A novel ENU-generated truncation mutation lacking the spectrin-binding and C-terminal regulatory domains of Ank1 models severe hemolytic hereditary spherocytosis
A novel ENU-generated truncation mutation lacking the spectrin-binding and C-terminal regulatory domains of Ank1 models severe hemolytic hereditary spherocytosis
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DOI：
10.1016/j.exphem.2010.12.009
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发表时间：
2011-03-01
影响因子：
2.6
通讯作者：
Stanford, William L.
中科院分区：
文献类型：
--
作者：
Hughes, Michael R.;Anderson, Nicole;Stanford, William L.
目标。遗传性球形红细胞增多症（HS）是一组异质性的自发产生和遗传性红细胞疾病，范围从非常轻微的亚临床病例到严重和危及生命的病例，其症状与分子水平上突变的严重程度直接相关。我们研究了一种新的小鼠模型，其中具有轻度HS诊断特征的杂合子和幸存的纯合子表型为严重溶血性HS。材料与方法。我们使用n -乙基-n -亚硝基脲诱变技术在小鼠基因组中产生随机点突变，并使用显性筛选技术鉴定人类造血疾病小鼠模型。HS菌株的基因定位显示，Ank1（菌株名称：Ank1(E924X)）的第27外显子单碱基翻转引起了一个独特的框内无义突变。采用常规的造血、病理、生化和细胞生物学分析，我们在生化、细胞和病理生理水平上对杂合子和纯合子Ank1（E924X）小鼠进行了表征。虽然Ank1(E924X/E924X)：红细胞内缺乏丰富的全长锚蛋白-1亚型，但n-末端表位锚蛋白-1抗体显示出与截断突变锚蛋白-1的理论大小一致的条带。使用结构域特异性抗体，我们进一步证明该蛋白缺乏谱蛋白结合结构域和c端调节结构域。最后，使用检测替代Ank1转录产物c端残基的抗血清，我们发现了在野生型或Ank1（E924X）杂合小鼠的红细胞中未观察到的独特免疫反应带，包括与全长锚蛋白大小相似的条带。Ank1（E924X）菌株为研究Ank1和HS模型提供了新的工具。(C) 2011 ISEH -血液学与干细胞学会。Elsevier Inc.出版。
Objective. Hereditary spherocytosis (HS) is a heterogeneous group of spontaneously arising and inherited red blood cell disorders ranging from very mild subclinical cases to severe and life-threatening cases, with symptoms linked directly to the severity of the mutation at the molecular level. We investigated a novel mouse model in which the heterozygotes present with the diagnostic hallmarks of mild HS and surviving homozygotes phenocopy severe hemolytic HS.Materials and Methods. We used N-ethyl-N-nitrosourea mutagenesis to generate random point mutations in the mouse genome and a dominant screen to identify mouse models of human hematopoietic disease. Gene mapping of the HS strain revealed a unique in-frame nonsense mutation arising from a single base transversion in exon 27 of Ank1 (strain designation: Ank1(E924X)). Employing conventional hematopoietic, pathological, biochemical, and cell biology assays, we characterized heterozygous and homozygous Ank1(E924X) mice at the biochemical, cellular, and pathophysiological levels.Results. Although Ank1(E924X/E924X): red blood cell ghosts lack abundant full-length ankyrin-1 isoforms, N-terminal epitope ankyrin-1 antibodies reveal a band consistent with the theoretical size of a truncated mutant ankyrin-1. Using domain-specific antibodies, we further show that this protein lacks both a spectrin-binding domain and a C-terminal regulatory domain. Finally, using antisera that detect C-terminal residues of the products of alternative Ank1 transcripts, we find unique immunoreactive bands not observed in red blood cell ghosts from wild-type or Ank1(E924X) heterozygous mice, including a band similar in size to full-length ankyrin-1.Conclusions. The Ank1(E924X) strain provides a novel tool to study Ank1 and model HS. (C) 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.