Pravitt Gourh

Genome-wide association study of systemic sclerosis identifies CD247 as a new susceptibility locus

Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and internal organs that leads to profound disability and premature death. To identify new SSc susceptibility loci, we conducted the first genome-wide association study in a population of European ancestry including a total of 2,296 individuals with SSc and 5,171 controls. Analysis of 279,621 autosomal SNPs followed by replication testing in an independent case-control set of European ancestry (2,753 individuals with SSc (cases) and 4,569 controls) identified a new susceptibility locus for systemic sclerosis at CD247 (1q22–23, rs2056626, P = 2.09 × 10−7 in the discovery samples, P = 3.39 × 10−9 in the combined analysis). Additionally, we confirm and firmly establish the role of the MHC (P = 2.31 × 10−18), IRF5 (P = 1.86 × 10−13) and STAT4 (P = 3.37 × 10−9) gene regions as SSc genetic risk factors.

Identification of novel genetic markers associated with clinical phenotypes of systemic sclerosis through a genome-wide association strategy

The aim of this study was to determine, through a genome-wide association study (GWAS), the genetic components contributing to different clinical sub-phenotypes of systemic sclerosis (SSc). We considered limited (lcSSc) and diffuse (dcSSc) cutaneous involvement, and the relationships with presence of the SSc-specific auto-antibodies, anti-centromere (ACA), and anti-topoisomerase I (ATA). Four GWAS cohorts, comprising 2,296 SSc patients and 5,171 healthy controls, were meta-analyzed looking for associations in the selected subgroups. Eighteen polymorphisms were further tested in nine independent cohorts comprising an additional 3,175 SSc patients and 4,971 controls. Conditional analysis for associated SNPs in the HLA region was performed to explore their independent association in antibody subgroups. Overall analysis showed that non-HLA polymorphism rs11642873 in IRF8 gene to be associated at GWAS level with lcSSc (P = 2.32×10−12, OR = 0.75). Also, rs12540874 in GRB10 gene (P = 1.27 × 10−6, OR = 1.15) and rs11047102 in SOX5 gene (P = 1.39×10−7, OR = 1.36) showed a suggestive association with lcSSc and ACA subgroups respectively. In the HLA region, we observed highly associated allelic combinations in the HLA-DQB1 locus with ACA (P = 1.79×10−61, OR = 2.48), in the HLA-DPA1/B1 loci with ATA (P = 4.57×10−76, OR = 8.84), and in NOTCH4 with ACA P = 8.84×10−21, OR = 0.55) and ATA (P = 1.14×10−8, OR = 0.54). We have identified three new non-HLA genes (IRF8, GRB10, and SOX5) associated with SSc clinical and auto-antibody subgroups. Within the HLA region, HLA-DQB1, HLA-DPA1/B1, and NOTCH4 associations with SSc are likely confined to specific auto-antibodies. These data emphasize the differential genetic components of subphenotypes of SSc.

Major histocompatibility complex (MHC) class II alleles, haplotypes and epitopes which confer susceptibility or protection in systemic sclerosis: analyses in 1300 Caucasian, African-American and Hispanic cases and 1000 controls

Objective To determine human leucocyte antigen-class II (HLA-class II) (DRB1, DQB1, DQA1 and DPB1) alleles, haplotypes and shared epitopes associated with scleroderma (systemic sclerosis (SSc)) and its subphenotypes in a large multi-ethnic US cohort by a case–control association study. Patients and methods 1300 SSc cases (961 white, 178 black and 161 Hispanic subjects) characterised for clinical skin forms (limited vs diffuse), SSc-specific autoantibodies (anticentromere (ACA), anti-topoisomerase I (ATA), anti-RNA polymerase III (ARA), anti-U3 ribonucleoprotein (fibrillarin)) and others were studied using molecular genotyping. Statistical analyses in SSc itself by ethnicity, gender, skin type and autoantibodies were performed using exact logistic regression modelling for dominant, additive and recessive effects from HLA. Results The strongest positive class II associations with SSc in white and Hispanic subjects were the DRB1*1104, DQA1*0501, DQB1*0301 haplotype and DQB1 alleles encoding a non-leucine residue at position 26 (DQB1 26 epi), while the DRB1*0701, DQA1*0201, DQB1*0202 haplotype and DRB1*1501 haplotype were negatively correlated and possibly protective in dominant and recessive models, respectively. These associations did not discriminate between limited and diffuse SSc. SSc in black subjects was associated with DRB1*0804, DQA1*0501, DQB1*0301 alleles. DPB1*1301 showed the highest odds ratio for ATA (OR = 14). Moreover, it showed no linkage disequilibrium or gene interaction with DR/DQ. ACA was best explained by DQB1*0501 and DQB1*26 epi alleles and ARA by DRB1*0404, DRB1*11 and DQB1*03 alleles in white and Hispanic subjects but DRB1*08 in black subjects. Conclusion These data indicate unique and multiple HLA-class II effects in SSc, especially on autoantibody markers of different subphenotypes.

Major Histocompatibility Complex (MHC) class II alleles, haplotypes, and epitopes which confer susceptibility or protection in the fibrosing autoimmune disease systemic sclerosis: analyses in 1300 Caucasian, African-American and Hispanic cases and 1000 controls

Objective To determine human leucocyte antigen-class II (HLA-class II) (DRB1, DQB1, DQA1 and DPB1) alleles, haplotypes and shared epitopes associated with scleroderma (systemic sclerosis (SSc)) and its subphenotypes in a large multi-ethnic US cohort by a case–control association study. Patients and methods 1300 SSc cases (961 white, 178 black and 161 Hispanic subjects) characterised for clinical skin forms (limited vs diffuse), SSc-specific autoantibodies (anticentromere (ACA), anti-topoisomerase I (ATA), anti-RNA polymerase III (ARA), anti-U3 ribonucleoprotein (fibrillarin)) and others were studied using molecular genotyping. Statistical analyses in SSc itself by ethnicity, gender, skin type and autoantibodies were performed using exact logistic regression modelling for dominant, additive and recessive effects from HLA. Results The strongest positive class II associations with SSc in white and Hispanic subjects were the DRB1*1104, DQA1*0501, DQB1*0301 haplotype and DQB1 alleles encoding a non-leucine residue at position 26 (DQB1 26 epi), while the DRB1*0701, DQA1*0201, DQB1*0202 haplotype and DRB1*1501 haplotype were negatively correlated and possibly protective in dominant and recessive models, respectively. These associations did not discriminate between limited and diffuse SSc. SSc in black subjects was associated with DRB1*0804, DQA1*0501, DQB1*0301 alleles. DPB1*1301 showed the highest odds ratio for ATA (OR = 14). Moreover, it showed no linkage disequilibrium or gene interaction with DR/DQ. ACA was best explained by DQB1*0501 and DQB1*26 epi alleles and ARA by DRB1*0404, DRB1*11 and DQB1*03 alleles in white and Hispanic subjects but DRB1*08 in black subjects. Conclusion These data indicate unique and multiple HLA-class II effects in SSc, especially on autoantibody markers of different subphenotypes.

Plasma cytokine profiles in systemic sclerosis: associations with autoantibody subsets and clinical manifestations

IntroductionSystemic sclerosis (SSc) (scleroderma) is a complex autoimmune disease that clinically manifests as progressive fibrosis of the skin and internal organs. Anti-centromere antibodies (ACAs), anti-topoisomerase antibodies (ATAs), and anti-RNA polymerase III antibodies (ARAs) are three mutually exclusive SSc-associated autoantibodies that correlate with distinct clinical subsets characterized by extent of cutaneous involvement and pattern of organ involvement. The current report sought to determine whether plasma cytokine profiles differ in SSc patients grouped according to these SSc-associated autoantibody subsets.MethodsPlasma from 444 SSc patients and 216 healthy controls was obtained from the Scleroderma Family Registry and University of Texas Rheumatology Division. Patients were classified according to the presence of ACAs, ATAs, ARAs, or none of the above (antibody-negative). Levels of 13 cytokines were determined using multiplex assays.ResultsCompared with females, healthy control males had higher plasma levels of IL-2 (P = 0.008), IL-5 (P = 0.01) and IL-8 (P = 0.01). In addition, in controls, IL-6 (P = 0.02) and IL-17 (P = 0.01) levels increased with advancing age. After adjusting for age and gender, SSc patients had higher circulating levels of TNFα (P < 0.0001), IL-6 (P < 0.0001), and IFNγ (P = 0.05) and lower IL-17 (P = 0.0005) and IL-23 (P = 0.014). Additional analyses demonstrated that disease duration also influenced these cytokine profiles. IL-6 was elevated in ATA-positive and ARA-positive patients, but not in ACA-positive patients. IL-8 was uniquely increased in the ATA-positive subset while both ATA-positive and ACA-positive subsets had elevated IFNγ and IL-10. IL-5 was only significantly increased in the ACA-positive subset. Lastly, patients with interstitial lung disease had elevated IL-6 and patients with pulmonary hypertension had elevated IL-6 and IL-13.ConclusionsPlasma cytokine profiles differ in SSc patients based on the presence of SSc-associated autoantibodies. Plasma cytokine profiles in SSc patients may also be affected by disease duration and the pattern of internal organ involvement.

Association of TNFSF4 (OX40L) polymorphisms with susceptibility to systemic sclerosis.

Objective It is increasingly being appreciated that multiple autoimmune diseases share common susceptibility genes. The tumour necrosis factor ligand superfamily member 4 gene (TNFSF4, OX40L), which encodes for the T cell costimulatory molecule OX40 ligand, has been identified as a susceptibility gene for the development of systemic lupus erythematosus (SLE). Accordingly, the aim of the current study was to investigate the possible association of the TNFSF4 gene region with systemic sclerosis (SSc), an autoimmune disease that leads to the development of cutaneous and visceral fibrosis. Methods A total of 9 single nucleotide polymorphisms (SNPs) in the TNFSF4 gene region, previously associated with susceptibility to SLE, were tested for association with SSc in a collection of 1059 patients with SSc and 698 controls. Results Case-control comparisons revealed a significant association between susceptibility to SSc and the minor alleles at SNPs rs1234314 (OR 1.20, 95% CI 1.04 to 1.4, pFDR=0.019), rs2205960 (OR 1.24, 95% CI 1.10 to 1.50, pFDR=0.019) and rs844648 (OR 1.16, 95% CI 1.01 to 1.30, pFDR=0.032). The minor allele at rs844644 was protective (OR 0.84, 95% CI 0.70 to 0.97, pFDR=0.038). Analysis of subsets of patients with SSc demonstrated significant associations of the TNFSF4 SNPs with limited and diffuse SSc as well as specific SNPs that were associated with SSc-associated autoantibodies. Finally, the analyses suggest a potential interaction between two TNFSF4 SNPs, rs2205960 and rs844648, with regards to SSc susceptibility. Conclusions Polymorphisms in the TNFSF4 gene region are associated with susceptibility to SSc and its clinical and autoantibody subsets. TNFSF4 may be another gene that confers risk to multiple autoimmune diseases.

Clinical and Genetic Factors Predictive of Mortality in Early Systemic Sclerosis

OBJECTIVE To investigate the clinical and genetic variables at initial presentation that predict survival in the Genetics versus Environment in Scleroderma Outcome Study (GENISOS) cohort. METHODS GENISOS is a prospective, observational study of a multiethnic early systemic sclerosis (SSc) cohort. To date, a total of 250 patients have been enrolled. In addition to clinical and laboratory data, electrocardiograms (EKGs), chest radiographs, and pulmonary function tests have been obtained from each patient. A modified Rodnan skin thickness score, HLA class II genotyping, and a Medsger Damage Index also have been collected. We performed multivariable analyses utilizing the Cox regression following a purposeful model building strategy. RESULTS The study analyzed 122 white, 47 African American, and 71 Hispanic SSc patients with an average disease duration of 2.6 years at enrollment. At the time of analysis, 52 (20.8%) of the 250 patients had died. In the final multivariable model excluding HLA genes, 7 variables emerged as significant predictors of mortality: age > or =65 years at enrollment, forced vital capacity <50% predicted, clinically significant arrhythmia on EKG, absence of anticentromere antibodies, hypertension, chest radiograph suggestive of pulmonary fibrosis, and low body mass index (BMI). In separate modeling that included HLA genes, HLA alleles DRB1*0802 and DQA1*0501 were significant predictors of mortality in addition to the predictors mentioned above. CONCLUSION A limited number of variables collected at presentation, including BMI, are able to predict mortality in patients with early SSc. In addition, some of the HLA genes associated with SSc susceptibility are useful for predicting SSc outcome.

Polymorphisms in TBX21 and STAT4 Increase the Risk of Systemic Sclerosis: Evidence of Possible Gene–Gene Interaction and Alterations in Th1/Th2 Cytokines

OBJECTIVE Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and internal organs. Dysregulation of the immune system, including the Th1/Th2 cytokine balance, is central to the pathogenesis of SSc. This study was undertaken to investigate the hypothesis that single-nucleotide polymorphisms (SNPs) in TBX21 and STAT4, both of which are critical transcription factors that regulate the Th1/Th2 balance, are associated with SSc susceptibility. METHODS We tested SNPs in TBX21 and STAT4 for association with SSc in 2 independent cohorts, the SSc Registry cohort (880 SSc cases and 507 controls) and the University of Texas SSc cohort (522 cases and 531 controls). Additional white control genotypes were obtained from public repositories. We also investigated for gene-gene interactions. Plasma cytokines and whole blood gene expression profiles were examined to determine functional effects of these SNPs. RESULTS Multiple SNPs in TBX21 and STAT4 were found to be associated with SSc. In a combined analysis of 902 SSc patients and 4,745 controls, TT genotyping of the TBX21 rs11650354 variant revealed a recessive pattern for disease susceptibility (Pcorr=1.4x10(-15), odds ratio 3.37, 95% confidence interval 2.4-4.6). In an analysis of 1,039 SSc patients and 3,322 controls, the A allele of the STAT4 variant rs11889341 was associated with increased SSc susceptibility in a dominant pattern (Pcorr=2.4x10(-5), odds ratio 1.29, 95% confidence interval 1.2-1.5). Furthermore, we identified gene-gene interaction among the TBX21 and STAT4 variants, such that the STAT4 genotype increased the risk of SSc only in the TBX21 CC genotype group. SSc patients carrying the TBX21 CC genotype had higher interleukin-6 (IL-6) and tumor necrosis factor alpha levels, and those with the TT genotype had elevated IL-2, IL-5, IL-4, and IL-13 (Th2) levels, compared with controls. Whole blood expression profiles revealed dysregulation of type I interferon pathways in the CC group and T cell pathways in the TT group of the TBX21 SNP. CONCLUSION The present results, from studies of 2 independent cohorts, indicate that SNPs in TBX21 and STAT4 contribute uniquely and interactively to SSc susceptibility, leading to altered cytokine balance and immune dysregulation.

BANK1 functional variants are associated with susceptibility to diffuse systemic sclerosis in Caucasians

Objective To investigate the possible association of the BANK1 gene with genetic susceptibility to systemic sclerosis (SSc) and its subphenotypes. Methods A large multicentre case–control association study including 2380 patients with SSc and 3270 healthy controls from six independent case–control sets of Caucasian ancestry (American, Spanish, Dutch, German, Swedish and Italian) was conducted. Three putative functional BANK1 polymorphisms (rs17266594 T/C, rs10516487 G/A, rs3733197 G/A) were selected as genetic markers and genotyped by Taqman 5´ allelic discrimination assay. Results A significant association of the rs10516487 G and rs17266594 T alleles with SSc susceptibility was observed (pooled OR=1.12, 95% CI 1.03 to 1.22; p=0.01 and pooled OR=1.14, 95% CI 1.05 to 1.25; p=0.003, respectively), whereas the rs3733197 genetic variant showed no statistically significant deviation. Stratification for cutaneous SSc phenotype showed that the BANK1 rs10516487 G, rs17266594 T and rs3733197 G alleles were strongly associated with susceptibility to diffuse SSc (dcSSc) (pooled OR=1.20, 95% CI 1.05 to 1.37, p=0.005; pooled OR=1.23, 95% CI 1.08 to 1.41, p=0.001; pooled OR=1.15, 95% CI 1.02 to 1.31, p=0.02, respectively). Similarly, stratification for specific SSc autoantibodies showed that the association of BANK1 rs10516487, rs17266594 and rs3733197 polymorphisms was restricted to the subgroup of patients carrying anti-topoisomerase I antibodies (pooled OR=1.20, 95% CI 1.02 to 1.41, p=0.03; pooled OR=1.24, 95% CI 1.05 to 1.46, p=0.01; pooled OR=1.26, 95% CI 1.07 to 1.47, p=0.004, respectively). Conclusion The results suggest that the BANK1 gene confers susceptibility to SSc in general, and specifically to the dcSSc and anti-topoisomerase I antibody subsets.

Primary biliary cirrhosis (PBC), PBC autoantibodies, and hepatic parameter abnormalities in a large population of systemic sclerosis patients.

Objective. To investigate the diagnostic accuracy of antimitochondrial antibodies (AMA), sp100, and gp210 antibodies for primary biliary cirrhosis (PBC) in a large population of patients with systemic sclerosis (SSc); to examine concordance of these antibodies with subsets of SSc. Further, to assess the association of SSc-related antibodies with hepatic parameter abnormalities. Methods. We obtained medical records to verify the diagnoses of SSc and PBC. Sera from all participants were examined for the presence of SSc- and PBC-related antibodies, as well as for abnormalities in hepatic parameters. Results. We examined 817 patients with SSc, of whom 16 (2%) had confirmed PBC. The sensitivity and specificity of AMA by a MIT3 ELISA for PBC were 81.3% and 94.6%, respectively. Sp100 had a sensitivity and specificity of 31.3% and 97.4%, respectively, while gp210 had an even lower sensitivity. We were able to detect all PBC cases using AMA(MIT3) and sp100 as a combined marker, resulting in a significantly improved sensitivity of 100% (p = 0.042) with an incremental decrease in specificity to 92.6%. Independent of AMA or sp100 status, there was an association of anticentromere B (CENP-B) and anti-topoisomerase antibodies (ATA) with higher alkaline phosphatase levels (p = 0.051 and p = 0.003, respectively) while anti-RNA polymerase III (anti-RNAP) was associated with lower alkaline phosphatase levels (p = 0.019) among the patients with SSc. Conclusion. Utilization of AMA(MIT3) and sp100 antibodies as a combined diagnostic marker leads to an improved detection of PBC in patients with SSc. CENP-B and ATA are associated with alkaline phosphatase elevation.