【作者】 颜士岩；

【导师】 施达仁； 周晓燕； 杜祥； 陆洪芬； 平波；

【作者基本信息】 复旦大学 ， 肿瘤学， 2008， 博士

【摘要】 遗传性非息肉病性结直肠癌(Hereditary nonpolyposis colorectal cancer,HNPCC)是一种常染色体显性遗传性恶性肿瘤综合征,外显率高达80%-90%,占所有结直肠癌的2%-15%。HNPCC与散发性结直肠癌(Sporadic colorectalcancer,SCRC)相比,无论在发病机制、临床特征、治疗方案的选择和随访方案的制定上均不尽相同。为此,区分HNPCC与SCRC不仅具有重要的临床意义,而且有利于HNPCC家系成员的遗传学咨询。目前,世界许多国家和地区都制定了一定的HNPCC临床诊断标准,如Amsterdam标准、Amsterdam修改标准、日本标准及Bethesda指导纲要等。然而,要确诊HNPCC家系和HNPCC患者仅仅依靠上述临床诊断标准显然是不够的,HNPCC的确诊依赖于错配修复(Mismatchrepair,MMR)基因胚系病理性突变的检出。目前,国内仅有小家系hMSH2和hMLH1基因胚系突变的研究及少数临床病例报道,没有进行较大HNPCC家系的分子遗传学筛检,可能会遗漏很大一部分HNPCC的确诊。本研究组先前进行了50余个符合不同临床标准的HNPCC家系胚系hMSH2和hMLH1基因突变方面的研究,发现国人HNPCC患者hMSH2和hMLH1基因的胚系突变率约为31%,明显低于文献报道的80%-90%。是否有其它MMR基因的突变或MMR基因的其它异常形式导致了HNPCC的发生?目前国内尚未见这方面的研究报道。为此,对HNPCC家系深入开展hMSH6基因胚系突变检测,hMSH2、hMLH1、hMSH6和hPMS2基因大片段缺失检测及hMLH1基因胚系启动子甲基化研究,对于HNPCC家系全面的分子遗传学筛选,防止遗漏部分HNPCC家系的诊断具有重要意义。故本研究对上述几个部分分别报告如下:第一部分中国人HNPCC家系先证者hMSH6基因胚系突变的测序研究目的:1.分析无hMSH2和hMLH1基因胚系突变的中国人HNPCC患者hMSH6基因胚系突变情况;2.评价hMSH6基因胚系突变检测在HNPCC家系分子遗传学筛选中的作用。方法:收集经过hMSH2和hMLH1基因胚系突变检测无hMSH2和hMLH1突变的HNPCC家系39个,其中符合Amsterdam标准的家系11个、符合日本标准的家系11个及17个符合Bethesda指导纲要的家系,分别收集各HNPCC先证者外周血样本,提取胚系基因组DNA;利用PCR技术分别扩增hMSH6基因的10外显子(计18个片段);PCR产物纯化后进行DNA测序;对测序结果进行生物信息学分析;对检测到的错义突变采用137个正常人基因组DNA进行PCR扩增和测序来排除是否为单核苷酸多态性(Single Nucleotide Polymorphism,SNP);对检测到的hMSH6基因的所有SNP进行分析。结果:在39个符合不同临床标准且无hMSH2和hMLH1基因胚系突变的HNPCC先证者中,共检测到6个hMSH6基因的胚系突变。其中H14患者的hMSH6基因第6外显子c.3488A＞T的错义突变在健康人群中的发生率约3.6%(5/137),为未报道过的新发现的SNP。其余5个是尚未报道的新突变,其中1个为位于第4外显子密码子493处c.1477G＞T的无义突变、1个为位于第9内含子的插入突变即c.4001-9InsTTTT,可能导致第十外显子的剪接异常、3个为错义突变分别为第4外显子c.1403G＞A、第4外显子c.1996T＞C及第9外显子c.3851C＞T,其中c.1403G＞A的突变发生在符合Amsterdam标准的H3患者。hMSH6基因胚系突变率约为12.8%(5/39)。hMSH6基因的SNP主要分布在第1外显子c.116 G＞A、第3外显子c.458-52 T＞G及第5外显子c.3306 T＞A,分别占33.7%(31/92)、18.5%(17/92)、31.5%(29/92),该3个位点及形式的SNP可能是HNPCC家系hMSH6基因SNP发生热点。结论:1.在无hMSH2和hMLH1基因胚系突变的HNPCC先证者中,hMSH6基因的胚系突变率较低约为12.8%,但对该部分家系进行hMSH6基因胚系突变的检测可能是必要的,有利于防止部分HNPCC家系的漏诊。2.hMSH6基因的胚系突变可以发生在具有典型HNPCC临床特征的Amsterdam标准的患者。3.hMSH6基因第1外显子c.116 G＞A、第3外显子c.458-52 T＞G及第5外显子c.3306 T＞A,这三个位点的SNP可能是HNPCC患者hMSH6基因SNP发生热点,对于hMSH6基因的SNP研究具有重要意义。4.hMSH6基因第6外显子c.3488A＞T的错义突变是新发现的国际上未报道的新的SNP。第二部分遗传性非息肉病性结直肠癌hMSH6蛋白表达的研究目的:1.评价第一部分检测到的6个hMSH6基因胚系新突变的功能。2.评价hMSH6基因蛋白表达异常的MMR缺陷表型与另一种MMR缺陷表型MSI之间的关系。方法:收集符合不同临床诊断标准的50个HNPCC家系(含检测到hMSH6基因胚系突变的6个家系)先证者的肿瘤组织,采用Envision二步法检测各患者肿瘤组织hMSH6蛋白的表达;收集并整理上述50个HNPCC患者的先前hMSH2、hMLH1和hMSH6基因胚系突变检测结果、微卫星检测结果及6个有hMSH6基因胚系突变的HNPCC家系的临床资料。结合IHC和MSI结果综合分析6个hMSH6基因胚系突变的功能并评价hMSH6蛋白表达与MSI的关系。结果:1.50个HNPCC家系基本资料:50个HNPCC家系中,符合Amsterdam标准的家系24个(1个符合ACⅡ),13个符合日本标准,剩余13个HNPCC家系符合BG标准。其中有hMSH2基因胚系突变的7例,有hMLH1基因胚系突变的11例,有hMSH6基因胚系突变的6例。除7位HNPCC先证者肿瘤组织显示微卫星稳定(Microsatellite stability,MSS)外,其余家系的先证者肿瘤组织均为高频微卫星不稳定(High frequency microsatellite instability,MSI-H)。2.6个有hMSH6基因胚系突变家系的临床资料:6个家系中1个符合AC标准、2个符合JC标准、3个符合BG标准。共有肿瘤患者20个,平均最初发病年龄为42.8岁,有异时多原发癌者3例,其中H61患者33岁时患左半结肠癌、45岁时患子宫内膜癌和卵巢癌、51岁时患右半结肠癌。右半结肠癌4个约占36.4%(4/11),左半结肠癌7个约占63.6%(7/11)。3.IHC检测结果:50个先证者中共检测到12个hMSH6蛋白表达异常(5个为弱表达,7个为失表达),其中6个有hMSH6基因胚系突变的患者5个为hMSH6蛋白失表达、1个为阳性表达(证实该例hMSH6突变为新发现的SNP);5个hMSH6蛋白弱表达患者均为具有MSI-H表型并有hMSH2基因胚系突变;最后2个hMSH6蛋白失表达病例分别为H45和H47患者,H45患者具有MSI-H表型、hMLH1基因胚系突变并有hMSH6基因Exon1缺失(第三部分检测结果),H47患者具有MSS表型和hMLH1基因胚系突变。其余38个患者均为hMSH6蛋白阳性表达(32个具有MSI-H表型,6个具有MSS表型)。结论:1.检测到的6个hMSH6基因胚系新突变中,5个有hMSH6蛋白功能的改变,为病理性的;2.hMSH6蛋白弱表达表型可能与hMSH2基因的胚系突变有关;3.hMSH6蛋白表达缺陷可发生在具有MSS表型的HNPCC患者;4.hMSH6蛋白弱表达表型并不能预示hMSH6基因的突变,而失表达表型可能预示hMSH6基因的突变。第三部分应用MLPA-Array技术检测中国人HNPCC患者MMR基因的大片段缺失与扩增目的:1.建立一种新的、简便的快速检测HNPCC家系MMR基因的缺失和扩增等基因拷贝数变化的检测方法;2.运用MLPA-Array技术检测符合不同临床诊断标准的HNPCC患者hMSH2、hMLH1、hMSH6和hPMS2基因大片段缺失和扩增情况,全面了解MMR基因大片段缺失和扩增在HNPCC分子遗传学筛选中的作用。方法:收集80个符合不同临床诊断标准的HNPCC家系,其中符合Amsterdam标准的家系28个、符合JC标准的家系19个、符合Bethesda指导纲要的家系33个。收集各家系先证者外周血提取胚系基因组DNA。应用MLPA-Array技术检测各患者hMSH2、hMLH1、hMSH6和hPMS2基因的大片段改变情况,每个样本重复一次MLPA-Array,对MLPA-Array检测结果应用传统的MLPA技术进行验证保证结果的准确性。分析中国人HNPCC家系总体MMR基因大片段异常情况及各MMR基因的大片段异常频率。结果:在80个符合不同临床标准的HNPCC家系的先证中应用MLPA-Array技术检测平台共发现19个MMR基因大片段缺失的患者,异常率为23.75%(19/80),其中8个符合AC标准、3个符合JC标准、8个符合BG标准。检测到的19个MMR基因的大片段异常,均为大片段缺失、未见扩增现象,其中hMSH2基因的大片段缺失9个占47.4%(9/19)、hMLH1基因的大片段缺失7个占36.8%(7/19)、hMSH6基因的大片段缺失3个占15.8%(3/19)、hPMS2基因的大片段缺失0个,hMSH2和hMLH1基因的大片段缺失共16个约占84.2%(16/19)。应用传统的MLPA技术验证上述结果发现两者100%符合。结论:1.MLPA-Array技术是一种简便、快速和高通量检测MMR基因大片段异常的技术方法,可以应用于我国HNPCC的分子遗传学筛选工作;2.HNPCC患者MMR基因的大片段异常主要表现为大片段缺失;3.中国人HNPCC患者MMR基因大片段缺失发生率约为23.75%(19/80),主要分布在符合AC标准和BG标准的患者;4.HNPCC患者MMR基因的大片段缺失以hMSH2和hMLH1基因多见约占84.2%(16/19),其次为hMSH6基因的大片段缺失占15.8%(3/19),而未见hPMS2基因的大片段缺失;5.对中国人HNPCC家系进行hMSH2、hMLH1和hMSH6基因大片段缺失的检测对于HNPCC的分子遗传学筛选是必要的。第四部分中国人遗传性非息肉病性结直肠癌家系hMLH1基因启动子甲基化研究目的:1.检测hMLH1基因胚系启动子甲基化状态在中国人HNPCC家系患者中的发生状况:2.评价hMLH1基因胚系启动子甲基化检测在HNPCC分子遗传学筛选中的价值。方法:收集具有MMR基因缺陷表型MSI-H,且经过hMSH2、hMLH1和hMSH6基因突变检测无突变的符合不同临床诊断标准的HNPCC家系18个,同时收集无hMSH2、hMLH1和hMSH6基因胚系突变并具有MSS表型的HNPCC患者6例作比较研究。收集各家系先证者外周血提取胚系基因组DNA,设计hMLH1基因甲基化及非甲基化特异性的PCR引物,采用甲基化特异PCR(MethylationSpecific PCR,MSP)的方法分别扩增各检测样品的甲基化和非甲基化目的条带,根据两者的出现状况判定各样品的甲基化状态,同时采用克隆测序的方法验证PCR扩增结果。为了保证结果的可靠性,采用已确定有hMSH2基因胚系c.2228C＞A无义突变的HNPCC患者(H65)的基因组DNA作为hMLH1基因甲基化的阴性对照,采用国际上公认有hMLH1基甲基化的SW48结肠腺癌细胞株的DNA作为甲基化的阳性对照,以水代替DNA模板作空白对照。同时采用Envision二步法检测有hMLH1基因甲基化的患者肿瘤组织hMLH1蛋白的表达,判断甲基化状态对hMLH1蛋白表达的影响。结果:在18例无hMSH2、hMLH1和hMSH6基因胚系突变且具有MSI-H表型的HNPCC患者中有5例患者显示hMLH1基因甲基化,其中完全甲基化2例、部分甲基化3例;13例未甲基化患者中8例为符合JC标准和BG标准的HNPCC先证者、5例符合AC标准,部分甲基化患者均为符合AC标准的HNPCC先证者,而2例完全甲基化患者分别为H10和H29,其分别符合HNPCC临床诊断标准JC及AC。可见,在具有MSI-H表型而无hMSH2、hMLH1及hMSH6基因胚系突变的HNPCC患者中,符合AC标准的患者hMLH1基因甲基化异常率22.2%(4/18)明显高于JC组5.6%(1/18)。而在无hMSH2、hMLH1和hMSH6基因胚系突变并具有MSS表型的6例HNPCC患者中未发现甲基化病例,可见hMLH1基因的甲基化可能与微卫星表型有关。Envision二步法检测hMLH1蛋白表达结果显示:只有完全甲基化的两例患者肿瘤组织显示hMLH1蛋白失表达,而部分甲基化的病例肿瘤组织hMLH1蛋白表达正常,提示完全甲基化方可导致hMLH1基因的表达缺陷。结论:1.具有MSI-H表型且无hMSH2、hMLH1和hMSH6基因胚系突变的HNPCC患者hMLH1基因胚系启动子甲基化异常率约为27.8%(5/18),而完全甲基化仅占11.1%(2/18)。在所有HNPCC患者中的发生率可能更低;2.hMLH1基因甲基化与MMR基因的微卫星表型有关,具有MSI-H表型的患者易于发生hMLH1基因的甲基化;3.hMLH1基因的完全甲基化能影响hMLH1基因的表达,而部分甲基化不一定影响hMLH1基因的表达;4.hMLH1基因胚系甲基化检测可以作为HNPCC的分子遗传学筛选的一部分,但应对病例进行选择,可能不宜作为普遍筛检指标。更多还原

【Abstract】 Hereditary nonpolyposis colorectal cancer（HNPCC） is one of the most common autosomal dominantly inherited cancers syndrome,accounts for 2%-15%of all colorectal cancers and the penetrance reach up to 80%-90%.Compare with sporadic colorectal cancer（SCRC）,HNPCC shows its own characteristics associated with the molecular mechanism,clinical features,the methods for treatment,and management of HNPCC kindreds.Therefore,there are many benefits to differentiate HNPCC from SCRC,which interests not only in the clinic but also in genetic counseling of HNPCC kindreds.Now,many countries and territories have established the clinical diagnostic criteria for HNPCC,such as,Amsterdam criteria,Amsterdam revised criteria, Japanese criteria and Bethesda guidelines and so on.However,there are not enough to make a final diagnosis families and the patients of HNPCC only rely on the above clinical diagnostic criteria.It is a gold criteria to make a final diagnosis for HNPCC dependents on the detection of pathological mutations of mismatch repair genes.There are parts of studies in germline mutations of hMSH2 and hMLH1 genes and less clinical reports in our domestic now.Up to now,it has not been reported in the molecular genetics screening of large HNPCC families and it can not make a final diagnosis to tremendous portion of HNPCC kindreds.Previously study showed that the rate of germline mutations was account for 31%and significantly less than other studies（80%-90%） in hMSH2 and hMLH1 genes of our HNPCC families fulfilling different clinical criteria.There are less reports about the other mismatch repair genes germline muations and another type abnormalities of MMR genes in the genesis of HNPCC.Therefore,it is urgent to conduct a comprehensive study in the fields,such as,the detection of germline mutation in hMSH6 gene,the detections of large genomic deletions in hMSH2/hMLH1/hMSH6/hPMS2 genes and the study of methylation of hMLH1 promoter in HNPCC families fitting with different clinical criteria.It is very important for full-scal molecular genetics screening HNPCC kindreds and avoiding omission the early diagnosis of parts of HNPCC families.The current research project is comprised of the following four parts.Part 1 Study on the germline mutation of hMSH6 gene in Chinese hereditary nonpolyposis colorectal cancer pedigrees using PCR based sequencingObjectives:To analysis the germline mutation of hMSH6 gene in unrelated HNPCC probands fufilling different clinical criteria in which hMSH2 and hMLH1 mutations were excluded.To evaluate the detection of hMSH6 gene germline mutation in the molecular genetics screening of HNPCC families.Methods:The peripheral blood was collected from the probands of 39 HNPCC families fulfilling different clinical criteria in which hMSH2 and hMLH1 mutations were excluded.11 families fulfilled ACⅠand ACⅡ,11 additional families fulfilled JC and the remaining 17 kindreds fit BG.Genomic DNA was extracted following the manufacturer’s protocol.18 sets of primers were designed to amplify the entire coding region,including 10 exons and each splicing site of hMSH6.PCR products were purified and used as a template for direct DNA sequencing.The results of sequencing were bioinformatic analyzed by different bioanalysis software.To further investigate the pathological effects of detected missense mutations,we analyzed the same exons of hMSH6 gene using PCR-based sequencing in 137 healthy persons with no family history.To analysis the single nucleotide polymorphism（SNP） by detected in HNPCC families.Results:In 39 unrelated HNPCC probands fulfilling different clinical criteria in which hMSH2 and hMLH1 mutations were excluded,six germline mutation were found after SNP reported were excluded.The missense mutation in the case H14 at c.3488A＞T of exon6 of hMSH6 gene was also found in the persons of control,the rate was approximately 3.6%（5/137）,it was a new unreported SNP.The remaining five mutations were new unreported mutations.Of them,one showed 1477G＞T at exon 4,which resulted in glutamine at codon 493 to a stop codon,this nonsense mutation would lead to a truncated hMSH6 protein whose normal function could be lost.One showed TTTT insertion at c4001-9 in intron 9,which was most likely result in splicing defect.The other three missense mutations are at codon 468（CGT＞ CAT,Arg＞His）,codon 666（TCT＞CCT,Ser＞Pro） and codon 1284（ACG＞ATG,Thr＞Met）,respectively.Of them,the missense mutation at the codon 468 of exon 4.3 of hMSH6 gene occurred in the H3 proband fufilling the typical AC HNPCC family.The SNP of germline hMSH6 gene was mainly distributed the 1^st,3^rd and 5^th exons in the probands of Chinese HNPCC families,they are at c.116G＞A,c.458-52T＞G and c.3306T＞A of hMSH6 gene,respectively.The three site and type of SNP might be the hot spot in the genesis of SNP in hMSH6 gene of Chinese HNPCC families.Conclusions:The rate of hMSH6 gene germline mutation is less account for 12.8%in the probands of Chinese HNPCC familes without hMSH2 and hMLH1 germline mutations,Perhaps,it is necessary to detect the germline mutation of hMSH6 gene for the above HNPCC families and its benefit to avoiding omission the early diagnosis parts of HNPCC pedigrees.The germline mutation of hMSH6 gene can occur in the patient with the typical clinical HNPCC features fulfilling Amsterdam criteria.The SNP of germline hMSH6 gene is mainly distributed the 1^st,3^rd and 5^th exons in the probands of Chinese HNPCC families,they are at c.116G＞A,e.458-52T＞G and c.3306T＞A,respectively,it is very important to study the SNP of hMSH6 gene.The SNP of c.3488A＞T at the codon 1163 of 6^th exon is a new SNP unreported previously.Part 2 Study on the expression of hMSH6 protein in the probands of hereditary nonpolyposis colorectal cancer familiesObjectives:To evaluate whether the six novel germline mutations in hMSH6 gene are pathological.To evaluate the relationship between the abnormal expression of hMSH6 protein and the another defect-phenotype of MMR gene（microsatellite instability,MSI）.Methods:Fifty tumor tissues were collected from the probands of HNPCC families fulfilling different clinical criteria containing six HNPCC families with hMSH6 gene germline mutations.Immunohistochemical staining of hMSH6 protein was performed using Envision two-step method.The data of germline mutations in hMSH2,hMLH1 and hMSH6 genes and the results of microsatellite instability status were collected in forty HNPCC families,and the clinical data were also collected about six HNPCC kindreds with hMSH6 gene germline mutations.The function of the six novel germline mutations of hMSH6 gene were evaluated by means of combining the results of IHC and MSI with the clinical information from the different HNPCC families. The relationship was evaluated between the expreesion of hMSH6 protein and the status of microsatellite instability.Results:The basic information of forty HNPCC families:In fifty unrelated families, there are twenty four families fufilling Amsterdam criteria（AC） including one （ACⅡ）,13 additional families fulfilling Japanese criteria（JC） and the remaining 13 patients fitting Bethesda guidelines（BG）.Of them,seven germline mutation of hMSH2 gene,11 germline mutation of hMLH1 gene and six germline mutation of hMSH6 gene were detected.All the probands of HNPCC families showed High frequency microsatellite instability in tumor tissues except seven probands with MSS phenotype.The clinical data about six HNPCC families with hMSH6 gene germline mutations:In six families,there were one proband fulfilling Amsterdam criteria,2 additional families fulfilling Japanese criteria and the remaining 3 patients fitting Bethesda guidelines.There were 20 persons to suffering HNPCC and concerning tumors,the average age of first onset CRC was 42.8 years old.Metachronous multiple CRCs occurred in 3 patients.The ease of H61 suffered from a left hemicolon cancer at age 33,had endometrial and ovarian cancer at age 45,and had a right hemicolon cancer at age 51.Four persons suffered from right hemicolon cancer account for 36.4%（4/11）,and 7 ones suffered from left hemicolon cancer account for 63.6%（7/11）. Results of IHC:Twelve abnormal expressions of hMSH6 protein were detected in 50 probands of HNPCC families including 5 weak expression and 7 lack expression.In 6 probands’ tumor tissures with hMSH6 gene germline mutation,5 patiens displayed the lack of hMSH6 protein expression,one displayed positive expression of hMSH6 protein（the proband with the mutation of hMSH6 gene was confirmed as a new unreported SNP）.Five patients with weak expression of hMSH6 protein displayed MSI-H phenotype and germline mutations in hMSH2 gene,and the remaining 2 patients with lack expression of hMSH6 protein were the cases of H45 and H47.The H45 case displayed MSI-H phenotype,germline mutation of hMLH1 gene and the deletion of Exonl in hMSH6 gene（See part3）,the H47 case displayed MSS phenotype and germline mutation of hMLH1 gene.The remaining 38 patients also displayed the positive expression of hMSH6 protein（Containing 32 patients with MSI-H phenotype and 6 persons with MSS phenotype）.Conclusions:Five out of six germline mutations in hMSH6 gene are preliminarily considered to be pathological ones owing to display abnoraml expression of hMSH6 protein.The phenotype with weak expression of hMSH6 protein can concern with the germline mutation of hMSH2 gene.The defect expression of hMSH6 protein can occur in the patients of HNPCC families with MSS phenotype.The weak expression of hMSH6 protein has not prognosticate the germline mutation of hMSH6 gene,but lack expression of hMSH6 protein may be prognosticate the germline mutation of hMSH6 gene.Part 3 Detection of large genomic deletions and duplications of mismatch repair genes in Chinese HNPCC patients using MLPA-ArrayObjectives:To set up a new convenient molecular method to screening the copy number changes and its clinical application for the rapid screening the deletions and duplications of MMR genes in HNPCC families.Detection of large genomic deletions and duplications in hMSH2,hMLH1,hMSH6 and hPMS2 genes of HNPCC patients fulfilling different clinical criteria using MLPA-Array method,and comprehensive its role in the molecular genetics screening HNPCC families.Methods:The peripheral blood was collected from the probands of 80 HNPCC families fulfilling different clinical criteria,28 families fulfilled ACⅠand ACⅡ,19 additional families fulfilled JC and the remaining 33 kindreds fit BG.Genomic DNA was extracted following the manufacturer’s protocol.The large genomic changes in hMSH2,hMLH1,hMSH6 and hPMS2 genes were detected by using MLPA-Array, every samples were tested again by the same method.The results of MLPA-Array detection were confirmed by the classic multiplex ligation-dependent probe amplification（MLPA）.Analysis the whole large genomic changes of MMR genes and abnormal frequency of large genomic changes in mismatch repair genes.in Chinese HNPCC familiesResults:Nineteen patients with large genomic deletions of MMR genes were found in the probands of HNPCC families fulfilling different clinical criteria by using MLPA-Array platform.The abnormal rate accouted for 23.75%（19/80）.Of them,8 patients fulfilling Amsterdam criteria,3 additional patients fulfilling Japanese criteria and the remaining 8 persons fitting Bethesda guidelines.Nineteen large genomic changes of mismatch repair genes were detected in 80 probands of HNPCC kindreds, they were all large genomic deletions not duplications.Among them,9 large genomic deletions were found in hMSH2 gene and they account for up to 47.4%（9/19）,7 large genomic deletions were found in hMLH1 gene and they account for 36.8%（7/19） and the remaining 3 ones were hMSH6 gene which only account for 15.8%（3/19）. However,large genomic deletions in hPMS2 gene were not found in all the probands of Chinese HNPCC families.Sixteen large genomic deletions of hMSH2 and hMLH1 genes were found（84.2%,16/19） in 80 HNPCC probands.The above results detected by MLPA-Array were confirmed by the classic MLPA analysis with 100% concordanceConclusions:MLPA-Array is a new convenient high-flux molecular method to rapid screening the deletions and duplications of MMR genes and it can apply for the molecular genetics screening of Chinese HNPCC families.The major type of large genomic changes is the deletion of MMR genes in the patients of HNPCC kindreds. The rate of large genomic deletions in MMR genes is approximately account for 23.75%（19/80）,they are main distribute in the patients fulfilling Amsterdam criteria and Bethesda guidelines.The large genomic deletions of hMSH2 and hMLH1 genes are more common changes and they accout for 84.2%（16/19）,the large deletions of hMSH6 gene is only account for 15.8%（3/19） and the large deletion of hPMS2 gene is not found.Therefore,it is necessary to detect the large genomic deletions in hMSH2, hMLH1 and hMSH6 genes for Chinese HNPCC families and it is very important to screening HNPCC kindreds.Part 4 Study on the methylation of hMLH1 gene promoter in the probands of Chinese hereditary nonpolyposis colorectal cancerObjectives:To detect the methylation of hMLH1 gene promoter in the patients of Chinese hereditary nonpolyposis colorectal cancer.To evaluate the role between the detection of methylation in hMLH1 gene promoter and the molecular genetics screening for HNPCC.Methods:Eighteen HNPCC families with the defect-phenotype of MMR gene（MSI-H） and without the germline muations in hMSH2,hMLH1 and hMSH6 genes were collected by us.At the same time,we collected 6 HNPCC kindreds with MSS phenotype and without germline mutations in hMSH2,hMLH1 and hMSH6 genes as the comparative group.The peripheral blood was collected from the probands of 24 HNPCC families fulfilling different clinical criteria.Genomic DNA was extracted following the manufacturer’s protocol.Design the specific primers for methylation specific PCR of hMLH1 gene.The specific straps of methylation and unmethylation of hMLH1 gene were amplified by using MSP in every samples.The status of methylation in hMLH1 gene was evaluated according to specific straps of methylation and unmethylation of hMLH1 gene.The results of MSP were confirmed by using clone sequencing of PCR products.To ensure the reliability of the result,the DNA of H65 case with the nonsense mutation at c.2228C＞A in germline hMSH2 gene was used as negative control of methylation in hMLH1 gene,the DNA of cell lines sw48 was used as known positive control in the methylation of hMLH1 gene and the water as DNA was used blank control.Immunohistochemical staining of hMLH1 protein was performed by using Envision two-step method in the tumor tissues of patients with hMLH1 gene methylation.To judge whether the status of methylation of hMLH1 gene effect the expression of hMLH1 protein.Results:Five hMLH1 gene methylation was found in the patients with MSI-H phenotype and without germline mutations in hMSH2,hMLH1 and hMSH6 genes in Chinese HNPCC families.Of them,2 patients displayed exhaustive-methylation and the remaining 3 patients displayed part-methylation.Eight out of 13 patients with unmethylation phenotype fulfilling Japanese criteria and Bethesda guidelines,only five patient fitting Amsterdam criteria.All the patients with part-methylation phenotype fulfilling Amsterdam criteria.The patients of cases H10 and H29 displayed exhaustive-methylation of hMLH1 gene,they fulfilling Japanese criteria and Amsterdam criteria,respectively.The rate of abnor-methylation in hMLH1 gene in AC group（22.2%,4/18） was higher than its in JC groups（5.6%,1/18） in the HNPCC families with MSI-H phenotype and without germline mutations of hMSH2,hMLH1 and hMSH6 genes.However,no patients with methylation of hMLH1 gene was found in the HNPCC families with MSS phenotype and without germline mutations of hMSH2,hMLH1 and hMSH6 genes.Therefore,we believed that the methylation of hMLH1 gene might be relationship with microsatellite phenotype.Results of IHC by using Envision two-step method:The tumor tissues of two patients with exhaustive-methylation phenotype displayed lack expression of hMLH1 protein, however,the tumor tissues of the patients with part-methylation phenotype displayed positive expression of hMLH1 protein.So,we could conclude that exhaustive-methylation of hMLH1 gene can cause the defect-expression of hMLH1 protein.Conclusions:The rate of abnor-methylation of hMLH1 gene is approximately 27.8%（5/18） in the patients with MSI-H phenotype and without germline mutations of hMSH2,hMLH1 and hMSH6 genes of Chinese HNPCC families,however, exhaustive-methylation rate is only accout for 11.1%（2/18）.Perhaps,the changes may be much lower in all the patients of HNPCC families.The methylation phenotype of hMLH1 gone concerns with microsatellite phenotype of MMR gone,the patient with MSI-H phenotype is easy to genesis the methylation of hMLH1 gene.The exhaustivemethylation of hMLH1 gene can effect the expression of hMLH1 protein,however, the part-methylation of hMLH1 gene has no effect with the expression of hMLH1 protein.hMLH1 promoter methylation analysis is a promising tool to molecular genetics screening for HNPCC but is not yet used in daily screening and diagnostics.更多还原