Identification of functional non-coding variants associated with orofacial cleft [MPRA]

Oral facial cleft (OFC) is a multifactorial disorder that can present as a cleft lip with or without cleft palate (CL/P) or a cleft palate only. Genome wide association studies (GWAS) of isolated OFC have identified common single nucleotide polymorphisms (SNPs) at the 1q32/IRF6 locus and many other loci where, like IRF6, the gene presumed to be relevant to OFC risk is expressed in embryonic oral epithelium. To identify the functional subset of SNPs at eight such loci we conducted a massively parallel reporter assay in a cell line derived from fetal oral epithelium, revealing SNPs with allele-specific effects on enhancer activity. Overall design: A massively parallel reporter library was constructed containing candidate cis-regulatory element (CRE) sequences each 161 bp in length, and with non-risk and risk alleles of 889 SNPs. The CREs were cloned upstream of a hsp68 minimal promoter. The MRA library was electroporated into fetal oral epithelial cells (GMSM-K) with four biological replicates. Four RNA libraries, the input plasmid DNA library and three DNA libraries (post-transfection) were sequenced.