GENE ANALYSIS IN HEMOPHILIA

血友病的基因分析

• 批准号: 3354409
• 负责人: catherine driscoll
• 金额: $ 13.08万
• 依托单位: MONTEFIORE MEDICAL CENTER (BRONX, NY)
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 1989-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3354409
• 关键词: autosomal recessive trait; biological polymorphism; blood coagulation; endonuclease; gel electrophoresis; gene expression; genetic manipulation; genetic markers; haploidy; hemophilia As; molecular cloning; molecular genetics; nucleic acid sequence; oligonucleotides; pancreatic ribonuclease; restriction mapping

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.

血友病、FVIII:C 和 FIX 缺陷是第二类 最常见的 X 连锁隐性遗传疾病。 他们的特点是 根据凝血百分比具有临床异质性 活性、交叉反应物质 (CRM) 以及存在 凝血蛋白抑制剂。 FVIII:C 的基因和 FIX最近已被克隆及其编码序列 决定。 本提案的目的是研究 血友病基因的分子突变 生成 FVIII:C 和 FIX 基因组和 cDNA 探针并 将分子缺陷与临床血友病表型相关联。 该提案的其他目标是检测机制 某些血友病自发突变的父母来源 谱系，检测 FVIII:C 和 FIX 中新的 DNA 多态性 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，分析多代血友病谱系 使用这些新工具的重组事件。 对此至关重要 研究是利用重组开发新方法 DNA技术筛选186 kb FVIII:C和34 kb 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、 使用核糖核酸酶 A 和现场进行 DNA:RNA 杂交双链体分析 反转凝胶电泳将用作筛选方法 破译血友病基因的突变位点。 进一步分析 突变位点 DNA 序列的分析将通过 将 FVIII:C 和 FIX 基因分子克隆到 lambda 噬菌体中 载体 Charon 21A 和 30、EMBL3 和 gamma gt10。 脱氧核糖核酸 FVIII:C或FIX重组体的序列将通过以下方式确定 亚克隆至 M13 噬菌体并生成单链 用于双脱氧法 DNA 测序的噬菌体和插入片段 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 基因以及 侧翼 RFLP 探针 Dx13、St14 和 52A 将用于 这些分析。 此外，新的链接RFLP的开发 探针，以及基因内 DNA 多态性的发现， 对于进一步确定重组位点至关重要 FVIII:C 和 FIX 基因或其染色体位点内。 最后， 这些研究应该能够改进遗传诊断和 血友病中的携带者检测。