Guido Plotz

Bethesda Guidelines: Relation to Microsatellite Instability and MLH1 Promoter Methylation in Patients with Colorectal Cancer

Hereditary nonpolyposis colorectal cancer (HNPCC) is a common, inherited cancer syndrome associated with an increased lifetime risk for cancer, predominantly colorectal and endometrial cancer (1). This familial cancer syndrome is frequently caused by germline mutations in MLH1 and MSH2; other DNA mismatch repair genes (MSH6, PMS1, PMS2) are rarely affected (24). Tumors in mutation carriers typically exhibit microsatellite instability, a characteristic phenotype that is caused by expansions or contractions of short nucleotide repeat sequences (58). In HNPCC, a single mutation in one allele of a mismatch repair gene is inherited in the germline; however, microsatellite instability only follows inactivation of the other allele. Clinical criteria facilitated the identification of the molecular basis of HNPCC (9). Because the original criteria (Amsterdam criteria) were considered too restrictive (10, 11), extended criteria were established (Amsterdam II criteria) that took into account other types of HNPCC-associated cancer, such as cancer of the endometrium, small bowel, ureter, and renal pelvis (Table 1) (1215). Because 85% to 90% of HNPCC-associated colorectal tumors are characterized by microsatellite instability (3, 5, 6, 16, 17), testing for microsatellite instability is an important tool to identify patients with hereditary colorectal cancer, especially in patients who do not meet the Amsterdam criteria. The Bethesda guidelines were introduced to identify patients with colorectal cancer who should be tested for microsatellite instability (10). These guidelines include features of the Amsterdam criteria and other phenotypic characteristics found more frequently in HNPCC-associated cancer (Table 1). Although microsatellite instability has also been observed in approximately 10% to 15% of sporadic cases of colorectal cancer without HNPCC (3, 5, 6, 8, 16, 17), somatic MLH1 or MSH2 mutations are rarely found in this subgroup of patients with colorectal cancer (18, 19). Table 1. Amsterdam and Amsterdam II Criteria and Bethesda Guidelines DNA mismatch repair activity can be impaired not only by somatic or germline mismatch repair gene mutations but also by epigenetic modifications, such as DNA methylation (Figure 1). Methylation of normally unmethylated cytidine phosphate guanosine (CpG) islands in the promoter regions of tumor suppressor and DNA repair genes is correlated with loss of expression of these genes in different tumors (2022). Silencing of MLH1 in association with CpG island methylation precedes microsatellite instability in colorectal tumors, and interactions between genetic and epigenetic events may advance tumor development (9, 19, 2226). MLH1 promoter methylation may be more prevalent in sporadic colorectal cancer with microsatellite instability than in HNPCC tumors with MLH1 mutations (27). However, the clinical impact of MLH1 promoter methylation in the evaluation of patients with suspected HNPCC-associated tumors is unknown. We therefore investigated the effectiveness of the Bethesda guidelines in detecting microsatellite instability and the correlation among microsatellite instability, mismatch repair gene mutations, and MLH1 promoter methylation in patients with colorectal cancer. Figure 1. Allelic MLH1 gene inactivation in colorectal cancer. Top. white circles MLH1 TC Middle. MLH1 Bottom. MLH1 black circles Methods Patients We conducted the study at the University of Frankfurt (Frankfurt, Germany) in the Department of Internal Medicine, which is a tertiary care referral center. The study was approved by the local ethics committee at our university hospital. Between February 1997 and March 2000, 125 unrelated patients with histologically verified colorectal cancer were referred for treatment evaluation or genetic counseling for suspected hereditary colorectal cancer. All patients provided written, informed consent and were consecutively enrolled. The patients (56 women, 69 men) were 22 to 95 years of age (mean age [SD] at diagnosis, 52.0 14.4 years). Seventy-eight patients had left-sided colorectal cancer (in the descending colon, sigmoid colon, or rectum), and 47 patients had right-sided colorectal cancer (in the transverse colon, ascending colon, or cecum). We carefully recorded the personal and family history of cancer and demographic information for all patients. The cohort had 147 total colorectal tumors, which included synchronous and metachronous colorectal cancer in 14 patients. In addition, 15 patients had a history of 18 total cases of extracolonic cancer. Clinical and histopathologic characteristics of the 125 patients are shown in Table 2. Table 2. Patient Characteristics, according to Fulfillment or Nonfulfillment of at Least One Criterion of the Bethesda Guidelines Analysis of Microsatellite Instability Colorectal tumor tissue and corresponding normal colonic epithelium were microdissected from representative 5-m sections of paraffin-embedded specimens, and DNA was extracted by using Triton X-100 (Sigma, St. Louis, Missouri). Between February 1997 and April 1998, seven dinucleotide repeat microsatellites (D2S119, D2S123, D2S136, D3S1266, D3S1298, D15S120, and D18S58) were successfully amplified in 69 patients, as described elsewhere (2831). Two mononucleotide microsatellites (BAT25 and BAT26) and three dinucleotide repeat microsatellites (D2S123, D5S346, and D17S250) were studied in the remaining 56 patients, according to international reference panel recommendations (32). Dietmaier and colleagues (30) and we (unpublished data) have found similar rates of microsatellite instability by using either panel. Microsatellite instability, which was defined as an alteration in at least two microsatellites (30, 33), was assessed by direct, semiautomatic comparison of each microsatellite motif between tumor and normal colonic tissue. Analysis of MLH1 Methylation MLH1 promoter methylation was studied in all colorectal tumor samples with microsatellite instability and in an International Union Against Cancer (UICC) stagematched control group of 20 patients with colorectal cancer but no instability. MSH2 promoter methylation was not assessed because the MSH2 promoter is not prone to DNA methylation in tumors that test positive for microsatellite instability (19, 25). The methylation pattern within the CpG islands of the MLH1 promoter was determined by sodium bisulfite modification of unmethylated cytosines to uracil. Methylation-specific polymerase chain reaction (PCR) was performed by using primers specific for methylated or modified unmethylated DNA (19, 20, 34, 35). The primer sequences for the unmethylated reaction were 5-TTTTGATGTAGATGTTTTATTAGGGTTGT-3 (forward primer) and 5-ACCACCTCATCATAACTACCCACA-3 (reverse primer), and the sequences for the methylated reaction were 5-ACGTAGACGTTTTATTAGGGTCGC-3 (forward primer) and 5-CCTCATCGTAACTACCCGCG-3 (reverse primer). DNA extracted from the SW48 and the HCT116 colon-cancer cell line served as positive and negative controls, respectively, for methylation. Polymerase chain reaction was performed in a final volume of 50 L that contained 3.5 mmol of MgCl2 per L (2 mmol/L for the methylated reaction); 15 mmol of (NH4)2SO4 per L; 60 mmol of Tris-HCl per L (pH 8.5); 250 mol each of dATP, dTTP, dCTP, and dGTP per L (Invitrogen, Leek, the Netherlands); 0.1 mol of forward and reverse primers per L (Biospring, Frankfurt, Germany); and 2.5 U of AmpliTaq Gold DNA polymerase (Applied Biosystems, Weiterstadt, Germany). This was done for 10 minutes at 95 C, followed by 55 cycles of 30 seconds each at 95 C, 30 seconds at 55 C, 1 minute at 72 C, and a final extension of 10 minutes at 72 C. The PCR products were subjected to electrophoresis on nondenaturing polyacrylamide gels (8%) and visualized by silver staining. Sequence Analysis of MLH1 and MSH2 Genomic DNA was isolated from peripheral blood mononuclear cells according to standard protocols (36). For mutation analysis of the complete coding region and the exonintron junctions of MLH1 and MSH2 in patients with microsatellite instability, we performed direct DNA sequencing after PCR amplification by using primers, as described elsewhere (3739). Statistical Analysis Data are expressed as the mean ( SD). The MannWhitney U test and Fisher exact test were used to compare means and proportions, respectively. We report two-sided P values and 95% CIs. Analyses were performed with the use of StatView5, version 5.0.1 (SAS Institute, Inc., Cary, North Carolina), and BiAS, version 7.04 (Epsilon Verlag, Darmstadt, Germany). Role of the Funding Source The study was supported by a research grant from the University of Frankfurt. The funding source had no role in the collection, analysis, or interpretation of data or in the decision to publish the results. Results Fifty-eight of the 125 patients with colorectal cancer (46% [95% CI, 37% to 56%]) fulfilled at least one of the seven Bethesda criteria, whereas 67 patients (54% [CI, 44% to 63%]) fulfilled none. Of the 58 patients who satisfied at least one Bethesda criterion, 31 met one criterion, 12 met 2 criteria, 8 met 3 criteria, and 7 met 4 criteria. Criterion B4 was the most commonly satisfied criterion. All 58 patients who fulfilled criteria of the Bethesda guidelines met criterion B1, criterion B2, criterion B3, criterion B4, or some combination of these criteria, whereas only 12 of these 58 patients (21%) additionally met criteria B5, B6, or B7. The distribution of sex, UICC tumor stage, and histopathologic grading were similar in the patients who did and did not satisfy Bethesda guidelines. Detection of Microsatellite Instability Microsatellite instability was detected in 22 of the 125 patients with colorectal cancer (17.6% [95% CI, 11.4% to 25.4%]) (5 had UICC stage I disease, 11 had stage II disease, and 6 had stage III disease). Seventeen of the 58 patients who met Bethesda criteria (29.3% [CI, 18.1% to 42.7%]) but only 5 of the 67 patients who met none of the Bethesd

Frequency and phenotypic spectrum of germline mutations in POLE and seven other polymerase genes in 266 patients with colorectal adenomas and carcinomas.

In a number of families with colorectal adenomatous polyposis or suspected Lynch syndrome/HNPCC, no germline alteration in the APC, MUTYH, or mismatch repair (MMR) genes are found. Missense mutations in the polymerase genes POLE and POLD1 have recently been identified as rare cause of multiple colorectal adenomas and carcinomas, a condition termed polymerase proofreading‐associated polyposis (PPAP). The aim of the present study was to evaluate the clinical relevance and phenotypic spectrum of polymerase germline mutations. Therefore, targeted sequencing of the polymerase genes POLD1, POLD2, POLD3, POLD4, POLE, POLE2, POLE3 and POLE4 was performed in 266 unrelated patients with polyposis or fulfilled Amsterdam criteria. The POLE mutation c.1270C>G;p.Leu424Val was detected in four unrelated patients. The mutation was present in 1.5% (4/266) of all patients, 4% (3/77) of all familial cases and 7% (2/30) of familial polyposis cases. The colorectal phenotype in 14 affected individuals ranged from typical adenomatous polyposis to a HNPCC phenotype, with high intrafamilial variability. Multiple colorectal carcinomas and duodenal adenomas were common, and one case of duodenal carcinoma was reported. Additionally, various extraintestinal lesions were evident. Nine further putative pathogenic variants were identified. The most promising was c.1306C>T;p.Pro436Ser in POLE. In conclusion, a PPAP was identified in a substantial number of polyposis and familial colorectal cancer patients. Screening for polymerase proofreading mutations should therefore be considered, particularly in unexplained familial cases. The present study broadens the phenotypic spectrum of PPAP to duodenal adenomas and carcinomas, and identified novel, potentially pathogenic variants in four polymerase genes.

The PMS2 Subunit of Human MutLα Contains a Metal Ion Binding Domain of the Iron-Dependent Repressor Protein Family

DNA mismatch repair (MMR) is responsible for correcting replication errors. MutLalpha, one of the main players in MMR, has been recently shown to harbor an endonuclease/metal-binding activity, which is important for its function in vivo. This endonuclease activity has been confined to the C-terminal domain of the hPMS2 subunit of the MutLalpha heterodimer. In this work, we identify a striking sequence-structure similarity of hPMS2 to the metal-binding/dimerization domain of the iron-dependent repressor protein family and present a structural model of the metal-binding domain of MutLalpha. According to our model, this domain of MutLalpha comprises at least three highly conserved sequence motifs, which are also present in most MutL homologs from bacteria that do not rely on the endonuclease activity of MutH for strand discrimination. Furthermore, based on our structural model, we predict that MutLalpha is a zinc ion binding protein and confirm this prediction by way of biochemical analysis of zinc ion binding using the full-length and C-terminal domain of MutLalpha. Finally, we demonstrate that the conserved residues of the metal ion binding domain are crucial for MMR activity of MutLalpha in vitro.

Transient mismatch repair gene transfection for functional analysis of genetic hMLH1 and hMSH2 variants

Background: Germline mutations in the mismatch repair (MMR) genes hMLH1 and hMSH2 can cause hereditary non-polyposis colorectal cancer (HNPCC). However, the functional in vitro analysis of hMLH1 and hMSH2 mutations remains difficult. Aims: To establish an in vitro method for the functional characterisation of hMLH1 and hMSH2 mutations. Methods:hMLH1 and hMSH2 wild type (wt) genes and several mutated subclones were transiently transfected in mismatch repair deficient cell lines (HCT-116 and LOVO). Apoptosis, proliferation, and regulation of mRNA expression and protein expression of interacting proteins were analysed by Hoechst staining, AlamarBlue staining, real time polymerase chain reaction, and western blotting, respectively. Results: The protein expression of hMLH1 and hMSH2 mutants was significantly decreased after transfection compared with wild type transfections. The hMLH1 and hMSH2 interacting proteins hPMS2 and hMSH6 became detectable only after transfection of the respective wild type genes. In parallel, hMSH6 mRNA levels were increased in hMSH2 wt transfected cells. However, hPMS2 mRNA levels were independent of the mutation status of its interacting partner hMLH1, indicating a post-transcriptional regulating pathway. In the hMLH1 deficient HCT-116 cell line apoptosis was not affected by transfection of any mismatch repair gene, whereas complementation of hMSH2 deficency in LOVO cells increased apoptosis. Conversely, proliferative activity of HCT-116 was decreased by complementation with hMLH1wt and unaffected in hMSH2 deficient LOVO cells. Conclusion: These data show that the cellular role of the MMR genes and its mutations are assessable in a simple transient transfection system and show the influence of MMR gene regulation on major cell growth regulating mechanisms. This method is applicable for the functional definition of mutations in hMLH1 and hMSH2 genes observed in patients with suspected HNPCC.

Identification of Lynch syndrome mutations in the MLH1–PMS2 interface that disturb dimerization and mismatch repair†

Missense alterations of the mismatch repair gene MLH1 have been identified in a significant proportion of individuals suspected of having Lynch syndrome, a hereditary syndrome that predisposes for cancer of colon and endometrium. The pathogenicity of many of these alterations, however, is unclear. A number of MLH1 alterations are located in the C‐terminal domain (CTD) of MLH1, which is responsible for constitutive dimerization with PMS2. We analyzed which alterations may result in pathogenic effects due to interference with dimerization. We used a structural model of CTD of MLH1–PMS2 heterodimer to select 19 MLH1 alterations located inside and outside two candidate dimerization interfaces in the MLH1–CTD. Three alterations (p.Gln542Leu, p.Leu749Pro, p.Tyr750X) caused decreased coexpression of PMS2, which is unstable in the absence of interaction with MLH1, suggesting that these alterations interfere with dimerization. All three alterations are located within the dimerization interface suggested by our model. They also compromised mismatch repair, suggesting that defects in dimerization abrogate repair and confirming that all three alterations are pathogenic. Additionally, we provided biochemical evidence that four alterations with uncertain pathogenicity (p.Ala586Pro, p.Leu636Pro, p.Thr662Pro, and p.Arg755Trp) are deleterious because of poor expression or poor repair efficiency, and confirm the deleterious effect of eight further alterations. Hum Mutat 31:975–982, 2010.

Characterization of the nuclear import of human MutLα

DNA mismatch repair (MMR) is essential for the maintenance of replication fidelity. Its major task is to recognize mismatches as well as insertion/deletion loops of newly synthesized DNA strands. Although different players of human MMR have been identified, the regulation of essential steps of MMR is poorly understood. Because MMR is initiated in the nucleus, nuclear import might be a mechanism to regulate MMR. Nuclear targeting is accomplished by conserved signal sequences called nuclear localization signals (NLS), which represent clusters of positively charged amino acids (aa). hMLH1 contains two clusters of positively charged amino acids, which are candidate NLS sequences (aa 469–472 and 496–499), while hPMS2 contains one (aa 574–580). To study the effect of these clusters on nuclear import, NLS mutants of hMLH1 and hPMS2 were generated and expressed in 293T cells. The subcellular localization of the mutant constructs was monitored by confocal laser microscopy. We demonstrated that missense mutations of two signal sequences, one in hMLH1 and one in hPMS2, lead to impaired nuclear import, which was especially prominent for mutants of the hMLH1 residues K471 and R472; and hPMS2 residues K577 and R578.

Refining the role of pms2 in Lynch syndrome: germline mutational analysis improved by comprehensive assessment of variants

Background and aim The majority of mismatch repair (MMR) gene mutations causing Lynch syndrome (LS) occur either in MLH1 or MSH2. However, the relative contribution of PMS2 is less well defined. The aim of this study was to evaluate the role of PMS2 in LS by assessing the pathogenicity of variants of unknown significance (VUS) detected in the mutational analysis of PMS2 in a series of Spanish patients. Methods From a cohort of 202 LS suspected patients, 13 patients showing loss of PMS2 expression in tumours were screened for germline mutations in PMS2, using a long range PCR based strategy and multiplex ligation dependent probe amplification (MLPA). Pathogenicity assessment of PMS2 VUS was performed evaluating clinicopathological data, frequency in control population and in silico and in vitro analyses at the RNA and protein level. Results Overall 25 different PMS2 DNA variants were detected. Fourteen were classified as polymorphisms. Nine variants were classified as pathogenic: seven alterations based on their molecular nature and two after demonstrating a functional defect (c.538-3C>G affected mRNA processing and c.137G>T impaired MMR activity). The c.1569C>G variant was classified as likely neutral while the c.384G>A remained as a VUS. We have also shown that the polymorphic variant c.59G>A is MMR proficient. Conclusions Pathogenic PMS2 mutations were detected in 69% of patients harbouring LS associated tumours with loss of PMS2 expression. In all, PMS2 mutations account for 6% of the LS cases identified. The comprehensive functional analysis shown here has been useful in the classification of PMS2 VUS and contributes to refining the role of PMS2 in LS.

Comprehensive functional assessment of MLH1 variants of unknown significance

Lynch syndrome is associated with germline mutations in DNA mismatch repair (MMR) genes. Up to 30% of DNA changes found are variants of unknown significance (VUS). Our aim was to assess the pathogenicity of eight MLH1 VUS identified in patients suspected of Lynch syndrome. All of them are novel or not previously characterized. For their classification, we followed a strategy that integrates family history, tumor pathology, and control frequency data with a variety of in silico and in vitro analyses at RNA and protein level, such as MMR assay, MLH1 and PMS2 expression, and subcellular localization. Five MLH1 VUS were classified as pathogenic: c.[248G>T(;)306G>C], c.[780C>G;788A>C], and c.791‐7T>A affected mRNA processing, whereas c.218T>C (p.L73P) and c.244G>A (p.T82A) impaired MMR activity. Two other VUS were considered likely neutral: the silent c.702G>A variant did not affect mRNA processing or stability, and c.974G>A (p.R325Q) did not influence MMR function. In contrast, variant c.25C>T (p.R9W) could not be classified, as it associated with intermediate levels of MMR activity. Comprehensive functional assessment of MLH1 variants was useful in their classification and became relevant in the diagnosis and genetic counseling of carrier families. Hum Mutat 33:1576–1588, 2012.

Expression Defect Size among Unclassified MLH1 Variants Determines Pathogenicity in Lynch Syndrome Diagnosis

Purpose: Lynch syndrome is caused by a germline mutation in a mismatch repair gene, most commonly the MLH1 gene. However, one third of the identified alterations are missense variants with unclear clinical significance. The functionality of these variants can be tested in the laboratory, but the results cannot be used for clinical diagnosis. We therefore aimed to establish a laboratory test that can be applied clinically. Experimental Design: We assessed the expression, stability, and mismatch repair activity of 38 MLH1 missense variants and determined the pathogenicity status of recurrent variants using clinical data. Results: Four recurrent variants were classified as neutral (K618A, H718Y, E578G, V716M) and three as pathogenic (A681T, L622H, P654L). All seven variants were proficient in mismatch repair but showed defects in expression. Quantitative PCR, pulse-chase, and thermal stability experiments confirmed decreases in protein stability, which were stronger in the pathogenic variants. The minimal cellular MLH1 concentration for mismatch repair was determined, which corroborated that strongly destabilized variants can cause repair deficiency. Loss of MLH1 tumor immunostaining is consistently reported in carriers of the pathogenic variants, showing the impact of this protein instability on these tumors. Conclusions: Expression defects are frequent among MLH1 missense variants, but only severe defects cause Lynch syndrome. The data obtained here enabled us to establish a threshold for distinguishing tolerable (clinically neutral) from pathogenic expression defects. This threshold allows the translation of laboratory results for uncertain MLH1 variants into pathogenicity statements for diagnosis, thereby improving the targeting of cancer prevention measures in affected families. Clin Cancer Res; 19(9); 2432–41. ©2013 AACR.

MUTYH gene expression and alternative splicing in controls and polyposis patients

Mutational loss of the human DNA repair gene MUTYH in the germline predisposes for colorectal polyposis and cancer, a recessively heritable disease called MUTYH‐associated polyposis. The MUTYH gene shows heavy alternative splicing, but the transcripts relevant for biological function and cancer prevention have not been determined. This knowledge is required to assess the consequences that germline variants of unknown functional significance may have. We therefore quantified expression and investigated patterns of alternative splicing in control individuals, tissue samples, and carriers of two frequent germline alterations. MUTYH expression differed organ dependently, correlating with proliferative activity. Alternative first exons were used tissue specifically; transcripts for mitochondrial proteins predominated in muscle tissues, while ascending colon and testes showed the highest fractions of transcripts for nuclear proteins. Colon cancer cell lines produced predominant transcripts for nuclear protein. Exon skipping was frequent and governed by splice‐site quality. Five transcripts were found to encode the biologically relevant products of the MUTYH gene. Carriers of the disease‐causing mutation c.1187G>A (p.Gly396Asp) showed normal transcript composition, but the frequent single‐nucleotide polymorphism rs3219468:G>C largely reduced one transcript species of MUTYH. Since this alteration decreases protein production of the gene, an increased cancer risk for compound heterozygous carriers is possible. Hum Mutat 33:1067–1074, 2012.