DNA methylation at IL32 in juvenile idiopathic arthritis.

Juvenile idiopathic arthritis (JIA) is the most common autoimmune rheumatic disease of childhood. We recently showed that DNA methylation at the gene encoding the pro-inflammatory cytokine interleukin-32 (IL32) is reduced in JIA CD4+ T cells. To extend this finding, we measured IL32 methylation in CD4+ T-cells from an additional sample of JIA cases and age- and sex-matched controls, and found a reduction in methylation associated with JIA consistent with the prior data (combined case-control dataset: 25.0% vs 37.7%, p = 0.0045). Further, JIA was associated with reduced IL32 methylation in CD8+ T cells (15.2% vs 25.5%, p = 0.034), suggesting disease-associated changes to a T cell precursor. Additionally, we measured regional SNPs, along with CD4+ T cell expression of total IL32, and the γ and ß isoforms. Several SNPs were associated with methylation. Two SNPs were also associated with JIA, and we found evidence of interaction such that methylation was only associated with JIA in minor allele carriers (e.g. rs10431961 p(interaction) = 0.011). Methylation at one measured CpG was inversely correlated with total IL32 expression (Spearman r = -0.73, p = 0.0009), but this was not a JIA-associated CpG. Overall, our data further confirms that reduced IL32 methylation is associated with JIA, and that SNPs play an interactive role.

Epistasis amongst PTPN2 and genes of the vitamin D pathway contributes to risk of juvenile idiopathic arthritis.

Juvenile idiopathic arthritis (JIA) is a leading cause of childhood-onset disability. Although epistasis (gene-gene interaction) is frequently cited as an important component of heritability in complex diseases such as JIA, there is little compelling evidence that demonstrates such interaction. PTPN2, a vitamin D responsive gene, is a confirmed susceptibility gene in JIA, and PTPN2 has been suggested to interact with vitamin D pathway genes in type 1 diabetes. We therefore, tested for evidence of epistasis amongst PTPN2 and the vitamin D pathway genes GC, VDR, CYP24A1, CYP2R1, and DHCR7 in two independent JIA case-control samples (discovery and replication). In the discovery sample (318 cases, 556 controls), we identified evidence in support of epistasis across six gene-gene combinations (e.g., GC rs1155563 and PTPN2 rs2542151, ORint=0.45, p=0.00085). Replication was obtained for three of these combinations. That is, for GC and PTPN2, CYP2R1 and VDR, and VDR and PTPN2, similar epistasis was observed using the same SNPs or correlated proxies in an independent JIA case-control sample (1008 cases, 9287 controls). Using SNP data imputed across a 4 MB region spanning each gene, we obtained highly significant evidence for epistasis amongst all 6 gene-gene combinations identified in the discovery sample (p-values ranging from 5.6×10(-9) to 7.5×10(-7)). This is the first report of epistasis in JIA risk. Epistasis amongst PTPN2 and vitamin D pathway genes was both demonstrated and replicated.

Independent confirmation of juvenile idiopathic arthritis genetic risk loci previously identified by immunochip array analysis.

BACKGROUND: Our understanding of the genetic factors underlying juvenile idiopathic arthritis (JIA) is growing, but remains incomplete. Recently, a number of novel genetic loci were reported to be associated with JIA at (or near) genome-wide significance in a large case-control discovery sample using the Immunochip genotyping array. However, independent replication of findings has yet to be performed. We therefore attempted to replicate these newly identified loci in the Australian CLARITY JIA case-control sample. FINDINGS: Genotyping was successfully performed on a total of 404 JIA cases (mean age 6.4 years, 68% female) and 676 healthy child controls (mean age 7.1 years, 42% female) across 19 SNPs previously associated with JIA. We replicated a significant association (p < 0.05, odds ratio (OR) in a direction consistent with the previous report) for seven loci, six replicated for the first time--C5orf56-IRF1 (rs4705862), ERAP2-LNPEP (rs27290), PRR5L (rs4755450), RUNX1 (rs9979383), RUNX3 (rs4648881), and UBE2L3 (rs2266959). CONCLUSIONS: We have carried out the first independent replication of association for six genes implicated in JIA susceptibility. Our data significantly strengthens the evidence that these loci harbor true disease associated variants. Thus, this study makes an important contribution to the growing body of international data that is revealing the genetic risk landscape of JIA.

Independent replication analysis of genetic loci with previous evidence of association with juvenile idiopathic arthritis.

BACKGROUND: Over the last five years, there have been numerous reports of association of juvenile idiopathic arthritis with single nucleotide polymorphisms (SNPs) at various loci outside the major histocompatibility complex (MHC) region. However, the majority of these association findings have been generated using a limited number of international cohorts, and thus there is benefit in further independent replication. To address this, we examined a total of 56 SNPs in 42 non-MHC gene regions previously reported to be associated with JIA, in the ChiLdhood Arthritis Risk factor Identification sTudY (CLARITY), a new Australian collection of cases and healthy child controls. FINDINGS: Genotyping was performed on a total of 324 JIA cases (mean age 9.7 years, 67.3% female) and 568 controls (mean age 7.8 years, 40.7% female). We demonstrated clear evidence for replication of association of JIA with SNPs in or around c12orf30, c3orf1, PTPN22, STAT4, and TRAF1-C5, confirming the involvement of these loci in disease risk. Further, we generated evidence supportive of replication of association of JIA with loci containing AFF3, CD226, MBL2, PSTPIP1, and RANTES (CCL5). These results were robust to sensitivity analyses for ethnicity. CONCLUSION: We have provided valuable independent data as to the underlying genetic architecture of this understudied pediatric autoimmune disease, further confirming five loci outside the MHC, and supporting a role for a further five loci in determining disease risk.

CLARITY - ChiLdhood Arthritis Risk factor Identification sTudY.

BACKGROUND: The aetiology of juvenile idiopathic arthritis (JIA) is largely unknown. We have established a JIA biobank in Melbourne, Australia called CLARITY - ChiLdhood Arthritis Risk factor Identification sTudY, with the broad aim of identifying genomic and environmental disease risk factors. We present here study protocols, and a comparison of socio-demographic, pregnancy, birth and early life characteristics of cases and controls collected over the first 3 years of the study. METHODS: Cases are children aged ≤18 years with a diagnosis of JIA by 16 years. Controls are healthy children aged ≤18 years, born in the state of Victoria, undergoing a minor elective surgical procedure. Participant families provide clinical, epidemiological and environmental data via questionnaire, and a blood sample is collected. RESULTS: Clinical characteristics of cases (n = 262) are similar to those previously reported. Demographically, cases were from families of higher socio-economic status. After taking this into account, the residual pregnancy and perinatal profiles of cases were similar to control children. No case-control differences in breastfeeding commencement or duration were detected, nor was there evidence of increased case exposure to tobacco smoke in utero. At interview, cases were less likely to be exposed to active parental smoking, but disease-related changes to parent behaviour may partly underlie this. CONCLUSIONS: We show that, after taking into account socio-economic status, CLARITY cases and controls are well matched on basic epidemiological characteristics. CLARITY represents a new study platform with which to generate new knowledge as to the environmental and biological risk factors for JIA.

Genome-scale case-control analysis of CD4+ T-cell DNA methylation in juvenile idiopathic arthritis reveals potential targets involved in disease.

BACKGROUND: Juvenile Idiopathic Arthritis (JIA) is a complex autoimmune rheumatic disease of largely unknown cause. Evidence is growing that epigenetic variation, particularly DNA methylation, is associated with autoimmune disease. However, nothing is currently known about the potential role of aberrant DNA methylation in JIA. As a first step to addressing this knowledge gap, we have profiled DNA methylation in purified CD4+ T cells from JIA subjects and controls. Genomic DNA was isolated from peripheral blood CD4+ T cells from 14 oligoarticular and polyarticular JIA cases with active disease, and healthy age- and sex-matched controls. Genome-scale methylation analysis was carried out using the Illumina Infinium HumanMethylation27 BeadChip. Methylation data at >25,000 CpGs was compared in a case-control study design. RESULTS: Methylation levels were significantly different (FDR adjusted p<0.1) at 145 loci. Removal of four samples exposed to methotrexate had a striking impact on the outcome of the analysis, reducing the number of differentially methylated loci to 11. The methotrexate-naive analysis identified reduced methylation at the gene encoding the pro-inflammatory cytokine IL32, which was subsequently replicated using a second analysis platform and a second set of case-control pairs. CONCLUSIONS: Our data suggests that differential T cell DNA methylation may be a feature of JIA, and that reduced methylation at IL32 is associated with this disease. Further work in larger prospective and longitudinal sample collections is required to confirm these findings, assess whether the identified differences are causal or consequential of disease, and further investigate the epigenetic modifying properties of therapeutic regimens.