GENETICS OF MYELODYSPLASTIC SYNDROMES

描述(由申请人提供)：该项目的长期目标是识别和表征与骨髓增生异常综合征(MDS)的发展和进展有关的基因。更好地了解这些基因可能会让我们为MDS患者创造有效的治疗方法，并改善结果。间质缺失涉及5号染色体长臂(5q)的一个拷贝，是从头开始和治疗相关MDS(25%-40%的患者)中最常见的细胞遗传学异常之一。我们发现HSPA9是一个候选基因，位于染色体5q31.2常见缺失区，与异常的造血功能有关。我们建议确定HSPA9水平降低引起的造血改变的机制，并分别使用原代人类造血细胞和杂合子Hspa9基因敲除小鼠(Hspa9+/-)来测试Hspa9单倍体不足是否有助于MDS的启动和进展，具体目的如下：特定目标1：我们将确定HSPA9基因敲除后观察到的加速细胞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物理上相互作用，我们将确定它们是否在原代人类CD34+细胞中相互作用。我们将使用报告实验测量HSPA9被敲除后CD34+细胞中P53的激活，并确定与表型相关的候选P53靶基因。我们将使用小鼠基因敲除模型和原代人类CD34+细胞中的RNA干扰来验证P53和候选基因的重要性。具体目标2：我们将在小鼠中确定Hspa9的单倍体不足是否与Rps14单倍体不足协同作用。缺乏一个拷贝的Rps14，一种del(5q)基因的小鼠，会患上大细胞性贫血和祖细胞减少。大多数染色体5q缺失导致Hspa9和Rps14基因的单倍性不足，这表明这两个基因的丢失可能在MDS中协同作用。为了直接测试Hspa9和Rps14单倍体缺陷在MDS启动过程中是否协同作用，我们将Hspa9+/-小鼠与缺乏一个Rps14拷贝的小鼠杂交，并综合表征单杂合或双杂合小鼠的造血功能。具体目标3：我们将确定Hspa9单倍体不足是否会使小鼠患上白血病。MDS的一个毁灭性的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。识别与del(5q31.2)相关白血病相关的基因对于我们理解这种疾病至关重要。为了直接测试Hspa9单倍体缺陷是否有助于白血病的发生，我们将用改良的Moloney鼠白血病病毒(MOL4070LTR逆转录病毒，AML模型)感染Hspa9+/-或野生型小鼠，并监测小鼠AML的发展。我们将使用小鼠模型来鉴定和研究候选的协同基因。

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.