Identification of a human mutation of DMT1 in a patient with microcytic anemia and iron overload

Blood ◽  
2005 ◽  
Vol 105 (3) ◽  
pp. 1337-1342 ◽  
Author(s):  
Martha P. Mims ◽  
Yongli Guan ◽  
Dagmar Pospisilova ◽  
Monika Priwitzerova ◽  
Karel Indrak ◽  
...  
Keyword(s):  
Iron Overload ◽  
Iron Absorption ◽  
Microcytic Anemia ◽  
Homozygous Mutation ◽  
Divalent Metal Transporter 1 ◽  
Hypochromic Microcytic Anemia ◽  
Nucleotide Mutation ◽  
Human Mutation ◽  
Processing Stability ◽  
Iron Utilization

Abstract Divalent metal transporter 1 (DMT1) is a transmembrane protein crucial for duodenal iron absorption and erythroid iron transport. DMT1 function has been elucidated largely in studies of the mk mouse and the Belgrade rat, which have an identical single nucleotide mutation of this gene that affects protein processing, stability, and function. These animals exhibit hypochromic microcytic anemia due to impaired intestinal iron absorption, and defective iron utilization in red cell precursors. We report here the first human mutation of DMT1 identified in a female with severe hypochromic microcytic anemia and iron overload. This homozygous mutation in the ultimate nucleotide of exon 12 codes for a conservative E399D amino acid substitution; however, its pre-dominant effect is preferential skipping of exon 12 during processing of pre–messenger RNA (mRNA). The lack of full-length mRNA would predict deficient iron absorption in the intestine and deficient iron utilization in erythroid precursors; however, unlike the animal models of DMT1 mutation, the patient is iron overloaded. This does not appear to be due to up-regulation of total DMT1 mRNA. DMT1 protein is easily detectable by immunoblotting in the patient's duodenum, but it is unclear whether the protein is properly processed or targeted.

Functional consequences of the human DMT1 (SLC11A2) mutation on protein expression and iron uptake

Blood ◽  
2005 ◽  
Vol 106 (12) ◽  
pp. 3985-3987 ◽  
Author(s):  
Monika Priwitzerova ◽  
Guangjun Nie ◽  
Alex D. Sheftel ◽  
Dagmar Pospisilova ◽  
Vladimir Divoky ◽  
...  
Keyword(s):  
Protein Expression ◽  
Iron Uptake ◽  
Chinese Hamster ◽  
Microcytic Anemia ◽  
Homozygous Mutation ◽  
Divalent Metal Transporter 1 ◽  
Divalent Metal Transporter ◽  
Hypochromic Microcytic Anemia ◽  
Functional Consequences ◽  
Uptake Activity

We have previously described a case of severe hypochromic microcytic anemia caused by a homozygous mutation in the divalent metal transporter 1 (DMT1 1285G > C). This mutation encodes for an amino acid substitution (E399D) and causes preferential skipping of exon 12 during processing of the DMT1 mRNA. To examine the functional consequences of this mutation, full-length DMT1 transcript with the patient's point mutation or a DMT1 transcript with exon 12 deleted was expressed in Chinese hamster ovary (CHO) cells. Our results demonstrate that the E399D substitution has no effect on protein expression and function. In contrast, deletion of exon 12 led to a decreased expression of the protein and disruption of its subcellular localization and iron uptake activity. We hypothesize that the residual protein in hematopoietic cells represents the functional E399D DMT1 variant, but because of its quantitative reduction, the iron uptake activity of DMT1 in the patient's erythroid cells is severely suppressed.

Effect of DMT1 Mutations at Birth and in the First Years of Life.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3586-3586
Author(s):  
Iolascon Achille ◽  
d’Apolito Maria ◽  
Servedio Veronica ◽  
De Falco Luigia ◽  
Piga Antonio ◽  
...  
Keyword(s):  
Iron Overload ◽  
Iron Uptake ◽  
Iron Absorption ◽  
Transferrin Saturation ◽  
Adult Life ◽  
Microcytic Anemia ◽  
Hepatic Iron ◽  
Liver Iron ◽  
Divalent Metal Transporter 1 ◽  
Divalent Metal Transporter

Abstract Divalent metal transporter 1 (DMT1) is involved in dietary iron uptake on the luminal side of duodenal enterocytes and transfers iron from the endosome to the cytosol in the marrow erythroblasts. Spontaneous (mk mice and Belgrade rats) or acquired (DMT1 -/- mice) inactivation of DMT1 in rodents produces a severe microcytic anemia at birth, caused by inefficient intestinal iron absorption and defective iron utilization in erythroid cells. The first reported patient with DMT1 mutations had microcytic anemia and iron overload in adult life. We here report the hematological phenotype of a newborn with a severe mycrocytic anemia (Hb 4 g/dL, MCV 71 fL) at birth and during the first months of life. Serum iron, transferrin saturation and serum ferritin were 160 microg/L, 100% and 846 ng/ml respectively at 3 months of age. Hepatic iron overload wad documented at the age of 5 years by both non invasive SQUID and liver biopsy. Sequence analysis of genomic DNA of the family revealed that the child was compound heterozygote for two novel DMT1 mutations, inherited by the asymptomatic parents. The first change deleted 3 bp (c.310 - 3_5del CTT) in intron 4 resulting in a splicing abnormality and the skipping of exon 5. The second was C>T 1246 substitution that causes arginine > cysteine replacement at position 416 (p. R416C) in the protein. This missense affects an highly conserved residue in one of the putative transmembrane domains. A striking reduction of the protein in peripheral blood cells of the proband was demonstrated by western blot using an anti-DMT1 antibody. The child required blood transfusions at birth and in the first two months of life. Thereafter, treatment with subcutaneous erythropoietin mantained hemoglobin levels between 7.5–9.5 g/dL, allowing transfusion-independence. The haematological phenotype of this patient highlights the essential role of DMT1 in erythropoiesis. The early and significant hepatic iron accumulation indicates that, as in animal models, DMT1 is dispensable for liver iron uptake. Finally DMT1 inactivation in the gut is likely bypassed by other pathways of iron absorption.

DMT1 Mutation in a Patient with Hypochromic Microcytic Anemia: Functional Consequences and Response to Erythropoietin.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3587-3587
Author(s):  
Monika Priwitzerova ◽  
Dagmar Pospisilova ◽  
Guangjun Nie ◽  
Alex D. Sheftel ◽  
Martha P. Mims ◽  
...  
Keyword(s):  
Iron Uptake ◽  
Chinese Hamster ◽  
Microcytic Anemia ◽  
Hemoglobin Level ◽  
Homozygous Mutation ◽  
Divalent Metal Transporter 1 ◽  
Divalent Metal Transporter ◽  
Hypochromic Microcytic Anemia ◽  
Functional Consequences ◽  
Uptake Activity

Abstract We have previously described a case of severe hypochromic microcytic anemia caused by a homozygous mutation in the divalent metal transporter 1 ( DMT1 1285G>C ). This mutation encodes for an amino acid substitution (E399D) and causes preferential skipping of exon 12 during processing of the DMT1 mRNA. To examine the functional consequences of this mutation, full length DMT1 transcript with the patient’s point mutation or a DMT1 transcript with exon 12 deleted was expressed in Chinese hamster ovary (CHO) cells. Our results demonstrate that the E399D substitution has no effect on protein expression and function. In contrast, deletion of exon 12 led to a decreased expression of the protein and disruption of its subcellular localization and iron uptake activity. We hypothesize that the residual protein in hematopoietic cells represents the functional E399D DMT1 variant, but because of its quantitative reduction, the iron uptake activity of DMT1 in the patient’s erythroid cells is severely suppressed. Because of the positive effect of erythropoietin (EPO) on the growth of patient’s BFU-Es in our in vitro studies, we have treated the patient with recombinant human EPO. The dose of 1.2 μg/kg given once a week did not lead to any improvement in hemoglobin level. However, doubling the dose of EPO (2.4 μg/kg) led to an increase in hemoglobin level from 75 to 91 g/L and to an improvement in the patient’s clinical condition.

scholarly journals Mutation of the gastric hydrogen-potassium ATPase alpha subunit causes iron-deficiency anemia in mice

Blood ◽  
2011 ◽  
Vol 118 (24) ◽  
pp. 6418-6425 ◽  
Author(s):  
Lara Krieg ◽  
Oren Milstein ◽  
Philippe Krebs ◽  
Yu Xia ◽  
Bruce Beutler ◽  
...  
Keyword(s):  
Iron Deficiency ◽  
Iron Deficiency Anemia ◽  
Iron Absorption ◽  
Osmotic Fragility ◽  
Microcytic Anemia ◽  
Deficiency Anemia ◽  
Α Subunit ◽  
Hypochromic Microcytic Anemia ◽  
Gastric Proton Pump ◽  
Potassium Atpase

Abstract Iron is an essential component of heme and hemoglobin, and therefore restriction of iron availability directly limits erythropoiesis. In the present study, we report a defect in iron absorption that results in iron-deficiency anemia, as revealed by an N-ethyl-N-nitrosourea–induced mouse phenotype called sublytic. Homozygous sublytic mice develop hypochromic microcytic anemia with reduced osmotic fragility of RBCs. The sublytic phenotype stems from impaired gastrointestinal iron absorption caused by a point mutation of the gastric hydrogen-potassium ATPase α subunit encoded by Atp4a, which results in achlorhydria. The anemia of sublytic homozygotes can be corrected by feeding with a high-iron diet or by parenteral injection of iron dextran; rescue can also be achieved by providing acidified drinking water to sublytic homozygotes. These findings establish the necessity of the gastric proton pump for iron absorption and effective erythropoiesis.

Two nonsense mutations in the TMPRSS6 gene in a patient with microcytic anemia and iron deficiency

Blood ◽  
2008 ◽  
Vol 112 (5) ◽  
pp. 2089-2091 ◽  
Author(s):  
Flavia Guillem ◽  
Sarah Lawson ◽  
Caroline Kannengiesser ◽  
Mark Westerman ◽  
Carole Beaumont ◽  
...  
Keyword(s):  
Iron Deficiency ◽  
Microcytic Anemia ◽  
Deficiency Anemia ◽  
Nonsense Mutations ◽  
Hepcidin Expression ◽  
Hypochromic Microcytic Anemia ◽  
Iv Iron ◽  
Membrane Bound ◽  
Inherited Mutations ◽  
Iron Utilization

Abstract Genetic causes of hypochromic microcytic anemia include thalassemias and some rare inherited diseases such as DMT1 deficiency. Here, we show that iron deficiency anemia with poor intestinal absorption and defective iron utilization of IV iron is caused by inherited mutations in TMPRSS6, a liver-expressed gene that encodes a membrane-bound serine protease of previously unknown role that was recently reported to be a regulator of hepcidin expression.

Two New Human DMT1 Mutations in a Compound Heterozygous Patient with Microcytic Anemia and Low Iron Stores.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3540-3540
Author(s):  
Carole Beaumont ◽  
Jean Delaunay ◽  
Gilles Hetet ◽  
Mariane de Montalembert ◽  
Bernard Grandchamp ◽  
...  
Keyword(s):  
Serum Ferritin ◽  
Transferrin Receptor ◽  
Serum Iron ◽  
Transmembrane Domain ◽  
Iron Absorption ◽  
Transferrin Saturation ◽  
Microcytic Anemia ◽  
The Other ◽  
Erythroid Cells ◽  
Hypochromic Microcytic Anemia

Abstract DMT1 is a divalent metal transporter with 12 transmembrane domains. It is expressed at the apical membrane of duodenal enterocytes, where it mediates pH-dependent uptake of Fe2+. In erythroid cells, it is found in the endosomal membrane where it transfers iron internalized through the transferrin-transferrin receptor pathway from the endosome to the cytosol. The same homozygous G>A substitution resulting in the G185A replacement is responsible for a severe hypochromic microcytic anemia in both the mk mouse and in the Belgrade rat. In humans, a homozygous G>C mutation has been described in a Czech patient, affecting the last nucleotide of exon 12. This mutation leads to the G399A replacement, without affecting the transport function of the protein. However, this mutation also induces a preferential in-frame skipping of exon 12, albeit not in all tissues. Accordingly, the patient has impaired iron acquisition in erythrocytes while duodenal iron absorption is increased leading to progressive iron overload. Here, we report a female patient born in 1996, with low birth weight and hypochromic microcytic anemia (Hb = 7.5 g/dl; MCV = 53 fL). She was transfused at day 0 and put on oral iron treatment. She was then lost to follow-up for five years. At the age of five, more extensive explorations showed a persistent microcytic anemia. The bone marrow displayed normal cellularity, 30% of nucleated cells were erythroid precursors with a moderate maturation defect, acidophilic forms being under-represented as compared to more immature forms. Soluble transferrin receptors were increased (8.3 mg/L; N = 0.83–1.76). Following oral iron therapy, serum ferritin levels remained low (15–25 μg/L; N = 14–197) despite an increase in transferrin saturation from 68 to 95 %. This high transferrin saturation resulted from the combination of reduced transferrin levels (1.64 g/L, N = 2.2–4.0) and increased serum iron levels (35 μmol/L; N = 11–24). On the other hand, hemoglobin raised from 7 to 9 g/dL only through increased number of RBC (5 to 5.7 T/L), since MCV and MCHC remained unchanged. We sequenced the entire transferrin receptor cDNA in this patient and found no mutation. We then sequenced the exons and the intron-exon boundaries of the DMT1 gene and found two heterozygous mutations. One mutation was a deletion of a GTG codon in exon 5, leading to the V114 in-frame deletion, in transmembrane domain 2. The other mutation is a G>T substitution in exon 8 leading to the G212V replacement in transmembrane domain 5. Both parents were asymptomatic, the father being heterozygous for the delV114 mutation and the mother heterozygous for the G212V mutation. This is the second patient described with a neonatal hypochromic microcytic anemia due to DMT1 mutations. Our data suggest that the two combined DMT1 mutations are responsible for the defect in iron utilization by erythroid cells, resulting in persistant microcytosis and impaired red cell maturation. The effect of the mutations on intestinal iron absorption is more difficult to evaluate since iron therapy allowed serum iron and transferrin saturation to increase but serum ferritin remained low and hemoglobin did not reach normal values.

scholarly journals Hereditary hypochromic microcytic anemia associated with loss-of-function DMT1 gene mutations and absence of liver iron overload

2017 ◽  
Vol 93 (3) ◽  
pp. E58-E60 ◽  
Author(s):  
Maddalena Casale ◽  
Adriana Borriello ◽  
Saverio Scianguetta ◽  
Domenico Roberti ◽  
Martina Caiazza ◽  
...  
Keyword(s):  
Iron Overload ◽  
Gene Mutations ◽  
Microcytic Anemia ◽  
Loss Of Function ◽  
Liver Iron ◽  
Hypochromic Microcytic Anemia ◽  
Liver Iron Overload

scholarly journals Iron Overload in the Face of Anemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. SCI-38-SCI-38
Author(s):  
Yatrik Shah
Keyword(s):  
Transcription Factors ◽  
Sickle Cell Disease ◽  
Iron Overload ◽  
Sickle Cell ◽  
Iron Absorption ◽  
Conflicts Of Interest ◽  
Iron Accumulation ◽  
Cell Disease ◽  
Divalent Metal Transporter 1 ◽  
Tissue Iron

Abstract Several distinct congenital disorders can lead to tissue-iron overload with anemia including β-thalassemia and sickle cell disease. We show that intestinal absorption of iron is highly increased and significantly contributes to tissue iron accumulation in these disorders. The present work describes a novel pathway by which oxygen sensing transcription factors are highly upregulated in iron overload anemias and are subsequently essential for the increase intestinal iron absorption. Oxygen signaling is mediated through well-conserved hypoxia driven transcription factors, hypoxia-inducible factor (HIF)1a and HIF2a. In the intestine, HIF2a directly activates divalent metal transporter 1 (DMT1), duodenal ferric reductase (DcytB), and Fpn1, which are iron transporters critical for adaptive changes in iron absorption. We demonstrate that HIF2a and its downstream target gene, DMT1 are essential for iron accumulation in mouse models of β-thalassemia and sickle cell disease. Furthermore, studies of thalassemic mouse model with established iron overload demonstrated that loss of intestinal HIF2a and DMT1 signaling led to decreased tissue iron accumulation in the livers. Interestingly, disrupting intestinal HIF2a not only improves tissue iron accumulation, but a marked improvement of anemia was also observed. These novel findings suggests that inhibition of HIF2a signaling pathway could be a novel and robust treatment strategy for several conditions that cause iron overload with anemia. Disclosures No relevant conflicts of interest to declare.

Kinetic of Iron Absorption and Expression of Iron Related Genes in Beta-Thalassemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3846-3846
Author(s):  
Laura Breda ◽  
Sara Gardenghi ◽  
Ella Guy ◽  
Ninette Amariglio ◽  
Konstantin Adamsky ◽  
...  
Keyword(s):  
Iron Overload ◽  
Iron Content ◽  
Iron Absorption ◽  
Globin Gene ◽  
Active Form ◽  
Beta Thalassemia ◽  
Post Transplantation ◽  
Divalent Metal Transporter 1 ◽  
Hematopoietic Stem ◽  
Divalent Metal Transporter

Abstract We generated the first transplantable adult mouse models of beta-thalassemia intermedia and major by infusing mouse hematopoietic-fetal-liver cells (HFLC) heterozygous or homozygous for a deletion of the beta-globin gene (respectively with th3/+ and th3/th3 cells) into lethally irradiated congenic C57BL/6 mice. Six to 8 weeks post transplantation, mice transplanted with th3/+ HFLCs show 7 to 9 g/dL of hemoglobin levels, splenomegaly, abnormal red cells and increased iron overload. Mice transplanted with th3/th3 HFLCs, unless blood transfused, die 8 to 10 weeks after engraftment showing profound anemia, massive splenomegaly and very rapid and dramatic iron overload. For this reason, we began a systematic study to compare iron content and the expression level of iron related genes in normal and thalassemic mice of varying ages and sex in different organs (liver, duodenum, spleen, kidney and heart). In liver, we observed that iron content increases proportionally with the level of anemia, age and if the blood transfusion is included. We are currently analyzing the other organs. The expression of hepcidin, ferroportin, Hfe, ferritin, transferrin, transferrin-receptor 1 and 2, ceruloplasmin, divalent metal transporter 1 and hemojuvelin are being tested also in all these organs. In particular, we observed that hepcidin is dramatically downregulated in liver of beta-thalassemic animals. Our hypothesis is that low expression of this gene leads to high iron content in these animals. We intend to demonstrate that administration or increasing hepcidin levels of this peptide can prevent iron absorption in beta-thalassemia. We developed two alternative strategies to test our hypothesis. In the first one, we synthesized the active form of the mouse hepcidin peptide that will be administered intraperitoneally to mice affected by beta-thalassemia. In the second, lentiviral vectors have been generated in order to constitutively secrete hepcidin in the bloodstream of animals affected by beta-thalassemia. These vectors were introduced into hematopoietic stem cells derived from mouse embryos of normal and mice affected by beta-thalassemia and engrafted in myeolablated normal mice. The engrafted mice express hepcidin 6 weeks post transplantation by RT PCR. These animals, along with the animals in which hepcidin will be administrated intraperitoneally, will be analyzed at the endpoint of the experiment (> 4 months) for their hematological values and iron content to see if the use of hepcidin can be used to prevent excessive iron absorption in beta-thalassemia.

Molecular and Functional Characterization of Two Novel Missense Mutations in Iron Transport Protein Causing Hypochromic Microcytic Anemia and Hemosiderosis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3202-3202
Author(s):  
Rekha Athiyarath ◽  
Neeraj Arora ◽  
Rayaz Ahmed ◽  
Biju George ◽  
Mammen Chandy ◽  
...  
Keyword(s):  
T Cells ◽  
Iron Overload ◽  
Serum Iron ◽  
Microcytic Anemia ◽  
Oral Iron ◽  
Hemoglobin F ◽  
Hypochromic Microcytic Anemia ◽  
Transferrin Gene ◽  
Hemoglobin A2

Abstract Abstract 3202 Atransferrinemia is a rare autosomal recessive disorder characterized by hypochromic microcytic anemia and iron overload. It is extremely rare with 10 cases reported worldwide and only half of them have been characterized at the genetic level. In this report we describe two novel mutations in the transferrin gene found in two patients in two unrelated families from India. The proband-I was a 9 year old girl who had severe hypochromic microcytic anemia requiring intermittent transfusions since the age of four years. She presented at 7 years of age with symptomatic anemia requiring blood transfusion not responding to oral iron therapy. She had pallor and mild hepato splenomegaly and her hemoglobin was 6.0g/dL with a MCV of 67.1fL. Her hemoglobin F (HbF) and hemoglobin A2 (HbA2) was normal and screening of beta globin gene did not reveal any mutation. Bone marrow examination done to rule out CDA & MDS showed a cellular marrow with absent iron stores. She didn't respond to oral iron supplements and her serum iron was 12ug/dL and TIBC was 110ug/dL with a ferritin of 3205ng/ml. Atransferrinemia was suspected because of a low TIBC and transferrin level of 5.7mg/dL confirmed the diagnosis. The proband-II was a 4 year old boy who had presented with hypochromic microcytic anemia and elevated ferritin. He was transfusion dependent from the age of two. His Hb was 6.4g/dl and MCV of 54.8fL with normal hemoglobin F (HbF) and hemoglobin A2 (HbA2) levels. His serum iron was 16ug/dL and TIBC 19 ug/dL with elevated ferritin (12910ng/ml). Low TIBC is correlated with very low transferrin level (TF-7.5mg/dl) found in this patient. DNA was extracted from the patients and parents by standard protocol. Both coding and adjacent regions of the exons in the transferrin gene (TF) were sequenced. Mutation analysis revealed the presence of a mutation c.1940 A>T in homozygous state resulting in a missense mutation p.Asp647Val in the C-lobe in proband-I. Both parents were heterozygous for this mutation and this change was absent in fifty healthy controls. Proband–II showed a homozygous c.1825 C>T variation in the transferrin gene resulting in p.Arg609Trp. Both parents were heterozygous for this mutation. These amino acids were highly conserved across species. When the protein sequence of TF was interrogated using SIFT and Polyphen algorithm for p.Asp647Val and p.Arg609Trp, both mutations were predicted to be pathological and deleterious. SPDBV modeling showed the p.Asp647Val will result in loss of interaction with a crucial amino acid Arg at 632 position which is essential for solvent accessibility and conformational change during iron release. There was a conformational change observed for the substitution of Arg by Trp at 609 position in TF protein by SPDBV modeling that may lead to instability or defective secretion and this has to be evaluated by invitro functional assays. To assess whether p.Asp647Val mutation leads to TF deficiency, in vitro TF production was assayed. T cells from the patient and controls were stimulated with phytohaemagglutinin and IL2 and the transferrin released was measured by ELISA. The amount of transferrin released from proband-I's T cells at 72hrs was 4.75ng/ml which was much lower when compared to transferrin secreted (72.14ng/ml) from control T cells. In vitro experimental observations clearly suggest that p.Asp647Val mutation in transferrin gene results in protein deficiency. As predicted, replacement of Aspartic acid with valine in C-lobe of transferrin leads to ineffective protein secretion. Proband-I was started on monthly plasma infusions. Her hemoglobin levels improved and she became transfusion independent. Oral iron chelation was started to reduce tissue iron overload. Proband-II has just been infused with plasma and the outcome is awaited. This is the first report from India where prevalence of thalassemia is high and identification of two cases underlines the importance of screening of atransferrinemia in cases with hypochromic microcytic anemia and iron overload in order to start appropriate treatment to reduce iron overload related complications and anemia. Disclosures: Edison: Department Of Biotechnology, Government of India: Research Funding.

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