Autosomal recessive congenital recessive (ARCI) refers to a large and genetically heterogeneous group of non-syndromic disorders of cornification featuring diffuse scaling. Ichthyosis, leukocyte vacuoles, alopecia and sclerosing cholangitis (ILVASC) syndrome is a rare autosomal recessive syndromic form of ichthyosis. The disease usually results from premature termination codon-causing pathogenic variants in CLDN1 encoding CLAUDIN-1 (CLDN1). Here, we aimed at delineating the genetic basis of autosomal recessive congenital ichthyosis in two adult siblings. The two patients displayed lamellar ichthyosis and one patient had underwent liver transplantation in early childhood due to biliary atresia. Her affected sister had normal liver function. No hair or dental anomalies were observed. Whole exome sequencing revealed a homozygous missense variant, c.242G>A (p.Arg81His), in CLDN1 in the two ARCI patients. Mutations in CLDN1 are known to be associated with ILVASC syndrome but have not been shown to cause ARCI. The ARCI-associated variant in CLDN1 resulted in decreased CLDN1 expression in patient skin. 3D protein modeling predicted that p.Arg81His induces deleterious conformational changes. Accordingly, HaCaT cells transfected with a construct expressing the mutant CLDN1 cDNA featured decreased levels and mis-location of CLDN1 as compared with cells expressing the wildtype cDNA. In conclusion, we describe the first pathogenic missense variant in CLDN1 shown to result in ARCI.

Inherited palmoplantar keratodermas (PPKs) refer to a large and heterogeneous group of conditions resulting from abnormal epidermal differentiation and featuring thickening of the skin of the palms and soles. Here, we aimed at delineating the genetic basis of an autosomal recessive form of PPK manifesting with erythematous hyperkeratotic plaques over the palms and soles, extending to non- palmoplantar areas and associated with peripheral peeling of the skin. Whole exome sequencing revealed homozygous nonsense variants in the SERPINA12 gene in affected individuals. SERPINA12 encodes vaspin, a serine protease inhibitor. The SERPINA12 disease-causing variants were found to result in reduced vaspin expression in patient skin biopsies. SERPINA12 downregulation in three- dimensional skin equivalents was associated with marked epidermal acanthosis and hyperkeratosis, replicating the human phenotype. Moreover, decreased SERPINA12 expression resulted in reduced vaspin-mediated inhibition of kallikrein 7 activity as well as decreased levels of desmoglein-1 and corneodesmosin, two known kallikrein 7 substrates which have been previously implicated in the pathogenesis of PPKs and peeling skin syndromes. Taken collectively, the present data demarcate a novel type of autosomal recessive PPK, attribute to vaspin a role in skin biology and emphasize the importance of mechanisms regulating proteolytic activity for normal epidermal differentiation.

Pemphigus vulgaris (PV) is a life-threatening autoimmune mucocutaneous blistering disease. We previously showed that a genetic variant within the ST18 promoter promotes ST18 up-regulation in a p53/p63-dependent manner and is associated with a 6-fold increased risk to develop PV. ST18 was also found to be overexpressed in the skin of PV patients. In addition, it has been shown that desmoglein 3 (DSG3) down-regulation is associated with increased p53 expression and activity. Based on these data, using a combination of reporter assays, Western blotting, confocal immunofluorescence microscopy, we investigated the possibility that ST18, DSG3 and p53 may be jointly involved on the pathogenesis of PV. First, we demonstrated that antibody-mediated DSG3 down-regulation results in enhanced expression of p53. Second, we showed that DSG3 down- regulation activates the ST18 promoter activity. Third, p53 silencing abolished the DSG3-mediated activation of the ST18 promoter activity. Finally, we demonstrated that ST18 overexpression in keratinocytes significantly augments antibody-mediated DSG3 down-regulation in keratinocytes. Taken collectively, these data indicate that ST18 up-regulation triggers a pathophysiological self- amplifying cycle involving DSG3 and p53 dysregulation, which may underlie the genetic association of ST18 variants with PV. Supporting the clinical relevance of these findings, a genetic variant causing increased ST18 promoter activity was found to be associated (p = 0.003) with a more severe phenotype in a cohort of 100 PV patients.

Pemphigus vulgaris (PV) is a life‐threatening mucocutaneous autoimmune blistering disease. We previously showed that genetic variants within the ST18 gene promoter area confer a sixfold increase in the propensity to develop PV. ST18, a transcription factor, was found to be overexpressed in the epidermis of patients with PV. In addition, it was found to promote autoantibody‐mediated abnormal epidermal cell–cell adhesion and secretion of proinflammatory mediators by keratinocytes.

Background Hypotrichosis simplex of the scalp (HSS) is characterized by progressive loss of scalp hair that results in almost complete baldness at a young age. HSS is often caused by dominant nonsense mutations in CDSN encoding corneodesmosin, leading to the formation of an amyloid-like material, which interferes with normal hair follicle cycle. Objectives As gentamicin has been shown to mediate ribosomal read-through, we aimed to ascertain its therapeutic efficacy in a small series of patients carrying a recurrent mutation in CDSN. Methods We used a green fluorescence reporter assay system, confocal microscopy and Western blot analysis to ascertain in vitro the ability of gentamicin to induce translational read through across a causative CDSN mutation. Results Using a reporter assay, we initially showed that gentamicin induces read-through activity across an HSS-causing nonsense mutation. Gentamicin was further shown to rescue corneodesmosin translation in primary keratinocytes obtained from a patient with HSS. To validate the in vitro data, we conducted a pilot clinical trial where the scalp of four patients was treated topically with gentamicin for 6 months, demonstrating significant improvement as ascertained by the Severity of Alopecia Tool score. Conclusions Our findings indicate that topical gentamicin should be considered as a potential therapeutic modality in HSS.

BACKGROUND:Pachyonychia congenita (PC) is a rare autosomal dominant disorder featuring palmoplantar keratoderma, nail dystrophy, oral leucokeratosis, pilosebaceous cysts and natal teeth. PC results from dominant mutations in one of five genes (KRT6A, KRT6B, KRT6C, KRT16, KRT17) encoding keratin proteins.AIM:To delineate the clinical and genetic features of PC in a series of Israeli patients.METHODS:We used direct sequencing of genomic DNA, and also used cDNA sequencing where applicable.RESULTS:We collected clinical information and molecular data in a cohort of Israeli families diagnosed with PC (n = 16). Most of the patients were Ashkenazi Jews and had a family history of PC. The most common clinical findings were painful focal plantar keratoderma (94%) accompanied by nail dystrophy (81%), pilosebaceous cysts (31%) and prenatal/natal teeth (13%). In contrast to the high prevalence of KRT6A mutations in other populations, we found that KRT16 mutations were the most common type among Israeli patients with PC (56%). Most (77%) of the Israeli patients with PC with KRT16 mutation carried the same variant (c.380G>A; p.R127H) and shared the same haplotype around the KRT16 locus, suggestive of a founder effect.CONCLUSION:The data gleaned from this study emphasizes the importance of population-specific tailored diagnostic strategies.

BACKGROUND:Palmoplantar keratoderma (PPK) refers to a large group of disorders characterized by extensive genetic and phenotypic heterogeneity. PPK diagnosis therefore increasingly relies upon genetic analysis.AIM:To delineate the genetic defect underlying a case of diffuse erythematous PPK associated with peeling of the skin.METHODS:Whole exome and direct sequencing, real-time quantitative PCR, protein modelling and a cathepsin B enzymatic assay were used.RESULTS:The patient studied had severe diffuse erythematous PPK transgrediens. Pedigree analysis suggested an autosomal dominant mode of inheritance. Whole exome sequencing revealed a heterozygous missense mutation in the CTSB gene, encoding the cysteine protease cathepsin B. Genomic duplications in a noncoding region, which regulates the expression of CTSB, were recently found to cause erythrokeratolysis hiemalis, a rare autosomal dominant disorder of cornification. This mutation affects a highly conserved residue, and is predicted to be pathogenic. Protein modelling indicated that the mutation is likely to lead to increased endopeptidase cathepsin B activity. Accordingly, the CTSB variant was found to result in increased cathepsin B proteolytic activity.CONCLUSION:In summary, we report the identification of the first gain-of-function missense mutation in CTSB, which was found to be associated in one individual with a dominant form of diffuse PPK.

Erythrokeratolysis hiemalis (EH) also known as keratolytic winter erythema is a rare autosomal-dominantly inherited disorder of cornification. It manifests with recurrent episodes of skin peeling and palmoplantar erythema, often more evident during the winter season. EH was recently found to be caused by genomic duplications in a non-coding region which regulates CTSB, a gene encoding the cysteine protease Cathepsin B. As a consequence, Cathepsin B expression is increased which in turn causes EH. We aimed at identifying the genetic defect underlying an atypical disorder of cornification in a family featuring severe diffuse transgradient hyperkeratosis involving the palms and soles accompanied by mild diffuse erythema of the palms. Pedigree analysis suggested autosomal dominant inheritance. Whole-exome sequencing revealed a heterozygous missense variant in CTSB. The variant affects a highly conserved residue and is predicted to be pathogenic. Protein modelling indicated that the variant is likely to lead to increased endopeptidase Cathepsin B activity. Accordingly, using a reporter assay expressing the variant, we found that it indeed results in increased Cathepsin B activity. Moreover, Cathepsin B expression was increased in a biopsy sample obtained from the patient. In summary, we report the identification of the first gain-of-function missense variant in CTSB which was found to be associated with an atypical EH-like phenotype.

Cole disease is a unique genodermatosis featuring combined abnormal pigmentation and keratinization. It is characterized by congenital or early-onset punctate keratoderma associated with irregularly shaped hypopigmented macules. Cole disease results from heterozygous mutations in ENPP1 encoding a protein that has been shown to play a critical role in bone mineralization and insulin resistance. Here, we aimed at investigating ENPP1 involvement in epidermal differentiation and pigmentation, which are abnormal in Cole disease. Using qPCR and Western blot analysis, we observed that ENPP1 is expressed in keratinocytes (KCs) in a calcium-dependent manner. In organotypic skin cultures, downregulation of ENPP1 was associated with hyperproliferation and decreased differentiation. In line with these data, immunostaining of ENPP1-silenced skin equivalents revealed a significant increase in KRT5 and a significant reduction in KRT10 and loricrin expression. Increase in KRT5 was also observed in hypopigmented macules of a patient carrying the c.530G>A mutation and in skin equivalents reconstructed with melanocytes from this patient. Furthermore, Ki67-positive cells were significantly more numerous in ENPP1-downregulated models. Downregulation of ENPP1 in MNT1 cells (a melanocytic cell line) resulted in decreased melanin synthesis as measured by immunohistochemistry and a melanin synthesis assay. Overexpression of c.530G>A mutation in primary melanocytes decreased the expression of TRP-1 and tyrosinase at the protein level. Primary melanocytes overexpressing the c.530G>A mutation induced hyperproliferation in skin equivalents. In summary, we found that ENPP1 not only regulates ectopic calcification, it also promotes keratinocyte differentiation and melanogenesis, which may in part contribute to the pathogenesis of Cole disease.

Central centrifugal cicatricial alopecia (CCCA) is the most common form of scarring alopecia among women of African ancestry (prevalence 2.7%-5.6%). The disease usually manifests clinically after intense hair grooming. Autosomal dominant inheritance of CCCA in some families suggests the existence of a genetically determined predisposition. Here, we aimed at identifying genetic risk variants for CCCA. Using whole exome sequencing in a discovery set of 16 patients, we identified one splice site and three missense heterozygous mutations in PADI3. PADI3 encodes peptidyl arginine deiminase, type III (PADI3), an enzyme that post-translationally modifies proteins that are essential to hair-shaft formation. All three CCCA-associated missense mutations in PADI3 affect highly conserved residues and are predicted to be pathogenic. Protein modeling suggested that the mutations result in protein misfolding. Accordingly, CCCA-associated mutations were found to result in reduced PADI3 expression, abnormal intracellular localization of the protein, and decreased PAD enzymatic activity. Immunofluorescence staining showed decreased expression of PADI3 in scalp skin obtained from CCCA patients. Finally, we directly sequenced PADI3 in a replication set of 42 patients. Combining the data sets, we found pathogenic variants in 24% of all CCCA patients. In conclusion, intense hair grooming habits combined with mutations in PADI3, which encodes a protein that is essential to proper hair shaft formation, may result in CCCA.