Quantification of sequence exchange events between PMS2 and PMS2CL provides a basis for improved mutation scanning of lynch syndrome patients

Heterozygous mutations in PMS2 are involved in Lynch syndrome, whereas biallelic mutations are found in Constitutional mismatch repair‐deficiency syndrome patients. Mutation detection is complicated by the occurrence of sequence exchange events between the duplicated regions of PMS2 and PMS2CL. We investigated the frequency of such events with a nonspecific polymerase chain reaction (PCR) strategy, coamplifying both PMS2 and PMS2CL sequences. This allowed us to score ratios between gene and pseudogene‐specific nucleotides at 29 PSV sites from exon 11 to the end of the gene. We found sequence transfer at all investigated PSVs from intron 12 to the 3′ end of the gene in 4 to 52% of DNA samples. Overall, sequence exchange between PMS2 and PMS2CL was observed in 69% (83/120) of individuals. We demonstrate that mutation scanning with PMS2‐specific PCR primers and MLPA probes, designed on PSVs, in the 3′ duplicated region is unreliable, and present an RNA‐based mutation detection strategy to improve reliability. Using this strategy, we found 19 different putative pathogenic PMS2 mutations. Four of these (21%) are lying in the region with frequent sequence transfer and are missed or called incorrectly as homozygous with several PSV‐based mutation detection methods. Hum Mutat 31:578–587, 2010. © 2010 Wiley‐Liss, Inc.     

Novel biallelic mutations in MSH6 and PMS2 genes: gene conversion as a likely cause of PMS2 gene inactivation

Since the first report by our group in 1999, more than 20 unrelated biallelic mutations in DNA mismatch repair genes (MMR) have been identified. In the present report, we describe two novel cases: one carrying compound heterozygous mutations in the MSH6 gene; and the other, compound heterozygous mutations in the PMS2 gene. Interestingly, the inactivation of one PMS2 allele was likely caused by gene conversion. Although gene conversion has been suggested to be a mutation mechanism underlying PMS2 inactivation, this is the first report of its involvement in a pathogenic mutation. The clinical features of biallelic mutation carriers were similar to other previously described patients, with the presence of café-au-lait spots (CALS), early onset of brain tumors, and colorectal neoplasia. Our data provide further evidence of the existence, although rare, of a distinct recessively inherited syndrome on the basis of MMR constitutional inactivation. The identification of this syndrome should be useful for genetic counseling, especially in families with atypical hereditary nonpolyposis colon cancer (HNPCC) associated with childhood cancers, and for the clinical surveillance of these mutation carriers.     

Early age of onset and broad cancer spectrum persist in MSH6- and PMS2-associated Lynch syndrome

PurposeThis study aimed to characterize MSH6/PMS2-associated mismatch repair–deficient (MMR-D)/microsatellite instability-high (MSI-H) tumors, given revised guidelines suggesting more modest phenotypes.MethodsPatients who consented to Institutional Review Board–approved protocols of tumor/germline sequencing or Lynch syndrome registry at a single institution from February 2005 to January 2021 with germline, heterozygous MSH6/PMS2 pathogenic/likely pathogenic variants were identified. Clinical data were abstracted and correlated with MMR/microsatellite instability status using nonparametric tests.ResultsWe identified 243 patients (133 sequencing, 110 registry) with germline MSH6/PMS2 pathogenic/likely pathogenic variants; 186 (77%) had >1 cancer. Of 261 pooled tumors, colorectal cancer (CRC) and endometrial cancer (EC) comprised 55% and 43% of cancers in MSH6 and PMS2, respectively; 192 tumors underwent molecular assessments and 122 (64%) were MMR-D/MSI-H (77 in MSH6, 45 in PMS2). MMR-D/MSI-H cancers included CRC (n = 56), EC (n = 35), small bowel cancer (n = 6), ovarian cancer (n = 6), urothelial cancer (n = 5), pancreas/biliary cancer (n = 4), gastric/esophageal cancer (n = 3), nonmelanoma skin tumors (n = 3), prostate cancer (n = 2), breast cancer (n = 1), and central nervous system/brain cancer (n = 1). Among MMR-D/MSI-H CRC and EC, median age of diagnosis was 51.5 (range = 27-80) and 55 (range = 39-74) years, respectively; 9 of 56 (16%) MMR-D/MSI-H CRCs were diagnosed at age <35 years.ConclusionMSH6/PMS2 heterozygotes remain at risk for a broad spectrum of cancers, with 16% of MMR-D/MSI-H CRCs presenting before upper threshold of initiation of colonoscopy per guidelines.     

Mismatch repair analysis of inherited MSH2 and/or MSH6 variation pairs found in cancer patients

Mismatch repair (MMR) malfunction causes the accumulation of mismatches in the genome leading to genomic instability and cancer. The inactivation of an MMR gene (MSH2, MSH6, MLH1, or PMS2) with an inherited mutation causes Lynch syndrome (LS), a dominant susceptibility to cancer. MMR gene variants of uncertain significance (VUS) may be pathogenic mutations, which cause LS, may result in moderately increased cancer risks, or may be harmless polymorphisms. Our study suggests that an inherited MMR VUS individually assessed as proficient may, however, in a pair with another MMR VUS found in the same colorectal cancer (CRC) patient have a concomitant contribution to the MMR deficiency. Here, eight pairs of MMR gene variants found in cancer patients were functionally analyzed in an in vitro MMR assay. Although the other pairs do not suggest a compound deficiency, the MSH2 VUS pair c.380A>G/c.982G>C (p.Asn127Ser/p.Ala328Pro), which nearly halves the repair capability of the wild-type MSH2 protein, is presumed to increase the cancer risk considerably. Moreover, two MSH6 variants, c.1304T>C (p.Leu435Pro) and c.1754T>C (p.Leu585Pro), were shown to be MMR deficient. The role of one of the most frequently reported MMR gene VUS, MSH2 c.380A>G (p.Asn127Ser), is especially interesting because its concomitant defect with another variant could finally explain its recurrent occurrence in CRC patients.     

Clinical analysis of PMS2: mutation detection and avoidance of pseudogenes

Germline mutation detection in PMS2, one of four mismatch repair genes associated with Lynch syndrome, is greatly complicated by the presence of numerous pseudogenes. We used a modification of a long‐range PCR method to evaluate PMS2 in 145 clinical samples. This modification avoids potential interference from the pseudogene PMS2CL by utilizing a long‐range product spanning exons 11–15, with the forward primer anchored in exon 10, an exon not shared by PMS2CL. Large deletions were identified by MLPA. Pathogenic PMS2 mutations were identified in 22 of 59 patients whose tumors showed isolated loss of PMS2 by immunohistochemistry (IHC), the IHC profile most commonly associated with a germline PMS2 mutation. Three additional patients with pathogenic mutations were identified from 53 samples without IHC data. Thirty‐seven percent of the identified mutations were large deletions encompassing one or more exons. In 27 patients whose tumors showed absence of either another protein or combination of proteins, no pathogenic mutations were identified. We conclude that modified long‐range PCR can be used to preferentially amplify the PMS2 gene and avoid pseudogene interference, thus providing a clinically useful germline analysis of PMS2. Our data also support the use of IHC screening to direct germline testing of PMS2. Hum Mutat 31:588–593, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

A new variant database for mismatch repair genes associated with Lynch syndrome

Mutations in some mismatch repair (MMR) genes are associated with Lynch syndrome (LS; also called hereditary nonpolyposis colorectal cancer [HNPCC]), an autosomal dominant cancer susceptibility syndrome. Colorectal cancer (CRC) is the most frequent cancer observed in LS. However, tumors occur at a variety of extracolonic sites and individuals may have multiple primary cancers. LS is the most common hereditary form of CRC, accounting for approximately 1% of all CRC. Since the first account of mutations in MSH2 causing this cancer susceptibility syndrome in 1993, mutations in three additional MMR genes, MLH1, MSH6, and PMS2, have been shown to cause LS. More than 1,500 different variants have been identified in these four genes and approximately 80% of the alterations have been identified in MLH1 and MSH2. There have been a few previous attempts to systematically record MMR variants associated with LS patients; however, they were not complete nor were they continuously updated. Thus, it was our goal to generate and maintain a comprehensive catalogue of MMR variants from genes known to be mutated in LS (http://www.med.mun.ca/MMRvariants; last accessed 8 February 2007). Providing such a resource should aid investigators in understanding the significance of the variants.     

Functional PMS2 hybrid alleles containing a pseudogene‐specific missense variant trace back to a single ancient intrachromosomal recombination event

Sequence exchange between PMS2 and its pseudogene PMS2CL, embedded in an inverted duplication on chromosome 7p22, has been reported to be an ongoing process that leads to functional PMS2 hybrid alleles containing PMS2‐ and PMS2CL‐specific sequence variants at the 5′‐and the 3′‐end, respectively. The frequency of PMS2 hybrid alleles, their biological significance, and the mechanisms underlying their formation are largely unknown. Here we show that overall hybrid alleles account for one‐third of 384 PMS2 alleles analyzed in individuals of different ethnic backgrounds. Depending on the population, 14–60% of hybrid alleles carry PMS2CL‐specific sequences in exons 13–15, the remainder only in exon 15. We show that exons 13–15 hybrid alleles, named H1 hybrid alleles, constitute different haplotypes but trace back to a single ancient intrachromosomal recombination event with crossover. Taking advantage of an ancestral sequence variant specific for all H1 alleles we developed a simple gDNA‐based polymerase chain reaction (PCR) assay that can be used to identify H1‐allele carriers with high sensitivity and specificity (100 and 99%, respectively). Because H1 hybrid alleles harbor missense variant p.N775S of so far unknown functional significance, we assessed the H1‐carrier frequency in 164 colorectal cancer patients. So far, we found no indication that the variant plays a major role with regard to cancer susceptibility. Hum Mutat 31:1–8, 2010. © 2010 Wiley‐Liss, Inc.     

A rapid and cell-free assay to test the activity of lynch syndrome-associated MSH2 and MSH6 missense variants

Lynch syndrome (LS) is an autosomal dominant disorder that predisposes to colon, endometrial, and other cancers. LS is caused by a heterozygous germline mutation in one of the DNA mismatch repair (MMR) genes. A significant proportion of all mutations found in suspected LS patients comprises single amino acid alterations. The pathogenicity of these variants of uncertain significance (VUS) is difficult to assess, precluding diagnosis of carriers and their relatives. Here we present a rapid cell-free assay to investigate MMR activity of MSH2 or MSH6 VUS. We used this assay to analyze a series of MSH2 and MSH6 VUS, selected from the Leiden Open Variation Database. Whereas a significant fraction of the MSH2 VUS has lost MMR activity, suggesting pathogenicity, the large majority of the MSH6 VUS appears MMR proficient. We anticipate that this assay will be an important tool in the development of a comprehensive and widely applicable diagnostic procedure for LS-associated VUS.     

Ten novel MSH2 and MLH1 germline mutations in families with HNPCC

Hereditary nonpolyposis colorectal cancer (HNPCC) is one of the most common hereditary cancer-susceptibility syndromes. Germline mutations in mismatch repair genes are associated with the clinical phenotype of HNPCC. We report ten novel germline mutations, three in MSH2 and seven in MLH1. All but one mutation have been found in families fulfilling criteria of the Bethesda guidelines; four of them additionally fulfilled the Amsterdam criteria I or II. Eight mutations were considered pathogenic and predictive diagnostics in healthy family members at risk shall be undertaken; these include five frameshift mutations leading to premature stop codons, in MSH2: c.1672delT (p.S558Xfs) and c.2466_2467delTG (p.C822X) and in MLH1: c.1023delG (p.R341Xfs), c.1127_1128dupAT (p.K377Xfs) and c.1310delC (p.P437Xfs); three mutations leading to splice aberrations, in MSH2: c.1661G>C (r.1511_1661del) and in MLH1: c.677+3A>C (r.589_677del) and c.1990-2A>G predicted to result in a splice site defect. The remaining two mutations are unclassified variants with assumed pathogenicity: one missense mutation in the highly conserved ATPase domain of MLH1 (c.122A>G [p.D41G]) and one in-frame insertion of twelve nucleotides in MLH1 (c.2155_2156insATGTGTTCCACA [p.I719delinsNVFHI]). These two mutations were not found in 102 alleles of healthy control individuals. The corresponding tumors from all patients showed a high level of microsatellite instability (MSI-H). Immunohistochemistry (IHC) revealed complete loss of expression of the affected protein in the tumor cells from all but three patients. The tumors from the patients with the mutations c.1127_1128dupAT and c.1990-2A>G showed a reduction of expression of the MLH1-protein, rather than complete loss. In the tumor from the patient with the missense mutation c.122A>G [p.D41G] a normal expression of the proteins coded by MLH1 and MSH2 was noticed.     

A common MSH2 mutation in English and North American HNPCC families: origin, phenotypic expression, and sex specific differences in colorectal cancer

The frequency, origin, and phenotypic expression of a germline MSH2 gene mutation previously identified in seven kindreds with hereditary non-polyposis cancer syndrome (HNPCC) was investigated. The mutation (A→T at nt943+3) disrupts the 3' splice site of exon 5 leading to the deletion of this exon from MSH2 mRNA and represents the only frequent MSH2 mutation so far reported. Although this mutation was initially detected in four of 33 colorectal cancer families analysed from eastern England, more extensive analysis has reduced the frequency to four of 52 (8%) English HNPCC kindreds analysed. In contrast, the MSH2 mutation was identified in 10 of 20 (50%) separately identified colorectal families from Newfoundland. To investigate the origin of this mutation in colorectal cancer families from England (n=4), Newfoundland (n=10), and the United States (n=3), haplotype analysis using microsatellite markers linked to MSH2 was performed. Within the English and US families there was little evidence for a recent common origin of the MSH2 splice site mutation in most families. In contrast, a common haplotype was identified at the two flanking markers (CA5 and D2S288) in eight of the Newfoundland families. These findings suggested a founder effect within Newfoundland similar to that reported by others for two MLH1 mutations in Finnish HNPCC families. We calculated age related risks of all, colorectal, endometrial, and ovarian cancers in nt943+3 A→T MSH2 mutation carriers (n=76) for all patients and for men and women separately. For both sexes combined, the penetrances at age 60 years for all cancers and for colorectal cancer were 0.86 and 0.57, respectively. The risk of colorectal cancer was significantly higher (p<0.01) in males than females (0.63 v 0.30 and 0.84 v 0.44 at ages 50 and 60 years, respectively). For females there was a high risk of endometrial cancer (0.5 at age 60 years) and premenopausal ovarian cancer (0.2 at 50 years). These intersex differences in colorectal cancer risks have implications for screening programmes and for attempts to identify colorectal cancer susceptibility modifiers.


Keywords: MSH2 mutation; HNPCC; colorectal cancer     

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.     

A large fraction of unclassified variants of the mismatch repair genes MLH1 and MSH2 is associated with splicing defects

Numerous unclassified variants (UVs) have been found in the mismatch repair genes MLH1 and MSH2 involved in hereditary nonpolyposis colorectal cancer (HNPCC or Lynch syndrome). Some of these variants may have an effect on pre-mRNA splicing, either by altering degenerate positions of splice site sequences or by affecting intronic or exonic splicing regulatory sequences such as exonic splicing enhancers (ESEs). In order to determine the consequences of UVs on splicing, we used a functional assay of exon inclusion. For each variant, mutant and wild-type exons to be tested were PCR-amplified from patient genomic DNA together with approximately 150 bp of flanking sequences and were inserted into a splicing reporter minigene. After transfection into HeLa cells, the effects on splicing were evaluated by RT-PCR analysis and systematic sequencing. A total of 22 UVs out of 85 different variant alleles examined in 82 families affected splicing, including four exonic variants that affected putative splicing regulatory elements. We analyzed short stretches spanning the latter variants by cloning them into the ESE-dependent central exon of a three-exon splicing minigene and we showed in cell transfection experiments that the wild-type sequences indeed contain functional ESEs. We then used this construct to query for ESE elements in the MLH1 or MSH2 regions affected by 14 previously reported exonic splicing mutations and showed that they also contain functional ESEs. These splicing assays represent a valuable tool for the interpretation of UVs and should contribute to the optimization of the molecular diagnosis of the Lynch syndrome and of other genetic diseases.     

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.     

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.     

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.