HNPCC mutations in the human DNA mismatch repair gene hMLH1 influence assembly of hMutLalpha and hMLH1-hEXO1 complexes.

Hereditary nonpolyposis colorectal cancer (HNPCC) is a common inherited form of neoplasia caused by germline mutations in DNA mismatch repair (MMR) genes. MMR proteins have been reported to associate with several proteins, including the human exonuclease 1 (hEXO1). We report here novel HNPCC-hMLH1 mutant proteins (T117M, Q426X and 1813insA) in Danish HNPCC patients. We demonstrate that these mutant HNPCC-hMLH1 proteins are unable to form complexes with hEXO1 and hPMS2 in vivo. The results indicate that mutations found in HNPCC gene carriers disrupt hMLH1-hEXO1 complex formation and hMutLalpha heterodimer assembly essential for MMR activity.     

错配修复基因hMLH3在家族性食管癌中的意义

目的：探讨错配修复基因hMLH3在家族性食管癌发生与发展中的作用。方法：应用聚合酶链反应（PCR）、变性高效液相色谱分析（DHPLC）和直接测序法对10个有遗传背景的食管癌家族（66名成员）,检测hMLH3基因所有外显子（共12个）的突变。对发现的基因改变,在家系内进行分离分析,并与在96例散发性食管癌患者和96例正常对照中发生的频率相比较。结果：在4个家系中共发现了4个错义突变和3个碱基多态性,而4个错义突变在各自家系中的发生频率均高于散发性食管癌患者和正常对照中的频率。在家系9（A2173C）和10（C2825T）中,hMLH3的突变可能具有致病性,但外显率下降;而在家系1（T3826C）和7（T3826C）的结果不支持它是单一的高风险基因。结论：hM-LH3基因在某些家族性食管癌中可能为高风险基因,但外显率下降,而在某些家系中可能仅为一低风险的基因,它诱导肿瘤的产生可能通过与其他基因相互叠加、共同起作用。     

Expression of the MutL homologue hMLH3 in human cells and its role in DNA mismatch repair

The human mismatch repair (MMR) proteins hMLH1 and hPMS2 function in MMR as a heterodimer. Cells lacking either protein have a strong mutator phenotype and display microsatellite instability, yet mutations in the hMLH1 gene account for approximately 50% of hereditary nonpolyposis colon cancer families, whereas hPMS2 mutations are substantially less frequent and less penetrant. Similarly, in the mouse model, Mlh1-/- animals are highly cancer prone and present with gastrointestinal tumors at an early age, whereas Pms2-/- mice succumb to cancer much later in life and do not present with gastrointestinal tumors. This evidence suggested that MLH1 might functionally interact with another MutL homologue, which compensates, at least in part, for a deficiency in PMS2. Sterility of Mlh1-/-, Pms2-/-, and Mlh3-/- mice implicated the Mlh1/Pms2 and Mlh1/Mlh3 heterodimers in meiotic recombination. We now show that the hMLH1/hMLH3 heterodimer, hMutLgamma, can also assist in the repair of base-base mismatches and single extrahelical nucleotides in vitro. Analysis of hMLH3 expression in colon cancer cell lines indicated that the protein levels vary substantially and independently of hMLH1. If hMLH3 participates in MMR in vivo, its partial redundancy with hPMS2, coupled with the fluctuating expression levels of hMLH3, may help explain the low penetrance of hPMS2 mutations in hereditary nonpolyposis colon cancer families.     

Analysis of mismatch repair gene mutations in Turkish HNPCC patients

Hereditary non-polyposis colorectal cancer (HNPCC or Lynch syndrome) is caused by the inheritance of a mutant allele of a DNA mismatch repair gene. We aimed to investigate types and frequencies of mismatch repair (MMR) gene mutations in Turkish patients with HNPCC and to identify specific biomarkers for early diagnosis of their non-symptomatic kindred’s. The molecular characteristics of 28 Turkish colorectal cancer patients at high-risk for HNPCC were investigated by analysis of microsatellite instability (MSI), immunohistochemistry and methylation-specific PCR in order to select tumors for mutation analysis. Ten cases (35.7%) were classified as MSI (+). Lack of expression of the main MMR proteins was observed in MSI (+) tumors. Hypermethylation of the MLH1 promoter region was observed in one tumor. Nine Lynch syndrome cases showed novel germ-line alterations of the MMR gene: two frame-shifts (MLH1 c.1843dupC and MLH1 c.1743delG) and three missense mutations (MLH1 c.293G>C, MLH1 c.954_955delinsTA and MSH2 c.2210G>A). Unclassified variants were evaluated as likely to be pathogenic by using the in-silico analyses. In addition, the MSH2 c.2210G>A alteration could be considered as a founder mutation for the Turkish population due to its identification in five different Lynch syndrome families and absence in control group. The present study adds new information about MMR gene mutation types and their role in Lynch syndrome. This is the first detailed research on Turkish Lynch syndrome families.     

ＤＮＡ错配修复基因ｈＭＬＨ１与肿瘤及化疗耐药相关性的研究进展

ＤＮＡ是生命活动最重要的遗传物质。ＤＮＡ复制产生的误差（碱基错配）是一种重要的损伤。若ＤＮＡ损伤得不到修复,将会导致基因的突变,其中一部分突变有利于物种的进化,而另一部分将导致细胞恶化和死亡。ＤＮＡ错配修复系统（ｍｉｓｍａｔｃｈｒｅｐａｉｒｓｙｓｔｅｍ,ＭＭＲ）是人体细胞中存在的一种能识别并修复ＤＮＡ碱基错配的安全保障系统。到目前为止,已从人体细胞中共分离克隆到９种ＭＭＲ基因。ｈＭＬＨ１基因是一系列ＤＮＡ错配修复基因中最重要的一种,也是ＭＭＲ系统的重要成分,其甲基化可导致ＭＭＲ缺陷。近年来的研究发现,ｈＭＬＨ１的失活与肿瘤发生发展有关,并可能因此导致肿瘤对某些抗肿瘤药物耐药。     

The Role of Expression of Mismatch Repair Proteins hMSH2 and hMLH1 in Gastric Carcinogenesis and its Clinical Significance

OBJECTIVE To investigate the expression of the mismatch repair proteins hMSH2 and hMLH1,and to examine the clinical significance of the intracellular expression site(ICES)in gastric carcinogenesis. METHODS Specimens from 172 cases of gastric cancer,151 tissues from paraneoplastic gastric mucosa and 34 from noncancerous gastric mucosa were collected in Dalain,China.An immunohistochemical method was used to determine the expression of the hMSH2,hMLH1 proteins and their ICES in the gastric mucosas. RESULTS The rate of hMSH2 expression in gastric cancers,paraneoplastic gastric mucosas and noncancerous gastric mucosas were respectively 69.8%,49.7%and 32.4%.The rate was significantly higher in gastric cancer compared to the latter two groups(P=0.000),but there was no obvious difference in the expression between the two latter groups(P=0.067). The hMLH1 protein expression rates were respectively 73.3%,57.6%and 41.2%in the above three groups.The expression was significantly higher in the gastric cancer group compared to the two latter groups(P=0.000),while there was no significant difference between the latter groups(P=0.082). There was no obvious correlation between the hMSH2 and hMLH1 protein expression rates and related factors,such as gender,age and differentiated level of gastric cancer etc.The cell-nuclear expression of the hMSH2 protein was respectively 70.0%,58.7%and 36.4%in the gastric cancer,paraneoplastic gastric mucosa and noncancerous gastric mucosa groups.The cytoplasmic expression rates were 30.0%,41.3%and 63.6%in the three groups. The cell-nuclear expression rate of the hMSH2 protein gradually decreased in the gastric mucosas in the fol owing order:cancer,paraneoplastic and noncancerous but cytoplasmic expression only increased slightly in these groups(r=0.161,P=0.020).There was no significant difference in the ICES of the hMLH1 protein among the three different gastric mucosas(P=0.659). CONCLUSION Simultaneous determination of the expression and ICES of the mismatch repair proteins hMSH2 and hMLH1 in the gastric mucosa may be helpful in detecting early gastric cancer.     

Nontruncating APC germ-line mutations and mismatch repair deficiency play a minor role in APC mutation-negative polyposis

Familial adenomatous polyposis, an autosomal-dominantly inherited colorectal cancer predisposition syndrome, is caused by germ-line mutations in the adenomatous polyposis coli (APC) gene. Despite the use of different screening methods, studies worldwide fail to identify APC mutations in 20-50% of all familial adenomatous polyposis patients (APC mutation-negatives). In this study, missense mutations in the coding region of the APC gene, which would have been missed by the protein truncation test, as well as mutations in the APC promoter and the 3' untranslated region, were determined by the single nucleotide polymorphism discovery assay and direct DNA sequencing in 31 mutation-negative polyposis patients. Seventeen gene alterations were identified, whereof four (12.9%) represent possibly pathogenic germ-line mutations: silent A290T (promoter) and A8822G (3' untranslated region) as well as missense R99W and E1317Q (coding region). The 27 remaining, truly APC mutation-negative polyposis patients displayed a significantly later age at diagnosis compared with APC mutation carriers (46.1 versus 35.2 years; P < 0.01). APC mutation-negative individuals with >100 colonic polyps were more likely to present with extracolonic disease (P < 0.05) than those with <100. Assessment of microsatellite instability (MSI), a hallmark of mismatch repair deficiency, in 68 tumors from 21 truly APC mutation-negative patients, identified 4 (5.9%) unstable tubulo-villous adenomas (3 MSI-High and 1 MSI-Low), stemming from 4 (19%) unrelated individuals and likely to be caused by hMLH1 promoter hypermethylation. In conclusion, only a small proportion of APC germ-line mutation carriers is missed by the protein truncation test, and mismatch repair deficiency does not seem to substantially contribute to tumor development in APC mutation-negative polyposis patients.     

Analysis for microsatellite instability and mutations of the DNA mismatch repair gene hMLH1 in familial gastric cancer

We examined 30 gastric-cancer patients with a varying degree of family history of stomach cancer and/or synchronous gastric tumors for microsatellite instability. We observed microsatellite instability at at least 1 of 8 loci tested in tumors of 14/30 patients; of these 14, 8 had single locus alterations and 6 had alterations at at least half of the 8 loci. Among the patients with microsatellite instability at >=4 loci, 3 patients showed a strong familial clustering of gastric cancer. Mutation analysis of the DNA mismatch repair gene hMLH1 on paired non-tumorous and tumor DNA from 10 patients 6 with microsatellite instability at 24 loci and 4 with an alteration at one locus, revealed a novel missense mutation, present in the normal and tumor DNA of one patient with microsatellite instability at multiple loci in his tumor. His family history of cancer included one second-degree relative affected with gastric cancer. These data suggest that germline mutations in the hMLH1 gene occur in some gastric-cancer patients and that in the majority of cases microsatellite instability in gastric tumors may be due to defects in other genes responsible for DNA replication fidelity than the hMLH1. © 1996 Wiley-Liss, Inc.     

大肠腺瘤及癌变组织hMLH1和hMSH2表达与细胞凋亡的研究

目的:探讨hMLH1和hMSH2基因在大肠腺瘤及其癌变中的作用,及其表达对细胞凋亡的影响.方法:采用免疫组织化学染色检测63例大肠腺瘤、20例腺瘤癌变和20例大肠癌组织hMLH1和hMSH2表达;同时采用TUNEL法检测其细胞凋亡指数(AI).结果:大肠腺瘤、腺瘤癌变和大肠癌组织错配修复基因hMLH1、hMSH2的表达率逐渐降低,与正常大肠相比相差显著,随腺瘤不典型增生程度增加其阳性率逐渐降低;大肠腺瘤、腺瘤癌变和大肠癌中hMLH1表达缺失者细胞凋亡指数较显著高于其阳性者,且大肠腺瘤不典型增生Ⅰ、Ⅱ、Ⅲ级hMLH1-与hMLH1+组存在差异显著;而hMSH2-与hMSH2-间AI仅大肠腺瘤组有显著性差异,不典型增生Ⅰ、Ⅱ级组hMSH2-与hMSH2+间AI差异显著,而不典型增生Ⅲ级组hMSH2蛋白的表达阴性与阳性间AI无统计学差异.结论:DNA错配修复基因突变或功能缺失与大肠癌的发生有关,可能系大肠癌发生过程中的早期事件,且可能与大肠肿瘤细胞凋亡活性增加相关.     

大肠腺瘤及癌变组织hMLH1和hMSH2表达与细胞凋亡的研究

目的：探讨hMLH1和hMSH2基因在大肠腺瘤及其癌变中的作用,及其表达对细胞凋亡的影响。方法：采用免疫组织化学染色检测63例大肠腺瘤、20例腺瘤癌变和20例大肠癌组织hMLH1和hMSH2表达;同时采用TUNEL法检测其细胞凋亡指数（AI）。结果：大肠腺瘤、腺瘤癌变和大肠癌组织错配修复基因hMLH1、hMSH2的表达率逐渐降低,与正常大肠相比相差显著,随腺瘤不典型增生程度增加其阳性率逐渐降低;大肠腺瘤、腺瘤癌变和大肠癌中hMLH1表达缺失者细胞凋亡指数较显著高于其阳性者,且大肠腺瘤不典型增生Ⅰ、Ⅱ、Ⅲ级hMLH1-与hMLH1＋组存在差异显著;而hMSH2-与hMSH2-间AI仅大肠腺瘤组有显著性差异,不典型增生Ⅰ、Ⅱ级组hMSH2-与hMSH2＋间AI差异显著,而不典型增生Ⅲ级组hMSH2蛋白的表达阴性与阳性间AI无统计学差异。结论：DNA错配修复基因突变或功能缺失与大肠癌的发生有关,可能系大肠癌发生过程中的早期事件,且可能与大肠肿瘤细胞凋亡活性增加相关。     

Microsatellite instability and protein expression of the DNA mismatch repair gene, hMLH1, of lung cancer in chromate-exposed workers

Our previous studies of lung cancer in chromate-exposed workers (chromate lung cancer) have revealed that the frequency of replication error (RER) in chromate lung cancer is very high. We examined whether the RER phenotype of chromate lung cancer is due to an abnormality of DNA mismatch repair protein. We investigated the expression of a DNA mismatch repair gene, hMLH1, and hMSH2 proteins using immunohistochemistry and microsatellite instability (MSI) in 35 chromate lung cancers and 26 nonchromate lung cancers. Lung cancer without MSI or with MSI at one locus was defined as "RER(-)," lung cancer with MSI at two loci was defined as "RER(+)," and lung cancer with MSI at three or more loci was defined as "RER(++)." The repression rate of hMLH1 and hMSH2 proteins in chromate lung cancer was significantly more than that of nonchromate lung cancer (hMLH1: 56% vs. 20%, P = 0.006, hMSH2: 74% vs. 23%, P < 0.0001). In chromate lung cancer, the repression rate for hMLH1 was 43% in RER(-), 40% in RER(+), and 90% in the RER(++) group. The repression rate of hMLH1 protein in the RER(++) group was significantly higher than that in the RER(-) and RER(+) groups (P = 0.039). The inactivation of hMLH1 expression strongly correlated with the microsatellite high instability phenotype in chromate lung cancer. The genetic instability of chromate lung cancer is due to the repression of hMLH1 protein.     

错配修复基因hMLH1和hMSH2在散发性大肠癌中的表达及其意义

目的 探讨错配修复基因hMLH1和hMSH2在散发性大肠癌(SCC)组织中的表达及其临床意义.方法 采用免疫组化Max Vision二步法对63例SCC标本中的癌组织、距癌灶3 cm以外的癌旁组织、距癌灶10 cm以外的正常组织中hMLH1和hMSH2蛋白的表达进行检测.结果 hMLH1蛋白在63例正常大肠组织、癌旁组织和SCC组织中的阳性表达率分别为95.2%、85.7%和81.0%,hMLH1蛋白在SCC组织中的阳性表达率明显低于正常大肠组织(P＜0.05).hMSH2蛋白在63例正常大肠组织、癌旁组织和SCC组织中的阳性表达率分别为76.2%、66.7%和52.4%,hMSH2蛋白在SCC组织中的阳性表达率明显低于正常大肠组织(P＜0.01).hMLH1蛋白在＜60岁的SCC患者组织中的阳性表达率(100%)明显高于≥60岁的SCC患者组织(75.0%,P＜0.05),在有淋巴结转移的SCC组织中的阳性表达率(50.0%)明显低于无淋巴结转移的SCC组织(93.3%,P＜0.05).hMSH2蛋白在＜60岁的SCC患者组织中的阳性表达率(80.0%)明显高于≥60岁的SCC患者组织(43.8%,P＜0.05),在癌组织浸润至肠壁浆膜层SCC组织中的阳性表达率(61.5%)明显高于浸润至黏膜下层和肌层的SCC组织(37.5%,P＜0.05).SCC组织中hMLH1和hMSH2蛋白的表达呈正相关关系(r=0.254,P＜0.01).结论 hMLH1和hMSH2蛋白在SCC组织中的表达均有一定的缺失,并且与患者的年龄、淋巴结转移和癌组织浸润的范围有关.hMLH1和hMSH2基因可以作为临床预测和判断SCC发生和发展有用的实验室指标.     

Functional role of DNA mismatch repair gene PMS2 in prostate cancer cells

DNA mismatch repair (MMR) enzymes act as proofreading complexes that maintains genomic integrity and MMR-deficient cells show an increased mutation rate. MMR has also been shown to influence cell signaling and the regulation of tumor development. MMR consists of various genes and includes post-meiotic segregation (PMS) 2 which is a vital component of mutL-alpha. In prostate, the functional role of this gene has never been reported and in this study, our aim was to investigate the effect of PMS2 on growth properties of prostate cancer (PCa) cells. Previous studies have shown PMS2 to be deficient in DU145 cells and this lack of expression was confirmed by Western blotting whereas normal prostatic PWR-1E and RWPE-1 cells expressed this gene. PMS2 effects on various growth properties of DU145 were then determined by creating stable gene transfectants. Interestingly, PMS2 caused decreased cell proliferation, migration, invasion, and in vivo growth; and increased apoptosis as compared to vector control. We further analyzed genes affected by PMS2 expression and observe the apoptosis-related TMS1 gene to be significantly upregulated whereas anti-apoptotic BCL2A1 was downregulated. These results demonstrate a functional role for PMS2 to protect against PCa progression by enhancing apoptosis of PCa cells.     

Roles of mismatch repair proteins hMSH2 and hMLH1 in the development of sporadic breast cancer

Defects in mismatch repair (MMR) genes, particularly the hMSH2 and hMLH1 genes, are associated with a variety of cancers including sporadic breast cancer. However, whether or not patient clinical background, e.g. age, progesterone receptor (PR), estrogen receptor (ER), tumor progression and stage, chemotherapy history, and menopausal status, influences MMR status is not understood. To address these issues, 83 archival breast cancer specimens were examined for expression of hMSH2 and hMLH1 by immunohistochemistry and the relationship between MMR protein expression and patient clinical background was analyzed. We detected lack of or reduced expression of hMSH2 and hMLH1 in 23 (27.7%) and 26 cases (31.3%), respectively, and hypermethylation of the hMLH1 promoter accounted for the majority of the cases with reduced expression of hMLH1. Statistical analysis revealed that (i) reduced expression of hMLH1 and hMSH2 seemed to confer advantage for the progression of breast tumors to more advanced stages; (ii) attenuated expression of hMLH1 correlated with history of chemotherapy, but not with age, menopause, or the status of PR and ER; (iii) hypermethylation of the hMLH1 promoter was linked with clinical stage and lymphatic metastasis. These analyses indicate that defective expression of MMR genes is closely associated with the development of sporadic breast cancer.     

Hypermethylation of the DNA mismatch repair gene hMLH1 and its association with lymph node metastasis and T1799A BRAF mutation in patients with papillary thyroid cancer

BACKGROUND: It remains to be investigated whether the aberrant methylation of DNA repair genes plays a pathogenic role in BRAF mutation-promoted tumorigenesis of papillary thyroid cancer (PTC). METHODS: In the current study, the promoter methylation status of 23 DNA repair genes in relation to clinicopathologic characteristics and BRAF mutation was examined in PTC tumors using methylation-specific polymerase chain reaction. RESULTS: Among the 38 PTC tumors examined, 3 of 23 DNA repair genes were hypermethylated, including the hMLH1 gene in 8 of 38 samples (21%), the PCNA gene in 5 of 38 samples (13%), and the OGG1 gene in 2 of 38 samples (5%). Methylation of these genes was also found in some thyroid cancer cell lines. Methylation of the hMLH1 gene in particular was found to be associated with lymph node metastasis of PTC (5 of 8 samples [63%] in the methylation group vs 3 of 30 samples [10%] in the nonmethylation group; P = .0049). Methylation of the hMLH1 gene was also found to be associated with the T1799A BRAF mutation in PTC (6 of 19 samples (32%) in the BRAF mutation-positive group vs 2 of 19 samples (11%) in the BRAF mutation-negative group; P = .042). CONCLUSIONS: The data from the current study suggest that, as shown previously in colon cancer, aberrant methylation of the hMLH1 gene may play a role in BRAF mutation-promoted thyroid tumorigenesis.     

Expression of the DNA mismatch repair proteins hMLH1 and hPMS2 in normal human tissues.

hMLH1 and hPMS2 are part of the DNA mismatch repair complex. Mutations in these genes have been linked to hereditary non-polyposis colon cancer; they also occur in a variety of sporadic cancers. Western blot analysis and immunohistochemistry demonstrated that hMLH1 and hPMS2 are widely expressed nuclear proteins with a distribution pattern very similar to that previously described for hMSH2. These observations showing similar localization of hMLH1 and hPMS2 with hMSH2 are consistent with the biochemical function of these proteins in DNA mismatch repair.     

错配修复基因hMSH2和hMLH1在结直肠癌组织中的表达及临床意义

  目的   评价hMLH1和hMSH2蛋白在结直肠癌中的表达及意义。   方法   选取2009年1月至2011年12月经病理学确诊的结肠癌手术切除标本72例, 所有患者在术前均未接受过放疗或化疗, 内镜活检取正常肠黏膜上皮25例。免疫组织化学检测hMLH1和hMSH2蛋白表达情况。   结果   结直肠癌组织中hMSH2缺失率为88.9% (64/72), 高于正常肠组织中hMSH2蛋白缺失率28.0% (7/25)。hMSH2蛋白缺失率随T分期增加而增加, 但差异无统计学意义(χ2=37.622, P < 0.001);hMSH2蛋白缺失率与N分期相关, 有淋巴结转移者的hMSH2蛋白缺失40例, 缺失率达97.6% (40/41), 无淋巴结转移患者的hMSH2蛋白缺失率为77.4% (24/31, χ2=7.251, P=0.007)。而hMLH1蛋白缺失率为90.3% (65/72), 高于正常组肠组织中hMLH1蛋白缺失率为32.0% (8/25, χ2=33.847, P < 0.001), 但与肿瘤部位、分期、分化程度均无关。   结论   在结直肠癌组织中存在错配修复基因hMSH2和hMLH1蛋白缺失, 且表达缺失与肿瘤分期有关。通过免疫组织化学方法检测hMLH1和hMSH2蛋白表达可以简便、准确地发现错配修复基因的突变, 从而对其后期的治疗和预后判断有参考价值。