IDENTIFICATION OF 2 MUTATIONS IN A COMPOUND HETEROZYGOUS CHILD WITH DIHYDROLIPOAMIDE DEHYDROGENASE-DEFICIENCY

An infant girl with elevated blood lactate, pyruvate, and plasma branc hed-chain amino acids was diagnosed with dihydrolipoamide dehydrogenas e (E3; dihydrolipoamide: NAD(+) oxidoreductase, EC 1.8.1.4) deficiency , Activities of the pyruvate dehydrogenase complex and E3 from patient were 26 and 2% of controls in blood lymphocytes, and 11 and 14% in cu ltured skin fibroblasts, respectively. Western blot analysis demonstra ted that the amount of E3 protein in fibroblasts from the patient and her father was about half of controls, while Northern blot analysis sh owed normal amounts of E3 RNA, DNA sequencing of cloned full-length E3 cDNAs from the patient revealed two mutations in separate alleles, On e is a single base insertion of an extra adenine in the last codon of the leader peptide sequence (TAC-->TAAC) leading to a nonsense mutatio n which results in the premature termination of the precursor E3 polyp eptide (Y35X), The other is a missense mutation due to substitution of guanine for adenine, causing an Arg-->Gly substitution at amino acid 460 of the mature protein (R460G) which triggers the loss of E3 activi ty probably by structural change in the E3 dimer, DNA sequencing of E3 cDNAs from the parents demonstrated that the nonsense mutation was in herited from the father and the missense mutation was inherited from t he mother.