Common Polymorphic Variants In Drug Metabolizing Enzymes And Characterization Of The Promotor Of The Human CYP4Z1 Gene

Genetic polymorphism is the occurrence of two or more genetically determined phenotypes within the same population,caused by the presence of two or more alleles at one or several gene loci.Genetic polymorphisms of cytochrome P450(abbreviated as CYPs or P450s)or uridine diphosphate glucuronyltransferase(UGT)genes can cause changes in the drug metabolizing capacity of an individual and in homeostasis of endocrine regulatory factors.In most cases,gene polymorphisms of drug-metabolizing enzymes are single nucleotide polymorphisms(SNPs)which show significant differences between individuals,races,and regions.The Exome Aggregation Consortium(Ex AC)has recently published allele frequency data obtained from more than 60,000 human exomes,which considerably exceeds previously available exome-wide variant databases.In this work,all common SNP variants were extracted from these data that lead to missense mutations in 57 CYPs and 22 UGTs as well as in their helping proteins,thus obtaining the frequencies of 582 CYP alleles and 123 UGT alles as well as those of 32 alleles of cytochrome P450 reductase(CPR),adrenodoxin reductase(Ad R)and adrenodoxin(Adx).With a total of 737 alleles from 83 genes a comprehensive overview of missense allele distributions in drug metabolizing enzymes is provided.In all variants the most common allele was always considered to be the wild-type,even if it was not identical to the *1-allele and/or the reference standard sequence of the Ref Seq project.Surprisingly,in 15 genes(Ad R,CYP2A7,CYP2C19,CYP2D6,CYP4A22,CYP4F11,CYP4F12,CYP4V2,CYP8B1,CYP20A1,UGT1A3,UGT1A7,UGT2A3,UGT2B7,and UGT2B15),the wild-type protein sequences encoded by the most common gene variants were found not to be identical with the reference standard sequences.The human CYP4Z1 enzyme is of interest because it is strongly overexpressed in breast cancer;however,the details of its expression regulation are not well known.In general,eukaryotic promoters are divided into three classes based on the RNA polymerase(RNA pol)types that use them,which are either RNA pol I,II,or III,respectively.The RNA pol II promotor is involved in the control of the expression of genes that code for proteins(such as CYP4Z1).Its basic structural feature is its location upstream of the transcription initiation site,where it regulates the initiation of transcription.The process of transcription initiation is influenced by a variety of DNA-binding proteins called transcription factors(TFs).A transcription factor is a protein that specifically binds to the upstream part of the 5’ end of a gene and enhances or decreases the stability of the transcription initiation complex.A transcription factor binding site(TFBS)is a small DNA sequence element in a promotor where transcription factors can bind.It was the aim of this study to provide a more detailed analysis of the human CYP4Z1 promotor.For this purpose,14 putative transcription factors and their binding sites within this promotor were identified using the world’s largest transcription factor database Genomatix Mat Inspector.Taking these binding sites into account,a series of CYP4Z1 promotor deletion variants were cloned into a vectorcontaining the luciferase-encoding reporter gene luc.The resulting plasmids were transiently transfected into the breast cancer cell line MCF-7 or the human normal breast cell line MCF-10 A,respectively,and luciferase activities were determined.These experiments corroborated the presence of several of the potential transcriptional activator binding sites;in addition,evidence for the involvement of a transcription repressor was obtained.However,further studies are needed to understand the complex regulation of this promotor and its differences in normal or cancerous breast cells.