Molecular Genetics of Hemophilia A

Abstract

Hemophilia A is one of the most common bleeding disorders in man. It affects approximately 1 male in 5,000-10,000 Caucasians and Chinese. Patients are due to the lack of factor VIII’s clotting activity and usually suffer from spontaneous bleeding to joints and soft tissues. Mutational detection in patients with Hemophilia A is complicated for the large size of the factor VIN gene (186 kb doding for 2351 amino acids) and the heterogeneity of its abnormal alleles, PCR-SSCP method (PCR-Single strand conformation polymorphism) was used because of its simplicity in screening all the factor VIII exons for mutated regions for subsequent DNA sequencing. A total of 102 individuals from 87 different families, accounting for nearly 10% total cases in Taiwan, have been analyzed. These include 10 patients (10 families) with mild-to-moderate and 92 patients (77 families) with severe hemophilia A. Of the 87 cases, 46% were point mutations, insertions and deletions in the coding regions but approximately 50-60% patients failed to reveal abnormal exons. Almost ＞ 90% of the latter were severe. Among the 46% (40 cases) whose mutations have been determined, 21 cases (24.1%) were single base changes resulting in 8 nonsense and 13 missense codons, 16 with deletion or insertion of 1-11 nucleotides (18.4%), and 3 with deletion of large DNA fragments (3.5%). As for those unidentified patients, their factor VIII genes were found to be disrupted as a result of intrachromosomal gene inversion. Examination of 112 patients by Southern blotting has estimated a 33% incidence for factor VIII gene inversion in all the patients with hemophilia A ) representing 37% severe cases). Taken together, PCR-SSCP and gene inversion analyses will serve approximately 79% (46% + 33%) hemophilia A patients. Since direct detection for factor VIII mutations can not be used in all Chinese families with hermophilia A, genetic markers such as the CA repeats located at intron 13 (CA-13) and intron 22 (CA-22), respectively, were investigated. Seven different alleles with 18-24 CAs have been identified for CA-13. Alleles of 20 and 21 CAs are the most common and their population frequency were 0.68% and 0.4, respectively. The CA-22 marker contained repetition of (GT)n(AG)n as was identified in the white European but not in the Canadian population. Alleles with 25 and 26 GT/AGs accounts for 18% and 75% this group of samples, 68% respectively. The expected rate. of heterozygosity for either CA markers was 68%, although a value of 57% was observed by haplotype analysis, indicating an association of the two repeat markers. nevertheless, the study of 62 females showed that with the combined use of CA-13 and CA-22 with BctI, approximately 71% would be informative for these markers. This number may increaseto 81% if XbaI polymorphism is added. In summary, we propose that a better genetic diagnosis procedure for Chinese individuals with hemophilia A would be first to look for the inversion mutation, and secondly for one of the intragenic markers, and then for the PCR-SSCP analysis.