Establishment and clinical application of the stable expression system of mutant gene for alpha-subunit of pyruvate dehydrogenase

丙酮酸脱氢酶α亚基突变基因稳定表达体系的建立及临床应用

• 批准号: 08670893
• 负责人: ITO Michinori
• 金额: $ 1.41万
• 依托单位: University of Tokushima
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08670893/
• 关键词: pyruvate dehydrogenase; lactic acidemia; expression system; mutation; pyruvate dehydrogenase deficiency

Pyruvate dehydrogenase alpha-subunit (E1alpha) deficiency is one of the most common causes of congenital lactic acidemia. Pyruvate dehydrogenase complex (PDHC) consists of six components, so that host cells which have normal other components except E1alpha, to establish the expression system with mutant E1alpha protein for confirmation of pathogenesity of mutation in E1alpha gene and exmination of functional abnormality of E1alpha in patients with E1alpha deficiency, are necessary. Then, we prepared the stable expression plasmid vector with CAG promoter and normal E1alpha cDNA for human cells and transfected this plasmid vector into lymphoblastoid cells with E1alpha deficiency. After transfection and selection, the lymphoblastoid cells with E1alpha deficiency showed the normal PDHC activity and normal amount of E1alpha protein. With this expression system, we became to confirm the pathogenesity of mutations in E1alpha gene and examine the functional abnormality of mutant E1alpha proteins. So, we prepared the expression vector containing mutant E1alpha cDNA found in patients with E1alpha deficiency. Furthermore, we found the new mutation, 105bp insertion, in a female patient with congenital lactic acidemia, having normal PDHC activity with the assay of deviation of X-chromosome inactivation, SSCP and the direct sequencing.

丙酮酸脱氢酶α亚基（E1α）缺乏是先天性乳酸血症的最常见原因之一。丙酮酸脱氢酶复合物(PDHC)由六种成分组成，因此需要在除E1α外其他成分正常的宿主细胞中建立突变型E1α蛋白的表达系统，以证实E1α基因突变的致病性，并排除E1α缺陷患者的E1α功能异常。然后，我们制备了带有CAG启动子和正常E1α cDNA的人细胞稳定表达质粒载体，并将该质粒载体转染到E1α缺陷的淋巴母细胞中。转染和选择后，E1α缺陷的类淋巴母细胞显示出正常的PDHC活性和正常量的E1α蛋白。利用该表达系统，我们可以确认E1α基因突变的致病性，并检查突变E1α蛋白的功能异常。因此，我们制备了含有在 E1α 缺乏症患者中发现的突变型 E1α cDNA 的表达载体。此外，我们通过X染色体失活偏差、SSCP和直接测序，在一名患有先天性乳酸血症的女性PDHC活性正常的患者中发现了新的突变，即105bp插入。

项目成果

Saijo T,Naito E,Ito M,Matsuda J,Yokota I,Kuroda Y: "Stable Restoration of Pyruvate dehydrogenase Complex in E1-Defective Human Lymphoblastoid Cells." Bioch Biophys Res Commun. 228. 446-45 (1996)

Saijo T、Naito E、Ito M、Matsuda J、Yokota I、Kuroda Y：“E1 缺陷的人淋巴母细胞中丙酮酸脱氢酶复合物的稳定恢复。”

Saijo T et al: "Stable Restoration of Pyruvate dehydrogenase Complex in E1-Defective Human Lymphoblastoid Cells" Bioch Biophy Res Commun. 228. 446-451 (1996)

Saijo T 等人：“E1 缺陷的人淋巴母细胞中丙酮酸脱氢酶复合物的稳定恢复”Bioch Biophy Res Commun。