MOLECULAR BASIS AND THERAPY OF HEMOPHILIA A

这个实验室的长期目标是：1)了解 凝血因子V11基因及其突变方式 导致血友病A，以及2)有助于改进治疗 血友病患者通过开发新的基于DNA的基因测试 通过制作血友病A的实验动物模型。 与拟议研究密切相关的初步结果包括 人因子V11基因的分离； 在因子V11基因中发现两个可能的基因； 描述两类导致血友病的突变；以及 开发灵敏的快速遗传预测方法 血友病A和突变的检测。 这项研究的四个具体目标如下： 1)发现并确定血友病的碱基变化(S)- 导致编码、调控和外显子剪接序列的突变 Vll因子基因。血友病患者基因组DNA序列分析 将被扩增并通过变性梯度筛选突变 凝胶电泳法。我们将检查突变的性质。 根据患者的因子V11活性、抗原和 临床表型。这项研究的一个重要成果将是 是为了在许多情况下确定突变的来源 新出现的血友病。 2)发现DNA序列的多态及其两侧 因子V11基因，以提高基因诊断水平， 开发一种快速、非放射性的检测方法，使基因 全世界都可以很容易地获得诊断结果。这个 还将通过变性来发现序列多态 梯度凝胶电泳法。 3)确定这两个基因的结构和功能 似乎存在于因子V11基因中，特别是关于 它们与因子Vll本身的关系。这将会实现的 主要是通过克隆、定位和测序。 4)建立血友病A的实验动物模型 使小鼠因子V11基因失活。转基因小鼠将成为 从胚胎干细胞中产生的缺陷因子 通过同源重组引入了V11基因。在 未来，这些小鼠将用于体内测试新的因子 Vll产品和疗法，以及体细胞基因 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.