Whole gene sequencing identifies deep-intronic variants with potential functional impact in patients with hypertrophic cardiomyopathy

PLoS One. 2017 Aug 10;12(8):e0182946. doi: 10.1371/journal.pone.0182946. eCollection 2017.


Background: High throughput sequencing technologies have revolutionized the identification of mutations responsible for genetic diseases such as hypertrophic cardiomyopathy (HCM). However, approximately 50% of individuals with a clinical diagnosis of HCM have no causal mutation identified. This may be due to the presence of pathogenic mutations located deep within the introns, which are not detected by conventional sequencing analysis restricted to exons and exon-intron boundaries.

Objective: The aim of this study was to develop a whole-gene sequencing strategy to prioritize deep intronic variants that may play a role in HCM pathogenesis.

Methods and results: The full genomic DNA sequence of 26 genes previously associated with HCM was analysed in 16 unrelated patients. We identified likely pathogenic deep intronic variants in VCL, PRKAG2 and TTN genes. These variants, which are predicted to act through disruption of either splicing or transcription factor binding sites, are 3-fold more frequent in our cohort of probands than in normal European populations. Moreover, we found a patient that is compound heterozygous for a splice site mutation in MYBPC3 and the deep intronic VCL variant. Analysis of family members revealed that carriers of the MYBPC3 mutation alone do not manifest the disease, while family members that are compound heterozygous are clinically affected.

Conclusion: This study provides a framework for scrutinizing variation along the complete intronic sequence of HCM-associated genes and prioritizing candidates for mechanistic and functional analysis. Our data suggest that deep intronic variation contributes to HCM phenotype.

MeSH terms

  • AMP-Activated Protein Kinases / genetics
  • Cardiomyopathy, Hypertrophic / genetics*
  • Carrier Proteins / genetics
  • Child
  • Connectin / genetics
  • Female
  • Genetic Variation*
  • Humans
  • Introns*
  • Male
  • Middle Aged
  • Pedigree
  • Polymorphism, Single Nucleotide
  • Sequence Analysis, DNA
  • Vinculin / genetics


  • Carrier Proteins
  • Connectin
  • TTN protein, human
  • VCL protein, human
  • myosin-binding protein C
  • Vinculin
  • PRKAG2 protein, human
  • AMP-Activated Protein Kinases

Grant support

This work was supported by Fundação para a Ciência e Tecnologia and FEDER, Portugal (Scholarship BD/82023/2011 to R.M.A, grant EXPL/BIM-MEC/0201/2013 to S.M., grant Programa de Actividades Conjuntas n.º 016394 to M.C.-F, and LISBOA-01-0145-FEDER-007391), and an unrestricted grant from Merck Sharp & Dohme to M.C.-F. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.