**2. Genetic causes of familial DCM**

Extensive studies on genetic basis of DCM underscored profound heterogeneous nature of DCM disease. DCM variants mutate genes encoding a broad spectrum of proteins with distinct functions and intracellular localization have been identified (**Figure 1** and **Table 1**). For example, mutations in genes encoding sarcomere protein involving in mechanosensing and force transmission, encoding nuclear envelope proteins required for the protection of biochemical forces, encoding desmosomal proteins required for maintaining the structural integrity of desomosome, encoding ion channels and molecules involving in calcium handling, encoding chaperone proteins, transcription factors and RNA-binding proteins have all been identified to cause DCM. Specific variants in these genes may alter various signaling pathways and cellular structures in cardiomyocyte that can disrupt the mechanism of cardiac muscle contraction and function, leading to common phenotypes of DCM.

#### **2.1 Sarcomere genes**

Sarcomere is the basic contractile unit mainly composed of thin and thick filaments. Mutations in sarcomere genes are one of the most important causes of DCM, which have been identified in about one third of DCM [19–21]. In addition to cause DCM, mutations in sarcomere genes often lead to overlapping phenotypes of hypertrophic cardiomyopathy (HCM), another major genetic type of cardiomyopathy characterized by ventricular wall thickness and diastolic dysfunction. Genes with definitive evidence, supported by studies from multiple centers/groups, with both

clinical linkage data and confirmative data from animal models, include *ACTC1, MYH7, TNNT2, TNNC1, TRM1,TTN*. Among which, *TTN, MYH7* and *TTNT2* are most commonly implicated in DCM, accounting for about 25%, 5% and 3% of familial DCM, respectively.

*TTN* gene encodes the titin, the largest known protein in human, consisting of 364 exons and about 35,000 amino acids that spans about a half of the sarcomere. It has long been recognized as a sarcomere scaffolding protein that serves as a blue print for sarcomerogenesis and myofibrillar assembly. In addition, titin also serves as a molecular spring that provides passive tension to regulate sarcomere contraction. With the advent of next-generation sequencing, human genetic analysis had revealed that *TTN*-truncating variants (*TTNtvs*) represent the most prevalent gene mutations in DCM patients, accounting for up to 25% of familial DCM cases [19]. Notebly, *TTNtvs* are also found in about 1% of general population [22, 23]. While not all of these healthy individuals with the *TTNtvs* are expected to develop DCM, they showed significantly increased left ventricular volumes and mild reduction of contractility [23]. Initially, it was hypothesized that *TTNtvs* caused DCM through a dominant negative mechanism [24]. This hypothesis is compromised by that truncated titin peptides were not detected from DCM hearts that harbored *TTNtvs*. Instead, there is evidence to support that premature truncations in the *TTN* transcripts trigger nonsense-mediated decay in rat models with *TTNtvs* [23]. Together with recently cumulative data derived from studies in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and human cardiac tissues, the hypothesis of haploinsufficiency mechanisms is now more widely recognized [25].

*MYH7* encodes the beta isoform of myosin heavy chain (MHC-β) that is predominantly expressed in the heart. MHC-β is a major component of the thick filament responsible for hydrolyzing ATP to produce force for cardiac muscle contraction. The Arg403Gln missense mutation of *MYH7* was first identified to cause HCM back to 1990 [12]. Since then, many *MYH7* variants were identified to associate with both HCM and DCM, accounting for approximately 40% of HCM and 5% of DCM cases, respectively. The definitive mechanisms underlying how *MYH7* mutations cause different types of cardiomyopathies remain to be elusive. Clinically, HCM is often characterized by hypercontractility. In contrast, DCM is characterized by hypocontractility. Mutations in myosin head domain consume more energy, leading to hypercontractility. One observation is that the *MYH7* variants associated with HCM are located more in the region encoding the myosin head domain, while *MYH7* mutations causal to DCM appear to disperse across the entire gene. The Arg403Gln variant causal to HCM, for example, increased energy usage due to impaired catalytic cycle of ATP hydrolysis, resulting in increased contractility [26]. In contrast, mutations associated with DCM showed an increased tension cost, with more energy consumption, have reduced force-generating capacity, thus causes a hypocontractility, leading to DCM [27, 28]. For example, the ASN1918LYS variant, causal to DCM, is located to the coiled-coil rod region, which is hypothesized to impair the incorporation of myosin into the myofilaments.

*TNNT2* encodes the cardiac muscle isoform of troponin T (cTnT). cTnT is one of the major tropomyosin-binding subunits of troponin on the thin filament that regulates cardiac muscle contraction. *TNNT2* variants are thought to account for approximately 5% of HCM, up to 3% of DCM, and a small fraction of arrhythmogenic cardiomyopathy (ACM). Many mutations in *TNNT2* causing DCM are located in both the middle and C-terminal regions of cTnT. These mutations mostly impair the cTnT's interaction with the thin filament regulatory system, myofilament calcium sensitivity, and/or the myosin ATPase activity, thus cause DCM.

*TPM1* encodes the alpha tropomyosin chain protein that belongs to the tropomyosin family of highly conserved thin filament proteins. In association with the

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troponin complex, tropomyosin mostly participates in the calcium regulation of cardiac muscle contraction and interaction of actin and myosin. Mutations in *TPM1* cause both HCM and DCM phenotypes [29, 30], accounting for about 5% of HCM and 1% of DCM cases, respectively. Functional characterization of *TPM1* mutations associated with both cardiomyopathies has led to a better understanding of the primary effects and consequence triggered by mutations in the long-range commu-

Mutations in gene encoding nucleus or nuclear envelope localized proteins that definitively link to DCM such as *LMNA, EMD* and *RBM20* were reported. *LMNA* is the second most common gene implicated in DCM, and *LMNA* variants account for about 6–10% of genetic DCM [32, 33]. *LMNA* encodes the nuclear envelope localized lamins A and C resulted from differential splicing at the 3′ end. Both lamins A and C are intermediate filament structural proteins, playing major roles in supporting cells with stability and strength. *LMNA* mutations linked to DCM can be both missense and frameshift across the coding region. Both dominant negative and haploinsufficiency mechanisms were proposed for *LMNA* mutations caused DCM. Beyond their roles as structural proteins, both lamins A and C involve in many different cellular process including regulation of gene expression, mechanosensing and nuclear to cytoplasmic transport. Functional study in animal models revealed that ERK1/2, JNK and p38 kinase pathways were drastically activated in *LMNA*-associated DCM. By targeting to the p38 kinase pathway through using a specific p38 kinase inhibitor ARRY-371797, Muchir and colleagues showed that LV dilation and deterioration of EF were effectively blocked, and *LMNA*-related severe biomechanical defects were significantly rescued in neonatal rat ventricular myocytes [34, 35]. Based on their encouraging and other related data, a clinical trial to study the protective effect of ARRY-371797 on patients with symptomatic DCM due to *LMNA* gene mutations was initiated (NCT03439514). This represents one of the first clinical trials involving

The RNA binding motif protein 20 (*RBM20)* gene encodes a nucleus localized RNA-binding protein. RBM20 protein mostly functions as a regulator of post-transcriptional splicing of a subset of genes involved in cardiomyopathy, ion-homeostasis, and sarcomere biology [36]. *RBM20* is predominantly expressed in skeletal and cardiac muscles. Loss of function mutations in the *RBM20* were firstly linked to familial DCM and account for 2–5% of DCM cases [37, 38]. Of the many targets regulated by *RBM20*, aberrant splicing of *TTN* is believed to be the main determinant of *RBM20* mutations caused DCM. Calcium/calmodulin-dependent kinase II delta (*CAMK2D*) is another pivotal cardiac gene transcriptionally regulated by *RBM20*. A recent study showed that *RBM20* mutations carriers also had increased risk of malignant ventricular arrhythmias and sudden cardiac death (SCD), likely

resultant from disturbed Ca2+ handling and arrhythmic Ca2+ cycling [39].

Other genes such as *DES, DMD* and *FLNC* that encode components of cytoskeleton localized proteins are also identified to link to DCM pathogenesis. Pathogenic mutations in these genes causing DCM often accompany with additional phenotypes, most notably skeletal myopathy. *DES* encodes desmin, a muscle-specific component of the intermediate filament presented at the Z-disk and intercalated discs that integrates sarcolemma, Z-disk and nuclear membrane to maintain the structural and functional integrity of sarcomeric contractile apparatus [40].

*DOI: http://dx.doi.org/10.5772/intechopen.94434*

**2.2 Nucleus and nuclear envelope genes**

genotype-specific therapy particularly for DCM.

**2.3 Cytoskeletal protein coding genes**

nication of the thin filament and specific phenotypes [31].

*Genetic Determinant of Familial Dilated Cardiomyopathy and Genotype-Targeted Therapeutic… DOI: http://dx.doi.org/10.5772/intechopen.94434*

troponin complex, tropomyosin mostly participates in the calcium regulation of cardiac muscle contraction and interaction of actin and myosin. Mutations in *TPM1* cause both HCM and DCM phenotypes [29, 30], accounting for about 5% of HCM and 1% of DCM cases, respectively. Functional characterization of *TPM1* mutations associated with both cardiomyopathies has led to a better understanding of the primary effects and consequence triggered by mutations in the long-range communication of the thin filament and specific phenotypes [31].

#### **2.2 Nucleus and nuclear envelope genes**

*Cardiac Diseases - Novel Aspects of Cardiac Risk, Cardiorenal Pathology and Cardiac Interventions*

clinical linkage data and confirmative data from animal models, include *ACTC1, MYH7, TNNT2, TNNC1, TRM1,TTN*. Among which, *TTN, MYH7* and *TTNT2* are most commonly implicated in DCM, accounting for about 25%, 5% and 3% of

*TTN* gene encodes the titin, the largest known protein in human, consisting of 364 exons and about 35,000 amino acids that spans about a half of the sarcomere. It has long been recognized as a sarcomere scaffolding protein that serves as a blue print for sarcomerogenesis and myofibrillar assembly. In addition, titin also serves as a molecular spring that provides passive tension to regulate sarcomere contraction. With the advent of next-generation sequencing, human genetic analysis had revealed that *TTN*-truncating variants (*TTNtvs*) represent the most prevalent gene mutations in DCM patients, accounting for up to 25% of familial DCM cases [19]. Notebly, *TTNtvs* are also found in about 1% of general population [22, 23]. While not all of these healthy individuals with the *TTNtvs* are expected to develop DCM, they showed significantly increased left ventricular volumes and mild reduction of contractility [23]. Initially, it was hypothesized that *TTNtvs* caused DCM through a dominant negative mechanism [24]. This hypothesis is compromised by that truncated titin peptides were not detected from DCM hearts that harbored *TTNtvs*. Instead, there is evidence to support that premature truncations in the *TTN* transcripts trigger nonsense-mediated decay in rat models with *TTNtvs* [23]. Together with recently cumulative data derived from studies in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and human cardiac tissues, the hypothesis of haploinsufficiency mechanisms is now more widely recognized [25]. *MYH7* encodes the beta isoform of myosin heavy chain (MHC-β) that is predominantly expressed in the heart. MHC-β is a major component of the thick filament responsible for hydrolyzing ATP to produce force for cardiac muscle contraction. The Arg403Gln missense mutation of *MYH7* was first identified to cause HCM back to 1990 [12]. Since then, many *MYH7* variants were identified to associate with both HCM and DCM, accounting for approximately 40% of HCM and 5% of DCM cases, respectively. The definitive mechanisms underlying how *MYH7* mutations cause different types of cardiomyopathies remain to be elusive. Clinically, HCM is often characterized by hypercontractility. In contrast, DCM is characterized by hypocontractility. Mutations in myosin head domain consume more energy, leading to hypercontractility. One observation is that the *MYH7* variants associated with HCM are located more in the region encoding the myosin head domain, while *MYH7* mutations causal to DCM appear to disperse across the entire gene. The Arg403Gln variant causal to HCM, for example, increased energy usage due to impaired catalytic cycle of ATP hydrolysis, resulting in increased contractility [26]. In contrast, mutations associated with DCM showed an increased tension cost, with more energy consumption, have reduced force-generating capacity, thus causes a hypocontractility, leading to DCM [27, 28]. For example, the ASN1918LYS variant, causal to DCM, is located to the coiled-coil rod region, which is hypothesized to

familial DCM, respectively.

**106**

impair the incorporation of myosin into the myofilaments.

*TNNT2* encodes the cardiac muscle isoform of troponin T (cTnT). cTnT is one of the major tropomyosin-binding subunits of troponin on the thin filament that regulates cardiac muscle contraction. *TNNT2* variants are thought to account for approximately 5% of HCM, up to 3% of DCM, and a small fraction of arrhythmogenic cardiomyopathy (ACM). Many mutations in *TNNT2* causing DCM are located in both the middle and C-terminal regions of cTnT. These mutations mostly impair the cTnT's interaction with the thin filament regulatory system, myofilament calcium sensitivity, and/or the myosin ATPase activity, thus cause DCM.

*TPM1* encodes the alpha tropomyosin chain protein that belongs to the tropomyosin family of highly conserved thin filament proteins. In association with the

Mutations in gene encoding nucleus or nuclear envelope localized proteins that definitively link to DCM such as *LMNA, EMD* and *RBM20* were reported. *LMNA* is the second most common gene implicated in DCM, and *LMNA* variants account for about 6–10% of genetic DCM [32, 33]. *LMNA* encodes the nuclear envelope localized lamins A and C resulted from differential splicing at the 3′ end. Both lamins A and C are intermediate filament structural proteins, playing major roles in supporting cells with stability and strength. *LMNA* mutations linked to DCM can be both missense and frameshift across the coding region. Both dominant negative and haploinsufficiency mechanisms were proposed for *LMNA* mutations caused DCM. Beyond their roles as structural proteins, both lamins A and C involve in many different cellular process including regulation of gene expression, mechanosensing and nuclear to cytoplasmic transport. Functional study in animal models revealed that ERK1/2, JNK and p38 kinase pathways were drastically activated in *LMNA*-associated DCM. By targeting to the p38 kinase pathway through using a specific p38 kinase inhibitor ARRY-371797, Muchir and colleagues showed that LV dilation and deterioration of EF were effectively blocked, and *LMNA*-related severe biomechanical defects were significantly rescued in neonatal rat ventricular myocytes [34, 35]. Based on their encouraging and other related data, a clinical trial to study the protective effect of ARRY-371797 on patients with symptomatic DCM due to *LMNA* gene mutations was initiated (NCT03439514). This represents one of the first clinical trials involving genotype-specific therapy particularly for DCM.

The RNA binding motif protein 20 (*RBM20)* gene encodes a nucleus localized RNA-binding protein. RBM20 protein mostly functions as a regulator of post-transcriptional splicing of a subset of genes involved in cardiomyopathy, ion-homeostasis, and sarcomere biology [36]. *RBM20* is predominantly expressed in skeletal and cardiac muscles. Loss of function mutations in the *RBM20* were firstly linked to familial DCM and account for 2–5% of DCM cases [37, 38]. Of the many targets regulated by *RBM20*, aberrant splicing of *TTN* is believed to be the main determinant of *RBM20* mutations caused DCM. Calcium/calmodulin-dependent kinase II delta (*CAMK2D*) is another pivotal cardiac gene transcriptionally regulated by *RBM20*. A recent study showed that *RBM20* mutations carriers also had increased risk of malignant ventricular arrhythmias and sudden cardiac death (SCD), likely resultant from disturbed Ca2+ handling and arrhythmic Ca2+ cycling [39].

#### **2.3 Cytoskeletal protein coding genes**

Other genes such as *DES, DMD* and *FLNC* that encode components of cytoskeleton localized proteins are also identified to link to DCM pathogenesis. Pathogenic mutations in these genes causing DCM often accompany with additional phenotypes, most notably skeletal myopathy. *DES* encodes desmin, a muscle-specific component of the intermediate filament presented at the Z-disk and intercalated discs that integrates sarcolemma, Z-disk and nuclear membrane to maintain the structural and functional integrity of sarcomeric contractile apparatus [40].

Mutations in *DES* have been associated with a spectrum of cardiomyopathies, mostly notably DCM, in about 1–2% of cases [41]. Overlapping phenotypes of *DES* mutations including arrhythmia, cardiac conduction diseases, and skeletal myopathy and smooth muscle defects are frequently observed.

Mutations in the gene encoding dystrophin (*DMD*) cause severe muscle weakness and DCM in Duchenne muscular dystrophy (DMD). Because the *DMD* gene is located in the short arm of X chromosome, pathogenic mutations causing DMD mostly affect boys. The frequency of *DMD* caused muscular dystrophy and DCM is rare, with an estimated incidence 1 in 3500 male births worldwide [42]. Dystrophin protein is a key component of dystrophin-glycoprotein complex and plays a critical role in maintaining the structural integrity of sarcolemma during repeated cycles of muscle contraction and relaxation [43]. Mutations in *DMD* result in loss of the dystrophin protein expression that causes primary muscular dystrophy in males presenting with progressive muscle wasting at early childhood. Subsequently, cardiac dysfunction is involved and more than 90% of affected individuals manifest DCM and patient often died of cardiac and respiratory muscle failure [44].

### **2.4 Z-disk gene**

The Z-disk is an anchoring plane for the actin (thin) filaments to attach and stabilize in the sarcomere. Mutations in many Z-disk-associated proteins coding genes result in cardiac disorders. *BAG3* encodes a highly conserved, Z-disk localized co-chaperone protein that is predominantly expressed in heart and skeletal muscle. BAG3 binds to the ATPase domain of the heat shock protein (Hsp) 70 and exerts multiple functions in regulating apoptosis, preserving the integrity of sarcomere, mediating unfolded protein response and autophagy. *BAG3* variants linked to DCM were firstly reported by two independent genome-wide association studies (GWASs). Later on*, BAG3* mutations were identified in 2–7% of DCM cases [45–47]. Genotype–phenotype correlation study revealed that DCM attributed by *BAG3* mutations is characterized by high penetrance in carriers more than 40 years of age. Patients with *BAG3* mutations are at a higher risk of developing a more severe and progressive heart failure compared with patients without *BAG3* mutations [46]. The level of BAG3 protein was reduced by about a half in both animal models of heart failure and DCM patients as well. Based on the evidence that truncation or deletion mutations in *BAG3* are associated with BAG3 haploinsufficiency which co-segregates with affected DCM family members, it was proposed that the decreased levels of BAG3 protein is the cause of DCM. BAG3 is also an independent heart failure risk factors associated with subclinical LV dysfunction. Thus, cumulative data support that *BAG3* as a bona-fide disease susceptibility gene for DCM [48].

#### **2.5 Ion channel gene**

Mutations in the ion channel coding gene *SCN5A* are identified to cause DCM with strong supporting evidence. *SCN5A* encodes the sodium channel Nav1.5 that is mainly expressed in the cardiac muscle [49]. Mutations in *SCN5A* are associated primarily with conduction disorder, arrhythmia and DCM. Incidence of pathogenic *SCN5A* variants is estimated to be 2–4% in all DCM cases [50]. Missense mutations such as R222Q variant located in a voltage-sensing domain exert activating effects on sodium channel function and were thought to cause DCM. While guidelinebased heart failure therapies have moderate effect, drugs that have sodium channelblocking properties such as amiodarone or flecainide could substantially reduce DCM phenotype in patients with R222Q carriers [51]. Moreover, a recent report showed that quinidine treatment of a DCM patient with R222Q mutation achieved

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a rapid and significant reduction of ventricular tachyarrhythmia and an improvement in the myocardial function [52]. These interesting genotype–phenotype association studies thus provide another successful example of elucidation of the genetic basis of familial DCM which can lead to effective genotype-tailored thera-

*PLN* encodes phosphlamban, a transmembrane protein localized to the sarcoplasmic reticulum. Mutations in *PLN* cause variable DCM phenotype, with underlying mechanisms proposed through inhibiting the sarcoplasmic reticulum Ca2 + -ATPase (SERCA2a) [53]. While founder mutation R14del mutation in *PLN* is associated with severe phenotype with high risk for lethal ventricular arrhythmias and end-stage heart failure in the European [54], a milder phenotype had been reported from others [55], suggesting that genetic background might have a big impact on modifying the disease progression associated with *PLN* mutations

Mutations in genes other encoding the sarcoglycans (α, β, γ, δ) were also identified to cause DCM. The sarcoglycans are transmembrane proteins mainly expressed in heart and skeletal muscle that interact with dystrophin. α-, β-, γ-, and δ-sarcoglycans form the sarcoglycan complex that is key components of the dystrophin-associated glycoprotein complex, conferring structural integrity and stability to the sarcolemma through connecting the muscle fiber cytoskeleton to the extracellular matrix, and protecting muscle fibers from mechanical stress during muscle contraction. Mutations in sarcoglycans coding genes cause primary limbgirdle muscular dystrophy presented with early onset muscle weakness and associate with significant DCM [56]. Notably, mutations in the δ- sarcoglycan coding gene lead to DCM without involvement of obvious muscular dystrophy phenotypes [57]. Mutations in nuclear encoded mitochondrial genes such as *TAZ* and *DNAJC19*

were also identified to cause DCM. *TAZ* encodes a mitochondrial localized Tafazzin protein that is predominantly expressed in cardiac and skeletal muscle. Tafazzin functions as a phospholipid transacylase that catalyzes the remodeling of cardiolipin that is required for oxidative phosphorylation. Mutations in the TAZ gene cause X-linked Barth syndrome and DCM, leading to premature death [58]. Mechanistically, mutations in *TAZ* result in Taffazin deficiency and cause mitochondrial dysfunction and impaired mitophagy and increased oxidative stress,

The advent of next-generation sequencing enables cost-effective genetic testing

in familial DCM which can define the precise genetic cause of disease. Genetic testing can also help optimize risk stratification and assess prognostics of patients and their relatives. With the identification of a pathogenic mutation and early diagnostic certainty, clinical management of affect individuals could be tailored and patients' survival can be improved. One best example is related to clinical practice of early intervention of DCM patients with L*MNA* mutations. *LMNA*-related DCM usually accompanied by significant conduction system disease, atrial fibrillation, ventricular tachycardia, and sudden cardiac death (SCD). Thus, *LMNA* mutations are often associated with a higher disease penetrance and more severe morbidity and high mortality [60]. Studies in several different cohorts of DCM patients with *LMNA* mutations identified non-missense mutations, LVEF<45% and higher

*DOI: http://dx.doi.org/10.5772/intechopen.94434*

peutic strategy.

caused DCM.

leading to DCM [59].

**3. Genetic testing**

**2.6 Other DCM genes**

#### *Genetic Determinant of Familial Dilated Cardiomyopathy and Genotype-Targeted Therapeutic… DOI: http://dx.doi.org/10.5772/intechopen.94434*

a rapid and significant reduction of ventricular tachyarrhythmia and an improvement in the myocardial function [52]. These interesting genotype–phenotype association studies thus provide another successful example of elucidation of the genetic basis of familial DCM which can lead to effective genotype-tailored therapeutic strategy.
