High proportion of large genomic rearrangements in hMSH2 in hereditary nonpolyposis colorectal cancer (HNPCC) families of the Basque Country

Hereditary nonpolyposis colorectal cancer (HNPCC), which represents the most common form of inherited colorectal cancer, results from germline alterations of the mismatch repair genes MSH2, MLH1 and MSH6. Rearrangements of MSH2 and MLH1 are involved in at least 10% and 4.3%, respectively, of the HNPCC families fulfilling the Amsterdam (AMS) criteria. We applied a recently developed method, multiplex ligation-dependent probe amplification (MLPA), to study MLH1/MSH2 copy number changes in 29 unrelated Basque Country HNPCC families. We detected six different genomic rearrangements in total (6/29=20.69%), four in MSH2 gene (13.79%), and two in MLH1 gene. All of the MSH2 rearrangements were genomic deletions involving several exons. The MLH1 rearrangements were initially detected as one deletion of exon 18 and one deletion of exon 19, but after sequencing analysis, these deletions were not confirmed and corresponded to base pair mutations. We conclude that MLPA is an excellent tool for detecting exon copy number changes in MLH1 and MSH2 in the DNA from HNPCC patients, although all detected rearrangements should be confirmed by an independent molecular methodology. Furthermore, our results in the Basque Country show higher percentages of rearrangements than previously published by other authors.     

Genomic rearrangements in MSH2 and MLH1 are rare mutational events in Spanish patients with hereditary nonpolyposis colorectal cancer

Colorectal cancer (CRC) is one of the most common neoplasms and a leading cause of death related to cancer worldwide. Hereditary nonpolyposis colorectal cancer (HNPCC) is the most frequent autosomal dominant predisposition to the development of CRC, accounting for approximately 2.5% of the total CRC burden in Spain. Genomic rearrangements in the MSH2 and MLH1 genes have been reported to account for an important proportion of the mutation spectrum in HNPCC, and DNA dosage techniques have been developed facilitating molecular screening of such deletions/duplications. We screened for MSH2 and MLH1 genomic rearrangements by multiplex ligation-dependent probe amplification (MLPA) in 142 Spanish patients at risk for HNPCC prior to the exon-by-exon mutation scanning and found a deletion encompassing exons 9-16 of MSH2 and a duplication encompassing exons 11-16 of MSH2, both only in one case. These results showed that MSH2/MLH1 rearrangements in Spanish patients at risk for HNPCC seem to be a less frequent mutational event than in other populations.     

Genomic rearrangements in MSH2, MLH1 or MSH6 are rare in HNPCC patients carrying point mutations

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant disease with high penetrance, caused by germline mutations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6, PMS2 and MLH3. Most reported pathogenic mutations are point mutations, comprising single base substitutions, small insertions and deletions. In addition, genomic rearrangements, such as large deletions and duplications not detectable by PCR and Sanger sequencing, have been identified in a significant proportion of HNPCC families, which do not carry a pathogenic MMR gene point mutation. To clarify whether genomic rearrangements in MLH1, MSH2 or MSH6 also occur in patients carrying a point mutation, we subjected normal tissue DNA of 137 colorectal cancer (CRC) patients to multiplex ligation-dependent probe amplification (MLPA) analysis. Patients fulfilled the following pre-requisites: all patients met at least one criterion of the Bethesda guidelines and their tumors exhibited high microsatellite instability (MSI-H) and/or showed loss of expression of MLH1, MSH2 or MSH6 proteins. PCR amplification and Sanger sequencing of all exons of at least one MMR gene, whose protein expression had been lost in the tumor tissue, identified 52 index patients without a point mutation (Group 1), 71 index patients with a pathogenic point mutation in MLH1 (n=38) or MSH2 (n=22) or MSH6 (n=11) (Group 2) and 14 patients with an unclassified variant in MLH1 (n=9) or MSH2 (n=3) or MSH6 (n=2) (Group 3). In 13 of 52 patients of group 1 deletions of at least one exon were identified. In addition, in group 3 one EX1_15del in MLH1 was found. No genomic rearrangement was identified in group 2 patients. Genomic rearrangements represent a significant proportion of pathogenic mutations of MMR genes in HNPCC patients. However, genomic rearrangements are rare in patients carrying point mutations in MMR genes. These findings suggest the use of genomic rearrangement tests in addition to Sanger sequencing in HNPCC patients.     

Partial loss of heterozygosity events at the mutated gene in tumors from MLH1/MSH2 large genomic rearrangement carriers

Background  

Depending on the population studied, large genomic rearrangements (LGRs) of the mismatch repair (MMR) genes constitute various proportions of the germline mutations that predispose to hereditary non-polyposis colorectal cancer (HNPCC). It has been reported that loss of heterozygosity (LOH) at the LGR region occurs through a gene conversion mechanism in tumors from MLH1/MSH2 deletion carriers; however, the converted tracts were delineated only by extragenic microsatellite markers. We sought to determine the frequency of LGRs in Slovak HNPCC patients and to study LOH in tumors from LGR carriers at the LGR region, as well as at other heterozygous markers within the gene to more precisely define conversion tracts.     

MSH2 in contrast to MLH1 and MSH6 is frequently inactivated by exonic and promoter rearrangements in hereditary nonpolyposis colorectal cancer

To estimate the relative frequency of mismatch repair genes, rearrangements in hereditary nonpolyposis colorectal cancer (HNPCC) families without detectable mutations in MSH2 or MLH1, we have analyzed by multiplex PCR of short fluorescent fragments MSH2, MLH1, and MSH6 in 61 families, either fulfilling Amsterdam criteria or including cases of multiple primary cancers belonging to the HNPCC spectrum. We detected 13 different genomic rearrangements of MSH2 in 14 families (23%), whereas we found no rearrangement of MLH1 and MSH6. Analysis of 31 other families, partially meeting Amsterdam criteria, revealed no additional rearrangement of MSH2. All of the MSH2 rearrangements, except one, corresponded to genomic deletions involving one or several exons. In 8 of 13 families with a MSH2 genomic deletion, the MSH2 promoter was also deleted, and the 5' breakpoint was located either within or upstream the MSH2 gene. This study demonstrates the heterogeneity of MSH2 exonic and promoter rearrangements and shows that, in HNPCC families without detectable MSH2 or MLH1 point mutation, one must consider the presence of MSH2 genomic rearrangements before the involvement of other mismatch repair genes. The simplicity and rapidity of their detection, using fluorescent multiplex PCR, led us to recommend to begin the molecular analysis in HNPCC by screening for MSH2 rearrangements.     

Identification and characterization of a novel MLH1 genomic rearrangement as the cause of HNPCC in a Tunisian family: evidence for a homologous Alu-mediated recombination

High rates of early colorectal cancers are observed in Tunisia suggesting high genetic susceptibility. Nevertheless, up to now no molecular studies have been performed. Hereditary nonpolyposis colorectal cancer (HNPCC) is the most frequent cause of inherited colorectal cancer. It is caused by constitutional mutations in the DNA mismatch repair (MMR) genes. Here, we investigated a Tunisian family highly suspected of hereditary nonpolyposis colorectal cancer (HNPCC). Six patients were diagnosed with a colorectal or an endometrial cancer at an early age, including one young female who developed a colorectal cancer at 22 years and we tested for germline mutations in MMR genes. MMR genes were tested for rearrangements by MLPA (MLH1, MSH2) and the presence of point mutations by sequencing (MLH1, MSH2, MSH6). Moreover, tumors were analyzed for microsatellite instability and expression of MMR proteins, as well as for somatic rearrangements in MLH1 and MSH2 by MLPA. MMR gene analysis by MLPA revealed the presence of a large deletion in MLH1 removing exon 6. Sequence analysis of the breakpoint region showed that this rearrangement resulted from a homologous unequal recombination mediated by a repetitive Alu sequence. Moreover, tumors harbored biallelic deletion of MLH1 exon 6 and loss of heterozygosity at MLH1 intragenic markers, suggesting duplication of the rearranged allele in the tumor. This germline MLH1 rearrangement was associated to a severe phenotype in this family. This is the first report of a molecular analysis in a Tunisian family with HNPCC.     

Genomic deletions of MSH2 and MLH1 in colorectal cancer families detected by a novel mutation detection approach

Hereditary non-polyposis colorectal cancer is an autosomal dominant condition due to germline mutations in DNA-mismatch-repair genes, in particular MLH1, MSH2 and MSH6. Here we describe the application of a novel technique for the detection of genomic deletions in MLH1 and MSH2. This method, called multiplex ligation-dependent probe amplification, is a quantitative multiplex PCR approach to determine the relative copy number of each MLH1 and MSH2 exon. Mutation screening of genes was performed in 126 colorectal cancer families selected on the basis of clinical criteria and in addition, for a subset of families, the presence of microsatellite instability (MSI-high) in tumours. Thirty-eight germline mutations were detected in 37 (29.4%) of these kindreds, 31 of which have a predicted pathogenic effect. Among families with MSI-high tumours 65.7% harboured germline gene defects. Genomic deletions accounted for 54.8% of the pathogenic mutations. A complete deletion of the MLH1 gene was detected in two families. The multiplex ligation-dependent probe amplification approach is a rapid method for the detection of genomic deletions in MLH1 and MSH2. In addition, it reveals alterations that might escape detection using conventional diagnostic techniques. Multiplex ligation-dependent probe amplification might be considered as an early step in the molecular diagnosis of hereditary non-polyposis colorectal cancer.     

Phenotype comparison of MLH1 and MSH2 mutation carriers in a cohort of 1,914 individuals undergoing clinical genetic testing in the United States

BACKGROUND AND AIMS: Lynch syndrome is caused by germ-line mismatch repair gene mutations. We examined the phenotypic differences between MLH1 and MSH2 gene mutation carriers and whether mutation type (point versus large rearrangement) affected phenotypic expression. METHODS: This is a cross-sectional prevalence study of 1,914 unrelated probands undergoing clinical genetic testing for MLH1 and MSH2 mutations at a commercial laboratory. RESULTS: Fifteen percent (285 of 1,914) of subjects had pathogenic mutations (112 MLH1, 173 MSH2). MLH1 carriers had a higher prevalence of colorectal cancer (79% versus 69%, P = 0.08) and younger mean age at diagnosis (42.2 versus 44.8 years, P = 0.03) than MSH2 carriers. Forty-one percent of female carriers had endometrial cancer and prevalence was similar in both groups. Other cancers were more frequent in MSH2 carriers (24% versus 9%, P = 0.001) and their families (P < 0.001). Multivariable analyses confirmed these associations. Of the 1,016 subjects who underwent Southern blot analysis, 42 had large rearrangements (7 MLH1, 35 MSH2). There were no phenotypic differences between carriers with large rearrangements and point mutations. CONCLUSIONS: In this large study of mismatch repair gene mutation carriers from the United States, MLH1 carriers had more colorectal cancer than MSH2 carriers whereas endometrial cancer prevalence was similar. Large genomic rearrangements were more frequent in the MSH2 gene. MSH2 carriers and their relatives have more extracolonic nonendometrial Lynch syndrome-associated cancers and may benefit from additional screening.     

MLH1 and MSH2 protein expression as a pre-screening marker in hereditary and non-hereditary endometrial hyperplasia and cancer

The predictive value of MLH1 or MSH2 protein expression for the presence of truncating germline mutations was examined in benign and (pre)malignant endometrial samples from 3 patient groups: (I) 10 endometrial cancer patients from hereditary non-polyposis colorectal cancer (HNPCC) families with (n = 6) or without (n = 4) a known germline mutation; (II) 15 women from HNPCC families with (n = 7) or without (n = 8) a known germline mutation, who underwent endometrial sampling for non-malignant reasons; (III) 38 endometrial cancer patients <50 years of age, without HNPCC family history. Immunostaining for MLH1 and MSH2 was performed on paraffin-embedded sections. In group III, tumor DNA was examined for microsatellite instability (MSI) and MLH1, MSH2 and MSH6 mutation analysis was carried out. In 6/6 MLH1/MSH2 mutation carriers with endometrial cancer (group I), concordance was found between protein loss in the tumor and the corresponding mutation. In 3 MLH1 mutation carriers, MLH1 protein loss was also observed in concurrent endometrial hyperplasia. In group II, no protein loss was detected in normal endometrial tissue samples; in 3/4 patients with endometrial hyperplasia, MLH1/MSH2 protein loss was observed. In group III, protein loss was detected in 12/38 patients (9 MLH1, 3 MSH2), while in 3/11 patients with concurrent endometrial hyperplasia protein loss was also observed in the hyperplasia. MSI analysis in group III revealed 26 MSI-low and 12 MSI-high tumors. Mutation analysis in 28/38 patients showed only 1 missense MSH6 and no MLH1 or MSH2 germline mutations. In group III, loss of MLH1/MSH2 protein expression was not related to the presence of MSI or MLH1/MSH2 germline mutations. In conclusion, MLH1 or MSH2 protein loss in HNPCC-related endometrial neoplasia is strongly related to corresponding germline mutations. This relation was not clearly present in young sporadic endometrial cancer patients. Immunohistochemical pre-screening of the MLH1 and MSH2 proteins in endometrial hyperplasia or cancer can thus be helpful in HNPCC families. Frequent loss of MLH1 or MSH2 protein in endometrial hyperplasia indicates that this loss is an early event in endometrial carcinogenesis.     

Immunohistochemistry and microsatellite instability testing for selecting MLH1, MSH2 and MSH6 mutation carriers in hereditary non-polyposis colorectal cancer

Hereditary non-polyposis colorectal cancer (HNPCC) represents 1-3% of all colorectal cancers. HNPCC is caused by a constitutional defect in a mismatch repair (MMR) gene, most commonly affecting the genes MLH1, MSH2 and MSH6. The MMR defect results in an increased cancer risk, with the greatest lifetime risk for colorectal cancer and other cancers associated to HNPCC. The HNPCC-associated tumor phenotype is generally characterized by microsatellite instability (MSI) and immunohistochemical loss of expression of the affected MMR protein. The aim of this study was to determine the sensitivity of IHC for MLH1, MSH2 and MSH6, and MSI analysis in tumors from known MMR gene mutation carriers. Fifty-eight paired normal and tumor samples from HNPCC families enrolled in our high-risk colorectal cancer registry were studied for the presence of germline mutations in MLH1, MSH2 and MSH6 by DGGE and direct sequencing. MSI analysis and immunostaining for MLH1, MSH2 and MSH6 were evaluated. Of the 28 patients with a real pathogenic mutation, loss of immunohistochemical expression for at least 1 of these MMR proteins was found, and all except 1 have MSI-H. Sensitivity by MSI analysis was 96%. IHC analysis had a sensitivity of 100% in detecting MMR deficiency in carriers of a pathogenic MMR mutation, and can be used to predict which gene is expected to harbor the mutation for MLH1, MSH2 and MSH6. This study suggests that both analyses are useful for selecting high-risk patients because most MLH1, MSH2 and MSH6 gene carriers will be detected by this 2-step approach. This practical method should have immediate application in the clinical work of patients with inherited colorectal cancer syndromes.     

Epitope-positive truncating MLH1 mutation and loss of PMS2: implications for IHC-directed genetic testing for lynch syndrome

We assessed mismatch repair by immunohistochemistry (IHC) and microsatellite instability (MSI) analysis in an early onset endometrial cancer and a sister’s colon cancer. We demonstrated high-level MSI and normal expression for MLH1, MSH2 and MSH6. PMS2 failed to stain in both tumors, strongly implicating a PMS2 defect. This family did not meet clinical criteria for Lynch syndrome. However, early onset endometrial cancers in the proband and her sister, a metachronous colorectal cancer in the sister as well as MSI in endometrial and colonic tumors suggested a heritable mismatch repair defect. PCR-based direct exonic sequencing and multiplex ligation-dependent probe amplification (MLPA) were undertaken to search for PMS2 mutations in the germline DNA from the proband and her sister. No mutation was identified in the PMS2 gene. However, PMS2 exons 3, 4, 13, 14, 15 were not evaluated by MLPA and as such, rearrangements involving those exons cannot be excluded. Clinical testing for MLH1 and MSH2 mutation revealed a germline deletion of MLH1 exons 14 and 15. This MLH1 germline deletion leads to an immunodetectable stable C-terminal truncated MLH1 protein which based on the IHC staining must abrogate PMS2 stabilization. To the best of our knowledge, loss of PMS2 in MLH1 truncating mutation carriers that express MLH1 in their tumors has not been previously reported. This family points to a potential limitation of IHC-directed gene testing for suspected Lynch syndrome and the need to consider comprehensive MLH1 testing for individuals whose tumors lack PMS2 but for whom PMS2 mutations are not identified.     

The Value of Multi-Modal Gene Screening in HNPCC in Quebec: Three Mutations in Mismatch Repair Genes that would have not been Correctly Identified by Genomic DNA Sequencing Alone

Hereditary non-polyposis colorectal cancer (HNPCC) is a dominantly inherited cancer syndrome caused by a mutation in one of the mismatch repair genes, most frequently MLH1 or MSH2. The rate of mutation detection is influenced by many factors, including the diagnostic methods used. Large deletions, which occur frequently in MLH1 and MSH2, are not detected by exon-by-exon screening methods. Here, we describe three mutations in mismatch repair genes detected using a screening protocol that combines protein truncation test (PTT) analysis and multiplex ligation-dependent probe amplification (MLPA) with genomic and cDNA sequencing. Two of these mutations consist of large deletions in MLH1 that were detected by both MLPA and PTT but that would have been missed by genomic DNA sequencing. The third is a large deletion in MSH2 that could not be detected by PTT because of its location relative to the primers used to amplify the cDNA, or by sequencing. This mutation was detected by MLPA.     

A polymorphism in the ATM gene modulates the penetrance of hereditary non-polyposis colorectal cancer

Germ-line mutations in MLH1 and MSH2 genes predispose to hereditary non-polyposis colorectal cancer (HNPCC) syndrome, but they do not predict a specific phenotype of the disease. We speculated that the ataxia-telangiectasia mutated gene (ATM) was a candidate gene to modulate the phenotypic expression of HNPCC, as heterozygous individuals for germ-line ATM mutations have been considered at higher risk of developing epithelial malignancies. The frequency of the ATM D1853N polymorphism was evaluated in 167 individuals from 20 HNPCC families in which MLH1 or MSH2 germ-line mutations co-segregated with the disease. Among the 67 MLH1 or MSH2 mutation carriers, the ATM 1853N variant was associated with a significantly higher incidence of colorectal and other HNPCC-related cancers, when compared with individuals carrying the ATM 1853D variant [12/13 (92%) vs. 31/54 (57.5%); p = 0.02]. MLH1 and MSH2 mutation carriers who concomitantly carried the ATM 1853N variant, had an 8 times increased risk of developing colorectal and other HNPCC-related cancers (OR: 8.9; p = 0.02), when compared with MLH1 or MSH2 mutation carriers with the ATM 1853D variant. Our results suggest that the ATM D1853N polymorphism modulates the penetrance of MLH1 and MSH2 germ-line mutations.     

Spectrum and frequencies of mutations in MSH2 and MLH1 identified in 1,721 German families suspected of hereditary nonpolyposis colorectal cancer

Mutations in DNA MMR genes, mainly MSH2 and MLH1, account for the majority of HNPCC, an autosomal dominant predisposition to colorectal cancer and other malignancies. The evaluation of many questions regarding HNPCC requires clinically and genetically well-characterized HNPCC patient cohorts of reasonable size. One main focus of this multicenter study is the evaluation of the mutation spectrum and mutation frequencies in a large HNPCC cohort in Germany; 1,721 unrelated patients, mainly of German descent, who met the Bethesda criteria were included in the study. In tumor samples of 1,377 patients, microsatellite analysis was successfully performed and the results were applied to select patients eligible for mutation analysis. In the patients meeting the strict Amsterdam criteria (AC) for HNPCC, 72% of the tumors exhibited high microsatellite instability (MSI-H) while only 37% of the tumors from patients fulfilling the less stringent criteria showed MSI-H; 454 index patients (406 MSI-H and 48 meeting the AC of whom no tumor samples were available) were screened for small mutations. In 134 index patients, a pathogenic MSH2 mutation, and in 118 patients, a pathogenic MLH1 mutation was identified (overall detection rate for pathogenic mutations 56%). One hundred sixty distinct mutations were detected, of which 86 are novel mutations. Noteworthy is that 2 mutations were over-represented in our patient series: MSH2,c.942+3A>T and MLH1,c.1489_1490insC, which account for 11% and 18% of the MSH2 and MLH1 mutations, respectively. A subset of 238 patients was screened for large genomic deletions. In 24 (10%) patients, a deletion was found. In 72 patients, only unspecified variants were found. Our findings demonstrate that preselection by microsatellite analysis substantially raises mutation detection rates in patients not meeting the AC. As a mutation detection strategy for German HNPCC patients, we recommend to start with screening for large genomic deletions and to continue by screening for common mutations in exon 5 of MSH2 and exon 13 of MLH1 before searching for small mutations in the remaining exons.     

Evaluation of microsatellite instability and immunohistochemistry for the prediction of germ-line MSH2 and MLH1 mutations in hereditary nonpolyposis colon cancer families

Forty-eight hereditary nonpolyposis colorectal carcinoma (HNPCC) families for which a tumor sample was available were evaluated for the presence of germ-line mutations in MSH2 and MLH1, tumor microsatellite instability (MSI), and where possible, expression of MSH2 and MLH1 in tumors by immunohistochemistry. Fourteen of 48 of the families had a germ-line mutation in either MSH2 or MLH1 that could be detected by genomic DNA sequencing, and 28 of 48 of the families had MSI-H tumors. Four additional families showed loss of expression of MSH2, and one additional family showed loss of expression of MLH1 but did not have germ-line mutations in MSH2 or MLH1 that could be detected by DNA sequencing. MSI-H, as defined using the National Cancer Institute recommended five-microsatellite panel, had a 100% sensitivity for identifying samples having MSH2 or MLH1 mutations or loss of expression. In contrast, loss of MSH2 and MLH1 expression did not identify all samples having germ-line mutations in MSH2 or MLH1, because in five cases, a mutant protein product was expressed that could be detected by IHC. A combination of the Bethesda criteria for HNPCC and an MSI-H phenotype defined the smallest number of cases having all of the germ-line MSH2 and MLH1 mutations that could be detected by DNA sequencing.     

MLH3 mutation in endometrial cancer

MLH3 is a recently described member of the DNA mismatch repair gene family. Based on its interaction with the MutL homologue MLH1, it was postulated that MLH3 might play a role in tumorigenesis. Germ line and somatic mutations in MLH3 have been identified in a small fraction of colorectal cancers, but the role of MLH3 in colorectal cancer tumorigenesis remains controversial. We investigated MLH3's role in endometrial tumorigenesis through analysis of tumor and germ line DNA from 57 endometrial cancer patients who were at increased risk for having inherited cancer susceptibility. Patients with known MSH2 or MSH6 mutations were excluded as well as those who had MLH1-methylated tumors. Sixteen different variants were identified by single-strand conformational variant analysis. Of the 12 missense changes identified, three were somatic mutations. One patient had a germ line missense variant and loss of heterozygosity (LOH) in her tumor specimen. There was no evidence of MLH3 promoter methylation based on combined bisulfite restriction analysis. The identification of inherited missense variants, somatic missense mutations (present in 3 of 57 tumors), and LOH in the tumor from a patient with a germ line missense change suggest a role for MLH3 in endometrial tumorigenesis.     

Detection of exon deletions and duplications of the mismatch repair genes in hereditary nonpolyposis colorectal cancer families using multiplex polymerase chain reaction of short fluorescent fragments

Large genomic deletions within the mismatch repair MLH1 and MSH2 genes have been identified in families with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome, and their detection represents a technical problem. To facilitate their detection, we developed a simple semiquantitative procedure based on the multiplex PCR of short fluorescent fragments. This method allowed us to confirm in HNPCC families three known deletions of MLH1 or MSH2 and to detect in 19 HNPCC families, in which analysis of mismatch repair genes using classical methods had revealed no alteration, a deletion of exon 5 and a duplication of MSH2 involving exons 9 and 10. The presence of such duplications, the frequency of which is probably underestimated, must be considered in HNPCC families in which conventional screening methods have failed to detect mutations.     

Hereditary nonpolyposis colorectal cancer in endometrial cancer patients

Endometrial cancer is the second most common cancer in hereditary nonpolyposis colorectal cancer (HNPCC). It has often been overlooked to explore the possibility of HNPCC in endometrial cancer patients. Our study was to investigate how many HNPCC patients existed among endometrial cancer patients. Among patients who underwent hysterectomy for endometrial cancer at Seoul National University Hospital from 1996 to 2004, 113 patients were included, whose family history and clinical data could be obtained and tumor specimens were available for microsatellite instability (MSI) testing and immunohistochemical (IHC) staining of MLH1, MSH2 and MSH6 proteins. There were 4 (3.5%) clinical HNPCC patients fulfilling the Amsterdam criteria II, and 2 (2/4, 50%) of them carried MSH2 germline mutations. There were also 8 (7.1%) suspected HNPCC (s-HNPCC) patients fulfilling the revised criteria for s-HNPCC, and one (1/8, 12.5%) of them revealed MLH1 germline mutation. In 101 patients, who were not clinical HNPCC or s-HNPCC, 11 patients showed both MSI-high and loss of expression of MLH1, MSH2 or MSH6 proteins, and 2 (2/11, 18.2%) of them showed MSH6 germline mutations. In 113 patients with endometrial cancer, we could find 5 (4.4%) HNPCC patients with MMR germline mutation and 2 (1.8%) clinical HNPCC patients without identified MMR gene mutation. Family history was critical in detecting 3 HNPCC patients with MMR germline mutation, and MSI testing with IHC staining for MLH1, MSH2 and MSH6 proteins was needed in the diagnosis of 2 HNPCC patients who were not clinical HNPCC or s-HNPCC, especially for MSH6 germline mutation.