Impact of the mitochondrial genetic background in complex III deficiency.
BACKGROUND
In recent years clinical evidence has emphasized the importance of the mtDNA genetic background that hosts a primary pathogenic mutation in the clinical expression of mitochondrial disorders, but little experimental confirmation has been provided. We have analyzed the pathogenic role of a novel homoplasmic mutation (m.15533 A>G) in the cytochrome b (MT-CYB) gene in a patient presenting with lactic acidosis, seizures, mild mental delay, and behaviour abnormalities.


METHODS
Spectrophotometric analyses of the respiratory chain enzyme activities were performed in different tissues, the whole muscle mitochondrial DNA of the patient was sequenced, and the novel mutation was confirmed by PCR-RFLP. Transmitochondrial cybrids were constructed to confirm the pathogenicity of the mutation, and assembly/stability studies were carried out in fibroblasts and cybrids by means of mitochondrial translation inhibition in combination with blue native gel electrophoresis.


RESULTS
Biochemical analyses revealed a decrease in respiratory chain complex III activity in patient's skeletal muscle, and a combined enzyme defect of complexes III and IV in fibroblasts. Mutant transmitochondrial cybrids restored normal enzyme activities and steady-state protein levels, the mutation was mildly conserved along evolution, and the proband's mother and maternal aunt, both clinically unaffected, also harboured the homoplasmic mutation. These data suggested a nuclear genetic origin of the disease. However, by forcing the de novo functioning of the OXPHOS system, a severe delay in the biogenesis of the respiratory chain complexes was observed in the mutants, which demonstrated a direct functional effect of the mitochondrial genetic background.


CONCLUSIONS
Our results point to possible pitfalls in the detection of pathogenic mitochondrial mutations, and highlight the role of the genetic mtDNA background in the development of mitochondrial disorders.
Gil Borlado
Mari Carmen
MC
Centro de Investigación, Hospital Universitario 12 de Octubre, Madrid, Spain.
Moreno Lastres
David
D
Gonzalez Hoyuela
Maritza
M
Moran
Maria
M
Blazquez
Alberto
A
Pello
Rosa
R
Marin Buera
Lorena
L
Gabaldon
Toni
T
Garcia Peñas
Juan Jose
JJ
Martín
Miguel A
MA
Arenas
Joaquin
J
Ugalde
Cristina
C
D000595:Amino Acid Sequence; D001483:Base Sequence; D045303:Cytochromes b / Q000235:genetics / Q000378:metabolism; D014450:Electron Transport Complex III / Q000172:deficiency* / Q000235:genetics; D003576:Electron Transport Complex IV / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D008928:Mitochondria / Q000235:genetics* / Q000378:metabolism; D028361:Mitochondrial Diseases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D017354:Point Mutation
D045303:Cytochromes b; D003576:Electron Transport Complex IV; D014450:Electron Transport Complex III