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Identification and Functional Analysis of Inherited Variation in the CYP3A4 Gene Regulatory Region.

Hamzeiy, Hossein. (2002) Identification and Functional Analysis of Inherited Variation in the CYP3A4 Gene Regulatory Region. Doctoral thesis, University of Surrey (United Kingdom)..

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Abstract

CYP3A4 is usually the major cytochrome P450 enzyme in adult human liver. It is known to metabolise a wide variety of xenobiotic and endogenous compounds. Substantial inter-individual variation in hepatic levels of CYP3A4 has been observed and although polymorphic mutations have been reported in both the 5' regulatory and coding regions of the CYP3A4 gene, those investigated so far do not appear to make a major contribution to CYP3A4 variation in the population as a whole. To determine whether regulatory mutations might occur in more distal regions of the promoter, I have performed a new population screening on a panel of 101 human DNA samples. 1141 bp of the proximal 5' regulatory region of the CYP3A4 gene and 300 bp of the distal enhancer region at -7. 9 kb, both containing numerous regulatory motifs, were amplified from genomic DNA. Screening for mutations in the resulting PCR products was carried out using non-radioactive single strand conformation polymorphism (SSCP) followed by confirmatory sequencing of both DNA strands. In addition to detection of the previously reported CYP3A4*1B allele in nine subjects, three novel alleles were found: CYP3A4*1E (having a T→A transversion at -369 in one subject), CYP3A4*1F (having a C→G transversion at -747 in 17 subjects) and CYP3A4*15B having a nine-nucleotide insertion between -845 and -844 linked to an A→G transition at -392 and a G→A transition in exon 6 (position 485 in the cDNA) in one subject. No mutations were found in the distal enhancer region indicating strong conservation of sequence in this region of the promoter. Functional analysis of the regulatory region mutants was performed in vitro using reporter DNA constructs in which the whole 1141 bp proximal promoter region from each mutant allele was inserted between a single copy of the 300 bp core distal enhancer sequence and the cDNA for human secreted alkaline phosphatase (SEAP). The individual reporter constructs were co-transfected with an hPXR expression vector into human liver (HepG2, HuH7) and intestinal (Caco-2) cell lines, in the presence or absence of classical xenobiotic inducers of CYP3A4. Modulation of transcriptional activation, as indicated by SEAP expression, was measured by chemiluminescent SEAP assay of the culture medium. Significant variation in promoter strength was found between the mutant CYP3A4 promoter alleles depending on the inducer used and the recipient cell line. While there was close similarity between the xenobiotic induction patterns for wild type and mutant promoters in HepG2 and HuH7 cells, the pattern in Caco-2 cells was substantially different. Identification of novel regulatory and coding region alleles of CYP3A4 will assist detailed investigation of the mechanistic basis of CYP3A4 regulation and help elucidate the relationship between genotype, xenobiotic metabolism and toxicity in the CYP3A family of isoenzymes.

Item Type: Thesis (Doctoral)
Divisions : Theses
Authors : Hamzeiy, Hossein.
Date : 2002
Additional Information : Thesis (Ph.D.)--University of Surrey (United Kingdom), 2002.
Depositing User : EPrints Services
Date Deposited : 24 Apr 2020 15:27
Last Modified : 24 Apr 2020 15:27
URI: http://epubs.surrey.ac.uk/id/eprint/855257

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