A novel de novo truncating variant in a Hungarian patient with CTNNB1 neurodevelopmental disorder.

PURPOSE: We aimed to elucidate the underlying disease in a Hungarian family, with only one affected family member, a 16-year-old male Hungarian patient, who developed global developmental delay, cognitive impairment, behavioral problems, short stature, intermittent headaches, recurrent dizziness, strabismus, hypermetropia, complex movement disorder and partial pituitary dysfunction. After years of detailed clinical investigations and careful pediatric care, the exact diagnosis of the patient and the cause of the disease was still unknown. METHODS: We aimed to perform whole exome sequencing (WES) in order to investigate whether the affected patient is suffering from a rare monogenic disease. RESULTS: Using WES, we identified a novel, de novo frameshift variant (c.1902dupG, p.Ala636SerfsTer12) of the catenin beta-1 (CTNNB1) gene. Assessment of the novel CTNNB1 variant suggested that it is a likely pathogenic one and raised the diagnosis of CTNNB1 neurodevelopmental disorder (OMIM 615,075). CONCLUSIONS: Our manuscript may contribute to the better understanding of the genetic background of the recently discovered CTNNB1 neurodevelopmental disorder and raise awareness among clinicians and geneticists. The affected Hungarian family demonstrates that based on the results of the clinical workup is difficult to establish the diagnosis and high-throughput genetic screening may help to solve these complex cases.

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.

Whole-Exome Sequencing Identified Two Novel Pathogenic Mutations in the PTCH1 Gene in BCNS.

Basal cell nevus syndrome (BCNS, OMIM 109400) is a familial cancer syndrome characterized by the development of numerous basal cell cancers and various other developmental abnormalities, including epidermal cysts of the skin, calcified dural folds, keratocysts of the jaw, palmar and plantar pits, ovarian fibromas, medulloblastomas, lymphomesenteric cysts, and fetal rhabdomyomas. BCNS shows autosomal dominant inheritance and is caused by mutations in the patched 1 (PTCH1) gene and the suppressor of the fused homolog (SUFU) gene. In a few cases, variants of patched 2 (PTCH2) have been found in patients who met the criteria for BCNS. In an investigation of 11 Hungarian families who fulfilled the diagnostic criteria for BCNS, whole-exome sequencing (WES) and multiplex ligation-dependent probe amplification (MLPA) identified two novel pathogenic variants (c.2994C>A; p.Cys998Ter and c.814_818del; p.Asn272SerfsTer11), one recently identified variant (c.1737_1745del p.Val580_Val582del), and three recurrent disease-causing variants of the PTCH1 gene with a diagnosis rate of 63.6%. Disease-causing variants were not found for the SUFU and PTCH2 genes. These applied methods could not fully elucidate the genetic background of all the BCNS cases that we investigated. To uncover the missing heritability of BCNS, whole-genome sequencing or an epigenetic approach might be considered in the future.

Genetic Screening of a Hungarian Cohort with Focal Dystonia Identified Several Novel Putative Pathogenic Gene Variants.

Dystonia is a rare movement disorder which is characterized by sustained or intermittent muscle contractions causing abnormal and often repetitive movements, postures, or both. The two most common forms of adult-onset focal dystonia are cervical dystonia (CD) and benign essential blepharospasm (BSP). A total of 121 patients (CD, 74; BSP, 47) were included in the study. The average age of the patients was 64 years. For the next-generation sequencing (NGS) approach, 30 genes were selected on the basis of a thorough search of the scientific literature. Assessment of 30 CD- and BSP-associated genes from 121 patients revealed a total of 209 different heterozygous variants in 24 genes. Established clinical and genetic validity was determined for nine heterozygous variations (three likely pathogenic and six variants of uncertain significance). Detailed genetic examination is an important part of the work-up for focal dystonia forms. To our knowledge, our investigation is the first such study to be carried out in the Middle-European region.

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.

Genetic Etiology of Nonsyndromic Hearing Loss in Hungarian Patients.

Hearing loss is the most prevalent sensory disorder worldwide. The majority of congenital nonsyndromic hearing loss (NSHL) cases are caused by hereditary factors. Previously, the majority of NSHL studies focused on the GJB2 gene; however, with the availability of next-generation sequencing (NGS) methods, the number of novel variants associated with NSHL has increased. The purpose of this study was to design effective genetic screening for a Hungarian population based on a pilot study with 139 NSHL patients. A stepwise, comprehensive genetic approach was developed, including bidirectional capillary sequencing, multiplex ligation-dependent probe amplification (MLPA), and an NGS panel of 108 hearing loss genes. With our results, a genetic diagnosis was possible for 92 patients. Sanger sequencing and MLPA identified the genetic background of 50% of these diagnosed cases, and the NGS panel identified another 16%. The vast majority (92%) of the diagnosed cases showed autosomal recessive inheritance and 76% were attributed to GJB2. The implementation of this stepwise analysis markedly increased our diagnostic yield and proved to be cost-effective as well.

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.

Could basement membrane alterations, resembling micro-wounds at the dermo-epidermal junction in psoriatic non-lesional skin, make the skin susceptible to lesion formation?

Current data suggest that tissue microenvironment control immune functions. Therefore, understanding the tissue environment in which immune activation occurs will enhance our capability to interfere with abnormal immune pathology. Here, we argue that studying the constitutively abnormal functions of clinically uninvolved psoriatic skin in patients with plaque type psoriasis is very important to better understand psoriasis pathobiology, because non-lesional skin provides the tissue environment in which the psoriatic lesion develops. A key question in psoriasis is what initiates the abnormal, uncontrolled immune activation in the first place and the answer may lie in the skin. In light of this concept, we summarize abnormalities at the dermal-epidermal junction region which shows a special "non-healing-like" micro-wound phenotype in the psoriatic non-lesional skin that may act as a crucial susceptibility factor in the development of the disease.

Passive Transfer of Blood Sera from ALS Patients with Identified Mutations Results in Elevated Motoneuronal Calcium Level and Loss of Motor Neurons in the Spinal Cord of Mice.

Introduction: Previously, we demonstrated the degeneration of axon terminals in mice after repeated injections of blood sera from amyotrophic lateral sclerosis (ALS) patients with identified mutations. However, whether a similar treatment affects the cell body of motor neurons (MNs) remained unresolved. Methods: Sera from healthy individuals or ALS patients with a mutation in different ALS-related genes were intraperitoneally injected into ten-week-old male Balb/c mice (n = 3/serum) for two days. Afterward, the perikaryal calcium level was measured using electron microscopy. Furthermore, the optical disector method was used to evaluate the number of lumbar MNs. Results: The cytoplasmic calcium level of the lumbar MNs of the ALS-serum-treated mice, compared to untreated and healthy-serum-treated controls, was significantly elevated. While injections of the healthy serum did not reduce the number of MNs compared to the untreated control group, ALS sera induced a remarkable loss of MNs. Discussion: Similarly to the distant motor axon terminals, the injection of blood sera of ALS patients has a rapid degenerative effect on MNs. Analogously, the magnitude of the evoked changes was specific to the type of mutation; furthermore, the degeneration was most pronounced in the group treated with sera from ALS patients with a mutation in the chromosome 9 open reading frame 72 gene.

[Consensus statement of the Hungarian Clinical Neurogenic Society about the therapy of adult SMA patients]. / A Magyar Klinikai Neurogenetikai Társaság konszenzusajánlása a felnottkori spinalis izomatrophia (SMA) kezeléséhez.

BACKGROUND AND PURPOSE: Background - Spinal muscular atrophy (SMA) is an autosomal recessive, progressive neuromuscular disorder resulting in a loss of lower motoneurons. Recently, new disease-modifying treatments (two drugs for splicing modification of SMN2 and one for SMN1 gene replacement) have become available. Purpose - The new drugs change the progression of SMA with neonatal and childhood onset. Increasing amount of data are available about the effects of these drugs in adult patients with SMA. In this article, we summarize the available data of new SMA therapies in adult patients. METHODS: Methods - Members of the Executive Committee of the Hungarian Clinical Neurogenetic Society surveyed the literature for palliative treatments, randomized controlled trials, and retrospective and prospective studies using disease modifying therapies in adult patients with SMA. Patients - We evaluated the outcomes of studies focused on treatments of adult patients mainly with SMA II and III. RESULTS: In this paper, we present our consensus statement in nine points covering palliative care, technical, medical and safety considerations, patient selection, and long-term monitoring of adult patients with SMA. CONCLUSION: This consensus statement aims to support the most efficient management of adult patients with SMA, and provides information about treatment efficacy and safety to be considered during personalized therapy. It also highlights open questions needed to be answered in future. Using this recommendation in clinical practice can result in optimization of therapy.

The association of HLA-C and ERAP1 polymorphisms in early and late onset psoriasis and psoriatic arthritis patients of Hungary.

INTRODUCTION: Single nucleotide polymorphisms (SNPs) of the HLA-C and ERAP1 genes were recently determined to contribute to psoriasis susceptibility. However, data regarding the association of these genes with specific subgroups of psoriasis are scarce. AIM: To examine the possible association of the HLA-C and ERAP-1 polymorphisms with early and late onset psoriasis and psoriatic arthritis. MATERIAL AND METHODS: Five ERAP1 SNPs and two HLA-C SNPs were genotyped in 105 psoriatic arthritis patients, 214 cutaneous psoriasis patients and 200 healthy individuals. Haplotypes were constructed for three ERAP1 SNPs (rs17482078, rs10050860, rs30187), and interaction between HLA-Cw*0602 and ERAP1 was also analysed. RESULTS: The HLA-Cw*0602 rs10484554 SNP was found to be a strong susceptibility factor for early onset cutaneous psoriasis and early onset psoriatic arthritis. ERAP1 SNPs (rs10050860, rs17482078, rs27525) appear to have a protective function for early onset psoriatic arthritis. The haplotype B was identified as a susceptibility factor for late onset psoriatic arthritis. In HLA-C positive individuals the rs27524 ERAP1 SNP was associated with a significantly increased risk of psoriatic arthritis development, whereas the rs27525 ERAP1 SNP had the opposite effect. CONCLUSIONS: These results suggest that the HLA-C and ERAP1 genes contribute to the pathogenesis of psoriasis and psoriatic arthritis in an age-dependent manner.

Identification of putative phenotype-modifying genetic factors associated with phenotypic diversity in Brooke-Spiegler syndrome.

Brooke-Spiegler syndrome (BSS, OMIM 605041) is a rare monogenic skin disease characterized by the development of skin appendage tumors caused by mutations in the cylindromatosis gene. We recently investigated a Hungarian and an Anglo-Saxon pedigrees affected by Brooke-Spiegler syndrome. Despite carrying the same disease-causing mutation (c.2806C>T, p.Arg936X) of the cylindromatosis (CYLD) gene, the affected family members of the two pedigrees exhibit striking differences in their phenotypes. To identify phenotype-modifying genetic factors, whole exome sequencing was performed and the data from the Hungarian and Anglo-Saxon BSS patients were compared. Three putative phenotype-modifying genetic variants were identified: the rs1053023 SNP of the signal transducer and activator of transcription 3 (STAT3) gene, the rs1131877 SNP of the tumor necrosis factor receptor-associated factor 3 (TRAF3) gene and the rs202122812 SNP of the neighbour of BRCA1 gene 1 (NBR1) gene. Our study contributes to the accumulating evidence for the clinical importance of phenotype-modifying genetic factors, which are potentially important for the elucidation of disease prognosis.

Differential role of D cyclins in the regulation of cell cycle by influencing Ki67 expression in HaCaT cells.

D-type cyclins are important regulatory proteins of the G1/S phase of the cell cycle however, their specific functions are only partially understood. We show that silencing of individual D-type cyclins has no effect on the proliferation and morphology of Immortalized non-tumorigenic human epidermal (HaCaT) cells, while double and triple D cyclin silencing results in the failure of the cytokinesis leading to the appearance of large multinucleated cells. Both CDC20 and Ki67 mRNA is downregulated in these cells. Ki67 mRNA silenced cells show similar multinucleated cellular phenotype as double or triple D cyclin silenced cells without affecting D cyclin expression, suggesting that Ki67 is necessary for normal G2/M transition. Our data have revealed that cyclin D1 may have a leading role in G1/S phase regulation and suggest an incomplete functional overlap among D cyclins. Our results indicate that beside their well-known functions during the G0-G1/S phase, D-type cyclins play a pivotal role in the regulation of mitosis via influencing Ki67 expression in a downstream manner probably through E2F1 activation in HaCaT cells.

Clinical and genetic differences between pustular psoriasis subtypes.

BACKGROUND: The term pustular psoriasis indicates a group of severe skin disorders characterized by eruptions of neutrophil-filled pustules. The disease, which often manifests with concurrent psoriasis vulgaris, can have an acute systemic (generalized pustular psoriasis [GPP]) or chronic localized (palmoplantar pustulosis [PPP] and acrodermatitis continua of Hallopeau [ACH]) presentation. Although mutations have been uncovered in IL36RN and AP1S3, the rarity of the disease has hindered the study of genotype-phenotype correlations. OBJECTIVE: We sought to characterize the clinical and genetic features of pustular psoriasis through the analysis of an extended patient cohort. METHODS: We ascertained a data set of unprecedented size, including 863 unrelated patients (251 with GPP, 560 with PPP, 28 with ACH, and 24 with multiple diagnoses). We undertook mutation screening in 473 cases. RESULTS: Psoriasis vulgaris concurrence was lowest in PPP (15.8% vs 54.4% in GPP and 46.2% in ACH, P < .0005 for both), whereas the mean age of onset was earliest in GPP (31.0 vs 43.7 years in PPP and 51.8 years in ACH, P < .0001 for both). The percentage of female patients was greater in PPP (77.0%) than in GPP (62.5%; P = 5.8 × 10-5). The same applied to the prevalence of smokers (79.8% vs 28.3%, P < 10-15). Although AP1S3 alleles had similar frequency (0.03-0.05) across disease subtypes, IL36RN mutations were less common in patients with PPP (0.03) than in those with GPP (0.19) and ACH (0.16; P = 1.9 × 10-14 and .002, respectively). Importantly, IL36RN disease alleles had a dose-dependent effect on age of onset in all forms of pustular psoriasis (P = .003). CONCLUSIONS: The analysis of an unparalleled resource revealed key clinical and genetic differences between patients with PPP and those with GPP.

Passive Transfer of Sera from ALS Patients with Identified Mutations Evokes an Increased Synaptic Vesicle Number and Elevation of Calcium Levels in Motor Axon Terminals, Similar to Sera from Sporadic Patients.

Previously, we demonstrated increased calcium levels and synaptic vesicle densities in the motor axon terminals (MATs) of sporadic amyotrophic lateral sclerosis (ALS) patients. Such alterations could be conferred to mice with an intraperitoneal injection of sera from these patients or with purified immunoglobulin G. Later, we confirmed the presence of similar alterations in the superoxide dismutase 1 G93A transgenic mouse strain model of familial ALS. These consistent observations suggested that calcium plays a central role in the pathomechanism of ALS. This may be further reinforced by completing a similar analytical study of the MATs of ALS patients with identified mutations. However, due to the low yield of muscle biopsy samples containing MATs, and the low incidence of ALS patients with the identified mutations, these examinations are not technically feasible. Alternatively, a passive transfer of sera from ALS patients with known mutations was used, and the MATs of the inoculated mice were tested for alterations in their calcium homeostasis and synaptic activity. Patients with 11 different ALS-related mutations participated in the study. Intraperitoneal injection of sera from these patients on two consecutive days resulted in elevated intracellular calcium levels and increased vesicle densities in the MATs of mice, which is comparable to the effect of the passive transfer from sporadic patients. Our results support the idea that the pathomechanism underlying the identical manifestation of the disease with or without identified mutations is based on a common final pathway, in which increasing calcium levels play a central role.

Consequences of cathepsin C inactivation for membrane exposure of proteinase 3, the target antigen in autoimmune vasculitis.

Membrane-bound proteinase 3 (PR3m) is the main target antigen of anti-neutrophil cytoplasmic autoantibodies (ANCA) in granulomatosis with polyangiitis, a systemic small-vessel vasculitis. Binding of ANCA to PR3m triggers neutrophil activation with the secretion of enzymatically active PR3 and related neutrophil serine proteases, thereby contributing to vascular damage. PR3 and related proteases are activated from pro-forms by the lysosomal cysteine protease cathepsin C (CatC) during neutrophil maturation. We hypothesized that pharmacological inhibition of CatC provides an effective measure to reduce PR3m and therefore has implications as a novel therapeutic approach in granulomatosis with polyangiitis. We first studied neutrophilic PR3 from 24 patients with Papillon-Lefèvre syndrome (PLS), a genetic form of CatC deficiency. PLS neutrophil lysates showed a largely reduced but still detectable (0.5-4%) PR3 activity when compared with healthy control cells. Despite extremely low levels of cellular PR3, the amount of constitutive PR3m expressed on the surface of quiescent neutrophils and the typical bimodal membrane distribution pattern were similar to what was observed in healthy neutrophils. However, following cell activation, there was no significant increase in the total amount of PR3m on PLS neutrophils, whereas the total amount of PR3m on healthy neutrophils was significantly increased. We then explored the effect of pharmacological CatC inhibition on PR3 stability in normal neutrophils using a potent cell-permeable CatC inhibitor and a CD34+ hematopoietic stem cell model. Human CD34+ hematopoietic stem cells were treated with the inhibitor during neutrophil differentiation over 10 days. We observed strong reductions in PR3m, cellular PR3 protein, and proteolytic PR3 activity, whereas neutrophil differentiation was not compromised.

Genetic investigation confirmed the clinical phenotype of congenital chloride diarrhea in a Hungarian patient: a case report.

BACKGROUND: Congenital chloride diarrhea (CCD, OMIM 214700) is a rare autosomal recessively inherited condition characterized by watery diarrhea, hypochloremia and metabolic alkalosis. Mutations of the solute carrier family 26, member 3 (SLC26A3, OMIM 126650) gene are responsible for the disease. The gene encodes a transmembrane protein, which is essential for intestinal chloride absorption. CASE PRESENTATION: Here we report a Hungarian boy, presenting the clinical phenotype of CCD. The patient born at 32 weeks of gestation and underwent surgery for abdominal distension and intestinal obstruction related to malrotation. After recovery, electrolyte replacement therapy was necessary due to several periods of diarrhea. After exclusion of other possible causes, increased chloride concentration in the feces supported the diagnosis of CCD. The diagnosis was confirmed by molecular genetic testing. Direct sequencing revealed compound-heterozygosity for a frameshift mutation c.1295delT (p.Leu432Argfs*11) and the known Polish founder mutation c.2024_2026dupTCA (p.Ile675_Arg676insLeu). CONCLUSIONS: Here we present the clinical symptoms of the first patient in Hungary diagnosed with CCD. Based on the clinical symptoms, stool analysis and genetic testing, the diagnosis of CCD was established. Our study provides expansion for the mutation spectrum of the SLC26A3 gene and the genetic background of CCD.

Experimental Motor Neuron Disease Induced in Mice with Long-Term Repeated Intraperitoneal Injections of Serum from ALS Patients.

In an earlier study, signs of commencing degeneration of spinal motor neurons were induced in mice with short-term intraperitoneal injections of immunoglobulin G (IgG) taken from patients with amyotrophic lateral sclerosis (ALS). Since in that study, neither weakness nor loss of motor neurons was noted, to test whether the ALS IgG in this paradigm has the potential to evoke relentless degeneration of motor neurons, treatment with repeated injections over a longer period was carried out. Mice were systematically injected intraperitoneally with serum taken from ALS patients over a 75-day period. At selected time points, the isometric force of the limbs, number of spinal motor neurons and their intracellular calcium levels were determined. Furthermore, markers of glial activation and the motoneuronal uptake of human IgG were monitored. During this period, gliosis and progressive motoneuronal degeneration developed, which led to gradual loss of spinal motor neurons, more than 40% at day 21, along with decreasing muscle strength in the limbs. The inclusion-like accumulation of IgG appeared in the perikarya with the increase of intracellular calcium in the cell bodies and motor nerve terminals. Our results demonstrate that ALS serum can transfer motor neuron disease to mice.

Analysis of psoriasis-relevant gene expression and exon usage alterations after silencing of SR-rich splicing regulators.

In our recent cDNA microarray experiment, three SR-rich splicing factors-SFRS18, PPIG and LUC7L3-were shown to exert altered responsiveness upon T-lymphokine stimulation of psoriatic non-involved and healthy epidermis samples. We have also demonstrated that double silencing LUC7L3 and SFRS18 efficiently decreased production of the psoriasis-associated EDA+ fibronectin isoform. These findings prompted the further investigation of signalling pathways affected by LUC7L3 and SFRS18. To detect gene expression and splicing pattern alterations upon double silencing of LUC7L3 and SFRS18 in an HPV-immortalised keratinocyte cell culture, paired-end RNA sequencing was carried out. Marked changes in exon usage were revealed, in contrast to the modest alterations detected in gene expression, providing a closer delineation of the potential targets of the examined splicing factors. The most prominent gene expression change was detected for IFI6, an interferon-inducible gene highly expressed in psoriasis. Interacting partners of IFI6 and certain psoriasis-associated transcripts also exhibited significantly increased expression upon silencing. In addition to elevated abundance of the EDA+ fibronectin interactor ITGA5, we confirmed decreased EDA domain inclusion, which agrees well with our prior experimental data. Furthermore, differential exon usage was established for the transcription element CREB1, along with HERC6 and CUL1, which are implicated in ubiquitination. Although immortalised keratinocytes express low levels of TINCR, a long non-coding RNA involved in terminal differentiation of keratinocytes, splicing alterations were successfully demonstrated for this RNA as well. We believe that the targeted investigation of mRNA maturation disturbances may help us gain deeper insight into the molecular pathogenesis of psoriasis.