Targeted Capture and Massively Parallel Sequencing in Pediatric Cardiomyopathy: Development of Novel Diagnostics

Pediatric cardiomyopathy is a genetically heterogeneous disease associated with significant morbidity. Although identification of underlying etiology is important for management, therapy, and screening of at risk family members, molecular diagnosis is difficult due to the large number of causative genes, the high rate of private mutations, and cost. In this study, we aimed to define the genetic basis of pediatric cardiomyopathy and test a novel diagnostic tool using a custom targeted microarray coupled to massively parallel sequencing. Three patients with cardiomyopathy were screened using a custom NimbleGen sequence capture array containing 110 genes and providing 99.9% coverage of the exons of interest. The sensitivity and specificity was over 99% as determined by comparison to long-range polymerase chain reaction (PCR)- based massively parallel sequencing, Sanger sequencing of missense variants, and single nucleotide polymorphisms genotyping using the Illumina Infinium Omni1 array. Overall, 99.73% of the targeted regions were captured and sequenced at over 10x coverage, allowing reliable mutation calling in all patients. Analysis identified a total of 165 non-synonymous coding single nucleotide polymorphisms (cSNPs) of which 89 were unique and 14 were novel. On average, each patient had 4 cSNPs predicted to be pathogenic. In conclusion, we report a cardiomyopathy sequencing array that allows simultaneous assessment of 110 genes. Comparison of targeted sequence capture versus PCR-based enrichment methods demonstrates that the former is more sensitive and efficient. Array-based sequence capture technology followed by massively parallel sequencing is promising as a robust and comprehensive tool for genetic screening of cardiomyopathy. These results provide important information about genetic architecture and indicate that improved annotation of variants and interpretation of clinical significance, particularly in cases with multiple rare variants, are important for clinical practice.