Molecular and cellular pathology of very-long-chain acyl-CoA dehydrogenase deficiency

Abstract

Background: Very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD) is diagnosed in the US through newborn screening (NBS). NBS often unequivocally identifies affected individuals, but a growing number of variant patterns can represent mild disease or heterozygous carriers.

Aims: To evaluate the validity of standard diagnostic procedures for VLCADD by using functional in vitro tools.

Methods: We retrospectively investigated 13 patient samples referred to our laboratory because of a suspicion of VLCADD but with some uncertainty to the diagnosis. All 13 patients were suspected of having VLCADD either because of abnormal NBS or suggestive clinical findings. ACADVL genomic DNA sequencing data were available for twelve of them. Ten of the patients had an abnormal NBS suggestive of VLCADD, with three samples showing equivocal results. Three exhibited suggestive clinical findings and blood acylcarnitine profile (two of them had a normal NBS and the third one was unscreened). Assay of VLCAD activity and immunoblotting or immunohistologic staining for VLCAD were performed on fibroblasts. Prokaryotic mutagenesis and expression studies were performed for nine uncharacterized ACADVL missense mutations.

Results: VLCAD activity was abnormal in fibroblast cells from 9 patients (8 identified through abnormal NBS, 1 through clinical symptoms). For these 9 patients, immunoblotting/staining showed the variable presence of VLCAD; all but one had two mutated alleles. Two patients with equivocal NBS results (and a heterozygous genotype) and the two patients with normal NBS exhibited normal VLCAD activity and normal VLCAD protein on immunoblotting/staining thus ruling out VLCAD deficiency. Nine pathogenic missense mutations were characterized with prokaryotic expression studies and showed a decrease in enzyme activity and variable stability of VLCAD antigen.

Conclusions: These results emphasize the importance of functional investigation of abnormal NBS or clinical testing suggestive but not diagnostic of VLCADD. A larger prospective study is necessary to better define the clinical and metabolic ramifications of the defects identified in such patients.

Figure 1

Characterization of wild type (WT) and patient fibroblast samples for VLCAD activity and antigen. The top line shows mutations identified in patient cell lines. Western blot results using VLCAD antisera are shown in the next line. The bottom line gives the VLCAD activity from fibroblast extracts presented as a percentage of the concurrent wild type control (ND is non-detectable). All samples were also tested for MCAD activity and antigen as a control and gave normal levels of both (not shown).

Figure 2

Immunofluorescent staining of wild type (A) and patient (B) fibroblasts. A. Wild type cells (WT). VLCAD antigen was visualized with green fluorescently tagged antibodies (top, middle panel) and mitochondrial cytochrome c oxidase (COX) was visualized with red fluorescently tagged antibodies (top, right panel). Nuclei were visualized with DAPI staining (blue; top, left panel). The merged image (bottom panel) shows colocalization of VLCAD and COX in mitochondria as yellow (white arrow). B. Patient fibroblasts. Each panel shows the merged only image of patients (PTs 1–6 and 8–11) with the corresponding Western blot results. The mutations identified in the cell line are shown at the top of each panel for convenience. Scale bar, 10.75 μm.

Figure 2

Immunofluorescent staining of wild type (A) and patient (B) fibroblasts. A. Wild type cells (WT). VLCAD antigen was visualized with green fluorescently tagged antibodies (top, middle panel) and mitochondrial cytochrome c oxidase (COX) was visualized with red fluorescently tagged antibodies (top, right panel). Nuclei were visualized with DAPI staining (blue; top, left panel). The merged image (bottom panel) shows colocalization of VLCAD and COX in mitochondria as yellow (white arrow). B. Patient fibroblasts. Each panel shows the merged only image of patients (PTs 1–6 and 8–11) with the corresponding Western blot results. The mutations identified in the cell line are shown at the top of each panel for convenience. Scale bar, 10.75 μm.

Figure 3

Prokaryotic mutagenesis and expression studies of 9 missense ACADVL mutations. Each mutation shown at the top of the figure was expressed in E. coli and the extract was analyzed by SDS-PAGE followed by western blotting with VLCAD antibodies (middle). Activity in cell-free extracts following prokaryotic expression is given on the bottom line. ND is non-detectable.

Figure 4

Results from the fatty acid oxidation probe assay in fibroblasts for 10 of the 13 patients. Since the analyses were performed at two different clinical laboratories, abnormal C12, C14 and C16 acylcarnitines species are expressed as fold of increase relative to upper limit of control values of the testing lab rather than as actual values.

Figure 5

Molecular model of VLCAD crystal structure and mutation positions. The molecular structure of VLCAD from PDB file 2UXW was visualized with the MolView viewer (Accelerys, Inc, San Diego, CA). Subunit A is color coded according to structural motif: red, α-helix; blue, β-sheet; white, random coil, orange, C-terminal domain. The B subunit is colored purple. The mutations described in this manuscript are represented as yellow balls. The stick structures of FAD and palmitoyl-CoA are colored yellow and green, respectively.