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


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.


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



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.


This research was supported by institutional funds.

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.


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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

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