The Y42H mutation in medium-chain acyl-CoA dehydrogenase, which is prevalent in babies identified by MS/MS-based newborn screening, is temperature sensitive.

Medium-chain acyl-CoA dehydrogenase (MCAD) is a homotetrameric flavoprotein which catalyses the initial step of the beta-oxidation of medium-chain fatty acids. Mutations in MCAD may cause disease in humans. A Y42H mutation is frequently found in babies identified by newborn screening with MS/MS, yet there are no reports of patients presenting clinically with this mutation. As a basis for judging its potential consequences we have examined the protein phenotype of the Y42H mutation and the common disease-associated K304E mutation. Our studies of the intracellular biogenesis of the variant proteins at different temperatures in isolated mitochondria after in vitro translation, together with studies of cultured patient cells, indicated that steady-state levels of the Y42H variant in comparison to wild-type were decreased at higher temperature though to a lesser extent than for the K304E variant. To distinguish between effects of temperature on folding/assembly and the stability of the native enzyme, the thermal stability of the variant proteins was studied after expression and purification by dye affinity chromatography. This showed that, compared with the wild-type enzyme, the thermostability of the Y42H variant was decreased, but not to the same degree as that of the K304E variant. Substrate binding, interaction with the natural electron acceptor, and the binding of the prosthetic group, FAD, were only slightly affected by the Y42H mutation. Our study suggests that Y42H is a temperature sensitive mutation, which is mild at low temperatures, but may have deleterious effects at increased temperatures.     

Molecular survey of a prevalent mutation, 985A-to-G transition, and identification of five infrequent mutations in the medium-chain Acyl-CoA dehydrogenase (MCAD) gene in 55 patients with MCAD deficiency.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is an inborn error of fatty-acid oxidation that is characterized by fasting intolerance and recurrent episodes of hypoglycemic coma which can be fatal. Its incidence is one of the highest among genetic metabolic disorders. Using a modified PCR and NcoI digestion method, we have surveyed 46 additional, unrelated MCAD-deficient patients for a prevalent mutation, an 985A-to-G transition (985A----G), that we previously identified in nine MCAD-deficient patients. Among the total of 55 studied, 44 were homozygous and 10 were heterozygous for the 985G allele, whereas one did not carry this mutant allele, indicating that the prevalence of the 985G allele is 89.1%. Furthermore, we identified five other types of mutation: one each in three of the compound heterozygotes and two in the single non-985G patient. An RFLP study of 12 985G-homozygotes showed that all 24 alleles fell into a single haplotype. A questionnaire regarding the ethnic and national origin of their patients was sent to all referring investigators. All 41 patients for whom this information was provided were Caucasians. Of 29 patients whose country of origin was specified, 19 and five were from the British Isles and Germany, respectively. These data suggest that 985A----G may have occurred in a single person in an ancient Germanic tribe.     

Molecular characterization of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency: identification of a lys329 to glu mutation in the MCAD gene,and expression of inactive mutant enzyme protein in E. coli

Summary A series of experiments has established the molecular defect in the medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) gene in a family with MCAD deficiency. Demonstration of intra-mitochondrial mature MCAD indistinguishable in size (42.5-kDa) from control MCAD, and of mRNA with the correct size of 2.4 kb, indicated a point-mutation in the coding region of the MCAD gene to be disease-causing. Consequently, cloning and DNA sequencing of polymerase chain reaction (PCR) amplified complementary DNA (cDNA) from messenger RNA of fibroblasts from the patient and family members were performed. All clones sequenced from the patient exhibited a single base substitution from adenine (A) to guanine (G) at position 985 in the MCAD cDNA as the only consistent base-variation compared with control cDNA. In contrast, the parents contained cDNA with the normal and the mutated sequence, revealing their obligate carrier status. Allelic homozygosity in the patient and heterozygosity for the mutation in the parents were established by a modified PCR reaction, introducing a cleavage site for the restriction endonuclease NcoI into amplified genomic DNA containing G⁹⁸⁵. The same assay consistently revealed A⁹⁸⁵ in genomic DNA from 26 control individuals. The A to G mutation was introduced into an E. coli expression vector producing mutant MCAD, which was demonstrated to be inactive, probably because of the inability to form active tetrameric MCAD. All the experiments are consistent with the contention that the G⁹⁸⁵ mutation, resulting in a lysine to glutamate shift at position 329 in the MCAD polypeptide chain, is the genetic cause of MCAD deficiency in this family. We found the same mutation in homozygous form in 11 out of 12 other patients with verified MCAD deficiency.