Abstract A13: Gene body methylation could regulate the expression of genes associated to papillary thyroid carcinomas
Authors: Beltrami Caroline Moraes , Barros-Filho Mateus Camargo , Reis Mariana Bisarro dos , Marchi Fábio Albuquerque , Kuasne Hellen , Ambatipudi Skirant , Herceg Zdenko , Kowalski Luiz Paulo , Rogatto Silvia Regina 
Published: May 2019

Background: Papillary thyroid carcinoma (PTC) is the histological subtype more frequently reported among the endocrine malignancies. Deregulation of DNA methylation has been extensively reported in cancer with hypermethylation usually found in promoter and hypomethylation in nonpromoter regions. The methylation profile of PTC revealed altered genome regions; however, its impact on the gene expression remains poorly understood. DNA methylation profile in PTC patients was performed, aiming to investigate the role of novel genic and intergenic regions on the expression of genes.

Patients and Methods: The methylation profile of 41 patients diagnosed with PTC was evaluated using the Methylation 450 Human Infinium®BeadChip platform (Illumina). The data were normalized and analyzed using SVA, watermelon, and LIMMA packages. Delta beta of 0.15 and adjusted p-value &lt;0.05 were used to identify differentially methylated probes between PTC and non-neoplastic thyroid tissue (NT). An integrative analysis was performed using the expression data obtained in 34 matched-cases. The results were submitted to a cross-validation with The Cancer Genome Atlas (TCGA) database. An in silico analysis was performed using genes/probes showing negative correlation and confirmed by TCGA using the Ingenuity Pathways Analysis. ERBB3, FGF1, FGFR2, GABRB2, HMGA2, and RDH5 were validated by pyrosequencing and RT-qPCR methods.

Results: Methylation analysis revealed 4,995 differentially methylated probes. A global hypomethylation was found in PTC samples (88% hypomethylated probes) mostly in nonpromoter regions (especially in gene body), distant from CGI and enriched by enhancers. The integrative analysis between gene expression and DNA methylation revealed 185 and 38 genes with negative and positive correlation, respectively. Genes showing negative correlation highlighted FGF and retinoic acid signaling as crucial pathways disrupted by DNA methylation in PTC. The selected genes were confirmed as altered in both methylation and expression levels. Interestingly, four of them (ERBB3, FGF1, GABRB2, and HMGA2) were mapped in gene body, suggesting that methylation in this region regulates the gene expression.

Conclusion: Loss of methylation was extensively observed in PTC, especially in nonpromoter, poor CGI, and enhancer-enriched regions. Gene body and promoter regions have the potential to influence the gene expression levels (both repressing and inducing). Genes potentially regulated by DNA methylation were identified from the integrative and cross-validation analyses. ERBB3, FGF1, FGFR2, GABRB2, HMGA2, and RDH5 biomarkers are regulated by methylation in PTC samples.

Citation Format: Caroline Moraes Beltrami, Mateus Camargo Barros-Filho, Mariana Bisarro dos Reis, Fábio Albuquerque Marchi, Hellen Kuasne, Skirant Ambatipudi, Zdenko Herceg, Luiz Paulo Kowalski, Silvia Regina Rogatto. Gene body methylation could regulate the expression of genes associated to papillary thyroid carcinomas [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A13.