- Email: [email protected]
Novel mutations in the NDUFS1 gene cause low residual activities in human complex I deficiencies
Molecular Genetics and Metabolism 100 (2010) 251–256
Contents lists available at ScienceDirect
Molecular Genetics and Metabolism journal homepage: www.elsevier.com/locate/ymgme
Novel mutations in the NDUFS1 gene cause low residual activities in human complex I deficiencies Saskia J.G. Hoefs a, Ola H. Skjeldal b, Richard J. Rodenburg a, Bård Nedregaard c, Edwin P.M. van Kaauwen a, Ute Spiekerkötter d, Jürgen-Christoph von Kleist-Retzow d, Jan A.M. Smeitink a, Leo G. Nijtmans a, Lambert P. van den Heuvel a,* a
Department of Pediatrics, Nijmegen Center for Mitochondrial Disorders, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands Department of Medical Research and Department of Pediatrics, Innlandet Hospital Thrust, Brumuddal, Norway Neuroradiological Department, Oslo University Hospital, Rikshospitalet, Oslo, Norway d Department of General Pediatrics, University Children’s Hospital, University of Köln, Köln, Germany b c
a r t i c l e
i n f o
Article history: Received 18 March 2010 Accepted 18 March 2010 Available online 21 March 2010 Keywords: OXPHOS Complex I NDUFS1 Assembly Residual activity
a b s t r a c t Mitochondrial complex I deficiency is the most frequently encountered defect of the oxidative phosphorylation system. To identify the genetic cause of the complex I deficiency, we screened the gene encoding the NDUFS1 subunit. We report 3 patients with low residual complex I activity expressed in cultured fibroblasts, which displayed novel mutations in the NDUFS1 gene. One mutation introduces a premature stop codon, 3 mutations cause a substitution of amino acids and another mutation a deletion of one amino acid. The fibroblasts of the patients display a decreased amount and activity of complex I. In addition, a disturbed assembly pattern was observed. These results suggest that NDUFS1 is a prime candidate to screen for disease-causing mutations in patients with a very low residual complex I activity in cultured fibroblasts. Ó 2010 Elsevier Inc. All rights reserved.
Introduction The oxidative phosphorylation (OXPHOS) system, embedded in the inner mitochondrial membrane is essential for the production of mitochondrial ATP. It is composed of 4 enzyme complexes (complex I–IV), which form the electron transport chain, and ATP synthase (complex V) [1]. Complex I and II transfer electrons from NADH and FADH2 to ubiquinone. Subsequently, complex III transfers the electrons from ubiquinol to cytochrome c, and complex IV transfers them to molecular oxygen. The transport of the electrons is coupled to the translocation of protons, which results in a proton gradient. Finally, this gradient can be used by complex V to produce ATP from ADP + Pi. Disturbances in the OXPHOS system cause mitochondrial disorders with an incidence of 1 in approximately 5000 births [2]. Isolated complex I deficiency is the most frequently diagnosed form accounting for almost 23% of all cases of childhood respiratorychain deficiency. Mitochondrial disorders may affect one or more tissues or organs, especially the ones with high metabolic energy rates. Therefore, mitochondrial diseases have a wide clinical vari-
* Corresponding author. Address: Department of Pediatrics, Nijmegen Center for Mitochondrial Disorders, Radboud University Nijmegen Medical Center, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Fax: +31 24 3618900. E-mail address: [email protected] (L.P. van den Heuvel). 1096-7192/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2010.03.015
ety [3,4]. This is also due to the bigenomic control of the OXPHOS system. Mutations in either the mitochondrial DNA (mtDNA) or nuclear encoded genes may cause complex I deficiency. NADH:ubiquinone oxidoreductase (E.C. 1.6.5.3.), or complex I, is the largest complex of the OXPHOS system, consisting of 45 subunits. Seven are encoded by the mtDNA and with 7 nuclear encoded subunits they form the ‘core subunits’ [5,6]. These are considered as the minimal constitution of complex I, since the subunits are involved in catalysis of electron transfer from NADH to ubiquinone and for generation of the membrane potential. One of the ‘core subunits’ is NDUFS1. This protein is the largest subunit of complex I and is located in the iron–sulfur fragment of the enzyme [7]. It contains 3 iron–sulfur clusters, N1b, N4 and N5 and plays an important role in the electron transfer [8]. Mutations in this gene are associated with isolated complex I deficiency. Benit and colleagues described 5 point mutations involving highly conserved amino acids (del222I, D252G, M707V, R241W, and R557X) and one large-scale deletion in this gene [9]. Additionally, 2 homozygous missense mutations (L231V and C1564A) were reported [10,11]. In this study we describe novel mutations in NDUFS1 in 3 unrelated patients with complex I deficiency expressed in cultured skin fibroblasts and we show that the assembly pattern of complex I is disturbed in these cells. Studies identifying new disease-causing mutations are necessary since molecular diagnosis is still lacking in many patients [12].
252
S.J.G. Hoefs et al. / Molecular Genetics and Metabolism 100 (2010) 251–256
Materials and methods Patients Patient 1 The patient, a girl, was born from healthy unrelated parents after a pregnancy complicated by gestational diabetes with a modest intrauterine growth retardation (weight 5th percentile). One older sib is a healthy boy. She had normal development in the first months of life, but started at age 8 months with abnormal crying and a regression of already acquired motor skills. At that age her brain MRI showed bilateral and symmetric leucodystrophic cystic lesions. Her clinical status worsened and she ultimately developed spasticity, microcephaly, mental retardation, and a progressive neuropathy. She died at the age of 12 years. Patient 2 The patient, a boy, was the third child of consanguineous parents (first cousins). One of the older sibs is a healthy boy, the other one was also affected. The index patient had problems with the central nervous system like leucoencephalopathy, episodic brainstem events, reduced spontaneous movement and he had an abnormal breathing pattern. His clinical status worsened and he had feeding problems. Furthermore he had muscle dystrophy and generalized hypotonia and died at the age of 8 months. The affected brother had a similar clinical picture. The age of onset was 4 months. In this patient the levels for lactate, pyruvate and alanine in both plasma and liquor indicate a mitochondrial disorder. He died at the age of 7 months.
blasts by standard procedures [15]. The nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence. The initiation codon is codon 1. BN–PAGE analysis and in-gel activity assay Mitochondria-enriched fractions were obtained by incubation with 2% (w/v) digitonin (Calbiochem) on ice for 10 min, followed by centrifugation at 10,000g and 4 °C for 10 min and washing with PBS, twice. Blue native-gradient gels (5–15%) were cast as described previously [16] and run with 40 lg (or 80 lg) of the solubilised mitochondrial protein. After electrophoresis, the gels were further processed for in-gel activity assay or second-dimension 10% SDS–PAGE followed by immunoblot analysis as described by Nijtmans et al. [17]. Antibodies and ECL detection Protein immunodetection was performed with primary antibodies directed against NDUFA9 (Invitrogen), NDUFS3 (Invitrogen), the CII 70 kDa subunit (SDHA; Invitrogen) and the CIII CORE2 subunit (UQCRC2; Invitrogen). Secondary antibodies used were peroxidise-conjugated anti-mouse IgGs (Invitrogen) and detection of the signal performed using ECL Plus (Amersham Biosciences) according to the manufacturer’s instructions. Results
Patient 3 This female patient was born after an unremarkable pregnancy, from non-related parents. The development was normal in the first months. Nystagmus was detected when she was 5 months of age. When she was 12 months of age she walked without support, but from that age her development stagnated motorically as well as mentally. After 15 months she started to lose neurological abilities and there was no doubt that she suffered from a neurological progressive disorder. Her clinical situation was dominated by a failure to thrive, crying, eating difficulties, spasticity, and mental retardation. She died at the age of 2 years.
Biochemical studies
Cell culture and biochemical analysis
MRI findings
Skin fibroblasts were cultured in M199 medium (Life Technologies) supplemented with 10% fetal calf serum and penicillin/streptomycin in a humified atmosphere of 95% air and 5% CO2 at 37 °C. Measurement of the mitochondrial respiratory-chain enzymes in skin fibroblasts and in muscle tissue were performed according to established procedures [13,14]. The values were expressed relative to the activity of cytochrome c oxidase and/or citrate synthase.
MRI pictures were available for patient 1 and 3. Patient 1: the first MRI was obtained at the age of 9 months. In the central white matter of both cerebral hemispheres, there were symmetric areas of marked hyperintensity on T2 and hypointensity of T1. The internal capsule was unaffected. The corpus callosum had normal signal, but was somewhat atrophic. The peripheral U-fibres were
Genetic analysis of the NDUFS1 gene Patient RNA was extracted from cultured fibroblasts by standard procedures. The RNA was reverse transcribed to cDNA by superscript II RNase H reverse transcriptase with oligo(dT) and random hexamer primers. Oligonucleotide primers were designed from the NDUFS1 gene sequence (Genbank Accession No. NM_005006). Primer sequences are available upon request. Polymerase chain reaction products were run on a 1.5% agarose gel and sequenced directly with the BigDye terminator cycle sequencing chemistry on a Applied Biosystems ABI PRISM 3130xl Genetic Analyzer. The mutations were also checked on genomic DNA level. Therefore, patient DNA was extracted from cultured skin fibro-
The activities of the respiratory-chain complexes were measured in skin fibroblasts of the 3 patients, in frozen muscle tissue of patient 1 and in fresh muscle tissue of patient 2, all displaying a marked decrease of complex I (Table 1). The fibroblast complex I enzymatic activities were 27%, 20%, and 24% of the lowest control value for the patients 1, 2 and 3, respectively. In fresh muscle tissue of patient 2, the enzymatic activity of complex III was decreased to 70% of the lowest control value. However, the activity of this complex in skin fibroblasts was within the control range.
Table 1 Respiratory-chain enzyme activities in skin fibroblasts, and frozen and fresh muscle tissue of the index patients. Complex Fibroblasts
I II III IV
Frozen muscle
Fresh muscle
P1
P2
P3
Control range
P1
Control range
P2
Control range
30 n.m. 1520 860
22 648 1522 1075
26 801 1867 953
110–206a 536–1027a 1270–2620a 680–1190b
53 n.m. n.m. 737
84–273c – – 520–2080c
7 119 1540 1305
70–250c 67–177c 2200–6610c 810–3120c
n.m. = not measured. Deficiencies are indicated in bold. a mU/U COX. b mU/mU CS. c mU/U CS.
S.J.G. Hoefs et al. / Molecular Genetics and Metabolism 100 (2010) 251–256
spared and the basal ganglia, brainstem and cerebellum were normal. The second MRI was done at the age of 11 months. There was at this stage a marked progression of the signal changes, which now were extending throughout the central white matter in the centrum semiovale. The temporal lobes were spared. There was progressive atrophy of the corpus callosum. The cortical grey matter, the basal ganglia, the brainstem, and cerebellum were still unaffected. The degree of signal change was very pronounced, the MRI signal not very different from that of the cerebrospinal fluid. The previously shown pathology, seen in the first MRI, now presented as ‘‘holes” within the central part of the new signal changes, having even more CSF-like signal (consistent with rarefaction). MR-spectroscopy was not performed. Patient 3: the first MRI was performed at the age of 5 months and was normal. At the age of 16 months a second MRI was done and following features were observed: extensive and confluent areas with markedly hyperintensity on T2 and hypointensity on T1 in subcortical and deep white matter of the cerebrum. It included the entire length of the corpus callosum, internal capsule (anterior limb and genu), extended through the cerebral peduncles and the ventral midbrain and ending in the central parts of pons from both sides. There was a striking sparing of the subcortical white matter of perirolandic gyri, as well as the central part of the corona radiata, together with the posterior limb of the internal
253
capsule and the optic radiations. The distribution of signal changes was symmetrical, dominating in the frontal and parietal lobes, but also affecting the periventricular areas in the temporal lobes. On diffusion sequence, there were multifocal areas with restricted diffusion scattered throughout the affected white matter, consistent with active demyelination. The last (third) MRI was obtained at the age of 17 months (Fig. 1). The affected white matter was slightly swollen, and the cerebellum still being normal. Furthermore, there was symmetrical, partial involvement of the nucleus caudatus. Otherwise the grey matter was normal. Multi-voxel MR-spectroscopy showed decreased NAA peaks and multifocal lactate peaks, anatomically corresponding to the areas of signal change. Molecular genetic studies in NDUFS1 gene We searched for the molecular defect causing these complex I deficiencies, by screening the cDNA of the NDUFS1 gene. In patient 1 we found a homozygous mutation, c.1855G>A, which introduced a substitution of amino acids (p.Asp619Asn). Verifying this mutation on genomic DNA revealed that this mutation was heterozygous (Fig. 2A). This suggests that one of the alleles results in an unstable RNA transcript making it undetectable on cDNA level. To detect a mutation on the second allele, the remaining exons of NDUFS1 were screened and this resulted in another heterozy-
Fig. 1. MRI findings of patient 3. (A) Cerebral T2-MRI of patient 3 at the age of 17 months shows marked white matter abnormalities. (B) Multi-voxel MRS shows decreased NAA signals and increased lactate peaks anatomically corresponding to the areas of signal change (left) compared to a normal spectrum from the gray matter of the same patient (right).
254
S.J.G. Hoefs et al. / Molecular Genetics and Metabolism 100 (2010) 251–256
gous mutation, c.1669C>T, which introduced a premature stop codon (p.Arg557X) (Fig. 2B). The father and brother were found to be heterozygous for the Arg557X mutation, and the mother for the Asp619Asn mutation. Sequence analysis of both cDNA and genomic DNA of patient 2 demonstrated the presence of a homozygous substitution of C to T (c.1222C>T; p.Arg408Cys) (Fig. 2C), which was also present in the other affected brother. Analysis of DNA of the third patient showed 2 heterozygous mutations (c.631–633delGAA; c.683T>C) (Fig. 2D and E). The first mutation causes a deletion of one amino acid (p.211delGlu), the second a substitution of ami-
no acids (p.Val228Ala). The father was found to be heterozygous for c.631–633delGAA and the mother heterozygous for c.683T>C. All above described mutations were not present in 100 healthy controls or 25 other patients with isolated complex I deficiency in cultured skin fibroblasts. Furthermore, all missense mutations and the deletion are present in strongly conserved amino acids. Computational modeling of the amino acid at position 228 reveals that this residue is located in a conserved region between two FeS clusters (N4 and N5) and that it is important for the structure around these clusters. The mutation p.Val228Ala changed a
Fig. 2. Molecular analysis of the NDUFS1 gene. (A–E) Electropherograms showing the wild-type sequence of NDUFS1 (top panels) and the nucleotide changes in the complex I deficient patients (bottom panels). Conservation of the altered amino acids is shown in Clustal W alignments. (A) Nucleotide change c.1669T>C, and amino acid change Arg557X in patient 1. (B) Nucleotide change c.1855G>A, and amino acid change Asp619Asn in patient 1. (C) Nucleotide change c.1222C>T, and amino acid change Arg408Cys in patient 2. (D) Nucleotide change c.631–622delGAA, and amino acid change 211delGlu in patient 3. (E) Nucleotide change c.683T>C, and amino acid changes Val228Ala in patient 3. (F) Overview of the NDUFS1 protein consisting of 727 amino acids, with the location of structural domains, according to InterProScan. The 5 different mutations that are described in this paper, are indicated in blue. The mutations described in earlier studies are indicated in black. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
S.J.G. Hoefs et al. / Molecular Genetics and Metabolism 100 (2010) 251–256
255
hydrofobe residue into a smaller but also hydrophobic residue, which results in a gap within the NDUFS1 subunit. Furthermore the structure around this residue will probably be less stable. This may have an effect on the binding and working of the FeS clusters and therefore on the transfer of the electrons. Modeling of the other amino acids changes was not possible. NDUFS1 contains several structural domains, as shown in Fig. 2F. All above described mutations are located in the NADH-quinone oxidoreductase chain G domain. Additionally, the mutations Arg408Cys, found in patient 2 and Arg557X and Asp619Asn, found in patient 1, are also located in the molybdopterin oxidoreductase domain (Fig. 2F). Effect of the mutations in NDUFS1 gene on complex I To study the effect of the mutations on the expression and activity complex I, we performed BN–PAGE analysis followed by Western blotting with an anti-NDUFA9 antibody and in-gel activity measurement with mitochondria-enriched fractions of cultured fibroblasts of the 3 patients. Fig. 3 shows severely reduced activities of complex I in the fibroblasts of the 3 patients compared to the control. Furthermore, the expression of subunit NDUFA9 was decreased in the patients compared to the control, suggesting a decrease in the amount of fully assembled complex I. Patients 2 and 3 showed also an extra band, which suggest the formation of subcomplexes. Together, these results show a severely reduced amount and activity of complex I in the patients’ fibroblasts. To further investigate the complex I assembly profile in the patient fibroblasts, we performed two-dimensional BN/SDS–PAGE analysis followed by immunoblotting with an antibody directed against NDUFS3. Fig. 4 displays less complex I and the formation of subcomplexes in fibroblasts of patients 1 and 3 compared to the control. Even more accumulation of subcomplexes was visible in cells of patient 2. These results suggest that the mutations in NDUFS1 cause a disturbance in the assembly and/or stability of complex I. Discussion In this report we describe 3 patients with complex I deficiency expressed in cultured fibroblasts harboring novel mutations in NDUFS1. Patient 1 was compound heterozygous for Arg557X and Asp619Asn. The first mutation has been previously reported in a
Fig. 3. The mutations in NDUFS1 cause decreased expression and activity of complex I. BN–PAGE analysis followed by in-gel activity measurement demonstrates a reduced complex I activity in the fibroblasts of the patients compared to the control. The expression of subunit NDUFA9 is also severely reduced, which indicates a decreased amount of whole complex I. Furthermore, the fibroblasts of patient 2 and 3 show an extra band, which suggest the formation of subcomplexes. The expression of the complexes II and III is not affected.
Fig. 4. The NDUFS1 mutations cause accumulation of subcomplexes. Two-dimensional BN/SDS–PAGE analysis followed by immunoblotting with an antibody directed against NDUFS3 shows less complex I in cells of patient 1 and 3 and the accumulation of subcomplexes in the fibroblasts of the three patients.
patient compound heterozygous for that mutation and the Arg241Trp mutation [9]. The second patient harbored a homozygous missense mutation (Arg408Cys) and the third patient was compound heterozygous for 211delGlu and Val228Ala. Several lines of evidence suggest that these mutations are the underlying cause of the complex I deficiencies in the patients: (1) the mutations cause nucleotide changes in conserved regions of the NDUFS1 gene; (2) the parents are heterozygous carriers and the mutations are not present in 100 healthy controls; (3) the patient fibroblasts show a decrease in amount and activity of complex I, measured by immunoblotting and in-gel activity assays; and (4) the assembly of complex I was also disturbed. The clinical features, especially the rapid progression of the disease, were similar in the patients. Patients 1 and 3 have a somewhat different distribution of signal changes. Common factors are the rapid progression of the disease, the central white matter distribution mostly sparing of the grey matter, and the degree of signal change with sharp demarcation. The extensive signal changes in the white matter with relative sparing of the gray matter inclusive the basal ganglia is different from the classical findings in Leigh disease (being dominant in gray matter). However, MRI findings in these patients are consistent with described cases in the literature for isolated complex I deficiency [18]. The lactate peaks in patient 3 are consistent with anaerobe metabolism and are strongly suggestive of mitochondrial disease. Complex I deficiency combined with a decrease in activity of complex III, like the enzyme activities in muscle tissue of patient 2, has been described before by Budde and colleagues [19]. These patients harbored a mutation in the NDUFS4 gene. An explanation for these combined deficiencies could be the formation of supercomplexes in which complex I and III occur together (CI/CIII2) [20]. This indicates that screening of complex I subunits including NDUFS1 should be considered not only in isolated complex I deficient patients but also in patients with combined complex I and III deficiency. Furthermore, it is remarkable that the activity of complex I in fibroblasts of the 3 patients was very low (<30% of the lowest control value). An earlier study of Loeffen et al. showed a mean residual activity of 49% of the lowest control value measured in a cohort of 27 patients with isolated complex I deficiency [4]. Since thera-
256
S.J.G. Hoefs et al. / Molecular Genetics and Metabolism 100 (2010) 251–256
peutic strategies for complex I deficiency are hardly available, it is important to elucidate the possible genetic causes for prenatal diagnostics and genetic counseling. A good pre selection is important, since more than 70 candidate genes for complex I deficiency are known at present. Therefore, above mentioned low residual complex I activities suggest that screening of the NDUFS1 gene is preferred when patients have very low complex I activities expressed in cultured fibroblasts. NDUFS1 is the largest subunit of complex I, and is suggested to be incorporated into this complex in stage 6 of the assembly process, as proposed by Vogel et al. [21]. In line with this idea is the fact that in patient 2 and 3 an additional band of about 800 kDa is visible on BN blots (Fig. 3) which is lacking in the control cells. Such a subcomplex also occurs in patients with mutations in NDUFS4, NDUFV1, and NDUFS6 and is believed to represent a complex I subassembly which lacks subunits NDUFV1, NDUFV2, NDUFS4, NDUFS1, and NDUFS6 [20]. Although all patients show decreased amounts of fully assembled complex I, this decrease is more pronounced in patient 1 and 3. Nevertheless, the similar decrease in enzyme activity as measured by spectrophotometrically and by in-gel activity (Fig. 3) is comparable in all patients suggesting that beside the decreased amount of complex I also the intrinsic enzyme activity of the remaining complex is also affected in these patients. Identifying the underlying cause of complex I deficiency is important to get more insight in the function of this complex. New strategies like genomic microarray screening or exome sequencing of the nuclear complex I and assembly genes may be possible in the near future. Research to identify new mutations causing complex I deficiency is still ongoing to improve genetic counseling and possibilities for prenatal diagnostics.
Web resources The URLs for data presented herein are as follows: Genbank, http://www.ncbi.nlm.gov/Genbank. InterProScan, http://www.ebi.ac.uk/Tools/InterProScan.
Acknowledgments This work was supported by the European Community’s sixth Framework Programme for Research, Priority 1 ‘‘Life sciences, genomics and biotechnology for health”, contract numbers LSHM-CT-2004-005260 (MITOCIRCLE). The authors would like to thank Hanka Venselaar (Centre for Molecular and Biomolecular Informatics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands) for the computational modeling study.
References [1] M. Saraste, Oxidative phosphorylation at the fin de siècle, Science 283 (1999) 1488–1493. [2] J. Smeitink, L. van den Heuvel, S. DiMauro, The genetics and pathology of oxidative phosphorylation, Nat. Rev. Genet. 2 (2001) 342–352. [3] S. Pitkanen, A. Feigenbaum, R. Laframboise, B.H. Robinson, NADH-coenzyme Q reductase (complex I) deficiency: heterogeneity in phenotype and biochemical findings, J. Inherit. Metab. Dis. 19 (1996) 675–686. [4] J.L. Loeffen, J.A. Smeitink, J.M. Trijbels, A.J. Janssen, R.H. Triepels, R.C. Sengers, L.P. van den Heuvel, Isolated complex I deficiency in children: clinical, biochemical and genetic aspects, Hum. Mutat. 15 (2000) 123–134. [5] J. Carroll, I.M. Fearnley, J.M. Skehel, R.J. Shannon, J. Hirst, J.E. Walker, Bovine complex I is a complex of 45 different subunits, J. Biol. Chem. 281 (2006) 32724–32727. [6] U. Brandt, Energy converting NADH:quinone oxidoreductase (complex I), Annu. Rev. Biochem. 75 (2006) 69–92. [7] J.A. Smeitink, J.L. Loeffen, R.H. Triepels, R.J. Smeets, J.M. Trijbels, L.P. Van den Heuvel, Nuclear genes of human complex I of the mitochondrial electron transport chain: state of the art, Hum. Mol. Genet. 7 (1998) 1573–1579. [8] M. Finel, Organization and evolution of structural elements within complex I, Biochim. Biophys. Acta 1364 (1998) 112–121. [9] P. Benit, D. Chretien, N. Kadhom, P. de Lonlay-Debeney, V. Cormier-Daire, A. Cabral, S. Peudenier, P. Rustin, A. Munnich, A. Rotig, Large-scale deletion and point mutations of the nuclear NDUFV1 and NDUFS1 genes in mitochondrial complex I deficiency, Am. J. Hum. Genet. 68 (2001) 1344–1352. [10] M.A. Martin, A. Blazquez, L.G. Gutierrez-Solana, D. Fernandez-Moreira, P. Briones, A.L. Andreu, R. Garesse, Y. Campos, J. Arenas, Leigh syndrome associated with mitochondrial complex I deficiency due to a novel mutation in the NDUFS1 gene, Arch. Neurol. 62 (2005) 659–661. [11] A. Iuso, S. Scacco, C. Piccoli, F. Bellomo, V. Petruzzella, R. Trentadue, M. Minuto, M. Ripoli, N. Capitanio, M. Zeviani, S. Papa, Dysfunctions of cellular oxidative metabolism in patients with mutations in the NDUFS1 and NDUFS4 genes of complex, J. Biol. Chem. 281 (2006) 10374–10380. [12] D.R. Thorburn, Mitochondrial disorders: prevalence, myths and advances, J. Inherit. Metab. Dis. 27 (2004) 349–362. [13] J. Smeitink, R. Sengers, F. Trijbels, L. van den Heuvel, Human NADH:ubiquinone oxidoreductase, J. Bioenerg. Biomembr. 33 (2001) 259–266. [14] A.J. Janssen, F.J. Trijbels, R.C. Sengers, L.T. Wintjes, W. Ruitenbeek, J.A. Smeitink, E. Morava, B.G. van Engelen, L.P. van den Heuvel, R.J. Rodenburg, Measurement of the energy-generating capacity of human muscle mitochondria: diagnostic procedure and application to human pathology, Clin. Chem. 52 (2006) 860– 871. [15] S.A. Miller, D.D. Dykes, H.F. Polesky, A simple salting out procedure for extracting DNA from human nucleated cells, Nucleic. Acids. Res. 16 (1988) 1215. [16] M.A. Calvaruso, J. Smeitink, L. Nijtmans, Electrophoresis techniques to investigate defects in oxidative phosphorylation, Methods 46 (2008) 281–287. [17] L.G. Nijtmans, N.S. Henderson, I.J. Holt, Blue native electrophoresis to study mitochondrial and other protein complexes, Methods 26 (2002) 327–334. [18] S. van der Knaap, J. Valk, Magnetic Resonance of Myelination and Myelin Disorders, third ed., Springer, 2005. [19] S.M. Budde, L.P. van den Heuvel, A.J. Janssen, R.J. Smeets, C.A. Buskens, L. DeMeirleir, R. van Coster, M. Baethmann, T. Voit, J.M. Trijbels, J.A. Smeitink, Combined enzymatic complex I and III deficiency associated with mutations in the nuclear encoded NDUFS4 gene, Biochem. Biophys. Res. Commun. 275 (2000) 63–68. [20] M. Lazarou, M. McKenzie, A. Ohtake, D.R. Thorburn, M.T. Ryan, Analysis of the assembly profiles for mitochondrial- and nuclear-DNA-encoded subunits into complex I, Mol. Cell. Biol. 27 (2007) 4228–4237. [21] R.O. Vogel, C.E. Dieteren, L.P. van den Heuvel, P.H. Willems, J.A. Smeitink, W.J. Koopman, L.G. Nijtmans, Identification of mitochondrial complex I assembly intermediates by tracing tagged NDUFS3 demonstrates the entry point of mitochondrial subunits, J. Biol. Chem. 282 (2007) 7582–7590.
Contents lists available at ScienceDirect
Molecular Genetics and Metabolism journal homepage: www.elsevier.com/locate/ymgme
Novel mutations in the NDUFS1 gene cause low residual activities in human complex I deficiencies Saskia J.G. Hoefs a, Ola H. Skjeldal b, Richard J. Rodenburg a, Bård Nedregaard c, Edwin P.M. van Kaauwen a, Ute Spiekerkötter d, Jürgen-Christoph von Kleist-Retzow d, Jan A.M. Smeitink a, Leo G. Nijtmans a, Lambert P. van den Heuvel a,* a
Department of Pediatrics, Nijmegen Center for Mitochondrial Disorders, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands Department of Medical Research and Department of Pediatrics, Innlandet Hospital Thrust, Brumuddal, Norway Neuroradiological Department, Oslo University Hospital, Rikshospitalet, Oslo, Norway d Department of General Pediatrics, University Children’s Hospital, University of Köln, Köln, Germany b c
a r t i c l e
i n f o
Article history: Received 18 March 2010 Accepted 18 March 2010 Available online 21 March 2010 Keywords: OXPHOS Complex I NDUFS1 Assembly Residual activity
a b s t r a c t Mitochondrial complex I deficiency is the most frequently encountered defect of the oxidative phosphorylation system. To identify the genetic cause of the complex I deficiency, we screened the gene encoding the NDUFS1 subunit. We report 3 patients with low residual complex I activity expressed in cultured fibroblasts, which displayed novel mutations in the NDUFS1 gene. One mutation introduces a premature stop codon, 3 mutations cause a substitution of amino acids and another mutation a deletion of one amino acid. The fibroblasts of the patients display a decreased amount and activity of complex I. In addition, a disturbed assembly pattern was observed. These results suggest that NDUFS1 is a prime candidate to screen for disease-causing mutations in patients with a very low residual complex I activity in cultured fibroblasts. Ó 2010 Elsevier Inc. All rights reserved.
Introduction The oxidative phosphorylation (OXPHOS) system, embedded in the inner mitochondrial membrane is essential for the production of mitochondrial ATP. It is composed of 4 enzyme complexes (complex I–IV), which form the electron transport chain, and ATP synthase (complex V) [1]. Complex I and II transfer electrons from NADH and FADH2 to ubiquinone. Subsequently, complex III transfers the electrons from ubiquinol to cytochrome c, and complex IV transfers them to molecular oxygen. The transport of the electrons is coupled to the translocation of protons, which results in a proton gradient. Finally, this gradient can be used by complex V to produce ATP from ADP + Pi. Disturbances in the OXPHOS system cause mitochondrial disorders with an incidence of 1 in approximately 5000 births [2]. Isolated complex I deficiency is the most frequently diagnosed form accounting for almost 23% of all cases of childhood respiratorychain deficiency. Mitochondrial disorders may affect one or more tissues or organs, especially the ones with high metabolic energy rates. Therefore, mitochondrial diseases have a wide clinical vari-
* Corresponding author. Address: Department of Pediatrics, Nijmegen Center for Mitochondrial Disorders, Radboud University Nijmegen Medical Center, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Fax: +31 24 3618900. E-mail address: [email protected] (L.P. van den Heuvel). 1096-7192/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2010.03.015
ety [3,4]. This is also due to the bigenomic control of the OXPHOS system. Mutations in either the mitochondrial DNA (mtDNA) or nuclear encoded genes may cause complex I deficiency. NADH:ubiquinone oxidoreductase (E.C. 1.6.5.3.), or complex I, is the largest complex of the OXPHOS system, consisting of 45 subunits. Seven are encoded by the mtDNA and with 7 nuclear encoded subunits they form the ‘core subunits’ [5,6]. These are considered as the minimal constitution of complex I, since the subunits are involved in catalysis of electron transfer from NADH to ubiquinone and for generation of the membrane potential. One of the ‘core subunits’ is NDUFS1. This protein is the largest subunit of complex I and is located in the iron–sulfur fragment of the enzyme [7]. It contains 3 iron–sulfur clusters, N1b, N4 and N5 and plays an important role in the electron transfer [8]. Mutations in this gene are associated with isolated complex I deficiency. Benit and colleagues described 5 point mutations involving highly conserved amino acids (del222I, D252G, M707V, R241W, and R557X) and one large-scale deletion in this gene [9]. Additionally, 2 homozygous missense mutations (L231V and C1564A) were reported [10,11]. In this study we describe novel mutations in NDUFS1 in 3 unrelated patients with complex I deficiency expressed in cultured skin fibroblasts and we show that the assembly pattern of complex I is disturbed in these cells. Studies identifying new disease-causing mutations are necessary since molecular diagnosis is still lacking in many patients [12].
252
S.J.G. Hoefs et al. / Molecular Genetics and Metabolism 100 (2010) 251–256
Materials and methods Patients Patient 1 The patient, a girl, was born from healthy unrelated parents after a pregnancy complicated by gestational diabetes with a modest intrauterine growth retardation (weight 5th percentile). One older sib is a healthy boy. She had normal development in the first months of life, but started at age 8 months with abnormal crying and a regression of already acquired motor skills. At that age her brain MRI showed bilateral and symmetric leucodystrophic cystic lesions. Her clinical status worsened and she ultimately developed spasticity, microcephaly, mental retardation, and a progressive neuropathy. She died at the age of 12 years. Patient 2 The patient, a boy, was the third child of consanguineous parents (first cousins). One of the older sibs is a healthy boy, the other one was also affected. The index patient had problems with the central nervous system like leucoencephalopathy, episodic brainstem events, reduced spontaneous movement and he had an abnormal breathing pattern. His clinical status worsened and he had feeding problems. Furthermore he had muscle dystrophy and generalized hypotonia and died at the age of 8 months. The affected brother had a similar clinical picture. The age of onset was 4 months. In this patient the levels for lactate, pyruvate and alanine in both plasma and liquor indicate a mitochondrial disorder. He died at the age of 7 months.
blasts by standard procedures [15]. The nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence. The initiation codon is codon 1. BN–PAGE analysis and in-gel activity assay Mitochondria-enriched fractions were obtained by incubation with 2% (w/v) digitonin (Calbiochem) on ice for 10 min, followed by centrifugation at 10,000g and 4 °C for 10 min and washing with PBS, twice. Blue native-gradient gels (5–15%) were cast as described previously [16] and run with 40 lg (or 80 lg) of the solubilised mitochondrial protein. After electrophoresis, the gels were further processed for in-gel activity assay or second-dimension 10% SDS–PAGE followed by immunoblot analysis as described by Nijtmans et al. [17]. Antibodies and ECL detection Protein immunodetection was performed with primary antibodies directed against NDUFA9 (Invitrogen), NDUFS3 (Invitrogen), the CII 70 kDa subunit (SDHA; Invitrogen) and the CIII CORE2 subunit (UQCRC2; Invitrogen). Secondary antibodies used were peroxidise-conjugated anti-mouse IgGs (Invitrogen) and detection of the signal performed using ECL Plus (Amersham Biosciences) according to the manufacturer’s instructions. Results
Patient 3 This female patient was born after an unremarkable pregnancy, from non-related parents. The development was normal in the first months. Nystagmus was detected when she was 5 months of age. When she was 12 months of age she walked without support, but from that age her development stagnated motorically as well as mentally. After 15 months she started to lose neurological abilities and there was no doubt that she suffered from a neurological progressive disorder. Her clinical situation was dominated by a failure to thrive, crying, eating difficulties, spasticity, and mental retardation. She died at the age of 2 years.
Biochemical studies
Cell culture and biochemical analysis
MRI findings
Skin fibroblasts were cultured in M199 medium (Life Technologies) supplemented with 10% fetal calf serum and penicillin/streptomycin in a humified atmosphere of 95% air and 5% CO2 at 37 °C. Measurement of the mitochondrial respiratory-chain enzymes in skin fibroblasts and in muscle tissue were performed according to established procedures [13,14]. The values were expressed relative to the activity of cytochrome c oxidase and/or citrate synthase.
MRI pictures were available for patient 1 and 3. Patient 1: the first MRI was obtained at the age of 9 months. In the central white matter of both cerebral hemispheres, there were symmetric areas of marked hyperintensity on T2 and hypointensity of T1. The internal capsule was unaffected. The corpus callosum had normal signal, but was somewhat atrophic. The peripheral U-fibres were
Genetic analysis of the NDUFS1 gene Patient RNA was extracted from cultured fibroblasts by standard procedures. The RNA was reverse transcribed to cDNA by superscript II RNase H reverse transcriptase with oligo(dT) and random hexamer primers. Oligonucleotide primers were designed from the NDUFS1 gene sequence (Genbank Accession No. NM_005006). Primer sequences are available upon request. Polymerase chain reaction products were run on a 1.5% agarose gel and sequenced directly with the BigDye terminator cycle sequencing chemistry on a Applied Biosystems ABI PRISM 3130xl Genetic Analyzer. The mutations were also checked on genomic DNA level. Therefore, patient DNA was extracted from cultured skin fibro-
The activities of the respiratory-chain complexes were measured in skin fibroblasts of the 3 patients, in frozen muscle tissue of patient 1 and in fresh muscle tissue of patient 2, all displaying a marked decrease of complex I (Table 1). The fibroblast complex I enzymatic activities were 27%, 20%, and 24% of the lowest control value for the patients 1, 2 and 3, respectively. In fresh muscle tissue of patient 2, the enzymatic activity of complex III was decreased to 70% of the lowest control value. However, the activity of this complex in skin fibroblasts was within the control range.
Table 1 Respiratory-chain enzyme activities in skin fibroblasts, and frozen and fresh muscle tissue of the index patients. Complex Fibroblasts
I II III IV
Frozen muscle
Fresh muscle
P1
P2
P3
Control range
P1
Control range
P2
Control range
30 n.m. 1520 860
22 648 1522 1075
26 801 1867 953
110–206a 536–1027a 1270–2620a 680–1190b
53 n.m. n.m. 737
84–273c – – 520–2080c
7 119 1540 1305
70–250c 67–177c 2200–6610c 810–3120c
n.m. = not measured. Deficiencies are indicated in bold. a mU/U COX. b mU/mU CS. c mU/U CS.
S.J.G. Hoefs et al. / Molecular Genetics and Metabolism 100 (2010) 251–256
spared and the basal ganglia, brainstem and cerebellum were normal. The second MRI was done at the age of 11 months. There was at this stage a marked progression of the signal changes, which now were extending throughout the central white matter in the centrum semiovale. The temporal lobes were spared. There was progressive atrophy of the corpus callosum. The cortical grey matter, the basal ganglia, the brainstem, and cerebellum were still unaffected. The degree of signal change was very pronounced, the MRI signal not very different from that of the cerebrospinal fluid. The previously shown pathology, seen in the first MRI, now presented as ‘‘holes” within the central part of the new signal changes, having even more CSF-like signal (consistent with rarefaction). MR-spectroscopy was not performed. Patient 3: the first MRI was performed at the age of 5 months and was normal. At the age of 16 months a second MRI was done and following features were observed: extensive and confluent areas with markedly hyperintensity on T2 and hypointensity on T1 in subcortical and deep white matter of the cerebrum. It included the entire length of the corpus callosum, internal capsule (anterior limb and genu), extended through the cerebral peduncles and the ventral midbrain and ending in the central parts of pons from both sides. There was a striking sparing of the subcortical white matter of perirolandic gyri, as well as the central part of the corona radiata, together with the posterior limb of the internal
253
capsule and the optic radiations. The distribution of signal changes was symmetrical, dominating in the frontal and parietal lobes, but also affecting the periventricular areas in the temporal lobes. On diffusion sequence, there were multifocal areas with restricted diffusion scattered throughout the affected white matter, consistent with active demyelination. The last (third) MRI was obtained at the age of 17 months (Fig. 1). The affected white matter was slightly swollen, and the cerebellum still being normal. Furthermore, there was symmetrical, partial involvement of the nucleus caudatus. Otherwise the grey matter was normal. Multi-voxel MR-spectroscopy showed decreased NAA peaks and multifocal lactate peaks, anatomically corresponding to the areas of signal change. Molecular genetic studies in NDUFS1 gene We searched for the molecular defect causing these complex I deficiencies, by screening the cDNA of the NDUFS1 gene. In patient 1 we found a homozygous mutation, c.1855G>A, which introduced a substitution of amino acids (p.Asp619Asn). Verifying this mutation on genomic DNA revealed that this mutation was heterozygous (Fig. 2A). This suggests that one of the alleles results in an unstable RNA transcript making it undetectable on cDNA level. To detect a mutation on the second allele, the remaining exons of NDUFS1 were screened and this resulted in another heterozy-
Fig. 1. MRI findings of patient 3. (A) Cerebral T2-MRI of patient 3 at the age of 17 months shows marked white matter abnormalities. (B) Multi-voxel MRS shows decreased NAA signals and increased lactate peaks anatomically corresponding to the areas of signal change (left) compared to a normal spectrum from the gray matter of the same patient (right).
254
S.J.G. Hoefs et al. / Molecular Genetics and Metabolism 100 (2010) 251–256
gous mutation, c.1669C>T, which introduced a premature stop codon (p.Arg557X) (Fig. 2B). The father and brother were found to be heterozygous for the Arg557X mutation, and the mother for the Asp619Asn mutation. Sequence analysis of both cDNA and genomic DNA of patient 2 demonstrated the presence of a homozygous substitution of C to T (c.1222C>T; p.Arg408Cys) (Fig. 2C), which was also present in the other affected brother. Analysis of DNA of the third patient showed 2 heterozygous mutations (c.631–633delGAA; c.683T>C) (Fig. 2D and E). The first mutation causes a deletion of one amino acid (p.211delGlu), the second a substitution of ami-
no acids (p.Val228Ala). The father was found to be heterozygous for c.631–633delGAA and the mother heterozygous for c.683T>C. All above described mutations were not present in 100 healthy controls or 25 other patients with isolated complex I deficiency in cultured skin fibroblasts. Furthermore, all missense mutations and the deletion are present in strongly conserved amino acids. Computational modeling of the amino acid at position 228 reveals that this residue is located in a conserved region between two FeS clusters (N4 and N5) and that it is important for the structure around these clusters. The mutation p.Val228Ala changed a
Fig. 2. Molecular analysis of the NDUFS1 gene. (A–E) Electropherograms showing the wild-type sequence of NDUFS1 (top panels) and the nucleotide changes in the complex I deficient patients (bottom panels). Conservation of the altered amino acids is shown in Clustal W alignments. (A) Nucleotide change c.1669T>C, and amino acid change Arg557X in patient 1. (B) Nucleotide change c.1855G>A, and amino acid change Asp619Asn in patient 1. (C) Nucleotide change c.1222C>T, and amino acid change Arg408Cys in patient 2. (D) Nucleotide change c.631–622delGAA, and amino acid change 211delGlu in patient 3. (E) Nucleotide change c.683T>C, and amino acid changes Val228Ala in patient 3. (F) Overview of the NDUFS1 protein consisting of 727 amino acids, with the location of structural domains, according to InterProScan. The 5 different mutations that are described in this paper, are indicated in blue. The mutations described in earlier studies are indicated in black. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
S.J.G. Hoefs et al. / Molecular Genetics and Metabolism 100 (2010) 251–256
255
hydrofobe residue into a smaller but also hydrophobic residue, which results in a gap within the NDUFS1 subunit. Furthermore the structure around this residue will probably be less stable. This may have an effect on the binding and working of the FeS clusters and therefore on the transfer of the electrons. Modeling of the other amino acids changes was not possible. NDUFS1 contains several structural domains, as shown in Fig. 2F. All above described mutations are located in the NADH-quinone oxidoreductase chain G domain. Additionally, the mutations Arg408Cys, found in patient 2 and Arg557X and Asp619Asn, found in patient 1, are also located in the molybdopterin oxidoreductase domain (Fig. 2F). Effect of the mutations in NDUFS1 gene on complex I To study the effect of the mutations on the expression and activity complex I, we performed BN–PAGE analysis followed by Western blotting with an anti-NDUFA9 antibody and in-gel activity measurement with mitochondria-enriched fractions of cultured fibroblasts of the 3 patients. Fig. 3 shows severely reduced activities of complex I in the fibroblasts of the 3 patients compared to the control. Furthermore, the expression of subunit NDUFA9 was decreased in the patients compared to the control, suggesting a decrease in the amount of fully assembled complex I. Patients 2 and 3 showed also an extra band, which suggest the formation of subcomplexes. Together, these results show a severely reduced amount and activity of complex I in the patients’ fibroblasts. To further investigate the complex I assembly profile in the patient fibroblasts, we performed two-dimensional BN/SDS–PAGE analysis followed by immunoblotting with an antibody directed against NDUFS3. Fig. 4 displays less complex I and the formation of subcomplexes in fibroblasts of patients 1 and 3 compared to the control. Even more accumulation of subcomplexes was visible in cells of patient 2. These results suggest that the mutations in NDUFS1 cause a disturbance in the assembly and/or stability of complex I. Discussion In this report we describe 3 patients with complex I deficiency expressed in cultured fibroblasts harboring novel mutations in NDUFS1. Patient 1 was compound heterozygous for Arg557X and Asp619Asn. The first mutation has been previously reported in a
Fig. 3. The mutations in NDUFS1 cause decreased expression and activity of complex I. BN–PAGE analysis followed by in-gel activity measurement demonstrates a reduced complex I activity in the fibroblasts of the patients compared to the control. The expression of subunit NDUFA9 is also severely reduced, which indicates a decreased amount of whole complex I. Furthermore, the fibroblasts of patient 2 and 3 show an extra band, which suggest the formation of subcomplexes. The expression of the complexes II and III is not affected.
Fig. 4. The NDUFS1 mutations cause accumulation of subcomplexes. Two-dimensional BN/SDS–PAGE analysis followed by immunoblotting with an antibody directed against NDUFS3 shows less complex I in cells of patient 1 and 3 and the accumulation of subcomplexes in the fibroblasts of the three patients.
patient compound heterozygous for that mutation and the Arg241Trp mutation [9]. The second patient harbored a homozygous missense mutation (Arg408Cys) and the third patient was compound heterozygous for 211delGlu and Val228Ala. Several lines of evidence suggest that these mutations are the underlying cause of the complex I deficiencies in the patients: (1) the mutations cause nucleotide changes in conserved regions of the NDUFS1 gene; (2) the parents are heterozygous carriers and the mutations are not present in 100 healthy controls; (3) the patient fibroblasts show a decrease in amount and activity of complex I, measured by immunoblotting and in-gel activity assays; and (4) the assembly of complex I was also disturbed. The clinical features, especially the rapid progression of the disease, were similar in the patients. Patients 1 and 3 have a somewhat different distribution of signal changes. Common factors are the rapid progression of the disease, the central white matter distribution mostly sparing of the grey matter, and the degree of signal change with sharp demarcation. The extensive signal changes in the white matter with relative sparing of the gray matter inclusive the basal ganglia is different from the classical findings in Leigh disease (being dominant in gray matter). However, MRI findings in these patients are consistent with described cases in the literature for isolated complex I deficiency [18]. The lactate peaks in patient 3 are consistent with anaerobe metabolism and are strongly suggestive of mitochondrial disease. Complex I deficiency combined with a decrease in activity of complex III, like the enzyme activities in muscle tissue of patient 2, has been described before by Budde and colleagues [19]. These patients harbored a mutation in the NDUFS4 gene. An explanation for these combined deficiencies could be the formation of supercomplexes in which complex I and III occur together (CI/CIII2) [20]. This indicates that screening of complex I subunits including NDUFS1 should be considered not only in isolated complex I deficient patients but also in patients with combined complex I and III deficiency. Furthermore, it is remarkable that the activity of complex I in fibroblasts of the 3 patients was very low (<30% of the lowest control value). An earlier study of Loeffen et al. showed a mean residual activity of 49% of the lowest control value measured in a cohort of 27 patients with isolated complex I deficiency [4]. Since thera-
256
S.J.G. Hoefs et al. / Molecular Genetics and Metabolism 100 (2010) 251–256
peutic strategies for complex I deficiency are hardly available, it is important to elucidate the possible genetic causes for prenatal diagnostics and genetic counseling. A good pre selection is important, since more than 70 candidate genes for complex I deficiency are known at present. Therefore, above mentioned low residual complex I activities suggest that screening of the NDUFS1 gene is preferred when patients have very low complex I activities expressed in cultured fibroblasts. NDUFS1 is the largest subunit of complex I, and is suggested to be incorporated into this complex in stage 6 of the assembly process, as proposed by Vogel et al. [21]. In line with this idea is the fact that in patient 2 and 3 an additional band of about 800 kDa is visible on BN blots (Fig. 3) which is lacking in the control cells. Such a subcomplex also occurs in patients with mutations in NDUFS4, NDUFV1, and NDUFS6 and is believed to represent a complex I subassembly which lacks subunits NDUFV1, NDUFV2, NDUFS4, NDUFS1, and NDUFS6 [20]. Although all patients show decreased amounts of fully assembled complex I, this decrease is more pronounced in patient 1 and 3. Nevertheless, the similar decrease in enzyme activity as measured by spectrophotometrically and by in-gel activity (Fig. 3) is comparable in all patients suggesting that beside the decreased amount of complex I also the intrinsic enzyme activity of the remaining complex is also affected in these patients. Identifying the underlying cause of complex I deficiency is important to get more insight in the function of this complex. New strategies like genomic microarray screening or exome sequencing of the nuclear complex I and assembly genes may be possible in the near future. Research to identify new mutations causing complex I deficiency is still ongoing to improve genetic counseling and possibilities for prenatal diagnostics.
Web resources The URLs for data presented herein are as follows: Genbank, http://www.ncbi.nlm.gov/Genbank. InterProScan, http://www.ebi.ac.uk/Tools/InterProScan.
Acknowledgments This work was supported by the European Community’s sixth Framework Programme for Research, Priority 1 ‘‘Life sciences, genomics and biotechnology for health”, contract numbers LSHM-CT-2004-005260 (MITOCIRCLE). The authors would like to thank Hanka Venselaar (Centre for Molecular and Biomolecular Informatics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands) for the computational modeling study.
References [1] M. Saraste, Oxidative phosphorylation at the fin de siècle, Science 283 (1999) 1488–1493. [2] J. Smeitink, L. van den Heuvel, S. DiMauro, The genetics and pathology of oxidative phosphorylation, Nat. Rev. Genet. 2 (2001) 342–352. [3] S. Pitkanen, A. Feigenbaum, R. Laframboise, B.H. Robinson, NADH-coenzyme Q reductase (complex I) deficiency: heterogeneity in phenotype and biochemical findings, J. Inherit. Metab. Dis. 19 (1996) 675–686. [4] J.L. Loeffen, J.A. Smeitink, J.M. Trijbels, A.J. Janssen, R.H. Triepels, R.C. Sengers, L.P. van den Heuvel, Isolated complex I deficiency in children: clinical, biochemical and genetic aspects, Hum. Mutat. 15 (2000) 123–134. [5] J. Carroll, I.M. Fearnley, J.M. Skehel, R.J. Shannon, J. Hirst, J.E. Walker, Bovine complex I is a complex of 45 different subunits, J. Biol. Chem. 281 (2006) 32724–32727. [6] U. Brandt, Energy converting NADH:quinone oxidoreductase (complex I), Annu. Rev. Biochem. 75 (2006) 69–92. [7] J.A. Smeitink, J.L. Loeffen, R.H. Triepels, R.J. Smeets, J.M. Trijbels, L.P. Van den Heuvel, Nuclear genes of human complex I of the mitochondrial electron transport chain: state of the art, Hum. Mol. Genet. 7 (1998) 1573–1579. [8] M. Finel, Organization and evolution of structural elements within complex I, Biochim. Biophys. Acta 1364 (1998) 112–121. [9] P. Benit, D. Chretien, N. Kadhom, P. de Lonlay-Debeney, V. Cormier-Daire, A. Cabral, S. Peudenier, P. Rustin, A. Munnich, A. Rotig, Large-scale deletion and point mutations of the nuclear NDUFV1 and NDUFS1 genes in mitochondrial complex I deficiency, Am. J. Hum. Genet. 68 (2001) 1344–1352. [10] M.A. Martin, A. Blazquez, L.G. Gutierrez-Solana, D. Fernandez-Moreira, P. Briones, A.L. Andreu, R. Garesse, Y. Campos, J. Arenas, Leigh syndrome associated with mitochondrial complex I deficiency due to a novel mutation in the NDUFS1 gene, Arch. Neurol. 62 (2005) 659–661. [11] A. Iuso, S. Scacco, C. Piccoli, F. Bellomo, V. Petruzzella, R. Trentadue, M. Minuto, M. Ripoli, N. Capitanio, M. Zeviani, S. Papa, Dysfunctions of cellular oxidative metabolism in patients with mutations in the NDUFS1 and NDUFS4 genes of complex, J. Biol. Chem. 281 (2006) 10374–10380. [12] D.R. Thorburn, Mitochondrial disorders: prevalence, myths and advances, J. Inherit. Metab. Dis. 27 (2004) 349–362. [13] J. Smeitink, R. Sengers, F. Trijbels, L. van den Heuvel, Human NADH:ubiquinone oxidoreductase, J. Bioenerg. Biomembr. 33 (2001) 259–266. [14] A.J. Janssen, F.J. Trijbels, R.C. Sengers, L.T. Wintjes, W. Ruitenbeek, J.A. Smeitink, E. Morava, B.G. van Engelen, L.P. van den Heuvel, R.J. Rodenburg, Measurement of the energy-generating capacity of human muscle mitochondria: diagnostic procedure and application to human pathology, Clin. Chem. 52 (2006) 860– 871. [15] S.A. Miller, D.D. Dykes, H.F. Polesky, A simple salting out procedure for extracting DNA from human nucleated cells, Nucleic. Acids. Res. 16 (1988) 1215. [16] M.A. Calvaruso, J. Smeitink, L. Nijtmans, Electrophoresis techniques to investigate defects in oxidative phosphorylation, Methods 46 (2008) 281–287. [17] L.G. Nijtmans, N.S. Henderson, I.J. Holt, Blue native electrophoresis to study mitochondrial and other protein complexes, Methods 26 (2002) 327–334. [18] S. van der Knaap, J. Valk, Magnetic Resonance of Myelination and Myelin Disorders, third ed., Springer, 2005. [19] S.M. Budde, L.P. van den Heuvel, A.J. Janssen, R.J. Smeets, C.A. Buskens, L. DeMeirleir, R. van Coster, M. Baethmann, T. Voit, J.M. Trijbels, J.A. Smeitink, Combined enzymatic complex I and III deficiency associated with mutations in the nuclear encoded NDUFS4 gene, Biochem. Biophys. Res. Commun. 275 (2000) 63–68. [20] M. Lazarou, M. McKenzie, A. Ohtake, D.R. Thorburn, M.T. Ryan, Analysis of the assembly profiles for mitochondrial- and nuclear-DNA-encoded subunits into complex I, Mol. Cell. Biol. 27 (2007) 4228–4237. [21] R.O. Vogel, C.E. Dieteren, L.P. van den Heuvel, P.H. Willems, J.A. Smeitink, W.J. Koopman, L.G. Nijtmans, Identification of mitochondrial complex I assembly intermediates by tracing tagged NDUFS3 demonstrates the entry point of mitochondrial subunits, J. Biol. Chem. 282 (2007) 7582–7590.