Unpredicted Aberrant Splicing Products Identified in Postmortem Sudden Cardiac Death Samples

Area of Science:

• Genetics
• Molecular Biology
• Cardiology

Background:

• Molecular screening for sudden cardiac death (SCD) genes frequently identifies variants of unknown significance (VUS), complicating interpretation.
• Genetic variants affecting splice sites are particularly difficult to classify, hindering accurate diagnosis and family follow-up.

Purpose of the Study:

• To investigate rare intronic variants in exonic flanking sequences in sudden cardiac death (SCD) cases.
• To validate if canonical intronic variants cause aberrant splicing and to assess the impact of VUS-predicted intronic variants on splicing products.

Main Methods:

• Analysis of 28 heart samples from SCD cases with rare intronic variants.
• Custom panel sequencing of 85 SCD-related genes.
• Experimental validation of splicing alterations caused by intronic variants.

Main Results:

• Rare intronic variants at canonical splice sites were predicted to affect splicing in 100% of cases, but 25% showed normal splicing.
• In silico predictions underestimated the impact, with experimental results revealing over 20% of unpredicted aberrant splicing from deep intronic variants.
• A significant portion of intronic variants, especially deep intronic ones, may be misclassified due to underestimation of their splicing effects.

Conclusions:

• Current in silico predictions may underestimate the pathogenic potential of intronic variants in sudden cardiac death (SCD) cases.
• Aberrant splicing caused by intronic variants, including those predicted as VUS, requires further experimental validation for accurate pathogenicity classification.
• Re-evaluation of intronic variant interpretation is crucial for improving diagnostic yield and family risk assessment in sudden cardiac death (SCD).