Functional evaluation allows ACMG/AMP-based re-classification of CNGA3 variants associated with achromatopsia.

PURPOSE:CNGA3 encoding the main subunit of the cyclic nucleotide-gated ion channel in cone photoreceptors is one of the major disease-associated genes for achromatopsia. Most CNGA3 variants are missense variants with the majority being functionally uncharacterized and therefore hampering genetic diagnosis. In light of potential gene therapy, objective variant pathogenicity assessment is essential. METHODS:We established a medium-throughput aequorin-based luminescence bioassay allowing mutant CNGA3 channel function assessment via quantification of CNGA3 channel-mediated calcium influx in a cell culture system, thereby enabling American College of Medical Genetics and Genomics/Association for Molecular Pathology-based variant re-classification. RESULTS:We provide functional read-out obtained for 150 yet uncharacterized CNGA3 missense substitutions of which 55 were previously categorized as variants of uncertain significance (VUS) identifying 25 as functionally normal and 125 as functionally abnormal. These data enabled the American College of Medical Genetics and Genomics/ Association for Molecular Pathology-based variant re-classification of 52/55 VUS as either benign, likely benign, or likely pathogenic reaching a VUS re-classification rate of 94.5%. CONCLUSION:Our aequorin-based bioassay allows functionally ensured clinical variant interpretation for 150 CNGA3 missense variants enabling and supporting VUS re-classification and assuring molecular diagnosis to patients affected by CNGA3-associated achromatopsia, hereby identifying patients eligible for future gene therapy trials on this disease.