Patients with epidermodysplasia verruciformis (EV) and biallelic null mutations of (encoding EVER1) or (EVER2) are selectively prone to disseminated skin lesions due to keratinocyte-tropic human β-papillomaviruses (β-HPVs), which lack E5 and E8. We describe EV patients homozygous for null mutations of the gene encoding calcium- and integrin-binding protein-1 (CIB1). CIB1 is strongly expressed in the skin and cultured keratinocytes of controls but not in those of patients. CIB1 forms a complex with EVER1 and EVER2, and CIB1 proteins are not expressed in EVER1- or EVER2-deficient cells. The known functions of EVER1 and EVER2 in human keratinocytes are not dependent on CIB1, and CIB1 deficiency does not impair keratinocyte adhesion or migration. In keratinocytes, the CIB1 protein interacts with the HPV E5 and E8 proteins encoded by α-HPV16 and γ-HPV4, respectively, suggesting that this protein acts as a restriction factor against HPVs. Collectively, these findings suggest that the disruption of CIB1-EVER1-EVER2-dependent keratinocyte-intrinsic immunity underlies the selective susceptibility to β-HPVs of EV patients.

Epidermodysplasia verruciformis (EV) is an autosomal recessive skin disorder with a phenotype conditional on human beta-papillomavirus (beta-HPV) infection. Such infections are common and asymptomatic in the general population, but in individuals with EV, they lead to the development of plane wart-like and red or brownish papules or pityriasis versicolor-like skin lesions, from childhood onwards. Most patients develop non-melanoma skin cancer (NMSC), mostly on areas of UV-exposed skin, from the twenties or thirties onwards. At least half of the cases of typical EV are caused by biallelic loss-of-function mutations of TMC6/EVER1 or TMC8/EVER2. The cellular and molecular basis of disease in TMC/EVER-deficient patients is unknown, but a defect of keratinocyte-intrinsic immunity to beta-HPV is suspected. Indeed, these patients are not susceptible to other infectious diseases and have apparently normal leukocyte development. In contrast, patients with an atypical form of EV due to inborn errors of T-cell immunity invariably develop clinical symptoms of EV in the context of other infectious diseases. The features of the typical and atypical forms of EV thus suggest that the control of beta-HPV infections requires both EVER1/EVER2-dependent keratinocyte-intrinsic immunity and T cell-dependent adaptive immunity.

The relatively high proportion of EV patients without mutation in TMC6/8 indicates the existence of EV-causing mutations in additional, presently unknown gene(s). However, a homozygous TMC8 splice site mutation in our patients resulted in aberrant transcripts which cannot retain the healthy phenotype. The literature review revealed that HPV-5 is the most commonly identified HPV in patients with EV, but HPV-3, HPV-14 and HPV-20 were unexpectedly identified more frequently than HPV-8.

Ichthyosis with confetti (IWC) is a genodermatosis associated with dominant‐negative variants in keratin 10 (KRT10) or keratin 1 (KRT1). These frameshift variants result in extended aberrant proteins, localized to the nucleus rather than the cytoplasm. This mislocalization is thought to occur as a result of the altered carboxy (C)‐terminus, from poly‐glycine to either a poly‐arginine or ‐alanine tail. Previous studies on the type of C‐terminus and subcellular localization of the respective mutant protein are divergent. In order to fully elucidate the pathomechanism of IWC, a greater understanding is critical. This study aimed to establish the consequences for localization and intermediate filament formation of altered keratin 10 (K10) C‐termini. To achieve this, plasmids expressing distinct KRT10 variants were generated. Sequences encoded all possible reading frames of the K10 C‐terminus as well as a nonsense variant. A keratinocyte line was transfected with these plasmids. Additionally, gene editing was utilized to introduce frameshift variants in exon 6 and exon 7 at the endogenous KRT10 locus. Cellular localization of aberrant K10 was observed via immunofluorescence using various antibodies. In each setting, immunofluorescence analysis demonstrated aberrant nuclear localization of K10 featuring an arginine‐rich C‐terminus. However, this was not observed with K10 featuring an alanine‐rich C‐terminus. Instead, the protein displayed cytoplasmic localization, consistent with wild‐type and truncated forms of K10. This study demonstrates that, of the various 3′ frameshift variants of KRT10, exclusively arginine‐rich C‐termini lead to nuclear localization of K10.

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Naegeli-Franceschetti-Jadassohn syndrome (NFJS) is a rare autosomal dominantly inherited genodermatosis. Only seven families with NFJS are currently reported in literature. Mutations in keratin 14 (KRT14) additionally cause dermatopathia pigmentosa reticularis (DPR), a phenotypically similar disease, as well as epidermolysis bullosa simplex (EBS). Our study aimed on identification of the causative mutation in an unknown family with NFJS and gain of knowledge on genotype-phenotype correlation in KRT14. We examined a five generation family with NFJS phenotype in eight individuals. Initially, the index patient visited our clinic because of difficulties in registration of her fingerprints. Since birth she has suffered from anhidrosis and palmoplantar keratoderma. Additionally, she had had patchy hyperpigmentations which disappeared over time. Clinical investigations on further family members revealed a heterogeneous severity of phenotype in the affected family members. We isolated gDNA from patients' lymphocytes and performed Sanger sequencing of exon 1 of KRT14. By that we identified a two base pair insertion leading to a frameshift and a premature stop codon after 10 amino acids. The same mutation, which is unknown in large databases like gnomAD (123,136 exome and 15,496 whole-genome sequences), has been found in further affected family members. The correlation between the genotype and phenotype in patients with KRT14 mutation is not completely understood. We present clinical and sequence data from this family and discuss them in context of the original NFJS family as well as other descriptions in literature.

Recessive dystrophic epidermolysis bullosa (RDEB) is a blistering disorder caused by mutations in COL7A1 that encodes for the anchoring fibrils of type VII collagen. Our lab has demonstrated that non-viral gene delivery by polymer is able to restore the expression of type VII collagen (C7) to human RDEB skin and could overcome the possibility of generating an immune response when compared to viral vectors. Silencing caused by viral promoters is a major problem for an episomal non-viral gene therapy. To increase the safety profile of COL7A1 therapy more focus needs to be in the DNA cassette itself as an important tool for resisting transgene silencing and extending C7 expression. The feasibility of this approach was demonstrated using a novel gene delivery polymer as a non-viral vector combined with minicircle (MC) DNA encoding full-length COL7A1 under the control of three different promoters, including human COL7A1 promoter (C7p) as a tissue-specific promoter, human elongation factor one alpha (EF1a) and cytomegalovirus (CMV). The three MC plasmid (MC-C7p-C7, MC-EF1a-C7 and MC-CMV-C7) were used to transfect human RDEB Keratinocytes (K) and Fibroblasts (F). Type VII collagen expression was detected and compared among the three promoters and confirmed in in vitro at the gene level using RT-qPCR as well as at the protein level using immuno-blotting. All the three MC constructs are capable of achieving expression levels comparable to native protein from healthy human primary cells, there is an indication of a 20 fold C7 expression change detected in transfected RDEBF cells compared to un-transfected cells. A multifaceted analysis of the promoters expression is being performed. In conclusion, our results suggest that an optimal promoter selection is an important enhancement that can be added to the minicircle vector in our non-viral system and therefore will enhance transgene expression and help to reduce immuneimmunogenicity.

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