Mutational Analysis Of ALD Gene In Patients With X-linked Adrenoleukodystrophy And Expression Of ALDP Mutants In E.coli

X-linked adrenoleukodystrophy (X-ALD), the most common peroxisomal disorder, causes damage in cerebral white matter and adrenal cortex, through mutations in ABCD1 gene on chromosome Xq28. The length of mature mRNA of the gene is about 3.7kb, which encodes a protein of 745 amino acid residues. Mutations in ABCD1 gene result in abnormal accumulation of saturated very long chain fatty acid (VLCFA), which in turn leads to demyelination of cerebral white matter and dysfunction of adrenal cortex in X-ALD patients. The clinical symptoms of X-ALD patient can not be reversed once manifested. Molecular analysis can reveal the mutations of ABCD1 gene and the genotypes of the family members. It can also provide reliable data for genetic consultation and prenatal diagnosis.Five unrelated Chinese ALD families were studied at both mRNA and genomic DNA level in this study. Four fragments covering the entire coding sequence of the ABCD1 gene from the patients were amplified by RT-PCR. The PCR products were directly sequenced by chain termination method. The result of sequencing was confirmed by restriction enzyme digestion or denaturing high performance liquid chromatography (DHPLC) of PCR products from genomic DNA. Human ABCD1 gene and ALDP were aligned with the homologs of rat, monkey, mouse and cattle by Clustal X 1.83. The sofewares of Motif Scan, TMpred, ESYpred3D were used to predict the domains, transmembrane helix regions and tertiary structure of ALDP. Four distinct missense mutations, one samesense mutation and one frame shift mutation were detected in the ABCD1 gene of the five patients. A mutation of GGC→AGC was detected at codon 512 of the ABCD1 gene from patient 1, resulting in the substitution of glycine for serine (G512S). A frame shift mutation was found at codon 471 of the second patient's gene, accompanied by subsequent change in amino acid sequence(fs Glu471). A mutation of AAG→GAG was found at codon 217 in one ABCD1 allele of the third patient, leading to the substitution of lysine for glutamic acid (K217E). A samesense mutation of GTG→GTA was also found at codon 489 in the ABCD1 allele of the third patient, without leading to the substitution of amino acid (V489V). A mutation of CAC→CGC was detected at codon 283 of the ABCD1 gene from patient 4, resulting in the replacement of histidine for arginine (H283R). A mutation of CAC→GAC was detected at codon 283 of the ABCD1 gene from patient 5, resulting in the replacement of histidine for aspartic acid (H283D). The H283R mutation was a novel mutation. The mutated amino acid residue (283H) was highly conservative in evolution, and caused a dramatic change in the structure of ALDP protein. The G512S mutation was de novo mutation, because the mutation was not detected in his parents. In pedigree 2, the same genotype was found in an elder brother of patient 2, who had not manifested any clinical symptoms. The mutations in patient 3 belonged to multiple mutations (K217E+V489V). In pedigree 4, three female patients heterozygous for X-ALD were first reported in China.After confirming the mutations in ABCD1 gene from probands, prenatal molecular diagnosis was carried out in three fetuses at high risk for X-ALD. The amniotic fluid cells from the fetus were obtained with the help of an obstetrician and the genomic DNA was isolated from them. Maternal DNA contamination was excluded by STR profiling. In the pedigree 1, the PCR product (407bp)of the fetus 1 and her mother could be digested with MaeI. The Q177X mutation, which was present in the index patient, was detected in the ABCD1 gene from the genomic DNA of the fetus 1 using direct sequencing. In the pedigree 2, the fs Glu 471 mutation was not found in the ABCD1 gene from the genomic DNA of the fetus 2 using direct sequencing. In the pedigree 3, the multiple mutations (K217E+V489V) was found in the ABCD1 gene from the genomic DNA of the fetus 3 using direct sequencing. The paternity test showed that fetus 1 and fetus 2 were female and fetus 3 was male. From these results, it could be deduced that fetus 1 was an X-ALD carrier, fetus 2 normal homozygotes, and fetus 3 was an X-ALD hemizygote.To construct prokaryotic recombinant vectors, the coding sequence of ATP-binding domain, transmembrane domain and the full-length ALDP were inserted into pGEX-4T-2 vector after the restrictive digestion by EcoRⅠand XhoⅠ, and expressed in E.coli BL-21. GST fusion proteins could be detected by 12%SDS polyacrylamide gel and could also be recognized by a mouse anti-human ALDP monoclonal antibody in Western blotting. To perform site-specific mutagenesis for three mutations (P508L, G512S and R617C) in the recombinant plasmid, a site-specific mutagenesis kit was used and the resultant mutants were expressed in E.coli BL-21. GST fused mutants could be detected by 12%SDS polyacrylamide gel electrophoresis and Western blotting. To purify GST fusion protein and ALDP mutants, we used MagneGSTTM protein purification system. The preparation of three ALDP mutants would lay the foundations for further study of X-ALD molecular pathogenesis and effects of ABCD1 gene mutation on the function or protein structure of ALDP.