| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1997: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1996: ¥1,500,000 (Direct Cost: ¥1,500,000)
|
|---|
| Research Abstract |
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.
|
