Structure, Biology, and Genetics of Factor VIII. Factor VIII Function, страница 13

Study of Mutations in CRM-Positive and CRM-Reduced Patients

The elucidation of mutations in this group of patients is highly instructive for understanding the importance of specific amino acid residues. A small number of such mutations have been described. MLID93194188  188 Since about 40% of CRM-positive mutations occur in the A2 domain, which consists of 228 amino acids or about 10% of the coding region of factor VIII, this region must be important in procoagulant activity. Most mutations, however, are CRM negative and probably affect the folding or stability, or both, of the protein. Since these mutations result in absence of secreted factor VIII and the in vitro functional studies depend on the analysis of the protein produced in eukaryotic cells after transfection with factor VIII cDNA, the mechanisms of action of these mutants will be difficult to elucidate.

Other Missense Mutations of Interest

Cases with two different mutations in the same amino acid are also of interest (Fig. 105-6). These are E272G, E272K; Y473C, Y473H; R531C, R531G; V634A, V634M; R1781C, R1781H; N1922D, N1922S; R1941L, R1941Q; R2209L, R2209Q; P2300L, P2300S; and R2307L, R2307Q. Mutations in the last 30 amino acids of factor VIII (C2 domain) may cause reduced phospholipid binding. Candidates are R2304L, R2307L, and R2307Q. The domains of factor VIII for binding to factors IX, X, and others have not been clearly elucidated.

Splicing Errors

A small number of potential splicing errors have been identified. 178 However, no formal proof that the mutations cause abnormal splicing has been obtained. Two mutations in the invariant GA of the acceptor splice site in introns 5 and 6 are associated, as expected, with severe hemophilia A. Four mutations occur in the donor splice site consensus and two in cryptic splice sites. No extensive functional analysis of these mutations has been done. It seems that in spite of the presence of >50 splice junctions in the factor VIII gene, splicing mutants do not account for a sizable fraction of hemophilia patients.

Promoter Mutations

No examples of mutations in the 5' untranslated region of factor VIII gene have been reported to date. If such mutations do occur, they are probably infrequent, since two laboratories failed to find any nucleotide substitutions in 530 nucleotides of the 5' flanking region of factor VIII in 227 patients with hemophilia A 74 (Gitschier J, Kogan S, Levinson B et al., unpublished data). Notably, however, the cis-regulatory elements for factor VIII gene expression are either unknown or poorly understood.

Factor VIII Inhibitors

Approximately 5–10% of patients with hemophilia A develop antibodies to factor VIII after treatment with exogenous factor VIII. 200 The problem is serious, since it represents an obstacle for the long-term treatment of patients with hemophilia. The etiology of development of inhibitors is not well understood. Epitope mapping of antibodies has shown specificities against the heavy or light chain, or both, in different patients. MLID87185841 MLID90001544  201,202 The analysis of many factor VIII mutations and their association with inhibitor development may uncover some rules concerning the contribution of the nature of mutations to the inhibitor formation. Almost all reported inhibitor cases have nonsense mutations or deletions in their factor VIII gene. MLID91334474  174 However, two missense mutations (R2209Q and W2229C) are associated with low levels of inhibitors. Plausibly, these mutations create such local structural variation that the wild-type sequence presents an immunologic epitope. Among the nonsense mutations, R1941X is associated with inhibitors in five of seven cases; R2147X in three of four and R2209X in three of five cases. Other nonsense mutations, however, are never associated with inhibitors. For example, in six cases with R336X that were not associated with inhibitors, exon skipping may be responsible for some factor VIII protein that "immunologically" protects from the development of inhibitors.