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.

1 型假性甲状旁腺功能减退症 (PHP) 是一种常染色体显性遗传疾病，其特征是由于靶组织对甲状旁腺激素 (PTH) 抵抗而导致生化甲状旁腺功能减退症。 已经描述了 PHP 类型 1 的两种根本不同的形式。在 PHP 1a 型中，GNAS1 基因的突变导致 G 蛋白 (Gs α) 的 α 亚基表达或活性降低，该蛋白将 PTH 和许多其他激素的七螺旋受体与腺苷酸环化酶的激活结合起来。 因此，Gs α 的广泛缺乏不仅与对 PTH 的抵抗有关，还与对通过刺激腺苷酸环化酶起作用的其他激素的抵抗有关。 相比之下，Gs α 在 PHP 1b 型中正常，激素抵抗仅限于 PTH 靶组织。 这些观察表明 PHP 1b 型的缺陷是 1 型 PTH 受体，但对该基因的分子分析尚未发现突变。 该项目的目标是识别和表征 PHP 1b 型的基因。 我们使用连锁分析将 PHP 类型 1b（指定为 PHP1b）的基因座分配给 10q26。 该区域包含一个编码 5 型 G 蛋白偶联受体激酶 (GRK 5) 的候选基因，GRK 5 是一种可以磷酸化 1 型 PTH 受体并使其脱敏的蛋白激酶。 为了确定 PHP1b 基因，将使用涉及以下方法的定位克隆策略：（1）首先通过对我们已确定的另外三个 PHP 1b 型家族进行单倍型分析来进一步细化 PHP1b 基因座，随后使用映射到该区域的其他多态性标记对所有家族进行进一步分析。 (2)筛选公开的YAC克隆并组装跨越位点的线性重叠群，提高物理图谱的分辨率。 (3) 通过审查公开的 EST 图谱、通过 GeneTrapper 方法筛选 cDNA 文库以及通过外显子捕获来鉴定该区域的编码序列。 (4) 通过评估与其他已知基因的同源性、组织特异性表达模式和跨物种保守性来表征转录本。 (5)对家族性和散发性病例的基因组DNA样本进行突变分析。 一旦确定了 PHP1b 基因，长期目标将是表征该基因产物的功能，检查基因型-表型相关性，并了解其在健康和疾病中调节 PTH 受体表达或作用的作用。