The role of regulatory variants in FZD6 and interacting genes in nonsyndromic cleft lip and palate

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Project Summary Nonsyndromic cleft lip with or without cleft palate (NSCLP) is the most common craniofacial birth defect resulting from incomplete fusion of the embryonic facial prominences which leaves a gap in the lip, primary palate and/or the secondary palate. The common birth prevalence of 1 per 700 newborns results in 135,000 NSCLP newborns worldwide each year. While multidisciplinary treatments now achieve satisfactory outcomes, there are many long-term health issues that persist, imposing significant economic and psychosocial burdens. Familial aggregation and segregation analyses suggest polygenic inheritance, but despite decades of study, only a small portion of the NSCLP genetic liability has been identified leaving a large knowledge gap. This gap could be the result of most genomic analyses aimed at identification of exonic/coding variants and rare mutations. Recently, noncoding functional variants that regulate gene expression have been shown to play a role in birth defects; our work and that of others demonstrated that dysregulation of genes and gene networks during craniofacial development contributes to NSCLP. This proposal takes advantage of the strong evidence that FZD6, LRP5, LRP6 and DKK1, genes at the receptor level of the WNT pathway are NSCLP candidate genes. Together, they act on the β-catenin mediated signaling: FZD6 forms a co-receptor complex with either LRP5 or LRP6 while DKK1 is an inhibitor of this co-receptor complex. These genes play a critical role in craniofacial development and the tight regulation of their expression is crucial for the normal formation and fusion of the upper lip and palate. The goal of this project is to identify functional noncoding genetic variants in FZD6, LRP5, LRP6 and DKK1 and their contribution to NSCLP. Towards accomplishing this goal, we initially prioritized 21 putatively functional variants in FZD6, LRP5, LRP6 and DKK1 and found 10 variants that bind transcription factors in an allele-specific manner. In this continuing work, luciferase reporter assays in embryonic cell lines and in vivo analysis with transgenic reporters in zebrafish will be used to define the role of these variants on gene expression. For variants with cell-specific and spatiotemporal effects during development, association analyses will be carried out in our large and well-characterized NSCLP families. Together, results from this work will determine both individual and combinatorial contributions of noncoding regulatory variants in genes controlling β-catenin Wnt signaling during orofacial development. These results will enhance our knowledge of the role of noncoding variants in NSCLP and have the potential to be translated into the clinical setting to aid in the assessment of NSCLP risk.