Restricted accessResearch articleFirst published online 2009-05
A Fast SSP-PCR Method for Genotyping the ATP-Binding Cassette Subfamily B Member 1 Gene C3435T and G2677T Polymorphisms in Chinese Transplant Recipients
P-glycoprotein, the product of the ATP-binding cassette subfamily B member 1 (ABCB1) gene (or the so-called multidrug resistance 1 gene), is an ATP-driven efflux pump contributing to the pharmacokinetics as well as the pharmacokinetics of drugs that are P-glycoprotein substrates, such as tacrolimus. This paper describes the development of a new method for detection of the 3435C/T and 2677G/T/A single nucleotide polymorphisms of the ABCB1 gene. The method is a simple sequence-specific primer polymerase chain reaction (SSP-PCR).
Methods
158 Chinese health checkup examinees and 214 transplant recipients were included in the study. Genomic DNA was extracted from peripheral blood and amplified with SSP-PCR to detect the 3435C/T and 2677G/T/A mutations in ABCB1. The SSP-PCR condition was optimized, and the PCR results were compared with those of DNA sequencing.
Results
In the optimized condition, the two polymorphisms could be clearly distinguished after one-step PCR and electrophoresis. The ABCB1 3435C/T and 2677G/T/A genotypes of the subjects were scanned, and allele-specific bands were successfully amplified by SSP-PCR, which were in full accordance with the results of sequencing.
Conclusion
As a fast, simple and inexpensive genotyping tool, the method would be practicable in large clinical studies on interindividual pharmacokinetics.
ThiebautF., TsuruoT., HamadaH., ThiebautF., GottesmanM.M., PastanI., WillinghamM.C.: Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues. Proc Natl Acad Sci USA, 84: 7735–7738, 1987.
2.
Cordon-CardoC., O'BrienJ.P., CasalsD., Rittman-GrauerL., BiedlerJ.L., MelamedM.R., BertinoJ.R.: Multidrug-resistance gene (P-glycoprotein) is expressed by endothelial cells at blood-brain barrier sites. Proc Natl Acad Sci USA, 86: 695–698, 1989.
3.
SeeligA.: A general pattern for substrate recognition by P-glycoprotein. Eur J Biochem, 251: 252–261, 1998.
4.
KimR.B.: Drugs as P-glycoprotein substrates, inhibitors, and inducers. Drug Metab Rev, 34: 47–54, 2002.
5.
AnglicheauD., VerstuyftC., Laurent-PuigP., BecquemontL., SchlageterM.H., CassinatB., BeauneP., LegendreC., ThervetE.: Association of the multidrug resistance-1 gene single-nucleotide polymorphisms with the tacrolimus dose requirements in renal transplant recipients. J Am Soc Nephrol, 14: 1889–1896, 2003.
6.
HoffmeyerS., BurkO., von RichterO., ArnoldH.P., Brock-mollerJ., JohneA., CascorbiI., GerloffT., RootsI., EichelbaumM., BrinkmannU.: Functional polymorphisms of the human multidrug-resistance gene: multiple sequence variations and correlation of one allele with P-glycoprotein expression and activity in vivo. Proc Natl Acad Sci USA, 97: 3473–3478, 2000.
7.
LiD., GuiR., JinL., HuangZ., XinminN.: Tacrolimus dosing in Chinese renal transplant patients is related to MDR1 gene C3435T polymorphisms. Transplant Proc, 38: 2850–2852, 2006.
8.
FooteC.J., GreerW., KiberdB.A., FraserA., LawenJ., NashanB., BelitskyP.: MDR1 C3435T polymorphisms correlate with cyclosporine levels in de novo renal recipients. Transplant Proc, 38: 2847–2849, 2006.
9.
TanabeM., IeiriI., NagataN., InoueK., ItoS., KanamoriY., TakahashiM., KurataY., KigawaJ., HiguchiS., TerakawaN., OtsuboK.: Expression of P-glycoprotein in human placenta: relation to genetic polymorphism of the multidrug resistance (MDR)-1 gene. J Pharmacol Exp Ther, 297: 1137–1143, 2001.
10.
KimR.B., LeakeB.F., ChooE.F., DresserG.K., KubbaS.V., SchwarzU.I., TaylorA., XieH.G., McKinseyJ., ZhouS., LanL.B., SchuetzJ.D., SchuetzE.G., WilkinsonG.R.: Identification of functionally variant MDR1 alleles among European Americans and African Americans. Clin Pharmacol Ther, 70: 189–199, 2001.
11.
AnglicheauD., ThervetE., EtienneI., Hurault De LignyB., Le MeurY., TouchardG., BüchlerM., Laurent-PuigP., TregouetD., BeauneP., DalyA., LegendreC., MarquetP.: CYP3A5 and MDR1 genetic polymorphisms and cyclosporine pharmacokinetics after renal transplantation. Clin Pharmacol Ther, 75: 422–433, 2004.
12.
SaitoK., MiyakeS., MoriyaH., YamazakiM., ItohF., ImaiK., KurosawaN., OwadaE., MiyamotoA.: Detection of the four sequence variations of MDR1 gene using TaqMan MGB probe based real-time PCR and haplotype analysis in healthy Japanese subjects. Clin Biochem, 36: 511–518, 2003.
13.
JohneA., KopkeK., GerloffT., MaiI., RietbrockS., MeiselC., HoffmeyerS., KerbR., FrommM.F., BrinkmannU., EichelbaumM., BrockmöllerJ., CascorbiI., RootsI.: Modulation of steady-state kinetics of digoxin by haplotypes of the P-glyco-protein MDR1 gene. Clin Pharmacol Ther, 72: 584–594, 2002.
14.
KurataY., IeiriI., KimuraM., MoritaT., IrieS., UraeA., OhdoS., OhtaniH., SawadaY., HiguchiS., OtauboK.: Role of human MDR1 gene polymorphism in bioavailability and interaction of digoxin, a substrate of P-glycoprotein. Clin Pharmacol Ther, 72: 209–219, 2002.
15.
ErlichH.A., GelfandD., SninskyJ.J.: Recent advances in the polymerase chain reaction. Science, 252: 1643–1651, 1991.
16.
HamajimaN., SaitoT., MatsuoK., MatsuoK., KozakiK., TakahashiT., TajimaK.: Polymerase chain reaction with confronting two-pair primers for polymorphism genotyping. Jpn J Cancer Res, 91: 865–868, 2000.
17.
HamajimaN.: PCR-CTPP: a new genotyping technique in the era of genetic epidemiology. Expert Rev Mol Diagn, 1: 119–123, 2001.
18.
HamajimaN., SaitoT., MatsuoK., TajimaK.: Competitive amplification and unspecific amplification in polymerase chain reaction with confronting two-pair primers. J Mol Diagn, 4: 103–107, 2002.
19.
SasamotoH., NagasakaT., NotoharaK., OzakiK., IsozakiH., TanakaN., MatsubaraN.: Allele-specific methylation analysis on upstream promoter region of H19 by methylation-specific PCR with confronting two-pair primers. Int J Oncol, 25: 1273–1278, 2004.
20.
ZhangW.X., ChenG.L., WeiZ., TanZ.R., JieL., GanZ., HuD.L., ZhouH.H.: MDR1 genotype do not influence the absorption of a single oral dose of 100 mg talinolol in healthy Chinese males. Clin Chim Acta, 359: 46–52, 2005.
21.
LeeS.S., KimS.Y., KimW.Y., Thi-LeH., YoonY.R., YeaS.S., ShinJ.G.: MDR1 genetic polymorphisms and comparison of MDR1 haplotype profiles in Korean and Vietnamese populations. Ther Drug Monit, 27: 531–535, 2005.
22.
CascorbiI., GerloffT., JohneA., MeiselC., HoffmeyerS., SchwabM., SchaeffelerE., EichelbaumM., BrinkmannU., RootsI.: Frequency of single nucleotide polymorphisms in the P-glycoprotein drug transporter MDR1 gene in white subjects. Clin Pharmacol Ther, 69: 169–174, 2001.
23.
HitzlM., DrescherS., van der KuipH., SchäffelerE., FischerJ., SchwabM., EichelbaumM., FrommM.F.: The C3435T mutation in the human MDR1 gene is associated with altered efflux of the P-glycoprotein substrate rhodamine 123 from CD56+ natural killer cells. Pharmacogenetics, 11: 293–298, 2001.
24.
PawlikA., Baskiewicz-MasiukM., MachalinskiB., KurzawskiM., Gawronska-SzklarzB.: Involvement of C3435T and G2677T multidrug resistance gene polymorphisms in release of cytokines from peripheral blood mononuclear cells treated with methotrexate and dexamethasone. Eur J Pharmacology, 528: 27–36, 2005
