Genetic studies of non-HLA loci in rheumatoid arthritis : expression and interaction of candidate genes

Genetic predisposition is an important contributor in development of human complex diseases, like rheumatoid arthritis (RA). In this thesis work, we present advances for involvement of non-HLA genetic risk factors for RA. In the same study, by using methods of genetic epidemiology and molecular genetics, we demonstrate how even moderate contribution from candidate genes could be found, interpreted and how this may affect important biological functions. The majority of the study has been performed in a large population based cohort of Swedish RA patients with matched controls and with additional cohorts from Norway, UK and the Netherlands. Data has been generated with both TaqMan allelic discrimination and DNA array-based genotyping. A subset of the cohort has been used for studying mRNA expression with quantitative PCR. Three risk loci have been investigated in this thesis: the MHC class II Transactivator (CIITA), the Dendritic Cell Immunoreceptor (DCIR) and Protein Tyrosine Phosphatase Non receptor 22 (PTPN22). For CIITA we aimed to produce further evidence for association with disease by replication and fine mapping of the locus. For DCIR and PTPN22 our aim was to examine the gene expression for finding potential regulatory differences. We present data that CIITA is a valid risk factor for RA and that this risk seems to be population specific. The risk for disease was higher in the subgroup defined by shared epitope (SE) positivity. We extensively analyzed a possible interaction effect for the risk of developing disease in four independent populations. However, no significant interaction between the CIITA and the HLA-DRB1 locus was found. When measuring expression of promoter isoforms of CIITA in cells from peripheral blood, we found that both CIITA_pIII and CIITA_pIV expression are associated with genetic variation in the locus. For DCIR we could establish that five splice forms were present in blood mononuclear cells, including a novel variant, which were down regulated upon immunostimulation. Transcript DCIR_v4 was associated with genetic variation in the locus. This correlation was similar for both RA patients and controls. Finally, we present a novel finding that the expression of PTPN22 splice forms is different for RA patients and healthy controls with more of the full-length, putatively more active, splice forms for patients and less of the alternative variant. This mixed effect was replicated in three independent cohorts. In conclusion, we present a framework for delineating genetic risk association signals by fine-mapping loci and combining with expression analysis of existing splice forms. More specifically, we give further insights for three genetic risk factors for RA that may lead to less expression of HLA class II (CIITA) and stronger inhibition of immune cell signaling (DCIR and PTPN22). A combined orchestrated effect of all this risk variants together with other risk factors known for RA may be what predisposes certain individuals for rheumatoid arthritis

The balance of expression of PTPN22 splice forms is significantly different in rheumatoid arthritis patients compared with controls

Complex disease is characterized by the interplay of multiple genetic and environmental factors. Rheumatoid arthritis (RA) is a complex autoimmune disease with a pronounced genetic component, mainly due to HLA-DRB1 gene, but also a multitude of loci outside the HLA region. In this work we strive to contribute to the understanding of the functional involvement of these susceptibility loci in the pathogenesis of RA. This study is based on a large material of whole blood samples and peripheral blood mononuclear cells (PBMCs) from RA patients and matched healthy controls from Sweden. The main methods used in this work included probe-based genotyping and gene-expression assays, cell cultures, RNA-sequencing, gene-gene interaction and pathway analysis, as well as a plethora of common molecular genetics and bioinformatics methods. We investigated the role of expression of known genetic risk factors PTPN22 and PTPN2 in RA, with a special attention to the splicing profile of these genes. Our data indicates significant differences in the expression ratio of splice variants for PTPN22 in whole blood samples from RA patients and healthy controls. For PTPN2 we demonstrate a significant difference in the relative mRNA expression of' transcript TC48 in PBMCs of healthy controls and RA patients. Additionally, we identified new susceptibility SNPs in the PTPN2 locus: rs657555 and rs11080606, by addressing the interaction of PTPN2 variants with HLA-DRB1 shared-epitope (SE) alleles in autoantibody positive RA patients in two independent cohorts. In this work, we also address the functional genetic role of the members of the MAP signaling pathway upstream of p38 and JNK – crucial enzymes in RA – with a regard to splicing profile and their connection to HLA-DRB1. We found a significant statistical interaction for rs10468473 from MAP2K4 locus with SE alleles in autoantibody-positive RA. Importantly, individuals heterozygous for rs10468473 demonstrated higher expression of total MAP2K4 mRNA in blood, compared to A-allele homozygous. We also describe a novel, putatively translated RNA splice form of MAP2K4, that is differentially expressed in peripheral blood mononuclear cells from 88 RA cases and controls, and is modulated in response to TNF in Jurkat cell line. Finally, we performed an expression analysis of multiple validated RA risk loci, and pathway analysis to assess functional relationship between RA susceptibility genes and predict new potential study candidates. New candidate molecules suggested by the pathway analysis, genes ERBB2 and HSPB1, as well as HLA-DRB1, were differentially expressed between RA patients and healthy individuals in RNA-seq data. ERBB2 expression profile was similar in whole blood of both treated and untreated patients compared to healthy individuals. A similar expression profile was replicated for ERBB2 in PBMCs in an independent material. In this work, we approached the task of elucidating the functional aspects of genetic susceptibility of RA, by integrating genetic epidemiology, transcriptomics, proteomics, cellmodels, and bioinformatics. We maintain, that such integrative approach provides the rationale to prioritize genes and genetic events for further functional studies. Our findings also outline the need to consider potential clinical significance of alternative splicing in gene expression studies

SNP Variants in Major Histocompatibility Complex Are Associate with Sarcoidosis Susceptibility - A Joint Analysis in Four European Populations

Sarcoidosis is a multiorgan inflammatory disorder with heritability estimates up to 66%. Previous studies have shown the major histocompatibility complex (MHC) region to be associated with sarcoidosis, suggesting a functional role for antigen-presenting molecules and immune mediators in the disease pathogenesis. To detect variants predisposing to sarcoidosis and to identify genetic differences between patient subgroups, we studied four genes in the MHC Class III region (LTA, TNF, AGER, BTNL2) and HLA-DRA with tag-SNPs and their relation to HLA-DRB1 alleles. We present results from a joint analysis of four study populations (Finnish, Swedish, Dutch, and Czech). Patients with sarcoidosis (n = 805) were further subdivided based on the disease activity and the presence of Lofgren's syndrome. In a joint analysis, seven SNPs were associated with non-Lofgren sarcoidosis (NL; the strongest association with rs3177928, P = 1.79E-07, OR = 1.9) and eight with Lofgren's syndrome [ Lofgren syndrome (LS); the strongest association with rs3129843, P = 3.44E-12, OR = 3.4] when compared with healthy controls (n = 870). Five SNPs were associated with sarcoidosis disease course (the strongest association with rs3177928, P = 0.003, OR = 1.9). The high linkage disequilibrium (LD) between SNPs and an HLA-DRB1 challenged the result interpretation. When the SNPs and HLA-DRB1 alleles were analyzed together, independent association was observed for four SNPs in the HLA-DRA/BTNL2 region: rs3135365 (NL; P = 0.015), rs3177928 (NL; P <0.001), rs6937545 (LS; P = 0.012), and rs5007259 (disease activity; P = 0.002). These SNPs act as expression quantitative trait loci (eQTL) for HLA-DRB1 and/or HLA-DRB5. In conclusion, we found novel SNPs in BTNL2 and HLA-DRA regions associating with sarcoidosis. Our finding further establishes that polymorphisms in the HLA-DRA and BTNL2 have a role in sarcoidosis susceptibility. This multi-population study demonstrates that at least a part of these associations are HLA-DRB1 independent (e.g., not due to LD) and shared across ancestral origins. The variants that were independent of HLA-DRB1 associations acted as eQTL for HLA-DRB1 and/or -DRB5, suggesting a role in regulating gene expression.Peer reviewe

Crispr/Cas Mediated Deletion of PTPN22 in Jurkat T Cells Enhances TCR Signaling and Production of IL-2

A single nucleotide polymorphism, C1858T, in the gene encoding the protein tyrosine phosphatase nonreceptor type 22 (PTPN22) results in one of the strongest genetic traits associated with autoimmune disease outside of the Major Histocompatibility Complex (MHC) genes. However, the consequences of this polymorphism, which introduces an arginine to tryptophan substitution at amino acid 620, for the function of PTPN22 protein is unclear and conflicting results have been obtained in human compared to mouse cells expressing this variant phosphatase. In mouse the variant appears to be a loss-of-function allele resembling a milder form of the null allele, while studies in human cells have reported it to be a gain-of-function mutation. To address whether the phosphatase has distinct functions in mouse vs. human T cells, we used CRISPR gene-editing to generate the first example of human PTPN22-KnockOut (KO) T cells. By comparing isogenic human T cells which express or lack PTPN22, we showed that PTPN22 KO T cells displayed enhanced expression of IL-2 and CD69 upon stimulation with cognate antigen. PTPN22 KO cells also showed increased Erk phosphorylation upon stimulation with weak antigen, but the difference was diminished in response to strong antigen, indicating that PTPN22 plays a more critical role in regulating weak-antigen responses. These data are in keeping with a role for PTPN22 in determining the threshold of stimulation required to activate T cells, a critical function of autoimmune pathogenesis. Our data indicate that PTPN22 has comparable functions in mouse and human T cells, and that the conflicting results in the literature regarding the impact of the point mutation are not due to differences in the activity of PTPN22 itself, but may be related to interactions with other proteins or splice variation

Interaction Analysis between HLA-DRB1 Shared Epitope Alleles and MHC Class II Transactivator CIITA Gene with Regard to Risk of Rheumatoid Arthritis

Abstract HLA-DRB1 shared epitope (SE) alleles are the strongest genetic determinants for autoantibody positive rheumatoid arthritis (RA). One of the key regulators in expression of HLA class II receptors is MHC class II transactivator (CIITA). A variant of the CIITA gene has been found to associate with inflammatory diseases. We wanted to explore whether the risk variant rs3087456 in the CIITA gene interacts with the HLA-DRB1 SE alleles regarding the risk of developing RA. We tested this hypothesis in a case-control study with 11767 individuals from four European Caucasian populations (6649 RA cases and 5118 controls). We found no significant additive interaction for risk alleles among Swedish Caucasians with RA (n = 3869, attributable proportion due to interaction (AP) = 0.2, 95%CI: 20.2-0.5) or when stratifying for anti-citrullinated protein antibodies (ACPA) presence (ACPA positive disease: n = 2945, AP = 0.3, 95%CI: 20.05-0.6, ACPA negative: n = 2268, AP = 20.2, 95%CI: 21.0-0.6). We further found no significant interaction between the main subgroups of SE alleles (DRB1*01, DRB1*04 or DRB1*10) and CIITA. Similar analysis of three independent RA cohorts from British, Dutch and Norwegian populations also indicated an absence of significant interaction between genetic variants in CIITA and SE alleles with regard to RA risk. Our data suggest that risk from the CIITA locus is independent of the major risk for RA from HLA-DRB1 SE alleles, given that no significant interaction between rs3087456 and SE alleles was observed. Since a biological link between products of these genes is evident, the genetic contribution from CIITA and class II antigens in the autoimmune process may involve additional unidentified factors

Protein tyrosine phosphatase non-receptor 22 and C-Src tyrosine kinase genes are down-regulated in patients with rheumatoid arthritis

Several protein tyrosine phosphatase non-receptor 22 (PTPN22) single-nucleotide polymorphisms (SNPs) have been significantly related with rheumatoid arthritis (RA) susceptibility. Nevertheless, its potential influence on PTPN22 expression in RA has not been completely elucidated. Furthermore, PTPN22 binds to C-Src tyrosine kinase (CSK) forming a key complex in autoimmunity. However, the information of CSK gene in RA is scarce. In this study, we analyzed the relative PTPN22 and CSK expression in peripheral blood from 89 RA patients and 43 controls to determine if the most relevant PTPN22 (rs2488457, rs2476601 and rs33996649) and CSK (rs34933034 and rs1378942) polymorphisms may influence on PTPN22 and CSK expression in RA. The association between PTPN22 and CSK expression in RA patients and their clinical characteristics was also evaluated. Our study shows for the first time a marked down-regulation of PTPN22 expression in RA patients carrying the risk alleles of PTPN22 rs2488457 and rs2476601 compared to controls (p?=?0.004 and p?=?0.007, respectively). Furthermore, CSK expression was significantly lower in RA patients than in controls (p?<?0.0001). Interestingly, a reduced PTPN22 expression was disclosed in RA patients with ischemic heart disease (p?=?0.009). The transcriptional suppression of this PTPN22/CSK complex may have a noteworthy clinical relevance in RA patients

Interaction Analysis between HLA-DRB1 Shared Epitope Alleles and MHC Class II Transactivator CIITA Gene with Regard to Risk of Rheumatoid Arthritis

HLA-DRB1 shared epitope (SE) alleles are the strongest genetic determinants for autoantibody positive rheumatoid arthritis (RA). One of the key regulators in expression of HLA class II receptors is MHC class II transactivator (CIITA). A variant of the CIITA gene has been found to associate with inflammatory diseases