Lynch syndrome (LS), an autosomal dominantly inherited disease previously known as hereditary non-polyposis colorectal cancer (HNPCC), leads to a high risk of colorectal cancer (CRC) as well as malignancy at certain sites including endometrium, ovary, stomach, and small bowel (Hampel et al., 2008; Lynch et al., 2009). Clinically, LS is considered the most common hereditary CRC-predisposing syndrome, accounting for about 3% of all CRC cases (Popat et al., 2005). LS is associated with mutations of DNA mismatch repair (MMR) genes such as MLH1, MSH2, MSH6, PMS2, and EPCAM (Ligtenberg et al., 2009; Lynch et al., 2009), which can trigger a high frequency of replication errors in both microsatellite regions and repetitive sequences in the coding regions of various cancer-related genes. Immunohistochemistry (IHC) tests followed by genetic analysis of these mutations play a significant role in diagnosis, treatment determination, and therapeutic response prediction of LS (Lynch et al., 2009; Alex et al., 2017; Ryan et al., 2017). Here, we report substitution of one base-pair in exon 1 of MLH3 (c.1397C>A) and a frameshift mutation in exon 19 of MLH1 (c.2250_2251ins AA) in a 43-year-old Chinese male with an LS pedigree.
Adult ; Asian People/genetics* ; China ; Colorectal Neoplasms, Hereditary Nonpolyposis/genetics* ; Exons ; Female ; Frameshift Mutation ; Germ-Line Mutation ; Humans ; Male ; MutL Protein Homolog 1/genetics* ; MutL Proteins/genetics* ; Pedigree

OBJECTIVETo investigate the expression of cyclooxygenase-2 (COX-2), human mut-l homologue 1 (hMLH1) and human mut-s homologue 2 (hMSH2) proteins in human paired gastric carcinoma (GC) and adjacent normal mucosa, and analyze their relationship with microsatellite instability (MSI).

METHODSThe protein expressions were examined by western blotting. Five MSI loci were assessed by PCR.

RESULTSIn 30 surgically excised GC tissues, the overexpression rate of COX-2, the low expression rate of hMLH1 and hMSH2 were 66.7%, 40% and 33.3%, respectively. Significant differences were found when compared with those of adjacent normal mucosa (P < 0.05). MSI was detected in 13 GC. The number of MSI-H (MSI-High, > or = 2 loci), MSI-L (MSI-Low, only one locus), and MSS (microsatellite stable) were 9, 4 and 17, respectively. The number of low expression rates of COX-2, hMLH1 and hMSH2 in MSI-H were 6, 8 and 5, respectively. There were significant differences compared to that of MSS (P < 0.05).

CONCLUSIONThe results suggest that microsatellite instability pathway is probably involved in the carcinogenesis of gastric carcinoma, which is frequently accompanied by low expression of hMLH1 and hMSH2, and may be also by low expression of COX-2.

Adaptor Proteins, Signal Transducing ; biosynthesis ; genetics ; Cyclooxygenase 2 ; biosynthesis ; genetics ; Humans ; Microsatellite Repeats ; genetics ; MutL Protein Homolog 1 ; MutL Proteins ; Neoplasm Proteins ; biosynthesis ; genetics ; Nuclear Proteins ; biosynthesis ; genetics ; Stomach Neoplasms ; genetics ; metabolism

OBJECTIVETo investigate the expression difference of DNA mismatch repair gene hMLH1 and hMSH2 between schistosomiasis-associated colorectal cancer and sporadic colorectal cancer.

METHODClinical and pathological data of colorectal cancer patients receiving operations in Zhejiang Cancer Hospital between January 2008 and December 2010 were retrospectively analyzed. Patients were divided into schistosomiasis group(n=80) and sporadic group (n=258) according to the preoperative history and pathologic results. Pathological specimens were collected and tissue chips were made to analyze the expression of hMLH1 and hMSH2 by immunohistochemistr.

RESULTSCompared with sporadic group, older age [(62.2 ± 9.6) year vs. (57.2 ± 11.7) year, P=0.000)], lower platelet level [(197.0 ± 59.6) × 10(9)/L vs. (217.0 ± 84.3) × 10(9)/L, P=0.02] and lower WBC level [(5.9 ± 1.9) × 10(9)/L vs. (6.6 ± 2.8) × 10(9)/L, P=0.02] were found in schistosomiasis group. Ratio of low differentiation-undifferentiation tumor was significantly higher in schistosomiasis group [44.2% (34/77) vs. 4.9% (12/247), P<0.05]. Lower positive rate of hMLH1 expression [77.5% (62/80) vs. 98.1% (253/258), P=0.000] and hMSH2 expression [75.0% (60/80) vs. 95.3% (246/258), P=0.000] was found in schistosomiasis group compared with sporadic group. Concurrent schistosomiasis was one of the risk factors of hMLH1/hMSH2 deficiency (RR: 0.913, 95% CI: 0.836-0.997, P=0.043), but not an independent factor (RR: 0.951, 95% CI: 0.867-1.043, P=0.286).

CONCLUSIONSchistosomiasis is associated with lower positive expression of hMLH1 and hMSH2, which indicates that hMLH1/hMSH2 deficiency may be a potential mechanism of schistosomiasis inducing carcinogenesis of colorectal cancer.

Adaptor Proteins, Signal Transducing ; Colorectal Neoplasms ; DNA Mismatch Repair ; Humans ; MutL Protein Homolog 1 ; MutS Homolog 2 Protein ; Nuclear Proteins ; Polymerase Chain Reaction ; Schistosomiasis

OBJECTIVETo illustrate the role of methylation level of hMLH1 gene promoter in different stages of gastric carcinogenesis by methylation-specific PCR (MSP) detection of samples from paracancerous tissue and gastric cancer tissue.

METHODSMethylation status of hMLH1 gene promoter of 40 patients undergoing radical stomach cancer operation in the Tumor Research Institute of China Medical University between January 2006 and August 2006 was detected by MSP. For each patient, 2 samples were chosen from the cancer site, paracancerous tissues of 1 cm, 3 cm, 5 cm away from the cancer site, separately. One sample was used in pathology examination, and the other in methylation detection.

RESULTSPositive rates of hMLH1 gene promoter methylation in the paracancerous tissues of 1 cm, 3 cm, 5 cm away from the cancer site were 10%(4/40), 12.5%(5/40) and 2.5%(1/40) respectively, which were significantly lower than 32.5%(13/40) in cancer site(all P<0.05). Pathological examination showed precancerous lesions in 23 samples of paracancerous 1 cm and 3 cm tissues and normal tissues in 24 samples of paracancerous 5 cm tissues. Positive rates of hMLH1 gene promoter methylation in the cancer site, paracancerous tissue and normal gastric tissue were 32.5%(13/40), 8.7%(2/23) and 0(0/24) (P<0.01). For cancer tissue penetrated the gastric serosa, 8 out of 14 tissue samples were positive methylation (57.1%), which was significantly higher compared with 5 out of 26 tissue samples without penetration of gastric serosa(19.2%). Positive rate of hMLH1 gene promoter methylation in tissue samples with 7 or more of metastatic lymphatic node number was 61.5%(8/13), which was higher compared to that with less than 7(5/27, 18.5%) (P<0.05). No significant differences of positive rate of hMLH1 gene promoter methylation were found between different tumor gross types, tumor grow pattern, tumor differentiation degree, patient age and sex(all P>0.05).

CONCLUSIONHypermethylation of hMLH1 gene promoter may be associated with the carcinogenesis stages and progression of human gastric cancer.

Adaptor Proteins, Signal Transducing ; Cell Transformation, Neoplastic ; China ; DNA Methylation ; Disease Progression ; Humans ; MutL Protein Homolog 1 ; Nuclear Proteins ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; Stomach Neoplasms

OBJECTIVETo study the protein expression pattern of DNA mismatch repair genes hMSH(2), hMLH(1) and the microsatellite instability (MSI) status in the tumor tissue from hereditary nonpolyposis colorectal cancer in the Chinese.

METHODSFifty-eight families fulfilling different clinical criteria including Amsterdam Criteria (AC) (22/24 families, 38 tumors), Japanese Criteria (JC) (12/15 families, 16 tumors) and Bethesda Guidelines (BG) (12/19 patients, 13 tumors) were studied. Monoclonal antibodies against hMSH(2), hMLH(1) proteins and a panel of microsatellite markers (5 loci) including BAT26, BAT25, D2S123, D5S346 and D17S250 were used for study.

RESULTSMSI-H was identified in all 22 (100%) AC tumors, with 81.8% (18/22) showing altered hMSH(2) or hMLH(1) expression; in 14/15 (93.8%) JC cancer, 1/1 (100%) JC adenoma, with 45.5% (5/11) showing altered hMSH(2) or hMLH(1) expression; and in 7/13 (53.8%) BG tumors, with 4/7 showing loss of hMSH(2) or hMLH(1) gene expression.

CONCLUSIONThe frequency of MSI-H and loss of mismatch repair protein are different in the families fulfilling different clinical criteria. Amsterdam Criteria and Japanese Criteria are the two most useful criterion systems for identifying mismatched repair defective tumors. However, Bethesda Guidelines should also be used for detecting more such tumors. The combination of immunohistochemical methods and microsatellite instability analysis is an effective strategy to detect the mismatch repair defective tumors. A close correlation does exist between hMSH(2), hMLH(1) protein expression pattern and MSI status.

Adaptor Proteins, Signal Transducing ; Base Pair Mismatch ; Carrier Proteins ; Colorectal Neoplasms, Hereditary Nonpolyposis ; genetics ; DNA Repair ; DNA-Binding Proteins ; Humans ; Immunohistochemistry ; Microsatellite Repeats ; MutL Protein Homolog 1 ; MutS Homolog 2 Protein ; Neoplasm Proteins ; genetics ; Nuclear Proteins ; Proto-Oncogene Proteins ; genetics

OBJECTIVESTo determine the germ-line mutations of hMSH2 and hMLH1 genes in Chinese hereditary nonpolyposis colorectal cancer (HNPCC) families' probands or in patients fulfilling different clinical criteria or guidelines; to clarify the nature and distribution of the mutations; to evaluate the sensitivity of different clinical criteria in mutation prediction.

METHODSThe entire coding regions (35 exons including exon-intron boundaries) of hMSH2 and hMLH1 genes were directly sequenced in 24 Amsterdam criteria (AC) probands, 15 Japanese criteria (JC) probands (except AC kindreds) and 19 Bethesda guidelines (BG) patients (except two former groups). All available affected and unaffected members from families of those with mutations were screened for mutation.

RESULTSIn 16 unrelated families selected by the different clinical criteria, 17 germ-line mutations were found with 11 (64.7%) of hMLH1 and 6 (35.3%) of hMSH2. Two mutations were identified in one of the families. Among the 17 germ-line mutations, 12 had not been reported previously. A diversified mutation spectrum was found, but 6 hMLH1 mutations were found to be concentrated in the region encompassing exon 14, 15 and 16. There was a wide spectrum of mutation type including frame shift, nonsense, splice site mutation, in frame insertion or deletion and missense mutations. The mutation detection rate of hMSH2 and hMLH1 in the AC group was significantly higher than that in the JC group (12/24 vs. 3/15). On the other hand, a low mutation rate (1/19) was detected in 19 BG patients. The mutation cosegregated with disease. Besides, three different genotypes in tumors from probands of mutation-positive families were found.

CONCLUSIONShMSH2 and hMLH1 mutations in Chinese HNPCC families show a wide spectrum. It seems that hMLH1 gene is involved more frequently than hMSH2 gene in Chinese HNPCC families. Different clinical criteria predict mutations with different sensitivities. The Amsterdam Criteria are most sensitive, while Japanese Criteria are highly practical and the Bethesda Guidelines are also practical to some extent. Gene mutations cosegregate with the disease phenotype. Carriers with no symptom in HNPCC families are most vulnerable groups, follow-ups are required for this group to get early diagnosis and to prevent the development of CRCs.

Adaptor Proteins, Signal Transducing ; Carrier Proteins ; Colorectal Neoplasms, Hereditary Nonpolyposis ; genetics ; DNA-Binding Proteins ; Germ-Line Mutation ; Humans ; Microsatellite Repeats ; MutL Protein Homolog 1 ; MutS Homolog 2 Protein ; Neoplasm Proteins ; genetics ; Nuclear Proteins ; Pedigree ; Proto-Oncogene Proteins ; genetics

OBJECTIVETo assess the role of methylated mismatch repair (MMR) genes (hMLH1, hMSH2 and hMSH3) in the carcinogenesis and progression of hepatocellular carcinoma (HCC).

METHODSSamples of 38 cases of HCC along with their corresponding noncancerous tissues, 2 samples of donated normal tissue and 6 cell lines were collected and subject to the methylation-specific PCR (MSP) to examine promoter methylation status of MLH1, MSH2 and MSH3. Six tumor cell lines were analyzed before and after 5-aza-2'-deoxycytidine treatment. In addition, alterations of mRNA expression of MMRs were investigated by quantitative reverse transcription-PCR.

RESULTSCpG island methylation of hMLH1 and hMSH2 was observed in 13.2% (5 of 38 samples) and 68.4% (26 of 38 samples) respectively in HCC, 2.6% (1 of 38 samples) and 55.3% (21 of 38) respectively in corresponding noncancerous tissues, but not in normal control tissues. Promoter methylation of the hMSH2 gene was present in 83.3% of cell lines tested (5/6), but none were observed for the hMLH1 gene. Promoter methylation of the hMSH3 gene was not identified in any tissue samples or cell lines. After 5-aza-2'-deoxycytidine treatment, hMSH2 methylation was induced or completely reversed, and its mRNA expression was increased in most cell lines.

CONCLUSIONSOur results suggest that promoter hypermethylation of hMLH1 and hMSH2 genes is common in HCC. Particularly, there is a high frequency of methylation of hMSH2 in both cancer and noncancerous tissues, but not in normal control tissue. Therefore, hypermethylation of MMR genes, especially hMSH2, may be involved in the carcinogenesis of HCC and may serve as an early diagnostic marker for HCC. The close correlation between hMSH2 methylation and low expression of its mRNA suggests that hMSH2 methylation is an important pathway in the regulation of gene expression.

Adaptor Proteins, Signal Transducing ; Azacitidine ; analogs & derivatives ; pharmacology ; Base Pair Mismatch ; genetics ; Carcinoma, Hepatocellular ; genetics ; Carrier Proteins ; biosynthesis ; genetics ; Cell Line, Tumor ; DNA Methylation ; DNA Modification Methylases ; antagonists & inhibitors ; DNA Repair ; genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Liver Neoplasms ; genetics ; MutL Protein Homolog 1 ; MutL Proteins ; Neoplasm Proteins ; biosynthesis ; genetics ; Nuclear Proteins ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics

Adaptor Proteins, Signal Transducing ; Animals ; Carrier Proteins ; CpG Islands ; DNA Methylation ; Genes, Tumor Suppressor ; Genes, p16 ; Genes, p53 ; Humans ; MutL Protein Homolog 1 ; Mutation ; Neoplasm Proteins ; genetics ; Neoplasms ; genetics ; Nuclear Proteins

OBJECTIVETo evaluate the application of mismatch repair (MMR) genes proteins expression to screen for Lynch syndrome in colorectal cancer patients.

METHODSOne hundred consecutive colorectal cancers cases collected from 2012 to 2013 were tested immunohistochemically for the protein expression of MLH1, MSH2, MSH6 and PMS2, and also by the ARMS method for the mutation status of BRAF genes in those cases lacking protein expression for MLH1.

RESULTSThe result of MMR immunocytochemistry showed that nine of 100 cases lacked MMR protein expression, including three cases each that were MLH1-/PMS2- and MSH2-/MSH6- respectively, two cases were MLH6- and one case was PMS2-; overall, the majority of these cases lacked protein expression of MLH1 and MSH2. The BRAF genes mutation test showed one case of mutation, indicating that the patient might have MLH1 gene methylation as a result of the mutation of BRAF genes, and that was a sporadic case. The age of onset for patients lacking MMR protein expression was lower than patients with sporadic colorectal cancer (P = 0.011). Colorectal cancers associated with the lack of MMR protein expression mostly occurred in the right colon (P = 0.001), and histomorphologically were often accompanied by mucinous adenocarcinoma (P = 0.010) and tumor lymphocytic infiltration.

CONCLUSIONImmunohistochemical staining for MMR proteins in patients with colorectal cancer, accompanied by testing for BRAF genes mutation, may be an effective approach to screen for Lynch syndrome.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Colorectal Neoplasms, Hereditary Nonpolyposis ; diagnosis ; genetics ; DNA Mismatch Repair ; Humans ; Immunohistochemistry ; MutL Protein Homolog 1 ; Mutation ; Nuclear Proteins ; genetics ; metabolism ; Proto-Oncogene Proteins B-raf ; genetics ; metabolism

BACKGROUND AND OBJECTIVEVarious factors affect the radioresistance of tumor cells, with unknown molecular mechanism(s). Many genes have been found to associate with the radioresistance of tumor cells, however, the precise mechanism of these genes have not been elucidated. This paper was to analyze the differential expressions of DNA repair genes in esophageal carcinoma cells at different time after X-ray irradiation, and to investigate the role of these DNA repair genes in radiation resistance.

METHODSEsophageal cancer parental cells Seg-1 were treated with continuous 2 Gy of fractionated irradiation until the total dose reached 60 Gy to establish the radioresistant cell line Seg-1R. Total RNA was extracted from each cell line at 0, 8, and 24 h after irradiation. Illumine Human-6 V3 microarray was used to identify differentially expressed genes between parental and radioresistant cells. Ten genes involved in DNA repair were obtained and their expressions at different time points after irradiation were analyzed by Gene Ontology analysis.

RESULTSTen DNA repair associated genes were found to be differentially expressed. Three of these genes, SLK, HMGB1, and PMS1, were not only differentially expressed between parental and radioresistant cell lines, but also expressed differently at different time points after irradiation in the same cell line.

CONCLUSIONSPMS1 may be an important factor involved in the mechanism of radioresistance of esophageal carcinoma cells.

Cell Line, Tumor ; radiation effects ; DNA Repair ; genetics ; DNA, Neoplasm ; genetics ; Esophageal Neoplasms ; genetics ; pathology ; Gene Expression Regulation, Neoplastic ; radiation effects ; Humans ; MutL Proteins ; Neoplasm Proteins ; genetics ; metabolism ; Oligonucleotide Array Sequence Analysis ; Radiation Tolerance ; Transcriptome ; X-Rays