CLONING OF THE PSEUDOHYPOPARATHYROIDISM TYPE 1B GENE

假性甲状旁腺功能减退症（PHP） 1型是一种常染色体显性遗传病，以靶组织对甲状旁腺激素（PTH）的抵抗为特征，表现为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α亚基（Gs α）的表达或活性降低，该亚基偶联PTH七螺旋受体和许多其他激素以激活腺苷酸环化酶。因此，Gs α的普遍缺乏不仅与对甲状旁腺激素的抗性有关，而且与其他通过刺激腺苷酸环化酶起作用的激素的抗性有关。相比之下，Gs α在1b型PHP中是正常的，激素抵抗仅限于PTH靶组织。这些观察结果表明，PHP 1b型的缺陷是1型PTH受体，但该基因的分子分析未显示突变。该项目的目标是鉴定和表征PHP 1b型的基因。我们使用连锁分析将PHP1b型（指定为PHP1b）的位点定位为10q26。该区域包含一个编码5型G蛋白偶联受体激酶（grk5）的候选基因，grk5是一种可以磷酸化和脱敏1型甲状旁腺激素受体的蛋白激酶。为了确定PHP1b基因，将使用定位克隆策略，包括以下方法：(1)首先通过对我们确定的另外三个PHP1b型家族的单倍型分析来进一步完善PHP1b位点，然后使用附加的多态性标记对该区域的所有家族进行进一步分析。(2)筛选公开可用的YAC克隆并组装一个跨越位点的线性配置，并提高物理地图的分辨率。(3)通过查阅公开的EST图谱、GeneTrapper方法筛选cDNA文库和外显子捕获来鉴定该区域的编码序列。(4)通过评估转录本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护来表征转录本。(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，并了解其在调节PTH受体在健康和疾病中的表达或作用中的作用。

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.