technologies for detecting genetic polymorphisms in pharmacogenomics

Molecular Diagnosis Vol. 4 No. 4 1999

Technologies for Detecting Genetic Polymorphisms in Pharmacogenomics

M I C H A E L M. SHI, MD, PhD, M I C H A E L R. B L E A V I N S , PhD,

F E L I X A. de la I G L E S I A , M D

A n n Arbor, Michigan

Background: Pharmacogenomics is an emerging scientific discipline examining the genetic basis for individual variations in response to therapeutics. Methods and Results: Genetic polymorphisms are a major cause of individual differences in drug response. Metabolic phenotyping can be accomplished by administrating a probe drug or substrate and measuring the metabolites and clinical outcomes. However, this approach tends to be labor intensive and requires repeated sample collection from the individual being tested. Alternatively, genotyping allows determination of individual DNA sequence differences for a particular trait. Commonly used genotyping methods include gel electrophoresis-based techniques, such as polymerase chain reaction (PCR) coupled with restriction fragment length polymorphism analysis, multiplex PCR, and allele-specific amplification. Fluorescent dye-based high-throughput genotyping procedures are increasing in popularity, including oligonucleotide ligation assay, direct heterozygote sequencing, and TaqMan (Perkin Elmer, Foster City, CA) allelic discrimination. High-density chip array and mass spectrometry technologies are the newest advances in the genotyping field, but their wide application is yet to be developed. Novel mutations/polymorphisms also can be identified by conformation-based mutation screening and direct high-throughput heterozygote sequencing. Conclusions: Rapid and accurate detection of genetic polymorphisms has great potential for application to drug development, animal toxicity studies, improve- ment of human clinical trials, and postmarket monitoring surveillance for drug efficacy and toxicity. Key words: pharmacogenomics, genotyping, phenotyping, polymorphisms.

Pharmacogenomics is an emerging scientific discipline that examines the genetic basis for individual variations in response to therapeutic agents or biotechno-

From the Genomic Pathology Laboratory, Pathology and Experimental Toxicology, Parke-Davis Pharmaceutical Research, Warner-Lambert Company; and the Department of Pathology, The University of Michigan Medical School, Ann Arbor, Michigan.

Reprint requests: Michael M. Shi, MD, PhD, Pathology and Experimental Toxicology, Parke-Davis Pharmaceutical Re- search, Warner-Lambert Company, 2800 Plymouth Road, Ann Arbor, MI 48105. Email: [email protected]

Copyright © 1999 by Churchill Livingstone ® 1084-8592/99/0404-0011 $10.00/0

logical interventions [1].Variations can result from genetic or environmental factors, such as age, sex, body size, diet, alcohol or tobacco consumption, pregnancy, kidney or liver dysfunction, concurrent disease states, and drug interactions [2]. Interindividual variations also result from genetically determined differences in absorption, distribution, metabolism, and excretion of therapeutic agents. These genetic factors can alter the effects of a given dose, leading to a spectrum of responses ranging from clinical benefits to adverse effects and therapeutic failures. Recent research shows that certain genetic polymorphisms cause significantly different pharmacological and toxicological effects among individuals on exposure to a particular drug [3]. Most genetic varia-

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344 Molecular Diagnosis Vol. 4 No. 4 December 1999

tions do not affect protein function and therefore have no phenotypic expression. However, other polymorphic genes encode proteins with dysfunctional or non- functional activities in comparison with normal function (wild-type). Thus, determination of these genetic polymorphisms may be of significant value in predict- ing drug efficacy or toxicity in a variety of therapeutic scenarios.

Genetic polymorphisms are usually determined by phenotyping or genotyping. Phenotypes constitute ob- servable characteristics of a cell or organism, usually monitored by direct observations or biochemical or functional analyses. In pharmacogenomics, phenotypes can be monitored at additional levels, such as low or ex- aggerated pharmacological effects, frequency of side effects, and metabolic rate for certain drugs. In contrast, genotyping determines individual DNA sequence differences for particular traits independently of their functional effects. This approach is increasingly being used in biomedical research and clinical molecular diagnostics to replace traditional serological testing to some extent.

Establishing the genotypic and phenotypic correla- tion through clinical pharmacogenomics will allow strat- ification of patient subpopulations according to different drug responses. These data offer the potential for wider and safer drug use based on individual genetic makeup. When applied to drug discovery and development, pharmacogenomics also may help the development of therapeutic interventions targeting the specific responder patient groups. Advanced technologies to identify genetic polymorphisms rapidly, accurately, and economically are becoming a priority in the imple- mentation of pharmacogenomics to drug development, clinical trials, and clinical monitoring for drug efficacy and toxicity.

Phenotyping in Pharmacogenomics

Genetic variations contribute to interpersonal differences in drug response at both the pharmacokinetic and pharmacodynamic levels. Over the past 2 decades, many genetic polymorphisms in drug-metabolizing enzymes have been described [3]. When these genetic variations significantly affect the enzyme's function, different clinical outcomes can occur among people exposed to the same drug. Phenotyping for these drug-metabolizing enzymes can be divided into functional phenotyping or metabolic phenotyping. Functional phenotyping is the direct measurement of the properties of a biomarker specific to a system, organ, or tissue, such as enzyme activities and drug-receptor interactions. Although di- rectly reflecting a protein's properties, functional phenotyping is usually hampered by tissue availability because

of the necessary invasive procedures for tissue sampling, such as liver biopsy.

In some cases, it may be possible to use surrogate tis- sues or peripheral blood for functional analysis. Sulfa- tion and glucuronidation are two major pathways for the metabolism of many drugs and are catalyzed by the liver phase II enzymes, sulfotransferases (SULTs), and uridine diphosphate glucuronosyltransferases (UGTs), re- spectively. Significant interindividual variations have been reported for both enzymes [4]. Deficiencies in UGT activity are responsible for the bilirubin level elevations seen in Crigler-Najjar type I and II [5] and Gilbert syn- dromes, which affect approximately 2% to 9% of the population [6]. Gilbert syndrome is a benign form of unconj ugated hyperbilirubinemia in the absence of liver disease or overt hemolysis. Patients with Gilbert syndrome have inherited abnormalities of UGT1A1 activity and, as a result, a defect in bilirubin glucuronide for- mation. This genetic impairment results in enhanced drug toxicities. For example, patients with Gilbert syndrome are predisposed to severe diarrhea and hemato- logic toxicities caused by the chemotherapeutic agent irinotecan because of defective UGTIA1 activity [7]. Irino- tecan is metabolized to its active metabolite, SN38, conju- gated by UGT1A1 to form SN38-glucuronide, and then se- creted into the bile. Gilbert syndrome often is undiagnosed because the enzymatic assays usually require liver biopsy specimens to measure the enzyme activity. Recent reports have shown that the enzyme activities of both SULT1A1 and UGT1A1 can be analyzed using circulating platelets [8,9]. Platelets are a readily accessible source of both enzymes, and the enzymatic activities of platelet UGTs have been reported to correlate with the activity found in the liver [9,10]. Functional phenotyping also has some limitation when enzymatic activities overlap substrate specificity. Although UGTIA1 is the primary isoform responsible for the bilirubin conjugation, other isoforms, such as UGT1A4, also are capable of catalyzing this reaction [11]. It therefore is difficult to specifically identify the protein subtype responsible for a particular phenotype or pathway using only functional phenotyping.

In general, a significant challenge resides in the identification of surrogate markers for specific phenotyping, such as drug-metabolizing enzymes. More commonly, phenotyping is accomplished by administrating a probe drug and measuring metabolites in plasma or urine (metabolic phenotyping [12,13]). To date, cytochrome P-4502D6 (CYP2D6) is an extensively characterized drug-metabolizing enzyme. This microsomal isozyme is responsible for the oxidative metabolism of approximately 50 clinically relevant drugs from multiple therapeutic classes. These substrates include antiarrhythmics, tricyclic antidepressants, beta-adrenergic blockers, neu- roleptics, and other classes of drugs frequently having narrow therapeutic windows. For these drugs, slightly

less than targeted plasma concentrations cannot have the desired therapeutic effect, whereas slightly greater concentrations can cause toxicity. CYP2D6 has three clinically distinct phenotypes: the normal (or extensive) metabolizer, slow (or poor) metabolizer, and fast (or ultraextensive) metabolizer. The same dose of a drug metabolized through CYP2D6 will result in plasma concentrations that vary greatly among individuals with these distinct phenotypes. In normal metabolizers, steady-state plasma concentrations are within the expected therapeutic range, and toxic effects are minimal or nonexistent. In fast metabolizers, steady-state drug levels are less than the therapeutic concentration, and efficacy is subtherapeutic. Poor metabolizers have greater- than-expected drug levels and are therefore susceptible to undesired toxicity. Debrisoquine hydroxylation is commonly used for CYP2D6 metabolic phenotyping [14]. For this assay, a dose of debrisoquine is adminis- tered to the subject, and the ratio between the parent drug and its metabolites is monitored in urine. The metabolic ratio is expressed as debrisoquine divided by 4- hydroxydebrisoquine, the major metabolite of the parent drug. If the metabolic ratio is greater than the normal range of 0.5 to 12.6, the individual is categorized as a slow or poor metabolizer. A metabolic ratio less than the ref- erence range indicates an ultraextensive metabolizer. Si- milar approaches are used for metabolic phenotyping of other cytochrome P-450 isoforms. The methods used for CYP1A2, 2C19, 2D6, 2El, and 3A4 are listed in Table 1.

Phenotyping methods were used long before molecular biological tools became available and therefore have been fully validated. However, these metabolic phenotyping procedures involve sophisticated analytic techniques and require significant time for obtaining results. Metabolic results also can be complicated by sample sta- bility, as well as the external factors of age, nutrition, disease status, and concurrent medications. In addition, different substrates of a polymorphic enzyme typically have different specificities, and the phenotype may not accurately represent the disposition of a particular drug substrate. Also, poor metabolizers may experience unpleas- ant side effects from the probe drugs, thus further limiting the clinical application of metabolic phenotyping.

Technologies for Pharmacogenomics • Shi et al. 345

Genotyping in Pharmacogenomics

A variety of genotyping techniques have been developed for identified polymorphisms. Commonly used methods include gel electrophoresis-based techniques, such as polymerase chain reaction (PCR) coupled with restriction fragment length polymorphism (RFLP) analysis, multiplex PCR, and allele-specific amplification. Fluorescent dye-based high-throughput genotyping procedures have gained increased popularity, including the oligonucleotide ligation assay (OLA), heterozygote sequencing, TaqMan (Perkin Elmer, Foster City, CA) allelic discrimination, and high-density chip array technology. Novel polymorphisms also can be identified by single-strand conformation polymorphism (SSCP) and direct heterozygote sequencing. Mass spectrometry (MS) genotyping similarly has been used for both known and unknown polymorphisms.

One significant advantage of genotyping over phenotyping is the less invasive nature of DNA sampling. Buc- cal swabs, mouthwashes, hair roots, and blood spot collec- tions make DNA sampling easier and more acceptable to patients. In contrast to phenotyping, genotyping results are not modulated by drug-drug or drug-food interactions and reliably identify heterozygotes with intermedi- ate phenotypes. This approach is not without limitations because many functionally important allelic variants are not yet identified or linked to phenotypic function. Never- theless, with increasing sequence information becoming available, more and more genetic polymorphisms are being characterized by genotype/phenotype association studies. Simple, reliable, high-throughput, and cost- effective DNA technologies are highly desirable for large-scale genotyping projects and association studies in pharmacogenomics.

Gel-based Genotyping. Gel-based genotyping methods include PCR-RFLP, allele-specific PCR, and multiplex PCR. PCR amplifies specific regions of DNA sequences between two custom-designed oligonucleotide primers, allowing small quantities of DNA containing the polymorphic site to be increased dramatically. These detectable levels of specific DNA then are di-

Table 1. Metabolic Phenotyping of Drug-Metabolizing Enzymes

Enzyme Probe Drug React ion References

CYPIA2 Caffeine 3N-Demethylation 15 CYP2D6 Debrisoquine Hydroxylation 14

Dextromethorphan N-Demethylation 16 CYP2E1 Chlorzoxazone 6-Hydroxylation 17 CYP3A4 Erythromycin 3N-Demethylation 18

Nifedipine Oxidation 19


gested with appropriate restriction enzymes, with vis- ualization by gel staining after electrophoresis. The restriction digestion pat tern differs if the genetic polymorphism results in a gain or loss of the restriction site. Many restriction enzymes exist that cleave at a wide variety of DNA sequences. A PCR-RFLP method to detect the CYP2D6*4 allele (B mutation) is described in Fig. 1. A guanosine (G) to adenosine (A) transition at the junction of the third intron and fourth exon (nucleotide 1934) causes a messenger RNA splic- ing site mutation. This results in no functional CYP2D6 enzyme in homozygous mutant individuals and the slow-metabolizer phenotype [20]. DNA is purified from human peripheral blood samples and subjected to PCR amplification of a 355-bp fragment of the gene spanning the polymorphic site. The wild-type allele PCR product is recognized by the restriction enzyme BstNI, which digests the D N A into 250-bp and 105-bp fragments. The G to A change in the mutant allele ren- ders the restriction site unrecognizable to BstNI, giv-

E < z a £1.

O O

= E

-355 bp -250 bp

-105 bp

Fig. 1. PCR-RFLP for genotyping CYP2D6*4, Forward primer: 5'-CCT TCG CCA ACC ACT CCG-3'; reverse primer: 5'-AAA TCC TGC TCT TCC GAG GC-3'. After PCR and restriction enzyme B.stNI digestion, the PCR products can be resolved by agarose gel electrophoresis and visual- ized under ultraviolet (UV) light after ethidium bromide staining. The presence of DNA bands of 250 and 105 bp indicates a CYP2D6 homozygous wild-type genotype (wt/wt). The presence of a single DNA 355-bp band indicates the absence of this restriction endonuclease site and a CYP2D6*4 homozygous mutant genotype (rout/rout). Subjects with 3 bands of 355, 250, and 105 bp indicate a heterozygous genotype (wt/mut).

ing rise to a single undigested band of 355 bp. Three genotypes (homozygous wild-type, heterozygous, and homozygous mutant) can be resolved by analyzing the digestion patterns after gel electrophoresis.

A significant limitation of the PCR-RFLP method is the requirement that the polymorphisms alter a restriction enzyme cutting site. To detect mutations that do not create or eliminate restriction sites, allele-specific PCR can be performed using mutation-specific amplification.This technique has been applied for identifying major CYP2D6 poor-metabolizer genotypes [21]. In contrast to poor metabolizers, approximately 7% of whites are ultraextensive metabolizers of CYP2D6. These individuals have alleles with duplication of CYP2D6 genes and ex- press excessive amounts of CYP2D6 enzymes. As a result, ultraextensive metabolizers require greater-than- average doses of drugs metabolized by CYP2D6 to reach therapeutic plasma concentrations. This C Y P 2 D 6

variation also can be identified by PCR amplification [22]. Allele-specific amplification methods depend on the nature of the mutation and the surrounding sequences, with guanine cytosine (GC)-rich regions making PCR assays difficult to optimize and multiplex.

Overall, gel-based genotyping assays are relatively straightforward and are useful when dealing with a small number of samples of no more than a few hundred. The methods are labor intensive and require experienced and skilled technical staff for final analysis. These requirements make gel-based techniques difficult to apply for large-scale genotyping in clinical trials and routine testing in diagnostic laboratories.

OLA Genotyping. The PCR-OLA takes advantage of fluorescent detection systems. The three oligonucleotides used in this assay are two allele-specific oligonucleotide probes (one specific for the wild-type allele and the other specific for the mutant allele) plus a fluorescent common probe. The 3'-ends of the allele-specific probes are immediately adjacent to the 5'-end of the common probe. To perform OLA, the gene fragment containing the polymorphic site is amplified by PCR and incubated with the probes. In the presence of thermally stable DNA ligase, ligation of the fluorescent-labeled probe to the allele-specific probe(s) only occurs when there is a perfect match between the mutant or the normal probe and the PCR product template. These ligation products are then separated by electrophoresis on a fluorescent-based DNA sequencer, which permits the recognition of the normal genotypes, mutants, heterozygotes, and unligated probes. By varying the combinations of color dyes and probe lengths, multiple mutations can be detected in a single reaction. This technology is par- ticularly useful for disorders in which there are many mutations causing a single disease, such as cystic fibrosis and familial hypercholesterolemia [23]. An OLA for genotyping CYP2D6 poor metabolizers is reported that

significantly reduces the time and cost associated with high-throughput testing [24].

Similar to allele-specific amplification, OLA relies on hybridization with specific oligonucleotide probes that can effectively discriminate between the wild-type and variant sequences. This method can be used to genotype a large panel of informative biallelic markers. The hybridization of allele-specific oligonucleotides is dependent on both the variant and surrounding sequences. Highly GC-rich DNA regions make the allele-specific ligation step in OLA difficult to optimize and multiplex.

Fluorescence Resonance Energy Transfer Genotyp- ing. Fluorescence resonance energy transfer (FRET) occurs when two fluorescent dyes are in close proximity to each other and one fluorophore's emission spectrum overlaps the other's excitation spectrum [25]. One application using this approach is the TaqMan Allelic Dis- crimination assay, which uses the 5'-nuclease activity of Taq polymerase to detect a fluorescent reporter signal generated during or after PCRs [26]. For genotyping single-nucleotide polymorphisms (SNPs), one pair of TaqMan probes and one pair of PCR primers are used. TaqMan probes typically consist of 20- to 40-bp oligonucleotides complementary to the polymorphic region, with the polymorphic nucleotide located at approximately the middle of the sequences. The assay uses two TaqMan probes that differ at the polymorphic site, with one probe


complementary to the wild-type allele and the other to the variant allele. A 5'-reporter dye (6-carboxy-4,7,2',7'- tetrachlorofluorescenin [TET]) and a 3'-quencher dye (6-carboxy-N,N,N',N'-tetrachlorofluorescein [TAMRA]) are covalently linked to the wild-type allele probe. Sim- ilarly, the variant allele probe is labeled with a 5'- reporter dye (6-carboxyfluorescein [FAM]) and the same 3'-quencher dye, TAMRA. When the probes are intact, fluorescence is quenched because of the physical proximity of the reporter and quencher dyes.

During the PCR annealing step, the TaqMan probes hybridize to the targeted polymorphic site. During the PCR extension phase, the 5'-reporter dye is cleaved by the 5'-nuclease activity of the Taq polymerase, leading to an increase in the reporter's characteristic fluorescence. Specific genotyping is determined by measuring the signal intensity of TET and FAM immediately after the PCR. A high-throughput CYP2D6*4 TaqMan genotyping assay also has been developed (Fig. 2). In addition to detecting SNPs, small gene deletions and insertions can be identified by this method.

FRET genotyping offers high sample throughput and accurate detection of SNPs. This fluorescence-based genotyping approach significantly reduces assay complexity by eliminating restriction enzyme digestion, gel electrophoresis, resolution of PCR products, and visual assessment of bands. In addition, the assay can use 96-well

Fig. 2. TaqMan genotyping for CYP2D6*4. Forward primer 5'-CGC CTI" CGC CAA CCA CT-3' and reverse primer 5'-CTT TGT CCA AGA GAC CGT TGG-3'. Allele-1 (mutant) TaqMan probe TET-CAC CCC CAA GAC GCC CCT TF-TAMRA, allele 2 (wild-type) probe 6FAM-CAC CCC CAG GAC GCC CCT-TAMRA. The CYP2D6 genotypes were determined according to the ratio of FAM and TET signal. Allele-i, A/A (mut/mut, FAM~TET); allele-2, G/G (wt/wt, FAM,,TET); allele-1 and 2, G/A (wt/mut, FAM --~ TET).


plate formats and closed-tube PCRs, minimizing the potential for contamination. The ability to automate data handling further enhances accuracy by eliminating oper- ator bias. However, because FRET (TaqMan) genotyping involves at least several steps to optimize conditions and is difficult to multiplex, it will not be the method of choice for determining multiple markers in a small number of samples. This assay, however, may be very useful for large-scale characterization of the same genetic polymorphism.

Conformation-based Mutation Screening. Mutation detection is a key step for the identification of new polymorphisms. A commonly used strategy for detecting SNPs is to PCR-amplify genes of interest and scan the PCR products for the presence of mutations by confir- mation-based mutation scanning methods, followed by sequencing-positive PCR products. One of the most widely used techniques for identifying unknown mutations is SSCP analysis. SSCP is based on the principle that the 3-dimensional conformation of a single- stranded DNA molecule has a specific sequence-based secondary structure in a nondenaturing gel matrix. SSCP involves three major steps: (1) PCR amplification of the region with potential polymorphisms, (2) forma- tion of single-stranded DNA by denaturing the double- stranded PCR products with formamide and heat, and (3) separation of the single-stranded DNA using a nondenaturing polyacrylamide gel. A fragment with a single-base modification generally forms a different conformer and migrates differently than wild-type DNA, and thus mutants can be easily identified by these mobility difference~ Whereas the original SSCP protocols incorporated radio- active labels for detection [27], fluorescence labeling now has become widely used [28].

SSCP can be used as a first-pass discrimination test for screening large numbers of samples and offers an economic way to detect new polymorphisms. The sensitivity of this method, however, varies for different DNA sequences and is a concern. The sensitivity of SSCP is typically 60% to 95% for fragments less than 250 bp [29]. This sensitivity can be increased to nearly 100% by coupling with either restriction enzyme fingerprinting [30] or dideoxy-sequencing fingerprinting [31]. Al- though these additional components improve sensitivity, they also increase assay complexity and decrease throughput.

Other popular mutation scanning methods include conformation-sensitive gel electrophoresis [32], chemi- cal or enzymatic mismatch cleavage detection [33-35], denaturing gradient gel electrophoresis [36], and denaturing high-performance liquid chromatography [37]. The underlying principle of these methods is that the melting characteristics of double-stranded DNA is largely defined by its sequence. Therefore, a single-base mismatch can produce conformation changes in the double

helix that causes the differential migration of homoduplexes and heteroduplexes containing base mismatches during gel electr0ph0resis. Conformation-sensitive gel electrophoresis has been developed to distinguish homoduplexes from heteroduplexes containing a single mismatched base pair by polyacrylamide gel electrophoresis in a mildly denaturing solvent system. This method has been shown to be highly sensitive to pick up mutations in areas of highly repetitive and GC-rich sequences [38].

Mismatch cleavage detection takes advantage of the sensitivity of mismatched bases to cleavage by enzymes or chemicals. After PCR amplification, the wild-type and mutant products are subjected to denaturation/ renaturation to create heteroduplex molecules. After in- cubation with the resolvases or chemicals, the products are resolved electrophoretically side by side to score the presence of mismatch cleaved molecules [33-35]. The DNA fragment could be up to 1,000 bp, with a sensitivity greater than 90%.

In denaturing gradient gel electrophoresis, genomic DNA are PCR amplified, and the products are then resolved in a polyacrylamide gel with an increasing denaturing gradient of formamide and urea under careful temperature control. Heteroduplex DNA fragments with a single mismatched base pair are shown as migra- tional differences from homoduplexes. The advantages of this method are its accuracy and relatively inexpen- sive cost. The disadvantages are its low throughput and difficulty to optimize [36].

Denaturing high-performance liquid chromatography is another form of heteroduplex analysis for detecting DNA mobility differences by chromatographic analysis in slightly denaturing conditions. Temperature-modulated heteroduplex analysis is a newly commercialized method from Transgenomic, Inc. (San Jose, CA) [39]. The mutant sample is first hybridized with wild-type DNA to form a mixture of homoduplexes and heteroduplexes. The heteroduplexes can be separated from the homoduplexes by column chromatography at a temperature to partially denature the mismatched DNA. The denaturing temperature can be selected for optimal resolution. The procedure is fully automated and could be applied for high-throughput mutation detection.

High-throughput DNA Sequencing. DNA sequencing is the most direct diagnostic method to determine a target gene and remains the gold standard for detecting mutations. In this mode, an individual's sequence can be compared with the wild-type sequence to identify polymorphisms. The major platform used by research and diagnostic laboratories is now fluorescent-based sequencing using multiple fluorochromes. With the im- provement of computer software and detection systems, fluorescent DNA sequencing has become fully automated in all phases of the sequencing process.

The fluorescent DNA sequencer also offers precise


allele sizing and quantitation of DNA fragments. These advances have set the stage for other molecular biological applications based on multicolor fluorescence, including microsatellite polymorphism sizing and tandem repeat genotyping [40], gene mapping [41], detection of loss of heterozygosity [42], and microsatellite instability [43]. The sensitivity and specificity of these fluorescent genotyping methods for detecting heterozygous genotypes are superior to conventional RFLP or allele-specific PCR. These approaches are emerging as vital techniques for disease gene mapping in research and in diagnostic laboratories.

DNA Microarray Chip. The DNA microarray chip is a hybridization-based genotyping technique that offers si- multaneous analysis of many polymorphisms. High- density microarrays are created by attaching hundreds of thousands of oligonucleotides to a solid silicon surface in an ordered array.The DNA sample of interest is PCR amplified to incorporate fluorescent-labeled nucleotides and then hybridized to the chip array. Each oligonucleotide in the high-density array acts as an allele-specific probe. Per- fectly matched sequences hybridize more efficiently to their corresponding oligomers on the array and therefore give stronger fluorescent signals over mismatched probe- target combinations. The hybridization signals are quanti- tated by high-resolution fluorescent scanning and analyzed by computer software. DNA alterations, such as heterozygous base-pair polymorphisms or mutations, insertions, and deletions, can be identified [44]. Cur- rently, the initial start-up and chip costs of the microarray procedures are high relative to other methods. As greater numbers of clinically important polymorphisms are identified, application of microarray chip technology to simultaneously monitor tens of thousands of genetic variations will provide significant advantages for genome-wide screening. The clinical usefulness of these chip-based genotyping technologies is currently under development and evaluation in several companies.

Although simple tandem repeats or microsatellite polymorphisms have been used in linkage analysis, it is becoming increasingly popular to use SNP analysis because SNPs are the most frequent DNA sequence variations found in the human genome. By creating high-density SNP maps, researchers will expand genomic capabilities for identification of critical diseases and drug response genes in nonfamilial studies. Creation of these maps now is feasible using high-throughput DNA sequencing [45] and chip hybridization [46].

MS. MS is an established method in the analytic arena and a new technology for high-throughput genotyping [47-50]. MS yields precise information on the molecular mass of the DNA fragments, the procedure can be fully automated, and both DNA strands can be analyzed in parallel. Large molecules now can be identified by MS through electrospray or matrix-assisted laser desorption

ionization (MALDI) and ion trap or time of flight (TOF) detectors. MALDI-TOF MS has been successfully applied to DNA analysis [49]. The mass determination process usually involves incorporation of internal standards into the MALDI preparation, and the internal standard signals are used to calibrate the mass spectrometer. These methods are more precise and quicker than traditional gel-based analyses [51]. One previous limiting factor for SNP analysis was the mass resolution required for discrim- inating the small-molecular-weight differences between adenosine and thymidine (9Da) in PCR-generated fragments. By generating very short defined fragments of PCR products using restriction endonuclease digestion and direct analysis by electrospray ionization MS, the method has been dramatically improved [51]. By incor- porating the restriction site into the PCR primers, no restriction recognition sites in the targeted DNA sequences are required. The size of the final endonuclease products can be as small as 7 bp and will allow accurate identification of all common DNA variations.

Unlike the fluorescent genotyping methods previously described, MS offers specificity and accuracy without requiring specially labeled probes or primers. This feature reduces start-up reagent costs. The current limitation of MS is the difficulty applying it to ultra-high- throughput genotyping because the current MS instru- ments only handle one sample at a time. Even with multiplex PCR, each MS instrument is capable of analyzing no more than a few thousand markers per day. In addition, MS genotyping requires purified samples free of ions and other impurities, thus increasing the technical time and sample-processing costs.

Conclusions

Pharmacogenomics has the potential to improve our understanding of interindividual variations in drug responses resulting from genetic polymorphisms. A major challenge we are facing today is to compare hundreds of thousands of polymorphisms among numerous individuals.Thus, the success of pharmacogenomics depends on the technologies that can detect polymorphisms rapidly and accurately in a cost-effective and user-friendly way on a large scale. Each of the methods previously dis- cussed has been proven to work in a variety of settings, but each method has its own limitations. Of all the op- tions available, non-gel-based and chip-based genotyping technologies will most likely evolve as the ultra-high- throughput detection systems to meet the requirements of pharmacogenomics. Once validated and proven to be cost effective, pharmacogenomics will revolutionize our concepts of modern medicine by allowing physicians to prescribe the most effective and safest drug based on the patient's genetic blueprint.


Acknowledgment

The authors thank Scott Myrand for technical assis- tance and efforts in developing multiple new genotyping techniques.

Received April 27, 1999. Received in revised form June 18, 1999.

Accepted August 10, 1999.

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