Molecular genetic analysis of the disease resulted from abnomal genomic imprinting

我们之前明确指出基因p57KIP2是导致beckwithwithwiedemann综合征的基因。这一次，我们进一步收集了患者。总的来说，收集了1例患者，分析了10例患者，发现了突变，频率为11%。p57KIP2基因的QT结构域在所有突变体中均缺失。为了了解突变在细胞水平上的功能，我们首先检查了该基因产物的细胞定位，因为我们在该蛋白的c端观察到核定位信号。此外，我们还利用在大肠杆菌中表达的产物，分析了体外磷酸化的抑制活性。结果显示野生型的核定位，但2个突变体患者6和8都不能进入细胞核，停留在细胞质中，表明所有患者的核定位可能都受到抑制。CDK抑制结构域被截断的患者6未观察到磷酸化抑制，但在抑制结构域完整的患者S中观察到磷酸化抑制。我们搜索了p57K1P2及其邻近基因mpt1、1PL和ORCTL2S的突变。胚胎性肿瘤包括肾母细胞瘤、肝母细胞瘤、rabdomyosarcoma和肾上腺皮质癌。分析41例病例。在Wilms肿瘤中未发现p57KIP2的缺失和重排，50例直接对肿瘤进行测序，未发现突变。然而，我们发现在Wilms肿瘤中有1例ORCTL2S基因错义突变，在肝母细胞瘤和肾上腺皮质癌中各有1例ORCTL2S基因错义突变。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测该区域的10千碱基。在人类基因组中根本没有发现甲基化，尽管这个基因是印迹的。另一方面，在小鼠基因组中观察到n父系甲基化，因为该基因是母系表达的。

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.