Pediatric Cardiology

, Volume 39, Issue 4, pp 682–689 | Cite as

Targeted Next-Generation Sequencing in Patients with Non-syndromic Congenital Heart Disease

  • Silvia Pulignani
  • Cecilia Vecoli
  • Andrea Borghini
  • Ilenia Foffa
  • Lamia Ait-Alì
  • Maria Grazia Andreassi
Original Article

Abstract

Congenital heart disease (CHD) is a genetically heterogeneous disease. Targeted next-generation sequencing (NGS) offers a unique opportunity to sequence multiple genes at lower cost and effort compared to Sanger sequencing. We tested a targeted NGS of a specific gene panel in a relatively large population of non-syndromic CHD patients. The patient cohort comprised 68 CHD patients (45 males; 8.3 ± 1.7 years). Amplicon libraries for 16 CHD-strictly related genes were generated using a TruSeq® Custom Amplicon kit (Illumina, CA) and sequenced using the Illumina MiSeq platform. Sequence data were processed through the MiSeq Reporter and wANNOVAR softwares. After applying stringent filtering criteria, 20 missense variants in 9 genes were predicted to be damaging and were validated by Sanger sequencing with 100% concordance. Fourteen variants were present in public databases with very rare allele frequency, of which four variants (p.Arg25Cys in NKX2-5, p.Val763Ile in ZFPM2, p.Arg1398Gln and Gly1826Asp in MYH6) have been previously linked to CHD or cardiomyopathy. The remaining six variants in four genes (GATA4, NKX2-5, NOTCH1, TBX1) were novel mutations, currently not found in public databases, and absent in 200 control alleles of healthy subjects. Four patients (5.8%) carried two missense variants (1 compound heterozygote in the same gene and 3 double heterozygotes in different genes), with possibly synergistic deleterious effects. Targeted NGS is a powerful and efficient tool to detect DNA sequence variants in multiple genes, providing the opportunity for discovery of the co-occurrence of two or more missense rare variants.

Keywords

Congenital heart disease Next-generation sequencing Gene panel Single-nucleotide variants 

Notes

Acknowledgements

We thank the patients and families who participated in this study. We are very grateful to all clinical staff of Pediatric Cardiology, Fondazione Toscana, Massa, Italy for kind support and assistance.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical Approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed Consent

Informed consent was obtained from all individual participants included in the study.

References

  1. 1.
    Fahed AC, Gelb BD, Seidman JG, Seidman CE (2013) Genetics of congenital heart disease: the glass half empty. Circ Res 112:707–720.  https://doi.org/10.1161/CIRCRESAHA.112.300853 CrossRefPubMedGoogle Scholar
  2. 2.
    Gelb BD, Chung WK (2014) Complex genetics and the etiology of human congenital heart disease. Cold Spring Harb Perspect Med 4:a013953.  https://doi.org/10.1101/cshperspect.a013953 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Vecoli C, Pulignani S, Foffa I, Andreassi MG (2014) Congenital heart disease: the crossroads of genetics, epigenetics and environment. Curr Genomics 15:390–399.  https://doi.org/10.2174/1389202915666140716175634 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Srivastava D, Olson EN (2000) A genetic blueprint for cardiac development. Nature 407:221–226.  https://doi.org/10.1038/35025190 CrossRefPubMedGoogle Scholar
  5. 5.
    Bruneau BG (2008) The developmental genetics of congenital heart disease. Nature 51:943–948.  https://doi.org/10.1038/nature06801 CrossRefGoogle Scholar
  6. 6.
    Nemer M (2008) Genetic insights into normal and abnormal heart development. Cardiovasc Pathol 17:48–54.  https://doi.org/10.1016/j.carpath.2007.06.005 CrossRefPubMedGoogle Scholar
  7. 7.
    Dorn C, Grunert M, Sperling SR (2014) Application of high-throughput sequencing for studying genomic variations in congenital heart disease. Brief Funct Genomics 13:51–65.  https://doi.org/10.1093/bfgp/elt040 CrossRefPubMedGoogle Scholar
  8. 8.
    Andreassi MG, Della Corte A (2016) Genetics of bicuspid aortic valve aortopathy. Curr Opin Cardiol 31:585–592.  https://doi.org/10.1097/HCO.0000000000000328 CrossRefPubMedGoogle Scholar
  9. 9.
    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, Sinke RJ (2013) Targeted next-generation sequencing can replace Sanger sequencing in clinical diagnostics. Hum Mutat 34:1035–1042.  https://doi.org/10.1002/humu.22332 CrossRefPubMedGoogle Scholar
  10. 10.
    Blue GM, Kirk EP, Giannoulatou E, Dunwoodie SL, Ho JW, Hilton DC, White SM, Sholler GF, Harvey RP, Winlaw DS (2014) Targeted next-generation sequencing identified pathogenic variants in familial congenital heart disease. J Am Coll Cardiol 64:2498–2506.  https://doi.org/10.1016/j.jacc.2014.09.048 CrossRefPubMedGoogle Scholar
  11. 11.
    Jia Y, Louw JJ, Breckpot J, Callewaert B, Barrea C, Sznajer Y, Gewillig M, Souche E, Dehaspe L, Vermeesch JR, Lambrechts D, Devriendt K, Corveleyn A (2015) The diagnostic value of next generation sequencing in familial nonsyndromic congenital heart defects. Am J Med Genet A 167A:1822–1829.  https://doi.org/10.1002/ajmg.a.37108 CrossRefPubMedGoogle Scholar
  12. 12.
    Yagi H, Furutani Y, Hamada H, Sasaki T, Asakawa S, Minoshima S, Ichida F, Joo K, Kimura M, Imamura S, Kamatani N, Momma K, Takao A, Nakazawa M, Shimizu N, Matsuoka R (2003) Role of TBX1 in human del22q11.2 syndrome. Lancet 362:1366–1373.  https://doi.org/10.1016/S0140-6736(03)14632-6 CrossRefPubMedGoogle Scholar
  13. 13.
    Gong W, Gottlieb S, Collins J, Blescia A, Dietz H, Goldmuntz E, McDonald-McGinn DM, Zackai EH, Emanuel BS, Driscoll DA, Budarf ML (2001) Mutation analysis of TBX1 in non-deleted patients with features of DGS/VCFS or isolated cardiovascular defects. J Med Genet 38:E45.  https://doi.org/10.1136/jmg.38.12.e45 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Zweier C, Sticht H, Aydin-Yaylagül I, Campbell CE, Rauch A (2007) Human TBX1 missense mutations cause gain of function resulting in the same phenotype as 22q11.2 deletions. Am J Hum Genet 80:510–517.  https://doi.org/10.1086/511993 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Cabuk F, Karabulut HG, Tuncali T, Karademir S, Bozdayi M, Tükün A (2007) TBX1 gene mutation screening in patients with non-syndromic Fallot tetralogy. Turk J Pediatr 49:61–68PubMedGoogle Scholar
  16. 16.
    Rauch R, Hofbeck M, Zweier C, Koch A, Zink S, Trautmann U, Hoyer J, Kaulitz R, Singer H, Rauch A (2009) Comprehensive genotype-phenotype analysis in 230 patients with Tetralogy of Fallot. J Med Genet 47:321–331.  https://doi.org/10.1136/jmg.2009.070391 CrossRefPubMedGoogle Scholar
  17. 17.
    Xu YJ, Chen S, Zhang J, Fang SH, Guo QQ, Wang J, Fu QH, Li F, Xu R, Sun K (2014) Novel TBX1 loss-of-function mutation causes isolated conotruncal heart defects in Chinese patients without 22q11.2 deletion. BMC Med Genet 15:78.  https://doi.org/10.1186/1471-2350-15-78 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Griffin HR, Töpf A, Glen E, Zweier C, Stuart AG, Parsons J, Peart I, Deanfield J, O’Sullivan J, Rauch A, Scambler P, Burn J, Cordell HJ, Keavney B, Goodship JA (2010) Systematic survey of variants in TBX1 in non-syndromic tetralogy of Fallot identifies a novel 57 bp deletion that reduces transcriptional activity but finds no evidence for association with common variants. Heart 96:1651–1655.  https://doi.org/10.1136/hrt.2010.200121 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Goldmuntz E, Geiger E, Benson DW (2001) NKX2.5 mutations in patients with tetralogy of Fallot. Circulation 104:2565–2568.  https://doi.org/10.1161/hc4601.098427 CrossRefPubMedGoogle Scholar
  20. 20.
    Akçaboy MI, Cengiz FB, Inceoğlu B, Uçar T, Atalay S, Tutar E, Tekin M (2008) The effect of p.Arg25Cys alteration in NKX2–5 on conotruncal heart anomalies: mutation or polymorphism? Pediatr Cardiol 29:126–129.  https://doi.org/10.1007/s00246-007-9058-2 CrossRefPubMedGoogle Scholar
  21. 21.
    Bottillo I, D’Angelantonio D, Caputo V, Paiardini A, Lipari M, De Bernardo C, Giannarelli D, Pizzuti A, Majore S, Castori M, Zachara E, Re F, Grammatico P (2015) Molecular analysis of sarcomeric and non-sarcomeric genes in patients with hypertrophic cardiomyopathy. Gene 577:227–235.  https://doi.org/10.1016/j.gene.2015.11.048 CrossRefPubMedGoogle Scholar
  22. 22.
    D’Alessandro LC, Al Turki S, Manickaraj AK, Manase D, Mulder BJ, Bergin L, Rosenberg HC, Mondal T, Gordon E, Lougheed J, Smythe J, Devriendt K, Bhattacharya S, Watkins H, Bentham J, Bowdin S, Hurles ME, Mital S (2016) Exome sequencing identifies rare variants in multiple genes in atrioventricular septal defect. Genet Med 18:189–198.  https://doi.org/10.1038/gim.2015.60 CrossRefPubMedGoogle Scholar
  23. 23.
    Chung IM, Rajakumar G (2016) Genetics of Congenital Heart Defects: The NKX2-5 Gene, a Key Player. Genes (Basel) 23:7.  https://doi.org/10.1038/gim.2015.60 Google Scholar
  24. 24.
    Lopes LR, Zekavati A, Syrris P, Hubank M, Giambartolomei C, Dalageorgou C, Jenkins S, McKenna W; Uk10k Consortium., Plagnol V, Elliott PM (2013) Genetic complexity in hypertrophic cardiomyopathy revealed by high-throughput sequencing. J Med Genet 50:228–239.  https://doi.org/10.1136/jmedgenet-2012-101270 CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Marian AJ (2012) Challenges in medical applications of whole exome/genome sequencing discoveries. Trends Cardiovasc Med 22:219–223.  https://doi.org/10.1016/j.tcm.2012.08.001 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Melum E, May S, Schilhabel MB, Thomsen I, Karlsen TH, Rosenstiel P, Schreiber S, Franke A (2010) SNP discovery performance of two second-generation sequencing platforms in the NOD2 gene region. Hum Mutat 31:875–885.  https://doi.org/10.1002/humu.21276 CrossRefPubMedGoogle Scholar
  27. 27.
    Sivakumaran TA, Husami A, Kissell D, Zhang W, Keddache M, Black AP, Tinkle BT, Greinwald JH Jr, Zhang K (2013) Performance evaluation of the next-generation sequencing approach for molecular diagnosis of hereditary hearing loss. Otolaryngol Head Neck Surg 148:1007–1016.  https://doi.org/10.1177/0194599813482294 CrossRefPubMedGoogle Scholar
  28. 28.
    Zaidi S, Choi M, Wakimoto H, Ma L, Jiang J, Overton JD, Romano-Adesman A, Bjornson RD, Breitbart RE, Brown KK, Carriero NJ, Cheung YH, Deanfield J, DePalma S, Fakhro KA, Glessner J, Hakonarson H, Italia MJ, Kaltman JR, Kaski J, Kim R, Kline JK, Lee T, Leipzig J, Lopez A, Mane SM, Mitchell LE, Newburger JW, Parfenov M, Pe’er I, Porter G, Roberts AE, Sachidanandam R, Sanders SJ, Seiden HS, State MW, Subramanian S, Tikhonova IR, Wang W, Warburton D, White PS, Williams IA, Zhao H, Seidman JG, Brueckner M, Chung WK, Gelb BD, Goldmuntz E, Seidman CE, Lifton RP (2013) De novo mutations in histone-modifying genes in congenital heart disease. Nature 498:220–223.  https://doi.org/10.1038/nature12141 CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Soemedi R, Wilson IJ, Bentham J, Darlay R, Töpf A, Zelenika D, Cosgrove C, Setchfield K, Thornborough C, Granados-Riveron J, Blue GM, Breckpot J, Hellens S, Zwolinkski S, Glen E, Mamasoula C, Rahman TJ, Hall D, Rauch A, Devriendt K, Gewillig M, O’ Sullivan J, Winlaw DS, Bu’Lock F, Brook JD, Bhattacharya S, Lathrop M, Santibanez-Koref M, Cordell HJ, Goodship JA, Keavney BD (2012) Contribution of global rare copy-number variants to the risk of sporadic congenital heart disease. Am J Hum Genet 91:489–501.  https://doi.org/10.1016/j.ajhg.2012.08.003 CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Glessner JT, Bick AG, Ito K, Homsy JG, Rodriguez-Murillo L, Fromer M, Mazaika E, Vardarajan B, Italia M, Leipzig J, DePalma SR, Golhar R, Sanders SJ, Yamrom B, Ronemus M, Iossifov I, Willsey AJ, State MW, Kaltman JR, White PS, Shen Y, Warburton D, Brueckner M, Seidman C, Goldmuntz E, Gelb BD, Lifton R, Seidman J, Hakonarson H, Chung WK (2014) Increased frequency of de novo copy number variants in congenital heart disease by integrative analysis of single nucleotide polymorphism array and exome sequence data. Circ Res 115:884–896.  https://doi.org/10.1161/CIRCRESAHA.115.304458 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Silvia Pulignani
    • 1
  • Cecilia Vecoli
    • 1
  • Andrea Borghini
    • 1
  • Ilenia Foffa
    • 1
  • Lamia Ait-Alì
    • 1
  • Maria Grazia Andreassi
    • 1
  1. 1.CNR Institute of Clinical PhysiologyPisaItaly

Personalised recommendations