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Ellis J Neufeld - One of the best experts on this subject based on the ideXlab platform.
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Systematic Molecular Genetic Analysis of Congenital Sideroblastic Anemia: Evidence for Genetic Heterogeneity and Identification of Novel Mutations
Pediatric Blood & Cancer, 2009Co-Authors: Anke K. Bergmann, Mark D Fleming, Dean R. Campagna, Erin M. Mcloughlin, Suneet Agarwal, Sylvia S. Bottomley, Ellis J NeufeldAbstract:Background Sideroblastic anemias are heterogeneous congenital and acquired bone marrow disorders characterized by pathologic iron deposits in mitochondria of erythroid precursors. Among the congenital sideroblastic anemias (CSAs), the most common form is X-linked sideroblastic anemia, due to mutations in 5-aminolevulinate synthase (ALAS2). A novel autosomal recessive CSA, caused by mutations in the erythroid specific mitochondrial Transporter SLC25A38, was recently defined. Other known etiologies include mutations in genes encoding the Thiamine Transporter (SLC19A2), the RNA-modifying enzyme pseudouridine synthase 1 (PUS1), a mitochondrial ATP-binding cassette Transporter (ABCB7), glutaredoxin 5 (GLRX5), as well as mitochondrial DNA deletions. Despite these known diverse causes, in a substantial portion of CSA cases a presumed genetic defect remains unknown.
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role of defective high affinity Thiamine Transporter slc19a2 in marrow from a mouse model of Thiamine responsive anemia syndrome evidence for defective deoxyribose and heme synthesis
Blood, 2005Co-Authors: Elena Tartaglini, Inderneel Sahai, Claudia M Montefusco, Judidth C Fleming, Laszlo G Boros, Gretchen Chick, Ellis J NeufeldAbstract:The rare autosomal recessive human disorder, TRMA syndrome (Thiamine-responsive anemia with diabetes and deafness, OMIM 249270), is caused by mutations in the high affinity vitamin B1 Transporter, SLC19A2. In human TRMA, ringed sideroblasts and megaloblastic changes are found in the marrow, suggesting potential defects in heme synthesis or mitochondrial function, and DNA synthesis, respectively. The mechanism whereby defective marrow Thiamine transport causes these findings in the marrow was examined in a mouse model for TRMA. We previously demonstrated in fibroblasts from humans with TRMA a defect in the rate and pathway used for de novo ribose synthesis using Stable Isotope Dynamic Metabolic Profiling (SIDMaP analysis - Boros et al, Blood102: 3556, 2003). To examine whether this fibroblast defect accurately defined the marrow pathophysiology, we have now examined this pathway in detail in mice defective for the orthologous slc19a2 gene by targeted gene disruption (Fleming et al, Mol Genet Metab80:234, 2003). Slc19a2 mutant (−/−) or wildtype (+/+) mice were subjected to normal mouse chow (22 mg/kg Thiamine), or Thiamine deficient chow diet. Diets were confirmed Thiamine deficient by chemical analysis. To examine heme biosynthesis, we used 55 Fe-transferrin, ex vivo , to label marrow from +/+ or −/− mice maintained for 10 days in Thiamine replete or deficient chow. Mutant mice had cell surface labeling indistinguishable from +/+ at 0°C (P>0.3), reflecting available transferrin receptors. Total cellular 55 Fe uptake at 37 C was equal to or slightly greater than that of +/+ marrow in Thiamine depleted state. However, incorporation of 55 Fe into heme lagged in −/− cells (7.3 +/− 0.4 pmol/10 6 cells/7 min) compared to +/+ (8.6 +/− 0.5 pmol/10 6 cells, P =0.018 in triplicate experiments). To examine deoxyribose (dRibose) synthesis, mice were maintained on Thiamine replete or depleted chow for 10 days, then [1,2 13 C] glucose was administered intraperitoneally overnight and 3 hours before sacrifice and marrow harvest. DNA from whole marrow and from sorted basophilic erythroblasts (CD71+/Ter119+) was isolated, hydrolized, derivatized, and dRibose analyzed by GC-MS. The initial rate of incorporation of 13 C into DNA dRibose, reflecting de novo synthetic rate, was reduced 34% in basophilic erythroblasts of −/− mice after Thiamine depletion (n=3 in duplicate experiments, P slc9a2 leads to two distinct erythropoietic defects. De novo DNA dRibose synthesis via transketolase is markedly impaired. Further, while iron uptake by marrow cells is maintained at normal levels, the heme biosynthetic rate is decreased in −/− mice in Thiamine-depleted status. This is the first direct demonstration of a role for Thiamine in normal erythrocyte heme and DNA metabolism. Together, these defects can account directly for the meglaoblastosis and sideroblasts of human SLC19A2 defects in TRMA.
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male infertility and Thiamine dependent erythroid hypoplasia in mice lacking Thiamine Transporter slc19a2
Molecular Genetics and Metabolism, 2003Co-Authors: Judith C Fleming, Elena Tartaglini, Ryosuke Kawatsuji, Mark D Fleming, Yuko Fujiwara, Jeffrey J Bednarski, Ellis J NeufeldAbstract:Abstract Thiamine-responsive megaloblastic anemia with diabetes and deafness (TRMA) is an autosomal recessive disease caused by mutations in the high-affinity Thiamine Transporter gene SLC19A2. To study the role of Thiamine transport in the pathophysiology of TRMA syndrome and of each of the component disorders, we created a targeted disruption of the Slc19a2 gene in mice. Slc19a2 −/− mice are viable and females are fertile. Male −/− mice on a pure 129/Sv background are infertile with small testes (testis/body weight=0.13 ± 0.04 knockout vs. 0.35 ± 0.05 wild type, P
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characterization of a murine high affinity Thiamine Transporter slc19a2
Molecular Genetics and Metabolism, 2001Co-Authors: Judith C Fleming, Elena Tartaglini, Mara P Steinkamp, Ryosuke Kawatsuji, Jack L Pinkus, Geraldine S Pinkus, Mark D Fleming, Ellis J NeufeldAbstract:Abstract Thiamine-responsive megaloblastic anemia with deafness and diabetes (TRMA) is a rare autosomal recessive disorder of Thiamine transport. Previous studies have demonstrated that the disease is caused by mutations in the SLC19A2 gene encoding a high-affinity Thiamine Transporter. We hypothesize that Thiamine transport, mediated by SLC19A2, plays a role in the development and or maintenance of several organ systems, in particular the erythropoietic, auditory, and glucose homeostasis systems. To investigate the Transporter further, we cloned the murine Slc19a2 locus and characterized the resulting protein. Murine Slc19a2 is a 498 amino acid protein, with 12 predicted transmembrane domains. The gene spans ∼13kb with 6 exons, structurally identical to that of the human homolog. We localized the Slc19a2 gene to mouse chromosome 1, a region syntenic to human chromosome 1q23 that contains the TRMA locus. Transient expression of Slc19a2 in HEK293T cells resulted in specific uptake of [ 3 H] Thiamine, confirming a Thiamine Transporter function. Western blot analysis of mouse tissues reveals a wide distribution of Slc19a2 protein. Immunohistochemistry studies indicate that Slc19a2 is expressed on the cell surface and intracellularly, and is specifically localized to a subpopulation of cells in cochlea, small intestine, and pancreas.
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the gene mutated in Thiamine responsive anaemia with diabetes and deafness trma encodes a functional Thiamine Transporter
Nature Genetics, 1999Co-Authors: Judith C Fleming, Elena Tartaglini, Mara P Steinkamp, Daniel F Schorderet, Nadine Cohen, Ellis J NeufeldAbstract:Thiamine-responsive megaloblastic anaemia with diabetes and deafness1 (TRMA; MIM 249270) is an autosomal recessive disease thought to be due to a defect in Thiamine (vitamin B1) transport2,3. Pharmacological doses of Thiamine correct the anaemia, and in some cases improve the diabetes, although progressive sensorineural deafness is irreversible4. Previous studies localized the TRMA gene to a 4-cM region on chromosome 1q23.3 (ref. 5), and fine-mapping has recently narrowed that region further6,7. We have previously demonstrated that fibroblasts from people with TRMA lack high-affinity Thiamine transport8. Expression of a gene encoding a known yeast Thiamine Transporter, THI10 (refs 8,9,10), in TRMA mutant cells prevents apoptotic cell death in Thiamine-depleted medium. On the basis of these studies, we hypothesized that a defective Thiamine Transporter causes TRMA. We undertook a candidate gene approach to identify putative Thiamine Transporters in the 1q23.3 critical region. Here we present evidence that the gene SLC19A2 (for solute carrier family 19 (Thiamine Transporter), member 2) encodes the first known mammalian Thiamine Transporter, which we designate Thiamine Transporter-1 (THTR-1).
Hamid M Said - One of the best experts on this subject based on the ideXlab platform.
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Novel nonsense mutation (p.Ile411Metfs*12) in the SLC19A2 gene causing Thiamine Responsive Megaloblastic Anemia in an Indian patient.
Clinica Chimica Acta, 2015Co-Authors: Paramasivam Manimaran, Hamid M Said, Veedamali S Subramanian, Sellamuthu Karthi, Krishnan Gandhimathi, Perumal Varalakshmi, Ramasamy Ganesh, Andiappan Rathinavel, Balasubramaniem AshokkumarAbstract:Thiamine-responsive megaloblastic anemia (TRMA), an autosomal recessive disorder, is caused by mutations in SLC19A2 gene encodes a high affinity Thiamine Transporter (THTR-1). The occurrence of TRMA is diagnosed by megaloblastic anemia, diabetes mellitus, and sensorineural deafness. Here, we report a female TRMA patient of Indian descent born to 4th degree consanguineous parents presented with retinitis pigmentosa and vision impairment, who had a novel homozygous mutation (c.1232delT/ter422; p.Ile411Metfs*12) in 5th exon of SLC19A2 gene that causes premature termination of hTHTR-1. PROSITE analysis predicted to abrogate GPCRs family-1 signature motif in the variant by this mutation c.1232delT/ter422, suggesting uncharacteristic rhodopsin function leading to cause RP clinically. Thiamine transport activity by the clinical variant was severely inhibited than wild-type THTR-1. Confocal imaging had shown that the variant p.I411Mfs*12 is targeted to the cell membrane and showed no discrepancy in membrane expression than wild-type. Our findings are the first report, to the best of our knowledge, on this novel nonsense mutation of hTHTR-1 causing TRMA in an Indian patient through functionally impaired Thiamine Transporter activity.
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association of tm4sf4 with the human Thiamine Transporter 2 in intestinal epithelial cells
Digestive Diseases and Sciences, 2014Co-Authors: Veedamali S Subramanian, Svetlana M Nabokina, Hamid M SaidAbstract:Background The human Thiamine Transporter-2 (hTHTR-2) is involved in the intestinal absorption of Thiamine. Recent studies with membrane Transporters of other nutrients/substrates have shown that they have associated proteins that affect different aspects of their physiology and cell biology. Nothing is known about protein(s) that interact with hTHTR-2 in intestinal epithelial cells and influence its physiological function and/or its cell biology.
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Adaptive regulation of human intestinal Thiamine uptake by extracellular substrate level: a role for THTR-2 transcriptional regulation
American Journal of Physiology-gastrointestinal and Liver Physiology, 2013Co-Authors: Svetlana M Nabokina, Veedamali S Subramanian, Judith E. Valle, Hamid M SaidAbstract:The intestinal Thiamine uptake process is adaptively regulated by the level of vitamin in the diet, but the molecular mechanism involved is not fully understood. Here we used the human intestinal epithelial Caco-2 cells exposed to different levels of extracellular Thiamine to delineate the molecular mechanism involved. Our results showed that maintaining Caco-2 cells in a Thiamine-deficient medium resulted in a specific and significant increase of [3H]Thiamine uptake compared with cell exposure to a high level of Thiamine (1 mM). This adaptive regulation was also associated with a higher level of mRNA expression of Thiamine Transporter-2 (THTR-2), but not Thiamine Transporter-1 (THTR-1), in the deficient condition and a higher level of promoter activity of gene encoding THTR-2 (SLC19A3). Using 5′-truncated promoter-luciferase constructs, we identified the Thiamine level-responsive region in the SLC19A3 promoter to be between −77 and −29 (using transcriptional start site as +1). By means of mutational analysis, a key role for a stimulating protein-1 (SP1)/guanosine cytidine box in mediating the effect of extracellular Thiamine level on SLC19A3 promoter was established. Furthermore, extracellular level of Thiamine was found to affect SP1 protein expression and binding pattern to the Thiamine level-responsive region of SLC19A3 promoter in Caco-2 cells as shown by Western blotting and electrophoretic mobility shift assay analysis, respectively. These studies demonstrate that the human intestinal Thiamine uptake is adaptively regulated by the extracellular substrate level via transcriptional regulation of the THTR-2 system, and report that SP1 transcriptional factor is involved in this regulation.
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tspan 1 interacts with the Thiamine Transporter 1 in human intestinal epithelial cells and modulates its stability
American Journal of Physiology-gastrointestinal and Liver Physiology, 2011Co-Authors: Svetlana M Nabokina, Sundar Rajan Senthilkumar, Hamid M SaidAbstract:The human Thiamine Transporter-1 (hTHTR-1) contributes to intestinal Thiamine uptake, and its function is regulated at both the transcriptional and posttranscriptional levels. Nothing, however, is known about the protein(s) that may interact with hTHTR-1 and affects its cell biology and physiology. We addressed this issue in the present investigation using a bacterial two-hybrid system to screen a human intestinal cDNA library with the complete coding sequence of hTHTR-1 as a bait. Our results showed that a member of the tetraspanin family of proteins, Tspan-1, interacts with hTHTR-1. Coimmunoprecipitation and glutathione S-transferase (GST)-pulldown assays confirmed the existence of such an interaction between hTspan-1 and hTHTR-1 in human intestinal epithelial Caco-2 cells. Furthermore, live cell confocal imaging demonstrated that hTspan-1 and hTHTR-1 colocalize in human intestinal epithelial HuTu-80 cells. The importance of the interaction between hTspan-1 and hTHTR-1 for cell biology of the Thiamine Transporter was examined in HuTu-80 cells stably expressing hTHTR-1. Coexpression of hTspan-1 in these cells led to a significant decrease in the rate of degradation of hTHTR-1 compared with cells expressing the hTHTR-1 alone; in fact the half-life of the hTHTR-1 protein was twice longer in the former cell type compared with the latter cell type (12 h vs. 6 h, respectively). This finding was also confirmed at the functional level when a significantly higher Thiamine uptake was observed in cycloheximide-treated (6 h) cells expressing hTHTR-1 together with hTspan-1 compared with those expressing hTHTR-1 alone. These studies demonstrate for the first time that Tspan-1 is an interacting partner with hTHTR-1 and that this interaction affects hTHTR-1 stability.
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Targeting and intracellular trafficking of clinically relevant hTHTR1 mutations in human cell lines.
Clinical Science, 2007Co-Authors: Veedamali S Subramanian, Jonathan S Marchant, Hamid M SaidAbstract:The micronutrient Thiamine is required for normal growth and development of human tissues, and is accumulated into cells through the activity of plasma membrane Thiamine Transporters, e.g. hTHTR1 (human Thiamine Transporter 1). Recent genetic evidence has linked mutations in hTHTR1 with the manifestation of TRMA (Thiamine-responsive megaloblastic anaemia), a condition also associated with diabetes mellitus, sensorineural deafness and retinal disorders. To examine how mutations in hTHTR1 impair Thiamine accumulation, we have investigated the targeting and functional properties of several different hTHTR1 mutants in human cell lines derived from epithelia relevant to Thiamine absorption or tissues implicated in TRMA pathology. These constructs encompassed two newly identified point mutations (P51L and T158R) and two truncations of hTHTR1 identical with those found in TRMA kindreds (W358X and Δ383fs). Our results reveal a spectrum of mutant phenotypes, underlining that TRMA can result from decreased Thiamine transport activity underpinned by changes in hTHTR1 expression levels, cellular targeting and/or protein transport activity.
Judith C Fleming - One of the best experts on this subject based on the ideXlab platform.
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male infertility and Thiamine dependent erythroid hypoplasia in mice lacking Thiamine Transporter slc19a2
Molecular Genetics and Metabolism, 2003Co-Authors: Judith C Fleming, Elena Tartaglini, Ryosuke Kawatsuji, Mark D Fleming, Yuko Fujiwara, Jeffrey J Bednarski, Ellis J NeufeldAbstract:Abstract Thiamine-responsive megaloblastic anemia with diabetes and deafness (TRMA) is an autosomal recessive disease caused by mutations in the high-affinity Thiamine Transporter gene SLC19A2. To study the role of Thiamine transport in the pathophysiology of TRMA syndrome and of each of the component disorders, we created a targeted disruption of the Slc19a2 gene in mice. Slc19a2 −/− mice are viable and females are fertile. Male −/− mice on a pure 129/Sv background are infertile with small testes (testis/body weight=0.13 ± 0.04 knockout vs. 0.35 ± 0.05 wild type, P
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characterization of a murine high affinity Thiamine Transporter slc19a2
Molecular Genetics and Metabolism, 2001Co-Authors: Judith C Fleming, Elena Tartaglini, Mara P Steinkamp, Ryosuke Kawatsuji, Jack L Pinkus, Geraldine S Pinkus, Mark D Fleming, Ellis J NeufeldAbstract:Abstract Thiamine-responsive megaloblastic anemia with deafness and diabetes (TRMA) is a rare autosomal recessive disorder of Thiamine transport. Previous studies have demonstrated that the disease is caused by mutations in the SLC19A2 gene encoding a high-affinity Thiamine Transporter. We hypothesize that Thiamine transport, mediated by SLC19A2, plays a role in the development and or maintenance of several organ systems, in particular the erythropoietic, auditory, and glucose homeostasis systems. To investigate the Transporter further, we cloned the murine Slc19a2 locus and characterized the resulting protein. Murine Slc19a2 is a 498 amino acid protein, with 12 predicted transmembrane domains. The gene spans ∼13kb with 6 exons, structurally identical to that of the human homolog. We localized the Slc19a2 gene to mouse chromosome 1, a region syntenic to human chromosome 1q23 that contains the TRMA locus. Transient expression of Slc19a2 in HEK293T cells resulted in specific uptake of [ 3 H] Thiamine, confirming a Thiamine Transporter function. Western blot analysis of mouse tissues reveals a wide distribution of Slc19a2 protein. Immunohistochemistry studies indicate that Slc19a2 is expressed on the cell surface and intracellularly, and is specifically localized to a subpopulation of cells in cochlea, small intestine, and pancreas.
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A novel mutation in the SLC19A2 gene in a Tunisian family with Thiamine-responsive megaloblastic anaemia, diabetes and deafness syndrome
British Journal of Haematology, 2001Co-Authors: Sami Gritli, Judith C Fleming, Elena Tartaglini, Mara P Steinkamp, Souheil Omar, Souha Guannouni, Charles I. Berul, Raouf Hafsia, Sarrah Baltagi-ben Jilani, Ali BelhaniAbstract:Thiamine-responsive megaloblastic anaemia (TRMA) syndrome with diabetes and deafness was found in two patients from a Tunisian kindred. The proband was homozygous for a novel mutation, 287delG, in the high-affinity Thiamine Transporter gene, SLC19A2. We demonstrated that fibroblasts from this patient exhibited defective Thiamine transport. These data confirm that the SLC19A2 gene is the high-affinity Thiamine carrier and that this novel mutation is responsible for TRMA syndrome.
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the gene mutated in Thiamine responsive anaemia with diabetes and deafness trma encodes a functional Thiamine Transporter
Nature Genetics, 1999Co-Authors: Judith C Fleming, Elena Tartaglini, Mara P Steinkamp, Daniel F Schorderet, Nadine Cohen, Ellis J NeufeldAbstract:Thiamine-responsive megaloblastic anaemia with diabetes and deafness1 (TRMA; MIM 249270) is an autosomal recessive disease thought to be due to a defect in Thiamine (vitamin B1) transport2,3. Pharmacological doses of Thiamine correct the anaemia, and in some cases improve the diabetes, although progressive sensorineural deafness is irreversible4. Previous studies localized the TRMA gene to a 4-cM region on chromosome 1q23.3 (ref. 5), and fine-mapping has recently narrowed that region further6,7. We have previously demonstrated that fibroblasts from people with TRMA lack high-affinity Thiamine transport8. Expression of a gene encoding a known yeast Thiamine Transporter, THI10 (refs 8,9,10), in TRMA mutant cells prevents apoptotic cell death in Thiamine-depleted medium. On the basis of these studies, we hypothesized that a defective Thiamine Transporter causes TRMA. We undertook a candidate gene approach to identify putative Thiamine Transporters in the 1q23.3 critical region. Here we present evidence that the gene SLC19A2 (for solute carrier family 19 (Thiamine Transporter), member 2) encodes the first known mammalian Thiamine Transporter, which we designate Thiamine Transporter-1 (THTR-1).
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defective high affinity Thiamine Transporter leads to cell death in Thiamine responsive megaloblastic anemia syndrome fibroblasts
Journal of Clinical Investigation, 1999Co-Authors: Amy Stagg, Judith C Fleming, Nadine Cohen, Meghan A Baker, Massayuki Sakamoto, Ellis J NeufeldAbstract: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/106 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. J. Clin. Invest. 103:723–729 (1999).
Rafael Artuch - One of the best experts on this subject based on the ideXlab platform.
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op6 2631 decreased free Thiamine in cerebro spinal fluid and fibroblasts is a sensitive marker of Thiamine Transporter 2 deficiency in leigh syndrome patients
European Journal of Paediatric Neurology, 2015Co-Authors: J Ortigoza D Escobar, Mercedes Serrano, Marta Moleroluis, Angela Arias, Niklas Darin, Mireia Tondo, Mercedes Casado, J A Mayr, A Ribes, Rafael ArtuchAbstract:Objectives Thiamine Transporter-2 (hTHTR2) deficiency due to SLC19A3 mutations is a potentially reversible cause of Leigh syndrome for which no biochemical markers are currently available. Our aim was to assess the sensitivity of Thiamine quantification in cerebrospinal fluid (CSF) and fibroblasts from patients with Leigh encephalopathy and SLC19A3 defects as compared with other causes of Leigh syndrome. Methods Thiamine vitamers (free-Thiamine, Thiamine monophosphate (TMP) and Thiamine diphosphate (TDP)) were analyzed by HPLC-fluorescence detection in whole-blood and cerebrospinal fluid (CSF) samples from 106 and 38 paediatric controls, respectively. Results were compared with patients with Leigh syndrome due to SLC19A3 defects (N=6) and mitochondrial respiratory chain defects (N=9). In all but one SLC19A3 patient, samples were collected before Thiamine supplementation. Thiamine vitamers were also analyzed by HPLC in fibroblasts from SLC19A3 patients (N=3) and patients with other metabolic defects (N=6). Results A negative correlation between Thiamine isoforms and age was detected in whole-blood and CSF, thus three reference intervals were established for free-Thiamine and two intervals for TMP and TDP. Free-Thiamine was severely reduced in five non-treated SLC19A3 patients CSF, but not TMP and TPP. The SLC19A3 patient under Thiamine supplementation showed Thiamine values above reference range. Nine Leigh patients with mitochondrial defects showed normal o slightly reduced values for CSF Thiamine. In SLC19A3 patient's fibroblasts, a reduction in free-Thiamine was detected as compared with control sample. These values normalized after Thiamine was added to the culture medium. Conclusion SLC19A3 patients show a profound deficiency of free-Thiamine in the CSF that allows their identification from other causes of Leigh syndrome. SLC19A3 is essential to maintain CSF Thiamine homeostasis and to prevent brain damage. Thiamine overload can supply the SLC19A3 defect and restore Thiamine values in fibroblasts and CSF, probably by an alternative transport system.
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OP6 – 2631: Decreased free-Thiamine in cerebro spinal fluid and fibroblasts is a sensitive marker of Thiamine Transporter 2 deficiency in Leigh syndrome patients
European Journal of Paediatric Neurology, 2015Co-Authors: J.d. Ortigoza Escobar, Mercedes Serrano, Angela Arias, Niklas Darin, Mireia Tondo, Marta Molero-luis, Mercedes Casado, J A Mayr, A Ribes, Rafael ArtuchAbstract:Objectives Thiamine Transporter-2 (hTHTR2) deficiency due to SLC19A3 mutations is a potentially reversible cause of Leigh syndrome for which no biochemical markers are currently available. Our aim was to assess the sensitivity of Thiamine quantification in cerebrospinal fluid (CSF) and fibroblasts from patients with Leigh encephalopathy and SLC19A3 defects as compared with other causes of Leigh syndrome. Methods Thiamine vitamers (free-Thiamine, Thiamine monophosphate (TMP) and Thiamine diphosphate (TDP)) were analyzed by HPLC-fluorescence detection in whole-blood and cerebrospinal fluid (CSF) samples from 106 and 38 paediatric controls, respectively. Results were compared with patients with Leigh syndrome due to SLC19A3 defects (N=6) and mitochondrial respiratory chain defects (N=9). In all but one SLC19A3 patient, samples were collected before Thiamine supplementation. Thiamine vitamers were also analyzed by HPLC in fibroblasts from SLC19A3 patients (N=3) and patients with other metabolic defects (N=6). Results A negative correlation between Thiamine isoforms and age was detected in whole-blood and CSF, thus three reference intervals were established for free-Thiamine and two intervals for TMP and TDP. Free-Thiamine was severely reduced in five non-treated SLC19A3 patients CSF, but not TMP and TPP. The SLC19A3 patient under Thiamine supplementation showed Thiamine values above reference range. Nine Leigh patients with mitochondrial defects showed normal o slightly reduced values for CSF Thiamine. In SLC19A3 patient's fibroblasts, a reduction in free-Thiamine was detected as compared with control sample. These values normalized after Thiamine was added to the culture medium. Conclusion SLC19A3 patients show a profound deficiency of free-Thiamine in the CSF that allows their identification from other causes of Leigh syndrome. SLC19A3 is essential to maintain CSF Thiamine homeostasis and to prevent brain damage. Thiamine overload can supply the SLC19A3 defect and restore Thiamine values in fibroblasts and CSF, probably by an alternative transport system.
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Thiamine Transporter 2 deficiency outcome and treatment monitoring
Orphanet Journal of Rare Diseases, 2014Co-Authors: Juan Dario Ortigozaescobar, Mercedes Serrano, Monica Rebollo, Jordi Muchart, Rafael Artuch, Alfonso Oyarzabal, Marta Molero, Pilar Rodriguezpombo, Belen PerezduenasAbstract:Background: The clinical characteristics distinguishing treatable Thiamine Transporter-2 deficiency (ThTR2) due to SLC19A3 genetic defects from the other devastating causes of Leigh syndrome are sparse. Methods: We report the clinical follow-up after Thiamine and biotin supplementation in four children with ThTR2 deficiency presenting with Leigh and biotin-Thiamine-responsive basal ganglia disease phenotypes. We established whole-blood Thiamine reference values in 106 non-neurological affected children and monitored Thiamine levels in SLC19A3 patients after the initiation of treatment. We compared our results with those of 69 patients with ThTR2 deficiency after a review of the literature. Results: At diagnosis, the patients were aged 1 month to 17 years, and all of them showed signs of acute encephalopathy, generalized dystonia, and brain lesions affecting the dorsal striatum and medial thalami. One patient died of septicemia, while the remaining patients evidenced clinical and radiological improvements shortly after the initiation of Thiamine. Upon follow-up, the patients received a combination of Thiamine (10–40 mg/kg/day) and biotin (1–2 mg/kg/day) and remained stable with residual dystonia and speech difficulties. After establishing reference values for the different age groups, whole-blood Thiamine quantification was a useful method for treatment monitoring. Conclusions: ThTR2 deficiency is a reversible cause of acute dystonia and Leigh encephalopathy in the pediatric years. Brain lesions affecting the dorsal striatum and medial thalami may be useful in the differential diagnosis of other causes of Leigh syndrome. Further studies are needed to validate the therapeutic doses of Thiamine and how to monitor them in these patients.
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reversible lactic acidosis in a newborn with Thiamine Transporter 2 deficiency
Pediatrics, 2013Co-Authors: Belen Perezduenas, Mercedes Serrano, Monica Rebollo, Jordi Muchart, Eva Gargallo, Celine Dupuits, Rafael ArtuchAbstract:Thiamine Transporter-2 deficiency is a recessive disease caused by mutations in the SLC19A3 gene. Patients manifest acute episodes of encephalopathy; symmetric lesions in the cortex, basal ganglia, thalami or periaqueductal gray matter, and a dramatic response to biotin or Thiamine. We report a 30-day-old patient with mutations in the SLC19A3 gene who presented with acute encephalopathy and increased level of lactate in the blood (8.6 mmol/L) and cerebrospinal fluid (7.12 mmol/L), a high excretion of α-ketoglutarate in the urine, and increased concentrations of the branched-chain amino acids leucine and isoleucine in the plasma. MRI detected bilateral and symmetric cortico-subcortical lesions involving the perirolandic area, bilateral putamina, and medial thalami. Some lesions showed low apparent diffusion coefficient values suggesting an acute evolution; others had high values likely to be subacute or chronic, most likely related to the perinatal period. After treatment with Thiamine and biotin, irritability and opisthotonus disappeared, and the patient recovered consciousness. Biochemical disturbances also disappeared within 48 hours. After discontinuing biotin, the patient remained stable for 6 months on Thiamine supplementation (20 mg/kg/day). The examination revealed subtle signs of neurologic sequelae, and MRI showed necrotic changes and volume loss in some affected areas. Our observations suggest that patients with Thiamine Transporter 2 deficiency may be vulnerable to metabolic decompensation during the perinatal period, when energy demands are high. Thiamine defects should be excluded in newborns and infants with lactic acidosis because prognosis largely depends on the time from diagnosis to Thiamine supplementation. * Abbreviations: ADC — : apparent diffusion coefficient hTHTR1 — : Thiamine Transporter 1 hTHTR2 — : Thiamine Transporter 2 RV — : reference values
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reversible generalized dystonia and encephalopathy from Thiamine Transporter 2 deficiency
Movement Disorders, 2012Co-Authors: Mercedes Serrano, Monica Rebollo, Jordi Muchart, Christel Depienne, Agnes Rastetter, Emilio Fernandezalvarez, Loreto Martorell, Rafael ArtuchAbstract:Background: Thiamine Transporter-2 deficiency, a condition resulting from mutations in the SLC19A3 gene, has been described in patients with subacute dystonia and striatal necrosis. The condition responds extremely well to treatment with biotin and has thus been named biotin-responsive basal ganglia disease. Recently, this deficiency has also been related to Wernicke's-like encephalopathy and atypical infantile spasms, showing heterogeneous responses to biotin and/or Thiamine. Methods: Two Spanish siblings with a biotin-responsive basal ganglia disease phenotype and mutations in SLC19A3 presented with acute episodes of generalized dystonia, rigidity, and symmetrical lesions involving the striatum, midline nuclei of the thalami, and the cortex of cerebral hemispheres as shown by magnetic resonance imaging. Results: The clinical features resolved rapidly after Thiamine administration. Conclusions: Despite the rarity of Thiamine Transporter–2 deficiency, it should be suspected in patients with acute dystonia and basal ganglia injury, as Thiamine can halt disease evolution and prevent further episodes. © 2012 Movement Disorder Society
Jeffrey A Moscow - One of the best experts on this subject based on the ideXlab platform.
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Can an enzyme cofactor be a factor in malignant progression
Cancer Biology & Therapy, 2010Co-Authors: Adam D. Richardson, Jeffrey A MoscowAbstract:Commentary to: Hypoxia induced up-regulation and function of the Thiamine Transporter, SLC19A3 in a breast cancer cell line Rebecca Sweet, Amber Paul and Jason Zastre
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Sensitivity of breast cancer cell lines to recombinant thiaminase I
Cancer Chemotherapy and Pharmacology, 2009Co-Authors: Noel R. Monks, Jeremiah W. Hanes, Tadhg P. Begley, Hui Yu, Jeffrey A MoscowAbstract:Purpose We have previously shown that the expression of the Thiamine Transporter THTR2 is decreased sevenfold in breast cancer, which may leave breast cancer cells vulnerable to acute Thiamine starvation. This concept was supported by the observation that MDA231 breast cancer xenografts demonstrated growth inhibition in mice fed a Thiamine-free diet.
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Thiamine Transporter gene expression and exogenous Thiamine modulate the expression of genes involved in drug and prostaglandin metabolism in breast cancer cells
Molecular Cancer Research, 2004Co-Authors: Arnold J Stromberg, Jeffrey A MoscowAbstract:In previous studies, we have shown that RNA levels of the Thiamine Transporter THTR2 were down-regulated in breast cancer tumors in comparison with normal tissues and that THTR2-mediated increases in Thiamine uptake activity contributed to increased apoptosis after exposure to ionizing radiation. To further understand the biological effects of the alteration of THTR2 expression, we conducted a DNA microarray study of gene expression in THTR2-transfected breast cancer cells and found that, in addition to increased expression of THTR2 attributable to the transgene, three other genes were up-regulated >2.5-fold in the transfected cells: cytochrome P450 isoform CYP4B1, 15-hydroxyprostaglandin dehydrogenase (15-PGDH), and transcription factor CRIP1. In addition, two genes were confirmed to be down-regulated in THTR2-transfected cells: trefoil factor 1 (TFF1) and Rho-GDP dissociation inhibitor (RGDI). Up-regulation of 15-PGDH and CYP4B1 expression was observed in other breast cancer cell lines transfected with THTR2, and down-regulation was observed after suppression of THTR2 with siRNA vectors. To determine the role of exogenous Thiamine in the expression of these genes, we analyzed THTR2-transfected breast cancer cells grown in Thiamine-depleted medium by quantitative reverse transcription-PCR and showed that three of these five genes showed evidence of regulation by exogenous Thiamine in a manner concordant with the effects of THTR2 overexpression. One of the genes up-regulated by THTR2 transfection was down-regulated by Thiamine depletion (CYP4B1), and two genes with decreased expression in THTR2-transfected breast cancer cells were up-regulated by Thiamine depletion (TFF1 and RGDI). In summary, these studies show unexpected relationships between Thiamine metabolism and genes that may be involved in the oncogenesis of breast and lung cancer.
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Thiamine Transporter gene expression and exogenous Thiamine modulate expression of genes in breast and lung cancers
Cancer Research, 2004Co-Authors: Arnold J Stromberg, Jeffrey A MoscowAbstract:1378 Eukaryotic cells possess specific mechanisms for the uptake of water-soluble vitamins, including folate and Thiamine. In previous studies we found that the RNA expression of the Thiamine Transporter THTR2 (SLC19A3) was down-regulated in breast and lung tumors in comparison to non-malignant surrounding tissue. To determine whether down-regulation of THTR2 expression may influence cancer progression, we analyzed gene expression in THTR2-transfected breast cancer cell line (MTXRZR75) compared to mock-transfected cells using a DNA microarray (Affymetrix). We found that cytochrome P450 isoform CYP4B1was 4-fold increased and 15-prostaglandin dehydrogenase (15-PGDH) was 2.7-fold increased in THTR2-transfected cells compared to control cells. Trefoil factor 1 (TFF1) was 2.6-fold decreased in THTR2-transfected cells. The microarray results were confirmed by quantitative RT PCR and western blot. To determine whether THTR2 expression had similar effects in other cell lines, we transfected THTR2 into MCF-7 and MDA-231 breast cancer cell lines. CYP4B1 RNA levels were 2.4-fold increased in THTR2-transfected MCF-7 cells compared to control cells. 15-PGDH RNA levels were 2.2-fold and 3.5-fold increased in transfected MCF-7 and MDA231cells, respectively. MDA231 cells did not have detectable expression of CYP4B1, so changes in its expression could not be measured. In addition, MDA231 cells also showed a 6.7-fold decrease in TFF1 gene expression after THTR2 transfection. To determine the role of exogenous Thiamine in the expression of these three genes, we analyzed RNA levels of these genes in these cell lines by growing them in Thiamine-depleted medium and demonstrated that two of three genes showed evidence of regulation by exogenous Thiamine in the direction predicted by THTR2 transfection studies. CYP4B1was down-regulated by Thiamine depletion and TFF1 was up-regulated by Thiamine depletion. 15-PGDH RNA levels were not affected by Thiamine depletion. Time-course studies of the effect of Thiamine depletion and repletion on CYP4B1 and TFF1 show a gradual change in gene expression over an 8-day period. Since 15-PDGH and CYP4B1 are potentially involved in carcinogenesis, we determined whether there was evidence of coordinate regulation of these genes with THTR2 in lung tumors. These studies provide evidence that THTR2, 15-PGDH and CYP4B1 are all down-regulated in lung tumors in comparison to non-malignant surrounding tissue, and that TFF1 is up-regulated in lung tumors. In summary, these studies suggest potential links between Thiamine metabolism and genes that may be involved in the oncogenesis of breast and lung cancer.
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down regulation of Thiamine Transporter thtr2 gene expression in breast cancer and its association with resistance to apoptosis
Molecular Cancer Research, 2003Co-Authors: Hui Huang, Xin Lu, Miroslaw Golinski, Sebastien Comesse, David S Watt, Robert B Grossman, Jeffrey A MoscowAbstract:The recent molecular identification of two Thiamine Transporters, SLC19A2 (THTR1) and SLC19A3 (THTR2), has provided the opportunity to study Thiamine Transporter gene expression in human malignancies. We compared RNA levels of both THTR1 and THTR2 in a panel of human breast tumors and corresponding normal tissues. THTR2 RNA levels were down-regulated in breast cancer to 14% of the level found in corresponding normal tissues, while THTR1 levels were unchanged. Both Thiamine transport genes were cloned and expressed in a breast cancer cell line to examine the impact of reconstituted Thiamine transport gene expression on drug and radiation sensitivity and on resistance to apoptosis. THTR2-transfected breast cancer cells showed a 2.5-fold increase in specific THTR2 activity and a 3-fold increase in cytotoxicity against a bromoacetyl ester derivative of Thiamine. Surprisingly, these cells also showed a 3-fold increase in sensitivity to doxorubicin and an increase in sensitivity to ionizing radiation, but no change in sensitivity to methotrexate or paclitaxel. TUNEL assays demonstrate an increase in apoptosis in THTR2-transfected cells exposed to doxorubicin and radiation, and Western blot analysis suggests that apoptosis associated with these cytotoxic stresses is mediated at least in part by a caspase-3-dependent pathway. Therefore, Thiamine Transporter THTR2 gene expression is down-regulated in breast cancer, which may contribute to resistance to apoptosis in these tumors.