An increasing number of gene-specific therapies for inherited retinal dystrophies are being developed and applied in clinical trials. It is therefore crucial to ensure that disease in such patients is truly (and exclusively) caused by mutations in the gene targeted by the therapy. The current practice for the selection of patients for gene therapeutic trials regarding their genotype or its validity is neither objective nor standardized. Recently, the American College of Medical Genetics & Genomics (ACMG) and the Association for Molecular Pathology have developed general guidelines for the interpretation of sequence variants in genetic diagnostics. However, there is a need to implement such recommendations in a standardized rule-based and expert-guided process, and thus to make an objective and reliable pathogenicity assessment for individual variants as well as for the actual genotype of individual patients as an essential criterion for the selection of patients in genotype-based therapies studies.

We want to use the ACMG scoring system for variants (and individual patient genotypes) as a proof-of-concept in the genes CNGA3 and CNGB3: Mutations in CNGA3 and CNGB3 are the major cause of autosomal  recessive inherited achromatopsia (ACHM), and clinical trials for gene replacement therapy for both genetic subtypes are currently being conducted at various sites. In fact the proposed project is a direct outcome of the experiences of the CNGA3-ACHM gene therapy study in Tübingen. We have genetic data from 971 patients whose disease is presumably caused by mutations in CNGA3 and CNGB3 and in this context have identified over 300 different allegedly pathogenic variants in CNGA3 and CNGB3. 50% of these variants are missense variants, but only about 1/3 of these variants have hitherto been functionally analysed in heterologous expression systems. CNGA3 and CNGB3 encode the cGMP-controlled (CNG) cation channel of the cone photoreceptors. We have established and validated an in vitro aequorin-based bioassay with which we want to test the functionality of CNG channels with previously uncharacterized, presumably disease-causing CNGA3 variants in a medium throughput format in HEK293T cells. We expect to deliver an objective functionally confirmed pathogenicity score for all CNGA3 variants observed in our patient cohort, literature and mutation databases, unequivocally identifying patients eligible for future gene therapy trials on ACHM.