Influence Of ABCB1 Genetic Polymorphism And Epigenetic Variance On P-Glycoprotein Function

OBJECTIVE1.To determine the frequency of common SNPs,haplotypes and the methylation status in promoter region of ABCB1 gene in Chinese Han population.2.To analysis the relationship between the genetic polymorphism and the methylation status of ABCB1 gene in healthy Han.3.To evaluate the impact of the genetic polymorphism and methylation status in ABCB1 promotor region on the function of P-glycoprotein in healthy Chinese Han.4.To estimate the effects of different dosage of the probe drug on the functional evaluation of P-glycoprotein.5.To observe the association between the ABCB1 genetic polymorphism and methylation status and the steady state cerebrospinal fluid concentration of antiepileptic drugs.METHODS1.The distribution of ABCB1 genetic polymorphism in healthy Chinese Han207 healthy Han were recruited in this study.2 mL periphery venous blood were collected for all the volunteers.DNA was extracted with a convenient extraction procedure,which developed based on the traditional phenol-chloroform method in our laboratory.A polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) assay for the detection of the genetic polymorphism of G1199A,C1236T, G2677T/A and C3435T of ABCB1 in Han population.The results of PCR-RFLP were confirmed by DNA sequencing with an automated ABI Prism 3730 DNA Analyzer.The frequency of ABCB1 SNPs and the linkage disequilibrium between the SNPs was analyzed by SHEsis,and the haplotyes were reconstructed by PHASE 2.1.1.2.Analysis of methylation status in ABCB1 promoterThe methylation status of CpG sites within the proximal promoter region of ABCB1 gene in 207 Han was confirmed using the combined bisulfite restriction analysis(COBRA)method.2μg of DNA were diluted to 50μl.DNA was denatured,and then incubated with 3M sodium bisulfite at 50℃for 16 hours.This combination of bisulfite treatment and PCR amplification results in the conversion of unmethylated cytosine residues to thymine and methylated cytosine residues to cytosine.This sequence conversion can lead to the methylation-dependent creation of new restriction enzyme sites.PCR was performed and PCR productes were digested with TaqI endonuclease that digests alleles which were methylated prior to bisulfite treatment and separated in nondenaturing polyacrylamide gels.Gels were stained with ethidium bromide.The proportion of methylated v.s unmethylated product was quantitated by densitometric analysis to determine the density of methylation.3.Impact of ABCB1 genetic polymorphism and methylation status in promoter region on the P-glycoprotein functionDigoxin,a typical probe drug of P-glycoprotein,was used as the mode drug to study the influence of ABCB1 genetic polymorphism and epigenetic variance on the P-glycoprotein function.Digoxin plasma concentrations were determined by a microparticle enzyme immunoassay (AxSYM Digoxinâ…¡Assay,Abbott Laboratories;USA).The standard cruve range is between 0.3～4.0μg·L^-1,the lower limit of quantification is 0.3μg·L^-1.Those samples,whose concentrations are more than 4.0μg·L^-1, were diluted before determination.The interassay coefficients of variation at plasma concentrations of 0.9,1.9,3.2,were 10.1%,6.8%and 5.3%, respectively.The pharmacokinetic parameters of digoxin were calculated with Drug and Statistics Software.4.Effect of ABCB1 genetic polymorphism and methylation status in promoter region on the steady state concentration of phenytoin and Phenobarbital in plasma and cerebrospinal fluid75(phenytoin 35;phenobarbital 40)patients were selected for this study.After the drugs were administered at least seven to ten days,6 mL venous blood and 2 mL cerebrospinal fluid were collected simultaneously. 2 mL blood was used for DNA extraction,and the other 4 mL was for the determination after centrifugation.The genetic polymorphism and methylation status of these patients were detected with the same methods as the part 1 and part 2.The concentrations of phenytoin and phenobarbital in plasma and cerebrospinal fluid(CSF)were determined by a high-performance liquid chromatography(HPLC)method.The HPLC apparatus consisted of: Ultimate^TMColumn AQ-C₁₈(250mm×4.6mm,5μm,USA),Column temperature:40℃,UV-wavelength:220nm,mobile phase:acetonitrile: 30mmol·L^-1NH₄AC(0.25%formic acid)(50:50,v/v),rate: 1.0ml·min^-1,injection volume:20μl.Samples were pretreated by a liquid-liquid extraction,alprazolam was used as internal standard.The retention times for Phenobarbital,phenytoin and internal standard were 4.8,7.1,and 9.2min,respectively.The standard curve ranges for phenobarbital and phenytoin were 1.0～62.5μg·mL^-1and 0.1～40.25μg·mL^-1,respectively.Then,the correlation between the genetic polymorphism or methylation status and the CSF drug concentration or CSF/plasma concentration ration was analyzsed.5.Statistical methodsGenotype frequencies for the variants SNPs were assessed for deviation from Hardy-Weinberg equilibrium using the Chi-square test. Statistical significance of LD between SNP pairs was assessed using Fisher's Exact Test.Nonparametric tests including Kruskal-Wallis method and Mann-Whitney U test were used to compare the methylation status among different genotypes.Spearman analysis were performed to investigate the correlation between Methylation Status in Promoter Region and the pharmacokinetic parameters.A p value＜0.05 was deemed to indicate a significant difference.All statistical analyses were performed using SPSS version 13.0.RESULTS1.The distribution of ABCB1 genetic polymorphism in healthy Chinese Han populationThe mutation of G1199A had not been found in our study though the frequency in Caucasians is approximately 6.5%～11%and it can change the transport activity in vitro,all subjects are homozygous for genotype 1199GG.Our study confirmed the allelic frequency distribution of the C3435T,G2677T/A,and C1236T,as well as haplotpes in Chinese Han subjects.The allelic frequency distribution of the C3435T,G2677T/A and C1236T genetic variants in our sample are 40.4%,41.6%,16.3%and 66.6%,respectively.This is in good agreement with other Chinese population studies and did not show significant deviation from Hardy-Weinberg equilibrium.The frequencies of homozygote of C3435T SNP wild type(CC)was 35.1%,and homozygote of C3435T SNP mutant type(TT)was 15.9%,heterozygots of C3435T SNP mutant type(CT)was 49.0%.The frequencies of homozygote of C1236T SNP wild type(CC) was 14.4%,and homozygote of C1236T SNP mutant type(TT)was 47.6%, heterozygots of C1236T SNP mutant type(CT)was 38.0%.The frequencies of homozygote of G2677T/A SNP wild type(GG)was 16.3%, and homozygote of G2677T/A SNP mutant type,including TT,TA,and AA was 16.8%,10.6%,4.8%,respectively;heterozygots of G2677T/A SNP mutant type,including GT and GA was 38.9%,12.5%,respectively. A haplotype comprised of three common SNPs was constructed for those 207 individuals.It revealed that the four haplotypes T-T-T,T-G-C, C-G-C and C-A-C(in order of 1236-2677-3435)composed more than 90%in Chinese Han population and an incomplete linkage between exon 26 and the variants in exon 21 and exon 12.2.Methylation status in promoter region of ABCB1 in healthy ChineseIn 207 healthy Chinese Han,the median of methylation status in ABCB1 promoter region is 13.2%(3.2%～28.7%),no gender difference was found.In this study a correlation between methylation status in ABCB1 promoter region and ABCB1 genetic polymorphism was found.It was revealed that a significant correlation between the methylation status and the different genotypes of C3435T and G2677T/A as well as haplotype derived from C3435T,G2677T/A and C1236T,but there is no significant difference between different genotypes of C1236T.3.Impact of ABCB1 genetic polymorphism on the pharmacokinetics of digoxin with different dosageall the volunteers in this study were administered digoxin with 0.25 mg(low level),0.5 mg(middle level)and 0.75 mg(high level).Apart from the difference in pharmacokinetics parameters,which root in different dosage,it cannot be found any significant difference of ABCB1 SNPs among the three dose levels,which was described as digoxin intestinal uptake.However,when the volunteers were re-grouped according to their haplotypes,AUC_0-4and C_maxin individuals with TGC-CGC were significantly lower than those with TTT-TTT in 0.25 mg digoxin group(p＜0.05).But,we did not find this difference among the groups with digoxin 0.5 mg or 0.75 mg.4.Correlation between the methylation status and digoxin pharmacokineticsIt was found that there was significant positive correlation between AUC_0-4and C_maxof 0.25 mg digoxin and ABCB1 Methylation Status in Promoter Region(p＜0.05).However,no correlation was found among the 0.5 mg and 0.75 mg digoxin groups.5.Effect of ABCB1 genetic polymorphism and methylation status in promoter region on phenytoin steady state cerebrospinal fluid concentration35 inpatients(male 22,femal 13),who taken phenytoin were selected for this study.A negative correlation was illustrated between the methylation status in ABCB1 promoter region and the ratio of phenytoin steady state cerebrospinal fluid concentration/plasma concentration (r=-0.407,p=0.015).No effect of ABCB1 SNPs and haplotypes on patients' phenytoin steady state cerebrospinal fluid concentration or the ratio of the CSF/plasma concentration was found.6.Effect of ABCB1 genetic polymorphism and methylation status in promoter region on phenytoin steady state cerebrospinal fluid concentration40 inpatients(male 24,femal 16),who using Phenobarbital up to 7-10 days,were selected for this study.However,no impact of the ABCB1 SNPs,haplotypes or the methylation status in ABCB1 promoter region on the steady state cerebrospinal fluid concentration and the ratio of CSF/plasma concentration of phenobarbital was observed.CONCLUSIONIt is the first time to approve that no mutation for G1199A of exon 11 in healthy Han was detected.It suggests that allele Gl199A is ethnic specific or geographically restricted and has no linkage with other SNPs. The frequencies and distribution of ABCB1 C1236T,G2677T/A,and C3435T polymorphisms,as well as haplotypes,in healthy Han subjects were similar with those results of the previous studies performed in Chinese.The distribution of the alleles is fit for Hardy-Weinberg equilibrium,that means the population in this study can represent Han population.It is the first study to analysis the methylaiton status in ABCB1 promoter region in a large-scale healthy Han.A correlation between the methylation status of the promoter region and ABCB1 genetic polymorphism was found in this study.Further investigations are needed to explore the clinical significant and molecular mechanism of this phenomenon.We observed a interesting phenomenon that ABCB1 genetic polymorphism and methylation status both have an effect on AUC_0-4and C_maxof 0.25 mg digoxin group;but neither the 0.5 mg nor 0.75 mg group. It reminds us that 0.25mg digoxin may be better to evaluate the P-glycoprotein function,and the dosage of the probe drug or substrate is very important for P-glycoprotein function evaluation since the protein is ATP-dependent and saturable.Except a correlation between methylation status in ABCB1 promoter region and ratio of steady state phenytoin CSF/plasma concentration,we did not find any effects of ABCB1 genetic polymorphism on the steady state CSF concentration of phenytoin and phenobarbital though they are both the substrates of P-gp.No influence of ABCB1 methylation status on phenobarbital steady state CSF concentration or the ration was observed in this study.Due to the absence of data about the expression of P-gp in brain of the patients,it is impossible to make a conclusion about the effect of P-gp on these drugs' CSF concentration.Instead,the present study illustrates the methodologic difficulties and complexity involved in performing these studies in human.