IDENTIFICATION OF 2 MISSENSE MUTATIONS IN A DIHYDROLIPOAMIDE DEHYDROGENASE-DEFICIENT PATIENT

The molecular basis of dihydrolipoamide dehydrogenase (E3; dihydrolipo amide:NAD+ oxidoreductase, EC 1.8.1.4) deficiency in an E3-deficient p atient was studied. Fibroblasts cultured from the patient contained on ly almost-equal-to 6% of the E3 activity of cells from a normal subjec t. Western and Northern blot analyses indicated that, compared to cont rol cells, the patient's cells had a reduced amount of protein but nor mal amounts of E3 mRNA. Direct sequencing of E3 cDNA derived from the patient's RNA as well as each of the subclones of the cDNA revealed th at the patient had two substitution mutations in the E3 coding region. One mutation changed a single nucleotide from A to G, resulting in su bstitution of Glu (GAA) for Lys-37 (AAA). The other point mutation was a nucleotide change from C to T, resulting in the substitution of Leu (CTG) for Pro-453 (CCG). These mutations appear to be significant in that they alter the active site and possibly the binding of FAD.