Abstract 4247: Identification of new hypermethylated candidate genes in colorectal cancer using reduced representation bisulfite next generation sequencing.
Authors: Ashktorab Hassan , Daremipouran Mohammad , Rahi Hamed , Lee Eward L. , Frederick Wayne , Laiyemo Adeyinka O. , Leavitt Ron , Sun Xueguang , Varma Sudhir , Brim Hassan 
Published: August 2014

Abstract
Background: The identification and validation of genes that are frequently methylated in CRC will have potential value for both diagnostic and therapeutic implications. However, the DNA methylation patterns in CRC especially in African Americans have not been well investigated. Here we aimed to determine the methylation status of CpG islands of candidate genes in critical pathways important in the initiation and development of CRC.
Materials and Methods: Genomic DNA from 7 individuals (1 normal, 2 adenomas, and 4 adenocarcinomas) was used for global methylation analysis using Reduced Representation Bisulfite Sequencing (RRBS). Based on literature review, differential methylation ratio, gene ontology, WNT, Notch, EGFR, RAS-MPK, PI3K, TGF-b, and P53 pathways analysis, RGS3, EID3, GNAS, ATXN7L1, GAS7, HNRNPF, GPR7, SOX15, and TNFAIP2 genes were selected for methylation validation . Established methylation status of NDRG4, BMP3, Septin 9, Vimentin, and B-Actin were used as validation controls. Forty-four paired colorectal tumors and normal adjacent colonic tissue samples were used for validation. Samples were first bisulfite converted and then amplified at specific loci using 48.48 Access Array (Fluidigm). Amplicon pools were barcoded and adapterized prior to 150-bp paired-end sequencing on the Illumina MiSeq Reads were de-multiplexed using the Fluidigm indexes and mapped back to the reference genome (Hg18). A paired t-test was used for p-value and meth-diff estimation.
Results: We validated the methylation status of 355 CpG sites located in 16 gene promoter regions associated with CpG islands. Fifty-nine CpG sites located on CpG islands of ATXN7L1 (2), BMP3 (7), EID3 (15), GAS7 (1), GPR75 (24), NDRG4 (2), Sept9 (6), and TNFAIP2 (1), were significantly methylated in tumor vs. normal (p&lt;0.05). Most of these genes showed significant hypermethylation in tumors compared to normal mucosa, According to gene ontology analysis, GAS7 methylation is associated with the cadherin signaling (WNT) pathway.
Conclusion: Methylation profiling based on RRBS is an effective method for screening aberrantly methylated genes in CRC. We identified novel methylated genes that are potential biomarkers for CRC. In consistence with other reports, our study showed that GAS7 is also hypermethylated in African American CRC patients. Investigations into the possible roles of this gene as a potential biomarker in the context of early diagnosis and prognosis of CRC are underway.
Citation Format: Hassan Ashktorab, Mohammad Daremipouran, Hamed Rahi, Eward L. Lee, Wayne Frederick, Adeyinka O. Laiyemo, Ron Leavitt, Xueguang Sun, Sudhir Varma, Hassan Brim. Identification of new hypermethylated candidate genes in colorectal cancer using reduced representation bisulfite next generation sequencing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4247. doi:10.1158/1538-7445.AM2013-4247