MOLECULAR GENETICS OF LOW-RENIN HYPERTENSION

拟议的研究旨在阐明分子遗传基础 两种遗传形式的低肾素儿童高血压：明显 盐皮质激素过多（AME；可能是一种常染色体隐性遗传病） 和地塞米松抑制性醛固酮增多症（DSH；和常染色体 显性障碍）。 患有这些疾病的患者将通过 详细的内分泌学研究以及患者和家人的 DNA 样本 将获得会员。 在特定目标 I 中，基因突变 编码皮质类固醇 11β-脱氢酶 (11-DH) 将作为 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 可能发生重大删除或重排 AME患者的基因将通过印迹杂交分析进行检测 DNA 样本。 患者 11-DH 基因的小突变将 通过克隆突变基因组基因的序列分析或 通过聚合酶链式反应扩增的片段。 正常人11- DH 酶可以通过细胞培养物中的 cDNA 表达 转染含有强启动子的质粒或通过感染 重组牛痘病毒。 确定每个突变的影响 使用体外诱变和上述之一在 AME 患者中检测到 表达系统。 如果怀疑有特定突变受影响 表达、正常和突变启动子活性将通过以下方式进行分析 将启动子连接至指示基因并转染至细胞中 表达内在 11-DH 活性的细胞系。 在特定目标 II 中，突变 类固醇 11-羟化酶 (P450cll) 基因（CYP11B1 和 CYP11B2）将是 研究作为 DSH 的可能原因。 DSH 的连锁分析将是 使用 DSH 患者的 P450cll cDNA 和 DNA 样本进行 他们的家人。 如果 CYP11B 多态性信息不足 连锁分析将扩展到其他多态性探针 位于 CYP11B1 和 B2 两侧的染色体 8q。 如果连锁分析是 与 DSH 是由 或附近的突变引起的假设一致 DSH 患者的 CYP11B 基因、突变 CYP11B 基因中的任何一个都会 被分离并测序以鉴定每个突变。 如果错义突变 被鉴定后，正常和突变的酶将在细胞培养物中表达 确定每个突变对 18-氧化酶调节的影响 活动。

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.