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).
