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BACKGROUND Peeling skin syndrome type 1 (PSS1) is a rare and severe autosomal recessive form of congenital ichthyosis. Patients are affected by pronounced erythroderma accompanied by pruritus and superficial generalized peeling of the skin. The disease is caused by nonsense mutations or complete deletion of the CDSN gene encoding for corneodesmosin (CDSN). PSS1 severely impairs quality of life and therapeutic approaches are totally unsatisfactory. OBJECTIVES The objective of this study was to develop the first steps towards a specific protein replacement therapy for CDSN deficiency. Using this approach, we aimed to restore the lack of CDSN and improve cell-cell cohesion in the transition area of the stratum granulosum (SG) to the stratum corneum. METHODS Human CDSN was recombinantly expressed in Escherichia coli. A liposome-based carrier system, prepared with a cationic lipopeptide to mediate the transport to the outer membrane of keratinocytes, was developed. This formulation was chosen for CDSN delivery into the skin. The liposomal carrier system was characterized with respect to size, stability and toxicity. Furthermore, the interaction with primary keratinocytes and human epidermal equivalents was investigated. RESULTS The liposomes showed an accumulation at the membranes of keratinocytes. CDSN-deficient epidermal equivalents that were treated with liposomal encapsulated CDSN demonstrated presence of CDSN in the SG. Finally, the penetration assay and histological examinations revealed an improved epidermal integrity for CDSN-deficient epidermal equivalents, if they were treated with liposomal encapsulated CDSN. CONCLUSIONS This study presents the first preclinical in vitro experiments for a future specific protein replacement therapy for patients affected by PSS1.

X-linked ichthyosis (XLI) is a keratinization disorder caused by deficient activity of steroidsulfatase. In contrast, ichthyosis vulgaris is due to semidominant mutations of the filaggrin gene ( FLG ). In view of phenotypic variations of these ichthyoses we speculated that XLI may be influenced by additional FLG mutations in a significant number of patients. We characterized a group of 51 patients with XLI and systematically analyzed them for additional FLG mutations (R501X, 2282del4, R2447X, S3247X). The study was complemented by morphological analyses. Full FLG sequencing for rare mutations was performed in special cases. Interestingly, prevalence of FLG mutations was significantly increased compared to a population-based control cohort of 1,377 individuals (17.6% vs. 8.4%, p=0.038). Palmoplantar hyperlinearity was significantly associated with the FLG mutation status. Ichthyosis severity score seemed to be increased in XLI with FLG mutations, but the difference was not significant (p=0.124). To our surprise, percentages of atopic manifestations were highly prevalent in both subgroups, 40% and 33% in XLI without and with filaggrin deficiency, respectively. Of note, reduction of filaggrin staining or keratohyalin could not be explained by FLG mutations in all patients. However, we conclude that FLG mutations represent a significant genetic modifier of XLI. [196 words]

Background Transglutaminase (TG)1 plays a key role in the formation of the cornified envelope and thus in the maintenance of the epidermal barrier. Patients with Netherton syndrome (LEKTI deficiency) have increased activity of both TG1 and serin proteases. Objectives To determine whether there is a functional biochemical link between TG1 and LEKTI and whether LEKTI domains could possibly serve as substrates for TG1. Methods We analysed the protein sequence of LEKTI for possible TG1 recognition sites using bioinformatics. Synthetic peptides and recombinant LEKTI domains D6, D7 and D8+9 were examined in vitro and in situ for possible substrate specificity. The recombinant LEKTI domains were studied for inhibitory activity in a kallikrein (KLK)5 activity test. Results We identified possible TG1 consensus sequences in LEKTI domains D6, D7 and D8+9, pointing to a novel biological link between these two proteins. Indeed, synthesized short peptides from these consensus sequences were incorporated into the TG1 activity zone of the epidermis. In vitro the entire recombinant domains of LEKTI showed substrate specificity for TG1, which was again confirmed in situ. The inhibitory activity of the recombinant LEKTI domains was confirmed by a KLK5 inhibition test. The strongest inhibition was observed for domains D8+9. Conclusions There are specific domains of LEKTI that are recognized and processed by TG1. LEKTI domains D6, D7 and D8+9 contribute to the formation and protection of the cornified envelope. These results impact the development of protein replacement therapy approaches for Netherton syndrome. What's already known about this topic? LEKTI and transglutaminase (TG)1 are key proteins involved in the terminal differentiation of the epidermis. Lack of LEKTI causes Netherton syndrome; TG1 deficiency causes lamellar ichthyosis. The serine protease inhibitor LEKTI is processed into different functional units. Among different target proteases, kallikrein (KLK)5 appears to be a key player in disease pathology. It has been demonstrated that LEKTI domain 6 inhibits KLK5 and KLK7; LEKTI domains 8-11 also inhibit KLK14. What does this study add? The single LEKTI domains 6, 7 and the functional unit of domains 8 and 9 contain recognition motifs for TG1. We show that these domains and unit are crosslinked into the epidermis by TG1. Functional analyses of the recombinant LEKTI domains revealed that LEKTI D8+9 has the strongest inhibitory effect on KLK5. What is the translational message? The novel functional link between LEKTI and TG1 should be taken into account when considering the development of a targeted topical protein therapy for Netherton syndrome. The unit of domains D8+9 may be sufficient for this purpose.

Background Ichthyosis vulgaris (IV) is caused by loss-of-function mutations in the profilaggrin (FLG) gene. Filaggrin drives complex interrelated functions, with strategic roles in establishing structural and chemical barrier function, hydration of the skin and maintaining epidermal homeostasis. Data on the effect of FLG mutations on epidermal barrier function in IV are very scarce. Objectives A primary aim of this study was to determine in vivo characteristics of epidermal permeability barrier function such as transepidermal water loss (TEWL), skin hydration and skin surface pH in homozygous or compound heterozygous (FLG−/−) and heterozygous (FLG+/−) subjects with IV. Methods We evaluated a cohort of 15 patients with IV, analysed epidermal ultrastructure and investigated epidermal barrier function by measurement of TEWL, skin surface pH and skin hydration. Mutations were screened by restriction enzyme analysis and/or complete sequencing. Ten patients were homozygous or compound heterozygous (FLG−/−), while five patients were heterozygous (FLG+/−). Twenty healthy individuals served as controls. Results In FLG−/− subjects, a moderate increase of TEWL from 5.41 ± 0.32–7.54 ± 0.90 g/m2h (P

International audience; Peeling skin syndromes (PSS) refer to a heterogeneous group of generalized and/or palmoplantar disorders. In­flammatory peeling skin disease (PSD) refers to PSS type B [MIM 270300] (1) and is an ichthyosiform erythro­derma characterized by recurrent patchy peeling of the skin with accompanying pruritus. The disease persists throughout life, is accompanied by significant atopic manifestations, and can be reminiscent of Netherton syn­drome (NTS).

Generalized peeling skin disease is an autosomal-recessive ichthyosiform erythroderma characterized by lifelong patchy peeling of the skin. After genome-wide linkage analysis, we have identified a homozygous nonsense mutation in CDSN in a large consanguineous family with generalized peeling skin, pruritus, and food allergies, which leads to a complete loss of corneodesmosin. In contrast to hypotrichosis simplex, which can be associated with specific dominant CDSN mutations, peeling skin disease is characterized by a complete loss of CDSN expression. The skin phenotype is consistent with a recent murine Cdsn knockout model. Using three-dimensional human skin models, we demonstrate that lack of corneodesmosin causes an epidermal barrier defect supposed to account for the predisposition to atopic diseases, and we confirm the role of corneodesmosin as a decisive epidermal adhesion molecule. Therefore, peeling skin disease will represent a new model disorder for atopic diseases, similarly to Netherton syndrome and ichthyosis vulgaris in the recent past.