SINGLE-STRAND CONFORMATION POLYMORPHISM (SSCP) ANALYSIS OF THE MOLECULAR PATHOLOGY OF HEMOPHILIA-B

In the present study, we report the application of polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis to the screening of seven functionally important factor IX gene (FIX) regions (total length 2.66 kb) in 9 unrelated haemophilia B patients of Portuguese or African origin. In eight of the patients an altered migration pattern of single-stranded DNA was observed. Direct sequencing of the relevant DNA fragments unveiled the following sequence alterations: two novel mutations, namely FIX(Barcelos) Thr-380-Pro and FIX(Lousada) 9bp insertion at position 31,309 or 31,318; five mutations previously reported in other ethnic groups (FIX(Porto) Arg-145-His, FIX(Luanda) Gly-207-Arg, FIX(Penafiel) Arg-248-Gln, FIX(Sesimbra) Arg-333-Gln, FIX(Cascais) Arg-333-Stop); and a normal variant, G --> T transvertion at position 6,596 in intron 2. We propose hypothetical models for the generation of the 9 bp duplication (FIX(Lousada). We have performed molecular modeling studies in order to predict the structure of the variant FIX molecules. (C) 1993 Wiley-Liss, Inc.