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

ObjectivesFollowing 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.MethodsWe 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.ResultsWe 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).ConclusionWe 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.

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A New Risk Variant for Multiple Sclerosis at 11q23.3 Locus Is Associated with Expansion of CXCR5+ Circulating Regulatory T Cells

Journal of Clinical Medicine ◽

10.3390/jcm9030625 ◽

2020 ◽

Vol 9 (3) ◽

pp. 625

Author(s):

Elia Gil-Varea ◽

Maria Fedetz ◽

Herena Eixarch ◽

Nino Spataro ◽

Luisa María Villar ◽

...

Keyword(s):

Multiple Sclerosis ◽

T Cells ◽

Regulatory T Cells ◽

Mononuclear Cells ◽

Association Studies ◽

Luciferase Reporter ◽

Regulatory Sequences ◽

Genome Wide Association Studies ◽

Enhancer Activity ◽

Peripheral Blood Mononuclear

Genome-wide association studies and meta-analysis have contributed to the identification of more than 200 loci associated with multiple sclerosis (MS). However, a proportion of MS heritability remains unknown. We aimed to uncover new genetic variants associated with MS and determine their functional effects. For this, we resequenced the exons and regulatory sequences of 14 MS risk genes in a cohort of MS patients and healthy individuals (n = 1070) and attempted to validate a selection of signals through genotyping in an independent cohort (n = 5138). We identified three new MS-associated variants at C-X-C motif chemokine receptor 5 (CXCR5), Ts translation elongation factor, mitochondrial (TSFM) and cytochrome P450 family 24 subfamily A member 1 (CYP24A1). Rs10892307 resulted in a new signal at the CXCR5 region that explains one of the associations with MS within the locus. This polymorphism and three others in high linkage disequilibrium mapped within regulatory regions. Of them, rs11602393 showed allele-dependent enhancer activity in the forward orientation as determined by luciferase reporter assays. Immunophenotyping using peripheral blood mononuclear cells from MS patients associated the minor allele of rs10892307 with increased percentage of regulatory T cells expressing CXCR5. This work reports a new signal for the CXCR5 MS risk locus and points to rs11602393 as the causal variant. The expansion of CXCR5+ circulating regulatory T cells induced by this variant could cause its MS association.

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Detection and Functional Evaluation of −262A/T and −188A/G Polymorphisms of SLAM Gene in Patients with Systemic Lupus Erythematosus

The Journal of Rheumatology ◽

10.3899/jrheum.091390 ◽

2010 ◽

Vol 37 (11) ◽

pp. 2268-2272 ◽

Cited By ~ 3

Author(s):

YI YOU ◽

ZHE WANG ◽

GUO-HONG DENG ◽

YI LIU ◽

FEI HAO

Keyword(s):

Systemic Lupus Erythematosus ◽

Lupus Erythematosus ◽

Mononuclear Cells ◽

Gene Promoter ◽

Chinese Patients ◽

Luciferase Reporter ◽

Functional Evaluation ◽

Reporter System ◽

Systemic Lupus ◽

Peripheral Blood Mononuclear

Objective.Signaling lymphocytic activation molecule (SLAM) has been related to the pathology of systemic lupus erythematosus (SLE) through regulation of T cell-dependent humoral immune responses. We investigated the functional associations of the −262A/T and −188A/G polymorphisms of SLAM in Chinese patients with SLE.Methods.Genotyping of −262A/T (rs2295614) and −188A/G (rs2295613) in SLAM was carried out in 248 cases and 278 controls. Promoter activities of haplotypes on the SLAM gene were evaluated with the dual-luciferase reporter system. The mRNA expressions of SLAM on peripheral blood mononuclear cells (PBMC) of SLE patients with different genotypes were determined by real-time polymerase chain reaction.Results.Frequencies of −262A allele and −188G allele were significantly higher in SLE patients than in controls. Haplotype analysis and multifactorial logistic regression analysis showed that individuals with the AG/AG haplotype had increased susceptibility to SLE (p = 0.002, OR 1.478, 95% CI 1.152–1.897). In response to PHA stimulation, the SLAM mRNA expression on PBMC of SLE patients was significantly higher in −262A-188G haplotype homozygotes compared with −262A-188G heterozygotes and individuals with other genotypes.Conclusion.Our findings suggest that −262A-188G haplotype in the SLAM gene promoter contributes to the risk of SLE by increasing the expression of SLAM.

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IL28B expression depends on a novel TT/-G polymorphism which improves HCV clearance prediction

Journal of Experimental Medicine ◽

10.1084/jem.20130012 ◽

2013 ◽

Vol 210 (6) ◽

pp. 1109-1116 ◽

Cited By ~ 156

Author(s):

Stéphanie Bibert ◽

Thierry Roger ◽

Thierry Calandra ◽

Murielle Bochud ◽

Andreas Cerny ◽

...

Keyword(s):

Mononuclear Cells ◽

Association Studies ◽

Antiviral Agents ◽

Genetic Regulation ◽

World Population ◽

Genome Wide Association Studies ◽

Peripheral Blood Mononuclear ◽

Genome Wide ◽

Inducible Protein ◽

Improve Patient Management

Approximately 3% of the world population is chronically infected with the hepatitis C virus (HCV), with potential development of cirrhosis and hepatocellular carcinoma. Despite the availability of new antiviral agents, treatment remains suboptimal. Genome-wide association studies (GWAS) identified rs12979860, a polymorphism nearby IL28B, as an important predictor of HCV clearance. We report the identification of a novel TT/-G polymorphism in the CpG region upstream of IL28B, which is a better predictor of HCV clearance than rs12979860. By using peripheral blood mononuclear cells (PBMCs) from individuals carrying different allelic combinations of the TT/-G and rs12979860 polymorphisms, we show that induction of IL28B and IFN-γ–inducible protein 10 (IP-10) mRNA relies on TT/-G, but not rs12979860, making TT/-G the only functional variant identified so far. This novel step in understanding the genetic regulation of IL28B may have important implications for clinical practice, as the use of TT/G genotyping instead of rs12979860 would improve patient management.

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Decreased miR-4512 Levels in Monocytes and Macrophages of Individuals With Systemic Lupus Erythematosus Contribute to Innate Immune Activation and Neutrsophil NETosis by Targeting TLR4 and CXCL2

Frontiers in Immunology ◽

10.3389/fimmu.2021.756825 ◽

2021 ◽

Vol 12 ◽

Author(s):

Binbin Yang ◽

Xinwei Huang ◽

Shuangyan Xu ◽

Li Li ◽

Wei Wu ◽

...

Keyword(s):

Systemic Lupus Erythematosus ◽

Lupus Erythematosus ◽

Target Genes ◽

Mononuclear Cells ◽

Therapeutic Potential ◽

Luciferase Reporter ◽

Systemic Lupus ◽

Peripheral Blood Mononuclear ◽

Monocytes And Macrophages

ObjectiveSystemic lupus erythematosus (SLE) is an autoimmune disease with complex etiology that is not yet entirely understood. We aimed to elucidate the mechanisms and therapeutic potential of microRNAs (miRNAs) in SLE in a Tibetan population.MethodsPeripheral blood mononuclear cells from SLE patients (n = 5) and healthy controls (n = 5) were used for miRNA–mRNA co-sequencing to detect miRNAs related to immune abnormalities associated with SLE. Luciferase reporter assay was used to identify potential targets of candidate miRNA. The target genes were verified in miRNA-agomir/antagomir transfection assays with multiple cells lines and by expression analysis. The effects of candidate miRNA on monocyte and macrophage activation were evaluated by multiple cytokine profiling. Neutrophil extracellular traps (NETs) formation was analyzed in vitro by cell stimulation with supernatants of monocytes and macrophages transfected with candidate miRNA. The rodent MRL/lpr lupus model was used to evaluate the therapeutic effect of CXCL2Ab on SLE and the regulation effect of immune disorders.ResultsIntegrated miRNA and mRNA expression profiling identified miRNA-4512 as a candidate miRNA involved in the regulation of neutrophil activation and chemokine-related pathways. MiR-4512 expression was significantly reduced in monocytes and macrophages from SLE patients. MiR-4512 suppressed the TLR4 pathway by targeting TLR4 and CXCL2. Decreased monocyte and macrophage miR-4512 levels led to the expression of multiple proinflammatory cytokines in vitro. Supernatants of miR-4512 antagomir-transfected monocytes and macrophages significantly promoted NETs formation (P < 0.05). Blocking of CXCL2 alleviated various pathogenic manifestations in MRL/lpr mice, including kidney damage and expression of immunological markers of SLE.ConclusionsWe here demonstrated the role of miR-4512 in innate immunity regulation in SLE. The effect of miR-4512 involves the regulation of monocytes, macrophages, and NETs formation by direct targeting of TLR4 and CXCL2, indicating the miR-4512-TLR4-CXCL2 axis as a potential novel therapeutic target in SLE.

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TTC7B Emerges as a Novel Risk Factor for Ischemic Stroke Through the Convergence of Several Genome-Wide Approaches

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2012.24 ◽

2012 ◽

Vol 32 (6) ◽

pp. 1061-1072 ◽

Cited By ~ 39

Author(s):

Tiago Krug ◽

João Paulo Gabriel ◽

Ricardo Taipa ◽

Benedita V Fonseca ◽

Sophie Domingues-Montanari ◽

...

Keyword(s):

Ischemic Stroke ◽

Mononuclear Cells ◽

Association Studies ◽

Differentially Expressed ◽

Data Sets ◽

Genome Wide Association Studies ◽

Peripheral Blood Mononuclear ◽

Pathogenic Potential ◽

Novel Approach ◽

Genome Wide

We hereby propose a novel approach to the identification of ischemic stroke (IS) susceptibility genes that involves converging data from several unbiased genetic and genomic tools. We tested the association between IS and genes differentially expressed between cases and controls, then determined which data mapped to previously reported linkage peaks and were nominally associated with stroke in published genome-wide association studies. We first performed gene expression profiling in peripheral blood mononuclear cells of 20 IS cases and 20 controls. Sixteen differentially expressed genes mapped to reported whole-genome linkage peaks, including the TTC7B gene, which has been associated with major cardiovascular disease. At the TTC7B locus, 46 tagging polymorphisms were tested for association in 565 Portuguese IS cases and 520 controls. Markers nominally associated in at least one test and defining associated haplotypes were then examined in 570 IS Spanish cases and 390 controls. Several polymorphisms and haplotypes in the intron 5–intron 6 region of TTC7B were also associated with IS risk in the Spanish and combined data sets. Multiple independent lines of evidence therefore support the role of TTC7B in stroke susceptibility, but further work is warranted to identify the exact risk variant and its pathogenic potential.

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Association ofTNFAIP3Polymorphism with Susceptibility to Systemic Lupus Erythematosus in a Japanese Population

Journal of Biomedicine and Biotechnology ◽

10.1155/2010/207578 ◽

2010 ◽

Vol 2010 ◽

pp. 1-5 ◽

Cited By ~ 25

Author(s):

Aya Kawasaki ◽

Ikue Ito ◽

Satoshi Ito ◽

Taichi Hayashi ◽

Daisuke Goto ◽

...

Keyword(s):

Systemic Lupus Erythematosus ◽

Lupus Erythematosus ◽

Japanese Population ◽

Association Studies ◽

Susceptibility Gene ◽

Allelic Association ◽

Genome Wide Association Studies ◽

Mrna Levels ◽

Nucleotide Polymorphisms ◽

Systemic Lupus

Recent genome-wide association studies demonstrated association of single nucleotide polymorphisms (SNPs) in theTNFAIP3region at 6q23 with systemic lupus erythematosus (SLE) in European-American populations. In this study, we investigated whether SNPs in theTNFAIP3region are associated with SLE also in a Japanese population. A case-control association study was performed on the SNPs rs13192841, rs2230926, and rs6922466 in 318 Japanese SLE patients and 444 healthy controls. Association of rs2230926 G allele with SLE was replicated in Japanese (allelic associationP=.033, odds ratio [OR] 1.47, recessive modelP=.023, OR 8.52). The association was preferentially observed in the SLE patients with nephritis. When theTNFAIP3mRNA levels of the HapMap samples were examined using GENEVAR database, the presence ofTNFAIP3rs2230926 G allele was associated with lower mRNA expression ofTNFAIP3(P=.013). These results indicated thatTNFAIP3is a susceptibility gene to SLE both in the Caucasian and Asian populations.

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The STAT4 SLE risk allele rs7574865[T] is associated with increased IL-12-induced IFN-γ production in T cells from patients with SLE

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2017-212794 ◽

2018 ◽

Vol 77 (7) ◽

pp. 1070-1077 ◽

Cited By ~ 25

Author(s):

Niklas Hagberg ◽

Martin Joelsson ◽

Dag Leonard ◽

Sarah Reid ◽

Maija-Leena Eloranta ◽

...

Keyword(s):

T Cells ◽

Immune Cells ◽

Lupus Erythematosus ◽

Kinase Inhibitors ◽

Mononuclear Cells ◽

Risk Allele ◽

Severe Disease ◽

Janus Kinase ◽

Peripheral Blood Mononuclear ◽

Ifn Γ

ObjectivesGenetic variants in the transcription factor STAT4 are associated with increased susceptibility to systemic lupus erythematosus (SLE) and a more severe disease phenotype. This study aimed to clarify how the SLE-associated intronic STAT4 risk allele rs7574865[T] affects the function of immune cells in SLE.MethodsPeripheral blood mononuclear cells (PBMCs) were isolated from 52 genotyped patients with SLE. Phosphorylation of STAT4 (pSTAT4) and STAT1 (pSTAT1) in response to interferon (IFN)-α, IFN-γ or interleukin (IL)-12, total levels of STAT4, STAT1 and T-bet, and frequency of IFN-γ+ cells on IL-12 stimulation were determined by flow cytometry in subsets of immune cells before and after preactivation of cells with phytohaemagglutinin (PHA) and IL-2. Cellular responses and phenotypes were correlated to STAT4 risk allele carriership. Janus kinase inhibitors (JAKi) selective for TYK2 (TYK2i) or JAK2 (JAK2i) were evaluated for inhibition of IL-12 or IFN-γ-induced activation of SLE PBMCs.ResultsIn resting PBMCs, the STAT4 risk allele was neither associated with total levels of STAT4 or STAT1, nor cytokine-induced pSTAT4 or pSTAT1. Following PHA/IL-2 activation, CD8+ T cells from STAT4 risk allele carriers displayed increased levels of STAT4 resulting in increased pSTAT4 in response to IL-12 and IFN-α, and an augmented IL-12-induced IFN-γ production in CD8+ and CD4+ T cells. The TYK2i and the JAK2i efficiently blocked IL-12 and IFN-γ-induced activation of PBMCs from STAT4 risk patients, respectively.ConclusionsT cells from patients with SLE carrying the STAT4 risk allele rs7574865[T] display an augmented response to IL-12 and IFN-α. This subset of patients may benefit from JAKi treatment.

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MiR-301a-3p Advances IRAK1-Mediated Differentiation of Th17 Cells to Promote the Progression of Systemic Lupus Erythematosus via Targeting PELI1

Journal of Healthcare Engineering ◽

10.1155/2021/2982924 ◽

2021 ◽

Vol 2021 ◽

pp. 1-7

Author(s):

Shuaihantian Luo ◽

Ruifang Wu ◽

Qianwen Li ◽

Guiying Zhang

Keyword(s):

Systemic Lupus Erythematosus ◽

Lupus Erythematosus ◽

Th17 Cells ◽

Mononuclear Cells ◽

Inflammatory Factors ◽

Luciferase Reporter ◽

Regulation Mechanism ◽

Systemic Lupus ◽

Peripheral Blood Mononuclear ◽

Aberrant Expression

Systemic lupus erythematosus (SLE) is a common autoimmune disease with high incidence in females. The pathogenesis of SLE is complex, and healing SLE has become a serious challenge for clinical treatment. Aberrant expression of miR-301a-3p involves the progressions of multiple diseases, and some studies have indicated that increased miR-301a-3p could induce the inflammatory injury of some organs. However, the role and molecular mechanism of miR-301a-3p in SLE remain unclear. In this study, the miR-301a-3p levels in peripheral blood mononuclear cells (PBMCs) of the patients with SLE and health subjects were measured with qRT-PCR. The ELISA assay was used to investigate the effect of miR-301a-3p on the levels of inflammatory factors in PBMCs, and flow cytometry assays were used to observe the effect of miR-301a-3p on the levels of CD4+ T cells and Th17 cells in PBMCs. Moreover, TargetScan, dual-luciferase reporter assay, and western blot were used to reveal the downstream targets and regulation mechanism of miR-301a-3p in SLE. The results showed that miR-301a-3p was significantly upregulated in PBMCs of the SLE patients, and increased miR-301a-3p could boost the expression of IL-6, IL-17, and INF-γ in PBMCs and promote the differentiation of Th17 cells. It was found that PELI1 was a target of miR-301a-3p, and PELI1 upregulation could effectively reverse the effect of miR-301a-3p on PBMCs. Besides, this study also found that miR-301a-3p could promote the expression of IRAK1 to involve the progression of SLE via targeting PELI1. In conclusion, this study suggests that increased miR-301a-3p serves as a pathogenic factor in SLE to promote IRAK1-mediated differentiation of Th17 cells via targeting PELI1.

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Genomic sequencing and functional analyses identify MAP4K3/GLK germline and somatic variants associated with systemic lupus erythematosus

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-221010 ◽

2021 ◽

pp. annrheumdis-2021-221010

Author(s):

Huai-Chia Chuang ◽

Wei-Ting Hung ◽

Yi-Ming Chen ◽

Pu-Ming Hsu ◽

Jeng-Hsien Yen ◽

...

Keyword(s):

Systemic Lupus Erythematosus ◽

T Cells ◽

Lupus Erythematosus ◽

Mononuclear Cells ◽

Site Directed Mutagenesis ◽

Mrna Levels ◽

Systemic Lupus ◽

Peripheral Blood Mononuclear ◽

Somatic Variant ◽

Germline Variant

ObjectivesMAP4K3 (GLK) overexpression in T cells induces interleukin (IL)-17A production and autoimmune responses. GLK overexpressing T-cell population is correlated with severity of human systemic lupus erythematosus (SLE); however, it is unclear how GLK is upregulated in patients with SLE.MethodsWe enrolled 181 patients with SLE and 250 individuals without SLE (93 healthy controls and 157 family members of patients with SLE) in two independent cohorts from different hospitals/cities. Genomic DNAs of peripheral blood mononuclear cells were subjected to next-generation sequencing to identify GLK gene variants. The functional consequences of the identified GLK germline or somatic variants were investigated using site-directed mutagenesis and cell transfection, followed by reporter assays, mass spectrometry, immunoblotting, coimmunoprecipitation, and in situ proximity ligation assays.ResultsWe identified 58 patients with SLE from Cohort #1 and #2 with higher frequencies of a somatic variant (chr2:39 477 124 A>G) in GLK 3′-untranslated region (UTR); these patients with SLE showed increased serum anti-double-stranded DNA levels and decreased serum C3/C4 levels. This somatic variant in 3′-UTR enhanced GLK mRNA levels in T cells. In addition, we identified five patients with SLE with GLK (A410T) germline variant in Cohort #1 and #2, as well as two other patients with SLE with GLK (K650R) germline variant in Cohort #1. Another GLK germline variant, A579T, was also detected in one patient with SLE from Cohort #2. Both GLK (A410T) and GLK (K650R) mutants inhibited GLK ubiquitination induced by the novel E3 ligase makorin ring-finger protein 4 (MKRN4), leading to GLK protein stabilisation.ConclusionsMultiple GLK germline and somatic variants cause GLK induction by increasing mRNA or protein stability in patients with SLE.

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Vitamin D receptor is negatively correlated with interferon-γ-inducible protein-10 in systemic lupus erythematosus with renal involvement

European Journal of Inflammation ◽

10.1177/2058739220942333 ◽

2020 ◽

Vol 18 ◽

pp. 205873922094233

Author(s):

Xueqin Wu ◽

Hao Zhang ◽

Shuang Zhang ◽

Jinzhong Yuan ◽

Jishi Liu ◽

...

Keyword(s):

Systemic Lupus Erythematosus ◽

Vitamin D ◽

Vitamin D Receptor ◽

Lupus Erythematosus ◽

Mononuclear Cells ◽

Renal Involvement ◽

Mrna Levels ◽

Systemic Lupus ◽

Peripheral Blood Mononuclear ◽

Inducible Protein

The vitamin D receptor (VDR) plays an important role in the pathogenesis of systemic lupus erythematosus (SLE) mainly due to its anti-inflammatory and immunomodulatory activities. Interferon-inducible protein-10 (IP-10) is a chemokine found to be increased in SLE patients and correlated with severity of lupus nephritis. In this case control study, we investigated the connection between VDR expression and chemokine IP-10 in peripheral blood mononuclear cells (PBMCs) of SLE patients with or without renal involvement. 62 SLE patients and 30 healthy subjects were enrolled between 2014 and 2016. PBMC VDR mRNA and IP-10 mRNA were quantified by real-time PCR. PBMC VDR protein was measured by Western blotting. PBMC VDR mRNA and protein were downregulated, whereas IP-10 mRNA was upregulated in SLE patients with renal involvement. VDR mRNA levels were negatively correlated with systemic lupus international collaborating clinics (SLICC) renal activity scores (r = −0.423, P = 0.016) and IP-10 mRNA levels (r = −0.428, P = 0.008) in SLE with renal involvement. This study demonstrates that PBMC VDR was negatively correlated with SLICC renal activity and chemokine IP-10 in SLE patients with renal involvement which suggests that VDR downregulation may be used as an indicator of renal injury in SLE patients.

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