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.
Summary
Haemophilia A (HA) is an X‐linked recessive bleeding disorder caused by defects in the F8 gene encoding the coagulation factor VIII. Mutation analysis in HA is important to confirm the diagnosis, genotype‐phenotype correlations and for genetic counselling and family study. The aim of this study was to detect causative mutations of F8 in severe HA patients in Korea and to correlate the mutation type with the risk of inhibitor development. A total of 100 unrelated Korean patients with severe HA were enrolled for this study. The Nijeman modification of the Bethesda assay was used to determine the presence of inhibitor. Molecular analysis of F8 was performed using a combination of molecular techniques, including long‐distance polymerase chain reaction, direct sequencing and multiplex ligation‐dependent probe amplification (MLPA). We identified causative mutations in 98% of severe HA patients (98/100). Inv22 and Inv1 mutations were detected in 30 patients and one patient, respectively. A total of 59 unique mutations were identified in 69 non‐inversion patients, including 24 novel mutations. The overall prevalence of inhibitor was 26%. Inhibitor risk was highest in patients with large deletion mutations identified using MLPA (100%). Among those with point mutations, the prevalence of inhibitor was highest when the mutation occurred in the A3 and C2 domains (60% and 50%, respectively). The molecular diagnostic strategy involving multiplex PCR, sequencing and dosage analyses identified causative mutations in most cases of severe HA. The high inhibitor risk was associated with large deletion mutations and point mutations in A3 and C2 domains.
To cite this article: Lee S-T, Kim H-J, Kim D-K, Schuit RJL, Kim S-H. Detection of large deletion mutations in the SERPINC1 gene causing hereditary antithrombin deficiency by multiplex ligation-dependent probe amplification (MLPA). J Thromb Haemost 2008; 6: 701-3.
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