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Supplementary figures legends

Supplementary materials and methods. Detailed bioinformatics analysis methods (softwares commands and parameters) used to analyse in silicon TCGA and OKG exam sequencing data.

Supplementary figures and tables for bioinformatic analyses. Fig S1 TCGA bioinformatics analysis workflow chart Fig S2 alignment plots of quality score accuracy Fig S3 Screenshot of region of interest on Chr 19 indicating genotype information on KIR2DS4 variant call Table SI Characteristics of the patients and control population used in this study Table SII Clinical data of 300 GBM patient obtained from The Cancer Genome Atlas (TCGA) project. Table SIII Description of the 2504 control population from 1000 Genome Project. Table SIV Antibodies used for Flow cytometry

By affecting immunological presentation, the presence of cytomegalovirus in some glioblastomas may impact progression. In this study, we examined a hypothesized role for natural killer (NK) cells in impacting disease progression in this setting. We characterized 108 glioblastoma patients and 454 healthy controls for HLA-A,-B,-C, NK-cell KIR receptors, and CMV-specific antibodies and correlated these metrics with clinical parameters. Exome sequences from a large validation set of glioblastoma patients and control individuals were examined from in silico databases. We demonstrated that the KIR allele KIR2DS4*00101 was independently prognostic of prolonged survival. KIR2DS4*00101 displayed 100% concordance with cognate HLA-C1 ligands in glioblastoma patients, but not controls. In the context of both HLA-C1/C2 ligands for the KIR2DS4 receptor, patient survival was further extended. Notably, all patients carrying KIR2DS4*00101 alleles were CMV seropositive, but not control individuals, and exhibited increased NK-cell subpopulations, which expressed the cytotoxicity receptors CD16, NKG2D, and CD94/NKG2C. Finally, healthy controls exhibited a reduced risk for developing glioblastoma if they carried two KIR2DS4*00101 alleles, where protection was greatest among Caucasian individuals. Our findings suggest that KIR2DS4*00101 may offer a molecular biomarker to identify intrinsically milder forms of glioblastoma. Cancer Res; 76(18); 5326–36. ©2016 AACR.

Extracellular vesicles (EVs) are implicated in the pathogenesis of rheumatoid arthritis (RA) but little is known about the composition of specific small EV (sEV) subpopulations. This study aimed to characterize the CD63, CD81 and CD9 tetraspanin profile in the membrane of single EVs in plasma from treatment naïve RA patients and assess potential discrepancies between methotrexate (MTX) responder groups. EVs isolated from plasma were characterized using transmission electron microscopy, and detection of surface markers (CD63, CD81 and CD9) on single EVs was performed on the ExoView platform. All RA patients (N = 8) were newly diagnosed, treatment naïve, females, ACPA positive and former smokers. The controls (N = 5) were matched for age and gender. After three months of MTX treatment, responders (N = 4) were defined as those with ΔDAS28 > 1.2 and DAS28 ≤ 3.2 post-treatment. The isolated EVs were 50–200 nm in size. The RA patients had a higher proportion of both CD9 and CD81 single positive sEVs compared to healthy controls, while there was a decrease in CD81/CD9 double positive sEVs in patients. Stratification of RA patients into MTX responders and non-responders revealed a distinctly higher proportion of CD81 single positive sEVs in the responder group. The proportion of CD81/CD9 double positive sEVs (anti-CD9 captured) was lower in the non-responders, but increased upon 3 months of MTX treatment. Our exploratory study revealed distinct tetraspanin profiles in RA patients suggesting their implication in RA pathophysiology and MTX treatment response.

This is an open access article under the CC BY license. The etiology of myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is unknown, but involvement of the immune system is one of the proposed underlying mechanisms. Human leukocyte antigen (HLA) associations are hallmarks of immune-mediated and autoimmune diseases. We have previously performed high resolution HLA genotyping and detected associations between ME/CFS and certain HLA class I and class II alleles. However, the HLA complex harbors numerous genes of immunological importance, and there is extensive and complex linkage disequilibrium across the region. In the current study, we aimed to fine map the association signals in the HLA complex by genotyping five additional classical HLA loci and 5,342 SNPs in 427 Norwegian ME/CFS patients, diagnosed according to the Canadian Consensus Criteria, and 480 healthy Norwegian controls. SNP association analysis revealed two distinct and independent association signals (p ≤ 0.001) tagged by rs4711249 in the HLA class I region and rs9275582 in the HLA class II region. Furthermore, the primary association signal in the HLA class II region was located within the HLA-DQ gene region, most likely due to HLA-DQB1, particularly the amino acid position 57 (aspartic acid/alanine) in the peptide binding groove, or an intergenic SNP upstream of HLA-DQB1. In the HLA class I region, the putative causal locus might map outside the classical HLA genes as the association signal spans several genes (DDR1, GTF2H4, VARS2, SFTA2 and DPCR1) with expression levels influenced by the ME/CFS associated SNP genotype. Taken together, our results implicate the involvement of the MHC, and in particular the HLA-DQB1 gene, in ME/CFS. These findings should be replicated in larger cohorts, particularly to verify the putative involvement of HLA-DQB1, a gene important for antigen-presentation to T cells and known to harbor alleles providing the largest risk for well–established autoimmune diseases.

We have established an in-house HLA-B*27 multiplex typing assay by using a combination of previously published, newly designed and commercial primers and probes for use with real-time PCR instruments. Hence, facilitating quick and large-scale HLA-B*27 typing.

Study ObjectivesNarcolepsy type 1 (NT1) is characterized by unstable sleep-wake and muscle tonus regulation during sleep. We characterized dream enactment and muscle activity during sleep in a cohort of post-H1N1 NT1 patients and their siblings, and analyzed whether clinical phenotypic characteristics and major risk factors are associated with increased muscle activity.MethodsRBD symptoms and polysomnography m. tibialis anterior electromyographical signals [long (0.5–15 s); short (0.1–0.49 s)] were compared between 114 post-H1N1 NT1 patients and 89 non-narcoleptic siblings. Association sub-analyses with RBD symptoms, narcoleptic symptoms, CSF hypocretin-1 levels, and major risk factors [H1N1-(Pandemrix)-vaccination, HLA-DQB1*06:02-positivity] were performed.ResultsRBD symptoms, REM and NREM long muscle activity indices and REM short muscle activity index were significantly higher in NT1 patients than siblings (all p < 0.001). Patients with undetectable CSF hypocretin-1 levels (ConclusionIncreased REM and NREM muscle activity and more RBD symptoms is characteristic of NT1, and muscle activity severity is predicted by hypocretin deficiency severity but not by H1N1-(Pandemrix)-vaccination status. In the patients’ non-narcoleptic siblings, neither RBD symptoms, core narcoleptic symptoms, nor the major NT1 risk factors is associated with muscle activity during sleep, hence not indicative of a phenotypic continuum Study Objectives Narcolepsy type 1 (NT1) is characterized by unstable sleep-wake and muscle tonus regulation during sleep. We characterized dream enactment and muscle activity during sleep in a cohort of post-H1N1 NT1 patients and their siblings, and analyzed whether clinical phenotypic characteristics and major risk factors are associated with increased muscle activity. Methods RBD symptoms and polysomnography m. tibialis anterior electromyographical signals [long (0.5-15 s); short (0.1-0.49 s)] were compared between 114 post-H1N1 NT1 patients and 89 non-narcoleptic siblings. Association sub-analyses with RBD symptoms, narcoleptic symptoms, CSF hypocretin-1 levels, and major risk factors [H1N1-(Pandemrix)-vaccination, HLA-DQB1*06:02-positivity] were performed. Results RBD symptoms, REM and NREM long muscle activity indices and REM short muscle activity index were significantly higher in NT1 patients than siblings (all p < 0.001). Patients with undetectable CSF hypocretin-1 levels (

Disability and distress caused by chronic low back pain (LBP) lacking clear pathoanatomical explanations cause huge problems both for patients and society. A subgroup of patients has Modic changes (MC), identifable by MRI as vertebral bone marrow lesions. The cause of such changes and their relationship to pain are not yet understood. We explored the pathobiology of these lesions using profling of gene expression in blood, coupled with an edema-sensitive MRI technique known as short tau inversion recovery (STIR) imaging. STIR images and total RNA from blood were collected from 96 patients with chronic LBP and MC type I, the most infammatory MC state. We found the expression of 37 genes signifcantly associated with STIR signal volume, ten genes with edema abundancy (a constructed combination of STIR signal volume, height, and intensity), and one gene with expression levels signifcantly associated with maximum STIR signal intensity. Gene sets related to interferon signaling, mitochondrial metabolism and defense response to virus were identifed as signifcantly enriched among the upregulated genes in all three analyses. Our results point to infammation and immunological defense as important players in MC biology in patients with chronic LBP. Tis work was supported by governmental organisations Helse Sør-Øst (Grant number 2015090); Helse Vest (Grant numbers HV 911938, HV 911891); and Klinbeforsk (Grant number 2017201).

Objective Juvenile idiopathic arthritis (JIA) is the most common chronic immune-mediated joint disease among children and encompasses a heterogeneous group of immune-mediated joint disorders classified into 7 subtypes according to clinical presentation. However, phenotype overlap and biologic evidence suggest a shared mechanistic basis between subtypes. This study was undertaken to systematically investigate shared genetic underpinnings of JIA subtypes. Methods We performed a heterogeneity-sensitive genome-wide association study encompassing a total of 1,245 JIA cases (classified into 7 subtypes) and 9,250 controls, followed by fine-mapping of candidate causal variants at each genome-wide significant locus, functional annotation, and pathway and network analysis. We further identified candidate drug targets and drug repurposing opportunities by in silico analyses. Results In addition to the major histocompatibility complex locus, we identified 15 genome-wide significant loci shared between at least 2 JIA subtypes, including 10 novel loci. Functional annotation indicated that candidate genes at these loci were expressed in diverse immune cell types. Conclusion This study identified novel genetic loci shared by JIA subtypes. Our findings identified candidate mechanisms underlying JIA subtypes and candidate targets with drug repurposing opportunities for JIA treatment.

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 similar to 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=2.1x10(-9)), more than the rs2476601-A missense variant in PTPN22 (OR=1.59, p=1.3x10(-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=6.6x10(-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. Funding Agencies|NORDFORSK [90825]; Swedish Research Council [2018-02803]; Swedish innovation Agency (Vinnova); Innovationsfonden; The Research Council of Norway; Region Stockholm-Karolinska Institutet; Region Vasterbotten (ALF); Danish Rheumatism Association [R194-A6956, A1923, A3037, A3570]; Swedish Brain Foundation; Nils and Bibbi Jensens Foundation; Knut and Alice Wallenberg Foundation; Margaretha af Ugglas Foundation; South-Eastern Heath Region of Norway; Health Research Fund of Central Denmark Region; Region of Southern Denmark; A.P. Moller Foundation for the Advancement of Medical Science; Colitis-Crohn Foreningen; Novo Nordisk Foundation [NNF15OC0016932]; Aase og Ejnar Danielsens Fond; Beckett-Fonden; Augustinus Fonden; Knud and Edith Eriksens Mindefond; Laege Sofus Carl Emil Friis and Hustru Olga Doris Friis Legat; Psoriasis Forskningsfonden; University of Aarhus; Region of Southern Denmarks PhD Fund [12/7725]; Department of Rheumatology, Frederiksberg Hospital; Research Council of Norway [229624, 223273]; South East and Western Norway Health Authorities; ERC AdG project SELECTionPREDISPOSED; Stiftelsen Kristian Gerhard Jebsen; Trond Mohn Foundation; Novo Nordisk Foundation; University of Bergen

Unlike chimeric antigen receptors, T-cell receptors (TCRs) can recognize intracellular targets presented on human leukocyte antigen (HLA) molecules. Here we demonstrate that T cells expressing TCRs specific for peptides from the intracellular lymphoid-specific enzyme terminal deoxynucleotidyl transferase (TdT), presented in the context of HLA-A*02:01, specifically eliminate primary acute lymphoblastic leukemia (ALL) cells of T- and B-cell origin in vitro and in three mouse models of disseminated B-ALL. By contrast, the treatment spares normal peripheral T- and B-cell repertoires and normal myeloid cells in vitro, and in vivo in humanized mice. TdT is an attractive cancer target as it is highly and homogeneously expressed in 80-94% of B- and T-ALLs, but only transiently expressed during normal lymphoid differentiation, limiting on-target toxicity of TdT-specific T cells. TCR-modified T cells targeting TdT may be a promising immunotherapy for B-ALL and T-ALL that preserves normal lymphocytes.Engineered T cells kill leukemic cells with little off-target toxicity.

Joint replacement surgery provides pain relief and restoration of mobility for millions of patients around the world each year.[1] However, the release of wear debris from implant surfaces can limit the lifespan of a prosthesis through the promotion of inflammatory responses.[2] Implants must therefore be constructed from materials with sufficient durability and biocompatibility. One such material is cobalt chrome alloy, which is used in the majority of joint replacements.[3] Unfortunately, it is recognised that some patients develop lymphocyte mediated delayed type hypersensitivity (DTH) responses to this material,[4] a response which may result in extensive bone and soft tissue destruction.[5] A genetic predisposition to DTH has been proposed[6], though specific genes have yet to be identified, or the effects quantified. Here we show that variation in HLA class II genotype influences an individual’s susceptibility to DTH. HLA-DQ haplotypes encoding peptide binding grooves with greater affinity for the N terminal peptide sequence of albumin (containing two recognised metal binding sites) confer a greater risk of DTH. We describe the development and validation of a machine learning algorithm to investigate the possibility that a patient’s genotype and basic clinical parameters may be used to predict DTH. Incorporating this novel finding, gradient boosted survival analysis machine learning models were trained and validated using results from 606 patients from three international units. These models were assessed using Uno’s c-index, time-dependent AUROCs, and integrated calibration index performance statistics. At present, there are no tests in widespread clinical use which use a patient’s genetic profile to guide implant selection or inform post-operative management. The algorithm described herein may address this issue.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a disease with a variety of symptoms such as post-exertional malaise, fatigue, and pain, but where aetiology and pathogenesis are unknown. An increasing number of studies have implicated the involvement of the immune system in ME/CFS. Furthermore, a hereditary component is suggested by the reported increased risk for disease in relatives, and genetic association studies are being performed to identify potential risk variants. We recently reported an association with the immunologically important human leucocyte antigen (HLA) genes HLA-C and HLA-DQB1 in ME/CFS. Furthermore, a genome-wide genetic association study in 42 ME/CFS patients reported significant association signals with two variants in the T cell receptor alpha (TRA) locus (P value −8). As the T cell receptors interact with the HLA molecules, we aimed to replicate the previously reported findings in the TRA locus using a large Norwegian ME/CFS cohort (409 cases and 810 controls) and data from the UK biobank (2105 cases and 4786 controls). We investigated numerous SNPs in the TRA locus, including the two previously ME/CFS-associated variants, rs11157573 and rs17255510. No associations were observed in the Norwegian cohort, and there was no significant association with the two previously reported SNPs in any of the cohorts. However, other SNPs showed signs of association (P value

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a disease of unknown etiology and pathogenesis, which manifests in a variety of symptoms like post-exertional malaise, brain fog, fatigue and pain. Hereditability is suggested by an increased disease risk in relatives, however, genome-wide association studies in ME/CFS have been limited by small sample sizes and broad diagnostic criteria, therefore no established risk loci exist to date. In this study, we have analyzed three ME/CFS cohorts: a Norwegian discovery cohort (N = 427), a Danish replication cohort (N = 460) and a replication dataset from the UK biobank (N = 2105). To the best of our knowledge, this is the first ME/CFS genome-wide association study of this magnitude incorporating 2532 patients for the genome-wide analyses and 460 patients for a targeted analysis. Even so, we did not find any ME/ CFS risk loci displaying genome-wide significance. In the Norwegian discovery cohort, the TPPP gene region showed the most significant association (rs115523291, P = 8.5 × 10− 7 ), but we could not replicate the top SNP. However, several other SNPs in the TPPP gene identified in the Norwegian discovery cohort showed modest association signals in the self-reported UK biobank CFS cohort, which was also present in the combined analysis of the Norwegian and UK biobank cohorts, TPPP (rs139264145; P = 0.00004). Interestingly, TPPP is expressed in brain tissues, hence it will be interesting to see whether this association, with time, will be verified in even larger cohorts. Taken together our study, despite being the largest to date, could not establish any ME/CFS risk loci, but comprises data for future studies to accumulate the power needed to reach genome-wide significance.

High-throughput sequencing is increasingly favoured to assay the presence and abundance of micro RNAs (miRNAs) in biological samples, even from low RNA amounts, and a number of commercial vendors now offer kits that allow miRNA sequencing from sub-nanogram (ng) inputs. However, although biases introduced during library preparation have been documented, the relative performance of current reagent kits has not been investigated in detail. Here, six commercial kits capable of handling

OBJECTIVE Circumstantial evidence links 25-hydroxy vitamin D [25(OH)D], vitamin D–binding protein (DBP), vitamin D–associated genes, and type 1 diabetes (T1D), but no studies have jointly analyzed these. We aimed to investigate whether DBP levels during pregnancy or at birth were associated with offspring T1D and whether vitamin D pathway genetic variants modified associations between DBP, 25(OH)D, and T1D. RESEARCH DESIGN AND METHODS From a cohort of >100,000 mother/child pairs, we analyzed 189 pairs where the child later developed T1D and 576 random control pairs. We measured 25(OH)D using liquid chromatography–tandem mass spectrometry, and DBP using polyclonal radioimmunoassay, in cord blood and maternal plasma samples collected at delivery and midpregnancy. We genotyped mother and child for variants in or near genes involved in vitamin D metabolism (GC, DHCR7, CYP2R1, CYP24A1, CYP27B1, and VDR). Logistic regression was used to estimate odds ratios (ORs) adjusted for potential confounders. RESULTS Higher maternal DBP levels at delivery, but not in other samples, were associated with lower offspring T1D risk (OR 0.86 [95% CI 0.74–0.98] per μmol/L increase). Higher cord blood 25(OH)D levels were associated with lower T1D risk (OR = 0.87 [95% CI 0.77–0.98] per 10 nmol/L increase) in children carrying the VDR rs11568820 G/G genotype (Pinteraction = 0.01 between 25(OH)D level and rs11568820). We did not detect other gene-environment interactions. CONCLUSIONS Higher maternal DBP level at delivery may decrease offspring T1D risk. Increased 25(OH)D levels at birth may decrease T1D risk, depending on VDR genotype. These findings should be replicated in other studies. Future studies of vitamin D and T1D should include VDR genotype and DBP levels.

Highly polymorphic interactions of KIR3DL1 and KIR3DS1 with HLA class I ligands modulates the effector functions of natural killer (NK) cells and some T cells. This genetically determined diversity affects severity of infections, immune-mediated diseases, and some cancers, and impacts the course of cancer treatment, including transplantation. KIR3DL1 is an inhibitory receptor, and KIR3DS1 is an activating receptor encoded by the KIR3DL1/S1 gene that has more than 200 diverse and divergent alleles. Determination of KIR3DL1/S1 genotypes for medical application is hampered by complex sequence and structural variation that distinguishes individuals and populations, requiring targeted approaches to generate and analyze high-resolution allele data. To overcome these obstacles, we developed and optimized a model for imputing KIR3DL1/S1 alleles at high-resolution from whole-genome SNP data, and designed to represent a substantial component of human genetic diversity. We show that our Global model is effective at imputing KIR3DL1/S1 alleles with an accuracy ranging from 89% in Africans to 97% in East Asians, with mean specificity of 99.8% and sensitivity of 99% for named alleles >1% frequency. We used the established algorithm of the HIBAG program, in a modification named Pulling Out Natural killer cell Genomics (PONG). Because HIBAG was designed to impute HLA alleles also from whole-genome SNP data, PONG allows combinatorial diversity of KIR3DL1/S1 and HLA-A and B to be analyzed using complementary techniques on a single data source. The use of PONG thus negates the need for targeted sequencing data in very large-scale association studies where such methods might not be tractable. All code, imputation models, test data and documentation are available at https://github.com/NormanLabUCD/PONG.Author SummaryNatural killer (NK) cells are cytotoxic lymphocytes that identify and kill infected or malignant cells and guide immune responses. The effector functions of NK cells are modulated through polymorphic interactions of KIR3DL1/S1 on their surface with the human leukocyte antigens (HLA) that are found on most other cell types in the body. KIR3DL1/S1 is highly polymorphic and differentiated across human populations, affecting susceptibility and course of multiple immune-mediated diseases and their treatments. Genotyping KIR3DL1/S1 for direct medical application or research has been encumbered by the complex sequence and structural variation, which requires targeted approaches and extensive domain expertise to generate and validate high-resolution allele calls. We therefore developed Pulling Out Natural Killer Cell Genomics (PONG) to impute KIR3DL1/S1 alleles from whole genome SNP data, and which we implemented as an open-source R package. We assessed imputation performance using data from five broad population groups that represent a substantial portion of human genetic diversity. We can impute KIR3DL1/S1 alleles with an accuracy ranging from 89% in Africans and South Asians to 97% in East Asians. Globally, imputation of KIR3DL1/S1 alleles having frequency >1% has a mean sensitivity of 94% and specificity of 99.8%. Thus, the PONG method both enables highly sensitive individual-level calling and makes large scale medical genetic studies of KIR3DL1/S1 possible.

Rheumatoid arthritis (RA) is a complex disease with a wide range of underlying susceptibility factors. Recently, dysregulation of microRNAs (miRNAs) in RA have been reported in several immune cell types from blood. However, B cells have not been studied in detail yet. Given the autoimmune nature of RA with the presence of autoantibodies, CD19+ B cells are a key cell type in RA pathogenesis and alterations in CD19+ B cell subpopulations have been observed in patient blood. Therefore, we aimed to reveal the global miRNA repertoire and to analyze miRNA expression profile differences in homogenous RA patient phenotypes in blood-derived CD19+ B cells. Small RNA sequencing was performed on CD19+ B cells of newly diagnosed untreated RA patients (n=10), successfully methotrexate (MTX) treated RA patients in remission (MTX treated RA patients, n=18) and healthy controls (n=9). The majority of miRNAs was detected across all phenotypes. However, significant expression differences between MTX treated RA patients and controls were observed for 27 miRNAs, while no significant differences were seen between the newly diagnosed patients and controls. Several of the differentially expressed miRNAs were previously found to be dysregulated in RA including miR-223-3p, miR-486-3p and miR-23a-3p. MiRNA target enrichment analysis, using the differentially expressed miRNAs and miRNA-target interactions from miRTarBase as input, revealed enriched target genes known to play important roles in B cell activation, differentiation and B cell receptor signaling, such as STAT3, PRDM1 and PTEN. Interestingly, many of those genes showed a high degree of correlated expression in CD19+ B cells in contrast to other immune cell types. Our results suggest important regulatory functions of miRNAs in blood-derived CD19+ B cells of MTX treated RA patients and motivate for future studies investigating the interactive mechanisms between miRNA and gene targets, as well as the possible predictive power of miRNAs for RA treatment response.

Evidence suggests a cell-mediated autoimmune pathogenesis for narcolepsy type 1 (NT1), but it is not clear whether the disease is associated with overall changes in T cell subsets. The increase in NT1 incidence after H1N1 vaccination campaign with the Pandemrix™ vaccine suggests that disease-relevant changes in the immune system following this vaccination were important. In this study, we aimed to investigate differentiated T cell subsets and levels of CD25 and CD69 activation markers in a cohort of mainly Pandemrix™-vaccinated NT1 patients compared with their vaccinated and unvaccinated siblings.Peripheral blood mononuclear cells were collected in parallel and analysed with flow cytometry in 31 NT1 patients with disease onset after the 2009 influenza A (H1N1) pandemic and/or Pandemrix™ vaccination and 45 of their non-narcoleptic siblings (29/31 and 34/45 vaccinated, respectively).We observed significantly lower effector memory CD4We confirm that NT1 is associated with lower levels of effector memory CD4

BackgroundThe impact of COVID-19 varies markedly, not only between individual patients but also between different populations. We hypothesised that differences in human leukocyte antigen (HLA) genes might influence this variation.MethodsUsing next generation sequencing, we analysed the class I and class II classical HLA genes of 147 white British patients with variable clinical outcomes. 49 of these patients were admitted to hospital with severe COVID infection. They had no significant pre-existing comorbidities. We compared the results to those obtained from a group of 69 asymptomatic hospital workers who evidence of COVID exposure based on blood antibody testing. Allelic frequencies in both the severe and asymptomatic groups were compared to local and national healthy controls with adjustments made for age and sex. With the inclusion of hospital staff who had reported localised symptoms only (limited to loss of smell/taste, n=13) or systemic symptoms not requiring hospital treatment (n=16), we carried out ordinal logistic regression modelling to determine the relative influence of age, BMI, sex and the presence of specific HLA genes on symptomatology.FindingsWe found a significant difference in the allelic frequency of HLA-DRB1*04:01 in the severe patient compared to the asymptomatic staff group (5.1% versus 16.7%, p=0.003 after adjustment for age and sex). There was a significantly lower frequency of the haplotype DQA1*01:01/DQB1*05:01/DRB1*01:01 in the asymptomatic group compared to the background population (p=0.007). Ordinal logistic regression modelling confirmed the significant influence of DRB1*04:01 on the clinical severity of COVID-19 observed in the cohorts.InterpretationThis study provides evidence that patient age, sex, BMI and HLA genotype interact to determine the clinical outcome of COVID-19 infection.Research in contextEvidence before this studyHLA genes are implicated in host resistance or susceptibility to a range of pathogens. No studies thus far have compared HLA allele frequencies in patients requiring hospital admission following COVID-19 exposure to a group of asymptomatic individuals.Added value of this studyThe results indicate that the presence of HLA-DRB1*04:01 might confer protection from the development of respiratory failure following exposure to COVID. Individuals remaining asymptomatic following exposure to COVID are less likely to carry the haplotype DQA1*01:01/DQB1*05:01/DRB1*01:01 compared to the background population. This may indicate a host defence pathway not primarily dependent on an IgG response for clearance of infection. These findings conflict with larger genome wide association studies which compared HLA allelic frequencies of severely unwell patients with the background population.Implications of all the available evidenceThe findings could have implications for targeted vaccination regimes as well as helping assess the impact of social restrictions on mortality rates in different populations.

Background In addition to being a tumour suppressor, TP53 is a suppressor of inflammation, and dysfunction of this gene has been related to autoimmune diseases. Patients with autoimmunity, such as rheumatoid arthritis (RA) have an increased risk of certain cancers, like lymphomas, indicating that some underlying mechanisms may modulate risk of both cancers and autoimmunity. Methods We genotyped 5 common genetic variants in TP53 and its main regulators MDM2 and MDM4 in a sample of 942 RA patients and 3,747 healthy controls, and mined previously published GWAS-data, to assess the potential impact of these variants on risk of RA. Results For the TP53 Arg72Pro polymorphism (rs1042522), MDM4 SNP34091 (rs4245739) and MDM2 SNP285C (rs117039649), we found no association to risk of RA. For MDM2 SNP309 (rs2279744), the minor G-allele was associated with a reduced risk of RA (OR: 0.87; CI: 0.79–0.97). This association was also seen in genotype models (OR: 0.86; CI: 0.74–0.99 and OR: 0.79; CI 0.63–0.99; dominant and recessive model, respectively), but was not validated in a large GWAS data set. For MDM2 del1518 (rs3730485), the minor del-allele was associated with an increased risk of RA in the dominant model (OR: 1.18; CI: 1.02–1.38). Stratifying RA cases and controls into phylogenetic subgroups according to the combined genotypes of all three MDM2 polymorphism, we found individuals with the del158-285–309 genotype del/ins-G/G-T/T to have an increased risk of RA as compared to those with the ins/ins-G/G-G/G genotype (OR: 1.56; CI: 1.18–2.06) indicating opposite effects of the del1518 del-allele and the SNP309 G-allele. Conclusion We find a potential association between the MDM2 del1518 variant and RA, and indications that combinatorial genotypes and haplotypes in the MDM2 locus may be related to RA.

Study objectives: Evidence suggests a cell-mediated autoimmune pathogenesis for narcolepsy type 1 (NT1), but it is not clear whether the disease is associated with overall changes in T cell subsets. The increase in NT1 incidence after H1N1 vaccination campaign with the Pandemrix™ vaccine suggests that disease-relevant changes in the immune system following this vaccination were important. In this study, we aimed to investigate differentiated T cell subsets and levels of CD25 and CD69 activation markers in a cohort of mainly Pandemrix™-vaccinated NT1 patients compared with their vaccinated and unvaccinated siblings. Methods: Peripheral blood mononuclear cells were collected in parallel and analysed with flow cytometry in 31 NT1 patients with disease onset after the 2009 influenza A (H1N1) pandemic and/or Pandemrix™ vaccination and 45 of their non-narcoleptic siblings (29/31 and 34/45 vaccinated, respectively). Results: We observed significantly lower effector memory CD4+ T cell levels in NT1 patients compared to their siblings, when controlling for HLA DQB1∗06:02 and vaccination status. Further, within the sibling group, vaccination status significantly affected frequencies of central memory and CD8+CD25+ T cells, and HLA DQB1∗06:02 status significantly affected frequencies of CD4+CD25+ T cells. Conclusion: We confirm that NT1 is associated with lower levels of effector memory CD4+ T cells in peripheral blood. Importantly, this finding was only significant when controlling for vaccination and HLA status in both patients and controls. We thus demonstrate the importance of characterizing such factors (eg HLA and vaccination) when studying T cell subsets in NT1. This might explain earlier conflicting results.

Study Objectives Narcolepsy type 1 (NT1) is associated with hypocretin neuron loss. However, there are still unexplained phenotypic NT1 features. We investigated the associations between clinical and sleep phenotypic characteristics, the NT1-associated P2RY11 polymorphism rs2305795, and P2Y11 protein levels in T lymphocytes in patients with NT1, their first-degree relatives and unrelated controls. Methods The P2RY11 SNP was genotyped in 100 patients (90/100 H1N1-(Pandemrix)-vaccinated), 119 related and 123 non-related controls. CD4 and CD8 T lymphocyte P2Y11 protein levels were quantified using flow cytometry in 167 patients and relatives. Symptoms and sleep recording parameters were also collected. Results We found an association between NT1 and the rs2305795 A allele (OR = 2, 95% CI (1.3, 3.0), p = 0.001). T lymphocyte P2Y11 protein levels were significantly lower in patients and relatives homozygous for the rs2305795 risk A allele (CD4: p = 0.012; CD8: p = 0.007). The nocturnal sleep fragmentation index was significantly negatively correlated with patients’ P2Y11 protein levels (CD4: p = 0.004; CD8: p = 0.006). Mean MSLT sleep latency, REM-sleep latency, and core clinical symptoms were not associated with P2Y11 protein levels. Conclusions We confirmed that the P2RY11 polymorphism rs2305795 is associated with NT1 also in a mainly H1N1-(Pandemrix)-vaccinated cohort. We demonstrated that homozygosity for the A risk allele is associated with lower P2Y11 protein levels. A high level of nocturnal sleep fragmentation was associated with low P2Y11 levels in patients. This suggests that P2Y11 has a previously unknown function in sleep-wake stabilization that affects the severity of NT1.

The impact of COVID-19 varies markedly, not only between individual patients but also between different populations. We hypothesised that differences in human leukocyte antigen (HLA) genes might influence this variation. Using next generation sequencing, we analysed the class I and class II classical HLA genes of 147 individuals of European descent experiencing variable clinical outcomes following COVID-19 infection. Forty-nine of these patients were admitted to hospital with severe respiratory disease. They had no significant pre-existing comorbidities. We compared the results to those obtained from a group of 69 asymptomatic hospital workers who evidence of COVID exposure based on blood antibody testing. Allele frequencies in both the severe and asymptomatic groups were compared to local and national healthy controls with adjustments made for age and sex. With the inclusion of hospital staff who had reported localised symptoms only (limited to loss of smell/taste, n = 13) or systemic symptoms not requiring hospital treatment (n = 16), we carried out ordinal logistic regression modelling to determine the relative influence of age, BMI, sex and the presence of specific HLA genes on symptomatology. We found a significant difference in the allele frequency of HLA-DRB1*04:01 in the severe patient compared to the asymptomatic staff group (5.1% vs. 16.7%, P = .003 after adjustment for age and sex). There was a significantly lower frequency of the haplotype DQA1*01:01-DQB1*05:01-DRB1*01:01 in the asymptomatic group compared to the background population (P = .007). Ordinal logistic regression modelling confirmed the significant influence of DRB1*04:01 on the clinical severity of COVID-19 observed in the cohorts. These alleles are found in greater frequencies in the North Western European population. This regional study provides evidence that HLA genotype influences clinical outcome in COVID-19 infection. Validation studies must take account of the complex genetic architecture of the immune system across different geographies and ethnicities.

Low back pain is common and a significant number of patients experience chronic low back pain. Current treatment options offer small to moderate effects. Patients with vertebral bone marrow lesions visualized as Modic changes on magnetic resonance imaging may represent a subgroup within the low back pain population. There is evidence for inflammatory mediators being involved in development of Modic changes; hence, suppression of inflammation could be a treatment strategy for these patients. This study examines the effect of anti-inflammatory treatment with the TNF-α inhibitor infliximab in patients with chronic low back pain and Modic changes.Methods/design: The BackToBasic trial is a multicenter, double blind, randomized controlled trial conducted at six hospitals in Norway, comparing intravenous infusions with infliximab with placebo. One hundred twenty-six patients aged 18–65 with chronic low back pain and type 1 Modic changes will be recruited from secondary care outpatients’ clinics. The primary outcome is back pain-specific disability at day 154 (5 months). The study is designed to detect a difference in change of 10 (SD 18) in the Oswestry Disability Index at day 154/ 5 months. The study also aims to refine MRI-assessment, investigate safety and cost-effectiveness and explore the underlying biological mechanisms of Modic changes..Discussion: Finding treatments that target underlying mechanisms could pose new treatment options for patients with low back pain. Suppression of inflammation could be a treatment strategy for patients with low back pain and Modic changes. This paper presents the design of the BackToBasic study, where we will assess the effect of an anti-inflammatory treatment versus placebo in patients with chronic low back pain and type 1 Modic changes. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data

Objectives: During pregnancy, small quantities of maternal cells are naturally transmitted to the fetus. This transmission, termed maternal microchimerism (MMc), has been implicated in autoimmune diseases but its potential role is unclear. We aimed to investigate if MMc at birth predicted childhood celiac disease (CD) risk, a common immune-mediated enteropathy often presenting in childhood. Methods: We designed a case-control study, nested in the Norwegian Mother, Father and Child Cohort. Participants were HLA class II typed to determine noninherited, nonshared maternal alleles (NIMA). Droplet digital (dd) PCR assays specific for common HLA class II NIMAs (HLA-DQB1∗03:01, ∗04:02 and ∗06:02/03) were used to estimate the quantity of maternal DNA, as a marker of maternal cells, in cord blood DNA from 124 children who later developed clinically diagnosed CD (median age at end of study 7.4 years, range 3.6–12.9) and 124 random controls. We tested whether presence of MMc was associated with CD using logistic regression, and compared ranks between cases and controls. Results: MMc, for example, maternal HLA antigens not inherited by the child, was found in 42% of cases and 43% of controls, and not associated with CD (odds ratio [OR] 0.97, 95% confidence interval [CI] 0.58–1.60). The ranks of MMc quantities in cases and controls were also similar (Mann-Whitney U-test, P = 0.71). The subgroup with HLA-DQB1:03∗01 as their NIMA had a potential association with MMc, where levels greater than median was associated with CD (OR 3.78, 95% CI 1.28–11.18). Conclusion: MMc measured in cord blood was not associated with later risk of CD.

Background The thymus is a highly specialized organ of the immune system where T cell precursors develop and differentiate into self-tolerant CD4+ or CD8+ T cells. No studies to date have investigated how the human transcriptome profiles differ, between T cells still residing in the thymus and T cells in the periphery. Results We have performed high-throughput RNA sequencing to characterize the transcriptomes of primary single positive (SP) CD4+ and CD8+ T cells from infant thymic tissue, as well as primary CD4+ and CD8+ T cells from infant and adult peripheral blood, to enable the comparisons across tissues and ages. In addition, we have assessed the expression of candidate genes related to autoimmune diseases in thymic CD4+ and CD8+ T cells. The thymic T cells showed the largest number of uniquely expressed genes, suggesting a more diverse transcription in thymic T cells. Comparing T cells of thymic and blood origin, revealed more differentially expressed genes, than between infant and adult blood. Functional enrichment analysis revealed an over-representation of genes involved in cell cycle and replication in thymic T cells, whereas infant blood T cells were dominated by immune related terms. Comparing adult and infant blood T cells, the former was enriched for inflammatory response, cytokine production and biological adhesion, while upregulated genes in infant blood T cells were associated with cell cycle, cell death and gene expression. Conclusion This study provides valuable insight into the transcriptomes of the human primary SP T cells still residing within the thymus, and offers a unique comparison to primary blood derived T cells. Interestingly, the majority of autoimmune disease associated genes were expressed in one or more T cell subset, however ~ 11% of these were not expressed in frequently studied adult peripheral blood.

High-throughput sequencing has emerged as the favoured method to study microRNA (miRNA) expression, but biases introduced during library preparation have been reported. To assist researchers choose the most appropriate library preparation kit, we recently compared the performance of six commercially-available kits on synthetic miRNAs and human RNA, where library preparation was performed by the vendors. We hereby supplement this study with data from two further commonly used kits (NEBNext, NEXTflex) whose manufacturers initially declined to participate. As before, performance was assessed with respect to sensitivity, reliability, titration response and differential expression. Despite NEXTflex employing partially-randomised adapter sequences to minimise bias, we reaffirm that biases in miRNA abundance are kit-specific, complicating the comparison of miRNA datasets generated using different kits.

The etiology and pathogenesis of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) are unknown, and autoimmunity is one of many proposed underlying mechanisms. Human Leukocyte Antigen (HLA) associations are hallmarks of autoimmune disease, and have not been thoroughly investigated in a large ME/CFS patient cohort. We performed high resolution HLA -A, -B, -C, -DRB1, -DQB1 and -DPB1 genotyping by next generation sequencing in 426 adult, Norwegian ME/CFS patients, diagnosed according to the Canadian Consensus Criteria. HLA associations were assessed by comparing to 4511 healthy and ethnically matched controls. Clinical information was collected through questionnaires completed by patients or relatives. We discovered two independent HLA associations, tagged by the alleles HLA-C*07:04 (OR 2.1 [95% CI 1.4–3.1]) and HLA-DQB1*03:03 (OR 1.5 [95% CI 1.1–2.0]). These alleles were carried by 7.7% and 12.7% of ME/CFS patients, respectively. The proportion of individuals carrying one or both of these alleles was 19.2% in the patient group and 12.2% in the control group (OR 1.7 [95% CI 1.3–2.2], pnc = 0.00003). ME/CFS is a complex disease, potentially with a substantial heterogeneity. We report novel HLA associations pointing toward the involvement of the immune system in ME/CFS pathogenesis.

Objective.We aimed to jointly investigate the role of antipeptidyl arginine deiminase type 4 antibodies (anti-PAD4) and polymorphisms in thePADI4gene together with clinical variables in rheumatoid arthritis (RA).Methods.Serum IgG autoantibodies to human recombinant PAD4 were identified by DELFIA technique in 745 patients with RA (366 available from previous studies). Genotyping ofPADI4was performed using TaqMan assays in 945 patients and 1118 controls. Clinical data, anticitrullinated protein antibodies (ACPA) status, shared epitope status, and a combined genetic risk score were also available.Results.Anti-PAD4 antibodies were detected in 193 (26%) of 745 patients with RA; 149 (77%) of these were also ACPA-positive. No association was observed between anti-PAD4 status and clinical characteristics,PADI4polymorphisms, or genetic risk scores after stratification for ACPA status.Conclusion.Taken together, the results from these combined serological, genetic, and clinical analyses suggest that anti-PAD4 appears to be a bystander autoantibody with no current clinical utility in RA.

Objective Several genetic polymorphisms determine vitamin D status. We aimed to estimate the strength of association of established 25-hydroyxvitamin D (25OHD)-associated variants in the mother and in the fetus, with 25OHD concentration in newborn umbilical cord plasma. Methods We randomly selected 578 mother and child dyads from the prospective Norwegian Mother and Child Cohort study. 25OHD was assayed in maternal samples taken shortly after delivery and in cord samples. We genotyped the mother and child for single nucleotide polymorphisms in or near GC, DHCR7, CYP2R1, and CYP24A1, previously confirmed to be associated with 25OHD, and computed genetic risk score (GRS). The genetic associations were replicated in an independent sample of 594 subjects. Results Although both fetal and maternal GRS were associated with cord 25OHD, only fetal GRS remained significantly associated with cord 25OHD in a regression model with maternal and fetal GRS simultaneously (1.6 nmol/L per fetal 25OHD-increasing allele; 95% confidence interval, 0.6 to 2.5, P = 0.0001). Two fetal single nucleotide polymorphisms in the GC gene (rs2282679 and rs222040) were the strongest genetic predictors of cord 25OHD [4.0 (2.1 to 5.9) and 3.0 (1.3 to 4.8) nmol/L per fetal allele, P < 0.001], followed by rs12785878 in DHCR7 [2.0 (0.1 to 3.8) nmol/L, P = 0.037]. The independent replication sample gave similar results. With fetal genotype included in a regression model with environmental factors, R2 for cord 25OHD was 0.28. Conclusions We show that fetal 25OHD-modifying genotype was a stronger predictor of cord 25OHD than corresponding maternal genotype. This raises interesting questions about fetal acquisition and placental transfer of 25OHD.

Primary sclerosing cholangitis (PSC) is strongly associated with several human leukocyte antigen (HLA) haplotypes. Due to extensive linkage disequilibrium and multiple polymorphic candidate genes in the HLA complex, identifying the alleles responsible for these associations has proven difficult. We aimed to evaluate whether studying populations of admixed or non-European descent could help in defining the causative HLA alleles. When assessing haplotypes carrying HLA-DRB1*13:01 (hypothesized to specifically increase the susceptibility to chronic cholangitis), we observed that every haplotype in the Scandinavian PSC population carried HLA-DQB1*06:03. In contrast, only 65% of HLA-DRB1*13:01 haplotypes in an admixed/non-European PSC population carried this allele, suggesting that further assessments of the PSC-associated haplotype HLA-DRB1*13:01-DQA1*01:03-DQB1*06:03 in admixed or multi-ethnic populations could aid in identifying the causative allele.