A Korean female patient with thiamine-responsive pyruvate dehydrogenase complex deficiency due to a novel point mutation (Y161C)in the PDHA1 gene.

Abstract

Pyruvate dehydrogenase complex (PDHC) deficiency is mostly due to mutations in the X-linked E1alpha subunit gene (PDHA1). Some of the patients with PDHC deficiency showed clinical improvements with thiamine treatment. We report the results of biochemical and molecular analysis in a female patient with lactic acidemia. The PDHC activity was assayed at different concentrations of thiamine pyrophosphate (TPP). The PDHC activity showed null activity at low TPP concentration (1 x 10(-3) mM), but significantly increased at a high TPP concentration (1 mM). Sequencing analysis of PDHA1 gene of the patient revealed a substitution of cysteine for tyrosine at position 161 (Y161C). Thiamine treatment resulted in reduction of the patient's serum lactate concentration and dramatic clinical improvement. Biochemical, molecular, and clinical data suggest that this patient has a thiamine-responsive PDHC deficiency due to a novel mutation, Y161C. Therefore, to detect the thiamine responsiveness it is necessary to measure activities of PDHC not only at high but also at low concentration of TPP.

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INTRODUCTION
The human pyruvate dehydrogenase complex (PDHC),
which is localized in the mitochondrial matrix, catalyzes the
thiamine-dependent decarboxylation of pyruvate to acetyl-
CoA. It plays an important role in energy metabolism of the
cell since it is an essential and rate-limiting enzyme connect-
ing glycolysis with the tricarboxylic acid cycle and oxidative
phosphorylation. This enzyme complex consists of three cat-
alytic components, pyruvate dehydrogenase (E1; EC 1.2.4.1),
dihydrolipoamide acetyltransferase (E2; EC 2.3.1.12), and
dihydrolipoamide dehydrogenase (E3; EC 1.8.1.4); two reg-
ulatory proteins, E1-kinase (EC 2.7.1.99) and phospho-E1-
phosphatase (EC 3.1.3.43) ; and an additional protein known
as the E3 binding protein (E3BP) (1). The first enzyme of
the complex, thiamine pyrophosphate dependent pyruvate
dehydrogenase (E1) which is a heterotetramer composed of
two and two subunits, contains a thiamine pyrophos-
phate (TPP) binding site (2). The great majority of PDHC
deficiencies result from mutations in the X-linked E1 sub-
unit gene (PDHA1), which is subjected to random inactiva-
tion. Highly variable X-inactivation contributes significantly
to the wide spectrum of clinical features in most heterozygous
females (3). Thiamine treatment is occasionally effective for
some patients with PDHC deficiency. Among these patients,
five mutations have been reported in a region outside the TPP
binding site of the PDHA1 gene. In this study, we describe
the results of biochemical and molecular analysis in a female
PDHC deficiency patient with thiamine-responsiveness and
a new missense mutation in the PDHA1gene.
MATERIALS AND METHODS
Subject
This female patient was born after 38 weeks of uncompli-
catedpregnancy to healthy nonconsanguineous parents. There
was no perinatal hypoxic ischemic insult. Family history of
neurodegenerative disorders was denied. Her neurodevelop-
ments; motor, language and cognitive development, were
delayed from the beginning. At 18 months of age, she was
diagnosed to have spastic cerebral palsy. She finally walked
at the age of 20 months. From this time, she occasionally had
episodic lethargy, decreased mental state, and temporary dete-
rioration of motor and cognitive function whenever she had
Eun-Ha Lee, Mi-Sun Ahn,
Jin-Soon Hwang, Kyung-Hwa Ryu,
Sun-Jun Kim*, Sung-Hwan Kim
Department of Pediatrics and Research Laboratory for
Human Mitochondrial Disorders, Ajou University School
of Medicine, Suwon; Department of Pediatrics*,
Chonbuk National University Medical School, Jeonju,
Korea
Address for correspondence
Sung-Hwan Kim, M.D.
Department of Pediatrics, Ajou University School of
Medicine, San 5 Wonchon-dong, Youngtong-gu,
Suwon 442-749, Korea
Tel : +82.31-219-5162, Fax : +82.31-219-5169
E-mail : pedkim@ajou.ac.kr
*This study was supported by Sooyeon Charity
Foundation, Suwon, Korea.
800
J Korean Med Sci 2006; 21: 800-4
ISSN 1011-8934
Copyright � The Korean Academy
of Medical Sciences
A Korean Female Patient with Thiamine-responsive Pyruvate
Dehydrogenase Complex Deficiency Due to a Novel Point Mutation
(Y161C) in the PDHA1 Gene
Pyruvate dehydrogenase complex (PDHC) deficiency is mostly due to mutations
in the X-linked E1 subunit gene (PDHA1). Some of the patients with PDHC defi-
ciency showed clinical improvements with thiamine treatment. We report the results
of biochemical and molecular analysis in a female patient with lactic acidemia. The
PDHC activity was assayed at different concentrations of thiamine pyrophosphate
(TPP). The PDHC activity showed null activity at low TPP concentration (1×
mM), but significantly increased at a high TPP concentration (1 mM). Sequencing
analysis of PDHA1gene of the patient revealed a substitution of cysteine for tyrosine
at position 161 (Y161C). Thiamine treatment resulted in reduction of the patient’s
serum lactate concentration and dramatic clinical improvement. Biochemical, molec-
ular, and clinical data suggest that this patient has a thiamine-responsive PDHC
deficiency due to a novel mutation, Y161C. Therefore, to detect the thiamine respon-
siveness it is necessary to measure activities of PDHC not only at high but also at
low concentration of TPP.
×10-3
Key Words : Pyruvate Dehydrogenase Complex Deficiency Disease; Pyruvate Dehydrogenase (Lipoamide);
E1Subunit; Thiamine Pyrophosphate
Received : 7 November 2005
Accepted : 15 February 2006

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Pyruvate Dehydrogenase Complex Deficiency and Thiamine-responsiveness
801
a viral illness. At 4 yr of age, she admitted due to recurrent
epileptic seizures. On admission, neurologic examination re-
vealed drowsy mental state, tremor and increase muscle tone
of both lower extremity. Laboratory tests revealed elevated
arterial blood lactic acid (4.14 mM/L, normal range 0.7-2.1
mM/L), pyruvic acid (0.18 mM/L, normal range 0.01-0.10
mM/L) and elevated alanine (798 nM/mL, normal range 152-
547 mM/L) concentration. Lactic acid level was also elevated
in the cerebrospinal fluid (7.33 mM/L, normal range 0.7-2.1
mM/L). T2-weighted brain MRI revealed high signal inten-
sity lesions in both basal ganglia suggestive of Leigh disease.
Epileptic seizures were controlled with anticonvulsant treat-
ments, and she finally recovered with some neurologic seque-
les such as persistent irritability and cognitive dysfunction.
She was referred to Ajou University Medical Center for the
evaluation of the lactic acidemia at the age of 5 yr. Neurologic
examination on admission revealed irritability, cognitive dys-
function, hyperreflexia, increased muscle tone in lower extre-
mity, and bilateral pathologic reflex. Laboratory tests revealed
elevated arterial blood lactic acid (3.9 mM/L) and pyruvic
acid (0.17 mM/L) concentration. L/P ratio (23) was elevat-
ed. Skin biopsy was done for the evaluation of PDHC and
mitochondrial respiratory chain enzyme assay. PDHC activ-
ity in cultured skin fibroblasts was mildly reduced to 0.804
nM/min/mg protein (normal: 1.13-6.67 nM/min/mg pro-
tein). All the mitochondrial respiratory chain enzyme acti-
vities were normal. Thiamine treatment at a dose of 10 mg/
kg/day was started. On 5 months of thiamine treatment, neu-
rologic manifestations which were observed on admission;
irritability, cognitive dysfunction, hyperreflexia, and bilater-
al pathologic reflex, were gradually improved with some re-
sidual clumsiness. Recently estimated arterial blood lactic
acid (2.1 mM/L) and pyruvic acid (0.082 mM/L) concentra-
tions were decreased to near normal range. Now she can attend
regular nursery school without difficulties.
Enzymatic assays
The PDHC activity was measured in cultured skin fibro-
blasts by measuring 14CO2 production from [1-14C]-labeled
pyruvate, modified from the method of Sheu et al. (4). Patient’s
fibroblasts was obtained from skin biopsy and cultured in
high-glucose Dulbecco’s modified Eagle’s medium contain-
ing 1×10-2mM thiamine-HCl. To compare the TPP respon-
siveness, we also tested the activity of the TPP unresponsive-
PDHC deficient fibroblasts with an AAGT duplication at
nucleotide 1162. The activity of the PDHC was assayed at
different concentrations of TPP (1×10-3, 1×10-2, 0.1 and
1.0 mM) after the activation of PDHC using sodium dichloro-
acetate (DCA).
Molecular analysis of the PDHC E1 gene (PDHA1)
Mutation analysis of PDHA1 was performed on genomic
DNA samples, which was extracted from whole blood cells
of the patient and also her mother. Amplification of individ-
ualexons of the PDHA1gene from genomic DNA was per-
formedusing primer pairs and conditions as described in (3).
Exonic PCR products were sequenced directly using appro-
priateamplification primers by an ABI 200 automated DNA
sequencer (Applied Biosystems, Foster City, CA, U.S.A.). To
confirm that Y161C is a disease-causing mutation, the sequ-
ences of exon 5 were analyzed from seventy independent DNA
samples in healthy children (35 males and 35 females). Y161C
created a site for the restriction enzyme Fau I, resulting in
two novel bands, therefore genomic PCR products of exon
5 were digested with FauI.
RESULTS
DCA-activated PDHC activity in cultured fibroblasts of
the patient was assayed at different concentrations of TPP
(1×10-3, 1×10-2, 0.1 and 1.0 mM). Cultured fibroblasts in
normal control and patient showed an exponential increase
in PDHC activity with a gradual increase of TPP concentra-
tion. Normal cells showed 6.6% of maximal PDHC activity
in low TPP concentration (1×10-3mM), and the addition
of 0.1 mM TPP restored full activity, with no further change
in PDHC activity between 0.1 and 1.0 mM TPP. However,
PDHC activity in the patient showed null activity in low
concentration of TPP (1×10-3mM), but her PDHC activi-
ties significantly increased up to 58% of the PDHC activity
of normal cells at a high concentration of TPP (1.0 mM), as
shown in Fig. 1. There was no increase of PDHC activity in
thiamine-unresponsive patient’s cell line which has an AAGT
duplication at nucleotide 1162.
Direct sequencing of entire coding region of the PDHA1
gene of the patient revealed both a A (normal) and G (mutant)
at nucleotide 482 in exon 5, resulting in the presence of a
PDHC activity (nM/min/mg protein)
3.0
2.0
1.0
10-3
10-2
10-1
1
Thiamine pyrophosphate (TPP) (mM)
Fig. 1. Effect of TPP on the activity of DCA-activated PDHC in cul-
tured fibroblasts from normal subject and two patients with PDHC
deficiency (Y161C; a patient with thiamine-responsive PDHC defi-
ciency, AAGT insertion at nt.1162; positive control which has non-
responsive PDHC deficiency).
Normal control
Y161C
AAGT insertion at nt.1162

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802
E.H. Lee, M.-S. Ahn, J.-S. Hwang, et al.
tyrosine (codon TAC) at amino acid position 161 of the wild
type E1 protein and a cysteine (codon TGC) in a mutant pro-
tein (Fig. 2). No other mutation except for Y161C was found
in the PDHA1 gene of this patient and Y161C was not de-
tected in the genomic DNA of the patient’s mother (Fig. 3).
To confirm that Y161C is disease-causing mutation, we screen-
ed the existence of this mutation from seventy unrelated heal-
thy controls. The PCR products of exon 5 were digested with
the restriction enzymes, Fau I, as a result this mutation was
not found in seventy unrelated healthy Korean.
DISCUSSION
The great majority of thiamine-responsive PDHC deficiency
results from mutations in the X-linked PDHA1 gene which
cause a decreased affinity of PDHC for TPP and a consequent
defective enzymatic activation. Clinical manifestations of PD-
HC deficiency in female are remarkably variable because muta-
tions are present in one of the two X-chromosomes in affected
females. Random patterns of X-inactivation will lead to vari-
able expression of the mutant and normal genes in different
tissues. Therefore, sufficient ATP is produced in normal cells
but severe ATP deficiency occurs in affected cells. The pro-
portion of mutant genes to normal genes in different tissues
determine variable phenotypic expression. Severe organ dys-
functions occur especially in the brain, muscle, and heart which
usually depend on aerobic oxidation of glucose for most of
its energy supply. There are no effective treatment modalities.
However, there are a few case reports of thiamine-responsive
PDHC deficiency in the literatures reported by Naito et al.
(5, 6). According to Naito et al., high concentrations of TPP
in vitro as well as treatment with thiamine in vivo is effec-
tive in thiamine-responsive PDHC deficiency. Therefore, it
is necessary to measure PDHC activity at low TPP concen-
tration in order to prevent diagnostic errors. Furthermore,
PDHC assays with serial TPP concentrations could detect
patients with thiamine-responsive PDHC deficiency (7).
We described here the identification of a novel mutation,
Y161C, in female patient with PDHC deficiency who showed
neuroradiological findings suggestive of Leigh disease, delayed
motor development and intermittent neurologic deteriora-
tion with intercurrent infection. In order to compare TPP
responsiveness, we used the 1162ins-mutant for positive con-
trol in PDHC activity analysis. The 1162ins-mutant is miss-
ing three C-terminal amino acids, Ser-Val-Ser, and these three
amino acids are known to play a critical role in the structural
integrity of the E1 component rather than in its catalytic acti-
vity. It is suggested that mutant E1 protein of 1162ins-mu-
tant causes mis-folding of both subunits leading to their mutu-
al destruction (8). However, in our patient thiamine respon-
siveness was found at serial TPP concentrations on contrary
to the 1162ins-mutant showing unresponsiveness (Fig. 1).
Therefore, the PDHC deficiency in this patient was not due
to mutual destruction of E1 component but due to a decreased
affinity of PDHC for TPP. Furthermore, clinical improvements
after thiamine administration correlated to in vitro enzymatic
studies in this patient. These results not only indicate the
presence of thiamine responsive PDHC deficiency in this
patient, but also support that the thiamine-responsive PDHC
deficiency should be diagnosed on the basis of the measure-
ment of PDHC activity under serial TPP concentrations.
Normal
Tyr
A
1234
Fig. 3. Detection of the 482 A-to-G substitution in the E1 gene. PCR-
amplified products (206 bp) of exon 5 were digested by Fau I,
which creates two fragments of 108 bp and 98 bp in the case of
the mutated sequence (Lane 1, Marker; Lane 2, Normal subject;
Lane 3, Patient (heterozygous); and Lane 4, Patient’s mother).
Fig. 2. Electropherograms of DNA sequencing for Y161C mutation. (A) Normal sequence of exon 5. (B) Patient sequence has both a A
(normal) and G (mutant) at nucleotide 482, resulting in the presence of a tyrosine (codon TAC) and cysteine (codon TGC) at position 161.
A
T CG GGG
Patient
Tyr+Cys
A
G
B
T CGG G G
206 bp
108 bp and
98 bp

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Pyruvate Dehydrogenase Complex Deficiency and Thiamine-responsiveness
803
However, there was a difference in the level of PDHC activ-
ities at low TPP concentrations compared to other reports.
According to Naito et al., normal cells showed approximately
50% of the full PDHC activity in the absence of TPP and
addition of 1×10-5mM TPP restored the full enzyme activ-
ity (6). In another report, normal cells displayed PDHC activi-
ty of 7% of the maximal level without TPP, and the addition
of 1×10-3mM TPP restored full PDHC activity (9). How-
ever, PDHC activity of normal cells showed 6% of the full
PDHC activity at 1×10-3mM TPP in this study (Fig. 1).
This difference could be derived from two possible explana-
tions. One possible explanation is that it may be due to an
absence of dephosphorylation step in our method (4). The
role of PDHC phosphatase is dephosphorylation of PDHC
with concomitant activation of this enzyme. Therefore, anal-
ysis of PDHC activity without dephosphorylation step might
lower the enzyme activity significantly. The other possible
explanation is that very low enzyme activity at low TPP con-
centration might be due to relatively higher TPP concentra-
tion in the culture medium. Naito et al. suggested that the
PDHC activity in the cultured cells with thiamine-respon-
sive mutations might depend not only on the concentration
of TPP in the reaction mixture, but also in the concentration
of thiamine in the culture medium or within the cultured
cells (5). TPP concentration in normal human blood is 1×
10-4mM but the concentration of thiamine-HCl in the cul-
ture medium used for our study was 1×10-2mM (10). It is
approximately 100 times higher than those in normal human
blood (5, 6). Therefore, it might be possible that the normal
cell lines would be adjusted to a higher TPP concentration
in culture medium and they could not react sensitively at low
TPP concentration in the reaction mixture. The PDHC activi-
ties of the 1162ins-mutant and patient showed null activities
in TPP concentration (1×10-3mM). These results might
also have been due to a difference in the method or the higher
TPP concentration in the culture medium, or the character-
istics of these mutations.
In relation to the TPP-binding sites, there are two types of
mutations: those directly interacting with the Ca++-pyrophos-
phate complex of TPP and those located outside the TPP-
binding site but inhibit TPP binding (5). The E1 subunit
has been carried the TPP binding motif GDG X26/27 NN
common to all TPP-utilizing enzyme, and it is thought to
locate in the region from amino acid 195 in exon 6 to amino
acid 225 in exon 7 (9). In the patients with PDHC deficiency
responding to TPP, five mutations in the region outside the
TPP-binding site of the E1 subunit gene have been reported
previously: H44R, R88S, G89S, R263G, and V3869fs (6,
12-14). On the contrary, in the case of a patient with Y243S
mutation, which is located outside the conserved TPP-bind-
ing motif, the PDHC activity in cultured fibroblasts was very
low even in the presence of high TPP concentration. Western
blot analysis showed that the immunoreative signals corre-
sponding to E1 and E1 subunit were weak (9). The Y243S
mutation led to structural consequences affecting the inter-
action and stability of the 2 2 heterotetramer. Therefore,
functional defects in the E1 enzyme could be quite differ-
ent according to not only the location of mutations but also
nature of amino acid subsititutions in the region outside the
TPP-binding site.
For this reason, we suggest that the Y161C mutation, which
is located in a region outside the conserved TPP-binding mo-
tif, probably induced a minimal alteration in the amino acid
sequence affecting only E1 protein folding rather than the
assembly of the 2 2 heterotetramer. However, expression
studies must be needed to know the important information
about the structural and functional role of the affected amino
acids in PDHC catabolism and in regulation or interaction
with the other components, especially the E1 subunit. They
may lead to an understanding of exact mechanism of thiamine
responsiveness in this patient with Y161C mutation.
PDHA1 gene is located on X-chromosome. In affected
females, mutations could be present in one of the two X-chro-
mosomes, and random patterns of X-inactivation will lead to
variable expression of the mutant and normal genes in differ-
ent tissues. Whether clinically manifested or not, it will de-
pend on the degree of residual PDHC activity reflecting the
proportion of cells expressing the normal E1 subunit. How-
ever, all the male havoring PDHA1 gene mutations always
present clinical features of PDHC deficiency. We did not
screened Y161C mutation in her father who did not showed
any clinical evidences of pyruvate metabolic disorder. In this
patient Y161C mutation may have occurred de novo.
In conclusion, we identified a new mutation, Y161C, caus-
ing thiamine-responsive PDHC deficiency. No other muta-
tions were found in the PDHA1gene and this mutation was
not present in the genomic DNA from seventy unrelated con-
trols. Clinical and biochemical parameters were improved by
a high dose of thiamine treatment in this patient. Thiamine-
responsive PDHC deficiency was confirmed by in vitro stud-
ies showing a thiamine-responsive functional defect in cul-
tured skin fibroblasts of the patient.
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