Abstract
Hemophilia B (HB) is an X-linked recessive bleeding disorder caused by mutations in the coagulation factor IX (FIX) gene. Genotyping patients with HB is essential for genetic counseling and provides useful information for patient management. In this study, the F9 gene from 23 patients with HB was analyzed by direct sequencing. Nineteen point mutations were identified, including a novel missense variant (c.520G > C, p.Val174Leu) in a patient with severe HB and a previously unreported homozygous missense mutation (c.571C > T, p.Arg191Cys) in a female patient with mild HB. Two large F9 gene deletions with defined breakpoints (g.10413_11363del, g.12163_23369del) were identified in two patients with severe HB using a primer walking strategy followed by sequencing. The flanking regions of the two breakpoints revealed recombination-associated elements (repetitive elements, non-B conformation forming motifs) with a 5-bp microhomology in the breakpoint junction of g.12163_23369del. These findings imply that non-homologous end joining and microhomology-mediated break-induced replication are the putative mechanisms for the deletions of the F9 gene. Because the g.12163_23369del deletion caused exons to be absent without a frameshift mutation occurring, a smaller FIX protein was observed in western blot analyses.
Highlights
The human coagulation factor IX (FIX) gene, mapped at chromosome Xq27.1-q27.2, is approximately 32.7 kilobases in length
Large deletions (> 50 bp) in the F9 gene, 90% of which are associated with the severe phenotype, occur in approximately 5% of patients with severe Hemophilia B (HB) and significantly increased risks for developing inhibitors[5,7,8]
It has been proposed that the underlying mechanisms may be non-allelic homologous recombination (NAHR), non-homologous end joining (NHEJ) or microhomology-mediated break-induced recombination (MMBIR) events[10]
Summary
To identify the junctions of the deletions, sequential primers were designed at the interval of approximately 0.5–1 kb to amplify the sequences (fragment by fragment, and the ends of the adjacent fragments were overlapping) on both sides of the breakpoints (from exon 1 to exon 4 in patient LXF and from exon 3 to exon 6 in patient CD) to narrow down the regions containing the breakpoints. The nomenclature of the F9 mutations was based on cDNA reference sequence NM_000133.3 and protein reference sequence NP_000124.1 in accordance with the recommendations of the Human Genome Variation Society (HGVS; http://www.hgvs.org/mutnomen/) guidelines. To evaluate the influence of the large deletions on plasma factor IX protein and blood coagulation, western blotting, ELISA and a thrombin generation test were performed using plasma samples from patient LXF and patient CD, and from CDM and CDS.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
