Functional assays identified aberrant splicing in 9 of 13 variants, enabling the reclassification of 8 variants of uncertain significance to likely pathogenic and 1 to likely benign.
Do functional assays improve the classification of suspected splice-altering variants of uncertain significance in Mendelian channelopathies?
Functional assays using minigenes and iPSC-derived cardiomyocytes successfully reclassify splice-altering variants outside canonical splice sites in Brugada and long QT syndrome genes, improving diagnostic certainty.
BACKGROUND: are major causes of Brugada syndrome and the congenital long QT syndrome. While splice-altering variants lying outside 2-bp canonical splice sites can cause these diseases, their role remains poorly described. We implemented 2 functional assays to assess 12 recently reported putative splice-altering variants of uncertain significance and 1 likely pathogenic variant without functional data observed in Brugada syndrome and long QT syndrome probands. METHODS: We deployed minigene assays to assess the splicing consequences of 10 variants. Three variants incompatible with the minigene approach were introduced into control induced pluripotent stem cells by CRISPR genome editing. We differentiated cells into induced pluripotent stem cell-derived cardiomyocytes and studied splicing outcomes by reverse transcription-polymerase chain reaction. We used the American College of Medical Genetics and Genomics functional assay criteria (PS3/BS3) to reclassify variants. RESULTS: We identified aberrant splicing, with presumed disruption of protein sequence, in 8/10 variants studied using the minigene assay and 1/3 studied in induced pluripotent stem cell-derived cardiomyocytes. We reclassified 8 variants of uncertain significance to likely pathogenic, 1 variant of uncertain significance to likely benign, and 1 likely pathogenic variant to pathogenic. CONCLUSIONS: Functional assays reclassified splice-altering variants outside canonical splice sites in Brugada Syndrome- and long QT syndrome-associated genes.
O’Neill et al. (Wed,) conducted a other in Brugada syndrome and long QT syndrome (n=13). Functional assays (minigene assays and CRISPR-edited iPSC-CMs) was evaluated on Aberrant splicing and variant reclassification. Functional assays identified aberrant splicing in 9 of 13 variants, enabling the reclassification of 8 variants of uncertain significance to likely pathogenic and 1 to likely benign.
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