- Email: [email protected]
Expanding the clinical and molecular spectrum of thiamine pyrophosphokinase deficiency: A treatable neurological disorder caused by TPK1 mutations
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
Article history: Received 28 June 2014 Received in revised form 15 September 2014 Accepted 15 September 2014 Available online 5 October 2014 Keywords: Thiamine TPK1 Thiamine pyrophosphokinase TPK deficiency Episodic encephalopathy type thiamine metabolism dysfunction
a b s t r a c t Thiamine pyrophosphokinase (TPK) produces thiamine pyrophosphate, a cofactor for a number of enzymes, including pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase. Episodic encephalopathy type thiamine metabolism dysfunction (OMIM 614458) due to TPK1 mutations is a recently described rare disorder. The mechanism of the disease, its phenotype and treatment are not entirely clear. We present two patients with novel homozygous TPK1 mutations (Patient 1 with p.Ser160Leu and Patient 2 with p.Asp222His). Unlike the previously described phenotype, Patient 2 presented with a Leigh syndrome like nonepisodic early-onset global developmental delay, thus extending the phenotypic spectrum of the disorder. We, therefore, propose that TPK deficiency may be a better name for the condition. The two cases help to further refine the neuroradiological features of TPK deficiency and show that MRI changes can be either fleeting or progressive and can affect either white or gray matter. We also show that in some cases lactic acidosis can be absent and 2-ketoglutaric aciduria may be the only biochemical marker. Furthermore, we have established the assays for 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.
1. Introduction 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: [email protected] (S. Banka).
http://dx.doi.org/10.1016/j.ymgme.2014.09.010 1096-7192/© 2014 Elsevier Inc. All rights reserved.
Thiamine pyrophosphokinase (TPK, EC 2.7.6.2.) transfers a pyrophosphate 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
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 dehydrogenase), 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 tendon reflexes. He recovered gradually. At 32 months he presented similarly 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 emotionally 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) images 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 decreased 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 immunoblotting were performed as described previously [3]. 3.1. Cloning of mutant TPK1 Human wild type TPK1 was expressed from the vector PRSEThTPK1 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 contains 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 expression 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 episode, 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
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 ultracentrifugation 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% trichloroacetic 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 supernatants were transferred into new tubes. Immediately prior to injection into High-performance Liquid Chromatography (HPLC), 50 μl of the supernatants was derivatized to thiochrome products by the
303
addition of 5 μl of a freshly prepared solution of 10 mmol/l potassium 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 immunoblot 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 dimerization 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. Polyacrylamide 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 Thiamine, thiamine monophosphate (TMP) and thiamine pyrophosphate (TPP) quantification. TMP
Thiamine
Frozen muscle 600 g sup. [nmol/g protein] 15.9 P1 Controls (n = 11), mean ± SD 117.8 ± 61.0 Controls (n = 11), range 75.9–278.5
TPP
0.5 4.9 ± 4.3 1.0–15.1
1.1 7.1 ± 14.5 0.3–49.5
Fresh muscle 600 g sup. [nmol/g protein] P2 11.3 Controls (n = 9), mean ± SD 58.7 ± 12.6 Controls (n = 9), range 41.6–81.6
0.5 1.1 ± 0.4 0.4–1.7
2.8 0.9 ± 1.4 0.2–4.4
Blood [nmol/l] P1 P2 Controls (n = 10), mean ± SD Controls (n = 10), range
4.4 3.0 6.2 ± 1.3 4.1–8.8
18.9 248.9a 10.7 ± 6.1 5.0–26.4
60.9 85.4 190.9 ± 41.5 132.2–271.2
Low TPP and TMP values outside the respective control ranges in the corresponding tissues are underlined. a On thiamine supplementation.
5. Treatment 500 mg thiamine hydrochloride supplementation was started for P1 after the diagnosis was reached. He has not had further encephalopathic episodes, even with infectious illnesses. He has shown gradual slow developmental progression with improvement in understanding, social interaction, language skills and motor abilities. He does not need the nasogastric tube anymore. He is in mainstream school with extra
educational support. He continues to have unclear speech and significant spasticity of all the limbs with exaggerated deep tendon reflexes. At 19 months of age P2 could cruise and understood simple words. The biochemical results prior to the genetic diagnosis had suggested a defect in pyruvate transport or a disorder of the cofactor metabolism and, therefore, ketogenic diet was started [5]. It induced severe metabolic acidosis and resulted in severe deterioration of P2's condition. She lost her head control and the ability to sit. Ketogenic diet was stopped after five days. Examination at seven years of age showed minor recovery. The TPK1 mutation was identified in P2 at the age of eight years but thiamine supplementation after that has not resulted in any significant improvement.
6. Discussion Five disorders of thiamine transport or metabolism are now known. Thiamine responsive megaloblastic anemia syndrome (OMIM 249270) caused by SLC19A2 mutations is characterized by megaloblastic anemia, diabetes mellitus and sensorineural deafness and varying degrees of response to thiamine treatment [6]. Biotin or thiamine responsive basal ganglia disease (OMIM 607483) caused by SLC19A3 mutations is characterized by sub-acute encephalopathy, coma, epilepsy, generalized dystonia and resolution of symptoms on high dose biotin or thiamin if the diagnosis is made early [7]. Two distinct phenotypes are associated with mutations in SLC25A19 that encodes the mitochondrial thiamine transporter. One is Amish microcephaly (OMIM 607196) 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); OHgroup 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 identified 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 components of the branched-chain alpha-ketoacid dehydrogenase complex) 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 mutation, detected via whole exome sequencing, was reported [10]. Clinical, radiological and biochemical features of all 9 patients (including the two reported in this paper) are summarized in Table S3. Our report 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 described patients. There is an interesting partial overlap of this presentation 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 thiamine 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 phenotypes 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 described 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 described 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 affects 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 diagnosis was confirmed by other means. We have established the laboratory methods to facilitate the confirmation of TPK deficiency. TPK enzyme activity has never been previously measured for human mutations. The technique described here establishes an assay for testing the pathogenicity of novel TPK1 mutations 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 muscle tissue can be used to measure TPP levels. It is especially important in clinical settings because fresh muscle tissue may not always be available. 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 homogenate 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 encephalopathy in P1. One of the previously described patients with TPK deficiency 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 supplementation truly results in improvement of developmental progression 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 mechanism for TPK deficiency. In all previously described patients, the mutations 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 experimentally 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 stability of the protein may differ in human cells. Remarkably, we found that in vitro TPK activity could be significantly 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 treatable disorder and there is a need for more work to improve the understanding 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.
[4]
[5]
[6]
[7]
Conflicts of interest [8]
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).
[9]
[10]
[11]
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.
[12]
References
[13]
[1] D.E. Timm, J. Liu, L.-J. Baker, R.A. Harris, Crystal structure of thiamin pyrophosphokinase, J. Mol. Biol. 310 (2001) 195–204, http://dx.doi.org/10.1006/ jmbi.2001.4727. [2] G. Brown, Defects of thiamine transport and metabolism, J. Inherit. Metab. Dis. (2014) 1–9, http://dx.doi.org/10.1007/s10545-014-9712-9. [3] J.A. Mayr, P. Freisinger, K. Schlachter, B. Rolinski, F.A. Zimmermann, T. Scheffner, et al., Thiamine pyrophosphokinase deficiency in encephalopathic children with
[14]
defects in the pyruvate oxidation pathway, Am. J. Hum. Genet. 89 (2011) 806–812, http://dx.doi.org/10.1016/j.ajhg.2011.11.007. M. Onozuka, K. Nosaka, Steady-state kinetics and mutational studies of recombinant human thiamin pyrophosphokinase, J. Nutr. Sci. Vitaminol. 49 (2003) 156–162. I.D. Wexler, S.G. Hemalatha, J. McConnell, N.R. Buist, H.H. Dahl, S.A. Berry, et al., Outcome of pyruvate dehydrogenase deficiency treated with ketogenic diets. Studies in patients with identical mutations, Neurology 49 (1997) 1655–1661. V. Labay, T. Raz, D. Baron, H. Mandel, H. Williams, T. Barrett, et al., Mutations in SLC19A2 cause thiamine-responsive megaloblastic anaemia associated with diabetes mellitus and deafness, Nat. Genet. 22 (1999) 300–304, http://dx.doi.org/10. 1038/10372. W.-Q. Zeng, E. Al-Yamani, J.S. Acierno, S. Slaugenhaupt, T. Gillis, M.E. MacDonald, et al., Biotin-responsive basal ganglia disease maps to 2q36.3 and is due to mutations in SLC19A3, Am. J. Hum. Genet. 77 (2005) 16–26, http://dx.doi.org/10.1086/ 431216. R.I. Kelley, D. Robinson, E.G. Puffenberger, K.A. Strauss, D.H. Morton, Amish lethal microcephaly: a new metabolic disorder with severe congenital microcephaly and 2‐ketoglutaric aciduria, Am. J. Med. Genet. 112 (2002) 318–326, http://dx.doi.org/ 10.1002/ajmg.10529. R. Spiegel, A. Shaag, S. Edvardson, H. Mandel, P. Stepensky, S.A. Shalev, et al., SLC25A19 mutation as a cause of neuropathy and bilateral striatal necrosis, Ann. Neurol. 66 (2009) 419–424, http://dx.doi.org/10.1002/ana.21752. J.L. Fraser, A. Vanderver, S. Yang, T. Chang, L. Cramp, G. Vezina, et al., Thiamine pyrophosphokinase deficiency causes a Leigh disease like phenotype in a sibling pair: identification through whole exome sequencing and management strategies, Mol. Genet. Metab. Rep. 1 (2014) 66–70, http://dx.doi.org/10.1016/j.ymgmr.2013. 12.007. G. Giribaldi, L. Doria-Lamba, R. Biancheri, M. Severino, A. Rossi, F.M. Santorelli, et al., Intermittent-relapsing pyruvate dehydrogenase complex deficiency: a case with clinical, biochemical, and neuroradiological reversibility: case report, Dev. Med. Child Neurol. 54 (2012) 472–476, http://dx.doi.org/10.1111/j.1469-8749.2011. 04151.x. T. Matsuda, Y. Yabushita, T. Doi, H. Iwata, Regional distribution of thiamin pyrophosphokinase in rat brain, Experientia 41 (1985) 924–925, http://dx.doi.org/ 10.1007/BF01970015. C.D.M. van Karnebeek, M. Shevell, J. Zschocke, J.B. Moeschler, S. Stockler, The metabolic evaluation of the child with an intellectual developmental disorder: diagnostic algorithm for identification of treatable causes and new digital resource, Mol. Genet. Metab. 111 (2014) 428–438, http://dx.doi.org/10.1016/j. ymgme.2014.01.011. M.S. Duchowny, Food for thought: the ketogenic diet and adverse effects in children, Epilepsy Curr. 5 (2005) 152–154, http://dx.doi.org/10.1111/j.1535-7511.2005. 00044.x.
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
Article history: Received 28 June 2014 Received in revised form 15 September 2014 Accepted 15 September 2014 Available online 5 October 2014 Keywords: Thiamine TPK1 Thiamine pyrophosphokinase TPK deficiency Episodic encephalopathy type thiamine metabolism dysfunction
a b s t r a c t Thiamine pyrophosphokinase (TPK) produces thiamine pyrophosphate, a cofactor for a number of enzymes, including pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase. Episodic encephalopathy type thiamine metabolism dysfunction (OMIM 614458) due to TPK1 mutations is a recently described rare disorder. The mechanism of the disease, its phenotype and treatment are not entirely clear. We present two patients with novel homozygous TPK1 mutations (Patient 1 with p.Ser160Leu and Patient 2 with p.Asp222His). Unlike the previously described phenotype, Patient 2 presented with a Leigh syndrome like nonepisodic early-onset global developmental delay, thus extending the phenotypic spectrum of the disorder. We, therefore, propose that TPK deficiency may be a better name for the condition. The two cases help to further refine the neuroradiological features of TPK deficiency and show that MRI changes can be either fleeting or progressive and can affect either white or gray matter. We also show that in some cases lactic acidosis can be absent and 2-ketoglutaric aciduria may be the only biochemical marker. Furthermore, we have established the assays for 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.
1. Introduction 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: [email protected] (S. Banka).
http://dx.doi.org/10.1016/j.ymgme.2014.09.010 1096-7192/© 2014 Elsevier Inc. All rights reserved.
Thiamine pyrophosphokinase (TPK, EC 2.7.6.2.) transfers a pyrophosphate 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
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 dehydrogenase), 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 tendon reflexes. He recovered gradually. At 32 months he presented similarly 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 emotionally 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) images 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 decreased 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 immunoblotting were performed as described previously [3]. 3.1. Cloning of mutant TPK1 Human wild type TPK1 was expressed from the vector PRSEThTPK1 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 contains 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 expression 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 episode, 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
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 ultracentrifugation 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% trichloroacetic 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 supernatants were transferred into new tubes. Immediately prior to injection into High-performance Liquid Chromatography (HPLC), 50 μl of the supernatants was derivatized to thiochrome products by the
303
addition of 5 μl of a freshly prepared solution of 10 mmol/l potassium 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 immunoblot 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 dimerization 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. Polyacrylamide 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 Thiamine, thiamine monophosphate (TMP) and thiamine pyrophosphate (TPP) quantification. TMP
Thiamine
Frozen muscle 600 g sup. [nmol/g protein] 15.9 P1 Controls (n = 11), mean ± SD 117.8 ± 61.0 Controls (n = 11), range 75.9–278.5
TPP
0.5 4.9 ± 4.3 1.0–15.1
1.1 7.1 ± 14.5 0.3–49.5
Fresh muscle 600 g sup. [nmol/g protein] P2 11.3 Controls (n = 9), mean ± SD 58.7 ± 12.6 Controls (n = 9), range 41.6–81.6
0.5 1.1 ± 0.4 0.4–1.7
2.8 0.9 ± 1.4 0.2–4.4
Blood [nmol/l] P1 P2 Controls (n = 10), mean ± SD Controls (n = 10), range
4.4 3.0 6.2 ± 1.3 4.1–8.8
18.9 248.9a 10.7 ± 6.1 5.0–26.4
60.9 85.4 190.9 ± 41.5 132.2–271.2
Low TPP and TMP values outside the respective control ranges in the corresponding tissues are underlined. a On thiamine supplementation.
5. Treatment 500 mg thiamine hydrochloride supplementation was started for P1 after the diagnosis was reached. He has not had further encephalopathic episodes, even with infectious illnesses. He has shown gradual slow developmental progression with improvement in understanding, social interaction, language skills and motor abilities. He does not need the nasogastric tube anymore. He is in mainstream school with extra
educational support. He continues to have unclear speech and significant spasticity of all the limbs with exaggerated deep tendon reflexes. At 19 months of age P2 could cruise and understood simple words. The biochemical results prior to the genetic diagnosis had suggested a defect in pyruvate transport or a disorder of the cofactor metabolism and, therefore, ketogenic diet was started [5]. It induced severe metabolic acidosis and resulted in severe deterioration of P2's condition. She lost her head control and the ability to sit. Ketogenic diet was stopped after five days. Examination at seven years of age showed minor recovery. The TPK1 mutation was identified in P2 at the age of eight years but thiamine supplementation after that has not resulted in any significant improvement.
6. Discussion Five disorders of thiamine transport or metabolism are now known. Thiamine responsive megaloblastic anemia syndrome (OMIM 249270) caused by SLC19A2 mutations is characterized by megaloblastic anemia, diabetes mellitus and sensorineural deafness and varying degrees of response to thiamine treatment [6]. Biotin or thiamine responsive basal ganglia disease (OMIM 607483) caused by SLC19A3 mutations is characterized by sub-acute encephalopathy, coma, epilepsy, generalized dystonia and resolution of symptoms on high dose biotin or thiamin if the diagnosis is made early [7]. Two distinct phenotypes are associated with mutations in SLC25A19 that encodes the mitochondrial thiamine transporter. One is Amish microcephaly (OMIM 607196) 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); OHgroup 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 identified 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 components of the branched-chain alpha-ketoacid dehydrogenase complex) 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 mutation, detected via whole exome sequencing, was reported [10]. Clinical, radiological and biochemical features of all 9 patients (including the two reported in this paper) are summarized in Table S3. Our report 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 described patients. There is an interesting partial overlap of this presentation 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 thiamine 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 phenotypes 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 described 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 described 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 affects 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 diagnosis was confirmed by other means. We have established the laboratory methods to facilitate the confirmation of TPK deficiency. TPK enzyme activity has never been previously measured for human mutations. The technique described here establishes an assay for testing the pathogenicity of novel TPK1 mutations 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 muscle tissue can be used to measure TPP levels. It is especially important in clinical settings because fresh muscle tissue may not always be available. 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 homogenate 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 encephalopathy in P1. One of the previously described patients with TPK deficiency 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 supplementation truly results in improvement of developmental progression 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 mechanism for TPK deficiency. In all previously described patients, the mutations 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 experimentally 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 stability of the protein may differ in human cells. Remarkably, we found that in vitro TPK activity could be significantly 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 treatable disorder and there is a need for more work to improve the understanding 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.
[4]
[5]
[6]
[7]
Conflicts of interest [8]
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).
[9]
[10]
[11]
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.
[12]
References
[13]
[1] D.E. Timm, J. Liu, L.-J. Baker, R.A. Harris, Crystal structure of thiamin pyrophosphokinase, J. Mol. Biol. 310 (2001) 195–204, http://dx.doi.org/10.1006/ jmbi.2001.4727. [2] G. Brown, Defects of thiamine transport and metabolism, J. Inherit. Metab. Dis. (2014) 1–9, http://dx.doi.org/10.1007/s10545-014-9712-9. [3] J.A. Mayr, P. Freisinger, K. Schlachter, B. Rolinski, F.A. Zimmermann, T. Scheffner, et al., Thiamine pyrophosphokinase deficiency in encephalopathic children with
[14]
defects in the pyruvate oxidation pathway, Am. J. Hum. Genet. 89 (2011) 806–812, http://dx.doi.org/10.1016/j.ajhg.2011.11.007. M. Onozuka, K. Nosaka, Steady-state kinetics and mutational studies of recombinant human thiamin pyrophosphokinase, J. Nutr. Sci. Vitaminol. 49 (2003) 156–162. I.D. Wexler, S.G. Hemalatha, J. McConnell, N.R. Buist, H.H. Dahl, S.A. Berry, et al., Outcome of pyruvate dehydrogenase deficiency treated with ketogenic diets. Studies in patients with identical mutations, Neurology 49 (1997) 1655–1661. V. Labay, T. Raz, D. Baron, H. Mandel, H. Williams, T. Barrett, et al., Mutations in SLC19A2 cause thiamine-responsive megaloblastic anaemia associated with diabetes mellitus and deafness, Nat. Genet. 22 (1999) 300–304, http://dx.doi.org/10. 1038/10372. W.-Q. Zeng, E. Al-Yamani, J.S. Acierno, S. Slaugenhaupt, T. Gillis, M.E. MacDonald, et al., Biotin-responsive basal ganglia disease maps to 2q36.3 and is due to mutations in SLC19A3, Am. J. Hum. Genet. 77 (2005) 16–26, http://dx.doi.org/10.1086/ 431216. R.I. Kelley, D. Robinson, E.G. Puffenberger, K.A. Strauss, D.H. Morton, Amish lethal microcephaly: a new metabolic disorder with severe congenital microcephaly and 2‐ketoglutaric aciduria, Am. J. Med. Genet. 112 (2002) 318–326, http://dx.doi.org/ 10.1002/ajmg.10529. R. Spiegel, A. Shaag, S. Edvardson, H. Mandel, P. Stepensky, S.A. Shalev, et al., SLC25A19 mutation as a cause of neuropathy and bilateral striatal necrosis, Ann. Neurol. 66 (2009) 419–424, http://dx.doi.org/10.1002/ana.21752. J.L. Fraser, A. Vanderver, S. Yang, T. Chang, L. Cramp, G. Vezina, et al., Thiamine pyrophosphokinase deficiency causes a Leigh disease like phenotype in a sibling pair: identification through whole exome sequencing and management strategies, Mol. Genet. Metab. Rep. 1 (2014) 66–70, http://dx.doi.org/10.1016/j.ymgmr.2013. 12.007. G. Giribaldi, L. Doria-Lamba, R. Biancheri, M. Severino, A. Rossi, F.M. Santorelli, et al., Intermittent-relapsing pyruvate dehydrogenase complex deficiency: a case with clinical, biochemical, and neuroradiological reversibility: case report, Dev. Med. Child Neurol. 54 (2012) 472–476, http://dx.doi.org/10.1111/j.1469-8749.2011. 04151.x. T. Matsuda, Y. Yabushita, T. Doi, H. Iwata, Regional distribution of thiamin pyrophosphokinase in rat brain, Experientia 41 (1985) 924–925, http://dx.doi.org/ 10.1007/BF01970015. C.D.M. van Karnebeek, M. Shevell, J. Zschocke, J.B. Moeschler, S. Stockler, The metabolic evaluation of the child with an intellectual developmental disorder: diagnostic algorithm for identification of treatable causes and new digital resource, Mol. Genet. Metab. 111 (2014) 428–438, http://dx.doi.org/10.1016/j. ymgme.2014.01.011. M.S. Duchowny, Food for thought: the ketogenic diet and adverse effects in children, Epilepsy Curr. 5 (2005) 152–154, http://dx.doi.org/10.1111/j.1535-7511.2005. 00044.x.