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

alas 2编码红系细胞特异性8-氨基喹啉酸合酶（ALAS 2），其是红系细胞中血红素生物合成中的第一个酶。在小鼠体内，alas 2缺失的原始成红细胞表现出成熟停滞以及大量的细胞质铁积累。然而，ALAS 2缺陷对确定性红细胞生成的影响仍然不清楚，因为ALAS 2缺陷小鼠在胚胎第11.5天在确定性红细胞生成形成之前在子宫内死亡。为了明确血红素缺乏对定形红系细胞分化的影响，在体外诱导缺乏alas 2基因的ES细胞特异性分化为红系谱系，并检查定形成红细胞的表型。与野生型成红细胞的红细胞团块相比，alas 2缺失的定形成红细胞由于血红素的显著缺乏而完全为白色，尽管形态与野生型成红细胞相似。与它们相似的形态学相一致，在alas 2缺失的定形成红细胞中红细胞特异性基因表达水平如加塔-1、NF-E2和TER 119也与野生型细胞相似，表明alas 2缺失和野生型成红细胞都发育到成熟定形成红细胞的阶段。然而，与它们相似的形态相反，alas 2缺失的定形成红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一致，与野生型成红细胞相比，alas 2-null定形成红细胞被更多氧化，这是通过增加过氧化代谢产物的形成来判断的。这些发现表明，alas 2缺乏本身并不影响红系分化，但诱导异常铁积累和氧化条件的定形成红细胞。

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.