Isolation of human BCAD gene and analysis of putative BCAD deficiency

The 2-methylbranched chain acyl-CoA dehydrogenase (BCAD) is a mitochondrial enzyme that catalyzes the third reaction in isoleucine and valine metabolism, the oxidation of 2-methylbutyryl-CoA and isobutyryl-CoA, respectively. BCAD deficiency would result in the accumulation of branched chain acyl-CoAs or their derivatives. Three patients with a putative defect in BCAD have been reported. This study consists of a molecular examination of one such patient as well as the characterization of the BCAD gene. In Northern blot analysis of human fibroblast RNA, the BCAD cDNA hybridized to two RNA species of 2.7 and 6.5 kb. The 2.7 kb band corresponds to the size of the BCAD cDNA, which consists of the entire coding region of 1.3 kb and a 3$ sp prime$ untranslated region of 1.4 kb. The coding regions of the BCAD gene span approximately 21 kb and consist of 12 exons and 11 introns. The exons range in size from 39 to 108 bp. In the analysis of the putative BCAD-deficient patient, no significant difference was observed at the level of DNA (Southern), RNA (Northern) or protein (Western) when compared to controls, suggesting that the BCAD gene in this patient did not contain any large insertions or deletions, or a frameshift mutation. The single strand conformation polymorphism (SSCP) technique and sequencing of the entire coding region did not reveal any disease-causing mutations but two polymorphisms were identified: one in exon 6 and the other in exon 10