inter - individual variability in drug absorption , distribution , metabolism and elimination ( adme ) , arising from both genetic and non - genetic factors , is a main cause of therapeutic failure or undesirable adverse effects . to optimize drug response , multiple strategies have been developed to tailor individual drug therapy , employing pharmacokinetic parameters based methods [ 13 ] , pharmacodynamic monitoring , toxicity based titration , and use of genetic biomarker tests . genetic variants in cyp2c9 , cyp2c19 and cyp2d6 are already clinically employed for predicting dosages and response for warfarin , clopidogrel , and many cyp2d6 substrate drugs ( see fda table of pharmacogenomic biomarkers in drug labels ) . while cyp3a isozymes , the most abundant and important drug metabolizing enzymes in the liver , display large inter - individual variability , valid genetic biomarkers for overall cyp3a activity have yet to be established . as pharmacogenomic biomarkers begin to play a prominent role in personalized medicine , predictive tests for cyp3a activity , in particular that of cyp3a4 and cyp3a5 could have substantial clinical utility . the four human cyp3a genes cluster on chromosome 7q21 in the order of cyp3a43 , cyp3a4 , cyp3a7 and cyp3a5   ( figure 1 ) . cyp3a4 is the most abundant isoform in liver and intestine , followed by cyp3a5 in individuals with cyp3a5 * 1/*1 genotype . cyp3a7 is highly and variably expressed in fetal livers , accounting for up to 50% of total cytochrome p450s , while the expression level of cyp3a7 decreases rapidly after birth and becomes undetectable in most of adult livers , except for individuals who carry two promoter variants , cyp3a7 * 1b and cyp3a71c . cyp3a43 is expressed at very low levels in adult livers , accounting for only 0.1%0.2% cyp3a4 transcripts . cyp3a4 and cyp3a5 have similar substrate specificity , while cyp3a7 has a smaller substrate spectrum whereas cyp3a43 does not appear to contribute substantially in drug metabolism . considerable inter - person variability in cyp3a expression and enzyme activity has been attributed to genetic and non - genetic factors [ 1821 ] . while each cyp3a member may contribute to overall cyp3a enzyme variability , cyp3a4 accounts for a majority the hepatic cyp3a activity in most individuals , followed by cyp3a5 . moreover , cyp3a4 has higher specific activity towards common cyp3a drug substrates than the other isozymes . in the case of cyp3a5 and cyp3a7 , known cyp3a5 alleles ( e.g. , * 3 and * 7 ) and cyp3a7 ( * 1b and * 1c ) largely account for variable expression in adult livers with clinical implications . however , despite large cyp3a4 variability , frequent polymorphisms affecting cyp3a4 activity were thought to be absent , except for the recently described * 22 allele . because the impact of polymorphisms in cyp3a5 and cyp3a7 on drug metabolism depends on the concomitant expression status of cyp3a4 , variants in cyp3a4 or in genes regulating cyp3a4 expression must be considered in developing a genetic biomarker panel for predicting cyp3a activity . overlaid onto such a genetic allele panel must be an assessment of relative substrate selectivity for each dug and isozymes ; in some instances , a drug could be predominantly metabolized by cyp3a5 or cyp3a7 so that cyp3a4 activity then has less impact . this review focuses on factors that determine variable cyp3a4 expression / enzyme activity , including common regulatory polymorphisms and rare coding region cyp3a4 polymorphisms , interaction between cyp3a4 and cyp3a5 alleles , and trans - acting factors that regulate cyp3a4 expression . we also discuss the clinical utilities of these factors as biomarkers for predicting cyp3a activity . because of the eminent role of cyp3a4 in drug metabolism , we discuss in detail all proposed functional variants ; even if they are rare , as new genotyping and sequencing methodologies enable rare variants with clear evidence for effect on expressed protein activity to be incorporated into predictive biomarker panels . previous studies have focused on promoter snp rs2740574 ( cyp3a4 * 1b , 392a > g , maf 5.4% in caucasians and 35% in non - caucasians ) . despite numerous clinical association studies , moreover , cyp3a4 * 1b is in ld with the fully active cyp3a5 * 1 reference allele in african americans , raising the possibility that cyp3a5 activity could have accounted for any clinical phenotype associated with cyp3a4 * 1b . proposed additional regulatory variants reside in intron 7 ( snp rs4646437 ) , intron 10 ( snp rs2242480 ) , and in putative enhancer regions . intron 7 snp rs4646437 was associated with cyp3a4 protein expression and enzyme activity in human liver microsomes , a finding observed only in males but not in females ; absent of a convincing mechanism , this result requires further study . the intron 10 snp cyp3a4 * 1 g ( rs2242480 ) has been associated with lipid - lowering efficacy of atorvastatin , tacrolimus pharmacokinetics in renal transplant patients , risk of coronary heart disease   and severity of withdrawal symptoms in methadone maintenance patients , all studies performed in asian populations . however , the effect of cyp3a4 * 1 g on mrna / protein level remains uncertain . in reporter gene assays , however , the g allele was associated with lower dose - adjusted blood levels ( auc ) of tacrolimus in vivo , implicating a gain of function ( although reporter gene assays in a heterologous tissue may not be predictive of in vivo effects ) . again , cyp3a4 * 1 g is in high ld with cyp3a5 * 1 in japanese individuals , confounding the analysis . variants in the enhancer region of cyp3a4 include rs2737418 ( 7,206 upstream ) , found to be associated with cyp3a4 enzyme activity in livers from african americans , but the results for enzyme activity and mrna levels were contradictory , the minor t allele being associated with higher enzyme activity but lower mrna levels . similarly , while reporter gene assays suggested an effect for a tgt insertion ( rs34401238 , 11 kb upstream ) on cyp3a4 transcription , any in vivo effect remains unknown . moreover , allelic rna expression in results in human livers argues against a detectable effect on rna expression for any of these regulatory snps mentioned above . the inconsistent clinical associations observed with these variants may be caused in part by ld with cyp3a5 * 1 , as suggested by a recent study . recently , we have identified an intron 6 snp ( rs35599367 , designated as * 22 ) in cyp3a4 that strongly affects hepatic expression and is associated with statin dose requirements . measuring allelic cyp3a4 heteronuclear rna ( hnrna ) and mrna expression in human livers using multiple marker snps , we found marked allelic expression imbalance in 13% of samples . genotyping and sequencing the cyp3a4 locus identified intron 6 snp rs35599367 , which fully accounted for the allelic cyp3a4 expression imbalance ; with the minor t allele expressed 1.65 folds less than the main c allele . consistently , cyp3a4 mrna and enzyme activity in livers with cc genotype were higher than ct and tt carriers . while the underlying molecular mechanism remains unclear , in vitro minigene transfection assays suggested that the t allele affect nascent rna elongation . consistent with reduced enzyme activity supported by cyp3a4 * 22 , others have reported that * 22 was associated with tacrolimus pharmacokinetics in kidney transplant recipients , cyclosporine and tacrolimus trough blood levels and dose requirements , simvastatin - mediated cholesterol reduction , and increased risk of delayed graft function and worse renal function in cyclosporine - treated kidney transplant patients . the allele frequency of * 22 is 3%6% in caucasian population and may be lower in subjects of african descent . because cyp3a4 metabolizes nearly 50% of clinically used drugs , * 22 could become a broadly useful biomarker to predict drug dosage , efficacy , and adverse effects . as cyp3a4 * 22 is embedded in main reference haplotype , lacking substantial ld with any other hapmap snps , it had escaped detection in genome - wide association studies built on tagging snp panels . more than 20 non - synonymous coding region snps have been reported for cyp3a4 with low minor allele frequency ( maf ) for each ( table 1 ) . * 2 , * 3 , * 7 , * 9 , * 10 , * 16 , * 17 and * 19 support similar protein expression compared to the reference allele * 1 , measured in a bacterial expression system . cyp3a4 * 3 , * 7 , * 9 , * 10 and * 19 also did not change enzyme activity , while cyp3a4 * 2 , * 7 , * 16 and * 17 caused substrate specific changes in enzyme activity [ 25,4749 ] cyp3a4 * 3 was associated with lower levels of low - density lipoprotein cholesterol in hypercholesterolemia patients but lacking a cogent mechanistic interpretation , and with a higher hdl increase upon fluvastatin treatment . on the other hand , cyp3a4 * 3 was not associated with changes in ldl level after atorvastatin treatment , a cyp3a4 substrate , arguing against a substantial effect on enzyme activity and hence drug effect . in japanese cancer patients receiving paclitaxel , * 1/*16 carriers showed a 20% lower median auc ratio of 3'-p - hydroxypaclitaxel to paclitaxel and a 2.4 fold higher median auc ratio of 6-hydroxypaclitaxel to paclitaxel , compared to * 1/*1 carriers , indicating * 16 is associated with reduced 3'-p - hydroxylation . cyp3a4 * 8 and * 13 did not yield detectable p450 holoproteins in a bacterial expression system , indicating they may affect protein translation or protein stability . similarly , cyp3a4 * 11 and * 12 showed less protein expression than * 1 in in vitro expression system , while cyp3a4 * 12 also altered substrate - specific enzyme activity . no in vivo effects or association studies have been reported , owing to the low allele frequencies of these variants ; therefore , it is premature to consider these alleles in clinical biomarker panels until more results are available , specifically on * 8 and * 13 expression in human cells . cyp3a4 * 4 , * 5 and * 6 occur only in asian populations with maf of 1.5%3% , < 1% , and < 1% , respectively . subjects heterozygous for * 4 , * 5 , and * 6 displayed decreased urinary ratios of 6-hydroxycortisol over cortisol as a measure of cyp3a4 activity , but the number of subjects was small and all were taking additional drugs such as ketoconazole , shedding doubt over any in vivo effect . in addition , after 4 weeks of oral administration of 20 mg simvastatin , * 4 carriers had greater reduction in triglycerides and total cholesterol than non - carriers , but again relying on a very small sample size . as cyp3a4 * 6 represents an a insertion causing a frameshift , it is likely to abolish cyp3a4 enzyme activity . one study found that individuals with * 15 also express cyp3a5 , indicating * 15 may be in ld with cyp3a5 * 1 . on the other hand , cyp3a4 * 18 is specific to asian population with maf of 2.8% . in vitro expression results indicate that * 18 expresses similar protein levels as * 1 and did not alter catalytic activity . yet , in 13 subjects heterozygous for * 18 and 26 control subjects , * 18 carriers showed diminished midazolam clearance compared to non - carriers ( and also with low lumbar spine bone mass density ) , suggesting * 18 is associated with decreased catalytic activity for midazolam . molecular modeling suggested structural change in substrate recognition sites in * 18 compared to * 1 . in contrast , * 18 was associated with decreased cmax and auc , and increased clearance of cyclosporine in healthy chinese subjects , suggesting higher enzymatic activity . similarly , * 18 was associated with increase clearance of tacrolimus in healthy chinese subjects , and in renal transplant recipients . yet , in japanese cancer patients , * 18 was not associated with paclitaxel pharmacokinetic parameters . these results are compatible with the hypothesis that * 18 has substrate - specific properties , but more direct evidence is needed to test the validity of this mechanism . in a cohort of 2125 korean women , * 18 was in high linkage disequilibrium ( ld ) with cyp3a5 * 3 ( d ' = 0.8 ) . as discussed earlier , high ld between haplotypes spanning cyp3a4 and cyp3a5 confounds conclusion about cyp3a4 activity by effects on cyp3a5 . cyp3a4 * 20 has a maf of < 0.6% in caucasian population . representing an a insertion , * 20 shifts the open reading frame and creates a premature stop codon yielding a truncated protein . in yeast and hek cell expression system , we surmise that the evidence for * 20 abolishing cyp3a4 activity is strong . in summary , non - synonymous snps with maf > 1% either did not change activity / protein expression or their functions have not been fully characterized ( * 2 , * 3 , * 7 , * 9 , * 10 , * 15 , * 17 , * 19 ) . cyp3a4 * 4 , * 5 , * 6 , * 8 , * 11 , * 12 , * 13 , * 16 and * 20 appear to decrease protein expression and/or enzyme activity , but display maf s < 1% , mostly in only one ethnic group , and with varying degree of evidence supported a robust effect on cyp3a4 activity . however , anticipating the arrival of large scale sequencing in clinical practice , it now becomes imperative to assess the impact of variants with low minor allele frequency , as these will become available when full sequences are being deployed . some rare coding region variants ( for example , snps that shift open reading frame or encode an unstable protein like * 6 , * 20 , * 8 , * 11 , * 12 and * 13 ) may have significant impact on drug metabolism in individuals who carry them and may become valuable biomarkers for predicting cyp3a activity in comprehensive biomarker panels . in table 2 , we present those alleles with currently the strongest evidence of functional variation , while additional studies are needed to validate them for clinical utility . because drugs metabolized by cyp3a4 are often also cyp3a5 substrates while cyp3a5 expression is comparable to that of cyp3a4 in some individuals , cyp3a5 could contribute substantially to inter - individual variability in cyp3a activity . the most frequent polymorphism in cyp3a5 ( * 3 , rs776746 ) , causing aberrant splicing and abolishing cyp3a5 mrna and enzyme activity   ( table 2 ) , is highly prevalent in caucasians ( > 80% ) but less frequent in african americans ( < 20% ) . since cyp3a4 * 22 and cyp3a5 * 3 are in very low ld , loss of cyp3a function caused by * 22 can be compensated by cyp3a5 expression in individual carrying the wild - type cyp3a5 * 1 allele . cyp3a5 * 3 has been associated with tacrolimus / cyclosporine pharmacokinetics , but the results are not always consistent , especially for cyclosporine . because tacrolimus is preferentially metabolized by cyp3a5 , and cyclosporine by cyp3a4 , it is possible that the inconsistent associations were caused by neglecting the activity and functional polymorphism in cyp3a4 . with discovery of the relatively common functional polymorphisms * 22 in cyp3a4 , it is now possible to test the in vivo effects of the combination of cyp3a4 and cyp3a5 alleles on cyp3a activity . for example , the association between * 22 and simvastatin - mediated cholesterol reduction is stronger when analyzed only in individuals who carry cyp3a5 * 3/*3 than in the entire cohort . similarly , the association between * 22 and tacrolimus pharmacokinetics and tacrolimus / cyclosporine dose requirements became stronger when cyp3a5 * 3 genotype was considered . moreover , cyp3a7 variants cyp3a7 * 1b and cyp3a7 * 1c may also contribute to inter - person variability in cyp3a activity for some substrates especially steroids in adult livers . however , the impact of cyp3a7 in clinical drug metabolism is still largely unknown . cyp3a4 is subject to regulation by several transcription factors , including liver enriched transcription factors and nuclear receptor family [ 6366 ] . the expression of cyp3a4 mrna is correlated with the expression of pxr , car , hnf4 , foxa2 , and others . therefore , polymorphisms present in these transcription factors could regulate the constitutive and inducible expression of cyp3a4 . however , coding region snps in pxr and car are rare , and any effect on cyp3a4 expression is unclear . common polymorphisms in promoter , intron or downstream regions have been identified in pxr , some of them significantly associated with cyp3a4 mrna expression or enzyme activity . in one study , rs2472677 has been associated with unboosted atazanavir clearance , while a pxr haplotype was found to be associated with cyp3a4 and abcb1 mrna expression and doxorubicin clearance in asian breast cancer patients . yet , further results failed to confirm associations between pxr , car and hnf4 genotype and docetaxel / doxorubicin pharmacokinetics . these studies suggest that polymorphisms in transcription factors have the potential to affect cyp3a4 expression , but likely account only for a portion of inter - individual variability in cyp3a4 expression and enzyme activity , being confounded by environmental conditions that impact multiple transcription factor expression . however , the polymorphisms identified by associations often lack a clear mechanistic role and likely represent only tagging snps , so that the effect size of true regulatory variants remains unclear . in addition , pxr , car and other transcriptions factors are subject to alternative splicing , with splice variants having different activities in dna binding or ligand binding . it is unknown whether splicing events are regulated by cis - acting polymorphisms ; therefore , any genetic trans - acting effect on cyp3a enzymes remains open for study . at present , genetic variants in transcription factors mir-27b binds to a cyp3a4 3'utr target sequence and decreases cyp3a4 mrna and protein expression , thereby reducing in vitro sensitivity of panc1 cells to cyclophosphamide . moreover , cyp3a4 expression can be regulated indirectly by mir-148a or mir-27b via targeting pxr   or vdr . furthermore , cyp3a4 expression appears to be regulated by epigenetic modifications directly or indirectly through transcription factor pxr . these regulatory events may contribute to the large inter - individual variability of hepatic cyp3a4 but can not be readily translated into a biomarker test . although coding region polymorphisms of cyp3a4 are included in the affymetrix drug - metabolizing enzymes and transporters panel ( dmet plus ) , they usually are not detected because of maf < 1% in caucasian or caucasian / african american mixed populations . therefore , their impact on clinical phenotypes can be assessed only by inference from in vitro studies , unless very large cohorts are studied  limiting their clinical utility . cyp3a4 * 18 is relatively frequent in asian population , showing some clinical associations , but its function on enzyme activity is controversial and the evidence insufficient for clinical decision making [ 48,49,53,5658 ] . also , because cyp3a4 * 18 is in ld with cyp3a5 * 1 , the independent effect of cyp3a4 * 18 may be limited . cyp3a5 * 3 was proposed to be a biomarker for predicting tacrolimus dose in combination with clinical factors , but a significant portion of observed inter - individual variability remains unaccounted for , possibly caused by neglecting cyp3a4 polymorphisms or using previously proposed regulatory cyp3a4 snps relying on ambiguous results [ 8688 ] . on the other hand , the recently discovered regulatory cyp3a4 * 22 allele ( rs35599367 )   has promise as a useful biomarker for predicting cyp3a4 enzyme activity . combined with cyp3a5 genotype ( * 1 , * 3 and * 7 ) , it is now possible to categorize individuals into poor , intermediate and extensive metabolizer phenotypes for overall cyp3a activity . indeed , a combination of cyp3a4 and cyp3a5 genotypes predicted tacrolimus / cyclosporine dose or pharmacokinetics parameters better than cyp3a4 or cyp3a5 genotype alone . although cyp3a4 expression is further regulated by polymorphisms in transcription factors and the expression of micrornas , their clinical utility is unknown at the present . in conclusion , multiple factors regulate cyp3a4 expression / enzyme activity . yet , at this point only one relatively common regulatory polymorphism rs35599367 ( cyp3a4 * 22 ) has promise as a biomarker . while the * 22 allele contributes a relatively small portion to overall population variance in cyp3a4 activity , owing to its relatively low allele frequency , it does have significant influence on those subjects carrying the minor allele . homozygous carriers of * 22 may be rare ( < < 1% ) , but given the large number of subjects taking cyp3a4 substrate drugs , such individuals may experience unusual drug effects . combined with well - established variants in cyp3a5 ( * 3 and * 7 ) , it is now possible to predict a larger portion of the genetic components contributing to overall cyp3a enzyme activity toward cyp3a substrate drugs across different racial groups . rare mutations with obvious and documented effect on enzyme activity can be incorporated into a predictive panel once clinical genotyping / sequencing is routinely performed on a broader scale .
cyp3a ranks among the most abundant cytochrome p450 enzymes in the liver , playing a dominant role in metabolic elimination of clinically used drugs . 
 a main member in cyp3a family , cyp3a4 expression and activity vary considerably among individuals , attributable to genetic and non - genetic factors , affecting drug dosage and efficacy . 
 however , the extent of genetic influence has remained unclear . 
 this review assesses current knowledge on the genetic factors influencing cyp3a4 activity . 
 coding region cyp3a4 polymorphisms are rare and account for only a small portion of inter - person variability in cyp3a metabolism . except for the promoter 
 allele cyp3a4 * 1b with ambiguous effect on expression , common cyp3a4 regulatory polymorphisms were thought to be lacking . 
 recent studies have identified a relatively common regulatory polymorphism , designated cyp3a4 * 22 with robust effects on hepatic cyp3a4 expression . 
 combining cyp3a4 * 22 with cyp3a5 alleles * 1 , * 3 and * 7 has promise as a biomarker predicting overall cyp3a activity . also contributing to variable expression , 
 the role of polymorphisms in transcription factors and micrornas is discussed .