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：明显的 盐皮质激素过量（AME;可能是一种常染色体隐性遗传病） 地塞米松抑制性醛固酮增多症（DSH）和常染色体 显性障碍）。 将通过以下方式确定患有这些疾病的患者： 详细的内分泌研究，以及患者和家人的DNA样本 成员将获得。 在特定目标I中，基因突变 编码皮质类固醇11 β-脱氢酶（11-DH）的基因将作为 因为AME。 编码人11-DH的基因组克隆的序列分析 将完成。 11-DH可能的重大缺失或重排 通过印迹杂交分析检测AME患者的基因 DNA样本。 患者的11-DH基因中的小突变将被 通过克隆的突变基因组基因或 通过聚合酶链反应扩增的片段。 普通人11- DH酶将在细胞培养物中从cDNA表达，或者通过 转染含有强启动子的质粒或通过感染 重组牛痘病毒。 为了确定每种突变的影响 使用体外诱变和上述之一在AME患者中检测到 表达系统。 如果怀疑某些突变会影响 表达、正常和突变启动子活性将通过 将启动子与指示基因连接并转染到细胞中 表达内在11-DH活性的细胞系。 在具体目标II中， 类固醇11-羟化酶（P450 c11）基因（CYP 11B 1和CYP 11B 2）将 被认为是DSH的可能原因。 DSH的连锁分析将 使用来自DSH患者的P450 c11 cDNA和DNA样品进行， 家人 如果CYP 11B多态性不足以提供信息， 连锁分析将扩展到其他多态性探针， 位于CYP 11B 1和B2侧翼的染色体8 q。 如果关联分析是 这与DSH是由突变引起的假设一致， 来自DSH患者的CYP 11B基因、突变CYP 11B基因中的任一个将 进行分离和测序以识别每个突变。 如果错义突变 正常和突变酶将在细胞培养物中表达 为了确定每个突变对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.