Analysis of the mitochondrial encoded subunits of complex I in 20 patients with a complex I deficiency

Summary

NADH-ubiquinone oxidoreductase or complex I deficiency is a frequently diagnosed enzyme defect of the oxidative phosphorylation (OXPHOS) system in humans. However, in many patients, with complex I deficiency and clinical symptoms suggestive of mitochondrial disease, often no genetic defect can be found after investigation of the most common mitochondrial DNA (mtDNA) mutations. In this study, 20 patients were selected with a biochemically documented complex I defect and no common mtDNA mutation. We used the Denaturing Gradient Gel Electrophoresis (DGGE) method with primers encompassing all mitochondrial encoded fragments, to search in a systematic manner for mutations in the mitochondrial genome of complex I. In our group of patients, we were able to detect a total of 96 nucleotide changes. We were not able to find any disease causing mutation in the mitochondrial encoded subunits of complex I. These results suggested that the complex I deficiency in this group of patients is most probably caused by a defect in one of the nuclear encoded structural genes of complex I, or in one of the genes involved in proper assembly of the enzyme.

Introduction

Mitochondrial cytopathies are a heterogeneous group of diseases, with a wide spectrum of clinical symptoms and multisystem involvement. They are caused by defects in the oxidative phosphorylation due to mutations in either the nuclear (n) or the mitochondrial DNA (mtDNA).1, 2, 3, 4, 5

In the last decade, almost 100 different point mutations and more than 100 deletions and rearrangements in mtDNA have been reported.³ The most common mtDNA mutations are associated with mitochondrial encephalomyopathy, lactic acidosis and stroke like episodes (MELAS, A3243G) in the tRNA^leu gene, myoclonic epilepsy with ragged red fibers (MERRF, A8344G) in the tRNA^lys gene, neurogenic muscle weakness ataxia and retinitis pigmentosa (NARP, T8933G/C) in the ATP 6 gene or Leber's hereditary optic neuropathy (LHON, G3460A-G11778A-T14484C) in complex I subunits ND 1-4-6.6, 7 Although mtDNA plays an important role in mitochondrial cytopathies, mutations in nuclear encoded genes are being increasingly associated with mitochondrial dysfunction.3, 5

Isolated complex I deficiency is one of the most common enzyme defect of the OXPHOS disorders. Complex I, or NADH-oxidoreductase, is the first of five energy transducing enzyme complexes of the respiratory chain in mitochondria and the point of entry for the major fraction of electrons that cross the respiratory chain. As a result of the electron transfer, protons are pumped from the matrix side to the intermembrane space of mitochondria eventually resulting in the reduction of oxygen. Complex I is composed of at least 43 different polypeptides. Of these subunits, only seven are encoded by the mitochondrial genome. Deficiencies of complex I are associated with a wide range of clinical presentations, but most often with LHON, Leigh or Leigh-like syndrome. It has been assumed that nuclear mutations account for the majority of cases, but so far in only a few patients mutations were identified in nuclear genes encoding the subunits NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS7, NDUFS8, NDUFV1 or NDUFV2. Pathogenic mtDNA mutations have been found in all seven NADH subunits.8, 9, 10, 11, 12, 13

In 20 of our patients suspected of having a mitochondrial cytopathy caused by a biochemically determined complex I impaired activity, no pathogenic mutations in any of the 22 mitochondrial tRNA genes, and no NARP mutations or large-scale rearrangements were found in the mitochondrial genome. Since genetic counseling can only be accurate when the genomic origin of the gene defect is known, the complete sequence of the mtDNA encoded subunits of complex I was analyzed for nucleotide alterations with DGGE. This technique, developed by Fischer and Lerman in 1983, is based on the melting behavior of a given double stranded DNA fragment and has proven, over the years, its high specificity and sensitivity to detect a nucleotide change in double stranded DNA.14, 15, 16, 17

The first step in our analysis consisted of optimizing an ad hoc DGGE protocol, starting from the protocol published by van Orsouw et al.¹⁸ We then re-analyzed patient and control DNA samples with known mutations and polymorphisms to determine the sensitivity and specificity of the protocol. In the third and last step we analyzed our 20 selected patients.