Expression Of Adrenoleukodystrophy Protein In E.coli And Molecular Diagnosis Of Adrenoleukodystrophy

X-linked adrenoleukodystrophy(ALD) is the most common peroxisomal disorder. The clinical phenotypes of X-ALD are childhood cerebral ALD, adolescent cerebral ALD, adult cerebral ALD, adrenomyeloneuropathy, Addison- only disease, heterozygous and asymptomatic phenotype based on the age of onset and the organs principally affected. ALD is characterized by abnormal accumulation of saturated very long chain fatty acids(VLCFA) in tissues, which is responsible for the lesion in cerebral white matter, adrenal cortex, and testis.X-ALD is caused by ABCD1 gene mutation, which results in the structural change and/or functional insufficiency of ABCD1 protein. The accumulation of VLCFA in plasma and tissues of patient is due to an impaired capacity to β- oxidize these fatty acids, which results in clinical symptoms of progressive cerebral demyelination and adrenocortical dysfunction. The clinical symptom of ALD patient cannot be reversed once manifested. The prognosis of childhood cerebral ALD patient is most severe because the life expectancy of the patient is about three years once clinical manifestations occur. Molecular analysis can reveal the ALD patients' mutation and the genotypes of the family members. It can also provide correct diagnosis for ALD patients and reliable evidence for genetic consultation, prenatal diagnosis, and gene therapy. Total RNA was extracted from the human peripheral blood leukocytes of normal individual. The coding sequence of ATP-binding domain was amplified after reverse transcription. To construct a prokaryotic recombinant vector, the PCR product was cloned into pGEX-4T-2 vector after the restrictive digestion by EcoRⅠ and XhoⅠ, and its expression was detected in E. coli BL-21. GST fusion protein was expressed at a high level after induction with IPTG and could be detected by 10%SDS polyacrylamide gel electrophoresis. And the GST fusion protein could also be recognized by a mouse anti-human ABCD1 protein monoclonal antibody in Western blotting. Anti- ATP-binding cassette domain sera were collected after rabbits were immunized by the purified GST fusion protein.Four unrelated Chinese ALD families were studied at both cDNA and genomic DNA level in this study. Total RNA was isolated from the peripheral blood leukocytes of patients and their mothers, using RNA blood Mini kit. After reverse transcription, cDNA was amplified in four overlapping segments. The PCR products were purified and directly sequenced. To confirm the mutations, the genomic DNA was extracted from the patients and their family members using DNA blood isolation kit, and analyzed by PCR-restrictive digestion, direct sequencing or amplification refractory mutation system. Four distinct missense mutations were detected in the ABCD1 gene of the four pedigrees. A mutation of CCC→CGC was detected at codon 534 of the ABCD1 gene from patient 1, resulting in the substitution of proline for arginine(P534R). A change of GGG→AGG was found at codon 266 of the second patient's gene, accompanied by the replacement of glycine by arginine(G266R). A mutation of CGC→GGC was found at codon 617 in one ABCD1 allele of the third patient's mother, leading to the substitution of arginine for glycine(R617G). A mutation of GGC→GTC was detected at codon 343 of the ABCD1 gene from patient 4, resulting in the replacement of glycine for valine(G343V). The P534R mutation and the G343V mutation were novel, and the other two mutations have been reported in patients from outside China. The four mutations were confirmed through restriction analysis, direct sequencing or amplification refractory mutation system. In pedigree 4, the same genotype was found in an elder brother of patient 4, who had not manifested any clinical symptoms.A fetus of pedigree 3 was subjected to prenatal molecular diagnosis. The amniotic fluid cells from the fetus were obtained with the help of a clinical doctor and the genomic DNA was isolated from them. Maternal DNA contamination was excluded by STR profiling. And the R617G mutation was examined using amplif...