**2. Titin**

Titin, the largest member of the superfamily of giant sarcomeric proteins, is a 3-4 MDa protein encoded by the single *TTN* gene. The 363 exons that make up *TTN* undergo extensive alter‐ native splicing resulting in the expression of several large variants of the protein [11]. A single titin molecule spans a half sarcomere, with its NH2-terminus anchored to the Z-disc and its COOH-terminus extending into the M-band [11]-[14]. Titin possesses a modular structure, composed mainly of immunoglobulin (Ig) and fibronectin type III (FN-III) domains. Specifi‐ cally, its Z-disc portion is composed of Ig domains along with ~45-residue long repeats unique to that region (Z-repeats) and possesses binding sites for several sarcomeric proteins (Figure 1) [7]. The portion of titin that spans the I-band is composed mainly of Ig domains intersected by titin specific N2A and N2B regions along with several ~30-residue long PEVK repeats (Figure 2). Titin's I-band region holds binding sites for actin and thin filament proteins, as well as docking sites for many signaling molecules [7]. Within its A-band portion, titin is organized in repeats containing numerous FN-III domains interspersed by Ig domains, which provide repetitive binding sites for myosin and thick filament associated proteins (Figure 3) [7]. The portion of titin that extends into the M-band begins with a Ser/Thr kinase domain followed by additional Ig domains and M-band specific insertions (Figure 4) [7]. Through this region, titin interacts with many other structural proteins to form a scaffold at the M-band.

Within the sarcomere, titin, through its PEVK domain, functions as a "molecular spring," contributing to the biomechanical properties and structural integrity of striated muscle cells during the contractile cycle [15], [16]. In addition, it acts as a "molecular blueprint" coordi‐ nating the assembly of structural, regulatory, and contractile proteins [17]. Given the elastic nature and scaffolding role of titin, it is not surprising that mutations along the length of *TTN* are intimately associated with the development of cardiomypathy.

presence of this mutation [18]. Sequencing of the DNA encoding the Z-disc region of titin in patients with HCM revealed a G to T transversion in codon 740 that is located within the 7th Z-repeat of titin and results in the replacement of an arginine residue with leucine (R740L) [19]. The mutation was not found in DNA from corresponding controls, suggesting it is not a polymorphism. Yeast two-hybrid assays showed that the mutation increased binding to αactinin by ~40% [19]. Interestingly the opposite biochemical effect was observed in a father and daughter with DCM where a point mutation in codon 743 resulting in an alanine to valine (A743V) conversion in the Zq region of titin was identified [18]. The A743V mutation, which is also localized within the α-actinin binding site on titin, significantly decreases the binding capacity of titin for α-actinin [18]. Additionally, a kindred with autosomal dominant DCM was analyzed and shown to have a point mutation in exon 18 encoding Ig4 that results in the conversion of a tryptophan residue to arginine (W976R), but the functional consequence of this mutation is currently unknown [20]. Recently, Golbus et al identified 3 indels within the Z-disc portion of titin in a large population of individuals exhibiting cardiac disease [5]. Due to the nature of this extensive study, phenotypic data regarding the subjects is unavailable,

**Figure 1.** Schematic representation of the Z-disc region of titin, illustrating its motifs and cardiomyopathy associated mutations. Insertions or deletions predicted to cause frame shifts (fs) and single amino acid deletions (del) are noted in blue and yellow, respectively. Missense mutations are shown with a magenta background. Mutations in the *TTN*

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**2.2. Cardiomyopathy linked mutations within the region of titin that spans the I-band region** To date, several mutations within the region of titin that spans the I-band have been linked to DCM and HCM. In particular, three mutations within titin's N2B region at the beginning of its I-band portion have been identified. A missense mutation in the N2B region of titin (S3753Y) was identified in two siblings with familial HCM [18], and shown to increase binding to fourand-a-half-LIM domain protein 2 (FLH2) by ~26% in a yeast two-hybrid assay [21]. In addition, in patients exhibiting a DCM phenotype a transversion of C to T in codon 4007 was found to result in the conversion of a glutamine residue to an early termination codon (Q4007X), and another missense mutation in codon 4417 was identified to replace a serine residue with an asparagine (S4417N) [18]. The premature stop codon (Q4007X) occurs just prior to the binding

however they have been linked to either DCM or HCM.

gene within this region are shown relative to the domains in which they are found.

To date, at least 107 mutations in *TTN* have been causally linked to HCM, DCM, and ARVC. Many of these mutations occur within essential binding sites along its length, with a high incidence of mutations occurring within the region that spans the A-band, disrupting the ability of titin to bind to myosin thick filaments. The remaining mutations are present within the extensible region of titin affecting its ability to respond to the constant stretching of the sarcomere during repeating cycles of contraction and relaxation. Notably, 84 of these muta‐ tions, found exclusively in the I- and A-band regions of titin, alter the length of the protein. Thus, mutations within *TTN* compromise the structural integrity of sarcomeres and lead to impaired contractile activity of cardiac muscle cells.

#### **2.1. Cardiomyopathy linked mutations within titin's Z-disc region**

The extreme NH2-terminus of titin is only mildly affected by cardiomyopathy-causing mutations; only 4 missense mutations and 3 insertion/deletion (indel) polymorphisms have been identified within the Z-disc portion of titin. A missense mutation identified in codon 54 leads to conversion of a valine residue to methionine (V54M) in the region encoding the first Ig domain of titin [18]. The V54M mutation is located in the telethonin-binding domain of titin and functional analysis revealed a decrease of titin's ability to interact with telethonin in the

**2. Titin**

64 Cardiomyopathies

Titin, the largest member of the superfamily of giant sarcomeric proteins, is a 3-4 MDa protein encoded by the single *TTN* gene. The 363 exons that make up *TTN* undergo extensive alter‐ native splicing resulting in the expression of several large variants of the protein [11]. A single titin molecule spans a half sarcomere, with its NH2-terminus anchored to the Z-disc and its COOH-terminus extending into the M-band [11]-[14]. Titin possesses a modular structure, composed mainly of immunoglobulin (Ig) and fibronectin type III (FN-III) domains. Specifi‐ cally, its Z-disc portion is composed of Ig domains along with ~45-residue long repeats unique to that region (Z-repeats) and possesses binding sites for several sarcomeric proteins (Figure 1) [7]. The portion of titin that spans the I-band is composed mainly of Ig domains intersected by titin specific N2A and N2B regions along with several ~30-residue long PEVK repeats (Figure 2). Titin's I-band region holds binding sites for actin and thin filament proteins, as well as docking sites for many signaling molecules [7]. Within its A-band portion, titin is organized in repeats containing numerous FN-III domains interspersed by Ig domains, which provide repetitive binding sites for myosin and thick filament associated proteins (Figure 3) [7]. The portion of titin that extends into the M-band begins with a Ser/Thr kinase domain followed by additional Ig domains and M-band specific insertions (Figure 4) [7]. Through this region, titin

interacts with many other structural proteins to form a scaffold at the M-band.

are intimately associated with the development of cardiomypathy.

**2.1. Cardiomyopathy linked mutations within titin's Z-disc region**

impaired contractile activity of cardiac muscle cells.

Within the sarcomere, titin, through its PEVK domain, functions as a "molecular spring," contributing to the biomechanical properties and structural integrity of striated muscle cells during the contractile cycle [15], [16]. In addition, it acts as a "molecular blueprint" coordi‐ nating the assembly of structural, regulatory, and contractile proteins [17]. Given the elastic nature and scaffolding role of titin, it is not surprising that mutations along the length of *TTN*

To date, at least 107 mutations in *TTN* have been causally linked to HCM, DCM, and ARVC. Many of these mutations occur within essential binding sites along its length, with a high incidence of mutations occurring within the region that spans the A-band, disrupting the ability of titin to bind to myosin thick filaments. The remaining mutations are present within the extensible region of titin affecting its ability to respond to the constant stretching of the sarcomere during repeating cycles of contraction and relaxation. Notably, 84 of these muta‐ tions, found exclusively in the I- and A-band regions of titin, alter the length of the protein. Thus, mutations within *TTN* compromise the structural integrity of sarcomeres and lead to

The extreme NH2-terminus of titin is only mildly affected by cardiomyopathy-causing mutations; only 4 missense mutations and 3 insertion/deletion (indel) polymorphisms have been identified within the Z-disc portion of titin. A missense mutation identified in codon 54 leads to conversion of a valine residue to methionine (V54M) in the region encoding the first Ig domain of titin [18]. The V54M mutation is located in the telethonin-binding domain of titin and functional analysis revealed a decrease of titin's ability to interact with telethonin in the

**Figure 1.** Schematic representation of the Z-disc region of titin, illustrating its motifs and cardiomyopathy associated mutations. Insertions or deletions predicted to cause frame shifts (fs) and single amino acid deletions (del) are noted in blue and yellow, respectively. Missense mutations are shown with a magenta background. Mutations in the *TTN* gene within this region are shown relative to the domains in which they are found.

presence of this mutation [18]. Sequencing of the DNA encoding the Z-disc region of titin in patients with HCM revealed a G to T transversion in codon 740 that is located within the 7th Z-repeat of titin and results in the replacement of an arginine residue with leucine (R740L) [19]. The mutation was not found in DNA from corresponding controls, suggesting it is not a polymorphism. Yeast two-hybrid assays showed that the mutation increased binding to αactinin by ~40% [19]. Interestingly the opposite biochemical effect was observed in a father and daughter with DCM where a point mutation in codon 743 resulting in an alanine to valine (A743V) conversion in the Zq region of titin was identified [18]. The A743V mutation, which is also localized within the α-actinin binding site on titin, significantly decreases the binding capacity of titin for α-actinin [18]. Additionally, a kindred with autosomal dominant DCM was analyzed and shown to have a point mutation in exon 18 encoding Ig4 that results in the conversion of a tryptophan residue to arginine (W976R), but the functional consequence of this mutation is currently unknown [20]. Recently, Golbus et al identified 3 indels within the Z-disc portion of titin in a large population of individuals exhibiting cardiac disease [5]. Due to the nature of this extensive study, phenotypic data regarding the subjects is unavailable, however they have been linked to either DCM or HCM.

#### **2.2. Cardiomyopathy linked mutations within the region of titin that spans the I-band region**

To date, several mutations within the region of titin that spans the I-band have been linked to DCM and HCM. In particular, three mutations within titin's N2B region at the beginning of its I-band portion have been identified. A missense mutation in the N2B region of titin (S3753Y) was identified in two siblings with familial HCM [18], and shown to increase binding to fourand-a-half-LIM domain protein 2 (FLH2) by ~26% in a yeast two-hybrid assay [21]. In addition, in patients exhibiting a DCM phenotype a transversion of C to T in codon 4007 was found to result in the conversion of a glutamine residue to an early termination codon (Q4007X), and another missense mutation in codon 4417 was identified to replace a serine residue with an asparagine (S4417N) [18]. The premature stop codon (Q4007X) occurs just prior to the binding site for FHL2 while the S4417N mutation decreases the binding capacity of titin for FHL2, as determined by yeast two-hybrid studies [21]. Within titin's PEVK region 3 missense mutations have been identified in DCM and HCM patients (G3470D, R8500H, and R8604Q) [22], [23]. Mutations R8500H and R8604Q were shown to increase the binding capacity of titin for cardiac ankyrin repeat protein (CARP) as determined by coimmunoprecipitation assays [22]. The pathogenicity of the G3470D is still unknown, however. Moreover, using population based studies of DCM and HCM patients, Golbus et al recently identified 9 indels within the portion of titin that spans the I-band [5].

In a large study, using next generation sequencing, Herman et al analyzed 203 and 231 patients with DCM and HCM, respectively and the corresponding control subjects for mutations in the *TTN* gene [6]. The frequency of *TTN* polymorphisms was significantly higher in DCM than HCM patients or normal subjects. Interestingly, of the mutations identified, all were shown to cause alterations in full-length titin, many of which caused early termination. Two of these truncations occurred as a result of missense mutations within the N2B region and also within Ig94 following the PEVK region. In addition, several splice site donor/acceptor mutations were found to cause truncations within the PEVK region and Ig85. The study also revealed 3 deletion and 2 insertion mutations affecting the I-band region of titin. Specifically, frameshift mutations within Ig11, 45, 61, and 85 alter the length of the protein, while a large duplication of exons 72-124 corresponding to Ig50 through the PEVK region increased *TTN*'s already large size by ~28kb. The functional significance of these mutations, which alter the length of full-length titin, is currently unknown.

Studies linking *TTN* to the development of DCM and HCM date back about a decade, however, it is only recently that *TTN* has been linked to ARVC. A recent study using DNA screening of patients diagnosed with ARVC revealed 3 missense mutations along the I-band region of titin [9]. The study identified a threonine to isoleucine transversion within codon 2896 (T2896I) located in Ig16 as well as two mutations within the PEVK region (Y8031C and H8848Y). Proteomic techniques revealed that the T2896I mutation reduces the structural stability of Ig16 and increases its propensity for degradation [9]. The pathogenicity of the other two mutations has not yet been determined.

tively [10]. In addition to the several length altering mutations noted within the I-band region of titin by Herman et al, many more have been found within the region spanning the A-band [6]. An astonishing 23 missense mutations have been identified to cause early termination within several of the Ig and FN-III domains throughout the A-band. Another 11 spice site donor/acceptor mutations were found to cause truncations throughout the A-band. In addition, 13 deletion, 4 insertion, and 2 insertion/deletion mutations have been shown to cause frameshifts in the coding region of titin, resulting in altered full-length titin protein. The functional significance of these mutations is not yet known, however. Moreover, Golbus et al recently identified 3 indels within the portion of titin that spans the A-band in a large popu‐ lation of individuals exhibiting cardiac disease [5]. Due to the nature of this extensive study phenotypic data regarding the subjects in unavailable, however they have been linked to either

**Figure 2.** Schematic representation of the I-band region of titin, illustrating its motifs and cardiomyopathy associated mutations. Mutations in the *TTN* gene within this region are shown relative to the domains in which they are found. Insertions or deletions predicted to cause frame shifts (fs) and single amino acid deletions (del) are noted in blue and yellow, respectively. Missense mutations are shown with a magenta background. A red background indicates non‐ sense mutations resulting in premature stop codons (ter). Splice site donor/acceptor mutations are shown in green.

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In addition, 4 missense mutations affecting patients with ARVC have been identified within the A-band region of titin. DNA screening of patients exhibiting signs of ARVC revealed 4 missense mutations affecting both FN-III and Ig domains along the A-band region of titin (I16949T, A18579T, A19309S, P30847L) [9]. The molecular effects of these mutations have not

DCM or HCM.

yet been determined.

#### **2.3. Cardiomyopathy linked mutations within titin's A-band region**

The region of titin spanning the A-band can be considered a "hot spot" for cardiomyopathylinked changes with an overwhelming 63 identified mutations. In the early 2000's, Gerull et al analyzed two siblings with autosomal dominant DCM and identified a unique mutation in titin [20]. A 2-bp insertion mutation in exon 326, caused a frameshift at K20995 within Ig115 resulting in a premature stop codon leading to proteolytic degradation of titin, probably near or within the PEVK domain, as determined by antibody labeling [20]. A few years later, the same group discovered a frameshift mutation at A27460 within FN-III107, also resulting in a premature stop codon, and proteolytic degradation of titin [24].

Recently, two additional insertions within FN-III domains present at the A-band have been identified in patients with DCM. The first causes a frame shift at S19628 and the second a frameshift at G26124 resulting in early termination within domains FN-III 42 and 97, respec‐

site for FHL2 while the S4417N mutation decreases the binding capacity of titin for FHL2, as determined by yeast two-hybrid studies [21]. Within titin's PEVK region 3 missense mutations have been identified in DCM and HCM patients (G3470D, R8500H, and R8604Q) [22], [23]. Mutations R8500H and R8604Q were shown to increase the binding capacity of titin for cardiac ankyrin repeat protein (CARP) as determined by coimmunoprecipitation assays [22]. The pathogenicity of the G3470D is still unknown, however. Moreover, using population based studies of DCM and HCM patients, Golbus et al recently identified 9 indels within the portion

In a large study, using next generation sequencing, Herman et al analyzed 203 and 231 patients with DCM and HCM, respectively and the corresponding control subjects for mutations in the *TTN* gene [6]. The frequency of *TTN* polymorphisms was significantly higher in DCM than HCM patients or normal subjects. Interestingly, of the mutations identified, all were shown to cause alterations in full-length titin, many of which caused early termination. Two of these truncations occurred as a result of missense mutations within the N2B region and also within Ig94 following the PEVK region. In addition, several splice site donor/acceptor mutations were found to cause truncations within the PEVK region and Ig85. The study also revealed 3 deletion and 2 insertion mutations affecting the I-band region of titin. Specifically, frameshift mutations within Ig11, 45, 61, and 85 alter the length of the protein, while a large duplication of exons 72-124 corresponding to Ig50 through the PEVK region increased *TTN*'s already large size by ~28kb. The functional significance of these mutations, which alter the length of full-length titin,

Studies linking *TTN* to the development of DCM and HCM date back about a decade, however, it is only recently that *TTN* has been linked to ARVC. A recent study using DNA screening of patients diagnosed with ARVC revealed 3 missense mutations along the I-band region of titin [9]. The study identified a threonine to isoleucine transversion within codon 2896 (T2896I) located in Ig16 as well as two mutations within the PEVK region (Y8031C and H8848Y). Proteomic techniques revealed that the T2896I mutation reduces the structural stability of Ig16 and increases its propensity for degradation [9]. The pathogenicity of the other two mutations

The region of titin spanning the A-band can be considered a "hot spot" for cardiomyopathylinked changes with an overwhelming 63 identified mutations. In the early 2000's, Gerull et al analyzed two siblings with autosomal dominant DCM and identified a unique mutation in titin [20]. A 2-bp insertion mutation in exon 326, caused a frameshift at K20995 within Ig115 resulting in a premature stop codon leading to proteolytic degradation of titin, probably near or within the PEVK domain, as determined by antibody labeling [20]. A few years later, the same group discovered a frameshift mutation at A27460 within FN-III107, also resulting in a

Recently, two additional insertions within FN-III domains present at the A-band have been identified in patients with DCM. The first causes a frame shift at S19628 and the second a frameshift at G26124 resulting in early termination within domains FN-III 42 and 97, respec‐

**2.3. Cardiomyopathy linked mutations within titin's A-band region**

premature stop codon, and proteolytic degradation of titin [24].

of titin that spans the I-band [5].

66 Cardiomyopathies

is currently unknown.

has not yet been determined.

**Figure 2.** Schematic representation of the I-band region of titin, illustrating its motifs and cardiomyopathy associated mutations. Mutations in the *TTN* gene within this region are shown relative to the domains in which they are found. Insertions or deletions predicted to cause frame shifts (fs) and single amino acid deletions (del) are noted in blue and yellow, respectively. Missense mutations are shown with a magenta background. A red background indicates non‐ sense mutations resulting in premature stop codons (ter). Splice site donor/acceptor mutations are shown in green.

tively [10]. In addition to the several length altering mutations noted within the I-band region of titin by Herman et al, many more have been found within the region spanning the A-band [6]. An astonishing 23 missense mutations have been identified to cause early termination within several of the Ig and FN-III domains throughout the A-band. Another 11 spice site donor/acceptor mutations were found to cause truncations throughout the A-band. In addition, 13 deletion, 4 insertion, and 2 insertion/deletion mutations have been shown to cause frameshifts in the coding region of titin, resulting in altered full-length titin protein. The functional significance of these mutations is not yet known, however. Moreover, Golbus et al recently identified 3 indels within the portion of titin that spans the A-band in a large popu‐ lation of individuals exhibiting cardiac disease [5]. Due to the nature of this extensive study phenotypic data regarding the subjects in unavailable, however they have been linked to either DCM or HCM.

In addition, 4 missense mutations affecting patients with ARVC have been identified within the A-band region of titin. DNA screening of patients exhibiting signs of ARVC revealed 4 missense mutations affecting both FN-III and Ig domains along the A-band region of titin (I16949T, A18579T, A19309S, P30847L) [9]. The molecular effects of these mutations have not yet been determined.

808 amino acids, respectively. Genetic analysis showed the defects in the *TTN* gene to be homozygous, leaving the heterozygote parents clinically unaffected. These mutations (1bp deletion in exon 360 and an 8bp deletion in exon 358) in the *TTN* gene are the first to be

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**Figure 4.** Schematic representation of the M-band region of titin, illustrating its motifs and cardiomyopathy-associat‐ ed mutations. Mutations in the *TTN* gene within this region are shown relative to the domains in which they can be found. Insertions or deletions predicted to cause frame shifts (fs) and single amino acid deletions (del) are noted in

Although titin has been implicated in cardiomyopathies for over a decade, only recently has its direct role begun to be expounded. Many of the follow-up results on identified cardiomy‐ opathy linked mutations of *TTN* indicate that these mutations can alter titin's binding capacity to its ligands, however, it remains to be proven that this is sufficient to cause DCM and HCM. Further study of the functional consequences of the *TTN* mutations*,* especially those causing truncated variants, using *in vivo* animal models is still necessary to elucidate titin's role in

Nebulin is a giant (~500-800 kDa) sarcomeric protein of striated muscles [26]. Similar to titin, nebulin is oriented longitudinally across the sarcomere, spanning the length of the thin filament [27]. Its NH2-terminus extends to the pointed ends of thin filaments in the sarcomeric I-band, and its COOH-terminus resides within the Z-disc [28]. The nebulin gene, *NEB*, contains 183 exons and is the product of extensive gene duplication, resulting in a protein of highly repetitive domain structure [29]. Nebulin is mostly composed of tandem nebulin-repeats with the central motifs organized as super-repeats. In addition, nebulin contains a glutamine rich region at its NH2-terminus, as well as a serine rich region and a Src Homology 3 (SH3) domain at its COOH-terminus. The organization of the nebulin repeats complements the periodicity of actin filaments [28]. Consistent with this, alternative splicing of the *NEB* gene generates proteins of different sizes, which correspond to thin filaments of various lengths [30], [31]. In addition to its role in stabilizing thin filaments, nebulin has also been implicated as a regulator

identified that produce both skeletal and cardiac muscle defects.

blue and yellow, respectively. Missense mutations are shown with a magenta background.

cardiomyopathies.

of thin filament length [32], [33].

**3. Nebulin**

**Figure 3.** Schematic representation of the A-band region of titin, illustrating its motifs and cardiomyopathy-associated mutations. Mutations in the *TTN* gene within this region are shown relative to the domains in which they are found. Insertions or deletions predicted to cause frame shifts (fs) and single amino acid deletions (del) are noted in blue and yellow, respectively. A red background indicates nonsense mutations resulting in premature stop codons (ter). Splice site donor/acceptor mutations are shown in green. Missense mutations are shown with a magenta background.

#### **2.4. Cardiomyopathy linked mutations within the region of titin that extends into the Mband**

Similar to the NH2-terminus of titin, the COOH-terminus remains relatively unaffected by cardiomyopathy causing mutations. A total of 6 mutations have been described within the portion of titin that extends into the M-band. Sequencing of DNA from patients with DCM and ARVC has identified 2 missense mutations localized to the M-band region of titin. Specifically, in two related individuals exhibiting late-onset DCM, an arginine to glutamine conversion at amino acid 32069 was identified (R32069Q) [21]. In addition, a patient diagnosed with ARVC possessed a methionine to threonine transition at codon 33291 (M33291T) [9]. These mutations localize to Ig146 and Ig152, respectively, however, their pathogenicity has not yet been determined. A recent study using population based analysis of DCM and HCM patients, identified 2 indels within the M-band portion of titin [5]. Phenotypic data regarding the subjects, as well as the mechanistic affects of the mutations are unavailable. Interestingly, 2 deletion mutations within the M-band region of titin were identified in 2 non-related families exhibiting early onset myopathy, affecting skeletal muscle, with fatal cardiomyopathy. Sequence analysis indicated a deletion mutation of 1 bp in exon 360 (Mex3) and an 8 bp deletion in exon 358 (Mex1) [25]. Both deletions left the titin kinase domain intact but resulted in premature stop codons at Ig domains 147 and 150 and a loss of the COOH-terminal 447 and 808 amino acids, respectively. Genetic analysis showed the defects in the *TTN* gene to be homozygous, leaving the heterozygote parents clinically unaffected. These mutations (1bp deletion in exon 360 and an 8bp deletion in exon 358) in the *TTN* gene are the first to be identified that produce both skeletal and cardiac muscle defects.

**Figure 4.** Schematic representation of the M-band region of titin, illustrating its motifs and cardiomyopathy-associat‐ ed mutations. Mutations in the *TTN* gene within this region are shown relative to the domains in which they can be found. Insertions or deletions predicted to cause frame shifts (fs) and single amino acid deletions (del) are noted in blue and yellow, respectively. Missense mutations are shown with a magenta background.

Although titin has been implicated in cardiomyopathies for over a decade, only recently has its direct role begun to be expounded. Many of the follow-up results on identified cardiomy‐ opathy linked mutations of *TTN* indicate that these mutations can alter titin's binding capacity to its ligands, however, it remains to be proven that this is sufficient to cause DCM and HCM. Further study of the functional consequences of the *TTN* mutations*,* especially those causing truncated variants, using *in vivo* animal models is still necessary to elucidate titin's role in cardiomyopathies.
