WO2010111600A1 - Polymorphism in cyp3a4 gene affecting drug metabolizing and uses thereof - Google Patents

Abstract

A method for predicting a subject's risk factors for CYP3A4-related disorders includes detecting the allelic status of a SNP in a nucleic acid sample of the subject.

Description

TITLE

POLYMORPHISM IN CYP3A4 GENE AFFECTING DRUG METABOLIZING AND USES THEREOF

Inventors: Wolfgang Sadee, Danxin Wang

RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application 61/163,555 filed March 26, 2009, the disclosure of which is incorporated herein by reference, in its entirety.

GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant Number NIH NIAID (1R21AI074399) awarded by the National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy, created on March 23, 2010, is named 604_50806_SEQLIST_OSURF-09032.txt and is 34,669 bytes in size.

BACKGROUND

[0004] Cytochrome P450 (CYP) enzymes metabolize endogenous and xenobiotic compounds. CYP3A4 belongs to the CYP3A subfamily and is the most abundant CYP enzyme. CYP3A4 is involved in metabolizing 45-60% of all currently used drugs (1), including several statins — cholesterol-lowering HMG-CoA reductase inhibitors. However, CYP3A4 activity shows wide inter-individual variation, influencing drug response and toxicity. While genetic factors are thought to be main contributors to inter- individual differences in CYP3A4 activity (2), currently known CYP3A4 polymorphisms cannot account for the observed variability.

[0005] Genetic variants in CYP3A4 that change the amino acid sequence are rare (<1%) (www.cypalleles.ki.se/cyp3a4.htm). A more common variant, CYP3A4*1B, in the 5'- flanking region, has been associated with drug response and diseases (3,4), but results are inconsistent (5-7), and its function remains controversial (3,8-10). Moreover, CYP3A4*1B is in linkage disequilibrium (LD) with the adjacent CYP3A5 (11), encoding a similar but usually less abundant CYP enzyme that could have accounted for any linked clinical phenotype (12).

[0006] Further suspected CYP3A4 polymorphisms include a TGT insertion (13), an enhancer region SNP (rs2737418) (14), and an intron7 SNP (rs4646437) (15). While reporter gene assays suggested an effect for the TGT insertion and for rs2737418, the in vivo significance of TGT remains unresolved (13), while results on CYP3A4 mRNA and enzyme activity were contradictory for rs2737418 (14). The intron7 SNP rs4646437 was found to be associated with CYP3A4 protein/enzyme activity, but only in livers from males (15). Therefore, the role of functional polymorphisms in CYP3A4 remains uncertain.

[0007] Single nucleotide polymorphisms (SNPs) are useful as biomarkers for predicting disease susceptibility or progression, or as a guide for individualized therapy, including drug therapy.

[0008] What are lacking are tools for predicting the likelihood that a particular patient will be responsive to a particular therapeutic agent, and in particular, identifying polymorphisms to which a CYP3A4 agent will be sensitive or resistant. Also lacking are tools for profiling genetic factors influencing sensitivity and resistance of patients to such therapeutic agents. Such tools, and the resulting gene expression profiles, would be predictive of treatment response of a patient to a particular drug, and would allow for increased predictability regarding efficacy, adverse drug reactions, chemosensitivity or chemoresistance of such patients to enable the design of optimal treatment regimens for patients, or to enable drug development in early clinical trials avoiding unexpected toxicities in poor metabolizers.

[0009] Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The objects and advantages of the invention may be realized and attained as particularly pointed out in the appended claims.

SUMMARY

[0010] In a first aspect, there is provided herein a method for predicting a subject's response to CYP3A4-metabolized compounds, including, for example efficacy and/or adverse drug reactions. The method includes detecting the allelic status of one or more polymorphisms in a nucleic acid sample of the subject, wherein the polymorphism is one or more of: i) CYP3A4-associated SNP (rs35599367 [SEQ ID NO: 152]) located in intronό (C>T); or, ii) a SNP in linkage disequilibrium therewith, wherein the allelic status of the polymorphism in the subject is predictive of the subject's risk for having or developing the CYP3A4-related disorder.

[0011] In another aspect, there is provided herein, a method of screening a subject for a prognostic biomarker of an CYP3A4-related disorder, comprising detecting the allelic status of one or more polymorphisms in a nucleic acid sample of the subject, wherein the polymorphism is one or more of: i) CYP3A4-associated SNP (rs35599367 [SEQ ID NO: 152]) located in intronό (C>T); or, ii) a SNP in linkage disequilibrium therewith, wherein the allelic status of the polymorphism in the subject is predictive of the prognostic outcome of the CYP3A4-related disorder.

[0012] In certain embodiments, the method further includes the step of correlating the allelic status of the polymorphism in the subject with the allelic status of the polymorphism in a reference population to predict the subject's risk for having or developing the CYP3A4-related disorder.

[0013] In certain embodiment, the method further includes the step of correlating the allelic status of the polymorphism in the subject with the allelic status of the polymorphism in a reference population to predict whether the subject has a more or less severe phenotype of the CYP3A4-related disorder.

[0014] In certain embodiment, the method further includes the step of correlating the allelic status of the polymorphism in the subject with the allelic status of the polymorphism in a reference population to predict the prognostic outcome of the disorder in the subject.

[0015] In certain embodiment, the method further includes the step of correlating the allelic status of the polymorphism in the subject with the allelic status of the polymorphism in a reference population to predict the subject's response to treatment, dosage and/or toxicity.

[0016] In certain embodiment, the CYP3A4-related disorder comprises a metabolic- related disorder.

[0017] In certain embodiment, the CYP3A4-metabolized compounds comprise one or more pharmaceuticals metabolized in the liver, including statin drugs.

[0018] In certain embodiment, the CYP3A4-metabolized compounds comprise one or more of: CYP3A4 inhibitors or CYP3A4 enhancers. [0019] In certain embodiment, the polymorphism comprises a CYP3A4-associated SNP (rs35599367 [SEQ ID NO: 152]) located in intronό (OT).

[0020] In certain embodiment, the polymorphism comprises rs35599367 [SEQ ID NO: 152], wherein the presence of the polymorphism in a subject is predictive of an increased risk for a CYP3A4-related disorder.

[0021] In certain embodiment, the presence of a minor allele of the polymorphism is predictive of lower levels of CYP3A4 in target tissue and is associated with a decreased CYP3A4 mRNA expression.

[0022] In another aspect, there is provided herein, a kit comprising an assay for detecting the allelic status of one or more polymorphisms in a nucleic acid sample of a subject, wherein the polymorphism is one or more of: i) a CYP3A4-associated SNP (rs35599367 [SEQ ID NO: 152]) located in intronό (OT); or, ii) a SNP in linkage disequilibrium therewith.

[0023] In certain embodiment, the kit further includes instructions for correlating the assay results with the subject's risk for having or developing a CYP3A4-related disorder.

[0024] In certain embodiment, the kit further includes instructions for correlating the assay results with the subject's prognostic outcome for the disorder.

[0025] In certain embodiment, the kit further includes instructions for correlating the assay results with the probability of success or failure of a particular drug treatment in the subject.

[0026] In another aspect, there is provided herein, a method for finding a functional polymorphism in a target gene implicated in an CYP3A4-related disorder, comprising: i) providing a sample of a target tissue expressing the target gene; ii) measuring the target gene's allelic mRNA expression imbalance (AEI) by: a) quantitatively measuring the relative amounts of mRNA generated from each of two alleles in a transcribed region of the target gene, and b) comparing the mRNA expression of one allele against the other allele to obtain an AEI ratio; and, iii) using the AEI ratio as a phenotype to scan the target gene for regions containing polymorphisms, wherein a significant association between the AEI ratio and the polymorphism indicates that the polymorphism is a functional polymorphism that can serve as a biomarker for the CYP3A4-related disorder.

[0027] In certain embodiment, the polymorphism resides in an intronic region.

[0028] In certain embodiment, the polymorphism is a SNP.

[0029] In certain embodiment, the biomarker affects gene transcription, mRNA processing, mRNA splicing, or a combination thereof.

[0030] In certain embodiment, the target gene is a CYP3A4 gene locus.

[0031] In another aspect, there is provided herein, a method for determining metabolism of a statin drug comprising screening for an intronό SNP.

[0032] In another aspect, there is provided herein, a method for determining metabolism of a statin drug comprising screening for a CYP3A4-associated SNP (rs35599367 [SEQ ID NO: 152]) located in intronό (OT).

[0033] In another aspect, there is provided herein a biomarker for detecting variability in CYP3A4 comprising intronό SNP.

[0034] In another aspect, there is provided herein a biomarker for detecting variability in CYP3A4 comprising a CYP3A4-associated SNP (rs35599367 [SEQ ID NO: 152]) located in intronό (C>T).

[0035] In another aspect, there is provided herein, a biomarker for determining a dosing requirement of a CYP3A4-metabolizing therapeutic agent comprising SNP (rs35599367 [SEQ ID NO: 152]) located in intronό (OT).

[0036] In another aspect, there is provided herein a biomarker for determining a response of a CYP3A4-metabolizing therapeutic agent comprising SNP (rs35599367 [SEQ ID NO: 152]) located in intronό (OT).

[0037] In another aspect, there is provided herein a biomarker for determining toxicity of a CYP3A4-metabolizing therapeutic agent comprising SNP (rs35599367 [SEQ ID NO: 152]) located in intronό (OT). In certain embodiment, the therapeutic agent is a statin drug. In certain embodiment, the therapeutic agent is an anti-cancer drug. In certain embodiment, the therapeutic agent is a drug having a narrowly defined dosage regimen.

[0038] In another aspect, there is provided herein a method of clinical pharmacogenomic screening comprising: a) screening a sample for the presence of at least one or more biomarkers described herein, where the presence of the one or more biomarkers is indicative of a patient with altered metabolism; and b) including a reference control in a random or predetermined manner in the screening, wherein the reference control comprises DNA comprising a biomarker indicative of a patient with altered metabolism, wherein the detection of the presence of one or more biomarkers in one or more drug- metabolizing genes in the reference control verifies that the screening is effective to detect the same one or more biomarkers in one or more drug-metabolizing genes in the sample. [0039] In another aspect, there is provided herein a method of personalized medical therapy, comprising: i) performing the method of screening described herein on samples from a target patient population to identify patients with a genetic profile comprising one or more mutations in the CYP3A4 or other gene associated with drug metabolism; and ii) treating patients identified in step i) as possessing a particular genetic profile with a therapy of interest particular to the identified genetic profile.

[0040] In certain embodiments, the genetic profile is indicative of a patient with altered metabolism.

[0041] In certain embodiments, the altered metabolism is selected from the group consisting of: poor metabolizer, intermediate metabolizer, extensive metabolizer, and ultra-rapid metabolizer.

[0042] In certain embodiments, the genetic profile is indicative of the effectiveness of the therapy of interest in the patient.

[0043] In certain embodiments, the genetic profile is indicative of a patient with a genetic disorder.

[0044] In certain embodiments, the genetic profile is indicative of a patient who should not be treated with a particular therapy.

[0045] In certain embodiments, the therapy of interest is used to treat a disease or disorder selected from the group consisting of: cancer, heart disease, neurological disorders, psychiatric disorders, autoimmune disorders, and metabolic disorders.

[0046] In certain embodiments, the one or more mutations comprises a mutation in CYP3A4 and wherein the therapy of interest comprises administration of at least one statin to the patient.

[0047] In another aspect, there is provided herein a method for identifying a cell that can be used to generate isolated genomic DNA suitable for use as a reference control, wherein the method comprises: a) prospectively screening a human volunteer for the presence of a human genomic DNA sequence comprising one or more polymorphisms in a nucleic acid sample of the volunteer associated with a genetic predisposition that determines a patient's predicted degree of response to a particular therapy; wherein the polymorphism is one or more of: i) CYP3A4-associated SNP (rs35599367 [SEQ ID NO: 152]) located in intron 6 (C>T); or, ii) a SNP in linkage disequilibrium therewith, wherein the allelic status of the polymorphism in the subject is predictive of the subject's risk for having or developing the CYP3A4-related disorder; b) isolating the cell from the volunteer that possesses the one or more mutations of interest; and c) subjecting DNA from the cell to a plurality of validated genomic screening assays.

BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The patent or application file may contain at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0049] The invention can be more fully understood from the following detailed description, the drawings and the Sequence Descriptions that form a part of this application. The Sequence Descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 CFR §§1.821-1.825. The Sequence Descriptions contain the three letter codes for amino acids as defined in 37 CFR §§1.821-1.825, which are incorporated herein by reference.

[0050] Figure 1: Allelic mRNA/hnRNA expression ratios of CYP3A4 in human livers measured with a primer extension assay (SNaPshot) using multiple marker SNPs (Figure 3) (Figure IA) or intronό SNP rs35599367 [SEQ ID NO: 152] only (Figure IB). Allelic RNA ratios were normalized to gDNA ratios set at 1. Data represent the average of 2-3 measurement using single or multiple marker SNPs. An arrow indicates samples with AEI ratios significantly different from 1 (P<0.05). All allelic RNA ratios in Panel B are significantly different from 1 (ANOVA with Dunnett post-test, P<0.05).

[0051] Figures 2A-2B: Box plot of CYP3A4 mRNA levels (Figure 2A) and enzyme activity (Figure 2B) in human liver samples, grouped by intronό SNP genotype.

[0052] Figure 3: Location and rs number of marker SNPs used for AEI measurements. Intronό SNP rs35599367 [SEQ ID NO: 152] is in red.

[0053] Figure 4: Association between genotypes and allelic RNA expression imbalance (AEI). Only intronό SNP rs35599367 [SEQ ID NO: 152] and much less strongly SNP rs2246709 associated with AEI with an adjusted P<0.05.

[0054] Figure 5: LD plot for 13 polymorphisms in CYP3A4. Detailed SNP information is provided in Table 1. SNP6 is intronό SNP rs35599367 [SEQ ID NO: 152], only partial LD with SNP 7 rs2246709.

[0055] Figures 6A-6D: Correlation between mRNA expression of four transcription factors and CYP3A4 in human livers. DETAILED DESCRIPTION OF THE INVENTION

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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 ds-acting regulatory factors in CYP3A4 that affect RNA expression, processing, or turnover.

[0060] The results described herein demonstrate that an intronό 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.

[0061] Moreover, intronό 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. [0062] EXAMPLE I

[0063] Methods

[0064] 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)

[0065] 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 statm for lipid control were selected. Enrollment and tπal 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.

[0066] DNA and RNA preparation. Preparation of genomic DNA, RNA and cDNA from tissues or blood samples were performed as descnbed (16-18)

[0067] 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 mtronic regions were used to measure allelic ratios of mature mRNA (3'UTR markers) or hnRNA (mtronic 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-actmg polymorphisms in CYP3A4 that affect mRNA expression levels

[0068] 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

[0069] Seven SNPs in CYP3A4/3A5 were genotyped in gDNA from 275 patients, as shown in Table 2.

[0070] PCR conditions and primer sequences are shown in Table 3, Table 4, Table 5.

[0071] Quantitative mRNA analysis of CYP 3 A4 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.

[0072] Table 3: Geno typing 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.

[0073] Table 4: Multiplex PCR and Snapshot assay for CYP3A4 and CYP3A5 SNP genotypmg. Column labeled "PCR pπmer 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.

[0074] 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 E) NOS 96-151, respectively, in order of appearance.

[0075] 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 Figure 16 - Table 10.

[0076] CYP3A4 enzymatic activity assay: CYP3A4 activities were quantified from liver microsomes with testosterone as a probe as described (23).

[0077] Data analysis: Association between discrete variables and genotypes were analyzed with Helix-Tree software (Golden Helix, Bozeman MT). 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.

[0078] Results:

[0079] Scanning for cis-acting CYP 3 A4 polymorphisms that affect mRNA levels:

[0080] Allelic mRNA expression of CYP3A4 was measured in human livers using 3 frequent 3' UTR marker SNPs (Figure 3). 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 (Figure IA). Therefore, a cis-acύng polymorphism(s) in CYP3A4 affects both mRNA and hnRNA levels equally.

[0081] To search for responsible polymorphism(s), 13 CYP3A4 polymorphisms (Table 1) were genotyped and the association between allelic RNA ratios and genotype tested.

[0082] A single SNP (rs35599367 [SEQ K) NO: 152]) located in intronό (OT) showed highly significant association with AEI (adjusted p value 9.12 x 10^"10) (Figure 4), while another SNP rs2246709 also scored with moderate significance (P = 0.034), a likely result of partial linkage disequilibrium (LD) with the intronό SNP (Figure 5).

[0083] Other SNPs including previously identified promoter SNP rs2740574, TGT insertion, rs2737418, and rs4646437 did not show significant association (P>0.05). These results indicated that intronό SNP is functional or in highly LD with a functional SNP.

To test this further, intronό SNP was used as a marker, and AEI was measured in 10 heterozygous samples, including 3 heterozygous only for intronό SNP. All 10 samples showed AEI with allelic ratios (minor T allele/major C allele) ranging from 0.16 to 0.62 (Figure IB), with no differences between males and females (P=O.314). Therefore, the minor T allele of intronό 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 (Figure 12 - Table 6; LD plot Figure 5).

[0084] 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 intronό 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.

[0085] 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 intronό SNP is functional.

[0086] Intronό SNP associates with decreased CYP3A4 mRNA level and enzyme activity in human livers:

[0087] 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 intronό 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, NR1I2), constitutive androstane receptor (CAR, NR1I3), retinoid receptor (RXRa), and hepatocyte nuclear factor (HNF4αlA). CYP3A4 mRNA expression positively correlated with all four transcription factors, as reported (26-29) (Figures 6A-6D).

[0088] After adjusting for age and transcription factors, intronό SNP remained significantly linked to CYP3A4 expression (1.67-fold CC over CT/TT (95%, CI 1.11- 2.46, p = 0.014) (Figure 2A), showing that the genotype effect is independent of the transcription factors.

[0089] CYP3A4 enzyme activity (testosterone 6β-hydroxylation) in 23 livers was 2.46 fold higher for intronό SNP CC than CT carriers, after adjusting for age (<15 child, >15 adult), sex, and use of inducers (phenobarbital, carbamazepine, nifedipine and dexamethasone) (two-sided p = 0.037, 95% CI 1.07-5.62) (Figure 2B).

[0090] Consistent with allelic mRNA expression, these results demonstrate that intronό 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>0.05). [0091] Intronό SNP (C>T) associates with statin dose requirement and lipid control outcome:

[0092] The in vivo effect of intronδ SNP was assessed as the dosage of CYP3A4- metabolized statins required for reaching a pre-determined LDL goal. Intronό 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, *77rs4987161, and *iδ rs28371759) and CYP3A5 (*3 rs776746 and *5 rs41303343) (Figure 11 - 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).

[0093] The allele frequency of intronό SNP is -5%, consistent with that reported in NCBI database. Absence of CYP3A4 SNPs *17 and *18 is consistent with reported low allele frequency.

[0094] 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 (< 20 mg, mean 16 ± 5 mg) and high-dose (> 40 mg, mean 53 ± 19 mg) (P<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<0.05) (Table 8). Table 8 shows the baseline characteristics of patients on CYP3A4-metabolized statins (atorvastatin, lovastatin and simvastatin).

[0095] The minor intronό 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<0.05, **P<0.01.

[0096] Multiple linear regression analysis shows that the stable statin dose for intronό 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 atorvastam and simvastatin separately (Table 10). Therefore, intronό SNP is linked to reduced statm dose requirement

[0097] For testing the association of intronό SNP with cholesterol control outcome, 198 patients with available LDL data were divided into those who reached the cholesterol goal (LDL cholesterol <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 (>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 intronό SNP had a 3 5 fold higher chance of reaching goal cholesterol control than non T-carners (P = 0 039) (Table 2) In contrast, other SNPs did not show such associations. [0098] Discussion of EXAMPLE I

[0099] Example I shows that intronό 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.

[00100] Since intronό SNP is embedded in the mam 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 mtron7 SNP rs4646437 had detectable effects on allelic mRNA expression, mRNA, and enzyme activity, arguing against a contribution of these SNPs to CYP3A4 variability.

[00101] The allele frequency of intronό 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 intronό SNP allele frequency ranges from 4 to 8% in various ethnic groups.

[00102] A common molecular mechanism for an mtromc 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 exomc and mtromc 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, intronό SNP could affect the folding of nascent RNA and hence elongation.

[00103] In silico RNA folding analysis shows that intronό SNP promotes a loop-to-stem structural change (not shown), possibly impeding the binding of regulatory proteins.

[00104] Consistent with reduced expression of the minor allele, intronό 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 intronό SNP T allele conveying reduced metabolism. In support of the inventors' 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.

[00105] The finding that patients carrying the intronό 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>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, intronό SNP is the only CYP 3 A polymorphism shown to affect statin therapy.

[00106] 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.

[00107] The results presented here show that a portion of the variability in CYP3A4 can be accounted for by intronό SNP. The clinical relevance of this finding is demonstrated by the impact of intronό 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 intronό 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.

[00108] Therefore, CYP3A4 intronό SNP is a valuable biomarker in clinical practice, and in drug discovery and development.

[00109] EXAMPLE II

[00110] 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.

[00111] 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.

[00112] 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's condition is not worsened before appropriate therapy is initiated. It is also essential that appropriate dosing regimens are selected.

[00113] 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.

[00114] 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.

[00115] 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.

[00116] 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.

[00117] 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.

[00118] 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

[00119] 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.

[00120] 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.

[00121] 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 pnoπty date of each claim of this application REFERENCES

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Claims

CLAIMSWhat is claimed is:

1. A method for predicting a subject's response to at least one CYP3A4- metabolized compound, the method comprising detecting the allelic status of one or more polymorphisms in a nucleic acid sample of the subject, wherein the polymorphism is one or more of: i) CYP3A4-associated SNP (rs35599367 [SEQ ID NO: 152]) located in intron 6 (OT); or, ii) a SNP in linkage disequilibrium therewith, wherein the allelic status of the polymorphism in the subject is predictive of the subject's risk for having or developing the CYP3A4-related disorder.

2. A method of screening a subject for a prognostic biomarker of an CYP3A4- related disorder, comprising detecting the allelic status of one or more polymorphisms in a nucleic acid sample of the subject, wherein the polymorphism is one or more of: i) CYP3A4-associated SNP (rs35599367 [SEQ ID NO: 152]) located in intron 6 (OT); or, ii) a SNP in linkage disequilibrium therewith, wherein the allelic status of the polymorphism in the subject is predictive of the prognostic outcome of the CYP3A4- related disorder.

3. The method of claim 1 or 2, further comprising the step of correlating the allelic status of the polymorphism in the subject with the allelic status of the polymorphism in a reference population to predict the subject's risk for having, or developing, the CYP3A4-related disorder.

4. The method of claim lor 2, further comprising the step of correlating the allelic status of the polymorphism in the subject with the allelic status of the polymorphism in a reference population to predict whether the subject has a more or less severe phenotype of the CYP3A4-related disorder.

5. The method of claim 1 or 2, further comprising the step of correlating the allelic status of the polymorphism in the subject with the allelic status of the polymorphism in a reference population to predict the prognostic outcome of the disorder in the subject.

6. The method of claim 1 or 2, further comprising the step of correlating the allelic status of the polymorphism in the subject with the allelic status of the polymorphism in a reference population to predict the subject's response to treatment.

7. The method of claim 1 or 2, wherein the CYP3A4-related disorder comprises a metabolic -related disorder.

8. The method of claim 1 or 2, wherein CYP3A4-metabolized compounds comprises one or more pharmaceuticals metabolized in the liver.

9. The method of claim 1 or 2, wherein CYP3A4-metabolized compounds comprises one or more statin drugs.

10. The method of claim 1 or 2, wherein CYP3A4-metabolized compounds comprise one or more of atorvastatin, lovastatin and simvastatin

11. The method of claim 1 or 2, wherein the CYP3 A4-metabolized compounds comprises one or more of: CYP3A4 inhibitors or CYP3A4 enhancers.

12. The method of any claim 1 or 2, wherein the polymorphism comprises a CYP3A4-associated SNP (rs35599367 [SEQ ID NO: 152]) located in intronβ (OT).

13. The method of claim 1 or 2, wherein the polymorphism comprises rs35599367 [SEQ ID NO: 152], wherein the presence of the polymorphism in a subject is predictive of an increased risk for a CYP3A4-related disorder.

14. The method of claim 1 or 2, wherein the presence of a minor allele of the polymorphism is predictive of lower levels of CYP3A4 in target tissue and is associated with a decreased CYP3A4 mRNA expression.

15. An assay system for detecting the presence of enzyme- specific metabolites in a biological sample, the sample obtained from an individual treated with a known amount of at least one probe substrate for at least one CYP3A4-metabolizable compound, specific for metabolic pathways of the metabolites, the assay comprising: i) means for receiving the biological sample, including a plurality of affinity complexation agents contained therein; ii) means for detecting presence of the enzyme-specific metabolites bound to the affinity complexation agents; and ii) means for quantifying ratios of the metabolites to provide corresponding phenotypic determinants; wherein the phenotypic determinants provide a metabolic phenotypic profile of the individual.

16. The assay system of claim 15, wherein the step ii) or step iii) is effected according to the method comprising: a) administering to an individual a probe substrate specific to metabolic pathway(s) for the CYP3A4-metabolizing drug; b) detecting metabolites of the metabolic pathway(s) in a biological sample from the individual in response to the probe substrate; and c) characterizing respective phenotypic determinants of the multi-determinant metabolic phenotype based on detected metabolites; wherein the probe substrate is at least one substrate known to be metabolized by the metabolic pathway, and wherein the probe substrate is other than an inducer or inhibitor of the metabolic pathway.

17. The assay system of claim 16, wherein the assay is a ligand binding assay.

18. The assay system of claim 17, wherein the ligand binding assay is selected from the group consisting of immunoassay, enzyme-linked immunosorbent assay (ELISA), microarray formatted immunoassay and microarray formatted ELISA.

19. The assay system of claim 15, wherein the means for receiving the biological sample is a multi-well microplate including the plurality of affinity complexation agents in each well.

20. The assay system of claim 19, wherein the plurality of affinity complexation agents are bound to each well in an array-based format.

21. The assay system of claim 15, wherein the means for detecting the presence of the metabolites bound to the binding agents is a charge -coupled device (CCD) imager.

22. The assay system of claim 15, wherein the means for the quantifying ratios of the metabolites is a densitometer.

23. A kit comprising an assay for detecting the allelic status of one or more polymorphisms in a nucleic acid sample of a subject, wherein the polymorphism is one or more of: i) a CYP3A4-associated SNP (rs35599367 [SEQ ID NO: 152]) located in intronό (OT); or, ii) a SNP in linkage disequilibrium therewith.

24. The kit of claim 23, further comprising instructions for correlating the assay results with the subject's risk for having or developing a CYP3A4-related disorder.

25. The kit of claim 23, further comprising instructions for correlating the assay results with the subject's prognostic outcome for the disorder.

26. The kit of claim 23, further comprising instructions for correlating the assay results with the probability of success or failure of a particular drug treatment in the subject.

27. A method for finding a functional polymorphism in a target gene implicated in an CYP3A4-related disorder, comprising: i) providing a sample of a target tissue expressing the target gene; ii) measuring the target gene's allelic mRNA expression imbalance (AEI) by: a) quantitatively measuring the relative amounts of mRNA generated from each of two alleles in a transcribed region of the target gene, and b) comparing the mRNA expression of one allele against the other allele to obtain an AEI ratio; and, iii) using the AEI ratio as a phenotype to scan the target gene for regions containing polymorphisms, wherein a significant association between the AEI ratio and the polymorphism indicates that the polymorphism is a functional polymorphism that can serve as a biomarker for the CYP3A4-related disorder.

28. The method of claim 27, wherein the polymorphism resides in an exonic region.

29. The method of claim 27, wherein the polymorphism is a SNP.

30. The method of claim 27, wherein the biomarker affects gene transcription, mRNA processing, mRNA splicing, or a combination thereof.

31. The method of claim 27, wherein the biomarker affects mRNA translation into protein.

32. The method of claim 27, wherein the target gene is a CYP3A4 gene locus.

33. A method for determining metabolism of a statin drug comprising screening for an intron 6 SNP.

34. A method for determining metabolism of a statin drug comprising screening for a CYP3A4-associated SNP (rs35599367 [SEQ ID NO: 152]) located in intronό (C>T).

35. A biomarker for detecting variability in CYP3A4 comprising intronό SNP.

36. A biomarker for detecting variability in CYP3A4 comprising a CYP3A4- associated SNP (rs35599367 [SEQ ID NO: 152]) located in intronό (OT).

37. A biomarker for determining a dosing requirement of a CYP3A4- metabollizing therapeutic agent comprising SNP (rs35599367 [SEQ ID NO: 152]) located in intronό (C>T).

38. A biomarker for determining a response of a CYP3A4-metabollizing therapeutic agent comprising SNP (rs35599367 [SEQ ID NO: 152]) located in intronδ (OT).

39. A biomarker for determining toxicity of a CYP3A4-metabollizing therapeutic agent comprising SNP (rs35599367 [SEQ ID NO: 152]) located in intronό (OT).

40. The biomarker of one of claims 35-39, wherein the therapeutic agent is a statin drug.

41. The biomarker of one of claims 35-39, wherein the therapeutic agent is an anti-cancer drug.

42. The biomarker of one of claims 35-39, wherein the therapeutic agent is a drug having a narrowly defined dosage regimen.

43. A method of clinical pharmacogenomic screening comprising: a) screening a sample for the presence of at least one or more biomarkers of claims 35-39, wherein the presence of the one or more biomarkers is indicative of a patient with altered metabolism; and b) including a reference control in a random or predetermined manner in the screening, wherein the reference control comprises DNA comprising a biomarker indicative of a patient with altered metabolism, wherein the detection of the presence of one or more biomarkers in one or more drug-metabolizing genes in the reference control verifies that the screening is effective to detect the same one or more biomarkers in one or more drug-metabolizing genes in the sample.

44. A method of personalized medical therapy, comprising: i) performing the method of screening of claim 43 on samples from a target patient population to identify patients with a genetic profile comprising one or more mutations in the CYP3A4 or other gene associated with drug metabolism; and ii) treating patients identified in step i) as possessing a particular genetic profile with a therapy of interest particular to the identified genetic profile.

45. The method of claim 44, wherein the genetic profile is indicative of a patient with altered metabolism.

46. The method of claim 44, wherein the altered metabolism is selected from the group consisting of: poor metabolizer, intermediate metabolizer, extensive metabolizer, and ultra-rapid metabolizer.

47. The method of claim 44, wherein the genetic profile is indicative of the effectiveness of the therapy of interest in the patient.

48. The method of claim 44, wherein the genetic profile is indicative of a patient with a genetic disorder.

49. The method of claim 44, wherein the genetic profile is indicative of a patient who should not be treated with a particular therapy.

50. The method of claim 44, wherein the therapy of interest is used to treat a disease or disorder selected from the group consisting of: cancer, heart disease, neurological disorders, psychiatric disorders, autoimmune disorders, and metabolic disorders.

51. The method of claim 44, wherein the one or more mutations comprises a mutation in CYP3A4 and wherein the therapy of interest comprises administration of at least one statin to the patient.

52. A method for identifying a cell that can be used to generate isolated genomic DNA suitable for use as a reference control, wherein the method comprises: a) prospectively screening a human volunteer for the presence of a human genomic DNA sequence comprising one or more polymorphisms in a nucleic acid sample of the volunteer associated with a genetic predisposition that determines a patient's predicted degree of response to a particular therapy; wherein the polymorphism is one or more of: i) CYP3A4-associated SNP (rs35599367 [SEQ ID NO: 152]) located in intron 6 (OT); or, ii) a SNP in linkage disequilibrium therewith, wherein the allelic status of the polymorphism in the subject is predictive of the subject's risk for having or developing the CYP3A4-related disorder; b) isolating the cell from the volunteer that possesses the one or more mutations of interest; and c) subjecting DNA from the cell to a plurality of validated genomic screening assays.