CLONING OF THE PSEUDOHYPOPARATHYROIDISM TYPE 1B GENE

1B 型假性甲状旁腺功能减退症基因的克隆

• 批准号: 6312282
• 负责人: MICHAEL ALAN LEVINE
• 金额: $ 7.57万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6312282
• 关键词: artificial chromosomes; clinical research; expression cloning; family genetics; gene mutation; genetic library; genetic mapping; genotype; hormone receptor; human subject; phenotype; pseudohypoparathyroidism

Pseudohypoparathyroidism (PHP) type 1 is an autosomal dominant disorder characterized by biochemical hypoparathyroidism due to resistance of target tissues to parathyroid hormone (PTH). Two fundamentally different forms of PHP type 1 have been described. In PHP type 1a, mutations in the GNAS1 gene lead to reduced expression or activity of the alpha subunit of the G protein (Gs alpha) that couples heptahelical receptors for PTH and many other hormones to activation of adenylyl cyclase. Accordingly, widespread deficiency of Gs alpha is associated with resistance not only to PTH but also to other hormones that act by stimulating adenylyl cyclase. By contrast, Gs alpha is normal in PHP type 1b, and hormone resistance is limited to PTH target tissues. These observations had suggested that the defect in PHP type 1b was the type 1 PTH receptor, but molecular analysis of this gene has not revealed mutations. The goal of this project is to identify and characterize the gene for PHP type 1b. We have used linkage analysis to assign the locus for PHP type 1b (designated PHP1b) to 10q26. This region contains a candidate gene that encodes the type 5 G protein-coupled receptor kinase (GRK 5), a protein kinase that can phosphorylate and desensitize the type 1 PTH receptor. To determine the PHP1b gene, a positional cloning strategy will be used involving the following approaches: (1) Further refine the PHP1b locus first by haplotype analysis of three additional PHP type 1b families we have ascertained, and subsequently by further analysis of all families using additional polymorphic markers mapped to this region. (2) Screen publicly available YAC clones and assemble a linear contig spanning the locus, and increase the resolution of the physical map. (3) Identify coding sequences in this region by review of publicly available EST maps, screening cDNA libraries by GeneTrapper methodology, and by exon-trapping. (4) Characterize transcripts through evaluation of homology to other known genes, patterns of tissue-specific expression, and cross-species conservation. (5) Perform mutational analysis of genomic DNA samples from familial and sporadic cases. Once the PHP1b gene is identified, the long-range goal will be to characterize the function of the gene product, to examine genotype-phenotype correlations, and to understand its role in regulating expression or action of the PTH receptor in health and disease.

假甲型甲状腺功能减退症（PHP）1型是一种常染色体显性疾病，其特征是靶组织对甲状旁腺激素（PTH）的耐药性，其特征是生化甲状旁腺功能减退症。 已经描述了两种根本不同形式的PHP 1型。在PHP 1A型中，GNAS1基因中的突变导致G蛋白（GSα）的α亚基的表达或活性降低，伴侣是PTH的甲基甲基受体，以及许多其他激素来激活腺苷酸环化酶。 因此，GS Alpha的广泛缺陷不仅与PTH的抗性有关，而且与其他通过刺激腺苷环酶起作用的激素有关。 相比之下，GSα在PHP 1B型中正常，激素耐药性仅限于PTH靶组织。 这些观察结果表明，PHP 1B型的缺陷是1型PTH受体，但是该基因的分子分析尚未揭示突变。 该项目的目的是识别和表征PHP类型1B的基因。 我们已经使用链接分析将PHP类型1B（指定的PHP1B）的基因座分配给10Q26。 该区域包含一个候选基因，该基因编码5G蛋白偶联受体激酶（GRK 5），这是一种蛋白激酶，可以磷酸化并脱敏1型PTH受体。 为了确定php1b基因，将使用涉及以下方法的位置克隆策略：（1）通过对我们已经确定的另外三个PHP 1B型家族的单倍型分析进一步完善PHP1B基因座，然后通过使用其他多态标记物映射到该区域的所有家族进行了进一步分析。 （2）屏幕公开可用的YAC克隆并组装跨越基因座的线性重叠群，并增加物理图的分辨率。 （3）通过审查公开可用的EST映射，通过Genetrapper方法筛选cDNA库以及外显子捕获来识别该区域中的编码序列。 （4）通过评估与其他已知基因的同源性，组织特异性表达的模式和跨物种保护来表征转录本。 （5）对来自家族性和零星病例的基因组DNA样品进行突变分析。 一旦确定了PHP1b基因，远程目标将是表征基因产物的功能，检查基因型 - 表型相关性，并了解其在调节PTH受体在健康和疾病中的表达或作用中的作用。