Next generation sequencing of 11 HLA loci characterises a diverse UK cord blood bank.

The introduction of next generation sequencing (NGS) for stem cell donor registry typing has contributed to faster identification of compatible stem cell donors. However, the successful search for a matched unrelated donor for some patient groups is still affected by their ethnicity. In this study, DNA samples from 714 National Health Service (NHS) Cord Blood Bank donors were typed for HLA-A, -B, -C, -DRB1, -DRB345, -DQA1, -DQB1, -DPA1 and -DPB1 by NGS. Analysis of the ethnic diversity showed a high level of diversity, with the cohort comprising of 62.3% European and 37.7% of either multi-ethnic or non-European donors, of which 12.3% were multi-ethnic. The HLA diversity was further confirmed using PyPop analysis, 405 distinct alleles were observed in the overall NHS-CBB cohort, of which 37 alleles are non-CWD, including A*31:14N, B*35:68:02, C*14:23 and DQA1*05:10. Furthermore, HLA-DQA1 and HLA-DPA1 analysis showed 12% and 10%, respectively, of the alleles currently submitted to IMGT, confirming further diversity of the NHS-CBB cohort. The application of 11 HLA loci resolution by NGS revealed a high level of diversity in the NHS-CBB cohort. The incorporation of this data coupled with ethnicity data could lead to improved donor selection, contributing to better clinical outcomes for patients.

Analysis of Anti-RNA Polymerase III Antibody-positive Systemic Sclerosis and Altered GPATCH2L and CTNND2 Expression in Scleroderma Renal Crisis.

OBJECTIVE: Scleroderma renal crisis (SRC) is a life-threatening complication of systemic sclerosis (SSc) strongly associated with anti-RNA polymerase III antibody (ARA) autoantibodies. We investigated genetic susceptibility and altered protein expression in renal biopsy specimens in ARA-positive patients with SRC. METHODS: ARA-positive patients (n = 99) with at least 5 years' follow-up (49% with a history of SRC) were selected from a well characterized SSc cohort (n = 2254). Cases were genotyped using the Illumina Human Omni-express chip. Based on initial regression analysis, 9 single-nucleotide polymorphisms (SNP) were chosen for validation in a separate cohort of 256 ARA-positive patients (40 with SRC). Immunostaining of tissue sections from SRC or control kidney was used to quantify expression of candidate proteins based upon genetic analysis of the discovery cohort. RESULTS: Analysis of 641,489 SNP suggested association of POU2F1 (rs2093658; P = 1.98 × 10-5), CTNND2 (rs1859082; P = 5.58 × 10-5), HECW2 (rs16849716; P = 1.2 × 10-4), and GPATCH2L (rs935332; P = 4.92 × 10-5) with SRC. Further, the validation cohort showed an association between rs935332 within the GPATCH2L region, with SRC (P = 0.025). Immunostaining of renal biopsy sections showed increased tubular expression of GPATCH2L (P = 0.026) and glomerular expression of CTNND2 (P = 0.026) in SRC samples (n = 8) compared with normal human kidney controls (n = 8), despite absence of any genetic replication for the associated SNP. CONCLUSION: Increased expression of 2 candidate proteins, GPATCH2L and CTNND2, in SRC compared with control kidney suggests a potential role in pathogenesis of SRC. For GPATCH2L, this may reflect genetic susceptibility in ARA-positive patients with SSc based upon 2 independent cohorts.

Next-generation HLA typing of 382 International Histocompatibility Working Group reference B-lymphoblastoid cell lines: Report from the 17th International HLA and Immunogenetics Workshop.

Extended molecular characterization of HLA genes in the IHWG reference B-lymphoblastoid cell lines (B-LCLs) was one of the major goals for the 17th International HLA and Immunogenetics Workshop (IHIW). Although reference B-LCLs have been examined extensively in previous workshops complete high-resolution typing was not completed for all the classical class I and class II HLA genes. To address this, we conducted a single-blind study where select panels of B-LCL genomic DNA samples were distributed to multiple laboratories for HLA genotyping by next-generation sequencing methods. Identical cell panels comprised of 24 and 346 samples were distributed and typed by at least four laboratories in order to derive accurate consensus HLA genotypes. Overall concordance rates calculated at both 2- and 4-field allele-level resolutions ranged from 90.4% to 100%. Concordance for the class I genes ranged from 91.7 to 100%, whereas concordance for class II genes was variable; the lowest observed at HLA-DRB3 (84.2%). At the maximum allele-resolution 78 B-LCLs were defined as homozygous for all 11 loci. We identified 11 novel exon polymorphisms in the entire cell panel. A comparison of the B-LCLs NGS HLA genotypes with the HLA genotypes catalogued in the IPD-IMGT/HLA Database Cell Repository, revealed an overall allele match at 68.4%. Typing discrepancies between the two datasets were mostly due to the lower-resolution historical typing methods resulting in incomplete HLA genotypes for some samples listed in the IPD-IMGT/HLA Database Cell Repository. Our approach of multiple-laboratory NGS HLA typing of the B-LCLs has provided accurate genotyping data. The data generated by the tremendous collaborative efforts of the 17th IHIW participants is useful for updating the current cell and sequence databases and will be a valuable resource for future studies.

Evaluation of Ion Torrent sequencing technology for rapid clinical human leucocyte antigen typing.

The development of techniques to define the human leucocyte antigen (HLA) region has proven to be challenging due to its high level of polymorphism. Within a clinical laboratory, a technique for high-resolution HLA typing, which is rapid and cost effective is essential. NGS has provided a rapid, high-resolution HLA typing solution, which has reduced the number of HLA ambiguities seen with other typing methods. In this study, the One Lambda NXType NGS kit was tested on the Ion Torrent PGM platform. A total of 362 registry donors from four ethnic populations (Europeans, South Asians, Africans and Chinese) were NGS HLA typed across 9-loci (HLA-A, -B, -C, -DRB1,-DRB345 -DQB1 and -DPB1). Concordance rates of 91%-98% were obtained (for HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1) when compared to historical PCR-SSO HLA types, and the identification of uncommon alleles such as A*24:07:01 and C*04:82 were observed. A turnaround time of four days was achieved for typing 44 samples. However, some limitations were observed; primer locations did not allow all ambiguities to be resolved for HLA Class II where Exon I and IV amplification are needed (HLA-DRB1*04:07:01/04:92, HLA-DRB1*09:01:02/*09:21 and HLA-DRB1*12:01:01/*12:10). This study has demonstrated high-resolution typing by NGS can be achieved in an acceptable turnaround time for a clinical laboratory; however, the Ion Torrent workflow has some technical limitations that should be addressed.

Molecular Basis for Dysregulated Activation of NKX2-5 in the Vascular Remodeling of Systemic Sclerosis.

OBJECTIVE: NKX2-5 is a homeobox transcription factor that is required for the formation of the heart and vessels during development, with significant postnatal down-regulation and reactivation in disease states, characterized by vascular remodeling. The purpose of this study was to investigate mechanisms that activate NKX2-5 expression in diseased vessels, such as systemic sclerosis (scleroderma; SSc)-associated pulmonary hypertension (PH), and to identify genetic variability that potentially underlies susceptibility to specific vascular complications. METHODS: We explored NKX2-5 expression in biopsy samples from patients with SSc-associated PH and in pulmonary artery smooth muscle cells (PASMCs) from patients with scleroderma. Disease-associated putative functional single-nucleotide polymorphisms (SNPs) at the NKX2-5 locus were cloned and studied in reporter gene assays. SNP function was further examined through protein-DNA binding assays, chromatin immunoprecipitation assays, and RNA silencing analyses. RESULTS: Increased NKX2-5 expression in biopsy samples from patients with SSc-associated PH was localized to remodeled vessels and PASMCs. Meta-analysis of 2 independent scleroderma cohorts revealed an association of rs3131917 with scleroderma (P = 0.029). We demonstrated that disease-associated SNPs are located in a novel functional enhancer, which increases NKX2-5 transcriptional activity through the binding of GATA-6, c-Jun, and myocyte-specific enhancer factor 2C. We also characterized an activator/coactivator transcription-enhancer factor domain 1 (TEAD1)/Yes-associated protein 1 (YAP1) complex, which was bound at rs3095870, another functional SNP, with TEAD1 binding the risk allele and activating the transcription of NKX2-5. CONCLUSION: NKX2-5 is genetically associated with scleroderma, pulmonary hypertension, and fibrosis. Functional evidence revealed a regulatory mechanism that results in NKX2-5 transcriptional activation in PASMCs through the interaction of an upstream promoter and a novel downstream enhancer. This mechanism can act as a model for NKX2-5 activation in cardiovascular disease characterized by vascular remodeling.

Influence of TYK2 in systemic sclerosis susceptibility: a new locus in the IL-12 pathway.

OBJECTIVES: TYK2 is a common genetic risk factor for several autoimmune diseases. This gene encodes a protein kinase involved in interleukin 12 (IL-12) pathway, which is a well-known player in the pathogenesis of systemic sclerosis (SSc). Therefore, we aimed to assess the possible role of this locus in SSc. METHODS: This study comprised a total of 7103 patients with SSc and 12 220 healthy controls of European ancestry from Spain, USA, Germany, the Netherlands, Italy and the UK. Four TYK2 single-nucleotide polymorphisms (V362F (rs2304256), P1104A (rs34536443), I684S (rs12720356) and A928V (rs35018800)) were selected for follow-up based on the results of an Immunochip screening phase of the locus. Association and dependence analyses were performed by the means of logistic regression and conditional logistic regression. Meta-analyses were performed using the inverse variance method. RESULTS: Genome-wide significance level was reached for TYK2 V362F common variant in our pooled analysis (p=3.08×10(-13), OR=0.83), while the association of P1104A, A928V and I684S rare and low-frequency missense variants remained significant with nominal signals (p=2.28×10(-3), OR=0.80; p=1.27×10(-3), OR=0.59; p=2.63×10(-5), OR=0.83, respectively). Interestingly, dependence and allelic combination analyses showed that the strong association observed for V362F with SSc, corresponded to a synthetic association dependent on the effect of the three previously mentioned TYK2 missense variants. CONCLUSIONS: We report for the first time the association of TYK2 with SSc and reinforce the relevance of the IL-12 pathway in SSc pathophysiology.

Multiplex cytokine analysis of dermal interstitial blister fluid defines local disease mechanisms in systemic sclerosis.

INTRODUCTION: Clinical diversity in systemic sclerosis (SSc) reflects multifaceted pathogenesis and the effect of key growth factors or cytokines operating within a disease-specific microenvironment. Dermal interstitial fluid sampling offers the potential to examine local mechanisms and identify proteins expressed within lesional tissue. We used multiplex cytokine analysis to profile the inflammatory and immune activity in the lesions of SSc patients. METHODS: Dermal interstitial fluid sample from the involved forearm skin, and synchronous plasma samples were collected from SSc patients (n = 26, diffuse cutaneous SSc (DcSSc) n = 20, limited cutaneous SSc (LcSSc) n = 6), and healthy controls (HC) (n = 10) and profiled by Luminex® array for inflammatory cytokines, chemokines, and growth factors. RESULTS: Luminex® profiling of the dermal blister fluid showed increased inflammatory cytokines (median interleukin ( IL)-6 in SSc 39.78 pg/ml, HC 5.51 pg/ml, p = 0.01, median IL-15 in SSc 6.27 pg/ml, HC 4.38 pg/ml, p = 0.03), chemokines (monocyte chemotactic protein (MCP)-3 9.81 pg/ml in SSc, 7.18 pg/ml HC, p = 0.04), and profibrotic growth factors (platelet derived growth factor (PDGF)-AA 10.38 pg/ml versus 6.94 pg/ml in HC, p = 0.03). In general dermal fluid and plasma cytokine levels did not correlate, consistent with predominantly local production of these factors within the dermal lesions, rather than leakage from the serum. In hierarchical clustering and network analysis IL-6 emerged as a key central mediator. CONCLUSIONS: Our data confirm that an immuno-inflammatory environment and aberrant vascular repair are intimately linked to fibroblast activation in lesional skin in SSc. This non-invasive method could be used to profile disease activity in the clinic, and identifies key inflammatory or pro-fibrotic proteins that might be targeted therapeutically. Distinct subgroups of SSc may be defined that show innate or adaptive immune cytokine signatures.

Trans-ancestral studies fine map the SLE-susceptibility locus TNFSF4.

We previously established an 80 kb haplotype upstream of TNFSF4 as a susceptibility locus in the autoimmune disease SLE. SLE-associated alleles at this locus are associated with inflammatory disorders, including atherosclerosis and ischaemic stroke. In Europeans, the TNFSF4 causal variants have remained elusive due to strong linkage disequilibrium exhibited by alleles spanning the region. Using a trans-ancestral approach to fine-map the locus, utilising 17,900 SLE and control subjects including Amerindian/Hispanics (1348 cases, 717 controls), African-Americans (AA) (1529, 2048) and better powered cohorts of Europeans and East Asians, we find strong association of risk alleles in all ethnicities; the AA association replicates in African-American Gullah (152,122). The best evidence of association comes from two adjacent markers: rs2205960-T (P=1.71 × 10(-34) , OR=1.43[1.26-1.60]) and rs1234317-T (P=1.16 × 10(-28) , OR=1.38[1.24-1.54]). Inference of fine-scale recombination rates for all populations tested finds the 80 kb risk and non-risk haplotypes in all except African-Americans. In this population the decay of recombination equates to an 11 kb risk haplotype, anchored in the 5' region proximal to TNFSF4 and tagged by rs2205960-T after 1000 Genomes phase 1 (v3) imputation. Conditional regression analyses delineate the 5' risk signal to rs2205960-T and the independent non-risk signal to rs1234314-C. Our case-only and SLE-control cohorts demonstrate robust association of rs2205960-T with autoantibody production. The rs2205960-T is predicted to form part of a decameric motif which binds NF-κBp65 with increased affinity compared to rs2205960-G. ChIP-seq data also indicate NF-κB interaction with the DNA sequence at this position in LCL cells. Our research suggests association of rs2205960-T with SLE across multiple groups and an independent non-risk signal at rs1234314-C. rs2205960-T is associated with autoantibody production and lymphopenia. Our data confirm a global signal at TNFSF4 and a role for the expressed product at multiple stages of lymphocyte dysregulation during SLE pathogenesis. We confirm the validity of trans-ancestral mapping in a complex trait.

The genetics of lupus: a functional perspective.

Systemic lupus erythematosus (SLE) is an autoimmune disease with a strong genetic component and is characterized by chronic inflammation and the production of anti-nuclear auto-antibodies. In the era of genome-wide association studies (GWASs), elucidating the genetic factors present in SLE has been a very successful endeavor; 28 confirmed disease susceptibility loci have been mapped. In this review, we summarize the current understanding of the genetics of lupus and focus on the strongest associated risk loci found to date (P <1.0 × 10-8). Although these loci account for less than 10% of the genetic heritability and therefore do not account for the bulk of the disease heritability, they do implicate important pathways, which contribute to SLE pathogenesis. Consequently, the main focus of the review is to outline the genetic variants in the known associated loci and then to explore the potential functional consequences of the associated variants. We also highlight the genetic overlap of these loci with other autoimmune diseases, which indicates common pathogenic mechanisms. The importance of developing functional assays will be discussed and each of them will be instrumental in furthering our understanding of these associated variants and loci. Finally, we indicate that performing a larger SLE GWAS and applying a more targeted set of methods, such as the ImmunoChip and next generation sequencing methodology, are important for identifying additional loci and enhancing our understanding of the pathogenesis of SLE.

Dense mapping of IL18 shows no association in SLE.

Systemic lupus erythematosus (SLE) is an autoimmune disease which behaves as a complex genetic trait. At least 20 SLE risk susceptibility loci have been mapped using both candidate gene and genome-wide association strategies. The gene encoding the pro-inflammatory cytokine, IL18, has been reported as a candidate gene showing an association with SLE. This pleiotropic cytokine is expressed in a range of immune cells and has been shown to induce interferon-γ and tumour necrosis factor-α. Serum interleukin-18 has been reported to be elevated in patients with SLE. Here we aimed to densely map single nucleotide polymorphisms (SNPs) across IL18 to investigate the association across this locus. We genotyped 36 across IL18 by Illumina bead express in 372 UK SLE trios. We also genotyped these SNPs in a further 508 non-trio UK cases and were able to accurately impute a dense marker set across IL18 in WTCCC2 controls with a total of 258 SNPs. To improve the study's power, we also imputed a total of 158 SNPs across the IL18 locus using data from an SLE genome-wide association study and performed association testing. In total, we analysed 1818 cases and 10 770 controls in this study. Our large well-powered study (98% to detect odds ratio = 1.5, with respect to rs360719) showed that no individual SNP or haplotype was associated with SLE in any of the cohorts studied. We conclude that we were unable to replicate the SLE association with rs360719 located upstream of IL18. No evidence for association with any other common variant at IL18 with SLE was found.