cfDNA methylation quantification of two DNA methyl trasferase genes in human papillary thyroid carcinoma

Papillary thyroid cancer (PTC) is the most common type of thyroid malignancies. The behavior of thistumor has changed over the last decade and some diagnostic biomarker for its management is extremely needed. Circulating tumor DNA could be considered in plasma from cancer patients by detecting tumor specific alterations, such as genetic or epigenetic modifications. Epigenetic alteration of some tumor suppressor genes can be an excellent indicator of PTC invasive behavior. Circulating cell free DNA (cfDNA) methylation can be a good epigenetic representative of tumor. In this research we quantified the cfDNA methylation status of seven promoter regions through two DNA methyl Trasferase (MGMT and DNMT1) genes in PTC cases and goiter patients. Both cfDNA and tissue genomic DNA of 57 PTC and 45 Goiter samples were isolated. After bisulfite treatment the quantitative methylation were done by Methylation-sensitive high resolution melting (MS-HRM) assay technique.A methylation cut-point was defined at > 12%. Sensitivity, specificity and area under ROC curve (AUC) of methylation in individual genes were examined. Four promoter region of MGMT and three promoter region of DNMT1 were targeted. Our result suggested that amongst seven candidate promoter region the MGMT (a), MGMT (c), MGMT (d), and DNMT1 (b) methylation status were meaningfully different between PTC cases and controls. By far the most differences were seen in MGMT (c) fully methylated : 25 (43.9 %) of PTC patients versus 2 (4. 4 % )in goiter samples, and then the MGMT (d) 11 ( 19.6 % ) in PTC versus 2 ( 4.5 % ) in Goiter of circulating DNA . Between two selected DNA methyl trasfease as the writers of the epigenome the MGMT as the maintenance methylatrasferase was more significantly different between PTC cases and goiter controls in comparison with DNMT1 as a de novo methyltrasferase. The high correlation of methylation between tissue and cfDNA was distinguished but several region of each DNA methyltransferase were not correlated about methylation status. The resulting areas under the ROC curve were 0.78 for MGMT (d) for PTC versus goiter samples. Among seven candidate region of cfDNA the MGMT (c) and MGMT (d) show higher sensitivity and specificity for PTC vs. goiter. Our findings suggest these two regions can be suitable for early PTC tumor detection.

Keywords:

Papillary Thyroid Cancer , MS-HRM , Methylation , MGMT , DNMT1