Transethnic meta-analysis identifies GSDMA and PRDM1 as susceptibility genes to systemic sclerosis.

OBJECTIVES: Systemic sclerosis (SSc) is an autoimmune disease characterised by skin and systemic fibrosis culminating in organ damage. Previous genetic studies including genome-wide association studies (GWAS) have identified 12 susceptibility loci satisfying genome-wide significance. Transethnic meta-analyses have successfully expanded the list of susceptibility genes and deepened biological insights for other autoimmune diseases. METHODS: We performed transethnic meta-analysis of GWAS in the Japanese and European populations, followed by a two-staged replication study comprising a total of 4436 cases and 14 751 controls. Associations between significant single nuclear polymorphisms (SNPs) and neighbouring genes were evaluated. Enrichment analysis of H3K4Me3, a representative histone mark for active promoter was conducted with an expanded list of SSc susceptibility genes. RESULTS: We identified two significant SNP in two loci, GSDMA and PRDM1, both of which are related to immune functions and associated with other autoimmune diseases (p=1.4×10-10 and 6.6×10-10, respectively). GSDMA also showed a significant association with limited cutaneous SSc. We also replicated the associations of previously reported loci including a non-GWAS locus, TNFAIP3. PRDM1 encodes BLIMP1, a transcription factor regulating T-cell proliferation and plasma cell differentiation. The top SNP in GSDMA was a missense variant and correlated with gene expression of neighbouring genes, and this could explain the association in this locus. We found different human leukocyte antigen (HLA) association patterns between the two populations. Enrichment analysis suggested the importance of CD4-naïve primary T cell. CONCLUSIONS: GSDMA and PRDM1 are associated with SSc. These findings provide enhanced insight into the genetic and biological basis of SSc.

Disease progression in systemic sclerosis-overlap syndrome is significantly different from limited and diffuse cutaneous systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc)-overlap syndromes are a very heterogeneous and remarkable subgroup of SSc-patients, who present at least two connective tissue diseases (CTD) at the same time, usually with a specific autoantibody status. OBJECTIVES: To determine whether patients, classified as overlap syndromes, show a disease course different from patients with limited SSc (lcSSc) or diffuse cutaneous SSc (dcSSc). METHODS: The data of 3240 prospectively included patients, registered in the database of the German Network for Systemic Scleroderma and followed between 2003 and 2013, were analysed. RESULTS: Among 3240 registered patients, 10% were diagnosed as SSc-overlap syndrome. Of these, 82.5% were female. SSc-overlap patients had a mean age of 48±1.2 years and carried significantly more often 'other antibodies' (68.0%; p<0.0001), including anti-U1RNP, -PmScl, -Ro, -La, as well as anti-Jo-1 and -Ku antibodies. These patients developed musculoskeletal involvement earlier and more frequently (62.5%) than patients diagnosed as lcSSc (32.2%) or dcSSc (43.3%) (p<0.0001). The onset of lung fibrosis and heart involvement in SSc-overlap patients was significantly earlier than in patients with lcSSc and occurred later than in patients with dcSSc. Oesophagus, kidney and PH progression was similar to lcSSc patients, whereas dcSSc patients had a significantly earlier onset. CONCLUSIONS: These data support the concept that SSc-overlap syndromes should be regarded as a separate SSc subset, distinct from lcSSc and dcSSc, due to a different progression of the disease, different proportional distribution of specific autoantibodies, and of different organ involvement.

A regulatory variant in CCR6 is associated with susceptibility to antitopoisomerase-positive systemic sclerosis.

OBJECTIVE: Recognition of the well-known pleiotropism of autoimmune genes supports the concept of a shared pathogenesis across autoimmune diseases such as rheumatoid arthritis (RA) and systemic sclerosis (SSc). Studies have reproducibly demonstrated an association between susceptibility to RA and polymorphisms of the CCR6 gene, a surface marker for Th17 cells, and the causal variant was recently identified. The present study was thus undertaken to investigate whether CCR6 polymorphisms could also be associated with susceptibility to SSc. METHODS: Twelve tag single-nucleotide polymorphisms (SNPs) of CCR6, including the known RA-associated SNP rs3093023, were genotyped in a total of 2,411 SSc patients and 7,084 healthy individuals from 3 European populations (France, Italy, and Germany). Meta-analyses of the data were performed to assess whether an association exists between CCR6 polymorphisms and susceptibility to SSc or its main subtypes. Direct sequencing of DNA was performed to ascertain whether the functional dinucleotide polymorphism of CCR6 previously identified in RA (CCR6DNP) was also present in SSc. RESULTS: Combined analyses revealed an association between the rs10946216 SNP and SSc susceptibility (odds ratio [OR] 1.13, 95% confidence interval [95% CI] 1.05-1.21, adjusted P [P(adj)] = 0.026). The rs3093023 A allele and rs10946216 T allele were in high linkage disequilibrium, and both were found to confer disease susceptibility in the antitopoisomerase-positive subset of SSc patients (OR 1.27, 95% CI 1.13-1.42, P(adj) = 1.5 × 10(-3) and OR 1.32, 95% CI 1.17-1.48, P(adj) = 9.0 × 10(-5), respectively, relative to healthy controls). Direct sequencing of the DNA of 78 individuals supported the hypothesis that the regulatory dinucleotide CCR6DNP could be the causal variant in SSc. CONCLUSION: The results of this study establish CCR6 as a new susceptibility factor for antitopoisomerase-positive SSc, as demonstrated in 3 European Caucasian populations, confirming the notion that SSc and RA could conceivably share autoimmune risk alleles. The results also suggest a potential role of the interleukin-17 pathway in SSc.

Genome-wide scan identifies TNIP1, PSORS1C1, and RHOB as novel risk loci for systemic sclerosis.

Systemic sclerosis (SSc) is an orphan, complex, inflammatory disease affecting the immune system and connective tissue. SSc stands out as a severely incapacitating and life-threatening inflammatory rheumatic disease, with a largely unknown pathogenesis. We have designed a two-stage genome-wide association study of SSc using case-control samples from France, Italy, Germany, and Northern Europe. The initial genome-wide scan was conducted in a French post quality-control sample of 564 cases and 1,776 controls, using almost 500 K SNPs. Two SNPs from the MHC region, together with the 6 loci outside MHC having at least one SNP with a P<10(-5) were selected for follow-up analysis. These markers were genotyped in a post-QC replication sample of 1,682 SSc cases and 3,926 controls. The three top SNPs are in strong linkage disequilibrium and located on 6p21, in the HLA-DQB1 gene: rs9275224, P = 9.18×10(-8), OR = 0.69, 95% CI [0.60-0.79]; rs6457617, P = 1.14×10(-7) and rs9275245, P = 1.39×10(-7). Within the MHC region, the next most associated SNP (rs3130573, P = 1.86×10(-5), OR = 1.36 [1.18-1.56]) is located in the PSORS1C1 gene. Outside the MHC region, our GWAS analysis revealed 7 top SNPs (P<10(-5)) that spanned 6 independent genomic regions. Follow-up of the 17 top SNPs in an independent sample of 1,682 SSc and 3,926 controls showed associations at PSORS1C1 (overall P = 5.70×10(-10), OR:1.25), TNIP1 (P = 4.68×10(-9), OR:1.31), and RHOB loci (P = 3.17×10(-6), OR:1.21). Because of its biological relevance, and previous reports of genetic association at this locus with connective tissue disorders, we investigated TNIP1 expression. A markedly reduced expression of the TNIP1 gene and also its protein product were observed both in lesional skin tissue and in cultured dermal fibroblasts from SSc patients. Furthermore, TNIP1 showed in vitro inhibitory effects on inflammatory cytokine-induced collagen production. The genetic signal of association with TNIP1 variants, together with tissular and cellular investigations, suggests that this pathway has a critical role in regulating autoimmunity and SSc pathogenesis.

The human AIRE gene at chromosome 21q22 is a genetic determinant for the predisposition to rheumatoid arthritis in Japanese population.

Rheumatoid arthritis (RA) is a typical complex trait and the major cause of chronic inflammation worldwide. Although multiple genetic loci have been shown for their association with the onset of RA, they cover only a part of its genetic components and are largely ethnicity-specific. To identify novel genetic factors related to the predisposition and prognosis of RA in Japanese, we conducted a large-scale genome-wide association (GWA) study. We performed a GWA analysis by scanning the genome of 1247 RA cases and 1486 controls for 277 420 single nucleotide polymorphisms (SNPs), followed by replication analysis using two independent sample sets consisting of 1865 cases and 1623 controls, and 2303 cases and 3380 controls. We identified two SNPs, rs2075876 and rs760426, in intron of the autoimmune regulator AIRE gene at chromosome 21q22 that showed strong associations with the disease (P= 3.6 × 10(-9) and P= 4.4 × 10(-8), respectively). Rs1800250, in exon7 of AIRE, was in strong linkage disequilibrium (r(2)= 0.94) with rs2075876 and introduced an amino acid alteration (S278R) in the SAND domain of the AIRE protein. In silico analysis showed the decreased transcription of AIRE by the risk allele of rs2075876 compared with the alternative allele (P= 6.8 × 10(-5)). No correlation was observed between the rs2075876 genotype and quantitative traits reflecting the progression of RA. As AIRE is a key molecule which regulates the expression and presentation of self-antigens in thymic negative selection, its downregulation by genetic polymorphisms may result in the survival of auto-reactive T cells to trigger auto-inflammation in RA.

TGFß receptor gene variants in systemic sclerosis-related pulmonary arterial hypertension: results from a multicentre EUSTAR study of European Caucasian patients.

INTRODUCTION: Systemic sclerosis (SSc)-related pulmonary arterial hypertension (PAH) has emerged as a major mortality prognostic factor. Mutations of transforming growth factor beta (TGFß) receptor genes strongly contribute to idiopathic and familial PAH. OBJECTIVE: To explore the genetic bases of SSc-PAH, we combined direct sequencing and genotyping of candidate genes encoding TGFß receptor family members. MATERIALS AND METHODS: TGFß receptor genes, BMPR2, ALK1, TGFR2 and ENG, were sequenced in 10 SSc-PAH patients, nine SSc and seven controls. In addition, 22 single-nucleotide polymorphisms (SNP) of these four candidate genes were tested for association in a first set of 824 French Caucasian SSc patients (including 54 SSc-PAH) and 939 controls. The replication set consisted of 1516 European SSc (including 219 SSc-PAH) and 3129 controls from the European League Against Rheumatism Scleroderma Trials and Research group network. RESULTS: No mutation was identified by direct sequencing. However, two repertoried SNP, ENG rs35400405 and ALK1 rs2277382, were found in SSc-PAH patients only. The genotyping of 22 SNP including the latter showed that only rs2277382 was associated with SSc-PAH (p=0.0066, OR 2.13, 95% CI 1.24 to 3.65). Nevertheless, this was not replicated with the following result in combined analysis: p=0.123, OR 0.79, 95% CI 0.59 to 1.07. CONCLUSIONS: This study demonstrates the lack of association between these TGFß receptor gene polymorphisms and SSc-PAH using both sequencing and genotyping methods.

A trans-ethnic genetic study of rheumatoid arthritis identified FCGR2A as a candidate common risk factor in Japanese and European populations.

Rheumatoid arthritis (RA) is a common systemic autoimmune disease and its onset and prognosis are controlled by genetic, immunological, and environmental factors. The HLA locus, particularly HLA-DRB1, is its strongest genetic risk determinant across ethnicities. Several other genes, including PTPN22 and PADI4, show modest association with RA. However, they cover only a part of its genetic components and their relative contribution is different between populations. To identify novel genetic determinants, we took a candidate gene approach in a trans-ethnic manner. After critical selection of 169 genes based on their immunological function, we performed SNP discovery of these genes by the resequencing of exons and surrounding areas using European and Japanese DNAs. We then generated a panel of 1,509 SNPs for case-control association study in both populations. The DerSimonian-Laird test for meta-analysis, using the combined results of the two populations, identified rs7551957 at the 5'-flanking region of the low-affinity Fc-gamma receptor IIa (FCGR2A) gene as the strongest candidate for the association (p = 8.6 × 10(-5), odds ratio = 1.58 with 95%CI 1.25-1.99). Suggestive signals were also obtained for three SNPs in the dihydropyrimidine dehydrogenase (DPYD) gene (rs6685859; p = 1.3 × 10(-4), rs7550959; p = 1.5 × 10(-4) and rs7531138; p = 1.7 × 10(-4)) and an intronic SNP, rs2269310, of the erythrocytic spectrin beta (SPTB) gene (p = 7.9 × 10(-4)).

Vasoactive Therapy in Systemic Sclerosis: Real-life Therapeutic Practice in More Than 3000 Patients.

OBJECTIVE: Vasculopathy is a key factor in the pathophysiology of systemic sclerosis (SSc) and the main cause for Raynaud phenomenon (RP), digital ulcers (DU), and/or pulmonary arterial hypertension (PAH). It is so far unknown how patients with SSc are treated with vasoactive agents in daily practice. To determine to which extent patients with SSc were treated with different vasoactive agents, we used data from the German Network for Systemic Scleroderma registry. METHODS: The data of 3248 patients with SSc were analyzed. RESULTS: Patients were treated with vasoactive drugs in 61.1% of cases (1984/3248). Of these, 47.6% received calcium channel inhibitors, followed by 34.2% treated with angiotensin-converting enzyme (ACE) inhibitors, 21.1% treated with intravenous (IV) prostanoids, 10.1% with pentoxifylline, 8.8% with angiotensin 1 receptor antagonists (AT1RA), 8.7% with endothelin 1 receptor antagonists (ET1RA), 4.1% with phosphodiesterase type 5 (PDE5) inhibitors, and 5.3% with others. Patients with RP received vasoactive therapy in 63.3% of cases, with DU in 70.1%, and with PAH in 78.2% of cases. Logistic regression analysis revealed that patients with PAH were significantly more often treated with PDE5 inhibitors and ET1RA, and those with DU with ET1RA and IV prostanoids. In addition, 41.8% of patients were treated with ACE inhibitors and/or AT1RA. Patients registered after 2009 received significantly more often ET1RA, AT1RA, and IV prostanoids compared with patients registered prior to 2005. CONCLUSION: These data clearly indicate that many patients with SSc do not yet receive sufficient vasoactive therapy. Further, in recent years, a marked change of treatment regimens can be observed.

Defective removal of ribonucleotides from DNA promotes systemic autoimmunity.

Genome integrity is continuously challenged by the DNA damage that arises during normal cell metabolism. Biallelic mutations in the genes encoding the genome surveillance enzyme ribonuclease H2 (RNase H2) cause Aicardi-Goutières syndrome (AGS), a pediatric disorder that shares features with the autoimmune disease systemic lupus erythematosus (SLE). Here we determined that heterozygous parents of AGS patients exhibit an intermediate autoimmune phenotype and demonstrated a genetic association between rare RNASEH2 sequence variants and SLE. Evaluation of patient cells revealed that SLE- and AGS-associated mutations impair RNase H2 function and result in accumulation of ribonucleotides in genomic DNA. The ensuing chronic low level of DNA damage triggered a DNA damage response characterized by constitutive p53 phosphorylation and senescence. Patient fibroblasts exhibited constitutive upregulation of IFN-stimulated genes and an enhanced type I IFN response to the immunostimulatory nucleic acid polyinosinic:polycytidylic acid and UV light irradiation, linking RNase H2 deficiency to potentiation of innate immune signaling. Moreover, UV-induced cyclobutane pyrimidine dimer formation was markedly enhanced in ribonucleotide-containing DNA, providing a mechanism for photosensitivity in RNase H2-associated SLE. Collectively, our findings implicate RNase H2 in the pathogenesis of SLE and suggest a role of DNA damage-associated pathways in the initiation of autoimmunity.

Monoclonal autoantibodies from patients with autoimmune diseases: specificity, affinity and crossreactivity of MAbs binding to cytoskeletal and nucleolar epitopes, cartilage antigens and mycobacterial heat-shock protein 60.

Serum autoantibodies produce typical immunofluorescence staining patterns on HEp-2 cells, which are frequently used for diagnostic purposes. These include antibodies recognizing cytoskeletal and nuclear epitopes. The detailed analysis of human monoclonal antibodies (MAbs) should help to understand which antigens or autoantigens were involved in the generation of these immune responses. Here, three MAbs are described staining HEp-2 cells in a characteristic pattern. They were derived from peripheral blood B cells of two patients with rheumatic diseases (rheumatoid arthritis and relapsing polychondritis). Their binding reactivities were characterized in detail in several assay systems and their affinities measured. Although the antibodies differed in their fine specificity and crossreactivity, all three MAbs (2 IgM, 1 IgA) bound to purified cytoskeletal antigens (desmin) and, in addition, to cartilage antigens (human collagen type II, proteoglycans). The binding to HEp-2 cells could be inhibited specifically with soluble antigens as shown by intracellular flow cytometry. The affinities for both groups of antigens were relatively high (examples: K(D) (desmin) = 0.1 x 10(-7) M; K(D) (collagen) = 3.5 x 10(-7) M). Two of the MAbs also bound to heat-shock protein 60 (HSP60) derived from Mycobacterium tuberculosis. The results prove that antibodies and B cells with reactivity to both intracellular cytoskeletal and nuclear antigens and exogenous antigens (e. g. HSP60) exist in patients with rheumatic diseases. Similar to an animal model such human B cells may be induced by the exogenous antigen (HSP60) and crossreact with local auto-antigens related to the disease (cartilage). In this way they might contribute to pathogenic processes. Due to their additional crossreactivity with intracellular cytoskeletal and nuclear antigens, these antibodies simultaneously can be detected in the HEp-2 immunofluorescence assay.