FACTOR-VIII GENE-MUTATIONS FOUND BY A COMPARATIVE-STUDY OF SSCP, DGGEAND CMC AND THEIR ANALYSIS ON A MOLECULAR-MODEL OF FACTOR-VIII PROTEIN

Screening of the factor VIII (FVIII) gene which spans 186 kb and codes for 26 exons, was originally hampered by its size but is now feasible because rapid DNA scanning methodologies have been developed. The pre sent study for the first time directly compares the three most widely applied screening methods, denaturing gradient gel electrophoresis (DG GE), single-stranded conformational polymorphism (SSCP) and chemical m ismatch cleavage (CMC) for their sensitivity of mutation detection in a selected group of ten haemophilia A patients. Nine of these patients are known to be cross-reacting material positive and eight exhibited a mild to moderate phenotype. Of the ten patients screened, we identif ied mutations in nine by all three screening methods. Of the mutations characterised, two are previously unpublished. T to C (S373P) and G t o A (D525N). In one mildly affected haemophiliac, we identified a seco nd T to C sequence change in the 5' untranslated region at -601 bp, pr obably having no effect on FVIII gene expression. Modelling studies we re performed on those mutations lying within the A domains of FVIII (D 525N, R527W, 1566T) to study the possible effect of these mutations on structure and/or function. When the three methods are performing opti mally and have been standardised, our experience is that CMC and DGGE are equally efficient at sequence variation detection while SSCP is sl ightly less sensitive.