manifested by the older proband, her younger brother’s presentation was of the hepatic type. Członkowska et al. (2009) furthermore described phenotypic discordance in two pairs of monozygotic twins. In the first pair of twins, both sisters were compound heterozygous for the H1069Q and N404Kfs mutations and were found to have evidence of previous hepatitis B infection, with active infection having been excluded. The proposita had presented relatively late at the age of 38, with a 4-year history of fatigue followed by progressive hepatic decompensation and then the onset on neuropsychiatric symptoms. In contrast, her twin was asymptomatic, with normal brain and liver imaging, the only biochemical abnormality detected being decreased serum levels of copper and ceruloplasmin. The second pair of twins described was homozygous for the common H1069Q mutation. The results of their diagnostic evaluation at the age of 28 were similar. Imaging revealed T2-weighted changes on brain magnetic resonance imaging (MRI) and hepatosplenomegaly on ultrasound scanning. Laboratory investigations identified evidence of abnormal copper metabolism, liver function test derangement, leukopenia, and thrombocytopenia. Whilst one twin had presented with neurologic symptoms, the other described problems with menstrual irregularity and recurrent nose bleeding but barring the finding of Kayser–Fleischer rings on examination was otherwise devoid of neurologic signs (Członkowska et al., 2009).

POPULATION ISOLATES Various population isolates exist in which Wilson disease occurs at a greater incidence and prevalence than would otherwise be expected based on worldwide figures. In a small mountain village next to Heraklion city in the island of Crete, the reported incidence of clinically and/or biochemically diagnosed Wilson disease was 6 out of 90 births over a 25-year period. Screening of

200 unrelated habitants revealed a carrier frequency of p.Q289X and 398delT mutations of 1 in 11 (Dedoussis et al., 2005). The relative genetic homogeneity within the Sardinian population is thought to arise from its marked isolation and history of inbreeding propagating a possible founder effect (Loudianos et al., 1999b). Figus et al. (1995) postulated a higher prevalence of Wilson disease within the Sardinian population in 1995 based on their identification of 10–12 new cases per year. Sixteen different haplotypes associated with Wilson disease chromosomes were identified in 39 individuals of Sardinian descent, of which haplotype IX (5 10 3 3) was the most common, observed at a frequency of 55% of Wilson disease chromosomes. Further analysis of the promoter and the 5’ UTR of the Wilson disease gene sequence from patients with the most common haplotype revealed a single mutation consisting of a 15-nt deletion from position –441 to position –427 relative to the translation start site, which accounted for 60.5% of the studied Wilson disease chromosomes (Loudianos et al., 1999b). These mutations are uncommon in patients of European ancestry outside of Sardinia (Cullen et al., 2003). However, screening of 5290 newborns in Sardinia revealed the presence of 122 individuals heterozygous for the – 441/–427 deletion, equating to an allelic frequency of 1.15%. With the inclusion of an allelic frequency of 0.77%, accounting for non- –441/–427 deletions, an overall Wilson disease mutation frequency of 1.92% was inferred. Assuming Hardy–Weinberg equilibrium, the prevalence of Wilson disease was calculated at 370 per million births, one of the highest in the world and considerably higher than that first quoted by Bachmann et al. (1979b) (Zappu et al., 2008). Yet it is comparable with the estimated prevalence of 366 per million calculated from a homozygosity index of 0.476 and inbreeding coefficient of 7.8  10–4 and applied to a set of 178 Sardinian individuals with clinically and molecularly diagnosed Wilson disease (Gialluisi et al., 2013). As six mutations account for around 85% of Wilson disease chromosomes within the Sardinian population, further efforts have included the targeted screening for these mutations using automated TaqMan screening (Zappu et al., 2010). The identification of heterozygotes through this initial screening process is followed by screening for the remaining 16 mutations detected in the Sardinian population, yielding a sensitivity of 94.6% with a specificity of 100% and potentially resulting in the diagnosis of an additional 5 cases per year (Lovicu et al., 2003; Zappu et al., 2008, 2010). A similarly high prevalence of Wilson disease of 385 per million was reported for the island of Gran Canaria, where the rare Leu708Pro mutation was observed in 18 out of 24 individuals with Wilson disease; 12 in the

EPIDEMIOLOGY AND INTRODUCTION TO THE CLINICAL PRESENTATION OF WILSON DISEASE

homozygous state, 4 as compound heterozygotes in conjunction with another established Wilson disease mutation, and 2 further individuals who only had one identifiable ATP7B mutation. A further 3 individuals who were compound heterozygous for previously established Wilson disease mutations were detected and no mutations were identified in 3 affected individuals from the same pedigree. Despite their common genotype, affected individuals with homozygous Leu708Pro mutations had marked phenotypic variability (Garcia-Villarreal et al., 2000). The same group who initiated newborn screening of the Sardinian population also screened 396 newborns on the similarly isolated Greek island of Kalymnos. The screening of 60% of newborns between 1999 and 2003 identified 18 newborns heterozygous for the H1069Q mutation, 9 heterozygous for the R969Q mutation, and 1 compound heterozygote. The allelic frequencies of the H1069Q and R969Q mutations were 2.4% and 1.3% respectively, yielding a carrier rate for the two mutations of 7% and, assuming Hardy–Weinberg equilibrium, an even higher prevalence than in Sardinia of 135 per million births (Zappu et al., 2008). The highest prevalence of genetically confirmed Wilson disease ever reported is 885 per million from within the mountainous region of Rucar in Romania. Its isolation and tradition of intraclan marriage are thought to have contributed to substantial levels of consanguinity. Screening for ATP7B mutations in 50 individuals from two large families, spanning six generations, identified compound heterozygous H1069Q/M769H– mutations in 5 symptomatic adults and 2 clinically asymptomatic children. Symptomatic individuals demonstrated strong phenotypic concordance with an age of symptom onset of 18  1 years. Initial symptoms included dysarthria and dysphagia and, in all symptomatic individuals, the presence of Kayser–Fleischer rings was observed (Cocos et al., 2014).

GENETIC VERSUS CLINICAL PREVALENCE Sequencing of all 21 ATP7B exons in over 1000 DNA samples and exons 8, 14, and 18 in over 5000 samples by Coffey et al. led to a revised figure of 0.040 or 1:25 as the frequency of heterozygote mutation carriers in the UK. Even with the exclusion of four class 2 single-nucleotide variants in the absence of in silico (computer-simulated) evidence of pathogenicity, the frequency of individuals predicted to carry two pathogenic ATP7B mutations was 142 per million, over four times the often-quoted prevalence figure of 30 per million for Wilson disease (Scheinberg and Sternlieb, 1984; Coffey et al., 2013).

11

There is a significant discrepancy between the number of individuals predicted on the basis of genetic studies to be affected with Wilson disease and those clinically diagnosed. Reduced penetrance of ATP7B mutations may offer an alternative explanation for the lower than expected number of individuals diagnosed clinically with Wilson disease. Indeed, Członkowska et al. (2008) described the indolent course of Wilson disease first diagnosed in a woman at the age of 54 when she was assessed during familial screening. At the time of her initial assessment she was asymptomatic, the only sign being Kayser–Fleischer rings, and she elected to refuse treatment. Genetic confirmation was later achieved when she was found to be homozygous for the H1069Q mutation. It was not until the age of 74 that she started to demonstrate evidence of liver dysfunction with a slight elevation in her liver enzymes and reduction in serum albumin. Thirty years after her original diagnosis, at the age of 84, she remained symptom free (Członkowska et al., 2008). Identification of such individuals may still be warranted with respect to the risk of transmission to potential children that they may have. Of greatest concern is the potential for there to be a large number of patients affected by Wilson disease who remain undiagnosed and without effective treatment, having either never presented to their medical practitioner or having presented with seemingly atypical features (Coffey et al., 2013; Bandmann et al., 2015). The absence of a “typical” family history suggestive of autosomal-recessive inheritance may also dissuade a clinician from considering the diagnosis of Wilson disease. It is important to be aware of less common mechanisms of inheritance, including the inheritance of three different ATP7B mutations, the potential for Wilson disease in successive generations, and the role of segmental uniparental isodisomy, as will now be outlined. The majority of patients with Wilson disease are compound heterozygotes with one mutation on each ATP7B allele (Schilsky, 2014a). There are nevertheless a number of patients who carry three different mutations. Dedoussis et al. (2005) described a 15-year-old boy with Wilson disease from a consanguineous pedigree who was compound heterozygous for the Q289X, I1148T, and G1176R mutations; the latter two mutations cosegregating in cis (i.e., in the same copy of the gene). Although his clinically asymptomatic younger brother carried a wild-type allele alongside the I1148T/G1176R mutations, biochemical indices demonstrated significantly lower copper and ceruloplasmin levels. In an ethnic Han Chinese population, with one out of 65 families reporting consanguinity, Mak et al. (2008) identified 4 patients who carried the Q1142H nonsense and I1148T missense mutations in cis, with another

12

C. LO AND O. BANDMANN

mutation on the trans allele (i.e., in the other copy of the gene). All mutations had previously been identified independently as disease-causing mutations (Loudianos et al., 1998, 1999a). Due to the frequency of their cosegregation in cis, the authors recommended further parental genotyping or exon sequencing upon the simultaneous discovery of Q1142H and I1148T mutations, in order to distinguish individuals bearing two mutations from those bearing three (Mak et al., 2008). Wang et al. (2011) subsequently described 2 patients who presented with neuropsychiatric symptoms and were found to carry three mutations. Fascinatingly, having previously been reported to cosegregate with another mutation in cis, the I1148T and Q1142H mutations were identified on the same allele, a finding also reported in another patient by Coffey et al. (2013). The I381S/ I1184T mutation and the N41S/I1021V mutations have also been reported to cosegregate in cis (Coffey et al., 2013). As an autosomal-recessively inherited condition, Wilson disease would normally not be expected in subsequent generations within a family. The phenomenon of “pseudodominance” is one that has been observed in other conditions, including Friedreich’s ataxia (Lamont et al., 1997) and ataxia with ocular apraxia type 2 (Schols et al., 2008). Cases of Wilson disease in two successive generations have also been reported. Loudianos et al. (2013) described two families from Sardinia. In the first family, Wilson disease was diagnosed after the proband presented following a suicide attempt. She was found to be compound heterozygous for the R919W and H1069Q mutations. Screening of her triplet siblings led to the diagnosis of her dizygotic sibling with Wilson disease, carrying both the R919W and T993W mutations. Their mother carried the R919W mutation in addition to a wild-type allele. A posthumous diagnosis was made in their father, who was postulated to have transmitted the H1069Q and T993W mutations, having died at the age of 39 with liver failure. The affected mother of another family was homozygous for the –441/–427 deletion, the most common regional mutation. Both of her monozygotic twins had inherited an allele with the –441/–427 deletion as well as the G869R mutation, which they inherited from their father. Despite being compound heterozygotes, the twins were clinically asymptomatic when they were diagnosed at the age of 33 (Loudianos et al., 2013). A similar pattern of inheritance, whereby an affected parent unknowingly had children with a heterozygous carrier resulting in a child with Wilson disease, was reported in three out of four French families described by Dufernez et al. (2013). In the fourth family, the identification of both the mother and her affected child as being homozygous for the same T569del mutation raised

the strong suspicion of consanguinity, later confirmed when the mother and father were found to be first cousins. Based on the much-quoted heterozygote carrier frequency of 1 in 90 (Scheinberg and Sternlieb, 1984), Bennett et al. (2013) calculated the probability of an individual with Wilson disease having an affected grandchild to be 0.003%. Yet such a family did they describe, with the proband’s mother, maternal aunt, and son having Wilson disease; this was due to all their reproductive partners having been heterozygote carriers. Dziezyc et al. (2014) identified nine nonconsanguineous families comprising 12 affected offspring from 9 probands from their screen of 294 individuals. A risk of 4.08% of Wilson disease in the offspring of probands was calculated, higher than the figure of 0.5% that had previously been estimated (Ala et al., 2007; Dziezyc et al., 2014). In the study by Coffey et al. (2013), three families with “pseudodominant” Wilson disease were identified. In one family, the proband and his wife remained concerned about the genetic risk to their asymptomatic children in spite of a very low risk having been quoted to them. Rather surprisingly, a novel variant, predicted by in silico analysis to be pathogenic, was identified in the proband’s wife, leading to the subsequent diagnosis of one of their two children as a compound heterozygote for the V1234F and R778W mutations (Coffey et al., 2013). Taken together, the comparatively frequent observation of families with Wilson disease in subsequent generations in different populations lends support to the assumption that heterozygote carrier status for ATP7B mutations may be more common than previously thought. Segmental uniparental isodisomy has also been proposed as a mechanism by which the direct transmission of a normally monogenically inherited disorder may occur between a single parent and his or her offspring (Loudianos et al., 1999a). Uniparental isodisomy for any chromosome is estimated to affect up to 1 in 3500 births and occurs when both copies of a chromosome or part thereof are inherited from a single parent with no contribution from the other. From their cohort of 181 index cases with Wilson disease, Coffey et al. (2013) reported the finding of 2 patients homozygous for different mutations, in the absence of biparental homologue contribution. Multiple microsatellite markers subsequently confirmed segmental isodisomy giving rise to autozygosity for mutations inherited from one parent, with potential significance with respect to genetic counseling of individuals (Coffey et al., 2013). Heterozygote carriers are often clinically asymptomatic. However, detailed examination may reveal subtle abnormalities. Ultrastructural examination of liver tissue using electron microscopy has revealed alterations in the

EPIDEMIOLOGY AND INTRODUCTION TO THE CLINICAL PRESENTATION OF WILSON DISEASE

mitochondria and endoplasmic reticulum of heterozygous individuals, in keeping with copper toxicity (Lough and Wiglesworth, 1976). Biochemical studies have reported various abnormalities of copper metabolism, including decreased levels of serum ceruloplasmin and copper and an increase in the biologic halftime for the clearance of the isotope 65Cu from the plasma pool (Marecek and Nevsimalova, 1984; Lyon et al., 1995; Merli et al., 1998; Tarnacka et al., 2009). Relative exchangeable copper determination may be better at distinguishing patients with Wilson disease from heterozygotes and controls (Trocello et al., 2014). In a study involving 12 heterozygous carriers, without pathologic changes on plain MRI, significantly higher ratios of Glx/Cr and Lip/Cr in 1H magnetic resonance spectroscopy in the pallidum and thalami were reported compared to controls, suggesting possible asymptomatic copper deposition (Tarnacka et al., 2009). Electroencephalographic abnormalities have also been described in heterozygous children. However, such studies, performed prior to the discovery of the ATP7B gene, relied upon the inferred heterozygous carrier state in immediate relatives of affected individuals. Out of 16 presumed heterozygotes, neurologic abnormalities were detected in 5 children. Abnormalities on electroencephalography were detected in 12 children (75%), 5 of which demonstrated epileptic features which did not correlate with clinical symptomatology (Marecek and Nevsimalova, 1984). The findings were replicated in a subsequent study but interestingly appeared to resolve by adulthood, at which time no significant pathologic electroencephalographic abnormalities were detected above that of the control group (Nevsimalova et al., 1986). The potential for heterozygous mutations in the Wilson disease gene to influence other, apparently unrelated disease processes has been suggested. Cocco et al. (2009) described a 37-year-old man with liver cirrhosis secondary to chronic hepatitis C presenting with encephalopathy, in whom treatment with penicillamine and trihexyphenidyl resulted in a marked improvement of his neuropsychiatric symptoms. The patient was later found to be a heterozygous carrier of the G1111A missense mutation, his response to treatment suggesting a subtle defect in brain copper metabolism (Cocco et al., 2009). In another case, screening of an otherwise asymptomatic 18-year-old man, the sibling of a patient recently diagnosed with Wilson disease, revealed deranged liver function tests. Indices of copper metabolism were normal and further investigation led to a diagnosis of Alagille syndrome, a rare autosomal dominantly inherited disorder of embryogenesis, affecting multiple organ systems, in which abnormalities in bile duct formation led to liver dysfunction. The associated JAG1 gene mutation was found to have arisen de novo but, interestingly, one

13

H1069Q mutation was also identified. The authors speculated on the unusual occurrence of two rare conditions within a single family and postulated a potential interplay between the mutations that might account for an unusually late presentation of Alagille syndrome in the absence of bile duct paucity typically observed on liver biopsy (Amson et al., 2012).

CLINICAL COURSE Three main patterns of presentation have been recognized: hepatic, neurologic, and psychiatric, the symptoms of the latter two groups sometimes being combined. The presentations observed by clinicians may be skewed by the specialties to which patients present. Unsurprisingly, neurologists report a greater proportion of patients presenting with neurologic symptoms (69.1%) compared to hepatic symptoms (14.9%), whereas the opposite is true with patients presenting to gastroenterologists, where hepatic features predominate (68.1%) (Taly et al., 2007; Weiss et al., 2011). In a registry of 627 patients with Wilson disease seen at a neurology department in Poland, 510 of whom were symptomatic at the point of diagnosis, a male preponderance was observed (55%). Neurologic and hepatic symptoms and signs were seen more frequently in men (60%) and women (57%) respectively. Overall, symptoms and signs were found to precede a diagnosis of Wilson disease by around 2 years. The mean age of symptom onset in the hepatic group was about 4 years earlier than in the group with neuropsychiatric symptoms (23.7  8 years versus 28.0  8 years) (Litwin et al., 2012). Similar findings were reported in an independent German cohort of 163 patients with Wilson disease, 137 of whom were symptomatic. Though the mean age of symptom onset was younger than that described in the Polish cohort, hepatic symptoms were again found to precede neurologic symptoms (15.5 compared to 20.2 years of age). The interval between symptom onset and diagnosis was reported to be almost three times longer in patients presenting with neurologic symptoms compared to hepatic symptoms (44.4 months vs. 14.4 months) (Merle et al., 2007). Individuals may present with a spectrum of hepatic symptoms, with 25.4% of patients described by Gheorghe et al. (2004) having clinically asymptomatic disease, 21.8% fulminant hepatic failure, and 52.8% chronic liver disease at presentation . Untreated, hepatic symptoms may remain self-limiting, yet may also progress and precede the development of neurologic sequelae. Reassuringly, in treated cases, the prognosis of hepatic Wilson disease is good (Schilsky, 2014b). Of patients presenting with neurologic symptoms, improvement in 53.5–58.2%, stability in 22.4–27.3%,

14

C. LO AND O. BANDMANN

and worsening in 3.6–24.1% may be expected after pharmacologic treatment (Merle et al., 2007; Bruha et al., 2011). Psychiatric symptoms have been reported in up to 51% of patients upon their initial presentation and 72% of patients established on treatment, although a pure psychiatric presentation is rare (Dening and Berrios, 1989; Svetel et al., 2009). The most commonly identified symptoms on the neuropsychiatric inventory include anxiety, depression, irritability, and apathy (Svetel et al., 2009). Cognitive changes, when present, may be subtle and prone to influence by changes in affect (Lang et al., 1990). Normalization may occur in response to treatment (Rosselli et al., 1987). Whilst symptomatic individuals typically present with hepatic, neurologic, or psychiatric manifestations of Wilson disease, less common presentations have been recognized, including presentation with an osseomuscular phenotype as well as renal, cardiovascular, endocrine, hematologic, and dermatologic involvement. The varied presentations of Wilson disease and the role of genetic and environmental modifiers will be detailed in subsequent chapters. With the ability to screen for mutations in the ATP7B gene, a diagnosis of Wilson disease is possible even in the absence of symptoms or definitive biochemical abnormalities. Diagnosis is therefore possible in the presymptomatic stage, even in the newborn. Shimizu et al. (1997) described an asymptomatic 8-month-old boy who was found on opportunistic screening to have low ceruloplasmin levels in the context of otherwise normal biochemical parameters. No signs were demonstrated on clinical examination. A diagnosis of Wilson disease was made on the basis of DNA sequencing analysis, which revealed him to be homozygous for the frameshift mutation 2302insC (Shimizu et al., 1997). A child of the same age was also reported to be the youngest individual to present with evidence of liver dysfunction, detected during a hospital admission with diarrhea. Clinical examination was normal, yet low ceruloplasmin levels raised the suspicion of Wilson disease. Other causes of elevated alanine aminotransferase and aspartate aminotransferase were excluded. Genetic testing identified him to be compound heterozygous for the missense mutations A874V and N1270S (Abuduxikuer et al., 2015). A similar presentation in a 9-month-old boy was reported by Kim et al. (2013). Biochemical evidence of liver dysfunction was investigated further with percutaneous liver biopsy. Elevated hepatic copper content (748 mg/g dry weight of liver tissue) was accompanied by histologic findings of mild lobular activity, mild portoperiportal activity, and periportal fibrosis. Molecular analysis confirmed the presence of the missense mutation G1186S and the frameshift mutation c.4006delA on separate alleles (Kim et al., 2013).

Although classically considered to be associated with an onset in the second or third decade of life, an increasing number of cases of late-onset Wilson disease are being reported (Członkowska and Rodo, 1981; Ala et al., 2005; Ferenci et al., 2007; Sohtaoglu et al., 2007; Członkowska et al., 2008). Two siblings, diagnosed in their eighth decade of life, were described by Ala et al. (2005). The elder sibling had presented at the age of 72 with a 5-year history of progressive neurologic disability. Her younger brother had had a mild hand tremor at the age of 45 that had been treated with a beta-blocker. He was diagnosed at the age of 70 during screening for Wilson disease; his only symptom was mild gait abnormalities. Both siblings were found to be compound heterozygous for the E1064A and H1069Q mutations with stabilization of their clinical course following treatment (Ala et al., 2005). Another similar case, described by Sohtaoglu et al. (2007), was of a woman who developed slurred speech, tremor, and postural instability at the age of 75, though her first symptoms of mild tremor and forgetfulness had begun 8 years earlier. The salient message from her case was of a marked deterioration following the initiation of penicillamine, prompting the authors to caution with respect to its use in older patients with neurologic Wilson disease.

CONCLUSION Considerable advances have been made since the first estimate of the prevalence of Wilson disease in 1968 (Scheinberg and Sternlieb, 1984). As diagnostic techniques have become increasingly sophisticated, so too has our ability to identify the causative mutations in individuals with Wilsondisease. Nonetheless, work from our own recent genetic prevalence study and others has raised concerns that Wilson disease may be considerably more common than previously thought (Ohura et al., 1999; Coffey et al., 2013; Gialluisi et al., 2013). Considerable phenotypic variation, the potential for reduced penetrance, and the diversity in the number of mutations and mechanisms of inheritance may all complicate the diagnosis. Thus, it is necessary to maintain an index of suspicion in individuals presenting with symptoms consistent with a diagnosis of Wilson disease, even though they may not conform to the stereotypic presentation of this disorder.

REFERENCES Abuduxikuer K, Li LT, Qiu YL et al. (2015). Wilson disease with hepatic presentation in an eight-month-old boy. World J Gastroenterol 21: 8981–8984. Ala A, Borjigin J, Rochwarger A et al. (2005). Wilson disease in septuagenarian siblings: raising the bar for diagnosis. Hepatology 41: 668–670.

EPIDEMIOLOGY AND INTRODUCTION TO THE CLINICAL PRESENTATION OF WILSON DISEASE Ala A, Walker AP, Ashkan K et al. (2007). Wilson’s disease. Lancet 369: 397–408. Amson M, Lamoureux E, Hilzenrat N et al. (2012). Alagille syndrome and Wilson disease in siblings: a diagnostic conundrum. Can J Gastroenterol 26: 330–332. Bachmann H, Lossner J, Biesold D (1979a). Wilson’s disease in the German Democratic Republic. I. Genetics and epidemiology. Z Gesamte Inn Med 34: 744–748. Bachmann H, Lossner J, Gruss B et al. (1979b). The epidemiology of Wilson’s disease in the German Democratic Republic and current problems from the viewpoint of population genetics. Psychiatr Neurol Med Psychol (Leipz) 31: 393–400. Bandmann O, Weiss KH, Kaler SG (2015). Wilson’s disease and other neurological copper disorders. Lancet Neurol 14: 103–113. Bennett JT, Schwarz KB, Swanson PD et al. (2013). An exceptional family with three consecutive generations affected by Wilson disease. JIMD Rep 10: 1–4. Bruha R, Marecek Z, Pospisilova L et al. (2011). Long-term follow-up of Wilson disease: natural history, treatment, mutations analysis and phenotypic correlation. Liver Int 31: 83–91. Chabik G, Litwin T, Członkowska A (2014). Concordance rates of Wilson’s disease phenotype among siblings. J Inherit Metab Dis 37: 131–135. Chuang LM, Wu HP, Jang MH et al. (1996). High frequency of two mutations in codon 778 in exon 8 of the ATP7B gene in Taiwanese families with Wilson disease. J Med Genet 33: 521–523. Cocco GA, Loudianos G, Pes GM et al. (2009). “Acquired” hepatocerebral degeneration in a patient heterozygote carrier for a novel mutation in ATP7B gene. Mov Disord 24: 1706–1708. Cocos R, Sendroiu A, Schipor S et al. (2014). Genotype– phenotype correlations in a mountain population community with high prevalence of Wilson’s disease: genetic and clinical homogeneity. PLoS One 9. e98520. Coffey AJ, Durkie M, Hague S et al. (2013). A genetic study of Wilson’s disease in the United Kingdom. Brain 136: 1476–1487. Cullen LM, Prat L, Cox DW (2003). Genetic variation in the promoter and 5’ UTR of the copper transporter, ATP7B, in patients with Wilson disease. Clin Genet 64: 429–432. Curtis D, Durkie M, Balac P et al. (1999). A study of Wilson disease mutations in Britain. Hum Mutat 14: 304–311. Członkowska A, Rodo M (1981). Late onset of Wilson’s disease. Report of a family. Arch Neurol 38: 729–730. Członkowska A, Rodo M, Gromadzka G (2008). Late onset Wilson’s disease: therapeutic implications. Mov Disord 23: 896–898. Członkowska A, Gromadzka G, Chabik G (2009). Monozygotic female twins discordant for phenotype of Wilson’s disease. Mov Disord 24: 1066–1069. Dedoussis GV, Genschel J, Sialvera TE et al. (2005). Wilson disease: high prevalence in a mountainous area of Crete. Ann Hum Genet 69: 268–274.

15

Dening TR, Berrios GE (1989). Wilson’s disease. Psychiatric symptoms in 195 cases. Arch Gen Psychiatry 46: 1126–1134. Dufernez F, Lachaux A, Chappuis P et al. (2013). Wilson disease in offspring of affected patients: report of four French families. Clin Res Hepatol Gastroenterol 37: 240–245. Dziezyc K, Litwin T, Chabik G et al. (2014). Families with Wilson’s disease in subsequent generations: clinical and genetic analysis. Mov Disord 29: 1828–1832. Ferenci P, Członkowska A, Merle U et al. (2007). Late-onset Wilson’s disease. Gastroenterology 132: 1294–1298. Figus A, Angius A, Loudianos G et al. (1995). Molecular pathology and haplotype analysis of Wilson disease in Mediterranean populations. Am J Hum Genet 57: 1318–1324. Garcia-Villarreal L, Daniels S, Shaw SH et al. (2000). High prevalence of the very rare Wilson disease gene mutation Leu708Pro in the Island of Gran Canaria (Canary Islands, Spain): a genetic and clinical study. Hepatology 32: 1329–1336. Gheorghe L, Popescu I, Iacob S et al. (2004). Wilson’s disease: a challenge of diagnosis. The 5-year experience of a tertiary centre. Rom J Gastroenterol 13: 179–185. Gialluisi A, Incollu S, Pippucci T et al. (2013). The homozygosity index (HI) approach reveals high allele frequency for Wilson disease in the Sardinian population. Eur J Hum Genet 21: 1308–1311. Gomes A, Dedoussis GV (2015). Geographic distribution of ATP7B mutations in Wilson disease. Ann Hum Biol: 1–8. Gromadzka G, Schmidt HH, Genschel J et al. (2006). p.H1069Q mutation in ATP7B and biochemical parameters of copper metabolism and clinical manifestation of Wilson’s disease. Mov Disord 21: 245–248. Hahn SH (2014). Population screening for Wilson’s disease. Ann N Y Acad Sci 1315: 64–69. Hahn SH, Lee SY, Jang YJ et al. (2002). Pilot study of mass screening for Wilson’s disease in Korea. Mol Genet Metab 76: 133–136. Iacob R, Iacob S, Nastase A et al. (2012). The His1069Gln mutation in the ATP7B gene in Romanian patients with Wilson’s disease referred to a tertiary gastroenterology center. J Gastrointestin Liver Dis 21: 181–185. Ivanova-Smolenskaya IA, Ovchinnikov IV, Karabanov AV et al. (1999). The His1069Gln mutation in the ATP7B gene in Russian patients with Wilson disease. J Med Genet 36: 174. Kim EK, Yoo OJ, Song KY et al. (1998). Identification of three novel mutations and a high frequency of the Arg778Leu mutation in Korean patients with Wilson disease. Hum Mutat 11: 275–278. Kim JW, Kim JH, Seo JK et al. (2013). Genetically confirmed Wilson disease in a 9-month old boy with elevations of aminotransferases. World J Hepatol 5: 156–159. Kroll CA, Ferber MJ, Dawson BD et al. (2006). Retrospective determination of ceruloplasmin in newborn screening blood spots of patients with Wilson disease. Mol Genet Metab 89: 134–138.

16

C. LO AND O. BANDMANN

Lamont PJ, Davis MB, Wood NW (1997). Identification and sizing of the GAA trinucleotide repeat expansion of Friedreich’s ataxia in 56 patients. Clinical and genetic correlates. Brain 120 (Pt 4): 673–680. Lang C, Muller D, Claus D et al. (1990). Neuropsychological findings in treated Wilson’s disease. Acta Neurol Scand 81: 75–81. Litwin T, Gromadzka G, Członkowska A (2012). Gender differences in Wilson’s disease. J Neurol Sci 312: 31–35. Liu XQ, Zhang YF, Liu TT et al. (2004). Correlation of ATP7B genotype with phenotype in Chinese patients with Wilson disease. World J Gastroenterol 10: 590–593. Loudianos G, Dessi V, Lovicu M et al. (1998). Haplotype and mutation analysis in Greek patients with Wilson disease. Eur J Hum Genet 6: 487–491. Loudianos G, Dessi V, Lovicu M et al. (1999a). Mutation analysis in patients of Mediterranean descent with Wilson disease: identification of 19 novel mutations. J Med Genet 36: 833–836. Loudianos G, Dessi V, Lovicu M et al. (1999b). Molecular characterization of Wilson disease in the Sardinian population – evidence of a founder effect. Hum Mutat 14: 294–303. Loudianos G, Zappu A, Lepori MB et al. (2013). Wilson’s disease in two consecutive generations: the detection of three mutated alleles in the ATP7B gene in two Sardinian families. Dig Liver Dis 45: 342–345. Lough J, Wiglesworth FW (1976). Wilson disease. Comparative ultrastructure in a sibship of nine. Arch Pathol Lab Med 100: 659–663. Lovicu M, Dessi V, Zappu A et al. (2003). Efficient strategy for molecular diagnosis of Wilson disease in the Sardinian population. Clin Chem 49: 496–498. Lyon TD, Fell GS, Gaffney D et al. (1995). Use of a stable copper isotope (65Cu) in the differential diagnosis of Wilson’s disease. Clin Sci (Lond) 88: 727–732. Mak CM, Lam CW, Tam S et al. (2008). Mutational analysis of 65 Wilson disease patients in Hong Kong Chinese: identification of 17 novel mutations and its genetic heterogeneity. J Hum Genet 53: 55–63. Marecek Z, Nevsimalova S (1984). Biochemical and clinical changes in Wilson’s disease heterozygotes. J Inherit Metab Dis 7: 41–45. Margarit E, Bach V, Gomez D et al. (2005). Mutation analysis of Wilson disease in the Spanish population – identification of a prevalent substitution and eight novel mutations in the ATP7B gene. Clin Genet 68: 61–68. Merle U, Schaefer M, Ferenci P et al. (2007). Clinical presentation, diagnosis and long-term outcome of Wilson’s disease: a cohort study. Gut 56: 115–120. Merle U, Weiss KH, Eisenbach C et al. (2010). Truncating mutations in the Wilson disease gene ATP7B are associated with very low serum ceruloplasmin oxidase activity and an early onset of Wilson disease. BMC Gastroenterol 10: 8. Merli M, Patriarca M, Loudianos G et al. (1998). Use of the stable isotope 65Cu test for the screening of Wilson’s disease in a family with two affected members. Ital J Gastroenterol Hepatol 30: 270–275.

Nakayama K, Kubota M, Katoh Y et al. (2008). Early and presymptomatic detection of Wilson’s disease at the mandatory 3-year-old medical health care examination in Hokkaido Prefecture with the use of a novel automated urinary ceruloplasmin assay. Mol Genet Metab 94: 363–367. Nanji MS, Nguyen VT, Kawasoe JH et al. (1997). Haplotype and mutation analysis in Japanese patients with Wilson disease. Am J Hum Genet 60: 1423–1429. Nevsimalova S, Marecek Z, Roth B (1986). An EEG study of Wilson’s disease. Findings in patients and heterozygous relatives. Electroencephalogr Clin Neurophysiol 64: 191–198. Ohura T, Abukawa D, Shiraishi H et al. (1999). Pilot study of screening for Wilson disease using dried blood spots obtained from children seen at outpatient clinics. J Inherit Metab Dis 22: 74–80. Owada M, Suzuki K, Fukushi M et al. (2002). Mass screening for Wilson’s disease by measuring urinary holoceruloplasmin. J Pediatr 140: 614–616. Park RH, McCabe P, Fell GS et al. (1991). Wilson’s disease in Scotland. Gut 32: 1541–1545. Przuntek H, Hoffmann E (1987). Epidemiologic study of Wilson’s disease in West Germany. Nervenarzt 58: 150–157. Reilly M, Daly L, Hutchinson M (1993). An epidemiological study of Wilson’s disease in the Republic of Ireland. J Neurol Neurosurg Psychiatry 56: 298–300. Rosselli M, Lorenzana P, Rosselli A et al. (1987). Wilson’s disease, a reversible dementia: case report. J Clin Exp Neuropsychol 9: 399–406. Saito T (1981). An assessment of efficiency in potential screening for Wilson’s disease. J Epidemiol Community Health 35: 274–280. Scheinberg IH, Sternlieb I (1984). Wilson’s disease, W.B. Saunders, Philadelphia. Schilsky ML (2014a). A century for progress in the diagnosis of Wilson disease. J Trace Elem Med Biol 28: 492–494. Schilsky ML (2014b). Long-term outcome for Wilson disease: 85% good. Clin Gastroenterol Hepatol 12: 690–691. Schols L, Arning L, Schule R et al. (2008). “Pseudodominant inheritance” of ataxia with ocular apraxia type 2 (AOA2). J Neurol 255: 495–501. Shah AB, Chernov I, Zhang HT et al. (1997). Identification and analysis of mutations in the Wilson disease gene (ATP7B): population frequencies, genotype–phenotype correlation, and functional analyses. Am J Hum Genet 61: 317–328. Shimizu N, Nakazono H, Watanabe A et al. (1997). Molecular diagnosis of Wilson’s disease. Lancet 349: 1811–1812. Sohtaoglu M, Ergin H, Ozekmekci S et al. (2007). Patient with late-onset Wilson’s disease: deterioration with penicillamine. Mov Disord 22: 290–291. Stapelbroek JM, Bollen CW, van Amstel JK et al. (2004). The H1069Q mutation in ATP7B is associated with late and neurologic presentation in Wilson disease: results of a meta-analysis. J Hepatol 41: 758–763. Sternlieb I, Scheinberg IH (1968). Prevention of Wilson’s disease in asymptomatic patients. N Engl J Med 278: 352–359. Svetel M, Potrebic A, Pekmezovic T et al. (2009). Neuropsychiatric aspects of treated Wilson’s disease. Parkinsonism Relat Disord 15: 772–775.

EPIDEMIOLOGY AND INTRODUCTION TO THE CLINICAL PRESENTATION OF WILSON DISEASE Taly AB, Meenakshi-Sundaram S, Sinha S et al. (2007). Wilson disease: description of 282 patients evaluated over 3 decades. Medicine (Baltimore) 86: 112–121. Tanzi RE, Petrukhin K, Chernov I et al. (1993). The Wilson disease gene is a copper transporting ATPase with homology to the Menkes disease gene. Nat Genet 5: 344–350. Tarnacka B, Gromadzka G, Rodo M et al. (2000). Frequency of His1069Gln and Gly1267Lys mutations in Polish Wilson’s disease population. Eur J Neurol 7: 495–498. Tarnacka B, Szeszkowski W, Buettner J et al. (2009). Heterozygous carriers for Wilson’s disease – magnetic spectroscopy changes in the brain. Metab Brain Dis 24: 463–468. Thomas GR, Jensson O, Gudmundsson G et al. (1995). Wilson disease in Iceland: a clinical and genetic study. Am J Hum Genet 56: 1140–1146. Trocello JM, El Balkhi S, Woimant F et al. (2014). Relative exchangeable copper: a promising tool for family screening in Wilson disease. Mov Disord 29: 558–562.

17

Wang LH, Huang YQ, Shang X et al. (2011). Mutation analysis of 73 southern Chinese Wilson’s disease patients: identification of 10 novel mutations and its clinical correlation. J Hum Genet 56: 660–665. Weiss KH, Gotthardt DN, Klemm D et al. (2011). Zinc monotherapy is not as effective as chelating agents in treatment of Wilson disease. Gastroenterology 140: 1189–1198. e1181. Yamaguchi Y, Aoki T, Arashima S et al. (1999). Mass screening for Wilson’s disease: results and recommendations. Pediatr Int 41: 405–408. Zappu A, Magli O, Lepori MB et al. (2008). High incidence and allelic homogeneity of Wilson disease in 2 isolated populations: a prerequisite for efficient disease prevention programs. J Pediatr Gastroenterol Nutr 47: 334–338. Zappu A, Lepori MB, Incollu S et al. (2010). Development of TaqMan allelic specific discrimination assay for detection of the most common Sardinian Wilson’s disease mutations. Implications for genetic screening. Mol Cell Probes 24: 233–235.

Handbook of Clinical Neurology, Vol. 142 (3rd series) Wilson Disease A. Członkowska and M.L. Schilsky, Editors http://dx.doi.org/10.1016/B978-0-444-63625-6.00003-3 © 2017 Elsevier B.V. All rights reserved

Chapter 3

The genetics of Wilson disease 1

IRENE J. CHANG1 AND SI HOUN HAHN2* Division of Medical Genetics, Department of Medicine, University of Washington School of Medicine, Seattle, WA, USA

2

Division of Genetic Medicine, Department of Pediatrics, University of Washington School of Medicine, Seattle Children’s Hospital, Seattle, WA, USA

Abstract Wilson disease (WD) is an autosomal-recessive disorder of hepatocellular copper deposition caused by pathogenic variants in the copper-transporting gene, ATP7B. Early detection and treatment are critical to prevent lifelong neuropsychiatric, hepatic, and systemic disabilities. Due to the marked heterogeneity in age of onset and clinical presentation, the diagnosis of Wilson disease remains challenging to physicians today. Direct sequencing of the ATP7B gene is the most sensitive and widely used confirmatory testing method, and concurrent biochemical testing improves diagnostic accuracy. More than 600 pathogenic variants in ATP7B have been identified, with single-nucleotide missense and nonsense mutations being the most common, followed by insertions/deletions, and, rarely, splice site mutations. The prevalence of Wilson disease varies by geographic region, with higher frequency of certain mutations occurring in specific ethnic groups. Wilson disease has poor genotype–phenotype correlation, although a few possible modifiers have been proposed. Improving molecular genetic studies continue to advance our understanding of the pathogenesis, diagnosis, and screening for Wilson disease.

INTRODUCTION In this chapter, we will discuss the inheritance, gene frequency, variants, genotype–phenotype correlation, and modifiers of the ATP7B gene, and the clinical molecular diagnosis and population screening for Wilson disease.

INHERITANCE Wilson disease is a monogenic autosomal-recessive condition and carriers do not manifest any symptoms. Autosomal-recessive conditions are not usually present in consecutive generations, but may occur in populations with particularly high carrier frequency of Wilson disease (Wu et al., 2015). Our group and others have reported the presence of Wilson disease in two or more successive generations within the same family, reflecting a “pseudo-dominant” inheritance (Dziezyc et al., 2011, 2014; Bennett et al., 2013; H. Park et al., 2015). Therefore, the diagnosis of Wilson disease should not be

excluded simply due to a misleading family history consistent with an autosomal-dominant inheritance pattern. Furthermore, recent studies have also identified Wilson disease due to atypical forms of inheritance, such as the presence of three concurrent mutations in a single patient or segmental uniparental disomy (Coffey et al., 2013). Uniparental disomy occurs when both homologs of a chromosome originate from a single parent. These findings have implications for clinical practice and genetic counseling, as clinicians may need to consider genotyping asymptomatic parents or obtaining full sequencing of ATP7B to confirm that pathogenic variants occur in trans.

ATP7B GENE AND ATPASE Wilson disease is caused by homozygous or compound heterozygous mutations in the ATP7B gene (OMIM# 606882), which encodes a transmembrane coppertransporting P-type ATPase of the same name. Currently,

*Correspondence to: Si Houn Hahn, MD, PhD, Department of Pediatrics, University of Washington School of Medicine, Seattle Children’s Hospital, Seattle WA 98105, USA. Tel: +1-206-987-7610, Fax: +1-206-987-5329, E-mail: [email protected]

20

I.J. CHANG AND S.H. HAHN

Fig. 3.1. Schematic representation of copper-induced relocalization of ATP7A and ATP7B. The left side of the diagram represents an enterocyte and the right side represents a hepatocyte. On both sides, copper enters the cell through copper transporter 1 (CTR1) and is escorted by copper chaperone antioxidant protein 1 (ATOX1) to ATP7A or ATP7B in the trans-Golgi network (TGN). When copper levels rise above a certain threshold, ATP7A and ATP7B excrete copper into the plasma on the basolateral side of the enterocyte and into the bile on the apical side of the hepatocyte. Defects in localization of ATP7B may lead to copper accumulation at the (1) TGN due to unresponsiveness, (2) cell periphery, and (3) endoplasmic reticulum (ER) due to misfolding. (Reproduced from de Bie et al., 2007.)

ATP7B is the only identified gene known to cause Wil son disease (Bull et al., 1993; Petrukhin et al., 1993; Tanzi et al., 1993). Mutations in the ATP7B gene have been reported in almost all exons. Previous studies have reported individuals with both biochemical and clinical diagnosis of Wilson disease in the absence of two ATP7B mutations, raising the possibility of a second causative gene (Lovicu et al., 2006; Kenney and Cox, 2007; S. Park et al., 2007; Mak and Lam, 2008; Nicastro et al., 2010; Coffey et al., 2013). Nonetheless, ATP7B remains the only known gene responsible for Wilson disease. Human dietary intake of copper is about 1.5–2.5 mg/day, which is absorbed in the stomach and duodenum, bound to circulating albumin, and transported to the liver for regulation and excretion (Culotta and Scott, 2016). The uptake of copper occurs on the basolateral side of hepatocytes via copper transporter 1 (CTR1), as illustrated in Figure 3.1. A specific copper chaperone, antioxidant protein 1 (ATOX1), delivers copper to the Wilson disease protein, ATP7B, by copperdependent protein–protein interactions (Walker et al., 2004). Within hepatocytes, ATP7B performs two important functions in either the trans-Golgi network (TGN) or in cytoplasmic vesicles. In the TGN, ATP7B activates ceruloplasmin by packaging six copper molecules into apoceruloplasmin, which is then secreted into the plasma. In the cytoplasm, ATP7B sequesters excess

copper into vesicles and excretes it via exocytosis across the apical canalicular membrane into bile (Bull et al., 1993; Tanzi et al., 1993; Yamaguchi et al., 1999; Cater et al., 2007). Due to the binary role of the ATP7B transporter in both the synthesis and excretion of copper, defects in its function lead to copper accumulation and the progressive features of Wilson disease (Fig. 3.1).

MOLECULAR STRUCTURE OF ATP7B ATP7B is located on 13q14.3 and contains 20 introns and 21 exons, for a total genomic length of 80 kb (Bull et al., 1993; Petrukhin et al., 1993; Tanzi et al., 1993). The gene is synthesized in the endoplasmic reticulum, then relocated to the TGN within hepatocytes. ATP7B is most highly expressed in the liver, but is also found in the kidney, placenta, mammary glands, brain, and lung.

ATP7B (P-TYPE ATPASE) PROTEIN STRUCTURE AND FUNCTION ATP7B belongs to class 1B (PIB) of the highly conserved P-type ATPase superfamily, which is responsible for the transport of copper and other heavy metals across cellular membranes (Gourdon et al., 2011). The protein contains 1465 amino acids, a phosphatase domain (A-domain), phosphorylation domain (P-domain, amino acid residues 971–1035), nucleotide-binding domain (N-domain, amino acid residues 1240–1291), and

THE GENETICS OF WILSON DISEASE 5’UTR

2

M

3

Tm

Tm

Tm

4

5 6 7

Tm

Tm

Tm

Tm

D K T G

COOH

ND

GV

Cu

9 10 11 12 13 14 15 16 17 18 19 20 21

GD

PD

Cu Cu

Tm

8

21

Hinge region

NH2 Phosphorylation domain

Cu

Cu Cu

SEHPL

N

D TG

ATP binding site

Fig. 3.2. Schematic representation of ATP7B gene and corresponding human ATP7B protein. Top diagram shows 5’UTR promoter region and exons separated by introns. Bottom diagram shows the domain organization of human copper ATPase. Conserved amino acid motifs are present at the core structure of each functional domain, i.e., TGDN and GDGVND at the A-domain, DKTG at the P-domain, and SEHPL in the N-domain. M, phospholipidic bilayer of the membrane; Cu, the metal-binding domains of the trasmembrane cation channel; Tm, transmembrane domains; PD, phosphatase domain. (Reproduced from Fanni et al., 2005.)

M-domain, which comprises eight transmembrane ion channels (Fig. 3.2) (Cater et al., 2004, 2007; Lenartowicz and Krzeptowski, 2010). Unique amino acid motifs are present at the core structure of each domain, such as TGEA at the A-domain, DKTGT at the P-domain, and SEHPL in the N-domain. Specifically, the N-terminal metal-binding domain (MBD) is composed of six copper-binding sites, each with the conserved sequence motif GMXCXXC (Fatemi and Sarkar, 2002; Sazinsky et al., 2006). These MBDs play a central role in accepting copper from copper chaperone ATOX1 through protein–protein interactions. Previous studies have demonstrated unequal impact of MBDs on ATP7B activity, with MBD 5 and 6 having stronger effects on the catalytic activation of ATP7B than MBDs 1–4 (Lutsenko et al.,1997). The active transport of copper across membranes is a complex process that begins with ATP7B binding copper at the N-terminal domain and transporting it across cellular membranes, using ATP as an energy source (Fig. 3.2). Next, free copper binds intracellularly to GG motifs in the MBDs, followed by transport on to the Cys-Pro-Cys (CPC) sequence motifs in MBD 6. Finally, dephosphorylation of acyl-phosphate at the A-domain discharges copper across the cellular membrane. Mutations causing copper accumulation may occur at any of these steps (Huster et al., 2006; Schushan et al., 2012).

Although the mechanism by which the histadinecontaining SEHPL motif affects copper transport remains to be elucidated, it is clear that histidine-toglutamate substitution at amino acid 1069 (p.H1069Q) in this motif is the most common cause of Wilson disease in northern Europeans. In the hepatocytes of patients homozygous for p.H1069Q, ATP7B was found in the endoplasmic reticulum instead of its usual TGN location, suggesting abnormal protein trafficking (Huster et al., 2003). Insect models with the p.H1069Q mutation in SF9 cells showed decreased ATP-mediated catalytic phosphorylation but no major protein misfolding, suggesting a role for p.H1069Q in the orientation of the ATP7B catalytic site for ATP binding prior to hydrolysis (Tsivkovskii et al., 2003).

VARIANTS IN THE ATP7B GENE More than 600 pathogenic variants in ATP7B have been identified, with single-nucleotide missense and nonsense mutations being the most common, followed by insertions/deletions and splice site mutations (Human Gene Mutation Database, accessed 29 April 2016; Stenson et al., 2014). Other rare genetic mechanisms that have been reported in the literature include whole-exon deletions, promoter region mutations, three concurrent pathogenic variants, and monogenic disomy (Coffey et al., 2013; Bandmann et al., 2015). Mutation “hotspots” in

22

I.J. CHANG AND S.H. HAHN Transmembrane (1-6)

Cu binding

2 p.C271* p.E122fs c.-441_-427del

3

4

5

7

p.M645R p.Q457* p.c.1708-1G>C p.G691R p.Y670*

8

9

10

p.R778L p.L795F p.G710S c.2299insC p.S774R p.W779X p.L708P p.M769fs p.W779* p.A1003T

11

12

13

14

15

16

c.3400delC p.L1083F p.A1135Q p.I1002T

p.G943D p.A874V p.P992H p.A1003V

p.V845fs p.K832R

Transmembrane (7-8)

ATP binding

p.H1069Q p.E1064K p.G1061E p.R969Q p.A1003T p.P992L p.P992H p.T977M p.N958fs

17

18

19

p.N1270S p.L1273S p.N1270S p.G1266R c.3903+6C>T

p.V1146M p.A11140V

20

21

c.4193delC p.Q1399R p.Q1399R p.S774R

p.G1351* p.L1371P

Fig. 3.3. Schematic of the ATP7B gene with common mutation sites, including p.H1069Q (rs76151636), p.R778L (rs28942074), p.E1064K (rs376910645), c.3400delC, and p.Ala1135fs (rs137853281). Please refer to Table 3.1 for more details.

ATP7B have also been reported to vary by geographic region (see regional gene frequency section, below). The majority of pathogenic mutations are located in the M- and N-domains in presymptomatic patients or in those with hepatic symptoms (S. Park et al., 2007). The common mutations in ATP7B seen in various populations are listed in Figure 3.3. The p.H1069Q mutation is one of the most common mutations, with a population allelic frequency of 10–40% (30–70% among Caucasians). Most patients are compound heterozygotes, carrying different mutations on each copy of the chromosome (Usta et al., 2014). The p.H1069Q mutation occurs when histidine of the conserved SEHPL motif in the N-domain of ATP7B is replaced by glutamic acid, resulting in N-domain protein misfolding, abnormal phosphorylation in the P-domain, and decreased ATP binding affinity (Rodriguez-Granillo et al., 2008). This mutation also leads to decreased heat stability and abnormal localization of the protein to the TGN (Ralle et al., 2010). Other common mutations in ATP7B include p. E1064A, p.R778L, p.G943S, and p.M769V. Mutations in p.E1064A, also found in the SEHPL motif, completely disable ATP binding affinity but do not result in protein misfolding, transport abnormalities, or thermal instability. The p.R778L mutation affects transmembrane transport of copper (Dmitriev et al., 2011). The p.G943S and p.M769V mutations result in defective copper metabolism but preserved ceruloplasmin levels (Okada et al., 2010). A substantial proportion of Wilson disease-associated missense mutations, including p.H1069Q and p.R778L, result in markedly decreased level of the protein caused by enhanced degradation (Payne et al., 1998; de Bie

et al., 2007; van den Berghe et al., 2009). Other prevalent mutations, such as protein-truncating nonsense mutations (13% of known point mutations) (Merle et al., 2010) and frameshift mutations (Vrabelova et al., 2005), are predicted to cause decay of mRNA (Mendell et al., 2004; Chang et al., 2007) or a severely truncated protein, resulting in absent or diminished levels of protein. It is therefore expected that most patients with Wilson disease have absent or significantly reduced levels of ATP7B.

REGIONAL GENE FREQUENCY The prevalence of Wilson disease varies by geographic region, with higher prevalence of specific mutations reported in certain populations (Ferenci, 2006) (see Chapter 2 for more details). A list of the common regional variants of ATP7B mutations and geographic clustering of mutations are shown in Table 3.1 and Figure 3.4, respectively.

GENOTYPE–PHENOTYPE CORRELATION Direct genotype–phenotype relationships in Wilson disease have been difficult to establish, despite several studies examining correlation (Panagiotakaki et al., 2004; Vrabelova et al., 2005; Nicastro et al., 2010; Cocoş et al., 2014; Usta et al., 2014). The numerous low-frequency and compound heterozygous nature of Wilson disease obfuscate the process of characterizing its numerous genetic variants and their clinical consequences. Descriptions of phenotypes are limited to age of onset and presenting symptoms, both of which may be affected by inaccurate diagnostic criteria, delayed diagnosis, and practitioner selection bias. Therefore,

Table 3.1 Regional distribution of common Wilson disease mutations by geographic location Prevalent mutations Region Europe Austria (Ferenci, 2006)

Benelux (Ferenci, 2006) Bulgaria (Todorov et al., 2005) Canary Islands (García Villarreal et al., 2000) Czech Republic (Vrabelova et al., 2005) Denmark (Møller et al., 2011) France (Bost et al., 2012) Germany (Ferenci, 2006) Germany (East, former) (Caca et al., 2000) Greece (Panagiotakaki et al., 2004; Dedoussis et al., 2005; Gomes and Dedoussis, 2016) Hungary (Firneisz et al., 2002; Folhoffer et al., 2007) Iceland (Thomas et al., 1995a; Hofer et al., 2012) Italy (Loudianos et al., 1999)

Netherlands (Stapelbroek et al., 2004)

AF (%)

Protein

Nucleotide

RS

Exon

Type

Domain

34.1 6.4 3.6 53 58.8 64

p.His1069Gln p.Gly710Ser p.Met769fs p.His1069Gln p.His1069Gln p.Leu708Pro

c.3207C > A c.2128G > A c.2298_2299insC c.3207C > A c.3207C > A c.2123 T > C

rs76151636

14 8 8 14 14 8

Missense Missense Premature stop Missense Missense Missense

ATP loop TM2 TM4 ATP loop ATP loop TM2

57

p.His1069Gln

c.3207C > A

rs76151636

14

Missense

ATP loop

18

p.His1069Gln

c.3207C > A

rs76151636

14

Missense

ATP loop

16 15

p.Trp779* p.His1069Gln

c.2336G > A c.3207C > A

rs137853283 rs76151636

8 14

Nonsense Missense

TM4 ATP loop

47.9

p.His1069Gln

c.3207C > A

rs76151636

14

Missense

ATP loop

63

p.His1069Gln

c.3207C > A

rs76151636

14

Missense

ATP loop

35

p.His1069Gln

c.3207C > A

rs76151636

14

Missense

ATP loop

12 42.9

p.Arg969Gln p.His1069Gln

c.2906G > A c.3207C > A

rs774028495 rs76151636

13 14

Missense Missense

TM6 ATP loop

100

p.Tyr670*

c.2007_2013del

7

Nonsense

TM1

17.5

p.His1069Gln

c.3207C > A

rs76151636

14

Missense

ATP loop

9 6 33

p.Val845fs p.Met769fs p.His1069Gln

c.2530delA c.2298_2299insC c.3207C > A

rs755709270 rs137853287 rs76151636

10 8 14

Premature stop Premature stop Missense

Td TM4 ATP loop

rs137853287 rs76151636 rs76151636

Continued

Table 3.1 Continued Prevalent mutations Region

AF (%)

Protein

Nucleotide

RS

Exon

Type

Domain

Poland (Gromadzka et al., 2005)

72

p.His1069Gln

c.3207C > A

rs76151636

14

Missense

ATP loop

7.3 3.7 38.1 49

p.Ala1135fs p.Gln1351* p.His1069Gln p.His1069Gln

c.3400delC c.4051C > T c.3207C > A c.3207C > A

rs137853281

15 20 14 14

Premature stop Nonsense Missense Missense

ATP loop

c.-441_-427del

5prime

Unknown

Promoter

Deletion Missense Missense

TM4/Td ATP loop ATP loop

Romania (Iacob et al., 2012) Russia (Ivanova-Smolenskaya et al., 1997) Sardinia (Figus et al., 1995)

Serbia (Tomi
c et al., 2013)

Spain (Margarit et al., 2005) Sweden (Shah et al., 1997) Turkey (Ferenci, 2006; Simsek Papur et al., 2013)

UK (Coffey et al., 2013) Yugoslavia (former) (Loudianos et al., 1999) Asia China (Gu et al., 2003; Z.-Y. Wu et al., 2003; Wang et al., 2011; Wei et al., 2014)

60.5

rs76151636 rs76151636

ATP loop ATP loop

8.5 7.9 38.4

p.Met822fs p.Val1146Met p.His1069Gln

c.2463delC c.3436G > A c.3207C > A

rs76151636

10 16 14

11.6 9.3 27

p.Met769fs p.Ala1003Thr p.Met645Arg

c.2304dupC c.3007G > A c.1934 T > G

rs1801247 rs121907998

8 13 6

Missense Missense Missense

TM4 TM6/Ph Cu6/TM1

38

p.His1069Gln

c.3207C > A

rs76151636

14

Missense

ATP loop

17.4

p.His1069Gln

c.3207C > A

rs76151636

14

Missense

ATP loop

5.3 4.53 19

p.Gly710Ser p.Gln457* p.His1069Gln

c.2128G > A c.1369C > T c.3207C > A

rs772595172 rs76151636

8 3 14

Missense Nonsense Missense

TM2 Cu4/Cu5 ATP loop

8 48.9

p.Met769Val p.His1069Gln

c.2305A > G c.3207C > A

rs76151636

8 14

Missense Missense

TM4 ATP loop

11.4

p.Met769fs

c.2298_2299insC

rs137853287

8

Premature stop

TM4

31

p.Arg778Leu

c.2332C > T

rs28942074

8

Missense

TM4

10 9.6 3.3 19

p.Pro992Leu p.Ile1148Thr p.Thr935Met p.Arg778Leu

c.2975C > T c.3443 T > C c.2804C > T c.2332C > T

rs201038679 rs60431989

13 16 12 8

Missense Missense Missense Missense

TM6/Ph ATP loop TM5 TM4

rs28942074

North India (S. Kumar et al., 2006; Gupta et al., 2007)

South India (Santhosh et al., 2006; S. S. Kumar et al., 2012)

East India (Gupta et al., 2005)

West India (Aggarwal and Bhatt, 2013; Aggarwal et al., 2013)

Japan (Okada et al., 2000; Tatsumi et al., 2010)

Korea (E. K. Kim et al., 1998; Yoo, 2002; G.-H. Kim et al., 2008; Song et al., 2012)

Lebanon (Usta et al., 2014) Saudi Arabia (Al Jumah et al., 2004; Majumdar et al., 2004) Taiwan (Lee et al., 2000; Wan et al., 2006)

12 9 11

p.Ile1102Thr p.Pro992His p.Ala1003Val

c.3305 T > C c.2975C > A c.3008C > T

rs560952220

15 13 13

Missense Missense Missense

ATP loop TM6/Ph TM6/Ph

11 9 9 16

p.Cys271* p.Pro768Leu p.Arg969Gln p.Cys271*

c.813C > A c.2303C > T c.2906G > A c.813C > A

rs572147914 rs121907996 rs572147914

2 8 13 2

Nonsense Missense Missense Nonsense

Cu3 TM4 TM6 Cu3

11 8.5 20

p.Gly1061Glu p.Cys271*

c.3182G > A c.1708-1G > C c.813C > A

rs137853280 rs572147914

14 5 2

Missense Splice Nonsense

ATP loop Cu6 Cu3

11

p.Glu122fs

2

Ins/Del

Cu1

6 6 17.95

p.Thr977Met p.Leu795Phe p.Asn958fs

rs72552255

13 9 13

Missense Missense Premature stop

TM6 TM4/Td TM5/TM6

16.7 10.5 37.9

p.Arg778Leu

rs28942074

p.Arg778Leu

c.2332C > T c.1708-5 T > G c.2332C > T

8 5 8

Missense Splice Missense

TM4 Cu6 TM4

12.1 9.4 8 44.7

p.Asn1270Ser p.Ala874Val p.Leu1083Phe p. Ala1003Thr

c.3809A > G c.2621C > T c.3247C > T c.2299insC

rs121907990 rs376355660

18 11 15 8

Missense Missense Missense Missense

ATP hinge TM5 ATP loop TM4

32

p.Gln1399Arg

c.4196A > G

21

Missense

After TM8

16 29.6

p.Ser774Arg p.Arg778Leu

c.2230 T > C c.2332C > T

rs535217574 rs28942074

21 8

Missense Missense

TM3 TM4

8.9 4.8

p.Pro992Leu p.Gly943Asp

c.2975C > T c.2828G > A

rs201038679

13 12

Missense Missense

TM6 TM5

c.365_366delins TTCGAAGC c.2930C > T c.2383C > T c.2871delC

rs28942074

rs137853287

Continued

Table 3.1 Continued Prevalent mutations Region

AF (%)

Protein

Nucleotide

RS

Exon

Type

Domain

Thailand (Panichareon et al., 2011)

10.52

p.Arg778Leu

c.2332C > T

rs28942074

8

Missense

TM4

7.89 19

p.Leu1371Pro p.His1069Gln

c.4112 T > C c.3207C > A

rs76151636

20 14

Missense Missense

TM8 ATP loop

42.2

IVS18 + 6 T > C

c.3903 + 6C > T

rs2282057

18

Splice

40.6 26.5

p.Ala11140Val p.Lys832Arg

c.3419C > T c.2495A > G

rs1061472

16 10

Missense Missense

ATP loop TM4/Td

40.3

p.His1069Gln

c.3207C > A

rs76151636

14

Missense

ATP loop

1.9 1.9 37.1

p.Asn1270Ser p.Gly1266Arg p.His1069Gln

c.3809A > G c.3796G > A c.3207C > A

rs121907990 rs121907992 rs76151636

18 18 14

Missense Missense Missense

ATP hinge ATP hinge ATP loop

31.25 11.4

c.3400delC c.3402delC c.2123 T > C c.3809A > G

rs137853281 rs137853281

61

p.Ala1135fs p.Ala1135GlnfsX13 p.Leu708Pro p.Asn1270Ser

rs121907990

15 15 8 18

Premature stop Premature stop Missense Missense

ATP loop ATP loop TM2 ATP hinge

26.9

p.Ala1135GlnfsX13

c.3402delC

rs137853281

15

Premature stop

ATP loop

9.6

p.Gly691Arg

c.2071G > A

7

Missense

TM2

Iran (Zali et al., 2011) Africa Egypt (Abdelghaffar et al., 2008; Abdel Ghaffar et al., 2011)

Americas USA (Kuppala et al., 2009)

Brazil (Deguti et al., 2004; Machado et al., 2008; Bem et al., 2013)

Costa Rica (Shah et al., 1997) Venezuela (Paradisi et al., 2015)

AF, allelic frequency; RS, Reference single nucleotide polymorphism (SNP) cluster identification number.

THE GENETICS OF WILSON DISEASE

27

Fig. 3.4. Prevalence of ATP7B mutation by geographic region; the darker the gradient, the higher the allelic frequency. (Reproduced from Gomes and Dedoussis, 2016, with permission from Taylor and Francis.)

the marked variability in phenotype of Wilson disease is likely attributable to an amalgamation of genetic, metabolic, and environmental factors (Leggio et al., 2006). The most consistent genotype–phenotype correlation in Wilson disease is that the most severe, early-onset disease with predominantly hepatic presentation is associated with mutations causing absent ATPase activity. Convincing studies have demonstrated fulminant hepatic disease in mouse models such as the toxic milk (tx) mouse and the Jackson tx mouse (txj), which harbor point mutations causing loss of ATP7B function, but not affecting ATP7B synthesis (Theophilos et al., 1996; Coronado et al., 2001; La Fontaine et al., 2001; Huster et al., 2006). Genetic polymorphisms in ATP7B, other genes, and epigenetic factors have been shown to impact disease phenotype by affecting ATP7B protein structure and function. Of the over 600 mutations associated with Wilson disease, the majority are missense mutations that completely inactivate the copper-transporting function of ATP7B (Lutsenko, 2014). In general, individuals with protein-truncating mutations have earlier onset of disease due to decreased protein stability and quantity (Merle et al., 2010). However, other studies have demonstrated

partial preservation of copper-transporting function, perhaps explaining the milder phenotypes associated with certain mutations (Rodriguez-Granillo et al., 2008; Dmitriev et al., 2011; Huster et al., 2012). Individuals with the R778L mutation have been shown to have an earlier onset of disease and predominantly hepatic presentation (Z. Y. Wu et al., 2003). In contrast, individuals with the H1069Q mutation have a mean onset of symptoms between 20–22 years old and predominantly neurologic phenotype (Stapelbroek et al., 2004; Kalita et al., 2010). There is also some evidence that Kayser– Fleischer rings are more common in H1069Q homozygous patients in Hungary at time of diagnosis than in compound heterozygous individuals (Folhoffer et al., 2007). Moreover, pathogenic variants may affect ATP7B targeting from the TGN to cytosolic vesicles. For instance, the p.Met875Val mutation results in a less stable protein and causes reversible ATP7B localization defects. Under a low-copper environment, the p.Gly875Arg variant is sequestered in the endoplasmic reticulum. However, addition of exogenous copper to the cellular growth medium stabilizes the protein, allowing it to complete

28

I.J. CHANG AND S.H. HAHN

its intended journey to the TGN and overcoming its disease-causing phenotype. Theoretically, patients with this specific variant may be more sensitive to dietary copper deficiency (Gupta et al., 2011). The timing and location of copper buildup can also preferentially alter the hepatic transcriptome, based on homozygous ATP7B–/– mouse models. Proteomic analyses of mRNA profiles at each of these disease stages reflect unique patterns (Huster et al., 2006; Ralle et al., 2010). In the initial stage, mRNA for proteins responsible for cell cycle regulation, splicing, and cholesterol synthesis is present (Burkhead et al., 2011). This leads to early accumulation of copper bound to metallothioneins in the cytosol and free copper in the nuclei. In the progressive stage, mRNA changes throughout the cell are present, including the endoplasmic reticulum, mitochondria, and endocytic pathways, causing copper to pathologically accumulate within hepatocytes. In the later stages, mRNA for lysosomal and endosomal proteins is upregulated. In these final stages, copper concentrations decrease in the cytosol and nuclei, and accumulate in the membranous cellular compartment, causing bile duct proliferation and hepatic neoplastic changes. Therefore, the location of copper accumulation may convey more specific prognostic information about disease progression rather than total copper levels. Other studies have compared homozygotes to compound heterozygotes of the same mutation to establish genotype–phenotype correlations. A study of 76 members of a large, consanguineous Lebanese family showed an association between c.2299insC and hepatic disease and between the p.Ala1003Thr mutation and neurologic disease (Usta et al., 2014). Other candidate polymorphisms that are thought to modify the clinical phenotype of Wilson disease include MTHFR (Gromadzka et al., 2005), COMMD1 (Weiss, 2006), ATOX1 (Simon, 2008), XIAP (Weiss et al., 2010), PNPLA3 and hepatic steatosis (St€attermayer et al., 2012), and DMT1 (Przybyłkowski et al., 2014), although none of these genes has been demonstrated to have significant diagnostic or predictive value. Significant phenotypic variation of Wilson disease exists between individuals with the same mutation, individuals within the same family, and even between monozygotic twins (Członkowska et al., 2009; Kegley et al., 2010). While some studies have documented high intrafamilial concordance of clinical symptoms and biochemical results (Hofer et al., 2012; Chabik et al., 2014; Ferenci et al., 2015), others have reported a wide range in age of onset and presenting symptoms amongst siblings (Ala et al., 2007; Taly et al., 2007) and families carrying the same mutation (Takeshita et al., 2002). Indeed, disparate clinical presentations in monozygotic twins

raise the suspicion for epigenetic modifiers in Wilson disease. See Chapter 4 for more details about the genetic and environmental modifiers of Wilson disease.

CLINICAL MOLECULAR DIAGNOSIS The current gold standard of diagnosis for Wilson disease is direct Sanger sequencing of the ATP7B gene or molecular testing for previously identified familial mutations. Historically, most pathogenic variants in ATP7B were identified using a combination of polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP), single-strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), temporal temperature gradient electrophoresis (TTGE), denaturing high-performance liquid chromatography (DHPLC), and Sanger sequencing (Loudianos et al., 1999; Shimizu et al., 1999; Margarit et al., 2005; Vrabelova et al., 2005; G. H. Kim et al., 2008). The critical demerits of this complex tiered approach are that the detection rate is not high enough to find most mutations and the turnaround time is often extended. Although regional clusters of specific mutations have been well described, a customized screening approach taking into account these regional variants may be complicated by ethnically diverse populations and inaccurate clinical information provided with samples. Biochemical results are often imprecise, as elevations in urinary copper excretion tend to occur late in the disease process and fewer than 40% of presymptomatic patients excrete copper less than 100 mg/day (Sternlieb and Scheinberg, 1968; Nakayama et al., 2008). For these reasons, direct sequencing of the ATP7B gene has become the preferred standard and provides the greatest yield in clinical molecular diagnosis. Please refer to Chapter 14 for details about the diagnosis of Wilson disease. Starting the diagnostic process with molecular testing may significantly reduce the need for invasive liver biopsy. Liver copper content alone was found to be insufficient to exclude Wilson disease, as levels may not be elevated in some affected patients. Based on several previous studies, biallelic pathogenic variants were identified in about 80% of patients with biochemical and clinical tests suggestive of Wilson disease. Currently available screening tests may not definitively rule out the disease, and no single test could permit de novo diagnosis. Of note, many patients may not possess the characteristic findings and may present when their clinical disease is relatively mild. Inappropriate treatment for false-positive cases has the potential of inducing copper deficiency, which can result in hematologic and neurologic sequelae (N. Kumar et al., 2003). These findings reinforce the need for reliable clinical diagnostic criteria

THE GENETICS OF WILSON DISEASE and underscore the benefits of DNA testing prior to invasive procedures (Ferenci, 2005). Multiplex PCR is used to amplify all 21 exons and splice sites of ATP7B, including promoter regions. Although the large deletions or duplications cannot be detected with this conventional Sanger sequencing method, the chance of these being present in Wilson disease appears low (Stenson et al., 2012). If clinical suspicion is still high with only one pathogenic variant identified, then multiplex ligation-dependent probe amplification (MLPA) test should be considered. Microarray-based comparative genomic hybridization is another option to evaluate partial or full gene deletions or duplications with higher sensitivity. Cases with only one pathogenic variant present should be carefully reviewed in the context of other biochemical and clinical findings. Molecular genetic testing using direct mutation analysis is very effective in identifying affected patients and presymptomatic siblings of probands (Manolaki et al., 2009). Wilson disease is an autosomal-recessive disorder, which means that there is a 25% chance that a full sibling of the index case is also affected. Once homozygous or compound heterozygous mutations in ATP7B have been established in the index patient, mutation detection becomes valuable in family screening. The same genotype in asymptomatic family members confirms diagnosis of the disease, thus allowing for early treatment before the onset of complications. In family members in whom clinical and biochemical features are uncertain, the demonstration of either heterozygous (carrier) or wild-type gene sequence prevents unnecessary treatment (Chang et al., 2007). If the proband has secured a diagnosis of Wilson disease on the basis of clinical and biochemical evidence, but testing for ATP7B mutations is not available, family screening can be done by haplotype analysis of polymorphic markers flanking the disease gene (Thomas et al., 1995b; Gupta et al., 2005; Przybyłkowski et al., 2014). In this instance, the rare possibility of recombination events (typically 0.5–5% of cases) needs to be considered. The rate of recombination is dependent on which flanking markers are studied. Microsatellite or singlenucleotide polymorphisms in the ATP7B lateral wing are used for haplotyping, which is useful for screening relatives of patients with previously identified familial mutations. False-positive results may occur if haplotyping is used on patients with low-probability gene recombinations. Genetic testing for ATP7B mutations can be valuable to confirm a diagnosis of Wilson disease, especially when presentation is unusual (Caprai et al., 2006). Attention has been drawn to this situation by the molecular confirmation of early-onset hepatic disease in a

29

3-year-old child (Wilson et al., 2000). Mutation analysis has also confirmed late-onset disease, including the case of two siblings in their 70s – the oldest reported patients so far at time of diagnosis (Nanji et al., 1997; Gupta et al., 2005; Perri et al., 2005; Weitzman et al., 2014). ATP7B mutation analysis makes an important contribution to clinical practice. Unfortunately, systematic genetic testing for Wilson disease is still difficult and fairly expensive due to the plethora of different mutations, the occurrence of regulatory mutations in noncoding sequence, the large size of the gene, and the limitations of currently available methods. However, technical advances allowing high-throughput screening could be applied to the disease (Bost et al., 2012; Lepori et al., 2012). This new apparatus can sequence six million basepairs of DNA per hour with accuracy greater than 99%. Such advances might permit specialized laboratories to detect all variants by sequencing the entire genomic Wilson disease gene from patients, including not only the translated exons, but also the important noncoding sequences that are not normally investigated. Interpretation of variants of uncertain significance has become a major challenge for accurate interpretation, genetic counseling, and prevention. Screening family members may help with the interpretation of variants of uncertain significance, but not all variants can be resolved with this approach. Functional analysis is often necessary; however, no clinical functional analysis is currently available. A computational approach to predict significance of mutations is often helpful, but a further concrete model is required to demonstrate the efficacy in guiding clinical decisions.

POPULATION SCREENING The purpose of newborn screening is to identify treatable congenital conditions that can affect a child’s long-term health and development. Recent tandem mass spectrometry (MS/MS) applications have markedly expanded the ability to screen for >50 metabolic diseases from a single dried blood spot. In addition to the original Wilson– Jungner classic screening criteria (Wilson and Jungner, 1968), the American College of Medical Genetics convened the Newborn Screening Expert Group to develop a uniform screening panel in 2006 (American College of Medical Genetics Newborn Screening Expert Group, 2006). Of the primary tenants, Wilson disease is an ideal target for screening, given its relatively high prevalence and availability of effective treatment (Hahn et al., 2002; Roberts et al., 2008). Unfortunately, despite extensive discussion on the need for population screening, no cost-effective biomarkers or methods for early detection have been developed for Wilson disease yet. Several

30

I.J. CHANG AND S.H. HAHN

small pilot studies have been conducted using ceruloplasmin as a biomarker for screening, with limited findings (Yamaguchi et al., 1999; Hahn et al., 2002; Owada et al., 2002; Schilsky and Shneider, 2002; Kroll et al., 2006). Ceruloplasmin alone is not sufficient to screen for Wilson disease in newborns, as a substantial number of newborns present with physiologically low ceruloplasmin. Ceruloplasmin assay around 3 years of age may be the most appropriate population-screening method, but mandatory health checkups at this age are not universally available in the USA and worldwide. Many treatable congenital disorders are caused by mutations that result in absent or diminished levels of proteins; thus, protein biomarkers have enormous potential in the diagnosis/screening of congenital disorders. Liquid chromatography mass spectrometry with multiple reaction monitoring (LC-MRM-MS) has emerged as a robust technology that enables highly precise, specific, multiplex quantification of signature proteotypic peptides as stoichiometric surrogates of biomarker proteins. Our lab is currently exploring the use of peptide immunoaffinity enrichment (Whiteaker et al., 2010, 2011) to quantify ATP7B in dried blood spot (DBS). These promising proof-of-concept data open up the possibility of screening for Wilson disease in newborns. Further clinical validation on a large-scale study will be required to determine the efficacy of the assay.

CONCLUSION Wilson disease is an autosomal-recessive disease due to pathogenic mutations in ATP7B. ATP7B is the only identified gene known to cause Wilson disease, and encodes a transmembrane copper-transporting ATPase of the same name. While biochemical testing and clinical criteria may assist in the early diagnosis and treatment, the current gold standard for Wilson disease diagnosis is direct Sanger sequencing of ATP7B or molecular testing for known familial mutations. Genotype–phenotype correlations have been studied extensively but direct causations remain nebulous. Modifier genes may affect the penetrance and phenotypes but a large-scale study for clinical validation is warranted. The overall worldwide prevalence of Wilson disease is 1 in 30 000 individuals, with significant geographic variation. The most common mutation in Northern America and Europe is the missense mutation p.H1069Q and the most common mutation in East Asian populations is the missense p.R778L. Ceruloplasmin alone is insufficient to screen for Wilson disease in newborns. While peptide immunoaffinity assays show promise for newborn screening, further large-scale clinical studies are required to determine efficacy of these population-based screening methods for Wilson’s disease.

REFERENCES Abdel Ghaffar TY, Elsayed SM, Elnaghy S et al. (2011). Phenotypic and genetic characterization of a cohort of pediatric Wilson disease patients. BMC Pediatr 11 (1): 56. Abdelghaffar TY, Elsayed SM, Elsobky E et al. (2008). Mutational analysis of ATP7B gene in Egyptian children with Wilson disease: 12 novel mutations. J Hum Genet 53 (8): 681–687. Aggarwal A, Bhatt M (2013). Update on Wilson disease. Int Rev Neurobiol 110: 313–348. Aggarwal A, Chandhok G, Todorov T et al. (2013). Wilson disease mutation pattern with genotype–phenotype correlations from Western India: confirmation of p.C271* as a common Indian mutation and identification of 14 novel mutations. Ann Hum Genet 77 (4): 299–307. Al Jumah M, Majumdar R, Al Rajeh S et al. (2004). A clinical and genetic study of 56 Saudi Wilson disease patients: identification of Saudi-specific mutations. Eur J Neurol 11 (2): 121–124. Ala A, Walker AP, Ashkan K et al. (2007). Wilson’s disease. Lancet (London, England) 369 (9559): 397–408. American College of Medical Genetics Newborn Screening Expert Group (2006). Newborn screening: toward a uniform screening panel and system – executive summary. Pediatrics 117 (5 Pt 2): S296–S307. Bandmann O, Weiss KH, Kaler SG (2015). Wilson’s disease and other neurological copper disorders. Lancet Neurol 14 (1): 103–113. Bem RS, Raskin S, Muzzillo DA, de et al. (2013). Wilson’s disease in Southern Brazil: genotype–phenotype correlation and description of two novel mutations in ATP7B gene. Arq Neuropsiquiatr 71 (8): 503–507. Bennett JT, Schwarz KB, Swanson PD et al. (2013). An exceptional family with three consecutive generations affected by Wilson disease. JIMD Reports 10 (Chapter 206): 1–4. Bost M, Piguet-Lacroix G, Parant F et al. (2012). Molecular analysis of Wilson patients: direct sequencing and MLPA analysis in the ATP7B gene and Atox1 and COMMD1 gene analysis. J Trace Elem Med Biol 26 (2–3): 97–101. Bull PC, Thomas GR, Rommens JM et al. (1993). The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene. Nat Genet 5 (4): 327–337. Burkhead JL, Ralle M, Wilmarth P et al. (2011). Elevated copper remodels hepatic RNA processing machinery in the mouse model of Wilson’s disease. J Mol Biol 406 (1): 44–58. Caca K, Ferenci P, Kuhn HJ et al. (2000). High prevalence of the ATP7B-H1069Q mutation in Wilson disease patients from Eastern Germany. J Hepatol 32: 134. Caprai S, Loudianos G, Massei F et al. (2006). Direct diagnosis of Wilson disease by molecular genetics. J Pediatr 148 (1): 138–140. Cater MA, Forbes J, La Fontaine S et al. (2004). Intracellular trafficking of the human Wilson protein: the role of the six N-terminal metal-binding sites. Biochem J 380 (3): 805–813.

THE GENETICS OF WILSON DISEASE Cater MA, La Fontaine S, Mercer JFB (2007). Copper binding to the N-terminal metal-binding sites or the CPC motif is not essential for copper-induced trafficking of the human Wilson protein (ATP7B). Biochem J 401 (1): 143–153. Chabik G, Litwin T, Członkowska A (2014). Concordance rates of Wilson’s disease phenotype among siblings. J Inherit Metab Dis 37 (1): 131–135. Chang Y-F, Imam JS, Wilkinson MF (2007). The nonsensemediated decay RNA surveillance pathway. Annu Rev Biochem 76 (1): 51–74. Cocos¸ R, Sendroiu A, Schipor S et al. (2014). Genotype– phenotype correlations in a mountain population community with high prevalence of Wilson’s disease: genetic and clinical homogeneity. PLoS One 9 (6): e98520. Coffey AJ, Durkie M, Hague S et al. (2013). A genetic study of Wilson’s disease in the United Kingdom. Brain 136 (Pt 5): 1476–1487. Coronado V, Nanji M, Cox DW (2001). The Jackson toxic milk mouse as a model for copper loading. Mamm Genome 12: 793–795. Culotta V, Scott RA (2016). Metals in Cells, John Wiley, Chichester. Członkowska A, Gromadzka G, Chabik G (2009). Monozygotic female twins discordant for phenotype of Wilson’s disease. Mov Disord 24 (7): 1066–1069. de Bie P, van de Sluis B, Burstein E et al. (2007). Distinct Wilson’s disease mutations in ATP7B are associated with enhanced binding to COMMD1 and reduced stability of ATP7B. Gastroenterology 133 (4): 1316–1326. Dedoussis GVZ, Genschel J, Sialvera TE et al. (2005). Wilson disease: high prevalence in a mountainous area of Crete. Ann Hum Genet 69 (Pt 3): 268–274. Deguti MM, Genschel J, Cancado EL et al. (2004). Wilson disease: novel mutations in the ATP7B gene and clinical correlation in Brazilian patients. Hum Mutat 23 (4): 398. Dmitriev OY, Bhattacharjee A, Nokhrin S et al. (2011). Difference in stability of the N-domain underlies distinct intracellular properties of the E1064A and H1069Q mutants of copper-transporting ATPase ATP7B. J Biol Chem 286 (18): 16355–16362. Dziezyc K, Groamadzka G, Czlonkowska A (2011). Wilson’s disease in consecutive generations of one family. Park Rel Dis 17: 577–578. Dziezyc K, Litwin T, Chabik G et al. (2014). Families with Wilson’s disease in subsequenr generations: clinical and genetic analysis. Mov Dis 29: 1828–1832. Fanni D, Pilloni L, Orru S et al. (2005). Expression of ATP7B in normal human liver. Eur J Histochem 49 (4): 371–378. Fatemi N, Sarkar B (2002). Molecular mechanism of copper transport in Wilson disease. Environ Health Perspect 110 (Suppl 5): 695–698. Ferenci P (2005). Wilson’s disease. Clin Gastroenterol Hepatol 3 (8): 726–733. Ferenci P (2006). Regional distribution of mutations of the ATP7B gene in patients with Wilson disease: impact on genetic testing. Hum Genet 120 (2): 151–159.

31

Ferenci P, Litwin T, Seniow J et al. (2015). Encephalopathy in Wilson disease: copper toxicity or liver failure? J Clin Exp Hepatol 5: S88–S95. Figus A, Arigius A, Loudianos G et al. (1995). Molecular pathology and haplotype analysis of Wilson disease in Mediterranean populations. Am J Hum Genet 57 (6): 1318–1324. Firneisz G, Lakatos PL, Szalay F et al. (2002). Common mutations of ATP7B in Wilson disease patients from Hungary. Am J Med Genet 108 (1): 23–28. Folhoffer A, Ferenci P, Csak T et al. (2007). Novel mutations of the ATP7B gene among 109 Hungarian patients with Wilson’s disease. Eur J Gastroenterol Hepatol 19 (2): 105–111. Garcı´a Villarreal L, Daniels S, Shaw SH et al. (2000). High prevalence of the very rare Wilson disease gene mutation Leu708Pro in the island of Gran Canaria (Canary Islands, Spain): a genetic and clinical study. Hepatology (Baltimore, Md) 32 (6): 1329–1336. Gomes A, Dedoussis GV (2016). Geographic distribution of ATP7B mutations in Wilson disease. Ann Hum Biol 43 (1): 1–8. Gourdon P, Liu XY, Skjorringe T et al. (2011). Crystal structure of a copper-transporting PIB-type ATPase. Nature 475 (7354): 59–64. Gromadzka G, Schmidt HH, Genschel J et al. (2005). Frameshift and nonsense mutations in the gene for ATPase7B are associated with severe impairment of copper metabolism and with an early clinical manifestation of Wilson’s disease. Clin Genet 68 (6): 524–532. Gu Y-H, Kodama H, Du SL et al. (2003). Mutation spectrum and polymorphisms in ATP7B identified on direct sequencing of all exons in Chinese Han and Hui ethnic patients with Wilson’s disease. Clin Genet 64 (6): 479–484. Gupta A, Aikath D, Neogi R et al. (2005). Molecular pathogenesis of Wilson disease: haplotype analysis, detection of prevalent mutations and genotype–phenotype correlation in Indian patients. Hum Genet 118 (1): 49–57. Gupta A, Chattopadhyay I, Dey S et al. (2007). Molecular pathogenesis of Wilson disease among Indians: a perspective on mutation spectrum in ATP7B gene, prevalent defects, clinical heterogeneity and implication towards diagnosis. Cell Mol Neurobiol 27 (8): 1023–1033. Gupta A, Bhattacharjee A, Dmitriev OY et al. (2011). Cellular copper levels determine the phenotype of the Arg875 variant of ATP7B/Wilson disease protein. Proc Natl Acad Sci U S A 108 (13): 5390–5395. Hahn SH, Lee SY, Jang YJ et al. (2002). Pilot study of mass screening for Wilson’s disease in Korea. Mol Genet Metab 76 (2): 133–136. Hofer H, Willheim-Polli C, Knoflauch P et al. (2012). Identification of a novel Wilson disease gene mutation frequent in Upper Austria: a genetic and clinical study. J Hum Genet 57 (9): 564–567. Huster D, Hoppert M, Lutsenko S et al. (2003). Defective cellular localization of mutant ATP7B in Wilson’s disease patients and hepatoma cell lines. Gastroenterology 124 (2): 335–345.

32

I.J. CHANG AND S.H. HAHN

Huster D, Finegold MJ, Morgan CT et al. (2006). Consequences of copper accumulation in the livers of the Atp7b–/– (Wilson disease gene) knockout mice. Am J Pathol 168 (2): 423–434. Huster D, Kuhne A, Bhattacharjee A et al. (2012). Diverse functional properties of Wilson disease ATP7B variants. Gastroenterology 142 (4): 947–956.e5. Iacob R, Iacob S, Nastase A et al. (2012). The His1069Gln mutation in the ATP7B gene in Romanian patients with Wilson’s disease referred to a tertiary gastroenterology center. J Gastrointestin Liver Dis 21 (2): 181–185. Ivanova-Smolenskaya I, Karabanov AV, Illarioshkin SN et al. (1997). 5-29-06 Molecular analysis in Russian families with Wilson’s disease. J Neurol Sci 150: S314. Kalita J, Somarajan BI, Misra UK et al. (2010). R778L, H1069Q, and I1102T mutation study in neurologic Wilson disease. Neurol India 58 (4): 627–630. Kegley KM, Sellers MA, Ferber MJ et al. (2010). Fulminant Wilson’s disease requiring liver transplantation in one monozygotic twin despite identical genetic mutation. Am J Transplant 10 (5): 1325–1329. Kenney SM, Cox DW (2007). Sequence variation database for the Wilson disease copper transporter, ATP7B. Hum Mutat 28 (12): 1171–1177. Kim EK, Yoo OJ, Song KY et al. (1998). Identification of three novel mutations and a high frequency of the Arg778Leu mutation in Korean patients with Wilson disease. Hum Mutat 11 (4): 275–278. Kim G-H, Jang JY, Park JY et al. (2008). Estimation of Wilson’s disease incidence and carrier frequency in the Korean population by screening ATP7B major mutations in newborn filter papers using the SYBR green intercalator method based on the amplification refractory mutation system. Genet Test 12 (3): 395–399. Kroll CA, Ferber MJ, Dawson BD et al. (2006). Retrospective determination of ceruloplasmin in newborn screening blood spots of patients with Wilson disease. Mol Genet Metab 89 (1–2): 134–138. Kumar N, Gross JB, Ahlskog JE (2003). Myelopathy due to copper deficiency. Neurology 61 (2): 273–274. Kumar S, Thapa B, Kaur G et al. (2006). Analysis of most common mutations R778G, R778L, R778W, I1102T and H1069Q in Indian Wilson disease patients: correlation between genotype/phenotype/copper ATPase activity. Mol Cell Biochem 294 (1–2): 1–10. Kumar SS, Kurian G, Eapen CE et al. (2012). Genetics of Wilson’s disease: a clinical perspective. Indian J Gastroenterol 31 (6): 285–293. Kuppala D, Deng J, Brewer GJ et al. (2009). Wilson disease mutations in the American population: identification of five novel mutations in ATP7B. Open Hepatol J 1 (1): 1–4. La Fontaine S, Theophilos MB, Firth SD et al. (2001). Effect of the toxic milk mutation (tx) on the function and intracellular localization of Wnd, the murine homologue of the Wilson copper ATPase. Hum Mol Genet 10 (4): 361–370. Lee CC, Wu JY, Tsai FJ et al. (2000). Molecular analysis of Wilson disease in Taiwan: identification of one novel

mutation and evidence of haplotype-mutation association. J Hum Genet 45 (5): 275–279. Leggio L, Addolorato G, Loudianos G et al. (2006). Genotype– phenotype correlation of the Wilson disease ATP7B gene. Am J Med Genet A 140A (8): 933. Lenartowicz M, Krzeptowski W (2010). Structure and function of ATP7A and ATP7B proteins – Cu-transporting ATPases. Postepy Biochem 56: 317–327. Lepori M-B, Zappu A, Incollu S et al. (2012). Mutation analysis of the ATP7B gene in a new group of Wilson’s disease patients: contribution to diagnosis. Mol Cell Probes 26 (4): 147–150. Loudianos G, Dessi V, Lovicu M et al. (1999). Mutation analysis in patients of Mediterranean descent with Wilson disease: identification of 19 novel mutations. J Med Genet 36 (11): 833–836. Lovicu M, Dessi V, Lepori MB et al. (2006). The canine copper toxicosis gene MURR1 is not implicated in the pathogenesis of Wilson disease. J Gastroenterol 41 (6): 582–587. Lutsenko S (2014). Modifying factors and phenotypic diversity in Wilson’s disease. Ann N Y Acad Sci 1315 (1): 56–63. Lutsenko S, Petrukhin K, Cooper MJ et al. (1997). N-terminal domains of human copper-transporting adenosine triphosphatases (the Wilson’s and Menkes disease proteins) bind copper selectively in vivo and in vitro with stoichiometry of one copper per metal-binding repeat. J Biol Chem 272 (30): 18939–18944. Machado AAC, Deguti MM, Genschel J et al. (2008). Neurological manifestations and ATP7B mutations in Wilson’s disease. Parkinsonism Relat Disord 14 (3): 246–249. Majumdar R, Al-Jumah M, Zaidan R (2004). A rare homozygous missense mutation in ATP7B exon 19 in a case of Wilson disease. Eur Neurol 51 (1): 52–54. Mak CM, Lam C-W (2008). Diagnosis of Wilson’s disease: a comprehensive review. Crit Rev Clin Lab Sci 45 (3): 263–290. Manolaki N, Nikolopoulou G, Daikos GL et al. (2009). Wilson disease in children: analysis of 57 cases. J Pediatr Gastroenterol Nutr 48 (1): 72–77. Margarit E, Bach V, Gomez D et al. (2005). Mutation analysis of Wilson disease in the Spanish population - identification of a prevalent substitution and eight novel mutations in the ATP7B gene. Clin Genet 68 (1): 61–68. Mendell JT, Sharifi NA, Meyers JL et al. (2004). Nonsense surveillance regulates expression of diverse classes of mammalian transcripts and mutes genomic noise. Nat Genet 36 (10): 1073–1078. Merle U, Weiss KH, Eisenbach C et al. (2010). Truncating mutations in the Wilson disease gene ATP7B are associated with very low serum ceruloplasmin oxidase activity and an early onset of Wilson disease. BMC Gastroenterol 10 (1): 8. Møller LB, Horn N, Jeppesen TD et al. (2011). Clinical presentation and mutations in Danish patients with Wilson disease. Eur J Hum Genet 19 (9): 935–941.

THE GENETICS OF WILSON DISEASE Nakayama K, Kubota M, Katoh Y et al. (2008). Early and presymptomatic detection of Wilson’s disease at the mandatory 3-year-old medical health care examination in Hokkaido Prefecture with the use of a novel automated urinary ceruloplasmin assay. Mol Genet Metab 94 (3): 363–367. Nanji MS, Nguyen VT, Kawasoe JH et al. (1997). Haplotype and mutation analysis in Japanese patients with Wilson disease. Am J Hum Genet 60 (6): 1423–1429. Nicastro E, Ranucci G, Vajro P et al. (2010). Re-evaluation of the diagnostic criteria for Wilson disease in children with mild liver disease. Hepatology (Baltimore, Md) 52 (6): 1948–1956. Okada T, Shiono Y, Hayashi H et al. (2000). Mutational analysis of ATP7B and genotype–phenotype correlation in Japanese with Wilson’s disease. Hum Mutat 15 (5): 454–462. Okada T, Shinono Y, Kaneko Y et al. (2010). High prevalence of fulminant hepatic failure among patients with mutant alleles for truncation of ATP7B in Wilson’s disease. Scand J Gastroenterol 45 (10): 1232–1237. Owada M, Suzuki K, Fukushi M et al. (2002). Mass screening for Wilson’s disease by measuring urinary holoceruloplasmin. J Pediatr 140 (5): 614–616. Panagiotakaki E, Tzetis M, Manolaki N et al. (2004). Genotype–phenotype correlations for a wide spectrum of mutations in the Wilson disease gene (ATP7B). Am J Med Genet A 131A (2): 168–173. Panichareon B, Taweechue K, Thongnoppakhun W et al. (2011). Six novel ATP7B mutations in Thai patients with Wilson disease. Eur J Med Genet 54 (2): 103–107. Paradisi I, De Freitas L, Arias S (2015). Most frequent mutation c.3402delC (p.Ala1135GlnfsX13) among Wilson disease patients in Venezuela has a wide distribution and two old origins. Eur J Med Genet 58 (2): 59–65. Park S, Park JY, Kim GH et al. (2007). Identification of novel ATP7Bgene mutations and their functional roles in Korean patients with Wilson disease. Hum Mutat 28 (11): 1108–1113. Park H, Park DK, Kim MS et al. (2015). Pseudo-dominant inheritance in Wilson’s disease. Neurol Sci 37 (1): 153–155. Payne AS, Kelly EJ, Gitlin JD (1998). Functional expression of the Wilson disease protein reveals mislocalization and impaired copper-dependent trafficking of the common H1069Q mutation. Proc Natl Acad Sci U S A 95 (18): 10854–10859. Perri RE, Hahn SH, Ferber MJ et al. (2005). Wilson disease – keeping the bar for diagnosis raised. Hepatology (Baltimore, Md) 42 (4): 974. Petrukhin K, Fischer SG, Pirastu M et al. (1993). Mapping, cloning and genetic characterization of the region containing the Wilson disease gene. Nat Genet 5 (4): 338–343. Przybyłkowski A, Gromadzka G, Członkowska A (2014). Polymorphisms of metal transporter genes DMT1 and ATP7A in Wilson’s disease. J Trace Elem Med Biol 28 (1): 8–12.

33

Ralle M, Huster D, Vogt S et al. (2010). Wilson disease at a single cell level: intracellular copper trafficking activates compartment-specific responses in hepatocytes. J Biol Chem 285 (40): 30875–30883. Roberts EA, Schilsky MLAmerican Association for Study of Liver Diseases (AASLD) (2008). Diagnosis and treatment of Wilson disease: an update. Hepatology (Baltimore, Md) 47 (6): 2089–2111. Rodriguez-Granillo A, Sedlak E, Wittung-Stafshede P (2008). Stability and ATP binding of the nucleotide-binding domain of the Wilson disease protein: effect of the common H1069Q mutation. J Mol Biol 383 (5): 1097–1111. Santhosh S, Shaji RV, Eapen CE et al. (2006). ATP7B mutations in families in a predominantly Southern Indian cohort of Wilson’s disease patients. Indian J Gastroenterol 25 (6): 277–282. Sazinsky MH, Mandal AK, Arguello JM et al. (2006). Structure of the ATP binding domain from the Archaeoglobus fulgidus Cu + -ATPase. J Biol Chem 281 (16): 11161–11166. Schilsky ML, Shneider B (2002). Population screening for Wilson’s disease. J Pediatr 140 (5): 499–501. Schushan M, Bhattacharjee A, Ben-Tai N et al. (2012). A structural model of the copper ATPase ATP7B to facilitate analysis of Wilson disease-causing mutations and studies of the transport mechanism. Metallomics : Integrated Biometal Science 4 (7): 669–678. Shah AB, Chernov I, Zhang HT et al. (1997). Identification and analysis of mutations in the Wilson disease gene (ATP7B): population frequencies, genotype–phenotype correlation, and functional analyses. Am J Hum Genet 61 (2): 317–328. Shimizu N, Nakazono H, Takeshita Y et al. (1999). Molecular analysis and diagnosis in Japanese patients with Wilson’s disease. Pediatr Int 41 (4): 409–413. Simon I (2008). Analysis of the human Atox 1 homologue in Wilson patients. World J Gastroenterol 14 (15): 2383. Simsek Papur O, Akman SA, Cakmur R et al. (2013). Mutation analysis of ATP7B gene in Turkish Wilson disease patients: identification of five novel mutations. Eur J Med Genet 56 (4): 175–179. Song M-J, Lee ST, Lee MK et al. (2012). Estimation of carrier frequencies of six autosomal-recessive Mendelian disorders in the Korean population. J Hum Genet 57 (2): 139–144. Stapelbroek JM, Bollen CW, van Amstel JK et al. (2004). The H1069Q mutation in ATP7B is associated with late and neurologic presentation in Wilson disease: results of a meta-analysis. J Hepatol 41 (5): 758–763. St€attermayer AF, Rutter K, Beinhardt S et al. (2012). Genetic factors associated with histologic features of the liver and treatment outcome in chronic hepatitis C patients. Z Gastroenterol 50 (05): P51. Stenson PD, Ball EV, Mort M et al. (2012). The Human Gene Mutation Database (HGMD) and its exploitation in the fields of personalized genomics and molecular evolution. Curr Protoc Bioinformatics. Chapter 1: Unit 1.13.

34

I.J. CHANG AND S.H. HAHN

Stenson PD, Mort M, Ball EV et al. (2014). The Human Gene Mutation Database: building a comprehensive mutation repository for clinical and molecular genetics, diagnostic testing and personalized genomic medicine. Hum Genet 133 (1): 1–9. Sternlieb I, Scheinberg IH (1968). Prevention of Wilson’s disease in asymptomatic patients. New Engl J Med 278: 352–359. Takeshita Y, Shimizu N, Yamaguchi Y et al. (2002). Two families with Wilson disease in which siblings showed different phenotypes. J Hum Genet 47 (10): 0543–0547. Taly AB, Meenakshi-Sundaram S, Sinha S et al. (2007). Wilson disease: description of 282 patients evaluated over 3 decades. Medicine 86 (2): 112–121. Tanzi RE, Petrukhin K, Chernov I et al. (1993). The Wilson disease gene is a copper transporting ATPase with homology to the Menkes disease gene. Nat Genet 5 (4): 344–350. Tatsumi Y, Hattori A, Hayashi H et al. (2010). Current state of Wilson disease patients in central Japan. Intern Med (Tokyo, Japan) 49 (9): 809–815. Theophilos MB, Cox DW, Mercer JF (1996). The toxic milk mouse is a murine model of Wilson disease. Hum Mol Genet 5 (10): 1619–1624. Thomas GR, Jensson O, Gudmundsson G et al. (1995a). Wilson disease in Iceland: a clinical and genetic study. Am J Hum Genet 56 (5): 1140–1146. Thomas GR, Roberts EA, Walshe JM et al. (1995b). Haplotypes and mutations in Wilson disease. Am J Hum Genet 56 (6): 1315–1319. Todorov T, Savov A, Jelev H et al. (2005). Spectrum of mutations in the Wilson disease gene (ATP7B) in the Bulgarian population. Clin Genet 68 (5): 474–476. Tomi
c A, Dobricic V, Novakovic I et al. (2013). Mutational analysis of ATP7B gene and the genotype–phenotype correlation in patients with Wilson’s disease in Serbia. Vojnosanit Pregl 70 (5): 457–462. Tsivkovskii R, Efremov RG, Lutsenko S (2003). The role of the invariant His-1069 in folding and function of the Wilson’s disease protein, the human copper-transporting ATPase ATP7B. J Biol Chem 278: 13302–13308. Usta J, Wehbeh A, Rida K et al. (2014). Phenotype–genotype correlation in Wilson disease in a large Lebanese family: association of c.2299insC with hepatic and of p. Ala1003Thr with neurologic phenotype. PLoS One 9 (11): e109727. van den Berghe PVE, Stapelbroek JM, Krieger E et al. (2009). Reduced expression of ATP7B affected by Wilson disease-causing mutations is rescued by pharmacological folding chaperones 4-phenylbutyrate and curcumin. Hepatology (Baltimore, Md) 50 (6): 1783–1795. Vrabelova S, Letocha O, Borsky M et al. (2005). Mutation analysis of the ATP7B gene and genotype/phenotype correlation in 227 patients with Wilson disease. Mol Genet Metab 86 (1–2): 277–285. Walker JM, Huster D, Ralle M et al. (2004). The N-terminal metal-binding site 2 of the Wilson’s disease protein plays

a key role in the transfer of copper from Atox1. J Biol Chem 279 (15): 15376–15384. Wan L, Tsai CH, Tsai Y et al. (2006). Mutation analysis of Taiwanese Wilson disease patients. Biochem Biophys Res Commun 345 (2): 734–738. Wang L-H, Huang YQ, Shang X et al. (2011). Mutation analysis of 73 southern Chinese Wilson’s disease patients: identification of 10 novel mutations and its clinical correlation. J Hum Genet 56 (9): 660–665. Wei Z, Huang Y, Liu A et al. (2014). Mutational characterization of ATP7B gene in 103 Wilson’s disease patients from Southern China: identification of three novel mutations. Neuroreport 25 (14): 1075–1080. Weiss KH (2006). Copper toxicosis gene MURR1 is not changed in Wilson disease patients with normal blood ceruloplasmin levels. World J Gastroenterol 12 (14): 2239. Weiss KH, Runz H, Noe B et al. (2010). Genetic analysis of BIRC4/XIAP as a putative modifier gene of Wilson disease. J Inherit Metab Dis 33 (3): 233–240. Weitzman E, Pappo O, Weiss P et al. (2014). Late onset fulminant Wilson’s disease: a case report and review of the literature. World J Gastroenterol 20 (46): 17656–17660. Whiteaker JR, Zhao L, Anderson L et al. (2010). An automated and multiplexed method for high throughput peptide immunoaffinity enrichment and multiple reaction monitoring mass spectrometry-based quantification of protein biomarkers. Mol Cell Proteomics 9 (1): 184–196. Whiteaker JR, Lin C, Kennedy J et al. (2011). A targeted proteomics-based pipeline for verification of biomarkers in plasma. Nat Biotechnol 29 (7): 625–634. Wilson JMG, Jungner G (1968). Principles and practice of screening for disease. Public Health Paper no. 34, World Health Organization, Geneva. Wilson DC, Phillips MJ, Cox DW et al. (2000). Severe hepatic Wilson’s disease in preschool-aged children. J Pediatr 137 (5): 719–722. Wu Z-Y, Lin MT, Murong SX et al. (2003). Molecular diagnosis and prophylactic therapy for presymptomatic Chinese patients with Wilson disease. Arch Neurol 60 (5): 737–741. Wu F, Wang J, Pu C et al. (2015). Wilson’s disease: a comprehensive review of the molecular mechanisms. Int J Mol Sci 16 (3): 6419–6431. Yamaguchi Y, Aoki T, Arashima S et al. (1999). Mass screening for Wilson’s disease: results and recommendations. Pediatr Int 41 (4): 405–408. Yoo HW (2002). Identification of novel mutations and the three most common mutations in the human ATP7B gene of Korean patients with Wilson disease. Genet Med 4 (6 Suppl): 43S–48S. Zali N, Mohebbi SR, Estaghamat S et al. (2011). Prevalence of ATP7B gene mutations in Iranian patients with Wilson disease. Hepatitis Monthly 11 (11): 890–894.

Handbook of Clinical Neurology, Vol. 142 (3rd series) Wilson Disease A. Członkowska and M.L. Schilsky, Editors http://dx.doi.org/10.1016/B978-0-444-63625-6.00004-5 © 2017 Elsevier B.V. All rights reserved

Chapter 4

Genetic and environmental modifiers of Wilson disease VALENTINA MEDICI1* AND KARL-HEINZ WEISS2 Division of Gastroenterology and Hepatology, Department of Internal Medicine, University of California Davis, Sacramento, CA, USA

1

2

Department of Gastroenterology and Hepatology, University Hospital of Heidelberg, Heidelberg, Germany

Abstract Wilson disease (WD) is characterized by remarkable variety in its phenotypic presentation. Patients with WD can present with hepatic, neurologic, and psychiatric symptoms combined in different and unpredictable ways. Importantly, no convincing phenotype–genotype correlation has ever been identified, opening the possibility that other genes, aside from ATPase copper-transporting beta (ATP7B), are involved in the pathogenesis of this condition. In addition, modifier genes, or genes that can affect the expression of other genes, may be involved. Clinical and basic science data indicate that environmental and dietary factors can potentially modify gene expression in WD and, consequently, its clinical presentation and course. In particular, previously studied genes include copper metabolism domain-containing 1 (COMMD1), antioxidant 1 copper chaperone (ATOX1), X-linked inhibitor of apoptosis (XIAP), apolipoprotein E (APOE), hemochromatosis (HFE), and 5,10-methylenetetrahydrofolate reductase (MTHFR). Dietary factors include iron and methyl group donors which could affect methionine metabolism and epigenetic mechanisms of gene expression regulation. Most of the work conducted in this field is in its initial stages but it has the potential to change the diagnosis and treatment of WD.

INTRODUCTION1

COMMD1 GENE

The major knowledge gap in Wilson disease (WD) is the lack of mechanistic understanding of the phenotype diversity and the responses to treatment despite the genetic inheritance of ATPase copper-transporting beta (ATP7B) mutations. Proteins other than ATP7B contribute to the molecular mechanism of copper excretion and mutations or polymorphisms of these proteins might contribute to WD, perhaps explaining the highly variable clinical presentation (Steindl et al., 1997; Riordan and Williams, 2001; Ala and Schilsky, 2011) and course of this disease. This chapter will review the published data on concomitant genes and modifier genes as well as environmental factors that can have a role in WD phenotypic expression.

An interesting candidate for a modifier gene is copper metabolism domain-containing 1 (COMMD1). Deficiency of COMMD1 causes canine copper toxicosis of Bedlington terriers (Twedt et al., 1979; van de Sluis et al., 2002; Klomp et al., 2003) that resembles WD, although ceruloplasmin levels are not decreased (Su et al., 1982) and there are no evident neurologic symptoms. The human orthologous gene was identified on chromosome 2p13-16 and is distinct from the ATP7B gene locus (van de Sluis et al., 1999). As several studies confirmed a physical interaction of COMMD1 with ATP7B (Tao et al., 2003; de Bie et al., 2006; Donadio et al., 2007), cohorts of WD patients were

1

Abbreviations used in the chapter are listed at the end of the chapter before References section.

*Correspondence to: Valentina Medici, MD, Department of Internal Medicine, Division of Gastroenterology and Hepatology, University of California Davis, 4150 V St., Suite 3500, Sacramento CA 95817, USA. Tel: +1-916-734-3751, E-mail: [email protected]

36

V. MEDICI AND K.-H. WEISS

screened for alterations in COMMD1. So far no exonic mutations of COMMD1 have been found in patients with WD (Stuehler et al., 2004) or other rare human copper overload diseases like Indian childhood cirrhosis, endemic Tyrolean infantile cirrhosis, or idiopathic copper toxicosis (Muller et al., 2003). Studies on known polymorphisms of COMMD1 in patients with WD revealed conflicting results. In WD patients homozygous for the most common ATP7B mutation H1069Q, an association between a COMMD1 polymorphism and onset of neurologic and hepatic symptoms was reported. Onset of disease was significantly earlier for heterozygous patients at codon Asn 164 (GAT/GAC) than for wild-type (GAT/GAT) (Stuehler et al., 2004). This could only partially be confirmed by others (Weiss et al., 2006; Bost et al., 2012).

ATOX1 GENE The human homologue antioxidant 1 copper chaperone (ATOX1) is an 8-kDa cytosolic protein that contains a single copy of the highly conserved MxCxxC motif in the amino-terminus which is repeated sixfold in ATP7B. This metallochaperone interacts directly with ATP7B (Hamza et al., 1999) and can regulate its copper occupancy (Walker et al., 2002). By modulating the amount of copper bound to the protein, ATOX1 has an impact on intracellular localization (DiDonato et al., 2000), as well as posttranslational modification and enzymatic activity of ATP7B as a copper-dependent transcription factor involved in cell proliferation (Vanderwerf et al., 2001; Itoh et al., 2008). However, in human cohort studies, no significant nonsynonymous coding variations in the ATOX1 gene were evident (Simon et al., 2008; Bost et al., 2012). A common polymorphism within ATOX1 (5’UTR -99 T > C) was detected at expected frequencies of 49–57%. Based on these data, no major role can be attributed to ATOX1 in the pathophysiology or clinical variation of WD.

XIAP GENE XIAP (X-linked inhibitor of apoptosis protein), a well-characterized antiapoptotic protein, has been proposed as a regulator of copper-induced cell injury. XIAP binds copper directly, followed by enhanced degradation and blunted inhibition of caspases. Therefore, in addition to copper overload, copper binding to XIAP sensitizes hepatocytes for apoptosis (Mufti et al., 2006), which suggests an unexpected new pathogenic mechanism for copper-induced cell damage (Mufti et al., 2007). It has also been hypothesized that XIAP plays a role in maintaining cellular copper homeostasis by interacting with COMMD1 and promoting its degradation (Burstein et al., 2004; Prohaska, 2008; Maine et al., 2009).

Correspondingly, reduced copper levels are found in cells and tissues of XIAP-deficient knockout mice (Burstein et al., 2004). Four nonsynonymous coding single-nucleotide polymorphisms (SNPs) have previously been described in the coding region of the XIAP gene (Salzer et al., 2008; Serre et al., 2008); their physiologic role however remains unclear. In 98 WD patients (Weiss et al., 2010), overall allele frequency did not differ significantly from previously reported panels. For all SNPs, statistical analysis did not reveal any correlation to age of onset, clinical presentation (neurologic vs. hepatic vs. mixed vs. asymptomatic), or presentation as fulminant WD (Weiss et al., 2010).

APOE GENE Similarly to WD, several studies suggest that copper dysfunction and ATP7B variants influence the phenotypes of neurodegenerative disorders such as Alzheimer’s disease (Squitti et al., 2013). The presence of apolipoprotein E (APOE) allele ε4 is associated with an increased vulnerability of the brain to disease effects, whereas the presence of APOE genotype ε3/3 appears to provide moderate neuroprotection. In line with this concept, Schiefermeier et al. (2000) reported that the homozygous APOE ε3 genotype is associated with delayed onset of WD signs and symptoms. In an independent cohort, Litwin et al. (2012b) reported that women with APOE ε4-positive genotypes presented earlier onset of WD symptoms, particularly among ATP7B p.H1069Q homozygous patients. However, data on an association between APOE genotype and WD clinical expression conflict with other authors who do not confirm these findings (Gu et al., 2005; Kocabay et al., 2009).

HFE GENE AND METAL TRANSPORTER GENES DMT1 AND ATP7A Data in patients and in animal models of WD indicate iron accumulation may contribute to the phenotype of this condition. HFE (hemochromatosis) gene mutations are associated with hereditary hemochromatosis and are related to increased intestinal iron uptake. Two initial case reports described concomitant presence of HFE and ATP7B gene polymorphisms with corresponding hepatic iron and copper accumulation (Hafkemeyer et al., 1994; Walshe and Cox, 1998). A study of 32 WD patients from Sardinia described iron and copper metabolism indices, HFE mutations, and liver biopsies (Sorbello et al., 2010). Six of the studied patients were heterozygous for the H63D mutation; none presented C282Y and S65C mutations. Patients with HFE polymorphisms presented with approximately double the hepatic iron concentration compared to patients without HFE polymorphisms. Long-term anticopper treatment,

GENETIC AND ENVIRONMENTAL MODIFIERS OF WILSON DISEASE both zinc salts and chelation, was associated with improved alanine aminotransferase (ALT) levels and decreased hepatic iron concentration in HFE wild-type patients, whereas ALT levels and hepatic iron remained stable in patients with HFE polymorphism. HFE mutations were also studied in 40 patients with WD compared to 295 healthy controls and there was no difference in allele frequencies for the C282Y and H63D mutations between the two populations (Erhardt et al., 2002). Similarly, a larger study on 143 WD patients explored HFE allele frequencies and did not find any significant difference between WD patients and the general population (Pfeiffenberger et al., 2012). The prevalence of divalent metal transporter 1 (DMT1) and ATPase copper-transporting alpha (ATP7A) mutations was also studied in patients with WD (Przybyłkowski et al., 2014), as DMT1 is involved in iron transport and ATP7A is involved in copper transport in the intestinal epithelium. The study, which included more than 100 patients with WD, found an increased frequency of the DMT1 IVS4 C(+) allele in patients with WD compared to healthy controls, but no differences according to hepatic or neurologic phenotype. ATP7A alleles did not present different prevalence between the two studied populations. The rationale for studying these genes resides in two previous findings: increased transcript levels of DMT1 in the brains of patients with Parkinson’s disease and in animal models of this condition (Salazar et al., 2008), and increased mRNA transcript and protein levels of duodenal ATP7A as a possible compensatory mechanism after copper accumulation (Przybyłkowski et al., 2013).

MTHFR GENE A key enzyme in folate and methionine metabolism is 5,10-methylenetetrahydrofolate reductase (MTHFR), which catalyzes the conversion of 5,10methylenetetrahydrofolate to 5-methyltetrahydrofolate, a co-substrate for homocysteine remethylation to methionine. Mutations of MTHFR are associated with increased homocysteine levels, which may contribute to greater severity of WD or to its phenotypic variability. Two-hundred and forty-five patients with WD were genotyped for the two MTHFR polymorphisms, C677T and A1298C, and genotype–phenotype correlations were studied. The C667T genotype was more frequent than expected according to Hardy–Weinberg equilibrium. The C677T allele was associated more frequently with hepatic onset. The A1298C allele was associated with younger age at presentation. MTHFR polymorphisms were not associated with any difference in copper metabolism (Gromadzka et al., 2011). Even though these initial observations were not explored in other populations and

37

hyperhomocysteinemia has not been described in WD, the possibility that homocysteine or aberrant methionine metabolism can affect the phenotype is interesting, as homocysteine can pass the blood–brain barrier and can also have neurotoxic effects by interacting with copper (White et al., 2001; Linnebank et al., 2006). In addition, methionine metabolism is closely related to mechanisms of DNA and histone methylation with potential consequences for gene expression regulation.

HUMAN PRION GENE Prion protein (PRNP) binds copper ion with low affinity (Brown et al., 1997) and may affect copper metabolism, especially in the central nervous system, where this protein is highly expressed (Ford et al., 2002). The interaction between copper and PRNP may have a protective effect on neurons (Gasperini et al., 2015). Given this background, Merle et al. (2006) studied the prevalence of the PRNP polymorphism at codon 129 (M129V) associated with age at disease onset and subsequent disease course in 134 patients with WD. The prevalence of the M129V genotype was similar in WD patients compared to a healthy control population. With respect to their clinical presentation, patients with PRNP codon 129 resulting in homozygous methionine (129 M/M) were about 5 years older at disease onset and had neurologic presentation an average of 7 years later compared to carriers of PRNP 129 V(+).

GENDER As pointed out by a study from Litwin et al. (2012a), neuropsychiatric presentation in WD is more common in men than women and women develop these symptoms at a later age than men. In a complex study on more than 1000 WD patients, Ferenci (2014) reported that gender and age modify disease presentation specifically. Young females present more often with fulminant liver disease (Ferenci et al., 2011) and males present more frequently with neurologic disease, whereas neurologic disease is rare among children with WD (Ferenci, 2014). These findings may be related to hormonal changes but also to differences in iron accumulation and metabolism (by menstrual iron loss), which are discussed below.

IRON Studies in animal models and in humans show evidence that copper accumulation and low ceruloplasmin levels as well as long-term treatment of WD are associated with iron accumulation. There is a close relation between iron and copper accumulation primarily derived from the fact that ceruloplasmin is a potent ferroxidase, catalyzing the conversion of Fe2+ to Fe3+, and this activity is required

38

V. MEDICI AND K.-H. WEISS

for iron cellular uptake (Attieh et al., 1999). Low ceruloplasmin levels as presented in WD may thus result in iron accumulation. The other mechanism possibly underlying iron accumulation in WD is related to long-term anticopper treatment or overtreatment, probably associated with reduced copper bioavailability, worsening hypoceruloplasminemia, and, ultimately, iron overload (Schilsky, 2001). In a genetic animal model of WD, the Long– Evans Cinnamon (LEC) rats, iron deprivation or phlebotomies prevented the development of acute liver failure and hepatocellular carcinoma, despite absence of hepatic copper treatment (Kato et al., 1996). In WD patients treated with penicillamine for 3–8.5 years, there was evidence of hepatic iron accumulation and reduced hepatic copper content over time. Phlebotomies in 2 patients achieved lower serum ferritin and ALT levels (Shiono et al., 2001). Iron parameters were studied for a group of 40 patients with WD compared to healthy control subjects. WD patients presented higher serum ferritin levels than controls and a subgroup of WD patients with low ceruloplasmin presented higher ferritin levels than those with normal ceruloplasmin (Erhardt et al., 2002). A larger study of hepatic iron concentrations on follow-up liver biopsies in patients with WD confirmed penicillamine treatment was associated with 3–10 times increased hepatic iron content compared to baseline, whereas zinc treatment was not associated with significant increase in hepatic iron concentration (Medici et al., 2007). However, iron studies were conducted on a subsequent larger study of 143 patients with WD (Pfeiffenberger et al., 2012). Interestingly, serum ferritin was lower in males with WD who also presented lower ceruloplasmin levels. Only 3 out of 27 patients who underwent liver biopsy presented hepatic iron concentration higher than the upper limit, all of them on chelating agents at the time of the biopsy. Collectively, the direct and indirect evidence points to the fact that iron may contribute to the phenotype of WD and monitoring of iron metabolism parameters should be considered in the long-term management of these patients.

METHYL GROUPS Hepatic methionine metabolism regulates DNA and histone methylation reactions with potential consequences on gene expression regulation and disease presentation and course. The central players in methionine metabolism are S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and the bidirectional SAH hydrolase (SAHH). SAM is irreversibly converted to SAH by donating its methyl moiety to DNA methyltransferases. The SAM-to-SAH ratio is considered a useful index of methylation capacity, as SAM is the universal methyl donor for methyltransferase reactions and

SAH is an inhibitor of almost all methylation reactions. SAHH is a bidirectional enzyme that regulates the production of homocysteine in the forward direction, while excess homocysteine promotes SAH in the reverse direction. Thus, SAHH may play a critical role in the regulation of methylation pathways; its inhibition could result in an increase of its substrate SAH, with resultant inhibition of gene methylation reactions, thereby potentially regulating the expression of genes involved in liver injury. Dietary methyl groups are provided by either betaine or choline which are abundant in spinach, beet, cereals or eggs, liver, almonds, and broccoli. Copper is a major regulator of methionine metabolism through its effect on SAHH (Bethin et al., 1995). Copper inhibits SAHH in a noncompetitive manner since its binding to this enzyme results in the release of NAD+ cofactors (Li et al., 2007). Sahh hepatic transcript levels are downregulated in a murine model of WD characterized by spontaneous hepatic copper accumulation (Medici et al., 2013; Le et al., 2014). In addition, maternal provision of methyl groups in the form of choline was able to increase Sahh transcripts to control levels, indicating maternal diet can have an effect on gene expression and epigenetic mechanisms of phenotype regulation in WD (Medici et al. 2014).

OTHER DIETARY FACTORS Another study on the LEC rat examined the effects of dietary fatty acids on peroxidative stress and histologic damage. Polyunsaturated fatty acids, both n-6 and n-3 type, were associated with fewer histologic features of hepatitis and lower liver enzymes. Cyclooxygenase-2 hepatic transcript levels, a marker of inflammation, were downregulated in the group of rats receiving polyunsaturated fatty acids (Du et al., 2004). Another study explored the effects of mild zinc deficiency on the same animal model and the major finding was that LEC rats exposed to a zinc-deficient diet for 4 weeks developed acute liver failure at a younger age compared to control rats (94 vs. 136 days of age) (Saito et al., 2007). A diet rich in histidine was associated with reduced hepatic copper levels by 47% and increased urinary copper excretion in LEC rats, with corresponding reduced activity of the antioxidant enzyme Cu, Zn-superoxide dismutase (Xu et al., 2003). LEC rats fed a soy protein-rich diet starting at 6 weeks of age presented 80% higher hepatic copper concentration and reduced survival compared to control mice (Yonezawa et al., 2003).

SUMMARYAND CONCLUSION It is becoming evident from clinical and basic science research that the phenotype of WD is affected by

GENETIC AND ENVIRONMENTAL MODIFIERS OF WILSON DISEASE genetic, epigenetic, and environmental factors. This can have implications for the neonatal screening, early diagnosis, and treatment of this condition. The fact that maternal diet can affect the onset and progression of disease supports the indication of neonatal screening of WD. In addition, current anticopper treatments could be optimized by modifying dietary factors and could be tailored according to the presence of certain mutations and gene modifiers, ultimately improving disease course and prognosis.

ABBREVIATIONS APOE, apolipoprotein E; ATOX1, antioxidant 1 copper chaperone; ATP7A, ATPase copper-transporting alpha; ATP7B, ATPase copper-transporting beta; COMMD1, copper metabolism domain-containing 1; DMT1, divalent metal transporter 1; HFE, hemochromatosis; LEC, Long–Evans Cinnamon; MTHFR, 5,10methylenetetrahydrofolate reductase; PRNP, prion protein; SAH, S-adenosylhomocysteine; SAHH, S-adenosylhomocysteine hydrolase; SAM, S-adenosylmethionine; WD, Wilson disease.

REFERENCES Ala A, Schilsky M (2011). Genetic modifiers of liver injury in hereditary liver disease. Semin Liver Dis 31: 208–214. Attieh ZK, Mukhopadhyay CK, Seshadri V et al. (1999). Ceruloplasmin ferroxidase activity stimulates cellular iron uptake by a trivalent cation-specific transport mechanism. J Biol Chem 274: 1116–1123. Bethin KE, Cimato TR, Ettinger MJ (1995). Copper binding to mouse liver S-adenosylhomocysteine hydrolase and the effects of copper on its levels. J Biol Chem 270: 20703–20711. Bost M, Piguet-Lacroix G, Parant F et al. (2012). Molecular analysis of Wilson patients: direct sequencing and MLPA analysis in the ATP7B gene and Atox1 and COMMD1 gene analysis. J Trace Elem Med Biol 26: 97–101. Brown DR, Qin K, Herms JW et al. (1997). The cellular prion protein binds copper in vivo. Nature 18–25; 390: 684–687. Burstein E, Ganesh L, Dick RD et al. (2004). A novel role for XIAP in copper homeostasis through regulation of MURR1. Embo J 23: 244–254. de Bie P, Burstein E, van de Sluis B et al. (2006). Several COMMD proteins interact with ATP7B; possible candidate genes for hepatic copper overload disorders with unknown etiology. Eur J Gastroenterol Hepatol 18: A52. DiDonato M, Hsu HF, Narindrasorasak S et al. (2000). Copper-induced conformational changes in the N-terminal domain of the Wilson disease copper-transporting ATPase. Biochemistry 39: 1890–1896. Donadio S, De Keukeleire J, Micoud J et al. (2007). Cellular localization and trafficking of endogenous ATP7B and COMMD1 proteins. Febs J 274: 106.

39

Du C, Fujii Y, Ito M et al. (2004). Dietary polyunsaturated fatty acids suppress acute hepatitis, alter gene expression and prolong survival of female Long–Evans Cinnamon rats, a model of Wilson disease. J Nutr Biochem 15: 273–280. Erhardt A, Hoffmann A, Hefter H et al. (2002). HFE gene mutations and iron metabolism in Wilson’s disease. Liver 22: 474–478. Ferenci P (2014). Phenotype–genotype correlations in patients with Wilson’s disease. Ann N Y Acad Sci 1315: 1–5. Ferenci P, Weiss KH, Członkowska A et al. (2011). Gender influences the clinical presentation of Wilson disease (WD). Gastroenterology 140: S939. Ford MJ, Burton LJ, Morris RJ et al. (2002). Selective expression of prion protein in peripheral tissues of the adult mouse. Neuroscience 113: 177–192. Gasperini L, Meneghetti E, Pastore B et al. (2015). Prion protein and copper cooperatively protect neurons by modulating NMDA receptor through S-nitrosylation. Antioxid Redox Signal 22: 772–784. Gromadzka G, Rudnicka M, Chabik G et al. (2011). Genetic variability in the methylenetetrahydrofolate reductase gene (MTHFR) affects clinical expression of Wilson’s disease. J Hepatol 55: 913–919. Gu YH, Kodama H, Du SL (2005). Apolipoprotein E genotype analysis in Chinese Han ethnic children with Wilson’s disease, with a concentration on those homozygous for R778L. Brain Dev 27: 551–553. Hafkemeyer P, Schupp M, Storch M et al. (1994). Excessive iron storage in a patient with Wilson’s disease. Clin Investig 72: 134–136. Hamza I, Schaefer M, Klomp LW et al. (1999). Interaction of the copper chaperone HAH1 with the Wilson disease protein is essential for copper homeostasis. Proc Natl Acad Sci U S A 96: 13363–13368. Itoh S, Kim HW, Nakagawa O et al. (2008). Novel role of antioxidant-1 (Atox1) as a copper-dependent transcription factor involved in cell proliferation. J Biol Chem 283: 9157–9167. Kato J, Kobune M, Kohgo Y et al. (1996). Hepatic iron deprivation prevents spontaneous development of fulminant hepatitis and liver cancer in Long–Evans Cinnamon rats. J Clin Invest 98: 923–929. Klomp AE, van de Sluis B, Klomp LW et al. (2003). The ubiquitously expressed MURR1 protein is absent in canine copper toxicosis. J Hepatol 39: 703–709. Kocabay G, Tutuncu Y, Yilmaz H et al. (2009). Impact of apolipoprotein E genotypes on phenotypic expression in Turkish patients with Wilson’s disease. Scand J Gastroenterol 44: 1270–1271. Le A, Shibata NM, French SW et al. (2014). Characterization of timed changes in hepatic copper concentrations, methionine metabolism, gene expression, and global DNA methylation in the Jackson Toxic Milk mouse model of Wilson disease. Int J Mol Sci 15: 8004–8023. Li M, Li Y, Chen J et al. (2007). Copper ions inhibit S-adenosylhomocysteine hydrolase by causing dissociation of NAD + cofactor. Biochemistry 46: 11451–11458.

40

V. MEDICI AND K.-H. WEISS

Linnebank M, Lutz H, Jarre E et al. (2006). Binding of copper is a mechanism of homocysteine toxicity leading to COX deficiency and apoptosis in primary neurons, PC12 and SHSY-5Y cells. Neurobiol Dis 23: 725–730. Litwin T, Gromadzka G, Członkowska A (2012a). Gender differences in Wilson’s disease. J Neurol Sci 312: 31–35. Litwin T, Gromadzka G, Członkowska A (2012b). Apolipoprotein E gene (APOE) genotype in Wilson’s disease: impact on clinical presentation. Parkinsonism Relat Disord 18: 367–369. Maine GN, Mao X, Muller PA et al. (2009). COMMD1 expression is controlled by critical residues that determine XIAP binding. Biochem J 417: 601–609. Medici V, Di Leo V, Lamboglia F et al. (2007). Effect of penicillamine and zinc on iron metabolism in Wilson’s disease. Scand J Gastroenterol 42: 1495–1500. Medici V, Shibata NM, Kharbanda KK et al. (2013). Wilson’s disease: changes in methionine metabolism and inflammation affect global DNA methylation in early liver disease. Hepatology 57: 555–565. Medici V, Shibata NM, Kharbanda KK et al. (2014). Maternal choline modifies fetal liver copper, gene expression, DNA methylation, and neonatal growth in the tx-j mouse model of Wilson disease. Epigenetics 9: 286–296. Merle U, Stremmel W, Gessner R (2006). Influence of homozygosity for methionine at codon 129 of the human prion gene on the onset of neurological and hepatic symptoms in Wilson disease. Arch Neurol 63: 982–985. Mufti AR, Burstein E, Csomos RA et al. (2006). XIAP Is a copper binding protein deregulated in Wilson’s disease and other copper toxicosis disorders. Mol Cell 21: 775–785. Mufti AR, Burstein E, Duckett CS (2007). XIAP: cell death regulation meets copper homeostasis. Arch Biochem Biophys 463: 168–174. Muller T, van de Sluis B, Zhernakova A et al. (2003). The canine copper toxicosis gene MURR1 does not cause non-Wilsonian hepatic copper toxicosis. J Hepatol 38: 164–168. Pfeiffenberger J, Gotthardt DN, Herrmann T et al. (2012). Iron metabolism and the role of HFE gene polymorphisms in Wilson’s disease. Liver Int 32: 165–170. Prohaska JR (2008). Role of copper transporters in copper homeostasis. Am J Clin Nutr 88: 826S–829S. Przybyłkowski A, Gromadzka G, Wawer A et al. (2013). Intestinal expression of metal transporters in Wilson’s disease. Biometals 26: 925–934. Przybyłkowski A, Gromadzka G, Członkowska A (2014). Polymorphisms of metal transporter genes DMT1 and ATP7A in Wilson’s disease. J Trace Elem Med Biol 28: 8–12. Riordan SM, Williams R (2001). The Wilson’s disease gene and phenotypic diversity. J Hepatol 34: 165–171. Saito A, Nakayama K, Hara H (2007). Mild zinc deficiency and dietary phytic acid accelerates the development of fulminant hepatitis in LEC rats. J Gastroenterol Hepatol 22: 150–157. Salazar J, Mena N, Hunot S et al. (2008). Divalent metal transporter 1 (DMT1) contributes to neurodegeneration in

animal models of Parkinson’s disease. Proc Natl Acad Sci U S A 105: 18578–18583. Salzer U, Hagena T, Webster DB et al. (2008). Sequence analysis of BIRC4/XIAP in male patients with common variable immunodeficiency. Int Arch Allergy Immunol 147: 147–151. Schiefermeier M, Kollegger H, Madl C et al. (2000). The impact of apolipoprotein E genotypes on age at onset of symptoms and phenotypic expression in Wilson’s disease. Brain 123: 585–590. Schilsky M (2001). The irony of treating Wilson’s disease. Am J Gastroenterol 96: 3055–3057. Serre D, Gurd S, Ge B et al. (2008). Differential allelic expression in the human genome: a robust approach to identify genetic and epigenetic cis-acting mechanisms regulating gene expression. PLoS Genet 4. e1000006. Shiono Y, Wakusawa S, Hayashi H et al. (2001). Iron accumulation in the liver of male patients with Wilson’s disease. Am J Gastroenterol 96: 3147–3151. Simon I, Schaefer M, Reichert J et al. (2008). Analysis of the human Atox 1 homologue in Wilson patients. World J Gastroenterol 14: 2383–2387. Sorbello O, Sini M, Civolani A et al. (2010). HFE gene mutations and Wilson’s disease in Sardinia. Dig Liver Dis 42: 216–219. Squitti R, Polimanti R, Siotto M et al. (2013). ATP7B variants as modulators of copper dyshomeostasis in Alzheimer’s disease. Neuromolecular Med 15: 515–522. Steindl P, Ferenci P, Dienes HP et al. (1997). Wilson’s disease in patients presenting with liver disease: a diagnostic challenge. Gastroenterology 113: 212–218. Stuehler B, Reichert J, Stremmel W et al. (2004). Analysis of the human homologue of the canine copper toxicosis gene MURR1 in Wilson disease patients. J Mol Med 82: 629–634. Su LC, Ravanshad S, Owen CA et al. (1982). A comparison of copper-loading disease in Bedlington terriers and Wilson’s disease in humans. Am J Physiol Gastrointest Liver Physiol 243: G226–G230. Tao TY, Liu F, Klomp L et al. (2003). The copper toxicosis gene product Murr1 directly interacts with the Wilson disease protein. J Biol Chem 278: 41593–41596. Twedt DC, Sternlieb I, Gilbertson SR (1979). Clinical, morphologic, and chemical studies on copper toxicosis of Bedlington terriers. J Am Vet Med Assoc 175: 269–275. van de Sluis B, Breen M, Nanji M et al. (1999). Genetic mapping of the copper toxicosis locus in Bedlington terriers to dog chromosome 10, in a region syntenic to human chromosome region 2p13-p16. Hum Mol Genet 8: 501–507. van de Sluis B, Rothuizen J, Pearson PL et al. (2002). Identification of a new copper metabolism gene by positional cloning in a purebred dog population. Hum Mol Genet 11: 165–173. Vanderwerf SM, Cooper MJ, Stetsenko IV et al. (2001). Copper specifically regulates intracellular phosphorylation of the Wilson’s disease protein, a human copper-transporting ATPase. J Biol Chem 276: 36289–36294. Walker JM, Tsivkovskii R, Lutsenko S (2002). Metallochaperone Atox1 transfers copper to the NH2-terminal domain of the

GENETIC AND ENVIRONMENTAL MODIFIERS OF WILSON DISEASE Wilson’s disease protein and regulates its catalytic activity. J Biol Chem 277: 27953–27959. Walshe JM, Cox DW (1998). Effect of treatment of Wilson’s disease on natural history of haemochromatosis. Lancet 352: 112–113. Weiss KH, Merle U, Schaefer M et al. (2006). Copper toxicosis gene MURR1 is not changed in Wilson disease patients with normal blood ceruloplasmin levels. World J Gastroenterol 12: 2239–2242. Weiss KH, Runz H, Noe B et al. (2010). Genetic analysis of BIRC4/XIAP as a putative modifier gene of Wilson disease. J Inherit Metab Dis 33: S233–S240.

41

White AR, Huang X, Jobling MF et al. (2001). Homocysteine potentiates copper- and amyloid beta peptide-mediated toxicity in primary neuronal cultures: possible risk factors in the Alzheimer’s-type neurodegenerative pathways. J Neurochem 76: 1509–1520. Xu H, Sakakibara S, Morifuji M et al. (2003). Excess dietary histidine decreases the liver copper level and serum alanine aminotransferase activity in Long–Evans Cinnamon rats. Br J Nutr 90: 573–579. Yonezawa K, Nunomiya S, Daigo M et al. (2003). Soy protein isolate enhances hepatic copper accumulation and cell damage in LEC rats. J Nutr 133: 1250–1254.

Handbook of Clinical Neurology, Vol. 142 (3rd series) Wilson Disease A. Członkowska and M.L. Schilsky, Editors http://dx.doi.org/10.1016/B978-0-444-63625-6.00005-7 © 2017 Elsevier B.V. All rights reserved

Chapter 5

Pathogenesis of Wilson disease 1

IVO FLORIN SCHEIBER1, RADAN BRŮHA2, AND PETR DUŠEK3* Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic

2

Fourth Department of Internal Medicine, Charles University in Prague, First Faculty of Medicine and General University Hospital in Prague, Prague, Czech Republic 3

Department of Neurology and Center of Clinical Neuroscience, Charles University in Prague, First Faculty of Medicine and General University Hospital in Prague, Prague, Czech Republic

Abstract Wilson disease is an autosomal-recessive disorder originating from a genetic defect in the coppertransporting ATPase ATP7B that is required for biliary copper secretion and loading of ceruloplasmin with copper. Impaired ATP7B function in Wilson disease results in excessive accumulation of copper in liver, brain, and other tissues. Toxic copper deposits may induce oxidative stress, modify expression of genes, directly inhibit proteins, and impair mitochondrial function, leading to hepatic, neuropsychiatric, renal, musculoskeletal, and other symptoms. Hepatocyte dysfunction initially manifests as steatosis and later may progress to other hepatic phenotypes such as acute liver failure, hepatitis, and fibrosis. In the brain, copper accumulates in astrocytes, leading to impairment of the blood–brain barrier and consequent damage to neurons and oligodendrocytes. Basal ganglia and brainstem are the brain regions with highest susceptibility to copper toxicity and their lesions lead to various combinations of movement and psychiatric disorders. This chapter will give an overview of the essential requirement of copper for biologic processes and the molecular mechanisms employed by cells to maintain their copper levels in a proper range. We will specify the physiologic functions of ATP7B and the consequences of its dysfunction and summarize the current knowledge on the pathogenesis of liver and neuropsychiatric disease. Finally, we will describe the consequences of copper overload in Wilson disease in other tissues.

INTRODUCTION Wilson disease (WD) is a rare, inherited autosomalrecessive disease of copper metabolism, caused by mutations in the copper-transporting ATPase ATP7B, responsible for biliary excretion of copper. During the course of the disease copper accumulates excessively in several organs, most notably in the liver, brain, and eye (Scheinberg and Sternlieb, 1996; Pfeiffer, 2007; Huster, 2010).WD may present with a variety of clinical features, including liver disease, ophthalmologic manifestations, neurologic disorders, neuropsychiatric symptoms, osteoarthritis, renal tubular dysfunction, and

cardiomyopathy (Lorincz, 2010; Bandmann et al., 2015; Wu et al., 2015). WD is fatal unless measures are taken to remove the excess copper from the body, either by copper chelation or blocking intestinal uptake of copper with zinc (Scheinberg and Sternlieb, 1996; Huster, 2010).

COPPER METABOLISM Copper represents the third most abundant essential transition metal in humans (Lewinska-Preis et al., 2011). In its function as a cofactor and/or as structural component

*Correspondence to: Petr Dušek, Neurologická klinika VFN a 1.LFUK, Katerinská 30, 128 01, Prague 2, Czech Republic. Tel: +420-224965528, E-mail: [email protected]

44

I.F. SCHEIBER ET AL.

Fig. 5.1. Cellular and systemic copper homeostasis. (A) Cellular copper uptake is primarily mediated by the copper transporter Ctr1. A yet unknown ecto-cuprireductase and/or extracellular ascorbate provide the reduced copper species for uptake by Ctr1. Accumulated copper is sequestered by glutathione (GSH) and/or stored in metallothioneins (MT). Specialized chaperons shuttle copper to its specific cellular targets: the copper chaperon for superoxide dismutase (CCS) to superoxide dismutase 1 (SOD1), Cox17 to Sco1/2 (not shown) and Cox 11 (not shown) for subsequent incorporation into cytochrome c oxidase, and antioxidant protein 1 (Atox1) to the copper-transporting ATPases ATP7A and ATP7B. Both ATP7A and ATP7B transport copper into the trans-Golgi network (TGN) for subsequent incorporation into copper-dependent enzymes: ATP7B provides copper for incorporation into ceruloplasmin (Cp), ATP7A is required for metallation of tyrosinase, peptidylglycine amidating monoxygenase, dopamine-b-monoxygenase, and lysyl oxidase (not shown). When cellular copper levels rise above certain thresholds (ATP7A and ATP7B) or in response to peripheral tissue demand (ATP7A expressed in liver), both ATPases translocate reversibly via vesicles toward the plasma membrane to export copper. In the liver ATP7B is required for biliary copper excretion within hepatocytes, whereas ATP7A appears to function in copper mobilization from hepatic stores into the blood stream during times of peripheral copper deficiency. (B) Dietary copper is taken up in the small intestine

for several enzymes, copper participates in many physiologic pathways, including energy metabolism, antioxidative defense, and iron metabolism (Scheiber et al., 2014). Furthermore, copper has been linked to important biologic processes, including angiogenesis, response to hypoxia, and neuromodulation (Scheiber et al., 2014). However, when in excess of cellular needs copper is deleterious. Unbound or loosely bound copper may induce oxidative stress and subsequent damage of cellular components (Uriu-Adams and Keen, 2005) and/or exert its toxicity by direct inhibition of protein functions (Boulard et al., 1972; Letelier et al., 2005, 2006, 2007; Pamp et al., 2005; Schwerdtle et al., 2007). Given the requirement for copper on the one hand and its potential toxicity on the other hand, cells have evolved mechanisms to maintain cellular copper concentrations in a proper range. Many components of the cellular copper homeostasis machinery have been described on the molecular level (Fig. 5.1A). The copper transport receptor 1 (Ctr1) is considered as the major entry pathway for copper into eukaryotic cells (Lee et al., 2002a, b). Although it is currently the sole known pathway for copper entry into the cell, the existence of a Ctr1independent copper uptake by a yet unknown transporter has been reported (Lee et al., 2002b). The accumulation of copper in the cytosol bears a risk for copper-mediated oxidative damage and binding of copper to essential biomolecules. However, under physiologic conditions the concentration of free copper within the cell is maintained at around 10–18 M (i.e., 0.06 pg/g) by binding of copper to metallothioneins (MTs) and ligands of low molecular mass such as glutathione (GSH; Rae et al., 1999). MTs and GSH also represent the major molecules involved in the intracellular sequestering and storing of excess copper. In addition, mitochondria have been suggested to contribute to the cellular copper buffering capacity (Cobine et al., 2004; Maxfield et al., 2004; Leary et al., 2009). A group of specialized proteins, termed copper chaperones, shuttle copper to copper-dependent enzymes and to organelles, thereby protecting it from being scavenged by MTs or GSH. Atox1 transfers Cu+ to the N-terminal metal-binding domains (MBDs) of by intestinal enterocytes by Ctr1 on the apical site and released into the portal circulation by ATP7A. Most of the newly absorbed copper is taken up by the liver, mediated primarily by Ctr1. Excess copper is excreted into the bile by ATP7B. When tissue demand is sensed, copper is mobilized from hepatic stores into the blood stream by ATP7A. Copper enters the brain via brain endothelial cells which comprise the blood– brain barrier (BBB). These cells take up copper from the blood by Ctr1 and release copper into the brain parenchyma by ATP7A. Copper transport into milk and to the developing fetus is mediated by ATP7B and ATP7A, respectively.

PATHOGENESIS OF WILSON DISEASE the copper-transporting P-type ATPases ATP7A and ATP7B, the copper chaperone for superoxide dismutase (CCS) is involved in the insertion of copper into superoxide dismutase 1 (SOD1; Culotta et al., 1997) and Cox17, Sco1, Sco2, and Cox11 participate in the insertion of copper ions into mammalian cytochrome c oxidase (Robinson and Winge, 2010). Transport of copper into mitochondria is accomplished by a yet unknown copper ligand (Cobine et al., 2004; Vest et al., 2013). Cellular copper export in mammals relies on the function of two proteins, ATP7A and ATP7B. These proteins belong to the protein family of P1B-type ATPases that use the energy of ATP hydrolysis to transport heavy metals across cellular membranes (Arguello et al., 2007). In addition to their critical function in the efflux of excess cellular copper, ATP7A and ATP7B shuttle copper to the secretory pathway for incorporation into copper-dependent enzymes such as tyrosinase, peptidylglycine amidating monoxygenase, dopamineb-monoxygenase, lysyl oxidase, and ceruloplasmin (Cp; Scheiber et al., 2014).

ATP7B Human ATP7B is a large multispanning membrane protein with its overall structure consisting of: (1) a cytosolic amino-terminus; (2) eight transmembrane helices; (3) an ATP-binding domain; (4) an actuator domain; and (5) a cytosolic carboxyl-terminus (Lutsenko et al., 2007a). The N-terminal tail of human ATP7B harbors six MBDs, each capable of binding one Cu+ ion (Banci et al., 2009). However, only the MBDs closest to the membrane, MBD5 and MBD6, are important for efficient copper transport (Cater et al., 2007), while MBD1–4 appear to primarily function in the regulation of the catalytic activity in response to copper (Lutsenko et al., 2007a). The eight transmembrane helices of ATP7B are involved in the formation of the copper translocation pathway (Lutsenko et al., 2007a). Specific residues within TM6–TM8 are thought to contribute to the intramembrane copper coordination sites required for copper transmembrane transport (Arguello et al., 2007; Lutsenko et al., 2007a). The ATP-binding domain of ATP7B, located between TM6 and TM7, comprises a nucleotide-binding domain (N-domain) and a phosphorylation domain containing the DKTG, TGDN, and GDGxND signature motifs of P-type ATPases (Bull et al., 1993).The Asp residue in the DKTG sequence motif of the P-domain is crucial for the catalytic cycle of P-type ATPases (Palmgren and Nissen, 2011). It accepts the g-phosphate from the ATP upon binding of ATP to the N-domain and copper to the intramembrane copper sites (Lutsenko et al., 2007b). The formation of this phosphorylated intermediate induces conformational changes that

45

allow the copper ion to be released on the other side of the membrane (Lutsenko et al., 2007b). The catalytic cycle is closed by the hydrolysis of the aspartyl phosphate bond and the return of the enzyme to its initial state (Lutsenko et al., 2007b). The dephosphorylation step is facilitated by the actuator domain linked to TM4 and TM5 (Lutsenko et al., 2007b). This domain harbors the TGE signature motif of the P-type ATPases that is strictly required for their phosphatase activity (Palmgren and Nissen, 2011). Consequently, mutations of the TGE motif in ATP7B result in hyperphosphorylated and catalytically inactive proteins (Cater et al., 2007).

Physiologic function ATP7B, abundantly expressed in the liver and at lower levels in kidney, placenta, brain, lung, and heart (Bull et al., 1993; Tanzi et al., 1993; Yamaguchi et al., 1993), participates in two important physiologic processes: the maintenance of systemic copper homeostasis and the supply of Cp with copper (Fig. 5.1A; La Fontaine and Mercer, 2007; Lutsenko et al., 2007a). Overall balance of copper in the body is achieved by regulation of the rate of uptake of copper in the small intestine and biliary excretion of copper (Fig. 5.1B; Scheiber et al., 2013). Current evidence suggests that the copper transporter Ctr1 is the major transporter involved in the apical uptake of copper, which activity is regulated in response to copper at the level of protein localization and abundance (Nevitt et al., 2012; Kim et al., 2013a; Ohrvik and Thiele, 2014). The copper efflux protein ATP7A is involved in the transport of copper across the basolateral surface of the enterocyte, thereby facilitating copper delivery to the portal circulation (Monty et al., 2005; Nyasae et al., 2007). ATP7B is the transporter responsible for efflux of copper from the liver into the bile. Excess copper in the hepatocyte stimulates trafficking of this protein from the trans-Golgi network, where it supplies copper to nascent Cp, to vesicles close to the apical membrane of the hepatocyte that abuts the biliary canaliculus (Cater et al., 2006; La Fontaine and Mercer, 2007; Lutsenko et al., 2007a). This copper-induced trafficking of ATP7B is the principal homeostatic mechanism for removing excess copper from the body (Scheiber et al., 2013), increasing the capacity of rapid copper sequestration from the cytosol and allowing for subsequent excretion of excess copper via exocytosis.

Consequences of dysfunction Inactivation of ATP7B in WD results in failure of biliary copper excretion by ATP7B that leads to copper overload in liver and other tissues as well as failure of loading of Cp with copper. A defect in the normal copper incorporation into Cp leads to the secretion of the peptide

46

I.F. SCHEIBER ET AL.

without copper that has a shorter half-life, leading to its reduced plasmatic concentration (Hellman and Gitlin, 2002). Hepatic copper concentrations in patients with WD are typically above 250 mg/g dry weight compared to 15–55 mg/g dry weight in healthy individuals (Mikol et al., 2005; Pfeiffer, 2007; Yang et al., 2015) and the copper content in brains of WD patients has been reported to be up to 450 mg/g dry weight compared to a control value of 7–60 mg/g dry weight (Horoupian et al., 1988; Faa et al., 2001; Mikol et al., 2005). In contrast, total serum copper levels typically are reduced in WD patients, reflecting reduced copper bound to Cp that normally represents 90% of total serum copper (Pfeiffer, 2007). The toxic effects of excess copper are considered as the primary cause of the symptoms seen in WD (Pfeiffer, 2007; Huster, 2010). Copper toxicity is in part a consequence of the redox activity of copper that may lead to subsequent damage of lipids, proteins, DNA, and RNA molecules. Redox cycling of copper in the presence of partially reduced oxygen species such as hydrogen peroxide and superoxide can catalyze the generation of highly reactive hydroxyl radicals via the Haber–Weiss cycle (Halliwell and Gutteridge, 1990; Gunther et al., 1995). Copper ions can also accelerate lipid peroxidation by splitting lipid hydroperoxides, in a reaction analogous to the Fenton reaction, giving alkoxyl and peroxyl radicals and thereby propagating the chain reaction (Britton, 1996; Burkitt, 2001; Halliwell, 2006). Lipid peroxidation and DNA damage have been observed in livers of WD patients, suggesting an involvement of oxidative stress in the etiology of this disease (Huster, 2014). Mitochondria are sensitive targets for copper-induced oxidative stress. Copper has been shown to induce cardiolipin fragmentation, important for mitochondrial membrane integrity and function, and thereby promoting mitochondrial damage (Yurkova et al., 2011). Severe mitochondrial dysfunction heralded by low activities of respiratory chain enzymes has been described in the livers of WD patients (Gu et al., 2000). In cultured astrocytes, copper-induced oxidative stress has been shown to induce mitochondrial permeability transition (Reddy et al., 2008). Severe mitochondrial alterations in human liver occur already in the early stages of the disease and mitochondrial damage is therefore considered as an initial event of hepatic injury in WD (Huster, 2014; Zischka and Lichtmannegger, 2014). Although the consequences of copper accumulation in WD are generally ascribed in large part to copperinduced oxidative stress, alternative mechanisms of copper toxicity, namely activation of acid sphingomyelinase and subsequent induction of apoptosis (Lang et al., 2007) and direct binding of copper to proteins, should be considered (Boulard et al., 1972; Vasic et al., 1999; Letelier

et al., 2005, 2006; Pamp et al., 2005; Schwerdtle et al., 2007; Hegde et al., 2010). In regard of the latter, binding of copper to the X-linked inhibitor of apoptosis has been shown to make cells more susceptible to apoptotic stimuli (Mufti et al., 2006). In addition, copper may unspecifically bind to thiol and amino groups in proteins not related to copper metabolism, thereby altering protein structure and modifying their biologic functions (Letelier et al., 2005). Inactivation of ATP7B in WD results not only in failure of biliary copper excretion by ATP7B but also in failure of loading of apo-Cp with copper that is necessary for gaining the ferroxidase activity. However, a role of decreased Cp oxidase activity and subsequent iron metabolism disturbance in the pathogenesis of WD appears to be negligible (Roeser et al., 1970; Pfeiffenberger et al., 2012; Dusek et al., 2015).

LIVER DISEASE The liver plays a central role in systemic copper homeostasis, serving as a central copper storage site and being required for the efflux of excess copper in the bile. Due to the tissue expression pattern of ATP7B and its function in biliary copper excretion, liver injury is the most common manifestation of WD. In fact, even all patients with nonhepatic WD manifestation (i.e., mainly neurologic symptoms) excessively accumulate copper in the liver and more than half of them present with cirrhosis (Ferenci, 2014; Przybylkowski et al., 2014). Several inbred rodent models of WD have been utilized to study copper toxicity in the liver. The toxic milk (tx) mouse and the Jackson tx mouse (txj) have missense mutations in ATP7B which affect ATP7B function (Theophilos et al., 1996), whereas the Long–Evans Cinnamon (LEC) rat has a 300-bp deletion in ATP7B, resulting in the loss of ATP7B (Wu et al., 1994). Although all these animals excessively accumulate copper in the liver, they present with a wide spectrum of liver abnormities ranging from normal liver in the tx mice to massive liver necrosis, fulminant hepatitis, and even hepatocellular carcinoma in the LEC rat (Huster et al., 2006). The variations in the observed liver pathology could point to a species-specific response to copper overload, but may also result from incomplete inactivation of ATP7B in the tx mice and/or the involvement of other genes mutated in these inbred animals (Huster et al., 2006). These problems have been overcome with the generation of the ATP7B–/– mouse, which possesses a well-defined genetic background (Buiakova et al., 1999). Biochemical characteristics and liver pathology of the ATP7B–/– mice resemble that of WD patients and can be directly traced back to the inactivation of ATP7B, making it an excellent animal model for WD (Huster et al., 2006).

PATHOGENESIS OF WILSON DISEASE Biochemical characteristics and morphologic changes in the liver of ATP7B–/– mice have been described in detail by Huster et al. (2006). ATP7B–/– mice hyperaccumulated copper in their livers compared to wild-type mice (Huster et al., 2006). While the highest level of copper was reached at 5–6 weeks of age, the excess of copper compared to wild-type mice increased from 18-fold at 5–6 weeks to 37-fold in 20-week-old mice due to a decrease of copper in wild-type mice (Huster et al., 2006). Liver pathology was either absent or mild (focal hepatocellular swelling, necrosis, inflammation, and depletion of glycogen stores) up to an age of 6 weeks but electron microscopy revealed focally distorted and enlarged mitochondria even in microscopically normal livers at this stage (Huster et al., 2006). At an age of 12–20 weeks severe hepatocellular injury with widespread necrosis and inflammation and remarkable nuclear enlargement with prominent nucleoli and vacuolization were observed (Huster et al., 2006). Fibrosis, cholangitis, scattered macrophages, and focal steatosis were detected in a limited number of animals (Huster et al., 2006). Between the age of 28 and 52 weeks the normal architecture of liver parenchyma was restored; however, morphologic changes in the bile duct consistent with cholangiocarcinoma were observed (Huster et al., 2006). Subcellular copper distribution was similar in livers of ATP7B–/– mice at the age of 6 weeks (mild liver pathology) and at the age of 20 weeks (severe liver pathology). Copper accumulated mainly in the cytoplasm but was also elevated in insoluble fractions enriched in nuclei, mitochondria, endosome, and plasma membrane. The degree of copper overload in the insoluble fractions was highest in nuclei and associated with an increase in nuclear size and appearance of a proliferation marker. Since both copper overload and alteration in nuclear morphology and function appeared before development of significant liver pathology, Huster et al. (2006) suggested that the nucleus is an important and early target of copper toxicity. The toxic effects of excess copper in hepatocytes have been considered to be in large part a consequence of the redox activity of copper. Mitochondria had been postulated to be initial targets of copper-mediated oxidative stress (Sokol et al., 1994). Indeed, severe mitochondrial alterations in human liver occur already in the initial stages of the disease (Huster, 2010; Zischka and Lichtmannegger, 2014) and hepatic steatosis, the most common liver pathology seen in early stages of WD, might be a direct consequence of mitochondrial damage (Ferenci, 2004). However, at least in presymptomatic 6-week-old ATP7B–/– mice, copper-mediated oxidative stress appears not to play a major role despite significant copper accumulation (Huster et al., 2007). Instead, accumulated copper selectively upregulated the molecular

47

machinery associated with the cell cycle and chromatin structure and downregulated lipid metabolism (Huster et al., 2007; Medici et al., 2014). The marked downregulation of genes involved in cholesterol biosynthesis and decreased cholesterol contents in the ATP7B–/– mouse are consistent with the development of steatosis (Huster et al., 2007). The downregulation of cholesterol metabolism in this animal model of WD was attributed to an inhibition of sterol regulatory-binding protein 2 (SREBP-2), a transcription factor which activates cholesterol biosynthesis by copper (Huster et al., 2007). Downregulation of SREBP-2 target genes has also been verified in liver samples of WD patients (Huster et al., 2007). Independently of copper accumulation, steatosis is more pronounced in WD patients carrying a single-nucleotide polymorphism in the patatin-like phospholipase domain-containing 3 (PNPLA3) gene known to be associated with nonalcoholic fatty liver disease in non-WD population (Stattermayer et al., 2015). While steatosis appears to be rather a consequence of altered lipid metabolism, other phenotypic manifestations of WD, including acute liver failure, hepatitis, and fibrosis, develop as a consequence of hepatocyte injury and cell death (Gitlin, 2003). Hepatocyte cell death may occur either by necrosis or apoptosis, the latter being more important in the pathology of a wide range of liver diseases (Luedde et al., 2014). In WD, coppermediated oxidative stress could trigger apoptosis via damage of mitochondria, leading to release of cytochrome c, activation of intrinsic caspase-dependent apoptotic cascade, and subsequent tissue inflammatory changes (Canbay et al., 2004; Crosas-Molist and Fabregat, 2015). In addition, copper has been shown to trigger hepatocyte apoptosis through activation of acid sphingomyelinase (Asm) and release of ceramide (Lang et al., 2007). Genetic deficiency or pharmacologic inhibition of Asm prevented copper-induced hepatocyte apoptosis and protected rats, genetically prone to develop WD, from acute hepatocyte death and liver failure (Lang et al., 2007). Elevated plasma levels of Asm and a constitutive increase of ceramide- and phosphatidylserine-positive erythrocytes have also been observed in patients with WD, suggesting a role of Asm activation and ceramide release in liver cirrhosis and anemia in WD (Lang et al., 2007). Regardless of etiology, the most serious consequence of chronic liver disease is fibrosis, which can progress to liver cirrhosis (Friedman, 2003). Fibrosis is the net accumulation of extracellular matrix due to its increased synthesis and/or decreased breakdown (Mederacke et al., 2013). Hepatic stellate cells (HSCs), nonparenchymal cells residing in the perisinusoidal space of Disse, play a key role in liver fibrogenesis. Under various stimuli

48

I.F. SCHEIBER ET AL.

linked to hepatocellular injury, HSCs undergo a transformation into contractile, proliferative, and fibrogenic myofibroblasts (Lee et al., 2015). This transformation may be triggered by products of oxidative stress, apoptotic bodies from hepatocytes, transforming growth factorb, and inflammatory signals from infiltrating immune cells (Puche et al., 2013). Activation of HSCs is thought to be a complex protective response with the purpose of limiting hepatic injury. However, in the case of chronic hepatic injury, the ongoing HSC activation can become maladaptive and lead to fibrosis with subsequent complications, including portal hypertension, variceal bleeding, ascites, encephalopathy, liver failure, and death (Wallace et al., 2015). Despite the core pathways of HSC activation that are common in chronic hepatopathy, there is growing evidence suggesting that activation pathways may be specific to certain liver diseases (Gao and Bataller, 2011; Min et al., 2012; Wang et al., 2013). Copper overload may hypothetically trigger HSC transformation through oxidative stress, transforming growth

factor-b, or apoptotic bodies, but whether there is a specific activation pathway related to WD still needs to be investigated.

NEUROPSYCHIATRIC DISEASE Neurologic symptoms and central nervous system damage are primarily a consequence of extrahepatic copper toxicity (Walshe and Potter, 1977; Brewer, 2005; Fig. 5.2). Under physiologic circumstances, copper concentration is not homogeneous across different brain regions. Highest copper concentrations are contained in the locus coeruleus, followed by substantia nigra, striatum, cerebral cortex, and globus pallidus (Cumings, 1968; Zecca et al., 2004; Krebs et al., 2014); although not well documented in humans, the hippocampus may also contain high amounts of copper (Pal et al., 2013; Manto, 2014). Brain copper levels in WD are rather nonselectively increased in all brain regions, including brainstem and subcortical white matter, reaching up to

Fig. 5.2. Mechanism and consequences of copper toxicity in Wilson disease. Liver is the central organ for systemic copper balance. Impairment of biliary copper excretion in Wilson disease (dashed arrow) caused by ATP7B dysfunction leads to copper accumulation in liver. Copper toxicity results in liver steatosis and apoptosis of hepatocytes by a not completely understood mechanism. Chronic hepatocytic injury activates hepatic stellate cells (not shown), a process that underlies liver fibrosis that may ultimately lead to complications such as portal hypertension, variceal bleeding, and hepatic encephalopathy. When the liver capacity to store copper gets exhausted, copper is overflown to the blood stream as nonceruloplasmin-bound “free” plasma copper and is deposited in various organs exerting extrahepatic copper toxicity. Copper accumulates in red blood cells and its toxic effects may ultimately lead to hemolysis and anemia. In the brain copper initially accumulates in astrocytes, causing dysfunction of blood– brain barrier (BBB). Hepatic encephalopathy may also contribute to this process (not shown). Long-term disruption of BBB ultimately leads to tissue necrosis in affected brain regions. Copper accumulates in skeletal muscle cells and in cardiomyocytes and causes rhabdomyolysis and cardiomyopathy (not shown). Copper accumulated in synovial membranes of large joints leads to osteoarthritis and accelerated degenerative changes. Osteoporosis is rather a consequence of renal calcium (Ca) and phosphate wasting. Free plasma copper is filtrated by renal tubular epithelium and excreted in large amounts via urine (thick arrow). Copper accumulated in renal parenchyma may cause renal tubular dysfunction with aminoaciduria or even renal failure.

PATHOGENESIS OF WILSON DISEASE 10 times higher values than in control subjects (Green, 1955; Scheinberg and Sternlieb, 1984; Walshe and Gibbs, 1987; Mikol et al., 2005; Litwin et al., 2013). A marked increase in brain and cerebrospinal fluid copper concentrations is detectable even after several years of chelation therapy (Weisner et al., 1987; Kodama et al., 1988; Stuerenburg, 2000) and a fair degree of correlation between tissue damage severity and cerebral copper content was observed in a neuropathologic study with 11 WD patients (Horoupian et al., 1988). Astrocytes, forming a part of the blood–brain barrier, are the major cells to buffer the toxic effects of copper (Aschner et al., 1999; Pal and Prasad, 2014). They possess the capacity to store large quantities of copper, probably by upregulation of MTs and GSH (Szerdahelyi and Kasa, 1986; Scheiber et al., 2010; Scheiber and Dringen, 2011; Tiffany-Castiglioni et al., 2011). Upon chronic copper intoxication astrocytes increase in numbers and undergo cellular swelling (Pal et al., 2013). Abnormal astrocytic cells referred to as Alzheimer type I glia and Opalski cells in postmortem WD brain tissue stain strongly for MT and copper (Bertrand et al., 2001; Mikol et al., 2005), supporting their role in copper buffering. During the course of continuous intracellular copper buildup, astrocytes’ storage capacity becomes exhausted, leading to an increase in copper concentration in other brain tissues. Copper can be histochemically detected in the adventitia and media of small vessels and as perivascular granules in postmortem WD brains (Green, 1955). Several mechanisms may be involved in the pathogenesis of neuronal dysfunction. First, neurons are exposed to direct toxic effect of copper after blood–brain barrier breakage. Second, astrocytes are necessary for normal neuronal function through glutamate uptake and metabolism, potassium uptake and spatial buffering, water excretion from the brain, modulation of blood–brain barrier function, defense against free radicals and other toxic substances such as ammonia (Parpura et al., 2012). Impairment of astrocyte functions may thus contribute to neuronal dysfunction (Pal and Prasad, 2014) accompanied by axonal swelling and spheroid formation (Anzil et al., 1974; Mikol et al., 2005). In WD oligodendrocytes appear to be affected at least to the same extent as neurons. Due to their enormous metabolic demands and high membranous lipid content in myelin sheaths, these cells are very prone to oxidative damage. Profound demyelination has been described in animal models of copper toxicosis (Vogel and Evans, 1961) as well as in human postmortem tissue, particularly affecting bundles passing through basal ganglia, subcortical frontal white matter, and pontine fibers (Richter, 1948; Schulman and Barbeau, 1963; Meenakshi-Sundaram et al., 2008). Hydropic swelling

49

of myelin sheaths can even be the first sign of copper toxicity (Vogel and Evans, 1961). Nevertheless, long-term exposure to high concentrations of copper results in nonselective necrosis of all parenchymal elements. At the biochemical level, copper toxicity leads to depletion of reduced GSH and SOD1 in brain tissue suggestive of a compromised antioxidative defense system (Ozcelik and Uzun, 2009). Low total serum antioxidant capacity, determined as serum peroxyl radical scavenging capacity, was found to be correlated with severity of neurologic symptoms in WD patients (Bruha et al., 2012). Altered activities of cuproenzymes such as dopamine-b-monoxygenase with subsequent disturbance in monoamine neurotransmission may also contribute to neurologic and psychiatric symptoms (Nyberg et al., 1982; Saito et al., 1996; Fujiwara et al., 2006). Distinct susceptibility to copper toxicity of different brain regions likely underlies the prevalence of neuropsychiatric symptoms. Macroscopic structural abnormalities are most often reported in basal ganglia, thalamus, and upper brainstem. The putamen is one of the most commonly affected brain regions in WD; and its lesion has been linked to dystonic symptoms (Starosta-Rubinstein et al., 1987; Svetel et al., 2001), parkinsonism (Starosta-Rubinstein et al., 1987), and, together with globus pallidus, to various dyskinesias (Oder et al., 1993). Connection of putaminal lesions and parkinsonism suggests a disruption of the postsynaptic part of the nigrostriatal pathway. However, neuropathologic reports show that dopaminergic neurons in substantia nigra may to some extent also be affected, suggesting impairment of the presynaptic part of the nigrostriatal pathway (Schulman and Barbeau, 1963; Mikol et al., 2005; Meenakshi-Sundaram et al., 2008). Finally, a concomitant deficit of both presynaptic and postsynaptic parts of the nigrostriatal pathway was confirmed by single-photon emission computed tomography (SPECT) studies using markers for dopamine transporter and dopamine receptor (Oertel et al., 1992; Oder et al., 1996; Jeon et al., 1998; Barthel et al., 2003). At the basal ganglia level, there are interactions of motor, cognitive, and motivational systems, including reward processes, motor learning, and working memory. Structural basal ganglia lesions together with neurotransmitter imbalances thus often lead to various psychiatric symptoms, including affective, behavioral, personality, cognitive, and other disturbances (Zimbrean and Schilsky, 2014). Specifically dysfunction of the corticostriatal pathways leads to cognitive deficits mostly affecting the executive domain (Frota et al., 2013; Iwanski et al., 2015). The typical tremor in WD, i.e., asymmetric coarse tremor of upper extremities with increasing amplitude during sustained posture holding (wing-beating tremor), has some resemblance to midbrain tremor. This type of

50

I.F. SCHEIBER ET AL.

tremor is caused by lesions in the midbrain, affecting nucleus ruber, its connections, and/or cerebellar outflow pathway in superior cerebellar peduncle (Krauss et al., 1995; Milanov, 2002). In other WD patients, tremor may resemble essential tremor, which has been associated with dysfunction of cerebello-brainstem pathways and subsequent propagation of oscillations from these circuits into thalamocortical loops (Helmich et al., 2013). It is thus likely that WD tremor is associated with lesions in the dentate rubro-olivary triangle and/or its output pathways in the thalamus (Hitoshi et al., 1991; Oder et al., 1993; Frucht et al., 1998; Matsuura et al., 1998; Sudmeyer et al., 2006; Pal et al., 2007). Other less frequent consequences of brainstem lesions are rapid eye movement sleep behavior disorder (Tribl et al., 2014) and oculomotor disturbances, particularly abnormalities of vertical saccades (Ingster-Moati et al., 2007; Lesniak et al., 2008). Cortex and subcortical white matter, especially in the frontal lobe, seem to be substantially affected only after prolonged exposure to copper toxicity (Richter, 1948), and these lesions are only sparsely reported in treated patients (Huang and Chu, 1995; Prashanth et al., 2010; Kim et al., 2013b). It is unclear whether other factors besides copper toxicity may contribute to the pathogenesis of central nervous system damage, namely ATP7B dysfunction, hepatic encephalopathy, and accumulation of iron. ATP7B is expressed in the human brain but its exact function is unknown (Davies et al., 2013). It seems that its mutation does not cause any neurologic symptoms per se and that WD patients develop symptoms only when copper deposits disrupt normal brain function. However, in ATP7B–/– mice neurons display morphologic abnormalities even without copper deposits (Dong et al., 2015). Several factors support the role of hepatic encephalopathy: the neuropathologic abnormalities in WD, namely presence of Alzheimer type I and II glia, resemble those seen in hepatic encephalopathy (Finlayson and Superville, 1981); magnetic resonance imaging (MRI) in some WD patients shows hyperintensities in T1-weighted image typical for manganese deposits seen in portosystemic shunt (Kozic et al., 2003); and fast clinical improvement of neuropsychiatric symptoms has been observed after liver transplantation (Guillaud et al., 2014; Ferenci et al., 2015). Iron accumulation in basal ganglia of WD patients has been documented in postmortem biochemical (Cumings, 1948), neuropathologic (Dusek et al., 2016), and in vivo iron citrate positron emission tomography (Bruehlmeier et al., 2000) studies. Several reports showing hypointensities on T2/T2*w MRI are also suggestive of increased paramagnetic effect of ferritin iron (Engelbrecht et al., 1995; Skowronska et al., 2013); however, the clinical consequences of these findings are unclear (Dusek et al., 2015).

OTHER SYMPTOMS Copper may accumulate in virtually any organ in human body, but the most affected tissues besides liver and brain are kidneys, bones, and blood cells (Fig. 5.2). The effects of copper toxicity are different in cases of rapid increase of its serum level and chronic buildup in tissues. Rapid release of hepatic copper into the blood stream caused by hepatic necrosis in WD may lead to similar consequences as acute copper poisoning, i.e., hemolytic anemia, rhabdomyolysis, and renal tubular damage (Walsh et al., 1977). Hemolysis is a consequence of dramatically increased free copper in the plasma exerting toxic effects on erythrocytes (Deiss et al., 1970; Forman et al., 1980). Copper inhibits sulfhydryl groups of glucose-6-phosphate dehydrogenase and GSH, reducing their free radical scavenging capacity and ultimately leading to oxidation of hemoglobin (Takeda et al., 2000; Gunay et al., 2006; Franchitto et al., 2008). Acute rhabdomyolysis results likely from copper-induced inhibition of Na+/K+-ATPase activity (Benders et al., 1994; Propst et al., 1995). Acute renal failure is presumably caused by a combination of toxic effects of copper on renal parenchyma, hemoglobinuria, myoglobinuria, and hypovolemia (Chugh et al., 1977; Rector et al., 1984; Zhuang et al., 2008). Pathologic changes of bone structure such as osteomalacia and osteoporosis with increased incidence of spontaneous fractures have been ascribed to renal wasting of calcium and phosphorus (Golding and Walshe, 1977). On the other hand, copper accumulation in the synovial membrane and cartilage has been suggested as a cause of osteoarthritis and accelerated degenerative changes affecting particularly larger joints (Menerey et al., 1988; Kramer et al., 1993). Penicillamine treatment is known to induce a lupus-like reaction and may contribute to arthropathy in some cases (Walshe and Golding, 1977). Myocardial copper accumulation can cause cardiomyopathy (Hlubocka et al., 2002) with interstitial and replacement fibrosis, intramyocardial small-vessel sclerosis, and focal inflammatory cell infiltration (Factor et al., 1982). These changes may lead to arrhythmias such as supraventricular tachycardia (Kuan, 1987; Meenakshi-Sundaram et al., 2004). Renal abnormalities, including aminoaciduria and nephrocalcinosis, are presumably a consequence of copper toxicity on tubular epithelium (Vogel, 1960) and they tend to improve with chelation therapy (Leu et al., 1970; Monro, 1970).

ACKNOWLEDGMENTS This work was supported by Charles University in Prague, PRVOUK P26/LF1/4, Czech Ministry of Health, 15-25602A, the European Social Fund, and the State Budget of the Czech Republic (project no. CZ.1.07/ 2.3.00/30.0061).

PATHOGENESIS OF WILSON DISEASE

REFERENCES Anzil AP, Herrlinger H, Blinzinger K et al. (1974). Ultrastructure of brain and nerve biopsy tissue in Wilson disease. Arch Neurol 31: 94–100. Arguello JM, Eren E, Gonzalez-Guerrero M (2007). The structure and function of heavy metal transport P1B-ATPases. Biometals 20: 233–248. Aschner M, Allen JW, Kimelberg HK et al. (1999). Glial cells in neurotoxicity development. Annu Rev Pharmacol Toxicol 39: 151–173. Banci L, Bertini I, Cantini F et al. (2009). An NMR study of the interaction of the N-terminal cytoplasmic tail of the Wilson disease protein with copper(I)-HAH1. J Biol Chem 284: 9354–9360. Bandmann O, Weiss KH, Kaler SG (2015). Wilson’s disease and other neurological copper disorders. Lancet Neurol 14: 103–113. Barthel H, Hermann W, Kluge R et al. (2003). Concordant preand postsynaptic deficits of dopaminergic neurotransmission in neurologic Wilson disease. AJNR Am J Neuroradiol 24: 234–238. Benders AA, Li J, Lock RA et al. (1994). Copper toxicity in cultured human skeletal muscle cells: the involvement of Na +/K(+)-ATPase and the Na +/Ca(2+)-exchanger. Pflugers Arch 428: 461–467. Bertrand E, Lewandowska E, Szpak GM et al. (2001). Neuropathological analysis of pathological forms of astroglia in Wilson’s disease. Folia Neuropathol 39: 73–79. Boulard M, Blume KG, Beutler E (1972). The effect of copper on red cell enzyme activities. J Clin Invest 51: 459–461. Brewer GJ (2005). Neurologically presenting Wilson’s disease: epidemiology, pathophysiology and treatment. CNS Drugs 19: 185–192. Britton RS (1996). Metal-induced hepatotoxicity. Semin Liver Dis 16: 3–12. Bruehlmeier M, Leenders KL, Vontobel P et al. (2000). Increased cerebral iron uptake in Wilson’s disease: a 52Fe-citrate PET study. J Nucl Med 41: 781–787. Bruha R, Vitek L, Marecek Z et al. (2012). Decreased serum antioxidant capacity in patients with Wilson disease is associated with neurological symptoms. J Inherit Metab Dis 35: 541–548. Buiakova OI, Xu J, Lutsenko S et al. (1999). Null mutation of the murine ATP7B (Wilson disease) gene results in intracellular copper accumulation and late-onset hepatic nodular transformation. Hum Mol Genet 8: 1665–1671. Bull PC, Thomas GR, Rommens JM et al. (1993). The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene. Nat Genet 5: 327–337. Burkitt MJ (2001). A critical overview of the chemistry of copper-dependent low density lipoprotein oxidation: roles of lipid hydroperoxides, alpha-tocopherol, thiols, and ceruloplasmin. Arch Biochem Biophys 394: 117–135. Canbay A, Friedman S, Gores GJ (2004). Apoptosis: the nexus of liver injury and fibrosis. Hepatology 39: 273–278. Cater MA, La Fontaine S, Shield K et al. (2006). ATP7B mediates vesicular sequestration of copper: insight into biliary copper excretion. Gastroenterology 130: 493–506.

51

Cater MA, La Fontaine S, Mercer JF (2007). Copper binding to the N-terminal metal-binding sites or the CPC motif is not essential for copper-induced trafficking of the human Wilson protein (ATP7B). Biochem J 401: 143–153. Chugh KS, Sharma BK, Singhal PC et al. (1977). Acute renal failure following copper sulphate intoxication. Postgrad Med J 53: 18–23. Cobine PA, Ojeda LD, Rigby KM et al. (2004). Yeast contains a non-proteinaceous pool of copper in the mitochondrial matrix. J Biol Chem 279: 14447–14455. Crosas-Molist E, Fabregat I (2015). Role of NADPH oxidases in the redox biology of liver fibrosis. Redox Biol 6: 106–111. Culotta VC, Klomp LW, Strain J et al. (1997). The copper chaperone for superoxide dismutase. J Biol Chem 272: 23469–23472. Cumings JN (1948). The copper and iron content of brain and liver in the normal and in hepato-lenticular degeneration. Brain 71: 410–415. Cumings JN (1968). Trace metals in the brain and in Wilson’s disease. J Clin Pathol 21: 1–7. Davies KM, Hare DJ, Cottam V et al. (2013). Localization of copper and copper transporters in the human brain. Metallomics 5: 43–51. Deiss A, Lee GR, Cartwright GE (1970). Hemolytic anemia in Wilson’s disease. Ann Intern Med 73: 413–418. Dong Y, Shi SS, Chen S et al. (2015). The discrepancy between the absence of copper deposition and the presence of neuronal damage in the brain of Atp7b(-/-) mice. Metallomics 7: 283–288. Dusek P, Roos PM, Litwin T et al. (2015). The neurotoxicity of iron, copper and manganese in Parkinson’s and Wilson’s diseases. J Trace Elem Med Biol 31: 193–203. Dusek P, Bahn E, Litwin T et al. (2016). Brain iron accumulation in Wilson disease: a post-mortem 7 Tesla MRI – histopathological study. Neuropathol Appl Neurobiol. http:// dx.doi.org/10.1111/nan.12341. Engelbrecht V, Schlaug G, Hefter H et al. (1995). MRI of the brain in Wilson disease: T2 signal loss under therapy. J Comput Assist Tomogr 19: 635–638. Faa G, Lisci M, Caria MP et al. (2001). Brain copper, iron, magnesium, zinc, calcium, sulfur and phosphorus storage in Wilson’s disease. J Trace Elem Med Biol 15: 155–160. Factor SM, Cho S, Sternlieb I et al. (1982). The cardiomyopathy of Wilson’s disease. Myocardial alterations in nine cases. Virchows Arch A Pathol Anat Histol 397: 301–311. Ferenci P (2004). Pathophysiology and clinical features of Wilson disease. Metab Brain Dis 19: 229–239. Ferenci P (2014). Phenotype–genotype correlations in patients with Wilson’s disease. Ann N Y Acad Sci 1315: 1–5. Ferenci P, Litwin T, Seniow J et al. (2015). Encephalopathy in Wilson disease: copper toxicity or liver failure? J Clin Exp Hepatol 5: S88–S95. Finlayson MH, Superville B (1981). Distribution of cerebral lesions in acquired hepatocerebral degeneration. Brain 104: 79–95. Forman SJ, Kumar KS, Redeker AG et al. (1980). Hemolytic anemia in Wilson disease: clinical findings and biochemical mechanisms. Am J Hematol 9: 269–275.

52

I.F. SCHEIBER ET AL.

Franchitto N, Gandia-Mailly P, Georges B et al. (2008). Acute copper sulphate poisoning: a case report and literature review. Resuscitation 78: 92–96. Friedman SL (2003). Liver fibrosis – from bench to bedside. J Hepatol 38 (Suppl 1): S38–S53. Frota NA, Barbosa ER, Porto CS et al. (2013). Cognitive impairment and magnetic resonance imaging correlations in Wilson’s disease. Acta Neurol Scand 127: 391–398. Frucht S, Sun D, Schiff N et al. (1998). Arm tremor secondary to Wilson’s disease. Mov Disord 13: 351–353. Fujiwara N, Iso H, Kitanaka N et al. (2006). Effects of copper metabolism on neurological functions in Wistar and Wilson’s disease model rats. Biochem Biophys Res Commun 349: 1079–1086. Gao B, Bataller R (2011). Alcoholic liver disease: pathogenesis and new therapeutic targets. Gastroenterology 141: 1572–1585. Gitlin JD (2003). Wilson disease. Gastroenterology 125: 1868–1877. Golding DN, Walshe JM (1977). Arthropathy of Wilson’s disease. Study of clinical and radiological features in 32 patients. Ann Rheum Dis 36: 99–111. Green CL (1955). Histochemical demonstration of copper in a case of hepatolenticular degeneration. Am J Pathol 31: 545–553. Gu M, Cooper JM, Butler P et al. (2000). Oxidativephosphorylation defects in liver of patients with Wilson’s disease. Lancet 356: 469–474. Guillaud O, Dumortier J, Sobesky R et al. (2014). Long term results of liver transplantation for Wilson’s disease: experience in France. J Hepatol 60: 579–589. Gunay N, Yildirim C, Karcioglu O et al. (2006). A series of patients in the emergency department diagnosed with copper poisoning: recognition equals treatment. Tohoku J Exp Med 209: 243–248. Gunther MR, Hanna PM, Mason RP et al. (1995). Hydroxyl radical formation from cuprous ion and hydrogen peroxide: a spin-trapping study. Arch Biochem Biophys 316: 515–522. Halliwell B (2006). Oxidative stress and neurodegeneration: where are we now? J Neurochem 97: 1634–1658. Halliwell B, Gutteridge JM (1990). Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol 186: 1–85. Hegde ML, Hegde PM, Holthauzen LMF et al. (2010). Specifc inhibition of NEIL-initiated repair of oxidized base damage in human genome by copper and iron: potential etiological linkage to neurodegenerative diseases. J Biol Chem 285: 28812–28825. Hellman NE, Gitlin JD (2002). Ceruloplasmin metabolism and function. Annu Rev Nutr 22: 439–458. Helmich RC, Toni I, Deuschl G et al. (2013). The pathophysiology of essential tremor and Parkinson’s tremor. Curr Neurol Neurosci Rep 13: 378. Hitoshi S, Iwata M, Yoshikawa K (1991). Mid-brain pathology of Wilson’s disease: MRI analysis of three cases. J Neurol Neurosurg Psychiatry 54: 624–626.

Hlubocka Z, Marecek Z, Linhart A et al. (2002). Cardiac involvement in Wilson disease. J Inherit Metab Dis 25: 269–277. Horoupian DS, Sternlieb I, Scheinberg IH (1988). Neuropathological findings in penicillamine-treated patients with Wilson’s disease. Clin Neuropathol 7: 62–67. Huang CC, Chu NS (1995). Psychosis and epileptic seizures in Wilson’s disease with predominantly white matter lesions in the frontal lobe. Parkinsonism Relat Disord 1: 53–58. Huster D (2010). Wilson disease. Best Pract Res Clin Gastroenterol 24: 531–539. Huster D (2014). Structural and metabolic changes in Atp7b-/mouse liver and potential for new interventions in Wilson’s disease. Ann N Y Acad Sci 1315: 37–44. Huster D, Finegold MJ, Morgan CT et al. (2006). Consequences of copper accumulation in the livers of the Atp7b-/- (Wilson disease gene) knockout mice. Am J Pathol 168: 423–434. Huster D, Purnat TD, Burkhead JL et al. (2007). High copper selectively alters lipid metabolism and cell cycle machinery in the mouse model of Wilson disease. J Biol Chem 282: 8343–8355. Ingster-Moati I, Bui Quoc E, Pless M et al. (2007). Ocular motility and Wilson’s disease: a study on 34 patients. J Neurol Neurosurg Psychiatry 78: 1199–1201. Iwanski S, Seniow J, Lesniak M et al. (2015). Diverse attention deficits in patients with neurologically symptomatic and asymptomatic Wilson’s disease. Neuropsychology 29: 25–30. Jeon B, Kim JM, Jeong JM et al. (1998). Dopamine transporter imaging with [123I]-beta-CIT demonstrates presynaptic nigrostriatal dopaminergic damage in Wilson’s disease. J Neurol Neurosurg Psychiatry 65: 60–64. Kim H, Wu X, Lee J (2013a). SLC31 (CTR) family of copper transporters in health and disease. Mol Aspects Med 34: 561–570. Kim YE, Yun JY, Yang HJ et al. (2013b). Unusual epileptic deterioration and extensive white matter lesion during treatment in Wilson’s disease. BMC Neurol 13: 127. Kodama H, Okabe I, Yanagisawa M et al. (1988). Does CSF copper level in Wilson disease reflect copper accumulation in the brain? Pediatr Neurol 4: 35–37. Kozic D, Svetel M, Petrovic B et al. (2003). MR imaging of the brain in patients with hepatic form of Wilson’s disease. Eur J Neurol 10: 587–592. Kramer U, Weinberger A, Yarom R et al. (1993). Synovial copper deposition as a possible explanation of arthropathy in Wilson’s disease. Bull Hosp Jt Dis 52: 46–49. Krauss JK, Wakhloo AK, Nobbe F et al. (1995). Lesion of dentatothalamic pathways in severe post-traumatic tremor. Neurol Res 17: 409–416. Krebs N, Langkammer C, Goessler W et al. (2014). Assessment of trace elements in human brain using inductively coupled plasma mass spectrometry. J Trace Elem Med Biol 28: 1–7. Kuan P (1987). Cardiac Wilson’s disease. Chest 91: 579–583.

PATHOGENESIS OF WILSON DISEASE La Fontaine S, Mercer JF (2007). Trafficking of the copper-ATPases, ATP7A and ATP7B: role in copper homeostasis. Arch Biochem Biophys 463: 149–167. Lang PA, Schenck M, Nicolay JP et al. (2007). Liver cell death and anemia in Wilson disease involve acid sphingomyelinase and ceramide. Nat Med 13: 164–170. Leary SC, Winge DR, Cobine PA (2009). “Pulling the plug” on cellular copper: the role of mitochondria in copper export. Biochim Biophys Acta 1793: 146–153. Lee J, Pena MM, Nose Y et al. (2002a). Biochemical characterization of the human copper transporter Ctr1. J Biol Chem 277: 4380–4387. Lee J, Petris MJ, Thiele DJ (2002b). Characterization of mouse embryonic cells deficient in the ctr1 high affinity copper transporter. Identification of a Ctr1-independent copper transport system. J Biol Chem 277: 40253–40259. Lee YA, Wallace MC, Friedman SL (2015). Pathobiology of liver fibrosis: a translational success story. Gut 64: 830–841. Lesniak M, Członkowska A, Seniow J (2008). Abnormal antisaccades and smooth pursuit eye movements in patients with Wilson’s disease. Mov Disord 23: 2067–2073. Letelier ME, Lepe AM, Faundez M et al. (2005). Possible mechanisms underlying copper-induced damage in biological membranes leading to cellular toxicity. Chem Biol Interact 151: 71–82. Letelier ME, Martinez M, Gonzalez-Lira V et al. (2006). Inhibition of cytosolic glutathione S-transferase activity from rat liver by copper. Chem Biol Interact 164: 39–48. Letelier ME, Lagos F, Faundez M et al. (2007). Copper modifies liver microsomal UDP-glucuronyltransferase activity through different and opposite mechanisms. Chem Biol Interact 167: 1–11. Leu ML, Strickland GT, Gutman RA (1970). Renal function in Wilson’s disease: response to penicillamine therapy. Am J Med Sci 260: 381–398. Lewinska-Preis L, Jablonska M, Fabianska MJ et al. (2011). Bioelements and mineral matter in human livers from the highly industrialized region of the Upper Silesia Coal Basin (Poland). Environ Geochem Health 33: 595–611. Litwin T, Gromadzka G, Szpak GM et al. (2013). Brain metal accumulation in Wilson’s disease. J Neurol Sci 329: 55–58. Lorincz MT (2010). Neurologic Wilson’s disease. Ann N Y Acad Sci 1184: 173–187. Luedde T, Kaplowitz N, Schwabe RF (2014). Cell death and cell death responses in liver disease: mechanisms and clinical relevance. Gastroenterology 147: 765–783. e764. Lutsenko S, Barnes NL, Bartee MY et al. (2007a). Function and regulation of human copper-transporting ATPases. Physiol Rev 87: 1011–1046. Lutsenko S, LeShane ES, Shinde U (2007b). Biochemical basis of regulation of human copper-transporting ATPases. Arch Biochem Biophys 463: 134–148. Manto M (2014). Abnormal copper homeostasis: mechanisms and roles in neurodegeneration. Toxics 2: 327–345. Matsuura T, Sasaki H, Tashiro K (1998). Atypical MR findings in Wilson’s disease: pronounced lesions in the dentate nucleus causing tremor. J Neurol Neurosurg Psychiatry 64: 161.

53

Maxfield AB, Heaton DN, Winge DR (2004). Cox17 is functional when tethered to the mitochondrial inner membrane. J Biol Chem 279: 5072–5080. Mederacke I, Hsu CC, Troeger JS et al. (2013). Fate tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis independent of its aetiology. Nat Commun 4: 2823. Medici V, Shibata NM, Kharbanda KK et al. (2014). Maternal choline modifies fetal liver copper, gene expression, DNA methylation, and neonatal growth in the tx-j mouse model of Wilson disease. Epigenetics 9: 286–296. Meenakshi-Sundaram S, Sinha S, Rao M et al. (2004). Cardiac involvement in Wilson’s disease – an electrocardiographic observation. J Assoc Physicians India 52: 294–296. Meenakshi-Sundaram S, Mahadevan A, Taly AB et al. (2008). Wilson’s disease: a clinico-neuropathological autopsy study. J Clin Neurosci 15: 409–417. Menerey KA, Eider W, Brewer GJ et al. (1988). The arthropathy of Wilson’s disease: clinical and pathologic features. J Rheumatol 15: 331–337. Mikol J, Vital C, Wassef M et al. (2005). Extensive corticosubcortical lesions in Wilson’s disease: clinico-pathological study of two cases. Acta Neuropathol 110: 451–458. Milanov I (2002). Clinical and electromyographic examinations of patients with midbrain and cerebellar tremor. Electromyogr Clin Neurophysiol 42: 105–112. Min HK, Kapoor A, Fuchs M et al. (2012). Increased hepatic synthesis and dysregulation of cholesterol metabolism is associated with the severity of nonalcoholic fatty liver disease. Cell Metab 15: 665–674. Monro P (1970). Effect of treatment on renal function in severe osteomalacia due to Wilson’s disease. J Clin Pathol 23: 487–491. Monty JF, Llanos RM, Mercer JF et al. (2005). Copper exposure induces trafficking of the menkes protein in intestinal epithelium of ATP7A transgenic mice. J Nutr 135: 2762–2766. Mufti AR, Burstein E, Csomos RA et al. (2006). XIAP is a copper binding protein deregulated in Wilson’s disease and other copper toxicosis disorders. Mol Cell 21: 775–785. Nevitt T, Ohrvik H, Thiele DJ (2012). Charting the travels of copper in eukaryotes from yeast to mammals. Biochim Biophys Acta 1823: 1580–1593. Nyasae L, Bustos R, Braiterman L et al. (2007). Dynamics of endogenous ATP7A (Menkes protein) in intestinal epithelial cells: copper-dependent redistribution between two intracellular sites. Am J Physiol Gastrointest Liver Physiol 292: G1181–G1194. Nyberg P, Gottfries CG, Holmgren G et al. (1982). Advanced catecholaminergic disturbances in the brain in a case of Wilson’s disease. Acta Neurol Scand 65: 71–75. Oder W, Prayer L, Grimm G et al. (1993). Wilson’s disease: evidence of subgroups derived from clinical findings and brain lesions. Neurology 43: 120–124. Oder W, Brucke T, Kollegger H et al. (1996). Dopamine D2 receptor binding is reduced in Wilson’s disease: correlation of neurological deficits with striatal 123I-iodobenzamide binding. J Neural Transm 103: 1093–1103.

54

I.F. SCHEIBER ET AL.

Oertel WH, Tatsch K, Schwarz J et al. (1992). Decrease of D2 receptors indicated by 123I-iodobenzamide single-photon emission computed tomography relates to neurological deficit in treated Wilson’s disease. Ann Neurol 32: 743–748. Ohrvik H, Thiele DJ (2014). How copper traverses cellular membranes through the mammalian copper transporter 1, Ctr1. Ann N Y Acad Sci 1314: 32–41. Ozcelik D, Uzun H (2009). Copper intoxication; antioxidant defenses and oxidative damage in rat brain. Biol Trace Elem Res 127: 45–52. Pal A, Prasad R (2014). Recent discoveries on the functions of astrocytes in the copper homeostasis of the brain: a brief update. Neurotox Res 26: 78–84. Pal PK, Sinha S, Pillai S et al. (2007). Successful treatment of tremor in Wilson’s disease by thalamotomy: a case report. Mov Disord 22: 2287–2290. Pal A, Badyal RK, Vasishta RK et al. (2013). Biochemical, histological, and memory impairment effects of chronic copper toxicity: a model for non-Wilsonian brain copper toxicosis in Wistar rat. Biol Trace Elem Res 153: 257–268. Palmgren MG, Nissen P (2011). P-type ATPases. Annu Rev Biophys 40: 243–266. Pamp K, Bramey T, Kirsch M et al. (2005). NAD(H) enhances the Cu(II)-mediated inactivation of lactate dehydrogenase by increasing the accessibility of sulfhydryl groups. Free Radic Res 39: 31–40. Parpura V, Heneka MT, Montana V et al. (2012). Glial cells in (patho)physiology. J Neurochem 121: 4–27. Pfeiffenberger J, Gotthardt DN, Herrmann T et al. (2012). Iron metabolism and the role of HFE gene polymorphisms in Wilson disease. Liver Int 32: 165–170. Pfeiffer RF (2007). Wilson’s disease. Semin Neurol 27: 123–132. Prashanth LK, Sinha S, Taly AB et al. (2010). Spectrum of epilepsy in Wilson’s disease with electroencephalographic, MR imaging and pathological correlates. J Neurol Sci 291: 44–51. Propst A, Propst T, Feichtinger H et al. (1995). Copper-induced acute rhabdomyolysis in Wilson’s disease. Gastroenterology 108: 885–887. Przybylkowski A, Gromadzka G, Chabik G et al. (2014). Liver cirrhosis in patients newly diagnosed with neurological phenotype of Wilson’s disease. Funct Neurol 29: 23–29. Puche JE, Saiman Y, Friedman SL (2013). Hepatic stellate cells and liver fibrosis. Compr Physiol 3: 1473–1492. Rae TD, Schmidt PJ, Pufahl RA et al. (1999). Undetectable intracellular free copper: the requirement of a copper chaperone for superoxide dismutase. Science 284: 805–808. Rector Jr WG, Uchida T, Kanel GC et al. (1984). Fulminant hepatic and renal failure complicating Wilson’s disease. Liver 4: 341–347. Reddy PV, Rao KV, Norenberg MD (2008). The mitochondrial permeability transition, and oxidative and nitrosative stress in the mechanism of copper toxicity in cultured neurons and astrocytes. Lab Invest 88: 816–830. Richter R (1948). The pallial component in hepato-lenticular degeneration. J Neuropathol Exp Neurol 7: 1–18.

Robinson NJ, Winge DR (2010). Copper metallochaperones. Annu Rev Biochem 79: 537–562. Roeser HP, Lee GR, Nacht S et al. (1970). The role of ceruloplasmin in iron metabolism. J Clin Invest 49: 2408–2417. Saito T, Nagao T, Okabe M et al. (1996). Neurochemical and histochemical evidence for an abnormal catecholamine metabolism in the cerebral cortex of the Long–Evans Cinnamon rat before excessive copper accumulation in the brain. Neurosci Lett 216: 195–198. Scheiber IF, Dringen R (2011). Copper-treatment increases the cellular GSH content and accelerates GSH export from cultured rat astrocytes. Neurosci Lett 498: 42–46. Scheiber IF, Schmidt MM, Dringen R (2010). Zinc prevents the copper-induced damage of cultured astrocytes. Neurochem Int 57: 314–322. Scheiber I, Dringen R, Mercer JF (2013). Copper: effects of deficiency and overload. Met Ions Life Sci 13: 359–387. Scheiber IF, Mercer JFB, Dringen R (2014). Metabolism and functions of copper in brain. Prog Neurobiol 116: 33–57. Scheinberg I, Sternlieb I (1984). Wilson’s disease, W. B. Saunders, Philadelphia. Scheinberg IH, Sternlieb I (1996). Wilson disease and idiopathic copper toxicosis. Am J Clin Nutr 63: 842S–845S. Schulman S, Barbeau A (1963). Wilson’s disease: a case with almost total loss of white matter. J Neuropathol Exp Neurol 22: 105–119. Schwerdtle T, Hamann I, Jahnke G et al. (2007). Impact of copper on the induction and repair of oxidative DNA damage, poly(ADP-ribosyl)ation and PARP-1 activity. Mol Nutr Food Res 51: 201–210. Skowronska M, Litwin T, Dziezyc K et al. (2013). Does brain degeneration in Wilson disease involve not only copper but also iron accumulation? Neurol Neurochir Pol 47: 542–546. Sokol RJ, Twedt D, McKim Jr JM et al. (1994). Oxidant injury to hepatic mitochondria in patients with Wilson’s disease and Bedlington terriers with copper toxicosis. Gastroenterology 107: 1788–1798. Starosta-Rubinstein S, Young AB, Kluin K et al. (1987). Clinical assessment of 31 patients with Wilson’s disease. Correlations with structural changes on magnetic resonance imaging. Arch Neurol 44: 365–370. Stattermayer AF, Traussnigg S, Dienes HP et al. (2015). Hepatic steatosis in Wilson disease – role of copper and PNPLA3 mutations. J Hepatol 63: 156–163. Stuerenburg HJ (2000). CSF copper concentrations, blood– brain barrier function, and coeruloplasmin synthesis during the treatment of Wilson’s disease. J Neural Transm 107: 321–329. Sudmeyer M, Pollok B, Hefter H et al. (2006). Synchronized brain network underlying postural tremor in Wilson’s disease. Mov Disord 21: 1935–1940. Svetel M, Kozic D, Stefanova E et al. (2001). Dystonia in Wilson’s disease. Mov Disord 16: 719–723. Szerdahelyi P, Kasa P (1986). Histochemical demonstration of copper in normal rat brain and spinal cord. Evidence of localization in glial cells. Histochemistry 85: 341–347.

PATHOGENESIS OF WILSON DISEASE Takeda T, Yukioka T, Shimazaki S (2000). Cupric sulfate intoxication with rhabdomyolysis, treated with chelating agents and blood purification. Intern Med 39: 253–255. Tanzi RE, Petrukhin K, Chernov I et al. (1993). The Wilson disease gene is a copper transporting ATPase with homology to the Menkes disease gene. Nat Genet 5: 344–350. Theophilos MB, Cox DW, Mercer JF (1996). The toxic milk mouse is a murine model of Wilson disease. Hum Mol Genet 5: 1619–1624. Tiffany-Castiglioni E, Hong S, Qian Y (2011). Copper handling by astrocytes: insights into neurodegenerative diseases. Int J Dev Neurosci 29: 811–818. Tribl GG, Bor-Seng-Shu E, Trindade MC et al. (2014). Wilson’s disease presenting as rapid eye movement sleep behavior disorder: a possible window to early treatment. Arq Neuropsiquiatr 72: 653–658. Uriu-Adams JY, Keen CL (2005). Copper, oxidative stress, and human health. Mol Aspects Med 26: 268–298. Vasic V, Jovanovic D, Krstic D et al. (1999). Prevention and recovery of CuSO4-induced inhibition of Na+/K+-ATPase and Mg2+-ATPase in rat brain synaptosomes by EDTA. Toxicol Lett 110: 95–104. Vest KE, Leary SC, Winge DR et al. (2013). Copper import into the mitochondrial matrix in Saccharomyces cerevisiae is mediated by Pic2, a mitochondrial carrier family protein. J Biol Chem 288: 23884–23892. Vogel FS (1960). Nephrotoxic properties of copper under experimental conditions in mice: with special reference to the pathogenesis of the renal alterations in Wilson’s disease. Am J Pathol 36: 699–711. Vogel FS, Evans JW (1961). Morphologic alterations produced by copper in neural tissues with consideration of the role of the metal in the pathogenesis of Wilson’s disease. J Exp Med 113: 997–1004. Wallace MC, Friedman SL, Mann DA (2015). Emerging and disease-specific mechanisms of hepatic stellate cell activation. Semin Liver Dis 35: 107–118. Walsh FM, Crosson FJ, Bayley M et al. (1977). Acute copper intoxication. Pathophysiology and therapy with a case report. Am J Dis Child 131: 149–151. Walshe JM, Gibbs KR (1987). Brain copper in Wilson’s disease. Lancet 2: 1030.

55

Walshe JM, Golding DN (1977). Penicillamine-induced arthropathy in Wilson’s disease. Proc R Soc Med 70 (Suppl 3): 4–6. Walshe JM, Potter G (1977). The pattern of the whole body distribution of radioactive copper (67Cu, 64Cu) in Wilson’s disease and various control groups. Q J Med 46: 445–462. Wang Y, Li J, Wang X et al. (2013). Hepatic stellate cells, liver innate immunity, and hepatitis C virus. J Gastroenterol Hepatol 28 (Suppl 1): 112–115. Weisner B, Hartard C, Dieu C (1987). CSF copper concentration: a new parameter for diagnosis and monitoring therapy of Wilson’s disease with cerebral manifestation. J Neurol Sci 79: 229–237. Wu J, Forbes JR, Chen HS et al. (1994). The LEC rat has a deletion in the copper transporting ATPase gene homologous to the Wilson disease gene. Nat Genet 7: 541–545. Wu F, Wang J, Pu C et al. (2015). Wilson’s disease: a comprehensive review of the molecular mechanisms. Int J Mol Sci 16: 6419–6431. Yamaguchi Y, Heiny ME, Gitlin JD (1993). Isolation and characterization of a human liver cDNA as a candidate gene for Wilson disease. Biochem Biophys Res Commun 197: 271–277. Yang X, Tang XP, Zhang YH et al. (2015). Prospective evaluation of the diagnostic accuracy of hepatic copper content, as determined using the entire core of a liver biopsy sample. Hepatology 62: 1732–1741. Yurkova IL, Arnhold J, Fitzl G et al. (2011). Fragmentation of mitochondrial cardiolipin by copper ions in the Atp7b-/mouse model of Wilson’s disease. Chem Phys Lipids 164: 393–400. Zecca L, Stroppolo A, Gatti A et al. (2004). The role of iron and copper molecules in the neuronal vulnerability of locus coeruleus and substantia nigra during aging. Proc Natl Acad Sci U S A 101: 9843–9848. Zhuang XH, Mo Y, Jiang XY et al. (2008). Analysis of renal impairment in children with Wilson’s disease. World J Pediatr 4: 102–105. Zimbrean PC, Schilsky ML (2014). Psychiatric aspects of Wilson disease: a review. Gen Hosp Psychiatry 36: 53–62. Zischka H, Lichtmannegger J (2014). Pathological mitochondrial copper overload in livers of Wilson’s disease patients and related animal models. Ann N Y Acad Sci 1315: 6–15.

Handbook of Clinical Neurology, Vol. 142 (3rd series) Wilson Disease A. Członkowska and M.L. Schilsky, Editors http://dx.doi.org/10.1016/B978-0-444-63625-6.00006-9 © 2017 Elsevier B.V. All rights reserved

Chapter 6

Animal models of Wilson disease 1

VALENTINA MEDICI1 AND DOMINIK HUSTER2* Division of Gastroenterology and Hepatology, Department of Internal Medicine, University of California Davis, Sacramento, CA, USA 2

Department of Gastroenterology and Oncology, Deaconess Hospital Leipzig, Leipzig, Germany

Abstract Wilson disease (WD) is caused by ATPase copper-transporting beta (ATP7B) mutations and results in copper toxicity in liver and brain. Although the defective gene was identified in 1993, the specific mechanisms underlying copper toxicity and the remarkable phenotypic diversity of the disease are still poorly understood. Animal models harboring defects in the ATP7B homolog have helped to reveal new insights into pathomechanisms of WD. Four rodent models with ATP7B gene defects have been described – the Long–Evans Cinnamon (LEC) rat, inbred mouse models (toxic milk (tx), the Jackson Laboratory toxic milk (tx-j)), and the genetically engineered ATP7B–/– (knockout) mouse – all of which develop liver disease to different extents. Copper accumulation in parts of the brain accompanied by some neurologic involvement was revealed in LEC rats and tx/tx-j mice, but the pathology is less severe than human neurologic WD. Several dogs show hepatic copper toxicity resembling WD; however, brain involvement has not been observed and the underlying genetic defect is different. These models are of great value for examination of copper distribution and metabolism, gene expression, and investigation of liver and brain pathology. The availability of disease models is essential for therapeutic interventions such as drug, gene, and cell therapy. Findings made by animal studies may facilitate the development of specific therapies to ameliorate WD progression.

INTRODUCTION1 Since the discovery of ATPase copper-transporting beta (ATP7B), our knowledge of the genetic basis and pathophysiology of Wilson disease (WD) has increased dramatically. However, deep insight into the biology and molecular physiology of the disease has been hampered due to certain limitations in human research, including the low prevalence of the disease and the difficult access to liver and brain samples from humans. Animal models have helped considerably to increase our knowledge of the underlying pathophysiology of WD.

In contrast to what is observed in some rodent models, copper deficiency at birth is not clinically relevant in human WD. Copper accumulates during childhood and, after an asymptomatic period, WD manifests itself as liver (usually between the end of the first and the end of the second decade of life) and/or neurologic disease (early adulthood) (Huster, 2010). Rodent models have been shown to provide valuable insights into presymptomatic molecular events and mechanisms of disease development. While early/past therapies of WD were experimental and involved a substantial risk for the treated patient, the current availability of animal models provides adequate and safe conditions for further

1

Abbreviations used in the chapter are listed at the end of the chapter before References section.

*Correspondence to: Dominik Huster, MD, Department of Gastroenterology and Oncology, Deaconess Hospital Leipzig, Georg-Schwarz-Str. 49, 04177 Leipzig, Germany. Tel: +49-341-444-3622, E-mail: [email protected]

V. MEDICI AND D. HUSTER

58

therapy developments. Indeed, several animal models have already contributed to the development of innovative treatments for WD (Howell, 1999). The purpose of this review is to summarize the discovery, development, current knowledge, and new insights of important animal (mainly rodent) models of copper accumulation and its consequences. Furthermore, a short overview about current and possible future therapeutic approaches will be provided.

COPPER TOXICITY AND LIVER DISEASE IN RODENT MODELS OF WILSON DISEASE Long–Evans Cinnamon rat DISCOVERY, GENE DEFECT, AND NATURAL COURSE OF DISEASE

Long–Evans Cinnamon (LEC) rats are an inbred strain of mutant rat originally isolated from a colony of Long– Evans rats (Sasaki et al., 1985). There is more than 80% identity in the amino acid sequence between the human and rat genes. The rat gene for ATP7B was cloned in 1994 (Wu et al., 1994) and it is characterized by a partial deletion that removes at least 900 bp of the coding region at the 30 end and at least 400 bp of the downstream untranslated region. The natural disease course in LEC rats is characterized by the development of a severe acute hepatitis at about 4 months of age with elevated liver enzymes and bilirubin, and submassive or massive hepatocyte necrosis with at least 30–40% animal mortality. Untreated rats present elevation in their liver enzymes with aspartate transaminase (AST) and alanine transaminase (ALT) greater than 500 U/L and bilirubin greater than 30 mg/dL. The rats surviving the hepatitis develop fibrosis with hepatocellular and cholangiocarcinoma by 1 year of age (Okuda, 1992; Jong-Hon et al., 1993). LEC rats present a spontaneous, abnormal accumulation of copper in the liver with associated low serum copper and low ceruloplasmin. Hepatic copper concentration reaches more than 1000 mg/g dry weight by the age of 8 weeks (Medici et al., 2005b). Another important feature is the concomitant hepatic iron accumulation which contributes significantly to the development and progression of liver pathology (Kato et al., 1993).

LIVER PATHOLOGY, ULTRASTRUCTURE, AND METABOLISM

At 6 weeks of age, liver histology shows changes in nuclear size, hepatocyte ballooning, and increased mitotic figures. Apoptosis, increased number of Kupffer cells and polymorphonucleate leukocytes, and giant cells are described in the early phases of the disease prior to

the appearance of jaundice. With severe jaundice and elevated bilirubin, liver pathology is also characterized by cholestasis, hepatocyte necrosis, and erythrophagocytosis (Klein et al., 2003). Microarray analysis demonstrated the progression of liver disease and hepatic copper accumulation in LEC rats is associated with upregulation of gene transcript levels related to cytochrome P-450, oxidative stress, DNA damage, and apoptosis (Klein et al., 2003). Cholangiofibrosis is associated with advanced liver disease and correlates with advanced animal age (Schilsky et al., 1998). Progression of liver damage has been related predominantly to mechanisms of oxidative stress with increased superoxide dismutase (SOD) activity, a reduced glutathione ratio (GSH/GSSG), and markedly elevated levels of hepatic thiobarbituric acid reactive substances (Samuele et al., 2005). Hepatic mitochondrial changes are a typical feature of liver pathology in LEC rats that makes them similar to patients with WD. Sternlieb et al. (1995) described electron microscope images in LEC rats characterized by marked mitochondrial pleomorphism and changes in matrix density, and abnormal cristae morphology with elongation, dilatation, stacking, or disappearance as well as matrix inclusions with electron-dense deposits. Mitochondrial changes in LEC rats mirror changes in patients with WD (Sternlieb, 1968), appear early in the disease development, and are among the earliest pathologic changes observed with copper accumulation. Proteomic analysis confirmed increased expression levels of mitochondrial matrix proteins (Lee et al., 2011). Mitochondria are primary targets of copper toxicity, which leads to cross-linking and disintegration of mitochondrial membranes, ultimately triggering hepatocyte death (Zischka et al., 2014). Changes in liver pathology and hepatic copper accumulation have been associated with changes in lipid metabolism. In particular, Levy et al. (2007) showed that 12-week-old LEC rats displayed increased hepatic triglycerides, free cholesterol, and cholesteryl ester. This increased hepatic lipid content was probably attributable to reduced fat excretion from the liver given the changes in microsomal triglyceride transfer protein, hypotriglyceridemia, and hypocholesterolemia. LEC rats have also been extensively studied as a model of hepatocellular carcinoma. The hepatocarcinogenic process in LEC rats is associated with reduced apoptosis, accumulation of DNA strand breaks, and high cellular proliferation (Jia et al., 2002). The transition from chronic hepatitis to hepatocellular carcinoma is characterized by increased levels and kinase activities of proteins related to cell cycle and proliferation, including cyclin D1, cyclin E, cyclin-dependent kinase 4, cyclin A, and Wee1-like protein kinase (Masaki et al., 2000a). Src-related protein tyrosine kinase, related to cell

ANIMAL MODELS OF WILSON DISEASE transformation, was found to be increased in hepatocytes from LEC rats and correlated with the development of liver cancer (Masaki et al., 2000b). Transcript levels of DNA methyltransferases, enzymes typically associated with liver cancer, were upregulated in liver tumor tissue from LEC rats (Miyoshi et al., 1993).

NEUROLOGIC CHANGES AND BRAIN PATHOLOGY The brain pathology and neurologic presentation in LEC rats have been less studied than the liver pathology. Copper accumulates in all brain regions of LEC rats, reaching a peak at about 24 weeks of age and significantly increased compared to control rats starting at 20 weeks of age. DNA single-strand breaks in the brain appeared at 24 weeks as well (Hayashi et al., 2006). Microarray analysis demonstrated changes in gene transcript levels related to neuronal development, oxidative stress, apoptosis, and inflammation. In particular, there was evidence of changes in the expression of succinate dehydrogenase complex assembly factor 2 (Sdhaf2) and NADH: ubiquinone oxidoreductase subunit B7 (Ndufb7), both central in the mitochondrial oxidative phosphorylation pathway (Lee et al., 2013). Substantia nigra and striatum showed significant increase in manganese-SOD in LEC rats compared to control rats, reflecting increased oxidative stress (Kim et al., 2005). Changes in monoaminergic neuron tyrosinase, as indicated by hydroxylase and 5-hydroxytryptamine immunoreactive fiber densities from 4- to 20-week old rats, were observed (Kawano et al., 2001). When behavioral tests were performed, LEC rats presented higher locomotor activity, decreased habituation to startle response, and lower prepulse inhibition compared to control rats (Fujiwara et al., 2006).

THERAPEUTIC INTERVENTIONS The LEC rat model has been extensively studied to explore therapeutic options in WD. Togashi et al. (1992) demonstrated the efficacy of 12-week oral penicillamine treatment in preventing the development of hepatitis in LEC rats as indicated by improvement of liver enzymes and hepatic histology. Copper chelation with trientine prevented the development of hepatitis, significantly improved liver histology, and, in the long term, reduced the development of hepatocellular carcinoma and cholangiofibrosis (Sone et al., 1996). Tetrathiomolybdate administered to LEC rats early after the onset of acute hepatitis improved liver damage by decreasing hepatic copper accumulation (Klein et al., 2004). Oral zinc administration prevented the development of hepatitis and improved survival in LEC rats compared to untreated rats (Medici et al., 2005b) through an underlying mechanism probably related to increased

59

hepatic transcript levels of metallothionein and reduced oxidative stress (Medici et al., 2002). Iron metabolism manipulation has also shown significant improvement of liver damage in LEC rats. In particular, Kato et al. (1996) demonstrated that by providing an iron-deficient diet, severe hepatitis and hepatocellular carcinoma were prevented, underscoring the importance of the interaction between copper and iron in the progression of liver damage in LEC rats. More recently, studies conducted on LEC rats elucidated the possibility of implementing new approaches to the treatment of WD based on hepatocyte and stem cell transplantation as well as gene therapy. Chen et al. showed that infusing LEC rat livers through the portal vein with ATP7B-transduced bone marrow mesenchymal stem cells was associated with increased expression of ATP7B, increased levels of ceruloplasmin, lower hepatic copper concentration, and lower AST and ALT levels. Optimal liver engraftment and repopulation were achieved by preconditioning with radiation and performing ischemia-reperfusion before the portal vein infusion (Chen et al., 2014). Gene therapy was attempted in LEC rats by lentiviral ATP7B gene transfer. The hepatic transgene expression was detected at 24 weeks of age and was associated with reduced hepatic copper levels and improved liver fibrosis (Merle et al., 2006). Cell therapy with hepatocytes from rats with normal copper metabolism was also successfully attempted in LEC rats. In particular, normal hepatocytes were transplanted intrasplenically into the liver and rats were treated with bile salts before transplantation to improve the efficacy of the transplant. Cell therapy was associated with reduced hepatic copper concentration and improved liver histology (Joseph et al., 2009). In summary, the LEC rat represents a model of rapidly developing hepatic copper accumulation with high mortality at 4 months of age due to severe hepatitis and also development of hepatocellular carcinoma later in life. LEC rats have been particularly appropriate for intervention studies due to the rapid progression of severe liver disease. The development of hepatocellular carcinoma is a feature that does not mirror WD in humans where liver cancer occurs rarely. Regardless, LEC rats are an excellent model to study the pathogenesis of liver cancer in general. The utility of LEC rats to study the neurologic manifestation of WD needs to be further assessed by functional studies.

Toxic milk mouse DISCOVERY, GENE DEFECT, AND NATURAL COURSE OF DISEASE

The first mouse model bearing a genetic defect similar to that of human WD was first observed by Rauch in 1983.

60

V. MEDICI AND D. HUSTER

In contrast to human disease, where copper deficiency is not a clinical issue in newborn individuals, the offspring of mutant female mice are characterized by poor growth, hypopigmentation, tremors, and death within weeks of birth due to a gestational copper deficit in the liver, which is exacerbated and accelerated postpartum because of the greatly reduced copper content of the dam’s breast milk. However, litters could be rescued by foster nursing on normal dams or by direct supplementation of copper. Because of this observation, the model is called toxic milk (tx) mouse. The underlying defect responsible for lack of copper in the milk was found to be a mislocalization of mutant ATP7B in the mammary gland of the tx mouse, leading to decreased secretion of copper with the milk (Michalczyk et al., 2000). Moreover, the impaired delivery of copper into breast milk was not ameliorated by dietary copper supplementation. In contrast to this and other mouse models with ATP7B gene defects, copper concentration in human breast milk is not affected in mothers with WD (Dorea, 2000), and copper deficiency is not a clinical issue in newborns. In 1995, 2 years after the discovery of the human ATP7B gene, Rauch and Wells reported the linkage of the tx mutation to mouse chromosome 8 and the mouse homolog of human ATP7B (Rauch and Wells, 1995). This assumption was confirmed by findings of Theophilos et al. (1996), who isolated cDNA clones encoding the murine homolog of the WD gene ATP7B, identified the mutation (methionine1356valine in the eighth transmembrane domain) causing the disease, and established the tx mouse as a valid model for human WD, despite there being some dissimilarities. Further basic research using in vitro model systems for ATP7B localization and copper transport provided evidence that the Met1386Val mutation found in the tx mouse caused mislocalization as well as loss of copper transport activity (La Fontaine et al., 2001; Voskoboinik et al., 2001). Comparable results have been shown for a number of human ATB7B variants, both in vitro and in vivo (Huster et al., 2003, 2012), which revealed significant similarities between the human disease and the animal model, and underline the importance of its availability.

LIVER PATHOLOGY, ULTRASTRUCTURE, AND METABOLISM

Early in life, tx mice accumulate large amounts of copper in their liver followed by liver damage. By 6 months of age, the livers show nodular fibrosis, bile duct hyperplasia, and inflammatory cell infiltration. Further investigation of disease development in these mice revealed numerous parallels in gross morphology, and histologic and ultrastructural abnormalities in livers between tx

mice and human hepatic WD (Biempica et al., 1988). Importantly, beside tremors in newborn mice, which could be attributed to copper deficiency, no obvious neurologic symptoms were observed in tx mice as seen in human WD. An important observation frequently made in human WD and an indicator of copper status is impaired copper incorporation and decreased level of ceruloplasmin (Gitlin, 2003). Rauch already reported in the first tx mouse publication the reduced ceruloplasmin activity (Rauch, 1983). Mercer et al. (1991) confirmed Rauch’s results of reduced ceruloplasmin concentrations in tx mouse serum and found that mRNA levels of ceruloplasmin were unaffected or even higher in pregnant female tx mice. Therefore, they concluded the lower activity was a result of failure of copper incorporation into apoceruloplasmin and predicted the primary gene defect is not related to the ceruloplasmin gene. Later, Howell and Mercer (1994) provided a detailed analysis of trace element status and involvement of other organs, and described copper accumulation in liver, kidney, spleen, muscle, brain, and red blood cells accompanied by hemolysis, another pathologic condition frequently found in human WD. An important defense mechanism against elevated copper concentration in the liver, kidney, and spleen is the overproduction of metallothionein, which was detected in tx mouse livers (Koropatnick and Cherian, 1993; Deng et al., 1998).

NEUROLOGIC CHANGES AND BRAIN PATHOLOGY As mentioned above, no rodent model with the ATP7B defect develops major neurologic pathology. Several studies examined the copper concentration in the brains of tx mice (Howell and Mercer, 1994; Ono et al., 1997). Mutant mice had significantly higher copper levels in cerebral cortex, corpus striatum, thalamus/hypothalamus, and brainstem than control mice. Furthermore, tx mice had significantly higher metallothionein levels in all brain regions compared to age-matched controls which might provide protection against copper toxicity and can explain the lack of symptoms due to brain copper toxicity. Despite significant differences in brain copper concentration, pathology, and phenotype between human WD and tx mice, further exploration of neuronal structure and injury in tx mice might reveal more parallels and improve the understanding of neuropathology in WD.

THERAPEUTIC INTERVENTIONS The tx mouse model has been utilized for various therapeutic interventions. One example is the successful drug treatment with tetrathiomolybdate (Czachor et al., 2002),

ANIMAL MODELS OF WILSON DISEASE a drug which is currently under investigation for use in humans. Liver cell transplantation has demonstrated partial correction of the underlying gene defect, i.e., significant decrease of copper concentration in the liver, kidney, and spleen; however, this therapeutic approach failed to decrease brain copper levels (Allen et al., 2004). In another therapeutic experiment utilizing the tx mouse model, Buck et al. (2008) performed bone marrow stem cell transplantation and demonstrated partial correction of copper metabolism after 5 months. However, this initial response was not maintained in the long term. No significant improvement of copper metabolism and liver histology was observed after 9 months. Nevertheless, these results have important implications for therapeutic research and underline the importance of this WD model.

Toxic milk mouse from the Jackson laboratory DISCOVERY, GENE DEFECT, AND NATURAL COURSE OF DISEASE

The toxic milk mouse (tx-j) model from the Jackson laboratory has a spontaneous recessive point mutation at position 2135 in exon 8, which leads to a G712D missense mutation, predicted to be in the second transmembrane region of ATP7B. This mutation results in a phenotypic disorder similar to WD (Coronado et al., 2001). Similar to the tx mouse, tx-j mouse breast milk has an insufficient concentration of copper to maintain neonatal growth and development, and therefore tx-j pups must be fostered to a control dam with normal copper metabolism.

LIVER PATHOLOGY, ULTRASTRUCTURE, AND METABOLISM

The natural history of liver disease in the tx-j mouse is characterized by slow progression of liver histology to cirrhosis. Liver histology is almost normal until 3–4 months of age (Roberts et al., 2008; Le et al., 2014). The presence of enlarged hepatocyte nuclei, about two times larger than control mice, is among the first abnormalities. Starting at 5 months, there is an increased inflammatory infiltrate and mild microvesicular steatosis. Mild fibrosis is present at 6–7 months of age and ultimately it will progress to cirrhosis (Terwel et al., 2011). Hepatic copper concentration rises to 40–50 times the normal level after 2 months of age and remains elevated through the first year of life. Changes in mitochondrial morphology and function are typical features associated with copper accumulation. Starting at 3 months of age, electron microscopic images

61

show cystic dilatation of the cristae, with increased citrate synthase activity and decreased complex IV activity at 5 months of age (Roberts et al., 2008). In addition, tx-j mice appear to be a good model to study the interactions between copper accumulation and methionine metabolism. In fact, tx-j mice showed a downregulation of the bidirectional enzyme S-adenosylhomocysteine (SAH) hydrolase, which is responsible for metabolizing SAH to homocysteine or vice versa according to the availability of the substrate. SAH elevation is associated with inhibition of DNA methylation confirmed by the finding of global DNA hypomethylation in tx-j livers compared to control mice (Medici et al., 2013). Changes in gene transcript levels related to lipid metabolism are described in tx-j mice starting from gestation day 17. Fetal livers from tx-j mice showed downregulation of the expression of sterol regulatory element-binding transcription factor 1 (Srebf1), central in lipogenesis; and carnitine palmitoyltransferase 1A (Cpt1A) and peroxisome proliferator-activated receptor alpha (Ppara), important in fatty acid oxidation, as well as genes related to methionine metabolism, including SAH hydrolase (Le et al., 2014).

NEUROLOGIC CHANGES AND BRAIN PATHOLOGY At 12 months of age, tx-j brains present increased copper concentration in the striatum, hippocampus, and cerebellum, but no changes in the cerebral cortex (Terwel et al., 2011). A subsequent study showed increased copper concentration also in the cerebral cortex, aside from striatum, hippocampus, and cerebellum (Przybylkowski et al., 2013). Neuroinflammatory changes were predominantly localized in the striatum, which was characterized by increased levels of markers of astro- and microglial activation as detected by immunohistochemistry. At the same time, there were increased gene transcript levels related to inflammation, including interleukin-1 beta (Il1b), interleukin 4 (Il4), tumor necrosis factoralpha (Tnfa), and nitric oxide synthase 2, inducible (Nos2). When analyzing their behavior, tx-j mice presented mild abnormalities, including a preference in forelimb usage, and were slower to reach maximal performance in the rotarod test compared to control mice. In addition, tx-j mice resulted in an inability to acquire spatial memory in the Morris water maze (Terwel et al., 2011). Behavioral tests on tx-j mice were confirmed to be abnormal by a later study that showed impaired locomotor performance. Interestingly, the expression of dopaminergic, noradrenergic, and serotoninergic-specific enzymes to demonstrate neuronal loss was not different between tx-j and control mice (Przybylkowski et al., 2013).

62

V. MEDICI AND D. HUSTER

THERAPEUTIC INTERVENTIONS Despite the fact the tx-j mouse is a relatively new model of WD, some important intervention and treatment studies have been performed using this model. Given the changes in methionine metabolism and DNA methylation potential described in adult mice, choline was administered to tx-j female mice before conception and during pregnancy, and fetal livers were studied for changes in gene expression. Choline administration was associated with improvement to control levels of the expression of selected genes related to lipid and methionine metabolism, indicating that environmental and in utero factors could affect the phenotype of WD in adult life (Medici et al., 2014). The only study describing the effects of copper chelator penicillamine on brain copper metabolism was conducted in tx-j mice. Penicillamine administration for up to 14 days was associated with increased free copper and reduced protein-bound copper concentration. Increased levels of oxidative stress biomarkers, including reduced GSH/GSSG and increased malondialdehyde, were observed in the cortex and basal ganglia (Chen et al., 2012), providing a possible explanation for frequent worsening of neurologic symptoms observed after starting penicillamine treatment in patients with WD. In summary, the tx-j mouse represents a valid model of WD, characterized by slow progression of liver damage over 1 year after birth with liver and brain metabolic changes that make it suitable for studies of copper metabolism and anticopper accumulation interventions.

ATP7B–/– mouse DISCOVERY, GENE DEFECT, AND NATURAL COURSE OF DISEASE

A genetically engineered strain of ATP7B null mice was generated by introduction of an early termination codon in exon 2 of wild-type mouse ATP7B mRNA (Buiakova et al., 1999). This ATP7B knockout mouse features properties of both human WD as well as tx mouse phenotypes. Comparable to tx mice, newborn litters are born copper-deficient (although copper in the placenta and lactating breast glands is elevated) and accumulate copper in the liver, kidneys, and brain to a level up to 60-fold greater than normal by 5 months of age. Slight neurologic abnormalities and growth retardation were also observed. The specific advantages of this model compared to the inbred strains (see above) are the well-defined genetic background and the availability of the original strain as control in comparative analyses.

LIVER PATHOLOGY, ULTRASTRUCTURE, AND METABOLISM

A detailed investigation and description of liver pathology revealed further parallels of human liver disease (Huster et al., 2006). ATP7B–/– mice have low serum ceruloplasmin activity, increased urinary copper excretion, intracellular and nuclear copper accumulation in the hepatocyte, and severe liver pathology, developing earlier and exceeding the other mouse models described above. Liver pathology includes ultrastructural changes, steatosis, and mild inflammation at early stages (6 weeks of age), followed by hepatitis, dysplasia, and necroinflammation in advanced stages (at 12–20 weeks of age). These changes are followed by bile duct proliferation, development of fibrosis, and even neoplastic proliferation later in life (at 36 weeks and older). Yet, the majority of mice survive severe hepatitis and show remarkable regeneration in large parts of the liver. Therefore ATP7B–/– mice not only are an interesting model for studying pathology of copper toxicity, but moreover give access for investigation of compensatory and defense machinery. However, in accordance with the other rodent models, no significant neurologic symptoms or specific brain pathology were observed in these knockout mice. Mouse models are important for studying the mechanism and effects of copper toxicity in cells and cell organelles. Free copper and the generation of reactive oxygen species have deleterious effects on lipid membranes, proteins, DNA, and RNA molecules (Tapiero et al., 2003; Valko et al., 2005). As mentioned above, Sternlieb (1980) described mitochondrial alterations in human WD livers. An advanced study using this model and mass spectrometry techniques examined the underlying mechanism and revealed fragmentation of cardiolipin, an essential mitochondrial lipid required for activity of several mitochondrial proteins, as one potential cause of mitochondrial injury due to copper accumulation in ATP7B–/– mice (Yurkova et al., 2011). In agreement with observations of other species with hepatic copper overload, metallothionein is significantly overexpressed in ATP7B–/– mouse livers (Huster et al., 2007). The importance of elevated metallothionein levels for copper binding and defense against oxidative stress is supported by the observation that the double knockout of ATP7B and metallothionein genes was lethal during embryonic development (DH, unpublished data). In healthy livers, excess copper is excreted via the bile in human and mice. In contrast, WD patients have increased copper concentration in the urine, suggesting an overflow or compensatory mechanism to release excess body copper. In a combined study on ATP7B–/– mice that included conventional and advanced techniques

ANIMAL MODELS OF WILSON DISEASE such as positron emission tomography (PET), Gray et al. (2012) demonstrated a complex response to copper accumulation leading to elevated urinary copper excretion. This response included downregulation of copper transporter 1 (CTR1) and the appearance of small copper carrier (SCC) in urine to maintain copper balance. Hepatic steatosis is a frequent observation in both human WD and mouse models of copper toxicity. Recent studies utilizing microarray technique and real-time polymerase chain reaction of gene transcripts in ATP7B–/– mice revealed downregulation of genes important for cholesterol and lipid metabolism (Huster et al., 2007). The rate-limiting enzyme 3-hydroxy-3methyl-glutaryl-coenzyme A (HMG-CoA) reductase and a further eight enzymes involved with cholesterol biosynthesis were downregulated in livers of knockout mice compared to wild-type mice at 6 weeks of age. Decreased cholesterol biosynthesis was observed as well as markedly lower levels of serum lipids (total cholesterol, high-density lipoprotein, very-low-density lipoprotein, and trigycerides: one-, two-, three-, and sixfold reduction, respectively). These results were confirmed in studies with older mice (at 30, 46, and 60 weeks) (Ralle et al., 2010) and were comparable to results found in LEC rats (Levy et al., 2007). Taken together, there is increasing evidence for a link between copper and cholesterol metabolism in the liver which was first established in ATP7B–/– mice (Huster and Lutsenko, 2007; Huster et al., 2007; Ralle et al., 2010; Wilmarth et al., 2012). Parallels between human WD and different model organisms as well as changes of whole metabolic pathways, accompanied by changes in serum lipids and hepatic lipid metabolism, became an attractive field of further investigation. Elevated copper levels in the liver due to the ATP7B gene defect also led to an alteration of cell cycle machinery (Huster et al., 2007). Interestingly, upregulation of enzymes involved in cell cycle control, DNA replication, chromosome condensation and assembly, nuclear and cellular division occurs at early stages of the disease when copper is highly elevated, but significant cellular injury or inflammation is absent. One explanation for these observations is these responses trigger immune and inflammatory processes. On the other hand, upregulation of cell cycle machinery provides the basis for defense mechanisms and regeneration which is observed in later phases of the disease. It can be speculated that imbalance of upregulated proliferative signals becomes detrimental due to inflammatory and immunologic cascades which are triggered by elevated cellular copper at later stages. The observation of massive bile duct proliferation accompanied by neoplastic regeneration gives reason for further exploration.

63

Copper distribution occurs nonuniformly between intracellular compartments (Huster et al., 2006). Early in disease development, copper levels are high in the nucleus and cytosol, probably bound to metallothioneins. However, consequences of nuclear copper entry, alteration of the protein synthesis machinery, and the correlation between copper elevation and specific nuclear responses remained puzzling. Burkhead et al. (2011) further dissected the development of disease on molecular and ultrastructural levels. This study revealed that copper did not influence nuclear ion content or lead to significant protein oxidation, but specifically resulted in modification of nuclear proteins associated with RNA processing. A systems approach integrated data from expression studies with analysis of the presymptomatic hepatic nuclear proteome and liver metabolites (Wilmarth et al., 2012). This examination revealed suppression of nuclear receptors farnesoid X receptor (FXR) and glucocorticoid receptor (GR) in copper-loaded livers, both involved in lipid metabolism, while molecules with a function in DNA repair and enzymes involved in oxidative defense were more abundant. In a very recent study of ATP7B–/– mice and WD patients, investigators found a decreased binding of the nuclear receptors FXR, retinoic acid receptor (RXR), hepatocyte nuclear factor 4 alpha (HNF4A), and liver receptor homolog 1 (LRH-1) to promoter response elements and decreased mRNA expression of nuclear receptor target genes (Wooton-Kee et al., 2015). The authors of this study concluded that disruption of nuclear receptor activity provides a new mechanism which may contribute to chronic hepatic copper accumulation in the pathology of WD and suggested the restoration of nuclear receptors or other transcription factor functions as an additional mechanism for the beneficial effects of zinc therapy. ATP7B–/– mice have been used to test advanced research technologies such as laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) to monitor hepatic copper distribution (Boaru et al., 2015) and in vivo fluorescence to detect copper (Hirayama et al., 2012). Other imaging techniques, such as PET, using new tracers to reveal trace element status, showed feasibility, provided novel insight into copper distribution, and gained more significance for further research (Peng et al., 2012). All these advanced research approaches provide fundamental new insights into age-dependent copper accumulation and dynamics of copper fluxes in living animals, and may accelerate the development for noninvasive, real-time test systems of copper metabolism and its deregulation. Based on the available literature, it seems liver pathology is most pronounced in the ATP7B–/– mouse model; however, the variety of the phenotype and the degree

64

V. MEDICI AND D. HUSTER

of regeneration in the liver are remarkable, in line with observations in human WD. So far the underlying cause of this variability has yet to be elucidated.

NEUROLOGIC CHANGES AND BRAIN PATHOLOGY To date, no detailed behavioral studies of ATP7B–/– mice comparable to tx-j mice have been published. However, elevated copper in the brains of ATP7B–/– mice was already described in the initial investigation (Buiakova et al., 1999). A more specific study using LA-ICP-MS imaging of the whole brain confirmed the increased copper accumulation in brain parenchyma by a factor of two in comparison to control mice at 11 months (Boaru et al., 2014). Copper was increased proportionately throughout the all-cerebral regions and the authors assumed a differential regional susceptibility to copper. On the other hand, copper was reduced in periventricular regions, suggesting an active copper transport into cerebral fluid. In contrast, another study (Dong et al., 2015) did not find significant differences in brain copper concentration among different compartments (basal ganglia, cerebral cortex, cerebellum, and whole brain). Interestingly, the same study revealed abnormal ultrastructural cell organelles of neurons in basal ganglia (mainly mitochondria, deformed microtubule, and axons). The authors explained the discrepancy between the absence of copper deposition and the presence of neuronal damage with unidentified pathogenetic factors which could enhance toxic effects of copper in ATP7B–/– brain. ATP7B is expressed in Purkinje cells of the mouse cerebellum and delivers copper to ceruloplasmin (Barnes et al., 2005). In ATP7B–/– mice, ceruloplasmin expression is shifted to Bergmann glial cells, which also express ATPase copper-transporting alpha (ATP7A, Menkes disease protein), and copper delivery to ceruloplasmin is restored, possibly reflecting a compensatory mechanism for the lack of ATP7B function. The authors of this study concluded the conspicuous absence of neurologic abnormalities found in the ATP7B–/– mouse might be explained by this and other functional compensation by ATP7A.

THERAPEUTIC INTERVENTIONS Very high levels of copper in the liver and striking phenotype make the ATP7B–/– mouse a valuable model for therapeutic interventions. Cheng et al. (2015) reported a successful liver transplantation using the ATP7B–/– mouse model. In another report, early gestational transfer of hepatocytes improved copper concentration and reduced the liver pathology in ATP7B–/– mice significantly (Roybal et al., 2012). This study provided proof of principle for effective in utero gene therapy in WD using lentiviral vectors containing the ATP7B gene.

Several experiments with a focus on drug therapy targeting the impaired copper and lipid metabolism of the ATP7B–/– mouse model are currently under way.

COMPARISON OF RODENT MODELS FOR WILSON DISEASE Rodent models have been useful for studying the various effects of copper imbalance and toxicity. A comparison of WD liver pathology between human, tx-j mouse, and ATP7B–/– mouse is shown in Figure 6.1. Each mouse model has specific properties, similarities, and dissimilarities to human WD. A common feature among all animal models of human WD is the exceptional diversity in tissue copper accumulation, phenotype, and even outcome of the disease. The availability of various rodent models with different defects in ATP7B will help the understanding of phenotypic diversity found in WD. The most important parallels and differences between WD and rodent models are summarized in Table 6.1.

OTHER ANIMAL MODELS OF COPPER ACCUMULATION AND TOXICITY IN THE LIVER AND BRAIN There are several other mammalian species with inherited defects in copper metabolism which show copper accumulation and toxicity in the liver. However, the underlying genetic defect is not the same as WD. Therefore their usefulness to serve as models for WD is limited. Bedlington terriers accumulate copper and show signs of hepatic copper toxicosis with liver fibrosis and cirrhosis (Favier et al., 2012; Fieten et al., 2014); however, the underlying genetic defect was identified in the copper metabolism domain containing 1 (COMMD1) gene (formerly known as MURR1) and not in the ATP7B gene (van De Sluis et al., 2002; Fieten et al., 2014). Hepatic copper accumulation and liver pathology resemble human WD, but, as in rodent models, overt neurologic defects are absent and the affected dogs have normal concentrations of ceruloplasmin. The North Ronaldsay sheep is another interesting mammalian model for hepatic and neurologic copper accumulation; however, as with the canine models, they are clinically and genetically different from WD (Haywood et al., 2004). The underlying gene defect is still unknown. Nevertheless, this model may have some importance for studying copper accumulation in the brain, the involvement of the blood–brain barrier, and the influence of environmental copper (Haywood et al., 2008; Haywood and Vaillant, 2014). Further models of copper-associated liver disease include different dog breeds (Doberman Pinscher, Skye terrier, Dalmatian, Labrador retriever), cats, and

ANIMAL MODELS OF WILSON DISEASE

Fig. 6.1. Liver pathology of human Wilson disease (WD) compared to mouse models. EM, electron microscope.

65

66

V. MEDICI AND D. HUSTER

Table 6.1 Important phenotypic similarities and differences of animal models compared to human Wilson disease Model animal and underlying gene defect

Common features

Differences

Long–Evans Cinnamon rat ATP7B deletion of 900 basepairs of the coding region at the 30 end and 400 basepairs of the downstream untranslated region

Hepatic copper accumulation Liver disease Copper increased in brain Mitochondrial abnormalities Lipid metabolism affected Decreased ceruloplasmin levels Increased metallothionein Hepatic copper accumulation Liver disease Copper increased in brain Mitochondrial abnormalities Decreased ceruloplasmin levels Increased metallothionein Hepatic copper accumulation Liver disease Copper increased in brain Mitochondrial abnormalities Ceruloplasmin? Metallothionein? Hepatic copper accumulation Liver disease Copper increased in brain Mitochondrial abnormalities Lipid metabolism affected Decreased ceruloplasmin levels Increased metallothionein

Only slight neurologic symptoms Liver tumors common

tx mouse Met1386Val mutation (A4066G, exon 20)

tx-j mouse Gly712Asp mutation G2135A, exon 8

ATP7B–/– mouse Multiple stop codons in exon 2 of ATP7B

ruminants, and are described in more detail elsewhere (Howell, 1999; Fuentealba and Aburto, 2003; Fieten et al., 2014).

RESEARCH PERSPECTIVES AND FUTURE THERAPY DEVELOPMENTS The discovery and detailed description of WD animal models over the last three decades paved the way for new therapeutic interventions. Although WD has become a treatable disorder with an overall fair prognosis, several problems, such as drug side-effects, neurologic deterioration after therapy initiation, residual symptoms, lack of compliance, or even failure of therapy, underline the importance of developing novel therapeutic approaches. Currently, liver transplantation is the only causal therapy form of WD and cures the hepatic disease. However, liver transplantation as treatment for neurologic disease is controversial and the procedure has a number of limitations (Schumacher et al., 1997; Brewer and Askari, 2000; Medici et al., 2005a). The availability of valid model organisms enables innovative research strategies such as cell and gene therapy (Filippi and Dhawan,

Relevant copper deficiency at birth, no copper deficiency in human breast milk Tremor in new born mice due to copper deficiency No neurologic symptoms Relevant copper deficiency at birth, no copper deficiency in human breast milk Only slight neurologic symptoms

Relevant copper deficiency at birth, no copper deficiency in human breast milk No neurologic symptoms Liver tumors/neoplastic proliferation of bile ducts

2014; Gupta, 2014) and drug development (Gateau and Delangle, 2014; Weiss and Stremmel, 2014). Detailed analysis of WD models has uncovered parallels to other liver and brain disorders, and provided novel insights in copper metabolism, inflammatory and neoplastic liver diseases, cholesterol metabolism, and even liver fibrosis and repair mechanism. These investigations and, even more importantly, the understanding of brain involvement and phenotypic diversity are just at the beginning (Lutsenko, 2014). Copper dyshomeostasis plays an unequivocal role in the pathogenesis of neurologic WD. Any innovative and targeted therapy approach is based on better understanding of metal homeostasis in the brain. Whereas neurologic symptoms due to copper accumulation in parts of the brain are a common phenomenon of human WD, to date little is known about the specific mechanism and the remarkable diversity in severity. Nevertheless, due to a broad scientific interest and the use of improved research tools, especially molecular approaches and neuroimaging, the available disease models have shown promising progress and will overcome past shortcomings.

ANIMAL MODELS OF WILSON DISEASE

67

Fig. 6.2. Current and future research targets and strategies using animal models for Wilson disease (WD).

An overview of the important research goals for a better understanding and treatment of WD is shown in Figure 6.2. WD as a model disease for oxidative stress, cell death, and tissue repair, and utilizing rodent models for basic and therapeutic research, will help to understand and treat WD, and possibly other hepatic and neurologic disorders.

ABBREVIATIONS ALT, alanine transaminase; AST, aspartate transaminase; ATP7A, ATPase copper-transporting alpha; ATP7B, ATPase copper-transporting beta; COMMD1, copper metabolism domain containing 1; Cpt1A, carnitine palmitoyltransferase 1A; CTR1, copper transporter 1; FXR, farnesoid X receptor; GR, glucocorticoid receptor; GSH/GSSG, glutathione ratio; HMG-CoA, 3-hydroxy3-methyl-glutaryl-coenzyme A; HNF4A, hepatocyte nuclear factor 4-alpha; Il1b, interleukin-1 beta; Il4, interleukin-4; LA-ICP-MS, laser ablation inductively coupled plasma mass spectrometry; LEC, Long–Evans Cinnamon; LRH-1, liver receptor homolog 1; Ndufb7, NADH: ubiquinone oxidoreductase subunit B7; Nos2, nitric oxide synthase 2, inducible; Ppara, peroxisome proliferator-activated receptor alpha; PET, positron emission tomography; RXR, retinoic acid receptor; SAH, S-adenosylhomocysteine; SCC, small copper carrier; Sdhaf2, succinate dehydrogenase complex assembly factor 2; SOD, superoxide dismutase; Srebf1, sterol regulatory element-binding transcription factor 1; Tnfa, tumor necrosis factor-alpha; tx mouse, toxic milk mouse; tx-j mouse, the Jackson laboratory toxic milk mouse; WD, Wilson disease.

ACKNOWLEDGMENT The authors thank Dr. G. Sawers for critical reading of the manuscript and useful comments. This work was partly supported by the Deutsche Forschungsgemeinschaft (HU 932/3-2).

REFERENCES Allen KJ, Cheah DM, Wright PF et al. (2004). Liver cell transplantation leads to repopulation and functional correction in a mouse model of Wilson’s disease. J Gastroenterol Hepatol 19: 1283–1290. Barnes N, Tsivkovskii R, Tsivkovskaia N et al. (2005). The copper-transporting ATPases, Menkes and Wilson disease proteins, have distinct roles in adult and developing cerebellum. J Biol Chem 280: 9640–9645. Biempica L, Rauch H, Quintana N et al. (1988). Morphologic and chemical studies on a murine mutation (toxic milk mice) resulting in hepatic copper toxicosis. Lab Invest 59: 500–508. Boaru SG, Merle U, Uerlings R et al. (2014). Simultaneous monitoring of cerebral metal accumulation in an experimental model of Wilson’s disease by laser ablation inductively coupled plasma mass spectrometry. BMC Neurosci 15: 98. Boaru SG, Merle U, Uerlings R et al. (2015). Laser ablation inductively coupled plasma mass spectrometry imaging of metals in experimental and clinical Wilson’s disease. J Cell Mol Med 19: 806–814. Brewer GJ, Askari F (2000). Transplant livers in Wilson’s disease for hepatic, not neurologic, indications. Liver Transpl 6: 662–664. Buck NE, Cheah DM, Elwood NJ et al. (2008). Correction of copper metabolism is not sustained long term in Wilson’s disease mice post bone marrow transplantation. Hepatol Int 2: 72–79.

68

V. MEDICI AND D. HUSTER

Buiakova OI, Xu J, Lutsenko S et al. (1999). Null mutation of the murine ATP7B (Wilson disease) gene results in intracellular copper accumulation and late-onset hepatic nodular transformation. Hum Mol Genet 8: 1665–1671. Burkhead JL, Ralle M, Wilmarth P et al. (2011). Elevated copper remodels hepatic RNA processing machinery in the mouse model of Wilson’s disease. J Mol Biol 406: 44–58. Chen DB, Feng L, Lin XP et al. (2012). Penicillamine increases free copper and enhances oxidative stress in the brain of toxic milk mice. PLoS One 7. e37709. Chen S, Shao C, Dong T et al. (2014). Transplantation of ATP7B-transduced bone marrow mesenchymal stem cells decreases copper overload in rats. PLoS One 9. e111425. Cheng Q, He SQ, Gao D et al. (2015). Early application of auxiliary partial orthotopic liver transplantation in murine model of Wilson disease. Transplantation 99: 2317–2324. Coronado V, Nanji M, Cox DW (2001). The Jackson toxic milk mouse as a model for copper loading. Mamm Genome 12: 793–795. Czachor JD, Cherian MG, Koropatnick J (2002). Reduction of copper and metallothionein in toxic milk mice by tetrathiomolybdate, but not deferiprone. J Inorg Biochem 88: 213–222. Deng DX, Ono S, Koropatnick J et al. (1998). Metallothionein and apoptosis in the toxic milk mutant mouse. Lab Invest 78: 175–183. Dong Y, Shi SS, Chen S et al. (2015). The discrepancy between the absence of copper deposition and the presence of neuronal damage in the brain of Atp7b(-/-) mice. Metallomics 7: 283–288. Dorea JG (2000). Iron and copper in human milk. Nutrition 16: 209–220. Favier RP, Spee B, Schotanus BA et al. (2012). COMMD1deficient dogs accumulate copper in hepatocytes and provide a good model for chronic hepatitis and fibrosis. PLoS One 7. e42158. Fieten H, Penning LC, Leegwater PA et al. (2014). New canine models of copper toxicosis: diagnosis, treatment, and genetics. Ann N Y Acad Sci 1314: 42–48. Filippi C, Dhawan A (2014). Current status of human hepatocyte transplantation and its potential for Wilson’s disease. Ann N Y Acad Sci 1315: 50–55. Fuentealba IC, Aburto EM (2003). Animal models of copper-associated liver disease. Comp Hepatol 2: 5. Fujiwara N, Iso H, Kitanaka N et al. (2006). Effects of copper metabolism on neurological functions in Wistar and Wilson’s disease model rats. Biochem Biophys Res Commun 349: 1079–1086. Gateau C, Delangle P (2014). Design of intrahepatocyte copper(I) chelators as drug candidates for Wilson’s disease. Ann N Y Acad Sci 1315: 30–36. Gitlin JD (2003). Wilson disease. Gastroenterology 125: 1868–1877. Gray LW, Peng F, Molloy SA et al. (2012). Urinary copper elevation in a mouse model of Wilson’s disease is a regulated process to specifically decrease the hepatic copper load. PLoS One 7. e38327. Gupta S (2014). Cell therapy to remove excess copper in Wilson’s disease. Ann N Y Acad Sci 1315: 70–80.

Hayashi M, Fuse S, Endoh D et al. (2006). Accumulation of copper induces DNA strand breaks in brain cells of Long-Evans Cinnamon (LEC) rats, an animal model for human Wilson disease. Exp Anim 55: 419–426. Haywood S, Vaillant C (2014). Overexpression of copper transporter CTR1 in the brain barrier of North Ronaldsay sheep: implications for the study of neurodegenerative disease. J Comp Pathol 150: 216–224. Haywood S, Muller T, Mackenzie AM et al. (2004). Copper-induced hepatotoxicosis with hepatic stellate cell activation and severe fibrosis in North Ronaldsay lambs: a model for non-Wilsonian hepatic copper toxicosis of infants. J Comp Pathol 130: 266–277. Haywood S, Paris J, Ryvar R et al. (2008). Brain copper elevation and neurological changes in North Ronaldsay sheep: a model for neurodegenerative disease? J Comp Pathol 139: 252–255. Hirayama T, Van de Bittner GC, Gray LW et al. (2012). Near-infrared fluorescent sensor for in vivo copper imaging in a murine Wilson disease model. Proc Natl Acad Sci U S A 109: 2228–2233. Howell JM (1999). Animal models of Wilson’s disease. Adv Exp Med Biol 448: 139–152. Howell JM, Mercer JF (1994). The pathology and trace element status of the toxic milk mutant mouse. J Comp Pathol 110: 37–47. Huster D (2010). Wilson disease. Best Pract Res Clin Gastroenterol 24: 531–539. Huster D, Lutsenko S (2007). Wilson disease: not just a copper disorder. Analysis of a Wilson disease model demonstrates the link between copper and lipid metabolism. Mol Biosyst 3: 816–824. Huster D, Hoppert M, Lutsenko S et al. (2003). Defective cellular localization of mutant ATP7B in Wilson’s disease patients and hepatoma cell lines. Gastroenterology 124: 335–345. Huster D, Finegold MJ, Morgan CT et al. (2006). Consequences of copper accumulation in the livers of the atp7b-/- (Wilson disease gene) knockout mice. Am J Pathol 168: 423–434. Huster D, Purnat TD, Burkhead JL et al. (2007). High copper selectively alters lipid metabolism and cell cycle machinery in the mouse model of Wilson disease. J Biol Chem 282: 8343–8355. Huster D, Kuhne A, Bhattacharjee A et al. (2012). Diverse functional properties of Wilson disease ATP7B variants. Gastroenterology 142: 947–956 e945. Jia G, Tohyama C, Sone H (2002). DNA damage triggers imbalance of proliferation and apoptosis during development of preneoplastic foci in the liver of Long–Evans Cinnamon rats. Int J Oncol 21: 755–761. Jong-Hon K, Togashi Y, Kasai H et al. (1993). Prevention of spontaneous hepatocellular carcinoma in Long–Evans cinnamon rats with hereditary hepatitis by the administration of D-penicillamine. Hepatology 18: 614–620. Joseph B, Kapoor S, Schilsky ML et al. (2009). Bile salt-induced pro-oxidant liver damage promotes transplanted cell proliferation for correcting Wilson disease in the Long–Evans Cinnamon rat model. Hepatology 49: 1616–1624.

ANIMAL MODELS OF WILSON DISEASE Kato J, Kohgo Y, Sugawara N et al. (1993). Abnormal hepatic iron accumulation in LEC rats. Jpn J Cancer Res 84: 219–222. Kato J, Kobune M, Kohgo Y et al. (1996). Hepatic iron deprivation prevents spontaneous development of fulminant hepatitis and liver cancer in Long–Evans Cinnamon rats. J Clin Invest 98: 923–929. Kawano H, Takeuchi Y, Yoshimoto K et al. (2001). Histological changes in monoaminergic neurons of Long–Evans Cinnamon rats. Brain Res 915: 25–31. Kim DW, Ahn TB, Kim JM et al. (2005). Enhanced Mn-SOD immunoreactivity in the dopaminergic neurons of Long– Evans Cinnamon rats. Neurochem Res 30: 475–478. Klein D, Lichtmannegger J, Finckh M et al. (2003). Gene expression in the liver of Long–Evans Cinnamon rats during the development of hepatitis. Arch Toxicol 77: 568–575. Klein D, Arora U, Lichtmannegger J et al. (2004). Tetrathiomolybdate in the treatment of acute hepatitis in an animal model for Wilson disease. J Hepatol 40: 409–416. Koropatnick J, Cherian MG (1993). A mutant mouse (tx) with increased hepatic metallothionein stability and accumulation [see comments]. Biochem J 296 (Pt 2): 443–449. La Fontaine SS, Theophilos MB, Firth SD et al. (2001). Effect of the toxic milk mutation (tx) on the function and intracellular localization of Wnd, the murine homologue of the Wilson copper ATPase. Hum Mol Genet 10: 361–370. Le A, Shibata NM, French SW et al. (2014). Characterization of timed changes in hepatic copper concentrations, methionine metabolism, gene expression, and global DNA methylation in the Jackson toxic milk mouse model of Wilson disease. Int J Mol Sci 15: 8004–8023. Lee BH, Kim JM, Heo SH et al. (2011). Proteomic analysis of the hepatic tissue of Long–Evans Cinnamon (LEC) rats according to the natural course of Wilson disease. Proteomics 11: 3698–3705. Lee BH, Kim JH, Kim JM et al. (2013). The early molecular processes underlying the neurological manifestations of an animal model of Wilson’s disease. Metallomics 5: 532–540. Levy E, Brunet S, Alvarez F et al. (2007). Abnormal hepatobiliary and circulating lipid metabolism in the Long–Evans Cinnamon rat model of Wilson’s disease. Life Sci 80: 1472–1483. Lutsenko S (2014). Modifying factors and phenotypic diversity in Wilson’s disease. Ann N Y Acad Sci 1315: 56–63. Masaki T, Shiratori Y, Rengifo W et al. (2000a). Hepatocellular carcinoma cell cycle: study of Long– Evans Cinnamon rats. Hepatology 32: 711–720. Masaki T, Tokuda M, Shiratori Y et al. (2000b). A possible novel src-related tyrosine kinase in cancer cells of LEC rats that develop hepatocellular carcinoma. J Hepatol 32: 92–99. Medici V, Santon A, Sturniolo GC et al. (2002). Metallothionein and antioxidant enzymes in Long–Evans Cinnamon rats treated with zinc. Arch Toxicol 76: 509–516.

69

Medici V, Mirante VG, Fassati LR et al. (2005a). Liver transplantation for Wilson’s disease: the burden of neurological and psychiatric disorders. Liver Transpl 11: 1056–1063. Medici V, Sturniolo GC, Santon A et al. (2005b). Efficacy of zinc supplementation in preventing acute hepatitis in Long–Evans Cinnamon rats. Liver Int 25: 888–895. Medici V, Shibata NM, Kharbanda KK et al. (2013). Wilson’s disease: changes in methionine metabolism and inflammation affect global DNA methylation in early liver disease. Hepatology 57: 555–565. Medici V, Shibata NM, Kharbanda KK et al. (2014). Maternal choline modifies fetal liver copper, gene expression, DNA methylation, and neonatal growth in the tx-j mouse model of Wilson disease. Epigenetics 9: 286–296. Mercer JF, Grimes A, Danks DM et al. (1991). Hepatic ceruloplasmin gene expression is unaltered in the toxic milk mouse. J Nutr 121: 894–899. Merle U, Encke J, Tuma S et al. (2006). Lentiviral gene transfer ameliorates disease progression in Long–Evans cinnamon rats: an animal model for Wilson disease. Scand J Gastroenterol 41: 974–982. Michalczyk AA, Rieger J, Allen KJ et al. (2000). Defective localization of the Wilson disease protein (ATP7B) in the mammary gland of the toxic milk mouse and the effects of copper supplementation. Biochem J 352: 565–571. Miyoshi E, Jain SK, Sugiyama T et al. (1993). Expression of DNA methyltransferase in LEC rats during hepatocarcinogenesis. Carcinogenesis 14: 603–605. Okuda K (1992). Hepatocellular carcinoma: recent progress. Hepatology 15: 948–963. Ono S, Koropatnick DJ, Cherian MG (1997). Regional brain distribution of metallothionein, zinc and copper in toxic milk mutant and transgenic mice. Toxicology 124: 1–10. Peng F, Lutsenko S, Sun X et al. (2012). Positron emission tomography of copper metabolism in the Atp7b-/knock-out mouse model of Wilson’s disease. Mol Imaging Biol 14: 70–78. Przybylkowski A, Gromadzka G, Wawer A et al. (2013). Neurochemical and behavioral characteristics of toxic milk mice: an animal model of Wilson’s disease. Neurochem Res 38: 2037–2045. Ralle M, Huster D, Vogt S et al. (2010). Wilson disease at a single cell level: intracellular copper trafficking activates compartment-specific responses in hepatocytes. J Biol Chem 285: 30875–30883. Rauch H (1983). Toxic milk, a new mutation affecting cooper metabolism in the mouse. J Hered 74: 141–144. Rauch H, Wells AJ (1995). The toxic milk mutation, tx, which results in a condition resembling Wilson disease in humans, is linked to mouse chromosome 8. Genomics 29: 551–552. Roberts EA, Robinson BH, Yang S (2008). Mitochondrial structure and function in the untreated Jackson toxic milk (tx-j) mouse, a model for Wilson disease. Mol Genet Metab 93: 54–65. Roybal JL, Endo M, Radu A et al. (2012). Early gestational gene transfer with targeted ATP7B expression in the liver

70

V. MEDICI AND D. HUSTER

improves phenotype in a murine model of Wilson’s disease. Gene Ther 19: 1085–1094. Samuele A, Mangiagalli A, Armentero MT et al. (2005). Oxidative stress and pro-apoptotic conditions in a rodent model of Wilson’s disease. Biochim Biophys Acta 1741: 325–330. Sasaki M, Yoshida MC, Kagami K et al. (1985). Spontaneous hepatitis in an inbred strain of Long-Evans rats. Rat News Letter 14: 4–6. Schilsky ML, Quintana N, Volenberg I et al. (1998). Spontaneous cholangiofibrosis in Long–Evans Cinnamon rats: a rodent model for Wilson’s disease. Lab Anim Sci 48: 156–161. Schumacher G, Platz KP, Mueller AR et al. (1997). Liver transplantation: treatment of choice for hepatic and neurological manifestation of Wilson’s disease. Clin Transplant 11: 217–224. Sone K, Maeda M, Wakabayashi K et al. (1996). Inhibition of hereditary hepatitis and liver tumor development in Long– Evans cinnamon rats by the copper-chelating agent trientine dihydrochloride. Hepatology 23: 764–770. Sternlieb I (1968). Mitochondrial and fatty changes in hepatocytes of patients with Wilson’s disease. Gastroenterology 55: 354–367. Sternlieb I (1980). Copper and the liver. Gastroenterology 78: 1615–1628. Sternlieb I, Quintana N, Volenberg I et al. (1995). An array of mitochondrial alterations in the hepatocytes of Long– Evans Cinnamon rats. Hepatology 22: 1782–1787. Tapiero H, Townsend DM, Tew KD (2003). Trace elements in human physiology and pathology. Copper. Biomed Pharmacother 57: 386–398. Terwel D, Loschmann YN, Schmidt HH et al. (2011). Neuroinflammatory and behavioural changes in the Atp7B mutant mouse model of Wilson’s disease. J Neurochem 118: 105–112.

Theophilos MB, Cox DW, Mercer JF (1996). The toxic milk mouse is a murine model of Wilson disease. Hum Mol Genet 5: 1619–1624. Togashi Y, Li Y, Kang JH et al. (1992). D-penicillamine prevents the development of hepatitis in Long–Evans Cinnamon rats with abnormal copper metabolism. Hepatology 15: 82–87. Valko M, Morris H, Cronin MT (2005). Metals, toxicity and oxidative stress. Curr Med Chem 12: 1161–1208. van De Sluis B, Rothuizen J, Pearson PL et al. (2002). Identification of a new copper metabolism gene by positional cloning in a purebred dog population. Hum Mol Genet 11: 165–173. Voskoboinik I, La Fontaine S, Mercer JF et al. (2001). Functional studies on the Wilson copper P-type ATPase and toxic milk mouse mutant. Biochem Biophys Res Commun 281: 966–970. Weiss KH, Stremmel W (2014). Clinical considerations for an effective medical therapy in Wilson’s disease. Ann N Y Acad Sci 1315: 81–85. Wilmarth PA, Short KK, Fiehn O et al. (2012). A systems approach implicates nuclear receptor targeting in the Atp7b(-/-) mouse model of Wilson’s disease. Metallomics 4: 660–668. Wooton-Kee CR, Jain AK, Wagner M et al. (2015). Elevated copper impairs hepatic nuclear receptor function in Wilson’s disease. J Clin Invest 125: 3449–3460. Wu J, Forbes JR, Chen HS et al. (1994). The LEC rat has a deletion in the copper transporting ATPase gene homologous to the Wilson disease gene. Nat Genet 7: 541–545. Yurkova IL, Arnhold J, Fitzl G et al. (2011). Fragmentation of mitochondrial cardiolipin by copper ions in the Atp7b-/mouse model of Wilson’s disease. Chem Phys Lipids 164: 393–400. Zischka H, Lichtmannegger J, Schmitt S et al. (2014). Liver mitochondrial membrane crosslinking and destruction in a rat model of Wilson disease. J Clin Invest 121: 1508–1518.

Handbook of Clinical Neurology, Vol. 142 (3rd series) Wilson Disease A. Członkowska and M.L. Schilsky, Editors http://dx.doi.org/10.1016/B978-0-444-63625-6.00007-0 © 2017 Elsevier B.V. All rights reserved

Chapter 7

Wilson disease – liver pathology MACIEJ PRONICKI* Department of Pathology, The Children’s Memorial Health Institute, Warsaw, Poland

Abstract The liver in Wilson disease may demonstrate a wide range of damage patterns. Some patients may present almost no detectable microscopic pathology, while others display lesions consistent with fulminant hepatitis or acute liver failure. Most liver biopsy specimens show moderate to severe steatosis, variable degree of portal and/or lobular inflammation, and fibrosis eventually progressing to cirrhosis. Additional findings include liver cell degeneration and ballooning, Mallory hyaline bodies, liver cell necrosis, and glycogenation of periportal hepatocytic nuclei. None of the above lesions are specific for Wilson disease and should be interpreted in a wider medical context and particular clinical setting. The main message concerning liver pathology is that Wilson disease may be microscopically misinterpreted as many other liver diseases, including viral or autoimmune hepatitis, alcoholic/nonalcoholic steatohepatitis, toxic liver injury, cryptogenic cirrhosis, metabolic liver disease, and many others. The possibility of Wilson disease should be considered in all patients, especially young ones presenting unexplained liver diseases with many variable patterns of microscopic liver involvement.

PATHOLOGY OF WILSON DISEASE In a proportion of patients, particularly children, teenagers, or young adults suffering from Wilson disease, liver disease constitutes the most frequently observed clinical manifestation. The condition of the liver as an organ readily accessible by biopsy is quite well studied in Wilson disease, and histologic findings may bring about clinically useful information. Liver pathology has been widely described in medical surgical pathologic literature and constitutes the basic handbook knowledge (Arroyo and Crawford, 2009; Desmet and Rosai, 2012; Thompson et al., 2012; Torbenson, 2015). Clinical pattern of liver involvement may attain the full spectrum of presentations. Symptoms usually do not appear before the age of 3–5 years. Some patients develop acute or fulminant liver failure; on the other hand, many children and adults may stay clinically asymptomatic for a long time. The reason for phenotypic variability is not clear. This topic is widely discussed in Chapters 2–5. The following light microscopic pathologic findings, isolated or combined

in diverse associations, have been reported in patients with Wilson disease.

Liver steatosis In general, fatty degeneration is considered rather a frequent and, for the most part, unspecific expression of the liver parenchymal cell damage. In Wilson disease it is considered by many as one of the most typical features (Figs 7.2–7.7). The range of fatty accumulation may be wide, from mild and focal to severe, and diffuse. Types of steatosis – microvesicular vs. macrovesicular – also may vary, or frequently coexist. Interestingly, in some patients liver steatosis and inflammation in Wilson disease closely resemble nonalcoholic fatty liver disease, as seen in metabolic syndrome, or other forms of steatohepatitis. This phenomenon should be kept in mind when evaluating patients with liver disease, presenting steatohepatitis in biopsy, particularly if patients are young and not overweight.

*Correspondence to: Maciej Pronicki, Department of Pathology, The Children’s Memorial Health Institute, al. Dzieci Polskich 20, 04-730 Warsaw, Poland. Tel: +48-22-815-19-60, E-mail: [email protected]

Fig. 7.1. Liver biopsy sample of Wilson disease patient showing no substantial microscopic pathology. In some patients the liver is normal, or presents only minimal lesions in light microscopy. Portal space visible in upper right corner. Central vein at the left lower margin. Hematoxylin and eosin; original optical magnification 200 .

Fig. 7.4. Moderate mixed steatosis with features of early fibrosis. No substantial hepatitis. Hematoxylin and eosin; original optical magnification 200  .

Fig. 7.2. Mixed, predominantly pericentral steatosis of moderate degree, minimal portal inflammatory infiltrate. No fibrosis. Hematoxylin and eosin; original optical magnification 200  .

Fig. 7.5. Mild steatosis and inflammation with early, thin fibrous bridges. Hematoxylin and eosin; original optical magnification 100  .

Fig. 7.3. Moderate to severe diffuse fatty degeneration, portal space widened by lymphocytic inflammatory infiltration. No interface inflammatory activity. Hematoxylin and eosin; original optical magnification 200  .

Fig. 7.6. Severe mixed fatty degeneration with no fibrosis and no hepatitis. Hematoxylin and eosin; original optical magnification 200  .

WILSON DISEASE – LIVER PATHOLOGY

Fig. 7.7. Diffuse fatty liver with moderate fibrosis and no inflammation. Hematoxylin and eosin; original optical magnification 100  .

Fig. 7.8. Severe active inflammation with parenchymal destruction and fibrosis, steatosis. Hematoxylin and eosin; original optical magnification 200  .

Inflammation Noninfectious hepatitis of any kind and degree may be observed in Wilson disease. In terms of histologic pattern, liver involvement may attain any form of acute or chronic inflammation, including forms mimicking viral, autoimmune, or other form of hepatitis (Fig. 7.8). Inflammatory infiltrates may occupy portal spaces, sometimes with accompanying active interface hepatitis. Inflammatory cells may be also seen in intralobular areas, leading to damage and necrosis of small groups of hepatocytes (Figs 7.9 and 7.10). Both portal and lobular hepatitis may coexist in individual patients (Fig. 7.11).

Fibrosis and cirrhosis Liver fibrosis usually progresses with the development of copper deposition and consequent liver damage (Fig. 7.12). Several authors discern precirrhotic and

73

Fig. 7.9. Massive liver cell necrosis with numerous neocholangioles and diffuse scarring, with no features of liver cell regeneration (explant). Hematoxylin and eosin; original optical magnification 100  .

Fig. 7.10. High-power magnification of Fig. 7.11 (explant). Irreversible liver cell damage, neocholangioles with bile plugs, diffuse fibrosis, and inflammation. Hematoxylin and eosin; original optical magnification 400  .

cirrhotic phases of liver disease in Wilsonian patients. Initially, fibrous tissue expands portal tracts and forms thin perisinusoidal fibrous strands, then fibrous bridges develop. Later in the disease, full-blown cirrhosis may develop (Fig. 7.13). The cirrhosis in Wilson disease is usually macronodular.

Glycogenated nuclei Glycogen deposition inside the liver cell nucleus is described as one of the typical findings in Wilson disease. Glycogenated nuclei may be observed both in light and electron microscope, and are visible in periportal areas of the liver lobule (Fig. 7.14). However, this observation is highly nonspecific, also occurring in many other liver diseases.

74

M. PRONICKI

Necrosis Individual hepatocytic cell necrosis may be observed in scattered cells in many cases of Wilson disease. In rare instances of fulminant liver failure, the necrosis is massive and diffuse. In such instances, the liver cell regeneration is usually nonexistent, with diffuse neocholangiolization and fibrosis.

Ballooning degeneration

Fig. 7.11. Intralobular foci of inflammatory liver cell dropout, mixed steatosis. Hematoxylin and eosin; original optical magnification 400  .

This type of liver cell injury consists of cell enlargement with cytoplasm clearing (Fig. 7.15). It is typically observed in some types of fatty liver disease and steatohepatitis, as well as liver inflammation. Thus, ballooned hepatocytes, although seen in Wilson disease, are not specific for this disorder.

Fig. 7.12. Severe fibrosis with nodular transformation and relatively scant inflammatory infiltrate. Moderate to severe mixed steatosis. Hematoxylin and eosin; original optical magnification 200  .

Fig. 7.14. Glycogenated liver cell nuclei of ballooned and steatotic liver cells. Hematoxylin and eosin; original optical magnification 600  . The arrow shows one of the glycogenated liver cell nuclei present in the microphotograph.

Fig. 7.13. Liver cirrhosis with dispersed mild inflammatory cells; original optical magnification 100 . Periodic–acid Schiff stain with diastase digestion (explant).

Fig. 7.15. Liver cell ballooning (center) and steatosis, leading to effacement of trabecular architecture. Hematoxylin and eosin; original optical magnification 600  .

WILSON DISEASE – LIVER PATHOLOGY

Mallory bodies Mallory hyaline bodies, of the same type and morphology as classically seen in ballooned hepatocytes in alcoholic fatty liver disease and other chronic liver diseases, may be observed in Wilson disease. They consist of intracytoplasmic eosinophilic inclusions built of cytoskeletal proteins, mainly keratins. All of the above-mentioned individual histologic findings in Wilson disease may combine to form several large “biopsy patterns” (Torbenson, 2015). The most typical and well-defined are: (1) predominantly steatosis/ steatohepatitis-type damage with variable degree of fibrosis; (2) unexplained cirrhosis or hepatic fibrosis; (3) acute or fulminant hepatitis; and (4) mild, borderline pathologic lesions or even normal liver histology (Fig. 7.1).

LIGHT MICROSCOPIC DETECTION OF COPPER DEPOSITS Copper deposits in the liver are not detectable in routine sections stained with hematoxylin and eosin. In light microscopy, accumulated copper and associated proteins may be visualized by special histochemical stains. The most widely used and recognized are rhodanine and orcein. The first stain shows orange-red material accumulated in hepatocytic cytoplasm; the second stains excessive copper binding protein in brown. However, due to the focal pattern of copper accumulation in the liver, the sensitivity of the above stainings in biopsy specimens may be unsatisfactory. Moreover, copper may accumulate in liver cells in disorders other than Wilson disease, e.g., chronic cholestatic states. In the author’s experience, copper staining does not appear useful as a diagnostic tool in everyday diagnostic lab work.

ULTRASTRUCTURE Although the golden era of transmission electron microscopy in pathologic diagnostics has already passed, it should be noticed that ultrastructure of liver in Wilson disease is considered quite typical, and recognition of its pattern may appear diagnostically useful (Phillips et al., 1987). The most striking are the mitochondrial lesions. The mitochondria are enlarged, have abnormal shapes, and characteristically dilated terminal cristae

75

sometimes containing floccular deposits. Bile canaliculi show elongation of microvilli, sometimes with features of luminal obstruction, as well as “fish-mouth” outline. Other lesions include electron-dense intracytoplasmic inclusions, lysosomal copper deposits, glycogenated periportal hepatocytic nuclei, steatosis, and Mallory bodies. All of the above features may be suggestive of Wilson disease when observed together, in corresponding clinical setting. None of the described ultrastructural observations is diagnostic as an isolated finding. More about the role of liver pathology in clinical symptoms and diagnosis of WD is given in Chapters 9 and 14.

SUMMARY Since the hepatolenticular degeneration is considered a treatable disease, one message should be kept in mind: any acute or chronic liver injury of unknown origin presenting any kind and degree of histologic damage, particularly in a child or young person, should raise the suspicion of Wilson disease and prompt proper differential diagnostic action. This is particularly important when liver disease coexists with neuropsychiatric symptoms.

REFERENCES Arroyo M, Crawford J (2009). Pediatric liver disease and inherited, metabolic, and developmental disorders of the pediatric and adult liver. In: R Odze, J Goldblum (Eds.), Surgical Pathology of the GI tract, Liver, Biliary Tract and Pancreas, Saunders Elsevier, Philadelphia, pp. 1281–1283. Desmet V, Rosai J (2012). Liver, non-neoplastic diseases, tumors and tumorlike conditions. In: J Rosai (Ed.), Rosai and Ackerman’s Surgical Pathology, Mosby Elsevier, Philadelphia, PA, p. 899. Phillips M, Poucell S, Patterson J et al. (1987). The liver: an ultrastructural atlas and text of liver diseases, Raven Press, New York, pp. 255–256. Thompson R, Portmann B, Roberts E (2012). Genetic and metabolic liver disease. In: A Burt, B Portmann, L Ferrell (Eds.), Mac Sween’s Pathology of the Liver, Edinburgh, Churchill Livingstone Elsevier, pp. 203–208. Torbenson M (2015). Biopsy interpretation of the liver, Wolters Kluwer, Philadelphia, pp. 234–327.

Handbook of Clinical Neurology, Vol. 142 (3rd series) Wilson Disease A. Członkowska and M.L. Schilsky, Editors http://dx.doi.org/10.1016/B978-0-444-63625-6.00008-2 © 2017 Elsevier B.V. All rights reserved

Chapter 8

Wilson disease: brain pathology
AURELIA POUJOIS1, JACQUELINE MIKOL2, AND FRANCE WOIMANT1* French National Reference Centre for Wilson Disease, Neurology Department, Lariboisière Hospital, Paris, France

1

2

Pathology Department, Paris Diderot University, Paris, France

Abstract In Wilson disease (WD), brain cellular damage is thought to be due to copper deposition. Striatal lesions are the most characteristic lesions found in the brain of patients with neurologic symptoms, as emphasized in the initial reports of S.A.K. Wilson. WD brain lesions can be more diffuse, including in the pons, midbrain, thalamus, dentate nucleus, and, less frequently, corpus callosum and cortex. In rare cases, extensive cortical-subcortical lesions have been reported. Increased cellularity is noted in the lesions due to the proliferation of modified astrocytes named Alzheimer types of glia and specific cells, called Opalski cells, that are characteristic of WD. Although abnormalities in the putamen predominate in patients with dystonic syndrome, clinicopathologic correlations are scarce. Furthermore, the cerebral copper content is not correlated with the severity of the neuropathologic abnormalities or with the neuropsychiatric symptomatology. This fact raises the question of factors other than copper toxicity that may contribute to the pathogenesis of WD neurologic disturbances.

INTRODUCTION S.A.K. Wilson, in his seminal report in Brain (1912), had extensively described the pathologic data of six cases published in the literature (Gowers, Ormerod, H€ omen, and three of his own). He also showed that the main lesion from “softening” to cavitation was in the putamen and that the globus pallidus and caudate nucleus could be involved but never to the same extent. In three cases, the central nervous system (CNS) was not affected. Furthermore, even an advanced degree of cirrhosis did not give rise to neurologic symptoms during life. Also Von Hesslin and Alzheimer reported a case of Westphal– Str€ umpell pseudosclerosis with liver cirrhosis characterized by loss of nervous tissue and abnormalities of astrocytes, subsequently referred to as Alzheimer types of astroglial cells (Von Hoesslin and Alzheimer, 1912). As early as 1914, according to Bostroem, the triad of juvenile multilobular cirrhosis, gait disturbances, and necrotic abnormalities of extrapyramidal systems was considered as the basis of a separate disorder (see review

in Boise et al., 2011) which became known as the hepatolenticular degeneration (Hall, 1921). In 1920, Spielmeyer emphasized the links between pseudosclerosis and Wilson’s cases and the presence in both disorders of the modified astrocytes that he named Alzheimer types of glia. In 1930, Opalski described a “special type of nervous cells,” now designated by his name. Extensive reviews (Hall, 1921; Greenfield et al., 1924; Konovalov, 1960; Denny-Brown, 1962; Schulman, 1968; Shiraki, 1968; Brewer and Yuzbasiyan-Gurkan, 1992; Meenakshi-Sundaram et al., 2008) and monographs (Boudin and P
epin, 1959; Scheinberg and Sternlieb, 1984; Hoogenraad et al., 1996) on these descriptions were subsequently published. The inherited nature of the disease as autosomal recessive was established. It was demonstrated that the brain lesions extended beyond the putaminal formation, involving the cerebral cortex, brainstem, and dentate nucleus. An excess of copper, already found in the cornea by Fleischer, was shown to be present in the brain

*Correspondence to: France Woimant, French National Reference Centre for Wilson Disease, Neurology Department, Lariboisière Hospital, Assistance Publique-H^opitaux de Paris APHP, 2 rue Ambroise Par
e, 75010 Paris, France. Tel: +33-1-49-95-65-27, E-mail: [email protected]

78

A. POUJOIS ET AL.

(Haurowitz, 1930; L€ uthy, 1931; Glazebrook, 1945; Cumings, 1948). Rather than hereditary hepatocerebral degeneration of Wilson–Westphal–Str€ umpell, the disease is now known as Wilson disease (WD), especially since cerebral involvement may be missing, particularly in hepatic forms of the disease. However, as early as 1914, Van Woerkom had reported an adult case that presented with tremor, somnolence, emotional instability, and liver cirrhosis. At postmortem examination, loss of nerve cells and gliosis were noted in the striatum without a destructive lesion in the putamen. This case and the following ones questioned for a long period the relationships of this disorder with WD. Subsequently, large series were reported (Victor et al., 1965; Stadler, cited in Adams, 1968). This led to the recognition of acquired hepatocerebral degeneration (AHCD) (see review in Adams, 1968), secondary to acute and chronic liver disease. Characteristics of the disease were fluctuating intellectual decline, intention tremor, lack of familial history, absence of Kayser–Fleischer ring, absence of copper storage, and no cavitation in the lenticular nuclei (see section on differential diagnosis, below).

PATHOLOGIC CHANGES Since the initial report of S.A.K. Wilson, no main change has been noted in the classic form, but rare subgroups have been segregated that we will describe in detail separately. On gross examination, the brain is not modified; sometimes it is slightly atrophic in long-lasting cases but it is not seen in hepatic forms of WD. The ventricles may be enlarged with flattening of the convexity of the head of the caudate nucleus. The main lesion is located in the middle zone of each putaminal nucleus, which becomes a brown-yellowish color. The nucleus appears atrophic, shrunken, and crumbly or, in most untreated patients, a cavitation occupies the central and anterior part of the nucleus; it is elongated and flat in vertical sections and more extensive in horizontal sections, extending close to the head of the globus pallidus and external capsule. On average the cavitation is 1 cm wide and 1–2.5 cm long (Fig. 8.1). In Wilson’s original report this lesion was described as a softening. In rare forms, cavitations may also be found in the thalamus (Wilson, 1912), subthalamic nucleus, red nucleus, dentate nucleus (Howard and Royce, 1919; Frets, 1938), cerebellar cortex (Schulman and Barbeau, 1963), and the white matter (WM) (Dymecki, cited in Hoogenraad et al., 1996). Central pontine myelinosis (CPM) (see review in Castan, 1967; MeenakshiSundaram et al., 2008) is frequently recognized after myelin staining and softening and/or cavitation of the WM (see reviews in Richter, 1948; Scheinberg and

Sternlieb, 1984; Mikol et al., 2005; MeenakshiSundaram et al., 2008) (Fig. 8.2). The lesions of the WM had already been described by H€omen and Anton (both cited in Wilson, 1912). Most cases were reported before 1970 in untreated patients, but they are also present in a few treated patients (Mikol et al., 2005; Meenakshi-Sundaram et al., 2008). The brain is soft with marked loss of superficial and deep WM and relative preservation of the superficial cortex (Fig. 8.1). The lesions involve primarily the frontal lobe, sometimes asymmetrically, but also the temporal lobe and, to a lesser degree, the parietal and occipital lobes, corona radiata, corpus callosum (Trocello et al., 2011), cerebellum, and optic radiations (Meenakshi-Sundaram et al., 2008). In one case almost total loss of cerebral WM was observed (Schulman and Barbeau, 1963). These lesions may be multifocal and diversely associated and are concomitant with basal ganglia involvement. In 10% of cases, WM changes may be more marked than those of the basal ganglia (Scheinberg and Sternlieb, 1984). Most of these lesions are only visualized by magnetic resonance imaging (MRI) (Starosta-Rubinstein et al., 1987; Prashanth et al., 2010; Trocello et al., 2013; Hermann, 2014). The first report of bilateral hypertrophic olivary degeneration, diagnosed by MRI, has been reported (Otto et al., 2013). On microscopic examination, there are several changes of different degrees in the putamen. A disintegration of the tissue appears to be the first step. In advanced cases, tiny foci of status spongiosus become confluent, resulting in a cavity formation (Fig. 8.3 A, B). The edges of the cavity are irregular and devoid of glial scaring. “Nerve cells and fibers which show little evidence of pathology may persist and in this way, such cavitation may be distinguished from ischemic infarction” (Denny-Brown, 1962). However, in many cases, regressive changes, such as shrinkage, pyknosis, and apoptotic nuclei, are present in small and/or large neurons of the putamen and infrequently in other involved structures. The myelinated fibers are less distinguishable. Spheroids are abundant in the lower brainstem sensory nuclei (Gallyas and K€ornyey, 1968). Increased cellularity is noted in the lesions due to the proliferation of astrocytes. The two types of astrocytes described by Von H€oblin and Alzheimer are present but their frequency is quite variable. In Alzheimer type 2, the nucleus is swollen with little chromatin, and the nuclear membrane is faint and segmented and may appear as rumpled, giving the impression of multiple nuclei; the cell has little cytoplasm. These astrocytes are now considered as evocative but not specific of hepatic encephalopathy. Type 1 astrocytes are labeled by glial fibrillary acidic protein

WILSON DISEASE: BRAIN PATHOLOGY

79

Fig. 8.1. Computed tomography (CT) scan and gross neuropathologic findings. (A) Brain CT scan: diffuse hypodensity in frontal lobes. (B) Gross neuropathologic findings: cavitation of the upper part of both frontal lobes (W€ oelcke myelin staining). (C) Coronal section showing diffuse pallor of the frontal white matter, softening and light-brown discoloration of the inferior part of the putamen. (D) Necrosis of the frontal white matter without preservation of the U fibers at the top of the gyri and limited destructive lesion of the putamen. (Hematoxylin and eosin.) (Reproduced from Mikol et al. (2005), with permission from Springer Science and Business Media.)

(GFAP), S-100 protein and metallothioneins (MTs) (Kimura and Budka, 1986; Mikol et al., 2005); they may be related to reactive astrogliosis (Ma et al., 1988). A positive reaction for copper has been observed in the cytoplasm of these cells (Bertrand et al., 2001). Type 2 astrocytes are reduced to the swollen nuclei with a nucleolus adjacent to the membrane on routine staining; they show immunoreactivity for S-100 protein but not for GFAP, and some lack both markers (Kimura and Budka, 1986) (Figs 8.3D and 8.4C). Using anti-MT antibodies, the cell cytoplasm is visualized but not the nuclei (Bertrand et al., 2001; Hoogenraad, 2001) (Fig. 8.4E). The topographic distribution of these cells has been intensively described in case reports, but no feature emerges from these reports, except that they are more frequent in gray matter and may vary in number. Oligodendrocytes are rarefied when the tissue is destroyed and macrophages are present. They are sometimes mixed with a few perivascular inflammatory

lymphoid cells. Opalski cells (OCs) are characteristic of WD. They are large cells up to 35–40 mm in diameter. Their cytoplasm is foamy with periodic acid–Schiff staining granules; the nucleus, when visible, is frequently eccentric. They are absent in hepatic forms of the disease (Meenakshi-Sundaram et al., 2008). OC origin is still a matter of debate: they are derived from an astrocytic GFAP-positive (Scheinberg and Sternlieb, 1984; Kimura and Budka, 1986), CD68-negative (MeenakshiSundaram et al., 2008), MT-positive (Hoogenraad, 2001), or histiocytic (Greenfield et al., 1958), CD68positive, ubiquitin-positive and IL-1-positive (Mikol et al., 2005) cell line (Fig. 8.4 F–I). Ramified and rod microglia are present in the white and gray matter (Wierzba-Bobowicz et al., 2002). The same lesions can occur, never to the same extent, in the globus pallidus, caudate nucleus, thalamus, subthalamic nucleus, dentate nucleus, midbrain, and spinal cord. Glial reaction, usually of type 2, and OC are frequently recognized in these areas

80

A. POUJOIS ET AL.

Fig. 8.2. Gross neuropathologic findings of the cerebellum, pons, and spinal cord. (A) Horizontal section showing spongy degeneration of the white matter in the lateral part of the cerebellum (arrow). (B) Horizontal section showing patchy degeneration of the Goll fasciculi at the level of the upper part of the first cervical spinal cord. (C, D) W€ oelcke staining showing two different aspects of central pontine myelinolysis.

A

D

B

50 µm

E

C

50 µm

F

25 µm

Fig. 8.3. Microscopic findings in the putamen of treated patients. Parts A, B, and C emphasize the three main stages of putaminal involvement. (A) Severe long-lasting hepatocerebral form of the disease: vacuolization of the tissue (hematin-phloxineLuxol staining). (B) Classic form of the disease: spongiotic foci (Masson trichrome staining). (C) Patient cured of neurologic symptoms, dead of cirrhosis: faint residual loss of parenchyma (hematoxylin and eosin staining) (Boudin et al., 1968). (D) Alzheimer type 2 astrocytes (arrow) (Hematoxylin and eosin staining). (E) Small iron deposits (Perls staining). (F) Copper deposits (diethyldithiocarbamate staining).

WILSON DISEASE: BRAIN PATHOLOGY

81

Fig. 8.4. Microscopic findings in a case of white-matter involvement. (A) White-matter coalescent spongiotic foci (hematoxylin and eosin staining). (B) Demyelination of the frontal cortex and the underlying white matter (W€ oelcke). (C) White-matter myelin and axonal loss; presence of an Alzheimer type 1 astrocyte (Bodian-Luxol staining). (D, E) Immunolabeling of white-matter astrocytes with antiglial fibrillary acidic protein (D) and antimetallothionein (E). (F) Opalski cell in white matter (hematoxylin and eosin staining). (G) Immunolabeling of Opalski cell with anti-CD68. (H) Opalski cell with antiubiquitin. (I) Immunolabeling with anti-interleukin-1. (Reproduced from Mikol et al. (2005), with permission from Springer Science and Business Media.)

associated with capillary proliferation. The cerebellum may be involved and, especially, the WM with or without extension to the dentate nucleus and/or the cerebellar cortex (Schulman and Barbeau, 1963) (Fig. 8.2A). CPM is located in the middle and rostral portions of the pons. The lesion is triangular in shape or transverse and is rarely cavitary (Fig. 8.2C, D). Histologically, the myelinated fibers appear discolored in the lesion where astrocytes, OC, and foamy macrophages are present. Usually pontine nuclei are preserved. CPM has been described in patients with severe liver disease (Adams et al., 1959). Most of the time it is related to a focal damage to brain myelin that occurs following brain disturbances and rapid correction of hyponatremia (Messert et al., 1979).

Accordingly, it could be considered to proceed from a similar mechanism in WD. However, its frequency in neurologic forms, treated or untreated, justifies its inclusion in the disease, although its origin remains unknown. WM lesions extend beyond the U fibers. They consist of small, coalescent spongiotic foci of the WM and the deep cortex forming cystic necrosis (Figs 8.1 and 8.4A–D). In the deep layers of the cortex, a few interneurons are still present (Mikol et al., 2005). The superficial cortex is better preserved. Myelin is destroyed and a few axons may be seen, leading to the description of a “demyelinating type of WD” (Miyakawa and Murayama, 1976) (Fig. 8.4B). Astrocytes and OC are present in the cortex and the WM and there is some degree of capillary proliferation.

82

A. POUJOIS ET AL.

These lesions are prominent in patients with neurologic manifestations and absent in patients with hepatic forms of WD. Neuropathologic studies of a few treated cases have been reported (Boudin et al., 1968; Horoupian et al., 1988). The neurologic symptoms of the patient published by Boudin et al. (1968) were cured: only very tiny spongiotic foci, limited to the putamen, and a few macrophages with hemosiderin pigments and glial cells were found (Fig. 8.3C). Seventeen patients treated for as long as 17 years were described in the series of Horoupian et al. (1988): 5 of them had complete resolution of neurologic symptomatology. Abnormal glial cells were seen in all of the brains; gross or microcavitary changes were present in the putamen of 8 patients. The authors found a correlation between the severity of the neuropathologic findings and the cerebral copper content (Horoupian et al., 1988). No post liver transplantation neuropathologic reports have been published to our knowledge. Ultrastructural studies are very limited and mostly restricted to previous old cases (Foncin, 1970; Anzil et al., 1974). In a frontal biopsy, spheroids were abundant, randomly distributed in the neuropil, and associated with Hirano bodies (Anzil et al., 1974). Neurons had excessive amounts of lipofuscin and intranuclear rod inclusions. Protoplasmic astrocytes occurred together with a low-density cytoplasm (Foncin, 1970; Anzil et al., 1974). An additional elongated cell type with protoplasmic processes was described as a “neuropil-based non-neuronal element” (“M”) or “intermediate cell” (Mossakowski et al., 1970). It contained cytoplasmic vacuoles filled with a flocculent material. In the nucleus, heterochromatin was clumped beneath the nuclear envelope. Recent data, involving immunohistochemistry, did not elicit this category of cells. Studies of postmortem tissue did not elucidate,but may show the morphology of the nuclei (Fig. 8.5). It is noteworthy that the simple eponym “Alzheimer protoplasmic astrocyte” was considered as characteristic of WD, according to Anzil et al. (1974). Activated microglial cells were also present in WD. They showed traits of indirect forms between activated microglial rods, ramified, and ameboid cells (Lewandowska et al., 2004). None of these data appeared to be specific. Likewise, correlations may be established with hepatic encephalopathy observed in patients treated with portocaval shunts (see below). In the biopsy, reported by Foncin (1970), astrocytes were enlarged; they showed cavities in the cytoplasm, 1–2 mm in diameter, without limiting membrane, glycogen, and many ribosomes. The astrocytic processes were modified close to the impaired swollen neurons. Lesions of astrocytes would precede, with neuronal involvement being secondary. According to Gruner (personal communication), this was comparable to what is noted in WD.

Fig. 8.5. Electron micrograph of type 2 astrocyte showing an infolded nucleus. Uranyl acetate-lead citrate staining. Original magnification
4320. Putaminal postmortem specimen.

Peripheral nerves have been rarely examined as their clinical involvement is not frequent. Abnormalities are mostly observed with the electron microscope. Myelin sheaths exhibited myelin fragments and/or membranous structures and a few denuded axons (Miyakawa et al., 1973). Tubulostructures and dense neurofilaments were observed in the Schwann cell cytoplasm and in some axons. This damage is characteristic of a primary demyelination with secondary change in the axons.

COPPER IN THE BRAIN Distribution of copper BRAIN COPPER IN NORMAL CONDITION Found in all living organisms, copper is an important microelement that is essential for enzymes. It participates in numerous physiologic processes, including mitochondrial respiratory chain, neurotransmitter synthesis, and iron metabolism. It has a key role in the development and function of the nervous system. Copper is too small to be seen with classical transmission microscopy. Many biochemical studies have shown a diffuse accumulation of copper in different parts of the brain, even devoid of apparent lesions. Different histologic methods have been used to visualize the metal, mostly without success, even on frozen sections (Okamoto and Utamara rubeanic acid, Okamoto rhodanin, Feigl benzidine, Waterhouse diethyldithiocarbamate, Shikata modified orcein) (Fig. 8.3 F). So far, dosage of copper remains the best method. The concentration of copper is highest in the human brain after that in the liver (Lech and Sadlik, 2007). Its estimated value is grossly 7–10% of total body copper, which corresponds to variable mean concentrations of 2.9–10.7 mg/g wet weight (Rongzhu et al., 2009) or 13–60 mg/g of dry tissue (Faa et al., 2001). Therefore,

WILSON DISEASE: BRAIN PATHOLOGY 83 the mean value of copper estimated from 14 brain areas in 10–15-fold than in normal conditions, but such a wide 42 normal subjects in a recent study by Ramos et al. range depends on different brain areas. A fair degree (2014) was 22  5 mg/g (range 10–37 mg/g) of dry tissue. of correlation has been found between the severity of Normal copper distribution in the brain is largely hetthe neuropathologic results and cerebral copper content erogeneous. In humans, general distribution of copper is (Horoupian et al., 1988), but not always between copper not easy to determine with the previous sensitive method concentration and the clinical picture (Faa et al., 2001; Litwin et al., 2013). If imaging studies have shown that due to the size of the human brain. So copper concentramany brain structures may be involved in symptomatic tion studies rely on comparisons of different brain areas patients, the most common lesion is observed in the basal instead of a global analysis. Most studies have demonganglia (Sinha et al., 2006). This brain region vulnerabilstrated that copper concentration in the human brain is ity is usually explained by its high metabolic rate, on average higher in gray matter than in the WM increased blood supply, and mitochondrial contents, (Cumings, 1948; Bush et al., 1995). An autopsy study which make it more sensitive to toxins and metal accumade in 27 normal human brains examined the copper content of cerebral gray matter, WM, and basal ganglia mulation (Belz and Mullins, 2010). using the sodium diethyldithiocarbamate method. The The first neuropathologic study of two cases of WD patients found high copper content in the basal authors found an average level of 3.33 mg/g (range ganglia – 28 and 31 mg/g wet weight (Haurowitz, 1.1–7.2 mg/g) for cortical gray matter and 3.06 mg/g 1930) – whereas Glazebrook (1945) in one case of dry weight (range 1.2–8.2 mg/g) for WM. With regard WD found 12.7 mg of copper/g wet weight in the basal to copper in basal ganglia examined in six normal ganglia and 7.8 mg of copper/g wet weight in the cortex. brains, globus pallidus had the highest concentration More recently, Faa et al. (2001) studied brain copper, (10.5–18.8 mg/g), then the putamen (6.1–12 mg/g), iron, magnesium, calcium, zinc, and phosphorus storage caudate nucleus (3.4–9.4 mg/g), and thalamus (3.1– in 28 brain samples of a WD patient who died of acute 12.4 mg/g) (Cumings, 1948). Substantia nigra and denhepatic failure without any neurologic sign but had a tate nucleus demonstrated also very high metal ion concentration, frequently higher than in the basal ganglia bilateral Kayser–Fleischer ring. The trace elements were (Krebs et al., 2014). In contrast, a study by Mikol et al. determined by ICP-AES. The authors showed that the (2005) in two normal brains, using inductively coupled mean copper concentration in the brain was markedly plasma atomic emission spectrometry (ICP-AES), found increased (125  19 mg/g of dry tissue; range that the copper concentration was higher in the WM than 82–167 mg/g) and that copper was unevenly distributed in the gray matter, with values between 17.1 and with marked differences even among adjacent areas 18.4 mg/g dry weight in the frontal lobe and 7 mg/g in and between the cerebral hemispheres. The lowest conthe putamen. Table 8.1 gives a review of published copcentrations were in the periventricular areas and the highper concentrations in normal brain. est in the subthalamic regions. Copper appeared also Copper correlation with age is not clear. Zatta et al. differently stored in the symmetric WM regions. In this (2005) showed that copper concentration rises with age study, no correlation was found between the increased and V€ olkl et al. (1974) observed that copper rose during copper content and the macroscopic and histologic the juvenile years in the gray matter whereas changes in lesions. Furthermore, the increase of copper concentraWM remained inconspicuous. They described typical tion was associated with depletion of all the other trace regional distributions that developed until this period elements studied (iron and magnesium were 20-fold and remained stable afterward with copper values indelower than reference values, zinc and calcium 10 times pendent of age between the fourth and seventh decades of lower, as well as phosphorus), which may suggest that life. But two others studies found a negative correlation other factors enhance copper toxicity. between brain copper levels and age in patients older Another recent analysis of metal accumulation in a than 50 (Bonilla et al., 1984; Ramos et al., 2014). cohort of 12 brains from WD patients (10 neuropsychiCopper concentration increases in neurodegenerative atric and two hepatic forms, with 2.4  2.3 years of disdiseases like Alzheimer’s disease and in amyotrophic lateral ease duration) showed that copper was increased almost sclerosis, where CNS copper was reported to be twofold eight times in WD brains compared to controls (41 vs. higher than in age-matched controls (Ahuja et al., 2015). 5.4 mg/g of dry tissue), with homogeneous copper accumulation in the examined structures (putamen, pons, dentate nucleus, and frontal cortex). The putamen had BRAIN COPPER IN WD the highest copper concentration (49.7  28 mg/g) and The copper content in the brain of WD patients with neuthe frontal cortex the lowest (34.3  17.5 mg/g). rologic symptoms is elevated with heterogeneous reparA positive correlation between the disease duration tition. Copper concentration has been reported to be and copper content in the putamen was found (r ¼ 0.61

Table 8.1 Review of copper concentrations in normal brain

Authors

Year Methods

Number of normal Number of human brains brain areas

Age (years)

Cumings Warren et al.

1948 AAS 1960 AAS

6 9

6 26

–

Harrison et al.

1968 AAS

1

10

–

V€ olkl et al. Smeyers-Verbeke et al. Goldberg and Allen Bonilla et al.

1974 AAS 1974 AAS

33 11

13 13

0–80 –

1981 AAS

3

6

–

1984 AAS

7

38

Riederer et al.

1989 AAS

4

8

Duflou et al. Andrasi et al.

1989 PIXE 1990 ICP-AES and INAA 1991 ICP-MS 1994 ICP-AES

3 11

46 12

Olfactory bulb > nucleus caudate > postcentral gyrus > cuneus > precentral gyrus > mammillary bodies > pineal gland > optic nerve > substantia nigra > cerebellum > putamen > globus pallidus > thalamus 73 (68–78) Substantia nigra > nucleus caudate > putamen > globus pallidus > cingulate gyrus > red nucleus > amygdala > reticular formation 7, 15, 69 Cerebellum > putamen > nucleus caudate > optic chiasma > tractus corticospinalis > corpus fornicis 65–75 Putamen > cerebral cortex > thalamus > corpus callosum

34 12

6 7

Andrasi et al. Deibel et al. Rajan et al. Rahil-Khazen et al. Mikol et al. Ramos et al.

1995 1996 1997 2002

ICP-AES INAA ICP-AES ICP-AES

20 11 8 30

10 7 12 2

75.5  2.7 Substantia nigra > cerebellum > putamen > nucleus caudate > cerebral cortex 41–91 Nucleus caudate > hypothalamus > cortex > amygdala > cingulate gyrus > corpus mammillare > hippocampus 70 Putamen > globus pallidus > nucleus caudate > occipital area > hippocampus 81 1,7 Cerebellar cortex > temporal and parietal lobes > amygdala > hippocampus Cerebellar cortex > cerebral cortex > hypothalamus > thalamus 50–60 17–87 Cerebellar cortex > frontal lobe

2005 ICP-MS 2014 ICP-AES

2 42

2 14

– 50–101

Krebs et al.

2014 ICP-MS

11

13

48–81

Dexter et al. Kornhuber et al.

Copper concentration: from highest to lowest Globus pallidus > putamen > nucleus caudate > cortical gray matter > thalamus > cortical white matter Locus coeruleus > substantia nigra > nucleus dentatus > parietal cortex > corpus callosum > pons > optic chiasma Cerebellar cortex > putamen > nucleus caudate > corpus callosum > thalamus > frontal and cerebellar white matter Substantia nigra > occipital cortex > neostriatum > nucleus dentatus > frontal cortex > thalamus Cerebellar cortex > occipital cortex > basal ganglia > frontal and temporal cortex > thalamus > central ovale > capsula interna > brainstem > corpus callosum Substantia nigra > > putamen > nucleus caudate > globus palllidus > cerebral cortex > hippocampus

11–75

Frontal lobe > putamen Putamen > inferior parietal lobule > nucleus caudate > midbrain > middle temporal gyrus > globus pallidus > cerebellum > occipital cortex > frontal cortex > cingulate gyrus > hippocampus > pons > medulla Substantia nigra > putamen > nucleus caudate > occipital cortex > globus pallidus > red nucleus > thalamus > frontal white matter > pons > corpus callosum

AAS, atomic absorption spectroscopy; PIXE, particle-induced X-ray emission analysis; ICP-AES, inductively coupled atomic emission spectrometry; INAA, instrumental neutron activation analysis; ICP-MS, inductively coupled plasma mass spectrometry.

WILSON DISEASE: BRAIN PATHOLOGY and p ¼ 0.03), whereas gender, phenotype presentation, type, and duration of WD treatment did not correlate with the metal accumulation (Litwin et al., 2013). In a few patients with severe neurologic presentations marked by epilepsy or cognitive impairment, WM overload of copper can dominate (Horoupian et al., 1988; Mikol et al., 2005). Indeed, a study by Mikol et al. (2005) in two WD brains, using ICP-AES, found that the copper concentration was slightly higher in the WM frontal lobe than in the gray matter (putamen), but the 2 patients had severe cortical involvement with cognitive decline and brain MRI cortical lesions. Molecular mechanisms of copper neurotoxicity include oxidative/nitrosative stress (free copper catalyzes the generation of hydroxyl radical, the most active reactive oxygen species), N-methyl-D-aspartate receptormediated excitotoxicity and inflammatory processes (see Chapter 5).

Brain localization of ATP7A and ATP7B ATP7A is widely present in the brain. Highly expressed in the endothelial cells of the blood–brain barrier (BBB) and the choroid plexus, it is also present in most CNS cells. In transgenic mice that overexpressed human ATP7A, the protein is primarily produced in the CA2 region of the hippocampus, the Purkinje neurons of the cerebellum, and the choroid plexus (Niciu et al., 2006). In human brain parenchyma, it is expressed in both neurons and nonneuronal cells, prominently in the cerebellum, the substantia nigra, and on the basolateral surface of the choroid plexus cells (Davies et al., 2013). ATP7A expression is developmentally regulated with a widespread expression in neurons and ependymal cells during embryonic and postnatal development in the mouse. The increase in ATP7A during the early postnatal period indicates a crucial role for copper during early development, and particularly in synaptogenesis and axon extension (Telianidis et al., 2013). ATP7B is expressed in many brain regions but its expression patterns and contribution to brain copper homeostasis are less clear than for ATP7A. In adult mice, it is observed in brain capillary endothelial cells, apical surface of choroidal epithelial cells, ependymal cells, hippocampus, granular cells of the dentate gyrus and pyramidal cells of the CA1 to CA4 layers, olfactory bulbs, Purkinje neurons of the cerebellum, pyramidal neurons of the cerebral cortex, and in several nuclei (pontine nuclei and lateral reticular nuclei) of the brainstem. In these brain regions, ATP7B correlates with copper distribution as determined by staining with the copper chelator bathocuproine disulfonic acid (Saito et al., 1999; Barnes et al., 2005; Niciu et al., 2006; Zheng and Monnot, 2012; Davies et al., 2013). In the human brain,

85

immunohistochemical staining reveals expression of the ATP7B protein in the visual cortex, cingulate cortex, caudate nucleus, putamen, substantia nigra, and cerebellum, with the most significant levels of ATP7B detected in the Purkinje neurons, cingulate cortex, caudate nucleus, and putamen (Davies et al., 2013). No correlation between ATP7A and ATP7B protein levels and copper levels in the brain regions investigated was found in this study. It is hypothesized that ATP7B expression begins postnatally, as brain copper levels in ATP7B–/– mouse continue to increase slightly throughout adult life (Telianidis et al., 2013). In contrast to ATP7A, there is a continuous ATP7B expression in the adult mouse cerebellar Purkinje neurons, and an age-dependent downregulation (Barnes et al., 2005).

Astrocytes: a key cell in brain copper balance Astrocytes, a subset of glial cells, have essential functions in the brain: they have a key role in extracellular ion homeostasis, metabolic supply to neurons, maintenance of the BBB, modulation of synaptic transmission and synaptic plasticity, and the defense of the brain against oxidative stress and toxins (Parpura et al., 2012; Scheiber et al., 2014). Moreover, their strategic location in the brain, between the endothelial cells of brain capillaries and the neurons of the brain parenchyma, allows them to be the first brain cells to encounter metal ions that cross the BBB (Mathiisen et al., 2010). Numerous studies on cell culture models have demonstrated that astrocytes are key regulators in the homeostasis of copper, as they efficiently take up, store, and export this metal in the brain. They protect other brain cells against copper toxicity but provide also copper to neurons and other neighboring cells in the brain (Tiffany-Castiglioni et al., 2011; Scheiber and Dringen, 2013; Dringen et al., 2013). The copper machinery in astrocytes is very similar to that in other brain cells (see Chapter 5).

IRON, MANGANESE, AND OTHER CAUSATIVE FACTORS An increasing number of studies suggest that iron metabolism is disturbed in WD. In WD postmortem studies, increased levels of iron have been reported in the striatum, gray matter, WM (Cumings, 1948), and the dentate nuclei of the cerebellum (Litwin et al., 2013). Small iron deposits are present in the softened areas of the putamen (Fig. 8.3E). According to Dringen et al. (2013), they are especially found in astrocytes. During the lifetime of WD patients, MRI studies documented T2 hypointensities in globus pallidus (Skowronska et al., 2013) and in other brain regions (caudate nucleus, putamen, thalamus, substantia nigra, and red nucleus) (Yang et al., 2015). The

86

A. POUJOIS ET AL.

exact nature of these deposits with paramagnetic properties is still unclear, but they could mainly be caused by iron deposition. Indeed, a positron emission tomography study with radioactively labeled iron in human subjects found that cerebral iron uptake was significantly higher in WD patients compared to healthy volunteers (Bruehlmeier et al., 2000). A combined neuropathologic and MRI study in postmortem WD brain tissue confirmed that iron accumulates in the lentiform nucleus of WD with neuropsychiatric presentation and that iron content in the putamen and globus pallidus correlates with the hypointensities on T2* MR images (Dusek et al., 2016). Factors contributing to iron accumulation in WD remain to be discussed. The consequences of the mutated ATP7B gene in WD patients are decreased ceruloplasmin (which is a link between copper and iron metabolism) and impaired incorporation of copper into enzymes. Copper-dependent enzymes play an important role in iron metabolism. Treatment could also explain brain iron accumulation during the course of WD. D-penicillamine chelation does not remove iron from the organism; conversely, it may lead to higher tissue iron accumulation because it decreases the availability of copper for ceruloplasmin production (Dusek et al., 2015). Unlike D-penicillamine, triethylenetetramine chelates copper, iron, and manganese. Manganese may also accumulate in WD brain. Liver dysfunction may lead to manganase accumulation in the CNS. Bilateral, symmetric hyperintensities of the globus pallidus on a T1-weighted sequence are reported in patients with chronic liver disease; there is a significant relationship between these MRI changes and the severity of the liver disease, and the presence and degree of portosystemic shunting of blood. These T1 hyperintensities are suggestive of manganese deposits (Klos et al., 2006; Sinha et al., 2007).

DIFFERENTIAL DIAGNOSIS Acquired hepatocerebral degeneration The differential diagnosis of WD is AHCD (Victor et al., 1965). On gross examination, a patchy brown band of necrosis is present at the junction between the cortex and the WM. It is distributed through the hemisphere with predominance in the parietal and occipital lobes. Few vascular lesions may be associated. Histologically, the lesion is made of small focal coalescent vacuoles easily visualized on myelin staining. The vacuoles appear empty or filled with macrophages with or without oligodendrocytes and astrocytes. The myelin sheaths are disrupted. Small spongiotic foci are seldom present in the basal ganglia, but never to the same degree as in WD. Neuronal loss and degeneration of parenchymal elements are mostly distributed in the cortex, the basal

ganglia and, also, in the thalamus, cerebellum, dentate nuclei, and infrequently in the brainstem. The most remarkable lesion is a massive, diffuse, reactive astrocytosis predominant in the cortex. Some of these glial cells have glycogenic nuclei. Astrocytes, of Alzheimer type, show the same characteristics as in WD; they are now considered to be symptomatic of hepatic encephalopathy. However, using perfused material of an experimental rat model of hepatic encephalopathy, Norenberg and Lapham (1974) have demonstrated, at ultrastructural level, that nuclear enlargement of type 2 astrocytes is an artefact of immersion fixation. There is hypertrophy and hyperplasia of the cells that exhibit an expanded cytoplasmic compartment associated with proliferation of mitochondria and endoplasmic reticulum. Glial fiber formation is conspicuously absent; it may be correlated with the absence of immunoreactivity of GFAP. The mechanism is supposed to be linked to toxic effect of manganese.

Aceruloplasminemia Aceruloplasminemia is linked to the mutation of the ceruloplasmin gene located on chromosome 3. Ceruloplasmin is a copper-binding oxidase that carries more than 95% of plasma copper. It plays the role of a ferroxidase, which converts ferrous to ferric ions. The clinical picture is associated with severe iron deposition in visceral organs and brain tissues, never observed in WD, at such intensity (Miyajima et al., 1987; Morita et al., 1995). Neurologic dysfunction includes cognitive impairment, cerebellar ataxia, extrapyramidal syndrome associated with retinal degeneration and diabetes. On gross examination, the basal ganglia, thalamus, and dentate nucleus also have rust-brown pigmentation and there is cavitation of the striatum (Morita et al., 1995; Kawanami et al., 1996; Kaneko et al., 2002; Chr
etien et al., 2006). The main lesion consists of iron deposition in the glial and nerve cells, and neuronal loss, mainly in the striatum. Marked loss of Purkinje cells is also noted. The hallmark of the disease is the presence of perivascular, bizarrely deformed astrocytes and numerous grumose or foamy spheroid bodies (Kaneko et al., 2002; Oide et al., 2006). If the macroscopic data of the disease are close to those noted in WD, the histology is different and the mechanisms are supposed to be related to the toxic effects of excess iron.

CONCLUSIONS Neuropathology of Wilson’s disease mostly relies on the description of old cases. Due to the results of efficient treatments, the lesions, if not totally absent, are far less pronounced. The main target is the putamen but lesions extend to the globus pallidus, thalamus, and the

WILSON DISEASE: BRAIN PATHOLOGY subthalamic structures. Lesions are of different degrees: they consist of the destruction of ground substance associated with modifications of astrocytes and presence of OCs. The modifications of astrocytes are comparable to those observed in hepatic encephalopathy. Their dysfunction as a buffer is still unclear and may play a fundamental role in copper toxicity. Iron and manganese may also participate in the neurodegeneration, and specially while acting synergistically with copper. However, some cases appear atypical, with WM lesions and CPM, whose pathogenesis is not yet explained. ATP7A and ATP7B are expressed in many brain regions and contribute to brain copper homeostasis. If copper overload is probably the main acting element, the molecular mechanisms of WD-related neurodegeneration remain essentially uncharacterized. The understanding of these mechanisms is still a matter of research, which is essential to treat patients.

ACKNOWLEDGMENT We thank Emeline Ruano for her excellent technical assistance in the preparation of this manuscript.

REFERENCES Adams RD (1968). Acquired hepatocerebral degeneration. Vinken and Bruyn Handbook of Clinical Neurology, Vol 6 Chap 12, Elsevier (North-Holland), Amsterdam, 279–297. Adams RD, Victor M, Mancall EL (1959). Central pontine myelinolysis: a hitherto undescribed disease occurring in alcoholic and malnourished patients. AMA Arch Neurol Psychiatr 81 (2): 154–172. Ahuja A, Dev K, Tanwar RS et al. (2015). Copper mediated neurological disorder: visions into amyotrophic lateral sclerosis. Alzheimer and Menkes disease. J Trace Elem Med Biol 29: 11–23. Andra´si E, Na´dasdi J, Molnar Z et al. (1990). Determination of main and trace element contents in human brain by NAA and ICP-AES methods. Biol Trace Elem Res 26–27: 691–698. Andra´si E, Farkas E, Scheibler H et al. (1995). Al, Zn, Cu, Mn and Fe levels in brain in Alzheimer’s disease. Arch Gerontol Geriatr 21 (1): 89–97. Anzil AP, Herrlinger H, Blinzinger K et al. (1974). Ultrastructure of brain and nerve biopsy tissue in Wilson disease. Arch Neurol 31: 94–100. Barnes N, Tsivkovskii R, Tsivkovskaia N et al. (2005). The copper-transporting ATPases, Menkes and Wilson disease proteins, have distinct roles in adult and developing cerebellum. J Biol Chem 280: 9640–9645. Belz EE, Mullins ME (2010). Radiological reasoning: hyperintensity of the basal ganglia and cortex on FLAIR and diffusion-weighted imaging. AJR Am J Roentgenol 195: 1–8. Bertrand E, Lewandowska E, Szpak GM et al. (2001). Neuropathological analysis of pathological forms of astroglia in Wilson’s disease. Folia Neuropathol 39: 73–79.

87

Boise P, Wagner A, Steinberg H (2011). Wilson-Krankheit und Westphal-Str€ umpell-Pseudosklerose. Nervenartz 82: 1335–1342. Bonilla E, Salazar E, Villasmil JJ et al. (1984). Copper distribution in the normal human brain. Neurochem Res 9 (11): 1543–1548. Boudin G, P
epin B (1959). D
eg
en
erescence H
epatolenticulaire, Masson, Paris. Boudin G, P
epin B, Milhaud M et al. (1968). Anatomo-clinical study of a case of Wilson’s disease treated with chelating agents for 5 years. Rev Neurol (Paris) 118 (1): 27–35. Brewer GJ, Yuzbasiyan-Gurkan V (1992). Wilson disease. Medicine (Baltimore) 71 (3): 139–164. Bruehlmeier M, Leenders KL, Vontobel P et al. (2000). Increased cerebral iron uptake in Wilson’s disease: a 52 Fe-citrate PET study. J Nucl Med 41 (5): 781–787. Bush VJ, Moyer TP, Batts KP et al. (1995). Essential and toxic element concentrations in fresh and formalin-fixed human autopsy tissues. Clin Chem 41: 284–294. Castan P (1967). The nervous manifestations of chronic uremia. Apropos of certain clinical, E.E.G. and neuropathologic aspects. Presse Med 75 (48): 2443–2447. Chr
etien F, Servan J, Mikol J et al. (2006). A 70-year-old man with extrapyramidal symptoms, dementia and hemosiderosis. Brain Pathol 16 (3): 235–236. Cumings JN (1948). The copper and iron content of brain and liver in the normal and in hepato-lenticular degeneration. Brain 71 (Pt. 4): 410–415. Davies KM, Hare DJ, Cottam V et al. (2013). Localization of copper and copper transporters in the human brain. Metallomics 5: 43–51. Deibel MA, Ehmann WD, Markesbery WR (1996). Copper, iron, and zinc imbalances in severely degenerated brain regions in Alzheimer’s disease: possible relation to oxidative stress. J Neurol Sci 143 (1–2): 137–142. Denny-Brown D (1962). The basal ganglia and their relation to disorders of movements, Oxford University Press, London, pp. 18–52. Dexter DT, Carayon A, Javoy-Agid F et al. (1991). Alterations in the levels of iron, ferritin and other trace metals in Parkinson’s disease and other neurodegenerative diseases affecting the basal ganglia. Brain 114 (Pt 4): 1953–1975. Dringen R, Scheiber IF, Mercer JF (2013). Copper metabolism of astrocytes. Front Aging Neurosci 14 (5): 9. Duflou H, Maenhaut W, De Reuck J (1989). Regional distribution of potassium, calcium, and six trace elements in normal human brain. Neurochem Res 14 (11): 1099–1112. Dusek P, Roos PM, Litwin T et al. (2015). The neurotoxicity of iron, copper and manganese in Parkinson’s and Wilson’s diseases. J Trace Elem Med Biol 31: 193–203. Dusek P, Bahn E, Litwin T et al. (2016). Brain iron accumulation in Wilson disease: a post-mortem 7 Tesla MRI – histopathological study. Neuropathol Appl Neurobiol. http://dx.doi.org/10.1111/nan.12341. Faa G, Lisci M, Caria MP et al. (2001). Brain copper, iron, magnesium, zinc, calcium, sulfur and phosphorus storage in Wilson’s disease. J Trace Elem Med Biol 15 (2-3): 155–160.

88

A. POUJOIS ET AL.

Foncin JF (1970). Pathologie ultrastructurale de la glie chez l’homme. In: Proceedings of the Sixth International Congress of Neuropathology, Masson, Paris, pp. 377–390. Frets GP (1938). Die Wilsonsche Krankheit und die Westphal-Str€ umpellsche Pseudosclerose. Psychiatrische en Neurologische Bladen 24: 310–334. Gallyas F, K€ornyey S (1968). Weiterer Beitrag zur Kenntnis der Hallervorden-Spatzchen Krankheit. Arch Psychiatr Nervenkr 212: 33–45. Glazebrook AJ (1945). Wilson’s disease. Medical Journal; Edinburgh 52: 83–87. Goldberg WJ, Allen N (1981). Determination of Cu, Mn, Fe, and Ca in six regions of normal human brain, by atomic absorption spectroscopy. Clin Chem 27 (4): 562–564. Greenfield JG, Poyton FJ, Walshe FMR (1924). On progressive lenticular degeneration. Quaterly Journal of Medicine 17: 385–404. Greenfield JG, Blackwood W, Mc Menemey WH (1958). Hepatolenticular degeneration Wilson’s disease, Vol. 8 E. Arnold Publishing, London, pp. 521–525. Hall HC (1921). La d
eg
en
erescence h
epato-lenticulaire: maladie de Wilson-pseudoscl
erose, Masson, Paris. Harrison WW, Netsky MG, Brown MD (1968). Trace elements in human brain: copper, zinc, iron, and magnesium. Clin Chim Acta 21 (1): 55–60. Haurowitz F (1930). Zeit Physiol Chemie 190: 72. Hermann W (2014). Morphological and functional imaging in neurological and non-neurological Wilson’s patients. Ann N Y Acad Sci 1315: 24–29. Hoogenraad TU (2001). Wilson’s disease, Intermed Medical Publisher, Amsterdam, pp. 45–70. Hoogenraad TU, Jansen GH, Van Hattum J (1996). Extrahepatic manifestations of hepatitis C virus infection. Ann Intern Med 125 (4): 345–346. Horoupian DS, Sternlieb I, Scheinberg IH (1988). Neuro-pathological findings in penicillamine-treated patients with Wilson’s disease. Clin Neuropathol 7: 62–67. Howard CP, Royce CE (1919). Progressive lenticular degeneration associated with cirrhosis of the liver (Wilson’s disease). Arch Int Med 24: 497. Kaneko K, Yoshida K, Arima K et al. (2002). Astrocytic deformity and globular structures are characteristic of the brains of patients with aceruloplasminemia. J Neuropathol Exp Neurol 61 (12): 1069–1077. Kawanami T, Kato T, Daimon M et al. (1996). Hereditary caeruloplasmin deficiency: clinicopathological study of a patient. J Neurol Neurosurg Psychiatry 61: 506–509. Kimura T, Budka H (1986). Glial fibrillary acidic protein and S-100 protein in human hepatic encephalopathy: immunocytochemical demonstration of dissociation of two gliaassociated proteins. Acta Neuropathol (Berl) 70: 17–21. Klos KJ, Ahlskog JE, Kumar N et al. (2006). Brain metal concentrations in chronic liver failure patients with pallidal T1 MRI hyperintensity. Neurology 67: 1984–1989. Konovalov NV (1960). Hepatocerebral Dystrophy, Russian Academy of Medical Sciences, Moscow. Kornhuber J, Lange KW, Kruzik P et al. (1994). Iron, copper, zinc, magnesium, and calcium in postmortem brain tissue from schizophrenic patients. Biol Psychiatry 36 (1): 31–34.

Krebs N, Langkammer C, Goessler W et al. (2014). Assessment of trace elements in human brain using inductively coupled plasma mass spectrometry. J Trace Elem Med Biol 28 (1): 1–7. Lech T, Sadlik JK (2007). Copper concentration in body tissues and fluids in normal subjects of southern Poland. Biol Trace Elem Res 118 (1): 10–15. Lewandowska E, Wierzba-Bobrowicz T, Kosno-Kruszewska E et al. (2004). Ultrastructural evaluation of activated forms of microglia in human brain in selected neurological diseases (SSPE, Wilson’s disease and Alzheimer’s disease). Folia Neuropathol 42: 81–91. Litwin T, Gromadzka G, Szpak GM et al. (2013). Brain metal accumulation in Wilson’s disease. J Neurol Sci 329 (1–2): 55–58. € L€ uthy F (1931). Uber die hepato-lentikul€are Degeneration (Wilson-Westphal-Str€ umpell). Dtsch Z Nervenheilk 123: 101. Ma KC, Ye ZR, Fang J, Wu JV (1988). Glial fibrillary acidic protein immunohistochemical study of Alzheimer I & II astrogliosis in Wilson’s disease. Acta Neurol Scand 78 (4): 290–296. Mathiisen TM, Lehre KP, Danbolt NC et al. (2010). The perivascular astroglial sheath provides a complete covering of the brain microvessels: an electron microscopic 3D reconstruction. Glia 58 (9): 1094–1103. Meenakshi-Sundaram S, Mahadevan A, Taly AB et al. (2008). Wilson’s disease: a clinico-neuropathological autopsy study. J Clin Neurosci 15 (4): 409–417. Messert B, Orrison WW, Hawkins MJ et al. (1979). Central pontine myelinolysis. Considerations on etiology, diagnosis, and treatment. Neurology 29 (2): 147–160. Mikol J, Vital C, Wassef M et al. (2005). Extensive cortico-subcortical lesions in Wilson’s disease: clinicopathological study of two cases. Acta Neuropathol 110 (5): 451–458. Miyajima H, Nishimura Y, Mizoguchi K et al. (1987). Familial apoceruloplasmin deficiency associated with blepharospasm and retinal degeneration. Neurology 37 (5): 761–767. Miyakawa T, Murayama E (1976). An autopsy case of the “demyelinating type” of Wilson’s disease. Acta Neuropathol 35 (3): 235–241. Miyakawa T, Murayama E, Sumiyoshi S et al. (1973). A biopsy case of Wilson’s disease. Pathological changes in peripheral nerves. Acta Neuropathol 24: 174–177. Morita H, Ikeda S, Yamamoto K et al. (1995). Hereditary ceruloplasmin deficiency with hemosiderosis: a clinicopathological study of a Japanese family. Ann Neurol 37 (5): 646–656. Mossakowski MJ, Renkawek K, Kras´nicka Z et al. (1970). Morphology and histochemistry of Wilsonian and hepatogenic gliopathy in tissue culture. Acta Neuropathol 16: 1–16. Niciu MJ, Ma XM, El-Meskini R et al. (2006). Developmental changes in the expression of ATP7A during a critical period in postnatal neurodevelopment. Neuroscience 139: 947–964. Norenberg MD, Lapham LW (1974). The astrocyte response in experimental portal-systemic encephalopathy: an electron

WILSON DISEASE: BRAIN PATHOLOGY microscopic study. J Neuropathol Exp Neurol 33 (3): 422–435. Oide T, Yoshida K, Kaneko K et al. (2006). Iron overload and antioxidative role of perivascular astrocytes in aceruloplasminemia. Neuropathol Appl Neurobiol 32 (2): 170–176. Otto J, Guenther P, Hoffmann KT (2013). Bilateral hypertrophic olivary degeneration in Wilson disease. Korean J Radiol 14: 316–320. Parpura V, Heneka MT, Montana V et al. (2012). Glial cells in (patho) physiology. J Neurochem 121 (1): 4–27. Prashanth LK, Sinha S, Taly AB et al. (2010). Do MRI features distinguish Wilson’s disease from other early onset extrapyramidal disorders? An analysis of 100 cases. Mov Disord 25 (6): 672–678. Rahil-Khazen R, Bolann BJ, Myking A et al. (2002). Multielement analysis of trace element levels in human autopsy tissues by using inductively coupled atomic emission spectrometry technique (ICP-AES). J Trace Elem Med Biol 16 (1): 15–25. Rajan MT, Jagannatha Rao KS, Mamatha BM et al. (1997). Quantification of trace elements in normal human brain by inductively coupled plasma atomic emission spectrometry. J Neurol Sci 146 (2): 153–166. Ramos P, Santos A, Pinto NR et al. (2014). Anatomical region differences and age-related changes in copper, zinc, and manganese levels in the human brain. Biol Trace Elem Res 161 (2): 190–201. Richter R (1948). The pallial component in hepato-lenticular degeneration. J Neuropathol Exp Neurol 7 (1): 1–18. Riederer P, Sofic E, Rausch WD et al. (1989). Transition metals, ferritin, glutathione, and ascorbic acid in parkinsonian brains. J Neurochem 52 (2): 515–520. Rongzhu L, Suhua W, Guangwei X et al. (2009). Zinc, copper, iron, and selenium levels in brain and liver of mice exposed to acrylonitrile. Biol Trace Elem Res 130 (1): 39–47. Saito T, Okabe M, Hosokawa T et al. (1999). Immunohistochemical determination of the Wilson copper- transporting P-type ATPase in the brain tissues of the rat. Neurosci Lett 266: 13–16. Scheiber IF, Dringen R (2013). Astrocyte functions in the copper homeostasis of the brain. Neurochem Int 62 (5): 556–565. Scheiber IF, Mercer JF, Dringen R (2014). Metabolism and functions of copper in brain. Prog Neurobiol 116: 33–57. Scheinberg IH, Sternlieb I (1984). Wilson’s Disease. Major Problems in Internal Medicine, W.B. Saunders, Philadelphia, Vol. 23. Schulman S (1968). Wilson’s disease. In: J Minckler (Ed.), Pathology of the Nervous System, McGraw-Hill, New York, pp. 1139–1151. Schulman S, Barbeau A (1963). Wilson’s disease: a case with almost total loss of white matter. J Neuropathol Exp Neurol 22: 105–119. Shiraki H (1968). Comparative neuropathologic study of Wilson’s disease and other types of hepatocerebral disease. Birth Defects Orig Artic Ser 4 (2): 64–73. Sinha S, Taly AB, Ravishankar S et al. (2006). Wilson’s disease: cranial MRI observations and clinical correlation. Neuroradiology 48 (9): 613–621.

89

Sinha S, Taly AB, Prashanth LK et al. (2007). Sequential MRI changes in Wilson’s disease with de-coppering therapy: a study of 50 patients. Br J Radiol 80 (957): 744–749. Skowronska M, Litwin T, Dziezyc K et al. (2013). Does brain degeneration in Wilson disease involve not only copper but also iron accumulation? Neurol Neurochir Pol 47 (6): 542–546. Smeyers-Verbeke J, Defrise-Gussenhoven E, Ebinger G et al. (1974). Distribution of Cu and Zn in human brain tissue. Clin Chim Acta 51 (3): 309–314. Starosta-Rubinstein S, Young AB, Kluin K et al. (1987). Clinical assessment of 31 patients with Wilson’s disease. Correlations with structural changes on magnetic resonance imaging. Arch Neurol 44: 365–370. Telianidis J, Hung YH, Materia S et al. (2013). Role of the P-type ATPases, ATP7A and ATP7B in brain copper homeostasis. Front Aging Neurosci 5: 44. Tiffany-Castiglioni E, Hong S, Qian Y (2011). Copper handling by astrocytes: insights into neurodegenerative diseases. Int J Dev Neurosci 29 (8): 811–818. Trocello JM, Guichard JP, Leyendecker A et al. (2011). Corpus callosum abnormalities in Wilson’s disease. J Neurol Neurosurg Psychiatry 82 (10): 1119–1121. Trocello JM, Woimant F, El Balkhi S et al. (2013). Extensive striatal, cortical, and white matter brain MRI abnormalities in Wilson disease. Neurology 81 (17): 1557. Victor M, Adams RD, Cole M (1965). The acquired (non-Wilsonian) type of chronic hepatocerebral degeneration. Medicine (Baltimore) 44 (5): 345–396. V€ olkl A, Berlet H, Ule G (1974). Trace elements (Cu, Fe, Mg, Zn) of the brain during childhood. Neuropadiatrie 5 (3): 236–242. Von Hoesslin C, Alzheimer A (1912). Ein Beitrag zur Klinik und pathologischen Anatomie der WestphalStr€ umpellschen Pseudosclerose. Zeitschrift f€ ur die gesamte Neurologie und Psychiatrie 8: 183–209. Warren PJ, Earl CJ, Thompson RH (1960). The distribution of copper in human brain. Brain 83: 709–717. Wierzba-Bobrowicz T, Gwiazda E, Kosno-Kruszewska E et al. (2002). Morphological analysis of active microglia – rod and ramified microglia in human brains affected by some neurological diseases (SSPE, Alzheimer’s disease and Wilson’s disease). Folia Neuropathol 40: 125–131. Wilson SAK (1912). Progressive lenticular degeneration: a familial nervous disease associated with cirrhosis of the liver. Brain 34: 295–509. Yang J, Li X, Yang R et al. (2015). Susceptibility-weighted imaging manifestations in the brain of Wilson’s disease patients. PLoS One 10 (4): e0125100. Zatta P, Raso M, Zambenedetti P et al. (2005). Copper and zinc dismetabolism in the mouse brain upon chronic cuprizone treatment. Cell Mol Life Sci 62 (13): 1502–1513. Zheng W, Monnot AD (2012). Regulation of brain iron and copper homeostasis by brain barrier systems: implication in neurodegenerative diseases. Pharmacol Ther 133 (2): 177–188.

Handbook of Clinical Neurology, Vol. 142 (3rd series) Wilson Disease A. Członkowska and M.L. Schilsky, Editors http://dx.doi.org/10.1016/B978-0-444-63625-6.00009-4 © 2017 Elsevier B.V. All rights reserved

Chapter 9

Hepatic features of Wilson disease 1

SALIH BOGA1, AFTAB ALA2,3, AND MICHAEL L. SCHILSKY4* Department of Gastroenterology, Sisli Etfal Education and Research Hospital, Istanbul, Turkey

2

Department of Clinical and Experimental Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, UK

3

Department of Gastroenterology and Hepatology, Royal Surrey County Hospital NHS Foundation Trust, Guildford, Surrey, UK

4

Section of Digestive Diseases and Transplantation and Immunology, Department of Medicine and Surgery, Yale University School of Medicine, New Haven, CT, USA

Abstract In Wilson disease (WD) defective AT7B function leads to biliary copper excretion and pathologic copper accumulation, particularly in liver and brain, where it induces cellular damage. Liver disease most often precedes neurologic or psychiatric manifestations. In most patients with neurologic or psychiatric symptoms there is some degree of liver disease at the time of disease presentation. Hepatic manifestations of WD can be extremely variable. Patients with clinically asymptomatic WD are often found by family screening or identified on routine laboratory testing. Others may have a clinical picture of chronic active hepatitis or of end-stage liver disease with cirrhosis. A minority present with acute liver failure, often on the background of advanced fibrosis. Complications from liver disease may be related to portal hypertension and concomitant liver disease may accelerate the course of liver disease. Liver cancer may occur in patients with WD, most commonly when cirrhosis and inflammation are present. The prognosis of patients with WD is excellent, especially for those without cirrhosis at the time of diagnosis, but requires timely initiation of appropriate therapy specific for WD and for the patient’s liver disease independent of WD.

HEPATIC FEATURES OF WILSON DISEASE Hepatic manifestations of Wilson disease (WD) can be extremely variable, and range from asymptomatic to liver failure. Complications from advanced liver disease may be related to portal hypertension but also include the development of liver cancers. WD patients with neurologic or psychiatric disease likely have some degree of hepatic disease, and concomitant liver diseases with WD may accelerate the progression of liver disease. Prognosis with treatment of WD is excellent, but requires timely diagnosis and initiation of appropriate therapy as well as attention to other potential complications of WD to achieve best outcomes.

Symptoms of WD usually present in the first decades of life, with the majority of cases occurring between the ages of 5 and 35 years old. Hepatic symptoms predominate in younger patients in their first decade of life, and neurologic symptoms occur in older patients as they approach the third decade of life, but there are numerous exceptions, with some having both hepatic and neuropsychiatric symptoms. WD rarely presents clinically in very young children less than 3–5 years old, but there are exceptions. Some examples of liver disease due to WD in very young patients include a 13-month-old who was evaluated for transaminitis (Iorio et al., 2003), a 3-year-old with cirrhosis (Wilson et al., 2000), and acute liver failure (ALF) in a 5-year-old patient (Kalach et al., 1993). Newer molecular diagnostic testing

*Correspondence to: Michael L. Schilsky, M.D., Section of Digestive Diseases and Transplantation and Immunology, Department of Medicine and Surgery, Yale University School of Medicine, Yale University Medical Center, 333 Cedar Street, LMP 1080, New Haven CT 06520, USA. Tel: +1-203-737-1592, Fax: +1-203-785-6645, E-mail: [email protected]

92

S. BOGA ET AL.

in parents has led to identification of children at risk for WD, and even diagnosis of some patients by molecular studies before signs or symptoms have ever appeared.

THE SPECTRUM OF LIVER DISEASE IN WILSON DISEASE WD may present with a wide spectrum of liver disease (Table 9.1). The variability in the age of onset of WD probably reflects differences in mutations and penetrance, extragenic factors, and environmental influences, including diet (Ala and Schilsky, 2004). Although an overall 40–50% of affected individuals will present with symptomatic liver disease (Gollan and Gollan, 1998), there is wide variability in the reported rates of the hepatic manifestations seen at the time of presentation (18–84%) (Asadi Pooya et al., 2005; Taly et al., 2007). In part, the wide variability in the estimates may be attributable to ascertainment bias based upon the clinical specialty to which the patient was referred (e.g., neurologists are likely to see patients with neurologic symptoms and thus report that the majority of patients with WD have neurologic involvement). Regardless of the clinical manifestations present initially, untreated patients often develop different manifestations as the disease progresses. Below, we address the types and characteristics of presentation of liver disease in WD patients. More patients with asymptomatic WD are being identified due to increased recognition of the need for family screening once a patient is identified, and improvement in diagnostic testing. As noted in Chapter 14, clinical and biochemical testing is now also aided by direct mutation analysis of ATP7B that is useful for screening and identification of affected first-degree relatives of newly diagnosed patients. In asymptomatic patients, particularly those diagnosed by family screening, many have no signs or stigmata of clinical liver disease and have abnormalities of liver tests alone. Asymptomatic WD patients with only Table 9.1 Hepatic presentations in patients with Wilson disease Asymptomatic hepatomegaly Isolated splenomegaly Persistent elevations in serum aminotransferases Jaundice Fatty liver Resembling autoimmune hepatitis Acute hepatitis Compensated cirrhosis Decompensated cirrhosis (with jaundice, ascites, varices, hepatic encephalopathy, hepatoma) Acute liver failure (with coagulopathy and hepatic encephalopathy)

mild elevations in liver tests occur with a frequency between 18% and 23% (Stremmel et al., 1991; Ferenci et al., 2003), and mild hepatomegaly may be their only finding on physical examination. However, a few of these patients may have more advanced liver disease that was unrecognized. In a study that included 18 asymptomatic patients detected through screening of an affected sibling, 2 (11%) already had cirrhosis at the time of diagnosis (Ferenci et al., 2005). Symptomatic patients present across the spectrum of liver disease, from mild disease to liver failure. In the earlier phase of the disease, there is progression from asymptomatic transaminitis to chronic active hepatitis (Schilsky et al., 1991), a clinical, biochemical, and pathologic condition associated with protracted inflammatory changes in the liver. An important factor that negatively impacts the prognosis of patients with chronic active hepatitis is advanced fibrosis. With fibrosis progression that occurs due to ongoing active inflammation, patients develop cirrhosis and sequelae of portal hypertension. Cirrhosis begins as compensated disease, but with progression and advancing portal hypertension there can be decompensation with variceal bleeding, ascites, or encephalopathy. When present, hepatic symptoms usually present in the first decade of life at an average age between 10 and 13 years (Merle et al., 2007). The most common hepatic signs and symptoms are jaundice, anorexia, vomiting (37–44%), followed by ascites/edema (23–26%) and hepatomegaly/splenomegaly (16–29%) (Stremmel et al., 1991; Ferenci et al., 2003; Merle et al., 2007). Some WD patients have transient episodes of jaundice due to hemolysis. Low-grade hemolysis may occur in WD even when liver disease is not clinically evident and in the absence of fulminant liver failure associated with WD. In one series, hemolysis was a presenting feature in 25 of 220 cases (11%). In these patients, hemolysis occurred as a single acute episode, intermittently, or was low-grade and chronic (Walshe, 1987). In a series of 283 Japanese patients with WD, only 3 presented with acute hemolysis alone, but one-quarter of those who presented with jaundice also had hemolysis (Saito, 1987). Other presenting symptoms of advanced liver disease include fatigue and confusion due to hepatic encephalopathy, and clinical signs of cirrhosis such as spider angiomas, gynecomastia, palmar erythema, easy bruising, easy bleeding (epistaxis, gingival), and muscle wasting. One important issue relevant for prognosis and clincal management is whether cirrhosis is present at the time of diagnosis of WD. In a retrospective study of 229 patients, 62% with hepatic manifestations of WD had cirrhosis (Beinhardt et al., 2014). By contrast only 11% of asymptomatic patients with WD had cirrhosis (Ferenci et al., 2005). In one series of 14 patients over the age of 40 years presenting with neurologic symptoms due to WD who

HEPATIC FEATURES OF WILSON DISEASE underwent liver biopsy, significant fibrosis was present in 10 (71%) (Ferenci et al., 2007), while another study of 34 patients with neurologic WD found that 41% of patients had cirrhosis (Ferenci et al., 2005). Similarly, in another study, 11/23 patients with predominantly neurologic WD had cirrhosis at presentation (Przbylkowski et al., 2014). The development of portal hypertension in the cirrhotic patient with WD is important with respect to the potential associated complications from the increased pressure. In WD, portal hypertension results from increased sinusoidal resistance as a consequence of collagen deposition at the sinusoidal level with chronic enlargement of the venae comitantes in an attempt to deliver portal blood flow to the liver, so assuming a leash-like cavernous appearence. Bleeding from the esophagogastric varices is the most common presentation of portal hypertension, but ascites and hepatic encephalopathy may also appear. The spleen is always enlarged and symptomless splenomegaly may be the earliest presentation, particularly in children. In a pilot series evaluating 14 patients with neurologic WD, severe portal hypertension and liver damage was reversible or failed to develop if chelating therapy was initiated 20 mg/dL, monoclonal gammopathies, multiple myeloma, arci senilis, and others (Fleming et al., 1977; Scheinberg, et al., 1986; Tzelikis et al., 2005; Jawairia et al., 2012)). This corneal phenotype may be due to copper deposition, or other metals, as well as bilirubin deposits located in other corneal layers. Therefore a diagnosis of WD based on the detection of a K-F ring (at early stages of the disease) warrants caution, with any examination performed by an experienced ophthalmologist using a slit lamp (EASL, 2012). The description of the sunflower cataract as a pathognomonic sign of WD was first made in 1922 by (Siemerling and Oloff, 1922), who noted a similar cataract in the eye of a WD patient, and patients presenting with intraocular foreign bodies that contained copper. In both cases the cataract was caused by copper deposits located under the lens capsule (not directly in the lens cortex or nucleus), which did not impair visual acuity, unlike other types of cataract. The characteristic pattern of sunflower cataracts comprises a central disc, with radiating petal-like fronds (Goyal and Tripathi, 2000; Langwinska-Wosko et al., 2015a). Data for sunflower cataract occurrence in WD have been, till now, notoriously variable, with figures of between 2% and 20% quoted (Wiebers et al., 1977; Pfeiffer, 2007; Huo et al., 2008). However, a current study performed by LangwinskaWosko et al., (2015a) suggested that sunflower cataracts could be a very rare ocular sign of WD. Further, contrary to K-F ring data, there are no reports that link sunflower cataracts explicitly to WD, although resolution of sunflower cataracts following decoppering treatment has been reported in WD (Litwin et al., 2015b).

109

Current studies assessing retina and optic nerve morphology and functions in WD (using spectral optical coherence tomography (OCT), electrophysiology (electroretinography (ERG), and pattern reversal visual evoked potentials (PVEP)) show that not only the cornea and lens are involved in WD, but that there are also neurodegenerative changes in the retina and optic nerve. Albrecht et al. (2012) identified a reduced thickness of the retinal nerve fiber layer, the paramacular region, the retinal ganglion cell/inner plexifrom layer, and the inner nuclear layer. Langwinska-Wosko et al., (2015b) documented not only PVEP abnormalities, which reflect a slow conduction velocity of the visual tract, but also ERG abnormalities showing defective retinal function. The degree of retinal and optic nerve abnormalities correlated with the presence of brain changes by MRI. It is suggested that the use of retinal OCT and electrophysiology tests may be of use in tracking neurodegeneration as well as recovery during treatment. Other, more complex aspects of ocular system involvement in WD include oculomotor abnormalities analyzed with oculography (Takahashi et al., 1993; Ingster-Moati et al., 2007; Lesniak et al., 2008). These studies document impaired voluntary control of saccades, with disturbed smooth-eye movement. This oculomotor disturbance possibly relates to cognitive disturbances (lesions in prefrontal cortex following atrophy) in the course of WD. The direct involvement of eyeball motor nerves in WD, as well as nystagmus, arises incidentally.

CLINICAL SCALES ASSESSING NEUROLOGIC DEFICITS IN WILSON DISEASE Although the severity of hepatic involvement in WD is based on well-accepted scales used in hepatology (Child–Pugh, Model for End-stage Liver Disease (MELD) score, described in detail in Chapters 7 and 9), neurologic severity has, until the last decade, been based on a subjective assessment made by the clinician (i.e., asymptomatic vs. improved vs. deteriorated vs. unchanged vs. dead) (Członkowska et al., 1996). Due to its wide spectrum, and the fluctuation of WD neurologic symptoms, there was a need to establish a scale inclusive of the spectrum of neurologic manifestations seen in WD. For WD patients, a detailed neurologic assessment combined with its impact on activities of daily living (ADL) are important criteria both at diagnosis and at follow-up. In response to this need, the UWDRS was instigated in 2007 (Członkowska et al., 2007; Leinweber et al., 2008). The UWDRS consists of three

110

A. CZŁONKOWSKA ET AL.

parts: (I) consciousness (1 item, scored 0–3 points); (II) a review of the patient’s ADL (10 items, scored 0–4 points); and (III) a detailed neurologic assessment (34 items, with a maximum score of 143 points). Further, definitions of neurologic improvement and deterioration were proposed according to UWDRS scores. Deterioration was defined as: (1) increase at least 4 points in UWDRS part III; or (2) any increase in UWDRS part two. Overall therapeutic success was proposed to be indicated by no deterioration of 2 points in UWDRS part II, and no deterioration of 4 points in UWDRS part III (Członkowska et al., 2014; Litwin et al., 2015a). Almost simultaneously, a second scale for WD, termed GAS, was established. Apart from neurologic deficits, GAS includes liver, cognitive/behavioral, as well as osseomuscular items, but without an ADL score (Aggarwal et al., 2009). The GAS comprises two tiers. Tier 1 scores global disability in the four domains mostly affected in WD patients: liver, cognition/behavior, motor, and osseomuscular. Each domain is scored in the range of 0–5 points. Tier 2 details neurologic deficits (total score 0–56). Currently, and mainly depending on the geographic localization of the treatment center, one of these two scales will be used in WD assessment and treatment, in concordance with the international guidelines of WD treatment (EASL, 2012).

NEUROIMAGING IN WILSON DISEASE Neuroimaging techniques (especially brain MRI) are important diagnostic techniques used in diagnosis and treatment monitoring for WD (EASL, 2012). The pathologic changes visualized in different neuroimaging techniques are seen in almost all patients with neurologic signs of WD, but also in some hepatic or presymptomatic cases. These pathologic changes are principally localized to the basal ganglia (caudate nucleus, putamen, globus pallidus), thalamus, and occasionally the brainstem. Broadly speaking, the neuroimaging techniques employed for WD assessment can be subdivided into: (1) structural brain analyses (CT previously, MRI currently); (2) brain metabolism analyses (MRS, singlephoton emission computed tomography (SPECT)); (3) techniques that use radioisotopes (positron emission tomography (PET)) to analyze neurotransmission (van Wassenaer-van Hall, 1997; EASL, 2012; Dusek et al., 2015a).

Brain computed tomography Most of the studies describing brain CT for WD patients were performed in the 1980s (before the MRI era). These studies documented ventricular dilation as well as hypotension in gray-matter nuclei (basal ganglia) (Wiliams and Walshe, 1981; van Wassenaer-van Hall, 1997)

Fig. 10.8. Hypointensive changes in both basal ganglia visualized on brain computed tomography.

(Fig. 10.8). Other findings, such as atrophy of the cortex, posterior fossa, brainstem, and white-matter changes, have been reported less frequently (van Wassenaervan Hall, 1997). Changes visualized in basal ganglia are more pronounced in patients with a severe neurologic presentation (van Wassenaer-van Hall, 1997) and may diminish after anticopper therapy. However, none of the described changes are pathognomonic for WD (van Wassenaer-van Hall, 1997). Currently, brain CT is not used in WD management apart from those cases where MRI is contraindicated (EASL, 2012; Dusek et al., 2015a).

Brain MRI Brain MRI is the most valuable examination of all the neuroimaging techniques used and tested in WD. Patients with neurologic phenotypes present with brain MRI abnormalities in almost all cases (StarostaRubinstein et al., 1987; Prayer et al., 1990; King et al., 1996; Saatci et al., 1997; Litwin et al., 2013c), while abnormalities in 42–70% of patients with hepatic symptoms can be identified (King et al., 1996; Kozic et al., 2003; Litwin et al., 2013c). MRI can even indicate brain abnormalities in almost 20% of presymptomatic patients (King et al., 1996; Litwin et al., 2013c). Other than brain atrophy, the pathologic changes visualized

WILSON DISEASE: NEUROLOGIC FEATURES

111

Fig. 10.9. Symmetric hyperintensive changes in T2-weighted sequences and fluid-attenuated inversion recovery sequences for the putamen, caudate, and thalamus.

Fig. 10.10. Hyperintensive changes in T2-weighted sequences, localized to the mesencephalon and pons.

on brain MRI are typically described as symmetric (in early stages these can be asymmetric), hyperintensive, or mixed-intensity changes in T2-weighted images, localized in the putamina, globi pallidi, caudate nuclei, thalami, and pons (Figs 10.9 and 10.10) (Prayer et al., 1990; King et al., 1996). The midbrain, cerebellum,

Fig. 10.11. “Giant panda” sign.

corticospinal tracts, as well as white matter could also be affected. These changes, when more advanced, are visualized as hypointensive regions in T1-weighted images. Hyperintensive changes in T1-weighted images, which occur rarely in WD, are due to manganese accumulation because of liver failure (Sinha et al., 2007; EASL, 2012).

112

A. CZŁONKOWSKA ET AL.

The characteristic brain MRI signs for WD were also described in T2-weighted images as: (1) the midbrain “face of the giant panda” (Fig. 10.11), which is described as a high signal in the tegmentum, with preservation of signal intensity of the lateral portion of the pars reticulata of the substantia nigra and red nuclei, with hypointensity of the superior colliculus; and (2) the pons “miniature panda” with hypointensity of central tegmental tracts and hyperintensity of the aqueductal opening to the fourth ventricle (Hitoshi et al., 1991; Sinha et al., 2007; Dusek et al., 2015a). These changes occur, in the main, for more advanced cases of the disease. In some WD patients, hypointensive changes in the globi pallidi in T2-weighted sequences have also been described (Sinha et al., 2007; Skowronska et al., 2013), which are similar to those seen in neurodegeneration with brain iron accumulation. This MRI pattern has also been described as the “eye of the tiger” (Litwin et al., 2014). Systemic iron metabolism disturbances in WD, and slightly elevated serum ferritin in male patients (Pfeiffenberg et al., 2012), have led to the hypothesis that iron is involved in the pathology of WD (Skowronska et al., 2013; Litwin et al., 2014; Walter et al., 2014; Dusek et al., 2015b). However, this possibility awaits confirmation in further studies. White-matter involvement in WD, as identified by brain MRI, is rarely described, although it is typically found in patients with a very severe course of disease, with epilepsy, myoclonus, and a poor outcome (Huang et al., 1997; Aikath et al., 2006; Grover et al., 2006; Kaver and Narayan, 2006; Barbosa et al., 2007; Prashanth et al., 2010a; Trocello et al., 2013). This discussion is relevant to cases for which demyelination is due to hypocupremia (WD overtreatment), or is a feature

of the natural course of severe disease (Barbosa et al., 2007; Trocello et al., 2013). There is no consensus for any correlates between neurologic presentation in WD and brain MRI pathology; some authors have attempted to draw relationships between dystonia and chorea with globi pallidi lesions, while others reported correlates with ataxia and tremor (Magalhaes et al., 1994; Sudmeyer et al., 2006; Sinha et al., 2007). However, other groups have failed to report any clear correlates for MRI changes and neurologic presentation, or the severity of neurologic symptoms (Prayer et al., 1990; Nazer, et al., 1993). Gender-related differences in phenotypic presentation, which include brain MRI pathology, have also been described (Litwin et al., 2013d). Women more frequently manifest with liver disease, and globi pallidi lesions. Men, on the other hand, more generally present with neurologic symptoms, with neurodegenerative changes and atrophy of the brain (Litwin et al., 2013d). As brain MRI provides specific pathologic data for WD, especially for neurologic patients, this examination is included in WD diagnosis algorithms such as the Ferenci score (EASL, 2012), and usefully distinguishes WD patients from those with other movement disorders (Prashanth et al., 2010b). Currently, brain MRI data for WD patients are also used to monitor recovery (Fig. 10.12) (Da Costa et al., 2009; EASL, 2012; Litwin et al., 2013c), although this use is neglected in some areas (Prayer et al., 1990). Finally, several new MRI techniques are currently being investigated. These include diffusion sequences to indicate edema via early astrocyte swelling (Sener, 2003), as well as gradient echo and susceptibility-weighted imaging, thought to identify iron accumulation during disease (Skowronska et al., 2013; Yang et al., 2015).

Fig. 10.12. Brain magnetic resonance imaging visualized in T2-weighted sequences: (A) at Wilson disease diagnosis – symmetric hyperintensive changes in the globi pallidi and caudate; (B) 2 years after liver transplantation, showing complete regression of the previous pathology.

WILSON DISEASE: NEUROLOGIC FEATURES Diffusion tensor imaging (Jadav et al., 2013), and more advanced 7 T imaging, which provides a noninvasive quantitative assessment of brain copper accumulation (Fritzsch et al., 2014; Dusek et al., 2016), are also being tested for WD. However, at present, these applications are restricted to research studies (EASL, 2012; Dusek et al., 2015a), and have yet to be put to clinical use.

Brain magnetic resonance spectroscopy MRS examinations based on noninvasive chemical analyses of brain tissue content (Alanen et al., 1999; Tarnacka et al., 2008; Sinha et al., 2010) have taken the form of phosphorous (31P-MRS) and proton (1H) MRS studies (Sinha et al., 2010). By analyzing different metabolites (as markers of neuron and glia activity), MRS examinations can provide useful data for the etiology of MR-identified changes (Alanen et al., 1999). The main limitation of MRS is that the assessed area should have the volume of at least 3 cm3 (the so-called volume of interest) (Alanen et al., 1999). MRS examinations show signal intensities for metabolites, with each reporting a different processes. N-acetylaspartate (NAA) is used as a marker of neuron activity (and decreases during neurodegeneration, including that seen in WD), choline (Cho) is a precursor of acetylcholine, and phosphatidylcholine is used as a marker of the intensity of membrane cell metabolism (which increases during hepatic encephalopathy (HE)). Myo-inositol (mI) is a marker of glia, and localizes to astrocytes (which decrease in signal in HE), lactate (Lac) is a marker of anaerobic glycolysis (e.g., necrosis factor), glutamine and glutamate (Glx) indicates astrocytes (which increases during astrocyte “protection” and in HE), and creatine (Cr) indicates energetic process (Alanen et al., 1999; Sinha et al., 2010). Thus far, in WD, MRS has only been used in the research context, without altering clinical practice (Tarnacka et al., 2008, 2009; Sinha et al., 2010). MRS studies have been used to assess, in vivo, brain metabolism at the cellular level during the course of WD in nontreated, as well as in chronically treated WD patients (with and without improvement). The results showed that in WD there is significant reduction of NAA/choline and NAA/Cr ratios in the striatum with decreased NAA indicating neuronal loss, and to a lesser degree, a decrease of choline as a collateral disturbance in metabolic processes, or as a direct paramagnetic effect (Tarnacka et al., 2008). Increased ratios of mI/Cr and Glx/Cr, as markers of astrocyte activation and proliferation, were also reported. While there were some differences between neurologic and hepatic patients, abnormalities have also been observed in asymptomatic WD patients, as well as in healthy

113

subjects heterozygous for the WD gene (Tarnacka et al., 2008, 2009; Sinha et al., 2010). During WD treatment, a correction of brain metabolism occurs (Tarnacka et al., 2008), with decreased ratios of: (1) NAA/Cr (reflecting normalization of neurons, reversible swelling, synaptogenesis, and possibly neuronal regeneration); and (2) mI/Cr ratios indicating normalization via astrocyte activation (Tarnacka et al., 2008). These observations suggest that (especially) the NAA/Cr ratio could be used as an early marker of sensitivity to anticopper treatment, including metabolic recovery, or deterioration prior to neurologic clinical signs and symptoms (Tarnacka et al., 2008). However, as mentioned above, there are no current international guidelines for the use of MRS in WD management (EASL, 2012).

SPECT and PET Both methodologies (SPECT and PET) are used exclusively in the research sphere for WD, with neither of any relevance to current practice, disease diagnosis, or treatment (Watanabe et al., 1995; Oder et al., 1996; Bruehleimer et al., 2000; Piga et al., 2008; Hermann, 2014). SPECT undertaken with different radiopharmaceutics could be used to analyze alterations in regional blood flow (with 99m Tc-ECD), as well as dopamine reuptake in the brain (dopamine transport measured with 123 I-beta-CIT), or selective receptor function (dopamine D2 receptors exposed to the antagonist 123 iodobenzamide) (Wang et al., 2012; Hermann, 2014). In the case of blood flow analyses, SPECT for WD patients have revealed hypoperfusion areas, especially in the caudate and lenticular nuclei, and cerebellum (Watanabe et al., 1995; Piga et al., 2008), but also, less frequently, in the temporal, frontal, and occipital lobes (Piga et al., 2008). Explanations for this hypoperfusion include changes in microvessel structure (thickening, fibrosis), which arise prior to structural changes to brain tissue that typify WD (Piga et al., 2008; Hermann, 2014). A comparison of SPECT with brain MRI confirmed these blood flow changes, suggesting that these anticipate later structural changes (Piga et al., 2008). When analyzing the results of dopamine neurotransmission in WD, there are, especially in neurologic patients, deficits of dopamine transport and reduced numbers of pre- and postsynaptic dopamine D2 receptors (Barthel et al., 2003; Hermann, 2014) that subsequently increase in number during decoppering treatment (Ishida et al., 2012; Hermann, 2014). These SPECT data could explain the extrapyramidal symptoms seen in the course of WD, and the comparable lack of benefit conferred by levodopa for the majority of cases. However, the

114

A. CZŁONKOWSKA ET AL.

heterogeneity of D2 receptor involvement in WD (in terms of severity) could provide a plausible explanation for the alleviation of neurologic symptoms using dopamine agonists in some WD patients (Litwin et al., 2013b). PET studies can assess brain glucose metabolism using markers such as [18F]-fluorodeoxyglucose (FDG), or brain dopamine D2 receptor density using [18F]methylspiperone (Hawkins et al., 1987; Bruehleimer et al., 2000; Piga et al., 2008; Loanne and Politis, 2011; Hermann, 2014). PET data collected for WD patients revealed a significant decrease of brain glucose metabolism in the basal ganglia (most severely in the caudate nucleus), as well as in cortical structures (Schlaug et al., 1994, 1996; Hermann, 2014), which could be ameliorated by anticopper treatment (Schlaug et al., 1996). Similarly, dopamine D2 receptor density was decreased prior to treatment (Schlaug et al., 1996; Bruehleimer et al., 2000; Hermann, 2014). Although these observations could be of use in assessing the dopaminergic system and its status in WD, the costs associated with either SPECT or PET (costs, access, isotope use) preclude their current use in published guidelines (EASL, 2012). Instead the administration of levodopa (see Chapter18) would be sufficient to assess the dopaminergic system in WD (Dusek et al., 2015b).

OTHER EXAMINATIONS Transcranial sonography (TCS) TCS is currently recognized as a sensitive tool with which to detect basal ganglia involvement in neurodegenerative disorders, mainly those of the substantia nigra in Parkinson’s disease (Walter et al., 2003). Thus far, for WD, only three studies have been performed. Each documented an increased ratio of the lenticular nuclei hyperechogenicity, especially in WD patients with a neurologic presentation (81.5–100%), which probably reflects copper accumulation (Walter et al., 2005; Svetel et al., 2012; Skowronska et al., 2013). The second, less frequent finding of TCS for WD patients with a neurologic presentation (up to 42%) is substantia nigra hyperechogenicity (Fig. 10.13), a feature that arises in Parkinson disease as a result of iron accumulation. The possibility of iron accumulation, in addition to copper, based on TCS and changes in brain MRI warrants further investigation (Walter et al., 2014).

Electrophysiology and electroencephalography Although EEG is not routinely performed for WD patients (EASL, 2012), this procedure can be helpful in informing epilepsy control (occurring in 6% of WD

Fig. 10.13. Transcranial Doppler (TCD) in Wilson disease: blue area shows hyperechogenicity of substantia nigra in Wilson disease patient; green shows brainstem visualized in TCD.

patients) and in terms of excluding differential diagnoses (Prashanth et al., 2010a; EASL, 2012). EEG abnormalities are commonly detected in WD, but these are mostly nonspecific (decreased a activity, increased y and d activities, with low voltage) and without clinical significance (Marecek and Nevsimalova, 1984; Chu et al., 1991). Currently EEG is not recommended as a diagnostic tool for these patients (apart from epilepsy and the suspicion of hepatic encephalopathy) (Fig. 10.14) (Prashanth et al., 2010a; EASL, 2012).

Evoked potentials Evoked potentials, including auditory evoked potentials, motor evoked potentials, and somatosensory potentials, have been examined in single studies in attempts to investigate the utility of these methods in WD (including the possibility of classification according to neurophysiologic parameters (Hermann et al., 2005)). Currently these methodologies have no clinical significance in either the diagnosis or treatment of WD (Grimm et al., 1992; Arendt et al., 1994; Hermann et al., 2003, 2005; Hsu et al., 2003; Das et al., 2007; Ecevit et al., 2012). That said, all of these studies have shown abnormalities in some WD patients, allowing their categorization according to phenotypic presentation (neurologic vs. hepatic). Additionally, even for patients who were asymptomatic in terms of neurologic presentation, subclinical brain damage could be detected using electrophysiologic studies (Laskowska-Studniarska et al., 1991; Grimm et al., 1992; Arendt et al., 1994; Hsu et al., 2003; Ecevit et al., 2012). On analyzing the electrophysiologic results in neurologic patients, the most common abnormalities were found in the auditory brainstem, followed by visual and then motor evoked potentials (Das et al., 2007).

WILSON DISEASE: NEUROLOGIC FEATURES

115

Fig. 10.14. Electroencephalogram (EEG) showing triphasic and biphasic waves (the EEG changes typical for hepatic encephalopathy).

CONCLUSIONS WD, as a neurodegenerative disorder, presents as a complex syndrome of neurologic and mainly extrapyramidal symptoms. It is implicated as a differential diagnosis when movement disorders present in young patients, especially those with liver disease. Ophthalmologic, as well as brain, MRI examinations should be performed during the course of WD diagnosis and treatment. The other neurologic examinations described in this chapter are less clinically significant, but can, nonetheless, contribute valuable information for pathogenesis, which could be of value in certain clinical situations.

REFERENCES Aggarwal A, Bhatt M (2014). The pragmatic treatment of Wilson’s disease. Mov Disord Clin Pract 1: 14–23. Aggarwal A, Aggarwal N, Nagral A et al. (2009). A novel global assessment scale for Wilson’s Diseases (GAS for WD). Mov Disord 2494: 509–518. Aikath D, Gupta A, Chattppadhyay I et al. (2006). Subcortical white matter abnormalities related to drug resistance in Wilson disease. Neurology 67: 878–880. Ala A, Borjigin J, Rochwarger et al. (2005). Wilson disease in septuagenarian siblings. Raising the bar for diagnosis. Hepatology 41: 668–670.

Alanen A, Komu M, Pentinen M et al. (1999). Magnetic resonance imaging and proton MR spectroscopy in Wilson’s disease. Br J Radiol 72: 749–756. Albrecht P, Muller AK, Ringelstein M et al. (2012). Retinal neurodegeneration in Wilson’s disease revealed by spectral domain optical coherence tomography. PLoS One 7, e49825. Arendt G, Hefter H, Stremmel W et al. (1994). The diagnostic value of multi-modality evoked potentials in Wilson’s disease. Electromyogr Clin Neurophysiol 34: 137–148. Bandmann O, Weiss KH, Kaler SG (2015). Wilson’s disease and other neurological copper disorders. Lancet 14: 103–113. Barbosa ER, Silveira-Moriyama L, Costa Machado A et al. (2007). Wilson’s disease with myoclonus and white matter lesions. Parkinsonism Relat Disord 13: 185–188. Barbosa AF, Souza Cde O, Chen J et al. (2015). The competition with a concurrent cognitive task affects posturographic measures in patients with Parkinson’s disease. Arq neuropsiquitr 73: 906–912. Barthel H, Hermann W, Kluge R et al. (2003). Concordant preand postsynaptic deficits of dopaminergic neurotransmission in neurologic Wilson disease. Am J Neuroradiol 23: 234–238. Belkin M, Chajek T, Zeimer R et al. (1976). Non-invasive quntitation of corneal copper in heptolenticular degeneration (Wilson’s disease). Lancet 21: 391–392.

116

A. CZŁONKOWSKA ET AL.

Benbir G, Gunduz A, Ertan S et al. (2010). Partial status epilepticus induced by hypocupremia in a patient with Wilson’s disease. Seizure 19: 602–604. Berry WR, Darley FL, Aronson AE (1974). Dysarthria in Wilson’s disease. J Speech Hear Res: 169–183. Boyce HW, Bakheet MR (2005). Sialorrhea a review of vexing, often unrecognized sign of oropharyngeal and esophageal disease. J Clin Gastroenterol 39: 89–97. Brewer GJ (2005). Neurologically presenting Wilson’s disease. CNS Drugs 19: 185–192. Brewer GJ, Askari FK (2005). Wilson’s disease: clinical management and therapy. J Hepatol 42: 13–21. Bruehleimer M, Leenders KL, Vontobel P et al. (2000). Increased cerebral iron uptake in Wilson’s disease: a 52Fe-citrate PET study. J Nucl Med 41: 781–787. Burke JF, Dayalu P, Nan B et al. (2011). Prognostic significance of neurologic examination findings in Wilson disease. Parkinsonism Relat Disord 17: 551–556. Cairns JE, Parry Wiliams H, Walshe JM (1969). “Sunflower cataract” in Wilson’s disease. British Med Journal 3: 95–96. Cairns JE, Parry Wiliams H, Walshe JM (1970). The Kayser Fleischer ring. Trans Ophtalmol Soc 90: 187–190. Chakor RT, Bharote H, Eklare N et al. (2015). Unilateral rubral tremors in Wilson’s disease treated with dimercaprol. Ann Indian Acad Neurol 18: 115–116. Chu NS, Chu CC, Tu SC et al. (1991). EEG spectral analysis and topographic mapping in Wilson’s disease. J Neurol Sci 106: 1–9. Chu EC, Chu N, Huang C (1997). Autonomic involvement in Wilson’s disease: a study of sympathetic skin response and RR interval variation. J Neurol Sci 149: 131–137. Crary MA, Carnaby-Mann GD, Faunce A (2007). Electrical stimulation therapy for dysphagia: descriptive results of two surveys. Dysphagia 22: 165–173. Członkowska A, Gajda J, Rodo M (1996). Effects of long-term treatment in Wilson’s disease with d-penicillamine and zinc sulphate. J Neurol 243: 269–273. Członkowska A, Litwin T, Karlinski M, et al. (2014). D-penicillamine versus zinc sulfate as first-line therapy for Wilson’s Disease. Eur J Neurol 21: 599–606. Członkowska A, Tarnacka B, Litwin T et al. (2005). Wilson’s disease – cause of mortality in 164 patients during 1992–2003 observation period. J Neurol 252: 698–703. Członkowska A, Tarnacka B, Moller JC et al. (2007). Unified Wilson’s Diseases rating scale - proposal for the neurological scoring of Wilson’s Diseases patients. Neurol Neurochir Pol 41: 1–12. Da Costa M, Spitz M, Baschesi L et al. (2009). Wilson’s disease: two treatment modalities. Correlations to pretreatment and post treatment brain MRI. Neuroradiology 51: 627–633. Da Silva-Junor FP, Carrasco AEAB, DaSilva Mendes AM et al. (2008). Swallowing dysfunction in Wilson’s disease: a scintigraphic study. Neurogstroenterol Motil 20: 285–290. Dalvi A (2014). Wilson’s disease: neurological and psychiatric manifestations. Dis Mon 60: 460–464.

Das M, Misra UK, Kalita J (2007). A study of clinical, MRI and multimodality evoked potentials in neurologic Wilson disease. Eur J Neurol 14: 498–504. Denning TR (1985). Psychiatric aspects of Wilson’s disease. Br J Psychiatr 147: 677–682. Denning TR, Berrios GE, Walshe JM (1988). Wilson’s disease and epilepsy. Brain 111: 1139–1155. Dening TR, Berrios GE (1989). Wilson’s diseases: clinical groups in 400 cases. Acta Neurol Scand 80: 527–534. Denny-Brown D (1962). The basal ganglia, Oxford University Press, London. Dusek P, Bahn E, Litwin T, et al. (2016). Brain iron accumulation in Wilson disease: a post-mortem 7 Tesla MRI histopathological study. Neuropathol Appl Neurobiol. http://dx.doi.org/10.1111/nan.12341. Dusek P, Litwin T, Członkowska A (2015a). Wilson disease and other neurodegenerations with metal accumulations. Neurol Clin 33: 175–204. Dusek P, Roos P, Litwin T et al. (2015b). The neurotoxicity of iron, copper and manganese in Parkinson’s and Wilson’s diseases. J Trace Elem Med Biol 31: 193–203. Dziezyc K, Litwin T, Sobanska A et al. (2014). Symptomatic copper deficiency in three Wilson’s disease patient’s treated with zinc sulphate. Neurol Neurochir Pol 48: 214–218. Dziezyc K, Litwin T, Chabik G et al. (2015). Frequencies of initial gait disturbances and falls in 100 Wilson’s diseases patients. Gait Posture 42: 601–603. Ecevit C, Ozgenc F, Gokcay F et al. (2012). The diagnostic value of multimodal evoked potentials in the determination of subclinical neurological involvement of Wilson’s disease. Eur J Gastroenterol Hepatol 24: 627–632. European Association For The Study of The Liver Disease (2012). EASL clinical practice guidelines: Wilson’s disease. J Hepatol 56: 671–685. Ferenci P, Caca K, Loudianos G et al. (2003). Diagnosis and phenotypic classification of Wilson disease. Liver Int 23: 139–142. Ferenci P, Członkowska A, Merle U et al. (2007). Late-onset Wilson’s disease. Gastroenterology 132: 1294–1298. Ferrazzoli D, Fasano A, Maestri R et al. (2015). Balance dysfunction in Parkinson’s disease: the role of posturography in developing a rehabilitation program. Parkinsons Dis 2015: 520128. Fleischer B (1903). Zwei weiterer Falle von grunlicher Verfarbung der Kornea. Klin Monatsbl Augenheilkd 41: 489–491. Fleischer B (1912). Uber eine der “Pseudosklerose” nhestehende bisher unbekannte Krankheit (gekennzeichnet durch tremor, psychische storungen, brunliche Pigmentierung bestimmer gewebe, inbesondere uch der Hornhautperipherie, Lebecirrhose). Dtsch Z Nervenheilkd 44: 179–201. Fleming CR, Dickson ER, Wahner HW et al. (1977). Corneal pigmented rings in patients with primary biliary cirrhosis. Gastroenterology 69: 220–225. Foubert-Samier A, Kzdi A, Rouanet M et al. (2009). Axonal sensory motor neuropathy in copper deficient Wilson’s disease. Muscle and Nerve 40: 294–296.

WILSON DISEASE: NEUROLOGIC FEATURES Fox CM, Ramig LO, Ciucci MR et al. (2006). The science and practice of LSVT/LOUD: neural plasticity-principled approach to treating individuals with Parkinson’s disease and other neurological disorders. Semin Speech Lang 27: 283–299. Fritzsch D, Reiss-Zimmermann M, Trampel R et al. (2014). Seven-tesla magnetic resonance imaging in Wilson disease using quantitative susceptibility mapping for measurement of copper accumulation. Invest Radiol 49: 299–306. Gage H, Storey L (2004). Rehabilitation for Parkinson’s disease: a systematic review of available evidence. Clin Rehabil 18: 463–482. Gondim FA, Araujo DF, Oliveira IS et al. (2013). Levodopa-responsive Parkinsonism and small fiber dysfunction in patients with Wilson’s disease. Movement Disord 28: S351–S352. Gondim F, de A, Arau´jo DF, Oliveira I et al. (2014). Small fiber dysfunction in patients with Wilson’s disease. Arq Neuropsiquiatr 72 (8): 592–595. Goyal V, Tripathi M (2000). Sunflower cataract in Wilson’s disease. J Neurol Neurosurg Psychiatry 69: 133. Grimm G, Madl C, Katzenschlager R et al. (1992). Detailed evaluation of evoked potentials in Wilson’s disease. Electroencephalogr Clin Neurophysiol 82: 119–124. Grover SB, Gupta P, Kumar A et al. (2006). Extensive gray and white matter abnormalities in Wilson’s disease: a case report. Ind J Radiol Imag 16: 91–94. Hall HC (1921). La degenerescence hepato-lenticulare (maladie de Wilson-pseudosclerose), Masson, Paris. Harry J, Tripahti R (1970). Kayser-Fleischer ring: a pathological study. Brit J Ophtal 54: 794–800. Hawkins RA, Mazziota JC, Phepls ME (1987). Wilson’s disease studied with FDG nd positron emission tomography. Neurology 37: 1707–1711. Henkin R, Keiser HR, Jaeffe I et al. (1967). Decreased taste sensitivity after d-penicillamine reversed by copper administration. Lancet 290: 1268–1271. Hermann W (2014). Morphological and functional imaging in neurological and non-neurological Wilson’s patients. Ann NY Acad Sci 1315: 24–29. Hermann W, Vililmann T, Wagner A (2003). Electrophysiological impairment profile of patients with Wilson’s disease. Nervenarzt 74: 881–887. Hermann W, Gunther P, Wagner et al. (2005). Classification of Wilson’s disease based on neurophysiological parameters. Nervenarzt 76: 733–739. Hitoshi S, Iwata M, Yoshikawa K (1991). Mid-brain pathology of Wilson’s disease: MRI analysis of three cases. J Neurol Neurosurg Psych 54: 624–626. Hoskovcova M, Dusek P, Sieger T et al. (2015). Predicting falls in Parkinson disease: what is the value of instrumented testing in off medication state. PloS ONE: e0139849. Hsu YS, Chng YC, Lee WT et al. (2003). The diagnostic value of sensory evoked potentials in pediatric Wilson disease. Pediatr Neurol 29: 42–45. Huang CC, Chu NS, Chen RS (1997). Asymmetric dystonia with frontal white matter lesions in Wilson’s disease. Eur J Neurol 4: 240–245.

117

Huo LJ, Liao RD, Chen XM (2008). Ophthalmic manifestations of Wilson’s disease. Zhonghua Yan Ke Za Zhi 44: 128–130. Ingster-Moati I, Quoc EB, Pless M et al. (2007). Ocular motility and Wilson’s disease: study on 34 patients. J Neurol Neurosurg Psychiatry 78: 1199–1201. Ishida S, Doi Y, Yamane et al. (2012). Resolution of cranial MRI and SPECT abnormalities in a patient with Wilson’s disease following oral zinc monotherapy. Intern Med 51: 1759–1763. Jadav R, Saini J, Sinha S et al. (2013). Diffusion tensor imaging (DTI) and its critical correlates in drug naive Wilson’s disease. Metab Brain Dis 28: 455–462. Jawairia M, Subhani M, Siddiqui G et al. (2012). Unexplained findings of Kayser-Fleischer-like rings in a patient with cryptogenic cirrhosis. Case Reports in Gastrointestinal Medicine. http://dx.doi.org/10.1153/2012/438525. Jung KH, Ahn TB, Jeon BS (2005). Wilson disease as an initial manifestation of polyneuropathy. Arch Neurol 62: 1628–1631. Kaveer N, Narayan SK (2006). CNS demyelination due to hypocupremia in Wilson’s disease from overzealous treatment. Neurol India 54: 110–111. Kayser B (1902). Uber einen Fall von angeborner grunlicher Verfarbung der Kornea. Klin Monatsbl Augenheilkd 40: 22–25. King AD, Walshe JM, Kendall BE et al. (1996). Cranial MR imaging in Wilson’s disease. Am J Roentgenol 167: 1579–1584. Konovalov NV (1960). Hepatocerebral dystrophy, Russian Academy of Medical Sciences, Moscow. Kontaxakis V, Stefanis C, Markidis M (1988). Neuroleptic malignant syndrome in a patient with Wilson’s disease. J Neurol Neurosurg Psych 51: 1001–1002. Kozic D, Svetel M, Petrovic B et al. (2003). MR imaging of the brain in patients with hepatic form of Wilson’s disease. Eur J Neurol 10: 587–592. Kumar S (2005). Severe autonomic dysfunction as a presenting feature of Wilson’s disease. J Postgrad Med 51: 75–76. Langwinska-Wosko E, Litwin T, Dziezyc K et al. (2015a). The sunflower cataract in Wilson’s disease: pathognomonic sign or rare finding? Act Neurol Belgica. http://dx.doi. org/10.1007/s13760-015-0566-1. Langwi nska-Wos´ko E, Litwin T, Szulborski K et al. (2015b). Optical coherence tomography and electrophysiology of retinal and visual pathways in Wilson’s disease. Metab Brain Dis. http://dx.doi.org/10.1007/s11011-0159776-8. Laskowska-Studniarska W, Niedzielska K, Walinowska E et al. (1991). Brainstem auditory evoked potentials and computed tomography in Wilson’s Disease. In: A Członkowska, CJA van den Hamer (Eds.), Proceedings of 5th Symposium on Wilson’s Disease, Ma˛dralin, Poland, 20–23 June 1990, Interfacultair Reactor Institute Technical Universitum Delft, The Netherlands. Lee SY, Yang HE, Yang HS et al. (2012). Neuromuscular electrical stimulation therapy for dysphagia caused by Wilson’s disease. Ann Rehabil Med 36: 409–413.

118

A. CZŁONKOWSKA ET AL.

Leinweber B, Moller JC, Scherag A et al. (2008). Evaluation of the Unified Wilson’s Disease Rating Scale (UWDRS) in German patients with treated Wilson’s disease. Mov Disord 23: 54–62. Lesniak M, Członkowska A, Seniow J (2008). Abnormal antisaccades and smooth pursuit eye movement in patients with Wilson’s disease. Mov Disord 23: 2067–2073. Litwin T, Członkowska A (2013). Wilson’s disease – factors affecting clinical presentation. Neurol Neurochir Pol 47: 161–169. Litwin T, Gromadzka G, Członkowska A (2012). Gender differences in Wilson disease. J Neurol Sci 312: 31–35. Litwin T, Chabik G, Członkowska A (2013a). Acute focal dystonia induced by tricyclic antidepressant in a patient with Wilson disease: a case report. Neurol Neurochir Pol 47: 502–506. Litwin T, Dusek P, Członkowska A (2016). Neurological manifestations in Wilson’s disease -possible treatment options for symptoms. Expert Opin. Orphan Drugs 4: 719–728. Litwin T, Gromadzka G, Samochowiec J et al. (2013b). Association of dopamine receptor gene polymorphisms with the clinical course of Wilson disease. JIMD Rep 8: 73–80. Litwin T, Dziezyc K, Poniatowska R et al. (2013c). Effect of liver transplantation on brain magnetic resonance imaging pathology in Wilson disease: a case report. Neurol Neurochir Pol 47: 393–397. Litwin T, Gromadzka G, Członkowska A et al. (2013d). The effect of gender on brain MRI pathology in Wilson’s disease. Metab Brain Dis 28: 69–75. Litwin T, Karlinski M, Skowronska M et al. (2014). MR image mimicking the “eye of the tiger” sign in Wilson’s disease. J Neurol 261: 1025–1027. Litwin T, Dziez˙yc K, Karlinski M et al. (2015a). Early neurological worsening in patients with Wilson’s Diseases. J Neurol Sci 355: 162–167. Litwin T, Langwinska-Wosko E, Dziezyc K et al. (2015b). Sunflower cataract: do not forget Wilson’s Diseases. Pract Neurol 15: 385–386. Loanne C, Politis M (2011). Positron emission tomography neuroimaging in Parkinson’s disease. Am J Transl Res 3: 323–341. Lorincz MT (2010). Neurologic Wilson’s disease. Ann N Y Sci 1184: 173–187. Lobner A, Lobner J, Bachmann H et al. (1986). The Kayser-Fleischer ring during long-term treatment in Wilson’s disease (hepatolenticular degeneration). Graefe’s Arch Clin Exp Ophtalmol 224: 152–155. Machado A, Chien HF, Deguti MM et al. (2006). Neurological manifestations in Wilson’s disease: Report of 119 cases. Mov Disord 21: 2192–2196. Magalhaes ACA, Caramelli P, Menezes JR et al. (1994). Wilson’s disease : MRI with clinical correlation. Neuroradiology 36: 97–100. Marecek Z, Nevsimalova S (1984). Biochemical and clinical changes in Wilson’s disease heterozygotes. J Inherit Metab Dis 7: 41–45. Marsden CD (1987). Wilson’s disease. QJM 248: 959–966.

Meenakshi-Sundaram S, Taly B, Kamath V et al. (2002). Autonomic dysfunction in Wilson’s disease – clinical and electrophysiological study. Clin Auton Res 12: 185–189. Mesholam RI, Moberg PJ, Mahr RN et al. (1998). Olfaction in neurodegenerative disease: meta-analysis of olfactory functioning in Alzheimer’s and Parkinson’s diseases. Arch Neurol 55: 84–90. Mueller A, Reuner U, Landis B et al. (2006). Extrapyramidal symptoms in Wilson’s disease re associated with olfactory dysfunction. Mov Disord 21: 1311–1316. Oder W, Grimm G, Kolleger H et al. (1991). Neurological and neuropsychiatric spectrum of Wilson’s disease: prospective study of 45 cases. J Neurol 238: 281–287. Oder W, Prayer L, Grimm G et al. (1993). Wilsons’ disease: evidence of subgroups derived from clinical findings and brain lesions. Neurology 43: 120–124. Oder W, Brucke T, Kolleger H et al. (1996). Dopamine receptor binding is reduced in Wilson’s disease: correlation of neurological deficits with striatal I-iodobenzamide binding. J Neural Transm 103: 1096–1103. Pestana Knight EM, Gilman S, Selwa L (2009). Status epilepticus in Wilson’s disease. Epileptic Disord 11: 138–143. Pfeiffenberg J, Gothardt DN, Hermann T et al. (2012). Iron metabolism and the role of HFE gene polymorphisms in Wilson disease. Liver Int 32: 165–170. Pfeiffer R (2007). Wilson’s disease. Semin Neurol 27: 123–132. Piga M, Murru A, Satta L et al. (2008). Brain MRI and SPECT in the diagnosis of early neurological involvement in Wilson’s disease. Eur J Nucl Med Mol Imaging 35: 716–724. Pool KD, Feit H, Kirkptrick J (1981). Penicillamine induced neuropathy in rheumatoid arthritis. Ann Intern Med 95: 457–458. Prashanth LK, Taly S, Sinha S et al. (2005). Prognostic factors in patient presenting with severe neurological forms of Wilson’s disease. Q J Med 98: 557–563. Prashanth LK, Sinha S, Taly AB et al. (2010a). Spectrum of epilepsy in Wilson’s disease with electroencephalographic, MR imaging and pathological correlates. J Neurol Sci 291: 44–51. Prashanth LK, Sinha S, Taly AB et al. (2010b). Do MRI distinguish Wilson’s disease from other early onset extrapyramidal disorders? An analysis of 100 cases. Mov Disord 25: 672–678. Prayer L, Wimberger J, Kramer J et al. (1990). Cranial MRI in Wilson’s disease. Neuroradiology 32: 211–214. Roberts E, Schilsky M (2008). Diagnosis and treatment of Wilson’s disease an update. Hepatology 47: 2089–2111. Saatci I, Topcu M, Baltaoglu F et al. (1997). Cranial MR findings in Wilson’s disease. Acta Radiol 38: 250–258. Scheinberg IH, Sternlieb I, Walshe JM (1986). Wilson’s disease and Kayser-Fleischer rings. Ann Neurol 19: 613–614. Schlaug G, Hefter H, Nebeling B et al. (1994). Dopamine D2 receptor binding and cerebral glucose metabolism recover after d-penicillamine-therapy in Wilson’s disease. J Neurol 241: 577–584. Schlaug G, Hefter H, Engelbrecht V et al. (1996). Neurological impairment and recovery in Wilson’s disease: evidence from PET and MRI. J Neurol Sci 136: 129–139.

WILSON DISEASE: NEUROLOGIC FEATURES Sener RN (2003). Diffusion MRI findings in Wilson’s disease. Comput Med Imaging Graph 27: 17–21. Siemerling E, Oloff H (1922). Pseudoskleroses (Westphal-Strumpell) mit Cornealring (Kayser-Fleicher) und doppelseitiger Scheinkatarakt, die nur bei seitlicher Beleuchtnung sichtbar ist und die der nach Verletzung durch Kupfersplitter entstehenden Katarakt anlich ist. Klin Wochenschr 1: 1087–1089. Sinha S, Taly AB, Prashanth LK et al. (2007). Sequential MRI changes in Wilson’s disease with de-coppering therapy: a study of 50 patients. Br J Radiol 80: 744–749. Sinha S, Taly AB, Rvinshakar S et al. (2010). Wilson’s disease 31P and 1H MR spectroscopy and clinical correlation. Neuroradiology 52: 977–985. Skowronska M, Litwin T, Dziezyc K et al. (2013). Does brain degeneration in Wilson Diseases involve not only copper but also iron accumulation? Neurol Neurochir Pol 47: 542–546. Soni D, Shukla G, Singh S et al. (2009). Cardiovascular and sudomotor autonomic dysfunction in Wilson’s diseaselimited correlation with disease severity. Aut Neurosci 151: 154–158. Starosta-Rubinstein S, Young AB, Kluin K et al. (1987). Clinical assessment of 31 patients with Wilson’s disease: correlations with structural changes on magnetic resonance imaging. Arch Neurol 44: 365–370. Stremmel W, Meyerrose KW, Niederau C et al. (1991). Wilson disease: clinical presentation, treatment, and survival. Ann Intern Med 115: 720–726. Sudmeyer M, Saleh A, Wojtecki L et al. (2006). Wilson’s disease tremor is associated with magnetic resonance imaging lesions in basal ganglia. Mov Disord 12: 2134–2139. Svetel M, Kozic D, Stefanova E et al. (2001). Dystonia in Wilson’s disease. Mov Disord 16: 719–723. Svetel M, Mijajlovic M, Tomic A et al. (2012). Transcranial sonography in Wilson’s disease. Parkinsonism Rel Disord 18: 234–238. Takahashi M, Kaneko M, Hattori T et al. (1993). Slow eye movements (slow saccades) in Wilson’s disease. Rinsho Shinkeigaku 33: 652–656. Taly AB, Meenakshi-Sundaram S, Sinha S et al. (2007). Wilson disease: description of 282 patients evaluated over 3 decades. Medicine 86: 112–121. Tarnacka B, Szeszkowski W, Gołebiowski M et al. (2008). MR spectroscopy in monitoring the treatment of Wilson’s Disease patients. Mov Disord 23: 1560–1566. Tarnacka B, Szeszkowski W, Gołebiowski M et al. (2009). Metabolic changes in 37 newly diagnosed Wilson’s Disease patients assessed by magnetic resonance spectroscopy. Park Rel Dis 15: 582–586. Teive HA, Kluppel LE, Munhoz RP et al. (2012). Jaw-opening oromandibular dystonia secondary to Wilson’s Disease treated with botulinum toxin type A. Arq Neuropsiquiatr 70: 407–409.

119

Trocello JM, Woimant F, El Balkhi S et al. (2013). Extensive striatal, cortical, and white matter brain MRI abnormalities in Wilson disease. Neurology 81: 1557. Trocello JM, Osmani K, Pernon M et al. (2015). Hypersialorrhea in Wilson’s disease 30: 489-495. Tzelikis PF, Laibson PR, Ribeiro MP et al. (2005). Ocular copper deposition associated with monoclonal gammopathy of undetermined significance; case report. Arq Bras Oftalmol 68: 539–541. Uzman LL, Jakus MA (1957). The Kayser-Fleischer ring. A histochemical and electron microscope study. Neurology 7: 341–355. Van Wassenaer-van Hall HN (1997). Neuroimaging in Wilson Disease. Met Brain Dis 12: 1997. Von Giesen H-J, Weiss P, Arendt G et al. (2003). Potential C-fiber damage in Wilson’s disease. Acta Neurol Scand 108: 257–261. Walshe JM (1986). Wilson’s disease. Handb Clin Neurol 49: 223–238. Walshe JM (2007). Cause of death in Wilson disease. Mov Disord 22: 2216–2220. Walshe JM, Yealland M (1992). Wilson’s disease: the problem of delayed diagnosis. J Neurol Neurosurg Psychiatry 55: 692–696. Walter U, Niehaus L, Probst T et al. (2003). Brain parenchyma sonography discriminates Parkinson’s disease and atypical parkinsonian syndromes. Neurology 60: 74–77. Walter U, Krolikowski K, Tarnacka B et al. (2005). Sonographic detection of basal ganglia lesions in asymptomatic and symptomatic Wilson disease. Neurology 64: 1726–1732. Walter U, Skowronska M, Litwin T et al. (2014). Lenticular nucleus hyperechogenicity in Wilson’s disease reflects local copper, but not iron accumulation. J Neural Transm 121: 1273–1279. Wang L, Zhang Q, Huanbin L et al. (2012). SPECT molecular imaging in Parkinson’s disease. J Biomed Biotechnol 412486. Watanabe N, Seto H, Shimizu M et al. (1995). Brain SPECT in Wilson’s disease. Clin Nucl Med 20: 1028–1030. Wiebers DO, Hollenhorst RW, Goldstein NP (1977). The ophthalmologic manifestations of Wilson’s disease. Mayo Clin Proc 52: 409–416. Wiliams JB, Walshe JM (1981). Wilson’s disease an analysis of the cranial computerized tomographic appearances found in 60 patients and the changes in response to treatment with chelating agents. Brain 104: 735–752. Wilson SA (1912). Progressive lenticular degeneration: a familial nervous disease associated with cirrhosis of the liver. Brain 34: 20–509. Yang J, Li X, Yang R et al. (2015). Susceptibility-weighted imaging manifestations in the brain of Wilson’s disease patients. PLoS One 10, e0125100. Zimbrean PC, Schilsky ML (2014). Psychiatric aspects of Wilson disease a review. Gen Hosp Psychiatry 36: 53–62.

Handbook of Clinical Neurology, Vol. 142 (3rd series) Wilson Disease A. Członkowska and M.L. Schilsky, Editors http://dx.doi.org/10.1016/B978-0-444-63625-6.00011-2 © 2017 Elsevier B.V. All rights reserved

Chapter 11

Cognitive and psychiatric symptoms in Wilson disease 1

PAULA ZIMBREAN1* AND JOANNA SENIÓW2 Departments of Psychiatry and Surgery (Transplant), Yale University, New Haven, CT, USA 2

Second Department of Neurology, Institute of Psychiatry and Neurology, Warsaw, Poland

Abstract Wilson disease – can present with such a variety of psychiatric and cognitive symptoms that it has been named the “great masquerader.” Symptoms may include cognitive deficits, impairment of executive function, mood disturbance or psychosis. These impairments may occur in different stages of the disease and with varying intensity in individual patients. This chapter reviews the literature and authors’ clinical experiences of the assessment, mechanism, and prevalence of cognitive and psychiatric pathology occurring in Wilson disease. Evidence of pharmacologic and nonpharmacologic treatments is also discussed.

INTRODUCTION Wilson disease (WD) can present with such a variety of psychiatric and cognitive symptoms that it has been named the “great masquerader” (Soltanzadeh et al., 2007). Psychiatric symptoms have been considered a part of the clinical presentation in WD since the disease was first described in 1912. Eight out of 12 cases in Wilson’s monograph had psychiatric symptoms (Kinnier-Wilson, 1912). Irreversible cognitive decline was considered common in WD and psychiatric textbooks often cited this disorder as one of the classic examples of subcortical dementia. Cognitive problems and psychiatric symptoms were considered inseparable from the neurologic findings. Studies published in the last three decades started to point out that cognitive, psychiatric, and neurologic findings can present and evolve separately in the course of WD. Longitudinal observations have challenged the general view that cognitive impairment is irreversible in WD. Successful symptomatic treatment of psychiatric symptoms has been described even in severe cases. In addition, improved diagnostic tests make it easier to reach the diagnosis of WD when the initial presentation is atypical.

PHYSIOLOGIC BASIS FOR PSYCHIATRIC AND COGNITIVE SYMPTOMS IN WILSON DISEASE Copper is considered essential for normal brain function (Madsen and Gitlin, 2007) and its role in various psychiatric illnesses has been explored for decades. As early as the 1940s, copper was postulated to play an essential role in schizophrenia (Beard, 1959), although the relationship between peripheral copper levels and the copper effects in the brain has never been clarified. Studies showed increased levels of copper in plasma and hair of patients with acute schizophrenia compared to controls (Yanik et al., 2004; Rahman et al., 2009). Copper serum values were also elevated in criminal schizophrenic men compared to noncriminal subjects (Tokdemir et al., 2003). More recently, Wolf et al. (2006) showed that patients with schizophrenia had serum copper levels that were increased by 23% and serum ceruloplasmin elevated by 20% compared to controls. The exact mechanism of how copper participates in the course of psychiatric illness is not known. It was postulated that excess of serum copper may affect dopamine activity through several enzymes which are copper-dependent (dopa-decarboxylase,

*Correspondence to: Paula Zimbrean, MD, Yale University, Departments of Psychiatry and Surgery (Transplant), 20 York St Rm F611, New Haven CT 06511, USA. Tel: +1-203-688-6266, Fax: +1-203-737-2221, E-mail: [email protected]

122 P. ZIMBREAN AND J. SENIÓW beta-hydroxylase, and monoamine oxidase) (Jeon the other, because they co-occur and influence each et al., 1998). other. Conscious, aim-directed, intelligent behavior is In addition to its role in the dopamine system, serotonin based on many fundamental cognitive abilities such as abnormalities have been described in WD, in particular perception, memory and learning, language abilities, reaabnormalities of the presynaptic serotonin transporter soning, praxis, and construction. The distinction between (SERT) density. A significant negative correlation was testing cognitive function, verbal abilities or behaviors is found between severity of depression in WD and SERT often artificial and intended to facilitate description and density in the thalamus–hypothalamus region (Eggers measurement. In psychology as well as in neuroscience, et al., 2003) and hypothalamus (Hesse et al., 2003). More each cognitive function is usually divided into more spedetails about brain pathology in WD and pathogenesis are cific subsystems, with characteristics of their parameters presented in Chapters 8 (on brain pathology) and 5 (on (e.g., speed, precision). pathogenesis). Differentiation of cognitive function uses various criBrain lesions identical to those in WD occur in teria developed by general and clinical psychology based patients with other varieties of cirrhosis in which copper on theoretic models and empiric research. metabolism is not affected (Duchen, 1984). One study In the dynamic organization of the brain, cognitive (Rathbun, 1996b) assessed simultaneously cognitive function is inseparably linked with volition, motivation, domains in a cohort of 34 WD patients, 25 with neuroand affective systems, as well as with the system controllogic and/or hepatic symptoms and 9 asymptomatics, ling intentional, aim-directed behavior, which is the and measured urinary copper. There was no significant executive system. The latter, being a theoretic construct, correlation between the copper level and degree, freincludes a series of abilities such as inhibitory control, quency, and nature of neuropsychologic variables in working memory, cognitive flexibility, problem solving, either symptomatic or asymptomatic patients. Since the and planning. It is rare to find a patient who has defects diverse pathologies of WD have been usually directly in just one aspect of executive functioning. Rather, defeclinked to copper toxicity, the absence of any significant tive executive behavior involves a cluster of deficiencies correlation in Rathbun’s sample has heuristic implicaof which one may be dominant. Such pathology is tions for additional clinical and experimental studies. very sensitive to prefrontal damage and to prefrontoThis finding might suggest that direct copper toxicity subcortical loop impairment, which usually occur in may not be the mediating factor in cognition in WD WD. Therefore it is very difficult, in relatively short clinpatients. Possible mechanisms may consist of brain damical assessment, to identify at what level of hierarchically age secondary to copper toxicity or alternative biochemorganized cognitive behavior the primary pathology ical influences produced from pathologically involved arises. organs such as the liver (Rathbun, 1996b). During the neuropsychologic assessment clinicians In another study (Goldstein et al., 1968) of 17 WD pay great attention to differentiate primary from secondpatients who underwent decoppering treatment, psychoary cognitive deficits, specifically those impairments due logic tests showed no specific changes, suggesting the to alteration of consciousness, physical illness, affective lack of a direct relationship between copper toxicity impairment, speech disorders, and sensorimotor deficits. and cognition. These findings contradict other studies The latter frequently occur in WD population. which showed that effective decoppering therapies The quality of psychometric evaluation of the cogniarrested or reversed psychiatric, neurologic, and hepatic tive state depends on the characteristics of the tests, their symptoms (Cartwright, 1978). reliability, sensitivity, temporal stability, and predictive validity. However, tests are rarely designed for a specific EVALUATION OF COGNITIVE clinical population that may have its own specific limitaIMPAIRMENT AND PSYCHIATRIC tions (e.g., motor impairment or dysarthria that occurs in SYMPTOMS IN WILSON DISEASE WD). This reduces the reliability of the evaluation because some of the variables (e.g., even mild extrapyraClinical assessment midal movement or dysarthria) are in practice difficult to Before reviewing the literature on cognitive functioning control for. and psychiatric symptoms in WD it is worth discussing Many inconsistencies concerning cognitive dysfuncthe challenges in measuring these findings. tions in WD originate from the differences between the Cognitive function is a general term, used both in clinassessment tools used by different researchers. Some ical and common language, usually to determine the corresearchers prefer screening tools which are less sensitive. rect, logical, and effective reasoning, manifested in Other authors use highly sensitive tests, but assess selecverbal statements or behavior. In any assessment of cogtively a narrow aspect of cognitive functioning (e.g., nition it is difficult to separate one function/ability from divided attention or information-processing speed). In

COGNITIVE AND PSYCHIATRIC SYMPTOMS IN WILSON DISEASE the presence of movement disorders typical for WD, measurement of cognitive abilities might be altered when tests chosen by a researcher engage manual skills. The rarity of WD underlines the weaknesses of some studies resulting from small groups of participants, which are, in addition, diverse as to the severity and duration of the disease, its treatment duration, the characteristics of the clinical picture, and the severity of the pathology of the liver and brain. This reduces the reliability of generalizations about the entire WD population and makes it impossible to predict development or reversibility of specific dysfunctions in the individual patient (treated vs. nontreated). Methodologic difficulties limiting the reliability of cognitive assessment in WD were summarized by Frota et al. (2013): “Despite being present in the first patients described by Wilson in 1912, cognitive impairment has been little studied and is still a matter of controversy until today.” Due to the fact that the psychiatric, cognitive, and neurologic symptoms were often described together as one entity, it was difficult to distinguish them until the late 1970s, when investigators started assessing the symptoms in more detail. Structured psychiatric measures started being used to specifically describe mood, psychotic or behavioral symptoms. All the limitations described above in relation to cognitive testing apply to an extent to psychiatric measures: many scales have not been validated in this specific population, results could be skewed by motor or verbal deficits, and limited longitudinal follow-up does not allow accounting for normal variations in mood or anxiety.

123

brainstem, and cerebellum (see Chapter 10 for further details). Neuroimaging can provide important information for treatment-resistant illness, such as a case with rapid deterioration on computed tomography (CT) scan of the brain (bilateral putamen necrosis 15 months after diagnosis of WD) (Awada et al., 1990). However, there are no studies looking at correlations between MRI studies and specific psychiatric presentations in WD. There are few studies correlating cognitive functions with MRI findings in WD. Seniów and coworkers (2002) compared cognitive functioning in 50 WD patients with different type and degree of neurologic symptoms, 17 WD neurologically asymptomatic (some of them with very mild hepatic dysfunction), and 50 healthy controls. Multifocal brain pathology with cortex atrophy revealed on MRI was associated with more severe cognitive impairment than pathology within selective basal ganglia lesions. Hegde et al. (2010) examined 12 treated Indian WD patients with neurologic symptoms to assess their cognitive abilities in relation to MRI findings. The researchers measured sustained attention, focused attention, mental speed, motor speed, category fluency, working memory, planning and problem solving, concept formation, abstract thinking, cognitive flexibility, verbal learning, visuoconstructive ability, and visual memory. Patients with changes in the putamen had cognitive impairment in more than six examined domains, while those without signal changes in the putamen had deficits in fewer than four domains. A deficit in visual and verbal learning was confirmed in all patients with pathology of putamen.

Laboratory data and neuroimaging COPPER AND CERULOPLASMIN LEVELS

EVENT-RELATED POTENTIALS (ERP)

At this time there is not enough information about the correlation between specific psychiatric symptoms and levels of copper or ceruloplasmin (Dening and Berrios, 1989).

It has been earlier shown that ERPs may reflect cognitive decline in patients with dementia (e.g., Lai et al., 2010). Gunther and coauthors (2011) assessed cognitive functioning of 35 WD patients by dividing them into two groups: 25 with neurologic symptoms /basal ganglionic and/or cerebellar/ and 10 with hepatic or asymptomatic type of WD. Results of cognitive measurement tools (Structured Interview for the Diagnosis of Dementia of the Alzheimer Type, multi-infarct dementia, and dementias of other etiology (SIDAM) and Mini Mental State Examination (MMSE)) were correlated with auditory ERPs for a neurophysiologic assessment of cerebral information processing underlying cognition. The auditory P300 ERPs were studied using the Nihon Kohden Neuropack m device. Binaural auditory stimuli were presented in a random series. Twenty percent of stimuli were rare target tones of 2000 Hz; the remaining 80% were frequent tones of 1000 Hz at 75 dB. For each patient analysis was performed twice.

ELECTROENCEPHALOGRAM (EEG) Small studies have suggested that the beta EEG generator was more anteriorly localized in patients with more pronounced psychiatric symptoms and cognitive deficits (Dierks et al., 1999). Other studies have described abnormalities on EEG in patients with WD; however, no clear correlation between EEG findings and psychiatric symptoms has been established (Arendt et al., 1994a).

NEUROIMAGING The most common finding on the brain magnetic resonance imaging (MRI) in WD is T2 hyperintensity signal lesions in basal ganglia, as well as in thalamus,

124

P. ZIMBREAN AND J. SENIÓW

For each set of signal-averaged data 30 target responses were required and series mode rejection was performed. The P300 was measured by quantifying its amplitude and latency. Results of ERPs were compared between the two WD groups and in regard to the literature, about P300 studies (Lai et al., 2010). Patients with a neurologic course of the disease by trend showed lower amplitudes, delayed latencies of P300, and lower values in the cognitive tests. In structured interview SIDAM concerning cognitive functioning, the median of neurologic patients was only mild, reduced to 52 of a possible 55 points. The nonneurologic group had a normal median result of 54.5 points. The median for all participants was reduced to 53 points. In the neurologic group the median in MMSE was minimally decreased to 29 of 30, which represents normal values. Median amplitude and latency of P300 were 326 ms and 9 mV within normal range in all patients. There were only slight differences between the median of the neurologic and nonneurologic group, showing a reduced amplitude with 7.8 mV and delayed latency of 333 ms in the neurologic group, compared to 11.3 mV and 305.5 ms in the nonneurologic group. The authors concluded that the neurologic WD group – as compared to the nonneurologic group – only by trend showed reduced cognitive functioning, lower amplitudes, and delayed latencies of P300, but all median results were still within a normal range in both WD groups. Therefore, WD did not influence significantly the auditory ERPs in this study. The examined cohort was formed of patients with very discrete mental deficits; therefore it might not be a representative WD sample. Furthermore, the applied tests were not sufficiently sensitive to reveal mild cognitive impairments usually present in neurologic WD patients, and it might have been more useful if they were assessed using specific neuropsychologic tools (Seniów et al., 2002).

GENETIC STUDIES Genetic studies looking at correlations between various genes and psychiatric aspects of patients with WD are promising, although limited at this time to small studies and to limited psychiatric symptoms. Twenty-six patients with WD were evaluated via the Comprehensive Psychopathological Rating Scale (CPRS) and Karolinska Personality Scales (KPS) and findings were analyzed against the gene mutations present in ATP7B. Patients with the Trp779Stop mutation scored the lowest on CPRS, while heterozygous patients had the highest score on CPRS. When the results of personality testing were studied, those homozygous for Trp779Stop mutation had the highest scores on the psychopathy versus

conformity scale and socialization scale, and low scores on the impulsiveness, avoidant, and detachment scales. Patients homozygous for Thr977Met scored high on the psychopathy versus conformity scale as well. Patients with heterogeneous mutation His1069Gln/Arg1319Stop had the lowest score on the psychopathy versus conformity scale. Of note, there was low agreement between the interview-based ratings and self-ratings. The significance of the correlations of psychiatric findings with ATP7B genotype is unclear, as the authors noted, because the cohort size was small and, at the time of study publication, there were at least 300 gene sequence variants known to occur in WD (Portala et al., 2008).

COGNITIVE FUNCTION IN PATIENTS WITH WILSON DISEASE The literature review presented in Table 11.1 summarizes the types of cognitive deficits disclosed in various psychometric tests in WD groups. Groups are numerically and clinically diverse as WD population is very heterogeneous. The reversibility of cognitive dysfunctions under the influence of treatment is difficult to predict. Cognitive impairment can be more easily recognized in the neuropsychiatric form of WD or in end-stage liver WD than in mild hepatic WD (Ferenci et al., 2015) and therefore mixing of these patients during examination blurs pathologic specifics. In such a diverse and dynamic disease, prediction of a specific pattern of cognitive pathology is doubtful, as sickness pathology overlaps with the highly individualized premorbid abilities. When assessing cognition in WD, patients at the extreme end of the intelligence spectrum are the most difficult to categorize. The highly intelligent may appear average and not impaired and may continue fulfilling their social roles. There are also WD patients with such severe intellectual impairment that they are even excluded from clinical trials. These patients could usefully be described in methodologically correct case studies, based largely on observation of their behavior, nonstandardized simple clinical tasks, and interviews with the family. The solution for a reliable assessment of cognitive functioning WD patients with specific disease symptoms, including somatic, motor, cognitive, emotional symptoms, and speech dysfunction, requires implementation of an interdisciplinary, multicenter study with standardized measurement tools of cognitive-behavioral variables. Only in such a model could the data be compared and generalized. Describing the real state of a heterogeneous patient population requires experimental, prospective, randomized studies, including large groups, and this is not easy to implement in a rare disease at a single medical center. The selection of cognitive tests must

Table 11.1 Review of research studies on cognitive functioning in the Wilson disease population Authors and year

Study design/ participants (n)

Medalia et al. (1988)

Comparative study WDN ¼ 19 WDA ¼ 12 The whole group ¼ 35 Healthy controls ¼ 15

Lang et al. (1990)

Comparative study WDN (11) + WDH (6) ¼ 17 Healthy controls ¼ 17

Medalia et al. (1992)

Comparative study WDNP ¼ 16 WDH (5) + WDA (3) ¼ 8

Arendt et al. (1994b)

Comparative study WDN ¼ 19 Healthy controls ¼ 19 Comparative study WDN ¼ 17 Healthy controls ¼ 17 Comparative study WDN + WDH ¼ 25 WDA ¼ 9 The whole WD group ¼ 34 Without control group (test results compared with Indian norms)

Littman et al. (1995) Rathbun (1996b)

Measurement tools

Conclusions/cognitive deficits

Wechsler Adult Intelligence Scale-R (with exclusion of two subtests: Vocabulary and Object Assembly); Wechsler Memory Scale; Dementia Rating Scale; Wisconsin Card Sorting Test; Boston Naming Test; Trail Making Test; Animal Naming Test Chosen subtests from Wechsler Adult Intelligence Scale-R: Digit Span, Picture arrangement, Arithmethics; Multiple Choice Vocabulary Test; Raven’s Progressive Matrices; Short-term memory, Attention and Perceptual Speed; Benton Visual Retention Test – multiple choice version; Chosen subtests from German Intelligence Test: verbal fluency, logical reasoning, visual perception, and spatial imaginary; two subtests from German Intelligenz Struktur Test, assessing perception and visual-spatial imaginary Wechsler Memory Scale; Minnesota Multiple Personality Inventory (MMPI); The Sickness Impact Profile (SIP)

The WDN patients, as compared to the WDA, and the controls, revealed mild and selective cognitive dysfunctions: the lower IQ, a worse memory quotient, worse scores in Dementia Rating Scale (although test scores were still within the low area of norms)

Multiple Choice Vocabulary Test; Ravens Progressive Matrices; The Short Cognitive Performance Test Memory scanning task; Visual scanning task (tasks based on the Sternberg fixed-set paradigm) Wechsler Adult Intelligence Scale-R; Hooper Visual Organization Test; Ravens Progressive Matrices; Benton Visual Retention Test; Symbol Digit Substitution (written and oral versions); Peabody Picture Vocabulary Test-R; Wechsler Memory Scale; Purdue Pegboard Test

The WDN patients, as compared to the healthy individuals, had diminished logical reasoning The whole WD group – lower speed in perceptual tasks

The WDNP group: lower scores on Wechsler Memory Scale. more psychopathologic features in MMPI; more subjective complaints regarding the impact of WD on everyday functioning (SIP) WDN, as compared with the healthy, did not reveal any significant cognitive impairment All WDN complained about depressed mood WDH did not slow down information processing Motor deficits had a clear impact on cognitive tests performance Symptomatic (neurologic and hepatic) patients, as compared to asymptomatic patients, were slightly lower results in all cognitive tests, except visual-spatial task

Continued

Table 11.1 Continued Authors and year

Study design/ participants (n)

Seniów et al. (2002)

Comparative study WDN ¼ 50 WDA ¼ 17 The whole WD group ¼ 67 Healthy controls ¼ 50 WDN ¼ 12 Without control group (test results compared with the norms)

Hegde et al. (2010)

Xu et al. (2010)

Comparative study WDS ¼ 20 Healthy controls ¼ 38

Frota et al. (2013)

Comparative study WDN ¼ 18 WDA ¼ 2 The whole WD group ¼ 20 Healthy controls ¼ 20

Measurement tools

Conclusions/cognitive deficits

Wechsler Adult Intelligence Scale; Rey’s Auditory Verbal Learning Test; Benton Visual Retention Test (multiple choice version); Raven’s Progressive Matrices

The WDN group, as compared to the controls, showed a mild impairment in all tested cognitive functions The WDA patients had no deficits in any measured cognitive functions

Digit Vigilance Test; Color Trial Test and Triads Test; Digit Symbol Substitution; Finger Tapping Test; Animal Names Test; Rey’s Auditory Verbal Learning Test; Rey’s Complex Figure Test. Verbal N-back Test; Tower of London; Wisconsin Card Sorting Test Clinical Dementia Rating Scale; Memory abilities-experimental computer tasks; Perceptual learning tasks in situation with low vs high external noise

The WDN group had deficits in: psychomotor speed, verbal working memory, sustained and focused attention, verbal learning, visuoconstructive ability, verbal memory, mental speed, verbal fluency, set-shifting ability, visual memory Only 1 patient (with normal MRI) did not reveal any cognitive deficits

Mini Mental State Examination; Digit span forward and backward task; Memory Test of Figures; Clock Drawing Test; Verbal Fluency Test; CERAD naming test; Stroop Test; Frontal Assessment Battery; Mattis Dementia Rating Scale; Wisconsin Card Sorting Test; Hooper Visual Organization Test; Block Design (a subtest from WAIS)

7 WDS patients had no deficits in general cognitive functioning, 13 patients revealed mild to moderate impairment All patients had deficits in both forms of category learning and in perceptual learning but only in high external noise; in low external noise these functions were relatively spared The WDN patients had various isolated cognitive impairments, especially concerning executive functions Only 10% of WD patients did not reveal any executive disturbances

Ma et al. (2013)

Comparative study WDN ¼ 30 Healthy controls ¼ 30

Wenisch et al. (2013)

Comparative study The whole WD group ¼ 31 WDN ¼ 18 WDH + WDA ¼ 13 Without control group (compared with test norms)

Han et al. (2014)

Comparative study WDN ¼ 35 Healthy controls ¼ 35

Iwa
nski et al. (2015)

Comparative study WDN ¼ 33 WDH + WDA ¼ 34 Healthy controls ¼ 43

Mini Mental State Examination; Wechsler Adult Intelligence Scale-R; Verbal Fluency Test; Digit Span Test (forward and backward); The Stroop Test; Wisconsin Card Sorting Test; Decision Making Tasks (under risk condition) Chosen subtests from Wechsler Adult Intelligence Scale-III: Information, Similarities, Digit Span, Digit Symbol; Symbol Search; Raven’s Progressive Matrices; California Verbal Learning Test; Modified Taylor Complex Figure-recall; Chosen subtests from Wechsler Memory Scale: Letter-Number Sequencing, Visual Span; Wisconsin Card Sorting Test; Stroop Test; Test Uwagi D2; Baddeley’s Double Test Task; Trial Making Test Wechsler Adult Intelligence Scale-R; Attention Network Test; Short-term memory and attention span (digits repetition forward and backwards); Verbal fluency task Test of Everyday Attention

The WDN patients had worse results in a task that requires decision making in a situation of risk and in tests: Verbal Fluency, Digit Span, The Stroop Test, Wisconsin Card Sorting Test (all tests are sensitive for frontal damage)

In all WD patients verbal intelligence and episodic memory and visuospatial functions were preserved In WDN patients pathology concerned more executive functions: working memory, abstract reasoning, and inhibitory process

The WDN patients had diminished alertness to what may secondarily disturb more complex attention subsystem and intellectual skills The other measured cognitive functions did not differ significantly from those in healthy individuals The WDN patients had deficiency in all attention subsystems (sustained, selective, divided, switching); however relatively the most disturbed was attention switching WDH + WDA group – an isolated deficit of sustained attention

WD, Wilson disease; WDS, WD symptomatic; WDA, WD clinically asymptomatic; WDH, WD hepatic; WDN, WD neurologic; WDNP, WD neuropsychiatric.

128

P. ZIMBREAN AND J. SENIÓW

be carefully matched with the typical symptoms of WD (accurate tests, responsive to change, reliable, functional, acceptable and understandable by the patient, and results that are not significantly influenced by neurologic symptoms). In clinical research it is worth determining in advance whether the measurement of cognitive functioning is done at the level of: (1) impairment; (2) disability; or (3) participation (e.g., social, cultural, economic). Such proceedings are recommended by the World Health Organization (1980) for neurorehabilitation. There is a need for more research to assess the impact of pharmacologic or behavioral treatment on cognitive functioning. It is also important to assess the preserved functions and well as the impaired ones. The failure of strenuous attempts to find the typical profile of cognitive and affective dysfunctions in WD patients appears discouraging. Sometimes it may be more valuable to describe individual patients and their preserved cognitive resources. It is important that rehabilitation programs are conducted in parallel with pharmacotherapy in order to assess outcomes. A few decades ago cognitive dysfunction in the neurologic form of WD was sometimes called subcortical dementia. This implied that the disease – especially if left untreated – frequently resulted in gradual intellectual deterioration which met the criteria for dementia (Cummings and Benson, 1984). However it seems that, when properly treated, the majority of WD patients reveal only selective, mild cognitive deficits, that do not meet the current criteria of dementia (Seniów et al., 2002; Frota et al., 2009; Wenisch et al., 2013). There is a relatively good prognosis for maintenance of cognition in asymptomatic patients with treated WD (Medalia et al., 1988; Seniów et al., 2002). None (Lang et al., 1990) or only mild and isolated cognitive deficits may occur in some individuals with WD with the hepatic form without overt encephalopathy (Tarter et al., 1987; Szutkowska-Hoser et al., 2005). They may include slightly diminishing visuopractic abilities, psychomotor speed, learning, and arithmetic skills.

PSYCHIATRIC PRESENTATIONS IN WILSON DISEASE Psychiatric symptoms have been described in all stages of WD. A particular focus has been placed on the presence of psychiatric symptoms at the initial stages of the disease, in the absence of hepatic or neurologic manifestations, since it has been linked with delays in diagnosis. A meta-analysis of the case reports when the psychiatric disease was the initial presentation of WD showed that average time between psychiatric symptoms and diagnosis of WD was 2.42 years (SD 2.97) (Zimbrean

and Schilsky, 2014). In comparison, the median time between initial symptoms and when the diagnosis of WD was first established was 1.5 years for neurologic WD and 0.5 years for hepatic disease (Oder et al., 1991). Cohort studies have reported that up to 64.8% of patients with WD reported psychiatric symptoms at their initial presentation (Akil et al., 1991) and up to 20% of patients had seen a psychiatrist prior to the formal diagnosis of WD (Dening and Berrios, 1989). Table 11.2 summarizes the case studies in which psychiatric presentations occurred in patients with WD disease prior to any neurologic or hepatic manifestation. Studies have indicated that up to 64.8% of patients had psychiatric symptoms in the beginning of illness (with or without hepatic or neurologic findings), while up to 70.7% of patients experienced neuropsychologic symptoms at some point in the course of the disease (Pan et al., 2005). The presence of multiple psychiatric conditions is common. A cross-sectional study of 50 patients using the Structured Clinical Diagnostic Interview (SCID) found that 40% met criteria for more than three psychiatric diagnoses (Svetel et al., 2009). Table 11.3 summarizes the prevalence of psychiatric symptoms in WD.

Mood disorders The occurrence of depressive symptoms has been well documented in patients with WD. The prevalence of major depressive disorder has been reported to be between 4% (assessed by the SCID: Shanmugiah et al., 2008) and 47.8% (assessed by Advanced Neuropsychiatric Tools and Assessment Schedule: Carta et al., 2012a). It is important to note that depression was often described in the beginning of the illness, therefore it could not be explained by the psychosocial consequences of a chronic medical disease. In one of the three prospective studies looking at psychiatric symptoms in the course of WD disease, depression remained present in a significant part of the group: 1 in 3 patients had depression on initial evaluation and 1 in 4 continued to have depression at follow-up (Dening and Berrios, 1990). At least two systematic studies have documented a high prevalence of bipolar disorders in WD patients: a cross-sectional study in which each patient was examined by two psychiatrists using SCID diagnosed bipolar disorder in 18% of 50 patients (Shanmugiah et al., 2008). A more recent case-control study of 23 patients using Mood Disorder Questionnaire (a screening instrument for mania or hypomania) showed that 30% of those studied met criteria for bipolar disorder, while 39% met criteria for mania or hypomania (Carta et al., 2012a).

Table 11.2 Psychiatric symptoms as the initial presentation of Wilson disease Days from onset of psychiatric symptoms to diagnosis

Psychiatric treatment

Author and year

Age (years)

Gender

Psychiatric symptom

Neurologic symptom

Carr and McDonnell (1986) Chung et al. (1986) Bouix et al. (1987)

13

M

Behavioral problems, academic decline

Involuntary limb and head movement

1460

No details

30 24

F M

Parkinsonism Dysarthria

14 3650

Haloperidol, chlorpromazine

20 16

F F

Psychosis Concentration problems, poor academic performance, catatonia, conversion Catatonic schizophrenia Personality change

Parkinsonism, dystonia Neuroleptic malignant syndrome

480 90

18

M

Parkinsonism

4380

No details; psychiatric hospitalization Amitriptyline, clomipramine, perphenazine Neuroleptics, chelation

12

M

22

Awada et al. (1990) Buckley et al. (1990) Saint-Laurent (1992) Davis and Borde (1993) Gwirtsman et al. (1993) Garnier et al. (1997) Shah and Kumar (1997) Walter and Lyndon (1997) Keller et al. (1999)

Tremor, drooling

365

Benzodiazepines, chelating agents

F

Psychosis (three episodes) Irritability, academic problems, catatonia Anorexia nervosa

None

1825

Eating disorder behavioral treatment

39

M

Psychosis

Akathisia, dysarthria, resting tremor

180

23

M

Schizophrenia

Dysarthria, ataxia, tremor

60

21

M

Major depressive disorder

Drooling, motor retardation

84

38

M

Extrapyramidal symptoms

180

Muller et al. (1998)

23

M

Major depressive disorder then mania after antidepressants were started Psychosis, depression

Neuroleptics, D-penicillinamine, zinc, antiparkinsonian Electroconvulsive therapy, haloperidol Tricyclic antidepressants, mianserine, lithium, decoppering agents Antidepressants, interpersonal therapy, neurorehabilitation

Parkinsonism, dysphagia

1215

Muller (1999)

13

M

Stiller et al. (2002)

18

M

Suicide attempts, mania, psychosis Depression

Stuttering

2555

Haloperidol, pipamperone, bipiriden, alprazolam Zuclopenthixol

2190

Psychotherapy Continued

Table 11.2 Continued

Author and year

Age (years)

Gender

Psychiatric symptom

Neurologic symptom

Days from onset of psychiatric symptoms to diagnosis

Stiller et al. (2002) (2) Campos Franco et al. (2003) Rodrigues and Dalgalarrondo (2003) Sagawa et al. (2003) Shah and Vankar (2003) Smit et al. (2004) Chan et al. (2005)

19

M

Psychosis, schizophrenia

Dysarthria

730

58

M

Depression, psychosis

26

M

Depression with psychosis

Bradykinesia

600

17

F M

Dysarthria and finger tremor, rigidity Muttering, tremor

2430

11

Psychosis (olfactory paranoia) Psychosis

30

Alprazolam, risperidone, divalproex

21 35

M F

Depression Depression, suicide attempt

Bradykinesia Parkinsonism

30 1460

11

F

Major depressive disorder

Abnormal movements

270

Fluoxetine Electroconvulsive therapy, selective serotonin reuptake inhibitors, haloperidol, eventually chelation Thioridazine

18

M

Mania

Extrapyramidal symptoms

120

Lithium

10

M

Drooling, seizures

600

Jukic et al. (2006) Barbosa et al. (2007)

26 16

F M

Attention deficit disorder, violent rages Psychosis Academic decline, aggression, irritability

Choreoathetosis Jerks of four limbs, unsteady gait, impaired coordination, action tremor of upper limbs, difficulty in swallowing solid foods

7 730

Carbamazepine, Penicillamine, vitamin B6, and zinc supplement Haloperidol Haloperidol, chlorpromazine

Benhamla et al. (2007) Kumawat et al. (2007b)

59

M

7

Elavil, haloperidol

17

M

180

Fluoxetine

Krishnakumar and Riyaz (2005) Chand and Murthy (2006) Lin et al. (2006)

Major depressive disorder with psychosis Obsessive-compulsive disorder

Psychiatric treatment Antipsychotics

6  365

None

Electroconvulsive therapy, risperidone, amytriptyline, thiaridazine

Machado et al. (2008)

26

M

Manic episode

Tremor in the upper limb (1 year later)

1215

Aggarwal and Bhatt (2009)

18

M

Dysarthria, dystonia, abnormal limb posture

1095

Aravind et al. (2009) Habek et al. (2009)

41

M

Valproic acid, chlorpromazine

F

Rigidity, bradykinesia, dysphagia, dysarthria None

1825

45

7

Tatay et al. (2010) Alva-Moncayo et al. (2011)

23 13

M M

Irritability, personality change, behavioral disturbance Mania (four episodes) Generalized anxiety disorder Bipolar I Inexpressive face, indifference

Lithium initially; after chelation treatment, patient was asymptomatic without psychiatric medications for years No details

Tremor Dystonia, bradykinesia, stiffness

365 730

No psychotropic medications; penicillamine only Lithium, risperidone No details

Silva et al. (2011)

5

M

Diadocokinesia

365

Methylphenidate

Nayak et al. (2012)

19

M

Attention-deficit hyperactivity disorder Catatonia

14

Aljukic et al. (2013)

18

M

Behavioral problems (lack of interest in the surroundings, decreased interactions with family)

Aphasia, drooling, tongue protrusion and cogwheel rigidity

Unspecified “prior history”

Electroconvulsive therapy, olanzapine trifluoperazine 15 mg/day, trihexyphenidyl 6 mg/day Clozapine, pyridoxine

Araujo-de-Freitas et al. (2014)

31

M

Drooling, cogwheeling

195

Yigit et al. (2014)

25

M

Basu et al. (2015)

19

F

Depression, psychotic symptoms after 6 months Psychosis; persecutory delusions, auditory and visual hallucinations Paranoia, catatonia

Zimbrean and Schilsky (2015)

38

F

Depression initially, mania and psychosis after diagnosis of Wilson disease

Perioral numbness

Days

365

365

Various antidepressants initially; escitalopram with paliperidone, mirtazapine No details

Risperidone, olanzapine, aripiprazole, quetiapine initially for psychosis without improvement; lorazepam when catatonia developed Escitalopram, quetiapine, clonazepam

Table 11.3 Prevalence of psychiatric problems in Wilson disease Findings

Author and year

Type of study

n

Measure

CSec Medalia and Scheinberg (1989)

24

MMPI

Dening and Berrios (1990)

P

129

Akil et al. (1991)

R

41

Oder et al. (1991)

P

45

HDS ZDS ADS MMSE

Huang and Chu (1992)

R

71

CE

Psychiatric symptoms Depression

Patients with neurologic impact scored higher on the Schizophrenia and depression scale AMDP system 51% had 15% psychopathologic features 20% had seen a psychiatrist prior to the diagnosis of WD CE 64.8% had psychiatric 27% symptoms in the beginning of the illness 26.6% mood symptoms; 31.1% affective instability Mean age for onset of symptoms 25.3  2.4 for WD with psychiatric features

Mania/ hypomania

Anxiety

Psychosis

Personality changes

Rare

Other

15% irritability 15% “incongruent behavior”

45.9%

6.7% psychosis

11.3% Schizophrenia; was 38.0% the initial diagnosis for 4.2%

Catatonia, cognitive changes, anxiety, psychosis 15.5% past suicide attempts; 20% impaired social judgment 24.4% belligerence

Rathbun (1996a) Portala et al. (2000)

CSec CSec

Bono et al. (2002)

CR

Pan et al. (2005)

R

Soltanzadeh et al. R (2007) Srinivas et al. (2008) R

Shanmugiah et al. (2008)

CSec

34 26

MNB CPRS KSP

26% Autonomic disturbances, muscular tension, fatigability, decreased sexual interest, lack of appropriate emotion, concentration difficulties, reduced sleep 21 CE 19% had initial psychiatric symptoms 41 CE 70.7% had neuropsychologic symptoms 44 CE 44% had psychiatric or sleep problems 4.2% had psychiatric 15 out of CE findings; cohort 33.3% of psychiatric of 350 symptoms improved with decoppering treatment alone 50 SCID, two 24% met diagnosis of a 4% MDD psychiatrists psychiatric disorder 2% dysthymia

20%

2.5% Bipolar

2.0%

1.4% Schizophrenia 0.8% Schizoaffective, sensitivity to neuroleptics

18% had bipolar disorder Continued

Table 11.3 Continued Findings

Author and year

Type of study

n

Measure

Psychiatric symptoms Depression

Svetel et al. (2009)

CSec

50

SCID NPI

Mercier-Jacquier et al. (2011)

R

19

CE

Depression 40% had >3 36% psychiatric diagnoses; 14% had two psychiatric diagnoses; 18% had one psychiatric diagnosis; 70% had one psychiatric symptom 21% had psychiatric 15.7% disorders depression

Netto et al. (2011)

CSec

24

PSQI

Nevsimalova et al. (2011)

CSec

55

ESS RBD-SQ MSLT (24)

Carta et al. (2012a)

CC

23

ANTAS MDQ

30% Bipolar disorder

47.8% MDD

Mania/ hypomania

Anxiety Anxiety 62%

Psychosis

Personality changes

Other Irritability 26%; disinhibition 24%; apathy 24%

One anorexia nervosa, two impulsivity PSQI worse in WD than in controls ( p ¼ 0.03) Lower sleep duration, decreased sleep efficiency, and increased wakefullness 39% mania/ 8.7% Panic hypomania disorder

CPRS, Comprehensive Psychopathologic Rating Scale; MMSE, Mini Mental Status Examination; MMPI, Minessota Personality Inventory; HDS, Hamilton Depression Scale; ZDS, Zeissen Depression Scale; ADS, Adjective Depression Scale; MNB, Michigan Neuropsychological Battery; KSP, Karolynskaya Scale of Personality; SCID, Structured Clinical Diagnostic Interview; CE, clinical examination; ANTAS, Advanced Neuropsychiatric Tools and Assessment Schedule; MDQ, Mood Disorders Questionnaire; PSQI, Pittsburg Sleep Quality Index; ESS, Epworth Sleepiness Scale; RBD-SQ, rapid eye movement behavior disorder questionnaire; MSLT, Multiple sleep latency test; MDD, major depressive disorder; CSer, case series; CSec, cross-sectional; CC, case control; CR, case registry; R, retrospective; P, prospective.

COGNITIVE AND PSYCHIATRIC SYMPTOMS IN WILSON DISEASE

135

Anxiety

Other psychiatric symptoms

Between 20% and 62% of patients with WD are reported to have significant anxiety symptoms (Rathbun, 1996a; Svetel et al., 2009), while 8.7% of patients met criteria for panic disorder (Carta et al., 2012a).

Other psychiatric symptoms reported in WD are obsessive compulsive disorder, both in the beginning of illness (Kumawat et al., 2007a), but also associated with treated WD (Sahu et al., 2013) and catatonia (Davis and Borde, 1993; Nayak et al., 2012; Basu et al., 2015). Probably more significant, in addition to wellconstituted psychiatric syndromes discussed above, patients with WD may present at various points in their lives with behavioral changes that do not meet criteria for specific psychiatric disease: irritability (prevalence 15–25%: Dening and Berrios, 1990; Svetel et al., 2009), impaired social judgment or disinhibition (Oder et al., 1991; Svetel et al., 2009), apathy (Svetel et al., 2009), “belligerence” (Oder et al., 1991), or “incongruent behavior” (Dening and Berrios, 1990). Personality changes are described in up to 57% of patients with WD (Dening and Berrios, 1990; Akil et al., 1991). Due to the atypical presentation, these patients rarely receive psychiatric or psychologic assistance, and their quality of life suffers.

Psychotic symptoms In a Chinese group, 11% of patients with WD met criteria for schizophrenia (Huang and Chu, 1992). The typical presentation is a patient with psychotic symptoms who is started on antipsychotic medications and develops significant neurologic symptoms, which are interpreted as being side-effects related to antipsychotics. When these side-effects take an unusual presentation, neurologic consultation is requested, and this sometimes leads to the diagnosis of WD. These particular cases are associated with significant delays in diagnosis and treatment. Cases have been reported in patients who spent years in psychiatric facilities only to be diagnosed with WD and have their psychiatric symptoms resolved when chelation therapy was instituted. Up to 4.2% of patients seen in WD clinic were reported to present with initial symptoms of psychosis (Huang and Chu, 1992). Psychotic symptoms may also occur during the treatment phase of WD in up to 40% of cases (McDonald and Lake, 1995; Aggarwal and Bhatt, 2012). Cases of new-onset psychosis and euphoria have been reported post liver transplantation for WD (Al-Hilou et al., 2012). One patient developed acute neuropsychiatric presentation (major depressive disorder with psychotic features, suicide attempt, involuntary placement of his arms, speech disturbance) immediately after general anesthesia and was subsequently diagnosed with WD and treated accordingly (Dikmen et al., 2008).

Sleep disturbances Poor quality of sleep, frequent nocturnal awakening, and other sleep disturbances like sleep paralysis or cataplexy have been described (Portala et al., 2002). Patients with WD tend to have worse daytime somnolence compared to controls (Netto et al., 2011). Another study compared 55 patients with WD disease with age- and sex-matched controls, and the results showed that patients with WD tended to have lower sleep time, decreased sleep efficiency, and increased wakefulness. Case reports have described the development of excessive somnolence confirmed on multiple sleep latency test (Amann et al., 2015); in one case, severe hypersomnia developed late in the course of the illness, 3 years after initiation of depression, and after 2 years of cognitive impairment (Kim et al., 2011).

TREATMENT OF PSYCHIATRIC SYMPTOMS IN WILSON DISEASE One approach to treatment of psychiatric symptoms in WD is to expect that primary treatment (namely decoppering therapy) will improve the psychiatric symptoms by addressing the primary disease process, without any additional intervention needed. Up to one-third of patients with psychiatric symptoms will improve with decoppering treatment alone (Modai et al., 1985; Bachmann et al., 1989; Srinivas et al., 2008). In support of this approach is the finding that D-penicillamine treatment is believed to impact brain metabolism (De Volder et al., 1988), although the clinical correlation of this finding has not yet been fully investigated. Liver transplantation did not always improve the neuropsychiatric symptoms, as improvement was noted only in a minority of patients (Geissler et al., 2003; Boeka et al., 2011; Al-Hilou et al., 2012). One case, however, reported resolution of psychosis in a patient with WD disease after liver transplantation (Sorbello et al., 2011). At this time liver transplantation is not recommended for WD patients with psychiatric symptoms in the absence of liver disease due to the lack of prospectively studied outcomes for this subset of patients. A second approach involves treating psychiatric symptoms with specific interventions. Multiple modalities of symptomatic treatment for psychiatric presentations in WD have been described in the literature, including biologic approaches and psychotherapy. At this time, in the USA there are no treatments approved

136

P. ZIMBREAN AND J. SENIÓW

by the Food and Drug Administration for psychiatric symptoms in WD; however, an increasing body of evidence supports the use of psychotropics and/or psychotherapy when symptoms are causing significant functional impairment. Examples of such situations include psychotic episodes, mania, or severe depression. The use of psychotropic medications in WD is supported by multiple cases that describe efficacy of these treatments, but also by the evidence regarding neurotransmitter abnormalities (such as abnormalities of SERT in patients with WD and depression: Eggers et al., 2003; Hesse et al., 2003), which provide a theoretic basis for the use of psychotropic agents. The use of lithium (Loganathan et al., 2008; Tatay et al., 2010; Rybakowski et al., 2013), divalproex (Shah and Vankar, 2003; Aravind et al., 2009), serotonin reuptake inhibitors (Smit et al., 2004; Kumawat et al., 2007b), trycyclic antidepressants (Buckley et al., 1990; Benhamla et al., 2007), methylphenidate (Silva et al., 2011), benzodiazepines (Muller, 1999; Shah and Vankar, 2003; Zimbrean and Schilsky, 2015), haloperidol (Chung et al., 1986), risperidone (Shah and Vankar, 2003), quetiapine (Kulaksizoglu and Polat, 2003), and clozapine (Krim and Barroso, 2001) with good treatment results has been described in patients with WD. Anecdotal successful use of cyproterone for hypersexuality associated with psychosis in a patient with WD has been described (Volpe and Tavares, 2000). Lithium is an attractive choice as a mood stabilizer considering that it is not metabolized by the liver and does not carry the risk of extrapyramidal side-effects. With the cognitive impact of lithium still being debated (Pfennig et al., 2014), it is prudent to monitor cognitive abilities during lithium therapy in patients with WD. Another interesting choice of psychotropic medication is quetiapine, which has the advantage of reduced risk of extrapyramidal side-effects. Neuroleptic malignant syndrome has been reported during therapy with the use of various neuroleptics in WD (Kontaxakis et al., 1988; Buckley et al., 1990; Chroni et al., 2001; Czlonkowska et al., 2005). Acute focal dystonic symptoms induced by clomipramine have been described (Litwin et al., 2013). We have however to remember that psychotropic medications must be used in WD with great caution, understanding metabolic disorders and potential adverse events which may cause rapid and severe clinical deterioration (see Table 11.4). Another biologic treatment effective for mood symptoms in patients with WD is electroconvulsive therapy (Negro and Louza Neto, 1995; Shah and Kumar, 1997; Rodrigues et al., 2004; Avasthi et al., 2010; Vaishnav et al., 2013). Cognitive-behavioral therapy (Kumawat et al., 2007a) and interpersonal therapy (Keller et al.,

Table 11.4 Precautions in prescribing psychotropic medications to patients with Wilson disease Avoid medications with potential hepatotoxic effect (e.g., valproic acid, duloxetine) Whenever possible, preference should be given to medications with minimal hepatic metabolism (e.g., lithium, gabapentin) When prescribing neuroleptics, preference should be given to those with reduced risk for causing extrapyramidal side-effects (e.g., quetiapine) in minimal effective dose Unless there is evidence of an independent psychiatric condition separate from Wilson disease or repeated recurrence of psychiatric symptoms, consideration should be given to tapering off psychotropic medications once full remission of symptoms has been achieved Consider monitoring of cognitive status when treating patients with Wilson disease with psychotropic agents that can impact cognition (e.g., benzodiazepines, lithium)

1999) have been used independently or in association with other interventions, with good results.

Significance of psychiatric symptoms in Wilson disease Some authors have hypothesized that psychiatric symptoms indicate a more severe or advanced disease, may signal irreversible brain damage, or are secondary to metabolic disturbances produced by the liver disease, such as hyperammonemia associated with hepatic encephalopathy (Rathbun, 1996a). The presence of psychiatric symptoms in general in WD has been associated with lower quality of life (Svetel et al., 2011; Petrovic et al., 2012). In particular, the presence of bipolar disorders in patients with WD has been associated with a poorer quality of life (Carta et al., 2012b) and with higher frequency of brain damage (Carta et al., 2015). Presence of psychiatric symptoms is feared to lead to nonadherence with medical treatment, which further worsens the overall prognosis of WD (Mrabet et al., 2013).

CONCLUSION Cognitive, emotional, psychotic, and other less specific psychiatric problems are common in the WD population and can occur at any stage of the illness. These symptoms can greatly impair a patient’s quality of life and delay proper diagnosis. Improvement or resolution of psychiatric WD symptoms is – besides hepatic and neurologic symptoms – usually the goal of treatment. Dementia, understood as severe and irreversible cognitive impairment, preventing independent functioning, is not expected in properly treated WD patients. New

COGNITIVE AND PSYCHIATRIC SYMPTOMS IN WILSON DISEASE psychotropic medications allow safer symptomatic treatment and can improve a patient’s quality of life, and should be considered (however, with caution) independently of primary treatment of WD.

REFERENCES Aggarwal A, Bhatt M (2009). Eye sign in an 18 year old man with psychosis. BMJ (Clinical research ed) 339: b3494. Aggarwal A, Bhatt M (2012). Recovery from severe neurological Wilson’s disease with copper chelation. Mov Disord 27: S71. Akil M, Schwartz JA, Dutchak D et al. (1991). The psychiatric presentations of Wilson’s disease. J Neuropsychiatry Clin Neurosci 3: 377–382. Al-Hilou H, Hebbar S, Gunson BK et al. (2012). Long term outcomes of liver transplantation for Wilson’s disease: a single centre experience. Gut 61: A210. Aljukic N, Sutovic A, Avdic L et al. (2013). A neuropsychiatric presentation of Wilson’s disease – case report. Conference: 21st European Congress of Psychiatry, EPA, 28. Eur Psychiatry. Alva-Moncayo E, Castro-Tarin M, Gonzalez-Serrano A (2011). Wilson disease. A case report and review of the literature Revista medica del Instituto Mexicano del Seguro Social 49: 331–334. Amann VC, Maru NK, Jain V (2015). Hypersomnolence in Wilson disease. J Clin Sleep Med 11: 1341–1343. Araujo-De-Freitas L, Rocha M, Gondim V et al. (2014). Major depression caused by Wilson’s disease. Rev Bras Psiquiatr 36: 184. Aravind VK, Krishnaram VD, Neethiarau V et al. (2009). Wilson’s disease – a rare psychiatric presentation. J Indian Med Assoc 107: 456–457. Arendt G, Hefter H, Stremmel W et al. (1994a). The diagnostic value of multi-modality evoked potentials in Wilson’s disease. Electromyography & Clinical Neurophysiology 34: 137–148. Arendt G, Hefter H, Stremmel W et al. (1994b). The diagnostic value of multi-modality evoked potentials in Wilson’s disease. Electromyogr Clin Neurophysiol 34: 137–148. Avasthi A, Sahoo M, Modi M et al. (2010). Psychiatric manifestations of Wilson’s disease and treatment with electroconvulsive therapy. Indian J Psychiatry 52(1): 66–68. http://dx.doi.org/10.4103/0019-5545.58898. Awada A, Al Rajeh S, Al Qorain A et al. (1990). Wilson’s disease in a Saudi Arabian female patient. Rapid changes in cerebral x-ray computed tomography. Rev Neurol 146: 306–307. Bachmann H, Lossner J, Kuhn HJ et al. (1989). Long-term care and management of Wilson’s disease in the GDR. Eur Neurol 29: 301–305. Barbosa ER, Silveira-Moriyama L, Machado AC et al. (2007). Wilson’s disease with myoclonus and white matter lesions. Parkinsonism Relat Disord 13: 185–188. Basu A, Thanapal S, Sood M et al. (2015). Catatonia: an unusual manifestation of Wilson’s disease. Journal of Neuropsychiatry and Clinical Neurosciences 27: 72–73.

137

Beard AW (1959). The association of hepatolenticular degeneration with schizophrenia. A review of the literature and case report. Acta Psychiatr Scand 34: 411–428. Benhamla T, Tirouche YD, Abaoub-Germain A et al. (2007). The onset of psychiatric disorders and Wilson’s disease. Enc
ephale 33: 924–932. Boeka AG, Solomon AC, Lokken K et al. (2011). A biopsychosocial approach to liver transplant evaluation in two patients with Wilson’s disease. Psychol Health Med 16: 268–275. Bono W, Moutie O, Benomar A et al. (2002). Wilson’s disease. Clinical presentation, treatment and evolution in 21 cases. Revue de Medecine Interne 23: 419–431. Bouix J, Courtine P, Alezrah C (1987). Wilson’s disease and psychiatry: about one case. L’Encephale: Revue de psychiatrie clinique biologique et therapeutique 13: 113–116. Buckley P, Carmody E, Hutchinson M (1990). Wilson’s disease and the neuroleptic malignant syndrome. Irish Journal of Psychotherapy & Psychosomatic Medicine 7: 138–139. Campos Franco J, Dominguez Santalla MJ, Tome Martinez De RITUERTOS et al. (2003). Late-onset Wilson’s disease. Anales de Medicina Interna (Madrid, Spain : 1984) 20: 416–418. Carr A, McDonnell DJ (1986). Wilson’s disease in an adolescent displaying an adjustment reaction to a series of life stressors: a case study. Journal of Child Psychology & Psychiatry & Allied Disciplines 27: 697–700. Carta MG, Farina GC, Sorbello O et al. (2012a). The risk of bipolar disorders and major depressive disorders in Wilson’s disease: results of a casecontrol study. Int Clin Psychopharmacol 28, e61. Carta MG, Mura G, Sorbello O et al. (2012b). Quality of life and psychiatric symptoms in Wilson’s disease: the relevance of bipolar disorders. Clinical Practice and Epidemiology in Mental Health 8: 102–109. Carta MG, Saba L, Moro MF et al. (2015). Homogeneous magnetic resonance imaging of brain abnormalities in bipolar spectrum disorders comorbid with Wilson’s disease. Gen Hosp Psychiatry 37: 134–138. Cartwright GE (1978). Diagnosis of treatable Wilson’s disease. N Engl J Med 298: 1347–1350. Chan KH, Cheung RT, Au-Yeung KM et al. (2005). Wilson’s disease with depression and parkinsonism. J Clin Neurosci 12: 303–305. Chand PK, Murthy P (2006). Mania as a presenting symptom of Wilson’s disease. Acta Neuropsychiatrica 18: 47–49. Chroni E, Lekka NP, Tsibri E et al. (2001). Acute, progressive akinetic rigid syndrome induced by neuroleptics in a case of Wilson’s disease. J Neuropsychiatry Clin Neurosci 13: 531–532. Chung YS, Ravi SD, Borge GF (1986). Psychosis in Wilson’s disease. Psychosomatics: Journal of Consultation Liaison Psychiatry 27: 65–66. Cummings JL, Benson DF (1984). Subcortical dementia – review of an emerging concept. Arch Neurol 41: 874–879.

138

P. ZIMBREAN AND J. SENIÓW

Czlonkowska A, Tarnacka B, Litwin T et al. (2005). Wilson’s disease – cause of mortality in 164 patients during 1992–2003 observation period. J Neurol 252: 698–703. Davis EJ, Borde M (1993). Wilson’s disease and catatonia. Br J Psychiatry 162: 256–259. Dening TR, Berrios GE (1989). Wilson’s disease. Psychiatric symptoms in 195 cases. Arch Gen Psychiatry 46: 1126–1134. Dening TR, Berrios GE (1990). Wilson’s disease: a longitudinal study of psychiatric symptoms. Biol Psychiatry 28: 255–265. De Volder A, Sindic CJ, Goffinet AM (1988). Effect of D-penicillamine treatment on brain metabolism in Wilson’s disease: a case study. J Neurol Neurosurg Psychiatry 51: 947–949. Dierks T, Kuhn W, Oberle S et al. (1999). Generators of brain electrical activity in patients with Wilson’s disease. European Archives of Psychiatry & Clinical Neuroscience 249: 15–20. Dikmen PY, Baybas S, Aydinlar EI et al. (2008). Case of Wilson’s disease with acute neuropsychiatric symptoms after general anesthesia. Noropsikiyatri Arsivi / Archives of Neuropsychiatry 45: 21–22. Duchen LW (1984). Nutritional deficiencies and metabolic disorders. In: JH Adams, LW Duchen (Eds.), Greenfield’s neuropathology, John Wiley, New York. Eggers B, Hermann W, Barthel H et al. (2003). The degree of depression in Hamilton rating scale is correlated with the density of presynaptic serotonin transporters in 23 patients with Wilson’s disease. J Neurol 250: 576–580. Ferenci P, Litwin T, Seniow J et al. (2015). Encephalopathy in Wilson disease: copper toxicity or liver failure? J Clin Exp Hepatol 5: S88–S95. Frota NAF, Caramelli P, Barbosa ER (2009). Cognitive impairment in Wilson’s disease. Demen Neuropsychol 3: 16–21. Frota NA, Barbosa ER, Porto CS et al. (2013). Cognitive impairment and magnetic resonance imaging correlations in Wilson’s disease. Acta Neurol Scand 127: 391–398. Garnier H, Diederich N, Pilloy W et al. (1997). Late form with psychiatric presentation of Wilson’s disease, with pseudo-compulsive stereotyped movements. Neuroradiological correlations. Rev Neurol 153: 124–128. Geissler I, Heinemann K, Rohm S et al. (2003). Liver transplantation for hepatic and neurological Wilson’s disease. Transplant Proc 35: 1445–1446. Goldstein NP, Ewert JC, Randall RV et al. (1968). Psychiatric aspects of Wilson’s disease (hepatolenticular degeneration) – results of psychometric tests during long-term therapy. Am J Psychiatry 124: 1555. Gwirtsman HE, Prager J, Henkin R (1993). Case report of anorexia nervosa associated with Wilson’s disease. International Journal of Eating Disorders 13: 241–244. Habek M, Gabeli T, Brinar VV et al. (2009). Late-onset Wilson’s disease presenting with general anxiety disorder. Acta Neuropsychiatrica 21: 267–268. Han Y, Zhang F, Tian Y et al. (2014). Selective impairment of attentional networks of alerting in Wilson’s disease. Plos One 9.

Hegde S, Sinha S, Rao SL et al. (2010). Cognitive profile and structural findings in Wilson’s disease: a neuropsychological and MRI-based study. Neurol India 58: 708–713. Hesse S, Barthel H, Hermann W et al. (2003). Regional serotonin transporter availability and depression are correlated in Wilson’s disease. J Neural Transm 110: 923–933. Huang CC, Chu NS (1992). Wilson’s disease: clinical analysis of 71 cases and comparison with previous Chinese series. Journal of the Formosan Medical Association ¼ Taiwan yi zhi 91: 502–507. Iwa
nski S, Senio´w J, Les´niak M et al. (2015). Diverse attention deficits in patients with neurologically symptomatic and asymptomatic Wilson’s disease. Neuropsychology 29: 25–30. Jeon B, Kim JM, Jeong JM et al. (1998). Dopamine transporter imaging with [123I]-beta-CIT demonstrates presynaptic nigrostriatal dopaminergic damage in Wilson’s disease. J Neurol Neurosurg Psychiatry 65: 60–64. Jukic I, Titlic M, Tonkic A et al. (2006). Psychosis and Wilson’s disease: a case report. Psychiatr Danub 18: 105–107. Keller R, Torta R, Lagget M et al. (1999). Psychiatric symptoms as late onset of Wilson’s disease: neuroradiological findings, clinical features and treatment. Ital J Neurol Sci 20: 49–54. Kim SY, Chang Y, Jang IM et al. (2011). Wilson’s disease presenting with the episodic hypersomnia. In: Neurodegenerative Diseases. Conference: 10th International Conference AD/PD Alzheimer’s and Parkinson’s Diseases: Advances, Concepts and New Challenges Barcelona Spain. Conference Start, p. 8. Kinnier-Wilson S (1912). Progressive lentivular degeneration – a familial nervous disease associated with cirrhosis of the liver Brain 34. Kontaxakis V, Stefanis C, Markidis M et al. (1988). Neuroleptic malignant syndrome in a patient with Wilson’s disease. J Neurol Neurosurg Psychiatry 51: 1001–1002. Krim E, Barroso B (2001). Psychiatric disorders treated with clozapine in a patient with Wilson’s disease. Presse Med 30: 738. Krishnakumar P, Riyaz A (2005). Wilson’s disease presenting as depressive disorder. Indian Pediatr 42: 1172–1173. Kulaksizoglu IB, Polat A (2003). Quetiapine for mania with Wilson’s disease. Psychosomatics: Journal of Consultation Liaison Psychiatry 44: 438–439. Kumawat B, Sharma C, Tripathi G et al. (2007a). Wilson’s disease presenting as isolated obsessive-compulsive disorder. Indian J Med Sci 61: 607–610. Kumawat BL, Sharma CM, Tripathi G et al. (2007b). Wilson’s disease presenting as isolated obsessive-compulsive disorder. Indian J Med Sci 61: 607–610. Lai C-L, Lin R-T, Liou L-M et al. (2010). The role of event-related potentials in cognitive decline in Alzheimer’s disease. Clin Neurophysiol 121: 194–199. Lang C, M€ uller D, Claus D et al. (1990). Neuropsychological findings in treated Wilson’s disease. Acta Neurol Scand 81: 75–81.

COGNITIVE AND PSYCHIATRIC SYMPTOMS IN WILSON DISEASE Lin JJ, Lin KL, Wang HS et al. (2006). Psychological presentations without hepatic involvement in Wilson disease. Pediatr Neurol 35: 284–286. Littman E, Medalia A, Senior G et al. (1995). Rate of information-processing in patients with Wilson’s disease. Journal of Neuropsychiatry and Clinical Neurosciences 7: 68–71. Litwin T, Chabik G, Czlonkowska A (2013). Acute focal dystonia induced by a tricyclic antidepressant in a patient with Wilson disease: a case report. Neurol Neurochir Pol 47: 502–506. Loganathan S, Nayak R, Sinha S et al. (2008). Treating mania in Wilson’s disease with lithium. J Neuropsychiatry Clin Neurosci 20: 487–489. Ma H, Lv X, Han Y et al. (2013). Decision-making impairments in patients with Wilson’s disease. J Clin Exp Neuropsychol 35: 472–479. Machado AC, Deguti MM, Caixeta L et al. (2008). Mania as the first manifestation of Wilson’s disease. Bipolar Disord 10: 447–450. Madsen E, Gitlin JD (2007). Copper and iron disorders of the brain. Annu Rev Neurosci 30: 317–337. McDonald LV, Lake CR (1995). Psychosis in an adolescent patient with Wilson’s disease: effects of chelation therapy. Psychosom Med 57: 202–204. Medalia A, Scheinberg IH (1989). Psychopathology in patients with Wilson’s disease. Am J Psychiatry 146: 662–664. Medalia A, Isaacs-Glaberman K, Scheinberg IH (1988). Neuropsychological impairment in Wilson’s disease. Arch Neurol 45: 502–504. Medalia A, Galynker I, Scheinberg IH (1992). The interaction of motor, memory, and emotional dysfunction in Wilson’s disease. Biol Psychiatry 31: 823–826. Mercier-Jacquier M, Bronowicki JP, Raabe JJ et al. (2011). Wilson’s disease in an adult. Rev Med Interne 32: 341–346. Modai I, Karp L, Liberman UA et al. (1985). Penicillamine therapy for schizophreniform psychosis in Wilson’s disease. Journal of Nervous & Mental Disease 173: 698–701. Mrabet S, Ellouze F, Mrad MF (2013). Wilson’s disease and psychiatric disorders: is there comorbidity or indicator of relapse? J Neurol Sci 333, e117. Muller JL (1999). Schizophrenia-like symptoms in the WestphalStrumpell form of Wilson’s disease. J Neuropsychiatry Clin Neurosci 11: 412. Muller J, Landgraf F, Trabert W (1998). Schizophrenia-like symptoms in the Westphal-Strumpell variation of Wilson disease. Nervenarzt 69: 264–268. Nayak RB, Shetageri VN, Bhogale GS et al. (2012). Catatonia: a rare presenting symptom of Wilson’s disease. J Neuropsychiatry Clin Neurosci 24: E34–E35. Negro PJ, Louza Neto MR (1995). Results of ECT for a case of depression in Wilson’s disease. J Neuropsychiatry Clin Neurosci 7: 384. Netto A, Sinha S, Taly A et al. (2011). Sleep in Wilson’s disease: questionnaire based study. Annals of Indian Academy of Neurology 14: 31–34.

139

Nevsimalova S, Buskova J, Bruha R et al. (2011). Sleep disorders in Wilson’s disease. Eur J Neurol 18: 184–190. Oder W, Grimm G, Kollegger H et al. (1991). Neurological and neuropsychiatric spectrum of Wilson’s disease: a prospective study of 45 cases. J Neurol 238: 281–287. Pan JJ, Chu CJ, Chang FY et al. (2005). The clinical experience of Chinese patients with Wilson’s disease. Hepatogastroenterology 52: 166–169. Petrovic I, Svetel M, Pekmezovic T et al. (2012). Quality of life in patients with Wilson’s disease in Serbia. Mov Disord 27: S76. Pfennig A, Alda M, Young T et al. (2014). Prophylactic lithium treatment and cognitive performance in patients with a long history of bipolar illness: no simple answers in complex disease–treatment interplay. Int J Bipolar Disord 2: 1. Portala K, Westermark K, Von Knorring L et al. (2000). Psychopathology in treated Wilson’s disease determined by means of CPRS expert and self-ratings. Acta Psychiatr Scand 101: 104–109. Portala K, Westermark K, Ekselius L et al. (2002). Sleep in patients with treated Wilson’s disease. A questionnaire study Nordic Journal of Psychiatry 56: 291–297. Portala K, Waldenstrom E, Von Knorring L et al. (2008). Psychopathology and personality traits in patients with treated Wilson disease grouped according to gene mutations. Ups J Med Sci 113: 79–94. Rahman A, Azad MA, Hossain I et al. (2009). Zinc, manganese, calcium, copper, and cadmium level in scalp hair samples of schizophrenic patients. Biol Trace Elem Res 127: 102–108. Rathbun JK (1996a). Neuropsychological aspects of Wilson’s disease. Int J Neurosci 85: 221–229. Rathbun JK (1996b). Neuropsychological aspects of Wilson’s disease. Int J Neurosci 85: 221–229. Rodrigues AC, Dalgalarrondo P (2003). Neuropsychiatric disturbances in Wilson’s disease and use of electroconvulsive therapy: case report. Arq Neuropsiquiatr 61: 876–880. Rodrigues ACT, Dalgalarrondo P, Banzato CEM (2004). Successful ECT in a patient with a psychiatric presentation of Wilson’s disease. Convuls Ther 20: 55. Rybakowski J, Litwin T, Chlopocka-Wozniak M et al. (2013). Lithium treatment of a bipolar patient with Wilson’s disease: a case report. Pharmacopsychiatry 46: 120–121. Sagawa M, Takao M, Nogawa S et al. (2003). Wilson’s disease associated with olfactory paranoid syndrome and idiopathic thrombocytopenic purpura. No to Shinkei Brain & Nerve 55: 899–902. Sahu JK, Singhi P, Malhotra S (2013). Late occurrence of isolated obsessive-compulsive behavior in a boy with Wilson’s disease on treatment. J Child Neurol 28: 277. Saint-Laurent M (1992). Schizophrenia and Wilson’s disease. Can J Psychiatry 37: 358–360. Senio´w J, Bak T, Gajda J et al. (2002). Cognitive functioning in neurologically symptomatic and asymptomatic forms of Wilson’s disease. Mov Disord 17: 1077–1083. Shah N, Kumar D (1997). Wilson’s disease, psychosis, and ECT. J ECT 13: 278–279.

140

P. ZIMBREAN AND J. SENIÓW

Shah H, Vankar GK (2003). Wilson’s disease : a case report. Indian Journal of Psychiatry 45: 253–254. Shanmugiah A, Sinha S, Taly AB et al. (2008). Psychiatric manifestations in Wilson’s disease: a cross-sectional analysis. Journal of Neuropsychiatry & Clinical Neurosciences 20: 81–85. Silva F, Nobre S, Campos AP et al. (2011). Behavioural and psychiatric disorders in paediatric Wilson’s disease. BMJ case reports2011. http://dx.doi.org/10.1136/bcr.05.2011.4249. Smit JP, De Graaf J, Tjabbes T et al. (2004). Depression as first symptom of Wilson’s disease. A case study Tijdschrift voor Psychiatrie 46: 255–258. Soltanzadeh A, Soltanzadeh P, Nafissi S et al. (2007). Wilson’s disease: a great masquerader. Eur Neurol 57: 80–85. Sorbello O, Riccio D, Sini M et al. (2011). Resolved psychosis after liver transplantation in a patient with Wilson’s disease. Clinical Practice and Epidemiology in Mental Health : CP & EMH 7: 182–184. Srinivas K, Sinha S, Taly AB et al. (2008). Dominant psychiatric manifestations in Wilson’s disease: a diagnostic and therapeutic challenge! J Neurol Sci 266: 104–108. Stiller P, Kassubek J, Schonfeldtlecuona C et al. (2002). Wilson’s disease in psychiatric patients. Psychiatry Clin Neurosci 56: 649. Svetel M, Potrebic A, Pekmezovic T et al. (2009). Neuropsychiatric aspects of treated Wilson’s disease. Parkinsonism Relat Disord 15: 772–775. Svetel M, Pekmezovic T, Tomic A et al. (2011). Quality of life in patients with treated and clinically stable Wilson’s disease. Mov Disord 26: 1503–1508. Szutkowska-Hoser J, Senio´w J, Członkowska A et al. (2005). Cognitive functioning and life activity in patients with hepatic form of Wilson’s disease. Pol Psychol Bull 36: 234–238. Tarter RE, Switala J, Carra J et al. (1987). Neuropsychological impairment associated with hepatolenticular degeneration (Wilson’s disease) in the absence of overt encephalopathy. Int J Neurosci 37: 67–71. Tatay A, Lloret M, Merino T et al. (2010). Mania as a first symptom of Wilson’s disease: a case report. In: European

Psychiatry. Conference: 18th European Congress of Psychiatry Munich Germany. Conference Start, 25. Tokdemir M, Polat SA, Acik Y et al. (2003). Blood zinc and copper concentrations in criminal and noncriminal schizophrenic men. Arch Androl 49: 365–368. Vaishnav P, Mistry K, Banwari G (2013). Electro convulsive therapy for control of psychiatric manifestations of Wilson’s disease. Indian Journal of Psychiatry 55: S111. Volpe FM, Tavares A (2000). Cyproterone for hypersexuality in a psychotic patient with Wilson’s disease. Australian and New Zealand Journal of Psychiatry 34: 878–879. Walter G, Lyndon B (1997). Depression in hepatolenticular degeneration (Wilson’s disease). Australian & New Zealand Journal of Psychiatry 31: 880–882. Wenisch E, De Tassigny A, Trocello JM et al. (2013). Cognitive profile in Wilson’s disease: a case series of 31 patients. Rev Neurol (Paris) 169: 944–949. Wolf TL, Kotun J, Meador-Woodruff JH (2006). Plasma copper, iron, ceruloplasmin and ferroxidase activity in schizophrenia. Schizophr Res 86: 167–171. World Health Organization (1980). International Classification of Impairment, Disabilities and Handicaps (ICIDH). WHO, Geneva. http://apps.who.int/iris/bitstream/10665/41003/1/ 9241541261_eng.pdf. Accessed 26 January 2017. Xu P, Lu ZL, Wang X et al. (2010). Category and perceptual learning in subjects with treated Wilson’s disease. PLoS One 5: e9635. Yanik M, Kocyigit A, Tutkun H et al. (2004). Plasma manganese, selenium, zinc, copper, and iron concentrations in patients with schizophrenia. Biol Trace Elem Res 98: 109–117. Yigit M, Ozkan D, Altunsoy-Sen N et al. (2014). Psychosis as the first manifestation of Wilson’s disease. Klinik Psikofarmakoloji Bulteni 24: S235. Zimbrean PC, Schilsky ML (2014). Psychiatric aspects of Wilson disease: a review. Gen Hosp Psychiatry 36: 53–62. Zimbrean PC, Schilsky ML (2015). The spectrum of psychiatric symptoms in Wilson’s disease: treatment and prognostic considerations. Am J Psychiatry 172: 1068–1072.

Handbook of Clinical Neurology, Vol. 142 (3rd series) Wilson Disease A. Członkowska and M.L. Schilsky, Editors http://dx.doi.org/10.1016/B978-0-444-63625-6.00012-4 © 2017 Elsevier B.V. All rights reserved

Chapter 12

Wilson disease in children EVE A. ROBERTS1* AND PIOTR SOCHA2 Departments of Paediatrics, Medicine and Pharmacology and Toxicology, University of Toronto, Toronto, Canada

1 2

Departments of Gastroenterology, Hepatology, Nutritional Disorders and Pediatrics, The Children’s Memorial Health Institute, Warsaw, Poland

Abstract Wilson disease (WD) is an inherited disorder mainly of hepatocellular copper disposition, due to dysfunction of the Wilson ATPase, a P1B-ATPase encoded by the gene ATP7B. In children, as in older age brackets, clinical disease is highly diverse. Although hepatic disease is the common presentation in children/ adolescents, neurologic, psychiatric, and hematologic clinical presentations do occur. Very young children may have clinically evident liver disease due to WD. Early diagnosis, preferably when the child/adolescent is asymptomatic, is most likely to result in near-normal longevity with generally good health so long as the patient tolerates effective medication, is adherent to the lifelong treatment regimen, and has consistent access to the medication. Apart from a lively index of clinical suspicion on the part of physicians, biochemical tests including liver tests, serum ceruloplasmin, and basal 24-hour urinary copper excretion and genotype determination are key to diagnosis. Oral chelation treatment remains central to medical management, although zinc appears to be an attractive option for the presymptomatic child. Pediatric patients presenting with Wilsonian fulminant hepatic failure must be differentiated from those with decompensated cirrhosis, since the latter may respond to intensive medical interventions and not require liver transplantation. Recently identified WD-mimic disorders reveal important aspects of WD pathogenesis.

Wilson disease (WD) is an inherited disorder of copper disposition in hepatocytes. Defective copper handling leads to hepatic copper retention and chronic liver injury. Subsequently other organs/tissues (mainly the brain, eyes, erythrocytes, and cartilage) may be damaged due to copper toxicity. The pattern of inheritance is autosomal recessive. Mutations in the gene ATP7B, which encodes the metal-transporting P1B-type ATPase known as the Wilson ATPase, are causal. The Wilson ATPase is a multifunctional membrane-spanning protein which monitors hepatocellular levels of copper, contributes to the production of holoceruloplasmin, and expedites biliary excretion of copper. WD has been considered as a disease of children and young adults. In Wilson’s own paper, among his cases and those he cited, the age of onset was approximately 14 years, with nearly equal gender distribution (Wilson, 1912). The classic focus

on adolescence and early adulthood has proven too narrow. WD symptomatic in childhood or diagnosed at an early age through newborn or first-degree relative screening poses special problems relating to diagnosis, management, and prognostication of disease. WD can present clinically with hepatic or neurologic disorders (hepatic WD and neuro-WD, respectively); the neuropsychiatric presentation may be difficult to diagnose in childhood. Availability of genetic diagnosis has certainly improved diagnostic efficacy but not necessarily shortened time to diagnosis. Early diagnosis, preferably in the presymptomatic period, is associated with normal, reasonably healthy longevity in patients who are adherent to an effective medical regimen. Ensuring accessibility and adherence to effective treatment has recently assumed prime importance in childhood WD.

*Correspondence to: Eve A. Roberts, M.D., Ph.D., FRCPC, Division of Gastroenterology, Hepatology and Nutrition, Room 8263, Black Wing, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8. Tel: +1-416-813-6171, Fax: +1-416-813-4972, E-mail: [email protected]

142

E.A. ROBERTS AND P. SOCHA

DEVELOPMENTAL PHYSIOLOGY OF COPPER The Wilson and Menkes ATPases both contribute to homeostasis of copper in the maternal–fetal unit. Both are expressed in placenta. Their localization differs somewhat: the Wilson ATPase is found only in the syncytiotrophoblast (Hardman et al., 2011). Likely the Menkes ATPase is involved in copper delivery to the fetus whereas the Wilson ATPase effects return of excess copper to the maternal circulation; both are subject to hormonal regulation (Hardman et al., 2007). Serum concentrations of ceruloplasmin are very low in the newborn period, in the 100–200 mg/L (10–20 mg/dL) range (Fryer et al., 1993). This accounts for the quip that all infants are a little “Wilsonian” at birth, an impression supported by finding relatively high concentrations of copper in the newborn liver, possibly due to its physiologic cholestasis. The predominant form of ceruloplasmin in the newborn period is apoceruloplasmin (Hellman and Gitlin, 2002). Neonatal hepatic ceruloplasmin production may be under hormonal control (Fitch et al., 1999). Serum ceruloplasmin levels then rise in the first years of life to levels (300–500 mg/L; 30–50 mg/dL) which can exceed the typical range in the adult (200–400 mg/L; 20–40 mg/dL); levels in adolescence resemble those of adulthood except for showing gender difference, higher in females.

AGE INCIDENCE WD can present with clinical disease in toddlers and the elderly, as well as in the classic 5–40-year-old age bracket. Age is no longer a relevant criterion for making the diagnosis. Since the report of a 3-year-old girl, homozygous for a severe mutation of ATP7B, who presented with decompensated cirrhosis resembling (but not qualifying as) Wilsonian fulminant hepatic failure (FHF) Wilson et al., 2000), instances of clinically apparent WD in children 3 years old or younger have been reported more frequently (Iorio et al., 2003; Marcellini et al., 2005; Beyersdorff and Findeisen, 2006; Jang et al., 2010; Wang et al., 2010; Kim et al., 2013; Beinhardt et al., 2014; Abuduxikuer et al., 2015). WD is also diagnosed in very young presymptomatic children by family screening (Manolaki et al., 2009). The broad diversity of liver disease in these very young children with WD is noteworthy. Almost any clinical pattern is possible, from asymptomatic through severe disease. Frequently, as in older WD patients, the hepatic abnormality is only biochemical: elevated serum aminotransferases. Some of these very young WD patients are identified only when liver tests are done during investigation for another health issue, such as

infection or diarrhea. WD needs to be featured in the differential diagnosis of type 2 autoimmune hepatitis (AIH) occurring in the toddler. Fatty liver with elevated serum aminotransferases was reported in a 4-year-old girl (Stillman and Rohr, 1982) and typical Wilsonian FHF in a 5-year-old girl (Kalach et al., 1993). In general, as shown by data from mouse models of WD, copper accumulation occurs rapidly in the first year of life, even if it produces no clinical abnormality in most affected individuals. Moreover, this hepatocellular copper is dispersed in the cytoplasm, not evident by histologic stains.

CLINICAL PRESENTATIONS WD has numerous clinical patterns. The generalization that its spectrum includes hepatic, neurologic, and psychiatric disorders holds for children/adolescents but requires fine-tuning. Hepatic WD tends to be evident at an earlier age than neuro-WD. In a recent large, highly inclusive Austrian series of children and adults with WD, mean age at diagnosis for hepatic WD was 21, statistically different from mean age (19 years) at diagnosis for neurologic WD; however, age at first manifestation extended into early childhood for both hepatic WD and neuro-WD (Beinhardt et al., 2014). Renal Fanconi syndrome is typical of childhood WD and may lead to nephrolithiasis. Problems with fertility and history of recurrent spontaneous abortion are features found mainly in the nonpediatric age bracket. Transient self-limited episodes of intravascular hemolysis are frequently reported in children, mainly as brief episodes of jaundice resolving spontaneously. Data from numerous pediatric clinical series (Table 12.1) indicate that gender distribution shows some male predominance (M:F ¼ 5:4) and that average age at diagnosis is 9–10 years old.

Liver disease The clinical symptoms of hepatic WD in children are frequently nonspecific, such as fatigue, anorexia, or abdominal pain. Jaundice is typical of more severe liver involvement but can be due to intravascular hemolysis. Epistaxis as a presenting complaint is frequently mentioned in pediatric series. Hepatic WD should be considered as a possible diagnosis in any child/adolescent who has hepatomegaly, elevated serum aminotransferases, or evidence of fatty liver. WD may also present as simple acute hepatitis, and in children it often closely resembles acute AIH (Milkiewicz et al., 2000). Laboratory features typically include elevated serum IgG and detectable antinuclear antibodies and smooth-muscle antibody: it is the pattern of type 1 AIH. In several different series of pediatric WD, hepatic steatosis was a prominent feature of otherwise mild liver disease (Roberts et al., 2004; Marcellini et al., 2005;

Table 12.1 Representative reports of pediatric Wilson disease (WD) worldwide, selected for geographic distribution and clarity/extent of data

n Sex Age at diagnosis, years, mean Age range, years Hepatic WD, total (asymptomatic) Neuro-WD Screening Other

Spain Melbourne Rome London Athens (1982–1996) (1971–1998) (1983–1993) (1967–2000) (1983–2004)

Taiwan (1996–2008)

Morocco (2003–2010)

São Paulo (1987–2009)

Cairo (1992–2009)

Costa Rica (1992–2006)

Toronto (1984–2004)

Pune (1980–2000)

26 11 F 15 M 9.8

17 9F 8M 10.9

22 12 F 10 M 6.2

74 29 F 45 M 11.2

57 25 F 32 M 9.3

11 2F 9M 10

20 7F 13 M 9

28 12 F 16 M 11 (median)

54 23 F 31 M 10 (median*)

35 11 F 24 M 10

39 21 F 18 M 11

124 ? 8.5

4–16 25 (18)

7–18 14

3–12 21 (20)

2–17 57

0.33–18 39 (19)

2.5–17.7 8

5–13 12

2–18 13 (5)

3–18 33

5–15 24

2–19 29

? 67 (?)

1 2 of the 26

2 1

0 1

(6 of 57)† 17

0 0 4 18 1 3 7 hematologic 2 hematologic 1 arthralgias

3 12

8 13

1 1 6 9 4 hematologic

2

?

0

1

?

6

5

22 of 57 ¼ 38%

?

14 (70%)

6 of 28 ¼ 21% 31%

?

18 of 35 ¼51%

Wilsonian FHF in 3 6 (5 F) 0 27 cohort, n K-F rings 3 of 15 of 3 of 17 of 57 17 ¼ 18% 17 ¼ 88% 22 ¼ 14% ¼30% Cirrhosis Reference

*

5 of 14 of 0 24 ¼ 21% 17 ¼ 82% Marcellini Gow et al. Sanchez(2000) et al. Albisua (2005) et al. (1999)

8 of 57 Dhawan et al. (2005)

12 of 2 of 11 ¼18% (80% by 7 of 17 57 ¼ 21% sonography) ¼ 41% Manolaki et al. Wang et al. Idrissi et al. Kleine et al. (2009) (2010) (2013) (2012)

Average of three median ages (for hepatic WD, neuro-WD, and screening) reported. Incidental to referral for hepatic WD. ? no data or unclear data; osseo, osseomuscular; FHF, fulminant hepatic failure; K-F, Kayser–Fleischer. †

28 19 10 osseo or hematologic 11 ?

5 of 21 ¼24% 9 of 30 ? ¼ 31% El-Karaksy Jimenez et al. Roberts et al. Pandit et al. et al. (2011) (2009) (2004) (2002)

3 of 6

144

E.A. ROBERTS AND P. SOCHA

Manolaki et al., 2009). In one series almost 40% had moderate to severe macrovesicular steatosis with fibrosis and another 27% had mild abnormalities  steatosis (Roberts et al., 2004). In the Spanish series, portal fibrosis plus steatosis was found in 29% of those biopsied (Sanchez-Albisua et al., 1999). Hepatic WD may be indistinguishable from nonalcoholic steatohepatitis in children. Occurrence of cirrhosis in pediatric WD is a complicated issue. Cirrhosis is uncommonly found in presymptomatic patients identified by first-degree-relative screening (Beinhardt et al., 2014). Whether cirrhosis develops is highly variable in children. It depends in part on the nature of ATP7B mutation(s). Prevalence of cirrhosis in pediatric reports may reflect variations in diagnostic practice in a retrospective review spanning several decades, ascertainment bias at a tertiary center providing liver transplantation, and the actual locality. In pediatric series (Table 12.1) with reasonably complete data reporting, the prevalence of cirrhosis was 34%. In certain places, such as Iran (Dehghani et al., 2013) and India (Simon et al., 2009), WD is a leading cause of cirrhosis in children. Wilsonian FHF poses both practical and conceptual challenges. It classically comprises a typical constellation of features: moderate to severe Coombs-negative intravascular hemolysis, fixed coagulopathy, some degree of encephalopathy, lowish (2000 U/L) serum aminotransferases from the very onset of clinical illness, and strikingly low serum alkaline phosphatase. Serum copper is greatly elevated and urinary copper excretion is likewise greatly increased. Early renal failure may supervene. It occurs more often in females than in males. The rationale for calling this condition “Wilsonian fulminant hepatic failure” is that it does not meet stringent criteria for acute liver failure, which it strongly resembles. In Wilsonian FHF, there is previous liver disease, even if it has been clinically inapparent. Typically the patient has cirrhosis, and copper deposits are found in the Kupffer cells, not in hepatocytes. The pre-existing cirrhosis may account for the relatively modest elevations of serum aminotransferases. A second conceptual issue arises only in the pediatric age bracket. If pediatric acute liver failure is defined as severe impairment of hepatic synthetic function (indicated by coagulopathy), a timeframe extending to 8 weeks, and no previously known liver disease – encephalopathy not required for the diagnosis – then Wilsonian FHF in a child meets this definition. However, decompensated cirrhosis in children with WD also meets this definition and it, unlike Wilsonian FHF, may often respond to intensive medical treatment. Differentiation between these two entities is important in pediatric WD since treatment strategies differ. How the recently

developed term acute-on-chronic liver disease applies to either of these severe presentations of WD in children is currently indeterminate because the connotations of this term remain uncertain.

Neurologic disease Although hepatic WD is the common presentation of WD in childhood, unquestionably neuro-WD occurs and affects children in their first decade and adolescents. An early report included 9 children with neuro-WD aged 12–19 years old; it further indicated that children presenting with hepatic WD but not treated developed neurologic symptoms 4–11 years later (median: 4 years, mean: 6 years) (Walshe, 1962). As is well known, neuro-WD tends to be either a movement disorder with dystonia or tremor or a reduction of movement resembling parkinsonian rigidity. Pseudobulbar palsy can occur with either pattern: typical features are drooling, dysarthria, and problems with swallowing. In a recent pediatric series from Pakistan (consisting of 50 children assessed in 2005–2008, all with Kayser–Fleischer rings), dystonia, dysarthria, and cognitive decline were the most frequent clinical presentations; dysphagia, drooling, and difficulty walking were also common (Noureen and Rana, 2011). Although this experience may represent the more severe end of the clinical spectrum, it is similar to the report from São Paolo where the predominant neurologic findings were dystonia, tremor, dysarthria, gait abnormalities, and a Parkinson-like movement disorder (Machado et al., 2006). In this series the youngest patients were a 7-year-old girl and a 9-year-old boy; all patients had Kayser–Fleischer rings. Seizures and/or hyperreflexia was described as atypical manifestations in some teenagers. Overall, dystonia and dysarthria appear to be the very common manifestations of pediatric neuro-WD. The difficulties of diagnosing neuro-WD in children and adolescents arise from the slow subtle onset of symptoms and from their very nonspecific nature. A 14-year-old boy is typical: over months he developed soft speech, then tremor, also cramps in hands and feet, then mildly antisocial behavior characterized by indecisiveness and immature actions, finally micrographia and deteriorated school performance (Ware et al., 2013). Some or all of these findings might have been ascribed to medical/social issues of early male adolescence. A similar problem is posed by children with attention deficit disorder and elevated serum aminotransferases or by those with apparent learning disability (Gronlund et al., 2006). Likewise it may be difficult to assess minor resting tremors in children. Gait disorders or clumsiness may be too nonspecific to attract much attention.

WILSON DISEASE IN CHILDREN

Psychiatric presentation in children and adolescents The sorts of psychiatric illness associated with WD are highly variable, ranging from depression to frank psychosis and including neurotic behaviors such as phobias or compulsive behaviors (Rathbun, 1996). Deteriorated school/occupational performance may also be classified as a psychiatric feature. Children may be affected (Matarazzo, 2002). A recent comprehensive review of published literature revealed depressive and manic episodes as initial features of psychiatric WD (Zimbrean and Schilsky, 2014). Psychosis, including frank delusional disorders or thought disorder resembling schizophrenia, may occur. Obsessive compulsive disorder is in the spectrum of psychiatric WD. This review identified 15 children (aged 5–18 years old) in whom a psychiatric problem was the initial presentation of WD: psychiatric diagnoses included psychosis, mania, major depressive disorder, attention deficit hyperactivity syndrome, and obsessive compulsive disorder, personality change, and poor school performance (Zimbrean and Schilsky, 2014). When WD presents as a psychiatric illness, diagnosis may be delayed. With hepatic WD, psychiatric features possibly eclipsed by the hepatic disease should be sought by direct questioning. Pediatric WD patients may develop psychiatric problems after diagnosis, and mental health requires attention during long-term follow-up. It is not certain whether psychiatric features portend a worse prognosis, although it has been pointed out that psychiatric aspects of WD can adversely affect management after liver transplant (Medici et al., 2005).

Other clinical features Hemolysis may be an initial presentation of WD. In some cases presenting with hepatic or neurologic features, medical history reveals one or more previously undiagnosed hemolytic episodes. As previously noted, Coombs-negative intravascular hemolysis is prominent in Wilsonian FHF. Hemolysis in WD is sometimes apparently precipitated by infection or drugs. Recent retrospective analysis of 321 WD patients revealed incidence of acute hemolysis in 7%. The patients’ age ranged from 7 to 20 years with an average onset of 12.6 years. Delayed diagnosis resulted in progression to severe hepatic disease and neurologic deterioration (Walshe, 2013). Chronic hemolytic anemia may also occur in pediatric WD (Wang et al., 2010). Arthritis may occur in WD, and it is attributed to synovial copper deposition. Patients from the Indian subcontinent, including teenagers, are often described as having an “osseomuscular” manifestation of WD. This includes arthralgia (more common than arthritis), proximal

145

muscle weakness, bony deformities, and pathologic fractures (Taly et al., 2007); bone pain may be dismissed as growing pains (Aggarwal and Bhatt, 2013). Myopathy has rarely been reported in the North American literature (Thapa et al., 2009; Rosen et al., 2013); debilitating muscle cramps may rarely occur (Listernick, 2011). Rhabdomyolysis has been reported (Propst et al., 1995). Other relatively rare organ involvement includes hypoparathyroidism and pancreatitis. In a Chinese series 5 children aged 4–11 years old presented with a renal disorder severe enough to cause pedal edema (Lin et al., 2014). Cardiac involvement may manifest as arrhythmias (Elkiran et al., 2013) or cardiomyopathy (Lin et al., 2014). Skin signs in children are relatively nonspecific but include abnormally dry skin (Seyhan et al., 2009). A clinical picture resembling Henoch–Sch€onlein purpura with palpable purpura has been reported in pediatric WD (Lin et al., 2014).

DIAGNOSIS Diagnosis of WD is regarded as difficult, possibly an unfair characterization. As with many disorders, diagnosis depends upon a composite of clinical features and laboratory findings. Relevant laboratory findings include serum ceruloplasmin and basal 24-hour urinary copper excretion, as reflecting nonceruloplasmin-bound copper. The latter might be measured directly, expressed as relative exchangeable copper (El Balkhi et al., 2011). Moreover, genotype determination demonstrating homozygosity for one disease-causing ATP7B alteration, or compound heterozygosity for two, is conclusive of the diagnosis, whatever the clinical scenario. Therefore what is the source of confusion? One problem is that the hallmark finding historically associated with WD, the Kayser–Fleischer ring, is inconstant, especially early in the disease course. Secondly, immunologically based analytic methods for measuring serum ceruloplasmin has changed how that parameter contributes to diagnosis of WD. Thirdly, for children and arguably for adults, the reference value of 100 mg/24 hours (¼1.6 mmol/ 24 hours) for basal 24-hour urinary copper excretion as the cutoff for diagnosing WD is too high. Another source of difficulty is lingering disputes over the reference value for copper accumulation in liver tissue. Finally, genetic diagnosis is not always straightforward, and currently it is resource-intensive. Kayser–Fleischer rings are often absent in children. Pediatric series show that they are found in 40% of children with WD (range 14–88%). In children with hepatic WD, they tend to be associated with more severe disease. Perhaps surprisingly, Kayser–Fleischer rings may also be absent with neuro-WD (Moller et al., 2011), although this is uncommon. Thus lack of Kayser–Fleischer ring

146

E.A. ROBERTS AND P. SOCHA

does not exclude WD. Contrariwise, in a child with no evidence of cholestatic liver disease, finding a Kayser–Fleischer ring serves as a strong rationale for aggressively pursuing the diagnosis of WD. Proficient slit-lamp examination is required for evaluating Kayser–Fleischer rings. Immunologic measurement of serum ceruloplasmin, routine for automated technology, measures apo- as well as holo- (copper-containing) ceruloplasmin. The Wilson ATPase is instrumental for the production of holoceruloplasmin, the fully functional ferroxidase. Thus enzymatic measurement addresses a function of the Wilson ATPase itself. Since apoceruloplasmin has a relatively short half-life of 5 hours, as opposed to 5.5 days for holoceruloplasmin (Hellman and Gitlin, 2002), serum levels tend to end up lower than normal. One approach is to take 140 mg/L (14.0 mg/dL) as the informative cutoff, based on receiver operating characteristic (ROC) analysis in genetically confirmed WD patients (Mak et al., 2008). As many as one-third of WD patients may have a normal serum ceruloplasmin. Serum ceruloplasmin level is elevated by inflammation or the oral contraceptive pill. With congenital nephrotic syndrome, serum ceruloplasmin may be subnormal, accompanied by anemia (Niel et al., 2011). Very low serum ceruloplasmin (40 mg/ 24 hours) is preferable. This recommendation is based on a series of 29 pediatric patients of whom 27 had basal 24-hour urinary copper excretion exceeding 0.6 mmol/24 hours; in that series 8 of 29 (28%) had values 0.6 mmol/24 hours (>40 mg/ 24 hours) and require genetic diagnosis. The penicillamine challenge test, in which 500 mg D-penicillamine is given as a 24-hour urine collection

is commenced and then again 12 hours later at the halfway point of the collection, was considered to be a valuable diagnostic test in children (Martins da Costa et al., 1992). Urinary copper excretion 25 mmol/24 hours was taken as diagnostic of WD. Further experience has provoked reassessment. In various series 40–50% or more of patients failed to meet that diagnostic criterion and yet were shown to have WD. It appears presymptomatic patients may not be detected (Muller et al., 2007). Less stringent criteria for assessing response to D-penicillamine challenge require further validation. Nearly everyone cites 250 mg/g dry weight as the hepatic parenchymal copper concentration diagnostic for WD. This value is based on an early study of a small number of subjects; however, many WD patients have hepatic parenchymal copper concentrations far above this value. Based on a large series of genetically confirmed patients, the cutoff of 70 mg/g dry weight has been suggested (Ferenci et al., 2005), whereas a more conservative value of 100 mg/g dry weight has also been suggested (Roberts and Schilsky, 2008). Nicastro et al. (2010) reported hepatic copper in 30 pediatric WD patients and 24 control subjects and found significant difference between both groups (813 vs. 38.4). Only 2 out of the 30 WD patients (7%) had a liver copper level 250 mg/g dry weight. The threshold of 75 mg/g dry weight was therefore proposed as a new optimal one in the differential diagnosis of WD in children. This criterion requires further evaluation in pediatric WD. Some risk of misdiagnosis of WD exists when liver copper content is used as the only test (Song and Chen, 2000). Importantly, any finding of hepatic parenchymal copper content in the 70- or 100–250-mg range merits further investigation, usually by genotyping. Normal hepatic parenchymal copper concentration (200 mg/L) 10–20 mg/dL (or 100–200 mg/L)