中国人遗传性非息肉病性结直肠癌MSH6基因胚系突变的测序研究

目的探讨中国人遗传性非息肉病性结直肠癌(hereditary nonpolyposis colorectal cancer,HNPCC)家系中MSH6基因胚系突变。方法采用PCR-直接测序的方法检测39个无胚系MSH2及MLH1基因突变、符合不同临床标准的中国人HNPCC家系先证者MSH6基因各外显子胚系突变;对137名正常人胚系基因组DNA进行错义突变相应外显子的测序分析。应用Envision二步法检测有突变的先证者肿瘤组织MSH6蛋白表达。结果在39个HNPCC先证者中共发现6个MSH6基因的胚系突变,分别位于第4、6、9和第10外显子;突变类型为4个错义突变、1个无义突变、1个剪接区的插入突变;对4个错义突变的相应外显子的测序分析显示:137名正常人胚系基因组DNA5例具有第6外显子1163密码子处的c.3488A>T的错义突变,约占3.65%(5/137),为单核苷酸多态性(single nucleotide polymorphism,SNP);其余错义突变在正常人群中均未发现。在6例有MSH6基因胚系突变家系的肿瘤组织中免疫组化染色除1例为SNP的肿瘤组织MSH6蛋白阳性表达外,其余均为阴性表达。经过查询国际HNPCC突变数据库及SNP数据库证实上述突变中5个为国际上尚未报道的病理性突变,1个为新发现的SNP。结论MSH6基因胚系突变在符合不同临床标准的中国人HNPCC中均起一定作用,对无MSH2及MLH1基因胚系突变的先证者行MSH6基因胚系突变的测序分析对确诊HNPCC家系是必要的。

Study on the germline mutation of MSH6 gene in Chinese hereditary nonpolyposis colorectal cancer pedigrees using PCR based sequencing

Objective To detect the germline mutation of mismatch repair gene(MSH6) in hereditary nonpolyposis colorectal cancer(HNPCC) kindreds fulfilling different clinical criteria.Methods The germline mutations of MSH6 gene were detected by PCR based DNA sequencing in 39 unrelated HNPCC probands fulfilling different clinical criteria in which MSH2 and MLH1 mutations were excluded.The exons with missense mutations were analyzed using PCR sequencing in the germline genomic DNA of 137 healthy persons.The expression of MSH6 protein was detected by Envision immunohistochemistry staining in the tumor tissues of the mutational probands.Results Six germline mutations of MSH6 gene were detected in 39 probands of Chinese HNPCC kindreds,and the mutations distributed in the exon 4,6,9 and 10.Four out of six mutations were missense mutation,one was nonsense mutation and the remaining one was insertion mutation in splice site.The results of sequecing for the exons with above four missense mutations in 137 healthy persons' genomic DNA showed that 5 of 137 persons had the missense mutation of c.3488 A>T at codon 1163 of the 6th exon.The mutational rate was approximately 3.65%(5/137),so the mutation could be a single nucleotide polymorphism(SNP).The remaining missense mutations were not found in any germline genomic DNA of 137 healthy persons.Positive expression of MSH6 protein had been identified in the tumor of the SNP proband while the tumors had negative MSH6 protein expression in the rest probands of germline mutation MSH6 gene.The types of mutations and their potential significance were determined by comparing the following databases:http://www.ncbi.nlm.nih.gov/,http://www.ensembl.org/homo-sapies,and http://www.insight-group.org.Five out of the six mutations had not been reported previously and they were new pathological mutations,the rest one was a new SNP.Conclusion Germline mutations of MSH6 gene may play an important role in Chinese HNPCC kindreds fulfilling different clinical criteria.It is necessary to analyze the germline mutations of MSH6 gene using sequencing to identify HNPCC families in the probands in which MSH2 and MLH1 mutation were excluded.