MOLECULAR BASIS AND THERAPY OF HEMOPHILIA A

该实验室的广泛长期目标是：1）了解 因子VIII基因和该基因中突变的方式 导致血友病A，2）有助于改善治疗 通过开发新的基于DNA的基因检测， 以及通过产生血友病A的实验室动物模型。 与拟议研究密切相关的初步结果包括 人因子VIII基因的分离; 在因子VIII基因中发现了两个推定的基因; 描述两类血友病致突变;以及 开发快速遗传预测的灵敏方法 血友病A和突变检测。 这项研究的四个具体目标如下： 1)发现和确定血友病的基础变化- 导致编码、调节和外显子剪接序列的突变 第VIII因子基因。 血友病患者的基因组DNA序列 将被扩增并通过变性梯度筛选突变 凝胶电泳 突变的性质将被检验 根据患者的因子VIII活性、抗原和 临床表型 这项研究的一个重要成果将 是为了确定突变的起源在许多情况下， 新生血友病。 2)为了发现DNA序列多态性及其侧翼， 因子VIII基因，以改善遗传诊断， 开发一种快速的非放射性检测方法， 全世界都可以随时得到诊断。 的 序列多态性也将通过变性 梯度凝胶电泳 3)为了确定这两个基因的结构和功能， 似乎位于因子VIII基因内，特别是关于 它们与VIII因子本身的关系 完成这项工作的方法是 主要通过cDNA克隆、定位和测序。 4)建立血友病A的实验动物模型， 使鼠因子VIII基因失活。 转基因小鼠将是 由胚胎干细胞产生， VIII基因已经通过同源重组导入。 在 未来，这些小鼠将用于新因子的体内测试， VIII产品和疗法，以及用于体细胞基因 治疗实验

The broad long-term goals of this laboratory are 1) to understand the factor Vlll gene and the manner by which mutations in this gene give rise to hemophilia A, and 2) to contribute to improved therapy for hemophilia patients by developing new DNA-based genetic tests and by producing a laboratory animal model for hemophilia A. The preliminary results germane to the proposed research include the following: the isolation of the human factor Vlll gene; the discovery of two putative genes within the factor Vlll gene; the description of two classes of hemophilia-causing mutations; and the development of sensitive methodologies for rapid genetic prediction of hemophilia A and detection of mutations. The four specific aims of this research are the following: 1) To discover and determine the base change(s) of hemophilia- causing mutations in coding, regulatory and exon-splicing sequences of the factor Vlll gene. Genomic DNA sequences from hemophiliacs will be amplified and screened for mutations by denaturing gradient gel electrophoresis. The nature of the mutation will be examined in light of the patient's factor Vlll activity, antigen, and clinical phenotype. One important outcome of this research will be to determine the origin of the mutations in the many cases of hemophilia that arise de novo. 2) To discover DNA sequence polymorphisms in and flanking the factor Vlll gene in order to improve genetic diagnosis, and to develop a rapid non-radioactive assay for these so that genetic diagnosis can be made readily available throughout the world. The sequence polymorphisms will also be discovered by denaturing gradient gel electrophoresis. 3) To determine the structure and function of the two genes that appear to lie within the factor Vlll gene, particularly regarding their relationship to factor Vlll itself. This will be achieved mainly by cDNA cloning, mapping and sequencing. 4) To create a laboratory animal model for hemophilia A by inactivating the murine factor Vlll gene. Transgenic mice will be produced from embryonic stem cells into which a defective factor Vlll gene has been introduced by homologous recombination. In the future, these mice will be used for in vivo testing of new factor Vlll products and therapies, as well as for somatic cell gene therapy experiments.