Evaluating the Effect of Unclassified Variants Identified in MMR Genes Using Phenotypic Features, Bioinformatics Prediction, and RNA Assays
2013; Elsevier BV; Volume: 15; Issue: 3 Linguagem: Inglês
10.1016/j.jmoldx.2013.02.003
ISSN1943-7811
AutoresLucía Pérez‐Cabornero, Mar Infante, Eladio A. Velasco, Enrique Lastra, Cristina Miner, M. Durán,
Tópico(s)RNA Research and Splicing
ResumoLynch syndrome is caused by mutations in one of the mismatch-repair system (MMR) genes. A major difficulty in diagnosis and management of Lynch syndrome is the existence of unclassified genetic variants (UVs) with unknown clinical significance, especially mutations with new descriptions and missense-type nucleotide substitutions. We evaluated the pathogenicity of 20 such mutations (6 in MLH1, 4 in MSH2, and 7 in MSH6) found in Spanish patients suspected of Lynch syndrome. The UVs were tested for evidence of MMR defect in tumor samples and were evaluated for co-occurrence with a pathogenic mutation, the cosegregation of the variant with the disease; where sufficient data were available, in silico resources at the protein level and mRNA analysis were used to assess the putative effect on the splicing mechanism. To evaluate the frequency of these UVs in the general population, a case–control study was also performed. Five variants were identified with similar frequencies in both cases and controls, suggesting a nonpathogenic effect in patients. In contrast, abnormal splicing mutations were detected in a high proportion of patients [3/20 (15%)]. In this study, we classified 15 of the 20 UVs: six variants with strong evidence of pathogenicity and nine variants that should be considered neutral variants. Clinical significance of the other five remains unknown. Lynch syndrome is caused by mutations in one of the mismatch-repair system (MMR) genes. A major difficulty in diagnosis and management of Lynch syndrome is the existence of unclassified genetic variants (UVs) with unknown clinical significance, especially mutations with new descriptions and missense-type nucleotide substitutions. We evaluated the pathogenicity of 20 such mutations (6 in MLH1, 4 in MSH2, and 7 in MSH6) found in Spanish patients suspected of Lynch syndrome. The UVs were tested for evidence of MMR defect in tumor samples and were evaluated for co-occurrence with a pathogenic mutation, the cosegregation of the variant with the disease; where sufficient data were available, in silico resources at the protein level and mRNA analysis were used to assess the putative effect on the splicing mechanism. To evaluate the frequency of these UVs in the general population, a case–control study was also performed. Five variants were identified with similar frequencies in both cases and controls, suggesting a nonpathogenic effect in patients. In contrast, abnormal splicing mutations were detected in a high proportion of patients [3/20 (15%)]. In this study, we classified 15 of the 20 UVs: six variants with strong evidence of pathogenicity and nine variants that should be considered neutral variants. Clinical significance of the other five remains unknown. CME Accreditation Statement: This activity (“JMD 2013 CME Program in Molecular Diagnostics”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity (“JMD 2013 CME Program in Molecular Diagnostics”) for a maximum of 48 AMA PRA Category 1 Credit(s)TM. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. CME Accreditation Statement: This activity (“JMD 2013 CME Program in Molecular Diagnostics”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity (“JMD 2013 CME Program in Molecular Diagnostics”) for a maximum of 48 AMA PRA Category 1 Credit(s)TM. Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Hereditary non–polyposis colorectal cancer (HNPCC) or Lynch syndrome is the most frequent autosomal dominant colorectal cancer susceptibility syndrome caused by mutations inactivating one of the genes of the mismatch-repair system (MMR), most frequently MLH1 (OMIM 120436, NM_000249.3) and MSH2 (OMIM 609309; NM_000251.1), and less often in MSH6 (OMIM 600678; NM_000179.2) and PMS2 (OMIM 600259; NM_000535).1Fishel R. Lescoe M.K. Rao M.R. Copeland N.G. Jenkins N.A. Garber J. Kane M. Kolodner R. The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer [Erratum appeared in Cell 1994, 77(1):1 p following 166].Cell. 1993; 75: 1027-1038Abstract Full Text PDF PubMed Scopus (2603) Google Scholar, 2Bronner C.E. Baker S.M. Morrison P.T. Warren G. Smith L.G. Lescoe M.K. Kane M. Earabino C. Lipford J. Lindblom A. Tannergård P. Bollag R.J. Godwin A.R. Ward D.C. Nordenskjøld M. Fishel R. Kolodner R. Liskay R.M. 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Genotype to phenotype: analyzing the effects of inherited mutations in colorectal cancer families.Mutat Res. 2010; 693: 32-45Crossref PubMed Scopus (42) Google Scholar that truncate and inactivate MMR genes. Mutations in three of these MMR genes (MLH1, MSH2, and MSH6) account for the majority of the patients with Lynch syndrome.7Peltomäki P. Deficient DNA mismatch repair: a common etiologic factor for colon cancer.Hum Mol Genet. 2001; 10: 735-740Crossref PubMed Scopus (403) Google Scholar Nonetheless, there are unclassified genetic variants (UVs) with unknown clinical significance; these are nucleotide substitutions (generally not truncating missense type) whose clinical interpretation can be difficult when detected in a family with suspected Lynch syndrome. 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Classification of ambiguous mutations in DNA mismatch repair genes identified in a population-based study of colorectal cancer.Hum Mutat. 2008; 29: 367-374Crossref PubMed Scopus (73) Google Scholar, 10Genuardi M. Carrara S. Anti M. Ponz de Leòn M. Viel A. Assessment of pathogenicity criteria for constitutional missense mutations of the hereditary nonpolyposis colorectal cancer genes MLH1 and MSH2.Eur J Hum Genet. 1999; 7: 778-782Crossref PubMed Scopus (29) Google Scholar co-occurrence (in trans) with deleterious mutations, the determination of the variant frequency in unaffected controls, amino acid polarity or size, and evolutionary conservation of the residue. However, the clinical phenotype of a nontruncating mutation may vary within different families, and cosegregation data are not always available. Functional assays have therefore been developed to clarify the activity of nontruncating MMR gene mutations. The analysis of a number of missense variants has shown that, rather than causing changes to a single amino acid as classically predicted, many variants are instead associated with defects in RNA splicing; some silent mutations have also been shown to cause splice defects.11Gorlov I.P. Gorlova O.Y. Frazier M.L. Amos C.I. Missense mutations in hMLH1 and hMSH2 are associated with exonic splicing enhancers.Am J Hum Genet. 2003; 73: 1157-1161Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 12Lastella P. Surdo N.C. Resta N. Guanti G. Stella A. In silico and in vivo splicing analysis of MLH1 and MSH2 missense mutations shows exon- and tissue-specific effects.BMC Genomics. 2006; 7: 243Crossref PubMed Scopus (54) Google Scholar, 13Auclair J. Busine M.P. Navarro C. Ruano E. Montmain G. Desseigne F. Saurin J.C. Lasset C. Bonadona V. Giraud S. Puisieux A. Wang Q. 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A large fraction of unclassified variants of the mismatch repair genes MLH1 and MSH2 is associated with splicing defects.Hum Mutat. 2008; 29: 1412-1424Crossref PubMed Scopus (135) Google Scholar To determine whether a change in the sequence may be a cause of disease or not, pathogenicity can be evaluated based on numerous criteria and using different approaches. Thus, for example, Barnetson et al9Barnetson R.A. Cartwright N. van Vliet A. Haq N. Drew K. Farrington S. Williams N. Warner J. Campbell H. Porteous M.E. Dunlop M.G. Classification of ambiguous mutations in DNA mismatch repair genes identified in a population-based study of colorectal cancer.Hum Mutat. 2008; 29: 367-374Crossref PubMed Scopus (73) Google Scholar applied a qualitative, point-based, integrative analysis of UVs; of 23 initially unclassified MLH1 or MSH2 missense variants, they classified 11 as benign and 2 as pathogenic. Kansikas et al20Kansikas M. Kariola R. Nystrom M. Verification of the three-step model in assessing the pathogenicity of mismatch repair gene variants.Hum Mutat. 2011; 32: 107-115Crossref PubMed Scopus (29) Google Scholar recently proposed a three-step assessment model. Nonetheless, classification of some of these described substitutions indicates conflict between the clinicopathological data set of the family carriers and contemporaneous data from either functional assay results or in silico approaches. We found different interpretations of the same variant, relative to its pathogenicity, in the MMR Gene Unclassified Variants Database (http://www.MMRuv.info, last accessed June 30, 2012).21Ou J. Niessen R.C. Vonk J. Westers H. Hofstra R.M. Sijmons R.H. A database to support the interpretation of human mismatch repair gene variants.Hum Mutat. 2008; 29: 1337-1341Crossref PubMed Scopus (48) Google Scholar Description of more carrier families is therefore needed to help clarify the role of a given UV in susceptibility to Lynch syndrome. In the present study, we performed a structured assessment of the pathogenicity of all ambiguous variants identified in our series of colorectal cancer cases, using a set of complementary approaches: phenotypic features in the families (co-occurrence with a pathogenic mutation, cosegregation with the disease, tumor MSI, and DNA mismatch repair protein expression analysis), bioinformatics assessment of the functional consequence of the amino acid change, mRNA analysis, and frequency in a control population (case–control comparisons). The ability to determine the likelihood that a given UV contributes to the disease phenotype is likely to have beneficial consequences for management of the patient. The 159 index cases were from unrelated families referred for MMR mutation analysis under the Junta de Castilla y León Cancer Genetic Counseling Program for the years 2007 to 2010. Informed consent was obtained from the subjects or their parents. DNA and RNA were purified from peripheral blood lymphocytes by using QIAamp DNA and RNA blood mini kits (Qiagen, Iberia SL, Madrid, Spain; Valencia, CA), respectively. To screen for MLH1, MSH2, or MSH6 point mutations, we used a method developed in our laboratory,22Velasco E. Infante M. Durán M. Esteban-Cardeñosa E. Lastra E. García-Girón C. Miner C. Rapid mutation detection in complex genes by heteroduplex analysis with capillary array electrophoresis.Electrophoresis. 2005; 26: 2539-2552Crossref PubMed Scopus (21) Google Scholar with validation for MMR genes as recently described.23Perez-Cabornero L. Velasco E. Infante M. Sanz D. Lastra E. Hernández L. Miner C. Duran M. A new strategy to screen MMR genes in Lynch syndrome: hA-CAE. MLPA and RT-PCR 5.Eur J Cancer. 2009; 45: 1485-1493Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar For each UV under study, detailed information was gathered on family history, tumor characteristics (IHC, MSI), cosegregation of the variant with disease in families, and co-occurrence with other pathological or UVs. MLH1 promoter hypermethylation was tested using methylation-sensitive multiplex ligation-dependent probe amplification (MS-MLPA; MRC-Holland, Amsterdam, the Netherlands). BRAF p.V600E hot-spot mutation (exon 15) was directly sequenced in both directions in tumor samples of all patients; this assay is characterized by a sensitivity of 10% to 20%. Primers were BRAF-Ex15-forward 5′-TCATAATGCTTGCTCTGATAGGA-3′ and BRAF-Ex15-reverse 5′-GGCCAAAAATTTAATCAGTGGA-3′. Tumor immunostaining was analyzed in all patients by a pathologist at the General Yagüe Hospital, Burgos, Spain. Tumor cells were judged to be deficient for protein expression only if they lacked staining in a sample in which normal tissue and stromal cells were stained. If no immunostaining of normal tissue could be demonstrated, the results were considered not evaluable. In brief, a BOND-III stainer system (Leica Biosystems, Barcelona, Spain; Wetzlar, Germany) was used with NCL-L-PMS2, PA0610-MLH1, PA0048-MSH2, and PA0597-MSH6 antibodies (Leica Biosystems), according to the manufacturer’s protocol. A panel of 478 controls (278 men, 200 women) was selected from the Spanish National DNA bank. These control subjects had no personal histories of cancer and had diagnoses unrelated to the variables of interest. The screening method used was the same as with the case samples, and MMR gene heteroduplex analysis by capillary array electrophoresis23Perez-Cabornero L. Velasco E. Infante M. Sanz D. Lastra E. Hernández L. Miner C. Duran M. A new strategy to screen MMR genes in Lynch syndrome: hA-CAE. MLPA and RT-PCR 5.Eur J Cancer. 2009; 45: 1485-1493Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar was performed. To assess whether the variant amino acids had been evolutionarily conserved across a number of phylogenetically diverse species, we used the T-Coffee tool (http://tcoffee.vital-it.ch/cgi-bin/Tcoffee/tcoffee_cgi/index.cgi). The alignment of homologous MLH1, MSH2, and MSH6 proteins was performed using the BLAST tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The following sequences were used for this purpose: Homo sapiens, Bos taurus, Rattus norvegicus, Mus musculus, Gallus gallus, Xenopus tropicalis, Danio rerio, Drosophila melanogaster, and Saccharomyces cerevisiae. The PolyPhen polymorphism phenotyping tool (http://genetics.bwh.harvard.edu/pph2/index.shtml, last accessed June 30, 2012)24Sunyaev S. Lathe 3rd, W. Bork P. Integration of genome data and protein structures: prediction of protein folds, protein interactions and “molecular phenotypes” of single nucleotide polymorphisms.Curr Opin Struct Biol. 2001; 11: 125-130Crossref PubMed Scopus (45) Google Scholar uses information from protein structure databases and three-dimensional structure databases to predict the effects on the secondary structure of the protein, interchain contacts, and functional sites. PolyPhen scores effects as 1 = benign, 2 = possibly damaging, and 3 = probably damaging. The bioinformatics tool SIFT (the name stands for “sorting intolerant from tolerant”; http://sift.bii.a-star.edu.sg, last accessed June 30, 2012) was also used. This tool predicts whether an amino acid substitution affects protein function, based on sequence homology and the physical properties of the amino acid.25Ng P.C. Henikoff S. Predicting deleterious amino acid substitutions.Genome Res. 2001; 11: 863-874Crossref PubMed Scopus (1902) Google Scholar SIFT can be applied to nonsynonymous polymorphisms, and the tool provides a binary classification: tolerated versus not tolerated (and therefore predicted to affect protein function). The Align-GVGD tool (http://agvgd.iarc.fr, last accessed June 30, 2012)26Tavtigian S.V. Deffenbaugh A.M. Yin L. Judkins T. Scholl T. Samollow P.B. de Silva D. Zharkikh A. Thomas A. Comprehensive statistical study of 452 BRCA1 missense substitutions with classification of eight recurrent substitutions as neutral.J Med Genet. 2006; 43: 295-305Crossref PubMed Scopus (527) Google Scholar combines biophysical characteristics of amino acids and proteins using multiple sequence alignments to predict where reversal substitutions in the genes of interest fall into a spectrum from enriched to deleterious to neutral. The output from Align-GVGD is an ordered series of grades ranging from C65 (most likely deleterious) to C0 (most likely neutral). Mutant and normal sequences were analyzed using two bioinformatics tools to identify potential splicing mutations. Disruption/creation of splice sites was evaluated with NNSplice (version 0.9; http://www.fruitfly.org/seq_tools/splice.html, last accessed June 30, 2012).27Reese M.G. Eeckman F.H. Kulp D. Haussler D. Improved splice site detection in Genie.J Comput Biol. 1997; 4: 311-323Crossref PubMed Scopus (1377) Google Scholar Analysis of putative splicing regulator elements was done with ESEfinder (version 3.0; http://rulai.cshl.edu/cgi-bin/tools/ESE3/esefinder.cgi?process=home, last accessed June 30, 2012).28Liu H.X. Zhang M. Krainer A.R. Identification of functional exonic splicing enhancer motifs recognized by individual SR proteins.Genes Dev. 1998; 12: 1998-2012Crossref PubMed Scopus (420) Google Scholar This tool is a web-based resource that facilitates rapid analysis of exon sequences to identify putative exonic splicing enhancers (ESEs) responsive to the human SR proteins SF2/ASF, SC35, SRp40, and SRp55, and to predict whether exonic mutations disrupt such elements. The synthesis of complementary DNA (cDNA) was performed with a high-capacity cDNA reverse transcription kit (Life Technologies–Applied Biosystems, Foster City, CA) using DNase-treated RNA in the presence of random primers. The nucleotide sequences used to confirm the splicing alteration of the mutation c.2634G>A (p.E878D) were MSH2-Ex14-forward 5′-CGATGGATTTGGGTTAGCAT-3′ and MSH2-Ex16-reverse 5′-AGGGCAQTTTGTTTCACCTTG-3′. Mutational screening of MMR genes was performed in samples from 159 cases of suspected Lynch syndrome. We identified 20 missense mutations or new intronic or synonymous alterations that were categorized of uncertain relevance for causing cancer. These variants are the focus of the present report. Clinicopathological features and molecular findings for the index patients of these variants are presented in Table 1. Mutation nomenclature is based on GenBank reference sequence NM_000249.3 (MLH1); NM_000251.1 (MSH2), and NM_000179.2 (MSH6). Nucleotide numbering reflects cDNA, with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence, according to Human Genome Variation Society nomenclature for the description of sequence variants (http://www.hgvs.org/mutnomen, last accessed June 30, 2012).Table 1Clinicopathologic data for 20 UVs of the MLH1, MSH2, and MSH6 Genes Identified in Colorectal Cancer CasesMutationFamily (n/N)∗Identifier for the family carrying a UV. n, number of affected carriers; N, total number of relatives tested.Cosegregation with diseaseIndex caseTumor analysisCo-occurrence with other pathogenic or UV mutationsOther cancers in family (onset†Age at onset (years).)Cancer (onset†Age at onset (years).)CriteriaBRAFMSIIHCMLH1 c.306+5G>AVA-44 (5/5)YesCRC (31)AmstWTMSI-HNENoFDR: colon (50); SDR: colon (39)VA-67 (3/3)YesCRC (59)AmstWTMSI-HMLH1 deficientNoFDR: endo (49), colon (66/72), SDR: endo (50), gastric (50)VA-167 (1/1)YesCRC (37)AmstNANANANoFDR: colon (54); SDR: colon (56), colon (68) c.545+40C>AVA-95 (2/2)YesCRC (42)AmstWTMSSNormalMSH6: c.4003A>C; (p.E335A)FDR: colon (69); SDR: colon (75); endo (45) c.1217G>A; p.S406NVA-140 (1/1)NECRC (39)BethWTMSSNormalNoFDR: colon (65); gastric (70) c.1574G>A; p.S505NVA-30 (1/1)NECRC (52)BethWTMSSNENoFDR: meln (80), HL (53); SDR: BC (56) c.1820T>A; p.L607HVA-152 (1/2)NoPolyps (40)AmstNANANANoFDR: colon (48), polyps (39), BC (41); SDR: BC (45) c.1865T>A; p.L622HVA-179 (1/1)NECRC (37)AmstNANANANoNot available c.1852_1853AA>GC; p.K618AVA-47 (1/3)NoCRC (45)AmstNANANANoFDR: colon (48), polyps (39), BC (41); SDR: coloNEsoph (42/72), colon (72), colon (35), colon (45), BC (37), BC (43)VA-55 (1/2)NECRC (43)BethV600EMSSMLH1 deficientNoNo other cancers in the familyVA-82 (1/1)NECRC/CRC (74/74)BethNANANANoFDR: coloNThyroid (81/86); SDR: gastric (70), colon (70)VA-108 (1/1)NECRC (44)BethNANANANoFDR: ovariaNColon (57/57); SDR: Blad (25), colon (48)VA-112 (1/1)NECRC (39)BethWTMSSNENoFDR: colon (75)VA-121 (1/1)NECRC (48)BethWTMSSNENoNo other cancers in the family c.2088C>G; p.T696TVA-199 (1/1)NEBiliary/CRC (50/52)AmstNANANAMSH2: c.2470C>T; (p.Gln824X)FDR: colon (36), ovarian (47), colon (40); SDR: CNS (63), gastric (52),colon/urethral (70/80) c.2146G>A; p.V716MVA-70 (1/1)NECRC (52)BethWTMSI-HMLH1 -MLH1 promoter methylationNo other cancers in the familyMSH2 c.212-5delTVA-43 (1/1)NECRC (72)AmstNANANANoNot available c.1661G>A; p.S554TVA-6 (7/46)YesCRC (47)AmstNAMSI-HMSH2/MSH6 deficientNoFDR: endo/colon/Blad (42/50/60), colon (45), endo (45); SDR: gastric/prostate (40/64) c.2634G>A; p.E878DVA-191 (1/1)NEEndo (40)AmstNAMSI-HMSH2/MSH6 deficientNoFDR: endo (30); SDR: colon/ovariaNColon (29/39/63), endo (30), endo/colon (46/51), endo (30) c.2651T>G; p.I884SVA-8 (2/2)YesCRC (20)AmstNAMSI-HMSH2/MSH6 deficientNoFDR: ovarian (45), endo (45).MSH6 c.98G>C; p.R33PVA-121 (1/1)NECRC (30)BethWTMSSNENoNo other cancers in the family c.431G>T; p.S144IVA-4 (8/12)YesCRC (36)AmstNAMSI-HMSH2/MSH6 deficientMSH2: exon7 delFDR: colon (32), panc (40); SDR: colon (35), colon/Blad (40/40), colon (41), colon (39)VA-94 (1/1)NEBC (52)BethNANANANoFDR: small intestine (40), coloNColon (69/73), c.457+53insTGVA-194 (1/1)NECRC (41)BethNAMSI-HMLH1/MSH6 deficientNoNo other cancers in the family c.2400T>C; p.V800VVA-22 (7/12)NECRC/panc (45/61)AmstNAMSI-HMSH2/MSH6 deficientMSH2: c.229_230delAGFDR: colon (53); SDR: colon (75); colon (50); colon (55); colon (60); gastric (70) c.2633T>C; p.V878Asame familyYesNA c.3425C>T; p.T1142MVA-85 (2/2)YesPolyps (27)BethNANANANoFDR: polyps (61) c.4004A>C; p.E1335AVA-95 (2/2)YesCRC (42)AmstWTMSSNormalMLH1: c.545+40C>AFDR: colon (69); SDR: colon (75); endo (45)Variants with new descriptions are highlighted in bold.Amst, fulfilled Amsterdam criteria; BC, breast cancer; Beth, fulfilled Bethesda criteria; Blad, bladder; CNS, central nervous system; CRC, colorectal cancer; del, deletion; endo, endometrial; esoph, esophageal; FDR, first-degree relatives; HL, Hodgkin’s lymphoma; meln, melanoma; MSI, microsatellite instability; MSI-H, microsatellite instability, high; MSS, microsatellite stability; NA, not available; NE, not evaluable; panc, pancreatic; SDR, second-degree relatives; WT, wild type.∗ Identifier for the family carrying a UV. n, number of affected carriers; N, total number of relatives tested.† Age at onset (years). Open table in a new tab Variants with new descriptions are highlighted in bold. Amst, fulfilled Amsterdam criteria; BC, breast cancer; Beth, fulfilled Bethesda criteria; Blad, bladder; CNS, central nervous system; CRC, colorectal cancer; del, deletion; endo, endometrial; esoph, esophageal; FDR, first-degree relatives; HL, Hodgkin’s lymphoma; meln, melanoma; MSI, microsatellite instability; MSI-H, microsatellite instability, high; MSS, microsatellite stability; NA, not available; NE, not evaluable; panc, pancreatic; SDR, second-degree relatives; WT, wild type. The 20 UVs considered here have been described previously: 9 in MLH1 (45%), 4 in MSH2 (20%), and 7 in MSH6 (35%). We present new descriptions for 11 of these variants (3 in MLH1, 3 in MSH2, and 5 in MSH6 ) (Table 1); the remainder appear in the Leiden Open Variation Database (LOVD 3.0; last accessed June 30, 2012) (LOVD 2.0; http://www.lovd.nl/2.0, last accessed June 30, 2012). Their pathogenicity, however, is still under discussion, because the data provided by different authors are contradictory. Thus the return to value in the present study using the system described here. Considered by type of mutation, the UVs included in the present study consist of 6 synonymous or intronic variants and 14 missense variants (Table 1). The coding variants that we studied are dispersed across different domains of the MLH1, MSH2, and MSH6 polypeptides (Figure 1). Two of the detected UVs are synonymous variants: MLH1 c.2088C>G (p.T696T) and MSH6 c.2400T>C (p.V800V). In our series, both variants cosegregated with a mutation previously described as pathological, so the clinicopathological features obtained from these variants seem to be produced by the one that is deleterious. Moreover, neither of them seemed to significantly alter the elements involved in splicing tested by bioinformatics tools (VA-199 and VA-22 families; Table 1). All intronic variants (MLH1 c.545+40C>A, c.306+5G>A, and MSH6 c.457+53insTG) have been analyzed for effects on splicing machinery, to account for their putative pathogenicity. MLH1 c.545+40C>A and MSH6 c.457+53insTG create a new enhancer for an SRp protein without relevant implications in the splicing, because the variants are relatively deeply intronic (VA-95 and VA-194 families; Table 1). However, the mutation in MLH1 c.306+5G>A creates an alternative 5′ donor site, causing an in-frame deletion of five nucleotides. The sequence of the deleted transcript obtained from RT-PCR was reported previously.23Perez-Cabornero L. Velasco E. Infante M. Sanz D. Lastra E. Hernández L. Miner C. Duran M. A new strategy to screen MMR genes in Lynch syndrome: hA-CAE. MLPA and RT-PCR 5.Eur J Cancer. 2009; 4
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