MODELING DIAMOND-BLACKFAN ANEMIA

描述(由申请人提供)： 四分之一的钻石-布莱克范贫血(DBA)家系存在RPS19的杂合突变，RPS19编码40S核糖体亚单位蛋白S19(William et at.1999年)。患有DBA的儿童有一种非再生性红细胞再生障碍性疾病，伴有不同的出生缺陷，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，核蛋白突变，称为分钟，是丰富的，并分散在整个基因组(Lambertsson 1998)。它们是纯合子致命性的，具有相似的杂合子表型，包括更小、更薄的刚毛和严重延迟的发育(Schultz 1929)。在早期的研究中，由于微小细胞在体细胞嵌合体中与野生型克隆竞争较差，会议纪要对定义细胞自主、克隆谱系和发育分区的概念起到了重要作用。除了人类DBA外，关于脊椎动物核糖体蛋白(RP)基因突变的研究知之甚少。最近，我的实验室发现小鼠的自发突变BST(腹点和尾巴)是由编码60S核糖体亚单位蛋白L24的Rpl24的基因内缺失引起的(Oliver等人。2004年)。BST/+小鼠有脊椎缺陷(尾部扭结)、轴前多指、腹中斑点和视网膜神经节细胞缺乏。纯合子在植入之前就会死亡。BST突变导致80%的RPL24转录本的异常剪接，L24蛋白的截断，以及体内核糖体生物发生的延迟。与野生型相比，BST/+成纤维细胞生长慢，处于G1期的时间更长，蛋白质合成的总体速率降低。小鼠BAG和人类的cdna转基因可以纠正这种表型。BST/+小鼠的造血能力还没有得到详细的研究。在这项建议中，我们的目标是：(1)使用精选的基因TRAP ES细胞系建立Rps19零突变和亚形突变小鼠；(2)研究正常和红系应激条件下Rps19基因敲除小鼠和自发核蛋白突变体Bst/+(Rpl24)和ts/+(Rpl38)的造血功能；以及(3)测试DBA突变骨髓特异性的两种替代机制--正常红系祖细胞表达相对较低的Rps19 mRNA，使他们对基因剂量减少特别敏感，并且S19具有促进红细胞分化的体外功能。

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.