Missing Heritability in Albinism: Deep Characterization of a Hungarian Albinism Cohort Raises the Possibility of the Digenic Genetic Background of the Disease.

Albinism is characterized by a variable degree of hypopigmentation affecting the skin and the hair, and causing ophthalmologic abnormalities. Its oculocutaneous, ocular and syndromic forms follow an autosomal or X-linked recessive mode of inheritance, and 22 disease-causing genes are implicated in their development. Our aim was to clarify the genetic background of a Hungarian albinism cohort. Using a 22-gene albinism panel, the genetic background of 11 of the 17 Hungarian patients was elucidated. In patients with unidentified genetic backgrounds (n = 6), whole exome sequencing was performed. Our investigations revealed a novel, previously unreported rare variant (N687S) of the two-pore channel two gene (TPCN2). The N687S variant of the encoded TPC2 protein is carried by a 15-year-old Hungarian male albinism patient and his clinically unaffected mother. Our segregational analysis and in vitro functional experiments suggest that the detected novel rare TPCN2 variant alone is not a disease-causing variant in albinism. Deep genetic analyses of the family revealed that the patient also carries a phenotype-modifying R305W variant of the OCA2 protein, and he is the only family member harboring this genotype. Our results raise the possibility that this digenic combination might contribute to the observed differences between the patient and the mother, and found the genetic background of the disease in his case.

Spinocerebellar Ataxia in a Hungarian Female Patient with a Novel Variant of Unknown Significance in the CCDC88C Gene.

Spinocerebellar ataxia (SCA) 40 is an extremely rare subtype of the phenotypically and genetically diverse autosomal dominant ataxias caused by mutations of the CCDC88C gene. Most reported cases of SCA40 are characterized by late-onset cerebellar ataxia and variable extrapyramidal features; however, there is a report of a patient with early-onset spastic paraparesis as well. Here, we describe a novel missense CCDC88C mutation (p.R203W) in the hook domain of the DAPLE protein encoded by the CCDC88C gene that was identified in a female patient who developed late-onset ataxia, dysmetria and intention tremor. To explore the molecular consequences of the newly identified and previously described CCDC88C mutations, we carried out in vitro functional tests. The CCDC88C alleles were expressed in HEK293 cells, and the impact of the mutant DAPLE protein variants on JNK pathway activation and apoptosis was assessed. Our results revealed only a small-scale activation of the JNK pathway by mutant DAPLE proteins; however, increased JNK1 phosphorylation could not be detected. Additionally, none of the examined mutations triggered proapoptotic effect. In conclusion, we identified a novel mutation of the CCDC88C gene from a patient with spinocerebellar ataxia. Our results are not in accord with previous observations and do not support the primary role of the CCDC88C mutations in induction of JNK pathway activation in ataxia. Therefore, we propose that CCDC88C mutations may exert their effects through different and possibly in much broader, yet unexplored, biological processes.

Psoriasis-Associated Inflammatory Conditions Induce IL-23 mRNA Expression in Normal Human Epidermal Keratinocytes.

Psoriasis is a multifactorial, chronic inflammatory skin disease, the development of which is affected by both genetic and environmental factors. Cytosolic nucleic acid fragments, recognized as pathogen- and danger-associated molecular patterns, are highly abundant in psoriatic skin. It is known that psoriatic skin exhibits increased levels of IL-23 compared to healthy skin. However, the relationship between free nucleic acid levels and IL-23 expression has not been clarified yet. To examine a molecular mechanism by which nucleic acids potentially modulate IL-23 levels, an in vitro system was developed to investigate the IL-23 mRNA expression of normal human epidermal keratinocytes under psoriasis-like circumstances. This system was established using synthetic nucleic acid analogues (poly(dA:dT) and poly(I:C)). Signaling pathways, receptor involvement and the effect of PRINS, a long non-coding RNA previously identified and characterized by our research group, were analyzed to better understand the regulation of IL-23 in keratinocytes. Our results indicate that free nucleic acids regulate epithelial IL-23 mRNA expression through the TLR3 receptor and specific signaling pathways, thereby, contributing to the development of an inflammatory milieu favorable for the appearance of psoriatic symptoms. A moderate negative correlation was confirmed between the nucleic-acid-induced IL-23 mRNA level and the rate of its decrease upon PRINS overexpression.

TRAF3 and NBR1 both influence the effect of the disease-causing CYLD(Arg936X) mutation on NF-κB activity.

Recently described Hungarian and Anglo-Saxon pedigrees that are affected by CYLD cutaneous syndrome (syn: Brooke-Spiegler syndrome (BSS)) carry the same disease-causing mutation (c.2806C>T, p.Arg936X) of the cylindromatosis (CYLD) gene but exhibit striking phenotypic differences. Using whole exome sequencing, missense genetic variants of the TRAF3 and NBR1 genes were identified in the affected family members of the Hungarian pedigree that are not present in the Anglo-Saxon pedigree. This suggested that the affected proteins (TRAF3 and NBR1) are putative phenotype-modifying factors. An in vitro experimental system was set up to clarify how wild type and mutant TRAF3 and NBR1 modify the effect of CYLD on the NF-κB signal transduction pathway. Our study revealed that the combined expression of mutant CYLD(Arg936X) with TRAF3 and NBR1 caused increased NF-κB activity, regardless of the presence or absence of mutations in TRAF3 and NBR1. We concluded that increased expression levels of these proteins further strengthen the effect of the CYLD(Arg936X) mutation on NF-κB activity in HEK293 cells and may explain the phenotype-modifying effect of these genes in CYLD cutaneous syndrome. These results raise the potential that detecting the levels of TRAF3 and NBR1 might help explaining phenotypic differences and prognosis of CCS.

Nod-Like Receptors in Host Defence and Disease at the Epidermal Barrier.

The nucleotide-binding domain and leucine-rich-repeat-containing family (NLRs) (sometimes called the NOD-like receptors, though the family contains few bona fide receptors) are a superfamily of multidomain-containing proteins that detect cellular stress and microbial infection. They constitute a critical arm of the innate immune response, though their functions are not restricted to pathogen recognition and members engage in controlling inflammasome activation, antigen-presentation, transcriptional regulation, cell death and also embryogenesis. NLRs are found from basal metazoans to plants, to zebrafish, mice and humans though functions of individual members can vary from species to species. NLRs also display highly wide-ranging tissue expression. Here, we discuss the importance of NLRs to the immune response at the epidermal barrier and summarise the known role of individual family members in the pathogenesis of skin disease.

Differential Inflammatory-Response Kinetics of Human Keratinocytes upon Cytosolic RNA- and DNA-Fragment Induction.

Keratinocytes are non-professional immune cells contributing actively to innate immune responses partially by reacting to a wide range of molecular patterns by activating pattern recognition receptors. Cytosolic nucleotide fragments as pathogen- or self-derived trigger factors are activating inflammasomes and inducing anti-viral signal transduction pathways as well as inducing expression of inflammatory cytokines. We aimed to compare the induced inflammatory reactions in three keratinocyte cell types-normal human epidermal keratinocytes, the HaCaT cell line and the HPV-KER cell line-upon exposure to the synthetic RNA and DNA analogues poly(I:C) and poly(dA:dT) to reveal the underlying signaling events. Both agents induced the expression of interleukin-6 and tumor necrosis factor α in all cell types; however, notable kinetic and expression level differences were found. Western blot analysis revealed rapid activation of the nuclear factor κB (NF-κB), mitogen activated protein kinase and signal transducers of activator of transcription (STAT) signal transduction pathways in keratinocytes upon poly(I:C) treatment, while poly(dA:dT) induced slower activation. Inhibition of NF-κB, p38, STAT-1 and STAT-3 signaling resulted in decreased cytokine expression, whereas inhibition of mitogen-activated protein kinase kinase 1/2 (MEK1/2) signaling showed a negative feedback role in both poly(I:C)- and poly(dA:dT)-induced cytokine expression. Based on our in vitro results nucleotide fragments are able to induce inflammatory reactions in keratinocytes, but with different rate and kinetics of cytokine expression, explained by faster activation of signaling routes by poly(I:C) than poly(dA:dT).

PRINS, a primate-specific long non-coding RNA, plays a role in the keratinocyte stress response and psoriasis pathogenesis.

In the last few years with the recent emergence of high-throughput technologies, thousands of long non-coding RNAs (lncRNAs) have been identified in the human genome. However, assigning functional annotation and determining cellular contexts for these RNAs are still in its infancy. As information gained about lncRNA structure, interacting partners, and roles in human diseases may be helpful in the characterization of novel lncRNAs, we review our knowledge on a selected group of lncRNAs that were identified serendipitously years ago by large-scale gene expression methods used to study human diseases. In particular, we focus on the Psoriasis-susceptibility-Related RNA Gene Induced by Stress (PRINS) lncRNA, first identified by our research group as a transcript highest expressed in psoriatic non-lesional epidermis. Results gathered for PRINS in the last 10 years indicate that it is conserved in primates and plays a role in keratinocyte stress response. Elevated levels of PRINS expression in psoriatic non-lesional keratinocytes alter the stress response of non-lesional epidermis and contribute to disease pathogenesis. Finally, we propose a categorization for the PRINS lncRNA based on a recently elaborated system for lncRNA classification.

[Telemedicine in dermatological practice: teledermatology]. / A telemedicina alkalmazása a borgyógyászatban: a teledermatológia.

Technological advances in the fields of information and telecommunication technologies have affected the health care system in the last decades, and lead to the emergence of a new discipline: telemedicine. The appearance and rise of internet and smart phones induced a rapid progression in telemedicine. Several new applications and mobile devices are published every hour even for medical purposes. Parallel to these changes in the technical fields, medical literature about telemedicine has grown rapidly. Due to its visual nature, dermatology is ideally suited to benefit from this new technology and teledermatology became one of the most dynamically evolving fields of telemedicine by now. Teledermatology is not routinely practiced in Hungary yet, however, it promises the health care system to become better, cheaper and faster, but we have to take notice on the experience and problems faced in teledermatologic applications so far, summarized in this review.

Stress-Related Regulation Is Abnormal in the Psoriatic Uninvolved Skin.

Keratinocyte stress-response of the uninvolved psoriatic epidermis is known to be altered compared to healthy cells. Therefore, we aimed to reveal potential mechanisms underlying this alteration. We compared the expression of annotated cell-stress-related proteins between uninvolved psoriatic and healthy skin using the protein array method. Data were analyzed by the Reactome over-representation test. We found that p27/CDKN1B and cytochrome C showed at least a two-fold increase, while cyclooxygenase-2, indolamine-2,3-dioxygenase-1, serum paraoxonase 1, serum paraoxonase 3, serine-46-phosphorylated tumor protein p53, and superoxide-dismutase-2 showed a two-fold decrease in expression in the uninvolved skin. Over-representation analysis suggested the Forkhead-box protein O (FOXO)-mediated transcription as the most significant pathway affected by the differently expressed cell-stress-related proteins (DECSRPs). DECSRPs indicate increased FOXO-mediated transcription of cell-cycle genes and reduced interleukin-signaling in the psoriatic uninvolved skin. Nuclear positivity of the FOXO-signaling-related p27/CDKN1B and FOXO1 are negatively correlated with the disease severity and showed increased expression in the uninvolved epidermis and also in healthy primary keratinocytes, which were grown on cartilage oligomeric matrix protein-coated surfaces. Our results indicate a cell-cycle inhibitory process, as a stress-related compensatory mechanism in the uninvolved epidermis, that could be responsible for blocking keratinocyte hyperproliferation in the psoriatic uninvolved skin, thus maintaining the symptomless skin phenotype.

The Psoriatic Nonlesional Skin: A Battlefield between Susceptibility and Protective Factors.

In the last two decades, large-scale gene-expression studies on psoriatic skin samples revealed that even though nonlesional skin is macroscopically identical to healthy skin, it harbors several molecular differences. Originally, these molecular differences were thought to represent susceptibility factors for plaque formation. However, we review in this paper the several factors of immune regulation and structural alteration that are specific for the nonlesional skin and serve as protective factors by counteracting plaque formation and contributing to the maintenance of the nonlesional phenotype.

Cartilage Oligomeric Matrix Protein Negatively Influences Keratinocyte Proliferation via α5ß1-Integrin: Potential Relevance of Altered Cartilage Oligomeric Matrix Protein Expression in Psoriasis.

In psoriasis, nonlesional skin shows alterations at the dermal-epidermal junction compared with healthy skin. Cartilage oligomeric matrix protein (COMP) is part of the papillary dermis of healthy skin, and its expression has not yet been studied in psoriatic skin. In this study, we found that COMP localization extended deeper into the dermis and formed a more continuous layer in psoriatic nonlesional skin compared with healthy skin, whereas in psoriatic lesions, COMP showed a partially discontinuous deposition at the dermal-epidermal junction. COMP and ß1-integrin showed strong colocalization in nonlesional skin, where the laminin layer within the basement membrane is discontinuous. In in vitro models, the presence of exogenous COMP decreased the proliferation rate of keratinocytes, and this proliferation-suppressing effect was diminished by blocking α5ß1-integrin. Our results suggest that COMP can interact with α5ß1-integrin of basal keratinocytes through the disrupted basement membrane, and this interaction might stabilize the epidermis in the nonlesional state by contributing to the suppression of keratinocyte proliferation. The antiproliferative effect of COMP is likely to be relevant to other skin diseases in which chronic nonhealing wounds are coupled with massive COMP accumulation.

Exosomal long non-coding RNAs as biomarkers in human diseases.

The intensive study of extracellular vesicles was started about a decade ago revealing alterations of their amount and content to several cellular stimuli, highly depending on the releasing cell type. Exosomes, a type of extracellular vesicles, are released by every cell type and are present in most body fluids, what makes them attractive targets of biomarker research. Several studies have indicated that their content - including proteins and coding, as well as non-coding nucleic acids - could represent the disease state and serves as specific disease biomarkers. Out of these molecules, a special interest was gained by long non-coding RNAs (lncRNAs). Just as exosomes, lncRNAs are specific to their cell of origin and often specific to diseases, also found extracellularly, mainly contained in extracellular vesicles. Thus, recent efforts in biomarker research has turned to circulating exosomal lncRNAs, which might lead to the development of highly specific disease markers. Here we summarize the current knowledge on disease-associated exosomal long non-coding RNAs. The intensive studies in this area have revealed numerous potential targets for biomarkers, and highlighted the potential of their combination with other exosomal markers to represent a highly sensitive and specific diagnostic tool. However, we believe that additional functional data on both exosomes and lncRNAs are necessary for understanding their deregulation in diseases and developing their use as diagnostic approaches.

Variable Ventilation Is Equally Effective as Conventional Pressure Control Ventilation for Optimizing Lung Function in a Rabbit Model of ARDS.

BACKGROUND: Introducing mathematically derived variability (MVV) into the otherwise monotonous conventional mechanical ventilation has been suggested to improve lung recruitment and gas exchange. Although the application of a ventilation pattern based on variations in physiological breathing (PVV) is beneficial for healthy lungs, its value in the presence of acute respiratory distress syndrome (ARDS) has not been characterized. We therefore aimed at comparing conventional pressure-controlled ventilation with (PCS) or without regular sighs (PCV) to MVV and PVV at two levels of positive end-expiratory pressure (PEEP) in a model of severe ARDS. METHODS: Anesthetised rabbits (n = 54) were mechanically ventilated and severe ARDS (PaO2/FiO2 ≤ 150 mmHg) was induced by combining whole lung lavage, i.v. endotoxin and injurious ventilation. Rabbits were then randomly assigned to be ventilated with PVV, MVV, PCV, or PCS for 5 h while maintaining either 6 or 9 cmH2O PEEP. Ventilation parameters, blood gas indices and respiratory mechanics (tissue damping, G, and elastance, H) were recorded hourly. Serum cytokine levels were assessed with ELISA and lung histology was analyzed. RESULTS: Although no progression of lung injury was observed after 5 h of ventilation at PEEP 6 cmH2O with PVV and PCV, values for G (58.8 ± 71.1[half-width of 95% CI]% and 40.8 ± 39.0%, respectively), H (54.5 ± 57.2%, 50.7 ± 28.3%), partial pressure of carbon-dioxide (PaCO2, 43.9 ± 23.8%, 46.2 ± 35.4%) and pH (-4.6 ± 3.3%, -4.6 ± 2.2%) worsened with PCS and MVV. Regardless of ventilation pattern, application of a higher PEEP improved lung function and precluded progression of lung injury and inflammation. Histology lung injury scores were elevated in all groups with no difference between groups at either PEEP level. CONCLUSION: At moderate PEEP, variable ventilation based on a pre-recorded physiological breathing pattern protected against progression of lung injury equally to the conventional pressure-controlled mode, whereas mathematical variability or application of regular sighs caused worsening in lung mechanics. This outcome may be related to the excessive increases in peak inspiratory pressure with the latter ventilation modes. However, a greater benefit on respiratory mechanics and gas exchange could be obtained by elevating PEEP, compared to the ventilation mode in severe ARDS.

Protective effects of glycerol and xylitol in keratinocytes exposed to hyperosmotic stress.

Purpose: Our goal was to study whether glycerol and xylitol provide protection against osmotic stress in keratinocytes. Methods: The experiments were performed on HaCaT keratinocytes. Hyperosmotic stress was induced by the addition of sorbitol (450, 500 and 600 mOsm). Both polyols were applied at two different concentrations (glycerol: 0.027% and 0.27%, xylitol: 0.045% and 0.45%). Cellular viability and cytotoxicity were assessed, intracellular Ca2+ concentration was measured, and the RNA expression of inflammatory cytokines was determined by means of PCR. Differences among groups were analyzed with one-way ANOVA and Holm-Sidak post-hoc test. When the normality test failed, Kruskal-Wallis one-way analysis of variance on ranks, followed by Dunn's method for pairwise multiple comparison was performed. Results: The higher concentrations of the polyols were effective. Glycerol ameliorated the cellular viability while xylitol prevented the rapid Ca2+ signal. Both polyols suppressed the expression of IL-1α but only glycerol decreased the expression of IL-1ß and NFAT5. Conclusions: Glycerol and xylitol protect keratinocytes against osmotic stress. Despite their similar chemical structure, the effect of these polyols displayed differences. Hence, joint application of glycerol and xylitol may be a useful therapeutic approach for different skin disorders.

Angiogenin mutations in Hungarian patients with amyotrophic lateral sclerosis: Clinical, genetic, computational, and functional analyses.

INTRODUCTION: Mutations in the angiogenin (ANG) gene are known to be associated with both familial and sporadic amyotrophic lateral sclerosis (ALS). The majority of disease-causing mutations of ANG result in loss of either ribonucleolytic activity, nuclear translocation activity or both. METHODS: We sequenced ANG gene from a total of 136 sporadic ALS patients and 112 healthy controls of Hungarian origin. To elucidate the role of the R33W mutation in the disease mechanism, computational, and functional analyses were performed. RESULTS: Mutation screening revealed a mutation located in the signal peptide (M-24I) and two mutations that affect the mature protein (R33W, V103I). The R33W mutation, which has not been previously detected in ALS patients, affects the key amino acid of the nuclear translocation signal of the ANG protein. Molecular dynamics simulations suggested that the R33W mutation results in partial loss of ribonucleolytic activity and reduced nuclear translocation activity. The ribonucleolytic assay and nuclear translocation assay of the R33W ANG protein confirmed the molecular dynamics results. CONCLUSIONS: In the Hungarian ALS population, the observed frequency of ANG mutations was 2.9%, which is higher than previously reported for sporadic cohorts. The evidence from computational and functional analyses support the deleterious effect of the novel R33W variant detected in this study.

Nuclear Factor κB Activation in a Type V Pityriasis Rubra Pilaris Patient Harboring Multiple CARD14 Variants.

Pityriasis rubra pilaris (PRP) is a rare papulosquamous skin disorder, which is phenotypically related to psoriasis. Some familial PRP cases show autosomal dominant inheritance due to CARD14 mutations leading to increased nuclear factor κB (NFκB) activation. Moreover, CARD14 polymorphisms have also been implicated in sporadic PRP. A Hungarian PRP patient with childhood onset disease showing worsening of the symptoms in adulthood with poor therapeutic response underwent genetic screening for the CARD14 gene, revealing four genetic variants (rs117918077, rs2066964, rs28674001, and rs11652075). To confirm that the identified genetic variants would result in altered NFκB activity in the patient, functional studies were carried out. Immunofluorescent staining of the NFκB p65 subunit and NFκB-luciferase reporter assay demonstrated significantly increased NFκB activity in skin samples and keratinocytes from the PRP patient compared to healthy samples. Characterization of the cytokine profile of the keratinocytes and peripheral blood mononuclear cells demonstrated that the higher NFκB activation in PRP cells induces enhanced responses to inflammatory stimuli. These higher inflammatory reactions could not be explained solely by the observed CARD14 or other inflammation-related gene variants (determined by whole exome sequencing). Thus our study indicates the importance of investigations on other genetic factors related to PRP and their further functional characterization to bring us closer to the understanding of cellular and molecular background of disease pathogenesis.

PRINS Non-Coding RNA Regulates Nucleic Acid-Induced Innate Immune Responses of Human Keratinocytes.

Cytosolic DNA fragments are recognized as pathogen- and danger-associated molecular patterns that induce a cascade of innate immune responses. Moreover, excessive cytosolic DNA can enhance chronic inflammation, predominantly by activating inflammasomes, and thereby contributing to the pathogenesis of chronic diseases, such as psoriasis. Psoriasis associated non-protein coding RNA induced by stress (PRINS) is a long non-coding RNA, which has been shown to be associated with psoriasis susceptibility and cellular stress responses; however, the precise mechanism of its action has not been determined. Here, we provide evidence that, in addition to inflammasome activation, cytosolic DNA induces intracellular inflammatory reactions while decreasing PRINS gene expression. Furthermore, PRINS overexpression can ameliorate the inflammatory-mediator production of keratinocytes induced by cytosolic DNA. Overexpression of PRINS resulted in decreased interleukin-6 (IL-6) and chemokine (C-C motif) ligand 5 (CCL-5) expression and secretion. In silico analysis predicted direct binding sites between PRINS and the mRNAs, which was confirmed by an in vitro binding assay and on cellular level. Our results indicated that PRINS binds directly to the mRNAs of IL-6 and CCL-5 at specific binding sites and eventually destabilizes these mRNAs, leading to a decrease in their expression and secretion of the corresponding proteins. These results may indicate a restrictive role for PRINS in inflammatory processes.

Keratinocytes express functional CARD18, a negative regulator of inflammasome activation, and its altered expression in psoriasis may contribute to disease pathogenesis.

Caspase recruitment domain family member 18 (CARD18, Iceberg) is known as a negative regulatory molecule that inhibits inflammatory events by terminating inflammasome activation due to a direct interaction with pro-caspase-1. During the investigation of molecular mechanisms in keratinocytes that contribute to the pathogenesis of psoriasis, we found that CARD18 expression differs in healthy and psoriatic skin; moreover, CARD18 demonstrated altered response under inflammatory conditions in healthy and psoriatic skin. In healthy skin, low basal CARD18 expression was detected, which showed significant elevation in response to inflammatory stimuli (lymphokine treatment or mechanical injury). In contrast, higher basal expression was observed in psoriatic non-involved skin, but no further induction could be detected. We demonstrated that keratinocytes express CARD18 both at mRNA and protein levels and the expression increased in parallel with differentiation. The investigation of cellular inflammatory processes revealed that psoriasis-associated danger signals triggered the expression of inflammasome components (AIM2, Caspase-1) and CARD18 as well as IL-1ß production of keratinocytes. Furthermore, gene-specific silencing of CARD18 in cells treated with cytosolic DNA (poly(dA:dT)) resulted in increased IL-1ß secretion, suggesting a negative regulatory role for CARD18 in keratinocyte inflammatory signaling. The differential regulation of CARD18 in healthy and psoriatic uninvolved epidermis may contribute to the susceptibility of psoriasis. Furthermore, our in vitro results indicate that CARD18 may contribute to the fine tuning of keratinocyte innate immune processes.