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Molecular genetic analysis of the disease resulted from abnomal genomic imprinting

异常基因组印记导致疾病的分子遗传学分析

基本信息

批准号：
09470048
负责人：
MUKAI Tsunehiro
金额：
$ 4.99万
依托单位：
Saga Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

We previously specified the gene p57KIP2 as a responsible gene for Beckwith-Wiedemann syndrome. This time, we further collected the patients. Over all, flinty one patients were collected, analized and mutaitons were found in ten patients, indicating the frequency of 11%. QT domain of p57KIP2 gen was always deleted in all of the mutant. In order to know a function of the mutation in a cellular level, we first examined the cellular localization of this gene product because we observed a nuclear localization signal in a C-terminus of this protein. In addition, we also analized an inhibitory activity of the phosphorylation in vitro using the product which was expressed in E.coli. The result showed the nuclear localization of the wild type, ut two mutants, patient 6 and 8, both could not enter into the nucleus and remained in the cytoplasm, suggesting the nuclear localization might be inhibited in all patients. Inhibition of the phosphorylation was not observed in the patient 6 whose CDK inhibitory domain is truncated, but did observed in the patient S whose inhibitory domain is left intact.We searched the mutation of p57K1P2 and the neighboring genes-MPT1, 1PL and ORCTL2S.in the embryonal tumor including Wilms tumor, hepatoblastoma, rabdomyosarcoma and adrenocortical carcinoma. 41 cases were analyzed. No deletion and rearrangement of the p57KIP2 was found in Wilms tumor and then tumors are directly sequenced in 50 cases, but no mutation found at all. We found, however, one case of missense mutation in Wilms tumor and each one case of LOR in hepatoblastoma and adrenocortical carcinoma in ORCTL2S gene.We analyzed the methylation status of the surrounding area of p57KIP2. Ten kilobase of the region was examined using methylation-sensitive enzyme. No methylation was found in the human genome at all, although this gen is imprinted. On the other hand, n paternal methylation was observed in the mouse genome because this gene is expressed maternally.

我们之前将基因 p57KIP2 指定为 Beckwith-Wiedemann 综合征的相关基因。这次，我们进一步收集了患者。总共收集并分析了 1 名患者，发现 10 名患者出现突变，频率为 11%。 p57KIP2 基因的 QT 结构域在所有突变体中均被删除。为了了解细胞水平上突变的功能，我们首先检查了该基因产物的细胞定位，因为我们在该蛋白质的 C 末端观察到了核定位信号。此外，我们还利用在大肠杆菌中表达的产物在体外分析了磷酸化的抑制活性。结果显示野生型的核定位，而两个突变体，患者6和8，均不能进入细胞核并保留在细胞质中，这表明所有患者的核定位可能受到抑制。在CDK抑制域被截短的患者6中未观察到磷酸化的抑制，但在抑制域完整的患者S中观察到了磷酸化的抑制。我们在胚胎肿瘤（包括肾母细胞瘤、肝母细胞瘤、横纹肌肉瘤和肾上腺皮质）中搜索了p57K1P2及其邻近基因MPT1、1PL和ORCTL2S的突变。癌。分析了41个案例。肾母细胞瘤中未发现p57KIP2的缺失和重排，对50例肿瘤进行直接测序，但根本没有发现突变。然而，我们在肾母细胞瘤中发现1例ORCTL2S基因出现错义突变，肝母细胞瘤和肾上腺皮质癌中各有1例ORCTL2S基因发生LOR。我们分析了p57KIP2周围区域的甲基化状态。使用甲基化敏感酶检查该区域的十个碱基。尽管人类基因组带有印记，但在人类基因组中根本没有发现甲基化。另一方面，在小鼠基因组中观察到 n 父系甲基化，因为该基因在母系表达。