GENETICS OF MYELODYSPLASTIC SYNDROMES

描述（由申请人提供）：该项目的长期目标是鉴定和表征导致骨髓增生异常综合征（MDS）发生和进展的基因。更好地了解这些基因可能使我们能够为MDS患者创造有效的治疗方法并改善预后。涉及5号染色体长臂（5q）的一个拷贝的间质缺失是新发和治疗相关MDS（25-40%的患者）中最常见的细胞遗传学异常之一。我们确定HSPA9是位于染色体5q31.2常缺失区域的候选基因，该区域有助于改变造血功能。我们提出明确HSPA9水平降低导致造血功能改变的机制，并分别使用原代人造血细胞和杂合HSPA9敲除小鼠（HSPA9 +/-）检测HSPA9单倍不足是否有助于MDS的发生和进展，具体目的如下：具体目的1：我们将确定HSPA9敲除后观察到的加速凋亡和细胞周期动力学改变是否依赖于p53。由于已知HSPA9在几种模型中与p53物理相互作用，我们将确定它们是否在原代人CD34+细胞中相互作用。在HSPA9基因敲除后，我们将使用报告基因法测量CD34+细胞中p53的激活情况，并确定导致表型的候选p53靶基因。我们将利用小鼠敲除模型和人原代CD34+细胞的RNA干扰来验证p53和候选基因的重要性。特异性目的2：我们将在小鼠中确定Hspa9单倍性不足是否与Rps14单倍性不足有协同作用。缺乏Rps14（一种del（5q）基因）拷贝的小鼠会出现大细胞性贫血和祖细胞减少。大多数染色体5q缺失会导致Hspa9和Rps14基因的单倍不足，这表明这两个基因的缺失可能在MDS中起作用。为了直接检测Hspa9单倍不足和Rps14是否在MDS启动过程中协同作用，我们将Hspa9+/-小鼠与缺少一个Rps14拷贝的小鼠交叉，全面表征单杂合或双杂合小鼠的造血功能。特异性目的3：我们将确定Hspa9单倍不足是否使小鼠易患白血病。MDS的一个毁灭性并发症是白血病的发展，这与跨越染色体5q31.2的缺失高度相关。识别导致del（5q31.2）相关白血病的基因对我们了解这种疾病至关重要。为了直接测试Hspa9的单倍不足是否与白血病的发展有关，我们将用改良的Moloney小鼠白血病病毒（MOL4070LTR逆转录病毒，一种成熟的AML模型）感染Hspa9+/-或野生型的小鼠，并监测小鼠的AML发展情况。我们将利用小鼠模型识别和研究候选的合作基因。

DESCRIPTION (provided by applicant): The long-term goal of this project is to identify and characterize genes that contribute to the development and progression of myelodysplastic syndromes (MDS). A better understanding of these genes may allow us to create effective therapies for patients with MDS and improve outcomes. An interstitial deletion involving one copy of the long arm of chromosome 5 (5q) is among the most common cytogenetic abnormalities in de novo and therapy-related MDS (25-40% of patients). We identified HSPA9 as a candidate gene located on the chromosome 5q31.2 commonly deleted region that contributes to altered hematopoiesis. We propose to define the mechanism of altered hematopoiesis caused by reduced levels of HSPA9 and to test whether Hspa9 haploinsufficiency contributes to MDS initiation and progression using primary human hematopoietic cells and heterozygous Hspa9 knock-out mice (Hspa9+/-), respectively, in the following specific aims: Specific Aim 1: We will determine whether the accelerated apoptosis and altered cell cycle kinetics observed following HSPA9 knockdown are p53-dependent. Because HSPA9 is known to physically interact with p53 in several models, we will determine whether they interact in primary human CD34+ cells. We will measure p53 activation in CD34+ cells following HSPA9 knockdown using a reporter assay and identify candidate p53-target genes responsible for the phenotype. We will validate the importance of p53 and the candidate genes using murine knockout models and RNA interference in primary human CD34+ cells. Specific Aim 2: We will establish whether haploinsufficiency of Hspa9 cooperates with Rps14 haploinsufficiency in mice. Mice lacking one copy of Rps14, a del(5q) gene, develop a macrocytic anemia and reduced progenitor cells. Most chromosome 5q deletions produce haploinsufficiency of both Hspa9 and Rps14 genes, suggesting that loss of both genes may cooperate in MDS. To directly test whether haploinsufficiency of Hspa9 and Rps14 cooperate during MDS initiation, we will intercross Hspa9+/- mice with mice lacking one copy of Rps14 and comprehensively characterize the hematopoiesis of singly or doubly heterozygous mice. Specific Aim 3: We will determine whether Hspa9 haploinsufficiency predisposes mice to develop leukemia. A devastating complication of MDS is the development of leukemia, which is highly-associated with deletions spanning chromosome 5q31.2. Identifying genes that contribute to del(5q31.2)-associated leukemia is vital to our understanding of this disease. To directly test whether haploinsufficiency of Hspa9 contributes to leukemia development, we will infect Hspa9+/- or wild-type littermate mice with a modified Moloney murine leukemia virus (MOL4070LTR retrovirus, a well established AML model) and monitor mice for the development of AML. We will identify and study candidate cooperating genes using mouse models.