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Alterations of copy number of methylation pattern in mismatch repair genes by methylation specific-multiplex ligation-dependent probe amplification in cases of colon cancer.

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 methylation-sensitive 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.

SUBMITTER: Onrat S

PROVIDER: S-EPMC3776700 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Alterations of copy number of methylation pattern in mismatch repair genes by methylation specific-multiplex ligation-dependent probe amplification in cases of colon cancer.

Onrat St S Ceken I I Ellidokuz E E Kupelioğlu A A

Balkan journal of medical genetics : BJMG 20111201 2

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 ...[more]

PMID: 24052709

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Project description:Our group has previously employed array Comparative Genomic Hybridization (aCGH) to assess the genomic patterns of BRCA1-mutated breast cancers. We have shown that the so-called BRCA1-like(aCGH) profile is also present in about half of all triple-negative sporadic breast cancers and is predictive for benefit from intensified alkylating chemotherapy. As aCGH is a rather complex method, we translated the BRCA1(aCGH) profile to a Multiplex Ligation-dependent Probe Amplification (MLPA) assay, to identify both BRCA1-mutated breast cancers and sporadic cases with a BRCA1-like(aCGH) profile.The most important genomic regions of the original aCGH based classifier (3q22-27, 5q12-14, 6p23-22, 12p13, 12q21-23, 13q31-34) were mapped to a set of 34 MLPA probes. The training set consisted of 39 BRCA1-like(aCGH) breast cancers and 45 non-BRCA1-like(aCGH) breast cancers, which had previously been analyzed by aCGH. The BRCA1-like(aCGH) group consisted of germline BRCA1-mutated cases and sporadic tumours with low BRCA1 gene expression and/or BRCA1 promoter methylation. We trained a shrunken centroids classifier on the training set and validation was performed on an independent test set of 40 BRCA1-like(aCGH) breast cancers and 32 non-BRCA1-like(aCGH) breast cancer tumours. In addition, we validated the set prospectively on 69 new triple-negative tumours.BRCAness in the training set of 84 tumours could accurately be predicted by prediction analysis of microarrays (PAM) (accuracy 94%). Application of this classifier on the independent validation set correctly predicted BRCA-like status of 62 out of 72 breast tumours (86%). Sensitivity and specificity were 85% and 87%, respectively. When the MLPA-test was subsequently applied to 46 breast tumour samples from a randomized clinical trial, the same survival benefit for BRCA1-like tumours associated with intensified alkylating chemotherapy was shown as was previously reported using the aCGH assay.Since the MLPA assay can identify BRCA1-deficient breast cancer patients, this method could be applied both for clinical genetic testing and as a predictor of treatment benefit. BRCA1-like tumours are highly sensitive to chemotherapy with DNA damaging agents, and most likely to poly ADP ribose polymerase (PARP)-inhibitors. The MLPA assay is rapid and robust, can easily be multiplexed, and works well with DNA derived from paraffin-embedded tissues.

Dataset Information

Alterations of copy number of methylation pattern in mismatch repair genes by methylation specific-multiplex ligation-dependent probe amplification in cases of colon cancer.

Publications

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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