Distinct Effector B Cells Induced by Unregulated Toll-like Receptor 7 Contribute to Pathogenic Responses in Systemic Lupus Erythematosus.

Systemic Lupus Erythematosus (SLE) is characterized by B cells lacking IgD and CD27 (double negative; DN). We show that DN cell expansions reflected a subset of CXCR5- CD11c+ cells (DN2) representing pre-plasma cells (PC). DN2 cells predominated in African-American patients with active disease and nephritis, anti-Smith and anti-RNA autoantibodies. They expressed a T-bet transcriptional network; increased Toll-like receptor-7 (TLR7); lacked the negative TLR regulator TRAF5; and were hyper-responsive to TLR7. DN2 cells shared with activated naive cells (aNAV), phenotypic and functional features, and similar transcriptomes. Their PC differentiation and autoantibody production was driven by TLR7 in an interleukin-21 (IL-21)-mediated fashion. An in vivo developmental link between aNAV, DN2 cells, and PC was demonstrated by clonal sharing. This study defines a distinct differentiation fate of autoreactive naive B cells into PC precursors with hyper-responsiveness to innate stimuli, as well as establishes prominence of extra-follicular B cell activation in SLE, and identifies therapeutic targets.

Axonal dysfunction is associated with interferon-γ levels in childhood-onset systemic lupus erythematosus: a multivoxel magnetic resonance spectroscopy study.

OBJECTIVE: Axonal/neuronal damage has been shown to be a pathological finding that precedes neuropsychiatric manifestations in SLE. The objective of this study was to determine the presence of axonal dysfunction in childhood-onset SLE patients (cSLE) and to determine clinical, immunological and treatment features associated with its occurrence. METHODS: We included 86 consecutive cSLE patients [median age 17 (range 5-28) years] and 71 controls [median age 18 (5-28) years]. We performed proton magnetic resonance spectroscopic imaging using point resolved spectroscopy sequence over the superior-posterior region of the corpus callosum and signals from N-acetylaspartate (NAA), choline-based (CHO), creatine-containing (Cr), myo-inositol (mI), glutamate, glutamine and lactate were measured and metabolites/Cr ratios were determined. Complete clinical, laboratory and neurological evaluations were performed in all subjects. Serum IL-4, IL-5, IL-6, IL-10, IL-12, IL-17, TNF-α and INF-γ cytokine levels, antiribosomal P protein antibodies (anti-P) and S100ß were measured by ELISA using commercial kits. Data were compared by non-parametric tests. RESULTS: NAA/Cr ratios (P = 0.035) and lactate/Cr ratios (P = 0.019) were significantly decreased in cSLE patients when compared with controls. In multivariate analysis, IFN-γ levels [odds ratio (OR) = 4.1; 95% CI: 2.01, 7.9] and depressive symptoms (OR = 1.9; 95% CI: 1.1, 3.2) were associated with NAA/Cr ratio. Increased CHO/Cr was associated with the presence of cognitive impairment (OR = 3.4; 95% CI: 2.034, 5.078; P < 0.001). mI/Cr ratio correlated with cumulative glucocorticoids dosage (r = 0.361, P = 0.014). CONCLUSION: NAA and CHO ratios may be useful as biomarkers in neuropsychiatric cSLE. Longitudinal studies are necessary to determine whether they predict structural damage.

Progressive Cellular Senescence Mediates Renal Dysfunction in Ischemic Nephropathy.

BACKGROUND: Peripheral vascular diseases may induce chronic ischemia and cellular injury distal to the arterial obstruction. Cellular senescence involves proliferation arrest in response to stress, which can damage neighboring cells. Renal artery stenosis (RAS) induces stenotic-kidney dysfunction and injury, but whether these arise from cellular senescenceand their temporal pattern remain unknown. METHODS: Chronic renal ischemia was induced in transgenic INK-ATTAC and wild type C57BL/6 mice by unilateral RAS, and kidney function (in vivo micro-MRI) and tissue damage were assessed. Mouse healthy and stenotic kidneys were analyzed using unbiased single-cell RNA-sequencing. To demonstrate translational relevance, cellular senescence was studied in human stenotic kidneys. RESULTS: Using intraperitoneal AP20187 injections starting 1, 2, or 4 weeks after RAS, selective clearance of cells highly expressing p16Ink4a attenuated cellular senescence and improved stenotic-kidney function; however, starting treatment immediately after RAS induction was unsuccessful. Broader clearance of senescent cells, using the oral senolytic combination dasatinib and quercetin, in C57BL/6 RAS mice was more effective in clearing cells positive for p21 (Cdkn1a) and alleviating renal dysfunction and damage. Unbiased, single-cell RNA sequencing in freshly dissociated cells from healthy and stenotic mouse kidneys identified stenotic-kidney epithelial cells undergoing both mesenchymal transition and senescence. As in mice, injured human stenotic kidneys exhibited cellular senescence, suggesting this process is conserved. CONCLUSIONS: Maladaptive tubular cell senescence, involving upregulated p16 (Cdkn2a), p19 (Cdkn2d), and p21 (Cdkn1a) expression, is associated with renal dysfunction and injury in chronic ischemia. These findings support development of senolytic strategies to delay chronic ischemic renal injury.

Clinical disease activity and flare in SLE: Current concepts and novel biomarkers.

Systemic lupus erythematosus (SLE) is a complex and heterogeneous systemic autoimmune disease associated with innate and adaptive immune dysregulation. SLE occurs primarily in females of childbearing age, with increased prevalence and severity in minority populations. Despite improvements in treatment modalities, SLE patients frequently experience periods of heightened disease activity and flare that can lead to permanent organ damage, increased morbidity, and early mortality. Such outcomes impair quality of life and inflict a significant socioeconomic burden. Predicting changes in SLE disease activity could allow for closer monitoring and preemptive treatment, but existing clinical, demographic and serologic markers have been only modestly predictive. Novel, proactive approaches to clinical disease management are thus critically needed. Panels of blood biomarkers can detect a breadth of immune pathway dysregulation that captures SLE heterogeneity and disease activity. Alterations in the balance of pro-inflammatory and regulatory soluble mediators have been associated with changes in clinical disease activity and are detectable several weeks prior to clinical flare occurrence. A soluble mediator score has been highly predictive of impending flare in both European American and African American SLE patients, and this score does not require a priori knowledge of specific pathway activation in the patient. We review current concepts of disease activity and flare in SLE, focusing on the potential of novel blood biomarkers to characterize and predict changes in disease activity. Measuring the disordered immune response in SLE in this way promises to improve disease management and prevent organ damage in SLE.

Novel mutation in the NRLP3 manifesting as an intermediate phenotype of cryopyrinopathies.

Cryopyrin-associated periodic syndromes (CAPS) are a group of autoinflammatory diseases associated with NLRP3 gain of function mutations. CAPS associated mutations are found predominantly in exon 3. The objective of this study is to describe a new variant on NRLP3 gene and its phenotype. Case report description of a new NRLP3 pathogenic variant and literature case-based search through INFEVERS database. A 21-year old male who presented multiple tonic-clonic seizures on his 3rd day of life. At age 2, he had recurrent central facial palsy, high fever (40 °C), painful and persistent oral ulcers, abdominal pain, nausea and vomiting, and delayed neuropsychomotor development, with polyarthritis in wrists and knees. Over the years, several symptoms were observed: livedo reticularis, Raynaud's phenomenon, positive pathergy test, heat allodynia, extremely painful genital ulcers, and sporadic conjunctivitis. Laboratory studies revealed persistently elevated inflammatory markers and serum amyloid protein A (30 µg/l). The genetic panel for autoinflammatory diseases revealed heterozygous mutation in the NLRP3, (c.2068G > C, p.E690Q) with 0% of frequency in the general population. The patient denies rash and did not have frontal bossing or patellar overgrowth. We found a positive familial history on mother and brother, who carried the same mutation. The patient was started on canakinumab which controlled his symptoms. Currently, 241 missense variants in the NLRP3 have been described. We presented a new mutation in exon 3 of the NRLP3 gene in a patient that fulfills clinical criteria for CAPS who had complete clinical response to Canakinumab, supporting the idea that this mutation is pathogenic.

Risk of Toxicity After Initiating Immune Checkpoint Inhibitor Treatment in Patients With Rheumatoid Arthritis.

INTRODUCTION: Immune checkpoint inhibitors (ICIs) are increasingly used to treat advanced cancer. Rheumatoid arthritis (RA) is associated with an increased risk of malignancies; however, patients with RA have been excluded from ICI trials. In this study, we evaluated risk of toxicity after initiation of ICI treatment in RA patients. METHODS: We conducted a single-institution, medical records review analysis to assess the incidence of immune-related adverse events (irAEs) and autoimmune disease (AID) flares among patients with AIDs treated with ICIs from 2011 to 2018. A subgroup analysis for RA patients was performed with frequencies of irAEs and AID flares reported. RESULTS: Twenty-two patients with RA who were treated with ICI for malignancy were identified. At the time of ICI initiation, 86% had inactive RA disease activity. Immune-related adverse events occurred in 7 (32%) of patients, with 2 (9%) developing grade 3 (i.e., severe) irAEs. Immune checkpoint inhibitors were temporarily discontinued because of irAEs in 5 patients (23%), and permanently in 1 patient. Rheumatoid arthritis flares occurred in 12 patients (55%). Of those, 10 (83%) received oral corticosteroids with an adequate treatment response. CONCLUSIONS: Our analysis suggests that irAEs following ICI treatment are not increased among RA patients compared with other cancer patients. Heightened RA disease activity during ICI treatment is common, but most adverse events are manageable with oral corticosteroids, and few require permanent ICI discontinuation. A close collaboration between the oncologist and rheumatologist is advisable when considering ICIs in patients with RA.

Novel genetic associations with interferon in systemic lupus erythematosus identified by replication and fine-mapping of trait-stratified genome-wide screen.

BACKGROUND/PURPOSE: High serum interferon alpha (IFN-α) is an important heritable phenotype in systemic lupus erythematosus (SLE) which is involved in primary disease pathogenesis. High vs. low levels of IFN-α are associated with disease severity and account for some of the biological heterogeneity between SLE patients. The aim of the study was to replicate and fine-map previously detected genetic associations with serum IFN-α in SLE. METHODS: We previously undertook a case-case genome-wide association study of SLE patients stratified by ancestry and extremes of phenotype in serum IFN-α. Single nucleotide polymorphisms (SNPs) in seven loci identified in this screen were selected for follow up in a large independent cohort of 1370 SLE patients (703 European-ancestry, 432 African ancestry, and 235 Amerindian ancestry). Each ancestral background was analyzed separately, and ancestry-informative markers were used to control for ancestry and admixture. RESULTS: We find a rare haplotype spanning the promoter region of EFNA5 that is strongly associated with serum IFN-α in both African-American and European-American SLE patients (OR = 3.0, p = 3.7 × 10-6). We also find SNPs in the PPM1H, PTPRM, and NRGN regions associated with IFN-α levels in European-American, Amerindian, and African-American SLE patients respectively. Many of these associations are within regulatory regions of the gene, suggesting an impact on transcription. CONCLUSION: This study demonstrates the power of molecular sub-phenotypes to reveal genetic factors involved in complex autoimmune disease. The distinct associations observed in different ancestral backgrounds emphasize the heterogeneity of molecular pathogenesis in SLE.

Lessons from precision medicine in rheumatology.

Systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) are two common autoimmune rheumatic diseases that vary in severity, clinical presentation, and disease course between individuals. Molecular and genetic studies of both diseases have identified candidate genes and molecular pathways that are linked to various disease outcomes and treatment responses. Currently, patients can be grouped into molecular subsets in each disease, and these molecular categories should enable precision medicine approaches to be applied in rheumatic diseases. In this article, we will review key lessons learned about disease heterogeneity and molecular characterization in rheumatology, which we hope will lead to personalized therapeutic strategies.

Manifestations of systemic lupus erythematosus in female patients with polysomy X: Possible roles of chromosome X.

Clinical manifestations of systemic lupus erythematosus (SLE) in female patients with polysomy X have been less characterized as compared to those in male patients. Here, we describe a 28-year-old woman with trisomy X (47,XXX) who developed SLE. She had polyarthritis, hemolytic anemia, and was positive for anti-nuclear and anti-dsDNA antibodies. We discuss the common SLE manifestations with female polysomy X and the possible link between the development of SLE and the presence of extra X-chromosomes.

Differential Expression of miR-4520a Associated With Pyrin Mutations in Familial Mediterranean Fever (FMF).

Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurrent, acute, and self-limiting attacks of fever. Mutations in MEFV gene encoding pyrin account for FMF, but the high number of heterozygote patients with typical symptoms of the disease has driven a number of alternative aetiopathogenic hypotheses. The MEFV gene was knocked down in human myelomonocytic cells that express endogenous pyrin to identify deregulated microRNAs (miRNAs). Microarray analyses revealed 29 significantly differentially expressed miRNAs implicated in pathways associated with cellular integrity and survival. Implementation of in silico gene network prediction algorithms and bioinformatics analyses showed that miR-4520a is predicted to target genes implicated in autophagy through regulation of RHEB/mTOR signaling. Differential expression levels of RHEB were confirmed by luciferase reporter gene assays providing further evidence that is directly targeted by miR-4520a. Although the relative expression levels of miR-4520a were variable among FMF patients, the statistical expression of miR-4520a was different between FMF mutation carriers and controls (P = 0.0061), indicating an association between miR-4520a expression and MEFV mutations. Comparison between FMF patients bearing the M694V mutation, associated with severe disease, and healthy controls showed a significant increase in miR-4520a expression levels (P = 0.00545). These data suggest that RHEB, the main activator of mTOR signaling, is a valid target of miR-4520a with the relative expression levels of the latter being significantly deregulated in FMF patients and highly dependent on the presence of pyrin mutations, especially of the M694V type. These results suggest a role of deregulated autophagy in the pathogenesis of FMF. J. Cell. Physiol. 232: 1326-1336, 2017. © 2016 Wiley Periodicals, Inc.

Genetics of human lupus nephritis.

Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease characterized by immune complex formation with multi-organ manifestations. Lupus nephritis (LN) is one of the most severe types of organ damage in SLE, and it clearly contributes to increased morbidity and mortality due to SLE. LN occurs more frequently and is more severe in non-European ancestral backgrounds, although the cause of this disparity remains largely unknown. Genetic factors play an important role in the pathogenesis of SLE. Although many SLE susceptibility genes have been identified, the genetic basis of LN is not as well understood. While some of the established general SLE susceptibility genes are associated with LN, recent discoveries highlight a number of genes with renal functions that are specifically associated with LN. Some of these genes associated with LN help to explain the disparity in the prevalence of nephritis between individuals with SLE, and also partially explain differences in LN between ancestral backgrounds. Moreover, not only the gene mutations, but also post-translational modifications seem to play important roles in the pathogenesis of LN. Overall it seems likely that a combination of general SLE susceptibility genes cooperate with LN specific risk genes to result in the genetic propensity for LN. In this review, we will outline the genetic contribution to LN and describe possible roles of LN susceptibility genes.

Two functional lupus-associated BLK promoter variants control cell-type- and developmental-stage-specific transcription.

Efforts to identify lupus-associated causal variants in the FAM167A/BLK locus on 8p21 are hampered by highly associated noncausal variants. In this report, we used a trans-population mapping and sequencing strategy to identify a common variant (rs922483) in the proximal BLK promoter and a tri-allelic variant (rs1382568) in the upstream alternative BLK promoter as putative causal variants for association with systemic lupus erythematosus. The risk allele (T) at rs922483 reduced proximal promoter activity and modulated alternative promoter usage. Allelic differences at rs1382568 resulted in altered promoter activity in B progenitor cell lines. Thus, our results demonstrated that both lupus-associated functional variants contribute to the autoimmune disease association by modulating transcription of BLK in B cells and thus potentially altering immune responses.

Combined role of vitamin D status and CYP24A1 in the transition to systemic lupus erythematosus.

OBJECTIVE: We examined whether measures of vitamin D were associated with transitioning to systemic lupus erythematosus (SLE) in individuals at risk for SLE. METHODS: 436 individuals who reported having a relative with SLE but who did not have SLE themselves were evaluated at baseline and again an average of 6.3 (±3.9) years later. Fifty-six individuals transitioned to SLE (≥4 cumulative American College of Rheumatology criteria). 25-Hydroxyvitamin D (25[OH]D) levels were measured by ELISA. Six single-nucleotide polymorphisms in four vitamin D genes were genotyped. Generalised estimating equations, adjusting for correlation within families, were used to test associations between the vitamin D variables and the outcome of transitioning to SLE. RESULTS: Mean baseline 25[OH]D levels (p=0.42) and vitamin D supplementation (p=0.65) were not different between those who did and did not transition to SLE. Vitamin D deficiency (25[OH]D <20 ng/mL) was greater in those who transitioned compared with those who did not transition to SLE (46% vs 33%, p=0.05). The association between 25[OH]D and SLE was modified by CYP24A1 rs4809959, where for each additional minor allele increased 25[OH]D was associated with decreased SLE risk: zero minor alleles (adjusted OR: 1.03, CI 0.98 to 1.09), one minor allele (adjusted OR: 1.01, CI 0.97 to 1.05) and two minor alleles (adjusted OR: 0.91, CI 0.84 to 0.98). Similarly, vitamin D deficiency significantly increased the risk of transitioning to SLE in those with two minor alleles at rs4809959 (adjusted OR: 4.90, CI 1.33 to 18.04). CONCLUSIONS: Vitamin D status and CYP24A1 may have a combined role in the transition to SLE in individuals at increased genetic risk for SLE.

The Thr300Ala variant in ATG16L1 is associated with improved survival in human colorectal cancer and enhanced production of type I interferon.

OBJECTIVE: ATG16L1 is an autophagy gene known to control host immune responses to viruses and bacteria. Recently, a non-synonymous single-nucleotide polymorphism in ATG16L1 (Thr300Ala), previously identified as a risk factor in Crohn's disease (CD), was associated with more favourable clinical outcomes in thyroid cancer. Mechanisms underlying this observation have not been proposed, nor is it clear whether an association between Thr300Ala and clinical outcomes will be observed in other cancers. We hypothesised that Thr300Ala influences clinical outcome in human colorectal cancer (CRC) and controls innate antiviral pathways in colon cancer cells. DESIGN: We genotyped 460 patients with CRC and assessed for an association between ATG16L1 Thr300Ala and overall survival and clinical stage. Human CRC cell lines were targeted by homologous recombination to examine the functional consequence of loss of ATG16L1, or introduction of the Thr300Ala variant. RESULTS: We found an association between longer overall survival, reduced metastasis and the ATG16L1 Ala/Ala genotype. Tumour sections from ATG16L1 Ala/Ala patients expressed elevated type I interferons (IFN-I)-inducible, MxA, suggesting that differences in cytokine production may influence disease progression. When introduced into human CRC cells by homologous recombination, the Thr300Ala variant did not affect bulk autophagy, but increased basal production of type I IFN. Introduction of Thr300Ala resulted in increased sensitivity to the dsRNA mimic poly(I:C) through a mitochondrial antiviral signalling (MAVS)-dependent pathway. CONCLUSIONS: The CD-risk allele, Thr300Ala, in ATG16L1 is associated with improved overall survival in human CRC, generating a rationale to genotype ATG16L1 Thr300Ala in patients with CRC. We found that Thr300A alters production of MAVS-dependent type I IFN in CRC cells, providing a mechanism that may influence clinical outcomes.

Combined protein- and nucleic acid-level effects of rs1143679 (R77H), a lupus-predisposing variant within ITGAM.

Integrin alpha M (ITGAM; CD11b) is a component of the macrophage-1 antigen complex, which mediates leukocyte adhesion, migration and phagocytosis as part of the immune system. We previously identified a missense polymorphism, rs1143679 (R77H), strongly associated with systemic lupus erythematosus (SLE). However, the molecular mechanisms of this variant are incompletely understood. A meta-analysis of published and novel data on 28 439 individuals with European, African, Hispanic and Asian ancestries reinforces genetic association between rs1143679 and SLE [Pmeta = 3.60 × 10(-90), odds ratio (OR) = 1.76]. Since rs1143679 is in the most active region of chromatin regulation and transcription factor binding in ITGAM, we quantitated ITGAM RNA and surface protein levels in monocytes from patients with each rs1143679 genotype. We observed that transcript levels significantly decreased for the risk allele ('A') relative to the non-risk allele ('G'), in a dose-dependent fashion: ('AA' < 'AG' < 'GG'). CD11b protein levels in patients' monocytes were directly correlated with RNA levels. Strikingly, heterozygous individuals express much lower (average 10- to 15-fold reduction) amounts of the 'A' transcript than 'G' transcript. We found that the non-risk sequence surrounding rs1143679 exhibits transcriptional enhancer activity in vivo and binds to Ku70/80, NFKB1 and EBF1 in vitro, functions that are significantly reduced with the risk allele. Mutant CD11b protein shows significantly reduced binding to fibrinogen and vitronectin, relative to non-risk, both in purified protein and in cellular models. This two-pronged contribution (nucleic acid- and protein-level) of the rs1143679 risk allele to decreasing ITGAM activity provides insight into the molecular mechanisms of its potent association with SLE.

Altered type II interferon precedes autoantibody accrual and elevated type I interferon activity prior to systemic lupus erythematosus classification.

OBJECTIVES: The relationship of immune dysregulation and autoantibody production that may contribute to systemic lupus erythematosus (SLE) pathogenesis is unknown. This study evaluates the individual and combined contributions of autoantibodies, type I interferon (IFN-α) activity, and IFN-associated soluble mediators to disease development leading to SLE. METHODS: Serial serum specimens from 55 individuals collected prior to SLE classification (average timespan=4.3 years) and unaffected healthy controls matched by age (±5 years), gender, race and time of sample procurement were obtained from the Department of Defense Serum Repository. Levels of serum IFN-α activity, IFN-associated mediators and autoantibodies were evaluated and temporal relationships assessed by growth curve modelling, path analysis, analysis of covariance and random forest models. RESULTS: In cases, but not matched controls, autoantibody specificities and IFN-associated mediators accumulated over a period of years, plateauing near the time of disease classification (p<0.001). Autoantibody positivity coincided with or followed type II IFN dysregulation, preceding IFN-α activity in growth curve models, with elevated IFN-α activity and B-lymphocyte stimulator levels occurring shortly before SLE classification (p≤0.005). Cases were distinguished by multivariate random forest models incorporating IFN-γ, macrophage chemoattractant protein (MCP)-3, anti-chromatin and anti-spliceosome antibodies (accuracy 93% >4 years pre-classification; 97% within 2 years of SLE classification). CONCLUSIONS: Years before SLE classification, enhancement of the type II IFN pathway allows for accumulation of autoantibodies and subsequent elevations in IFN-α activity immediately preceding SLE classification. Perturbations in select immunological processes may help identify at-risk individuals for further clinical evaluation or participation in prospective intervention trials.

Increased pretreatment serum IFN-ß/α ratio predicts non-response to tumour necrosis factor α inhibition in rheumatoid arthritis.

OBJECTIVE: Studies suggest that circulating type I interferon (IFN) may predict response to biological agents in rheumatoid arthritis (RA). Prediction of response prior to initiating therapy would represent a major advancement. METHODS: We studied sera from a test set of 32 patients with RA from the Auto-immune Biomarkers Collaborative Network Consortium and a validation set of 92 patients with RA from the Treatment Efficacy and Toxicity in Rheumatoid Arthritis Database and Repository registry. The test set included those with good response or no response to tumour necrosis factor (TNF) inhibitors at 14 weeks by European League Against Rheumatism criteria. The validation set included subjects with good, moderate or no response at 12 weeks. Total serum type I IFN activity, IFN-α and IFN-ß activity were measured using a functional reporter cell assay. RESULTS: In the test set, an increased ratio of IFN-ß to IFN-α (IFN-ß/α activity ratio) in pretreatment serum associated with lack of response to TNF inhibition (p=0.013). Anti-cyclic citrullinated peptide antibody titre and class of TNF inhibitor did not influence this relationship. A receiver-operator curve supported a ratio of 1.3 as the optimal cut-off. In the validation set, subjects with an IFN-ß/α activity ratio >1.3 were significantly more likely to have non-response than good response (OR=6.67, p=0.018). The test had 77% specificity and 45% sensitivity for prediction of non-response compared with moderate or good response. Meta-analysis of test and validation sets confirmed strong predictive capacity of IFN-ß/α activity ratio (p=0.005). CONCLUSIONS: Increased pretreatment serum IFN-ß/α ratio strongly associated with non-response to TNF inhibition. This study supports further investigation of serum type I IFN in predicting outcome of TNF inhibition in RA.

Decreased SMG7 expression associates with lupus-risk variants and elevated antinuclear antibody production.

OBJECTIVES: Following up the systemic lupus erythematosus (SLE) genome-wide association studies (GWAS) identification of NMNAT2 at rs2022013, we fine-mapped its 150 kb flanking regions containing NMNAT2 and SMG7 in a 15 292 case-control multi-ancestry population and tested functions of identified variants. METHODS: We performed genotyping using custom array, imputation by IMPUTE 2.1.2 and allele specific functions using quantitative real-time PCR and luciferase reporter transfections. SLE peripheral blood mononuclear cells (PBMCs) were cultured with small interfering RNAs to measure antinuclear antibody (ANA) and cyto/chemokine levels in supernatants using ELISA. RESULTS: We confirmed association at NMNAT2 in European American (EA) and Amerindian/Hispanic ancestries, and identified independent signal at SMG7 tagged by rs2702178 in EA only (p=2.4×10-8, OR=1.23 (95% CI 1.14 to 1.32)). In complete linkage disequilibrium with rs2702178, rs2275675 in the promoter region robustly associated with SMG7 mRNA levels in multiple expression quantitative trait locus (eQTL) datasets. Its risk allele was dose-dependently associated with decreased SMG7 mRNA levels in PBMCs of 86 patients with SLE and 119 controls (p=1.1×10-3 and 6.8×10-8, respectively) and conferred reduced transcription activity in transfected HEK-293 (human embryonic kidney cell line) and Raji cells (p=0.0035 and 0.0037, respectively). As a critical component in the nonsense-mediated mRNA decay pathway, SMG7 could regulate autoantigens including ribonucleoprotein (RNP) and Smith (Sm). We showed SMG7 mRNA levels in PBMCs correlated inversely with ANA titres of patients with SLE (r=-0.31, p=0.01), and SMG7 knockdown increased levels of ANA IgG and chemokine (C-C motif) ligand 19 in SLE PBMCs (p=2.0×10-5 and 2.0×10-4, respectively). CONCLUSION: We confirmed NMNAT2 and identified independent SMG7 association with SLE. The inverse relationship between levels of the risk allele-associated SMG7 mRNAs and ANA suggested the novel contribution of mRNA surveillance pathway to SLE pathogenesis.