HEME AND GLOBIN SYNTHESIS IN INFANTS AND CHILDREN

这项研究项目的目的是调查 儿科血液病及其相关基因的表达 造血细胞。这个项目的范围已经从 包括红细胞紊乱在内的血红蛋白疾病的研究， 巨核细胞和凝血。先前关于引起突变的研究 β-地中海贫血、三角洲-地中海贫血、凝血酶原缺乏症和 血栓减少症将继续，并继续进行新的调查 定义先天性红细胞增多症--第九因子莱登的遗传缺陷 和先天性再生障碍性贫血。我们的具体目标是1) 人类遗传缺陷导致红细胞疾病的研究， 包括地中海贫血，2)确定特定的遗传缺陷在 凝血蛋白与包括血小板在内的人类疾病有关 糖蛋白与血浆凝血因子、凝血酶原和凝血因子 IX和3)基因转录控制的特征 对于血小板蛋白、血小板因子4和抗体受体 FcGammariia.确定所有基因突变的方法将是 包括基因组DNA的PCR扩增，然后是基于荧光的 测序。相对较小的基因(1-2kb)，如β-和Delta- 珠蛋白，将被完全测序，而5‘侧翼 较大基因的区域外显子和外显子/内含子边界将被扩增 并对其进行了分析。扩增和测序的自动化 将采取步骤，以便能够更快地分析数据。 聚合酶链式反应产物的荧光标记及复合带分析 模式将应用于现有的突变扫描技术， 例如异源双链错配的化学切割。促进的 对错配的分析将允许减少每个基因的测序 目标区域，进一步增加突变检测。最终， 将设计基于荧光的等位基因特异性多重PCR分析 用于诊断特定疾病的突变。研究也将是 巨核细胞生成与分子生物学关系的研究进展 红细胞生成，检查表达的基因的控制区 在血小板中：血小板因子4和抗体受体FCGammaRIIA。 对这些控制区的精细结构分析将表明 顺式作用元件与无处不在的或特定于骨架的相互作用 反式作用因子导致观察到的表达模式。 这些实验将提供更多关于正常 基因表达的调控，决定了几个分子基础 有利于遗传性血液疾病的诊断和治疗 这些疾病。

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.