PROJECT SUMMARY: Disorders of keratinization (DOK) are severe, rare genetic skin disorders in which the central barrier function of the skin is disrupted and a compensatory pathway of hyper-proliferation is activated in either a localized area or throughout the entire skin surface. This results in a grossly compromised epidermis that fails to adequately protect against bacterial, chemical, and mechanical assault or to prevent transepidermal water loss. In early life, the consequences of these disorders can be life-threatening, with increased susceptibility to infection due to blistering and fissures, dramatically increased metabolic demands due to evaporative heat loss and increased epidermal turnover rate, and various associated comorbid conditions and systemic abnormalities which can persist throughout life. We and others have identified over 70 diverse genes for these disorders, yet clinical experience and our data show that these genes explain only a portion of heritability for DOK, which demonstrate marked locus and phenotypic heterogeneity, with greater than 15% of subjects without mutation in known genes. In the last funding period, we identified five new genes for DOK, provided evidence for phenotypic expansion in two others, and identified a novel pathogenesis-directed therapy for one disorder. We now propose to expand our large cohort of well-phenotyped DOK kindreds, to screen for mutations in known causative genes, and to employ exome and genome sequencing in those subjects without mutation in known DOK genes to discover novel genetic causes of these disorders. We will employ patient-derived cells and tissue to interrogate the function of identified novel genes, and will generate transgenic skin equivalents to study and prove pathogenesis of identified mutations. For a limited number of novel genes not previously implicated in DOK, we will generate mouse models using CRISPR technology to further examine the effect of identified mutations on cutaneous function. These studies will continue to identify molecular pathways central to the complex processes of epidermal differentiation and self-renewal, and will provide critical context for more detailed future biologic studies.