Original Research Received: April 27, 2015 Accepted after revision: September 3, 2015 Published online: October 28, 2015
Cardiology 2016;133:91–96 DOI: 10.1159/000440877
A Double Heterozygous Mutation of TNNI3 Causes Hypertrophic Cardiomyopathy in a Han Chinese Family Hua Zheng a Huajie Huang b Zhisong Ji b Qi Yang b Qiuxia Yu b Fan Shen b Cuixian Liu b Fu Xiong b
Department of Cardiovascular Medicine, Nanfang Hospital, Southern Medical University, and b Department of Medical Genetics, School of Basic Medicine Sciences, Southern Medical University, Guangzhou, China
Key Words Hypertrophic cardiomyopathy · Heterozygous mutation · Cardiac troponin I · Pathophysiology
Abstract Objectives: To investigate the variations in the TNNI3 gene in a Chinese Han family affected by hypertrophic cardiomyopathy (HCM) and the potential molecular mechanism linking these mutations with disease. Methods: Peripheral venous blood was acquired from family members, and TNNI3 mutations were identified by DNA sequencing. The pathophysiology of TNNI3 mutations was investigated using bioinformatics, subcellular localization determination and Western blotting. Results: Sanger sequencing revealed that the proband possessed 2 heterozygous mutations, c.235C>T and c.470C>T, located at exons 4 and 6 of the TNNI3 gene. The proband (II-2) and her brother (II-1), who had been previously diagnosed with HCM, harbored both mutations whereas their healthy parents harbored only 1. Alignment of the TNNI3 amino acid sequence indicated that the two Pro residues were highly conserved across species. Subcellular localization showed that both wild-type (WT) and mutant
© 2015 S. Karger AG, Basel 0008–6312/15/1332–0091$39.50/0 E-Mail [email protected] www.karger.com/crd
TNNI3 proteins were localized at the cell nucleus. Western blot analysis of expression in human embryonic kidney 293T cells showed that the intracellular levels of the mutant proteins were significantly decreased compared to WT TNNI3 (p < 0.01). Conclusions: Our findings showed that a double heterozygous mutation in the TNNI3 gene is involved in the pathogenesis of HCM via haploinsufficiency. These results will inspire further studies to investigating the link between the TNNI3 gene and HCM. © 2015 S. Karger AG, Basel
Introduction
Hypertrophic cardiomyopathy (HCM) is the most common autosomal-dominant monogenic cardiac disease with an estimated prevalence of 1 in 500 of the population [1, 2]. Clinically, HCM is characterized by hypertrophy of the left ventricle (LV) walls, especially the septum, usually in an asymmetric manner, and the absence
H. Zheng and H. Huang contributed equally to this article.
Fu Xiong Department of Medical Genetics, School of Basic Medical Sciences Southern Medical University, 1838 Guangzhou Dadao Rd. Guangzhou 510515 (China) E-Mail xiongfu @ smu.edu.cn
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a
Methods Patients This study was approved by the Southern Medical University ethics committee. Informed consent was obtained from patients and relatives before analysis. Patients were recruited from outpatient clinics. The proband (II-2), a 21-year-old girl, was referred to the Cardiothoracic Surgery at Nanfang Hospital for consultation concerning chest pain and syncope. Clinical examination of all family members was performed by an experienced cardiac surgeon, and unexplained LV hypertrophy in cardiac imaging studies was investigated. The 200 normal controls matched by gender and ethnic origin were selected and recruited from healthy individuals at the department of Medical Genetics, School of Basic Medicine Sciences, Southern Medical University. Mutation Screening Peripheral blood was acquired from all family members, and standard phenol/chloroform extraction was performed to extract genomic DNA. Target capture and next-generation sequencing of the TNNT2, MYPBC3, MYH7, TPM1 and TNNI3 genes from the
92
Cardiology 2016;133:91–96 DOI: 10.1159/000440877
proband, all of which have been linked to HCM, were performed by KeyBay Co., Ltd. Two significant mutations within exons 4 and 6 of the TNNI3 gene were identified and verified using Sanger sequencing. PCR primers were designed using Primer3 (http:// frodo.wi.mit.edu/) to amplify regions including exons 4 and 6 to allow identification of these 2 mutations in family members. PCR products were analyzed by agarose gel electrophoresis and verified by Sanger sequencing. The PCR primers were as follows: F1: 5′-CGCCTGGTCTTTATCCTGAA-3′ and R1: 5′-TAGAAACCTCGCATCCTTGG-3′ for exon 4, and F2: 5′-AGTACCCACCCCCTCGTTT-3′ and R2: 5′-CCTCAGCATCCTCTTTCCTG-3′ for exon 6. Bioinformatics To provide further insight into TNNI3 function, secondary structure of the mutant TNNI3 proteins were predicted using the SOPMA method, and the 3-dimensional structures of wild-type (WT) and mutant TNNI3 proteins were predicted using the RCSB PDB (Research Collaboratory for Structural Bioinformatics Protein Data Bank). PolyPhen2 software was used to predict the harmfulness of mutations and analyze the conservation of TNNI3 across species. The SIFT program was used for predicting the function of the mutant proteins. Cellular Localization Recombinant plasmids were transfected into COS-7 cells to investigate the subcellular localization of the TNNI3 protein. The WT coding sequence of the TNNI3 gene was cloned into the HindIII and KpnI sites of the pEGFP-C1 vector (GENEWIZ, Inc. South Plainfield, N.J., USA), and this plasmid was used for generation of mutant plasmids by site-directed mutagenesis with the QuikChange site-directed mutagenesis kit (Stratagene). COS-7 cells were cultured in 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum. Recombinant plasmids were transfected into COS-7 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. At 24 h after transfection, cells were rinsed 3 times with PBS and stained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma, St. Louis, Mo., USA). Fluorescence microscopy (Nikon, Eclipse Ti-U, Tokyo, Japan) was used to visualize transfected cells. The primers used for the mutations were as follows: F1: 5′-CGCGCTCTGAGCACCTGCTGCCAGCCGCTGG-3′, R1: 5′-GCGCGAGACTCGTGGACGACGGTCGGCGACC-3′, F2: 5′-ATGCCATGATGCAGGTGCTGCTGGGGGCCCG-3′ and R2: 5′-TACGGTACTACGTCCACGACGACCCCCGGGC-3′. TNNI3 Expression in Human Embryonic Kidney 293T Cells To measure the expression of WT and mutant TNNI3, human embryonic kidney (HEK)293T cells were transfected with recombinant plasmids using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions, and then cultured in DMEM medium (Invitrogen) supplemented with 10% fetal bovine serum. The cells produced WT or mutant TnI-GFP fusions. At 24 h after transfection, cells were harvested and homogenized in RIPA lysis buffer containing protease inhibitor cocktail (Sigma). Blots were incubated with rabbit anti-GFP (1:1,000, Santa Cruz) overnight at 4 ° C, followed by goat anti-rabbit IgGHRP (1: 2,000, Santa Cruz) at room temperature for 1 h. Detection was performed with Immobilon Western chemiluminescent HRP substrate (Millipore). Fusion proteins were detected using Western blotting.
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of other cardiac or systemic disease that leads to secondary hypertrophy. The disease has a varied clinical course and outcome; many patients have little or no discernible cardiovascular symptoms whereas others have profound exercise limitation and recurrent arrhythmias [3]. The heritable nature and autosomal dominant pattern of inheritance of HCM have been long established [2]. Mutations in genes encoding the cardiac myosin heavy chain (βMHC), cardiac myosin-binding protein C (MyBPC), cardiac troponin T (TnT), cardiac troponin I (TnI), α-tropomyosin (αTM), essential and regulatory light chains and cardiac actin have all been correlated with the disease [4]. The troponin complex is a component of skeletal and cardiac muscle thin filaments that consists of three subunits: troponin C (calcium-binding protein), troponin T (tropomyosin-binding protein) and troponin I (inhibitory protein) that together play a crucial role in muscle activity [5, 6]. TnI, encoded by the TNNI3 gene, is the inhibitory subunit that blocks actin-myosin interactions and thereby mediates striated muscle relaxation [7–9]. Here, we investigated a Han Chinese family with HCM and found that a double heterozygous mutation in TNNI3 causes the disease. We studied the function and potential pathogenic mechanism of the mutated gene. Our results showed that the double heterozygous mutation results in haploinsufficiency of TNNI3 that ultimately causes HCM. This study may provide a reference model for future studies of the potential pathogenic mechanism by which mutations in TNNI3 cause HCM.
Color version available online
a
b
c
d
Fig. 1. Two-dimensional echocardiogram
and continuous-wave Doppler of HCM patients. a, b The interventricular septum (29 mm) of the proband II-2 is clearly thickened. c Similarly, the interventricular septum (28 mm) was also thickened in the proband’s brother II-1. d The velocity of blood flow in the LVOT of II-1 was increased to 219 cm/s, and the pressure gradient of the LVOT was increased to 19 mm Hg.
Table 1. Clinical features and genotypes of the proband and her family Subject
TNNI3 mutation
Father c.235C>T Mother c.470C>T II-2 c.235C>T, c.470C>T II-1 c.235C>T, c.470C>T
Protein alteration
Age, years
Age at onset, years
Clinical symptoms
Electrocardiogram
Echocardiography
chest pain
dyspnea
syncope systolic murmur
LVH + ST
AF
IVS, mm
Max LVWT, LVOT, mm mm
LADD, SAM mm
R79C A157C R79C, A157C R79C, A157C
49 48 21
– – 8
no no yes
– – –
no no yes
no no yes
– – –
– – –
11 11 29
11 10 12
19 18 10.2
41 37 43
no no yes
23
–
no
–
no
yes
–
–
28
12
10
49
yes
AF = Atrial fibrillation; IVS = interventricular septum; LADD = left atrial diastolic diameter; LVH + ST = LV hypertrophy combined with ST-T changes; Max LVWT = maximum LV wall thickness; SAM = systolic anterior motion of the mitral valve.
Clinical Features The clinical features of the proband and her family members are shown in table 1. She had experienced onset at the age of 8 years and exhibited symptoms including chest pain and syncope; systolic murmur was detected and she had an abnormal electrocardiogram (fig. 1; table 1). However, the other family members were asymptomatic. Color Doppler revealed that II-1 and II-2 suffered from LV outflow tract (LVOT) obstruction whereas their parents were normal in this regard. Echocardiography confirmed the diagnosis of II-1 and II-2 as HCM with A Double Heterozygous Mutation of TNNI3 Causes HCM
mitral and tricuspid regurgitation, and their parents had decreased LV compliance. In addition, the interventricular septum of II-1 and II-2 was 28 mm, but that of their parents was 11 mm, i.e. within the normal range of T
Normal
c.235C>T
c.470C>T
CAGGCGCTG
CAGGCGCTG
Normal
c.470C>T
I
Exon 4
1
2
Color version available online
b
a
II 1
Exon 6
2
c
p.RମC
p.AମV
Human Guinea pig Dolphin Mouse Rat Cow Horse Baboon Pika Dipodomys Bushbaby Platypus Shrew Wallaby
Human Guinea pig Dolphin Mouse Rat Cow Horse Baboon Pika Dipodomys Bushbaby Platypus Shrew Wallaby exon 4
WT
c.235C>T
c.470C>T
Fig. 2. Mutation screening and bioinformatics analysis. a Family pedigree. The arrow denotes the proband. b DNA sequences of
c Alignment of WT and mutant TNNI3 amino acid sequences. d Prediction of WT (left) and mutant (middle and right) protein
WT (left) and mutant (right) were validated by Sanger sequencing.
topology by RCSB PDB.
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d
exon 6
Color version available online
TNNI3 GAPDH EGFP
DAPI
Merge
b
M1
M2
M1/M2
WT
WT
1.5
*** ***
c.470C>T
EGFP-TNN3:GAPDH
a
c
Fig. 3. Functional studies on the mutated proteins. a COS-7 cells
transfected with constructs expressing WT and mutant TnI-GFP fusion proteins for cellular localization studies. b Western blot of HEK293T cells transfected constructs expressing WT and mutant (M) TnI-GFP fusion proteins. The upper band was incubated with
*** 1.0
0.5
0
M1
M2
M1/M2
WT
rabbit anti-GFP, and the lower band was incubated with mouse anti-GAPDH. c Protein expression in HEK293T cells transfected with TnI single or double mutant (M) plasmids was significantly decreased compared with cells transfected with a WT plasmid. *** p < 0.01.
family members was performed, and 7 mutations were identified. Five of these were synonymous mutations which had not been previously connected to pathogenicity, while 2 significant mutations, c.235C>T (rs3729712) and c.470C>T (CM031379) within exons 4 and 6 of TNNI3, had been previously identified [10]. These mutations resulted in the mutated proteins p.R79C and p.A157V, respectively. These variants were not identified in 200 normal control samples [10]. Sanger sequencing revealed that the proband and her brother presented both double heterozygous mutations in TNNI3 whereas their father harbored the c.235C>T mutation in exon 4 and their mother the c.470C>T mutation in exon 6 (fig. 2). Bioinformatics analysis was subsequently performed, and PolyPhen2 software predicted that both mutations are likely to be harmful to humans. SOPMA predicted that the mutations could alter the protein secondary structure and SIFT predicted the probable effects on protein function. Additionally, sequence alignment showed that these 2 mutations are located in evolutionarily conserved regions.
Protein Expression Analysis The subcellular localization of WT pEGFP-C1-TnI and mutant pEGFP-C1-ΔcTnI in COS-7 cells was assessed, and both were localized at the cell nucleus. Thus, mutation did not appear to affect subcellular localization (fig. 3). An identically sized band (approx. 51 kDa) was observed in cells expressing both WT and mutant GFPtagged TnI fusion proteins. However, Western blotting showed that the expression of the mutants was lower than that of the WT protein.
A Double Heterozygous Mutation of TNNI3 Causes HCM
Cardiology 2016;133:91–96 DOI: 10.1159/000440877
Discussion
We investigated the clinical findings of HCM patients carrying 2 heterozygous mutations in the TNNI3 gene that resulted in the R79C and A157V TnI variants. Relatives of the patients were compared to clarify whether the mutations caused differences in expression. These 2 mu95
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c.235C>T
tations appear to be disease-causing since the patients carrying them suffered from HCM, and mutations in 4 different genes (TNNT2, MYPBC3, MYH7 and TPM1) were excluded from involvement in HCM. Clinical diagnosis indicated that the proband II-2 and her brother II-1 both suffered from HCM whereas their parents, who each carried only 1 mutation in TNNI3, showed no symptoms of this disease. Previous studies reported that clinical symptoms in patients with >1 mutation are more severe, and double mutations are accompanied by a greater risk of sudden death and LV hypertrophy [11, 12]. The proband experienced disease onset at the age of 8 years whereas her brother displayed no clinical symptoms, but did produce abnormal echocardiograms. Most patients with HCM have few symptoms, and diagnosis is usually made incidentally or during family screening [2]. Therefore, we agree that patients with the same mutation may present different disease phenotypes [12]. From the results of subcellular localization, GFP-TnI was located at the nucleus of COS-7 and HEK293T cells, while it is predicted to be located in the whole cell in GeneCards (http://www.genecards.org/). This may be explained by the fact that GFP-TnI has different localization in different cells. Western blotting showed that expression of the mutant proteins was lower than that of WT TnI. Previous studies indicated that the pathogenesis of HCM involves a dominant negative function, an imbalance of myocardial energetic metabolism and haploinsufficiency [13]. In this family, HCM caused by the 2 mutations may be explained by haploinsufficiency for the decreased expression of abnormal protein; the protein expression analysis
proved that the mutants could not produce enough to bring about a WT condition. Bioinformatics analysis showed that the mutations were located in evolutionarily conserved regions and could alter the protein secondary structure. Therefore, we deduced that the mutants produce unstable protein or the mutant protein could interfere with the normal mechanical and electrophysiological function of cardiomyocytes. In conclusion, we identified a compound heterozygous mutation in the TNNI3 gene that is involved in HCM pathogenesis. Harboring only 1 of 2 mutations cannot lead to the disorder. These results are therefore consistent with other studies that suggested that compound single-nucleotide polymorphisms, in particular genes, can cause genetic disorders. These findings provide a new insight into the pathogenesis of HCM and provide a basis for future studies into the molecular mechanisms of disease-causing mutations.
Acknowledgement We thank the patients and their family members for consenting to this research. This work was partially supported by a grant from the National Natural Science Foundation of China (31371279), the Science and Technology Program of Guangzhou (201300000095) and Natural Science Foundation of Guangdong (2015A030313305).
Conflicts of Interest The authors declare that they have no competing interests.
References
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7 Mogensen J, Kruse TA, Børglum AD: Assignment of the human cardiac troponin I gene (TNNI3) to chromosome 19q13.4 by radiation hybrid mapping. Cytogenet Cell Genet 1997;79:272–273. 8 Kimura A, Harada H, Park JE, Nishi H, Satoh M, Takahashi M, et al: Mutations in the cardiac troponin I gene associated with hypertrophic cardiomyopathy. Nat Genet 1997; 16: 379–382. 9 Parvatiyar MS, Pinto JR, Dweck D, Potter JD: Cardiac troponin mutations and restrictive cardiomyopathy. J Biomed Biotechnol 2010; 2010:350706.
10 Santos S, Marques V, Pires M, Silveira L, Oliverira H, Lanca V, et al: High resolution melting: improvements in the genetic diagnosis of hypertrophic cardiomyopathy in a Portuguese cohort. BMC Med Genet 2012;13:17. 11 Zou Y, Wang J, Liu X, Wang Y, Chen Y, Sun K, et al: Multiple gene mutations, not the type of mutation, are the modifier of left ventricle hypertrophy in patients with hypertrophic cardiomyopathy. Mol Biol Rep 2013; 40: 3969–3976. 12 Marsiglia JD, Pereira AC: Hypertrophic cardiomyopathy: how do mutations lead to disease? Arq Bras Cardiol 2014;102:295–304. 13 Tian T, Liu Y, Zhou X, Song L: Progress in the molecular genetics of hypertrophic cardiomyopathy: a mini-review. Gerontology 2013; 59:199–205.
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1 Marian AJ, Mares A Jr, Kelly DP, Yu QT, Abchee AB, Hill R, et al: Sudden cardiac death in hypertrophic cardiomyopathy. Variability in phenotypic expression of beta-myosin heavy chain mutations. Eur Heart J 1995; 16: 368– 376. 2 Ho CY: Hypertrophic cardiomyopathy. Heart Fail Clin 2010;6:141–159. 3 Elliott P, McKenna WJ: Hypertrophic cardiomyopathy. Lancet 2004;363:1881–1891. 4 Arad M, Seidman JG, Seidman CE: Phenotypic diversity in hypertrophic cardiomyopathy. Hum Mol Genet 2002;11:2499–2450. 5 Katrukha IA: Human cardiac troponin complex. Structure and functions. Biochemistry (Mosc) 2013;78:1447–1465. 6 Dewantoro O, Harun S: Troponin in heart disorder and chronic renal failure. Acta Med Indones 2005;37:218–223.
Cardiology 2016;133:91–96 DOI: 10.1159/000440877
A Double Heterozygous Mutation of TNNI3 Causes Hypertrophic Cardiomyopathy in a Han Chinese Family Hua Zheng a Huajie Huang b Zhisong Ji b Qi Yang b Qiuxia Yu b Fan Shen b Cuixian Liu b Fu Xiong b
Department of Cardiovascular Medicine, Nanfang Hospital, Southern Medical University, and b Department of Medical Genetics, School of Basic Medicine Sciences, Southern Medical University, Guangzhou, China
Key Words Hypertrophic cardiomyopathy · Heterozygous mutation · Cardiac troponin I · Pathophysiology
Abstract Objectives: To investigate the variations in the TNNI3 gene in a Chinese Han family affected by hypertrophic cardiomyopathy (HCM) and the potential molecular mechanism linking these mutations with disease. Methods: Peripheral venous blood was acquired from family members, and TNNI3 mutations were identified by DNA sequencing. The pathophysiology of TNNI3 mutations was investigated using bioinformatics, subcellular localization determination and Western blotting. Results: Sanger sequencing revealed that the proband possessed 2 heterozygous mutations, c.235C>T and c.470C>T, located at exons 4 and 6 of the TNNI3 gene. The proband (II-2) and her brother (II-1), who had been previously diagnosed with HCM, harbored both mutations whereas their healthy parents harbored only 1. Alignment of the TNNI3 amino acid sequence indicated that the two Pro residues were highly conserved across species. Subcellular localization showed that both wild-type (WT) and mutant
© 2015 S. Karger AG, Basel 0008–6312/15/1332–0091$39.50/0 E-Mail [email protected] www.karger.com/crd
TNNI3 proteins were localized at the cell nucleus. Western blot analysis of expression in human embryonic kidney 293T cells showed that the intracellular levels of the mutant proteins were significantly decreased compared to WT TNNI3 (p < 0.01). Conclusions: Our findings showed that a double heterozygous mutation in the TNNI3 gene is involved in the pathogenesis of HCM via haploinsufficiency. These results will inspire further studies to investigating the link between the TNNI3 gene and HCM. © 2015 S. Karger AG, Basel
Introduction
Hypertrophic cardiomyopathy (HCM) is the most common autosomal-dominant monogenic cardiac disease with an estimated prevalence of 1 in 500 of the population [1, 2]. Clinically, HCM is characterized by hypertrophy of the left ventricle (LV) walls, especially the septum, usually in an asymmetric manner, and the absence
H. Zheng and H. Huang contributed equally to this article.
Fu Xiong Department of Medical Genetics, School of Basic Medical Sciences Southern Medical University, 1838 Guangzhou Dadao Rd. Guangzhou 510515 (China) E-Mail xiongfu @ smu.edu.cn
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a
Methods Patients This study was approved by the Southern Medical University ethics committee. Informed consent was obtained from patients and relatives before analysis. Patients were recruited from outpatient clinics. The proband (II-2), a 21-year-old girl, was referred to the Cardiothoracic Surgery at Nanfang Hospital for consultation concerning chest pain and syncope. Clinical examination of all family members was performed by an experienced cardiac surgeon, and unexplained LV hypertrophy in cardiac imaging studies was investigated. The 200 normal controls matched by gender and ethnic origin were selected and recruited from healthy individuals at the department of Medical Genetics, School of Basic Medicine Sciences, Southern Medical University. Mutation Screening Peripheral blood was acquired from all family members, and standard phenol/chloroform extraction was performed to extract genomic DNA. Target capture and next-generation sequencing of the TNNT2, MYPBC3, MYH7, TPM1 and TNNI3 genes from the
92
Cardiology 2016;133:91–96 DOI: 10.1159/000440877
proband, all of which have been linked to HCM, were performed by KeyBay Co., Ltd. Two significant mutations within exons 4 and 6 of the TNNI3 gene were identified and verified using Sanger sequencing. PCR primers were designed using Primer3 (http:// frodo.wi.mit.edu/) to amplify regions including exons 4 and 6 to allow identification of these 2 mutations in family members. PCR products were analyzed by agarose gel electrophoresis and verified by Sanger sequencing. The PCR primers were as follows: F1: 5′-CGCCTGGTCTTTATCCTGAA-3′ and R1: 5′-TAGAAACCTCGCATCCTTGG-3′ for exon 4, and F2: 5′-AGTACCCACCCCCTCGTTT-3′ and R2: 5′-CCTCAGCATCCTCTTTCCTG-3′ for exon 6. Bioinformatics To provide further insight into TNNI3 function, secondary structure of the mutant TNNI3 proteins were predicted using the SOPMA method, and the 3-dimensional structures of wild-type (WT) and mutant TNNI3 proteins were predicted using the RCSB PDB (Research Collaboratory for Structural Bioinformatics Protein Data Bank). PolyPhen2 software was used to predict the harmfulness of mutations and analyze the conservation of TNNI3 across species. The SIFT program was used for predicting the function of the mutant proteins. Cellular Localization Recombinant plasmids were transfected into COS-7 cells to investigate the subcellular localization of the TNNI3 protein. The WT coding sequence of the TNNI3 gene was cloned into the HindIII and KpnI sites of the pEGFP-C1 vector (GENEWIZ, Inc. South Plainfield, N.J., USA), and this plasmid was used for generation of mutant plasmids by site-directed mutagenesis with the QuikChange site-directed mutagenesis kit (Stratagene). COS-7 cells were cultured in 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum. Recombinant plasmids were transfected into COS-7 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. At 24 h after transfection, cells were rinsed 3 times with PBS and stained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma, St. Louis, Mo., USA). Fluorescence microscopy (Nikon, Eclipse Ti-U, Tokyo, Japan) was used to visualize transfected cells. The primers used for the mutations were as follows: F1: 5′-CGCGCTCTGAGCACCTGCTGCCAGCCGCTGG-3′, R1: 5′-GCGCGAGACTCGTGGACGACGGTCGGCGACC-3′, F2: 5′-ATGCCATGATGCAGGTGCTGCTGGGGGCCCG-3′ and R2: 5′-TACGGTACTACGTCCACGACGACCCCCGGGC-3′. TNNI3 Expression in Human Embryonic Kidney 293T Cells To measure the expression of WT and mutant TNNI3, human embryonic kidney (HEK)293T cells were transfected with recombinant plasmids using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions, and then cultured in DMEM medium (Invitrogen) supplemented with 10% fetal bovine serum. The cells produced WT or mutant TnI-GFP fusions. At 24 h after transfection, cells were harvested and homogenized in RIPA lysis buffer containing protease inhibitor cocktail (Sigma). Blots were incubated with rabbit anti-GFP (1:1,000, Santa Cruz) overnight at 4 ° C, followed by goat anti-rabbit IgGHRP (1: 2,000, Santa Cruz) at room temperature for 1 h. Detection was performed with Immobilon Western chemiluminescent HRP substrate (Millipore). Fusion proteins were detected using Western blotting.
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of other cardiac or systemic disease that leads to secondary hypertrophy. The disease has a varied clinical course and outcome; many patients have little or no discernible cardiovascular symptoms whereas others have profound exercise limitation and recurrent arrhythmias [3]. The heritable nature and autosomal dominant pattern of inheritance of HCM have been long established [2]. Mutations in genes encoding the cardiac myosin heavy chain (βMHC), cardiac myosin-binding protein C (MyBPC), cardiac troponin T (TnT), cardiac troponin I (TnI), α-tropomyosin (αTM), essential and regulatory light chains and cardiac actin have all been correlated with the disease [4]. The troponin complex is a component of skeletal and cardiac muscle thin filaments that consists of three subunits: troponin C (calcium-binding protein), troponin T (tropomyosin-binding protein) and troponin I (inhibitory protein) that together play a crucial role in muscle activity [5, 6]. TnI, encoded by the TNNI3 gene, is the inhibitory subunit that blocks actin-myosin interactions and thereby mediates striated muscle relaxation [7–9]. Here, we investigated a Han Chinese family with HCM and found that a double heterozygous mutation in TNNI3 causes the disease. We studied the function and potential pathogenic mechanism of the mutated gene. Our results showed that the double heterozygous mutation results in haploinsufficiency of TNNI3 that ultimately causes HCM. This study may provide a reference model for future studies of the potential pathogenic mechanism by which mutations in TNNI3 cause HCM.
Color version available online
a
b
c
d
Fig. 1. Two-dimensional echocardiogram
and continuous-wave Doppler of HCM patients. a, b The interventricular septum (29 mm) of the proband II-2 is clearly thickened. c Similarly, the interventricular septum (28 mm) was also thickened in the proband’s brother II-1. d The velocity of blood flow in the LVOT of II-1 was increased to 219 cm/s, and the pressure gradient of the LVOT was increased to 19 mm Hg.
Table 1. Clinical features and genotypes of the proband and her family Subject
TNNI3 mutation
Father c.235C>T Mother c.470C>T II-2 c.235C>T, c.470C>T II-1 c.235C>T, c.470C>T
Protein alteration
Age, years
Age at onset, years
Clinical symptoms
Electrocardiogram
Echocardiography
chest pain
dyspnea
syncope systolic murmur
LVH + ST
AF
IVS, mm
Max LVWT, LVOT, mm mm
LADD, SAM mm
R79C A157C R79C, A157C R79C, A157C
49 48 21
– – 8
no no yes
– – –
no no yes
no no yes
– – –
– – –
11 11 29
11 10 12
19 18 10.2
41 37 43
no no yes
23
–
no
–
no
yes
–
–
28
12
10
49
yes
AF = Atrial fibrillation; IVS = interventricular septum; LADD = left atrial diastolic diameter; LVH + ST = LV hypertrophy combined with ST-T changes; Max LVWT = maximum LV wall thickness; SAM = systolic anterior motion of the mitral valve.
Clinical Features The clinical features of the proband and her family members are shown in table 1. She had experienced onset at the age of 8 years and exhibited symptoms including chest pain and syncope; systolic murmur was detected and she had an abnormal electrocardiogram (fig. 1; table 1). However, the other family members were asymptomatic. Color Doppler revealed that II-1 and II-2 suffered from LV outflow tract (LVOT) obstruction whereas their parents were normal in this regard. Echocardiography confirmed the diagnosis of II-1 and II-2 as HCM with A Double Heterozygous Mutation of TNNI3 Causes HCM
mitral and tricuspid regurgitation, and their parents had decreased LV compliance. In addition, the interventricular septum of II-1 and II-2 was 28 mm, but that of their parents was 11 mm, i.e. within the normal range of T
Normal
c.235C>T
c.470C>T
CAGGCGCTG
CAGGCGCTG
Normal
c.470C>T
I
Exon 4
1
2
Color version available online
b
a
II 1
Exon 6
2
c
p.RମC
p.AମV
Human Guinea pig Dolphin Mouse Rat Cow Horse Baboon Pika Dipodomys Bushbaby Platypus Shrew Wallaby
Human Guinea pig Dolphin Mouse Rat Cow Horse Baboon Pika Dipodomys Bushbaby Platypus Shrew Wallaby exon 4
WT
c.235C>T
c.470C>T
Fig. 2. Mutation screening and bioinformatics analysis. a Family pedigree. The arrow denotes the proband. b DNA sequences of
c Alignment of WT and mutant TNNI3 amino acid sequences. d Prediction of WT (left) and mutant (middle and right) protein
WT (left) and mutant (right) were validated by Sanger sequencing.
topology by RCSB PDB.
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Cardiology 2016;133:91–96 DOI: 10.1159/000440877
Zheng/Huang/Ji/Yang/Yu/Shen/Liu/ Xiong
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d
exon 6
Color version available online
TNNI3 GAPDH EGFP
DAPI
Merge
b
M1
M2
M1/M2
WT
WT
1.5
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c.470C>T
EGFP-TNN3:GAPDH
a
c
Fig. 3. Functional studies on the mutated proteins. a COS-7 cells
transfected with constructs expressing WT and mutant TnI-GFP fusion proteins for cellular localization studies. b Western blot of HEK293T cells transfected constructs expressing WT and mutant (M) TnI-GFP fusion proteins. The upper band was incubated with
*** 1.0
0.5
0
M1
M2
M1/M2
WT
rabbit anti-GFP, and the lower band was incubated with mouse anti-GAPDH. c Protein expression in HEK293T cells transfected with TnI single or double mutant (M) plasmids was significantly decreased compared with cells transfected with a WT plasmid. *** p < 0.01.
family members was performed, and 7 mutations were identified. Five of these were synonymous mutations which had not been previously connected to pathogenicity, while 2 significant mutations, c.235C>T (rs3729712) and c.470C>T (CM031379) within exons 4 and 6 of TNNI3, had been previously identified [10]. These mutations resulted in the mutated proteins p.R79C and p.A157V, respectively. These variants were not identified in 200 normal control samples [10]. Sanger sequencing revealed that the proband and her brother presented both double heterozygous mutations in TNNI3 whereas their father harbored the c.235C>T mutation in exon 4 and their mother the c.470C>T mutation in exon 6 (fig. 2). Bioinformatics analysis was subsequently performed, and PolyPhen2 software predicted that both mutations are likely to be harmful to humans. SOPMA predicted that the mutations could alter the protein secondary structure and SIFT predicted the probable effects on protein function. Additionally, sequence alignment showed that these 2 mutations are located in evolutionarily conserved regions.
Protein Expression Analysis The subcellular localization of WT pEGFP-C1-TnI and mutant pEGFP-C1-ΔcTnI in COS-7 cells was assessed, and both were localized at the cell nucleus. Thus, mutation did not appear to affect subcellular localization (fig. 3). An identically sized band (approx. 51 kDa) was observed in cells expressing both WT and mutant GFPtagged TnI fusion proteins. However, Western blotting showed that the expression of the mutants was lower than that of the WT protein.
A Double Heterozygous Mutation of TNNI3 Causes HCM
Cardiology 2016;133:91–96 DOI: 10.1159/000440877
Discussion
We investigated the clinical findings of HCM patients carrying 2 heterozygous mutations in the TNNI3 gene that resulted in the R79C and A157V TnI variants. Relatives of the patients were compared to clarify whether the mutations caused differences in expression. These 2 mu95
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c.235C>T
tations appear to be disease-causing since the patients carrying them suffered from HCM, and mutations in 4 different genes (TNNT2, MYPBC3, MYH7 and TPM1) were excluded from involvement in HCM. Clinical diagnosis indicated that the proband II-2 and her brother II-1 both suffered from HCM whereas their parents, who each carried only 1 mutation in TNNI3, showed no symptoms of this disease. Previous studies reported that clinical symptoms in patients with >1 mutation are more severe, and double mutations are accompanied by a greater risk of sudden death and LV hypertrophy [11, 12]. The proband experienced disease onset at the age of 8 years whereas her brother displayed no clinical symptoms, but did produce abnormal echocardiograms. Most patients with HCM have few symptoms, and diagnosis is usually made incidentally or during family screening [2]. Therefore, we agree that patients with the same mutation may present different disease phenotypes [12]. From the results of subcellular localization, GFP-TnI was located at the nucleus of COS-7 and HEK293T cells, while it is predicted to be located in the whole cell in GeneCards (http://www.genecards.org/). This may be explained by the fact that GFP-TnI has different localization in different cells. Western blotting showed that expression of the mutant proteins was lower than that of WT TnI. Previous studies indicated that the pathogenesis of HCM involves a dominant negative function, an imbalance of myocardial energetic metabolism and haploinsufficiency [13]. In this family, HCM caused by the 2 mutations may be explained by haploinsufficiency for the decreased expression of abnormal protein; the protein expression analysis
proved that the mutants could not produce enough to bring about a WT condition. Bioinformatics analysis showed that the mutations were located in evolutionarily conserved regions and could alter the protein secondary structure. Therefore, we deduced that the mutants produce unstable protein or the mutant protein could interfere with the normal mechanical and electrophysiological function of cardiomyocytes. In conclusion, we identified a compound heterozygous mutation in the TNNI3 gene that is involved in HCM pathogenesis. Harboring only 1 of 2 mutations cannot lead to the disorder. These results are therefore consistent with other studies that suggested that compound single-nucleotide polymorphisms, in particular genes, can cause genetic disorders. These findings provide a new insight into the pathogenesis of HCM and provide a basis for future studies into the molecular mechanisms of disease-causing mutations.
Acknowledgement We thank the patients and their family members for consenting to this research. This work was partially supported by a grant from the National Natural Science Foundation of China (31371279), the Science and Technology Program of Guangzhou (201300000095) and Natural Science Foundation of Guangdong (2015A030313305).
Conflicts of Interest The authors declare that they have no competing interests.
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Cardiology 2016;133:91–96 DOI: 10.1159/000440877
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