Deficiency of the MCAD enzyme

Scientists

Medium-chain acyl-CoA dehydrogenase (MCAD) is a mitochondrial enzyme that participates in the degradation of medium chain length fatty acids. Deficiencies of this enzyme are the most frequently diagnosed defect of mitochondrial beta-oxidation. The patients show metabolic crisis, characterised by hypoglycemia, lethargy and seizures, when first exposed to viral infections or challenged by fasting. About 20% of the affected infants die. MCAD deficiency results in accumulation of medium-chain acylcarnitines in the urine, which can be analysed by mass-spectroscopy. A large newborn screen showed an incidence from 1:15,000 in the US population.

Molecular cause and consequences

The major reason for deficiency is a K304E missense mutation leading to a less active protein. The newborn screening project identified a 362C->T missense mutation in exon 5 of the MCAD gene that causes exon skipping and subsequent degradation of the mRNA by nonsense-mediated decay. This mutation disrupts a splicing enhancer that is highly similar to the SF2/ASF enhancer in the SMN2 exon 7.

Cotransfection experiments demonstrate that an increase of the SF2/ASF concentration promotes inclusion of the mutated exon, suggesting that the mutation weakens an SF2/ASF-dependent enhancer.

Interestingly, a synonymous mutation 351A->C was identified 11 nucleotides upstream in the same exon. This mutation affects an hnRNP A1 dependent silencer. Since the exon is constitutively used in the absence of the 362C->T SF2/ASF enhancer mutation, it has no effect on splicing.

However, in the presence of the 362C->T enhancer mutation, it promotes exon inclusion, which antagonised the exon-skipping effect of the 362C->T SF2/ASF enhancer mutation.

This example illustrates the fine-tuned balance of positive and negative acting factors that exists in splicing regulation. It also shows that seemingly irrelevant mutations can have an effect on splicing when they are combined with other mutations. Finally, the similarities between the regulation of MCAD exon 5 and SMN2 exon 7 suggest that there are degenerate ‘building blocks’ or ‘regulatory modules’ in the splicing code.