Functional Cloning

Functional cloning describes the identification of a human disease gene through knowledge of its protein product. From the amino-acid sequence of a protein, oligonucleotide probes could be synthesized to act as probes for screening complementary DNA (cDNA) libraries (p. 56).

An alternative approach was to generate an antibody to the protein for screening of a cDNA expression library.

Use of Animal Models

The recognition of phenotypic features in a model organism, such as the mouse, which are similar to those seen in persons affected with an inherited disorder, allowed the possibility of cloning the gene in the model organism to lead to more rapid identification of the gene responsible in humans. An example of this approach was the mapping of the gene responsible for the inherited disorder of pigmentation and deafness known as Waardenburg syndrome (p. 91) to the long arm of human chromosome 2. This region of chromosome 2 shows extensive homology, or what is known as synteny, to the region of mouse chromosome 1 to which the gene for the murine pigmentary mutant known as Splotch had been assigned. The mapping of the murine Pax3 gene, which codes for a transcription factor expressed in the developing nervous system, to this region suggested it as a positional candidate gene for the disorder. It was suggested that the pigmentary abnormalities could arise on the basis that melanocytes, in which melanin synthesis takes place, are derived from the neural crest. Identification of mutations in PAX3, the human homolog, confirmed it as the gene responsible for Waardenburg syndrome.

Mapping Trinucleotide Repeat Disorders

A number of human diseases are attributable to expansions of trinucleotide repeats, and in particular CAG repeat expansions which cause extended polyglutamate tracts in Huntington disease and many forms of spinocerebellar ataxia. A method developed to seek novel trinucleotide repeat expansions in genomic DNA from affected patients led to the successful identification of a CTG repeat expansion in patients with spinocerebellar ataxia type 8.

Next-Generation ‘Clonal’ Sequencing

This new sequencing technology shows great promise for elucidating the remaining ~55% of single gene disorders where the genetic aetiology remains unknown (Figure 5.2). The first success was in the identification of mutations in the DHODH gene that cause Miller syndrome by ‘exome’ sequencing. Around 164,000 regions encompassing exons and their conserved splice sites (a total of 27 Mb) were sequenced in a pair of affected siblings and probands from two additional families. Non-synonymous variants, splice donor/acceptor, or coding insertion/deletion mutations were identified in nearly 5000 genes in each of the two affected siblings. Filtering these variants against public databases (dbSNP and HapMap) yielded novel variants in less than 500 genes. Analysis of pooled data from the four affected patients revealed just one gene, DHODH, which contained two mutated alleles in each of the four individuals.

FIGURE 5.2 A strategy for disease gene identification using exome sequencing.

Linkage Analysis

Genetic mapping, or linkage analysis (p. 137), is based on genetic distances that are measured in centimorgans (cM). A genetic distance of 1 cM is the distance between two genes that show 1% recombination, that is, in 1% of meioses the genes will not be co-inherited and is equivalent to approximately 1 Mb (1 million bases). Linkage analysis is the first step in positional cloning that defines a genetic interval for further analysis.

Linkage analysis can be performed for a single, large family or for multiple families, although this assumes that there is no genetic heterogeneity (p. 378). The use of genetic markers located throughout the genome is described as a genome-wide scan. In the 1990s, genome-wide scans used microsatellite markers (a commercial set of 350 markers was popular), but microarrays with several million SNPs now provide greater statistical power.

Autozygosity mapping (also known as homozygosity mapping) is a powerful form of linkage analysis used to map autosomal recessive disorders in consanguineous pedigrees (p. 269). Autozygosity occurs when affected members of a family are homozygous at particular loci because they are identical by descent from a common ancestor.

Linkage of cystic fibrosis (CF) to chromosome 7 was found by testing nearly 50 white families with hundreds of DNA markers. The gene was mapped to a region of 500 kilobases (kb) between markers MET and D7S8 at chromosome band 7q31-32, when it became evident that the majority of CF chromosomes had a particular set of alleles for these markers (shared haplotype) that was found in only 25% of non-CF chromosomes. This finding is described as linkage disequilibrium and suggests a common mutation from a founder effect (p. 378). Extensive physical mapping studies eventually led to the identification of four genes within the genetic interval identified by linkage analysis, and in 1989 a 3-bp deletion was found within the cystic fibrosis transmembrane receptor (CFTR) gene. This mutation (p.Phe508del) was present in approximately 70% of CF chromosomes and 2% to 3% of non-CF chromosomes, consistent with the carrier frequency of 1 in 25 in whites.

Chromosome Abnormalities

Occasionally, individuals are recognized with single-gene disorders who are also found to have structural chromosomal abnormalities. The first clue that the gene responsible for Duchenne muscular dystrophy (DMD) (p. 307) was located on the short arm of the X chromosome was the identification of a number of females with DMD who were also found to have a chromosomal rearrangement between an autosome and a specific region of the short arm of one of their X chromosomes. Isolation of DNA clones spanning the region of the X chromosome involved in the rearrangement led in one such female to more detailed gene-mapping information as well as to the eventual cloning of the DMD or dystrophin gene (p. 307).

At the same time as these observations, a male was reported with three X-linked disorders: DMD, chronic granulomatous disease, and retinitis pigmentosa. He also had an unusual X-linked red cell group known as the McLeod phenotype. It was suggested that he could have a deletion of a number of genes on the short arm of his X chromosome, including the DMD gene, or what is now termed a contiguous gene syndrome. Detailed prometaphase chromosome analysis revealed this to be the case. DNA from this individual was used in vast excess to hybridize in competitive reassociation, under special conditions, with DNA from persons with multiple X chromosomes to enrich for DNA sequences that he lacked, the so-called phenol enhanced reassociation technique, or pERT, which allowed isolation of DNA clones containing portions of the DMD gene.

The occurrence of a chromosome abnormality and a single-gene disorder is rare, but identification of such individuals is important as it has led to the cloning of several other important disease genes in humans, such as tuberous sclerosis (p. 316) and familial adenomatous polyposis (p. 221).

Candidate Genes

Searching databases for genes with a function likely to be involved in the pathogenesis of the inherited disorder can also suggest what are known as candidate genes. If a disease has been mapped to a particular chromosomal region, any gene mapping to that region is a positional candidate gene. Data on the pattern of expression, the timing, and the distribution of tissue and cells types may suggest that a certain positional candidate gene or genes is more likely to be responsible for the phenotypic features seen in persons affected with a particular single-gene disorder. Several computer programs have been developed that can search genomic DNA sequence databases for sequence homology to known genes, as well as DNA sequences specific to all genes, such as the conserved intron–exon splice junctions, promoter sequences, polyadenylation sites and stretches of open reading frames (ORFs).

Identification of a gene with homology to a known gene causing a recognized inherited disorder can suggest it as a possible candidate gene for other inherited disorders with a similar phenotype. For example, the identification of mutations in the connexin 26 gene, which codes for one of the proteins that constitute the gap junctions between cells causing sensorineural hearing impairment or deafness, has led to the identification of other connexins responsible for inherited hearing impairment or deafness.

Confirmatory Testing that a Candidate Gene Is a Disease Gene

Mutations in candidate genes can be screened for by a variety of methods (p. 59) and confirmed by DNA sequencing (p. 61). Finding loss-of-function mutations or multiple different mutations that result in the same phenotype provides convincing evidence that a potential candidate gene is associated with a disorder. For example, in the absence of functional data to demonstrate the effect of the p.Phe508del mutation on the CFTR protein, confirmation that mutations in the CFTR gene caused cystic fibrosis was provided by the nonsense mutation p.Gly542X.

Further support is provided by the observation that the candidate gene is expressed in the appropriate tissues and at the relevant stages of development. The production of a transgenic animal model by the targeted introduction of the mutation into the homologous gene in another species that is shown to exhibit phenotypic features similar to those seen in persons affected with the disorder, or restoration of the normal phenotype by transfection of the normal gene into a cell line, provides final proof that the candidate gene and the disease gene are one and the same.

The Human Gene Map

The rate at which single-gene disorders and their genes are being mapped in humans is increasing exponentially (see Figure 1.6, p. 7). Many of the more common and clinically important monogenic disorders have been mapped to produce the ‘morbid anatomy of the human genome’ (Figure 5.3).

Beginning and Organization of the Human Genome Project

The concept of a map of the human genome was proposed as long ago as 1969 by Victor McKusick (see Figure 1.5, p. 7), one of the founding fathers of medical genetics. Human gene mapping workshops were held regularly from 1973 to collate the mapping data. The idea of a dedicated human genome project came from a meeting organized by the US Department of Energy at Sante Fe, New Mexico, in 1986. The US Human Genome Project started in 1991 and is estimated to have cost around 2.7 billion US dollars. Other nations, notably France, the UK, and Japan, soon followed with their own major national human genome programs and were subsequently joined by a number of other countries. These individual national projects were all coordinated by the Human Genome Organization, which has three centers, one for the Americas based in Bethesda, Maryland, one for Europe located in London, and one for the Pacific in Tokyo.

Although the key objective of the Human Genome Project was to sequence all 3 × 10⁹ base pairs of the human genome, this was just one of the six main objectives/areas of work of the Human Genome Project.

Human Gene Maps and Mapping of Human Inherited Diseases

Designated genome mapping centers with ear-marked funding were involved in the coordination and production of genetic or recombination and physical maps of the human genome. The genetic maps initially involved the production of fairly low-level resolution index, skeleton or framework maps, which were based on polymorphic variable-number di-, tri-, and tetranucleotide tandem repeats (p. 17) spaced at approximately 10-cM intervals throughout the genome.

The mapping information from these genetic maps was integrated with high-resolution physical maps (Figure 5.4). Access to the detailed information from these high-resolution genetic and physical maps allowed individual research groups, often interested in a specific or particular inherited disease or group of diseases, rapidly and precisely to localize or map a disease gene to a specific region of a chromosome.

Figure 5.4 A summary map of human chromosome 3, estimated to be 210 Mb in size, which integrates physical mapping data covered by 24 YAC contigs and the Genethon genetic map with cumulative map distances.

(From Gemmill RM, Chumakov I, Scott P, et al 1995 A second-generation YAC contig map of human chromosome 3. Nature 377:299–319; with permission.)

Ethical, Legal, and Social Issues of the Human Genome Project

The rapid advances in the science and application of developments from the Human Genome Project have presented complex ethical issues for both the individual and society. These issues include ones of immediate practical relevance, such as who owns and should control genetic information with respect to privacy and confidentiality; who is entitled to access to it and how; whether it should be used by employers, schools, etc.; the psychological impact and potential stigmatization of persons positive for genetic testing; and the use of genetic testing in reproductive decision making. Other issues include the concept of disability/differences that have a genetic basis in relation to the treatment of genetic disorders or diseases by gene therapy and the possibility of genetic enhancement (i.e., using gene therapy to supply certain characteristics, such as height.) Last, issues need to be resolved with regard to the appropriateness and fairness of the use of the genetic technologies that come out of the Human Genome Project, with prioritization of the use of public resources and commercial involvement and property rights, especially with regard to patenting.