Hemophilia A is due to the functional deficiency of factor VIII (FVIII, gene locus F8C). Although half the patients have no delectable FVIII protein in their plasma, the more rare patients (∼5%) have normal levels of a dysfunctional FVIII and are termed cross-reacting material (CRM)-positive. More commonly (∼45%), patients have plasma FVIII protein reduced to an extent roughly comparable to the level of FVIII activity and are designated CRM-reduced. We used denaturing gradient gel electrophoresis to screen for mutations within the F8C gene of 11 patients (6 CRM-positive, 5 CRM-reduced) and identified 9 different mutations in 9 patients after analyses of all 26 exons, the promoter region, and the polyadenylation site. Six mutations have not been described previously. Five were missense (Ser289Leu, Scer558Pbe, Val634Ala, Val634-Met, Asn1441Lys), and the sixth was a 3-bp deletion (ΔPhe652). A review of the literature and the assay of FVIII antigen in 5 hemophilia A patients with previously identified missense mutations from this laboratory yielded a total of 20 other unique CRM-reduced and CRM-positive mutations. Almost all CRM-positive/reduced mutations (24/26) were missense, and many (12/26) occurred at CpG dinucleotides. We examined 19 missense mutations for evolutionary conservation using the portions of the porcine and murine F8C sequences that are known, and 18/19 amino acid residues altered by mutation in these patients were conserved. Almost 50% of mutations (11/26) clustered in the A2 domain, suggesting that this region is critical for the function of FVIII. The results indicate a nonrandom distribution of mutations and suggest that mutations in a limited number of FVIII regions may cause CRM-positive and CRM-reduced hemophilia A.
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