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Systems Analysis Implicates WAVE2&Nbsp JACC: BASIC TO TRANSLATIONAL SCIENCE VOL.5,NO.4,2020 ª 2020 THE AUTHORS. PUBLISHED BY ELSEVIER ON BEHALF OF THE AMERICAN COLLEGE OF CARDIOLOGY FOUNDATION. THIS IS AN OPEN ACCESS ARTICLE UNDER THE CC BY-NC-ND LICENSE (http://creativecommons.org/licenses/by-nc-nd/4.0/). PRECLINICAL RESEARCH Systems Analysis Implicates WAVE2 Complex in the Pathogenesis of Developmental Left-Sided Obstructive Heart Defects a b b b Jonathan J. Edwards, MD, Andrew D. Rouillard, PHD, Nicolas F. Fernandez, PHD, Zichen Wang, PHD, b c d d Alexander Lachmann, PHD, Sunita S. Shankaran, PHD, Brent W. Bisgrove, PHD, Bradley Demarest, MS, e f g h Nahid Turan, PHD, Deepak Srivastava, MD, Daniel Bernstein, MD, John Deanfield, MD, h i j k Alessandro Giardini, MD, PHD, George Porter, MD, PHD, Richard Kim, MD, Amy E. Roberts, MD, k l m m,n Jane W. Newburger, MD, MPH, Elizabeth Goldmuntz, MD, Martina Brueckner, MD, Richard P. Lifton, MD, PHD, o,p,q r,s t d Christine E. Seidman, MD, Wendy K. Chung, MD, PHD, Martin Tristani-Firouzi, MD, H. Joseph Yost, PHD, b u,v Avi Ma’ayan, PHD, Bruce D. Gelb, MD VISUAL ABSTRACT Edwards, J.J. et al. J Am Coll Cardiol Basic Trans Science. 2020;5(4):376–86. ISSN 2452-302X https://doi.org/10.1016/j.jacbts.2020.01.012 JACC: BASIC TO TRANSLATIONALSCIENCEVOL.5,NO.4,2020 Edwards et al. 377 APRIL 2020:376– 86 WAVE2 Complex in LVOTO HIGHLIGHTS ABBREVIATIONS AND ACRONYMS Combining CHD phenotype–driven gene set enrichment and CRISPR knockdown screening in zebrafish is an effective approach to identifying novel CHD genes. CHD = congenital heart disease Mutations affecting genes coding for the WAVE2 protein complex and small GTPase-mediated signaling CORUM = Comprehensive are associated with LVOTO lesions. Resource of Mammalian WAVE2 complex genes brk1, nckap1,andwasf2 and regulators of small GTPase signaling cul3a and racgap1 Protein Complexes are critical to zebrafish heart development. CRISPR = clustered regularly interspaced short palindromic repeats CTD = conotruncal defect SUMMARY GOBP = Gene Ontology biological processes Genetic variants are the primary driver of congenital heart disease (CHD) pathogenesis. However, our ability to HHE = high heart expression fi identify causative variants is limited. To identify causal CHD genes that are associated with speci c molecular HLHS = hypoplastic left heart functions, the study used prior knowledge to filter de novo variants from 2,881 probands with sporadic severe syndrome CHD. This approach enabled the authors to identify an association between left ventricular outflow tract HTX = heterotaxy obstruction lesions and genes associated with the WAVE2 complex and regulation of small GTPase-mediated LVOTO = left ventricular signal transduction. Using CRISPR zebrafish knockdowns, the study confirmed that WAVE2 complex proteins outflow tract obstruction brk1, nckap1, and wasf2 and the regulators of small GTPase signaling cul3a and racgap1 are critical to cardiac MGI = Mouse Genome development. (J Am Coll Cardiol Basic Trans Science 2020;5:376–86) © 2020 The Authors. Published by Informatics Elsevier on behalf of the American College of Cardiology Foundation. This is an open access article under the PCGC = Pediatric Cardiac Genomics Consortium CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). PPI = protein-protein interaction From the aDepartment of Pediatrics, Division of Pediatric Cardiology, Children’s Hospital of Philadelphia, Philadelphia, Penn- sylvania; bDepartment of Pharmacological Sciences, Mount Sinai Center for Bioinformatics, LINCS-BD2K DCIC, Icahn School of Medicine at Mount Sinai, New York, New York; cDepartment of Molecular Physiology and Biophysics, Vanderbilt School of Medicine, Nashville, Tennessee; dMolecular Medicine Program, University of Utah School of Medicine, Salt Lake City, Utah; eCoriell Institute, Camden, New Jersey; fGladstone Institute of Cardiovascular Disease, San Francisco, California; gDivision of Pediatric Cardiology, Stanford University School of Medicine, Stanford University, Stanford, California; hDepartment of Cardiol- ogy, Great Ormond Street Hospital, University College London, London, United Kingdom; iDepartment of Pediatrics, University of Rochester Medical Center, University of Rochester School of Medicine and Dentistry, Rochester, New York; jSection of Cardio- thoracic Surgery, Keck School of Medicine of USC, University of Southern California, Los Angeles, California; kDepartment of Cardiology, Children’s Hospital Boston, Boston, Massachusetts; lDepartment of Pediatrics, Perelman School of Medicine, Uni- versity of Pennsylvania, Philadelphia, Pennsylvania; mDepartment of Genetics, Yale School of Medicine, New Haven, Connecticut; nHoward Hughes Medical Institute, Yale University, New Haven, Connecticut; oDepartment of Genetics, Harvard Medical School, Boston, Massachusetts; pHoward Hughes Medical Institute, Harvard University, Boston, Massachusetts; qCardiovascular Division, Brigham and Women’s Hospital, Harvard University, Boston, Massachusetts; rDepartment of Pediatrics, Columbia University Medical Center, New York, New York; sDepartment of Medicine, Columbia University Medical Center, New York, New York; tNora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah School of Medicine, Salt Lake City, Utah; uMindich Child Health and Development Institute, Icahn School of Medicine at Mount Sinai, New York, New York; and the vDepartment of Pediatrics, Icahn School of Medicine at Mount Sinai, New York, New York. This work was supported by a grant from the National Center for Research Resources and the National Center for Advancing Translational Sciences (U01 HL098153), National Institutes of Health grants to the Pediatric Cardiac Genomics Consortium (U01-HL098188, U01-HL098147, U01-HL098153, U01-HL098163, U01-HL098123, U01-HL098162, and U01-HL098160), and the National Institutes of Health Centers for Mendelian Genomics (5U54HG006504). Dr. Edwards was supported by National Institutes of Health Grant No. 5T32HL007915. Drs. Lifton and Seidman were supported by the Howard Hughes Medical Institute. Dr. Chung was supported by the Simons Foundation. Dr. Srivastava is co-founder and has served on the scientific advisory board for Tenaya Therapeutics. Dr. Lifton is director of Roche; has served on the scientific advisory board for Regeneron; and has served as a consultant for Johnson and Johnson. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose. The authors attest they are in compliance with human studies committees and animal welfare regulations of the authors’ in- stitutions and Food and Drug Administration guidelines, including patient consent where appropriate. For more information, visit the JACC: Basic to Translational Science author instructions page. Manuscript received November 19, 2019; revised manuscript received January 23, 2020, accepted January 24, 2020. 378 Edwards et al. JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 5, NO. 4, 2020 WAVE2 Complex in LVOTO APRIL 2020:376– 86 ongenital heart disease (CHD) is the most sequencing from 900 control trios in Simon’s Foun- C common clinically devastating birth defect dation Autism Research Initiative Simplex Collection (1). Among the multiple factors that drive (6). The research protocols were approved by the CHD pathogenesis, genetic variants appear to be a Institutional Review Boards at each participating primary driver (2,3). Despite greater understanding center—Boston’sChildren’s Hospital, Brigham and of the molecular mechanisms of heart development, Women’s Hospital, Great Ormond Street Hospital, our ability to definitively identify specificgenetic Children’s Hospital of Los Angeles, Children’sHos- causes for most patients has progressed more slowly. pital of Philadelphia, Columbia University Irving A barrier to unraveling genotype-phenotype asso- Medical Center, Icahn School of Medicine at Mount ciations is the high degree of genetic heterogeneity, Sinai, Rochester School of Medicine and Dentistry, such that analysis of even a moderate-sized cohort Steven and Alexandra Cohen Children’sMedical results in a relatively low number of genes observed Center of New York, and Yale School of Medicine. to be mutated recurrently (4,5). Hence, identifying Briefly, sequencing for case and control trios was novel CHD genes is well suited for network analysis, performed at Yale Center for Genome Analysis using wherein discrete mutations and affected genes can be NimbleGen v2.0 exome capture reagent (Roche, connected based on prior knowledge of gene-gene Basel, Switzerland) and Illumina HiSeq 2000, 75-bp functional networks such as associations with devel- paired-end reads (Illumina, San Diego, California). opmental pathways and known protein-protein Three independent analysis pipelines were used to interactions. Together, these functional data can process reads and were mapped to hg19 using provide a platform to identify molecular pathways Novoalign (Novocraft Technologies, Selangor, that link variants to prioritize identification and lead Malaysia) and Genome Analysis Toolkit 3.0 (Broad to the discovery of novel candidate causal CHD genes. Institute, Cambridge, Massachusetts) best practices at Validation of selected genes in model systems will Harvard Medical School and BWA-mem at Yale School ultimately expand our ability to detect genetic of Medicine and Columbia University Medical Center etiologies for patients. (8,9), Variant calls were made using GATK Hap- Initial whole exome sequencing from the Pediatric lotypeCaller
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