Investigating the Molecular Defects Contributing to Skin Fragility in Two Ectodermal Dysplasias

Abstract

Ankyloblepharon ectodermal dysplasia and cleft lip/palate (AEC) and ectrodactyly-ectodermal dysplasia cleft lip/palate syndrome (EEC) are rare genetic skin disorders caused by missense mutations in TP63. AEC is caused by mutations to the sterile alpha motif domain of TP63, while EEC is caused by mutations in the DNA binding domain. TP63 is an essential component in epidermal and epidermal appendage development and maintenance. Mutations to TP63 cause a wide variety of ectodermal defects, including abnormal development to the skin, hair, nails, and limbs. The most severe manifestations are extensive skin erosions that can be life-threatening and decrease the patient's quality of life. Additionally, these erosions have no therapeutic options. To develop novel therapies, we need to first understand how TP63 mutations lead to skin erosions. Our laboratory aims to uncover the pathological mechanisms that contribute to the devastating skin fragility in AEC and EEC. To investigate this, we have generated complementary in vitro model systems. First, we have generated patient-derived induced pluripotent stem cells (iPSC) which can be differentiated into keratinocytes (iPSC-K), one of the cell types affected in AEC and EEC. Additionally, with CRISPR/Cas we have corrected the disease-causing mutation, generating conisogenic pairs of keratinocytes. This is an ideal model as they have the same genetic background as the patients and only differ in the presence or absence of the single point mutation. Additionally, using the commercially available immortalized keratinocyte line, NTERT, and lentiviral constructs harboring AEC- or EEC-mutant TP63, we generated an additional disease-relevant model (AEC-NTERT and EEC-NTERT). RNA sequencing was performed with subsequent in silico pathway analysis on the conisogenic AEC iPSC-K and EEC iPSC-K pairs. A comparison of AEC iPSC-K to their respective gene-corrected (GC) iPSC-K showed “integrin cell surface receptors” and “cell adhesion genes” as abnormally expressed. However, EEC iPSC-K, compared to their GC iPSC-K, had “epidermal differentiation” as a significant defect, suggesting different pathological mechanisms. AEC iPSC-K RNA and protein analyses show reduced expression of several cell-cell and cell-extracellular matrix (ECM) adhesion structures, including the desmosome, hemidesmosome, and focal adhesion. Additionally, AEC-NTERT keratinocytes revealed significantly reduced adhesion and migration on several ECMs. The inability to adhere and migrate over ECMs could explain long-lasting wounds in AEC patients, suggesting wound healing might be impaired. AEC patient skin samples validated focal downregulation of several cell adhesion structures. However, EEC iPSC-K did not show abnormalities to hemidesmsosmal or focal adhesion genes. Instead, we observed inappropriate expression of genes associated with epidermal terminal differentiation in keratinocytes cultured under proliferating conditions. Further, 3D epidermal equivalents generated from EEC-NTERT showed increased differentiation programs which was validated in EEC patient skin. Altogether, our data suggests that skin fragility in AEC and EEC is due to different molecular mechanisms. AEC epidermal defects can be at least in part attributed to the cooperative downregulation of cell-cell and cell-ECM components while EEC epidermal defects show accelerated terminal differentiation. Further understanding these molecular mechanisms may eventually lead to the development of novel therapies for these patient populations and improve patient quality of life.