Identification of a novel non-coding mutation in C1qB in a Dutch child with C1q deficiency associated with recurrent infections.

INTRODUCTION: C1q deficiency is a rare genetic disorder that is strongly associated with development of systemic lupus erythematosus (SLE). Several mutations in the coding regions of the C1q genes have been described that result in stop-codons or other genetic abnormalities ultimately leading to C1q deficiency. Here we report on a Dutch boy suffering from recurrent infections with a complete C1q deficiency, without any SLE symptoms. METHODS: The presence of C1q in serum was assessed using ELISA and hemolytic assay. By western blot we examined the different C1q chains in cell lysates. We identified the mutation using deep-sequencing. By qPCR we studied the mRNA expression of C1qA, C1qB and C1qC in the PBMCs of the patient. RESULTS: Deep-sequencing revealed a homozygous mutation in the non-coding region of C1qB in the patient, whereas both parents were heterozygous. The mutation is located two nucleotides before the splice site of the second exon. In-silico analyses predict a complete abrogation of this natural splice site. Analyses of in vitro cultured cells from the patient revealed a lack of production of C1q and intracellular absence of C1qB in the presence of C1qA and C1qC peptides. Quantitative PCR analysis revealed total absence of C1qB mRNA, a reduced level of C1qA mRNA and normal levels of C1qC mRNA. CONCLUSION: In this study we report a new mutation in the non-coding region of C1qB that is associated with C1q deficiency.

Genetic risk scores and number of autoantibodies in patients with rheumatoid arthritis.

OBJECTIVE: Certain HLA-DRB1 alleles and single-nucleotide polymorphisms (SNPs) are associated with rheumatoid arthritis (RA). Our objective was to examine the combined effect of these associated variants, calculated as a cumulative genetic risk score (GRS) on RA predisposition, as well as the number of autoantibodies (none, one or two present). METHOD: We calculated four GRSs in 4956 patients and 4983 controls from four European countries. All four scores contained data on 22 non-HLA-risk SNPs, and three scores also contained HLA-DRB1 genotypes but had different HLA typing resolution. Most patients had data on both rheumatoid factor (RF) and anti-citrullinated proteins antibodies (ACPA). The GRSs were standardised (std.GRS) to account for population heterogeneity. Discrimination between patients and controls was examined by receiveroperating characteristics curves, and the four std.GRSs were compared across subgroups according to autoantibody status. RESULTS: The std.GRS improved its discriminatory ability between patients and controls when HLA-DRB1 data of higher resolution were added to the combined score. Patients had higher mean std.GRS than controls (p=7.9×10(-156)), and this score was significantly higher in patients with autoantibodies (shown for both RF and ACPA). Mean std.GRS was also higher in those with two versus one autoantibody (p=3.7×10(-23)) but was similar in patients without autoantibodies and controls (p=0.12). CONCLUSIONS: The GRS was associated with the number of autoantibodies and to both RF and ACPA positivity. ACPA play a more important role than RF with regards to the genetic risk profile, but stratification of patients according to both RF and ACPA may optimise future genetic studies.

A genetic variant in osteoprotegerin is associated with progression of joint destruction in rheumatoid arthritis.

INTRODUCTION: Progression of joint destruction in rheumatoid arthritis (RA) is partly heritably; 45 to 58% of the variance in joint destruction is estimated to be explained by genetic factors. The binding of RANKL (Receptor Activator for Nuclear Factor κ B Ligand) to RANK results in the activation of TRAF6 (tumor necrosis factor (TNF) receptor associated factor-6), and osteoclast formation ultimately leading to enhanced bone resorption. This bone resorption is inhibited by osteoprotegerin (OPG) which prevents RANKL-RANK interactions. The OPG/RANK/RANKL/TRAF6 pathway plays an important role in bone remodeling. Therefore, we investigated whether genetic variants in OPG, RANK, RANKL and TRAF6 are associated with the rate of joint destruction in RA. METHODS: 1,418 patients with 4,885 X-rays of hands and feet derived from four independent data-sets were studied. In each data-set the relative increase of the progression rate per year in the presence of a genotype was assessed. First, explorative analyses were performed on 600 RA-patients from Leiden. 109 SNPs, tagging OPG, RANK, RANKL and TRAF6, were tested. Single nucleotide polymorphisms (SNPs) significantly associated in phase-1 were genotyped in data-sets from Groningen (Netherlands), Sheffield (United Kingdom) and Lund (Switzerland). Data were summarized in an inverse weighted variance meta-analysis. Bonferonni correction for multiple testing was applied. RESULTS: We found that 33 SNPs were significantly associated with the rate of joint destruction in phase-1. In phase-2, six SNPs in OPG and four SNPs in RANK were associated with progression of joint destruction with P-value <0.05. In the meta-analyses of all four data-sets, RA-patients with the minor allele of OPG-rs1485305 expressed higher rates of joint destruction compared to patients without these risk variants (P = 2.35x10-4). This variant was also significant after Bonferroni correction. CONCLUSIONS: These results indicate that a genetic variant in OPG is associated with a more severe rate of joint destruction in RA.

Genetic studies on components of the Wnt signalling pathway and the severity of joint destruction in rheumatoid arthritis.

BACKGROUND: Progression of joint destruction in rheumatoid arthritis (RA) is partly heritable; knowledge of genetic factors may increase our understanding of the mechanisms underlying joint destruction. The activity of the Wnt/ß-catenin pathway influences osteoblast differentiation. Dickkopf-1 (Dkk-1) and sclerostin (Sost) are negative regulators and lipoprotein receptor-related protein-5 (LRP-5) and Kremen-1 are transmembrane receptors involved in this pathway. OBJECTIVE: To study variants in the genes encoding these proteins in relation to progression of joint destruction. METHODS: 1418 patients with RA of four cohorts with 4885 sets of hands and feet x-rays were studied. Explorative analyses were performed on 600 patients with RA from Leiden on single nucleotide polymorphisms (SNPs) tagging Dkk-1, Sost, Kremen-1 and LRP-5. SNPs significantly associating with joint damage progression were subsequently genotyped in cohorts from Groningen (NL), Sheffield (UK) and Lund (Sweden). Data were summarised in meta-analyses. Serum levels of functional Dkk-1 and sclerostin were measured and studied in relation to genotypes. RESULTS: In the first cohort, six Dkk-1, three Sost, one Kremen-1 and 10 LRP-5 SNPs were significantly associated with radiological progression of joint destruction. Three Dkk-1 SNPs were associated significantly with progression of joint damage in the meta-analysis, also after correction for multiple testing (rs1896368, rs1896367 and rs1528873). Two Sost SNPs tended to significance (rs4792909 and rs6503475, p=0.07 after false discovery rate correction). Gene-gene interactions between SNPs on Dkk-1 and Sost were seen. Serum levels of Dkk-1 were significantly correlated with the genotypes in rs1896368 (p=0.02). CONCLUSIONS: Patients with RA carrying risk alleles of genetic variants in Dkk-1 have higher serum levels of functional Dkk-1 and more progressive joint destruction over time.

Interaction analysis between HLA-DRB1 shared epitope alleles and MHC class II transactivator CIITA gene with regard to risk of rheumatoid arthritis.

HLA-DRB1 shared epitope (SE) alleles are the strongest genetic determinants for autoantibody positive rheumatoid arthritis (RA). One of the key regulators in expression of HLA class II receptors is MHC class II transactivator (CIITA). A variant of the CIITA gene has been found to associate with inflammatory diseases.We wanted to explore whether the risk variant rs3087456 in the CIITA gene interacts with the HLA-DRB1 SE alleles regarding the risk of developing RA. We tested this hypothesis in a case-control study with 11767 individuals from four European Caucasian populations (6649 RA cases and 5118 controls).We found no significant additive interaction for risk alleles among Swedish Caucasians with RA (n = 3869, attributable proportion due to interaction (AP) = 0.2, 95%CI: -0.2-0.5) or when stratifying for anti-citrullinated protein antibodies (ACPA) presence (ACPA positive disease: n = 2945, AP = 0.3, 95%CI: -0.05-0.6, ACPA negative: n = 2268, AP = -0.2, 95%CI: -1.0-0.6). We further found no significant interaction between the main subgroups of SE alleles (DRB1*01, DRB1*04 or DRB1*10) and CIITA. Similar analysis of three independent RA cohorts from British, Dutch and Norwegian populations also indicated an absence of significant interaction between genetic variants in CIITA and SE alleles with regard to RA risk.Our data suggest that risk from the CIITA locus is independent of the major risk for RA from HLA-DRB1 SE alleles, given that no significant interaction between rs3087456 and SE alleles was observed. Since a biological link between products of these genes is evident, the genetic contribution from CIITA and class II antigens in the autoimmune process may involve additional unidentified factors.

Novel genetic association of the VTCN1 region with rheumatoid arthritis.

OBJECTIVE: Based upon findings in juvenile idiopathic arthritis, the genetic contribution of the VTCN1 region to rheumatoid arthritis (RA) susceptibility and anticitrullinated protein antibody (ACPA) status was investigated. VTCN1 is known to play a pivotal role in regulation of the immune system and, in soluble form, has previously been associated with higher disease activity. METHODS: Ten VTCN1 polymorphisms were genotyped in 1237 Dutch patients with RA and 1055 healthy controls. Significant findings were replicated in two independent RA populations of northern European descent consisting of 2826 patients and 2122 healthy controls. Allele distribution was analysed using a χ(2) test and combined analysis of all studies was performed using the Mantel-Haenszel fixed effects method. RESULTS: A significant association with two polymorphisms was observed in the Dutch RA population. Replication of these findings showed an overall significant association with rs4376721 and rs10923217 (OR 1.13, 95% CI 1.03 to 1.24, p=0.013 and OR 0.78, 95% CI 0.67 to 0.91, p=0.0011, respectively). Stratification for ACPA status revealed an association in the ACPA-negative subset for rs4376721 (OR 1.19, 95% CI 1.05 to 1.35, p=0.0071), while no overall significance could be observed in the ACPA-positive population. rs10923217 was associated with both subsets of the disease. CONCLUSION: These results indicate a novel genetic association with the VTCN1 region in RA susceptibility.

Complement activation by (auto-) antibodies.

The complement system is a key part of the innate immune system and plays an important role in the clearance of pathogens and apoptotic cells upon its activation. It is well known that both IgG and IgM can activate complement via the classical pathway by binding of C1q to the Fc regions of these immunoglobulins. Recent advances have shown that also IgA is capable of activating the complement system. Besides, more insight is gained into an additional role for antibodies in the activation of both the alternative and the lectin pathways. Mouse models have shown that auto-antibodies can activate the alternative pathway and induce in cell lysis and tissue damage. Besides the role of antibodies in complement activation, complement may also be a target for recognition by antibodies directed against autologous complement components. These auto-antibodies play a role in several diseases, especially vascular diseases. Understanding how antibodies interact with the complement system will allow the manipulation of this interaction to diminish pathological consequences of auto-antibodies and optimize the effect of therapeutic antibodies. In the current review, we discuss complement activation by (auto-) antibodies by the different pathways.

Rheumatoid arthritis: Are ACPA-positive and ACPA-negative RA the same disease?

Seemingly contrasting genetic backgrounds in anti-citrullinated-protein antibody (ACPA)-positive and ACPA-negative rheumatoid arthritis (RA) support the notion that these are in fact two distinct disease subsets, with different underlying pathogenesis, that might need tailored treatment strategies.

Variants of gene for microsomal prostaglandin E2 synthase show association with disease and severe inflammation in rheumatoid arthritis.

Microsomal PGE synthase 1 (mPGES-1) is the terminal enzyme in the induced state of prostaglandin E(2) (PGE(2)) synthesis and constitutes a therapeutic target for rheumatoid arthritis (RA) treatment. We examined the role of the prostaglandin E synthase (PTGES) gene polymorphism in susceptibility to and severity of RA and related variations in the gene to its function. The PTGES gene polymorphism was analyzed in 3081 RA patients and 1900 controls from two study populations: Swedish Epidemiological Investigation of Rheumatoid Arthritis (EIRA) and the Leiden Early Arthritis Clinic (Leiden EAC). Baseline disease activity score (DAS28) was employed as a disease severity measure. mPGES-1 expression was analyzed in synovial tissue from RA patients with known genotypes using immunohistochemistry. In the Swedish study population, among women a significant association with risk for RA was observed for PTGES single-nucleotide polymorphisms (SNPs) in univariate analysis and for the distinct haplotype. These results were substantiated by meta-analysis of data from EIRA and Leiden EAC studies with overall OR 1.31 (95% confidence interval 1.11-1.56). Several PTGES SNPs were associated with earlier onset of disease or with higher DAS28 in women with RA. Patients with the genotype associated with higher DAS28 exhibited significantly higher mPGES-1 expression in synovial tissue. Our data reveal a possible influence of PTGES polymorphism on the pathogenesis of RA and on disease severity through upregulation of mPGES-1 at the sites of inflammation. Genetically predisposed individuals may develop earlier and more active disease owing to this mechanism.

PADI4 polymorphism predisposes male smokers to rheumatoid arthritis.

OBJECTIVE: To elucidate the differential role of peptidyl arginine deiminase 4 (PADI4) polymorphism in rheumatoid arthritis (RA) between Asian and European populations, possible gene-environmental interactions among the PADI4 polymorphism, sex and smoking status were analysed. METHODS: Three independent sets of case-control samples were genotyped for single-nucleotide polymorphisms in PADI4; Japanese samples (first set, 1019 RA patients, 907 controls; second set, 999 RA patients, 1128 controls) using TaqMan assays and Dutch samples (635 RA patients, 391 controls) using Sequenom MassARRAY platform. The association of PADI4 with RA susceptibility was evaluated by smoking status and sex in contingency tables and logistic regression models. RESULTS: In the first set of Japanese samples, PADI4 polymorphism (rs1748033) showed a greater risk in men (OR(allele) 1.39; 95% CI 1.10 to 1.76; p(trend)=0.0054) than in women and in ever-smokers (OR(allele) 1.25; 95% CI 1.02 to 1.53; p(trend)=0.032) than in never-smokers. Moreover, the highest risk was seen in male ever-smokers (OR(allele) 1.46; 95% CI 1.12 to 1.90; p(trend)=0.0047). Similar trends were observed in the second set of Japanese samples as well as in Dutch samples. CONCLUSION: PADI4 polymorphism highly predisposes male smokers to RA, and the genetic heterogeneity observed between Asian and European populations may be partly explained by differences in smoking prevalence among men.

Evidence for interaction between 5-hydroxytryptamine (serotonin) receptor 2A and MHC type II molecules in the development of rheumatoid arthritis.

It has repeatedly been suggested that the development of complex diseases can be elucidated by gene-gene interactions. Recently, we found that HTR2A, a member of the serotonin receptor family, is associated with rheumatoid arthritis (RA). This study was aimed to investigate the possibility of a gene-gene interaction between HTR2A and the major genetic risk factor for RA, HLA-DRB1 shared epitope (SE) alleles. We studied 4095 RA cases and 3223 controls from three different populations - from Sweden, the United States and the Netherlands - to test for interaction between the protective HTR2A haplotype and HLA-DRB1 SE alleles. Further, we analyzed mRNA and/or protein expression of HTR2A and HLA-DR in biopsy samples and in synovial fibroblasts from RA patients. The interaction was defined as departure from additivity of effects using attributable proportion due to interaction. First, we could demonstrate and further replicate an interaction between a protective haplotype in HTR2A and HLA-DRB1 SE alleles regarding risk of developing autoantibody-positive RA. Second, we could show that both genes are constitutively expressed in fibroblasts from synovial tissue of RA patients, and, by double immunofluorescence staining, we demonstrated that these two proteins are colocalized in these cells. In conclusion, our data demonstrate a statistical interaction between HTR2A and HLA-DRB1 SE alleles and colocalization of the product of these two genes in inflamed synovial tissue, which suggest a possible biological relationship between these two proteins. This finding may lead to the development of treatment based on enhancing the protective features of 5-HT2A in individuals with a certain HLA genotype.

Confirmation of STAT4, IL2/IL21, and CTLA4 polymorphisms in rheumatoid arthritis.

OBJECTIVE: Recent advances have led to novel identification of genetic polymorphisms that are associated with susceptibility to rheumatoid arthritis (RA). Currently, 5 loci (HLA, PTPN22, TRAF1/C5, TNFAIP3, and STAT4) have been consistently reported, whereas others have been observed less systematically. The aim of the present study was to independently replicate 3 recently described RA susceptibility loci, STAT4, IL2/IL21, and CTLA4, in a large Dutch case-control cohort, and to perform a meta-analysis of all published studies to date and investigate the relevance of the findings in clinically well-defined subgroups of RA patients with or without autoantibodies. METHODS: The STAT4, IL2/IL21, and CTLA4 gene polymorphisms (rs7574865, rs6822844, and rs3087243, respectively) were genotyped in 877 RA patients and 866 healthy individuals. A meta-analysis of all published studies of disease association with these polymorphisms was performed using the Mantel-Haenszel fixed-effects method. RESULTS: An association of STAT4, IL2/IL21, and CTLA4 with RA was detected in Dutch patients (odds ratio [OR] 1.19 [P=0.031], OR 0.84 [P=0.051], and OR 0.87 [P=0.041], respectively). Results from the meta-analysis confirmed an association of all 3 polymorphisms with RA in Caucasians (OR 1.24 [P=1.66x10(-11)], OR 0.78 [P=5.6x10(-5)], and OR 0.91 [P=1.8x10(-3)], respectively). The meta-analysis also revealed that STAT4 predisposed to disease development equally in patients with autoantibodies and those without autoantibodies, and that CTLA4 enhanced the development of anti-citrullinated protein antibody (ACPA)-positive RA as compared with ACPA-negative RA. CONCLUSION: Our results replicate and firmly establish the association of STAT4 and CTLA4 with RA and provide highly suggestive evidence for IL2/IL21 loci as a risk factor for RA. Given the strong statistical power of our meta-analysis to confirm a true-positive association, these findings provide considerable support for the involvement of CTLA4 in distinct subsets of RA patients.