Isolated 2-methylbutyrylglycinuria caused by short/branched-chain acyl-CoAdehydrogenase deficiency: Identification of a new enzyme defect, resolution of its molecular basis, and evidence for distinct acyl-CoA dehydrogenasesin isoleucine and valine metabolism

Acyl-CoA dehydrogenase (ACAD) defects in isoleucine and valine catabolism h ave been proposed in clinically diverse patients with an abnormal pattern o f metabolites in their urine, but they have not been proved enzymatically o r genetically, and it is unknown whether one or two ACADs are involved. We investigated a patient with isolated 2-methylbutyrylglycinuria, suggestive of a defect in isoleucine catabolism. Enzyme assay of the patient's fibrobl asts, using 2-methylbutyryl-CoA as substrate, confirmed the defect. Sequenc e analysis of candidate ACADs revealed heterozygosity for the common short- chain ACAD A625 variant allele and no mutations in ACAD-8 but a 100-bp dele tion in short/branched-chain ACAD (SBCAD) cDNA from the patient. Our identi fication of the SBCAD gene structure (11 exons; >20 kb) enabled analysis of genomic DNA. This showed that the deletion was caused by skipping of exon 10, because of homozygosity for a 1228G-->A mutation in the patient. This m utation was not present in 118 control chromosomes. In vitro transcription/ translation experiments and overexpression in COS cells confirmed the disea se-causing nature of the mutant SBCAD protein and showed that ACAD-8 is an isobutyryl-CoA dehydrogenase and that both wild-type proteins are imported into mitochondria and form tetramers. In conclusion, we report the first mu tation in the SBCAD gene, show that it results in an isolated defect in iso leucine catabolism, and indicate that ACAD-8 is a mitochondrial enzyme that functions in valine catabolism.