Structure, Biology, and Genetics of Factor VIII. Factor VIII Function, страница 12

Exon Skipping due to Nonsense Mutations

An important observation concerning the pathophysiology of nonsense mutations has recently been made in the factor VIII gene and independently in the fibrillin and OAT genes. MLID93258342 MLID93157831  190,194 In some cases a nonsense codon mutation can lead to abnormal RNA processing, in which the exon containing the mutation is skipped. This observation was made after the introduction of RT-PCR in the mutation detection methodology. In one case of Glu, 1987 to Stop mutation in exon 19 all detectable RNA lacked the sequences of this exon. In the second case of Arg 2,116 to Stop mutation of exon 22, there was about 50% of RNA without the sequences of exon 22, while the remaining 50% of RNA was of normal size. The junctions of exons 18–20 and 21–23 do not result in translational frameshift. The mechanism, significance, and frequency of the exon skipping due to nonsense mutations are presently unknown.

Missense Mutations

The study of missense mutations (i.e., nucleotide substitutions that result in amino acid substitutions) is important for understanding the function of the protein and the pathophysiology of the disease. A total of 92 mutations leading to amino acid substitutions have been described (Fig. 105-7). These mutations are spread throughout the different domains of the gene except for exon 14; this exon encodes for the B domain which is devoid of amino acid substitutions that cause hemophilia A. In spite of knowledge of amino acid substitutions, the mode of action of most of these mutations in producing reduced factor VIII activity in plasma is unknown. However, several mutations have been identified that alter thrombin cleavage sites or the VWF-binding site, or otherwise introduce or destroy N-linked glycosylation sites.

Natural mutations in patients with CRM-positive hemophilia A affect the thrombin cleavage needed for activation of the molecule. Mutations R372H and R372C have been shown in vitro to abolish the normal cleavage by thrombin in the heavy chain. MLID88327107 MLID90227237  195–197 It is not clear whether the S373L mutation has an effect in thrombin cleavage. Mutations R1689C and R1689H abolish thrombin cleavage at the light chain. MLID90105723  173,198 These mutations also lead to CRM-positive hemophilia A in which there is a normal amount of nonfunctional factor VIII in plasma.

Two sulfated tyrosine residues (Y1664 and Y1680) are found in the region of factor VIII between amino acids K1673 and E1684 in which a VWF-binding site has been localized. A natural mutation (Y1680F) has been observed in patients with moderate, CRM-reduced hemophilia A. MLID90152691  72 Site-directed mutagenesis of Y1680F results in a molecule that has lost high-affinity binding to VWF, presumably because the phenylalanine residue cannot be sulfated. MLID91093266  70,71

Two other CRM-positive mutations produce severe hemophilia A by creating new N-glycosylation sites in the protein. MLID92279241  199 The first, I566T, creates a new such site in N564 (NQI to NQT) in the A2 domain of the heavy chain. The second new site is in the A3 domain of the light chain; the mutation is M1772T, changing the N1770 (NIM to NIT). In both cases factor VIII is present at normal levels in plasma, but it is completely inactive. When the plasma of either patient is deglycosylated, factor VIII activity is restored to a significant degree. The significance of a S577P mutation that in theory eliminates a potential N-glycosylation site at N575 is unknown.