Functional Variants Affecting Tacrolimus Blood Concentration And Side Effects In Chinese Renal Transplant Recipients

Background and ObjectiveRenal transplant is the treatment of choice for patients with end-stage renal disease（ESRD）and substantially improve the survival and life quality of patients.Immunological rejection is the leading cause of renal allograft dysfunction after transplant,and immune-suppression medications play the main role in preventing against it.Calcineurin inhibitors（CNIs）including tacrolimus（TAC）and cyclosporine A（CsA）are widely used in solid organ transplant,and TAC has became the main CNI as it is reportedly more effective than CsA at improving allograft survival and preventing acute rejection.The mechanism of action of TAC is inhibiting the early phase of T-cell activation.TAC binds to FK506 binding protein-12 in T cells to form a FK506-FKBP-12 complex,which then binds to calcineurin and inhibits its activity of phosphatase,so preventing the critical dephosphorylation reactions necessary for the gene transcription of early lymphokine（e.g.interleukin-2,interleukin-4 and y-interferon）.TAC is absorbed by small intestine and subject to substantial intestinal and hepatic first-pass effects.TAC is metabolized by the cytochrome p450 enzymes,particularly CYP3A subfamily（CYP3A4 and CYP3A5）and degradation of TAC by CYP3A occurs largely in the liver,and CYP3A existing in enterocytes also play a considerable role in it.The therapeutic range for TAC is narrow,underexposure to TAC may lead to acute rejection（AR）in transplant recipients,whereas overexposure to TAC puts patients at risk of TAC-related toxicities including nephrotoxicity,neurotoxicity,infectious complications,and disturbances in lipid metabolism.Oral bioavailability of TAC is generally poor（mean approximately 17%-22%depending on the type of transplantation）and display a large inter-individual variability range 4%-93%,which result from many factors and genetic variability play the dominant role in it.Because of this variability in conjunction with its narrow therapeutic range,therapeutic drug monitoring（TDM）is routinely performed in renal allograft recipients,which guide doctors to adjust TAC dose to keep the blood concentration within therapeutic range and achieve optimal efficacy while minimizing the risk of toxicity.But there are some limitations for TDM.Firstly,TDM is not useful to determine the optimal starting TAC-dose for patients.Secondly,TDM can not achieve stable TAC-dose in a short time,which is especially dangerous for patients in the early stage after transplantation.Therefore,there is a necessity for searching genetic marker affecting TAC metabolism to guide individualized administration of TAC.It has been reported previously that inter-individual differences in corresponding coding genes expression and enzyme activity of CYP3A4 and CYP3A5 are the key factors contributing to the inter-individual differences in TAC pharmacokinetic.A number of studies have demonstrated that CYP3A5*3 located in intron 3 of CYP3A5 gene was an important functional variant affecting TAC metabolism.CYP3A5*3 causes alternative splicing,which results in unstable protein truncation and a severe decrease of functional CYP3A5 enzyme.Carriers of homozygous CYP3A5*3 allele called CYP3A5 low-expressers and carriers of one or two CYP3A5*1 alleles called CYP3A5 expressers,and the later have a higher CYP3A5 expression compared with the former.We found from GTEx database that gene expression of CYP3A5 still varies significantly among patients with same CYP3A5*3 genotype.This result suggested that there may be other variants affecting the TAC metabolism.Therefore,the objective of this study was to search novel functional SNPs and analyze their molecular mechanism affecting TAC blood concentration and side effects.Study Design and MethodsStudy cohort＆population819 recipients of renal transplant between 1998 and 2017 at Nanfang Hospital were selected for this study as the Chinese renal transplant cohort.Dose-adjusted TAC trough blood concentration（Co/D）was the ratio of measured TAC C0 divided by corresponding daily TAC dose expressed as mg/kg bodyweight,and TAC C0 without normal distribution was log transformed to attain normality and was used for statistical analysis.Candidate SNPs selectionCandidate SNPs possibly affecting TAC blood concentration and side effects were selected in three ways.First,we selected SNPs previously reported as functional variants affecting TAC pharmacokinetics.Second,we selected SNPs in two reported genome-wide association studies（GWAS）cohorts which analyzed the association between genetic factors and CYP3A gene expression or TAC metabolism.Third,significant expression quantitative trait loci（eQTLs）associated with CYP3A gene expression were selected from GTEx database.Finally,51 candidate SNPs were included in our study.SNPs genotypingWhole blood-based genomic DNA was extracted using the standard phenol/chloroform method.51 candidate SNPs were genotyped by high-resolution melting（HRM）assays and SNPscan genotyping assays,respectively.Statistical analysisStatistical analyses were performed using R software version 3.4.1.For each candidate SNP,we analyzed the association between genotype and TAC log（C0/D）and TAC related side effects in five genetic models using R SNPassoc package.Survival analysis of acute rejection in patients with different genotype were performed by GraphPad prism.Relationship between TAC log（C0/D）and clinical factors were analyzed using multivariate linear regression model.Molecular and genetic mechanism studyVariants within genomic regions with the Histone Marks associated with transcription activation were selected as candidate functional SNPs.We constructed recombined pGL4.23 vector inserted enhancer containing candidate functional SNPs.Recombined pGL4.23 vector and pRL-TK vector were cotransfected to HEK 293T and Huh7 cell and analyzed molecular mechanism of candidate functional SNPs affecting TAC pharmacokinetic by Dual-luciferase reporter assay.And we downloaded the original genotype data and CYP3A5 transcript isoforms TPM data from GTEx database.The proportion of different CYP3A5 isoforms were compared among the carriers with different genotypes of rs4646450.Results1.51 candidate SNPs possibly affecting TAC blood concentration and side effects were selected into our study.2.We found that rs4646450,rs26 87 1 34,rs6945984,rs1419745,rs17161774 and rs34880695 were significantly associated with TAC log（C0/D）after adjusted by CYP3A5*3.3.rs554238,rs10274646 and rs4729523 were found to be significantly associated with acute rejection.4.Age of recipients,Cr and HCT were significantly associated with TAC log（C0/D）.5.Dual-luciferase reporter assay indicated that rs4646450 G allele were significantly associated with higher normalized luciferase activity ratios.6.Carriers of rs4646450 A allele expressed higher CYP3A5-001 isoform which encoding functional CYP3A5 enzyme and lower CYP3A5-007 and CYP3A5-009 isoform.Conclusions1.rs4646450,rs2687134,rs6945984,rs1419745,rs17161774 and rs34880695 may serve as significant genetic markers affecting TAC blood concentration independently of CYP3A5*3.2.rs4646450 may be a Splicing QTL regulating alternative splicing of CYP3A5.3.rs554238,rs10274646 and rs4729523 may be the important genetic markers affecting acute rejection.4.Clinical factors also play important roles in inter-individual variability of TAC.