Whole genome sequencing in patients with retinitis pigmentosa reveals pathogenic DNA structural changes and NEK2 as a new disease gene.

We performed whole genome sequencing in 16 unrelated patients with autosomal recessive retinitis pigmentosa (ARRP), a disease characterized by progressive retinal degeneration and caused by mutations in over 50 genes, in search of pathogenic DNA variants. Eight patients were from North America, whereas eight were Japanese, a population for which ARRP seems to have different genetic drivers. Using a specific workflow, we assessed both the coding and noncoding regions of the human genome, including the evaluation of highly polymorphic SNPs, structural and copy number variations, as well as 69 control genomes sequenced by the same procedures. We detected homozygous or compound heterozygous mutations in 7 genes associated with ARRP (USH2A, RDH12, CNGB1, EYS, PDE6B, DFNB31, and CERKL) in eight patients, three Japanese and five Americans. Fourteen of the 16 mutant alleles identified were previously unknown. Among these, there was a 2.3-kb deletion in USH2A and an inverted duplication of ∼446 kb in EYS, which would have likely escaped conventional screening techniques or exome sequencing. Moreover, in another Japanese patient, we identified a homozygous frameshift (p.L206fs), absent in more than 2,500 chromosomes from ethnically matched controls, in the ciliary gene NEK2, encoding a serine/threonine-protein kinase. Inactivation of this gene in zebrafish induced retinal photoreceptor defects that were rescued by human NEK2 mRNA. In addition to identifying a previously undescribed ARRP gene, our study highlights the importance of rare structural DNA variations in Mendelian diseases and advocates the need for screening approaches that transcend the analysis of the coding sequences of the human genome.

The pathogenic role of tissue-resident immune cells in psoriasis.

Psoriasis is a common chronic inflammatory skin disease, the study of which might also be of considerable value to the understanding of other inflammatory and autoimmune-type diseases, such as rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis and diabetes mellitus. There is clear evidence that T cells and dendritic cells have a central role in psoriasis. Based on recent data from humans and animal models, we propose that a psoriasis lesion can be triggered and sustained by the local network of skin-resident immune cells. This concept focuses attention on local, rather than systemic, components of the immune system for rationalized therapeutic approaches of psoriasis and possibly also other chronic inflammatory diseases.

Recurrent stroke predictors differ in medically treated patients with pathogenic vs. other PFOs.

OBJECTIVE: To examine predictors of stroke recurrence in patients with a high vs a low likelihood of having an incidental patent foramen ovale (PFO) as defined by the Risk of Paradoxical Embolism (RoPE) score. METHODS: Patients in the RoPE database with cryptogenic stroke (CS) and PFO were classified as having a probable PFO-related stroke (RoPE score of >6, n = 647) and others (RoPE score of ≤6 points, n = 677). We tested 15 clinical, 5 radiologic, and 3 echocardiographic variables for associations with stroke recurrence using Cox survival models with component database as a stratification factor. An interaction with RoPE score was checked for the variables that were significant. RESULTS: Follow-up was available for 92%, 79%, and 57% at 1, 2, and 3 years. Overall, a higher recurrence risk was associated with an index TIA. For all other predictors, effects were significantly different in the 2 RoPE score categories. For the low RoPE score group, but not the high RoPE score group, older age and antiplatelet (vs warfarin) treatment predicted recurrence. Conversely, echocardiographic features (septal hypermobility and a small shunt) and a prior (clinical) stroke/TIA were significant predictors in the high but not low RoPE score group. CONCLUSION: Predictors of recurrence differ when PFO relatedness is classified by the RoPE score, suggesting that patients with CS and PFO form a heterogeneous group with different stroke mechanisms. Echocardiographic features were only associated with recurrence in the high RoPE score group.

Proteinaceous infectious behavior in non-pathogenic proteins is controlled by molecular chaperones.

External stresses or mutations may cause labile proteins to lose their distinct native conformations and seek alternatively stable aggregated forms. Molecular chaperones that specifically act on protein aggregates were used here as a tool to address the biochemical nature of stable homo- and hetero-aggregates from non-pathogenic proteins formed by heat-stress. Confirmed by sedimentation and activity measurements, chaperones demonstrated that a single polypeptide chain can form different species of aggregates, depending on the denaturing conditions. Indicative of a cascade reaction, sub-stoichiometric amounts of one fast-aggregating protein strongly accelerated the conversion of another soluble, slow-aggregating protein into insoluble, chaperone-resistant aggregates. Chaperones strongly inhibited seed-induced protein aggregation, suggesting that they can prevent and cure proteinaceous infectious behavior in homo- and hetero-aggregates from common and disease-associated proteins in the cell.

Pathogenic Vibrio activate NLRP3 inflammasome via cytotoxins and TLR/nucleotide-binding oligomerization domain-mediated NF-kappa B signaling.

Vibrio vulnificus and Vibrio cholerae are Gram-negative pathogens that cause serious infectious disease in humans. The beta form of pro-IL-1 is thought to be involved in inflammatory responses and disease development during infection with these pathogens, but the mechanism of beta form of pro-IL-1 production remains poorly defined. In this study, we demonstrate that infection of mouse macrophages with two pathogenic Vibrio triggers the activation of caspase-1 via the NLRP3 inflammasome. Activation of the NLRP3 inflammasome was mediated by hemolysins and multifunctional repeat-in-toxins produced by the pathogenic bacteria. NLRP3 activation in response to V. vulnificus infection required NF-kappaB activation, which was mediated via TLR signaling. V. cholerae-induced NLRP3 activation also required NF-kappaB activation but was independent of TLR stimulation. Studies with purified V. cholerae hemolysin revealed that toxin-stimulated NLRP3 activation was induced by TLR and nucleotide-binding oligomerization domain 1/2 ligand-mediated NF-kappaB activation. Our results identify the NLRP3 inflammasome as a sensor of Vibrio infections through the action of bacterial cytotoxins and differential activation of innate signaling pathways acting upstream of NF-kappaB.

Gene expression profiling in the human pathogenic dermatophyte Trichophyton rubrum during growth on proteins.

Dermatophytes are highly specialized filamentous fungi which cause the majority of superficial mycoses in humans and animals. The high secreted proteolytic activity of these microorganisms during growth on proteins is assumed to be linked to their particular ability to exclusively infect keratinized host structures such as the skin stratum corneum, hair, and nails. Individual secreted dermatophyte proteases were recently described and linked with the in vitro digestion of keratin. However, the overall adaptation and transcriptional response of dermatophytes during protein degradation are largely unknown. To address this question, we constructed a cDNA microarray for the human pathogenic dermatophyte Trichophyton rubrum that was based on transcripts of the fungus grown on proteins. Profiles of gene expression during the growth of T. rubrum on soy and keratin protein displayed the activation of a large set of genes that encode secreted endo- and exoproteases. In addition, other specifically induced factors potentially implicated in protein utilization were identified, including heat shock proteins, transporters, metabolic enzymes, transcription factors, and hypothetical proteins with unknown functions. Of particular interest is the strong upregulation of key enzymes of the glyoxylate cycle in T. rubrum during growth on soy and keratin, namely, isocitrate lyase and malate synthase. This broad-scale transcriptional analysis of dermatophytes during growth on proteins reveals new putative pathogenicity-related host adaptation mechanisms of these human pathogenic fungi.

Enrichment of pathogenic alleles in the brittle cornea gene, ZNF469, in keratoconus.

Keratoconus, a common inherited ocular disorder resulting in progressive corneal thinning, is the leading indication for corneal transplantation in the developed world. Genome-wide association studies have identified common SNPs 100 kb upstream of ZNF469 strongly associated with corneal thickness. Homozygous mutations in ZNF469 and PR domain-containing protein 5 (PRDM5) genes result in brittle cornea syndrome (BCS) Types 1 and 2, respectively. BCS is an autosomal recessive generalized connective tissue disorder associated with extreme corneal thinning and a high risk of corneal rupture. Some individuals with heterozygous PRDM5 mutations demonstrate a carrier ocular phenotype, which includes a mildly reduced corneal thickness, keratoconus and blue sclera. We hypothesized that heterozygous variants in PRDM5 and ZNF469 predispose to the development of isolated keratoconus. We found a significant enrichment of potentially pathologic heterozygous alleles in ZNF469 associated with the development of keratoconus (P = 0.00102) resulting in a relative risk of 12.0. This enrichment of rare potentially pathogenic alleles in ZNF469 in 12.5% of keratoconus patients represents a significant mutational load and highlights ZNF469 as the most significant genetic factor responsible for keratoconus identified to date.

Evaluation of the susceptibility of pathogenic Candida species to fluconazole. Fluconazole Global Susceptibility Study Group.

A fluconazole 25 microg disk diffusion test was used to test 2230 consecutively isolated Candida strains from 42 different hospital laboratories in 23 countries. Ninety seven percent of 1634 Candida albicans isolates and 83.4% of 596 non-Candida albicans isolates were susceptible to fluconazole, applying the proposed breakpoints (> or = 26 mm for susceptible strains and 18-25 mm for dose-dependent susceptible strains). This is the first hospital laboratory study to evaluate a large number and wide range of sequential Candida isolates from patients with all types of hospital infections. The fluconazole disk diffusion test appears to be a low-cost, reproducible, and accurate means of assessing the in vitro susceptibility of Candida isolates.

Novel pathogenic pathways in diabetic neuropathy.

Diabetic peripheral neuropathy (DPN) is a common complication affecting more than one third of diabetes mellitus (DM) patients. Although all cellular components participating in peripheral nerve function are exposed to and affected by the metabolic consequences of DM, nodal regions, areas of intense interactions between Schwann cells and axons, may be particularly sensitive to DM-induced alterations. Nodes are enriched in insulin receptors, glucose transporters, Na(+) and K(+) channels, and mitochondria, all implicated in the development and progression of DPN. Latest results particularly reinforce the idea that changes in ion-channel function and energy metabolism, both of which depend on axon-glia crosstalk, are among the important contributors to DPN. These insights provide a basis for new therapeutic approaches aimed at delaying or reversing DPN.

Two Cases of Interferon-Alpha-Induced Sarcoidosis Koebnerized along Venous Drainage Lines: New Pathogenic Insights and Review of the Literature of Interferon-Induced Sarcoidosis.

Sarcoidosis is a systemic granulomatous disorder of unknown origin commonly affecting the lung, the lymphoid system and the skin. We report here two cases of cutaneous sarcoidosis in two former intravenous drug users following interferon (IFN)-α and ribavirin therapy for chronic hepatitis C. Both patients developed skin sarcoidosis along venous drainage lines of both forearms, coinciding with the areas of prior drug injections. The unique distribution of the skin lesions suggests that tissue damage induced by repeated percutaneous drug injections represents a trigger for the local skin manifestation of sarcoidosis. Interestingly, skin damage was recently found to induce the local expression IFN-α, a well-known trigger of sarcoidosis in predisposed individuals. Here we review the literature on sarcoidosis elicited in the context of IFN-α therapy and propose a new link between the endogenous expression of IFN-α and the induction of disease manifestations in injured skin. © 2013 S. Karger AG, Basel.

Comparative genomics suggests that the human pathogenic fungus Pneumocystis jirovecii acquired obligate biotrophy through gene loss.

Pneumocystis jirovecii is a fungal parasite that colonizes specifically humans and turns into an opportunistic pathogen in immunodeficient individuals. The fungus is able to reproduce extracellularly in host lungs without eliciting massive cellular death. The molecular mechanisms that govern this process are poorly understood, in part because of the lack of an in vitro culture system for Pneumocystis spp. In this study, we explored the origin and evolution of the putative biotrophy of P. jirovecii through comparative genomics and reconstruction of ancestral gene repertoires. We used the maximum parsimony method and genomes of related fungi of the Taphrinomycotina subphylum. Our results suggest that the last common ancestor of Pneumocystis spp. lost 2,324 genes in relation to the acquisition of obligate biotrophy. These losses may result from neutral drift and affect the biosyntheses of amino acids and thiamine, the assimilation of inorganic nitrogen and sulfur, and the catabolism of purines. In addition, P. jirovecii shows a reduced panel of lytic proteases and has lost the RNA interference machinery, which might contribute to its genome plasticity. Together with other characteristics, that is, a sex life cycle within the host, the absence of massive destruction of host cells, difficult culturing, and the lack of virulence factors, these gene losses constitute a unique combination of characteristics which are hallmarks of both obligate biotrophs and animal parasites. These findings suggest that Pneumocystis spp. should be considered as the first described obligate biotrophs of animals, whose evolution has been marked by gene losses.

Gene expression profiling in human pathogenic dermatophytes in vitro and in vivo

Objectives: Dermatophytes are highly specialized fungi which are the most common agents of superficial mycoses in humans and animals. The particular ability of these microorganisms to invade and multiply within keratinized host structures is presumably linked to their secreted keratinolytic activity, which is therefore a major putative virulence attribute of these fungi. The overall adaptation and transcriptional response of dermatophytes during protein degradation and/or infection is largely unknown. Methods: A Trichophyton rubrum cDNA microarray was developed and used for the transcriptional analysis of T. rubrum and Arthroderma benhamiae cells during growth on protein substrates. Moreover, the gene expression profile in A. benhamiae cells was monitored during infection of guinea pigs. Results: T. rubrum and A. benhamiae cells activate a large set of genes encoding secreted endo- and exoproteases during growth on soy and keratin. In addition, other specifically induced factors with potential implication in protein utilization were identified, e.g. multiple transporters, metabolic enzymes, transcription factors and hypothetical proteins with unknown function. Notably however, the protease gene expression profile in the fungal cells during infection was significantly different from the pattern elicited during in vitro growth on keratin. Conclusions: Our results suggest specific functions of individual proteases during infection, which may not be restricted to the degradation of keratin. This first, broad in vivo transcriptional profiling approach in dermatophytes gives new molecular insights into pathogenicity associated adaptation mechanisms that make these microorganisms the most successful causitive agents of superficial mycoses.

Pathogenic role of basic calcium phosphate crystals in destructive arthropathies.

BACKGROUND: basic calcium phosphate (BCP) crystals are commonly found in osteoarthritis (OA) and are associated with cartilage destruction. BCP crystals induce in vitro catabolic responses with the production of metalloproteases and inflammatory cytokines such as interleukin-1 (IL-1). In vivo, IL-1 production induced by BCP crystals is both dependant and independent of NLRP3 inflammasome. We aimed to clarify 1/ the role of BCP crystals in cartilage destruction and 2/ the role of IL-1 and NLRP3 inflammasome in cartilage degradation related to BCP crystals. METHODOLOGY PRINCIPAL FINDINGS: synovial membranes isolated from OA knees were analysed by alizarin Red and FTIR. Pyrogen free BCP crystals were injected into right knees of WT, NLRP3 -/-, ASC -/-, IL-1α -/- and IL-1β-/- mice and PBS was injected into left knees. To assess the role of IL-1, WT mice were treated by intra-peritoneal injections of anakinra, the IL-1Ra recombinant protein, or PBS. Articular destruction was studied at d4, d17 and d30 assessing synovial inflammation, proteoglycan loss and chondrocyte apoptosis. BCP crystals were frequently found in OA synovial membranes including low grade OA. BCP crystals injected into murine knee joints provoked synovial inflammation characterized by synovial macrophage infiltration that persisted at day 30, cartilage degradation as evidenced by loss of proteoglycan staining by Safranin-O and concomitant expression of VDIPEN epitopes, and increased chondrocyte apoptosis. BCP crystal-induced synovitis was totally independent of IL-1α and IL-1β signalling and no alterations of inflammation were observed in mice deficient for components of the NLRP3-inflammasome, IL-1α or IL-1β. Similarly, treatment with anakinra did not prevent BCP crystal effects. In vitro, BCP crystals elicited enhanced transcription of matrix degrading and pro-inflammatory genes in macrophages. CONCLUSIONS SIGNIFICANCE: intra-articular BCP crystals can elicit synovial inflammation and cartilage degradation suggesting that BCP crystals have a direct pathogenic role in OA. The effects are independent of IL-1 and NLRP3 inflammasome.