Original articleAnalysis of the mitochondrial encoded subunits of complex I in 20 patients with a complex I deficiency
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 tRNAleu gene, myoclonic epilepsy with ragged red fibers (MERRF, A8344G) in the tRNAlys 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.
Section snippets
Patients
Table 1 summarizes the clinical, biochemical and histochemical features of the patients analyzed in this study.
DNA samples
DNA of patients was extracted from leukocytes, using the QIAamp DNA blood Maxi Kit (Westburg, Leusden, The Netherlands), and from muscle, fibroblasts and liver samples using an in house protocol. DNA, extracted from blood of a healthy person was used as a reference. Unlike some other protocols, we did not first separate the mtDNA from the nDNA by a long-distance PCR protocol,
Results
The aim of this study was to detect nucleotide changes in the mtDNA encoded subunits of complex I in 20 patients with clinical and biochemical features associated with mitochondrial dysfunction of complex I (see Table 1). Direct sequencing of fragments showing abnormal DGGE patterns revealed a total of 96 nucleotide alterations with respect to the reference sequence CRSr. No heteroplasmic sequence variations were found. All of the mtDNA alterations were homoplasmic according to their DGGE and
Discussion
Complex I deficiency is one of the most frequently diagnosed defects of the mitochondrial OXPHOS system. It is associated with a spectrum of severe metabolic disorders like Leigh's syndrome, cardiomyopathy and progressive encephalomyopathy. So far, pathogenic mutations have been reported in all mitochondrial encoded subunits of complex I but only in eight of the at least 36 nuclear encoded genes.
We selected 20 patients with a decreased catalytic activity of complex I in muscle tissue and/or
Acknowledgements
This work has been performed in the center of Medical Genetics with the support of grants of the OZR (887), FWO (G.0119.02), ABMM and GEPTS foundation.
References (29)
- et al.
Nuclear genes in mitochondrial disorders
Curr Opin Genet Dev
(2003) - et al.
Length-independent separation of DNA restriction fragments in two-dimensional gel electrophoresis
Cell
(1979) - et al.
Mutational scanning of mitochondrial DNA by two-dimensional electrophoresis
Genomics
(1998) - et al.
A mitochondrial DNA mutation co segregates with the pathophysiological U wave
Biochem Biophys Res Commun
(1999) - et al.
Human mitochondrial DNA disease
Adv Drug Deliv Rev
(2002) - et al.
Mitochondrial disorders
Curr Opin Neurol
(2003) - et al.
Mitochondrial respiratory-chain diseases
N Engl J Med
(2003) - et al.
Clinical spectrum and diagnosis of mitochondrial disorders
Am J Med Genet
(2001) - et al.
The mitochondrial ND6 gene is a hot spot for mutations that cause Leber's hereditary optic neuropathy
Brain
(2001) - et al.
Leber's hereditary optic neuropathy
J Med Genet
(2002)
Isolated complex I deficiency in children: clinical, biochemical and genetic aspects
Hum Mutat
Differences in assembly or stability of complex I and other mitochondrial OXPHOS complexes in inherited complex I deficiency
Hum Mol Genet
Mutant NDUFS3 subunit of mitochondrial complex I causes Leigh syndrome
J Med Genet
De novo mutations in the mitochondrial ND3 gene as a cause of infantile mitochondrial encephalopathy and complex I deficiency
Ann Neurol
Cited by (7)
Degenerative Disorders of the Newborn
2018, Volpe's Neurology of the NewbornHypotonia and Weakness: Level of the Muscle
2018, Volpe's Neurology of the NewbornDegenerative Disorders of the Newborn
2017, Volpe's Neurology of the NewbornMuscle Involvement and Restricted Disorders
2017, Volpe's Neurology of the NewbornNeurology of the Newborn
2008, Neurology of the NewbornAnalysis of mitochondrial DNA sequences in patients with isolated or combined oxidative phosphorylation system deficiency
2006, Journal of Medical Genetics