Functional Genomic Dissection of Refractory Anemia

描述(申请人提供)：骨髓增生异常综合征(MDS)的特征是无效的造血，最常见的是红系，导致一种称为难治性贫血的表型。在5q综合征中，MDS的一个亚型，一个单一的遗传病变，5q染色体的杂合子，间质缺失，导致高度可重复性的临床表型，尽管该表型的分子基础先前尚不清楚。在之前的资助期间，我们发现了一个蛋白质编码基因RPS14和一个miRNA miR-145，它们与5q综合征的异常造血有关。RPS14单倍体不足导致5q综合征的主要表型红细胞生成受阻，这一发现在5q综合征和钻石黑扇贫血之间建立了以前不为人知的联系，钻石黑扇贫血是一种具有类似表型的先天性疾病，也是由编码核糖体蛋白的基因的一个等位基因失活引起的。我们发现miR-145的单倍体不足导致关键靶基因Fli-1表达增加，导致巨核细胞产生增加，并在这种综合征中发现特征的小巨核细胞减数。在这一新的应用中，我们的目标是了解RPS14单倍体功能不全和RPS14和miR-145联合单倍体功能不全影响的分子基础，并检测这些损伤对造血干细胞的影响。此外，在建立了一种在染色体缺失中识别关键MDS基因的方法后，我们将应用我们的方法来识别7q缺失中的关键基因，这是MDS中另一种常见的遗传损伤。在目标1中，我们将研究核糖体单倍体不足导致红细胞生成受损的机制。目前的证据支持两个非排他性假设。第一种可能性是，红系细胞中P53的选择性激活导致细胞周期停滞和细胞凋亡，导致大细胞性贫血。或者，或者另外，核糖体生物发生异常可能导致mRNA翻译失调和特定蛋白质的产生改变。我们将在原代人类骨髓祖细胞中检验这两种假说。在目标2中，我们将在RPS14、miR-145或RPS14和miR-145组合的条件单倍性功能不全的基因工程小鼠模型中检测造血功能。特别是，我们将使用这些模型来检查每个病变对造血干细胞功能的影响。在目标3中，我们将扩展我们的RNA干扰筛选方法，以确定更多对MDS发病机制至关重要的基因。在证明了使用这种方法识别染色体5q上的单倍体功能不全疾病基因的能力后，我们接下来将把这项技术集中在识别染色体7q上的新的MDS基因上。总而言之，这些实验将为骨髓增生异常综合征的分子基础提供关键的见解。

DESCRIPTION (provided by applicant): Myelodysplastic syndrome (MDS) is characterized by ineffective hematopoiesis, most commonly of the erythroid lineage, resulting in a phenotype termed refractory anemia. In the 5q- syndrome, a subtype of MDS, a single genetic lesion, a heterozygous, interstitial deletion of Chromosome 5q, causes a highly reproducible clinical phenotype, though the molecular basis of this phenotype was previously unknown. In the previous funding period, we identified one protein-coding gene, RPS14, and one miRNA, miR-145, that contribute to abnormal hematopoiesis in the 5q- syndrome. The finding that RPS14 haploinsufficiency causes a block in erythropoiesis, the dominant phenotype of the 5q- syndrome, established a previously unrecognized link between the 5q- syndrome and Diamond Blackfan Anemia, a congenital disorder with a similar phenotype that is also caused by genetic inactivation of one allele of genes encoding ribosomal proteins. We found that haploinsufficiency for miR-145 causes increased expression of a key target gene, FLI-1, leading to increased megakaryocyte production and the characteristic hypolobated micromegakaryocytes found in this syndrome. In this renewal application, we aim to understand the molecular basis for the effects of RPS14 haploinsufficiency and combined haploinsufficiency for RPS14 and miR-145, and to examine the effects of these lesions on hematopoietic stem cells. In addition, having established an approach to the identification of key MDS genes within chromosomal deletions, we will apply our methodology to identify a key gene within the 7q deletion, another common genetic lesion in MDS. In Aim 1, we will investigate the mechanism whereby ribosomal haploinsufficiency leads to impaired erythropoiesis. Current evidence supports two non-exclusive hypotheses. The first possibility is that selective activation of p53 in the erythroid lineage causes cell cycle arrest and apoptosis, resulting in macrocytic anemia. Alternatively, or additionally, abnormal ribosome biogenesis could lead to dysregulated mRNA translation and altered production of specific proteins. We will examine both hypotheses in primary human bone marrow progenitor cells. In Aim 2, we will examine hematopoiesis in genetically engineered murine models with conditional haploinsufficiency of RPS14, miR-145, or the combination of RPS14 and miR-145. In particular, we will use these models to examine the effect of each lesion on hematopoietic stem cell function. In Aim 3, we will extend our RNA interference screening approach to identify additional genes that are critical for the pathogenesis of MDS. Having demonstrated the ability to use this approach to identify haploinsufficiency disease genes on Chromosome 5q, we will next focus this technology towards the identification of novel MDS genes on Chromosome 7q. In aggregate, these experiments will provide critical insight into the molecular basis of myelodysplastic syndrome.