Suzanne de Bruijn

38 Chapter 1.2 IDENTIFICATION OF GENES ASSOCIATED WITH HEARING LOSS AND RETINAL DYSTROPHY Linkage analysis The first HL- and RD-associated genes were identified using linkage analysis and candidate gene strategies in the early 90s. 10-12 Examples of candidate gene approaches include analysis of candidate disease-associated genes based on their function, gene expression or animal model studies (discussed in (13)). Linkage analysis was used to pinpoint a genomic region of interest likely to encompass the disease gene. The strategy is based on the key principle that a disease haplotype is shared between affected individuals within a family but is not present in unaffected individuals. The shared haplotype cosegregates with the disease according to the observed mode of inheritance. Initially, linkage regions were mapped using laborious genotyping of polymorphic microsatellite markers, but the process was optimized when microarray technologies became available. Microarrays, such as SNP-arrays, allow rapid genotyping of thousands of single nucleotide polymorphisms (SNPs) that are present across the genome and have a variant allele frequency higher than 1% in the healthy population. The higher the density of the SNPs on the array and the more SNPs that reside within the region showing linkage disequilibrium, themore precise the determination of a possible disease haplotype is. The distance between two SNPs can be expressed in centimorgans (cM). One cM is defined as the distance between chromosomal positions that have a 1% chance of being separated by chromosomal recombination during meiosis. A logarithm of the odds (LOD) score can be calculated to estimate the odds that two loci, or a locus and a disease-associated gene, are located at an assumed distance from each other (expressed as the recombination fraction theta). A LOD-score of 3.3 or higher is considered evidence for linkage in a genome-wide manner, with a probability of 95%. 14 Nowadays, several tools (e.g. GENEHUNTER 15 and PLINK 16 ) are available to calculate the LOD-score and identify a linkage region. However, large family pedigrees and sufficient participating family members are required to reach a statistically significant linkage. When a disease-associated locus is defined, Sanger sequencing can be performed to evaluate the genomic region for causative variants. In this way, the linkage analysis strategy has been applied very effectively for disease gene identification for many years (reviewed in (13, 17)). Despite the introduction of higher throughput sequencing techniques, SNP genotyping can still be very useful to determine regions of genotype- sharing even in small families, especially to reduce the number of candidate variants.