Evaluation of genetic markers linked to hemophilia A locus: an Indian experience

Abstract

Hemophilia A is an X-linked recessive bleeding disorder caused by defects in factor VIII gene (F8). Our study examines variations of single nucleotide polymorphism (SNP) in F8 in the Indian population and establishes the utility of a combination of SNP and microsatellite markers for the successful identification of carriers in the affected families. The disease affects 1 in 5,000–10,000 males worldwide. Due to the large size of F8 (186 Kb), direct mutation analysis is not realistic. Therefore, linkage analysis using informative markers is the most practical approach where family members of the suspect, including an affected member, are available. Single nucleotide polymorphisms (SNPs) are the preferred markers because of their dense coverage of the genome and relatively easy assay format including commonly used restriction fragment length polymorphism (RFLP). SNPs are known to have variable allele frequencies in different populations. Ideally, informative markers should be highly heterozygous in the test population and the set of markers selected for use should not be in linkage disequilibrium (LD) for maximum efficiency of the linkage-based diagnostic test for carrier detection. We recently reported a remarkable variation in the informativeness of RFLP markers linked to hemophilia B locus in several Indian population groups.1 In this context, we examined several published studies from across different regions of India which have shown variable levels of heterozygosity and informativeness of the F8 markers.2–4 However, all of them had been conducted on a regional basis and on a relatively small number of samples. Until now, no exhaustive study has been carried out to verify the variability of informativeness of markers in F8 across different population groups in India. To assess the heterozygosity of the SNPs and to evaluate the linkage status among them in the Indian population, 1,486 unrelated individuals recruited as part of the Indian Genome Variation project5 were included in the present study. These individuals belonged to 54 distinct ethnic groups living in 6 different geographic regions (north, northeast, east, south, west and central) of mainland India. They also included the four major linguistic families of the Indian population i.e. Indo-European (IE), Tibeto-Burman (TB), Austro-Asiatic (AA) and Dravidian (DR). The internal review committee on research using human subjects reviewed and approved the project according to the regulations of the Indian Council of Medical Research. Four SNPS, rs5987079 (SNP1), rs1936645 (SNP2), rs4898352 (SNP3) and rs4898399 (SNP4), were selected (Figure 1, panel A) spanning the entire F8 with almost equal spacing to reduce the possibility of them being in linkage disequilibrium (LD). Three (rs5987079, rs1936645 and rs4898352) of the four SNPs selected were observed to have high minor allele frequencies (MAF) ranging between 0.34–0.42. These could, therefore, be used as markers to distinguish between a mutant and a normal F8 chromosome in the Indian population. However, high LD values (r>0.5) between the three SNP markers (i.e. SNP #1-3) in all four linguistic groups suggest that any one of these three SNPs is sufficient to be used successfully as a marker for carrier diagnosis (Figure 1, panel B). The fourth SNP (#4; rs4898399), not in LD with other SNPs, showed low MAFs (0.01–0.1) in all the four linguistic groups and could not be used as a marker. Therefore, we selected the most widely used SNP (#3; rs4898352) having an RFLP assay format using Bcl I restriction enzyme.