TRANSFECTION SCREENING FOR PRIMARY DEFECTS IN THE PYRUVATE-DEHYDROGENASE E1-ALPHA SUBUNIT GENE

In a significant number of patients with biochemical evidence of a def ect in the E1 (pyruvate dehydrogenase) component of the pyruvate dehyd rogenase complex, it has not proved possible to identify a mutation in the gene coding regions, To assess the need for more extensive geneti c analysis in these patients and to establish a test system in which t o study the biochemical consequences of mutations in the E1 alpha subu nit gene (which is responsible for the great majority of defined cases of pyruvate dehydrogenase deficiency), we have developed a method to screen for E1 alpha gene defects based on complementation of the enzym e deficiency in transformed fibroblast cell lines following transfecti on and expression of the normal cDNA. Using this system, cell lines fr om patients with a variety of different defined mutations in the E1 al pha gene show restoration of enzyme activity, A number of patients hav e been identified in whom deficient enzyme activity is not restored by expression of the normal cDNA indicating that an alternative explanat ion for the enzyme defect must be sought.