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Clinical and genetic spectrum of pyruvate dehydrogenase deficiency Dihydrolipoamide acetyltransferase (E2) deficiency.

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Clinical and Genetic Spectrum of Pyruvate
Dehydrogenase Deficiency:
Dihydrolipoamide Acetyltransferase
(E2) Deficiency
Rosemary A. Head, MA,1 Ruth M. Brown, MSc,1 Zarazuela Zolkipli, MRCPCH,2
Raveen Shahdadpuri, MRCP(I),3 Mary D. King, FRCP(I), FRCPCH,3
Peter T. Clayton, MD, FRCP, FRCPCH,2 and Garry K. Brown, FRCP, PhD1
Pyruvate dehydrogenase deficiency is a major cause of primary lactic acidosis and neurological dysfunction in infancy
and early childhood. Most cases are caused by mutations in the X-linked gene for the E1␣ subunit of the complex.
Mutations in DLAT, the gene encoding dihydrolipoamide acetyltransferase, the E2 core component of the complex, have
not been described previously. We report two unrelated patients with pyruvate dehydrogenase deficiency caused by
defects in the E2 subunit. Both patients are less severely affected than typical patients with E1␣ mutations and both have
survived well into childhood. Episodic dystonia was the major neurological manifestation, with other more common
features of pyruvate dehydrogenase deficiency, such as hypotonia and ataxia, being less prominent. The patients had
neuroradiological evidence of discrete lesions restricted to the globus pallidus, and both are homozygous for different
mutations in the DLAT gene. The clinical presentation and neuroradiological findings are not typical of pyruvate dehydrogenase deficiency and extend the clinical and mutational spectrum of this condition.
Ann Neurol 2005;58:234 –241
Pyruvate dehydrogenase (PDH) deficiency is a welldefined inborn error of metabolism with a broad spectrum of clinical manifestations.1,2 The majority of patients have severe, often fatal, neonatal or infantile
lactic acidosis or a more chronic neurodegenerative disease with extensive neuropathology, but often little or
no systemic acidosis. The characteristic clinical and
neuropathological features of Leigh syndrome develop
in some patients. Less commonly, patients have episodes of lactic acidosis, usually associated with recurrent ataxia. The defined clinical spectrum of PDH deficiency continues to expand because some patients
have been identified with relatively normal mental ability and prolonged survival into childhood and even
adulthood.3,4 Episodic dystonia, developing during
childhood, also has been described recently.5
PDH is a large mitochondrial enzyme complex that
catalyzes the conversion of pyruvate to acetyl coenzyme
A. The complex contains multiple copies of three
enzymes: E1 (PDH, an ␣2␤2 heterotetramer), E2 (dihydrolipoamide acetyltransferase), and E3 (dihydro-
lipoamide dehydrogenase). Dihydrolipoamide acetyltransferase accepts acetyl groups formed by the
oxidative decarboxylation of pyruvate, catalyzed by E1,
and transfers them to coenzyme A.6 This transfer is
mediated by a lipoic acid cofactor that is reduced in
the course of the reaction. The cofactor subsequently is
reoxidized by E3, allowing the reaction cycle to continue.
In addition to its catalytic function, the E2 enzyme
forms the structural core of the PDH complex, and in
the mammalian enzyme, 60 subunits are arranged as a
dodecahedron to which the other subunits are attached.7 Each E2 molecule has two lipoyl domains in
which a lipoic acid cofactor is attached to a lysine residue, as well as a catalytic domain and binding domains for other subunits. In addition to the E1, E2,
and E3 enzymes, the complex also contains the E3
binding protein (E3BP) and specific PDH kinase and
phosphatase enzymes that regulate the activity of the
complex through phosphorylation and dephosphorylation of serine residues on the E1␣ subunit.6
From the 1Genetics Unit, Department of Biochemistry, University
of Oxford, Oxford; 2Institute of Child Health, University College
London, London, United Kingdom; and 3Department of Paediatric
Neurology, Children’s University Hospital, Dublin, Ireland.
Published online Jul 27, 2005, in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/ana.20550
Received Feb 23, 2005, and in revised form May 11. Accepted for
publication May 21, 2005.
234
Address correspondence to Dr Brown, Genetics Unit, Department
of Biochemistry, University of Oxford, South Parks Road, Oxford,
OX1 3QU, United Kingdom. E-mail: garry.brown@bioch.ox.ac.uk
© 2005 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
The most common cause of PDH deficiency is mutation in PDHA1, the gene for the E1␣ subunit.8 Mutations have been described in some of the other subunits of the complex, but at a much lower frequency.
There are more than 20 reported cases of E3 deficiency, with a common mutation found particularly in
Ashkenazi Jewish patients.9 There are only about 12
reported patients with E3BP deficiency, although this
condition is being described with increasing frequency.4,10 Two unrelated patients with mutation in
the E1␤ subunit recently have been identified,11 and
there are reports of several patients with PDH phosphatase deficiency.12 No patients have previously been
reported with mutation in DLAT, the gene for the E2
subunit, which is located on chromosome 11 at position q23.1.
We describe the clinical, biochemical, and genetic
findings in two unrelated patients who are homozygous
for different mutations in the gene for the E2 subunit.
The first patient has a deletion of a glutamic acid residue in the outer lipoyl domain of the protein, whereas
the second patient has a missense mutation that leads
to substitution of leucine for phenylalanine in the catalytic site. The pathogenic significance of these mutations was confirmed by functional complementation after expression of the normal E2 coding sequence in
cultured cells from the patients. Both patients have
clinical and biochemical findings that further extend
the recognized presentations of PDH deficiency.
Subjects and Methods
Patient 1
Patient 1 is a male patient born at term to first cousin parents. Pregnancy and delivery were normal, birth weight was
3.1kg, and there were no problems in the neonatal period.
At age 1 month, he was started on phenobarbitone for inconsolable crying at night. At age 5 months, he experienced
development of nystagmus, jerky head movements and episodic clenching of his hands. By age 6 months, he had
marked head lag and was generally floppy. Multivitamins
and monthly vitamin B12 injections were administered, and
his parents described an improvement in his nystagmus,
which was sustained for a few months. He crawled at age 13
months, walked with knee-foot orthoses at age 14 months,
and had said four single words at age 18 months. Examination at 4 years confirmed that he was ataxic with gross and
fine motor delay. He had mild intermittent ptosis and an
intermittent convergent squint with full abduction in each
eye. He had pendular nystagmus and reduced, abnormal eye
movements; eye movement studies confirmed saccade initiation failure (oculomotor apraxia). He drooled constantly and
had dystonic movements of the facial muscles and of his
hands and feet. Passive movements demonstrated variable
tone in the limbs, mostly somewhat reduced. Tendon reflexes were brisk, with some recruitment in the upper limbs.
He could say 12 words.
Normal investigations included plasma amino acids, transferrin isoforms, creatine kinase, very long chain fatty acids,
biotinidase, and purines and pyrimidines. There were no vacuolated lymphocytes, white cell lysosomal enzymes were normal, and there was a normal karyotype. Urinary organic acids and cerebrospinal fluid (CSF) amino acids and
neurotransmitters also were normal. On one occasion
(3 hours postprandial), the blood lactate was 1.47mmol/L
(normal, 1.1–2.2), the pyruvate was 0.137mmol/L, and the
lactate/pyruvate ratio 11 (normal, ⬍25). On a second occasion (2 hours postprandial), plasma lactate was 2.3mmol/L
(normal, ⬍1.8) and the CSF lactate was 3.2mmol/L (normal, ⬍2.0). Magnetic resonance imaging (MRI) of the brain
at 1 year was normal, but a repeat at 6 years showed lesions
in the globus pallidus with low T1 signal and bright T2 signal, compatible with an abnormality of energy metabolism
(Fig 1). Magnetic resonance spectroscopy showed accumula-
Fig 1. Magnetic resonance images of the brain of Patient 1. Two T2-weighted images taken at age 6 years showing high signal
indicative of lesions in the globus pallidus bilaterally.
Head et al: Pyruvate Dehydrogenase E2 Deficiency
235
tion of lactic acid in the lentiform nuclei, dorsal midbrain,
and localized areas of the cortex. Electromyographic and
electroencephalographic results were normal. The clinical
and radiological features and the accumulation of lactate in
the brain and CSF led to studies of PDH in cultured skin
fibroblasts.
Treatment with thiamine (100mg daily) led to a reduction
in drooling but no change in muscle tone or movements. In
response to administration of coenzyme Q10 (60mg twice
daily), lipoic acid (100mg daily), riboflavin (100mg daily),
vitamin E (400mg daily), and selenium (250mg twice
weekly), there was also some improvement in the drooling
and a slight reduction in choreoathetoid movements. When
lipoic acid (120mg three times daily) and thiamine (100mg
daily) were given together with a ketogenic diet, there was a
striking improvement in motor function such that 9 months
after starting the diet, he was walking steadily, speaking fairly
clearly, and was able to build a tower of eight bricks and
copy a circle. At age 11 years, he can still walk independently
without orthoses, and peripheral tone and power are normal.
He continues to have some mild coordination difficulties,
mainly in his upper limbs. There is a mild convergent
squint, but no nystagmus. He is now able to hold conversations and understands four different languages, but he has
difficulty writing. He attends mainstream school and is
growing along the 0.4th centile for height and weight. When
he adhered to the diet, his lactate, pyruvate, and lactate/
pyruvate ratio were normal.
Patient 2
This male patient is the first of three children born to first
cousin parents. The two younger siblings are well, and there
is no family history of neurological or developmental problems. He developed normally until age 11 months, when he
presented with episodes of arching of the body, stiffening of
the limbs with flexion of the wrists and inversion of the feet,
eye-rolling, and distress, without loss of consciousness and
lasting up to 3 hours several times daily. Extensive investigations, including plasma and CSF lactate concentrations, were
negative apart from abnormal signal in the globus pallidus
bilaterally on brain MRI. The question of pantothenate kinase associated neurodegeneration was raised, but subsequent
PANK2 mutation analysis was negative.13 A specific cause for
the paroxysmal dystonia was not found. There was no response to carbamazepine, benzhexol, or L-dopa.
After the onset of episodes of dystonia, developmental
progress slowed, but he continued to acquire new skills. At
age 8 years, the paroxysmal dystonia continues but has decreased in frequency (two or three times a week) and duration (15– 60 minutes) over the last year. The episodes are
triggered particularly by stress or fever. His neurodevelopment is delayed, he bottom shuffles, he is unable to weight
bear, and he is wheelchair bound. He can self-feed with difficulty with a spoon and drink from a cup using both hands.
He has poor pronunciation and tends to drool. Cognitive
assessment shows him to be functioning in the borderline
range. He has generalized dystonia with tightening of the
Achilles tendon and brisk tendon reflexes.
Levels of plasma amino acids, transferrin isoforms, very
long chain fatty acids, biotinidase, copper, ceruloplasmin,
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carnitines, lysosomal enzymes, urine organic and amino acids, CSF amino acids and neurotransmitters, and muscle histology, electron microscopy, and respiratory chain enzyme
analysis were all normal. Brainstem and visual-evoked responses and electroretinogram were normal. Blood film results were normal. Fasting plasma lactate level was
1.28mmol/L (normal, ⬍1.8) and the CSF lactate level was
1.65mmol/L (normal, ⬍2.0). Corresponding pyruvate concentrations were not available. Repeated electroencephalographic studies showed a slow waking background without
any epileptiform abnormality.
MRI of the brain at 15 months showed focal signal abnormality in the basal ganglia with high T2 signal and low
T1 signal in the globus pallidus bilaterally. Repeat MRI
scans at age 2 and 6 years confirmed persistent but unchanged signal abnormalities in the globus pallidus bilaterally
(Fig 2). Despite the normal lactate concentrations, the clinical and imaging findings, considered in conjunction with
recent observations of patients with PDH deficiency with
dystonia,5 led to this diagnosis being pursued.
Biochemical Characterization
Fibroblast cultures from the patients and both sets of parents
were established from skin biopsies, which were taken with
informed consent. They were grown in Dulbecco’s modified
Eagle medium–nutrient mixture F-12 Ham (DMEM/F12)
with 10% fetal calf serum, 100U/ml penicillin, and
100␮g/ml streptomycin. Overall PDH complex activity was
measured using [1-14C]-pyruvate as substrate after maximal
activation of the enzyme complex with dichloroacetate as described previously.14 Immunochemical analysis was performed by Western blot with monoclonal antibodies to the
E1␣ and E2 subunits of the PDH complex.15
Mutation Analysis
Fibroblast RNA, extracted using a Roche High Pure RNA
Isolation Kit (Roche Diagnostics GmbH, Mannheim, Germany), was converted to complementary DNA (cDNA) using a Qiagen Omniscript RT Kit (Qiagen, Chatsworth, CA).
Overlapping segments of the PDH E1␣, E3 binding protein,
and E2 cDNA were amplified by polymerase chain reaction
(PCR); products were assessed by analyzing an aliquot on
1% agarose gels, purified using a Qiagen QIAquick PCR Purification Kit (Qiagen), and sequenced using ABI Big Dye
Terminators (ABI, Foster City, CA). Genomic DNA was
prepared from fibroblasts using a Nucleon BACC2 Kit
(Tepnel Life Sciences, Manchester, UK). Exon 2 or 13 of the
PDH E2 gene and adjacent intronic sequences were amplified by PCR; the products were assessed on agarose gels, then
purified and sequenced as described earlier.
Expression of the Normal Pyruvate Dehydrogenase E2
Sequence in Patient Cells
A cDNA segment covering the complete PDH E2 coding
sequence was amplified using Pfx DNA polymerase (Invitrogen, La Jolla, CA), cloned initially into pCR-Blunt II-TOPO
(Invitrogen), and then into the mammalian expression vector, pcDNA3.1 (Invitrogen). After confirmation of the sequence, the construct was transfected into patient primary
fibroblasts using Lipofectamine 2000 (Invitrogen). The cells
Fig 2. Magnetic resonance (MR) images of the brain of Patient 2. (A–C) Sequential T2-weighted MR images taken at the same
level at 15 months (A), 2 years (B), and 6 years old (C) demonstrating abnormal high signal in the globus pallidus bilaterally. Of
note is the lack of change in the signal abnormality over this period. (D–F) MR images at age 6 years. (D) T2-weighted image at
level of basal ganglia. There is abnormal high signal in the globus pallidus bilaterally. This area should be as dark as the remainder of the lentiform nucleus. (E) A second T2-weighted image at a slightly higher level. (F) T1-weighted image showing a slightly
smaller area of abnormal, high T1 signal in each globus pallidus.
were analyzed for PDH enzyme activity 3 to 6 days after
transfection. Efficiency of transfection was assessed by cotransfection of the cells with a green fluorescent protein
(GFP) construct and fluorescence microscopy.
Results
Biochemical and Genetic Analysis
PDH activity in fibroblasts of Patient 1 was
0.27nmol/mg protein/min after dichloroacetate activation, compared with the reference range of 0.7 to 1.1.
Native PDH activity was 0.22nmol/mg protein/min.
In fibroblasts from the mother and father, the activated
activity was 0.55 and 0.50nmol/mg protein/min, respectively. Assay of the enzyme under conditions favoring activation by PDH phosphatase (5mM dichloroacetate, 2mM Ca2⫹), or inactivation by PDH kinase
(0.2mM adenosine triphosphate, 0.5mM EGTA, 5mM
NaF) demonstrated that the mutant enzyme responded
to the same extent as the normal enzyme (results not
shown). A Western blot showed an apparent reduction
in E2 immunoreactive protein relative to the E1␣ band
in the patient compared with normal control subjects
(Fig 3), with possibly also a smaller reduction in the
parents. This was confirmed by densitometry measurements that indicated a 50% reduction in the E2/E1␣
ratio in the patient’s cells and a 20 to 25% reduction
in the parents’ cells.
In cultured fibroblasts from Patient 2, the PDH activity was 0.36nmol/mg protein/min with dichloroacetate activation and 0.22nmol/mg protein without.
The activated activity in fibroblasts from his mother
Head et al: Pyruvate Dehydrogenase E2 Deficiency
237
Fig 3. Immunochemical analysis of pyruvate dehydrogenase (PDH) subunits. Fibroblast protein extracts from both patients, their
parents, and two healthy control subjects were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis, transferred to
Immobilon-P filters (Millipore, Bedford, MA) and probed with monoclonal antibodies to the E2 and E1␣ subunits of the PDH
complex. Track 1, healthy control subject; track 2, Patient 1; track 3, mother of Patient 1; track 4, father of Patient 1; track 5,
healthy control subject; track 6, Patient 2; track 7, mother of Patient 2; track 8, father of Patient 2.
and father was 0.57 and 0.65nmol/mg protein/min, respectively. Immunochemical analysis of PDH subunits
in this patient showed a normal amount of PDH E2
immunoreactive protein (see Fig 3).
Given the parental consanguinity, the reduction in
E2 immunoreactive protein, and the autosomal location of the E2 gene, cDNA corresponding to this subunit was sequenced in Patient 1. This showed a deletion of three nucleotides from position c361, which
would result in deletion of glutamic acid residue 121.
In genomic DNA, the patient appeared to be homozygous for this mutation (located in exon 2 of the gene);
this was confirmed by demonstrating heterozygosity in
both parents. Because there was no indication from the
immunochemical analysis as to which subunit might be
responsible for the enzyme deficiency in Patient 2,
cDNA corresponding to the E1␣ subunit was sequenced first and found to be normal. In view of the
parental consanguinity, attention was then turned to
the E3-binding protein; in our experience, the E3binding protein is the second most common cause of
PDH deficiency. However, cDNA corresponding to
this subunit was also normal. Subsequently, sequence
analysis of the coding region of the E2 gene showed a
single base substitution, c1728 C ⬎ A, which would
change phenylalanine 576 to leucine. In genomic
DNA, the patient appeared to be homozygous for this
mutation (in exon 13), and this was confirmed by
analysis of the parents who are both heterozygous.
Functional Complementation of the Defect
Neither of these mutations has been identified in 50
other DLAT alleles analyzed in our laboratory, nor are
they found in any DLAT cDNA or genomic sequences
in the human EST and genomic DNA databases. Nevertheless, to demonstrate that the mutations are the
cause of the PDH deficiency, the normal E2 coding
sequence was cloned into an expression vector and
transfected into patient cells. Transfected primary fibroblasts from both patients were analyzed for PDH
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activity after 6 days, and the results are presented in
the Table. Similar results were obtained using SV40
transformed fibroblasts16 from Patient 1 with electroporation of the expression construct (results not
shown). PDH activity increased significantly after
transfection, although this was not observed before 3
days. This is in contrast with previous experiments
with E1␣ and E1␤ constructs where an increase in activity in deficient cells could be readily demonstrated
within 24 to 48 hours of transfection.11,16 The recovery of activity is corrected for efficiency of transfection,
which was usually 40 to 50%.
Discussion
These patients have PDH deficiency caused by mutations in DLAT, the gene for the E2 subunit of the
complex. In both cases, the clinical presentation was
relatively mild compared with the most common form
of PDH deficiency caused by mutation in the E1␣
subunit gene. Both patients have particular features
that should be noted.
In Patient 1, there is good mental function and an
excellent response to a ketogenic diet. In early life, the
main clinical manifestations were ataxia and dystonia.
After introduction of the diet, these resolved to a significant extent, and the main residual problem is now
Table. Functional Complementation by Expression of the
Normal PDH E2 Sequence
Subject
Normal controls
Patient 1
Patient 2
Activity after
Expression of E2
PDH Activity
Construct
(nmol/mg protein/min)
0.7–1.1
0.27 ⫾ 0.05
0.36 ⫾ 0.07
0.61 ⫾ 0.04a
0.81 ⫾ 0.10a
Activity corrected for efficiency of transfection, with mean ⫾ SD of
three experiments.
a
PDH ⫽ pyruvate dehydrogenase.
fine-motor incoordination. Prolonged survival with relative sparing of cognitive function also has been described in patients with E3BP deficiency.4,10 These patients also have significant residual activity of the PDH
complex because some assembly of E3 onto the E2
core is possible, even in the complete absence of
E3BP.17
In Patient 2, the clinical presentation is dominated
by episodic dystonia, although there is also significant
developmental delay. Despite the neurological involvement, it is particularly notable that he had a normal
lactate concentration in both blood and CSF. Dystonia, either as the main clinical feature or in combination with other neurological manifestations, is increasingly being recognized in patients with PDH
deficiency.5,18 However, this is still an infrequent presentation, and currently too few patients exist to determine whether it is particularly associated with specific
mutations in some of the subunits. Of more practical
importance is the finding of a normal lactate concentration, particularly in CSF. Increased CSF lactate concentration is considered a key diagnostic finding in patients with impaired neurological function resulting
from defects in cerebral energy metabolism, and it is
often the indication for more detailed biochemical and
genetic analysis.19 Although the finding of normal CSF
lactate concentration would not usually suggest the diagnosis, the combination of MRI signal abnormalities
in the globus pallidus with episodes of paroxysmal dystonia prompted further investigation for PDH deficiency in this case.
The findings in serial MRI studies of these two patients are particularly noteworthy. The neuropathology
of PDH deficiency is quite variable. In some patients,
particularly those presenting with severe neonatal lactic
acidosis, there are minimal changes, often a minor degree of cerebral atrophy, and some slight developmental anomaly.20 At the other extreme is the extensive
neuropathology commonly seen in female patients who
are heterozygous for mutations in the E1␣ subunit
gene which result in complete deficiency of the protein
product. In these patients, gross cerebral atrophy and
ventricular dilatation often is accompanied by a number of developmental anomalies such as agenesis of the
corpus callosum, absence of the medullary pyramids,
and inferior olive ectopia.21 Between these two groups
are patients with Leigh syndrome with the characteristic symmetrical necrotic lesions in the basal ganglia and
brainstem.22 By contrast, MRI in these two patients
with E2 deficiency showed circumscribed lesions restricted to the globus pallidus bilaterally. This pattern
would be unusual in Leigh syndrome, where lesions in
the putamen are more common,23–28 and it would not
be expected in other forms of dystonia. Although it is
unlikely that this localized neuroradiological finding
will be a consistent feature of PDH E2 deficiency, it is
sufficiently distinctive to be useful diagnostically in patients with similar clinical presentations, even when the
CSF lactate concentration is not increased.
The mutation in Patient 1 is in the outer of the two
lipoyl domains of the E2 enzyme, toward the
N-terminal end of the protein. The deleted glutamic
acid residue is highly conserved across a wide range of
species and is near to lysine 132, the site of lipoic acid
attachment. The lipoic acid cofactors are attached to
flexible arms, which are critical for the reaction mechanism, and alterations in the local structure could have
a significant effect on function.29 However, the biochemical defect cannot simply be related to the mutation. Studies of the E2 enzyme in other species suggest
that this lipoyl domain may not be essential for enzyme function. The Saccharomyces cerevisiae enzyme has
only a single lipoyl domain,30 whereas one or two of
the three lipoyl domains in the Escherichia coli enzyme
can be deleted without loss of activity.31 Recently, interactions between the E2 lipoyl domains and PDH
kinase and phosphatase isoforms have been shown to
be important for the regulation of the complex,32 but
the most significant effects appear to be mediated
through the inner lipoyl domain. However, these in
vitro studies may not accurately reflect the in vivo situation because there are growth differences between
isogenic strains of E. coli with either one or three lipoyl
domains on the E2 enzyme,33 and some isoforms of
PDH kinase, particularly isoform 4, appear to bind
more strongly to the outer lipoyl domain of the mammalian enzyme.32,34 Direct examination of the susceptibility of the enzyme in cultured fibroblasts to the activity of PDH kinase and phosphatase showed no
significant difference from normal cells.
There was an approximately 50% reduction in immunoreactive E2 protein in the fibroblasts from Patient 1 compared with normal control subjects and an
intermediate reduction in cells from both parents. This
raises the possibility that the mutation interferes with
assembly of the complex or results in instability of the
E2 protein. However, these studies were performed
with a monoclonal antibody, and the reduction may
simply reflect reduced avidity of binding of this antibody to an altered epitope.
Because available data do not provide an explanation
for the functional consequences of this amino acid deletion, expression studies were performed to determine
whether the enzyme deficiency could be complemented
by the normal E2 protein. Functional complementation was demonstrated, although full restoration of activity was not achieved and significant recovery of activity was not observed before 3 days after transfection.
Labeling studies in muscle indicate a much slower
turnover of the E2 protein compared with the other
subunits of the complex: a half-life of 7.7 days compared with 2.5 and 2.6 days for the E1␣ and E1␤ sub-
Head et al: Pyruvate Dehydrogenase E2 Deficiency
239
units, respectively.35 This may account for the slow recovery of activity compared with results obtained in
transfection and expression studies in cells from patients with E1␣ and E1␤ defects, where restoration of
activity can be demonstrated within 24 to 48 hours.
Another factor may be the pre-existence in the patient’s
cells of significant amounts of mutant E2 protein that
may be able to join with newly synthesized subunits in
heteromeric complexes with reduced activity or stability. This might make it impossible to complement the
biochemical defect fully.
It is easier to account for the pathogenic consequences of the mutation in Patient 2 because a highly
conserved amino acid residue in the catalytic site of the
enzyme is altered. This position is occupied by phenylalanine in all species studied to date, including animals,
plants, fungi, and bacteria. The crystal structure of the
E2 catalytic domain has been determined in several
bacterial species, and the sequence conservation allows
a direct analysis of the consequences of the mutation.29,36 –38 From structural analysis and mutagenesis
experiments, the phenylalanine corresponding to position 576 in the human E2 appears to be particularly
important in determining substrate specificity. In the
corresponding position in the E2 components of
␣-ketoglutarate and branched chain ␣-ketoacid dehydrogenase complexes, this position usually is occupied
by serine, and it is proposed that the larger phenylalanine restricts access to the active site to the smaller
acetyl group substrate of PDH E2.38 Although the mutation in Patient 2 provided a plausible explanation for
the enzyme defect, this was also confirmed by functional complementation.
It appears that mutation in the gene for the E2 component of the PDH complex is an extremely rare cause
of PDH deficiency as these are the first cases to be
defined genetically. One patient has been reported previously with reduced PDH activity and undetectable
E2 immunoreactive protein, but the genetic defect was
not identified.39 This patient experienced development
of severe lactic acidosis in the newborn period, made
virtually no developmental progress, and was microcephalic. Despite a high-fat, low-carbohydrate diet, she
had a persistent hyperlactatemia.
Given the rarity of PDH E2 deficiency and the fact
that it is inherited as an autosomal recessive trait, it is
highly likely that there will be parental consanguinity.
As with the two patients described in this report, the
symptoms may be generally milder than those commonly associated with mutations in the gene for the
E1␣ subunit and may be more likely to respond to
therapy with a ketogenic diet (as in the case of Patient
1). They may also respond significantly to dichloroacetate administration, although this is difficult to assess.
PDH activity increased in the fibroblasts of both patients after incubation with dichloroacetate, with a pro-
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portionally greater response in the cells from Patient 2
(64% increase compared with 23% in cells from Patient 1). The response in the cells from Patient 2 is
comparable with that found with normal fibroblasts in
our laboratory. Reports of in vitro activation of normal
fibroblast PDH by dichloroacetate are highly variable,
with increases ranging from 15 to 60%, suggesting that
this cannot be used as a reliable indicator of in vivo
efficacy.40,41 The response is even more variable, as expected, in patients with defects in the E1␣ subunit, the
target for phosphorylation and inactivation by PDH
kinase.42
The most significant determinant of the outcome in
these patients is likely to be the position and nature of
the underlying mutations that leave substantial residual
enzyme activity. The E2 enzyme forms the structural
core of the PDH complex, and mutations that result in
complete deficiency, or have a major effect on the
structure and function of the complex, are expected to
be incompatible with fetal development. The manifestations of PDH E2 deficiency may be more similar to
those of E3BP deficiency, in which a high proportion
of patients have prolonged survival and less severe neurological symptoms. If this is the case, it may be that
some patients have not been identified in the past because their clinical features were not considered to fall
within the spectrum of PDH deficiency.
We thank Dr I. Boubriak for assistance with the immunochemical
analysis and Drs J. G. Lindsay and M. V. Squier for helpful discussions.
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Head et al: Pyruvate Dehydrogenase E2 Deficiency
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