高分辨率熔解曲线分析在血友病BF9基因突变检测中的应用  

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

作　　者：郁婷婷[1] 戴菁[2] 傅启华[1] 王学锋[2] 

机构地区：[1]上海交通大学医学院附属上海儿童医学中心检验科,200127 [2]上海交通大学医学院附属瑞金医院检验科

出　　处：《中华检验医学杂志》2012年第7期620-624,共5页Chinese Journal of Laboratory Medicine

摘　　要：目的利用高分辨率熔解曲线（HRM）技术建立简便有效的血友病B（HB）基因诊断的方法。方法收集2005年1月至2010年6月就诊于上海瑞金医院的55例HB患者的外周血标本，抽提外周血基因组DNA。采用PCR扩增结合测序的方法对40例HB患者进行F9基因突变检测，利用其中21例带有阳基因1～7号外显子突变的HB患者标本建立相应的HRM突变筛查方法。采用阳基因1～7号外显子的PCR—HRM突变筛查方法及8号外显子的DNA直接测序方法，对15例未知F、9基因突变的HB患者进行基因诊断。结果通过PCR扩增结合测序，40例HB患者均检测到F9基因突变。21例带有D基因1～7号外显子突变的HB患者标本中，19例（90％）患者的突变可通过PCR．HRM技术进行检测。通过D基因1～7号外显子PCR—HRM突变筛查及8号外显子的DNA直接测序，15例未知F、9基因突变的HB患者均检测到F9基因突变。55例HB患者中共检测到34种F9基因突变。结论HB基因诊断的新方法，即PCR．HRM筛查阳基因1～7号外显子突变结合8号外显子的DNA测序法操作简便、结果可靠。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 exonl to exon7 of F9 gene. Mutation scanning of exonl 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 exonl 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 exonl 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）

关 键 词：血友病B 因子IX 突变 聚合酶链反应 

分 类 号：R554.1[医药卫生—血液循环系统疾病]