HEME AND GLOBIN SYNTHESIS IN INFANTS AND CHILDREN

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The goals of this research project have been the investigation of hematologic disorders in pediatrics and the expression of genes in hematopoietic cells. The scope of this project has expanded from the study of hemoglobinopathies to include disorders of erythrocytes, megakaryocytes and coagulation. Previous studies on mutations causing beta-thalassemia, delta--thalassemia, prothrombin deficiency and thrombasthenia will be continued, and new investigations pursued to define the genetic defects in factor IX Leyden, congenital erythrocytosis and congenital hypoplastic anemia. Our specific aims are 1) investigation of human genetic defects causing disorders of erythrocytes, including thalassemia, 2) identification of specific genetic defects in coagulation proteins involved in human disease including platelet glycoproteins and the plasma coagulation factors prothrombin and factor IX and 3) characterization of the transcriptional control of the genes for the platelet proteins platelet factor 4 and the antibody receptor FcgammaRIIA. The approach to defining mutations in all of the genes will involve PCR amplification of genomic DNA followed by fluorescence-based sequencing. Relatively small genes (1-2 kb), such as beta- and delta- globin, will be sequenced in their entirety, while the 5'-flanking regions exons and exon/intron borders of larger genes will be amplified in clusters and analyzed. Automation of the amplification and sequencing steps will be implemented to permit more rapid analysis of the data. Fluorescent labelling of PCR products and analysis of complex banding patterns will be applied to existing techniques for mutation scanning, such as chemical cleavage of heteroduplex mismatches. Facilitated analysis of mismatches will permit sequencing for each gene to be reduced to targeted areas and further increase mutation detection. Ultimately, fluorescence-based allele-specific multiplex PCR assays will be designed for diagnosis of mutations for a given disorder. Studies will also be conducted on the molecular relationship of megakaryopoiesis and erythropoiesis, examining control regions for genes that are expressed in platelets: platelet factor 4 and the antibody receptor FCgammaRIIA. Fine-structure analysis of these control regions will indicate how interaction between cis-acting elements and ubiquitous or linage specific trans-acting factors results in the observed patterns of expression. These experiments will yield further information concerning the normal regulation of gene expression, determine the molecular basis of several inherited blood disorders and facilitate the diagnosis and treatment of these diseases.