Elisabet Svenungsson

A regulatory polymorphism in PDCD1 is associated with susceptibility to systemic lupus erythematosus in humans

Systemic lupus erythematosus (SLE, OMIM 152700) is a complex autoimmune disease that affects 0.05% of the Western population, predominantly women. A number of susceptibility loci for SLE have been suggested in different populations, but the nature of the susceptibility genes and mutations is yet to be identified. We previously reported a susceptibility locus (SLEB2) for Nordic multi-case families. Within this locus, the programmed cell death 1 gene (PDCD1, also called PD-1) was considered the strongest candidate for association with the disease. Here, we analyzed 2,510 individuals, including members of five independent sets of families as well as unrelated individuals affected with SLE, for single-nucleotide polymorphisms (SNPs) that we identified in PDCD1. We show that one intronic SNP in PDCD1 is associated with development of SLE in Europeans (found in 12% of affected individuals versus 5% of controls; P = 0.00001, r.r. (relative risk) = 2.6) and Mexicans (found in 7% of affected individuals versus 2% of controls; P = 0.0009, r.r. = 3.5). The associated allele of this SNP alters a binding site for the runt-related transcription factor 1 (RUNX1, also called AML1) located in an intronic enhancer, suggesting a mechanism through which it can contribute to the development of SLE in humans.

A large-scale replication study identifies TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL10 as risk loci for systemic lupus erythematosus

Genome-wide association studies have recently identified at least 15 susceptibility loci for systemic lupus erythematosus (SLE). To confirm additional risk loci, we selected SNPs from 2,466 regions that showed nominal evidence of association to SLE (P < 0.05) in a genome-wide study and genotyped them in an independent sample of 1,963 cases and 4,329 controls. This replication effort identified five new SLE susceptibility loci (P < 5 × 10−8): TNIP1 (odds ratio (OR) = 1.27), PRDM1 (OR = 1.20), JAZF1 (OR = 1.20), UHRF1BP1 (OR = 1.17) and IL10 (OR = 1.19). We identified 21 additional candidate loci with P≤ 1 × 10−5. A candidate screen of alleles previously associated with other autoimmune diseases suggested five loci (P < 1 × 10−3) that may contribute to SLE: IFIH1, CFB, CLEC16A, IL12B and SH2B3. These results expand the number of confirmed and candidate SLE susceptibility loci and implicate several key immunologic pathways in SLE pathogenesis.

Functional variants in the B-cell gene BANK1 are associated with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by production of autoantibodies and complex genetic inheritance. In a genome-wide scan using 85,042 SNPs, we identified an association between SLE and a nonsynonymous substitution (rs10516487, R61H) in the B-cell scaffold protein with ankyrin repeats gene, BANK1. We replicated the association in four independent case-control sets (combined P = 3.7 × 10−10; OR = 1.38). We analyzed BANK1 cDNA and found two isoforms, one full-length and the other alternatively spliced and lacking exon 2 (Δ2), encoding a protein without a putative IP3R-binding domain. The transcripts were differentially expressed depending on a branch point–site SNP, rs17266594, in strong linkage disequilibrium (LD) with rs10516487. A third associated variant was found in the ankyrin domain (rs3733197, A383T). Our findings implicate BANK1 as a susceptibility gene for SLE, with variants affecting regulatory sites and key functional domains. The disease-associated variants could contribute to sustained B cell–receptor signaling and B-cell hyperactivity characteristic of this disease.

Three functional variants of IFN regulatory factor 5 (IRF5) define risk and protective haplotypes for human lupus

Systematic genome-wide studies to map genomic regions associated with human diseases are becoming more practical. Increasingly, efforts will be focused on the identification of the specific functional variants responsible for the disease. The challenges of identifying causal variants include the need for complete ascertainment of genetic variants and the need to consider the possibility of multiple causal alleles. We recently reported that risk of systemic lupus erythematosus (SLE) is strongly associated with a common SNP in IFN regulatory factor 5 (IRF5), and that this variant altered spicing in a way that might provide a functional explanation for the reproducible association to SLE risk. Here, by resequencing and genotyping in patients with SLE, we find evidence for three functional alleles of IRF5: the previously described exon 1B splice site variant, a 30-bp in-frame insertion/deletion variant of exon 6 that alters a proline-, glutamic acid-, serine- and threonine-rich domain region, and a variant in a conserved polyA+ signal sequence that alters the length of the 3′ UTR and stability of IRF5 mRNAs. Haplotypes of these three variants define at least three distinct levels of risk to SLE. Understanding how combinations of variants influence IRF5 function may offer etiological and therapeutic insights in SLE; more generally, IRF5 and SLE illustrates how multiple common variants of the same gene can together influence risk of common disease.

A risk haplotype of STAT4 for systemic lupus erythematosus is over-expressed, correlates with anti-dsDNA and shows additive effects with two risk alleles of IRF5

Systemic lupus erythematosus (SLE) is the prototype autoimmune disease where genes regulated by type I interferon (IFN) are over-expressed and contribute to the disease pathogenesis. Because signal transducer and activator of transcription 4 (STAT4) plays a key role in the type I IFN receptor signaling, we performed a candidate gene study of a comprehensive set of single nucleotide polymorphism (SNPs) in STAT4 in Swedish patients with SLE. We found that 10 out of 53 analyzed SNPs in STAT4 were associated with SLE, with the strongest signal of association (P = 7.1 × 10−8) for two perfectly linked SNPs rs10181656 and rs7582694. The risk alleles of these 10 SNPs form a common risk haplotype for SLE (P = 1.7 × 10−5). According to conditional logistic regression analysis the SNP rs10181656 or rs7582694 accounts for all of the observed association signal. By quantitative analysis of the allelic expression of STAT4 we found that the risk allele of STAT4 was over-expressed in primary human cells of mesenchymal origin, but not in B-cells, and that the risk allele of STAT4 was over-expressed (P = 8.4 × 10−5) in cells carrying the risk haplotype for SLE compared with cells with a non-risk haplotype. The risk allele of the SNP rs7582694 in STAT4 correlated to production of anti-dsDNA (double-stranded DNA) antibodies and displayed a multiplicatively increased, 1.82-fold risk of SLE with two independent risk alleles of the IRF5 (interferon regulatory factor 5) gene.

Periodontitis Increases the Risk of a First Myocardial Infarction: A Report From the PAROKRANK Study

Background— The relationship between periodontitis (PD) and cardiovascular disease is debated. PD is common in patients with cardiovascular disease. It has been postulated that PD could be causally related to the risk for cardiovascular disease, a hypothesis tested in the Periodontitis and Its Relation to Coronary Artery Disease (PAROKRANK) study. Methods and Results— Eight hundred five patients (<75 years of age) with a first myocardial infarction (MI) and 805 age- (mean 62±8), sex- (male 81%), and area-matched controls without MI underwent standardized dental examination including panoramic x-ray. The periodontal status was defined as healthy (≥80% remaining bone) or as mild-moderate (from 79% to 66%) or severe PD (<66%). Great efforts were made to collect information on possibly related confounders (≈100 variables). Statistical comparisons included the Student pairwise t test and the McNemar test in 2×2 contingency tables. Contingency tables exceeding 2×2 with ranked alternatives were tested by Wilcoxon signed rank test. Odds ratios (95% confidence intervals) were calculated by conditional logistic regression. PD was more common (43%) in patients than in controls (33%; P<0.001). There was an increased risk for MI among those with PD (odds ratio, 1.49; 95% confidence interval, 1.21–1.83), which remained significant (odds ratio, 1.28; 95% confidence interval, 1.03–1.60) after adjusting for variables that differed between patients and controls (smoking habits, diabetes mellitus, years of education, and marital status). Conclusions— In this large case–control study of PD, verified by radiographic bone loss and with a careful consideration of potential confounders, the risk of a first MI was significantly increased in patients with PD even after adjustment for confounding factors. These findings strengthen the possibility of an independent relationship between PD and MI.Background— The relationship between periodontitis (PD) and cardiovascular disease is debated. PD is common in patients with cardiovascular disease. It has been postulated that PD could be causally related to the risk for cardiovascular disease, a hypothesis tested in the Periodontitis and Its Relation to Coronary Artery Disease (PAROKRANK) study. Methods and Results— Eight hundred five patients (<75 years of age) with a first myocardial infarction (MI) and 805 age- (mean 62±8), sex- (male 81%), and area-matched controls without MI underwent standardized dental examination including panoramic x-ray. The periodontal status was defined as healthy (≥80% remaining bone) or as mild-moderate (from 79% to 66%) or severe PD (<66%). Great efforts were made to collect information on possibly related confounders (≈100 variables). Statistical comparisons included the Student pairwise t test and the McNemar test in 2×2 contingency tables. Contingency tables exceeding 2×2 with ranked alternatives were tested by Wilcoxon signed rank test. Odds ratios (95% confidence intervals) were calculated by conditional logistic regression. PD was more common (43%) in patients than in controls (33%; P <0.001). There was an increased risk for MI among those with PD (odds ratio, 1.49; 95% confidence interval, 1.21–1.83), which remained significant (odds ratio, 1.28; 95% confidence interval, 1.03–1.60) after adjusting for variables that differed between patients and controls (smoking habits, diabetes mellitus, years of education, and marital status). Conclusions— In this large case–control study of PD, verified by radiographic bone loss and with a careful consideration of potential confounders, the risk of a first MI was significantly increased in patients with PD even after adjustment for confounding factors. These findings strengthen the possibility of an independent relationship between PD and MI. # CLINICAL PERSPECTIVE {#article-title-44}

TNF-α: a link between hypertriglyceridaemia and inflammation in SLE patients with cardiovascular disease:

Patients with systemic lupus erythematosus (SLE) are at high risk of cardiovascular disease (CVD). Tumour necrosisfactor-a (TNF-α) has been implicatedin the pathophysiologicalprocessesof both SLE and CVD. This study focuses on the role of TNF-α and its soluble receptors in SLE-related CVD. In summary, 26 women (52 + 8.2 years) with SLE and a history of CVD (SLE cases)were compared with 26 age-matched women with SLE and no clinical manifestations of CVD (SLE controls) and 26 age-matched population-based control women (population controls). Plasma concentrations of circulating TNF-α, TNF-α receptor 1 (sTNFR1) and TNF-α receptor 2 (sTNFR2) were determined by ELISA. TNF-α, sTNFR1 and sTNFR2 were raised in SLE cases as compared to SLE controls (P = 0.009; P = 0.001; P = 0.001, respectively), and SLE controls had higher levels than population controls (P = 0.001; P = 0.02; P = 0.001, respectively). Exclusively in the SLE case group there was a striking positivecorrelationbetweenTNF-α and plasma triglycerides(r = 0.57, P < 0.002), VLDL triglycerides (r = 0.54, P = 0.004) and VLDL cholesterol (r = 0.58, P = 0.002). Furthermore, TNF-α correlated with the waist-hip ratio but not with estimated insulin resistance. TNF-α may thus be a major factor in SLE-related CVD acting both by contributing to hypertriglyceridaemia and by promoting atherosclerosis-relatedinflammation. sTNFR1 and sTNFR2 are strongly associated with CVD in SLE but their exact roles in disease development remain to be elucidated.

Predictors of the first cardiovascular event in patients with systemic lupus erythematosus - a prospective cohort study

IntroductionCardiovascular disease (CVD) is a major cause of premature mortality among Systemic lupus erythematosus (SLE) patients. Many studies have measured and evaluated risk factors for premature subclinical atherosclerosis, but few studies are prospective and few have evaluated risk factors for hard endpoints, i.e. clinically important cardiovascular events (CVE). We investigated the impact of traditional and lupus associated risk factors for the first ever CVE in a longitudinal cohort of SLE patients.MethodsA total of 182 SLE patients (mean age 43.9 years) selected to be free of CVE were included. Cardiovascular and autoimmune biomarkers were measured on samples collected after overnight fasting at baseline. Clinical information was collected at baseline and at follow up. End point was the first ever CVE (ischemic heart, cerebrovascular or peripheral vascular disease or death due to CVD). Impact of baseline characteristics/biomarkers on the risk of having a first CVE was evaluated with Cox regression.ResultsFollow up was 99.5% after a mean time of 8.3 years. Twenty-four patients (13%) had a first CVE. In age-adjusted Cox regression, any positive antiphospholipid antibody (aPL), elevated markers of endothelial activation (von Willebrand factor (vWf), soluble vascular cellular adhesion molecule-1 (sVCAM-1)) and fibrinogen predicted CVEs. Of SLE manifestations, arthritis, pleuritis and previous venous occlusion were positively associated with future CVEs while thrombocytopenia was negatively associated. Among traditional risk factors only age and smoking were significant predictors. In a multivariable Cox regression model age, any positive aPL, vWf and absence of thrombocytopenia were all predictors of the first CVE.ConclusionsIn addition to age, positive aPL, biomarkers indicating increased endothelial cell activity/damage, and absence of thrombocytopenia were independent predictors of CVEs in this prospective study. Our results indicate that activation of the endothelium and the coagulation system are important features in SLE related CVD. Furthermore, we observed that the risk of CVEs seems to differ between subgroups of SLE patients.

Copy number, linkage disequilibrium and disease association in the FCGR locus

The response of a leukocyte to immune complexes (ICs) is modulated by receptors for the Fc region of IgG (FcγRs), and alterations in their affinity or function have been associated with risk of autoimmune diseases, including systemic lupus erythematosus (SLE). The low-affinity FcγR genomic locus is complex, containing regions of copy number variation (CNV) which can alter receptor expression and leukocyte responses to IgG. Combined paralogue ratio tests (PRTs) were used to distinguish three intervals within the FCGR locus which undergo CNV, and to determine FCGR gene copy number (CN). There were significant differences in FCGR3B and FCGR3A CNV profiles between Caucasian, East Asian and Kenyan populations. A previously noted association of low FCGR3B CN with SLE in Caucasians was supported [OR = 1.57 (1.08–2.27), P = 0.018], and replicated in Chinese [OR = 1.65 (1.25–2.18), P = 4 × 10−4]. There was no association of FCGR3B CNV with vasculitis, nor with malarial or bacterial infection. Linkage disequilibrium (LD) between multi-allelic FCGR3B CNV and SLE-associated SNPs in the FCGR locus was defined for the first time. Despite LD between FCGR3B CNV and a variant in FcγRIIB (I232T) which abolishes inhibitory function, both reduced CN of FCGR3B and homozygosity of the FcγRIIB-232T allele were individually strongly associated with SLE risk. Thus CN of FCGR3B, which controls IC responses and uptake by neutrophils, and variations in FCGR2B, which controls factors such as antibody production and macrophage activation, are important in SLE pathogenesis. Further interpretations of contributions to pathogenesis by FcγRs must be made in the context of LD involving CNV regions.

Risk factors for cardiovascular mortality in patients with systemic lupus erythematosus, a prospective cohort study

IntroductionSystemic lupus erythematosus (SLE) is a chronic autoimmune disease. Cardiovascular disease (CVD) is common and a major cause of mortality. Studies on cardiovascular morbidity are abundant, whereas mortality studies focusing on cardiovascular outcomes are scarce. The aim of this study was to investigate causes of death and baseline predictors of overall (OM), non-vascular (N-VM), and specifically cardiovascular (CVM) mortality in SLE, and to evaluate systematic coronary risk evaluation (SCORE).Methods208 SLE patients were included 1995-1999 and followed up after 12 years. Clinical evaluation, CVD risk factors, and biomarkers were recorded at inclusion. Death certificates and autopsy protocols were collected. Causes of death were divided into CVM (ischemic vascular and general atherosclerotic diseases), N-VM and death due to pulmonary hypertension. Predictors of mortality were investigated using multivariable Cox regression. SCORE and standardized mortality ratio (SMR) were calculated.ResultsDuring follow-up 42 patients died at mean age of 62 years. SMR 2.4 (CI 1.7-3.0). 48% of deaths were caused by CVM. SCORE underestimated CVM but not to a significant level. Age, high cystatin C levels and established arterial disease were the strongest predictors for all- cause mortality. After adjusting for these in multivariable analyses, only smoking among traditional risk factors, and high soluble vascular cell adhesion molecule-1 (sVCAM-1), high sensitivity C-reactive protein (hsCRP), anti-beta2 glycoprotein-1 (abeta2GP1) and any antiphospholipid antibody (aPL) among biomarkers, remained predictive of CVM.ConclusionWith the exception of smoking, traditional risk factors do not capture the main underlying risk factors for CVM in SLE. Rather, cystatin C levels, inflammatory and endothelial markers, and antiphospholipid antibodies (aPL) differentiate patients with favorable versus severe cardiovascular prognosis. Our results suggest that these new biomarkers are useful in evaluating the future risk of cardiovascular mortality in SLE patients.