Evaluation of MLH1 variants of unclear significance.

Inactivating mutations in the MLH1 gene cause the cancer predisposition Lynch syndrome, but for small coding genetic variants it is mostly unclear if they are inactivating or not. Nine such MLH1 variants have been identified in South American colorectal cancer (CRC) patients (p.Tyr97Asp, p.His112Gln, p.Pro141Ala, p.Arg265Pro, p.Asn338Ser, p.Ile501del, p.Arg575Lys, p.Lys618del, p.Leu676Pro), and evidence of pathogenicity or neutrality was not available for the majority of these variants. We therefore performed biochemical laboratory testing of the variant proteins and compared the results to protein in silico predictions on structure and conservation. Additionally, we collected all available clinical information of the families to come to a conclusion concerning their pathogenic potential and facilitate clinical diagnosis in the affected families. We provide evidence that four of the alterations are causative for Lynch syndrome, four are likely neutral and one shows compromised activity which can currently not be classified with respect to its pathogenic potential. The work demonstrates that biochemical testing, corroborated by congruent evolutionary and structural information, can serve to reliably classify uncertain variants when other data are insufficient.

Features of Patients With Hereditary Mixed Polyposis Syndrome Caused by Duplication of GREM1 and Implications for Screening and Surveillance.

Hereditary mixed polyposis syndrome is a rare colon cancer predisposition syndrome caused by a duplication of a noncoding sequence near the gremlin 1, DAN family BMP antagonist gene (GREM1) originally described in Ashkenazi Jews. Few families with GREM1 duplications have been described, so there are many questions about detection and management. We report 4 extended families with the duplication near GREM1 previously found in Ashkenazi Jews; 3 families were identified at cancer genetic clinics in Israel and 1 family was identified in a cohort of patients with familial colorectal cancer. Their clinical features include extracolonic tumors, onset of polyps in adolescence, and rapid progression of some polyps to advanced adenomas. One family met diagnostic criteria for Lynch syndrome. Expansion of the hereditary mixed polyposis syndrome phenotype can inform surveillance strategies for carriers of GREM1 duplications.

Missed opportunities: Genetic counseling and testing among an ethnically diverse cohort of women with endometrial cancer.

OBJECTIVES: Lynch syndrome (LS) accounts for the majority of inherited endometrial cancers (EC), and the identification of probands presents a unique opportunity to treat and prevent multiple cancers. The diagnosis of EC can provide the indication for women with specific risk factors to undergo genetic testing (GT). We sought to evaluate genetic counseling referrals (GCR) and subsequent GT rates in an ethnically diverse group of high-risk women. METHODS: All women diagnosed with EC between 2011 and 2016 were identified. Risk factors for LS including age, family and personal histories of Lynch-related cancers and loss of tumor mismatch repair (MMR) protein expression were identified from laboratory and medical records. Standard two-sided statistical tests were used. RESULTS: Of 583 women diagnosed with EC, 184 (31.6%) were found to have at least one high-risk characteristic for LS. Among these high-risk women, 58% were given GCR and resulting in only 35% undergoing GT. Ten of the 65 high-risk women who had GT (15.4%) were diagnosed with Lynch syndrome, and all ten met high-risk criteria. Two women of Asian race had tumors exhibiting retained MMR protein expression despite germline testing demonstrating Lynch syndrome. CONCLUSIONS: Many high-risk women do not receive GCR despite a high rate of germline mutations among these women. Improving GCR among high-risk women will lead to more subsequent GT to identify more Lynch syndrome families and prevent additional cancers. Among our ethnically diverse cohort, two women diagnosed with LS had retained MMR protein expression. GCR should be offered to women who possess high-risk characteristics despite normal MMR protein expression.

DNA mismatch repair deficiency and hereditary syndromes in Latino patients with colorectal cancer.

BACKGROUND: The landscape of hereditary syndromes and clinicopathologic characteristics among US Latino/Hispanic individuals with colorectal cancer (CRC) remains poorly understood. METHODS: A total of 265 patients with CRC who were enrolled in the Hispanic Colorectal Cancer Study were included in the current study. Information regarding CRC risk factors was elicited through interviews, and treatment and survival data were abstracted from clinical charts. Tumor studies and germline genetic testing results were collected from medical records or performed using standard molecular methods. RESULTS: The mean age of the patients at the time of diagnosis was 53.7 years (standard deviation, 10.3 years), and 48.3% were female. Overall, 21.2% of patients reported a first-degree or second-degree relative with CRC; 3.4% met Amsterdam I/II criteria. With respect to Bethesda guidelines, 38.5% of patients met at least 1 criterion. Of the 161 individuals who had immunohistochemistry and/or microsatellite instability testing performed, 21 (13.0%) had mismatch repair (MMR)-deficient (dMMR) tumors. dMMR tumors were associated with female sex (61.9%), earlier age at the time of diagnosis (50.4 ± 12.4 years), proximal location (61.9%), and first-degree (23.8%) or second-degree (9.5%) family history of CRC. Among individuals with dMMR tumors, 13 (61.9%) had a germline MMR mutation (MutL homolog 1 [MLH1] in 6 patients; MutS homolog 2 [MSH2] in 4 patients; MutS homolog 6 [MHS6] in 2 patients; and PMS1 homolog 2, mismatch repair system component [PMS2] in 1 patient). The authors identified 2 additional MLH1 mutation carriers by genetic testing who had not received immunohistochemistry/microsatellite instability testing. In total, 5.7% of the entire cohort were confirmed to have Lynch syndrome. In addition, 6 individuals (2.3%) had a polyposis phenotype. CONCLUSIONS: The percentage of dMMR tumors noted among Latino individuals (13%) is similar to estimates in non-Hispanic white individuals. In the current study, the majority of individuals with dMMR tumors were confirmed to have Lynch syndrome. Cancer 2017. © 2017 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society. Cancer 2017;123:3732-3743. © 2017 American Cancer Society.

Mismatch repair deficiency screening via immunohistochemical staining in young Asians with colorectal cancers.

BACKGROUND: The incidence of mismatch repair deficiency in colorectal cancer (CRC) in young people remains unknown in Asians. The present study assessed the clinicopathological features and efficacy of immunohistochemistry screening for Lynch syndrome in young Asian CRC patients. MATERIAL AND METHODS: This was a retrospective review conducted in Singapore General Hospital between January 2006 and December 2010 of 240 unrelated patients under the age of 50. All patients had immunohistochemical (IHC) staining for mismatch repair proteins in resected CRC specimen data retrieved from a prospective computerized database. RESULTS: A total of 21 % (n = 51) of the patients had abnormal IHC staining. Loss of staining for MLH1, MSH2, MSH6, and PMS2 proteins was observed in 10, 4, 6, and 13 % of tumors, respectively. Of the 22 patients who had abnormal staining of MLH1, 13 had concomitant abnormal staining for PMS2. One tumor specimen had abnormal staining in all four proteins. If the Amsterdam criteria alone were to be used, 86 % (n = 44) of the cohort would have not been detected for mismatch repair gene defects. CONCLUSIONS: The overall burden of germline mismatch repair deficiency in the Singapore population may be as high as 21 %. The Amsterdam criteria alone are inadequate to detect Lynch syndrome patients. The use of IHC staining of at least four mismatch repair proteins is a useful screening strategy for Lynch syndrome diagnosis. Routine screening of mismatch repair deficiency may be recommended for all young Asian CRC patients.

High risk of colorectal and endometrial cancer in Ashkenazi families with the MSH2 A636P founder mutation.

BACKGROUND & AIMS: The MSH2 A636P mutation is a founder mutation in Ashkenazi Jews that causes Lynch syndrome, with a prevalence of 0.4%-0.7%. Estimates of age-specific cumulative risk and lifetime risk for colorectal cancer (CRC) and endometrial cancer (EC) specific to carriers of this mutation are not available. METHODS: We studied 27 families with MSH2 A636P gene mutations identified in Israel; 13 were identified via a population-based, case-control study and 14 were identified from a clinical genetics service. Age-specific cumulative risks (penetrance) and hazard ratio (HR) estimates of CRC and EC risks were calculated and compared with the general Ashkenazi population using modified segregation analysis. An ascertainment-corrected likelihood that combined population-based and clinic-based sampling provided a powerful analysis for estimating penetrance. We analyzed 74 cases of CRC (40 in the clinic series and 34 in the population-based series), diagnosed at median ages of 50 years (men) and 49 years (women) in the combined sample. RESULTS: The cumulative risk of CRC at age 70 was 61.62% for men (95% confidence interval [CI], 37.49%-76.45%) and 61.08% for women (95% CI, 39.39%-75.14%), with overall HRs of 31.8 (19.9-51.0) and 41.8 (27.4-64.0), respectively. There were 28 cases of EC, diagnosed at a median age of 53.0 years. The cumulative risk of EC was 55.64% (95% CI, 33.07%-70.58%) with an overall HR of 66.7 (41.7-106.7). CONCLUSIONS: Lifetime risks of CRC and EC in MSH2 A636P carriers are high even after adjusting for ascertainment. These estimates are valuable for patients and providers; specialized cancer screening is necessary for carriers of this mutation.

Risk and epidemiological time trends of gastric cancer in Lynch syndrome carriers in the Netherlands.

BACKGROUND & AIMS: Although gastric cancer forms part of the Lynch syndrome tumor spectrum, the risk of developing gastric cancer in Lynch syndrome families is unknown, resulting in a lack of clear guidelines for surveillance. The aim of this study was to evaluate incidence trends and risk of developing gastric cancer among Lynch syndrome mutation carriers in a Western population. METHODS: Lynch syndrome mutation carriers were selected from the Dutch Hereditary Cancer Registry. The gastric cancer incidence in Lynch syndrome mutation carriers was compared to the gastric cancer incidence in the Dutch population between 1970 and 2003. Standardized incidence ratios were calculated by a Poisson model. Cumulative risks were calculated by Kaplan-Meier analysis. RESULTS: Overall, 2014 Lynch syndrome mutation carriers were identified. Gastric cancer was diagnosed in 32 (1.6%) subjects (male/female: 21/11), 22 (69%) of them had a negative family history of gastric cancer. The standardized incidence ratios of gastric cancer was 3.4 (95% confidence interval, 2.1-5.2) and showed a nonsignificant decline between 1970 and 2003 (P = .30). Absolute risk of developing gastric cancer also showed no significant change over time (P = .51). Lifetime risk of developing gastric cancer was 8.0% in males vs 5.3% in females (P = .02), and 4.8% and 9% for MLH1 and MSH2 carriers, respectively. None of the 378 MSH6 carriers developed gastric cancer (P = .002 vs MLH1 and MSH2 combined lifetime risk). CONCLUSIONS: Lynch syndrome mutation carriers have a substantial risk for gastric cancer, in particular patients with an MLH1 or MSH2 mutation. Family history for gastric cancer is a poor indicator for individual risk. Surveillance gastroscopy for Lynch syndrome patients carrying an MLH1 or MSH2 mutation should therefore be considered.

Lynch syndrome-associated extracolonic tumors are rare in two extended families with the same EPCAM deletion.

OBJECTIVES: The Lynch syndrome (LS) is an inherited cancer syndrome showing a preponderance of colorectal cancer (CRC) in context with endometrial cancer and several other extracolonic cancers, which is due to pathogenic mutations in the mismatch repair (MMR) genes, MLH1, MSH2, MSH6, and PMS2. Some families were found to show a LS phenotype without an identified MMR mutation, although there was microsatellite instability and absence of MSH2 expression by immunohistochemistry. Studies of a subset of these families found a deletion at the 3' end of the epithelial cell adhesion molecule (EPCAM) gene, causing transcription read-through resulting in silencing of MSH2 through hypermethylation of its promoter. The tumor spectrum of such families appears to differ from classical LS. METHODS: Our study of two large families (USA Family R and Dutch Family A) with an EPCAM deletion was carried out using each institution's standard family study protocol. DNA was extracted from peripheral blood and EPCAM deletion analysis was performed. RESULTS: Both families were found to harbor the same deletion at the 3' end of EPCAM. Analysis showed that the deletion originated from a common ancestor. Family R and Family A members showed segregation of CRC with the presence of this EPCAM mutation. Compared with classic LS, there were almost no extracolonic cancers. CONCLUSIONS: Members of Family R and Family A, all with the same EPCAM deletion, predominantly presented with CRC but no LS-associated endometrial cancer, confirming findings seen in other, smaller, LS families with EPCAM mutations. In these EPCAM mutation carriers, cancer surveillance should be focused on CRC.

Characterization of two Ashkenazi Jewish founder mutations in MSH6 gene causing Lynch syndrome.

Founder mutations are an important cause of Lynch syndrome and facilitate genetic testing in specific ethnic populations. Two putative founder mutations in MSH6 were analyzed in 2685 colorectal cancer (CRC) cases, 337 endometrial cancer (EnCa) cases and 3310 healthy controls of Ashkenazi Jewish (AJ) descent from population-based and hospital-based case­control studies in Israel, Canada and the United States. The carriers were haplotyped and the age of the mutations was estimated. MSH6*c.3984_3987dupGTCA was found in 8/2685 CRC cases, 2/337 EnCa cases, and 1/3310 controls, consistent with a high risk of CRC (odds ratio (OR) = 9.9, 95% confidence interval (CI) = 1.2­78.9, p = 0.0079) and a very high risk of EnCa (OR = 19.6, 95% CI = 1.8­217.2, p = 0.0006). MSH6*c.3959_3962delCAAG was identified in 3/2685 CRC cases, 2/337 EnCa cases and no controls. Each mutation was observed on separate conserved haplotypes. MSH6*c.3984_3987dupGTCA and MSH6*c.3959_3962delCAAG probably arose around 585 CE and 685 CE, respectively. No carriers were identified in Sephardi Jews (450 cases and 490 controls). Truncating mutations MSH6*c.3984_3987dupGTCA and MSH6*c.3959_3962delCAAG cause Lynch syndrome and are founder mutations in Ashkenazi Jews. Together with other AJ founder mutations, they contribute substantially to the incidence of CRC and EnCa and are important tools for the early diagnosis and appropriate management of AJ Lynch syndrome patients.

Germline Predisposition to Prostate Cancer in Diverse Populations.

There remains a paucity of data related to germline genetic alterations predisposing patients to prostate cancer. Recent data suggest that African American, Hispanic, and Asian and Pacific Islander men exhibit genetic alterations in both highly penetrant germline genes, including BRCA1/2, ATM, and CHEK2, and the mismatch repair genes associated with Lynch syndrome, as well as low-penetrant single-nucleotide polymorphisms. However, cohort sizes remain small in many studies limiting the ability to determine clinical significance, appropriate risk stratification, and treatment implications in these diverse populations.

Cumulative risks of colorectal cancer in Han Chinese patients with Lynch syndrome in Taiwan.

Patients with Lynch syndrome have a high risk of colorectal cancer (CRC). In this study, we estimated the age- and sex-specific cumulative risks of CRC in Han Chinese patients with Lynch syndrome caused by the pathogenic germline mutations in MLH1 or MSH2 in Taiwan. Based on 321 mutation carriers and 419 non-mutation carriers from 75 pedigrees collected in an Amsterdam criteria family registry in Taiwan, the age- and sex-specific cumulative risks of CRC in male carriers of mutation in MLH1 and MSH2 at the age of 70 years were 60.3% (95% confidence interval (CI) = 31.1%-89.9%) and 76.7% (95% CI = 37.2%-99.0%), respectively. For females, the cumulative risks of CRC at the age of 70 were estimated to be 30.6% (95% CI = 14.3%-57.7%) and 49.3% (95% CI = 21.9%-84.5%) in the carriers of MLH1 and MSH2 germline mutations, respectively. In conclusion, the cumulative risks of CRC at the age of 70 in the Han Chinese patients is higher in mutation carriers than non-mutation carriers and male mutation carriers have a higher cumulative risk of developing CRC than the female mutation carriers.

Implementation of Universal Colorectal Cancer Screening for Lynch Syndrome in Hispanics Living in Puerto Rico.

OBJECTIVE: Colorectal cancer is the leading cause of cancer death in Puerto Rico and third among Hispanics in the USA. Up to 2-4% of colorectal cancer cases are a result of Lynch syndrome (LS), a hereditary cancer syndrome caused by a germline mutation in at least one of the DNA mismatch repair genes. The objective of this study was to determine the prevalence of LS in colorectal tumors during the first 15-months after the implementation of universal tumor-based screening for LS in Puerto Rico. METHODS: A total of 317 colorectal tumors were evaluated in a large private pathology laboratory from September 2014 to December 2015. Clinical characteristics were obtained from the pathology reports. Unadjusted and adjusted logistic regression models were used to estimate the magnitude of association (odds ratio [OR] with 95% confidence intervals [CI]) between absent MMR protein expression and patient characteristics. RESULTS: Most cases (93.4%) were analyzed by immunohistochemistry; 11.8% (35 of 296) had deficient mismatch repair protein expression. While 29 of the 317 cases were subjected to PCR-based microsatellite instability analysis of which 10.3% (3 of 317) had microsatellite instability. In total, 11.0% of the tumors were reported MMR deficient. These tumors were more likely from females and more likely localized in the proximal colon compared to those with proficient MMR expression. CONCLUSIONS: Our data is consistent with the results from other studies including US Hispanics, where approximately 10% of Hispanic individuals with colorectal cancer have microsatellite instability. Our results support universal tumor-based screening for LS among Hispanics in accordance with National Comprehensive Cancer Network guidelines.

Assessment of a Polygenic Risk Score for Colorectal Cancer to Predict Risk of Lynch Syndrome Colorectal Cancer.

It was not known whether the polygenic risk scores (PRSs) that predict colorectal cancer could predict colorectal cancer for people with inherited pathogenic variants in DNA mismatch repair genes-people with Lynch syndrome. We tested a PRS comprising 107 established single-nucleotide polymorphisms associated with colorectal cancer in European populations for 826 European-descent carriers of pathogenic variants in DNA mismatch repair genes (293 MLH1, 314 MSH2, 126 MSH6, 71 PMS2, and 22 EPCAM) from the Colon Cancer Family Registry, of whom 504 had colorectal cancer. There was no evidence of an association between the PRS and colorectal cancer risk, irrespective of which DNA mismatch repair gene was mutated, or sex (all 2-sided P > .05). The hazard ratio per standard deviation of the PRS for colorectal cancer was 0.97 (95% confidence interval = 0.88 to 1.06; 2-sided P = .51). Whereas PRSs are predictive of colorectal cancer in the general population, they do not predict Lynch syndrome colorectal cancer.

Population-based genetic testing for Women's cancer prevention.

Germline mutations in cancer-susceptibility-genes (CSG) can dramatically increase womens' lifetime risk of ovarian, endometrial, breast and bowel cancers. Identification of unaffected carriers is important to enable proactive engagement with highly effective screening and preventive options to minimise cancer risk. Currently, a family-history model is used to identify individuals with CSGs. Complex regional referral guidelines specify the family-history criteria required before an individual is eligible for genetic-testing. This model is ineffective, resource intense, misses >50% CSG carriers, is associated with underutilisation of genetic-testing services and delays detection of mutation carriers. Although awareness and detection of CSG-carriers has improved, over 97% carriers remain unidentified. This reflects significant missed opportunities for precision-prevention. Population-based genetic-testing (PBGT) represents a novel healthcare strategy with the potential to dramatically improve detection of unaffected CSG-carriers along with enabling population risk-stratification for cancer precision-prevention. Several research studies have assessed the impact, feasibility, acceptability, long-term psychological outcomes and cost-effectiveness of population-based BRCA-testing in the Ashkenazi-Jewish population. Initial data on PBGT in the general-population is beginning to emerge and large implementation studies investigating PBGT in the general-population are needed. This review will summarise the current research into the clinical, psycho-social, health-economic, societal and ethical consequences of a PBGT model for women's cancer precision-prevention.

Effectiveness of each Bethesda marker in defining microsatellite instability when screening for Lynch syndrome.

BACKGROUND/AIMS: A panel of five markers (two mononuclotide markers and three dinucleotide markers; the so-called Bethesda markers) has been proposed to define MSI status. However reducing these 5 markers into one or two markers have been suggested to be sufficient for detection of the MLH1- or MSH2-mutated Lynch syndrome. We attempted to examine the effectiveness of each Bethesda marker for the determination of MSI status clinically relevant to Lynch syndrome. METHODOLOGY: We compared the MSI status obtained using each or a combination of two Bethesda markers to those obtained using all five Bethesda markers in 1,531 non-selected colorectal cancer patients. RESULTS: At least one mononucleotide marker was unstable in 94% of the MSI-H tumors defined by the Bethesda markers (126 of 134). After sequencing MLH1 and MSH2 genes from 31 of 86 patients eligible for the genetic test, 18 germline mutations were detected. Seventeen of these mutations were from high MSI tumors, and 1 was from a MSS tumor defined by 5 Bethesda markers. A combination of two mononucleotide markers was able to identify all 17 mutation-positive individuals with MSI-H tumors defined by the five Bethesda markers. CONCLUSIONS: Instability in two mononucleotide markers, BAT26 and BAT25, was most effective markers at defining MSI status. Sensitivity is only slightly impaired by using two mononucleotide markers instead of five markers.

A novel MSH2 germline mutation in a Druze HNPCC family.

Germline mutations in DNA mismatch repair (DNA-MMR) genes, mainly MLH1, MSH2, and MSH6, underlie Hereditary non-polyposis colorectal cancer (HNPCC) and are mostly family-specific, with few reported founder mutations in MSH2 (Ashkenazim) MLH1 (Finnish). No mutations in colon cancer susceptibility genes have ever been reported in Druze individuals, a Moslem related faith encompassing approximately 1,000,000 individuals worldwide. A novel MSH2 mutation is described in a Druze HNPCC family: a multigenerational family with 10 members in 4 generations affected with colorectal cancer (mean age of diagnosis 46.5 years), two with gastric cancer and one--endometrial cancer. Mutational analysis of the MSH2 gene using denaturing gradient gel electrophoresis (DGGE) and direct sequencing revealed the c.702delA mutation in codon 234 of exon 4 of the MSH2 gene leading to a premature early stop in codon 245, p.Thr234fsX245. Analysis of mutation-carrying or presumed carriers individuals' offspring, revealed 11/42 asymptomatic mutation carriers, age range 17-50 years. The mutation was not present in two additional Druze HNPCC families and 20 Druze sporadic colon cancer patients. This is the first mutation ever reported in a colon cancer susceptibility gene in a Druze family and it appears not to be a founder mutation in Druze individuals with HNPCC.

Gynecologic malignancies in Ashkenazi families with the MSH2 A636P founder mutation.

OBJECTIVE: A founder mutation A636P in the MSH2 gene was found to be related to hereditary nonpolyposis colorectal cancer in Ashkenazi Jews. Although the incidence of colorectal cancer in carriers is relatively well established, the frequency of other tumors is less clear. STUDY DESIGN: We studied a consecutive series of 19 carrier families that were cared for by the Clalit Health Studies National Familial Cancer Consultation Service, most of whom were identified through a population-based case-control study of colorectal cancer in northern Israel. RESULTS: Gynecologic cancers, 88% of which (28 cases) were endometrial cancers, were diagnosed in 78.9% of the carrier families and in 26.2% of the women who were at risk, with a mean age at diagnosis of 51.2 years. Forty-six percent of the women with endometrial cancer reported at least 1 other primary tumor. CONCLUSION: Genetic counseling and testing for the MSH2 A636P mutation is indicated for Ashkenazi Jewish women with an endometrial cancer, especially if the cancer is detected before the age of 70 years in women with a personal or family history of colorectal cancer.

MLH1 promoter germline-methylation in selected probands of Chinese hereditary non-polyposis colorectal cancer families.

AIM: To detect the MLH1 gene promoter germline-methylation in probands of Chinese hereditary nonpolyposis colorectal cancer (HNPCC), and to evaluate the role of methylation in MLH1 gene promoter and molecular genetics in screening for HNPCC. METHODS: The promoter germline methylation of MLH1 gene was detected by methylation-specific PCR (MSP) in 18 probands from unrelated HNPCC families with high microsatellite-instability (MSI-H) phenotype but without germline mutations in MSH2, MLH1 and MSH6 genes. At the same time, 6 kindreds were collected with microsatellite-stability (MSS) phenotype but without germline mutations in MSH2, MLH1 and MSH6 genes as controls. The results of MSP were confirmed by clone sequencing. To ensure the reliability of the results, family H65 with nonsense germline mutation at c.2228C > A in MSH2 gene was used as the negative control and the cell line sw48 was used as the known positive control along with water as the blank control. Immunochemical staining of MLH1 protein was performed with Envision two-step method in those patients with aberrant methylation to judge whether the status of MLH1 gene methylation affects the expression of MLH1 protein. RESULTS: Five probands with MLH1 gene promoter methylation were detected in 18 Chinese HNPCC families with MSI-H phenotype but without germline mutations in MSH2, MLH1 and MSH6 genes. Two of the five probands from families H10 and H29 displayed exhaustive-methylation, fulfilling the Japanese criteria (JC) and the Amsterdam criteria (AC), respectively. The other 3 probands presented part-methylation fulfilling the AC. Of the 13 probands with unmethylation phenotype, 8 fulfilled the JC and the Bethesda guidelines (BG), 5 fulfilled the AC. The rate of aberrant methylation in MLH1 gene in the AC group (22.2%, 4/18) was higher than that in the JC/BG groups (5.6%, 1/18) in all HNPCC families with MSI-H phenotype but without germline mutations in MSH2, MLH1 and MSH6 genes. However, no proband with methylation in MLH1 gene was found in the families with MSS phenotype and without germline mutations in MSH2, MLH1 and MSH6 genes. No expression of MLH1 protein was found in tumor tissues from two patients with exhaustive-methylation phenotype, whereas positive expression of MLH1 protein was observed in tumor tissues from patients with partial methylation phenotype (excluding family H42 without tumor tissue), indicating that exhaustive-methylation of MLH1 gene can cause defective expression of MLH1 protein. CONCLUSION: Methylation phenotype of MLH1 gene is correlated with microsatellite phenotype of MMR genes, especially with MSI-H. Exhaustive-methylation of MLH1 gene can silence the expression of MLH1 protein. MLH1 promoter methylation analysis is a promising tool for molecular genetics screening for HNPCC.

Detection of hMSH2 and hMLH1 mutations in Chinese hereditary non-polyposis colorectal cancer kindreds.

AIM: To establish and validate the mutation testing for identification and characterization of hereditary non-polyposis colorectal cancer (HNPCC) in suspected Chinese patients. METHODS: Five independent Chinese kindreds with HNPCC fulfilling the classical Amsterdam criteria were collected. Genomic DNA was extracted after informed consent was obtained. The coding region of hMSH2 and hMLH1 genes was detected by polymerase chain reaction (PCR) and denaturing high-performance liquid chromatography (DHPLC). Mutations identified in the proband by DHPLC were directly sequenced using a 377 DNA sequencer, analyzed with a basic local alignment tool (BLAST), and tested in the corresponding family members by direct DNA sequencing. RESULTS: Mutations were identified in two Chinese HNPCC kindreds. One was the missense mutation of hMSH2 c.1808A-->G resulting in Asp 603 Gly identified in the proband of the fifth HNPCC (HNPCC5) kindred. In the HNP5 kindred, three family members were found to have this mutation and two of them had colorectal cancer. The other mutation of hMLH1 c.1882A-->G was identified in the HNP2 kindred's proband, which might be the nonsense mutation analyzed by BLAST. CONCLUSION: Pedigree investigation and mutation testing of hMSH2 and hMLH1 are the practical methods to identify high-risk HNPCC patients in China.

Clinical features and hMSH2/hMLH1 germ-line mutations in Chinese patients with hereditary nonpolyposis colorectal cancer.

BACKGROUND: At least five mismatch repair (MMR) genes, including hMSH2, hMLH1, hPMS, hPMS2, and hMSH6/GTBP, are associated with hereditary nonpolyposis colorectal cancer (HNPCC). More than 90% of families with HNPCC harbor the hMSH2 and hMLH1 gene mutations. We have analyzed the clinical features of HNPCC among Chinese patients and report the results of screening for mutations in the hMSH2 and hMLH1 genes. METHODS: The data concerning gender, site of colorectal cancer (CRC), age at diagnosis, history of synchronous and/or metachronous colorectal cancer, instance of extracolonic cancers, and histopathology of tumors for 126 patients from 28 independent families with HNPCC were collected. Fifteen of the families met the Amsterdam I criteria, and 13 met the Japanese clinical criteria for diagnosis. Genomic DNA was extracted from the peripheral lymphocytes. Polymerase chain reaction (PCR) and denaturing high-performance liquid chromatography (DHPLC) were used to screen the coding region of the hMSH2 and hMLH1 genes. Samples showing abnormal DHPLC profiles were sequenced. RESULTS: One hundred and seventy malignant neoplasms were found in the 126 patients, of whom 23 had multiple cancers. Ninety-eight of the patients (77.8%) had colorectal cancers, with an average age at onset of 45.9 years and a right-sided predominance. Eight hMSH2 or hMLH1 gene sequence variations were found in 12 families, and a germ-line G204X nonsense mutation in the third exon of hMSH2 was found, representing the first mutation in an MMR gene ever found in people of Chinese Mongolian ethnicity. CONCLUSIONS: HNPCC is a typical autosomally dominant hereditary disease, characterized by early onset, a predominance of proximal colorectal cancer, and multiple synchronous and metachronous colorectal cancers. DHPLC is a powerful tool for detecting mutations in the hMSH2 and hMLH1 genes. Mutations in the first nine exons of the hMLH1 gene were more common in Chinese patients.