Clonal haematopoiesis across the age spectrum of vasculitis patients with Takayasu's arteritis, ANCA-associated vasculitis and giant cell arteritis.

OBJECTIVES: Ageing and inflammation are associated with clonal haematopoiesis (CH), the emergence of somatic mutations in haematopoietic cells. This study details CH in patients with systemic vasculitis in association with clinical, haematological and immunological parameters. METHODS: Patients with three forms of vasculitis were screened for CH in peripheral blood by error-corrected sequencing. Relative contributions of age and vasculitis on CH prevalence were calculated using multivariable logistic regression. Clonal hierarchies were assessed by proteogenomic single-cell DNA sequencing, and functional experiments were performed in association with CH status. RESULTS: Patients with Takayasu's arteritis (TAK; n=70; mean age=33.2 years), antineutrophil cytoplasmic antibody-associated vasculitis (AAV; n=47; mean age=55.3 years) and giant cell arteritis (GCA; n=59; mean age=71.2 years) were studied. CH, most commonly in DNMT3A and TET2, was detected in 34% (60/176) of patients versus 18% (28/151) of age-matched controls (p<0.01). Prevalence of CH was independently associated with age (standardised B=0.96, p<0.01) and vasculitis (standardised B=0.46, p<0.01), occurring in 61%, 32% and 13% of patients with GCA, AAV and TAK, respectively. Both branched and linear clonal trajectories showed myeloid-lineage bias, and CH was associated with markers of cellular activation. In GCA, mutations were detected in temporal artery biopsies, and clinical relapse correlated with CH in a dose-dependent relationship with clone size. CONCLUSIONS: Age was more strongly associated with CH prevalence than inflammation in systemic vasculitis. Clonal profile was dominated by DNMT3A mutations which were associated with relapse in GCA. CH is not likely a primary causal factor in systemic vasculitis but may contribute to inflammation.

Associations of residential greenness with bone mineral density and osteoporosis: the modifying effect of genetic susceptibility.

OBJECTIVES: To investigate the associations of residential greenness with bone mineral density and incident osteoporosis, and further evaluate the potential modifying effect of genetic susceptibility. METHODS: We used the Normalised Difference Vegetation Index (NDVI) at various buffer distances, including 300 m (NDVI300m), 500 m (NDVI500m), 1000 m (NDVI1000m) and 1500 m (NDVI1500m), to serve as indicators of greenness. We fitted linear regression, logistic regression and Cox proportional hazard models to assess the associations of residential greenness with estimated bone mineral density (eBMD), prevalent osteoporosis and incident osteoporosis, respectively. With the Polygenic Risk Score (PRS) for osteoporosis, we further assessed the joint effects of genetic risk and greenness on the risk of osteoporosis. We conducted causal mediation analyses to explore potential mediators. RESULTS: Each IQR increase in NDVI300m was associated with 0.0007 (95% CI 0.0002 to 0.0013) increase in eBMD, 6% lower risk of prevalent osteoporosis (OR 0.94; 95% CI 0.92 to 0.97) and 5% lower risk of incident osteoporosis (HR 0.95; 95% CI 0.93 to 0.98). The joint effects of greenness and PRS on the risk of osteoporosis displayed a clear dose-response pattern. Compared with individuals exposed to low NDVI levels and high genetic risk, those exposed to high NDVI levels and low genetic risk had a 56% (95% CI 51% to 61%) lower risk of osteoporosis. The primary mediators in the association between greenness and incident osteoporosis were identified as PM2.5 and NO2. CONCLUSIONS: Residential greenness was associated with higher bone mineral density and decreased risk of incident osteoporosis.

Expression quantitative trait loci analysis in rheumatoid arthritis identifies tissue specific variants associated with severity and outcome.

OBJECTIVE: Genome-wide association studies have successfully identified more than 100 loci associated with susceptibility to rheumatoid arthritis (RA). However, our understanding of the functional effects of genetic variants in causing RA and their effects on disease severity and response to treatment remains limited. METHODS: In this study, we conducted expression quantitative trait locus (eQTL) analysis to dissect the link between genetic variants and gene expression comparing the disease tissue against blood using RNA-Sequencing of synovial biopsies (n=85) and blood samples (n=51) from treatment-naïve patients with RA from the Pathobiology of Early Arthritis Cohort. RESULTS: This identified 898 eQTL genes in synovium and genes loci in blood, with 232 genes in common to both synovium and blood, although notably many eQTL were tissue specific. Examining the HLA region, we uncovered a specific eQTL at HLA-DPB2 with the critical triad of single-nucleotide polymorphisms (SNPs) rs3128921 driving synovial HLA-DPB2 expression, and both rs3128921 and HLA-DPB2 gene expression correlating with clinical severity and increasing probability of the lympho-myeloid pathotype. CONCLUSIONS: This analysis highlights the need to explore functional consequences of genetic associations in disease tissue. HLA-DPB2 SNP rs3128921 could potentially be used to stratify patients to more aggressive treatment immediately at diagnosis.

Haematopoietic stem cell-derived immune cells have reduced X chromosome inactivation skewing in systemic lupus erythematosus.

OBJECTIVES: Systemic lupus erythematosus (SLE) shows a marked female bias in prevalence. X chromosome inactivation (XCI) is the mechanism which randomly silences one X chromosome to equalise gene expression between 46, XX females and 46, XY males. Though XCI is expected to result in a random pattern of mosaicism across tissues, some females display a significantly skewed ratio in immune cells, termed XCI-skew. We tested whether XCI was abnormal in females with SLE and hence contributes to sexual dimorphism. METHODS: We assayed XCI in whole blood DNA in 181 female SLE cases, 796 female healthy controls and 10 twin pairs discordant for SLE. Using regression modelling and intra-twin comparisons, we assessed the effect of SLE on XCI and combined clinical, cellular and genetic data via a polygenic score to explore underlying mechanisms. RESULTS: Accommodating the powerful confounder of age, XCI-skew was reduced in females with SLE compared with controls (p=1.3×10-5), with the greatest effect seen in those with more severe disease. Applying an XCI threshold of >80%, we observed XCI-skew in 6.6% of SLE cases compared with 22% of controls. This difference was not explained by differential white cell counts, medication or genetic susceptibility to SLE. Instead, XCI-skew correlated with a biomarker for type I interferon-regulated gene expression. CONCLUSIONS: These results refute current views on XCI-skew in autoimmunity and suggest, in lupus, XCI patterns of immune cells reflect the impact of disease state, specifically interferon signalling, on the haematopoietic stem cells from which they derive.

KLF2 enhancer variant rs4808485 increases lupus risk by modulating inflammasome machinery and cellular homoeostasis.

OBJECTIVE: A recent genome-wide association study linked KLF2 as a novel Asian-specific locus for systemic lupus erythematosus (SLE) susceptibility. However, the underlying causal functional variant(s), cognate target gene(s) and genetic mechanisms associated with SLE risk are unknown. METHODS: We used bioinformatics to prioritise likely functional variants and validated the best candidate with diverse experimental techniques, including genome editing. Gene expression was compared between healthy controls (HCs) and patients with SLE with or without lupus nephritis (LN+, LN-). RESULTS: Through bioinformatics and expression quantitative trait locus analyses, we prioritised rs4808485 in active chromatin, predicted to modulate KLF2 expression. Luciferase reporter assays and chromatin immunoprecipitation-qPCR demonstrated differential allele-specific enhancer activity and binding of active histone marks (H3K27ac, H3K4me3 and H3K4me1), Pol II, CTCF, P300 and the transcription factor PARP1. Chromosome conformation capture-qPCR revealed long-range chromatin interactions between rs4808485 and the KLF2 promoter. These were directly validated by CRISPR-based genetic and epigenetic editing in Jurkat and lymphoblastoid cells. Deleting the rs4808485 enhancer in Jurkat (KO) cells disrupted NLRP3 inflammasome machinery by reducing KLF2 and increasing CASPASE1, IL-1ß and GSDMD levels. Knockout cells also exhibited higher proliferation and cell-cycle progression than wild type. RNA-seq validated interplay between KLF2 and inflammasome machinery in HC, LN+ and LN-. CONCLUSIONS: We demonstrate how rs4808485 modulates the inflammasome and cellular homoeostasis through regulating KLF2 expression. This establishes mechanistic connections between rs4808485 and SLE susceptibility.

Evaluation of European-based polygenic risk score for breast cancer in Ashkenazi Jewish women in Israel.

BACKGROUND: Polygenic risk score (PRS), calculated based on genome-wide association studies (GWASs), can improve breast cancer (BC) risk assessment. To date, most BC GWASs have been performed in individuals of European (EUR) ancestry, and the generalisation of EUR-based PRS to other populations is a major challenge. In this study, we examined the performance of EUR-based BC PRS models in Ashkenazi Jewish (AJ) women. METHODS: We generated PRSs based on data on EUR women from the Breast Cancer Association Consortium (BCAC). We tested the performance of the PRSs in a cohort of 2161 AJ women from Israel (1437 cases and 724 controls) from BCAC (BCAC cohort from Israel (BCAC-IL)). In addition, we tested the performance of these EUR-based BC PRSs, as well as the established 313-SNP EUR BC PRS, in an independent cohort of 181 AJ women from Hadassah Medical Center (HMC) in Israel. RESULTS: In the BCAC-IL cohort, the highest OR per 1 SD was 1.56 (±0.09). The OR for AJ women at the top 10% of the PRS distribution compared with the middle quintile was 2.10 (±0.24). In the HMC cohort, the OR per 1 SD of the EUR-based PRS that performed best in the BCAC-IL cohort was 1.58±0.27. The OR per 1 SD of the commonly used 313-SNP BC PRS was 1.64 (±0.28). CONCLUSIONS: Extant EUR GWAS data can be used for generating PRSs that identify AJ women with markedly elevated risk of BC and therefore hold promise for improving BC risk assessment in AJ women.

Clinical applicability of the Polygenic Risk Score for breast cancer risk prediction in familial cases.

BACKGROUND: Common low-risk variants are presently not used to guide clinical management of familial breast cancer (BC). We explored the additive impact of a 313-variant-based Polygenic Risk Score (PRS313) relative to standard gene testing in non-BRCA1/2 Dutch BC families. METHODS: We included 3918 BC cases from 3492 Dutch non-BRCA1/2 BC families and 3474 Dutch population controls. The association of the standardised PRS313 with BC was estimated using a logistic regression model, adjusted for pedigree-based family history. Family history of the controls was imputed for this analysis. SEs were corrected to account for relatedness of individuals. Using the BOADICEA (Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm) V.5 model, lifetime risks were retrospectively calculated with and without individual PRS313. For 2586 cases and 2584 controls, the carrier status of pathogenic variants (PVs) in ATM, CHEK2 and PALB2 was known. RESULTS: The family history-adjusted PRS313 was significantly associated with BC (per SD OR=1.97, 95% CI 1.84 to 2.11). Including the PRS313 in BOADICEA family-based risk prediction would have changed screening recommendations in up to 27%, 36% and 34% of cases according to BC screening guidelines from the USA, UK and the Netherlands (National Comprehensive Cancer Network, National Institute for Health and Care Excellence, and Netherlands Comprehensive Cancer Organisation), respectively. For the population controls, without information on family history, this was up to 39%, 44% and 58%, respectively. Among carriers of PVs in known moderate BC susceptibility genes, the PRS313 had the largest impact for CHEK2 and ATM. CONCLUSIONS: Our results support the application of the PRS313 in risk prediction for genetically uninformative BC families and families with a PV in moderate BC risk genes.

Identification of new risk loci shared across systemic vasculitides points towards potential target genes for drug repurposing.

OBJECTIVES: The number of susceptibility loci currently associated with vasculitis is lower than in other immune-mediated diseases due in part to small cohort sizes, a consequence of the low prevalence of vasculitides. This study aimed to identify new genetic risk loci for the main systemic vasculitides through a comprehensive analysis of their genetic overlap. METHODS: Genome-wide data from 8467 patients with any of the main forms of vasculitis and 29 795 healthy controls were meta-analysed using ASSET. Pleiotropic variants were functionally annotated and linked to their target genes. Prioritised genes were queried in DrugBank to identify potentially repositionable drugs for the treatment of vasculitis. RESULTS: Sixteen variants were independently associated with two or more vasculitides, 15 of them representing new shared risk loci. Two of these pleiotropic signals, located close to CTLA4 and CPLX1, emerged as novel genetic risk loci in vasculitis. Most of these polymorphisms appeared to affect vasculitis by regulating gene expression. In this regard, for some of these common signals, potential causal genes were prioritised based on functional annotation, including CTLA4, RNF145, IL12B, IL5, IRF1, IFNGR1, PTK2B, TRIM35, EGR2 and ETS2, each of which has key roles in inflammation. In addition, drug repositioning analysis showed that several drugs, including abatacept and ustekinumab, could be potentially repurposed in the management of the analysed vasculitides. CONCLUSIONS: We identified new shared risk loci with functional impact in vasculitis and pinpointed potential causal genes, some of which could represent promising targets for the treatment of vasculitis.

Spanish cohort of VEXAS syndrome: clinical manifestations, outcome of treatments and novel evidences about UBA1 mosaicism.

BACKGROUND: The vacuoles, E1-enzyme, X linked, autoinflammatory and somatic (VEXAS) syndrome is an adult-onset autoinflammatory disease (AID) due to postzygotic UBA1 variants. OBJECTIVES: To investigate the presence of VEXAS syndrome among patients with adult-onset undiagnosed AID. Additional studies evaluated the mosaicism distribution and the circulating cytokines. METHODS: Gene analyses were performed by both Sanger and amplicon-based deep sequencing. Patients' data were collected from their medical charts. Cytokines were quantified by Luminex. RESULTS: Genetic analyses of enrolled patients (n=42) identified 30 patients carrying UBA1 pathogenic variants, with frequencies compatible for postzygotic variants. All patients were male individuals who presented with a late-onset disease (mean 67.5 years; median 67.0 years) characterised by cutaneous lesions (90%), fever (66.7%), pulmonary manifestations (66.7%) and arthritis (53.3%). Macrocytic anaemia and increased erythrocyte sedimentation rate and ferritin were the most relevant analytical abnormalities. Glucocorticoids ameliorated the inflammatory manifestations, but most patients became glucocorticoid-dependent. Positive responses were obtained when targeting the haematopoietic component of the disease with either decitabine or allogeneic haematopoietic stem cell transplantation. Additional analyses detected the UBA1 variants in both haematopoietic and non-haematopoietic tissues. Finally, analysis of circulating cytokines did not identify inflammatory mediators of the disease. CONCLUSION: Thirty patients with adult-onset AID were definitively diagnosed with VEXAS syndrome through genetic analyses. Despite minor interindividual differences, their main characteristics were in concordance with previous reports. We detected for the first time the UBA1 mosaicism in non-haematopoietic tissue, which questions the previous concept of myeloid-restricted mosaicism and may have conceptual consequences for the disease mechanisms.

Clinical and genetic factors associated with radiographic damage in patients with ankylosing spondylitis.

OBJECTIVES: To identify clinical and genetic factors associated with severe radiographic damage in patients with ankylosing spondylitis (AS). METHODS: We newly generated genome-wide single nucleotide polymorphism data (833K) for 444 patients with AS. The severity of radiographic damage was assessed using the modified Stoke Ankylosing Spondylitis Spinal Score (mSASSS). To identify clinical and genetic factors associated with severe radiographic damage, multiple linear regression analyses were performed. Human AS-osteoprogenitor and control-osteoprogenitor cells were used for functional validation. RESULTS: The significant clinical factors of final mSASSS were baseline mSASSS (ß=0.796, p=3.22×10-75), peripheral joint arthritis (ß=-0.246, p=6.85×10-6), uveitis (ß=0.157, p=1.95×10-3), and smoking (ß=0.130, p=2.72×10-2) after adjusting for sex, age and disease duration. After adjusting significant clinical factors, the Ryanodine receptor 3 (RYR3) gene was associated with severe radiographic damage (p=1.00×10-6). For pathway analysis, the PI3K-Akt signalling pathway was associated with severe radiographic damage in AS (p=2.21×10-4, false discovery rate=0.040). Treatment with rhodamine B, a ligand of RYR3, dose-dependently induced matrix mineralisation of AS osteoprogenitors. However, the rhodamine B-induced accelerated matrix mineralisation was not definitive in control osteoprogenitors. Knockdown of RYR3 inhibited matrix mineralisation in SaOS2 cell lines. CONCLUSIONS: This study identified clinical and genetic factors that contributed to better understanding of the pathogenesis and biology associated with radiographic damage in AS.

A meta-analysis and a functional study support the influence of mtDNA variant m.16519C on the risk of rapid progression of knee osteoarthritis.

OBJECTIVES: To identify mitochondrial DNA (mtDNA) genetic variants associated with the risk of rapid progression of knee osteoarthritis (OA) and to characterise their functional significance using a cellular model of transmitochondrial cybrids. METHODS: Three prospective cohorts contributed participants. The osteoarthritis initiative (OAI) included 1095 subjects, the Cohort Hip and Cohort Knee included 373 and 326 came from the PROspective Cohort of Osteoarthritis from A Coruña. mtDNA variants were screened in an initial subset of 450 subjects from the OAI by in-depth sequencing of mtDNA. A meta-analysis of the three cohorts was performed. A model of cybrids was constructed to study the functional consequences of harbouring the risk mtDNA variant by assessing: mtDNA copy number, mitochondrial biosynthesis, mitochondrial fission and fusion, mitochondrial reactive oxygen species (ROS), oxidative stress, autophagy and a whole transcriptome analysis by RNA-sequencing. RESULTS: mtDNA variant m.16519C is over-represented in rapid progressors (combined OR 1.546; 95% CI 1.163 to 2.054; p=0.0027). Cybrids with this variant show increased mtDNA copy number and decreased mitochondrial biosynthesis; they produce higher amounts of mitochondrial ROS, are less resistant to oxidative stress, show a lower expression of the mitochondrial fission-related gene fission mitochondrial 1 and an impairment of autophagic flux. In addition, its presence modulates the transcriptome of cybrids, especially in terms of inflammation, where interleukin 6 emerges as one of the most differentially expressed genes. CONCLUSIONS: The presence of the mtDNA variant m.16519C increases the risk of rapid progression of knee OA. Among the most modulated biological processes associated with this variant, inflammation and negative regulation of cellular process stand out. The design of therapies based on the maintenance of mitochondrial function is recommended.

Meta-analysis of erosive hand osteoarthritis identifies four common variants that associate with relatively large effect.

OBJECTIVES: Erosive hand osteoarthritis (EHOA) is a severe subset of hand osteoarthritis (OA). It is unclear if EHOA is genetically different from other forms of OA. Sequence variants at ten loci have been associated with hand OA but none with EHOA. METHODS: We performed meta-analysis of EHOA in 1484 cases and 550 680 controls, from 5 populations. To identify causal genes, we performed eQTL and plasma pQTL analyses, and developed one zebrafish mutant. We analysed associations of variants with other traits and estimated shared genetics between EHOA and other traits. RESULTS: Four common sequence variants associated with EHOA, all with relatively high effect. Rs17013495 (SPP1/MEPE, OR=1.40, p=8.4×10-14) and rs11243284 (6p24.3, OR=1.35, p=4.2×10-11) have not been associated with OA, whereas rs11631127 (ALDH1A2, OR=1.46, p=7.1×10-18), and rs1800801 (MGP, OR=1.37, p=3.6×10-13) have previously been associated with hand OA. The association of rs1800801 (MGP) was consistent with a recessive mode of inheritance in contrast to its additive association with hand OA (OR homozygotes vs non-carriers=2.01, 95% CI 1.71 to 2.37). All four variants associated nominally with finger OA, although with substantially lower effect. We found shared genetic components between EHOA and other OA measures, grip strength, urate levels and gout, but not rheumatoid arthritis. We identified ALDH1A2, MGP and BMP6 as causal genes for EHOA, with loss-of-function Bmp6 zebrafish mutants displaying EHOA-like phenotypes. CONCLUSIONS: We report on significant genetic associations with EHOA. The results support the view of EHOA as a form of severe hand OA and partly separate it from OA in larger joints.

Pathogenesis of systemic lupus erythematosus: risks, mechanisms and therapeutic targets.

Research elucidating the pathogenesis of systemic lupus erythematosus (SLE) has defined two critical families of mediators, type I interferon (IFN-I) and autoantibodies targeting nucleic acids and nucleic acid-binding proteins, as fundamental contributors to the disease. On the fertile background of significant genetic risk, a triggering stimulus, perhaps microbial, induces IFN-I, autoantibody production or most likely both. When innate and adaptive immune system cells are engaged and collaborate in the autoimmune response, clinical SLE can develop. This review describes recent data from genetic analyses of patients with SLE, along with current studies of innate and adaptive immune function that contribute to sustained IFN-I pathway activation, immune activation and autoantibody production, generation of inflammatory mediators and tissue damage. The goal of these studies is to understand disease mechanisms, identify therapeutic targets and stimulate development of therapeutics that can achieve improved outcomes for patients.

Genome-wide association meta-analysis of knee and hip osteoarthritis uncovers genetic differences between patients treated with joint replacement and patients without joint replacement.

OBJECTIVES: Osteoarthritis is a common and severe, multifactorial disease with a well-established genetic component. However, little is known about how genetics affect disease progression, and thereby the need for joint placement. Therefore, we aimed to investigate whether the genetic associations of knee and hip osteoarthritis differ between patients treated with joint replacement and patients without joint replacement. METHODS: We included knee and hip osteoarthritis cases along with healthy controls, altogether counting >700 000 individuals. The cases were divided into two groups based on joint replacement status (surgical vs non-surgical) and included in four genome-wide association meta-analyses: surgical knee osteoarthritis (N = 22 525), non-surgical knee osteoarthritis (N = 38 626), surgical hip osteoarthritis (N = 20 221) and non-surgical hip osteoarthritis (N = 17 847). In addition, we tested for genetic correlation between the osteoarthritis groups and the pain phenotypes intervertebral disc disorder, dorsalgia, fibromyalgia, migraine and joint pain. RESULTS: We identified 52 sequence variants associated with knee osteoarthritis (surgical: 17, non-surgical: 3) or hip osteoarthritis (surgical: 34, non-surgical: 1). For the surgical phenotypes, we identified 10 novel variants, including genes involved in autophagy (rs2447606 in ATG7) and mechanotransduction (rs202127176 in PIEZO1). One variant, rs13107325 in SLC39A8, associated more strongly with non-surgical knee osteoarthritis than surgical knee osteoarthritis. For all other variants, significance and effect sizes were higher for the surgical phenotypes. In contrast, genetic correlations with pain phenotypes tended to be stronger in the non-surgical groups. CONCLUSIONS: Our results indicate differences in genetic associations between knee and hip osteoarthritis depending on joint replacement status.

Multiomics analysis of rheumatoid arthritis yields sequence variants that have large effects on risk of the seropositive subset.

OBJECTIVES: To find causal genes for rheumatoid arthritis (RA) and its seropositive (RF and/or ACPA positive) and seronegative subsets. METHODS: We performed a genome-wide association study (GWAS) of 31 313 RA cases (68% seropositive) and ~1 million controls from Northwestern Europe. We searched for causal genes outside the HLA-locus through effect on coding, mRNA expression in several tissues and/or levels of plasma proteins (SomaScan) and did network analysis (Qiagen). RESULTS: We found 25 sequence variants for RA overall, 33 for seropositive and 2 for seronegative RA, altogether 37 sequence variants at 34 non-HLA loci, of which 15 are novel. Genomic, transcriptomic and proteomic analysis of these yielded 25 causal genes in seropositive RA and additional two overall. Most encode proteins in the network of interferon-alpha/beta and IL-12/23 that signal through the JAK/STAT-pathway. Highlighting those with largest effect on seropositive RA, a rare missense variant in STAT4 (rs140675301-A) that is independent of reported non-coding STAT4-variants, increases the risk of seropositive RA 2.27-fold (p=2.1×10-9), more than the rs2476601-A missense variant in PTPN22 (OR=1.59, p=1.3×10-160). STAT4 rs140675301-A replaces hydrophilic glutamic acid with hydrophobic valine (Glu128Val) in a conserved, surface-exposed loop. A stop-mutation (rs76428106-C) in FLT3 increases seropositive RA risk (OR=1.35, p=6.6×10-11). Independent missense variants in TYK2 (rs34536443-C, rs12720356-C, rs35018800-A, latter two novel) associate with decreased risk of seropositive RA (ORs=0.63-0.87, p=10-9-10-27) and decreased plasma levels of interferon-alpha/beta receptor 1 that signals through TYK2/JAK1/STAT4. CONCLUSION: Sequence variants pointing to causal genes in the JAK/STAT pathway have largest effect on seropositive RA, while associations with seronegative RA remain scarce.

Auxilin is a novel susceptibility gene for congenital heart block which directly impacts fetal heart function.

OBJECTIVE: Neonatal lupus erythematosus (NLE) may develop after transplacental transfer of maternal autoantibodies with cardiac manifestations (congenital heart block, CHB) including atrioventricular block, atrial and ventricular arrhythmias, and cardiomyopathies. The association with anti-Ro/SSA antibodies is well established, but a recurrence rate of only 12%-16% despite persisting maternal autoantibodies suggests that additional factors are required for CHB development. Here, we identify fetal genetic variants conferring risk of CHB and elucidate their effects on cardiac function. METHODS: A genome-wide association study was performed in families with at least one case of CHB. Gene expression was analysed by microarrays, RNA sequencing and PCR and protein expression by western blot, immunohistochemistry, immunofluorescence and flow cytometry. Calcium regulation and connectivity were analysed in primary cardiomyocytes and cells induced from pleuripotent stem cells. Fetal heart performance was analysed by Doppler/echocardiography. RESULTS: We identified DNAJC6 as a novel fetal susceptibility gene, with decreased cardiac expression of DNAJC6 associated with the disease risk genotype. We further demonstrate that fetal cardiomyocytes deficient in auxilin, the protein encoded by DNAJC6, have abnormal connectivity and Ca2+ homoeostasis in culture, as well as decreased cell surface expression of the Cav1.3 calcium channel. Doppler echocardiography of auxilin-deficient fetal mice revealed cardiac NLE abnormalities in utero, including abnormal heart rhythm with atrial and ventricular ectopias, as well as a prolonged atrioventricular time intervals. CONCLUSIONS: Our study identifies auxilin as the first genetic susceptibility factor in NLE modulating cardiac function, opening new avenues for the development of screening and therapeutic strategies in CHB.

Biological insights into systemic lupus erythematosus through an immune cell-specific transcriptome-wide association study.

OBJECTIVE: Genome-wide association studies (GWAS) have identified >100 risk loci for systemic lupus erythematosus (SLE), but the disease genes at most loci remain unclear, hampering translation of these genetic discoveries. We aimed to prioritise genes underlying the 110 SLE loci that were identified in the latest East Asian GWAS meta-analysis. METHODS: We built gene expression predictive models in blood B cells, CD4+ and CD8+ T cells, monocytes, natural killer cells and peripheral blood cells of 105 Japanese individuals. We performed a transcriptome-wide association study (TWAS) using data from the latest genome-wide association meta-analysis of 208 370 East Asians and searched for candidate genes using TWAS and three data-driven computational approaches. RESULTS: TWAS identified 171 genes for SLE (p<1.0×10-5); 114 (66.7%) showed significance only in a single cell type; 127 (74.3%) were in SLE GWAS loci. TWAS identified a strong association between CD83 and SLE (p<7.7×10-8). Meta-analysis of genetic associations in the existing 208 370 East Asian and additional 1498 cases and 3330 controls found a novel single-variant association at rs72836542 (OR=1.11, p=4.5×10-9) around CD83. For the 110 SLE loci, we identified 276 gene candidates, including 104 genes at recently-identified SLE novel loci. We demonstrated in vitro that putative causal variant rs61759532 exhibited an allele-specific regulatory effect on ACAP1, and that presence of the SLE risk allele decreased ACAP1 expression. CONCLUSIONS: Cell-level TWAS in six types of immune cells complemented SLE gene discovery and guided the identification of novel genetic associations. The gene findings shed biological insights into SLE genetic associations.

Factors predicting axial spondyloarthritis among first-degree relatives of probands with ankylosing spondylitis: a family study spanning 35 years.

OBJECTIVE: Factors predicting axial spondyloarthritis (axSpA) among first-degree relatives (FDRs) of ankylosing spondylitis (AS) patients need to be defined. We investigated the predictive value of the probands' HLA-B27 and radiographic sacroiliitis status on disease occurrence among their FDR. We also assessed the predictive value of features of the clinical history, including chronic inflammatory back pain (CIBP) and acute anterior uveitis (AAU), among the FDR and how they can be used to improve classification and diagnosis of axSpA. METHODS: In 1985, we studied 363 AS probands and 806 FDR who underwent rheumatologic examination, completed questionnaires, provided blood samples for HLA-typing and underwent radiography of sacroiliac joints. At follow-up in 2018-2019, 125 patients and 360 FDR were available for study, and completed a postal questionnaire about axSpA features. FDRs were asked to report whether after 1985 they had been diagnosed by Swiss rheumatologists as having axSpA. RESULTS: Among HLA-B27(+) FDR, axSpA occurred in 25.4%-26.3%, independent of the radiographic sacroiliitis status of the proband. AAU occurred in 13/34 (38.2%) FDR with axSpA vs 29/251 (11.6%) FDR without axSpA (p=0.00004, OR=4.74 95% CI 2.15 to 10.47). The presence of CIBP at baseline did not predict later occurrence of axSpA but combining CIBP and pain/discomfort at the thoracic spine and at anterior (ventral) chest wall ever, assessed at follow-up in 2018-2019, provided 83.1% sensitivity and 87.2% specificity for current axSpA. CONCLUSION: Occurrence of AAU among FDR of axSpA probands should prompt screening for axSpA. Moreover, co-occurrence of CIBP and pain/discomfort in the thoracic spine and at anterior chest wall as a three-question tool may further enhance clinical suspicion of axSpA among these FDR.

NOD/RIPK2 signalling pathway contributes to osteoarthritis susceptibility.

OBJECTIVES: How inflammatory signalling contributes to osteoarthritis (OA) susceptibility is undetermined. An allele encoding a hyperactive form of the Receptor Interacting Protein Kinase 2 (RIPK2) proinflammatory signalling intermediate has been associated with familial OA. To test whether altered nucleotide-binding oligomerisation domain (NOD)/RIPK2 pathway activity causes heightened OA susceptibility, we investigated whether variants affecting additional pathway components are associated with familial OA. To determine whether the Ripk2104Asp disease allele is sufficient to account for the familial phenotype, we determined the effect of the allele on mice. METHODS: Genomic analysis of 150 independent families with dominant inheritance of OA affecting diverse joints was used to identify coding variants that segregated strictly with occurrence of OA. Genome editing was used to introduce the OA-associated RIPK2 (p.Asn104Asp) allele into the genome of inbred mice. The consequences of the Ripk2104Asp disease allele on physiology and OA susceptibility in mice were measured by histology, immunohistochemistry, serum cytokine levels and gene expression. RESULTS: We identified six novel variants affecting components of the NOD/RIPK2 inflammatory signalling pathway that are associated with familial OA affecting the hand, shoulder or foot. The Ripk2104Asp allele acts dominantly to alter basal physiology and response to trauma in the mouse knee. Whereas the knees of uninjured Ripk2Asp104 mice appear normal histologically, the joints exhibit a set of marked gene expression changes reminiscent of overt OA. Although the Ripk2104Asp mice lack evidence of chronically elevated systemic inflammation, they do exhibit significantly increased susceptibility to post-traumatic OA (PTOA). CONCLUSIONS: Two types of data support the hypothesis that altered NOD/RIPK2 signalling confers susceptibility to OA.

Hypomethylation of miR-17-92 cluster in lupus T cells and no significant role for genetic factors in the lupus-associated DNA methylation signature.

OBJECTIVES: Lupus T cells demonstrate aberrant DNA methylation patterns dominated by hypomethylation of interferon-regulated genes. The objective of this study was to identify additional lupus-associated DNA methylation changes and determine the genetic contribution to epigenetic changes characteristic of lupus. METHODS: Genome-wide DNA methylation was assessed in naïve CD4+ T cells from 74 patients with lupus and 74 age-matched, sex-matched and race-matched healthy controls. We applied a trend deviation analysis approach, comparing methylation data in our cohort with over 16 500 samples. Methylation quantitative trait loci (meQTL) analysis was performed by integrating methylation profiles with genome-wide genotyping data. RESULTS: In addition to the previously reported epigenetic signature in interferon-regulated genes, we observed hypomethylation in the promoter region of the miR-17-92 cluster in patients with lupus. Members of this microRNA cluster play an important role in regulating T cell proliferation and differentiation. Expression of two microRNAs in this cluster, miR-19b1 and miR-18a, showed a significant positive correlation with lupus disease activity. Among miR-18a target genes, TNFAIP3, which encodes a negative regulator of nuclear factor kappa B, was downregulated in lupus CD4+ T cells. MeQTL identified in lupus patients showed overlap with genetic risk loci for lupus, including CFB and IRF7. The lupus risk allele in IRF7 (rs1131665) was associated with significant IRF7 hypomethylation. However, <1% of differentially methylated CpG sites in patients with lupus were associated with an meQTL, suggesting minimal genetic contribution to lupus-associated epigenotypes. CONCLUSION: The lupus defining epigenetic signature, characterised by robust hypomethylation of interferon-regulated genes, does not appear to be determined by genetic factors. Hypomethylation of the miR-17-92 cluster that plays an important role in T cell activation is a novel epigenetic locus for lupus.