Sequence analysis of mitochondrial and nuclear candidate genes of complex I in children with deficiency of this complex and exhibiting Leigh-like syndrome has revealed, in one of them, a novel mutation in the NDUFS4 gene encoding the 18 kDa subunit. Phosphorylation of this subunit by cAMP-dependent protein kinase has previously been found to activate the complex. The present mutation consists of a homozygous G→A transition at nucleotide position +44 of the coding sequence of the gene, resulting in the change of a tryptophan codon to a stop codon. Such mutation causes premature termination of the protein after only 14 amino acids of the putative mitochondrial targeting peptide. Fibroblast cultures from the patient exhibited severe reduction of the rotenone-sensitive NADH→UQ oxidoreductase activity of complex I, which was insensitive to cAMP stimulation. Two-dimensional electrophoresis showed the absence of detectable normally assembled complex I in the inner mitochondrial membrane. These findings show that the expression of the NDUFS4 gene is essential for the assembly of a functional complex I.

INTRODUCTION

Human diseases associated with disorders in the mitochondrial energy metabolism occur with an estimated incidence of 1 in 10 000 live births. Complex I (NADH→ubiquinone oxidoreductase; E.C.1.6.5.3) deficiency represents one of the most frequent of these disorders (1). Loeffen et al. (2), on examining numerous tissue specimens from children with suspected oxidative phosphorylation (OXPHOS) disorders, found complex I deficiency in ∼50% of the patients.

In mammalian mitochondria, complex I catalyses the proton-motive oxidation of NADH by ubiquinone. It consists of 43 subunits (3,4). Seven subunits (ND1–ND6 and ND4L) are encoded by the mitochondrial genome (5,6), the others by nuclear genes. The cDNAs of 35 of the human nuclear genes coding for complex I subunits have been sequenced (7,8), but for most of them the function is unknown (9). The NDUFS4 gene product is a complex I 18 kDa subunit of 175 amino acids, which appears to be highly conserved in all the known mammalian sequences (1012). The mature form of 133 amino acids does not bind to any prosthetic group. In mammals it has, at position 129–131, a canonical cAMP-dependent protein kinase phosphorylation consensus site (RVS) in which the serine residue is phosphorylated (Fig. 1) (13,14). The protein has a leader sequence, removed after import into mitochondria (10), that also contains a phosphorylation consensus site (RTS) in the human protein, at positions –7 and –5. Papa et al. (15,16) have found that cAMP promotes serine phosphorylation in the nuclear-encoded 18 kDa subunit of complex I, which results in activation of the normal, rotenone-sensitive NADHUQ oxidoreductase of the complex (13,17). Smeitink et al. (11) have identified, in patients with complex I deficiency, one case with a 5 bp duplication in the cDNA of the NDUFS4 gene coding for the 18 kDa subunit of complex I and two cases with premature termination of the same subunit (18), all leading to destruction of the phosphorylation consensus site present in the C-terminal region of the protein. The 5 bp duplication has been found to abolish the cAMP-dependent phosphorylation of the protein and activation of complex I in fibroblast cultures from the patient (19). These observations assign a critical role to the NDUFS4 gene in the regulation of the activity of complex I. Since complex I is, at least under certain conditions, the rate limiting enzyme of the respiratory chain, cAMP can regulate, through modulation of complex I, the overall NAD-linked respiration in response to a variety of neuro-hormone effectors (13).

Here, we report on the identification of a novel homozygous G→A transition at nucleotide +44 of the coding sequence of the NDUFS4 gene in a patient with complex I deficiency affected by a Leigh-like syndrome. This mutation, which results in the change of the tryptophan codon (TGG) to a stop codon (TAG), causes the premature termination of the protein after only 14 amino acids of the putative mitochondrial targeting peptide (Fig. 1). Fibroblast cultures from the patient exhibited severe reduction of the normal rotenone-sensitive NADH→UQ oxidoreductase activity, which was completely insensitive to cAMP. Two-dimensional gel electrophoresis showed the absence of detectable normally assembled complex I in the inner mitochondrial membrane.

RESULTS

Analysis of candidate genes in complex I deficiency

Fibroblast cultures from three patients (1136, 1119 and 743) affected by complex I deficiency, deriving from a collection of OXPHOS patients at the ‘C. Besta’ Institute, Milan, Italy, were processed for mutational analysis of candidate genes encoding different subunits of the complex. A search for most of the currently known mutations associated with complex I deficiency was first performed by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis in both mitochondrial and nuclear DNA. The following pathogenic mitochondrial DNA mutations were absent in all three patients: 14484 T→C (ND-6), 14459 G→A (ND-6), 11778 C→A (ND-4), 8356 T→C (tRNA Lys), 8344 A→G (tRNA Lys), 3460 G→A (ND-1), 3271 T→C (tRNA Leu) and 3243 A→G (tRNA Leu) (20,21). Likewise, negative results were obtained by searching for the following mutations in nuclear genes: A341V, R59X and T423M in the NDUFVI gene (22); V122M in the NDUFS7 gene (23); P79L and R102H in the NDUFS8 gene (24); and a 5 bp duplication (AAGTC at positions 466–470) in the NDUFS4 gene (11).

A systematic analysis of RT–PCRed cDNAs encoding evolutionary conserved subunits of complex I (3,25) or subunits with putative functions (8) was carried out by automated sequencing. Sequences stored in the MitoPick database (http://www-dsv.cea.fr/thema/mitoPick/Default.html) were used for comparative analysis. NDUFS7, NDUFS8, NDUFV1, NDUFV3 and NDUFS4 cDNAs were completely sequenced in the three patients. The analysis revealed the presence of a homozygous transition G→A at nucleotide +44 of the coding sequence of the NDUFS4-18 kDa subunit in patient 1136 (Figs 1 and 2). The G→A mutation changes a codon for tryptophan to a stop codon and causes a premature termination of the protein after 14 amino acids in the mitochondrial leader peptide (Fig. 1). Immunoblot analysis with a polyclonal antibody against the 18 kDa subunit of complex I in fact showed this to be present in control fibroblasts, but to be missing in the patient’s fibroblasts (Fig. 3). Direct sequencing of an RT–PCRed region encompassing the mutation of the NDUFS4 cDNAs from both the parents revealed the presence of the G→A transition in heterozygosity (Fig. 2). Sequence and RFLP analysis on genomic DNA from the patient and both parents confirmed that the +44 mutation was homozygous in the patient and heterozygous in the parents (Fig. 4), indicating the mutation to be consistent with an autosomal recessive mode of inheritance. The mutation was absent in the other two patients analysed.

Additional substitutions found in the three patients are listed in Table 1. Two nucleotide changes were found in the NDUFS4 cDNA: the C198A in patient 1136 and the G312A in patients 1136 and 743. Using the BLASTN option of basic local alignment search tool (BLAST) and searching the expressed sequence tag database (dbEST), both changes were found in 3 and 8 of 50 human ESTs, respectively, suggesting them to be polymorphisms. Likewise, we found the heterozygous T68C transition, introducing an MspI restriction site in the NDUFS7 gene (23), in patient 1136, and an A→C transversion, 13 nucleotides after the stop codon, in all the three patients examined. The T68C transition, which has been described with comparable distribution in complex I-deficient patients and in the control population (23), occurs in 7 of 12 ESTs and, the second change, in 11 of 12 ESTs. Both substitutions were, presumably, polymorphisms. Conversely, two changes in the NDUFV3 gene compared with the sequence (26) and reported in MitoPick were found in all three patients and appeared in all the 11 ESTs present in the dbEST, thus indicating errors in the sequence of this gene presently reported in MitoPick. A single nucleotide change, a C→T transition at position 14 in the 3′ UTR of the NDUFS8 cDNA, has been found in patients 1119 and 743 but not in dbEST. No variants were found in the coding sequence for the NDUFV1 gene in all three patients analysed.

Activity and two-dimensional polyacrylamide gel electrophoresis (PAGE) pattern of complex I

Screening of the activities of OXPHOS complexes in muscle homogenates and in skin fibroblast cultures from the NDUFS4-mutant patient 1136 revealed a marked depression in the activity of complex I in both tissues (Table 2). In order to better characterize the complex I deficiency in the NDUFS4-mutant patient, we analysed the activity of the NADH→UQ oxidoreductase and cytochrome c oxidase in fibroblast cultures. Cells were grown under two conditions: serum starvation, which was previously found to downregulate cAMP-dependent activation of the complex, and cholera toxin-treated cells, in which production of intracellular cAMP maximally activates the complex (17). The Vmax and Km values of rotenone-sensitive NADH→UQ oxidoreductase activity presented in Table 3 show that in the fibroblasts from the patient the rotenone-sensitive NADH→UQ oxidoreductase activity was approximately zero both in the absence and presence of cholera toxin. The rotenone-insensitive NADH→UQ oxidoreductase activity in the patient’s fibroblast (∼20% of the total) was equal to that exhibited by control fibroblasts. Spectrophotometric measurement of the cytochrome c oxidase activity in the serum-starved fibroblasts from the patient showed this to be practically comparable to that of control fibroblasts.

Two-dimensional blue native (BN)–PAGE/SDS–PAGE (27) showed, in the mitoplast fraction of control fibroblasts, an assembled complex I, in which the 18 kDa subunit was immunodetected by the specific antibody. No 18 kDa subunit, as expected, nor a detectable band corresponding to normally assembled complex I was, on the contrary, found in mitoplasts from the patient’s fibroblasts (Fig. 3).

DISCUSSION

The new mutation in the cDNA of the NDUFS4 gene coding for the 18 kDa subunit of complex I, described in this paper, consists of a G→A transition at nucleotide +44 of the coding sequence and changes a codon for tryptophan to a stop codon, resulting in premature termination of the protein (Fig. 1). It is segregated in the patient’s family with an autosomal recessive mode of inheritance.

This nonsense mutation completely suppressed the normal rotenone-sensitive NADH→UQ activity of the complex, both in the absence and presence of cholera toxin-induced cAMP production, and prevented its normal assembly in the inner mitochondrial membrane. Interestingly, the 18 kDa subunit is apparently located in a strategic position within the complex, at the junction between the peripheral mass protruding in the matrix and the membrane moiety of complex I (3). The present observations thus show that the 18 kDa subunit, in addition to its role in regulation of the activity of complex I (13,17), also plays a critical role in the assembly of the complex. In Neurospora crassa, inactivation of the nuo21 gene coding for the 21 kDa subunit of complex I, considered to be orthologous to the mammalian NDUFS4 18 kDa subunit (28), did produce alterations in the catalytic activity and subunit assembly of complex I, which need to be further clarified (29). In an animal model of mitochondrial myopathy and cardiomyopathy, recently created by inactivation of the heart/muscle-specific isoform of the adenine nucleotide translocator (30), an upregulation of the expression of the 18 kDa subunit has been reported, suggesting that NDUFS4 is an important gene involved in mitochondrial biogenesis and function (31). It is remarkable that, although the nonsense mutation in the NDUFS4 gene resulted in suppression of the normal assembly of a functional complex I, the patient with such a defect survived until the age of 7 months. Furthermore, the other patients, with different mutations in the same gene, survived a few months after birth (11,18). It is conceivable that, in these patients, a metabolic condition is set up in which the glycerol-phosphate shuttle, which mediates mitochondrial oxidation of glycolitic NADH bypassing complex I, is able to replace complex I in supporting mitochondrial energy metabolism, at least in part and under the limited functional activities in the first months of life. Evidence for a significant contribution of NADH shuttles in sustaining mitochondrial energy metabolism and glucose-induced insulin secretion in pancreatic islets has been obtained in transgenic mice (32).

The finding, in a relatively short time, of four families bearing mutations in the coding region of the NDUFS4 gene, which is localized on chromosome 5 (33), shows this to represent a hotspot of mutations in the genetic apparatus of oxidative phosphorylation. This might be related to the finding of complex I deficiency in ageing (34) and in certain human diseases (35), as well as to its apparent involvement in apoptosis (36).

MATERIALS AND METHODS

Case report

The patient 1136, in which the nonsense mutation in the NDUFS4 gene was discovered, was a baby girl, born at term by caesarean section from apparently non-consanguineous healthy parents, after a pregnancy complicated by a pre-eclamptic syndrome. The Apgar score at birth was 10/10, but lingual fasciculations were noted. From the first 2 weeks after birth the patient suffered from tonic–clonic seizures, persistent vomiting and failure to thrive. The clinical picture was characterized by a rapidly downhill course with severe, progressive psychomotor delay, loss of contact, profound hypotonia and seizures. Additional findings were severe lactic acidosis, severe hypertrophic cardiomyopathy and bilateral hyperecogenic signals at the ultrasound scan of the basal ganglia. The latter features suggested a diagnosis of Leigh syndrome. A muscle biopsy revealed neither ragged-red nor cytochrome c oxidase-negative fibres. However, a diffuse lipid accumulation was suggestive of a mitochondrial abnormality. This was confirmed by the identification of a severe defect of complex I in both muscle homogenate and cultured skin fibroblasts (Table 2). A prolonged apnoeic episode with cyanosis was followed by a profound, persistent comatose status leading to death at 7 months of age.

Fibroblast cell cultures

Primary fibroblast lines were established from skin biopsies of three children showing a severe deficiency in complex I activity. The fibroblasts were grown in high glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (EuroClone), 110 µg/ml pyruvate and 50 µg/ml uridine. In the experiments for kinetic characterization of complex I, once the cells were at 75% confluence, the medium was replaced with DMEM with 0.5% FBS. After 3 days of serum starvation, cells were incubated at 37°C and treated, where indicated, for 3 h with 1 µg/ml cholera toxin (Sigma-Aldrich) and 100 µM 3-isobutyl-1-methylxantine (IBMX). The fibroblasts were harvested from Petri dishes with 0.05% trypsin, 0.02% EDTA and phosphatase inhibitors (5 mM NaF, 500 nM okadaic acid and 1 mM Na-orthovanadate) (37) and washed in PBS pH 7.4 with 5% calf serum.

Biochemical analysis

Screening of mitochondrial enzymes was performed on fibroblasts from patient’s skin biopsy and cultivated in the above medium at semi-confluency, as described by Tiranti et al. (38). The Vmax and Km values of the NADH→UQ oxidoreductase activity of complex I were obtained from typical Lineweaver–Burk plots of the normal rotenone-sensitive NADH→UQ oxidoreductase in serum-starved fibroblasts, previously exposed to ultrasonic energy to eliminate permeability barriers for exogenous NADH and decylubiquinone as described by Scacco et al. (17). Elevation of intracellular cAMP concentration was produced by exposure of fibroblast cultures to cholera toxin (17,39).

Mutational analysis of candidate genes of complex I subunits

Poly(A)+ mRNA was extracted from patients’ cultured fibroblasts (patients 1136, 1119 and 743) and from the lymphocytes of the parents of patient 1136 using the MicrofastTrack kit (Invitrogen). An aliquot of poly(A)+ mRNA was reverse transcribed by using the random-primer method of the First Strand cDNA Synthesis kit for RT–PCR (AMV) (Roche Molecular Biochemicals). Five microlitres of cDNAs from patients 1136, 1119 and 743 was used for PCR amplification to generate fragments containing the open reading frames (ORFs) for NDUFS7, NDUFS8, NDUFV1, NDUFV3 and NDUFS4. For NDUFS7 cDNA, oligonucleotides for PCR amplification were designed according to human ESTs (GenBank accession no. BE794266) and to Triepels et al. (23). Primers for NDUFS8 cDNA amplification were as described by Loeffen et al. (24). PCR amplification conditions for both cDNAs were as described by Triepels et al. (23) and Loeffen et al. (24). NDUFV1 cDNA was amplified using the following primer pair to obtain a fragment 1497 bp in length encompassing the 1392 bp ORF: forward (5′-CAGCCTCAGTGCTATGAAGGTG-3′); reverse (5′-CCAGCATTCCACATGGATAGAC-3′). The amplification was performed for 35 cycles (1 min at 94°C, 1 min at 63°C and 1 min and 30 s at 72°C), preceded by a denaturation of 5 min at 94°C and followed by an extension of 7 min at 72°C. A 469 bp fragment containing the 324 bp ORF of the NDUFV3 cDNA was amplified with the following primer pair: forward (5′-AAGCTGCTGTGGCCCTGCTTG-3′); reverse (5′-CGATTATGCACAGCGGGCCTTG-3′), for 35 cycles (30 s at 94°C, 30 s at 66°C and 30 s at 72°C), preceded by a denaturation of 5 min at 94°C and followed by an extension of 7 min at 72°C. A 529 bp fragment encompassing most of the coding region of the NDUFS4 gene, using as forward primer, 18k-F (5′-AAGATGGCGGCGGTCTCAATGTC-3′) and as reverse primer, 18k-R1 (5′-TATTTTGTGGATACTCTTGTTC-3′), was generated. After an initial denaturation step of 5 min at 94°C, the PCR was carried out for 35 cycles (30 s at 94°C, 30 s at 53°C and 1 min at 72°C), followed by an extension of 7 min at 72°C. From parents’ cDNAs, a 117 bp fragment was amplified using, as forward primer, 18k-F and as reverse primer, 18k-R122 (5′-GGAAGTCCTCAACGACCTGGTC-3′), with an initial denaturation step of 5 min at 94°C, for 35 cycles (30 s at 94°C, 30 s at 55°C and 30 s at 72°C) followed by an extension of 7 min at 72°C. The PCR products were analysed by electrophoresis on an agarose gel of a percentage (w/v) depending on the different sizes of the amplified fragments, with 0.5 µg/ml ethidium bromide in 40 mM Tris, 20 mM sodium acetate and 2 mM EDTA pH 7.8 (1× TAE). The bands of expected sizes for the different cDNAs were cut from the gel and eluted by Concert extraction kit (Gibco BRL). Cycle sequencing of the gel purified fragments was performed on an automated ABI 310 sequencer using Big Dye Termination kit according to the manufacturer’s instructions (PE Applied Biosystems). For NDUFS7 and NDUFS8 cDNAs, oligonucleotides for sequence analysis were the same as described by Triepels et al. (23) and Loeffen et al. (24). For NDUFV1 cDNA, the following primers were used: 5′-CTACAATGAGGCCTCCAATCTGC-3′, 5′-TTGCACTGTGGAGGAGGAGATG-3′, 5′-ATAGCCAGAGCCACAAGCATTC-3′ and 5′-TGCCATCACCTTGTTCATCCAG-3′. For NDUFV3 and NDUFS4 cDNAs, sequence analysis was performed with the same primers used for PCR amplification.

Previously reported mutations were searched in the NDUFV1 gene as described by Schuelke et al. (22), in the NDUFS7 gene as described by Triepels et al. (23), in NDUFS8 as described by Loeffen et al. (24) and in the 5 bp duplication in NDUFS4 as described by van den Heuvel et al. (11).

DNA analysis

DNA was extracted from the cultured fibroblasts of patient 1136 and from the parents’ lymphocytes according to a standard protocol including SDS lysis, Proteinase K digestion and phenol/chloroform extraction. PCR amplification of the region encompassing the G→A mutation in the NDUFS4 gene was performed using as forward primer, 18k-F and as reverse primer, 18k-R-108 (5′-ACCTGGTCGGAACCCTGGAAAC-3′) with 35 cycles of 30 s at 94°C, 30 s at 55°C and 30 s at 72°C, preceded by an initial denaturation step of 5 min at 94°C and followed by an extension of 7 min at 72°C. The PCR product was analysed on a 3% agarose gel with 0.5 µg/ml ethidium bromide in 1× TAE. A band of the expected size was cut from the gel and eluted by using the Concert extraction kit (Gibco BRL). Cycle sequencing of the fragment was performed using the same primers as for the PCR amplification.

For PCR–RFLP analysis of the G44A transition, a reverse mismatch primer, 5′-CGGAAAGGGCAGCTACAGCCACTGCCCTTCTCAGC-3′ (mismatch underlined), was constructed creating an AluI restriction site (5′-AG+CT-3′) in the case of G→A mutation (the symbol + indicates the break point as created by the restriction enzyme). The mutation present in our patient introduces the restriction site. A forward primer, 18k-F-76 (5′-GCACTGGCTTGAGAACGAAGGAAG-3′), at nucleotide –76 from the initiation codon, was synthesized on the basis of the genomic contig sequence GenBank accession no. AC024569 which contains the entire NDUFS4 human gene. A 155 bp fragment was produced by PCR amplification with 35 cycles of 30 s at 94°C, 30 s at 57°C and 30 s at 72°C, preceded by an initial denaturation step of 5 min at 94°C, followed by an extension of 7 min at 72°C. The product was digested with AluI restriction enzyme into fragments of 34 and 121 bp in the presence of the mutation. Resulting RFLP was separated on a 3% agarose–nusieve gel (1:2) in the presence of 0.5 µg/ml ethidium bromide in 1× TAE.

Analysis of mutations of mitochondrial DNA was performed according to Santorelli et al. (40).

Mitoplast preparation, electrophoresis and immunoblotting

Mitoplasts were prepared from freshly harvested fibroblasts exposed to 0.5 mg of digitonin per mg of cellular protein for 15 min on ice as described by Scacco et al. (17). The mitoplasts were then re-suspended in 750 mM aminocaproic acid, 50 mM Bis–Tris, 0.5 mM EDTA pH 7.0 containing 0.3% (w/v) lauryl maltoside instead of 1%. Separation of OXPHOS complexes was performed by two-dimensional BN/SDS–PAGE of mitoplasts as described by Schagger (27). Immunoblotting was performed as described by Scacco et al. (17) with polyclonal antibody specific for the 18 kDa subunit of complex I produced by Neosystem.

ACKNOWLEDGEMENTS

We are grateful to Drs T. Cocco and S. Scacco for helpful discussion and technical advice. This work was financially supported by grants from the National Project on ‘Bioenergetics and Biomembranes’, the Project on ‘Molecular, Cellular, Diagnostic and Epidemiological Analysis of Pediatric and Neurologic Diseases’ (Cluster 04) of the Italian Ministry for the University and Scientific and Technological Research (MURST) and from the finalized Project for Biotechnology of the Italian Research Council (CNR, Rome) Project nos 99.00430.PF49 and 99.03622.PF49.

+

To whom correspondence should be addressed. Tel: +39 080 5478428; Fax: +39 080 5478429; Email: papabchm@cimedoc.uniba.it

Figure 1. Nucleotide sequence coding for the NDUFS4 18 kDa subunit precursor and predicted amino acid sequence of the 18 kDa subunit precursor. The locations of all the mutations found along the NDUFS4 cDNA sequence are shown. Arrow, the pathogenic mutation; asterisk, silent polymorphisms found by us. The mutations reported by van den Heuvel et al. (11) and Budde et al. (18) are indicated with a small circle. It should be noted that the 5 bp duplication of the sequence at positions 466–470 (aagtc) results in a frameshift at codon K158 and destroys the phosphorylation site RVS at position 129–131 (11) The phosphorylation sites in the mitochondrial leader sequence (position –7/–5) and in the mature protein (position 129–131) of the predicted protein sequence are underlined. The mitochondrial targeting signal peptide at position 1–42 (amino acids), corresponding to 1–126 (nucleotides), is in italics.

Figure 1. Nucleotide sequence coding for the NDUFS4 18 kDa subunit precursor and predicted amino acid sequence of the 18 kDa subunit precursor. The locations of all the mutations found along the NDUFS4 cDNA sequence are shown. Arrow, the pathogenic mutation; asterisk, silent polymorphisms found by us. The mutations reported by van den Heuvel et al. (11) and Budde et al. (18) are indicated with a small circle. It should be noted that the 5 bp duplication of the sequence at positions 466–470 (aagtc) results in a frameshift at codon K158 and destroys the phosphorylation site RVS at position 129–131 (11) The phosphorylation sites in the mitochondrial leader sequence (position –7/–5) and in the mature protein (position 129–131) of the predicted protein sequence are underlined. The mitochondrial targeting signal peptide at position 1–42 (amino acids), corresponding to 1–126 (nucleotides), is in italics.

Figure 2. Sequence analysis of NDUFS4 cDNA. Chromatograms represent the forward sequence of NDUFS4 cDNA showing the region spanning the G44A mutation in a control sample in the patient and in both parents. The mutation is indicated by an arrow. For further details see Materials and Methods.

Figure 2. Sequence analysis of NDUFS4 cDNA. Chromatograms represent the forward sequence of NDUFS4 cDNA showing the region spanning the G44A mutation in a control sample in the patient and in both parents. The mutation is indicated by an arrow. For further details see Materials and Methods.

Figure 3. BN–SDS two-dimensional gel separating the constituent subunits of respiratory chain complexes and ATP synthase. Protein aliquots of mitoplasts from control and patient fibroblasts were separated, in the first dimension, by BN–PAGE and, in the second dimension, by SDS–PAGE. (A) The position of respiratory complexes I (CI), III (CIII), IV (CIV) and ATP synthase (CV) was revealed by silver staining. (B) Immunoblot analysis of the 18 kDa subunit of complex I in the fractions separated by two-dimensional BN/SDS–PAGE of mitoplasts. For further details see Materials and Methods.

Figure 3. BN–SDS two-dimensional gel separating the constituent subunits of respiratory chain complexes and ATP synthase. Protein aliquots of mitoplasts from control and patient fibroblasts were separated, in the first dimension, by BN–PAGE and, in the second dimension, by SDS–PAGE. (A) The position of respiratory complexes I (CI), III (CIII), IV (CIV) and ATP synthase (CV) was revealed by silver staining. (B) Immunoblot analysis of the 18 kDa subunit of complex I in the fractions separated by two-dimensional BN/SDS–PAGE of mitoplasts. For further details see Materials and Methods.

Figure 4. Segregation of the NDUFS4 nonsense mutation within the family, as revealed by PCR–RFLP analysis of genomic DNA. Using a reverse mismatch primer, the presence of the G44A transition results in the digestion by AluI restriction enzyme of the 155 bp PCR product into two fragments, a 121 bp (arrow) and 34 bp (not shown). This analysis shows that both parents are heterozygous for the G44A transition, whereas the affected baby is homozygous. For further details see Materials and Methods.

Figure 4. Segregation of the NDUFS4 nonsense mutation within the family, as revealed by PCR–RFLP analysis of genomic DNA. Using a reverse mismatch primer, the presence of the G44A transition results in the digestion by AluI restriction enzyme of the 155 bp PCR product into two fragments, a 121 bp (arrow) and 34 bp (not shown). This analysis shows that both parents are heterozygous for the G44A transition, whereas the affected baby is homozygous. For further details see Materials and Methods.

Table 1.

Nucleotide changes found in the cDNAs of NDUFS4, NDUFS7, NDUFV3 and NDUFS8 genes sequenced in the three patients with complex I deficiency

Gene  Mutations (cDNA position)  Change  Patients 
NDUFS4 (18 kDa)  +198 C→A  Silent  1136 
  +312 G→A  Silent  1136, 743 
NDUFS7 (20 kDa)  +68 T→C heterozygous  Leu→Pro  1136 
  3′ UTR +13 A→C  –  1136, 1119, 743 
NDUFV3 (10 kDa)  +22 A→C  Silent  1136, 1119, 743 
  +25 A→C  Lys→Gln  1136, 1119, 743 
NDUFS8 (23 kDa)  3′ UTR +14 C→T  –  1119, 743 
Gene  Mutations (cDNA position)  Change  Patients 
NDUFS4 (18 kDa)  +198 C→A  Silent  1136 
  +312 G→A  Silent  1136, 743 
NDUFS7 (20 kDa)  +68 T→C heterozygous  Leu→Pro  1136 
  3′ UTR +13 A→C  –  1136, 1119, 743 
NDUFV3 (10 kDa)  +22 A→C  Silent  1136, 1119, 743 
  +25 A→C  Lys→Gln  1136, 1119, 743 
NDUFS8 (23 kDa)  3′ UTR +14 C→T  –  1119, 743 

For details see Materials and Methods.

Table 2.

Activities of NADH→UQ oxidoreductase of complex I and cytochrome c oxidase in skeletal muscle and cultured fibroblasts from controls and patient 1136

  CI/citrate synthase  CIV/citrate synthase  Citrate synthase (nmoles substrate/min/mg protein) 
Control muscle (n = 30)  24 ± 4  130 ± 25  150 ± 30 
Patient’s muscle  8.5  151  91 
Control fibroblasts (n = 30)  25.5 ± 6.3  90 ± 15  110 ± 25 
Patient’s fibroblasts  4.1  114  160 
  CI/citrate synthase  CIV/citrate synthase  Citrate synthase (nmoles substrate/min/mg protein) 
Control muscle (n = 30)  24 ± 4  130 ± 25  150 ± 30 
Patient’s muscle  8.5  151  91 
Control fibroblasts (n = 30)  25.5 ± 6.3  90 ± 15  110 ± 25 
Patient’s fibroblasts  4.1  114  160 

NADH→UQ oxidoreductase of complex I (CI) and cytochrome c oxidase (CIV) activities were measured by spectrophotometric assay and normalized to citrate synthase, a mitochondrial matrix marker.

Activities of controls are expressed as mean values of different determinations on 30 samples ± standard error.

For details see Materials and Methods.

Table 3.

NADH-UQ oxidoreductase of complex I and cytochrome c oxidase activities in mitoplasts from serum-starved and cholera toxin-treated control and patient fibroblasts

  Serum-starved        +Cholera toxin       
  Control    Patient    Control    Patient   
  Vmax  Km  Vmax  Km  Vmax  Km  Vmax  Km 
  (fmol/min/cell)  (µM NADH)  (fmol/min/cell)  (µM NADH)  (fmol/min/cell)  (µM NADH)  (fmol/min/cell)  (µM NADH) 
NADH→UQ oxidoreductase  2.39 ± 0.15 (8)  2.77 ± 0.40 (8)  ∼0  Not measurable  6.46 ± 0.37 (8) P < 0.001  6.51 ± 1.13 (8) Not significant  ∼0  Not measurable 
Cytochrome c oxidase  7.76 ± 0.92 (5)    6.50 (2)    12.06 ± 1.02 (4) Not significant    6.42   
  Serum-starved        +Cholera toxin       
  Control    Patient    Control    Patient   
  Vmax  Km  Vmax  Km  Vmax  Km  Vmax  Km 
  (fmol/min/cell)  (µM NADH)  (fmol/min/cell)  (µM NADH)  (fmol/min/cell)  (µM NADH)  (fmol/min/cell)  (µM NADH) 
NADH→UQ oxidoreductase  2.39 ± 0.15 (8)  2.77 ± 0.40 (8)  ∼0  Not measurable  6.46 ± 0.37 (8) P < 0.001  6.51 ± 1.13 (8) Not significant  ∼0  Not measurable 
Cytochrome c oxidase  7.76 ± 0.92 (5)    6.50 (2)    12.06 ± 1.02 (4) Not significant    6.42   

The effect of cAMP on NADH→UQ oxidoreductase and cytochrome c oxidase activities in serum-starved mitoplasts from control and patient 1136 fibroblasts was tested in serum-starved cultures incubated for 3 h with cholera toxin + IBMX.

Activities are expressed as mean values of different (n) experiments ± standard error.

For details see Materials and Methods.

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