Molecular Definition of Pseudohypoparathyroidism

假性甲状旁腺功能减退症Ib型(PHP-lb)的特点是低钙血症和高磷血症 肾脏对甲状旁腺激素(PTH)的抵抗。常染色体显性遗传性PHP-lb(AD-PHP-lb)为 定位于染色体20q13.3，显示父系印记，以及GNAS外显子A/B甲基化缺失。完毕 在过去的资助期间，我们发现了A/B外显子上游约220kb的两个不同的微缺失 去除STX16外显子2-4或4-6。在一个AD-PHP-lb中发现了4.4 kb的缺失(S7X76del2-4 家系；3-kb缺失(STX76del4-6)在&gt；30个无关AD-PHP-lb家系中发现。我们确认了 还有两个类似的GNAS缺失，它们移除了外显子NESP55和反义外显子3和4；两者都导致了 失去了所有母体的甲基化印记。目前还不确定这些缺失是如何导致甲基化变化的， 以及这些药物如何减少近端肾小管中GSA的表达。有趣的是，都是非家族性的，零星的 例PHP-1b(零星PHP-lb)没有携带任何已知的缺失，但这些患者显示出甲基化 通常影响所有差异甲基化的GNAS区域的变化。可以想象，这些患者 在GNAS中有从头缺失，或者他们有常染色体隐性形式的PHP-lb。对于 在下一个资助期，我们计划扩大我们对GNAS基因座的突变分析，以确定新的 Spphp-lb的微缺失/突变(目标1)，预计将提供有关以下方面的补充信息 建立或维持GNAS甲基化印记需要哪个基因组区域；这些 可以努力确定与调节DMA甲基化有关的控制因素。我们还收集了更多 来自一个常染色体隐性遗传性PHP-lb形式(AR-PHP-lb)的家庭的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 STX16或GNAS突变(目标2)。然后，遗传连锁研究可能有助于识别涉及的新基因 在调节GNAS甲基化方面。在目标3中，我们计划确定缺乏外显子Nesp55和 母亲的邻近区域(Nesp55del/AS3-4del)，而不是父亲的等位基因，会产生PTH抗性。 我们计划确定原代或克隆性近端肾小管细胞是否与母亲的，而不是父亲的， Nesp55del/AS3-4del细胞GSA表达降低/缺失。然后对这些细胞进行分析 基因芯片实验确定哪些蛋白质参与GNAS甲基化和沉默 GSA在近端肾小管和可能的其他组织/细胞类型中的表达。目标4将决定如何 S7X76del4-6或STX76del2-4调控人类外显子A/B的甲基化，其中同等的调控 区域位于鼠标中。这些研究预计将为我们提供对 调节A/B甲基化从而沉默母体等位基因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.