IADR Abstract Archives
SALIVARY METHYLOME ANALYSIS in ORAL CANCER to UNRAVEL DIFFERENTIALLY METHYLATED REGIONS for NONINVASIVE DIAGNOSIS
Objectives: Genome-wide methylation analysis in oral cavity squamous cell carcinoma (OSCC) for liquid biopsy is often performed using array platforms with restricted CpG site interrogation and tissue samples which may not represent ‘dynamic’ alterations. This study aims to identify differentially methylated CpG regions that are diagnostic for OSCC in saliva at the genome scale.
Methods: Saliva samples of eight patients (4 OSCC and 4 oral mucosa disease) were randomly selected from the specimen biobank prospectively obtained for methylation analysis and verification. Genomic DNA extraction was performed on cell pellets and subjected to whole-genome methylation analysis at base-pair resolution using reduced-representation bisulfite sequencing (RRBS). Following quality control, filtered reads were aligned to the hg38 reference genome assembly, and differential methylation analysis was performed on CpG sites and 100bp regions with a minimum of 10X coverage that was common to all samples. Aberrantly methylated sites and regions were selected using a mean methylation difference > 10 and q-values < 0.01. Further, Gene Ontology terms based on the Biological process and KEGG pathway analysis were used to enrich differentially methylated regions (DMRs) annotated to genes.
Results: The analysis identified a total of 148,143 CpG sites that were differentially methylated in OSCC. Furthermore, 1823 DMRs annotated to 1066 genes were identified which were significantly hypermethylated (66.5%) than hypomethylated (33.5%). DMRs were frequently annotated to the promoter region (38%) compared to intergenic, intronic, or exonic regions (18 – 22%). DMR-related genes are enriched in the regulation of cell junction assembly, pattern specification process, and axonogenesis in the Biological Process of Gene Ontology terms as well as the Hippo signaling cascade following KEGG pathway analysis.
Conclusions: Overall, this study suggests that the aberrant methylation of genes involved in cell-cell and cell-matrix adhesion are detectable in the saliva of OSCC patients and is likely to be utilizable for disease detection.
Methods: Saliva samples of eight patients (4 OSCC and 4 oral mucosa disease) were randomly selected from the specimen biobank prospectively obtained for methylation analysis and verification. Genomic DNA extraction was performed on cell pellets and subjected to whole-genome methylation analysis at base-pair resolution using reduced-representation bisulfite sequencing (RRBS). Following quality control, filtered reads were aligned to the hg38 reference genome assembly, and differential methylation analysis was performed on CpG sites and 100bp regions with a minimum of 10X coverage that was common to all samples. Aberrantly methylated sites and regions were selected using a mean methylation difference > 10 and q-values < 0.01. Further, Gene Ontology terms based on the Biological process and KEGG pathway analysis were used to enrich differentially methylated regions (DMRs) annotated to genes.
Results: The analysis identified a total of 148,143 CpG sites that were differentially methylated in OSCC. Furthermore, 1823 DMRs annotated to 1066 genes were identified which were significantly hypermethylated (66.5%) than hypomethylated (33.5%). DMRs were frequently annotated to the promoter region (38%) compared to intergenic, intronic, or exonic regions (18 – 22%). DMR-related genes are enriched in the regulation of cell junction assembly, pattern specification process, and axonogenesis in the Biological Process of Gene Ontology terms as well as the Hippo signaling cascade following KEGG pathway analysis.
Conclusions: Overall, this study suggests that the aberrant methylation of genes involved in cell-cell and cell-matrix adhesion are detectable in the saliva of OSCC patients and is likely to be utilizable for disease detection.