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1.  New insight on the Xq28 association with systemic sclerosis 
Annals of the rheumatic diseases  2013;72(12):10.1136/annrheumdis-2012-202742.
Objective
To evaluate whether the systemic sclerosis (SSc)-associated IRAK1 non-synonymous single-nucleotide polymorphism rs1059702 is responsible for the Xq28 association with SSc or whether there are other independent signals in the nearby methyl-CpG-binding protein 2 gene (MECP2).
Methods
We analysed a total of 3065 women with SSc and 2630 unaffected controls from five independent Caucasian cohorts. Four tag single-nucleotide polymorphisms of MECP2 (rs3027935, rs17435, rs5987201 and rs5945175) and the IRAK1 variant rs1059702 were genotyped using TaqMan predesigned assays. A meta-analysis including all cohorts was performed to test the overall effect of these Xq28 polymorphisms on SSc.
Results
IRAK1 rs1059702 and MECP2 rs17435 were associated specifically with diffuse cutaneous SSc (PFDR=4.12×10−3, OR=1.27, 95% CI 1.09 to 1.47, and PFDR=5.26×10−4, OR=1.30, 95% CI 1.14 to 1.48, respectively), but conditional logistic regression analysis showed that the association of IRAK1 rs1059702 with this subtype was explained by that of MECP2 rs17435. On the other hand, IRAK1 rs1059702 was consistently associated with presence of pulmonary fibrosis (PF), because statistical significance was observed when comparing SSc patients PF+ versus controls (PFDR=0.039, OR=1.30, 95% CI 1.07 to 1.58) and SSc patients PF+ versus SSc patients PF− (p=0.025, OR=1.26, 95% CI 1.03 to 1.55).
Conclusions
Our data clearly suggest the existence of two independent signals within the Xq28 region, one located in IRAK1 related to PF and another in MECP2 related to diffuse cutaneous SSc, indicating that both genes may have an impact on the clinical outcome of the disease.
doi:10.1136/annrheumdis-2012-202742
PMCID: PMC3818491  PMID: 23444193
2.  Classification Criteria for Systemic Sclerosis: An ACR-EULAR Collaborative Initiative 
Arthritis and rheumatism  2013;65(11):2737-2747.
Background
The 1980 classification criteria for systemic sclerosis (SSc) lack sensitivity in early SSc and limited cutaneous SSc. A joint ACR-EULAR committee was established to develop new classification criteria for SSc.
Methods
Using consensus methods, 23 candidate items were arranged in a multi-criteria additive point system with a threshold to classify cases as SSc. The classification system was reduced by clustering items, and simplifying weights. The system was tested by: a) determining specificity and sensitivity in SSc cases and controls with scleroderma-like disorders; b) validating against the combined view of a group of experts on a set of cases with or without SSc.
Results
Skin thickening of the fingers extending proximal to the MCPs is sufficient to be classified as SSc, if that is not present, seven additive items apply with varying weights for each: skin thickening of the fingers, finger tip lesions, telangiectasia, abnormal nailfold capillaries, interstitial lung disease or pulmonary arterial hypertension, Raynaud's phenomenon, and SSc-related autoantibodies. Sensitivity and specificity in the validation sample were 0.91 and 0.92 for the new classification criteria and 0.75 and 0.72 for the 1980 ARA classification criteria. All selected cases were classified in accordance with consensus-based expert opinion. All cases classified as SSc by the 1980 ARA criteria were classified with the new criteria, and several additional cases were now considered to be SSc.
Conclusion
The ACR-EULAR classification criteria for SSc performed better than the 1980 ARA Criteria for SSc and should allow for more patients to be classified correctly as SSc.
doi:10.1002/art.38098
PMCID: PMC3930146  PMID: 24122180
Systemic Sclerosis; Scleroderma; Classification Criteria; Conjoint Analysis; Multi Criteria Additive Point System; Validation; ACR-EULAR
3.  A systemic sclerosis and systemic lupus erythematosus pan-meta-GWAS reveals new shared susceptibility loci 
Human Molecular Genetics  2013;22(19):4021-4029.
Systemic sclerosis (SSc) and systemic lupus erythematosus (SLE) are two archetypal systemic autoimmune diseases which have been shown to share multiple genetic susceptibility loci. In order to gain insight into the genetic basis of these diseases, we performed a pan-meta-analysis of two genome-wide association studies (GWASs) together with a replication stage including additional SSc and SLE cohorts. This increased the sample size to a total of 21 109 (6835 cases and 14 274 controls). We selected for replication 19 SNPs from the GWAS data. We were able to validate KIAA0319L (P = 3.31 × 10−11, OR = 1.49) as novel susceptibility loci for SSc and SLE. Furthermore, we also determined that the previously described SLE susceptibility loci PXK (P = 3.27 × 10−11, OR = 1.20) and JAZF1 (P = 1.11 × 10−8, OR = 1.13) are shared with SSc. Supporting these new discoveries, we observed that KIAA0319L was overexpressed in peripheral blood cells of SSc and SLE patients compared with healthy controls. With these, we add three (KIAA0319L, PXK and JAZF1) and one (KIAA0319L) new susceptibility loci for SSc and SLE, respectively, increasing significantly the knowledge of the genetic basis of autoimmunity.
doi:10.1093/hmg/ddt248
PMCID: PMC3766185  PMID: 23740937
4.  Osteoprotegerin CGA Haplotype Protection against Cerebrovascular Complications in Anti-CCP Negative Patients with Rheumatoid Arthritis 
PLoS ONE  2014;9(9):e106823.
Introduction
Rheumatoid arthritis is an inflammatory disease with high incidence of cardiovascular disease due to accelerated atherosclerosis. Osteoprotegerin (OPG) has been associated with increased risk of atherosclerotic disease in the general population. Several polymorphisms in the OPG gene with functional effects on cardiovascular disease in non-rheumatic individuals have been described. Therefore, we aimed to analyze the effect of three of these functional OPG polymorphisms on the risk of cardiovascular disease in a large and well-characterized cohort of Spanish patients with rheumatoid arthritis.
Methods
Three OPG gene variants (rs3134063, rs2073618 and rs3134069) were genotyped by TaqMan assays in 2027 Spanish patients with rheumatoid arthritis. Anti-cyclic citrullinated peptide (anti-CCP) antibody testing was positive in 997 of 1714 tested. Also, 18.3% of the whole series had experienced cardiovascular events, including 5.4% with cerebrovascular accidents. The relationship between OPG variants and cardiovascular events was assessed using Cox regression.
Results
No association between OPG gene variants and cardiovascular disease was observed in the whole group of rheumatoid arthritis patients or in anti-CCP positive patients. Nevertheless, a protective effect of CGA haplotype on the risk of cardiovascular disease in general, and specifically in the risk of cerebrovascular complications after adjusting for sex, age at disease diagnosis and traditional cardiovascular risk factors was disclosed in anti-CCP negative patients (HR = 0.54; 95%CI: 0.31–0.95; p = 0.032 and HR = 0.17; 95%CI: 0.04–0.78; p = 0.022, respectively).
Conclusion
Our results indicate a protective effect of the OPG CGA haplotype on cardiovascular risk, mainly due to a protective effect against cerebrovascular events in anti-CCP negative rheumatoid arthritis patients.
doi:10.1371/journal.pone.0106823
PMCID: PMC4153690  PMID: 25184828
5.  Interferon regulatory factor 5 genetic variants are associated with cardiovascular disease in patients with rheumatoid arthritis 
Arthritis Research & Therapy  2014;16(4):R146.
Introduction
Rheumatoid arthritis (RA) is a complex polygenic inflammatory disease associated with accelerated atherosclerosis and increased cardiovascular (CV) disease risk. Interferon regulatory factor 5 (IRF5) is a regulator of type I interferon induction. Recently, researchers have described an association between multiple single-nucleotide polymorphisms of the IRF5 gene and some rheumatic disorders. In this study, we aimed to evaluate whether three different haplotype blocks within the IRF5 locus which have been shown to alter the protein function are involved in the risk of CV events occurring in Spanish RA patients.
Methods
Three IRF5 polymorphisms (rs2004640, rs2070197 and rs10954213) representative of each haplotype group were genotyped by performing TaqMan assays using a 7900HT Fast Real-Time PCR System with tissue from a total of 2,137 Spanish patients diagnosed with RA. Among them, 390 (18.2%) had experienced CV events. The relationship of IRF5 genotypes and haplotypes to CV events was tested using Cox regression.
Results
Male sex, age at RA diagnosis and most traditional risk factors (hypertension, dyslipidemia and smoking habit) were associated with increased risk for CV events in the RA population. Interestingly, a protective effect of both IRF5 rs2004640 GG and IRF5 rs10954213 GG genotypes against the risk for CV events after adjusting the results for sex, age at RA diagnosis and traditional CV disease risk factors was observed (hazard ratio (HR) = 0.6, 95% confidence interval (CI) = 0.38 to 0.92, P = 0.02; and HR = 0.58, 95% CI = 0.36 to 0.95, P = 0.03, respectively). Moreover, we detected a protective effect of the GTG haplotype against the risk for CV events after adjusting the results for potential confounding factors (HR = 0.72, 95% CI = 0.56 to 0.93, P = 0.012).
Conclusions
Our results reveal that IRF5 gene variants are associated with risk of CV events in patients with RA.
doi:10.1186/ar4608
PMCID: PMC4227041  PMID: 25011482
6.  Implication of IL-2/IL-21 region in systemic sclerosis genetic susceptibility 
Annals of the rheumatic diseases  2012;72(7):10.1136/annrheumdis-2012-202357.
Objective
The interleukin 2 (IL-2) and interleukin 21 (IL-21) locus at chromosome 4q27 has been associated with several autoimmune diseases, and both genes are related to immune system functions. The aim of this study was to evaluate the role of the IL-2/IL-21 locus in systemic sclerosis (SSc).
Patients and methods
The case control study included 4493 SSc Caucasian patients and 5856 healthy controls from eight Caucasian populations (Spain, Germany, The Netherlands, USA, Italy, Sweden, UK and Norway). Four single nucleotide polymorphisms (rs2069762, rs6822844, rs6835457 and rs907715) were genotyped using TaqMan allelic discrimination assays.
Results
We observed evidence of association of the rs6822844 and rs907715 variants with global SSc (pc=6.6E-4 and pc=7.2E-3, respectively). Similar statistically significant associations were observed for the limited cutaneous form of the disease. The conditional regression analysis suggested that the most likely genetic variation responsible for the association was the rs6822844 polymorphism. Consistently, the rs2069762A-rs6822844T-rs6835457G-rs907715T allelic combination showed evidence of association with SSc and limited cutaneous SSc subtype (pc=1.7E-03 and pc=8E-4, respectively).
Conclusions
These results suggested that the IL-2/IL-21 locus influences the genetic susceptibility to SSc. Moreover, this study provided further support for the IL-2/IL-21 locus as a common genetic factor in autoimmune diseases.
doi:10.1136/annrheumdis-2012-202357
PMCID: PMC3887514  PMID: 23172754
7.  Lack of replication of higher genetic risk load in men than in women with systemic lupus erythematosus 
Arthritis Research & Therapy  2014;16(3):R128.
Introduction
We aimed to replicate a recent study which showed higher genetic risk load at 15 loci in men than in women with systemic lupus erythematosus (SLE). This difference was very significant, and it was interpreted as indicating that men require more genetic susceptibility than women to develop SLE.
Methods
Nineteen SLE-associated loci (thirteen of which are shared with the previous study) were analyzed in 1,457 SLE patients and 1,728 healthy controls of European ancestry. Genetic risk load was calculated as sex-specific sum genetic risk scores (GRSs).
Results
Our results did not replicate those of the previous study at either the level of individual loci or the global level of GRSs. GRSs were larger in women than in men (4.20 ± 1.07 in women vs. 3.27 ± 0.98 in men). This very significant difference (P < 10−16) was more dependent on the six new loci not included in the previous study (59% of the difference) than on the thirteen loci that are shared (the remaining 41%). However, the 13 shared loci also showed a higher genetic risk load in women than in men in our study (P = 6.6 × 10−7), suggesting that heterogeneity of participants, in addition to different loci, contributed to the opposite results.
Conclusion
Our results show the lack of a clear trend toward higher genetic risk in one of the sexes for the analyzed SLE loci. They also highlight several limitations of assessments of genetic risk load, including the possibility of ascertainment bias with loci discovered in studies that have included mainly women.
doi:10.1186/ar4585
PMCID: PMC4095681  PMID: 24946689
8.  The wide expansion of hepatitis delta virus-like ribozymes throughout trypanosomatid genomes is linked to the spreading of L1Tc/ingi clade mobile elements 
BMC Genomics  2014;15(1):340.
Background
Hepatitis Delta Virus (HDV)-like ribozymes have recently been found in many mobile elements in which they take part in a mechanism that releases intermediate RNAs from cellular co-transcripts. L1Tc in Trypanosoma cruzi is one of the elements in which such a ribozyme is located. It lies in the so-called Pr77-hallmark, a conserved region shared by retrotransposons belonging to the trypanosomatid L1Tc/ingi clade. The wide distribution of the Pr77-hallmark detected in trypanosomatid retrotransposons renders the potential catalytic activity of these elements worthy of study: their distribution might contribute to host genetic regulation at the mRNA level. Indeed, in Leishmania spp, the pervasive presence of these HDV-like ribozyme-containing mobile elements in certain 3′-untranslated regions of protein-coding genes has been linked to mRNA downregulation.
Results
Intensive screening of publicly available trypanosomatid genomes, combined with manual folding analyses, allowed the isolation of putatively Pr77-hallmarks with HDV-like ribozyme activity. This work describes the conservation of an HDV-like ribozyme structure in the Pr77 sequence of retrotransposons in a wide range of trypanosomatids, the catalytic function of which is maintained in the majority.
These results are consistent with the previously suggested common phylogenetic origin of the elements that belong to this clade, although in some cases loss of functionality appears to have occurred and/or perhaps molecular domestication by the host.
Conclusions
These HDV-like ribozymes are widely distributed within retrotransposons across trypanosomatid genomes. This type of ribozyme was once thought to be rare in nature, but in fact it would seem to be abundant in trypanosomatid transcripts. It can even form part of the pool of mRNA 3′-untranslated regions, particularly in Leishmania spp. Its putative regulatory role in host genetic expression is discussed.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-340) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-340
PMCID: PMC4035085  PMID: 24884364
Retrotransposon; LINE; SINE; HDV-like ribozyme; Pr77; Trypanosoma; Leishmania; L1Tc; SIDER; ingi
9.  Confirmation of TNIP1 but not RHOB and PSORS1C1 as systemic sclerosis risk factors in a large independent replication study 
Annals of the rheumatic diseases  2012;72(4):10.1136/annrheumdis-2012-201888.
Introduction
A recent genome-wide association study in European systemic sclerosis (SSc) patients identified three loci (PSORS1C1, TNIP1 and RHOB) as novel genetic risk factors for the disease. The aim of this study was to replicate the previously mentioned findings in a large multicentre independent SSc cohort of Caucasian ancestry.
Methods
4389 SSc patients and 7611 healthy controls from different European countries and the USA were included in the study. Six single nucleotide polymorphisms (SNP): rs342070, rs13021401 (RHOB), rs2233287, rs4958881, rs3792783 (TNIP1) and rs3130573 (PSORS1C1) were analysed. Overall significance was calculated by pooled analysis of all the cohorts. Haplotype analyses and conditional logistic regression analyses were carried out to explore further the genetic structure of the tested loci.
Results
Pooled analyses of all the analysed SNPs in TNIP1 revealed significant association with the whole disease (rs2233287 pMH=1.94×10−4, OR 1.19; rs4958881 pMH=3.26×10−5, OR 1.19; rs3792783 pMH=2.16×10−4, OR 1.19). These associations were maintained in all the subgroups considered. PSORS1C1 comparison showed association with the complete set of patients and all the subsets except for the anti-centromere-positive patients. However, the association was dependent on different HLA class II alleles. The variants in the RHOB gene were not associated with SSc or any of its subsets.
Conclusions
These data confirmed the influence of TNIP1 on an increased susceptibility to SSc and reinforced this locus as a common autoimmunity risk factor.
doi:10.1136/annrheumdis-2012-201888
PMCID: PMC3887516  PMID: 22896740
10.  Lack of Association between ABO, PPAP2B, ADAMST7, PIK3CG, and EDNRA and Carotid Intima-Media Thickness, Carotid Plaques, and Cardiovascular Disease in Patients with Rheumatoid Arthritis 
Mediators of Inflammation  2014;2014:756279.
Introduction. Rheumatoid arthritis (RA) is a polygenic disease associated with accelerated atherosclerosis and increased cardiovascular (CV) mortality. Recent studies have identified the ABO rs579459, PPAP2B rs17114036, and ADAMTS7 rs3825807 polymorphisms as genetic variants associated with coronary artery disease and the PIK3CG rs17398575 and EDNRA rs1878406 polymorphisms as the most significant signals related to the presence of carotid plaque in nonrheumatic Caucasian individuals. Accordingly, we evaluated the potential relationship between these 5 polymorphisms and subclinical atherosclerosis (assessed by carotid intima-media thickness (cIMT) and presence/absence of carotid plaques) and CV disease in RA. Material and Methods. 2140 Spanish RA patients were genotyped for the 5 polymorphisms by TaqMan assays. Subclinical atherosclerosis was evaluated in 620 of these patients by carotid ultrasonography technology. Results. No statistically significant differences were found when each polymorphism was assessed according to cIMT values and presence/absence of carotid plaques in RA, after adjusting the results for potential confounders. Moreover, no significant differences were obtained when RA patients were stratified according to the presence/absence of CV disease after adjusting for potential confounders. Conclusion. Our results do not confirm association between ABO rs579459, PPAP2B rs17114036, ADAMTS7 rs3825807, PIK3CG rs17398575, and EDNRA rs1878406 and subclinical atherosclerosis and CV disease in RA.
doi:10.1155/2014/756279
PMCID: PMC3984817  PMID: 24795506
11.  Lack of validation of genetic variants associated with anti–tumor necrosis factor therapy response in rheumatoid arthritis: a genome-wide association study replication and meta-analysis 
Introduction
In this study, our aim was to elucidate the role of four polymorphisms identified in a prior large genome-wide association study (GWAS) in which the investigators analyzed the responses of patients with rheumatoid arthritis (RA) to treatment with tumor necrosis factor inhibitors (TNFi). The authors of that study reported that the four genetic variants were significantly associated. However, none of the associations reached GWAS significance, and two subsequent studies failed to replicate these associations.
Methods
The four polymorphisms (rs12081765, rs1532269, rs17301249 and rs7305646) were genotyped in a total of 634 TNFi-treated RA patients of Spanish Caucasian origin. Four outcomes were evaluated: changes in the Disease Activity Score in 28 joints (DAS28) after 6 and 12 months of treatment and classification according to the European League Against Rheumatism (EULAR) response criteria at the same time points. Association with DAS28 changes was assessed by linear regression using an additive genetic model. Contingency tables of genotype and allele frequencies between EULAR responder and nonresponder patients were compared. In addition, we combined our data with those of previously reported studies in a meta-analysis including 2,998 RA patients.
Results
None of the four genetic variants showed an association with response to TNFi in any of the four outcomes analyzed in our Spanish patients. In addition, only rs1532269 yielded a suggestive association (P = 0.0033) with the response to TNFi when available data from previous studies were combined in the meta-analysis.
Conclusion
Our data suggest that the rs12081765, rs1532269, rs17301249 and rs7305646 genetic variants do not have a role as genetic predictors of TNFi treatment outcomes.
doi:10.1186/ar4504
PMCID: PMC4060376  PMID: 24612463
13.  SMAD3 rs17228212 Gene Polymorphism Is Associated with Reduced Risk to Cerebrovascular Accidents and Subclinical Atherosclerosis in Anti-CCP Negative Spanish Rheumatoid Arthritis Patients 
PLoS ONE  2013;8(10):e77695.
Rheumatoid arthritis (RA) is a complex polygenic inflammatory disease associated with accelerated atherosclerosis and increased risk of cardiovascular (CV) disease. Previous genome-wide association studies have described SMAD3 rs17228212 polymorphism as an important signal associated with CV events. The aim of the present study was to evaluate for the first time the relationship between this gene polymorphism and the susceptibility to CV manifestations and its potential association with the presence of subclinical atherosclerosis assessed by the evaluation of carotid intima-media thickness (cIMT) in patients with RA.
Methods
One thousand eight hundred and ninety-seven patients fulfilling classification criteria for RA were genotyped for SMAD3 rs17228212 gene polymorphism through TaqMan genotyping assay. Also, subclinical atherosclerosis determined by the assessment of cIMT was analyzed in a subgroup of these patients by carotid ultrasonography.
Results
No statistically significant differences were observed when allele frequencies of RA patients with or without CV events were compared. Nevertheless, when RA patients were stratified according to anti-cyclic citrullinated peptide (anti-CCP) status, we found that in RA patients who were negative for anti-CCP antibodies, the presence of C allele of SMAD3 rs17228212 polymorphism conferred a protective effect against the risk of cerebrovascular accident (CVA) after adjustment for demographic and classic CV risk factors (HR [95%CI]=0.36 [0.14–0.94], p=0.038) in a Cox regression model. Additionally, correlation between the presence of C allele of SMAD3 rs17228212 polymorphism and lower values of cIMT was found after adjustment for demographic and classic CV risk factors (p-value=0.0094) in the anti-CCP negative RA patients.
Conclusions
Our results revealed that SMAD3 rs17228212 gene variant is associated with lower risk of CVA and less severe subclinical atherosclerosis in RA patients negative for anti-CCP antibodies. These findings may have importance to establish predictive models of CV disease in RA patients according to anti-CCP status.
doi:10.1371/journal.pone.0077695
PMCID: PMC3804609  PMID: 24204921
14.  Identification of CSK as a systemic sclerosis genetic risk factor through Genome Wide Association Study follow-up 
Human Molecular Genetics  2012;21(12):2825-2835.
Systemic sclerosis (SSc) is complex autoimmune disease affecting the connective tissue; influenced by genetic and environmental components. Recently, we performed the first successful genome-wide association study (GWAS) of SSc. Here, we perform a large replication study to better dissect the genetic component of SSc. We selected 768 polymorphisms from the previous GWAS and genotyped them in seven replication cohorts from Europe. Overall significance was calculated for replicated significant SNPs by meta-analysis of the replication cohorts and replication-GWAS cohorts (3237 cases and 6097 controls). Six SNPs in regions not previously associated with SSc were selected for validation in another five independent cohorts, up to a total of 5270 SSc patients and 8326 controls. We found evidence for replication and overall genome-wide significance for one novel SSc genetic risk locus: CSK [P-value = 5.04 × 10−12, odds ratio (OR) = 1.20]. Additionally, we found suggestive association in the loci PSD3 (P-value = 3.18 × 10−7, OR = 1.36) and NFKB1 (P-value = 1.03 × 10−6, OR = 1.14). Additionally, we strengthened the evidence for previously confirmed associations. This study significantly increases the number of known putative genetic risk factors for SSc, including the genes CSK, PSD3 and NFKB1, and further confirms six previously described ones.
doi:10.1093/hmg/dds099
PMCID: PMC3368627  PMID: 22407130
15.  Inhibition of focal adhesion kinase prevents experimental lung fibrosis and myofibroblast formation 
Arthritis and Rheumatism  2012;64(5):1653-1664.
Objective
Enhanced adhesive signaling including activation of the focal adhesion kinase (FAK) is a hallmark of fibroblasts from lung fibrosis patients, and FAK has been therefore hypothesized to be a key mediator of this disease. This study was undertaken to characterize the contribution of FAK to the development of pulmonary fibrosis both in vivo and in vitro.
Methods
FAK expression and activity were analyzed in lung tissue samples from lung fibrosis patients by immunohistochemistry. Mice orally treated with the FAK inhibitor, PF-562,271, or with siRNA-mediated silencing of FAK, were exposed to intratracheally instilled bleomycin to induce lung fibrosis, and the lungs were harvested for histological and biochemical analysis. Using endothelin-1 (ET-1) as stimulus, cell adhesion and contraction, as well as profibrotic gene expression were studied in fibroblasts isolated from wild type and FAK-deficient mouse embryos. ET-1-mediated FAK activation and gene expression were studied in primary mouse lung fibroblasts, as well as in wild type and integrin β1-deficient fibroblasts.
Results
Increased FAK expression and activity are upregulated in fibroblast foci and remodeled vessels in lung fibrosis patients. Pharmacological or siRNA-mediated targeting of FAK resulted in marked abrogation of bleomycin-induced lung fibrosis. Loss of FAK impaired the acquisition of a profibrotic phenotype in response to ET-1. Profibrotic gene expression leading to myofibroblast differentiation required cell adhesion, and was driven by Jun N-terminal kinase activation through integrin β1/FAK signaling.
Conclusion
These results implicate FAK as a central mediator of fibrogenesis, and highlight this kinase as a potential therapeutic target in fibrotic diseases.
doi:10.1002/art.33482
PMCID: PMC3338902  PMID: 22492165
16.  Items for developing revised classification criteria in systemic sclerosis: results of a consensus exercise with the ACR/EULAR working committee for classification criteria in systemic sclerosis 
Arthritis care & research  2012;64(3):351-357.
Background
Classification criteria for systemic sclerosis (SSc) are being updated.
Objective
To select a set of items potentially useful for the classification of SSc using consensus procedures including the Delphi and nominal group techniques (NGT).
Methods
Items were identified through two independent consensus exercises performed by the Scleroderma Clinical Trials Consortium (SCTC) and the EULAR Scleroderma Trials and Research Group (EUSTAR). The first-round items from both exercises were collated and redundancies were removed leaving 168 items. A 3-round Delphi exercise was performed using a 1–9 scale (1=completely inappropriate and 9=completely appropriate) and a consensus meeting using NGT. During the last Delphi, the items were ranked on a 1–10 scale.
Results
Round 1: 106 experts rated the 168 items. Those with a median score <4 were removed, resulting in a list of 102 items. Round 2: The items were again rated for appropriateness and subjected to a consensus meeting using NGT by European and North American SSc experts (n=16), resulting in 23 items. Round 3: SSc experts (n=26) then individually scored each of the 23 items in a last Delphi round, using an appropriateness score (1–9) and ranking their 10 most appropriate items for classification of SSc. Presence of skin thickening, SSc-specific autoantibodies, abnormal nailfold capillary pattern and Raynaud’s phenomenon ranked highest in the final list that also included items indicating internal organ involvement.
Conclusion
The Delphi exercise and NGT resulted in a set of 23 items for classification of SSc which will be assessed for their discriminative properties in a prospective study.
doi:10.1002/acr.20679
PMCID: PMC3288452  PMID: 22052558
Delphi technique; nominal group technique; systemic sclerosis; scleroderma; classification; classification criteria
17.  Validation of potential classification criteria for systemic sclerosis 
Arthritis care & research  2012;64(3):358-367.
Background
Classification criteria for systemic sclerosis (SSc) are being updated jointly by ACR and EULAR. Potential items for classification were reduced to 23 using Delphi and Nominal Group Techniques. We evaluated the face, discriminant and construct validity of the items to be further studied as potential criteria.
Methods
Face validity was evaluated using the frequency of items in patients sampled from the Canadian Scleroderma Research Group, 1000 Faces of Lupus, the Pittsburgh, Toronto, Madrid and Berlin CTD databases. SSc (n=783) were compared to 1071 patients with diseases similar to SSc (mimickers): SLE (n=499), myositis (n=171), Sjögren’s syndrome (n=95), Raynaud’s phenomenon (RP) (n=228), MCTD (n=29), and idiopathic PAH (n=49). Discriminant validity was evaluated using odds ratios (OR). For construct validity, empiric ranking was compared to expert ranking.
Results
Compared to mimickers, SSc are more likely to have skin thickening (OR=427), telangiectasias (OR=91), anti-RNA polymerase III antibody (OR=75), puffy fingers (OR=35), finger flexion contractures (OR=29), tendon/bursal friction rubs (OR=27), anti-topoisomerase-I antibody (OR=25), RP (OR=24), finger tip ulcers/pitting scars (OR=19), anti-centromere antibody(OR=14), abnormal nailfold capillaries (OR=10), GERD symptoms (OR=8), and ANA, calcinosis, dysphagia, esophageal dilation (all OR=6), interstitial lung disease/pulmonary fibrosis (OR=5) and anti-PM-Scl antibody (OR=2). Reduced DLCO, PAH, and reduced FVC had OR<2. Renal crisis and digital pulp loss/acro-osteolysis did not occur in SSc mimickers (OR not estimated). Empiric and expert ranking were correlated (Spearman rho 0.53, p=0.01).
Conclusion
The candidate items have good face, discriminant and construct validity. Further item reduction will be evaluated in prospective SSc and mimicker cases.
doi:10.1002/acr.20684
PMCID: PMC3376721  PMID: 22052658
Systemic Sclerosis; Scleroderma; Classification Criteria; Validity; Bayesian
18.  A GWAS follow-up study reveals the association of the IL12RB2 gene with systemic sclerosis in Caucasian populations 
Human Molecular Genetics  2011;21(4):926-933.
A single-nucleotide polymorphism (SNP) at the IL12RB2 locus showed a suggestive association signal in a previously published genome-wide association study (GWAS) in systemic sclerosis (SSc). Aiming to reveal the possible implication of the IL12RB2 gene in SSc, we conducted a follow-up study of this locus in different Caucasian cohorts. We analyzed 10 GWAS-genotyped SNPs in the IL12RB2 region (2309 SSc patients and 5161 controls). We then selected three SNPs (rs3790567, rs3790566 and rs924080) based on their significance level in the GWAS, for follow-up in an independent European cohort comprising 3344 SSc and 3848 controls. The most-associated SNP (rs3790567) was further tested in an independent cohort comprising 597 SSc patients and 1139 controls from the USA. After conditional logistic regression analysis of the GWAS data, we selected rs3790567 [PMH= 1.92 × 10−5 odds ratio (OR) = 1.19] as the genetic variant with the firmest independent association observed in the analyzed GWAS peak of association. After the first follow-up phase, only the association of rs3790567 was consistent (PMH= 4.84 × 10−3 OR = 1.12). The second follow-up phase confirmed this finding (Pχ2 = 2.82 × 10−4 OR = 1.34). After performing overall pooled-analysis of all the cohorts included in the present study, the association found for the rs3790567 SNP in the IL12RB2 gene region reached GWAS-level significant association (PMH= 2.82 × 10−9 OR = 1.17). Our data clearly support the IL12RB2 genetic association with SSc, and suggest a relevant role of the interleukin 12 signaling pathway in SSc pathogenesis.
doi:10.1093/hmg/ddr522
PMCID: PMC3298110  PMID: 22076442
19.  The Systemic Lupus Erythematosus IRF5 Risk Haplotype Is Associated with Systemic Sclerosis 
PLoS ONE  2013;8(1):e54419.
Systemic sclerosis (SSc) is a fibrotic autoimmune disease in which the genetic component plays an important role. One of the strongest SSc association signals outside the human leukocyte antigen (HLA) region corresponds to interferon (IFN) regulatory factor 5 (IRF5), a major regulator of the type I IFN pathway. In this study we aimed to evaluate whether three different haplotypic blocks within this locus, which have been shown to alter the protein function influencing systemic lupus erythematosus (SLE) susceptibility, are involved in SSc susceptibility and clinical phenotypes. For that purpose, we genotyped one representative single-nucleotide polymorphism (SNP) of each block (rs10488631, rs2004640, and rs4728142) in a total of 3,361 SSc patients and 4,012 unaffected controls of Caucasian origin from Spain, Germany, The Netherlands, Italy and United Kingdom. A meta-analysis of the allele frequencies was performed to analyse the overall effect of these IRF5 genetic variants on SSc. Allelic combination and dependency tests were also carried out. The three SNPs showed strong associations with the global disease (rs4728142: P  = 1.34×10−8, OR  = 1.22, CI 95%  = 1.14–1.30; rs2004640: P  = 4.60×10−7, OR  = 0.84, CI 95%  = 0.78–0.90; rs10488631: P  = 7.53×10−20, OR  = 1.63, CI 95%  = 1.47–1.81). However, the association of rs2004640 with SSc was not independent of rs4728142 (conditioned P  = 0.598). The haplotype containing the risk alleles (rs4728142*A-rs2004640*T-rs10488631*C: P  = 9.04×10−22, OR  = 1.75, CI 95%  = 1.56–1.97) better explained the observed association (likelihood P-value  = 1.48×10−4), suggesting an additive effect of the three haplotypic blocks. No statistical significance was observed in the comparisons amongst SSc patients with and without the main clinical characteristics. Our data clearly indicate that the SLE risk haplotype also influences SSc predisposition, and that this association is not sub-phenotype-specific.
doi:10.1371/journal.pone.0054419
PMCID: PMC3553151  PMID: 23372721
20.  Analysis of Class II human leucocyte antigens in Italian and Spanish systemic sclerosis 
Rheumatology (Oxford, England)  2011;51(1):52-59.
Objective. To determine the role of Class II HLAs in SSc patients from Italy and Spain and in SSc patients of Caucasian ancestry.
Methods. Nine hundred and forty-four SSc patients (Italy 392 patients; Spain 452 patients) and 1320 ethnically matched healthy controls (Italy 398 patients; Spain 922 patients) were genotyped up to the fourth digit by PCR with sequence-specific oligonucleotides for HLA-DRB1, DQA1 and DQB1 loci. Patients included 390 ACA-positive and 254 anti-topo I-positive subjects. Associations between SSc or SSc-specific antibodies and HLA alleles or HLA haplotypes were sought via the chi-square test after 10 000-fold permutation testing. A meta-analysis including this study cohort and other Caucasoids samples was also conducted.
Results. In both the cohorts, the strongest association was observed between the HLA-DRB1*1104 allele and SSc or anti-topo I antibodies. The HLA-DRB1*1104 -DQA1*0501 -DQB1*0301 haplotype was overrepresented in Italian [odds ratio (OR) = 2.069, 95% asymptotic CIs (CI95) 1.486, 2.881; P < 0.001] and in Spanish patients (OR = 6.707, CI95 3.974, 11.319; P < 0.001) as well as in anti-topo-positive patients: Italy (OR = 2.642, CI95 1.78, 3.924; P < 0.001) and Spain (OR = 20.625, CI95 11.536, 36.876; P < 0.001). In both the populations we also identified an additional risk allele (HLA-DQB1*03) and a protective allele (HLA-DQB1*0501) in anti-topo-positive patients. The meta-analysis showed different statistically significant associations, the most interesting being the differential association between HLA-DRB1*01 alleles and ACAs (OR = 1.724, CI95 1.482, 2.005; P < 0.001) or topo I antibodies (OR = 0.5, CI95 0.384, 0.651; P < 0.001).
Conclusions. We describe multiple robust associations between SSc and HLA Class II antigens in Caucasoids that may help to understand the genetic architecture of SSc.
doi:10.1093/rheumatology/ker335
PMCID: PMC3276293  PMID: 22087014
systemic sclerosis; HLA; genetics; epidemiology
21.  Novel identification of the IRF7 region as an anticentromere autoantibody propensity locus in systemic sclerosis 
Annals of the Rheumatic Diseases  2011;71(1):114-119.
Objective
Systemic sclerosis (SSc) and systemic lupus erythematosus (SLE) are related chronic autoimmune diseases of complex aetiology in which the interferon (IFN) pathway plays a key role. Recent studies have reported an association between IRF7 and SLE which confers a risk to autoantibody production. A study was undertaken to investigate whether the IRF7 genomic region is also involved in susceptibility to SSc and the main clinical features.
Methods
Two case-control sets of Caucasian origin from the USA and Spain, comprising a total of 2316 cases of SSc and 2347 healthy controls, were included in the study. Five single nucleotide polymorphisms (SNPs) in the PHRF1-IRF7-CDHR5 locus were genotyped using TaqMan allelic discrimination technology. A meta-analysis was performed to test the overall effect of these genetic variants on SSc.
Results
Four out of five analysed SNPs were Significantly associated with the presence of anticentromere autoantibodies (ACA) in the patients with SSc in the combined analysis (rs1131665: pFDR=6.14 × 10−4, OR=0.78; rs4963128: pFDR=6.14 × 10−4, OR=0.79; rs702966: pFDR=3.83 × 10−3, OR=0.82; and rs2246614: pFDR=3.83 × 10−3, OR=0.83). Significant p values were also obtained when the disease was tested globally; however, the statistical significance was lost when the ACA-positive patients were excluded from the study, suggesting that these associations rely on ACA positivity. Conditional logistic regression and allelic combination analyses suggested that the functional IRF7 SNP rs1131665 is the most likely causal variant.
Conclusions
The results show that variation in the IRF7 genomic region is associated with the presence of ACA in patients with SSc, supporting other evidence that this locus represents a common risk factor for autoantibody production in autoimmune diseases.
doi:10.1136/annrheumdis-2011-200275
PMCID: PMC3369428  PMID: 21926187
22.  KCNA5 gene is not confirmed as a systemic sclerosis-related pulmonary arterial hypertension genetic susceptibility factor 
Arthritis Research & Therapy  2012;14(6):R273.
Introduction
Potassium voltage-gated channel shaker-related subfamily member 5 (KCNA5) is implicated in vascular tone regulation, and its inhibition during hypoxia produces pulmonary vasoconstriction. Recently, a protective association of the KCNA5 locus with systemic sclerosis (SSc) patients with pulmonary arterial hypertension (PAH) was reported. Hence, the aim of this study was to replicate these findings in an independent multicenter Caucasian SSc cohort.
Methods
The 2,343 SSc cases (179 PAH positive, confirmed by right-heart catheterization) and 2,690 matched healthy controls from five European countries were included in this study. Rs10744676 single-nucleotide polymorphism (SNP) was genotyped by using a TaqMan SNP genotyping assay.
Results
Individual population analyses of the selected KCNA5 genetic variant did not show significant association with SSc or any of the defined subsets (for example, limited cutaneous SSc, diffuse cutaneous SSc, anti-centromere autoantibody positive and anti-topoisomerase autoantibody positive). Furthermore, pooled analyses revealed no significant evidence of association with the disease or any of the subsets, not even the PAH-positive group. The comparison of PAH-positive patients with PAH-negative patients showed no significant differences among patients.
Conclusions
Our data do not support an important role of KCNA5 as an SSc-susceptibility factor or as a PAH-development genetic marker for SSc patients.
doi:10.1186/ar4124
PMCID: PMC3674598  PMID: 23270786
23.  Further Evidence of Subphenotype Association with Systemic Lupus Erythematosus Susceptibility Loci: A European Cases Only Study 
PLoS ONE  2012;7(9):e45356.
Introduction
Systemic Lupus Erythematosus (SLE) shows a spectrum of clinical manifestations that complicate its diagnosis, treatment and research. This variability is likely related with environmental exposures and genetic factors among which known SLE susceptibility loci are prime candidates. The first published analyses seem to indicate that this is the case for some of them, but results are still inconclusive and we aimed to further explore this question.
Methods
European SLE patients, 1444, recruited at 17 centres from 10 countries were analyzed. Genotypes for 26 SLE associated SNPs were compared between patients with and without each of 11 clinical features: ten of the American College of Rheumatology (ACR) classification criteria (except ANAs) and age of disease onset. These analyses were adjusted for centre of recruitment, top ancestry informative markers, gender and time of follow-up. Overlap of samples with previous studies was excluded for assessing replication.
Results
There were three new associations: the SNPs in XKR6 and in FAM167A-BLK were associated with lupus nephritis (OR = 0.76 and 1.30, Pcorr = 0.007 and 0.03, respectively) and the SNP of MECP2, which is in chromosome X, with earlier age of disease onset in men. The previously reported association of STAT4 with early age of disease onset was replicated. Some other results were suggestive of the presence of additional associations. Together, the association signals provided support to some previous findings and to the characterization of lupus nephritis, autoantibodies and age of disease onset as the clinical features more associated with SLE loci.
Conclusion
Some of the SLE loci shape the disease phenotype in addition to increase susceptibility to SLE. This influence is more prominent for some clinical features than for others. However, results are only partially consistent between studies and subphenotype specific GWAS are needed to unravel their genetic component.
doi:10.1371/journal.pone.0045356
PMCID: PMC3458859  PMID: 23049788
24.  Analysis of the association between CD40 and CD40 ligand polymorphisms and systemic sclerosis 
Arthritis Research & Therapy  2012;14(3):R154.
Introduction
The aim of the present study was to investigate the possible role of CD40 and CD40 ligand (CD40LG) genes in the susceptibility and phenotype expression of systemic sclerosis (SSc).
Methods
In total, 2,670 SSc patients and 3,245 healthy individuals from four European populations (Spain, Germany, The Netherlands, and Italy) were included in the study. Five single-nucleotide polymorphisms (SNPs) of CD40 (rs1883832, rs4810485, rs1535045) and CD40LG (rs3092952, rs3092920) were genotyped by using a predesigned TaqMan allele-discrimination assay technology. Meta-analysis was assessed to determine whether an association exists between the genetic variants and SSc or its main clinical subtypes.
Results
No evidence of association between CD40 and CD40LG genes variants and susceptibility to SSc was observed. Similarly, no significant statistical differences were observed when SSc patients were stratified by the clinical subtypes, the serologic features, and pulmonary fibrosis.
Conclusions
Our results do not suggest an important role of CD40 and CD40LG gene polymorphisms in the susceptibility to or clinical expression of SSc.
doi:10.1186/ar3890
PMCID: PMC3446540  PMID: 22731751
25.  Bias in effect size of systemic lupus erythematosus susceptibility loci across Europe: a case-control study 
Introduction
We aimed to investigate whether the effect size of the systemic lupus erythematosus (SLE) risk alleles varies across European subpopulations.
Methods
European SLE patients (n = 1,742) and ethnically matched healthy controls (n = 2,101) were recruited at 17 centres from 10 different countries. Only individuals with self-reported ancestry from the country of origin were included. In addition, participants were genotyped for top ancestry informative markers and for 25 SLE associated SNPs. The results were used to compare effect sizes between the Central Eureopan and Southern European subgroups.
Results
Twenty of the 25 SNPs showed independent association with SLE, These SNPs showed a significant bias to larger effect sizes in the Southern subgroup, with 15/20 showing this trend (P = 0.019) and a larger mean odds ratio of the 20 SNPs (1.46 vs. 1.34, P = 0.02) as well as a larger difference in the number of risk alleles (2.06 vs. 1.63, P = 0.027) between SLE patients and controls than for Central Europeans. This bias was reflected in a very significant difference in the cumulative genetic risk score (4.31 vs. 3.48, P = 1.8 × 10-32). Effect size bias was accompanied by a lower number of SLE risk alleles in the Southern subjects, both patients and controls, the difference being more marked between the controls (P = 1.1 × 10-8) than between the Southern and Central European patients (P = 0.016). Seven of these SNPs showed significant allele frequency clines.
Conclusion
Our findings showed a bias to larger effect sizes of SLE loci in the Southern Europeans relative to the Central Europeans together with clines of SLE risk allele frequencies. These results indicate the need to study risk allele clines and the implications of the polygenic model of inheritance in SLE.
doi:10.1186/ar3818
PMCID: PMC3446468  PMID: 22541939

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