The m.14597A>G (p.I26T) variant in MT-ND6 has been reported in three unrelated individuals with primary mitochondrial disease (PS4_supporting; PMIDs: 31669237, 34045482, 26633545). Clinical features in affected individuals include Leber Hereditary Optic Neuropathy (LHON, PMID: 31669237, this 39-year-old man was in haplogroup T2 and heteroplasmy levels were 25% in blood, 76% in skin fibroblasts, and 87% in urine), Leigh syndrome and optic atrophy (PMID: 34045482, this was a Japanese boy with 91% heteroplasmy in skin fibroblasts), and dystonia, dysarthria and prominent perivascular spaces (PMID: 26633545; the heteroplasmy levels in this man were 49% although the tissue was not specified). The variant segregated with clinical features in at least one family (the variant was found at 66% in blood in the sister of the boy with Leigh syndrome and at 17% in his mother’s blood, however blood heteroplasmy was not reported for the proband; PMID: 34045482). The variant was reported to occur de novo in at least one case, however technical details and tissues tested were not provided (PMID: 26633545). This variant is present in population databases (absent in Mitomap GenBank sequences and gnomAD v3.1.2; one heteroplasmic occurrence in the Helix dataset; PM2_supporting). The computation predictor APOGEE predicts this variant to be deleterious (score 0.67 in APOGEE1 and 0.63 in APOGEE2; PP3). Cybrid studies support the functional impact of this variant (PS3_supporting; PMIDs: 34045482) as significantly reduced complex I activity, respiration rate, and ATP content in three cybrid fibroblast cell lines were demonstrated. Furthermore, complex I deficiency was noted in skin fibroblasts of an affected individual in whom nuclear DNA etiologies were excluded (PP4, PMID: 26741492). Complex I deficiency was also reported in muscle, however the activity of complex I was reported to be 0% in this tissue, raising concern for the validity of this result. A different amino acid change at this position has been reported (m.14596A>T, p.I26M), however this was classified as a variant of uncertain significance, precluding formal consideration of this as evidence of pathogenicity for the m.14597A>G (p.I26T) variant. In summary, this variant meets criteria to be classified as uncertain significance for primary mitochondrial disease however, after extensive discussion, this Expert Panel elected to modify the classification to likely pathogenic given the consistent biochemical phenotype, compelling functional validation, and that other nearby variants are highly concerning to be causative of primary mitochondrial disease. This classification was approved by the NICHD/NINDS U24 ClinGen Mitochondrial Disease Variant Curation Expert Panel on August 14, 2023. Mitochondrial DNA-specific ACMG/AMP criteria applied (PMID: 32906214): PM2_supporting, PS3_supporting, PS4_supporting, PP3, PP4.