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Jo Field Tuesday, 12 th January 2010

Population Genetics Describe the principles of autozygosity mapping and how it can be used to identify disease loci. Jo Field Tuesday, 12 th January 2010. Introduction to autozygosity mapping.

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Jo Field Tuesday, 12 th January 2010

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  1. Population GeneticsDescribe the principles of autozygosity mapping and how it can be used to identify disease loci Jo Field Tuesday, 12th January 2010

  2. Introduction to autozygosity mapping • Autozygosity is defined as homozygosity for an allele where both copies of the allele are inherited from a single ancestor • alleles are termed identical by descent (IBD) • common in consanguineous families • can also occur in apparently non-consanguineous families, especially if from isolated populations – due to shared common ancestors. • The probability that the child of a consanguineous marriage will be homozygous for a specific gene derived from a common ancestor is termed the coefficient of inbreeding (F) • Where the parents are first cousins, F = 1/16

  3. Introduction to autozygosity mapping (2) • Autozygosity mapping has been widely used for gene mapping in autosomal recessive disorders, mainly using data from consanguineous families. • Autozygosity mapping involves detection of a locus for an autosomal recessive disease • based on homozygosity (autozygosity) for genetic markers adjacent to the disease locus in affected individuals • For linkage, a single affected child of a first-cousin marriage has been shown to contain the same information as a non-consanguineous nuclear family with three affected children • Autozygosity mapping techniques – initially used restriction fragment length polymorphisms (RFLPs) as genetic markers • more recently, microsatellite markers were used • current studies generally use SNP genotyping using genome-wide SNP array chips (advantages - rapid, high-resolution) • can combine techniques – e.g. microsatellite markers can be used to further characterise regions of interest identified by SNP genotyping

  4. Illustration of autozygosity mapping • see http://autozygosity.org • Affected individuals will be homozygous (autozygous) for microsatellite marker/SNP alleles around disease locus • Unaffected individuals not homozygous for the same marker alleles in the gene region – (heterozygous or homozygous for another allele) – this information can be used to exclude candidate regions. • Ian Carr at Leeds Centre for Autozygosity Mapping has designed software tools for the interpretation of autozygosity mapping data, e.g. IBDfinder

  5. Examples of autozygosity mapping • Autozygosity mapping in autosomal recessive deafness • particularly useful because of the extreme genetic heterogeneity in autosomal recessive forms of deafness • several families may include more than one gene for AR deafness segregating (especially because of marriages between deaf people), which complicates other linkage approaches • In consanguineous families – less likely that more than one deafness gene is segregating • Chaib et al (1996), Hum Mol Genet 5, 155-158,used LOD score linkage analysis in a large consanguineous Lebanese family to map the DFNB9 locus to 2p22-23 • Chaib et al (1996), Hum Mol Genet 5, 1061-1064, used the same approach in a consanguineous Sunni family living in Syria to map the DFNB12 locus to 10q21-22. • DFNB1, DFNB2, DFNB4 and DFNB8previously mapped using autozygosity mapping

  6. Autozygosity mapping in AR deafness - 1 • Boxed areas indicate haplotype associated with DFNB9 in each individual • Significant linkage found with AFM242yd8 (D2S171) – maximum LOD score = 5.9 (=0.03) • Genotyping of adjacent markers indicates DFNB9 locus should be between AFMa052yb5 and D2S158

  7. Autozygosity mapping in AR deafness - 1 • DFNB9 locus further studied using autozygosity mapping in three additional unrelated consanguineous Lebanese families • locus refined to a 1 cM region between D2S158 and D2S174 • DFNB9locus shown to correspond to otoferlin gene using a candidate gene approach (Yasunaga et al, 1999, Nature Genet 21, 363-639). • Sequencing of the otoferlin gene detected the same nonsensemutation in each of the four families studied • role of otoferlin gene in deafness supported by gene function and mouse expression studies

  8. Autozygosity mapping in AR deafness - 2 • Boxed areas indicate haplotype associated with DFNB12 in each individual • Significant linkage found with D10S535 (maximum LOD score = 6.64 at =0.03) • Analysis of surrounding markers indicates DFNB12 locus should be between D10S529 and D10S532

  9. Autozygosity mapping in AR deafness - 2 • DFNB12 locus further studied using linkage analysis in several additional consanguineous families showing AR deafness linked to 10q21-22 (Bork et al, 2001, Am J Hum Genet 68, 26-37) • locus refined to a critical region ~0.55 cM apart • Candidate genes in the region were sequenced • Identified homozygous missense and nonsense mutations in the CDH23 (cadherin-23) gene • CDH23 found to be expressed in cochlea • mutations in CDH23 also found in a syndromic form of deafness (Usher syndrome)

  10. Examples of autozygosity mapping - 3 • Parry et al (2009) used autozygosity mapping in a consanguineous Brazilian family with recessive cone-rod dystrophy (CRD) linked to 8p11 (CORD9 locus) • CRD shows genetic heterogeneity and shows AD, AR and X-linked inheritance patterns • Initial haplotype analysis showed a 12 Mb segment with two putative autozygous segments • Locus further refined by autozygosity mapping, genotyping >200 microsatellite markers and SNPs in two affected individuals • localised to a 2.95 Mb homozygous (autozygous segment) between two SNPs, which contained 34 genes • Candidate genes chosen based on gene function/expression predictions • Direct sequencing of 10 genes revealed a splice mutation in the ADAM9 metalloprotease gene

  11. Examples of autozygosity mapping – 3 (cont) • Further autozygosity mapping with Affymetrix 10K and 250K SNP arrays revealed three additional consanguineous CORD9-linked families • also found nonsense/splice mutations in ADAM9 in these three additional families • Role of ADAM9 in photoreceptor function supported by mouse knockout studies • implicated in several cellular functions

  12. Factors affecting autozygosity mapping • Number of informative affected and unaffected individuals • useful technique if more than one sibship linked by consanguinity and multiple affected individuals are available • less informative in smaller families (fewer meioses) • Degree of consanguinity • if common ancestor is far removed from the affected individual: • smaller proportion of the genome shows autozygosity • greater significance of detecting that patients share segments of DNA that are identical by descent • however, there is then a greater population that a second independent allele (disease or marker allele) will enter the family from elsewhere, so it is less likely that homozygosity for an allele represents autozygosity. • affected individuals may be compound heterozygotes • more likely to encounter allelic heterogeneity –which may occur even in small consanguineous families – and thus affect linkage results

  13. Factors affecting autozygosity mapping - 2 • Value of coefficient of inbreeding (F) may be underestimated in complex consanguineous families (e.g. if “founder” individuals of pedigree are already related) • may result in artificial inflation of calculated LOD scores and therefore false positive linkage results • may result in segments of autozygosity that are unrelated to disease (especially when using whole genome scans to detect regions of homozygosity) • Frequency of marker alleles being genotyped • the less common the marker allele, the more powerful the approach • as individuals may be homozygous for common marker alleles which have not been inherited from the common ancestor (identical by state [IBS] rather than identical by descent [IBD])

  14. Further applications/Related techniques - 1 • Autozygosity may influence cancer predisposition – consanguinity has been linked to higher rates of cancer • Autozygosity mapping has been used in some cases for gene mapping in apparently non-consanguineous families • E.g. in families drawn from isolated populations, who may share common ancestry • – mapping of a gene for benign recurrent intrahepatic cholestasis (BRIC1) to chromosome 18 using three distantly related patients from an isolated Dutch population (Houwen et al, 1994, Nature Genet 8, 380-386 • = linkage disequilibrium (LD) mapping or “search for shared segments” • distinguish between IBS shared segments and IBD (autozygous) shared segments by typing additional markers in the shared segments

  15. Related techniques - 2 • Recent publications have used whole genome SNP screens to identify runs of homozygosity (ROH) in non-consanguineous populations – whole genome homozygosity association (WGHA) • ROHs <4 Mb are common in outbred individuals • = function of linkage disequilibrium (LD) • individuals with a higher coefficient of inbreeding (F) have larger ROHs (may represent autozygous regions). • Approach used to identify recessive genetic risk loci for schizophrenia (Lencz et al (2007), PNAS 104, 19942-19947) • nine ROHs were significantly different in cases compared to controls • 4/9 contained genes associated with schizophrenia • risk ROHs generally at low frequencies in general population

  16. Additional references • Strachan and Read (2004). Human Molecular Genetics (Third Edition). Garland Science, London and New York (2004). • Lander and Botstein (1987). Homozygosity Mapping: A Way to Map Human Recessive Traits with the DNA of Inbred Children. Science 236, 1567-1570. • Gibbs and Singleton (2006). Application of Genome-Wide Single Nucleotide Polymorphism Typing: Simple Association and Beyond. PLoS Genetics 2, 1511-1517 • McQuillan et al (2008). Runs of Homozygosity in European Populations. Am J Hum Genet 83, 359-372. • Miano et al (2000). Pitfalls in Homozygosity Mapping. Am J Hum Genet 67, 1348-1351. • Bacolod et al (2009). Emerging paradigms in cancer genetics: some important findings from high-density single nucleotide polymorphism array studies. Cancer Res 69, 723-727

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