Genetic Variations In Exonic And 5′ Regulatory Region Of Cyp2C19 And Their Functional Importance As Explored In South Indian Population
Creators: name: Uppugunduri S Chakradhara Rao
Publisher: name: Zenodo
Year: 2010
Subjects: 5′ regulatory region
CYP2C19
Genotype
Promoter region
Phenotype
Proguanil
Pharmacogenetics
Soun Indian Population

description: CYP2C19 is involved in the metabolism of clinically important drugs such as proton pump inhibitors, proguanil, anti depressants, nelfinavir, voriconazole, etc. Various studies have established the genotype-phenotype correlation of CYP2C19 in different populations. Discrepancies were found in these studies; where no correlation was observed between the CYP2C19 genotype (variations in the exonic region) and phenotype in some individuals, indicating the presence of variations in 5' flanking region or intronic region. The objective of this study was to sequence and functionally characterize the 5'regulatory region of CYP2C19 in South Indian individuals. In this study, the 5' regulatory region of CYP2C19 was sequenced from 58 unrelated healthy volunteers of South Indian origin, and 8 novel variations were identified. All the sequences of 5' regulatory region of CYP2C19 were<br> submitted to Genbank database and are available in public domain (Accession ID:EU369428 to EU 369485). Identified novel variations and their percentage frequencies were: -779A&gt;C (16.4), -828T&gt;A (2.6), -934del&gt;T (3.5),-1051T&gt;C (1.72), -1289T&gt;G (3.4),<br> -1442T&gt;C (12.1), -1498T&gt;G (25.0) and -1558T&gt;G (2.6). The reported variations found in the study population and their frequencies were: -98T&gt;C (28.4),-806C&gt;T (2.6), -833del&gt;T (9.5), 889T&gt;G (10.3) and -1418C&gt;T (1.7). Some of these variations were found to be<br> present in the transcription factors binding site such as ATF-2, GATA 1, CREB, CEBPB and CDP repressor protein etc. All the subjects were also explored for exonic region polymorphisms of CYP2C19, 681G&gt;A (*2 allele) and 636G&gt;A (*3 allele) and were<br> detected at 0.371 and 0.025 frequencies, respectively. 43 different haplotypes were constructed with all the variations observed in this population, of which frequency of 13 haplotypes were found to be more than 1%. Phenotyping of 50 subjects was also<br> performed by using proguanil as a probe drug (subjects were phenotyped by measuring the plasma concentrations of proguanil and cycloguanil by reverse phase HPLC after a single oral dose (200mg) of proguanil), and correlated with the variations in exonic region and 5' flanking region. The gene-dose effect of CYP2C19 and proguanil metabolic ratio (progunail/cycloguanil) was found in this population. The mean proguanil metabolic ratios (proguanil/cycloguanil) were highest in CYP2C19*1/*2 or *1/*3subjects (9.6 ± 2.6;<br> p=0.03) and in *2/*2 or *2/*3 (9.9 ± 3.7; p=0.04) as compared to normal subjects (*1/*1; 4.7 ± 1.2), but still some overlapping of the metabolic ratios were found between different genotypes, which may be due to the variations found in the promoter region. Further<br> analysis showed that variations -98T&gt;C and -806 C&gt;T have influenced the proguanil metabolic ratio irrespective of their exonic region variations. In silico docking analysis of transcription factors to its binding sites in CYP2C19 5' regulatory region with and without variations revealed the alteration in the interaction of transcription factors to their binding sites in the presence of variant alleles. The 5' regulatory region of samples with 13 different inferred haplotypes were cloned into pGL-3 basic reporter vectors for studying the impact of the promoter variations on expression of luciferase gene in human HepG2 cell lines. Transfection of pGL-3 fused vectors with CYP2C19 5' regulatory region using lipofectamine reagent into HepG2 cell lines and luciferase assay have shown that variations present in the cis-acting elements of the CYP2C19 promoter region such as -1442T&gt;C, -779A&gt;C and -98T&gt;C -1498T&gt;G and -828del&gt;T alter the transcription of the gene. Electrophoretic mobility shift assays had shown the sequence specific binding of proteins from HepG2 nuclear extracts. These results indicate the influence of these variations on gene expression which can explain the Interindividual differences in CYP2C19 gene expression. This also helps us to understand the genotype dependant<br> differences in enzyme induction and inhibition in a clinical setting. The results also suggest the role of activating transcription factor-2, cyclic AMP response element binding protein and CCAAT displacement repressor protein on CYP2C19 gene transcription.
description: Supported by funding from : 1) ICMR-INSERM Joint Research project (ICMR sanction order no. 50/6/2004 dated 05/10/06). 2) JIPMER Institutional intramural research fund (Edn.7 (1)/2010 dated 04.02.2010) 3) Indo-Swiss Research Fellowship- 2009 (ISJRP Grant-23)