New insight into keratin 16 function in palmoplantar epidermis and the pathophysiology of pachyonychia congenita

Summary: Pachyonychia congenita (PC) is a rare genetic disorder with palmoplantar keratoderma (PPK) as its most debilitating symptom, manifesting as painful, thick calluses on palmar and plantar skin. The disorder is caused by dominantly-acting variants in the <i>KRT6A, KRT6B, KRT6C, KRT16, </i>and <i>KRT17</i> genes. Previous studies in our laboratory demonstrated that <i>Krt16</i> null mice develop PC-like PPK in footpad skin, at a histological and molecular levels. Unfortunately, the <i>Krt16</i> null strain is not genetically representative of most PC cases, which predominantly involve missense variants. To address this limitation, we utilized CRISPR-Cas9 to introduce a R123C variant, which is implicated in over 20% of <i>KRT16</i>-related PC cases, in the mouse <i>Krt16</i> gene. Whole genome sequencing confirmed the successful knock-in of the R123C mutation. Beginning at 15-16 weeks post-birth, homozygous mutant mice (<i>Krt16^{R123C*/R123C*}</i>) exhibit footpad skin lesions that closely resemble those seen in <i>Krt16</i> null mice and the PPK lesions arising in individuals with PC. Notably, these lesions display an upregulation of K16 protein, paralleling observations in PC individuals. <i>In vitro</i> studies further revealed that cells transfected with an epitope-tagged mutant K16 plasmid form distinct aggregates or puncta, as opposed to the extensive filament arrays forming in cells expressing wildtype K16. This <i>Krt16^R123C*</i>mouse model uniquely expresses the mutant <i>Krt16</i> mRNA and K16 protein, representing an advanced tool for developing mutant allele-specific treatments beyond palliative care. Furthermore, it provides a valuable biological context to explore the role of K16 and the contributions of its structural domains in skin physiology and pathology. Amanda C. Orosco^{1, 2}, Beau Su², Craig Johnson², Pierre A. Coulombe^{2, 3, 4} 1. Program in Cellular and Molecular Biology, University of Michigan Medical School, Ann Arbor, MI, United States. 2. Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, United States. 3. Departent of Dermatology, University of Michigan Medical School, Ann Arbor, MI, United States. 4. Rogel Cancer Center, University of Michigan Medical School, Ann Arbor, MI, United States. Genetic Disease, Gene Regulation, Gene Therapy & Epigenetics