Raul A Chavez

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

BackgroundJuvenile 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.ResultsMethylation 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.ConclusionsOur 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.

CLARITY – ChiLdhood Arthritis Risk factor Identification sTudY

BackgroundThe aetiology of juvenile idiopathic arthritis (JIA) is largely unknown. We have established a JIA biobank in Melbourne, Australia called CLARITY – C hiL dhood A rthritis R isk factor I dentification sT udY, 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.MethodsCases 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.ResultsClinical 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.ConclusionsWe 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.

Effects of NRG1 and DAOA genetic variation on transition to psychosis in individuals at ultra-high risk for psychosis

Prospective studies have suggested genetic variation in the neuregulin 1 (NRG1) and D-amino-acid oxidase activator (DAOA) genes may assist in differentiating high-risk individuals who will or will not transition to psychosis. In a prospective cohort (follow-up=2.4–14.9 years) of 225 individuals at ultra-high risk (UHR) for psychosis, we assessed haplotype-tagging single-nucleotide polymorphisms (htSNPs) spanning NRG1 and DAOA for their association with transition to psychosis, using Cox regression analysis. Two NRG1 htSNPs (rs12155594 and rs4281084) predicted transition to psychosis. Carriers of the rs12155594 T/T or T/C genotype had a 2.34 (95% confidence interval (CI)=1.37–4.00) times greater risk of transition compared with C/C carriers. For every rs4281084 A-allele the risk of transition increased by 1.55 (95% CI=1.05–2.27). For every additional rs4281084-A and/or rs12155594-T allele carried the risk increased ∼1.5-fold, with 71.4% of those carrying a combination of ⩾3 of these alleles transitioning to psychosis. None of the assessed DAOA htSNPs were associated with transition. Our findings suggest NRG1 genetic variation may improve our ability to identify UHR individuals at risk for transition to psychosis.

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 β 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 pinteraction = 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.

The association of PTPN22 rs2476601 with juvenile idiopathic arthritis is specific to females

A preponderance of females develop autoimmune disease, including juvenile idiopathic arthritis (JIA), yet the reason for this bias remains elusive. Evidence suggests that genetic risk of disease may be influenced by sex. PTPN22 rs2476601 is associated with JIA and numerous other autoimmune diseases, and has been reported to show female-specific association with type 1 diabetes. We performed main effect and sex-stratified association analyses to determine whether a sex-specific association exists in JIA. As expected, rs2476601 was associated with JIA in our discovery (413 cases and 690 controls) and replication (1008 cases and 9284 controls) samples. Discovery sample sex-stratified analyses demonstrated an association specifically in females (odds ratio (OR)=2.35, 95% confidence interval (CI)=1.52-3.63, P=0.00011) but not males (OR=0.91, 95% CI=0.52–1.60, P=0.75). This was similarly observed in the replication sample. There was evidence for genotype-by-sex interaction (Pinteraction=0.009). The association between rs2476601 and JIA appears restricted to females, partly accounting for the predominance of females with this disease.

Genes controlling postural changes in blood pressure: comprehensive association analysis of ATP-sensitive potassium channel genes KCNJ8 and ABCC9

Buffering of blood pressure during change of posture such as standing is controlled largely by the baroreflex. In our population-based Victorian Family Heart Study (VFHS), we previously demonstrated that, on average, systolic blood pressure (SBP) changes very little on standing; however, interindividual variation is substantial and shows familial aggregation, with approximately 25% of the variance attributable to genetic factors. Our genomewide linkage analysis suggests a region on chromosome 12p that harbors two strong candidate genes, KCNJ8 and ABCC9, encoding the channel-forming inward rectifier subunit Kir6.1 and the ATP-sensitive binding cassette SUR2B, respectively. These are key components of smooth muscle ATP-sensitive potassium (K(ATP)) channels, important regulators of arterial tone and blood flow and central to autonomic baroreceptor control of changes in total peripheral resistance. We performed a comprehensive association analysis of 47 tag single nucleotide polymorphisms (SNPs) spanning the KCNJ8 and ABCC9 gene regions with postural change in SBP (DeltaSBP). To augment power, we took a selective genotyping approach in which we compared allele and genotype frequencies between 150 unrelated individuals with high (positive) DeltaSBP (> or = 7 mmHg) and 150 unrelated individuals with low (negative) DeltaSBP (< or = -7 mmHg) drawn from the offspring generation (18-30 yr) of the VFHS. Association analyses showed that no SNPs demonstrated statistically significant differences in genotype frequencies between groups, particularly after adjustments for multiple testing. We conclude that sequence variants in KCNJ8 and ABCC9 are unlikely to contribute to variation in DeltaSBP. Other genes in the identified chromosome 12p region warrant investigation.

PReS-FINAL-2149: Interaction of PTPN2 and vitamin D GC sequence variants and risk of juvenile idiopathic arthritis

The causes of autoimmune diseases, including juvenile idiopathic arthritis (JIA) are presumed to involve the interplay of genes and environment. Vitamin D interacts with genes via vitamin D response elements (vdres). The gene PTPN2, associated with various autoimmune diseases including JIA, carries a VDRE and is regulated by vitamin D. Recently, an interaction between PTPN2 and VDR (vitamin D receptor gene) was reported for type 1 diabetes, such that disease association with VDR differed by PTPN2 genotype.

CLARITY: ChiLdhood Arthritis Risk Factor Identification STudY. Investigating the role of aberrant DNA methylation in juvenile idiopathic arthritis

Background Juvenile Idiopathic Arthritis (JIA) is a complex autoimmune disorder likely to be determined by multiple genetic and environmental factors. Mounting evidence shows that epigenetic variation influences autoimmune disease risk. The most well-studied epigenetic mark is DNA methylation; increased methylation of CpG dinucleotides can reduce gene expression. Nothing is currently known about the role of methylation in JIA. In 2008, we established CLARITY, a JIA Biobank that is collecting biospecimens and extensive information about environment from cases presenting to the Royal Children’s Hospital (RCH), Melbourne, Australia. A control sample of healthy children attending the RCH Day Surgery Unit has also been collected. Aims To identify key differences in methylation patterns across the genome associated with the most common subtypes of JIA. Methods