Splicing variants in DEPDC5-related epilepsies: From functional characterization to correction

This study aims to investigate the role of splicing variants in the DEPDC5 gene, which is commonly associated with familial focal epilepsies. Although heterozygous germline variants in genes encoding components of the GAP activity toward Rags 1 (GATOR1) complex (DEPDC5, NPRL2, and NPRL3) have been frequently identified in these cases, the effects of most previously identified variants on splicing remain unstudied. We focused on analyzing both intronic and exonic splicing variants and developing a potential correction strategy.

Gene panel, whole-exome sequencing (WES), or whole-genome sequencing (WGS) was used to identify pathogenic variants in four familial cases with DEPDC5-related epilepsy, whereas RNA analysis evaluated their impact on splicing. For seven previously described variants, a functional analysis using a minigene expression system assessed their effects on splicing. A correction system using modified small nuclear RNAs (snRNAs) was developed to target one of the identified splicing variants.

Two canonical splice-site variants, missense and synonymous variants, were identified in DEPDC5 across affected families. Pathogenicity was confirmed for the exonic variants, and the molecular mechanisms of splicing disruption were elucidated for all four variants. Analysis of previously published DEPDC5 variants revealed that 13.6% of reported single nucleotide variants may affect splicing, including both intronic and exonic variants. Notably, approximately half of non-canonical intronic splice region variants were predicted to have no impact on splicing, indicating a high likelihood of misannotation. Five missense and two nonsense previously reported DEPDC5 variants were functionally analyzed for their effect on splicing, revealing diverse mechanisms such as activation of cryptic splice sites and disruption of exonic splicing enhancers. For the c.3264G>A patient variant, a correction system using modified snRNAs successfully rescued normal splicing.

This study enhances understanding of splicing variants in the DEPDC5 gene and their role in focal epilepsies. The development of a correction system using modified snRNAs represents a novel therapeutic approach, laying the groundwork for personalized treatment strategies.