Targeted sequence capture and GS-FLX Titanium sequencing of 23 hypertrophic and dilated cardiomyopathy genes: implementation into diagnostics

J Med Genet. 2013 Sep;50(9):614-26. doi: 10.1136/jmedgenet-2012-101231. Epub 2013 Jun 19.

Abstract

Background: Genetic evaluation of cardiomyopathies poses a challenge. Multiple genes are involved but no clear genotype-phenotype correlations have been found so far. In the past, genetic evaluation for hypertrophic (HCM) and dilated (DCM) cardiomyopathies was performed by sequential screening of a very limited number of genes. Recent developments in sequencing have increased the throughput, enabling simultaneous screening of multiple genes for multiple patients in a single sequencing run.

Objective: Development and implementation of a next generation sequencing (NGS) based genetic test as replacement for Sanger sequencing.

Methods and results: In order to increase the number of genes that can be screened in a shorter time period, we enriched all exons of 23 of the most relevant HCM and DCM related genes using on-array multiplexed sequence capture followed by massively parallel pyrosequencing on the GS-FLX Titanium. After optimisation of array based sequence capture it was feasible to reliably detect a large panel of known and unknown variants in HCM and DCM patients, whereby the unknown variants could be confirmed by Sanger sequencing.

Conclusions: The rate of detection of (pathogenic) variants in both HCM and DCM patients was increased due to a larger number of genes studied. Array based target enrichment followed by NGS showed the same accuracy as Sanger sequencing. Therefore, NGS is ready for implementation in a diagnostic setting.

Keywords: Cardiomyopathy; Diagnostics; Genetics; Molecular genetics.

MeSH terms

  • Adult
  • Aged
  • Cardiomegaly / genetics*
  • Cardiomyopathy, Dilated / genetics
  • Female
  • Genetic Testing / methods*
  • Humans
  • Male
  • Middle Aged
  • Polymorphism, Single Nucleotide
  • Sequence Alignment
  • Sequence Analysis, DNA / methods*
  • Titanium / chemistry*

Substances

  • Titanium