Variant-specific RNA testing resolves variants of uncertain significance in exome testing

Background Variants of uncertain significance (VUS) are a substantial barrier to clinical care in genetics, and VUS that are suspected to affect splicing can be particularly difficult to resolve. We used exome sequencing (ES) followed by variant-specific RNA testing to resolve VUS in four separate cases resulting in reclassification to Likely Pathogenic and a positive molecular diagnosis in all cases. Results The variants in these cases include a homozygous variant in ATP6V0A2 in a trio exome case, heterozygous variants in SLC20A2 and in CSF1R in adult proband-only cases, and a heterozygous variant in COL1A1 in a family. All variants were associated with aberrant splicing resulting in loss of function or a major alteration to the protein sequence. Conclusions These findings demonstrate the power of targeted RNA testing for reclassifying VUS that have limited direct evidence across a broad range of indications for exome testing.