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亚单位(E1pha)缺乏是先天性乳酸血症最常见的原因之一。丙酮酸脱氢酶复合体(PDHC)由6个组分组成，因此，对于除E1a外其他成分正常的宿主细胞，建立突变的E1a蛋白表达系统，以确认E1a基因突变的致病作用，并检测E1a缺乏症患者E1a的功能异常，是必要的。然后，我们为人细胞制备了含有CAG启动子和正常E1a基因的稳定表达载体，并将该载体导入E1a缺陷的淋巴母细胞。经转染和筛选后，E1pha缺失的淋巴母细胞显示出正常的PDHC活性和正常的E1pha蛋白含量。有了这个表达系统，我们就可以确认E1pha基因突变的致病性，并检测突变的E1pha蛋白的功能异常。因此，我们制备了含有E1a缺乏症患者突变E1pha基因的表达载体。此外，我们还通过X染色体失活偏差分析、SSCP和直接测序，在1例女性先天性乳酸血症患者中发现了新的105bp插入突变，具有正常的PDHC活性。

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.