Dominant-negative GCMB mutations cause hypoparathyroidism

描述（由申请人提供）： GCMB是一种仅在甲状旁腺细胞中表达的转录因子，是甲状旁腺发育所必需的。这些腺体产生甲状旁腺激素（PTH），这是钙稳态和骨代谢的最重要的调节剂之一。我的长期目标是阐明GCMB在成熟甲状旁腺中的作用及其在治疗原发性和继发性甲状旁腺功能亢进的治疗策略中的应用潜力。 两种哺乳动物直系同源基因之一的Gcm 2纯合子消融的小鼠缺乏甲状旁腺，并发生低钙血症和高磷血症。与小鼠中的这些发现一致，Gcm2的人类同源物GCMB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。我现在已经在两个不相关的常染色体显性遗传性甲状旁腺功能减退症家族中发现了受影响的杂合子GCMB突变，但没有发现未受影响的成员。两种杂合GCMB突变都是GCMB外显子5内的单核苷酸缺失，其在GCMB的C末端部分内引入变化，其包含推定的反式激活结构域。缺失导致分别在氨基酸残基463和467处的开放阅读框的移位，以及分别添加65和63个不相关蛋白质序列的氨基酸。初步数据表明，GCMB的突变形式抑制了野生型蛋白在体外对荧光素酶报告基因构建体的作用，表明突变GCMB具有显性负特性。 为了探索导致PTH合成和分泌受损的潜在机制，我将确定杂合GCMB突变导致常染色体显性甲状旁腺功能减退症（目的1）的机制，使用GCMB反应报告体外，以及鉴定的显性阴性GCMB突变体是否影响原代甲状旁腺细胞培养物中PTH的合成和分泌（目的2）。此外，我将调查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（目的3）。 甲状旁腺产生甲状旁腺激素（PTH），这是必要的钙调节和骨骼健康。在两个甲状旁腺活性不足的家庭中，我们已经确定了GCMB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.