错配修复、微卫星不稳定性与胃癌

胃癌(gastric cancer，GC)是常见的恶性肿瘤，我国是胃病的高发地区，仅次于日本。发病率的不同不单单是地域环境的不同所引起的，还与其相关的分子学特点有关^[1]。现已发现的与胃癌密切相关的癌基因主要有ras、TPRmet、cerbB一2、β—catenln和Bcl-2基因等。     

错配修复基因与消化道肿瘤

错配修复是DNA复制后的一种修复机制，对维持基因组稳定方面起重要作用，错配修复系统主要由hMSH2,hMLH1,hPMS1,hPMS2组织，是遗传性非息肉性大肠癌（HNPCC）的遗传易感基因，与HNPCC及部分散发性肿瘤的发生相关，错配修复缺陷是继癌基因激活，抑癌基因失活之后的又一癌变机制，本就错配修复基因的结构，作用机理，与消化道肿瘤发生的关系及检测方法等作一综述。     

错配修复基因hMSH2启动子甲基化与胃癌的关系

目的:探讨hMSH2基因启动子区5CpG岛高甲基化在胃癌发生过程中的作用.方法:应用甲基化特异性PCR(methylationspecific PCR,MSP)方法检测胃癌及非癌组织中hMSH2基因启动子区甲基化状态.结果:40例胃癌中hMSH2基因启动子区高甲基化24例(60%),其癌旁黏膜组织中有15例(37.5%)发生甲基化,14例慢性萎缩性胃炎组织中有5例(35.7%)发生甲基化,6例慢性浅表性胃炎组织中未见甲基化.四组甲基化水平相比,差别有统计意义(P＜0.05).胃癌组甲基化水平高于癌旁组,差别有统计意义(P＜0.05).癌旁组、慢性萎缩性胃炎组、慢性浅表性胃炎组三组甲基化水平相比,差别无统计意义.胃癌各临床病理参数组之间相比差别无统计意义.结论:胃癌组织中hMSH2基因启动子区高甲基化可能是导致其错配修复功能缺陷的重要原因之一;而错配修复功能缺陷在胃癌的发生中起着重要作用,但可能与其发展关系不大.     

微卫星不稳定性与胃癌的发展

DNA错配修复(MMR)缺陷与许多癌症发生有关。细胞错配修复的缺陷会导致广泛的遗传突变和肿瘤的发生和发展。MMR缺陷肿瘤的一项重要的诊断学特征就是核苷酸重复区域的高突变率的累积,而这些突变就是微卫星不稳定性(MSI)。应用一种标准的标记物检测MSI 肿瘤,并可把肿瘤分为高度、低度MSI或微卫星稳定性(MSI-H)。MSI-H肿瘤特征性显示一种或多种DNAMMR重要蛋白的缺失,如MSH1或MSH2,以及比较少见的MSH6和MSH3。本文就近年来国内外关于MSI在胃癌发生发展的研究进展作一概述。     

错配修复基因hMSH2在散发性胰岛素瘤中的表达及临床意义

目的 探讨错配修复基因hMSH2在散发性胰岛素瘤发生中的作用,以及hMSH2表达能否作为胰岛素瘤良、恶性鉴别的标志物.方法 以取自50例散发性胰岛素瘤患者的55个肿瘤(良性40个,恶性15个)作为研究对象,用免疫组化方法检测hMSH2蛋白表达.提取显微切割组织DNA,用PCR-LOH方法测定hMSH2的杂合缺失.在3条染色体上选择6个微卫星位点,PCR法测定肿瘤组织有无微卫星不稳定(MSI).并与患者的临床、病理资料进行相关分析.结果 13%的散发性胰岛素瘤hMSH2蛋白表达下调.55个胰岛素瘤均未发生hMSH2基因杂合缺失.36%(20/55)的胰岛素瘤中发现高频MSI(MSI-H,即6个位点中至少2个位点发生(MSI).15个恶性肿瘤中有33%存在hMSH2表达下调,而40个良性肿瘤中hMSH2表达下调仅为5%(P=0.019).结论 错配修复基因hMSH2表达丢失或下降可能与胰岛素瘤的恶性程度相关.测定肿瘤中hMSH2表达下调可作为判断该肿瘤良、恶性的潜在标志物.     

错配修复基因hMLH1表达与胰腺癌临床病理特征的相关性

环境中的有害因素会导致DNA突变和错配，机体正常的DNA复制过程也会发生碱基错配。正常细胞具有纠正DNA复制错误的错配修复系统。hMLH1是DNA错配修复的控制基因。已有研究发现，错配修复基因hMLH1的改变与一部分肿瘤尤其是消化道肿瘤有密切关系。hMLH1基因表达是否与胰腺癌的发生及临床病理特征存在相关性，值得讨论。本研究通过检测胰腺癌错配修复基因hMLH1蛋白表达及启动子甲基化，探讨其与胰腺癌临床及病理特征的关系。     

人源DNA错配修复基因和微卫星不稳定与老年结肠癌的相关性

目的探讨老年结肠癌患者肿瘤组织的人源DNA错配修复基因(h MLH)1和微卫星不稳定性型情况。方法通过甲基化特异性聚合酶链反应(PCR)检测老年结肠癌样本中基因h MLH1启动子甲基化修饰的异常;研究结肠癌样本中微卫星标志物杂合型丢失频率;确定结肠癌患者中的变异与结肠癌的相关性。结果 18.0%的老年结肠癌样本中出现h MLH1启动子高甲基化;51.2%出现微卫星标记D22S280或D22S929的杂合型缺失,25.6%出现D22S280和D22S929的杂合型缺失,并且杂合型缺失与基因h MLH1启动子高甲基化状态相互独立。基因h MLH1启动子甲基化可作为独立的变量预测肿瘤的预后。结论根据h MLH1启动子甲基化状态对高甲基化状态结肠癌进行分类,基因h MLH1可作为微卫星不稳定型老年结肠癌潜在预后标志物之一。     

错配修复基因与消化道肿瘤

错配修复是DNA复制后的一种修复机制,对维持基因组稳定方面起重要作用。错配修复系统主要由hMSH_2、hMLH_1、hPMS_1、hPMS_2组成,是遗传性非息肉性大肠癌(HNPCC)的遗传易感基因,与HNPCC及部分散发性肿瘤的发生相关。错配修复缺陷是继癌基因激活、抑癌基因失活之后的又一癌变机制。本文就错配修复基因的结构、作用机理、与消化道肿瘤发生的关系及检测方法等作一综述。     

散发性大肠癌患者中错配修复基因胚系突变筛查策略

目的探讨在散发性大肠癌患者中筛查错配修复(MMR)基因胚系突变的可行性和策略。方法以150例散发性大肠癌患者为研究对象,以微卫星不稳定性(MSI)检测和免疫组化(IHC)检测作为初筛方法,对MSI-H表型或IHC检测MMR蛋白缺失的患者行hMLH1和hMSH2基因测序,检测MMR基因胚系突变。结果150例散发性大肠癌中MSI-H表型20例,IHC示22例MMR蛋白缺失。共发现3例MMR基因突变。MSI检测和IHC检测结果具有很好的一致性。结论散发性大肠癌患者中分子生物学筛查方法能够有效鉴别出MMR基因胚系突变。     

错配修复基因hMLH1甲基化与胃癌的关系

目的:通过研究散发性胃癌中错配修复基因hMLH1 mRNA的表达及其启动子区5'CpG岛甲基化状态,探讨hMLH1基因异常甲基化在胃癌发生过程中的作用.方法:应用甲基化特异性PCR(methylation specific PCR,MSP)检测60例胃癌组织及其癌旁黏膜组织中hMLH1基因启动子区的甲基化状态,逆转录PCR(RT-PCR)检测两种组织中hMLH1 mRNA表达情况.结果:胃癌组织hMLH1 mRNA表达水平明显低于癌旁组织(t=4.082,P<0.01),hMLH1基因启动子区高甲基化18例(30%),其癌旁黏膜组织中未发现有甲基化.hMLH1基因启动子区甲基化与胃癌的临床病理参数之间无明显的相关性.hMLH1 mRNA表达阴性的21例病例中,17例(81%)发生甲基化,而hMLH1 mRNA表达阳性的39例中仅有1例(2.5%)发生甲基化,hMLH1 mRNA表达降低与甲基化之间存在明显的相关性(χ2=8.0182,P=0.0046).结论:胃癌组织中hMLH1基因启动子区甲基化与其mRNA表达缺失密切相关,是导致hMLH1基因错配修复功能缺陷的重要原因之一.     

Relationship between grade of microsatellite instability and target genes of mismatch repair pathways in sporadic colorectal carcinoma

Microsatellite instability (MSI) induces carcinoma through the alteration of target genes; TGF- RII, BAX, IGFIIR, hMSH3, and hMSH6. The grade of MSI is classified into two categories according to the number of positive microsatellite markers; MSI-H and MSI-L. To elucidate the relationship between the propriety of grading MSI, target genes and clinical features of sporadic colorectal cancers, MSI and frameshift mutation of target genes were examined in 132 colorectal cancer patients. Fourteen and 12 cases showed MSI-H and MSI-L, respectively. Of the 14 MSI-H cases, 13 cases showed alteration of target genes. However, of the 12 MSI-L cases, only one case showed alteration of one target gene. Furthermore, all 14 MSI-H patients had cancers of the right colon and were typically older in age than the MSI-negative patients. These results imply a strong relationship between MSI-H and carcinogenesis by the frameshift mutation of target genes.     

High level of microsatellite instability but not hypermethylation of mismatch repair genes in therapy-related and secondary acute myeloid leukaemia and myelodysplastic syndrome

Microsatellite instability (MSI) is associated with defects in the DNA mismatch repair (MMR) system, such as mutation or epigenetic silencing of the genes by promoter hypermethylation. We investigated the presence of MSI and promoter hypermethylation of hMLH1 and hMSH2 genes in 82 patients (68 acute myeloid leukaemia, AML; 14 myelodysplastic syndromes, MDS). Twelve separate microsatellite loci, including three mononucleotide repeat markers, were used. Mutator phenotype (RER+) was detected in 20 AML (29.4%) and 3 MDS (21.4%) patients. RER+ rate was much higher in the therapy-related and secondary cases compared with the de novo cases. Three out of 7 (42.9%) secondary (s-AML) and 8 out of 17 (47.1%) therapy-related (t-AML) showed RER+ in comparison with 9 out of 44 (20.5%) de novo cases. Similar rates were detected in MDS patients (2/2 therapy-related and 1/12 de novo). The promoter hypermethylation was found in three hMLH1 (3.7%) and two hMSH2 (2.4%) genes. All these five patients had AML and were older than 60 years of age. Two of them had s-AML and one had t-AML. RER+ was detected in three of these five patients. Our data suggest that genetic instability is associated with AML and MDS, especially t-AML and s-AML. In addition, our results indicate that the hMSH2 and hMLH1 promoter hypermethylation is not a common event in these malignancies, but may play a role in the development of AML in elderly patients.     

Correlation between polymorphisms in DNA mismatch repair genes and the risk of primary hepatocellular carcinoma for the Han population in northern China

Abstract

Purpose: This study investigated correlations between polymorphisms in DNA mismatch repair (MMR) genes and the risk of primary hepatocellular carcinoma (PHC). Methods. Single nucleotide polymorphisms (SNPs) in the DNA MMR genes MLH3 (rs175080), PMS1 (rs5742933), PMS2 (rs1059060), MSH3 (rs26279), MSH5 (rs1150793, rs2075789) and MSH6 (rs1042821) were detected using the SNaPshot method in 250 PHC cases and in 308 patients without PHC in the Han population in northern China. Results. The AA genotype in MLH3 (rs175080) increased the risk of PHC (odds ratio [OR] = 3.424; 95% confidence interval [CI]: 1.097–10.689). The AG and GG genotypes in MSH3 (rs26279) increased the risk of PHC (OR: 1.644 and 3.300; 95% CI: 1.112–2.428 and 1.765–6.168, respectively). The AA genotype in MSH5 (rs2075789) increased the risk of PHC (OR: 9.229; 95% CI: 1.174–72.535). The CT genotype in MSH6 (rs1042821) reduced the risk of PHC (OR: 0.629; 95% CI: 0.428–0.924). Conclusions. Our study suggests that polymorphisms in MLH3 (rs175080), MSH3 (rs26279), MSH5 (rs2075789) and MSH6 (rs1042821) may be independent risk factors for PHC.     

DNA microsatellite instability and mismatch repair protein loss in adenomas presenting in hereditary non-polyposis colorectal cancer

BACKGROUND AND AIM: Hereditary non-polyposis colorectal cancer (HNPCC), as its name implies, is associated with few adenomas, and the early evolution of colorectal neoplasia is poorly understood. In this study our aim was to clarify the genetic profiles of benign polyps in subjects with HNPCC using a combined molecular and immunohistochemical approach. METHODS: Thirty adenomas and 17 hyperplastic polyps were obtained from 24 affected HNPCC subjects. DNA was extracted from paraffin embedded tissue by microdissection and analysed for the presence of microsatellite instability (MSI) and mutations in five genes known to be targets in mismatch repair deficiency (TGFbetaRII, IGF2R, BAX, hMSH3, and hMSH6). Serial sections were stained by immunohistochemistry for hMLH1 and hMSH2. RESULTS: Twenty four (80%) of 30 adenomas showed MSI. Of MSI positive adenomas, 66.7% showed MSI at more than 40% of markers (high level of MSI (MSI-H)). Two of 17 hyperplastic polyps revealed MSI at one marker (low level of MSI (MSI-L)). A significant association was found between MSI-H and high grade dysplasia in adenomas (p=0.004). Eight of nine adenomas with mutations of coding sequences revealed high grade dysplasia and all nine were MSI-H. Four of the nine ranged in size from 2 to 5 mm. The presence of the hMSH6 mutation was significantly correlated with high levels of MSI (80% of markers) (p<0.02). Twenty four adenomas gave evaluable results with immunohistochemistry. One of six (17%) microsatellite stable, six of seven (86%) MSI-L, and 11 of 11 (100%) MSI-H adenomas showed loss of either hMLH1 or hMSH2. CONCLUSIONS: Most adenomas in subjects with a definite diagnosis of HNPCC show MSI (80%). The finding of MSI-L is usually associated with loss of expression of hMLH1 or hMSH2, unlike the situation in MSI-L sporadic colorectal cancer. The transition from MSI-L to MSI-H correlated with the finding of high grade dysplasia and mutation of coding sequences and may be driven by mutation of secondary mutators such as hMSH3 and hMSH6. Advanced genetic changes may be present in adenomas of minute size.     

Choice of management strategy for colorectal cancer based on a diagnostic immunohistochemical test for defective mismatch repair

BACKGROUND: Despite intensive research into the molecular abnormalities associated with colorectal cancer (CRC), no diagnostic tests have emerged which usefully complement standard histopathological assessments. AIMS: To assess the feasibility of using immunohistochemistry to detect replication error (RER) positive CRCs and determine the incidence of RER positivity within distinct patient subgroups. METHODS: 502 CRCs were analysed for RER positivity (at least two markers affected) and/or expression of hMSH2 and hMLH1. RESULTS: There were 15/30 (50%) patients with metachronous CRCs, 16/51 (31%) with synchronous CRCs, 14/45 (31%) with a proximal colon carcinoma, and 4/23 (17%) who developed a CRC under the age of 50 showed RER positivity. However, 0/54 patients who developed a solitary carcinoma of the rectum/left colon over the age of 50 showed RER positivity. Immunohistochemical analysis revealed that 66/66 (100%) RER positive carcinomas were associated with complete lack of expression of either hMSH2 or hMLH1. This correlation was confirmed using a further 101 proximal colon carcinomas. Patients with a mismatch repair defective carcinoma showed improved survival but a 5.54 times relative risk of developing a metachronous CRC. A prospective immunohistochemical study revealed 13/117 (11%) patients had a mismatch repair defective carcinoma. A fivefold excess of hMLH1 defective cases was noted. CONCLUSIONS: All RER positive carcinomas were identified by the immunohistochemical test. This is the first simple laboratory test which can be performed routinely on all CRCs. It will provide a method for selecting patients who should be investigated for HNPCC, offered long term follow up, and who may not respond to standard chemotherapy regimens.     

Mutational analysis of the DNA mismatch repair gene hMLH1 in myeloid leukaemias

Mutations of the DNA mismatch repair (MMR) gene hMLH1 have recently been linked to the development of some hereditary and sporadic cancers which frequently display widespread microsatellite instability (MSI). Conflicting results regarding the extent of MSI in myeloid leukaemias prompted us to perform mutational analysis of all 19 exons of the hMLH1 gene by polymerase chain reaction-single-stranded conformation polymorphism (PCR-SSCP) and sequence analysis in a total of 133 patients with acute and chronic myeloid leukaemia. Apart from one exonic and one intronic polymorphism, no mutations were detected in any of the samples indicating that the major MMR gene hMLH1 is not involved in the pathogenesis or progression of myeloid malignancies.     

错配修复基因和骨保护素在胃癌中的表达及意义

目的 研究胃癌组织中错配修复基因MSH2和骨保护素(OPG)的表达、两者间的相关性及与胃癌生物学行为的关系.方法 制备组织芯片,应用原位杂交和免疫组化技术检测220例胃癌组织中MSH2和OPG的表达情况.结果 MSH2 mRNA、OPG mRNA、MSH2蛋白和OPG蛋白在胃癌组织中的阳性表达率分别为57.3%、56.4%、59.6%和55.9%.MSH2和OPG表达均与胃痛的TNM分期、浸润深度、脉管侵犯、淋巴结转移和远处转移呈正相关,与分化程度呈负相关.胃癌中MSH2和OPG的表达呈正相关.MSH2和OPG阳性表达病例的平均生存时间和5年生存率明显低于阴性者.结论 胃癌中MSH2和OPG的表达上凋,且两者呈正相关.MSH2和OPG的表达与胃癌的临床病理特征相关,两者的联合测定有利于胃癌诊断和判断预后.     

p53基因突变的霍奇金淋巴瘤R-S细胞错配修复基因蛋白的表达及意义

目的探讨错配修复基因在霍奇金淋巴瘤发生中的作用。方法采用免疫组化SP法检测霍奇金淋巴瘤标本中突变型p53蛋白及错配修复基因hMLH1、hMSH2蛋白表达情况。结果霍奇金淋巴瘤组织中R-S细胞hMSH2、hMLH1表达阳性率分别为50.00%、70.00%,未表现出错配修复基因hMSH2、hMLH1蛋白表达减低或缺失,突变型p53表达阳性率为100%。结论错配修复基因系统缺陷可能不是引发伴有p53基因突变的霍奇金淋巴瘤发生、发展的途径。