遗传性非息肉性结直肠癌家系的MLH1基因两个胚系新突变

目的初步评价遗传性非息肉性结直肠癌（HNPCC）胚系MLH1基因突变中新突变的病理性。方法收集符合AmsterdamⅡ标准的12个不同家系的12例患者外周血,用特异引物和耐热性逆转录酶特异地逆转录MLH1的mRNA;利用长模板PCR扩增酶扩增逆转录产物（cDNA）;测序分析扩增产物;利用PCR-Genescan技术和免疫组织化学染色分别检测有新突变患者肿瘤组织的5个微卫星位点（BAT26,BAT25,D5S346,D2S123和Mfd15）和MLH1蛋白的表达。结果在4例患者中检出4个MLH1突变,其中2个突变为第12外显子的第384密码子（1151bp处）GTT→GAT的突变,该突变是已报道的病理性突变;另外2个突变分别是第8外显子的第217密码子（649bp处）CGC→TGC突变和第16外显子的第581密码子（1742bp处）CC→CTG突变;后两者为尚未报道的新突变。2个新突变的患者肿瘤组织均呈高度微卫星不稳定性,两者的瘤组织MLH1蛋白均失表达。结论MLH1第8外显子的第217密码子突变和第16外显子的第581密码子的两个新突变很可能为病理性突变。     

MLH1、MSH2基因 mRNA突变分析与遗传性非息肉性结直肠癌的基因诊断

目的检测胚系MLH1和MSH2基因mRNA突变，确立遗传性非息肉性结直肠癌（hereditary nonpolyposis colorectal cancer，HNPCC）家系。方法收集符合Amsterdam标准Ⅱ的12个家系14名家庭成员外周血，用特异引物和耐热性逆转录酶特异地逆转录MLH1和MSH2的RNA；利用长模板PCR扩增酶扩增逆转录产物（cDNA）；测序分析扩增产物。提取外周血的DNA，设计与利用上述方法检测出突变对应外显子的特异性引物，利用Taq DNA聚合酶扩增测序，以检测上述方法的有效性。结果利用基于外周血mRNA的方法，在6个家系中检出6个胚系突变，4个MLH1突变和2个MSH2突变，MLH1突变分别位于第8、12、16和第19外显子；MSH2突变分别位于第1和第2外显子。利用基于外周血DNA的方法，上述突变均在MLH1和MSH2相应的外显子中得到验证。突变类型为4个错义突变、1个同义突变和1个非编码区突变；其中5个突变国际上尚未报道；6个突变中有5个为病理性，分布于5个不同家系，该5个家系被确诊为HNPCC家系。结论基于外周血MLH1和MSH2 mRNA异常的检测能确诊HNPCC家系；该方法敏感、省时、节约成本。     

一个遗传性非息肉性结直肠癌大家系调查及种系突变研究

目的探讨一个中国人遗传性非息肉性结直肠癌（heraditary nonpolyposis colorectal cancer，HNPCC）大家系的临床特点，报告基因突变筛查结果。方法调查一个HNPCC大家系，记录的数据包括患者性别，结直肠癌发生的部位，诊断年龄，是否具有同时和（或）异时结直肠癌及结肠外癌，肿瘤的组织病理特点等。抽取家族成员外周血，采用聚合酶链反应和扩增产物直接测序进行基因检测。结果该家系符合阿姆斯特丹Ⅰ标准，4代31人中17例患者共诊断21例次恶性肿瘤。12例（70．6％）患者患有直肠癌，且发病年龄早（平均42．9岁），右半结肠癌多见。基因检测发现一种国内外尚未见报道的MSH2基因的新突变。该突变位于MSH2基因的第7外显子中，由于4个核苷酸（CCGA）的重复导致移码突变，形成截短蛋白。结论HNPCC患者是恶性肿瘤（尤其是结直肠癌）的高发人群。新的MSH2基因突变（MSH2：C．1215-1218dupCCGA）导致该家系遗传性非息肉性结直肠癌的发生。     

遗传性非息肉病性结直肠癌的分子生物学研究

目的：探索遗传性非息肉病性结直肠癌（HNPCC）基因突变规律。方法：用聚合酶链式反应和PCR-SSCP对9例HN-PCC患者及其家系成员4例政审对照的hMSH2、hHLH1基因进行检测。结果：9例来自不同家庭的患者中,7例出现电泳条带异常;4例来自上述家系的无症状成员,其中2例出现电泳条带异常。结论：聚合酶链式反应和PCR-SSCP联合应用,可用于 hMSH2、hMLH1基因突变的检测。     

中国人遗传性非息肉病性结直肠癌错配修复基因大片段缺失研究

目的了解中国人遗传性非息肉病性结直肠癌(hereditary nonpolyposis colorectal cancer．HNPCC)家系中MSH和MLH1基因大片段缺失情况及特点，以进一步完善中国人HNPCC家系遗传检测内容。方法取14个符合中国人HNPCC诊断标准的HNPCC家系肿瘤先证者外周血DNA，用荧光标记多重PCR技术结合GeneScan分析系统检测MSH2和MLH1基因大片段缺失。结果14例患者中有1例检测到MSH2基因第1～7外显子缺失，该家系另1例大肠癌患者和3个家系成员有同样的基因片段缺失。结论中国人HNPCC家系错配修复基因大片段缺失可能以MSH2比较常见。建议在中国人HNPCC家系遗传检测中常规包含错配修复基因大片段缺失检测。     

两个遗传性非患肉性结直肠癌家系中MLH1和MSH2基因的突变检测

目的 确定两个遗传性非息肉性结直肠癌(hereditary nonpolyposis colorectal cancer,HNPCC)家系的致病基因,选择MLH1基因和MSH2基因进行突变检测.方法 采用聚合酶链反应结合DNA直接测序法,对两个遗传性非息肉性结直肠癌家系的患者进行MLH1基因和MSH2基因的突变检测;发现变异后,采用PCR-限制性片段长度多态性或直接测序法鉴定此变异是否属于突变.结果 在家系A的患者中发现了位于MLH1基因第3外显子内的新突变c.243_244 insA;在家系B的患者中发现了MSH2基因第7外显子内的c.1215_1218dupCCGA突变,这两个突变都导致了编码蛋白的提前终止.结论 MLH1基因的c.243_244insA突变和MSH2基因的c.1215_1218dupCCGA突变分别是导致家系A和家系B发生遗传性非息肉性结直肠癌的致病突变.     

遗传性非息肉病性结直肠癌

遗传性非息肉病性结直肠癌 (HNPCC)是一常染色体显性遗传性疾病 ,肿瘤主要发生在右半结肠 ,同时性和异时性癌发生率高 ,并较常发生结肠外肿瘤。HNPCC的发生因于干细胞错配修复基因(MMR)的突变 ,目前为止 ,已克隆出 6种人类MMR ,其中 4种与HNPCC相关 ,并且MMR基因突变的内容(基因型 )与临床表现 (表型 )有联系。由于分子遗传学的进展 ,对HNPCC的诊断 ,建立了Bethesda指南。在癌发之前 ,基因检测使个体得到对癌的监控和处理方法的选择 ,对有癌风险的家庭成员作出较准确的判断 ,对癌患者 ,MMR检测能为外科手术方式的选择提供依据。     

典型遗传性非息肉病性结直肠癌家系临床病理及分子遗传学分析

目的 了解国人遗传性非息肉病性结直肠癌（HNPCC）的临床病理及分子遗传学特征。方法 用微解剖、微卫星不稳定性分析、免疫组织化学及直接DNA测序方法,检测4例HNPCC患者的肿瘤组织微卫星不稳定性状态、错配修复基因hMSH2及hMLH1蛋白水平的表达变化以及生殖细胞突变。结果 4例先证者5个肿瘤组织均表现为高度微卫星不稳定性,3例表现为hMSH2蛋白表达异常,1例表现为hMLH1蛋白表达异常。检测出3个生殖细胞病理性突变。结论 中国人典型HNPCC病例中错配修复基因突变率较高。高度微卫星不稳定性、错配修复基因hMSH2及hMLH1蛋白表达异常与错配修复基因生殖细胞突变密切相关。微卫星不稳定性和错配修复基因蛋白分析可作为DNA测序前的筛选手段。     

遗传性非息肉性大肠癌患者中hMLH1和hMSH2基因遗传性突变的分析

目的通过分析遗传性非息肉性大肠癌（ＨＮＰＣＣ）患者错配修复基因的遗传性突变，对患者的家族成员进行遗传咨询和症状前的基因诊断。方法用ＰＣＲ－异源双链体形成、ＰＣＲ－ＳＳＣＰ和ＤＮＡ序列分析技术，检测１４例ＨＮＰＣＣ、１０例有家族史大肠癌患者的外周血细胞ＤＮＡ，分析其错配修复基因ｈＭＬＨ１、ｈＭＳＨ２的所有３５个外显子。结果确认４／１４的ＨＮＰＣＣ、１／１０有家族史的大肠癌患者携带遗传性错配修复基因的突变，其中２例见于ｈＭＬＨ１基因，３例ｈＭＳＨ２基因。突变类型：３例由碱基缺失导致的移码突变，１例无义突变，１例错义突变。结论ＨＮＰＣＣ的发生与错配修复基因的突变密切相关；在大肠癌患者中检测遗传性错配修复基因的突变不宜仅限于严格符合临床诊断标准的ＨＮＰＣＣ患者。     

An appendix carcinoid tumor in a patient with hereditary nonpolyposis colorectal cancer

Gastrointestinal carcinoid tumors are often associated with other tumors, particularly colon adenocarcinomas; but the association between carcinoid tumors and hereditary nonpolyposis colorectal cancer (HNPCC) syndrome has not yet been explored. We report an unusual case of a 28-year-old woman with HNPCC who underwent surgery for a transverse colon adenocarcinoma in whom an appendix carcinoid tumor was incidentally found. To assess whether the carcinoid tumor displayed the characteristic molecular features of HNPCC tumors, we investigated the expression of mismatch-repair (MMR) proteins and microsatellite instability (MSI) status in both tumors. Both tumors demonstrated normal expression of the MMR proteins hMLH1, hMSH2, hMSH6, and hPMS2. Interestingly, the adenocarcinoma exhibited an MSI phenotype but the carcinoid tumor did not, indicating that these 2 tumors arose through different molecular pathways.     

Mononucleotide microsatellite instability and germline MSH6 mutation analysis in early onset colorectal cancer.

Germline mutations in the MSH2 and MLH1 mismatch repair genes account for most cases of hereditary non-polyposis colon cancer syndrome (HNPCC). In addition, germline MSH2 and MLH1 mutations have been detected in patients with non-HNPCC early onset colorectal cancer. Germline MSH6 mutations appear to be rare in classical HNPCC families, but their frequency in young colorectal cancer cases has not been studied previously. In a population based study of early onset colorectal cancer (<50 years) investigated for tumour microsatellite instability (MSI), we identified a subgroup of tumours with MSI for mono- but not dinucleotide repeat markers (m-MSI+ group). In contrast to tumours with classical MSI for dinucleotide markers (d-MSI+), the m-MSI+ group cancers were mainly left sided (6/7). As MSH6 mutations in yeast and human cell lines are associated with weak (and preferential mononucleotide) MSI, the complete MSH6 gene coding region was sequenced in blood DNA from the five m-MSI+ cases available for analysis. A germline nonsense mutation was identified in an isolated case of early onset colorectal cancer (age 43 years). These results support previous findings that germline MSH6 mutations may not be associated with classical MSI and suggest a role for germline MSH6 mutations in isolated early onset colorectal cancer.     

中国汉族和韩国朝鲜族非患肉性结直肠癌家系临床及遗传学表型的比较分析

目的 分析和比较汉族和朝鲜族遗传性非息肉性结直肠癌(hereditary nonpolyposis colorectal cancer,HNPCC)家系的临床及遗传学表型的异同点.方法 收集31个中国的汉族家系和63个韩国的朝鲜族家系先证者和家系成员的各项临床资料,对先证者外周血DNA进行相关基因hMLH和hMSH2的种系突变检测.应用聚合酶链反应-单链构象多态性分析或变性高效液相色谱法筛查突变,对结果异常的样本进行DNA测序.结果 31个汉族家系中共发生136例次恶性肿瘤,其中结直肠癌106例次,占所有肿瘤患者的77.9%,诊断年龄平均为(48.6±29.0)岁;其次为胃癌共14例.经突变检测,31例汉族先证者中有7例被检出含有hMLH1(3个)或hMSH2(4个)基因的病理性突变,总突变率为22.6%.其中错义突变2个、无义突变2个、移码突变2个、大片段缺失1个.63个朝鲜族家系中共发生293例次恶性肿瘤,其巾结直肠癌242例次,占所有肿瘤患者的82.6%,诊断年龄平均为(45.9±11.0)岁;胃癌同样也是第2大常见肿瘤类型,共发生21例.63例朝鲜族先证者中有19例被检测出含有hMLH1(17个)或hMSH2(2个)基因的突变,总突变率为30.2%.其中12个为移码突变,5个为错义突变,1个为无义突变,1个为剪接位点的碱基改变导致异常剪接.结论 (1)汉族与朝鲜族HNPCC家系在临床表现上相似,均有发病年龄轻、以远端结肠癌和直肠癌多见、多原发大肠癌发生率较两方国家低、肠外肿瘤以胃癌最多见等特点.(2)遗传表型方面,汉族与朝鲜族HNPCC家系的总突变率相似,但均低于西方国家的报道.两个种族的家系中的突变基因、突变类型和突变分布上存在差异及各自的特征.     

Medullary carcinoma of the pancreas in a man with hereditary nonpolyposis colorectal cancer due to a mutation of the MSH2 mismatch repair gene

Pancreatic adenocarcinoma has been reported in kindreds with hereditary nonpolyposis colorectal cancer (HNPCC). Medullary carcinoma of the pancreas is a recently described rare variant of pancreatic adenocarcinoma. We describe a man with colorectal carcinoma who subsequently developed pancreatic medullary carcinoma. The tumor displayed microsatellite instability and loss of expression of the mismatch repair proteins MSH2 and MSH6. Mutational analysis of the mismatch repair genes MLH1 and MSH2 demonstrated a pathogenic nonsense mutation within the MSH2 gene, which is consistent with a diagnosis of HNPCC. This report adds support to an association between HNPCC and pancreatic adenocarcinoma displaying the medullary phenotype, suggesting that medullary features in a pancreatic carcinoma may point toward a genetic cancer predisposition. To our knowledge, this is the first reported case of medullary carcinoma of the pancreas in a patient with HNPCC due to a mutation of the MSH2 gene.     

MLH1 and MSH2 protein immunohistochemistry is useful for detection of hereditary non-polyposis colorectal cancer in young patients

AIMS: Hereditary non-polyposis colorectal cancer is related to germline mutations of DNA mismatch repair genes MLH1 and MSH2, which result in microsatellite instability and loss of protein expression of the corresponding mutated gene in the tumour tissue. METHODS AND RESULTS: MLH1 and MSH2 protein expression was studied by immunohistochemistry in paraffin-embedded surgical samples of 100 colorectal adenocarcinomas occurring before 50 years of age. Absence of tumour cell nuclear staining with positive internal control (normal mucosa, lymphoid follicles) was considered negative. Loss of MLH1 or MSH2 expression was found in 20 cases with microsatellite instability in 15 cases. Twelve of these patients had a family history of colorectal cancer. Compared with MLH1- and MSH2-positive cases, MLH1- or MSH2-deficient colorectal adenocarcinomas were significantly associated on multivariate analysis with a younger age (38 vs. 43 years, P;0.0224), a larger tumour size (60 +/- 6 vs. 46 +/- 2 mm, P=0.0291), an expanding margin (85% vs. 51%, P=0.0159), a higher number of tumour-infiltrating lymphocytes assessed by CD3 immunostaining (202 +/- 48 vs. 33 +/- 4 CD3+ lymphocytes/10 high-power fields, P=0.0039), and a grade 2 Crohn's like lymphoid reaction (70% vs. 9%, P=0.0037). The two groups were not different for tumour site, differentiation, pTNM stage, vascular and perineural invasion, peripheral adenomatous residue, and 5-year survival rates. CONCLUSIONS: MLH1- or MSH2-deficient colorectal carcinomas of young patients exhibit pathological and molecular features similar to hereditary non-polyposis colorectal cancer. This suggests that MLH1 and MSH2 immunohistochemistry is valuable for detecting hereditary non-polyposis colorectal cancer in young patients.