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2.  High resolution identity testing of inactivated poliovirus vaccines 
Vaccine  2015;33(30):3533-3541.
Highlights
•Identity testing is a critical step in the quality control process.•Serological testing is the current approved method, but has certain limitations.•Existing molecular methods (qPCR) provide information about small genomic regions.•Random amplification and shotgun sequencing provide full genome coverage.•Distinction of highly similar viruses, and manufacturer-specific differences is possible.
Background
Definitive identification of poliovirus strains in vaccines is essential for quality control, particularly where multiple wild-type and Sabin strains are produced in the same facility. Sequence-based identification provides the ultimate in identity testing and would offer several advantages over serological methods.
Methods
We employed random RT-PCR and high throughput sequencing to recover full-length genome sequences from monovalent and trivalent poliovirus vaccine products at various stages of the manufacturing process.
Results
All expected strains were detected in previously characterised products and the method permitted identification of strains comprising as little as 0.1% of sequence reads. Highly similar Mahoney and Sabin 1 strains were readily discriminated on the basis of specific variant positions. Analysis of a product known to contain incorrect strains demonstrated that the method correctly identified the contaminants.
Conclusion
Random RT-PCR and shotgun sequencing provided high resolution identification of vaccine components. In addition to the recovery of full-length genome sequences, the method could also be easily adapted to the characterisation of minor variant frequencies and distinction of closely related products on the basis of distinguishing consensus and low frequency polymorphisms.
doi:10.1016/j.vaccine.2015.05.052
PMCID: PMC4504004  PMID: 26049003
Inactivated poliovirus vaccine; IPV; Vaccine contamination; Deep sequencing
3.  High-Density Genotyping of Immune Loci in Koreans and Europeans Identifies Eight New Rheumatoid Arthritis Risk Loci 
Objective
A highly polygenic etiology and high degree of allele-sharing between ancestries have been well-elucidated in genetic studies of rheumatoid arthritis. Recently, the high-density genotyping array Immunochip for immune disease loci identified 14 new rheumatoid arthritis risk loci among individuals of European ancestry. Here, we aimed to identify new rheumatoid arthritis risk loci using Korean-specific Immunochip data.
Methods
We analyzed Korean rheumatoid arthritis case-control samples using the Immunochip and GWAS array to search for new risk alleles of rheumatoid arthritis with anti-citrullinated peptide antibodies. To increase power, we performed a meta-analysis of Korean data with previously published European Immunochip and GWAS data, for a total sample size of 9,299 Korean and 45,790 European case-control samples.
Results
We identified 8 new rheumatoid arthritis susceptibility loci (TNFSF4, LBH, EOMES, ETS1–FLI1, COG6, RAD51B, UBASH3A and SYNGR1) that passed a genome-wide significance threshold (p<5×10−8), with evidence for three independent risk alleles at 1q25/TNFSF4. The risk alleles from the 7 new loci except for the TNFSF4 locus (monomorphic in Koreans), together with risk alleles from previously established RA risk loci, exhibited a high correlation of effect sizes between ancestries. Further, we refined the number of SNPs that represent potentially causal variants through a trans-ethnic comparison of densely genotyped SNPs.
Conclusion
This study demonstrates the advantage of dense-mapping and trans-ancestral analysis for identification of potentially causal SNPs. In addition, our findings support the importance of T cells in the pathogenesis and the fact of frequent overlap of risk loci among diverse autoimmune diseases.
doi:10.1136/annrheumdis-2013-204749
PMCID: PMC4467986  PMID: 24532676
Rheumatoid arthritis; Gene polymorphism; Anti-CCP
4.  Clinical Effects of Liraglutide in a Real-World Setting in Spain: eDiabetes-Monitor SEEN Diabetes Mellitus Working Group Study 
Diabetes Therapy  2015;6(2):173-185.
Introduction
A limitation with randomized controlled trials is that, while they provide unbiased evidence of the efficacy of interventions, they do so under unreal conditions and in a very limited and highly selected patient population. Our aim was to provide data about the effectiveness of liraglutide treatment in a real-world and clinical practice setting.
Methods
In a retrospective and observational study, data from 753 patients with type 2 diabetes were recorded through an online tool (eDiabetes-Monitor).
Results
Mean baseline glycated hemoglobin (HbA1c) was 8.4 ± 1.4% and mean body mass index (BMI) was 38.6 ± 5.4 kg/m2. After 3–6 months of treatment with liraglutide, we observed a change in HbA1c of −1.1 ± 1.2%, −4.6 ± 5.3 kg in weight and −1.7 ± 2.0 kg/m2 in BMI (p < 0.001 for all). Compared to baseline, there was a significant reduction in systolic blood pressure (−5.9 mmHg, p < 0.001), diastolic blood pressure (−3.2 mmHg, p < 0.001), LDL cholesterol (−0.189 mmol/l, p < 0.001) and triglycerides (−0.09 mmol/l, p = 0.021). In patients switched from DPP-4 inhibitors, liraglutide induced a decrease of −1.0% in HbA1c (p < 0.001) and a reduction in weight (−4.5 kg, p < 0.001). In patients treated with liraglutide as an add-on therapy to insulin a decrease of −1.08% in HbA1c (p < 0.001) and a weight reduction of −4.15 kg (p < 0.001) were observed.
Conclusion
Our study confirms the effectiveness of liraglutide in a real-life and clinical practice setting.
Funding
Spanish Society of Endocrinology and Nutrition.
Electronic supplementary material
The online version of this article (doi:10.1007/s13300-015-0112-4) contains supplementary material, which is available to authorized users.
doi:10.1007/s13300-015-0112-4
PMCID: PMC4478178  PMID: 26055216
Effectiveness; Liraglutide; Real life; Type 2 diabetes
5.  Major histocompatibility complex associations of ankylosing spondylitis are complex and involve further epistasis with ERAP1 
Nature Communications  2015;6:7146.
Ankylosing spondylitis (AS) is a common, highly heritable, inflammatory arthritis for which HLA-B*27 is the major genetic risk factor, although its role in the aetiology of AS remains elusive. To better understand the genetic basis of the MHC susceptibility loci, we genotyped 7,264 MHC SNPs in 22,647 AS cases and controls of European descent. We impute SNPs, classical HLA alleles and amino-acid residues within HLA proteins, and tested these for association to AS status. Here we show that in addition to effects due to HLA-B*27 alleles, several other HLA-B alleles also affect susceptibility. After controlling for the associated haplotypes in HLA-B, we observe independent associations with variants in the HLA-A, HLA-DPB1 and HLA-DRB1 loci. We also demonstrate that the ERAP1 SNP rs30187 association is not restricted only to carriers of HLA-B*27 but also found in HLA-B*40:01 carriers independently of HLA-B*27 genotype.
Ankylosing spondylitis is a common, highly inheritable inflammatory arthritis with poorly understood biology. Here Brown, Cortes and colleagues use fine mapping of the major histocompatibility complex and identify novel associations, and identify other HLA alleles that like HLA-B27 interact with ERAP1 variants to influence disease risk.
doi:10.1038/ncomms8146
PMCID: PMC4443427  PMID: 25994336
7.  TYK2 Protein-Coding Variants Protect against Rheumatoid Arthritis and Autoimmunity, with No Evidence of Major Pleiotropic Effects on Non-Autoimmune Complex Traits 
PLoS ONE  2015;10(4):e0122271.
Despite the success of genome-wide association studies (GWAS) in detecting a large number of loci for complex phenotypes such as rheumatoid arthritis (RA) susceptibility, the lack of information on the causal genes leaves important challenges to interpret GWAS results in the context of the disease biology. Here, we genetically fine-map the RA risk locus at 19p13 to define causal variants, and explore the pleiotropic effects of these same variants in other complex traits. First, we combined Immunochip dense genotyping (n = 23,092 case/control samples), Exomechip genotyping (n = 18,409 case/control samples) and targeted exon-sequencing (n = 2,236 case/controls samples) to demonstrate that three protein-coding variants in TYK2 (tyrosine kinase 2) independently protect against RA: P1104A (rs34536443, OR = 0.66, P = 2.3x10-21), A928V (rs35018800, OR = 0.53, P = 1.2x10-9), and I684S (rs12720356, OR = 0.86, P = 4.6x10-7). Second, we show that the same three TYK2 variants protect against systemic lupus erythematosus (SLE, Pomnibus = 6x10-18), and provide suggestive evidence that two of the TYK2 variants (P1104A and A928V) may also protect against inflammatory bowel disease (IBD; Pomnibus = 0.005). Finally, in a phenome-wide association study (PheWAS) assessing >500 phenotypes using electronic medical records (EMR) in >29,000 subjects, we found no convincing evidence for association of P1104A and A928V with complex phenotypes other than autoimmune diseases such as RA, SLE and IBD. Together, our results demonstrate the role of TYK2 in the pathogenesis of RA, SLE and IBD, and provide supporting evidence for TYK2 as a promising drug target for the treatment of autoimmune diseases.
doi:10.1371/journal.pone.0122271
PMCID: PMC4388675  PMID: 25849893
8.  Effect of Formaldehyde Inactivation on Poliovirus 
Journal of Virology  2014;88(20):11955-11964.
ABSTRACT
Inactivated polio vaccines, which have been used in many countries for more than 50 years, are produced by treating live poliovirus (PV) with formaldehyde. However, the molecular mechanisms underlying virus inactivation are not well understood. Infection by PV is initiated by virus binding to specific cell receptors, which results in viral particles undergoing sequential conformational changes that generate altered structural forms (135S and 80S particles) and leads to virus cell entry. We have analyzed the ability of inactivated PV to bind to the human poliovirus receptor (hPVR) using various techniques such as ultracentrifugation, fluorescence-activated cell sorting flow cytometry and real-time reverse transcription-PCR (RT-PCR). The results showed that although retaining the ability to bind to hPVR, inactivated PV bound less efficiently in comparison to live PV. We also found that inactivated PV showed resistance to structural conversion in vitro, as judged by measuring changes in antigenicity, the ability to bind to hPVR, and viral RNA release at high temperature. Furthermore, viral RNA from inactivated PV was shown to be modified, since cDNA yields obtained by RT-PCR amplification were severely reduced and no infectious virus was recovered after RNA transfection into susceptible cells.
IMPORTANCE This study represents a novel insight into the molecular mechanisms responsible for poliovirus inactivation. We show that inactivation with formaldehyde has an effect on early steps of viral replication as it reduces the ability of PV to bind to hPVR, decreases the sensitivity of PV to convert to 135S particles, and abolishes the infectivity of its viral RNA. These changes are likely responsible for the loss of infectivity shown by PV following inactivation. Techniques used in this study represent new approaches for the characterization of inactivated PV products and could be useful in developing improved methods for the production and quality control testing of inactivated polio vaccines. Measuring the antigenicity, capsid stability, and RNA integrity of inactivated PV samples could help establishing the optimal balance between the loss of infectivity and the preservation of virus antigenicity during inactivation.
doi:10.1128/JVI.01809-14
PMCID: PMC4178759  PMID: 25100844
9.  Rheumatoid arthritis response to treatment across IgG1 allotype – anti-TNF incompatibility: a case-only study 
Introduction
We have hypothesized that incompatibility between the G1m genotype of the patient and the G1m1 and G1m17 allotypes carried by infliximab (INX) and adalimumab (ADM) could decrease the efficacy of these anti-tumor necrosis factor (anti-TNF) antibodies in the treatment of rheumatoid arthritis (RA).
Methods
The G1m genotypes were analyzed in three collections of patients with RA totaling 1037 subjects. The first, used for discovery, comprised 215 Spanish patients. The second and third were successively used for replication. They included 429 British and Greek patients and 393 Spanish and British patients, respectively. Two outcomes were considered: change in the Disease Activity Score in 28 joint (ΔDAS28) and the European League Against Rheumatism (EULAR) response criteria.
Results
An association between less response to INX and incompatibility of the G1m1,17 allotype was found in the discovery collection at 6 months of treatment (P = 0.03). This association was confirmed in the replications (P = 0.02 and 0.08, respectively) leading to a global association (P = 0.001) that involved a mean difference in ΔDAS28 of 0.4 units between compatible and incompatible patients (2.3 ± 1.5 in compatible patients vs. 1.9 ± 1.5 in incompatible patients) and an increase in responders and decrease in non-responders according to the EULAR criteria (P = 0.03). A similar association was suggested for patients treated with ADM in the discovery collection, but it was not supported by replication.
Conclusions
Our results suggest that G1m1,17 allotypes are associated with response to INX and could aid improved therapeutic targeting in RA.
Electronic supplementary material
The online version of this article (doi:10.1186/s13075-015-0571-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s13075-015-0571-z
PMCID: PMC4411723  PMID: 25885039
10.  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
11.  Genetics of rheumatoid arthritis contributes to biology and drug discovery 
Okada, Yukinori | Wu, Di | Trynka, Gosia | Raj, Towfique | Terao, Chikashi | Ikari, Katsunori | Kochi, Yuta | Ohmura, Koichiro | Suzuki, Akari | Yoshida, Shinji | Graham, Robert R. | Manoharan, Arun | Ortmann, Ward | Bhangale, Tushar | Denny, Joshua C. | Carroll, Robert J. | Eyler, Anne E. | Greenberg, Jeffrey D. | Kremer, Joel M. | Pappas, Dimitrios A. | Jiang, Lei | Yin, Jian | Ye, Lingying | Su, Ding-Feng | Yang, Jian | Xie, Gang | Keystone, Ed | Westra, Harm-Jan | Esko, Tõnu | Metspalu, Andres | Zhou, Xuezhong | Gupta, Namrata | Mirel, Daniel | Stahl, Eli A. | Diogo, Dorothée | Cui, Jing | Liao, Katherine | Guo, Michael H. | Myouzen, Keiko | Kawaguchi, Takahisa | Coenen, Marieke J.H. | van Riel, Piet L.C.M. | van de Laar, Mart A.F.J. | Guchelaar, Henk-Jan | Huizinga, Tom W.J. | Dieudé, Philippe | Mariette, Xavier | Bridges, S. Louis | Zhernakova, Alexandra | Toes, Rene E.M. | Tak, Paul P. | Miceli-Richard, Corinne | Bang, So-Young | Lee, Hye-Soon | Martin, Javier | Gonzalez-Gay, Miguel A. | Rodriguez-Rodriguez, Luis | Rantapää-Dahlqvist, Solbritt | Ärlestig, Lisbeth | Choi, Hyon K. | Kamatani, Yoichiro | Galan, Pilar | Lathrop, Mark | Eyre, Steve | Bowes, John | Barton, Anne | de Vries, Niek | Moreland, Larry W. | Criswell, Lindsey A. | Karlson, Elizabeth W. | Taniguchi, Atsuo | Yamada, Ryo | Kubo, Michiaki | Liu, Jun S. | Bae, Sang-Cheol | Worthington, Jane | Padyukov, Leonid | Klareskog, Lars | Gregersen, Peter K. | Raychaudhuri, Soumya | Stranger, Barbara E. | De Jager, Philip L. | Franke, Lude | Visscher, Peter M. | Brown, Matthew A. | Yamanaka, Hisashi | Mimori, Tsuneyo | Takahashi, Atsushi | Xu, Huji | Behrens, Timothy W. | Siminovitch, Katherine A. | Momohara, Shigeki | Matsuda, Fumihiko | Yamamoto, Kazuhiko | Plenge, Robert M.
Nature  2013;506(7488):376-381.
A major challenge in human genetics is to devise a systematic strategy to integrate disease-associated variants with diverse genomic and biological datasets to provide insight into disease pathogenesis and guide drug discovery for complex traits such as rheumatoid arthritis (RA)1. Here, we performed a genome-wide association study (GWAS) meta-analysis in a total of >100,000 subjects of European and Asian ancestries (29,880 RA cases and 73,758 controls), by evaluating ~10 million single nucleotide polymorphisms (SNPs). We discovered 42 novel RA risk loci at a genome-wide level of significance, bringing the total to 1012–4. We devised an in-silico pipeline using established bioinformatics methods based on functional annotation5, cis-acting expression quantitative trait loci (cis-eQTL)6, and pathway analyses7–9 – as well as novel methods based on genetic overlap with human primary immunodeficiency (PID), hematological cancer somatic mutations and knock-out mouse phenotypes – to identify 98 biological candidate genes at these 101 risk loci. We demonstrate that these genes are the targets of approved therapies for RA, and further suggest that drugs approved for other indications may be repurposed for the treatment of RA. Together, this comprehensive genetic study sheds light on fundamental genes, pathways and cell types that contribute to RA pathogenesis, and provides empirical evidence that the genetics of RA can provide important information for drug discovery.
doi:10.1038/nature12873
PMCID: PMC3944098  PMID: 24390342
12.  Emerging Strategies and Integrated Systems Microbiology Technologies for Biodiscovery of Marine Bioactive Compounds 
Marine Drugs  2014;12(6):3516-3559.
Marine microorganisms continue to be a source of structurally and biologically novel compounds with potential use in the biotechnology industry. The unique physiochemical properties of the marine environment (such as pH, pressure, temperature, osmolarity) and uncommon functional groups (such as isonitrile, dichloroimine, isocyanate, and halogenated functional groups) are frequently found in marine metabolites. These facts have resulted in the production of bioactive substances with different properties than those found in terrestrial habitats. In fact, the marine environment contains a relatively untapped reservoir of bioactivity. Recent advances in genomics, metagenomics, proteomics, combinatorial biosynthesis, synthetic biology, screening methods, expression systems, bioinformatics, and the ever increasing availability of sequenced genomes provides us with more opportunities than ever in the discovery of novel bioactive compounds and biocatalysts. The combination of these advanced techniques with traditional techniques, together with the use of dereplication strategies to eliminate known compounds, provides a powerful tool in the discovery of novel marine bioactive compounds. This review outlines and discusses the emerging strategies for the biodiscovery of these bioactive compounds.
doi:10.3390/md12063516
PMCID: PMC4071589  PMID: 24918453
marine bioactive compounds; metagenomics; synthetic biology; biocatalyst discovery; metaproteomic; dereplication; omic approaches
13.  Caries-free subjects have high levels of urease and arginine deiminase activity 
Journal of Applied Oral Science  2014;22(3):235-240.
Objectives
This study investigated the relationship between urease and arginine deiminase system (ADS) activities and dental caries through a cross-sectional study.
Material and Methods
Urease and ADS activities were measured in saliva and plaque samples from 10 caries-free subjects and 13 caries-active. Urease activity was obtained from the ammonia produced by incubation of plaque and saliva samples in urea. ADS activity was obtained from the ammonia generated by the arginine-HCl and Tris-maleate buffer. Specific activity was defined as micromoles of ammonia per minute per milligram of protein. Shapiro-Wilk statistical test was used to analyze the distribution of the data, and Mann-Whitney test was used to determine the significance of the data.
Results
The specific urease activity in saliva and plaque was significantly higher in individuals with low DMFT scores. ADS activity in saliva (6.050 vs 1.350, p=0.0154) and plaque (8.830 vs 1.210, p=0.025) was also higher in individuals with low DMFT scores.
Conclusions
Caries-free subjects had a higher ammonia generation activity by urease and arginine deiminase system for both saliva and plaque samples than low caries-active subjects. High levels of alkali production in oral environment were related to caries-free subjects.
doi:10.1590/1678-775720130591
PMCID: PMC4072275  PMID: 25025565
Alkali; Arginine; Dental plaque; Saliva; Urease; Dental caries
14.  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
15.  A genome-wide association study follow-up suggests a possible role for PPARG in systemic sclerosis susceptibility 
Introduction
A recent genome-wide association study (GWAS) comprising a French cohort of systemic sclerosis (SSc) reported several non-HLA single-nucleotide polymorphisms (SNPs) showing a nominal association in the discovery phase. We aimed to identify previously overlooked susceptibility variants by using a follow-up strategy.
Methods
Sixty-six non-HLA SNPs showing a P value <10-4 in the discovery phase of the French SSc GWAS were analyzed in the first step of this study, performing a meta-analysis that combined data from the two published SSc GWASs. A total of 2,921 SSc patients and 6,963 healthy controls were included in this first phase. Two SNPs, PPARG rs310746 and CHRNA9 rs6832151, were selected for genotyping in the replication cohort (1,068 SSc patients and 6,762 healthy controls) based on the results of the first step. Genotyping was performed by using TaqMan SNP genotyping assays.
Results
We observed nominal associations for both PPARG rs310746 (PMH = 1.90 × 10-6, OR, 1.28) and CHRNA9 rs6832151 (PMH = 4.30 × 10-6, OR, 1.17) genetic variants with SSc in the first step of our study. In the replication phase, we observed a trend of association for PPARG rs310746 (P value = 0.066; OR, 1.17). The combined overall Mantel-Haenszel meta-analysis of all the cohorts included in the present study revealed that PPARG rs310746 remained associated with SSc with a nominal non-genome-wide significant P value (PMH = 5.00 × 10-7; OR, 1.25). No evidence of association was observed for CHRNA9 rs6832151 either in the replication phase or in the overall pooled analysis.
Conclusion
Our results suggest a role of PPARG gene in the development of SSc.
doi:10.1186/ar4432
PMCID: PMC3978735  PMID: 24401602
16.  STAT4 Associates with SLE Through Two Independent Effects that Correlate with Gene Expression and Act Additively with IRF5 to Increase Risk 
Annals of the rheumatic diseases  2008;68(11):10.1136/ard.2008.097642.
Objectives
To confirm and define the genetic association of STAT4 and systemic lupus erythematosus, investigate the possibility of correlations with differential splicing and/or expression levels, and genetic interaction with IRF5.
Methods
30 tag SNPs were genotyped in an independent set of Spanish cases and controls. SNPs surviving correction for multiple tests were genotyped in 5 new sets of cases and controls for replication. STAT4 cDNA was analyzed by 5’-RACE PCR and sequencing. Expression levels were measured by quantitative PCR.
Results
In the fine-mapping, four SNPs were significant after correction for multiple testing, with rs3821236 and rs3024866 as the strongest signals, followed by the previously associated rs7574865, and by rs1467199. Association was replicated in all cohorts. After conditional regression analyses, two major independent signals represented by SNPs rs3821236 and rs7574865, remained significant across the sets. These SNPs belong to separate haplotype blocks. High levels of STAT4 expression correlated with SNPs rs3821236, rs3024866 (both in the same haplotype block) and rs7574865 but not with other SNPs. We also detected transcription of alternative tissue-specific exons 1, indicating presence of tissue-specific promoters of potential importance in the expression of STAT4. No interaction with associated SNPs of IRF5 was observed using regression analysis.
Conclusions
These data confirm STAT4 as a susceptibility gene for SLE and suggest the presence of at least two functional variants affecting levels of STAT4. Our results also indicate that both genes STAT4 and IRF5 act additively to increase risk for SLE.
doi:10.1136/ard.2008.097642
PMCID: PMC3878433  PMID: 19019891
Association studies; systemic lupus erythematosus; STAT4 transcription factor; Interferon regulatory factor; genetic predisposition to disease
17.  Replication of the TNFSF4 (OX40L) Promoter Region Association with Systemic Lupus Erythematosus 
Genes and immunity  2008;10(3):10.1038/gene.2008.95.
The tumor necrosis factor ligand superfamily member 4 gene (TNFSF4) encodes the OX40 ligand (OX40L), a co-stimulatory molecule involved in T-cell activation. A recent study demonstrated the association ofTNFSF4 haplotypes located in the upstream region with risk for- or protection from Systemic Lupus Erythematosus (SLE) (Graham et al, 2008). In order to replicate this association, five single nucleotide polymorphisms (SNPs) tagging the previously associated haplotypes and passing the proper quality control filters were tested in 1312 cases and 1801 controls from Germany, Italy, Spain, and Argentina. The association of TNFSF4 with SLE was replicated in all the sets except Spain. There was a unique risk haplotype tagged by the minor alleles of the SNPs rs1234317 (pooled OR=1.39, p=0.0009) and rs12039904 (pooled OR=1.38, p=0.0012). We did not observe association to a single protective marker (rs844644) or haplotype as the first study reported; instead, we observed different protective haplotypes, all carrying the major alleles of both SNPs rs1234317 and rs12039904. Association analysis conditioning on the haplotypic background confirmed that these two SNPs explain the entire haplotype effect. This is the first replication study that confirms the association of genetic variation in the upstream region of TNFSF4 with susceptibility to SLE.
doi:10.1038/gene.2008.95
PMCID: PMC3867640  PMID: 19092840
Systemic lupus erythematosus; TNFSF4; OX40L; genetic association study
18.  Two Independent Functional Risk Haplotypes in TNIP1 are Associated with Systemic Lupus Erythematosus 
Arthritis and rheumatism  2012;64(11):3695-3705.
Objective
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoantibody production and altered type I interferon expression. Genetic surveys and genome-wide association studies have identified more than 30 SLE susceptibility genes. One of these genes, TNIP1, encodes the ABIN1 protein. ABIN1 functions in the immune system by restricting the NF-κB signaling. In order to better understand the genetic factors that influence association with SLE in genes that regulate the NF-κB pathway, we analyzed a dense set of genetic markers spanning TNIP1 and TAX1BP1, as well as the TNIP1 homolog, TNIP2, in case-control sets of diverse ethnic origins.
Methods
We fine-mapped TNIP1, TNIP2, and TAX1BP1 in a total of 8372 SLE cases and 7492 healthy controls from European-ancestry, African-American, Hispanic, East Asian, and African-American Gullah populations. Levels of TNIP1 mRNA and ABIN1 protein were analyzed using quantitative RT-PCR and Western blotting, respectively, in EBV-transformed human B cell lines.
Results
We found significant associations between genetic variants within TNIP1 and SLE but not in TNIP2 or TAX1BP1. After resequencing and imputation, we identified two independent risk haplotypes within TNIP1 in individuals of European-ancestry that were also present in African-American and Hispanic populations. These risk haplotypes produced lower levels of TNIP1 mRNA and ABIN1 protein suggesting they harbor hypomorphic functional variants that influence susceptibility to SLE by restricting ABIN1 expression.
Conclusion
Our results confirmed the association signals between SLE and TNIP1 variants in multiple populations and provide new insight into the mechanism by which TNIP1 variants may contribute to SLE pathogenesis.
doi:10.1002/art.34642
PMCID: PMC3485412  PMID: 22833143
19.  Impact of Genetic Ancestry and Socio-Demographic Status on the Clinical Expression of Systemic Lupus Erythematosus in Amerindian-European Populations 
Arthritis and rheumatism  2012;64(11):3687-3694.
Objective
Amerindian-Europeans, Asians and African-Americans have an excess morbidity from SLE and higher prevalence of lupus nephritis than Caucasians. The aim of this study was to analyze the relationship between genetic ancestry and socio-demographic characteristics and clinical features in a large cohort of Amerindian-European SLE patients.
Methods
A total of 2116 SLE patients of Amerindian-European origin and 4001 SLE patients of European descent with clinical data were used in the study. Genotyping of 253 continental ancestry informative markers was performed on the Illumina platform. The STRUCTURE and ADMIXTURE software were used to determine genetic ancestry of each individual. Correlation between ancestry and socio-demographic and clinical data were analyzed using logistic regression.
Results
The average Amerindian genetic ancestry of 2116 SLE patients was 40.7%. There was an increased risk of having renal involvement (P<0.0001, OR= 3.50 95%CI 2.63-4.63) and an early age of onset with the presence of Amerindian genetic ancestry (P<0.0001). Amerindian ancestry protected against photosensitivity (P<0.0001, OR= 0.58 95%CI 0.44-0.76), oral ulcers (P<0.0001, OR= 0.55 95%CI 0.42-0.72), and serositis (P<0.0001, OR= 0.56 95%CI 0.41-0.75) after adjustment by age, gender and age of onset. However, gender and age of onset had stronger effects on malar rash, discoid rash, arthritis and neurological involvement than genetic ancestry.
Conclusion
In general, genetic Amerindian ancestry correlates with lower socio-demographic status and increases the risk for developing renal involvement and SLE at an earlier age of onset.
doi:10.1002/art.34650
PMCID: PMC3485439  PMID: 22886787
20.  Preferential Binding to Elk-1 by SLE-Associated IL10 Risk Allele Upregulates IL10 Expression 
PLoS Genetics  2013;9(10):e1003870.
Immunoregulatory cytokine interleukin-10 (IL-10) is elevated in sera from patients with systemic lupus erythematosus (SLE) correlating with disease activity. The established association of IL10 with SLE and other autoimmune diseases led us to fine map causal variant(s) and to explore underlying mechanisms. We assessed 19 tag SNPs, covering the IL10 gene cluster including IL19, IL20 and IL24, for association with SLE in 15,533 case and control subjects from four ancestries. The previously reported IL10 variant, rs3024505 located at 1 kb downstream of IL10, exhibited the strongest association signal and was confirmed for association with SLE in European American (EA) (P = 2.7×10−8, OR = 1.30), but not in non-EA ancestries. SNP imputation conducted in EA dataset identified three additional SLE-associated SNPs tagged by rs3024505 (rs3122605, rs3024493 and rs3024495 located at 9.2 kb upstream, intron 3 and 4 of IL10, respectively), and SLE-risk alleles of these SNPs were dose-dependently associated with elevated levels of IL10 mRNA in PBMCs and circulating IL-10 protein in SLE patients and controls. Using nuclear extracts of peripheral blood cells from SLE patients for electrophoretic mobility shift assays, we identified specific binding of transcription factor Elk-1 to oligodeoxynucleotides containing the risk (G) allele of rs3122605, suggesting rs3122605 as the most likely causal variant regulating IL10 expression. Elk-1 is known to be activated by phosphorylation and nuclear localization to induce transcription. Of interest, phosphorylated Elk-1 (p-Elk-1) detected only in nuclear extracts of SLE PBMCs appeared to increase with disease activity. Co-expression levels of p-Elk-1 and IL-10 were elevated in SLE T, B cells and monocytes, associated with increased disease activity in SLE B cells, and were best downregulated by ERK inhibitor. Taken together, our data suggest that preferential binding of activated Elk-1 to the IL10 rs3122605-G allele upregulates IL10 expression and confers increased risk for SLE in European Americans.
Author Summary
Systemic lupus erythematosus (SLE), a debilitating autoimmune disease characterized by the production of pathogenic autoantibodies, has a strong genetic basis. Variants of the IL10 gene, which encodes cytokine interleukin-10 (IL-10) with known function of promoting B cell hyperactivity and autoantibody production, are associated with SLE and other autoimmune diseases, and serum IL-10 levels are elevated in SLE patients correlating with increased disease activity. In this study, to discover SLE-predisposing causal variant(s), we assessed variants within the genomic region containing IL10 and its gene family member IL19, IL20 and IL24 for association with SLE in case and control subjects from diverse ancestries. We identified SLE-associated SNP rs3122605 located at 9.2 kb upstream of IL10 as the most likely causal variant in subjects of European ancestry. The SLE-risk allele of rs3122605 was dose-dependently associated with elevated IL10 expression at both mRNA and protein levels in peripheral blood samples from SLE patients and controls, which could be explained, at least in part, by its preferential binding to Elk-1, a transcription factor activated in B cells during active disease of SLE patients. Elk-1-mediated IL-10 overexpression could be downregulated by inhibiting activation of mitogen-activated protein kinases, suggesting a potential therapeutic target for SLE.
doi:10.1371/journal.pgen.1003870
PMCID: PMC3794920  PMID: 24130510
21.  Identification of multiple risk variants for ankylosing spondylitis through high-density genotyping of immune-related loci 
Nature genetics  2013;45(7):730-738.
Ankylosing spondylitis is a common, highly heritable inflammatory arthritis affecting primarily the spine and pelvis. In addition to HLA-B*27 alleles, 12 loci have previously been identified that are associated with ankylosing spondylitis in populations of European ancestry, and 2 associated loci have been identified in Asians. In this study, we used the Illumina Immunochip microarray to perform a case-control association study involving 10,619 individuals with ankylosing spondylitis (cases) and 15,145 controls. We identified 13 new risk loci and 12 additional ankylosing spondylitis–associated haplotypes at 11 loci. Two ankylosing spondylitis–associated regions have now been identified encoding four aminopeptidases that are involved in peptide processing before major histocompatibility complex (MHC) class I presentation. Protective variants at two of these loci are associated both with reduced aminopeptidase function and with MHC class I cell surface expression.
doi:10.1038/ng.2667
PMCID: PMC3757343  PMID: 23749187
22.  Trans-Ancestral Studies Fine Map the SLE-Susceptibility Locus TNFSF4 
PLoS Genetics  2013;9(7):e1003554.
We previously established an 80 kb haplotype upstream of TNFSF4 as a susceptibility locus in the autoimmune disease SLE. SLE-associated alleles at this locus are associated with inflammatory disorders, including atherosclerosis and ischaemic stroke. In Europeans, the TNFSF4 causal variants have remained elusive due to strong linkage disequilibrium exhibited by alleles spanning the region. Using a trans-ancestral approach to fine-map the locus, utilising 17,900 SLE and control subjects including Amerindian/Hispanics (1348 cases, 717 controls), African-Americans (AA) (1529, 2048) and better powered cohorts of Europeans and East Asians, we find strong association of risk alleles in all ethnicities; the AA association replicates in African-American Gullah (152,122). The best evidence of association comes from two adjacent markers: rs2205960-T (P = 1.71×10−34, OR = 1.43[1.26–1.60]) and rs1234317-T (P = 1.16×10−28, OR = 1.38[1.24–1.54]). Inference of fine-scale recombination rates for all populations tested finds the 80 kb risk and non-risk haplotypes in all except African-Americans. In this population the decay of recombination equates to an 11 kb risk haplotype, anchored in the 5′ region proximal to TNFSF4 and tagged by rs2205960-T after 1000 Genomes phase 1 (v3) imputation. Conditional regression analyses delineate the 5′ risk signal to rs2205960-T and the independent non-risk signal to rs1234314-C. Our case-only and SLE-control cohorts demonstrate robust association of rs2205960-T with autoantibody production. The rs2205960-T is predicted to form part of a decameric motif which binds NF-κBp65 with increased affinity compared to rs2205960-G. ChIP-seq data also indicate NF-κB interaction with the DNA sequence at this position in LCL cells. Our research suggests association of rs2205960-T with SLE across multiple groups and an independent non-risk signal at rs1234314-C. rs2205960-T is associated with autoantibody production and lymphopenia. Our data confirm a global signal at TNFSF4 and a role for the expressed product at multiple stages of lymphocyte dysregulation during SLE pathogenesis. We confirm the validity of trans-ancestral mapping in a complex trait.
Author Summary
Systemic lupus erythematosus (SLE/lupus) is a complex disease in which the body's immune cells cause inflammation in one or more systems to cause the associated morbidity. Hormones, the environment and genes are all causal contributors to SLE and over the past several years the genetic component of SLE has been firmly established. Several genes which are regulators of the immune system are associated with disease risk. We have established one of these, the tumour-necrosis family superfamily member 4 (TNFSF4) gene, as a lupus susceptibility gene in Northern Europeans. A major obstacle in pinpointing the marker(s) at TNFSF4 which best explain the risk of SLE has been the strong correlation (linkage disequilibrium, LD) between adjacent markers across the TNFSF4 region in this population. To address this, we have typed polymorphisms in several populations in addition to the European groups. The mixed ancestry of these populations gives a different LD pattern than that found in Europeans, presenting a method of pinpointing the section of the TNFSF4 region which results in SLE susceptibility. The Non-European populations have allowed identification of a polymorphism likely to regulate expression of TNFSF4 to increase susceptibility to SLE.
doi:10.1371/journal.pgen.1003554
PMCID: PMC3715547  PMID: 23874208
23.  Association of CD247 Polymorphisms with Rheumatoid Arthritis: A Replication Study and a Meta-Analysis 
PLoS ONE  2013;8(7):e68295.
Given the role of CD247 in the response of the T cells, its entailment in autoimmune diseases and in order to better clarify the role of this gene in RA susceptibility, we aimed to analyze CD247 gene variants previously associated with other autoimmune diseases (rs1052237, rs2056626 and rs864537) in a large independent European Caucasian population. However, no evidence of association was found for the analyzed CD247 single-nucleotide polymorphisms (SNPs) with RA and with the presence/absence of anti-cyclic citrullinated polypeptide. We performed a meta-analysis including previously published GWAS data from the rs864537 variant, revealing an overall genome-wide significant association between this CD247 SNP and RA with anti-CCP (OR = 0.90, CI 95% = 0.87–0.93, Poverall = 2.1×10−10). Our results show for first time a GWAS-level association between this CD247 polymorphism and RA risk.
doi:10.1371/journal.pone.0068295
PMCID: PMC3702579  PMID: 23861880
24.  IRF5 polymorphism predicts prognosis in patients with systemic sclerosis 
Annals of the Rheumatic Diseases  2012;71(7):1197-1202.
Objective
The first genome-wide association study (GWAS) of systemic sclerosis (SSc) demonstrated three non-major histocompatibility complex (MHC) susceptibility loci. The goal of this study was to investigate the impact of these gene variants on survival and severity of interstitial lung disease (ILD) in SSc.
Methods
The authors examined 1443 Caucasian SSc patients enrolled in the Genetics versus Environment In Scleroderma Outcome Study (GENISOS) and Scleroderma Family Registry (n = 914 – discovery cohort) and The Johns Hopkins Scleroderma Cohort (n = 529 – replication cohort). Forced vital capacity (FVC)% predicted was used as a surrogate for ILD severity. Five single nucleotide polymorphisms, IRF5 (rs10488631, rs12537284, rs4728142), STAT4 (rs3821236), CD247 (rs2056626) reached genome-wide significance in the SSc-GWAS and were examined in the current study.
Results
Overall, 15.5% of the patients had died over the follow-up period of 5.5 years. The IRF5 rs4728142 minor allele was predictive of longer survival in the discovery cohort (p = 0.021) and in the independent replication cohort (p = 0.047) and combined group (HR: 0.75, 95% CI 0.62 to 0.90, p = 0.002). The association of this SNP with survival was independent of age at disease onset, disease type and autoantibody profile (anticentromere and antitopoisomerase antibodies). The minor allele frequency of IRF5 rs4728142 was 49.4%.
Moreover, IRF5 rs4728142 minor allele correlated with higher FVC% predicted at enrolment (p = 0.019). Finally, the IRF5 rs4728142 minor allele was associated with lower IRF5 transcript expression in patients and controls (p = 0.016 and p = 0.034, respectively), suggesting that the IRF5, rs4728142 SNP, may be functionally relevant.
Conclusion
An SNP in the IRF5 promoter region (rs4728142), associated with lower IRF5 transcript levels, was predictive of longer survival and milder ILD in patients with SSc.
doi:10.1136/annrheumdis-2011-200901
PMCID: PMC3375372  PMID: 22440820
25.  Independent Replication and Metaanalysis of Association Studies Establish TNFSF4 as a Susceptibility Gene Preferentially Associated with the Subset of Anticentromere-positive Patients with Systemic Sclerosis 
The Journal of rheumatology  2012;39(5):997-1003.
Objective
Independent replication with large cohorts and metaanalysis of genetic associations are necessary to validate genetic susceptibility factors. The known tumor necrosis factor (ligand) superfamily, member 4 gene (TNFSF4) systemic lupus erythematosus (SLE) risk locus has been found to be associated with systemic sclerosis (SSc) in 2 studies, but with discrepancies between them for genotype-phenotype correlation. Our objective was to validate TNFSF4 association with SSc and determine the subset with the higher risk.
Methods
Known SLE and SSc TNFSF4 susceptibility variants (rs2205960, rs1234317, rs12039904, rs10912580, and rs844648) were genotyped in 1031 patients with SSc and 1014 controls of French white ancestry. Genotype-phenotype association analysis and metaanalysis of available data were performed, providing a population study of 4989 patients with SSc and 4661 controls, all of European white ancestry.
Results
Allelic and genotypic associations were observed for the 5 single-nucleotide polymorphisms (SNP) with the subset of patients with SSc who are positive for anticentromere antibodies (ACA) and only a trend for association with SSc and limited cutaneous SSc. Rs2205960 exhibited the strongest allelic association in ACA+ patients with SSc [p = 0.0015; OR 1.37 (1.12–1.66)], with significant intracohort association when compared to patients with SSc positive for ACA. Metaanalysis confirmed overall association with SSc but also raised preferential association with the ACA+ subset and strongest effect with rs2205960 [T allele p = 0.00013; OR 1.33 (1.15–1.54) and TT genotype p = 0.00046; OR 2.02 (1.36–2.98)].
Conclusion
We confirm TNFSF4 as an SSc susceptibility gene and rs2205960 as a putative causal variant with preferential association in the ACA+ SSc subphenotype. (First Release March 15 2012; J Rheumatol 2012;39:997–1003; doi:10.3899/jrheum.111270)
doi:10.3899/jrheum.111270
PMCID: PMC3687343  PMID: 22422496
SYSTEMIC SCLEROSIS; TNFSF4; AUTOIMMUNITY; AUTOANTIBODIES

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