Alterations of Copy Number of Methylation Pattern in Mismatch Repair Genes by Methylation Specific-Multiplex Ligation-Dependent Probe Amplification in Cases of Colon Cancer

S Onrat; I Çeken; E Ellidokuz; A Kupelioğlu

doi:10.2478/v10034-011-0044-x

Abstract

Genetic alterations and changes in genomic DNA cytosine methylation patterns are associated with all types of cancer and are caused by germline mutations in DNA mismatch repair (MMR) genes, predominantly MLH1 (MutL homolog 1, 19 exons) and MSH2 (MutS homolog 2, 16 exons). Genomic DNA was extracted from tissue samples embedded in paraffin from 49 patients with adenocarcinoma and from 21 patients with carcinoma for the study group; genomic DNA was extracted from lymphocytes from 10 healthy donors for the control group. We used methylation specific multiplex ligation-dependent probe amplification (MS-ML-PA), which allows the detection of copy number changes and unusual methylation levels of 10 to 50 different sequences in one reaction by use of the methylationsensitive restriction enzyme HhaI and sequence-specific capillary electrophoresis for the study of 24 genes.

We found the mean methylation rates for MLH1 (97.14%), MSH2 (24.28%), MSH6 (MutS homolog 6) (67.14%), MSH3 (MutS homolog 3) (78.57%), MLH3 (MutL homolog 3) (75.71%), PMS2 (postmeiotic segregation increased 2) (65.71%), MGMT(O-6-methylguanine-DNA methyltransferase) (82.85%). We conclude that the mismatch repair (MMR) system is critical for the maintenance of genomic stability.

References

Peltomäki P. Role of DNA mismatch repair defects in the pathogenesis of human cancer. J Clin Oncol. 2003; 21(6): 1174-1179.10.1200/JCO.2003.04.06012637487
Search in Google Scholar
Chan TL, Yuen ST, Ho JW, Chan AS, Kwan K, Chung LP, Lam PW, Tse CW, Leung SY. A novel germline 1.8-kb deletion of hMLH1 mimicking alternative splicing: a founder mutation in the Chinese population. Oncogene. 2001; 20(23): 2976-2981.10.1038/sj.onc.120437611420710
Search in Google Scholar
Li L, McVety S, Younan R, Liang P, Du Sart D, Gordon PH, Hutter P, Hogervorst FB, Chong G, Foulkes WD. Distinct patterns of germline deletions in MLH1 and MSH2: the implication of Alu repetitive element in the genetic etiology of Lynch syndrome (HNPCC). Hum Mutat. 2006; 27(4): 388.10.1002/humu.941716541406
Search in Google Scholar
Jeuken JW, Cornelissen JBS, Vriezen M, Dekkers MMG, Errami A, Sijben A, Boots-Sprenger SHE, Wesseling P. MS-MLPA: an attractive alternative laboratory assay for robust, reliable, and semiquantitative detection of MGMT promoter hypermethylation in gliomas. Lab Invest. 2007; 87(10): 1055-1065.10.1038/labinvest.370066417700563
Search in Google Scholar
Kunkel TA, Erie DA. DNA mismatch repair. Annu Rev Biochem. 2005; 74: 681-710.10.1146/annurev.biochem.74.082803.13324315952900
Search in Google Scholar
Strand M, Prolla TA, Liskay RM, Petes TD. Destabilization of tracts of simple repetitive DNA in yeast by mutations affecting DNA mismatch repair. Nature. 1993; 365(6443): 274-276.10.1038/365274a08371783
Search in Google Scholar
Sia EA, Kokoska RJ, Dominska M, Greenwell P, Petes TD. Microsatellite instability in yeast:dependence on repeat unit size and DNA mismatch repair genes. Mol Cell Biol. 1997; 17(5): 2851-2858.10.1128/MCB.17.5.28512321379111357
Search in Google Scholar
Peltomäki P, Aaltonen LA, Sistonen P, Pylkkanen L, Mecklin JP, Järvinen H, Green JS, Jass JR, Weber JL, Leach FS, Petersen GM, Hamilton SR, de la Chapelle A, Vogelstein B. Genetic mapping of a locus predisposing to human colorectal cancer. Science. 1993; 260(5109): 810-812.10.1126/science.84841208484120
Search in Google Scholar
Peltomäki P. Lynch syndrome genes. Fam Cancer. 2005; 4(3): 227-232.10.1007/s10689-004-7993-016136382
Search in Google Scholar
Liu T, Yan H, Kuismanen S, Percesepe A, Bisgaard ML, Pedroni M, Benatti P, Kinzler KW, Vogelstein B, Ponz de Leon M, Peltomäki P, Lindblom A. The role of hPMS1 and hPMS2 in predisposing to colorectal cancer. Cancer Res. 2001; 61(21):7798-7802.
Search in Google Scholar
Huang J, Kuismanen SA, Liu T, Chadwick RB, Johnson CK, Stevens MW, Richards SK, Meek JE, Gao X, Wright FA, Mecklin J-P, Järvinen HJ, Grönberg H, Bisgaard ML, Lindblom A, Peltomäki P. MSH6 and MSH3 are rarely involved in genetic predisposition to nonpolypotic colon cancer. Cancer Res. 2001; 61(4): 1619-1623.
Search in Google Scholar
Peltomäki P, Vasen H. Mutations associated with HNPCC predisposition - Update of ICGHNPCC/INSiGHT mutation database. Dis Markers. 2004; 20(4-5): 269-276.10.1155/2004/305058383939715528792
Search in Google Scholar
Tannergård P, Lipfordd JR, Kolodner R, Frodin RJE, Nordenskjöld M, Lindblom A. Mutation screening in the hMLH1 gene in Swedish hereditary nonpolyposis colon cancer families. Cancer Res. 1995; 55(24): 6092-6096.
Search in Google Scholar
Vasen. HFA., Wijnen J, Menko FH, Kleibeuker JH, Taal BG, Griffioen G, Nagengast FM, Meijers-Heijboer EJ, Bertario L, Varesco L, Bisgaard M, Mohr J, Fodde R, Meera Khan P. Cancer risk in families with hereditary nonpolyposis colorectal cancer diagnosed by mutation analysis. Gastroenterology. 1996; 110(4): 1020-1027.10.1053/gast.1996.v110.pm8612988
Search in Google Scholar
Kane MF, Loda M, Gaida GM, Lipman J, Mishra R, Goldman H, Jessup JM, Kolodner, R. Methylation of the hMLH1 promoter correlates with lack of expression oh hMLH1 in sporadic colon tumors and mismatch repair-defective human tumor cell lines. Cancer Res. 1997; 57(5): 808-811.
Search in Google Scholar
Herman JG, Umar A, Plyak K, Graff JR, Ahuja N, Issa J-PJ, Markowitz S, Willson JKV, Hamilton SR, Kinzler KW, Kane MF, Kolodner RD, Vogelstein B, Kunkel TA, Baylin SB. Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carsinoma. Proc Natl Acad Sci USA 1998; 95(12): 6870-6875.10.1073/pnas.95.12.6870
Search in Google Scholar
Gisselsson D. Chromosome instability in cancer: how, when, and why? Adv Cancer Res. 2003; 87: 1-29.10.1016/S0065-230X(03)87164-6
Search in Google Scholar
Esteller M, Herman JG. Cancer as an epigenetic disease: DNA methylation and chromatin alterations in human tumours. J Pathol. 2002; 196(1): 1-7.10.1002/path.1024
Search in Google Scholar
Esteller M. Relevance of DNA methylation in the management of cancer. Lancet Oncol. 2003; 4(6): 351-358.10.1016/S1470-2045(03)01115-X
Search in Google Scholar
Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D, Diepvens F, Pals G. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res. 2002; 30(12): e57.10.1093/nar/gnf05611729912060695
Search in Google Scholar
MRC-Holland, Amsterdam, The Netherlands. http://www.mrcholland.com
Search in Google Scholar
Jeuken JWM, Cornelissen S, Boots-Sprenger SHE, Gijsen S, Wesseling P. Multiplex ligation-dependent probe amplification (MLPA): a diagnostic tool for simultaneous identification of different genetic markers in glial tumors. J Mol Diagnostics. 2006; 8(4): 433-443.10.2353/jmoldx.2006.060012186761516931583
Search in Google Scholar
Morak M, Schackert HK, Rahner N, Betz B, Ebert M, Walldorf C, Royer-Pokora B, Schulmann K, von Knebel-Doeberitz M, Dietmaier W, Keller G, Kerker B. Further evidence for heritability of an epimutation in one of 12 cases with MLH1 promoter methylation in blood cells clinically displaying HNPCC. Eur J Hum Genet. 2008; 16(7): 804-811.10.1038/ejhg.2008.2518301449
Search in Google Scholar
Nyström-Lahti M, Wu Y, Moisio A-L, Hoftra RMW, Osinga J, Mecklin J-P, Järvinen HJ, Leisti J, Buys CHCM, 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(6): 763-769.10.1093/hmg/5.6.7638776590
Search in Google Scholar
Nyström-Lahti M, Kristo P, Nicolaides NC, Chang SY, Aaltonen LA, Moisio AL, Järvinen HJ, Mecklin JP, Kinzler KW, Vogelstein B, de la Chapelle A, Peltomäki P. Founding mutations and Alu mediated recombination in hereditary colon cancer. Nat Med. 1995; 1(11): 1203-1206.10.1038/nm1195-12037584997
Search in Google Scholar
Moisio AL, Sistonen P, Weissenbach J, de la Chapelle A, Peltomäki P. Age and origin of two common MLH1 mutations predisposing to hereditary colon cancer. Am J Hum Genet. 1996; 59(6): 1243-1251.
Search in Google Scholar
Liu HX, Zhou XL, Liu T, Werelius B, Lindmark G, Dahl N, Lindblom A. The role of hMLH3 in familial colorectal cancer. Cancer Res. 2003; 63(8): 1894-1899.
Search in Google Scholar
Kariola R, Raevaara TE, Lonnqvist KE, Nyström-Lahti M. Functional analysis of MSH6 mutations linked to kindreds with putative hereditary non-polyposis colorectal cancer syndrome. Hum Mol Genet. 2002; 11(11): 1303-1310.10.1093/hmg/11.11.130312019211
Search in Google Scholar
Kariola R, Hampel H, Frankel WL, Raevaara TE, de la Chapelle A, Nyström-Lahti M. MSH6 missense mutations are often associated with no or low cancer susceptibility. Br J Cancer. 2004; 91(7): 1287-1292.10.1038/sj.bjc.6602129240991215354210
Search in Google Scholar
Deng G, Peng E, Gum J, Terdiman J, Sleisenger M, Kim YS. Methylation of hMLH1 promoter correlates with the gene silencing with a region-specific manner in colorectal cancer. Br J Cancer. 2002; 86(4): 574-579.10.1038/sj.bjc.6600148237527711870540
Search in Google Scholar
Boland CR, Goel A. The silence of the genes: matching mismatch repair defects with tumors. Cancer. 2003; 98(1): 2091-2094.
Search in Google Scholar
Jubb AM, Bell SM, Quirke P. Mythylation and colorectal cancer. J Pathol. 2001; 195(1): 111-134.10.1002/path.92311568897
Search in Google Scholar
Deng G, Chen A, Hong J, Chae HS, Kim YS. Methylation of CpG in a small region of the hMLH1 promoter invariably correlates with the absence of gene expression. Cancer Res. 1999; 59(9): 2029-2033.
Search in Google Scholar
Hawkins N, Norrie M, Cheong K, Mokany E, Ku SL, Meagher A, O'Connor T, Ward R. CpG island methylation in sporadic colorectal cancers and its relationship to microsatellite instability. Gastroenterology. 2002; 122(5): 1376-1387.10.1053/gast.2002.3299711984524
Search in Google Scholar

Alterations of Copy Number of Methylation Pattern in Mismatch Repair Genes by Methylation Specific-Multiplex Ligation-Dependent Probe Amplification in Cases of Colon Cancer

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