Figure 2. Detection and Functional Effects of the m.5789T>C Sequence Variant.

(A) Sequencing chromatograms from the patient's skeletal muscle and blood. Filled arrow indicates the heteroplasmic m.5789T>C variant, whereas empty arrows show the homoplasmic m.5785T>C variant. (B) Restriction fragment length polymorphism (RFLP) analysis of the patient's skeletal muscle biopsy sample (MB), blood (BL), urine (UR), and fibroblasts (FB). M, molecular weight marker. wt, wild type; mut, mutant. (C) Secondary structure of the human mitochondrial tRNA cysteine adapted from the Mamit-tRNA database.²³ Filled arrow indicates the position of the novel m.5789T>C variant. Note that the replacement of a Watson-Crick base pair by a U-G base pair generates 2 adjacent wobble base pairs in the anticodon stem. Stars indicate the anticodon. Dash, Watson-Crick base pair; dot, wobble base pair. (D) Bee swarm diagram of m.5789T>C mutation loads in COX-negative and COX-positive single skeletal muscle fibers as determined by RFLP. Note that fibers with 78% mutation load or less show COX activity. (E) Northern blot analysis of skeletal muscle RNA of the patient (P) and 3 controls (C1–3) as well as aminoacylated (AC) and deaminoacylated (deAC) human osteosarcoma cells 143B RNA. The overall intensity of the tRNA^Cys signal is decreased in comparison to tRNA^SerUCN, whereas the proportions of aminoacylated and deaminoacylated RNA species are similar in the patient and in controls. COX = cytochrome c oxidase.