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- Herausgeber: John Wiley & Sons
- Kategorie: Fachliteratur
- Sprache: Englisch
Comprehensive textbook on molecular pathology for newcomers to the field, covering history, analytics, types of cancers, and more
Molecular Pathology delivers an in-depth outline of molecular pathology appropriate for the undergraduate, postgraduate, or early-stage career lab scientist, drawing from authoritative sources such as NICE, the World Health Organisation, the Office of National Statistics, clinical guidelines, research publications and the National Genomic Test Directory. The text contains numerous tables, diagrams, and photos in each chapter to help elucidate key concepts.
The opening chapter provides a general introduction to the subject with a historical perspective of key findings that have led to our current view of molecular pathology. The introduction is followed by a look at the scope of chromosomal disease (such as Down’s syndrome) and genetic disease (such as sickle cell disease). Chapter two describes the numerous analytical tools required in molecular pathology, and how complex genetic data is interpreted – i.e., bioinformatics. The remaining three chapters review the molecular pathology of focused groups of disease, including solid tumor cancer, blood cancer, and metabolic and other genetic diseases. Each of these three chapters explains the role of molecular pathology in the etiology, diagnosis, and management of each particular condition.
Sample topics explored in Molecular Pathology include:
- Generating DNA and RNA for analysis, amplification by PCR, and DNA sequencing through micro-arrays, libraries, and Next Generation Sequencing
- Chromosome structure, covering histones and the centromere, abnormal chromosome numbers, and errors within chromosomes such as translocations and deletions
- Myelofibrosis, myelodysplasia, essential thrombocythemia, polycythaemia, leukemia, lymphoma, myeloma, and haemopoiesis and maturation arrest
- Molecular pathology of single organ/organ system disease, such as Huntington’s and cardiomyopathy, and multi-organ disease such as Barth syndrome
Molecular Pathology serves as an excellent introduction to the subject for first to third year undergraduate students. Healthcare professionals and biomedical scientists and researchers will also find it valuable.
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Table of Contents
Cover
Table of Contents
Title Page
Copyright
Preface
Acknowledgements
Chapter 1: Introduction to Molecular Pathology
1.1 A Historical Perspective
1.2 The Scope of Human Disease
1.3 Chromosomal Abnormalities and Disease
1.4 The Practice of Molecular Pathology
1.5 Conclusion and a View Forwards
Bibliography
Websites
Chapter 2: Methods in Molecular Pathology 1
2.1 Sample Acquisition and Preparation
2.2 Cytogenetics and Karyotyping
2.3 Fluorescence In situ Hybridisation
2.4 Preparation of Nucleic Acids for Analysis
2.5 DNA Sequencing
2.6 Microarrays
2.7 Summary of First-generation Techniques
Bibliography
Chapter 3: Methods in Molecular Pathology 2
3.1 Next-generation Sequencing
3.2 Third-generation Sequencing
3.3 Bioinformatics
3.4 Chapter Summary and Conclusions
Further Reading
Websites
Chapter 4: Solid Tumour Cancer 1: Carcinoma
4.1 Introduction to Cancer
4.2 The Cellular Basis of Cancer
4.3 Lung Cancer
4.4 Colorectal Cancer
4.5 Prostate Cancer
4.6 Breast Cancer
4.7 Pancreatic Cancer
4.8 Other Carcinomas
4.9 Chapter Summary
Further reading
Websites
Chapter 5: Solid Organ Cancers Part 2: Sarcoma, Neurological, Paediatric, Dermal and Others
5.1 Sarcomas
5.2 Tumours of the Central Nervous System
5.3 Paediatric Cancers
5.4 Dermal Cancers
5.5 Other Cancers
5.6 Summary
Bibliography
Websites
Chapter 6: Blood Cancer
6.1 Lymphoma
6.2 Leukaemia
6.3 Myeloma and Related Diseases
6.4 Other Blood Cancer
6.5 Summary
Bibliography
Websites
Chapter 7: Rare, Inherited, Metabolic and Other Diseases
7.1 Cardiology
7.2 Developmental Disorders
7.3 Endocrinology
7.4 Ophthalmology and Audiology
7.5 Foetal and Non-invasive Prenatal Detection
7.6 Gastrohepatology
7.7 Haematology
7.8 Immunology
7.9 Lipid and Metabolic Diseases
7.10 Mitochondrial Diseases
7.11 Musculoskeletal Diseases
7.12 Neurological Diseases
7.13 Renal Diseases
7.14 Respiratory Diseases
7.15 Dermatological Diseases
7.16 Summary
Bibliography
Websites
Genes Index
Text Index
End User License Agreement
List of Illustrations
Preface
Figure 1 Impact of molecular pathology on other pathology disciplines.
Chapter 1
Figure 1.1 PubMed hits for molecular biology and molecular pathology. Squares: m...
Figure 1.2 Karyogram of the human chromosome complement. The numbering system g...
Figure 1.3 Structure and banding of chromosome 20 showing the location of certa...
Figure 1.4 Chromosome translocations. Upper panel: a reciprocal translocation, w...
Figure 1.5 Chromosome inversions and duplications. Upper panels: inversions in t...
Figure 1.6 Chromosome deletions and insertions. Upper panel: deletions of the t...
Figure 1.7 Falling cost of sequencing the human genome over time.
Chapter 2
Figure 2.1 Early steps in practical molecular pathology. FISH, fluorescence in s...
Figure 2.2 A metaphase spread.
Figure 2.3 The Philadelphia chromosome. From left to right: a normal chromosome...
Figure 2.4 Variants of fluorescence in situ hybridisation. (A) illustrates the b...
Figure 2.5 FISH and a translocation. Red and green fluorochromes are linked to p...
Figure 2.6 Gene amplification detected by FISH. An interphase spread probed by F...
Figure 2.7 Production of DNA and RNA.
Figure 2.8 Quantifying DNA amplification. On the left, the SYBR Green method pro...
Figure 2.9 Real-time increase in fluorescence in an rtPCR reaction. The four lin...
Figure 2.10 A thermal cycler showing rtPCR sigmoidal curves. The display shows s...
Figure 2.11 Principle of multiplex ligation-dependent probe amplification. In st...
Figure 2.12 Fred Sanger.
Figure 2.13 Fluorochrome printout of a Sanger sequence. Each of the four nucleot...
Figure 2.14 Basics of microarray analysis. (a) A simple representation of key st...
Figure 2.15 Microarray analysis. The upper panel shows (in red) the binding of t...
Figure 2.16 An Affymetrix GeneChip.
Chapter 3
Figure 3.1 Nucleotide chemistry. The two nucleotides on the left form a dimer on...
Figure 3.2 The basis of pyrosequencing. A nucleotide is added to the developing...
Figure 3.3 A pyrogram. The vertical axis is light intensity, and the horizontal...
Figure 3.4 Ion Torrent sequencing. On the left, the sample dsDNA is stripped to...
Figure 3.5 Essentials of Illumina sequencing. The process starts at the top left...
Figure 3.6 A Manhattan plot. In this very simplistic sketch, each dot represents...
Figure 3.7 Basics of nanopore analysis. On the left, the DNA sample, complexed w...
Figure 3.8 Basics of Pacific Biosystems sequencing. The process begins (top left...
Figure 3.9 Nanoball technology. The DNA sample is fragmented and loose ends are...
Figure 3.10 Sequence alignment. A simple view of the alignment of nine short sec...
Chapter 4
Figure 4.1 Simplified representation of a signal transduction pathway.
Chapter 5
Figure 5.1 An acute lymphoblastic leukaemia. Blood film: blasts are stained purple.
Figure 5.2 A glioblastoma.
Figure 5.3 A Wilms tumour. https://pediatricimaging.org/diseases/wilms-tumor/.
Figure 5.4 A retinoblastoma. Note the blood vessels over the tumour demonstratin...
Figure 5.5 A melanoma. https://www.nhs.uk/conditions/melanoma-skin-cancer/symptoms/
Chapter 6
Figure 6.1 Myelopoiesis and lymphopoiesis. The four stands of haemopoiesis: fro...
Figure 6.2 A blood film in AML.
Figure 6.3 Formation of
BCR
::
ABL1
.
Figure 6.4 A blood film in CML.
Figure 6.5 A blood film in ALL.
Figure 6.6 A blood film in CLL.
Figure 6.7 A blood film in HCL.
Figure 6.8 A blood film in monocytic leukaemia.
Figure 6.9 The complex relationship between the MPNs. An illustration of overlap...
Chapter 7
Figure 7.1 Overlaps in rare, inherited, metabolic and other diseases. Some disea...
Figure 7.2 Molecular pathology of the red blood cell. At the top, section 7.7.4...
Figure 7.3 Glucose/glycogen metabolism. Types refer to the group of glycogen sto...
List of Tables
Chapter 1
Table 1.1 Selected focused analytical kits.
Table 1.2 The ICD-11 classification of diseases.
Table 1.3 Leading causes of death in England and Wales, 2023.
Table 1.4 Selected features of chromosomes.
Table 1.5 Selected chronology of molecular genetics and molecular pathology.
Table 1.6 Examples of molecular pathology of single genes.
Chapter 2
Table 2.1 Common cytogenetic deletion investigations.
Table 2.2 Selected translocations, their genes and linked pathology.
Chapter 3
Table 3.1 Examples of the use of genome-wide association studies.
Table 3.2 Key features of leading NGS platforms.
Table 3.3 Key features of major platforms.
Chapter 4
Table 4.1 Frequency of leading malignant cancers in England and Wales in 2023.
Table 4.2 Selected genes linked to cancer.
Table 4.3 The frequency of genes linked to non-small cell lung cancer.
Table 4.4 Options for tyrosine kinase inhibitors in advanced NSCLC.
Table 4.5 Leading candidate genes for breast cancer.
Table 4.6 The Oncotype DX panel.
Table 4.7 The EndoPredict panel.
Table 4.8 Genes, and their products, forming the Prosigna panel.
Table 4.9 Links between
BRCA1
and
BRCA2
and cancer.
Table 4.10 Links between gene mutations and certain carcinomas.
Chapter 5
Table 5.1 Genetics of selected bone-related sarcomas.
Table 5.2 Genetics of selected soft-tissue sarcomas.
Table 5.3 Childhood and teenage cancers.
Table 5.4 Prevalence of acute lymphoblastic and myeloid leukaemia.
Table 5.5 Deaths linked to all cancers and selected blood cancers in those age...
Chapter 6
Table 6.1 Selected genes mutated in AML.
Table 6.2 Leading translocations and inversions in AML.
Table 6.3 Genes mutated in ALL.
Table 6.4 Leading translocations in ALL.
Table 6.5 Genes of value in assessing MRD in leukaemia.
Table 6.6 Gene frequencies in myeloma.
Table 6.7 Genetics of the MGUS–SMM–MM pathway.
Table 6.8 Selected translocations in MPNs.
Table 6.9 Frequency of leading gene mutations in pMF, ET and PV.
Chapter 7
Table 7.1 Frequences of leading trisomies.
Table 7.2 Leading subtypes of MODY.
Table 7.3 Genes linked to premature ovarian insufficiency.
Table 7.4 Selected defined cases where NIPD may be appropriate.
Table 7.5 Autosomal recessive genes in screening for carrier status.
Table 7.6 Global frequences of major haemoglobinopathies.
Table 7.7 Genes influencing sickle cell disease phenotype.
Table 7.8 Molecular pathology of haemoglobinopathy.
Table 7.9 Genes involved in haem synthesis.
Table 7.10 Prevalence and relative risk data for the classical inherited thromb...
Table 7.11 Leading mucopolysaccharidoses.
Table 7.12 Inborn errors of metabolism.
Guide
Cover
Table of Contents
Title Page
Copyright
Preface
Acknowledgements
Begin Reading
Genes Index
Text Index
End User License Agreement
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Molecular Pathology
A Primer for Laboratory Scientists
Andrew Blann
School of Applied Sciences
Huddersfield University
Huddersfield, UK
This edition first published 2025
© 2025, John Wiley & Sons Ltd.
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Library of Congress Cataloging-in-Publication Data
Names: Blann, Andrew D. author
Title: Molecular pathology : a primer for the laboratory scientist / Dr. Andrew Blann, University Department of Medicine, City Hospital NHS Trust, Birmingham, UK.
Description: Hoboken, NJ, USA : Wiley, 2025. | Includes bibliographical references and index.
Identifiers: LCCN 2024055147 | ISBN 9781394254637 paperback | ISBN 9781394254651 adobe pdf | ISBN 9781394254644 epub
Subjects: LCSH: Pathology, Molecular–Textbooks | Diseases–Molecular aspects–Textbooks
Classification: LCC RB113 .B553 2025 | DDC 616.07–dc23/eng/20250226
LC record available at https://lccn.loc.gov/2024055147
Cover Design: Wiley
Cover Images: © Anna/stock.adobe.com, © Love Employee/Shutterstock
Preface
The understandable desire to fully come to grips with the functions of our bodies has led to the development of molecular genetics. This discipline of medical science explains the form and function of genes at the level, not of the individual (although this naturally follows), but at the level of the molecule. The principal molecule in this investigation is the macromolecular deoxyribonucleic acid (DNA), followed very closely by its near relative, ribonucleic acid (RNA, of which there are several forms).
However, scientific knowledge is nothing without application. Molecular pathology may be seen as a point at which decades of biomedical research into molecular genetics has a direct impact on the practice of medicine. This is pertinent as perhaps 1–2% of the population carries a genetic variant that brings a high lifetime risk of cardiovascular disease or cancer, while many more have a variant that predisposes them to many other diseases and conditions. As a developing discipline, molecular pathology has a place in each of the other major pathology disciplines (Figure 1).
Figure 1 Impact of molecular pathology on other pathology disciplines.
The purpose of this book is to provide a sound and broad outline of the subject for the laboratory scientist, which will be laid out in a series of chapters.
What This Book Is…
This book will provide awareness among health-care professionals, both in training and in practice, with a solid understanding of the role of molecular pathology in modern medicine.
What This Book Is Not…
Molecular genetics and pathology are complex branches of biomedical science and require a basic knowledge of cell biology, physiology and pathology. A basic knowledge of these subdivisions is expected of the reader, and numerous texts on the basics of these three aspects of biology are commonly available, the contents of which are essential prerequisites.
How This Book Is Organised…
Many readers find molecular genetics and molecular pathology particularly taxing. Accordingly, the opening chapter of the book provides a general introduction to molecular pathology, with a historical perspective of the key findings leading to our current view of molecular pathology in 2024. This will be followed by a look at the scope of chromosomal conditions (such as Down’s syndrome) and genetic diseases (such as sickle cell disease), which will provide information about the material in chapters to come, and an introduction to the modern practice of the discipline.
Having refreshed ourselves on the basics of nucleic acid biology, we move to the laboratory. The complex nature of many analyses demands a full explanation of each, and as there are many such platforms, it is convenient to describe them in the broad order in which they were developed. Thus, Chapter 2 will describe the numerous analytical tools in the early stages of molecular pathology. Again, a historical perspective will serve to explain how modern technology has evolved from techniques invented in previous decades as a tool to solve research questions. Many of these fundamental methods are still in use in academic settings, but few are used in today’s routine molecular pathology labs. Chapter 3 will further explore two groups of methods – the so-called New Generation Sequencing and Third Generation Techniques. The chapter will conclude with a section on how complex genetic data is interpreted – i.e. the field of bioinformatics.
The remaining chapters will look at the molecular pathology of different groups of diseases, taking a clinical approach. Since cancer is the consequence of chromosomal and genetic abnormalities and is the leading cause of death in the United Kingdom, it calls for three chapters. Chapter 4 serves as an introduction to cancer, and then focuses on the leading solid tumour cancer of adults, these being carcinomas. The other forms of cancer are discussed in Chapter 5, which also discusses paediatric cancer. Blood cancer is a simple grouping and is the subject of Chapter 6. The book concludes with the remaining conditions, which may variously be described as rare, inherited, metabolic, and those that fail to fall into any of these groups. However, there is considerable overlap between the groups – many metabolic diseases are both rare and inherited.
Each of these latter four chapters will explain the role of molecular pathology in the aetiology, diagnosis, and management of each particular condition.
Current Practice of Molecular Pathology
In the UK, the public health aspect of molecular pathology lies with each of the four nations: England, Northern Ireland, Scotland and Wales. Each has its own directory of genomic testing, listing diseases in various groupings, the nature of the test, target genes, technology and further eligibility criteria. These provide a part template for the layout of the chapters of this text. A second aspect of the choice of material for this book is to acknowledge the role of the UK’s National Institute for Health and Care Excellence (NICE). This body offers guidance on the choice of diagnostics and treatment for various conditions, and some of the former involve gene variants, and so a role in molecular pathology.
Conflict of Interest Statement
The author has not taken any fees, stipends, hospitality, grants or any other commercial link with any of the suppliers of analysers, reagents or software described in this volume.
Acknowledgements
I am very glad to be able to acknowledge the support and contributions of Rob Dunn, Robert Baker, Jennifer Henson, Susanne Kricke, Katya Mokretar, Amy Newton, Ben Poskitt, Helen Warwicker, Isla Henderson and Richard Matthews.
Chapter 1Introduction to Molecular Pathology
A large component of human disease has a clear genetic component and can be described in a number of ways. Some of us (some say most of us) harbour abnormal genes within us that we have inherited from our parents, which may cause disease, perhaps evident at birth, perhaps developing years in the future. These are described as ‘germline’ or perhaps ‘constitutive’. Alternatively, disease may arise from normal genes that have become abnormal due to the action of an external factor, such as ionising radiation. This may occur only within a particular organ (such as the breast or prostate), in which case the abnormalities should not be present elsewhere in the body or, indeed, in phenotypically normal tissues within the same organ. Accordingly, in certain cases, both normal and abnormal tissues may be sent for analysis and so compared. Molecular pathology is crucial in determining the cellular basis of all of these abnormalities, and in many cases, suggestions as to treatment. A third aspect of genetic analysis is the precise recognition of an infectious agent such as a bacterium or a virus.
This chapter will provide a sound and basic introduction for the chapters to follow that will address the following three factors. Section 1.1 will present a brief historical perspective of key developments in biomedical science that led to the development of molecular genetics, and then the application of this knowledge to the study of disease, i.e. molecular pathology. We then move to Section 1.2, an examination of the different types of human disease and how they come about (i.e. their aetiology). This is followed, in Section 1.3, by a view of the broad nature of the practice of molecular pathology. The chapter concludes, in Section 1.4, with an introduction to the modern practice of molecular pathology.
Learning Outcomes
After studying this chapter, you should confidently be able to…
Describe major landmarks in the development of molecular pathology
Recall the various forms of human disease and the leading causes of death
Comment upon major features of chromosomal structure
List common pathological alterations in sections of chromosomes
Explain the importance of single nucleotide polymorphisms
Describe the development of the practice of molecular pathology
1.1 A Historical Perspective
The modern practice of molecular pathology has only become possible through the slow and steady increase in our knowledge of the many facets of cell biology and deoxyribonucleic acid (DNA). We can view this as part of the development of our scientific understanding of the composition and working of the cell, the nucleus, heredity, chromosomes, genes and DNA (i.e. physiology). Only then can these be used as tools to address disease (i.e. pathology).
1.1.1 The Development of Molecular Genetics
The Nineteenth Century
Although it was well known from the Middle Ages (and probably earlier) that certain physiological features and diseases were present in families, and that these moved down the generations, perhaps one place to start is in the Victorian era with Darwin and Mendel. The former observed amendable hereditary features and postulated links with survival, publishing his seminal work ‘The Origin of Species’ in 1859. Mendel provided the mechanisms for the transfer of these features, which we now know as genes, and published his findings in 1865 and 1866, although it was not widely disseminated until the early 1900s. Neither was aware of each other’s work.
Miescher is credited with the first description, in 1871, of a substance he named nuclein, obtained from the nuclei of white blood cells, whilst in 1882, Flemming further analysed this substance, describing it as chromatin, a material we now know to be the basis of chromosomes (Greek: coloured body). He subsequently observed the replication of chromosomes in dividing cells and concluded that these molecules were the source of information passed from generation to generation. By the end of the century, Kossel had isolated adenine, cytosine, guanine, thymine and uracil from nuclein, for which he was awarded a Nobel Prize.
The Twentieth Century – Part 1
The last century opened with Bateson’s coining of the word genetics in 1906, followed by Johannsen’s introduction of the term gene in 1909, emphasising the distinction between phenotypic characteristics of individuals and the material units of heredity that determine them. Levene and others extended the work of Kossel, showing that the nucleic acid component was dominated by only four of the complex molecules described by Kossel and that they were connected to each other by a sugar, the basics of nucleotide chemistry. This was followed by the recognition by Boveri, Sutton and others that chromosomes were responsible for heredity, whilst Griffith, in 1928, showed that physical characteristics of pneumococci can be transferred horizontally (i.e. not merely from parent to offspring) between different strains of the bacterium.
The 1940s were a key decade. McClintock did crucial and pioneering work on cytogenetics, such as the demonstration of transposable elements (which we now call transposons) that move from one chromosomal position to another, which was recognised by the award of a Nobel Prize in 1983. Ris and Mirsky described chromosomes as a complex system of non-histone protein, (DNA) and histone with a definite structure, whilst Avery and colleagues considered DNA a ‘transforming factor’ in the phenotypes of strains of pneumococcus. Beadle and Tatum published data supporting the hypothesis that genes can exert an effect on development and function through enzymes, whilst the Avery–MacLeod–McCarty experiment showed the hereditable material in chromosomes to be DNA, although some considered that heredity lay in the protein component. Smith coined the term ‘genomics’ to describe the total chromosomal and genetic component of an individual (the genome) in 1943, whilst decades later, in 1995, Wasinger and colleagues used ‘the proteome’ as a shorthand to describe the complete protein component of a genome present within a cell, a tissue, an organ, or an organism.
In 1950, Chargaff and colleagues showed that the amount of adenine in a sample of DNA was equal to the amount of thymine, and that the amounts of guanine and cytosine were also equivalent, a key component of the structure of DNA and the genes therein. In 1952, Hershey and Chase reported their now classic and crucial experiment where a virus infecting a bacterium unequivocally showed that the hereditable material was not the protein component of the chromosome but was indeed DNA. The Lederbergs did fundamental work in virology and bacterial genetics, showing the transfer of DNA between bacteria.
Although the term ‘molecular biology’ was introduced by Weaver in 1938, it was later developed by Astbury in 1950, whilst Butel and Melnick used the term in 1970 to describe the ability of the virus SV40 to induce in cells a transformation to a malignant phenotype. Although at the time the expression had wide usage in the exploration of any molecule, by the 1990s its use was becoming focused specifically on the gene. A key feature of molecular biology is that of the development and refinement of analytical techniques that drove the embryo discipline of biomedical science (as it was developing into) from cell-based studies to the more refined study of individual macro- (as in DNA) and micro-molecules (such as penicillin and insulin). An example of such a technique is that of X-ray crystallography, a necessity in the discovery of the structure of DNA by Watson and Crick in 1953, and of the structure of vitamin B12 by Hodgkin in 1955. The following year saw Tjio and Levan using cytogenetics to report the human diploid chromosome number to be 46.
The Twentieth Century – Part 2
The work of Watson and Crick led directly to knowledge of the transcription of DNA into RNA, and the translation of RNA into protein at the ribosome, with the description of ribosomal RNA in 1955 and transfer RNA in 1957. The central dogma of molecular biology was described in 1958, whilst proof of the long-hypothesised existence of messenger RNA (mRNA) was published in 1961. Other steps demonstrated the molecular basis of DNA in its four nucleotides (adenine [A], cytosine [C], guanine [G] and thymine [T]), and that a triplet sequence of these nucleotides defined an amino acid – the genetic code. For example, the triplet GGC ultimately codes (through RNA, where uracil is substituted for thymine) for glycine. Thus, a sequence of GGC-TTA-GTT in the DNA becomes GGC-UUA-GUU in the mRNA, and thereby codes for the tripeptide glycine–leucine–valine, and, by extrapolation, a protein.
The discovery in the 1950s by Luria, Bertani and colleagues of microbial enzymes able to cut specific sections of viral DNA led directly to their development in the 1960s and 1970s by Smith, Kelly, Berg and others as tools for analysing sections of DNA. These restriction endonucleases, such as EcoRI, cut DNA only at specific sites in the nucleotide sequence and provided the impetus for the major breakthrough of genetic engineering. Advances in RNA included the description of small nuclear RNA in 1968 and circular RNA in 1976 – the precursors of whole families of non-coding RNAs (ncRNAs). The 1970s also saw the discovery of methyl- groups on the DNA, a feature now known to be important in gene regulation and in cancer, and the publication in 1971 of Knudson’s two-hit hypothesis of carcinogenesis, which later laid the groundwork for the description of tumour suppressors. A later development of the two-hit aspect of carcinogenesis is the escalation of a slowly developing cancer into one that is more aggressive, potentially caused by a further external factor.
Subsequent advances included DNA hybridisation, gene cloning, Southern blotting, the development of recombinant DNA techniques for vaccines (such as for the hepatitis B virus) and hormones (such as insulin and growth hormone). This phase also saw widespread use of ligating enzymes to join synthetic sections of DNA onto the ends of samples of DNA to be analysed – the process of ligation. This simple technique was to prove crucial in DNA sequencing with the development of primers.
Further steps saw the development of methods to determine the sequence of the nucleotide make-up of a DNA chain by Sanger and colleagues, and by Maxam and Gilbert (as explained in Chapter 2), used to elucidate the entire DNA sequences of a virus (1977) and a human mitochondrion (1981). This period also saw the development of fluorescence in situ hybridisation (FISH), whose origins can be traced back a decade to the use of radiolabeled probes, and the establishment of hybridising microarray technology. Much of this work was enabled by the development of several important technical methods in the 1980s, such as the polymerase chain reaction (PCR, explained in detail in Chapter 2). Other advances include the determination of repeated sections of DNA (such as dimers of T and G) described as microsatellites or tandem repeats, the number of which varied from site to site.
Technical development continued in the 1990s: Govan et al. described a method for fine-mapping DNA damage and repair in specific genome segments, whilst others developed FISH-based microarray technology, which could monitor the expression of many genes in parallel. The genomes of other organisms were subsequently reported: a bacterium (1995), a yeast (1996) and the nematode worm Caenorhabditis elegans (1998). This period also saw the development of DNA ‘chips’, often described as a ‘lab on a chip’, which reflected the increased use of complex analysers and robots, the technique of comparative genomic hybridisation (CGH), which was initially used to determine any gains or losses in DNA sequences, and the automated use of capillary electrophoresis in place of slab gel electrophoresis. These methodological advances are described in Chapter 2.
Key Point 1.1
The Human Genome Project effectively kick-started ‘industrial’ gene sequencing, and promoted the development of next-generation sequencing and bioinformatics.
A key development of this decade was the start of the Human Genome Project, which aimed to sequence an entire genome, an ambition that called for considerable international co-operation. The project demanded numerous technical innovations, one being the large-scale fragmentation of the genome into sections around 150 kbps long. These were inserted into bacterial and yeast artificial chromosomes, amplified and sequenced, according to a ‘shotgun’ approach, developed in 1981. This generated thousands of overlapping sequences that required the development of complex software needed for interpretation and which prompted the birth of bioinformatics. By 1997, an estimate of 90,000 human genes was postulated, and 1999 saw the landmark first report of the sequence of human chromosome 22.
The Twenty-First Century
The present century saw the publication of the DNA sequence of an insect (2000), the mouse (2002), the rat (2004), the chimpanzee (2005) and the zebrafish (2013). The mode of inheritance of eye colour was clarified in the early 2000s with studies pointing to a role for genes such as OCA2, although up to 50 other genes (including HERC2, TYRP1, ASIP, ALC42A5) are also implicated. The clear hereditability of height is considerably more complex, with over 12,000 very small variations implicated, although a small number dominate, and, of course, there is a considerable environmental effect of adequate nutrition.
Nomenclature Part 1
By consensus, genes are described in italics, and their protein product in standard notation. Thus, INS codes for insulin, and TYRP1 for tyrosinase-related protein-1. However, in many cases, the link is not as clear as MUTYH codes for mutY DNA glycosylase, and some genes are named from their discovery, such as a developmental gene in the Drosophila thorax or wings, which has no parallel in our own species.
In 2001, the Human Genome Project published an initial sequencing of the genome, estimated as being composed of 30,000–40,000 genes, and the location of numerous disease genes, such as BRCA2, which brings a susceptibility to breast cancer. Landmarks included the sequences of chromosomes 2, 6, 7, 13, 14 and Y by 2004. Despite these advances, there remained gaps in the sequencing, and in 2022, a leading scientific journal, ‘Science’, dedicated an entire issue on the completion of the genome.
The current view is that the human haploid genome consists of a little over 3000 million nucleotide base pairs with a little under 20,000 protein-coding genes. By comparison, the genome of the bacterium Escherichia coli has a single chromosome of some 5 million base pairs that include over 4000 protein-coding genes, whilst that of C elegans (commonly used in eukaryote gene research) has six chromosomes that together have around 100 million base pairs that, in turn, give rise to over 20,000 protein-coding genes. However, in our own species, the genome generates thousands of RNA molecules that do not participate in translation but seem likely to have a role in the regulation of mRNAs and DNA. These include around 14,500 species of long ncRNAs, 4800 small ncRNAs and 1700 micro-RNAs (miRNAs), to be discussed in sections to come.
Self-check 1.1
What do you consider to be the most important steps in the historical development of molecular genetics?
1.1.2 The Development of Molecular Pathology
As with the development of molecular genetics, certain steps in the development of molecular pathology are precise, whilst others are the sum of several small changes. For example, with surprising prescience, in 1902, Boveri suggested that malignant tumours may be the result of an abnormal condition of the chromosomes, a hypothesis now completely accepted.
The Fruit Fly and Cytogenetics
A key early step in the evolution of ‘academic’ molecular genetics into clinical molecular pathology was the work of Morgan, Muller and colleagues, who in the first half of the last century studied different phenotypes of the fruit fly Drosophila melanogaster. One of the first was the demonstration, in 1916, by Morgan of a link between the white eye phenotype and the X chromosome in female flies. Initially used to explore how genes are inherited, the use of X-rays greatly increased the rate of generation of different variants (mutations) of the fly. When linked to later advances in chromosome identification, this demonstrated the association between chromosomal abnormalities and variant phenotypes that was invaluable to the development of cancer genetics in our own species.
During the 1940s and 1950s, further advances in cell biology and microscopy enabled the identification of chromosomes and variants in their structure. The key event in clinical cytology was the 1959 report by Lejeune and colleagues of an extra chromosome in Down syndrome. By the end of the 1960s, cytogenetic studies had shown chromosomal abnormalities in numerous cancers, which subsequently extended to specific genes in cancer, as is explored in Chapters 3, 4 and 5.
Key Point 1.2
Early research on ionising radiation and chemical mutagens shows their effect on chromosomes in the development of cancer.
Virology
A further cause of cancer is certain viruses. In 1910, Peyton Rous reported that an avian cancer in the form of a sarcoma, a tumour of connective tissues such as muscle and bone, could be transmitted by an unknown soluble factor, smaller than a cell. Decades later, this cancer was shown to be caused by a gene in a virus, hence the Rous Sarcoma Virus (RSV). In the 1960s, Eddy and colleagues, and Huebner and Todaro, postulated viral genes as a cause of cancer (i.e. are oncogenes), with the subsequent discovery of oncogenes linked to specific tumours. The latter included src so named (at the time) after the sarcoma caused by RSV (more latterly known as SRC), and myc a transforming gene derived from the avian leukocytosis virus, causing myelocytomatosis.
Nomenclature Part 2
It is appropriate at this stage to discuss in more detail the systems for naming genes that often link to their product (as in part 1). There are numerous examples where, with improved knowledge, a gene is renamed, and so may be referred to by different writers in alternative forms as they are discovered. An example of this is MYC, originally (as myc) thought to be a single gene but now known to have isoforms and so a family of genes, c-MYC, l-MYC and n-MYC, all present on different chromosomes. Accordingly, these may be described in slightly different terms, such as MYCN, N-MYC and n-Myc. So, although confusing, in the current text, genes will be referred to in the shorthand used by the particular publication. With intellectual flexibility, this should not present too much of a problem in our understanding of molecular pathology.
It was later shown that certain other genes, such as ras, first described in a rat sarcoma virus (and later developed as Kirsten rat sarcoma, then KRAS, named after its discoverer, Werner Kirsten) and TP53 (sometimes described as tp53), so named because its protein product was first thought to have a relative molecular mass of 53 kDa. The latter is an example of a gene whose product normally prevents the formation of cancers, i.e. are tumour suppressor genes. Thus, the loss of function of these genes permitted cancers to develop, an important step in understanding carcinogenesis. It has since been shown that the products of these genes have key roles in cell physiology, such as control of the cell cycle and apoptosis. Although Paul and Hickey pointed to the importance of molecular pathology in cancer in 1974, it was haematology that used this developing field to investigate certain clinical conditions.
Blood Cancer
During the 1960s and 1970s, cytogenetics showed abnormal chromosome structure in the most common forms of blood cancer (lymphoma and leukaemia), observations that were extended by the demonstrations of increased numbers of chromosomes and the transfer of sections between chromosomes, described as a translocation. By the 1980s, genetic analysis of one such transfer placed an oncogene next to an immunoglobulin heavy chain gene; changes were subsequently found in numerous lymphomas and leukaemias. Perhaps the leading example of this is from 1982, with the translocation of a form of myc (i.e. c-myc) next to a gene for part of an immunoglobulin molecule being linked to Burkitt lymphoma. In 1987, Weiss et al. reported the presence of the Epstein–Barr virus (EBV) in tissues of Hodgkin lymphoma, extended in 1990 by Herbst and colleagues, who showed that its DNA can be detected in paraffin-embedded tissues, both pointing to a potential causal effect and the ability to probe archived tissues. Blood cancers are further described in Chapter 6.
However, one of the most far-reaching examples of the power of molecular pathology in this setting is the demonstration of a genetic lesion in the Philadelphia chromosome. First identified using cytogenetics by Nowell and Hungerford in 1960, Rowley showed a decade later a more precise transfer of sections of chromatin between chromosome 9 and chromosome 22. The decade that followed saw the now classic report that this transfer brings together two otherwise non-pathological genes to form a new oncogene (i.e. a neo-oncogene) that causes chronic myeloid leukaemia. Subsequent research identified the protein product, and, in the decades that followed, the design of drugs to target and (in many cases) effect a cure. These observations will be revisited in more detail in Chapter 6.
Genetic analysis of other leukaemias followed. The Philadelphia chromosome was also demonstrated in cases of acute lymphoid leukaemia (as it was then described), and in chronic myeloid leukaemia. The abnormality was detected using leukaemic-specific mRNA sequences amplified in vitro. c-fms (with origins in a feline leukaemia virus, and since renamed CSF1R) was confirmed as a molecular marker for certain types of acute myeloid leukaemia in 1987. The presence of oncogene ETS2 on chromosome 21, which when translocated to chromosome 8, was found to be associated with a type of acute myeloid leukaemia, whilst others reported deletions in part of chromosome 7 in myelodysplasia and other myeloid disorders. In 1990, Becher et al. reported a case series of duplication of part of chromosome 17 in leukaemia, and by the middle of the decade, all forms of blood cancer had been linked to a genetic lesion.
By the 1990s, advances in molecular genetics were being used in routine clinical practice. An example of this is the use of PCR technology to probe for the presence of any instances of the neo-oncogene in the Philadelphia chromosome being present in suspected relapsed leukaemia after transplantation, for minimal residual disease in the treatment of acute lymphoblastic leukaemia, and the use of FISH for the presence of the Philadelphia chromosome on glass slide smears.
Self-check 1.2
Give examples of the value of virology in the development of molecular pathology.
Solid Organ Cancers
Studies from the 1960s and earlier demonstrating familial breast cancer strongly suggested a genetic component, ultimately proven. The 1980s also saw the expansion of molecular pathology into cancer of a number of diverse organs, with the development of techniques such as PCR permitting further analyses. Dubeau and colleagues showed that re-arranged genes can be detected within tissues that are embedded in paraffin wax. In 1984, others provided firm evidence that the human papillomavirus was the causal factor in many genital organ cancers, especially the cervix. Subsequent research used a DNA probe to find viral DNA in oral tissues and certain dermal lesions.
A key advance in molecular pathology in 1987 was the finding that several different types of malignant cells produce increased levels of one of the alternative forms of basic fibroblast growth factor to stimulate their own growth and that of other cells. Subsequent work showed upregulation of its gene (FGF2) in transformed cells and its interaction with a cell surface receptor. The late 1980s also saw the cloning and subsequent analysis of sections of chromosome 22, showing the presence of genes that had the potential to be an oncogene, i.e. proto-oncogenes. Examples include SIS and EWSR1, the latter linked to many cases of Ewing sarcoma.
In 1990, research into liver and ovarian cancer was enhanced by reports of, respectively, the detection of hepatitis B virus DNA and PCR for c-myc in formalin-fixed paraffin wax-embedded tissue. In the same year, Rodenhuis et al. reported activation of a ras oncogene in non-small cell lung cancer. By the end of the decade, genetic abnormalities had been reported in all major cancers, with many instances of several genes linked to the same disease. Furthermore, TP53 was shown to be the most frequently mutated gene in human cancer, underlining the importance of tumour suppressor genes, explained in more detail in section 2.3.2. In 1994, several groups reported using the technique of CGH to show altered DNA sequence copy numbers and chromosomes in breast cancer tumours and cell lines, in primary small cell lung cancers, renal cell carcinomas and malignant gliomas. BRCA1 and BRCA2 were reported, in 1994 and 1995, to be responsible for hereditary breast and ovarian cancer, and subsequently mapped to chromosome 17 and chromosome 13, respectively, whilst in 1996, DeRisi and colleagues used a microarray of over 1000 DNA elements to analyse tumour suppressor gene expression in melanoma.
The molecular pathology of solid organ cancers is further discussed in Chapters 4 and 5.
Other Diseases
The list of diseases with a genetic component (some being hereditary) is considerable. As is discussed in detail in Section 1.3.4 and in Chapter 7, the various forms of haemoglobinopathy constitute the leading global hereditary disease. During the 1960s and 1970s, Weatherall, Orkin and others showed that the disease, known to be caused by an abnormal amino acid sequence in a haemoglobin molecule, was subsequently shown, in turn, to be due to mutations in the genes for this molecule. Wexler and colleagues were amongst the first to point to the location of a gene for a particular disease – that being Huntington disease on chromosome 4, and named HTT, coding for huntingtin. The leading form of familial hypercholesterolaemia (FH), in its heterozygous form, and more so in its homozygous form, brings high levels of serum cholesterol and a markedly increased risk of premature cardiovascular disease. Goldstein and Brown, and their colleagues, showed this condition to be due to defects in the receptor for low-density lipoprotein cholesterol, and in 1984 assigned the gene for this molecule to chromosome 19. The presence of mutations in this gene (LDLR) unequivocally defines the disease. Further research showed the effects of other genes that interact with the low-density lipoprotein cholesterol receptor, and which cause increased plasma cholesterol. Retinoblastoma is the leading ocular cancer, molecular pathology showing most cases resulting from a loss of function, or deletion, of RB1, found on chromosome 13. Further advances in virology included the sequencing of numerous pathogenic viruses, setting the way for the development of rapid diagnosis kits for use in routine diagnostic pathology (Table 1.1).
Table 1.1 Selected focused analytical kits.
Pathology of interest
Examples of focused kit (pathogen or genes determined)
Infectious diseases
Influenza panel: influenza A and B, pandemic H1 influenza virus, H3 influenza virus, respiratory syncytial virus.
Neuro panel: enterovirus, EBV, simplex virus, human adenovirus, human cytomegalovirus, human herpesvirus 6 and 7, human parechovirus, human parvovirus B19, Varicella Zoster virus.
Oncology
Myeloproliferative neoplasms:
BCR
::
ABL
1
, JAK2, CALR, MPL, ABL1
.
Acute lymphoblastic leukaemia:
BCR
::
ABL1
,
TEL
::
AML1, E2A
::
PBX1
,
MLL
::
AF4, MLL
::
AF9, MLL
::
ENL, ABL1.
Coagulation/thrombophilia
Prothrombin (
G2021A
), Factor V Leiden (
FVL
), MTHFR (
C677T
and
A1298C
).
In 1987, Robakis and colleagues cloned and sequenced the gene for amyloid and localised it to the long arm of chromosome 21, thus providing a huge leap forward in our understanding of Alzheimer disease. The year 1990 saw a report of in vitro amplification of DNA for CYP21B, coding for 21-hydroxylase, in the prenatal diagnosis of congenital adrenal hyperplasia, and that Hunter syndrome, a form of mucopolysaccharidosis, is caused by mutations in the gene for iduronate-2-sulphatase on the X chromosome.
Advances in molecular genetics were not confined to the diseases in man. In 1990, Golemboski et al. used genetic engineering in botany with their report of inducing resistance to tobacco mosaic virus into Nicotiana tabacum, whilst Whetter et al. brought the technology to the veterinarian world with the cloning of an equine infectious anaemia virus.
Epigenetics
Mutations are not only found in the DNA nucleotide sequence: epigenetics considers other changes to chromosomes that do not directly involve the nucleotide sequence. In many cases, there are changes to the exposed, outermost section of DNA, often a methylation, and in others there are changes to histone proteins within the chromatin, mostly acetylations. Enzymes that cause these changes (e.g. DNA methyltransferase) can be inhibited with drugs such as 5-azacitidine and decitabine. These (and several other) drugs have been licenced for use in diseases that include myelodysplastic syndromes and acute myeloid leukaemia, whilst inhibitors of histone acetylation, such as vorinostat and pracinostat, have been approved for cutaneous T cell lymphoma and acute myeloid leukaemia, respectively. Other targets for pharmacotherapy include lysine-specific demethylase-1, which acts on histones and may be an effective therapy in prostate cancer.
Summary
The previous section has provided an outline of certain events likely to be recognised as representative of importance in the early days of the development of molecular pathology. By the mid-1990s, the sub-discipline of molecular diagnostics was becoming established, as were the publications of dedicated learned journals in which new findings could be disseminated. In the early 2000s, roles for miRNAs in cancer were becoming recognised, opening a vast new field of investigation. As Figure 1.1 shows, using key words of ‘Molecular Biology’ and ‘Molecular Pathology’ as cited by ‘PubMed’, the depository of the National Library of Medicine of the USA, the number of research papers increased exponentially from around 1990 to 2024, and is likely to continue.
Figure 1.1 PubMed hits for molecular biology and molecular pathology. Squares: molecular biology, circles: molecular pathology.
Source: National Library of Medicine / Public domain / https://pubmed.ncbi.nlm.nih.gov/.
Key Point 1.3
Molecular genetics, in the form of molecular pathology, has enabled scientists to link abnormal genes with a mechanism likely to point to the causes of a particular disease and so to potential treatments.
1.2 The Scope of Human Disease
In order to study the various forms of human disease, we must understand the basis of their classification. They may be defined as those that are evident at, or shortly after, birth, in which case they are described as congenital. Of the remainder, some may arise at any time, and these may also be classified as those developing from within the individual, and those linked to external factors. Many diseases can also be inherited. In all of these groupings, molecular pathology can be very useful in establishing the nature of the disease.
1.2.1 The Classification of Disease
The International Classification of Disease (ICD) provides a system by which diseases can be collected into sections, based mostly on organs and organ systems, as shown in Table 1.2.
Table 1.2 The ICD-11 classification of diseases.
Adapted from https://www.who.int/standards/classifications/classification-of-diseases.
Disease group
Examples
Infectious and parasitic diseases
Tuberculosis, bacterial diseases, viral infections (viral hepatitis and HIV), mycoses and protozoal diseases
Cancer (neoplasms)
Melanoma, cancer of the breast, prostate, blood, brain and other parts of the central nervous system
Blood and blood-forming organs
Nutritional, haemolytic, aplastic and other anaemias, coagulation defects
The immune system
Primary immunodeficiencies, autoinflammatory disorders and diseases of the thymus
Endocrine, nutritional and metabolic diseases
Diabetes mellitus, disorders of the thyroid and pancreas, nutritional deficiencies, obesity
Mental and behavioural disorders
Schizophrenia and delusional disorders, mood disorders, neuroses, disorders of personality
The nervous system
Inflammatory and atrophic diseases of the central nervous system, movement disorders, stroke
The eye
Disorders of the conjunctiva, sclera, cornea, iris and ciliary body, lens, choroid and retina
The ear
Diseases of external, middle and inner ear
The circulatory (cardiovascular) system
Hypertensive and ischaemic heart diseases, pulmonary heart disease
The respiratory system
Acute upper respiratory infections, influenza and pneumonia, chronic lower respiratory diseases
The digestive system
Diseases of the salivary glands oesophagus, stomach and duodenum, liver and gallbladder
The skin
Infections of the skin and subcutaneous tissue, bullous disorders, dermatitis, eczema, urticaria
The musculoskeletal system
Arthropathies, systemic connective tissue disorders, soft tissue disorders, osteopathies
The genitourinary system
Renal diseases, urolithiasis, ureter, bladder, male and female genital organs and breast disorders
The perinatal period
Foetus and newborn affected by maternal factors, complications of pregnancy and delivery
Developmental anomalies
Congenital and chromosomal abnormalities (duplications and trisomies)
Some diseases have both an internal and external precipitant. An example of the latter being cardiovascular disease, where many cases result from the effects of risk factors, such as smoking, hypertension and diabetes, whilst hypercholesterolaemia may have a genetic component that is frequently inherited. Other causes of disease that arises within the body include autoimmune diseases (although some may be linked to an external stimulus) and cancer.
However, there are numerous examples of malignancy being the consequence of an external factor, such as the hepatitis B virus and the hepatitis C virus being linked to liver cancer, and radiation causing blood cancer. Nevertheless, many cancers appear without a clear scientific precipitant and are due to seemingly spontaneous genetic aberration.
1.2.2 Morbidity and Mortality
The preceding section and the ICD system make no comment on the extent to which disease brings an increased likelihood of mortality. For example, although relatively common in an elderly population, osteoarthritis is a major morbidity, but is rarely directly fatal, but may be indirectly. Similarly, asthma is relatively common, as are ocular problems, with a high proportion of people wearing reading and/or long-distance spectacles, but these are infrequently listed on death certificates.
One of the leading UK government data-collecting bodies is the Office for National Statistics (ONS). This organisation publishes a set of data of the various forms of death, which includes those from accidents and crime as well as from disease. The reporting of the types of death follows that of ICD and shows that the most common cause of death is cancer (Table 1.3).
Table 1.3 Leading causes of death in England and Wales, 2023.
Source: Adapted from Nomis web / https://www.ons.gov.uk/peoplepopulationandcommunity/birthsdeathsandmarriages/deaths/methodologies/mortalitystatisticsinenglandandwalesqmi / Last accessed at 08/09/2024.
Cause
Number of deaths
Percentage (%)
All causes
580,108
.
100
Cancer
152,418
26.3
Disease of the circulatory system
139,984
24.1
Respiratory disease
1
71,144
12.3
Mental and behavioral disorders
1
44,000
1
7.4
Nervous system disease
1
42,709
1
7.4
Digestive system disease
1
30,858
1
5.3
COVID-19
a
1
11,818
1
2.0
Endocrine, nutritional and metabolic disease
1
10,706
1
1.8
Genitourinary disease
1,1
9049
1
1.6
Infectious and parasitic disease
1,1
7695
1
1.3
Musculoskeletal system disease
1,1
4360
1
0.8
Disease of the skin and subcutaneous tissue
1,1
2544
1
0.4
Congenital malformations, deformations and chromosomal abnormalities
1,1
1516
1
0.2
Figures do not sum to 100% as only selected categories are presented.
a In 2021, this caused 67,057 (11.5%) of total deaths and in 2022, was linked 22,396 deaths (3.9%) and may be linked to fewer deaths in 2024.
Whilst there can be many causes of cancer, the link between this disease and its clinical development (generally, a tumour) is an abnormality in the DNA sequence of a particular cell within the ‘host’ organ involved. This therefore provides the justification for the investigation of the DNA in these cancers, and so the development of clinical aspects of molecular pathology.
Key Point 1.4
The ICD provides a globally recognised common standard for the investigation and reporting of disease.
Despite the focus on cancer, there are a great number of other diseases that have a chromosomal or genetic component, and which are not necessarily fatal but bring a considerable burden of morbidity, an example being Down syndrome (as discussed in Chapter 7). Knowledge of an individual’s genetic profile can predict ill health and can be useful in focusing on the treatment of those with the greatest risk of developing disease, as in the case of Huntington disease. The presence of the mutant Factor V Leiden, a variant of ‘normal’ coagulation factor V, may bring the need for anticoagulation in certain circumstances. Thus, the knowledge of potential or actual health issues represents a major step forward in medical care.
Self-check 1.3
What are the leading causes of death in England and Wales? – Which of these can be prevented, and how can this be achieved?
1.3 Chromosomal Abnormalities and Disease
The astute laboratory scientist working in molecular pathology will appreciate numerous aspects of the physiology and pathology of the cell, and of the nucleus in detail. The objective of this section is to summarise gross concepts in both chromosomal and genetic disease, and in doing so, prepare the path for chapters to come. The key laboratory method used to determine the number of chromosomes and certain chromosomal abnormalities is cytogenetics. The practical aspects are described in Chapter 2.
1.3.1 Chromosomes in Health and Disease
Normal Chromosome Structure and Function
The leading component of chromosomes, chromatin, is a complex of DNA and histones, the latter a collection of five forms (H1–H5) of a globular protein. An octamer core complex of H2–H4 that forms a template around which around 146 base pairs of DNA can coil is a nucleosome, whilst H1 and H5 link adjacent nucleosomes. This system allows for more efficient packaging of the 2 m of DNA in our nucleus, and also its unwinding for transcription and replication.
Each chromosome is numbered by size from 1 to 22 (the autosomes), and in alphabetic groups (such as group E for chromosomes 16, 17 and 18), whilst the sex chromosomes are named X and Y. Each chromosome has a different density of histones and frequency of nucleotides whose pattern can be visualised by staining with dyes, such as quinacrine and Giemsa, hence Q-bands and G-bands. The latter is more commonly used: dark staining G-bands are more likely to localise dense chromatin enriched in thymine and adenine, whilst sections with a high component of cytosine and guanine, and which are loosely packed, stain lighter. However, before staining, chromosomes must be freed from the tight constraints of the nucleus, best done by inducing an in-vitro culture of cells to undergo mitosis, and then arresting the process before the new cells are formed. Careful manipulation of these ‘poisoned’ cells allows the cell to burst and release its 46 chromosomes, which can then be stained and visualised as a karyogram (Figure 1.2).
Figure 1.2 Karyogram of the human chromosome complement. The numbering system goes from left to right and top to bottom, so the top row is pairs of chromosomes 1–5, one each from the mother and father. The bottom row is chromosomes 19−22, with, at the bottom right, the unpaired X and Y chromosomes.
Not all the DNA in a chromosome codes for a functioning protein. Two such regions are the telomere (at the ends of each chromosome) and centromere. The former are composed of up to 800 repeats of the nucleotide sequence TTAGGG, which protect the two ends of the chromosome from degradation each time the cell replicates. The centromere is composed of densely packed heterochromatin and is involved in sister chromatid binding in mitosis and is the site of the attachment of kinetophores. The exact position of the centromere varies between chromosomes and allows a classification:
