Partial tandem duplication of mtDNA-tRNA(Phe) impairs mtDNA translation in late-onset mitochondrial myopathy

Abstract

An 80-year-old woman (PI) has been suffering of late onset progressive weakness and wasting of lower-limb muscles, accompanied by high creatine kinase levels in blood. A muscle biopsy, performed at 63 years, showed myopathic features with partial deficiency of cytochrome c oxidase. A second biopsy taken 7 years later confirmed the presence of a mitochondrial myopathy but also of vacuolar degeneration and other morphological features resembling inclusion body myopathy. Her 46-year-old daughter (PII) and 50-year-old son (PIII) are clinically normal, but the creatine kinase levels were moderately elevated and the EMG was consistently myopathic in both. Analysis of mitochondrial DNA sequence revealed in all three patients a novel, homoplasmic 15 bp tandem duplication adjacent to the 5' end of mitochondrial tRNA(Phe) gene, encompassing the first 11 nucleotides of this gene and the four terminal nucleotides of the adjacent D-loop region. Both mutant fibroblasts and cybrids showed low oxygen consumption rate, reduced mitochondrial protein synthesis, and decreased mitochondrial tRNA(Phe) amount. These findings are consistent with an unconventional pathogenic mechanism causing the tandem duplication to interfere with the maturation of the mitochondrial tRNA(Phe) transcript.

Fig. 1

Morphological analysis: PI muscle biopsies at 63 (A) and 70 (B) years of age. Panels a, d: H&E; note the fiber size variation and numerous centralized nuclei in a, with severe worsening in d. Panels b, e: Gomori trichrome; a pre-ragged red fiber is present in b, whereas several fibers contain rimmed vacuoles in e. Panel c, f: COX + SDH histochemistry; numerous SDH-positive, COX-depleted blue fibers are present in both samples. Magnification of panels A (a, b, c), referring to the first biopsy, is 20×. Magnification of panels B (d, e, f), referring to the second biopsy, is 40×, due to the scarcity of muscle tissue.

Fig. 2

MtDNA analysis: (A) Electropherogram and schematic representation of the 15 bp duplication. Arrows indicate the H1 and H2 promoters with the corresponding nucleotide positions. Underlined letters indicate the duplicated stretch. The same color code is used in the scheme and sequence letters to indicate the D-loop (ocre) and tRNA^Phe gene (lilac). (B) Scheme of the human mtDNA. Hooked arrows indicate the two promoters of the heavy strand (H₁, H₂) and that of the light strand (L₁). The mt-tRNAs are denominated as the corresponding aminoacid residues expressed in the single-letter code.

Fig. 3

Biochemical analysis: (A) RC complex activities normalized to CS and expressed as percentage of the mean control value. (B) Oxygen consumption rate (OCR) in cells. Note that for fibroblasts, values were measured as O₂ fmol/min/cell, whereas for cybrids values were measured as O₂ fmol/min normalized to the amount of mtDNA. Ctrl: control. Vertical bars indicate the standard deviations. Unpaired, two-tail Student’s t test: *p < 0.05; **p < 0.0005.

Fig. 4

In organello translation and tRNA analysis: (A) Mitochondrial protein synthesis. [³⁵S]-labeled mtDNA translation products. ND: subunits of complex I; CO: subunits of complex IV (COX); cyb: cytochrome b (complex III); ATPase: subunits of complex V (ATP synthase). Note the reduced intensity of the bands from mutant cybrids compared to 143B cells. (B) Northern-blot analysis of total and aminoacylated mt-tRNA transcripts. Top panel: Northern-blot analysis indicating the total amount of each mt-tRNA transcript. Bottom panel: aminoacylation assay. Analysis of the aminoacylation capacity in mutant and control cells performed using ³H-labeled amino acids. (C) Quantitative analysis of mt-RNA transcripts. Quantitative real-time PCR of mitochondrial H-strand transcripts relative to ND6 mRNA in mutant cybrids and 143B control cells.