Functional Characterization and Pathogenicity Classification of PRRT2 Splice Variants in PRRT2-Related Disorders.

Objective: Paroxysmal kinesigenic dyskinesia (PKD) is the most common hereditary paroxysmal movement disorder. The PRRT2 gene is the first identified causative gene and accounts for the majority of PKD. In this study, we investigated the pathogenicity of PRRT2 variants in the splice regions.

Methods: Patients with clinically suspected PKD and no detectable pathogenic variants in the PRRT2 gene were included. Targeted next-generation sequencing technology was used to screen the full-length sequence of PRRT2. In silico analyses were performed on splice region variants identified in our cohort and compiled from the Human Gene Mutation Database (HGMD). Subsequently, a minigene system carrying these variants was constructed and introduced into HEK293T cells for functional assays to assess the pathogenicity.

Results: Fourteen PRRT2 variants were analyzed, including four identified in patients with clinically suspected PKD from our center and 10 retrieved from HGMD. These variants comprised 10 intronic variants, two synonymous variants, one deletion, and one missense variant. In silico predictions suggested that all variants, except for one deep intronic variant, had the potential to affect normal splicing. Functional assays showed that 11 PRRT2 variants, including missense and intronic variants, caused aberrant splicing events, such as exon skipping and intron retention. The two synonymous variants and one deep intronic variant exhibited no splicing abnormalities. Based on these results, five patients with PRRT2 variants previously classified as variants of uncertain significance can now be genetically diagnosed with PKD or other PRRT2-related disorders.

Interpretation: Combining in silico analyses with functional assays is essential for determining the pathogenicity of splice variants. It can help confirm the diagnosis of patients with clinically suspected PKD and other PRRT2-related disorders.