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Background: Atopic dermatitis (AD) is a highly prevalent chronic inflammatory skin disease that is known to be, at least in part, genetically determined. Mutations in caspase recruitment domain-containing protein 14 (CARD14) have been shown to result in various forms of psoriasis and related disorders. Objective: We aimed to identify rare DNA variants conferring a significant risk for AD through genetic and functional studies in a cohort of patients affected with severe AD. Methods: Whole-exome and direct gene sequencing, immunohistochemistry, real-time PCR, ELISA, and functional assays in human keratinocytes were used. Results: In a cohort of patients referred with severe AD, DNA sequencing revealed in 4 patients 2 rare heterozygous missense mutations in the gene encoding CARD14, a major regulator of nuclear factor kB (NF-kB). A dual luciferase reporter assay demonstrated that both mutations exert a dominant loss-offunction effect and result in decreased NF-kB signaling.

Summary 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.

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Halophilic archaea were found to contain in their cytoplasm millimolar concentrations of ␥-glutamylcysteine (␥GC) instead of glutathione. Previous analysis of the genome sequence of the archaeon Halobacterium sp. strain NRC-1 has indicated the presence of a sequence homologous to sequences known to encode the glutamate-cysteine ligase GshA. We report here the identification of the gshA gene in the extremely halophilic archaeon Haloferax volcanii and show that H. volcanii gshA directs in vivo the synthesis and accumulation of ␥GC. We also show that the H. volcanii gene when expressed in an Escherichia coli strain lacking functional GshA is able to restore synthesis of glutathione.Many organisms contain millimolar concentrations of lowmolecular-weight thiol compounds that participate in a number of important biological functions involving thiol-disulfide exchanges (7). In particular, they serve to maintain an intracellular reducing environment, to provide reducing power for key reductive enzymes, to combat the effects of oxidative and disulfide stress, and to detoxify xenobiotic compounds (7). Glutathione (GSH), a cysteine-containing tripeptide, L-␥-glutamyl-L-cysteinylglycine, is the best-characterized low-molecular-weight thiol (7,19,21). GSH is made in a highly conserved two-step ATP-dependent process by two unrelated peptide bond-forming enzymes (3, 21). The ␥-carboxyl group of Lglutamate and the amino group of L-cysteine are ligated by the enzyme glutamylcysteine (GC) ligase EC 6.3.2.2 (GshA, encoded by gshA), which is then condensed with glycine in a reaction catalyzed by GSH synthetase (GshB, encoded by gshB) to form GSH (10,38). GSH is found primarily in gramnegative bacteria and eukaryotes and only rarely in grampositive bacteria (26). Fahey and coworkers showed that GSH is absent from the high-GC gram-positive actinomycetes which produce, as the major low-molecular-weight thiol, mycothiol, (13,(26)(27)(28)35). GSH is also absent in Archaea. In Pyrococcus furiosus, coenzyme A SH (CoASH) is the main thiol (11), whereas in Halobacterium salinarum, ␥GC is the predominant thiol and the organism possesses bis-␥GC reductase activity (30,36). Similarly, Leuconostoc kimchi and Leuconostoc mesenteroides, gram-positive lactic acid bacterial species, were recently found to contain ␥GC rather than GSH (15). To date, these are the sole procaryotic species reported to naturally produce ␥GC but not GSH (6,30). In this report, we describe the identification of the gshA gene in the extremely halophilic archaeon Haloferax volcanii. Copley and Dhillon (6) previously identified, using bioinformatic tools, an open reading frame (ORF) (gene VNG1397C) in Halobacterium sp. strain NRC-1 with limited sequence relatedness to known GshA proteins (6). However, no genetic or biochemical evidence was presented to substantiate their conclusion. Here, we show that Haloferax volcanii strain DS2 (1, 25) contains an ORF that directs in vivo the synthesis and accumulation of ␥GC. We also show that the H. volcanii ORF, when expressed in Esche...

Ribosome biogenesis is a highly energy-demanding process in eukaryotes which requires the concerted action of all three RNA polymerases. In RNA polymerase II transcription, the general transcription factor TFIIH is recruited by TFIIE to the initiation site of protein-coding genes. Distinct mutations in TFIIH or TFIIE lead to the rare autosomal recessive disorder trichothiodystrophy (TTD) characterized by a variety of symptoms including brittle hair, ichthyosis, and premature aging symptoms. While TFIIH is known to play an additional role in the production of ribosomal RNA (rRNA) by RNA polymerase I, the involvement of TFIIE in the RNA polymerase I transcription remains unknown. Here we uncovered an unexpected role of TFIIE in rRNA synthesis by RNA polymerase I. With high resolution microscopy we detected TFIIE in the nucleolus, at the site of active rRNA production. With specific inhibition of the RNA polymerase I transcription, TFIIE de-localizes from the nucleolus, implying RNA polymerase I transcription-dependent localization of TFIIE to the nucleolus. Mutations in TFIIE affects gene-occupancy of RNA polymerase I, rRNA maturation, ribosomal assembly and performance. In consequence, the elevated translational error rate with imbalanced protein synthesis and turnover results in an increase in heat-sensitive proteins. The results of our study unravel a novel role of TFIIE in RNA polymerase I transcription and identify loss of proteostasis as a possible pathomechanism in TTD.

Autosomal recessive congenital ichthyosis (ARCI) is a rare and heterogeneous skin cornification disorder presenting with generalized scaling and varying degrees of erythema. Clinical manifestations range from lamellar ichthyosis (LI), congenital ichthyosiform erythroderma (CIE) through the most severe form of ARCI, Harlequin ichthyosis (HI). We used homozygosity mapping, whole-exome and direct sequencing to delineate the relative distribution of pathogenic variants as well as identify genotypephenotype correlations in a cohort of 62 Middle Eastern families with ARCI of various ethnic backgrounds. Pathogenic variants were identified in most ARCI-associated genes including TGM1 (21%), CYP4F22 (18%), ALOX12B (14%), ABCA12 (10%), ALOXE3 (6%), NIPAL4 (5%), PNPLA1 (3%), LIPN (2%) and SDR9C7 (2%). In 19% of cases, no | 1291 MOHAMAD et Al.