变性高效液相色谱法筛检hMLH1和hMSH2微小突变技术

目的:建立基于变性高效液相色谱法(DHPLC)的快速筛检错配修复基因hMLH1和hMSH2微小突变的技术平台。方法:自行设计PCR扩增hMLH1和hMSH2各外显子的引物,应用DHPLC检测26个遗传性非息肉病性结直肠癌(HNPCC)家系的先证者hMLH1和hMSH2种系微小突变,并与先前进行的DNA直接测序结果相比较。结果:hMLH1与hMSH2各外显子的PCR扩增引物,均能很好地扩增出相应的外显子及剪接区;DHPLC检出了所有已知突变,突变阳性筛检与阴性筛检的灵敏度和特异性均为100%;hMLH1的扩增子12A和hMSH2的扩增子2、3、7、5中相应外显子的剪接区跨越2个温度,而且相差较大(2.2-8.5℃):与DNA直接测序相比较,DHPLC具有快速、高效、低劳动强度、费用低、人为误差小、灵敏度和特异性高等优点。结论:基于DH- PLC的突变筛检平台,能够有效地筛检hMLH1和hMSH2微小突变,并具有较高的费用效率比。     

四种错配修复基因蛋白在结直肠癌中的表达及临床意义

目的:分析hMLH1、hMSH2、hMSH6和hPMS2四种错配修复基因蛋白在结直肠癌中的表达及其临床意义。方法:随机选取2013年1月至2015年12月广州医科大学附属第三医院结直肠癌患者标本177例,采用免疫组织化学法检测hMLH1、hMSH2、hMSH6和hPMS2蛋白的表达情况,并分析蛋白表达与临床参数间关系。结果:177例结直肠癌组织中,hMLH1蛋白的缺失率为6.2%(11/177),hMSH2蛋白的缺失率为4.0%(7/177),hMSH6蛋白的缺失率为1.7%(3/177),hPMS2蛋白的缺失率为8.0%(14/177),四者之和占所有结直肠癌病例的19.8%(35/177)。四种错配修复基因蛋白表达缺失均与肿瘤发生部位有关(P0.05),另外,hMLH1及hPMS2蛋白的表达缺失还与肿瘤分化程度相关(P0.05),hMSH6蛋白的表达缺失还与肿瘤浸润深度相关(P0.05);而缺失均与年龄、性别、淋巴结转移和远处转移无关(P0.05)。结论:错配修复蛋白的表达在部分结直肠癌组织中出现缺失现象,且与肿瘤部位及分化程度密切相关。hMLH1、hMSH2、hMSH6和hPMS2四种基因的突变,为临床判断预后及拟定治疗方案提供一个有参考价值的依据。     

错配修复系统hMSH2基因IVS12（-6）T〉C多态及饮食习惯与结直肠癌的易感性

目的：研究江苏省结直肠癌发病与hMSH2基因IVS12（-6）T〉C多态及饮食习惯的相关性。方法：对江苏省金坛、泰兴和淮安地区近2年来新发结直肠癌患者共计108例以及配对180例健康体检者的饮食等生活习惯因素进行调查,提取外周血DNA,应用PCR-DHPLC和DNA序列分析的方法,采用病例对照研究结合现场收集的流行病学资料统计分hMSH2基因IVS12（-6）T〉C与结直肠癌发病的关系。结果：hMSH2基因IVS12（-6）T〉C在家族性结直肠癌患者中检出率较高,且与散发性结直肠癌病例之间差异有统计学意义（P〈0.05）,在喜食油炸和腌渍食物组,hMSH2基因IVS12（-6）T〉CC突变与正常人群之间也存在明显的统计学差异（P〈0.05）。结论：hMSH2基因IVS12（-6）T〉C突变,通过遗传性和后天获得性等多重因素影响,使得该突变的携带者有更高的结直肠癌发病风险。     

脑胶质瘤MGMT、hMLH1和hMSH2基因启动子甲基化状态研究

目的：探讨脑胶质瘤患者O6-甲基鸟嘌呤-DNA甲基转移酶基因MGMT和错配修复基因hMLH1、hMSH2启动子CpG岛甲基化状态,及其在烷化剂化疗中的意义。方法：采用甲基化特异性PCR（MSP）方法检测39例脑胶质瘤和6例正常脑组织MGMT、hMLH1和hMSH2基因启动子区的甲基化状态,免疫组化方法测定蛋白表达。结果：脑胶质瘤患者组织MGMT、hMLH1和hMSH2基因启动子区甲基化发生率分别为46.2%、10.3%和20.5%,3种基因启动子未甲基化模式与其对应蛋白表达模式相似,并与患者性别、年龄、病理类型和病理分级无明显相关性。回顾性分析患者资料,显示39例脑胶质瘤患者中,MGMT基因甲基化的患者生存期显著高于MGMT基因未甲基化患者（P〈0.05,Log-rank检验）。结论：MGMT及错配修复基因甲基化是脑胶质瘤发生过程中常见的分子事件,可能与肿瘤的发生有关;检测MGMT、hMLH1和hMSH2基因启动子甲基化状态,在判断脑胶质瘤患者预后和预测烷化剂化疗耐药性中可能具有重要意义。     

错配修复系统hMSH2基因IVS12(-6)T>C多态及饮食习惯 与结直肠癌的易感性

目的:研究江苏省结直肠癌发病与hMSH2基因IVS12(-6)T>C多态及饮食习惯的相关性。方法:对江苏省金坛、泰兴和淮安地区近2年来新发结直肠癌患者共计108例以及配对180例健康体检者的饮食等生活习惯因素进行调查,提取外周血DNA,应用PCR-DHPLC和DNA序列分析的方法,采用病例对照研究结合现场收集的流行病学资料统计分hMSH2基因IVS12(-6)T>C与结直肠癌发病的关系。结果:hMSH2基因IVS12(-6)T>C在家族性结直肠癌患者中检出率较高,且与散发性结直肠癌病例之间差异有统计学意义(P<0.05),在喜食油炸和腌渍食物组,hMSH2基因IVS12(-6)T>CC突变与正常人群之间也存在明显的统计学差异(P<0.05)。结论:hMSH2基因IVS12(-6)T>C突变,通过遗传性和后天获得性等多重因素影响,使得该突变的携带者有更高的结直肠癌发病风险。     

食管鳞状细胞癌癌组织中hMLH1、hMSH2的表达及其意义

目的:探讨错配修复基因hMSH2、hMLH1在食管鳞癌组织中的表达及其临床意义。方法:采用sP免疫组化的方法检测144例食管鳞癌手术切除标本、30例正常粘膜组织中hMSH2、hMLH1表达情况,并与临床病理参数作相关分析。结果:食管鳞癌组织hMSH2、hMLH1基因阳性表达明显下降,与正常食道粘膜相比相差显著(P<0.01),hMSH2、hMLH1表达缺失与年龄、性别、肿瘤大小、淋巴结转移、浸润深度无显著相关,与肿瘤的分化程度有关(P<0.01)。结论:提示hMLH1和hMSH2基因突变所致的蛋白表达异常缺失与食管鳞状细胞癌发生有关,可能为一种早期事件。     

子宫内膜异位症中hMLH1、hMSH2基因启动子区的甲基化研究

目的：探讨错配修复基因1（hMLH1）、错配修复基因2（hMSH2）启动子区甲基化与子宫内膜异位症的关系。方法：采用甲基化特异性PCR方法检测23例子宫内膜异位症组织和20例正常子宫内膜中hMLH1、hMSH2基因启动子区的甲基化状态。结果：hMLH1基因在子宫内膜异位症中的甲基化率为39％（9/23）,在正常子宫内膜组织中的甲基化率为5％（1／20）,两者比较,差异有统计学意义（P〈0．05）;hMSH2基因在子宫内膜异位症中的甲基化率为8％（2／23）,在正常子宫内膜组织中的甲基化率为5％（1／20）,两者比较,无明显差异（P〉0．05）。结论：hMLH1基因启动子区甲基化可能与子宫内膜异位症发病有关;hMSH2基因启动子区甲基化与子宫内膜异位症之间不存在显著联系。     

脑胶质瘤患者组织和血清MGMT、hMLH1和hMSH2基因启动子甲基化状态检测

目的探讨脑胶质瘤患者组织和血清中MGMT、hMLH1和hMSH2基因启动子CpG岛甲基化发生率及相关性。方法甲基化特异性PCR（MSP）检测39例脑胶质瘤组织样本及32例预处理的脑胶质瘤血清样本中MGMT、hMLH1和hMSH2基因启动子区的甲基化状态。结果脑胶质瘤组织MGMT、hMLH1和hMSH2基因启动子区甲基化发生率分别为46．2％、10．3％和20．5％,肿瘤组织中至少有一种基因甲基化的发生率为64．1％（25／39）;在脑胶质瘤患者外周循环血液中检测到了相关基因甲基化系列,并且与组织中基因甲基化发生率明显相关。结论MGMT、hMLH1和hMSH2基因启动子甲基化是脑胶质瘤发生过程中常见的分子事件,血清中相关基因DNA甲基化检测有可能为脑胶质瘤诊断和个体化化疗提供一种稳定的无创性检测指标。     

涎腺粘液表皮样癌中hMLH1、hMSH2与p53基因的表达及意义

目的:探索hMLH1、hMSH2基因在涎腺粘液表皮样癌中的表达及其与p53表达的关系.方法:采用免疫组织化学S-P法对涎腺粘液表皮样癌及正常涎腺组织石蜡切片进行hMLH1、hMSH2及p53表达的检测.结果:(1)hMLH1、hMSH2及p53在癌组织中表达阳性率均显著高于正常组织(P＜0.01).(2)hMLH1、hMSH2的表达与癌的分化程度、患者年龄、性别、肿块大小、浸润深度以及有无淋巴结转移均无关(P＞0.05).(3)hMLH1、hMSH2基因均与p53表达呈正相关.结论:检测hMLH1、hMSH2、p53将有助于判断涎腺粘液表皮样癌的恶性程度和相关的生物学行为.     

错配修复蛋白hMSH2、hMLH1在涎腺粘液表皮样癌中的表达

目的:探讨人类错配修复基因2 the human mutS homolog 2(hMSH2)人类错配修复基因1human mutL homolog 1(hMLH1)在涎腺粘液表皮样癌(salivary gland mucoepidermoid carcinoma-SMEC)表达水平及临床病理意义。重点研究人类错配修复基因2和人类错配修复基因1与目标肿瘤发生的相关性。方法:采用HE染色方法筛选共计47例典型病例,采用免疫组织化学染色分析37例SMEC、10例正常组织涎腺中hMSH2、hMLH1的表达水平,结合计算机辅助高清晰图像分析处理技术做出综合评价。结果:hMLH1的表达与SMEC分化呈负相关(P0.05);hMLH1的低表达或表达缺失在低分化的SMEC中较普遍,在中分化和高分化中表达逐步增强;hMSH2的表达与SMEC分化不相关(P0.05)。结论:hMSH2、hMLH1异常表达与涎腺黏液表皮样的发生、演进存在相关性,以hMLH1、hMSH2为切入点为涎腺黏液表皮样癌治疗与预防提供参考依据。     

Use of SSCP analysis to identify germline mutations in HNPCC families fulfilling the Amsterdam criteria

Hereditary non-polyposis colorectal cancer (HNPCC) is a clinical syndrome characterised by an inherited predisposition to early onset colorectal and uterine cancers and an increased incidence of other cancers. It is caused by germline defects in the human mismatch repair genes. Defects in two of the known mismatch repair genes (namely hMSH2 and hMLH1) account for over 90% of mutations found in HNPCC families. In this study we have identified 14 families that fulfilled the clinical criteria for HNPCC and screened the hMSH2 and hMLH1 genes for germline mutations using single-strand conformational polymorphism (SSCP) analysis and DNA sequencing. Seven mutations were identified. Of these, there were five frameshifts, one missense mutation and a further novel mutation that involved separate transition and transversion changes in successive amino acid residues. Three of the mutations were in hMSH2 and four in hMLH1. The identification of germ-line mutations in an HNPCC family enables targeted surveillance and the possibility of early curative intervention. SSCP is a simple and effective method for identifying most mutations in the human mismatch repair genes using DNA from fresh, frozen or archival material. Received: 24 July 1996 / Revised: 26 September 1996     

Endometrial Cancer as a Familial Tumor: Pathology and Molecular Carcinogenesis (Review)

Some cases of endometrial cancer are associated with a familial tumor and are referred to as hereditary nonpolyposis colorectal cancer (HNPCC or Lynch syndrome). Such tumors are thought to be induced by germline mutation of the DNA mismatch repair (MMR) gene, but many aspects of the pathology of familial endometrial cancer are unclear and no effective screening method has been established. However, the pathology of endometrial cancer with familial tumor has been progressively clarified in recent studies. At present, about 0.5% of all cases of endometrial cancers meet the clinical diagnostic criteria for HNPCC. A recent analysis of the three MMR genes (hMLH1, hMSH2 and hMSH6) revealed germline mutations in 18 of 120 cases (15.0%) of endometrial cancer with familial accumulation of cancer or double cancer, with a frameshift mutation of the hMSH6 gene being the most common. Many cases with mutation did not meet the current clinical diagnostic criteria for HNPCC, indicating that familial endometrial cancer is often not diagnosed as HNPCC. The results suggest that the hMSH6 gene mutation may be important in carcinogenesis in endometrial cancer and germline mutations of the MMR gene may be more prevalent in cases associated with familial accumulation of cancer. An international large-scale muticenter study is required to obtain further information about the pathology of endometrial cancer as a familial tumor.Key Words: HNPCC, Endometrial cancer, DNA mismatch repair gene, hMLH1, hMSH6.     

Missense mutations in hMLH1 associated with colorectal cancer

One of the most prevalent hereditary syndromes associated with colorectal cancer is hereditary nonpolyposis colorectal cancer (HNPCC). The inherited gene defects in HNPCC have been shown to reside in DNA mismatch repair genes, mostly hMSH2 or hMLH1. Most HNPCC patients are heterozygous with regard to the relevant mismatch repair gene; they have one normal and one mutated allele, and mismatch repair in normal somatic cells is functional. Cancer predisposition in HNPCC is believed to be associated with the loss of the wild-type allele in somatic cells, resulting in defective DNA mismatch repair. This gives rise to DNA microsatellite instability (MSI), an increased somatic mutation rate, and eventually, to the accumulation of mutations in genes involved in colorectal carcinogenesis. In support of this theory, colorectal tumors in HNPCC patients and in mice deficient for hMSH2 or hMLH1 show MSI. Here, we describe two missense mutations in hMLH1 exon 16 associated with colorectal cancer. Interestingly, the tumors do not show MSI. This raises some potentially important issues. First, even microsatellite-negative colorectal tumors can be associated with germline mutations and these will be missed if an MSI test is used to select patients for mutation screening. Second, the lack of MSI in these cases suggests that the mechanism involved in carcinogenesis could be different from that generally hypothesized.     

Hereditary nonpolyposis colorectal cancer in 95 families: differences and similarities between mutation-positive and mutation-negative kindreds

Hereditary nonpolyposis colorectal cancer (HNPCC) describes the condition of a disparate group of families that have in common a predisposition to colorectal cancer in the absence of a premalignant phenotype. The genetic basis of this disease has been linked to mutations in genes associated with DNA mismatch repair. A large proportion of families harbor changes in one of two genes, hMSH2 and hMLH1. Approximately 35% of families in which the diagnosis is based on the Amsterdam criteria do not appear to harbor mutations in DNA-mismatch-repair genes. In this report we present data from a large series of families with HNPCC and indicate that there are subtle differences between families that harbor germline changes in hMSH2 and families that harbor hMLH1 mutations. Furthermore, there are differences between the mutation-positive group (hMSH2 and hMLH1 combined) of families and the mutation-negative group of families. The major findings identified in this study focus primarily on the extracolonic disease profile observed between the mutation-positive families and the mutation-negative families. Breast cancer was not significantly overrepresented in the hMSH2 mutation-positive group but was overrepresented in the hMLH1 mutation-positive group and in the mutation-negative group. Prostate cancer was not overrepresented in the mutation-positive groups but was overrepresented in the mutation-negative group. In age at diagnosis of colorectal cancer, there was no difference between the hMSH2 mutation-positive group and the hMLH1 mutation-positive group, but there was a significant difference between these two groups and the mutation-negative group.     

Hereditary nonpolyposis colorectal cancer families not complying with the Amsterdam criteria show extremely low frequency of mismatch-repair-gene mutations.

Hereditary nonpolyposis colorectal cancer (HNPCC) is a common autosomal dominant cancer-susceptibility condition characterized by early onset colorectal cancer. Germ-line mutations in one of four DNA mismatch repair (MMR) genes, hMSH2, hMLH1, hPMS1, or hPMS2, are known to cause HNPCC. Although many mutations in these genes have been found in HNPCC kindreds complying with the so-called Amsterdam criteria, little is known about the involvement of these genes in families not satisfying these criteria but showing clear-cut familial clustering of colorectal cancer and other cancers. Here, we applied denaturing gradient-gel electrophoresis to screen for hMSH2 and hMLH1 mutations in two sets of HNPCC families, one set comprising families strictly complying with the Amsterdam criteria and another set in which at least one of the criteria was not satisfied. Interestingly, hMSH2 and hMLH1 mutations were found in 49% of the kindreds fully complying with the Amsterdam criteria, whereas a disease-causing mutation could be identified in only 8% of the families in which the criteria were not satisfied fully. In correspondence with these findings, 4 of 6 colorectal tumors from patients belonging to kindreds meeting the criteria showed microsatellite instability, whereas only 3 of 11 tumors from the other set of families demonstrated this instability. Although the number of tumors included in the study admittedly is small, the frequencies of mutations in the MMR genes show obvious differences between the two clinical sets of families. These results also emphasize the practical importance of the Amsterdam criteria, which provide a valid clinical subdivision between families, on the basis of their chance of carrying an hMSH2 or an hMLH1 mutation, and which bear important consequences for genetic testing and counseling and for the management of colorectal cancer families.     

DNA mismatch repair defects: role in colorectal carcinogenesis

The inactivation of the DNA mismah repair (MMR) system, which is associated with the predisposition to the hereditary non-polyposis colorectal cancer (HNPCC), has also been documented in nearly 20% of the sporadic colorectal cancers. These tumors are characterized by a high frequency of microsatellite instability (MSI(+) phenotype), resulting from the accumulation of small insertions or deletions that frequently arise during replication of these short repeated sequences. A germline mutation of one of the two major MMR genes (hMSH2 or hMLH1) is found in half to two-thirds of the patients with HNPCC, whereas in sporadic cases hypermethylation of the hMLH1 promoter is the major cause of the MMR defect. Germline mutations in hMSH6 are rare and rather confer predisposition to late-onset familial colorectal cancer, and frequent extracolonic tumors. Yet, the genetic background of a number of HNPCC patients remains unexplained, indicating that other genes participate in MMR and play important roles in cancer susceptibility. The tumor-suppressor genes that are potential targets for the MSI-driven mutations because they contain hypermutable repeated sequences are likely to contribute to the etiology and tissue specificity of the MSI-associated carcinogenesis. Because the prognosis and the chemosensitivity of the MSI(+) colorectal tumors differ from those without instability, the determination of the MSI phenotype is expected to improve the clinical management of patients. This review gives an overview of various aspects of the biochemistry and genetics of the DNA mismah repair system, with particular emphasis in its role in colorectal carcinogenesis.     

Enhanced detection of deleterious and other germline mutations of hMSH2 and hMLH1 in Japanese hereditary nonpolyposis colorectal cancer kindreds

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal, dominantly inherited cancer-prone syndrome. Here, we describe a novel and efficient approach for screening mutations of two major HNPCC susceptibility genes, hMSH2 and hMLH1. The system consists of RNA extraction from whole blood treated with the translation inhibitor, followed by long RT-PCR of the entire coding regions combined with direct sequencing. In analysis of 15 kindreds suspicious for HNPCC, 8 samples were subjected to analysis after puromycin treatment and 7 samples were analyzed without puromycin treatment. Three deleterious mutations were detected in the kindreds with puromycin treatment, while none were observed in those without puromycin. Signals from mutated alleles were enhanced after puromycin treatment and easily distinguished from the wild-type allele, achieved by suppression of nonsense-mediated mRNA decay. Furthermore, 12 other mutations were detected in 15 kindreds. The system is considered to be a reliable and useful approach for detecting germline mutations of hMSH2 and hMLH1 in HNPCC kindreds.     

Mutation analysis of hMSH2 and hMLH1 in colorectal cancer patients in India

The aim of this work was to study the mutation profile in hMSH2 and hMLH1 genes in hereditary nonpolyposis colorectal cancer (HNPCC) patients in India. On the basis of the Bethesda criteria, 31 colorectal cancer patients were studied first for microsatellite instability, using the five markers recommended by the Bethesda guidelines. Twelve of 31 tumor samples were found to be MSI-H, 9 of 31 were MSI-L, and the rest were MSS. The 12 patients with MSI-H were analyzed for mutations in hMSH2 and hMLH1 genes using PCR-denaturing high-performance liquid chromatography (dHPLC), followed by sequencing of samples showing abnormal peaks. Of the five mutations detected, three were found to be deleterious mutations (hMSH2-R680X, hMLH1-E671X, and a splice junction mutation IVS16-2A --> G); one had a mutation of probable significance (hMLH1-C680G) and one was of unknown significance (hMSH2-R171K). This study has also shown that most of the early-onset colon (4/7) and early-onset rectal (15/21) cancers are MSS or MSI-L. This is the first study to describe the mutation in hMSH2 and hMLH1 in Indian patients, a low incidence region for colorectal cancer. A two-stage procedure using MSI testing followed by PCR-dHPLC was found to be an efficient method in studying the mutation profile in high-risk patients.     

Age at diagnosis of cancer as predictor of mutation occurrence in families suspected of HNPCC

Analysis of significance of age at cancer diagnosis as a factor allowing identification of a subgroup of patients with a high frequency of hMSH2 and hMLH1 mutations among families that fulfil suspected HNPCC criteria was performed. DNA from thirty-one unrelated patients affected by colorectal cancer from families matching the above criteria were studied by direct sequencing for occurrence of hMSH2 and hMLH1 gene mutations. Seven unequivocal constitutional mutations were detected: five in the hMLH1 gene and two in the hMSH2 gene. Additionally, one hMLH1 alteration of unknown significance was found. All seven mutations were found in a subgroup of 19 patients with cancer diagnosed before the age of 50 years. In a subgroup of 12 patients with cancer diagnosed at an older age only one case with hMLH1 alteration of unknown significance was detected. Our results indicate that early age at cancer diagnosis seems to be a crucial pedigree factor in discrimination of patients with hMSH2 or hMLH1 mutations among families suspected of HNPCC and matching criteria I of ICG-HNPCC.