Analysis of DNA methylation in canine osteosarcoma using next generation sequencing technology

Osteosarcoma is the most common type of malignant bone cancer found in dogs (Canis familiaris), with in excess of 25,000 cases diagnosed annually. DNA methylation is a form of epigenetic modification that is essential for normal development and gene transcription regulation; however, aberrant DNA methylation can result in gene expression disturbances which are frequently observed in numerous forms of cancer. In this project, DNA methylation patterns in canine osteosarcoma were investigated to provide a greater understanding of the role of epigenetics in canine osteosarcoma. Epigenetics affects heritable changes in gene expression and function without altering the genome sequence. To enable the comparison of DNA methylation in a canine osteosarcoma cell line to that of reference genomes, publicly available sequence data was retrieved, and a canine osteosarcoma cell line (OSCA-29) was sequenced using Nanopore sequencing. Oxford Nanopore sequencing is a sequencing method that measures the variation in ionic current through a biological nanopore as a single-stranded nucleic acid is ratcheted through. These signals can then be used to determine DNA methylation states bioinformatically. This project focused on the fifth canine chromosome, and using the software tool Metilene, has identified several differences in the methylation status of dogs with canine osteosarcoma to those without. Notably, four genes (PRDM16, ADAMTS15, OPCML and NTM) display a difference in methylation status. Within all four genes, the methylation differences are observed within the introns. These genes were subject to further scrutiny using Nanopore data and RT-qPCR, where all genes were found to be downregulated in canine osteosarcoma cells compared to osteoblasts from healthy dogs. This project has developed a bioinformatics workflow for analysing DNA methylation in canine osteosarcoma using Next Generation Sequencing Datasets. Preliminary results have revealed differently methylated regions in the cancer-related genes OPCML, NTM, PRDM16 and ADAMTS15, however, the precise role of these genes in the development of canine osteosarcoma remains to be investigated.
This project aids in furthering the understanding of how epigenetics regulates the development of osteosarcoma in dogs, which in turn aids the continued research into both preventative and curative treatments of osteosarcoma for both dogs and humans.