PROJECT SUMMARY Orofacial clefts (OFCs) are the most common craniofacial birth defect in humans and are caused by multiple genetic and environmental risk factors. Elucidating the etiology of OFCs is critical not only for our knowledge of developmental biology and for how clefts arise, but ultimately for improved prevention, treatment, and prognosis for individuals affected by OFCs. Clinical applications of genome sequencing are growing, but the usability for OFCs is hindered by a substantial missing fraction of heritable risk and a poor understanding of how variants in non-coding regulatory elements contribute to OFC risk. These elements include (but are not limited to) enhancers, promoters, noncoding RNAs (e.g., microRNA), and topologically associated domain boundaries. Despite the mounting evidence that non-coding regulatory variants are important contributors to human disease, widespread analysis of such variants in OFCs has been limited by the lack of a large resource of whole genome sequences in individuals with OFCs and difficulties annotating craniofacial regulatory variants. In this proposal, we will take advantage of several recent initiatives that directly address these barriers by analyzing rare de novo, inherited, and structural variants from over 1,300 case-parent trios with OFCs sequenced through the Gabriella Miller Kids First Research Program. We will integrate these data with genomic atlases of chromatin profiling, microRNAs, and RNA-seq data generated from human and mouse craniofacial tissues. These analyses will provide key insights into the genetic architecture of OFCs and will reveal the full potential of WGS data for OFCs.