MOLECULAR BASIS OF HEMOPHILIA B: IDENTIFICATION OF THE DEFECT IN FACTOR IX VANCOUVER

Factor IX Vancouver (fIX-V) is the cause of a moderate form of hemophilia B. An individual presenting with this disorder had 2.6% of normal procoagulant activity in his plasma but had 62% of the normal factor IX antigen level. Specific antibodies showed that fIX-V contains epitopes for both the heavy and light chains of factor IXa. To identify the defect involved, DNA was isolated from the lymphocytes of the male hemophiliac. Southern blot analysis using a full-length factor IX cDNA as a hybridization probe showed no gross differences between the fIX-V gene and the normal factor IX gene. The DNA from the hemophiliac was then partially digested with Sau3A and the resulting fragments (10-20kbp in size) were ligated into the BamHI site of λEMBL3. The DNA was then packaged into phage particles in vitro, and the recombinant phage were screened with the factor IX cDNA as a probe. Eight phage were isolated that contained overlapping DNA covering the complete gene for fIX-V. DNA sequence analysis of the protein-encoding regions, the intron/exon junctions and 5'-and 3'-flanking sequences revealed a single nucleotide change from the normal factor IX gene. The codon for amino acid 397 was changed from ATA (lie) to ACA (Thr). This mutation is in the catalytic domain of factor IXa and is novel amongst those hemophilia B mutations reported to date. Based on the known three dimensional structures of the pancreatic serine proteases, trypsin, elastase and chymotrypsin, models have been constructed for the structures of the catalytic domains of both the normal and Thr-397 mutant of factor IXa. These results suggest that the Thr-397 mutation may alter the conformation of the substrate binding region in the active site of factor IXa Vancouver through the formation of a hydrogen bond between the hydroxyl group of the Thr-397 side chain and the main chain carbonyl group of Trp-385. The postulated conformational change would lead to reduced binding affinity for the factor IXa substrate resulting in a reduction in the catalytic activity of fIXa-Vancouver.

Supported in part by grants from the Medical Research Council of Canada (to GDB and RTAM).