Dominant-negative GCMB mutations cause hypoparathyroidism

描述(由申请人提供)： GCMB是一种转录因子，仅在甲状旁腺细胞中表达，是甲状旁腺发育所必需的。这些腺体产生甲状旁腺激素(PTH)，它是钙稳态和骨代谢最重要的调节器之一。我的长期目标是阐明GCMB在成熟的甲状旁腺中的作用(S)，以及它在治疗原发性和继发性甲状旁腺功能亢进症的治疗策略中的潜力。 纯合子切除Gcm2(两种哺乳动物同源基因之一)的小鼠缺乏甲状旁腺，并出现低钙血症和高磷血症。与在小鼠身上的这些发现一致，GCMB(人类的Gcm2同源物)内的纯合缺失先前被揭示为常染色体隐性形式的甲状旁腺功能减退症的原因。我现在已经在受影响的患者中发现了GCMB杂合突变，但在两个常染色体显性遗传性甲状旁腺功能减退症家系中没有发现未受影响的成员。这两个GCMB杂合性突变都是GCMB外显子5中的单核苷酸缺失，导致GCMB的C末端部分发生变化，该部分包含假定的反式激活结构域。这些缺失分别导致463和467个氨基酸残基的开放阅读框发生移位，并分别增加了65个和63个氨基酸的非相关蛋白序列。初步数据表明，突变形式的GCMB在体外抑制了野生型蛋白对荧光素酶报告结构的作用，这表明突变的GCMB具有显性-负性。 为了探讨导致甲状旁腺激素合成和分泌受损的潜在机制，我将在体外使用GCMB反应报告，确定GCMB杂合突变导致常染色体显性遗传性甲状旁腺功能减退症(目标1)的机制，以及已确定的显性-阴性GCMB突变是否影响原代甲状旁腺细胞培养物的甲状旁腺激素合成和分泌(目标2)。此外，我将调查这两个常染色体显性遗传性甲状旁腺功能减退症家族的其他成员，并确定GCMB的天然靶基因(目标3)。 甲状旁腺产生甲状旁腺激素(PTH)，这是钙调节和骨骼健康所必需的。在两个甲状旁腺活性不足的家系中，我们发现了GCMB的突变，这是甲状旁腺发育所必需的一个因素。这些突变的特征将有助于提高我们对甲状旁腺生物学的理解。

DESCRIPTION (provided by applicant): GCMB is a transcription factor that is exclusively expressed in parathyroid cells and that is necessary for parathyroid gland development. These glands produce parathyroid hormone (PTH), which is one of the most important regulators of calcium homeostasis and bone metabolism. My long-term goal is to elucidate the role(s) of GCMB in the mature parathyroid gland and its potential for use in therapeutic strategies to treat primary and secondary hyperparathyroidism. Mice with homozygous ablation of Gcm2, one of the two mammalian orthologs, lack parathyroid glands and develop hypocalcemia and hyperphosphatemia. Consistent with these findings in mice, a homozygous deletion within GCMB, the human homolog of Gcm2, was previously revealed as a cause of an autosomal recessive form of hypoparathyroidism. I have now identified heterozygous GCMB mutations in the affected, but not the unaffected members of two unrelated families with autosomal dominant hypoparathyroidism. Both heterozygous GCMB mutations are single nucleotide deletions within GCMB exon 5 that introduce changes within the C-terminal portion of GCMB, which contains the putative transactivation domain. The deletions lead to a shift in open reading frame at amino acid residues 463 and 467, respectively, and the addition of 65 and 63 amino acids, respectively, of unrelated protein sequence. Preliminary data demonstrate that the mutant forms of GCMB inhibited the actions of the wild-type protein on a luciferase reporter construct in vitro suggesting that the mutant GCMB has dominant-negative properties. To explore the underlying mechanisms leading to impaired PTH synthesis and secretion, I will determine the mechanism through which heterozygous GCMB mutations cause autosomal-dominant hypoparathyroidism (Aim 1), using a GCMB-responsive reporter in vitro, and whether the identified dominant-negative GCMB mutants affect PTH synthesis and secretion in primary parathyroid cell cultures (Aim 2). Furthermore, I will investigate additional members of the two families with autosomal dominant hypoparathyroidism, and I will identify the natural target genes for GCMB(Aim 3). Parathyroid glands produce parathyroid hormone (PTH), which is necessary for calcium regulation and bone health. In two families with insufficient parathyroid gland activity, we have identified mutations in GCMB, a factor necessary for the development of parathyroid glands. The characterization of these mutations will help improve our understanding of parathyroid gland biology.