A Rapid, High-Quality, Cost-Effective, Comprehensive and Expandable Targeted Next-Generation Sequencing Assay for Inherited Heart Diseases

Abstract

Rationale Thousands of mutations across more than 50 genes have been implicated in inherited cardiomyopathies. However, options for sequencing this rapidly evolving gene set are limited as many sequencing services and off-the-shelf kits suffer from slow turnaround, inefficient capture of genomic DNA, and/or high cost. Furthermore, customization of these assays to cover emerging targets and to suit individual needs is often expensive and time-consuming. Objective We sought to develop a custom high throughput, clinical-grade next generation sequencing (NGS) assay for detecting cardiac disease gene mutations with improved accuracy, flexibility, turnaround, and cost. Methods and Results We employed double-stranded probes (complementary long “padlock” probes (cLPPs)), an inexpensive and customizable capture technology, to efficiently capture and amplify the entire coding region and flanking intronic and regulatory sequences of 88 genes and 40 microRNAs (miRNA) associated with inherited cardiomyopathies, congenital heart disease (CHD), and cardiac development. Multiplexing 11 samples per sequencing run resulted in a mean base coverage of 420, of which 97% had >20× coverage and >99% were concordant with known heterozygous single nucleotide polymorphisms (SNPs). The assay correctly detected germline variants in 24 individuals and revealed several polymorphic regions in miR-499. Total run time was three days at an approximate cost of $100 per sample. Conclusions Accurate, high throughput detection of mutations across numerous cardiac genes is achievable with cLPP technology. Moreover, this format allows facile insertion of additional probes as more cardiomyopathy and CHD genes are discovered, giving researchers a powerful new tool for DNA mutation detection and discovery.

New Management Strategies in Heart Failure

“…134 Increasing availability of high throughput, affordable next generation sequencing platforms will promote investigation of these theories with subsequent testing of newly discovered genes/mutations in patient-specific models. 135 The therapeutic implications of being able to deeply phenotype and target research and subsequent pharmacological therapy to at-risk individuals or those in early stages of disease will be an exciting frontier of investigation and treatment in the coming years.…”

Circulation Research “In This Issue” Anthology

“…cLPP NGS Assay for Inherited Heart Diseases (p 603) 46 Wilson et al unveil a high-quality, fast, flexible and cheap way to screen for heart disease related mutations.…”

Making a heart: advances in understanding the mechanisms of cardiac development

“…Wilson et al . [30] reported ‘a rapid, high-quality cost-effective comprehensive and expandable targeted next-generation sequencing assay for inheritable heart disease’ with a total run time of 3 days and cost of $100 per sample. The expandability of the assay allows for new probes to be added as they are discovered for cardiomyopathies and perhaps one day for congenital heart defects.…”