Seven novel MLH1 and MSH2 germline mutations in hereditary nonpolyposis colorectal cancer

Hereditary nonpolyposis colorectal cancer (HNPCC) is the most frequent hereditary form of colorectal cancer and is caused by germline mutations in mismatch repair (MMR) genes. The majority of mutations occur in MLH1 and MSH2. We report hereby seven novel germline mutations in these two genes (five in MLH1 and two in MSH2). All mutations have been found in families fulfilling criteria of the Bethesda guidelines and four of which also fulfilled the Amsterdam criteria. We identified three insertions or deletions of 1 bp leading to premature stop codons (MLH1: c.341delC, c.1413‐1414insA; MSH2: c.1119delG) and three nonsense mutations (MLH1: c.67G>T [E23X], c.436C>T [Q146X]; MSH2: c.1857T>G [Y619X]). The corresponding tumors showed a high level of microsatellite instability (MSI‐H) and a complete loss of expression of the affected protein. In addition, a missense mutation in MLH1 was identified (c.1984A>C [T662P]). The respective tumor also showed a high level of microsatellite instability but a reduced, rather then lost, expression of the MLH1‐protein. This missense mutation was not found in 107 healthy control individuals and in 54 HNPCC patients. © 2001 Wiley‐Liss, Inc.     

Novel MLH1 mutations and a novel MSH2 polymorphism identified by SSCP and DHPLC in Portuguese HNPCC families

Mismatch repair genes MSH2 and MLH1 are the two major genes implicated in hereditary nonpolyposis colorectal cancer. For the past years, we have successfully searched for mutations in both genes in affected Portuguese families, by SSCP and DNA sequencing analysis but because of the advantages that DHPLC offers, we have established conditions in our laboratory to use this new method. While screening for mutations by both methods, in 35 individuals belonging to HNPCC Portuguese families, 4 novel MLH1 mutations (c.307-1G>C; c.1023delG [p.R341fsX366]; c.2154_2155delCA [p.H718fsX721], c.2154_2155dupCA [p.I719fsX782]), an unclassified variant (c.-28A>T) and one silent MSH2 polymorphism (c.2766T>C) have been identified.     

Eight novel germline MLH1 and MSH2 mutations in hereditary non‐polyposis colorectal cancer families from Spain

Germline mutations in the MLH1 and MSH2 genes, account for the majority of HNPCC families. We have screened such families from Spain by using DGGE analysis and subsequent direct sequencing techniques. In eight families we identified six novel MLH1 and two novel MSH2 mutations comprising one frame shift mutation (c.1420 del C), two missense mutations (L622H and R687W), two splice site mutations (c.1990‐1 G>A and c.453+2 T>C and one nonsense mutation (K329X) in the MLH1 gene as well as two frame shift mutations (c.1979‐1980 del AT and c.1704‐1705 del AG) in the MSH2 gene. Our analysis contributes to the further characterization of the mutational spectrum of MLH1 and MSH2 genes in HNPCC families. © 2001 Wiley‐Liss, Inc.     

Tumours from MSH2 mutation carriers show loss of MSH2 expression but many tumours from MLH1 mutation carriers exhibit weak positive MLH1 staining

Microsatellite analysis (MSA) in tumour tissue is useful for pre-selection of hereditary non-polyposis colorectal cancer (HNPCC) patients for mutation screening, but is time-consuming and cost-intensive. Immunohistochemistry (IHC) for expression of MLH1 and MSH2 proteins is simple, fast, and indicates the affected gene. IHC has therefore been proposed as an alternative pre-screening method. However, some authors report a lower sensitivity of IHC compared with MSA. The present study reports IHC results for MSH2 and MLH1 performed in 82 tumours with high microsatellite instability (MSI-H) from 81 carriers of pathogenic mutations in MSH2 or MLH1. One hundred per cent (38/38) of the tumours from MSH2 mutation carriers showed loss of MSH2 staining; in all cases, the affected MSH2 gene was predicted correctly by IHC. Complete loss of MLH1 expression was observed in 66% (29/44) of MLH1 mutation carriers. Weak positive MLH1 staining was observed in 14 (32%) cases and, in one case, normal MLH1 staining was seen. The pathologist was aware of the weak staining pattern as an indicator of an MLH1 mutation; 98% of the MLH1 mutations were predicted correctly. To evaluate whether weak positive MLH1 staining is observed more often with in-frame or missense mutations, IHC data from 23 MSI-H tumours from carriers of unspecified variants were added and mutations were grouped into truncating mutations, large non-truncating deletions, and small non-truncating mutations. Weak MLH1 staining was observed in all three categories and it is postulated that other factors, such as mutation of the second allele, also influence protein expression. In conclusion, IHC can be regarded as a very useful method for selecting HNPCC patients for mutation analysis, as long as it is interpreted by an experienced pathologist. The high specificity of IHC in terms of indicating the affected gene is useful for evaluating unspecified variants. However, the staining pattern does not predict whether the underlying germ-line mutation is truncating or not.     

A modified multiplex PCR assay for detection of large deletions in MSH2 and MLH1

A method for detection of large genomic deletions in the MSH2 and MLH1 genes based on multiplex PCR and quantitative evaluation of PCR products is presented. All 35 exons of MSH2 and MLH1 were screened simultaneously in seven PCR reactions, each of them including primers for both genes. The method is reliable for uncovering large genomic deletions in patients suspected of HNPCC. With this method, six novel deletions were identified, two in MSH2: EX1_10del and EX1_16del (representing deletion of the entire MSH2 gene); and four in MLH1: EX1_10del in two unrelated patients, EX3_5del, and EX4del. The deletions were detected in 18 unrelated patients in whom no germline mutation had been identified by SSCP and DHPLC. These results indicate that our modified multiplex PCR assay is suited for the detection of large deletions both in the MSH2 and MLH1 gene and therefore represents an additional valuable tool for mutation screening in HNPCC families.     

Germline MSH2 and MLH1 mutational spectrum including large rearrangements in HNPCC families from Poland (update study)

Germline mutations in the DNA mismatch repair genes MSH2 and MLH1 account for a significant proportion of hereditary non-polyposis colorectal cancer (HNPCC) families. One approach by which development of an efficient DNA-testing procedure can be implemented is to describe the nature and frequency of common mutations in particular ethnic groups. Two hundred and twenty-six patients from families matching the Amsterdam II diagnostic criteria or suspected HNPCC criteria were screened for MSH2 and MLH1 germline mutations. Fifty different pathogenic mutations were found, 25 in MSH2 and 25 in MLH1. Twenty-four of these had not previously been described in other populations. Among our 78 families with MSH2 or MLH1 mutations, 54 (69.2%) were affected by recurrent mutations including 38 found at least twice in our own series. Two of the most frequent alterations were a substitution of A to T at the splice donor site of intron 5 of MSH2 and a missense change (A681T) of MLH1 found in 10 and eight families, respectively. Among large deletions detected by the multiplex ligation-dependent probe amplification assay, exon 9 deletions in the MSH2 gene were found in two families. Our results indicate that a screening protocol specific for the Polish population that is limited to the detection of all reported mutations will result in the identification of the majority of changes present in MLH1 and MSH2 genes in Polish HNPCC kindreds.     

Four new mutations in the DNA mismatch repair gene MLH1 in colorectal cancers with microsatellite instability

Hereditary nonpolyposis colorectal cancer (HNPCC) is frequently associated with inherited mutation in one of four DNA mismatch repair genes. Somatic mutations in the same genes are also found in a subset of sporadic colorectal cancers. A defect in DNA mismatch repair results in an RER (replication error) tumor phenotype. We screened 110 archival and 11 prospectively acquired colorectal cancers for the RER phenotype. A total of 22 cancers were RER-positive. RER-positive tumors were investigated for mutations in the DNA mismatch repair gene MLH1 using single-strand-conformation-polymorphism (SSCP) analysis. We identified four previously undescribed mutations in four different samples. Three mutations were exonic: a point mutation at codon 69 (AGG→AAG [arg→lys]); a single base pair deletion at codon 42/43 (GCAAAATCC→GCAAATCC) leading to a new stop codon downstream; and a point mutation at codon 757 (TAA→TAT) [termination→tyr] which extend the MLH1 peptide by 36 amino acids. The fourth mutation was a 1 base pair insertion six base pairs 5′ to the start of exon 14 (tttgtttt→tttggtttt). The mutations were not seen in the patients' constitutional DNA. The somatic MLH1 mutations identified appear to be causally associated with the RER phenotype. Hum Mutat 12:73, 1998. © 1998 Wiley-Liss, Inc.     

Assessing How Reduced Expression Levels of the Mismatch Repair Genes MLH1, MSH2, and MSH6 Affect Repair Efficiency

Lynch syndrome (LS), the most common familial colon cancer, is associated with mismatch repair (MMR) malfunction. As mutation carriers inherit one normal and one defected MMR gene allele, cancer risk can be considered as limited amount of normal MMR gene product. How reductions in different MMR gene expressions affect MMR capability is, however, not known. The in vitro MMR assay is a method for the pathogenicity assessment of MMR gene variants causing functional or expressional defects and thus also suitable to evaluate the effects of reduced expression of normal mRNA. Here, the assay was applied to quantify repair efficiencies of human cells retaining varying expression levels (25%/50%/75%) of the main LS susceptibility genes MLH1, MSH2, or MSH6. Compared with the shRNA knockdown control, already a 50% reduction in mRNA levels could be detected as decreased MMR function although without statistical significance in MLH1. In MSH2 and MLH1, total loss of MMR was achieved with 25% expression, whereas in MSH6 and MSH2, the repair capability decreased significantly already with 75% expression. Our results provide a preliminary indication of relative expressions required for wild‐type function and suggest that the in vitro MMR assay could be used to recognize expression levels indicative of LS.     

DNA错配修复基因MSH2和MLH1单核苷酸多态性与食管癌发生风险相关性研究

目的:探讨DNA错配修复基因MSH2和MLH1单核苷酸多态性对于食管癌易感性的潜在作用。方法:采用医院为基础的病例-对照研究方法,应用PCR-RFLP检测包括正常对照132例,食管癌患者169例MSH2c.2063TG和MLH1IVS14-19AG两个基因多态性位点的基因型。通过Logistic回归分析计算出比值比(OR)和95%置信区间(95%CI),估计不同基因型频率分布与食管癌发生风险的关系。结果:MSH2c.2063TG携带突变等位基因个体发生食管癌的风险是非携带者的3.24倍。MLH1IVS14-19AG突变等位基因携带者发生食管癌风险是非携带者的1.58倍。对MSH2和MLH1基因交互作用分析发现两突变基因型携带者发生食管癌风险大大增加并具有显著的统计学意义。结论:DNA错配修复基因MSH2c.2063G突变等位基因和MLH1IVS14-19G突变等位基因可能在促成食管癌发生过程起到一定作用。     

Polymorphisms and HNPCC: PMS2‐MLH1 protein interactions diminished by single nucleotide polymorphisms

Hereditary nonpolyposis colorectal cancer (HNPCC) is one of the most common autosomal dominant inherited diseases. Mutations in the human mismatch repair (MMR) proteins MLH1, MSH2, MSH6, PMS1, and PMS2 have been found to co‐segregate with HNPCC. The MLH1 and MSH2 proteins have been demonstrated to interact with PMS1, PMS2, and MSH6 proteins. A previous study reported that missense mutations in specific regions of MLH1 can lead to defects in protein–protein interactions with PMS2. Here we report that three missense alterations previously identified as single nucleotide polymorphisms (SNPs) in PMS2 (P511K, T597S, and M622I) cause defective protein–protein interactions with MLH1, even though the alterations are not in the previously reported interaction domain. These results suggest that an additional domain in PMS2 affects MLH1–PMS2 interaction. This study also demonstrates that SNPs can result in gene alterations that indeed have a functional effect on protein phenotype. Thus, these three SNPs may ultimately represent variants with an increased risk factor for tumorgenesis in HNPCC. This study is one of the first to use a functional assay to appraise the role of SNPs and suggests that traditional definitions of polymorphisms and mutations are in need of reconsideration. Hum Mutat 19:108–113, 2002. © 2002 Wiley‐Liss, Inc.     

两个遗传性非患肉性结直肠癌家系中MLH1和MSH2基因的突变检测

目的 确定两个遗传性非息肉性结直肠癌(hereditary nonpolyposis colorectal cancer,HNPCC)家系的致病基因,选择MLH1基因和MSH2基因进行突变检测.方法 采用聚合酶链反应结合DNA直接测序法,对两个遗传性非息肉性结直肠癌家系的患者进行MLH1基因和MSH2基因的突变检测;发现变异后,采用PCR-限制性片段长度多态性或直接测序法鉴定此变异是否属于突变.结果 在家系A的患者中发现了位于MLH1基因第3外显子内的新突变c.243_244 insA;在家系B的患者中发现了MSH2基因第7外显子内的c.1215_1218dupCCGA突变,这两个突变都导致了编码蛋白的提前终止.结论 MLH1基因的c.243_244insA突变和MSH2基因的c.1215_1218dupCCGA突变分别是导致家系A和家系B发生遗传性非息肉性结直肠癌的致病突变.     

A mononucleotide markers panel to identify hMLH1/hMSH2 germline mutations

Hereditary NonPolyposis Colorectal Cancer (Lynch syndrome) is an autosomal dominant disease caused by germline mutations in a class of genes deputed to maintain genomic integrity during cell replication, mutations result in a generalized genomic instability, particularly evident at microsatellite loci (Microsatellite Instability, MSI). MSI is present in 85-90% of colorectal cancers that occur in Lynch Syndrome. To standardize the molecular diagnosis of MSI, a panel of 5 microsatellite markers was proposed (known as the "Bethesda panel"). Aim of our study is to evaluate if MSI testing with two mononucleotide markers, such as BAT25 and BAT26, was sufficient to identify patients with hMLH1/hMSH2 germline mutations. We tested 105 tumours for MSI using both the Bethesda markers and the two mononucleotide markers BAT25 and BAT26. Moreover, immunohistochemical evaluation of MLH1 and MSH2 proteins was executed on the tumours with at least one unstable microsatellite, whereas germline hMLH1/hMSH2 mutations were searched for all cases showing two or more unstable microsatellites. The Bethesda panel detected more MSI(+) tumors than the mononucleotide panel (49.5% and 28.6%, respectively). However, the mononucleotide panel was more efficient to detect MSI(+) tumours with lack of expression of Mismatch Repair proteins (93% vs 54%). Germline mutations were detected in almost all patients whose tumours showed MSI and no expression of MLH1/MSH2 proteins. No germline mutations were found in patients with MSI(+) tumour defined only through dinucleotide markers. In conclusion, the proposed mononucleotide markers panel seems to have a higher predictive value to identify hMLH1 and hMSH2 mutation-positive patients with Lynch syndrome. Moreover, this panel showed increased specificity, thus improving the cost/effectiveness ratio of the biomolecular analyses.     

Germ line MLH1 and MSH2 mutations in Taiwanese Lynch syndrome families: characterization of a founder genomic mutation in the MLH1 gene

This multicenter study evaluated the mutation spectrum and frequencies of the MLH1 and MSH2 genes and determined the occurrence of large genomic deletions in 93 unrelated Taiwanese families that fulfilled the Amsterdam criteria II by denaturing high-performance liquid chromatography analysis, DNA sequencing for aberrant chromatograms, and multiplex ligation-dependent probe amplification analysis. In total, 38 pathogenic mutations (10 large deletions and 28 point mutations or small deletion/insertions) in the MSH2 or MLH1 gene were identified in 61 of the 93 families (66%). Three of the 10 large deletions and 14 of the 28 point mutations or small insertions/deletions have not been reported elsewhere. Three mutations in the MLH1 gene, the MLH1 c.1846_1848delAAG (5 families), deletion exons 11–15 (4 unrelated families), and MLH1 c.793C>T (13 unrelated families), accounted for 35% of all cases with pathogenic mutations. Haplotype analysis indicated that mutant c.793C>T alleles were derived from two distinct common founders that might be inherited from a single ancestor of presumably Chinese origin. As a mutation detection strategy for Taiwanese Lynch syndrome patients, we recommend that diagnosis starts with screening for large genomic deletions and continues by screening for common mutations in exons 10 and 16 of the MLH1 gene prior to searching for small mutations in the remaining exons.     

Mutation sharing,predominant involvement of the MLH1 gene and description of four novel mutations in hereditary nonpolyposis colorectal cancer

Worldwide, the DNA mismatch repair genes MSH2 and MLH1 account for a major share and almost equal proportions of hereditary nonpolyposis colorectal cancer (HNPCC). Furthermore, the predisposing mutation usually varies from kindred to kindred. In this study, we screened 29 verified or putative HNPCC kindreds from Finland for mutations in these two genes and found 8 different mutations, 7 in MLH1 and 1 in MSH2, occurring in 13 families. Four of these mutations were novel. Altogether, we have to date studied 81 kindreds for mutations and 12 different mutations in 52 families have been identified, 10 in MLH1 and 2 in MSH2. These data show that Finnish HNPCC kindreds are characterized by the predominant involvement of MLH1 (49/52, 94% of the families) and a high rate of shared mutations (5/12, 42%) offering unique possibilities for mutation screening for both research and diagnostic purposes. Hum Mutat 11:482–483, 1998. © 1998 Wiley-Liss, Inc.     

Germline novel MSH2 deletions and a founder MSH2 deletion associated with anticipation effects in HNPCC

Hereditary non-polyposis colorectal cancer (HNPCC) is caused by inactivating mutations of DNA mismatch repair genes. Large genomic rearrangements in these genes have been increasingly recognized as important causes of HNPCC. Using multiplex ligation-dependent probe amplification, we identified three MSH2 deletions in Italian patients with HNPCC (proband A: exons 1-3, proband M: exon 8, and proband C: exons 1-6). Deletion breakpoint sequencing allowed us to develop rapid polymerase chain reaction-based mutation screening, which confirmed the presence of the deletions in affected and asymptomatic individuals of families A, C, and M. While the exon 8 and exon 1-3 deletions appear to be novel, the MSH2 1-6 deletion found in family C is identical to the one recently documented in two branches of another unrelated Italian family (family V+Va). Haplotype analysis showed that the kindreds C and V+Va (both from northeastern Italy, both displaying clinical features of the Muir-Torre syndrome) shared a seven-locus haplotype, indicating that the MSH2 1-6 deletion is probably a founder mutation. Families A, C, M, and V+Va all showed progressively earlier cancer-onset ages in successive generations. Analysis of 23 affected parent-child pairs in the four kindreds showed median anticipation of 12 years in offsprings' onset of cancer (p = 0.0001). No birth cohort effect was found. This is the first significant evidence of anticipation effects in HNPCC families carrying MSH2 deletions.     

Relationship between MUC5AC and altered expression of MLH1 protein in mucinous and non-mucinous colorectal carcinomas

The main purpose of this study was to examine the expression of mucins and mismatch repair proteins in colorectal carcinomas. The immunohistochemical distribution of apomucins MUC2, MUC5AC, and the expression of MLH1 and MSH2 proteins were examined in 76 mucinous and 60 non-mucinous colorectal carcinomas. MUC2 was noted in all mucinous carcinomas, whereas MUC5AC was present in 41 cases only (54%). In non-mucinous carcinomas, MUC2 was expressed in 61.7% of the tumors; by contrast, MUC5AC was present in 20% of the cases. The expression level of apomucins was significantly different in mucinous and non-mucinous lesions (p<0.001). Twenty-seven (35.5%) of the mucinous carcinomas showed no MLH1 expression, whereas 11 (18.3%) of the non-mucinous tumors did. This difference was statistically significant (p<0.005). Altered expression of MSH2 protein was never observed. The lack of MLH1 expression was considerably more frequent in carcinomas with secretion of MUC5AC (p<0.005). Our study has demonstrated this close relationship by immunohistochemical methods. In summary, our data show: (1) differences in the expression of mucins between mucinous and non-mucinous tumors; (2) a high frequency of altered MLH1 protein expression (35.5%) in mucinous carcinomas; (3) a significant relationship between the presence of MUC5AC and the altered expression of MLH1 protein in colorectal carcinomas.     

Genomic deletions in MSH2 or MLH1 are a frequent cause of hereditary non-polyposis colorectal cancer: identification of novel and recurrent deletions by MLPA

Gene dosage abnormalities account for a significant proportion of the mutations in genes tested in DNA diagnostic laboratories. Detection of these changes has proved a challenge as the methods available to date are time consuming or unreliable. The multiplex ligation-dependent probe assay (MLPA) is a new technique allowing relative quantification of up to 40 different nucleic acid sequences in a single reaction tube. We have evaluated MLPA for potential use in the diagnostic setting against the following criteria: accuracy, reagent cost, hands-on time, reliability, and retests required. A total of 215 UK patients referred for genetic testing on the basis of a family history consistent with autosomal dominant hereditary non-polyposis colorectal cancer (HNPCC or Lynch syndrome) were tested by MLPA. Of these, 12 cases with deletions of one or more exons were identified, six with MLH1 deletions and six with MSH2 deletions. Test failure rates were less than 5% and overall mutation detection sensitivity in this series was increased by approximately 50% by the inclusion of MLPA for an additional testing cost of about 10%. Two novel mutations in MSH2 and 10 novel point mutations in MLH1 were also identified during the course of this study. We conclude that MLPA is a cost effective and robust gene dosage method that can be readily adopted by diagnostic services. Comprehensive mutation scanning for MSH2 and MLH1 is incomplete without gene dosage analysis.     

Prognostic significance of DNA repair proteins MLH1, MSH2 and MGMT expression in non-small-cell lung cancer and precursor lesions

Aims:  To investigate the role of DNA repair proteins and their prognostic significance in non-small-cell lung cancer (NSCLC).
Methods and results:  A retrospective analysis of 108 cases of stage I–II NSCLC was undertaken. Immunohistochemical expression of DNA repair proteins MLH1, MSH2 and MGMT was assessed using tissue microarrays of paraffin-embedded samples of invasive carcinoma and precursor lesions. Results were analysed in relation to clinicopathological parameters and patient survival. Reduced expression of MLH1 was found in 58.5% of tumours and occurred less frequently in poorly differentiated tumours ( P  = 0.044) and large cell carcinomas ( P  = 0.004). MSH2 and MGMT expression was reduced in 18.1% and 77.8% of cases, respectively. There was an inverse relationship between MLH1 and MSH2 expression ( P  = 0.012). Normal expression of MLH1, MSH2 and MGMT was found in all cases of squamous metaplasia and squamous dysplasia. Only a single case of carcinoma in situ (12.5%) showed reduced MLH1, none showed reduced MSH2 and 25% showed reduced MGMT. Survival analyses showed no prognostic significance based on expression of MLH1 ( P  = 0.92), MSH2 ( P  = 0.78) or MGMT ( P  = 0.57).
Conclusions:  Reduction in expression of DNA repair proteins MLH1, MSH2 and MGMT is relatively common in NSCLC, appears to be a late event in the development of invasive malignancy and does not influence survival in this patient cohort.