Genetic and Epigenetic Contributors to Cleft Laterality: Evidence from Monozygotic Mirror Twins and Replication Cohorts

Nonsyndromic cleft lip (nsCL) exhibits a non-random laterality pattern, with left-sided clefts occurring approximately twice as frequently as right-sided clefts. The molecular mechanisms underlying this laterality bias remain poorly understood. We performed whole-genome sequencing and methylation profiling on a family comprising monozygotic twins with mirror-image nsCL, their affected mother, and unaffected father and brother. We conducted three independent replications via (1) publicly available whole genome data; (2) genome-wide methylation analysis in 38 individuals with unilateral cleft; and (3) validation of methylation results in the top 3 candidate genes in 385 unrelated individuals with unilateral clefts (DNA from blood or saliva). We identified a variant in FGF20 (p.Ile79Val) shared by the twins and their mother. We observed laterality and severity-associated methylation differences in three main genes. ARID5B showed higher methylation in left clefts (saliva, p=0.001; blood, p=0.032). ZFP57 demonstrated a strong cleft-extent effect, with cleft lip and palate (CLP) showing markedly higher methylation than cleft lip only (CL) (LCLP vs. RCL padj=0.0004; LCLP vs. LCL padj = 0.019). HOOK2 displayed a cross-tissue cleft-extent effect in the opposite direction -- CLP subtypes were hypomethylated relative to CL-only subtypes in blood (p<0.0001) and saliva p=0.0008). This study provides evidence that DNA methylation patterns plays a role in both the laterality and severity of cleft lip. ARID5B provides a consistent laterality signal across tissues, while ZFP57 and HOOK2 track palatal involvement independently of side. Together, these findings suggest that epigenetic variation acts downstream of genetic predisposition to shape cleft phenotypes.