Next-generation sequencing using microfluidic PCR enrichment for molecular autopsy.

Area of Science:

• Genetics
• Cardiology
• Molecular Biology

Background:

• Sudden arrhythmic death syndrome (SADS) poses a significant public health challenge.
• Molecular autopsy is crucial for identifying genetic causes of SADS.
• Low-cost, high-throughput technologies are needed to improve SADS genetic testing.

Purpose of the Study:

• To determine the mutation yield and clinical applicability of molecular autopsy in SADS.
• To validate and utilize next-generation sequencing (NGS) for SADS genetic analysis.
• To assess the cost-effectiveness and diagnostic accuracy of NGS in SADS.

Main Methods:

• Validated and optimized a Fluidigm Access Array PCR-enrichment with Illumina HiSeq 2000 NGS platform.
• Compared NGS with Sanger sequencing for major arrhythmia risk genes in 46 patients.
• Sequenced a large, multi-ethnic SADS cohort (197 cases) using the validated NGS platform.

Main Results:

• NGS demonstrated 100% sensitivity for pathogenic variants and high specificity for substitutions.
• The overall molecular yield in SADS cases was 5.1%, identifying 10 cases with pathogenic mutations.
• Positive predictive value for rare variants by NGS was 16.0%, requiring Sanger confirmation.

Conclusions:

• Molecular autopsy using NGS offers moderate diagnostic yield for SADS.
• The validated NGS approach is applicable for identifying genetic risk factors in SADS.
• Confirmatory Sanger sequencing remains necessary for mutational variants identified by NGS.