Abstract:
Understanding how genetic variants influence disease risk through molecular mechanisms remains a central challenge in complex disease genetics. Nonsyndromic orofacial clefts (OFCs) exemplify this challenge, with most risk loci residing in non-coding regions. We hypothesized that common genetic variants influence OFC risk by modulating DNA methylation at regulatory elements through methylation quantitative trait loci (meQTLs). We analyzed 10 OFC-associated SNPs against genome-wide DNA methylation profiles in 409 cases and 456 controls, identifying 23 potential meQTLs. Findings were validated using 358 cleft-discordant sibling pairs with MethyLight assays. We performed formal mediation analysis, genotype-tissue interaction and cross-referenced with the mQTL Database to assess developmental timing. Nine meQTLs were validated, including rs987525 (8q24)–cg16561172 (
MYC
) (
P
= 9.6 × 10⁻⁶), which mapped to a mesendoderm-active enhancer upstream of
MYC
. Genotype × tissue interaction confirmed tissue-specificity (
P
= 1.00 × 10
− 3
), with stronger effects in oral-derived tissue (saliva). Additional validated SNP-CpG associations involved
MAFB
–
PLCG1
,
NOG
–
PPM1E
,
FOXE1
–
FRZB
, and
SPRY2
–
LGR4
. While effect sizes correlated between tissues (
r
= 0.81), formal mediation analysis indicated individual CpG sites do not fully mediate SNP-phenotype relationships, suggesting coordinated epigenetic mechanisms. Most associations showed peak effects during childhood, while 8q24 showed unique adult-specific patterns. We identified genetic variants influencing methylation at craniofacial regulatory elements, and provided a mechanistic link for a major risk locus, 8q24, with tissue-specific effects in saliva. While individual CpG sites did not fully mediate the genetic risk, our findings identified specific regulatory regions where coordinated epigenetic changes may contribute to OFC susceptibility.
