The m.12316G>A variant in MT-TL2 has been reported in three unrelated individuals with primary mitochondrial disease. The three individuals had chronic progressive external ophthalmoplegia (CPEO). Other features seen included peripheral neuropathy, myopathy, and exercise intolerance. Muscle biopsy in affected individuals revealed ragged red fibers, COX-negative fibers, and decreased activities of respiratory chain complexes I and IV. The levels of the variant in affected individuals ranged from 40-55.6% in muscle when reported. Levels in other tissues varied from undetectable to 10% (PS4_supporting; PMIDs: 23847141, 20163808, 18603265). There are no large families reported in the medical literature to consider for evidence of segregation. There are no reported de novo occurrences of this variant to our knowledge. The computational predictor MitoTIP suggests this variant impacts the function of this tRNA (83.7 percentile) as does HmtVar with a score of 0.6 (PP3). This variant is absent in the GenBank dataset, Helix dataset, and gnomAD v3.1.2 (PM2_supporting). Single fiber testing showed a higher degree of heteroplasmy in ragged red fibers (87.4% ± 8.5, n = 4) and COX-deficient fibers (59.7% ± 27.8, n = 4) compared to COX-positive muscle fibers (wild type mtDNA only, n = 6, Fig. 2). The difference between COX-positive and ragged red fibers was statistically significant (p<0.00001; PMID: 20163808; PS3_supporting). 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 February 27, 2023. Mitochondrial DNA-specific ACMG/AMP criteria applied (PMID: 32906214): PS4_supporting, PM2_supporting, PP3, PS3_supporting.