A Novel Double Mutation in the ABCD1 Gene in a Patient with X-linked Adrenoleukodystrophy: Analysis of the Stability and Function of the Mutant ABCD1 Protein

Abstract

We diagnosed an adrenomyeloneuropathy (AMN) patient with a double novel missense mutation, c.284C>A (p.A95D) and c.290A>T (p.H97L) in a single ABCD1 allele. In skin fibroblasts from the patient, no ABCD1 protein was detected by immunoblot analysis, and the C24:0 β-oxidation activity was decreased to a level at which the ABCD1 protein was absent. To determine the responsible gene mutation in the patient, we constructed three kinds of mutated ABCD1 gene expression vectors (c.284C>A, c.290A>T or c.284C>A/c.290A>T) and transfected them into CHO cells stably expressing GFP-SKL (CHO/GFP-SKL cells) or CADDS fibroblasts lacking the ABCD1 gene. ABCD1 (p.H97L) displayed the correct peroxisomal localization in CHO/GFP-SKL cells, but ABCD1 (p.A95D) and ABCD1 (p.A95D/p.H97L) were diffuse in the cytosol. Furthermore, ABCD1 (p.H97L) was detected by immunoblot analysis and restored the C24:0 β-oxidation activity in the CADDS fibroblasts, as the wild type ABCD1 did. On the other hand, ABCD1 (p.A95D) and ABCD1 (p.A95D/p.H97L) were not detected and the C24:0 β-oxidation activity was not restored. These results clearly show that c.284C>A is the responsible gene mutation, whereas c.290A>T is a novel polymorphism.

Fig. 1

An MRI image in the X-ALD patient. (a) A T1-weighted midsagittal image shows mild atrophy of the cerebellum. (b) A T2-weighted axial image shows there are no definite signal intensity changes in the cerebral white matter

Fig. 2

Immunofluorescence analysis of ABCD1 expression in normal, X-ALD, and CADDS fibroblasts. Expression of mutant ABCD1 proteins in fibroblasts was analyzed by immunofluorescence analysis. Normal, X-ALD, and CADDS fibroblasts were stained with anti-ABCD3 and anti-ABCD1 antibodies followed by secondary antibodies conjugated with the dye reagents. Fluorescent dots were observed under confocal microscopy. Bar = 20 μm. ABCD1 protein was not detected in X-ALD and CADDS fibroblasts

Fig. 3

VLCFA β-oxidation activity and ABCD1 protein expression in X-ALD fibroblasts. VLCFA β-oxidation activity in the normal, ALD, βand CADDS fibroblasts was measured using [1-¹⁴C]C24:0 as substrate (a). Results are the means ± S.D.; n = 3. Expression of ABCD1 proteins in each of the cells was analyzed by immunoblot analysis (b). The C24:0 β-oxidation was less than 20% of the normal fibroblasts and the ABCD1 protein was not detected in the X-ALD fibroblasts

Fig. 4

Expression of mutant ABCD1 proteins in CHO/GFP-SKL cells. CHO/GFP-SKL cells were transfected with empty vector, pcDNA4/WT ABCD1 (wild type), or pcDNA4/mutant ABCD1 (p.A95D, p.H97L, or p.A95D/p.H97L). After the transfection, cells on the coverslips were fixed and subjected to immunofluorescence analysis. The expressed GFP-SKL proteins that localized in the peroxisomes were detected as green fluorescent dots. The expressed ABCD1 proteins were detected using an anti-human ABCD1 antibody followed by secondary antibodies conjugated with the dye reagent. Bar = 20 μm. The mutant ABCD1 (p.H97L) was localized to peroxisomes, but the mutants ABCD1 (p.A95D) and ABCD1 (p.A95D/p.H97L) were diffuse in the cytosol

Fig. 5

C24:0 β-oxidation activity in CADDS fibroblasts expressing mutant ABCD1 proteins. C24:0 β-oxidation activity in the CADDS fibroblasts expressing mutant ABCD1s was analyzed as in Fig. 3. CADDS fibroblasts were transfected with empty vector, pcDNA4/WT ABCD1 (wild type) or each pcDNA4/mutant ABCD1 (p.A95D, p.H97L, p.A95D/p.H97L). After 3-day transfection, the cells were harvested for determination of the C24:0 β-oxidation activities (a) and immunoblotting (b). Results are means ± S.D.; n = 3. The mutant ABCD1 (p.H97L) was expressed in CADDS fibroblasts and the C24:0 β-oxidation activity was recovered as wild type ABCD1. In contrast, the mutants ABCD1 (p.A95D) and ABCD1 (p.A95D/p.H97L) were scarcely expressed and the C24:0 β-oxidation activities were not recovered