Reliable and sensitive SCN5A high resolution melting curve analysis as a primary gene scanning assay for genetic diagnosis of Brugada syndrome

Brugada syndrome (BrS) is an autosomal dominant inherited primary cardiac arrhythmia in a structurally normal heart, with a propensity to sudden cardiac death. Genetic defects have mainly been attributed to mutations in the alpha-subunit of the sodium channel gene (SCN5A), which account for approximately 20% of cases. The clinical diagnosis is based on an anamnesis of the patient and a baseline and/or infusion electrocardiogram, which should be confirmed genetically by the mutation analysis of the SCN5A gene.
To allow a convenient and cost-effective diagnostic genetic testing for BrS a primary indirect gene scanning assay by high resolution melting curve analysis (HRMCA) of the SCN5A gene was developed.
To assess the sensitivity and specificity of the HRMCA assay over 100 clinically diagnosed BrS patients were analyzed in parallel by bidirectional cycle sequencing of the 28 SCN5A exons and by HRMCA analysis of 24 exons. Sensitivity of the HRMCA could be increased by using spike-DNA completely homozygous in the amplified regions and by discriminatory analysis of melting patterns with dual melting domains. All of the Sanger sequencing confirmed mutations and SNPs could be detected through HRMCA, with the exception of a deep intronic mutation lying 8 nucleotides downstream of the 3'end of the forward primer. Specificity of the assay met expectations.
This study demonstrates that SCN5A HRMCA analysis can be implemented as a cost-effective, high-throughput, user-friendly primary gene scanning method within the framework of the molecular diagnosis of BrS.