不同测序技术在结节性硬化相关肾血管平滑肌脂肪瘤基因诊断中的应用

Different mutation analysis of TSC1 and TSC2 genes in tuberous sclerosis complex patients

目的:评估一代测序(PCR-SSCP)与目标序列捕获二代测序技术(NGS)在结节性硬化相关肾血管平滑肌脂肪瘤患者基因诊断中的应用价值。方法:分析2015年1月~2017年6月中国人民解放军总医院收治的47例临床诊断为结节性硬化相关肾血管平滑肌脂肪瘤患者及32例散发性肾血管平滑肌脂肪瘤患者的临床资料。其中男19例,女60例,平均32.8岁(18~53岁)。77患者诊断为肾脏多发血管平滑肌脂肪瘤,2例病理证实为恶性肾上皮样血管平滑肌脂肪瘤,签署知情同意书后,采外周血10 ml提取基因组DNA,分别进行PCR-SSCP一代测序和目标序列捕获二代测序,靶基因TSC1和TSC2,对二代测序应用Sanger测序技术完成验证。结果:一代测序中66例(83.5%)患者检测到TSC1或TSC2基因突变,其中TSC1 19例,TSC2 29例,两者同时突变18例;其中结节性硬化组40例(85.1%),散发组26例(81.3%)检测到基因突变,两组差异无统计学意义。目标序列捕获二代测序中39例(49.4%)患者检测到TSC1或TSC2基因突变,其中TSC1 6例,TSC2 31例,两者同时突变2例,其中结节性硬化组34例(72.3%),散发性组5例(15.6%)检测到基因突变,两组差异有统计学意义;同时检测到6种既往未报道的疑似致病性突变,主要突变类型包括:无义突变、错义突变、剪接突变、大片段缺失突变及框移突变等。结论:尽管一代测序技术突变率较高,但目标序列捕获二代测序技术能更加准确的检测出TSC1/2基因的致病性突变,对结节性硬化相关肾血管平滑肌脂肪瘤患者的基因诊断具有重要的临床和学术意义。

Objective: To evaluate the value of the single strand conformation polymorphism( PCR-SSCP) and targeted second-generation sequencing( NGS) in genetic diagnosis of tuberous sclerosis complex( TSC) associated with renal angiomyolipoma( AML). M ethods: From Jan. 2 0 1 5 to June 2 0 1 7,4 7 TSC-AML patients and 3 2 sporadic AML patients from the P LA General Hospital were enrolled in this study,including 1 9 females and 6 0 males.The average age was 3 2. 8 years old( ranging from 1 8 to 5 3). All patients were diagnosed as multiple bilateral renal AML and malignant epitheloid angiomyolipoma was found in two cases. TSC 1/2 gene was detected by P CRSSCP and NGS. Results: The TSC 1/2 mutation rate was 8 3. 5 %( 6 6/7 9) in P CR-SSCP,included 1 9 TSC1 mutations,2 9 TSC2 mutations and 1 8 both TSC1 and TSC2 mutations. The mutation rate was 8 5. 1 %( 4 0/4 7)in TSC-AML group and 8 1. 3 %( 2 6/3 2) in sporadic AML group. There was no significant difference between two groups. The TSC 1/2 mutation rate was 49. 4%( 39/79) in NGS,including 16 TSC1 mutations. 31 TSC2 mutations and two both TSC1 and TSC2 mutations. The mutation rate was 72. 3%( 34/47) in TSC-AML and 15. 6%(5/32) in sporadic AML group,and there was significant difference between two groups. Conclusions: Compared with PCR-SSCP,NGS had a higher accuracy for TSC 1/2 gene detection. The TSC1/2 gene mutation analysis was very important for the diagnosis and researches of TSC-AML.