Structure, Biology, and Genetics of Factor VIII. Factor VIII Function, страница 14

Gross deletions of factor VIII result in a fivefold greater incidence of inhibitors than for patients without detectable deletions. 178 However, no clear picture has emerged as to the correlation between the size or the breakpoints of the deletions and the development of inhibitors.

Carrier and Antenatal Diagnosis

The cloning of factor VIII gene, the discovery of DNA polymorphic markers within and closely linked to the gene, and the elucidation of molecular defects in many patients with hemophilia A dramatically changed the practice of diagnosis of carriers and affected fetuses. The discovery of the common partial inversion of the factor VIII gene provided a means of diagnosis using Southern blot analysis. 176 This defect accounts for approximately 45% of severe hemophilia A. The diagnosis of the exact molecular defect in the remaining families is still not practical even in sophisticated laboratories. Because of the enormous variety of the remaining mutations, DNA diagnosis is almost always limited to indirect detection using linked DNA polymorphisms. Figure 105-8 shows the location and informativeness of DNA polymorphisms within factor VIII gene. Families requesting carrier or prenatal diagnosis, or both (after the initial screening for the detection of the inversion), are usually asked to supply blood samples of a number of family members for linkage analysis. The affected factor VIII gene is marked within the family using polymorphic markers both within MLID91092638 MLID85296150 MLID86232589  203–206 and without MLID84244819 MLID85137718  207,208 the gene (Fig. 105-9). Short sequence repeats have been found in two introns, and additional ones will be soon identified. MLID91295681 MLID93271395  209,210 Nearly all families are informative, but 20–30% for extragenic polymorphisms only. In those families the chance of error is 2–5% depending on the polymorphism used. When an intragenic polymorphism is used, the chance or error is negligible (certainly <1%).

The indirect detection of mutant genes is not feasible when only one male offspring is available and the carrier status of the mother is unknown. In these cases direct detection of the molecular defect should be employed. However, because of the large number of different molecular defects, the considerable size of the gene, and the sophistication of the mutation detection methodology, direct diagnosis is not available for all families. The simple test for recognizing the common partial inversion of factor VIII, which accounts for about 45% of cases of severe hemophilia A, has dramatically changed the situation. Few laboratories will deal with the remaining molecular defects. It seems that the method of choice is the RT-PCR amplification of illegitimate transcripts and their analysis using chemical cleavage or another mutation screening method.