Molecular Genetics and Metabolism 113 (2014) 301–306

Contents lists available at ScienceDirect

Molecular Genetics and Metabolism

journal homepage: www.elsevier.com/locate/ymgme

Expanding the clinical and molecular spectrum of thiamine

pyrophosphokinase deficiency: A treatable neurological disorder caused
by TPK1 mutations
Siddharth Banka a,b,⁎, Christian de Goede c, Wyatt W. Yue d, Andrew A.M. Morris b, Beate von Bremen e,
Kate E. Chandler b, René G. Feichtinger f, Claire Hart b, Nasaim Khan b, Verena Lunzer f, Lavinija Mataković f,
Thorsten Marquardt g, Christine Makowski h, Holger Prokisch i, Otfried Debus j, Kazuto Nosaka k,
Hemant Sonwalkar l, Franz A. Zimmermann f, Wolfgang Sperl f, Johannes A. Mayr f
a
Faculty of Medical and Human Sciences, Manchester Centre for Genomic Medicine, Institute of Human Development, University of Manchester, Manchester Academic Health Science Centre
(MAHSC), Manchester, UK
b
Manchester Centre for Genomic Medicine, Central Manchester University Hospitals NHS Foundation Trust, MAHSC, Manchester, UK
c
Department of Paediatric Neurology, Royal Preston Hospital, Preston, UK
d
Structural Genomics Consortium, Old Road Campus Research Building, University of Oxford, Oxford, UK
e
Department of Paediatrics, Royal Blackburn Hospital, Blackburn, UK
f
Department of Paediatrics, Paracelsus Medical University, Salzburg, Austria
g
Department of General Paediatrics, University Children's Hospital Münster, Germany
h
Department of Paediatrics, Technische Universität München, Munich, Germany
i
Institute of Human Genetics, Helmholtz Center Munich, German Research Center for Environmental Health, Neuherberg, Germany
j
Clemenshospital, Children's Hospital, Münster, Germany
k
Department of Chemistry, Hyogo College of Medicine, Nishinomiya, Japan
l
Department of Radiology, Royal Preston Hospital, Preston, UK

a r t i c l e i n f o a b s t r a c t

Article history: Thiamine pyrophosphokinase (TPK) produces thiamine pyrophosphate, a cofactor for a number of enzymes,
Received 28 June 2014 including pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase. Episodic encephalopathy type thiamine
Received in revised form 15 September 2014 metabolism dysfunction (OMIM 614458) due to TPK1 mutations is a recently described rare disorder. The mech-
Accepted 15 September 2014
anism of the disease, its phenotype and treatment are not entirely clear.
Available online 5 October 2014
We present two patients with novel homozygous TPK1 mutations (Patient 1 with p.Ser160Leu and Patient 2 with
Keywords:
p.Asp222His). Unlike the previously described phenotype, Patient 2 presented with a Leigh syndrome like non-
Thiamine episodic early-onset global developmental delay, thus extending the phenotypic spectrum of the disorder. We,
TPK1 therefore, propose that TPK deficiency may be a better name for the condition. The two cases help to further
Thiamine pyrophosphokinase refine the neuroradiological features of TPK deficiency and show that MRI changes can be either fleeting or pro-
TPK deficiency gressive and can affect either white or gray matter. We also show that in some cases lactic acidosis can be absent
Episodic encephalopathy type thiamine and 2-ketoglutaric aciduria may be the only biochemical marker. Furthermore, we have established the assays for
metabolism dysfunction TPK enzyme activity measurement and thiamine pyrophosphate quantification in frozen muscle and blood.
These tests will help to diagnose or confirm the diagnosis of TPK deficiency in a clinical setting.
Early thiamine supplementation prevented encephalopathic episodes and improved developmental progression
of Patient 1, emphasizing the importance of early diagnosis and treatment of TPK deficiency. We present evidence
suggesting that thiamine supplementation may rescue TPK enzyme activity.
Lastly, in silico protein structural analysis shows that the p.Ser160Leu mutation is predicted to interfere with TPK
dimerization, which may be a novel mechanism for the disease.
© 2014 Elsevier Inc. All rights reserved.

Abbreviations: ATP, adenosine triphosphate; MRI, magnetic resonance imaging;
THDM5, episodic encephalopathy type thiamine metabolism dysfunction; TPK, thiamine
pyrophosphokinase; TPP, thiamine pyrophosphate.
⁎ Corresponding author at: Manchester Centre for Genomic Medicine, St Mary's
Hospital, University of Manchester, Manchester M13 9WL, UK.
E-mail address: Siddharth.Banka@manchester.ac.uk (S. Banka).
1. Introduction
Thiamine pyrophosphokinase (TPK, EC 2.7.6.2.) transfers a pyro-
phosphate group from adenosine triphosphate (ATP) to thiamine to
produce its active form, thiamine pyrophosphate (TPP) in the cytosol
[1]. TPP is a cofactor for enzymes important in a range of fundamental

http://dx.doi.org/10.1016/j.ymgme.2014.09.010
1096-7192/© 2014 Elsevier Inc. All rights reserved.
302 S. Banka et al. / Molecular Genetics and Metabolism 113 (2014) 301–306

processes such as cellular respiration (pyruvate dehydrogenase and

2-ketoglutarate dehydrogenase) and in providing substrates for
synthesis of nucleic acids, nucleotides, fatty acids and steroids
(transketolase in the pentose phosphate pathway). It is needed for
the catabolism of amino acids (branched-chain α-keto acid dehy-
drogenase), phytanic acid and 2-hydroxy straight chain fatty acids
(2-hydroxyphytanoyl-CoA lyase). A number of defects in thiamine
transport and metabolism are now known [2] and TPK1 mutations
resulting in episodic encephalopathy type thiamine metabolism
dysfunction (THDM5, OMIM 614458) is the most recently described
disorder of this group [3]. We present two new patients with TPK1
mutations providing novel clinical and biological insights into the
condition.

2. Case histories

Patient 1 (P1) is a male child born to first cousin parents of Indian

origin. He presented at 30 months of age during a viral illness with
loss of ability to walk and ataxia. On examination he had brisk deep ten-
don reflexes. He recovered gradually. At 32 months he presented simi-
larly with chicken pox, followed by development of extra-pyramidal
features, upper motor neuron signs and fluctuating hypertonia during
recovery. His vocabulary reduced to ten words and he became emotion-
ally labile. At 36 months he developed encephalopathy and fluctuating
awareness during an episode of gastroenteritis. During recovery his
vocabulary reduced to three words and he became naso-gastric tube
dependent.
Patient 2 (P2) is an eight-year-old daughter of unrelated German
parents. She presented during infancy with feeding difficulties, delayed
motor development, severe truncal hypotonia, hypertonia of the limbs
and brisk reflexes.
Investigations performed in P1 and P2 are summarized in Tables S1
and S2 respectively. Their brain magnetic resonance imaging (MRI) im-
ages are shown in Fig. 1. 2-Ketoglutaric aciduria was observed in both
patients. Lactate levels were elevated in P2 (with low lactate:pyruvate
ratio) but were normal in P1. Muscle biopsy studies of P2 revealed de-
creased utilization of pyruvate but normal pyruvate dehydrogenase
complex (PDHC) activity, suggesting a defect in pyruvate transport or
a disorder of the cofactor metabolism.

3. Materials and methods

TPK1 Sanger sequencing, TPP quantification and TPK immunoblot-

ting were performed as described previously [3].

3.1. Cloning of mutant TPK1

Human wild type TPK1 was expressed from the vector PRSET-
hTPK1 in Escherichia coli as reported previously [4]. Mutant TPK1
was amplified from patient cDNA and PCR amplification with the
forward primer 5′-cgggatccgATGGAGCATGCCTTTACC-3′ that con-
tains a BamHI site and the reverse primer 5′-gaagatctTTAGCTTTTG
ATGGCCATGG-3′ that contains a BglII site. The mutant TPK1 was
cloned into this vector by replacing the wild type sequence. The
final constructs were sequenced and the mutations, c.479CNT
(p.Ser160Leu, Patient 1) and c.664GNC (p.Asp222His, Patient 2)
were confirmed.
3.2. Expression and purification of TPK1 protein
The E. coli strain BL21(DE3)pLysS (Promega) was transformed with
either mutant or wild type TPK1 on pRSET B and grown on LB medium
containing 50 μl/ml chloramphenicol and 100 μl/ml ampicillin. For ex-
pression of the recombinant protein, 200 ml of this medium containing
1 mmol/l isopropyl-β-D-thiogalactopyranoside (IPTG) was inoculated
with an overnight preculture at an optical density at 600 nm of 0.1
Fig. 1. Axial T2 brain magnetic resonance imaging. A) Hyper-intense signals in the dentate
nucleus (P1, first episode, 30 months). B) Resolution of the earlier changes (P1, second ep-
isode, 32 months). C and D) Pyramidal tract signal changes in the medulla and left dentate
nucleus (P1, third episode, 36 months). E and F) Delayed myelination and altered density
in the basal ganglia (P2, 16 months). G and H) Enlarged extra-axial CSF spaces suggesting
cortical atrophy (P2, 41 months).
and grown for 2 h. The cells were harvested by centrifugation, washed
once and resuspended in 20 ml lysis buffer/wash buffer (50 mmol/l
sodium phosphate, 300 mmol/l sodium chloride, 10 mmol/l imidazole,
pH 7.4). Cells were disrupted by sonication and crude protein extracts
were obtained by collecting the supernatant after centrifugation for
5 min at 10,000 g. Sonication and all following steps were performed
under cooling with ice. Since the recombinant human TPK contains an
N-terminal His-tag we purified the crude extracts with HisPur cobalt
spin columns (1 ml columns, Pierce Biotechnology) according to the
manufacturer's instructions. After washing with 50 resin volumes of
 S. Banka et al. / Molecular Genetics and Metabolism 113 (2014) 301–306 303

buffer/wash buffer the His-tagged protein was eluted by 5 ml of
50 mmol/l sodium phosphate, 300 mmol/l sodium chloride, and
150 mmol/l imidazole, pH 7.4. The buffer was replaced with
30 mmol/l NaCl and 30 mmol/l Tris, pH 7.5 followed by ultracentrifu-
gation in an Amicon Ultra-15 (Millipore) centrifugal filter unit with
the molecular weight cut-off of 10 kDa.
3.3. Measurement of TPK activity
The activity of TPK was determined in a mixture containing
20 mmol/l Tris buffer (pH 7.5), 1 mmol/l ATP, 1 mmol/l MgCl2 and
variable concentrations of thiamin (10–1000 nmol/l). An equal
amount of either mutated or wild type recombinant purified TPK
(10 μl, approx. 10 ng) was added followed by incubation at 37 °C
for 30 min. The reaction was stopped by adding 20 μl of 50% trichlo-
roacetic acid. Samples were then incubated on ice for 15 min and
centrifuged for 5 min at 10,000 g. The supernatants obtained after
centrifugation were transferred into new tubes, extracted twice
with 400 μl of water-saturated diethyl ether, centrifuged for 10 s at
2500 g and finally incubated for 15 min at 37 °C to remove residual
ether. The samples were centrifuged at 10,000 g for 5 min, and super-
natants were transferred into new tubes. Immediately prior to injec-
tion into High-performance Liquid Chromatography (HPLC), 50 μl of
the supernatants was derivatized to thiochrome products by the
addition of 5 μl of a freshly prepared solution of 10 mmol/l potassi-
um ferricyanide in 15% NaOH.
4. Results
TPK1 Sanger sequencing revealed novel homozygous c.479CNT
(p.Ser160Leu) and c.664GNC (p.Asp222His) mutations in P1 and P2
respectively (Fig. 2A). Both mutations affect highly conserved residues
(Fig. S1). TPP levels were significantly decreased in patients' blood and
muscle samples (Table 1). Activities of the two recombinant mutant
TPK enzymes were significantly lower than that of the wild-type TPK
(Fig. 2B). Similar to what has been described previously [3] the immu-
noblot analysis showed marked reduction in the level of the TPK protein
in P2's muscle extract but, remarkably, not in P1's sample (Fig. 2C).
These results suggested that both the mutations were pathogenic and
in contrast to the previously reported cases, loss of TPK activity in P1
is not due to lower enzyme levels but possibly another mechanism.
Inspection of the available human TPK1 structure (PDB code 3S4Y)
revealed that the p.Ser160Leu mutation likely disrupts the TPK dimer-
ization interface (Fig. 3A) thereby possibly affecting its enzyme activity.
Similar analysis for P2's mutation showed that the Asp222 residue is
close to the thiamine-binding domain at the C-terminus of the enzyme
(Fig. 3B).

Fig. 2. Results of genetic and biochemical investigations. A) Sequencing chromatograms showing the homozygous c.479CNT mutation in P1 and c.664GNC in P2. B) TPK activity
analysis with recombinant TPK1 protein showing decreased enzyme activity for mutations seen in P1 and P2, most pronounced at lower thiamine concentrations. Polyacryl-
amide electrophoresis and staining with Coomassie brilliant blue showing the high purity of the purified recombinant proteins. C) Human TPK1 and GAPDH western blots in
muscle extracts showing no clear decrease of TPK levels in P1 when compared to controls (C1, C2, C3, C4). TPK levels are decreased in P2 and disease control (DC), a previously
published patient with p.[Arg60Lysfs*52] + [Asn219Ser] TPK1 mutations [3].
304 S. Banka et al. / Molecular Genetics and Metabolism 113 (2014) 301–306

Table 1 educational support. He continues to have unclear speech and signifi-

Thiamine, thiamine monophosphate (TMP) and thiamine pyrophosphate (TPP) cant spasticity of all the limbs with exaggerated deep tendon reflexes.
quantification.
At 19 months of age P2 could cruise and understood simple words.
TPP TMP Thiamine The biochemical results prior to the genetic diagnosis had suggested a
Frozen muscle 600 g sup. [nmol/g protein] defect in pyruvate transport or a disorder of the cofactor metabolism
P1 15.9 0.5 1.1 and, therefore, ketogenic diet was started [5]. It induced severe meta-
Controls (n = 11), mean ± SD 117.8 ± 61.0 4.9 ± 4.3 7.1 ± 14.5 bolic acidosis and resulted in severe deterioration of P2's condition.
Controls (n = 11), range 75.9–278.5 1.0–15.1 0.3–49.5
She lost her head control and the ability to sit. Ketogenic diet was
Fresh muscle 600 g sup. [nmol/g protein] stopped after five days. Examination at seven years of age showed
P2 11.3 0.5 2.8 minor recovery. The TPK1 mutation was identified in P2 at the age of
Controls (n = 9), mean ± SD 58.7 ± 12.6 1.1 ± 0.4 0.9 ± 1.4
eight years but thiamine supplementation after that has not resulted
Controls (n = 9), range 41.6–81.6 0.4–1.7 0.2–4.4
in any significant improvement.
Blood [nmol/l]
P1 60.9 4.4 18.9
P2 85.4 3.0 248.9a 6. Discussion
Controls (n = 10), mean ± SD 190.9 ± 41.5 6.2 ± 1.3 10.7 ± 6.1
Controls (n = 10), range 132.2–271.2 4.1–8.8 5.0–26.4
Five disorders of thiamine transport or metabolism are now known.
Low TPP and TMP values outside the respective control ranges in the corresponding tis-
Thiamine responsive megaloblastic anemia syndrome (OMIM 249270)
sues are underlined.
a
On thiamine supplementation. caused by SLC19A2 mutations is characterized by megaloblastic anemia,
diabetes mellitus and sensorineural deafness and varying degrees of
5. Treatment response to thiamine treatment [6]. Biotin or thiamine responsive
basal ganglia disease (OMIM 607483) caused by SLC19A3 mutations is
500 mg thiamine hydrochloride supplementation was started for P1 characterized by sub-acute encephalopathy, coma, epilepsy, general-
after the diagnosis was reached. He has not had further encephalopathic ized dystonia and resolution of symptoms on high dose biotin or
episodes, even with infectious illnesses. He has shown gradual slow thiamin if the diagnosis is made early [7]. Two distinct phenotypes are
developmental progression with improvement in understanding, social associated with mutations in SLC25A19 that encodes the mitochondrial
interaction, language skills and motor abilities. He does not need the thiamine transporter. One is Amish microcephaly (OMIM 607196)
nasogastric tube anymore. He is in mainstream school with extra characterized by congenital severe microcephaly, profound global

Fig. 3. Results of protein structural analysis for p.Ser160Leu and p.Asp222His. A) Structure of human TPK1 showing: the dimerization interface highlighted in red box (left panel); OH-
group of Ser160A in one subunit (green) forming a H-bond with Phe132B amide nitrogen of the neighboring dimeric subunit (blue); the snugly packed interface between Ser160A
(shown in surface representation) and Phe132B, which will preclude the substitution of a large non-polar amino acid at the Ser160 site, due to steric clash and loss of the H-bond
(right panel). B) Asp222 is towards the C-terminus of the TPK1 enzyme (PDB code 3S4Y), shown as a dimer (subunits A in green and B in blue). The Asp222 residue (magenta spheres)
is located at a surface-exposed loop, ~3–5 aa downstream from a β-strand that contributes to part of the thiamine binding site (thiamine shown in black line). Asp222 appears to stabilize
the conformation of the loop, and its substitution to Histidine may disrupt this conformation.
 S. Banka et al. / Molecular Genetics and Metabolism 113 (2014) 301–306
developmental delay, brain malformations, episodic encephalopathy
associated with lactic acidosis and alpha-ketoglutaric aciduria [8]. The
second phenotype associated with SLC25A19 mutations is bilateral
striatal degeneration and progressive polyneuropathy (OMIM 613710)
that is characterized by childhood onset of episodic encephalopathy
with febrile illnesses resulting in transient neurologic dysfunction and
slowly progressive axonal polyneuropathy [9]. The most recently iden-
tified condition in this group is episodic encephalopathy type thiamine
metabolism dysfunction caused by TPK1 mutations [3]. Some patients
with PDHC deficiency (OMIM 312170) and maple syrup urine disease
(OMIM 248600) (caused by mutations in genes encoding catalytic com-
ponents of the branched-chain alpha-ketoacid dehydrogenase com-
plex) are thiamine responsive [2].
Only five patients from three families with TPK1 mutations have
been previously described [3]. While this manuscript was being
reviewed, another sibling pair with a novel homozygous TPK1 muta-
tion, detected via whole exome sequencing, was reported [10]. Clini-
cal, radiological and biochemical features of all 9 patients (including
the two reported in this paper) are summarized in Table S3. Our re-
port expands the known phenotype of the condition. In P1 the early
development was slower than his siblings, but overall unremarkable.
His neurological episodes were triggered by infectious illnesses
followed by slow and incomplete recovery leading to a step-wise
deterioration and regression, which is similar to the previously de-
scribed patients. There is an interesting partial overlap of this presen-
tation with what is encountered in biotin or thiamine responsive basal
ganglia disease, bilateral striatal degeneration and progressive
polyneuropathy, maple syrup urine disease and in some patients
with PDHC deficiency. However, careful review of biochemical and
neuroradiological features may help to distinguish between these
phenotypes. Presenting features of P2 show that TPK1 mutations
may also result in a non-episodic early-onset global developmental
delay. The phenotype described by Fraser et al.[10] is similar to that
of P2. The current nomenclature of episodic encephalopathy type thi-
amine metabolism dysfunction for the disease should be therefore,
reconsidered. We propose that ‘TPK deficiency’ is a more appropriate
name. Interestingly, existence of two remarkably different pheno-
types with mutations in SLC25A19 (congenital Amish microcephaly
and childhood onset encephalopathic disorder of bilateral striatal
degeneration and progressive polyneuropathy) is known. Similarly,
deficiency of PDHC, of which TPP is a cofactor, can present in either
chronic progressive or intermittent-relapsing forms [11].
The signal changes seen in P1's left dentate nucleus during the first
presentation had completely resolved by the time of the second episode
(Fig. 1). During the third episode changes were noticed in the pyramidal
tracts in the medulla and left dentate nucleus. In an appropriate clinical
scenario, fleeting changes in the brain could be a clue to the diagnosis.
A similar pattern has been described in PDHC [11]. This stresses the
importance of performing and repeating neuroimaging during acute
clinical episodes. Interestingly, the location of the signal changes
seen in P1 matches the expression pattern of TPK in the rat brain
[12]. P2's neuroimaging showed Leigh syndrome like altered basal
ganglia density, delayed myelination and progressive cortical atrophy
(Fig. 1). Similar features were seen in the two siblings described by
Fraser et al.[10]. Comparison of MRI images of our patients with
those described previously [3] suggests that the neuroradiological
changes in TPK deficiency are variable and can be either fleeting or
progressive and can affect either white or gray matter.
Lactic acidosis was noted in P2 and has been seen in previously de-
scribed cases [3,10]. However, investigations in P1 show that the lactate
levels in blood and cerebrospinal fluid, even during an episode, may not
be reliable. 2-Ketoglutaric aciduria was observed in both patients de-
scribed here. 2-Ketoglutaric aciduria was also described in the siblings
reported by Fraser et al. and in the younger patient the lactic acid levels
were only mildly elevated. 2-Ketoglutaric acid may, therefore, be a good
marker for the disease. Repeated analyses may be needed to check
305
specifically for abnormal 2-ketoglutaric acid excretion. The biochemical
profiles of our patients suggest that in vivo TPK deficiency primarily af-
fects the function of PDHC and 2-ketoglutarate dehydrogenase complex.
Of note, PDHC activity was found to be normal in muscle biopsy from P2
because excess TPP is added in the standard laboratory assay. We have
previously shown that TPP depletion does not affect the stability of
PDHC and TPP-unstimulated:TPP-stimulated PDHC activity ratios are a
better in vitro marker for PDHC dysfunction in TPK deficiency [3]. This
assay was not performed in either of the cases described here as the di-
agnosis was confirmed by other means.
We have established the laboratory methods to facilitate the confir-
mation of TPK deficiency. TPK enzyme activity has never been previous-
ly measured for human mutations. The technique described here
establishes an assay for testing the pathogenicity of novel TPK1 muta-
tions and can be utilized in clinical settings. We have shown reduced
TPP levels in a frozen muscle biopsy sample from P1 and determined
the reference range for the assay, thus demonstrating that frozen mus-
cle tissue can be used to measure TPP levels. It is especially important in
clinical settings because fresh muscle tissue may not always be avail-
able. To the best of our knowledge, previous TPP levels have only ever
been measured in fresh muscle samples. Notably, TPP levels in frozen
muscle were approximately two-fold higher than those in muscle ho-
mogenate prepared from fresh tissue [3]. Higher amounts of thiamine
species in frozen muscle could be due to freezing and thawing resulting
in disintegration of the cell membranes.
Thiamine supplementation prevented further episodes of enceph-
alopathy in P1. One of the previously described patients with TPK de-
ficiency attends normal school and has normal development [3].
Hence, TPK deficiency can be added to the list of thiamine responsive
disorders [2] and potentially a treatable inborn error of metabolism
[13]. Lack of response to thiamine in P2 emphasizes the importance
of early diagnosis and treatment for better outcome. However, larger
long-term studies will be needed to determine if thiamine supple-
mentation truly results in improvement of developmental progres-
sion in TPK deficiency. Ketogenic diet is recommended in PDHC
deficiency because it provides alternative sources of acetyl-CoA [5].
In P2 ketogenic diet induced severe metabolic acidosis and resulted
in an adverse outcome. The underlying mechanism for this response
is unclear but metabolic acidosis is a known complication of ketogenic
diet [14]. Importantly, P2 did not receive thiamine supplementation
while on ketogenic diet (because the genetic diagnosis was not
known at the time of the start of dietary management). Of note, the
younger sibling described by Fraser et al. received ketogenic
diet along with thiamine supplementation and was reported to
make some developmental progress. Based on our clinical experience,
we recommend high dose thiamine supplementation as the mainstay
of the treatment and to avoid use of ketogenic diet on its own.
The p.Ser160Leu mutation reveals a possible novel disease mecha-
nism for TPK deficiency. In all previously described patients, the muta-
tions resulted in a significant decrease in the amount of protein. In
contrast, in silico analysis suggests that the loss of the enzyme activity
in P1 could be due to impaired dimerization of TPK. This could not be ex-
perimentally verified because of lack of a specific antibody suitable for
non-denatured TPK protein. Additionally, it is important to note that
we used an E. coli based system to express the mutant proteins. The sta-
bility of the protein may differ in human cells.
Remarkably, we found that in vitro TPK activity could be significant-
ly rescued by increasing the concentration of thiamine (Fig. 2B) close to
the serum level that can be reached by intensive supplementation with
thiamine (unpublished observation). Similar results were observed
with P2's mutation. This may represent a common basis for the clinical
response to thiamine in TPK deficiency.
In summary, we have reported novel clinical and biological insights
into a rare disease. It is important for clinicians to be aware of this treat-
able disorder and there is a need for more work to improve the under-
standing of TPK deficiency.
306 S. Banka et al. / Molecular Genetics and Metabolism 113 (2014) 301–306
Contributions
SB and JAM diagnosed the patients, set up the study and wrote the
paper. SB, WS and JAM analyzed data. CdG, AM, BvB, KEC, NK, HS, TM,
CM, and OD provided the clinical details. WY performed protein
structural analysis. CH and RGF performed biochemical analysis.
FAZ performed western blotting. LM and KN performed thiamine
quantification and protein expression. VL, HP, and KN performed
genetic investigations and cloning. All authors read and approved
the manuscript.
Conflicts of interest
None of the authors declare any conflicts of interest.
Acknowledgments
We acknowledge the support of Manchester Biomedical Research
Centre. This work was supported by the Marie Curie Initial Training
Network MEET supported by the European Union (LM, HP, JAM) and
the E-Rare project GENOMIT FWF I 920-B13 (VL, FAZ, RGF, WS).
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.ymgme.2014.09.010.
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