Analysis of DNA Methylation in a Three-Generation Family Reveals Widespread Genetic Influence on Epigenetic Regulation

Abstract

The methylation of cytosines in CpG dinucleotides is essential for cellular differentiation and the progression of many cancers, and it plays an important role in gametic imprinting. To assess variation and inheritance of genome-wide patterns of DNA methylation simultaneously in humans, we applied reduced representation bisulfite sequencing (RRBS) to somatic DNA from six members of a three-generation family. We observed that 8.1% of heterozygous SNPs are associated with differential methylation in cis, which provides a robust signature for Mendelian transmission and relatedness. The vast majority of differential methylation between homologous chromosomes (>92%) occurs on a particular haplotype as opposed to being associated with the gender of the parent of origin, indicating that genotype affects DNA methylation of far more loci than does gametic imprinting. We found that 75% of genotype-dependent differential methylation events in the family are also seen in unrelated individuals and that overall genotype can explain 80% of the variation in DNA methylation. These events are under-represented in CpG islands, enriched in intergenic regions, and located in regions of low evolutionary conservation. Even though they are generally not in functionally constrained regions, 22% (twice as many as expected by chance) of genes harboring genotype-dependent DNA methylation exhibited allele-specific gene expression as measured by RNA-seq of a lymphoblastoid cell line, indicating that some of these events are associated with gene expression differences. Overall, our results demonstrate that the influence of genotype on patterns of DNA methylation is widespread in the genome and greatly exceeds the influence of imprinting on genome-wide methylation patterns.

Author Summary

DNA methylation is a dynamic epigenetic mark that is essential for mammalian organismal development. DNA methylation levels can be influenced by environment, a chromosome's parental origin, and genome sequence. In this study, we evaluated the impact that DNA sequence has on DNA methylation by analyzing methylation levels in a three-generation family as well as unrelated individuals. By following DNA methylation patterns through the family along with nearby SNPs, we found that allelic differences between chromosomes play a much larger role in determining DNA methylation than the parental origin of the chromosome, indicating that DNA sequence has a larger impact on DNA methylation than gametic imprinting. We also found that allelic differences in DNA methylation found in the family can also be observed in unrelated individuals. In fact, the majority of variation in DNA methylation can be explained by genotype. Our results emphasize the importance of genome sequence in setting patterns of DNA methylation and indicate that genotype will need to be taken into account when assessing DNA methylation in the context of disease.