Investigations In Pathogenesis Of X-liked Adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is the most common peroxisomal disorder, which is characterized pathologically by progressive demyelination of the central nervous system and the adrenal dysfunction. ABCD1 is the gene responsible for X-ALD, which lies on chromosome Xq28 and encodes a peroxisomal membrane protein of 745 amino acid residues called adrenoleukodystrophy protein (ALDP). Defect in the ABCD1 gene impairs the peroxisomal capacity to metabolize very long chain saturated fatty acids (VLCFAs) viaβ-oxidation, leading to accumulation of VLCFAs in all parts of the body, including the brain and adrenal glands.From 2001 to 2010, the ABCD1 gene from more than 30 unrelated Chinese X-ALD families were analyzed in our laboratory. Here, we report the identification of 4 asymptomatic males with X-ALD from 3 pedigrees, 2 de novo mutations from 2 pedigrees and 2 heterozygous female patients from 1 pedigree, and the results of the X chromosome inactivation analysis showed that skewed X chromosome inactivation in favor of the mutant allele would be associated with the manifestation of heterozygous symptoms.To study the effects of the ABCD1 mutations on the structure and function of ALDP, different computational algorithms were used. The results showed that all of the missense mutations identified in our laboratory were at conserved codons, and the mutant ALDP might be degraded by proteasomes because of the abnormal folding, suggesting that all the mutations would be classified as pathogenic.To evaluate the accuracy of the bioinformatic predictions, functional studies on two variants (R280L and L576P) were carried out. The wild type and mutant recombinant eukaryotic expression vectors were constructed, named pEGFP-N2-ABCD1, pEGFP-N2-ABCD1-R280L and pEGFP-N2-ABCD1-L576P. Immunofluorescence study and western blotting analysis showed the expression of wild type ALDP-GFP in HeLa cells, while the mutant ALDP-GFP could not be detected, which might be due to rapid degradation. Therefore, the results of functional experiments were consistent with the computational predictions. The present study provided experimental data for the improvement of diagnosis, prevention and treatment of X-ALD, and furthers our understanding on the pathogenic mechanism of monogene disorders.