A study of 21 noncanonical CNGB3 gene variants found that 16 disrupted RNA splicing, prompting the reclassification of most as disease causing. The findings are expected to enhance genetic diagnosis and clarify eligibility for gene therapy in achromatopsia.
The CNGB3 gene encodes a subunit of a cone photoreceptor channel essential for color vision. Mutations in this gene are a major cause of autosomal recessive achromatopsia, a rare inherited retinal disorder marked by poor color discrimination, low visual acuity, and photosensitivity.
Noncanonical variants—those located outside the conserved ± 1 or 2 splice site positions—are challenging to interpret and often labeled as variants of uncertain significance (VUS). This classification limits their clinical and therapeutic utility.
To determine the functional effects of these variants, the research team used vitro minigene splice assays in cultured cells, followed by cDNA sequencing and fragment analysis.
“Twelve of fourteen (86%) variants initially categorized as VUS were upgraded to likely pathogenic or pathogenic, enhancing the clinical utility of the genetic data,” said lead study author Katharina Rawnsley, of the Centre for Ophthalmology at the University Hospital Tübingen in Germany, and colleagues.
The most common aberrant splicing events included exon skipping, activation of cryptic splice sites, and pseudoexon inclusion. One deep intronic variant led to a 98-base pair pseudoexon insertion, whereas another produced a 32-base pair inclusion. Several variants initially classified as synonymous or missense were shown to disrupt splicing, indicating their primary effect was at the RNA level rather than through amino acid substitution.
Three variants yielded only small amounts of aberrant transcripts. These were categorized as hypomorphic and were not sufficient to support reclassification.
The researchers also evaluated the accuracy of four splicing prediction tools. SpliceAI showed the highest concordance with experimental results (95%), followed by TraP Score (90%). MaxEntScan and NNSplice were less reliable.
Prior to the functional analysis, 14 of the 21 variants were classified as VUS. Following experimental validation, seven were reclassified as likely pathogenic and five as pathogenic. Three variants previously deemed likely pathogenic were upgraded to pathogenic. Two remained VUS, and two were confirmed as likely benign. These results underscored the importance of functional assays in supplementing in silico predictions and improving variant interpretation.
The study highlighted the role of functional splice assays in resolving ambiguous genetic findings, particularly in inherited retinal disorders where therapeutic options depend on definitive molecular diagnoses.
No conflicts of interest were reported.
Source: The Journal of Pathology
