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Research for the transcriptional regulation of Gsα protein gene

Gsα蛋白基因转录调控研究

基本信息

批准号：
14570724
负责人：
MINAGAWA Masanori
金额：
$ 2.24万
依托单位：
CHIBA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

The transcriptional regulation of Gsα gene (GNAS1) is under control of genetic and epigenetic mechanism and subjects to tissue-specific genomic imprinting. In order to clarify this mechanism, the experimental and clinical study was performed Allele-specific expression of GNAS1 mRNA was studied by RT-PCR using cultured tubular cells isolated from human urine which were considered to be proximal tubule origin. Despite both normal and abnormal methylation patterns in the promoter region, mRNA of Gsα protein was biallelically expressed in these cells, therefore tissue-specific genomic imprinting requires both DNA methylation in GNAS1 and other trans-acting factors produced in highly differentiated status. In all 14 cases with sporadic pseudohypoparathyroidism type Ib (PHP-Ib), the maternal methylation pattern was lost in three promoter regions (NESP55, AS, exon1A) of GNAS1 and the methylation pattern in XLas region was different in each case. Among these cases, eight showed loss of methylation in all sites in XLas region examined by Southern hybridization and included four cases with some specific clinical features like mental retardation. Methylation status of each CpG site in 5' and 3' flanking region of exon XL was examined by bisulfite PCR No specific methylation pattern for clinical features was shown and loss of allelic contiguity in CpG methylation was observed in PHP-Ib. These results suggest that the epigenetic abnormality in other region is responsible for the abnormal methylation pattern in XLas region.

Gsα基因（GNAS 1）的转录调控受遗传和表观遗传机制的控制，并受组织特异性基因组印记的影响。为了阐明这一机制，进行了实验和临床研究，使用从人尿中分离的被认为是近端小管起源的培养的肾小管细胞，通过RT-PCR研究了GNAS 1 mRNA的等位基因特异性表达。尽管启动子区存在正常和异常的甲基化模式，但Gsα蛋白的mRNA在这些细胞中是双等位基因表达的，因此组织特异性基因组印迹需要GNAS 1和高度分化状态下产生的其他反式作用因子中的DNA甲基化。在14例散发性假性甲状旁腺功能减退症Ib型（PHP-Ib）患者中，GNAS 1基因3个启动子区域（NESP 55、AS、exon 1A）的甲基化模式均丢失，XLas区域甲基化模式各不相同。在这些病例中，有8例在Southern杂交检测的XLas区域的所有位点均显示甲基化缺失，其中4例具有某些特定的临床特征，如精神发育迟滞。用亚硫酸氢盐PCR法检测外显子XL 5'和3'侧翼区各CpG位点的甲基化状态。在PHP-Ib中未观察到与临床特征相关的特异性甲基化模式，并且观察到CpG甲基化等位基因连续性的丧失。这些结果表明，其他区域的表观遗传异常是导致XLas区域异常甲基化模式的原因。