Defective RNA ribose synthesis in fibroblasts from patients with thiamine-responsive megaloblastic anemia (TRMA)

Abstract

Fibroblasts from patients with thiamineresponsive megaloblastic anemia (TRMA) syndrome with diabetes and deafness undergo apoptotic cell death in the absence of supplemental thiamine in their cultures. The basis of megaloblastosis in these patients has not been determined. Here we use the stable [1,2-13 C 2 ]glucose isotope-based dynamic metabolic profiling technique to demonstrate that defective high-affinity thiamine transport primarily affects the synthesis of nucleic acid ribose via the nonoxidative branch of the pentose cycle. RNA ribose isolated from TRMA fibroblasts in thiamine-depleted cultures shows a time-dependent decrease in the fraction of ribose derived via transketolase, a thiamine-dependent enzyme in the pentose cycle. The fractional rate of de novo ribose synthesis from glucose is decreased several fold 2 to 4 days after removal of thiamine from the culture medium. No such metabolic changes are observed in wild-type fibroblasts or in TRMA mutant cells in thiaminecontaining medium. Fluxes through glycolysis are similar in TRMA versus control fibroblasts in the pentose and TCA cycles. We conclude that reduced nucleic acid production through impaired transketolase catalysis is the underlying biochemical disturbance that likely induces cell cycle arrest or apoptosis in bone marrow cells and leads to the TRMA syndrome in patients with defective high-affinity thiamine transport. IntroductionThe pentose cycle, or "pentose-monophosphate shunt," allows de novo synthesis of ribose from glucose by either or both of 2 possible routes: a nonoxidative pathway via transaldolase and transketolase, and an oxidative pathway, via glucose-6-phosphate (G6P) and 6-phosphogluconate dehydrogenase (G6PDH). The oxidative pathway also generates nicotinamide adenine dinucleotide phosphate-positive (NADPH ϩ ) reducing equivalents. Whereas the oxidative branch of the pathway predominates in circulating red blood cells, in many tissues the nonoxidative branch is quantitatively more important for ribose synthesis destined for nucleic acids. 1-3 Thiamine pyrophosphate is a cofactor for transketolase, a key rate-limiting enzyme in the nonoxidative pathway of the pentose cycle. Defective thiamine uptake in fibroblasts isolated from patients with thiamine-responsive megaloblastic anemia (TRMA) is associated with the mutation and loss of function of the high-affinity, low-capacity thiamine transporter. 4 Fibroblasts isolated from patients with TRMA show cycle arrest and increased apoptosis in low-thiamine-containing media. 5 Although thiamine deficiency has been known to cause apoptosis of cells in sensitive brain regions including the thalamus, mammillary, and medial geniculate nuclei, 6 the biochemical disturbance resulting from defective high-affinity thiamine transport in human cells is yet to be elucidated.Metabolic pathway flux changes appear to be critical factors in cell differentiation, in apoptosis, and in mediating the antiproliferative effect of imatinib mesylate in Bcr-Abl-positive myeloid leukemia cells in culture. 7...

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