Orofacial clefts (OFC) such as cleft lip and/or palate (CL/P) are among the most common congenital structural anomalies. Most OFC cases are non-syndromic with complex genetic mechanisms that are yet to be elucidated. The majority of heritable OFC risk is expected to reside in rare or de novo variations. With expanding clinical use of prenatal DNA tests, there is a pressing need to improve the genetic interpretation of whole exome/genome sequence data. A major challenge is assignment of pathogenicity to both coding and non-coding variants. The gene encoding transcription factor IRF6 is strongly associated with non-syndromic CL/P, plus mutations in IRF6 cause the most common form of syndromic CL/P. We and others have shown that genes in the IRF6 pathway are good candidates to harbor rare variants that influence risk for CL/P, such as GRHL3, ARHGAP29 and KLF4. In this proposal, we extend this successful strategy to elucidate CL/P pathogenesis with a comprehensive and deep analysis of the IRF6 downstream gene pathway. We employed a rigorous gene prioritization strategy where critical IRF6 transcriptional target genes were identified via ChIP-seq from wild type embryos, enriched by subtraction against irf6 mutant dataset. The target genes were then selected for differential expression between wild type and irf6 mutants via RNA-seq. This IRF6 candidate target gene list was then cross-referenced for spatiotemporal expression patterns relevant for craniofacial development with zebrafish WISH and mouse gene expression data from the FaceBase project. Finally we selected genes associated with human CL/P pathology from a recent 800 CL/P case-parent trios WGS dataset from the Gabriella Miller Kids First sequencing project. Our central hypothesis is that IRF6 target genes are critical for palate development, and that rare and de novo mutations in such genes, whether coding or non-coding, are present in patients with non-syndromic OFC. To test this hypothesis, we propose three complementary aims to 1) gain new biological insight from known (Tfap2a) and newly identified (Dact1) genes in craniofacial development, 2) gain new functional and clinical insight of de novo coding gene variants important for OFC, 3) develop methodology and analyze non-coding gene variants implicated for OFC. The expected impact of this work will be to bridge the gap between WGS data and biological insight, an essential step to meaningfully translate genetic research data to inform clinical decisions.

Public Health Relevance

Orofacial clefts are among the most common structural birth anomalies with a strong but complex genetic basis. This proposal leverages robust preliminary data, advanced human genetics and experimental approaches, and a team of investigators with complementary expertise to gain biological and clinical insights into cleft pathogenesis.

National Institute of Health (NIH)
National Institute of Dental & Craniofacial Research (NIDCR)
Research Project (R01)
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Therapeutic Approaches to Genetic Diseases Study Section (TAG)
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Stein, Kathryn K
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Massachusetts General Hospital
United States
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