Majority of hMLH1 mutations responsible for hereditary nonpolyposis colorectal cancer cluster at the exonic region 15-16.

Hereditary nonpolyposis colorectal cancer (HNPCC) is a common autosomal dominant cancer susceptibility condition. Inherited mutations in at least four DNA mismatch repair genes, hMSH2, hMLH1, hPMS1, and hPMS2, are known to cause HNPCC. In this study we used denaturing gradient gel electrophoresis (DGGE) to screen for hMLH1 mutations in 34 unrelated HNPCC families (30 Dutch, 3 Italian, and 1 Danish). Ten novel pathogenic germ-line mutations (seven affecting splice sites, two frameshifts, and one in-frame deletion of a single amino acid) have been identified in 12 (35%) of these families. In a previous study, hMSH2 mutations were found in 21% of the same families. While the spectrum of mutations at the hMSH2 gene among HNPCC patients appears heterogeneous, a cluster of hMLH1 mutations has been found in the region encompassing exons 15 and 16, which accounts for 50% of all the independent hMLH1 mutations described to date and for > 20% of the unrelated HNPCC kindreds here analyzed. This unexpected finding has a great practical value in the clinical scenario of genetic services.     

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.     

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.     

CpG dinucleotides in the hMSH2 and hMLHI genes are hotspots for HNPCC mutations

Hereditary nonpolyposis colon cancer (HN-PCC) is an autosomally inherited predisposition to cancer that has recently been linked to defects in the human mismatch repair genes hMSH2 and hMLHI. The identification of the causative mutations in HNPCC families is desirable, since it confirms the diagnosis and allows the carrier status of unaffected relatives at risk to be determined. We report six different new mutations identified in the hMSH2 and hMLH1 genes of Russian and Moldavian HNPCC families. Three of these mutations occur in CpG dinucleotides and lead to a premature stop codon, a splicing defect or an amino-acid substitution in an evolutionary conserved residue. Analysis of a compilation of published mutations including our new data suggests that CpG dinucleotides within the coding regions of the hMSH2 and hMLH1 genes are hotspots for single base-pair substitutions.     

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.     

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     

DHPLC mutation analysis of the hereditary nonpolyposis colon cancer (HNPCC) genes hMLH1 and hMSH2

Denaturing high-performance liquid chromatography (DHPLC) is an efficient method for detection of mutations involving a single or few numbers of nucleotides, and it has been successfully used for mutation detection in disease-related genes. Colorectal cancer is one of the most common cancers, and mutations in the genes for hereditary nonpolyposis colon cancer (HNPCC), hMLH1 and hMSH2, also involve mainly point mutations. Sequence analysis is supposed to be a screening method with high sensitivity; however, it is time-consuming and expensive. We therefore decided to test sensitivity and reproducibility of DHPLC for 71 sequence variants in hMLH1 and hMSH2 initially found by sequence analysis in DNA samples of German HNPCC patients. DHPLC conditions of the PCR products were based on the melting pattern of the wild-type sequence of the corresponding PCR fragments. All but one of the 71 mutations was detected using DHPLC (sensitivity of 97%). Running time per sample averaged only 7 min, and the system is highly automated. Thus DHPLC is a rapid and sensitive method for the detection of hMLH1 and hMSH2 sequence variants.     

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.     

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.     

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.     

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.     

Analysis of aneuploidy frequencies in sperm from patients with hereditary nonpolyposis colon cancer and an hMSH2 mutation

Hereditary nonpolyposis colon cancer (HNPCC) has been shown to be caused by mutations in the mismatch repair genes hMSH2, hMLH1, hPMS1, and hPMS2. Recent evidence has demonstrated that mutations in mismatch repair genes disrupt meiosis in mice. A large HNPCC kindred in Newfoundland, Canada, has an hMSH2 mutation-an A-->T transversion at the +3 position of the splice-donor site of exon 5. We have studied sperm from men with this hMSH2 mutation, since it is possible that mismatch repair mutations in humans might also have an effect on meiosis and normal segregation of chromosomes. The frequencies of aneuploid and diploid sperm were determined in 10 men with the hMSH2 mutation, by use of multicolor FISH analysis for chromosomes 13, 21, X, and Y. A minimum of 10,000 sperm per man was studied per chromosome probe. Control individuals consisted of men in the same kindred with HNPCC who did not carry the mutation and of other normal men from Newfoundland. A total of 321,663 sperm were analyzed: 200,905 sperm were from men carrying the hMSH2 mutation and 120,758 sperm were from control men. There was a significantly increased frequency of disomy 13, disomy 21, XX, and diploidy in mutation carriers compared with control men. These results suggest that the hMSH2 mutation may affect meiosis in humans.     

Distinct mutations in MLH1 and MSH2 genes in hereditary non-polyposis colorectal cancer (HNPCC) families from China

Hereditary non-polyposis Colorectal Cancer (HNPCC) is an autosomal dominant inheritance syndrome. HNPCC is the most common hereditary variant of colorectal cancer (CRC), which accounts for 2-5% CRCs, mainly due to hMLH1 and hMSH2 mutations that impair DNA repair functions. Our study aimed to identify the patterns of hMSH2 and hMLH1 mutations in Chinese HNPCC patients. Ninety-eight unrelated families from China meeting Amsterdam or Bethesda criteria were included in our study. Germline mutations in MLH1 and MSH2 genes, located in the exons and the splice-site junctions, were screened in the 98 probands by direct sequencing. Eleven mutations were found in ten patients (11%), with six in MLH1 (54.5%) and five in MSH2 (45.5%) genes. One patient had mutations in both MLH1 and MSH2 genes. Three novel mutations in MLH1 gene (c.157_160delGAGG, c.2157dupT and c.-64G>T) were found for the first time, and one suspected hotspot in MSH2 (c.1168C>T) was revealed.     

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.     

Muir-Torre phenotype has a frequency of DNA mismatch-repair-gene mutations similar to that in hereditary nonpolyposis colorectal cancer families defined by the Amsterdam criteria.

Muir-Torre syndrome (MTS) is an autosomal dominant disease defined by the coincidence of at least one sebaceous skin tumor and one internal malignancy. About half of MTS patients are affected by colorectal cancer. In a subgroup of MTS patients the disease has an underlying DNA mismatch-repair (MMR) defect and thus is allelic to hereditary nonpolyposis colorectal cancer (HNPCC). The purpose of this study was to examine to what extent germ-line mutations in DNA MMR genes are the underlying cause of the MTS phenotype. We ascertained 16 MTS patients with sebaceous skin tumors and colorectal cancer, and we examined their skin and visceral tumors for microsatellite instability. All the patients exhibited high genomic instability in at least one tumor. The search for germ-line mutations in the hMSH2 and hMLH1 genes in 13 of the MTS patients revealed truncating mutations in 9 (69%): eight mutations in the hMSH2 gene and one in the hMLH1 gene. This is the first systematic search for germ-line mutations in patients ascertained on the basis of sebaceous skin tumors. Our results indicate that (1) MTS patients exhibit significantly more mutations in the hMSH2 gene than in the hMLH1 gene; and (2) the subpopulation of MTS patients who are also affected by colorectal cancer, irrespective of family history and age at onset of tumors, may have a likelihood for an underlying DNA MMR defect similar to that for patients with a family history fulfilling the strict clinical criteria for HNPCC.     

Interactions of Human hMSH2 with hMSH3 and hMSH2 with hMSH6: Examination of Mutations Found in Hereditary Nonpolyposis Colorectal Cancer

Mutations in the human mismatch repair protein hMSH2 have been found to cosegregate with hereditary nonpolyposis colorectal cancer (HNPCC). Previous biochemical and physical studies have shown that hMSH2 forms specific mispair binding complexes with hMSH3 and hMSH6. We have further characterized these protein interactions by mapping the contact regions within the hMSH2-hMSH3 and the hMSH2-hMSH6 heterodimers. We demonstrate that there are at least two distinct interaction regions of hMSH2 with hMSH3 and hMSH2 with hMSH6. Interestingly, the interaction regions of hMSH2 with either hMSH3 or hMSH6 are identical and there is a coordinated linear orientation of these regions. We examined several missense alterations of hMSH2 found in HNPCC kindreds that are contained within the consensus interaction regions. None of these missense mutations displayed a defect in protein-protein interaction. These data support the notion that these HNPCC-associated mutations may affect some other function of the heterodimeric complexes than simply the static interaction of hMSH2 with hMSH3 or hMSH2 with hMSH6.     

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.