CHAPTER OUTLINE

If we distinguish the actual combination of genes possessed by an individual, that is the genotype, from the observable activity of those genes, the phenotype, then study of inherited disease in clinical biochemistry laboratories has traditionally been concerned exclusively with analysis of phenotypes. The last two decades have witnessed a dramatic change in this situation: molecular biological techniques are now a much more common part of the repertoire of clinical biochemistry laboratories. Initially, the identification of genes responsible for inherited diseases involved heroic efforts, requiring expensive, complex and extremely time-consuming procedures plus a few strokes of luck. Once a gene has been identified, however, modern analytical techniques make detection of mutations more straightforward than before.

Although each nucleated human cell contains about two metres of deoxyribonucleic acid (DNA) (around 3 billion bases), it is the fundamental simplicity of DNA – its building blocks comprise just four nucleotides – that favours its automated analysis. With a few exceptions, every cell in the body of an individual contains a complete copy of their DNA (or genome). For this reason, genetic analysis can be carried out on almost any nucleated cell type (such as lymphocytes or buccal mucosal cells) that can conveniently be collected. The application of DNA analysis now extends well beyond diagnosis of the classic inherited diseases to include, for example, the diagnosis and prognosis of cancer.

A common working definition is that a gene is a sequence of nucleotide bases in DNA that codes for a single polypeptide, but the complexity of genomic organization is such that it is probably unwise to adhere rigidly to any one definition of the gene. Towards the end of the 19th century, it was already accepted that linear groups of ‘invisible self-propagating vital units’ were present in chromosomes. Mendel’s discovery (1865) that inheritance is particulate was rediscovered and publicized at the beginning of the 20th century, and the term gene was introduced to describe Mendel’s ‘particulate elements’ in 1909. By 1911, a specific gene (for colour blindness) had already been assigned to a particular chromosome (the X chromosome). With the work of Garrod, who first presented his studies on alkaptonuria in 1902, the association of specific diseases with inherited Mendelian traits became established.

The Human Genome Project (HGP) represents an outstanding piece of multinational cooperation to map the entire human DNA sequence. The project, started in 1990, had several aims, the first of which was to determine the entire base pair sequence. The sequence of 3 billion base pairs was announced in draft form in 2000 and the complete sequence in 2003. It was thought initially that the human genome would consist of approximately 100 000 different coding genes. As the HGP neared completion, it emerged that the actual number would be closer to 30 000.

Protein coding sequences and introns account for about 20% of DNA. The function of the remainder is being elucidated but in 2012, the initial findings of the ‘Encode’ project, which had been examining what had previously been called ‘junk’ DNA, were published. It appears that the remaining 80% does have a function within the genome, with much of the non-protein coding DNA appearing to code for RNA transcripts that may have other regulatory functions alongside those of gene enhancers and promoters.

That the number of genes is much lower than expected appears to be because many genes can perform multiple functions, and it now appears that these functions may be regulated by the remainder of the genome. These discoveries have implications for the investigation and diagnosis of genetic disease, and issues concerned with gene expression will have a growing role in clinical genetics.

The differentiated properties of each cell are determined by the pattern of genes in the cell that are active or dormant. In any one cell, only a small percentage of genes are likely to be actively engaged in directing synthesis of RNA at any one time. Many of these are ‘housekeeping’ genes, which are expressed in virtually all cell types. Much remains to be learned concerning the factors that determine whether or not a gene is expressed, but regulation of gene expression is clearly determined by proteins that interact with DNA.

In higher (eukaryotic) organisms, DNA is found within the nucleus and the mitochondria, although the mitochondria have a very small percentage of the total DNA within the cell and a very small number of genes. Nuclear DNA is complexed with basic proteins (histones) to form chromatin. At regular intervals, DNA is wrapped around complexes of eight histones to form nucleosomes. The copying of DNA into RNA (transcription) is performed by RNA polymerase, which initiates transcription by interacting with the promoter region of a gene. Gene expression is inhibited if nucleosomes cover a promoter region and many factors that regulate transcription probably do so by competing with histones for binding to the promoter region. Within each promoter region, there are several elements that bind specific proteins capable of interacting with RNA polymerase and associated proteins. One of the key proteins in this group binds to the so-called ‘TATA box’ element (actually a TATAAAA or related sequence), which is found in most eukaryotic promoters and is usually located around 30 base pairs (bp) upstream from the transcription start site. Other conserved sequences, such as CAAT, also bind transcription factors and are found within the promoter region. Another class of regulatory sequence in DNA, the enhancer region, binds regulatory molecules, which include the steroid hormone receptors. Enhancer sequences may be some distance from the gene that they regulate, but the proteins that bind to them may nevertheless interact with the transcriptional apparatus as a result of looping in the DNA molecule.

Finally, the process of translation involves the activity of ribosomes, transfer RNA and a variety of other molecules which synthesize a protein using the mRNA code as a template. A group of three nucleotides (a codon) specifies an amino acid and most amino acids are coded for by more than one codon (i.e. the genetic code is degenerate). In principle, each RNA sequence can be decoded in three different reading frames, depending on which triplet is chosen as the first codon. In practice, the reading frame is determined by the site of initiation, which always occurs at an AUG codon (AUG codes for methionine, but the initiating methionine is cleaved from proteins in eukaryotic cells). Translation stops at any one of three stop codons (UAA, UAG or UGA). Any subsequent modification of a protein, such as proteolytic cleavage or addition of carbohydrate, is known as post-translational modification.

The accepted terminology, which referred to the ‘normal’ gene in a population as the ‘wild type’, has changed and the terms ‘normal’ and ‘mutant’ (or ‘variant’ if pathogenicity is unclear or questionable) are now preferred. However, the genetic constitution of populations is in a constant state of flux with new genes appearing as a result of mutation and deleterious genes being removed by natural selection. Mutations can be broadly divided into those that change the genetic code at a specific location (point mutations or single nucleotide polymorphisms, SNPs) and those that result in gain or loss of genetic material (deletions, duplications and insertions). Point mutations can result from incorrect insertion of a base during DNA replication by DNA polymerase or spontaneous decomposition reactions such as depurination and deamination. Mutagenic chemicals that increase this error rate include those that mimic the natural bases or distort the structure of DNA and those that chemically modify DNA. Ultraviolet light also causes point mutations, particularly by formation of pyrimidine dimers. Point mutations in which a purine is replaced by another purine (e.g. A replaced by G) or a pyrimidine is replaced by another pyrimidine are known as transitions, while replacement of a purine by pyrimidine (e.g. G replaced by C) or vice versa is known as a transversion. Gain or loss of genetic material can result from various errors, including chromosomal breakage and unequal crossing over. Insertion of viral sequences into DNA can also disrupt the genetic code and the rate of spontaneous chromosomal breakage can be markedly increased by ionizing radiation. Whenever the number of bases deleted or inserted is not a multiple of three, the reading frame of the mRNA is altered (frameshift mutation) and the RNA sequence subsequent to the mutation becomes nonsense.

Accumulated damage to DNA would rapidly overwhelm the organism, but repair mechanisms recognize and repair damaged DNA so that fewer than 1 in every 1000 accidental base changes results in a stable mutation. It is estimated that stable point mutations are acquired at a rate of about 1 in every 10⁹ base pairs during each cell generation. Consequently, an average gene of about 10³ coding base pairs is likely to acquire a mutation once in every 10⁶ cell generations. As might be expected, individuals with inherited defects in the enzymes responsible for DNA repair are markedly more susceptible to the effects of environmental mutagens.

A significant proportion of germline point mutations are thought to be caused by modification of methylated cytosine residues. DNA methylation, restricted in eukaryotic cells to cytosine residues, usually at CpG dinucleotides (CpG denotes C-phosphate-G in a linear sequence, in distinction from a CG base pair) is not present in all such organisms but is thought to play an important role in ensuring stable inheritance of expression patterns when cells divide. Spontaneous deamination of the 5-methylcytosine creates thymidine, and problems can then arise when the normal guanine on the complementary strand becomes an adenine and the mutation cannot be detected by DNA repair mechanisms. Consequently, methylation of cytosine can produce mutation ‘hot spots’ (i.e. sequences associated with an unusually high frequency of mutations or recombination).

By convention, the DNA strand that has the same sequence as mRNA (except that it possesses T instead of U) is represented. This strand is known as the coding strand, although it is the ‘anticoding’ strand that is complementary to mRNA and therefore provides the template for mRNA synthesis.

An individual’s genotype is determined at the time of fertilization, when the chromosomes of the gametes (i.e. the male sperm and female egg) are combined. The formation of gametes (gametogenesis) is particularly relevant to an understanding of the detection of inherited disease because it is at this stage that ‘shuffling’ of genes occurs.

Normal cell division, or mitosis, involves a simple copying of each chromosome, with one identical copy being passed on to each daughter cell. To avoid a doubling in the number of chromosomes in each generation, gametogenesis involves a reduction (by half) of the chromosome number during two specialized cell divisions known as meiosis. Since the chromosome complement of parent and offspring must be equivalent, the reduction in chromosome number cannot be arbitrary: parental contributions must be equal and equivalent. This requirement can be met because each somatic cell of an individual is diploid, containing corresponding (homologous) pairs of chromosomes, one derived from the mother and the other from the father. Meiosis consists of two cell divisions: in the first, after duplication of DNA, pairing of homologous chromosomes occurs, and to ensure that each gamete receives just one member of each homologous pair, the duplicated paternal chromosome is distributed to one, the duplicated maternal chromosome to the other. The assortment between the two cells appears to be random so that each cell acquires some maternal and some paternal chromosomes. The second cell division is like ordinary mitosis, except that it is not preceded by duplication of chromosomes. As a consequence, the gametes produced are haploid, with half the normal number of chromosomes.

In females, gametogenesis is initiated during fetal development and, at birth, the germ cells are in a phase of arrested maturation of the first stage of meiosis, which is not completed until ovulation. The increased risk of chromosomal abnormalities in older mothers may be explained by the fact that completion of meiosis occurs only after ovulation, when the second stage of meiosis (which is similar to ordinary mitotic division) occurs and during which fertilization can take place. This may happen up to 50 years after formation of the germ cells. In males, sperm production continues from the time of sexual maturity into old age and the large number of cell divisions involved is probably the cause for the increased number of new single gene mutations that seem to occur in the children of older men.

When both alleles are expressed in a heterozygote, each producing its phenotype independently, inheritance is said to be codominant. This type of inheritance is seen most clearly when phenotypes are determined by immunological or biochemical tests – for example, in testing for blood groups and in restriction fragment length polymorphism (RFLP) analysis and, more recently, in the growing field of pharmacogenetics (see later), where it is possible to distinguish both alleles at each locus.

Of the inherited diseases currently recognized, the overwhelming majority are due to problems in nuclear DNA and are autosomal dominant, autosomal recessive or sex linked. However, it is now known that some diseases are associated with the small amount of DNA that is present in mitochondria. These diseases are maternally inherited, since the mitochondria in the fertilized egg are of maternal origin.

The relative frequency of inherited disease varies markedly between populations: for example, cystic fibrosis and α₁-antitrypsin deficiency are associated primarily with northern Europeans, while red cell disorders (thalassaemia, sickle cell anaemia and glucose 6-phosphate dehydrogenase deficiency) are found primarily in people of Mediterranean, Oriental or African origin, and Tay–Sachs disease is found primarily in Ashkenazi Jews. In some populations, the prevalence of an inherited disease is due to a ‘founder effect’, as with variegate porphyria in South Africans, which can be traced to a single couple who emigrated from Holland in the 1680s. Autosomal recessive diseases that are particularly widespread in larger populations are likely to represent a balanced polymorphism in which disadvantage to homozygotes is balanced by an advantage to the larger number of heterozygotes. With some red cell disorders (e.g. HbS, the sickle cell trait), the balanced polymorphism is clearly a response to the environment, in this case with heterozygosity conferring resistance to malaria caused by Plasmodium falciparum.

In a large population, the relative frequencies of different alleles tend to remain constant and a simple mathematical formula allows calculation of the frequency of different genotypes. If two alleles, A and a, occur at a given locus and their frequencies are p and q, respectively, then:

It can be shown that the genotypes AA, Aa and aa have frequencies p², 2pq and q², respectively (the Hardy–Weinberg law). Use of this law allows simple calculation of carrier frequencies for autosomal traits. For example, if the homozygote frequency (q²) for cystic fibrosis is about 1 in 2500, q is 1/50, p is 1 − q or 49/50 (~ 1) and the heterozygote frequency (2pq) is about 1/25.

Several factors dictate that each genetic disease is associated with symptoms of quite variable nature and severity.

It is often naively assumed that all mutations of a particular gene will have identical consequences for the organism, but this is far from being true. Different mutations in a given gene are quite likely to give rise to different phenotypes, just as a particular phenotype can result from many different mutations in the same gene or even in different genes. Since many different mutations that have deleterious effects on a particular gene are present in most populations, it is not surprising that molecular analysis often reveals that individuals who have been described as homozygous are in fact compound heterozygotes, that is, they are affected by two different deleterious alleles.

Analysis of DNA is heavily dependent on the availability of techniques to identify specific nucleotide sequences. Fortunately, the function of DNA has resulted in the evolution of proteins capable of recognizing specific DNA sequences and it is inherent in the structure of DNA that one strand should recognize and bind (hybridize) specifically to its complementary strand. Most of the techniques currently used in DNA technology exploit one or other of these properties.

In addition to allowing reproducible cleavage of DNA to a manageable size, restriction enzymes are also valuable tools for analysing molecular diversity and identifying the individuality of DNA sequences. Differences in DNA sequences between individuals may create or destroy sites for restriction enzymes (i.e. there is polymorphism of restriction sites). Thus, the distance between restriction sites will often differ between individuals and between the maternal and paternal strands of DNA. The pattern of restriction sites can therefore provide a ‘signature’ for each individual strand of DNA. The different populations of DNA fragments produced on digestion by an enzyme are known as restriction fragment length polymorphisms or RFLPs.

Restriction enzymes have more recently acquired a new use in preparing genomic DNA for the various techniques employed in ‘next generation’ sequencing.

A probe is a sequence of DNA (or RNA) that has been labelled in order to identify complementary base sequences by molecular hybridization. The two strands of DNA can be dissociated (‘denatured’ or ‘melted’) in various ways, such as by heating or addition of alkali. Denaturation for a given fragment of DNA occurs at a specific temperature, and the temperature at which 50% of the duplex is dissociated is known as the T_m. When the temperature is lowered to just below the T_m, hydrogen bonds begin to reform between complementary bases, a process known as annealing or renaturation. If the probe and target DNA are mixed before reannealing is allowed to occur, the probe can be used to ‘find’ its complementary sequence. The conditions under which reannealing occurs (in particular salt concentration and temperature) determine the degree of stringency of the hybridization.

Probes can be used to detect their complementary sequences, traditionally after DNA (or RNA) fragments have been separated by electrophoresis. Digestion of genomic DNA with a restriction enzyme will produce a million or so fragments of different sizes and electrophoresis has the great advantage of allowing simple determination of the size of fragments detected by probes. The now classic technique of Southern blotting involves the transfer of electrophoretically separated bands of DNA to a sheet of nitrocellulose or nylon. Complementary DNA sequences are then detected by hybridization with labelled probes. The technique was originated by Dr (later Professor) E M Southern. Subsequently, the terms Northern and Western blotting have been used for processes in which RNA or proteins, respectively, are transferred.

In the past, all DNA analysis required some technique of visualizing the products of the reaction, and with probe hybridization this involves labelling the probe in some way. ³²Phosphorous radioactive labelling, although still in use for blotting protocols, was superseded by the use of fluorescent dyes, particularly for gene sequencing, a system which allowed the development of large, high-throughput, DNA analysers, as well as techniques such as real-time PCR. However, some of the technologies employed in ‘next generation’ sequencing systems require neither gels nor dyes, as will be discussed later. Some of the newer systems allow for an electrical signal to be generated when hybridization of probe to target occurs, which may eventually lead to the development of point-of-care systems.

Probes, generally of a few thousand bases, may be sequences cut from genomic DNA or they may have been produced by making complementary DNA (cDNA) to an mRNA species. The latter procedure uses the enzyme reverse transcriptase, which transmits genetic information in the ‘reverse’ direction, that is, from RNA to DNA. Genomic and cDNA sequences differ, particularly in the absence of intron sequences from the latter. The tolerance of probes for mismatching of base sequences will depend on their size and on the stringency of hybridization. Shorter oligonucleotide probes are often more useful for direct identification of point mutations since conditions can be chosen such that hybridization occurs only when there is complete complementarity between probe and target. Because hybridization of a relatively smaller proportion of bases in the probe is required, larger genomic or cDNA probes will usually recognize corresponding sequences of DNA from different individuals in a population or even different but related genes.

Cloning, where the sequence of interest was ‘grown’ in bacteria such as E. coli after being inserted into the bacterial genome using bacteriophage viruses, has been largely superseded as a method of producing probes by the ability to create synthetic oligonucleotide sequences, although cloning as a technique is still employed in areas of research.

Stringent control of conditions, such as the concentrations of magnesium and nucleotides in the reaction mixture, have usually been needed to maintain the specificity of a PCR although conditions that maximize polymerase fidelity may reduce PCR efficiency. Where such manipulation was necessary, it was predominantly dependent on the sequences involved, especially that of the primers. However, the ability to amplify longer sections of DNA means that primers can be ‘picked’ to suit the conditions required, allowing the use of universal ‘master mixes’ and thus a greater standardization of PCR assays. PCR is so sensitive that it has been used to amplify the DNA from a single cell and it can be used with samples obtained from materials as diverse as ancient mummies, fossils, hair follicles, preimplantation embryos and fixed pathological specimens. The large quantities of amplified DNA produced by PCR, which can be detected using a variety of visual labels, have eliminated the need for radiolabelling. The extreme sensitivity of the technique is also the source of one of its disadvantages – contamination by extraneous DNA (e.g. from the operator) can create havoc, so that strict precautions must be taken to avoid artefacts.

Non-isotopic fluorescent labels are employed in the widely used technique of real-time PCR. There are several versions of this technique but the basic principle is that a PCR reaction is followed, in real time, by monitoring the signals produced by dye-labelled probes that bind to the accumulating products. Various modifications to the technique allow real-time PCR to be used for quantitation of a target sequence (hence, its other, abbreviated name of qPCR) and determining dosage (the number of copies of a gene in a cell) and gene duplication as well as for mutation detection. Quantitative analysis has become increasingly important as a tool for microbiologists for determining such things as viral load in samples from patients suffering from diseases such as human immunodeficiency virus infection.

Not to be confused with real-time PCR, reverse transcriptase PCR (rtPCR) uses purified RNA as the start point and uses the enzyme reverse transcriptase to produce cDNA, which can then be amplified using conventional PCR and used to examine the protein expressed by the gene of interest. Potentially, this allows investigation of both the effect of any sequence variants in a gene on its protein product, and the possible phenotypic consequences.