MOLECULAR GENETICS OF LOW-RENIN HYPERTENSION

拟议的研究旨在阐明分子遗传学基础。 两种遗传性低肾素儿童高血压：明显 盐皮质激素过多(AME；可能是一种常染色体隐性遗传病) 和地塞米松可抑制的醛固酮增多症(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，以及患者和家属的DNA样本 将获得成员。在特定的目标I中，基因的突变 编码皮质类固醇11β-脱氢酶(11-DH)将作为一种 原因是AME。人11-水解酶基因克隆的序列分析 将会完成。11-卫生署可能的主要删除或重新编排 斑点杂交分析将检测AME患者的基因 DNA样本。患者11-DH基因的小突变将是 通过对克隆的突变基因组基因进行序列分析或 聚合酶链式反应扩增片段。普通人11岁- 水解酶将在细胞培养中从cDNA中表达，或者通过 通过含有强启动子的质粒或通过感染 重组痘苗病毒。为了确定每种突变的影响 在AME患者中使用体外诱变和上述之一进行检测 表达系统。如果怀疑特定的突变会影响 表达、正常启动子和突变启动子活性将通过 将启动子连接到指示基因并将其导入细胞 表达内在11-水解酶活性的品系。在特定的目标II中，突变 类固醇11-羟基酶(P450Cl1)基因(CYP11B1和CYP11B2)将是 被研究为DSH的可能原因。DSH的连锁分析将是 用DSH和DSH患者的P450cl1基因和DNA样本进行检测 他们的家人。如果细胞色素P11B基因多态性不能提供足够的信息 连锁分析将扩展到额外的多态探针 染色体8q位于CYP11B1和B2两侧。如果链接分析是 与DSH是由DSH中或附近的突变引起的假设一致的 来自DSH患者的CYP11B基因、突变的CYP11B基因中的任何一个将 进行分离和测序，以鉴定每个突变。如果错义突变 正常和突变的酶将在细胞培养中表达 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.