Role of ABCB7 in RARS Figure 2. Confirmation of the microarray data for the ABCB7 gene using real-time quantitative PCR. doi:10.1371/journal.pone.0001970.g002 Discussion We have examined the role of ABCB7, the gene involved in the hereditary XLSA/A, in the acquired RARS syndrome. The hypothesis under consideration is that XLSA/A and acquired RARS may have a shared molecular basis. Interestingly, there is an excess of erythrocyte protoporphyrin in XLSA/A (in contrast with other types of genetic sideroblastic anemia) as also found in RARS [20]. We have determined the ABCB7 gene expression levels from our microarray dataset on CD34+ cells in 122 MDS patients and 16 healthy controls. The expression levels of ABCB7 are significantly lower in the RARS group than in healthy controls, RA or RAEB. In contrast there is no significant difference in ABCB7 expression between the latter three groups. The definition of RARS includes more than 15% ring sideroblasts in the bone marrow. However, we find not only lower levels of ABCB7 in RARS than other MDS but also a progressive reduction of ABCB7 expression as the percentage of ring sideroblasts increases. In addition, we have studied the ABCB7 gene expression levels in erythroblast cultures and confirmed the low levels of ABCB7 gene expression levels in RARS erythroblasts in comparison to those of RA and healthy controls. The gene expression levels of ABCB7 were validated and confirmed in a subset of MDS patients and healthy controls using real-time quantitative PCR. Direct sequencing of ABCB7, including the promoter region, was performed on DNA from 13 RARS patients and did not show any mutations in any of the patients. Similarly, Steensma et al, in a recent study, found no coding mutations of ABCB7 in patients with RARS [20]. We have investigated the methylation status of the ABCB7 promoter region and the results showed .80% methylation in all samples, with no significant difference between RARS patients and healthy controls. Gene promoters can be divided into three groups according to their CpG ratio, GC content and length of CpG-rich region: high-CpG, intermediate-CpG and low-CpG promoters [21]. Based on this classification, the ABCB7 promoter region is defined as an intermediate-CpG promoter. Intermediate- CpG promoters show a high frequency of DNA methylation [21]. Our data fit with the definition of the ABCB7 promoter region as an intermediate CpG promoter, but suggest that promoter methylation is unlikely to be the mechanism underlying the reduction in expression levels of ABCB7 observed in RARS. No genomic abnormality in the ABCB7 gene was identified in our cases, suggesting that there could be a trans-acting factor responsible for the low levels of ABCB7 in RARS (analogous to the down regulation of the alpha-globin genes in the acquired HbH associated with MDS caused by mutations in the ATRX gene) [22]. Therefore, we examined our microarray dataset for genes with expression levels correlated either strongly positively ($0.8) or inversely (#20.8) with ABCB7 gene expression levels. While no other gene had a strong inverse correlation with ABCB7, interestingly two genes showed a positive correlation: ELP2 (0.82) and RTF1 (0.80), both of which are associated with RNA Polymerase II [23,24]. Deregulation of these factors may cause defective gene transcription through chromatin [25]. The existence of an inherited disorder XLSA/A caused by inactivating mutations in ABCB7 is, of course, very informative of the probable consequences of reduced expression of the same gene in acquired disorders. Many of the key properties of ABCB7 have already been established by gene silencing studies and by conditional gene targeting in mice [13,14]. All of these experiments are consistent with the importance of ABCB7 in hematopoiesis and with the observed phenotype in XLSA/A. Therefore, it would be predicted that erythroblasts with low ABCB7 levels would have abnormal mitochondrial iron homeo- stasis. Our finding of a strong association between RARS and low gene expression levels of ABCB7 also raises the possibility of therapeutic approaches for RARS using drugs which induce the expression of ABCB7, for example, carbamazepine has been shown to induce ABCB7 in the liver of epileptic patients treated with the drug [26]. We suggest that down-regulation of the iron transporter ABCB7 plays an important role in the molecular pathogenesis of RARS, making an intriguing link between the inherited and acquired forms of sideroblastic anemia. Acknowledgments We are grateful to the Tayside Cancer Tissue Bank (Dundee, UK) for samples. Author Contributions Conceived and designed the experiments: EH JB MC JW. Performed the experiments: AG AP MN BP CF TL LM MD MJ SK DB. Analyzed the data: JB AP HC JW. Wrote the paper: JB AP JW. PLoS ONE | www.plosone.org 4 April 2008 | Volume 3 | Issue 4 | e1970