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 autosomal 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.

A great deal of progress has recently been made in the discovery and understanding of the genetics of familial dilated cardiomyopathy (FDC). A consensus has emerged that with a new diagnosis of idiopathic dilated cardiomyopathy (IDC), the clinical screening of 1st degree family members will reveal FDC in at least 20-35% of cases. Point mutations in 31 autosomal and 2 X-linked genes representing diverse gene ontogeny have been implicated in causing FDC, but account for only 30-35% of genetic cause. Next generation sequencing (NGS) methods have dramatically decreased sequencing costs, making clinical genetic testing feasible for extensive panels of DCM genes. NGS also provides opportunities to discover additional genetic cause of FDC and IDC. Guidelines for evaluation and testing of FDC and IDC are now available, and when combined with FDC genetic testing and counseling will bring FDC/IDC genetics to the forefront of cardiovascular genetic medicine.

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Background-Rare variants in Ͼ30 genes have been shown to cause idiopathic or familial dilated cardiomyopathy (DCM), but the frequency of genetic causation remains poorly understood. We have previously resequenced 9 genes in a cohort of idiopathic or familial DCM probands for rare variants, and now we report resequencing results for 5 more genes with established relationships to DCM. Methods and Results-Blood samples were collected, and DNA specimens were prepared from 312 patients, 181 with familial DCM and 131 with idiopathic DCM. Genomic DNA underwent bidirectional sequencing, and DNA of additional family members underwent analysis when a rare variant was identified. We identified rare variants in 34 probands (10.9% overall), including 29 unique protein-altering rare variants and 2 splicing variants that were absent in 246 control subjects (492 chromosomes). These variants were 12 MYBPC3 (myosin-binding protein C) in 13 (4.2%) probands, 8 MYH6 (␣-myosin heavy chain) in 10 (3.2%), 6 TPM1 (tropomyosin) in 6 (1.9%), 4 TNNC1 (cardiac troponin C) in 4 (1.3%), and 1 TNNI3 (cardiac troponin I) in 2 (0.6%). Variants were classified as likely or possibly disease causing in 13 and 20 probands, respectively (nϭ33; 10.6% overall). One MYH6 variant was classified as unlikely to be disease causing. Conclusion-Rare variants in these 5 genes likely or possibly caused 10.6% of DCM in this cohort. When combined with our prior resequencing reports, Ϸ27% of DCM probands had possible or likely disease-causing variants identified.(Circ Cardiovasc Genet. 2010;3:155-161.)

IntroductionDilated cardiomyopathy (DCM; OMIM 115200) is a debilitating primary cardiac muscle disease with a 5-year mortality approaching 50% following diagnosis.1,2 Whether familial or sporadic, DCM shows remarkably high genetic heterogeneity. 3,4 To date, the molecular basis of most of DCM cases remains unknown despite the fact that mutations in more than 30 genes have been shown to be disease causing or disease associated. 5,6 Because of this marked locus heterogeneity, the fraction of DCM patients who have a mutation in any one gene is small, and ranged from 0.3% to 5.9% in our recent resequencing studies. [7][8][9][10] Most of the genes with mutations causing DCM encode for sarcomeric proteins involved in contraction, or cytoskeletal proteins important for cell structure or force transduction. 5,6 Exceptions to this include a mutation identifi ed in the eye absent transcription factor 4 (EYA4) in a family with both DCM and sensorineural hearing loss, 11 and mutations in the genes encoding presenilin 1 and 2 ( PSEN1 and PSEN2 ) that we recently identifi ed in DCM families.10 Although the pathogenic mechanisms of EYA4 and the presenilins in DCM remain to be defi ned, both are known to play regulatory roles in the heart even though they are neither essential structural components nor contractile proteins.A recent genetic linkage and gene-mapping study demonstrated that mutations in RBM20 , a ribonucleic acid (RNA)-binding protein gene, cause DCM.12 Th is discovery is intriguing in several aspects. To our knowledge, this is the fi rst report to suggest that a genetic abnormality of an RNA-binding protein can lead to cardiomyopathy.13 It is also noteworthy that RBM20 mutations were associated with severe cases of DCM with high mortality and patients needing heart transplantation. Moreover, the fi ve mutations identifi ed in the fi rst report were concentrated in a small region of RBM20, spanning only fi ve amino acids, and encoded by a single exon. 12To further evaluate the role of RBM20 in DCM pathogenesis and the DCM clinical characteristics caused by RBM20 mutations, we genetically screened a cohort of 312 DCM probands, and their family members when a mutation was identifi ed. We found six unique mutations in six unrelated probands, four of which were novel. Th ese results expand the RBM20 mutation spectrum in DCM and further emphasize the importance of RBM20 in the myocardial disease. Materials and Methods Clinical evaluationWritten, informed consent was obtained from all subjects, and the Institutional Review Boards at the Oregon Health & Science University and the University of Miami approved the study. Th e investigation included 312 probands (290 Caucasians, of whom 7 were of Hispanic descent; 16 African-Americans, 3 Asians and 3 Native Americans/Alaskan Natives) and used methods of clinical categorization of DCM as previously described.14 Clinical data were obtained through our own evaluations, which included minimally a history and physical examination, an electrocardiogram (ECG) and an echocardiogram...