Cardiomyopathy is a major cause of heart failure and sudden cardiac death; several mutations in sarcomeric protein genes have been associated with this disease. Our aim in the present study is to investigate the genetic variations in Troponin T (cTnT) gene and its association with dilated cardiomyopathy (DCM) in south-Indian patients. Analyses of all the exons and exon-intron boundaries of cTnT in 147 DCM and in 207 healthy controls had revealed a total of 15 SNPs and a 5 bp INDEL; of which, polymorphic SNPs were compared with the HapMap population data. Interestingly, a novel R144W mutation, that substitutes polar-neutral tryptophan for a highly conserved basic arginine in cTnT, altering the charge drastically, was identified in a DCM, with a family history of sudden-cardiac death (SCD). This mutation was found within the tropomyosin (TPM1) binding domain, and was evolutionarily conserved across species, therefore it is expected to have a significant impact on the structure and function of the protein. Family studies had revealed that the R144W is co-segregating with disease in the family as an autosomal dominant trait, but it was completely absent in 207 healthy controls and in 162 previously studied HCM patients. Further screening of the proband and three of his family members (positive for R144W mutant) with eight other genes β-MYH7, MYBPC3, TPM1, TNNI3, TTN, ACTC, MYL2 and MYL3, did not reveal any disease causing mutation, proposing the absence of compound heterozygosity. Therefore, we strongly suggest that the novel R144W unique/private mutant identified in this study is associated with FDCM. This is furthermore signifying the unique genetic architecture of Indian population.
Citation: Rani DS, Dhandapany PS, Nallari P, Narasimhan C, Thangaraj K (2014) A Novel Arginine to Tryptophan (R144W) Mutation in Troponin T (cTnT) Gene in an Indian Multigenerational Family with Dilated Cardiomyopathy (FDCM). PLoS ONE 9(7): e101451. https://doi.org/10.1371/journal.pone.0101451
Editor: Niyaz Ahmed, University of Hyderabad, India
Received: March 5, 2014; Accepted: June 6, 2014; Published: July 3, 2014
Copyright: © 2014 Rani et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: DSR and KT, supported by the Council of Scientific and Industrial Research (CSIR), Centre for Cellular and Molecular Biology (CCMB), Hyderabad, Telangana, India. KT supported by Network project grant (CardioMed-BSC0122), Council of Scientific and Industrial Research (CSIR), Government of India, New Delhi, India. DSR and PN acknowledge OU-DST-PURSE, New Delhi, India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Dilated cardiomyopathy (DCM: OMIM 115200), is characterized by cardiac left ventricular dilation and systolic dysfunction, affects at least 1 in 2500 individuals , and a major cause for morbidity and mortality , including heart failure (HF) and sudden cardiac death (SCD) –. Familial DCM (FDCM) is a genetically heterogeneous disease , whereas Idiopathic DCM (IDCM) is diagnosed when clinically detectable causes of DCM are excluded. Genetic screening of first-degree relatives had revealed, approximately 20 to 35% of idiopathic cases, were due to genetic defects –. More than 30 nuclear genes, encoding for sarcomere (contractile apparatus), cytoskeletal and calcium homeostasis proteins of diverse functions, have been reported to cause FDCM . To date, mutations in LMNA, MYH7, MYBPC3, TNNT2, SCN5A, and MYH6 genes have been accounted for approximately 75% of FDCM . Most of the genes implicated in genetics of DCM/FDCM follow autosomal dominant mode of inheritance , though a few follow autosomal recessive, X-linked , – and mitochondrial , . Recent studies had suggested that the double and triple mutations identified in sarcomere protein genes were found to be associated with early onset of HCM , .
Indian populations are reported to be more prone to cardiac disorders, which might be due to their high effective population size (Ne) and lifestyle, resulting a unique genetic structure –. Our previous study on cardiac Troponin I3 (TNNI3) ,  and Troponin T2 (TNNT2)  in hypertrophic cardiomyopathy (HCM), and cardiac actin (ACTC) , myosin binding protein C (MyBPC3) , had revealed few variants, of which a 25 bp deletion was found to be associated with both HCM and DCM in India and south Asia . Unfortunately, not many studies have been conducted on Indian patients to explore the genetic etiology of the disease, particularly with reference to the sarcomere protein genes. Our aim in the present study is to investigate the genetic variations in Troponin T (cTnT) gene, and its association with DCM in South Indian cohorts.
Sequencing of all the exons and the exon-intron boundaries (5373 bp) of Troponin T2 (cTnT) gene in 147 DCM patients along with 207 healthy controls had revealed a total of 15 SNPs and a 5 bp INDEL (Fig. 1A to 1M and Table 1).
Mutation sites were shown with arrows. Fig. 1A. R144W [rs483352832]: Electropherogram (arrow) showing a novel missense mutation (R144W) at the nucleotide position g.14351 of human cTnT gene. The upper lane showing sequences of homozygous wild type allele ‘C’ in a control individual. The middle and the lower lanes were showing the sequences of heterozygous (C/T = Y) alleles in two individuals (a DCM patient and his relative, respectively). Fig. 1B. G>A [IVS11-1G] [rs483352835]: Electropherogram (arrow) showing a variant at splice acceptor site of human cTnT gene at nucleotide position g.16283, the electropherogram of a upper lane showing sequence of heterozygous (A/G = R) variant in a DCM patient, the lower lane showing sequence of control individual having wild type allele ‘G’ (homozygous). Fig. 1C. N164N [rs483352833]: Electropherogram (arrow) showing a novel synonumous mutation (N164) at the nucleotide position g.15304 of human cTnT gene in 2 DCM patients. The upper lane shows the sequences of heterozygous (C/T = Y) transition in a DCM patient. The middle lane was the sequences of a control individual showing the wild type allele ‘C’ (homozygous). The lower lane sequences showing heterozygous (C/T = Y) transition was from a 2nd DCM patient. Fig. 1D. [rs3729842]: Electropherogram showing (arrow) a single nucleotide polymorphism at the nucleotide position g.10636 (C/T = Y) in intron 5 of human cTnT gene. The upper and the middle lanes were sequences showing heterozygous (C/T = Y) transition in DCM patients, the lower lane showing homozygous wild type (C/C) allele in a control individual. Fig. 1E. [rs3729845]: Electropherogram showing (arrow) at the nucleotide position g.13011 of human cTnT gene. The upper lane showing sequences of the heterozygous (A/G = R) transition, and the lower lane showing homozygous wild type (G/G) allele of a control. Fig. 1F. [rs1104859]: Electropherogram showing (arrow) at the nucleotide position g.11643 (A/C = M) in Intron 11 of human cTnT gene. The upper lane sequences showing the heterozygous (A/C = M) transversion, the middle lane showing homozygous wild type (G/G), and the lower lane sequences showing mutant homozygous (C/C) allele. Fig.1G. SNP-rs3729843: Electropherogram showing (arrow) a SNP at the nucleotide position g.10822 (G/A = R) in intron 5 of human cTnT gene. The upper lane sequences showing mutant homozygous (A/A) allele. The middle lane sequences showing heterozygous (G/A = R) transition allele, and the lower lane showing sequences of homozygous wild type (G/G) allele in a control individual. Fig. 1H. [rs45576939]: Electropherogram showing (arrow) a novel mutation G>A at nucleotide position g.10370 in intron 4 of human cTnT gene, the upper lane displaying homozygous mutant (A/A) allele, and the lower lane showing sequences of a wild type allele (G/G). Fig. 1I. [rs45576635]: Electropherogram showing (arrow) a SNP at the nucleotide position g.14492 (C/T = Y) in intron 15 of human cTnT gene, the upper and the middle lanes sequences displaying heterozygous (C/T = Y) transition, and the lower lane sequences showing homozygous wild type (C/C) allele. Fig. 1J. [rs3729547]: Electropherogram showing (arrow) a polymorphic variant at the nucleotide position g.13424 of human cTnT gene, the upper lane displaying sequences of the heterozygous (C/T = Y) transition, the middle lane sequences showing homozygous wild type (C/C) allele, and the lower lane displaying sequences of the homozygous mutant (T/T) allele. Fig. 1K. [rs483352834]. Electropherogram (arrow) showing a novel mutation at the nucleotide position g.15179 C>T in intron 11 of human cTnT gene, the upper lane displaying sequences of a DCM patient having heterozygous (C/T) transition, and the lower lane exhibiting sequences of a control individual having homozygous wild type allele (C/C). Fig. 1L. K276K. [rs483352836]: Electropherogram (arrow) exhibiting novel synonumous (K276) variant at the nucleotide position g.19429 of human cTnT gene in a DCM patient, the DCM patient displaying heterozygous (G/A = R) transition. Fig. 1M. Sequence electropherogram showing (CTTCT) 5 bp Polymorphism. Ma. Presence of two copies of CTTCT (Insertion/Insertion – homozygous insertion) in both the chromosomes, Mb. Absence of one copy of CTTCT (Deletion/Deletion – homozygous deletion in both the chromosomes, Mc. Presence of 2 copies of CTTCT in one chromosome and presence of one copy of CTTCT in another chromosome (Insertion/deletion – heterozygous allele). g.6626-30 (5 bp).
Arginine to Tryptophan substitution at residue 144 (R144W) of cTnT gene
Of the 15 SNPs, a unique c.430 C>T transition (GenBank No. NM_000364) in exon 10 of TNNT2 gene, identified in a 29 years old male DCM patient, is of great interest, as the mutation replaces the highly conserved basic amino acid arginine at residue 144 to polar-neutral tryptophan R144W [rs483352832] (Fig. 1A). The R144W mutation has resulted with loss of restriction sites; Mbil 19, Acil 19, BsrBl 19, AccBSl 19. Subsequent, screening of this (R144W) mutation with available family members had revealed its presence in three other individuals with DCM phenotype (Fig. 2). However, this mutation was absent in 207 healthy unrelated controls, and in 162 HCM patients . Multiple alignment of the amino acid with different species had revealed that the arginine at 144 in human cTnT is evolutionarily conserved across species; including mammals, birds, reptiles, and nematode (Fig. 3).
Squares indicate males; circles, females; open symbols, normal individuals; solid symbols, affected individuals, Slanted bars indicate deceased members of family. Plus signs indicate the presence of R144W mutation in cTnT; minus signs suggest the absence of mutation R144W in cTnT.
While interacting with the family members of the proband, a history of sudden cardiac deaths (SCD) in the family was noted. Two individuals in the family, who were diagnosed with DCM, had died due to severe congestive heart failure at the age of 45 and 25 years. However, a 66-year-old individual in the same family with DCM having mild symptoms have also been noticed. Thus indicating that the age of onset, and the severity of the disease are highly variable within the family (Fig. 2 and, Table 2).
A novel splice acceptor site variant
We have also identified a novel splice acceptor site variant (G→A) in intron 12 of cTnT gene [rs483352835], in a 63 years old male DCM patient (Fig. 1B and, Table 1). This patient had both dilated LV/LA, with EF 25%, global hypokinesia, grade III systolic dysfunction, and IVS thinned out 7 mm. Unfortunately, we were unable to get the family samples for additional analyses.
Two novel synonymous mutations
We further identified two novel synonymous mutations, N164N (C→T; [rs483352833]) and K276K (G→A; [rs483352836]) in cTnT gene (Fig. 1C and 1L, Table 1) exclusively in DCM. Of which, N164N (Fig. 1C) was observed in 2 DCM (2/147 = 1.4%) patients with EF of 35% (a 35 year old female) and 30% (39 year old male). The codon bias analysis had revealed a replacement of more frequently used (wild type) codon (AAC: 64%) with a less frequent one (AAT: 36%) (Table 3). The female patient showed both dilated left ventricle and atrium, moderate mitral regurgitation and moderate LV systolic dysfunction, while the male patient showed LV dilation and moderate LV systolic dysfunction.
The K276K synonymous mutation (Fig. 1L; rs483352836) was observed in 2 DCM patients (2/147 = 1.4%), which replaces very frequent codon (71%; AAG) with the less frequent codon (29%; AAA) (Table 3). Though these two (N164N; K276K) mutations were synonymous, its exclusive presence in dilated cardiomyopathy patients, illustrates its possible role in disease pathogenesis, however, they need to be studied further.
Two intronic SNPs and their splicing patterns
We found two intronic SNPs of cTnT gene (G→A; g.10370_ [rs45576939] and C→T; g.15179- [rs483352834]), exclusively in DCM patients. In silico analyses had predicted abnormal splicing pattern (Table 4). The G→A variant was found to create an additional binding site for hnRNP. K1K2 (Fig. 1H and, Table 4), while the C→ T variant was also causes drastic changes by altering a total of 4 binding sites, 2 each in hnRNPs and SR proteins (SRP20 ASF/SF2, SC35 and U2AF65) (Fig. 1K and, Table 4), indicating its regulatory role, however, its clinical significance need to be studied further.
The chi-square and fisher exact probability test was done to test the significance of polymorphic SNPs that were observed in this study (Table 5). We have compared the genotype and allele frequencies of these SNPs (NCBI database; www.ncbi.nlm.nih.gov/projects/SNP/snp), with HapMap population's data, (HER_ASIAN-PANEL; HER_HISP-PANEL; HER_CEPH-PANEL; HER_YORUB-PANE).
a) SNP-rs3729842: The homozygous mutant allele was exclusively observed in DCM and completely absent in the normal controls and HapMap populations (ASW, CHB, LWK, MKK) (Fig. 1D and, Fig. 4A and 4B). b) SNP-rs3729843: The allele frequencies of DCM have matched only with MXL, TSI, HapMap populations. The minor allele frequency was low in CEU population, while it was completely absent in two (LWK and YRI) HapMap populations (Fig. 1G and, Fig. 4A and 4B). c) SNP-rs3729845: About 4%of heterozygous genotype was observed in DCM, but it was completely absent in the controls, and two (CHB, JPT) of the HapMap populations (Fig. 1E and, Fig. 4A and 4B). d) SNP-rs3729547: The frequency of mutant homozygous allele was 7% in DCM as seen in Gujarati Indians GIH (Hap-map sample), but it was as low as 3% in controls (Fig. 1J and, Fig. 4A and 4B). e) SNP-rs1104859: The percentage of homozygous mutant allele was 13% in DCM, it was very low (6%) in controls. The frequency of the heterozygous genotype was found to be high in CHB, CHD, JPT, HapMap population's (Fig. 1F and, Fig. 4 A and B).
B. The Allele frequencies of SNPs (rs3729842, rs3729843, rs3729845, rs3729547, rs1104859) in the present study were compared with HapMap samples (various populations). HapMap samples (various populations)- ASW, African Ancestry in SW USA; CEU, CEPH Collection; CHB, Han Chinese in Beijing, China; CHD, Chinese in Metropolitan Denver, CO; GIH, Gujarati Indians in Houston, TX; JPT, Japanese in Tokyo, Japan; LWK, Luhya in Webuye, Kenya; MEX, Mexican Ancestry in LA,CA; MKK, Maasai in Kinyawa, Kenya; TSI, Toscani in Italia; YRI, Yoruba in Ibadan, Nigeria; CON-controls; HCM-hypertrophic cardiomyopathy; DCM-dilated cardiomyopathy.
Plotting of all the SNPs observed in the present study had revealed a strong linkage disequilibrium among three SNPs; rs3729547 (C/T), rs3729843 (G/A), rs3729842 (C/T), (Fig. 1J, 1G and, 1D and Table 1), respectively, which were about 2.0 kb apart, in both HCM  and DCM (Fig. 5).
The bright red color indicates very strong LD (LOD = 2D′ = 1), white color no LD (LOD<2D′<1), and pink (LOD = 2D′<1) and blue (LOD<2D′ = 1) indicate intermediate LD (the standard color scheme is used to display LD). The values in the LD blocks show the r2 values in percentages or multiplied by 100.
A 5 bp INDEL (CTTCT) polymorphism
A 5 bp (CTTCT) polymorphism (Fig. 1M;a-c) that results in skipping of exon 4 of TNNT2 during splicing was not significant, when compared to normal controls, it was found to be almost equal in DCM however the deletion frequency was high in HCM . We have also further compared the 5 bp (CTTCT) polymorphic frequencies in 2092 randomly selected individuals belonging to 39 ethnic and endogamous populations from 19 states of India (Table 6), with DCM and HCM  (Fig. 6 A and B).
B. The Allele frequency of 5 bp polymorphism observed in Troponin T (cTnT) gene of DCM, HCM and controls in the present study were compared with the randomly selected individuals from 19 states of India. Individuals from Rajasthan showed high frequency of Deletion allele, whereas the individuals of northeastern states and HER-YORUB-PANEL of Hap Map population showed high frequency of Insertion allele. AP, Andhra Pradesh; KA, Karnataka; TN, Tamil Nadu; MP, Madhya Pradesh; UP, Uttar Pradesh; MH, Maharashtra; GJ, Gujarat; RJ, Rajasthan; CG, Chhattisgarh; WB, West Bengal; HR, Haryana; NL, Nagaland; MZ, Mizoram; JH, Jharkhand; UK, Uttaranchal; JK, Jammu & Kashmir; OD, Orissa; AI, Andaman Islands; NI, Nicobar Islands; AS, Arunachal Pradesh; KE, Kerala; CN, normal controls; HC, Hypertrophic Cardiomyopathy; DC, Dilated Cardiomyopathy.
It has been shown initially that the mutations in the cTnT gene are responsible for approximately 15% cases of familial hypertrophic cardiomyopathy (FHCM) . However, subsequent studies have identified cTnT gene mutations in familial dilated (FDCM) , restrictive (RCM) , and left ventricular noncompaction , cardiomyopathies. Interestingly, our study of cTnT gene in 147 dilated cardiomyopathy (DCM) patients against 207 ethnically matched healthy controls had revealed a total of 15 SNPs and a 5 bp INDEL, including a novel heterozygous C→T at nucleotide g.14351 in exon 10 of cTnT gene in a DCM patient. The mutation had substituted polar-neutral amino acid tryptophan for a highly conserved wild type basic amino acid arginine within the amino terminal tail at residue 144 (R144W) of cTnT.
The R144W mutation was found to be within the tropomyosin-binding domain of cTnT and alters the charge of the residue, so it is expected to have a significant impact on the structure and function of the protein. Later, screening of this mutation in all the available members of a large four generations family had revealed the presence of this heterozygous R144W mutation in three affected individuals of the family (Fig. 2), suggesting that it is an autosomal dominant trait. However, evaluation of 207 unrelated healthy control individuals and 162 HCM patients  did not show this (R144W) mutation.
The proband and 3 individuals positive for R144W mutation had showed clinical features, that are typical for DCM, specifically, left ventricular dilatation and depressed contractile function (Table 2). The sudden cardiac death (SCD) was also been reported in the family, two individuals, who were diagnosed with dilated cardiomyopathy (DCM), had died due to severe congestive heart failure at the age of 45 and 25 years, these deceased individuals were developed their cardiac condition in the second and third decades of their life, respectively. However, a 66-year-old individual in the same family has started having mild symptoms only at his sixties (Fig. 2). Thus, the age of onset and the severity of the disease are highly variable within the family, suggesting that, in many cases, the scenario is more complex, if the secondary etiological factors, such as lifestyle and environment are involved (Fig. 2).
In addition, this amino acid tail residue arginine at 144 in human cTnT is evolutionarily conserved across species, including mouse, rat, chicken, quail, and nematode etc. (Fig. 3). It appears that the amino-terminal tail of cTnT is essential for assembly and anchoring of the troponin-tropomyosin complex onto the thin filament –. The troponin-tropomyosin complex is a Ca2+ sensitive switch that regulates actin-myosin interaction. The troponin complex (Troponins T, I, and C) is anchored to tropomyosin predominantly by troponin T and to a lesser extent by troponin I, and Troponin C interacts with these two troponins T and I . During systole, Ca2+ binds to cTnC and initiates conformational changes of the troponin complex that attenuate the inhibitory effect of cTnI. Results in the release of active sites of the actin gene and this enables the myosin head of the thick filament to interact with it and generate force. The Ca2+ concentration controls cTnC-cTnT interaction, which is important for regulating sliding velocity between thick and thin filaments. Interestingly, recent studies have proposed that cTnT is critical, not only for the structural integrity of the troponin complex, but also for sarcomere assembly and cardiac contractility –.
In general, most of the reported mutations that were responsible for the disease phenotype of dilated (DCM) were in the amino-terminal tail of cTnT (exons 10 and 13) –. Moreover, no mutations responsible for familial hypertrophic cardiomyopathies have ever been identified in either of these exons, 10 and 13 . Study of  some of the published mutations [(R131W  and R141W  in exon 10), and (Lys 210 del [34,35], R205L  in exon 13)], in the amino-terminal tail of TNNT2 gene reported to be responsible for dilated cardiomyopathy (DCM); along with other 4 thin filaments mutations, reconstituted with a 1∶1 ratio of mutant∶wild type proteins, all showed reduced Ca2+ sensitivity of activation in ATPase and motility assays, and all showed lower maximum Ca2+ activation.
Integration of the cTnT mutations (R141W  and R205L , into skinned guinea pig cardiac trabeculae also reduced Ca2+ sensitivity of force generation . Therefore, diverse thin filament DCM mutations appeared to affect different aspects of regulatory function, nevertheless changing contractility in a consistent manner. Further stated that the DCM mutations depressed myofibrillar function, an effect opposite to that of HCM-causing thin filament mutations, and suggested that decreased contractility might trigger pathways that ultimately lead to the clinical phenotype. Generated knock-in mice  with a reported mutation, K210-del ,  in exon 13 of cTnT gene, and found that cardiac muscle fibers from mutant mice showed significantly lower Ca2+ sensitivity in force generation than those from wild type mice .
Compound heterozygosity (double and triple mutations) had been reported to cause HCM phenotype , . Therefore, we have further analyzed the patient and three of his family members carrying R144W mutation having DCM phenotype with eight other genes (β-MYH7, MYBPC3, TPM1, TNNI3, TTN, ACTC, MYL2 and MYL3), to rule out compound heterozygosity. Our analysis revealed that none of these 4 individuals showed any disease causing mutations in eight of the above-mentioned genes, except with few polymorphic variants. This had further confirmed that the missense mutation R144W in cTnT gene is essentially responsible for FDCM phenotype in our study family.
Of 15 SNPs, we have identified a novel splice acceptor site mutation (G→A) at g.16283 in intron 12 (rs) of cTnT gene in a 63-year-old male DCM patient (Table 1; Fig. 1B). Unfortunately, we were unable to get the family samples for further analysis. The splice acceptor site variant might create an alternative acceptor site for splicing, which may results in the inclusion or exclusion of amino acid (glutamine) or the complete skipping of the exon (9 nucleotides). As a result, this alternately spliced transcript might form isoforms, which may be expressed in the human heart are expected to be responsible for the disease phenotype; however, this need to be studied further.
Interestingly, we also found a variant C→T at g.15179 in intron11 of cTnT gene exclusively in a DCM, was predicted to affect splicing. But we have unable to collect the family samples. We have compared the genotype and allele frequencies of polymorphic SNPs observed in this study with HapMap (NCBI database; www.ncbi.nlm.nih.gov/projects/SNP/snp) populations (HER_ASIAN-PANEL; HER_HISP-PANEL;HER_CEPH-PANEL; HER_YORUB-PANE) (Fig.).
We have compared the 5 bp INDEL frequencies in 147 DCM against 207 healthy controls along with 2092 randomly selected individuals belonging to 39 ethnic and endogamous populations inhabited in 19 states of India (Table 6). Our study revealed that the 5 bp INDEL frequencies were found to be almost same in DCM and the controls; nevertheless this 5 bp INDEL frequency was high in South and the Northwest regions of Indian populations, and HCM  (Fig. 6B).
In conclusion, we strongly suggest that the novel unique/private R144W mutation identified in our present study is associated with FDCM. The high level of endogamy in Indian populations along with the influence of evolutionary forces such as genetic drift, fragmentation and long-term isolation, has kept the Indian populations diverse and distant . Hence, the unique mutation observed in this study is not surprising. Our study further suggests that it is important to understand the fundamental genetics (mutation) cause and its impact on disease phenotype, this will certainly lead to adopt novel approaches for the diagnosis and treatment of disease.
Materials and Methods
All of the DNA samples analyzed in the present study were derived from blood samples that were collected with the informed written consent of the donors. The Institutional Ethics Committee of Care Hospitals, Hyderabad, India; and the CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India, have approved the study. This study conforms to the principles outlined in the Declaration of Helsinki (WMA World Medical Association Declaration of Helsinki). The study subjects were all South Indian patients with dilated cardiomyopathy (DCM), diagnosed based on the NYHA (New York Heart Association, 1994), and WHO (www.who.int/cardiovascular_diseases) guidelines.
Dilated cardiomyopathy (DCM) is characterized by left ventricular enlargement (LVE), and when echocardiography demonstrated a depressed systolic dysfunction with an ejection fraction (LVEF) <45–50% and/or fractional shortening <25%.
Patients with concomitant disease like; autoimmune disease, cancer, as well as patients with coronary artery disease (CAD), ventricular outflow tract obstructions and with advanced chronic renal failure (CRF), were excluded.
We have sequenced all the exons, including the exon-intron boundaries (5373 bp length) of Troponin T2 (cTnT) gene (Table S1), of clinically well-characterized 147 DCM against ethnically matched 207 healthy controls. (Text S1)
In silico analysis
To evaluate whether the SNPs observed exclusively in DCM have any potential cause for the defect in splicing, we have analyzed these sites with ASD Workbench wrapper (http://www.ebi.ac.uk/asd-srv/wb.cgi) tools, such as poly-pyrimidine tract (PPT), and branch-points (BP). The novel SNPs observed in this study were subjected to identify the presence of PPT and BP binding sites for splicing factors, and exonic splicing enhancers/silencers (ESE/ESS) or intronic splicing enhancers/silencers (ISE/ISS), respectively. Splicing Rainbow tool searches for the SR proteins (serine/arginine-rich) as well as hnRNP motifs.
We thank all the patients and their family members, and the healthy individuals who have participated in the study. DSR and KT thank the CCMB, Council of Scientific and Industrial Research (CSIR), India. KT was supported by Network project grant (CardioMed-BSC0122) from CSIR, Government of India. DSR and PN acknowledge the help of Department of Genetics, Osmania University, Hyderabad.
Conceived and designed the experiments: DSR PN KT. Performed the experiments: DSR PSD. Analyzed the data: DSR KT. Contributed reagents/materials/analysis tools: DSR PSD PN CN KT. Wrote the paper: DSR. Provided input on manuscript writing: KT.
- 1. Codd MB, Sugrue DD, Gersh BJ, Melton LJ 3rd (1989) Epidemiology of idiopathic dilated and hypertrophic cardiomyopathy. A population-based study in Olmsted County, Minnesota, 1975–1984. Circulation 80: 564–572.
- 2. Knoll R, Hoshijima M, Hoffman HM, Person V, Lorenzen-Schmidt I, et al. (2002) The cardiac mechanical stretch sensor machinery involves a Z disc complex that is defective in a subset of human dilated cardiomyopathy. Cell 111: 943–955.
- 3. Ackerman MJ, Priori SG, Willems S, Berul C, Brugada R, et al. (2011) HRS/EHRA expert consensus statement on the state of genetic testing for the channelopathies and cardiomyopathies: this document was developed as a partnership between the Heart Rhythm Society (HRS) and the European Heart Rhythm Association (EHRA). Europace 13: 1077–1109.
- 4. Sugrue DD, Rodeheffer RJ, Codd MB, Ballard DJ, Fuster V, et al. (1992) The clinical course of idiopathic dilated cardiomyopathy. A population-based study. Ann Intern Med 117: 117–123.
- 5. Wiles HB, McArthur PD, Taylor AB, Gillette PC, Fyfe DA, et al. (1991) Prognostic features of children with idiopathic dilated cardiomyopathy. Am J Cardiol 68: 1372–1376.
- 6. Hershberger RE, Morales A, Siegfried JD (2010) Clinical and genetic issues in dilated cardiomyopathy: a review for genetics professionals. Genet Med 12: 655–667.
- 7. Grunig E, Tasman JA, Kucherer H, Franz W, Kubler W, et al. (1998) Frequency and phenotypes of familial dilated cardiomyopathy. J Am Coll Cardiol 31: 186–194.
- 8. Jefferies JL, Towbin JA (2010) Dilated cardiomyopathy. Lancet 375: 752–762.
- 9. Michels VV, Moll PP, Miller FA, Tajik AJ, Chu JS, et al. (1992) The frequency of familial dilated cardiomyopathy in a series of patients with idiopathic dilated cardiomyopathy. N Engl J Med 326: 77–82.
- 10. Burkett EL, Hershberger RE (2005) Clinical and genetic issues in familial dilated cardiomyopathy. J Am Coll Cardiol 45: 969–981.
- 11. Judge DP (2009) Use of genetics in the clinical evaluation of cardiomyopathy. JAMA 302: 2471–2476.
- 12. Hershberger RE, Norton N, Morales A, Li D, Siegfried JD, et al. (2010) Coding sequence rare variants identified in MYBPC3, MYH6, TPM1, TNNC1, and TNNI3 from 312 patients with familial or idiopathic dilated cardiomyopathy. Circ Cardiovasc Genet 3: 155–161.
- 13. Hershberger RE, Cowan J, Morales A, Siegfried JD (2009) Progress with genetic cardiomyopathies: screening, counseling, and testing in dilated, hypertrophic, and arrhythmogenic right ventricular dysplasia/cardiomyopathy. Circ Heart Fail 2: 253–261.
- 14. Hershberger RE, Lindenfeld J, Mestroni L, Seidman CE, Taylor MR, et al. (2009) Genetic evaluation of cardiomyopathy—a Heart Failure Society of America practice guideline. J Card Fail 15: 83–97.
- 15. Serio A, Narula N, Kodama T, Favalli V, Arbustini E (2012) Familial dilated cardiomyopathy. Clinical and genetic characteristics. Herz 37: 822–829.
- 16. Santorelli FM, Mak SC, El-Schahawi M, Casali C, Shanske S, et al. (1996) Maternally inherited cardiomyopathy and hearing loss associated with a novel mutation in the mitochondrial tRNA(Lys) gene (G8363A). Am J Hum Genet 58: 933–939.
- 17. Li YY, Maisch B, Rose ML, Hengstenberg C (1997) Point mutations in mitochondrial DNA of patients with dilated cardiomyopathy. J Mol Cell Cardiol 29: 2699–2709.
- 18. Ingles J, Doolan A, Chiu C, Seidman J, Seidman C, et al. (2005) Compound and double mutations in patients with hypertrophic cardiomyopathy: implications for genetic testing and counselling. J Med Genet 42: e59.
- 19. Girolami F, Ho CY, Semsarian C, Baldi M, Will ML, et al. (2010) Clinical features and outcome of hypertrophic cardiomyopathy associated with triple sarcomere protein gene mutations. J Am Coll Cardiol 55: 1444–1453.
- 20. Dhandapany PS, Sadayappan S, Xue Y, Powell GT, Rani DS, et al. (2009) A common MYBPC3 (cardiac myosin binding protein C) variant associated with cardiomyopathies in South Asia. Nat Genet 41: 187–191.
- 21. Reich D, Thangaraj K, Patterson N, Price AL, Singh L (2009) Reconstructing Indian population history. Nature 461: 489–494.
- 22. Metspalu M, Romero IG, Yunusbayev B, Chaubey G, Mallick CB, et al. (2011) Shared and unique components of human population structure and genome-wide signals of positive selection in South Asia. Am J Hum Genet 89: 731–744.
- 23. Rani DS, Nallari P, Priyamvada S, Narasimhan C, Singh L, et al. (2012) High prevalence of Arginine to Glutamine substitution at 98, 141 and 162 positions in Troponin I (TNNI3) associated with hypertrophic cardiomyopathy among Indians. BMC Med Genet 13: 69.
- 24. Ramachandran G, Kumar M, Selvi Rani D, Annanthapur V, Calambur N, et al. (2013) An in silico analysis of troponin I mutations in hypertrophic cardiomyopathy of Indian origin. PLoS One 8: e70704.
- 25. Rani DS, Nallari P, Dhandapany PS, Tamilarasi S, Shah A, et al. (2012) Cardiac Troponin T (TNNT2) mutations are less prevalent in Indian hypertrophic cardiomyopathy patients. DNA Cell Biol 31: 616–624.
- 26. Rangaraju A, Rani DS, Satyanarayana M, Calambur N, Swapna N, et al. (2012) Genetic variations of alpha-cardiac actin and cardiac muscle LIM protein in hypertrophic cardiomyopathy in South India. Exp Clin Cardiol 17: 26–29.
- 27. Watkins H, McKenna WJ, Thierfelder L, Suk HJ, Anan R, et al. (1995) Mutations in the genes for cardiac troponin T and alpha-tropomyosin in hypertrophic cardiomyopathy. N Engl J Med 332: 1058–1064.
- 28. Hershberger RE, Pinto JR, Parks SB, Kushner JD, Li D, et al. (2009) Clinical and functional characterization of TNNT2 mutations identified in patients with dilated cardiomyopathy. Circ Cardiovasc Genet 2: 306–313.
- 29. Pinto JR, Parvatiyar MS, Jones MA, Liang J, Potter JD (2008) A troponin T mutation that causes infantile restrictive cardiomyopathy increases Ca2+ sensitivity of force development and impairs the inhibitory properties of troponin. J Biol Chem 283: 2156–2166.
- 30. Luedde M, Ehlermann P, Weichenhan D, Will R, Zeller R, et al. (2010) Severe familial left ventricular non-compaction cardiomyopathy due to a novel troponin T (TNNT2) mutation. Cardiovasc Res 86: 452–460.
- 31. Hinkle A, Goranson A, Butters CA, Tobacman LS (1999) Roles for the troponin tail domain in thin filament assembly and regulation. A deletional study of cardiac troponin T. J Biol Chem 274: 7157–7164.
- 32. Hinkle A, Tobacman LS (2003) Folding and function of the troponin tail domain. Effects of cardiomyopathic troponin T mutations. J Biol Chem 278: 506–513.
- 33. Sehnert AJ, Huq A, Weinstein BM, Walker C, Fishman M, et al. (2002) Cardiac troponin T is essential in sarcomere assembly and cardiac contractility. Nat Genet 31: 106–110.
- 34. Kamisago M, Sharma SD, DePalma SR, Solomon S, Sharma P, et al. (2000) Mutations in sarcomere protein genes as a cause of dilated cardiomyopathy. N Engl J Med 343: 1688–1696.
- 35. Mogensen J, Murphy RT, Shaw T, Bahl A, Redwood C, et al. (2004) Severe disease expression of cardiac troponin C and T mutations in patients with idiopathic dilated cardiomyopathy. J Am Coll Cardiol 44: 2033–2040.
- 36. Li D, Czernuszewicz GZ, Gonzalez O, Tapscott T, Karibe A, et al. (2001) Novel cardiac troponin T mutation as a cause of familial dilated cardiomyopathy. Circulation 104: 2188–2193.
- 37. Mirza M, Marston S, Willott R, Ashley C, Mogensen J, et al. (2005) Dilated cardiomyopathy mutations in three thin filament regulatory proteins result in a common functional phenotype. J Biol Chem 280: 28498–28506.
- 38. Du CK, Morimoto S, Nishii K, Minakami R, Ohta M, et al. (2007) Knock-in mouse model of dilated cardiomyopathy caused by troponin mutation. Circ Res 101: 185–194.
- 39. Chaubey G, Metspalu M, Kivisild T, Villems R (2007) Peopling of South Asia: investigating the caste-tribe continuum in India. Bioessays 29: 91–100.