Familial dilated cardiomyopathy caused by an alpha-tropomyosin mutation: the distinctive natural history of sarcomeric dilated cardiomyopathy

Abstract

Objectives: We sought to further define the role of sarcomere mutations in dilated cardiomyopathy (DCM) and associated clinical phenotypes.

Background: Mutations in several contractile proteins contribute to DCM, but definitive evidence for the roles of most sarcomere genes remains limited by the lack of robust genetic support.

Methods: Direct sequencing of 6 sarcomere genes was performed on 334 probands with DCM. A novel D230N missense mutation in the gene encoding alpha-tropomyosin (TPM1) was identified. Functional assessment was performed by the use of an in vitro reconstituted sarcomere complex to evaluate ATPase regulation and Ca(2+) affinity as correlates of contractility.

Results: TPM1 D230N segregated with DCM in 2 large unrelated families. This mutation altered an evolutionarily conserved residue and was absent in >1,000 control chromosomes. In vitro studies demonstrated major inhibitory effects on sarcomere function with reduced Ca(2+) sensitivity, maximum activation, and Ca(2+) affinity compared with wild-type TPM1. Clinical manifestations ranged from decompensated heart failure or sudden death in those presenting early in life to asymptomatic left ventricular dysfunction in those diagnosed during adulthood. Notably, several affected infants had remarkable improvement.

Conclusions: Genetic segregation in 2 unrelated families and functional analyses conclusively establish a pathogenic role for TPM1 mutations in DCM. In vitro results demonstrate contrasting effects of DCM and hypertrophic cardiomyopathy mutations in TPM1, suggesting that specific functional consequences shape cardiac remodeling. Along with previous reports, our data support a distinctive, age-dependent phenotype with sarcomere-associated DCM where presentation early in life is associated with severe, sometimes lethal, disease. These observations have implications for the management of familial DCM.

Figure 1. Molecular Model of α-Tropomyosin

α-tropomyosin is an alpha-helical coiled-coil which interacts with actin (violet functional domains) and troponin T (blue). The locations of mutations associated with DCM (yellow) and HCM (green) are indicated. The D230N mutation is shown in red.

Figure 2. Pedigrees and Haplotype Analysis of Families with Autosomal Dominant DCM due to D230N Mutation in α-Tropomyosin

A., B. Pedigrees are shown for 2 unrelated families with DCM. Squares indicate males, circles females, black symbols DCM, open symbols unaffected, green symbols uncertain clinical status, slashes deceased individuals. Genotype results are indicated by (+) = D230N present, (−) =mutation absent. * = sudden cardiac death. C. Haplotype analysis indicates that the families are unrelated and that the D230N (688G>A) TPM1 mutation arose independently in these families. Several family members from each kindred were genotyped at four loci in the TPM1 gene (rs4775613, rs4775614, rs1071646, rs4774472), comprising 4 common haplotypes in Caucasians (A, B, C, D) as described by HapMap (www.hapmap.org). The D230N mutation is shown in red with *.

Figure 2. Pedigrees and Haplotype Analysis of Families with Autosomal Dominant DCM due to D230N Mutation in α-Tropomyosin

A., B. Pedigrees are shown for 2 unrelated families with DCM. Squares indicate males, circles females, black symbols DCM, open symbols unaffected, green symbols uncertain clinical status, slashes deceased individuals. Genotype results are indicated by (+) = D230N present, (−) =mutation absent. * = sudden cardiac death. C. Haplotype analysis indicates that the families are unrelated and that the D230N (688G>A) TPM1 mutation arose independently in these families. Several family members from each kindred were genotyped at four loci in the TPM1 gene (rs4775613, rs4775614, rs1071646, rs4774472), comprising 4 common haplotypes in Caucasians (A, B, C, D) as described by HapMap (www.hapmap.org). The D230N mutation is shown in red with *.

Figure 3. Functional Properties of the D230N Mutation in α-Tropomyosin

In vitro functional analyses of the D230N mutant α-tropomyosin show that at any activating-level of [Ca²⁺], the D230N mutation resulted in diminished sarcomere function and calcium affinity. A. Ca²⁺ sensitivity of thin filament regulation of actin-tropomyosin-activated myosin ATPase activity. Compared to wildtype, maximal ATPase turnover and pCa₅₀ were significantly reduced (p<0.05) in the DCM mutant α-tropomyosin (D230N). In contrast, both parameters were significantly increased (p<0.05) in the HCM mutant protein (D175N). B. Ca²⁺ binding to the thin filament causes a decrease in spectrofluorescent intensity. The Ca²⁺ affinity of reconstituted thin filaments containing D230N α-tropomyosin DCM-mutant was significantly reduced (p<0.001) compared to wildtype α-tropomyosin. In contrast the D175N HCM-mutant was associated with significantly increased Ca²⁺ affinity (p<0.05).

Figure 3. Functional Properties of the D230N Mutation in α-Tropomyosin

In vitro functional analyses of the D230N mutant α-tropomyosin show that at any activating-level of [Ca²⁺], the D230N mutation resulted in diminished sarcomere function and calcium affinity. A. Ca²⁺ sensitivity of thin filament regulation of actin-tropomyosin-activated myosin ATPase activity. Compared to wildtype, maximal ATPase turnover and pCa₅₀ were significantly reduced (p<0.05) in the DCM mutant α-tropomyosin (D230N). In contrast, both parameters were significantly increased (p<0.05) in the HCM mutant protein (D175N). B. Ca²⁺ binding to the thin filament causes a decrease in spectrofluorescent intensity. The Ca²⁺ affinity of reconstituted thin filaments containing D230N α-tropomyosin DCM-mutant was significantly reduced (p<0.001) compared to wildtype α-tropomyosin. In contrast the D175N HCM-mutant was associated with significantly increased Ca²⁺ affinity (p<0.05).

Figure 4. Clinical Outcomes in Carriers of the D230N α-Tropomyosin Mutation

The presence of TPM1 D230N is associated with a high risk for developing DCM, but over a wide spectrum of ages, ranging from infancy to the 6^th decade (Black curve). DCM includes both symptomatic and asymptomatic left ventricular systolic dilation and/or dysfunction. Severe clinical outcomes, cardiac death and transplantation, were confined to young family members and distinctly absent from those presenting in adulthood (red curve). * Individuals who suffered cardiac death prior to genotyping were assumed to be mutation carriers. G⁺ = mutation carriers.