Krisztina Andrea Szigeti1, Orsolya Galamb dr.2, Alexandra Kalmár dr.2, Gábor Valcz dr.1,2, Barnabás Wichmann dr.1,2, Sára Zsigrai dr.1, Zsófia Brigitta Nagy1, Barbara Kinga Barták dr.1, Zsolt Tulassay dr.1,2, Péter Igaz dr.1,2, Béla Molnár dr.1,2
1 2nd Department of Internal Medicine, Semmelweis University, Budapest
2 Molecular Medicine Research Unit, Hungarian Academy of Sciences, Budapest
Introduction: Besides promoter DNA hypermethylation of tumor suppressor genes, global DNA hypomethylation is also characteristic in cancerous diseases. Global DNA hypomethylation can cause genetic instability through the regulation of mobile genetic elements. Numerous studies have observed the hypomethylation of LINE-1 retrotransposon in the development of cancerous diseases. Copies of LINE-1 retrotransposon compose 17% of the human genome, thereby the methylation level of the whole genome could be estimated by determining DNA methylation level of LINE-1.
Aims: We aimed to examine the global DNA methylation level in tissue and plasma samples of colorectal normal-adenoma-carcinoma sequence, inflammatory bowel disease patients for diagnostic purposes.
Method: Bisulfite treatment was performed on the genomic DNA isolated from 30 normal (N), 10 adenoma (Ad), 10 colorectal carcinoma (CRC) and 10 ulcerative colitis (UC) tissue samples and on 11 N, 10 Ad, 15 CRC and 12 UC plasma samples. The LINE-1 bisulfite-specific PCR product was generated and pyrosequenced. The in situ tissue appearance of 5-methylcytosine was analyzed by immunohistochemistry staining (IHC).
Results: According to the LINE-1 bisulfite sequencing results, significant DNA hypomethylation was found in CRC (62,9±8,7%; p<0,001) and Ad (66,7±5,1%; p<0,001) tissue samples in comparison with the normal tissues (72±1,4%). Moreover, significant decrease of DNA methylation was observed in CRC (78,8±1,7%; p<0,02) and Ad (80,1±1,7%; p<0,02) plasma samples compared to normal plasma specimens (82,2±1,8%). Global DNA hypomethylation was not detected in the case of UC. 5-mC labelling of normal samples was strong (scoring values: +2 and +3), however, in adenomas (p<0,0039) and CRC samples (p<0.0015) significantly lower 5-mC level was observed than in normal tissue samples.
Conclusion: A significant decrease in DNA methylation level was found in the tissue and liquid biopsy samples of colorectal normal-adenoma-carcinoma sequences, but not in UC specimens. Our results suggest that determination of DNA hypomethylation could have prognostic and diagnostic value.
Doctoral School: Clinical Medicine
Supervisor: Béla Molnár
E-mail address: firstname.lastname@example.org