Unpredicted Aberrant Splicing Products Identified in Postmortem Sudden Cardiac Death Samples

Molecular screening for pathogenic mutations in sudden cardiac death (SCD)-related genes is common practice for SCD cases. However, test results may lead to uncertainty because of the identification of variants of unknown significance (VUS) occurring in up to 70% of total identified variants due to a lack of experimental studies. Genetic variants affecting potential splice site variants are among the most difficult to interpret. The aim of this study was to examine rare intronic variants identified in the exonic flanking sequence to meet two main objectives: first, to validate that canonical intronic variants produce aberrant splicing; second, to determine whether rare intronic variants predicted as VUS may affect the splicing product. To achieve these objectives, 28 heart samples of cases of SCD carrying rare intronic variants were studied. Samples were analyzed using 85 SCD genes in custom panel sequencing. Our results showed that rare intronic variants affecting the most canonical splice sites displayed in 100% of cases that they would affect the splicing product, possibly causing aberrant isoforms. However, 25% of these cases (1/4) showed normal splicing, contradicting the in silico results. On the contrary, in silico results predicted an effect in 0% of cases, and experimental results showed >20% (3/14) unpredicted aberrant splicing. Thus, deep intron variants are likely predicted to not have an effect, which, based on our results, might be an underestimation of their effect and, therefore, of their pathogenicity classification and family members' follow-up.

Automated summary

This study examined rare intronic variants in SCD cases to determine their effect on splicing. The results showed that variants affecting canonical splice sites almost always caused aberrant splicing, but a small percentage of cases still showed normal splicing. In contrast, in silico predictions failed to accurately predict the effects, with unexpected aberrant splicing occurring in some cases. These findings suggest that deep intronic variants may have a greater impact than previously thought, highlighting the importance of accurate classification and follow-up for family members.