1621: Genome-wide Association Study in African American Systemic Sclerosis Patients Identifies a Novel Target - Transforming Growth Factor-β3 (TGFβ3)

Urvashi Kaundal¹, Julia Hartman², Emilee Stenson¹, Sarah Safran¹, Chloe Borden¹, Mousumi Sahu¹, Janet Wang¹, Andrea Conte¹, Ami Shah³, Maureen Mayes⁴, Ayo Doumatey⁵, Amy Bentley⁵, Daniel Shriner⁵, Robyn Domsic⁶, Thomas Medsger⁷, Paula Ramos⁸, Richard Silver⁸, Virginia Steen⁹, John Varga¹⁰, Vivien Hsu¹¹, Lesley Ann Saketkoo¹², Elena Schiopu¹³, Dinesh Khanna¹⁴, Jessica Gordon¹⁵, Lindsey Criswell¹⁶, Heather Gladue¹⁷, Chris Derk¹⁸, Elana Bernstein¹⁹, S. Louis Bridges, Jr.¹⁵, Victoria Shanmugam²⁰, Lorinda Chung²¹, Suzanne Kafaja²², Reem Jan²³, Marcin Trojanowski²⁴, Avram Goldberg²⁵, Benjamin Korman²⁶, Settara Chandrasekharappa⁵, Yongbing Zhao²⁷, Stephen Brooks²⁷, Stefania Dell'Orso¹, Adebowale Adeyemo⁵, Charles Rotimi⁵, Elaine Remmers⁵, Daniel Kastner⁵, Francesco Boin²⁸, Rafael Casellas²⁷, Fredrick Wigley²⁹ and Pravitt Gourh³⁰, ¹National Institutes of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), National Institutes of Health (NIH), Bethesda, MD, ²National Institutes of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), National Institutes of Health (NIH), Washington, DC, ³Johns Hopkins Rheumatology, Baltimore, MD, ⁴Division of Rheumatology and Clinical Immunogenetics, University of Texas McGovern Medical School, Houston, TX, ⁵National Human Genome Research Institute, Bethesda, MD, ⁶University of Pittsburgh, Pittsburgh, PA, ⁷University of Pittsburgh School of Medicine, Pittsburgh, PA, ⁸Medical University of South Carolina, Charleston, SC, ⁹Georgetown University School of Medicine, Washington, DC, ¹⁰University of Michigan, Ann Arbor, MI, ¹¹Rutgers-RWJ Medical School, South Plainfield, NJ, ¹²University Medical Center - Comprehensive Pulmonary Hypertension Center and ILD Clinic Programs // New Orleans Scleroderma and Sarcoidosis Patient Care & Research Centeris, New Orleans, LA, ¹³Michigan Medicine, Ann Arbor, MI, ¹⁴Division of Rheumatology, Department of Internal Medicine, Scleroderma Program, University of Michigan, Ann Arbor, MI, ¹⁵Hospital for Special Surgery, New York, NY, ¹⁶National Human Genome Research Institute, NIH, Bethesda, MD, ¹⁷Arthritis & Osteoporosis Consultants of the Carolinas, Charlotte, NC, ¹⁸University of Pennsylvania, Philadelphia, PA, ¹⁹Columbia University, New York, NY, ²⁰George Washington University, Great Falls, VA, ²¹Stanford University, Stanford, CA, ²²UCLA Department of Medicine, Division of Rheumatology, Los Angeles, CA, ²³University of Chicago, Chicago, IL, ²⁴Boston University School of Medicine, Boston, MA, ²⁵NYU Langone Medical Center - NYU Hospital for Joint Diseases, Lake Success, NY, ²⁶University of Rochester, Rochester, NY, ²⁷National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, MD, ²⁸Cedars-Sinai Medical Center, Los Angeles, CA, ²⁹Johns Hopkins University, Baltimore, MD, ³⁰National Institutes of Health, Bethesda, MD

Background/Purpose: Transforming Growth Factor-β (TGFβ) isoforms play an important role in extracellular matrix biology. Increased TGFβ-regulated gene signature has been observed in lesional skin and lung tissues from patients with systemic sclerosis (SSc). A genome-wide association study (GWAS) from the Genome Research in African American Scleroderma Patients (GRASP) cohort has identified the top non-HLA locus, that is previously unreported, African ancestry-specific, and downstream of the TGFβ3 gene (Fig. 1A). The objective of this study is to elucidate the functional role of these variants and their role in SSc pathogenesis.

Methods: The Illumina MEGA array was used for genotyping and after quality control filtering, remaining variants were imputed into the 1000 Genome reference panel. Data from ENCODE and Genotype-Tissue Expression (GTEx) were used for Hi-C and expression quantitative trait loci (eQTL) analysis. Lymphoblastoid cell lines (LCLs) from the 1000 Genomes Project that were wildtype (WT) and homozygous (HOM) for the rs56032403 variant were selected and reprogrammed into induced pluripotent stem cells (iPSC). These LCL-iPSC lines were further differentiated into fibroblasts and validated by immunocytochemistry. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) was performed to confirm chromatin accessibility at the variant site and RNA-sequencing (RNA-Seq) was performed to analyze expression of TGFβ3. The transcription factor (TF) binding database, PROMO, was used for predicting TF binding. Gene expression data of SSc patients were obtained from GEO databases GSE181549 and GSE58095. Stimulation experiments were performed on normal human dermal fibroblasts (NHDF) using rTGFβ3.

Results: The rs56032403 variant was in the same topologically associated domain (TAD) as TGFβ3 and was an eQTL with TGFβ3 in fibroblasts (Fig. 1B, 1C). LCL-iPSC-derived fibroblasts were confirmed by fibroblast markers- Vimentin, a-SMA, Actin, Hsp47, and S100A4 (Fig. 2A). ATAC-seq highlighted that the chromatin at the site of the variant was accessible in HOM but not in WT samples (Fig. 2B). Higher expression of TGFβ3 was observed in HOM as compared to WT samples by RNA-Seq (Fig. 2C). The TF GATA2, which is expressed in fibroblasts, was predicted to bind to the alternate allele but not to the reference allele (Fig. 2D). Gene expression profiles from multiple published studies have shown increased TGFβ3 expression in the skin of SSc patients (Fig. 3 A-D). TGFb3 stimulation of NHDF resulted in increased expression of SERPINE1, COL1A1, and COMP genes that are increased in fibrosis and are previously reported to be upregulated in SSc (Fig. 3 E-G).

Conclusion: We report a novel, African ancestry-specific, non-coding variant located in the same TAD as TGFβ3 gene and provide evidence for the mechanism by which this variant regulates TGFβ3 expression and is involved in the pathogenesis of SSc. TGFβ3 is expressed in mesenchymal cells and is upregulated in SSc and other fibrotic conditions (i.e., idiopathic pulmonary fibrosis and non-alcoholic steatohepatitis). Targeting the TGFβ3 isoform specifically may be of therapeutic benefit in SSc and other fibrotic conditions.
Fig 1. A. Manhattan plot for association with SSc versus controls; B. Topologically associated domain (TAD) comprising the variant, rs56032403 (green line) and the TGFB3 promoter; C. rs56032403 eQTL analysis for LCLs. * normalized expression; WT- wild type; Het- heterozygous.

Fig 2. A. Fluorescent microscopy image of iPSC-LCL-derived fibroblasts stained for Vimentin, a-SMA, Actin, Hsp47, S100A4 (green) and nuclei (blue); B. ATAC-seq tracks for LCL-derived fibroblasts homozygous and WT for rs56032403 variants (top variant in green); C. RNA-seq for TGFB3 expression D. Transcription factor binding prediction at site of rs56032403 variant. *normalized expression; hom- homozygous LCL-iPSC-derived fibroblasts; WT- wild type LCL-iPSC-derived fibroblasts.

Fig 3. Differential expression of TGFB3 in skin samples of A, C. SSc patients and healthy controls, B, D. African American SSc patients and healthy controls. Differential expression of E. SERPINE1, F. COL1A1, G. COMP in untreated (-rTGFB3) or rTGFB3 treated (+ rTGFB3) human dermal fibroblasts. *normalized expression.
Disclosures: U. Kaundal, None; J. Hartman, None; E. Stenson, None; S. Safran, None; C. Borden, None; M. Sahu, None; J. Wang, None; A. Conte, None; A. Shah, Arena Pharmaceuticals, Medpace/Eicos, Kadmon Corporation; M. Mayes, Actelion Pharma, Mitsubishi-Tanabe, Boehringer Ingelheim, EICOS, Horizon Pharma, Prometheus, Corbus, Medtelligence; A. Doumatey, None; A. Bentley, None; D. Shriner, None; R. Domsic, None; T. Medsger, None; P. Ramos, None; R. Silver, None; V. Steen, None; J. Varga, Boehringer-Ingelheim; V. Hsu, None; L. Saketkoo, None; E. Schiopu, None; D. Khanna, Boehringer Ingelheim, Genentech, Prometheus, Horizon, Chemomab, Talaris, Gesynta, Amgen, Acceleron, Actelion, Bayer, CSL Behring, Paracrine Cell Therapy, Mitsubishi Tanabe, Theraly, Eicos Sciences; J. Gordon, None; L. Criswell, None; H. Gladue, GlaxoSmithKlein(GSK), AstraZeneca; C. Derk, None; E. Bernstein, Boehringer-Ingelheim, Kadmon, Pfizer; S. Bridges, Jr., Bristol Myers Squibb; V. Shanmugam, None; L. Chung, Kyverna, Mitsubishi Tanabe, Eicos, Boehringer-Ingelheim, Jasper, Genentech; S. Kafaja, None; R. Jan, None; M. Trojanowski, None; A. Goldberg, None; B. Korman, None; S. Chandrasekharappa, None; Y. Zhao, None; S. Brooks, None; S. Dell'Orso, None; A. Adeyemo, None; C. Rotimi, None; E. Remmers, None; D. Kastner, None; F. Boin, None; R. Casellas, None; F. Wigley, None; P. Gourh, None.