We have demonstrated that TRMA fibroblasts are uniquely sensitive to thiamine depletion. Cells die after several days in thi– medium, with DNA fragmentation as detected by TUNEL assay consistent with apoptosis or programmed cell death. To our knowledge, this is the first report of a genetic disorder of intermediary metabolism causing apoptosis. Labeling studies using [3H]thiamine of high specific activity demonstrate that TRMA fibroblasts take up ~5%–10% of wild-type amounts of thiamine at nanomolar concentrations. Nonspecific (unsaturable) thiamine uptake is not impaired in the mutants. Thus, in standard tissue culture medium (3 μM thiamine plus the rich supply in serum), the metabolic defect is obscured, whereas in thi– medium, characteristic organic acids accumulate faster in TRMA cells than in normal controls. Our results support the hypothesis that the primary defect in TRMA is a recessive mutation in the gene encoding a high-affinity thiamine transporter. Rescue of the lethal phenotype by a known yeast transporter provides additional support for this hypothesis.
Rindi and colleagues (9
) have previously defined a subtle defect of thiamine uptake in erythrocytes of patients with TRMA, ~60% of control. There are at least two possible explanations for the differences observed between their studies and the present work, in which we have found greater than 10-fold reduction in [3
H]thiamine uptake at low concentrations. First, the erythrocyte and fibroblast high-affinity systems might be similar or identical, but experimental variables allow the defect to be more readily discerned in the present fibroblast studies. For example, we have used thiamine of specific activity 20-fold higher than that used in previous studies (15 vs. 0.75 Ci/mmol; refs. 9
). This facilitates assays at low substrate concentrations, at which differences between mutant and normal cells are greatest (Fig. ). The Km
of the high-affinity system described here, ~0.5 μΜ, is at the lower end of concentrations studied by Rindi et al
). Second, there may be more than one specific thiamine uptake system, operating together or individually in different cell types. If erythrocytes normally have two systems, and fibroblasts only one, the degree of abnormality would appear more severe in the fibroblasts. The current data do not distinguish these possibilities.
Cell death by apparent apoptosis in severely thiamine-depleted TRMA fibroblasts raises several questions about the pathogenesis of the disease. First, why are patients with TRMA not severely ill without pharmacologic thiamine therapy? With a single exception (19
), subjects with TRMA have not had symptoms suggestive of severe tissue thiamine deficiency, and they have not had organic aciduria (5
). Red blood cell transketolase is also normal. We find that 10–30 nM thiamine is enough to rescue fibroblasts (Table ) and that thiamine depletion does lead to organic acidosis in the cells from patients after a few days of thiamine depletion (Table ). Normal plasma levels of free thiamine are on the order of 18–33 nM (5
). This implies that patients with TRMA on an adequate diet without pharmacologic supplementation are metabolically stable, with thiamine uptake solely by the low-affinity system.
Second, why is the deafness in TRMA progressive, whereas the anemia, and to some degree the diabetes, are reversible? We postulate that the thiamine requirement of cochlea or acoustic nerve cells is substantially higher than that of fibroblasts, which have very low energy requirements compared with neurons, myocytes, and other cell types. (We have not examined this phenomenon in other TRMA cell types.) If so, even pharmacologic doses of enteral thiamine may not prevent a thiamine-deficient phenotype in cell types with high energy usage at all times. On the basis of our observations in fibroblasts, we propose that this would lead to occasional cell death in the most sensitive tissues. This, in turn, may result in optic atrophy and progressive deafness. Bone marrow, in contrast, may be less sensitive to this effect for two reasons. First, as long as the pluripotent hematopoietic stem cells are not sensitive to cell death, the erythroid lineage can be repopulated when patients are thiamine replete. Persistent macrocytosis (2
) and poor erythroid colony growth in vitro
) suggest that even thiamine-replete erythroid progenitors are somewhat sensitive to the thiamine transport defect. Diabetes in TRMA patients has been variably reversible with thiamine. It is reasonable to conclude that the beta cells of the pancreas, or the target tissues, are of intermediate sensitivity to cellular thiamine deficiency. Other tissues, notably liver and muscles, which would be responsible for organic acidemia in severe thiamine deficiency, mus t be resistant to cell toxicity in the ambient thiamine concentration found in untreated patients with TRMA.
It is conceivable that deafness in patients with TRMA could be prevented by parenteral, early (even prenatal) thiamine therapy. Laforenza and colleagues (8
) have recently reported a thiamine uptake system from human intestinal biopsies (maximum rate in duodenum but present throughout the gut), with apparent Km
~4 μM. Whether this system is due to the same gene product defective in TRMA remains to be determined. The transport across the intestinal epithelium into the abluminal space was not measured. If either of these processes depends on the TRMA gene, it is possible that even pharmacologic doses of oral thiamine cannot provide supranormal levels of the vitamin in the blood and tissues. For cells with high energy requirements, this may not be sufficient in TRMA. We propose that if transintestinal transport is also defective in TRMA, patients may do substantially better if treated from birth (or even prenatally) with parenteral thiamine.
It is not yet clear how the vitamin transport defect in TRMA is linked to programmed cell death. Matsushima et al
) recently reported apoptotic cell death in the thalamus of thiamine-deficient rats. This suggests that the phenomenon is not limited to TRMA. However, it does not clarify the relationship between cellular thiamine deficiency and activation of apoptosis pathways. Fibroblasts in thi– medium release organic acids expected from defects in the TPP requiring enzymes, i.e.
, branched chain α-ketoacids and pyruvate (Table ). This suggests that thiamine deprivation can lead to dysfunction in the Krebs cycle, shutting down mitochondrial energy production. We speculate that TRMA cells in thi– medium will be relatively more impaired at these steps than normal fibroblasts. Mitochondrial changes, in turn, may play a pivotal role in activation of apoptosis. A number of possible mechanisms have been proposed (reviewed in ref. 21
). Mitochondrial dysfunction might lead to leakage of caspase activators, cytochrome C, or apoptosis-inducing factor, for example. It will be of interest in the future to examine whether TRMA cells from other kindreds exhibit the same uptake kinetics and thiamine-dependent survival as cells from the Alaskan patients tested here. Independent mutations have apparently arisen on different haplotypes in each TRMA family (2
), and it is possible that differences in phenotype will correlate with different genotypes.
In light of our present results, we believe that a candidate gene for TRMA would be a thiamine transporter, responsible for vitamin uptake at low extracellular thiamine concentration. Database homology searches have not yielded such a gene. Isolation of the TRMA gene will facilitate further studies of this unique biochemical disorder.