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Cytochrome c oxidase deficiency in leigh syndrome.

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Cytochrome c Oxldase
Deficiency in Leigh Syndrome
Salvatore DiMauro, MD," Serenella Servidei, MD," Massimo Zeviani, MD," Maja DiRocco, MD,"
Darryl C. DeVivo, MD," Stefan0 DiDonato, MD,t Graziella Uziel, MD,? Kenneth Berry, MD,$
George Hoganson, MD,§ Stanley D. Johnsen, MD," and Peter C. Johnson, MDil
We studied 6 mitochondrial enzymes in crude extracts and isolated mitochondria from 5 children with pathologically
proven subacute necrotizing encephalomyelopathy (Leigh syndrome). Samples were taken from brain ( 5 patients),
skeletal muscle (4 patients), liver (4 patients), kidney (4 patients), heart (1 patient), and cultured fibroblasts ( 3 patients).
An isolated defect of cytochrome c oxidase (COX) activity was found in brain (decrease of activity to 15 to 39% of the
normal mean), muscle (9 to 20%), kidney (1 to 67%), and in the 1 available heart (4%)from a patient with cardiopathy.
COX activity was also decreased in liver of 3 patients (2 to 13% of normal) and in culitured fibroblasts of 2 patients ( 18
and 27%), but it was normal in both liver and fibroblasts from 1patient. Immunotitration using polyclonal antibodies
against human heart COX showed essentially normal amounts of cross-reacting enzyme protein in various tissues from
different patients. Electrophoresis of COX immunoprecipitated from brain mitochondrial extracts showed normal
patterns of COX subunits in 2 patients. This study confirms the theory that COX deficiency is an important cause of
Leigh syndrome.
DiMauro S, Servidei S, Zeviani M, DiRocco M, DeVivo DC, DiDonato S, Uziel G, Berry K,
Hoganson G, Johnsen SD, Johnson PC: Cytochrome c oxidase deficiency
in Leigh syndrome. A n n Neurol 22:478-506, 1787
In 1951 Leigh described the clinical and pathological
features of a 7-month-old infant who died shortly after
the onset of a neurological disorder characterized by
somnolence, blindness, deafness, and spasticity [11. In
the numerous subsequent reports of Leigh disease, or
subacute necrotizing encephalomyelopathy (SNE), the
clinical picture has varied considerably. Symptoms usually begin in early infancy, but congenital, juvenile, and
even adult-onset cases have been described, and the
course can be acute, as in Leigh's patient, or intermittently progressive for several years [2). The diagnosis
rests on SNEs distinctive neuropathology, which consists of focal, symmetrical necrotic lesions extending
from thalamus to pons and involving the inferior olives
and the posterior columns of the spinal cord. Microscopically, there is necrosis, demyelination, vascular
proliferation, and astrocytosis.
Lactic acidosis, first noted by Worsely and associates
[31, is a consistent finding in Leigh syndrome, suggesting that a disorder of pyruvate metabolism may be the
primary biochemical defect. Pyruvate carboxylase (PC)
deficiency has been reported in 4 infants with the characteristic neuropatholcgical lesions (4-71, but the enzyme defect has not been documented unequivocally
[S- lo]. Defects of the pyruvate dehydrogenase complex (PDHC) have allso been described in some patients with Leigh syndrome { 10, l l}.
In 1977, Willems and associates [I21 described the
first case of Leigh syndrome with cytochrome c oxidase
(COX) deficiency; the patient died of respiratory
insufficiency at age 6 years after suffering from a syndrome that included ataxia, dementia, and optic atrophy. Muscle histochemistry was normal, but electron
microscopy showed su bsarcolemmal deposits of mitochondria. C O X activity was laclung in muscle mitochondria, decreased in the heart, but normal in the
liver. COX deficiency was also documented in 3 other
cases [13, 141.
We now report CO X deficiency in multiple tissues
from 5 unrelated children, including Hoganson's patient [141, with pathologically confirmed Leigh syndrome.
From the "H. Houston Merrin Clinical Research Center for Muscular Dystrophy and Related Diseases, and the Division of Pediatric
and Surgeons, New York, NY; tIstituto Neurologico "C. Besra," Milan,
Italy; $Vancouver General Hospital, Vancouver, BC, Canada; the
§Clinical Genetics Center, University of Wisconsin, Madison, WI;
and the "Barrow Neurological Institute, St. Joseph's Hospital and
Medical Center, Phoenix, AZ.
Received Oct 2 7 , 1986, and in revised form Feb 13, 1987. Accepted
for publication Feb 20. 1987.
Address correspondence to Dr DiMauro, 4-420, College of physicians and Surgeons, 630 West 168 St, New York, NY 10032.
Case Reports
Patient 1
Patient 1 was a normal boy until he was 1Y2 years old, when
he started losing skills such as walking and talking. An older
sibling was normal and there was no parental consanguinity.
At age 3, he had generalized hypotonia, ophthalmoplegia,
ataxia, sighing respirations, and hirsutism.
Blood lactate and pyruvate levels ranged from 1.9 to 7.0
mM (normal ? SD, 1.0 ? 0.4), and 0.11 to 0.25 mM (normal, 0.11 t 0.03) respectively, with lactate/pyruvate ratios
of 14 to 35 (normal, 10 to 18). Cerebrospinal fluid (CSF)
lactate was 4.7 m~ (normal, 0.5 to 1.3) and pyruvate was
0.24 mM (normal, 0.040 to 0.067). Beta-hydroxy-butyrate,
acetoacetate, and lactate were intermittently present in the
urine, and there was increased urinary excretion of glycine
and alanine.
Computed tomography (CT) scan showed cystic degeneration and cerebellar atrophy. Brainstem auditory responses
were abnormal, and electromyography suggested a myopathy. A muscle biopsy was normal, except for increased
number of lipid droplets and lack of stain with the COX
reaction 1141. There were no ragged-red fibers. Electron microscopy showed subtle alterations of mitochondrial size and
shape. The patient’s course was relentlessly downhill, and he
died at 41/2 years of respiratory arrest.
Autopsy findings were confined to the brain. Symmetrical
cystic lesions lined by necrotic tissue were seen in the
periventricular area, brainstem, and cerebellum. Histologically, there was neuronal loss, astrocytosis, and proliferation
of capillaries.
Patient 2
Patient 2 was a normal boy until he was 1 year old, when his
gait deteriorated. H e developed tremor of the arms and the
head and had jerky eye movements. By age 4, he had limitation of upward gaze, expressive dysarthria, diffuse wasting and weakness, hyporeflexia, limb ataxia, and intention
At age 5, he was unable to walk and sat only with support.
H e vomited several times a day, had an abnormal breathing
pattern, and poor articulation. The patient had a mental age
of 2 to 3 years by Gesell assessment. There was bilateral
temporal pallor of the optic discs. Eye movements showed
limited abduction bilaterally, with horizontal nystagmus.
There was diffuse hypotonia and weakness, and tendon
reflexes were absent. H e had mild lactic acidosis, nerve conduction velocities were slowed, and muscle biopsy showed
osmiophilic inclusions in mitochondria. Brainstem auditory
responses were abnormal. The child died at 5 years and 2
months, and autopsy was performed 3 hours after death.
Neuropathological examination showed the typical lesions
of SNE, affecting mostly the brainstem and the cerebellum,
but sparing the basal ganglia, thalamus, and cortex. There
was rarefaction of cerebral tissue (spongy change) with fine
glial meshwork and, in some areas, dense gliosis.
Patient 3
Patient 3 was a normal boy until 1 year of age, when his
parents noted loss of motor skills and stunted growth. Three
older siblings were normal. There was no consanguinity. At
34 months he could not walk and sat only with support. He
had superficial breathing, tachypnea, truncal ataxia, ptosis of
the right eyelid, sluggish pupillary reaction to light, bilateral
optic atrophy, and areflexia.
Serum lactate was 4.0 mM (normal, 0.47 to 1.58) and
pyruvate was 0.19 mM (normal, 0.1 1 5 0.03). Serum alanine
was increased. CSF lactate was 1.91 mM (normal, 0.5 to 1.3)
and pyruvate 0.093 mM (normal, 0.040 to 0.067). CT scan
showed cerebellar atrophy. Motor nerve conduction velocities were borderline. Electroencephalogram (EEG), electrocardiogram, echocardiogram, and muscle biopsy were
normal. The child developed nystagmus, ophthalmoplegia,
intellectual regression, and respiratory insufficiency, and died
at age 4 .
Autopsy showed symmetrical foci of spongiosis, with astrocytosis and proliferation of capillaries, affecting especially
the dentate nucleus, the inferior olives, and the medial reticular substance.
Patient 4
Patient 4 was a girl with delayed early development. She held
her head up at 5 months, sat at 14 months, spoke her first
words at 18 months, and walked at 3 years. An older brother
was normal and there was no consanguinity. At age 3 years,
she developed postural and intentional tremor, ataxia, bilateral hearing loss, and motor regression. EEG and brain C T
scan were normal. Lactic acidosis was noted at 3% years. At 4
years, she was small and hirsute, with ptosis and ophthalmoplegia, hypotonia, facial paresis, dysarthria, gross intentional
tremor, and hyporeflexia. Fundi, EEG, and muscle biopsy
were normal. Serum lactate was 2.7 m~ (normal, less than
2.2). She died at 5 years of respiratory failure. Autopsy
showed bilateral areas of necrosis in the mesencephalon,
pons, and medulla.
Patient 5
Fatient 5 was a boy and the first child of an unrelated Apache
Indian couple. He was floppy at birth and had irregular respirations. He improved in the next 3 months; the abnormal
respirations stopped, he could fix and follow objects and
smile, but he remained hypotonic.
At age 5 months, he stopped crying; at 6 months, he could
no longer fix and follow objects. Blood, CSF chemistries,
and EEG were normal. CT scan showed symmetrical lowdensity lesions in the thalamus. At age 7 months, there was a
sudden deterioration, with lethargy and irregular breathing.
Blood tests showed increased lactic acid (6.4 to 11.9 mM;
normal, 0.5 to 2.2) but normal pyruvate (0.10 to 0.19 mM;
normal, 0.11 2 0.03). CT and magnetic resonance imaging
scans showed lesions in the midbrain and thalamus. A muscle
biopsy was normal.
The child developed cardiomegaly and congestive heart
failure. Electrocardiogram showed right axis deviation, right
biventricular hypertrophy, and ST segment shift, compatible
with subendocardial ischemia. Echocardiogram showed poor
contractility of the ventricles with hypertrophy of the left
ventricle and interventricular septum, and mild hypertrophy
of the free wall of the right ventricle. There was mitral and
tricuspid insufficiency, and mildly reduced aortic and pulmonary flow. The child died of cardiac failure at 7 % months.
DiMauro et al: Cytochrome c Oxidase in Leigh Syndrome 433
Autopsy showed bilaterally symmetrical zones of grey discoloration in the periaqueductal grey matter and, to a lesser
extent, in the substantia nigra and lateral tegmentum. Microscopically, there was prominent capillary proliferation and
vacuolation of the extracellular space extending down into
the tegmentum of the medulla to the level of the inferior
olives. The heart was enlarged (50 gm; normal, less than 39)
but did not show microscopic abnormalities.
Materials and Methods
Muscle biopsies (patients 1, 2, and 5 ) and specimens of muscle, liver, kidney, brain, and heart obtained at autopsy (performed between 2 and 5 hours postmortem) were immed$tely frozen in liquid nitrogen for biochemical studies,
shipped on solid carbon dioxide, and stored in liquid nitrogen. Control muscle was obtained by diagnostic biopsy from
patients, including infants, who were free of neuromuscular
diseases. Control autopsy tissues were obtained through the
Tissue Bank of Columbia-Presbyterian Medical Center
(New York, NY) and from the National Diabetes Research
Interchange (Philadelphia, PA) from patients without clinical
or pathological involvement of the organ requested. The age
of patients at death varied, but children were always included. Tissues were stored in liquid nitrogen or in a deep
freeze (- 70°C) for up to 1 year.
Biacbemistty and Tisstle Preparation
Tissues were homogenized in 9 volumes of 0.15
M KC1, 50
mM Tris-HC1, pH 7.4, in all-glass, motor-driven homogenizers, and mitochondrial enzymes were measured in supernatants after centrifugation at 750 g for 15 minutes.
Mitochondria were isolated from frozen postmortem muscle, heart, liver, and kidney by the procedure of Bookelman
and associates {lS], and from frozen brain tissue by the
method of Lai and Clark {lb].
Determination of Enzymes and Cytochrome Spectra
Described spectrophotometric assays were used to measure succinate-cytochrome c reductase and rotenone (0.3
mM)-sensitive reduced nicotinamide adenine dinucleotide
(NADH)-cytochrome c reductase { 171, citrate synthase {18),
NADH-dehydrogenase { 193, and succinate dehydrogenase
[20]. COX was also determined spectrophotometrically by
the decrease in absorbance at 550 pm of reduced cytochrome c {21]. Reduced cytochrome c was prepared fresh
before each experiment by adding a few grains of sodium
hydrosulfide (dithionite) to a 1% solution in 10 mM Kphosphate buffer, p H 7.0.
Reduced-minus-oxidized spectra of cytochromes were recorded at room temperature in a Perkin-Elmer 572 doublebeam spectrophotometer. Cytochromes were reduced by
adding 10 mM KCN and 10 m~ succinate, or by adding a
few grains of dithionite. The concentrations of cytochromes
were calculated using the extinction coefficients of Bookelman and associates {l5]. Protein in mitochondrial preparations was measured by the method of Lowry and associates
500 Annals of Neurology
Vol 22
No 4
October 1987
Sodium Do&cyl Stlvatc?Poluactykzmide
Gel Electrophoresis
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) was performed o n a discontinuous gel by a
modification of the meirhod of Laemmli [23], as described
Immunological Studies
COX was purified from normal human heart as described
1241. Antibodies against the purified holoenzyme were
rised in rabbits by intradermal injection of 1 mg enzyme
protein mixed with complete Freund’s adjuvant. A booster
injection was given after 3 weeks. A week later, the animals
were bled, and serum was stored frozen in small aliquots.
The presence of immiinologically cross-reacting material
(CRM) was estimated by the enzyme-linked immunosorbent
assay (ELISA), using niitochondrial suspensions or crude
muscle extracts immobilized antigens, as described 1.24,
251. Immunoprecipitation of COX from mitochondrial extracts was performed by a modification of the procedure
of Merle and Kadenbach {26], as described 1241. The
immunoprecipitates were washed twice with 0.2 M Kphosphate buffer, p H 7.2, and solubilized for SDS-PAGE.
Several mitochondrial enzymes were measured in
brain of all 5 patiems; in muscle, liver, and kidney
from patients 1, 2, 3, and 5 ; and in the heart from
patient 5. Activities were determined in crude extracts
and, whenever possible, in isolated mitochondria.
T h e main alteration was a decrease of C O X activity
to approximately 30% of normal in brain, and to 15%
in muscle of all patients tested (Table 1). In liver, C O X
activity was about 10% of normal in 3 patients, but
normal in 1, who also had normal enzyme activity in
cultured slun fibroblasts (Table 1). In kidney, C O X
activity was decreased in all patients tested, but the
residual activity varied considerably in different patients (see Table l).
Cardiac tissue was obtained only from patient 5,
who had clinical card iomyopathy ; C O X activity was
very low (see Table 11.
Although there was some variability among patients,
residual activities were lower in muscle and liver than
in brain and kidney (see Table 1). In brain, C O X activity was never below 15% and averaged 32% of the
normal mean.
No inhibition of COX was seen in mixing experiments in which tissue extracts from patients were
added to control extracts.
Other mitochondrial enzymes had generally normal
activities (Fig 1). Occasional low values of individual
enzymes were found in single patients, o r in one preparation (e.g., crude extracts) but not in the other (e.g.,
isolated mitochondria).
Pyruvate dehydrogenase complex activity in skin
Table 1 . Cytocbrome c Oxirlase Actizfity in Crude Extracts and Isolated Iliitochondriaa
Crude Extract
9% of Mean Control'
2,800 2 520 (71)
1,340 5 320 (23)
1,620 ? 518 (17)
400 2 110 (5)
21 1
1,500 2 620
Crude Extract
9% of Mean Control'
% of Mean Control'
Crude Extract
% of Mean Control'
Crude Extract
95 of Mean Control"
Crude Extract
?h of Mean Control'
168 (13)
* 1,590(10)
"Activities are nmoles cytochrome c oxidizedlmidgm tissue (crude extracts) or nmoledmidmg protein (isolated mitochondria and fibroblasts).
bControl values are mean 2 SD; number of controls are in parentheses.
'Residual cytochrome c oxidase activities are also expressed as percent of mean control values; percent residual activities for crude extracts and
isolated mitochondria are averaged.
''Value kindly provided by Dr Brian Robinson, Hospital for Sick Children, Toronto, Ontario.
not done.
Fig 1. Activities of cytochrome c oxidase (COX),succinate-cyiochrome c reductase (Succ-CyT.CRed.), rotenone-sensitive reduced
nicotinamide adenine dinuclwtide (NADH)-cytocbromec reductase (NADH-CyTc Red.). and succinate debydrogenase (SDH)
in isolated muscle mitochondriafrom 3 patients with Lpigh syn-
drome and controls. Activities are nmoles substrate utilizedlminl
m g protein. Control activities are the mean ( * SD) of 23 values
for COX, 34 values for succinate-cytocbmme c reductase, 28
valuesfor NADH-qtocbmme c reductase, and 21 values for
DiMauro et al: Cytochrome c Oxidase in Leigh Syndrome
Log Ab Dilutions
i --$
Log Ah Dilutions
Fig 2. immunoreactivity by ELISA of mitochondrial extracts
(I 0 p.g proteinlmli from skeletal muscle (A) and brain (B) of
controls (open symbols) and patients (closed symbols).
fibroblasts cultured in a variety of laboratories was normal: patient 1, 1.78 nmoles COZ liberatedminlmg protein (mean & SD of 15 controls, 2.36
0.76); patient
2 (3 determinations), 2.29 ? 0.06 ( 3 controls, 1.71 &
0.11); patient 3, 1.92 (20 controls, 3.96 & 1.15); patient 5 , 1.55 (47 controls, 1.61 ? 0.37). In patient 4,
PDHC activity was normal in muscle (2.97 nmoled
midmg noncollagen protein; normal, 2.49 k 0.77)
and in brain (13.60 nmoledmidmg protein; normal,
11.21 f 2.00).
The spectra of reduced-minus-oxidized cytochromes
showed lack of the cytochrome aa3 peak in muscle
mitochondria from patient 1 using substrate-dependent reduction [14]. However, using dithionite as a
reducing agent, cytochrome aa3 peaks were clearly visible in brain and muscle mitochondria from patient 2.
Immunotitration by ELISA revealed CRM in all tissues of all patients, but the amount of CRM varied
from patient to patient. The amount was normal in
brain mitochondria from patient 2, but
decreased in brain mitochondria from patients and
mitochondria from patients 2, and
and in
(Fig 2). SDS-PAGE of COX immunoprecipitated
from liver mitochondria in patient 1 (not shown) and
patient 2 (Fig 3) showed a normal subunit pattern.
There was no evidence of missing or abnormal bands.
We studied 5 children with neuropathologically
proven Leigh syndrome. Four of them were normal at
birth and during the first year of life, and then developed a subacute neurodegenerative disorder characterized by psychomotor regression and signs of brainstem
and cerebella lesions (Table 2). All died after 3 to 4
years of intermittently progressive course. The fifth
child was floppy at birth and showed earlier and more
rapidly progressive neurological abnormalities. The co502 Annals of Neurology Vol 22
No 4 October 1987
Fig 3. Sodium dodql s.ulfate-polyacrylarnia2gel electrophoresis of
human heart cytochron,?ox,&se (lane A), and of irnrnunoprecipitates ofsolubilized livw mitochondriafrom a control (lane
B) and from patient 2 (lane C ) .Subunits are labeled according
to ~~~l~andKaAnbach{j31..
existence of cardiomyopathy, reported in some patients with Leigh syndrome [27,28], probably contributed to his early death at 7 % months (see Table 2). All
5 patients had lactic acidosis. Muscle biopsy was normal in all patients by light microscopy (see Table 2),
but ultrastructural examination in 2 patients showed
discrete mitochondrial changes, such as increased size,
bizarre shape, osmiophilic inclusions, and disoriented
cristae. Muscle that was normal by light microscopy
but had ultrastructurally abnormal mitochondria was
also described by Willems and associates in their patient with Leigh syndrome and COX deficiency [l2].
The only reported patient with ragged-red fibers [29}
Table 2. Clinical and Laboratory Features of J Patients with Leigh Syndrome
Age at Onset
Age at Death
Psychomotor Regression
Optic Atrophy
Hyporeflexia or Areflexia
Abnormal Breathing
Lactic Acidosis
Abnormal Baer’s
Normal Muscle Histochemistry
Family History
4% yr
1 Yr
1 Yr
4 yr
5 Yr
7% mo
= male; F = female;
condition present; - = condition not present; ND
probably did not have Leigh syndrome but KearnsSayre syndrome, another mitochondrial encephalomyopathy 1301.
Leigh syndrome has diverse biochemical causes. Because the histopathological features and the distribution of the lesions are similar to those of WernickeKorsakoff encephalopathy, an inherited disturbance
of thiamine metabolism was suspected. An inhibitor of
the brain enzyme that catalyzes the synthesis of thiamine triphosphate has been found in the urine of some
patients 1311, but the validity of that assay has been
questioned 1321. Negative results were obtained in the
only patient of ours (patient 2) whose urine was tested
for the inhibitor.
The association of PC deficiency and Leigh syndrome has been reported in 4 cases with characteristic
neuropathological features in the patients or affected
siblings 14-71. One, whose sibling was affected 151,
probably did not have Leigh syndrome [81; in another
141, PC was normal in later studies 191; 2 tests have
been questioned because PC, a labile enzyme 191, was
measured in liver obtained at autopsy. Conversely,
several patients with pathologically proven Leigh syndrome had normal PC, and many patients with documented PC deficiency lacked the neuropathological
features of Leigh syndrome [8, 91. It seems safe to
conclude that PC deficiency is rarely, if ever, associated with Leigh syndrome.
The role of pyruvate dehydrogenase deficiency in
the pathogenesis of Leigh syndrome appears more
convincing. The pyruvate dehydrogenase complex is
composed of 5 enzymes. Two of them, a kinase and a
phosphatase, regulate enzyme activity by phosphoryla-
not described.
tion (deactivation) and dephosphorylation (activation)
of the first enzyme component, pyruvate decarboxylase (El). The other 2 enzyme components are dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3) 1111.
Defects of PDHC affecting either the enzyme activity directly C33-38) or the activating mechanism
[39, 401 have been reported in 14 patients with
the neuropathological features of Leigh syndrome. Although in most cases the enzyme defect was shown
in fibroblasts, there has been no evidence of tissuespecrfic isozymes of PDHC, suggesting that the biochemical error was generalized. This was confirmed in
a few patients by studies of liver, brain, muscle, and
platelets 135-37, 391.
The association of COX deficiency and Leigh syndrome has been reported in 3 patients: a girl who died
at 7 years of age 1121, and 2 brothers, one of whom
died at 16 months while the other was still alive at 8
years [13]. In the girl, COX activity was undetectable
in muscle mitochondria, decreased in the h e m (8.5%
of normal), but normal in liver 112). In the brothers,
residual COX activity was 18% of normal in liver and
muscle, 15% in brain, and 25% in fibroblasts 113, 37).
The relative importance of COX deficiency, compared
to PDHC or PC deficiency, as a cause of Leigh syndrome is difficult to assess at the present time, but can
be estimated from systematic enzymatic analysis of cultured skin fibroblasts. We have studied 8 mitochondrial enzymes in fibroblasts from 23 patients who were
considered clinically (23 patients) or pathologically (5
patients) to have Leigh syndrome. All enzymes were
normal in 18 cell lines, 2 patients had COX deficiency,
DiMauro et al: Cytochrome c Oxidase in Leigh Syndrome
1 had PDHC deficiency, and 2 had intermediate PC
activities [41). Similar results were obtained in surveys
of fibroblasts from patients with lactic acidosis: in a
series of 28 patients, Miyabayashi and associates ( 3 7 )
found 2 with COX deficiency and 4 with PDHC
deficiency, and in a series of 95 patients, Robinson and
associates ( 4 2 ) found 6 with COX deficiency and 4
with complex I deficiency.
We have found COX deficiency in multiple tissues
of 5 children with Leigh syndrome. The defect of
COX seemed to be specific because 5 other mitochondrial enzymes were normal or showed only random
abnormal values. The defect was not due to postmortem changes because (1) it was observed in muscle
biopsies and cultured fibroblasts; (2) tissues were frozen 2 to 5 hours after death and compared to control
specimens obtained under similar conditions; and (3)
COX activity was not decreased in control tissues obtained 12 to 16 hours postmortem. The enzyme defect
was observed both in crude tissue extracts and in
isolated mitochondria, and the amount of residual activity was comparable in the 2 preparations (see Table
1). This observation suggests a primary mitochondrial
defect and excludes the presence of a cytoplasmic inhibitor of COX. Mixing experiments with crude extracts from patients and controls also failed to show
COX inhibition.
The enzyme defect was partial. Residual COX activity varied from tissue to tissue, but, except for kidney,
it was remarkably similar in the same tissues from different patients (see Table 1). The finding of higher
residual activity in brain than in liver or muscle is in
apparent contrast to the severe clinical and pathological involvement of the brain in Leigh syndrome.
However, brain is highly dependent on oxidative metabolism and, therefore, probably more vulnerable
than other tissues to even partial defects of key oxidative enzymes like COX. Furthermore, COX activity
was measured in specimens of brain cortex, not in severely affected regions such as the brainstem. COX
activity may normally be lower in certain areas of the
brain, as shown for PDHC [43), explaining the regional vulnerability to partial enzyme defects.
COX deficiency was generalized in 3 of 4 patients in
whom brain, muscle, liver, and kidney were studied. In
one of our own cases and another case 1127, however,
the liver was spared. In our patient, there were clinical
signs of cardiomyopathy, and COX deficiency was also
documented in the heart. Thus, COX deficiency can
explain the association of cardiopathy and Leigh syndrome described in a few patients (27, 281, although
the enzyme defect can also be clinically silent, as in
Willems’s patient 112) and, probably, in the 4 other
patients described here.
The expression of COX deficiency in cultured skin
fibroblasts in 2 of 3 patients has important practical
504 Annals of Neurology Vol 22 No 4 October 1987
implications: prenatal diagnosis may be possible. However, the normal activity in fibroblasts from l patient
(who also had normal liver enzyme) indicates biochemical heterogeneity and warns that false-negative results
are possible when enzyme studies in fibroblasts are
used for the diagnosis of CO X deficiency in Leigh
syndrome. However, studies of cultured amniocytes
should be reliable for prenatal diagnosis when COX
deficiency has already been documented in fibroblasts
from an affected sibling.
COX is a complex enzyme, composed of 13 subunits. The 3 larger subunits (1-111) that are responsible
for the essential properties of the enzyme (electron
transport and proton translocation) are encoded by mitochondrial deoxyribonucleic acid (DNA), while the
other 10 subunits ar12encoded by nuclear DNA, synthesized in the cytoplasm, and transported into the
mitochondria 144). The functions of the nuclearencoded subunits have not been fully elucidated, but a
regulatory role has been proposed by Kuhn-Nentwig
and Kadenbach 1451, who found that most, if not all,
of these subunits occur in tissue-specific isoforms.
In keeping with the complexity of the enzyme, there
is clinical and biochemical heterogeneity of COX
deficiency (46). Clinical phenotypes fall into 2 main
groups, one in which myopathy is the predominant or
exclusive manifestation, the other in which brain dysfunction predominates (46).
Among the disorders dominated by central nervous
system involvement, Leigh syndrome seems to be the
most common, but COX deficiency (42% and 10% of
normal) was also shown in muscle biopsies from 2
unrelated patients with progressive poliodystrophy
(Alpers disease) 147). A decrease of COX activity
in muscle (7% of normal) and platelets (16%) was
reported in a child who, at age 2 years, developed
psychomotor regression, hypotonia, ataxia, ophthalmoparesis, and nystrtgmus 148). He had lactic aciduria,
abnormal auditory-evoked potentials, and a muscle
biopsy showed increased number of lipid droplets and
subsarcolemmal NADH-tetrazolium positive rims. Because CT had been normal at age 3 and the patient
was still alive at age 8, the diagnosis of Leigh syndrome
was considered unlikely 148) but remains a possibility.
The molecular defect of COX in Leigh syndrome is
unknown. The autclsomal recessive transmission suggests involvement of 1 or more of the nuclear-encoded
subunits. The apparently generalized nature of the defect in 3 patients further suggests that the affected
subunit should be common to all tissues. The finding
of normal or only mildly decreased immunologically
reactive protein and of normal subunit pattern by electrophoresis indicates that the enzyme complex is assembled normally, excluding a defect of synthesis or
translocation of nucliear-encoded subunits from the cytoplasm into the mitochondria.
A structural mutation could cause loss of activity
and, possibly, accelerated degradation of the complex.
Increased thermolability of COX activity was reported
by Miyabayashi and associates in crude liver extracts
and isolated fibroblast mitochondria in 2 patients with
Leigh syndrome C 131. However, we found normal sensitivity to heat inactivation in crude muscle extract
from patient 5 and in liver mitochondria from patient 2
(data not shown). Other properties of the mutant enzyme, such as Km for cytochrome c, remain to be
The observation of normal COX activity in liver and
fibroblasts of 1 patient suggests genetic heterogeneity
even within this group of patients. The molecular basis
for the sparing of 2 tissues is not known. However, if
most of the nuclear-encoded COX subunits are tissue
specific, then it is conceivable that different genetic
defects may cause involvement of single tissues or
combinations of tissues. Isolated defects of muscle
COX have been documented in patients with fatal infantile myopathy 1247, while combined defects of muscle and kidney COX or muscle and heart COX have
been shown in patients with myopathy and renal dysfunction [49, 501 or myopathy and cardiopathy {Sl).
The genes encoding the cytoplasmically synthesized
COX subunits are being cloned 1521, and these genes
can be used as probes to identify the molecular lesions
in the different forms of COX deficiency.
Irrespective of the exact molecular lesion, COX
deficiency appears to be one important cause of Leigh
syndrome, in addition to PDHC deficiency. The diagnosis can be established by enzyme studies of cultured
fibroblasts in most patients, but should be confirmed
by muscle biopsy. Muscle may appear normal by
routine histochemistry, but lack of histochemical stain
for COX and decreased COX activity in crude extract
or isolated mitochondria will document the enzyme
Supported by center grants N S 11766 from the National Institute of
Neurological and Communicative Disorders and Stroke and from
the Muscular Dystrophy Association. Dr Servidei was supported by
a fellowship from the Unione Italiana Lotta alla Distrofia Muscolare,
Sezione Laziale “Giulia Testore,” and Dr DiRocco by a fellowship
from the Fondazione Giannina Gaslini.
Dr Margaret G. Norman, British Columbia’s Children’s Hospital,
Vancouver, British Columbia, provided pathological data on patient
2; Dr Brian Robinson, Hospital for Sick Children, Toronto, Ontario,
and Dr Douglas Kerr, Case Western Reserve University, Cleveland,
OH, generously provided biochemical data for patients 1 and 2; Dr
Lewis P. Rowland kindly revised the paper; and Ms Mary Tortorelis
typed the manuscript.
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After this paper was accepted, 2 more patients with Leigh
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