1126: Functional NOTCH4 Variants Increase Notch Signaling and Susceptibility for Systemic Sclerosis

Urvashi Kaundal¹, Emilee Stenson¹, Mousumi Sahu¹, Krishan Kumar Thakur¹, Janet Wang¹, Ami Shah², Maureen Mayes³, Ayo Doumatey⁴, Amy Bentley⁴, Daniel Shriner⁴, Robyn Domsic⁵, Thomas Medsger⁶, Paula Ramos⁷, Richard Silver⁷, Virginia Steen⁸, John Varga⁹, Vivien Hsu¹⁰, Lesley Ann Saketkoo¹¹, Elena Schiopu¹², Dinesh Khanna¹³, Jessica Gordon¹⁴, Lindsey Criswell¹⁵, Heather Gladue¹⁶, Chris Derk¹⁷, Elana Bernstein¹⁸, S. Louis Bridges, Jr.¹⁴, Victoria Shanmugam¹⁹, Lorinda Chung²⁰, Suzanne Kafaja²¹, Reem Jan²², Marcin Trojanowski²³, Avram Goldberg²⁴, Benjamin Korman²⁵, Jim Mullikin⁴, Stefania Dell'Orso¹, Adebowale Adeyemo⁴, Charles Rotimi⁴, Elaine Remmers⁴, Daniel Kastner⁴, Fredrick Wigley²⁶, Francesco Boin²⁷ and Pravitt Gourh²⁸, ¹National Institutes of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), National Institutes of Health (NIH), Bethesda, MD, ²Johns Hopkins Rheumatology, Baltimore, MD, ³Division of Rheumatology and Clinical Immunogenetics, University of Texas McGovern Medical School, Houston, TX, ⁴National Human Genome Research Institute, Bethesda, MD, ⁵University of Pittsburgh, Pittsburgh, PA, ⁶University of Pittsburgh School of Medicine, Pittsburgh, PA, ⁷Medical University of South Carolina, Charleston, SC, ⁸Georgetown University School of Medicine, Washington, DC, ⁹University of Michigan, Ann Arbor, MI, ¹⁰Rutgers-RWJ Medical School, South Plainfield, NJ, ¹¹University Medical Center - Comprehensive Pulmonary Hypertension Center and ILD Clinic Programs // New Orleans Scleroderma and Sarcoidosis Patient Care & Research Centeris, New Orleans, LA, ¹²Michigan Medicine, Ann Arbor, MI, ¹³Division of Rheumatology, Department of Internal Medicine, Scleroderma Program, University of Michigan, Ann Arbor, MI, ¹⁴Hospital for Special Surgery, New York, NY, ¹⁵National Human Genome Research Institute, NIH, Bethesda, MD, ¹⁶Arthritis & Osteoporosis Consultants of the Carolinas, Charlotte, NC, ¹⁷University of Pennsylvania, Philadelphia, PA, ¹⁸Columbia University, New York, NY, ¹⁹George Washington University, Great Falls, VA, ²⁰Stanford University, Stanford, CA, ²¹UCLA Department of Medicine, Division of Rheumatology, Los Angeles, CA, ²²University of Chicago, Chicago, IL, ²³Boston University School of Medicine, Boston, MA, ²⁴NYU Langone Medical Center - NYU Hospital for Joint Diseases, Lake Success, NY, ²⁵University of Rochester, Rochester, NY, ²⁶Johns Hopkins University, Baltimore, MD, ²⁷Cedars-Sinai Medical Center, Los Angeles, CA, ²⁸National Institutes of Health, Bethesda, MD

Background/Purpose: Genome wide association studies (GWAS) in systemic sclerosis (SSc) have identified several genetic loci, but the search for the causal variant and gene continues. In this study, using the Genome Research in African American Scleroderma Patients (GRASP) cohort, we evaluated previously reported genes from GWAS in SSc, primarily conducted in European ancestral populations. We further characterized the functional role of two variants in the Neurogenic Locus Notch Homolog Protein 4 (NOTCH4) gene.

Methods: We identified 32 genes associated with SSc susceptibility at p-value < 10^-6 in the GWAS catalog. The gene-based sequence kernel association test (SKAT) test was used for association analysis and Bonferroni's correction for multiple testing. Expression quantitative trait loci (eQTL) analysis was performed using the Genotype-Tissue Expression (GTEx) data. Lymphoblastoid cell lines (LCLs) from the 1000 Genomes Project that were wildtype (WT) and heterozygous (HET) for the rs8192564 (a 5' UTR variant with CADD score >15) and rs17604492 (missense) were used to confirm the expression of NOTCH4 genes using RT-PCR and ELISA. Human lung microvascular endothelial cells (HULEC) were stimulated with NOTCH4 ligand-DLL4 to replicate increased signaling via this pathway.

Results: On comparing 379 AA SSc patients and 411 controls, only the NOTCH4 gene remained significant after multiple testing correction (Table 1A). This NOTCH4 association remained significant and was independent of the HLA variants, after conditional analysis. To validate these results, we examined an independent cohort of 590 patients and 360 controls and the NOTCH4 association remained significant. After multiple testing correction, diffuse cutaneous SSc, interstitial lung disease, and anti-fibrillarin antibody subsets remained statistically significant (Table 1B). eQTL analysis using GTEx data showed that both variants were associated with increased NOTCH4 expression in the HET LCLs and the rs17604492 variant also increased HEY2, (a downstream, nuclear signaling molecule for the Notch pathway) (Fig. 1B-D). Increased expression of NOTCH4 was confirmed in rs8192564 HET LCLs by RT-PCR (Fig. 1E). ELISA and western blot analysis using the lysates from LCLs confirmed the increased expression of NOTCH4 in LCLs carrying the risk alleles (Fig. 1F, G). Increased expression of the HEY2 and ACTA2 genes was observed on stimulating HULECs with DLL4 (Fig. 1 H, I). A previously published study has shown increased NOTCH4 expression in the skin of SSc patients (Fig. 2 A-D).

Conclusion: Functional variants in the NOTCH4 gene are associated with AA SSc patients and are independent of the HLA genes. Increased Notch signaling in endothelial cells can induce endothelial-to-mesenchymal transition and increased ACTA2 expression along with neoangiogenic circulation with sparse branching and dilated capillaries. SSc associated variants are associated with increased NOTCH4 expression and constitutively increase Notch4 signaling. Inhibitors of the gamma-secretase complex that cause cleavage of Notch intracellular domain have been shown to have a potent anti-fibrotic effect in different murine models of SSc and could be a potential therapeutic agent in SSc.


Fig. 1. A. NOTCH4 rs17604492 variant alters glycine to arginine at position 942; B. rs8192564 eQTL analysis for wild type (WT) and heterozygous (Het) LCLs; C, D. rs17604492 eQTL analysis for WT and het LCLs; E. RT-PCR for NOTCH4 expression in LCLs WT and het for risk variants; F. ELISA for NOTCH4 protein in LCLs WT and het for risk variants; G. Western blot for NOTCH4 protein in LCLs and endothelial cells; H. RNA-seq for HEY2; I. RNA-seq for ACTA2. *normalized expression

Fig. 2. A, B. Differential expression of NOTCH4 and HEY2 in SSc and normal skin; C, D. Differential expression of NOTCH4 and HEY2 in AA SSc and AA normal skin. *normalized expression.
Disclosures: U. Kaundal, None; E. Stenson, None; M. Sahu, None; K. Thakur, None; J. Wang, 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; J. Mullikin, None; S. Dell'Orso, None; A. Adeyemo, None; C. Rotimi, None; E. Remmers, None; D. Kastner, None; F. Wigley, None; F. Boin, None; P. Gourh, None.