Functional Genomic Dissection of Refractory Anemia

描述（由申请人提供）：骨髓增生异常综合征（MDS）的特点是造血功能无效，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的一种亚型，即5q染色体的单遗传病变，杂合间质缺失，导致高度可重复的临床表型，尽管这种表型的分子基础以前是未知的。在之前的资助期内，我们发现了一个蛋白质编码基因RPS14和一个miRNA miR-145，它们有助于5q-综合征的异常造血。研究发现，RPS14单倍不全导致5q-综合征的主要表型红细胞生成障碍，这一发现在5q-综合征和Diamond Blackfan贫血之间建立了以前未被认识到的联系。Diamond Blackfan贫血是一种先天性疾病，具有相似的表型，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的单倍不足导致关键靶基因FLI-1的表达增加，导致巨核细胞的产生增加，并在该综合征中发现特征性的微巨核细胞。在这项更新应用中，我们旨在了解RPS14单倍功能不全以及RPS14和miR-145联合单倍功能不全影响的分子基础，并检查这些病变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MDS关键基因的方法之后，我们将应用我们的方法来识别7q缺失中的关键基因，7q缺失是MDS中另一种常见的遗传病变。在目的1中，我们将研究核糖体单倍体功能不全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的假设。第一种可能是红细胞系中p53的选择性激活导致细胞周期阻滞和细胞凋亡，从而导致大细胞性贫血。另外，异常的核糖体生物发生可能导致mRNA翻译失调和特定蛋白质的产生改变。我们将在原代人骨髓祖细胞中检验这两种假设。在Aim 2中，我们将在RPS14、miR-145或RPS14和miR-145的组合条件单倍不足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种病变对造血干细胞功能的影响。在Aim 3中，我们将扩展我们的RNA干扰筛选方法，以识别对MDS发病机制至关重要的其他基因。在证明了使用这种方法鉴定5q染色体上的单倍体功能不全疾病基因的能力之后，我们下一步将把这项技术的重点放在鉴定7q染色体上的新型MDS基因上。总的来说，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. PUBLIC HEALTH RELEVANCE: Myelodysplastic syndrome (MDS) is a disease of abnormal blood production that frequently progresses to acute leukemia and afflicts over 10,000 patients per year in the United States. Two of the most common genetic abnormalities in MDS are deletions of parts of Chromosomes 5 and 7. We aim to understand how these deletions cause anemia and MDS and to identify novel therapeutic strategies for patients with these genetic deletions.