MOLECULAR DIAGNOSIS AND CHARACTERIZATION OF MEDIUM-CHAIN ACYL-COA DEHYDROGENASE-DEFICIENCY

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most comm on defect in mitochondrial beta-oxidation in humans. It is an autosoma l recessive disorder which usually presents in infancy. The disease ma nifests itself in periods of metabolic stress to the beta-oxidation sy stem and may be fatal. Four years ago we identified a prevalent diseas e-causing mutation (G985) which causes an amino acid change (K304E) in the mature MCAD protein. Using a Polymerase Chain Reaction (PCR) base d assay for this mutation we have demonstrated: 1. that the G985 mutat ion is present in 90% of the disease alleles from patients from all ov er the world; 2. that the allele frequency of G985 in the general popu lation from most European countries is very high (the carrier frequenc y ranges from 1/68 to 1/333); 3. that MCAD deficiency is not, as has p reviously been suggested, related to Sudden Infant Death Syndrome (SID S). Moreover, investigation by Restriction Fragment Length Polymorphis m (RFLP) analysis of several families with diagnosed MCAD deficiency r evealed that the G985 mutation is only present in chromosomes of a par ticular RFLP haplotype, suggesting a common chromosomal background for this mutation. The other mutations in the MCAD gene are distributed t o all known MCAD RFLP haplotypes. Because 80% of the patients are homo zygous for the G985 mutation, DNA based diagnosis of most patients is now fast and easy. In order to make DNA based diagnosis possible for t he remaining 20% of patients we have set up PCR/solid-phase based semi -automated sequencing of all 12 exons of the MCAD gene. We have so far identified the mutation in 33 of 45 non-G985 homozygous families with verified MCAD deficiency, thereby bringing the number of known mutati ons in the MCAD gene up to 26. In order to investigate in detail the m olecular defects of the mutant MCAD proteins we overexpressed them in COS-7 and in an E. coli based expression system with and without co-ov erexpression of the molecular chaperones GroES and GroEL. The expressi on studies revealed that the primary effect of all the identified muta tions is on formation of correct enzyme structure, and does not direct ly affect the catalytically active regions of the enzyme. We find that our diagnostic set up, consisting of an initial testing by the G985 a ssay, followed by semi-automated sequencing of DNA from those patients who were indicated to be compound heterozygous, is an important impro vement to the diagnosis of MCAD deficiency. Furthermore, the identific ation of new mutations and characterization of their biochemical conse quences contribute significantly to a better understanding of the mole cular pathology of this disorder.