Abstract
Hemophilia B, an X-linked recessive bleeding disorder, is caused by heterogeneous mutations in the factor IX (F9) gene. Hence, carriers of the disease are usually detected by F9 gene linked RFLP analysis. We aimed to test a set of RFLP markers (DdeI, XmnI, MnlI, TaqI & HhaI), used worldwide for carrier detection, to estimate its heterozygosity in different population groups of India, and identify additional single nucleotide polymorphisms (SNPs) if necessary. A total of 8 population groups encompassing different regions of India, consisting of 107 unrelated normal females without any history of hemophilia B in the family and 13 unrelated obligate carriers were recruited in the study. Regions of F9 gene were amplified by PCR from genomic DNA of the donors followed by restriction enzyme digestion and/or sequencing as appropriate. Combined informativeness for the markers varied between 52–86% among normal females belonging to different geographical locations of India. Haplotype analysis revealed that the most prevalent haplotype lacked the restriction sites for all five RFLP markers. Screening regions of F9 gene that harbor 10 SNPs reported in dbSNP yielded only two SNPs, which increased the overall informativeness in each population group and heterozygosity in the obligate carriers for the disease from 38% to 69%. Our data show that heterozygosity of commonly used RFLP markers is remarkably variable across different regions of India. Thus prudent selection of the markers based on specific population groups including usage of additional markers is recommended for efficient carrier detection.
Highlights
Hemophilia B is an X-linked recessive bleeding disorder affecting approximately 1 in 30,000 males and caused by deficient clotting factor IX (FIX) ac-tivity [1]
The prevalence of hemophilia B is similar worldwide and the disease is caused by frequent de novo mutations spanning throughout the F9 gene, which is 30–50% of all patients [4,5]
The PCR based RFLP markers commonly advocated for use in carrier detection of hemophilia B were originally based on the studies primarily conducted on the Caucasians
Summary
Hemophilia B is an X-linked recessive bleeding disorder affecting approximately 1 in 30,000 males and caused by deficient clotting factor IX (FIX) ac-tivity [1]. The mutational heterogeneity and the inability of the immunoassay results to detect carriers necessitated an indirect approach for DNA based carrier analysis and prenatal diagnosis in the affected families, which involves tracking the mutant chromosome by analysis of intragenic markers at hemophilia B locus. This strategy fails if the markers are not informative in the family members. In 1993, WHO published a list of markers [10] for efficient carrier detection based on early reports on high heterozygosity of these markers in Caucasian population. We report identification of two new F9 polymorphisms with high levels of heterozygosity, which improve the efficiency of carrier detection for hemophilia B
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