Abstract:
The CNGA3 gene encodes the major subunit of the cyclic nucleotide-gated ion channel (CNG channel) in cone photoreceptors and is one of the major disease-causative genes for achromatopsia. Most CNGA3 variants are missense variants, the majority of which have not been functionally characterized, thereby significantly hampering the establishment of a definitive genetic diagnosis in achromatopsia patients. However, an objective assessment of the pathogenicity of CNGA3 variants is essential with regard to potential gene therapies.
The aim of this study was to: i. establish a CNGA3 variant database and to provide an expert-based assessment of sequence variants in the CNGA3 gene, ii. develop a medium-throughput bioassay to test the functionality of CNGA3 channels carrying functionally yet uncharacterized missense variants versus wild-type channels; and iii. to incorporate the obtained functional data into the existing ACMG/AMP classification enabling revision or confirmation of the classification.
In this doctoral thesis, the CNGA3 variant spectrum was substantially expanded to a total of 316 sequence alterations. From these, 244 variants could be interpreted as “likely disease-causing” following extensive in silico variant assessment, ACMG/AMP-based classification, and manual/expert-based variant categorization. The predominant variant type among the “likely disease-causing” variants were missense substitutions displaying the highest frequency of 66.8%.
Furthermore, I have developed an aequorin-based luminescence bioassay that enables the assessment of the function of mutated CNGA3 channels by quantifying the CNGA3 channel-mediated calcium influx in a cell culture system, thereby facilitating ACMG/AMP-based re-classification of these variants. Using this approach, I functionally analyzed a substantial number of 150 missense substitutions in the CNGA3 gene, 55 of which had previously been classified as variants of uncertain significance (VUS). Of the 150 missense substitutions analyzed, 25 exhibited channel function characteristics similar to those of the wild-type channels, and were considered “functionally normal”. In contrast, the remaining 125 missense substitutions displayed (severely) impaired or absent channel function, thus classifying them as “functionally abnormal”. These functional data enabled the ACMG/AMP-based re-classification of 52 out of 55 VUS as benign, likely benign, or likely pathogenic. This resulted in a VUS re-classification rate of 94.5%.
In summary, the expansion of the CNGA3 variant spectrum, functional evaluation, and subsequent ACMG/AMP-based variant re-classification presented in this doctoral thesis will greatly facilitate the clinical variant interpretation with respect to the analyzed CNGA3 missense variants ensuring a molecular genetic diagnosis for CNGA3-associated achromatopsia patients, hereby identifying patients eligible for future gene therapeutic trials and treatments.
Achromatopsia