RNA Analysis Enables Resolution and Reclassification of Reportedly Benign Synonymous Variants

ABSTRACT Synonymous variants can significantly impact protein levels and function, particularly through alterations in RNA processing. Consequently, variant classification must consider the broader impact on RNA splicing. We present three cases where synonymous variants were detected through exome sequencing. The variants in LARS1 and POLE were located at the last nucleotide of the exon (i.e., splice donor site), while the COL2A1 variant was located three nucleotides downstream of the splice acceptor site. Two variants were previously classified in ClinVar as “likely benign.” Segregation analysis confirmed segregation of the variants with the phenotype in available family members, and RNA studies revealed exon skipping in conserved regions of the protein, leading to reclassification of these variants as “likely pathogenic” and ultimately improving clinical management. These findings highlight the importance of incorporating RNA‐based testing to directly evaluate splicing effects and demonstrate the critical role of RNA analysis in the accurate interpretation of variants and their implications for diagnosis and treatment.