GENE ANALYSIS IN HEMOPHILIA

血友病、FVIII：C和FIX缺乏症是第二种 最常见的X连锁隐性疾病。他们的特点是 根据凝血百分比，临床上是异质性的 活性、交叉反应材料(CRM)和是否存在 凝血蛋白的抑制剂。FVIII：C和FVIII的基因 FIX是最近克隆的，它们的编码序列 下定决心。这项建议的目的是研究 血友病基因的分子突变 生成FVIII：C和FIX基因组和cDNA探针，并 分子缺陷与临床血友病表型的相关性。 这项提议的其他目标是检测机制 一些血友病的自发突变的亲本来源 家系，检测FVIII：C中新的DNA多态并修复 基因，开发新的限制性片段长度多态性 Xq27-28亚染色体区域的探针(RFLP) 最后，分析多代血友病家系 使用这些新工具的重组事件。对此至关重要 研究是利用重组的新方法的开发 用DNA技术筛选186kb FVIII：C和34kb FIX基因 用于点突变和缺失。限制方法 核酸内切酶分析，合成寡核苷酸探针分析， 核糖核酸酶A与FIELD进行DNA：RNA杂交双链分析 反转凝胶电泳法将被用作筛选方法 破译血友病基因中的突变位点。进一步分析 突变部位的DNA序列将由 FVIII：C和FIX基因在λ噬菌体中的克隆 矢量Charon 21A和30、EMBL3和Gamma gt10。DNA FVIII：C或FIX重组子的序列将通过以下方式确定 M13噬菌体的亚克隆及其单链基因的产生 双脱氧法DNA测序用噬菌体和插入体 链终止。自发性血友病的起源 突变和重组频率可以通过 使用多个DNA标记进行单倍型分析。脱氧核糖核酸 FVIII：C和FIX基因的多态位点以及 侧翼RFLP探针Dx13、St14和52a将用于 这些分析。此外，新的关联RFLP的开发 探针，以及基因内DNA多态的发现， 对于进一步定义重组位点也是必不可少的 在FVIII：C内，并固定基因或其染色体位置。最后， 这些研究应该允许改进遗传诊断和 血友病患者的携带者检测。

The hemophilias, FVIII:C and FIX deficiencies, are the second commonest X-linked, recessive disorders. They are characterized as being clinically heterogenous based on percent clotting activity, cross-reactive material (CRM), and the presence of inhibitors to the coagulation protein. The genes for FVIII:C and FIX have been recently cloned and their coding sequences determined. The objective of this proposal is to study the molecular mutations in hemophilia genes using the recently generated FVIII:C and FIX genomic and cDNA probes and to correlate molecular defects to the clinical hemophilia phenotype. Other objectives of this proposal are to detect the mechanisms and parental source of spontaneous mutations in some hemophilia pedigrees, detect new DNA polymorphisms in the FVIII:C and FIX gene, develop new restriction fragment-length polymorphism probes (RFLPs) for the Xq27-28 subchromosomal region and, finally, to analyze multigenerational hemophilia pedigrees for recombinational events using these new tools. Essential to this study is the development of new approaches using recombinant DNA technology to screen the 186 kb FVIII:C and 34 kb FIX genes for point mutations and deletions. Methods of restriction endonuclease analysis, synthetic oligonucleotide probe analysis, DNA:RNA hybrid duplex analysis with ribonuclease A and field inversion gel electrophoresis will be utilized as screening methods to decipher mutation sites in hemophilia genes. Further analyses of the DNA sequences at the mutation sites will be performed by molecular cloning of FVIII:C and FIX genes into the lambda phage vectors Charon 21A and 30, EMBL3, and gamma gt10. The DNA sequences of FVIII:C or FIX recombinants will be determined by subcloning into M13 phage and generation of single-stranded phage and inserts for DNA sequencing by the method of dideoxy chain termination. The origin of spontaneous hemophilia mutations and recombinational frequencies can be studied by the use of haplotype analysis using multiple DNA markers. DNA polymorphic sites in both the FVIII:C and FIX genes as well as in the flanking RFLPs probes, Dx13, St14 and 52A will be used for these analyses. In addition, the development of new linked RFLP probes, as well as the discovery of intragenic DNA polymorphs, will be essential to further define the recombination sites either within FVIII:C and FIX genes or their chromosomal locus. Finally, these studies should permit improved genetic diagnostic and carrier detection in the hemophilias.