PMC

Molecular analysis of the NDUFA10 gene. (a) Electropherograms showing the wild-type sequence of NDUFA10 (top) and the nucleotide changes in fibroblasts of the patient (bottom). Arrows represent the nucleotide substitutions, c.1A>G (left) and c.425A>G (right). (b) Conservation of the altered amino acid (p.Gln142Arg) is shown in a Clustal W alignment. Abbreviations : Wt, wild-type; P, index patient.

The mutations in NDUFA10 result in accumulation of subcomplexes of complex I. Two-dimensional BN/SDS-PAGE analysis followed by immunoblotting with antibodies directed against NDUFS3 to investigate the early steps of the complex I assembly, and ND1 to examine the incorporation of the mtDNA encoded subunits, shows the accumulation of mainly the subcomplexes 4 and 5 in the index patient. The subcomplexes are numbered according the assembly model described by Vogel and colleagues. Abbreviations: C, control; P-A10, patient with NDUFA10 mutations; C I, complex I.

The mutations in NDUFA10 result in decreased activity and amount of complex I. (a) BN-PAGE analysis followed by an in-gel activity assay demonstrates a decreased complex I activity in fibroblasts of the patient compared with control cells. The amount of subunit NDUFA9 is also severely reduced, indicating a decreased amount of whole complex I. The amount of complex II is not affected. (b) SDS-PAGE analysis and immunoblotting with an antibody directed against NDUFA10 shows that the amount of NDUFA10 is decreased in patient fibroblasts. The amount of NDUFA9 is also reduced, suggesting a reduction in amount of complex I. The amount of complex II is not affected. Abbreviations: C, control; P-A10, patient with NDUFA10 mutations; C, I, II, complexes I, II; IGA, in-gel activity; SDHA, succinate dehydrogenase complex subunit A.