Genetic Modifers of Sickle Cell Disease

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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.