Genotyping of ABCG2 34G>A polymorphism by allele-specific PCR

Abstract

Abstract: AIM: To establish an allele-specific PCR method to genotype ABCG2 34 G>A polymorphism and verify it using 100 healthy subjects gDNA samples. METHODS: Allele-specific PCR primers with or without an additional mismatch nucleotide artificially introduced within the two bases closest to the 3' end were designed, and with different primer pairs, AS-PCR was used to detect ABCG2 34 allele of 100 gDNA samples. The Genotyping results were compared to pyrosequencing results of the same samples we did before. RESULTS: The discrimination ability of 34F/34Rn2 primer pairs was significantly better than the others, and the frequency of ABCG2 34AA, 34GA and 34GG of the 100 gDNA samples were 7%, 32% and 61% respectively, which was consistent with pyrosequencing. CONCLUSION: The results suggest the discrimination ability of AS-PCR method established in this study has been greatly improved and that will facilitate this time-saving, rapid and inexpensive technique into clinical application.

Key words: allele-specific PCR, ABCG2, single nucleotide polymorphism, BCRP

CLC Number:

R968

LI Tai-lin, XIE Hai-tang, XU Biao-bo, PENG Yan, WAN Zi-rui, SUN Hong, ZENG Ying, SHEN Jie, WANG Guo, ZHU Yuan-shan. Genotyping of ABCG2 34G>A polymorphism by allele-specific PCR[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2014, 19(10): 1111-1115.

References 15

[1]	Kobayashi D, Ieiri I, Hirota T, et al.Functional assessment of ABCG2 (BCRP) gene polymorphisms to protein expression in human placenta[J]. Drug Metab Dispos,2005, 33(1): 94-101.
[2]	Backstrom G, Taipalensuu J, Melhus H, et al.Genetic variation in the ATP-binding cassette transporter gene ABCG2 (BCRP) in a Swedish population[J]. Eur J Pharm Sci,2003, 18(5): 359-364.
[3]	Iida A, Saito S, Sekine A, et al.Catalog of 605 single-nucleotide polymorphisms (SNPs) among 13 genes encoding human ATP-binding cassette transporters: ABCA4, ABCA7, ABCA8, ABCD1, ABCD3, ABCD4, ABCE1, ABCF1, ABCG1, ABCG2, ABCG4, ABCG5, and ABCG8[J]. J Hum Genet, 2002, 47(6): 285-310.
[4]	Zamber CP, Lamba JK, Yasuda K, et al.Natural allelic variants of breast cancer resistance protein (BCRP) and their relationship to BCRP expression in human intestine[J]. Pharmacogenetics,2003, 13(1):19-28.
[5]	Mizuarai S, Aozasa N, Kotani H.Single nucleotide polymorphisms result in impaired membrane localization and reduced atpase activity in multidrug transporter ABCG2[J]. Int J Cancer, 2004,109(2): p. 238-246.
[6]	肖宇山,胡丽莉. ABCG2基因单核苷酸多态性研究进展[J]. 广东药学院学报, 2010(5): 540-545.
[7]	万子睿, 谢海棠, 俞竞, 等. 焦磷酸测序分析ABCG2基因多态性方法的建立[J]. 中国临床药理学与治疗学,2012, 17(7):779-784.
[8]	Huang, MM, Arnheim N, Goodman MF. Extension of base mispairs by Taq DNA polymerase: implications for single nucleotide discrimination in PCR[J]. Nucleic Acids Res,1992, 20(17): 4567-4573.
[9]	Newton CR, Graham A, Heptinstall LE, et al.Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS)[J]. Nucleic Acids Res,1989, 17(7): 2503-2516.
[10]	Taylor GR.Laboratory Methods for the Detection of Mutations and Polymorphisms in DNA. [M]// Little S. ARMS analysis of point mutations. Boca Raton, FL: CRC Press, 1997:45-51.
[11]	Sparreboom A, Nooter K.Does P-glycoprotein play a role in anticancer drug pharmacokinetics?[J]. Drug Resistance Updates, 2000, 3(6):357-363.
[12]	Keskitalo JE, Pasanen MK, Neuvonen PJ, et al.Different effects of the ABCG2 c. 421C>A SNP on the pharmacokinetics of fluvastatin, pravastatin and simvastatin[J]. Pharmacogenomics, 2009, 10(10):1617-1624.
[13]	Urquhart BL, Ware JA, Tirona RG, et al.Breast cancer resistance protein (ABCG2) and drug disposition: intestinal expression, polymorphisms and sulfasalazine as an in vivo probe[J]. Pharmacogenet Genomics, 2008, 18(5):439-448.
[14]	王潇潇. BCRP基因的单核苷酸多态性研究:弥漫大B细胞性淋巴瘤的易感性与生存分析[D]. 广州:中山大学, 2007.
[15]	胡丽莉. MDR l及BCRP基因多态与肿瘤易感及预后的关系[D]. 广州:中山大学, 2007.