Fifty-two Genetic Loci Influencing Myocardial Mass Pim van der Harst1,2,3*†, MD, PhD, Jessica van Setten4,5†, PhD, Niek Verweij1†, PhD, Georg Vogler6†, PhD, Lude Franke2†, PhD, Matthew T. Maurano7,8,9,10†, PhD, Xinchen Wang11†, BSc, Irene Mateo Leach1†, PhD, Mark Eijgelsheim12,13, MD, PhD, Nona Sotoodehnia14 MD, MPH, Caroline Hayward15, PhD, Rossella Sorice16, PhD, Osorio Meirelles17, PhD, Leo-Pekka Lyytikäinen18,19, MD, Ozren Polašek20,21, MD, PhD, Toshiko Tanaka22, PhD, Dan E. Arking23, PhD, Sheila Ulivi24, PhD, Stella Trompet25,26, PhD, Martina Müller-Nurasyid27,28,29,30, PhD, Albert V. Smith31,32, PhD, Marcus Dörr33,34, MD, Kathleen F. Kerr35, PhD, Jared W. Magnani36, MD, MSc, Fabiola Del Greco M.37, PhD, Weihua Zhang38,39, PhD, Ilja M. Nolte40, PhD, Claudia T. Silva41,42,43, MSc, Sandosh Padmanabhan44, MD, PhD, Vinicius Tragante4,5, PhD, Tõnu Esko45,46, PhD, Gonçalo R. Abecasis47, PhD, Michiel E. Adriaens48,49, PhD, Karl Andersen32,50, PhD, Phil Barnett51, PhD, Joshua C. Bis52, PhD, Rolf Bodmer6, PhD, Brendan M. Buckley53, MD, PhD, Harry Campbell20, MD, Megan V. Cannon1, PhD, Aravinda Chakravarti23, PhD, Lin Y. Chen54, MD, MS, Alessandro Delitala55, PhD, Richard B. Devereux56, MD, Pieter A. Doevendans5, MD, PhD, Anna F. Dominiczak44, MD, FRCP, FAHA, FESC, FRSE, FMediSci, Luigi Ferrucci22, MD, PhD, Ian Ford57, PhD, Christian Gieger29,58,59, PhD, Tamara B. Harris60, PhD, Eric Haugen7, Matthias Heinig61,62,63, PhD, Dena G. Hernandez64, MSc, Hans L. Hillege1, MD, PhD, Joel N. Hirschhorn46,65,66,MD, PhD, Albert Hofman12,13, MD, PhD, Norbert Hubner61,67, MD, Shih-Jen Hwang68, PhD, Annamaria Iorio69, MD, Mika Kähönen70,71, MD, PhD, Manolis Kellis72,73, PhD, Ivana Kolcic21, MD, PhD, Ishminder K. Kooner39, MD, PhD, Jaspal S. Kooner39,74, MBBS, FRCP, MD, Jan A. Kors75, PhD, Edward G. Lakatta76, MD, Kasper Lage73,77,78, PhD, Lenore J. Launer60, PhD, Daniel Levy79, MD, Alicia Lundby80,81, PhD, Peter W. Macfarlane82, DSc, FRSE, Dalit May83, MD, Thomas 1 Meitinger30,84,85, MD, Andres Metspalu45, PhD, Stefania Nappo16, MSc, Silvia Naitza55, PhD, Shane Neph7, BS, Alex S. Nord86,87, PhD, Teresa Nutile16, PhD, Peter M. Okin56, MD, Jesper V. Olsen80, PhD, Ben A. Oostra41, PhD, Josef M. Penninger88, MD, Len A. Pennacchio86,89, PhD, Tune H. Pers46,65,90,91, PhD, Siegfried Perz58,92, MSc, Annette Peters30,58, PhD, Yigal M. Pinto48, MD, PhD, Arne Pfeufer37,93, MD, MSc, Maria Grazia Pilia55, PhD, Peter P. Pramstaller37,94,95, MD, Bram P. Prins96, MSc, Olli T. Raitakari97,98, MD, PhD, Soumya Raychaudhuri99,100, MD, PhD, Ken M. Rice35, PhD, Elizabeth J. Rossin78,101, MD, PhD, Jerome I. Rotter102, MD, Sebastian Schafer61,67,103, PhD, David Schlessinger17, PhD, Carsten O. Schmidt104, PhD, Jobanpreet Sehmi39,74, MRCP, PhD, MD, Herman H.W. Silljé1, PhD, Gianfranco Sinagra69, MD, FESC, Moritz F. Sinner27 MD, MPH, Kamil Slowikowski105, BS, Elsayed Z. Soliman106, MD, MSc, Timothy D. Spector107, MBBS, MRCP, MD, MSc, FRCP, Wilko Spiering108, MD, PhD, John A. Stamatoyannopoulos7, MD, Ronald P. Stolk40, MD, PhD, Konstantin Strauch28,29, PhD, Sian-Tsung Tan39,74, MD, Kirill V. Tarasov76, MD, PhD, Bosco Trinh6, BSc, Andre G. Uitterlinden12,13, PhD, Malou van den Boogaard51, MD, Cornelia M. van Duijn41, PhD, Wiek H. van Gilst1, PhD, Jorma S. Viikari109,110, MD, PhD, Peter M. Visscher111,112, PhD, Veronique Vitart15, PhD, Uwe Völker34,113, PhD, Melanie Waldenberger58,59, PhD, MPH, Christian X. Weichenberger37, PhD, Harm-Jan Westra65,114,115, PhD, Cisca Wijmenga2, PhD, Bruce H. Wolffenbuttel116, MD, PhD, Jian Yang111, PhD, Connie R. Bezzina48, PhD, Patricia B. Munroe117,118, PhD, Harold Snieder40, PhD, Alan F. Wright15, MD, PhD, Igor Rudan20, MD, PhD, Laurie A. Boyer11, PhD, Folkert W. Asselbergs3,5,119, MD, PhD, Dirk J. van Veldhuisen1, MD, PhD, Bruno H. Stricker12,13, MD, PhD, Bruce M. Psaty120,121, MD, PhD, Marina Ciullo16,122, PhD, Serena Sanna55, PhD, Terho Lehtimäki18,19, MD, PhD, James F. Wilson15,20, PhD, Stefania Bandinelli123, MSc, Alvaro Alonso124, MD, PhD, Paolo Gasparini24,125,126, MD, J. Wouter Jukema25,127, MD, PhD, Stefan Kääb27,30, MD, PhD, Vilmundur Gudnason31,32, MD, 2 PhD, Stephan B. Felix33,34, MD, Susan R. Heckbert121,128, MD, PhD, Rudolf A. de Boer1, MD, PhD, Christopher Newton-Cheh65,129,130, MD, PHD, Andrew A. Hicks37, PhD, John C. Chambers38,39†, BA, MBBS, FRCP, PhD, Yalda Jamshidi96†, PhD, Axel Visel86,89,131†, PhD, Vincent M. Christoffels51†, PhD, Aaron Isaacs41,132†, PhD, Nilesh J. Samani133,134†, MD, FRCP, Paul I.W. de Bakker4,135†, PhD Affiliations: are provided at the end of the manuscript Word count: 4978 * To whom correspondence should be addressed: Pim van der Harst University of Groningen, University Medical Center Groningen, Department of Cardiology & Department of Genetics Hanzeplein 1, 9700RB Groningen, The Netherlands Email: [email protected] 3 Abstract BACKGROUND: Myocardial mass is a key determinant of cardiac muscle function and hypertrophy. Myocardial depolarization leading to cardiac muscle contraction is reflected by the amplitude and duration of the QRS complex on the electrocardiogram (ECG). Abnormal QRS amplitude or duration reflect changes in myocardial mass and conduction, and are associated with increased risk of heart failure and death. OBJECTIVE: To gain insights into the genetic determinants of myocardial mass, METHODS: We carried out a genome-wide association meta-analysis of 4 QRS traits in up to 73,518 individuals of European ancestry, followed by extensive biological and functional assessment. RESULTS: We identified 52 genomic loci, of which 32 are novel, reliably associated with one or more QRS phenotypes at P<1×10-8. These loci are enriched in regions of open chromatin, histone modifications, and transcription factor binding suggesting that they represent regions of the genome that are actively transcribed in the human heart. Pathway analyses provide evidence that these loci play a role in cardiac hypertrophy. We further highlight 67 candidate genes at the identified loci that are preferentially expressed in cardiac tissue and associated with cardiac abnormalities in Drosophila melanogaster and Mus musculus. We validated the regulatory function of a novel variant in the SCN5A/SCN10A locus in vitro and in vivo. CONCLUSIONS: Taken together, our findings provide new insights into genes and biological pathways controlling myocardial mass and may help identify novel therapeutic targets. Keywords: Genetic association study, QRS, left ventricular hypertrophy, heart failure, electrocardiogram, genes 4 Abbreviations list DHS Deoxyribonuclease hypersensitivity sites DNase Deoxyribonuclease ECG Electrocardiogram eQTL Expression quantitative trait locus FDR False Discovery Rate GWAS Genome-wide association study LD Linkage Disequilibrium RNAi Ribonucleic acid interference SNP Single Nucleotide Polymorphisms TF Transcription Factor 5 Introduction The role of the heart is to provide adequate circulation of blood to meet the body's requirements of oxygen and nutrients. The QRS complex on the ECG is the most widely used measurement of cardiac depolarization, which causes the ventricular muscle to contract, resulting in pulsatile blood flow. The amplitude and duration of the QRS complex reflects the conduction through the left ventricle and is well correlated with left ventricular mass as measured by echocardiography (1,2). ECG measurements of the QRS complex are important in clinical and preclinical cardiovascular diseases such as cardiac hypertrophy, heart failure, and various cardiomyopathies, and can also predict cardiovascular mortality(3-6). Identification of specific genes influencing the QRS complex may thus enhance our understanding of the human heart and ultimately lead to prevention of cardiovascular disease and death. To further our understanding of the genetic factors influencing the QRS complex, we carried out a large scale GWAS and replication study of 4 related and clinically used QRS traits: the Sokolow-Lyon, Cornell and 12- lead-voltage duration products (12-leadsum), and QRS duration. We identified 52 loci that were subsequently interrogated using bioinformatics and experimental approaches to gain more insights into the biological mechanisms regulating cardiac mass and QRS parameters. 6 Methods Additional details on the methods can be found in the Supplementary Note. Genome wide analyses and replication testing Our study design is summarized in Fig. S1. Briefly, we combined summary statistics from 24 studies for up to 2,766,983 autosomal SNPs using an inverse-variance fixed-effects meta-analysis for each QRS trait. We performed replication testing for loci showing suggestive association (1×10-8<P<5×10-7) (table S1 and S2). The threshold for genome-wide significance was set at P<1×10-8. DNA Functional elements, coding variation and enrichment analyses We performed an intersection between SNPs and regions of DHSs, covalently modified histones and genomic features (ChromHMM) of cardiac tissues mapped by the National Institutes of Health Roadmap Epigenomics Program, as well as various cardiac transcription factor binding sites (GATA4, MEF2, SRF, TBX5, TBX3, GATA4 and Nkx2-5) measured by Chip-seq. Experimental cardiac enhancer studies Single cell suspensions of human ventricular tissue was obtained by dissociation with IKA Ultra Turrax T5 FU, followed by dounce homogenization. 4C templates were mixed and sequenced simultaneously in one Illumina HiSeq 2000 lane. Enhancer candidate regions with major and minor allele for rs6781009 were obtained by PCR from human control DNA and cloned into the