Intergenic HLA Variants in African American Patients with Systemic Sclerosis Regulate Expression of HLA-DRB1

Urvashi Kaundal¹, Julia Hartman², Chloe Borden², Janet Wang³, Ami Shah⁴, Maureen Mayes⁵, Ayo Doumatey⁶, Amy Bentley⁷, Daniel Shriner⁶, Robyn Domsic⁸, Thomas Medsger⁹, Paula Ramos¹⁰, Richard Silver¹¹, Virginia Steen¹², John Varga¹³, Vivien Hsu¹⁴, Lesley Ann Saketkoo¹⁵, Elena Schiopu¹⁶, Dinesh Khanna¹⁷, Jessica Gordon¹⁸, Lindsey Criswell¹⁹, Heather Gladue²⁰, Chris Derk²¹, Elana Bernstein²², S. Louis Bridges²³, Victoria Shanmugam²⁴, Kathleen Kolstad²⁵, Lorinda Chung²⁶, Suzanne Kafaja²⁷, Reem Jan²⁸, Marcin Trojanowski²⁹, Avram Goldberg³⁰, Benjamin Korman³¹, Monique Hinchcliff³², Settara Chandrasekharappa⁶, Massimo Gadina², Davide Randazzo², Stefania Dell'Orso², Adebowale Adeyemo⁶, Charles Rotimi⁶, Elaine Remmers⁶, Fredrick Wigley³³, Rafael Casellas², Daniel Kastner⁶, Francesco Boin³⁴ and Pravitt Gourh¹, ¹National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), NIH, Bethesda, ²National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), NIH, Bethesda, MD, ³National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), NIH, Beachwood, OH, ⁴Johns Hopkins University School of Medicine, Ellicott City, MD, ⁵University of Texas Houston McGovern Medical School, Division of Rheumatology and Clinical Immunogenetics, Houston, TX, ⁶National Human Genome Research Institute (NHGRI), NIH, Bethesda, MD, ⁷National Human Genome Research Institute (NHGRI), NIH, Bethedsa, MD, ⁸University of Pittsburgh School of Medicine, Pittsburgh, PA, ⁹University of Pittsburgh School of Medicine, Verona, PA, ¹⁰Medical University of South Carolina, Charleston, SC, ¹¹Medical University of South Carolina, Charleston, ¹²Division of Rheumatology, Department of Medicine, MedStar Georgetown University Hospital, Washington, DC, ¹³Northwestern University, Chicago, IL, ¹⁴Rutgers-RWJ Medical School, South Plainfield, NJ, ¹⁵Scleroderma Patient Care and Research Center, Tulane University, New Orleans, LA, ¹⁶Michigan Medicine, Ann Arbor, MI, ¹⁷University of Michigan, Ann Arbor, MI, ¹⁸Hospital for Special Surgery, New York, NY, ¹⁹Rosalind Russell/Ephraim P. Engleman Rheumatology Research Center, University of California San Francisco, San Francisco, CA, ²⁰Arthritis and Osteoporosis Consultants of the Carolinas, Charlotte, NC, ²¹University of Pennsylvania, Philadelphia, PA, ²²Columbia University, New York, NY, ²³University of Alabama at Birmingham, Mountain Brk, AL, ²⁴The George Washington University, Washington, DC, ²⁵Division of Immunology & Rheumatology, Stanford University School of Medicine, Palo Alto, CA, ²⁶Stanford University School of Medicine and Palo Alto VA Health Care System, Palo Alto, CA, ²⁷David Geffen School of Medicine, UCLA, Los Angeles, CA, ²⁸Pritzker School of Medicine, University of Chicago, Chicago, IL, ²⁹Boston University Medical Center, BOSTON, MA, ³⁰NYU Langone Medical Center - NYU Hospital for Joint Diseases, Lake Success, NY, ³¹Department of Medicine, University of Rochester Medical Center, Rochester, NY, ³²Yale School of Medicine, Westport, CT, ³³Johns Hopkins University School of Medicine, Baltimore, MD, ³⁴University of California San Francisco, Cedars-Sinai, West Hollywood, CA

Meeting: ACR Convergence 2020

Keywords: B-Lymphocyte, Epigenetics, Gene Expression, Genome Wide Association Studies, Scleroderma

Session Information

Date: Sunday, November 8, 2020

Title: Systemic Sclerosis & Related Disorders – Basic Science (1522–1526)

Session Type: Abstract Session

Session Time: 3:00PM-3:50PM

Background/Purpose: Genome-wide association study (GWAS) from the Genome Research in African American Scleroderma Patients (GRASP) cohort has identified the human leukocyte antigen (HLA) region as the strongest genetic risk factor in SSc. The functional roles of HLA variants in SSc susceptibility are largely unknown. We hypothesize that SSc-associated HLA variants overlap with one of the classes of cis-regulatory modules (CRMs) such as enhancer to regulate gene expression. CRMs can be mapped using DNase I hypersensitive site sequencing (DNase-Seq) or assay for transposase-accessible chromatin with high throughput sequencing (ATAC-Seq) giving information regarding chromatin accessibility. Thus, chromatin accessibility in the SSc-relevant cell types would be crucial in interpreting the function of these variants.

Methods: Genotyping data using the Illumina Multi-Ethnic Global array (MEGA) from the GRASP cohort were analyzed to identify the top HLA variant. Expression quantitative trait loci (eQTL) analysis was performed for the risk variants using RNA sequencing data from the Genotype-Tissue Expression (GTEx) project. Epstein-Barr virus-transformed B (EBV-B) cells with wild type, heterozygous and homozygous alleles for the most highly associated HLA variant were used to confirm the expression of HLA-II genes using RNA-sequencing. Protein expression was confirmed by flow cytometry and confocal microscopy. Chromatin accessibility at the variant position in EBV-B cells is being identified using ATAC-seq.

Results: The top variant in the African American GWAS was rs9469201, near the HLA-DQA1 gene with an odds ratio of 2.13 (P=4.5×10^-20). eQTL analysis suggested decreased expression of HLA-DQA1/-DQB1 (in whole blood) and increased expression of HLA-DRB1 (in EBV-B cells) for the risk allele (Figure 1). This is particularly intriguing because we have previously reported two HLA-DRB1 alleles (HLA-DRB1*08:04 and -DRB1*11:02) as independent risk factors in African American SSc. The rs9469201 variant was found not to be in linkage disequilibrium with the HLA-DRB1*08:04 allele. Flow cytometry (Figure 2) and confocal microscopy (Figure 3) confirmed the expression of HLA-DRB1 and HLA-DQB1. ATAC-Seq analysis of the EBV-B cells is currently ongoing.

Conclusion: Our preliminary findings suggest that peptide presentation by HLA molecules as well as the relative expression of the specific HLA molecule presenting the peptide are both important in SSc pathogenesis. Future directions will involve CRISPR/Cas9 based deletion of CRMs-with overlapping HLA variants, and subsequent evaluation of the changes in HLA-II gene expression. Transcription factor binding at the HLA variant site will be accessed using chromatin immunoprecipitation with sequencing (ChIP-seq) in EBV-B cells. This study will elucidate the functional role of the HLA variants in allele-specific HLA-II gene expression in SSc.

Figure 1. GTEx HLA-DRB1, HLA-DQA1 and HLA-DQB1 expression by rs9469201 variant

Figure 2. Flow cytometry plots for EBV-B cells with wild type allele stained for HLA-DRB1 and HLA-DQB1 protein using fluorochrome conjugated antibodies.

Figure 3. Confocal microscopy image for HLA-DRB1 and HLA-DQB1 stained cells for the wild type allele.

Disclosure: U. Kaundal, None; J. Hartman, None; C. Borden, None; J. Wang, None; A. Shah, None; M. Mayes, Actelion Pharmaceuticals, 1, Boehringer Ingelheim, 1, 2, Corbus, 1, Eicos Sciences, 1, Galapagos, 1; A. Doumatey, None; A. Bentley, None; D. Shriner, None; R. Domsic, Formation Biologics, 5, Eicos Sciences, Inc, 5, Corbus Pharmaceutical Holdings, 5; T. Medsger, None; P. Ramos, None; R. Silver, Boehringer Ingelheim, 1, 2, Actelion, 1, Corbus, 1, Forbius, 1; V. Steen, Boehringer Ingelheim, 2, 5, corbus, 2, 5, eicos, 2, 5, genetech, 2, forbius, 5, galapagos, 5; J. Varga, None; V. Hsu, None; L. Saketkoo, None; E. Schiopu, Octapharma, 2; D. Khanna, Bayer, 2, BMS, 2, Horizon, 2, Pfizer, 2, NIH, 2, Immune Tolerance Network, 2, Eicos Sciences Inc, 4, Acceleron,, 5, Actelion,, 5, Abbvie,, 5, Amgen,, 5, Bayer,, 5, Boehringer Ingelheim, 5, CSL Behring, 5, Corbus,, 5, Galapagos,, 5, Genentech/Roche, 5, GSK, 5, Horizon, 5, Merck,, 5, Mitsubishi Tanabe Pharma, 5, Sanofi-Aventis, 5, United Therapeutics, 5, Impact PH, 9, Scleroderma Development, 6, CiviBioPharma/Eicos Sciences Inc, 6; J. Gordon, None; L. Criswell, None; H. Gladue, None; C. Derk, None; E. Bernstein, None; S. Bridges, None; V. Shanmugam, None; K. Kolstad, None; L. Chung, Eicos, 1, Reata, 1, Boehringer Ingelheim, 1, 2, Mitsubishi Tanabe, 1; S. Kafaja, None; R. Jan, None; M. Trojanowski, None; A. Goldberg, None; B. Korman, None; M. Hinchcliff, None; S. Chandrasekharappa, None; M. Gadina, None; D. Randazzo, None; S. Dell'Orso, None; A. Adeyemo, None; C. Rotimi, None; E. Remmers, None; F. Wigley, None; R. Casellas, None; D. Kastner, None; F. Boin, None; P. Gourh, None.

To cite this abstract in AMA style:

Kaundal U, Hartman J, Borden C, Wang J, Shah A, Mayes M, Doumatey A, Bentley A, Shriner D, Domsic R, Medsger T, Ramos P, Silver R, Steen V, Varga J, Hsu V, Saketkoo L, Schiopu E, Khanna D, Gordon J, Criswell L, Gladue H, Derk C, Bernstein E, Bridges S, Shanmugam V, Kolstad K, Chung L, Kafaja S, Jan R, Trojanowski M, Goldberg A, Korman B, Hinchcliff M, Chandrasekharappa S, Gadina M, Randazzo D, Dell'Orso S, Adeyemo A, Rotimi C, Remmers E, Wigley F, Casellas R, Kastner D, Boin F, Gourh P. Intergenic HLA Variants in African American Patients with Systemic Sclerosis Regulate Expression of HLA-DRB1 [abstract]. Arthritis Rheumatol. 2020; 72 (suppl 10). https://acrabstracts.org/abstract/intergenic-hla-variants-in-african-american-patients-with-systemic-sclerosis-regulate-expression-of-hla-drb1/. Accessed .

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