Identification of mutations in the F9 gene including exon deletion by multiplex ligation-dependent probe amplification in 33 unrelated Korean patients with haemophilia B.

Haemophilia B (HB) is a rare X-linked recessive bleeding disorder caused by a mutation in the F9 gene. The aims of this study were to characterize the mutation spectrum of F9 in Korean patients with HB to establish the optimal molecular diagnostic strategy and to find genotype-phenotype correlations. Study subjects consisted of 33 unrelated Korean patients with HB. We performed polymerase chain reaction (PCR) amplification and direct sequencing of all exons and flanking sequences of F9. When large deletion was suspected from PCR failure, exon dosage test using multiplex ligation-dependent probe amplification (MLPA) was performed. We identified disease-causing mutations in 32 out of 33 patients by direct sequencing analyses (mutation detection rate, 97%). A total of 28 unique mutations were detected, including 7 novel ones. Six mutations were recurrent but observed in no more than two patients. In the remaining one patient, exon 1 was not amplified, and MLPA analysis confirmed a large deletion involving exon 1. The genotype-phenotype correlations between the type of mutation and the severity of factor deficiency were not consistent, as has been previously reported. One patient developed inhibitor, and he was the patient with exon 1 deletion. Based on our results from 33 Korean patients with HB, which showed no hotspot for mutations, direct sequencing of all exons with flanking sequences is needed as the first-line test. MLPA can be a feasible platform at clinical laboratories to detect large deletion mutations in suspected cases.