Abstract

Mutations in SCO2, a cytochrome c oxidase (COX) assembly gene located on chromosome 22, have recently been reported in patients with fatal infantile cardio­encephalomyopathy and severe COX deficiency in heart and skeletal muscle. The Sco2 protein is thought to function as a copper chaperone. To investigate the extent to which mutations in SCO2 are responsible for this phenotype, a complete sequence analysis of the gene was performed on ten patients in nine families. Mutations in SCO2 were found in three patients in two unrelated families. We detected two missense mutations, one of which (G1541A) results in an E140K substitution adjacent to the highly conserved CxxxC metal-binding site. The other (C1634T) results in an R171W substitution more distant from the copper-binding site. A nonsense codon was found on one allele in two siblings presenting with a rapidly progressive fatal cardio­encephalomyopathy. Interestingly, all patients so far reported are compound heterozygotes for the G1541A mutation, suggesting that this is either an ancient allele or a mutational hotspot. The COX deficiency in patient fibroblasts (∼50%) did not result in a measurable decrease in the steady-state levels of COX complex polypeptide subunits and could be rescued by trans­ferring chromosome 22, but not other chromosomes. These data indicate that mutations in SCO2 cause a fatal infantile mitochondrial disorder characterized by hypertrophic cardiomyopathy and encephalopathy, and point to the presence of one or more other genes, perhaps in the copper delivery pathway, in this clinical phenotype.

Received 29 November 1999; Revised and 17 Accepted January 2000.

INTRODUCTION

The clinical phenotypes associated with cytochrome c oxidase (COX) deficiency encompass a wide and heterogeneous spectrum of mostly severe multisystemic disorders, primarily affecting those organs with high energy demand, such as the brain, skeletal muscle, heart and kidney (reviewed in ref. 1). COX (Complex IV), the terminal step in the electron transport chain, is embedded in the inner mitochondrial membrane where it catalyzes the transfer of reducing equivalents from cytochrome c to molecular oxygen. The holoenzyme complex is made up of 13 subunits, three of which (subunits I–III) are encoded by mitochondrial DNA (mtDNA). The mtDNA-encoded subunits are composed of a number of highly conserved hydrophobic transmembrane domains containing two copper-binding sites (CuA and CuB) and two hemes (heme a and a3) (24). The 10 less conserved nuclear-encoded subunits are thought to modulate enzyme activity. A substantial number of other nuclear genes are required for the assembly and maintenance of the COX complex (5).

Pedigree studies suggest that the majority of genetic defects in fatal infantile COX deficiency disorders are of nuclear origin and inherited as autosomal recessive traits; however, defects in nuclear genes coding for the COX subunits themselves have not been reported (6,7). The first autosomal recessive inherited disease gene, causing isolated, systemic COX deficiency has recently been identified (8,9). This mitochondrial COX assembly gene, SURF1, was found to be mutated in patients with typical Leigh syndrome and COX deficiency, but not in other diseases associated with COX deficiency (10).

Using a candidate gene approach, a second COX assembly gene, SCO2, was reported to cause hypertrophic cardio‐ myopathy and encephalopathy, associated with severe COX deficiency in skeletal muscle and relatively high residual activity in fibroblasts (11). SCO2 is a homolog of two yeast genes (Sco1 and Sco2) that are involved in mitochondrial copper delivery. The delivery of copper to COX in yeast mitochondria is thought to involve the transfer of copper from the plasma membrane transporter to the cytosolic protein COX17, which then delivers it to Sco1p in the inner membrane (12,13). In support of this model, overexpression of Sco1p can rescue a COX17 deletion in the presence of elevated copper in the medium (13). The function of the second closely related Sco2 gene is unclear as its deletion does not produce a respiratory chain deficiency, nor can its overexpression rescue an Sco1 deletion (13).

This study focuses on a systematic mutation analysis of SCO2 in 10 patients with fatal hypertrophic cardiomyopathy from nine families, and a restriction fragment length polymorphism (RFLP) analysis for a common SCO2 mutation in 42 patients with isolated COX deficiency and 45 sudden infant death syndrome (SIDS) infants. We detected SCO2 mutations in three infants from two families. The causal involvement of SCO2 was confirmed by microcell-mediated chromosome transfer of chromosome 22, which was able to rescue the enzymatic defect in the patients’ fibroblasts. Normal SCO2 sequences in other patients with nearly identical clinical features suggest genetic heterogeneity in these cases, perhaps involving the other components of the copper delivery pathway to mitochondria.

RESULTS

Clinical classification of patients

The patients were classified into three groups according to their clinical features. The first group was characterized by a combination of hypertrophic cardiomyopathy, muscle hypo‐ tonia, lactic acidosis and severely decreased COX activity in skeletal muscle with a relatively high residual activity in fibroblasts (Table 1). The second group had a variety of different clinical symptoms (summarized in Table 2), but with the common feature of a COX deficiency in skeletal muscle. The third group was a collection of 45 infants from the South Bavarian part of Germany who died from SIDS in the years 1993–1995 with no measurements on respiratory chain function (post-mortem tissue).

Biochemistry

Biochemical measurement of respiratory chain activities in skeletal muscle biopsy specimens of index patient A at age 3.5 months revealed an enhanced activity of the mitochondrial marker enzyme citrate synthase (CS) consistent with the morphological demonstration of mitochondrial proliferation. In skeletal muscle, the activity of COX was undetectable, whereas the normalized (to CS) activities of complexes I–III were within the control range. In contrast, normalized COX activity in fibroblasts was ∼50% of the lowest reference range (Table 1). In index cases B1 and B2, both siblings had low CS (50% of control activity), normal activities of complexes I–III and severely reduced normalized COX activities, 10 and 12% of controls for B1 and B2, respectively (Table 1).

Biochemical measurements of respiratory chain complex activities revealed COX deficiency in all index patients from group 1 (Table 1). All patients from group 2 had COX deficiency of variable severity (Table 2). No measurements could be performed in post-mortem tissues from the SIDS group.

Microcell-mediated chromosome transfer

Microcell-mediated chromosome transfer was performed on a fibroblast cell line from index patient A. Approximately 4 weeks after cell-fusion experiments, enzyme activities of COX and CS of hygromycin-resistant clones were determined spectro­photometrically. Compared with the original untreated cells, COX activity was unchanged in selected clones after transfer of chromosomes 1, 5, 6, 9 and 17. In contrast, COX activity returned to 95% of control levels after transfer of chromosome 22. The ratio of COX:CS activity in patient cells was 1.1, 1.80 after transfer of chromosome 22 and 1.90 in control cells. The decrease in COX activity in patient fibroblasts was not associated with a measurable change in the steady-state levels of either mtDNA- or nuclear-encoded COX subunits on western blot analysis, which were also unaltered by transfer of chromosome 22 (Fig. 1).

Sequencing of SCO2

The entire SCO2 gene (GenBank accession no. AF177385) was sequenced in the index patients and in seven additional patients in group 1 (Table 1). The index patients A, B1 and B2 were all compound heterozygotes for a G1541A mutation, which predicts an E140K substitution immediately adjacent to the copper-binding site (Figs 2 and 3a). In patient A, a heterozygous C1634T transition mutation, predicting an R171W substitution, was identified on the other allele (Figs 2 and 3b). The father of patient A was positive for the G1541A mutation and the C1634T mutation was present in the mother (Fig. 3a). The C1634T mutation was absent in 100 healthy controls. In family B, the G1541A mutation was present in the father and the mother was positive for a C1391T mutation, creating a nonsense codon R90X, just downstream of the putative N-terminal membrane anchor. Their sons carried both mutations (Figs 1 and 3). No pathogenic mutations in the remaining seven patients of group 1 were found in the coding region or in the adjacent intronic regions. Because all patients reported here and by Papadopoulou et al. (11) are compound heterozygotes for the G1541A mutation, we searched for this mutation by RFLP analysis in group 2 and 3 patients, but all were negative. Both groups were also negative for the C1634T mutation. Polymorphic sequence changes are shown in Table 1 in parentheses. The R20P sequence variant (G1182C) occurs in the N-terminal mito­chondrial targeting sequence in several patients, including patient A in whom two pathogenic alleles were identified. It was also identified in the patients and controls reported by Papadopoulou et al. (11), suggesting that it is a common polymorphism. The predicted amino acid sequence of Sco2 with the position of known pathogenic mutations is shown in Figure 4.

SURF1 mutations were excluded in all patients with COX deficiency (groups 1 and 2). mtDNA mutations associated with MELAS (nucleotides 3243, 3250 and 3271), MERRF (nucleotides 8344 and 8356) and Leigh syndrome (nucleotide 8993) were excluded by standard RFLP analysis, and large-scale rearrangements were ruled out using Southern blot analysis.

Copper supplementation

Enzyme activities of COX and CS were determined spectro‐ photometrically in fibroblasts of index patient A after supplementation of the medium with copper. No increase of COX activity was found at any copper concentration (data not shown).

DISCUSSION

In addition to defective genes coding for structural components of the cardiac contractile apparatus (14), the development of hypertrophic cardiomyopathy is frequently found in patients with respiratory chain deficiencies (15). Very recently, mutations in SCO2, a COX assembly gene, have been described in association with a fatal infantile form of hypertrophic cardiomyopathy (11). Here we present additional data on two unrelated families and two novel mutations in the SCO2 gene. The pathogenicity of these mutations is strongly supported by the following findings: (i) they are associated with a distinct phenotype of a cardioencephalomyopathy and a fatal outcome, similar to that reported by Papadopoulou et al. (11); (ii) functional rescue of the COX deficiency in the patient fibroblasts occurred after transfer of chromosome 22, but not after transfer of other chromosomes; (iii) nonsense mutations (R90X) were found in patients B1 and B2; (iv) the missense mutations result in non-conservative amino acid substitutions and are located near the highly conserved copper-binding site of the predicted protein; and (v) the G1541A transition mutation is found in all patients described so far (but not in control subjects), suggesting that it is either an ancient mutation or a mutational hotspot.

The pathways for intracellular copper transport have been most intensively investigated in yeast, in which specific chaperone proteins have been identified for copper delivery to a post-Golgi compartment: Cu/Zn superoxide dismutase (SOD) and COX (16,17). The delivery of copper to COX in the inner mitochondrial membrane of yeast involves at least two chaperone proteins: Cox17p and Sco1p. Cox17p is found in the cytoplasm and mitochondrial intermembrane space (12,18), and has been suggested to function in delivering copper to Sco1p in the inner mitochondrial membrane (13) and perhaps directly to the CuA site on subunit II of COX (16). Consistent with this function, Cox17p is reported to bind copper as a binuclear cuprous-thiolate cluster (19). Cox17p function can, however, be abrogated by supplementation of the medium with copper (12). Sco1p contains the highly conserved CxxxC metal-binding motif near the C-terminal end of the protein, which protrudes into the intermembrane space (18). Deletion of Sco1 cannot be rescued by increased medium copper or by overexpression of Cox17p, suggesting that it is an essential and specific chaperone for the delivery of copper to COX (13). A third protein, Sco2p, has significant homology with Sco1p and a similar topology in the inner membrane, but its role in copper trafficking is less clear. Deletion of Sco2 does not impair respiratory chain function and overexpression cannot rescue an Sco1 null mutant (13). Overexpression of Sco2 can, however, partially rescue point mutations in Sco1, suggesting that Sco1 and Sco2 have partially overlapping functions (13).

The two human SCO genes are clearly homologs of the yeast genes, although the data presented here and by Papadopoulou et al. (11) suggest that the functions of SCO1 and SCO2 do not overlap significantly, as there is no evidence that SCO1 can functionally substitute for loss of function mutations in SCO2. Supplementation of the cell culture medium with copper failed to restore COX activity in fibroblasts from patients with SCO2 mutations, similar to results obtained with the yeast Sco1 deletion (12). Neither of the human genes can rescue an Sco1 deletion in yeast, but experiments with chimeric proteins have demonstrated that a protein with the N-terminal portion of yeast Sco1p and the C-terminal portion of SCO1, but not SCO2, can substitute for Sco1p function (20). Defining the roles of the two SCO genes in both human and yeast will require further investigation.

In patients with SCO2 mutations, it is of interest that skeletal and cardiac muscle show a severe COX deficiency, whereas fibroblasts exhibit a relatively high residual enzyme activity, despite the fact that SCO2 is ubiquitously expressed. Tissue-specific deficiencies in COX activity have also been reported in a distinct autosomal recessive form of Leigh syndrome in the French Canadian population (21). In these patients, residual COX activities are very low in liver and brain, and levels are relatively high in muscle and fibroblasts. Although the gene defect has not yet been identified in this syndrome, it does not involve any known tissue-specific structural component of the complex, but rather appears to result from a failure of COX assembly. In contrast to these results, there is no evidence for a marked tissue-specific effect in classical COX-deficient Leigh syndrome patients with mutations in SURF1, another ubiquitously expressed COX assembly factor (2224). Immuno‐ blot analysis also suggests that the steady-state levels of the COX polypeptide subunits are relatively unaffected in SCO2 patient fibroblasts, whereas immunocytochemical analysis suggests a substantial reduction in subunits I and II in skeletal muscle (11). The reasons for these differences are not clear. In the case of SCO2, it could reflect a reduced copper recruitment and turnover in fibroblasts, which have a lower COX activity than striated muscle, alternate tissue-specific pathways for copper delivery or, as previously suggested, different amounts of the Sco2 protein in different tissues (11).

The functional impairment of Sco2 is likely to decrease copper transport or delivery to COX subunits I and II. Such a mechanism would imply a primary copper deficiency of COX with a resulting defect in catalytic function. Copper-deficient rats exhibiting low levels of the cuproenzymes lysyl oxidase, COX and Cu/Zn SOD develop cardiac hypertrophy (25). The development of a cardiomyopathy is also a known pathophysiological effect in copper deficiency in humans and may be present in Menkes disease (26). In fact, some clinical symptoms in patient A resemble in part those found in Menkes disease, a disorder caused by cellular copper deficiency due to a defect in ATP7a, a copper transporting protein (reviewed in ref. 27). Most clinical features in Menkes disease can be explained by a defective synthesis of copper-requiring enzymes. The secondary deficiency of COX is probably an important factor in the develop­ment of a profound neurological disorder characterized by seizures, neurological deterioration with cerebral and cerebellar atrophy, focal cerebral necrosis and gliosis (28) or in the development of cardiomyopathy (26), although the latter is a rare finding in Menkes patients.

In Menkes disease, copper and ceruloplasmin are severely decreased in serum. This can be explained by a defective copper transport from the intestinal cell to the blood. Defects in Sco2 function are likely to be restricted to mitochondrial copper transport and do not therefore appear as systemic copper or ceruloplasmin derangements. Consistent with this, blood samples taken at different time points in patient B2 for copper and ceruloplasmin measurements were within the normal range at age 2 and 4 weeks.

Sequence analysis of seven patients presenting with an almost identical clinical and biochemical phenotype as those in whom we found SCO2 mutations did not reveal pathogenic mutations in the SCO2 coding or adjacent intronic regions. Although we cannot exclude a mutation in the promoter region of SCO2, it is more likely that there is genetic heterogeneity associated with this clinical phenotype, possibly involving other genes in mitochondrial copper transport and delivery. Sequence analysis of the genes in this pathway in more infants with hypertrophic cardiomyopathy will be useful in establishing the importance of this pathway for cardiac function.

PATIENTS AND METHODS

Case reports

Family A.

The index patient of family A was the first male child delivered to non-consanguineous, healthy parents after a full-term uneventful pregnancy. In the first 4 weeks of life, no complications were registered. He then developed severe muscle hypotonia with respiration difficulties presenting as inspiratory and expiratory stridor at age 6 weeks, when he was admitted to hospital. On examination, psychomotor retardation, muscular hypotonia and laxity of joints were the most prominent findings, whereas the echocardiographic and electrocardiographic examinations of the heart were normal. Owing to the respiratory insufficiency, he needed immediate ventilatory support. Progressive deterioration with the occurrence of multiple seizures occurred over the following days. At that time, electroencephalography (EEG) and cranial magnetic resonance imaging (MRI) results were normal. Bronchoscopy revealed a severe tracheomalazia. Enzymatic testing showed a severe COX deficiency in skeletal muscle. At age 2 months, he developed a rapid progressive hypertrophic obstructive cardiomyopathy with increasing respiration difficulties. Laboratory findings were normal except for lactate, which was highly elevated in blood and cerebrospinal fluid (CSF). The child died from cardiopulmonary arrest at age 4 months.

The pathological–anatomical examination revealed a severe obstructive hypertrophic cardiomyopathy of the left ventricle with an organ weight of 90 g. The trachea showed an increased mobility, explaining the inspiratory and expiratory stridor. Most organs showed hyperemia with an enlarged liver (organ weight 300 g) and with edema in the lungs (organ weight 50 g each) and brain (organ weight 565 g), likely to be the result of cardiac insufficiency. On macroscopic examination, the brain showed a focal lesion in the hippocampal region in addition to the edema. Morphological and ultrastructural examination of the myo‐ cardium and skeletal muscle showed a proliferation of abnormal mitochondria with vacuolated cytoplasm. No ragged red fibers were found. Microscopic analysis of the brain showed atrophic cells and increased gliosis in most sections investigated. Focal neuronal lesions were found in the hippocampal region.

Family B.

Index patients B1 and B2 were two male siblings from non-consanguineous healthy parents. One older child from a different father is unaffected. Prior to two full-term preg‐ nancies with her sons B1 and B2, the mother had two miscarriages in the 11th and 12th week of pregnancy. Both pregnancies were complicated by increased formation of amniotic fluid (hydramnion). At birth, both siblings (B1 and B2) developed a progressive respiratory insufficiency with muscle hypotonia and severe metabolic acidosis. Lactic acid was highly elevated in blood (11 mmol/l in B1 and 10 mmol/l in B2) and increased 3-fold in CSF. Owing to progressive respiration difficulties, both infants needed immediate ventilatory support. Both showed seizure-like episodes without characteristic EEG alterations. In index patient B2, cranial MRI revealed cerebellar atrophy with alterations in frontal brain regions pointing to maturation disturbances. In addition, a cystic infratentorial enlargement of the arachnoidea was described. No density changes were seen in the basal ganglia or in the brain stem. On echocardiography, hypertrophic cardiomyopathy was detectable in both infants at age 3 weeks. In patient B2, copper and ceruloplasmin values in blood were within the reference range at age 2 and 4 weeks, respectively. The first child died from aspiration after self-extubation (B1) and the second child from cardiorespiratory failure (B2) at age 4 weeks.

A pathological–anatomical examination was available for index patient B1. A severe obstructive biventricular hyper‐ trophic cardiomyopathy with a heart weight of 44 g (maximal normal weight 25 g) and an edema of the lungs was found. No cerebral and cerebellar abnormalities were seen on macroscopic examination.

Biochemistry

Complex I–IV activities were determined spectrophotometrically in postnuclear supernatants of fresh frozen muscle biopsy specimens according to the methods described by Fischer et al. (29). COX activity measurements in fibroblasts from patients 1–8 were performed as described (30) and for patient 9 as described by Merante et al. (21). The COX activities from patient 10 have been reported previously (31).

Immunoblot analysis

The steady-state levels of the COX II and COX IV subunits were determined by immunoblot analysis in comparison with porin as described by Yao and Shoubridge (24).

Fibroblast cell culture

Prior to performing the microcell-mediated chromosome transfers, the patients’ fibroblasts were transduced with a retroviral vector expressing E6/E7 (8,32) in order to extend their life span. The cells were grown in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Gibco BRL, Gaithersberg, MD) supplemented with 10% fetal bovine serum (Gibco BRL).

Microcell-mediated chromosome transfer

Microcell-mediated chromosome transfer was performed as described previously (8) in index case A.

DNA analysis

DNA was extracted from skeletal muscle, leukocytes and fibroblasts according to standard purification protocols (Qiagen, Mississauga, Ontario). The entire SCO2 coding sequence was amplified with the forward and backward intronic primers using the PCR conditions as described (11). Cycle sequencing of gel-purified fragments was performed using the ABI PRISM Big Dye Termination kit (Perkin Elmer, Foster City, CA) with nested sequencing primers. The mutations were then confirmed by additional RFLP analyses. The entire SCO2 gene was sequenced in the three index patients and in seven additional patients presenting with similar symptoms and with COX deficiency.

The 1541 and 1634 mutations were screened in other patients with COX deficiency, but without hypertrophic cardiomyopathy (n = 42), in 45 infants who died from SIDS and in healthy controls (n = 100). We used the forward and backward mismatch primer as described (11), as well as a novel mismatch primer abolishing a BsrBI restriction site in the case of the 1541 mutation (5′-GCA CCA CCT GCA CCA GCT TCT CCC GC-3′) to confirm the results. The 1634 mutation abolishes a natural BsrBI restriction site. We used primers F9/B5 (11) to amplify a 595 bp fragment. In case of the wild-type, the fragment is cut into two smaller fragments of 268 and 327 bp in size. To confirm the heterozygous nonsense mutation at position 1391, we designed a backward mismatch primer (5′-GGC GCA GGG CTT CTG ATC-3′) for amplification with F6 (11). The resulting fragment is cut into two small fragments of 18 and 83 bp and larger fragments of 101 and 228 bp (including one natural restriction site) with BclI in the presence of the heterozygous mutation. Resulting RFLPs were separated on a 4% agarose-nu sieve gel (1:2) in the case of the 1541 mutation and on a 2% normal agarose gel in the case of the 1634 mutations and analyzed after ethidium bromide staining. RFLP analysis for the 1391 mutation was carried out on 10% polyacrylamide gels and the bands were detected by silver staining.

Copper supplementation

Several final concentrations of CuSO4 (250, 125, 62.5, 25, 2.5 and 1.25 µM) were made up in DMEM medium and added to patient fibroblasts. The fibroblasts were then incubated for 10 days with a medium change at days 4 and 8.

ACKNOWLEDGEMENTS

We greatly appreciate the clinical and pathological investigations of Dr K. Dumke and Prof. Wünsch. A. Zimmermann, I. Kaus and T. Johns provided excellent technical assistance. We thank B. Robinson and N. Kennaway for providing patient cell lines, A. Cuthbert for the monochromosomal hybrid lines and the families whose collaboration made this study possible. This work was supported by grants from the Deutsche Forschungs­gemeinschaft (Ja 802/1-2) (M.J.), the Friedrich-Baur-Stiftung (M.J.), the Hospital for Sick Children, Toronto, Canada (E.A.S.) and March of Dimes Birth Defects Association (1-FY99-243) (E.A.S.). E.A.S. is an MNI Killam Scholar.

+

To whom correspondence should be addressed. Tel: +1 514 398 1997; Fax: +1 514 398 1509; Email: eric@ericpc.mni.mcgill.ca

Figure 1. Immunoblot analysis of COX subunits in fibroblasts from patient A. Lane 1, control fibroblasts; lane 2, patient A fibroblasts; lane 3, patient fibroblasts after chromosome transfer but no change in COX activity; lane 4, patient fibroblasts after transfer of chromosome 22 and restoration of COX activity to near control levels. The same amount of mitochondrial protein was loaded in each lane (2.5 µg) and porin, an outer mitochondrial membrane protein, was used as an internal loading control.

Figure 1. Immunoblot analysis of COX subunits in fibroblasts from patient A. Lane 1, control fibroblasts; lane 2, patient A fibroblasts; lane 3, patient fibroblasts after chromosome transfer but no change in COX activity; lane 4, patient fibroblasts after transfer of chromosome 22 and restoration of COX activity to near control levels. The same amount of mitochondrial protein was loaded in each lane (2.5 µg) and porin, an outer mitochondrial membrane protein, was used as an internal loading control.

Figure 2. Sequence analysis of SCO2. SCO2 gene mutations found in patients A (a and b) and B1 (a and c) and B2. The mutation at position 1634 is shown on the non-coding strand.

Figure 2. Sequence analysis of SCO2. SCO2 gene mutations found in patients A (a and b) and B1 (a and c) and B2. The mutation at position 1634 is shown on the non-coding strand.

Figure 3. RFLP analysis of mutations in SCO2. (a) Analysis of the heterozygous G1541A mutation. Using a reverse mismatch primer, the 203 bp PCR product is cleaved into two fragments of 178 and 25 bp by BsrBI in the case of wild-type DNA. Lane 1, control; lane 2, mother (family A); lane 3, index patient (family A); lane 4, father (family A); lane 5, control; lane 6, mother (family B); lane 7, index patient B1 (family B); lane 8, index patient B2 (family B); lane 9, father (family B). (b) Analysis of the heterozygous C1634T mutation. The 595 bp fragment is cut into two smaller fragments of 268 and 327 bp by BsrBI in the case of wild-type DNA. Lane 1, control; lane 2, mother (family A); lane 3, index patient A (family A); lane 4, father (family A). (c) Analysis of the heterozygous C1391T mutation. Lane 1, control; lane 2, mother (family B); lanes 3 and 4, index cases family B; lane 5, father (family B). The 339 bp PCR product is cut into fragments of 228, 101, 83 and 18 bp in the case of the mutant allele.

Figure 3. RFLP analysis of mutations in SCO2. (a) Analysis of the heterozygous G1541A mutation. Using a reverse mismatch primer, the 203 bp PCR product is cleaved into two fragments of 178 and 25 bp by BsrBI in the case of wild-type DNA. Lane 1, control; lane 2, mother (family A); lane 3, index patient (family A); lane 4, father (family A); lane 5, control; lane 6, mother (family B); lane 7, index patient B1 (family B); lane 8, index patient B2 (family B); lane 9, father (family B). (b) Analysis of the heterozygous C1634T mutation. The 595 bp fragment is cut into two smaller fragments of 268 and 327 bp by BsrBI in the case of wild-type DNA. Lane 1, control; lane 2, mother (family A); lane 3, index patient A (family A); lane 4, father (family A). (c) Analysis of the heterozygous C1391T mutation. Lane 1, control; lane 2, mother (family B); lanes 3 and 4, index cases family B; lane 5, father (family B). The 339 bp PCR product is cut into fragments of 228, 101, 83 and 18 bp in the case of the mutant allele.

Figure 4. Predicted amino acid sequence of Sco2 showing the predicted mitochondrial leader sequence (underlined), putative copper-binding site (boxed) and the sites of the known mutations. The R20P substitution appears to be a common polymorphism.

Figure 4. Predicted amino acid sequence of Sco2 showing the predicted mitochondrial leader sequence (underlined), putative copper-binding site (boxed) and the sites of the known mutations. The R20P substitution appears to be a common polymorphism.

Table 1.

Genotype–phenotype correlations in patients with hypertrophic cardiomyopathy and COX deficiency

Patient no. Gender Onset/death Clinical presentation/family history COX(M)a (%) COX(F)a (%) Mutation (variants) 
1 (A) Male Birth/4 months HCMP, seizures, MH, respiratory insufficiency 50 G1541A, EK 
      C1634T, RW 
      (G1182C, RP, h; A1756C, silent, h
2 (B1) Male Birth/4 weeks HCMP, seizures, MH, respiratory insufficiency 10 No data G1541A, EK 
      C1391T, stop 
      (A1756C, silent, h;G1182C, RP, h
3 (B2) Male Birth/4 weeks HCMP, seizures, MH, respiratory insufficiency 12 No data G1541A, EK 
      C1391T, stop 
      (A1756C, silent, h
Male 6 weeks/alive HCMP, seizures, MRI+, respiratory insufficiency, MH, low copper and ceruloplasmin, probably Menkes disease 60 No data –/– 
Male Birth/6 weeks HCMP, MH, respiratory insufficiency, consanguinity? 10 Normal (G1182C, RP, H; A1756C, silent, H
Male Birth/2 weeks HCMP, MH, respiratory insufficiency, MRI+, seizures, consanguinity 20 95 (A1756C, silent, h; C1130T, silent, H; G1182C, RP, h
Male Birth/4 months HCMP, anemia, MH, consanguinity 17 No data (C1130T, silent, H; G1182C, RP, H
Female 4 weeks/1 year HCMP, MH, seizures 15 Normal (A1756C, silent, H; C1831T, silent, h
Female Birth/26 days HCMP, lactic acidosis 15 40 (G1182C, RP, H)10 
10 Female Perinatal HCMP, seizures, lactic acidosis, MH No data (G1182C, RP, h; A1756C, silent, h
Patient no. Gender Onset/death Clinical presentation/family history COX(M)a (%) COX(F)a (%) Mutation (variants) 
1 (A) Male Birth/4 months HCMP, seizures, MH, respiratory insufficiency 50 G1541A, EK 
      C1634T, RW 
      (G1182C, RP, h; A1756C, silent, h
2 (B1) Male Birth/4 weeks HCMP, seizures, MH, respiratory insufficiency 10 No data G1541A, EK 
      C1391T, stop 
      (A1756C, silent, h;G1182C, RP, h
3 (B2) Male Birth/4 weeks HCMP, seizures, MH, respiratory insufficiency 12 No data G1541A, EK 
      C1391T, stop 
      (A1756C, silent, h
Male 6 weeks/alive HCMP, seizures, MRI+, respiratory insufficiency, MH, low copper and ceruloplasmin, probably Menkes disease 60 No data –/– 
Male Birth/6 weeks HCMP, MH, respiratory insufficiency, consanguinity? 10 Normal (G1182C, RP, H; A1756C, silent, H
Male Birth/2 weeks HCMP, MH, respiratory insufficiency, MRI+, seizures, consanguinity 20 95 (A1756C, silent, h; C1130T, silent, H; G1182C, RP, h
Male Birth/4 months HCMP, anemia, MH, consanguinity 17 No data (C1130T, silent, H; G1182C, RP, H
Female 4 weeks/1 year HCMP, MH, seizures 15 Normal (A1756C, silent, H; C1831T, silent, h
Female Birth/26 days HCMP, lactic acidosis 15 40 (G1182C, RP, H)10 
10 Female Perinatal HCMP, seizures, lactic acidosis, MH No data (G1182C, RP, h; A1756C, silent, h

aActivities of COX are related to the mitochondrial marker enzyme CS and are expressed in % residual activity, when compared with the lowest reference value (patients 1–8), or to mean control COX activity (patients 9 and 10).

HCMP, hypertrophic cardiomyopathy; MRI+, typical Leigh syndrome pathology on neuroradiological examination; MH, muscle hypotonia; h, heterozygous; H, homozygous. Pathogenic mutations are indicated in bold.

Table 2.

Summary of the clinical features of group 2 patients (n = 42) with isolated COX deficiency in skeletal muscle

Patients Diagnoses Average age at onset (range) COX, average residual activity in skeletal muscle (range) (%) 
1–9 Leigh syndrome without SURF1 mutation 15 months (birth–4 years) 42 (9–60) 
10–14 Severe congenital lactic acidosis Birth 32 (23–44) 
15–21 Muscle hypotonia and pathological EEG 8 months (birth–3 years) 62 (32–80) 
22–34 Syndromal encephalomyopathy 17 years (12–29 years) 68 (41–85) 
35–42 Other clinical symptoms including sepsis, brain atrophy, hepatopathy, tetraparesis 3 years (birth–8 years) 34 (15–75) 
Patients Diagnoses Average age at onset (range) COX, average residual activity in skeletal muscle (range) (%) 
1–9 Leigh syndrome without SURF1 mutation 15 months (birth–4 years) 42 (9–60) 
10–14 Severe congenital lactic acidosis Birth 32 (23–44) 
15–21 Muscle hypotonia and pathological EEG 8 months (birth–3 years) 62 (32–80) 
22–34 Syndromal encephalomyopathy 17 years (12–29 years) 68 (41–85) 
35–42 Other clinical symptoms including sepsis, brain atrophy, hepatopathy, tetraparesis 3 years (birth–8 years) 34 (15–75) 

Consanguinity of the parents was present in eight cases.

References

1 Zeviani, M., Tiranti, V. and Piantadosi, C. (
1998
) Mitochondrial disorders.
Medicine (Baltimore)
 ,
77
,
59
–72.
2 Capaldi, R.A. (
1990
) Structure and function of cytochrome c oxidase.
Annu. Rev. Biochem.
 ,
59
,
569
–596.
3 Taanman, J.W. (
1997
) Human cytochrome c oxidase: structure, function, and deficiency.
J. Bioenerg. Biomembr.
 ,
29
,
151
–163.
4 Yoshikawa, S., Shinzawa-Itoh, K. and Tsukihara, T. (
1998
) Crystal structure of bovine heart cytochrome c oxidase at 2.8 Å resolution.
J. Bioenerg. Biomembr.
 ,
30
,
7
–14.
5 Grivell, L.A. et al. (
1999
) Mitochondrial assembly in yeast.
FEBS Lett.
 ,
452
,
57
–60.
6 Adams, P.L., Lightowlers, R.N. and Turnbull, D.M. (
1997
) Molecular analysis of cytochrome c oxidase deficiency in Leigh’s syndrome.
Ann. Neurol.
 ,
41
,
268
–270.
7 Jaksch, M. et al. (
1998
) A systematic mutation screen of 10 nuclear and 25 mitochondrial candidate genes in 21 patients with cytochrome c oxidase (COX) deficiency shows tRNA(Ser)(UCN) mutations in a subgroup with syndromal encephalopathy.
J. Med. Genet.
 ,
35
,
895
–900.
8 Zhu, Z. et al. (
1998
) SURF1, encoding a factor involved in the biogenesis of cytochrome c oxidase, is mutated in Leigh syndrome.
Nature Genet.
 ,
20
,
337
–343.
9 Tiranti, V. et al. (
1998
) Mutations of SURF-1 in Leigh disease associated with cytochrome c oxidase deficiency.
Am. J. Hum. Genet.
 ,
63
,
1609
–1621.
10 Tiranti, V. et al. (
1999
) Loss-of-function mutations of SURF-1 are specifically associated with Leigh syndrome with cytochrome c oxidase deficiency.
Ann. Neurol.
 ,
46
,
161
–166.
11 Papadopoulou, L.C. et al. (
1999
) Fatal infantile cardioencephalomyopathy with COX deficiency and mutations in SCO2, a COX assembly gene.
Nature Genet.
 ,
23
,
333
–337.
12 Glerum, D.M., Shtanko, A. and Tzagoloff, A. (
1996
) Characterization of COX17, a yeast gene involved in copper metabolism and assembly of cytochrome oxidase.
J. Biol. Chem.
 ,
271
,
14504
–14509.
13 Glerum, D.M., Shtanko, A. and Tzagoloff, A. (
1996
) SCO1 and SCO2 act as high copy suppressors of a mitochondrial copper recruitment defect in Saccharomyces cerevisiae.
J. Biol. Chem.
 ,
271
,
20531
–20535.
14 McKenna, W.J., Coccolo, F. and Elliott, P.M. (
1998
) Genes and disease expression in hypertrophic cardiomyopathy.
Lancet
 ,
352
,
1162
–1163.
15 Zeviani, M. et al. (
1995
) OXPHOS defects and mitochondrial DNA mutations in cardiomyopathy.
Muscle Nerve
 ,
3
,
S170
–S174.
16 Harrison, M.D., Jones, C.E. and Dameron, C.T. (
1999
) Copper chaperones: function, structure and copper-binding properties.
J. Biol. Inorg. Chem.
 ,
4
,
145
–153.
17 Valentine, J.S. and Gralla, E.B. (
1997
) Delivering copper inside yeast and human cells.
Science
 ,
278
,
817
–818.
18 Beers, J., Glerum, D.M. and Tzagoloff, A. (
1997
) Purification, characterization, and localization of yeast Cox17p, a mitochondrial copper shuttle.
J. Biol. Chem.
 ,
272
,
33191
–33196.
19 Srinivasan, C., Posewitz, M.C., George, G.N. and Winge, D.R. (
1998
) Characterization of the copper chaperone Cox17 of Saccharomyces cerevisiae.
Biochemistry
 ,
37
,
7572
–7577.
20 Paret, C., Ostermann, K., Krause-Buchholz, U., Rentzsch, A. and Rodel, G. (
1999
) Human members of the SCO1 gene family: complementation analysis in yeast and intracellular localization.
FEBS Lett.
 ,
447
,
65
–70.
21 Merante, F. et al. (
1993
) A biochemically distinct form of cytochrome oxidase (COX) deficiency in the Saguenay-Lac-Saint-Jean region of Quebec.
Am. J. Hum. Genet.
 ,
53
,
481
–487.
22 Glerum, D.M., Yanamura, W., Capaldi, R.A. and Robinson, B.H. (
1988
) Characterization of cytochrome-c oxidase mutants in human fibroblasts.
FEBS Lett.
 ,
236
,
100
–104.
23 Lombes, A. et al. (
1991
) Biochemical and molecular analysis of cytochrome c oxidase deficiency in Leigh’s syndrome.
Neurology
 ,
41
,
491
–498.
24 Yao, J. and Shoubridge, E.A. (
1999
) Expression and functional analysis of SURF1 in Leigh syndrome patients with cytochrome c oxidase deficiency.
Hum. Mol. Genet.
 ,
8
,
2541
–2549.
25 Medeiros, D.M. and Wildman, R.E. (
1997
) Newer findings on a unified perspective of copper restriction and cardiomyopathy.
Proc. Soc. Exp. Biol. Med.
 ,
215
,
299
–313.
26 Nath, R. (
1997
) Copper deficiency and heart disease: molecular basis, recent advances and current concepts.
Int. J. Biochem. Cell Biol.
 ,
29
,
1245
–1254.
27 DiDonato, M. and Sarkar, B. (
1997
) Copper transport and its alterations in Menkes and Wilson diseases.
Biochim. Biophys. Acta
 ,
1360
,
3
–16.
28 Kaler, S.G. (
1998
) Diagnosis and therapy of Menkes syndrome, a genetic form of copper deficiency.
Am. J. Clin. Nutr.
 ,
67
,
1029S
–1034S.
29 Fischer, J.C. et al. (
1986
) A mitochondrial encephalomyopathy: the first case with an established defect at the level of coenzyme Q.
Eur. J. Pediatr.
 ,
144
,
441
–444.
30 Capaldi, R.A., Marusich, M.F. and Taanman, J.W. (
1995
) Mammalian cytochrome-c oxidase: characterization of enzyme and immunological detection of subunits in tissue extracts and whole cells.
Methods Enzymol.
 ,
260
,
117
–132.
31 Kennaway, N.G. et al. (
1990
) Isoforms of mammalian cytochrome c oxidase: correlation with human cytochrome c oxidase deficiency.
Pediatr. Res.
 ,
28
,
529
–535.
32 Lochmuller, H., Johns, T. and Shoubridge, E.A. (
1999
) Expression of the E6 and E7 genes of human papillomavirus (HPV16) extends the life span of human myoblasts.
Exp. Cell Res.
 ,
248
,
186
–193.