[Double mutation, a 2-bp deletion and Val211Ile, in the blood coagulation factor IX gene of a patient with severe hemophilia B].

Hemophilia B is an X-linked bleeding disorder caused by the absence or reduced activity of coagulation factor IX (FIX). Here, we report a double mutation in the FIX gene (F9) in a Japanese patient with severe hemophilia B. FIX activity (FIX:C) was measured with a one-step functional assay. FIX antigen (FIX:Ag) levels were determined by ELISA. Genomic DNA was amplified by PCR with Taq DNA polymerase. Purified products were sequenced with a Thermo Sequenase Pre-mixed Cycle Sequencing Kit. To determine whether the sequence changes were a mutation or polymorphism, PCR products from 54 Japanese individuals were investigated using PCR-Ase I restriction enzyme digestion. Both FIX:C and FIX:Ag levels in the patient were below 1%. Levels of FIX:C and FIX:Ag in the patient's mother were 42% and 46% of normal, respectively. Sequence analysis of F9 of the patient revealed two distinct mutations. The first mutation was a G-to-A transition at position 30084 in exon7, which caused a Val211Ile in the region which encodes the protease domain of FIX. The patient's mother was heterozygous for this mutation. This substitution was not detected by restriction enzyme digestion from 54 Japanese alleles. The second mutation was a 2-bp deletion in exon 2 (nt. 6396-6399, del. AG). The patient's mother was also heterozygous for this deletion. The authors identified a rare double mutation of a 2-bp deletion and Val211Ile in the F9 of a patient with severe hemophilia B. The Val211Ile was confirmed as a novel missense mutation of F9.