Human Leukocyte Antigen Risk Alleles Research Articles

Recombinant Interleukin-1 Receptor Antagonist Is an Effective First-Line Treatment Strategy in New-Onset Systemic Juvenile Idiopathic Arthritis, Irrespective of HLA-DRB1 Background and IL1RN Variants.

Human leukocyte antigen (HLA)-DRB1*15:01 has been recently associated with interstitial lung disease (LD), eosinophilia, and drug reactions in systemic juvenile idiopathic arthritis (sJIA). Additionally, genetic variants in IL1RN have been linked to poor response to anakinra. We sought to reproduce these findings in a prospective cohort study of patients with new-onset sJIA treated with anakinra as first-line therapy. HLA and IL1RN risk alleles were identified via whole-genome sequencing. Treatment responses and complications were compared between carriers versus noncarriers. Seventeen of 65 patients (26%) carried HLA-DRB1*15:01, comparable with the general population, and there was enrichment for HLA-DRB1*11:01, a known risk locus for sJIA. The rates of clinical inactive disease (CID) at 6 months, 1 year, and 2 years were generally high, irrespective of HLA-DRB1 or IL1RN variants, but significantly lower in carriers of an HLA-DRB1*11:01 allele. One patient, an HLA-DRB1*15:01 carrier, developed sJIA-LD. Of the three patients with severe drug reactions to biologics, one carried HLA-DRB1*15:01. The prevalence of eosinophilia did not significantly differ between HLA-DRB1*15:01 carriers and noncarriers at disease onset (6.2% vs 14.9%, P = 0.67) nor after the start of anakinra (35.3% vs 37.5% in the first 2 years of disease). We observed high rates of CID using anakinra as first-line treatment irrespective of HLA-DRB1 or IL1RN variants. Only one of the 17 HLA-DRB1*15:01 carriers developed sJIA-LD, and of the three patients with drug reactions to biologics, only one carried HLA-DRB1*15:01. Although thorough monitoring for the development of drug hypersensitivity and refractory disease courses in sJIA, including sJIA-LD, remains important, our data support the early start of biologic therapy in patients with new-onset sJIA irrespective of HLA-DRB1 background or IL1RN variants.

Association between HLA alleles and beta-lactam antibiotics-related severe cutaneous adverse reactions.

Introduction: Beta-lactam antibiotics are one of the most common causes of antibiotics-related severe cutaneous adverse reactions (SCARs) including Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN), drug reactions with eosinophilia and systemic symptoms (DRESS), and acute generalized exanthematous pustulosis (AGEP). Recent evidence demonstrated that the human leukocyte antigen (HLA) polymorphisms play important roles in the development of drug-related SCARs. This study aimed to extensively characterize the associations between HLA genetic polymorphisms and several phenotypes of SCARs related to beta-lactam antibiotics. Methods: Thirty-one Thai patients with beta-lactam antibiotics-related SCARs were enrolled in the study. A total of 183 unrelated native Thai subjects without any evidence of drug allergy were recruited as the control group. Genotyping of HLA class I and class II alleles was performed. Results: Six HLA alleles including HLA-A*01:01, HLA-B*50:01, HLA-C*06:02, HLA-DRB1*15:01, HLA-DQA1*03:01, and HLA-DQB1*03:02, were significantly associated with beta-lactam antibiotics-related SCARs. The highest risk of SCARs was observed in patients with the HLA-B*50:01 allele (OR = 12.6, 95% CI = 1.1-142.9, p = 0.042), followed by the HLA-DQB1*03:02 allele (OR = 5.8, 95% CI = 1.5-22.0, p = 0.012) and the HLA-C*06:02 allele (OR = 5.7, 95% CI = 1.6-19.9, p = 0.011). According to the phenotypes of SCARs related to beta-lactam antibiotics, the higher risk of SJS/TEN was observed in patients with HLA-A*03:02, HLA-B*46:02 (OR = 17.5, 95% CI = 1.5-201.6, p = 0.033), HLA-A*02:06, HLA-B*57:01 (OR = 9.5, 95% CI = 1.3-71.5, p = 0.028), HLA-DQB1*03:02 (OR = 7.5, 95% CI = 1.8-30.9, p = 0.008), or HLA-C*06:02 (OR = 4.9, 95% CI = 1.1-21.4, p = 0.008). While eight HLA alleles including HLA-A*02:05, HLA-A*02:11, HLA-B*37:01, HLA-B*38:01, HLA-B*50:01, HLA-C*06:02, HLA-C*03:09, and HLA-DRB1*15:01 were associated with AGEP, the highest risk of AGEP was observed in patients with the HLA-B*50:01 allele (OR = 60.7, 95% CI = 4.8-765.00, p = 0.005). Among the four HLA alleles associated with DRESS including HLA-C*04:06, HLA-DRB1*04:05, HLA-DRB1*11:01, and HLA-DQB1*04:01, the HLA-C*04:06 allele had the highest risk of beta-lactam antibiotics-related DRESS (OR = 60.0, 95% CI = 3.0-1202.1, p = 0.043). However, these associations did not achieve statistical significance after Bonferroni's correction. Apart from the HLA risk alleles, the HLA-A*02:07 allele appeared to be a protective factor against beta-lactam antibiotic-related SCARs (OR = 0.1, 95% CI = 0.0-0.5, p = 3.7 × 10-4, Pc = 0.012). Conclusion: This study demonstrated the candidate HLA alleles that are significantly associated with several phenotypes of beta-lactam antibiotics-related SCARs. However, whether the HLA alleles observed in this study can be used as valid genetic markers for SCARs related to beta-lactam antibiotics needs to be further explored in other ethnicities and larger cohort studies.

Susceptibility and Severity of COVID-19 Are Both Associated With Lower Overall Viral-Peptide Binding Repertoire of HLA Class I Molecules, Especially in Younger People.

An important number of studies have been conducted on the potential association between human leukocyte antigen (HLA) genes and COVID-19 susceptibility and severity since the beginning of the pandemic. However, case–control and peptide-binding prediction methods tended to provide inconsistent conclusions on risk and protective HLA alleles, whereas some researchers suggested the importance of considering the overall capacity of an individual’s HLA Class I molecules to present SARS-CoV-2-derived peptides. To close the gap between these approaches, we explored the distributions of HLA-A, -B, -C, and -DRB1 1st-field alleles in 142 Iranian patients with COVID-19 and 143 ethnically matched healthy controls, and applied in silico predictions of bound viral peptides for each individual’s HLA molecules. Frequency comparison revealed the possible predisposing roles of HLA-A*03, B*35, and DRB1*16 alleles and the protective effect of HLA-A*32, B*58, B*55, and DRB1*14 alleles in the viral infection. None of these results remained significant after multiple testing corrections, except HLA-A*03, and no allele was associated with severity, either. Compared to peptide repertoires of individual HLA molecules that are more likely population-specific, the overall coverage of virus-derived peptides by one’s HLA Class I molecules seemed to be a more prominent factor associated with both COVID-19 susceptibility and severity, which was independent of affinity index and threshold chosen, especially for people under 60 years old. Our results highlight the effect of the binding capacity of different HLA Class I molecules as a whole, and the more essential role of HLA-A compared to HLA-B and -C genes in immune responses against SARS-CoV-2 infection.

HLA autoimmune risk alleles restrict the hypervariable region of T cell receptors.

Polymorphisms in the human leukocyte antigen (HLA) genes strongly influence autoimmune disease risk. HLA risk alleles may influence thymic selection to increase the frequency of T cell receptors (TCRs) reactive to autoantigens (central hypothesis). However, research in human autoimmunity has provided little evidence supporting the central hypothesis. Here we investigated the influence of HLA alleles on TCR composition at the highly diverse complementarity determining region 3 (CDR3), which confers antigen recognition. We observed unexpectedly strong HLA-CDR3 associations. The strongest association was found at HLA-DRB1 amino acid position 13, the position that mediates genetic risk for multiple autoimmune diseases. We identified multiple CDR3 amino acid features enriched by HLA risk alleles. Moreover, the CDR3 features promoted by HLA risk alleles are more enriched in candidate pathogenic TCRs than control TCRs (e.g., citrullinated-epitope-specific TCRs in rheumatoid arthritis patients). Together, these results provide novel genetic evidence supporting the central hypothesis.

Genotyping for HLA Risk Alleles to Prevent Drug Hypersensitivity Reactions: Impact Analysis.

Human Leukocyte Antigen (HLA) variants can be a risk factor for developing potentially fatal drug hypersensitivity reactions. Our aim was to estimate the potential impact of genotyping for the HLA risk alleles incorporated in the Dutch Pharmacogenetics Working Group (DPWG) guidelines in The Netherlands. We estimated the number of hypersensitivity reactions and associated deaths that can be avoided annually by genotyping for these HLA risk alleles. Additionally, the cost-effectiveness was estimated. Nationwide implementation of genotyping HLA risk alleles before initiating drugs with an actionable drug–gene interaction can potentially save the life of seven allopurinol initiators and two flucloxacillin initiators each year in The Netherlands. Besides these deaths, 28 cases of abacavir hypersensitivity, 24 cases of allopurinol induced SCARs, 6 cases of carbamazepine induced DRESS and 22 cases of flucloxacillin induced DILI can be prevented. Genotyping HLA-B*5701 in abacavir initiators has a number needed to genotype of 31 to prevent one case of abacavir hypersensitivity and is cost-saving. Genotyping HLA-B*5801 in allopurinol initiators has a number needed to genotype of 1149 to prevent one case of SCAR but is still cost-effective. Genotyping before initiating antiepileptic drugs or flucloxacillin is not cost-effective. Our results confirm the need for mandatory testing of HLA-B*5701 in abacavir initiators, as indicated in the drug label, and show genotyping of HLA-B*5801 in allopurinol initiators should be considered.

HLA Expression in Relation to HLA Type in Classic Hodgkin Lymphoma Patients.

Simple SummaryClassic Hodgkin lymphoma (cHL) is a B-cell malignancy with involvement of Epstein–Barr virus (EBV) in about 30% of the European population. The risk to develop cHL is strongly linked to genetic variants in the human leukocyte antigen (HLA) genomic region and to certain HLA alleles. This may be caused by the function of HLA alleles, or by genetic linkage to non-HLA genes. HLA can present EBV-derived and tumour-cell specific antigens and this may lead to anti-tumour immune responses. However, the tumour cells downregulate HLA expression in a proportion of the cases, which may result in immune escape. In this study, we tested whether the loss of HLA expression is related to the presence of certain protective HLA alleles. We found that loss and retention of HLA expression is indeed associated with presence of known susceptibility HLA alleles. These findings suggest that HLA itself is involved in development of cHL.Several human leukocyte antigen (HLA) alleles are strongly associated with susceptibility to classic Hodgkin lymphoma (cHL), also in subgroups stratified for presence of the Epstein–Barr virus (EBV). We tested the hypothesis that the pressure on cHL tumour cells to lose HLA expression is associated with HLA susceptibility alleles. A meta-analysis was carried out to identify consistent protective and risk HLA alleles in a combined cohort of 839 cHL patients from the Netherlands and the United Kingdom. Tumour cell HLA expression was studied in 338 cHL cases from these two cohorts and correlated to the presence of specific susceptibility HLA alleles. Carriers of the HLA-DRB1*07 protective allele frequently lost HLA class II expression in cHL overall. Patients carrying the HLA-DRB1*15/16 (DR2) risk allele retained HLA class II expression in EBV− cHL and patients with the HLA-B*37 risk allele retained HLA class I expression more frequently than non-carriers in EBV+ cHL. The other susceptibility alleles showed no significant differences in expression. Thus, HLA expression by tumour cells is associated with a subset of the protective and risk alleles. This strongly suggests that HLA associations in cHL are related to peptide binding capacities of specific HLA alleles.

Visual Genomics Analysis Studio as a Tool to Analyze Multiomic Data.

Type B adverse drug reactions (ADRs) are iatrogenic immune-mediated syndromes with mechanistic etiologies that remain incompletely understood. Some of the most severe ADRs, including delayed drug hypersensitivity reactions, are T-cell mediated, restricted by specific human leukocyte antigen risk alleles and sometimes by public or oligoclonal T-cell receptors (TCRs), central to the immunopathogenesis of tissue-damaging response. However, the specific cellular signatures of effector, regulatory, and accessory immune populations that mediate disease, define reaction phenotype, and determine severity have not been defined. Recent development of single-cell platforms bringing together advances in genomics and immunology provides the tools to simultaneously examine the full transcriptome, TCRs, and surface protein markers of highly heterogeneous immune cell populations at the site of the pathological response at a single-cell level. However, the requirement for advanced bioinformatics expertise and computational hardware and software has often limited the ability of investigators with the understanding of diseases and biological models to exploit these new approaches. Here we describe the features and use of a state-of-the-art, fully integrated application for analysis and visualization of multiomic single-cell data called Visual Genomics Analysis Studio (VGAS). This unique user-friendly, Windows-based graphical user interface is specifically designed to enable investigators to interrogate their own data. While VGAS also includes tools for sequence alignment and identification of associations with host or organism genetic polymorphisms, in this review we focus on its application for analysis of single-cell TCR–RNA–Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE)-seq, enabling holistic cellular characterization by unbiased transcriptome and select surface proteome. Critically, VGAS does not require user-directed coding or access to high-performance computers, instead incorporating performance-optimized hidden code to provide application-based fast and intuitive tools for data analyses and production of high-resolution publication-ready graphics on standard specification laptops. Specifically, it allows analyses of comprehensive single-cell TCR sequencing (scTCR-seq) data, detailing (i) functional pairings of α–β heterodimer TCRs, (ii) one-click histograms to display entropy and gene rearrangements, and (iii) Circos and Sankey plots to visualize clonality and dominance. For unbiased single-cell RNA sequencing (scRNA-seq) analyses, users extract cell transcriptome signatures according to global structure via principal component analysis, t-distributed stochastic neighborhood embedding, or uniform manifold approximation and projection plots, with overlay of scTCR-seq enabling identification and selection of the immunodominant TCR-expressing populations. Further integration with similar sequence-based detection of surface protein markers using oligo-labeled antibodies (CITE-seq) provides comparative understanding of surface protein expression, with differential gene or protein analyses visualized using volcano plot or heatmap functions. These data can be compared to reference cell atlases or suitable controls to reveal discrete disease-specific subsets, from epithelial to tissue-resident memory T-cells, and activation status, from senescence through exhaustion, with more finite transcript expression displayed as violin and box plots. Importantly, guided tutorial videos are available, as are regular application updates based on the latest advances in bioinformatics and user feedback.

HLA Risk Alleles in Aromatic Antiepileptic Drug-Induced Maculopapular Exanthema.

To characterize human leukocyte antigen (HLA) loci as risk factors in aromatic antiepileptic drug-induced maculopapular exanthema (AED-MPE). A case-control study was performed to investigate HLA loci involved in AED-MPE in a southern Han Chinese population. Between January 2007 and June 2019, 267 patients with carbamazepine (CBZ), oxcarbazepine (OXC), or lamotrigine (LTG) associated MPE and 387 matched drug-tolerant controls from six centers were enrolled. HLA-A/B/C/DRB1 genotypes were determined using sequence-based typing. Potential risk alleles were validated by meta-analysis using data from different populations and in silico analysis of protein-drug interactions. HLA-DRB1*04:06 was significantly associated with OXC-MPE (p = 0.002, p c = 0.04). HLA-B*38:02 was associated with CBZ-MPE (p = 0.03). When pooled, HLA-A*24:02, HLA-A*30:01, and HLA-B*35:01 additionally revealed significant association with AED-MPE. Logistic regression analysis showed a multiplicative interaction between HLA-A*24:02 and HLA-B*38:02 in CBZ-MPE. Meta-analysis of data from different populations revealed that HLA-24*:02 and HLA-A*30:01 were associated with AED-MPE (p = 0.02 and p = 0.04, respectively). In silico analysis of protein-drug interaction demonstrated that HLA-A*24:02 and HLA-A*30:01 had higher affinities with the three aromatic AEDs than the risk-free HLA-A allele. HLA-DRB1*04:06 showed relatively specific high affinity with S-monohydroxy derivative of OXC. HLA-DRB1*04:06 is a specific risk allele for OXC-induced MPE in the Southern Han Chinese. HLA-A*24:02, possibly HLA-A*30:01, are common risk factors for AED-MPE. The multiplicative risk potential between HLA-A*24:02 and HLA-B*38:02 suggests that patients with two risk alleles are at greater risk than those with one risk allele. Inclusion of these HLA alleles in pre-treatment screening would help estimating the risk of AED-MPE.

Transethnic analysis of the human leukocyte antigen region for ulcerative colitis reveals not only shared but also ethnicity-specific disease associations.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gut. Genetic association studies have identified the highly variable human leukocyte antigen (HLA) region as the strongest susceptibility locus for IBD and specifically DRB1*01:03 as a determining factor for ulcerative colitis (UC). However, for most of the association signal such as delineation could not be made because of tight structures of linkage disequilibrium within the HLA. The aim of this study was therefore to further characterize the HLA signal using a transethnic approach. We performed a comprehensive fine mapping of single HLA alleles in UC in a cohort of 9272 individuals with African American, East Asian, Puerto Rican, Indian and Iranian descent and 40 691 previously analyzed Caucasians, additionally analyzing whole HLA haplotypes. We computationally characterized the binding of associated HLA alleles to human self-peptides and analyzed the physicochemical properties of the HLA proteins and predicted self-peptidomes. Highlighting alleles of the HLA-DRB1*15 group and their correlated HLA-DQ-DR haplotypes, we not only identified consistent associations (regarding effects directions/magnitudes) across different ethnicities but also identified population-specific signals (regarding differences in allele frequencies). We observed that DRB1*01:03 is mostly present in individuals of Western European descent and hardly present in non-Caucasian individuals. We found peptides predicted to bind to risk HLA alleles to be rich in positively charged amino acids. We conclude that the HLA plays an important role for UC susceptibility across different ethnicities. This research further implicates specific features of peptides that are predicted to bind risk and protective HLA proteins.

Towards Identifying the Target of Autoimmunity in Aplastic Anemia

Acquired aplastic anemia (AA) is an autoimmune bone marrow aplasia caused by T cell-mediated destruction of hematopoietic stem and progenitor cells. The antigenic target in AA remains unknown. Recently, we and others identified frequent somatic loss of function of several human leukocyte antigen (HLA) class I alleles in hematopoietic cells that survive the AA immune attack, suggesting these alleles ("risk alleles") present autoantigen in the affected patients. We hypothesize that risk alleles share structural features and peptide-binding characteristics that may inform our understanding of the immune mechanism of AA. To identify additional AA HLA class I risk alleles, we have partnered with two multi-institutional consortia, the North American Pediatric Aplastic Anemia Consortium (NAPAAC) and the Center for International Blood and Marrow Transplant Research (CIBMTR), to evaluate 507 AA patients for somatic HLA loss. Using a combination of targeted massively parallel sequencing of HLA class I genes and single nucleotide polymorphism array genotyping, we identified HLA loss in 19% of the NAPAAC cohort (30 of 156 unselected AA patients) and 13% of the CIBMTR cohort (46 of 351 patients selected to maximize discovery of less common risk alleles). Copy number-neutral loss of heterozygosity of chromosome arm 6p (6p CN-LOH) was the most common cause of HLA loss, occurring in 50 patients (10% of total cohort, 12% NAAPAC and 9% CIBMTR). Somatic mutations were present in 45 patients (9% of total cohort, 12% of NAPAAC and 7% CIBMTR). Patients with HLA loss had a median of 1 mutant clone per patient (range 1-7). Of the somatic mutations, 80% were predicted to disrupt expression of the affected allele (through loss of start, nonsense, or frameshift mutations). The other 20% were missense mutations affecting residues in the peptide binding groove (6 mutations) and the α3 domain (9 mutations) of the HLA class I protein. A total of 19 distinct AA HLA risk alleles were identified, of which 13 were newly identified in this study. Several risk alleles (HLA-B*14:02, HLA-B*40:02, HLA-A*02:01) were enriched in AA patients compared to ethnicity matched controls. Among the 507 patients in the study, at least one risk allele was present in 436/493 (88%) of AA patients and in 34/39 (87%) of the 6p CN-LOH events. Mutations clustered in several groups of alleles (supertypes) known to bind overlapping peptide repertoires, with alleles in B27 and B44 supertypes being most commonly affected. There were no mutations in A01, A01A03, A01A24, A24, B58, B62, or in any of the HLA-C alleles analyzed. Using several metrics to estimate the strength of the autoimmune selection on a given allele, including the frequency of somatic loss and similarity to other HLA risk alleles, we developed an AA HLA risk allele pathogenicity index ranging from very high (B*14:02 and B*40:02) and high pathogenicity (A*33:03, B*08:01, B*13:02, B*14:01, B*27:03, B*27:05, B*38:02, B*41:02 and B*49:01) to those unlikely to be pathogenic. Interestingly, even within high risk supertypes such as B44, there was a wide variation in predicted pathogenicity, ranging from high (for B*40:02 and B*41:02) to low pathogenicity (for B*44:02 and B*44:03), suggesting differences in autopeptide binding. Our study provides a comprehensive analysis of AA HLA risk alleles in a large diverse cohort of AA patients. Our results suggest that HLA risk alleles have shared autoantigen binding specificities that define their pathogenicity in AA, which can be used to identify candidate AA autoantigens. HLA risk allele pathogenicity may have future clinical utility as an adjunctive diagnostic test, as well as for prognostic assessment and haploidentical donor selection. Disclosures Lee: AstraZeneca: Research Funding; Kadmon: Research Funding; Takeda: Research Funding; Novartis: Research Funding; Amgen: Research Funding; Syndax: Research Funding; Pfizer: Consultancy, Research Funding; Incyte: Consultancy, Research Funding. Monos:Omixon: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Understanding Idiosyncratic Toxicity: Lessons Learned from Drug-Induced Liver Injury.

Idiosyncratic adverse drug reactions (IADRs) encompass a diverse group of toxicities that can vary by drug and patient. The complex and unpredictable nature of IADRs combined with the fact that they are rare makes them particularly difficult to predict, diagnose, and treat. Common clinical characteristics, the identification of human leukocyte antigen risk alleles, and drug-induced proliferation of lymphocytes isolated from patients support a role for the adaptive immune system in the pathogenesis of IADRs. Significant evidence also suggests a requirement for direct, drug-induced stress, neoantigen formation, and stimulation of an innate response, which can be influenced by properties intrinsic to both the drug and the patient. This Perspective will provide an overview of the clinical profile, mechanisms, and risk factors underlying IADRs as well as new approaches to study these reactions, focusing on idiosyncratic drug-induced liver injury.

Psoriatic arthritis: clinical patterns, rheumatoid factor, anti-cyclic citrullinated peptide and human leukocyte antigen risk alleles

Psoriatic arthritis: clinical patterns, rheumatoid factor, anti-cyclic citrullinated peptide and human leukocyte antigen risk alleles

Application of in Vitro T Cell Assay Using Human Leukocyte Antigen-Typed Healthy Donors for the Assessment of Drug Immunogenicity.

It is unclear whether priming of naïve T cells to drugs is detectable in healthy human donors expressing different human leukocyte antigen (HLA) alleles. Thus, we examined T cell priming with drugs associated with HLA risk alleles and control compounds in 14 HLA-typed donors. Nitroso sulfamethoxazole and piperacillin activated T cells from all donors, whereas responses to carbamazepine and oxypurinol were only seen in donors expressing HLA-B*15:02 and HLA-B*58:01, respectively. Weak flucloxacillin-specific T cell responses were detected in donors expressing HLA-B*57:01 and HLA-B*58:01. These data show that the priming of T cells with certain drugs is skewed toward donors expressing specific HLA alleles.

Protein-altering variants of PTPN2 in childhood-onset Type 1A diabetes.

To examine the contribution of PTPN2 coding variants to the risk of childhood-onset Type 1A diabetes. PTPN2 mutation analysis was carried out for 169 unrelated Japanese people with childhood-onset Type 1A diabetes. We searched for coding variants that were absent or extremely rare in the general population and were scored as damaging by multiple in silico programs. We performed mRNA analysis and three-dimensional structural prediction of the detected variants, when possible. We also examined possible physical links between these variants and previously reported risk SNPs as well as clinical information from variant-positive children. One frameshift variant (p.Q286Yfs*24) and two probably damaging missense substitutions (p.C232W and p.R350Q) were identified in one child each. Of these, p.Q286Yfs*24 and p.C232W were hitherto unreported, while p.R350Q accounted for 2/121,122 alleles of the exome datasets. The p.Q286Yfs*24 variant did not encode stable mRNA, and p.C232W appeared to affect the structure of the tyrosine-protein phosphatase domain. The three variants were physically unrelated to known risk SNPs. The variant-positive children manifested Type 1A diabetes without additional clinical features and invariably carried risk human leukocyte antigen alleles. The results provide the first indication that PTPN2 variants contribute to the risk of Type 1A diabetes, independently of known risk SNPs. PTPN2 coding variants possibly induce non-specific Type 1A diabetes phenotypes in individuals with human leukocyte antigen-mediated disease susceptibility. Our findings warrant further validation.

Comparison of HLA DQA1 and DQB1 Genes by Multiplex Ligation-Dependent Probe Amplification and Next Generation Sequencing Technologies for Celiac Disease Risk Determination

Introduction: Celiac disease is a systemic autoimmune disease with chronic gastrointestinal disorder caused by intolerance to gluten proteins. The most dominant genetic risk factors are the specific genotypes encoding the Human Leukocyte Antigen (HLA) class II molecule genes, DQA1 and DQB1. The DQ2.5 variant (DQA1*05/DQB1*02 alleles) is present in 90-95% of patients with celiac disease. The DQ8 variant (DQA1*03/DQB1*0302) and DQ2.2 variant (DQA1*02/DQB1*02) are found in most of the remaining patients. A positive test is indicative of genetic susceptibility but does not necessarily mean disease development. A negative test has a more significant value because gluten intolerance rarely occurs in the absence of specific HLA predisposing alleles. In the current study we compared Multiplex Ligation-dependent Probe Amplification (MLPA) and Next Generation Sequencing (NGS) technologies for identifying Celiac disease risk HLA alleles. Methods: DNA was extracted from either saliva or whole-blood archival samples obtained from commercial vendors using Qiagen DNA mini extraction kits. MLPA assay probe mix designed to detect the HLA alleles implicated in Celiac disease consists of 29 specific fluorescent labeled amplification products. The PCR products are separated by capillary gel electrophoresis on the ABI 3500 Genetic Analyzer yielding a specific electropherogram. Based on the presence and relative height of individual probe, as compared to the relative probe peak height in various reference samples, the HLA allele in the patient sample was determined. For Next Generation sequencing assay, long range PCR products of HLA DQA1 and DQB1 genes were pooled to prepare bar coded NGS libraries. Equimolar pools of the barcoded libraries were sequenced on the Illumina MiSeq platform by paired end 151 bp sequencing. Demultiplexed FASTQ files were aligned to HLA variant database and genotypes were identified. Results: A total of 26 samples with different HLA variants were analyzed by both MLPA and NGS assay panel. The samples included DQ2.5, DQ8, DQ2.2, both heterozygous and homozygous variants, negatives and positive control cell line DNA samples. Data is summarized in Table 1. There was 100% concordance between the two technologies in determining the Celiac disease risk alleles. DNA dilution studies showed that both technologies were optimal with a minimum of 40 ng of DNA input. The workflow for MLPA was simple with sample to data generation completed within 24 hours. NGS assay was laborious with highly technical library preparation, sequencing and data analysis with sample to data in 48 hours. However, NGS technology results in a high resolution genotyping whereas MLPA assay is limited to 5 alleles of DQA1 and DQB1 genes.Table: Table. HLA DQA1 and DQB1 genotyping data summary- MLPA and NGS dataConclusion: Both methods are reliable and efficient to provide necessary information for celiac disease risk evaluation. In the future if high resolution HLA genotyping is needed, NGS may be more applicable.

SNP-based HLA allele tagging, imputation and association with antiepileptic drug-induced cutaneous reactions in Hong Kong Han Chinese.

Human leukocyte antigen (HLA) genes control the regulation of the human immune system and are involved in immune-related diseases. Population surveys on relationships between single nucleotide polymorphisms (SNP) and HLA alleles are essential to conduct genetic association between HLA variants and diseases. Samples were obtained from our in-house database for epilepsy genetics and pharmacogenetics research. Using 184 epilepsy patients with both genome-wide SNP array and HLA-A/B candidate gene sequencing data, we sought tagging SNPs that completely represent sixHLA risk alleles; in addition, a Hong Kong population-specific reference panel was constructed for SNP-based HLA imputation. The performance of our new panel was compared to a recent Han Chinese panel. Finally, genetic associations of HLA variants with mild skin rash were performed on the combined sample of 408 patients. Common SNPs rs2571375 and rs144295468 were found to successfully tag HLA risk alleles A*31:01 and B*13:01, respectively. HLA-B*15:02 can be predicted by rs144012689 with >95% sensitivity and specificity. The imputation reference panel for the Hong Kong population had comparable performance to the Han Chinese panel due to the large sample size for common HLA alleles, though it retained discordance for imputing rare alleles. No significant genetic associations were found between HLA genetic variants and mild skin rash induced by aromatic antiepileptic drugs. This study provides new information on the genetic structure of HLA regions in the Hong Kong population by identifying tagging SNPs and serving as a reference panel. Moreover, our comprehensive genetic analyses revealed no significant association between HLA alleles and mild skin rash in Hong Kong Han Chinese.

Characterization of amoxicillin‐ and clavulanic acid‐specific T cells in patients with amoxicillin‐clavulanate–induced liver injury

Drug-induced liver injury (DILI) frequently has a delayed onset with several human leukocyte antigen (HLA) genotypes affecting susceptibility, indicating a potential role for the adaptive immune system in the disease. The aim of this study was to investigate whether drug-responsive T lymphocytes are detectable in patients who developed DILI with the combination, antimicrobial amoxicillin-clavulanate. Lymphocytes from 6 of 7 patients were found to proliferate and/or secrete interferon-gamma (IFN-γ) when cultured with amoxicillin and/or clavulanic acid. Amoxicillin (n = 105) and clavulanic acid (n = 16) responsive CD4(+) and CD8(+) T-cell clones expressing CCR, chemokine (C-C motif) receptor 4, CCR9, and chemokine (C-X-C motif) receptor 3 were generated from patients with and without HLA risk alleles; no cross-reactivity was observed between the two drug antigens. Amoxicillin clones were found to secrete a heterogeneous panel of mediators, including IFN-γ, interleukin-22 and cytolytic molecules. In contrast, cytokine secretion by the clavulanic acid clones was more restricted. CD4(+) and CD8(+) clones were major histocompatability complex class II and I restricted, respectively, with the drug antigen being presented to CD4(+) clones in the context of HLA-DR molecules. Several pieces of evidence indicate that the clones were activated by a hapten mechanism: First, professional antigen-presenting cells (APCs) were required for optimal activation; second, pulsing APCs for 4-16 hours activated the clones; and third, inhibition of processing abrogated the proliferative response and cytokine release. Both amoxicillin- and clavulanic acid-specific T cells participate in the liver injury that develops in certain patients exposed to amoxicillin-clavulanate.

The polymorphisms of human leukocyte antigen loci may contribute to the susceptibility and severity of severe aplastic anemia in Chinese patients

The human leukocyte antigen (HLA) system has been reported to be involved in the development of aplastic anemia (AA). We compared and analyzed HLA-A, B, C, DRB1 and DQB1 alleles in 96 Chinese severe AA (SAA) patients to those in 600 healthy people chosen randomly from the China Marrow Donor Program to investigate the association of HLA class I and II allele polymorphisms with disposition of SAA and its severity degree in Chinese population. The DNA of patients was extracted and HLA high-resolution genotyping was conducted using polymerase chain reaction-sequence based typing technique. The gene frequencies of A∗02:01, A∗02:06, B∗13:01, DRB1∗07:01, DRB1∗09:01, DRB1∗15:01 and DQB1∗06:02 in SAA patients were significantly higher than in controls (all P<0.05), while the allelic frequencies of A∗02:07, A∗11:01 and B∗40:01 were notably lower in SAA patients than those in the controls (P=0.001, 0.002, 0.005, respectively). Comparison among different severity of SAA groups exhibited significant increases of DRB1∗15:01 (P=0.027) and DQB1∗06:02 (P=0.013), but obviously lower frequencies of B∗46:01 (P=0.023) and DRB1∗09:01 (P=0.020) in non-VSAA patients than in VSAA patients. Thus, our results identified several risk and protective HLA alleles for Chinese SAA patients. Moreover, DRB1∗15:01, DQB1∗06:02, B∗46:01 and DRB1∗09:01 may be associated with severity of SAA.

Genome-wide expression analysis suggests unique disease-promoting and disease-preventing signatures in Pemphigus vulgaris

To evaluate pathogenetic mechanisms underlying disease development and progression in the autoimmune skin disease Pemphigus vulgaris (PV), we examined global peripheral blood gene expression in patients and healthy controls. Our goals were to: (1) assign blood gene expression signatures to patients and controls; (2) identify differentially expressed genes (DEGs) and investigate functional pathways associated with these signatures; and (3) evaluate the distribution of DEGs across the genome to identify transcriptional 'hot spots'. Unbiased hierarchical clustering clearly separated patients from human leukocyte antigen (HLA)-matched controls (MCRs; 'disease' signature), and active from remittent patients ('activity' signature). DEGs associated with these signatures are involved in immune response, cytoskeletal reorganization, mitogen-activated protein kinase (MAPK) signaling, oxidation-reduction and apoptosis. We further found that MCRs carrying the PV-associated HLA risk alleles cluster distinctly from unmatched controls (UMCR) revealing an HLA-associated 'control' signature. A subset of DEGs within the 'control' signature overlap with the 'disease' signature, but are inversely regulated in MCR when compared with either PV patients or UMCR, suggesting the existence of a 'protection' signature in healthy individuals carrying the PV HLA genetic risk elements. Finally, we identified 19 transcriptional 'hot spots' across the signatures, which may guide future studies aimed at pinpointing disease risk genes.

OC-090 Correlation between Distribution of HLA Haplotypes and Severity of Mucosal Damages

<h3>Introduction</h3> Celiac disease is a chronic intestinal inflammation resulting in different degree of intestinal mucosal damages. The disease occurs in genetically predisposed individuals and associated with human leukocyte antigen (HLA)-DQ2/8 heterodimers. The aim of this study is to evaluate the correlation between distribution of HLA haplotypes and the severity of mucosal damages. <h3>Methods</h3> Fifty nineIranian celiac disease patients were evaluated to predict the HLA-DQA1 and -DQB1 genes, using tagging SNPs method. All patients had positive tTGA and/or EMA antibodies and histology according to Modified Marsh classification (Marsh I-IIIc) by Rostami <i>et al</i> <h3>Results</h3> The result of this study show that 86.4% of cases were carriers of HLA-DQ2 and/or HLA-DQ8 heterodimers, either homozygous or heterozygous. Marsh IIIa lesions were seen in 20 cases, Marsh IIIb in 9 and Marsh IIIc in 12 cases. Marsh IIIa is associated with HLA-DQ2 haplotype in 10 cases, IIIb with 4 and IIIc with 10 cases respectively. Marsh IIIa is correlated with HLA-DQ2.5/2.5 in 4 cases followed by HLA-DQ2.5/DQ8 and HLA-DQ2.5/DQX each in 3 cases respectively. <h3>Conclusion</h3> According to high prevalence of HLA-DQ2 haplotype in Iranian population, we can conclude that there is a strong link between severity of mucosal damages and presence of HLA-DQ 2 haplotype. Celiac disease cases with Marsh III (a-c) carry more HLA risk alleles compared to those with Marsh I-II and this difference were statistically significant for DQ2.5 haplotype distribution (P = 0.0001). <h3>Disclosure of Interest</h3> None Declared