Different molecular mechanisms underlie skin fragility in AEC and EEC

Summary: Abstract Body: Ankyloblepharon ectodermal dysplasia and cleft lip/palate (AEC) and ectrodactyly ectodermal dysplasia and cleft lip/palate (EEC) are two genetic disorders caused by mutations in the transcription factor TP63. AEC mutations localize to the sterile alpha motif domain, a putative protein-protein interaction domain, while EEC mutations are clustered in the DNA binding domain. Both disorders are associated with severe skin erosions and have no treatment options. Our group is investigating the pathological mechanisms underlying skin fragility. To this end, we developed complementary in vitro systems. First, we created a patient-derived induced pluripotent stem cell (iPSC) system with matched gene-corrected pairs that can be differentiated into keratinocytes (iPSC-K). Using the commercially available immortalized keratinocyte line, NTERT, and lentiviral constructs harboring AEC- or EEC-mutant TP63, we generated an additional disease-relevant model. RNA sequencing performed on AEC and EEC iPSC-K, and in silico pathway analysis identified defects to “integrin receptors” and “cell adhesion” in AEC iPSC-K while EEC iPSC-K had a “epidermal differentiation” defect. AEC iPSC-K RNA and protein analysis show reduced expression of desmosomal genes (DSG3, DSC3, DSG1, DSC1), hemidesmosomal (HD) genes (ITGA6, ITGB4, COL17, DST, LM332), and focal adhesion (FA) genes (ITGA2, ITGA3, VINC, PAX, ILK). However, EEC iPSC-K did not show abnormalities to HD or FA genes. Instead, we observed inappropriate expression of genes associated with epidermal terminal differentiation in keratinocytes cultured under proliferating conditions. Upregulated genes included TGM1, DSG1, DSC1, ZNF750, and FLG. EEC iPSC-K also expressed several genes not typically expressed in keratinocytes, including KRT13 and POSTN. The results were confirmed in patient skin. Our findings reveal different pathological mechanisms underlie skin fragility in AEC and EEC. Further understanding of the link between the location of TP63 mutations and different pathological outcomes may facilitate therapeutics for both disorders. Maddison N. Salois¹, Saiphone Webb¹, S. R. Price¹, Maranke I. Koster¹ 1. Biochemistry and Molecular Biology, East Carolina University, Greenville, NC, United States. Genetic Disease, Gene Regulation, Gene Therapy & Epigenetics