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 separately 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, suggesting distinct context-dependent mutation patterns for unmethylated and methylated cytosines. Our analysis further reveals that the context effects are 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 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 on the chimpanzee lineage, point to recent evolutionary changes, possibly in context-specificity of proteins governing DNA demethylation and repair processes.