Abstract
Objectives
This study aims to develop a simple polymerase chain reaction (PCR) sequence-specific primer method, which will be used to genotype HLA-B*15:02, HLA-B*15:13, and HLA-B*15:21.
Methods
DNA was extracted from whole blood using a commercial DNA extraction kit. New specific primers were designed. The PCR was optimized for reproducibility and specificity. Parameters investigated included concentrations of MgCl2, primers concentration, and annealing temperature, to produce specific bands of interest. Known DNA samples were selected at random and tested using the PCR method.
Results
Simultaneously six duplex PCRs were successfully optimized using 1.0 mM MgCl2 and an annealing temperature of 62 °C. The method was also reproducible and specific. The results from the method showed 100% concordance with known DNA samples.
Conclusions
This study has successfully developed a simple and specific method to simultaneously genotype HLA-B*15:02, HLA-B*15:13, and HLA-B*15:21 using in-house developed PCR-SSP.
Introduction
Severe cutaneous adverse drug reaction (SCAR) is among the many forms of adverse drug reactions (ADRs) [1]. Although SCARs are rare, they contribute to a high mortality rate. Steven-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN), drug reaction with eosinophilia and systemic symptoms (DRESS), and acute generalised exanthematous pustulosis (AGEP) are among the common SCARs [2, 3]. The incidence of SCARs is commonly associated with drugs such as antiepileptic drugs, allopurinol, antibiotics, and antiretroviral drugs [1]. Several genetic variations, especially in the gene encodes for the human leukocyte antigen B, HLA-B, have been significantly associated with a high risk of developing SCAR. The most common allele markers associated with SCARs are the HLA-B*15:02, HLA-B*15:13, HLA-B*15:21, and HLA-A*31:01; each is prevalent in different populations [4], [5], [6]. Moreover, a guideline for HLA allele screening has been recommended by the Clinical Pharmacogenetics Implementation Consortium to prevent the use of carbamazepine and phenytoin among Asian patients with risky alleles [7]. Such screening program has been shown to be successful and cost-effective in reducing the rate of SJS/TEN in Taiwan and Singapore [8, 9]. To reduce the likelihood of SCARs brought on by a particular medicine, an optimised HLA genotyping procedure is essential.
Polymerase chain reaction-sequence specific primer (PCR-SSP)
The basis of the PCR-SSP technique is that a perfectly matched primer is more efficient in a PCR reaction than one or more mismatched primers. Specificity is determined using sequence-specific primers, where a 3′ single-base mismatch inhibits the initiation of a non-specific reaction. Therefore, only the required allele will be amplified, and then the amplified product can be detected by agarose gel electrophoresis.
PCR-SSP has been used for genotyping the patients specifically for the identification of HLA-B*15:02 [10], HLA-B*15:13, and HLA-B*15:21 (Supplementary Material 1). Genomic DNAs were extracted from whole blood using NucleoSpin® Blood Genomic DNA Purification Kit (MACHEREY-NAGEL GmbH & Co. KG, Germany). For the genotyping of HLA-B*15:02, four pairs of primers were used based on the study by Bunce et al. Meanwhile, for the other two alleles, two pairs of SSP were designed to distinguish the carrier of HLA-B*15:13 and HLA-B*15:21. Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast) was used to verify the sequences for their specificity (Supplementary Material 2). Table 1 shows the sequences of HLA-specific primers and control primers that were utilised in this genotyping process.
The HLA-specific primers and control primers that were utilised in the HLA genotyping process for this study.
| Primers | Sequence varianta | ||
|---|---|---|---|
| F1R1 | Forward primer 1 (F1) | 5′-CGA GAG AGC CTG CGG AAC-3′ | None |
| Reverse primer 1 (R1) | 5′-GCC CAC TTC TGG AAG GTT CT-3′ | ||
| F2R2 | Forward primer 2 (F2) | 5′-CGC GAG TCC GAG GAT GGC-3′ | 204A>G, 205G>A, 206A>T, 209A>C |
| Reverse primer 2 (R2) | 5′-GCA GGT TCC GCA GGC TCT-3′ | None | |
| F3R3 | Forward primer 3 (F3) | 5′-ACC GGA ACA CAC AGA TCT C-3′ | 272A>C |
| Reverse primer 3 (R3) | 5′-GAG CCA CTC CAC GCA CAG-3′ | 560A>T, 559G>C | |
| F4R4 | Forward primer 4 (F4) | 5′-GGA GTA TTG GGA CCG GAA C-3′ | None |
| Reverse primer 4 (R4) | 5′-GCC ATA CAT CCT CTG GAT GA-3′ | 369C>T, 363C>G, 355C>A, 353C>T | |
| F5R5 | Forward primer 5 (F5) | 5′-GAG GTA TTT CTA CAC CGC CA-3′ | 103T>G, 106G>A |
| Reverse primer 5 (R5) | 5′-TGT AGT AGC GGA GCG CGA-3′ | 319G>C, 317G>T, 314T>C, 313C>G, 311A>T | |
| F6R3 | Forward primer 6 (F6) | 5′-ACC GGA ACA CAC AGA TCT G-3′ | 272A>G |
| Reverse primer 3 (R3) | 5′-GAG CCA CTC CAC GCA CAG-3′ | 560A>T, 559G>C | |
| Control primer | Forward primer (FC) | 5′-TGC CAA GTG GAG CAC CCA A-3′ | HLA-DRB1 |
| Reverse primer (RC) | 5′-GCA TCT TGC TCT GTG CAG AT-3′ | ||
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aThe sequence variants compared to the IMGT/HLA reference sequence, B*07:02:01:01 for the HLA-B gene. The bold nucleotides are the position of the variants in the sequence.
In this study, six separated duplex PCRs were developed and optimized. The initial PCR condition was based on a previous study [11]. A temperature gradient was performed to obtain the optimum temperature for the annealing temperature (Ta). Other factors, such as the concentration of MgCl2 and primers, were also evaluated. The PCR reactions were carried out using Biorad T100 thermal cycler (CA, USA). All PCR reagents and chemicals used were from Promega (Wisconsin, USA). PCR products were analyzed by separation on 2% agarose gels stained with green DNA dye, followed by direct visualization over an ultraviolet transilluminator. The specificity of the test method was also tested using known DNA samples donated from Mahidol University, Bangkok, Thailand.
All parameters, such as the Ta, concentration of MgCl2, and primers, were successfully optimized (Figure 1). The optimized PCR reagents are shown in Table 2. Basically, the PCR mixture was split into two parts: The master mix and the primer mix. The master mix and the primer mix were combined into a PCR tube before the required amount of DNA sample was added at the final stage of preparation. The optimized thermal cycling conditions are summarized in Table 3. All amplicons were successfully amplified simultaneously and in 100% concordance with previously known samples. The result interpretation for each genotype is shown in Supplementary Material 3. This method represents an important prerequisite for further evaluating the clinical impact of the HLA-B genotypes in a larger population.
PCR optimization. (A) Shows the effects of different concentrations of MgCl2 used in this study. (B) Shows the effect of annealing temperature. Lane 7–100 bp DNA ladder; lane 1,8 is for F1R1 (1331 bp); lane 2,9 F2R2 (125 bp); lane 3,10 F3R3 (568 bp); lane 4,11 F4R4 (371 bp); lane 5,12 F5R5 (242 bp) and lane 6,13 F6R3 (568 bp). All the reactions were included with the control primer pair (785 bp). These DNA samples resulted in genotype HLA-B*15:02.
PCR mixture.
| PCR ingredients | Final concentration | Volume per reaction, µL | ||
|---|---|---|---|---|
| Master mix (10 µL) |
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| dH2O | – | 3.3 | ||
| PCR buffer (5 X) | 1 X | 5.0 | ||
| MgCl2 (25 mM) | 1.0 mM | 1.0 | ||
| dNTPs (10 mM) | 0.2 mM | 0.5 | ||
| Taq polymerase (5 U) | 1.0 U | 0.2 | ||
|
|
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| Primer mix (F1R1, 13 µL) | ||||
|
|
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| dH2O | – | 7.5 | ||
| FC | 0.15 µM | 0.75 | ||
| RC | 0.15 µM | 0.75 | ||
| F1 | 0.40 µM | 2.0 | ||
| R1 | 0.40 µM | 2.0 | ||
|
|
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| Primer mix (F2R2, 13 µL) | ||||
|
|
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| dH2O | – | 9.0 | ||
| FC | 0.15 µM | 0.75 | ||
| RC | 0.15 µM | 0.75 | ||
| F2 | 0.25 µM | 1.25 | ||
| R2 | 0.25 µM | 1.25 | ||
|
|
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| Primer mix (F3R3, 13 µL) | ||||
|
|
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| dH2O | – | 9.0 | ||
| FC | 0.15 µM | 0.75 | ||
| RC | 0.15 µM | 0.75 | ||
| F3 | 0.25 µM | 1.25 | ||
| R3 | 0.25 µM | 1.25 | ||
|
|
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| Primer mix (F4R4, 13 µL) | ||||
|
|
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| dH2O | – | 7.5 | ||
| FC | 0.15 µM | 0.75 | ||
| RC | 0.15 µM | 0.75 | ||
| F4 | 0.40 µM | 2.0 | ||
| R4 | 0.40 µM | 2.0 | ||
|
|
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| Primer mix (F5R5, 13 µL) | ||||
|
|
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| dH2O | – | 7.5 | ||
| FC | 0.15 µM | 0.75 | ||
| RC | 0.15 µM | 0.75 | ||
| F5 | 0.40 µM | 2.0 | ||
| R5 | 0.40 µM | 2.0 | ||
|
|
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| Primer mix (F6R3, 13 µL) | ||||
|
|
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| dH2O | – | 9.0 | ||
| FC | 0.15 µM | 0.75 | ||
| RC | 0.15 µM | 0.75 | ||
| F6 | 0.25 µM | 1.25 | ||
| R3 | 0.25 µM | 1.25 | ||
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The reaction mixture was prepared in a total volume of 25 µL with 2 µL of DNA samples added per reaction. The initial stock primer concentration was 5 µM.
Thermal cycling conditions for PCR.
| PCR profile | Temp, °C | Time | Cycles |
|---|---|---|---|
| Pre-denaturation | 96 | 1 min | 1 |
| Denaturation | 96 | 20 s | |
| Annealing | 70 | 45 s | 5 |
| Extension | 72 | 25 s | |
| Denaturation | 96 | 25 s | |
| Annealing | 62 | 50 s | 25 |
| Extension | 72 | 30 s | |
| Denaturation | 96 | 20 s | |
| Annealing | 55 | 60 s | 5 |
| Extension | 72 | 90 s | |
| Hold | 12 | ∞ | – |
Funding source: Universiti Sains Malaysia Short Term Grant
Award Identifier / Grant number: 304/PFARMASI/6315398
Acknowledgments
We would like to thank Prof. Dr. Chonlaphat Sukasem and his team members from the Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand, for providing DNA with known genotypes using the PCR-sequence-specific oligonucleotide probes method. We also would like to thank the Director General of Health Malaysia for his permission to publish this article.
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Research funding: Short Term Grant, Universiti Sains Malaysia (304/PFARMASI/6315398).
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Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
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Competing interests: Authors state no conflict of interest.
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Informed consent: Informed consent was obtained from all individuals included in this study.
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Ethical approval: Medical Review & Ethics Committee (MREC) (NMRR-19-4092-45982) and Human Research Ethics Committee Universiti Sains Malaysia (JEPEM) (USM/JEPeM/20050287).
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Supplementary Material
This article contains supplementary material (https://doi.org/10.1515/tjb-2022-0202).
© 2023 the author(s), published by De Gruyter, Berlin/Boston
This work is licensed under the Creative Commons Attribution 4.0 International License.
