increase of iron accumulation in the mitochondria that was poorly available to mitochondrial ferritin. We have investigated the expression levels of the ABCB7 gene using data from our large microarray dataset on CD34+ cells from MDS patients and healthy controls in order to determine whether the ABCB7 gene is implicated in the common acquired sideroblastic anemia, RARS. Materials and Methods Sample collection and cell separation 122 patients with MDS and 16 healthy controls were included in the study. Classification of MDS patients was according to the French-American-British (FAB) criteria [15] and the patients were selected solely on the basis of having MDS. The MDS patient samples were obtained from several sources: Oxford, Bourne- mouth and Dundee (United Kingdom), Stockholm (Sweden), Duisburg (Germany) and Pavia (Italy). At the time of investigation, 35 patients had Refractory Anemia (RA), 33 RARS and 54 Refractory Anemia with Excess Blasts (RAEB). The study was approved by the ethical committees of the John Radcliffe Hospital (Oxford), the Karolinska Institutet (Stockholm), the IRCCS Policlinico S. Matteo (Pavia), the Royal Bournemouth Hospital (Bournemouth), the St Johannes Hospital (Duisburg) and the Ninewells Hospital (Dundee); written informed consent was obtained from study participants. CD34+ cells were isolated from the bone marrow samples of MDS patients and healthy controls using MACS magnetic cell separation columns (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s recommendations. Erythroblast cell cultures CD34+ cells isolated from the bone marrow of 8 patients with RARS, 9 patients with RA and 8 healthy controls were cultured according to a method developed to study the generation of erythroblasts [16,17]. Gene expression profiling experiments were performed on cultured cells at day 7. Microarray experiments and data analysis Gene expression profiling experiments and data analysis were performed as previously described [18]. Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays (47,000 transcripts) were used. Gene correlations were calculated using Pearson correlation. Real-time quantitative PCR Real-time quantitative PCR for the ABCB7 gene was performed using cDNA made from the unamplified RNA from CD34+ cells of 7 patients with RARS, 8 patients with RA, 6 patients with RAEB and 7 healthy controls. Beta-2-microglobulin was used to normalize for differences in input cDNA. Reactions were performed as previously described [16] and expression ratios were calculated using the DDCT method [19]. Statistical analysis Statistical analysis and box plots were created using StatView software. Kruskal-Wallis test was used for comparison between three or more groups and Mann-Whitney U test for comparisons between any two groups. A P-value ,0.01 was considered significant. DNA sequencing Direct sequencing of ABCB7 was performed on DNA from 13 RARS patients using Applied Biosystems Big dye terminator kit v1.1. Role of ABCB7 in RARS Promoter methylation studies We looked at promoter methylation of ABCB7 in DNA samples from 6 RARS patients (2 bone marrow, 2 neutrophil and 2 CD34+ samples) and from 5 healthy controls (1 bone marrow, 1 neutrophil, 1 mononuclear cell and 2 CD34+ samples). The ABCB7 promoter was predicted using PromoterScan and CpGPlot software. For investigation of methylation status we used bisulphite sequencing. Bisulphite modification of the genomic DNA was carried out using the EpiTect Bisulphite kit (Qiagen). Primers to amplify the modified DNA for bisulphite sequencing were designed using Primer3 software and were as follows: 59- GTTTGGGGTTTTGGGAAAAT-39 (ABCB7 forward primer) and 59-TTCCATATCAATAACATCTCACCTAA-39 (ABCB7 reverse primer). The PCR products were purified with the Wizard PCR Clean-Up System and Vacuum Manifold (Promega, Madison, WI) according to the manufacturer’s instructions. PCR products were cloned using the pGEM-T Easy cloning system (Clontech) and sequenced with Applied Biosystems Big dye terminator kit v1.1 and the ABCB7 forward primer. Results ABCB7 expression levels in MDS subtypes We have determined the ABCB7 gene expression levels from our microarray dataset on CD34+ cells in 122 MDS patients and 16 healthy controls. We have compared the gene expression levels of ABCB7 between 35 patients with refractory anemia (RA), 33 patients with RARS, 54 patients with RA and excess blasts (RAEB), and 16 healthy controls (Table 1; Figure 1A). The expression levels of ABCB7 are lowest in the RARS group compared to RA (p,0.0001), RAEB (p,0.0001) and healthy controls (p,0.0001). No significant difference was observed between the RA, RAEB and healthy control groups. ABCB7 expression levels and percentage of bone marrow ring sideroblasts The 101 of 122 MDS cases, where data was available on the exact percentage of bone marrow ring sideroblasts, have been divided in three groups by percentage of bone marrow ring sideroblasts (0–14%, 15–40% and .41%) (Table 1; Figure 1B). It can be seen that the median expression level of ABCB7 falls progressively across the three groups. As shown in Table 1, the differences in ABCB7 expression between the three groups are all statistically significant. ABCB7 expression levels in erythroblast cultures In addition, we have studied the ABCB7 gene expression levels in erythroblast cultures derived from the CD34+ cells of 8 patients with RARS, 9 patients with RA and 8 healthy controls. Cells were cultured according to a method developed to study the generation of erythroblasts [16,17] and gene expression profiling experiments were performed on cultured cells at day 7. We confirmed the low levels of ABCB7 gene expression levels in RARS erythroblasts in comparison to those of RA and healthy controls (Table 1; Figure 1C). Confirmation of ABCB7 expression data The microarray-generated expression data of the ABCB7 gene were validated and confirmed in a subset of 21 MDS patients (7 patients with RARS, 8 patients with RA, 6 patients with RAEB) and 7 healthy controls using real-time quantitative PCR (Figure 2). A high concordance was observed between the expression levels obtained with Affymetrix chips and with real-time quantitative PCR, indicating a good level of agreement between the two assays. PLoS ONE | www.plosone.org 2 April 2008 | Volume 3 | Issue 4 | e1970