Sequence context and methylation interact to shape germline mutation rate variation at CpG sites

A prominent example of sequence context-dependent mutation rate variation is the elevated transition rate at CpG sites, which is largely attributed to cytosine methylation. CpGs with different flanking sequences also exhibit mutation rate variation, but this variation is only partially correlated with context-specific methylation level. Here, we quantify the CpG mutation rate and mutagenic effect of methylation across sequence contexts. Using a regression framework that accounts for recurrent mutations, we analyze human polymorphisms from the gnomAD dataset to estimate mutation rates of unmethylated and methylated CpGs in each unique 4-mer or 6-mer context. We find that CpG mutation rate variation in the human genome is shaped by methylation at the focal cytosine, the flanking nucleotides, and interactions between them. Our analysis reveals distinct context-dependent mutation patterns for unmethylated and methylated cytosines, driven by largely independent effects of upstream and downstream sequences. Notably, an upstream adenine markedly increases CpG mutation rates regardless of methylation status or downstream sequences. Furthermore, upstream and downstream sequences have qualitatively similar effects in chimpanzee and rhesus macaque, indicating that some conserved, intrinsic sequence features shape CpG mutability. On the other hand, some inter-species differences, which are especially pronounced at methylated sites, point to recent evolutionary changes, possibly in context-specificity of proteins governing DNA demethylation and repair processes. Author SummaryThe DNA sequence surrounding a nucleotide strongly influences how likely it is to mutate. An extreme example is the CpG dinucleotide: cytosines in CpGs mutate far more frequently than other sites in the human genome. This is related to DNA methylation, a chemical modification that occurs almost exclusively at CpGs in vertebrates and makes cytosines more prone to mutations. However, CpGs in different sequence contexts also vary in their mutation rates, and methylation level alone cannot explain this variation. To gain insight into what processes drive this variation, we estimate mutation rates for methylated and unmethylated CpGs in different sequence contexts using human genetic variation data. We find that methylation and neighboring bases interact to influence CpG mutation rates, and that the DNA sequence on either side of the CpG exerts largely independent effects. Extending our analysis to other primates reveals both conserved and species-specific patterns, with differences being especially pronounced at methylated sites. Together, our results suggest that while intrinsic DNA sequence features underlie some conserved context effects, other differences reflect recent evolutionary changes in the mechanisms that regulate DNA demethylation and repair.