Wainscoat1 Abstract The Role of the Iron Transporter ABCB7 in Refractory Anemia with Ring Sideroblasts Jacqueline Boultwood1.*, Andrea Pellagatti1., Maryam Nikpour2, Beena Pushkaran 1 , Carrie Fidler1, Helen Cattan 1 , Tim J. Littlewood1, Luca Malcovati3, Matteo G. Della Porta3, Martin Ja¨ dersten 2 , Sally Killick4, Aristoteles Giagounidis5, David Bowen6, Eva Hellstro¨ m-Lindberg2, Mario Cazzola3, James S. 1 LRF Molecular Haematology Unit, Nuffield Department of Clinical Laboratory Sciences (NDCLS), John Radcliffe Hospital, Oxford, United Kingdom, 2 Division of Hematology, Department of Medicine, Karolinska Institutet, Stockholm, Sweden, 3 Division of Hematology, University of Pavia Medical School, IRCCS Policlinico S. Matteo, Pavia, Italy, 4 Department of Haematology, Royal Bournemouth Hospital, Bournemouth, United Kingdom, 5 Medizinische Klinik II, St. Johannes Hospital, Duisburg, Germany, 6 Haematology Department, Leeds Teaching Hospitals, Leeds, United Kingdom Refractory Anemia with Ring Sideroblasts (RARS) is an acquired myelodysplastic syndrome (MDS) characterized by an excess iron accumulation in the mitochondria of erythroblasts. The pathogenesis of RARS and the cause of this unusual pattern of iron deposition remain unknown. We considered that the inherited X-linked sideroblastic anemia with ataxia (XLSA/A) might be informative for the acquired disorder, RARS. XLSA/A is caused by partial inactivating mutations of the ABCB7 ATP-binding cassette transporter gene, which functions to enable transport of iron from the mitochondria to the cytoplasm. Furthermore, ABCB7 gene silencing in HeLa cells causes an accumulation of iron in the mitochondria. We have studied the role of ABCB7 in RARS by DNA sequencing, methylation studies, and gene expression studies in primary CD34+ cells and in cultured erythroblasts. The DNA sequence of the ABCB7 gene is normal in patients with RARS. We have investigated ABCB7 gene expression levels in the CD34+ cells of 122 MDS cases, comprising 35 patients with refractory anemia (RA), 33 patients with RARS and 54 patients with RA with excess blasts (RAEB), and in the CD34+ cells of 16 healthy controls. We found that the expression levels of ABCB7 are significantly lower in the RARS group. RARS is thus characterized by lower levels of ABCB7 gene expression in comparison to other MDS subtypes. Moreover, we find a strong relationship between increasing percentage of bone marrow ring sideroblasts and decreasing ABCB7 gene expression levels. Erythroblast cell cultures confirm the low levels of ABCB7 gene expression levels in RARS. These data provide an important link between inherited and acquired forms of sideroblastic anemia and indicate that ABCB7 is a strong candidate gene for RARS. Citation: Boultwood J, Pellagatti A, Nikpour M, Pushkaran B, Fidler C, et al (2008) The Role of the Iron Transporter ABCB7 in Refractory Anemia with Ring Sideroblasts. PLoS ONE 3(4): e1970. doi:10.1371/journal.pone.0001970 Editor: Mikhail V. Blagosklonny, Ordway Research Institute, United States of America Received January 24, 2008; Accepted March 5, 2008; Published April 9, 2008 Copyright: 2008 Boultwood et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Leukaemia Research Fund of the UK, and in part by grants from AIRC (Associazione Italiana per la Ricerca sul Cancro), Milan, and Fondazione IRCCS Policlinico San Matteo, Pavia, to M.C. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jacqueline.boultwood@ndcls.ox.ac.uk . These authors contributed equally to this work. Introduction The myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic malignancies, characterized by blood cytopenias, ineffective hematopoiesis and a hypercellular bone marrow [1]. Refractory Anemia with Ring Sideroblasts (RARS) is a subtype of MDS in which excess iron accumulates in the form of aberrant mitochondrial ferritin in the mitochondria of the erythroid precursors [2,3]. RARS is a myeloid malignancy and transforms to acute leukemia in approximately 10–20% of cases [4]. The molecular genetic basis of RARS remains unknown. The hereditary sideroblastic anemias are a heterogeneous group of disorders characterized by the presence of ring sideroblasts in the bone marrow, microcytic hypochromic anemia and typically show X-linked inheritance [5–7]. Many of the reported cases of inherited X-linked sideroblastic anemia have been found to be caused by mutations in the erythroid-specific delta-aminolevuli- nate synthase gene (ALAS2) [8]. A rare inherited X-linked sideroblastic anemia with ataxia (XLSA/A) is caused by mutations of the mitochondrial ATP-binding cassette transporter ABCB7 [9,10]. The ABCB7 gene is the functional orthologue of the yeast Atm1p gene [11], which is required for mitochondrial iron homeostasis [12] and has been implicated in the transport of a component required for the maturation of iron-sulfur (Fe-S) cluster proteins out of mitochondria [13]. It has been demonstrated that XLSA/A is due to partial loss of function mutations in ABCB7 that directly or indirectly inhibit heme biosynthesis [13]. The anemia in XLSA/A is caused by the accumulation of iron in a form that is not readily usable for heme synthesis [14]. Moreover, ABCB7 has been demonstrated to be essential for hematopoiesis in experi- ments using conditional gene targeting in mice [13]. In an important experiment, ABCB7 has been silenced in HeLa cells by sequential transfection experiments with siRNAs [14]. The phenotype of the ABCB7-deficient cells was characterized by a reduction in proliferation rate that was not rescued by iron supplementation, by signs of iron deficiency, and by a large PLoS ONE | www.plosone.org 1 April 2008 | Volume 3 | Issue 4 | e1970