21. Johnson M. The beta-adrenoceptor.

Am J Resp Crit Care Med

1998;158:S146S153.
22. Hayes A, Williams DA. Contractile properties of clenbuterol-
treated mdx muscle are enhanced by low-intensity swimming.
J Appl Physiol 1997;82:435439.
23. Dupont-Versteegden EE. Exercise and clenbuterol as strate-
gies to decrease the progression of muscular dystrophy in mdx
mice. J Appl Physiol 1996;80:734741.
24. Lynch GS, Hinkle RT, Faulkner JA. Year-long clenbuterol
treatment of mice increases mass, but not specific force or
normalized power, of skeletal muscles. Clin Exp Pharmacol
Physiol 1999;26:117120.
Homozygosity (E140K) in SCO2 causes
delayed infantile onset of cardiomyopathy
and neuropathy
M. Jaksch, MD; R. Horvath, MD; N. Horn, PhD; D.P. Auer, MD; C. Macmillan, MD; J. Peters, MD;
K.D. Gerbitz, MD; I. KraegelohMann, MD; A. Muntau, MD; V. Karcagi, PhD; R. Kalmanchey, MD;
H. Lochmuller, MD; E.A. Shoubridge, PhD; and P. Freisinger, MD
Article abstractObjective: To report three unrelated infants with a distinctive phenotype of Leigh-like syndrome,
neurogenic muscular atrophy, and hypertrophic obstructive cardiomyopathy. The patients all had a homozygous missense
mutation in SCO2. Background: SCO2 encodes a mitochondrial inner membrane protein, thought to function as a copper
transporter to cytochrome c oxidase (COX), the terminal enzyme of the respiratory chain. Mutations in SCO2 have been
described in patients with severe COX deficiency and early onset fatal infantile hypertrophic cardioencephalomyopathy.
All patients so far reported are compound heterozygotes for a missense mutation (E140K) near the predicted CxxxC metal
binding motif; however, recent functional studies of the homologous mutation in yeast failed to demonstrate an effect on
respiration. Methods: Here we present clinical, biochemical, morphologic, functional, MRI, and MRS data in two infants,
and a short report in an additional patient, all carrying a homozygous G1541A transition (E140K). Results: The disease
onset and symptoms differed significantly from those in compound heterozygotes. MRI and muscle morphology demon-
strated an age-dependent progression of disease with predominant involvement of white matter, late appearance of basal
ganglia lesions, and neurogenic muscular atrophy in addition to the relatively late onset of hypertrophic cardiomyopathy.
The copper uptake of cultured fibroblasts was significantly increased. Conclusions: The clinical spectrum of SCO2
deficiency includes the delayed development of hypertrophic obstructive cardiomyopathy and severe neurogenic muscular
atrophy. There is increased copper uptake in patients fibroblasts indicating that the G1541A mutation effects cellular
copper metabolism.
NEUROLOGY 2001;57:14401446
Cytochrome c oxidase (COX) deficiency is found in
patients with different clinical phenotypes primarily
affecting organs with high energy demand such as
the brain, skeletal muscle, heart, and kidney.
1
Pedi-
gree studies suggest an autosomal recessive inheri-
tance in most cases; however, mutations in nuclear
genes coding for the COX subunits themselves have
not been reported.
2,3
Several nuclear genes are
known from yeast studies to be essential factors for
assembly and maintenance of the COX complex.
4
The
human homologues of four such assembly genes
SURF1,
5,6
SCO2,
7-9
COX10,
10
and SCO1
11
have now
been identified in human COX deficiency disorders.
SURF1 was found to be mutated in a series of pa-
tients with typical Leigh syndrome and COX
deficiency.
12
Mutations in SCO2 have so far been described in
six infants with a fatal disorder with hypertrophic
From the Metabolic Disease Centre Munich (Drs. Jaksch, Horvath, Peters, Gerbitz, and Freisinger) and Institute of Clinical Chemistry, Molecular
Diagnostics and Mitochondrial Genetics (Drs. Jaksch, Horvath, and Gerbitz), Munich; Childrens Hospital of the Technical University (Drs. Peters and
Freisinger), Munich; Max Planck Institute of Psychiatry (Dr. Auer), Munich; Childrens Hospital of the Ludwig Maximilians University (Dr. Muntau),
Munich; Genzentrum and Friedrich-Baur-Institut of the Ludwig-Maximilians-University (Dr. Lochmuller), Munich, Germany; The John F. Kennedy
Institute (Dr. Horn), Glostrup, Denmark; Departments of Pediatrics and Neurology (Dr. Macmillan), University of Illinois, Chicago; Dept. of Neuropediatrics,
Childrens Hospital (Dr. Kraegeloh-Mann), University of Tuebingen, Germany; National Institute of Public Health (Dr. Karcagi), Budapest; Department of
Pediatrics II (Dr. Kalmanchy), Semmelweis University, Budapest, Hungary; and Montreal Neurological Institute and Department of Human Genetics (Dr.
Shoubridge), McGill University, Montreal, Quebec, Canada.
Supported by grants from the Deutsche Forschungsgemeinschaft (Ja 802/1-2) (M.J.), the Friedrich-Baur-Stiftung (M.J., H.L.), the Ernst und Berta Grimmke
Stiftung (M.J., R.H.), the CIHR (E.A.S.), and the March of Dimes Birth Defects Association (E.A.S.). E.A.S. is an MNI Killam Scholar.
Received January 29, 2001. Accepted in final form July 4, 2001.
Address correspondence and reprint requests to Dr. M. Jaksch, Metabolic Disease Centre Munich-Schwabing, Koelner Platz 1, 80804 Muenchen, Germany;
e-mail: Michaela.Jaksch@lrz.uni-muenchen.de
1440 Copyright 2001 by AAN Enterprises, Inc.
obstructive cardiomyopathy (HCMP) as the predomi-
nant symptom.
7-9
As all patients with SCO2 muta-
tions carried a G1541A mutation on one allele with
different mutations, including stop and missense
mutations, on the other allele, it was suggested that
the G1541A mutation might represent an ancient
founder allele or a mutational hotspot.
8
The G1541A
mutation is located next to the CxxxC copper binding
motif of Sco2, changing a conserved glutamate to
lysine (E140K). This amino acid substitution was
proposed to alter the copper binding properties of the
protein, but the exact mechanism of pathogenesis
remains unknown. Evidence for a functional COX
impairment due to this mutation in humans was
obtained via restoration of COX activity after intro-
ducing a normal chromosome 22 into COX deficient
skin fibroblasts of an index patient with compound
heterozygous mutations at nucleotide positions
G1541A and C1634T.
8
In contrast, SCO2 mutations
engineered in yeast failed to demonstrate an effect
on respiratory function.
13,14
Here we present clinical, biochemical, functional,
and MRI and MRS data in two infants carrying a
homozygous G1541A transition (E140K) and briefly
report on a third case presenting with similar clini-
cal symptoms.
Patient and methods. Patient A. This girl was the
second child of nonconsanguineous parents (mother from
Poland, father from Hungary). There was no family history
of neurologic disease. Her older sister, an extremely pre-
mature baby, died after 3 days. The patient was born by
spontaneous delivery in the 38th week of gestation. Apgar
scores were 9/10/10. Persistent feeding difficulties
prompted a pediatric consultation at the age of 4 months,
when muscular hypotonia was noted. At age 7 months,
weight was 6200 g (3rd percentile), length was 65 cm
(3rd percentile), and head circumference was 42.5 cm
(10th percentile). She was unable to lift her head when
prone and at rest was hypotonic, but when upset she as-
sumed an opisthotonic posture. Clinical examinations of
the respiratory, cardiac, and abdominal organs were nor-
mal. A severe low frequency resting and action tremor of
the upper extremities and trunk were noted. Tongue fas-
ciculations and ptosis developed, and her cognitive devel-
opment plateaued. She developed breath-holding spells
and inspiratory stridor when upset. Abdominal ultra-
sound, EEG, and echocardiography were normal. Specific
laboratory tests and metabolic screening in blood and
urine were first performed at age 7 months. Copper con-
centrations were found repeatedly decreased at age 7 and
8 months (Cu: 0.38, 0.45; reference range 1.12.5 mg/L)
but ceruloplasmin, iron, transferrin, and ferritin were nor-
mal. Lactate was elevated (2.6; reference range 0.52.2
mmol/L) and free serum carnitine was slightly decreased.
CSF was normal except for a slightly elevated lactate (2.3;
reference range up to 2 mmol/L). Electromyography (EMG)
and nerve conduction studies at age 7 and 10 months re-
vealed a neurogenic pattern and active denervation. Se-
rum copper improved but did not normalize (0.61 [at age 9
months], 0.57 [9.5 months], 0.47 [10 months], 1.23 [11
months], 0.66 [12 months], 0.63 [12.5 months]) after start-
ing oral copper-orotate (930 g copper/day) supplementa-
tion at age 9 months and subcutaneous copper-histidinase
(500 g/day) supplementation at age 12 months. Urine
copper excretion was normal at the beginning of the oral
copper supplementation. Therapy with carnitine, ascor-
bate, coenzyme Q10, copper, riboflavin, L-valine, aspartate,
vitamin K, histidine, and diazepam was also instituted. At
age 8 months she was admitted twice to the pediatric in-
tensive care unit. Intubation was required for worsening
respiratory embarrassment and metabolic acidosis. During
this period HCMP developed and repeated echocardio-
graphies were done. At 11 months, left ventricular myocar-
dial thickening and a hypomobile intraventricular septum
was noted. At that time, lactate was highly elevated in
blood (7.8 mmol/L) and in CSF (8.0 mmol/L).
She was treated with propranolol and furosemide, but
the HCMP progressed rapidly. Two weeks later, echocar-
diogram demonstrated obstructive cardiomyopathy with a
thickness of the ventricular septum of 1.0 to 1.3 cm. At age
13 months, she died of cardiac failure.
Patient B. This girl is the second child born to noncon-
sanguineous German parents with no family history of
neurologic disease. Delivery was normal. Birthweight was
3430 g and Apgar scores were 10/10/10. Feeding difficulties
were noted shortly after birth and were persistent. At age
6 months developmental delay was first noted. At age 9
months she presented with dystrophy and severe general-
ized muscle hypotonia; she was unable to sit unsupported.
Impaired ocular movements, progressing bilateral ptosis,
and facial weakness were described. Chest was clear and
heart sounds were normal. EEG showed reduced back-
ground amplitude and generalized nonspecific cortical ac-
tivity changes. Echocardiography, MRI, EMG, and nerve
conduction velocities (NCV) were all normal at that time.
Laboratory investigation and metabolic screening in blood
and urine showed normal values except for mildly in-
creased lactate values.
At age 11 months she developed respiratory failure ne-
cessitating pediatric intensive care unit admission and in-
tubation, from which she cannot be weaned. Several
periods of lactic acidosis with maximum lactate peaks of 12
mmol/L have been observed, most of which have been asso-
ciated with respiratory infections.
At age 18 months mild HCMP was first noted (septum
thickness 0.8 cm), which progressed over the following 4
months (septum thickness 1.2 cm). After the diagnosis of
SCO2 mutations at age 21 months subcutaneous copper-
histidinase (500 g/day) supplementation was started, but
clinically no beneficial effect was observed. At age 23
months EMG did not detect any significant potentials and
NCV examination showed no conduction at all. During the
next 4 weeks (up to her current age of 25 months) her
HCMP worsened (septum thickness 1.5 cm); however,
blood pressure was normal or slightly elevated. Currently,
she has no detectable active movements except for very
discrete perioral contractions.
Patient C. This girl was the second child born to Hun-
garian nonconsanguineous parents who had no family his-
tory of neurologic disease. Her older sibling is healthy. She
was small for gestational age (weight 2650 g, length 50 cm
at term). At 3 months of age, her parents noted feeding
difficulties and developmental delay. The developmental
delay was documented at pediatric consultation at 4
October (2 of 2) 2001 NEUROLOGY 57 1441
months. MRI of the brain, EMG, and NCV were normal at
that time. She underwent physiotherapy; however, her
symptoms progressed.
At 7 months she had limited extraocular movements,
bilateral ptosis, inspiratory stridor, axial hypotonia, and
limb spasticity more of the lower than the upper extremi-
ties. Laboratory investigation and metabolic screening in
blood and urine showed normal values except for increased
lactate values both in blood (5.26 mmol/L) and in CSF (3.4
mmol/L). EEG demonstrated generalized high amplitude
slow waves. NCV revealed decreased motor conduction ve-
locities in both peroneal nerves and EMG showed polypha-
sic potentials.
At age 8 months severe generalized muscle hypotonia
occurred and she needed intensive care for respiratory in-
sufficiency. Symptoms of cardiac failure developed
promptly and 5 days later she died. At autopsy there was
severe HCMP, which was believed to be the immediate
cause of death.
MRI and
1
H-MRS. Patient A. Two MR examinations
(1.5 T Signa Echospeed, GE Medical Systems, Milwaukee,
WI) were performed at age 7 and 13 months including
standard imaging protocols (T1- and T2-weighted). In addi-
tion, localized proton spectroscopy (PRESS: repetition time
[TR] 2000/echo time [TE] 144 msec, 128 averages) of
the left putamen and right periventricular parietal white
matter (6.5 and 8 mL voxel sizes) were obtained and ana-
lyzed using a prior knowledge fitting procedure
(LCModel).
15
Patient B. The second patient was examined at age 20
months with the protocol as above.
1
H-MRS was obtained
from left putamen, white matter, and parietal cortex using
short echo and long echo times (PRESS: TR 2000/TE
144 msec or 35 msec, 128 averages).
Copper uptake in cultured skin fibroblasts. Copper up-
take and retention in fibroblasts of Patient A were mea-
sured using JFKs standard protocol.
16
In brief, the cells
were pulsed for 20 hours in 10M
64
CuCl
2
added to me-
dium F12 supplemented with 20% fetal calf serum fol-
lowed by a 24-hour pulse chase in nonlabeled medium.
Muscle histology and biochemistry. At age 7 and 11
months (Patient A), at age 9 months (Patient B), and at
age 7 months (Patient C), open muscle biopsies were per-
formed and used for histochemistry and biochemistry. Six-
micrometer serial cross-sections were obtained for
histochemical stainings according to standard proce-
dures.
17
A frozen part of each biopsy was used for
biochemistry.
Respiratory chain (RC) complexes IIV activities were
determined in skeletal muscle and in fibroblasts (Patient A
only) as described.
18
Skin and a second muscle biopsy in
Patient B were refused by the parents.
DNA analysis. DNA was extracted from skeletal mus-
cle and leukocytes according to standard purification pro-
tocols (Qiagen, Hilden, Germany). The SCO2 gene was
amplified and sequenced as described.
7,8
The G1541A mu-
tation was analyzed by additional restriction fragment
length polymorphism (RFLP) analysis in the index patient
and her parents as well as in 400 control subjects. SURF1,
as well as COX17 and SCO1 mutations
19
and frequent
mitochondrial DNA mutations at nucleotide positions
3243, 3250, 3271, 7512, 7472, 8344, 8356, and 8993, were
analyzed by standard RFLP, Southern blot, and sequence
analysis. We did not sequence the ATP7A gene because of
the normal copper retention in fibroblasts in Patient A.
Results. MRI and MRS. Patient A. Initially (7
months), nonspecific MRI findings with moderate frontal
atrophy, delay of myelination, and rare irregular T2 hyper-
intensities (HI) in the white matter were noted.
1
H-MRS
investigations showed moderately elevated lactate in the
left putamen only with otherwise unremarkable metabo-
lites (figure 1, a and b). At 13 months, the atrophy had
markedly progressed and there were multiple, symmetric
T2 hyper- and T1 hypointense lesions in the periventricu-
lar white matter, rostral mesencephalon, internal pallida,
and medial thalami, as well as diffuse T2 hyperintensities
of both striata, the dorsal pons, and large parts of the
white matter with sparing of the u-fibers (figure 1e). Lac-
tate had further increased in the putamen and was simi-
larly elevated in the partially necrotic appearing parietal
white matter in addition to mildly elevated choline-
containing compounds and reduced N-acetylaspartate
(NAA) (figure 1, c and d).
Patient B. MRI at 20 months showed marked ventric-
ular enlargement and bilateral HI in atrophic basal gan-
glia and thalami as well as in periventricular white matter
and diffuse subcortical HI, predominantly in the frontal
lobes. There was neither necrosis nor brainstem involve-
ment (figure 1f). MRS revealed severe changes with high
lactate increase and NAA reduction most pronounced in
the parietal cortex, but also in white matter and basal gan-
glia. In addition, choline and alanine were mildly elevated
and glucose was markedly elevated (data not shown).
Patient C. MRI was only performed at age 4 months
and showed no abnormalities.
Muscle histology and biochemistry. Patient A. At age
7 months, some degenerating, myopathic fibers were found
in a scattered distribution. In addition, a few small and
angular fibers were present, indicating additional neuro-
genic changes (figure 2B
1
). Histochemistry showed an
overall reduction of COX activity in the majority of fibers;
however, no COX-negative or ragged red fibers were seen
(figure 2B
2
). Biochemical measurements of a muscle ho-
mogenate showed reduced COX activity with 37 U (refer-
ence range: 90281 U, U/gram noncollagen protein) and to
citrate synthase (CS) with 85.5 U resulting in a ratio of
0.46 (reference range: 0.94.7). Other complexes of the RC
were normal. Residual activity of COX in fibroblasts was
68% compared to the lowest reference range. At age 11
months, a strong progression of the neurogenic changes
was noted compared to the first biopsy. The majority of
fibers showed an angular shape and there was a severe
reduction in fiber size (figure 2C
1
). On histochemical stain-
ing, COX activity was not detectable in most fibers (figure
2C
2
). Biochemically, COX activity was further reduced
when related to noncollagen protein (NCP) (12 U) and to
CS (37) with a ratio of 0.3. Other complexes of the RC were
normal.
Patient B. At age 9 months most of the fibers were
small and some degenerating fibers were also found in a
scattered distribution (figure 2D
1
). On histochemistry all
fibers were COX negative (figure 2D
2
) Biochemically, COX
activity was severely reduced when related to NCP (16 U)
and to CS (111) with a ratio of 0.14. Other complexes of the
RC were normal.
1442 NEUROLOGY 57 October (2 of 2) 2001
Figure 1. (ad)
1
H-MRS of the left putamen
(a, c) and right parietal white matter (b, d)
at 7 (a, b) and 13 months (c, d). Note the in-
crease of the inverted doublet at 1.3 ppm rep-
resenting lactate. (e) Patient A. Axial T2-
weighted MRI scans at 13 months show
marked atrophy and a chronic subdural he-
matoma on the left. Note the bilateral ne-
crotic lesions in the rostral mesencephalon,
internal globi pallida, and periventricular
white matter (arrows), and diffuse hyperin-
tensities in the basal ganglia and white mat-
ter. (f) Patient B. Axial T2-weighted MRI
scans at 20 months. Note the marked ven-
tricular enlargement and bilateral hyperin-
tensities in atrophic basal ganglia, thalami,
and in periventricular white matter.
October (2 of 2) 2001 NEUROLOGY 57 1443
Patient C. Muscle biopsy at age 7 months showed gen-
eralized atrophic fibers and some normal size fibers and
also some degenerating fibers in a scattered distribution
(data not shown). On histochemistry an overall reduction
of COX activity was observed (data not shown).
Copper uptake in cultured fibroblasts. The copper up-
take was performed on fibroblasts from Patient A and was
significantly increased in repeated experiments (patient
45.0; Menkes patients [n 47] median 82.6, range 42.6
127.6 ng,
64
Cu per mg protein per 20 hours; controls [n
18] median 19.9, range 8.230.8); however, the retention
value was completely normal (patient 18.7%; Menkes pa-
tients median 65.8%, range 40.394.5%; controls median
16.3%, range 9.722.7%), indicating that she did not have
Menkes disease. Increased copper uptake has now been
confirmed in other patients with compound heterozygous
mutations in SCO2 (unpublished data, 2001).
DNA analysis. A homozygous G1541A transition mu-
tation, predicting an E140K substitution, was identified on
both alleles of SCO2 in all three patients (figure 3A). The
parents of all patients were heterozygous for the G1541A
mutation (figure 3B, data not shown for Patient C). No
heterozygous carriers of the 1541 mutation were identified
in 400 controls. SURF1, as well as COX17 and SCO1 mu-
tations
19
and frequent mitochondrial DNA mutations at
nucleotide positions 3243, 3250, 3271, 7512, 7472, 8344,
8356, and 8993 and large-scale rearrangements, were
excluded.
Discussion. We here describe a distinctive (and
probably underdiagnosed) phenotype in three nonre-
lated patients with a homozygous E140K mutation.
All patients so far reported with SCO2 mutations are
compound heterozygotes for the E140K mutation,
the other allele being either a nonsense (Q53X,
R90X) or missense mutation (S225F, R171W).
7,8
These mutations result in a severe reduction in COX
Figure 2. (AD) Muscle histology. Magnification 100. Panels AD show H-E staining (A
1
D
1
) and COX staining (A
2
D
2
) for
each biopsy. A
1
, Normal control H-E staining; A
2
, COX staining. Patient A with biopsies taken at age 7 months (panel B)
and 11 months (panel C), and Patient B (panel D). Panel B (Patient A): At age 7 months, some degenerating fibers show
loss of myofibrils, or invasion of mononuclear cells was noted (B
1
). Residual COX activity (B
2
). Panel C (Patient A): At
age 11 months, the majority of fibers showed an angular shape and were severely reduced in fiber size, indicating a
strong progression of the neurogenic changes (C
1
). No COX activity (C
2
). Panel D (Patient B): At age 9 months, some de-
generating fibers show loss of myofibrils or invasion of mononuclear cells and small, angular-shaped fibers were noted
(D
1
). No COX activity (D
2
).
Figure 3. (AB) Sequence and restriction fragment length
polymorphism analysis. Sequencing of SCO2 in the pa-
tients showed a homozygous G1541A mutation (A). Re-
striction fragment length polymorphism analysis (B)
confirmed the homozygosity in the index Patients A and B
(lanes 1 and 2) and heterozygosity in both parents of index
Patients A and B (lanes 36) and the negative control
(lane 7). (Data not shown for Patient C.)
1444 NEUROLOGY 57 October (2 of 2) 2001
activity and early onset HCMP (table). The clinical
course and the predominant symptoms differ signifi-
cantly from all previously reported SCO2 cases in all
three of our patients. First, the onset of symptoms
occurred later and the clinical course was less pro-
gressive. Second, predominant involvement of the
peripheral nervous system, including demyelination
and denervation, was observed. In both Patients A
and C, a spinal muscular atrophy (SMA) variant was
considered histologically. We are therefore currently
investigating more samples genetically tested to be
negative for SMA for the occurrence of the common
SCO2 mutation. Third, there was an unusual pat-
tern of cerebral lesions, with predominant involve-
ment of white matter and late appearance of
moderate lesions in the basal ganglia and thalamus
(Leigh-like syndrome). Finally, the fatal HCMP oc-
curred late at age 10 months (Patient A), 18 months
(Patient B), and 8 months (Patient C). Thus, the
progressive cardiomyopathy in SCO2 patients is not
necessarily an early symptom of this disorder, but it
is indicative of a specific aerobic energy supply
threshold for cardiac function. The complex neuro-
logic phenotype observed in our patients suggests
that there may be significant clinical heterogeneity
associated with particular SCO2 mutations. Defects
of SCO2 should therefore be considered in patients
with COX deficiency and a Leigh-like syndrome, or
with prominent peripheral neuropathy and CNS in-
volvement, and also in patients with a muscle histol-
ogy suggestive for a SMA variant.
Patient A showed a mild specific COX deficiency
(~50% of the lowest reference in repeated experi-
ments) in muscle at age 7 months that declined to
about 30% on reinvestigation at 11 months. Al-
though such a decrease in COX could result from the
long immobilization of the patient, a 50% decrease in
the mitochondrial marker-enzyme CS was also ob-
served at 11 months and as COX activity was nor-
malized to CS, this excludes an artificial deficiency
of COX. A severe COX deficiency was detected in
Patient B at age 9 months, although she presented
with mild symptoms at that time. In support of the
different biochemical findings, histochemistry
showed a similar picture with higher levels of COX
activity in Patient A compared to Patient B. Thus, no
exact correlation can be found between COX activity
and clinical symptoms in our cases, which could re-
flect genetic background or environmental influ-
ences. On the other hand, our reference range for
COX activity assumes a normal fiber distribution
and the progressive neurogenic atrophy could influ-
ence the interpretation of COX biochemistry.
The mechanism by which the E140K affects Sco2
function is unknown; however, its close proximity to
the putative CxxxC copper binding site suggests that
it may impair copper binding or copper release to
COX. It is thought that the cysteine residues in the
CxxxC motif are directly involved in the insertion of
copper into the Cu
A
center of COX subunit 2 and this
may occur through an enzymatic function involving
direct metal ion exchange between the two CxxxC
domains.
20,21
Yeast functional studies on the homolo-
gous mutation (E155K) failed to demonstrate a defi-
ciency in COX activity in haploid yeast.
14
In contrast,
another mutation homologous to S225F in human
Sco2 produced a defect in COX assembly that re-
sulted in respiratory incompetence in these cells.
These data predict that the functional consequences
of E140K mutation in humans are less severe than
the other mutations described in SCO2, and this is
supported by the clinical outcome in our patients
Table Genotypephenotype correlation in patients with SCO2 mutations
Mutation
Onset/death,
mo
COX residual activity
related to CS, %
Main symptoms occurring
in chronological order
Onset of
HCMP References
E140K/R90X 0/1 10 MH, RI, seiz, HCMP 3 wk 8
E140K/R90X 0/1 12 MH, RI, seiz, HCMP 3 wk 8
E140K/Q53X 0/2 18 MH, HCMP, RI Neonatal 7
E140K/Q53X ND/3 4 RI, HCMP, MH 6 wk 7
E140K/R173W 1/4 0 MH, RI, seiz, HCMP 8 wk 8
E140K/S225F 2.5/6 4 HCMP, RI, MH, NP, BA 10 wk 7
E140K/E140K 4/13 51 MH, LLS, ptosis, NP Patient A (at age 7 mo)
30 MH, LLS, ptosis, NP, RI,
BA, HCMP
8 mo Patient A (between age
811 mo)
E140K/E140K 6/alive, 24 mo 16 MH, ptosis, RI, NP Patient B (at age 9 mo)
MH, ptosis, RI, NP, LLS 17 mo Patient B (at age 17
mo)
HCMP
E140K/E140K 3/8 ND Ptosis, MH, LLS, NP, RI,
HCMP
8 mo Patient C
MH muscle hypotonia; RI respiratory insufficiency; seiz seizures; HCMP hypertrophic cardiomyopathy; NP neuropathy;
LLS Leigh-like syndrome; BA brain atrophy.
October (2 of 2) 2001 NEUROLOGY 57 1445
(see the table). The very severe COX defect that is
seen in some compound heterozygotes, however, re-
mains unexplained. Although the sample size is not
large, the extent of the deficiency in these cases is
usually greater in those in which the E140K allele
occurs with another missense allele. Such a result
might be explained if the Sco2 protein functions as a
dimer or higher order oligomer.
The observation of an increased copper uptake in
fibroblasts of Patient A in the face of normal reten-
tion values demonstrates that function of the copper
export pump ATP7A is unchanged compared to con-
trols and thus may be viewed as a compensatory
mechanism to overcome impaired Sco2 function. A
higher copper turnover in cells might also explain
the low serum copper values found in Patient A.
Detailed functional analyses on patient cells with
different SCO2 mutations are needed to investigate
their role in the intramitochondrial copper transport.
Patients A and B presented here were the first
cases with SCO2 mutations being treated with cop-
per starting in a late stage of the disease at age 9
months and 21 months, respectively. As expected, no
beneficial effects on clinical progression were ob-
served, probably owing to irreversible damage on
heart and nervous system. Earlier treatment might
be considered in other patients with SCO2
mutations.
SCO2, like the other COX assembly factors that
have been implicated in human disease (Surf1, Sco1,
Cox10), is ubiquitously expressed in human tissues,
and there remains no satisfactory explanation for
the tissue-specific differences in COX deficiency, or
the clinical involvement of particular cell types that
characterize mutations in this gene. The develop-
ment of the fatal HCMP seems to be relatively spe-
cific for SCO2 defects as it has been reported only in
a single patient with SURF1 mutations so far,
22
and
the reported cases suggest that the onset of this
symptom reflects the severity of the mutation (see
the table). The fact that two nonsense mutations in
SCO2 have not been observed suggests that a Sco2
null phenotype might be associated with prenatal
lethality. Analysis of patient cells and other models
may reveal further insights in the biochemical conse-
quences of the common E140K mutation and the
pathogenesis of Sco2 defects.
Acknowledgment
The authors appreciate the clinical and pathologic investigations
of Prof. D. Pongratz and PD Dr. W. MuellerFelber, Dr. E. Siska,
and the helpful discussions with Prof. Sperl. A. Zimmermann pro-
vided excellent technical assistance. They thank the families
whose collaboration made this study possible.
References
1. Zeviani M, Tiranti V, Piantadosi C. Mitochondrial disorders.
Medicine 1998;77:5972.
2. Adams PL, Lightowlers RN, Turnbull DM. Molecular analysis
of cytochrome c oxidase deficiency in Leighs syndrome. Ann
Neurol 1997;41:268270.
3. Jaksch M, Hofmann S, Kleinle S, et al. A systematic screen of
nuclear and mitochondrial candidate genes in 21 patients with
cytochrome c oxidase (COX) deficiency reveals mitochondrial
tRNA
Ser(UCN)
mutations in a subgroup with syndromal en-
cephalopathy. J Med Genet 1998;35:895900.
4. Petruzzella V, Tiranti V, Fernandez P, Ianna P, Carrozzo R,
Zeviani M. Identification and characterization of human cD-
NAs specific to BCS1, PET112, SCO1, COX15, and COX11,
five genes involved in the formation and function of the mito-
chondrial respiratory chain. Genomics 1998;54:494504.
5. Zhu Z, Yao J, Johns T, et al. Surf1, a factor involved in the
biogenesis of cytochrome c oxidase, is mutated in Leigh syn-
drome. Nat Genet 1998;20:337343.
6. Tiranti V, Hoertnagel K, Carrozzo R, et al. Mutations of
SURF-1 in Leigh disease associated with cytochrome c oxidase
deficiency. Am J Hum Genet 1998;63:16091621.
7. Papadopoulou LC, Sue CM, Davidson M, et al. Fatal infantile
cardioencephalomyopathy with cytochrome c oxidase (COX)
deficiency and mutations in SCO2, a human COX assembly
gene. Nat Genet 1999;23:333337.
8. Jaksch M, Ogilvie I, Kortenhaus G, Bresser HG, Gerbitz KD,
Shoubridge EA. Mutations in SCO2 are associated with a
distinct form of hypertrophic cardiomyopathy and cytochrome
c oxidase deficiency. Hum Mol Genet 2000;9:795801.
9. Sue CM, Karadimas C, Checcarelli N, et al. Differential fea-
tures of patients with mutations in two COX assembly genes,
SURF-1 and SCO2. Ann Neurol 2000;47:589595.
10. Valnot I, von Kleist-Retzow JC, Barrientos A, et al. A muta-
tion in the human heme A:farnesyltransferase gene (COX10)
causes cytochrome c oxidase deficiency. Hum Mol Genet 2000;
9:12451249.
11. Valnot I, Osmond S, Gigarel N, et al. Mutations of the SCO1
gene in mitochondrial cytochrome c oxidase (COX) deficiency
with neonatal-onset hepatic failure and encephalopathy. Am J
Hum Genet 2000;67:11041109.
12. Tiranti V, Jaksch M, Hofmann S, et al. Loss-of-function muta-
tions of SURF-1 are specifically associated with Leigh syn-
drome with cytochrome c oxidase deficiency. Ann Neurol 1999;
46:161166.
13. Glerum DM, Shtanko A, Tzagoloff A. SCO1 and SCO2 act as
high copy suppressors of a mitochondrial copper recruitment
defect in Saccharomyces cerevisiae. J Biol Chem 1996;271:
2053120535.
14. Dickinson EK, Adams DL, Schon EA, Glerum DM. A human
SCO2 mutation helps define the role of Sco1p in the cyto-
chrome oxidase assembly pathway. J Biol Chem 2000;275:
2678026785.
15. Provencher SW. Estimation of metabolite concentrations from
localized in vivo proton NMR spectra. Magn Reson Med 1993;
30:672679.
16. Tumer Z, Horn N. Menkes disease: recent advances and new
aspects. J Med Genet 1997;34:265274.
17. Dubowitz V. Muscle biopsy: a practical approach. 2nd edition.
London: Bailliere Tindall, 1985.
18. Fischer JC, Ruitenbeek W, Gabreels FJ, et al. A mitochondrial
encephalomyopathy: the first case with an established defect
at the level of coenzyme Q Eur J Pediatr 1986;144:441444.
19. Horvath R, Lochmuller H, Stucka R, et al. Characterization of
human SCO1 and COX17 genes in mitochondrial cytochrome-
c-oxidase deficiency. Biochem Biophys Res Commun 2000;24:
530533.
20. Chinenov YV. Cytochrome c oxidase assembly factors with a
thioredoxin fold are conserved among prokaryotes and eu-
karyotes. J Mol Med 2000;78:239242.
21. Wernimont AK, Huffman DL, Lamb AL, OHalloran TV,
Rosenzweig AC. Structural basis for copper transfer by the
metallochaperone for the Menkes/Wilson disease proteins.
Nat Struct Biol 2000;7:766771.
22. Poyau A, Buchet K, Bouzidi MF, et al. Missense mutations in
SURF1 associated with deficient cytochrome c oxidase assem-
bly in Leigh syndrome patients. Hum Genet 2000;106:194
205.
1446 NEUROLOGY 57 October (2 of 2) 2001