Palmoplantar keratoderma caused by a missense variant in CTSB encoding cathepsin B.

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.

ST18 affects cell-cell adhesion in pemphigus vulgaris in a tumour necrosis factor-α-dependent fashion.

BACKGROUND: 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. OBJECTIVES: To delineate the mechanism through which ST18 contributes to destabilization of cell-cell adhesion. METHODS: We used quantitative reverse-transcriptase polymerase chain reaction, immunofluorescence microscopy, a luciferase reporter system, site-directed mutagenesis, chromatin immunoprecipitation (ChIP) and the dispase dissociation assay. RESULTS: The ChIP and luciferase reporter assays showed that ST18 directly binds and activates the TNF promoter. Accordingly, increased ST18 expression contributes to PV pathogenesis by destabilizing cell-cell adhesion in a tumour necrosis factor (TNF)-α-dependent fashion. In addition, dual immunofluorescence staining showed increased expression of both ST18 and TNF-α in the skin of patients with PV carrying an ST18-associated PV risk variant, which was found to be associated with a more extensive PV phenotype. CONCLUSIONS: Our findings suggest a role for TNF-α in mediating the deleterious effect of increased ST18 expression in PV skin.

Molecular epidemiology of pachyonychia congenita in the Israeli population.

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.

Treatment of hereditary hypotrichosis simplex of the scalp with topical gentamicin.

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. What's already known about this topic? Hypotrichosis simplex of the scalp (HSS) is caused by nonsense mutations in CDSN encoding corneodesmosin. The mutant corneodesmosin has been hypothesized to be toxic to the hair follicles, leading to hypotrichosis. Disorders caused by nonsense mutations are amenable to ribosomal read-through using gentamicin. What does this study add? Gentamicin enhanced read-through activity and promoted full-length corneodesmosin synthesis in primary keratinocytes derived from patients carrying a nonsense mutation in CDSN. Topical treatment with gentamicin was found to rescue the hypotrichosis phenotype partially in four patients with HSS. What is the translational message? Topical gentamicin should be considered as a potential treatment for HSS.

Quantitative and qualitative control of cytotoxic preparations by HPLC-UV in a centralized parenteral preparations unit.

The constantly growing incidence of cancer and long-term treatment are leading to an increasing number of cytotoxic preparations in hospital pharmacies. Security and quality standards of cytotoxic preparations are essential to assure treatment efficiency and limit iatrogenic toxicity. In order to secure the process of cytotoxic preparations; we decided to install a quantitative and qualitative High Performance Liquid Chromatography (HPLC) control of cytotoxic preparations carried inside our pharmacotechnic unit. A 100 microl sample of each preparation was assayed by HPLC with ultraviolet/visible-diode array detection, which enabled the identification of all cytotoxic agents thanks to their characteristic UV spectra. We developed rapid and specific HPLC assays that determined qualitatively and quantitatively the presence of 21 different cytotoxic agents in less than 3.5 min. A fifteen per cent tolerance from the theoretical concentration was chosen in agreement with preparation and dosage bias, and a first period control of more than 4400 preparations revealed that around 7.7% preparations did not conform. The main objective of these controls was to avoid the administration of defective chemotherapies to patients and finally to use their results to identify error factors; as a result we will take corrective measures in order to reduce error frequency.