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Disease may manifest with two copies of the functional variant

... or one copy of the functional variant

... or no copy of the functional variant

Figure 2.1 Linkage. Recombination arises following crossing over events during meiosis. Examples are shown for simple family pedigrees showing autosomal dominant (A) or autosomal recessive (B) inheritance. In the autosomal dominant pedigree, a single copy of the functional variant (mutation) is sufficient to cause disease. Transmission of the functional variant occurs with a closely linked genetic marker; in many linkage analyses polymorphic microsatellites are used as genetic markers. Recombination between chromatids leads to reshuffling of DNA and this can be informative for linkage analysis when a set of different genetic markers are used, the marker showing strongest evidence of linkage (from the lod score) localizing the likely site of the functional variant. In the figure, two genetic markers are shown, one recombinant (solid line, lying close to functional variant) and one non-recombinant (serrated line). The resolution achieved by linkage analysis is, however, modest and typically requires finer scale mapping. (C) In a common multifactorial disease such as asthma, the occurrence of a functional variant is neither sufficient nor necessary for disease to occur but may be an important determinant of disease susceptibility; typically several variants involving different gene loci are involved, each with a relatively modest magnitude of effect. This significantly limits the power of applying a linkage-based analysis.

to define the genetic basis of relatively rare, highly penetrant diseases showing a minimally ambiguous phenotype and clear evidence of familial segregation, in most cases showing dominant, codominant, recessive, or X-linked mendelian pattern of inheritance (Botstein and Risch 2003). Integral to the success of linkage analysis was the use of positional cloning (Box 2.5) to identify specific genes within genomic regions resolved by linkage analysis, and of DNA sequencing among affected and unaffected individuals to identify specific causal mutations.

The combination of linkage analysis and a positional cloning approach is illustrated by the pioneering studies in cystic fibrosis and Treacher Collins syndrome. Other examples of application to specific diseases are described elsewhere in the book including haemochromatosis (Section 12.6), Duchenne muscular dystrophy (Box 3.7), and Huntington's disease (Box 7.13).

Box 2.3 Recombination fraction and genetic distance

The recombination fraction describes the probability of recombination between two loci at meiosis. Each recombination event or crossing over involves two of the four chromatids such that a single crossover event can lead to only 50% recombinants. Thus even for very widely spaced loci on a chromosome, the maximum recombination fraction is 50%. Genetic distance can be defined based on the probability of a crossover event. One centimorgan (cM) describes a region in which a crossover event is expected to occur once in every 100 meioses and approximates in physical distance to 1 million bases (1 Mb), but this varies depending on genomic location as the recombination rate differs between regions of the genome.

Box 2.4 Lod score

The lod score is a measure of linkage between loci. The score uses the observed recombination fraction in order to derive the likelihood ratio in comparison with the null hypothesis of no linkage being present. Mathematically the lod score, z9, can be defined as log base 10 of the likelihood ratios between observed linkage (with a recombination fraction 9) and that of no linkage (9 = 0.5):

where p(r;9) is the probability of data r when the true recombination fraction is 9 (Chotai 1984; Dawn Teare and Barrett 2005).

A higher positive lod score provides evidence of linkage; when above 3 this is regarded as significant, and equivalent to a P value of less than 0.0001. For genome-wide significance, a threshold of 3.3 is proposed for parametric linkage analysis to give a genome-wide type I error (risk of a false positive result or rejecting a true null hypothesis of no linkage) of 0.05 (Dawn Teare and Barrett 2005). A lod score of less than -2 provides significant evidence against linkage.

Box 2.5 Positional cloning

Positional cloning refers to the cloning or identification of a gene based on its chromosomal location rather than identification based on knowledge of the encoded protein. It has also been described as 'reverse genetics' and contrasts with the situation seen with sickle cell anaemia where knowledge of the haemoglobin protein allowed the amino acid change to be defined (Section 1.2), or other instances where knowledge of the amino acid sequence or availability of antibodies to a protein has allowed screening of complementary DNA (cDNA) libraries and identification of a specific gene. cDNA refers to DNA generated by reverse transcriptase from a single strand of mature fully spliced mRNA.

2.3.1 Cystic fibrosis and the delta-F508 mutation

The identification of the role of the CFTR gene in cystic fibrosis (Box 2.6) by positional cloning was a landmark in the field, demonstrating the power of this approach (Kerem et al. 1989; Riordan et al. 1989; Rommens et al. 1989). A substantial body of work from many independent groups had established linkage to chromosome 7q31 (Buchwald et al. 1989), including a large collaborative study of more than 200 families (Beaudet et al. 1986). Cloning the DNA sequence for this linkage region and looking for candidate gene sequences was a very considerable undertaking at the time this work was done. Rommens and colleagues adopted a chromosome walking and jumping approach to assemble a contiguous 280 kb region spanning the putative gene locus (Rommens et al. 1989). Chromosome walking was based on overlapping the ends of clone segments, jumping to bypass unclonable regions, then beginning bidirectional walks after each jump.

The limited resources available at the time meant that the identification of possible gene sequences was based on several analyses including cross species hybridization to indicate evolutionary conservation (zoo blots), detection of CpG islands, RNA hybridization experiments, isolation of cDNA, and DNA sequencing (Rommens et al. 1989). cDNA screening of many different libraries using specific DNA segments as probes allowed the first exon of the putative cystic fibrosis gene to be identified from a cDNA library originating from epithelial cells of a sweat gland. Further screening with additional clones allowed the mRNA of the putative gene to be resolved. Expression of a 6.5 kb transcript was seen in a number of tissues, notably the lung, colon, and sweat glands, which was consistent with tissues known to be involved in the disease process (Riordan et al. 1989). The investigators defined a gene spanning some 250 kb with 24 exons based on hybridization to genomic DNA; specific motifs suggested membrane association and adenosine triphosphate (ATP) binding (Riordan et al. 1989). This is reflected in the current nomenclature referring to the gene as the cystic fibrosis transmembrane conductor regulator gene, CFTR, which encodes an ATP-binding cassette (ABC) transporter protein.

In order to identify associated mutations, the cDNA sequences from cystic fibrosis patients and unaffected individuals were compared. A 3 bp deletion was noted on two clones derived from a cystic fibrosis cDNA library which was predicted to result in loss of the amino acid phenylalanine at position 508 of the encoded polypep-tide (Riordan et al. 1989). Kerem and colleagues found the deletion was present in 145 out of 214 (68%) cystic fibrosis chromosomes derived from individuals in a general clinic population (Kerem et al. 1989). Further studies have confirmed that the 'delta-F508 mutation', a three nucleotide deletion in exon 10 of the CFTR gene on chromosome 7q31.2 denoted 'p.F508del', is the most common mutation leading to cystic fibrosis. The deletion results in retention and degradation of the CFTR protein within the endoplasmic reticulum. It is particularly associated with pancreatic insufficiency, which

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