MOLECULAR GENETICS OF LOW-RENIN HYPERTENSION

低肾素高血压的分子遗传学

• 批准号: 3243221
• 负责人: PERRIN C WHITE
• 金额: $ 16.65万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-08-15 至 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3243221
• 关键词: angiotensin /renin /aldosterone hypertension; autosomal dominant trait; autosomal recessive trait; blood chemistry; child (0-11); cytochrome P450; dexamethasone suppression test; familial hypertension; family genetics; gene deletion mutation; gene expression; genetic polymorphism; genetic promoter element; human genetic material tag; human subject; hydroxysteroid dehydrogenases; hyperaldosteronism; linkage mapping; molecular cloning; polymerase chain reaction; steroid 11beta monooxygenase; tissue /cell culture; transfection; urinalysis

The proposed studies are aimed at elucidating the molecular genetic bases of two inherited forms of low-renin childhood hypertension: apparent mineralocorticoid excess (AME; probably an autosomal recessive disorder) and dexamethasone-suppressible hyperaldosteronism (DSH; and autosomal dominant disorder). Patients with these disorders will be identified by detailed endocrinologic studies, and DNA samples from patients and family members will be obtained. In Specific Aim I, mutations in the gene encoding corticosteroid 11beta-dehydrogenase (11-DH) will be studied as a cause of AME. Sequence analysis of genomic clones encoding human 11-DH will be completed. Possible major deletions or rearrangements of the 11-DH genes in patients with AME will be detected by blot hybridization analysis of DNA samples. Small mutations in the 11-DH genes of patients will be identified by sequence analysis of cloned mutant genomic genes or of segments amplified by the polymerase chain reaction. The normal human 11- DH enzyme will be expressed from cDNA in cell culture, either by transfection of a plasmid containing a strong promoter or by infection of recombinant vaccinia virus. To determine the effects of each mutation detected in AME patients using in vitro mutagenesis and one of the above expression systems. If particular mutations are suspected to affected expression, normal and mutant promoter activities will be analyzed by ligating the promoters to an indicator gene and transfecting into a cell line expressing intrinsic 11-DH activity. In Specific Aim II, mutations in the steroid 11-hydroxylase (P450cll) gene (CYP11B1 and CYP11B2) will be studied as a possible cause of DSH. A linkage analysis of DSH will be carried out using P450cll cDNA and DNA samples from patients with DSH and their families. If CYP11B polymorphisms are not sufficiently informative the linkage analysis will be extended to additional polymorphic probes on chromosome 8q that flank CYP11B1 and B2. If the linkage analysis is consistent with the hypothesis that DSH is caused by a mutation in or near either of the CYP11B genes, mutant CYP11B genes from patients with DSH will be isolated and sequenced to identify each mutation. If missense mutations are identified, normal and mutant enzymes will be expressed in cell culture to determine the effect of each mutation on regulation of 18-oxidase activity.

拟议的研究旨在阐明分子遗传基础 在两种遗传形式的低肾上腺素儿童高血压中：明显 矿物皮质激素过量（AME；可能是常染色体隐性疾病） 和地塞米松供抑制的高醛溶作用（DSH；和常染色体 主导障碍）。 这些疾病的患者将通过 详细的内分泌研究和患者和家族的DNA样品 会员将获得。 在特定目的I中，基因中的突变 编码皮质类固醇11Beta-脱水酶（11-DH）将被研究为 AME的原因。 编码人11-DH的基因组克隆的序列分析 将完成。 11-DH可能的主要删除或重排 AME患者的基因将通过印迹杂交分析检测 DNA样品。 患者11-DH基因的小突变将是 通过克隆突变基因组基因的序列分析或 通过聚合酶链反应扩增的片段。 正常人11- DH酶将在细胞培养中从cDNA表达 转染含有强启动子或感染的质粒 重组疫苗病毒。 确定每个突变的影响 在AME患者中使用体外诱变和上述一种检测 表达系统。 如果怀疑特定的突变会受到影响 表达，正常和突变启动子活动将由 将启动子连接到指示基因并转染到细胞中 表达内在11-DH活性的线。 在特定的目标II中，突变 类固醇11-羟化酶（P450CLL）基因（CYP11B1和CYP11B2）为 研究是DSH的可能原因。 DSH的链接分析将是 使用DSH患者的P450CLL cDNA和DNA样品进行 他们的家人。 如果CYP11B多态性不足以提供信息 链接分析将扩展到对上的其他多态探针 CYP11B1和B2侧面的8Q染色体。 如果链接分析是 与DSH是由或附近突变引起的假设一致的 任何一个CYP11b基因，DSH患者的突变CYP11b基因将 分离并测序以识别每个突变。 如果错义突变 鉴定出，正常和突变酶将在细胞培养中表达 确定每个突变对调节18氧化酶的影响 活动。