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（e1α）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（Pyruvate dehydrogenase complex， PDHC）由6个组分组成，因此在除E1alpha外其他组分均正常的宿主细胞中，建立突变型E1alpha蛋白表达体系，以确定E1alpha基因突变的致病性，并检测E1alpha缺乏患者的E1alpha功能异常。然后，制备了CAG启动子和正常E1alpha cDNA稳定表达人细胞的质粒载体，并将该质粒载体转染到E1alpha缺乏的淋巴母细胞样细胞中。转染筛选后，缺乏E1alpha的淋巴母细胞PDHC活性正常，E1alpha蛋白含量正常。利用这一表达系统，我们开始确认E1alpha基因突变的致病性，并检测突变E1alpha蛋白的功能异常。因此，我们制备了含有E1alpha缺乏症患者突变体E1alpha cDNA的表达载体。通过x染色体失活偏差分析、SSCP和直接测序，在1例先天性乳酸血症女性患者中发现PDHC活性正常的新突变105bp插入。

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.