Functional analysis of genetic-epigenetic interplay in orofacial clefts

About This Grant

Project Summary Nonsyndromic orofacial cleft (OFC) is the most common craniofacial birth defect, affecting 1 in 700 individuals worldwide. OFCs cause significant psychosocial burden, increase risks for early life complications, cancer predisposition, and mental health disorders, while reducing quality of life and increasing mortality rates. OFC’s complex etiology involves multiple genetic and environmental factors, with over 60 identified risk loci accounting for only a minority of estimated risk. While family history exists in 23% of cases, monozygotic twins show only 50% concordance, suggesting factors beyond genetic risk. Epigenetic factors, such as differential DNA methylation (DNAm) are associated with increased OFC risk, can alter susceptibility to different cleft types and modify OFC penetrance; however, the overall role of epigenetics in OFC etiology remains understudied. DNAm is a covalent addition of a methyl (CH3) group to the nucleotide cytosine (CpG) that can lead to changes in expression of the target gene. DNAm can be affected by environmental influences and genetic variation via methylation quantitative loci (meQTLs). Our group recently showed evidence that differential methylation can contribute to phenotypic variability in patients with Van der Woude syndrome and nonsyndromic clefts, including monozygotic twins. Other studies have also shown that differentially methylated regions implicated in OFC etiology cluster near genes previously implicated in palatogenesis and some differential methylation is likely driven by genetic variation. These premises highlight the need for functional studies to test the role of the DNAm in OFC etiology. We hypothesize that aberrant DNAm and the resulting alterations in gene expression play a key role in the etiology of OFCs, and that certain common genetic variants that affect OFC risk do so by influencing DNAm. Based on our preliminary identification and replication of 9 meQTLs, we have implemented an in vitro CRISPR-based, gene- editing pipeline using relevant cell lines for functional meQTL validation. Our main goal is to leverage both our 9 confirmed meQTLs and our in vitro pipeline to investigate meQTL pathogenic mechanisms that lead to abnormal gene expression resulting in OFCs, via aims designed to (1) Functionally validate the impact of the meQTL (SNP) on the CpG methylation levels; and (2) Confirm CpG-target genes. Our pipeline has the potential to elucidate the biological mechanisms underlying statistical associations, particularly for non- coding SNPs with unclear functional relevance. The successful completion of these aims will lay the foundation for future R01 proposals designed to further investigate gene-epigenome networks in craniofacial development, including the functional impact of meQTLs on orofacial development using in vivo.