Genome-wide studies of copy number variation and exome sequencing identify rare variants in BAG3 as a cause of dilated cardiomyopathy

Abstract

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.

Figure 1

Pedigrees of BAG3-Associated Dilated Cardiomyopathy Pedigrees have been labeled by letter, which correspond to their respective mutation as shown in Table 3. Squares represent males and circles represent females. An arrowhead denotes the proband. A diagonal line marks deceased individuals. Solid symbols denote dilated cardiomyopathy with other causes except genetic ruled out. Open symbols denoteunaffected individuals. The presence or absence of the pedigree's BAG3 mutation is indicated by a + or − symbol, respectively; obligate carriers are noted in parenthesis, (+). Individuals who underwent exome sequencing are denoted (exome), as are those who underwent comparative genomic hybridization (CGH).

Figure 2

Summary of BAG3 Function Adapted from McCollum et al. and annotated with mutations identified in DCM families.

Figure 3

Morpholino Knockdown of bag3 in Zebrafish Causes Cardiac Phenotypes Whole-mount brightfield images of 72hpf zebrafish embros. (A) wild-type, (B) bag3ATG morpholino-injected, (C) exon 2 splice donor-blocking morpholino-injected embryos. Measurement of cardiac performance comparing wild-type and morphant embryos for (D) fractional shortening and (E) peak flow velocity averaged across all data points from onset of systole. ATG morpholino injected at 0.5 mM and exon 2 morpholino injected at 0.3 mM. Arrows mark pericardial effusion. The following abbreviations are used: WT, wild-type; MO, morpholino; ATG, translation initiation blocking. Bars in (D) correspond to minimum and maximum values. Boundaries of each box correspond to 25th and 75th percentiles and horizontal lines within boxes correspond to the median value.