Targeted capture sequencing identifies mutations in a substantial proportion of prior genotype negative - phenotype positive patients with inherited primary arrhythmia syndrome or cardiomyopathy

Background: In inherited primary arrhythmia syndromes (PAS) and cardiomyopathies (CMP) the yield of genetic testing varies between 20% and 75% in different diseases. These numbers are mainly derived from studies evaluating only the most frequently affected genes. Next generation sequencing (NGS) allowed us to design and validate a panel of 75 PAS and CMP susceptibility genes for targeted capture and massive parallel sequencing. Purpose: We evaluated the additional yield of NGS based panel testing in PAS and CMP patients and determined if genetic retesting was worthwhile in previously genotype negative – phenotype positive probands. Methods: We examined the database of our hereditary heart disease clinic and selected patients with a clear phenotype who were genotype negative after genetic analysis of the main genes implicated in their specific phenotype. Targeted sequencing of the coding regions of 75 genes implicated in PAS and CMP was performed. Variant interpretation and classification was done according a stringent scoring system implementing different in-silico analyses, population frequencies and paralogous and orthologous conservation. Sanger sequencing was performed to confirm the presence of class 3 (variant of unknown significance), 4 (probably pathogenic) and 5 (pathogenic) variants. Co-segregation was done when DNA and clinical data of family members was available. Results: 96 patients were included: 25 with LQTS, 8 with BrS, 4 with CPVT, 1 with SSS and familial sudden death, 1 with AF, 6 with idiopathic VF, 42 with HCM, 7 with DCM, 1 with restrictive CMP and 1 with ARVC. A total of 41 variants of class 3, 4 and 5 were identified. Co-segregation was performed on 20 variants. Three class 3/4 variants were downgraded to a benign variant due to lack of co-segregation. In contrast, 12 were upgraded to class 4 or 5 after critical evaluation of published functional studies or co-segregation analysis. In total we identified 22 variants of class 3 and 16 mutations (class 4/5) in 15 patients, resulting in a genetic yield of 16% (14% in CMP and 18% in PAS). The initial detection failures had several causes: detection of a mutation in a new gene in 8 cases, allelic dropout with DHPLC in 2, not reported variant by an external lab in 2, functional reclassification in 1, heterozygous calling with sequencing failed in 1 and a wrong initial diagnosis in 1. Conclusion: Genetic retesting in robust PAS and CMP cases, who were genotype negative with older scanning techniques, resulted in a genetic diagnosis in up to 16% of the cases and clearly supports genetic retesting with NGS based panels.status: publishe