hMLH1/hMSH2基因启动子变异在遗传性非息肉病性结直肠癌发生中的作用

目的 探讨hMLH1及hMSH2基因启动子变异在遗传性非息肉病性结直肠癌(HNPCC)发生中的作用。方法 PCR法扩增25例HNPCC患、20例散发性结直肠癌和10例非肿瘤患的hMLH1及hMSH2基因启动子序列，对PCR产物进行测序。对发现携带突变患的肿瘤标本进行hMLH1及hMSH2基因表达研究和微卫星不稳定(MSI)检测，同时用测序方法检测该患hMLH1及hMSH2基因编码序列的改变。结果55例检测样本中，C3．1及C3．2发现hMLH1启动子在-342位和-337位2处A插入变异，免疫组化检测该患hMLH1表达阴性，肿瘤MSI检测为MSI—H，而编码序列的检测未发现异常。结论 hMLH1／hMSH2基因启动子种系突变可能导致该基因的不表达，从而导致HNPCC结直肠癌的发生。错配修复基因编码序列正常的HNPCC患中可能存在该基因启动子突变。     

膀胱移行细胞癌中DNA错配修复基因hMLH1、hMSH2和hMSH3启动子区甲基化状态研究

目的探讨DNA错配修复基因启动子区CpG岛甲基化状态及其在膀胱移行细胞癌（BTCC）中的表达。方法应用MSP技术检测膀BTCC中hMLH1、hMSH2和hMSH3基因启动子区甲基化状态,RT-PCR法检测其mRNA表达水平。结果36例BTCC组织中hMSH1、hMSH2的甲基化阳性率分别为38.9％（14／36）、52.8％（19／36）,所有标本中均未发现有hMSH3启动子甲基化。hMLH1、hMSH2、hMSH3 mRNA在BTCC中表达率分别为47.2％（17／36）、38.9％（14／36）、16.7％（6／36）,在正常组织中分别为22.2％（8／36）、63.9％（23／36）、0％（0／36）,差异均有统计学意义（P〈0.01）,并且随肿瘤病理分级的增加呈下降趋势（P〈0.05）,均与临床分期不相关（P〉0.05）。结论hMLH1、hMSH2和hMSH3基因启动子异常甲基化可能导致该基因转录表达失活,使其mRNA表达减少甚至缺失,这可能是导致BTCC发生、发展的原因之一。     

中国人遗传性非息肉病性结直肠癌hMLH1与hMSH2基因的种系突变研究

目的 探讨中国人遗传性非息肉病性结直肠癌（HNPCC）患hMLH1与hMSH2基因的种系突变特点。方法 对诊治的6个HNPCC家系的先证用聚合边反应（PCR）的方法扩增其hMHL1及hMSH2的35个外显子，SSCP方法检测其变异，对可疑突变进行自动测序以确定突变类型。结果 6例先证SSCP检测在3例患中发现4处异常条带，自动测序证实hMSH211外显子有1处插入突变和1处错义突变、13外显子有1处错义突变，1例在hMLH118外显子及hMSH215外显子均有插入突变。结论 中国人HNPCC的错配修复基因突变以hMSH2为主，插入造成的移码突变和错义突变仍为主要病理突变类型。     

结直肠癌环氧化酶-2、错配修复酶-1微卫星不稳定与基因调控的关系

目的 检测临床手术切除42例结直肠癌及相应正常组织环氧化酶-2(COX-2)、错配修复酶-1(hMLH1)微卫星不稳定状态(MSI)及二种基因启动子甲基化,探讨它们与结直肠癌发生的关系.方法 采用聚合酶链反应(PCR)技术检测5个位点的MSI状态;甲基化特异性PCR(MSP)方法检测结直肠癌及正常组织COX-2、hMLH1二种基因启动子CpG岛甲基化状态.结果 42例结直肠癌中MSI总检出率为42.86%(18/42),5个位点的MSI检出率差异无统计学意义(P＞0.05).COX-2和hMLH1基因启动子CpG岛甲基化在42例结直肠癌中分别有13例和15例.MSI-H组中COX-2基因启动子CpG岛甲基化8例,且有6例结直肠癌同时出现hMLH1和COX-2基因启动子CpG岛甲基化.结论 MSI结直肠癌hMLH1基因启动子CpG岛甲基化率高于MSS结直肠癌,hMLH1基因启动子CpG岛甲基化有助于判断肿瘤类型.     

脑胶质瘤中hMLH1、hMSH2基因启动子区的甲基化状态研究

目的 检测脑胶质瘤中hMLH1、hMSH2基因启动子区的甲基化状态,探讨其在肿瘤发生中的作用.方法 采用甲基化特异性聚合酶链反应(MSP)方法,检测51例脑胶质瘤标本及人脑胶质瘤U251细胞株的hMLH1、hMSH2基因启动子C_PG岛甲基化状态.结果 在7例正常脑组织中,用MSP法检测的hMLH1、hMSH2基因启动子区均未发现甲基化,51例脑胶质瘤组织中,hMLH1、hMSH2甲基化率分别为13.7%(7/51)、35.3%(18/51),且为甲基化和非甲基化共存,人脑胶质瘤U251细胞中未发生hMLH1启动子甲基化,而发生了hMSI-12启动子甲基化,hMLH1、hMSH2基因启动子区甲基化与性别、年龄、病理类型无明显相关.但hMLH1在低级别(Ⅰ～Ⅱ级)患者标本中甲基化率为0.0%(0/25),高级别(Ⅲ～Ⅳ级)患者标本中甲基化率为26.9%(7/26),差异有统计学意义(χ~2=5.69,P<0.05).结论 hMLH1、hMSH2基因启动子区甲基化状态与性别、年龄、病理类型无明显相关.hMSH2基因启动子区的CpG岛在脑胶质瘤中有中等频率的甲基化,是脑胶质瘤发生过程中的早期事件,可能与脑胶质瘤的发生相关;脑胶质瘤中hMLH1基因启动子区甲基化率较低,但hMLH1启动子区甲基化状态与病理分级有关;人脑胶质瘤U251细胞株中hMSH2基因启动子C_PG岛高甲基化.     

膀胱移行细胞癌错配修复基因hMLH1、hMSH2、hMSH3启动子甲基化状态的研究

目的 探讨错配修复基因hMLH1、hMSH2、hMSH3在膀胱移行细胞癌中启动子甲基化和蛋白表达的相关性.方法 采用甲基化特异聚合酶链反应(MSP)检测正常膀胱上皮组织(13例)及膀胱移行细胞癌组织(57例)hMLH1、hMSH2、hMSH3启动子甲基化状态,免疫组织化学检测其蛋白表达.结果 在正常膀胱上皮中,hMLH1、hMSH2、hMSH3均未发现启动子甲基化;在肿瘤组织中,hMSH2未发现启动子甲基化,hMLH1、hMSH3启动子甲基化阳性率分别为36.8%、49.1%,与正常组织比较差异均有统计学意义(P＜0.01),但在不同肿瘤分级(G1、G2、G3)间及不同临床分期(浅表性、浸润性)间比较差异无统计学意义(P＞0.05).免疫组织化学显示:hMLH1、hMSH2、hMSH3蛋白表达在正常膀胱上皮组织中,阳性表达率分别为100%、100%、92.3%,与肿瘤组织阳性表达率分别为59.6%、52.6%、40.4%,两者比较差异有统计学意义(P＜0.01),但在不同临床分期(浅表性、浸润性)间比较差异无统计学意义(P＞0.05),且hMSH2、hMSH3蛋白阳性表达率随肿瘤分级的增加显著降低,G1与G3比较差异有统计学意义(P＜0.05).在肿瘤组织中,hMLH1和hMSH3启动子甲基化与蛋白表达具有极显著相关性(P＜0.01).结论 hMLH1、hMSH3蛋白表达受其启动子甲基化的调控,hMSH2蛋白表达不受其启动子甲基化的调控.错配修复基因hMLH1、hMSH2、hMSH3蛋白表达的缺失可能参与了膀胱移行细胞癌的发生.     

典型和非典型遗传性非息肉病性大肠癌的研究

目的：研究中国人遗传非息肉病性大肠癌（HNPCC）的临床、病理及其hMLH1和hMSH2基因种系突变的特点。方法：随访13个典型HNPCC家系54例患者,并与19个非典型HNPCC家系的38例患者进行比较。对典型和非典型HNPCC各6个家系的先证者进行了hMLH1和hMSH2基因的PCR-SSCP检测,对异常者进行测序确定突变类型。结果：典型HNPCC盲肠、升结肠肿瘤占39.7％,横结肠肝区癌为5.0％;非典型组直肠癌占65.8％,2组差异无显著性意义。异时性多原发癌为11.5％,典型HNPCC患者的3、5、10年的生存率分别为64.0％、45.3％和31.2％,而非典型HNPCC组分别为54.4％、42.3％和26.8％,2组差异无显著性意义。12例先证者中,PCR-SSCP共检测到MLH111外显子（c3、c1）、hMLH1 12外显子（c8）、hMLH1 18外显子（c4）、hMSH2 11外显子（c13）、hMSH2 1外显子（c6）,hMSH2 13外显子（c11）和hMSH2 15外显子（c4）的异常条带。除MLH1 11外显子（c3）为内含子多态性外,6例（50％）发现7个外显子的突变,测序证实错义突变4处、插入突变7外,无义突变1处。结论：中国人的典型HNPCC是一种发病年龄早、近段结肠多见预后较好的大肠癌,而通过比较,发现中国非典型HNPCC与典型HNPCC非常相似。中国人的典型HNPCC家系的错配修复基因突变hMSH2多见,而非典型的hMLH1突变多见。     

散发性结直肠癌的hMSH3和hMSH6基因突变

目的：研究错配修复基因hMSH3和hMSH6在散发性结直肠癌发生中的作用。方法：应用放射性同位素为基础的PCR技术，检测了48例散发性结直肠癌和20例癌旁组织中hMSH3和hMSH6基因的突变。结果hMSH3和hMSH6在散发性结直肠癌中的突变率分别为5／48（10．4％）和12／48（25％），在癌旁组织中未见突变。结论：错配修复基因hMSH3和hMSH6参与了部发散发性结直肠癌的发生过程。     

胰腺癌微卫星不稳定性、hMLH1启动子甲基化及其蛋白表达研究

目的探讨胰腺癌中微卫星不稳定性（MSI）与hMLH1启动子甲基化及蛋白表达之间的联系，揭示胰腺癌发生的分子机制。方法从35例胰腺癌患者的正常胰腺组织、癌组织中提取DNA；SSCP法检测标本中微卫星不稳定性发生情况；免疫组织化学法检测错配修复基因hMLH1在胰腺癌中的表达情况；MSP法检测hMLH1基因启动子甲基化状态。结果35例胰腺癌中微卫星高度不稳定7例，低度不稳定14例，稳定11例，正常组织中没有出现微卫星不稳定，两者之间差异有统计学意义（P〈0．05）。hMLH1在微卫星不稳定胰腺癌组织中常为缺失表达。在微卫星稳定胰腺癌组织中呈正常表达。35例胰腺癌中hMLH1启动子CpG岛甲基化发生率为60％（21/35）。正常组织中未发现甲基化，两者之间差异有统计学意义（P〈0．05）。结论与胰腺癌有关的错配修复基因hMLH1启动子CpG岛甲基化是hMLH1基因失活的重要机制，而hMLH1的表达失活则可能导致MSI的产生，促进胰腺癌的发生。     

错配修复基因启动子甲基化与胰腺癌发生的关系

目的通过对胰腺癌错配修复基因启动子甲基化及蛋白表达的检测与微卫星不稳定性的分析,探讨胰腺癌发病的分子机制。方法从35例胰腺癌病人的正常胰腺组织、癌组织中提取DNA;MSP法检测hMLH1及hMSH2基因启动子甲基化状态;SSCP法检测标本中微卫星不稳定性发生情况;免疫组化法检测错配修复基因hMLH1及hMSH2在胰腺癌中的表达情况。结果35例胰腺癌中hMLH1启动子甲基化发生率为60％（21／35）,正常组织中未发现甲基化,两者之间有统计学差异（P〈0.05）;而对于hMSH2仅有1例胰腺癌出现启动子甲基化;35例胰腺癌中微卫星高度不稳定7例,低度不稳定14例,稳定11例,正常组织中没有出现微卫星不稳定,两者之间有统计学差异（P〈0.05）;在微卫星不稳定的21例胰腺癌组织中有19例（90％）出现hMLH1启动子甲基化现象,微卫星稳定的ll例胰腺癌组织中仅有2例（6％）出现hMLH1启动子甲基化。hMLH1启动子甲基化在微卫星不稳定胰腺癌组织中常为缺失表达或低表达,在微卫星稳定胰腺癌组织中呈正常表达。hMSH2无论在MSI-H或MSI-L胰腺癌与正常胰腺组织中均无缺失表达,仅部分低表达。结论胰腺癌组织中错配修复基因的缺陷主要是hMLH1启动子甲基化,与胰腺癌微卫星不稳定性及蛋白表达缺失有关,在胰腺癌发生过程中起重要作用,是胰腺癌发生的重要机制之一。     

Implications of mismatch repair genes hMLH1 and hMSH2 in patients with sporadic renal cell carcinoma

OBJECTIVES

To analyse the implications of DNA mismatch repair genes hMLH1 and hMSH2 in sporadic renal cell carcinoma (RCC).

MATERIALS AND METHODS

Specimens of tumour and healthy renal tissue were collected from 89 patients treated for sporadic RCC. Another 95 blood samples taken from individuals with no history of cancer were also analysed. After DNA extraction and PCR amplification, microsatellite instability (MSI) was determined using the Bethesda microsatellite panel, two exonic microsatellites of the TGFbRII and BAX genes, and the microsatellite D3S1611. The promoter methylation status of hMLH1 was investigated using the HpaII and MspI restriction enzymes. In addition, a sequencing analysis of complete coding region of hMLH1 and hMSH2 genes was performed.

RESULTS

MSI and promoter hypermethylation of hMLH1 were not detected. Interestingly, loss of heterozygosity (LOH) was common among patients with RCC, particularly in microsatellite D3S1611 (34.9%). Mutations were identified in eight patients: K618A and V716M in gene hMLH1; and I145V, G322D, and the novel mutation P349A, in gene hMSH2. The mutations also appeared in healthy renal tissue and therefore, were considered as germline DNA sequence variations. There were G322D and K618A changes in >1% of the healthy control subjects, suggesting that they are DNA polymorphisms.

CONCLUSIONS

Our data show that loss of function of both hMLH1 and hMSH2 is not involved in sporadic RCC, either by promoter methylation or mutation in their exons. However, LOH indicated that chromosomal instability affecting large fragments of DNA was the main genetic alteration we detected associated with RCC.     

Immunohistochemistry for hMLH1 and hMSH2: a practical test for DNA mismatch repair-deficient tumors.

Inactivation of deoxyribonucleic acid (DNA) mismatch repair genes, most commonly human mutL homologue 1 (hMLH1) or human mutS homologue 2 (hMSH2), is a recently described alternate pathway in cancer development and progression. The resulting genetic instability is characterized by widespread somatic mutations in tumor DNA, and is termed high-frequency microsatellite instability (MSI-H). Although described in a variety of tumors, mismatch repair deficiency has been studied predominantly in colorectal carcinoma. Most MSI-H colorectal carcinomas are sporadic, but some occur in patients with hereditary nonpolyposis colorectal cancer (HNPCC), and are associated with germline mutations in mismatch repair genes. Until now, the identification of MSI-H cancers has required molecular testing. To evaluate the role of immunohistochemistry as a new screening tool for mismatch repair-deficient neoplasms, the authors studied the expression of hMLH1 and hMSH2, using commercially available monoclonal antibodies, in 72 formalin-fixed, paraffin-embedded tumors that had been tested previously for microsatellite instability. They compared immunohistochemical patterns of 38 MSI-H neoplasms, including 16 cases from HNPCC patients with known germline mutations in hMLH1 or hMSH2, with 34 neoplasms that did not show microsatellite instability. Thirty-seven of 38 MSI-H neoplasms were predicted to have a mismatch repair gene defect, as demonstrated by the absence of hMLH1 and/or hMSH2 expression. This included correspondence with all 16 cases with germline mutations. All 34 microsatellite-stable cancers had intact staining with both antibodies. These findings clearly demonstrate that immunohistochemistry can discriminate accurately between MSI-H and microsatellite-stable tumors, providing a practical new technique with important clinical and research applications.     

A meta-analysis of the prevalence of somatic mutations in the hMLH1 and hMSH2 genes in colorectal cancer

Aim The study aimed to understand better the somatic mutations in the human MutL Homolog 1 (hMLH1) and human MutS Homolog 2 (hMSH2) genes in colorectal cancer (CRC) and to investigate the differences derived from ethnicity, family history, detection method and microsatellite instability (MSI). Method The terms ‘hMSH2’ or ‘hMLH1’and‘colorectal cancer’‘colorectal carcinoma’ or ‘colorectal tumour’ were searched in the PubMed, Springer, Lippincott, Williams & Wilkins and HighWire Press databases for the publication period December 1993 to September 2010. The Comprehensive Meta Analysis V2 software (Biostat Inc.) was used to explore the prevalence and 95% confidence intervals. Results The prevalence of somatic mutations in the hMLH1 and hMSH2 genes in CRC was 0.15 (95% CI 0.10–0.22) and 0.10 (95% CI 0.07–0.16), respectively. A higher prevalence of somatic mutations in hMSH2 was found in hereditary non‐polyposis CRC than in sporadic CRC: 0.36 (95% CI 0.14–0.67) and 0.10 (95% CI 0.07–0.13) respectively. In addition, a higher prevalence of somatic mutations in the hMLH1 gene was observed relative to hMSH2 in the European group. The prevalence was higher in the high‐level instability (MSI‐H) group than in both the low‐level instability (MSI‐L) and the microsatellite stable (MSS) groups. Conclusion Somatic mutations in the hMLH1 and hMSH2 genes play a vital role in CRC and a high prevalence was found in this meta‐analysis. Furthermore, more studies are needed which focus on somatic mutations in the American population and in patients with MSI‐L and MSS.     

Prognostic value of hMLH1 methylation and microsatellite instability in pancreatic endocrine neoplasms

BACKGROUND: The aberrant promoter methylation of the mismatch repair gene, hMLH1, is associated with microsatellite instability (MSI) in cancer cells and often is associated with a favorable prognosis. METHODS: Pancreatic endocrine neoplasms (PENs) were obtained from 48 patients who underwent surgical resection. Methylation-specific polymerase chain reaction was used to detect methylation in the hMLH1 promoter. Tumor MSI at loci BAT26, BAT25, D2S123, D5S346, and D17S250 was determined with microsatellite polymerase chain reaction. RESULTS: Hypermethylation of the hMLH1 promoter was present in 11 of 48 PENs (23%). Five of the 11 hMLH1-methylated PENs were found to be microsatellite unstable, and MSI was restricted to PENs with hMLH1 hypermethylation. Tumor recurrence at 2 years after surgical resection was significantly less common among the hMLH1-methylated PENs (11%), compared with the unmethylated PENs (35%; P=.038). Patients with hMLH1-methylated PENs experienced improved 5-year survival (100%) compared with patients with unmethylated tumors (56%; P=.010). Likewise, MSI-positive PENs were associated with improved survival compared with MSI-negative tumors (100% vs 59%; P=.017) at 5 years. CONCLUSION: As in hereditary nonpolyposis colorectal cancer in which MSI is associated with improved survival, methylation of hMLH1 leads to MSI in PENs and affords a favorable prognosis.     

hMLH1及hMSH2蛋白免疫组化结合微卫星不稳定性检测在遗传性非息肉病性结直肠癌家系筛选中的作用

目的 探讨hMLH1及hMSH2蛋白免疫组化结合微卫星不稳定性检测在遗传性非息肉病性结直肠癌家系筛选中的敏感性、特异性及临床应用价值。方法 对12例符合Amsterdam标准的HNPCC患者和16例散发性结直肠癌患者的肿瘤标本进行hMLH1及hMSH2蛋白免疫组化检查和微卫星不稳定性检测。结果 hMLH1及hMSH2蛋白免疫组化筛选HNPCC家系的敏感性为91．7％,特异性为87．5％;微卫星不稳定性检测的敏感性为100％,特异性为75．0％;两者结合敏感性为91．7％,特异性为93．8％。结论 hMLH1及hMSH2蛋白免疫组化结合微卫星不稳定性检测筛选HNPCC家系敏感性和特异性明显提高,而且方法简单、经济,适合在临床广泛应用。     

"Null pattern" of immunoreactivity in a Lynch syndrome-associated colon cancer due to germline MSH2 mutation and somatic MLH1 hypermethylation

Lynch syndrome accounts for approximately 3% of newly diagnosed colorectal cancers and is caused by germline defects in DNA mismatch repair genes. Screening of patients for Lynch syndrome can be done by immunohistochemical staining for a panel of mismatch repair proteins and/or DNA testing for microsatellite instability. We describe a unique "null" immunophenotype in a Lynch syndrome associated colon cancer in a 71-year-old woman who also had a personal history of ureteral cancer and a strong family history of various malignancies. Immunohistochemical stains for MLH1, MSH2, PMS2, and MSH6 were completely negative in the tumor cells, with positive staining in stromal and inflammatory cells. Mutation analysis using peripheral blood showed a germline G587R mutation in the MSH2 gene. Further testing revealed the tumor to be positive for MLH1 promoter hypermethylation. Normal colonic mucosa adjacent to the tumor was negative for MLH1 promoter methylation. The lack of immunostaining for any of the 4 DNA mismatch repair proteins in this extremely unusual case was, therefore, related to a germline MSH2 mutation and somatic MLH1 promoter hypermethylation.     

Immunohistochemical staining for mismatch repair proteins, and its relevance in the diagnosis of hereditary non-polyposis colorectal cancer

BACKGROUND: Hereditary non-polyposis colorectal cancer (HNPCC) arises mostly from germline mutations of the mismatch repair genes MSH2 and MLH1. The diagnosis of HNPCC is based on a set of clinical criteria that may be too restrictive to identify all affected patients. Immunohistochemical staining (IHC) for the mismatch repair proteins, MutS homologue 2 (MSH2) and MutL homologue 1 (MLH1), reliably identifies the microsatellite instability phenotype. This study evaluated the ability of IHC to detect germline mutations in an unselected group of patients with colorectal cancer (CRC). METHODS: All patients with CRC operated on between July 2000 and March 2003, and demonstrating a loss of protein, were contacted. Following informed consent, searchs for germline mutation and methylation of the promoter were performed on normal and tumoral DNA. RESULTS: Thirty patients agreed to participate, four of whom fulfilled the Amsterdam II criteria. Loss of expression of MLH1 was found in 20 patients, and loss of expression of MSH2 in ten patients. Four of the MLH1-deficient patients had a germline MLH1 point mutation (positive predictive value (PPV) 20 (95 per cent confidence interval (c.i.) 2 to 38 per cent) and 11 had promoter methylation. Seven of the MSH2-deficient patients had a germline MSH2 point mutation (PPV 70 (95 per cent c.i. 54 to 96 per cent), and none showed promoter methylation. CONCLUSION: MLH1-deficient patients who are young or have a positive family history of cancer should be referred for genetic testing and counselling, whereas MSH2-deficient patients should be counselled in the same way as patients with HNPCC.