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1.  Phenome-wide association study (PheWAS) in EMR-linked pediatric cohorts, genetically links PLCL1 to speech language development and IL5-IL13 to Eosinophilic Esophagitis 
Frontiers in Genetics  2014;5:401.
Objective: We report the first pediatric specific Phenome-Wide Association Study (PheWAS) using electronic medical records (EMRs). Given the early success of PheWAS in adult populations, we investigated the feasibility of this approach in pediatric cohorts in which associations between a previously known genetic variant and a wide range of clinical or physiological traits were evaluated. Although computationally intensive, this approach has potential to reveal disease mechanistic relationships between a variant and a network of phenotypes.
Method: Data on 5049 samples of European ancestry were obtained from the EMRs of two large academic centers in five different genotyped cohorts. Recently, these samples have undergone whole genome imputation. After standard quality controls, removing missing data and outliers based on principal components analyses (PCA), 4268 samples were used for the PheWAS study. We scanned for associations between 2476 single-nucleotide polymorphisms (SNP) with available genotyping data from previously published GWAS studies and 539 EMR-derived phenotypes. The false discovery rate was calculated and, for any new PheWAS findings, a permutation approach (with up to 1,000,000 trials) was implemented.
Results: This PheWAS found a variety of common variants (MAF > 10%) with prior GWAS associations in our pediatric cohorts including Juvenile Rheumatoid Arthritis (JRA), Asthma, Autism and Pervasive Developmental Disorder (PDD) and Type 1 Diabetes with a false discovery rate < 0.05 and power of study above 80%. In addition, several new PheWAS findings were identified including a cluster of association near the NDFIP1 gene for mental retardation (best SNP rs10057309, p = 4.33 × 10−7, OR = 1.70, 95%CI = 1.38 − 2.09); association near PLCL1 gene for developmental delays and speech disorder [best SNP rs1595825, p = 1.13 × 10−8, OR = 0.65(0.57 − 0.76)]; a cluster of associations in the IL5-IL13 region with Eosinophilic Esophagitis (EoE) [best at rs12653750, p = 3.03 × 10−9, OR = 1.73 95%CI = (1.44 − 2.07)], previously implicated in asthma, allergy, and eosinophilia; and association of variants in GCKR and JAZF1 with allergic rhinitis in our pediatric cohorts [best SNP rs780093, p = 2.18 × 10−5, OR = 1.39, 95%CI = (1.19 − 1.61)], previously demonstrated in metabolic disease and diabetes in adults.
Conclusion: The PheWAS approach with re-mapping ICD-9 structured codes for our European-origin pediatric cohorts, as with the previous adult studies, finds many previously reported associations as well as presents the discovery of associations with potentially important clinical implications.
doi:10.3389/fgene.2014.00401
PMCID: PMC4235428  PMID: 25477900
PheWAS; ICD-9 code; genetic polymorphism
2.  Genome-wide association analysis of juvenile idiopathic arthritis identifies a new susceptibility locus at chromosomal region 3q13 
Arthritis and rheumatism  2012;64(8):2781-2791.
Objective
We have conducted a GWAS in a Caucasian cohort of juvenile idiopathic arthritis (JIA) patients and have previously published findings limited to autoimmune loci shared with other diseases. The goal of this study was to identify novel JIA predisposing loci using genome-wide approaches.
Methods
The Discovery cohort consisted of Caucasian JIA cases (814) and local controls (658) genotyped on the Affymetrix SNP 6.0 Array along with 2400 out-of-study controls. A replication study consisted of 10 SNPs genotyped in 1744 cases and 7010 controls from the US and Europe.
Results
Analysis within the Discovery cohort provided evidence of associations at 3q13 within C3orf1 and near CD80 (rs4688011, OR=1.37, P=1.88×10−6), and 10q21 near the gene JMJD1C [rs6479891, odds ratio (OR) =1.59, P=6.1×10−8; rs12411988, OR=1.57, P=1.16×10−7 and rs10995450, OR = 1.31, P=6.74×10−5]. Meta-analysis continued to provide evidence for association for these 4 SNPs (rs4688011, P=3.6×10−7, rs6479891, P=4.33×10−5; rs12411988, P=2.71×10−5; and rs10995450, 5.39×10−5;). Gene expression data from 68 JIA cases and 23 local controls showed cis eQTL associations for C3orf1 SNP rs4688011 (P=0.024 or P=0.034, depending on probe set) and the JMJD1C SNPs rs6479891 and rs12411988 (P=0.01 and P=0.008, respectively). A variance component liability model estimated that common SNP variation accounts for ~1/3 of JIA susceptibility.
Conclusions
Genetic association results and correlated gene expression findings provide evidence of association at 3q13 and 10q21 for JIA and offer novel genes as plausible candidates in disease pathology.
doi:10.1002/art.34429
PMCID: PMC3366043  PMID: 22354554
3.  Secondary analysis of APPLE study suggests atorvastatin may reduce atherosclerosis progression in pubertal lupus patients with higher C reactive protein 
Annals of the rheumatic diseases  2013;73(3):557-566.
Objective
Participants in the Atherosclerosis Prevention in Paediatric Lupus Erythematosus (APPLE) trial were randomised to placebo or atorvastatin for 36 months. The primary endpoint, reduced carotid intima medial thickness (CIMT) progression, was not met but atorvastatin-treated participants showed a trend of slower CIMT progression. Post-hoc analyses were performed to assess subgroup benefit from atorvastatin therapy.
Methods
Subgroups were prespecified and defined by age (> or ≤15.5 years), systemic lupus erythematosus (SLE) duration (> or ≤24 months), pubertal status (Tanner score ≥4 as post-pubertal or <4 as pre-pubertal), low density lipoprotein cholesterol (LDL) (≥ or <110 mg/dl) and high-sensitivity C reactive protein (hsCRP) (≥ or <1.5 mg/l). A combined subgroup (post-pubertal and hsCRP≥1.5 mg/l) was compared to all others. Longitudinal linear mixed-effects models were developed using 12 CIMT and other secondary APPLE outcomes (lipids, hsCRP, disease activity and damage, and quality of life). Three way interaction effects were assessed for models.
Results
Significant interaction effects with trends of less CIMT progression in atorvastatin-treated participants were observed in pubertal (3 CIMT segments), high hsCRP (2 CIMT segments), and the combined high hsCRP and pubertal group (5 CIMT segments). No significant treatment effect trends were observed across subgroups defined by age, SLE duration, LDL for CIMT or other outcome measures.
Conclusions
Pubertal status and higher hsCRP were linked to lower CIMT progression in atorvastatin-treated subjects, with most consistent decreases in CIMT progression in the combined pubertal and high hsCRP group. While secondary analyses must be interpreted cautiously, results suggest further research is needed to determine whether pubertal lupus patients with high CRP benefit from statin therapy.
ClinicalTrials.gov identifier
NCT00065806.
doi:10.1136/annrheumdis-2012-202315
PMCID: PMC4104199  PMID: 23436914
4.  Susceptibility to childhood onset rheumatoid arthritis: Investigation of a weighted genetic risk score that integrates cumulative effects of variants at five genetic loci 
Arthritis and rheumatism  2013;65(6):1663-1667.
Objectives
Children with rheumatoid-factor or anti-citrullinated peptide antibody positive juvenile idiopathic arthritis represent the childhood onset of RA (CORA). To test the hypothesis that adult-onset RA-associated variants are also associated with CORA, we investigated RA-associated variants at five loci in our CORA cohort. We also assessed the cumulative association of these variants in the susceptibility to CORA using a weighted genetic risk score (wGRS).
Methods
155 children with CORA and 684 healthy controls were genotyped for five variants in PTPN22, TRAF1/C5, STAT4, and TNFAIP3 loci. High-resolution HLA-DRB1 genotypes were available for 149 cases and 373 controls. We tested each locus for association with CORA via logistic regression. We also computed a wGRS for each subject, with weights based on the natural log of the published odds ratios for the alleles investigated, and used logistic regression to test the wGRS for association with CORA.
Results
CORA was associated with TNFAIP3-rs10499194 [OR 0.60 (95%CI 0.44–0.83)], PTPN22-rs2476601 [OR 1.61 (1.11–2.31)], and STAT4-rs7574865 [OR 1.41 (1.06–1.87)] variants. The wGRS was significantly different between cases and controls (P<2×10−16). Individuals in the third to fifth quintiles of wGRS had a significantly increased disease risk compared to the baseline. Higher wGRS associated with increased risk of CORA, especially among males.
Conclusions
TNFAIP3, STAT4 and PTPN22 variants are associated with CORA in a similar magnitude and direction as in RA, suggesting that adult-onset RA and CORA share common genetic risk factors. Utilizing a wGRS, we have demonstrated the cumulative association of RA-associated variants in the susceptibility to CORA.
doi:10.1002/art.37913
PMCID: PMC3683854  PMID: 23450725
juvenile idiopathic arthritis; rheumatoid arthritis; genetics; association
8.  Follistatin-like Protein 1 and the Ferritin/Erythrocyte Sedimentation Rate Ratio Are Potential Biomarkers for Dysregulated Gene Expression and Macrophage Activation Syndrome in Systemic Juvenile Idiopathic Arthritis 
The Journal of rheumatology  2013;40(7):10.3899/jrheum.121131.
Objective
Follistatin-like protein 1 (FSTL-1) is a secreted glycoprotein overexpressed in certain inflammatory diseases. Our objective was to correlate FSTL-1 levels with gene expression, known biomarkers, and measures of disease activity in systemic juvenile idiopathic arthritis (sJIA), including macrophage activation syndrome (MAS).
Methods
FSTL-1 serum levels were measured by ELISA in 28 patients with sJIA, including 7 patients who developed MAS, and 30 healthy controls. Levels were correlated with erythrocyte sedimentation rate (ESR), ferritin, and soluble interleukin-2 receptor-α (sIL-2Rα). Gene expression based on FSTL-1 levels was analyzed in peripheral blood mononuclear cells (PBMC).
Results
Serum levels of FSTL-1 were elevated at time of presentation of sJIA (mean 200.7 ng/ml) and decreased to normal (mean 133.7 ng/ml) over 24 months (p < 0.01). FSTL-1 levels were markedly elevated during acute MAS (mean 279.8 ng/ml) and decreased to normal following treatment (p < 0.001). FSTL-1 levels correlated with serum markers of inflammation, including sIL-2Rα and ferritin. Ferritin/ESR ratio was superior to ferritin, sIL-2Rα, and FSTL-1 in discriminating MAS from new-onset sJIA. PBMC from patients with FSTL-1 levels > 200 ng/ml showed altered expression of genes related to innate immunity, erythropoiesis, and natural killer cell dysfunction. Two patients with the highest FSTL-1 levels at disease onset (> 300 ng/ml) ultimately developed MAS.
Conclusion
Elevated pretreatment serum FSTL-1 levels in sJIA are associated with dysregulated gene expression suggestive of occult MAS, and may have utility in predicting progression to overt MAS. Ferritin/ESR ratio may be superior to ferritin alone in discriminating overt MAS from new-onset sJIA. (First Release May 15 2013; J Rheumatol 2013;40:1191–9; doi:10.3899/jrheum.121131)
doi:10.3899/jrheum.121131
PMCID: PMC3885333  PMID: 23678162
SYSTEMIC JUVENILE IDIOPATHIC ARTHRITIS; SENSITIVITY; SPECIFICITY; MACROPHAGE ACTIVATION SYNDROME; FOLLISTATIN-LIKE PROTEIN 1; BIOMARKERS
9.  Dense genotyping of immune-related disease regions identifies 14 new susceptibility loci for juvenile idiopathic arthritis 
Nature genetics  2013;45(6):664-669.
Analysis of the ImmunoChip single nucleotide polymorphism (SNP) array in 2816 individuals, comprising the most common subtypes (oligoarticular and RF negative polyarticular) of juvenile idiopathic arthritis (JIA) and 13056 controls strengthens the evidence for association to three known JIA-risk loci (HLA, PTPN22 and PTPN2) and has identified fourteen risk loci reaching genome-wide significance (p < 5 × 10-8) for the first time. Eleven additional novel regions showed suggestive evidence for association with JIA (p < 1 × 10-6). Dense-mapping of loci along with bioinformatic analysis has refined the association to one gene for eight regions, highlighting crucial pathways, including the IL-2 pathway, in JIA disease pathogenesis. The entire ImmunoChip loci, HLA region and the top 27 loci (p < 1 × 10-6) explain an estimated 18%, 13% and 6% risk of JIA, respectively. Analysis of the ImmunoChip dataset, the largest cohort of JIA cases investigated to date, provides new insight in understanding the genetic basis for this childhood autoimmune disease.
doi:10.1038/ng.2614
PMCID: PMC3673707  PMID: 23603761
10.  Multiple juvenile idiopathic arthritis subtypes demonstrate pro-inflammatory IgG glycosylation 
Arthritis and rheumatism  2012;64(9):3025-3033.
OBJECTIVES
Rheumatoid arthritis is associated with an excess of hypogalactosylated (G0) IgG that is considered relatively pro-inflammatory. Assessment of this association in juvenile idiopathic arthritis (JIA) is complicated by age-dependent IgG glycan variation. We undertook the first large-scale survey of IgG glycans in normal children and in patients with JIA, with a focus on early childhood, the time of peak JIA incidence.
METHODS
IgG glycans from healthy children and DMARD-naïve JIA patients were characterized using high-performance liquid chromatography (HPLC). Pro-inflammatory G0 glycans were quantitated with reference to monogalactosylated (G1) species. Associations were sought between G0/G1 and disease characteristics.
RESULTS
Among healthy children aged 9 months-16 years (n=165), G0/G1 was highly age-dependent, peaking in children <3 years old at 1.19 and declining to a nadir of 0.83 after age 10 years (Spearman ρ=0.60, p<0.0001). In patients with JIA (n=141), G0/G1 was elevated compared with controls (G0/G1 1.32 vs. 1.02, p<0.0001). Corrected for age, G0/G1 was abnormally high in all JIA subtypes (enthesitis-related arthritis not assessed), most strikingly in systemic JIA. Glycosylation aberrancy was comparable in patients with or without ANA and in both early- and late-onset disease, and exhibited at most a weak correlation with inflammatory markers.
CONCLUSIONS
IgG glycosylation is skewed toward pro-inflammatory G0 variants in healthy children, in particular during the first few years of life. This deviation is exaggerated in patients with JIA. The role for IgG glycan variation in immune function in children, including the predilection of JIA for early childhood, remains to be defined.
doi:10.1002/art.34507
PMCID: PMC3429730  PMID: 22549726
11.  Autoinflammatory gene polymorphisms and susceptibility to UK juvenile idiopathic arthritis 
Background
To investigate the autoinflammatory hereditary periodic fever syndrome genes MVK and TNFRSF1A, and the NLRP1 and IL1 genes, for association with juvenile idiopathic arthritis (JIA).
Methods
For MVK, TNFRSF1A and NLRP1 pair-wise tagging SNPs across each gene were selected and for IL1A SNPs from a prior meta-analysis were included. 1054 UK Caucasian JIA patients were genotyped by Sequenom iPlex MassARRAY and allele and genotype frequencies compared with 5380 unrelated healthy UK Caucasian controls.
Results
Four SNPs were significantly associated with UK JIA: rs2071374 within intron 4 of IL1A (ptrend=0.006), rs2228576 3’ of TNFRSF1A (ptrend=0.009) and 2 SNPs, rs11836136 and rs7957619, within MVK (ptrend=0.006, ptrend=0.005 respectively). In all cases the association appeared to be driven by the systemic-onset JIA (SoJIA) subtype. Genotype data for the two MVK SNPs was available in a validation cohort of 814 JIA (oligoarticular and RF negative polyarticular) cases and 3058 controls from the US. Replication was not confirmed, however, further suggesting that this association is specific to SoJIA.
Conclusions
These findings extend the observations of the relevance of studying monogenic loci as candidates for complex diseases. We provide novel evidence of association of MVK and TNFRSF1A with UK JIA, specifically driven by association with SoJIA and further confirm that the IL1A SNP association with SoJIA is subtype specific. Replication is required in independent cohorts.
doi:10.1186/1546-0096-11-14
PMCID: PMC3621775  PMID: 23547563
Autoinflammatory genes; Juvenile idiopathic arthritis; MVK; TNFRSF1A; IL1A
12.  Hierarchy of risk of childhood onset rheumatoid arthritis conferred by HLA-DRB1 alleles encoding the shared epitope 
Arthritis and Rheumatism  2012;64(3):925-930.
Objective
Associations between shared epitope (SE)-encoding HLA-DRB1 alleles and rheumatoid arthritis (RA) are well established but only a limited number of studies have investigated these alleles in childhood onset RA (CORA), defined as rheumatoid factor (RF) and/or anti-citrullinated protein antibody (ACPA) positive juvenile idiopathic arthritis. We sought to investigate the largest cohort of CORA for association with SE alleles, and to determine whether there was a hierarchy of risk based on the amino acid sequence of the SE.
Methods
High resolution HLA-DRB1 genotypes were obtained for 204 children with CORA and 373 healthy controls. Odds ratios (OR) and 95% CI were calculated for different SE-encoding HLA-DRB1 alleles. We also calculated genotypic OR for combinations of SE alleles classified into S2, S3P or L alleles, based on amino acids in positions 70-74 of the DRβ1 chain as proposed by Tezenas Du Montcel et al (2005).
Results
We confirmed associations between HLA-DRB1 SE alleles and CORA (76% of cases versus 46% of controls; OR=3.81 (95%CI 2.4-6.0), p <1×10−7). We also found associations between individual SE alleles (HLA-DRB1*0101, *0401, *0404, *0405, *0408 and *1001) and CORA. Genotype-specific risk estimates suggested a hierarchy of risk, with the highest risk among those heterozygous for S2/S3P (OR=22.3 (9.9-50.5) p <0.0001).
Conclusions
We confirm the association between SE-encoding HLA-DRB1 alleles and susceptibility to CORA. The excess risk conferred by individuals who carry the combination of S2/S3P risk alleles suggests that children with DRβ1 chains containing KRAA and Q/RRRAA are especially prone to RA.
doi:10.1002/art.33376
PMCID: PMC3276774  PMID: 21953520
juvenile idiopathic arthritis; rheumatoid arthritis; shared epitope; association
13.  ImmunoChip Study Implicates Antigen Presentation to T Cells in Narcolepsy 
PLoS Genetics  2013;9(2):e1003270.
Recent advances in the identification of susceptibility genes and environmental exposures provide broad support for a post-infectious autoimmune basis for narcolepsy/hypocretin (orexin) deficiency. We genotyped loci associated with other autoimmune and inflammatory diseases in 1,886 individuals with hypocretin-deficient narcolepsy and 10,421 controls, all of European ancestry, using a custom genotyping array (ImmunoChip). Three loci located outside the Human Leukocyte Antigen (HLA) region on chromosome 6 were significantly associated with disease risk. In addition to a strong signal in the T cell receptor alpha (TRA@), variants in two additional narcolepsy loci, Cathepsin H (CTSH) and Tumor necrosis factor (ligand) superfamily member 4 (TNFSF4, also called OX40L), attained genome-wide significance. These findings underline the importance of antigen presentation by HLA Class II to T cells in the pathophysiology of this autoimmune disease.
Author Summary
While there is now broad consensus that narcolepsy-hypocretin deficiency results from a highly specific autoimmune attack on hypocretin cells, little is understood regarding the initiation and progression of the underlying autoimmune process. We have taken advantage of a unique high-density genotyping platform (the ImmunoChip) designed to study variants in genes known to be important to autoimmune and inflammatory diseases. Our study of nearly 2000 narcolepsy cases compared to 10,000 controls underscored important roles for HLA DQB1*06:02 and the T cell receptor alpha genes and implicated two additional genes, Cathepsin H and TNFSF4/OX40L, in disease pathogenesis. These findings are particularly important, as these encoded proteins have key roles in antigen processing, presentation, and T cell response, and they suggest that specific interactions at the immunological synapse constitute the pathway to the disease. Further studies of these genes and encoded proteins may therefore reveal the mechanism leading to this highly selective and unique autoimmune disease.
doi:10.1371/journal.pgen.1003270
PMCID: PMC3573113  PMID: 23459209
14.  Rare and functional SIAE variants are not associated with autoimmune disease risk in up to 66,924 individuals of European ancestry 
Nature genetics  2011;44(1):3-5.
doi:10.1038/ng.1037
PMCID: PMC3287292  PMID: 22200769
15.  Biologic similarities based on age at onset in oligoarticular and polyarticular subtypes of juvenile idiopathic arthritis 
Arthritis and rheumatism  2010;62(11):3249-3258.
Objective
To explore biologic correlates to age at onset in patients with juvenile idiopathic arthritis (JIA) using peripheral blood mononuclear cell (PBMC) gene expression analysis.
Methods
PBMCs were isolated from 56 healthy controls and 104 patients with recent-onset JIA (39 with persistent oligoarticular JIA, 45 with rheumatoid factor–negative polyarticular JIA, and 20 with systemic JIA). RNA was amplified and labeled using NuGEN Ovation, and gene expression was assessed with Affymetrix HG-U133 Plus 2.0 GeneChips.
Results
A total of 832 probe sets revealed gene expression differences (false discovery rate 5%) in PBMCs from children with oligoarticular JIA whose disease began before age 6 years (early-onset disease) compared with those whose disease began at or after age 6 years (late-onset disease). In patients with early-onset disease, there was greater expression of genes related to B cells and less expression of genes related to cells of the myeloid lineage. Support vector machine analyses identified samples from patients with early- or late-onset oligoarticular JIA (with 97% accuracy) or from patients with early- or late-onset polyarticular JIA (with 89% accuracy), but not from patients with systemic JIA or healthy controls. Principal components analysis showed that age at onset was the major classifier of samples from patients with oligoarticular JIA and patients with polyarticular JIA.
Conclusion
PBMC gene expression analysis reveals biologic differences between patients with early-and late-onset JIA, independent of classification based on the number of joints involved. These data suggest that age at onset may be an important parameter to consider in JIA classification. Furthermore, pathologic mechanisms may vary with age at onset, and understanding these processes may lead to improved treatment of JIA.
doi:10.1002/art.27657
PMCID: PMC3018072  PMID: 20662067
16.  Generation of novel pharmacogenomic candidates in the response to methotrexate in juvenile idiopathic arthritis: correlation between gene expression and genotype 
Pharmacogenetics and genomics  2010;20(11):665-676.
Objectives
Little is known about mechanisms of efficacy of methotrexate (MTX) in childhood arthritis, or genetic influences upon response to MTX. The aims of this study were to use gene expression profiling to identify novel pathways/genes altered by MTX and then investigate these genes for genotype associations with response to MTX treatment.
Methods
Gene expression profiling before and after MTX treatment was performed on 11 children with juvenile idiopathic arthritis (JIA) treated with MTX, in whom response at 6 months of treatment was defined. Genes showing the most differential gene expression after treatment were selected for SNP genotyping. Genotype frequencies were compared between non-responders and responders (ACR-Ped70). An independent cohort was available for validation.
Results
Gene expression profiling before and after MTX treatment revealed 1222 differentially expressed probes sets (fold change >1.7, p< 0.05) and 1065 when restricted to full responder cases only. Six highly differentially expressed genes were analysed for genetic association to response to MTX. Three SNPs in the SLC16A7 gene showed significant association with MTX response. One SNP showed validated association in an independent cohort.
Conclusions
This study is the first, to our knowledge, to evaluate gene expression profiles in children with JIA before and after MTX, and to analyse genetic variation in differentially expressed genes. We have identified a gene which may contribute to genetic variability in MTX response in JIA, and established as proof of principle that genes which are differentially expressed at mRNA level after drug administration may also be good candidates for genetic analysis.
doi:10.1097/FPC.0b013e32833f2cd0
PMCID: PMC2963015  PMID: 20827233
Methotrexate (MTX); pharmacogenomics; treatment; juvenile idiopathic arthritis (JIA); autoimmunity; transcriptional profiling; microarray
17.  A Functional RANKL Polymorphism Associated with Younger Age at Onset of Rheumatoid Arthritis 
Arthritis and rheumatism  2010;62(10):2864-2875.
Objective
We previously reported association of co-occurrence of HLA-DRB1 shared epitope (SE) and RANKL SNPs with younger age of RA onset in 182 rheumatoid factor positive (RF) European American (EA) early RA patients. Here, we fine-mapped the 48 kb RANKL region in the extended 210 EA RF-positive early RA cohort, sought replication of RA-associated SNPs in additional 501 EA and 298 African-Americans (AA) RA cohorts, and explored functional consequences of RA-associated SNPs.
Methods
SNP genotyping was conducted using pyrosequencing or TaqMan PCR assays. Associations of rs7984870 with RANKL expression in plasma, PBMC and isolated T cells were quantified using ELISA and RT-PCR. Site-directed mutagenesis of rs7984870 within the 2kb RANKL promoter was performed to drive the luciferase reporter gene in osteoblast and stromal cell lines. Interaction of DNA and protein was determined by electrophoretic mobility shift assay.
Results
A single promoter SNP rs7984870 was consistently significantly associated with earlier age of RA onset in 3 independent seropositive (RF or anti-cyclic citrullinated peptide antibody positive) RA cohorts but not in seronegative RA patients. The risk C allele of rs7984870 conferred 2-fold higher plasma RANKL levels in RF-positive RA patients, significantly elevated RANKL mRNA expression in activated normal T cells, and increased promoter activity after stimulation in vitro via differential binding to transcription factor SOX5.
Conclusion
The RANKL promoter allele that increased transcriptional levels upon stimulation might promote interaction between activated T cells and dendritic cells, predisposing to younger RA onset in seropositive EA and/or AA individuals.
doi:10.1002/art.27589
PMCID: PMC2944013  PMID: 20533289
18.  Gene Expression Profiles from Peripheral Blood Mononuclear Cells Are Sensitive to Short Processing Delays 
Biopreservation and biobanking  2010;8(3):153-162.
In the analysis of peripheral blood gene expression, timely processing of samples is essential to ensure that measurements reflect in vivo biology, rather than ex vivo sample processing variables. The effect of processing delays on global gene expression patterns in peripheral blood mononuclear cells (PBMCs) was assessed by isolating and stabilizing PBMC-derived RNA from 3 individuals either immediately after phlebotomy or after a 4 h delay. RNA was labeled using NuGEN Ovation labeling and probed using the Affymetrix HG U133 Plus 2.0 GeneChip®. Comparison of gene expression levels (≥2-fold expression change and P < 0.05) identified 307 probe sets representing genes with increased expression and 46 indicating decreased expression after 4 h. These differentially expressed genes include many that are important to inflammatory, immunologic, and cancer pathways. Among others, CCR2, CCR5, TLR10, CD180, and IL-16 have decreased expression, whereas VEGF, IL8, SOCS2, SOCS3, CD69, and CD83 have increased expression after a 4 h processing delay. The trends in expression patterns associated with delayed processing were also apparent in an independent set of 276 arrays of RNA from human PBMC samples with varying processing times. These data indicate that the time between sample acquisition, initiation of processing, and when the RNA is stabilized should be a prime consideration when designing protocols for translational studies involving PBMC gene expression analysis.
doi:10.1089/bio.2010.0009
PMCID: PMC3129811  PMID: 21743826
19.  Gene Expression Profiles from Peripheral Blood Mononuclear Cells Are Sensitive to Short Processing Delays 
Biopreservation and Biobanking  2010;8(3):153-162.
In the analysis of peripheral blood gene expression, timely processing of samples is essential to ensure that measurements reflect in vivo biology, rather than ex vivo sample processing variables. The effect of processing delays on global gene expression patterns in peripheral blood mononuclear cells (PBMCs) was assessed by isolating and stabilizing PBMC-derived RNA from 3 individuals either immediately after phlebotomy or after a 4 h delay. RNA was labeled using NuGEN Ovation labeling and probed using the Affymetrix HG U133 Plus 2.0 GeneChip®. Comparison of gene expression levels (≥2-fold expression change and P < 0.05) identified 307 probe sets representing genes with increased expression and 46 indicating decreased expression after 4 h. These differentially expressed genes include many that are important to inflammatory, immunologic, and cancer pathways. Among others, CCR2, CCR5, TLR10, CD180, and IL-16 have decreased expression, whereas VEGF, IL8, SOCS2, SOCS3, CD69, and CD83 have increased expression after a 4 h processing delay. The trends in expression patterns associated with delayed processing were also apparent in an independent set of 276 arrays of RNA from human PBMC samples with varying processing times. These data indicate that the time between sample acquisition, initiation of processing, and when the RNA is stabilized should be a prime consideration when designing protocols for translational studies involving PBMC gene expression analysis.
doi:10.1089/bio.2010.0009
PMCID: PMC3129811  PMID: 21743826
20.  Follistatin-like-protein-1 is a mesenchyme-derived inflammatory protein and may represent a biomarker for systemic-onset juvenile rheumatoid arthritis 
Arthritis and rheumatism  2010;62(8):2510-2516.
Objective
To examine both the source of follistatin-like protein 1 (FSTL-1) and factors that induce its expression in arthritis and to determine whether juvenile rheumatoid arthritis (JRA) is characterized by over-expression of FSTL-1.
Methods
FSTL-1 expression patterns were analyzed by immunohistochemistry on joints derived from mice with collagen-induced arthritis. Induction of FSTL-1 secretion was assessed in osteoblasts, adipocytes, and human fibroblast-like synoviocytes in response to TGF-β, IL-1β, TNF-α, and IL-6. Finally, sera and synovial fluids from children with oligoarticular, polyarticular, and systemic-onset JRA were assayed for FSTL-1 using a custom ELISA. FSTL-1 concentrations were correlated with erythrocyte sedimentation rate (ESR) and platelet count.
Results
Immunohistochemical staining of joint sections demonstrated expression of FSTL-1 in all cell types of the mesenchymal lineage, including osteocytes, chondrocytes, adipocytes, and fibroblasts. FSTL-1 could be induced in osteoblasts, adipocytes, and human fibroblast-like synoviocytes by TGF-β, IL-1β, TNF-α, and IL-6. The IL-1β response was significantly greater than the TNF-α response (p < 0.05). Only the systemic-onset JRA subtype had elevated serum and synovial fluid FSTL-1 concentrations. Synovial fluid concentrations were 2–3-fold higher than serum concentrations. The elevation in serum FSTL-1 concentrations seen in systemic-onset JRA correlated closely with elevated ESR and platelet count.
Conclusion
These findings demonstrate that the arthritic joint matrix is a major source of FSTL-1 and that IL-1β is a central mediator of FSTL-1 secretion. Furthermore, FSTL-1 may represent a useful biomarker of disease activity in systemic-onset JRA.
doi:10.1002/art.27485
PMCID: PMC2921021  PMID: 20506332
21.  Synovial Fluid Proteins Differentiate Between the Subtypes of Juvenile Idiopathic Arthritis 
Arthritis and rheumatism  2010;62(6):1813-1823.
Objective
Juvenile Idiopathic Arthritis (JIA) is a heterogeneous group of inflammatory of diseases and there no clinically useful prognostic markers to predict disease outcome in these children. Synovial fluid is likely a reflection of the proteins present in the inflamed synovium. The purpose of this study was to delineate the synovial fluid proteome and determine whether there are differences in the protein expression in subtypes of JIA.
Methods
Synovial fluid from children with oligoarticular, polyarticular and systemic JIA were compared. Two dimensional gel electrophoresis for protein separation and Matrix associated laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) and Quadripole time of flight mass spectrometry (Q-TOF-MS) for protein identification were used for this study. Synovial fluid cells were analyzed by PCR for the presence of haptoglobin mRNA.
Results
The synovial fluid proteome of the samples was delineated. The majority of proteins showed overexpression in JIA synovial fluid as compared to non-inflammatory controls. There were 24 statistically significant differentially expressed spots (> 2 fold change and p<.05) between the subtypes of JIA. PCR analysis revealed haptoglobin mRNA suggesting that haptoglobin is locally produced in an inflamed joint in JIA.
Conclusions
Despite similar histological appearance of inflamed joints in subtypes of JIA, there are differences in protein expression in the subtypes of JIA. Haptoglobin is differentially expressed between the subtypes of JIA and is locally produced in an inflamed joint in JIA. Haptoglobin and other differentially expressed proteins may be potential biomarkers in JIA.
doi:10.1002/art.27447
PMCID: PMC2918379  PMID: 20222116
22.  Association of the 5-aminoimidazole-4-carboxamide ribonucleotide transformylase gene with response to methotrexate in juvenile idiopathic arthritis 
Annals of the Rheumatic Diseases  2011;70(8):1395-1400.
Objectives
Methotrexate (MTX) is the mainstay treatment for juvenile idiopathic arthritis (JIA), however approximately 30% of children will fail to respond to the drug. Identification of genetic predictors of response to MTX would be invaluable in developing optimal treatment strategies for JIA. Using a candidate gene approach, single nucleotide polymorphisms (SNPs) within genes in the metabolic pathway of MTX, were investigated for association with response to treatment in JIA cases.
Methods
Tagging SNPs were selected across 13 MTX metabolic pathway genes and were genotyped using Sequenom genotyping technology in subjects recruited from the Sparks Childhood Arthritis Response to Medication Study. Response to MTX was defined using the American College of Rheumatology (ACR) paediatric response criteria and SNP genotype frequencies were compared between the worst and best responders (ACR-Ped70) to MTX. An independent cohort of US JIA cases was available for validation of initial findings.
Results
One SNP within the inosine triphosphate pyrophosphatase gene (ITPA) and two SNPs within 5-aminoimidazole-4-carboxamide ribonucleotide transformylase gene (ATIC) were significantly associated with a poor response to MTX. One of the ATIC SNPs showed a trend towards association with MTX response in an independent cohort of US JIA cases. Meta-analysis of the two studies strengthened this association (combined p value=0.002).
Conclusions
This study presents association of a SNP in the ATIC gene with response to MTX in JIA. There is now growing evidence to support a role of the ATIC gene with response to MTX treatment. These results could contribute towards a better understanding of and ability to predict MTX response in JIA.
doi:10.1136/ard.2010.146191
PMCID: PMC3128324  PMID: 21515602
23.  Association of the IL2RA/CD25 gene with juvenile idiopathic arthritis (JIA) 
Arthritis and rheumatism  2009;60(1):251-257.
Objective
The interleukin 2 receptor alpha (IL2RA/CD25) gene is emerging as a general susceptibility gene for autoimmune diseases due to its role in development and function of T regulatory cells and the association of SNPs within this gene with type 1 diabetes, Graves' disease, RA and multiple sclerosis. The aim of this study was to determine whether SNPs within the IL2RA/CD25 gene are associated with juvenile idiopathic arthritis (JIA).
Methods
Three SNPs within the IL2RA/CD25 gene, that showed previous evidence for association with either rheumatoid arthritis, multiple sclerosis or type 1 diabetes, were selected for genotyping in UK JIA cases (n=654) and controls (n=3849). Data for one SNP (rs2104286) was also available from North American JIA cases (n=747) and controls (n=1161). Association analysis was performed using PLINK.
Results
The SNP (rs2104286) within the IL2RA/CD25 gene was significantly associated with UK JIA cases (allelic OR 0.76 95% CI 0.66-0.88, trend p=0.0002). A second SNP (rs41295061) also showed modest evidence for association with JIA (OR 0.80 95% CI 0.63-1.0, p=0.05). Association with rs2104286 was convincingly replicated in a second JIA cohort (OR 0.84 95% CI 0.65-1.0, trend p=0.05). Meta-analysis of the two cohorts yielded highly significant evidence for association (OR 0.76 95% CI 0.62-0.88, p=4.9 × 10−6).
Conclusion
These results provide strong evidence that the IL2RA/CD25 gene represents a JIA susceptibility locus. Further investigation of the gene using both genetic and functional approaches is now required.
doi:10.1002/art.24187
PMCID: PMC2963023  PMID: 19116909
24.  SEQUENCE FEATURE VARIANT TYPE (SFVT) ANALYSIS OF THE HLA GENETIC ASSOCIATION IN JUVENILE IDIOPATHIC ARTHRITIS 
The immune response HLA class II DRB1 gene provides the major genetic contribution to Juvenile Idiopathic Arthritis (JIA), with a hierarchy of predisposing through intermediate to protective effects. With JIA, and the many other HLA associated diseases, it is difficult to identify the combinations of biologically relevant amino acid (AA) residues directly involved in disease due to the high level of HLA polymorphism, the pattern of AA variability, including varying degrees of linkage disequilibrium (LD), and the fact that most HLA variation occurs at functionally important sites. In a subset of JIA patients with the clinical phenotype oligoarticular-persistent (OP), we have applied a recently developed novel approach to genetic association analyses with genes/proteins sub-divided into biologically relevant smaller sequence features (SFs), and their “alleles” which are called variant types (VTs). With SFVT analysis, association tests are performed on variation at biologically relevant SFs based on structural (e.g., beta-strand 1) and functional (e.g., peptide binding site) features of the protein. We have extended the SFVT analysis pipeline to additionally include pairwise comparisons of DRB1 alleles within serogroup classes, our extension of the Salamon Unique Combinations algorithm, and LD patterns of AA variability to evaluate the SFVT results; all of which contributed additional complementary information. With JIA-OP, we identified a set of single AA SFs, and SFs in which they occur, particularly pockets of the peptide binding site, that account for the major disease risk attributable to HLA DRB1. These are (in numeric order): AAs 13 (pockets 4 and 6), 37 and 57 (both pocket 9), 67 (pocket 7), 74 (pocket 4), and 86 (pocket 1), and to a lesser extent 30 (pockets 6 and 7) and 71 (pockets 4, 5, and 7).
PMCID: PMC2958177  PMID: 19908388
25.  Association of two functional polymorphisms in the CCR5 gene with juvenile rheumatoid arthritis 
Genes and immunity  2006;7(6):468-475.
Juvenile rheumatoid arthritis (JRA) is mediated by Th1-immune responses. In children with JRA, synovial T-cells express high levels of the Th1-chemokine receptor CCR5, which has been implicated in susceptibility to rheumatoid arthritis. To test the hypothesis that genetic variation in CCR5 is associated with susceptibility to JRA, we analyzed patterns of variation in the 5'cis-regulatory region of CCR5 in 124 multiplex families from a JRA affected-sibpair registry. After sequencing the upstream region of CCR5, variants were tested for association with JRA by transmission disequilibrium testing. A single nucleotide polymorphism, C-1835T, was significantly under-transmitted to children with early-onset JRA (p<0.01). C-1835T was genotyped in 424 additional simplex and multiplex families. CCR5-1835T allele was under-transmitted in the cohort of all probands with JRA (p<0.02), as well as in those with early-onset (p<0.01) or pauciarticular JRA (p<0.05). Another variant, a 32-bp deletion in the open reading frame of CCR5, (CCR5-Δ32), was also tested in ~700 simplex and multiplex families. CCR5-Δ32 was also significantly under-transmitted to probands with early-onset JRA (p<0.05). Both variants are in regions under natural selection, and result in functional consequences. Our results suggest these CCR5 variants are protective against early-onset JRA.
doi:10.1038/sj.gene.6364317
PMCID: PMC2927816  PMID: 16775617
Juvenile arthritis; CCR5; association; TDT; autoimmunity; chemokines

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