Myeloid Tumor Suppressor Gene Discovery from Chromosome Band 7q22

7号染色体的缺失（7号单体）和长臂[del（7 q）]的一段缺失是 新发和治疗相关的骨髓恶性肿瘤中复发的细胞遗传学异常， 与预后不良有关。在该奖项支持的先前研究中，细胞遗传学分析 描绘了一个常见的缺失节段（CDS）的骨髓疾病患者的特点是， del（7 q）位于带q22内，占大多数情况，第二个CDS位于带q32-34中。使用 有序酵母人工染色体克隆集为探针，荧光原位杂交 然后在具有7 q22内的缺失断点的白血病上进行实验。这些研究 暗示一个~2.5 Mb的CDS含有一个在骨髓中失活的肿瘤抑制基因（TSG）， 恶性肿瘤。在此支持期间，我们对该CDS进行了广泛的表征， 克隆了19个已知的和新的基因，分析了白血病样本中的突变， 候选TSG，并进行Taqman真实的时间定量聚合酶链反应实验， 测量正常和白血病人骨髓中的表达水平。这些研究并没有揭示 致病性突变或任何候选TSG中表达水平的一致降低。我们也 利用染色体工程技术，在~2 Mb同线区间两侧引入loxP位点， 在小鼠5号染色体上，并产生了条件和种系A5小鼠。我们目前正在 寻求一些策略来鉴定这些菌株中的候选TSG和协作基因。我们 基本假设是：（1）髓系TSG位于7 q22 CDS或DNA中， 片段近端的CDS;和，（2）该基因是失活或证明减少 在具有单体7或缺失（7 q）的骨髓恶性肿瘤中表达。我们将利用我们 已经产生了各种各样的白血病样本，通过两个目标来追求这一假设： (1)使用杂合和纯合A5小鼠来鉴定候选人7 q22 TSG和其他 在白血病发生中与A5缺失合作的基因;和（2）整合由以下研究开发的新数据： 项目2和3以及其他研究人员在间隔期间优先考虑候选骨髓TSG 紧邻7 q22 CDS，可能与7 q22 CDS的丢失协同作用。我们将 立即询问人类白血病标本中位于2.1 Mb间隔内的基因突变 在当前CDS的近端，并开发用于模拟这种更近端段缺失的试剂， 鼠标项目4与项目2和项目3高度互动;这些项目共同努力， 基因突变谱和导致烷化剂诱导的t-AML的遗传途径。

Loss of chromosome 7 (monosomy 7) and deletion of a segment of the long arm [del(7q)] are recurring cytogenetic abnormalities in de novo and therapy-related myeloid malignancies that are associated with a poor prognosis. In previous studies supported by this award, cytogenetic analysis delineated a commonly-deleted segment (CDS) in patients with myeloid disorders characterized by a del(7q) within band q22 that accounts for most cases, and a second CDS in bands q32-34. Using an ordered set of yeast artificial chromosome clones as probes, fluorescence in situ hybridization experiments were then performed on leukemias with deletion breakpoints within 7q22. These studies implicated a ~2.5 Mb CDS as harboring a tumor suppressor gene (TSG) that is inactivated in myeloid malignancies. During this period of support, we have extensively characterized this CDS, identified and cloned 19 known and novel genes from the interval, analyzed leukemia samples for mutations in these candidate TSGs, and performed Taqman real time quantitative polymerase chain reaction experiments to measure expression levels in normal and leukemic human bone marrows. These studies did not uncover either pathogenic mutations or a consistent reduction in expression levels in any candidate TSG. We also harnessed chromosome engineering technology to introduce loxP sites flanking a ~2 Mb syntenic interval on mouse chromosome 5, and have generated conditional and germline A5 mice. We are currently pursuing a number of strategies to identify candidate TSGs and cooperating genes in these strains. Our underlying hypotheses are: (1) that a myeloid TSG resides either within this 7q22 CDS or in a DNA segment proximal to this CDS; and, (2) that this gene is either inactivated or demonstrates reduced expression in myeloid malignancies with monosomy 7 or by a del(7q). We will exploit reagents that we have generated and a diverse collection of leukemia samples to pursue this hypothesis through two aims: (1) to use heterozygous and homozygous A5 mice to identify candidate human 7q22 TSGs and other genes that cooperate with the A5 deletion in leukemogenesis; and (2) to integrate new data developed by Projects 2 and 3 and by other researchers to prioritize candidate myeloid TSGs in the interval immediately proximal to the 7q22 CDS that might cooperate with loss of the 7q22 CDS. We will interrogate human leukemia specimens for mutations in genes located in a 2.1 Mb interval immediately proximal to the current CDS, and develop reagents for modeling loss of this more proximal segment in the mouse. Project 4 is highly interactive with Projects 2 and 3; together, these projects seek to identify the spectrum of genetic mutations, and genetic pathways leading to alkylating-agent induced t-AML.