HLA-DRA Research Articles

Identification of gene signature markers in gestational hypertension and early-onset pre-eclampsia

IntroductionHypertensive disorders in pregnancy (HDP) are the leading cause of perinatal mortality worldwide. Inflammatory responses induced by insufficient placental perfusion have become a focal point in understanding the pathogenesis and aetiology of HDP and developing reliable and consistent biomarkers. Therefore, this study aims to identify gene signatures linked to the pathophysiology of HDP (gestational hypertension and early and late-onset pre-eclampsia). MethodsRNA was extracted from the maternal serum from the blood samples collected from different groups of HDP patients. A multiplex inflammation panel (255 inflammatory and housekeeping genes) and further gene expression analysis using NanoString Digital Direct Detection were done. The prominent expressions of these genes were further validated through qPCR techniques. ResultsNanoString analysis identified nine unique, significantly expressed genes (MAPK1, MAPK3, MAFF, HLA-DRA, IL12B, RHOA, MASP2, MEF2A and NR3C1) between specific group comparisons of different HPD classes and the normotensive groups. The qPCR showed that the HLA-DRA gene was significantly upregulated in the early-onset pre-eclamptic and gestational hypertensive group compared to its respective normotensive group. In contrast, MAFF and MEF2A were significantly downregulated in both HDPs compared to their controls. The MAPK1 gene was significantly higher in the early-onset group compared to the gestational hypertensive and normotensive groups. DiscussionThe upregulation of these distinctive genes in hypertensive groups compared to normotensives confirmed their diagnostic potential. Therefore, HLA-DRA, MAFF and MEF2A could be candidate markers of HDP, while the MAPK1 gene could be a differentiating marker between early-onset pre-eclampsia and gestational hypertension.

Genome-wide association study identifies multiple HLA loci for sarcoidosis susceptibility.

Sarcoidosis is a complex systemic disease. Our study aimed to (1) identify novel alleles associated with sarcoidosis susceptibility; (2) provide an in-depth evaluation of HLA alleles and sarcoidosis susceptibility and (3) integrate genetic and transcription data to identify risk loci that may more directly impact disease pathogenesis. We report a genome-wide association study of 1335 sarcoidosis cases and 1264 controls of European descent (EA) and investigate associated alleles in a study of African Americans (AA: 1487 cases and 1504 controls). The EA and AA cohort was recruited from multiple United States sites. HLA alleles were imputed and tested for association with sarcoidosis susceptibility. Expression quantitative locus and colocalization analysis were performed using a subset of subjects with transcriptome data. Forty-nine SNPs in the HLA region in HLA-DRA, -DRB9, -DRB5, -DQA1 and BRD2 genes were significantly associated with sarcoidosis susceptibility in EA, rs3129888 was also a risk variant for sarcoidosis in AA. Classical HLA alleles DRB1*0101, DQA1*0101 and DQB1*0501, which are highly correlated, were also associated with sarcoidosis. rs3135287 near HLA-DRA was associated with HLA-DRA expression in peripheral blood mononuclear cells and bronchoalveolar lavage from subjects and lung tissue and whole blood from GTEx. We identified six novel SNPs (out of the seven SNPs representing the 49 significant SNPs) and nine HLA alleles associated with sarcoidosis susceptibility in the largest EA population. We also replicated our findings in an AA population. Our study reiterates the potential role of antigen recognition and/or presentation HLA class II genes in sarcoidosis pathogenesis.

Investigation of molecular interaction and conformational stability of disease concomitant to HLA-DRβ3

Human leucocyte antigen DRβ3 is associated with specific autoimmune thyroid disease and plays a vital role in the progression of Grave’s disease. The available crystallographic structure of the HLA DRA, DRβ3*0101, was selected and used to generate mutation at position 57 from valine amino acid to Aspartic acid (D), Glutamic acid (E), Alanine (A), and Serine (S) amino acids by computational modeling approach. Mutant models were minimized, and stable conformation was chosen based on the lowest root mean square deviation value. Molecular docking assessed the best binding affinity of ligands C1, C2, C3, and C4 with wild-type and mutant HLA-DRβ3 models. Molecular dynamics simulation studies were executed to evaluate the stability of selected hits with wild-type and mutant dock complexes. The C3 has shown good binding affinity with wild-type and selected mutants; V57A, V57E, and V57D. Structural and molecular dynamics insights reveal the differences between wild-type and mutant-type HLA-DRβ3, which could help design novel antagonist molecules against autoimmune thyroid disorder. Communicated by Ramaswamy H. Sarma

HLA-DRA Gene Polymorphisms Are Associated with Graves' Disease as an Autoimmune Thyroid Disease.

Background Autoimmune thyroid disease (AITD), one of the most prevalent organ-specific autoimmune diseases, mainly includes Graves' disease (GD) and Hashimoto's thyroiditis (HT). This study was aimed at researching the association between AITD and single nucleotide polymorphisms (SNPs) of the HLA-DRA gene. Methods Using Hi-SNP high-throughput sequencing technology, we detected the distribution of three SNPs (rs3177928, rs7197, and rs3129878) of HLA-DRA genotypes in 1033 AITD patients (634 GD and 399 HT ones) and 791 healthy volunteers in Chinese Han Population. Chi-square test, multivariate logistic regression, and haplotype analysis were performed by SPSS and Haploview software to analyze the relationship between HLA-DRA gene polymorphisms and AITD. Results The results show that allele frequency and genotype distribution of rs3177928 and rs7197 were correlated with AITD and GD compared with the healthy control group, but not with HT. Rs3177928 and rs7197 were correlated with AITD and HT in the allele model, dominant model, and overdominant model before and after gender and age adjustment, but not with HT. In addition, we found that two loci (rs3177928 and rs7197) constituted a linkage disequilibrium (LD) region, and haplotype AA was associated with AITD and GD. However, we found no association between rs3129878 and AITD. Conclusion Our study is the first to find that rs3177928 and rs7197 of HLA-DRA are significantly correlated with AITD and GD in the Chinese Han population. This will help further reveal the pathogenesis of AITD and provide new candidate genes for the prediction or treatment of AITD.

Abstract 6344: HLA and non-HLA associations with childhood and adolescent Hodgkin lymphoma risk

Abstract The genetic etiology for Hodgkin lymphoma (HL) has been investigated with findings reported in both human leukocyte antigens (HLA) and non-HLA genomic regions. However, results of genetic association studies that focus on HL developed among children and adolescent individuals are limited. Given the historically high survival rate, it is reasonable to leverage available genetic data for survivors of childhood/adolescent HL for this investigation. Utilizing existing data from the St. Jude Lifetime Cohort, the Childhood Cancer Survivor Study (CCSS) original cohort and CCSS expansion cohort, we performed a fixed-effects meta-analysis of three genome-wide association studies for a total of 1,286 HL cases, 6,824 other childhood cancer cases and 373 non-cancer controls, all of whom are of European ancestry. Three independent SNPs in the HLA locus (rs28383311, Odds Ratio [OR] = 1.80, P = 2.14×10-21; rs3129198, OR = 1.52, P = 2.05×10-14; rs3129890, OR = 1.51, P = 6.21×10-13) were identified using step-wise conditional logistic regression. HLA SNP rs28383311 had a moderate correlation (r2 = 0.46) with rs2858870 reported previously by Cozen et al. (Blood, 2012) in the HLA-DRA gene region in adolescent/young adults onset HL. HLA SNP rs3129198 had a perfect correlation (r2 = 1.0) with rs2281389 (HLA-DPB1) reported previously by Moutsianas et al. (Blood, 2011) in mostly adults-onset HL. In contrast, SNP rs3129890 had a low correlation (r2 = 0.15) with rs2395185 (HLA-DRA) reported previously by Urayama et al. (JNCI, 2012) in HL cases diagnosed across a wide age range between 16 and 80 years old. Our analysis also identified additional SNPs for non-HLA loci at genome-wide significance level including: 1) rs1432297 (OR = 1.30, P = 2.5×10-8), which is in moderate correlation (r2=0.52) with previously reported rs1432295 (REL); 2) rs13279159 (OR = 1.32, P = 1.7×10-8), which is in high correlation (r2=0.75) with previously reported rs2019960 (PVT1); and 3) rs3824662 (OR = 1.52, P = 3.9×10-10), which had a high correlation (r2 = 0.91) with previously reported rs3781093 (GATA3). In addition, a novel uncommon SNP with a large effect size (rs117361561, OR = 1.95, P = 2.50×10-8, minor allele frequency = 0.02) was identified and mapped to PDGFD, a plausible gene for HL susceptibility. Twelve out 18 (67%) previously reported genome-wide significant non-HLA SNPs were replicated with P < 0.05 based on our meta-analysis. By analyzing early onset HL within pediatric and adolescent age range, we further dissected the associations in HLA locus by replicating three independent SNPs. We also found a novel non-HLA locus and replicated the majority of previous findings in the non-HLA region, suggesting mostly overlapping as well as potentially unique genetic etiology for HL risk across different ages of onset. Citation Format: Cheng Chen, Nan Song, Xiaojun Sun, Qian Dong, Zhenghong Li, Hui Wang, Smita Bhatia, Heather Mulder, John Easton, Jinghui Zhang, Yutaka Yasui, Gregory T. Armstrong, Kirsten K. Ness, Melissa M. Hudson, Leslie L. Robison, Zhaoming Wang. HLA and non-HLA associations with childhood and adolescent Hodgkin lymphoma risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6344.

1267-P: Fine-Mapping Type 1 Diabetes Risk at the MHC Locus

The Major Histocompatibility Complex (MHC) locus regulates the adaptive immune response and self-recognition and is the largest contributor to genetic risk of type 1 diabetes (T1D) . Protein-coding alleles in MHC class II and class I genes have long been known to contribute substantial T1D risk, yet whether additional variants affect T1D risk at this locus particularly in the non-coding genome has not been exhaustively evaluated. We therefore performed high-resolution fine-mapping of the MHC locus to identify additional coding and non-coding variants that contribute to T1D risk. We imputed genotypes for 39,270 samples from six European or American cohorts of T1D cases and controls into the T1DGC reference panel using SNP2HLA. We then performed two analyses to fine-map causal variants, first, stepwise conditional analysis using Firth bias-corrected logistic likelihood ratio test and second, iterative Bayesian stepwise selection analysis using the Sum of Single Effects (SuSiE) method. We identified 33 signals in stepwise conditional analysis with p<1×10-6 and 30 signals in SuSiE, where the results were highly concordant between methods. Both methods identified the known high-risk amino acid residue 57 of HLA-DQB1 as the top signal, as well as most known and several novel protein-coding risk and protective alleles in MHC class I and II genes. In addition, we identified multiple signals where the likely causal variant was non-coding and not linked to a protein-coding variant, including those that mapped to the LST1, HLA-DRA and NOTCH4 genes. We further annotated these non-coding signals using accessible chromatin, and several likely causal variants mapped in immune cell and/or pancreatic islet accessible chromatin. In ongoing analyses, we are replicating these signals in independent cohorts and performing molecular validation studies of their regulatory effects in relevant cell types. In total, high-resolution fine-mapping of the MHC locus identified additional T1D risk including in the non-coding genome. Disclosure C.Mcgrail: None. J.Chiou: Employee; Pfizer Inc. P.Benaglio: None. K.J.Gaulton: Consultant; Genentech, Inc., Stock/Shareholder; Neurocrine Biosciences, Inc., Vertex Pharmaceuticals Incorporated. Funding National Institutes of Health (DK120429) National Institutes of Health (DK122607)

Transcriptomic Evaluation of Juvenile Localized Scleroderma Skin With Histologic and Clinical Correlation.

Juvenile localized scleroderma (LS) is an autoimmune disease of the skin whose pathogenesis is not well understood due to the rarity of the disease. This study was undertaken to determine the skin transcriptome in skin biopsy tissue from children with juvenile LS compared to pediatric healthy controls, with identification of significant molecular targets using RNA sequencing (RNA-Seq). In this study, differentially expressed genes (DEGs) were assessed for correlations with histopathologic and clinical features in children with juvenile LS, and were used to group the children into distinct genetic clusters based on immunophenotype. RNA-Seq was performed on sections of paraffin-embedded skin tissue obtained from 28 children with juvenile LS and 10 pediatric healthy controls. RNA-Seq was carried out using an Illumina HTS TruSeq RNA Access library prep kit, with data aligned using STAR and data analysis using a DESeq2 platform. A standardized histologic scoring system was used to score skin sections for the severity of inflammation and levels of collagen deposition. Histologic scoring was completed by 2 pathologists who were blinded with regard to the status of each sample. Spearman's rank correlation coefficients were used to assess significant correlations between DEG expression profiles and skin histologic findings in patients with juvenile LS. We identified 589 significant DEGs in children with juvenile LS as compared to healthy controls. Hierarchical clustering was used to demonstrate 3 distinct juvenile LS immunophenotype clusters. The histologic scores of skin inflammation (based on numbers and categories of inflammatory cell infiltrates) were significantly correlated with the expression levels of HLA-DPB1, HLA-DQA2, HLA-DRA, and STAT1 genes (rs > 0.5, P < 0.01). Collagen thickness correlated with the expression levels of collagen organization genes as well as with genes found to be correlated with the severity of inflammation, including genes for major histocompatibility complex (MHC) class I, MHC class II, and interferon-γ signaling. Among children with juvenile LS, 3 distinct genetic signatures, or clusters, were identified. In one cluster, inflammation-related pathways were up-regulated, corresponding to the histologic skin inflammation score. In the second cluster, fibrosis-related pathways were up-regulated. In the third cluster, gene expression in the skin corresponded to the patterns seen in healthy controls. Up-regulation of HLA class II genes was observed within the first cluster (characterized by predominant inflammation), a feature that has also been observed in the peripheral blood of patients with morphea and in the skin of patients with systemic sclerosis.

Abstract 2755: Single-cell RNA sequencing reveals priming professional antigen-presenting macrophages and chemokine expressing T cells in tumor microenvironment by AXL inhibitor, SKI-G-801

Abstract The mechanistic investigations have defined that AXL inhibition reprograms the immunological microenvironment leading to increased T cells and antigen-presenting cells. However, previous reports did not clearly demonstrate the linchpin mechanism of anti-tumor effects with AXL inhibitors. It may be possible to elucidate the immunological mechanism of AXL inhibitor based on a comprehensive analysis of cell type-specific transcriptomic changes using scRNA-seq. The human CD34-NSG mice were engrafted LUSC tumor. These mice were divided into four dose groups: Vehicle, SKI-G-801, pembrolizumab, and combination groups. The tumor-associated cells were collected for flow cytometry and scRNA-seq analysis, and FFPE sample of tumor tissue was analyzed by a multispectral imager. The combination group demonstrated the superior anti-cancer efficacy to vehicle, pembrolizumab, and SKI-G-801 groups (all p&amp;lt;0.05). The scRNA-seq was performed with Seurat 4.0. The cells were separated into total 9 clusters. Following SKI-G-801 treatment, the T cells showed higher expressions of CD2, CCL5, CCL4, GZMA, and GZMB compared to both pembrolizumab and vehicle groups (both p&amp;lt;0.05). The gene expressions of CD2 and GZMA were higher than pembrolizumab and vehicle groups (both p&amp;lt;0.05). The CDR3 length differences of TCRs and diversity of T cell clones were higher increased in SKI-G-801, pembrolizumab and combination groups than vehicle group (p&amp;lt;0.05). In the macrophage cluster, the HLA-A, HLA-DRA, HLA-DRB1, and IL-1B genes were commonly expressed in SKI-G-801 and combination group (p&amp;lt;0.05). Mostly, the professional antigen-presenting macrophages (MHC class 2 protein complex high, p&amp;lt;0.05) were increased in the combination group. The combination effects between pembrolizumab and SKI-G-801 can be addressed by enhanced antigen presenting machinery, T cell activation and unique T cell clones. These transcriptomic results were highly correlated with the results of multispectral imaging and flow cytometry. Tumor infiltrations of helper T cells and cytotoxic T cells significantly increased in combination and SKI-G-801 groups (p &amp;lt; 0.01, p &amp;lt; 0.001 respectively). In multiplex IHC analysis, the proportion of CD4+ and CD8+ T cells were significantly increased in the tumor nest (both p&amp;lt;0.05), but the Tregs were not changed in the tumor nest which observations were validated by further flow cytometry analysis. A novel AXL inhibitor, SKI-G-801 drives priming of professional antigen-presenting cells and tumor-infiltrating T cells, leading to immunological synapsis for tumor killing, which is significantly enhanced by the combination with pembrolizumab. These results suggest the inhibition of AXL signal pathway by SKI-G-801 could confer a solid rationale for clinical investigation of lung cancer cells. Citation Format: Kyung-Ho Pyo, Hee Kyu Lee, Ha Ni Jo, Wongeun Lee, Seong Gu Heo, Sang Bin Lim, Dong Kwon Kim, Chun-Bong Synn, Youngseon Byeon, Young Seob Kim, Beung-Chul Ahn, Min Hee Hong, Sun Min Lim, Hye Ryun Kim, Byoung Chul Cho. Single-cell RNA sequencing reveals priming professional antigen-presenting macrophages and chemokine expressing T cells in tumor microenvironment by AXL inhibitor, SKI-G-801 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2755.

Analysis of Hub Genes Involved in Distinction Between Aged and Fetal Bone Marrow Mesenchymal Stem Cells by Robust Rank Aggregation and Multiple Functional Annotation Methods.

Stem cells from fetal tissue protect against aging and possess greater proliferative capacity than their adult counterparts. These cells can more readily expand in vitro and senesce later in culture. However, the underlying molecular mechanisms for these differences are still not fully understood. In this study, we used a robust rank aggregation (RRA) method to discover robust differentially expressed genes (DEGs) between fetal bone marrow mesenchymal stem cells (fMSCs) and aged adult bone marrow mesenchymal stem cells (aMSCs). Multiple methods, including gene set enrichment analysis (GSEA), Gene Ontology (GO) analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed for functional annotation of the robust DEGs, and the results were visualized using the R software. The hub genes and other genes with which they interacted directly were detected by protein–protein interaction (PPI) network analysis. Correlation of gene expression was measured by Pearson correlation coefficient. A total of 388 up-regulated and 289 down-regulated DEGs were identified between aMSCs and fMSCs. We found that the down-regulated genes were mainly involved in the cell cycle, telomerase activity, and stem cell proliferation. The up-regulated DEGs were associated with cell adhesion molecules, extracellular matrix (ECM)–receptor interactions, and the immune response. We screened out four hub genes, MYC, KIF20A, HLA-DRA, and HLA-DPA1, through PPI-network analysis. The MYC gene was negatively correlated with TXNIP, an age-related gene, and KIF20A was extensively involved in the cell cycle. The results suggested that MSCs derived from the bone marrow of an elderly donor present a pro-inflammatory phenotype compared with that of fMSCs, and the HLA-DRA and HLA-DPA1 genes are related to the immune response. These findings provide new insights into the differences between aMSCs and fMSCs and may suggest novel strategies for ex vivo expansion and application of adult MSCs.

A splice acceptor variant in HLA-DRA affects the conformation and cellular localization of the class II DR alpha-chain.

Class II human leucocyte antigen (HLA) proteins are involved in the immune response by presenting pathogen-derived peptides to CD4+ T lymphocytes. At the molecular level, they are constituted by α/β-heterodimers on the surface of professional antigen-presenting cells. Here, we report that the acceptor variant (rs8084) in the HLA-DRA gene mediates the transcription of an alternative version of the α-chain lacking 25 amino acids in its extracellular domain. Molecular dynamics simulations suggest this isoform undergoes structural refolding which in turn affects its stability and cellular trafficking. The short HLA-DRA isoform cannot reach the cell surface, although it is still able to bind the corresponding β-chain. Conversely, it remains entrapped within the endoplasmic reticulum where it is targeted for degradation. Furthermore, we demonstrate that the short isoform can be transported to the cell membrane via interactions with the peptide-binding site of canonical HLA heterodimers. Altogether, our findings indicate that short HLA-DRA functions as a novel intact antigen for class II HLA molecules.

Analysis of Single Nucleotide Polymorphisms in HLA-DRA, IL2RA , and HMGB1 Genes in Multiple Sclerosis.

Multiple sclerosis (MS) is a common demyelinating neurodegenerative disorder with significant heritability. Previous studies have associated genetic variants in human leukocyte antigen (HLA) complex, IL2RA , and HMGB1 genes with the pathophysiology of MS. In order to investigate the gene association in the Iranian population, we performed a genotyping study of 36 variants in the mentioned genes using Sanger sequencing in 102 MS patients and 113 healthy controls. Our results identified significant associations as well as significant allele frequency differences in some of the studied single-nucleotide polymorphisms including rs4935356, rs3177928, and rs7197 from HLA-DRA gene, and rs12722489 and rs12722490 variants from IL2RA gene (p< 0.05). Moreover, the strong linkage disequilibrium of two common haplotypes was estimated from the HLA-DRA gene. This association study may suggest the role of these polymorphisms in the genetic susceptibility of MS in the Iranian population and would facilitate the recognition of causative variants in this disease.

The rs3129882/rs4248166 in HLA-DRA and rs34372695 in SYT11 are not associated with sporadic Parkinson’s disease in Central Chinese population

Background Parkinson’s disease (PD) is a common progressive neurodegenerative disorder. Up to now, several single-nucleotide polymorphisms (SNPs) located in virulence gene sites have been reported linked to PD. Candidate gene association studies and genome-wide association studies have identified rs3129882, rs4248166 in HLA-DRA and rs34372695 in SYT11 as risk factors for familial or sporadic PD. However, the association between variants of HLA-DRA, SYT11 and PD are still controversial, especially in the Central Chinese population. We here performed a case-control study to investigate whether HLA-DRA and SYT11 genes could predispose to sporadic PD in the Chinese population. Methods We investigate 486 PD patients and 457 age- and sex-matched controls from Central China to assess this association. Results In the allele model, the odds ratio (OR) result of rs3129882 was 0.905 (p = 0.287). Moreover, no significant difference was observed in the association between rs424816 (OR = 0.864, p = 0.106) and rs34372695 (p = 1.0) with PD risk. Genotypic analysis in SNP rs3129882, rs4248166 and rs34372695 indicated no significant association with PD. Subgroup analysis of our data showed age-onset and gender were not associated with either genotype or minor allele frequencies of rs3129882 and rs4248166. Moreover, the negative results were also observed in a meta-analysis of studies of rs3129882 from mainland China and Taiwanese population. Conclusions Our results reveal that rs3129882, rs4248166 and rs34372695 do not confer significant risks for sporadic PD in the Central Chinese population.

Dynamic changes in human HLA-DRA gene expression and Th cell subsets in sepsis: Indications of immunosuppression and associated outcomes.

Sepsis is a life-threatening disease that is an immune disorder response that causes multiple organ dysfunction. In this study, we investigated the dynamic changes in mRNA expression of HLA-DRA gene and the specific transcription factor of helper T cell subsets to explore long-term immunophenotyping and its relationship with prognosis. Seventy-eight sepsis patients and twelve healthy controls were recruited in this study. Blood samples were collected at eight-time points during their septic course and were assayed for the gene expression of HLA-DRA and T helper cell subset-specific transcription factors (T-bet: Th1, GATA3: Th2, Foxp3: Treg, RORC: Th17). The levels of HLA-DRA in survivors gradually increased but were maintained at lower levels in non-survivors. The specific transcription factor of Th1 and Th2 cells, T-bet and GATA-3 were significantly lower in sepsis patients than in normal controls, and the non-survivors showed significantly lower levels than the survivors (P<.05). RORC and FOXP3, the specific transcription factor of Treg and Th17 were significantly higher in survivors than in non-survivors and normal controls (P<.05). T-bet and GATA-3 had a linear correlation with HLA-DRA expression (P<.01). The dynamic changes in HLA-DRA expression in peripheral blood could accurately reflect the immune status of sepsis patients, and the reduction in HLA-DRA may be an important reason for abnormal T cell differentiation. The sustained low levels of the Th cell subsets (Th1 and Th2) suggest the suppression of adaptive immunity, and this persistent immunosuppression may be the leading cause of death in septic patients.

Insights into the polymorphism in HLA-DRA and its evolutionary relationship with HLA haplotypes.

HLA-DRA encodes the alpha chain of the HLA-DR protein, one of the classical HLA class II molecules. Reported polymorphism within HLA-DRA is currently limited compared with other HLA genes, as only a single polymorphism encodes an amino acid difference in the translated protein. Since this SNP (rs7192, HLA00662.1:g.4276G>T p.Val217Leu) lies within exon 4, in the region encoding the cytoplasmic tail, the resulting protein is effectively monomorphic. For this reason, in-depth studies on HLA-DRA and its function have been limited. However, analysis of sequences from the 1000 Genomes Project and preliminary data from our lab reveals unrepresented polymorphism within HLA-DRA, suggesting a more complex role within the MHC than previously assumed. This study focuses on elucidating the extent of HLA-DRA polymorphism, and extending our understanding of the gene's role in HLA-DR~HLA-DQ haplotypes. Ninety-eight samples were sequenced for full-length HLA-DRA, and from this analysis, we identified 20 novel SNP positions in the intronic sequences within the 5711 bp region represented in IPD-IMGT/HLA. This polymorphism gives rise to at least 22 novel HLA-DRA alleles, and the patterns of intronic and 3' UTR polymorphism correspond to HLA-DRA~HLA-DRB345~HLA-DRB1~HLA-DQB1 haplotypes. The current understanding of the organization of the genes within the HLA-DR region assumes a single lineage for the HLA-DRA gene, as opposed to multiple gene lineages, such as in HLA-DRB. This study suggests that the intron and 3' UTR polymorphism of HLA-DRA indicates different lineages, and represents the HLA-DRA~HLA-DRB345~HLA-DRB1~HLA-DQB1 haplotypes.

Insulin resistance in obese adolescents affects the expression of genes associated with immune response.

The development of obesity and its metabolic complications is associated with dysregulation of various intrinsic mechanisms, which control basic metabolic processes through changes in the expression of numerous regulatory genes. The expression level of HLA-DRA, HLA-DRB1, HLA-G, HLA-F, and NFX1 genes as well as miR-190b was measured in the blood of obese adolescents without signs of resistance to insulin and with insulin resistance in comparison with the group of relative healthy control individuals without signs of obesity. It was shown that obesity without signs of insulin resistance is associated with upregulation of the expression level of HLA-DRA and HLA-DRB1 genes, but with down-regulation of HLA-G gene expression in the blood as compared to control group of relative healthy adolescents. At the same time, no significant changes were observed in the expression level of HLA-F and NFX1 genes in the blood of this group of obese adolescents. Development of insulin resistance in obese individuals leads to significant down-regulation of HLA-DRA, HLA-DRB1, HLA-G, and HLA-F gene expressions as well as to up-regulation of NFX1 gene as well as microRNA miR-190b in the blood as compared to obese patients without signs of insulin resistance. Results of this study provide evidence that obesity affects the expression of the subset of genes related to immune response in the blood and that development of insulin resistance in obese adolescents is associated with strong down-regulation of the expressions of HLA-DRA, HLA-DRB1, HLA-F, and HLA-G genes, which may be contribute to the development of obesity complications. It is possible that transcription factor NFX1 and miR-190b participate in downregulation of HLA-DRA gene expression in the blood of obese adolescents with insulin resistance.

Association Study and Fine-Mapping MajorHistocompatibility Complex Analysisof Pemphigus Vulgaris in aHanChinese Population.

To identify possible additional genetic susceptibility loci for pemphigus vulgaris (PV), we performed a genome-wide association study of 240 PV patients and 1,031 control individuals, and we selected the top single nucleotide polymorphisms for replication in independent samples, with 252 patient samples and 1,852 control samples. We identified rs11218708 (P= 3.1× 10-8, odds ratio= 1.54) at chromosome locus 11q24.1 as significantly associated with PV. A fine-mapping analysis of PV risk in the major histocompatibility complex region showed three independent variants predisposed to PV using stepwise analysis: HLA-DRB1*14:04 (P= 2.47× 10-38, odds ratio= 6.28), rs7454108 at the TAP2 gene (P= 2.78× 10-12, odds ratio= 3.25), and rs1051336 at the HLA-DRA gene (P= 3.06× 10-6, odds ratio= 0.33). A systematic evaluation using gene- and pathway-based analyses showed a high tendency for PV susceptibility genes to be associated with autoimmunity. Our study highlights the involvement of immune-mediated processes in the pathophysiology of PV and illustrates the value of imputation to identify variants in the major histocompatibility complex region.

841 Association study and fine mapping major histocompatibility complex analysis in pemphigus vulgaris of Han Chinese population

To identify additional genetic susceptibility loci for pemphigus vulgaris (PV), we performed the first genome-wide association study in 240 PV cases and 1,031 controls, and we validated in independent samples of 252 cases and 1,852 controls. We identified rs11218708 (P= 3.1 × 10-8, OR=1.54) at 11q24.1 to be significantly associated with PV. Further fine-mapping analysis of PV risk in the MHC region demonstrated three independent variants predisposed to PV, HLA-DRB1*14:04 (P = 2.47 × 10-38; OR = 6.28), rs7454108 at the TAP2 gene (P = 2.78 × 10-12; OR = 3.25), and rs1051336 at the HLA-DRA gene (P = 3.06 × 10-6; OR = 0.33), by stepwise analysis. A systematic evaluation by gene- and pathway-based analyses demonstrated a high tendency for PV susceptibility genes to be associated with autoimmunity. Our study highlighted that immune-mediated processes were involved in the pathophysiology of PV and illustrated the value of imputation to identify variants in the MHC region.

Abstract P1-05-02: CRISPR/Cas9-guided editing of spliceosome factors enhances major histocompatibility complex proteins in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) exhibits an extraordinary plasticity allowing adaptation to unfamiliar microenvironments and survival despite hindrances imposed by aggressive therapeutic approaches and the immune system. Alternative mRNA splicing (AS), catalyzed by spliceosome factors (SFs), is a major source of transcriptional diversity and phenotypic plasticity for normal and cancer cells. We ascertained that patients with TNBC present up-regulation of specific subsets of SFs mainly involving the epithelial splicing regulatory proteins 1 and 2 (ESRP1/2) and the polypyrimidine tract binding proteins (PTBP1/2). Methods and Results: Through the integration of in-house and publicly-available gene expression profiles (n=890), we evaluated the correlation between the levels of 290 SFs in patients with TNBC. This analysis identified nine subsets of TNBC based on SFs expression profiles with diverse clinical and pathological characteristics. These findings were further validated using data generated by the TCGA-BRCA (n=1,105) and METABRIC projects (n=2,509). Interestingly, up-regulation of PTBP1 was significantly associated with a shorter relapse-free survival interval for patients with TNBC (n=305; HR=1.58 (1.07 − 2.33); p-value=0.02). To systematically identify PTBP1-regulated AS events, we generated and clonally selected CRISPR/Cas9-guided PTBP1 knock-out (KO) TNBC cell lines. Analysis of our RNA-sequencing data at the gene level revealed a significant enrichment of inflammatory response and antigen presentation pathways. Evaluation of potential upstream transcriptional regulators for the enriched molecular pathways and predicted PTBP1 targets identified SMARCA4, a member of the SWI/SNF chromatin remodeling complex, to be significantly activated after PTBP1KO (q-value&amp;lt;0.01). Integration of ENCODE ChIP-sequencing and JASPAR transcription factor binding profile databases revealed clusters of SMARCA4 binding sites upstream of several members of the major histocompatibility (MHC) class I and MHC class II genes. Mechanistically, we identified that PTBP1 induces SMARCA4 exon 30 retention leading to the full length transcript variant 1, which has lower affinity for HLA gene promoter regions. Significant up-regulation of HLA-A, HLA-B, HLA-DPA1, and HLA-DRA genes in CRIPSR-guided PTBP1KO TNBC cells was further demonstrated by either western blot or indirect immunofluorescence. Finally, a negative correlation between PTBP1 and HLA genes expression was also identified in multiple breast cancer gene expression datasets. Conclusions: This study suggests that alterations in the PTBP1-associated splicing programming lead to a reduction of the antigen presentation capability of TNBC cells. Due to the limited therapeutic alternatives for patients with TNBC, beyond chemotherapy, further understanding and modulation of this novel alteration may expand the applications of immunotherapy for patients with TNBC. Citation Format: Bustos MA, Orozco JIJ, Salomon MP, Hoon DSB, Marzese DM. CRISPR/Cas9-guided editing of spliceosome factors enhances major histocompatibility complex proteins in triple-negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-05-02.

OR21 The novel functional role of HLA DRA and DRB5 encoded mirna transcripts

Aim Our recent research demonstrates the existence of experimentally validated, functional novel microRNA (miRNA) transcripts that are encoded within the HLA-DRA and HLA-DRB5 loci. MicroRNAs are known to regulate the post-transcriptional expression of targeted genes. In order to better understand the physiological role of these miRNAs, we have identified computationally predicted miRNA target sites on a genome-wide scale, revealing numerous targeted mRNA transcripts and physiological processes mediated by HLA encoded miRNAs. Methods Computationally predicted miRNA target sites for two HLA encoded miRNAs (HLA-DRA, HLA-DRB5) were identified on every known gene (GENCODE 25) using miRanda and filtered to contain only those heteroduplexes with a G ⩽ −20Kcal/mol. Statistically enriched biological processes and pathways of targeted transcripts was determined using DAVID. Results The miRNA encoded within the HLA-DRA and HLA-DRB5 loci were predicted to target 930 and 181 transcripts respectively. Although these targeted transcripts are involved in numerous physiological processes, both miRNAs are found to target transcripts that are statistically enriched within biological processes related to nervous system development, including the β-amyloid (Aβ)-degrading enzyme, membrane metalloendopeptidase (MME) gene, which has been associated with Alzhemier’s disease. Both HLA-DRA and HLA-DRB have also previously been associated with Alzheimer’s disease, suggesting a potential functional link between HLA encoded miRNA and neurodegenerative processes leading to disease. Conclusions Our work demonstrates a novel functional role for the HLA genes, mediated by encoded miRNA transcripts. Our findings suggest that two miRNA encoded within the HLA-DRA and HLA-DRB5 genes, target hundreds of transcripts involved in a variety of metabolic processes, with both sets of target genes being significantly enriched within neurological processes in particular. Both HLA-DRA and the HLA-DRB5 have been associated with Alzheimer’s disease in GWAS studies and both encoded miRNA are found to target the MME gene, which has also been associated with Alzheimer’s disease. Together, these finding suggest a functional link between the class II HLA region and the etiology of Alzheimer’s disease.

New variants near RHOJ and C2, HLA-DRA region and susceptibility to endometriosis in the Polish population—The genome-wide association study

ObjectiveEndometriosis is a common gynaecological disease, associated with severe pelvic pain and reduced fertility; however, molecular mechanisms remain largely unknown. Genome-wide association studies (GWAS) are able to identify genetic loci, which can play significant role during endometriosis development. AimThe study aimed at localisation of new genes and chromosomal loci, the nucleotide variants of which determine the level of susceptibility to endometriosis. Study designBlood samples from 171 patients with endometriosis were used as material for studies. The patients were recruited to the study at the Department of Operative Gynaecology of the Institute of the Polish Mother’s Memorial Hospital in Lodz. A control group (n=2934) came from the POPULOUS collection registered at Biobank Lab, Department of Molecular Biophysics, University of Lodz. DNA of the patients with endometriosis was compared with DNA of women free from that disease, the comparison being supported by GWAS. ResultsGenome-wide significant correlation was identified between one new, not previously described, single nucleotide polymorphism (SNP), rs10129516, localised on chromosome 14 in intergenic region between PARP1P2 and RHOJ genes (p=1.44×10−10, OR=3.104, 95% CI=2.329–4.136) and endometriosis. We have also identified significant association with endometriosis of 18 SNPs localised on chromosome 6 in position range 31883957 − 32681631 (C2 and HLA-DRA genes region) with the lowest observed p value for rs644045 in C2 gene (p=2.04×10−8, OR=1.955, 95% CI=1.541–2.480). ConclusionReported GWAS identified the novel loci associated with endometriosis in Polish women, not previously reported. The most interesting observation shown in our study are regions associated with susceptibility to endometriosis of loci located near C2, HLA-DRA and RHOJ genes. Resultsof that study did not correspond to previously published data about polymorphism in that regions and further evaluations are necessary in groups with higher numbers of patients to explain whether the above-mentioned genetic variant may be the risk factor for pathogenesis of endometriosis.