Genetic Modifers of Sickle Cell Disease

描述（由申请人提供）： 将利用基于微阵列的表达谱分析和定位克隆 来确定改变镰状细胞病严重程度的基因。初始 实验将在镰状细胞病的小鼠模型中进行， 再现了大多数（如果不是全部的话）病症的病理学（科学278：873- 877）。 876）。该模型是通过靶向删除以下基因创建的： 球蛋白基因随后引入人类α、γ和β镰状细胞 珠蛋白转基因”进入生殖细胞系。这些动物只合成人类 成人红细胞血红蛋白。有趣的是，据观察，在人类中， 这些远系杂交的镰状小鼠的疾病严重程度范围是惊人的。基因 通过比较基因表达， 严重贫血的小鼠与不太严重的动物的概况 贫血最初的实验将确定血液和 肾脏的50只动物，具有广泛的疾病严重程度。 表达数据的层次聚类分析将定义表达数据的组。 动物，这些群体将与疾病的严重程度相关。一个主要 目的是确定预测严重疾病的表达谱。因此，我们认为， 将通过克隆固定多个严重受累动物的基因型; 也就是说，将从研究中的所有50只动物中培养原代成纤维细胞 并将细胞核移植到去核卵中， 发展为严重疾病和非严重影响的对照。表达 然后将在10、20和30日龄时测定曲线，以确定 贫血和肾脏病变之前的特征该分析将定义一个 预测严重疾病的特征。表型比较 克隆的兄弟姐妹也将揭示基因和 环境/随机因素对疾病进展和严重程度的影响。 贫血与SNPs的连锁分析也将用于定位基因， 改变疾病。回交到C57 B]/6上的镰状小鼠;1 背景一致发展为严重贫血。这些动物将与 镰状动物是回交！我发现了一种菌株 严重贫血 将对F2动物进行连锁分析，以确定 SNPS与严重疾病相关。候选修饰基因与 SNP将在公共和私人数据库中识别， 这些基因的功能意义将通过ES中的修饰来检查 镰状小鼠的细胞。表达谱也将是 测定人类镰状病人的血液。一种“镰刀芯片”， 在小鼠中鉴定的修饰基因的人类同源物将被 制作。 这些镰状芯片将用血液RNA进行探测， 三到六个月大的镰刀病人，在父母同意后。 表达 将确定I的特征并随访疾病进展 纵向。预测严重疾病的特征将提供重要的 早期干预的信息。

DESCRIPTION (provided by applicant): Microarray based expression profiling and positional cloning will be utilized to define genes that modify the severity of sickle cell disease. Initial experiments will be performed in a mouse model of sickle cell disease that reproduces most if not all of the pathology of the disorder (Science 278: 873- 876). The model was created by targeted deletion of the: mouse alpha and beta globin genes followed by introduction of human alpha, gamma and beta sickle globin transgenes" into the germline. These animals synthesize only human hemoglobin in adult red blood cells. Interestingly, as observed, in humans, the range of disease severity in these outbred sickle mice is dramatic. Genes that modify disease severity will be defined by comparing the gene expression profiles of mice that are severely anemic with animals that are less' severely anemic. Initial experiments will define expression profiles of blood and kidney for 50 animals that have a broad range of disease severity. Hierarchical cluster analysis of expression data will define groups of animals, and these groups will be correlated with disease severity. A major goal is to define expression profiles that predict severe disease. Therefore, the genotypes of multiple, severely affected animals will be fixed by cloning; that is, primary fibroblasts will be cultured from all 50 animals in the study and nuclear transfers into enucleated eggs will be performed for animals that develop severe disease and for non-severely affected controls. Expression profiles will then be determined at 10, 20 and 30 days of age to define profiles that precede anemia and kidney pathology. This analysis will define a profile(s), that predicts severe disease. Comparison of the phenotypes of cloned siblings will also reveal the relative contributions' of genetic and environmental/ stochastic influences on disease progression and severity. Linkage analysis of anemia with SNPs will also be used to map genes that modify the disease. Sickle mice that are backcrossed onto the C57B]/6;1 background uniformly develop severe anemia. These animals will be bred with sickle animals that are backcrossed! I onto a strain that results in less severe anemia. Linkage analysis will be performed on F2 animals to define SNPS linked, to severe disease. Candidate modifying genes that are linked to the SNPs will be identified in the public and private, databases and the functional significance of these genes will be examined by modification in ES cells derived from the sickle mice. Expression profiles will also be determined for blood of human sickle patients. A "SickleChip" which contains the human homologues of modifier genes identified in the mouse will be produced. These SickleChips will be probed with blood RNA collected from three to six month old sickle patients after parental consent. Expression profiles I will be determined and disease progression will be followed longitudinally. Profiles that predict severe disease will1i provide important information for early intervention.