Clinical and functional characterization of TNNT2 mutations identified in patients with dilated cardiomyopathy

Abstract

Background: A key issue for cardiovascular genetic medicine is ascertaining if a putative mutation indeed causes dilated cardiomyopathy (DCM). This is critically important as genetic DCM, usually presenting with advanced, life-threatening disease, may be preventable with early intervention in relatives known to carry the mutation.

Methods and results: We recently undertook bidirectional resequencing of TNNT2, the cardiac troponin T gene, in 313 probands with DCM. We identified 6 TNNT2 protein-altering variants in 9 probands, all who had early onset, aggressive disease. Additional family members of mutation carriers were then studied when available. Four of the 9 probands had DCM without a family history, and 5 probands had familial DCM. Only 1 mutation (Lys210del) could be attributed as definitively causative from previous reports. Four of the 5 missense mutations were novel (Arg134Gly, Arg151Cys, Arg159Gln, and Arg205Trp), and one was previously reported with hypertrophic cardiomyopathy (Glu244Asp). Based on the clinical, pedigree, and molecular genetic data, these 5 mutations were considered possibly or likely disease causing. To further clarify their potential pathophysiologic impact, we undertook functional studies of these mutations in cardiac myocytes reconstituted with mutant troponin T proteins. We observed decreased Ca(2+) sensitivity of force development, a hallmark of DCM, in support of the conclusion that these mutations are disease causing.

Conclusions: We conclude that the combination of clinical, pedigree, molecular genetic, and functional data strengthen the interpretation of TNNT2 mutations in DCM.

Figure 1. The TNNT2, cardiac troponin T gene structure, cardiac troponin T amino acid conservation, and pedigrees of cardiac troponin T associated cardiomyopathy

A. The TNNT2, cardiac troponin T gene structure. The TNNT2 gene is shown, and the adult isoform of cardiac troponin T, encoded by exons 2-17, is a total of 288 amino acids in length. The locations of the 6 mutations presented are labeled with their respective letters (A through I); other published DCM mutations are labeled J through N. Mutations reported to cause hypertrophic cardiomyopathy are labeled 1 through 32. Glu244Asp, previously reported in association HCM and reported here in association with DCM is identified as both (I, 24). Exon numbering and amino acid numbering is per prior reports. Binding regions for troponin C, troponin I, tropomyosin and cardiac actin are shown. HCM mutations are shown in Supplemental Table A. B. Amino acid conservation. Altered amino acids are shown for each missense mutation on the first line. The wild-type human sequence is shown, followed by rat, mouse, chicken, zebrafish and drosophila. All variants were conserved to zebrafish or drosophila except Arg159Gln (rat, mouse). C. Pedigrees of cardiac troponin T associated cardiomyopathy. Pedigrees have been labeled by letter, which correspond to their respective mutation as shown in panel A and given in Tables 1 and 2. Squares represent males, circles females. An arrowhead denotes the proband. A diagonal line marks deceased individuals. Solid symbols indicate idiopathic dilated cardiomyopathy with or without heart failure; shaded symbols represent any cardiovascular abnormality. Open symbols represent unaffected individuals. The presence or absence of the pedigree's TNNT2 mutation is indicated by a + or - symbol, respectively. Obligate carriers are noted in parenthesis, (+). Pedigree G, previously published by others, is not shown.

Figure 1. The TNNT2, cardiac troponin T gene structure, cardiac troponin T amino acid conservation, and pedigrees of cardiac troponin T associated cardiomyopathy

A. The TNNT2, cardiac troponin T gene structure. The TNNT2 gene is shown, and the adult isoform of cardiac troponin T, encoded by exons 2-17, is a total of 288 amino acids in length. The locations of the 6 mutations presented are labeled with their respective letters (A through I); other published DCM mutations are labeled J through N. Mutations reported to cause hypertrophic cardiomyopathy are labeled 1 through 32. Glu244Asp, previously reported in association HCM and reported here in association with DCM is identified as both (I, 24). Exon numbering and amino acid numbering is per prior reports. Binding regions for troponin C, troponin I, tropomyosin and cardiac actin are shown. HCM mutations are shown in Supplemental Table A. B. Amino acid conservation. Altered amino acids are shown for each missense mutation on the first line. The wild-type human sequence is shown, followed by rat, mouse, chicken, zebrafish and drosophila. All variants were conserved to zebrafish or drosophila except Arg159Gln (rat, mouse). C. Pedigrees of cardiac troponin T associated cardiomyopathy. Pedigrees have been labeled by letter, which correspond to their respective mutation as shown in panel A and given in Tables 1 and 2. Squares represent males, circles females. An arrowhead denotes the proband. A diagonal line marks deceased individuals. Solid symbols indicate idiopathic dilated cardiomyopathy with or without heart failure; shaded symbols represent any cardiovascular abnormality. Open symbols represent unaffected individuals. The presence or absence of the pedigree's TNNT2 mutation is indicated by a + or - symbol, respectively. Obligate carriers are noted in parenthesis, (+). Pedigree G, previously published by others, is not shown.

Figure 1. The TNNT2, cardiac troponin T gene structure, cardiac troponin T amino acid conservation, and pedigrees of cardiac troponin T associated cardiomyopathy

A. The TNNT2, cardiac troponin T gene structure. The TNNT2 gene is shown, and the adult isoform of cardiac troponin T, encoded by exons 2-17, is a total of 288 amino acids in length. The locations of the 6 mutations presented are labeled with their respective letters (A through I); other published DCM mutations are labeled J through N. Mutations reported to cause hypertrophic cardiomyopathy are labeled 1 through 32. Glu244Asp, previously reported in association HCM and reported here in association with DCM is identified as both (I, 24). Exon numbering and amino acid numbering is per prior reports. Binding regions for troponin C, troponin I, tropomyosin and cardiac actin are shown. HCM mutations are shown in Supplemental Table A. B. Amino acid conservation. Altered amino acids are shown for each missense mutation on the first line. The wild-type human sequence is shown, followed by rat, mouse, chicken, zebrafish and drosophila. All variants were conserved to zebrafish or drosophila except Arg159Gln (rat, mouse). C. Pedigrees of cardiac troponin T associated cardiomyopathy. Pedigrees have been labeled by letter, which correspond to their respective mutation as shown in panel A and given in Tables 1 and 2. Squares represent males, circles females. An arrowhead denotes the proband. A diagonal line marks deceased individuals. Solid symbols indicate idiopathic dilated cardiomyopathy with or without heart failure; shaded symbols represent any cardiovascular abnormality. Open symbols represent unaffected individuals. The presence or absence of the pedigree's TNNT2 mutation is indicated by a + or - symbol, respectively. Obligate carriers are noted in parenthesis, (+). Pedigree G, previously published by others, is not shown.

Figure 2. Normalized pCa-Force relationship in skinned cardiac muscle fibers

The wildtype (WT) and DCM-HCTnTs replaced native CTnT in the procedure shown in Figure 2.A) HCTnT-WT containing fibers were compared to DCM-mutant HCTnT-R134G B) HCTnT-WT containing fibers compared to DCM-mutant HCTnT-R151C C) HCTnT-WT containing fibers compared to DCM-mutant HCTnT-R159Q D) HCTnT-WT containing fibers compared to DCM-mutant HCTnT-R205W. The Ca²⁺ dependence of force development remained unchanged in native porcine fibers and after HCTnT-WT substitution (See Table 3). Data in each panel is an average of 7-9 experiments and the mean is shown as mean ± S.E.