Drug Metabolism and Disposition Fast Forward
First published on May 5, 2006; DOI: 10.1124/dmd.106.009548
0090-9556/06/3408-1411-1416$20.00
DMD 34:1411-1416, 2006
NEW CYTOCHROME P450 2D6*56 ALLELE IDENTIFIED BY GENOTYPE/PHENOTYPE ANALYSIS OF CRYOPRESERVED HUMAN HEPATOCYTES
Li Li,
Run-Mei Pan,
Todd D. Porter,
Neil S. Jensen,
Paul Silber,
Guy Russo,
John A. Tine,
John Heim,
Barbara Ring, and
Peter J. Wedlund
The College of Pharmacy, Departments of Pharmaceutical Sciences, University of Kentucky, Lexington, Kentucky (L.L., R.-M.P., T.D.P., P.J.W.); In Vitro Technologies, Inc., Baltimore, Maryland (N.S.J., P.S.); Center for Functional Genomics, University at Albany, Rensselaer, New York (G.R., J.A.T.); and Eli Lilly and Company, Indianapolis, Indiana (J.H., B.R.)
(Received January 31, 2006;
accepted April 27, 2006)
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Abstract
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Genotype/phenotype analysis with human hepatocytes has identified a new inactive CYP2D6 allele, CYP2D6*56. Cryopreserved human hepatocytes from 51 livers were evaluated for CYP2D6 activity with dextromethorphan as the probe substrate. Hepatocyte lots that lacked CYP2D6 activity were further evaluated for CYP2D6 expression and known genetic variations, including CYP2D6*2, *3, *4, *5, *6, *7, *8, *9, *10, *11, *14, *15, *17, *18, *19, *20, *25, *26, *29, *30, *35, *40, *41, *43, and various multiple copy CYP2D6 alleles (*1xn, *2xn, and *4xn) by the AmpliChip CYP450 prototype microarray (Roche Molecular Systems, Inc., Branchburg, NJ). Two discrepancies were uncovered between the CYP2D6 genotype and activity by this approach. In one sample, a previously unreported 3201C 224 T transition in exon 7 resulted in Arg344(CGA) being replaced by a stop codon (TGA), resulting in a CYP2D6 enzyme lacking the terminal 153 amino acids. This allele was given the designation of CYP2D6*56 and the GenBank accession number DQ282162
[GenBank]
. The lack of CYP2D6 activity in cryopreserved hepatocytes and microsomes found in the second sample, despite a normal level of CYP2D6 expression and a genotype (*10/*1) predictive of normal CYP2D6 activity, was attributed to enzyme inactivation by an unknown metabolite. The identification and characterization of the CYP2D6*56 allele indicates that commercial cryopreserved human hepatocytes may provide a valuable means to rapidly identify genetic variations with functional relevance. This integrated approach of identifying alleles and examining allele relationships to gene expression and function could be of tremendous value to understanding the mechanism responsible for functional differences in gene variation. The commercial availability of human cryopreserved hepatocytes also makes this potential readily available to any who are interested in it, not just those with access to private liver banks.
There have been several methods by which new gene variations have typically been discovered in human drug metabolism enzymes. DNA repositories have been sequenced for variations in a gene and from these sequences new variations in genes have been identified (Chevalier et al., 2001
; Kiyotani et al., 2002; Solus et al., 2004; Soyama et al., 2005). Although effective in identifying new gene variations, many gene sequences must be evaluated that have no functional variations, and some important variations are probably missed because the entire gene and regulatory region is seldom sequenced, particularly for very large genes. Furthermore, unless the new gene variations that are discovered produce an obvious effect on gene expression, the importance of the new gene variation is often unclear. Even the linkage between one gene variation and another variation in a different part of a gene (allele haplotype) is often unknown (Fernandes-Salguero et al., 1995; Marez et al., 1997; Chevalier et al., 2001; Solus et al., 2004). A second approach has identified new alleles by evaluating a specific gene in individuals who exhibit an obvious functional difference in a gene product (de Morais et al., 1994; Oscarson et al., 1997; Ferguson et al., 1998; Ibeanu et al., 1999; Dickmann et al., 2001; Kidd et al., 2001; Pitarque et al., 2001; Oscarson et al., 2002; Gaedigk et al., 2003; Gaedigk et al., 2005). This is a far more directed approach. The focus resides with evaluating only genes from individuals who have already been defined as very different from the rest of the population. A specific phenotypic trait is directly associated with a specific allele. However, the discovery of these alleles typically requires administering a drug or other substance to humans and assessing how a specific phenotype affects its disposition. The phenotype depends on the use of probes that are safe to administer to humans and that are substantially affected by the allele that produces the altered function. The discovery of these alleles seldom allows one to address the mechanism by which a functional change is produced (altered gene product, level of gene expression, mRNA level, etc.). Those issues must often be addressed by in vitro methods (Sullivan-Klose et al., 1996; Ariyoshi et al., 2001; Pitarque et al., 2001; Ramamoorthy et al., 2001) that may or may not provide a totally satisfactory answer about the mechanism. Fewer efforts have been directed toward using human liver tissue to identify dysfunctional gene variations (Marez et al., 1997; Hustert et al., 2001; Haberl et al., 2005), and when such tissue has been used, it has come from liver banks not generally accessible to the average investigator. Only recently has the entire process of using stored human liver tissue to evaluate gene expression, function, mRNA, and gene sequence been truly integrated to understand genetic variation in a P450 gene (Haberl et al., 2005). Although other investigators have suggested that human hepatocytes might be useful for examining CYP2D6 genetic polymorphisms (Komura and Iwaki, 2005), their actual application to identify new CYP2D6 alleles and the molecular basis for their functional effects has yet to be realized or generally appreciated for their broader application to functional genomics research.
Since the completion of the human genome project, the next great challenge rests with the efficient discovery and characterization of alleles in the human genome that are dysfunctional. This discovery and characterization would be facilitated if it were possible to evaluate genes and the mechanism(s) responsible for how or whether a new allele elicited a functional change in the gene product. The current studies were undertaken to determine whether commercially available cryopreserved human hepatocytes could be used in genomic research of metabolic enzymes. Specifically, we wanted to determine whether enzyme activity from human cryopreserved hepatic tissues correlates with genotype information from that same tissue: whether it is possible to relate enzyme level with activity in cryopreserved human hepatocytes, and whether detailed in vitro information about a gene, its expression, and its function could provide a viable approach for more efficient discovery of new gene variations of functional relevance in human metabolic enzymes. Our results indicate that this approach provides a promising means to approach the search for new alleles of a gene expressed in the liver, based on differences in in vitro protein activity and expression as the driver for locating new alleles with a dysfunctional effect.
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Materials and Methods
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Human Hepatocytes. Investigational Review Board approval was obtained from the University of Kentucky for exemption of commercially available cryopreserved human hepatocytes from the need to obtain a consent form before their purchase and testing for gene variations in metabolic enzymes. In Vitro Technologies, Inc. (IVT) (Baltimore, MD) supplied 51 different cryopreserved human hepatocyte lots previously evaluated for CYP2D6 and CYP2A6 enzyme activities with the standard probes (dextromethorphan O-demethylase and coumarin 7-hydroxylation, respectively). Hepatocyte enzyme activities for each hepatocyte lot are available from the IVT web site at http://www.invitrotech.com/characterizationtab.cfm#2. IVT has undertaken an extensive evaluation of their cryopreserved human hepatocytes to confirm that enzyme activities are reproducible and that fresh and cryopreserved human hepatocytes (n = 30) are strongly correlated (slope of approximately 1.0) (Pham et al., 2000). This has eliminated the need to perform these assessments by individual laboratories.
Chemical Supplies. Midazolam, propranolol, and NADPH were purchased from Sigma Chemical Co. (St. Louis, MO). Bufuralol was obtained from Ultrafine Chemicals (Isle of Palms, SC). Deuterium labeled 1'OH midazolam was synthesized by Eli Lilly Company.
Human Microsomes. Cryopreserved human hepatocytes (5 x 106 cells) were thawed in 37°C water bath and centrifuged at 500g for 5 min. The resulting pellets were re-suspended in 1 ml ice-cold buffer A (100 mM Tris-HCl; pH 7.5, 0.5 mM EDTA) and homogenized with homogenizer.
The cell debris was removed at 35,000g (30 min) and microsomes prepared from the 105,000g fraction before suspending in 100 µl buffer A. Protein concentration was measured by a Coomassie protein assay and the microsomes frozen. Microsomes from one hepatocyte lot (PFM) were further evaluated for CYP2D6 and CYP3A4 activity.
Bufuralol and Midazolam Assays. Human microsomal formation of 1'OH bufuralol and 1'OH midazolam from the PFM hepatocyte lot was compared with these same activities in three separate microsome preparations from different human livers purchased from Xenotech (Kansas City, KS). Enzyme activities were compared at Vmax concentrations for bufuralol (100 µM) and midazolam (50 µM) at 0.1 or 0.05 mg protein/ml, respectively. Microsomes were preincubated at 37°C for 3 min before initiation of the reaction with 1 mM NADPH. After a 30-min bufuralol incubation and a 1-min midazolam incubation (linear conditions for both metabolites with protein and time), the reaction was stopped with 1 ml of methanol. Samples were mixed and centrifuged and a 30-µl volume was mixed with either 170 µl of deuterated 1'OH midazolam in 25% methanol (1'OH midazolam assay) or propranolol (1'OH bufuralol assay) in water. 1'OH Bufurolol and 1'OH midazolam were assayed by liquid chromatography/mass spectrometry (Sciex API, San Francisco, CA). 1'OH Bufuralol was assayed using an ODS column and a gradient mobile phase containing mobile phase A (5 mM ammonium acetate in 95% water/5% methanol) and mobile phase B (5 mM ammonium acetate in 95% methanol/5% water). The two mobile phases were mixed in proportions of 90% A/10% B (0–0.25 min hold), and then a gradient from 10% B to 80% B from 0.25 to 1.5 min at a flow rate of 0.22 ml/min. Mass spectrum monitoring was carried out for 1'OH bufuralol at m/z 278/186 and propranolol at m/z 260/116.2. 1'OH Midazolam was assayed with an ODS column and a gradient mobile phase consisting of mobile phase A (50 mM ammonium acetate in 95% water/5% methanol) and mobile phase B (50 mM ammonium acetate in 95% methanol/5% water). The two mobile phases were mixed in proportions of 35% A/65% B (0–0.4 min hold, and then a gradient from 65% B to 95% B from 0.4 to 3.0 min at a flow rate of 0.22 ml/min). Mass spectrum monitoring was carried out for 1'OH midazolam at m/z 342/324 and for its internal standard at m/z 347/329.
DNA Isolation and Genetic Testing. Human cryopreserved hepatocyte lots were rapidly thawed at 37°C and immediately diluted into 49 ml of thawing buffer (InVitroGRO HT medium; In Vitro Technologies, Inc.). The thawed hepatocytes were centrifuged at 2000 to 3000g and the thawing buffer was removed. The hepatocyte cellular pellet was immediately processed and DNA was isolated from the thawed hepatocytes with a QIAGEN (Valencia, CA) DNA mini kit for tissue DNA extraction according to the manufacturer's instructions. Typically, 150 to 200 µl was recovered from a single vial of human hepatocytes (5000–15,000 DNA copies per µl). The isolated genomic DNA was initially characterized for CYP2D6 alleles *1xn, *2xn, *4xn, *2, *3, *4, *5, *6, *7, *9, *10,*17, *30, *35,*40, *41, and *43 according to published methods (Lovlie et al., 1996; Chou et al., 2003; Cai et al., 2006) and the AmpliChip CYP450 prototype microarray (Roche Molecular Systems, Inc., Branchburg, NJ). For comparative purposes CYP2A6*2,*3,*4,*5, *9, and *12 alleles were also assessed in these samples (Fernandes-Salguero et al., 1995; Rao et al., 2000; Paschke et al., 2001; Goods and Tynsdale, 2002; Oscarson et al., 2002). Two samples (WWM and PFM) were further evaluated by the CYP450 AmpliChip prototype microarray for an extended group of additional CYP2D6 alleles (*8, *11, *14, *15, *18, *19, *20, *25, *26, *29) when CYP2D6 activity and genotype did not match.
Immunoassays. Additional hepatocytes were obtained from In Vitro Technologies, Inc. for purposes of qualitatively assessing cytochrome P450 enzyme expression. Supersomes expressing either cytochrome P450 2D6 or CYP2A6 enzyme were used as a reference, and immunoassay kits for each enzyme (Western immunoblotting kits) were purchased from BD Bioscience (San Jose, CA). Hepatocytes were used to make a microsomal pellet as described before and assayed by the Coomassie method for protein amounts (Loffler and Kunze, 1989). Twenty-five micrograms of microsomal protein was fractionated by electrophoresis on a 12% SDS-polyacrylamide gel for 2 h at 120 V. Proteins were electroblotted to nitrocellulose, and the nitrocellulose was blocked with 2% powdered nonfat milk and probed by immunospecific antibodies for cytochrome P450 2D6 or CYP2A6 according to the manufacturer's instructions.
Cloning, Isolation, and Sequencing. A 6-kilobase fragment containing the complete CYP2D6 gene was amplified from genomic DNA by long-range PCR with the GeneAmp XL PCR kit (Applied Biosystems, Branchburg, NJ) and the primers CYP2D6F (5'-AGCTTTGTCGACGAATTCAAGACCAGCCTGGACAACTTGG) and CYP2D6R (5'-AAAACGCGGCCGCTCAGCCTCAACGTACCCCTGTCTCAAATG). The reactions consisted of 100 ng of genomic DNA template, a 200 nM concentration of each primer, and reaction buffer containing 0.9 mM Mg(OAc)2, 200 µM each deoxynucleoside-5'-triphosphate, and 2 U of rTth DNA Polymerase, XL. The cycling parameters for these reactions consisted of 1 cycle at 94°C for 1 min, followed by 16 cycles of 94°C for 0.5 min, and 68°C for 6.5 min, followed by 14 cycles of 94°C for 0.5 min and 68°C for 6.5 min, with 15 s added to each subsequent 68°C extension step. Amplified fragments were resolved on preparative 1% agarose gels, and the 6.0-kilobase CYP2D6 amplicon was excised and purified by use of the QIAEX II protocol (QIAGEN) according to manufacturer's instructions. The purified amplicon was subsequently cloned into the pCR4BluntTOPO vector (Invitrogen, Carlsbad CA) and transformed into chemically competent TOP10 cells (Invitrogen).
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FIG. 1. Histogram of 51 human hepatocyte lots based on their CYP2D6 activity and divided into three groups based on the predicted activity from each lot of CYP2D6 genotype. Cross-hatched bars are hepatocytes with no functional genes for CYP2D6 (*3/*4 or *4/*4) genotype (far left bar in figure). Gray bars represent hepatocytes with at least one inactive gene (*4/*1, *4/*2, *3/*1, etc.) or two alleles with diminished activity (*41/*10, *10/*10), and the remaining stippled black bars represent hepatocytes with two functional CYP2D6 alleles (*1,*2, *35) or one functional and one allele with only somewhat diminished activity (*1/*10; *2/*41, *35/*9, etc.). Where predicted and observed enzyme activity did not match, they were marked by an asterisk. The CYP2D6*1 allele is the default wild-type allele that is assumed to be present when tests for all other alleles are negative.
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Clones were screened by evaluating each CYP2D6 clone at position 100 in exon 1 by PCR-restriction fragment length polymorphism analysis. PCRs were performed with 20 ng of plasmid DNA template and the primers CYP2D610F (5'-GTGTGTCCAGAGGAGCCCAT) and CYP2D610R (5'-TCTCAGCCTGGCTTCTGGTC), resulting in the amplification of a 310-bp fragment. The reactions included 1.8 mM MgCl, 200 µM each deoxynucleoside-5'-triphosphate, and 0.75 U of AmpliTaq Gold (Applied Biosystems). The cycling parameters for these reactions consisted of 1 cycle at 95°C for 5 min, 30 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s, and a final cycle at 72°C for 10 min. Aliquots of the PCR products were digested with HphI (5 U; New England Biolabs, Beverly, MA), and the digestions were analyzed on a 2% Metaphor gel (Cambrex Bioscience, Rockland, ME). Clones containing the wild-type C at position 100 (*2 allele, WWM; or *1 allele, PFM) led to cleavage of the 310-bp fragment into subfragments of 230 and 80 bp, whereas clones with a T at position 100 (*4 allele, WWM; or *10 allele, PFM) were not cleaved.
DNA sequence analysis of 100°C clones (*2 allele, WWM; and *1 allele, PFM) were performed by cycle sequencing with BigDye v3.0 (Applied Biosystems) chemistry and custom oligonucleotide primers. Reactions were analyzed on an ABI Prism 3700 DNA Analyzer (Applied Biosystems), and data were assembled with the Sequencer analysis software. Sequencing was carried out in the forward and reverse directions, and each SNP was reevaluated to confirm the accuracy of the final sequence.
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Results
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The distribution of hepatocyte CYP2D6 activity exhibited a bimodal distribution, with 12% of the hepatocyte lots exhibiting virtually no CYP2D6 activity (<2 pmol/min/106 cells; log (CYP2D6 activity) <0.3), whereas the remaining 88% of hepatocytes expressed a dextromethorphan O-demethylase activity that ranged from 5 to 47 pmol/min/106 cells (18 ± 9 pmol/min/106 cells; log (CYP2D6 activity = 1.3); Fig. 1). The majority of the hepatocyte lots (four of six; 67%) without significant CYP2D6 activity were identified by genetic tests to contain two nonfunctional CYP2D6 alleles (*4/*4 and *3/*4; Fig. 1, cross-hatched bars). However, an extensive CYP2D6 genotype analysis could not explain why the remaining two lots from two Caucasian samples [PFM (*10/*1; Fig. 1, stippled black bar); and WWM (*4/*2; Fig. 1, gray bar)] had no CYP2D6 activity. Immunoblots indicated that the PFM (*10/*1) hepatocyte lot expressed significant quantities of CYP2D6 enzyme, but efforts to measure CYP2D6 activity showed a consistent lack of dextromethorphan metabolism in these hepatocytes. Microsomal measurements from this lot showed significant CYP3A4/A5 activity (1'OH midazolam, 591 pmol/min/mg protein), but no measurable 1'OH bufuralol formation, a specific indicator of CYP2D6 activity (<5.8 pmol/min/mg protein, limit of assay quantification). No CYP2D6 activity was detected in hepatocyte lot WWM (*4/*2) (Fig. 2A), although significant quantities and activity of CYP2A6 were observed in this lot and the PFM lot (Fig. 2B). The initial classification of the WWM hepatocyte lot as expressing a CYP2D6*2 allele was based on the SNP pattern in this allele being typical of a CYP2D6*2 allele (–1584G, –1235G, –740T, –678A; CYP2D7 gene conversion in intron 1: 1661C, 2850T, 3384C, 3584A, 3790T, 4180C). However, allele cloning and sequencing on separate occasions confirmed an additional point mutation at position 3201 in exon 7 that changed 3201C 224 T and the CGA(Arg344) 224 TGA(Stop) codon (Fig. 3), explaining the lack of CYP2D6 enzyme expression in Fig. 2A for this hepatocyte lot. The PFM *1 allele had the typical profile expected for a *1 allele (–1235A, 310G, 746C, 843T, 1661G, 3384A, 4180G), and no other variations in any exons or at intron-exon borders that would account for the complete lack of CYP2D6 enzyme activity in this lot. The only questionable variation was a G deletion in about the middle of intron 7, a variation unlikely to affect CYP2D6 expression or activity since it fell in the run GGGTGGGGGGT in intron 7.
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FIG. 2. A, immunoblot of CYP2D6 levels from hepatocyte lots with no CYP2D6 activity [the asterisked sample; CYP2D6*10/*1; and WWM, CYP2D6*4/*2) indicate that PFM has a normal level of CYP2D6, whereas WWM does not express this enzyme. These results suggest that the allele in sample WWM is not a functional CYP2D6*2 allele. B, results from the analysis of CYP2A6 expression, activity, and genotype. The immunoblot shows that the lot WWM is not devoid of all P450 protein, but has normal amounts of CYP2A6. Samples marked EVY and RML would be candidates for further study since their CYP2A6 genotypes, enzyme activities, and immunodetectable protein levels suggest that there is one or more null alleles present in these samples. Samples deficient in one P450 enzyme were not deficient in other P450 enzymes, suggesting that low enzyme expression is not a general phenomenon in these hepatocyte lots.
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The majority of hepatocyte lots exhibited significant overlap between the CYP2D6 genotype and predicted CYP2D6 activity (Fig. 1; *3/*1, *4/*1, *3/*2, *4/*2, *10/*10, *41/*10, gray bars; moderate activity; and *1/*1, *1/*2, *1/*35, *2xn/*1, *2/*2, stippled black bars; normal to high activity). However, moderate CYP2D6 activity genotypes expressed a lower average CYP2D6 activity [Fig. 2B, gray bars; mean = 13 ± 7 pmol/min/106 cells; log (CYP2D6 activity) = 1.1] relative to the hepatocyte lots with genotypes predicting a higher rate of CYP2D6 activity [Fig. 1, stippled black bars; mean = 22 ± 10 pmol/min/106 cells; log (CYP2D6 activity) = 1.3; p < 0.01 by Wilcoxan rank sum test]. The association between genotype predicted and quantified CYP2D6 activity makes outliers in a group of hepatocyte lots (like the PFM and WWM) immediately apparent (Fig. 1, samples marked with an asterisk). A similar bimodal distribution was seen for other P450 enzyme activities (CYP1A2, CYP2A6, CYP2C9; data are not presented, but it is easily obtained from the IVT website, which lists enzyme activities for various P450 enzymes in their banks).
The distribution of CYP2A6 activity also exhibited a bimodal shape, with 12% of the hepatocyte lots exhibiting little CYP2A6 activity (<5 pmol/min/106 cells; log (CYP2A6 activity <0.7) and the remaining 88% of hepatocyte lots expressing a coumarin 7-hydroxylase activity ranging from 9 to 135 pmol/min/106 cells [53 ± 32 pmol/min/106 cells; log (CYP2A6 activity) = 1.7]. The lots exhibiting low CYP2A6 activity were not the same lots that expressed low CYP2D6 activity. Only two of six (33%) of the low CYP2A6 activity hepatocyte lots could be explained based on the CYP2A6 alleles tested (*1/*5 and *9/*9). This finding suggests that additional undefined CYP2A6 alleles may be present in the remaining four hepatocyte lots with low CYP2A6 activity. In the case of two lots (EVY and RML), the low CYP2A6 activity was also associated with virtually no detectable CYP2A6 expression (Fig. 2B). This indicates that the default allele designation of CYP2A6*1 is incorrect. The CYP2A6 evaluations were done to provide evidence that differences in hepatocyte lot activity did not represent a general depression of all P450 enzyme function, and to show that this approach need not be limited to just CYP2D6 activity and genotype assessments (a single gene product).
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Discussion
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The identification of a new CYP2D6 allele and its functional relevance was greatly facilitated by the ability to screen hepatocytes for enzyme activity, enzyme expression, and the known dysfunctional alleles in each lot. The new CYP2D6 allele (in lot WWM) is expected to be a rare allele, since it has not been previously reported in GenBank, the CYP450 website (Ingleman-Sundberg et al., 2005), or the SNP database, and despite several large efforts aimed at sequencing CYP2D6 exons, this specific variation has never previously been observed. All known inactive CYP2D6 alleles related to the CYP2D6*2A allele reported to date (*11, *12, *19, *20, *21) are rare (<0.1% in the population) and race-specific. However, it will be desirable to use banked human DNA to establish the frequency of newly identified dysfunctional alleles after these are identified by such in vitro methodologies. We opted not to do that in this instance because of the anticipated low frequency for the occurrence of this new allele.
The inability to detect CYP2D6 activity in hepatocyte lot PFM, which contained a normal CYP2D6 allele, is more difficult to explain. It is hypothesized that the lack of activity in this lot resulted from selective inactivation of the CYP2D6 enzyme. This hepatocyte lot came from a 57-year-old Caucasian female patient with multiple medical problems (heart valve replacement, hysterectomy, type II diabetes, and hypertension). Her medical information also indicates that she did not smoke, drink, or use illicit drugs. The likelihood is that this patient was being treated with a variety of drugs for her multiple medical problems, and one of these medications was biotransformed into a selective and reactive metabolite that irreversibly bound to the CYP2D6 enzyme and inactivated it. This would account for the relatively normal levels of CYP2D6 enzyme or even slightly elevated levels, but a CYP2D6 activity that was absent based on separate hepatocyte (n = 2) and microsomal (n = 3) assessments. Normal or even elevated levels of CYP3A enzyme after treatment of humans with drugs that irreversibly inactivate the CYP3A enzyme were reported decades ago (Larrey et al., 1983; Watkins et al., 1985). This would also explain why other P450 enzymes (CYP2A6 and CYP3A4) had fairly normal activity. More detailed assessments of the CYP2D6 enzyme from this hepatocyte lot to uncover the specific mechanism for this isozyme-specific inactivation were beyond the scope of this research. It was also unlikely that mRNA levels would have been useful in understanding the low CYP2D6 activity in sample PFM, since alternative splicing or diminished CYP2D6 mRNA would have lowered and not resulted in normal or elevated CYP2D6 protein amounts in this hepatocyte lot. Quantification of mRNA will have the most value when the mechanism responsible for poor gene expression is unclear.
The identification of a rare new CYP2D6 allele illustrates the value of this approach to functional genomics. The ability to compare and relate enzyme activities from cryopreserved human hepatocytes with hepatocyte genotype and level of enzyme expression provides a simple means to quickly identify samples with gene variations producing dysfunctional effects. In 100% of hepatocyte lots without measurable CYP2D6 enzyme expression or activity, the effect could be predicted by alleles preventing enzyme expression. In one hepatocyte lot (PFM), where enzyme expression and activity were not related, the absence of CYP2D6 enzyme activity could be hypothesized to result from concurrent prescribing of multiple medications. In general, hepatocyte lots with diminished CYP2D6 enzyme activity had genotypes that were predictive of less enzyme expression, whereas hepatocyte lots with the most CYP2D6 enzyme activity had genotypes indicative of normal or elevated enzyme levels. When searching for the proverbial "needle in a haystack" (i.e., rare or unrecognized alleles with functional relevance), the search is facilitated tremendously by using an approach that allows one to readily identify phenotypic enzyme variation while simultaneously addressing the mechanism responsible for that variation. Therein are the two major advantages of this in vitro method over many other approaches. It is the greater information obtained and the broader application of the method that embodies the main attributes of this particular methodological approach. The focus and relevance do not reside with "what was found" but rather "how it was found".
It was decided not to quantify mRNA in this study to address the mechanism for either of the two hepatocyte lots without CYP2D6 activity (WWM and PFM). However, quantification of a specific mRNA could be done to help further elucidate mechanisms responsible for differences in allele expression when interpretation of the precise genetic change or basis for the differences in expression are unclear (McConnachie et al., 2004). This is typically difficult or impossible to address with other approaches used today in genetics. It is this complete range of issues to which human hepatocytes lend themselves (testing for gene variations, mRNA, protein level, and function) that makes cryopreserved human hepatocytes a promising resource for future research aimed at identifying and defining genetic variation(s) producing functional changes. Even today, nearly half of all P450 alleles are of undefined functional relevance (Ingelman-Sundberg et al., 2005) because the means for defining their functional relevance has not been possible or is extremely laborious. As efforts to understand the relevance of genetic variation in the human genome increases, more efficient methods to evaluate and address alleles with functional relevance are essential. It is hoped that this work serves as a stimulus for that future research.
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Conclusions
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Human hepatocytes are a potentially valuable resource for genomic research. Considerable research in the last four decades has already gone into testing and evaluating methods to most optimally cryopreserve human hepatic tissue. This commercial tissue should now be seriously considered for its potential to identify and characterize dysfunctional alleles. Integrating genomics, proteomics, proteomic function, and gene expression profiling, together with carefully cryopreserved human tissue, may provide the critical information needed to identify the presence of new alleles and the likely mechanism by which such alleles affect gene expression. Linking alleles with their functional effects remains critical to the eventual clinical application of genomic information.
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Acknowledgments
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We acknowledge Drs. Walter Koch and Maureen Fairchild at Roche Molecular Systems, Inc. (Branchburg, NJ) for testing some of the DNA samples with their AmpliChip CYP450 prototype microarray for CYP2D6 alleles.
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Footnotes
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This research was supported by discretionary funds of the investigators, by support of the Department of Pharmaceutical Sciences, and by the generous help and support of IVT, which provided all the human hepatocyte lots used for this work.
Presented in abstract form at the American Association of Pharmaceutical Scientists Meeting in Baltimore, MD, November 8–10, 2004.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.106.009548.
ABBREVIATIONS: IVT, In Vitro Technologies, Inc.; PCR, polymerase chain reaction; bp, base pair(s); SNP, single-nucleotide polymorphism.
Address correspondence to: Dr. Peter J. Wedlund, College of Pharmacy, 745 Rose Street, University of Kentucky, Lexington, KY 40536-0082. pjwedl1{at}email.uky.edu
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Abstract
Materials and Methods
T changes the codon CGA (Arg344)