The molecular basis of hemophilia B : identification of the defect in factor IXVancouver

Hemophilia BVancouver, is a moderately severe hereditary disorder in which the factor IX antigen is present in relatively normal amounts but the biological activity of factor IX is markedly reduced. Previous studies have demonstrated that although the patient has 62% of the normal factor IX antigen level in his plasma, he shows only 2.6% of normal factor IX procoagulant activity. In addition, radioimmunoassays have shown that epitopes on both the heavy and light chains of activated factor IX are present. These two results were taken as an indication that the molecular defect causing the hemophilia may be a point mutation involving an amino acid change in the protein. In order to identify the mutation involved, DNA was isolated from lymphocytes in a blood sample from the patient. This DNA was used to construct a genomic library in the λ vector EMBL3. One million of the resultant clones were screened with a labelled factor IX cDNA probe to identify those clones containing portions of the factor IX gene. DNA inserts from three λ clones, which together span the entire gene, were subcloned to facilitate sequence analysis of the exons and intron / exon junctions of the factor IX gene. The nucleotide sequences of the coding regions were found to match the published sequence of the normal gene, except for one nucleotide. A single mutation was found at nucleotide 31,311 of the factor IX gene (Yoshitake et al.,1985), corresponding to amino acid 397 of the mature protein. This alteration, which changes an isoleucine codon, ATA, to a threonine codon, ACA, is novel among the mutations which have been reported to cause hemophilia B. A three dimensional model of the protease domain of factor IXа, which was prepared on the basis of its homology to the pancreatic serine proteases, was examined in the vicinity of residue 397. The position of threonine 397 in this model suggests that this mutation could alter the hydrogen bonding between factor IXа and its substrate, factor X. Taken together, these data suggest that this mutation is the cause of the hemophilia in this patient.Medicine, Faculty ofMedical Genetics, Department ofGraduat