Investigating the molecular etiology of Keratinocyte Differentiation Factor 1 (KDF1) in disease phenotypes of the ectoderm.

Abstract Keratinocyte Differentiation Factor 1 (KDF1) is an ectodermal dysplasia (ED) disease gene with a wide range in phenotype severity. Individuals with putatively damaging heterozygous variants in KDF1 present with ED and severe tooth agenesis, or milder non-syndromic tooth agenesis. Identifying KDF1 genotype-phenotype correlations that will predict the severity of oral phenotypes will result in earlier intervention with dental implants and improved prognosis for affected families. Our work has broad future applications, as five other ED disease genes display the same range in severity of syndromic and non-syndromic tooth agenesis (NSTA) phenotypes, yet the mechanism driving this variable expression remains unclear. Investigating the mechanism underlying the range in phenotype expression in KDF1-disease biology will have a profound impact on diagnostic variant interpretation in the clinic and individualized treatment plans for individuals with pathogenic variants in KDF1. Preliminary analyses suggest that individuals with pathogenic missense variants within the central domain (CD) of the KDF1 protein develop ED and severe tooth agenesis, while patients with missense variants outside the CD develop the attenuated NSTA phenotype with milder tooth agenesis. The CD is critical for KDF1 to bind and deubiquitinate IKKα, resulting in IKKα stabilization and downstream transcriptional regulation of keratinocyte and tooth germ development. Understanding the functional consequences of KDF1 variation is critical to inform disease management and potential therapeutic development. My central hypothesis is that the position of variants within KDF1 mediates the phenotype of ED or NSTA through impacts on IKKα stability. To address this hypothesis, I will first determine if severe tooth agenesis and the presence of ED phenotypes are dependent upon variant position within KDF1 by statistically interrogating well-characterized cohorts of severe and more tolerated KDF1 variation. I will then quantify the effects of KDF1 variants associated with syndromic tooth agenesis on keratinocyte differentiation and IKKα stability and abundance by creating and characterizing CRISPR/Cas9-edited cell lines and performing co-immunoprecipitation assays and western blot analysis. The findings of these studies will elucidate the mechanism driving severe KDF1-disease, advance our understanding of ED pathogenesis, and inform predictive disease management practices. The research proposed in this application aims to foster my development in becoming a successful independent researcher and clinical molecular geneticist. My research environment within the Posey laboratory, the Texas Medical Center, and the Baylor College of Medicine GREGoR Consortium research center offers an exceptional foundation for achieving these aims.