A novel mitochondrial ATP8 gene mutation in a patient with apical hypertrophic cardiomyopathy and neuropathy

Abstract

To identify the biochemical and molecular genetic defect in a 16-year-old patient presenting with apical hypertrophic cardiomyopathy and neuropathy suspected for a mitochondrial disorder.Measurement of the mitochondrial energy-generating system (MEGS) capacity in muscle and enzyme analysis in muscle and fibroblasts were performed. Relevant parts of the mitochondrial DNA were analysed by sequencing.A homoplasmic nonsense mutation m.8529G→A (p.Trp55X) was found in the mitochondrial ATP8 gene in the patient's fibroblasts and muscle tissue. Reduced complex V activity was measured in the patient's fibroblasts and muscle tissue, and was confirmed in cybrid clones containing patient-derived mitochondrial DNAWe describe the first pathogenic mutation in the mitochondrial ATP8 gene, resulting in an improper assembly and reduced activity of the complex V holoenzyme.

Figure 1. Echocardiography showing apical left ventricular hypertrophy.

LA, left atrium; RA, right atrium; LV, left ventricle; RV, right ventricle; arrows, myocardial hypertrophy.

Figure 2. The C-terminal domain of mitochondrial ATP synthase subunit 8 is a conserved region in mammals.

p.Trp55X, mutation site at the protein level.

Figure 3. Molecular genetic analysis of the cybrid clones containing patient-derived mtDNA shows the mitochondrial nonsense mutation m.8529G→A (arrow).

Top row: control cybrid cell line; bottom row: patient cybrid clone. Sequence analysis showed that all the clones are homoplasmic for the mutation (data not shown).

Figure 4. Immunoblotting after blue native polyacrylamide gel electrophoresis shows increased amounts of subcomplexes of mitochondrial ATP synthase in the patient cybrid clones and muscle.

The control cybrid and muscle show only the complex V holoenzyme and dimer. Antibodies used are against ATP synthase subunit alpha and CoII-70 kDa (complex II, loading control). Lane 1, control cybrid cell line (coCy); lanes 2–8, patient cybrid clones (1–7); lane 9, patient muscle homogenate (ptMu); lane 10, control muscle homogenate (coMu).

Figure 5. The in-gel activity (IGA) assay of ATP hydrolysis shows activity of free F₁-ATPase in the patient cybrid clones and muscle. Lane 1, control cybrid cell line (coCy); lanes 2–8, patient cybrid clones (1–7); lane 9, patient muscle homogenate (ptMu); lane 10, control muscle homogenate (coMu).