Mutation Analysis Of The ABCD1Gene In Patients With X-linked Adrenoleukodystrophy And Expression Of ALDP Mutants In Eukaryotic Cells

X-linked adrenoleukodystrophy (X-ALD) is a genetically heterogeneous disease.The disease is caused by a defect in the ABCD1gene, which maps to Xq28andencodes a peroxisomal member of ATP-binding cassette (ABC) transporter subfamilyD called ALDP (adrenoleukodystrophy protein). Defective peroxisomal β-oxidationand accumulation of saturated very long chain saturated fatty acids (VLCFAs) are themain biochemical features of X-ALD. The consequential accumulation of these fattyacids in different tissues and body fluids leads to progressive central and peripheraldemyelination, as well as adrenocortical insufficiency. The major clinicalmanifestations are progressive impairment of hearing, vision, cognition, and motorfunction. The prognosis for patients with X-ALD is infaust. There is no effectivetherapy for neurologic abnormalities in X-ALD, though hematopoietic stem celltransplantation (HSCT) is shown to be the only therapy with long-term benefit, butonly when offered in the early stages of the illness. Prenatal diagnosis orpre-implantation genetic diagnosis of female carriers is an effective way to preventunnecessary new cases of this devastating disease.Mutation analysis of the ABCD1gene for X-ALD patients and their familymembers not only provides a clear diagnosis, but also provides reliable data forgenetic consultation and prenatal diagnosis. In this study, mutation analysis of theprobands and fetuses from5unrelated X-ALD pedigrees were performed. The codingregion of ABCD1cDNA of5patients was amplified and sequenced. To confirm themutations，the genomic DNA of the patients and their family members was analyzedby direct sequencing. Maternal contamination was excluded by fluorescent STRprofiling. The genomic DNA of amniotic fluid cells (AFC) of5fetuses was analyzedby PCR-restrictive digestion or direct sequencing. Five base substitutions (c.796G>A,c.521A>G, c.1523C>T, c.1534G> A and c.1252C>T) were identified in five X-ALD pedigrees, which resulted in five missense mutations (p.Gly266Arg, p.Tyr174Cys,p.Pro508Leu, p.Gly512Ser and p.Arg418Trp). The5fetuses were males. The samemutation as the proband was detected in the ABCD1gene of fetus1, fetus2, fetus3and fetus4. The same mutation as the proband was not detected in fetus5. Takentogether, it could be deduced that fetus1, fetus2, fetus3and fetus4were ALDhemizygotes, and fetus5was a normal hemizygote.To study the effects of the ABCD1mutations on the expression and function ofALDP, bioinformatic predictions and functional studies on two variants (H283R andP534R) were carried out. Firstly, we used different computational algorithms topredict the pathogenicity and the structural stability of the mutants. The resultsshowed that both mutations were at conserved codons, and the mutant ALDP might bedestabilizing, suggesting that the mutations would be pathogenic. Then molecularcloning techniques were used to study the function of ALDP mutants. We constructedthe wild type and mutant recombinant eukaryotic expression vectors, namedPLEX-ABCD1, PLEX-ABCD1-H283R and PLEX-ABCD1-P534R. The recombinantplasmids and two packaging vectors co-transfected293T cells, and successfullyobtained virus which subsequently infected host cells. Immunofluorescence study andwestern blotting analysis showed the overexpression of the wild type ALDP and thelower expression of the mutant ALDP, while no subcellular mislocalization of themutant ALDP was detected.In the present study, we assessed the effects of the ABCD1mutations on theexpression and localization of ALDP by functional experiments, which furthers ourunderstanding on the pathogenic mechanism of X-ALD. Moreover, it providedexperimental data for the improvement of diagnosis, prevention and treatment ofX-ALD.