bioRxiv preprint doi: https://doi.org/10.1101/2021.01.25.428086; this version posted January 28, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Genome-wide analysis of mitochondrial DNA copy number reveals multiple loci 2 implicated in nucleotide metabolism, platelet activation, and megakaryocyte proliferation 3 Longchamps RJ1*, Yang SY1*, Castellani CA1,2, Shi W1, Lane J3, Grove ML4, Bartz 4 TM5, Sarnowski C6, Burrows K7,8, Guyatt AL9, Gaunt TR7,8, Kacprowski T10, Yang 5 J11, De Jager PL12,13, Yu L11, CHARGE Aging and Longevity Group, Bergman A14, 6 Xia R15, Fornage M15,16, Feitosa MF17, Wojczynski MK17, Kraja AT 17, Province MA 17, Amin 7 N 18, Rivadeneira F19, Tiemeier H18,20, Uitterlinden AG18,19, Broer L19, Van Meurs JBJ18,19, Van 8 Duijn CM18, Raffield LM21, Lange L22, Rich SS23, Lemaitre RN24, Goodarzi 9 MO25, Sitlani CM24, Mak ACY26, Bennett DA11, Rodriguez S7,8, Murabito JM27, Lunetta 10 KL6, Sotoodehnia N28, Atzmon G29, Kenny Y30, Barzilai N31, Brody JA32, Psaty BM33, Taylor 11 KD34, Rotter JI34, Boerwinkle E4,35, Pankratz N3, Arking DE1 12 13 * Indicates authors contributed equally to this work. 14 1 McKusick-Nathans Institute, Department of Genetic Medicine, Johns 15 Hopkins University School of Medicine, Baltimore, MD 16 2 Department of Pathology and Laboratory Medicine, Western University, London, ON, Canada 17 3 Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, 18 Minneapolis, MN 19 4 Human Genetics Center, Department of Epidemiology, Human Genetics, and Environmental 20 Sciences, School of Public Health, The University of Texas Health Science Center at Houston, 21 Houston, TX 22 5 Cardiovascular Health Research Unit, Departments of Medicine and Biostatistics, University of 23 Washington, Seattle, WA 24 6 Department of Biostatistics, Boston University School of Public Health, Boston, MA 25 7 MRC Integrative Epidemiology Unit at the University of Bristol, University of Bristol, Oakfield 26 House, Oakfield Grove, Bristol, UK bioRxiv preprint doi: https://doi.org/10.1101/2021.01.25.428086; this version posted January 28, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 27 8 Population Health Sciences, Bristol Medical School, University of Bristol, Oakfield House, 28 Oakfield Grove, Bristol, UK 29 9 Department of Health Sciences, University of Leicester, University Road, Leicester, UK 30 10 Department of Functional Genomics, Interfaculty Institute for Genetics and Functional 31 Genomics, University of Greifswald, Greifswald, Germany; Division Data Science in 32 Biomedicine, Peter L. Reichertz Institute for Medical Informatics, TU Braunschweig and 33 Hannover Medical School, Brunswick, Germany 34 11 Rush Alzheimer’s Disease Center & Department of Neurological Sciences, Rush University 35 Medical Center, Chicago, IL 36 12 Center for Translational and Systems Neuroimmunology, Department of Neurology, 37 Columbia University Medical Center, New York, NY 38 13 Program in Medical and Population Genetics, Broad Institute, Cambridge, MA 39 14 Department of Systems and Computational Biology, Albert Einstein College of Medicine, 40 Bronx, New York, USA 41 15 Institute of Molecular Medicine, The University of Texas health Science Center at Houston, 42 Houston TX 43 16 Human Genetics Center, The University of Texas Health Science Center at Houston, 44 Houston 45 17 Division of Statistical Genomics, Department of Genetics, Washington University School of 46 Medicine 47 18 Department of Epidemiology, Erasmus Medical Center, Rotterdam, The Netherlands 48 19 Department of Internal Medicine, Erasmus Medical Center, Rotterdam, The Netherlands 49 20 Department of Social and Behavioral Science, Harvard T.H. School of Public Health, Boston, 50 USA 51 21 Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC bioRxiv preprint doi: https://doi.org/10.1101/2021.01.25.428086; this version posted January 28, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 52 22 Department of Medicine, University of Colorado Denver, Anschutz Medical Campus, Aurora, 53 CO 54 23 Center for Public Health Genomics, University of Virginia, Charlottesville, Virginia, USA 55 24 Cardiovascular Health Research Unit, Department of Medicine, University of Washington, 56 Seattle, WA 57 25 Division of Endocrinology, Diabetes and Metabolism, Cedars-Sinai Medical Center, Los 58 Angeles, CA 59 26 Cardiovascular Research Institute and Institute for Human Genetics, University of California, 60 San Francisco, California 61 27 Boston University School of Medicine, Boston University, Boston, MA 62 28 Cardiovascular Health Research Unit, Division of Cardiology, University of Washington, 63 Seattle, WA 64 29 Department of Natural science, University of Haifa, Haifa, Israel; Departments of Medicine 65 and Genetics, Albert Einstein College of Medicine, Bronx, NY, 10461, USA. 66 30 Department of Epidemiology and Population Health, Albert Einstein College of Medicine, 67 Bronx, NY, 10461, USA. 68 31 Departments of Medicine and Genetics, Albert Einstein College of Medicine, Bronx, NY, 69 10461, USA. 70 32 Cardiovascular Health Research Unit, Department of Medicine, University of Washington, 71 Seattle, WA 72 33 Cardiovascular Health Research Unit, Departments of Epidemiology, Medicine and Health 73 Services, University of Washington, Seattle, WA 74 34 The Institute for Translational Genomics and Population Sciences, Department of Pediatrics, 75 The Lundquist Institute for Biomedical Innovation at Harbor-UCLA Medical Center, Torrance, 76 CA 77 35 Baylor College of Medicine, Human Genome Sequencing Center, Houston, TX bioRxiv preprint doi: https://doi.org/10.1101/2021.01.25.428086; this version posted January 28, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 78 79 Abstract 80 Blood-derived mitochondrial DNA copy number (mtDNA-CN) is a minimally invasive 81 proxy measure of mitochondrial function that exhibits both inter-individual and intercellular 82 variation. While mtDNA-CN has been previously associated with various aging-related diseases, 83 little is known about the genetic factors that may modulate this phenotype. We performed a 84 genome-wide association study (GWAS) in 465,809 individuals of White (European) ancestry 85 from the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) 86 consortium and the UK Biobank (UKB). We identified 129 SNPs with statistically significant, 87 independent effects associated with mtDNA-CN across 96 loci. A combination of fine-mapping, 88 variant annotation, co-localization, and gene set enrichment analyses were used to prioritize 89 genes within each of the 129 independent sites. Putative causal genes were enriched for known 90 mitochondrial DNA depletion syndromes (p = 3.09 x 10-15) and the gene ontology (GO) terms for 91 mtDNA metabolism (p = 1.43 x 10-8) and mtDNA replication (p = 1.2 x 10-7). A clustering 92 approach leveraged pleiotropy between mtDNA-CN associated SNPs and 42 mtDNA-CN 93 associated phenotypes to identify functional domains, revealing five distinct groups, including 94 platelet activation, megakaryocyte proliferation, and mtDNA metabolism. In conclusion, in a 95 GWAS of mtDNA-CN conducted in >450,000 individuals, we identified SNPs within loci that 96 implicate novel pathways that provide a framework for defining the underlying mechanisms 97 involved in genetic control of mtDNA-CN. 98 99 100 Introduction 101 Mitochondria are the cellular organelles primarily responsible for producing the chemical energy 102 required for metabolism, as well as signaling the apoptotic process, maintaining homeostasis, 103 and synthesizing several macromolecules such as lipids, heme and iron-sulfur clusters1,2. bioRxiv preprint doi: https://doi.org/10.1101/2021.01.25.428086; this version posted January 28, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 104 Mitochondria possess their own genome (mtDNA); a circular, intron-free, double-stranded, 105 haploid, ~ 16.6 kb maternally inherited molecule encoding 37 genes vital for proper 106 mitochondrial function. Due to the integral role of mitochondria in cellular metabolism, 107 mitochondrial dysfunction is known to play a critical role in the underlying etiology of several 108 aging-related diseases3–5. 109 110 Unlike the nuclear genome, a large amount of variation exists in the number of copies of mtDNA 111 present within cells, tissues, and individuals. The relative copy number of mtDNA (mtDNA-CN) 112 has been shown to be positively correlated with oxidative stress6, energy reserves, and 113 mitochondrial membrane potential7. As a minimally invasive