The m.12294G>A variant in MT-TL2 has been reported in two unrelated individuals with primary mitochondrial disease. These two affected individuals had myopathy and ophthalmoplegia. Age of onset ranged from childhood to teens. Muscle biopsies revealed ragged red fibers, COX-deficient fibers, and combined respiratory chain enzyme activity deficiencies. The variant was identified in muscle in both individuals (one with the variant at 59.8% heteroplasmy and the other at 75% heteroplasmy) and was absent in other tissues tested (blood, urine, buccal, primary myoblasts; PS4_supporting, PMIDs: 14581685, 29052516). There are no large families reported in the medical literature to consider for evidence of segregation. The variant was confirmed to have occurred de novo in one of the reported individuals (absent in healthy mother’s muscle; PMID: 29052516, PM6_supporting). In silico predictors do not agree as the computational predictor MitoTIP suggests this variant is pathogenic (71.4 percentile) and HmtVAR predicts it to be likely polymorphic (0.15). This variant is absent in the Genbank dataset, Helix dataset and gnomAD v3.1.2 (PM2_supporting). Single fiber testing showed higher levels of the variant in COX-negative fibers (92.9% ± 7.4, n=10) than in COX-positive fibers (48.3% ± 22.19, n=18, p<0.00001; PS3_supporting, PMID: 14581685). In summary, this variant meets criteria to be classified as uncertain significance for primary mitochondrial disease inherited in a mitochondrial manner. This classification was approved by the NICHD/NINDS U24 ClinGen Mitochondrial Disease Variant Curation Expert Panel on October 24, 2022. Mitochondrial DNA-specific ACMG/AMP criteria applied (PMID: 32906214): PS4_supporting, PM6_supporting, PM2_supporting, PS3_supporting.