Mutations in the Gene Encoding DMT1: Clinical Presentation and Treatment

Mutations in the Gene Encoding DMT1: Clinical Presentation and Treatment Achille Iolascon and Luigia De Falco Divalent metal transporter 1 (DMT1) is the protein that allows elemental iron entry into the duodenal cell. It is expressed ubiquitously and it also allows the iron exit from the endosomes. This protein plays a central role in iron metabolism and it is strictly regulated. Several animal models elucidate its role in physiology. Recently three patients affected with DMT1 deficiency have been described. This recessively inherited condition appears at birth with severe microcytic anemia. Serum markers could be particularly useful to establish a correct diagnosis: high serum iron, normal total iron-binding capacity (TIBC), increased saturation of transferrin (Tf), slightly elevated ferritin, and increased soluble transferrin receptor (sTfR). Increased free erythrocyte protoporphyrins (FEPs) could address the diagnosis to iron-deficient anemia. All patients appeared to respond to erythropoietin (Epo) administration. Because mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) did not change during Epo treatment, it was concluded that Epo did not improve iron utilization of the erythroblasts but likely reduced the degree or intensity of apoptosis, affecting erythropoiesis. Moreover liver iron overload was present and documented in all of the affected patients. In this review we analyze the role of DMT1 in iron metabolism and the major causes of reduction and their consequences in animal models as well in humans, and we attempt to define the correct treatment for human mutants. Semin Hematol 46:358 –370. © 2009 Elsevier Inc. All rights reserved.

I

ron is a trace element. As the name suggests, “trace” elements are minerals that are needed by the body in very small amounts. They include chromium, copper, iodine, iron, selenium, and zinc. Most people effortlessly meet the daily requirements from their food if they have a balanced diet so deficiencies are rare. Low iron stores are associated with poor concentration, decreased resistance to infection, and decreased work performance. If dietary iron intake is consistently low, iron stores become depleted, which results in iron deficiency anemia.1–3 The best source of iron is lean red meat. Alternatively, eggs, fortified breakfast cereal, whole-meal bread, broccoli, spinach, prunes, and apricots are also good iron sources. Vitamin C can double or even CEINGE, Advanced Biotechnologies, Naples; and Department of Biochemistry and Medical Biotechnologies, University Federico II, Naples, Italy. This work is supported by Italian Ministero dell’Università e della Ricerca, project PS 35–126/IND, by Telethon (A.I.) (Italy), and “grants Convenzione CEINGE-Regione Campania-Ass. Sanità. Address correspondence to Achille Iolascon, MD, PhD, CEINGE, Advanced Biotechnologies, Via Comunale Margherita 482, 80145 Naples, Italy. E-mail: [email protected] 0037-1963/09/$ - see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1053/j.seminhematol.2009.06.005

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triple iron absorption. On the other hand, iron absorption is hampered by some nutrients in the diet, eg, phytates, calcium, and tannin.2 A regular diet provides roughly 10 mg of iron per day, of which 10% is absorbed to replace the losses. Iron is an essential element for humans because it is involved in oxygen delivery (as heme cofactor mainly in hemoglobin and myoglobin) and in enzymatic electron transfer (as in cytochromes, ribonucleotide reductase, and other enzymes). Too little introduction of iron causes functional deficiencies, mainly in hemoglobin (Hb) and DNA synthesis, cytochromes, and oxidase/reductase enzymes, and the appearance of anemia and cognitive deficiencies. On the other hand, too much iron is also harmful because it causes organ toxicity through the formation of oxygen radicals, lipid peroxidation, DNA damage, and tissue fibrosis. To avoid these two opposite conditions a correct balance of iron metabolism is mandatory: each day approximately 1 mg enters the body and the same amount is lost. This involves a control in absorption (duodenal cells) and regulation in cellular content. Iron absorption is strictly regulated and in response to iron status, erythropoietic demand, hypoxia, and inflammation, the intestine may increase the absorption rate up to 10 times. Unfortunately, in humans there is no regulatory mechanism to get rid of

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Mutations in the gene encoding DMT1

excess iron, and at steady state, absorption must be equal to losses; elimination is effected only through bleeding, sloughing of skin, and mucosal cells.4 Surplus iron is generally stored in hepatocytes and macrophages. To maintain the steady state of iron, four interconnected levels of iron metabolism regulation are involved: intestinal (absorption) in duodenal cells, hepatic (storage cells) in liver cells, erythroid (which use a large quantity) in erythroid precursors, and recycling red blood cell (RBC) destruction and iron reutilization in macrophage cells (Figure 1). Intestinal iron absorption involves elemental iron uptake that occurs in enterocytes of the duodenum. Iron is largely in the form of iron oxy-hydroxides and Fe3⫹ must be solubilized and reduced to Fe2⫹ by duodenal cytochrome b (DCYTB). Divalent metal trans-

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porter 1 (DMT1) transports Fe2⫹ at the apical side of the cells. In addition, mammalian divalent cation export systems have been described for zinc and copper, but a mammalian uptake system has not yet been identified. For those who consume meat, heme is the main dietary source of bioavailable iron. In recent years, a mammalian heme transporter has been discovered, namely, PCFT/HCP1. It acts as a direct transfer process across plasma membranes. At this early stage of our knowledge, the physiological relevance of these transporters is unclear. Interestingly, PCFT/HCP1 has been independently characterized as a folate/proton symporter and appears to play a key role in intestinal folate absorption.5,6 Iron can be stored as ferritin and eventually sloughed into the waste stream. Ferroportin (FPN) exports Fe2⫹ at the basolateral side of duodenal cells where it is oxidized

Figure 1. Four interconnected levels of iron metabolism regulation are involved to maintain the steady state of iron level: intestinal (absorption) in duodenal cells, hepatic (storage cells) in liver cells, erythroid (which use a large quantity) in erythroid precursors, and recycling (RBC destruction and iron reutilization) in macrophage cells. Adapted from Kemna EHJM, Tjalsma H, Willems HL, Swinkels DW. Hepcidin: from discovery to differential diagnosis. Haematologica. 2008;93:90 –7; and Graham RM, Chua ACG, Herbison CE, Olynyk JK, Trinder D. Liver iron transport. World J Gastroenterol. 2007;13:4725–36, with modifications.

360 to Fe3⫹ to bind transferrin (Tf) to circulate in the body. The latter reaction is operated by the ferroxidase hephaestin. Each molecule of Tf binds two Fe3⫹ molecules. Macrophage iron recycling is the major source of iron for erythropoiesis, which requires approximately 25 mg per day. To do this, macrophages engulf old or damaged RBCs and release heme from Hb. Heme oxygenase (HO) releases iron from heme and recovered iron can be stored as ferritin or exported to the plasma by ferroportin, where it is oxidized by ceruloplasmin, binds to Tf, and becomes available for erythopoiesis. Liver cells may be considered one of the main iron storage cells and for this they are implicated in hemochromatosis. In these cells, iron is recruited by means of transferrin receptor (TfR). In some diseases of iron overload or transfusion-dependent thalassemias, not all of the iron can be bound by Tf, which results in a pool of non–transferrin-bound iron (NTBI). NTBI uptake is increased in cells in which the DMT1 mRNA and protein expression are upregulated. It seems that DMT1 is the major transporter of NTBI in hepatocytes.7 Erythroid cells use the largest quantity of iron for hemoglobin synthesis. For this they are the main cause of the absence of Tf–Fe3⫹ in circulation. This complex in plasma is transported into cells principally through transferrin receptor 1 (TfR1), which is expressed on all dividing cells and is particularly abundant on erythroid precursor membrane. TfR2, encoded by a different gene, is expressed primarily in the liver and binds the Tf–Fe3⫹ complex at a much lower affinity than TfR1; it appears to be a signaling molecule rather than an iron transporter.8 At the pH of the cell surface, TfR1 binds only diferric Tf. The Tf–Fe3⫹/TfR1 complex is internalized into a clathrin-coated pit that, assisted by an adaptor protein complex designated AP-2,9 rapidly matures to a proton-pumping, pH-lowering endosome. At the lowered pH, iron is released from Tf and is subsequently reduced to Fe2⫹ and transported across the endosomal membrane by DMT1.10 Iron serum levels could play a role in regulating the expression of several relevant proteins involved in iron metabolism such as ferritin H and L, TfR, DMT1, aconitase 2 (ACO2), 5-aminolevulinate synthase 2 (ALAS2), succinate dehydrogenase (SDH), hypoxia inducible factor 2 (HIF2), FPN, and others by means of the iron regulatory protein (IRP)/iron regulatory element (IRE) system. In this review we will analyze the role of DMT1 in iron metabolism and the molecular basis of deficiency and consequences in animal models as well as in humans, and we will attempt to define possible treatments for human mutants.

THE GENE AND THE PROTEIN DMT1, also known as Nramp2 or Slc11a2, is a proton-coupled divalent metal transporter found at

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the plasma membrane and at the endosomal membrane of both intestinal and peripheral tissue cells. Nramp2 gene was cloned in 199511 and mapped on mouse chromosome 15, while Vidal and coworkers cloned and characterized the human form, mapping it by fluorescence in situ hybridization on chromosome 12q13.12 Nramp2 mRNAs were found to be ubiquitously expressed at low levels, most notably in the proximal duodenum,11 and its expression increases in conditions of iron deficiency.13 The characterization of Nramp2 (DMT1) as a divalent-cation transporter started with DCT1 cloning in the rat.13 DCT1 (DMT1) mediates active transport that is proton-coupled and depends on the cell membrane potential. In addition to its involvement in iron absorption, it also accepts a variety of divalent metal ions, including Zn2⫹, Mn2⫹, Co2⫹, Cd2⫹, Cu2⫹, Ni2⫹, and Pb2⫹.14 Although this gene belongs to the Nramp family, its role in resistance to infection remains unknown. In fact, Nramp2 mRNA expression is ubiquitous and does not seem particularly associated with organs or cell types implicated in host defenses.11 The human DMT1 gene consists of 17 exons, spanning more than 36 kb and encoding four variant mRNA transcripts, differing for alternative promoter at 5= and alternative splicing at 3= UTR15,16 (Figure 2A). Alternate promoter use produces isoform 1A, which starts from exon 1A localized 1.9 kb upstream of exon 1B, followed by a consensus splice sequence that connects it directly to exon 2. Isoform 1B starts from exon 1B, which is not translated, and translation begins in exon 2.15,16 This first ATG use produces a DMT1 with an additional 29 amino acids (1A), which give rise to a protein able to be inserted in cellular external membrane. This is mainly expressed in duodenum and kidney, whereas isoform 1B is expressed in all tissues in endosomal membranes.16 Furthermore, two alternatively spliced mRNA at 3= UTR differ by a specific sequence either containing or lacking an IRE (⫹IRE and ⫺IRE, respectively). They encode two transporters with distinct carboxy-termini: DMT1 ⫹IRE isoform has 18 amino acids, while the ⫺IRE form is seven amino acids longer17 (Figure 2B). The expression of these isoforms appears to be tissuespecific and they are differently regulated (as shown in the next section). Moreover, other minor isoforms were observed due to skipping of exons 10 and 12.17 The expression of Nramp2 mRNA lacking exon 10 was observed in hemochromatosis patients at levels much lower than the full-length mRNA, whereas Mims et al observed that, in normal reticulocytes, isoform lacking exon 12 represents 10% of total DMT1 mRNA.17,18 Skipping of either exon 10 or 12 did not abolish the correct mRNA

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Figure 2. DMT1 gene sequence (A) in humans, which gives four different mRNA transcripts. Alternative promoter at 5= produces isoform 1A, which starts from exon 1A localized 1.9 kb upstream of exon1B and is followed by a consensus splice sequence that connects it directly to exon 2. Isoform 1B starts from exon 1B, which is not translated and translation begins in exon 2. Furthermore, two alternatively spliced mRNA at 3= UTR differ by a specific sequence either containing or lacking an iron regulatory element (⫹IRE and ⫺IRE, respectively). This first ATG use produces a DMT1 with an additional 29 amino acids (1A), which give rise to a protein able to be inserted in cellular external membrane. This is mainly expressed in duodenum and kidney, whereas isoform 1B is expressed in all tissues in endosomal membranes (B). The alternative splicing at 3= gives rise to two transporters with distinct carboxy-termini: DMT1 ⫹IRE isoform has 18 amino acids, while the ⫺IRE form is seven amino acids longer.

frame and probably results in an Nramp2 protein that lacked a single transmembrane domain (TMs; domains 7 and 8, respectively), which would have severe topological consequences.17 DMT1 gene sequence gives rise to cDNA molecules of more than 4 kb, which encoded a 561/568/590/597– amino acid protein with a predicted structure containing 12 TMs, two asparagine-linked glycosylation signals in an extracytoplasmic loop, membrane targeting motifs, and a consensus transport motif common to homologous cation transport proteins found in other species.19 This structural unit defines a protein family highly conserved from bacteria to man, including the closely related macrophage-specific mammalian homologue Nramp1 (78% similarity).13,20 Sequence conserva-

tion is particularly high in the predicted TMs of the protein (90%), suggesting that these regions play a key role in the structural and functional aspects common to both proteins.21 Little is known about the structure, folding, and assembly of DMT1 protein in plasma membrane. TM4 of DMT1 has been shown to be crucial for its biological function. Using model peptides like circular dychroism analysis and nuclear magnetic resonance spectroscopy, it has been speculated that TM4s may self-assemble to form a channel through which metal ions may be transported, and that mutations which affect this domain could cause a different self assembly of the peptide that alters DMT1 function in vivo.22–24

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DMT1 LOCALIZATION AND EXPRESSION IN DIFFERENT TISSUES DMT1 isoforms have cell type–specific expression patterns and distinct subcellular localizations.25 However, the simultaneous expression of both DMT1 isoforms ⫹IRE and ⫺IRE mRNAs has been observed in several tissues, including kidney, thymus, and liver.13,25,26 Isoform ⫹IRE is expressed at the plasma membrane of certain epithelial cells, including duodenum brush border. Isoform ⫺IRE is expressed in many cells, particularly in erythroid precursors and macrophages, and it is essential for the acquisition of iron from acidified endosomes.10,27 In vitro studies in transfected cells have shown that isoform ⫺IRE is rapidly internalized along with the TfR from the plasma membrane by a clathrin- and dynamindependent process,28 whereas ⫹IRE isoform is not efficiently recycled upon internalization and is ultimately targeted to lysosomes.29 Isoform 1A is mainly present in duodenal and kidney cells, whereas 1B is ubiquitously expressed.15 The 1A/ IRE(⫹) and 1B/IRE(⫹) isoforms have an identical substrate profile and operate by identical kinetic mechanisms. That is to say, the additional 29 –amino acid region at the N-terminus of the 1A isoform does not confer any change in substrate selectivity or other functional properties of DMT1. All four isoforms transport Fe2⫹ at the same turnover rate and exhibit no differences in their functional properties, permeant ions, or rate-limiting steps.16 Therefore, these observed isoform variations could act as tissue-specific signals or cues to direct DMT1 to the appropriate subcellular compartments (eg, in erythroid cells) or the plasma membrane (eg, in intestine and kidney cells).16

Regulation of DMT1 Expression Because DMT1 expression is critical to maintain both systemic and cellular iron homeostasis,16,17 changes in cellular and systemic iron levels directly or indirectly control DMT1 expression. When systemic iron requirements are augmented, such as in iron deficiency, hypoxia, or stimulated erythropoiesis, the expression of DMT1 mRNA and protein in the duodenum is increased,9,10 and more iron is absorbed. DMT1 expression may be controlled at transcriptional, post-transcriptional, and post-translational levels. Transcriptional modulation of gene expression could be operated on promoter sequences by transcriptional factors. The gene for the 1B form of DMT1 is a member of a small class of genes in which the CCAAT box lies just upstream of the transcription start site and appears to be regulated by nuclear factor-kB.30,31 This observation started the definition of the signaling mechanisms responsible for regulating transcription of 1B isoforms in mice. The 5= regulatory region of Nramp2 also contains five potential metal response elements (MREs) that are

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similar to the MREs found in the metallothionein-IIA gene, three potential SP1 binding sites, and a single ␥-interferon regulatory element.17 DMT1 has been reported to contain two motifs (CCAAAGTGCTGGG) that are similar to HIF1 binding sites in the 5= regulatory region between ⫺412 and ⫺570.32 Lis et al33 demonstrated that hypoxia selectively increases expression of exon 1A, with lesser increases in either the ⫹IRE or ⫺IRE isoforms in rat pheochromocytoma (PC12) cells,33 while Li et al32 observed a decrease in expression of the DMT1 ⫹IRE induced by hypoxia in HepG2 cell lines. Very recently, intestinal HIF-2 signaling was shown to be critical in regulating DMT1 and DcytB expression following iron deprivation in a mouse model. These findings underline an essential role of intestinal HIF-2 in response to nutritional iron deficiency and provide further molecular evidence for a critical link between iron levels and iron absorption.34 Both 5= promoter/exon 1A region and the IRE-containing terminal exon participate in iron regulation of DMT1 expression, which operates in a tissue-specific way,15,35 while the expression of the ⫺IRE form was not altered significantly by iron treatment.35 Several studies in cell lines and animal models as well in human tissues demonstrated that ⫹IRE DMT1 isoform regulation involves the IRP system, which acts as an iron sensor modulating changes in protein levels in response to iron availability.36,37 IRE/IRP complex formation within the 5= UTR of mRNA (eg, FTH1, FTL, ALAS2, ACO2, FPN) inhibits translation, whereas IRP binding to IREs in the 3= UTR of TFR1 or DMT1 mRNA prevents its degradation. The combined IRE-binding activity of both IRPs is high in iron-deficient cells and low in iron-repleted cells.38 IRP1 is an iron-sulfur cluster protein that loses its RNA binding activity in iron-replete conditions through acquisition of a 4Fe– 4S cluster, whereas IRP2 is oxidized, ubiquinated, and subsequently degraded by the proteasome.38 There is considerable evidence indicating that the ⫺IRE isoform is involved in endosomal exit of iron.39 Unexpectedly, the ⫺IRE isoform appears in the nuclei of cells with neuronal properties.39,40 While the function of ⫺IRE DMT1 in the nucleus is speculative, one may safely infer that this localization identifies new role(s) for this multifunctional transporter. Very recently it has been shown that the ⫺IRE isoform is regulated by microRNA (miRNA) Let7D.41 miRNAs are single-stranded RNAs of approximately 22 nucleotides in length, and they constitute a novel class of gene regulators. In animals, miRNAs exert their regulatory effects by binding to imperfect complementary sites within the 3= UTRs of their mRNA targets affecting protein translation. The mechanisms of the post-translational regulation of DMT1 involve protein modification by ubiquitina-

Mutations in the gene encoding DMT1

tion and proteasome degradation. Foot et al have demonstrated that Ndfips regulate DMT1 by acting as adaptors to recruit the ubiquitin ligase WWP2 and enhancing the ubiquitination of DMT1 and subsequent degradation via the lysosome and proteasome.42

DMT1 Animal Models Mammalian animal models of hypochromic anemia that have mutations in DMT1 have clarified the role of this transporter in intestinal iron uptake and in defective iron metabolism in peripheral tissues, including erythroid precursors. DMT1 mutations were first described in microcytic anemia (mk) mice19 and belgrade (b) rats,43 both models that carry the same glycine to arginine (Gly185Arg) substitution in the TM4 of the protein, which is highly conserved among Nramp family proteins.19,43 These rodents exhibit severe microcytic, hypochromic anemia due to a defect in iron uptake in the intestine but also in iron acquisition and utilization in peripheral tissues, including RBC precursors.44,45 The zebrafish mutant chardonnay (cdy) also has a mutation in the DMT1 ortolog gene identified through a combination of positional and candidate cloning approaches.46 Although zebrafish cdy showed a truncation mutation distal to TM6, these animals are viable and have a hypochromic, microcytic anemia. The authors suggest that cdy zebrafish have an alternate protein or pathway for absorption and utilization of iron and that this alternate mechanism could also exist in mammals.46 Because the missense G185R mutation did not cause a complete loss of function of DMT1 protein, Gunshin et al have completely inactivated murine DMT1 gene both globally and in selected tissues in order to study DMT1 functions and to determine the presence of other possible iron import mechanisms.47 These animals showed a more severe phenotype than that observed in mk mouse or b rat. Furthermore, they found that fetal Slc11a2 is not needed for maternofetal iron transfer and established that at least one efficient, alternative iron uptake pathway must be active in the placenta, in hepatocytes, and possibly in other somatic cell types.46

DMT1 Deficiency in Humans DMT1 deficiency is a very rare cause of microcytic anemia, and to date only three affected families have been described.18,48 –51 It is an autosomal recessive condition and, as expected in this type of inherited disease, a consanguinity could be implicated. In fact, the first case described was in a Czech patient from a consanguinous union.18,48 The other cases were an Italian boy and a French girl from non-consanguinous parents.49,51 In view of the very few cases, we will try to define a

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natural history of this condition, but a true phenotype– genotype relationship is very difficult to obtain.52 Anemia was present since the first day of life and characterized by a severe reduction of MCV and MCH. Overall, patients required transfusional support during the first weeks of life and after until erythropoietin (Epo) treatment was introduced.48 –51 In mk mouse and b rats, anemia appeared at birth with pallor, marked anisocytosis, poikilocytosis, and hypochromia with target cells and polychromatophilia. These findings are reminiscent of those observed in humans. It is interesting to note that initial iron stores are established through iron transfer from the mother to the developing fetus. DMT1 is expressed in placenta, where it has been implicated in maternofetal iron transfer.53,54 Global inactivation of DMT1 gene was obtained by Gunshin et al,47 and these mice presented a phenotype more severe than that seen in animals homozygous for the G185R mutation. They were anemic and none survived more than 7 days. However, these mice were not iron-deficient in all tissues. These data confirm that placenta iron transfer was efficient and they established that at least one alternative iron uptake pathway must be active in placenta, hepatocytes, and possibly other somatic cell types. The fact that human mutants experienced reduced fetal growth seems to indicate that DMT1 in placenta is not essential but must play a relevant role during the fetal period. DMT1 seems to have relevance during postnatal life, mainly in four different cell compartments, which could explain the clinical findings. The duodenal form is used for the introduction of ferrous iron, but the defect in this form did not completely abolish the introduction of iron in humans because the known human mutants did not avoid DMT1 existence and moreover because part of the iron requirements could enter by means of the heme transporter. Furthermore, the reduction of iron availability could cause an increased expression of iron genes, including DMT1, Dcytb, TfR1, and HO1.55 Reduced expression or nonfunctional forms of DMT1 in duodenal cells seems to be important in the genesis of anemia, as demonstrated in cre mice by the absence of DMT1 only in the enterocytes that developed anemia 12 days after birth.47 Iron absorption is present mainly in the apical cell of villi responsive to IRP activity; by contrast, precursor cells in the crypts cannot take up dietary iron but acquire it from the blood via TfR1. In this way, precursor cells are informed about the iron status of the body and program their absorption activity accordingly.56 Erythrocytes are the unique blood lineage involved in this defect. In fact, anemia and microcytosis seem to be due to the defect of iron availability in erythroid progenitors. As expected, peripheral blood smears from KO mice transplanted with wild-type hematopoietic stem cells showed abnormal erythropoiesis morphology consistent with iron-deficiency erythropoiesis.47 However,

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this animal model progressively accumulated excess iron in the liver, which suggests either a compensatory increase in intestinal iron absorption or a redistribution of iron that was effectively utilized by erythroid cells. Blood smear observation may suggest some other clinical conditions that have to be excluded in differential diagnosis: alpha thalassemia, hereditary pyropoikilocytosis, and sideroblastic anemia. These could be excluded by means of molecular studies, spectrin dimer evaluation, and Perls’ staining of bone marrow smears. Serum markers could be particularly useful to establish a correct diagnosis: high serum iron, normal total iron-binding capacity (TIBC), increased saturation of Tf, slightly elevated ferritin, and highly increased soluble transferrin receptor (sTfR). Increased free erythrocyte protoporphyrins (FEPs) or elevation of protoporphyrins in feces could address the diagnosis of iron-deficient anemia. Comparative evaluation of Tf saturation and FEPs could suggest the correct diagnosis: a defect in iron availability at the erythroid compartment level (Table 1). Iron-deficient anemia may cause a growth defect. This is demonstrated by the low birth weight and growth insufficiency in chronic iron deficiency patients. The DMT1-deficient individuals appeared hypotrophic at birth, but the Italian subject described earlier, followed since birth and for 10 years, had growth curves constantly between the 30th and 50th centiles. Epo treatment avoids transfusions in DMT1 deficiencies. All three patients described to date appeared to respond to Epo administration.52 Because MCV and MCH did not change during Epo treatment, it was concluded that this hormone did not improve iron utilization of the erythroblasts, but likely reduced the

Table 1. Clinical and Laboratory Findings of DMT1 Mutations

MCV Serum iron Tf saturation sTfR Bone marrow sideroblasts FEP Liver iron Neonatal appearance Effect of oral Fe Effect of intravenous Fe Inheritance Therapy

45–55 ⫹⫹ ⫹⫹ ⫹⫹ ⫺ ⫹ ⫹⫹⫹ Yes No No Autosomal recessive Epo

Abbreviations: MCV, mean corpuscular volume; Tf, transferrin; sTfR, soluble transferrin receptor; FEP, free erythrocyte protoporphyrin; Fe, iron; Epo, erythropoietin.

degree or intensity of apoptosis, affecting erythropoiesis. Based on the clinical course of first patient, the Epo treatment maintained for 4 years did not prevent liver iron accumulation. However, serum ferritin levels were drastically reduced after 3 months of Epo therapy. Erythropoiesis in these subjects is Epo-dependent; in fact, two attempts at interruption in the Italian patient failed. Priwitzerova et al analyzed isolated erythroid progenitors from peripheral blood and bone marrow of the patient using an in vitro colony-forming assay.47 The numbers and the size of the patient’s erythroid colonies were smaller compared with the healthy controls and exhibited low cell content and abnormal morphology. Addition of iron chelates to the cultures might correct the poor growth of the patient’s erythroid colonies. Unfortunately this could not be used as a therapeutic approach due to the hepatic iron overload. The aforementioned French patient received oral iron supplementation during the first 2 years of life. This resulted in a rise of 10 to 20 g/L in Hb concentration, but there was a persistent discrepancy between high Tf saturation and low ferritin levels. Severe iron overload led to the interruption of iron treatment. Darbopoetin is a second generation of Epo characterized by the addition of two N-linked glycosylation chains to the native protein, an approach that also required the substitution of five amino acids.57 Elimination half-life following intravenous administration is approximately 25.3 hours (v 8.5 hours for epoetin). Pospisilova et al illustrated the patient’s response to darbopoetin.50 Hb level did not improve following administration of 100 ␮g of darbopoetin weekly for 3 months, but they demonstrated that 200 ␮g weekly improves and maintains the Hb level. The patient reported an increased sense of well-being and there was no change in other parameters, including hepcidin level, which remained significantly low. In the Italian patient we administered darbopoietin treatment for 1 year, resulting in an increase in Hb level of approximately 5–10 g/L (Iolascon A, unpublished data). Continuous erythropoietin receptor activator (CERA), which was synthesized by incorporation of a 30-kd methoxy-polyethylene glycol polymer chain into the erythropoietin molecule, has a molecular weight of approximately 60 kd and an elimination half-life of about 130 hours, and it may be efficacious with administration schedules of once every 3 or 4 weeks, by either subcutaneous or intravenous routes.58 New third-generation Epo could further improve Hb levels in DMT1 patients by assuring a persistent activation of Epo receptors. Liver iron overload is present and documented in all the affected patients. The Czech patient received less than 4 g of iron via transfusions in her lifetime, but gradually developed liver hemosiderosis with liver iron content of 16.2 mg/g dry weight at the age of 19 years. The Italian patient received transfusional support only

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during the first week of life, but at 5 years superconductium quantum interference device (SQUID) biomagnetic susceptometry demonstrated a remarkably increased liver iron concentration (2,536 ␮g/ hepatic wet weight) that was equivalent to 14.2 mg/g liver dry weight. Perls’ staining of a liver biopsy demonstrated a severe iron overload (grade 3 according to Sciot), involving hepatocytes and Küpffer cells. In the French patient, magnetic resonance imaging showed severe iron overload with 250 ␮mol/g liver (normal value ⬍36). Thus, one of the characteristics of these patients is the discrepancy between high Tf saturation and low ferritin, which suggests the necessity for an evaluation of liver iron stores to reveal liver iron overload. Hepatocytes take up serum iron by a TfR-dependent process. DMT1 in normal liver was predominantly located in the cytoplasm and was scarce on the cell surface. This supports the idea that DMT1 of hepatocytes mainly transports Tf-bound iron from recycling endosomes to cytoplasm and its expression is unchanged by iron overload. An additional iron transport system takes up NTBI from serum. The role of NTBI in the normal individual is limited because most serum iron is normally bound to Tf. However, under conditions in which Tf is fully iron saturated, such as hemochromatosis, atransferrinemia and DMT1 deficiency, a substantial amount of serum NTBI may be present. NTBI in serum is rapidly cleared by the liver, mainly by hepatocytes, and this could be one possible mechanism of iron overload in these patients (Figure 1). Hepcidin, a liver produced peptide hormone, is the main regulator of body iron metabolism. Its activity is exerted by binding ferroportin, which is present on macrophages and at the basolateral site of duodenal cells, but also in hepatocytes. This binding induces the internalization and degradation of ferroportin. Normal to low urinary hepcidin levels with regard to the levels of iron stores have been found in these patients, probably accounting for the increased intestinal iron absorption. This may have some consequences on iron absorption with an increase in ferroportin gene and could be one possible cause of the iron hepatic overload. This overload does not seem to be present in the heart. SQUID analysis of one of these subjects demonstrated that cardiac iron overload was not present. In fact, iron status had no significant effect on DMT1 (⫹IRE) and DMT1 (⫺IRE) mRNAs expression in the heart, although it can significantly influence heart TfR mRNA expression.59 Deferoxamine treatment was attempted without success in the Italian patient. Chelation therapy aiming at reducing the liver iron burden was either ineffective or led to a prompt drop in Hb level and thus was interrupted in the French girl (Tchernia G, Beaumont C, unpublished data). Further studies would be required to establish if new chelating agents, such as

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deferiprone or deferasirox, could be efficacious in this condition. Differences in clinical data between animals and humans regarding iron overload could be due to the difference in nutrition; in fact, heme iron was not present in animals. However, liver iron stores were elevated at birth in Slc11a2-/- mice compared with wild-type littermates.47 Thompson et al60 demonstrated that while DMT1 is involved in non-heme iron absorption of the mature intestine of weanling rats, the functional defect of the b rat’s DMT1 gene does not appear to significantly impair Fe uptake in suckling rat pups. Further insights into iron absorption during lactation come from the observation of Lopez et al61 that DMT1 is mislocalized during late gestation, minimally expressed during early life, and predominantly expressed in its deglycosylated form until postnatal day 20 (in mice). The immunolocalization and abundant protein expression of lactoferrin receptor (mLfR) suggests that accrual of iron from Lf may be the principal iron uptake pathway at this age. In conclusion, their findings support the notion that until the development-dependent expression of DMT1 in the intestine is induced, mLfR could serve as an alternative iron uptake pathway. Molecular defects of DMT1 in humans and genotype–phenotype relationships are listed in Table 2. The mildest anemia was found in a Czech patient with 1285G¡C mutation affecting the last nucleotide of exon 12, leading to an E399D replacement and preferential skipping of exon 12.18 The latter, expected to remove TM8, occurs physiologically at a minimal rate but prevails in the proband reticulocytes and duodenal cells (90%). It is unknown whether the RNA variant results in a shorter protein (no abnormal protein was demonstrated on Western blotting). Functional studies demonstrated that E399D protein is normally expressed and fully functional,62 but 90% of DMT1 transcripts in the patient’s erythroid cells lacked exon 12 and encoded a shorter and probably unstable version of the protein.63 This amino acid substitution is in the fourth intracellular loop of DMT1 and is part of a highly conserved transport signature. Lam-Yuk-Tseung et al demonstrated that this mutant is fully functional with respect to stability, targeting, and trafficking to the membrane and that it is transport-competent.29 Therefore, this mutation is not a complete loss of function but rather a reduction in protein quantity. The Italian patient was a compound heterozygote, including a 3-bp deletion (delCTT) in intron 4 that partially impaired normal splicing and an amino acid substitution (R416C) at a conserved residue in TM9.49 Interestingly, in this case there is a normal ratio between the two isoforms of DMT1, derived from splicing of exon 12. Functional studies of this protein in LLC-PK1 kidney cells demonstrated that it causes multiple functional deficiencies, including defective protein processing, loss of transport activity, impaired cell

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Table 2. Mutations and Clinical Features of the Three DMT1 Patients

Hb at Birth (g/L)

sTfR (mg/L)

Liver Iron

Czech (homozygous) G1285C, D399E (cytosolic loop) Exon 12 skipping

74

38 (N ⫽ 1.9–4.4)

⫹⫹⫹ (19 yr old)

Italian (compound heterozygous)

delCTT, intron 4 R416C, TM9

40

French (compound heterozygous)

del Val 114, TM2 G212V, TM5

83

Mutation

Urinary Hepcidin (ng/mg creatinine)

Functional Studies of the Mutation

1–2 (N ⫽10–200)

Reduced stability of del exon 13 mutant, normal targeting and function of E399D mutant 6.77 (N ⫽ 0.83–1.76) 2,536 ␮g/g liver 98–102 (N ⫽ 45–115) R416C complete loss (N ⫽ 0–400) of function (defective processing and targeting, ER retention, loss of transport function) 8.29 (N ⫽ 0.83–1.76) 250 ⫾ 50 ␮mol/g 19–43 (on 2 separate Not studied. G212V liver (9 yr old) occasions) (N ⫽ 45– probably 66 (after 3 mo 115) conservative Epo) (N ⬍36) mutation

Abbreviations: Hb, hemoglobin; sTFR, soluble transferrin receptor; Epo, erythropoietin; ER, endoplasmic reticulum. From Iolascon et al.52

A. Iolascon and L. De Falco

Mutations in the gene encoding DMT1

surface targeting, and recycling through endosomes, concomitant with retention of the transporter in the endoplasmic reticulum.29 It therefore represents a complete loss-of-function. The 3-bp deletion in intron 4 causes the skipping of exon 5 in approximately 60% of the transcripts. The predicted protein, lacking 40 amino acids of the first putative TM domain, might be destabilized, nonfunctional, and degraded. Thus, a quantitative reduction in DMT1 expression could be the cause of this patient’s severe anemia. The French patient was a compound heterozygote for two DMT1 mutations. The first is a GTG deletion in exon 5, leading to the V114 in frame deletion in TM2, and the second is a G¡T substitution in exon 8 leading to the G212V replacement in TM5. Although no functional studies were performed on the mutated proteins, it can be speculated that the milder phenotype reflects residual transport activity of the rather conservative G212V mutation in TM5, whereas the delVal114 is likely to disrupt the TM structure.51 DMT1 transports Fe2⫹, but it plays a similar role also for other metals: Mn2⫹, Cu2⫹, Zn2⫹, Co2⫹, and probably others. Unfortunately, we did not know if this is relevant in their physiology and if some clinical findings of DMT1 mutants could be due to a metal deficiency other than Fe. Could DMT1 be involved in other forms of microcytic anemias or inherited iron abnormalities as modifier gene? Gunshin et al have demonstrated that mk mice fail to develop iron overload when the diseaserelated hemochromatosis gene (Hfe) is inactivated.47,64 This observation suggested that iron overload in HFE hemochromatosis involves increased iron transport through a pathway involving DMT1. Animals with deletion of DMT1 gene showed a significant survival improvement when only one Hfe allele was inactivated and this improvement was more pronounced in animals with both Hfe alleles inactivated. Polymorphisms usually present in the promoter region or in the coding sequence of a gene could affect expression. In many cases these polymorphisms could act as modifying factors for inherited diseases. Five single-nucleotide polymorphisms were identified within the DMT1 gene.17 One of these, 1303C/A, occurs in the coding region of DMT1 and results in an amino acid change from leucine to isoleucine. A synonymous polymorphism (1254T/C) and three polymorphisms located within introns (IVS2111A/G, IVS4144C/A, and IVS61538G/Gdel) were known. In addition, a microsatellite TATATCTATATATC (TA)6–7 (CA)10–11 CCCCCTATA(TATC)3 (TCTG)5 TCCG (TCTA)6 was identified in intron 3.17 The exact functional effect of these polymorphisms on the expression is still unknown. It is possible to hypothesize that these polymorphisms of DMT1 may play a role in iron deficiency in celiac disease or in acquired iron deficiency. To date there are no published studies addressing this specific point. Restless legs syndrome (RLS) is a common neu-

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ropsychiatric disorder that manifests as an imperative urge to move with or without abnormal sensations in the legs.65 Several studies demonstrated negative correlations between the RLS symptoms and serum ferritin66,67 or Tf saturation levels.68 Moreover, RLS lymphocytes showed an increase in ferroportin, implying increased cellular iron excretion, despite the increased iron need (increased TfR1 and DMT1).69 Based on the genetic information and biological functions of the DMT1 gene, Xiong et al considered it an excellent functional, as well as a potential positional, candidate gene.70 They therefore undertook a series of molecular genetic studies to identify any DMT1 genetic variant(s) implicated in RLS patients and families. Unfortunately, they failed to demonstrate this hypothesis, but the problem of RLS and iron deficiency is still unsolved. DMT1 is also very important in the physiology of testis, kidney, and brain. Regarding the testis, in the adult rat this protein is localized in Sertoli cells, spermatocytes, and spermatids. Expression of DMT1 in the adult rat testis is cell-specific, and highly coordinated with spermatogenic cycle.71 These data imply that germ cells need a precisely targeted and timed supply of iron, and that iron plays an important role in male fertility. None of the KO mice survived more than 7 days, and the effect on testis was not appreciated.47 Unfortunately, there is no information available on fertility in male b or mk animals. The kidney expresses relatively high levels of DMT1 compared with other tissues. This expression is mostly concentrated in the cortex, particularly in the proximal tubule and to a lesser extent in the distal tubule and cortical collecting duct.72 These data suggest that apical endocytosis of Tf (by cubilin-mediated endocytosis) followed by transport of iron out of late endosomes/ lysosomes may constitute a physiological mechanism of iron acquisition by the proximal tubular cells. Renal functionality controlled at 8 years in the Italian patient failed to demonstrate any abnormality. DMT1 expression is high in the striatum, cerebellum, thalamus, ependymal cells lining the third ventricule, and vascular cells throughout the brain.73 This means that DMT1 has an important function in the brain. There are several human neurological disorders in which brain iron concentrations are abnormal, such as Parkinson disease, Alzheimer disease, RLS, Huntington’s chorea, and Hallervorden-Spatz syndrome. These disorders may have defective iron transport or altered iron metabolism as a component of their etiology. Neurodegeneration observed in IRP2-deficient mice could be due to the effect of this on DMT1 and SFT/UbcH5A.74

CONCLUSIONS Microcytic anemias are very frequent in children and adolescents. It is a very heterogeneous group of dis-

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eases that may be acquired (mostly due to nutritional iron deficiency) or inherited. In recent years, human patients and animal models have highlighted the existence of new conditions involved in the pathogenesis of hereditary microcytic anemias in the presence or not of iron overload. So far, only three human patients have been described with DMT1 mutations, and although this has led to a surge in the number of patients identified as having severe neonatal microcytic anemia in several countries, no other mutated gene has been identified in humans so far. These patients also showed some unexpected clinical features that raised interesting questions regarding iron metabolism. Despite a molecular defect in a ubiquitous iron transporter, they developed iron overload, suggesting that DMT1 is indispensable for both intestinal iron absorption and iron storage in the liver. The identification of the mutated genes in similar cases of inherited microcytic anemias should be of great interest for our understanding of iron homeostasis.

A. Iolascon and L. De Falco

12.

13.

14.

15.

16.

17.

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Mutations in the Gene Encoding DMT1: Clinical Presentation and Treatment Achille Iolascon and Luigia De Falco Divalent metal transporter 1 (DMT1) is the protein that allows elemental iron entry into the duodenal cell. It is expressed ubiquitously and it also allows the iron exit from the endosomes. This protein plays a central role in iron metabolism and it is strictly regulated. Several animal models elucidate its role in physiology. Recently three patients affected with DMT1 deficiency have been described. This recessively inherited condition appears at birth with severe microcytic anemia. Serum markers could be particularly useful to establish a correct diagnosis: high serum iron, normal total iron-binding capacity (TIBC), increased saturation of transferrin (Tf), slightly elevated ferritin, and increased soluble transferrin receptor (sTfR). Increased free erythrocyte protoporphyrins (FEPs) could address the diagnosis to iron-deficient anemia. All patients appeared to respond to erythropoietin (Epo) administration. Because mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) did not change during Epo treatment, it was concluded that Epo did not improve iron utilization of the erythroblasts but likely reduced the degree or intensity of apoptosis, affecting erythropoiesis. Moreover liver iron overload was present and documented in all of the affected patients. In this review we analyze the role of DMT1 in iron metabolism and the major causes of reduction and their consequences in animal models as well in humans, and we attempt to define the correct treatment for human mutants. Semin Hematol 46:358 –370. © 2009 Elsevier Inc. All rights reserved.

I

ron is a trace element. As the name suggests, “trace” elements are minerals that are needed by the body in very small amounts. They include chromium, copper, iodine, iron, selenium, and zinc. Most people effortlessly meet the daily requirements from their food if they have a balanced diet so deficiencies are rare. Low iron stores are associated with poor concentration, decreased resistance to infection, and decreased work performance. If dietary iron intake is consistently low, iron stores become depleted, which results in iron deficiency anemia.1–3 The best source of iron is lean red meat. Alternatively, eggs, fortified breakfast cereal, whole-meal bread, broccoli, spinach, prunes, and apricots are also good iron sources. Vitamin C can double or even CEINGE, Advanced Biotechnologies, Naples; and Department of Biochemistry and Medical Biotechnologies, University Federico II, Naples, Italy. This work is supported by Italian Ministero dell’Università e della Ricerca, project PS 35–126/IND, by Telethon (A.I.) (Italy), and “grants Convenzione CEINGE-Regione Campania-Ass. Sanità. Address correspondence to Achille Iolascon, MD, PhD, CEINGE, Advanced Biotechnologies, Via Comunale Margherita 482, 80145 Naples, Italy. E-mail: [email protected] 0037-1963/09/$ - see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1053/j.seminhematol.2009.06.005

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triple iron absorption. On the other hand, iron absorption is hampered by some nutrients in the diet, eg, phytates, calcium, and tannin.2 A regular diet provides roughly 10 mg of iron per day, of which 10% is absorbed to replace the losses. Iron is an essential element for humans because it is involved in oxygen delivery (as heme cofactor mainly in hemoglobin and myoglobin) and in enzymatic electron transfer (as in cytochromes, ribonucleotide reductase, and other enzymes). Too little introduction of iron causes functional deficiencies, mainly in hemoglobin (Hb) and DNA synthesis, cytochromes, and oxidase/reductase enzymes, and the appearance of anemia and cognitive deficiencies. On the other hand, too much iron is also harmful because it causes organ toxicity through the formation of oxygen radicals, lipid peroxidation, DNA damage, and tissue fibrosis. To avoid these two opposite conditions a correct balance of iron metabolism is mandatory: each day approximately 1 mg enters the body and the same amount is lost. This involves a control in absorption (duodenal cells) and regulation in cellular content. Iron absorption is strictly regulated and in response to iron status, erythropoietic demand, hypoxia, and inflammation, the intestine may increase the absorption rate up to 10 times. Unfortunately, in humans there is no regulatory mechanism to get rid of

Seminars in Hematology, Vol 46, No 4, October 2009, pp 358 –370