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疾病，其特征是由于靶组织对甲状旁腺激素（PTH）的抵抗而导致的生化甲状旁腺功能减退。 已经描述了PHP类型1的两种根本不同的形式。在PHP 1a型中，GNAS 1基因突变导致G蛋白α亚基（Gs α）的表达或活性降低，G蛋白α亚基将PTH和许多其他激素的七螺旋受体偶联到腺苷酸环化酶的活化。 因此，Gs α的广泛缺乏不仅与PTH的抗性有关，而且与通过刺激腺苷酸环化酶起作用的其他激素的抗性有关。 相比之下，Gs α在PHP 1b型中是正常的，并且激素抵抗仅限于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 1b型（命名为PHP 1b）的基因座分配到10 q26。 该区域包含编码5型G蛋白偶联受体激酶（GRK 5）的候选基因，该蛋白激酶可以磷酸化1型PTH受体并使其脱敏。 为了确定PHP 1b基因，将使用包括以下方法的定位克隆策略：（1）首先通过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细化PHP 1b基因座，随后通过使用映射到该区域的另外的多态性标记进一步分析所有家族。(2)筛选公共可用的YAC克隆并组装跨越该基因座的线性重叠群，并提高物理图谱的分辨率。(3)通过查阅公开的EST图谱、用GeneTrapper方法筛选cDNA文库和外显子捕获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PHP 1b基因被鉴定，长期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。

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.