Patent Application: US-201013259514-A

Abstract:
a method for predicting a subject &# 39 ; s risk factors for cyp3a4 - related disorders includes detecting the allelic status of a snp in a nucleic acid sample of the subject .

Description:
the present invention will now be described with occasional reference to the specific embodiments of the invention . this invention may , however , be embodied in different forms and should not be construed as limited to the embodiments set forth herein . rather , these embodiments are provided so that this disclosure will be thorough and complete , and will fully convey the scope of the invention to those skilled in the art . all publications , patent applications , patents , and other references mentioned herein are incorporated by reference in their entirety . the disclosure of all patents , patent applications ( and any patents that issue thereon , as well as any corresponding published foreign patent applications ), genbank and other accession numbers and associated data , and publications mentioned throughout this description are hereby incorporated by reference herein . it is expressly not admitted , however , that any of the documents incorporated by reference herein teach or disclose the present invention . the present invention may be understood more readily by reference to the following detailed description of the embodiments of the invention and the examples included herein . however , before the present methods , compounds and compositions are disclosed and described , it is to be understood that this invention is not limited to specific methods , specific cell types , specific host cells or specific conditions , etc ., as such may , of course , vary , and the numerous modifications and variations therein will be apparent to those skilled in the art . it is also to be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting . in a broad aspect , the inventors herein found common polymorphisms in cyp3a4 by measuring allelic hnrna / mrna expression in human autopsy livers . a detectable allelic rna expression imbalance ( aei ) is a direct measure of cis - acting regulatory factors in cyp3a4 that affect rna expression , processing , or turnover . the results described herein demonstrate that an intron6 snp of cyp3a4 fully accounts for the observed allelic mrna expression pattern and correlates with cyp3a4 enzyme activity in human livers , while previously suggested polymorphisms had no effect . moreover , intron6 snp was significantly associated with stable statin dosage taken for cholesterol control and with achievement of therapeutic ldl goal in a cohort of cad patients . tissue samples . 133 liver autopsy / biopsy samples were obtained from the cooperative human tissue network midwestern and western division , under a protocol approved by the ohio state university institutional review board ( osu irb ). patients . subjects were participants in the ohio state university coronary artery disease study , with patients presenting to the osu heart center with symptomatic cardiovascular disease requiring pci calls . 275 patients documented to be taking stable doses of a statin for lipid control were selected . enrollment and trial conditions have been approved by the osu irb , with written informed consent obtained from each patient . the study population reflects demographics of the columbus area and surrounding rural counties of ohio . dna and rna preparation . preparation of genomic dna , rna and cdna from tissues or blood samples were performed as described ( 16 - 18 ). quantitative analysis of allelic ratios in genomic dna and rna using snapshot . the detailed method has been published ( 16 , 17 ). briefly , a fragment of dna or rna ( after conversion to cdna ) surrounding a marker snp was pcr amplified , followed by a primer extension assay ( snapshot ) that targets the polymorphic site . seven marker snps located in either 3 ′ utr or intronic regions were used to measure allelic ratios of mature mrna ( 3 ′ utr markers ) or hnrna ( intronic markers ) in 73 out of the 133 livers heterozygous for at least one marker snp . genomic dna ( gdna ) allelic ratios , normalized to 1 , served as internal control ; none of the subjects displayed gdna copy number variants , detectable by a significant deviation from unity . deviations of allelic rna ratios from 1 ( after normalization to dna ratios ), i . e ., allelic expression imbalance ( aei ), indicates the presence of cis - acting polymorphisms in cyp3a4 that affect mrna expression levels . genotyping : thirteen snps in cyp3a4 ( including the 7 marker snps ) were genotyped in gdna from liver samples with a multiplex snapshot assay ( 19 ) or allele specific real - time pcr ( 20 ), as shown in table 1 . quantitative mrna analysis of cyp3a4 and transcription factors pxr , rxra , car , and hnf4a in human livers : mrna levels were measured with real - time pcr ( 16 , 21 ) using gene - specific primers ( 22 ) ( table 3 , table 4 , table 5 ) and sybr green ( applied biosystems ), with gadph mrna as an internal control . table 3 : genotyping or aei assays . column labeled “ sequence of pcr primers and assay condition ” discloses seq id nos 1 - 42 , respectively , in order of appearance and column labeled “ primer extension primers and condition ” discloses seq id nos 43 - 56 , respectively , in order of appearance . table 4 : multiplex pcr and snapshot assay for cyp3a4 and cyp3a5 snp genotyping . column labeled “ pcr primer sequence ” discloses seq id nos 57 - 68 , respectively , in order of appearance and column labeled “ primer extension primers ” discloses seq id nos 69 - 75 , respectively , in order of appearance . table 5 : primers and sequencing primers . pcr primers disclosed as seq id nos 76 - 95 , respectively , in order of appearance and sequencing primers disclosed as seq id nos 96 - 151 , respectively , in order of appearance . sequencing cyp3a4 : the region from ˜ 10 , 000 bp upstream of transcription start site to the last exon ( from 50013 to 89410 in af280107 , total length 39 , 397 bp ) was sequenced in two liver samples that showed allele - specific rna expression . pcr and sequencing primers are shown in fig1 - table 10 . cyp3a4 enzymatic activity assay : cyp3a4 activities were quantified from liver microsomes with testosterone as a probe as described ( 23 ). data analysis : association between discrete variables and genotypes were analyzed with helix - tree software ( golden helix , bozeman mont .). ld plots were generated using haploview . multiple linear regression analysis was used for testing genotype effects on rna expression , enzyme activity , and statin dose requirement using spss or minitab software . allelic mrna expression of cyp3a4 was measured in human livers using 3 frequent 3 ′ utr marker snps ( fig3 ). because of high cyp3a4 expression , 4 intronic snps also served to measure allelic expression of cyp3a4 hnrna ( 24 ). among 133 liver samples screened , 73 were heterozygous for at least one of the seven marker snps and therefore suitable for aei measurement . of the 73 samples , 7 were identified showing strong allelic expression imbalance ( aei ), with the main allele expressed less than the minor allele ( 0 . 2 to 0 . 5 times ), observed with both intronic and exonic marker snps ( fig1 a ). therefore , a cis - acting polymorphism ( s ) in cyp3a4 affects both mrna and hnrna levels equally . to search for responsible polymorphism ( s ), 13 cyp3a4 polymorphisms ( table 1 ) were genotyped and the association between allelic rna ratios and genotype tested . a single snp ( rs35599367 [ seq id no : 152 ]) located in intron6 ( c & gt ; t ) showed highly significant association with aei ( adjusted p value 9 . 12 ×) 10 − 10 ( fig4 ), while another snp rs2246709 also scored with moderate significance ( p = 0 . 034 ), a likely result of partial linkage disequilibrium ( ld ) with the intron6 snp ( fig5 ). other snps including previously identified promoter snp rs2740574 , tgt insertion , rs2737418 , and rs4646437 did not show significant association ( p & gt ; 0 . 05 ). these results indicated that intron6 snp is functional or in highly ld with a functional snp . to test this further , intron6 snp was used as a marker , and aei was measured in 10 heterozygous samples , including 3 heterozygous only for intron6 snp . all 10 samples showed aei with allelic ratios ( minor t allele / major c allele ) ranging from 0 . 16 to 0 . 62 ( fig1 b ), with no differences between males and females ( p = 0 . 314 ). therefore , the minor t allele of intron6 snp is linked to reduced mrna / hnrna levels ( the inverse allelic mrna ratio of major c / minor t ranges from 1 . 6 to 6 . 25 ). the minor t allele is exclusively linked to the main cyp3a4 haplotype , except for low ld with rs2246709 ( fig1 - table 6 ; ld plot fig5 ). table 6 : haplotype structure and estimated frequency of 13 snps tested in livers . detailed snp information is provided in table 1 . in haplotype 5 , the minor t allele of intron6 snp (# 6 from the left ) is exclusively linked to the major haplotype 1 ( in bold ), whereas it has no detectable ld with any of the other snps . sequencing of the entire cyp3a4 locus in two aei - positive samples did not implicate any other polymorphisms that would have to be heterozygous in both samples , indicating that intron6 snp is functional . intron6 snp associates with decreased cyp3a4 mrna level and enzyme activity in human livers : total cyp3a4 mrna levels were measured in 93 liver samples . while mrna levels did not differ between caucasians and african americans , females had − 1 . 3 fold higher levels than males ( 95 % cl 1 . 00 - 1 . 68 , two - sided p = 0 . 042 ) as reported ( 25 ). livers with the main cc genotype of intron6 snp had 1 . 71 - fold ( 95 % confidence interval ( ci ) 1 . 06 - 2 . 76 ) higher levels than ct / tt carriers ( t test , two - sided p = 0 . 028 ), with no interactions between genotypes and sex . to test the effect of cyp3a4 transcription factors ( 26 - 29 ), mrna levels were also measured for pregnane x receptor ( pxr , nr112 ), constitutive androstane receptor ( car , nr113 ), retinoid receptor ( rxra ), and hepatocyte nuclear factor ( hnf4α1a ). cyp3a4 mrna expression positively correlated with all four transcription factors , as reported ( 26 - 29 ) ( fig6 a - 6d ). after adjusting for age and transcription factors , intron6 snp remained significantly linked to cyp3a4 expression ( 1 . 67 - fold cc over ct / tt ( 95 %, ci 1 . 11 - 2 . 46 , p = 0 . 014 ) ( fig2 a ), showing that the genotype effect is independent of the transcription factors . cyp3a4 enzyme activity ( testosterone 6β - hydroxylation ) in 23 livers was 2 . 46 fold higher for intron6 snp cc than ct carriers , after adjusting for age (& lt ; 15 child , & gt ; 15 adult ), sex , and use of inducers ( phenobarbital , carbamazepine , nifedipine and dexamethasone ) ( two - sided p = 0 . 037 , 95 % ci 1 . 07 - 5 . 62 ) ( fig2 b ). consistent with allelic mrna expression , these results demonstrate that intron6 snp decreases cyp3a4 mrna and protein levels in vivo . in contrast , cyp3a4 * 1b , tgt insertion , rs2737418 , and rs4646437 had no effect on total cyp3a4 mrna level or enzyme activity ( p & gt ; 0 . 05 ). intron6 snp ( c & gt ; t ) associates with statin dose requirement and lipid control outcome : the in vivo effect of intron6 snp was assessed as the dosage of cyp3a4 - metabolized statins required for reaching a pre - determined ldl goal . intron6 snp was genotyped in 275 patients on stable doses of atorvastatin , lovastatin , and simvastatin , or the non - cyp3a4 substrates rosuvastatin and pravastatin . additional snps in cyp3a4 (* 1b rs2740574 , tgt insertion , * 17 rs4987161 , and * 18 rs28371759 ) and cyp3a5 (* 3 rs776746 and * 5 rs41303343 ) ( fig1 - table 5 ) were also genotyped . three snps deviated from hardy - weinberg equilibrium ( hwe ), because of different allele frequency in caucasian and african american populations , in which all snps followed hwe when analyzed separately ( table 7 ). a subset of 237 patients were on cyp3a4 - metabolized statins ( atorvastatin , lovastatin , simvastatin ) with doses ranging from 5 mg to 80 mg . the statins were either evaluated together , or each separately to account for potency differences . patients on all statins were divided into low - dose (& lt ; 20 mg , mean 16 ± 5 mg ) and high - dose (& gt ; 40 mg , mean 53 ± 19 mg ) ( p & lt ; 0 . 001 ) groups . baseline characteristics and lipid levels did not differ between low - and high - dose groups , except for maximum triglyceride and stable hdl levels ( p & lt ; 0 . 05 ) ( table 8 ). table 8 shows the baseline characteristics of patients on cyp3a4 - metabolized statins ( atorvastatin , lovastatin and simvastatin ). the minor intron6 snp t allele was significantly associated with a lower stable statin dose ( odd ratios = 3 . 80 , p = 0 . 005 ). none of the other snps were significant . table 9 shows the association of cyp3a4 / 3a5 snp genotypes with statins dose requirement and cholesterol control outcome . * p & lt ; 0 . 05 , ** p & lt ; 0 . 01 . multiple linear regression analysis shows that the stable statin dose for intron6 t allele carriers was only 0 . 27 that for non t - carriers ( p = 0 . 019 ), after adjusting for maximum lipid levels ( table 10 ). similar results were obtained when analyzing patients on atorvastain and simvastatin separately ( table 10 ). therefore , intron6 snp is linked to reduced statin dose requirement . for testing the association of intron6 snp with cholesterol control outcome , 198 patients with available ldl data were divided into those who reached the cholesterol goal ( ldl cholesterol & lt ; 70 mg / dl ; or 71 - 99 mg / dl with low / moderate risk ; or 100 - 129 mg / dl with low risk ) and those not at goal (& gt ; 130 mg / dl ; or 100 - 129 mg / dl with moderate / high risk ; or 71 - 99 mg / dl with high risk ) according to adult treatment panel iii guidelines ( 30 ). individuals bearing the t allele of intron6 snp had a 3 . 5 fold higher chance of reaching goal cholesterol control than non t - carriers ( p = 0 . 039 ) ( table 2 ). in contrast , other snps did not show such associations . example i shows that intron6 snp rs35599367 [ seq id no : 152 ] is significantly linked to reduced cyp3a4 mrna expression and enzyme activity in human livers , and moreover , fully accounts for differences in allelic mrna expression . since intron6 snp is embedded in the main haplotype of cyp3a4 , lacking substantial ld to any other snps , it had escaped detection by association studies using haplotype tag snps ( 14 , 15 ). none of the previously reported cyp3a4 snps , including promoter * 1b , enhancer tgt insertion , enhancer rs2737418 , and intron7 snp rs4646437 had detectable effects on allelic mrna expression , mrna , and enzyme activity , arguing against a contribution of these snps to cyp3a4 variability . the allele frequency of intron6 snp in the examined cohort was ˜ 5 %, resulting in ˜ 10 % heterozygocity . previously reported allele frequencies ( 35 ) were 0 . 043 , 0 . 043 , and 0 . 083 , for african americans , chinese , and caucasians , respectively , indicating that intron6 snp allele frequency ranges from 4 to 8 % in various ethnic groups . a common molecular mechanism for an intronic snp to alter mrna levels is to affect rna expression , elongation , splicing , or maturation . since the allelic ratios were similar for mrna and hnrna in livers heterozygous for both exonic and intronic marker snps , splicing and mrna turnover can be ruled out as main mechanisms . moreover , cyp3a4 mrna and hnrna levels were shown to vary in parallel in human livers ( 36 ), arguing for an early event in expression and processing . for example , intron6 snp could affect the folding of nascent rna and hence elongation . in silico rna folding analysis shows that intron6 snp promotes a loop - to - stem structural change ( not shown ), possibly impeding the binding of regulatory proteins . consistent with reduced expression of the minor allele , intron6 snp was significantly associated with reduced stable dose requirements of statin drugs that are mainly metabolized by cyp3a4 ( atorvastatin , lovastatin and simvastatin ). since statin doses are titrated to reach a desired ldl , this can be achieved at lower doses in carriers of the intron6 snp t allele conveying reduced metabolism . in support of the inventors &# 39 ; belief , pharmacokinetics studies showed that inhibition of cyp3a4 activity drastically increased plasma concentrations of simvastatin and lovastatin ( 31 , 32 ), suggesting cyp3a4 activity is a major determinant of serum concentration of cyp3a4 metabolized statins . the finding that patients carrying the intron6 t allele are also more likely to reach ldl goal may be related to insufficient dosage titration in subjects with normal cyp3a4 metabolism , or with lower fluctuations in statin levels between doses in t allele carriers . the previously identified cyp3a4 promoter snp (* 1b , − 392 a & gt ; g ) did not associate with lipid - lowering efficacy and safety of simvastin treatment ( 33 ), consistent with the results here showing this promoter snp has no effect on hepatic mrna expression . further , the current results indicate that snps in cyp3a5 do not affect statin dose or treatment outcomes , consistent with reports that cyp3a5 does not play a major role in statin metabolism ( 34 ). therefore , intron6 snp is the only cyp3a polymorphism shown to affect statin therapy . even though cyp3a4 activity shows considerable inter - individual variability , new drugs are often targeted for metabolism by cyp3a4 , to avoid problems arising from null mutations in other drug metabolizing cyp enzymes , such as cyp2d6 . the results presented here show that a portion of the variability in cyp3a4 can be accounted for by intron6 snp . the clinical relevance of this finding is demonstrated by the impact of intron6 snp on the titrated dose of two statin drugs that depend on cyp3a4 for their elimination . because cyp3a4 is involved in the metabolism of approximately half of all clinically used drugs , the intron6 snp is now believed by the inventors herein to affect dosing requirements , response , and toxicity of numerous drugs , including anticancer agents with narrowly defined dosage regimens . therefore , cyp3a4 intron6 snp is a valuable biomarker in clinical practice , and in drug discovery and development . the dna isolated from cells can be used as positive human genomic reference controls ( i . e ., they have mutations present ) or negative controls ( i . e ., they represent the normal or wild - type ), in particular , for human cyp3a4 genes . this can ensure accurate and reliable clinical diagnostic testing for these genes . the reference controls can be used , for example , in genotyping assays performed during clinical trials . where the reference controls include a genetic variation typical of a patient who does not respond to therapy , the use of reference controls helps ensure that the genotyping assay used performs reliably such that non - responders are properly identified and data regarding the ineffectiveness of the investigative therapy for non - responders is properly identified . similarly , where the reference controls include a genetic variation typical of a patient who metabolizes drugs at a different rate than normal patients ( i . e ., patients with mutant cytochrome p450 genes ), the use of reference controls helps ensure the validity of the genetic variation so that these patients are properly identified and properly dosed and adverse drug reactions or ineffective therapies are avoided . the reference controls can also be used in patient care . as with their use in clinical trials , it is essential that effective therapy is identified in a time - sensitive manner , so that the patient &# 39 ; s condition is not worsened before appropriate therapy is initiated . it is also essential that appropriate dosing regimens are selected . accordingly , one aspect of the present invention relates to a method of testing a plurality of patients for their genetic predisposition to respond to a particular therapy . in this aspect , one or more reference controls are tested as “ samples ”— with known expected results . these controls can include a genetic variant associated with patients who show a predisposition to not respond to the therapy ( positive controls ) or can include a normal / wild type variant ( negative controls ), associated with patients who show a predisposition to respond to the therapy . thus , the reference controls can be used by testing laboratories to ensure that their diagnostics assays are performing correctly and identify the genetic variations that convey resistance to drug therapy or reduced metabolic state . to ensure that non - responders are properly identified , testing laboratories can include reference controls in each assay to determine the validity of the assay , and hence , patient results . the reference controls can be used at random , or at pre - determined intervals . in the same respect , testing laboratories can use the reference controls as panels to evaluate the accuracy of their laboratory staff . accordingly , another aspect of the present invention relates to a method of testing a plurality of patients for their genetic predisposition to show rapid or slowed metabolism , so that proper dosing regimens can be set . in this aspect , one or more samples that are tested are reference controls that include a genetic variant associated with patients having a predisposition to be “ rapid ” or “ slow ” metabolizers . these polymorphisms are expressed in a number of phenotypes in the population such as , for example , a “ poor ” metabolizer , an “ intermediate ” metabolizer , an “ extensive ” metabolizer , and an “ ultra - rapid ” metabolizer . in certain situations , the “ extensive ” metabolizers can have at least one , and no more than two , normal functional alleles ; the “ intermediate ” metabolizers can possess one reduced activity allele and one null allele ; and , the “ poor ” metabolizers can carry two mutant alleles which result in complete loss of enzyme activity . in certain situations , the ultra - rapid metabolizers can carry multiple copies of functional alleles , and thus produce excess enzymatic activity . thus , when certain drugs are administered , a “ poor ” metabolizer may not obtain a significant benefit from the drugs , but rather , experience exaggerated drug response and side effects when they receive standard doses . that is , if a metabolite is the active therapeutic moiety , “ poor ” metabolizer may show no therapeutic response . on the other hand , an “ ultra - rapid ” metabolizer may fail to respond to standard doses . various methods for pre - screening samples may be used . for example , biological samples can be pre - screened to ensure that they have the mutation of interest . patient populations can be pre - screened , based on a variety of factors , to minimize the sample size needed to identify individuals that include the mutation . after performing an initial genomic screening on the samples to identify one or more samples which include the mutation of interest , the patients with these mutations can optionally be recalled to obtain additional biological material . this material can optionally be thoroughly sequenced to confirm the presence of the mutation of interest . the biological material can be immortalized , so it can provide a steady , on - demand source of the reference controls , or , alternatively , the cells themselves can be the reference controls . various types of biological samples can be used , such as , but not limited to human genomic dna present in any nucleic acid - containing sample of tissues , bodily fluids ( for example , blood , serum , plasma , saliva , urine , tears , semen , vaginal secretions , lymph fluid , cerebrospinal fluid or mucosa secretions ), individual cells or extracts of the such sources that contain the nucleic acid of the same , and subcellular structures such as mitochondria or chloroplasts , using protocols well established within the art . in certain embodiments , the nucleic acid has been obtained from a human to be pre - screened for the presence of one or more genetic sequences that can be diagnostic for , or predispose the subject to , a medical condition or disease . as an alternative to prescreening patient populations , one can screen cell cultures commonly available for the genetic sequence of interest , and then expand and preserve the cell clones for future continuous supply there are a number of known genomic assay methods for which the reference control can be used in testing where most involve hybridizing a primer with a dna sample that may or may not include a snp of interest . a diagnostic primer and / or probe can be tagged to permit rapid identification . once hybridization has occurred , the dna can be amplified , and the tagged primer and / or probe are detected . the validated primers can be used to confirm the validity of reference controls . once the reference controls are validated , they can be used in commercially available assays as a reference control , and can be used to validate primers that are designed for use in these or other assays to determine the presence or absence of a particular mutation . thus , while exemplary assay methods are described herein , the invention is not so limited . while the invention has been described with reference to various and preferred embodiments , it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the essential scope of the invention . in addition , many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof . therefore , it is intended that the invention not be limited to the particular embodiment disclosed herein contemplated for carrying out this invention , but that the invention will include all embodiments falling within the scope of the claims . the citation of any reference herein is not an admission that such reference is available as prior art to the instant invention . any publications mentioned in this specification are herein incorporated by reference . any discussion of documents , acts , materials , devices , articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention . it is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application . 1 . danielson , p . b . 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