MODELING DIAMOND-BLACKFAN ANEMIA

塑造钻石-黑粉丝贫血症

• 批准号: 6878323
• 负责人: Thomas M. Glaser
• 金额: $ 14.77万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6878323
• 关键词: alleles; aminohydrolases; biomarker; blood cell count; cell differentiation; cell line; cell proliferation; congenital aplastic anemia; disease /disorder etiology; disease /disorder model; embryonic stem cell; erythropoietin; flow cytometry; gene expression; gene mutation; genetic models; genetically modified animals; hematopoiesis; hemoglobin F; laboratory mouse; model design /development; phenylhydrazines; reticulocytes; ribosomal proteins; transposon /insertion element

DESCRIPTION (provided by applicant): One-quarter of families with Diamond-Blackfan anemia (DBA) have heterozygous mutations in RPS19, which encodes the 40S ribosomal subunit protein S19 (Willig et at. 1999). Children with DBA have a nonregenerative red blood cell aplasia with variable birth defects, including growth retardation, craniofacial malformations, triphalangeal thumbs and preaxial polydactyly. In view of the universal cellular requirement for ribosomes, the tissue specificity of RPS19 phenotypes is puzzling. The mechanism of erythroid marrow failure in DBA is unknown. In Drosophila, riboprotein mutations, termed Minutes, are abundant and dispersed across the genome (Lambertsson 1998). They are homozygous lethal and have similar heterozygous phenotypes consisting of smaller, thinner bristles and profoundly delayed development (Schultz 1929). Minutes were instrumental in early studies that defined concepts of cell autonomy, clonal lineage and developmental compartmentation, as Minute cells compete poorly with wild type clones in somatic mosaics. Apart from human DBA, little is known about ribosomal protein (RP) gene mutations in vertebrates. Recently, my laboratory discovered that the spontaneous mouse mutation Bst (belly spot and tail) is caused by an intragenic deletion in Rpl24, which encodes the 60S ribosomal subunit protein L24 (Oliver et al. 2004). Bst/+ mice have vertebral defects (tail kinks), preaxial polydactyly, midventral spotting and a deficiency of retinal ganglion cells. Homozygotes die before implantation. The Bst mutation causes aberrant splicing in 80% of Rpl24 transcripts, truncation of L24 protein, and delayed ribosome biogenesis in vivo. Bst/+ fibroblasts grow slower than wild type, remain longer in G1 phase, and exhibit decreased overall rates of protein synthesis. Mouse BAG and human cDNA transgenes correct this phenotype. The hematopoietic capacity of Bst/+ mice has not been explored in detail. In this proposal, we aim to: (1) generate mice with null and hypomorphic mutations in Rps19, using selected gene trap ES cell lines; (2) characterize hematopoiesis in Rps19 knockout mice and the spontaneous riboprotein mutants Bst/+ (Rpl24) and Ts/+ (Rpl38), under normal and erythroid stress conditions; and (3) test two alternative mechanisms for the marrow specificity of DBA mutations - that normal erythroid progenitors express relatively low levels of Rps19 mRNA, making them particularly sensitive to reduced gene dosage, and that S19 has an extraribosomal function to promote red cell differentiation.

描述（由申请人提供）： 四分之一患有 Diamond-Blackfan 贫血 (DBA) 的家族在 RPS19 中存在杂合突变，RPS19 编码 40S 核糖体亚基蛋白 S19（Willig 等，1999）。患有 DBA 的儿童患有非再生性红细胞再生障碍性贫血，并伴有多种出生缺陷，包括生长迟缓、颅面畸形、三指拇指和轴前多指畸形。鉴于细胞对核糖体的普遍需求，RPS19 表型的组织特异性令人费解。 DBA 红系骨髓衰竭的机制尚不清楚。在果蝇中，核糖蛋白突变（称为“分钟”）丰富且分散在整个基因组中（Lambertsson 1998）。它们是纯合致死的，并具有相似的杂合表型，包括更小、更薄的刚毛和严重延迟的发育（Schultz 1929）。分钟在定义细胞自主性、克隆谱系和发育区划概念的早期研究中发挥了重要作用，因为分钟细胞与体细胞嵌合体中的野生型克隆竞争较差。除了人类 DBA 之外，人们对脊椎动物核糖体蛋白 (RP) 基因突变知之甚少。最近，我的实验室发现小鼠自发突变 Bst（腹斑和尾部）是由 Rpl24 基因内缺失引起的，Rpl24 编码 60S 核糖体亚基蛋白 L24 (Oliver et al. 2004)。 Bst/+ 小鼠有脊椎缺陷（尾部扭结）、轴前多指畸形、中腹斑点和视网膜神经节细胞缺陷。纯合子在植入前死亡。 Bst 突变导致 80% 的 Rpl24 转录本出现异常剪接、L24 蛋白截短以及体内核糖体生物合成延迟。 Bst/+ 成纤维细胞比野生型生长得更慢，在 G1 期停留的时间更长，并且蛋白质合成的总体速率降低。小鼠 BAG 和人类 cDNA 转基因纠正了这种表型。 Bst/+小鼠的造血能力尚未得到详细研究。在本提案中，我们的目标是：（1）使用选定的基因捕获 ES 细胞系，产生 Rps19 无效突变和低等态突变的小鼠； (2) 表征正常和红细胞应激条件下 Rps19 敲除小鼠和自发核糖蛋白突变体 Bst/+ (Rpl24) 和 Ts/+ (Rpl38) 的造血功能； (3)测试DBA突变骨髓特异性的两种替代机制——正常红细胞祖细胞表达相对较低水平的Rps19 mRNA，使它们对减少的基因剂量特别敏感，并且S19具有促进红细胞分化的核糖体外功能。