Biallelic mutations in ACO2, encoding the mitochondrial aconitase 2, have been identified in individuals with neurodegenerative syndromes, including infantile cerebellar retinal degeneration and recessive optic neuropathies (locus OPA9). By screening European cohorts of individuals with genetically unsolved inherited optic neuropathies, we identified 61 cases harbouring variants in ACO2, among whom 50 carried dominant mutations, emphasizing for the first time the important contribution of ACO2 monoallelic pathogenic variants to dominant optic atrophy. Analysis of the ophthalmological and clinical data revealed that recessive cases are affected more severely than dominant cases, while not significantly earlier. In addition, 27% of the recessive cases and 11% of the dominant cases manifested with extraocular features in addition to optic atrophy. In silico analyses of ACO2 variants predicted their deleterious impacts on ACO2 biophysical properties. Skin derived fibroblasts from patients harbouring dominant and recessive ACO2 mutations revealed a reduction of ACO2 abundance and enzymatic activity, and the impairment of the mitochondrial respiration using citrate and pyruvate as substrates, while the addition of other Krebs cycle intermediates restored a normal respiration, suggesting a possible short-cut adaptation of the tricarboxylic citric acid cycle. Analysis of the mitochondrial genome abundance disclosed a significant reduction of the mitochondrial DNA amount in all ACO2 fibroblasts. Overall, our data position ACO2 as the third most frequently mutated gene in autosomal inherited optic neuropathies, after OPA1 and WFS1, and emphasize the crucial involvement of the first steps of the Krebs cycle in the maintenance and survival of retinal ganglion cells.

Autosomal dominant optic atrophy is one of the most common inherited optic neuropathies. This disease is genetically heterogeneous, but most cases are due to pathogenic variants in the OPA1 gene: depending on the population studied, 32-90% of cases harbor pathogenic variants in this gene. The aim of this study was to provide a comprehensive overview of the entire spectrum of likely pathogenic variants in the OPA1 gene in a large cohort of patients. Over a period of 20 years, 755 unrelated probands with a diagnosis of bilateral optic atrophy were referred to our laboratory for molecular genetic investigation. Genetic testing of the OPA1 gene was initially performed by a combined analysis using either single-strand conformation polymorphism or denaturing high performance liquid chromatography followed by Sanger sequencing to validate aberrant bands or melting profiles. The presence of copy number variations was assessed using multiplex ligation-dependent probe amplification. Since 2012, genetic testing was based on next-generation sequencing platforms. Genetic screening of the OPA1 gene revealed putatively pathogenic variants in 278 unrelated probands which represent 36.8% of the entire cohort. A total of 156 unique variants were identified, 78% of which can be considered null alleles. Variant c.2708_2711del/p.(V903Gfs*3) was found to constitute 14% of all disease-causing alleles. Special emphasis was placed on the validation of splice variants either by analyzing cDNA derived from patients´ blood samples or by heterologous splice assays using minigenes. Splicing analysis revealed different aberrant splicing events, including exon skipping, activation of exonic or intronic cryptic splice sites, and the inclusion of pseudoexons. Forty-eight variants that we identified were novel. Nine of them were classified as pathogenic, 34 as likely pathogenic and five as variant of uncertain significance. Our study adds a significant number of novel variants to the mutation spectrum of the OPA1 gene and will thereby facilitate genetic diagnostics of patients with suspected dominant optic atrophy.