Functional Significance and Clinical Phenotype of Nontruncating Mismatch Repair Variants of MLH1
2005; Elsevier BV; Volume: 129; Issue: 2 Linguagem: Inglês
10.1053/j.gastro.2005.06.005
ISSN1528-0012
AutoresTiina Raevaara, Mari Korhonen, Hannes Lohi, Heather Hampel, Elly Lynch, Karin E. Lönnqvist, Elke Holinski‐Feder, Christian Sutter, Wendy McKinnon, Sekhar Duraisamy, Anne–Marie Gerdes, Païvi Peltomäki, Maija Kohonen-Ccorish, Elisabeth Mangold, Finlay Macrae, Marc S. Greenblatt, Albert de la Chapelle, Minna Nyström,
Tópico(s)Cancer Genomics and Diagnostics
ResumoBackground & Aims: Germline mutations in mismatch repair genes are associated with hereditary nonpolyposis colorectal cancer. A significant proportion of mutations are nontruncating and associated with a variability of clinical phenotype and microsatellite instability and with occasional presence of residual protein in tumor tissue that suggests impaired functional activity but not total lack of mismatch repair. To address pathogenic significance and mechanism of pathogenicity, we studied the functionality of 31 nontruncating MLH1 mutations found in clinically characterized colorectal cancer families and 3 other variations listed in a mutation database. Methods: Mutations constructed by site-directed mutagenesis were studied for protein expression/stability, subcellular localization, protein-protein interaction, and repair efficiency. The genetic and biochemical data were correlated with clinical data. Finally, comparative sequence analysis was performed to assess the value of sequence homology as a tool for predicting functional results. Results: Altogether, 22 mutations were pathogenic in more than one assay, 2 variants were impaired in one assay, and 10 variants acted like wild-type protein. Twenty of 34 mutations affected the quantity of MLH1 protein, whereas only 15 mainly amino-terminal mutations were defective in an in vitro repair assay. Comparative sequence analysis correctly predicted functional studies for 82% of variants. Conclusions: Pathogenic nontruncating alterations in MLH1 may interfere with different biochemical mechanisms but generally more than one. The severe biochemical defects are mirrored by phenotypic characteristics such as early age at onset and high microsatellite instability, whereas variants with no or mild defects in functionality are associated with variable clinical phenotypes. Background & Aims: Germline mutations in mismatch repair genes are associated with hereditary nonpolyposis colorectal cancer. A significant proportion of mutations are nontruncating and associated with a variability of clinical phenotype and microsatellite instability and with occasional presence of residual protein in tumor tissue that suggests impaired functional activity but not total lack of mismatch repair. To address pathogenic significance and mechanism of pathogenicity, we studied the functionality of 31 nontruncating MLH1 mutations found in clinically characterized colorectal cancer families and 3 other variations listed in a mutation database. Methods: Mutations constructed by site-directed mutagenesis were studied for protein expression/stability, subcellular localization, protein-protein interaction, and repair efficiency. The genetic and biochemical data were correlated with clinical data. Finally, comparative sequence analysis was performed to assess the value of sequence homology as a tool for predicting functional results. Results: Altogether, 22 mutations were pathogenic in more than one assay, 2 variants were impaired in one assay, and 10 variants acted like wild-type protein. Twenty of 34 mutations affected the quantity of MLH1 protein, whereas only 15 mainly amino-terminal mutations were defective in an in vitro repair assay. Comparative sequence analysis correctly predicted functional studies for 82% of variants. Conclusions: Pathogenic nontruncating alterations in MLH1 may interfere with different biochemical mechanisms but generally more than one. The severe biochemical defects are mirrored by phenotypic characteristics such as early age at onset and high microsatellite instability, whereas variants with no or mild defects in functionality are associated with variable clinical phenotypes. Hereditary nonpolyposis colorectal cancer (HNPCC) is associated with dominantly inherited mutations in mismatch repair (MMR) genes. Even in the presence of uniform genetic predisposition, the clinical phenotypes, age at onset, and tumor spectrum vary significantly among families and even among patients from the same family. Furthermore, most but not all tumors show lack of the mutated protein and high microsatellite instability (MSI) in the genome, the hallmarks of HNPCC tumors. The first international criteria (Amsterdam I) for the diagnosis of HNPCC were developed in 1991 and required (1) histologically verified colorectal cancer in 3 or more relatives, one of whom is a first-degree relative of the other two; (2) colorectal cancer involving at least 2 generations; and (3) one or more colorectal cancer cases diagnosed before the age of 50 years.1Vasen H.F. Mecklin J.P. Khan P.M. Lynch H.T. The International Collaborative Group on Hereditary Non-Polyposis Colorectal Cancer (ICG-HNPCC).Dis Colon Rectum. 1991; 34: 424-425Google Scholar However, many putative HNPCC families do not fulfill the original criteria; new, less stringent criteria were developed that incorporate extracolonic cancers, notably endometrial carcinomas in addition to colorectal carcinomas (modified Amsterdam criteria, Amsterdam criteria II).2Vasen H.F. Watson P. Mecklin J.P. Lynch H.T. New clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by the International Collaborative Group on HNPCC.Gastroenterology. 1999; 116: 1453-1456Google Scholar Sometimes HNPCC should be considered even in the absence of a family history of cancer if the patient is young and has high MSI in tumor tissue (Bethesda guidelines).3Rodriguez-Bigas M.A. Boland C.R. Hamilton S.R. Henson D.E. Jass J.R. Khan P.M. Lynch H. Perucho M. Smyrk T. Sobin L. Srivastava S. Workshop on Hereditary Nonpolyposis Colorectal Cancer Syndrome meeting highlights and Bethesda guidelines.J Natl Cancer Inst. 1997; 89: 1758-1762Google Scholar, 4Umar A. Boland C.R. Terdiman J.P. Syngal S. de la Chapelle A. Ruschoff J. Fishel R. Lindor N.M. Burgart L.J. Hamelin R. Hamilton S.R. Hiatt R.A. Jass J. Lindblom A. Lynch H.T. Peltomäki P. Ramsey S.D. Rodriguez-Bigas M.A. Vasen H.F. Hawk E.T. Barrett J.C. Freedman A.N. Srivastava S. Revised Bethesda Guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability.J Natl Cancer Inst. 2004; 96: 261-268Google Scholar Remarkably, even susceptibility to sporadic, microsatellite-stable colorectal carcinoma may be linked to germline variants of MMR genes.5Lipkin S.M. Rozek L.S. Rennert G. Yang W. Chen P.C. Hacia J. Hunt N. Shin B. Fodor S. Kokoris M. Greenson J.K. Fearon E. Lynch H. Collins F. Gruber S.B. The MLH1 D132H variant is associated with susceptibility to sporadic colorectal cancer.Nat Genet. 2004; 36: 694-699Google Scholar Such a wide variety of clinical phenotypes complicates diagnostics, counseling, and design of appropriate follow-up and treatment strategies for individuals carrying the predisposing mutations. Biologic tools to predict the pathogenicity of the different mutations would be of prime clinical importance. To date, the database of sequence variations found in HNPCC or putative HNPCC kindreds (http://www.insight-group.org/) contains information on 448 different germline alterations that are likely to be pathogenic and 108 nonpathogenic alterations, affecting mostly the MMR genes MLH1 (50%), MSH2 (39%), and MSH6 (7%).6Peltomäki P. Vasen H. Mutations associated with HNPCC predisposition—update of ICG-HNPCC/InSiGHT mutation database.Dis Markers. 2004; 20: 269-276Google Scholar Germline mutations in the 2 major HNPCC-associated genes, MLH1 and MSH2, are found in two thirds of classic HNPCC that fulfill the stringent Amsterdam criteria and display MSI in tumor tissue,7Liu B. Parsons R. Papadopoulos N. Nicolaides N.C. Lynch H.T. Watson P. Jass J.R. Dunlop M. Wyllie A. Peltomäki P. de la Chapelle A. Hamilton S.R. Vogelstein B. Kinzler K.W. Analysis of mismatch repair genes in hereditary non-polyposis colorectal cancer patients.Nat Med. 1996; 2: 169-174Google Scholar compared with only 8%–47% of the less typical colon cancer families with late age at onset, atypical tumor spectrum, and/or lack of MSI.8Nyström-Lahti M. Wu Y. Moisio A.L. Hofstra R.M. Osinga J. Mecklin J.P. Järvinen H.J. Leisti J. Buys C.H. de la Chapelle A. Peltomäki P. DNA mismatch repair gene mutations in 55 kindreds with verified or putative hereditary non-polyposis colorectal cancer.Hum Mol Genet. 1996; 5: 763-769Google Scholar, 9Wijnen J. Khan P.M. Vasen H. van der Klift H. Mulder A. van Leeuwen-Cornelisse I. Bakker B. Losekoot M. Moller 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: 329-335Google Scholar Despite certain "typical" features, even the former group does not show a uniform clinical picture. Instead, the gene involved and the site and type of mutation seem to have an effect on the phenotype.10Vasen H.F. Wijnen J.T. Menko F.H. Kleibeuker J.H. Taal B.G. Griffioen G. Nagengast F.M. Meijers-Heijboer E.H. Bertario L. Varesco L. Bisgaard M.L. Mohr J. Fodde R. Khan P.M. Cancer risk in families with hereditary nonpolyposis colorectal cancer diagnosed by mutation analysis.Gastroenterology. 1996; 110: 1020-1027Google Scholar, 11Jäger A.C. Bisgaard M.L. Myrhoej T. Bernstein I. Rehfeld J.F. Nielsen F.C. Reduced frequency of extracolonic cancer families with monoallelic hMLH1 expression.Am J Hum Genet. 1997; 61: 129-138Google Scholar, 12Nyström-Lahti M. Perrera C. Raschle M. Panyushkina-Seiler E. Marra G. Curci A. Quaresima B. Costanzo F. D'Urso M. Venuta S. Jiricny J. Functional analysis of MLH1 mutations linked to hereditary nonpolyposis colon cancer.Genes Chromosomes Cancer. 2002; 33: 160-167Google Scholar In particular, missense mutations leading to single amino acid substitutions appear to be associated with a wide range of clinical phenotypes,13Peltomäki P. Vasen H.F. The International Collaborative Group on Hereditary Nonpolyposis Colorectal CancerMutations predisposing to hereditary nonpolyposis colorectal cancer database and results of a collaborative study.Gastroenterology. 1997; 113: 1146-1158Abstract Full Text Full Text PDF Scopus (683) Google Scholar and in many cases their pathogenicity is difficult to interpret. To address the pathogenic significance and mechanism of pathogenicity, we studied 34 nontruncating MLH1 mutations for expression/stability, nuclear localization, and functionality of the respective mutated protein. We also assessed how well comparative sequence analysis using alignments of eukaryotic MLH1 sequences predicts the results of functional assays. The genetic and biochemical data were then correlated with clinical data. Characterization of the biochemical defects facilitated the definition of functional domains of the MLH1 protein and revealed different mechanisms through which the pathogenic effects of the mutations were mediated. Our classification of the investigated variants as either pathogenic or nonpathogenic based on our in vitro results was supported by the clinical associations. The present study comprised 27 MLH1 missense mutations and 4 amino acid deletions found in 52 putative HNPCC families that have been subjected to molecular genetic and clinical studies suitable for prospective genotype-phenotype analysis. Table 1 shows the mutations and clinical characteristics of the index patients and their families collected through an international HNPCC collaboration. Because the HNPCC families included in the study were found and analyzed by many research groups, different methods were used for mutation detection (Table 1). The alterations are scattered throughout the MLH1 polypeptide but are mainly clustered in its amino-terminus, which is responsible for adenosine triphosphate binding and hydrolysis,14Ban C. Juno M. Yang W. Transformation of MutL by ATP binding and hydrolysis a switch in DNA mismatch repair.Cell. 1999; 97: 85-97Scopus (345) Google Scholar, 15Tran P.T. Liskay R.M. Functional studies on the candidate ATPase domains of Saccharomyces cerevisiae MutLalpha.Mol Cell Biol. 2000; 20: 6390-6398Google Scholar, 16Räschle M. Dufner P. Marra G. Jiricny J. Mutations within the hMLH1 and hPMS2 subunits of the human MutLalpha mismatch repair factor affect its ATPase activity, but not its ability to interact with hMutSalpha.J Biol Chem. 2002; 277: 21810-21820Google Scholar and in the carboxyl-terminus, which contains the region where MLH1 interacts with its counterparts, PMS2, MLH3, and PMS1 (Figure 1).17Guerrette S. Acharya S. Fishel R. The interaction of the human MutL homologues in hereditary nonpolyposis colon cancer.J Biol Chem. 1999; 274: 6336-6341Google Scholar, 18Kondo E. Horii A. Fukushige S. The interaction domains of three MutL heterodimers in man hMLH1 interacts with 36 homologous amino acid residues within hMLH3, hPMS1 and hPMS2.Nucleic Acids Res. 2001; 29: 1695-1708Google Scholar, 19Plotz G. Raedle J. Brieger A. Trojan J. Zeuzem S. N-terminus of hMLH1 confers interaction of hMutLalpha and hMutLbeta with hMutSalpha.Nucleic Acids Res. 2003; 31: 3217-3226Google ScholarTable 1Genetic and Clinical Data of HNPCC FamiliesFamily IDMLH1 mutationNo. of Affected patientsaAffected patients with HNPCC tumors.Amsterdam criteria IIndex patient: age at onset (y)/cancer siteAll affected patientsaAffected patients with HNPCC tumors.: mean age at onset (y)Method for mutation analysisMSI statusbFor the MSI analysis, the Bethesda panel was applied (BAT25, BAT26, D2S123, D5S346, and D17S250, or in some cases D18S69).42 Tumors with 2 or more unstable markers were considered to have a high degree of MSI.ImmunohistochemistryMLH1MSH2MSH652P28L4+30/CRC37DHPLCHighNANANA413P28L2−27/CRC29DHPLCHighNo lossNo lossNA1744A29S4+37/CRC47DSHighLossNo lossNA790TS145-47CF4+32/CRC43DHPLCNANANo lossNAB410D63E3−44/CRC41DSHighDecreasedNo lossNA487G67R1−36/CRC36DHPLCHighNANANA267Del718−24/CRC27Reference 24Raevaara T.E. Timoharju T. Lönnqvist K.E. Kariola R. Steinhoff M. Hofstra R.M.W. Mangold E. Vos Y.J. Nyström-Lahti M. Description and functional analysis of a novel in frame mutation linked to hereditary non-polyposis colorectal cancer.J Med Genet. 2002; 39: 747-750Google ScholarHighLossNo lossNAN:1Del719+19/CRC36Reference 24Raevaara T.E. Timoharju T. Lönnqvist K.E. Kariola R. Steinhoff M. Hofstra R.M.W. Mangold E. Vos Y.J. Nyström-Lahti M. Description and functional analysis of a novel in frame mutation linked to hereditary non-polyposis colorectal cancer.J Med Genet. 2002; 39: 747-750Google ScholarHighNANANA26C77R10+22/CRC32Reference 12Nyström-Lahti M. Perrera C. Raschle M. Panyushkina-Seiler E. Marra G. Curci A. Quaresima B. Costanzo F. D'Urso M. Venuta S. Jiricny J. Functional analysis of MLH1 mutations linked to hereditary nonpolyposis colon cancer.Genes Chromosomes Cancer. 2002; 33: 160-167Google ScholarHighNANANA1F80V3+51/CRC52DHPLCHighNo lossNo lossNA171K84E2−32/CRC36DHPLCHighNANANAI:1S93G13+70/CRC65Reference 39Quaresima B. Grandinetti C. Baudi F. Tassone P. Barbieri V. Conforti S. Avvedimento E.V. Costanzo F. Venuta S. Hereditary nonpolyposis colorectal cancer identification of novel germline mutations in two kindreds not fulfilling the Amsterdam criteria.Hum Mutat. 1998; 12: 433Google ScholarNANANANA105I107R7+46/EC49Reference 8Nyström-Lahti M. Wu Y. Moisio A.L. Hofstra R.M. Osinga J. Mecklin J.P. Järvinen H.J. Leisti J. Buys C.H. de la Chapelle A. Peltomäki P. DNA mismatch repair gene mutations in 55 kindreds with verified or putative hereditary non-polyposis colorectal cancer.Hum Mol Genet. 1996; 5: 763-769Google ScholarHighLossNANA67I107R7+53/CRC51Reference 8Nyström-Lahti M. Wu Y. Moisio A.L. Hofstra R.M. Osinga J. Mecklin J.P. Järvinen H.J. Leisti J. Buys C.H. de la Chapelle A. Peltomäki P. DNA mismatch repair gene mutations in 55 kindreds with verified or putative hereditary non-polyposis colorectal cancer.Hum Mol Genet. 1996; 5: 763-769Google ScholarHighLossNANA51I107R7+46/CA46Reference 8Nyström-Lahti M. Wu Y. Moisio A.L. Hofstra R.M. Osinga J. Mecklin J.P. Järvinen H.J. Leisti J. Buys C.H. de la Chapelle A. Peltomäki P. DNA mismatch repair gene mutations in 55 kindreds with verified or putative hereditary non-polyposis colorectal cancer.Hum Mol Genet. 1996; 5: 763-769Google ScholarNANANANA28I107R4+30/CRC54Reference 8Nyström-Lahti M. Wu Y. Moisio A.L. Hofstra R.M. Osinga J. Mecklin J.P. Järvinen H.J. Leisti J. Buys C.H. de la Chapelle A. Peltomäki P. DNA mismatch repair gene mutations in 55 kindreds with verified or putative hereditary non-polyposis colorectal cancer.Hum Mol Genet. 1996; 5: 763-769Google ScholarHighLossNANA194L155R5+33/CRC41DHPLCHighLossNo lossNAA:1V185G8+43/CRC44DSHighDecreasedNo lossNo loss169V213M3−64/CRC58DS, MLPALowNo lossNo lossLoss1020V213M4−44/EC53DS, MLPAHighNo lossLossLoss1219V213M1−67/CRC67DS, MLPAHighLossNo lossNo loss1364V213M2−46/CRC42DS, MLPAHighNo lossNo lossNo loss327S247P10+42/CRC49DHPLCHighDecreasedNo lossNA823S247P4−43/CRC44DHPLCNANANANA2H329P8+32/CRC44DHPLCHighLossNo lossNAA:2Del3306+29/CRC41DSHighLossNo lossNo loss129K443Q8−57/CRC62DS, MLPAHighLossNo lossNo loss545L550P3−45/CRC35DHPLCHighLossNo lossNA506A589D3−34/CRC34DHPLCHighLossNo lossNA777Del6125+47/CRC51DHPLCNANANANA4401Del61612+44/CRC50Reference 25Raevaara T.E. Vaccaro C. Abdel-Rahman W.M. Mocetti E. Bala S. Lönnqvist K.E. Kariola R. Lynch H.T. Peltomäki P. Nyström-Lahti M. Pathogenicity of the hereditary colorectal cancer mutation hMLH1 del616 linked to shortage of the functional protein.Gastroenterology. 2003; 125: 501-509Google ScholarHighLossNo lossNo loss539K618A2−43/CRC41DHPLCHighNo lossNo lossNA595K618A3 (+2)+44/CRC60DHPLCMSSNo lossNo lossNA4K618A3−33/CRC43DHPLCNANANANA171K618A2−76/CRC77DS, MLPALowLossNo lossNo loss793K618A1−72/CRC72DS, MLPAHighLossNo lossNo loss797K618A1−69/CRC69DS, MLPALowNo lossNo lossNo loss132cFamily 132 carries 2 MLH1 mutations; K618A and R659Q.K618A10+32/CRC38DS, MLPAHighLossNo lossNo loss283Y646C1−36/CRC36DS, MLPAHighNo lossNo lossNo loss5P648L4−43/CRC43DHPLCHighNo lossNo lossNAD:1P648S11+54/CRC50Reference 40Bisgaard M.L. Jäger A.C. Myrhoj T. Bernstein I. Nielsen F.C. Hereditary non-polyposis colorectal cancer (HNPCC) phenotype-genotype correlation between patients with and without identified mutation.Hum Mutat. 2002; 20: 20-27Google ScholarHighLossNo lossNo loss473P654L3−35/CRC53DHPLCNANANo lossNA15P654L6−31/CRC50DHPLCNALossNANAB439P654L7+41/CRC50DSHighLossNo lossNA585P654L2−38/CRC54DHPLCHighLossNo lossNA7R659P7+35/CRC45Reference 8Nyström-Lahti M. Wu Y. Moisio A.L. Hofstra R.M. Osinga J. Mecklin J.P. Järvinen H.J. Leisti J. Buys C.H. de la Chapelle A. Peltomäki P. DNA mismatch repair gene mutations in 55 kindreds with verified or putative hereditary non-polyposis colorectal cancer.Hum Mol Genet. 1996; 5: 763-769Google ScholarHighLossNANA132cFamily 132 carries 2 MLH1 mutations; K618A and R659Q.R659Q10+32/CRC38DS, MLPAHighLossNo lossNo loss652A681T1−38/CRC38DHPLCHighNANo lossNA8V716M3+43/CRC57DHPLCHighLossNo lossNA9V716M6+52/CRC46DHPLCMSSNANo lossNA55426V716M2−65/CRC67DS, MLPALowNo lossNo lossNo loss74V716M12−39/EC45DS, MLPAHighLossNo lossNo lossNOTE. The primary antibodies used in immunohistochemistry were anti-MLH1 (clone G168-15, BD Pharmingen, San Diego, CA), anti-MSH2 (clone FE11, Calbiochem/oncogene Research Products, San Diego, CA), and anti-MSH6 (clone 44, Transduction Laboratories, Lexington, KY). The percentage of tumor cells staining with the antibodies was recorded for each sample as recommended.41Boland C.R. Thibodeau S.N. Hamilton S.R. Sidransky D. Eshleman J.R. Burt R.W. Meltzer S.J. Rodriguez-Bigas M.A. Fodde R. Ranzani G.N. Srivastava S. A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition development of international criteria for the determination of microsatellite instability in colorectal cancer.Cancer Res. 1998; 58: 5248-5257Google ScholarCRC, colorectal cancer; EC, endometrial cancer; CA, colorectal adenoma; DHPLC, denaturing high-performance liquid chromatography; DS, direct sequencing; MLPA, multiplex ligation-dependent probe amplification; MSS, microsatellite stable; NA, not available.a Affected patients with HNPCC tumors.b For the MSI analysis, the Bethesda panel was applied (BAT25, BAT26, D2S123, D5S346, and D17S250, or in some cases D18S69).42Müller W. Burgart L.J. Krause-Paulus R. Thibodeau S.N. Almeida M. Edmonston T.B. Boland C.R. Sutter C. Jass J.R. Lindblom A. Lubinski J. MacDermot K. Sanders D.S. Morreau H. Muller A. Oliani C. Orntoft T. Ponz De Leon M. Rosty C. Rodriguez-Bigas M. Ruschoff J. Ruszkiewicz A. Sabourin J. Salovaara R. Moslein G. ICG-HNPCC (International Collaborative Group)The reliability of immunohistochemistry as a prescreening method for the diagnosis of hereditary nonpolyposis colorectal cancer (HNPCC)—results of an international collaborative study.Fam Cancer. 2001; 1: 87-92Google Scholar Tumors with 2 or more unstable markers were considered to have a high degree of MSI.c Family 132 carries 2 MLH1 mutations; K618A and R659Q. Open table in a new tab NOTE. The primary antibodies used in immunohistochemistry were anti-MLH1 (clone G168-15, BD Pharmingen, San Diego, CA), anti-MSH2 (clone FE11, Calbiochem/oncogene Research Products, San Diego, CA), and anti-MSH6 (clone 44, Transduction Laboratories, Lexington, KY). The percentage of tumor cells staining with the antibodies was recorded for each sample as recommended.41Boland C.R. Thibodeau S.N. Hamilton S.R. Sidransky D. Eshleman J.R. Burt R.W. Meltzer S.J. Rodriguez-Bigas M.A. Fodde R. Ranzani G.N. Srivastava S. A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition development of international criteria for the determination of microsatellite instability in colorectal cancer.Cancer Res. 1998; 58: 5248-5257Google Scholar CRC, colorectal cancer; EC, endometrial cancer; CA, colorectal adenoma; DHPLC, denaturing high-performance liquid chromatography; DS, direct sequencing; MLPA, multiplex ligation-dependent probe amplification; MSS, microsatellite stable; NA, not available. Half of the families did not fulfill the Amsterdam criteria. Mean age at onset of HNPCC-related tumors within the families varied from 27 to 77 years, and age at onset of index patients varied from 19 to 76 years. Altogether, 69 verified mutation carriers have had only colorectal carcinoma(s), 8 had colorectal carcinoma and endometrial carcinoma, and 5 had only endometrial carcinoma (data not shown). MSI phenotypes were high in 38, low in 4, and stable in 2 tumors originating from mutation carriers in different families (Table 1). Immunohistochemical staining was performed in tumors from 38 families, and MLH1 protein was lost in 24, decreased in 3, and present in 11 tumors (Table 1). In 4 tumors associated with mutations V213M, K618A, and V716M, MLH1 staining was positive and MSI status was low or stable. In addition to 31 mutations found in HNPCC families, 3 MLH1 variations listed in the international HNPCC mutation database (http://www.insight-group.org/) were studied: (1) I219V, which has been shown to be nonpathogenic in previous functional assays20Shimodaira H. Filosi N. Shibata H. Suzuki T. Radice P. Kanamaru R. Friend S.H. Kolodner R.D. Ishioka C. Functional analysis of human MLH1 mutations in Saccharomyces cerevisiae.Nat Genet. 1998; 19: 384-389Google Scholar, 21Ellison A.R. Lofing J. Bitter G.A. Functional analysis of human MLH1 and MSH2 missense variants and hybrid human-yeast MLH1 proteins in Saccharomyces cerevisiae.Hum Mol Genet. 2001; 10: 1889-1900Google Scholar, 22Trojan J. Zeuzem S. Randolph A. Hemmerle C. Brieger A. Raedle J. Plotz G. Jiricny J. Marra G. Functional analysis of hMLH1 variants and HNPCC-related mutations using a human expression system.Gastroenterology. 2002; 122: 211-219Abstract Full Text Full Text PDF Scopus (173) Google Scholar, 23Kondo E. Suzuki H. Horii A. Fukushige S. A yeast two-hybrid assay provides a simple way to evaluate the vast majority of hMLH1 germ-line mutations.Cancer Res. 2003; 63: 3302-3308Google Scholar and used here as a functional control; (2) K618T, whose pathogenicity according to previous functional assays has remained partly unsettled17Guerrette S. Acharya S. Fishel R. The interaction of the human MutL homologues in hereditary nonpolyposis colon cancer.J Biol Chem. 1999; 274: 6336-6341Google Scholar, 20Shimodaira H. Filosi N. Shibata H. Suzuki T. Radice P. Kanamaru R. Friend S.H. Kolodner R.D. Ishioka C. Functional analysis of human MLH1 mutations in Saccharomyces cerevisiae.Nat Genet. 1998; 19: 384-389Google Scholar, 22Trojan J. Zeuzem S. Randolph A. Hemmerle C. Brieger A. Raedle J. Plotz G. Jiricny J. Marra G. Functional analysis of hMLH1 variants and HNPCC-related mutations using a human expression system.Gastroenterology. 2002; 122: 211-219Abstract Full Text Full Text PDF Scopus (173) Google Scholar, 23Kondo E. Suzuki H. Horii A. Fukushige S. A yeast two-hybrid assay provides a simple way to evaluate the vast majority of hMLH1 germ-line mutations.Cancer Res. 2003; 63: 3302-3308Google Scholar; and (3) an in-frame deletion of codons 633–663, comprising exon 17, which we previously found to be pathogenic12Nyström-Lahti M. Perrera C. Raschle M. Panyushkina-Seiler E. Marra G. Curci A. Quaresima B. Costanzo F. D'Urso M. Venuta S. Jiricny J. Functional analysis of MLH1 mutations linked to hereditary nonpolyposis colon cancer.Genes Chromosomes Cancer. 2002; 33: 160-167Google Scholar and used here as a nonfunctional control. All studies were approved by the institutional review boards of the collaborating universities or local ethical committees. The MLH1 variants were created using site-directed mutagenesis as described previously.12Nyström-Lahti M. Perrera C. Raschle M. Panyushkina-Seiler E. Marra G. Curci A. Quaresima B. Costanzo F. D'Urso M. Venuta S. Jiricny J. Functional analysis of MLH1 mutations linked to hereditary nonpolyposis colon cancer.Genes Chromosomes Cancer. 2002; 33: 160-167Google Scholar, 24Raevaara T.E. Timoharju T. Lönnqvist K.E. Kariola R. Steinhoff M. Hofstra R.M.W. Mangold E. Vos Y.J. Nyström-Lahti M. Description and functional analysis of a novel in frame mutation linked to hereditary non-polyposis colorectal cancer.J Med Genet. 2002; 39: 747-750Google Scholar All of the primer sequences, polymerase chain reaction (PCR) parameters, and restriction sites are available from the authors upon request. In the first PCR, wild-type (WT) MLH1 complementary DNA (cDNA) cloned into pFastBac1 plasmid (Invitrogen, Carlsbad, CA) was used as template; the fragment A was created using primer pair forward A (fA) and reverse A (rA) and the fragment B was with primers fB and rB. In the second PCR, the primers fA and rB were used to complete the PCR products. The fragment containing the mutation was cloned into the original plasmid between the appropriate restriction sites, and the cloned fragments were verified by sequencing (AbiPrism 3100 Genetic Analyzer; Applied Biosystems, Foster City, CA). The recombinant baculoviruses for protein production in Spodoptera frugiperda 9 (Sf 9) insect cells were generated using the Bac-to-Bac system according to the manufacturer's instructions (Invitrogen). For protein expression in human cells, the mutated fragments were cloned into pMLH1-N1 plasmid (modified from pEGFP-N1 plasmid; BD Biosciences, Palo Alto, CA) as previously described.25Raevaara T.E. Vaccaro C. Abdel-Rahman W.M. Mocetti E. Bala S. Lönnqvist K.E. Kariola R. Lynch H.T. Peltomäki P. Nyström-Lahti M. Pathogenicity of the hereditary colorectal cancer mutation hMLH1 del616 linked to shortage of the functional protein.Gastroenterology. 2003; 125: 501-509Google Scholar, 26Raevaara T.E. Gerdes A.-M. Lönnqvist K.E. Tybjærg-Hansen A. Abdel-Rahman W.M. Kariola R. Peltomäki P. Nyström-Lahti M. HNPCC mutation MLH1 P648S makes the functional protein unstable and homozygosity predisposes to mild neurofibromatosis type 1.Genes Chromosomes Cancer. 2004; 40: 261-265Google Scholar For fluorescent protein production, MLH1 and PMS2 were fused to enhanced green fluorescent protein gene (EGFP). For cloning, stop codons were removed and the appropriate restriction sites generated into MLH1 and PMS2 cDNAs by the site-directed mutagenesis method described previously using pFastBac1-MLH1 and pFastBac1-PMS2 as templates. An NheI restriction site was generated upstream of the MLH1 start codon. A SacI site was generated to replace the MLH1 stop codon. The products of the second PCRs were cloned into the pFastBac1-MLH1 vector between BamHI and PvuII or NsiI and NotI sites, respectively. In PMS2, the stop codon was replaced by an AgeI restriction site. The product of the second PCR was cloned into the pFastBac1-PMS2 vector between restriction sites SpeI and NotI. The MLH1 and PMS2 cDNAs without stop codons were then cloned into the pEGFP-N1 vector (BD Biosciences) between NheI and SacI and BamHI and AgeI sites, respectively. The resulting constructs expressing MLH1-EGFP and PMS2-EGFP fluorescent fusion proteins are mentioned here as pMLH1-EGFP and pPMS2-EGFP. The variants A29S, TSI45-47CF, D63E, and G67R were generated to the pMLH1-EGFP plasmid between the NheI and EcoNI sites by site-directed mutagenesis as described previously. All other variants were cloned from pFastBac1-MLH1 into pMLH1-EGFP between the BglII and AccIII sites. For protein production in insect cells, 2 × 107 Sf 9 cells were coinfected with WT or mutated MLH1 together with WT PMS2 recombinant baculoviruses. After 48 hours of culture, the total protein extracts (TEs) including overexpressed MLH1 and PMS2 proteins were prepared as described previously.12Nyström-Lahti M. Perrera C. Raschle M. Panyushkina-Seiler E. Marra G. Curci A. Quaresima B. Costanzo F. D'Urso M. Venuta S. Jiricny J. Functional analysis of MLH1 mutations linked to hereditary nonpolyposis colon cancer.Genes Chromosomes Cancer. 2002; 33: 160-167Google Scholar, 24Raevaara T.E
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