Marte K. Viken

Association analysis of the 1858C>T polymorphism in the PTPN22 gene in juvenile idiopathic arthritis and other autoimmune diseases.

A functional single nucleotide polymorphism, 1858C>T, in the PTPN22 gene, encoding a tyrosine phosphatase, has been reported to be associated with type I diabetes and some other autoimmune diseases. To further investigate whether this polymorphism may be a general susceptibility factor for autoimmunity, we performed an association study in five different autoimmune diseases, three previously not tested. We found an association with juvenile idiopathic arthritis (OR=1.41; P=0.04), not previously reported, and a tendency for an association with coeliac disease (OR=1.35; P=0.08). In primary sclerosing cholangitis, no association was observed (OR=0.95; P=0.8). Furthermore, we confirmed the increased risk in rheumatoid arthritis (OR=1.58; P=0.001), but could not find support for an association with systemic lupus erythematosus (OR=0.94; P=0.8). Altogether, we have provided further evidence of an association between autoimmune diseases and the 1858C>T polymorphism in PTPN22.

Association analysis of the interleukin 17A gene in Caucasian rheumatoid arthritis patients from Norway and New Zealand

OBJECTIVE Elevated levels of IL-17A have been detected in the inflamed synovium of RA patients, and murine arthritis models deficient in IL17A have shown reduced inflammation. Our aim was to investigate IL17A as a candidate gene for RA, and to assess correlations between risk variants and disease phenotypes. METHODS Five single nucleotide polymorphisms (SNPs) were selected to tag the genetic variability of the IL17A region and were genotyped by TaqMan technology on 950 RA cases and 933 random controls from Norway. Associations to progression of radiographic damage and presence of autoantibodies were examined in a 10-yr follow-up cohort of early RA. In addition, 580 RA patients and 504 controls from New Zealand were used as a replication data set. RESULTS A weak association between RA and the promoter SNP rs2275913 [odds ratio (OR) = 1.17; 95% CI 1.02, 1.34; P = 0.02] was found in the Norwegian population. The association was also evident at the genotype level where it indicated a recessive model. The allelic association was not replicated in the RA cohort from New Zealand (OR = 0.96; 95% CI 0.81, 1.16; P = 0.69). However, combined analysis suggested a weak recessive association (OR = 1.19; 95% CI 1.02, 1.37; P = 0.02). No significant associations were observed with radiographic progression, anti-cyclic citrullinated peptide or IgM-RF. CONCLUSIONS Modest evidence of an association with IL17A in Norwegian RA patients was observed. Although, our findings were not replicated in an independent RA material from New Zealand, a significant common risk estimate indicated that IL17A warrants further investigation in RA.

Seed 1-Cysteine Peroxiredoxin Antioxidants Are Not Involved in Dormancy, But Contribute to Inhibition of Germination during Stress

Peroxiredoxins (Prx) are thiol-dependent antioxidants containing one (1-cysteine [-Cys]) or two (2-Cys) conserved Cys residues that protect lipids, enzymes, and DNA against reactive oxygen species. In plants, the 1-Cys Prxs are highly expressed during late seed development, and the expression pattern is dormancy related in mature seeds. We have expressed the Arabidopsis 1-Cys Prx AtPER1 in Escherichia coli and show that this protein has antioxidant activity in vitro and protects E. coli in vivo against the toxic oxidant cumene hydroperoxide. Although some 1-Cys Prxs are targeted to the nucleus, a green fluorescent protein-AtPER1 fusion protein was also localized to the cytoplasm in an onion epidermis subcellular localization assay. It has been proposed that seed Prxs are involved in maintenance of dormancy and/or protect the embryo and aleurone layer surviving desiccation against damage caused by reactive oxygen species. These hypotheses were tested using transgenic Arabidopsis lines overexpressing the barley (Hordeum vulgare) 1-Cys PER1 protein and lines with reduced levels of AtPER1 due to antisensing or RNA interference. We found no correlation between Prx levels and the duration of the after-ripening period required before germination. Thus, Prxs are unlikely to contribute to maintenance of dormancy. RNA interference lines almost devoid of AtPER1 protein developed and germinated normally under standard growth room conditions. However, seeds from lines overexpressing PER1 were less inclined to germinate than wild-type seeds in the presence of NaCl, mannitol, and methyl viologen, suggesting that Prx can sense harsh environmental surroundings and play a part in the inhibition of germination under unfavorable conditions.

Dominant Mutations in the Autoimmune Regulator AIRE Are Associated with Common Organ-Specific Autoimmune Diseases.

The autoimmune regulator (AIRE) gene is crucial for establishing central immunological tolerance and preventing autoimmunity. Mutations in AIRE cause a rare autosomal-recessive disease, autoimmune polyendocrine syndrome type 1 (APS-1), distinguished by multi-organ autoimmunity. We have identified multiple cases and families with mono-allelic mutations in the first plant homeodomain (PHD1) zinc finger of AIRE that followed dominant inheritance, typically characterized by later onset, milder phenotypes, and reduced penetrance compared to classical APS-1. These missense PHD1 mutations suppressed gene expression driven by wild-type AIRE in a dominant-negative manner, unlike CARD or truncated AIRE mutants that lacked such dominant capacity. Exome array analysis revealed that the PHD1 dominant mutants were found with relatively high frequency (>0.0008) in mixed populations. Our results provide insight into the molecular action of AIRE and demonstrate that disease-causing mutations in the AIRE locus are more common than previously appreciated and cause more variable autoimmune phenotypes.

Late Onset Myasthenia Gravis Is Associated with HLA DRB1*15:01 in the Norwegian Population

Background Acquired myasthenia gravis (MG) is a rare antibody-mediated autoimmune disease caused by impaired neuromuscular transmission, leading to abnormal muscle fatigability. The aetiology is complex, including genetic risk factors of the human leukocyte antigen (HLA) complex and unknown environmental factors. Although associations between the HLA complex and MG are well established, not all involved components of the HLA predisposition to this heterogeneous disease have been revealed. Well-powered and comprehensive HLA analyses of subgroups in MG are warranted, especially in late onset MG. Methodology/Principal Findings This case-control association study is of a large population-based Norwegian cohort of 369 MG patients and 651 healthy controls. We performed comprehensive genotyping of four classical HLA loci (HLA-A, -B, -C and -DRB1) and showed that the DRB1*15:01 allele conferred the strongest risk in late onset MG (LOMG; onset ≥60years) (OR 2.38, pc7.4×10−5). DRB1*13:01 was found to be a protective allele for both early onset MG (EOMG) and LOMG (OR 0.31, pc 4.71×10−4), a finding not previously described. No significant association was found to the DRB1*07:01 allele (pnc = 0.18) in a subset of nonthymomatous anti-titin antibody positive LOMG as reported by others. HLA-B*08 was mapped to give the strongest contribution to EOMG, supporting previous studies. Conclusion The results from this study provide important new information concerning the susceptibility of HLA alleles in Caucasian MG, with highlights on DRB1*15:01 as being a major risk allele in LOMG.

ABI3 mediates expression of the peroxiredoxin antioxidant AtPER1 gene and induction by oxidative stress

The peroxiredoxin antioxidant gene AtPER1 has been considered to be specifically expressed in the embryo and aleurone layer during maturation and desiccation stages of development, and in the mature seed, typically for late embryogenesis-abundant (lea) transcripts. In the abscisic acid-insensitive abi3-1 mutant, the AtPER1 transcript level is strongly reduced, suggesting ABI3 to be a prime regulator of AtPER1. We have studied the expression pattern and regulation of AtPER1 with a series of nine promoter::GUS constructs with deletions and/or mutations in putative regulatory elements. Arabidopsis lines harbouring these constructs revealed AtPER1 promoter activity in the endosperm, especially the chalazal cyst, already when the embryo is in the late globular stage, in the embryo from the late torpedo stage, and in distinct cells of unfertilized and fertilized ovules. Early expression seems to be dependent on a putative antioxidant-responsive promoter element (ARE), while from the bent cotyledon stage endosperm and embryo expression is dependent on an ABA-responsive element (ABRE) likely to bind ABI5. The shortest promoter fragment (113 bp), devoid of ARE, ABRE and without an intact RY/Sph element thought to bind ABI3 did not drive GUS expression. The AtPER1::GUSconstruct also revealed expression in cotyledons, meristems and stem branching points. In general, seed and vegetative expression coincided with the expression pattern of ABI3. In plants ectopically expressing ABI3, AtPER1::GUS expression was found in true leaves, and AtPER1 could be induced by exogenous ABA and oxidative stress (H2O2 and hydroquinone). ABI3-mediated oxidative stress induction was dependent on the presence of an intact ARE element.

Associations between the PTPN22 1858C→T polymorphism and radiographic joint destruction in patients with rheumatoid arthritis: results from a 10‐year longitudinal study

Objective: To investigate whether the PTPN22 1858T risk variant is associated with the rate of radiographic progression in rheumatoid arthritis (RA). Methods: A longitudinally followed cohort of 238 Norwegian patients with RA (the EURIDISS cohort) was genotyped for the PTPN22 1858C→T polymorphism. Radiographic damage was assessed by hand radiographs at baseline and after 1, 2, 5 and 10 years, and the radiographs were scored with the Sharp method modified by van der Heijde (Sharp–van der Heijde score) by a single experienced reader. Baseline serum levels of rheumatoid factor and anti-cyclic citrullinated peptide autoantibodies were also examined. Results: The reported association between RA susceptibility and carriage of the T allele (34.4% in patients vs 21.4% in controls; odds ratio 1.92, 95% confidence interval 1.36 to 2.71, p = 0.0002) was confirmed. An association between annual progression rate of Sharp–van der Heijde score and T-allele carriers (p = 0.01),was also found, which was also present when only patients positive for the shared epitope were analysed (p = 0.03). This association was also maintained in multivariate analyses adjusting for shared epitope and demographic variables. Conclusions: An association between the PTPN22 risk variant and increased progression rate for structural damage was found. The results indicate that the PTPN22 gene may not only be associated with disease susceptibility, but also with disease progression.

Reproducible association with type 1 diabetes in the extended class I region of the major histocompatibility complex

The high-risk human leukocyte antigen (HLA)-DRB1, DQA1 and DQB1 alleles cannot explain the entire type 1 diabetes (T1D) association observed within the extended major histocompatibility complex. We have earlier identified an association with D6S2223, located 2.3 Mb telomeric of HLA-A, on the DRB1*03-DQA1*0501-DQB1*0201 haplotype, and this study aimed to fine-map the associated region also on the DRB1*0401-DQA1*03-DQB1*0302 haplotype, characterized by less extensive linkage disequilibrium. To exclude associations secondary to DRB1-DQA1-DQB1 haplotypes, 205 families with at least one parent homozygous for these loci, were genotyped for 137 polymorphisms. We found novel associations on the DRB1*0401-DQA1*03-DQB1*0302 haplotypic background with eight single nucleotide polymorphisms (SNPs) located within or near the PRSS16 gene. In addition, association at the butyrophilin (BTN)-gene cluster, particularly the BTN3A2 gene, was observed by multilocus analyses. We replicated the associations with SNPs in the PRSS16 region and, albeit weaker, to the BTN3A2 region, in an independent material of 725 families obtained from the Type 1 Diabetes Genetics Consortium. It is important to note that these associations were independent of the HLA-DRB1-DQA1-DQB1 genes, as well as of associations observed at HLA-A, -B and -C. Taken together, our results identify PRSS16 and BTN3A2, two genes thought to play important roles in regulating the immune response, as potentially novel susceptibility genes for T1D.

Exploring the CLEC16A gene reveals a MS-associated variant with correlation to the relative expression of CLEC16A isoforms in thymus.

Genomewide association studies have implicated the CLEC16A gene in several autoimmune diseases, including multiple sclerosis (MS) and type 1 diabetes. However, the most associated single-nucleotide polymorphism (SNP) varies, and causal variants are still to be defined. In MS, two SNPs in partial linkage disequilibrium with each other, rs6498169 and rs12708716, have been validated at genomewide significance level. To explore the CLEC16A association in MS in more detail, we genotyped 57 SNPs in 807 Norwegian MS patients and 1027 Norwegian controls. Six highly associated SNPs emerged and were then replicated in two large independent sample sets (Norwegian and British), together including 1153 MS trios, 2308 MS patients and 4044 healthy controls. In combined analyses, SNP rs12708716 gave the strongest association signal in MS (P=5.3 × 10−8, odds ratio 1.18, 95% confidence interval=1.11–1.25), and was found to be superior to the other SNP associations in conditional logistic regression analyses. Expression analysis revealed that rs12708716 genotype was significantly associated with the relative expression levels of two different CLEC16A transcripts in thymus (P=0.004), but not in blood, possibly implying a thymus- or cell-specific splice regulation.

High-throughput T-cell receptor sequencing across chronic liver diseases reveals distinct disease-associated repertoires.

Hepatic T‐cell infiltrates and a strong genetic human leukocyte antigen association represent characteristic features of various immune‐mediated liver diseases. Conceptually the presence of disease‐associated antigens is predicted to be reflected in T‐cell receptor (TCR) repertoires. Here, we aimed to determine if disease‐associated TCRs could be identified in the nonviral chronic liver diseases primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), and alcoholic liver disease (ALD). We performed high‐throughput sequencing of the TCRβ chain complementarity‐determining region 3 of liver‐infiltrating T cells from PSC (n = 20), PBC (n = 10), and ALD (n = 10) patients, alongside genomic human leukocyte antigen typing. The frequency of TCRβ nucleotide sequences was significantly higher in PSC samples (2.53 ± 0.80, mean ± standard error of the mean) compared to PBC samples (1.13 ± 0.17, P < 0.0001) and ALD samples (0.62 ± 0.10, P < 0.0001). An average clonotype overlap of 0.85% was detected among PSC samples, significantly higher compared to the average overlap of 0.77% seen within the PBC (P = 0.024) and ALD groups (0.40%, P < 0.0001). From eight to 42 clonotypes were uniquely detected in each of the three disease groups (≥30% of the respective patient samples). Multiple, unique sequences using different variable family genes encoded the same amino acid clonotypes, providing additional support for antigen‐driven selection. In PSC and PBC, disease‐associated clonotypes were detected among patients with human leukocyte antigen susceptibility alleles. Conclusion: We demonstrate liver‐infiltrating disease–associated clonotypes in all three diseases evaluated, and evidence for antigen‐driven clonal expansions. Our findings indicate that differential TCR signatures, as determined by high‐throughput sequencing, may represent an imprint of distinctive antigenic repertoires present in the different chronic liver diseases; this thereby opens up the prospect of studying disease‐relevant T cells in order to better understand and treat liver disease. (Hepatology 2016;63:1608‐1619)