多重PCR靶向富集结合高通量测序筛查结直肠癌中MMR基因的突变  

Distinct mutations of MMRgene in colorectal cancer by targeted enrichment and high-throughput next generation sequencing

作　　者：黄凯[1,2] 陈慧杰[3] 刘方奇[4] 徐烨[4] 李轩[5] 南蓬[1] 

机构地区：[1]复旦大学生命科学学院生物多样性与生态工程教育部重点实验室,上海200433 [2]上海同达科信生物技术发展有限公司,上海200080 [3]上海生物信息技术研究中心,上海201203 [4]复旦大学附属肿瘤医院大肠外科,上海200032 [5]中国科学院上海生命科学研究院植物生理生态研究所,上海200032

出　　处：《复旦学报（医学版）》2016年第2期205-210,共6页Fudan University Journal of Medical Sciences

基　　金：国家重点基础研究发展计划(973计划)项目(2012CB316501);国家高技术研究发展计划(863计划)项目(2012AA02A602);国家自然科学基金(31271409;31401128)~~

摘　　要：目的探讨多重PCR靶向富集结合高通量测序技术在结直肠癌(colorectal cancer,CRC)中检测错配修复(mismatch repair,MMR)基因种系突变的应用。方法收集17例CRC患者和14例正常人的血液并提取基因组DNA;设计和优化寡聚核苷酸探针,使其能对5个基因MLHl、MSH2、PMS1、PMS2和MSH6的73个外显子序列进行有效的PCR扩增和富集;应用多重PCR技术靶向富集样本MMR基因的外显子序列;扩增产物进行文库构建和高通量测序,检测MLHl、MSH2、PMS1、PMS2和MSH6基因的突变情况。结果 31个样本共得到2.7Gb的数据,平均reads数为287 048,测序数据中平均82.18%可与参考序列进行比对,外显子序列平均覆盖度为99.9%,平均测序深度为2 282。在MMR基因的外显子区域共发现13种非同义单核苷酸变异(single nucleotide variation,SNV)、2种同义SNV,其中MSH6的c.G3205C:p.G1069R为未见报道SNV。检测结果经Sanger法测序验证,结果一致。结论多重PCR靶向富集结合高通量测序技术是一套通量高、速度快、费用低、准确性高的MMR基因突变筛查方法。Objective To detect the germline mutations of mismatch repair（MMR）genes in colorectal cancer（CRC）using targeted enrichment and high-throughput next generation sequencing,and to explore its applications in research and clinical diagnosis of Lynch syndrome. MethodsGenomic DNA was extracted from17 patients diagnosed with colorectal cancer and 14 healthy adults,Primers,which could amplify all the 73 exons and flanking regions of 5 MMR genes（MLH1,MSH2,MSH6,PMS1,PMS2）by multiplex PCR were designed and optimized,PCR products were then sequenced by Illumina Miseq sequencer. Results We obtained approximately2.7 giga-base sequence data,and the average reads number of individual samples was 287048.On average,82.18% of the reads could be mapped to the reference human genome（HG19）.Average coverage and sequencing depth of targeted regions were 99.9% and 2282-fold respectively.After bioinformatic analysis,we found 14 previously annotated single-nucleotide variants（SNVs）in5 mismatch repair（MMR）genes and1 novel mutations in MSH6 genes（c.G3205C：p.G1069R）.These results were confirmed by sanger sequencing. Conclusions Targeted enrichment combined with high-throughput next generation sequencing can be used to detect mutations in MMR genes with high sensitivity and lower cost than conventional methods.

关 键 词：错配修复基因 LYNCH综合征 靶向富集 高通量测序 

分 类 号：R735.3[医药卫生—肿瘤] Q781[医药卫生—临床医学]