Functional Genomic Dissection of Refractory Anemia

描述（申请人提供）：骨髓增生异常综合征（MDS）的特征是无效造血，最常见的是红系造血，导致称为难治性贫血的表型。在5 q综合征中，MDS的一种亚型，一种单一的遗传病变，一种杂合的染色体5 q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。在上一个资助期，我们确定了一个蛋白质编码基因RPS 14和一个miRNA miR-145，它们导致5 q综合征的异常造血。RPS 14单倍不足导致红细胞生成阻滞（5 q综合征的显性表型）的发现建立了5 q综合征与Diamond Blackfan贫血之间先前未被认识的联系，Diamond Blackfan贫血是一种具有相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的遗传失活引起的。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，导致巨核细胞产生增加和在该综合征中发现的特征性低叶小巨核细胞。在这项更新申请中，我们的目标是了解RPS 14单倍不足以及RPS 14和miR-145联合单倍不足影响的分子基础，并检查这些病变对造血干细胞的影响。此外，已经建立了一种方法来确定染色体缺失内的关键MDS基因，我们将应用我们的方法来确定7 q缺失内的关键基因，这是MDS中另一种常见的遗传病变。在目标1中，我们将研究核糖体单倍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的假设。第一种可能性是红细胞系中p53的选择性激活导致细胞周期停滞和凋亡，导致大红细胞性贫血。或者，或另外，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。在目的2中，我们将检查具有RPS 14、miR-145或RPS 14和miR-145的组合的条件性单倍不足的基因工程小鼠模型中的造血。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，以确定额外的基因是至关重要的MDS的发病机制。已经证明了使用这种方法鉴定染色体5 q上的单倍不足疾病基因的能力，我们接下来将把这种技术的重点放在染色体7 q上的新MDS基因的鉴定上。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重要的见解。 公共卫生相关性：骨髓增生异常综合征（MDS）是一种血液产生异常的疾病，其经常进展为急性白血病，并且在美国每年折磨超过10，000名患者。MDS中两种最常见的遗传异常是5号和7号染色体的部分缺失。我们的目的是了解这些缺失如何导致贫血和MDS，并确定新的治疗策略，这些基因缺失的患者。

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.