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Sunflower (Helianthus annuus L.) is the third largest grain crop by area planted in South Africa (SA). The annual yield is negatively affected by sunflower rust caused by Puccinia helianthi Schw. (Phe). Four Phe races were described in SA in the middle 1990s, but since then, no new race descriptions have been conducted. This has resulted in an information gap on the current Phe population, making it difficult to explain increased disease incidence and loss of resistance in previously resistant hybrids. To address this, 114 Phe field isolates along with 23 historic isolates were phenotyped using the international set of 11 sunflower differentials containing the R1, R2/R10, R3, R4a, R4b, R4c, R4d, R5, Pu6, and Radv resistance genes. Three new Phe races were identified, bringing the total number of South African races recorded to seven. No avirulence was detected attributable to the R1 gene, with the R4d and Radv genes remaining effective. Four main genetic lineages were detected with no obvious correlation between phenotype and genotype. The detection of three genetic lineages consisting exclusively of field isolates collected post-2006 suggested the possible recent entry of exotic introductions into SA. This, combined with the fact that one lineage consisted exclusively of the most virulent race Phe7721, confirmed a clear shift in the Phe population that could explain the increased virulence and occurrence of the disease in SA.

Chronic inflammatory arthritis in childhood is heterogeneous in presentation and course. Most forms exhibit clinical and genetic similarity to arthritis of adult onset, although at least one phenotype might be restricted to children. Nevertheless, paediatric and adult rheumatologists have historically addressed disease classification separately, yielding a juvenile idiopathic arthritis (JIA) nomenclature that exhibits no terminological overlap with adult-onset arthritis. Accumulating clinical, genetic and mechanistic data reveal the critical limitations of this strategy, necessitating a new approach to defining biological categories within JIA. In this Review, we provide an overview of the current evidence for biological subgroups of arthritis in children, delineate forms that seem contiguous with adult-onset arthritis, and consider integrative genetic and bioinformatic strategies to identify discrete entities within inflammatory arthritis across all ages. Childhood-onset arthritis has historically been treated as a separate entity to adult-onset arthritis, with its own nomenclature and classification system. Biological evidence has revealed the limitations of the current approach, necessitating a fresh look at the classification of paediatric arthritis.

This article describes three wilt diseases of sunflower (Helianthus annuus L.), charcoal rot, Phialophora yellows, and Verticillium wilt, caused by soilborne fungal pathogens that initiate infections through the roots. We provide information on methods and techniques for identifying and distinguishing these diseases with signs, symptoms, and biological characteristics described for each of the causal pathogens. Accurate diagnosis of diseases and knowledge of their causal agents on sunflower are critical steps for developing and recommending disease management strategies.

Objective Juvenile idiopathic arthritis (JIA) is the most common chronic immune-mediated joint disease among children and encompasses a heterogeneous group of immune-mediated joint disorders classified into 7 subtypes according to clinical presentation. However, phenotype overlap and biologic evidence suggest a shared mechanistic basis between subtypes. This study was undertaken to systematically investigate shared genetic underpinnings of JIA subtypes. Methods We performed a heterogeneity-sensitive genome-wide association study encompassing a total of 1,245 JIA cases (classified into 7 subtypes) and 9,250 controls, followed by fine-mapping of candidate causal variants at each genome-wide significant locus, functional annotation, and pathway and network analysis. We further identified candidate drug targets and drug repurposing opportunities by in silico analyses. Results In addition to the major histocompatibility complex locus, we identified 15 genome-wide significant loci shared between at least 2 JIA subtypes, including 10 novel loci. Functional annotation indicated that candidate genes at these loci were expressed in diverse immune cell types. Conclusion This study identified novel genetic loci shared by JIA subtypes. Our findings identified candidate mechanisms underlying JIA subtypes and candidate targets with drug repurposing opportunities for JIA treatment.

ObjectiveT helper cells develop into discrete Th1, Th2 or Th17 lineages that selectively express IFNγ, IL-4/IL-5/IL-13, or IL-17, respectively and actively silence signature cytokines expressed by opposing lineages. Our objective was to compare Th1, Th2 and Th17 polarization in cell culture models using JIA patient samples.MethodsPeripheral blood mononuclear cells were isolated from JIA or healthy prepubescent children. T cell naïve and memory phenotypes were assessed by flow cytometry. T cell proliferation was measured using a fluorescence-based assay. Th cell cultures were generated in vitro and IFNγ, IL-17, and TNFα measured by ELISA and flow cytometry.ResultsJIA Th1 cells produced increased IFNγ and inappropriately produced IL-17. JIA Th17 cells produced increased IL-17. JIA Th1 cell cultures develop dual producers of IFNγ and IL-17, which are Th1.17 cells. JIA Th1 cultures expressed elevated levels of both T-bet and RORγT. RNA sequencing confirmed activation of immune responses and inappropriate activation of IL-17 signaling pathways in Th1 cultures. A subset of JIA patient samples was disproportionally responsible for the enhanced IFNγ and IL-17 phenotype and Th1.17 phenotype.ConclusionsThis study reveals that JIA patient uncommitted T cell precursors, but not healthy children, inappropriately develop into inflammatory effector Th1.17 and Th17 cells under Th1 polarizing conditions.Rheumatology key messagesTh1 differentiation of JIA PBMCs generates high IFNγ, IL-17, and dual IFNγ-IL-17 producing cells.JIA Th1 differentiation increases master transcription factor expression for Tbet and RORγT.Enhanced JIA Th1 IFNγ and IL-17 production occurs in a subset of JIA patients.

Between 2002 and 2015, a comprehensive survey of sunflower fields across seven Midwestern U.S. states was conducted 12 times and continues to be conducted every other year. The surveyors collected data on yield, agronomic management factors, disease, insect, weed, and bird damage. All surveyors were volunteers and came from universities (extension and research staff), USDA-ARS, and seed and chemical companies. In the 12 years the survey was conducted, data from 2,267 fields were collected. The results are presented annually at the National Sunflower Association Research Forum and are used to set sunflower research priorities. While 10 diseases are surveyed annually, we focus this article on the importance, findings, implications, and impacts of the five most important: downy mildew, Phomopsis stem canker, rust, Rhizopus head rot, and Sclerotinia head rot. This survey is unique among field crops in both scope and scale, and this manuscript discusses salient and clandestine benefits of intense and long-term disease surveys.

Objective Variants in the SLCO1B1 gene, encoding a hepatic methotrexate (MTX) transporter, affect clearance of high-dose MTX. We tested whether in the *14 and *15 alleles of SLCO1B1 influenced the response to low-dose MTX in juvenile idiopathic arthritis (JIA) patients. Methods The study included 310 JIA patients genotyped for three single nucleotide polymorphisms (SNPs) in SLCO1B1 (rs4149056, rs2306283, and rs11045819). A patient's SLCO1B1 diplotype was determined by combining the SNPs into the *1a, *1b, *4, *5, *14, and *15 alleles. Number of active joints at follow-up (visit closest to 6 months of treatment and prior to starting a tumor necrosis factor inhibitor) was used as the dependent variable in a negative binomial regression model that included active joint count at baseline as a covariate. Results The SLCO1B1*14 allele was associated with less response to MTX (P = 0.024) and the *15 allele was not associated with response to MTX (P = 0.392). Conclusion SLCO1B1 alleles may be associated with poor response to MTX in JIA patients. The *14 allele has been associated with fast clearance (low exposure) after high-dose MTX in patients with leukemia. Thus, the SLCO1B1 gene may be informative for precision dosing of MTX in JIA patients. Patients carrying the *14 allele may require a higher dose than noncarriers to achieve a similar response to MTX.

ObjectiveTo ascertain the role of platelet glycoprotein Ib α‐chain (GPIbα) plasma protein levels in cardiovascular, autoimmune, and autoinflammatory diseases and whether its effects are mediated by platelet count.MethodsWe performed a two‐sample Mendelian randomization (MR) study, using both a cis‐acting protein quantitative trait locus (cis‐pQTL) and trans‐pQTL near the GP1BA and BRAP genes as instruments. To assess if platelet count mediated the effect, we then performed a two‐step MR study. Putative associations (GPIbα/platelet count/disease) detected by MR analyses were subsequently assessed using multiple‐trait colocalization analyses.ResultsAfter correction for multiple testing (Bonferroni‐corrected threshold P ≤ 2 × 10−3), GPIbα, instrumented by either cis‐pQTL or trans‐pQTL, was causally implicated with an increased risk of oligoarticular and rheumatoid factor (RF)–negative polyarticular juvenile idiopathic arthritis (JIA). These effects of GPIbα appeared to be mediated by platelet count and were supported by strong evidence of colocalization (probability of all 3 traits sharing a common causal variant ≥0.80). GPIbα instrumented by cis‐pQTL did not appear to affect cardiovascular risk, although the GPIbα trans‐pQTL was associated with an increased risk of cardiovascular diseases and autoimmune diseases but a decreased risk of autoinflammatory diseases, suggesting that this trans‐acting instrument operates through other pathways.ConclusionThe role of platelets in thrombosis is well‐established; however, our findings provide some novel genetic evidence that platelets may be causally implicated in the development of oligoarticular and RF‐negative polyarticular JIA, and indicate that GPIbα may serve as a putative therapeutic target for these JIA subtypes.

ObjectivesJuvenile idiopathic arthritis (JIA) is the most prevalent form of juvenile rheumatic disease. Our understanding of the genetic risk factors for this disease is limited due to low disease prevalence and extensive clinical heterogeneity. The objective of this research is to identify novel JIA susceptibility variants and link these variants to target genes, which is essential to facilitate the translation of genetic discoveries to clinical benefit.MethodsWe performed a genome-wide association study (GWAS) in 3305 patients and 9196 healthy controls, and used a Bayesian model selection approach to systematically investigate specificity and sharing of associated loci across JIA clinical subtypes. Suggestive signals were followed-up for meta-analysis with a previous GWAS (2751 cases/15 886 controls). We tested for enrichment of association signals in a broad range of functional annotations, and integrated statistical fine-mapping and experimental data to identify target genes.ResultsOur analysis provides evidence to support joint analysis of all JIA subtypes with the identification of five novel significant loci. Fine-mapping nominated causal single nucleotide polymorphisms with posterior inclusion probabilities ≥50% in five JIA loci. Enrichment analysis identified RELA and EBF1 as key transcription factors contributing to disease risk. Our integrative approach provided compelling evidence to prioritise target genes at six loci, highlighting mechanistic insights for the disease biology andIL6STas a potential drug target.ConclusionsIn a large JIA GWAS, we identify five novel risk loci and describe potential function of JIA association signals that will be informative for future experimental works and therapeutic strategies.

Background Juvenile idiopathic arthritis (JIA) is a clinically heterogeneous group of childhood onset inflammatory joint diseases with strong evidence to support a genetic contribution to susceptibility. JIA is divided into seven clinical subgroups based on observed patterns of clinical symptoms using the International League of Associations for Rheumatology (ILAR) classification system. The genetic overlap between these groups is not completely understood and this lack of knowledge typically leads to the different ILAR groups being analysed as discrete entities potentially reducing the power of genetic association studies. The aim of this study was to conduct a large case-control association study on susceptibility to JIA to identify novel susceptibility loci and to investigate differences in these associations between the different ILAR groups for the purposes of maximising power. Methods JIA participants were genotyped on the Illumina Infinium CoreExome or OmniExpress arrays. UK population control genotype data was obtained from the Understanding Society Longitudinal Study. Quality control of data was performed conforming to conventional standards and imputation was performed using the Haplotype Reference Consortium panel on the Michigan Imputation Server. Association testing of all SNPs was performed with an additive model incorporating imputation uncertainty using SNPTEST. Testing for specificity and sharing across ILAR groups was performed using Bayesian multinomial regression in the Trinculo software package. Loci passing the suggestive significance threshold (5x10-6) were selected for meta-analysis with previously published JIA GWAS data assuming fixed effects and weighted by inverse-variance using the software package GWAMA. Results Following quality control the dataset consisted of > 7.4 million SNPs for 3305 JIA cases and 9196 controls. We found no strong evidence to support association of any SNP to a specific ILAR group with most of the SNPs showing evidence for sharing across multiple groups. Association testing in a combined dataset of all ILAR groups identified seven SNPs associated at genome-wide significance (5x10-8); six of these have previously been reported for JIA while one is a novel association. The novel association (rs497523, p-value = 7.12x10-9) maps to chromosome 16p11 and is located within intron one of the CCDC101 gene. Meta-analysis identified association to rs7647909 which is an intronic variant in the FOXP1 gene (p-value 2.0x10-9) and rs2614258 within the AHI1 gene (p-value 9.5x10-12). Conclusion We identified novel associations to SNPs in the CCDC101 and FOXP1 genes. In addition, we report a genome-wide significant association to AHI1, previously reported at suggestive significance levels. Fine mapping with the integration of genomic data will be required to identify the true causal gene at each of these loci. The results provide little evidence to support ILAR subgroup specificity for any of the associated variants and combined analysis of data across subgroups maximised power to identify novel associations. Disclosures J. Bowes None. M.C. Marion None. S. Smith None. A. Yarwood None. M. Sudman None. D. Tarasek None. C.D. Langefeld None. S.D. Thompson None. W. Thomson None.

Although wild annual sunflower (Helianthus annuus L.) is indigenous to North America, it has been both inadvertently and intentionally introduced into other countries, such as Australia, where it has become naturalized. Prior to this research, there were no collections of naturalized H. annuus from Australia in the USDA National Plant Germplasm System gene bank. The possibility exists that naturalized Australian populations may contain distinct traits, such as oil content and fatty-acid composition, differing from their North American progenitors due to the different environments, diseases, and insect-pest complexes. The objective of this study was to collect naturalized H. annuus populations from the five states of Australia and to analyze them for oil content and fatty-acid composition. Fifty-six populations were collected throughout the broad distributional range of this species and then analyzed for oil content and fatty-acid composition. The mean oil content of the H. annuus populations was 28%, ranging from a low of 19% to a high of 31%, similar to populations of this species in its native North American habitats. Fatty-acid profiles were also similar for North American wild populations, Australian naturalized populations, and improved cultivars. Oil concentration of hybrids can be rapidly increased to acceptable levels by backcrossing with cultivated sunflower. Based on this fact, there should be little concern about the relatively low oil content of these naturalized populations when they are used as a source of other unique traits for cultivated sunflower improvement.

This article describes three distinct sunflower stalk diseases that are caused by pathogens that primarily begin as foliar infections. Included are two similar fungal diseases: Phoma black stem and Phomopsis stem canker, caused by Phoma macdonaldii and two species of Diaporthe (helianthi and gulyae), respectively. The bacterial stalk rot disease caused by Pectobacterium carotovorum subsp. carotovorum and Pectobacterium atrosepticum is also included for comparison. We provide information on how to identify the diseases and distinguish them from each other with signs, symptoms, and biological characteristics of the pathogens. It is important to learn to recognize and differentiate these three diseases, because only one (Phomopsis stem canker) is considered to be economically damaging. Misidentification could result in utilizing unneeded disease management techniques.

Current classification of primary inflammatory arthritis begins from the assumption that adults and children are different. No form of juvenile idiopathic arthritis bears the same name as an adult arthritis, a nomenclature gap with implications for both clinical care and research. Recent genetic data have raised questions regarding this adult/pediatric divide, revealing instead broad patterns that span the age spectrum. Combining these genetic patterns with demographic and clinical data, we propose that inflammatory arthritis can be segregated into 4 main clusters, largely irrespective of pediatric or adult onset: seropositive, seronegative (likely including a distinct group that usually begins in early childhood), spondyloarthritis, and systemic. Each of these broad clusters is internally heterogeneous, highlighting the need for further study to resolve etiologically discrete entities. Eliminating divisions based on arbitrary age cutoffs will enhance opportunities for collaboration between adult and pediatric rheumatologists, thereby helping to promote the understanding and treatment of arthritis.

Objective Juvenile idiopathic arthritis (JIA) is the most common childhood rheumatic disease and has a strong genomic component. To date, JIA genetic association studies have had limited sample sizes, used heterogeneous patient populations, or included only candidate regions. The aim of this study was to identify new associations between JIA patients with oligoarticular disease and those with IgM rheumatoid factor (RF)−negative polyarticular disease, which are clinically similar and the most prevalent JIA disease subtypes. Methods Three cohorts comprising 2,751 patients with oligoarticular or RF-negative polyarticular JIA were genotyped using the Affymetrix Genome-Wide SNP Array 6.0 or the Illumina HumanCoreExome-12+ Array. Overall, 15,886 local and out-of-study controls, typed on these platforms or the Illumina HumanOmni2.5, were used for association analyses. High-quality single-nucleotide polymorphisms (SNPs) were used for imputation to 1000 Genomes prior to SNP association analysis. Results Meta-analysis showed evidence of association (P < 1 × 10−6) at 9 regions: PRR9_LOR (P = 5.12 × 10−8), ILDR1_CD86 (P = 6.73 × 10−8), WDFY4 (P = 1.79 × 10−7), PTH1R (P = 1.87 × 10−7), RNF215 (P = 3.09 × 10−7), AHI1_LINC00271 (P = 3.48 × 10−7), JAK1 (P = 4.18 × 10−7), LINC00951 (P = 5.80 × 10−7), and HBP1 (P = 7.29 × 10−7). Of these, PRR9_LOR, ILDR1_CD86, RNF215, LINC00951, and HBP1 were shown, for the first time, to be autoimmune disease susceptibility loci. Furthermore, associated SNPs included cis expression quantitative trait loci for WDFY4, CCDC12, MTP18, SF3A1, AHI1, COG5, HBP1, and GPR22. Conclusion This study provides evidence of both unique JIA risk loci and risk loci overlapping between JIA and other autoimmune diseases. These newly associated SNPs are shown to influence gene expression, and their bounding regions tie into molecular pathways of immunologic relevance. Thus, they likely represent regions that contribute to the pathology of oligoarticular JIA and RF-negative polyarticular JIA.

Annals of the rheumatic diseases 76(5), 906-913 (2017). doi:10.1136/annrheumdis-2016-210324, Published by BMJ Publ. Group, London

Objective Variants in the SLCO1B1 gene, encoding a hepatic methotrexate (MTX) transporter, affect clearance of high‐dose MTX. We tested whether in the *14 and *15 alleles of SLCO1B1 influenced the response to low‐dose MTX in juvenile idiopathic arthritis (JIA) patients. Methods The study included 310 JIA patients genotyped for three single nucleotide polymorphisms (SNPs) in SLCO1B1 (rs4149056, rs2306283, and rs11045819). A patient's SLCO1B1 diplotype was determined by combining the SNPs into the *1a, *1b, *4, *5, *14, and *15 alleles. Number of active joints at follow‐up (visit closest to 6 months of treatment and prior to starting a tumor necrosis factor inhibitor) was used as the dependent variable in a negative binomial regression model that included active joint count at baseline as a covariate. Results The SLCO1B1*14 allele was associated with less response to MTX (P = 0.024) and the *15 allele was not associated with response to MTX (P = 0.392). Conclusion SLCO1B1 alleles may be associated with poor response to MTX in JIA patients. The *14 allele has been associated with fast clearance (low exposure) after high‐dose MTX in patients with leukemia. Thus, the SLCO1B1 gene may be informative for precision dosing of MTX in JIA patients. Patients carrying the *14 allele may require a higher dose than noncarriers to achieve a similar response to MTX.

Purpose of review We assess the implications of recent advances in the genetics of juvenile idiopathic arthritis (JIA) for the evolving understanding of inflammatory arthritis in children. Recent findings JIA exhibits prominent genetic associations with the human leukocyte antigen (HLA) region, extending perhaps surprisingly even to the hyperinflammatory systemic JIA category. Some HLA associations resemble those for adult-onset inflammatory arthritides, providing evidence for pathogenic continuity across the age spectrum. Genome-wide association studies have defined an increasing number of JIA-linked non-HLA loci, many again shared with adult-onset arthritis. As most risk loci contain only noncoding variants, new experimental methods such as SNP-seq and innovative big-data strategies help identify responsible causative mutations, termed functional SNPs (fSNPs). Alternately, gene hunting in multiplex families implicates new genes in monogenic childhood arthritis, including MYD88 and the intriguing innate immune gene LACC1. Summary Genetic data indicate a continuity between JIA and adult arthritis poorly reflected in current nomenclature. Advancing methodologies will help to identify new pathogenic mechanisms that inform the understanding of biologic subdivisions within JIA. Resulting insights will facilitate the application of lessons learned across the age spectrum to the treatment of arthritis in children and adults.

Objective Systemic lupus erythematosus (SLE) features high frequency of cardiovascular disease (CVD) and fluctuating complement levels. The clinical trial Atherosclerosis Prevention in Pediatric Lupus Erythematosus (APPLE) aimed to evaluate whether atorvastatin treatment reduced the progression of atherosclerosis in 221 patients with childhood-onset SLE (cSLE), using carotid intima media thickness (CIMT) as surrogates. We leveraged APPLE biorepository and trial data to investigate the relationship between complement and CVD in cSLE. Methods Gene copy numbers (GCNs) for total C4, C4A and C4B were measured by TaqMan-based real-time PCR and Southern blotting, and analysed with laboratory and clinical parameters through Student’s t-test and χ2 analyses. Effects of total C4, C4A and C4B GCNs on the response to placebo or atorvastatin treatment and progression of CIMT were examined by regression analyses. Results At baseline, C4 protein levels strongly correlated with GCNs of total C4 (p=1.8×10−6). Each copy of C4 gene increased mean serum C4 by 3.28 mg/dL. Compared with those without hypertension (N=142), individuals with hypertension demonstrated significantly elevated serum levels for C4 and C3 at baseline and serially (C4: P=5.0×10−25; C3: P=5.84×10−20). Individuals with ≥2 C4B genes had 2.5 times the odds of having hypertension (p=0.016) and higher diastolic blood pressure (p=0.015) compared with those with C4B deficiency. At the study end, subjects with ≥2 C4B and atorvastatin treatment had significantly slower increase in CIMT compared with those treated with placebo (p=0.018). Conclusions cSLE with hypertension had elevated serum levels of C4 and C3 and higher GCN of C4B; cSLE with ≥2 C4B genes would benefit from statins therapy to prevent atherosclerosis.

Polyarticular juvenile idiopathic arthritis (JIA) is among the most challenging of the JIA subtypes to treat. Even with current biologic therapies, the disease remains difficult to control in a substantial subset of patients, highlighting the need for new therapies. The aim of this study was to use the high dimensionality afforded by mass cytometry with phospho-specific antibodies to delineate signaling abnormalities in immune cells from treatment-naive polyarticular JIA patients. Peripheral blood mononuclear cells were isolated from 17 treatment-naive polyarticular JIA patients, 10 of the patients after achieving clinical remission, and 19 healthy controls. Samples were stimulated for 15 minutes with IL-6 or IFN-γ and analyzed by mass cytometry. Following IFN-γ stimulation, increased STAT1 and/or STAT3 phosphorylation was observed in subsets of CD4 T cells and classical monocytes from treatment-naive patients. The enhanced IFN-γ signaling was associated with increased expression of JAK1 and SOCS1 in CD4 T cells. Furthermore, substantial heterogeneity in surface marker expression was observed among the subsets of CD4 T cells and classical monocytes with increased IFN-γ responsiveness. The identification of enhanced IFN-γ signaling in CD4 T cells and classical monocytes from treatment-naive polyarticular JIA patients provides mechanistic support for investigations into therapies that attenuate IFN-γ signaling in this disease.

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.

Objective: The mechanisms that determine the efficacy or inefficacy of MTX in JIA are ill-defined. The objective of this study was to identify a gene expression transcriptional signature associated with poor response to MTX in patients with JIA. Methods: RNA sequencing was used to measure gene expression in peripheral blood mononuclear cells collected from 47 patients with JIA prior to MTX treatment and 14 age-matched controls. Differentially expressed baseline genes between responders and non-responders were evaluated. Biological differences between all JIA patients and controls were explored by constructing a signature of differentially expressed genes. Unsupervised clustering and pathway analysis was performed. Results: A signature of 99 differentially expressed genes (Bonferroni-corrected P < 0.05) capturing the biological differences between all JIA patients and controls was identified. Unsupervised clustering of samples based on this list of 99 genes produced subgroups enriched for MTX response status. Comparing this gene signature with reference signatures from sorted cell populations revealed high concordance between the expression signatures of monocytes and of MTX non-responders. CXCL8 (IL-8) was the most significantly differentially expressed gene transcript comparing all JIA patients with controls (Bonferroni-corrected P = 4.12 × 10-10). Conclusion: Variability in clinical response to MTX in JIA patients is associated with differences in gene transcripts modulated in monocytes. These gene expression profiles may provide a basis for biomarkers predictive of treatment response.

Systemic lupus erythematosus (SLE) is an autoimmune disease with marked gender and ethnic disparities. We report a large transancestral association study of SLE using Immunochip genotype data from 27,574 individuals of European (EA), African (AA) and Hispanic Amerindian (HA) ancestry. We identify 58 distinct non-HLA regions in EA, 9 in AA and 16 in HA (∼50% of these regions have multiple independent associations); these include 24 novel SLE regions (P, Systemic lupus erythematosus (SLE) is an autoimmune disease with a strong ethnic and gender bias. In a transancestral genetic association study, Langefeld et al. identify 24 novel regions associated with risk to lupus and propose a cumulative hits hypothesis for loci conferring risk to SLE.

Purpose of review In juvenile idiopathic arthritis (JIA), there are now more than 25 regions represented by single nucleotide polymorphisms that show strong genetic associations. The causal variants and corresponding functions have not yet been defined for the majority of these regions. Here, we review current JIA association findings and the recent progress in the annotation of noncoding portion of the human genome as well as the new technologies necessary to apply this knowledge to JIA association findings. Recent findings An international collaboration was able to amass sufficient numbers of JIA and control samples to identify significantly robust genetic associations for JIA. The Encyclopedia of DNA Elements project and the National Institutes of Health (NIH) Roadmap Epigenetics Program have now annotated more than 80% of the noncoding genome, important in understanding the impact of risk loci, the majority of which fall outside of protein coding regions. Recent technological advances in high throughput sequencing, chromatin structure determination, transcription factor and enhancer binding site mapping and genome editing will likely provide a basis for understanding JIA genetic risk. Summary Understanding the role of genetic variation in the cause of JIA will provide insight for disease mechanism and may explain disease heterogeneity between JIA subtypes and between autoimmune diseases in general.

ObjectiveSystemic lupus erythematosus (SLE) features high frequency of cardiovascular disease (CVD) and fluctuating complement levels. The clinical trial Atherosclerosis Prevention in Pediatric Lupus Erythematosus (APPLE) aimed to evaluate whether atorvastatin treatment reduced the progression of atherosclerosis in 221 patients with childhood-onset SLE (cSLE), using carotid intima media thickness (CIMT) as surrogates. We leveraged APPLE biorepository and trial data to investigate the relationship between complement and CVD in cSLE.MethodsGene copy numbers (GCNs) for total C4, C4A and C4B were measured by TaqMan-based real-time PCR and Southern blotting, and analysed with laboratory and clinical parameters through Student’s t-test and χ2 analyses. Effects of total C4, C4A and C4B GCNs on the response to placebo or atorvastatin treatment and progression of CIMT were examined by regression analyses.ResultsAt baseline, C4 protein levels strongly correlated with GCNs of total C4 (p=1.8×10−6). Each copy of C4 gene increased mean serum C4 by 3.28 mg/dL. Compared with those without hypertension (N=142), individuals with hypertension demonstrated significantly elevated serum levels for C4 and C3 at baseline and serially (C4: P=5.0×10−25; C3: P=5.84×10−20). Individuals with ≥2 C4B genes had 2.5 times the odds of having hypertension (p=0.016) and higher diastolic blood pressure (p=0.015) compared with those with C4B deficiency. At the study end, subjects with ≥2 C4B and atorvastatin treatment had significantly slower increase in CIMT compared with those treated with placebo (p=0.018).ConclusionscSLE with hypertension had elevated serum levels of C4 and C3 and higher GCN of C4B; cSLE with ≥2 C4B genes would benefit from statins therapy to prevent atherosclerosis.

Objective Children with childhood-onset rheumatoid arthritis (RA) include those with rheumatoid factor or anti–citrullinated protein antibody–positive juvenile idiopathic arthritis. To test the hypothesis that adult-onset RA–associated variants are also associated with childhood-onset RA, we investigated RA-associated variants at 5 loci in a cohort of patients with childhood-onset RA. We also assessed the cumulative association of these variants in susceptibility to childhood-onset RA using a weighted genetic risk score (wGRS). Methods A total of 155 children with childhood-onset RA and 684 healthy controls were genotyped for 5 variants in the 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 childhood-onset RA via logistic regression. We also computed a wGRS for each subject, with weights based on the natural log of the published odds ratios (ORs) for the alleles investigated, and used logistic regression to test the wGRS for association with childhood-onset RA. Results Childhood-onset RA was associated with TNFAIP3 rs10499194 (OR 0.60 [95% confidence interval 0.44–0.83]), PTPN22 rs2476601 (OR 1.61 [95% confidence interval 1.11–2.31]), and STAT4 rs7574865 (OR 1.41 [95% confidence interval 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 baseline (individuals in the first quintile). Higher wGRS was associated with increased risk of childhood-onset RA, especially among males. Conclusion The magnitude and direction of the association between TNFAIP3, STAT4, and PTPN22 variants and childhood-onset RA are similar to those observed in RA, suggesting that adult-onset RA and childhood-onset RA share common genetic risk factors. Using a wGRS, we have demonstrated the cumulative association of RA-associated variants with susceptibility to childhood-onset RA.

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.

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.

ObjectiveParticipants 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.MethodsSubgroups 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 ResultsSignificant 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.ConclusionsPubertal 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.

Author(s): Fotis, Lampros; Shaikh, Nur; Baszis, Kevin; French, Anthony; Tarr, Phillip; Grevich, Sriharsha; Lee, Peggy; Ringold, Sarah; Leroux, Brian; Leahey, Hannah; Yuasa, Megan; Foster, Jessica; Sokolove, Jeremy; Lahey, Lauren; Robinson, William; Newsom, Joshua; Stevens, Anne; Karasawa, Rie; Tamaki, Mayumi; Tanaka, Megumi; Sato, Toshiko; Yudoh, Kazuo; Jarvis, James N; Moncrieffe, Halima; Bennett, Mark F; Tsoras, Monica; Luyrink, Lorie; Xu, Huan; Prahalad, Sampath; Morris, Paula; Dare, Jason; Nigrovic, Peter A; Rosenkranz, Margalit; Becker, Mara; O’Neil, Kathleen M; Griffin, Thomas; Lovell, Daniel J; Grom, Alexei A; Medvedovic, Mario; Thompson, Susan D; Zhu, Lisha; Jiang, Kaiyu; Wong, Laiping; Buck, Michael J; Chen, Yanmin; Moncrieffe, Halima; Brungs, Laura; Liu, Tao; Wang, Ting; Jarvis, James N; Alsaeid, Khaled; Alfailakawi, Jasim; Alenezi, Hamid; Alsaeed, Hazim; Beukelman, Tim; Natter, Marc; Ilowite, Norm; Mieszkalski, Kelly; Burrell, Grendel; Best, Brian; Bristow, Helen; Carr, Shannon; Dennos, Anne; Kaufmann, Rachel; Kimura, Yukiko; Schanberg, Laura; Blier, Peter R; Boneparth, Alexis; Wenderfer, Scott E; Moorthy, L Nandini; Radhakrishna, Suhas M; Sagcal-Gironella, Anna Carmela P; von Scheven, Emily; Gedik, Kader Cetin; Siddique, Salma; Aguiar, Cassyanne L; Erkan, Doruk; Cohen, Ezra; Lee, Yvonne; Dossett, Michelle; Mehta, Darshan; Davis, Roger; Gilbert, Mileka; Goilav, Beatrice; Meidan, Esra

Rheumatoid arthritis (RA), which is estimated to affect ~1% of the adult population, is the most common form of inflammatory arthritis in the world. A substantial majority of individuals with RA have rheumatoid factor (RF) and/or anti-citrullinated protein antibodies (ACPA), and the presence of these antibodies has been associated with a worse prognosis. Some children with juvenile idiopathic arthritis (JIA) have a disease that phenotypically resembles RA, characterized by chronic inflammatory arthritis and the presence of the characteristic biomarkers seen in RA including RF and ACPA Such JIA patients presumably represent a subset of RA patients with childhood onset of RA (CORA), but it is unclear if CORA shares the same risk factors associated with adult-onset RA, which has its peak age of onset in the fifth decade, or if patients with CORA have additional risk factors that, once identified, will offer important insight into the etiopathogenesis of RA. Both genetic and environmental factors have been associated with the risk of developing RA in adults. A recent meta-analysis of genome-wide associations studies of RA brought the total number of loci with confirmed genetic association to 31(1). The strongest association observed to date has been with a subset of alleles encoding the class II Human leukocyte antigens (HLA). RA associated HLA-DRB1 alleles encode DRβ1 chains with conserved amino acid sequence in positions 70-74. The molecular structure encoding the susceptibility residues is termed the shared epitope (SE)(2). While associations between SE and risk of RA are well established, some alleles (e.g. HLA-DRB1*0401) appear to confer a greater degree of risk than others (HLA-DRB1*0101)(3). Tezenas Du Montcel et al proposed a classification based on the amino acid sequence at positions 70-74, and suggested that some classes of SE alleles conferred greater susceptibility than others (4, 5). While SE alleles have been evaluated in childhood cases of RA, often referred to in the literature as “seropositive juvenile rheumatoid arthritis” or “polyarticular RF+ juvenile idiopathic arthritis”, most prior cohorts were small, used low resolution HLA typing, and thus have been limited to evaluation of the presence or absence of SE (6-11). Our objectives were to investigate the association between SE-encoding HLA-DRB1 alleles and CORA, and to explore a possible hierarchy of risk conferred by SE alleles based on the amino-acid sequence in position 70-74, in a large cohort of CORA patients. We also sought to compare the frequencies of SE genotypes in children with CORA versus published frequencies in adults with RA.

Objective More than 80% of autoimmune disease predominantly affects females, but the mechanism for this female bias is poorly understood. We suspected that an X chromosome dose effect accounts for this, and we undertook this study to test our hypothesis that trisomy X (47,XXX; occurring in ∼1 in 1,000 live female births) would be increased in patients with female-predominant diseases (systemic lupus erythematosus [SLE], primary Sjogren's syndrome [SS], primary biliary cirrhosis, and rheumatoid arthritis [RA]) compared to patients with diseases without female predominance (sarcoidosis) and compared to controls. Methods All subjects in this study were female. We identified subjects with 47,XXX using aggregate data from single-nucleotide polymorphism arrays, and, when possible, we confirmed the presence of 47,XXX using fluorescence in situ hybridization or quantitative polymerase chain reaction. Results We found 47,XXX in 7 of 2,826 SLE patients and in 3 of 1,033 SS patients, but in only 2 of 7,074 controls (odds ratio in the SLE and primary SS groups 8.78 [95% confidence interval 1.67–86.79], P = 0.003 and odds ratio 10.29 [95% confidence interval 1.18–123.47], P = 0.02, respectively). One in 404 women with SLE and 1 in 344 women with SS had 47,XXX. There was an excess of 47,XXX among SLE and SS patients. Conclusion The estimated prevalence of SLE and SS in women with 47,XXX was ∼2.5 and ∼2.9 times higher, respectively, than that in women with 46,XX and ∼25 and ∼41 times higher, respectively, than that in men with 46,XY. No statistically significant increase of 47,XXX was observed in other female-biased diseases (primary biliary cirrhosis or RA), supporting the idea of multiple pathways to sex bias in autoimmunity.

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

In July 2016, peanuts (Arachis hypogaea L. cvs. Holt, Kairi) with symptoms of stem and peg dieback were collected from Kingaroy, Childers, and Bundaberg regions in Queensland, Australia. Pycnidia of Diaporthe spp. were visible in brown lesions on stems and on live and shriveled pegs. Affected tissues were surface sterilized in 1% v/v NaOCl for 3 min, rinsed with sterile distilled water, incubated on water agar with 100 µg/ml streptomycin sulfate at 23 to 25°C under ambient light. After 1 to 14 days, conidia were streaked onto potato dextrose agar with 100 µg/ml streptomycin sulfate (PDAS) and incubated as above. After 24 h, single germinating conidia were transferred to PDAS. Mycelial DNA was extracted from 80 single conidial isolates using the Bioline Isolate II Genomic DNA extraction kit. Primers for the internal transcribed spacer region (ITS) and partial region of the translation elongation factor 1-alpha (TEF) were as described in Thompson et al. (2015). Both ITS and TEF loci were amplified with Biolabs Hot Start Taq 2x Master Mix Enzyme. PCR products were purified using Bioline Sure Clean Plus and sequenced by Macrogen Incorporated (Seoul, Korea) with the amplification primers. Sequences were assembled with Geneious v.9.1. Species morphological descriptions are in Thompson et al. 2011, 2015, and Gao et al. 2016. Using BLAST sequence analysis and culture morphology, species were identified as D. kongii (four isolates), D. masirevicii (42), D. ueckerae (syn. D. miriciae) (27), or Diaporthe sp. (seven) (Gao et al. 2016; Thompson et al. 2011, 2015). A representative isolate of D. kongii (BRIP 64964d), D. masirevicii (BRIP 64786a), and D. ueckerae (BRIP 64785a) was deposited into the Queensland Plant Pathology Herbarium (Brisbane, Australia) and was used for pathogenicity testing. To verify pathogenicity, early podfill peanut plants (cv. Taabinga) grown at 21 to 25°C day, 14 to 18°C night, were stem slit inoculated (Thompson et al. 2011), using three plants per isolate. D. gulyae (BRIP 63295), a species pathogenic on sunflower and common in eastern Australia, was a positive control with negative controls mock inoculated with water agar. At the full seed to early maturity growth stage, lesions were rated on a 1 to 5 severity scale (SR) (Thompson et al. 2011). Inoculated stems were harvested, surface sterilized, and incubated as above. Isolates identified as D. kongii, D. masirevicii, and D. ueckerae were reisolated from pycnidia in lesions located 2 cm of the point of inoculation. In all cases, the reisolated culture morphology matched the inoculating culture. Negative controls were disease free. D. masirevicii and D. ueckerae were pathogenic on peanuts (cv. Taabinga) with moderate virulence (SR 3), D. kongii had low virulence (SR 2), and D. gulyae was pathogenic with high virulence (SR 4), causing significant stem dieback. Few Diaporthe (syn. Phomopsis) species have been recorded from peanuts. In India, Sharma (1974) isolated Phomopsis sp. from peanut leaf spots. Sanogo and Etarock (2009) reported P. longicolla causing peanut stem blight in New Mexico. To our knowledge, these are the first reports of D. kongii, D. masirevicii, and D. ueckerae on peanuts and are of significance as stem and peg infections potentially limit yield. Although not yet recorded on peanuts, D. gulyae is potentially a serious pathogen of peanuts.

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.

Objective We previously observed the association of the co-occurrence of the HLA–DRB1 shared epitope (SE) and RANKL single-nucleotide polymorphisms (SNPs) with younger age at the onset of rheumatoid arthritis (RA) in 182 rheumatoid factor (RF)–positive European American patients with early-onset RA. The aim of this study was to fine-map the 48-kb RANKL region in the extended cohort of 210 European American RF-positive patients with early RA, to seek replication of RA-associated SNPs in additional RA cohorts of 501 European Americans and 298 African Americans, and to explore the functional consequences of RA-associated SNPs. Methods SNP genotyping was conducted using pyrosequencing or TaqMan polymerase chain reaction (PCR) assays. Associations of rs7984870 with RANKL expression in plasma, peripheral blood mononuclear cells, and isolated T cells were quantified using enzyme-linked immunosorbent assay and reverse transcription–PCR. Site-directed mutagenesis of rs7984870 within the 2-kb 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 at the onset of RA in 3 independent seropositive (RF or anti–cyclic citrullinated peptide antibody) RA cohorts but not in seronegative RA patients. The C risk allele of rs7984870 conferred 2-fold higher plasma RANKL levels in RF-positive patients with RA, significantly elevated RANKL messenger RNA expression in activated normal T cells, and increased promoter activity after stimulation in vitro via differential binding to the transcription factor SOX5. Conclusion The RANKL promoter allele that increased transcription levels upon stimulation might promote interaction between activated T cells and dendritic cells, predisposing to a younger age at the onset of RA in seropositive European American and African American patients.

Objective Juvenile idiopathic arthritis (JIA) is a heterogeneous group of inflammatory diseases, and no clinically useful prognostic markers to predict disease outcome in children with JIA are currently available. Synovial fluid likely reflects the proteins present in the inflamed synovium. The purpose of this study was to delineate the synovial fluid proteome and determine whether protein expression differs in the different subtypes of JIA. Methods Synovial fluid samples obtained from children with oligoarticular JIA, polyarticular JIA, or systemic JIA were compared. Two-dimensional gel electrophoresis for protein separation and matrix-assisted laser desorption ionization−time-of-flight mass spectrometry and quadripole time-of-flight mass spectrometry for protein identification were used for this study. Synovial fluid cells were analyzed by polymerase chain reaction (PCR) for the presence of haptoglobin messenger RNA (mRNA). Results The synovial fluid proteome of the samples was delineated. The majority of proteins showed overexpression in JIA synovial fluid as compared with noninflammatory control samples. There were 24 statistically significantly differentially expressed spots (>2-fold change; P < 0.05) between the subtypes of JIA. PCR analysis revealed haptoglobin mRNA, suggesting that haptoglobin is locally produced in an inflamed joint in JIA. Conclusion Despite the similar histologic appearance of inflamed joints in patients with different subtypes of JIA, there are differences in protein expression according to the subtype 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.

Objective The aim of this study was to quantitate risk and to examine heterogeneity for HLA at high resolution in patients with the most common subtypes of juvenile idiopathic arthritis (JIA), IgM rheumatoid factor–negative polyarticular JIA and oligoarticular JIA. Use of 4-digit comprehensive HLA typing enabled great precision, and a large cohort allowed for consideration of both age at disease onset and disease subtype. Methods Polymerase chain reaction–based high-resolution HLA typing for class I and class II loci was accomplished for 820 patients with JIA and 273 control subjects. Specific HLA epitopes, potential interactions of alleles at specific loci and between loci (accounting for linkage disequilibrium and haplotypic associations), and an assessment of the current International League of Associations for Rheumatology classification criteria were considered. Results An HLA–DRB1/DQB1 effect was shown to be exclusively attributable to DRB1 and was similar between patients with oligoarticular JIA and a younger subgroup of patients with polyarticular JIA. Furthermore, patients with polyarticular JIA showed age-specific related effects, with disease susceptibility in the group older than age 6 years limited to an effect of the HLA–DRB1*08 haplotype, which is markedly different from the additional susceptibility haplotypes, HLA–DRB1*1103/1104, found in the group with oligoarticular JIA and the group of younger patients with polyarticular JIA. Also in contrast to findings for oligoarticular JIA, patients with polyarticular arthritis had no evidence of an HLA class I effect. Markers associated with a reduced risk of disease included DRB1*1501, DRB1*0401, and DRB1*0701. DRB1*1501 was shown to reduce risk across the whole cohort, whereas DRB1*0401 and DRB1*0701 were protective for selected JIA subtypes. Surprisingly, the disease predisposition mediated by DPB1*0201 in individuals without any disease-predisposing DRB1 alleles was great enough to overcome even the very strong protective effect observed for DRB1*1501. Conclusion Inherited HLA factors in JIA show similarities overall as well as differences between JIA subtypes.

Macrophage Activation Syndrome (MAS) is a severe, potentially fatal condition associated with excessive activation of macrophages and T cells leading to an overwhelming inflammatory reaction. The main manifestations of MAS include fever, hepatosplenomegaly, lymphadenopathy, severe cytopenias, serious liver disease, and disseminated intravascular coagulation [1,2]. The pathognomonic feature of MAS is often found in bone marrow: numerous, well-differentiated macrophages phagocytosing hematopoietic elements. Although MAS has been reported in patients with different rheumatic diseases, it is most strongly associated with Systemic Juvenile Idiopathic Arthritis (SJIA). In fact, it accounts for much of the morbidity and mortality seen in this form of JIA. At least 10% of the patients with SJIA develop MAS [3]. The true incidence of MAS might be much higher since there are no validated diagnostic criteria and mild instances of MAS are not always recognized [4,5]. It is now recognized that MAS bears close resemblance to a group of histiocytic disorders collectively known as hemophagocytic lymphohistiocytosis (HLH) [2,6]. HLH is a term that describes a spectrum of disease processes characterized by accumulations of well-differentiated mononuclear cells with a macrophage phenotype [7]. In the contemporary classification of histiocytic disorders, HLH is further subdivided into primary, or familial HLH, and secondary, or reactive HLH (ReHLH) [7]. Clinically, however, they may be difficult to distinguish from each other [9]. Familial hemophagocytic lymphohistiocytosis (FHLH) is a constellation of rare autosomal recessive immune disorders. The clinical symptoms of FHLH usually become evident within the first 2 months of life although initial presentation as late as 22 years of age has been reported [8]. Secondary HLH tends to occur in older children and more often is associated with an identifiable infectious episode, most notably Epstein-Barr virus (EBV) or cytomegalovirus (CMV) infection. The exact pathophysiological relationship between MAS and HLH is not understood. Some pediatric rheumatologists view MAS as ReHLH occurring in a setting of a rheumatologic disease [6]. The pathological mechanisms of HLH/MAS are not fully understood. In HLH, there is uncontrolled proliferation of T cells and macrophages that has been linked to decreased NK-cell and cytotoxic T-cell function [10] often due to mutations in the gene encoding perforin [11]. Perforin is a protein which cytolytic cells utilize to induce apoptosis of target cells such as tumor cells or cells infected by viruses. It has been hypothesized by some authors that abnormal cytotoxic cells may fail to provide appropriate apoptotic signals for removal of activated macrophages and T cells during the contraction stage of certain immune responses [12]. Our recent observations suggest that as in HLH, MAS patients have profoundly depressed NK-cell function, often associated with abnormal perforin expression [13]. More recently, mutations in another gene, MUNC13-4, have been implicated in the development of hemophagocytic lymphohistiocytosis in about 10-30% of patients with inherited HLH [14]. The protein encoded by the MUNC13-4 gene is an essential effector of the cytolytic secretory pathway. MUNC13-4 protein is involved in vesicle priming function which follows granule docking and precedes plasma granule membrane fusion [14]. Therefore, it is an important player in the intracellular transport of perforin. Although the cytolytic cells of the patients with FHLH caused by MUNC13-4 mutations produce sufficient amounts of perforin, the poor ability to deliver perforin to the surface of the cells leads to profoundly decreased cytolytic activity against target cells. Here we present new data that suggests an association between MAS in SJIA and specific mutations and/or haplotypes in MUNC13-4 gene.

Juvenile idiopathic arthritis (JIA) is an autoimmune disorder mediated by Th1 immune responses. CTLA-4, expressed on the T cell surface, plays a negative role in regulating T cell activation. Single-nucleotide polymorphisms (SNPs) in CTLA4 have been implicated in susceptibility to several autoimmune disorders, including JIA. This study was undertaken to test 3 functional CTLA4 variants for association with JIA.Families of 531 children with JIA were genotyped for SNPs located in the promoter region (C-318T), exon 1 (A49G), and the 3'-untranslated region (CT60) of CTLA4 by polymerase chain reaction amplification and digestion. Family Based Association Testing (FBAT) was used to test CTLA4 SNPs and haplotypes for association with JIA. A second independent cohort of300 children with JIA and500 controls were genotyped for case-control analyses. Case-control analyses of the combined cohorts, as well as meta-analyses of published association studies of CTLA4 and JIA, were performed.There were no deviations of transmission of any of the CTLA4 variants to children with JIA, or JIA subtypes, determined by FBAT. No significant associations between CTLA4 C-318T or A49G SNPs and JIA were found in 650 JIA cases and 350 controls. Similarly, no significant associations with CT60 variants were found in800 JIA cases and500 controls. The meta-analysis also failed to confirm an association between JIA and CTLA4 variants.These results suggest that C-318T, A49G, CT60, and haplotypes tagged by these CTLA4 SNPs are not associated with JIA or major JIA subtypes.

Objective Systemic juvenile idiopathic arthritis (JIA) is frequently associated with the development of macrophage activation syndrome. This study was undertaken to better understand the relationship between systemic JIA and macrophage activation syndrome. Methods Gene expression profiles were examined in 17 patients with untreated new-onset systemic JIA, 5 of whom showed evidence of subclinical macrophage activation syndrome (of whom 2 eventually developed overt macrophage activation syndrome). Peripheral blood mononuclear cells (PBMCs) were separated on Ficoll gradients, and purified RNA was analyzed using Affymetrix GeneChip expression arrays. A fraction of the PBMCs were used for flow cytometry to define the cellular composition of the samples. Results Two hundred twenty-five differentially expressed genes (P < 0.05) that distinguished patients with systemic JIA from healthy controls (n = 30) were identified. Clustering analysis indicated that expression patterns correlated with serum ferritin levels. Three main clusters distinguished systemic JIA patients with highly elevated ferritin levels (including those with subclinical macrophage activation syndrome) from those with normal or only moderately elevated ferritin levels. The first cluster comprised genes involved in the synthesis of hemoglobins and structural proteins of erythrocytes. This transcriptional profile was consistent with immature nucleated red blood cells, likely reflective of high red blood cell turnover. Also included were transcripts indicating immature granulocytes. The second cluster was enriched for genes involved in cell cycle regulation. The third cluster was enriched for genes involved in innate immune responses, including those involved in the negative regulation of Toll-like receptor/interleukin-1 receptor–triggered inflammatory cascades and markers of the alternative pathway of macrophage differentiation. Additional differentially expressed genes of interest were those involved in the cytolytic pathway, including SH2D1A and Rab27a. Conclusion These data indicate that gene expression profiling can be a useful tool for identifying early macrophage activation syndrome in patients with systemic JIA.

Systemic juvenile idiopathic arthritis (sJIA) is an often severe, potentially life-threatening childhood inflammatory disease, the pathophysiology of which is poorly understood. To determine whether genetic variation within the MHC locus on chromosome 6 influences sJIA susceptibility, we performed an association study of 982 children with sJIA and 8,010 healthy control subjects from nine countries. Using meta-analysis of directly observed and imputed SNP genotypes and imputed classic HLA types, we identified the MHC locus as a bona fide susceptibility locus with effects on sJIA risk that transcended geographically defined strata. The strongest sJIA-associated SNP, rs151043342 [P = 2.8 x 10(-17), odds ratio (OR) 2.6 (2.1, 3.3)], was part of a cluster of 482 sJIA-associated SNPs that spanned a 400-kb region and included the class II HLA region. Conditional analysis controlling for the effect of rs151043342 found that rs12722051 independently influenced sJIA risk [P = 1.0 x 10(-5), OR 0.7 (0.6, 0.8)]. Meta-analysis of imputed classic HLA-type associations in six study populations of Western European ancestry revealed that HLA-DRB1*11 and its defining amino acid residue, glutamate 58, were strongly associated with sJIA [P = 2.7 x 10(-16), OR 2.3 (1.9, 2.8)], as was the HLA-DRB1*11-HLA-DQA1*05-HLA-DQB1*03 haplotype [6.4 x 10(-17), OR 2.3 (1.9, 2.9)]. By examining the MHC locus in the largest collection of sJIA patients assembled to date, this study solidifies the relationship between the class II HLA region and sJIA, implicating adaptive immune molecules in the pathogenesis of sJIA.