GENETIC ANALYSIS--DYSERYTHROPOIETIC ANEMIA IN ZEBRAFISH

遗传分析--斑马鱼红细胞生成障碍性贫血

• 批准号: 2905017
• 负责人: BARRY H PAW
• 金额: $ 12.04万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2905017
• 关键词: alternatives to animals in research; artificial chromosomes; chromosome walking; comparative genomic hybridization; developmental genetics; dyserythropoietic anemia; embryo /fetus culture; gene expression; gene mutation; genetic markers; genetic models; hematopoiesis; hematopoietic stem cells; human genetic material tag; human tissue; in situ hybridization; molecular cloning; organ culture; phenotype; single strand conformation polymorphism; zebrafish

DESCRIPTION (adapted from the application) Hematopoiesis is a cascade of events leading from a pluripotent stem cell to terminally differentiated blood cells. Defects in this signaling cascade can results in dyserythropoiesis and reveal important molecular events and underlying processes in erythrocyte differentiation. Dyserythropoiesis is observed in a variety of congenital and acquired anemias indicative of bone marrow stress, such as congenital dyserythropoietic anemias (CDA), myelodysplastic syndromes, leukemia megaloblastic anemia, HIV infection, recovery from bone marrow transplantation, and hemolytic anemia. A full understanding of this marrow stress response awaits identification of genes involved in this cascade of events. To achieve this understanding, we propose to take an approach using zebrafish (Danio rerio) as a genetic model system. The combined genetic and embryological advantages of zebrafish make it an ideal model system to study the developmental events of hematopoiesis. Nearly 45 zebrafish mutations, representing about 25 genes, defective in hematopoiesis have been identified. One of these zebrafish blood mutations called retsina is an autosomal recessive disorder which results in a profound anemia at 3 days post-fertilization. Using molecular markers and histologic features, we showed that myeloid and lymphoid lineages are unaffected by this mutation. Retsina mutants that survive to adulthood show complete maturation arrest at the polychromatophilic erythroblast stage with a large percentage of cells have bilobed nuclei. The striking histologic feature of retsina mutant erythroblast are most reminiscent of smears from human patients with dyserythropoietic anemias, particularly HEMPAS (Hereditary Erythroblastic Multinuclearity with Positive Acidified Serum, CDA type 2). The specific aim of this proposal is to clone the retsina gene by a positional cloning strategy in zebrafish. I have mapped the retsina locus with a closely linked marker (0.7 cM based on 10/1380 meiotic recombinants). Candidate genes have been isolated and excluded for retsina using linkage analysis. Genetically linked YAC, BAC and PAC clones have been isolated, and a chromosome walk has been undertaken. After the isolation of the retsina gene in zebrafish, its human homolog will be cloned and examined in human patients with CDA and other dyserythropoietic anemias. The isolation of the human gene will be facilitated by the extensive synteny within this region of the zebrafish and human genome. The cloning of the retsina gene would further our understanding of molecular events in both normal erythrocyte differentiation and dyserythropoietic processes in CDA, myelodysplasia.

描述（改编自应用程序） 造血是由多能干细胞引发的一系列事件 至终末分化的血细胞。该信号的缺陷 级联反应可导致红细胞生成障碍并揭示重要的分子机制 红细胞分化的事件和潜在过程。 红细胞生成障碍可见于多种先天性和后天性 贫血表明骨髓应激，例如先天性贫血 红细胞生成障碍性贫血 (CDA)、骨髓增生异常综合征、白血病 巨幼细胞贫血、HIV 感染、骨髓恢复 移植、溶血性贫血。充分了解这个骨髓 应激反应等待参与这一级联反应的基因的鉴定 事件。为了实现这一理解，我们建议采取一种方法 使用斑马鱼（Danio rerio）作为遗传模型系统。合并后的 斑马鱼的遗传和胚胎学优势使其成为理想的模型 研究造血发育事件的系统。近 45 斑马鱼突变，代表大约 25 个基因，有缺陷 造血功能已被鉴定。这些斑马鱼血液突变之一 retsina 是一种常染色体隐性遗传疾病，会导致 受精后3天出现严重贫血。使用分子标记和 组织学特征，我们表明骨髓和淋巴谱系是 不受此突变的影响。能存活到成年的视网膜突变体 显示多染性成红细胞完全成熟停滞 大部分细胞具有双叶细胞核的阶段。引人注目的 retsina 突变型成红细胞的组织学特征最让人想起 来自患有红细胞生成障碍性贫血的人类患者的涂片，特别是 HEMPAS（具有阳性酸化的遗传性成红细胞多核性 血清，CDA 型 2)。该提案的具体目标是克隆 retsina 基因通过定位克隆策略在斑马鱼中进行。我已经映射了 具有紧密连锁标记的 retsina 基因座（0.7 cM，基于 10/1380 减数分裂重组体）。候选基因已被分离并排除 使用连锁分析的 retsina。基因连锁的 YAC、BAC 和 PAC 克隆 已被分离，并且已经进行了染色体行走。之后 在斑马鱼中分离retsina基因，其人类同源物将是 在患有 CDA 和其他红细胞生成障碍的人类患者中进行克隆和检查 贫血。人类基因的分离将有利于 斑马鱼和人类基因组的该区域内存在广泛的同线性。 retsina 基因的克隆将进一步加深我们对 正常红细胞分化和 CDA 中的红细胞生成障碍、骨髓增生异常。