The Germline MLH1 K618A Variant and Susceptibility to Lynch Syndrome-Associated Tumors
2012; Elsevier BV; Volume: 14; Issue: 3 Linguagem: Inglês
10.1016/j.jmoldx.2012.01.006
ISSN1943-7811
AutoresFabíola Medeiros, Noralane M. Lindor, Fergus J. Couch, W. Edward Highsmith,
Tópico(s)Cancer Genomics and Diagnostics
ResumoMissense variants discovered during sequencing of cancer susceptibility genes can be problematic for clinical interpretation. MLH1 K618A, which results from a 2-bp alteration (AAG→GCG) leading to a substitution of lysine to alanine in codon 618, has variously been interpreted as a pathogenic mutation, a variant of unknown significance, and a benign polymorphism. We evaluated the role of MLH1 K618A in predisposition to cancer by genotyping 1512 control subjects to assess its frequency in the general population. We also reviewed the literature concerning MLH1 K618A in families with colorectal cancer. The measured allele frequency of the K618A variant was 0.40%, which is remarkably close to the 0.44% summarized from 2491 control subjects in the literature. K618A was over-represented in families with suspected Lynch syndrome. In 1366 families, the allele frequency was 0.88% (OR = 2.1, 95% CI = 1.3 to 3.5; P = 0.006). In studies of sporadic cancers of the type associated with Lynch syndrome, K618A was over-represented in 1742 cases (allele frequency of 0.83) (OR = 2.0, 95% CI = 1.2 to 3.2; P = 0.008). We conclude that MLH1 K618A is not a fully penetrant Lynch syndrome mutation, although it is not without effect, appearing to increase the risk of Lynch syndrome-associated tumors approximately twofold. Our systematic assessment approach may be useful for variants in other genes. Missense variants discovered during sequencing of cancer susceptibility genes can be problematic for clinical interpretation. MLH1 K618A, which results from a 2-bp alteration (AAG→GCG) leading to a substitution of lysine to alanine in codon 618, has variously been interpreted as a pathogenic mutation, a variant of unknown significance, and a benign polymorphism. We evaluated the role of MLH1 K618A in predisposition to cancer by genotyping 1512 control subjects to assess its frequency in the general population. We also reviewed the literature concerning MLH1 K618A in families with colorectal cancer. The measured allele frequency of the K618A variant was 0.40%, which is remarkably close to the 0.44% summarized from 2491 control subjects in the literature. K618A was over-represented in families with suspected Lynch syndrome. In 1366 families, the allele frequency was 0.88% (OR = 2.1, 95% CI = 1.3 to 3.5; P = 0.006). In studies of sporadic cancers of the type associated with Lynch syndrome, K618A was over-represented in 1742 cases (allele frequency of 0.83) (OR = 2.0, 95% CI = 1.2 to 3.2; P = 0.008). We conclude that MLH1 K618A is not a fully penetrant Lynch syndrome mutation, although it is not without effect, appearing to increase the risk of Lynch syndrome-associated tumors approximately twofold. Our systematic assessment approach may be useful for variants in other genes. Lynch syndrome, or mismatch repair deficiency syndrome, previously termed hereditary nonpolyposis colorectal cancer (HNPCC), is an autosomal dominant cancer predisposition syndrome caused by mutations in DNA mismatch repair (MMR) genes, most commonly MLH1, MSH2, MSH6, or PMS2, and accounts for approximately 2% to 2.5% of colorectal cancers1Cunningham J.M. Kim C.Y. Christensen E.R. Tester D.J. Parc Y. Burgart L.J. Halling K.C. McDonnell S.K. Schaid D.J. Walsh Vockley C. Kubly V. Nelson H. Michels V.V. Thibodeau S.N. The frequency of hereditary defective mismatch repair in a prospective series of unselected colorectal carcinomas [Erratum appeared in Am J Hum Genet 2001, 69:1160].Am J Hum Genet. 2001; 69: 780-790Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 2Hampel H. Frankel W. Martin E. Arnold M. Khanduja K. Kuebler P. Nakagawa H. Sotamaa K. Prior T.W. Westman J. Panescu J. Fix D. Lockman J. Comeras I. de la Chapelle A. Screening for the Lynch syndrome (hereditary nonpolyposis colorectal cancer).N Engl J Med. 2005; 352: 1851-1860Crossref PubMed Scopus (1116) Google Scholar, 3Salovaara R. Loukola A. Kristo P. KÄäriäinen H. Ahtola H. Eskelinen M. Härkönen N. Julkunen R. Kangas E. Ojala S. Tulikoura J. Valkamo E. Järvinen H. Mecklin J.P. Aaltonen L. de la Chapelle A. Population-based molecular detection of hereditary nonpolyposis colorectal cancer [Erratum appeared in J Clin Oncol 2000, 18:3456].J Clin Oncol. 2000; 18: 2193-2200Crossref PubMed Scopus (447) Google Scholar and approximately 2% of endometrial cancers.4Hampel H. Frankel W. Panescu J. Lockman J. Sotamaa K. Fix D. Comeras I. La Jeunesse J. Nakagawa H. Westman J.A. Prior T.W. Clendenning M. Penzone P. Lombardi J. Dunn P. Cohn D.E. Copeland L. Eaton L. Fowler J. Lewandowski G. Vaccarello L. Bell J. Reid G. DE la Chapelle A. Screening for Lynch syndrome (hereditary nonpolyposis colorectal cancer) among endometrial cancer patients.Cancer Res. 2006; 66: 7810-7817Crossref PubMed Scopus (491) Google Scholar MLH1 inactivating mutations lead to an approximately 20-fold increased risk of cancer development and account for 50% of Lynch syndrome cases.5Peltomäki P. Vasen H. Mutations associated with HNPCC predisposition—Update of ICG-HNPCC/INSiGHT mutation database.Dis Markers. 2004; 20: 269-276Crossref PubMed Scopus (368) Google Scholar, 6Thibodeau S.N. French A.J. Cunningham J.M. Tester D.J. Burgart L.J. Roche P.C. McDonnell S.K. Schaid D.J. Vockley C.W. Michels V.V. Farr Jr, G.H. O'Connell M.J. Microsatellite instability in colorectal cancer: different mutator phenotypes and the principal involvement of hMLH1.Cancer Res. 1998; 58: 1713-1718PubMed Google Scholar However, several germline missense alterations have been identified in MLH1 whose role in the pathogenesis of colonic epithelial neoplasia is less clear. MLH1 K618A is a prominent example of this problematic category. This variant results from a 2-bp alteration in exon 16, involving nucleotides 1852 and 1853 (AAG→GCG) and leading to a substitution of lysine to alanine in codon 618. It was first identified in a mutation screening study of Swedish families with suspected HNPCC, performed in 1995.7Tannergård P. Lipford J.R. Kolodner R. Frödin J.E. Nordenskjöld M. Lindblom A. Mutation screening in the hMLH1 gene in Swedish hereditary nonpolyposis colon cancer families.Cancer Res. 1995; 55: 6092-6096PubMed Google Scholar Since then, the significance of MLH1 K618A has been debatable, being reported variously in the peer-reviewed literature as a pathogenic mutation, a sequence of unknown significance, and a benign polymorphism. In most instances, this variant has been found incidentally in genetic screening, and no previous study has focused specifically on the significance of MLH1 K618A in hereditary colorectal patients. The K618A variant has been included in several studies evaluating the biochemical function of various missense changes in MLH1. For example, functional studies tabulated at the Mismatch Repair Gene Unclassified Variant Database include eight published studies that report on 28 individual assays for K618A-bearing MLH1 protein. Of these, 6 assays were interpreted as showing pathogenicity, 22 were interpreted as demonstrating that this variant was benign, and 10 were inconclusive (http://www.mmruv.info, last accessed April 20, 2011). Thus, the cancer susceptibility of individuals and families identified with MLH1 K618A is not understood.8Ou 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 In the present study, we evaluated more precisely the role of MLH1 K618A in predisposition to cancer. We genotyped 1512 control subjects to assess the frequency of this variant in the general population. We also reviewed the experience of the Mayo Clinic Molecular Genetics Laboratory (MGL) in clinical Lynch syndrome testing and identified two cases in which individuals carried both the K618A variant and another, more typical MMR gene mutation. In addition, we performed a critical review of literature concerning MLH1 K618A in families with colorectal cancer and summarized the reported frequency of this variant in control subjects and in individuals with colorectal cancer. This study was reviewed and approved by the Mayo Clinic Institutional Review Board. The study sample was 1512 control subjects of European origin who had samples submitted to the Mayo Clinic MGL for cystic fibrosis carrier screening. DNA was isolated from whole blood using a QIAamp 96 DNA blood kit (Qiagen, Valencia, CA). After completion of the ordered service, samples annotated as Caucasian or Northern European ethnicity were selected and strictly anonymized. Briefly, the labels were removed from the sample tubes and the tubes were scrambled. After that, 10% of the samples were discarded and the DNA from the remaining samples was aliquoted into 96-well microtiter trays labeled numerically, starting simply at 1. Although no information other than the ethnicity of the sample collection was retained, the collection was heavily weighted toward women of childbearing age. Analysis of the K618A variant was performed using TaqMan chemistry (ABI Assays-on-Demand; Applied Biosystems, Foster City, CA) at the Mayo Clinic Genotyping Shared Resource. An anonymized sample known to be positive was provided as a positive heterozygous control. MLH1 whole-gene sequencing and dosage analysis are part of the clinical services offered by the Mayo Clinic MGL for the diagnosis of Lynch syndrome. Under procedures in place since 2005, all 16 exons of the MLH1 gene are amplified in four to five multiplex PCR reactions and are sequenced using ABI BigDye terminators and internal, exon-specific sequencing primers. Data analysis was performed using Mutation Surveyor (SoftGenetics, College Station, PA). For some cases, the ordering physician requested both MLH1 and MSH2 sequencing. In these cases, the MSH2 gene was also sequenced using a similar method. Before 2005, MLH1 (and MSH2) mutation scanning was done by conformation-sensitive gel electrophoresis,9Ganguly A. Rock M. Prockop D. Conformation-sensitive gel electrophoresis for rapid detection of single-base differences in double-stranded PCR products and DNA fragments: Evidence for solvent-induced bends in DNA heteroduplexes [Erratum appeared in Proc Natl Acad Sci USA 1994 May 24;91(11):5217].Proc Natl Acad Sci USA. 1993; 90: 10325-10329Crossref PubMed Scopus (619) Google Scholar followed by sequencing with 33P and manual interpretation of the autoradiographs. Before 2008, microsatellite instability (MSI) testing was done using a laboratory-developed panel of 10 short tandem repeat markers, as described by Baudhuin et al.10Baudhuin L.M. Burgart L.J. Leontovich O. Thibodeau S.N. Use of microsatellite instability and immunohistochemistry testing for the identification of individuals at risk for Lynch syndrome.Fam Cancer. 2005; 4: 255-265Crossref PubMed Scopus (90) Google Scholar This panel contained mono- and dinucleotide markers. Since June 2008, the laboratory has used the MSI Analysis System from Promega (Madison, WI).11Murphy K.M. Zhang S. Geiger T. Hafez M.J. Bacher J. Berg K.D. Eshleman J.R. Comparison of the microsatellite instability analysis system and the Bethesda panel for the determination of microsatellite instability in colorectal cancers.J Mol Diagn. 2006; 8: 305-311Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar The protein sequences of MLH1 homologs from 25 species (6 mammals, 4 other vertebrates, 1 echinoderm, 3 insects, 2 protozoan parasites, 1 nematode, 6 fungi, and 2 flowering plants) were aligned with ClustalW2 software (http://www.ebi.ac.uk/clustalw; last accessed May 9, 2011). The online sequence variation analysis packages SIFT (sort intolerant from tolerant; http://blocks.fhcrc.org/sift/SIFT.html; last accessed May 9, 2011), PolyPhen (polymorphism phenotype; http://www.bork.embl-heidelberg.de/PolyPhen; last accessed May 9, 2011), Align GVGD (http://agvgd.iarc.fr/agvgd_input.php; last accessed May 9, 2011), and PMut (http://mmb2.pcb.ub.es:8080/Pmut; last accessed May 9, 2011) were used according to the authors' instructions. The BLOSUM62 matrix was retrieved from the ExPASy website (http://ca.expasy.org; last accessed May 9, 2011). The significance of differences between groups was determined using Fisher's exact test. All articles mentioning MLH1 K618A listed in the InSIGHT mismatch repair gene variant database8Ou 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 and the Mismatch Repair Gene Unclassified Variant Database8Ou 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 were reviewed. A PubMed search yielded several additional references. The review was focused on the following aspects of the scientific articles: i) Reported frequency of MLH1 K618A in colorectal cancer patients with suspected diagnosis of Lynch syndrome; ii) reported frequency of MLH1 K618A in control subjects and in patients with cancers of the type seen in Lynch syndrome (ie, colorectal, gastric, and endometrial cancers) who were not selected on the basis of any high-risk criteria or prior suspicion of having Lynch syndrome (unselected cancers); iii) classification of MLH1 K618A variant as pathogenic mutation, sequence of unknown significance, or benign polymorphism; iv) results for MSI status and MMR protein immunohistochemistry in MLH1 K618A-related colorectal adenocarcinomas when available; and v) findings from functional studies. Of a total of 1512 control subjects genotyped, 12 individuals were identified with MLH1 K618A. The resulting allele frequency is 0.40%, with a carrier rate of 1/126. Over the past 8 years, out of approximately 600 MLH1 tests, the MGL at the Mayo Clinic has identified 13 cases carrying the K618A variant (Table 1). Because the MGL typically (but not exclusively) performs MLH1 mutation scanning after an individual's tumor has tested positive for MSI or loss of MLH1 protein by IHC, or both, this set is biased toward cases with high MSI (MSI-H).Table 1Individuals with Colorectal Cancer in Whom Germline MLH1 K618 Was Seen at the Mayo Clinic Molecular Genetics LaboratoryCase no.MSIIHCOther reasons for referralSequencing result1MSI-HLoss of MLH1 and PMS2UnknownMLH1 K618A2MSI-HNormalUnknownMLH1 K618A3MSI-LLoss of MLH1 and PMS2UnknownMLH1 K618A4MSI-HLoss of PMS2UnknownMLH1 K618A5UnknownUnknownHNPCC, family hxMLH1 K618A6UnknownUnknownHNPCC, clinical dxMLH1 K618A and MSH2 R487X7UnknownUnknownHNPCC, clinical dxMLH1 K618A and MLH1 V506A8UnknownUnknownUnknownMLH1 K618A and MSH2 2204delT9MSSNormalColon cancer, family hxMLH1 K618A10MSSNormalColon cancer, family hxMLH1 K618A11MSSNormalColon cancer, family hxMLH1 K618A12MSSNormalColon cancer, family hxMLH1 K618A13Not doneLoss of MLH1Colon cancer, family hxMLH1 K618Adx, diagnosis; HNPCC, hereditary nonpolyposis colorectal cancer; hx, history; MSI-H, microsatellite instability, high; MSI-L, microsatellite instability, low; MSS, microsatellite stable. Open table in a new tab dx, diagnosis; HNPCC, hereditary nonpolyposis colorectal cancer; hx, history; MSI-H, microsatellite instability, high; MSI-L, microsatellite instability, low; MSS, microsatellite stable. Eight of the 13 K618A-positive cases (cases 1 to 8) were tested for clinical purposes. Six of these had MLH1 testing only (cases 1 to 6) and the other two (cases 7 and 8) were tested for MSH2 mutations as well. Cases 1 and 2 were tested by conformation-sensitive gel electrophoresis with follow-up sequencing of positive exons. Of these eight cases, one was referred for MSI-H and absence of MLH1 and PMS2 by IHC, one for MSI-H only, one for low MSI (MSI-L) and absence of MLH1 and PMS2 by IHC, one for MSI-H and absence of PMS2 by IHC, one for a suspected family history of Lynch syndrome, and two for a clinical diagnosis of probable Lynch syndrome; for one case, the reason for testing was not given. Case 6 had a known pathogenic MLH1 mutation, R487X, in addition to the K618A variant. Case 7 had a second MLH1 variant of uncertain significance, V506A, and case 8 had a pathogenic MSH2 mutation, 2204delT. In addition to the clinical laboratory testing, the Mayo Clinic Colon Cancer Family Registry lists five cases with K618A (cases 9 to 13). Colorectal tumors from four of these cases were microsatellite stable (MSS) and had normal IHC; the fifth case exhibited loss of MLH1 by IHC. The lysine (K) at the positions corresponding to the human codon 618 is reasonably well conserved across species, diverging only in the fungi, protozoan parasites, and the worm. Of note, this position is also lysine in the two flowering plants for which a protein sequence was available (Table 2).Table 2Alignment of MLH1 HomologsOrganismMLH1 homologMammals Human (Homo sapiens)…KKKAEML… Chimpanzee (Pan troglodytes)…KKKAEML… Dog (Canis familiaris)…KKKAEML… Opossum (Didelphis marsupialis virginiana)…KRKAEML… Mouse (Mus musculus)…KKKAEML… Rat (Rattus norvegicus)…KKKAEML…Other vertebrates Chicken (Gallus gallus)…KKKTEML… Frog (Xenopus laevis)…KKKTEML… Zebrafish (Danio rerio)…KQKAEML… Pufferfish (Tetraodon nigroviridis)…KRKSEML…Other animals Sea urchin (Strongylocentrotus purpuratus)…KSKADML…Insects Drosophila melanogaster…LKKAPIM… Honeybee (Apis mellifera)…LEKADML… Mosquito (Anopheles gambiae)…VSKAPVL…Protozoan parasites, nematodes Plasmodium falciparum…YTYNEMY… Trypanosoma brucei…CNWRYML… Caenorhabditis elegans…AEHADLL…Fungi Saccharomyces cerevisiae…WDMSSML… Ashbya gossypii…WDMREML… Neurospora crassa…VERREML… Aspergillus fumigatus…IDRREML… Cryptococcus neoformans…RDRQEML… Ustilago maydis…LENAEML…Flowering plants Arabidopsis thaliana…KEKAEML… Rice (Oryza sativa)…KEKAEML… Open table in a new tab The results using the online analysis packages were mixed. When the 25 protein sequences described above were loaded, SIFT predicted that all sequence variants were tolerated. PolyPhen predicted that the alanine (A) change was probably damaging; PolyPhen performs its own BLAST search using the input protein identification number. For Swiss Prot ID P40692, the PolyPhen program retrieved 44 sequences, including 23 of the 25 listed above, with additional fungal sequences. The flowering plant sequences were not retrieved. PMut predicted the alanine substitution would be pathological, with a confidence rating of 6 (on a scale from 0, low confidence, to 9, high confidence). Align GVGD predicted that K618A would be a neutral variant. The BLOSUM62 matrix indicated that lysine to alanine changes had a score of −1, corresponding to a moderately low probability of occurrence. The results of the in silico analyses are summarized in Table 3.Table 3Online Mutation Classification Tool Analyses of MLH1 K618AOnline tool (source)Interpretation for K618ANumber of aligned sequencesSIFT (http://blocks.fhcrc.org/sift/SIFT.html)Tolerated25 (user-selected)PolyPhen (http://www.bork.embl-heidelberg.de/PolyPhen)Probably damaging44 (not user-selected)PMut (http://mmb2.pcb.ub.es:8080/Pmut)Pathological25 (user-selected)AlignGVGD (http://agvgd.iarc.fr/agvgd_input.php)Neutral25 (user-selected)BLOSUM62 (http://ca.expasy.org)Low probability of occurrenceNot available Open table in a new tab A total of 28 articles were identified with studies that had patients or control subjects harboring the MLH1 K618A variant: 19 articles concerned the identification of MMR variants in colorectal cancer families with suspected diagnosis of Lynch syndrome and 11 articles evaluated the frequency of MLH1 mutations (including K618A) in cohorts of patients with unselected carcinomas of various sites and normal control subjects. The mutation testing methods varied widely across studies, but most of the studies used direct sequencing or screening techniques followed by direct sequencing. In addition, six functional studies were available. Many published studies used some or all of the online computational tools that we used in the present work (Table 3). Two articles, however, carried the informatics analyses substantially further. We identified 19 articles reporting a total of 1545 individuals with suspected diagnosis of Lynch syndrome (Table 4). The studies used different inclusion criteria: either the Amsterdam, modified Amsterdam, or Bethesda criteria.30Bellacosa A. Genuardi M. Anti M. Viel A. de Leon P. Hereditary nonpolyposis colorectal cancer: review of clinical, molecular genetics, and counseling aspects.Am J Med Genet. 1996; 62: 353-364Crossref PubMed Scopus (76) Google Scholar, 31Benatti P. Sassatelli R. Roncucci L. Pedroni M. Fante R. Di Gregorio C. 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New clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by the International Collaborative Group on HNPCC.Gastroenterology. 1999; 116: 1453-1456Abstract Full Text Full Text PDF PubMed Scopus (2081) Google Scholar MLH1 K618A was encountered in 24 of 1366 subjects, providing an allele frequency of 0.88%. For age at onset, sufficient information was not available to allow comparing K618A-positive cases with all cases.Table 4MLH1 K618A in Cases with a Strong Family History of Lynch SyndromeReferenceTotal families (no.)MLH1 K618A (no.)Additional alterationsMSIIHCClassificationTannergård et al7Tannergård P. Lipford J.R. Kolodner R. Frödin J.E. Nordenskjöld M. Lindblom A. Mutation screening in the hMLH1 gene in Swedish hereditary nonpolyposis colon cancer families.Cancer Res. 1995; 55: 6092-6096PubMed Google Scholar391NANAVUSMauillon et al12Mauillon J.L. Michel P. Limacher J.M. Latouche J.B. Dechelotte P. Charbonnier F. Martin C. Moreau V. Metayer J. Paillot B. Frebourt T. Identification of novel germline hMLH1 mutations including a 22 kb Alu-mediated deletion in patients with familial colorectal cancer.Cancer Res. 1996; 56: 5728-5733PubMed Google Scholar171NANAMutationWeber et al13Weber T.K. Conlon W. Petrelli N.J. Rodriguez-Bigas M. Keitz B. Pazik J. Farrell C. O'Malley L. Oshalim M. Abdo M. Anderson G. Stoler D. Yandell D. Genomic DNA-based hMSH2 and hMLH1 mutation screening in 32 Eastern United States hereditary nonpolyposis colorectal cancer pedigrees.Cancer Res. 1997; 57: 3798-3803PubMed Google Scholar321NANAMutationWijnen et al14Wijnen J. Khan P.M. Vasen H. van der Klift H. Mulder A. van Leeuwen-Cornelisse I. Bakker B. Losekoot M. Møller P. Fodde R. Hereditary nonpolyposis colorectal cancer families not complying with the Amsterdam criteria show extremely low frequency of mismatch-repair-gene mutations.Am J Hum Genet. 1997; 61: 329-335Abstract Full Text PDF PubMed Scopus (189) Google Scholar1251NANAMutationWahlberg et al15Wahlberg S. Liu T. Lindblom P. Lindblom A. Various mutation screening techniques in the DNA mismatch repair genes hMSH2 and hMLH1.Genet Test. 1999; 3: 259-264Crossref PubMed Scopus (40) Google Scholar1421NANAMutationSyngal et al16Syngal S. Fox E.A. Eng C. Kolodner R.D. Garber J.E. Sensitivity and specificity of clinical criteria for hereditary non-polyposis colorectal cancer associated mutations in MSH2 and MLH1.J Med Genet. 2000; 37: 641-645Crossref PubMed Scopus (237) Google Scholar701MLH1 V506ANANAVUSFidalgo et al17Fidalgo P. Almeida M.R. West S. Gaspar C. Maia L. Wignen J. Albuquerque C. Curtis A. Cravo M. Fodde R. Leitao C.N. Burn J. Detection of mutations in mismatch repair genes in Portuguese families with hereditary non-polyposis colorectal cancer (HNPCC) by a multi-method approach.Eur J Hum Genet. 2000; 8: 49-53Crossref PubMed Scopus (32) Google Scholar201NANAMutationScott et al18Scott R.J. McPhillips M. Meldrum C.J. Fitzgerald P.E. Adams K. Spigelman A. du Sart D. Tucker K. Kirk J. Hereditary nonpolyposis colorectal cancer in 95 families: differences and similarities between mutation-positive and mutation-negative kindreds [Erratum appeared in Am J Hum Genet 2001 Feb;68(2):557].Am J Hum Genet. 2001; 68: 118-127Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar951NANAMutationSalahshor et al19Salahshor S. Koelble K. Rubio C. Lindblom A. Microsatellite instability and hMLH1 and hMSH2 expression analysis in familial and sporadic colorectal cancer.Lab Invest. 2001; 81: 535-541Crossref PubMed Scopus (97) Google Scholar151NANormalMutationMüller-Koch et al20Müller-Koch Y. Kopp R. Lohse P. Baretton G. Stoetzer A. Aust D. Daum J. Kerker B. Gross M. Dietmeier W. Holinski-Feder E. Sixteen rare sequence variants of the hMLH1 and hMSH2 genes found in a cohort of 254 suspected HNPCC (hereditary non-polyposis colorectal cancer) patients: mutations or polymorphisms?.Eur J Med Res. 2001; 6: 473-482PubMed Google Scholar2541NANAMutationGille et al21Gille J.J. Hogervorst F.B. Pals G. Wignen J.T. van Schooten R.J. Dommering C.J. Meijer G.A. Craanen M.E. Nederlof P.M. de Jong D. McElgunn C.J. Schouten J.P. Menko F.H. Genomic deletions of MSH2 and MLH1 in colorectal cancer families detected by a novel mutation detection approach.Br J Cancer. 2002; 87: 892-897Crossref PubMed Scopus (137) Google Scholar1261MSSNAVUSCaldes et al22Caldes T. Godino J. de la Hoya M. Garcia Carbonero I. Perez Segura P. Eng C. Benito M. Diaz-Rubio E. Prevalence of germline mutations of MLH1 and MSH2 in hereditary nonpolyposis colorectal cancer families from Spain.Int J Cancer. 2002; 98: 774-779Crossref PubMed Scopus (44) Google Scholar562MLH1 V716MMSI-HNAMutationScartozzi et al23Scartozzi M. Bianchi F. Rosati S. Galizia E. Antolini A. Loretelli C. Piga A. Bearzi I. Cellerino R. Porfiri E. Mutations of hMLH1 and hMSH2 in patients with suspected hereditary nonpolyposis colorectal cancer: correlation with microsatellite instability and abnormalities of mismatch repair protein expression.J Clin Oncol. 2002; 20: 1203-1208Crossref PubMed Scopus (68) Google Scholar371MSI-LLossMutationWard et al24Ward R. Meldrum C. Williams R. Mokany E. Scott R. Turner J. Hawkins N. Burgess B. Groombridge C. Spigelman A. Impact of microsatellite testing and mismatch repair protein expression on the clinical interpretation of genetic testing in hereditary non-polyposis colorectal cancer.J Cancer Res Clin Oncol. 2002; 128: 403-411Crossref PubMed Scopus (29) Google Scholar281MSSNormalVUSCaldes et al25Caldés T. Godino J. Sanchez A. Corbacho C. de la Hoya M. Lopez Asenjo J. Saez C. Sanz J. Benito M. Ramon y Cajal S. Diaz-Rubio E. Immunohistochemistry and microsatellite instability testing for selecting MLH1, MSH2, and MSH6 mutation carriers in hereditary non-polyposis colorectal cancer.Oncol Rep. 2004; 12: 621-629PubMed Google Scholar585MSH6 IVS5-16C>T1 MSI-LNormalVUSSame4 MSSWolf et al26Wolf B. Henglmueller S. Janschek E. Ilencikova D. Ludwig-Papst C. Bergmann M. Mannhalter C. Wrba F. Karner-Hanusch J. Spectrum of germ-line MLH1 and MSH2 mutations in Austrian patients with hereditary nonpolyposis colorectal cancer.Wien Klin Wochenschr. 2005; 117: 269-277Crossref PubMed Scopus (10) Google Scholar461MSSNAVUSHegde et al27Hegde M. Blazo M. Chong B. Prior T.W. Richards C. Assay validation for identification of hereditary nonpolyposis colon cancer-causing mutations in mismatch repair genes MLH1, MSH2, and MSH6.J Mol Diagn. 2005; 7: 525-534Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar231MutationBelvederesi et al28Belvederesi L. Bianchi F. Loretelli C. Gagliardini D. Galizia E. Bracci R. Rosati S. Bearzi I. Viel A. Cellerino R. Porfiri E. Assessing the pathogenicity of MLH1 missense mutations in patients with suspected hereditary nonpolyposis colorectal cancer: correlation with clinical, genetic and functional features.Eur J Hum Genet. 2006; 14: 853-859Crossref PubMed Scopus (25) Google Scholar841MSI-LLossVUSBianchi et al29Bianchi F. Galizia E. Bracci R. Belvederesi L. Catalani R. Loretelli C. Giorgetti G. Ferretti C. Bearzi I. Porfiri E. Cellerino R. Effectiveness of the CRCAPRO program in identifying patients suspected for HNPCC.Clin Genet. 2007; 71: 158-164Crossref PubMed Scopus (11) Google Scholar991NALossMutationMSI-H, microsatellite instability-high; MSI-L, microsatellite instability-low; MSS, microsatellite stable; NA, not available; VUS, variant of uncertain significance. Open table in a new tab MSI-H, microsatellite instability-high; MSI-L, microsatellite instability-low; MSS, microsatellite stable; NA, not available; VUS, variant of uncertain significance. In all 19 articles, the authors attempted to classify the pathogenicity of MLH1 K618A: in 12 articles it was categorized as a mutation, and in 7 articles as a variant of unknown significance. No time-related trend was noted. Of 24 individuals from colorectal cancer families found to carry MLH1 K618A, 11 were evaluated for tumor MSI status; 3 colonic tumors were MSI-H, 2 were MSI-L, and 6 were MSS. Immunohistochemistry for MLH1 analysis was available for 10 cases; 7 had normal results and 3 had loss of MLH1 exp
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