Comparison between MassARRAY and pyrosequencing for CYP2C19 and ABCB1 gene variants of clopidogrel efficiency genotyping

Abstract

Clopidogrel is one of the most frequently used drugs in patients to reduce cardiovascular events. Since patients with different genetic variations respond quite differently to clopidogrel therapy, the related genetic testing plays a vital role in its dosage and genetic testing related to clopidogrel therapy is currently considered as routine test worldwide. In this study, we aim to use two different methods MALDI-TOF mass spectrometry and pyrosequencing to detect gene variant of CYP2C19 and ABCB1. Six single nucleotides polymorphisms (SNP) within CYP2C19 (Ã 2, Ã 3, Ã 4, Ã 5, Ã 17) and ABCB1 C3435T in 458 Chinese Han patients were determined using both MassARRAY and Pyrosequencing. Sanger sequencing was used for verification. Results of both methods were analyzed and compared. Allele frequencies of each SNP and distribution of different genotypes were calculated based on the MassARRAY and Sanger sequencing results. Both methods provided 100% call rates for gene variants, while results of six samples were different with two methods. With Sanger sequencing as the reference results, MassARRAY generated all the same results. The minor allele frequencies of the above six SNPs were 27.1% (CYP2C19 Ã), 5.9% (CYP2C19 Ã 3), 0% (CYP2C19 Ã 4), 0% (CYP2C19 Ã 5), 1.1% (CYP2C19 Ã 17), 40.9% (ABCB1), respectively. MassARRAY provides accurate clopidogrel related genotyping with relatively high cost-efficiency, throughput and short time when compared with pyrosequencing.

Development and validation of T‐ARMS‐PCR to detect CYP2C19*17 allele

“…In this study, no TT genotype sample was found in the 93 samples. The genotype and allele frequencies of CYP2C19*17 in our study were very similar to those of two Chinese populations . The results of T‐ARMS‐PCR and DNA sequencing were completely consistent, which further confirmed the reliability of the T‐ARMS‐PCR for CYP2C19*17 genotyping.…”

“…Primers FO, RI, and RO in Table were designed by PRIMER1 online software, and primer FI was from Scott et al T‐ARMS‐PCR was performed in a total volume of 10 μL, including 5 μL of 2 × Taq master mix, 0.4 μM of primer FO, 0.4 μM of primer RI, 0.8 μM of primer FI, 0.4 μM of primer RO, 1 μL of DNA template (including genomic DNA and plasmid DNA), and 2 μL of ddH 2 O. The PCR conditions started with the initial denaturation at 94℃ for 3 minutes, 35 cycles of 94℃ for 20 seconds, 56℃ for 20 seconds, and 72℃ for 20 seconds, followed by a final extension at 72℃ for 5 minutes.…”

“…Currently, CYP2C19*17 can be detected by polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP), TaqMan assay, allele‐specific PCR (ASP‐PCR), high‐resolution melting (HRM), pyrosequencing, MassARRAY, and DNA direct sequencing . All these methods have their respective merits and shortcomings.…”

Genetic Polymorphisms, LDL-C and sdLDL-C Help Optimize Coronary Heart Disease (CHD) Therapy

“…Before use, SLCO1B1 (rs4149056, 521T>C), CYP2C19*2 (rs4244285, c.681G>A), and CYP2C19*3 (rs4986893, c.636G>A) were detected to ensure the best treatment was administered [21][22][23]. In this study, the SNPs of these genes were detected by a Agena Bioscience MassARRAY system, which had the advantages of detecting dozens of gene loci in one sample [24], requiring fewer samples, having a lower cost, and obtaining results in a shorter time than the Sanger sequencing method [28]. The results showed that there were three genotypes associated with SLCO1B1 (521TT, 521TC, 521CC), CYP2C19*2 (681GG, 681GA, 681AA) and two genotypes associated with CYP2C19*3 (636GG, 636GA) in these 208 CHD patients (Figure 1B).…”

Molecular Genetic Techniques in Biomarker Analysis Relevant for Drugs Centrally Approved in Europe

“…The process does not require labeling the primers as it measures the molecular weight differences after primer extension [32,33]. Compared to pyrosequencing and other costly sequencing methods, it can provide a cost-efficient option for accurate targeted genotyping in guided drug therapy [34][35][36]. MLPA is a semiquantitative PCR-based method that is suitable for the assessment of genetic loci for detection of CNVs in several types of disorders [37].…”