50 Original Article

Genetic variation in exon 5 of - I in hypertrophic cases

S. D. Annapurna, T. R. Reena, Pratibha Nallari, Narasimhan Calambur Department of Genetics, Osmania University, Hyderabad - 500 007, Cardiology Unit, CARE Hospital, The Institute of Medical Sciences, Nampally, Hyderabad - 500 001, Andhra Pradesh, India

hypertrophy and myofi brillar disarray and is commonly BACKGROUND: are a heterogeneous group of heart muscle disorders and are classifi ed as manifested as dyspnea, palpitations, chest pain and 1) Hypertrophic Cardiomyopathy (HCM) 2) Dilated syncope/pre syncope. cardiomyopathy (DCM) 3) Restrictive cardiomyopathy (RCM) and 4) Arrhythmogenic right ventricular dysplasia It is an autosomal dominant disorder with variable (ARVD) as per WHO classifi cation, of which HCM and DCM expression and penetrance with a prevalence of about are common. HCM is a complex but relatively common 0.2% in general population[1] and is associated with form of inherited heart muscle disease with prevalence of 1 in 500 individuals and is commonly associated with 16 nuclear and 3 mitochondrial gene mutations. Most sarcomeric gene mutations. of these encode for both sarcomeric and non (TNNI-3) is one such sarcomeric and is a subunit of the thin fi lament-associated troponin- complex sarcomeric as listed in Table 1. It is further involved in regulation of skeletal and cardiac classifi ed into several sub types as asymmetric septal . Mutations in this gene were found to hypertrophy (with and without obstruction), ventricular be associated with a history of sudden cardiac death in HCM patients. hypertrophy, hypertrophy of left ventricular posterior wall AIM: Therefore the present study aims to identify for and apical hypertrophy based on their morphology and mutations associated with troponin I gene in a set of HCM patients from Indian population. MATERIALS AND METHODS: Mutational analyses of 92 Table 1: Genes involved in hypertrophic cardiomyopathy HCM cases were carried out following PCR based SSCP and their chromosomal locations analysis. genes RESULTS: The study revealed band pattern variation in 3 Beta- heavy chain (β-MYH7) 14q12 cases from a group of 92 HCM patients. This band pattern variation, on sequencing revealed base changes, one at nt Myosin binding protein C (MYBPC3) 11p11 2560 with G>T transversion in exon-5 region with a wobble Cardiac (TNNT2) 1q32 and others at nt 2479 and nt 2478 with G>C(www.medknow.com). and C>G Cardiac troponin I (TNNI3) 19q13 transversions in the intronic region upstream of the exon Cardiac (TNNC1) 3p21-14 5 on sequencing. Further analysis showed that one of the Alpha tropomyosin (TPM1) 15q22.1 probands showed apical form of hypertrophy, two others Essential myosin light chain (MYL3) 3q21 showing asymmetricThis septal PDFa sitehypertrophy. is hosted available Two of by these forMedknow free download Publications from Regulatory myosin light chain (MYL2) 12q23-q24 probands showed family history of the condition. CONCLUSIONS: Hence, the study supports earlier reports Cardiac (ACTC) 15q14 of involvement of TNNI-3 in the causation of apical and (TTN) 2q24.3 asymmetrical forms of hypertrophy. Telethonin 17q12 Alpha- myosin heavy chain 14q12 Key words: Genetic variation, hypertrophic cardiomyopathy, Non sarcomeric genes sudden cardiac death, troponin-I AMP- activated protein kinase (PRKAG2) 7q36 Cardiac muscle LIM protein (CLP) 11p15.1 Introduction Cardiac myosin light-peptide kinase 20q13.3 3p25 Mitochondrial genes Hypertrophic cardiomyopathy (HCM) is a myocardial Mitochondrial tRNA –glycine disorder characterized by unexplained ventricular Mitochondrial tRNA- and leucine

Address for Correspondence: Dr. Pratiibha Nallari, Department of Genetics, Osmania University, Hyderabad, India. E-mail: [email protected]

Indian Journal of Human Genetics May-August 2007 Volume 13 Issue 2 Annapurna, et al.: Genetic variation in exon 5 of TNNI3 gene in HCM 51 their association with different gene mutations. silver staining according to the protocol of Orita et al.[6] One of the sarcomeric genes, Cardiac muscle troponin On a stained SSCP gel, mobility shift was recognized I (TNNI-3) is a basic globular protein, expressed only in as an aberrant band pattern compared to the control the heart.[2] It is a subunit of the thin fi lament-associated sample’s band pattern. PCR products showing an troponin-tropomyosin complex involved in calcium aberrant band pattern on SSCP gels were later purifi ed regulation of skeletal and cardiac muscle contraction. and commercially sequenced on a 373 DNA analyzer; Eight coding exons of TNNI-3 code for a polypeptide of Macrogen (Korea) for the detection of either SNPs and/or 210 amino acids[3,4] and mutations in this gene account mutations. for a total of 5% HCM cases, with a history of sudden cardiac death and poor prognosis. Hence this gene Results and Discussion was screened for mutations in a group of clinically well- characterized HCM patients. PCR based SSCP analysis [Figure 1A] of 92 HCM cases was carried out on troponin-I gene. Of the 92 Materials and Methods individuals screened, 3 probands (FHC 52, 139, 145) revealed a band pattern variation in exon-5. These A total of 92 cases confi rmed for HCM based on samples on sequencing revealed heterozygous base medical history, physical examination, electrocardiogram changes at nt 2560 with G>T transversion (at codon and echocardiogram fi ndings were included in the study. 68 CGG >CGT) leading for synonymous change for These cases were referred to the cardiology units of CARE hospitals, Hyderabad and KEM Hospital, Mumbai. A In addition to these cases, 100 voluntary blood donors with no history/family history of any cardiac disorders were included as controls in the study. Informed consent was obtained from all the participating individuals along with Institutional ethics committee clearance. B Isolation of total genomic DNA from whole blood[5] of patients and controls was carried out followed by PCR based SSCP analysis.[6] Primer sequences for PCR were obtained based on available database of Seidman.[7] A PCR mix of fi nal volume 25µl containing 10x(www.medknow.com). PCR buffer (10 mM Tris Hcl; 50 mM Kcl), 1.5 mM Mgcl2, 50 p moles of forward and reverse primers, 200 µM dNTPs and 1U of Taq DNA polymeraseThis PDFa was site used. is hosted available All PCR reagentsby forMedknow free download Publications from were mixed together in a 0.2 µl PCR tube, which was C then placed in a thermal cycler (Eppendorf Master Cycler gradient, Germany) at an annealing temperature of 58oC for 25 cycles. Amplifi ed products were mixed with equal volumes of formamide loading buffer (formamide 0.9 g/ml, 10 mM NaOH, 11 mM EDTA), denatured at 95°C for 10 min and quenched on ice for 5 min prior to loading. Figure 1: A) SSCP gel showing abnormal band 8 µl of diluted samples were loaded onto 10% non- pattern (indicated by an arrow) B) Sequence denaturing polyacrylamide gel and placed in Consortium electrophoretograms showing base changes (indicated by an arrow) C) Pedigrees of FHC 52, 139 showing electrophoresis unit at 150 Volts and 15 mA current. affected (black symbols) and unaffected individuals. After electrophoresis, band pattern was visualized by circles- Females; squares-Males 52 Annapurna, et al.: Genetic variation in exon 5 of TNNI3 gene in HCM

Arginine [Figure 1B], with other two base changes at nt modulator infl uence on the calcium-dependent actin- 2479 and nt 2478 with G>C and C>G transversions, 26 myosin interaction. The region in which the variations are bases upstream of the exon-5. found in the present study is between the sites interacting The proband (FHC 52) was 38 years old at the time with Tn T. Signifi cance of variations in this region in the of diagnosis with apical hypertophic cardiomyopathy, a pathogenesis of the condition needs to be studied by in rare form from the Indian context. Echocardiographic silico analysis. fi ndings showed a thickness of 22mm of interventricular Codon bias, the preference for some of the synonymous septum and a diameter of 56 mm x 34 mm of left ventricle. codons encoding the same amino acid exists in the Of the 15 family members available, two members had expression of the genes across organisms. The a positive history [Figure 1C]. These two relatives (I-2, synonymous changes may exert an effect on the II-6) were unavailable for ECG and Echo diagnosis but expression by codon-anticodon interaction during on physical examination were found to have symptoms translation, which may have effect on translational of HCM. effi ciency.[12] However this needs to be confi rmed by FHC 139 (37 years) had obstructive type of functional studies. cardiomyopathy with LV thickness being 39 mm x 28 mm Base changes in any of the consensus nucleotides of and 20 mm IVS. When the proband’s family was screened an intron involved in spliceosome complexes other than for similar base changes, his daughter was found to have the required GT and AG pairs can have a regulatory effect similar band pattern variation and base changes. on RNA processing, as reported in beta thalassemia.[13] Family members of FHC 145 (64 yrs; LV-32 mm x The base changes observed in present study (26 bases 23 mm; IVS-14 mm) were unavailable for clinical or upstream of intron-exon junction) fall near the region mutational analysis. involved in secondary lariat structure formation required for splicing. Since the base changes observed in the All the three probands showed clinically divergent patients (FHC 52, 139,145) were not found in any of forms of hypertrophy (FHC 52-Apical form) (FHC-139- the 100 controls screened, it can be suspected that Asymmetric septal Hypertrophy (ASH) with obstruction) disruption of spliceosome formation may be involved in and (FHC-145-ASH without obstruction) but possesed disease pathogenesis in these individuals. This needs to same set of base changes. This shows that there be further confi rmed by Insilico/invitro studies. could be different underlying primary mutations whose expression could have been modifi ed by these changes Conclusion in exon-5 and intron-4. (www.medknow.com). Six mutations have been originally described in troponin-I, of which, 5 were missense mutations and TNNI-3 base changes may also account for rare apical [8] hypertrophy, further strengthening the observations one deletion withThis no disruption PDFa site isof hostedthe available reading frame.by forMedknow free download Publications from Mutations in troponin I gene have been considered as made from other populations. The study shows that the an infrequent cause of HCM although more mutations DNA sequence variations found in our population may have recently been reported.[9,10] Unusual patterns of differ from the changes observed in other populations. hypertrophy, including a predominant apical involvement Acknowledgement have been associated with troponin I defects.[9,11] Based on the morphologic pattern of disease, troponin I mutations may be more prevalent in populations with Authors are grateful to the Department of Biotechnology a high incidence of apical HCM, as reported in Japan.[8] (DBT), New Delhi for their fi nancial assistance. Therefore the present finding further supports the involvement of multiple base changes in TNNI-3 in the References pathogenesis of apical and asymmetrical septal forms of HCM. 1. Maron BJ, Spirito P, Roman MJ, Paranicas M, Okin PM, Best LG, et al. Prevalence of hypertrophic cardiomyopathy Troponin I, a sub unit of troponin complex exerts a in a population-based sample of American Indians aged Annapurna, et al.: Genetic variation in exon 5 of TNNI3 gene in HCM 53

51 to 77 years (the Strong Heart Study). Am J Cardiol functional defects. Circ Res 1998;83:580-93. 2004;93:1510-4. 9. Kimura A, Harada H, Park JE, Nishi H, Satoh M, Takahashi 2. Bhavsar PK, Brand NJ, Yacoub MH, Barton PJ. Isolation M, et al. Mutations in the cardiac troponin I gene associated and characterization of the human cardiac troponin I gene with hypertrophic cardiomyopathy. Nat Genet 1997;16:379- (TNNI3). Genomics 1996;35:11-23. 82. 3. Potter JD, Gergely J. Troponin, tropomyosin and actin 10. Pascale R, Charron P, Carrier L. Distribution of disease interactions in the Ca2+ regulation of muscle contraction. gene in 102 genotyped families with hypertrophic Biochemistry 1974;13:2697-703. cardiomyopathy. Circulation 2001;104:II521. 4. Hitchcock SE. Regulation of muscle contraction: Bindings 11. Woo A, Rakowski H, Liew J. Hypertrophic cardiomyopathy: of troponin and its components to actin and tropomyosin. Genotypic and phenotypic heterogeneity. Circulation 2000; Eur J Biochem 1975;52:255-63. 102:169-78. 5. Lahiri D, Nurnberger J. A rapid non-enzymatic method for 12. Lavner Y, Kotlar D. 2005. Codon bias as a factor in the preparation of HMW DNA from blood for RFLP studies. regulating expression via translation rate in the human Nucleic Acids Res 1991;19:5444. genome. Gene. Dec 24. 6. Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya 13. Orkin S. A review of beta thalassemias: the spectrum of T. Detection of Polymorphisms of Human DNA by Gel gene mutations. In: “Recombinant DNA applications to Electrophoresis as SSCPs. Proc Natl Acad Sci U S A human disease”. Caskey CT, White RL, editors, Cold Spring 1989;86:2766-70. Harbor Laboratory: 1983. p. 19-28. 7. Seidman 2001. Available from: http://www.genetics.med. harvard.edu/~seidman/cg3/genes/TNNI3. 8. Bonne G, Carrier L, Richard P, Hainque B, Schwartz K. Source of Support: Nil, Confl ict of Interest: None declared. Familial hypertrophic cardiomyopathy: From mutations to

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