Novel CNKSR2 Splice Variant Shown to Skip Exon 16 in Boy with Epileptic Encephalopathy

A previously unreported de novo variant in the CNKSR2 gene has been functionally characterized for the first time at a noncanonical splice-site position, according to a study published in BMC Medical Genomics. The variant, NM_014927.5:c.1831–3 C>G, was identified in a 7-year-old boy with developmental delay, severe language impairment, intellectual disability, and developmental and epileptic encephalopathy. Whole-exome sequencing revealed the hemizygous change, and Sanger sequencing confirmed it was absent from both parents, establishing its de novo origin on the X chromosome.

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 make up an important proportion of pathogenic CNKSR2 variants, all previously described intronic variants had affected canonical splice-site nucleotides. The pathogenic significance of variants at noncanonical splice-site positions has remained difficult to establish without experimental evidence, a gap this study set out to address by determining the molecular consequence of the newly found intronic variant.

To predict the splicing effect of c.1831–3 C>G, the researchers used two in silico tools, MaxEntScan and SpliceAI. Both predicted disruption of the splice acceptor site upstream of exon 16. Because computational predictions alone cannot establish pathogenicity at noncanonical positions, the team proceeded to experimental validation using two independent minigene splicing assays, based on the pcMINI and pcDNA3.1 systems, carried out in HeLa and HEK293T cells.

The functional results were consistent across both minigene systems. In the mutant constructs, skipping of exon 16 was markedly increased, whereas the wild-type constructs predominantly produced the correctly spliced transcript. Aberrant transcripts were characterized by RT-PCR followed by Sanger sequencing. The loss of exon 16 shifts the reading frame and introduces a premature termination codon, predicted to yield a truncated protein designated p.Asp611CysfsTer8, a molecular outcome consistent with a loss-of-function mechanism.

On the basis of these functional data, the variant was reclassified from a variant of uncertain significance to likely pathogenic. The authors report that this is the first functional characterization of a CNKSR2 variant at a noncanonical splice-site position, demonstrating that c.1831–3 C>G causes markedly increased exon 16 skipping and loss of normal protein function. The finding expands the known mutational spectrum of CNKSR2 and supports the view that aberrant splicing is a major pathogenic mechanism underlying CNKSR2-related neurodevelopmental disorders.

The study also carries a clinical dimension. Following the introduction and dose titration of perampanel, an antiseizure medication, the patient’s seizure duration and frequency were substantially reduced, although seizures were not completely eliminated. This observation concerns a single patient and reflects a treatment response rather than a controlled trial result, but it documents the clinical course associated with this specific molecular diagnosis.

Beyond the individual case, the authors highlight the value of functional RNA assays for interpreting noncanonical intronic variants encountered in clinical genetic testing. Variants falling outside canonical splice dinucleotides are frequently classified as uncertain because in silico prediction alone is insufficient evidence of pathogenicity. Minigene assays that directly demonstrate aberrant splicing can therefore provide the experimental evidence needed to move such variants toward a pathogenic classification, with direct consequences for genetic counseling and diagnosis in families affected by X-linked neurodevelopmental conditions.

Subject of Research: Functional characterization of a novel noncanonical splice-site variant in CNKSR2 causing exon 16 skipping in X-linked syndromic intellectual developmental disorder

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

Article References: Dong, X., Peng, B., He, R., Dong, Y., Meng, Y., & Cai, X. (2026). A novel splice-site variant in CNKSR2 causes exon 16 skipping and X-linked syndromic intellectual developmental disorder. BMC Medical Genomics. https://doi.org/10.1186/s12920-026-02493-5

Image Credits: AI Generated

DOI: 10.1186/s12920-026-02493-5

Keywords: CNKSR2, splice-site variant, exon 16 skipping, X-linked intellectual disability, developmental and epileptic encephalopathy, minigene assay, whole-exome sequencing, loss of function, perampanel, variant reclassification, BMC Medical Genomics, novel