Academic Endocrine Unit, Nuffield Department of Clinical Medicine, University of Oxford, Oxford Centre for Diabetes, Endocrinology, and Metabolism (OCDEM), Churchill Hospital, Headington Oxford OX3 7LJ, UK.
J Clin Endocrinol Metab. 2010 Jul;95(7):3512-6. doi: 10.1210/jc.2009-2532. Epub 2010 May 12.
Glial cells missing B (GCMB), the mammalian homolog of the Drosophila GCM gene, encodes a 506-amino-acid parathyroid-specific transcription factor. To date, only two different heterozygous GCMB mutations have been reported in three kindreds with autosomal dominant hypoparathyroidism.
Our objective was to investigate a family with autosomal dominant hypoparathyroidism for PTH, CaSR, and GCMB mutations.
Leukocyte DNA was used with exon-specific primers for PCR amplification and the DNA sequences of the PCR products determined. Functional analyses using fluorescence microscopy, EMSAs, and luciferase reporter assays were undertaken. Informed consent was obtained using protocols approved by a national ethical committee.
DNA sequence analysis revealed an A to C transversion at codon 502 of GCMB, which altered the wild-type asparagine (Asn) to histidine (His). Functional studies, using transient transfections of COS7 cells with GCMB wild-type and mutant (Asn502His) tagged constructs, demonstrated that the wild-type and mutant proteins localized to the nucleus and retained the ability to bind the GCM-consensus DNA recognition motif. However, a luciferase reporter assay demonstrated that the Asn502His mutation resulted in a reduction in gene transactivation. Moreover, cotransfection of the wild-type with mutant did not lead to an increase in luciferase activity, thereby demonstrating a dominant-negative effect of the Asn502His mutant that would be consistent with an autosomal dominant inheritance.
Our results, which have identified the first dominant missense GCMB mutation, help to increase our understanding of the mechanism underlying gene transactivation that is a prerequisite for the function of this parathyroid gland-specific transcription factor.
神经胶质细胞缺失 B（GCMB）是果蝇 GCM 基因的哺乳动物同源物，编码甲状旁腺特异性转录因子。迄今为止，只有三种常染色体显性低钙血症中报道了两种不同的杂合 GCMB 突变。
我们的目的是研究一个常染色体显性低钙血症家族的 PTH、CaSR 和 GCMB 突变。
使用白细胞 DNA 进行外显子特异性引物 PCR 扩增，然后确定 PCR 产物的 DNA 序列。使用荧光显微镜、EMSA 和荧光素酶报告基因分析进行功能分析。使用国家伦理委员会批准的方案获得了知情同意。
DNA 序列分析显示 GCMB 密码子 502 处的 A 到 C 颠换，将野生型天冬酰胺（Asn）改变为组氨酸（His）。使用 GCMB 野生型和突变（Asn502His）标记构建体的 COS7 细胞瞬时转染进行的功能研究表明，野生型和突变蛋白定位于细胞核并保留结合 GCM 共识 DNA 识别基序的能力。然而，荧光素酶报告基因分析表明，Asn502His 突变导致基因转录激活减少。此外，野生型和突变型共转染不会导致荧光素酶活性增加，从而证明 Asn502His 突变具有显性负效应，这与常染色体显性遗传一致。
我们的研究结果确定了第一个显性错义 GCMB 突变，有助于增加我们对基因转录激活机制的理解，这是这种甲状旁腺特异性转录因子功能的前提。
