Multi-omic analyses of a twin pair with mirror image cleft lip identifies pathogenic variant in FGF20 modified by differential methylation upstream of ZFP57

BackgroundDisturbances in the intricate processes that control craniofacial morphogenesis can result in birth defects, most common of which are orofacial clefts (OFCs). Nonsyndromic cleft lip (nsCL), one of the phenotypic forms amongst OFCs, has a non-random laterality presentation with the left side being affected twice as often compared to the right side. This study investigates the etiology of nsCL and the factors contributing to its laterality using a pair of monozygotic twins with mirror-image cleft lip. MethodsWe conducted whole-genome sequencing (WGS) analyses in a female twin pair with mirror image nsCL, their affected mother and unaffected father to identify etiopathogenic variants. Additionally, to identify possible cleft lip laterality modifiers, DNA-methylome analysis was conducted to test for differential methylation patterns between the mirror twins. Lastly, DNA methylation patterns were also analyzed on an independent cohort of female cases with unilateral cleft lip (left=22; right=17) for replication purposes. ResultsWe identified a protein-altering variant in FGF20 (p.Ile79Val) within the fibroblast growth factor interacting family domain segregating with the nsCL in this family. Concurrently, DNA-methylome analysis identified differential methylation regions (DMRs) upstream of Zinc-finger transcription factor ZFP57 ({Delta}{beta} > 5%). Replication of these results on an independent cohort, confirmed these DMRs, emphasizing their biological significance (p<0.05). Enrichment analysis indicated that these DMRs are involved in DNA methylation during early embryo development (FDR adjusted p-value = 1.3241E-13). Further bioinformatics analyses showed one of these DMRs acting as a binding site for transcription factor AP2A (TFAP2A), a key player in craniofacial development. Interactome analysis also suggested a potential role for ZFP57 in left/right axis specification, thus emphasizing its significance in cleft laterality. ConclusionThis study provides novel insights into the etiology of nsCL and its laterality, suggesting an interplay between etiopathogenic variants and DNA methylation in cleft laterality. Our findings elucidate the intricate mechanisms underlying OFCs development. Understanding these factors may offer new tools for prevention and management of OFCs, alleviating the burden on affected individuals, their families and global health.