ARTICLE Genome-wide Studies of Copy Number Variation and Exome Sequencing Identify Rare Variants in BAG3 as a Cause of Dilated Cardiomyopathy Nadine Norton, 1 Duanxiang Li, 1 Mark J. Rieder, 2 Jill D. Siegfried, 1 Evadnie Rampersaud, 3 Stephan Zu ¨chner, 3 Steve Mangos, 4 Jorge Gonzalez-Quintana, 1 Libin Wang, 1 Sean McGee, 2 Jochen Reiser, 4 Eden Martin, 3 Deborah A. Nickerson, 2 and Ray E. Hershberger 1, * Dilated cardiomyopathy commonly causes heart failure and is the most frequent precipitating cause of heart transplantation. Familial dilated cardiomyopathy has been shown to be caused by rare variant mutations in more than 30 genes but only ~35% of its genetic cause has been identified, principally by using linkage-based or candidate gene discovery approaches. In a multigenerational family with auto- somal dominant transmission, we employed whole-exome sequencing in a proband and three of his affected family members, and genome-wide copy number variation in the proband and his affected father and unaffected mother. Exome sequencing identified 428 single point variants resulting in missense, nonsense, or splice site changes. Genome-wide copy number analysis identified 51 insertion deletions and 440 copy number variants > 1 kb. Of these, a 8733 bp deletion, encompassing exon 4 of the heat shock protein cochaperone BCL2-associated athanogene 3 (BAG3), was found in seven affected family members and was absent in 355 controls. To establish the relevance of variants in this protein class in genetic DCM, we sequenced the coding exons in BAG3 in 311 other unrelated DCM probands and identified one frameshift, two nonsense, and four missense rare variants absent in 355 control DNAs, four of which were familial and segregated with disease. Knockdown of bag3 in a zebrafish model recapitulated DCM and heart failure. We conclude that new comprehensive genomic approaches have identified rare variants in BAG3 as causative of DCM. Introduction Dilated cardiomyopathy, (DCM [MIM 115200]), a common primary myocardial disease, causes systolic dysfunction and heart failure, a major public health problem. Familial dilated cardiomyopathy has been shown to be caused by rare variant mutations in more than 30 genes, most of which encode sarcomeric or sarcomeric-associated proteins. 1,2 However, mutations in the known genes only account for ~35% of genetic causes, leaving the majority of them unknown. This highlights two observations: (1) additional rare variants in other genes remain to be found and (2) rare copy number variants (large deletions and duplications > 1 kb) have been largely ignored in DCM genetics. These types of genetic variation would be missed by traditional sequencing approaches to coding regions because of the background presence of the wild-type allele. At this time, no systematic study of the contribution of copy number variation to DCM has been performed. On these bases, we simultaneously performed exome sequencing and a high-resolution genome-wide study of copy number variation on a large multiplex family (Figure 1) with DCM. The family was previously screened for coding mutations in 15 known DCM genes and was mutation negative. 3–7 Exome sequencing was performed with NimbleGen solution-based capture and Illumina 76 bp read next-generation sequencing. Copy number variation was assessed with NimbleGen 2.1M catalog arrays, followed by NimbleGen 135K custom arrays, allow- ing detection of deletions and duplications at the resolu- tion of a single exon. Our approach identified a deletion of exon 4 of BCL2-associated athanogene 3 (BAG3) (MIM 603883) as a rare variant causative of DCM. Furthermore, post hoc analyses of our genomic data suggest that both point mutations and rare copy number variants can be identified from a single genomic platform (Figures S3A and S3B, available online), further development of which will have a significant impact on the discovery of unknown genetic causes of cardiovascular disease. Material and Methods Subjects Written, informed consent was obtained from all subjects, and the institutional review boards at the Oregon Health and Science University and the University of Miami approved the study. The investigation included 311 probands with DCM as previously described. 5,6,8 The investigation also included a total of 355 popu- lation controls: 45% male, 97% white, and 3.0% African Amer- ican. Genomic DNA was extracted from whole blood according to a standard salting-out procedure. Prior to the current study, we sequenced the coding and flanking intronic regions of 15 DCM genes in genomic DNA from these probands. 3–7 Exome Sequencing Three to five micrograms of DNA from four family members affected with DCM (pedigree A, Figure 1) were sent to the 1 Cardiovascular Division, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136-1015, USA; 2 Department of Genome Sciences, University of Washington, Seattle, WA 98195-5065, USA; 3 Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, FL 33136-1015, USA; 4 Division of Nephrology, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136-1015, USA *Correspondence: rhershberger@med.miami.edu DOI 10.1016/j.ajhg.2011.01.016. Ó2011 by The American Society of Human Genetics. All rights reserved. The American Journal of Human Genetics 88, 273–282, March 11, 2011 273