The genome comprises approximately 3.1 billion bp of DNA. Humans are diploid organisms, meaning that each nucleus contains two copies of the genome, visible microscopically as 22 identical chromosomal pairs – the autosomes – named 1 to 22 in descending size order (see Fig. 3.11, p. 57), and two sex chromosomes (XX in females and XY in males). Each DNA strand consists of a linear sequence of four bases – guanine (G), cytosine (C), adenine (A) and thymine (T) – covalently linked by phosphate bonds. The sequence of one strand of double-stranded DNA determines the sequence of the opposite strand because the helix is held together by hydrogen bonds between adenine and thymine or guanine and cytosine nucleotides. The accessibility of promoters to RNA polymerase II depends on the structural configuration of chromatin. Transcriptionally active regions have decondensed (or ‘open’) chromatin (euchromatin). Conversely, transcriptionally silent regions are associated with densely packed chromatin called heterochromatin. Chemical modification of both the DNA and core histone proteins allows heterochromatic regions to be distinguished from open chromatin. DNA can be modified by addition of a methyl group to cytosine molecules (methylation). In promoter regions, this silences transcription, since methyl cytosines are usually not available for transcription factor binding or RNA transcription. The core histones can also be modified via methylation, phosphorylation, acetylation or sumoylation at specific amino acid residues in a pattern that reflects the functional state of the chromatin; this is called the histone code – reflecting an emerging understanding of the ‘rules’ by which specific modifications mark transcriptionally activating (trimethylation of lysine 4 on histone H3; acetylation of many histone residues) or silencing (methylation of lysine 9 on histone H4; deacetylation of many histone residues) effects. Such DNA and protein modifications are termed epigenetic, as they do not alter the primary sequence of the DNA code but have biological significance in chromosomal function. Abnormal epigenetic changes are increasingly recognised as important events in the progression of cancer, allowing expression of genes which are normally silenced during development to support cancer cell de-differentiation (see Box 3.3, p. 54). They also afford therapeutic targets. For instance, the histone deacetylase inhibitor vorinostat has been successfully used to treat cutaneous T-cell lymphoma, due to the re-expression of genes that had previously been silenced in the tumour. These genes encode transcription factors which promote T-cell cell differentiation as opposed to proliferation, thereby causing tumour regression. Each mitochondrion contains 2–10 copies of a 16 kilobase (kB) double-stranded circular DNA molecule (mtRNA). mtDNA contains 13 protein-coding genes, all involved in the respiratory chain, and the ncRNA genes required for protein synthesis within the mitochondria (see Fig. 3.3). The mutational rate of mtDNA is relatively high due to the lack of protection by chromatin. Several mtDNA diseases characterised by defects in ATP production have been described. mtDNA diseases are inherited exclusively via the maternal line (see Fig. 3.7, p. 51). This unusual inheritance pattern exists because all mtDNA in an individual is derived from that person’s mother via the egg cell, as sperm contribute no mitochondria to the zygote. Mitochondria are most numerous in cells with high metabolic demands, such as muscle, retina and the basal ganglia, and these tissues tend to be the ones most severely affected in mitochondrial diseases (Box 3.1). There are many other mitochondrial diseases that are caused by mutations in nuclear genes, which encode proteins that are then imported into the mitochondrion and are critical for energy production: for example, Leigh’s syndrome and complex I deficiency. • ion channel-linked receptors (glutamate and the nicotinic acetylcholine receptor) • G protein-coupled receptors (GnRH, rhodopsin, olfactory receptors, parathyroid hormone receptor) • receptors with kinase activity (insulin receptor, erythropoietin receptor, growth factor receptors) • receptors which have no kinase activity, but interact with kinases via their intracellular domain when activated by ligand (TNF receptor) (see Figure 3.5 and Box 3.2). In normal tissues, molecules such as hormones, growth factors and cytokines provide the signal to activate the cell cycle, a controlled programme of biochemical events that culminates in cell division. During the first phase, G1, synthesis of the cellular components necessary to complete cell division occurs. In S phase, the cell produces an identical copy of each chromosome – which carries the cell’s genetic information – via a process called DNA replication. The cell then enters G2, when any errors in the replicated DNA are repaired before proceeding to mitosis, in which identical copies of all chromosomes are segregated to the daughter cells. The progression from one phase to the next is tightly controlled by cell cycle checkpoints. For example, the checkpoint between G2 and mitosis ensures that all damaged DNA is repaired prior to segregation of the chromosomes. Failure of these control processes is a crucial driver in the pathogenesis of cancer, as discussed in Chapter 11 (p. 262). Five modes of genetic disease inheritance are discussed below and illustrated in Figures 3.7 and 3.8.
Molecular and genetic factors in disease
Functional anatomy and physiology
DNA, chromosomes and chromatin
Genes and transcription
Gene transcription involves binding of RNA polymerase II to the promoter of genes being transcribed with other proteins (transcription factors) that regulate the transcription rate. The primary RNA transcript is a copy of the whole gene and includes both introns and exons, but the introns are removed within the nucleus by splicing and the exons are joined to form the messenger RNA (mRNA). Prior to export from the nucleus, a methylated guanosine nucleotide is added to the 5′ end of the RNA (‘cap’) and a string of adenine nucleotides is added to the 3′ (‘poly A tail’). This protects the RNA from degradation and facilitates transport into the cytoplasm. In the cytoplasm, the mRNA binds to ribosomes and forms a template for protein production.
Mitochondria and energy production
A Mitochondrial structure. There is a smooth outer membrane surrounding a convoluted inner membrane, which has inward projections called cristae. The membranes create two compartments: the inter-membrane compartment, which plays a crucial role in the electron transport chain, and the inner compartment (or matrix), which contains mitochondrial DNA and the enzymes responsible for the citric acid (Krebs) cycle and the fatty acid β-oxidation cycle. B Mitochondrial DNA. The mitochondrion contains several copies of a circular double-stranded DNA molecule, which has a non-coding region, and a coding region which encodes the genes responsible for energy production, mitochondrial tRNA molecules and mitochondrial rRNA molecules. ATP = adenosine triphosphate; NADH = nicotinamide adenine dinucleotide. C Mitochondrial energy production. Fatty acids enter the mitochondrion conjugated to carnitine by carnitine-palmityl transferase type 1 (CPT I) and, once inside the matrix, are unconjugated by CPT II to release free fatty acids (FFA). These are broken down by the β-oxidation cycle to produce acetyl-CoA. Pyruvate can enter the mitochondrion directly and is metabolised by pyruvate dehydrogenase (PDH) to produce acetyl-CoA. The acetyl-CoA enters the Krebs cycle, leading to the production of NADH and flavine adenine dinucleotide (reduced form) (FADH2), which are used by proteins in the electron transport chain to generate a hydrogen ion gradient across the inter-membrane compartment. Reduction of NADH and FADH2 by proteins I and II respectively releases electrons (e), and the energy released is used to pump protons into the inter-membrane compartment. As these electrons are exchanged between proteins in the chain, more protons are pumped across the membrane, until the electrons reach complex IV (cytochrome oxidase), which uses the energy to reduce oxygen to water. The hydrogen ion gradient is used to produce ATP by the enzyme ATP synthase, which consists of a proton channel and catalytic sites for the synthesis of ATP from ADP. When the channel opens, hydrogen ions enter the matrix down the concentration gradient, and energy is released that is used to make ATP.
The cell membrane and cytoskeleton
The basic cell components required for function within a tissue: (1) cell-to-cell communication taking place via gap junctions and the various types of receptor that receive signals from the extracellular environment and transduce these into intracellular messengers; (2) the nucleus containing the chromosomal DNA; (3) intracellular organelles, including the mechanisms for proteins and lipid catabolism; (4) the cellular mechanisms for import and export of molecules across the cell membrane. (ABC = ATP-binding cassette transporters; ATP = adenosine triphosphate: cAMP = cyclic adenosine monophosphate; CFTR = cystic fibrosis transmembrane regulator; CREB = cAMP response element-binding protein; GDP/GTP = guanine diphosphate/triphosphate; LDL = low-density lipoproteins; LH/FSH = luteinising hormone/follicle-stimulating hormone; PTH = parathyroid hormone; TSH = thyroid-stimulating hormone)
Receptors, cellular communication and intracellular signalling
TNF binds to its receptor, forming a trimeric complex in the cell membrane. Various receptor-associated factors are attracted to the intracellular domain of the receptor, including TNF-receptor-associated protein 6 (TRAF6) and tumour necrosis factor receptor type 1-associated death domain protein (TRADD). These proteins modulate activity of downstream signalling proteins, the most important of which are IKKγ (which in turn modulates activity of IKKα and IKKβ). These proteins cause phosphorylation of IκB, which is targeted for degradation by the proteasome, releasing NFκB, which translocates to the nucleus to activate gene expression. The signalling pathway is further regulated in a negative manner by cylindromatosis (CYLD), which de-ubiquitinates TRAF6, thereby impairing its ability to activate downstream signalling.
Cell division, differentiation and migration
Genetic disease and inheritance
Meiosis
The main chromosomal stages of meiosis in both males and females. A single homologous pair of chromosomes is represented in different colours. The final step is the production of haploid germ cells. Each round of meiosis in the male results in four sperm cells; in the female, however, only one egg cell is produced, as the other divisions are sequestered on the periphery of the mature egg as peripheral polar bodies.
Patterns of disease inheritance
A The main symbols used to represent pedigrees in diagrammatic form. B The main modes of disease inheritance (see text for details).
Several regions of the genome exhibit the phenomenon of imprinting, whereby expression of one or a group of genes is influenced by whether the chromosome is derived from the mother or the father; one such region lies on chromosome 15. A Normal imprinting. Under normal circumstances, expression of several genes is suppressed (silenced) on the maternal chromosome (red), whereas these genes are expressed normally by the paternal chromosome (blue). However, two genes in the paternal chromosome (UBE3 and ATP10A) are silenced. B In sporadic Prader–Willi syndrome, there is a non-disjunction defect on chromosome 15, and both copies of the chromosomal region are derived from the mother (maternal uniparental disomy). In this case, Prader–Willi syndrome occurs because there is loss of function of several paternally expressed genes, including MKRN3, MAGEK2, NDN, PWRN2, C15ORF2 and SNURF-SNRNP. C In sporadic Angelman’s syndrome, both chromosomal regions are derived from the father (paternal uniparental disomy) due to non-disjunction during paternal meiosis. As a result, both copies of the UBE3 gene are silenced and this causes Angelman’s syndrome. Note that the syndrome can also be caused by deletion of this region on the maternal chromosome or a loss-of-function mutation on the maternal copy of UBE3, causing an inherited form of Angelman’s, as illustrated in panel D. D Pedigree of a family with inherited Angelman’s syndrome due to a loss-of-function mutation in UBE3. Inheriting this mutation from a father causes no disease (because the gene is normally silenced in the paternal chromosome) (see individuals I-1, II-1, II-3, III-6), but the same mutation inherited from the mother causes the syndrome (individuals III-3, III-4, IV-4), as this is the only copy expressed and the UBE3 gene is mutated.
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Molecular and genetic factors in disease
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