Identification of novel mutations including a double mutation in patients with inherited cardiomyopathy by a targeted sequencing approach using the Ion Torrent PGM system

Zhao,Yue; Cao,Hong; Song,Yindi; Feng,Yue; Ding,Xiaoxue; Pang,Mingjie; Zhang,Yunmei; Zhang,Hong; Ding,Jiahuan; Xia,Xueshan

doi:10.3892/ijmm.2016.2565

Identification of novel mutations including a double mutation in patients with inherited cardiomyopathy by a targeted sequencing approach using the Ion Torrent PGM system

Authors:
- Yue Zhao
- Hong Cao
- Yindi Song
- Yue Feng
- Xiaoxue Ding
- Mingjie Pang
- Yunmei Zhang
- Hong Zhang
- Jiahuan Ding
- Xueshan Xia
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Affiliations: Faculty of Life Science and Technology, Research Center for Molecular Medicine in Yunnan Province, Kunming University of Science and Technology, Kunming, Yunnan 650500, P.R. China, Department of Cardiology, The First Hospital of Yunnan Province, Kunming, Yunnan 650034, P.R. China
Published online on: April 14, 2016 https://doi.org/10.3892/ijmm.2016.2565
Pages: 1511-1520
Copyright: © Zhao et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Inherited cardiomyopathy is the major cause of sudden cardiac death (SCD) and heart failure (HF). The disease is associated with extensive genetic heterogeneity; pathogenic mutations in cardiac sarcomere protein genes, cytoskeletal protein genes and nuclear envelope protein genes have been linked to its etiology. Early diagnosis is conducive to clinical monitoring and allows for presymptomatic interventions as needed. In the present study, the entire coding sequences and flanking regions of 12 major disease (cardiomyopathy)-related genes [namely myosin, heavy chain 7, cardiac muscle, β (MYH7); myosin binding protein C, cardiac (MYBPC3); lamin A/C (LMNA); troponin I type 3 (cardiac) (TNNI3); troponin T type 2 (cardiac) (TNNT2); actin, α, cardiac muscle 1 (ACTC1); tropomyosin 1 (α) (TPM1); sodium channel, voltage gated, type V alpha subunit (SCN5A); myosin, light chain 2, regulatory, cardiac, slow (MYL2); myosin, heavy chain 6, cardiac muscle, α (MYH6); myosin, light chain 3, alkali, ventricular, skeletal, slow (MYL3); and protein kinase, AMP-activated, gamma 2 non-catalytic subunit (PRKAG2)] in 8 patients with dilated cardiomyopathy (DCM) and in 8 patients with hypertrophic cardiomyopathy (HCM) were amplified and then sequenced using the Ion Torrent Personal Genome Machine (PGM) system. As a result, a novel heterozygous mutation (MYH7, p.Asn885Thr) and a variant of uncertain significance (TNNT2, p.Arg296His) were identified in 2 patients with HCM. These 2 missense mutations, which were absent in the samples obtained from the 200 healthy control subjects, altered the amino acid that was evolutionarily conserved among a number of vertebrate species; this illustrates that these 2 non-synonymous mutations play a role in the pathogenesis of HCM. Moreover, a double heterozygous mutation (PRKAG2, p.Gly100Ser plus MYH7, p.Arg719Trp) was identified in a patient with severe familial HCM, for the first time to the best of our knowledge. This patient provided us with more information regarding the genotype-phenotype correlation between mutations of MYH7 and PRKAG2. Taken together, these findings provide insight into the molecular mechanisms underlying inherited cardiomyopathy. The mutations identified in this study may be further investigated in the future in order to improve the diagnosis and treatment of patients with inherited cardiomyopathy. Furthermore, our findings indicated that sequencing using the Ion Torrent PGM system is a useful approach for the identification of pathogenic mutations associated with inherited cardiomyopathy, and it may be used for the risk evaluation of individuals with a possible susceptibility to inherited cardiomyopathy.

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1	Callis TE, Jensen BC, Weck KE and Willis MS: Evolving molecular diagnostics for familial cardiomyopathies: at the heart of it all. Expert Rev Mol Diagn. 10:329–351. 2010. View Article : Google Scholar : PubMed/NCBI
2	Hughes SE and McKenna WJ: New insights into the pathology of inherited cardiomyopathy. Heart. 91:257–264. 2005. View Article : Google Scholar : PubMed/NCBI
3	Hershberger RE, Hedges DJ and Morales A: Dilated cardiomyopathy: the complexity of a diverse genetic architecture. Nat Rev Cardiol. 10:531–547. 2013. View Article : Google Scholar : PubMed/NCBI
4	Hinson JT, Chopra A, Nafissi N, Polacheck WJ, Benson CC, Swist S, Gorham J, Yang L, Schafer S, Sheng CC, et al: HEART DISEASE. Titin mutations in iPS cells define sarcomere insufficiency as a cause of dilated cardiomyopathy. Science. 349:982–986. 2015. View Article : Google Scholar : PubMed/NCBI
5	van Spaendonck-Zwarts KY, Posafalvi A, van den Berg MP, Hilfiker-Kleiner D, Bollen IA, Sliwa K, Alders M, Almomani R, van Langen IM, van der Meer P, et al: Titin gene mutations are common in families with both peripartum cardiomyopathy and dilated cardiomyopathy. Eur Heart J. 35:2165–2173. 2014. View Article : Google Scholar : PubMed/NCBI
6	Zhou YM, Dai XY, Qiu XB, Yuan F, Li RG, Xu YJ, Qu XK, Huang RT, Xue S and Yang YQ: HAND1 loss-of-function mutation associated with familial dilated cardiomyopathy. Clin Chem Lab Med. Nov 18–2015.Epub ahead of print. View Article : Google Scholar : PubMed/NCBI
7	Zhao CM, Bing-Sun, Song HM, Wang J, Xu WJ, Jiang JF, Qiu XB, Yuan F, Xu JH and Yang YQ: TBX20 loss-of-function mutation associated with familial dilated cardiomyopathy. Clin Chem Lab Med. 54:325–332. 2016. View Article : Google Scholar
8	Qu XK, Yuan F, Li RG, Xu L, Jing WF, Liu H, Xu YJ, Zhang M, Liu X, Fang WY, et al: Prevalence and spectrum of LRRC10 mutations associated with idiopathic dilated cardiomyopathy. Mol Med Rep. 12:3718–3724. 2015.PubMed/NCBI
9	Zhang XL, Qiu XB, Yuan F, Wang J, Zhao CM, Li RG, Xu L, Xu YJ, Shi HY, Hou XM, et al: TBX5 loss-of-function mutation contributes to familial dilated cardiomyopathy. Biochem Biophys Res Commun. 459:166–171. 2015. View Article : Google Scholar : PubMed/NCBI
10	Zhou W, Zhao L, Jiang JQ, Jiang WF, Yang YQ and Qiu XB: A novel TBX5 loss-of-function mutation associated with sporadic dilated cardiomyopathy. Int J Mol Med. 36:282–288. 2015.PubMed/NCBI
11	Zhang XL, Dai N, Tang K, Chen YQ, Chen W, Wang J, Zhao CM, Yuan F, Qiu XB, Qu XK, et al: GATA5 loss-of-function mutation in familial dilated cardiomyopathy. Int J Mol Med. 35:763–770. 2015.
12	Xu L, Zhao L, Yuan F, Jiang WF, Liu H, Li RG, Xu YJ, Zhang M, Fang WY, Qu XK, et al: GATA6 loss-of-function mutations contribute to familial dilated cardiomyopathy. Int J Mol Med. 34:1315–1322. 2014.PubMed/NCBI
13	Zhao L, Xu JH, Xu WJ, Yu H, Wang Q, Zheng HZ, Jiang WF, Jiang JF and Yang YQ: A novel GATA4 loss-of-function mutation responsible for familial dilated cardiomyopathy. Int J Mol Med. 33:654–660. 2014.
14	Li J, Liu WD, Yang ZL, Yuan F, Xu L, Li RG and Yang YQ: Prevalence and spectrum of GATA4 mutations associated with sporadic dilated cardiomyopathy. Gene. 548:174–181. 2014. View Article : Google Scholar : PubMed/NCBI
15	Reinstein E, Orvin K, Tayeb-Fligelman E, Stiebel-Kalish H, Tzur S, Pimienta AL, Bazak L, Bengal T, Cohen L, Gaton DD, et al: Mutations in TAX1BP3 cause dilated cardiomyopathy with septo-optic dysplasia. Hum Mutat. 36:439–442. 2015. View Article : Google Scholar : PubMed/NCBI
16	Dhandapany PS, Razzaque MA, Muthusami U, Kunnoth S, Edwards JJ, Mulero-Navarro S, Riess I, Pardo S, Sheng J, Rani DS, et al: RAF1 mutations in childhood-onset dilated cardiomyopathy. Nat Genet. 46:635–639. 2014. View Article : Google Scholar : PubMed/NCBI
17	Zou Y, Song L, Wang Z, Ma A, Liu T, Gu H, Lu S, Wu P, Zhang Y, Shen L, et al: Prevalence of idiopathic hypertrophic cardiomyopathy in China: A population-based echocardiographic analysis of 8080 adults. Am J Med. 116:14–18. 2004. View Article : Google Scholar : PubMed/NCBI
18	Maron BJ, Gardin JM, Flack JM, Gidding SS, Kurosaki TT and Bild DE: Prevalence of hypertrophic cardiomyopathy in a general population of young adults. Echocardiographic analysis of 4111 subjects in the CARDIA Study. Coronary Artery Risk Development in (Young) Adults. Circulation. 92:785–789. 1995. View Article : Google Scholar : PubMed/NCBI
19	Yuan F, Qiu XB, Li RG, Qu XK, Wang J, Xu YJ, Liu X, Fang WY, Yang YQ and Liao DN: A novel NKX2-5 loss-of-function mutation predisposes to familial dilated cardiomyopathy and arrhythmias. Int J Mol Med. 35:478–486. 2015.
20	Rothberg JM, Hinz W, Rearick TM, Schultz J, Mileski W, Davey M, Leamon JH, Johnson K, Milgrew MJ, Edwards M, et al: An integrated semiconductor device enabling non-optical genome sequencing. Nature. 475:348–352. 2011. View Article : Google Scholar : PubMed/NCBI
21	Gersh BJ, Maron BJ, Bonow RO, Dearani JA, Fifer MA, Link MS, Naidu SS, Nishimura RA, Ommen SR and Rakowski H; American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines: 2011 ACCF/AHA Guideline for the Diagnosis and Treatment of Hypertrophic Cardiomyopathy a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. Developed in collaboration with the American Association for Thoracic Surgery, American Society of Echocardiography, American Society of Nuclear Cardiology, Heart Failure Society of America, Heart Rhythm Society, Society for Cardiovascular Angiography and Interventions, and Society of Thoracic Surgeons. J Am Coll Cardiol. 58:e212–260. 2011. View Article : Google Scholar : PubMed/NCBI
22	Mestroni L, Maisch B, McKenna WJ, Schwartz K, Charron P, Rocco C, Tesson F, Richter A, Wilke A and Komajda M: Collaborative Research Group of the European Human and Capital Mobility Project on Familial Dilated Cardiomyopathy: Guidelines for the study of familial dilated cardiomyopathies. Eur Heart J. 20:93–102. 1999. View Article : Google Scholar : PubMed/NCBI
23	Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES, Getz G and Mesirov JP: Integrative genomics viewer. Nat Biotechnol. 29:24–26. 2011. View Article : Google Scholar : PubMed/NCBI
24	Richards CS, Bale S, Bellissimo DB, Das S, Grody WW, Hegde MR, Lyon E and Ward BE; Collaborative Research Group of the European Human and Capital Mobility Project on Familial Dilated Cardiomyopathy: ACMG recommendations for standards for interpretation and reporting of sequence variations: Revisions 2007. Genet Med. 10:294–300. 2008. View Article : Google Scholar : PubMed/NCBI
25	Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, Kondrashov AS and Sunyaev SR: A method and server for predicting damaging missense mutations. Nat Methods. 7:248–249. 2010. View Article : Google Scholar : PubMed/NCBI
26	Kumar P, Henikoff S and Ng PC: Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc. 4:1073–1081. 2009. View Article : Google Scholar : PubMed/NCBI
27	Schwarz JM, Rödelsperger C, Schuelke M and Seelow D: MutationTaster evaluates disease-causing potential of sequence alterations. Nat Methods. 7:575–576. 2010. View Article : Google Scholar : PubMed/NCBI
28	Sikkema-Raddatz B, Johansson LF, de Boer EN, Almomani R, Boven LG, van den Berg MP, van Spaendonck-Zwarts KY, van Tintelen JP, Sijmons RH, Jongbloed JD and Sinke RJ: Targeted next-generation sequencing can replace Sanger sequencing in clinical diagnostics. Hum Mutat. 34:1035–1042. 2013. View Article : Google Scholar : PubMed/NCBI
29	Tarabeux J, Zeitouni B, Moncoutier V, Tenreiro H, Abidallah K, Lair S, Legoix-Né P, Leroy Q, Rouleau E, Golmard L, et al: Streamlined ion torrent PGM-based diagnostics: BRCA1 and BRCA2 genes as a model. Eur J Hum Genet. 22:535–541. 2014. View Article : Google Scholar :
30	Costa JL, Sousa S, Justino A, Kay T, Fernandes S, Cirnes L, Schmitt F and Machado JC: Nonoptical massive parallel DNA sequencing of BRCA1 and BRCA2 genes in a diagnostic setting. Hum Mutat. 34:629–635. 2013. View Article : Google Scholar : PubMed/NCBI
31	Li X, Buckton AJ, Wilkinson SL, John S, Walsh R, Novotny T, Valaskova I, Gupta M, Game L, Barton PJ, et al: Towards clinical molecular diagnosis of inherited cardiac conditions: a comparison of bench-top genome DNA sequencers. PLoS One. 8:e677442013. View Article : Google Scholar : PubMed/NCBI
32	Lakdawala NK, Funke BH, Baxter S, Cirino AL, Roberts AE, Judge DP, Johnson N, Mendelsohn NJ, Morel C, Care M, et al: Genetic testing for dilated cardiomyopathy in clinical practice. J Card Fail. 18:296–303. 2012. View Article : Google Scholar : PubMed/NCBI
33	Brito D, Miltenberger-Miltenyi G, Vale Pereira S, Silva D, Diogo AN and Madeira H: Sarcomeric hypertrophic cardio-myopathy: genetic profile in a Portuguese population. Rev Port Cardiol. 31:577–587. 2012.PubMed/NCBI
34	Zou Y, Wang J, Liu X, Wang Y, Chen Y, Sun K, Gao S, Zhang C, Wang Z, Zhang Y, 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. 40:3969–3976. 2013. View Article : Google Scholar : PubMed/NCBI
35	Zhao Y, Feng Y, Zhang YM, Ding XX, Song YZ, Zhang AM, Liu L, Zhang H, Ding JH and Xia XS: Targeted next-generation sequencing of candidate genes reveals novel mutations in patients with dilated cardiomyopathy. Int J Mol Med. 36:1479–1486. 2015.PubMed/NCBI
36	Das KJ, Ingles J, Bagnall RD and Semsarian C: Determining pathogenicity of genetic variants in hypertrophic cardiomyopathy: importance of periodic reassessment. Genet Med. 16:286–293. 2014. View Article : Google Scholar
37	Hassel D, Dahme T, Erdmann J, Meder B, Huge A, Stoll M, Just S, Hess A, Ehlermann P, Weichenhan D, et al: Nexilin mutations destabilize cardiac Z-disks and lead to dilated cardiomyopathy. Nat Med. 15:1281–1288. 2009. View Article : Google Scholar : PubMed/NCBI
38	Geisterfer-Lowrance AA, Christe M, Conner DA, Ingwall JS, Schoen FJ, Seidman CE and Seidman JG: A mouse model of familial hypertrophic cardiomyopathy. Science. 272:731–734. 1996. View Article : Google Scholar : PubMed/NCBI
39	Cheng J, Morales A, Siegfried JD, Li D, Norton N, Song J, Gonzalez-Quintana J, Makielski JC and Hershberger RE: SCN5A rare variants in familial dilated cardiomyopathy decrease peak sodium current depending on the common polymorphism H558R and common splice variant Q1077del. Clin Transl Sci. 3:287–294. 2010. View Article : Google Scholar : PubMed/NCBI
40	Chen L, Zhang W, Fang C, Jiang S, Shu C, Cheng H, Li F and Li H: Polymorphism H558R in the human cardiac sodium channel SCN5A gene is associated with atrial fibrillation. J Int Med Res. 39:1908–1916. 2011. View Article : Google Scholar : PubMed/NCBI
41	Nikulina SY, Chernova AA, Shulman VA, Maksimov VN, Gavrilyuk OA, Tretyakova SS and Marilovceva OV: An investigation of the association of the H558R polymorphism of the SCN5A gene with idiopathic cardiac conduction disorders. Genet Test Mol Biomarkers. 19:288–294. 2015. View Article : Google Scholar : PubMed/NCBI
42	Marangoni S, Di Resta C, Rocchetti M, Barile L, Rizzetto R, Summa A, Severi S, Sommariva E, Pappone C, Ferrari M, et al: A Brugada syndrome mutation (p.S216L) and its modulation by p.H558R polymorphism: standard and dynamic characterization. Cardiovasc Res. 91:606–616. 2011. View Article : Google Scholar : PubMed/NCBI
43	Roncarati R, Viviani Anselmi C, Krawitz P, Lattanzi G, von Kodolitsch Y, Perrot A, di Pasquale E, Papa L, Portararo P, Columbaro M, et al: Doubly heterozygous LMNA and TTN mutations revealed by exome sequencing in a severe form of dilated cardiomyopathy. Eur J Hum Genet. 21:1105–1111. 2013. View Article : Google Scholar : PubMed/NCBI
44	Lekanne Deprez RH, Muurling-Vlietman JJ, Hruda J, Baars MJ, Wijnaendts LC, Stolte-Dijkstra I, Alders M and van Hagen JM: Two cases of severe neonatal hypertrophic cardiomyopathy caused by compound heterozygous mutations in the MYBPC3 gene. J Med Genet. 43:829–832. 2006. View Article : Google Scholar : PubMed/NCBI
45	Ho CY: Genetics and clinical destiny: improving care in hyper-trophic cardiomyopathy. Circulation. 122:2430–2440. 2010. View Article : Google Scholar
46	Anan R, Greve G, Thierfelder L, Watkins H, McKenna WJ, Solomon S, Vecchio C, Shono H, Nakao S and Tanaka H: Prognostic implications of novel beta cardiac myosin heavy chain gene mutations that cause familial hypertrophic cardiomyopathy. J Clin Invest. 93:280–285. 1994. View Article : Google Scholar : PubMed/NCBI
47	Zhang BL, Xu RL, Zhang J, Zhao XX, Wu H, Ma LP, Hu JQ, Zhang JL, Ye Z, Zheng X and Qin YW: Identification and functional analysis of a novel PRKAG2 mutation responsible for Chinese PRKAG2 cardiac syndrome reveal an important role of non-CBS domains in regulating the AMPK pathway. J Cardiol. 62:241–248. 2013. View Article : Google Scholar : PubMed/NCBI
48	Van Driest SL, Vasile VC, Ommen SR, Will ML, Tajik AJ, Gersh BJ and Ackerman MJ: Myosin binding protein C mutations and compound heterozygosity in hypertrophic cardiomyopathy. J Am Coll Cardiol. 44:1903–1910. 2004. View Article : Google Scholar : PubMed/NCBI
49	Zhao Y, Feng Y, Zhang YM, Ding XX, Song YZ, Zhang AM, Liu L, Zhang H, Ding JH and Xia XS: Targeted next-generation sequencing reveals hot spots and doubly heterozygous mutations in Chinese patients with familial cardiomyopathy. BioMed Res Int. 2015:5618192015. View Article : Google Scholar : PubMed/NCBI
50	Brisca G, Fiorillo C, Nesti C, Trucco F, Derchi M, Andaloro A, Assereto S, Morcaldi G, Pedemonte M, Minetti C, et al: Early onset cardiomyopathy associated with the mitochondrial tRNALeu((UUR)) 3271T>C MELAS mutation. Biochem Biophys Res Commun. 458:601–604. 2015. View Article : Google Scholar : PubMed/NCBI
51	Wang AL, Kong DH, Chen DX, Wan J and Yu YX: Mutation of V896M in cardiac myosin binding protein-c gene in two Chinese families with hypertrophic cardiomyopathy. Mol Med Rep. 3:759–763. 2010.
52	Millat G, Chanavat V and Rousson R: Evaluation of a new NGS method based on a custom AmpliSeq library and Ion Torrent PGM sequencing for the fast detection of genetic variations in cardiomyopathies. Clin Chim Acta. 433:266–271. 2014. View Article : Google Scholar : PubMed/NCBI
53	Glotov AS, Kazakov SV, Zhukova EA, Alexandrov AV, Glotov OS, Pakin VS, Danilova MM, Poliakova IV, Niyazova SS, Chakova NN, et al: Targeted next-generation sequencing (NGS) of nine candidate genes with custom AmpliSeq in patients and a cardiomyopathy risk group. Clin Chim Acta. 446:132–140. 2015. View Article : Google Scholar : PubMed/NCBI