A Novel Mutation of SLC19A2 in a Chinese Zhuang Ethnic Family with Thiamine-Responsive Megaloblastic Anemia.

BACKGROUND/AIMS: Thiamine-responsive megaloblastic anemia syndrome is a rare autosomal recessive disorder resulting from mutations in SLC19A2, and is mainly characterized by megaloblastic anemia, diabetes, and progressive sensorineural hearing loss. METHODS: We study a Chinese Zhuang ethnicity family with thiamine-responsive megaloblastic anemia. The proband of the study presented with anemia and diabetes, similar to his late brother, as well as visual impairment. All clinical manifestations were corrected with thiamine (30 mg/d) supplementation for 1-3 months, except for visual impairment, which was irreversible. The presence of mutations in all exons and the flanking sequences of the SLC19A2 gene were analyzed in this family based on the proband's and his brother's clinical data. Computer analysis and prediction of the protein conformation of mutant THTR-1. The relative concentration of thiamine pyrophosphate in the proband's whole blood before and after initiation of thiamine supplement was measured by high performance liquid chromatography (HPLC). RESULTS: Gene sequencing showed a homozygous mutation in exon 6 of the SLC19A2 gene (c.1409insT) in the proband. His parents and sister were diagnosed as heterozygous carriers of the c.1409insT mutation. Computer simulation showed that the mutations caused a change in protein conformation. HPLC results suggested that the relative concentration of thiamine pyrophosphate in the proband's whole blood after thiamine supplement was significantly different (P=0.016) from that at baseline. CONCLUSIONS: This novel homozygous mutation (c.1409insT) caused the onset of thiamine-responsive megaloblastic anemia in the proband.

Folate deficiency increases chromosomal damage and mutations in hematopoietic cells in the transgenic mutamouse model.

Folate deficiency causes megaloblastic anemia and neural tube defects, and is also associated with some cancers. In vitro, folate deficiency increases mutation frequency and genome instability, as well as exacerbates the mutagenic potential of known environmental mutagens. Conversely, it remains unclear whether or not elevated folic acid (FA) intakes are beneficial or detrimental to the induction of DNA mutations and by proxy human health. We used the MutaMouse transgenic model to examine the in vivo effects of FA deficient, control, and supplemented diets on somatic DNA mutant frequency (MF) and genome instability in hematopoietic cells. We also examined the interaction between FA intake and exposure to the known mutagen N-ethyl-N-nitrosourea (ENU) on MF. Male mice were fed the experimental diets for 20 weeks from weaning. Half of the mice from each diet group were gavaged with 50 mg/kg body weight ENU after 10 weeks on diet and remained on their respective diet for an additional 10 weeks. Mice fed a FA-deficient diet had a 1.3-fold increase in normochromatic erythrocyte micronucleus (MN) frequency (P = 0.034), and a doubling of bone marrow lacZ MF (P = 0.035), compared to control-fed mice. Mice exposed to ENU showed significantly higher bone marrow lacZ and Pig-a MF, but there was no effect of FA intake on ENU-induced MF. These data indicate that FA deficiency increases mutations and MN formation in highly proliferative somatic cells, but that FA intake does not mitigate ENU-induced mutations. Also, FA intake above adequacy had no beneficial or detrimental effect on mutations or MN formation. Environ. Mol. Mutagen. 59:366-374, 2018. © 2018 Her Majesty the Queen in Right of Canada 2018.

Beta cell function and clinical course in three siblings with thiamine-responsive megaloblastic anemia (TRMA) treated with thiamine supplementation.

Three siblings with thiamine-responsive megaloblastic anemia (TRMA) with a homozygous c.454delGGCATinsAT mutation in SLC19A2 are described. The index case presented at 14 months' old with severe non-ketotic hyperglycemia, dehydration, seizures and sinovenous thrombosis. She was started on insulin and developed sensorineural hearing loss around 2 years old. Two siblings were found to have the same mutation and were started on thiamine. One sibling developed transient hyperglycemia after several years of thiamine supplementation of 12 mg/kg that resolved with an increased thiamine dose (23 mg/kg). A younger sibling continues to remain diabetes-free on thiamine (24 mg/kg). The clinical course in this family suggests that there is an effect of thiamine on pancreatic beta cell function in patients with TRMA given the resolution of impaired fasting glucose with increasing thiamine dose in one sibling and the lack of diabetes to date in the siblings that were treated early with thiamine.

Characterization and review of MTHFD1 deficiency: four new patients, cellular delineation and response to folic and folinic acid treatment.

In the folate cycle MTHFD1, encoded by MTHFD1, is a trifunctional enzyme containing 5,10-methylenetetrahydrofolate dehydrogenase, 5,10-methenyltetrahydrofolate cyclohydrolase and 10-formyltetrahydrofolate synthetase activity. To date, only one patient with MTHFD1 deficiency, presenting with hyperhomocysteinemia, megaloblastic anaemia, hemolytic uremic syndrome (HUS) and severe combined immunodeficiency, has been identified (Watkins et al J Med Genet 48:590-2, 2011). We now describe four additional patients from two different families. The second patient presented with hyperhomocysteinemia, megaloblastic anaemia, HUS, microangiopathy and retinopathy; all except the retinopathy resolved after treatment with hydroxocobalamin, betaine and folinic acid. The third patient developed megaloblastic anaemia, infection, autoimmune disease and moderate liver fibrosis but not hyperhomocysteinemia, and was successfully treated with a regime that included and was eventually reduced to folic acid. The other two, elder siblings of the third patient, died at 9 weeks of age with megaloblastic anaemia, infection and severe acidosis and had MTFHD1 deficiency diagnosed retrospectively. We identified a missense mutation (c.806C > T, p.Thr296Ile) and a splice site mutation (c.1674G > A) leading to exon skipping in the second patient, while the other three harboured a missense mutation (c.146C > T, p.Ser49Phe) and a premature stop mutation (c.673G > T, p.Glu225*), all of which were novel. Patient fibroblast studies revealed severely reduced methionine formation from [(14)C]-formate, which did not increase in cobalamin supplemented culture medium but was responsive to folic and folinic acid. These additional cases increase the clinical spectrum of this intriguing defect, provide in vitro evidence of disturbed methionine synthesis and substantiate the effectiveness of folic or folinic acid treatment.

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

Fibroblasts from patients with thiamine-responsive 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 [1,2-13C2]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 thiamine-containing 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.

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.