ANALYTICALBIOCHEMISTRY

Analytical Biochemistry 360 (2007) 105–113

www.elsevier.com/locate/yabio

Genotyping of eight polymorphic genes encoding drug-metabolizing enzymes and transporters using a customized oligonucleotide array

Yi Lu a, Shirley Kow-Yin Kham a, Toon-Chai Foo a, AriYn Hany b, Thuan-Chong Quah a, Allen Eng-Juh Yeoh a,¤

a Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119074b Department of Pediatrics, University of Malaya Medical Center, Kuala Lumpur, Malaysia

Received 8 September 2006Available online 26 October 2006

Abstract

Polymorphisms in drug-metabolizing genes may lead to the production of dysfunctional proteins and consequently aVect therapeuticeYcacy and toxicity of drugs. DiVerent frequencies of polymorphic alleles among the races have been postulated to account for theobserved ethnic variations in drug responses. In the current study, we aimed to estimate the frequencies of 14 polymorphisms in eightgenes (TPMT, NQO1, MTHFR, GSTP1, CYP1A1, CYP2D6, ABCB1, and SLC19A1) in the Singapore multiracial populations byscreening 371 cord blood samples from healthy newborns. To improve genotyping eYcacy, we designed an oligonucleotide array based onthe principle of allele-speciWc primer extension (AsPEX) that was capable of detecting the 14 polymorphisms simultaneously. Cross-vali-dation using conventional polymerase chain reaction–restriction fragment-length polymorphism (PCR–RFLP) assays demonstrated 99%concordant results. Measurements on the Xuorescent intensity displayed clear distinctions among diVerent genotypes. Statistical analysesshowed signiWcantly diVerent allele distributions in several genes among the three races, namely Chinese, Malays, and Indians. Compar-ing the allelic frequencies in Chinese with previous studies in Caucasian populations, NQO1 609C > T and SLC19A1 80G > A were dis-tinctly diVerent, whereas close similarity was observed for MTHFR 677C > T. We have demonstrated an array-based methodology forrapid multiplex detection of genetic polymorphisms. The allelic frequencies reported in this study may have important therapeutic andprognostic implications in the clinical use of relevant drugs.© 2006 Elsevier Inc. All rights reserved.

Keywords: Genetic polymorphism; Drug-metabolizing enzyme; Drug transporter; Genotyping; Allele-speciWc primer extension

Ethnicity, a multidimensional classiWcation that encom- development of pharmacogenetics as a predictive tool for

passes shared origins, social background, culture, and envi-ronment [1], is an important determinant of drugmetabolism and response and therefore contributes tointerindividual variability [2]. The ability to handle chemi-cal xenobiotics depends on numerous factors, including theenvironment, age, gender, nutrition, and genetics [3]. Theoverall contribution of genetic variation to drug metabo-lisms and transportation has been recognized as an impor-tant determinant on both clinical outcome and the future

* Corresponding author. Fax: +65 6779 7486.E-mail address: paeyej@nus.edu.sg (A.E. Yeoh).

0003-2697/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.ab.2006.10.006

drug discovery and patient care [4].Phenotypes resulting from the genetic variation contrib-

ute to the diVerences in drug responses, of which thiopurinemethyltransferase (TPMT) and cytochrome P450 (CYP)1

2D6 are two well-studied examples [5]. Although polymor-phisms are ubiquitous, their speciWc frequencies can diVergreatly among ethnic groups. For example, in the TPMTgene, the mutant allelic frequencies were found to be 10.1%

1 Abbreviations used: CYP, cytochrome P450; P-gp, P-glycoprotein; As-PEX, allele-speciWc primer extension; TAMRA, tetramethylrhodamine;SDS, sodium dodecyl sulfate; PBS, phosphate-buVered saline; OR, oddsratio; CI, conWdence interval; RFLP, restriction fragment-length polymor-phism; ASO, allele-speciWc oligonucleotides.

106 Genotyping polymorphic genes using oligonucleotide array / Y. Lu et al. / Anal. Biochem. 360 (2007) 105–113

in Caucasians, 2.0% in Southwest Asians, and 4.7% in Chi-nese [6]. Moreover, TPMT*2 (238G > C) and TPMT*3A(460G > A and 719A > G) were predominantly present inthe Caucasian population, whereas TPMT*3C (719A > G)was the most common polymorphism in the Asian popula-tions [7]. Likewise, CYP2D6 demonstrates ethnic-speciWcfrequencies; the CYP2D6*10 allele occurs at high frequen-cies in Asian populations, whereas CYP2D6*17 andCYP2D6*29, both associated with reduced activity, werefound to be more frequent in African Americans who werepoor metabolizers of CYP2D6 substrates [3]. Ethnic diVer-ences in allelic frequencies have also been observed in manyother genes encoding drug-metabolizing enzymes or trans-porters, including NAD(P)H dehydrogenase, quinone 1(NQO1) [8], CYP1A1 [9], glutathione S-transferase pi(GSTP1) [10], methylenetetrahydrofolate reductase(MTHFR) [11], and P-glycoprotein (P-gp) [12]. Such inter-ethnic diVerences in allelic frequencies may be useful indi-cators of the genetic variation, and drug administration canbe optimized based on ethnicity for a better therapeuticeVect.

However, compared with large cohort studies in Cauca-sian populations, allelic frequencies in genes encoding met-abolic enzymes or transporters have not been wellelucidated in Southeast Asian populations, especially inMalays. One of the diYculties in carrying out a large popu-lation genetics study is to develop a rapid and cost-eVectivegenotyping platform. High-throughput techniques may becommercially available but expensive, and they are notdesigned for particular polymorphisms of interests. There-fore, in the current study, we aimed to determine the fre-quencies of 14 polymorphisms in eight genes encodingmetabolic enzymes or transporters that have knownimpacts on enzymatic activities or potential inXuences onpharmacokinetics of related substrates and have been cor-related to particular diseases (Table 1). To facilitate geno-typing, we customized an oligonucleotide chip based on theprinciple of allele-speciWc primer extension (AsPEX) toscreen all 14 polymorphisms simultaneously in the Singa-pore multiracial population, comprising mainly Chinese,Malays, and Indians.

Materials and methods

DNA samples

A total of 371 anonymized cord blood samples were col-lected from unrelated healthy newborns (166 Chinese, 104Malays, and 101 Indians) at the National University Hospi-tal in Singapore. Genomic DNA was extracted using stan-dard phenol–chloroform methodology and was stored at¡80 °C.

Principle of AsPEX

We designed a genotyping strategy integrating a multi-plex single nucleotide AsPEX reaction and in situ hybrid-

ization on a glass slide, enabling us to screen all 14polymorphisms simultaneously. For each polymorphism,two homogeneous AsPEX querying primers that diVeredonly at their 3� nucleotides were matched to wild-type andmutant alleles, respectively. AsPEX primers for the wild-type or mutant alleles were pooled to make two primermixes. Multiplex primer extensions were carried out foreach sample using two primer mixes separately with Xuo-rescent (tetramethylrhodamine [TAMRA]) ddNTPs. AnAsPEX primer would be incorporated with a particularTAMRA ddNTP only when it matched the sequence com-pletely. Products after AsPEX reaction subsequently wereloaded onto the glass chip and hybridized with complemen-tary oligonucleotide tags. The Xuorescent pattern emittedby labeled primers would reveal the genotypes in the imag-ing system. The principle of AsPEX is illustrated in Fig. 1.

PCR ampliWcation and product puriWcation

Polymerase chain reaction (PCR) primer pairs weredesigned to initially amplify the sequences carrying thepolymorphisms in the respective genes. Primer sequenceswere analyzed using Oligo 6 software (Molecular BiologyInsight, USA) to avoid hairpin loop formation and ampliW-cation of pseudogenes. Three multiplex PCRs and one sin-gle PCR were carried out in a 30-�L reaction volume. Eachmultiplex reaction mixture contained 500 ng genomicDNA, 0.5 �mol/L of each primer, 250 �mol/L of eachdNTP, 1.5 mmol/L Mg2+, and 0.2 units of Platinum TaqDNA polymerase in 1£ supplied 10£PCR buVer (Invitro-gen, Singapore), whereas the single PCR mixture contained200 ng genomic DNA, 0.5 �mol/L of each primer, 100�mol/L of each dNTP, 1.5 mmol/L Mg2+, and 0.1 units of Plati-num Taq DNA polymerase in 1£ supplied 10£PCR buVer.All ampliWcations adopted the same protocol, consisting ofan initial prewarming step at 95 °C for 4 min, followed by35 cycles at 95 °C for 60 s, 55 °C for 30 s, and 72 °C for 30 s,and a Wnal extension at 72 °C for 5 min. Products from fourPCRs were pooled and puriWed using a QIAquick PCRPuriWcation Kit (Qiagen, Germany) according to the proto-col provided by the manufacturer. PuriWed PCR productwas ready for subsequent reaction or was stored at ¡20 °Cuntil use. (The sequences and multiplex combinations ofPCR primers are available in the supplementary material[Table S1].)

Multiplex single nucleotide AsPEX

AsPEX detecting primers were designed using Oligo 6.Two AsPEX detecting primer mixes, labeled as wild-typeand mutant, were prepared by pooling correspondingprimers at various concentrations. Of the reaction mix-ture, 10 �L contained 100 ng puriWed PCR product,1.0 �L wild-type or mutant primer mix, 1.0 �L TAMRA–ddNTP mix (0.2 �mol/L TAMRA–ddATP, 0.15 �mol/LTAMRA–ddCTP, 0.5 �mol/L TAMRA–ddGTP, and0.2 �mol/L TAMRA–ddUTP), 0.25 units of Thermo

Genotyping polymorphic genes using oligonucleotide array / Y. Lu et al. / Anal. Biochem. 360 (2007) 105–113 107

Sequenase DNA polymerase (Amersham Biosciences,USA), and 1.0 �L supplied 10£ reaction buVer. Cyclicmultiplex single nucleotide AsPEX was carried out usingthe protocol consisting of an initial prewarming step at95 °C for 3 min, followed by 60 cycles at 95 °C for 20 sand 60 °C for 20 s. After the reaction, 5 �L self-made 3£hybridization buVer (18£ SSC, 3£ TE) were immediatelyadded into 10-�L products to prevent side reactions. Theproducts were then ready for subsequent hybridization orwere stored at 4 °C until use. (The sequences of detectingprimers are available in the supplementary material[Table S2a].)

Array preparation

Oligonucleotide tags that were complementary toAsPEX primers were diluted in ArrayIt Micro Spotting

Solution Plus (TeleChem International, USA) at a Wnalconcentration of 20 �mol/L. Tags were loaded into a 384-well plate and spotted onto CSS-100 silylated slides (CELAssociates, USA) by contact printing using a ProSys5510A instrument (Cartesian Technologies, USA). Tagswere coupled to the slide surface by forming covalentbonds between amino residues and SchiV base aldehydeafter incubating spotted slides in a humid chamber at37 °C overnight. Using a High-Throughput Wash Station(TeleChem International) on a magnetic stirrer, slides afterincubation were washed in 0.2% sodium dodecyl sulfate(SDS) for 3 min, in sodium borohydride reducing solution(dissolved 1.0 g sodium borohydride [Sigma–Aldrich,USA] in 300 ml phosphate-buVered saline [PBS] and thenadded to 100 ml of 100% ethanol) for 10 min, in 0.2% SDSagain for 3 min, and in double-distilled water for a simplerinse. The slides were dried by centrifugation in a Microarray

Table 1Polymorphisms in genes encoding metabolic enzymes or transporters

a CYP1A1/2D6, cytochrome P450, family 1/2, subfamily A/D, polypeptide 1/6; TPMT, thiopurine methyltransferase; NQO1, NAD(P)H dehydrogenase,quinone 1; GSTP1, glutathione S-transferase pi; ABCB1, ATP-binding cassette, subfamily B (MDR/TAP), member 1; also known as multidrug resistantgene (MDR1); SLC19A1, solute carrier family 19 (folate transporter), member 1; also known as reduced folate carrier (RFC1); MTHFR, methylenetetra-hydrofolate reductase.

b Information obtained from the Pharmacogenetics and Pharmacogenomics Knowledge Base (PharmGKB, www.pharmgkb.org).

Genesa Polymorphisms RefSeq ID (GenBank) and nucleotide position

Drug/substrateb Related disease/therapy

Phase I metabolismCYP1A1 *2A (6235T > C) D12525/400 Estrogen, nicotine, phenacetin Lung cancer [24]

*2B (4889A > G and 6235T > C) X02612/6819 and D12525/400 Squamous cell carcinoma [25]*4 (4887C > A) X02612/6817

CYP2D6 *3 (2637delA) M33388/4168 Amitriptyline, antidepressants, antipsychotics, cocaine, codeine, debrisoquine, desipramine, sparteine

Tardive dyskinesia [26]*4 (1934G > A) M33388/3465 Age-related macular degeneration [27]

Bladder cancer [28]

Phase II metabolismTPMT *3A (460G > A and 719A > G) AB045146/16954 and 25270 6-Mercaptopurine,

6-thioguanine, azathioprineALL [29]

*3C (719A > G)*6 (539A > T)

AB045146/25270AB045146/22108

Immunosuppression after organ transplantations [30–32]

NQO1 *2 (609C > T) AY281093/9144 Etoposide, mitomycin c, vitamin K

Pancreatic cancer [33]Lung cancer [34]Cervical cancer [35]Infant ALL [36]

GSTP1 *B (1578A > G) AY324387/3292 Daunorubicin, doxorubicin, etoposide, methotrexate, prednisone, vincristine, ethacrynic acid

Bladder cancer [37]Endometriosis [38]Endometrial cancer [39]Childhood ALL [40]

Drug transporterABCB1 3435C > T M29445/176 Large number of anticancer

drugsChildhood ALL [40]Colon cancer [41]Parkinson’s disease [42]Major depression [43]

SLC19A1 80G > A U92869/135 Reduced folate (e.g., methotrexate)

Childhood ALL [44]Neural tube defects [45]

ReductaseMTHFR 677C > T AY338232/8747 5,10-Methylenetetrahydrofolate Colorectal adenomas [46]

1298A > C AY338232/10649 Breast cancer [47]Childhood ALL [48]Late-onset Alzheimer’s disease [49]

108 Genotyping polymorphic genes using oligonucleotide array / Y. Lu et al. / Anal. Biochem. 360 (2007) 105–113

High Speed Centrifuge (TeleChem International) and werestored at 4 °C until use. (The sequences of tags are avail-able in the supplementary material [Table S2b].)

Hybridization

For hybridization, 5�L of AsPEX products (premixedwith hybridization buVer) was loaded. Small glass coverslips were used to diminish evaporation. The chip wassealed in the ArrayIt Hybridization Cassette (TeleChemInternational) and was placed in a 50 °C water bath for 2 h.Then it was washed in 2£SSC (300 mmol/L NaCl,30 mmol/L Na citrate, pH 7.0) for 2 min, followed byanother 2 min wash in 1£SSC and a simple rinse in double-distilled water. After drying by centrifugation, the hybrid-ized chip was ready for signal detection or could be storedin the dark at 4 °C for up to 24 h.

Fluorescence detection and analysis

Fluorescent signals were detected in a ScanArray 5000scanning instrument with ScanArray 3.1 (Packard BioSci-ence, UK) using 543-nm excitation wavelengths forTAMRA and a scanning rate of 5�m/s. Both the laserpower and the photomultiplier tube gain were kept con-stant at 95%. The acquired images were analyzed usingQuantArray 3.0 (Packard BioScience). Signal intensitieswere adjusted by deducting the average background inten-sities around the spots.

Statistical analyses

A chi-square test was used to examine the diVerences inallelic frequencies among Chinese, Malay, and Indian sam-ples. The level of signiWcance was calculated by the Fisherexact test (two-sided). The crude odds ratio (OR) was givenwith a 95% conWdence interval (CI). All statistical analyseswere done in SPSS 12 (SPSS, USA).

Results

Genotyping using customized oligonucleotide chip

In our preliminary experiment, negative control contain-ing all reactants except for the puriWed DNA templates wastested for possible false Xuorescent signals. No false signalswere observed, indicating that there were no confoundingside reactions. Therefore, we could conduct our genotypingin cord blood DNA samples. An example is demonstratedin Fig. 2.

To validate the accuracy, all samples genotyped by thechip were cross-validated using published PCR–restrictionfragment-length polymorphism (RFLP) methods and/ordirect sequencing in double-blind tests. Two polymor-phisms, CYP1A1*4 and CYP2D6*3, were validated only ina part of samples due to DNA availability. The cross-vali-dation showed high concordance between DNA chip andPCR–RFLP: 100% for TPMT*3A, TPMT*3C, TPMT*6,NQO1*2, CYP1A1*2B, CYP1A1*4, CYP2D6*3,CYP2D6*4, MTHFR 677C > T, ABCB1 3435C > A, andSLC19A1 80G > A; 98.9% for CYP1A1*2A; 98.7% forMTHFR 1298A > C; and 99.1% for GSTP1*B.

Analyses of Xuorescence intensity

To demonstrate the power of discrimination amongwild-type, heterozygous, and homozygous mutant alleles,we randomly chose 15–20 samples of each genotype (ifavailable) for each polymorphic site, measured their Xuo-rescent intensities at the particular locus, and comparedthem with negative controls. Fig. 3 shows two examples ofsuch comparisons, demonstrating distinct Xuorescent inten-sity for diVerent genotypes. (Completed intensity analysesfor all studied loci are available in the supplementarymaterial [Fig. S].)

To standardize the genotype assignment, we set twocutoV values for each tested polymorphic locus to

Fig. 1. Principle of single nucleotide AsPEX. Each polymorphic locus is queried by a pair of AsPEX detecting primers. Homozygosity will display a singleXuorescent spot, whereas heterozygosity will display two Xuorescent spots.

Genotyping polymorphic genes using oligonucleotide array / Y. Lu et al. / Anal. Biochem. 360 (2007) 105–113 109

discriminate the diVerent genotypes. These two cutoV val-ues varied among diVerent polymorphic loci, for example,(6.2, 5.9) plus (4.2, 4.4) for NQO1 609C > T and (5.8, 5.8)plus (4.0, 4.0) for MTHFR 677C > T. (CutoV values forother loci are available in the supplementary material[Fig. S].) No such values were set for CYP2D6 2637delAbecause no polymorphism was found in our study.

Genotype/allelic frequencies

Cord blood DNA samples from 166 Chinese, 104 Malay,and 101 Indian healthy newborns were screened for the 14polymorphisms using our oligonucleotide chip. The geno-type frequencies of these polymorphisms in diVerent popu-lations are summarized in Table 2.

Chi-square tests showed that signiWcant diVerences werefound for three polymorphic loci between Chinese andMalays, eight loci between Chinese and Indians, and fourloci between Malays and Indians (Table 3a). Table 3bshows the comparisons of NQO1 609C > T allelic frequen-cies among three ethnic groups, exemplifying our statisticalanalyses. (Completed analyses for all studied loci are avail-able in the supplementary material [Table S3].)

Discussion

In this study, we successfully designed a chip-basedgenotyping strategy according to the principle of singlenucleotide AsPEX. With this new tool, we were able tosimultaneously screen 14 polymorphisms in eight genes

Fig. 2. Detection of 14 polymorphisms in eight genes encoding drug-metabolizing enzymes and transporters using customized oligonucleotide chip. Twoidentical arrays on the same chip correspond to two alleles. For a particular polymorphism, the Xuorescence on the wild-type and/or mutant panels indi-cates whether the sequence carries a mutation. Homozygosis displays single Xuorescence, whereas heterozygosis displays Xuorescence on both panels.

Fig. 3. Fluorescent intensity for diVerent genotypes at NQO1 609C > T (left) and MTHFR 677C > T (right). Intensity values are transformed to the corre-sponding logarithms of 2 and are labeled on the x and y axes. Two cutoV coordinates shown on each plot are used to compartmentalize the region for thegenotype speciWed. Spots out of genotype areas are considered to be ambiguous results and thus may require repeated tests to conWrm their genotypes.

110 Genotyping polymorphic genes using oligonucleotide array / Y. Lu et al. / Anal. Biochem. 360 (2007) 105–113

encoding metabolic enzymes and transporters in 371healthy newborns (166 Chinese, 104 Malays, and 101Indians).

Technical advantages

Accuracy and throughput are two important aspectsthat need to be taken into account when choosing a suit-able genotyping platform. The major advantage of

Table 2Genotype frequencies (and percentages) of 14 polymorphisms amongthree ethnic groups

Note. Percentages are in parentheses.

Polymorphic locus Genotype Ethnic group

Chinese Malays Indians

TPMT 460G > A Wild-type 166 (100) 104 (100) 93 (98.9)Heterozygosis 0 0 1 (1.1)Homozygosis 0 0 0

TPMT 539A > T Wild-type 166 (100) 104 (100) 94 (100)Heterozygosis 0 0 0Homozygosis 0 0 0

TPMT 719A > G Wild-type 163 (98.2) 99 (95.2) 89 (94.7)Heterozygosis 3 (1.8) 5 (4.8) 5 (5.3)Homozygosis 0 0 0

NQO1 609C > T Wild-type 29 (17.5) 42 (40.4) 45 (44.6)Heterozygosis 87 (52.4) 50 (48.1) 42 (41.6)Homozygosis 50 (30.1) 12 (11.5) 14 (13.8)

MTHFR 677C > T Wild-type 76 (45.8) 74 (71.2) 80 (79.2)Heterozygosis 69 (41.6) 30 (28.8) 21 (20.8)Homozygosis 21 (12.7) 0 0

MTHFR 1298A > C Wild-type 91 (54.8) 53 (51.0) 38 (37.6)Heterozygosis 66 (39.8) 38 (36.5) 48 (47.5)Homozygosis 9 (5.4) 13 (12.5) 15 (14.9)

CYP1A1 4887C > A Wild-type 165 (100) 101 (98.1) 96 (95.0)Heterozygosis 0 2 (1.9) 5 (5.0)Homozygosis 0 0 0

CYP1A1 4889A > G Wild-type 90 (55.2) 65 (62.5) 69 (68.3)Heterozygosis 58 (35.6) 34 (32.7) 31 (30.7)Homozygosis 15 (9.2) 5 (4.8) 1 (1.0)

CYP1A1 6235T > C Wild-type 40 (24.1) 26 (25.0) 40 (39.6)Heterozygosis 102 (61.4) 57 (54.8) 48 (47.5)Homozygosis 24 (14.5) 21 (20.2) 13 (12.9)

CYP2D6 1934G > A Wild-type 163 (100) 93 (94.9) 82 (82.0)Heterozygosis 0 5 (5.1) 17 (17.0)Homozygosis 0 0 1 (1.0)

CYP2D6 2637delA Wild-type 163 (100) 93 (100) 101 (100)Heterozygosis 0 0 0Homozygosis 0 0 0

GSTP1 1578A > G Wild-type 99 (59.6) 61 (58.7) 52 (51.5)Heterozygosis 63 (38.0) 37 (35.6) 42 (41.6)Homozygosis 4 (2.4) 6 (5.7) 7 (6.9)

ABCB1 3435C > T Wild-type 62 (39.7) 37 (35.9) 22 (22.0)Heterozygosis 67 (42.9) 50 (48.5) 51 (51.0)Homozygosis 27 (17.4) 16 (15.6) 27 (27.0)

SLC19A1 80G > A Wild-type 18 (12.0) 22 (21.4) 38 (38.0)Heterozygosis 87 (58.0) 48 (46.6) 42 (42.0)Homozygosis 45 (30.0) 33 (32.0) 20 (20.0)

AsPEX reaction is that the discrimination of two allelesis based on the correct 3� end base pairing for primerextension rather than the diVerences in thermal stabilitybetween mismatched and perfectly matched hybrids orthe recognition of speciWc DNA sequence by a particularrestriction enzyme. The latter two usually will requirediVerent reaction conditions to achieve the desired levelof stringency, and hence they are diYcult to multiplex.Therefore, AsPEX is able to achieve excellent distinctionbetween homozygotes and heterozygotes. To furtherimprove its speciWcity, we replaced commonly useddNTP with ddNTP, the latter of which terminates primerextension after single nucleotide incorporation. This willgreatly reduce false primer extension because it is diYcultfor mismatched primer to form a stable 3� end duplexwith a single nucleotide addition. Because the polymor-phic locus is the position of interest, the extension of addNTP suits the purpose, saving reagent costs and short-ening the reaction time.

Normally, it is diYcult to carry out allele-speciWc oligo-nucleotide (ASO) hybridization for more than 10 diVerentpolymorphisms simultaneously because this requires strin-gent washing of all probes at the same temperature in addi-tion to the enzyme-speciWc RFLP. Spotting oligonucleotideon the glass support allows parallel screening of a largenumber of polymorphic loci. The design of detecting primeris straightforward and is applicable to nearly all types ofgenetic polymorphisms except large deletions (e.g., dele-tions in GSTM1 and GSTT1 genes) that can be detectedsimply by a single PCR. Therefore, it is easy to extend thisplatform to screen for new polymorphisms other than theones in our study.

These features can help to minimize the eVort requiredfor assay design and optimization, making AsPEX conve-nient and reliable to use. According to our experience, toscreen these 14 polymorphisms in the same set of samples,our chip could be at least 10 times faster than methodsdevised previously [7,12–15] without compromising accu-racy.

Allelic frequencies in Chinese, Malays, and Indians

Among the 14 polymorphic loci in our study, weobserved that there were 3 polymorphisms that showed sta-tistically distinct allelic frequencies between Chinese andMalays, 8 between Chinese and Indians, and 4 betweenMalays and Indians (Table 3a). The level of similarityamong these three races may be explained partly by theprogress of human evolution. Chinese and Indians divergedat a relatively early stage in the history of human diversiW-cation [16], whereas the current structure of SoutheastAsian populations was formed mainly from two separatemigrations from West Asia [17] and South China [18]. Thesimilarities between Malays and Chinese or Indians may beinterpreted by the founder eVect or the result of intermar-riage, which reshapes allele distributions in the Malay Pen-insula through generations.

Genotyping polymorphic genes using oligonucleotide array / Y. Lu et al. / Anal. Biochem. 360 (2007) 105–113 111

Our group previously reported allelic frequencies in theTPMT gene [7] using a larger sample set (200 samples ineach race). All TPMT genotypes obtained by the DNA chipwere consistent with our previous results, although allelicfrequencies were slightly diVerent, probably due to sam-pling bias of the smaller sample size in the current study.Our report on CYP2D6*3 was also consistent with otherpopulation studies, showing that this mutation was veryrare in Asian populations [19,20]. It is also notable that eth-nic allelic frequencies of ABCB1 3435C > T reported in ourstudy were slightly diVerent from those reported in an ear-lier report on the Singapore multiracial populations [12],but the conclusions were consistent; that is, the frequenciesof the C and T alleles were similar between Chinese andMalays, whereas Indians had a higher frequency of the Tallele. In addition, respective allelic frequencies of CYP1A14889A > G in Chinese and Indians were very close to thedata reported in Chowbay and coworkers’ review [21]except in Malays. A further study with a larger sample sizeis warranted to reestimate the frequency of such polymor-phisms.

We also compared some allelic frequencies in Chineseand corresponding Wndings in Caucasians reported else-where. We observed that Chinese had a signiWcantly higherNQO1*2 T allele frequency of 56.3% (17.4% CC, 52.7% CT,and 29.9% TT) compared with 18.8% (66.8% CC, 28.8% CT,and 4.4% TT) in Caucasians [22]. This was similar toanother report in which T allele frequency was found to be49.0% in Chinese and 16.0% in Caucasians [8]. Anotherpolymorphic locus showing distinct allelic frequenciesbetween Chinese and Caucasians was SLC19A1 80G > A.Chango and coworkers [23] identiWed 52.7% G alleles and47.3% A alleles (27.2% GG, 50.9% GA, and 21.9% AA) in169 samples from French healthy adults; in our study, thecorresponding frequencies were 41.0% for G alleles and59.0% for A alleles (12.0% GG, 58.0% GA, and 30.0% AA).On the contrary, Chinese and Caucasians had nearly identi-cal allelic frequencies—even genotype distributions—atMTHFR 677C > T locus. We detected 66.2% C alleles and

33.8% T alleles (45.5% CC, 41.3% CT, and 13.2% TT),whereas Krajinovic and coworkers reported 63.3% C allelesand 36.7% T alleles (42.0% CC, 42.7% CT, and 15.3% TT) in300 healthy French Canadians [14]. The impact of intereth-nic diVerences of allelic frequencies in epidemiology of thediseases remains unclear. It is also unknown whether suchdiVerences will determine diVerent baselines of enzymeactivities and thus inXuence pharmacokinetics or pharma-codynamics. If they do, dose requirements of certain drugs(especially those with narrow therapeutic indexes) that areoptimal for one population might be suboptimal foranother population.

In conclusion, we have demonstrated a useful genotyp-ing strategy that integrates high Wdelity of single nucleotideAsPEX reaction and multiplexing capacity of oligonucleo-tide chip. A parallel molecular analysis of 14 polymor-phisms in eight genes encoding metabolic enzymes andtransporters was conducted successfully using this method.Because expressions of polymorphic genes may be impli-cated in the predisposition to a disease or the variability inmetabolisms of drugs, the optimal doses may diVer for vari-ous ethnic groups due to distinct allelic frequencies ofresponsible genes. This provides the basis and impetus for acomprehensive survey of polymorphisms in genes encodingmetabolic enzymes and transporters in diVerent popula-tions.

Acknowledgments

We thank Chan Yiong Huak (Yong Loo Lin School ofMedicine, National University of Singapore) for his kindassistance on statistical analyses. This work was supportedby a grant from the Singapore Cancer Syndicate (SCSEN14).

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at doi:10.1016/j.ab.2006.10.006.

Table 3Allele with signiWcantly diVerent frequency (Completed analyses are available in Supplementary Material, Table S3)

Note. C, Chinese; M, Malays; I, Indians; —, not applicable.

(a) Polymorphisms with signiWcantly diVerent allelic frequencies among Chinese, Malays, and Indians

Alleles with signiWcantly diVerent frequency

C vs. M NQO1 609C > T MTHFR 677C > T CYP2D6 1934G > AC vs. I NQO1 609C > T MTHFR 677C > T MTHFR 1298A > C CYP1A1 4887C > A

CYP1A1 4889A > G CYP2D6 1934G > A ABCB1 3435C > T SLC19A1 80G > AM vs. I CYP1A1 6235T > C CYP2D6 1934G > A ABCB1 3435C > T SLC19A1 80G > A

(b) Comparison of allelic frequencies of NQO1 609C > T among 3 ethnic groups. In this case, Chinese are more likely to carry T alleles than either Malayor Indian while Malays and Indians show no diVerence

Allele Ethnic group OR (95% CI) P value

Chinese Malays Indians C vs. M C vs. I M vs. I C vs. M C vs. I M vs. I

C 145 (43.7%) 134 (64.4%) 132 (65.3%) 1.0 (reference) 1.0 (reference) 1.0 (reference) — — —T 187 (56.3%) 74 (35.6%) 70 (34.7%) 2.335 (1.634–3.338) 2.432 (1.693–3.493) 1.041 (0.694–1.562) <0.001 <0.001 0.918

112 Genotyping polymorphic genes using oligonucleotide array / Y. Lu et al. / Anal. Biochem. 360 (2007) 105–113

References

[1] P.A. Senior, R. Bhopal, Ethnicity as a variable in epidemiologicalresearch, Br. Med. J. 309 (1994) 327–330.

[2] H.G. Xie, R.B. Kim, A.J. Wood, C.M. Stein, Molecular basis of ethnicdiVerences in drug disposition and response, Annu. Rev. Pharmacol.Toxicol. 41 (2001) 815–850.

[3] J.F. Solus, B.J. Arietta, J.R. Harris, D.P. Sexton, J.Q. Steward, C.McMunn, P. Ihrie, J.M. Mehall, T.L. Edwards, E.P. Dawson, Geneticvariation in eleven phase I drug metabolism genes in an ethnicallydiverse population, Pharmacogenomics 5 (2004) 895–931.

[4] A.D. Roses, Pharmacogenetics and drug development: the path tosafer and more eVective drugs, Nat. Rev. Genet. 5 (2004) 645–656.

[5] R. Weinshilboum, L. Wang, Pharmacogenomics: bench to bedside,Nat. Rev. Drug Discov. 3 (2004) 739–748.

[6] E.S. Collie-Duguid, S.C. Pritchard, R.H. Powrie, J. Sludden, D.A. Col-lier, T. Li, H.L. McLeod, The frequency and distribution of thiopurinemethyltransferase alleles in Caucasian and Asian populations, Phar-macogenetics 9 (1999) 37–42.

[7] S.K. Kham, P.L. Tan, A.H. Tay, C.K. Heng, A.E. Yeoh, T.C. Quah,Thiopurine methyltransferase polymorphisms in a multiracial Asianpopulation and children with acute lymphoblastic leukemia, J. Pedi-atr. Hematol. Oncol. 24 (2002) 353–359.

[8] A. Gaedigk, R.F. Tyndale, M. Jurima-Romet, E.M. Sellers, D.M.Grant, J.S. Leeder, NAD(P)H:quinone oxidoreductase: polymor-phisms and allele frequencies in Caucasian, Chinese, and CanadianNative Indian and Inuit populations, Pharmacogenetics 8 (1998) 305–313.

[9] K. Inoue, T. Asao, T. Shimada, Ethnic-related diVerences in the fre-quency distribution of genetic polymorphisms in the CYP1A1 andCYP1B1 genes in Japanese and Caucasian populations, Xenobiotica30 (2000) 285–295.

[10] H. Enokida, H. Shiina, S. Urakami, M. Igawa, T. Ogishima, D. Poo-kot, L.C. Li, Z.L. Tabatabai, M. Kawahara, M. Nakagawa, C.J. Kane,P.R. Carroll, R. Dahiya, Ethnic group-related diVerences in CpGhypermethylation of the GSTP1 gene promoter among African-American, Caucasian, and Asian patients with prostate cancer, Intl. J.Cancer 116 (2005) 174–181.

[11] S. Ogino, R.B. Wilson, Genotype and haplotype distributions ofMTHFR677C > T and 1298A > C single nucleotide polymorphisms:a meta-analysis, J. Hum. Genet. 48 (2003) 1–7.

[12] C. Balram, A. Sharma, C. Sivathasan, E.J. Lee, Frequency of C3435Tsingle nucleotide MDR1 genetic polymorphism in an Asian popula-tion: phenotypic–genotypic correlates, Br. J. Clin. Pharmacol. 56(2003) 78–83.

[13] M. Krajinovic, D. Labuda, G. Mathonnet, M. Labuda, A. Moghrabi,J. Champagne, D. Sinnett, Polymorphisms in genes encoding drugsand xenobiotic metabolizing enzymes, DNA repair enzymes, andresponse to treatment of childhood acute lymphoblastic leukemia,Clin. Cancer Res. 8 (2002) 802–810.

[14] M. Krajinovic, S. Lamothe, D. Labuda, E. Lemieux-Blanchard, Y.Theoret, A. Moghrabi, D. Sinnett, Role of MTHFR genetic polymor-phisms in the susceptibility to childhood acute lymphoblastic leuke-mia, Blood 103 (2004) 252–257.

[15] C. Laverdiere, S. Chiasson, I. Costea, A. Moghrabi, M. Krajinovic,Polymorphism G80A in the reduced folate carrier gene and its rela-tionship to methotrexate plasma levels and outcome of childhoodacute lymphoblastic leukemia, Blood 100 (2002) 3832–3834.

[16] M. Nei, A.K. Roychoudhury, Evolutionary relationships of humanpopulations on a global scale, Mol. Biol. Evol. 10 (1993) 927–943.

[17] S. Barnabas, B. Joshi, C.G. Suresh, Indian–Asian relationship:mtDNA reveals more, Naturwissenschaften 87 (2000) 180–183.

[18] Y.G. Yao, W.S. Watkins, Y.P. Zhang, Evolutionary history of themtDNA 9-bp deletion in Chinese populations and its relevance to thepeopling of east and southeast Asia, Hum. Genet. 107 (2000) 504–512.

[19] R. Ismail, L.K. Teh, J. Amir, Z. Alwi, C.G. Lopez, Genetic polymor-phism of CYP2D6 in Chinese subjects in Malaysia, J. Clin. Pharm.Ther. 28 (2003) 279–284.

[20] M. Kitada, Genetic polymorphism of cytochrome P450 enzymes inAsian populations: focus on CYP2D6, Intl. J. Clin. Pharmacol. Res.23 (2003) 31–35.

[21] B. Chowbay, S. Zhou, E.J. Lee, An interethnic comparison of poly-morphisms of the genes encoding drug-metabolizing enzymes anddrug transporters: experience in Singapore, Drug Metab. Rev. 37(2005) 327–378.

[22] M. Krajinovic, H. Sinnett, C. Richer, D. Labuda, D. Sinnett, Role ofNQO1, MPO, and CYP2E1 genetic polymorphisms in the susceptibil-ity to childhood acute lymphoblastic leukemia, Intl. J. Cancer 97(2002) 230–236.

[23] A. Chango, N. Emery-Fillon, G.P. de Courcy, D. Lambert, M. PWster,D.S. Rosenblatt, J.P. Nicolas, A polymorphism (80G- > A) in thereduced folate carrier gene and its associations with folate status andhomocysteinemia, Mol. Genet. Metab. 70 (2000) 310–315.

[24] M. Adonis, V. Martinez, P. Marin, L. Gil, CYP1A1 and GSTM1genetic polymorphisms in lung cancer populations exposed to arsenicin drinking water, Xenobiotica 35 (2005) 519–530.

[25] M. Gajecka, M. Rydzanicz, R. Jaskula-Sztul, M. Kujawski, W. Szyf-ter, K. Szyfter, CYP1A1, CYP2D6, CYP2E1, NAT2, GSTM1, andGSTT1 polymorphisms or their combinations are associated with theincreased risk of the laryngeal squamous cell carcinoma, Mutat. Res.574 (2005) 112–123.

[26] N.A. Patsopoulos, E.E. Ntzani, E. Zintzaras, J.P. Ioannidis, CYP2D6polymorphisms and the risk of tardive dyskinesia in schizophrenia: ameta-analysis, Pharmacogenet. Genom. 15 (2005) 151–158.

[27] H. Esfandiary, U. Chakravarthy, C. Patterson, I. Young, A.E. Hughes,Association study of detoxiWcation genes in age related maculardegeneration, Br. J. Ophthalmol. 89 (2005) 470–474.

[28] R.C. Sobti, A.I. Al-Badran, S. Sharma, S.K. Sharma, A. Krishan, H.Mohan, Genetic polymorphisms of CYP2D6, GSTM1, and GSTT1genes and bladder cancer risk in north India, Cancer Genet. Cytoge-net. 156 (2005) 68–73.

[29] M. Stanulla, E. SchaeVeler, T. Flohr, G. Cario, A. Schrauder, M. Zim-mermann, K. Welte, W.D. Ludwig, C.R. Bartram, U.M. Zanger, M.Eichelbaum, M. Schrappe, M. Schwab, Thiopurine methyltransferase(TPMT) genotype and early treatment response to mercaptopurine inchildhood acute lymphoblastic leukemia, JAMA 293 (2005) 1485–1489.

[30] M. Kurzawski, K. Dziewanowski, B. Gawronska-Szklarz, L. Doman-ski, M. Drozdzik, The impact of thiopurine S-methyltransferase poly-morphism on azathioprine-induced myelotoxicity in renal transplantrecipients, Ther. Drug Monit. 27 (2005) 435–441.

[31] M. Kurzawski, K. Dziewanowski, K. Ciechanowski, M. Drozdzik,Severe azathioprine-induced myelotoxicity in a kidney transplantpatient with thiopurine S-methyltransferase-deWcient genotype(TPMT*3A/*3C), Transpl. Intl. 18 (2005) 623–625.

[32] D.P. Breen, A.M. Marinaki, M. Arenas, P.C. Hayes, Pharmacogeneticassociation with adverse drug reactions to azathioprine immunosup-pressive therapy following liver transplantation, Liver Transpl. 11(2005) 826–833.

[33] A.M. Lewis, M. Ough, M.M. Hinkhouse, M.S. Tsao, L.W. Oberley, J.J.Cullen, Targeting NAD(P)H:quinone oxidoreductase (NQO1) inpancreatic cancer, Mol. Carcinog. 43 (2005) 215–224.

[34] S.J. Saldivar, Y. Wang, H. Zhao, L. Shao, J. Lin, M.R. Spitz, X. Wu,An association between a NQO1 genetic polymorphism and risk oflung cancer, Mutat. Res. 582 (2005) 71–78.

[35] Y. Niwa, K. Hirose, T. Nakanishi, A. Nawa, K. Kuzuya, K. Tajima, N.Hamajima, Association of the NAD(P)H:quinone oxidoreductaseC609T polymorphism and the risk of cervical cancer in Japanese sub-jects, Gynecol. Oncol. 96 (2005) 423–429.

[36] M. Lanciotti, C. Dufour, L. Corral, P. Di Michele, S. Pigullo, G. DeRossi, G. Basso, A. Leszl, M. Luciani, L. Lo Nigro, C. Micalizzi, M.G.Valsecchi, A. Biondi, R. Haupt, Genetic polymorphism ofNAD(P)H:quinone oxidoreductase is associated with an increasedrisk of infant acute lymphoblastic leukemia without MLL gene rear-rangements, Leukemia 19 (2005) 214–216.

[37] D.S. Srivastava, D.K. Mishra, A. Mandhani, B. Mittal, A. Kumar,R.D. Mittal, Association of genetic polymorphism of glutathione S-

Genotyping polymorphic genes using oligonucleotide array / Y. Lu et al. / Anal. Biochem. 360 (2007) 105–113 113

transferase M1, T1, P1, and susceptibility to bladder cancer, Eur.Urol. 48 (2005) 339–344.

[38] D. Ertunc, M. Aban, E.C. Tok, L. Tamer, M. Arslan, S. Dilek, Gluta-thione-S-transferase P1 gene polymorphism and susceptibility toendometriosis, Hum. Reprod. 20 (2005) 2157–2161.

[39] Q.K. Chan, U.S. Khoo, H.Y. Ngan, C.Q. Yang, W.C. Xue, K.Y. Chan,P.M. Chiu, P.P. Ip, A.N. Cheung, Single nucleotide polymorphism ofpi-class glutathione S-transferase and susceptibility to endometrialcarcinoma, Clin. Cancer Res. 11 (2005) 2981–2985.

[40] M. Stanulla, E. SchaVeler, S. Arens, A. Rathmann, A. Schrauder, K.Welte, M. Eichelbaum, U.M. Zanger, M. Schrappe, M. Schwab, GSTP1and MDR1 genotypes and central nervous system relapse in childhoodacute lymphoblastic leukemia, Intl. J. Hematol. 81 (2005) 39–44.

[41] M. Kurzawski, M. Drozdzik, J. Suchy, G. Kurzawski, M. Bialecka, W.Gornik, J. Lubinski, Polymorphism in the P-glycoprotein drug trans-porter MDR1 gene in colon cancer patients, Eur. J. Clin. Pharmacol.61 (2005) 389–394.

[42] E.K. Tan, D.K. Chan, P.W. Ng, J. Woo, Y.Y. Teo, K. Tang, L.P.Wong, S.S. Chong, C. Tan, H. Shen, Y. Zhao, C.G. Lee, EVect ofMDR1 haplotype on risk of Parkinson disease, Arch. Neurol. 62(2005) 460–464.

[43] R.L. Roberts, P.R. Joyce, R.T. Mulder, E.J. Begg, M.A. Kennedy, Acommon P-glycoprotein polymorphism is associated with nortripty-line-induced postural hypotension in patients treated for majordepression, Pharmacogenomics J. 2 (2002) 191–196.

[44] L. Zhang, J.W. Taub, M. Williamson, S.C. Wong, B. Hukku, J. Pul-len, Y. Ravindranath, L.H. Matherly, Reduced folate carrier geneexpression in childhood acute lymphoblastic leukemia: relation-ship to immunophenotype and ploidy, Clin. Cancer Res. 4 (1998)2169–2177.

[45] C.L. Relton, C.S. Wilding, P.A. Jonas, S.A. Lynch, E.J. Tawn, J. Burn,Genetic susceptibility to neural tube defect pregnancy varies withoVspring phenotype, Clin. Genet. 64 (2003) 424–428.

[46] M. Hirose, S. Kono, S. Tabata, S. Ogawa, K. Yamaguchi, M.Mineshita, T. Hagiwara, G. Yin, K.Y. Lee, A. Tsuji, N. Ikeda,Genetic polymorphisms of methylenetetrahydrofolate reductaseand aldehyde dehydrogenase 2, alcohol use, and risk of colorectaladenomas: Self-Defense Forces Health Study, Cancer Sci. 96 (2005)513–518.

[47] U. Deligezer, E.E. Akisik, N. Dalay, Homozygosity at the C677T ofthe MTHFR gene is associated with increased breast cancer risk inthe Turkish population, In Vivo 19 (2005) 889–893.

[48] E. Oliveira, S. Alves, S. Quental, F. Ferreira, L. Norton, V. Costa, A.Amorim, M.J. Prata, The MTHFR C677T and A1298C polymor-phisms and susceptibility to childhood acute lymphoblastic leukemiain Portugal, J. Pediatr. Hematol. Oncol. 27 (2005) 425–429.

[49] B. Wang, F. Jin, R. Kan, S. Ji, C. Zhang, Z. Lu, C. Zheng, Z. Yang, L.Wang, Association of MTHFR gene polymorphism C677T with sus-ceptibility to late-onset Alzheimer’s disease, J. Mol. Neurosci. 27(2005) 23–28.