Genetic Pathway Analysis of Abnormal Facial Development in Nonsyndromic Cleft Lip and Palate

Nonsyndromic cleft lip with or without cleft palate (NSCLP) is the most common craniofacial birth defect resulting from incomplete fusion of the facial prominences during development, which leaves a gap in the lip, primary palate and/or the secondary palate. NSCLP affects 135,000 NSCLP newborns worldwide each year based on a birth prevalence of 1 per 700 live births. While surgical treatments have dramatically improved, many long-term health issues persist, imposing significant medical, psychosocial and economic burdens. Familial aggregation and segregation analyses suggest genetic contributions underlie NSCLP, but despite decades of study, only a small portion of the NSCLP genetic liability has been identified leaving a large knowledge gap. Following a pathway-based approach to identify NSCLP etiologic genes, this dissertation examined gene networks regulating facial morphogenesis. Three different pathways were assessed and found to have etiologic roles in NSCLP. The PBX pathway, implicated in murine midfacial development, was confirmed to be associated with NSCLP in our family-based and case-control datasets. The second gene, identified in the CRISPLD2 network, was found to play a novel role in regulating oral and facial development, with perturbation causing abnormal oral morphogenesis in zebrafish. The final study used bioinformatic, cell-based and transgenic zebrafish approaches together to identify noncoding variants in FZD6, LRP5, LRP6 and DKK1 driving allele-specific expression during craniofacial development. Testing of these variants in our extensive family-based NSCLP dataset identified, for the first time, associations between LRP5 and DKK1 and NSCLP, and confirmed the previously identified association with FZD6 and LRP6. These results support the analysis of gene networks rather than individual genes to identify the missing heritability underlying NSCLP. This approach is critical towards understanding the polygenic contributions that are known to underlie NSCLP and other complex disorders. The goal of these studies is to construct and map all of the noncoding and coding variants contributing to NSCLP, with the ultimate goal of determining individual and family risks, so that the information can be used in the clinic setting