hMLH1和hMSH2种系突变携带者累积患癌风险研究

目的：探讨遗传性非息肉病性结直肠癌（HNPCC）家系中错配修复基因hMLH1和hMSH2种系突变携带者发生HNPCC相关恶性肿瘤的累积风险度。方法：通过随访14个HNPCC家系中222例hMLH1或hMSH2种系突变携带者与非携带者,应用SPSS14.0统计软件包分析种系突变携带者在不同年龄点发生HNPCC相关恶性肿瘤的累积风险度及两种基因种系突变累积患癌风险的差异。结果：hMLH1或hMSH2种系突变携带者肿瘤发生率为63.8%（60/94）,非突变携带者肿瘤发生率为0.8%（1/128）,种系突变携带者发生恶性肿瘤的相对危险度为非突变携带者的317.6倍;种系突变携带者发生各种HNPCC相关恶性肿瘤的累积风险度随年龄的增加逐渐增大,在60岁时发生各种HNPCC相关恶性肿瘤、结直肠癌、胃癌等的平均累积风险度分别为92.4%、81.3%、29.6%,40岁以前发生胃癌的平均风险度较低（6.1%）;hMLH1与hMSH2种系突变携带者发生各种HNPCC相关恶性肿瘤、结直肠癌、胃癌等累积风险度的差异无统计学意义（均为P＆gt;0.05）。结论：hMLH1或hMSH2种系突变携带者为HNPCC家系中患癌高危人群,发生HNPCC相关恶性肿瘤的风险度随年龄的增加而增大,最常发生恶性肿瘤的部位为胃和结直肠;hMLH1与hMSH2种系突变携带者发生各种HNPCC相关恶性肿瘤的累积风险度无明显差异。     

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.     

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.     

Mismatch repair defects in cancer

Post-replicative mismatch repair in humans utilises the hMSH2, hMSH6, hMSH3, hMLH1 and hPMS2 genes and possibly the newly identified hMLH3 gene. Recently, a link has been established between hMSH6 mutations and 'atypical' hereditary non-polyposis colon cancer (HNPCC) with an increased incidence of endometrial cancers. To satisfy the need for a diagnostic test capable of differentiating between pathogenic mutations and polymorphisms, several functional assays that fulfil these criteria have been described. These should allow for better diagnosis of HNPCC.     

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     

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.     

Identification of mismatch repair protein complexes in HeLa nuclear extracts and their interaction with heteroduplex DNA

Deficiencies in DNA mismatch repair (MMR) have been found in hereditary colon cancers (hereditary non-polyposis colon cancer, HNPCC) as well as in sporadic cancers, illustrating the importance of MMR in maintaining genomic integrity. We have examined the interactions of specific mismatch repair proteins in human nuclear extracts. Western blot and co-immunoprecipitation studies indicate two complexes as follows: one consisting of hMSH2, hMSH6, hMLH1, and hPMS2 and the other consisting of hMSH2, hMSH6, hMLH1, and hPMS1. These interactions occur without the addition of ATP. Furthermore, the protein complexes specifically bind to mismatched DNA and not to a similar homoduplex oligonucleotide. The protein complex-DNA interactions occur primarily through hMSH6, although hMSH2 can also become cross-linked to the mismatched substrate when not participating in the MMR protein complex. In the presence of ATP the binding of hMSH6 to mismatched DNA is decreased. In addition, hMLH1, hPMS2, and hPMS1 no longer interact with each other or with the hMutSalpha complex (hMSH2 and hMSH6). However, the ability of hMLH1 to co-immunoprecipitate mismatched DNA increases in the presence of ATP. This interaction is dependent on the presence of the mismatch and does not appear to involve a direct binding of hMLH1 to the DNA.     

Seven New Mutations in hMSH2, an HNPCC Gene, Identified by Denaturing Gradient-Gel Electrophoresis

Hereditary nonpolyposis colorectal cancer (HNPCC) is a relatively common autosomal dominant cancer-susceptibility condition. The recent isolation of the DNA mismatch repair genes (hMSH2, hMLH1, hPMS1, and hPMS2) responsible for HNPCC has allowed the search for germ-line mutations in affected individuals. In this study we used denaturing gradient-gel electrophoresis to screen for mutations in the hMSH2 gene. Analysis of all the 16 exons of hMSH2, in 34 unrelated HNPCC kindreds, has revealed seven novel pathogenic germ-line mutations resulting in stop codons either directly or through frameshifts. Additionally, nucleotide substitutions giving rise to one missense, two silent, and one useful polymorphism have been identified. The proportion of families in which hMSH2 mutations were found is 21%. Although the spectrum of mutations spread at the hMSH2 gene among HNPCC patients appears extremely heterogeneous, we were not able to establish any correlation between the site of the individual mutations and the corresponding tumor spectrum. Our results indicate that, given the genomic size and organization of the hMSH2 gene and the heterogeneity of its mutation spectrum, a rapid and efficient mutation detection procedure is necessary for routine molecular diagnosis and presymptomatic detection of the disease in a clinical setup.     

Integrative Analysis of Hereditary Nonpolyposis Colorectal Cancer: the Contribution of Allele-Specific Expression and Other Assays to Diagnostic Algorithms

The identification of germline variants predisposing to hereditary nonpolyposis colorectal cancer (HNPCC) is crucial for clinical management of carriers, but several probands remain negative for such variants or bear variants of uncertain significance (VUS). Here we describe the results of integrative molecular analyses in 132 HNPCC patients providing evidences for improved genetic testing of HNPCC with traditional or next generation methods. Patients were screened for: germline allele-specific expression (ASE), nucleotide variants, rearrangements and promoter methylation of mismatch repair (MMR) genes; germline EPCAM rearrangements; tumor microsatellite instability (MSI) and immunohistochemical (IHC) MMR protein expression. Probands negative for pathogenic variants of MMR genes were screened for germline APC and MUTYH sequence variants. Most germline defects identified were sequence variants and rearrangements of MMR genes. Remarkably, altered germline ASE of MMR genes was detected in 8/22 (36.5%) probands analyzed, including 3 cases negative at other screenings. Moreover, ASE provided evidence for the pathogenic role and guided the characterization of a VUS shared by 2 additional probands. No germline MMR gene promoter methylation was observed and only one EPCAM rearrangement was detected. In several cases, tumor IHC and MSI diverged from germline screening results. Notably, APC or biallelic MUTYH germline defects were identified in 2/19 probands negative for pathogenic variants of MMR genes. Our results show that ASE complements gDNA-based analyses in the identification of MMR defects and in the characterization of VUS affecting gene expression, increasing the number of germline alterations detected. An appreciable fraction of probands negative for MMR gene variants harbors APC or MUTYH variants. These results indicate that germline ASE analysis and screening for APC and MUTYH defects should be included in HNPCC diagnostic algorithms.     

Germline and somatic mutations in hMSH6 and hMSH3 in gastrointestinal cancers of the microsatellite mutator phenotype

Hereditary and sporadic gastrointestinal cancer of the microsatellite mutator phenotype (MMP) is characterized by a remarkable genomic instability at simple repeated sequences. The genomic instability is often caused by germline and somatic mutations in DNA mismatch repair (MMR) genes hMSH2 and hMLH1. The MMP can be also caused by epigenetic inactivation of hMLH1. The MMP generates many somatic frameshift mutations in genes containing mononucleotide repeats. We previously reported that in MMP tumors the hMSH6 and hMSH3 MMR genes often carry frameshift mutations in their (C)(8) and (A)(8) tracks, respectively. We proposed that these 'secondary mutator mutations' contribute to a gradual manifestation of the MMP. Here we report the detection of other frameshift, nonsense, and missense mutations in these genes in colon and gastric cancers of the MMP. A germline frameshift mutation was found in hMSH6 in a colon tumor harboring another somatic frameshift mutation. Several germline sequence variants and somatic missense mutations at conserved residues were detected in hMSH6 and only one was detected in hMSH3. Of the three hMSH6 germline variants in conserved residues, one coexisted with a somatic mutation at the (C)(8) track and another had a somatic missense mutation. We suggest that some of these germline and somatic missense variants are pathogenic. While biallelic hMSH6 and hMSH3 frameshift mutations were found in some tumors, many tumors seemed to contain only monoallelic mutations. In some tumors, these somatic monoallelic frameshift mutations at the (C)(8) and (A)(8) tracks were found to coexist with other somatic mutations in the other allele, supporting their functionality during tumorigenesis. However, the low incidence of these additional somatic mutations in hMSH6 and hMSH3 leaves many tumors with only monoallelic mutations. The impact of the frameshift mutations in gene expression was studied by comparative analysis of RNA and protein expression in different tumor cell clones with different genotypes. The results show that the hMSH6 (C)(8) frameshift mutation abolishes protein expression, ruling out a dominant negative effect by a truncated protein. We suggest the functionality of these secondary monoallelic mutator mutations in the context of an accumulative haploinsufficiency model.     

Do MSH6 mutations contribute to double primary cancers of the colorectum and endometrium?

Mismatch repair (MMR) gene mutations cause hereditary nonpolyposis colorectal cancer (HNPCC), a common form of familial colorectal cancer. Among MMR genes, germline MSH6 mutations are often observed in HNPCC-like families with an increased frequency of endometrial cancer. We have previously shown that a proportion of women affected with double primary cancers of the colorectum and endometrium carry germline MSH2 or MLH1 mutations and, thus, belong to HNPCC families. In this study, we have investigated the specific contribution of MSH6 defects to such double primary patients. By sequence analysis of the entire coding region of MSH6, three putative missense mutations were identified in patients with atypical family histories that do not meet HNPCC criteria. Moreover, one of these mutations, a novel substitution Arg901 His, was found in a patient previously shown to carry a truncating germline MLH1 mutation. Thus, MSH6 mutations are likely to contribute to the etiology of double primary cancers of the colorectum and endometrium.     

Hereditary nonpolyposis colorectal cancer: causative role of a germline missense mutation in the hMLH1 gene confirmed by the independent occurrence of the same somatic mutation in tumour tissue

Evaluation of the causative role of germline mutations in DNA mismatch repair genes in hereditary nonpolyposis colorectal cancer (HNPCC) families can be difficult. Whereas nonsense, frameshift or splice-site mutations are presumed to lead to dysfunctional gene products and thus are generally considered to be causative, the evaluation of missense mutations often remains uncertain. We observed a novel germline mutation in the hMLH1 gene (His→Pro at codon 329) in an HNPCC family. The same missense mutation also occurred as a somatic event in the colonic tumours of two other HNPCC patients who had germline mutations at different sites of the hMLH1gene. Thus, the H329P mutation present in the germline can be considered as having an aetiological role in this HNPCC family. Received: 1 April 1997 / Accepted: 13 May 1997     

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。     

Nuclear localization of human DNA mismatch repair protein exonuclease 1 (hEXO1)

Human exonuclease 1 (hEXO1) is implicated in DNA mismatch repair (MMR) and mutations in hEXO1 may be associated with hereditary nonpolyposis colorectal cancer (HNPCC). Since the subcellular localization of MMR proteins is essential for proper MMR function, we characterized possible nuclear localization signals (NLSs) in hEXO1. Using fluorescent fusion proteins, we show that the sequence ⁴¹⁸KRPR⁴²¹, which exhibit strong homology to other monopartite NLS sequences, is responsible for correct nuclear localization of hEXO1. This NLS sequence is located in a region that is also required for hEXO1 interaction with hMLH1 and we show that defective nuclear localization of hEXO1 mutant proteins could be rescued by hMLH1 or hMSH2. Both hEXO1 and hMLH1 form complexes with the nuclear import factors importin β/α1,3,7 whereas hMSH2 specifically recognizes importin β/α3. Taken together, we infer that hEXO1, hMLH1 and hMSH2 form complexes and are imported to the nucleus together, and that redundant NLS import signals in the proteins may safeguard nuclear import and thereby MMR activity.     

MUTYH and the mismatch repair system: partners in crime?

Biallelic germline mutations of MUTYH—a gene encoding a base excision repair protein—are associated with an increased susceptibility of colorectal cancer. Whether monoallelic MUTYH mutations also increase cancer risk is not yet clear, although there is some evidence suggesting a slight increase of risk. As the MUTYH protein interacts with the mismatch repair (MMR) system, we hypothesised that the combination of a monoallelic MUTYH mutation with an MMR gene mutation increases cancer risk. We therefore investigated the prevalence of monoallelic MUTYH mutations in carriers of a germline MMR mutation: 40 carriers of a truncating mutation (group I) and 36 of a missense mutation (group II). These patients had been diagnosed with either colorectal or endometrial cancer. We compared their MUTYH mutation frequencies with those observed in a group of 134 Dutch colorectal and endometrial cancer patients without an MMR gene mutation (0.7%) and those reported for Caucasian controls (1.5%). In group I one monoallelic MUTYH mutation was found (2.5%). In group II five monoallelic germline MUTYH mutations were found (14%), four of them in MSH6 missense mutation carriers (20%). Of all patients with an MMR gene mutation, only those with a missense mutation showed a significantly higher frequency of (monoallelic) MUTYH mutations than the Dutch cancer patients without MMR gene mutations (P=0.002) and the published controls (P=0.001). These results warrant further study to test the hypothesis of mutations in MMR genes (in particular MSH6) and MUTYH acting together to increase cancer risk.     

Mutator phenotypes of common polymorphisms and missense mutations in MSH2.

Hereditary non-polyposis colorectal cancer (HNPCC) is associated with germline mutations in the DNA mismatch repair gene hMSH2 [1], the human homologue of the Escherichia coli MutS gene. These are mostly nonsense, frameshift or deletion mutations that result in loss of intact protein and complete inactivation of DNA mismatch repair. However, cancer is also associated with hMSH2 missense mutations that are merely inferred to be deleterious because they result in non-conservative substitutions of amino acids that are highly conserved among MutS family proteins. Moreover, sequence polymorphisms exist in hMSH2 that also change conserved amino acids but whose functional consequences and relationship to cancer are uncertain. Here, we show that yeast strains harboring putative equivalents of three hMSH2 polymorphisms have elevated mutation rates. Mutator effects were also observed for yeast equivalents of hMSH2 missense mutations found in HNPCC families and in an early onset colon tumor. Several distinct phenotypes were observed, indicating that these missense mutations have differential effects on MSH2 function(s). The results suggest that cancer may be associated with even partial loss of hMSH2 function and they are consistent with the hypothesis that polymorphisms in hMSH2 might predispose humans to disease.     

Mutational analysis of the hMSH6 gene in familial and early-onset colorectal and endometrial cancer in Israeli patients

Familial colorectal cancer (CRC) is noted in about 15% of incident CRC cases, and at times is hallmarked by an age at diagnosis less than 50 years. Familial adenomatous polyposis (FAP) and hereditary non-polyposis colon cancer (HNPCC) account for about 40% of familial cases. Thus, the majority of familial and early-onset CRC remain genetically elusive. Similarly, the majority of familial and early onset endometrial cancer (EC), the most prevalent extracolonic tumor in HNPCC, are genetically undefined. An attractive candidate is the hMSH6 gene. Israeli patients with early onset (age under 50 years) (n = 44) and familial nonsyndromic (n = 23) CRC, and women with familial clustering of EC or CRC (n = 12), and those diagnosed with EC at, or under, the age of 50 years (n = 5) were genotyped for germ-line mutations within the hMSH6 gene. Exon-specific PCR was followed by denaturing gradient gel electrophoresis (DGGE) analysis, complemented by DNA sequencing of abnormally migrating fragments. No patients displayed a truncating mutation, and 1 CRC patient harbored a novel missense mutation (V878A). In addition, 6 previously described polymorphisms were detected. In conclusion, mutations in the hMSH6 gene occur uncommonly in Israeli patients with familial and early-onset CRC and EC.