Molecular Biological Studies Of X-linked Adrenoleukodystrophy

X-linked adrenoleukodystrophy(X-ALD), the most common peroxisomal disorder,mainly invades cerebral white matter and adrenal cortex. The clinical phenotypes of X-ALD are asymoptomatic type with normal MRI, asymoptomatic type with abnormal MRI, Addison disease only (MRI normal), Addison disease only (MRI abnormal), mild cerebral type without AMN, severe cerebral type without AMN, pure AMN, cerebral AMN, and cerebellar type. According to Hugo, approximately 47~48 percent of all the X-ALD patients belong to Cerebral phenotype.X-ALD results from 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 amo acid residues. Mutations in ABCD1 gene results 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 cannot 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 evidence for genetic consultation and prenatal diagnosis.Six unrelated Chinese X-ALD pedigrees were studied at both cDNA and genomic DNA level in this study. Total RNA was isolated from the peripheral blood leukocytes of patients. After reverse transcription into cDNA , the entire coding region of ABCD1 gene was amplified by PCR. The PCR products were purified and directly sequenced. To confirm the mutations, the genomic DNA was extracted from peripheral blood of the patients and their family members, and analyzed by denaturing high performance liquid chromatography (DHPLC) or direct sequencing. Six distinct mutations were detected in the ABCD1 gene of the six pedigrees. A mutation of CAG→TAG was detected at codon 177 of the ABCD1 gene from patient 1, resulting in the substitution of glutame by a stop codon (Q177X). A change of CGG→CAG was found at codon 401 of the sond patient's gene, accompanied by the replacement of arginine by glutame (R401Q). A mutation of AAG→GAG was found at codon 276 in the ABCD1 gene from patient 3, leading to the substitution of lysine for glutamic acid(K276E). A mutation of TAC→TGC was detected at codon 174 of the ABCD1 gene from patient 4, resulting in the substitution of tyrosine for cysteine (Y174C). A change of AAG→TAG was found at codon 602 of the ABCD1 gene from patient 5, accompanied by the replacement of lysine by a stop codon (K602X). A mutation of AAG→GAG was detected at codon 314 in the ABCD1gene from patient 6, leading to the substitution of alanine for proline(A314P). K602X and A314P mutations had not been reported.After confirmg the mutations in ABCD1 gene from probands, prenatal molecular diagnosis was carried out in four fetuses at high risk for X-ALD. The amniotic fluid was obtained with the help of an obstetrician and genemic DNA was isolated from amniotic fluid cell. Maternal contamation was evaluated by paternity test. PCR-RFLP , sequencing and DHPLC were used to detect the ABCD1 gene of fetal genome. In the pedigree 1, the PCR product (799bp)of the fetus 1 and her father (normal control)could be digested with Bcn I. No P560L 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 PCR product (232bp)was analysized with the DHPLC, and the pattern of elution peaks of the fetus 2 and her father was similar, but different from that of the proband. No Q177X mutation , which was present in the proband,was detected in the ABCD1 gene from the genomic DNA of the fetus 2 using direct sequencing. In the pedigree 3, the PCR product (271bp)was digested with Aci I, the pattern of the fetus 3 and the propositus being the same, and the R617C mutation was found in the ABCD1 gene from the genomic DNA of the fetus 3 using direct sequencing. In the pedigree 4, the PCR product (269bp)was analysized with the DHPLC, and the pattern of elution peaks of the fetus 4 and her father was similar, but different from that of the propositus. No K276E mutation was detectable in the ABCD1 gene from the genomic DNA of the fetus 4 by using direct sequencing. The paternity test also showed that fetus 1 and fetus 2 were female and fetus 3 and fetus 4 were male. From results described above, it could be deduced that both fetus 1 and fetus 2 were normal homozygotes, fetus 3 was an ALD hemizygote, and fetus 4 was a normal hemizygote.Total RNA was extracted from the peripheral blood leukocytes of normal individual and X-ALD patients (with K276E mutation and P560L mutation). Primers at both ends of the entire coding region of the normal ABCD1 gene were designed to amplify the cDNA from normal individual and X-ALD patients after reverse transcription. The PCR products were ligated with enhanced green fluorescent protein vector-- pEGFP-N2 after restrictive digestion with EcoRⅠand Xho I. The ligation products were introduced into JM109, the recombinant bacteria selected with 100μg/ml kanamycin, and the recombinant plasmids isolated from them. To identify the recombinant plasmids, the recombinant plasmids were digested with EcoRⅠand XhoⅠ, and the inserted segments were sequenced. Our data showed that the 2253bp fragments, as expected, could be released from the recombinant plasmids with the digestion of EcoRⅠand XhoⅠ. The sequence of inserted segment in one recombinant plasmid was identical to that of entire coding region of normal ABCD1 gene, that of another one carried the K276E mutation, and that of the third plasmid had the P560L mutation. These recombinant plasmids were named pEGFP-N2-ABCD1, pEGFP-N2-ABCD1(K276E)and pEGFP-N2-ABCD1(P560L)respectively.In summmary, methods for molecular diagnosis of X-linked adrenoleukodystrophy was established and two novel mutations ( K602X and A314P) were detected in Chinese X-ALD patients. A new protocol for X-ALD prenatal molecular diagnosis was put forward, which would ensure the accuracy of prenatal diagnosis. The eukaryotic expression vectors of normal and mutated ABCD1 gene were constructed successfully, which would lay the foundations for the further study of X-ALD molecular pathogenesis.