DISEASE-CAUSING MUTATIONS IN EXON-II OF THE MEDIUM-CHAIN ACYL-COA DEHYDROGENASE GENE

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most comm only recognized defect of the mitochondrial beta-oxidation in humans. It is a potentially fatal, autosomal recessive inherited defect. Most patients with MCAD deficiency are homozygous for a single disease-caus ing mutation (G985), causing a change from lysine to glutamate at posi tion 304 (K304E) in the mature MCAD. Only seven non-G985 mutations, al l of which are rare, have been reported. Because the G985 mutation and three of the non-G985 mutations are located in exon 11, it has been s uggested that this exon may be a mutational hot spot. Here we describe the results from sequence analysis of exon 11 and part of the flankin g introns from 36 compound heterozygous patients with MCAD deficiency. We have identified four previously unknown disease-causing mutations (M301T, S311R, R324X, and E359X) and two silent mutations in exon 11. Our results show that exon 11 is not especially mutation prone. We dem onstrate that two of the identified disease-causing mutations can be d etected by restriction enzyme digestion of the PCR product from the as say for the G985 mutation, a discovery that makes this assay even more useful than before. On the basis of expression of wild-type and mutan t MCAD protein in COS-7 cells, we show that the identified mutations a bolish MCAD enzyme activity and that they therefore must be disease ca using. The M301T, S311R, and K304E mutations are located in helix H, w hich makes up part of the dimer-dimer interface of the MCAD tetramer. On the basis of the three-dimensional structure of MCAD and the result s from the COS-7 expression experiments, we speculate that the primary effect of the M301T and S311R mutations is on correct folding/tetrame r assembly, as it has previously been observed for the K304E mutation.