P2RX7 gene variants associate with altered inflammasome assembly and reduced pyroptosis in chronic nonbacterial osteomyelitis (CNO).

Chronic nonbacterial osteomyelitis (CNO), an autoinflammatory bone disease primarily affecting children, can cause pain, hyperostosis and fractures, affecting quality-of-life and psychomotor development. This study investigated CNO-associated variants in P2RX7, encoding for the ATP-dependent trans-membrane K+ channel P2X7, and their effects on NLRP3 inflammasome assembly. Whole exome sequencing in two related transgenerational CNO patients, and target sequencing of P2RX7 in a large CNO cohort (N = 190) were conducted. Results were compared with publicly available datasets and regional controls (N = 1873). Findings were integrated with demographic and clinical data. Patient-derived monocytes and genetically modified THP-1 cells were used to investigate potassium flux, inflammasome assembly, pyroptosis, and cytokine release. Rare presumably damaging P2RX7 variants were identified in two related CNO patients. Targeted P2RX7 sequencing identified 62 CNO patients with rare variants (32.4%), 11 of which (5.8%) carried presumably damaging variants (MAF <1%, SIFT "deleterious", Polyphen "probably damaging", CADD >20). This compared to 83 of 1873 controls (4.4%), 36 with rare and presumably damaging variants (1.9%). Across the CNO cohort, rare variants unique to one (Median: 42 versus 3.7) or more (≤11 patients) participants were over-represented when compared to 190 randomly selected controls. Patients with rare damaging variants more frequently experienced gastrointestinal symptoms and lymphadenopathy while having less spinal, joint and skin involvement (psoriasis). Monocyte-derived macrophages from patients, and genetically modified THP-1-derived macrophages reconstituted with CNO-associated P2RX7 variants exhibited altered potassium flux, inflammasome assembly, IL-1ß and IL-18 release, and pyroptosis. Damaging P2RX7 variants occur in a small subset of CNO patients, and rare P2RX7 variants may represent a CNO risk factor. Observations argue for inflammasome inhibition and/or cytokine blockade and may allow future patient stratification and individualized care.

Loss-of-Function Myeloperoxidase Mutations Are Associated with Increased Neutrophil Counts and Pustular Skin Disease.

The identification of disease alleles underlying human autoinflammatory diseases can provide important insights into the mechanisms that maintain neutrophil homeostasis. Here, we focused our attention on generalized pustular psoriasis (GPP), a potentially life-threatening disorder presenting with cutaneous and systemic neutrophilia. Following the whole-exome sequencing of 19 unrelated affected individuals, we identified a subject harboring a homozygous splice-site mutation (c.2031-2A>C) in MPO. This encodes myeloperoxidase, an essential component of neutrophil azurophil granules. MPO screening in conditions phenotypically related to GPP uncovered further disease alleles in one subject with acral pustular psoriasis (c.2031-2A>C;c.2031-2A>C) and in two individuals with acute generalized exanthematous pustulosis (c.1705C>T;c.2031-2A>C and c.1552_1565del;c.1552_1565del). A subsequent analysis of UK Biobank data demonstrated that the c.2031-2A>C and c.1705C>T (p.Arg569Trp) disease alleles were also associated with increased neutrophil abundance in the general population (p = 5.1 × 10-6 and p = 3.6 × 10-5, respectively). The same applied to three further deleterious variants that had been genotyped in the cohort, with two alleles (c.995C>T [p.Ala332Val] and c.752T>C [p.Met251Thr]) yielding p values < 10-10. Finally, treatment of healthy neutrophils with an MPO inhibitor (4-Aminobenzoic acid hydrazide) increased cell viability and delayed apoptosis, highlighting a mechanism whereby MPO mutations affect granulocyte numbers. These findings identify MPO as a genetic determinant of pustular skin disease and neutrophil abundance. Given the recent interest in the development of MPO antagonists for the treatment of neurodegenerative disease, our results also suggest that the pro-inflammatory effects of these agents should be closely monitored.

Myeloperoxidase Modulates Inflammation in Generalized Pustular Psoriasis and Additional Rare Pustular Skin Diseases.

Generalized pustular psoriasis (GPP) is a severe multi-systemic inflammatory disease characterized by neutrophilic pustulosis and triggered by pro-inflammatory IL-36 cytokines in skin. While 19%-41% of affected individuals harbor bi-allelic mutations in IL36RN, the genetic cause is not known in most cases. To identify and characterize new pathways involved in the pathogenesis of GPP, we performed whole-exome sequencing in 31 individuals with GPP and demonstrated effects of mutations in MPO encoding the neutrophilic enzyme myeloperoxidase (MPO). We discovered eight MPO mutations resulting in MPO -deficiency in neutrophils and monocytes. MPO mutations, primarily those resulting in complete MPO deficiency, cumulatively associated with GPP (p = 1.85E-08; OR = 6.47). The number of mutant MPO alleles significantly differed between 82 affected individuals and >4,900 control subjects (p = 1.04E-09); this effect was stronger when including IL36RN mutations (1.48E-13) and correlated with a younger age of onset (p = 0.0018). The activity of four proteases, previously implicated as activating enzymes of IL-36 precursors, correlated with MPO deficiency. Phorbol-myristate-acetate-induced formation of neutrophil extracellular traps (NETs) was reduced in affected cells (p = 0.015), and phagocytosis assays in MPO-deficient mice and human cells revealed altered neutrophil function and impaired clearance of neutrophils by monocytes (efferocytosis) allowing prolonged neutrophil persistence in inflammatory skin. MPO mutations contribute significantly to GPP's pathogenesis. We implicate MPO as an inflammatory modulator in humans that regulates protease activity and NET formation and modifies efferocytosis. Our findings indicate possible implications for the application of MPO inhibitors in cardiovascular diseases. MPO and affected pathways represent attractive targets for inducing resolution of inflammation in neutrophil-mediated skin diseases.

VEXAS-Syndrome, a newly described autoinflammatory systemic disease with dermatologic manifestations.

VEXAS syndrome is a recently identified autoinflammatory systemic disease caused by an acquired somatic mutation of the X-linked UBA1 gene, the key enzyme of the first step of ubiquitylation. The acronym VEXAS stands for the characteristics Vacuoles, E1 enzyme, X-linked, autoinflammatory and somatic. The disease occurs in advanced adulthood preferentially in men and is characterized by hematological, rheumatological and dermatological symptoms. The latter include neutrophil-rich lesions reminiscent of Sweet's syndrome, erythema nodosum- and panniculitis-like skin manifestations and recurrent polychondritis of the nose and auricles. The presence of cytoplasmic vacuoles in myeloid and erythroid precursors in the bone marrow is characteristic. In up to half of the cases, VEXAS syndrome is associated with myelodysplastic syndrome. Dermatologists should be familiar with the clinical picture, as skin symptoms are often the first indicator of the disease. Molecular diagnostics are essential for confirming the diagnosis and risk stratification of affected patients. In this minireview we provide an overview of the pathophysiology, diagnosis and therapy of VEXAS syndrome and illustrate its clinical picture with two own cases.

ZMYND11 variants are a novel cause of centrotemporal and generalised epilepsies with neurodevelopmental disorder.

ZMYND11 is the critical gene in chromosome 10p15.3 microdeletion syndrome, a syndromic cause of intellectual disability. The phenotype of ZMYND11 variants has recently been extended to autism and seizures. We expand on the epilepsy phenotype of 20 individuals with pathogenic variants in ZMYND11. We obtained clinical descriptions of 16 new and nine published individuals, plus detailed case history of two children. New individuals were identified through GeneMatcher, ClinVar and the European Network for Therapies in Rare Epilepsy (NETRE). Genetic evaluation was performed using gene panels or exome sequencing; variants were classified using American College of Medical Genetics (ACMG) criteria. Individuals with ZMYND11 associated epilepsy fell into three groups: (i) atypical benign partial epilepsy or idiopathic focal epilepsy (n = 8); (ii) generalised epilepsies/infantile epileptic encephalopathy (n = 4); (iii) unclassified (n = 8). Seizure prognosis ranged from spontaneous remission to drug resistant. Neurodevelopmental deficits were invariable. Dysmorphic features were variable. Variants were distributed across the gene and mostly de novo with no precise genotype-phenotype correlation. ZMYND11 is one of a small group of chromatin reader genes associated in the pathogenesis of epilepsy, and specifically ABPE. More detailed epilepsy descriptions of larger cohorts and functional studies might reveal genotype-phenotype correlation. The epileptogenic mechanism may be linked to interaction with histone H3.3.

Genetic variants in FBLIM1 gene do not contribute to SAPHO syndrome and chronic recurrent multifocal osteomyelitis in typical patient groups.

BACKGROUND: Syndrome of synovitis acne pustulosis hyperostosis osteitis (SAPHO) and chronic recurrent multifocal osteomyelitis (CRMO) present two diseases of a dermatologic and rheumatologic spectrum that are variable in manifestation und therapeutic response. Genetic risk factors have long been assumed in both diseases, but no single reliable factor has been identified yet. Therefore, we aimed to clinically characterize a patient group with syndrome of synovitis acne pustulosis hyperostosis osteitis (SAPHO) (n = 47) and chronic recurrent multifocal osteomyelitis (CRMO)/ chronic non-bacterial osteomyelitis (CNO) (n = 9) and analyze a CRMO candidate gene. METHODS: Clinical data of all patients were collected and assessed for different combinations of clinical symptoms. SAPHO patients were grouped into categories according to the acronym; disease-contribution by pathogens was evaluated. We sequenced coding exons of FBLIM1. RESULTS: Palmoplantar pustular psoriasis (PPP) was the most common skin manifestation in CRMO/CNO and SAPHO patients; most SAPHO patients had sterno-costo-clavicular hyperostosis. The most common clinical category of the acronym was S_PHO (n = 26). Lack of pathogen detection from bone biopsies was more common than microbial isolation. We did not identify autosomal-recessive FBLIM1 variants. CONCLUSIONS: S_PHO is the most common combination of symptoms of its acronym. Genetic analyses of FBLIM1 did not provide evidence that this gene is relevant in our patient group. Our study indicates the need to elucidate SAPHO's and CRMO/CNO's pathogenesis.

Exome-wide association study reveals novel psoriasis susceptibility locus at TNFSF15 and rare protective alleles in genes contributing to type I IFN signalling.

Psoriasis is a common inflammatory skin disorder for which multiple genetic susceptibility loci have been identified, but few resolved to specific functional variants. In this study, we sought to identify common and rare psoriasis-associated gene-centric variation. Using exome arrays we genotyped four independent cohorts, totalling 11 861 psoriasis cases and 28 610 controls, aggregating the dataset through statistical meta-analysis. Single variant analysis detected a previously unreported risk locus at TNFSF15 (rs6478108; P = 1.50 × 10-8, OR = 1.10), and association of common protein-altering variants at 11 loci previously implicated in psoriasis susceptibility. We validate previous reports of protective low-frequency protein-altering variants within IFIH1 (encoding an innate antiviral receptor) and TYK2 (encoding a Janus kinase), in each case establishing a further series of protective rare variants (minor allele frequency < 0.01) via gene-wide aggregation testing (IFIH1: pburden = 2.53 × 10-7, OR = 0.707; TYK2: pburden = 6.17 × 10-4, OR = 0.744). Both genes play significant roles in type I interferon (IFN) production and signalling. Several of the protective rare and low-frequency variants in IFIH1 and TYK2 disrupt conserved protein domains, highlighting potential mechanisms through which their effect may be exerted.

Genome-wide association and targeted analysis of copy number variants with psoriatic arthritis in German patients.

BACKGROUND: Psoriatic Arthritis (PsA) is a chronic inflammatory disease of the joints. PsA is etiologically complex, and 11 susceptibility loci have been identified so far. Most of these overlap with loci associated with psoriasis vulgaris (PsV), the most common psoriatic skin manifestation which is also frequently seen in PsA patients. In addition, two copy number variants (CNVs) are associated with PsV, one of which, located within the LCE3 gene cluster, is also associated with PsA. Finally, an intergenic deletion has been reported as a PsA-specific CNV. METHODS: We performed a genome-wide association study (GWAS) of CNVs in PsA and assessed the contribution to disease risk by CNVs at known psoriasis susceptibility loci. RESULTS: After stringent quality assessment and validation of CNVs of the GWAS with an alternative quantitative method, two significantly associated CNVs remained, one near UXS1, the other one at the TRB locus. However, MLPA analysis did not confirm the CN state in ~1/3 of individuals, and an analysis of an independent case-control-study failed to confirm the initial associations. Furthermore, detailed PCR-based analysis of the sequence at TRB revealed the existence of a more complex genomic sequence most accurately represented by freeze hg18 which accordingly failed to confirm the hg19 sequence. Only rare CNVs were detected at psoriasis susceptibility loci. At three of 12 susceptibility loci with CNVs (CSMD1, IL12B, RYR2), CN variability was confirmed independently by MLPA. Overall, the rate of CNV confirmation by MLPA was strongly dependent upon CNV type, CNV size and the number of array markers involved in a CNV. CONCLUSION: Although we identified PsA associations at several loci and confirmed that the common CNVs at these sites were real, ~1/3 of the common CNV states could not be reproduced. Furthermore, replication analysis failed to confirm the original association. Furthermore, SNP array-based analyses of CNVs were found to be more reliable for deletions than duplications, independent of the respective CNV allele frequency. CNVs are thus good candidate disease variants, while the methods to detect them should be applied cautiously and reproduced by an independent method.

PTPN22 is associated with susceptibility to psoriatic arthritis but not psoriasis: evidence for a further PsA-specific risk locus.

OBJECTIVES: Psoriatic arthritis (PsA) is a chronic inflammatory arthritis associated with psoriasis; it has a higher estimated genetic component than psoriasis alone, however most genetic susceptibility loci identified for PsA to date are also shared with psoriasis. Here we attempt to validate novel single nucleotide polymorphisms selected from our recent PsA Immunochip study and determine specificity to PsA. METHODS: A total of 15 single nucleotide polymorphisms were selected (PImmunochip <1×10(-4)) for validation genotyping in 1177 cases and 2155 controls using TaqMan. Meta-analysis of Immunochip and validation data sets consisted of 3139 PsA cases and 11 078 controls. Novel PsA susceptibility loci were compared with data from two large psoriasis studies (WTCCC2 and Immunochip) to determine PsA specificity. RESULTS: We found genome-wide significant association to rs2476601, mapping to PTPN22 (p=1.49×10(-9), OR=1.32), but no evidence for association in the psoriasis cohort (p=0.34) and the effect estimates were significantly different between PsA and psoriasis (p=3.2×10(-4)). Additionally, we found genome-wide significant association to the previously reported psoriasis risk loci; NOS2 (rs4795067, p=5.27×10(-9)). CONCLUSIONS: For the first time, we report genome-wide significant association of PTPN22 (rs2476601) to PsA susceptibility, but no evidence for association to psoriasis.

Variants in RUNX3 contribute to susceptibility to psoriatic arthritis, exhibiting further common ground with ankylosing spondylitis.

OBJECTIVE: Psoriatic arthritis (PsA) is a common inflammatory joint disease distinct from other chronic arthritides and frequently accompanied by psoriasis vulgaris. In a first genome-wide association study (GWAS), we were able to identify several genetic risk factors. However, even combined with previously identified factors, the genetic contribution to disease was not fully explained. Therefore, we undertook this study to investigate further 17 loci from our GWAS that did not reach genome-wide significance levels of association in the initial analysis. METHODS: Twenty-one of 22 single-nucleotide polymorphisms were successfully genotyped in independent cohorts of 1,398 PsA patients and 6,389 controls and in a group of 964 German patients with psoriasis vulgaris. RESULTS: Association with a RUNX3 variant, rs4649038, was replicated in independent patients and controls and resulted in a combined P value of 1.40 × 10(-8) by Cochran-Mantel-Haenszel test and an odds ratio (OR) of 1.24 (95% confidence interval [95% CI] 1.15-1.33). Further analyses based on linkage disequilibrium (LD) at RUNX3 refined the most significant association to an LD block located in the first intron of one isoform. Weaker evidence for association was detected in German patients with psoriasis vulgaris (P = 5.89 × 10(-2) ; OR 1.13 [95% CI 1.00-1.28]), indicating a role in the skin manifestations of psoriasis. CONCLUSION: Our analyses identified variants in RUNX3 as susceptibility factors for PsA. RUNX3 has already been implicated in susceptibility to ankylosing spondylitis, another spondyloarthritis, although its risk allele is independent from the one for PsA. RUNX-3 is involved in CD8+ T lymphocyte differentiation and is therefore a good candidate for involvement in PsA and psoriasis vulgaris as T cell-mediated diseases.

Confirmation and expansion of the phenotype of the TCEAL1-related neurodevelopmental disorder.

Numerous contiguous gene deletion syndromes causing neurodevelopmental disorders have previously been defined using cytogenetics for which only in the current genomic era the disease-causing genes have become elucidated. One such example is deletion at Xq22.2, previously associated with a neurodevelopmental disorder which has more recently been found to be caused by de novo loss-of-function variants in TCEAL1. So far, a single study reported six unrelated individuals with this monogenetic disorder, presenting with syndromic features including developmental delay especially affecting expressive speech, intellectual disability, autistic-like behaviors, hypotonia, gait abnormalities and mild facial dysmorphism, in addition to ocular, gastrointestinal, and immunologic abnormalities. Here we report on four previously undescribed individuals, including two adults, with de novo truncating variants in TCEAL1, identified through trio exome or genome sequencing, further delineating the phenotype of the TCEAL1-related disorder. Whereas overall we identify similar features compared to the original report, we also highlight features in our adult individuals including hyperphagia, obesity, and endocrine abnormalities including hyperinsulinemia, hyperandrogenemia, and polycystic ovarian syndrome. X chromosome inactivation and RNA-seq studies further provide functional insights in the molecular mechanisms. Together this report expands the phenotypic and molecular spectrum of the TCEAL1-related disorder which will be useful for counseling of newly identified individuals and their families.

Genetic underpinnings of the psoriatic spectrum.

The psoriatic field includes both rare and common subtypes. Common complex forms include psoriasis vulgaris and psoriatic arthritis. In these subtypes, certain HLA alleles remain the most relevant genetic factors, although genome-wide association studies lead to the detection of more than 80 susceptibility loci. They mainly affect innate and adaptive immunity and explain over 28 % of the heritability. Pustular psoriasis comprises a group of rarer subtypes. Using exome sequencing, several disease genes were identified for mainly generalized pustular psoriasis, and an oligogenic inheritance is likely. Treatment studies based on the affected IL-36 pathway indicate a high response rate in this subtype further supporting the pathophysiological relevance of the affected gene products.

Expanding the phenotype of 12q21 deletions: A role of BTG1 in speech development?

We report on a female individual with feeding difficulties, constipation, poor overall growth, periventricular lesions resembling gliosis in brain MRI, recurrent otitis media with palsy of facial nerve, distinct facial features, and pronounced delay in speech development. The latter was the most prominent feature. Molecular karyotyping revealed a heterozygous de novo deletion of 4.353 Mb at chromosome 12q21.33q22. This report expands the number of described individuals with heterozygous deletions at 12q21.33, their clinical spectrum and highlights the clinical variability, even in individuals with deletion of the same genes. Furthermore, our findings indicate a role of BTG1 (OMIM *109580) in speech development.

Microdeletions at 19p13.11p12 in five individuals with neurodevelopmental delay.

Only few copy number variants at chromosome 19p13.11 have been reported, thus associated clinical information is scarce. Proximal to these copy number losses, we now identified deletions in five unrelated individuals with neurodevelopmental disorders. They presented with psychomotor delay as well as behavioral and sleeping disorders, while complex cardiovascular, skeletal, and various other malformations were more variable. Dysmorphic features were rather unspecific and not considered as a recognizable gestalt. Neither of the analyzed parents carried their offsprings' deletions, indicating de novo occurrence. The deletion sizes ranged between 0.7 and 5.2 Mb, were located between 18 and 24 megabases from the telomere, and contained a variable number of protein-coding genes (n = 25-68). Although not all microdeletions shared a common region, the smallest common overlap of some of the deletions provided interesting insights in the chromosomal region 19p13.11p12. Diligent literature review using OMIM and Pubmed did not identify a satisfying candidate gene for neurodevelopmental disorders. In the literature, a de novo in-frame deletion in MAU2 was considered pathogenic in an individual with Cornelia de Lange syndrome. Therefore, the clinical differential diagnosis of this latter syndrome in one individual and the encompassment of MAU2 in three individuals' deletions suggest clinical and genetic overlap with this specific syndrome. Three of the four here reported individuals with deletion encompassing GDF1 had different congenital heart defects, suggesting that this gene's haploinsufficiency might contribute to the cardiovascular phenotype, however, with reduced penetrance. Our findings indicate an association of microdeletions at 19p13.11/ 19p13.11p12 with neurodevelopmental disorders, variable symptoms, and malformations, and delineate the phenotypic spectrum of deletions within this genomic region.

Tumor necrosis factor promoter polymorphism TNF*-857 is a risk allele for psoriatic arthritis independent of the PSORS1 locus.

OBJECTIVE: The strongest susceptibility locus of psoriatic arthritis (PsA) is within the major histocompatibility complex (MHC) region (psoriasis susceptibility region 1, or PSORS1), and HLA-Cw*06:02 has been reported as the PSORS1 susceptibility allele. Non-HLA genes within the MHC region have also been implicated in PsA, but because of the strong linkage disequilibrium at chromosome 6p21, it is difficult to make a distinction between susceptibility alleles and linked markers. Recent studies have demonstrated that the association between PsA and the tumor necrosis factor (TNF) promoter polymorphism TNF*-857 is independent of PSORS1. The aim of this study was to replicate the independent association of TNF*-857 in patients with PsA. METHODS: A total of 909 patients with PsA and 1,315 healthy controls originating from the UK, Germany, and Italy were typed for TNF*-857 and for the estimated risk alleles of HLA-Cw*06:02. RESULTS: Overall, the results of genotyping in these 3 case-control cohorts replicated the finding that the frequency of carriers of TNF*-857 TT/CT who were negative for the PSORS1 risk allele was significantly higher among patients with PsA compared with control subjects (30% versus 21%; P = 9.17 × 10(-5)). CONCLUSION: The results of this collaborative study indicate that TNF*-857T is a susceptibility allele for PsA independent of the PSORS1 allele.

Deletion of LCE3C and LCE3B is a susceptibility factor for psoriatic arthritis: a study in Spanish and Italian populations and meta-analysis.

OBJECTIVE: The LCE3C_LCE3B-del variant is associated with psoriasis and rheumatoid arthritis. Its role in psoriatic arthritis (PsA) is unclear, however, as shown by 3 recent studies with contradictory results. In order to investigate whether LCE3C_LCE3B-del constitutes a risk factor for PsA susceptibility, we first tested this variant in patients with PsA from Spanish and Italian populations and then performed a meta-analysis including the previous case-control studies. METHODS: We genotyped LCE3C_LCE3B-del and its tag single-nucleotide polymorphism (SNP), rs4112788, in an original discovery cohort of 424 Italian patients with PsA and 450 unaffected control subjects. A Spanish replication cohort consisting of 225 patients with PsA and 469 control subjects was also genotyped. A meta-analysis considering 7,758 control subjects and 2,325 patients with PsA was also performed. RESULTS: We observed a significant association between PsA and the LCE3C_LCE3B-del tag SNP in the Italian and Spanish cohorts, with an overall corrected P value of 0.00019 and a corresponding odds ratio of 1.35 (95% confidence interval 1.14-1.59). Stratified analyses by subphenotype indicated a stronger association for patients with oligoarticular disease. Meta-analysis including data from all previous published studies confirmed an association of PsA with the LCE3C_LCE3B-del tag SNP. CONCLUSION: LCE3C_LCE3B-del is a susceptibility factor for PsA, confirming the existence of a shared risk factor involving the epidermal skin barrier in autoimmune disorders.