Second-generation antipsychotics (SGAs), or atypical antipsychotics, have been widely adopted in the treatment of schizophrenia, bipolar disorder and related psychiatric disorders. SGAs possess pharmacological binding affinity to various kinds of receptor systems, including dopaminergic, histaminic, serotonergic, muscarinic and α-adrenergic receptors. Compared with first-generation antipsychotics, SGAs have higher affinity for 5-hydroxytryptamine, and lower affinity for dopamine D2 receptors.[2,3] Thus, SGAs have a lower risk of side effects, such as extrapyramidal symptoms and prolonged elevations of serum prolactin. The former manifestation includes acute dystonia, akathisia, parkinsonism and tardive syndromes, whereas the latter one may contribute to hyperprolactinemia.[5,6] It has been reported that single nucleotide polymorphisms (SNPs) in SGAs-related metabolic genes may lead to antipsychotic-induced weight gain, tardive dyskinesia and other adverse drug events. Accordingly, pharmacogenomics tests are crucial in SGAs medication instructions.
Both quetiapine and aripiprazole are metabolized by cytochrome P450 (CYP450) enzymes, which are encoded by the CYP450 superfamily. This superfamily consists of numerous subfamilies, including CYP1A2, CYP3A5 and CYP3A4, and they all interact with the metabolism of quetiapine nd aripiprazole in vivo. The SNP CYP1A2*1F, an A to C variation, could increase the activity of metabolic enzymes. The variation form G to A in CYP3A5*3 leads to a truncated protein with loss of enzyme activity. Variation from C to T in CYP3A4*1G is a high frequency allele in Asians and contributes to a higher enzyme activity. Aripiprazole is also a partial agonist at dopamine D2 receptors. Ankyrin repeat and kinase domain containing 1 (ANKK1) is closely linked to the dopamine D2 receptor, meanwhile, ANKK1 Taq1A polymorphism reduces the expression of numerous proteins associated with drug metabolism.[12,13] There is a strong body of evidence that suggests these 4 SNP alleles are crucial for the metabolism of quetiapine and aripiprazole. Therefore, the development of convenient and efficient methods for the simultaneous detection of these SNP alleles would be significant.
Various kinds of SNP detection methods can be adopted to perform pharmacogenomics tests, including Sanger sequencing, allele-specific polymerase chain reaction (PCR) and biological mass spectrometry. Every method has its own advantage and disadvantage. High-resolution melting (HRM) and multicolor melting curve assay (MMCA) are ideal methods in SNP detection. These two methods are fast, automated and have closed-tube formats, allowing homozygous and heterozygous variation to be identified in a single tube. HRM, which uses a saturated fluorescent dye, is cost-saving because no fluorescence labeled probes or special instrument are needed. However, fluorescence labeled probes are needed in MMCA, which raises the price of this method. Unlike TaqMan assay, only one kind of probe is enough to detect one SNP allele. Because of this characteristic, probes targeting different SNP alleles can be labeled with different colors, so that they can be detected in different fluorescence channels.
The aim of the present study was to develop a rapid, reliable and operationally convenient technique to detect four SNP alleles: CYP1A2*1F (rs762551), CYP3A5*3 (rs776746), CYP3A4*1G (rs2242480), ANKK1 Taq1A (rs1800497). The former two SNPs were related to the metabolism of quetiapine, whereas the latter 2 were related to the metabolism of aripiprazole. HRM and MMCA were demonstrated as suitable methods in this report to perform the pharmacogenomics detection of these two drugs.
Materials and methods
Human blood samples were obtained with written informed consent. All experimental protocols were approved by the Ethics Committee of the Shanghai Children's Medical Center, China (SCMC-201015) on November 22, 2010 and carried out in accordance with the Declaration of Helsinki.
This study was based on 240 whole genome DNA samples from healthy people without genetic diseases (107 females and 133 males). All whole blood samples were obtained from Shanghai Children's Medical Center with EDTA as anticoagulant. Genomic DNA was extracted from a 500-μL whole blood by automatic DNA extraction system Lab-Aid 820 (Zeesan Biotech Co., Xiamen, Fujian Province, China). The extracted DNA was quantified using Nanodrop 2000 (Thermo Fisher, Waltham, MA, USA).
Primers and probes
The Primer3 (v.0.4.0) online software (http://bioinfo.ut.ee/primer3-0.4.0/primer3/) was used to design primers for HRM and MMCA detection. The Mfold online software (http://unafold.rna.albany.edu/?q=mfold) was used to predict the secondary structures of molecular beacons of the four alleles. The uMELT online software (https://www.dna.utah.edu/umelt/umelt.html) was used to predict amplicon melting curves of HRM. The primer sequences used in HRM and MMCA are shown in Tables 1–3. The primers were synthetized by Sangon Biotech (Shanghai, China).
Both the PCR and HRM steps were performed on the Rotor-gene Q (Qiagen, Duesseldorf, Germany). PCR was performed in 10 μL volumes containing 2 mM Mg2+, 200 μM of each dNTP, 0.4U of Klentaq1™ polymerase (Ab Peptides, St. Louis, MO, USA), 1× LCGreen® Plus dye (BioFire Defense, Salt Lake City, UT, USA), 0.5 μM of one of primer pairs and genomic DNA was 25 ng. The PCR amplification protocol began with an initial denaturation at 95°C for 3 minutes, followed by 40 cycles of 10 seconds denaturation at 95°C and 25 seconds annealing-extension at 65°C. After amplification, the samples were cooled to 55°C then melted by raising the temperature from 65°C to 90°C at the rate of 0.15°C/s, and the fluorescence intensity was recorded every 0.3°C to plot the HRM curve.
Both the PCR and melting curve steps were performed on the MicPCR instrument (Bio Molecular Systems, Queensland, Australia). PCR was performed in 10 μL volumes containing 2 mM Mg2+, 200 μM of each dNTP, 0.4U of Klentaq1™ polymerase (Ab Peptides), 0.9 μM of CYP1A2*1F forward primer, 0.09 μM of CYP1A2*1F reverse primer, 0.27 μM of CYP1A2*1F probe, 0.09 μM of CYP3A5*3 forward primer, 0.9 μM of CYP3A5*3 reverse primer, 0.09 μM CYP3A5*3 probe, 0.9 μM of CYP3A4*1G forward primer, 0.09 μM of CYP3A4*1G reverse primer, 0.27 μM of CYP3A4*1G probe, 0.9 μM of ANKK1 Taq1A forward primer, 0.09 μM of ANKK1 Taq1A reverse primer, 0.09 μM of ANKK1 Taq1A probe, and genomic DNA was 25ng. The PCR amplification protocol began with an initial denaturation at 95°C for 3 minutes, followed by 55 cycles of 15 seconds denaturation at 95°C, 15 seconds annealing at 55°C and 20 seconds extension at 76°C, then held at 76°C for 3 minutes after the cycles. After amplification, the samples were held at 95°C for 1minute and cooled to 45°C for 3 minutes, then melted by raising the temperature from 45°C to 85°C at the rate of 0.15°C/s, and the fluorescence intensity was recorded every 0.3°C to plot the melting curve.
The primers for Sanger sequencing are shown in Table 4. PCR was performed on EDC-810 (Eastwin, Beijing, China) in 25 μL volumes, containing 12.5 μL Premix Ex Taq™ and 0.4 μM of one of the primer pairs and genomic DNA was 25ng. Sequencing was performed by fluorescent dideoxy-nucleotide termination on the ABI Prism 3730xl DNA Sequencer (Applied Biosystems, Waltham, MA, USA) at Map Biotech (Shanghai, China). The sequence data were analyzed with Bioedit V188.8.131.52 (http://www.mbio.ncsu.edu/bioedit/bioedit.html).
Before HRM assay, samples were amplified by simplex PCR, and consequently products encompassed fragments of one specific SNP allele. Each sample was amplified in four independent reactions to product fragments containing all the SNP alleles and immediately all products were simultaneously detected by the HRM procedure. The shape of the melting curve is dependent on thermal stability which is affected by single nucleotide variations. Data were analyzed and plotted in Figure 1, which clearly distinguishes the wild type, heterozygous and homozygous samples with reference genotypes AA or TT.
In this study, a molecular beacon was chosen as the fluorescent probes in MMCA. It is a type of dual-labeled DNA probe, which has a fluorophore and a quencher on either side. To make sure the 5′ side fluorophore and the 3′ side quencher are close to each other in the free state, Mfold was tasked to predict the secondary structure of the probes. Thus, quenching certainly occurs in the free state. Each probe was labeled with a different colored fluorophore, which was detected in each corresponding channel of MicPCR (Table 3). Thus, quenching certainly occurs in the free state. Accordingly, the four target SNP alleles can be amplified and genotyped in one reaction (Fig. 2) but this required a quadruplex PCR and a 4 times per detection to record all the SNP alleles data. A primer ratio of 10:1 was adopted for each of the primer pairs to acquire more DNA single strands matched to the probes. Only one negative derivative peak appeared in the wild type and homozygous samples. Compared with wild type samples, peaks of homozygous samples were shifted 5 to 9°C because of a mismatch or match of the hybridization. In contrast, heterozygous samples have two negative derivative peaks, either at the same position as the wild type or homozygous peak, because two kinds of nucleotide existed in the same SNP site. Consequently, three kinds of genotypes can be identified clearly.
Genotyping results of HRM and MMCA were verified by Sanger Sequencing, the gold standard of SNP detection. Both methods have 100% coincidence with Sanger sequencing. Distribution of the 4 SNP alleles is shown in Table 5.
Two SNP detection methods, HRM and MMCA, were used to genotype these 4 alleles. A saturated fluorescent dye was adopted to produce the fluorescent signal in HRM, the fluorescent intensity enhanced when bound to DNA double strands and weakened in the free state. Alternatives to HRM have been developed where the fluorescent dye was replaced by labeled probes, for example, self-quenched probe and molecular beacon. In this study, a molecular beacon was adopted to perform SNP detection.
A molecular beacon is a kind of dual-labeled probe that has a stem-loop secondary structure. In the free state, a molecular beacon has a stem-loop structure to maintain the fluorophore and quencher close to each other. This assures that the fluorescence signal will be quenched. When the molecular beacon hybridizes with single-strand DNA, the subsequent double helix leads to the destruction of fluorescence quenching and raises the fluorescent signal. To increase the hybridization efficiency between the molecular beacon and single-strand DNA, asymmetric PCR was used to produce more single-strand DNA. Molecular beacons designed for the four SNP alleles were labeled by diverse fluorophores, and each labeled probe was detected by a corresponding channel. Thus, all SNP alleles could be detected separately in different fluorescence channels. Two kinds of quenchers, BHQ1 and BHQ2, were used, depending on its quenching range. The range of BHQ1, 480 to 580 nm, corresponded to FAM and HEX, whereas 520 to 600 nm of BHQ2 corresponded to ROX and Cy5.
The variation in thermodynamic stability is the key factor for SNP detection based on melting curves. In HRM assay, base pair changing between A-T and G-C would contribute to apparent variation in melting curves because of the number of hydrogen bonds changing between 2 and 3. However, base changing between A and T or G and C is not ideal in this kind of amplicon HRM assay because the thermodynamic stability changed little. Fortunately, in this study, none of these four SNP alleles changed without hydrogen bond variation, so amplicon HRM could be used to perform the detection. This type of limitation does not occur in MMCA. No matter how the single base site changes, a mismatch will happen between single strand amplicons and fluorescent probes. This results in great changes in thermodynamic stability.
In HRM assay, short amplicons can increase the proportion of the single base and facilitate more obvious variations of the melting curves for easier SNP detection. Thus, we shortened the length of all amplicons as much as possible to between 88 and 100 bp for HRM. Longer amplicons are acceptable in MMCA because the melting curves depend on the area of the hybridizing fluorescent probes. Because long amplicons can expand the distance between SNP sites and probes, the probes and primers will not overlap, thus avoiding hinderance to the amplification. Compared with fluorescent probes, fluorescent dyes can combine with nonspecific PCR products and produce fluorescent signals in HRM. To reduce the number of nonspecific products, a high annealing temperature was adopted.
In MMCA, the Tm difference of four primer pairs was less than 5°C, so all SNP alleles could be amplified in the same reaction. Although it is a single target method, choosing primer pairs with a similar Tm enabled a design that achieved identical conditions for each detection. Thus, four targets can be detected simultaneously. As asymmetric PCR is inefficient, the annealing temperature was decreased to 55°C to produce more amplicons. In the annealing stage, molecular beacons may hybridize with DNA templates, leading to hindrance of the next extension stage. Therefore, a slightly higher extension temperature (76°C) than conventional (72°C) was used to relieve the inhibition.
Sanger sequencing, often regarded as the gold standard in SNP detection, was used to validate the results of the double-blind studies. In all 240 samples tested, the results of HRM and MMCA had 100% concordance with Sanger sequencing. It takes nearly 96 minutes to perform an HRM assay, whereas MMCA needs 133 minutes. The time difference results from the more cycles to increase amplicons needed by asymmetric PCR. HRM and MMCA, 2 closed-tube methods, were not only time-saving compared with Sanger sequencing, but also easy to operate and were free of aerosol pollution because the processes took place in sealed closed tubes. Both are also cost-saving methods because no special reagents or special instruments are needed apart from standard PCR systems and instruments. In particular, the usage of fluorescent dyes in HRM is cheaper than probes used in MMCA, and the HRM method is easy to be developed. Therefore, HRM is less cost when a few sample needs to be detected. The advantage of MMCA is it is a multiplex assay, 4 SNP alleles were genotyped in one reaction, which significantly increased the efficiency of detection.
In conclusion, 2 SNP detection methods were developed to genotype four SNP alleles related to the metabolism of two SGA drugs: quetiapine and aripiprazole. Because fluorescent probes cost much than primer pairs and fluorescent dyes, HRM is more economical than MMCA. However, MMCA is suitable for multiplex assay owing to its multiple detection channels. Both methods have high throughput, are rapid, convenient, inexpensive and accurate, and eminently suitable for medication guidance of quetiapine and aripiprazole.
QF and LZ contributed to the conception and design of the study. XyZ and LZ contributed to the HRM experiment. GY and XqZ analyzed the data and wrote the manuscript. All authors approved the final version of the manuscript.
Institutional review board statement
This study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Shanghai Children's Medical Center, China (SCMC-201015) on November 22, 2010.
Declaration of participant consent
The authors certify that they have obtained all appropriate consent forms from the participants. In the forms, the participants have given their consent for their images and other clinical information to be reported in the journal. The participants understand that their names and initials will not be published and due efforts will be made to conceal their identity.
Conflicts of interest
The authors declare that they have no conflicts of interest.
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