Identification of the causative gene for inherited sideroblastic anemia and analysis of sideroblastis induced in vitro

alas2编码红系特异性8-氨酰yuiinate合成酶（alas2），这是红系细胞血红素生物合成的第一个酶。在小鼠体内，alas2缺失的原始红母细胞表现出成熟阻滞和大量的细胞质铁积累。然而，ALAS2缺乏对最终红细胞生成的影响仍然不清楚，因为ALAS2缺失小鼠在胚胎第11.5天在子宫内死亡，最终红细胞生成发生。为了明确血红素缺乏对最终红系细胞分化的影响，我们在体外诱导缺乏alas2基因的ES细胞特异性分化为红系，并检测了最终红系细胞的表型。与野生型红母细胞的红细胞颗粒相比，alas2缺失的最终型红母细胞由于血红素的明显缺乏而完全呈白色，尽管其形态与野生型红母细胞相似。与形态相似的是，alas2缺失的终型红母细胞中GATA-1、NF-E2和ter119等红细胞特异性基因的表达水平也与野生型细胞相似，这表明alas2缺失和野生型红母细胞都发育到了成熟的终型红母细胞阶段。然而，与它们相似的形态相反，alas2缺失的最终红母细胞中的铁含量是野生型细胞的两倍。与铁含量的异常增加相一致，通过过氧化代谢物的增加判断，alas2缺失的终代红母细胞比野生型红母细胞更容易被氧化。这些发现表明，alas2缺乏本身并不影响红细胞的分化，但在最终的红细胞中引起异常的铁积累和氧化状态。

alas2 encodes the erythroid specific 8-aniinoleyuIinate synthase (ALAS2), the first enzyme in heme biosynthesis in erythroid cells. In vivo in mice, alas2-null primitive erythroblasts showed a maturation arrest as well as massive cytoplasmic iron accumulation. However, the effect of ALAS2 deficiency, on definitive erythropoiesis still remains unclear, because alas2-null mice died in utero by embryonic day 11.5 before definitive erythropoiesis developed. To clarity the effect of heme deficiency on differentiation of definitive erythroid cells, ES cells lacking the alas2 gene were induced to differentiate specifically into erythroid lineage in vitro, and phenotypes of definitive erythroblasts were examined. In comparison to red cell pellets of Wild-type erythroblasts, alas2-null definitive erythroblasts were totally white due to a marked deficiency of heme, although the morphology was similar to wild-type erythroblasts. Consistent with their similar morphology, levels of erythroid-specific gene expression such as GATA-1, NF-E2 andTER119 in alas2-null definitive erythroblasts were also similar to those of wild-type cells, indicating that both alas2-null and wild-type erythroblasts developed to the stage of mature definitive erythroblasts. In contrast to their similar morphology, however, iron content in alas2-null definitive erythroblasts was twice more than that of wild-type, cells. Consistent with the aberrant increase in iron content, alas2-null definitive erythroblasts were more oxidized compared with wild-type erythroblasts as judged by increased formation of peroxidized metabolites. These findings suggest that alas2 deficiency does not influence erythroid differentiation per se, but induces aberrant iron accumulation and oxidized condition in definitive erythroblasts.