Figure 7. .

Molecular analysis of ACAD9-deficient patients. a, ACAD9 consisting of 22 exons and generating two predominant molecular species through alternative splicing. The schematic drawing illustrates that the basic ACAD domain structure of both translated proteins is conserved (ACDN, ACDM, and ACDC); however, in one, a nuclear targeting signal (N) is substituted for the mitochondrial targeting signal (M). This variant message also contains a longer exon 1, giving a longer N-terminus (U) and a shorter exon 18 along with additional exons 19–22. b and c, Amplification of ACAD9 sequences from control (C), patient 1 (P1), and patient 2 (P2) mRNA. PCRs with control fibroblast cDNA as template readily amplified full-length ACAD9 coding sequences (b, lane 6; c, lane 2 ) and overlapping subfragments (b, lane 2, exons 1–7; b, lane 4, exons 6–18). Full-length message (exons 1–18) could not be amplified from mRNA either from patient 1 (b, lane 7) or patient 2 (c, lane 3). Shorter-than-expected fragments were amplified from patient 1 cDNA (b, lane 3, exons 1–7; lane 5, exons 6–18). d, Sequence of ACAD9 UTR from genomic DNA and cDNA. Sequencing chromatograms of genomic DNA from patient 1 (left panel) identified a dual pattern due to a 4-bp insertion. Patient cDNA in that region showed only the normal sequence, without the insertion (right panel). e, Diagrams of ACAD9 species amplified from cDNA from patient 1. One consisted of exon 1 joined to exon 6 (α), whereas the other consisted of exon 11 jointed to exon 18 (β).