Development of keratinocyte culture models for epidermodysplasia verruciformis and ichthyosis with confetti

Abstract

Epidermodysplasia verruciformis (EV) and ichthyosis with confetti (IWC) are rare genodermatoses. EV is a autosomal recessive disease primarily characterized by an inability of keratinocytes to control cutaneous β-HPV infections and a high risk for non-melanoma skin cancer (NMSC). IWC is caused by heterozygous frameshift mutations in keratin 1 (K1) and keratin 10 (K10) leading to an arginine-rich carboxyl-terminus instead of the wildtype glycine-rich carboxyl-terminus and transport of the mutated keratin into the nucleus. Characteristic for skin of IWC patients are the numerous small spots of pale skin with postzygotic loss of heterozygosity on chromosome 12q or 17q leading to a loss of the mutated allele without loss of genetic material. A cell culture model for EV was developed for functional studies in keratinocytes. Using CRISPR/Cas9, nine knockout and nine wildtype clones were generated originating from an immortalized human keratinocyte line. Differential gene expression analysis using RNA-Seq showed that the effect of deficiency of an EV-causing gene is small, consistent with the narrow phenotype of EV patients. A few genes with slight differences in expression level provide insight into the potential effects of the introduced knockout. To study subcellular localization of keratins, a cell culture model for IWC was developed. Two clones with arginine-rich carboxyl-terminus were identified. Epidermal models grown with these clones allowed to observe that interaction partners of the mutated K10 are co-transported to the nucleus. This study has contributed to the discovery of a novel genetic etiology for EV, has provided cell culture models for EV and IWC, and has thereby given insights into gene expression changes of EV-related genes and subcellular localization of keratins in IWC.