The application of high-resolution melting curve analysis in the detection of F9 gene mutations in haemophilia B patients

Objective To establish an effective method for F9 gene mutation detection by using high resolution melting (HRM) curve analysis. Methods Peripheral blood samples of 55 hemophilia B （HB） patients were collected from Shanghai Ruijin Hospital during January 2005 to June 2010.Genomic DNA was extracted from the peripheral blood.PCR amplification combined with sequencing was used to identify the F9 gene mutations in 40 patients.HRM assay was established on the 21 DNA samples with known mutations in exon1 to exon7 of F9 gene.Mutation scanning of exon1 to exon7 by HRM combined with direct sequencing of exon8 was used in the molecular diagnosis of 15 HB patients with unknown F9 gene mutations. Results F9 gene mutation was detected in each of the 40 HB patients by direct sequencing.By HRM, the different melting curve patterns were identified in 19 out of 21 cases.The detection rate was about 90%.Through mutation scanning of exon1 to exon7 by HRM combined with direct sequencing of exon8, F9 gene mutations were detected in all the 15 HB patients with unknown F9 gene mutations.Thirty-four F9 gene mutations had been identified in the 55 HB patients. Conclusions A new strategy of HB genetic diagnosis, scanning mutations of exon1 to exon7 combined with DNA sequencing of exon8 of F9 gene, is established in this study.The new strategy is efficient and reliable.(Chin J Lab Med, 2012，35：620-624) Key words: Hemophilia B; Factor Ⅸ; Mutation; Polymerase chain reaction