| 1. |
- Houtman, Miranda, et al.
(författare)
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Five commercially-available antibodies react differentially with allelic forms of human HLA-DR beta chain
- 2022
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Ingår i: Molecular Immunology. - : Elsevier BV. - 0161-5890 .- 1872-9142. ; 152, s. 106-110
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Tidskriftsartikel (refereegranskat)abstract
- Allelic variants of HLA-DRB1 have been associated with a variety of autoimmune and infectious diseases. Although the precise molecular mechanisms by which HLA-DRB1 alleles predispose to a particular disease are currently unclear, it has been shown that mRNA expression levels of HLA-DRB1 are dependent on the different alleles. We aimed to measure HLA-DR beta chain levels in peripheral blood mononuclear cells of individuals carrying HLA-DRB1*03:01/*04:01 and HLA-DRB1*03:01/*15:01 alleles by western blotting, using five commercially-available HLA-DRB antibodies. We observed highly heterogeneous binding of the tested antibodies to the different allelic forms of the HLA-DR beta chain. Overall, we show that current immunological research that employs available antibodies to detect HLA-DR beta chains is biased towards detection of specific variants of the protein; this may cause significant discrepancy in quantification of protein expression in a heterogeneous human population.
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| 2. |
- Houtman, Miranda, et al.
(författare)
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Haplotype-Specific Expression Analysis of MHC Class II Genes in Healthy Individuals and Rheumatoid Arthritis Patients
- 2021
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Ingår i: Frontiers in Immunology. - : Frontiers Media S.A.. - 1664-3224. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- HLA-DRB1 alleles have been associated with several autoimmune diseases. For anti-citrullinated protein antibody positive rheumatoid arthritis (RA), HLA-DRB1 shared epitope (SE) alleles are the major genetic risk factors. In order to study the genetic regulation of major histocompatibility complex (MHC) Class II gene expression in immune cells, we investigated transcriptomic profiles of a variety of immune cells from healthy individuals carrying different HLA-DRB1 alleles. Sequencing libraries from peripheral blood mononuclear cells, CD4+ T cells, CD8+ T cells, and CD14+ monocytes of 32 genetically pre-selected healthy female individuals were generated, sequenced and reads were aligned to the standard reference. For the MHC region, reads were mapped to available MHC reference haplotypes and AltHapAlignR was used to estimate gene expression. Using this method, HLA-DRB and HLA-DQ were found to be differentially expressed in different immune cells of healthy individuals as well as in whole blood samples of RA patients carrying HLA-DRB1 SE-positive versus SE-negative alleles. In contrast, no genes outside the MHC region were differentially expressed between individuals carrying HLA-DRB1 SE-positive and SE-negative alleles, thus HLA-DRB1 SE alleles have a strong cis effect on gene expression. Altogether, our findings suggest that immune effects associated with different allelic forms of HLA-DR and HLA-DQ may be associated not only with differences in the structure of these proteins, but also with differences in their expression levels.
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| 3. |
- Thorlacius, Guðný Ella, et al.
(författare)
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Genetic and clinical basis for two distinct subtypes of primary Sjögren's syndrome
- 2021
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Ingår i: Rheumatology. - : Oxford University Press. - 1462-0324 .- 1462-0332. ; 60:2, s. 837-848
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Tidskriftsartikel (refereegranskat)abstract
- ObjectivesClinical presentation of primary Sjögren’s syndrome (pSS) varies considerably. A shortage of evidence-based objective markers hinders efficient drug development and most clinical trials have failed to reach primary endpoints.MethodsWe performed a multicentre study to identify patient subgroups based on clinical, immunological and genetic features. Targeted DNA sequencing of 1853 autoimmune-related loci was performed. After quality control, 918 patients with pSS, 1264 controls and 107 045 single nucleotide variants remained for analysis. Replication was performed in 177 patients with pSS and 7672 controls.ResultsWe found strong signals of association with pSS in the HLA region. Principal component analysis of clinical data distinguished two patient subgroups defined by the presence of SSA/SSB antibodies. We observed an unprecedented high risk of pSS for an association in the HLA-DQA1 locus of odds ratio 6.10 (95% CI: 4.93, 7.54, P=2.2×10−62) in the SSA/SSB-positive subgroup, while absent in the antibody negative group. Three independent signals within the MHC were observed. The two most significant variants in MHC class I and II respectively, identified patients with a higher risk of hypergammaglobulinaemia, leukopenia, anaemia, purpura, major salivary gland swelling and lymphadenopathy. Replication confirmed the association with both MHC class I and II signals confined to SSA/SSB antibody positive pSS.ConclusionTwo subgroups of patients with pSS with distinct clinical manifestations can be defined by the presence or absence of SSA/SSB antibodies and genetic markers in the HLA locus. These subgroups should be considered in clinical follow-up, drug development and trial outcomes, for the benefit of both subgroups.
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| 4. |
- Berggren, Olof, et al.
(författare)
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Activation of plasmacytoid dendritic cells and B cells with two structurally different Toll-like receptor 7 agonists
- 2020
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Ingår i: Scandinavian Journal of Immunology. - : Wiley. - 0300-9475 .- 1365-3083. ; 91:6
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Tidskriftsartikel (refereegranskat)abstract
- Synthetic Toll-like receptor (TLR) 7 agonists have been suggested as immune modulators in a range of conditions. In contrast, self-derived TLR7 activators, such as RNA-containing immune complexes (RNA-IC), can contribute to autoimmune diseases due to endogenous immune activation. The exact difference in immune cell response between synthetic and endogenous TLR7 triggers is only partly known. An understanding of these differences could aid in the development of new therapeutic agents and provide insights into autoimmune disease mechanisms. We therefore compared the stimulatory capacity of two TLR7 agonists, RNA-IC and a synthetic small molecule DSR-6434, on blood leucocytes, plasmacytoid dendritic cells (pDCs) and B cells from healthy individuals. IFN-α, IL-6, IL-8 and TNF levels were measured by immunoassays, and gene expression in pDCs was analysed by an expression array. DSR-6434 triggered 20-fold lower levels of IFN-α by pDCs, but higher production of IL-6, IL-8 and TNF, compared to RNA-IC. Furthermore, IFN-α and TNF production were increased with exogenous IFN-α2b priming, whereas IL-8 synthesis by B cells was reduced for both stimuli. Cocultivation of pDCs and B cells increased the RNA-IC-stimulated IFN-α and TNF levels, while only IL-6 production was enhanced in the DSR-6434-stimulated cocultures. When comparing pDCs stimulated with RNA-IC and DSR-6434, twelve genes were differentially expressed (log2 fold change >2, adjusted P-value <.05). In conclusion, RNA-IC, which mimics an endogenous TLR7 stimulator, and the synthetic TLR7 agonist DSR-6434 trigger distinct inflammatory profiles in immune cells. This demonstrates the importance of using relevant stimuli when targeting the TLR7 pathway for therapeutic purposes.
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| 5. |
- Bianchi, Matteo, et al.
(författare)
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Contribution of rare genetic variation to disease susceptibility in a large Scandinavian myositis cohort
- 2022
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Ingår i: Arthritis & Rheumatology. - : John Wiley & Sons. - 2326-5191 .- 2326-5205. ; 74:2, s. 342-352
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
- Bolin, Karin
(författare)
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Lupus Nephritis – Genetic Impact on Clinical Phenotypes, Disease Severity and Renal Outcome
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Systemic lupus erythematosus (SLE) is an autoimmune inflammatory disease that can affect every organ system. Lupus nephritis (LN) is one of the more serious SLE manifestations. Whilst the genetic background of SLE has been thoroughly investigated, less is known about the background of LN. The aim of this thesis was to further elucidate the genetic background of LN, its subtypes and outcome.In paper I, we analysed genetic variations for association with LN, its severe form proliferative nephritis and renal outcome, in two SLE cohorts. Patients and controls were genotyped and association analyses were performed for patients versus controls and for patients with or without a specific clinical manifestation. In the case-control analysis of cohort I, four highly linked risk alleles in the STAT4 gene were associated with LN with genome-wide significance. In the case-only meta-analysis of the two cohorts, a STAT4 risk allele was associated with severe renal insufficiency. We conclude that genetic variations in STAT4 predispose to LN and a worse outcome with severe renal insufficiency.In paper II, we describe a case of severe SLE on the basis of C1q deficiency. By sequencing, a mutation in the C1qC gene leading to a premature stop codon was found. The patient was also found to carry risk alleles in several SLE-associated variants. Interferon alpha (IFN-α) levels were analysed over time in patient serum, and were found to correlate with disease activity. The patient’s serum had a strong interferogenic capacity when stimulating peripheral blood mononuclear cells from healthy individuals. With this study, we further emphasise the role of IFN-α in C1q deficiency and highlight the need to consider inherited impairments in the complement system in SLE with childhood onset.In paper III, we studied the impact of sex on disease manifestations in SLE. Female SLE patients more often presented with malar rash, photosensitivity, oral ulcers and arthritis, whilst the frequency of serositis, renal disorder and immunologic disorder were higher among male patients. Women were younger at LN onset, whereas men had a higher risk for progression into end-stage renal disease.In paper IV, we analysed genetic variations for association with LN and its subtypes in three SLE cohorts. Patients were genotyped and association analyses were performed for patients with versus without different phenotypes. We found genetic variations in the BANK1 gene to be associated with LN.In conclusion, this thesis provides further insight into the genetic background of renal manifestations in patients with SLE.
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| 7. |
- Bolin, Karin, et al.
(författare)
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Variants in BANK1 are associated with lupus nephritis of European ancestry
- 2021
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Ingår i: Genes and Immunity. - : Springer Nature. - 1466-4879 .- 1476-5470. ; 22:3, s. 194-202
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Tidskriftsartikel (refereegranskat)abstract
- The genetic background of lupus nephritis (LN) has not been completely elucidated. We performed a case-only study of 2886 SLE patients, including 947 (33%) with LN. Renal biopsies were available from 396 patients. The discovery cohort (Sweden, n = 1091) and replication cohort 1 (US, n = 962) were genotyped on the Immunochip and replication cohort 2 (Denmark/Norway, n = 833) on a custom array. Patients with LN, proliferative nephritis, or LN with end-stage renal disease were compared with SLE without nephritis. Six loci were associated with LN (p < 1 × 10−4, NFKBIA, CACNA1S, ITGA1, BANK1, OR2Y, and ACER3) in the discovery cohort. Variants in BANK1 showed the strongest association with LN in replication cohort 1 (p = 9.5 × 10−4) and proliferative nephritis in a meta-analysis of discovery and replication cohort 1. There was a weak association between BANK1 and LN in replication cohort 2 (p = 0.052), and in the meta-analysis of all three cohorts the association was strengthened (p = 2.2 × 10−7). DNA methylation data in 180 LN patients demonstrated methylation quantitative trait loci (meQTL) effects between a CpG site and BANK1 variants. To conclude, we describe genetic variations in BANK1 associated with LN and evidence for genetic regulation of DNA methylation within the BANK1 locus. This indicates a role for BANK1 in LN pathogenesis.
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| 8. |
- Burska, Agata, et al.
(författare)
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Type I interferon pathway assays in studies of rheumatic and musculoskeletal diseases : a systematic literature review informing EULAR points to consider
- 2023
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Ingår i: RMD Open. - : BMJ Publishing Group Ltd. - 2056-5933. ; 9:1
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Forskningsöversikt (refereegranskat)abstract
- ObjectivesTo systematically review the literature for assay methods that aim to evaluate type I interferon (IFN-I) pathway activation and to harmonise-related terminology.MethodsThree databases were searched for reports of IFN-I and rheumatic musculoskeletal diseases. Information about the performance metrics of assays measuring IFN-I and measures of truth were extracted and summarised. A EULAR task force panel assessed feasibility and developed consensus terminology.ResultsOf 10 037 abstracts, 276 fulfilled eligibility criteria for data extraction. Some reported more than one technique to measure IFN-I pathway activation. Hence, 276 papers generated data on 412 methods. IFN-I pathway activation was measured using: qPCR (n=121), immunoassays (n=101), microarray (n=69), reporter cell assay (n=38), DNA methylation (n=14), flow cytometry (n=14), cytopathic effect assay (n=11), RNA sequencing (n=9), plaque reduction assay (n=8), Nanostring (n=5), bisulphite sequencing (n=3). Principles of each assay are summarised for content validity. Concurrent validity (correlation with other IFN assays) was presented for n=150/412 assays. Reliability data were variable and provided for 13 assays. Gene expression and immunoassays were considered most feasible. Consensus terminology to define different aspects of IFN-I research and practice was produced.ConclusionsDiverse methods have been reported as IFN-I assays and these differ in what elements or aspects of IFN-I pathway activation they measure and how. No 'gold standard' represents the entirety of the IFN pathway, some may not be specific for IFN-I. Data on reliability or comparing assays were limited, and feasibility is a challenge for many assays. Consensus terminology should improve consistency of reporting.
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| 9. |
- Carlsson Almlöf, Jonas, et al.
(författare)
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Contributions of de novo variants to systemic lupus erythematosus
- 2021
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Ingår i: European Journal of Human Genetics. - : Springer Nature. - 1018-4813 .- 1476-5438. ; 29:1, s. 184-193
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Tidskriftsartikel (refereegranskat)abstract
- By performing whole-genome sequencing in a Swedish cohort of 71 parent-offspring trios, in which the child in each family is affected by systemic lupus erythematosus (SLE, OMIM 152700), we investigated the contribution of de novo variants to risk of SLE. We found de novo single nucleotide variants (SNVs) to be significantly enriched in gene promoters in SLE patients compared with healthy controls at a level corresponding to 26 de novo promoter SNVs more in each patient than expected. We identified 12 de novo SNVs in promoter regions of genes that have been previously implicated in SLE, or that have functions that could be of relevance to SLE. Furthermore, we detected three missense de novo SNVs, five de novo insertion-deletions, and three de novo structural variants with potential to affect the expression of genes that are relevant for SLE. Based on enrichment analysis, disease-affecting de novo SNVs are expected to occur in one-third of SLE patients. This study shows that de novo variants in promoters commonly contribute to the genetic risk of SLE. The fact that de novo SNVs in SLE were enriched to promoter regions highlights the importance of using whole-genome sequencing for identification of de novo variants.
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| 10. |
- Carlsson, Henrik, et al.
(författare)
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Measurement of hydroxychloroquine in blood from SLE patients using LC-HRMS : Evaluation of whole blood, plasma and serum as sample matrices
- 2020
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Ingår i: Arthritis Research & Therapy. - : BioMed Central. - 1478-6354 .- 1478-6362. ; 22:1
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Tidskriftsartikel (refereegranskat)abstract
- Background Hydroxychloroquine (HCQ) is the standard of care in the treatment of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and other inflammatory rheumatic diseases and potentially for the treatment in COVID-19 patients. Determination of HCQ for therapeutic drug monitoring (TDM) can be performed in whole blood (WB), serum, and plasma. Direct comparisons of WB, serum, and plasma levels of HCQ in patients with SLE have not previously been reported. We describe a method for the determination of HCQ in human blood using liquid chromatography-high-resolution mass spectrometry (LC-HRMS) and compare the suitability of the three sample matrices. Methods A method for the determination of HCQ in human blood using LC-HRMS was developed, validated, and applied for the determination of HCQ levels in WB, serum, and plasma from 26 SLE patients. The reproducibility of the method, in the three matrices, was evaluated using quality control samples and repeated preparations and measurements of patient samples. The performance of the developed method for HCQ measurement in serum was further evaluated by comparison with two previously reported extraction methods. Results The performance of the presented method demonstrated high accuracy and precision. A large range of HCQ concentrations was observed for the SLE patients in all three matrices (WB, serum, and plasma). The mean levels in WB were approximately two-fold the levels in serum and plasma (813 ng/mL compared to 436 ng/mL and 362 ng/mL, respectively). Spiked quality controls showed high reproducibility for all matrices (coefficient of variation, CV, approx. 5%), whereas in patient samples, equally high-precision was only found using WB as the matrix (CV 3%). The CV for serum and plasma was 14% and 39%, respectively. Two alternative methods applied to serum samples did not demonstrate improved precision. Conclusions A LC-HRMS method for the measurement of HCQ in human blood was developed and validated. Whole blood was found to be the superior sample matrix in terms of sample reproducibility. Thus, whole blood samples should be used for HCQ analysis when patients are monitored for HCQ treatment effects. The assay is in clinical use to monitor levels of HCQ in patients.
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