Molecular Definition of Pseudohypoparathyroidism

假性甲状旁腺功能低下Ib型（PHP-Ib）的特征是低钙血症和高磷血症， 肾抵抗甲状旁腺激素（PTH）。常染色体显性PHP-lb（AD-PHP-lb）为 定位于染色体20 q13.3，显示了父系印记和GNAS外显子A/B甲基化的缺失。超过 在过去的资助期间，我们发现了两种不同的外显子A/B上游约220 kb的微缺失， 去除STX 16外显子2-4或4-6。在一个AD-PHP-lb中鉴定出4.4-kb缺失（S7 X76 del 2 -4）， 在>30个不相关的AD-PHP-lb激酶中发现3-kb缺失（STX 76 del 4 -6）。我们确定 还有两个类似的GNAS缺失，去除了外显子NESP 55和反义外显子3和4;两者都导致 所有母体甲基化印记的丢失。目前还不确定缺失如何导致甲基化变化， 以及它们如何降低近端肾小管中Gsa的表达。有趣的是，所有非家族性，散发性 例PHP-lb（sporPHP-lb）不携带任何已知的缺失，但这些患者显示甲基化 这些变化通常影响所有差异甲基化的GNAS区域。可以想象这些病人 在GNAS内具有从头缺失，或者它们具有PHP-lb的常染色体隐性形式。为 下一个资助期，我们计划扩大我们对GNAS基因座的突变分析， sporPHP-lb（Aim 1）中的微缺失/突变，预计将提供有关 哪个基因组区域是建立或维持GNAS甲基化印记所需的;这些 这些努力可以鉴定参与调节DMA甲基化的控制元件。我们还收集了 来自一个具有常染色体隐性PHP-lb形式（AR-PHP-lb）的家族的DNA，并将搜索其他 有两个或更多兄弟姐妹受PHP-lb影响的家庭，他们有广泛的GNAS甲基化变化，但缺乏 STX 16或GNAS突变（Aim 2）。遗传连锁研究可能有助于确定一个新的基因参与 在调节GNAS甲基化中的作用。在目标3中，我们计划确定缺乏外显子Nesp 55的小鼠和 母体等位基因上的相邻区域（Nesp 55 del/AS 3 -4del）而不是父本等位基因上的相邻区域产生PTH抗性。 我们计划确定原代或克隆近端肾小管细胞是否与母体，而不是父亲， Nesp 55 del/AS 3 -4del显示Gsa表达减少/缺失。这些细胞将通过 微阵列实验，以确定哪些蛋白质参与了Gnas甲基化和沉默， 近端肾小管和可能的其他组织/细胞类型中的Gsa表达。目标4将决定如何 S7 X76 del 4 -6或STX 76 del 2 -4调节人外显子A/B处的甲基化，并且其中等效的调节基因是 区域位于鼠标中。预计这些研究将提供更多的见解 调节A/B甲基化并因此沉默来自母体等位基因的Gsa表达的机制。

Pseudohypoparathyroidism type Ib (PHP-lb) is characterized by hypocalcemia and hyperphosphatemia due to renal resistance towards parathyroid hormone (PTH). Autosomal dominant PHP-lb (AD-PHP-lb) was mapped to chromosome 20q13.3, shows paternal imprinting, and loss of GNAS exon A/B methylation. Over the past funding period, we identified two different microdeletions about 220 kb up-stream of exon A/B, which remove STX16 exons 2-4 or 4-6. The 4.4-kb deletion (S7X76del2-4) was identified in one AD-PHP-lb kindred; the 3-kb deletion (STX76del4-6) was found in >30 unrelated AD-PHP-lb kindreds. We identified also two similar GNAS deletions, which remove exon NESP55 and antisense exons 3 and 4; both lead to loss of all maternal methylation imprints. It is uncertain how the deletions contribute to methylation changes, and how these reduce Gsa expression in proximal renal tubules. Interestingly, all non-familial, sporadic cases of PHP-lb (sporPHP-lb) do not carry any of the known deletions, yet these patients show methylation changes that usually affect all differentially methylated GNAS regions. It is conceivable that these patients have a de novo deletion within GNAS, or that they have an autosomal recessive form of PHP-lb. For the next funding period, we plan to expand our mutational analysis of the GNAS locus to identify novel microdeletions/mutations in sporPHP-lb (Aim 1), which is expected to provide additional information as to which genomic region is required for establishing or maintaining the GNAS methylation imprints; these efforts may identify control elements involved in regulating DMA methylation. We have furthermore collected DNA from one family with an autosomal recessive PHP-lb form (AR-PHP-lb) and will search for other families with two or more siblings affected by PHP-lb, who sjiow broad GNAS methylation changes, yet lack STX16 or GNAS mutations (Aim 2). Genetic linkage studies may then help identify of a novel gene involved in the regulation of GNAS methylation. In Aim 3, we plan to determine how mice lacking exon Nesp55 and adjacent regions (Nesp55del/AS3-4del) on the maternal, but not the paternal allele, develop PTH-resistance. We plan to determine whether primary or clonal proximal tubular cells with maternal, but not paternal, Nesp55del/AS3-4del show reduced/absent Gsa expression. These cells will then be analyzed through microarray experiments to determine which proteins are involved in Gnas methylation and the silencing of Gsa expression in proximal renal tubules and possibly other tissues/cell types. Aim 4 will determine how S7X76del4-6 or STX76del2-4 regulate methylation at human exon A/B, and where the equivalent regulatory region is located in the mouse. These studies are expected to provide additional insights into the mechanisms regulating A/B methylation and thus silencing of Gsa expression from the maternal allele.