BACKGROUND:CYLD cutaneous syndrome (CCS) skin tumours develop from puberty onwards, can number in the hundreds and progressively grow over time. Patients with CCS lack medical therapies and require repeated surgery to control tumour burden. CYLD loss of heterozygosity drives tumour growth, and CCS tumours have previously been shown to demonstrate increased canonical nuclear factor kappa B (NF-κB) and Wnt signalling. OBJECTIVES:To investigate NF-κB signalling in CCS tumours and CCS tumour keratinocytes, with the aim of identifying druggable targets. METHODS:We used complementary bulk transcriptomics and proteomics in patient-derived CCS tumour cell fractions, as well as single-cell RNA sequencing of CCS tumour cells to investigate aberrant NF-κB signalling. We developed a patient-derived CCS tumour spheroid culture model to determine the utility of targeting aberrant NF-κB cell signalling. RESULTS:We demonstrate evidence of non-canonical NF-κB signalling in CCS tumour keratinocytes, with increased p100 to p52 processing and RelB protein expression compared with normal skin. We identify IκB kinase alpha (IKKα) as a candidate target in the noncanonical NF-κB signalling pathway. A novel, highly selective IKKα inhibitor (SU1644) used in patient-derived CCS tumour spheroid cultures demonstrated that IKKα inhibition reduced tumour spheroid viability. CONCLUSIONS:These data provide the preclinical rationale for the assessment of topical IKKα inhibitors as a novel preventive treatment for CCS.

Chemokines play an important role in the pathogenesis of skin diseases, such as psoriasis, atopic dermatitis, vitiligo, and alopecia areata. Recently literature data supports the theory that alternatively spliced isoforms of these molecules may serve as potential regulators in these diseases. Since self-derived nucleic acids are main culprits in chronic skin diseases we compared the effects of synthetic RNA- and DNA-induced inflammation on the expression levels of chemokines in human keratinocytes. We found that cytoplasmic nucleic acids are potent inducers of monocyte chemoattractant protein-1 (CCL2), interferon gamma inducible protein-10 (CXCL10) and fractalkine (CX3CL1) mRNA-expression, mainly through NF-κB activation, but the pattern recognition receptors responsible for inducing this activation are still unknown. Alternative splicing of these chemokines in keratinocytes was not detected, suggesting other regulatory mechanisms for chemokine activity.

Circadian clocks rely on transcriptional/translational feedback loops involving clock genes and their corresponding proteins. While the primary oscillations originate from gene expression, the precise control of clock protein stability plays a pivotal role in establishing the 24-hour circadian rhythms. Most clock proteins are degraded through the ubiquitin/26S proteasome pathway, yet the enzymes responsible for ubiquitination and deubiquitination remain poorly characterised. We identified a missense allele (ubp12-3, S327F) of the UBP12 gene/protein in Arabidopsis. Despite ubp12-3 exhibited a short period phenotype similar to that of a loss-of-function allele, molecular analysis indicated elevated protease activity in ubp12-3. We demonstrated that early flowering of ubp12 mutants is a result of the shortened circadian period rather than a direct alteration of UBP12 function. Analysis of protease activity of non-phosphorylatable (S327A, S327F) and phosphomimetic (S327D) derivatives in bacteria suggested that phosphorylation of serine 327 inhibits UBP12 enzymatic activity, which could explain the over-functioning of S327F in vivo. We showed that phosphomimetic mutations of the conserved serine in the Neurospora and human orthologues reduced ubiquitin cleavage activity suggesting that not only the primary structures of UBP12-like enzymes are phylogenetically conserved across a wide range of species, but also the molecular mechanisms governing their enzymatic activity.

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.

Circadian clocks are biochemical timers regulating many physiological and molecular processes according to the day/night cycles. The function of the oscillator relies on negative transcriptional/translational feedback loops operated by the so-called clock genes and the encoded clock proteins. Previously, we identified the small GTPase LIGHT INSENSITIVE PERIOD 1 (LIP1) as a circadian-clock-associated protein that regulates light input to the clock in the model plant Arabidopsis thaliana. We showed that LIP1 is also required for suppressing red and blue light-mediated photomorphogenesis, pavement cell shape determination and tolerance to salt stress. Here, we demonstrate that LIP1 is present in a complex of clock proteins GIGANTEA (GI), ZEITLUPE (ZTL) and TIMING OF CAB 1 (TOC1). LIP1 participates in this complex via GUANINE EX-CHANGE FACTOR 7. Analysis of genetic interactions proved that LIP1 affects the oscillator via modulating the function of GI. We show that LIP1 and GI independently and additively regulate photomorphogenesis and salt stress responses, whereas controlling cell shape and photoperiodic flowering are not shared functions of LIP1 and GI. Collectively, our results suggest that LIP1 affects a specific function of GI, possibly by altering binding of GI to downstream signalling components.

Light affects almost every aspect of plant development. It is perceived by photoreceptors, among which phytochromes (PHY) are responsible for monitoring the red and far-red spectrum. Arabidopsis thaliana possesses five phytochrome genes (phyA-phyE). Whereas functions of phyA and phyB are extensively studied, our knowledge of other phytochromes is still rudimentary. To analyze phyD function, we expressed it at high levels in different phytochrome-deficient genetic backgrounds. Overexpressed phyD-YFP can govern effective light signaling but only at low temperatures and in cooperation with functional phyC. Under these conditions, phyD-YFP accumulates to high levels, and opposite to phyB, this pool is stable in light. By comparing the photoconvertible phyD-YFP and phyB levels and their signaling in continuous and pulsed irradiation, we showed that phyD-YFP is a less efficient photoreceptor than phyB. This conclusion is supported by the facts that only a part of the phyD-YFP pool is photoconvertible and that thermal reversion of phyD-YFP is faster than that of phyB. Our data suggest that the temperature-dependent function of phyD is based on the amount of phyD protein and not on its Pfr stability, as described for phyB. We also found that phyD-YFP and phyB-GFP are associated with strongly overlapping genomic locations and are able to mediate similar changes in gene expression; however, the efficiency of phyD-YFP is lower. Based on these data, we propose that under certain conditions, synergistic interaction of phyD and phyC can substitute phyB function in seedlings and in adult plants and thus increases the ability of plants to respond more flexibly to environmental changes.

The research of the genodermatoses began with the support of Professor Lajos Kemény at the Department of Dermatology and Allergology, University of Szeged (Szeged, Hungary) in 2010. Over the years, this research topic has become an increasingly complex one. Our results have been summarized in 28 scientific publications and in 5 PhD and 1 DSc degrees. We are thankful and grateful to Professor Lajos Kemény for supporting and participating in such a high quality and complex research. We hope that our results contribute to the better understanding of monogenic skin diseases, in exploring genotype-phenotype correlations and in clarifying disease mechanisms and in providing solid basis for the development of novel therapeutic modalities.

The skin tumor syndrome, CYLD cutaneous syndrome (CCS) is featured by the development of multiple variable adnexal tumors. CCS develops as a consequence of the pathogenic variants of a tumor suppressor gene, cylindromatosis (CYLD) encoding a deubiquitinase enzyme known as a negative regulator of the NF-κB signaling. Nonsense mutations of the CYLD gene cause the largest phenotypic diversity of the disease and nearly half of them (i.e. 40%) are recurrent. To elucidate the molecular and cellular consequences of three pathogenic variants of the CYLD protein, an in vitro experimental system was used. The recurrent CYLD(Arg936X) and CYLD(Arg758X) truncated derivatives and the novel CYLD(Tyr602X) mutant variant identified by our group in a Hungarian CCS patient were investigated. In silico and in vitro functional analyses were performed to compare the structure, stability and signaling of these truncated CYLD variants. CYLD(Arg936X) had the most profound effect on NF-κB activity. The other two protein variants showed altered stability, the CYLD(Arg758X) being an extremely unstable, while the CYLD(Tyr602X) being the most stable variant, both showing minor effects on NF-κB activity. Our results correlate with the phenotype spectrum caused by the three investigated CYLD mutations. Thus, we conclude that they might contribute to the development of CCS with distinct mechanisms.

Psoriasis is a multifactorial skin disease, caused by the disturbed homeostasis between the innate and adaptive immune cells, consequently leading to hyperproliferation of keratinocytes. 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 fractalkine (CX3CL1), a chemokine recruiting leukocytes. Our recent large-scale study showed that RNA- and DNA-fragments induce significant transcriptional changes in CX3CL1 expression, thus we aimed to identify the molecular background behind its expression. To induce inflammatory processes, normal human epidermal keratinocytes were transiently transfected with synthetic dsDNA analogue poly(dA:dT) and dsRNA analogue poly(I:C) or were treated with psoriasis-related cytokines. The role of pattern recognition receptors was studied by siRNA-based silencing, signaling pathways were studied by specific inhibitors, while CX3CL1 mRNA expression was monitored by RT-qPCR. Nucleic acid analogues caused elevated CX3CL1 mRNA expression, but to different degree, however cytokine treatments had no effect on it. According to our results, RIG-I, IFIH-1 and cGAS receptors play a role in nucleic-acid induced CX3CL1 expression, through activating p38- and STAT-pathways. Based on the NCBI Database CX3CL1 has two splice variants with the presence (full-length form) or absence of its chemokine motive, possibly altering its chemoattractant properties. We studied the expression of both splice-variants by PCR, using several primer pair combinations, but only the expression of the full-length variant could be verified in keratinocytes. Our results suggest that nucleic acids induce epithelial CX3CL1 production, possibly contributing to the recruitment of professional immune cells into the skin. Although databases predict the existence of multiple splice-variants with different functions, the expression of only the full-length CX3CL1 could be confirmed in keratinocytes.

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.

Skin adnexal tumors develop as a consequence of germline pathogenic variants in the cylindomatosis(CYLD) gene in patients with Brooke-Spiegler syndrome (BSS), a clinical variant of the CYLD cutaneous syndrome. The first tumors typically present at puberty and they progressively accumulate through adulthood. To improve understanding of the development of the skin tumors, we performed here the first genome-wide methylation analysis on blood DNA samples of 16 BSS patients and 6 controls using Infinium Methylation EPIC arrays. Among the observed DNA methylation alterations, we detected significant methylation differences in the BAG6 gene of BSS patients, which encodes a negative regulator of the LPS-induced NF-κB signaling through the CYLD interactor TRAF6 protein. Gene expression analysis demonstrated significantly altered BAG6 mRNA expression in the investigated patients. Our results raise the possibility that the altered methylation pattern and expression of BAG6 contribute to BSS pathogenesis via altering skin cell inducibility by microbial compounds.

Albinism follows recessive mode of inheritance and 20 disease-causing genes were implicated in its development. Using 20 gene panel, the genetic background of 11 out of 17 Hungarian patients were elucidated. In patients with unidentified genetic background (n=6) whole exome sequencing was performed. A novel pathogenic mutation (N687S) of the two pore channel two gene (TPCN2)was identified in a 15-year-old patient confirming the dominant type of albinism. Our discovery together with the recently described first Chinese case revolutionizes our thinking about albinism, which has been so far known as a disorder with exclusively recessive inheritance and emphasize that from now either recessive or dominant inheritance should be considered in the everyday clinical practice.

The human skin is a tightly-organized ecosystem composed of a dense network of thick structural appendices surrounding naturally-resident and recruited immune cells. Routine clinical assays, such as conventional immunohistochemistry, fail to resolve the regional heterogeneity and immune topology of inflammatory skin conditions, which can lead to false interpretation. Here we introduce MANTIS (Multiplexed Annotated Tissue Imaging System), a flexible analytical system specifically-designed for spatially-resolved immune-phenotyping of the skin in thick experimental samples or large clinical cohorts. Combining multiplexed 3-D imaging, machine learning and unsupervised bioinformatics, MANTIS automatically projects a representative digital immune landscape, while enabling interactive gating of anatomical regions and concomitant single-cell data quantification of biomarkers. We leverage these properties to analyze skin biopsies from healthy and pathological conditions.

CYLD cutaneous syndrome (CCS) is a rare monogenic skin disease characterized by the development of skin appendage tumors caused by mutations in the cylindromatosis (CYLD) gene. Recently described Hungarian and Anglo-Saxon pedigrees that are affected by CCS carry the same disease-causing truncating mutation of the CYLD gene (Arg936X) 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 have a more severe phenotype that are not present in the Anglo-Saxon pedigree. Our in vitro functional study revealed that the combined expression of mutant CYLD (Arg936X) with TRAF3 and NBR1 caused increased NF-kB activity, regardless of the presence or absence of mutations in TRAF3 and NBR1. To generalize this we broadened our investigations and included a newly identified (Tyr602X) and already described (Arg758X and Arg936X) disease causing CYLD mutations leading to the formation of premature stop codons causing truncations with different length of the ubiquitin-specific protease (USP) catalytic domain of the CYLD protein. The effect of these nonsense mutations on NF-kB activity in HEK293 cells as a consequence of the dysfunction of the CYLD protein was compared. Contrary to our hypothesis, truncated CYLD protein which lacks the entire USP domain caused no elevated NF-kB activation in our assay as compared to the effect of the overexpression of wt CYLD. We assume that the truncated CYLD proteins have altered characteristics (structure and interaction with partner molecules) that would explain our findings. To improve understanding, we have commenced in silico molecule structure analysis of the truncated proteins.

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.

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. To generalize this concept we broadened our investigations and included other – newly identified and already described – disease causing CYLD mutations and are currently working on clarifying their effect together with elevated TRAF3 and NBR1 levels on NF-κB activity.