Establishment of a forward primers-superposed amplification analysis for accurate aspirin pharmacogenomic measurement

Genotyping of gDNA rs12041331 (PEAR1), rs6065 (GP1BA), and rs730012 (LTC4S) can provide systematic guidance on the use of aspirin. However, an accurate, reliable and economical approach to simultaneous detection of the above single nucleotide polymorphisms (SNPs) is not reported. Herein, we designed and substantiated an allele-specific (AS) forward primer-superposed amplification analysis for measurement of the SNPs in PEAR1, GP1BA and LTC4S genes, in which the values of ∆Cq (differences in threshold cycles between the wild-type forward primer-based assay and the mutated-type forward primer-based assay) were employed to decide genotype. Mismatch AS forward primers were screened with the singleplex amplification analysis. Moreover, Cq extension optimized by AS forward primer superposition was observed in the selected forward primer-based triplex analysis. Further, robustness assessment of the triplex analysis showed the amplification efficiency ranging from 0.9 to 1.1. Precision test demonstrated the coefficient of variation of less than 2%. And the detective results of 189 DNA samples was completely concordant with that of commercial Sanger sequencing. In summary, we developed a simple, accurate and economical approach to genotyping of rs12041331 (PEAR1), rs6065 (GP1BA) and rs730012 (LTC4S) to provide a valuable pharmacogenomics tool for guidance of aspirin delivery.

of the above single nucleotide polymorphisms (SNPs) is not reported.Herein, we designed and substantiated an allele-specific (AS) forward primer-superposed amplification analysis for discrimination of the SNPs in PEAR1 (rs12041331), GP1BA (rs6065) and LTC4S (rs730012) genes.The results insinuated that this analysis is a valuable tool to facilitate personalized antiplatelet therapy.

Design strategy
First, mismatch AS F-primers were screened with singleplex amplification analysis.Next, the selected F-primersbased triplicate analysis was optimized by F-primer superposition to avoid undetermined results.Then, the optimized analysis was validated by robustness assessment and precision evaluation, as well as agreement analysis compared with Sanger sequencing.The values of ∆Cq (differences in threshold cycles between the wild-type F-primer-based amplification assay and the mutated-type F-primer-based amplification assay) were calculated to decide the outcomes.

DNA extraction from buccal swab
The human buccal swab samples used in this study involved 189 Chinese volunteers, which was not a train set classified by aspirin resistance.This study was approved by the Biomedical Research Ethic Committee of Shandong Provincial Hospital (No.2023-417) and has been conducted in accordance with ethical standards and guidelines of the Biomedical Research Ethic Committee of Shandong Provincial Hospital.Authors of this work extend a statement assuring that this work was conducted in accordance with the Declaration of Helsinki and obtained informed consent from all participants.Genomic DNA was extracted by using the QIAamp DNA Mini kit (Cat No. 51304, QIAGEN, Dusseldorf, Germany), and the procedure was carried out according to the instructions.DNA concentration was examined with a NanoPhotometer P360 (Implen GmbH, Munich, Germany).The quality was determined by using OD 260 / 280 ratio.Sanger sequencing was conducted by Personal Biotechnology Co., Ltd (Qingdao, China).

Primers and probes
AS F-primers, reverse primers and hydrolysis probes were designed using Primer Express 3.0, based on the information of the whole gene sequence.The second or fifth mismatch base was introduced at the 3′ end of F-primers, which were screened in a subsequent process.Probes for rs12041331 (PEAR1), rs6065 (GP1BA) and rs730012 (LTC4S) were labelled at the 5′ end with the fluorescent dye FAM, VIC, and NED, respectively, and at the 3′ end with the quencher BHQ1, BHQ1, and BHQ3, respectively.The oligonucleotide was synthesized by Sangon Biotech (Shanghai, China).

Real-time amplification assay
Triplex amplification analysis (TaqMan qPCR) was executed in a total of 20 μL reaction mixture, which contained 10 μL AceQ® Universal U + Probe Master Mix V2 (Vazyme, Nanjing, China), 0.2 μM of each wild/mutated-type F-primer, 0.2 μM of each reverse primer, 0.1 μM of hydrolysis probe and 10 ng DNA template.The singleplex amplification analysis was conducted according to the same protocol.A robotic liquid handling workstation (epMotion 5075 vt, Germany) was utilized to dispense the mix.The reaction protocols started with a contamination digestion step for 2 min at 37℃ and a pre-denaturation step for 5 min at 95 ℃, followed by 45 cycles of 95 ℃ for 10 s, and 60 ℃ for 35 s.Fluorescence data were collected at 60 ℃.These amplifications were performed on the ABI7500 Real-Time PCR Instrument (ThermoFisher Scientific Inc., MA, USA).

Data analysis
GraphPad Prism software version 9.5 (GraphPad Software, Inc., San Diego, CA) was used to conduct data analysis and graphing.

Screening of mismatch AS F-primers by singleplex amplification analysis
AS F-primers with a second or fifth mismatch base at 3' terminus were screened by detection of homozygote/heterozygote using singleplex real-time amplification analysis (10 ng DNA/test).And ΔCq (differences in threshold cycles between the wild-type F-primer-based amplification assay and the mutated-type F-primer-based amplification assay) was utilized to determine genotype.The principle for selection of the F-primer is the following: (a) no undetermined result was observed.(b) the Cq value was approximately 35 when wild homozygotes were detected in mutated-type F-primer-based amplification assay or when mutated homozygotes were measured in wild-type F-primer-based amplification assay.The original Cq values obtained from singleplex amplification analysis were shown in Table 1.And the selected sequences were shown in Table 2.
PCR-based analysis is a convenient tool to discriminate SNPs.Genetic polymorphism specific-binding molecules in PCR-based analysis comprise dsDNA-binding dye, AS probe and primer 33,34 .The dsDNA-binding dyebased high-resolution dissolution curve (HRM) assay needs specific equipment module.Besides diseconomy, it is time-consuming and laborious to discover appropriate Minor Groove Binder (MGB) probe 31,35 .For enhancement of AS primer specificity, base mismatch is more economic than locked nucleic acid (LNA) decoration 32 .In present study, the mismatch AS primers as polymorphism specific-binding molecules were screened and utilized to discriminate homozygotes/heterozygotes for rs12041331 (PEAR1), rs6065 (GP1BA) and rs730012 (LTC4S).

Development of triplex amplification analysis optimized by F-primer superposition
Based on the selected mismatch AS F-primers, we developed a triplex amplification analysis optimized by F-primer superposition.Undetermined results were observed when homozygotes were measured by un-optimized triplex amplification analysis (Fig. 1A).The extension of Cq was forced by the mismatch AS F-primer superposition, which was implemented with the addition of 0.01 μM mutated/wild-type F-primer into 0.2 μM wild/mutated-type F-primer-based amplification assay (Fig. 1B).The results showed that all outcomes of homozygotes were positive, suggesting that the mismatch AS F-primer superposition can improve detective convenience via omitting positive controls in the triplex amplification analysis.

Robustness assessment of triplex amplification analysis
To assess robustness of the triplex amplification analysis, the heterozygote was gradually reduced to generate gDNA samples at levels of 40 ng, 20 ng, 10 ng, 5 ng, 2.5 ng and 1.25 ng.Reactions were run in duplicate with three independent experiments.We used the following formula to calculate the amplification efficiency: 10 −1/ slope − 1, when the logarithm of the template concentration was plotted on the x-axis and Cq was plotted on the  www.nature.com/scientificreports/y-axis.The results demonstrated that the amplification efficiency calculated from standard curve ranged from 0.9 to 1.1 (Fig. 2), and limit of detection (LOD) was at least 1.25 ng/test.

Precision evaluation of triplex amplification analysis
The precision of the triplex amplification analysis was evaluated by detection of genomic DNA at 10 ng/test and 2.5 ng/test levels.Each specimen was tested in eight plicates by two operators with two reagent lots every day over 5 days (n = 80/specimen) at one site.A total of eighty Cq values were collected to calculate the coefficient of variance (CV).The results revealed that CV value was < 2% for all days, specimens, replicates, operators and reagent lots combined.Figure 3 shows the intra-day CV for PEAR1(rs12041331), GP1BA (rs6065) and LTC4S (rs730012).

Agreement analysis between triplex amplification analysis and Sanger sequencing
We conducted the triplex amplification analysis on each of 189 samples, in which 89, 165, and 136 specimens were defined as homozygotes for rs12041331 (PEAR1), rs6065 (GP1BA) and rs730012 (LTC4S), respectively, while 100, 24, and 53 samples were defined as heterozygotes for rs12041331 (PEAR1), rs6065 (GP1BA) and rs730012 (LTC4S), respectively.The cut-off values for genotyping were described in Table 3. Pharmacogenomics appears that some SNP s are more likely to initiate AR and adverse reactions 36,37 .Behaving as a kind of platelet transmembrane protein, the platelet endothelial aggregation receptor 1 (PEAR1) plays an important role in platelet aggregation.And genetic polymorphism of the rs12041331 in PEAR1 gene can obviously affect the inhibitive effect of aspirin on platelet aggregation 38 .Glycoprotein Ib-alpha (GP1BA) gene encodes platelet surface membrane glycoprotein (GPIb) that is a heterodimer consisting of bisulfide-linked α and β subunits, and acts as a receptor for von Willebrand factor(VWF) 39 .Genetic polymorphism of the rs6065 in GP1BA gene was evidenced to correlate aspirin resistance 40,41 .It was documented that patient carrying C-type allele for rs730012 in leukotriene C4 synthase (LTC4S) gene are prone to aspirin-induced urticaria 42,43 .Consulting to a scoring table proposed by Guangdong Pharmaceutical Association (Guangzhou, China) 32 (Table 4), a triplex amplification analysis to detect genetic polymorphism of gDNA rs12041331 (PEAR1), rs6065 (GP1BA) and rs730012 (LTC4S) was designed and substantiated in this study.The results of the agreement analysis indicated the genotyping outlined by the triplex amplification analysis is consistent with the results obtained from Sanger sequencing.In summary, we established a simple, efficient and accurate approach to the determination of genetic polymorphism of gDNA rs12041331 (PEAR1), rs6065 (GP1BA) and rs730012 (LTC4S), which can be used to guide aspirin delivery to reduce AR and adverse reaction.Adverse reaction 0 point: low risk, medication safe, no prompts 1 point: certain risk, this suggests some risk of an allergic reaction and the patient is instructed to be closely observed 2 points: high risk, it is recommended to pay close attention to the risk of adverse reactions or switch to other drugs if they occur 14:880 | https://doi.org/10.1038/s41598-024-51458-0www.nature.com/scientificreports/ F-primer: TTC TGC TGT CTC ACC TCC G FAM-CTG CTC CCC TAG AGC CCA CACTC-BHQ1 mutated-type F-primer: CTT CTG CTG TCT CAC GTC CA R-primer: CTC ACT GTG CCC CAACC GP1BA, rs6065 wild-type F-primer: CCC CAG GGC TCC TGCC VIC-ACA ACA ACT TGA CTG AGC TCC CCG C-BHQ1 mutated-type F-primer: CCC CAG GGC TCG TGAT R-primer: GAG ATT CTC CAG CCC ATT C LTC4S, rs730012 wild-type F-primer: CAG CCT GGA TGG GGATA NED-AGG TGG GTG GAG GAG TTA GCC GGG -BHQ2 mutated-type F-primer: GCC TGG ATG GTG ACC R-primer: ATG CTG GAG CCA GCCC Vol:.(1234567890)Scientific Reports | (2024) 14:880 | https://doi.org/10.1038/s41598-024-51458-0

Figure 1 .
Figure 1.Amplification plots of the triplex amplification analysis.The wild-type F-primer-based amplification assay and the mutated-type F-primer-based amplification assay were used to detect wild-type, mutated-type and heterozygous gDNA.(A) Amplification plots of un-optimized triplex amplification analyses.(B) Amplification plots of optimized triplex amplification analyses.Represented amplification plots are shown.The reaction was run in duplicate, in which 10 ng genomic DNA was inputted.Wt gDNA, wild-type genomic DNA; Mut gDNA, mutated-type genomic DNA; Het gDNA, heterozygote genomic DNA; WF primer, wild-type forward primer; MF primer, mutated-type forward primer.

Figure 2 .
Figure 2. Robustness assessment of the triplex amplification analysis.The robustness assessment was executed by employing mismatch allele-specific forward primers targeting single nucleotide polymorphism to simultaneously detect heterozygotes for PEAR1 (rs12041331), GP1BA (rs6065) and LTC4S (rs730012).Serial dilutions of heterozygote (1.25-40 ng) were measured by the triplex amplification analysis.Reactions were run in duplicate with three independent experiments.(A) Standard curve of the triplex amplification analysis.Amplification efficiency (Eff) % and R 2 are shown.Data are expressed as mean ± SE. (B) Amplification plots of the robustness assessment.Represented amplification plots are shown.WF primer, wild-type forward primer; MF primer, mutated-type forward primer.

Figure 3 .
Figure 3. Intra-day coefficient of variant (CV) for PEAR1 (rs12041331), GP1BA (rs6065) and LTC4S (rs730012).Genomic DNA at 10 ng (A) or 2.5 ng (B) level was measured in eight plicates by two operators with two reagent lots every day over 5 days (n = 80/specimen) at one site.Data were expressed as CV median in wild (a) or mutated-type (b) forward primer-based amplification assay.

Table 2 .
Sequences of primer and probe.

Table 3 .
The cut-off value for genotyping.wCq: Cq value in wild-type forward primer-based amplification assay; mCq: Cq value in mutated-type forward primer-based amplification assay