Modern issues in molecular diagnostics: manual 9786010412712

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1.1. Brief on history and main principles of molecular diagnostics

KAZAKH NATIONAL UNIVERSITY AFTER AL-FARABI

A. I. Zhussupova N. Zh. Omirbekova Z. M. Biyasheva

MODERN ISSUES IN MOLECULAR DIAGNOSTICS Manual

Almaty «Qazaq university» 2015

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Chapter 1. Introduction into molecular diagnostics of hereditary diseases

UDC 577.2(075.8) LBC 28.070a73 Zh 62 Recommended for publication by the decision of the Academic Council of the School of Biology and Biotechnology, Editorial and Publishing Council of the National University of Kazakhstan named after Al-Farabi (Protocol №3 4 march 2015) Reviewers: Candidate of biological sciences, Associate Professor A.V. Goncharova Doctor of biological sciences, Professor A.A. Nurzhanova

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Zhussupova A. I. Modern issues in molecular diagnostics: manual / A.I. Zhussupova, N.Zh. Omirbekova, Z.M. Biyasheva. – Almaty: Qazaq university, 2015. – 102 p. ISBN 978-601-04-1271-2 Mаnuаl соmprisеs bаsiс thеоrеtiсаl quеstiоns оf mоdеrn PСR-diаgnоstiсs, including its components and stages, its detection and analysis, primer and probes design, аnd its prасtiсаl аppliсаtiоn in thе fiеld оf mоlесulаr biоlоgy, gеnеtiс еnginееring аnd mеdiсinе, and in thе fiеld оf lаbоrаtоry diаgnоstiсs оf hеrеditаry аnd infесtiоus disеаsеs in pаrtiсulаr, control questions and sample tests; is wеll illustrаtеd with sсhеmеs аnd figurеs. Manual is aimed at master and doctoral students, specialty «Biology». Учебное издание содержит аналитический обзор теоретических вопросов совре­менной ДНК-диагностики, включая ее компоненты и стадии, применение для обнаружения и изучения целевого материала, дизайн праймеров и проб, ее прак­ тическое применение в области молекулярной биологии, генетической инженерии и медицины, в частности, в области лабораторной диагностики наследственных и инфекционных заболеваний, а также контрольные вопросы и образцы тестов. Пособие также хорошо проиллюстрировано схемами и рисунками. Учебное издание, в первую очередь, предназначено для магистрантов и док­ торантов специальности «Биология».

UDC 577.2(075.8) LBC 28.070a73 ISBN 978-601-04-1271-2

© Zhussupova A.I., Omirbekova N.Zh., Biyasheva Z.M., 2015 © Al-Farabi KazNU, 2015

FOREWORD With the development of technologies of molecular and genetic researches it became clear that variability of a genome causes not only evolutionary and biological diversity, but also defines development of different forms of pathologies. The beginning of the XXI century was marked by almost full interpretation of human genome. This break in human biology changes diagnostic opportunities of medicine, creates prerequisites for further development of molecular medicine, which considers emergence and pathogenesis of diseases at the molecular level: from prerequisites of developing of an illness and primary products of mutant genes to pathological metabolites. On the basis of knowledge of molecular events in norm and pathogenesis essentially new approaches to diagnostics, personalized treatment and prevention of diseases are developed. Practical potential of molecular diagnostics is most brightly shown in the biomedical researches directed on: identification of the genes responsible for developing of diseases; analysis of genetic polymorphism defining drug resistance; detection of genetic defects at the level of the whole genome; creation of models of various pathologies, etc. Also it is a little known of the difficult diseases, which are not submitting to simple Mendelian laws of inheritance, and relating to group of diseases with hereditary predisposition (cancer, psychiatric diseases, diabetes, asthma, atherosclerosis, hypertension, etc.), dependent on environmental conditions, which is the new direction of human genetics. Manual comprises the basic theoretical questions of modern molecular diagnostics, including modern concepts on molecular mechanisms of diseases and immune systems of the body; molecular mechanisms of apoptosis; molecular and genetic markers of diseases; classification of hereditary diseases and their characteristics; basic

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Modern issues in molecular diagnostics

methods of diagnostics and study of the mechanisms of diseases; principles of therapy and analysis of genetic susceptibility; current state and new achievements of the modern molecular diagnostics and their practical application, as well as guidelines for the implementation of practical training and independent work of students, with some examples of their works. Manual also includes the glossary with explanations of terms used in the text. It might be recommended as main literature for specific courses, such as «Molecular diagnostics» and «Molecular basics of apoptosis» as well as an additional literature for general courses in the field of biology and biotechnology. Manual might also be interesting for a broader group of readers. Authors are open for comments and suggestions benefitting the content of the manual.

1.1. Brief on history and main principles of molecular diagnostics

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INTRODUCTION INTO MOLECULAR DIAGNOSTICS OF HEREDITARY DISEASES

1.1. Brief on history and main principles of molecular diagnostics 1.1.1. Role of molecular diagnostics in modern life The term molecular diagnostics can be used to mean the detection and/or analysis of nucleic acid molecules (DNA or RNA) to provide clinical information for: pathogen detection (searching for exogenous, non-human nucleic acids) and genetic testing (searching for endogenous, host-derived nucleic acids). Molecular diagnostics combines laboratory medicine with the knowledge and technology of molecular genetics and has been enormously revolutionized over the last decades, benefiting from the discoveries in the field of molecular biology (see Fig. 1.1.1.1). The first seeds of molecular diagnostics were provided in the early days of recombinant DNA technology. cDNA cloning and sequencing were invaluable tools for providing the basic knowledge on the primary sequence of various genes. DNA sequencing provided a number of DNA probes, allowing the analysis via southern blotting of genomic regions, leading to the concept and application of restriction fragment length polymorphism (RFLP) track a mutant allele from heterozygous parents to a high-risk pregnancy. In 1976, Kan and colleagues carried

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Chapter 1. Introduction into molecular diagnostics of hereditary diseases

out, for the first time, prenatal diagnosis of α-thalassemia, using hybridization on DNA isolated from fetal fibroblasts. In 1978 RFLP analysis was implemented to pinpoint sickle cell alleles of African descent. This breakthrough provided the means of establishing similar diagnostic approaches for the characterization of other genetic diseases, such as phenylketonurea, cystic fibrosis, etc.

Figure 1.1.1.1. The timeline of the principal discoveries in the field of molecular biology, which influenced the development of molecular diagnostics (Patrinos, 2005)

Molecular diagnostics is a dynamic and transformative area of diagnostics, leading to insights in research and treatment in many disease states that are revolutionizing health care. It detects and measures the presence of genetic material or proteins associated with a specific health condition or disease, helping to uncover the underlying mechanisms of disease and enabling clinicians to tailor care at an individual level – facilitating the practice of personalized medicine (see Fig. 1.1.1.2).

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What’s so great about molecular diagnostics? Some benefits include: –– As many as 5,000 diseases have direct genetic causes; –– High sensitivity and increased specificity for most tests adds diagnostic utility; –– Potential for simple standardized procedures and rapid automated throughput; –– Increased number of techniques for infectious diseases and tumor diagnostics.

Figure 1.1.1.2. Molecular diagnostics uses techniques such as mass spectrometry and gene chips to capture the expression patterns of genes and proteins: http://www.prlog.org/12304778-molecular-diagnostics-market-expected-to-reachusd-87-billion-through-2019-tmr.html

Thereby, molecular diagnosis of human disorders is referred to as the detection of the various pathogenic mutations in DNA and / or RNA samples in order to facilitate detection, diagnosis, subclassification, prognosis, and monitoring response to therapy; use of molecular biology techniques to expand scientific knowledge of the natural history of diseases, identify people who are at risk for acquiring specific diseases, and diagnose human diseases at the nucleic acid level. Molecular diagnostics combines laboratory medicine with the knowledge and technology of molecular genetics. It has been revolutionized over the last decades, benefiting from the discoveries in the field of molecular biology.

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Chapter 1. Introduction into molecular diagnostics of hereditary diseases

1.1.2. Pathology and pathogenesis Pathology The study of pathology, including the detailed examination of the body, including dissection and inquiry into specific maladies, dates back to antiquity. Rudimentary understanding of many conditions was present in most early societies and is attested to in the records of the earliest historical societies, including those of the Middle East, India, and China. By the Hellenic period of ancient Greece, a concerted causal study of disease was underway with many notable early physicians (such as Hippocrates, for whom the modern Hippocratic Oath is named) having developed methods of diagnosis and prognosis for a number of diseases. The medical practices of the Romans and those of the Byzantines continued from these Greek roots, but, as with many areas of scientific inquiry, growth in understanding of medicine stagnated some after the Classical Era, but continued to slowly develop throughout numerous cultures. Many advances were made in the medieval era of Islam, during which numerous texts of complex pathologies were developed, also based on the Greek tradition. Even so, growth in complex understanding of disease mostly languished until knowledge and experimentation again began to proliferate in the Renaissance, Enlightenment, and Baroque eras, following the resurgence of the empirical method at new centers of scholarship. By the 17th century, the study of micrography was underway and examination of tissues had led British Royal Society member Robert Hooke to coin the word cell, setting the stage for later germ theory. Modern pathology began to develop as a distinct field of inquiry during the 19th Century through natural philosophers and physicians that studied disease and the informal study of what they termed pathological anatomy or morbid anatomy. However, pathology as a formal area of specialty was not fully developed until the late 19th and early 20th centuries, with the advent of detailed study of microbiology. In the 19th century, physicians had begun to understand that disease-causing pathogens, or germs (a catch-all for disease-causing, or pathogenic, microbes, such as bacteria, viruses, fungi, amoebae, molds, protists, and prions) existed and were capable of reproduction and multiplication, replacing earlier beliefs in humors or even spiritual agents, that had dominated for much of the previous 1,500 years in European medicine.

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With the new understanding of causative agents, physicians began to compare the characteristics of one germ’s symptoms as they developed within an affected individual to another germ’s characteristics and symptoms. This realization led to the foundational understanding that diseases are able to replicate themselves, and that they can have many profound and varied effects on the human host. In order to determine causes of diseases, medical experts used the most common and widely accepted assumptions or symptoms of their times, a general principal of approach that persists into modern medicine. Modern medicine was particularly advanced by further develop­ ments of the microscope to analyze tissues, to which Rudolf Virchow gave a significant contribution, leading to a slew of research devel­ opments. By the late 1920s to early 1930s pathology was deemed as medical specialty. Combined with developments in the understanding of general physiology, by the beginning of the 20th century, the study of pathology had begun to split into a number of rarefied fields and resulting in the development of large number of modern specialties within pathology and related disciplines of diagnostic medicine. Anatomic pathology is a medical specialty that is concerned with the diagnosis of disease based on the gross, microscopic, chemical, immunologic and molecular examination of organs, tissues, and whole bodies (as in a general examination or an autopsy), most basically breast, genitourinary, gynecological, gastrointestinal, bone, endocrine, head and neck, soft-tissue, and kidney. Anatomic pathology is itself divided into subfields, the main ones being surgical pathology, cytopathology, and forensic pathology. Anatomic pathology is one of two main divisions of the medical practice of pathology, the other being clinical pathology, the diagnosis of disease through the laboratory analysis of bodily fluids and tissues. Anatomic pathology relates to the processing, examination, and diagnosis of surgical specimens by a physician trained in pathological diagnosis. Clinical pathology is the division that processes the test requests more familiar to the general public; such as blood cell counts, coagulation studies, urinalysis, blood glucose level determinations and throat cultures. Its subsections include chemistry, hematology, microbiology, immunology, urinalysis and blood bank. Sometimes, pathologists practice both anatomic and clinical pathology, a

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Chapter 1. Introduction into molecular diagnostics of hereditary diseases

combination known as general pathology. At times, a surgical pathology diagnosis also includes an analysis of proteins expressed (immunohistochemistry) in the tissue, or the fine structure of individual cells (electron microscopy). Cytopathology (sometimes referred to as cytology) is a branch of pathology that studies and diagnoses diseases on the cellular level. It is usually used to aid in the diagnosis of cancer, but also helps in the diagnosis of certain infectious diseases and other inflammatory conditions as well as thyroid lesions, diseases involving sterile body cavities (peritoneal, pleural, and cerebrospinal), and a wide range of other body sites. Cytopathology is generally used on samples of free cells or tissue fragments, in contrast to histopathology, which studies whole tissues and cytopathologic tests are sometimes called smear tests because the samples may be smeared across a glass microscope slide for subsequent staining and microscopic examination. Dermatopathology is a subspecialty of anatomic pathology that focuses on the skin and the rest of the integumentary system as an organ. One of the greatest challenges of dermatopathology is its scope. More than 1500 different disorders of the skin exist, including coetaneous eruptions (rashes) and neoplasms. Dermatologists are able to recognize most skin diseases based on their appearances, anatomic distributions, and behavior. Sometimes, however, those criteria do not allow a conclusive diagnosis to be made, and a skin biopsy is taken to be examined under the microscope using usual histological tests. In some cases, additional specialized testing needs to be performed on biopsies, including immunofluorescence, immunohistochemistry, electron microscopy, flow cytometry, and molecular-pathologic analysis. Forensic pathology focuses on determining the cause of death by post-mortem examination of a corpse or partial remains. An autopsy is typically performed by a coroner or medical examiner, often during the criminal law investigations; in this role, coroners and medical examiners are also frequently asked to confirm the identity of a corpse. The requirements for becoming a licensed practitioner of forensic pathology varies from country to country (and even within a given nation), but typically a minimal requirement is a medical doctorate with a specialty in general or anatomical pathology with subsequent study in forensic medicine. Methods utilized by forensic scientists to determine death include examination of tissue specimens in order

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to identify the presence or absence of natural disease and other microscopic findings, interpretations of toxicology on body tissues and fluids to determine the chemical cause of overdoses, poisonings or other cases involving toxic agents, and the examinations of physical trauma. Histopathology refers to the microscopic examination of various forms of human tissue. Specifically, in clinical medicine, histopathology refers to the examination of a biopsy or surgical specimen by a patho­ logist, after the specimen has been processed and histological sections have been placed onto glass slides. Histopathological examination of tissues starts with surgery, biopsy, or autopsy. The tissue is removed from the body of an organism and then placed in a fixative which stabilizes the tissues to prevent decay. The most common fixative is formalin, although frozen section fixing is also common. To see the tissue under a microscope, the sections are stained with one or more pigments. The aim of staining is to reveal cellular components; counterstains are used to provide contrast. Histochemistry refers to the science of using chemical reactions between laboratory chemicals and components within tissue. The histological slides are then interpreted diagnostically and the resulting pathology report describes the histological findings and the opinion of the pathologist. In the case of cancer, this represents the tissue diagnosis required for most treatment protocols. Neuropathology is the study of disease of nervous system tissue, usually in the form of either small surgical biopsies or sometimes whole brains in the case of autopsy. Neuropathology is a subspecialty of anatomic pathology, neurology, and neurosurgery. If a disease of the nervous system is suspected, and the diagnosis cannot be made by less invasive methods, a biopsy of nervous tissue is taken from the brain or spinal cord to aid in diagnosis. Biopsy is usually requested after a mass is detected by medical imaging. With autopsies, the principal work of the neuropathologist is to help in the post-mortem diagnosis of various conditions that affect the central nervous system. Biopsies can also consist of the skin. Epidermal nerve fiber density testing is a more recently developed neuropathology test in which a punch skin biopsy is taken to identify small fiber neuropathies by analyzing the nerve fibers of the skin. This test is becoming available in select labs as well as many universities; it replaces the traditional nerve biopsy test as less invasive.

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Chapter 1. Introduction into molecular diagnostics of hereditary diseases

Pulmonary pathology is the subspecialty of anatomic (and especially surgical) pathology which deals with the diagnosis and characterization of neoplastic and non-neoplastic diseases of the lungs and thoracic pleura. Diagnostic specimens are often obtained via bronchoscopic transbronchial biopsy or video-assisted thoracic surgery. These tests can be necessary to diagnose between infection, inflammation, or fibrotic conditions. Renal pathology is a subspecialty of anatomic pathology that deals with the diagnosis and characterization of disease of the kidneys. The renal pathologist must synthesize findings from traditional microscope histology, electron microscopy, and immunofluorescence to obtain a definitive diagnosis. Medical renal diseases may affect the glomerulus, the tubules and interstitium, the vessels, or a combination of these compartments. Surgical pathology is one of the primary areas of practice for most anatomical pathologists. Often an excised tissue sample is the best and most definitive evidence of disease (or lack thereof) in cases where tissue is surgically removed from a patient. These determinations are usually accomplished by a combination of gross (i.e., macroscopic) and histological (i.e., microscopic) examination of the tissue, and may involve evaluations of molecular properties of the tissue by immunohistochemistry or other laboratory tests. There are two major types of specimens submitted for surgical pathology analysis: biopsies and surgical resections. A biopsy is a small piece of tissue removed primarily for the purposes of surgical pathology analysis, most often in order to render a definitive diagnosis. Types of biopsies include core biopsies, which are obtained through the use of large-bore needles, sometimes under the guidance of radiological techniques, such as ultrasound, magnetic resonance imaging. Incisional biopsies are obtained through diagnostic surgical procedures that remove part of a suspicious lesion, whereas excisional biopsies remove the entire lesion, and are similar to therapeutic surgical resections. Excisional biopsies of skin lesions and gastrointestinal polyps are very common. The pathologist’s interpretation of a biopsy is critical to establishing the diagnosis of a benign or malignant tumor, and can differentiate between different types and grades of cancer, as well as determining the activity of specific molecular pathways in the tumor. Surgical resection specimens are obtained by the therapeutic surgical removal of an entire diseased area or organ (and occasionally

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multiple organs). These procedures are often intended as definitive surgical treatment of a disease in which the diagnosis is already known or strongly suspected, but pathological analysis of these specimens remains important in confirming the previous diagnosis. Clinical pathology is a medical specialty that is concerned with the diagnosis of disease based on the laboratory analysis of bodily fluids such as blood and urine, as well as tissues, using the tools of chemistry, clinical microbiology, hematology and molecular pathology. Clinical pathologist learns to administer a number of visual and microscopic tests and an especially large variety of tests of the biophysical properties of tissue samples involving automated analyzers and cultures. The clinical laboratory diagnostics world is divided into two service businesses: clinical diagnostics and in vitro diagnostics. There are large service reference laboratories in Japan, Singapore, Korea, Australia, India, Brazil, Argentina, Colombia, Mexico and Canada. Europe has smaller reference laboratories in all countries, but the continent’s clinical service business is more concentrated in larger hospitals and academic centers. For U.S. market for this sector is close to $38 billion, while global market is estimated to comprise $70 billion with the average annual growth rate is around 15%. Molecular diagnostics testing is comprised of genetic, infectious disease, cancer, and companion diagnostics or pharmacogenomic tests. The in vitro diagnostics service business produces all manufactured kits and instrument. In U.S., these kits are generally FDA-approved, or have received approval by similar regulatory agencies (Leomics online Tutorial; http://www.leomics.com/our-space-molecular-diagnostics. html). Hematopathology is the study of diseases of blood cells (including constituents such as white blood cells, red blood cells, and platelets), organs and tissues that produce and/or primarily host hematopoietic cells and includes bone marrow, the lymph nodes, thymus, spleen, and other lymphoid tissues. The hematopathologist reviews biopsies of lymph nodes, bone marrows and other tissues involved by an infiltrate of cells of the hematopoietic system, and, sometimes flow cytometric and/or molecular hematopathology studies. The pathogenesis of a disease is the mechanism that causes the disease. The term can also describe the origin and development of the disease, and whether it is acute, chronic, or recurrent. The word

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Chapter 1. Introduction into molecular diagnostics of hereditary diseases

comes from the Greek pathos («disease») and genesis (creation). Types of pathogenesis include microbial infection, inflammation, malignancy and tissue breakdown. For example bacterial pathogenesis is the mechanism by which bacteria cause infectious illness. Four aspects of disease process: etiology, pathogenesis, morphological changes, clinical significance. Etiology is the cause, which might be determining (specifically known to be the sole cause of disease such pathogenic organism, e.g. HIV) or predisposing (leading indirectly to disease such as genetic predisposition). Often, a potential etiology is identified by epidemiological observations before a pathological link can be drawn between the cause and the disease (see Fig. 1.1.1.3).

Figure 1.1.2.3. Molecular basis of diseases: environment and genes (Ehlers, 2010)

Recently, pathological approach can be directly integrated into epidemiological approach in the interdisciplinary field of molecular pathological epidemiology, which can help to assess pathogenesis and causality by means of linking a potential etiologic factor to molecular pathologic signatures of a disease. (from the Ancient Greek roots of pathos (πάθος), meaning experience or suffering, and -logia (-λογία), an account of) is a significant component of the causal study of disease and a major field in modern medicine and diagnosis. The term pathology itself may be used broadly to refer to the study of disease in general,

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incorporating a wide range of bioscience research fields and medical practices, or more narrowly to describe work within the contemporary medical field of general pathology, which includes a number of distinct but inter-related medical specialties which diagnose disease mostly through the analysis of tissue, cell, and body fluid samples. 1.2. Human hereditary diseases: classification and characteristics 1.2.1. Basic stages and characteristics of a common disease A disease is a particular abnormal, pathological condition that affects part or all of an organism; medical condition associated with specific symptoms and signs. Disease might be: congenital and hereditary, inflammatory, degene­ rative, metabolic, neoplastic. It may be caused by factors originally from an external source, such as infectious disease, or it may be caused by internal dysfunctions, such as autoimmune diseases. In humans, disease is often used more broadly to refer to any condition that causes pain, dysfunction, distress, social problems, or death to the person afflicted (death by natural causes), or similar problems for those in contact with the person. In this broader sense, it sometimes includes injuries, disabilities, disorders, syndromes, infections, isolated symptoms, deviant beha­ viors, and atypical variations of structure (morphological changes can be morbid – visible to the naked eye and histological – visible under the microscope) and function, while in other contexts and for other purposes these may be considered distinguishable categories. Diseases can also be classified as communicable and non-commu­ nicable. The deadliest disease in humans is ischemic heart disease (blood flow obstruction), followed by cerebrovascular disease and lower respiratory infections, respectively. In an infectious disease, the incubation period is the time between infection and the appearance of symptoms. The latency period is the time between infection and the ability of the disease to spread to another person, which may precede, follow, or be simultaneous with the appearance of symptoms. Some viruses also exhibit a dormant phase, called viral latency, in which the virus hides in the body in an inactive state. For example,

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varicella zoster virus causes chickenpox in the acute phase; after recovery from chickenpox, the virus may remain dormant in nerve cells for many years, and later cause herpes. Six characteristics of infectious disease agents which determine whether or not an infectious disease will be transmitted to and infect a host, influencing the severity of the disease and even the outcome of the resulting infection: infectivity, pathogenecity, virulence, toxigenicity, resistance, antigenicity. Acute disease is a short-lived disease. Examples include a broken bone, a burn, and a neck injury while playing. Acute on chronic inflammation is a term sometimes used in pathology to describe a pattern of inflammation, which is a mixture of chronic and acute inflammation. It may be seen in asthma, rheumatoid arthritis, chronic peptic ulcer, chronic periodontitis, tuberculosis, tonsillitis and other conditions. Basically there are 3 stages: 1: acute or inflammatory stage (day 1-3), 2: sub-acute (day 4 - 3 weeks), 3: chronic (3 weeks - years). Chronic disease is one that lasts for a long time, usually at least six months. During that time, it may be constantly present, or it may go into remission and periodically relapse. A chronic disease may be stable (does not get any worse) or it may be progressive (gets worse over time). Some chronic diseases can be permanently cured. Most chronic diseases can be beneficially treated, even if they cannot be permanently cured. Examples of chronic conditions include osteoporosis, asthma, heart disease, osteoarthritis, kidney disease and diabetes. Many illnesses can occur in both acute and chronic form. For example, acute renal failure occurs when an event, such as dehydration, blood loss or taking medicines, leads to kidney malfunction. Chronic kidney disease, however, is caused by long-term conditions, such as high blood pressure or diabetes, and involves the gradual damage of the kidneys over time. Acute inflammation has macroscopic (redness, hotness, swelling, pain, tenderness, loss of function) and microscopic (local vascular change) signs. Based on the types of exudates acute inflammation might be: catarrhal, serous, fibrinous, membranous, hemorrhagic, gangrenous, allergic, suppurative or purulent. 1.2.2. Types of genetic disorders A genetic disorder is an illness caused by one or more abnormalities in the genome, especially a condition that is present from birth

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(congenital). Most genetic disorders are quite rare and affect one person in every several thousands or millions. Genetic disorders may or may not be heritable, i.e., passed down from the parents’ genes. In non-heritable genetic disorders, defects may be caused by new mutations or changes to the DNA. In such cases, the defect will only be heritable if it occurs in the germ line. The same disease, such as some forms of cancer, may be caused by an inherited genetic condition in some people, by new mutations in other people, and mainly by environmental causes in still other people. Whether, when and to what extent a person with the genetic defect or abnormality will actually suffer from the disease is almost always affected by environmental factors and events in the person’s development. Below is a list of genetic disorders and if known, causal type of mutation and the chromosome involved, where P – Point mutation, or insertion/deletion entirely inside one gene; D – Deletion of a gene or genes; C – Whole chromosome extra, missing, or both; T – Trinucleotide repeat disorders: gene is extended in length (Table 1; http://en.wikipedia.org/wiki/List_of_genetic_disorders; with re­ ductions). Table 1.2.2.1 Common genetic disorders with their causal mutations Disorder 1 Angelman syndrome Color blindness Cri du chat Cystic fibrosis Duchenne muscular dystrophy Haemophilia Klinefelter syndrome Phenylketonuria Polycystic kidney disease Prader–Willi syndrome Sickle-cell disease Tay–Sachs disease Turner syndrome

Mutation 2 DCP P D P D P C P P DC P P C

Chromosome 3 15 X 5 7q Xp X X 12q 16 (PKD1) or 4 (PKD2) 15 11p 15 X

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Chapter 1. Introduction into molecular diagnostics of hereditary diseases

Some types of recessive gene disorders confer an advantage in certain environments when only one copy of the gene is present: 1.Single gene disorders including Mendelian Disorders (i.e., following Mendelian order of inheritance, i.e. autosomal, X-linked and Y-linked) and non-Mendelian disorders (i.e., not following Mendelian order of inheritance, e.g. mitochondrial inheritance); 2. Multifactorial and polygenic disorders; 3. Disorders with variable modes of transmission; 4. Cytogenetic disorders: including autosomal disorders and sex chromosome disorders (Appendix 1, Fig. 1.2.2.1). 1.2.3. Single gene disorders The traits produced by a gene can be characterized as dominant or recessive. Dominant traits can be expressed when only one copy of the gene for that trait is present. Recessive traits carried on autosomal chromosomes can be expressed only when two copies of the gene for that trait are present because the corresponding gene that is not for that trait on the paired chromosome is usually expressed instead. People with one copy of an abnormal gene for a recessive trait (and who thus do not have the disorder) are called carriers. With codominant traits, both copies of a gene are expressed to some extent. An example of a codominant trait is blood type. If a person has one gene coding for blood type A and one gene coding for blood type B, the person has both blood types (blood type AB). Whether a gene is X-linked (sex-linked) also determines expression. Among males, almost all genes on the X chromosome, whether the trait is dominant or recessive, are expressed because there is no paired gene to offset their expression. Penetrance refers to how often a trait is expressed in people with the gene for that trait: complete or incomplete (not always expressed even when the trait it produces is dominant or when the trait is recessive and present on both chromosomes). If half the people with a gene show its trait, its penetrance is said to be 50%. Expressivity refers to if a trait is greatly, moderately, or mildly affects a person. Genomic imprinting and uniparental disomy may affect inheritance patterns. Sickle-cell anemia for instance is considered a recessive condition, but carriers that have it by half along with the normal gene have increased immunity to malaria in early childhood, which could

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be described as a related dominant condition. See Fig. 1.2.3.1 for its basic characteristics.

Figure 1.2.3.1. Sickle-cell anemia basic characteristics

Figure 1.2.3.2. Autosomal-dominant type of inheritance

Subclasses of single gene disorders are as follows: Autosomal dominant. Only one mutated copy of the gene is needed for a person to be affected by an autosomal dominant disorder. Each affected person usually has one affected parent (see Fig. 1.2.3.2).

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Chapter 1. Introduction into molecular diagnostics of hereditary diseases

Autosomal recessive. Two copies of the gene must be mutated for a person to be affected by an autosomal recessive disorder (see Fig. 1.2.3.3).

Figure 1.2.3.3. Autosomal-recessive type of inheritance

An affected person usually has unaffected parents who each carry a single copy of the mutated gene (and are referred to as carriers). Two unaffected people who each carry one copy of the mutated gene have a 25% chance with each pregnancy of having a child affected by the disorder, e.g. cystic fibrosis, spinal muscular atrophy. X-linked dominant. X-linked dominant disorders are caused by mutations in genes on the X chromosome. Only a few disorders have this inheritance pattern. Males are more frequently affected than females, and the chance of passing on an X-linked dominant disorder differs between men and women. The sons of a man with an X-linked dominant disorder will not be affected, and his daughters will all inherit the condition. A woman with an X-linked dominant disorder has a 50% chance of having an affected daughter or son with each pregnancy. Some X-linked dominant conditions, such as Aicardi Syndrome, are fatal to boys, so only girls have them. X-linked recessive. X-linked recessive disorders are also caused by mutations in genes on the X chromosome. Males are more frequently affected than females, and the chance of passing on the disorder differs between men and women. The sons of a man with an X-linked recessive disorder will not be affected, and his daughters will carry one copy of the mutated gene. With each pregnancy, a woman who carries an X-linked recessive disorder has a 50% chance of having sons who are affected and a 50% chance of having daughters who carry one copy of the mutated gene. General scheme for X-chromosomal type of inheritance is shown on Fig. 1.2.3.4. For instance, hemophilia A, color blindness, muscular dystrophy, androgenetic alopecia, glucose-6-phosphate dehydrogenase deficiency.

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Y-linked. Y-linked disorders are caused by mutations on the Y chromosome. Only males can get them, and all of the sons of an affected father are affected. Since the Y chromosome is very small, Y-linked disorders only cause infertility, and may be circumvented with the help of some fertility treatments; e.g. male Infertility

Figure 1.2.3.4. X-chromosomal type of inheritance

Mitochondrial. This type of inheritance, also known as maternal inheritance, applies to genes in mitochondrial DNA. Because only egg cells contribute mitochondria to the developing embryo, only females can pass on mitochondrial conditions to their children; e.g. Leber’s hereditary optic neuropathy (LHON) 1.2.4. Multifactorial and polygenic disorders Genetic disorders may also be complex, multifactorial or polygenic; this means that they are likely associated with the effects of multiple genes in combination with lifestyle and environmental factors. Multifactorial disorders include heart disease and diabetes. Although complex disorders often cluster in families, they do not have a clear-cut pattern of inheritance. This makes it difficult to determine a person’s risk of inheriting or passing on these disorders (see Figure 1.2.4.1).

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Chapter 1. Introduction into molecular diagnostics of hereditary diseases

Complex disorders are also difficult to study and treat because the specific factors that cause most of these disorders have not yet been identified. On a pedigree, polygenic diseases do tend to run in families, but the inheritance does not fit simple patterns as with Mendelian diseases. But this does not mean that the genes cannot eventually be located and studied. There is also a strong environmental component to many of them (e.g., blood pressure).

Figure 1.2.4.1. Integrated health care for monogenic and complex disorders

For instance, gout: genetic/acquired disorder of uric acid metabolism that leads to hyperuricemia and consequent acute and chronic arthritis. The recurrent but transient attacks of acute arthritis are triggered by the precipitation of monosodium

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urate crystals into joints from supersaturated body fluids which accumulate in and around the joints and other tissues causing inflammation. Cause of gout: unknown enyme defects or known enzyme defects leading to overproduction of uric acid like partial deficiency of hypoxanthine guanine phosphoribosyl transferase (HGPRT) enzyme (as person lacks the genes to produce this enzyme). Also high dietary intakes of purines as in pulses, as purines are metabolized to uric acid. Thus it has both a genetic (due to enzyme malfunction) and environmental predisposition (such as diet) and hence multifactorial.

Figure 1.2.4.2. Breast cancer as an example of a complex disorder

Other examples are heart disease, hypertension, diabetes, obesity, cancers (see Figure 1.2.4.2).

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Chapter 1. Introduction into molecular diagnostics of hereditary diseases

1.2.5. Disorders with variable modes of transmission Hereditary malformations are congenital malformations which may be familial and genetic or may be acquired by exposure to teratogenic agents in the uterus; are associated with several modes of transmission. Certain congenital malformations might be multifactorial or by a single mutant gene. For instance, Ehlers-Danlos syndrome: characterized by defects in collagen synthesis and structure. Causes include either of the following: deficiency of the enzyme lysyl hydroxylase, deficient synthesis of type 3 collagen due to mutations in coding genes, deficient conversion of procollagen type 1 to collagen due to mutation in the type 1 collagen gene. 1.2.6. Cytogenetic disorders May occur from alterations in the number or structure of the chromosomes, affect autosomes or sex chromosomes, e.g. fragile X chromosome. Characterized by mental retardation and an inducible cytogenetic abnormality in the X chromosome. Induced by certain culture conditions and is seen as a discontinuity of staining or constriction of in the long arm of the X-chromosome. Most known is the Down’s syndrome in which the number of chromosomes is increased by a third 21st chromosome and hence a total of 47 chromosomes occur (see Fig. 1.2.6.1).

Figure 1.2.6.1. Karyotype for Down syndrome: Notice the three copies of chromosome 21, caused by a failure of the 21st chromosome to separate during egg or sperm development

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1.3. Modern molecular diagnostics clinical laboratory Increasing knowledge of the molecular basis of disease and advances in technology for analyzing nucleic acids and gene products are changing pathology practice. Pathology residency programs are incorporating molecular pathology education into their curricula to prepare newly trained pathologists for the future, yet little guidance has been available regarding the important components of molecular pathology training, including recommendations for the acquisition of both basic knowledge in human genetics and molecular biology and specific skills relevant to microbiology, molecular oncology, genetics, histocompatibility, and identity determination. Integration of the data gained via nucleic acid based-technology with other laboratory and clinical information available in the care of patients is significant Several important steps need to be followed to ensure that a quality service is offered by a molecular laboratory. The quality of the test result is linked to a number of factors. It is reliant on activities that both directly and indirectly impact on the quality of the test ensuring that reliable and accurate results are obtained. There are several benefits to having a quality system in place, it allows for monitoring of the entire system, detects and limits errors, improves consistency among different testing sites and helps to contain costs. Quality control (QC) remains one of the most important tasks of the medical laboratory to ensure the reliability and accuracy of reported patient results. Whenever results are sent to physicians that need to be corrected, or any time prolonged quality troubleshooting is necessary within the laboratory, it can affect patient safety, laboratory credibility, operating costs, turnaround times, and regulatory or accreditation compliance. When applied properly, risk management can help minimize the risk of reporting incorrect patient test results. Minimizing the risk of reporting incorrect patient test results starts with good laboratory protocols, including proper calibration and maintenance of laboratory instruments. Good Clinical Practice (GCP) is an international ethical and scientific quality standard for designing, conducting, recording and reporting trials that involve the participation of human subjects. Compliance with this standard provides public assurance that the rights, safety and well-being of trial subjects are protected; consistent

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Chapter 1. Introduction into molecular diagnostics of hereditary diseases

with the principles that have their origin in the Declaration of Helsinki, and that the clinical trial data is credible. The conduct of the laboratory work involving diagnostic testing requires a hybrid of GLP and GCP requirements referred to as Good Clinical Laboratory Practice (GCLP). This would revolve around the application of those GLP principles that are relevant to the analyses of samples while ensuring the purpose and objectives of the GCP principles are maintained. General GCLP principles, which also hold for Molecular GCLP, such as: Facilities, Organization and Personnel Responsibilities, Personnel (significant number of well qualified people to perform the assays), Data Management (includes information flow scheme and a data collection and management system), Standard Operating Procedures (SOP; ensures that assay techniques and processes in the laboratory are standardized thereby contributing to reproducibility, each for one task, regularly checked and approved by the laboratory manager), Document Control, Stock Management system (for efficient management of reagents and consumables to ensure the continued ability to perform the assays the laboratory offers). Methods used should be validated, and appropriate quality control measures established and followed. To ensure all of the above mentioned steps are followed it is important there be a management review process, errors should be recorded (corrective actions), and all processes in the laboratory monitored through audits (both internal and external). This forms part of the Quality Assurance (QA) process. QA is defined as a team of persons charged with assuring management that Good Clinical Laboratory Practice compliance has been attained in the test facility as a whole and in each individual study. QA must be independent of the operational conduct of the studies, and functions as a witness to the entire process. Over the past decade there has been an expansion in molecular based technologies in the diagnostic environment. These molecular based technologies almost always involve Polymerase Chain Reaction of either DNA (PCR) or RNA (RT-PCR), but can also include isothermal amplification and/or sequencing. These molecular tests can be used for rapid qualitative or quantitative analysis for: the detection of infectious disease, viral load monitoring, HIV diagnosis in pediatrics, translocations, mutations, gene rearrangements, forensic medicine. Biological assays such as PCR-ELISA or fluorescence in situ hybridization can be used for detection of a marker of disease or

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risk in a sample taken from a patient. The detection of the marker might use real time PCR, direct sequencing, or microarrays - prefabricated chips that test many markers at once. The same principle applies to the proteome and the genome. High-throughput protein arrays can use complementary DNA or antibodies to bind and hence can detect many different proteins in parallel. Modern laboratories are under pressure from many directions: reducing turnaround times, managing staffing shortages, assimilating new laboratory automation options to handle burgeoning test volumes, and coping with leaner budgets. Laboratories now rely on automated quality control software programs to provide fast and reliable evaluation of results. These requirements are a direct result of the basis of the molecular technologies which use the ability of PCR to make millions of amplicons of the desired gene of interest (see Fig. 1.3.1).

Figure 1.3.1. Kary Mullis’ Polymerase Chain Reaction – The PCR method – a copying machine for DNA molecules; http://nobelprize.org/nobel_prizes/ chemistry/laureates/1993/illpres/pcr.html

Final size of the molecular diagnostics laboratory depends on the number of assays and associated techniques, being offered to customers based on clinical need.

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Chapter 1 Introduction into molecular diagnostics of hereditary diseases

Such in 1990 had 3 areas: sample preparation, pre-PCR and post-PCR. This was driven by the fear of contamination during the three common steps in the molecular assay: nucleic acid extraction (preparation), amplification usually by PCR (pre-PCR), detection or quantization of amplified product (post-PCR). Introduction of contamination safeguards, closed amplification instruments and fully automated systems has permitted many molecular techniques to be performed outside this traditional molecular laboratory. Laboratory Information Systems (LIS) functionality may be useful to increase workflow efficiency and tracking for surgical specimens during a molecular diagnostics evaluation. LIS focus on providing functionality for high-volume lab sections. For example, Gomah et al. describe a project to define workflow in molecular oncology that used surgical specimens as their source of nucleic acid. Diagnosis: looking at the disease from the small molecules point of view; elucidates the causes of the disease (viruses, hereditary disruptions of the normal control processes, such as the cell-cycle, apoptosis, etc.); provides a more comprehensive understanding of a disease, its history and progression; provides an understanding of the overall complexity of the disease. Prognosis: associates specific molecules or a set of molecules with the probable outcome of a disease. Treatment: The idea of personalized medicine is simple enough: create tailor-made treatments using DNA testing, implying the advantage of computational modeling, incorporating the wealth of genomic and proteomic information, in order to predict how each person will respond to the medication. Although still in developments, results from this study are a major step toward personalized medicine, which could potentially revolutionize the way that diseases are treated. Sample problem sets on Introduction into molecular diagnostics of hereditary diseases 1. Women have sex chromosomes of XX, and men have sex chromosomes of XY. Which of a woman’s grandparents could not be the source of any of the genes on either of her X-chromosomes? A) mother’s father B) father’s mother C) mother’s mother

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D) father’s father E) mother’s mother and mother’s father 2. Red-green color blindness is X-linked in humans. If a male is red-green color blind, and both parents have normal color vision, which of the male’s grandparents is most likely to be red-green color blind? A) maternal grandmother B) maternal grandfather C) paternal grandmother D) paternal grandfather E) either grandfather is equally likely 3. Which of the following sexually transmitted diseases may cause infertility? A) genital warts B) chlamydia C) human immunodeficiency virus 4. Which of the following sexually transmitted diseases is present in most diagnosed cervical cancers? A) human papilloma virus B) herpes C) human immunodeficiency virus D) gonorrhea 5. Hemophilia in humans is due to an X-chromosome mutation. What will be the results of mating between a normal (non-carrier) female and a hemophilac male? A) half of daughters are normal and half of sons are hemophilic B) all sons are normal and all daughters are carriers C) half of sons are normal and half are hemophilic; all daughters are carriers D) all daughters are normal and all sons are carriers E) half of daughters are hemophilic and half of daughters are carriers; all sons are normal 6. A couple has a female child with Tay Sachs disease, and three unaffected children. Neither parent nor any of the four biological grandparents of the affected child has had this disease. The most likely genetic explanation is that Tay Sachs disease is inherited as which kind of disease? A) autosomal dominant B) autosomal recessive C) sex-linked recessive D) sex-linked dominant E) cannot make a reasonable guess from this information 7. Diagnosis of chromosome aneuploidy of unborn children is normally done by a combination of amniocentesis, cell culture, and: A) enzyme assay

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Chapter 1 Introduction into molecular diagnostics of hereditary diseases B) RFLP analysis C) pedigree analysis D) karyotyping E) somatic cell fusion 8. Which statement about Down’s syndrome is false? A) frequency increases dramatically in mothers over the age of 40 B) cause is a non-disjunction when chromosomes do not separate during the first meiotic division C) affected individuals have an extra autosome D) long time lag between onset of meiosis in ovarian tissue (during fetal development) and its completion (at ovulation) is most likely the reason for increased incidence in older mothers E) none, all statements are true Questions for the individual work of students on Introduction into molecular diagnostics of hereditary diseases 1. Give the definition to pathological metabolites. 2. Give the definition to the personalized treatment. 3. Analyze use of biochemical methods in study of human hereditary diseases. 4. Give the definition for simple Mendelian laws of inheritance. 5. Give definition for genetic imprinting. 6. Give the definition for hereditary predisposition. 7. Give the definition for the monogenic diseases. 8. Give the definition for diseases with hereditary predisposition. 9. Name basic principles of therapy and analysis of genetic susceptibility. 10. Define the modern concepts on molecular mechanisms of diseases. 11. Name theoretical and applied issues of molecular medicine. 12. Define basic characteristics of human hereditary diseases. 13. Select the three characteristics of a disease agent that you believe place those exposed to it at a greater risk and justify your position. 14. Briefly discuss why the remaining three characteristics of infectious disease pathogens were not considered by you to be as significant as those you identified above. 15. What is aneuploidy and how would you diagnose it? 16. What does hemizygous mean? Give an example. 17. What is a Barr body? And how would you identify one? 18. How are somatic cell hybrids used to determine which human chromosome a particular gene is located on? 19. Give examples of at least four inherited diseases in which triplet repeat sequences are implicated in the inheritance of the disease.

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2

HUMAN GENOME PROJECT: METHODS, PROSPECTS

2.1. Human Genome Project goals and milestones The extensive genetic variation present in humans represents an invaluable resource for molecular biology and for medical investigation. Human Genome Project (HGP) was one of the great feats of exploration in history - an inward voyage of discovery rather than an outward exploration of the planet or the cosmos; an international research effort to sequence and map all of the genes - together known as the genome - of members of our species, Homo sapiens. Completed in April 2003, the HGP gave us the ability, for the first time, to read nature’s complete genetic blueprint for building a human being. Goals: –– identify all the approximate 30,000 genes in human DNA; –– determine the sequences of the 3 billion chemical base pairs that make up human DNA; –– store this information in databases; –– improve tools for data analysis; –– transfer related technologies to the private sector; –– address the ethical, legal, and social issues that may arise from the project. –– Milestones: –– 1990: P roject initiated as joint effort of U.S. Department of Energy and the National Institutes of Health;

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Chapter 2. Human genome project: methods, prospects

–– June 2000: Completion of a working draft of the entire human genome; –– February 2001: Analyses of the working draft are published; –– April 2003: HGP sequencing is completed and Project is declared finished two years ahead of schedule. The timeline of the HGP, as shown in Appendix 2, Fig. 2.1.1: 1. Much of the work that made sequencing the human genome possible actually took place something like more than a century ago, starting with the elucidation of the nature of inheritance, subsequent localization of heredity to chromosomes, then confirmation of DNA as the genetic material and finally the discovery of the double helical structure of DNA. This was then followed by discovering how DNA replication took place, how DNA specifies protein sequences and further insights from the role of genes and proteins in development. 2. Along the way, we learned how variations in genes can contribute to disease, starting with the implication of genes in Huntington’s chorea and the discovery of the variant gene that causes Duchenne’s muscular dystrophy. The development of Sanger sequencing was a landmark. 3. Sequencing the genomes of several model organisms along the way is very significant in my opinion because it allows us to use genetic data from those organisms in relation to our own so that they can be of import to human studies and biology. We already know of genes such as Pax6 which are universally conserved in the development of animal eyes, for instance. We are able to carry out precise comparison between gene sequences in order to identify what genetic differences are responsible for phenetic differences. 4. The immense amounts of cooperation seen, with the publicly funded project actively involving large amounts of international partners is an indicator of what could be achieved if only people were willing to look beyond the barriers of nationalism, in my opinion. The fact that we had sequencing centers from North America, Europe and Asia is also an indicator of how important one could hold the common heritage that we all share to be. 5. The completion of sequencing of the first chromosome in the HGP, namely Chromosome 22 was a significant landmark since it showed it could be done and was only a matter of time. 6. The announcement of a completed draft in 2003 was a beautiful moment in the history of science, since a thirteen year project had now officially ended with all its goals achieved.

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Upon publication of the majority of the genome in February 2001, Francis Collins, the director of NHGRI, noted that the genome could be thought of in terms of a book with multiple uses: It’s a history book – a narrative of the journey of our species through time. It’s a shop manual, with an incredibly detailed blueprint for building every human cell. And it’s a transformative textbook of medicine, with insights that will give health care providers immense new powers to treat, prevent and cure disease. 2.2. Basic instruments of the Human Genome Project In light of the project’s main goal - to map the location of all the genes on every chromosome and to determine the exact sequence of nucleotides of the entire genome - two types of maps are being made. One of these is a physical map that measures the distance between two genes in terms of nucleotides. A very detailed physical map is needed before real sequencing can be done. Sequencing is the precise order of the nucleotides. The other map type is called a genetic linkage map and it measures the distance between two genes in terms of how frequently the genes are inherited together. This is important since the closer genes are to each other on a chromosome; the more likely they are to be inherited together. The human genome presented unique challenges for the development of a clone-based physical map. Its size of 3.2 gigabases (Gb), which is 25 times as large as any previously mapped genome, meant that proportionately more clones had to be analysed. Its greater complexity also made it more difficult to distinguish true overlaps, which was further complicated by the repeat-rich nature of the genome. Early efforts to construct clone-based regional and even chromosomal physical maps of the human genome using cosmid libraries derived from isolated human chromosomes met with limited success. By contrast, maps based on sequence-tagged site (STS) landmarks provided greater coverage of the genome, as did genetic maps based on variations in simple sequence repeats in STS landmarks. The development of P1-artificial chromosome (PAC) and bacterial artificial chromosome (BAC) cloning systems was pivotal to the success of the whole-genome map. They provided larger inserts, more stable clones and better coverage of the genome. Clone-based maps similar to that described here have been important in the sequencing of most large genomes, including those of Saccharomyces cerevisiae, Caenorhabditis elegans and Arabidopsis

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Chapter 2. Human genome project: methods, prospects

thaliana. A clone-based map also contributed to the sequencing of the Drosophila melanogaster genome and a combined mapping and sequencing strategy is being applied to the mouse genome. This work illustrates the benefit of using the clone-based map in the assembly of the human genome sequence. The pilot phase of the sequencing project began in 1995, at which time efforts were renewed to develop clone-based maps covering specific regions of the genome. To construct these regional maps, we screened PAC and BAC clones for STS markers, fingerprinted the positive clones, integrated them into the existing maps, and selected the largest, intact clones with minimal overlap for sequencing. To keep pace with the ramping up of the sequencing effort in 1998, the ongoing efforts to construct the whole-genome BAC map were increased approximately tenfold. The whole-genome BAC map was constructed in several steps. First fingerprint data for a large sample of random clones from a genome-wide BAC library; then assembled the BAC map, first by using the fingerprint data to cluster highly related clones automatically, then by further refining them manually, and last by merging contigs with related clones at their ends; finally, in parallel with construction of the BAC map, the chromosomal positions of individual clones mapped on the basis of landmarks from existing landmark maps. In October 1998, fingerprinting 300,000 BACs started from the RPCI-11 library19 (http://www.chori.org/bacpac/). Redundancy of sam­ pling was vital to achieve high continuity in the final map14. Assuming an average BAC insert size of 150,000 base pairs (bp) and a genome size of 3.2 Gb, this level of fingerprinting would provide roughly 15-fold coverage of the genome. The library was derived from male DNA, providing full coverage of all 24 human chromosomes but with half as much coverage of the sex chromosomes as of the autosomes. The RPCI-11 library was one of the first libraries to meet the informed consent criteria in accordance with the NHGRI policy for the Use of Human Subjects in Large Scale Sequencing (Figure 2.2.1). Clone-by-clone sequencing means that the chromosomes were mapped and then split up into sections. A rough map was drawn for each of these sections, and then the sections themselves were split into smaller bits, with plenty of overlap between each of the bits. Each of these smaller bits would be sequenced, and the overlapping bits would be used to put the genome jigsaw back together again (Figure 2.2.2).

2.2. Basic instruments of the Human Genome Project

Figure 2.2.1. Gene Sequencing Computers

Figure 2.2.2. Clone-by-clone method

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Chapter 2. Human genome project: methods, prospects

First, by mapping the genome, researchers produce, at an early stage, a genetic resource that can be used to map genes. In addition, because every DNA sequence is derived from a known region, it is relatively easy to keep track of the project and to determine where there are gaps in the sequence. Moreover, assembly of relatively short regions of DNA is an efficient step. However, mapping can be a time-consuming, and costly, process. –– A genomic library (a collection of clones) is developed –– Physical maps are prepared –– Clones are organized into overlapping groups –– DNA cut with restriction enzymes –– Each clone is sequenced and software assembles sequence from the libraries (Figure 2.2.3).

Figure 2.2.3. Application of different sequencing methods during the project

The alternative to the clone-by-clone approach is the ‘bottom-up’ whole genome shotgun (WGS) sequencing. Shotgun sequencing was developed by Fred Sanger in 1982. First, all the DNA is first broken into fragments. The fragments are then sequenced at random and assembled together by looking for overlaps. In recent approaches, libraries have been made from DNA fragments of 2000 base pairs and of 10,000 base pairs in length. The

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two sizes of fragment provide complementary results; by sequencing the ends of the fragments, each provides information about DNA sequences separated by known distances. Computer analysis is used to search the sequences for overlaps. The advantage of the whole-genome shotgun is that it requires no prior mapping. Its disadvantage is that large genomes need vast amounts of computing power and sophisticated software to reassemble the genome from its fragments. To sequence the genome from a mammal (billions of bases long), you need about about 60,000,000 individual DNA sequence reads. Reassembling these sequenced fragments requires huge investments in IT, and, unlike the clone-by-clone approach, assemblies can’t be produced until the end of the project (Fig. 2.2.4).

Figure 2.2.4. The Book of Life displayed at the Wellcome Collection, London (photo from flickr.com)

Whole genome shotgun for large genomes is especially valuable if there is an existing ‘scaffold’ of organized sequences, localized to

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Chapter 2. Human genome project: methods, prospects

the genome, derived from other projects. When the whole genome shotgun data are laid on the ‘scaffold’ sequence, it is easier to resolve ambiguities. Today, whole genome shotgun is used for most bacterial genomes and as a ‘top-up’ of sequence data for many other genome projects (Figure 2.2.4; Appendix 3, Fig. 2.2.5, 2.2.6). Ethical, legal, and social implications of the human genome project –– Use of genetic information –– Privacy/confidentiality –– Psychological impact –– Genetic testing –– Reproductive options/issues –– Education, standards, and quality control –– Commercialization –– Conceptual and philosophical implications 2.3. Prospects and future challenges of the Human Genome Project The sequencing of the human genome holds benefits for many fields, from molecular medicine to human evolution. The Human Genome Project, through its sequencing of the DNA, can help us understand diseases including: genotyping of specific viruses to direct appropriate treatment; identification of oncogenes and mutations linked to different forms of cancer; detection of genetic predispositions to disease; the design of medication and more accurate prediction of their effects; advancement in forensic applied sciences; biofuels and other energy applications; microbial genome research (disease-causing microbes, biofuels, monitor environments to detect pollutants, protect citizenry from biological and chemical warfare); agriculture, livestock breeding, bioprocessing; risk assessment; better understanding of evolution and human migration (maternal inheritance, mutations on the Y chromosome to trace lineage and migration of males, compare breakpoints in the evolution of mutations with ages of populations and historical events). Another proposed benefit is the commercial development of genomics research related to DNA based products, a multibillion dollar industry. Future challenges (what we still don’t know): –– More accurate risk assessment –– Gene number, exact locations, and functions

2.3. Prospects and future challenges of the Human Genome Project

–– –– –– ––

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Gene regulation DNA sequence organization Chromosomal structure and organization Noncoding DNA types, amount, distribution, information content, and functions –– Coordination of gene expression, protein synthesis, and posttranslational events –– Interaction of proteins in complex molecular machines –– Predicted vs experimentally determined gene function –– Evolutionary conservation among organisms –– Protein conservation (structure and function) –– Proteomes (total protein content and function) in organisms –– Correlation of SNPs (single-base DNA variations among individuals) with health and disease –– Disease-susceptibility prediction based on gene sequence variation –– Genes involved in complex traits and multigene diseases –– Complex systems biology including microbial consortia useful for environmental restoration –– Developmental genetics, genomics We are now able to see the individuality of tumors at the level of gene expression programs that determine much of their behavior and capabilities,» says HHMI investigator Patrick O. Brown. By arraying nearly 18,000.00 genes on a glass chip about twice the size of a postage stamp and recording the expression patterns of those genes, researchers have obtained detailed molecular portraits of a form of lymphoma. The gene expression profiling experiments revealed that diffuse large-cell B-cell lymphoma (DLBCL) is actually at least two distinct forms of cancer. This work shows that the molecular portrait of a tumor that we get from DNA microarray analysis can actually be interpreted as a much clearer, more detailed picture of the tumor’s biology and that the new things we can see in this picture really make a difference for the patient (Figure 2.3). The industrialization of molecular biology assay tools has made it practical to use them in clinics. Miniaturization into a single handheld device can bring medical diagnostics into the clinic and into the office or home. The clinical laboratory requires high standards of reliability; diagnostics may require accreditation or fall under medical device regulations.

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Chapter 2. Human genome project: methods, prospects

Figure 2.3. The DLBCL expression matrix, with rows and columns ordered according to a hierarchical clustering applied separately to the rows and columns

Molecular diagnostics uses biological assays such as PCR-ELISA or fluorescence in situ hybridization. The assay detects a molecule, often in low concentrations, that is a marker of disease or risk in a sample taken from a patient. Preservation of the sample before analysis is critical. Manual handling should be minimized. The fragile RNA molecule poses certain challenges. As part of the cellular process of expressing genes as proteins, it offers a measure of gene expression but it is vulnerable to hydrolysis and breakdown by ever-present RNAse enzymes. Samples can be snap-frozen in liquid nitrogen or incubated in preservation agents. Because molecular diagnostics can detect slighter markers, it is less intrusive than a biopsy. For example, because cell-free nucleic acids exist in human plasma, a simple blood sample can be enough to sample genetic information from tumors, transplants or an unborn fetus. Molecular diagnostics based on nucleic acids use polymerase

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chain reaction to vastly increase the number of nucleic acid molecules and amplify the target. The detection of the marker might use real time PCR, direct sequencing, or microarrays – prefabricated chips that test many markers at once. The same principle applies to the proteome and the genome. High-throughput protein arrays can use complementary DNA or antibodies to bind and hence can detect many different proteins in parallel (Fig. 2.4).

Figure 2.4. A microarray chip contains complementary DNA (cDNA) to many sequences of interest. The cDNA fluoresces when it hybridizes with a matching DNA fragment in the sample

Conventional prenatal tests for chromosomal abnormalities such as Down syndrome rely on analyzing the number and appearance of the chromosomes – the karyotype. Molecular diagnostics tests such as microarray comparative genomic hybridization test a sample of DNA instead, and because of cell-free DNA in plasma, could be less invasive, but as of 2013 it is still an adjunct to the conventional tests. Some of a patient’s single nucleotide polymorphisms – slight differences in their DNA – can help predict how quickly they will metabolize particular drugs; this is called pharmacogenomics. For example, the enzyme CYP2C19 metabolizes several drugs, such as the anti-clotting agent Clopidogrel, into their active forms. Some patients possess polymorphisms in specific places on the 2C19 gene

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Chapter 2. Human genome project: methods, prospects

that make poor metabolizes of those drugs; physicians can test for these polymorphisms and find out whether the drugs will be fully effective for that patient. Advances in molecular biology have helped show that some syndromes that were previously classed as a single disease are actually multiple subtypes with entirely different causes and treatments. Molecular diagnostics can help diagnose the subtype – for example of infections and cancers - or the genetic analysis of a disease with an inherited component, such as Silver-Russell syndrome. Molecular diagnostics is used to identify infectious diseases such as chlamydia, influenza virus and tuberculosis; or specific strains such as H1N1 virus. Genetic identification can be swift; for example a loopmediated isothermal amplification test diagnoses the malaria parasite and is rugged enough for developing countries. But despite these advances in genome analysis, in 2013 infections are still more often identified by other means – their proteome, bacteriophage, or chromatographic profile. Molecular diagnostics are also used to understand the specific strain of the pathogen – for example by detecting which drug resistance genes it possesses – and hence which therapies to avoid. A patient’s genome may include an inherited or random mutation which affects the probability of developing a disease in the future. For example, Lynch syndrome is a genetic disease that predisposes patients to colorectal and other cancers; early detection can lead to close monitoring that improves the patient’s chances of a good outcome. Cardiovascular risk is indicated by biological markers and screening can measure the risk that a child will be born with a genetic disease such as Cystic fibrosis. Genetic testing is ethically complex: patients may not want the stress of knowing their risk. In countries without universal healthcare, a known risk may raise insurance premiums. Cancer is a change in the cellular processes that cause a tumor to grow out of control. Cancerous cells sometimes have mutations in oncogenes, such as KRAS and CTNNB1 (β-catenin). Analyzing the molecular signature of cancerous cells – the DNA and its levels of expression via messenger RNA – enables physicians to characterize the cancer and to choose the best therapy for their patients. As of 2010, assays that incorporate an array of antibodies against specific protein marker molecules are an emerging technology; there are hopes for these multiplex assays that could measure many markers at once. Other potential future biomarkers include micro RNA molecules, which cancerous cells express more of than healthy ones.

Figure 2.5. Part of the diseasome oversimplification. This tool links diseases by shared gene expression. That is, a particular gene may be consistently overexpressed or underexpressed in two diseases, compared to the healthy condition. The conditions are not necessarily inherited because gene expression changes in all situations. Shading for a symptom (Lewis, 2009)

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Chapter 2 Human genome project: methods, prospects

The rate of disease gene discovery is increasing exponentially, which facilitates the understanding diseases at molecular level. Molecular understanding of disease is translated into diagnostic testing, therapeutics, and eventually preventive therapies. To face the new century, the medical practitioner not only understand molecular biology, but must also embrace the use of this rapidly expanding body of information in his medical practice, whether practicing family medicine, oncology, obstetrics and gynecology, pathology, or any other medical specialty (see Fig. 2.5). Conventional prenatal tests for chromosomal abnormalities such as Down syndrome rely on analyzing the number and appearance of the chromosomes – the karyotype. Molecular diagnostics tests such as microarray comparative genomic hybridization test a sample of DNA instead, and because of cell-free DNA in plasma, could be less invasive, but as of 2013 it is still an adjunct to the conventional tests. Sample problem sets on Human Genome Project: Methods, Prospects 1. When RFLP analysis is used to search for a human gene, the strategy is to first locate? A) a known gene on the same chromosome B) an homozygous individual with a simple RFLP pattern C) a DNA sequence anywhere on the same chromosome D) any DNA marker co-inherited with the genetic trait of interest E) an exon of the disease gene 2. Which of the following is not one of the objectives of the Human Genome Project? A) create a detailed genetic map of every human chromosome, with an average of 2-5% recombination frequency between markers B) obtain a detailed physical map of every human chromosome, based on overlapping recombinant DNA molecules cloned as yeast artificial chromosomes C) clone human beings D) determine the sequence of all expressed human genes by cDNA cloning and sequencing E) determine the complete DNA sequence of each human chromosome 3. Which of the following is a useful marker for genetic or physical mapping of human chromosomes? (More than one answer could be correct) A) RFLPs, restriction fragment length polymorphisms

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B) ESTs, expressed sequence tags C) STRPs, short tandem repeat polymorphisms D) STSs, sequence tagged sites E) all of the above 4. Which of the following describes a type of polymorphism that occurs within the gene that causes Huntington’s disease? A) RFLPs, restriction fragment length polymorphisms B) ESTs, expressed sequence tags C) STRPs, short tandem repeat polymorphisms D) STSs, sequence tagged sites E) none of the above 5. Which of the following type(s) of polymorphism are commonly detected by using the polymerase chain reaction (PCR) and specific oligonucleotide primers? A) RFLPs, restriction fragment length polymorphisms B) ESTs, expressed sequence tags C) STRPs, short tandem repeat polymorphisms D) STSs, sequence tagged sites 6. Which of the following is a marker for regions of the DNA expressed as mRNA transcripts, and characterized by cDNA cloning? A) RFLPs, restriction fragment length polymorphisms B) ESTs, expressed sequence tags C) STRPs, short tandem repeat polymorphisms D) STSs, sequence tagged sites E) all of the above 7. The human MLH1 gene, a DNA mismatch repair protein, is mutated in a form of familial colon cancer. Go to LocusLink and retrieve the entry for human MLH1. Link to the variations mapped for this gene. (You can click on the Var link in the full report or the V in the summary.) What non-synonymous substitutions are reported? Note the Isoleucine-Valine polymorphism at position 32 of the protein. Could such a substitution affect the structure and function of MLH1? Some light can be shed on this question by examining the sequences and structures of homologous proteins from other organisms. You can view the E. coli mutL structure and align its sequence with the human MLH1 in Cn3D. Do this by following the BL link (BLink) from the MLH1 LocusLink report. Once you have the BLink report, click on the 3D structures button. Then retrieve the E. coli structure and sequence alignment by clicking on the blue dot next to 1B63A. What amino acid does the E. coli protein have at the equivalent position to Ile-32 in the human protein? 8. Working in mice, you discover a gene, LC1, that when mutant decreases the incidence of cancer in mice. This gene is normally expressed in the lymphatic

46

Chapter 2 Human genome project: methods, prospects tissues of mice. You know the sequence of this gene, and you really want to see if there is a similar gene is in humans. So, you set out to find the human homolog of LC1 by screening a human library. What kind of human library would you use? Explain. How would you screen the human library for the homolog of LC1? Questions for the individual work of students on Human Genome Project: Methods, Prospects 1. 2. 3. 4. 5. 6.

What is the world’s largest collaborative biological project? Name the basic milestones in HGP. Name current technologies for genome annotation. What is the Moore’s Law and how it benefits to HGP? Give definition to combined reference genome. Analyze the key findings of the draft (2001) and complete (2004) genome sequences. 7. Define genetic techniques for phylogenetic relationships on a broad evolutionary scale. 8. Speak on ethical, legal and social implications of HGP. 9. Give definition to single-nucleotide polymorphism. 10. What is the mitochondrial Eve? 11. What are the segmental duplications and their role in creating new primatespecific genes? 12. Why HGP is considered a Mega Project? 13. Give definition and characterize the bacterial artificial chromosomes. 14. Give definition to the hierarchical shotgun approach. 15. Speak on public versus private approaches on HGP. 16. What are the countries in IHGSC and their input? 17. Who was the single individual who had his entire genome unveiled for the first time ever? 18. Analyze the methods and possible benefits of HapMap and ENCODE Projects. 19. What other Sequencing Projects do you know?

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3

MOLECULAR BASICS OF DISEASES

3.1. Molecular markers of diseases Identification of the tissue of origin of a tumor is vital to its mana­ gement. Rosenwald et al. showed tissue-specific expression patterns of microRNA and suggested that their profiling would be useful in addressing this diagnostic challenge. They developed a clinical test for the identification of the tumor tissue of origin based on a standardized protocol and defined the classification criteria (see Fig. 3.1.1).

Figure 3.1.1. Structure of the binary decision tree

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A series of binary branching points (‘nodes’), starting at node #1 and moving downward along the branches and through the nodes, leads to one of the possible tumor types (classes). Each node (numbered 1-25) represents a binary decision between two sets of samples, those on the left and right branches of the node. Decisions are made at consecutive nodes using microRNA expression levels, until an end point (‘leaf’ of the tree) is reached, indicating the predicted class for this sample. The biliary tract is represented in both the hepatic branch (under node 2) and in the gastrointestinal branch (under node No. 15). The tree, therefore, represents 25 classes in 26 leaves. Developing a different classifier for male and female cases or for different tumor sites would inefficiently exploit measured data and would require unwieldy numbers of samples. Instead, exceptions were noted for several special cases. For samples from female patients, testis or prostate origins were excluded from the KNN database, and the right branch was automatically taken in node 3 and node 16 in the decision tree. For samples from male patients, ovary origin was excluded and the right branch taken at node 17. For samples that were indicated as metastases to the liver, liver origin (hepatocellular carcinoma and biliary tract carcinomas from within the liver) was excluded and the right branch taken at node 1. For samples indicated as brain metastases, brain origin was excluded and the right branch taken at node 7. Additional information is thus incorporated into the classification decision without loss of generality or need to retrain the classifier. In medicine, a biomarker is a measurable indicator of the severity or presence of some disease state. More generally a biomarker is anything that can be used as an indicator of a particular disease state or some other physiological state of an organism. A biomarker can be a substance that is introduced into an organism as a means to examine organ function or other aspects of health. For example, rubidium chloride is used in isotopic labeling to evaluate perfusion of heart muscle. It can also be a substance whose detection indicates a particular disease state, for example, the presence of an antibody may indicate an infection. More specifically, a biomarker indicates a change in expression or state of a protein that correlates with the risk or progression of a disease, or with the susceptibility of the disease to a given treatment. Biomarkers can be characteristic biological properties or molecules

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that can be detected and measured in parts of the body like the blood or tissue. They may indicate either normal or diseased processes in the body. Biomarkers can be specific cells, molecules, or genes, gene products, enzymes, or hormones. Complex organ functions or general characteristic changes in biological structures can also serve as biomarkers. Although the term biomarker is relatively new, biomarkers have been used in pre-clinical research and clinical diagnosis for a considerable time. For example, body temperature is a well-known biomarker for fever. Blood pressure is used to determine the risk of stroke. It is also widely known that cholesterol values are a biomarker and risk indicator for coronary and vascular disease, and that C-reactive protein (CRP) is a marker for inflammation. Biomarkers are useful in a number of ways, including measuring the progress of disease, evaluating the most effective therapeutic regimes for a particular cancer type, and establishing long-term susceptibility to cancer or its recurrence. The parameter can be chemical, physical or biological. In molecular terms biomarker is the subset of markers that might be discovered using genomics, proteomics technologies or imaging technologies. Biomarkers play major roles in medicinal biology. Biomarkers help in early diagnosis, disease prevention, drug target identification, drug response etc. Several biomarkers have been identified for many diseases such as serum LDL for cholesterol, blood pressure, and p53 gene and MMPs as tumor markers for cancer. A tumor marker is a biomarker found in the blood, urine, or body tissues that can be elevated in cancer, among other tissue types. There are many different tumor markers, each indicative of a particular disease process, and they are used in oncology to help detect the presence of cancer. An elevated level of a tumor marker can indicate cancer; however, there can also be other causes of the elevation. Tumor markers can be produced directly by the tumor or by non-tumor cells as a response to the presence of a tumor. Most tumor markers are tumor antigens, but not all tumor antigens can be used as tumor markers. Tumor markers can be detected by immunohistochemistry. If repeated measurements of tumor marker are needed, some clinical testing laboratories provide a special reporting mechanism, a serial monitor, that links test results and other data pertaining to the person being tested. Although mammography, ultrasonography, computed tomography, magnetic resonance imaging scans, and tumor marker assays help in

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the staging and treatment of the cancer, they are usually not definitive diagnostic tests. The diagnosis is confirmed by biopsy. Some tumor markers for associated primary tumors are shown in Table 3.1.1. Table 3.1.1 Some tumor markers for testicular or other germ cell cancers Tu m o u r Associated marker tumour CA 27.29

CEA

CA 19-9 AFP b-hCG

CA-125

PSA

Breast cancer.

primary Other conditions which may yield positive results Colonic, gastric, hepatic, lung, pancreatic, ovarian and prostate cancers. Breast, liver and kidney disorders, ovarian cysts.

Lung, gastric, pancreatic, breast, bladder cancers, medullary thyroid and other head and neck, cervical and hepatic cancers, lymphoma, Colorectal cancer. melanoma. Cigarette smoking, peptic ulcers, inflammatory bowel disease, pancreatitis, hypothyroidism, cirrhosis, biliary obstruction. Colonic, oesophageal and hepatic Pancreatic and biliary tract cancers, pancreatitis, biliary disease, cancers. cirrhosis. Hepatocellular carcinoma, Gastric, biliary and pancreatic cancers, nonseminomatous germ cell cirrhosis, viral hepatitis, pregnancy. tumours. Nonseminomatous germ Rarely elevated in gastrointestinal wcell tumours, gestational cancers, hypogonadal states and trophoblastic disease. marijuana use. Endometrial, Fallopian tube, breast, lung, oesophageal, gastric, hepatic and pancreatic cancers, menstruation, Ovarian cancer. pregnancy, fibroids, ovarian cysts, pelvic inflammation, cirrhosis, ascites, pleural and pericardial effusions, endometriosis. Prostate cancer.

Prostatitis, benign prostatic hypertrophy, prostatic trauma, after ejaculation.

In certain situations, the use of a combination of tumour markers may be appropriate such as: measurement of both human chorionic

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gonadotrophin (hCG) and alpha-fetoprotein (AFP) is mandatory in patients in whom testicular or other germ cell cancers are strongly suspected (these markers are not raised in all such patients); measurement of AFP and hCG is mandatory in the management of germ cell tumours; in some high-risk patients, measurement of AFP, CA-125, or CA 19-9 may aid early detection of hepatocellular carcinoma, ovarian cancer or pancreatic cancer. Detection of antigenic markers in both experimental and clinical settings is limited by both the selectivity of antibodies for the markers and the sensitivity of the detection methods. New methods for in vitro selection of highly selective artificial ligands have the potential to replace antibodies, since the new ligands are not limited by either toxicity or antigenicity of the targets in vivo. In addition, the new molecular tools have the capacity for exponential amplification in assays, making it theoretically possible to detect relatively few molecules in a sample. 3.2. Molecular basics of apoptosis The term apoptosis (a-po-toe-sis) was first used in a now-classic paper by Kerr, Wyllie, and Currie in 1972 to describe a morphologically distinct form of cell death, although certain components of the apoptosis concept had been explicitly described many years previously (Kerr et al., 1972; Paweletz, 2001; Kerr, 2002). Our understanding of the mechanisms involved in the process of apoptosis in mammalian cells transpired from the investigation of programmed cell death that occurs during the development of the nematode Caenorhabditis elegans (Horvitz, 1999). In this organism 1090 somatic cells are generated in the formation of the adult worm, of which 131 of these cells undergo apoptosis or programmed cell death. These 131 cells die at particular points during the development process, which is essentially invariant between worms, demonstrating the remarkable accuracy and control in this system. Apoptosis has since been recognized and accepted as a distinctive and important mode of programmed cell death, which involves the genetically determined elimination of cells. However, it is important to note that other forms of programmed cell death have been described and other forms of programmed cell death may yet be discovered (Formigli et al., 2000; Sperandio et al., 2000; Debnath et al., 2005). Many of the genes that control the killing and engulfment processes of programmed cell death have been identified, and the

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molecular mechanisms underlying these processes have proven to be evolutionarily conserved (Metzstein et al., 1998). Until recently, apoptosis has traditionally been considered an irreversible process with caspase activation committing a cell to death and the engulfment genes serving the purpose of dead cell removal. However, the uptake and clearance of apoptotic cells by macrophages may involve more than just the removal of cell debris. Hoeppner et al. have shown that blocking engulfment genes in C. elegans embryos enhances cell survival when cells are subjected to weak pro-apoptotic signals (Hoeppner et al., 2001). Reddien et al. demonstrated that, in C. elegans, mutations that cause partial loss of function of killer genes allow the survival of some cells that are programmed to die via apoptosis, and mutations in engulfment genes enhance the frequency of this cell survival. Moreover, mutations in engulfment genes alone allowed the survival and differentiation of some cells that were otherwise destined to die via apoptosis (Reddien et al., 2001). These findings suggest that genes that mediate corpse removal can also function to actively kill cells. In other words, the engulfing cells may act to ensure that cells triggered to undergo apoptosis will die rather than recover after the initial stages of death. In vertebrates, there is some evidence of a potential role for macrophages in promoting the death of cells in some tissues. Elimination of macrophages in the anterior chamber of the rat eye resulted in the survival of vascular endothelial cells that normally undergo apoptosis (Diez-Roux and Lang, 1997). Other studies have demonstrated that inhibition of macrophages can disrupt the remodeling of tissues in the mouse eye or in the tadpole tail during regression; two processes that involve apoptosis (Lang and Bishop, 1993; Little and Flores, 1993). Geske and coworkers (2001) demonstrated that early p53induced apoptotic cells can be rescued from the apoptotic program if the apoptotic stimulus is removed. Their research suggests that DNA repair is activated early in the p53-induced apoptotic process and that this DNA repair may be involved in reversing the cell death pathway in some circumstances. Apoptosis occurs normally during development and aging and as a homeostatic mechanism to maintain cell populations in tissues. Apoptosis also occurs as a defense mechanism such as in immune reactions or when cells are damaged by disease or noxious agents

3.2. Molecular basics of apoptosis

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(Norbury and Hickson, 2001). Although there are a wide variety of stimuli and conditions, both physiological and pathological, that can trigger apoptosis, not all cells will necessarily die in response to the same stimulus. Irradiation or drugs used for cancer chemotherapy results in DNA damage in some cells, which can lead to apoptotic death through a p53-dependent pathway. Some hormones, such as corticosteroids, may lead to apoptotic death in some cells (e.g., thymocytes) although other cells are unaffected or even stimulated.

Figure 3.2.1. Some of the factors influencing a cell’s decision to undergo apoptosis. Please note: lines that end with arrowheads indicate activation of another factor or process, whereas lines ending with a perpendicular bar indicate inhibition of another factor of process

Some cells express Fas or TNF receptors that can lead to apoptosis via ligand binding and protein cross-linking. Other cells have a default death pathway that must be blocked by a survival factor such as a hormone or growth factor. There is also the issue of distinguishing apoptosis from necrosis, two processes that can occur independently, sequentially, as well as simultaneously (Hirsch, 1997; Zeiss, 2003). In some cases it’s the type of stimuli and/or the degree of stimuli that determines if cells die by apoptosis or necrosis. At low doses, a variety

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of injurious stimuli such as heat, radiation, hypoxia and cytotoxic anticancer drugs can induce apoptosis but these same stimuli can result in necrosis at higher doses. Finally, apoptosis is a coordinated and often energy-dependent process that involves the activation of a group of cysteine proteases called caspases and a complex cascade of events that link the initiating stimuli to the final demise of the cell (Fig. 3.2.1).

Figure 3.2.2. Schematic representation of apoptotic events

The two main pathways of apoptosis are extrinsic and intrinsic as well as a perforin/granzyme pathway. Each requires specific triggering signals to begin an energy-dependent cascade of molecular events. Each pathway activates its own initiator caspase (8, 9, 10) which in turn will activate the executioner caspase-3. However, granzyme A works in a caspaseindependent fashion. The execution pathway results in characteristic cytomorphological features including cell shrinkage, chromatin con­ densation, formation of cytoplasmic blebs and apoptotic bodies and finally phagocytosis of the apoptotic bodies by adjacent parenchymal cells, neoplastic cells or macrophages (Fig. 3.2.2). Sample of case – study presentation on Apoptosis of erythrocytes is given in Appendix 4.

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There are some specific points that require specific attention: 1. Haploid cells contain only one copy of each chromosome. Diploid cells contain two copies, one from the organism’s father, and one from the mother. 2. Cell cycle comprises S, G1, M, and G2 phases. G2, S, and G1 phases together constitute interphase. DNA is replicated in S phase, so that each chromosome becomes a pair of identical chromatids. The cell physically divides in mitosis, or M phase. 3. Mitosis comprises prophase, prometaphase, metaphase, anap­ hase, telophase, and cytokinesis. The end result of mitosis is two dip­ loid cells whose chromosome complement is the same as that of the original cell before it underwent S phase. 4. Meiosis generates haploid germ cells: in vertebrates, eggs and sperm. Like mitosis, it follows an S phase but comprises two cycles of cell division, so that the end result is four cells whose chromosome complement is only half that of the original cell before it underwent S phase. 5. During meiosis I homologous chromosomes undergo recom­ bination, a physical resplicing of homologous chromosomes that allows information on chromosomes originating from father and mother to be mixed. 6. Recessive genes are usually those that fail to make functional protein. Examples are the gene for blue eyes and the gene for phenylketonurea. The functional gene is called dominant because an individual need inherit only one copy to be able to make functional protein. 7. In a few cases a defective gene, for example, the gene for fami­ lial Creutzfeldt–Jacob disease, is dominant over a normal one. 8. Cells enter mitosis when cyclin-dependent kinase 1 is active. This in turn requires that cyclin B be present in high enough concentration and that cyclin-dependent kinase 1 be dephosphorylated by Cdc25. Once mitosis is initiated, cyclin-dependent kinase 1 is rapidly turned off through phosphorylation by WEE1 in parallel with the proteolytic destruction of cyclin B. 9. The entry of cells into S phase is a more complex decision involving cyclin dependent kinases 2, 4, and 6. The main effect of active cyclin-dependent kinase 4 is to phosphorylate RB, causing it to release the transcription factor E2F-1 and hence allowing the synthesis of proteins required for DNA synthesis. Important components of the decision are a raised concentration of cyclinDas a result ofMAPkinase

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Chapter 3 Molecular basics of diseases

activity and a low concentration of CKIs. Cell–cell contact upregulates CKIs so that when an organ has filled the space available it stops growing. 10. p53 is continually produced in cells but is as quickly destroyed. An increase in the concentration of p53 follows DNA damage or other cell stress and has three main effects: (a) activation of DNA repair mechanisms, (b) synthesis of CKIs, preventing cell division, and (c) activation of apoptosis. 11. In contrast to necrosis, which causes inflammation, apoptosis is a regulated mechanism of cell suicide that has little effect on the surrounding tissue. The final effectors of apoptosis are a family of proteases called caspases. 12. Apoptosis can be triggered in three ways: (a) binding of ligand to death domain receptors, (b) denial of growth factors, and (c) cell stress. Sample problem sets on Molecular basics of diseases 1. Several changes occur during cancer progression. Number each of the following events to indicate their most likely order of occurrence, with 1 indicating the earliest stage and 6 indicating the most advanced stage. At which of the above stages does mutation of one or more genes play a significant role? A) cells in a state of metastasis B) cells intrude upon basement membrane C) hyperplasia D) cells form benign tumor, less than 1 mm in diameter E) cells in a state of homeostasis F) tumor cells induce angiogenesis 2. Which of the following is NOT a typical event associated with cell signaling? A) activation of G-proteins by exchanging GTP for GDP B) production of the second messengers cAMP and IP3 C) activation of protein kinases D) release of calcium ions from cell membranes E) stimulation of apoptosis 3. Some receptors for growth factors activate a protein kinase cascade, with the participation of multiple enzymes to effect a change in gene expression. Which of the following statements about a protein kinase cascade are true? A) multiple steps allow the amplification of the signal B) external signals can lead to changes in gene expression

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C) multiple steps leading to kinase activations can result in cells having different responses, depending on the presence or absence of target proteins D) multiple steps in an activation mean that abnormal stimulation of a cell response such as growth can occur with mutations in more than one gene E) all of the above are true 4. In some cancer cells, CDK6 enhances phosphorylation the retinoblastoma protein. Indole-3-carbinol (I3C), a compound found in vegetables such as Brussels sprouts, inhibits CDK6 activity. Knowing the cell cycle as you do, would you expect I3C to promote, suppress or have no net effect on the progression of cancerous cells? 5. The p53 gene is frequently mutated in human cancers. However, a subset of tumors possesses two wild type copies of the p53 gene, expresses normal level of p53 mRNA, but no p53 protein can be detected. Karyotyping analysis of these tumors reveals over-amplification of a locus that is unlinked from the p53 locus. Based on what you have learned about p53, propose a model to explain the above observations. 6. Which is more likely to be associated with the development and progression of cancer: an increase in the amount of apoptosis or a decrease in the amount of apoptosis among cells of the human body? Explain your answer in 15 words or fewer. 7. The sputum (fluid coughed up from the lungs) of many smokers contain cells with mutations in the genes for p53. The smoking induced mutations appear to be an early signal showing that cancer of the lungs will follow. What is the likely relationship between early p53 mutation and the development of lung cancer? A) p53 with a mutation directly stimulates the growth of cancer cells B) mutations in p53 would prevent abnormal cells from dying by apoptosis C) mutant p53 triggers the M phase of the cell cycle leading to abnormal cell division D) p53 causes a cell to enter G0, blocking cell division 8. Alternative splicing during expression of the Down Syndrome Cell Adhesion Molecule (DSCAM) permits the formation of approximately 38,000 different protein products. In contrast, V(D)J somatic recombination permits the formation of approximately 144,000 different antibody heavy chain proteins. How many different kinds of protein can be made by a single cell that is actively expressing either heavy chains or DSCAM? 9. HIV infection proceeds by a mechanism that requires a specific interaction between the virus and the CD4 protein on the surface of helper T cells. Imagine that you were able to develop an animal model to study HIV infection. When you expose normal animals to HIV, you find that the number of HIV particles

58

Chapter 3 Molecular basics of diseases (virions) circulating in their blood increases dramatically, and the animals typically die within 3 months from opportunistic infections. In transgenic animals, you eliminate the genes encoding CD4 protein, so HIV is unable to infect helper T cells. When you expose these animals to HIV, you find that no virus particles accumulate in their blood. You are elated! Then, much to your dismay, you find that most of the animals die anyway within 3 months from opportunistic infections. Explain this result in 15 words or fewer. 10. A human gene called the CF gene (for cystic fibrosis) encodes a protein that functions in the transport of chloride ions across the cell membrane. Most people have two copies of a functional CF gene and do not have cystic fibrosis. However, there is a mutant version of the cystic fibrosis gene. If a person has two mutant copies of the gene, he or she develops the disease known as cystic fibrosis. Below are the examples of which of the following: molecular, cellular, organismal, or populational level? A) people with cystic fibrosis have lung problems due to a buildup of mucus in their lungs B) mutant CF gene encodes a chloride transporter that doesn’t transport chloride ions well C) defect in the chloride transporter causes a salt imbalance in lung cells D) scientists have wondered why the mutant cystic fibrosis gene is relatively common. It is the most common mutant gene that causes a severe disease in Caucasians. Usually, mutant genes that cause severe diseases are relatively rare. One explanation why the CF gene is so common is that people who have one copy of the functional CF gene and one copy of the mutant gene are resistant to diarrheal diseases. Therefore, even though individuals with two mutant copies are very sick, people with one mutant copy and one functional copy are more disease resistant than people with two functional copies of the gene. 11. Suppose you have a unique fragment of DNA that has been mapped on human chromosome 5. Think about what you have to do to find out whether there are RFLPs at this locus in the human population. Hints: you need DNA from many people, you need many different restriction enzymes, you need to run many gels and perform Southern blots. Questions for the individual work of students on Molecular basics of diseases 1. 2. 3. 4. 5. 6. 7.

Name and describe different types of cell death. Reveal the basic ways of apoptosis regulation. Compare apoptosis in normal and pathological development. Define the morphological traits of apoptosis. Reveal the biochemical characteristics of apoptosis. Name genes responsible for apoptosis. Characterize the stages of apoptosis.

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8. Speak on Ca2+-dependent endonucleases. 9. Analyze apoptosis specific DNA-fragmentation. 10. Draw a scheme for the formation of apoptotic bodies and phagocytosis. 11. Name the methods of apoptosis detection. 12. Reveal the regulation of apoptosis by cell itself. 12. Draw a scheme for the mitochondrial ways of apoptosis regulation. 13. Characterize the role of nuclear transcriptional factors in the induction of apoptosis. 14. Reveal the role of cell signaling system in regulation of apoptosis. 15. Characterize the killer receptors. 16. Define the role of caspases in realization of apoptosis. 17. Give the definition to oncogene. Give an example. 18. Give the definition a tumor suppressor. Give an example. 19. Analyze the apoptosis regulation genes mutations as a reason for cancer development. 20. Analyze the apoptosis regulation genes as tumor suppressor genes and oncogenes. 21. Speak on the main effect of CDK4. 22. Characterize three main effects of p53 concentration increase. 23. Name three basic ways that apoptosis can be triggered and characterize them.

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CHAPTER

4

MOLECULAR DIAGNOSTICS IS TRANSFORMING MEDICINE

Molecular diagnostics, the use of diagnostic testing to understand the molecular mechanisms of an individual patient’s disease, will be pivotal in the delivery of safe and effective therapy for many diseases in the future. Role of molecular diagnostics in personalized medicine covers the following aspects: –– early detection and selection of appropriate treatment determined to be safe and effective on the basis of molecular diagnostics; –– integration of molecular diagnostics with therapeutics; –– monitoring therapy as well as determining prognosis. In parallel with two important components of personalized medicine – pharmacogenetics and pharmacogenomics there are two types of tests relevant to personalized medicine. 1. A pharmacogenomic test is an assay intended to study inter­ individual variations in whole genome single nucleotide polymorphism (SNP) maps and haplotype markers, alterations in gene expression, or inactivation that may be correlated with pharmacological function and therapeutic response. In some cases the pattern or profile of the change rather than the individual biomarker is relevant to diag­ nosis. 2. A pharmacogenetic test is an assay intended to study interindividual variations in DNA sequence related to drug absorption and disposition (pharmacokinetics), including polymorphic variations in genes that encode

Chapter 4. Molecular diagnostics is transforming medicine

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the functions of transporters, metabolizing enzymes, receptors, and other proteins.

Figure 4.1. Use of molecular medicine in a complex approach to improved quality of life and higher survival rates

Examples of molecular diagnostic technologies used for perso­ nalized medicine include: –– Polymerase chain reaction (PCR)-based methods –– Restriction fragment length polymorphism –– Single-strand conformational polymorphism –– Arrayed primer extension –– Enzyme mutation detection –– Locked nucleic acid technology –– Peptide nucleic acid technology –– Transcription-mediated amplification –– Gene chip and microfluidic microarrays –– Nanodiagnostics –– Nanoparticle-based integration of diagnostics with therapeutics –– Nanotechnology-based refinement of diagnostics for pharmacoge­ netics –– Toxicogenomics –– Single nucleotide polymorphism genotyping

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Modern issues in molecular diagnostics

–– DNA methylation studies –– Gene expression based tests –– DNA sequencing –– Multiplex DNA sequencing –– Sequencing in microfabricated high-density picoliter reactors –– Whole genome sequencing –– Cytogenetics –– Comparative genomic hybridization (CGH) –– Fluorescent in situ hybridization –– Proteomic-based methods –– Fluorescent in situ protein detection –– Protein biochip technology –– Toxicoproteomics –– MicroRNA-based diagnostics –– Molecular imaging –– Functional MRI with nanoparticle contrast –– FDG-PET –– Optical imaging –– Point-of-care diagnostics Several research and clinical laboratories are now using DNA/ RNA sequencing technology for the following applications that are relevant to personalized medicine: HIV resistance sequence analysis, HCV genotyping, genetic diseases. Most new sequencing techniques simulate aspects of natural DNA synthesis to identify the bases on a DNA strand of interest either by base extension or ligation. Both approaches depend on repeated cycles of chemical reactions. However, cost can be lowered and speed is increased by miniaturization to reduce the amount of chemicals used and to read millions of DNA sequences simultaneously. Biochip is a broad term indicating the use of microchip technology in molecular biology and can be defined as arrays of selected biomolecules immobilized on a surface. DNA microarray is a rapid method of sequencing and analyzing genes. An array is an orderly arrangement of samples. The sample spot sizes in microarray are usually less than 200 mm in diameter. It is comprised of DNA probes formatted on a microscale (biochips) plus the instruments needed to handle samples (automated robotics), read the reporter molecules (scanners), and analyze the data (bioinformatic tools).

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Selected applications of biochip technology relevant to personalized medicine are listed below: –– Rapid DNA sequencing –– Drug discovery and development –– High-throughput drug screening –– Design and stratification of clinical trials –– Drug safety: applications in pharmacogenetics –– Toxicogenomics –– Clinical drug safety –– Molecular diagnostics –– Genetic screening –– Detection of mutations –– Inherited disorders –– Identification of pathogens and resistance in infections –– Molecular oncology –– Cancer prognosis –– Cancer diagnosis –– Pharmacogenomics –– Gene identification –– Genetic mapping –– Gene expression profiling –– Detection of single nucleotide polymorphisms –– For storage of the patient’s genomic information –– Integration of diagnosis and therapeutics Microarrays allow scientists to look at very subtle changes in many genes simultaneously. They provide a snapshot of what genes are expressed or active, in normal and diseased cells. When normal cells or tissues are compared to those known to be diseased, patterns of gene expression can emerge, enabling scientists to classify the severity of the disease and to identify the genes that can be targeted for therapy. Exciting advances in fluorescent in situ hybridization (FISH) and array-based techniques are changing the nature of cytogenetics, in both basic research and molecular diagnostics. Cytogenetic analysis now extends beyond the simple description of the chromosomal status of a genome and allows the study of fundamental biological questions, such as the nature of inherited syndromes, the genomic changes that are involved in carcinogenesis, and the 3D organization of the human genome. The high resolution that is achieved by

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Modern issues in molecular diagnostics

these techniques, particularly by microarray technologies such as array comparative genomic hybridization, is blurring the traditional distinction between cytogenetics and molecular biology. Classic cytogenetics has evolved from black and white to technicolor images of chromosomes as a result of advances in FISH techniques, and is now called molecular cytogenetics. Improvements in the quality and diversity of probes suitable for FISH, coupled with advances in computerized image analysis, now permit the genome or tissue of interest to be analyzed in detail on a glass slide. It is evident that the growing list of options for cytogenetic analysis has improved the understanding of chromosomal changes in disease initiation, progression, and response to treatment. In addition, differential molecular cell phenotypes between diseased and healthy cells provide molecular data patterns for (a) predictive medicine by cytomics or for (b) drug discovery purposes using reverse engineering of the data patterns by biomedical cell systems biology. Molecular pathways can be explored in this way including the detection of suitable target molecules, without detailed a priori knowledge of specific disease mechanisms. This is useful during the analysis of complex diseases such as infections, allergies, rheumatoid diseases, diabetes, or malignancies. The top-down approach reaching from single cell heterogeneity in cell systems and tissues down to the molecular level seems suitable for a human cytome project to systematically explore the molecular biocomplexity of human organisms. High-resolution genome-wide association studies using panels of 300,000 to 1 million SNPs aim to define genetic risk profiles of common diseases. These studies provide an opportunity to explore pathomechanism of human diseases and are unbiased by previous hypotheses or assumptions about the nature of genes that influence complex diseases. Many genetic variants identified as risk factors for diseases by such studies have been localized to previously unsuspected pathways, to genes without a known function. An alternative approach to SNP genotyping is haplotyping. Haplotyping information makes it possible to highlight the structure of the genome, notably through haploblocks which correspond to segments of chromosomes unlikely to undergo a crossing-over event. Haplotyping is a way of characterizing combinations of SNPs that might influence response and is considered to be a more accurate measure of phenotypic variation.

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In the absence of functional information about which poly­ morphisms are biologically significant, it is desirable to test the potential effect of all polymorphismson drug response. Potential uses of SNP markers include drug discovery and prediction of adverse effects of drugs: –– Digital Genetic Analysis –– DNA chips and microarrays –– DNA sequencing –– Electrochemical DNA detection –– Solution-borne ferrocene-modified DNAs –– Redox-active intercalators –– Surface-bound molecular beacon-like DNA –– Fluorescence-detected 5¢-exonuclease assays –– Hybridization assays –– Allele-specific oligomer hybridization –– Hybridization with PNA probes –– Invader assay –– Mass spectrometry (MS) –– Competitive Oligonucleotide Single Base Extension –– Nanoparticle probes –– Oligomer-specific ligation assays –– PCR-based methods –– PCR-CTPP (confronting two-pair primers) –– Degenerate oligonucleotide primed (DOP)-PCR –– TaqMan real-time PCR –– Smart amplification process version 2 –– Peptide nucleic acid (PNA) probes –– Primer extension –– Pyrosequencing –– Single base extension-tag array on glass slides (SBE-TAGS) –– Single molecular fluorescence technology –– Triplex Assay (Genetic Technologies, Inc.) However, SNP-based tests have greater power when the number of causative SNPs (a subset of the total set of SNPs) is smaller than the total number of haplotypes. One limitation of haplotyping is that haplotypes need to be determined for each individual, as SNPs detected from a pool of DNA from a number of individuals cannot yield haplotypes. Until whole-genome sequencing of individual patients becomes feasible clinically, the identification of SNPs and haplotypes will prove

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instrumental in efforts to use genomic medicine to individualize health care. When an extensive inventory of genome-wide SNP scans has been assembled across diverse population samples, maps using SNP and/or haplotypes will dictate that it will not be necessary to identify the precise genes involved in determining therapeutic efficacy or an adverse reaction. Linkage disequilibrium (LD) methods can provide robust statistical correlations between a patients response/risk index for a given drug class and a specific LD-SNP/haplotype profile. Candidate gene-based haplotype approach has been applied to the pharmacogenetics of drug response and adverse events. Clinical trials using haplotyped individuals were the first genetically personalized medical treatments. Compared to the map of the human genome, which provides a route finder in genetics, a haplotype map will show the sites along the way. HapMap, a public resource created by the International HapMap Project (www.hapmap.org), is a catalog of genetic variants that are common in human populations. It will enable efficient and large scale studies in genetics and show common variants that cause disease. The HapMap project is the first major post-genomic initiative and is built on the experience gained from sequencing the human genome. The results will provide the physicians with basics of pharmacogenomics to enable them to give personalized treatments to their patients. HapMap will accelerate the discovery of genes related to common diseases, such as asthma, cancer, diabetes, and heart disease. This information will aid researchers searching for the genetic factors that affect health, disease, and responses to drugs and the environment. HapMap is a shortcut to scanning through millions of SNPs. One need only to find blocks into which the genome is organized, each of which may contain several SNPs. SNPs in a haplotype block are inherited together and the pattern of SNPs in a haplotype block is unique for an individual. Currently this information is being used for the development of genetic panels to be used in pharmacogenomic and disease risk assessment studies. HapMap would be useful in the US where little is known of the geneology of the population. Some population groups, however, share haplotype patterns from their common ancestors. HapMap program would be superfluous in Iceland, where it is possible to isolate disease genes in the highly structured genealogy of Iceland for any disease with a prevalence of more than 0.2%.

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Nanotechnology is the creation and utilization of materials, devices, and systems through the control of matter on the nanometer-length scale, i.e., at the level of atoms, molecules, and supramolecular structures. It is the popular term for the construction and utilization of functional structures with at least one characteristic dimension measured in nanometers (a nanometer is one billionth of a meter (10–9 m). Application of nanobiotechnology in molecular diagnostics is called nanodiagnostics. Advances in nanotechnology are providing nanofabricated devices that are small, sensitive and inexpensive enough to facilitate direct observation, manipulation, and analysis of a single biological molecule from a single cell. This opens new opportunities and provides powerful tools in the fields such as genomics, proteomics, molecular diagnostics, and high throughput screening. It seems quite likely that there will be numerous applications of inorganic nanostructures in biology and medicine as markers. Given the inherent nanoscale of receptors, pores, and other functional components of living cells, the detailed monitoring and analysis of these components will be made possible by the development of a new class of nanoscale probes. Biological tests measuring the presence or activity of selected substances become quicker, more sensitive, and more flexible when certain nanoscale particles are put to work as tags or labels. Nanoparticles are the most versatile material for developing diagnostics. Nanomaterials can be assembled into massively parallel arrays at much higher densities than is achievable with current sensor array platforms and in a format compatible with current microfluidic systems. Currently, quantum dot technology is the most widely employed nanotechnology for diagnostic developments. Among the recently emerging technologies, the one using cantilevers is the most promising. This technology complements and extends current DNA and protein microarray methods, because nanomechanical detection requires no labels, optical excitation, or external probes and is rapid, highly specific, sensitive, and portable. This will have applications in genomic analysis, proteomics, and molecular diagnostics. Nanotechnology has potential advantages in applications in point-of-care (POC) diagnosis: on patient’s bedside, self-diagnostics for use in the home, integration of diagnostics with therapeutics, and for the development of personalized medicines. An innovative method based on cantilevers has been developed for the rapid and sensitive detection of disease- and treatment-relevant

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genes. This method detects active genes directly by measuring their mRNA transcripts, which represent the intermediate step and link to protein synthesis. Short complementary nucleic acid segments (sensors) are attached to silicon cantilevers, which are 450 nm thick and therefore react with extraordinary sensitivity. Binding of the targeted gene transcript to its matching counterpart on one of the cantilevers results in optically measurable mechanical bending. Differential gene expression of the gene 1–8U, a potential marker for cancer progression or viral infections, could be observed in a complex background. The measurements provide results within minutes at the picomolar level without target amplification, and are sensitive to base mismatches. An array of different gene transcripts can even be measured in parallel by aligning appropriately coated cantilevers alongside each other like the teeth of a comb. The new method complements current molecular diagnostic techniques such as the gene chip and real- PCR. It could be used as a real-time sensor for continuously monitoring various clinical parameters or for detecting rapidly replicating pathogens that require prompt diagnosis. These findings qualify the technology as a rapid method to validate biomarkers that reveal disease risk, disease progression, or therapy response. Cantilever arrays have potential as a tool to evaluate treatment response efficacy for personalized medical diagnostics. Discovery of the genetic sequence encoding a protein by nucleic acid technologies is not sufficient to predict the size or biological nature of a protein. Studies at the messenger RNA level can assess the expression profiles of transcripts but these analyses measure only the relative amount of an mRNA encoding a protein and not the actual amount of protein in a tissue. To address this area, several proteinbased analysis technologies have been developed. Proteomicsbased assays are considered to be a distinct group within molecular diagnostics and should not be confused with immunoassays although some proteomic technologies are antibody-based. Technologies with the greatest potential are 2D PAGE, antibody-based screening, protein-binding assays, and protein biochips. 2D PAGE is combined with mass spectroscopy-based sequencing techniques, which identify both the amino acid sequences of proteins and their posttranslational appendages. This approach is combined with database search algorithms to sequence and characterize individual proteins.

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The activity of a gene, so called gene expression means that its DNA is used as a blueprint to produce a specific protein. The first step of gene expression is transcription, the process by which the sequence of DNA bases within a gene is used as a template to synthesize mRNA. Following transcription, the nascent mRNA is processed and transported out of the nucleus and into the cytoplasm of the cell. Once in the cytoplasm, the mature mRNA is engaged in the last step in gene expression, translation − the process by which proteins are synthesized. Finally there is posttranslational modification of proteins into mature forms. Each of these steps in gene expression is subject to precise cellular controls that collectively allow the cell to respond to changing needs. Less than half of all genes are expressed in a typical human cell, but the expressed genes vary from one cell to another and from one individual to another. Selected methods for gene expression profiling: –– Whole genome expression array –– Serial analysis of gene expression (SAGE) –– Expressed sequence tags (ESTs) analysis –– Gene expression profiling based on alternative RNA splicing –– Tangerine expression profiling –– Individual sequences –– Real time and competitive RT-PCR –– RNase protection assay –– T-cell receptor expression analysis –– Analysis of single-cell gene expression –– RNA amplification –– Monitoring in vivo gene expression –– Magnetic resonance imaging Gene expression is used for studying gene function. Gene expression profiling, therefore, is relevant to personalized medicine. The temporal, developmental, typographical, histological, and physiological patterns in which a gene is expressed provide clues to its biological role. All functions of cells, tissues, and organs are controlled by differential gene expression. Malfunctioning of genes is involved in most diseases, not only inherited ones. Knowledge of which genes are expressed in healthy and diseased tissues would allow us to identify both the protein required for normal function and the abnormalities causing disease. This information will help in the development of new

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diagnostic tests for various illnesses, as well as new drugs to alter the activity of the affected genes or proteins. Gene expression profiling is relevant to development of personalized medicine and some of the technologies used will be described briefly. Analysis of single-cell gene expression promises a more precise understanding of human disease pathogenesis and has important diagnostic applications. Single cell isolation methods include flow cytometry cell sorting and laser capture microdissection. Besides the gene expression analysis, the following nucleic acid amplification methods are suitable for single-cell analysis: single cell phenotyping, homomeric tailed PCR, which allows unbiased amplification of RNA, RNA amplification. Gene expression analysis of single cells is providing new insights into disease pathogenesis, and has applications in clinical diagnosis. Molecular signatures of some diseases can best be discerned by analysis of cell subpopulations. Studies in disease-relevant cell populations that identify important mRNA (and protein) differences between health and disease should allow earlier diagnosis, better therapeutic intervention, and more sensitive monitoring of treatment efficacy. This will facilitate the development of personalized medicine on the basis of the molecular signatures of the diseased cell population. Current assays for gene expression destroy the structural context. By combining advances in computational fluorescence microscopy with multiplex probe design, it is possible that expression of many genes can be visualized simultaneously inside single cells with high spatial and temporal resolution. Use of the nucleus as the substrate for parallel gene analysis can provide a platform for the fusion of genomics and cell biology and it is termed cellular genomics. This technique takes a snapshot of genes that are switched on in a single cell. Used on a breast biopsy or suspect skin mole, it could pick out the first one or two cells that have harmful genes and become malignant. Alterations in RNA splicing have a significant impact on drug action and can be exploited to generate pharmacogenomics tools in several ways: –– Analyses of RNA splicing might provide a rapid method for detection of polymorphisms across the whole gene. –– Alteration of alternative RNA splicing events triggered by drug or chemicals action constitutes a route through which

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relevant candidate genes can be selected for further genotyping because these genes are likely to lie within crucial pathways of drug action. –– RNA splicing alteration libraries between responders and nonresponders would constitute a discovery tool for SNPs that are relevant to pharmacogenomics. Positron emission tomography (PET) is the most sensitive and specific technique for imaging molecular pathways in vivo in humans. PET uses positron emitting radionuclides to label molecules, which can then be imaged in vivo. The inherent sensitivity and specificity of PET is the major strength of this technique. Indeed, PET can image molecular interactions and pathways, providing quantitative kinetic information down to subpicomolar levels. Generally, the isotopes used are shortlived. Once the molecule is labeled, it is injected into the patient. The positrons that are emitted from the isotopes then interact locally with negatively charged electrons and emit what is called annihilating radiation. This radiation is detected by an external ring of detectors. It is the timing and position of the detection that indicates the position of the molecule in time and space. Images can then be constructed tomographically, and regional time activities can be derived. The kinetic data produced provide information about the biological activity of the molecule. Molecular imaging provides in vivo information in contrast to the in vitro diagnostics. Moreover, it provides a direct method for the study of the effect of a drug in the human body. Molecular imaging plays a key role in the discovery and treatment process for neurological diseases such as Alzheimer’s disease and cancer. The ability to image biological and pathological processes at a molecular level using PET imaging offers an unparalleled opportunity to radically reform the manner in which a disease is diagnosed and managed. Its translation into clinical practice will impact upon personalized medicine. Combination of diagnosis with therapeutics, wrongly referred to as theranostic is an important component of personalized medicine. A more appropriate term is pharmacodiagnostic. The diagnostics is linked to the therapeutic substance to select patients who would be suitable for treatment by a particular drug. The drug and the diagnostic test are marketed together. There are several such combinations in the market particularly for the treatment of cancer. Applications of point-of-care diagnosis: –– Emergency room testing for various pathogens in ‘untested’ blood donations

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–– Rapid tests in emergency departments for microorganisms in severe infections –– Intensive care –– Operating room –– In the physician’s office –– Testing for viruses causing coughs and colds –– Detection of bacterial infections to select appropriate antibiotic –– Screening for cancer –– In field studies –– Screening of populations for genetic disorders –– Testing of patients in clinical trials –– Detection of microorganisms that are associated with bioterrorism –– Identification of patients with communicable diseases at the point of immigration. –– Food testing –– In the home –– Self testing by the patient –– Testing at home by visiting healthcare personnel. A large number of companies offer tests to screen for diseases with a genetic component or to identify those at risk of developing a certain disease. Some of the companies developing genetic tests are mentioned in other categories such as those involved in prenatal and cancer diagnostics. Commercialization of genetic technologies is expanding the horizons for the marketing and sales of direct-to-consumer (DTC) genetic tests. Several companies are involved in this activity. A selection of companies offers genetic screening tests directly to consumer, usually via Internet. This list does not include companies offering genetic testing only for paternity, athletic ability, etc. At least three companies − 23andMe, DeCode Genetics, and Navigenics/Affymetrix − have made available DTC personal genome services that rely on the same arrays of 500,000 to 1 million SNPs used in genome-wide association studies. The best organized program is that of Navigenics/Affymetrix, which also provides genetic counseling. There are several different types of approaches to gene therapy. One common approach involves simply replacing a faulty gene with a good gene with the use of manipulated vectors, such as viruses, or even microsurgery, using nanotechnology. Another method involves editing the chromosome itself in order to remove any defective genes.

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Genes causing a disease can be switched on or off. Genes could also be reverse-mutated back into normal genes. There are two basic types of gene therapy. Germ line Gene Therapy - involves altering the genetic makeup of a gene of either an egg or a sperm cell before fertilization, or altering the genetic composition of a blastomere during an early stage of its division. Somatic Gene Therapy - involves altering the genetic code or chromosomes of a person’s somatic cells, or body cells. It is mostly performed in fully grown organisms. Two ways for delivering a gene are shown on Figure 4.2.

Figure 4.2. Ex-vivo and in-vivo gene therapy

In ex-vivo gene therapy, cells are first cultured or synthesized outside of an organism (for gene therapy), and then inserted into the organism to provide the treatment. It significantly reduces many risks involved with gene therapy. At the same time, however, it holds some limitations - indirectly introducing the desired-containing cells into an organism may trigger immune responses. The cells also may not function as desired, malfunction, or not entirely work at all. In in-vivo gene therapy, the gene is directly delivered to the organism, such as through a vector or other means. In-vivo gene therapy is less commonly used than ex-vivo gene therapy. In-vivo gene therapy holds more risks than its ex-vivo counterpart, such as a possible immune reaction from the organism. Vectors used in in-vivo gene therapy include viruses, bacterial plasmids, nanoparticles, and more.

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Thus, advances in molecular and cell biology have provided us with an understanding of the mechanisms of disease at the molecular and genetic levels. This understanding can now be translated into diagnostic, prognostic, and therapeutic applications in modern medicine. Abnormal molecules not only provide a signature for the presence of a disease, but may also provide the indication for a drug targeting the specific abnormal function (Figure 4.3.).

Figure 4.3. Total number of publications describing novel biomarkers by cancer type

Concurrently, the role of diagnostic pathology has expanded from mere morphologic observation into comprehensive tissue analysis through combined histological, immunohistochemical and molecular evaluations. Questions for the individual work of students on Molecular diagnostics is transforming medicine 1. Name basic enzymes applied in molecular diagnostics 2. Define a role of molecular diagnostics in criminalistics 3. Name and describe the functions of basic enzymes applied in molecular diagnostics 4. Define the modern concepts on molecular mechanisms of disease. 5. Name theoretical issues of molecular medicine 6. Name applied issues of molecular medicine 7. Analyze application of biochemical methods in study of human hereditary diseases

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8. Define basic characteristics of human hereditary diseases 9. Give classification of human hereditary diseases 10. Give definition to genetic imprinting 11. Name basic methods applied in the Human Genome Project 12. Give the definition for the restriction fragments length polymorphism 13. Give the definition to genomic polymorphism 14. Name and describe the basic morphogenetic reactions of cells 15. Define DNA methylation and its epigenetic role in cancer and other diseases 16. Analyze the role of telomerase in cell senescence 17. Draw a scheme for genetic linkage of human genes 18. Draw a scheme for basic stages in mapping of human genes 19. Draw a scheme for identification of the genes responsible for developing of diseases 20. Draw a scheme for analysis of genetic polymorphism defining drug resis­tance 21. Draw a scheme for detection of genetic defects at the level of the whole genome 22. Draw s scheme for basic stages in creation of a model of any pathology 23. Draw a scheme for basic directions of human genetics 24. Draw a scheme for mapping the locus of genetic disease in certain region of chromosome 25. Draw a scheme for human genetics as a basis for new trends of medicine 26. Draw a scheme for influence of mutagenic factors on genotype 27. Draw a scheme for human diseases genes cloning 28. Draw a scheme for the common characteristics of somatic cell diseases 29. Draw a scheme for the common reasons of somatic cell diseases 30. Draw a scheme for mother and fetus genetic incompatibility 31. Draw a scheme for ex vivo gene therapy 32. Draw a scheme for in vivo gene therap

LIST OF ABBREVIATIONS BAC GCLP GCP GWAS HGP PCR QA QC NHGRI PAC RFLP SNP STS

– bacterial artificial chromosome – Good Clinical Laboratory Practice – Good Clinical Practice – Genome Wide Association Studies – Human Genome Project – Polymerase Chain Reaction – Quality Assurance – Quality control – National Human Genome Research Institute – P1-artificial chromosome – Restriction Fragment Length Polymorphism – Single Nucleotide Polymorphism – Sequence-tagged site

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GLOSSARY AFFINITY CHROMATOGRAPHY – which the components of the sample are separated on the basis of chemical affinity for a substance. ALPHA FETOPROTEIN – specific fetoglobulin synthesized by the liver and secreted in fetal serum during the fetal life and the neonatal period; measurement used in prenatal diagnosis of genetic diseases. AMPLIFICATION – increase in the number of copies of a specific DNA fragment; can take place in vivo or in vitro. ANTIGENICITY – refers to the ability of the agent to induce antibody production in the host. Agents that have a high antigenicity have a very low reinfection rate, whereas, agents with a low antigenicity rate have a higher reinfection rate. ARRAYED LIBRARY – individual primary recombinant clones (hosted in phage, cosmid, YAC, or other vector) that are placed in two-dimensional arrays in microtiter dishes. Each primary clone can be identified by the identity of the plate and the clone location (row and column) on that plate. Can be used for many applications, including screening for a specific gene or genomic region of interest as well as for physical mapping. Information gathered on individual clones from various genetic linkage and physical map analyses is entered into a relational database and used to construct physical and genetic linkage maps simultaneously; clone identifiers serve to interrelate the multilevel maps. ARTIFICIAL GENE – double-stranded DNA molecule, carrying a specific sequence, coding for a specific amino acid sequence produced in vitro. AUTORADIOGRAPHY – uses X- ray film to visualize radioactively labeled molecules or fragments of molecules; used in analyzing length and number of DNA fragments after they are separated by gel electrophoresis. AUTOSOME – chromosome not involved in sex determination. The diploid human genome consists of 46 chromosomes, 22 pairs of autosomes, and 1 pair of sex chromosomes : the X and Y chromosomes. BRCA1 / BRCA2 – The first breast cancer genes to be identified. Mutated forms of these genes are believed to be responsible for about half the cases of inherited breast cancer, especially those that occur in younger women. Both are tumor suppressor genes.

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Modern issues in molecular diagnostics CANDIDATE GENE – suspected to be involved in the etiology of a disease. CELL LINE – cultured cell type that can be reproduced indefinitely, or is immortalized. CHROMOSOME WALKING – sequential isolation of molecular clones in order to span large intervals on the chromosome. CIS-TRANS POSITION EFFECT – terms cis and trans describe the gene position on homologue chromosomes in double heterozygote individuals. When two alleles are located near each other on the same chromosome, they are in cis position. When they are located on different homologue chromosomes they are in trans position. Cis and trans are analogue to coupling and repulsion. CLINICAL HETEROGENECITY – different phenotypes due to mutations in the same gene. CLINICAL HISTORY – important part of the evaluation consists of: history of the patient’s current illness, past medical history, family history, review of systems. CLUB FOOT – congenital deformation of the foot. COMPLEMENTARY DNA – cDNA is synthesized from a messenger RNA template; the single-stranded form is often used as a probe in physical mapping. CONDITIONAL MUTATION – mutation expressed only under certain conditions. Lethal mutations exist in a cell but in the homozygote state under conditional forms, like for instance if they are expressed at certain temperatures. CONGENITAL – present at birth (not always genetically determined - e.g. congenital syphilis, toxoplasmosis), please notice that not all genetical diseases are congenital, e.g. Huntington disease - 3rd to 4th decade of life CONTAGIOUS DISEASE – subset of infectious diseases. COPY-NUMBER VARIATION – alteration of the DNA that results in the cell having abnormal or in some cases normal variation in the number of copies of one or more sections of the DNA, might be beneficial or associated with a disease. CRYPTOGENIC DISEASE – cause is currently unknown. CYCLIN – protein family important in the regulation of cell division. CYST – abnormal closed cavity, of various sizes in which there is a liquid collection of infectious or embryological origin. CYSTIC FIBROSIS, MUCOVISCIDOSIS – disease affecting the pancreas, gastro intestinal and pulmonary functions and due to several different mutations in the CFTR gene located on chromosome 7 at the 7q31.2 locus. CURE – end of a medical condition or a treatment that is very likely to end it, while remission refers to the disappearance, possibly temporarily, of symptoms. Complete remission is the best possible outcome for incurable diseases. DAPI – chromosome stain derived from fluorescent stain < 4’, 6-diamidino-2phenylindole> that stains preferentially heterochromatin of chromosomes 9, 15, Y.

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DEFORMATION – malformation due to an abnormal position of a limb or an abnormal pressure. DERMATOGLYPHICS – study of the surface markings of the skin. Dermatoglyphic studies are used in a number of malformation syndromes due to a chromosomal aberration as in trisomy 21 or Down syndrome. DISCORDANT – twin pair (or set of individuals) in which one member exhibits a certain trait and the other does not. DISRUPTION – in the disruption sequence the fetus is subject to a destructive problem. It may be a vascular, infectious or mechanical problem leading to a malformation. One example is the effect of an amniotic band. DISSEMINATED DISEASE – has spread to other parts, with cancer, this is usually called metastatic disease. DYSMORPHISM – developmental anomaly seen in a variety of syndromes with a genetic or environmental origin. DYSPLASIA – abnormality of development, or an epithelial anomaly of growth and differentiation, at macroscopic or microscopic level. EFFUSION – abnormal presence of fluid in a tissue or cavity, e.g. pleural. ELECTROPHORESIS – method of separating large molecules (such as DNA fragments or proteins) from a mixture of similar molecules. An electric current is passed through a medium containing the mixture, and each kind of molecule travels through the medium (most often agarose or polyacrylamide) at a different rate, depending on its electrical charge and size. ELECTROPORATION – used to facilitate the penetration of DNA in cells based on the use of electric pulsions to increase the membrane permeability. EMBRYO - fetus before the end of the third month. EMBRYONIC BIOPSY – biopsy of embryonic tissues usually performed on spontaneous miscarriage material. ENDONUCLEASE – enzyme that cleaves its nucleic acid substrate at internal sites in the nucleotide sequence. EPIDEMIOLOGY – study of the different factors that intervene in the onset and evolution of diseases. EPIGENETIC – term that refers to any factor that can affect the phenotype without change in the genotype. EUCHROMATIN – active region of chromosomes that have a normal cycle of spiralisation - despiralisation by opposition to heterochromatin. EUPLOIDY – is the state of a cell or organism having an integral multiple of the monoploid number, possibly excluding the sex-determining chromosomes; a human with abnormal, but integral, multiples of this full set (e.g. 69 chromosomes) would also be considered as euploid. EXONUCLEASE – enzyme that cleaves nucleotides sequentially from free ends of a linear nucleic acid substrate. FAMILIAL – transmitted in the gametes through generations, relevant to a trait that is more frequent in members of an affected individual family than in the general population. This may be caused by genetic or environmental factors, or both.

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Modern issues in molecular diagnostics FAMILY HISTORY – health of the patient’s parents or other relatives, as some diseases, like diabetes and heart diseases tend to occur in families. FINGER PRINTING – analytical method that supplies an exact identity card of a protein molecule containing electrophoretic and chromatographic properties of various polypeptide segments of the molecule. FISH – physical mapping approach that uses fluorescent tags to detect hybridization of probes with metaphase chromosomes and with the lesscondensed somatic interphase chromatin. FLARE-UP – recurrence of symptoms or an onset of more severe symptoms. FLOW CYTOMETRY – automated sorting devices, used to fractionate samples, sort successive droplets of the analyzed stream into different fractions depending on the fluorescence emitted by each droplet, might be used in karyotyping. FOUNDER EFFECT – high frequency of a mutant gene in a rapidly expanding population founded by a small ancestral group when one or more of the founders were, by chance, carriers of the mutant gene. FRAGILE X – Common form of X linked mental retardation associated with a fragile site on X chromosome. The genetic defect has been identified as an abnormal amplification or more than 60 times of triple CGG at the Xq27.3 locus. If the child has the disease the mother can have a pre-mutation with an abnormal amplification between 60-200 repeats of the CGG triplet. The amplification hampers the expression of gene FMR-1. FRAXA site is not far from FRAXE site, a variant of the syndrome. FRAMESHIFT – shift in the reading frame used to translate the base sequence of mRNA. It is caused by the addition or deletion of one or more bases. GENE BANK – collection of clones made from a set of randomly generated overlapping DNA fragments representing the entire genome of an organism. GENE CANDIDATE – gene suspected to be responsible for the occurrence of a genetic disease. GENE MAPPING - determination of the relative positions of genes on a DNA molecule, chromosome or plasmid, and of the distance, in linkage units or physical units, between them. GENE PRODUCT – biochemical material, either RNA or protein, resulting from expression of a gene. The amount of gene product is used to measure how active a gene is; abnormal amounts can be correlated with disease causing alleles. GENE TAGGING – insertion of a genetic marker in or close to a gene. GENE THERAPY – insertion of normal DNA directly into cells to correct a genetic defect. GENETIC DIAGNOSIS – detection of genes of an organism by hybridization of its genome with specific molecular probes. GENETIC DISEASE – disease due to the mutation of one or several genes. When only one gene is involved, we refer to a monogenic disease. GENETIC FOOTPRINT – fine structural characteristic of a specific DNA region allowing to identify a specific cell and its association. GENETIC HETEROGENECITY – presence of similar phenotypes due to different genetic mechanisms.

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GENETIC LOAD – hereditary defects which lower life expectancy or reduce reproduction capacity. GENETIC POLYMORPHISM – regular and simultaneous occurrence in the same population of two or more alleles at a genetic locus, with at least one minor allele having a frequency greater than 1%. GENOME SEQUENCING – determination of the order in which the bases are arranged within a length of DNA or RNA or, the sequence of amino acids that make up a protein. HEMATOPOIETIC – related to the formation of blood cells, a process that occurs mainly in the bone marrow. HEMIZYGOSITY – presence of a single gene copy in a diploid cell, e.g. X and Y in the male. In abnormal situations there may be a deletion on a chromosome or entire loss of that chromosome leading to a partial or complete deletion, ex Turner 45X syndrome or the Cri du Chat 5p- anomaly. HOLANDRIC – hereditary trait due to the presence of a gene on Y chromosome; rare; transmission of the trait is exclusively from father to son. HOMOLOGOUS CHROMOSOMES – pair of chromosomes containing the same linear gene sequences, each derived from one parent. HOUSEKEEPING GENE – regulates vital functions of all cell types. HIGH PRESSURE LIQUID CHROMATOGRAPHY – chromatographic technology used to separate and quantitate mixtures of substances in solution, allows a rapid analysis of complex mixtures. HUMAN GENE THERAPY – insertion of normal DNA directly into cells to correct a genetic defect. HYDROCEPHALY – abnormal enlargement of cerebral ventricles. HYPOPLOIDY – state of having lost one or more chromosomes. IMMUNOFLUORESCENCE TECHNIQUE – determining the location of antigen or antibody in tissue by the pattern of fluorescence resulting when the tissue is exposed to the specific antibody or antigen labeled with a fluorochrome. IMMUNOSUPPRESSION – artificial prevention or diminution of the immune response, by irradiation or administration of anti-metabolites, antilymphocyte serum, or specific antibody. INBORN ERROR OF METABOLISM – genetic metabolic disorder in which a protein defect produces a metabolic block which may have a deleterious effect. INCIDENCE – number of new patients or individuals who acquired the disease during a certain period of time in a specific population. INFECTIVITY – ability of the agent to enter a host and multiply to an infectious dose, thereby producing the infection or disease. IN SITU HYBRIDIZATION – use of a DNA or RNA probe to detect the presence of cDNA sequence in cloned bacterial or cultured eukaryote cells. INTERFERON – is synthesized by animal cells in response to viral infection and non specifically inhibits replication of the viruses; found in serum almost at the onset of the infection and long before the production of antibody.

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Modern issues in molecular diagnostics ISOELECTRIC FOCUSING – analytical separation procedure similar to gel electrophoresis; used to separate e.g. proteins on the basis of their isoelectric point. KARYOTYPE – photomicrograph of individual chromosomes arranged in a standard format showing the number, size, and shape of each chromosome type. LETHAL FACTOR – abnormality of the genome that leads to death in utero. LINKAGE MAP – relative positions of genetic loci on a chromosome, determined on the basis of how often the loci are inherited together. LOCALIZED DISEASE – affects only one part of the body, such as athlete’s foot or an eye infection. MALFORMATION – morphological defect present at birth, apparent or not. MARKER GENE - gene expression, allows sorting of cells where it’s found. MENDELIAN INHERITANCE – classical heredity, described by Gregory Mendel in 1866. METHYLATION – chemical reaction adding a methyl group to a compound. Note the hypermethylation in the FRA X syndrome leading to FMR1 gene inactivation. May be involved in the regulation of gene expression. MITOTIC INDEX – ratio of dividing cells in relation to all cells analyzed. MONOCLONAL ANTIBODIES – homogeneous antibodies produced by a clone of B lymphocytes originating from a unique mother cell that will generally detect only one genetic determinant. MUCOLIPIDOSIS – hereditary metabolic defects due to the deficiency of enzymes essential for the degradation of oligosaccharides. Skeletal, cardiac, ocular, systemic deleterious effects are observed in the affected individuals. MULTIPLEXING – sequencing approach that uses several pooled samples simultaneously, greatly increasing sequencing speed. NEAREST NEIGHBOR SEQUENCE ANALYSIS – biochemical technique for estimating the frequencies that pairs are next to one another. NEURONAL APOPTOSIS INHIBITORY PROTEIN – protein that inhibits the programmed neuronal death. NEUROTROPIC FACTORS – have some affinity for the nervous tissues. NORTHERN BLOT, RNA TRANSFER – gel base procedure locating mRNA sequences on a gel complementary to a piece of DNA used as a probe. NUCLEIC ACID SEQUENCING – determination of the linear order of the nucleic basic chains. NUCLEIC PROBE – DNA or RNA sequence marked by a fluorescent isotope or enzyme used to detect homologue sequences by in situ or in vitro hybridization. NULL MUTATION – allele that results in either the absence of the gene product or the absence of any function at the phenotypic level. OLIGOGENIC DISEASES – result from the effects of relatively few genes some of which have relatively large effects. ONCOGENE – gene, one or more forms of which is associated with cancer. PARENTAL COEFFICIENT - probability that an individual has received both alleles of a pair from an identical ancestral source.

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PARENTAL IMPRINTING – differential expression of genetic material, at either a chromosomal or an allelic level, depending on whether the genetic material has been inherited from the male or female patient. PATHOGENECITY – ability of the agent to cause disease in an infected host. PERACUTE - very acute or violent. PHYSICAL MAP – map of the locations of identifiable landmarks on DNA (e.g., restriction enzyme cutting sites, genes), regardless of inheritance. Distance is measured in base pairs. For the human genome, it is the banding patterns on the 24 different chromosomes including X and Y chromosomes the highest- resolution map would be the complete nucleotide sequence of the chromosome. PROBAND – family member through whom the family is ascertained. If the propositus is affected, he may be called the index case. PROGRESSIVE DISEASE – whose typical natural course is the worsening of the disease until death, serious debility, or organ failure occurs. Slowly progressive diseases are also chronic diseases; many are also degenerative diseases. PROTOONCOGEN – normal gene that with a slight alteration by mutation or other mechanism becomes an oncogene. RECOMBINANT DNA TECHNOLOGIES – procedures used to join together DNA segments in a cell-free system. Under appropriate conditions, a recombinant DNA molecule can enter a cell and replicate there, either autonomously or after it has become integrated into a cellular chromosome. REFRACTORY DISEASE – resists treatment, especially an individual case that resists treatment more than is normal for the specific disease in question. RESISTANCE – refers to the ability of the agent to survive under adverse environmental conditions. It is also a measure of the agent’s fragility. RESISTANCE FACTOR, R PLASMID – codes for one or more enzymes inactivating one or more toxic agents or antibiotics. RESTRICTION ENZYME CUTTING SITE – specific nucleotide sequence of DNA at which a particular restriction enzyme cuts the DNA. Some sites occur frequently in DNA (e.g., every several hundred base pairs), others much less frequently (rare- cutter; e.g., every 10,000 base pairs). RESTRICTION ENDONUCLEASE – protein that recognizes specific, short nucleotide sequences and cuts DNA at those sites. Bacteria contain over 400 such enzymes that recognize and cut over 100 different DNA sequences. RESTRICTION FRAGMENT LENGTH POLYMORPHISM – variation between individuals in DNA fragment sizes cut by specific restriction enzymes; usually caused by mutation at a cutting site polymorphic sequences that result in RFLPs are used as markers on both physical maps and genetic linkage maps. RETINOBLASTOMA – embryonic tumor of the retina. The gene locus for the retinoblastoma is located in the 13q14.1-14.2 region. In the proximal region of the long arm of chromosome 13. The tumor can be hereditary due to a germinal mutation or sporadic due to a somatic mutation. REVIEW OF SYSTEMS – presence of symptoms other than disclosed in the history of the present illness, might suggest disease affecting other parts of the body.

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Modern issues in molecular diagnostics SHOTGUN CLONING – cloning of DNA fragments randomly generated from a genome, without knowledge of where the piece originally came from. This can be contrasted with directed strategies, in which pieces of DNA from known chromosomal locations are sequenced. Because there are advantages to both strategies, researchers use both random (or shotgun) and directed strategies in combination to sequence the human genome. SIBSHIP – all the sibs (siblings) in a family. SILENT MUTATION – mutant gene that has no phenotypic effect. SINGLE-GENE DISORDER – hereditary disorder caused by a mutant allele of a single gene, e.g. Duchene muscular dystrophy, retinoblastoma, sickle cell. SOCIAL HISTORY - patient’s occupation, habits, etc. SOUTHERN BLOTTING – transfer by absorption of DNA fragments, separated in electrophoretic gels, to membrane filters for the detection of specific base sequences by radio labeled complementary probes. STABLE DISEASE, STATIC DISEASE – a medical condition that exists, but does not get better or worse. STEM CELL - pluripotent cell giving rise to cells with a different function. The origin can also be embryonic or germinal; e.g. undifferentiated cells in the bone marrow having ability both to multiply and to differentiate into specific blood cells. STRUCTURAL GENE – coding for any RNA or protein product. STRUCTURAL GENOMICS – determine the 3D structures of large numbers of proteins using both experimental techniques and computer simulation. SYNDROME – group or recognizable pattern of symptoms or abnormalities that indicate a particular trait or disease. SYNGENIC – genetically identical members of the same species. SYSTEMIC DISEASE – affects the entire body, such as influenza or high blood pressure TERATOMA – tumoral formation of embryonic origin in which nervous cells, hair, teeth that have no connection with the surrounding tissues might be found. TOXIGENICITY – refers to the ability of the agent to produce a toxin. The resulting illness or disease is from the toxin produced by the organism/agent and not the microorganism itself. TUMOR SUPPRESSOR GENE – normal gene involved in the regulation of cell growth; recessive mutations can lead to tumor development, as in the retinoblastoma gene or the p53 gene. TUMOR TRANSFORMATION – conversion of eucaryotic cells into a state of unrestrained growth in culture resembling or identical with the tumorigenic condition. UNIPARENTAL DISOMY – presence in a diploid cell of two homologue chromosomes inherited from the same parent. The most common mechanism is probably the correction of a trisomic cell. VIRULENCE – refers to the severity of the infection. A highly virulent strain of a disease agent will most always produce severe cases or death.

REFERENCES 1. Patrinos G. P., Ansorge W. Molecular diagnostics: past, present and future. – Elsevier Inc., 2005. – Ch. 1. – 12 p. 2. Buckingham L. Molecular diagnostics: fundamentals, methods and clinical applications. – F.A. Davis Company, 2011. – 2nd ed. – 528 p. 3. Burnett D. et al. Clinical pathology accreditation: standards for the medical laboratory // J. Clin. Pathol, 2002. – Vol. 55. – pp. 729-733. 4. Pierce B. A. The family genetic sourcebook. – New York: John Wiley & Sons, 1990. – 340 p. 5. Ehlers S., Kaufmann S. H. E. Infection, inflammation, and chronic diseases: consequences of a modern lifestyle // Trends in immunology. – Cell Press, 2010. – Vol. 31, Issue 5. – pp. 184-190. 6. Watson J. D. Molecular biology of the gene: 5th ed. San Francisco: Pearson Education, 2004. – 755 p. 7. Moss L. What genes can’t do? Cambridge, MA: The MIT Press, 2002. – 241 p. 8. Lodish H. Molecular cell biology. – USA: Freeman, 2003. – 5th ed. – 967 p. 9. Elmore S. Apoptosis: A Review of Programmed Cell Death // Toxicologic Pathology, 2007. – Vol. 35. – pp. 495–516. 10. Ashkenazi A., Dixit V. M. Death receptors: signaling and modulation // Science, 1998. – Vol. 281. – pp. 1305-1308. 11. Harris T. J., McCormick F. The molecular pathology of cancer // Nat Rev Clin Oncol, 2010. – Vol. 7, Issue 5. – pp. 251–265. 12. Ogino S. et al. Molecular pathological epidemiology of epigenetics: emerging integrative science to analyze environment, host, and disease // Mod Pathol, 2013. – Vol. 26. – pp. 465-84. 13. Lewis R. Human Genetics: Concepts and Applications. – McGraw-Hill, 2009. – 9th ed. – 475 p.

ADDITIONAL LITERATURE 1. Kan Y.W., Globus M.S., Dozy A.M. (1976) Prenatal diagnosis of a-thalassemia: clinical application of molecular hybridization. N. Engl. J. Med., 295, 1161–1169. 2. Kan Y.W., Dozy A.M. Polymorphism of DNA sequence adjacent to human beta-globin structural gene: relationship to sickle mutation. Proc Natl AcadSci U S A. 1978; 75(11):5631-5. 3. Kan Y.W., Lee, K.Y., Furbetta M., Angius A., Cao A. (1980). Polymorphism of DNA Sequence in the β-Globin Gene Region. New England Journal of| Medicine 302 (4): 185-188. 4. Machevsky, Alberto; Wick, MR (2004). Evidence-based Medicine, Medical Decision Analysis, and Pathology. Human Pathology 35 (10): 1179–88. 5. Leong, F.J. and Leong, A.S. (2003) Digital imaging applications in anatomic pathology. Adv. Anat. Pathol. 10: 88-95. 6. Kiechle FL, Main RI. The Hitchhiker’s Guide to Improving Efficiency in the Clinical Laboratory. AACC Press: Washington DC. 2002: 40-41. 7. Grody W.W. et al. Molecular Diagnostics: Techniques and Applications for the Clinical Laboratory. Boston MA: Academic Press Inc., 2010 8. Dieffenbach CW, Dveksler GS. Setting up a PCR laboratory. Genome Res 1993; 3:52-57. 9. Kiechle FL, Holland CA, Karcher R. Laboratory design. J Clin Ligand Assay 2005; 28:186-197. 10. Gomah ME, Turley JP, Lu H, Jones D. Modeling complex workflow in molecular diagnostics. Design specifications of laboratory software for support of personalized medicine. J Molecular Diagnostics. 2010; 12:51-57. 11. Campbell, D.A., Carmichael, J. and Chopra, R. (2004) Molecular pathology in oncology – the AstraZeneca perspective. Pharmacogenomics 5: 1167-1173. 12. Green, D.M. (2005) Improving health care and laboratory medicine: the past, present, and future of molecular diagnostics. Proc. Bayl. Univ. Med. Cent. 18: 125-129. 13. Greinacher, A. and Warkentin, T.E. (2005) Transfusion medicine in the era of genomics and proteomics. Transfus. Med. Rev. 19: 288–294. 14. Horvath, A.R. and Pewsner, D. (2004) Systematic reviews in laboratory medicine: principles, processes and practical considerations. Clin. Chim. Acta 342: 23-39. 15. Kubik, T., Bogunia-Kubik, K. and Sugisaka, M. (2005) Nanotechnology on duty in medical applications. Curr. Pharm. Biotechnol. 6: 17–33.

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16. Rosenwald Sh., Gilad Sh., Benjamin Sh., and others. Validation of a microRNA based qRT-PCR test for accurate identification of tumor tissue origin. Modern Pathology (2010) 23, 814-823. 17. Narayanan, S. (2000) Technology and laboratory instrumentation in the next decade. MLO Med. Lab. Obs. 32: 24-27, 30-31. 18. Plebani, M. (2005) Proteomics: the next revolution on laboratory medicine?. Clin. Chim. Acta 357: 113-122. 19. Robertson, B.H. and Nicholson, J.K. (2005) New microbiology tools for public health and their implications. Annu. Rev. Publ. Health 26: 281-302. 20. Valdes, R., Jr., Linder, M.W. and Jortani, S.A. (2003) What is next in phara­ ma­­co­ge­nomics? Translating it to clinical practice. Pharmacogenomics 4: 499-505. 21. Wills, S. (2000) The 21st century laboratory: information technology and health care. Clin. Leadersh. Man. Rev. 14: 289-291. 22. Harris TJ, McCormick F. (2010). The molecular pathology of cancer. Nat Rev Clin Oncol 7 (5): 251-265. 23. de Sanctis, Jorgelina T. еt al. Culture-negative endocarditis and the use of molecular diagnostics: a case report. Infectious diseases in clinical practice, 2010, Vol. 18, Issue 2, pp 120-123. 24. Hoffman N.G., Cookson B.T. Case 8-2007: a man with chest pain followed by cardiac arrest. New England Journal of Medicine, 2007, Vol. 356, p. 2652-2653. 25. Demeunynck M. Small molecule DNAand RNAbinders: from small molecules to drugs. – Germany, 2002. 26. Poste, G. (2001). Molecular diagnostics: A powerful new component of the healthcare value chain. Expert Review of Molecular Diagnostics 1 (1): 1-5. 27. Huser, V; Sincan, M; Cimino, J. J. (2014). Developing genomic knowledge bases and databases to support clinical management: Current perspectives. Pharmacogenomics and Personalized Medicine 7: 275-83. 28. Hamburg, M. A.; Collins, F. S. (2010). The Path to Personalized Medicine. New England Journal of Medicine 363 (4): 301–304. 29. MacKinnon, A. C.; Wang, Y. L.; Sahota, A.; Yeung, C. C.; Weck, K. E. (2012). Certification in Molecular Pathology in the United States. The Journal of Molecular Diagnostics 14 (6): 541-549. 30. Hammerling J.A. (2012). A review of medical errors in laboratory diagnostics and where we are today. Laboratory medicine 43 (2): 41-44. 31. Desta, Z.; Zhao, X.; Shin, J. G.; Flockhart, D. A. (2002). Clinical Significance of the Cytochrome P450 2C19 Genetic Polymorphism. Clinical Pharmacokinetics 41 (12): 913-958. 32. Eggermann, T.; Spengler, S.; Gogiel, M.; Begemann, M.; Elbracht, M. (2012). Epigenetic and genetic diagnosis of Silver-Russell syndrome. Expert Review of Molecular Diagnostics 12 (5): 459-471. 33. Shrimpton, A. E. (2002). Molecular diagnosis of cystic fibrosis. Expert Review of Molecular Diagnostics 2 (3): 240-256. 34. Minamoto, T.; Ougolkov, A. V.; Mai, M. (2002). Detection of oncogenes in the diagnosis of cancers with active oncogenic signaling. Expert Review of Molecular Diagnostics 2 (6): 565–575.

INTERNET-RESOURCES 1. http://www.sciencemag.org 2. http://www.who.int/genomics/elsi/en/ 3. http://ghr.nlm.nih.gov/ 4. http://www.science.com 5. http://www.pathologyoutlines.com/ 6. http://www.pathsoc.org/conversations/index.php? option=com_ content&view=frontpage&Itemid=1 7. http://www.thedoctorsdoctor.com/f_home.html 8. http://ezinearticles.com/?Classification-of-Hereditary-and-Genetic Disorders&id=650049 9. http://encyclopedia2.thefreedictionary.com/Hereditary+Diseases 10. http://www.cap.org/apps/docs/reference/myBiopsy/index2.html 11. http://www.leomics.com/our-space-clinical-laboratory-diagnostics.html 12. http://www.austincc.edu/mlt/mdfund/md_links.htm 13. http://www.genome.gov/12011238 14. http://ghr.nlm.nih.gov/handbook/hgp/description 15. http://geneontology.org/page/reference-genome-annotation-project 16. http://www.nlm.nih.gov/research/visible/visible_human.html

Figure 1.2.2.1. Concept map of genetic disorders patterns (http://cmapspublic.ihmc.us/ rid=1211337068109_647275002_32489/genetic%20disorders.cmap), with IHMC Cmap Tools: http://cmap.ihmc.us/

Appendix Appendix 1

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Figure 2.1.1. Timeline of the Human Genome Project: https://exploreable.wordpress. com/2011/05/03/the-story-of-the-human-genome-project-a-short-narration/

90 Modern issues in molecular diagnostics Appendix 2

(http://maaz.ihmc.us/rid=1177590640046_107008151_7542/Chapter%2020.cmap), with IHMC Cmap Tools: http://cmap.ihmc.us/

Figure 2.2.5. Concept map of the basic connections of DNA technology and genomics

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Appendix 3

Figure 2.2.6. Genomic achievements since the Human Genome Project. From: Green E.D., Guyer M.S., National Human Genome Research Institute. Charting a course for genomic medicine from base pairs to bedside. Nature, Vol. 470, Issue 7333, pp. 204–213.

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Appendix Sample of case – study presentation

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Appendix 4

Apoptosis of erythrocytes

Made by Almaganbetov Zhassulan Specialty “6M070100 - Biotechnology”

• Erythrocytes are produced by a complex and finely regulated process of erythropoiesis • Erythropoiesis ends with the mature circulating red cell, which is a nonnucleated biconcave disc, performing its function of oxygen delivery • It is now well established that erythropoietin stimulates erythropoiesis, at least in part, by protecting erythroblasts from apoptosis

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• Human mature erythrocytes are terminally differentiated cells that are devoid of mitochondria, as well as of nucleus and other organelles • The erythrocyte limited lifespan implies that, as in other cells, life and death are well regulated for erythrocytes, in spite of their lack of capacity for protein synthesis

• It is well known that eukaryotic cells use a death program • Instead, human mature erythrocytes have been considered as unable to undergo programmed cell death due to their lack of mitochondria, nucleus, and other organelles • Moreover, erythrocyte precursors, which are true organellecontaining cells, are susceptible to apoptosis induction • Erythrocyte death is characterized by some features that are shared by apoptosis (cell shrinkage, plasma membrane microvesiculation, shape changes, cytoskeleton alterations associated with protein degradation, and loss of plasma membrane phospholipid)

Appendix

• Erythrocyte lifespan is limited to approximately 120 days and is ended by a process of senescence during which aging erythrocytes suffer changes that display molecules that are recognized by macrophages • The knowledge of the mechanism of the erythrocyte death is of the highest importance since, apart from its association with anemia, it could lead to improvements of the storage conditions in blood banks by increasing the time of viability of stored red blood cells

Mechanism of erythrocyte death • Mature erythrocytes can undergo a rapid selfdestruction process leading to increased intracellular calcium content, modifications of the erythrocyte morphology, metabolic disruption, membrane protein modifications, and externalization of phosphatidylserine, thereby activating a clearance mechanism involving heterophagic removal in the reticuloendothelial system

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Enzyme activity • Mature erythrocytes contain considerable amounts of caspase-3 and caspase-8 whereas other essential components of the mitochondrial apoptotic cascade such as caspase-9, Apaf-1 and cytochrome c are absent • Strikingly, although caspase-3 and -8 were functionally active in vitro, they did not become activated by various proapoptotic stimuli • Cysteine protease inhibitors prevented programmed erythrocyte death induced by Ca2+ influx, and allowed erythrocyte survival in vitro and in vivo • However, the cysteine proteases involved seem not to be caspases, since caspase-3, while present in erythrocytes, was not activated during cell death, and cytochrome c, a critical component of the apoptosome, was lacking • Therefore, Ca2+-induced erythrocyte death appeared to proceed in the absence of caspase activation

• The volume of red cells decreases with cell aging and substantial amount of hemoglobin is lost from circulating erythrocytes during total lifespan • This is probably due to loss of potassium and to loss of membrane via microvesiculation, resulting in cellular dehydration, membrane protein removal, and increased density Altered shapes of erythrocytes subjected to proeryptotic agents --The normal discoid biconcave shape (top) turned to spherocyte with microvesiculation due to increased intracellular calcium concentration (left bottom) or to stomatocyte induced by oxidative stress (right bottom)

Appendix

Processes that induce premature erythrocyte death • Eryptosis can be triggered by different injuries such Energy depletion as energy depletion, osmotic shock or oxidative stress • The reduced calcium-ATPase activity due to energy depletion leads to decreased calcium efflux and this in turn accelerates the transmembrane movement of potassium and chloride, resulting in cell dehydration

Osmotic shock • Osmotic shock is found among the well-known inducers of apoptotic cell death • Erythrocytes incubated in a hyperosmotic environment released prostaglandin E2 (PGE2),which in turn activated nonselective cation channels and increased the cytosolic Ca2+ concentration

Oxidative stress • Increasing intracellular oxidants by altering ambient oxygen concentrations or lowering antioxidant levels accelerates the onset of erythrocyte senescence whereas lowering ambient oxygen or increasing reactive oxygen species (ROS) scavenging appears to delay senescence

Osmotic shock • Osmotic shock is found among the well-known inducers of apoptotic cell death • Erythrocytes incubated in a hyperosmotic environment released prostaglandin E2 (PGE2),which in turn activated nonselective cation channels and increased the cytosolic Ca2+ concentration

Oxidative stress • Increasing intracellular oxidants by altering ambient oxygen concentrations or lowering antioxidant levels accelerates the onset of erythrocyte senescence whereas lowering ambient oxygen or increasing reactive oxygen species (ROS) scavenging appears to delay senescence

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Conclusion • Senescence of red blood cells occurs along their lifespan in the vascular system • During aging, erythrocytes display molecules that lead to recognition and removal of old damaged cells by the immune system • Current evidence indicates that neoantigens on altered band 3 and phosphatidylserine exposed at the outside of erythrocytes are the main signals for cell removal and phagocytosis

Foreword

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CONTENT Foreword.........................................................................................................3 Chapter 1. Introduction into molecular diagnostics of hereditary diseases....................5 1.1. Brief on history and main principles of molecular diagnostics....5 1.1.1. Role of molecular diagnostics in modern life....................5 1.1.2. Pathology and pathogenesis...............................................8 1.2. Human hereditary diseases: classification and characteristics.....15 1.2.1. Basic stages and characteristics of a common disease.......15 1.2.2. Types of genetic disorders..................................................16 1.2.3. Single gene disorders.........................................................18 1.2.4. Multifactorial and polygenic disorders..............................21 1.2.5. Disorders with variable modes of transmission.................24 1.2.6. Cytogenetic disorders.........................................................25 1.3. Modern molecular diagnostics clinical laboratory.......................25 Sample problem sets on Introduction into molecular diagnostics of hereditary diseases..........................................28 Questions for the individual work of students on Introduction into molecular diagnostics of hereditary diseases...........................................30 Chapter 2. Human genome project: methods, prospects........................................................31 2.1. Human Genome Project goals and milestones.............................31 2.2. Basic instruments of the Human Genome Project...........................................................................................33 2.3. Prospects and future challenges of the Human Genome Project.....................................................38 Sample problem sets on Human Genome Project: Methods, Prospects..........................................................................................44 Questions for the individual work of students on Human Genome Project: Methods, Prospects............................................46

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Modern issues in molecular diagnostics Chapter 3. MOLECULAR BASICS OF DISEASESS............................. 47 3.1. .Molecular markers of diseases................................................... 47 3.2. .Molecular basics of apoptosis.................................................... 51 Sample problem sets on Molecular basics of diseases.................................. 56 Questions for the individual work of students on Molecular basics of diseases......................................................................... 58 Chapter 4. Molecular diagnostics is transforming medicine........................................... 60 Questions for the individual work of students on Molecular diagnostics is transforming medicine........................................................... 74 List of abbreviations................................................................................... 76 Glossary........................................................................................................77 References................................................................................................... 85 Additional literature..................................................................................... 86 Internet-resources......................................................................................... 88 Appendix 1. Concept map of genetic disorders patterns.............................. 89 Appendix 2. Timeline of the Human Genome Project................................. 90 Appendix 3. Concept map of the basic connections of DNA technology and genomics. Genomic achievements since the Human Genome Project................................................................ 91 Appendix 4. Sample of case – study presentation Apoptosis of erythrocytes............................................................................. 93

Foreword

Educational issue Aizhan Izbasarovna Zhussupova Nazgul Zhapparovna Omirbekova Zarema Maratovna Biyasheva

MODERN ISSUES IN MOLECULAR DIAGNOSTICS Manual Computer page makeup: N. Bazarbaeva Cover designer: K. Umirbekova _www.maths.york.ac.uk

IS No. 8323

Signed for publishing 12.06.15. Format 60x84 1/16. Offset paper. Digital printing. Volume 6,3 printer’s sheet. Edition: 100. Order No 1482. Publishing house «Qazaq university» Al-Farabi Kazakh National University KazNU, 71 Al-Farabi, 050040, Almaty Printed in the printing office of the «Qazaq universitety» publishing house

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