New Approach for Detection of Normal Alternative Splicing Events and Aberrant Spliceogenic Transcripts with Long-Range PCR and Deep RNA Sequencing

SIMPLE SUMMARY: RNA splicing defects, caused by genetic variants, are a common molecular mechanism of disease. To detect variants that cause splicing impairment, mRNA-based studies must be performed. Classical mRNA assays are time-consuming, which is why we have validated a new reliable straightforward approach to detect normal alternative splicing events and also splicing aberrations. Using our approach, we were able to reclassify three variants of uncertain significance in NBN and STK11 genes, which is of great importance for a proper clinical management of the patients. ABSTRACT: RNA sequencing is a promising technique for detecting normal and aberrant RNA isoforms. Here, we present a new single-gene, straightforward 1-day hands-on protocol for detection of splicing alterations with deep RNA sequencing from blood. We have validated our method’s accuracy by detecting previously published normal splicing isoforms of STK11 gene. Additionally, the same technique was used to provide the first comprehensive catalogue of naturally occurring alternative splicing events of the NBN gene in blood. Furthermore, we demonstrate that our approach can be used for detection of splicing impairment caused by genetic variants. Therefore, we were able to reclassify three variants of uncertain significance: NBN:c.584G>A, STK11:c.863-5_863-3delCTC and STK11:c.615G>A. Due to the simplicity of our approach, it can be incorporated into any molecular diagnostics laboratory for determination of variant’s impact on splicing.