Myeloid Tumor Suppressor Gene Discovery from Chromosome Band 7q22

7号染色体的丢失(7号单体)和一段长臂[del(7q)]的缺失 新发和与治疗相关的髓系恶性肿瘤中反复出现的细胞遗传学异常 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 描述了髓系疾病患者的共同缺失片段(CDS)，其特征是 Del(7q)在Q22带内，占大多数情况，第二个CDS在Q32-34带。使用 以有序酵母人工染色体克隆为探针的荧光原位杂交 然后对缺失断裂点在7q22内的白血病进行了实验。这些研究 推测~2.5Mb的CDS含有在髓系中失活的肿瘤抑制基因(TSG) 恶性肿瘤。在此支持期间，我们对此CDS进行了广泛的表征，确定了 克隆了19个已知和新的基因，分析了白血病样本中这些基因的突变 并进行Taqman实时定量聚合酶链式反应实验 测量正常人和白血病人骨髓中的表达水平。这些研究没有发现 在任何候选TSG中，要么是致病突变，要么是表达水平的一致降低。我们也 利用染色体工程技术在~2Mb共线区间两侧引入loxP位点 在小鼠5号染色体上，并产生了条件性和生殖系A5小鼠。我们目前正在 寻求一些策略来识别候选的TSG并在这些菌株中合作基因。我们的 潜在的假设是：(1)髓系TSG存在于这个7q22 CDS或DNA中 CDS附近的片段；以及，(2)该基因要么失活，要么显示减少 7号单体或Del(7q)在髓系恶性肿瘤中的表达。我们将利用我们能够 已经产生和收集了不同的白血病样本，通过两个目标来追求这一假说： (1)利用杂合子和纯合子A5小鼠鉴定候选的人7q22 TSGs和其他 在白血病发生中与A5缺失协同作用的基因；以及(2)整合由 项目2和3以及其他研究人员在间隔时间内对候选髓系TSG进行优先排序 紧邻7q22 CDS的近端，可能与7q22 CDS的丢失有关。我们会 立即询问人类白血病样本中位于2.1Mb区间的基因突变 目前的CDS的近端，并开发试剂来模拟这一更近端的节段在 那只老鼠。项目4与项目2和3具有高度的互动性；这些项目一起寻求确定 基因突变谱，以及导致烷化剂诱发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.