Identification of novel hyper- or hypo-methylated CpG sites and genes associated with anthracycline-induced cardiomyopathy

Genome wide DNA methylation profiling of peripheral blood DNA from childhood cancer survivors with and without anthracycline induced cardiomyopathy. DNA methylation status of 850,000 CpG sites was analyzed using the Illumina HumanMethylation EPIC BeadChip arrays. Matched case-control study (COG-ALTE03N1); 52 non-Hispanic White, anthracycline-exposed childhood cancer survivors with cardiomyopathy were matched 1:1 with 52 survivors with no cardiomyopathy. Anthracycline-induced cardiomyopathy is a leading cause of late morbidity in childhood cancers survivors. Aberrant DNA methylation plays a role in de novo cardiovascular disease. Epigenetic processes could play a role in anthracycline-induced cardiomyopathy, but remain unstudied. We sought to examine if genome-wide differential methylation at ‘CpG’ sites in peripheral blood DNA is associated with anthracycline-induced cardiomyopathy. We observed significant differences in DNA methylation between anthracycline-exposed childhood cancer survivors with and without cardiomyopathy, implicating differential DNA methylation in anthracycline-induced cardiomyopathy. Genomic DNA (1 μg) was bisulfite-converted using EZ DNA Methylation kit (Zymo Research Corp, Orange, CA, cat. no. D5004), and a cyclic denaturation step during the conversion reaction. Four microliters of bisulfite-converted DNA was used for hybridization on Infinium HumanMethylation 850 BeadChip. According to the Illumina Infinium HD Methylation protocol, the following steps were performed: whole-genome amplification, end-point fragmentation, precipitation and resuspension. The resuspended samples were hybridized onto HumanMethylation 850 BeadChips at 48°C for 18 hours. Then unhybridized and nonspecifically hybridized DNA were washed away, followed by a single nucleotide extension and repeated rounds of staining. Finally, the BeadChip was washed, coated and scanned. After scanning, the intensities of images were extracted using GenomeStudio Methylation module software (Illumina, Inc.).