Establishment and clinical application of the stable expression system of mutant gene for alpha-subunit of pyruvate dehydrogenase
| Project/Area Number |
08670893
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Pediatrics
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| Research Institution | University of Tokushima |
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Principal Investigator |
ITO Michinori School of Medicine, University of Tokushima Lecturer, 医学部, 講師 (40211057)
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| Co-Investigator(Kenkyū-buntansha) |
KURODA Yasuhiro School of Medicine, University of Tokushima Professor, 医学部, 教授 (20035471)
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| Project Period (FY) |
1996 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| 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)
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| Keywords | pyruvate dehydrogenase / lactic acidemia / expression system / mutation / pyruvate dehydrogenase deficiency |
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| 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.
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Report
(3 results)
Research Products
(4 results)
