A novel splice-site variant in CNKSR2 causes exon 16 skipping and X-linked syndromic intellectual developmental disorder

Abstract

CNKSR2 -related neurodevelopmental disorder is an X-linked condition characterized by developmental delay, severe language impairment, intellectual disability, and early-onset epilepsy. Although splice-altering variants constitute an important proportion of pathogenic CNKSR2 variants, all previously described intronic variants have affected canonical splice-site nucleotides. The pathogenic significance of variants at noncanonical splice-site positions remains difficult to establish without experimental evidence. We aimed to determine the molecular consequence of a novel intronic CNKSR2 variant identified in a patient with developmental and epileptic encephalopathy. A 7-year-old boy with developmental delay, severe language impairment, intellectual disability, and developmental and epileptic encephalopathy underwent whole-exome sequencing followed by Sanger sequencing for familial segregation analysis. The splicing effect of the identified variant, NM_014927.5:c.1831–3 C > G (dn), was predicted using MaxEntScan and SpliceAI. Functional validation was performed using two independent minigene splicing assays (pcMINI and pcDNA3.1) in HeLa and HEK293T cells. Aberrant transcripts were characterized by RT-PCR and Sanger sequencing. Whole-exome sequencing identified a previously unreported de novo hemizygous CNKSR2 variant, NM_014927.5:c.1831–3 C > G. Bioinformatic analyses predicted disruption of the splice acceptor site upstream of exon 16. Both minigene systems consistently demonstrated markedly increased skipping of exon 16 in the mutant construct, whereas the wild-type construct predominantly produced the correctly spliced transcript. Exon 16 skipping resulted in a frameshift and premature termination codon (p.Asp611CysfsTer8), consistent with a loss-of-function mechanism. These functional data supported reclassification of the variant from a variant of uncertain significance to likely pathogenic. Following introduction and dose titration of perampanel, seizure duration and frequency were substantially reduced, although seizures were not completely eliminated. We provide the first functional characterization of a variant at a noncanonical splice-site position in CNKSR2 , demonstrating that c.1831–3 C > G causes markedly increased exon 16 skipping and loss of normal protein function. Our findings expand the mutational spectrum of CNKSR2 , strengthen the evidence that aberrant splicing is a major pathogenic mechanism underlying CNKSR2 -related neurodevelopmental disorders, and highlight the value of functional RNA assays for interpreting noncanonical intronic variants encountered in clinical genetic testing.