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Technical Briefs |
Departments of
1
Biochemistry,
3 Pharmacology, and
4 Pediatrics, National University of Singapore, Singapore 117597, Singapore
2 Division of Medical Sciences, National Cancer Center, Singapore 169610, Singapore
Departments of
5
Pediatrics and
6 Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205
aaddress correspondence to this author at: Division of Medical Sciences, National Cancer Center, Level 6, Lab 5, 11 Hospital Dr., Singapore 169610, Singapore; fax 65-6224-1778, e-mail bchleec{at}nus.edu.sg
Responses to different drugs can vary widely among different individuals as a result of genetic variations in drug-metabolizing enzymes, transporters, receptors, and/or other cofactors. The multidrug resistance 1 (MDR1) transporter, a well-characterized member of the ATP-binding cassette superfamily, was shown to efflux a wide variety of structurally and functionally unrelated drugs, including anticancer, antiarrhythmic, antidepressant, antipsychotic, and antiviral agents. The pharmacogenetics of the MDR1 multidrug transporter have recently received much scientific attention. Several single-nucleotide polymorphisms (SNPs) have been identified in the MDR1 gene; some occur only in specific ethnic groups, whereas others occur in all ethnic groups but at significantly different allele frequencies among the different races [see Ref. (1) and references therein]. Nonetheless, the functional significance of these SNPs remains unclear. Various functional associations, some paradoxical, have been observed between the synonymous SNP (exon 26 3435C
T) and MDR1 protein expression and plasma drug concentrations (2)(3)(4)(5), drug-induced side effects (6), and drug response (7). The SNP exon 26 3435T allele has been associated with lower MDR1 expression in the duodenum (2), leukocytes (5), and placental tissues (8), leading to lower rhodamine efflux (5) and increased plasma digoxin concentrations (2). In addition, early-onset Parkinson patients have higher frequency of the SNP exon 26 3435T allele compared with late-onset patients or unaffected controls (9). However, although this same allele has been associated with lower MDR1 expression in peripheral blood mononuclear cells and better response to HIV-1 drugs, it has also been associated with lower plasma concentrations of nelfinavir (7). Additionally, the SNP exon 26 3435T allele has been associated with an increased risk of nortriptyline-induced postural hypertension, although blood concentrations of nortriptyline in these individuals were not significantly different from those in individuals carrying the C allele (6). Furthermore, no association has been demonstrated between the SNP exon 26 3435CT polymorphism and cyclosporin A efficacy in renal transplant patients (10) or cyclosporin A pharmacokinetics (11) in 14 healthy individuals. Together, these studies suggest that SNP exon 26 3435CT itself may not be the causal variant producing these observed functional differences.
Analyses of other SNPs within the MDR1 gene have revealed haplotype frequency differences among populations (1)(3). The linkage disequilibrium (LD) spanning the 40 kb between SNPs exon 12 1236TC and exon 26 3435CT was also found to differ among the Chinese, Malays, and Indians (1). Variations in LD blocks among different ethnic groups and the existence of ethnic-specific SNPs suggest that the current confusing association of SNP exon 26 3435CT with different functional changes may be attributable to strong LD between SNP exon 26 3435CT and different, as yet unidentified, causal SNPs within the different LD blocks in the different study populations. Analyses of MDR1 haplotypes rather than genotypes may provide additional insights in determining associations with functional differences and may assist in discriminating between surrogate SNPs and causative variants. It would be useful to determine the haplotype structure of the entire 100-kb MDR1 gene in the different ethnic populations and to study the relationship between MDR1 haplotypes and drug response.
In this report, we describe a rapid and robust assay to simultaneously genotype seven SNPs of the MDR1 gene. The seven SNPs span 100 kb of the gene and are located in five genomic regions that are potentially functional (promoter and exons 12, 21, 26, and 28).
The five genomic segments containing the seven SNPs were amplified in a single multiplex PCR reaction. We amplified 20 ng of genomic DNA in a T3 thermal cycler (Biometra) in a total volume of 10 µL containing 0.15 pmol/µL each of the 10 primers (Table 1A
), 5 mM MgCl2, 200 µM each of the four deoxynucleotide triphosphates (dNTPs), and 0.75 U of HotStarTaq polymerase in the PCR buffer that was supplied (Qiagen). The reaction mixture was subjected to initial denaturation at 94 °C for 15 min followed by 40 step-cycles of denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, and extension at 72 °C for 1 min. This was followed by a final extension at 72 °C for 5 min. The expected PCR fragments and their sizes are shown in Fig. 1A
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Unincorporated dNTPs and excess primers were inactivated and degraded in a single-step reaction by the addition of 5 U of exonuclease I and 0.5 U of shrimp alkaline phosphatase (SAP; United States Biochemical), respectively, to 1.3 µL of the PCR product in a final volume of 2 µL. The reaction mixture was incubated at 37 °C for 15 min, and the enzymes were subsequently inactivated at 80 °C for 15 min.
The treated PCR products were then subjected to a multiplex minisequencing reaction to interrogate the seven SNP loci simultaneously. SNP-specific probing primers (or minisequencing primers) were designed to anneal to template DNA next to each SNP site such that extension by DNA polymerase added a single dideoxyribonucleoside triphosphate (ddNTP) complementary to the nucleotide at the polymorphic site (Fig. 1B
). Each of the four ddNTPs was labeled with a spectrally distinct fluorophore. To facilitate the examination of the seven SNPs simultaneously, each SNP-specific primer was designed to be a different length by the addition of variable lengths of nonhomologous d(GACT) polynucleotide tails to the 5' end of the primer. This enabled differentiation of the SNP loci based on length of the different ddNTP-extended primers (Fig. 1B
). The addition of nonhomologous tails simplified the standardization of annealing temperatures for all primers regardless of their total primer lengths. Minisequencing primers longer than 40 bases were purified by HPLC to remove incomplete primer synthesis products. Table 1B
details the sequences of the minisequencing primers and their concentrations in the final minisequencing reaction mixture.
The multiplex minisequencing reaction contained the treated multiplex PCR product (2 µL), various concentrations of minisequencing primers, and 1.3 µL of SNaPshotTM Multiplex Ready Reaction Mix (Applied Biosystems) in a total reaction volume of 5 µL. The reaction mixture was subjected to 25 single-base extension cycles of denaturation at 96 °C for 10 s, primer annealing at 53 °C for 5 s, and primer extension at 60 °C for 30 s. Thereafter, unincorporated fluorescent ddNTPs were inactivated enzymatically with 0.5 U of SAP at 37 °C for 1 h, followed by SAP deactivation at 80 °C for 15 min. The multiplex minisequencing products (0.8 µL) were then mixed with 9 µL of HiDiTM formamide and 0.5 µL of GeneScan-120 LIZ size standard (Applied Biosystems), and resolved by automated capillary electrophoresis for 25 min on an ABI PRISM 3100® Genetic Analyzer (Applied Biosystems). Analyses were performed with the GeneScanTM 3.7 application software (Applied Biosystems). The relative position of each primer peak indicated the SNP locus, whereas the peak color(s) specified the genotype. Fig. 1C
provides a diagrammatic depiction of the various colored peaks denoting the alleles at the various SNP loci and their positions relative to one another.
As illustrated in Fig. 1D
, we were able to unambiguously genotype different DNA samples at the seven MDR1 SNP loci. In samples homozygous at a particular SNP locus, either of the alternative dye-terminators attached to the SNP-specific primer, producing a single primer peak at that site on the electropherogram. Samples heterozygous for a particular locus had two different dye-terminators attach to the minisequencing primer, producing two, different-colored peaks. Migration of this same minisequencing primer differed depending on the molecular weight differences of the nucleotide–fluorescent ddNTP combinations. For example, the A-allele peak of SNP exon 21 2677GT/A migrated more slowly than the G-allele peak, so that SNP exon 21 2677GA heterozygous samples displayed two allele peaks with minimal overlap (Fig. 1Df
). However, the T-allele peak did not migrate very differently from the A-allele peak; hence SNP exon 21 2677TA heterozygous sample displayed overlapping allele peaks (Fig. 1Dd
). Peak heights of the different alleles could also differ significantly because of differences in the fluorescence intensities of the different fluorophores (Fig. 1D
). Accurate genotypes were obtained from all 100 genotype type-known samples at all seven loci that were tested (data not shown).
A major advantage of this multiplex minisequencing method of genotype determination is its cost-effectiveness. By reducing the volumes and multiplexing both the PCR and the minisequencing steps, we were able to achieve reproducible results for the seven SNPs spanning 100 kb of the MDR1 gene, starting with only 20 ng of genomic DNA and one-fourth of the recommended SNaPshot Mix, reducing the cost per reaction to approximately US $2 or less than 30 cents per SNP. Additionally, the multiplex PCR and minisequencing assay as described here is relatively rapid, with results for 96 DNA samples obtained within a single day.
Acknowledgments
This study was supported by a grant from the National Medical Research Council, Singapore (NMRC/0657/2002) to C.G.L. Lee, S.S. Chong, and E.J.D. Lee.
References
The following articles in journals at HighWire Press have cited this article:
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  Z. Wang, J. Wang, E. Tantoso, B. Wang, A. Y.P. Tai, L. L.P.J. Ooi, S. S. Chong, and C. G.L. Lee Signatures of recent positive selection at the ATP-binding cassette drug transporter superfamily gene loci Hum. Mol. Genet., June 1, 2007; 16(11): 1367 - 1380. [Abstract] [Full Text] [PDF]
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Z. Wang, B. Wang, K. Tang, E. J.D. Lee, S. S. Chong, and C. G.L. Lee A functional polymorphism within the MRP1 gene locus identified through its genomic signature of positive selection Hum. Mol. Genet., July 15, 2005; 14(14): 2075 - 2087. [Abstract] [Full Text] [PDF]
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 E.-K. Tan, D. K.-Y. Chan, P.-W. Ng, J. Woo, Y. Y. Teo, K. Tang, L.-P. Wong, S. S. Chong, C. Tan, H. Shen, et al. Effect of MDR1 Haplotype on Risk of Parkinson Disease Arch Neurol, March 1, 2005; 62(3): 460 - 464. [Abstract] [Full Text] [PDF]
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 A. M. Knaapen, H. B. Ketelslegers, R. W.H. Gottschalk, R. G.J.H. Janssen, A. D.C. Paulussen, H. J.M. Smeets, R. W.L. Godschalk, F. J. Van Schooten, J. C.S. Kleinjans, and J. H.M. Van Delft Simultaneous Genotyping of Nine Polymorphisms in Xenobiotic-Metabolizing Enzymes by Multiplex PCR Amplification and Single Base Extension Clin. Chem., September 1, 2004; 50(9): 1664 - 1668. [Full Text] [PDF]
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 C G L Lee, K Tang, Y B Cheung, L P Wong, C Tan, H Shen, Y Zhao, R Pavanni, E J D Lee, M-C Wong, et al. MDR1, the blood-brain barrier transporter, is associated with Parkinson's disease in ethnic Chinese J. Med. Genet., May 1, 2004; 41(5): e60 - e60. [Full Text] [PDF]
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K. Tang, L. P. Wong, E. J.D. Lee, S. S. Chong, and C. G.L. Lee Genomic evidence for recent positive selection at the human MDR1 gene locus Hum. Mol. Genet., April 15, 2004; 13(8): 783 - 797. [Abstract] [Full Text] [PDF]
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