Genetics of BAG3: A Paradigm for Developing Precision Therapies for Dilated Cardiomyopathies

ABSTRACT

Nonischemic dilated cardiomyopathy is a common form of heart muscle disease in which genetic factors play a critical etiological role. In this regard, both rare disease‐causing mutations and common disease‐susceptible variants, in the Bcl‐2–associated athanogene 3 (BAG3) gene have been reported, highlighting the critical role of BAG3 in cardiomyocytes and in the development of dilated cardiomyopathy. The phenotypic effects of the BAG3 mutations help investigators understand the structure and function of the BAG3 gene. Indeed, we report herein that all of the known pathogenic/likely pathogenic variants affect at least 1 of 3 protein functional domains, ie, the WW domain, the second IPV (Ile‐Pro‐Val) domain, or the BAG domain, whereas none of the missense nontruncating pathogenic/likely pathogenic variants affect the proline‐rich repeat (PXXP) domain. A common variant, p.Cys151Arg, associated with reduced susceptibility to dilated cardiomyopathy demonstrated a significant difference in allele frequencies among diverse human populations, suggesting evolutionary selective pressure. As BAG3‐related therapies for heart failure move from the laboratory to the clinic, the ability to provide precision medicine will depend in large part on having a thorough understanding of the potential effects of both common and uncommon genetic variants on these target proteins. The current review article provides a roadmap that investigators can utilize to determine the potential interactions between a patient's genotype, their phenotype, and their response to therapeutic interventions with both gene delivery and small molecules.

Nonstandard Abbreviations and Acronyms

ACMG

American College of Medical Genetics and Genomics

B/LB

benign/likely benign

BAG3

Bcl‐2–associated athanogene 3

CAPZB

capping actin protein of muscle Z‐line subunit beta

DCM

dilated cardiomyopathy

eQTL

expression quantitative trait locus

gnomAD

Genome Aggregation Database

GTEx

Genotype‐Tissue Expression

Hsc70

heat shock cognate

Hsp70

70 kilo‐dalton heat shock protein

IPV

Ile‐Pro‐Val

LAMP‐2

lysosomal associated membrane protein 2

LTCC

L‐type voltage‐gated calcium channel Cav1.2

LD

linkage disequilibrium

NF‐κB

nuclear factor kappa B

P/LP

pathogenic/likely pathogenic

PDZGEF2

PDZ domain containing guanine nucleotide exchange factor 2

PPCM

peripartum cardiomyopathy

PXXP

proline‐rich repeat

SH3

Src homology 3

sHsps

small heat shock proteins

SIFT

Sorting Intolerant From Tolerant

VUS

variant of uncertain significance

Cardiomyopathies are a heterogeneous group of diseases of cardiac muscle ¹ that include dilated, hypertrophic, restrictive, constrictive and arrhythmogenic right ventricular cardiomyopathies. ² Dilated cardiomyopathies (DCMs) have been historically separated into those that are secondary to ischemic heart disease (ischemic DCM) and those that are nonischemic, with ischemic DCM being somewhat more common. Genetic variants were first shown to be etiologic in the development of hypertrophic cardiomyopathy ³ , ⁴ and novel therapies have been developed, using gene therapy ⁵ or small molecules. ⁶ By contrast, the cause of DCM has remained more obscure. Recent studies have shown convincingly that both heritable and sporadic genetic variants play a pivotal role in the development of many forms of DCM, the most common being loss of function variants in Titin, the largest protein found in humans. The Titin myofilament is one of the 3 major myofilaments found in the cardiac sarcomere (in addition to actin and myosin) and is critical for restoring the ability of the cardiac sarcomere to maintain optimal function of the left ventricle during diastole. ⁷ Truncating variants of Titin are the most common genetic variants, found in as many as 25% of patients with both heritable and sporadic forms of DCM, as well as in a significant number of patients with peripartum cardiomyopathy. ⁸ , ⁹ The discovery of genetic mutations causing DCM has enhanced our knowledge regarding the pathobiology of the disease and has provided an opportunity for more precise diagnosis, better risk prediction, and potential for improved treatment outcomes using both gene therapy and small molecules. ¹⁰

In addition to Titin, genetic variants in >30 genes have been reported to be associated with the development of DCMs. To date, genes related to DCM identified in patients or demonstrated in animal models have greatly expanded the biomolecular knowledge involving the pathogenesis of DMCs. They also highlight important therapeutic targets. Examples of pathogenic genetic variants have been found in the genes encoding β‐adrenergic receptors (β1 ¹¹ and β2 ¹² ), the calcium voltage–gated channel subunit alpha1 C gene encoding the L‐type voltage‐gated calcium channel Ca_v1.2 (LTCC), ¹³ and the mitochondrial calcium uniporter gene. ¹⁴ One of the most interesting of this group of DCM‐associated genes is Bcl‐2–associated athanogene‐3 (BAG3). BAG3 encodes a multifunctional protein that regulates or facilitates numerous biological processes in the heart and other tissues including stabilization of the sarcomere, ¹⁵ maintaining the integrity of the nuclear envelope, ¹⁶ coupling the β‐adrenergic receptor and the L‐type Ca²⁺ channel, activating autophagy and inhibiting apoptosis, ¹⁷ and modulating the cardiac inflammasome. ¹⁸ However, we know far more about the biology of less common proteins than we do about BAG‐3. The work detailed in this review was undertaken to bring order to the large number of reports detailing both informative and noninformative genetic variants in BAG3, so as to better understand how these variants alter the cardiomyopathy phenotype.

BAG3 GENE

BAG3 at Chr10q26.11 encodes a cochaperone, also known as Bcl‐2–associated athanogene 3, which was first recognized for its ability to bind to Bcl‐2 and effect an antiapoptotic function. The gene has ubiquitous expression in human tissues, with the highest level in heart, skeletal muscle, and the central nervous system (https://www.ncbi.nlm.nih.gov/gene/9531#gene‐expression). High levels of BAG3 have also been found in many cancers with perhaps the highest levels being found in pancreatic tumors. ¹⁹ The encoded protein, BAG3, comprises 575 aa and includes 4 known functional domains (National Center for Biotechnology Information reference NP_004272.2, Figure). ²⁰ , ²¹ Initially, BAG3 was found to serve as a cochaperone with the Hsp70 (70 kilo‐dalton heat shock protein) at the Z‐disc in striated muscle. ²² Molecular chaperones, eg, Hsps, are functionally related proteins assisting protein folding under both physiological and stressful conditions. ²³ Hsp70s are the most ubiquitous members of the family of Hsps, and are found in all major subcellular compartments ²⁴ where they are involved in a wide range of protein folding processes, including the folding of newly synthesized proteins and refolding of misfolded and aggregated proteins. ²⁵

Figure 1. The Bcl‐2–associated athanogene 3 (BAG3) gene model.

The gene model was based on the annotation of the Ensemble project and the studies by Fuchs et al ²⁰ and Doong et al ³⁴ and the review by McCollum et al.²¹ The encoded protein contains 4 types of protein domains, including 1 WW (Trp‐Trp) domain (19–55 aa), 2 IPV (Ile‐Pro‐Val) motifs (87–101, 200–213 aa), 1 proline‐rich repeat (PXXP) region (302–412 aa), and 1 BAG domain (420–499 aa).

BAG3 Structure

The BAG3 protein comprises 4 types of protein functional domains (Figure), as delineated below:

1. The WW (Trp‐Trp) domain seen in a number of unrelated proteins has known function in protein binding and signal transduction processes. ²⁶ The WW domain binds with the PPDY motif of the PDZ domain containing guanine nucleotide exchange factor 2 (PDZGEF2), which induces activation of Small GTPase Rap1 and promotes integrin‐mediated cell adhesion. ²⁷ The role of BAG3 in cell adhesion mediated by the WW domain is critical for cell vitality and its antiapoptotic function. ²⁸ One small nontruncating indel p.Ile30_Asp31delinsAsnHis in the WW domain has been reported as likely pathogenic in DCM. ²⁹

2. Two IPV (Ile‐Pro‐Val) motifs bind with the molecular chaperones, sHsps (small heat shock proteins) HspB8/HspB6, and allow BAG3 to function as a scaffolding protein, linking Hsp70 to HspB8/HspB6 to form a ternary complex. ³⁰ BAG3 facilitates the ability of sHsps to bind selectively with misfolded proteins and facilitates their refolding or degradation, the latter being comediated by Hsp70. ³¹ The importance of IPV in cardiomyopathies has been highlighted by disease‐causing (classified as pathogenic/likely pathogenic [P/LP]) point mutations at the Pro209 position in the second IPV motif of BAG3. ³² The mutant protein of the toxic gain‐of‐function mutation p.Pro209Leu in BAG3 forms aggregates with the wild‐type protein and causes BAG3 insufficiency. ³³

3. The proline‐rich repeat (PXXP) region binds to SH3 (Src homology 3) motifs and mediates protein–protein interaction, eg, with phospholipase C‐γ. ³⁴ The activity of BAG3 is regulated by its phosphorylation status. ³⁵ The binding of BAG3 with phospholipase C‐γ enables the regulation of the activity of phosphorylated BAG3. ³⁴ However, the role of PXXP in DCM is unclear. To date, 22 truncating mutations and 1 splicing mutation are reported in the PXXP region by the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar/, accessed by Aug 25, 2022). In contrast, there are no missense nontruncating P/LP variants related to DCM reported in the PXXP region. Moreover, two of the benign/likely benign (B/LB) variants (p.Pro407Leu and p.Pro380Ser) in the PXXP region recorded by the ClinVar database do not support the functional importance of this domain in DCM. Taken together, the pathogenicity of truncating/splicing variants are likely caused by lack of the BAG domain at the protein level.

4. The BAG domain mediates the binding of BAG3 with Hsp70. The BAG domain contains 3 antiparallel α‐helices, while the second and third α‐helices interact with the ATP‐binding pocket of Hsp70 or the Hsc70 (heat shock cognate 70). ³⁶ Hsp70 proteins have 3 major functional domains: an N‐terminal ATPase domain, a substrate binding domain, and a C‐terminal domain. ³⁷ The interaction between BAG‐3 and Hsp70 is mediated by the binding of the BAG domain of BAG3 and the N‐terminal ATPase domain of Hsp70. ³⁶ In addition, the BAG domain binds to the Bcl‐2 Homology 4 domain of Bcl‐2 to inhibit mitochondrial‐dependent (intrinsic) apoptosis. ³⁸ Bcl‐2 promotes cell survival by inhibiting cleavage (activation) of the caspases, which are essential mediators of apoptosis, eg, apoptotic protease activating factor 1. ³⁹ Bcl‐2 also inhibits the mitochondrial release of cytochrome c, which, when bound to apoptotic protease activating factor 1, activates the initiator executioner caspase‐9 and the subsequent activation of the executioner caspases‐3 and ‐7, which is the last step in the pathway leading to apoptosis. ⁴⁰

Functions of BAG3 in Cardiomyocytes

BAG3 has been shown to play ritical roles in cardiomyocytes, with effects mediated by Hsp70 ²² and Bcl‐2 ³⁸ actions. These critical functions include:

1. Maintaining sarcomere integrity: Under acute cellular stress, expression of HspB8/HspB6, Hsp70, and BAG3 are upregulated. ³⁵ , ⁴¹ After the BAG‐3‐sHsps (HspB8/HspB6) complex binds selectively with misfolded proteins, Hsp70 displaces sHsps in the complex. ³¹ The actin capping protein CapZ, a heterodimer with α and β subunits, ⁴² binds the barbed ends of actin at sarcomeric Z‐lines, and regulates thin filament assembly. ⁴² The β‐subunit at Z‐line is the β1 isoform encoded by the CAPZB (capping actin protein of muscle Z‐line subunit beta) gene. ⁴³ At the Z‐disc, BAG3 promotes the association between Hsc70 and CapZβ1 and stabilizes the structural integrity of the sarcomere during mechanical stress. ⁴⁴ Bag3 knockdown mice are shown to be susceptible to myofibrillar degeneration, and myofibrillar disarray has also been reported in humans with BAG3 haploinsufficiency. ⁴⁴

2. Regulating macroautophagy: The Hsp70‐BAG3 complex mediates the retrograde transport of misfolded proteins along microtubules to the microtubule‐based inclusion bodies, known as aggresomes, at the microtubule organizing center. ³⁵ , ⁴⁵ Subsequently, BAG3 interacts directly with the macroautophagy receptor protein p62 (also known as sequestosome‐1) and induces selective macroautophagy while lysosomes uptake and degrade protein aggregates. ³⁵ , ⁴⁶ BAG3‐mediated selective macroautophagy plays critical roles in the cellular protein quality control system. ³⁵

3. Antiapoptosis: With BAG3 as an athanogene, the Hsp70‐BAG3 complex protects antiapoptotic Bcl‐2 proteins from proteasomal degradation and activates the antiapoptotic nuclear factor kappa B (NF‐κB) pathway. ⁴⁷ The activation of the NF‐κB pathway consequently promotes the expression of Bcl‐2. ⁴⁸

4. β‐Adrenergic receptor/LTCC regulation: LTCC triggers intracellular Ca²⁺ release during excitation‐contraction coupling. ⁴⁹ The β‐adrenergic/cAMP signaling pathway is the major mediator of LTCC activation. ⁵⁰ A previous study identified that BAG3 coimmunoprecipitated with β1‐adrenergic receptor and LTCC. ⁵¹ Haploinsufficiency of BAG3 in mice results in β‐adrenergic insensitivity of LTCC. ⁵²

5. Mitochondrial function: Mitochondrial quality control in cardiomyocytes plays a critical role in the progression of cardiovascular diseases. ⁵³ Critical roles of BAG3 in cardiomyocytes involve mitochondrial quality control by regulating both endogenous and exogenous expression of Parkin, a major regulator of mitophagy in mitochondrial homeostasis control. ⁵⁴

PATHOGENESIS AND CLINICAL PRESENTATIONS OF GENETIC VARIANTS OF BAG3

Dilated Cardiomyopathy

Genetic mutations of BAG3 have been demonstrated to affect both the myocardium and the skeletal muscle leading to DCM and myofibrillar myopathy, respectively. ⁵⁵ It has been observed that impaired function of BAG3 by genetic mutation causes disrupted Z‐disc and enhanced sensitivity to apoptosis in cardiomyocytes and skeletal muscles, ²² disrupted interaction between BAG3 and HSP70 leading to decreased levels of sHsps, ⁵⁶ impaired BAG3‐mediated sarcomeric protein turnover leading to reduced myofilament maximal force‐generating capacity, ¹⁵ and cardiomyocyte accumulation of the preamyloid oligomers and activation of p38 signaling. ⁵⁷ The phenotypic effects of BAG3 mutations may be mediated by either haploinsufficiency ⁵⁷ or a dominant negative mechanism. ⁵⁸ Impaired selective macroautophagy has been highlighted as being critical in the pathogenesis in DCM and myofibrillar myopathy. In addition to the effects mediated by BAG3, the role of selective macroautophagy has also been emphasized by the previous finding that primary deficiency of the lysosomal structural protein LAMP‐2 (lysosomal associated membrane protein 2) attributable to genetic variants causes X‐linked vacuolar cardiomyopathy and myofibrillar myopathy (Danon disease). ⁵⁹

According to a large study of 2538 patients with DCM by Mazzarotto et al, truncating variants of BAG3 were identified in 0.3% of DCM cases. ⁶⁰ Clinically, BAG3 mutations present with diverse cardiovascular phenotypes, ⁶¹ and with variable age of onset from 18 years ⁶¹ to 64 years. ⁶² According to the large sample study of 129 individuals with BAG3 mutations (86% are truncating) by Domínguez et al, 68.4% patients had DCM, and the mean age of diagnosis was 36.9 years with an early age of onset in men (≈6 years in average). ²⁹ ECG abnormalities were observed with the diagnosis and progression of DCM, including prolonged QRS duration, as well as negative T waves in 34.0% patients. ²⁹ Echocardiogram may disclose decreased left ventricular (LV) ejection fraction, increased LV end‐diastolic diameter, and decreased score of tricuspid annular plane systolic excursion. ²⁹ LV noncompaction has also been observed in patients with BAG3 mutations. ⁶³ Clinical end points, including cardiac death, heart transplant, LV assist device, aborted sudden cardiac death, and serious ventricular arrhythmia, were observed in 30.1% patients with the incidence of 5.1% per year. ²⁹ It was also observed that acute‐onset DCM may be triggered by infection in patients with BAG3 mutations. ⁶⁴ Patients with DCM who have BAG3 mutations are less responsive to treatment than those with other genetic forms of DCM, while only 2.9% of patients with DCM at first evaluation have normalized LV ejection fraction during follow‐up. ²⁹ In addition to disease‐causing mutations, some genetic variants in BAG3 identified in individuals of African ancestry that were not causative of disease were associated with a negative outcome in patients with DCM. ⁶⁵

Myocarditis

To date, a causal link between viral myocarditis and the development of DCM has also been recognized. ⁶⁶ , ⁶⁷ The critical roles of BAG3 in cardiomyocytes imply its involvement in viral insults of cardiomyocytes, eg, in COVID‐19 infection and influenza. BAG3 mutations have been shown to result in dysregulation of mitochondrial dynamics and activation of p38 signaling in inflammatory response and apoptosis. ⁵⁷ Correlation between BAG3 mutations and myocarditis has been identified by recent studies. ⁶⁸ , ⁶⁹ These previous studies implied the possible correlation between BAG3 mutation and myocarditis caused by COVID‐19 infection, which warrants further investigation.

Peripartum Cardiomyopathy

Peripartum cardiomyopathy (PPCM) is a rare life‐threatening cardiomyopathy of unknown cause, which develops cardiac failure in the last month of pregnancy or within 5 months after delivery without history of a previous heart disease. ⁷⁰ PPCM can be the initial manifestation of familial DCM. ⁷¹ Shared genetic predisposition in PPCM and DCM has been demonstrated in previous studies, including BAG3 mutation. ⁸ , ⁹

Glycogen Storage Diseases

Autophagy mediates critical cytoprotective effects. ⁷² Defective autophagy causes the accumulation of aggregated/misfolded proteins, damaged organelles, and abnormal glycogen storage. ⁷³ Myopathy may be caused by abnormal glycogen storage. ⁷⁴ As discussed above, BAG3 plays a critical role in regulating autophagy. Glycogen accumulations have been observed in a patient with myofibrillar myopathy and polyneuropathy caused by the BAG3 mutation p.Pro209Gln. ⁷⁵

Oncogenesis and Cancer Chemotherapy

In addition to the roles of BAG3 in heart and DCM, BAG3 has been demonstrated to play a role in tumorigenesis by mediating the effects of Hsp70 on Src signaling in cancer initiation and progression. ⁷⁶ , ⁷⁷ Bortezomib was the first proteasome inhibitor approved for anticancer therapy. ⁷⁸ Bortezomib upregulates the level of BAG3, while BAG3 gene silencing sensitizes leukemic cells to Bortezomib‐induced apoptosis and enhances the therapeutic potency of Bortezomib. ⁷⁹ BAG3 may also downmodulate apoptotic response to tumor necrosis factor–related apoptosis‐inducing ligand in cancer cells. ⁸⁰ Targeting at BAG3 with a small‐molecule inhibitor YM‐1 to block binding of Hsp70 to BAG3 has been shown to suppress tumor growth in mice, highlighting Bag3 as an appealing anticancer target. ⁷⁶ Thus far, however, no genetic association has been identified between common variants of BAG3 and cancers.

RARE MUTATIONS AND PHENOTYPIC EFFECTS

To date, a number of disease‐related variants in BAG3 have been reported (Table S1). ⁸ , ²⁹ , ⁶⁰ , ⁶² , ⁸¹ , ⁸² Among these variants, 596 genetic variants in the BAG3 gene related to cardiomyopathy or myofibrillar myopathy are archived in the ClinVar database (accessed April 6, 2022). Among these variants, 429 are annotated as coding variants, including 11 deletions, 33 nonsense (truncating) variants, 33 frame‐shift (truncating) variants, and 351 missense (nontruncating) variants (Table 1). Besides these coding variants, a splicing mutation (truncating) NM_004281.4(BAG3):c.508‐2A>G has been classified as pathogenic. ²⁹ In addition to the above variants, 29 gross copy number variations encompassing BAG3 have been classified as P/LP in ClinVar, with 23 (79.31%) P/LP gross copy number variations as copy number gain (Table S1). However, none of these copy number gain copy number variations are reported to be associated with DCM or myofibrillar myopathy. In contrast to the duplications or gain‐of‐function variants of BAG3, all P/LP variants related to DCM are loss‐of‐function variants.

Table 1.

Clinical Significance of Different Types of Variants

Coding variant type	Clinical significance	Count (%)
Deletion	P/LP	7 (63.64)
	VUS	4 (36.36)
Nonsense (truncating)	P/LP	31 (93.94)
	VUS	1 (3.03)
	Conflicting interpretations of pathogenicity	1 (3.03)
Frame‐shift (truncating)	P/LP	28 (84.85)
	VUS	5 (15.15)
Missense (nontruncating)	B/LB	9 (2.56)
	P/LP	6 (1.70)
	VUS	308 (87.50)
	Conflicting interpretations of pathogenicity	28 (7.95)

B/LB indicates benign/likely benign; P/LP, pathogenic/likely pathogenic; and VUS, variant of uncertain significance.

Deletions

Among the 11 deletions reported in ClinVar, 7 (63.64%) are predicted as P/LP and 4 (36.36%) as variant of uncertain significance (VUS). For the 7 P/LP deletions, 6 result in gross deletions of the coding region and 1 other deletion (21bp deletion, NM_004281.4[BAG3]:c.508‐14_514del) causes damage of a splice acceptor site. For the 4 VUS deletions, one is an in‐frame deletion (NC_000010.11:g.[?_119669831]_[119670197_?]del, missing exon 2 of BAG3 with the first IPV motif), while the other 3 are single amino acid deletions. Two of the 3 single amino acid deletions involve the WW domain and the PXXP domain, respectively. Nonetheless, no deletion in the BAG3 coding region was classified as B/LB.

In addition to the ClinVar records, other deletions related to DCM have been reported in the literature. Two gross deletions are predicted as pathogenic by the American College of Medical Genetics and Genomics (ACMG) guidelines, both resulting in loss of the critical BAG domain, ie, c.508_1728del causing deletion of exons 3 and 4, as reported in DCM ²⁹ ; c.1070_1660del causing deletion of aa.357–533 in exon 4. ⁸¹ In addition, a small nontruncating indel c.89_91delTCGinsACC (p.Ile30_Asp31delinsAsnHis), which locates at the WW domain, was reported in DCM and predicted as LP. ²⁹ Importantly, all of the gross deletions involving the BAG domain identified to date are classified as pathogenic or LP. Potential importance of the WW domain in DCM is also supported by the reported small nontruncating LP deletion p.Ile30_Asp31delinsAsnHis. ²⁹

Nonsense Truncating Variants

Among the 33 nonsense truncating variants (producing a premature termination codon) registered in ClinVar, 31(93.94%) variants are classified as P/LP. ⁹ , ²⁹ , ⁶² , ⁸¹ , ⁸³ Only 1 (3.03%) nonsense variant p.Arg473Ter (with the BAG domain partially truncated, by missing 27 aa at the C‐terminus of the BAG domain) is classified as having conflicting status of pathogenicity, and 1 (3.03%) nonsense variant p.Gln511Ter is classified as VUS without involving any of the protein functional domains. Without exception, all of the P/LP nonsense variants are located upstream of the conflicting and VUS variants. The first P/LP nonsense truncating variant is p.Trp26Ter, which may fail to express any protein from the variant allele because of increased mRNA decay, ⁸⁴ with resultant haploinsufficiency. The last P/LP nonsense truncating variant that has been identified is p.Tyr451Ter. Its placement at amino acid 451 justifies classifying any newly identified nonsense truncating variant at an earlier amino acid as P/LP.

Frame‐Shift Truncating Variants

Among the 33 frame‐shift truncating variants recorded in ClinVar, 28 (84.85%) variants are classified as P/LP ²⁹ , ⁶² , ⁸⁵ and 5 (15.15%) are classified as VUS. The first frame‐shift variant is p.Ser14fs and the last is p.Ala474fs. The distribution of the P/LP frame‐shift variants are consistent with the distribution of nonsense variants as described above. In addition to the ClinVar recorded frame‐shift variants, another frame‐shift variant p.Glu57Lysfs*154 Pathogenic has been reported to be associated with DCM and is predicted to be pathologic by ACMG criteria. ²⁹ Without exception, all of the P/LP truncating variants reside upstream of the VUS variants in amino acid position. The last P/LP frame‐shift variant is p.Ala474fs, which is consistant with the distribution of nonsense/nonframe‐shift truncating variants. All VUS frame‐shift variants occur after amino acid position 507, which is posterior to the BAG domain.

Missense (Nontruncating) Variants

Compared with the deletions and truncating variants, missense variants causing a single amino acid substitution in BAG3 are less deleterious. For the majority of the reported missense variants, functional classification has been VUS (308 [87.50%]) or with conflicting information regarding pathogenicity (28 [7.95%]) (Table S2). Among the 351 missense variants, only 6 (1.70%) are classified as P/LP. All 6 P/LP variants are located in a functional domain, ie, the amino acid 209 position at the second IPV domain, and amino acid positions at the BAG domain. ²⁹ , ³² , ⁸⁶ Interestingly, the missense variant p.Pro209Leu was initially identified as having an association with myofibrillar myopathy and restrictive cardiomyopathy or hypertrophic cardiomyopathy, ⁸⁶ , ⁸⁷ , ⁸⁸ while later studies also reported the phenotype of DCM in relation with p.Pro209Leu. ³² , ⁸⁸ In Zebrafish models, p.Pro209Leu was demonstrated as a toxic gain‐of‐function mutation. ³³ Loss of BAG3 may result in myofibrillar disintegration, while the mutant protein of p.Pro209Leu can rescue the myofibrillar disintegration phenotype. ³³ However, the mutant protein may cause BAG3 insufficiency as a result of forming aggregates with the wild‐type protein as a dominant‐negative effect. ³³

In contrast to the P/LP missense variants, two of the B/LB variants (p.Pro407Leu and p.Pro380Ser) are located in the PXXP region, while both have relatively common allele frequencies in the general population, ie, p.Pro407Leu MAF=0.126 and p.Pro380Ser MAF=0.003 according to the Genome Aggregation Database (gnomAD, version 2.1.1). ⁸⁹ The amino acid substitutions caused by the 2 variants are significant with Grantham biochemical distances of 98 and 74, respectively. ⁹⁰ These two B/LB variants thus undermine the functional importance of the PXXP region in DCM. Nonetheless, the co‐occurrence of 2 missense variants––p.Pro63Ala (rs144041999) and p.Pro380Ser (rs144692954)––on the same chromosome was identified in individuals of African ancestry with cardiomyopathy, and shown to have dominant‐negative effect in mouse model. ⁶⁵ , ⁹¹ These 2 variants are most common in the African population with a frequency of 0.011 and in high linkage disequilibrium (LD) with r ²=1 (https://ldlink.nci.nih.gov/). The p.Pro63Ala is near the WW domain and classified as benign by ClinVar. Further study on a larger sample of patients with cardiomyopathy and African ancestry may help to establish the causal relationship of the haplotype with cardiomyopathy.

Allele frequencies of the variants are highly informative for the differentiation of the P/LP variants versus the B/LB variants. Without exception, all 6 P/LP variants are absent from controls in the exome sequencing data of gnomAD ⁸⁹ (ACMG Moderate Pathogenic Evidence Level: PM2). In contrast, 8 of the 9 B/LB variants are relatively common in the gnomAD (ACMG Benign Evidence Levels: benign stand alone BA1 or strong BS1). The other B/LB variant p.Glu553Asp is classified as likely benign with multiple lines of benign computational evidence (ACMG Evidence BP4). ²²

COMMON VARIANTS OF BAG3

Nonsynonymous coding variant p.Cys151Arg

Common variants from a number of genetic loci have been reported in association with DCM. In a study by Villard et al, ⁹² a common nonsynonymous coding variant, rs2234962 at BAG3 (p.Cys151Arg), was reported in association with DCM, while the minor allele (allele C encoding Arg151) is protective (odds ratio, 0.64 [95% CI, 0.55–0.74]). The association of rs2234962 and DCM was consequently replicated in independent studies. ⁹³ , ⁹⁴ In the genome‐wide association meta‐analysis comprising 47 309 cases and 930 014 controls of European ancestry, Shah et al identified the strongest association signal with DCM from the intronic SNP (single‐nucleotide polymorphism) rs17617337. ⁹⁴ The SNP rs17617337 is in high LD with the nonsynonymous rs2234962 (r ²=0.99), thus representing the same association signal. ⁹⁴ One study also showed that rs2234962 was associated with LV end‐diastolic diameter in both patients with DCM and those with ischemic heart failure. ⁹⁵ In the general population, rs2234962 is associated with LV functional and structural parameters, including LV end‐diastolic volume, ⁹⁶ LV end‐systolic volume, ⁹⁶ and LV ejection fraction. ⁹⁶ , ⁹⁷ In addition, a recent study showed that rs2234962 is associated with elevated levels of serum troponin, a marker of cardiomyocyte damage. ⁹⁸

The nonsynonymous p.Cys151Arg variant, located in between the 2 IPV motifs, is classified as B/LB by both ClinVar and the ACMG criteria, mainly because of its common allele frequency (Table S2). Bioinformatic approaches predict p.Cys151Arg with conflicting results, ie, damaging by SIFT (Sorting Intolerant From Tolerant), probably damaging by Polyphen‐HumDiv (correlated with evolutionary conservation), but benign by Polyphen‐HumVar (correlated with allele frequency). In addition to the protective effect, the position of p.Cys151Arg without involving a functional domain supports a benign classification, while all P/LP mutations of BAG3 (disease‐causing) involve a functional domain. Significant differences in allele frequencies of p.Cys151Arg is seen among different racial populations. For example the putatively protective allele Arg151 is extremely rare in East Asian populations (https://www.ncbi.nlm.nih.gov/snp/rs2234962), which suggests evolutionary selective pressure related to the pleiotropic function of BAG3. No expression quantitative trait loci (eQTLs) tagged by rs2234962 in heart or other tissues have been observed by the Genotype‐Tissue Expression (GTEx) project (https://www.gtexportal.org/). Concerning the molecular mechanisms underlying the genetic association of p.Cys151Arg, a recent study by Perez‐Bermejo et al showed that p.Cys151Arg enhanced the recruitment of factors critical to the maintenance of myofibril integrity and improved response to proteotoxic stress in cardiomyocytes. ⁹⁹

eQTLs of BAG3

According to GTEx project data, a number of SNPs have been identified as having an association with the expression levels of BAG3 in different tissues (Table 2). As shown by the harmonized data in the MR‐Base platform (https://www.mrbase.org/), 5 eQTL SNPs are also associated with primary cardiomyopathies by the FinnGen biobank genome‐wide association study (id:finn‐b‐I9_CARDMPRI, https://gwas.mrcieu.ac.uk/datasets/finn‐b‐I9_CARDMPRI/; https://www.finngen.fi/fi), which includes 2221 cases and 156 711 controls. The eQTL effects are seen in human tissues, including the tibial artery, sun‐exposed skin in the lower leg, the sigmoid colon, skeletal muscle, and subcutaneous adipose tissue. These eQTL SNPs have low LD r ² values with the nonsynonymous coding variant p.Cys151Arg (Figure S1, plotted by the NCI LDmatrix Tool, https://analysistools.cancer.gov/LDlink/?tab=ldmatrix). Two SNPs, rs196334 and rs196336, are in tight LD with r ²=1 in the European population, representing the same genetic signal. No eQTL is identified in the left ventricle of the heart. Interestingly, these eQTLs are associated with upregulated expression of BAG3 in noncardiac tissues, but are associated with increased risk of cardiomyopathies. Mechanistic and phenotypic study of these eQTLs and their roles in cardiomyopathies is warranted as they represent a unique opportunity to study the potential therapeutic as well as the possible detrimental effects of manipulating BAG3 expression.

Table 2.

The eQTLs of BAG3 and Their Associations With Cardiomyopathies

SNP	Expression of BAG3	pval.eQTL	pval.cardiomyopathy	effect_allele.eQTL	beta.eQTL	beta.cardiomyopathy	EAF	r2 with p.Cys151Arg
rs196334	Artery tibial	7.29E‐06	2.85E‐08	T	−0.176	−0.177	0.546	0.139
rs196336	Skin sun‐exposed lower leg	3.93E‐19	2.93E‐08	C	−0.346	−0.178	0.546	0.139
rs196298	Colon sigmoid	2.74E‐06	8.62E‐07	A	−0.316	−0.178	0.264	0.063
rs171910	Muscle skeletal	2.00E‐06	5.01E‐03	G	−0.224	−0.101	0.738	0.076
rs17099080	Adipose subcutaneous	1.06E‐05	7.24E‐03	C	0.224	0.098	0.253	0.034
rs111384142	Heart atrial appendage	1.25E‐06	0.103	T	−1.084	−0.266	0.010	0.016
rs11598594	Skin not sun exposed suprapubic	1.01E‐05	0.851	C	−0.799	−0.038	0.006	0.005
rs3887073	Brain anterior cingulate cortex BA24	4.32E‐07	0.938	T	0.398	−0.004	0.103	0.005

BAG3 indicates Bcl‐2–associated athanogene 3; EAF, effect allele frequency; eQTL, expression quantitative trait locus; and SNP, single nucleotide polymorphism.

CONCLUSIONS

Genetic analysis, including most importantly the phenotypic effect analysis of DNA variants, has been a successful approach to understand gene structure and function. A number of mutations in the BAG3 gene have been reported in relation to DCM causality, highlighting the critical role of BAG3 in cardiomyocytes. The observed phenotypic effects of the BAG3 mutations have provided new knowledge about essential functional domains. For instance, all of the truncating mutations before the 451th amino acid position in the BAG domain are P/LP variants, which indicates the essential/functional length of the BAG3 protein. All of the P/LP deletions involve the WW domain or the BAG domain, and all of the P/LP nsSNPs involve the second IPV domain or the BAG domain, highlighting the importance of these 3 protein domains in the function of BAG3 in the heart, whereas B/LB variants undermine the importance of the PXXP region.

Because of its critical role in cardiomyocytes in DCM, BAG3 is a clear major candidate gene in secondary DCM, including but not limited to peripartum DCM and severe cardiotoxicity of the proteasome inhibitor Bortezomib for cancer chemotherapy. A correlation between BAG3 mutations and myocarditis that was identified in a recent study ⁶⁸ implies that BAG3 may also be involved in viral insults of cardiomyocytes. In the era of precision medicine and genomic medicine, genetic information acquired by DNA sequencing may help to identify high‐risk individuals, with the goal of preventing adverse events related to BAG3. Patients with a positive family history of DCM and presence of P/LP genetic variants of the BAG3 gene, warrant extra attention for the incidence of secondary DCM‐like PPCM, cardiotoxicity of cancer chemotherapy, or myocarditis in viral infections. In contrast, decreased susceptibility of secondary heart failure or viral myocarditis might be expected in patients with the protective allele of BAG3. These possibilities warrant further research.

In contrast to its protective roles in cardiomyocytes, BAG3 has been identified as favoring tumor growth and antiapoptotic effects in cancerous cells. ⁷⁶ , ⁷⁹ This has led to BAG3 being considered as a target of anticancer therapy, particularly in pancreatic cancer and heme‐malignancies. ⁷⁶ This conundrum can be obviated by developing therapeutic strategies in heart failure with reduced ejection fraction that do not cause incorporation of the exogenous BAG3 into the native genome. ¹⁰⁰ Additional studies are warranted to dissect the relationship between PPCM and BAG3.

Sources of Funding

The study was supported by Institutional Development Funds from the Children's Hospital of Philadelphia to the Center for Applied Genomics and The Children's Hospital of Philadelphia Endowed Chair in Genomic Research (H.H.). Grant/award number: not applicable.

Disclosures

Dr Feldman is the founder of Renovacor, Inc., a publicly traded biotechnology company that is developing gene therapy for treatment of patients with cardiovascular and central nervous system diseases attributable to genetic variants in BAG3. He holds equity in the company and receives consultation fees, and is a member of the scientific advisory board of the company and serves as the Chief Scientific Advisor. Although he holds a grant from the company that funds the research in his laboratory in part, the work presented here was not funded by Renovacor. The remaining authors have no disclosures to report.

Supporting information

Tables S1–S2

Figure S1