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OBJECTIVE: Idiopathic inflammatory myopathies (IIMs) are a heterogeneous group of complex autoimmune conditions characterized by inflammation in skeletal muscle and extramuscular compartments, and interferon (IFN) system activation. We undertook this study to examine the contribution of genetic variation to disease susceptibility and to identify novel avenues for research in IIMs.METHODS: Targeted DNA sequencing was used to mine coding and potentially regulatory single nucleotide variants from ~1,900 immune-related genes in a Scandinavian case-control cohort of 454 IIM patients and 1,024 healthy controls. Gene-based aggregate testing, together with rare variant- and gene-level enrichment analyses, was implemented to explore genotype-phenotype relations.RESULTS: Gene-based aggregate tests of all variants, including rare variants, identified IFI35 as a potential genetic risk locus for IIMs, suggesting a genetic signature of type I IFN pathway activation. Functional annotation of the IFI35 locus highlighted a regulatory network linked to the skeletal muscle-specific gene PTGES3L, as a potential candidate for IIM pathogenesis. Aggregate genetic associations with AGER and PSMB8 in the major histocompatibility complex locus were detected in the antisynthetase syndrome subgroup, which also showed a less marked genetic signature of the type I IFN pathway. Enrichment analyses indicated a burden of synonymous and noncoding rare variants in IIM patients, suggesting increased disease predisposition associated with these classes of rare variants.CONCLUSION: Our study suggests the contribution of rare genetic variation to disease susceptibility in IIM and specific patient subgroups, and pinpoints genetic associations consistent with previous findings by gene expression profiling. These features highlight genetic profiles that are potentially relevant to disease pathogenesis.

Background Programmed cell death 1 (PD-1) and its ligands PD-L1 and PD-L2, as well as Indoleamine 2,3-deoxygenase (IDO1) can be expressed both by tumor and microenvironmental cells and are crucial for tumor immune escape. We aimed to evaluate the role of PD-1, its ligands and IDO1 in a cohort of patients with primary diffuse large B-cell lymphoma of the CNS (PCNSL). Material and methods Tissue microarrays (TMAs) were constructed in 45 PCNSL cases. RNA extraction from whole tissue sections and RNA sequencing were successfully performed in 33 cases. Immunohistochemical stainings for PD-1, PD-L1/paired box protein 5 (PAX-5), PD-L2/PAX-5 and IDO1, and Epstein-Barr virus encoding RNA (EBER) in situ hybridization were analyzed. Results High proportions of PD-L1 and PD-L2 positive tumor cells were observed in 11% and 9% of cases, respectively. High proportions of PD-L1 and PD-L2 positive leukocytes were observed in 55% and 51% of cases, respectively. RNA sequencing revealed that gene expression of IDO1 was high in patients with high proportion of PD-L1 positive leukocytes (p = .01). Protein expression of IDO1 in leukocytes was detected in 14/45 cases, in 79% of these cases a high proportion of PD-L1 positive leukocytes was observed. Gene expression of IDO1 was high in EBER-positive cases (p = .0009) and protein expression of IDO1 was detected in five of six EBER-positive cases. Conclusion Our study shows a significant association between gene and protein expression of IDO1 and protein expression of PD-L1 in the tumor microenvironment of PCNSL, possibly of importance for prediction of response to immunotherapies. Title in Thesis: PD-L1 and IDO1 are important immunosuppressive molecules in primary diffuse large B-cell lymphoma of the CNS

ObjectivesFatigue is common and severe in primary Sjögren’s syndrome (pSS). The aim of this study was to identify genetic determinants of fatigue in pSS through a genome-wide association study.MethodsPatients with pSS from Norway, Sweden, UK and USA with fatigue and genotype data available were included. After genotype imputation and quality control, 682 patients and 4 966 157 genetic markers were available. Association analysis in each cohort using linear regression with fatigue as a continuous variable and meta-analyses between the cohorts were performed.ResultsMeta-analysis of the Norwegian and Swedish cohorts identified five polymorphisms within the same linkage disequilibrium block at the receptor transporter protein 4 (RTP4)/MASP1 locus associated with fatigue with genome-wide significance (GWS) (p−8). Patients homozygous for the major allele scored 25 mm higher on the fatigue Visual Analogue Scale than patients homozygous for the minor allele. There were no variants associated with fatigue with GWS in meta-analyses of the US/UK cohorts, or all four cohorts. RTP4 expression in pSS B cells was upregulated and positively correlated with the type I interferon score. Expression quantitative trait loci effects in whole blood for fatigue-associated variants at RTP4/MASP1 and levels of RTP4 and MASP1 expression were identified.ConclusionGenetic variations at RTP4/MASP1 are associated with fatigue in Scandinavian pSS patients. RTP4 encodes a Golgi chaperone that influences opioid pain receptor function and MASP1 is involved in complement activation. These results add evidence for genetic influence over fatigue in pSS.

ObjectiveTo investigate how genetics influence the risk of smoking-related systemic lupus erythematosus (SLE) manifestations.MethodsPatients with SLE (ndiscovery cohort=776, nreplication cohort=836) were genotyped using the 200K Immunochip single nucleotide polymorphisms (SNP) Array (Illumina) and a custom array. Sixty SNPs with SLE association (p−8) were analysed. Signal transducer and activator of transcription 4 (STAT4) activation was assessed in in vitro stimulated peripheral blood mononuclear cells from healthy controls (n=45).ResultsIn the discovery cohort, smoking was associated with myocardial infarction (MI) (OR 1.96 (95% CI 1.09 to 3.55)), with a greater effect in patients carrying any rs11889341 STAT4 risk allele (OR 2.72 (95% CI 1.24 to 6.00)) or two risk alleles (OR 8.27 (95% CI 1.48 to 46.27)).Smokers carrying the risk allele also displayed an increased risk of nephritis (OR 1.47 (95% CI 1.06 to 2.03)). In the replication cohort, the high risk of MI in smokers carrying the risk allele and the association between the STAT4 risk allele and nephritis in smokers were confirmed (OR 6.19 (95% CI 1.29 to 29.79) and 1.84 (95% CI 1.05 to 3.29), respectively).The interaction between smoking and the STAT4 risk allele resulted in further increase in the risk of MI (OR 2.14 (95% CI 1.01 to 4.62)) and nephritis (OR 1.53 (95% CI 1.08 to 2.17)), with 54% (MI) and 34% (nephritis) of the risk attributable to the interaction. Levels of interleukin-12-induced phosphorylation of STAT4 in CD8+ T cells were higher in smokers than in non-smokers (mean geometric fluorescence intensity 1063 vs 565, p=0.0063).Lastly, the IL12A rs564799 risk allele displayed association with MI in both cohorts (OR 1.53 (95% CI 1.01 to 2.31) and 2.15 (95% CI 1.08 to 4.26), respectively).ConclusionsSmoking in the presence of the STAT4 risk gene variant appears to increase the risk of MI and nephritis in SLE. Our results also highlight the role of the IL12−STAT4 pathway in SLE-cardiovascular morbidity.

Background Patients with SLE have increased morbidity and mortality due to cardiovascular disease. Here, we construct and validate a polygenic risk score (PRS) for myocardial infarction (MI) in SLE. Methods Patients with SLE (European decent, ≥4 ACR-criteria) were genotyped using a 200K Immunochip SNP array (discovery cohort, Sweden, n=776) and custom MassARRAY assays (replication cohort, Norway/Denmark, n=890). In the discovery cohort, 57 SNPs with previously established association with SLE development (p Results Four SLE-risk genes were found to be associated with a decreased time until the first MI; PTPN22 (OR 1.61, p=0.041), NCF2 (OR 2.47, p=2.1×10-3), STAT4 (OR 1.66, p=5.2×10-3) and IL12A (OR 1.45, p=0.047) and were included in a PRS. The PRS was associated with a higher cumulative prevalence of MI in both the discovery cohort (p=1.1×10-5, fig 1A) and replication cohort (p=7.7×10-3, fig 1B). Exploring the PRS further in the replication cohort, patients in the high, compared to the low, PRS-quartile were more often male (p=1.3×10-3), and displayed higher prevalence of the ACR-1982 nephritis and immunological criteria (p=4.1×10-4 and p=0.036) (fig1C). Analyzing combinations of the identified SNPs, we found the prevalence of MI to be further increased in patients homozygous for both NCF2+STAT4 (pdiscovery=1.6×10-3, preplication=0.015) or STAT4+IL12A (pdiscovery=3.0×10-5, preplication=0.036) (fig1D). Conclusion A high polygenic risk score for MI in SLE is associated with an increased prevalence of myocardial infarction. If confirmed in prospective studies, our results suggest that genetic profiling may be useful for predicting MI in patients with SLE.

Background Lupus Nephritis (LN) is a cause of significant morbidity in SLE. While the genetic background to SLE has been well characterized, less is known about genes predisposing to LN. Methods The study consisted of 2886 SLE patients, including 947 (33%) with LN. The discovery cohort (Sweden, n=1091) and replication cohort 1 (US, n=962) were genotyped on the Immunochip and replication cohort 2 (Norway/Denmark, n=833) on a custom array chip. Allele frequencies were compared between patients with LN and LN-negative patients. SNPs with a p-value Results In the discovery cohort, strong association to LN was found with several highly linked SNPs in BANK1, with the top signal in the intronic SNP rs4699261 (p=9.9×10-5, OR 0.66). The association was also present in replication cohort 1 (p=9.5×10-4). In a meta-analysis of the discovery and replication cohort 1, a total of 20 SNPs in BANK1 were associated to LN with the highest signal in rs4699261 (p=3.3×10-7). There was a tendency towards association in replication cohort 2 (p=0.05) and in a meta-analysis of all cohorts, the association with BANK1 was strengthened (p=1.7×10-7). Conclusion BANK1 variations are associated with LN in patients with SLE. Upregulated BANK1 expression in renal biopsies from LN patients has previously been shown, however the exact role of BANK1 in LN pathogenesis remains to be elucidated.

Background Systemic lupus erythematosus (SLE) is a chronic, autoimmune disease with a complex genetic etiology. Over 100 risk genes for SLE have been identified at genome-wide significance, but their overall effect on disease severity has not previously been studied. We therefore assessed the relationship between a high genetic risk score and the development of organ damage in SLE. Methods Patients with SLE, who met 4 ACR criteria (n=1001), and healthy controls (n=2802) were genotyped using a 200K Immunochip SNP Array (Illumina). A genetic risk score (GRS) was assigned to each individual based on 57 SLE risk loci which have previously shown association (p Results SLE prevalence increased with increasing GRS (figure 1A) and was higher in the highest compared to the lowest GRS-quartile (OR 12.32 (9.5315.71) p=7.9×10–86). SLE onset occurred 5 years earlier in the high compared to the low quartile (figure 1B). The OR for organ damage increased with increasing GRS (figure 1C) and was significantly higher in the high compared to the low GRS-quartile (OR 1.47 (1.062.04) p=2.0×10–2). Moreover, patients in the high quartile had an increased prevalence of nephritis (OR 2.22 (1.503.27), p=5.9×10–5), end-stage kidney disease (ESKD) (OR 5.58 (1.5020.79), p=1.0×10–2) anti-dsDNA antibodies (OR 1.83 (1.192.81), p=6.1×10–03), anti-cardiolipin-IgG (OR 2.16 (1.303.59), p=2.8×10–03) and anti-2-glycoprotein-I (OR 1.69 (1.042.74), p=3.3×10–02). Analysis of renal biopsy data showed that the prevalence of proliferative nephritis was significantly higher in the high, compared to the low, quartile (OR 2.42 (1.304.49), p=5.1×10–03). Moreover, the patients in the high GRS-quartile displayed decreased survival until their first organ damage (HR 1.51 (1.042.25), p=3.7×10–02), first cardiovascular event (HR 1.65 (1.032.64), p=2.6×10–02), nephritis onset (HR 2.53 (1.723.71) p=9.6×10–7) and ESKD (6.78 (1.7826.86), p=6.5×10–3). Lastly, OR for mortality increased with increasing GRS (figure 1D), with a 5 year decrease in overall survival in the high compared to the low quartile (HR 1.82 (1.043.19), p=2.4×10–2). Conclusions A high genetic risk score is associated with earlier disease onset, increased risk of organ damage and impaired survival. Our results indicate that genetic profiling may be useful for predicting outcomes in patients with SLE. Funding Source(s): None

Background Systemic lupus erythematosus (SLE) is a chronic, autoimmune disease with a complex genetic aetiology. The overall effect of hereditary risk on organ damage has so far not been studied. We therefore assessed the relationship between genetic risk and development of organ damage in SLE. Methods Patients with SLE (Sweden, n=1001) were genotyped using a 200K Immunochip single nucleotide polymorphism (SNP) Array (Illumina). The Immunochip was HLA imputed using HLA*IMP:02. A non-HLA (58 SNPs) and a HLA (5 SNPs) genetic risk score (GRS) was assigned to each patient based on SNPs which in previous studies have shown association (p et al. 2017). For each SNP, the natural logarithm of the odds ratio (OR) for SLE susceptibility was multiplied by the number of risk alleles in each individual. The sum of all products for each patient was defined as the GRS. Clinical data, including the Systemic Lupus International Collaborating Clinics Damage Index (SLICC-DI), was retrieved from medical records. The relationship between GRS and SLICC-DI was analysed using an ordinal regression model. Results A higher non-HLA GRS was associated with increased organ damage (OR 1.10 (1.00–1.21), p=4.2×10–2), nephritis (OR 1.26 (1.13–1.41), p=2.8×10–5), anti-dsDNA (OR 1.33 (1.17–1.52), p=1.0×10–5) and a younger age at diagnosis (OR 1.33 (1.14–1.54), p=1.7×10–4). When analysing the relationship between individual SNPs (n=63) and SLICC-DI, we observed positive associations between SLICC-DI and rs6568431 (ATG5, OR 1.28 (1.08–1.51), p=3.6×10–3) and rs11889341 (STAT4, OR 1.27 (1.07–1.50), p=5.0×10–3). Rs1269852 (TNXB-ATF6B, OR 0.80 (0.66–0.98), p=2.7×10–2) and rs1132200 (TMEM39A, OR 0.72 (0.56–0.91), p=6.7×10–3) were negatively associated with SLICC-DI. Using a Kendall Tau correlation model, a positive correlation between the TNXB-ATF6B risk allele and the HLA DRB1*03:01 haplotype was observed (τ=0.91, p Conclusion In patients with SLE, a high genetic risk score is linked to increased organ damage and a younger age of disease onset. Further, the ATG5 and STAT4 risk alleles were associated with increased organ damage whereas the TNXB-ATF6B and TMEM39A risk alleles were associated with less organ damage. Consequently, genetic profiling of patients with SLE may provide a tool for predicting severity of the disease.

Purpose Childhood onset systemic lupus erythematosus (SLE) is associated with a more aggressive disease course and higher mortality risk than adult onset SLE. It has been suggested that juvenile onset SLE cases could have a more genetically determined disease. To identify genetic risk loci in juvenile onset SLE we performed targeted DNA resequencing in a cohort of Swedish SLE patients and control individuals. Methods Coding and regulatory regions of 1853 genes selected from pathways involved in immunological diseases were resequenced in 958 patients with SLE and in 1030 healthy individuals. All patients fulfilled at least four ACR 1982 classification criteria for SLE. For 117 of the SLE patients the disease onset was at age 18 or younger, 105 of whom were women and 12 men. Capturing of the targeted genes was performed with a Roche NimbleGen custom-made liquid capture library followed by Illumina HiSeq2500 sequencing. 97 264 single nucleotide variants (SNVs) passed quality control and had a minor allele frequency of at least 1%. Results Single variant case-control association analysis revealed that 40 SNVs were associated with juvenile onset SLE (false-discovery rate Conclusion Using targeted sequencing we have identified coding SNVs in novel candidate risk loci in juvenile onset SLE. Our finding suggests differences in the genetic risk for childhood and adult onset SLE and provides insight into the genetic aetiology of juvenile onset SLE.

The type I interferon (IFN) system is persistently activated in systemic lupus erythematosus (SLE) and many other systemic autoimmune diseases. Studies have shown an association between SLE and several gene variants within the type I IFN system. We investigated whether single-nucleotide polymorphisms (SNPs) associated with SLE and other autoimmune diseases affect the IFN-α production in healthy individuals. Plasmacytoid dendritic cells (pDCs), B cells and NK cells were isolated from peripheral blood of healthy individuals and stimulated with RNA-containing immune complexes (ICs), herpes simplex virus (HSV) or the oligonucleotide ODN2216. IFN-α production by pDCs alone or in cocultures with B or NK cells was measured by an immunoassay. All donors were genotyped with the 200K ImmunoChip, and a 5 bp CGGGG length polymorphism in the IFN regulatory factor 5 gene (IRF5) was genotyped by PCR. We found associations between IFN-α production and 18-86 SNPs (P ≤ 0.001), depending on the combination of the stimulated cell types. However, only three of these associated SNPs were shared between the cell-type combinations. Several SNPs showed novel associations to the type I IFN system among all the associated SNPs, whereas some loci have been described earlier for their association with SLE. Furthermore, we found that the SLE-risk variant of the IRF5 CGGGG-indel was associated with lower IFN-α production. We conclude that the genetic variants affecting the IFN-α production highlight the intricate regulation of the type I IFN system and the importance of understanding the mechanisms behind the dysregulated type I IFN system in SLE.

Patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) have increased risk of cardiovascular disease (CVD). We investigated whether single nucleotide polymorphisms (SNPs) at autoimmunity risk loci were associated with CVD in SLE and RA.Patients with SLE (n=1045) were genotyped using the 200K Immunochip SNP array (Illumina). The allele frequency was compared between patients with and without different manifestations of CVD. Results were replicated in a second SLE cohort (n=1043) and in an RA cohort (n=824). We analysed publicly available genetic data from general population, performed electrophoretic mobility shift assays and measured cytokine levels and occurrence of antiphospholipid antibodies (aPLs).We identified two new putative risk loci associated with increased risk for CVD in two SLE populations, which remained after adjustment for traditional CVD risk factors. AnThe

Objectives Systemic lupus erythematosus (SLE) is a chronic autoimmune condition with heterogeneous presentation and complex aetiology where DNA methylation changes are emerging as a contributing factor. In order to discover novel epigenetic associations and investigate their relationship to genetic risk for SLE, we analysed DNA methylation profiles in a large collection of patients with SLE and healthy individuals. Methods DNA extracted from blood from 548 patients with SLE and 587 healthy controls were analysed on the Illumina HumanMethylation 450 k BeadChip, which targets 485 000 CpG sites across the genome. Single nucleotide polymorphism (SNP) genotype data for 196 524 SNPs on the Illumina ImmunoChip from the same individuals were utilised for methylation quantitative trait loci (cis-meQTLs) analyses. Results We identified and replicated differentially methylated CpGs (DMCs) in SLE at 7245 CpG sites in the genome. The largest methylation differences were observed at type I interferon-regulated genes which exhibited decreased methylation in SLE. We mapped cis-meQTLs and identified genetic regulation of methylation levels at 466 of the DMCs in SLE. The meQTLs for DMCs in SLE were enriched for genetic association to SLE, and included seven SLE genome-wide association study (GWAS) loci: PTPRC (CD45), MHC-class III, UHRF1BP1, IRF5, IRF7, IKZF3 and UBE2L3. In addition, we observed association between genotype and variance of methylation at 20 DMCs in SLE, including at the HLA-DQB2 locus. Conclusions Our results suggest that several of the genetic risk variants for SLE may exert their influence on the phenotype through alteration of DNA methylation levels at regulatory regions of target genes.

Genome-wide association studies have identified risk loci for SLE, but a large proportion of the genetic contribution to SLE still remains unexplained. To detect novel risk genes, and to predict an individuals SLE risk we designed a random forest classifier using SNP genotype data generated on the "Immunochip" from 1,160 patients with SLE and 2,711 controls. Using gene importance scores defined by the random forest classifier, we identified 15 potential novel risk genes for SLE. Of them 12 are associated with other autoimmune diseases than SLE, whereas three genes (ZNF804A, CDK1, and MANF) have not previously been associated with autoimmunity. Random forest classification also allowed prediction of patients at risk for lupus nephritis with an area under the curve of 0.94. By allele-specific gene expression analysis we detected cis-regulatory SNPs that affect the expression levels of six of the top 40 genes designed by the random forest analysis, indicating a regulatory role for the identified risk variants. The 40 top genes from the prediction were overrepresented for differential expression in B and T cells according to RNA-sequencing of samples from five healthy donors, with more frequent over-expression in B cells compared to T cells. Funding Agencies|Knut and Alice Wallenberg Foundation; Swedish Research Council for Medicine and Health [521-2014-2263, 521-2013-2830]; Swedish Rheumatism Association; King Gustaf V 80-year Foundation; COMBINE

Cochlear implants (CI) have been used for several decades to treat patients with profound hearing loss. Nevertheless, results vary between individuals, and fine hearing is generally poor due to the lack of discrete neural stimulation from the individual receptor hair cells. A major problem is the deliverance of independent stimulation signals to individual auditory neurons. Fine hearing requires significantly more stimulation contacts with intimate neuron/electrode interphases from ordered axonal re-growth, something current CI technology cannot provide. Here, we demonstrate the potential application of micro-textured nanocrystalline diamond (NCD) surfaces on CI electrode arrays. Such textured NCD surfaces consist of micrometer-sized nail-head-shaped pillars (size 5×5μm(2)) made with sequences of micro/nano-fabrication processes, including sputtering, photolithography and plasma etching. The results show that human and murine inner-ear ganglion neurites and, potentially, neural progenitor cells can attach to patterned NCD surfaces without an extracellular matrix coating. Microscopic methods revealed adhesion and neural growth, specifically along the nail-head-shaped NCD pillars in an ordered manner, rather than in non-textured areas. This pattern was established when the inter-NCD pillar distance varied between 4 and 9μm. The findings demonstrate that regenerating auditory neurons show a strong affinity to the NCD pillars, and the technique could be used for neural guidance and the creation of new neural networks. Together with the NCD's unique anti-bacterial and electrical properties, patterned NCD surfaces could provide designed neural/electrode interfaces to create independent electrical stimulation signals in CI electrode arrays for the neural population.Cochlear implant is currently a successful way to treat sensorineural hearing loss and deafness especially in children. Although clinically successful, patients' fine hearing cannot be completely restored. One problem is the amount of the electrodes; 12-20 electrodes are used to replace the function of 3400 inner hair cells. Intense research is ongoing aiming to increase the number of electrodes. This study demonstrates the use of nanocrystalline diamond as a potential nerve-electrode interface. Micrometer-sized nanocrystalline diamond pillars showed high affinity to regenerated human neurons, which grew into a pre-defined network based on the pillar design. Our findings are of particular interest since they can be applied on any silicon-based implant to increase electrode count and to achieve individual neuron stimulation patterns.