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Spectrum of mutations in CRM-positive and CRM-reduced hemophilia A
M J McGinniss1, H H Kazazian, L W Hoyer
1Center for Medical Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland.
Abstract:
Hemophilia A is due to the functional deficiency of factor VII (FVIII, gene locus F8C). Although half the patients have no detectable FVIII protein in their plasma, the more rare patients (approximately 5%) have normal levels of a dysfunctional FVIII and are termed cross-reacting material (CRM)-positive. More commonly (approximately 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, Ser558Phe, Val634Ala, Val634-Met, Asn1441Lys), and the sixth was a 3-bp deletion (delta Phe652). 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Researchers identified novel mutations in the F8C gene causing Hemophilia A, particularly in cross-reacting material (CRM)-positive and CRM-reduced patients. Many mutations were missense and conserved across species, with a significant cluster in the A2 domain, highlighting its functional importance.
Area of Science:
- Genetics and Molecular Biology
- Hematology
- Biochemistry
Background:
- Hemophilia A results from factor VIII (FVIII) deficiency, with distinct patient groups including CRM-positive (dysfunctional FVIII) and CRM-reduced (low FVIII levels).
- Understanding the genetic basis of these variations is crucial for diagnosis and potential therapeutic strategies.
Purpose of the Study:
- To identify and characterize mutations in the F8C gene responsible for CRM-positive and CRM-reduced Hemophilia A.
- To investigate the prevalence and nature of these mutations, including their location and evolutionary conservation.
Main Methods:
- Denaturing gradient gel electrophoresis was employed to screen the F8C gene (exons, promoter, polyadenylation site) in 11 Hemophilia A patients.
- Literature review and FVIII antigen assays were conducted for additional patients with identified missense mutations.
Main Results:
- Nine novel mutations were identified in 9 patients, including six previously undescribed mutations (five missense, one deletion).
- A total of 26 unique CRM-positive/reduced mutations were compiled, with the majority (24/26) being missense mutations, often occurring at CpG dinucleotides (12/26).
- Evolutionary conservation analysis showed 18 out of 19 mutated residues were conserved, and nearly 50% (11/26) of mutations clustered in the FVIII A2 domain.
Conclusions:
- The F8C gene harbors diverse mutations leading to Hemophilia A, particularly in CRM-positive and CRM-reduced phenotypes.
- Missense mutations are predominant in these patient groups, and their conservation suggests critical functional roles.
- The A2 domain appears to be a functionally important region for FVIII, as indicated by the clustering of mutations.