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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

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.

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.

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.

Background Type 1 diabetes (T1D) and celiac disease (CeD) are 2 distinct diseases, but there is an increased risk of developing CeD for T1D patients. Both diseases are associated with HLA-class II alleles, such as DQB1 *02:01 and DQB1 *03:02; however, their risk contribution vary between the diseases. Materials and Methods We genotyped HLA-DRB1 and – DQB1 in 215 patients with coexisting T1D and CeD identified from a T1D cohort, and compared them to patients with T1D (N = 487) and CeD (N = 327), as well as healthy controls (N = 368). Results The patients with coexisting T1D and CeD had an intermediate carrier frequency (72.8%) of the DRB1 *03:01- DQB1 *02:01- DQA1 *05:01 haplotype compared to T1D (64.1%) and CeD (88.7%) patients. The DRB1 *03:01- DQB1 *02:01- DQA1 *05:01/ DRB1 *04- DQB1 *03:02- DQA1 *03 haplotype combination, encoding DQ2.5 and DQ8 molecules, was equally frequent among patients with both T1D and CeD (52.6%) and T1D patients (46.8%) but significantly lower in CeD patients (9.5%). Conclusion Overall, the patients with coexisting T1D and CeD had an HLA profile more similar to T1D patients than CeD patients.

Genetic polymorphisms in the endoplasmic reticulum aminopeptidase (ERAP)1 and ERAP2 genes have been associated with several autoimmune diseases (AIDs) at a genome-wide significance level. In this study, we performed a cis expression quantitative trait locus (eQTL) screen to investigate whether seven fine-mapped AID single-nucleotide polymorphisms (SNPs) in the ERAP-region influence the gene-expression levels of ERAP1 and ERAP2 in thymus. After quality control, we identified six significant eQTLs. We further assessed the peak eQTL signals, and both genes showed highly significant and independent thymic eQTL signals (P=2.16 × 10-15 and P=8.22 × 10-23, respectively). Interestingly, the peak eQTL signal overlapped with the AID risk loci in ERAP2 (r2>0.94), but were distinct in ERAP1 (r2

We aimed to jointly investigate the role of antipeptidyl arginine deiminase type 4 antibodies (anti-PAD4) and polymorphisms in theSerum IgG autoantibodies to human recombinant PAD4 were identified by DELFIA technique in 745 patients with RA (366 available from previous studies). Genotyping ofAnti-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,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 The Norwegian nationwide MCTD cohort was established to obtain unbiased data on key disease issues, and thereby reappraise the concept of MCTD. In the current study, the aims were to obtain detailed HLA profile data on the large Norwegian MCTD cohort and compare these with the HLA profiles of ethnically matched healthy controls and related CTD controls. METHODS HLA profiles, determined by sequence-based typing of HLA-B* and DRB1*, were compared between four control groups of Norwegian ancestry, SLE (n = 96), SSc (n = 95), PM/DM (n = 84), healthy individuals (n = 282), the complete MCTD cohort (n = 155) and MCTD subsets defined by key clinical parameters. RESULTS HLA-B*08 [odds ratio (OR) 2.05, P = 1.31 × 10(-4)) and DRB1*04:01 (OR 2.82, P = 3.64 × 10(-8)) were identified as risk alleles for MCTD, while DRB1*04:04, DRB1*13:01 and DRB1*13:02 were protective. Risk alleles for SLE and PM/DM were B*08 and DRB1*03:01. SSc risk was associated with DRB1*08:01. Analyses of MCTD subsets identified B*18 [OR 3.32 (95% CI 1.38, 8.01)] and DRB1*03:01 [OR 1.83 (95% CI 1.03, 3.25)] as independent risk factors for lung fibrosis. CONCLUSION Novel HLA alleles associated with MCTD and disease subsets were identified and DRB1*04:01 was confirmed as a major risk allele. Altogether, the data reinforce the notion of MCTD as a disease entity distinct from SLE, SSc and PM/DM.

Antigen presenting cells (APCs) in the thymus play an essential role in the establishment of central tolerance, i.e. the generation of a repertoire of functional and self-tolerant T cells to prevent autoimmunity. In this study, we have compared the transcriptomes of four primary APCs from human thymus (mTECs, CD19+ B cells, CD141+ and CD123+ DCs). We investigated a set of genes including the HLA genes, genes encoding transcriptional regulators and finally, tissue-enriched genes, i.e, genes with a five-fold higher expression in a particular human tissue. We show that thymic CD141+ DCs express the highest levels of all classical HLA genes and 67% (14/21) of the HLA class I and II pathway genes investigated in this study. CD141+ DCs also expressed the highest levels of the transcriptional regulator DEAF1, whereas AIRE and FEZF2 expression were mainly found in primary human mTECs. We found expression of “tissue enriched genes” from the Human Protein Atlas (HPA) in all four APC types, but the mTECs were clearly dominating in the number of uniquely expressed tissue enriched genes (20% in mTECs, 7% in CD19+ B cells, 4% in CD123+ DCs and 2% in CD141+ DCs). The tissue enriched genes also overlapped with reported human autoantigens. This is, to our knowledge, the first study that performs RNA sequencing of mTECs, CD19+ B cells, CD141+ and CD123+ DCs isolated from the same individuals and provides insight into the transcriptomes of these human thymic APCs.

Genome-wide association studies (GWAS) have boosted our knowledge of genetic risk variants in autoimmune diseases (AIDs). Most risk variants are located within or near genes with immunological functions, and the majority is found to be non-coding, pointing towards a regulatory role. In this study, we performed a cis expression quantitative trait locus (eQTL) screen restricted to 353 AID associated risk variants selected from the GWAS catalog to investigate whether these single nucleotide polymorphisms (SNPs) influence gene expression in thymus. Genotypes were obtained by Immunochip (Ichip) and tested against expression of surrounding genes (±1 Mb) in human thymic tissue (n = 42). We identified eight significant eQTLs located within seven genetic regions (FCRL3, RNASET2, C2orf74, NPIPB8, SIRPG, SYS1 and AJ006998.2) where the expression was associated with AID risk SNPs at a study-wide level of significance (P < 2.7 × 10-5). In NPIPB8 and AJ006998.2, the eQTL signals appeared to be thymus-specific. Furthermore, many AID risk SNPs from GWAS have been subsequently fine-mapped in recent Ichip projects, and fine-mapped AID SNPs overlapped with the thymic eQTLs within RNASET2 and SIRPG Finally, in all the eQTL regions, except C2orf74, SNPs underlying the thymic eQTLs were predicted to interfere with transcription factors important in T cell development. Our study therefore reveals autoimmune risk variants that act as eQTLs in thymus, and suggest that thymic gene regulation may play a functional role at some AID risk loci.

Available evidence suggests a Polynesian origin of the Easter Island population. We recently found that some native Easter Islanders also carried some common American Indian (Amerindian) human leukocyte antigen (HLA) alleles, which probably were introduced before Europeans discovered the island in 1722. In this study, we report molecular genetic investigations of 21 other selected native Easter Islanders. Analysis of mitochondrial DNA and Y chromosome markers showed no traces of an Amerindian contribution. However, high-resolution genomic HLA typing showed that two individuals carried some other common Amerindian HLA alleles, different from those found in our previous investigations. The new data support our previous evidence of an Amerindian contribution to the gene pool on Easter Island.

This study reports extensive genomic data for both human leukocyte antigen (HLA) class I and II loci in Norwegian Sami, a native population living in the northwest of Europe. The Sami have a distinct culture and their own languages, which belong to the Uralic linguistic family. Norwegian Sami (n = 200) were typed at the DNA level for the HLA-A, -C, -B, -DRB1 and -DQB1 loci, and compared with a non- Sami Norwegian population (n = 576). The two populations exhibited some common genetic features but also differed significantly at all HLA loci. The most significantly deviating allele frequencies were an increase of HLA-A*03, -B*27, -DRB1*08 and -DQB1*04 and a decrease of HLA-A*01, C*01, -DRB1*04 and -DQB1*02 among Sami compared with non-Sami Norwegians. The Sami showed no deviation from Hardy–Weinberg equilibrium. The hypothesis of selective neutrality was rejected at all loci except for the A- and C- loci for the Sami. HLA haplotype frequencies also differed between the two populations. The most common extended HLA haplotypes were A*02-B*27-C*01-DR*08-DQB1*04 in the Sami and A*01-B*08-C*07-DR*03- DQB1*02 in the other Norwegians. Genetic distance analyses indicated that theNorwegian Sami were highly differentiated from other Europeans and were most closely related to Finns whose language also belongs to the Uralic linguistic family. In conclusion, the Norwegian Sami and the non-Sami Norwegians were significantly different at all HLA loci. Our results can be explained by the fact that the two populations have different origins and that the Sami population has remained smaller and more isolated than its neighbors.

We have previously mapped a separate type 1 diabetes (T1D) association in the extended MHC class I region, marked by D6S2223, on the DRB1*03-DQA1*0501-DQB1*0201 haplotype. The associated region encompasses a gene encoding a thymus-specific serine protease (PRSS16), presumably involved in positive selection of T cells or in T-cell regulation. Fourteen PRSS16 polymorphisms were genotyped in two steps using a total of six T1D family data sets, as well as case-control materials for both T1D and celiac disease (CD). An association with a 15 base-pair deletion in exon 12 of PRSS16 was found on the DRB1*03-DQA1*0501-DQB1*0201 haplotype for both T1D and CD, but it could not explain the more pronounced disease associations observed at marker D6S2223. We compared the performance of the 14 tested PRSS16 polymorphisms, selected after our previous comprehensive screen, against HapMap selected tag SNPs. Use of a HapMap based SNP selection strategy would result in loss of a large proportion of the genetic variation in PRSS16. Our data suggest that it is unlikely that polymorphisms within the PRSS16 gene are involved in the predisposition to T1D. However, we cannot rule out that regulatory polymorphisms located some distance away from the gene may be involved.

BackgroundAcquired 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 findingsThis 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 ≥ 60 years) (OR 2.38, p(c)7.4 × 10(-5)). DRB1*13:01 was found to be a protective allele for both early onset MG (EOMG) and LOMG (OR 0.31, p(c) 4.71 × 10(-4)), a finding not previously described. No significant association was found to the DRB1*07:01 allele (p(nc) = 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.ConclusionThe 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.

The protein tyrosine phosphatase nonreceptor 22 (PTPN22) gene has, during the last 2 years, been recognized as a susceptibility gene for numerous autoimmune diseases, including rheumatoid arthritis (RA) and type 1 diabetes. An association between the exonic 1858C>T single nucleotide polymorphism (SNP) and RA has repeatedly been replicated in several Caucasian populations. The SNP is not associated with autoimmune diseases in Asian populations, as the 1858T allele is almost absent. Recently, a promoter polymorphism -1123G>C was proposed to be associated with acute-onset type 1 diabetes in Japanese and Korean populations. Furthermore, in Caucasian populations, the presence of additional PTPN22 risk variants has been suggested, indicating that the 1858C>T risk variant cannot explain the entire disease association observed in the region. In this study, we wanted to jointly address and integrate these separate findings to further elucidate the association between the PTPN22 gene and RA in a Norwegian material of 861 RA patients and 559 healthy controls. Our results revealed that the strength of the association with the PTPN22 promoter polymorphism, -1123G>C, is analogous to that observed for 1858C>T. As the -1123G>C variant is also polymorphic in Asian populations, our data underpin the need to further explore the association between this variant and autoimmune diseases in different populations.

Summary Background Rapa Nui (Easter Island), located in the easternmost corner of the Polynesian Triangle, is one of the most isolated locations on the planet inhabited by humans. Archaeological and genetic evidence suggests that the island was first colonized by Polynesians around AD 1200, during their eastward expansion. Although it remains contentious whether Polynesians reached South America, suggestive evidence has been brought forward supporting the possibility of Native American contact prior to the European "discovery" of the island in AD 1722. Results We generated genome-wide data for 27 Rapanui. We found a mostly Polynesian ancestry among Rapanui and detected genome-wide patterns consistent with Native American and European admixture. By considering the distribution of local ancestry tracts of eight unrelated Rapanui, we found statistical support for Native American admixture dating to AD 1280–1495 and European admixture dating to AD 1850–1895. Conclusions These genetic results can be explained by one or more pre-European trans-Pacific contacts.