The m.586G>A variant in MT-TF has been reported in two unrelated individuals with primary mitochondrial disease (PS4_supporting; PMIDs: 21060018, 31463198). One individual was a 57-year-old woman with hearing loss onset in her mid 40s that progressed to profound hearing loss in her 50s, myopathy and progressive exercise intolerance with reduced mobility and falls onset in her late 40s, and spasticity, facial grimacing, dementia, and psychiatric manifestations in her mid 50s (PMID: 21060018). Brain imaging showed generalized cerebral, cerebellar, and brainstem atrophy. Cerebrospinal fluid lactate was elevated. Muscle biopsy showed ragged red fibers and COX-deficient fibers. The variant was present in muscle at 85% heteroplasmy, urinary epithelia at 29%, and blood at 3%. Her mother had died however DNA from paraffin-embedded breast tissue was obtained and revealed the variant at 13% heteroplasmy. The other individual was a 14-year-old boy with exercise intolerance and muscle weakness that followed a stable course (PMID: 31463198). Brain imaging was normal. Lactate was elevated in blood. Muscle biopsy showed ragged red fibers, COX-negative fibers, and mitochondrial respiratory chain complexes I, III, and IV deficiency. The variant was present at >90% in muscle, 40% in urinary epithelia, and was present at trace levels in blood. The variant was undetectable in his mother’s blood and urinary epithelia (PM6_supporting; PMID: 31463198). This variant is absent in the GenBank dataset, Helix dataset, and gnomAD v3.1.2 (PM2_supporting). The computational predictor MitoTIP suggests this variant is pathogenic (89.7 percentile) and HmtVAR predicts it to be pathogenic score of 0.6 (PP3). Single fiber testing showed higher levels of the variant in COX-deficient fibers (99.4±0.2%, n=11) than in COX-positive fibers (72.1±8.5%, n=9; p<0.001; PS3_supporting, PMID: 21060018). 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 June 10, 2024. Mitochondrial DNA-specific ACMG/AMP criteria applied (PMID: 32906214): PS4_supporting, PM6_supporting, PP3, PS3_supporting, PM2_supporting.