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Pharmacogenetics of human cytochrome P450 3A (CYP3A) enzymes

thesis
posted on 2024-09-02, 19:20 authored by Anna Westlind Johnsson

The cytochrome P450 3A (CYP3A) subfamily encompasses the most important CYP enzymes with respect to drug metabolism, in particular CYP3A4. responsible for the metabolism of a majority of drugs being metabolized in order to be excreted. There is a substantial interindividual variability in activity and levels of expression of CYP3A, hence metabolic capability of substances being substrates of CYP3As differs between individuals. In this thesis, we have investigated the influence of genetic factors that could contribute to the variable expression seen in CYP3A expression and metabolism.

The proximal promoter of the CYP3A4 gene in a pool of human liver samples were sequenced in search of genetic variations. CYP3A4*1B was found in three of 39 livers. No evident effect on the enzyme activity by this polymorphism was seen. The functional significance of the site of the polymorphism was investigated by EMSA. Competition experiments revealed an unspecific binding of nuclear proteins to the polymorphic site, although having lower affinity to the CYP3A4*1B form. These findings suggested no influence of this site on regulation, expression and activity and questions previous data describing a proposed causal relationship between the presence of the CYP3A4*1B allele and incidence of cancer in the prostate and leukemia.

Through homology searches of CYP3A related sequences in the dbEST database, a fragment of a CYP3A related cDNA was identified. A full length sequence of a new member of the CYP3A subfamily, CYP3A43, was amplified and cloned from human liver cDNA. RT-PCR on cDNA from extrahepatic tissues showed, with the exception of the testis, only very low levels of CYP3A43 expression. In contrast to CYP3A4, heterologously expressed CYP3A43 protein in yeast and mammalian cells failed to produce a functional enzyme. It was concluded that CYP3A43 would not contribute significantly to human drug metabolism.

Realtime PCR analysis of the CYP3A4, CYP3A5, CYP3A43 and PXR mRNA levels in 46 Caucasian human liver tissue samples, revealed correlation between all CYP3A and PXR mRNA transcripts measured suggesting common regulatory mechanism(s) at the transcriptional level for the corresponding genes. In addition, protein expression in relation to the CYP3A5*1 genotype was investigated. The CYP3A5 protein was expressed at quantifiable levels in 5 (10.9%) of the livers. In total, CYP3A5 only contributed with 2% of the combined pool of CYP3A protein among all samples. Thus, the results suggests that CYP3A expression and activity in Caucasians are dominated by CYP3A4 with only minor contribution from CYP3A5.

An individual of Caucasian origin phenotyped with midazolam and having very low activity was found to carry a SNP in the CYP3A4 gene in exon 13 causing a P488T frameshift and a premature stop yielding a protein with a ten amino acid truncation and an altered sequence of the six most C-terminal amino acids. Heterologous expression of this variant allele, CYP3A4*20, in yeast and HEK 293 cells suggested that the protein did not fold properly and was inactive.

In summary, a new member of the CYP3A subfamily has been identified, cloned and characterized. Two different polymorphisms, CYP3A4*1B and the novel CYP3A4*20 allele, have been functionally characterized and it is concluded that in contrast to other reports, CYP3A5 does not contribute significantly to CYP3A expression and activity in Caucasians.

List of scientific papers

I. Westlind A, Lofberg L, Tindberg N, Andersson TB, Ingelman-Sundberg M (1999). Interindividual differences in hepatic expression of CYP3A4: relationship to genetic polymorphism in the 5-upstream regulatory region. Biochem Biophys Res Commun. 259(1): 201-5.
https://pubmed.ncbi.nlm.nih.gov/10334940

II. Westlind A, Malmebo S, Johansson I, Otter C, Andersson TB, Ingelman-Sundberg M, Oscarson M (2001). Cloning and tissue distribution of a novel human cytochrome p450 of the CYP3A subfamily, CYP3A43. Biochem Biophys Res Commun. 281(5): 1349-55.
https://pubmed.ncbi.nlm.nih.gov/11243885

III. Westlind-Johnsson A, Malmebo S, Johansson A, Otter C, Andersson TB, Johansson I, Edwards RJ, Boobis AR, Ingelman-Sundberg M (2003). Comparative analysis of cyp3a expression in human liver suggests only a minor role for cyp3a5 in drug metabolism. Drug Metab Dispos. 31(6): 755-61.
https://pubmed.ncbi.nlm.nih.gov/12756208

IV. Westlind-Johnsson A, Ingelman-Sandberg M, von Richter O (2003). Identification and charcterization of a novel defect CYP3A4 allele with impotant implications for drug response in vivo. [Manuscript]

History

Defence date

2003-11-14

Department

  • Institute of Environmental Medicine

Publication year

2003

Thesis type

  • Doctoral thesis

ISBN-10

91-7349-688-X

Number of supporting papers

4

Language

  • eng

Original publication date

2003-10-24

Author name in thesis

Westlind Johnsson, Anna

Original department name

Institute of Enviromental Medicine

Place of publication

Stockholm

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