利用目标基因测序技术发现马方综合征FBN1新突变  

Targeted next-generation sequencing in Marfan syndrome identifies mutations in FBN1

作　　者：郭俊[1] 蔡伦[1] 李小燕[1] 王绿娅[1] 杜杰[1] 

机构地区：[1]首都医科大学附属北京安贞医院-北京市心肺血管疾病研究所,心血管重塑相关疾病教育部重点实验室,100029

出　　处：《心肺血管病杂志》2014年第4期596-598,603,共4页Journal of Cardiovascular and Pulmonary Diseases

基　　金：国际科技合作项目(2010DFB30040);教育部长江创新团队(IRT1074)

摘　　要：目的：建立目标基因捕获结合第二代测序技术,对马方综合征（Marfan syndrome,MFS）患者的原纤维蛋白-1（fibrillin-1,FBN1）基因进行突变筛查,探讨MFS与FBN1基因突变的关系.方法：提取5例MFS患者外周血全基因组DNA,利用GenCap目标基因捕获技术（北京迈基诺公司）,设计FBN1的65个外显子区域特异性捕获探针,与基因组DNA文库进行杂交,将目标基因组区域的DNA片段进行富集后,再利用illumina hiseq2000第二代测序仪进行测序,通过数据分析,确定突变位点,用Sanger测序法对突变位点进行验证.结果：设计合成的目标基因特异性捕获探针可有效地捕捉并富集基因组DNA的目标靶片段.5例患者目标区域平均测序深度为173.85 ～ 280.73,97.10％ ～ 98.00％目标区域＞4×覆盖度.经过数据分析及Sanger测序验证,发现1个新的无义突变c.5968 C＞T（p.Gln1990X）.结论：本研究所建立的GenCap目标基因捕获技术结合illumina hiseq2000第二代测序技术成功的发现了FBN1的新突变.该方法快速而有效,可以使我们对MFS分子病因学有更好的认识.Objective：To develop an approach based on targeted gene capture and next-generation sequencing to determine the genetic defects in patients with marfan syndrome precisely and effectively and confirm the role of FBN1 in the pathogenesis of marfan syndrome.Methods：We analyzed five Marfan syndrome patients.The patients were diagnosed as Marfan syndrome according to the revised Ghent nosology based on their reported family history and clinical features.Peripheral blood was collected and genomic DNA was isolated.FBN1 were selected by a gene capture strategy,using GenCap custom enrichment kit.The enrichment libraries were sequenced on Illumina HiSeq 2000 sequencer for paired read 90bp to determine the mutation frequency in FBN1.Variants which have been reported in dbSNP137 or 1000 genome and in-house database were filtered.Selected variants were further validated by PCR and Sanger sequencing.Results：For the samples subjected to targeted next-generation sequencing,the average sequencing depths on the targeted regions were yielded from 173.85 to 280.73.Meanwhile,coverage of targeted exons for ＞ 4 reads were ranged from 97.10％ to 98.00％.We identified one stopgain SNP in FBN1：c.5968 C ＞ T （p.Gln1990X）,which was validated by Sanger sequencing.Conclusion：Our results confirm the role of FBN1 in the pathogenesis of marfan syndrome and demonstrated the robustness of targeted next-generation sequencing to precisely and rapidly determine genetic defects.The methodology provides a reliable strategy for routine gene diagnosis of marfan syndrome.

关 键 词：马方综合征 原纤维蛋白-1 目标基因捕获 第二代测序 

分 类 号：R54[医药卫生—心血管疾病]