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High blood pressure (BP) is the major risk factor for cardiovascular disease. Genome-wide association studies have identified genetic variants for BP, but functional insights into causality and related molecular mechanisms lag behind. We functionally characterize 4,608 genetic variants in linkage with 135 BP loci in vascular smooth muscle cells and cardiomyocytes by massively parallel reporter assays. High densities of regulatory variants at BP loci (i.e., ULK4, MAP4, CFDP1, PDE5A) indicate that multiple variants drive genetic association. Regulatory variants are enriched in repeats, alter cardiovascular-related transcription factor motifs, and spatially converge with genes controlling specific cardiovascular pathways. Using heuristic scoring, we define likely causal variants, and CRISPR prime editing finally determines causal variants for KCNK9, SFXN2, and PCGF6, which are candidates for developing high BP. Our systems-level approach provides a catalog of functionally relevant variants and their genomic architecture in two trait-relevant cell lines for a better understanding of BP gene regulation. We thank the Melé and Maass labs for intellectual input, Dr. Steven Erwood from Dr. Ronald Cohn’s lab for guidance in applying CRISPR prime editing, The Centre for Applied Genomics, The Structural & Biophysical Core Facility, and The Imaging Facility, The Hospital for Sick Children, Toronto, Canada, for assistance with high-throughput sequencing, luminescence detection, and imaging. We thank Dovetail Genomics, LLC, 100 Enterprise Way, Suite A101, Scotts Valley, CA 95066, USA, for generating Omni-C libraries and for the collaborative support throughout the project. W.O. was supported by a Fundació la Marató grant (ref. 321/C/2019), K.K. was supported by an OGS fellowship, J.W.L.B. was supported by a CGS-D fellowship, and D.F.L. was supported by an Ontario Genomics-CANSSI Ontario Postdoctoral Fellowship in Genome Data Science. This project was supported by Canada’s New Frontiers in Research Fund (NFRFE-2018-01305), the Canadian Institutes of Health Research (CIHR PJT 173542 [P.G.M.] and PJT 175034 [S.M., J.E.]), CIHR ENP 161429 under the frame of ERA PerMed (S.M.), the Ted Rogers Centre for Heart Research (S.M., J.E.), and the Heart and Stroke Foundation of Canada. J.E. holds a Canada Research Chair Tier 1 in Stem Cell Models of Childhood Disease, S.M. holds the Heart and Stroke Foundation of Canada & Robert M. Freedom Chair in Cardiovascular Science, M. Melé was supported by a Ramon y Cajal fellowship (RYC-2017-22249), and P.G.M. holds a Canada Research Chair Tier 2 in Non-coding Disease Mechanisms. Peer Reviewed "Article signat per 14 autors/es: Winona Oliveros, Kate Delfosse, Daniella F. Lato , Katerina Kiriakopulos, Milad Mokhtaridoost, Abdelrahman Said, Brandon J. McMurray, Jared W.L. Browning, Kaia Mattioli, Guoliang Meng, James Ellis, Seema Mital, Marta Melé, Philipp G. Maass"

Cardiomyopathy (CMP) is a heritable disorder. Over 50% of cases are gene-elusive on clinical gene panel testing. The contribution of variants in non-coding DNA elements that result in cryptic splicing and regulate gene expression has not been explored. We analyzed whole-genome sequencing (WGS) data in a discovery cohort of 209 pediatric CMP patients and 1953 independent replication genomes and exomes. We searched for protein-coding variants, and non-coding variants predicted to affect the function or expression of genes. Thirty-nine percent of cases harbored pathogenic coding variants in known CMP genes, and 5% harbored high-risk loss-of-function (LoF) variants in additional candidate CMP genes. Fifteen percent harbored high-risk regulatory variants in promoters and enhancers of CMP genes (odds ratio 2.25, p = 6.70 × 10−7 versus controls). Genes involved in α-dystroglycan glycosylation (FKTN, DTNA) and desmosomal signaling (DSC2, DSG2) were most highly enriched for regulatory variants (odds ratio 6.7–58.1). Functional effects were confirmed in patient myocardium and reporter assays in human cardiomyocytes, and in zebrafish CRISPR knockouts. We provide strong evidence for the genomic contribution of functionally active variants in new genes and in regulatory elements of known CMP genes to early onset CMP. This project was supported by the Ted Rogers Centre for Heart Research (SM, JE), the Canadian Institutes of Health Research (PJT 175034) (SM, JE) and by the Canadian Institutes of Health Research (ENP 161429), under the frame of ERA PerMed (SM). SM holds the Heart and Stroke Foundation of Canada & Robert M Freedom Chair in Cardiovascular Science. SWS holds the GlaxoSmithKline Endowed Chair in Genome Sciences at the Hospital for Sick Children and the University of Toronto. PGM holds a Canada Research Chair Tier 2 in Non-coding Disease Mechanisms. PGM acknowledges the support of the Government of Canada’s New Frontiers in Research Fund (NFRF), [NFRFE-2018-01305]. EO holds the Bitove Family Professorship of Adult Congenital Heart Disease. MM holds a Ramon y Cajal grant from the Spanish Ministry of Science and Innovation (RYC-2017-22249). WO is supported by funding from Fundació La Marató (321/C/2019). JB is funded by a Frans Van de Werf fellowship for clinical cardiovascular research, and by a senior clinical investigator fellowship of the FWO Flanders. KM was a National Science Foundation Graduate Research Fellow under grant no. DGE1144152 during the majority of the project. CS is the recipient of a National Health and Medical Research Council (NHMRC) Practitioner Fellowship (1154992). JI is the recipient of an NHMRC Career Development Fellowship (1162929). RDB is the recipient of a New South Wales Health Cardiovascular Disease Senior Scientist Grant. PSD is supported by the DBT/Wellcome Trust- Indian Alliance. We acknowledge the Labatt Family Heart Centre Biobank at the Hospital for Sick Children for access to DNA samples, and The Centre for Applied Genomics at the Hospital for Sick Children for performing WGS. We thank Xiucheng Cui and Emanuela Pannia for performing the zebrafish experiments at the SickKids Zebrafish Genetics and Disease Models Core (CRISPR-Cas9 and gRNA syntheses, zebrafish embryo microinjections, gRNA PCR validation, qRT-PCR, cardiac imaging). This research was made possible through access to the data and findings generated by the 100,000 Genomes Project. The 100,000 Genomes Project is managed by Genomics England Limited (a wholly owned company of the Department of Health and Social Care). The 100,000 Genomes Project is funded by the National Institute for Health Research and NHS England. The Wellcome Trust, Cancer Research UK and the Medical Research Council have also funded research infrastructure. The 100,000 Genomes Project uses data provided by patients and collected by the National Health Service as part of their care and support. We thank members of the ICGC/PCAWG working groups for generating the variant calls used in our case-control burden analyses. Peer Reviewed "Article signat per 38 autors/es: Robert Lesurf, Abdelrahman Said, Oyediran Akinrinade, Jeroen Breckpot, Kathleen Delfosse, Ting Liu, Roderick Yao, Gabrielle Persad, Fintan McKenna, Ramil R. Noche, Winona Oliveros, Kaia Mattioli, Shreya Shah, Anastasia Miron, Qian Yang, Guoliang Meng, Michelle Chan Seng Yue, Wilson W. L. Sung, Bhooma Thiruvahindrapuram, Jane Lougheed, Erwin Oechslin, Tapas Mondal, Lynn Bergin, John Smythe, Shashank Jayappa, Vinay J. Rao, Jayaprakash Shenthar, Perundurai S. Dhandapany, Christopher Semsarian, Robert G. Weintraub, Richard D. Bagnall, Jodie Ingles, Genomics England Research Consortium, Marta Melé, Philipp G. Maass, James Ellis, Stephen W. Scherer & Seema Mital"