Expression of the human CYP2D6 reporter constructs in t

Systematic functional study of cytochrome P450 2D6 promoter polymorphisms in the Chinese Han population. Gong X, Liu Y, Zhang X, Wei Z, Huo R, Shen L, He L, Qin S - PLoS ONE (2013) Bottom Line: Using site-directed mutagenesis, all the five SNPs identified and ten haplotypes with a frequency equal to or greater than 0.01 in the population were constructed on a luciferase reporter system.The activity produced by Haplo3(-2183G>A, -1775A>G, -1589G>C, -1431C>T, -1000G>A, -678A>G), Haplo8(-2065G>A, -2058T>G, -1775A>G, -1589G>C, -1235G>A, -678A>G) and MU3(-498C>A) was 0.7-, 0.7-, 1.2- times respectively compared with the wild type in human hepatoma cell lines(p<0.05).These findings might be useful for optimizing pharmacotherapy and the design of personalized medicine. View Article: PubMed Central - PubMed Affiliation: Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (Ministry of Education), Bio-X Institutes, Shanghai Jiao Tong University, Shanghai, China. ABSTRACT The promoter polymorphisms of drug-metabolizing genes can lead to interindividual differences in gene expression, which may result in adverse drug effects and therapeutic failure. Based on the database of CYP2D6 gene polymorphisms in the Chinese Han population established by our group, we functionally characterized the single nucleotide polymorphisms (SNPs) of the promoter region and corresponding haplotypes in this population. Using site-directed mutagenesis, all the five SNPs identified and ten haplotypes with a frequency equal to or greater than 0.01 in the population were constructed on a luciferase reporter system. Dual luciferase reporter systems were used to analyze regulatory activity. The activity produced by Haplo3(-2183G>A, -1775A>G, -1589G>C, -1431C>T, -1000G>A, -678A>G), Haplo8(-2065G>A, -2058T>G, -1775A>G, -1589G>C, -1235G>A, -678A>G) and MU3(-498C>A) was 0.7-, 0.7-, 1.2- times respectively compared with the wild type in human hepatoma cell lines(p<0.05). These findings might be useful for optimizing pharmacotherapy and the design of personalized medicine. Show MeSH Major Asian Continental Ancestry Group/ethnology/genetics Cytochrome P-450 CYP2D6/genetics/metabolism Ethnic Groups/genetics* Polymorphism, Single Nucleotide* Promoter Regions, Genetic/genetics* Minor Animals Computational Biology Genes, Reporter/genetics Hep G2 Cells Humans Luciferases/genetics Mutagenesis, Site-Directed Transcription Factors/metabolism Related in: MedlinePlus	© Copyright Policy Related In: Results - Collection Show All Figures getmorefigures.php?uid=PMC3585152&req=5 pone-0057764-g001: Expression of the human CYP2D6 reporter constructs in the human hepatoma cell line (HepG2).The relative luciferase activities were normalized against the human wild type construct. The results are the average of at least three independent experiments. Comparisons among groups were done with the aid of the ANOVA statistical procedure. Significantly different from Wild type transfected cells (P<0.05). Significantly different from Wild type transfected cells (P<0.01), Significantly different from Wild type transfected cells (P<0.001). Mentions:** The functional significance of all 5 novel SNPs and 10 haplotypes was determined by the Renilla/Firefly luciferase assay after they had been transfected into human hepatoma cell lines. The Firefly/Renilla luciferase activity ratio was a good indicator of the relative activity of the CYP2D6 5′-upstream regulatory region. We conducted systematic analysis on all constructs with seven independent experiments using 8 replicates for every sample in each independent experiment. 2 haplotype constructs, namely pGL3-Haplo3 (AGTGCTGACGCCTA) and pGL3-Haplo8 (GAGGCCAGCGCCTA), both exhibited lower luciferase activity (both were 70%) compared to the wild type construct pGL3-WT (P<0.05), whereas the single point mutation MU3 exhibited higher luciferase activity (120%) compared with the wild type(p<0.05) (Figure 1).

Systematic functional study of cytochrome P450 2D6 promoter polymorphisms in the Chinese Han population.

Gong X, Liu Y, Zhang X, Wei Z, Huo R, Shen L, He L, Qin S - PLoS ONE (2013)

Bottom Line: Using site-directed mutagenesis, all the five SNPs identified and ten haplotypes with a frequency equal to or greater than 0.01 in the population were constructed on a luciferase reporter system.The activity produced by Haplo3(-2183G>A, -1775A>G, -1589G>C, -1431C>T, -1000G>A, -678A>G), Haplo8(-2065G>A, -2058T>G, -1775A>G, -1589G>C, -1235G>A, -678A>G) and MU3(-498C>A) was 0.7-, 0.7-, 1.2- times respectively compared with the wild type in human hepatoma cell lines(p<0.05).These findings might be useful for optimizing pharmacotherapy and the design of personalized medicine.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (Ministry of Education), Bio-X Institutes, Shanghai Jiao Tong University, Shanghai, China.

ABSTRACT

The promoter polymorphisms of drug-metabolizing genes can lead to interindividual differences in gene expression, which may result in adverse drug effects and therapeutic failure. Based on the database of CYP2D6 gene polymorphisms in the Chinese Han population established by our group, we functionally characterized the single nucleotide polymorphisms (SNPs) of the promoter region and corresponding haplotypes in this population. Using site-directed mutagenesis, all the five SNPs identified and ten haplotypes with a frequency equal to or greater than 0.01 in the population were constructed on a luciferase reporter system. Dual luciferase reporter systems were used to analyze regulatory activity. The activity produced by Haplo3(-2183G>A, -1775A>G, -1589G>C, -1431C>T, -1000G>A, -678A>G), Haplo8(-2065G>A, -2058T>G, -1775A>G, -1589G>C, -1235G>A, -678A>G) and MU3(-498C>A) was 0.7-, 0.7-, 1.2- times respectively compared with the wild type in human hepatoma cell lines(p<0.05). These findings might be useful for optimizing pharmacotherapy and the design of personalized medicine.

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Related in: MedlinePlus

Expression of the human CYP2D6 reporter constructs in the human hepatoma cell line (HepG2).The relative luciferase activities were normalized against the human wild type construct. The results are the average of at least three independent experiments. Comparisons among groups were done with the aid of the ANOVA statistical procedure. *Significantly different from Wild type transfected cells (P<0.05). **Significantly different from Wild type transfected cells (P<0.01), ***Significantly different from Wild type transfected cells (P<0.001).

Related In: Results - Collection

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pone-0057764-g001: Expression of the human CYP2D6 reporter constructs in the human hepatoma cell line (HepG2).The relative luciferase activities were normalized against the human wild type construct. The results are the average of at least three independent experiments. Comparisons among groups were done with the aid of the ANOVA statistical procedure. *Significantly different from Wild type transfected cells (P<0.05). **Significantly different from Wild type transfected cells (P<0.01), ***Significantly different from Wild type transfected cells (P<0.001).

Mentions: The functional significance of all 5 novel SNPs and 10 haplotypes was determined by the Renilla/Firefly luciferase assay after they had been transfected into human hepatoma cell lines. The Firefly/Renilla luciferase activity ratio was a good indicator of the relative activity of the CYP2D6 5′-upstream regulatory region. We conducted systematic analysis on all constructs with seven independent experiments using 8 replicates for every sample in each independent experiment. 2 haplotype constructs, namely pGL3-Haplo3 (AGTGCTGACGCCTA) and pGL3-Haplo8 (GAGGCCAGCGCCTA), both exhibited lower luciferase activity (both were 70%) compared to the wild type construct pGL3-WT (P<0.05), whereas the single point mutation MU3 exhibited higher luciferase activity (120%) compared with the wild type(p<0.05) (Figure 1).