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

Abstract

Summary. 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 ligationdependent 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 genotypephenotype 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.

Heterogeneous lengths of copy number mutations in human coagulopathy revealed by genome-wide high-density SNP array

“…MLPA experiments were performed using commercial kits SALSA MLPA P178 F8, P207 F9, P265 PROS1, and P227 SERPINC1 (MRC-Holland, Amsterdam, The Netherlands) as described previously. 16 The results of MLPA analyses in 7 of the study patients had been reported previously (Table 1).…”

“…The patient with HB who had high-titer inhibitor (H8) was previously reported to be the only patient with inhibitor among 33 HB patients. 16 Two patients with HB who were negative for inhibitor were a 2-and 3-year old boy, respectively, and thus careful management is mandatory in these young, high-risk patients.…”

Chapter 1 Profiling of Mutations in the F8 and F9, Causative Genes of Hemophilia A and Hemophilia B"

“…As in HA, the MLPA technique is a robust molecular test to detect mutations of large exon deletion affecting 1 or more exons in F9 (Kwon et al, 2008). The principle and method of interpretation of MLPA results are similar to that for F8.…”

“…As in HA, the mutations are scattered throughout the gene, thus, sequencing analyses need to cover the coding sequences and flanking intronic sequences of all 8 exons (Kwon et al, 2008). The strategy for direct sequencing analysis is largely similar to that for HA, but is simpler and less costly because the F9 gene is smaller and is encoded by a smaller number of exons.…”

“…Large deletion mutations, which can be detected by MLPA analyses, should be suspected when 1 or more reactions to amplify a genomic segment fail. Below is an example of a sequencing result with a missense mutation in a Korean HB (Kwon et al, 2008). Fig.…”