[an error occurred while processing this directive]
en

Duplex genotyping of CYP2C19*2 and CYP2C19*3 by high-resolution melting curve analysis

Expand
  • State Key Laboratory of Genetic Engineering, School of Life Science, Fudan University, Shanghai 200433, China

Received date: 2013-04-01

  Revised date: 2013-04-25

  Online published: 2013-07-25

Abstract

Clopidogrel is a widely used anti-platelet agent for the prevention of arterial thrombosis. It has been suggested that clopidogrel may be less effective in inhibiting platelet aggregation among patients who are carriers of CYP2C19*2 and CYP2C19*3, two loss-of-function CYP2C19 alleles, which are associated with reduced conversion of clopidogrel to its active metabolite. The objective of this research was to develop a simple and accurate method for genotyping of CYP2C19*2 and CYP2C19*3 simultaneously in one closed-tube using high-resolution melting curve (HRM) analysis. Two amplicons bracketing CYP2C19*2 and CYP2C19*3 gene variants were designed, and AT- or GC-rich 5′ tails were added to selected primers to ensure two different amplicons with non-overlapping melting curves. Sixty-four random DNA samples were all fast and sensitively genotyped by HRM analysis. This method was validated by DNA sequencingtechnique, and genotypes obtained using the HRM approach perfectly matched the genotypes obtained by DNA sequencing technique. Therefore, this HRM-based assay allows simple and accurate duplex genotyping of CYP2C19*2 and CYP2C19*3 simultaneously in one closed-tube. This method is expected to be applied in clinical laboratory to guide indi-vidual dosage design of clopidogrel.

Cite this article

CAO Chun-Ge SUN Hai-Yan ZHOU Fang-Fang WANG Shi-Ming CHEN Hong-Yan LU Da-Ru . Duplex genotyping of CYP2C19*2 and CYP2C19*3 by high-resolution melting curve analysis[J]. Hereditas(Beijing), 2013 , 35(7) : 923 -930 . DOI: 10.3724/SP.J.1005.2013.00923

References

[1] Connolly SJ, Yusuf S, Camm J, Chrolavicius S, Commer-ford P, Flather M, Hart RG, Hohnloser SH, Joyner C, Pfeffer M, Gaudin C, Blumenthal M, Marchese C, Pogue J, Hart R, Hohnloser S, Anand I, Arthur H, Avezum A, Budaj A, Zoble RG. Effect of clopidogrel added to aspirin in pa-tients with atrial fibrillation. N Engl J Med, 2009, 360(20): 2066-2078.
[2] Yusuf S. Effects of clopidogrel in addition to aspirin in patients with acute coronary syndromes without ST-segment elevation. N Engl J Med, 2001, 345(23): 1716-1716.
[3] Savi P, Combalbert J, Gaich C, Rouchon MC, Maffrand JP, Berger Y, Herbert JM. The antiaggregating activity of clopidogrel is due to a metabolic-activation by the hepatic cytochrome P450-1A. Thromb Haemost, 1994, 72(2): 313-317.
[4] Ferreiro JL, Angiolillo DJ. Clopidogrel response variability: Current status and future directions. Thromb Haemost, 2009, 102(1): 7-14.
[5] De Morais SMF, Goldstein JA, Xie HG, Huang SL, Lu YQ, Xia H, Xiao ZS, Ile N, Zhou HH. Genetic-analysis of the s-mephenytoin polymorphism in a chinese population. Clin Pharmacol Ther, 1995, 58(4): 404-411.
[6] Demorais SMF, Wilkinson GR, Blaisdell J, Nakamura K, Meyer UA, Goldstein JA. The major genetic-defect responsible for the polymorphism of s-mephenytoin metabolism in humans. J Biol Chem, 1994, 269(22): 15419-15422.
[7] Xiao ZS, Goldstein JA, Xie HG, Blaisdell J, Wang W, Ji-ang CH, Yan FX, He N, Huang SL, Xu ZH, Zhou HH. Differences in the incidence of the CYP2C19 polymorphism affecting the S-mephenytoin phenotype in Chinese Han and Bai populations and identification of a new rare CYP2C19 mutant allele. J Pharmacol Exp Ther, 1997, 281(1): 604-609.
[8] Wittwer CT. High-resolution DNA melting analysis: advancements and limitations. Hum Mutat, 2009, 30(6): 857-859.
[9] Liew M, Pryor R, Palais R, Meadows C, Erali M, Lyon E, Wittwer C. Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. Clin Chem, 2004, 50(7): 1156-1164.
[10] Gonen D, Veenstra-VanderWeele J, Yang Z, Leventhal BL, Cook EH. High throughput fluorescent CE-SSCP SNP genotyping. Mol Psychiatry, 1999, 4(4): 339-343.
[11] Fukuen S, Fukuda T, Maune H, Ikenaga Y, Yamamoto I, Inaba T, Azuma J. Novel detection assay by PCR-RFLP and frequency of the CYP3A5 SNPs, CYP3A5*3 and *6, in a Japanese population. Pharmacogenetics, 2002, 12(4): 331-334.
[12] Wolford JK, Blunt D, Ballecer C, Prochazka M. High-throughput SNP detection by using DNA pooling and denaturing high performance liquid chromatography (DHPLC). Hum Genet, 2000, 107(5): 483-487.
[13] Steemers FJ, Chang WH, Lee G, Barker DL, Shen R, Gunderson KL. Whole-genome genotyping with the single-base extension assay. Nat Methods, 2006, 3(1): 31-33.
[14] Iannone MA, Taylor JD, Chen JW, Li MS, Rivers P, Slentz-Kesler KA, Weiner MP. Multiplexed single nucleo-tide polymorphism genotyping by oligonucleotide ligation and flow cytometry. Cytometry, 2000, 39(2): 131-140.
[15] Hirotsu N, Murakami N, Kashiwagi T, Ujiie K, Ishimaru K. Protocol: a simple gel-free method for SNP genotyping using allele-specific primers in rice and other plant species. Plant Methods, 2010, 6(12), doi: 10.1186/1746-4811-6-12.
[16] Griffin TJ, Smith LM. Single-nucleotide polymorphism analysis by MALDI-TOF mass spectrometry. Trends Bio-technol, 2000, 18(2): 77-84.
[17] Mhlanga MM, Malmberg L. Using molecular beacons to detect single-nucleotide polymorphisms with real-time PCR. Methods, 2001, 25(4): 463-471.
[18] Nomoto K, Tsuta K, Takano T, Fukui T, Yokozawa K, Sakamoto H, Yoshida T, Maeshima AM, Shibata T, Furuta K, Ohe Y, Matsuno Y. Detection of EGFR mutations in ar-chived cytologic specimens of non-small cell lung cancer using high-resolution melting analysis. Am J Clin Pathol, 2006, 126(4): 608-615.
[19] Lochlainn SO, Amoah S, Graham NS, Alamer K, Rios JJ, Kurup S, Stoute A, Hammond JP, Ostergaard L, King GJ, White PJ, Broadley MR. High Resolution Melt (HRM) ana
Outlines

/