Case report of two affected siblings in a family with thiamine metabolism dysfunction syndrome 5: a rare, but treatable neurodegenerative disease.

BACKGROUND: Thiamine metabolism dysfunction syndrome 5 (THMD5) is a rare inherited metabolic disorder due to thiamine pyrophosphokinase 1(TPK1) deficiency, caused by mutations in TPK1. The core symptoms of the disease is acute or subacute onset encephalopathy, ataxia, muscle hypotonia, and regression of developmental milestones in early infancy, repeatedly triggered by acute infectious illness. However, we report two brothers of THMD5 with compound heterozygous for the mutations c.614-1G > A,c.224 T > A p.(Ile75Asn), but the prognosis is quite different if thiamine suppled. According to our current knowledge, the missense variant c.224 T > A p.(Ile75Asn) was not published previously. CASE PRESENTATION: Here, we describe two affected siblings in a Chinese family, after an uneventful pregnancy to non-consanguineous and healthy parents. The older brother presented with normal development during the first 6 months of life, but developed regression of developmental milestones after, accompanied with muscle hypotonia, and chronic encephalopathy, and died at 1 year and 6 months old. The younger brother presented with acute onset encephalopathy, ataxia, muscle hypotonia, repeatedly triggered by acute infectious illness. He was compound heterozygous for the mutations c.614-1G > A,c.224 T > A p.(Ile75Asn) identified by whole exome sequencing. He was diagnosed of THMD5 when he was 11 month. Oral supplementation of thiamine 100 mg/day, the symptoms gradually disappeared. At the age of 2 years and 4 months, he stoped thiamine, his symptoms returned and were once again relieved by oral supplementation of thiamine 100 mg/day. CONCLUSIONS: THMD5 is a rare, but treatable neurodegenerative disease, the clinical phenotype ranges from mild to severe. Massive-dose of thiamine supplementation may ameliorate the course of TPK1 deficiency. When similar clinical cases appear, gene detection is particularly important, which is conducive to early diagnosis. Treatment with thiamine while awaiting the outcome of diagnostic tests may be a good choice.

Identification and characterisation of thiamine pyrophosphate (TPP) riboswitch in Elaeis guineensis.

The oil palm (Elaeis guineensis) is an important crop in Malaysia but its productivity is hampered by various biotic and abiotic stresses. Recent studies suggest the importance of signalling molecules in plants in coping against stresses, which includes thiamine (vitamin B1). Thiamine is an essential microelement that is synthesized de novo by plants and microorganisms. The active form of thiamine, thiamine pyrophosphate (TPP), plays a prominent role in metabolic activities particularly as an enzymatic cofactor. Recently, thiamine biosynthesis pathways in oil palm have been characterised but the search of novel regulatory element known as riboswitch is yet to be done. Previous studies showed that thiamine biosynthesis pathway is regulated by an RNA element known as riboswitch. Riboswitch binds a small molecule, resulting in a change in production of the proteins encoded by the mRNA. TPP binds specifically to TPP riboswitch to regulate thiamine biosynthesis through a variety of mechanisms found in archaea, bacteria and eukaryotes. This study was carried out to hunt for TPP riboswitch in oil palm thiamine biosynthesis gene. Riboswitch detection software like RiboSW, RibEx, Riboswitch Scanner and Denison Riboswitch Detector were utilised in order to locate putative TPP riboswitch in oil palm ThiC gene sequence that encodes for the first enzyme in the pyrimidine branch of the pathway. The analysis revealed a 192 bp putative TPP riboswitch located at the 3' untranslated region (UTR) of the mRNA. Further comparative gene analysis showed that the 92-nucleotide aptamer region, where the metabolite binds was conserved inter-species. The secondary structure analysis was also carried out using Mfold Web server and it showed a stem-loop structure manifested with stems (P1-P5) with minimum free energy of -12.26 kcal/mol. Besides that, the interaction of riboswitch and its ligand was determined using isothermal titration calorimetry (ITC) and it yielded an exothermic reaction with 1:1 stoichiometry interaction with binding affinities of 0.178 nM, at 30°C. To further evaluate the ability of riboswitch to control the pathway, exogenous thiamine was applied to four months old of oil palm seedlings and sampling of spear leaves tissue was carried out at days 0, 1, 2 and 3 post-treatment for expression analysis of ThiC gene fragment via quantitative polymerase chain reaction (qPCR). Results showed an approximately 5-fold decrease in ThiC gene expression upon application of exogenous thiamine. Quantification of thiamine and its derivatives was carried out via HPLC and the results showed that it was correlated to the down regulation of ThiC gene expression. The application of exogenous thiamine to oil palm affected ThiC gene expression, which supported the prediction of the presence of TPP riboswitch in the gene. Overall, this study provides the first evidence on the presence, binding and the functionality of TPP riboswitch in oil palm. This study is hoped to pave a way for better understanding on the regulation of thiamine biosynthesis pathway in oil palm, which can later be exploited for various purposes especially in manipulation of thiamine biosynthesis pathways in combating stresses in oil palm.

Functional analysis of the third identified SLC25A19 mutation causative for the thiamine metabolism dysfunction syndrome 4.

Thiamine metabolism dysfunction syndrome-4 (THMD4) includes episodic encephalopathy, often associated with a febrile illness, causing transient neurologic dysfunction and a slowly progressive axonal polyneuropathy. Until now only two mutations (G125S and S194P) have been reported in the SLC25A19 gene as causative for this disease and a third mutation (G177A) as related to the Amish lethal microcephaly. In this work, we describe the clinical and molecular features of a patient carrying a novel mutation (c.576G>C; Q192H) on SLC25A19 gene. Functional studies on this mutation were performed explaining the pathogenetic role of c.576G>C in affecting the translational efficiency and/or stability of hMTPPT protein instead of the mRNA expression. These findings support the pathogenetic role of Q192H (c.576G>C) mutation on SLC25A19 gene. Moreover, despite in other patients the thiamine supplementation leaded to a substantial improvement of peripheral neuropathy, our patient did not show a clinical improvement.

Reduced thiamine binding is a novel mechanism for TPK deficiency disorder.

Thiamine pyrophosphokinase (TPK) converts thiamine (vitamin B1) into thiamine pyrophosphate (TPP), an essential cofactor for many important enzymes. TPK1 mutations lead to a rare disorder: episodic encephalopathy type thiamine metabolism dysfunction. Yet, the molecular mechanism of the disease is not entirely clear. Here we report an individual case of episodic encephalopathy, with familial history carrying a novel homozygous TPK1 mutation (p.L28S). The L28S mutation leads to reduced enzymatic activity, both in vitro and in vivo, without impairing thiamine binding and protein stability. Thiamine supplementation averted encephalopathic episodes and restored the patient's developmental progression. Biochemical characterization of reported TPK1 missense mutations suggested reduced thiamine binding as a new disease mechanism. Importantly, many disease mutants are directly or indirectly involved in thiamine binding. Thus, our study provided a novel rationale for thiamine supplementation, so far the major therapeutic intervention in TPK deficiency.

The vitamin-sensitive promoter PTHI11 enables pre-defined autonomous induction of recombinant protein production in Pichia pastoris.

The methylotrophic yeast Pichia pastoris is widely used for production of recombinant proteins. Here we characterize a vitamin-sensitive regulatory sequence, which can be controlled independently of the main culture medium compounds such as carbon, nitrogen, or phosphor source. The THI11 promoter (PTHI11 ) sequence derives from a gene involved in biosynthesis of thiamine. For characterization, a P. pastoris strain expressing recombinant human serum albumin under control of PTHI11 was grown in the controlled environment of a bioreactor. The thiamine sensitivity of PTHI11 was proven and specified in batch cultures containing different amounts of extracellular thiamine. Under non-repressing conditions PTHI11 offers a constitutive expression pattern with growth rate dependent product formation. Furthermore, promoter activity and thus product formation can be repressed for a desired period of time by supplementing the culture with a pre-defined amount of exogenous thiamine. Once a threshold of biomass is reached, PTHI11 driven expression starts autonomously without external intervention. Based on these findings a tailor-made process strategy was developed and experimentally verified. Additionally, we compared the THI11 promoter with the commonly used GAP promoter. In conclusion, the THI11 promoter is a versatile and easy to control regulatory sequence which enables the realization of novel protein production strategies. Biotechnol. Bioeng. 2016;113: 2633-2643. © 2016 Wiley Periodicals, Inc.

Treatment of genetic defects of thiamine transport and metabolism.

INTRODUCTION: Thiamine is a key cofactor for energy metabolism in brain tissue. There are four major genetic defects (SLC19A2, SLC19A3, SLC25A19 and TPK1) involved in the metabolism and transport of thiamine through cellular and mitochondrial membranes. Neurological involvement predominates in three of them (SLC19A3, SCL25A19 and TPK1), whereas patients with SLC19A2 mutations mainly present extra-neurological features (e.g. diabetes mellitus, megaloblastic anaemia and sensori-neural hearing loss). These genetic defects may be amenable to therapeutic intervention with vitamins supplementation and hence, constitutes a main area of research. AREAS COVERED: We conducted a literature review of all reported cases with these genetic defects, and focused our paper on treatment efficacy and safety, adverse effects, dosing and treatment monitoring. Expert commentary: Doses of thiamine vary according to the genetic defect: for SLC19A2, the usual dose is 25-200 mg/day (1-4 mg/kg per day), for SLC19A3, 10-40 mg/kg per day, and for TPK1, 30 mg/kg per day. Thiamine supplementation in SLC19A3-mutated patients restores CSF and intracellular thiamine levels, resulting in successful clinical benefits. In conclusion, evidence collected so far suggests that the administration of thiamine improves outcome in SLC19A-2, SLC19A3- and TPK1-mutated patients, so most efforts should be aimed at early diagnosis of these disorders.

Analysis of thiamine transporter genes in sporadic beriberi.

OBJECTIVE: Thiamine or vitamin B1 deficiency diminishes thiamine-dependent enzymatic activity, alters mitochondrial function, impairs oxidative metabolism, and causes selective neuronal death. We analyzed for the first time, the role of all known mutations within three specific thiamine carrier genes, SLC19 A2, SLC19 A3, and SLC25 A19, in a patient with atrophic beriberi, a multiorgan nutritional disease caused by thiamine deficiency. METHODS: A 44-year-old male alcoholic patient from Morocco developed massive bilateral leg edema, a subacute sensorimotor neuropathy, and incontinence. Despite normal vitamin B1 serum levels, his clinical picture was rapidly reverted by high-dose intramuscular thiamine treatment, suggesting a possible genetic resistance. We used polymerase chain reaction followed by amplicon sequencing to study all the known thiamine-related gene mutations identified within the Human Gene Mutation Database. RESULTS: Thirty-seven mutations were tested: 29 in SLC19 A2, 6 in SLC19 A3, and 2 in SLC25 A19. Mutational analyses showed a wild-type genotype for all sequences investigated. CONCLUSION: This is the first genetic study in beriberi disease. We did not detect any known mutation in any of the three genes in a sporadic dry beriberi patient. We cannot exclude a role for other known or unknown mutations, in the same genes or in other thiamine-associated genes, in the occurrence of this nutritional neuropathy.

The role of thiamine in cancer: possible genetic and cellular signaling mechanisms.

The relationship between supplemental vitamins and various types of cancer has been the focus of recent investigation, and supplemental vitamins have been reported to modulate cancer rates. A significant association has been demonstrated between cancer and low levels of thiamine in the serum. Genetic studies have helped identify a number of factors that link thiamine to cancer, including the solute carrier transporter (SLC19) gene, transketolase, transcription factor p53, poly(ADP-ribose) polymerase-1 gene, and the reduced form of nicotinamide adenine dinucleotide phosphate. Thiamine supplementation may contribute to a high rate of tumor cell survival, proliferation and chemotherapy resistance. Thiamine has also been implicated in cancer through its effects on matrix metalloproteinases, prostaglandins, cyclooxygenase-2, reactive oxygen species, and nitric oxide synthase. However, some studies have suggested that thiamine may exhibit some antitumor effects. The role of thiamine in cancer is controversial. However, thiamine deficiency may occur in patients with cancer and cause serious disorders, including Wernicke's encephalopathy, that require parenteral thiamine supplementation. A very high dose of thiamine produces a growth-inhibitory effect in cancer. Therefore, further investigations of thiamine in cancer are needed to clarify this relationship.

Genetic diversity of thiamin and folate in primitive cultivated and wild potato (Solanum) species.

Biofortification of staple crops like potato via breeding is an attractive strategy to reduce human micronutrient deficiencies. A prerequisite is metabolic phenotyping of genetically diverse material which can potentially be used as parents in breeding programs. Thus, the natural genetic diversity of thiamin and folate contents was investigated in indigenous cultivated potatoes (Solanum tuberosum group Andigenum) and wild potato species (Solanum section Petota). Significant differences were found among clones and species. For about 50% of the clones there were variations in thiamin and folate contents between years. Genotypes which contained over 2-fold the thiamin and 4-fold the folate content compared to the modern variety Russet Burbank were identified and should be useful material to integrate in breeding programs which aim to enhance the nutritional value of potato. Primitive cultivars and wild species with widely different amounts of thiamin and folate will also be valuable tools to explore their respective metabolic regulation.

[Subtractive gene cloning and gene-disruption for elucidation of pseudohyphal formation in Candida tropicalis].

The dimorphic transition from yeast to pseudohyphae in the petroleum-assimilating yeast Candida tropicalis occurs following the addition of ethanol to glucose semi-defined medium. Subtractive gene cloning was performed on the cDNA from the yeast-growing control culture and on that from the ethanol-supplemented one (the ethanol culture). A homologue of Schizosaccharomyces pombe nmt1+ or Saccharomyces cerevisiae THI5 was isolated from the cDNA fraction as a preferentially expressed gene for the ethanol culture. This homologue was tentatively called Ctnmt1+, since exogenous thiamine repressed its expression in C. tropicalis growth media. The ethanol culture showed a biphasic pattern of growth phases and the expression of Ctnmt1+ occurred at the first growth phase. The supplementation of thiamine to the ethanol culture at the first phase was followed by repression of Ctnmt1+ expression and also delay of pseudohyphal growth: filamentous growth was inhibited and chains of yeast cells were formed. A Ctnmt1+ disruptant of this organism did not show thiamine auxotrophy and produced pseudohyphal filaments even in the control culture. The supplementation of oxythiamine, an analog of thiamine, to the control culture was followed by the appearance of pseudohyphal filaments, indicating the participation of thiamine during the process of pseudohyphal growth in this organism.

Defective high-affinity thiamine transporter leads to cell death in thiamine-responsive megaloblastic anemia syndrome fibroblasts.

We have investigated the cellular pathology of the syndrome called thiamine-responsive megaloblastic anemia (TRMA) with diabetes and deafness. Cultured diploid fibroblasts were grown in thiamine-free medium and dialyzed serum. Normal fibroblasts survived indefinitely without supplemental thiamine, whereas patient cells died in 5-14 days (mean 9.5 days), and heterozygous cells survived for more than 30 days. TRMA fibroblasts were rescued from death with 10-30 nM thiamine (in the range of normal plasma thiamine concentrations). Positive terminal deoxynucleotide transferase-mediated dUTP nick end-labeling (TUNEL) staining suggested that cell death was due to apoptosis. We assessed cellular uptake of [3H]thiamine at submicromolar concentrations. Normal fibroblasts exhibited saturable, high-affinity thiamine uptake (Km 400-550 nM; Vmax 11 pmol/min/10(6) cells) in addition to a low-affinity unsaturable component. Mutant cells lacked detectable high-affinity uptake. At 30 nM thiamine, the rate of uptake of thiamine by TRMA fibroblasts was 10-fold less than that of wild-type, and cells from obligate heterozygotes had an intermediate phenotype. Transfection of TRMA fibroblasts with the yeast thiamine transporter gene THI10 prevented cell death when cells were grown in the absence of supplemental thiamine. We therefore propose that the primary abnormality in TRMA is absence of a high-affinity thiamine transporter and that low intracellular thiamine concentrations in the mutant cells cause biochemical abnormalities that lead to apoptotic cell death.