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Deficiency of medium-chain acyl-CoA dehydrogenase (MCAD) is a common inherited defect in energy metabolism. Characterization of the mRNA encoding MCAD in a Dutch MCAD-deficient patient revealed an A----G change at nucleotide position 985 of the MCAD mRNA coding region. This point mutation results in the substitution of a glutamic acid for a lysine at amino acid position 304 of the mature protein. The single base change was not found in any wild-type MCAD mRNAs. A mutant allele-specific oligonucleotide probe was used in a hybridization analysis of amplified genomic DNA of MCAD-deficient family members, a carrier, and normal individuals. The hybridization analysis specifically identified individuals who were heterozygotes or homozygotes. In addition to the point mutation, a significant proportion of the index patient's MCAD mRNA contained a variety of deletions and insertions as a result of exon skipping and intron retention. The missplicing occurred in multiple regions throughout the MCAD mRNA. Analysis of the patient's MCAD gene in the regions where the missplicing occurred most frequently did not reveal a mutation in the splicing acceptor or donor sites. Therefore, the molecular characterization of this family revealed a crucial point mutation in the MCAD gene and an unusual abnormality in MCAD pre-mRNA splicing.