MODELING DIAMOND-BLACKFAN ANEMIA

描述（由申请人提供）： 四分之一的Diamond-Blackfan贫血（DBA）家族在编码40 S核糖体亚基蛋白S19的RPS 19中存在杂合突变（Willig et at. 1999年）。患有DBA的儿童具有非再生性红细胞发育不全，伴有各种出生缺陷，包括生长迟缓、颅面畸形、三趾拇指和轴前多指（趾）畸形。鉴于细胞对核糖体的普遍需求，RPS 19表型的组织特异性令人困惑。DBA中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，是丰富的，分散在整个基因组（Lambertsson 1998）。它们是纯合致死的，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）。在早期的研究中，分钟是有用的，这些研究定义了细胞自主性、克隆谱系和发育区室化的概念，因为分钟细胞在体细胞嵌合体中与野生型克隆的竞争很差。除了人类DBA之外，对脊椎动物中的核糖体蛋白（RP）基因突变知之甚少。最近，我的实验室发现自发的小鼠突变Bst（腹部斑点和尾部）是由编码60 S核糖体亚基蛋白L24的Rp 124中的基因内缺失引起的（奥利弗等人，2004）。Bst/+小鼠有脊椎缺陷（尾巴扭结），轴前多趾，中腹斑点和视网膜神经节细胞缺乏。纯合子在植入前死亡。Bst突变导致80%的Rp 124转录物的异常剪接、L24蛋白的截短和体内核糖体生物合成延迟。Bst/+成纤维细胞生长慢于野生型，在G1期停留时间更长，并表现出蛋白质合成的总体速率降低。小鼠BAG和人cDNA转基因纠正这种表型。尚未详细研究Bst/+小鼠的造血能力。（2）在正常和红细胞应激条件下，表征Rps 19敲除小鼠和自发核糖蛋白突变体Bst/+（Rpl 24）和Ts/+（Rpl 38）中的造血;和（3）测试DBA突变的骨髓特异性的两种替代机制-正常红系祖细胞表达相对低水平的Rps 19 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。

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.