Distribution of CYP2D6 multiplication, CYP2D6*5, and clinical implications in postoperative patients receiving tramadol analgesia in the Minangkabau ethnic group, Indonesia

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Desriani" } ], "publisher": { "@type": "Organization", "name": "F1000Research", "logo": { "@type": "ImageObject", "url": "https://f1000research.com/img/AMP/F1000Research_image.png", "height": 480, "width": 60 } }, "image": { "@type": "ImageObject", "url": "https://f1000research.com/img/AMP/F1000Research_image.png", "height": 1200, "width": 150 }, "description": " Background Cytochrome P450 2D6 (CYP2D6) is an important enzyme that metabolizes commonly used drugs, such as tramadol hydrochloride. Genetic polymorphisms of CYP2D6 have been shown to influence the pharmacodynamic properties of administered drugs. This study aimed to screen postoperative patients (wild-type, CYP2D6*5, and CYP2D6 multiplication) of Minangkabau ethnicity in West Sumatera, Indonesia, who received tramadol using a modified long PCR method, and to investigate the clinical impact of tramadol on these patients. This study had a retrospective clinical trial registry number NCT06642480, and a total of 65 patients participated. Methods The Reported Long PCR was modified using different DNA polymerases, optimizing annealing, and the number of step PCR to detect wild-type, CYP2D6*5, and multiplication CYP2D6. To ensure accurate PCR, the size of the PCR product was monitored: wild-type (5kb), CYP2D6*5 (6kb), and CYP2D6 multiplication genotype (10 kb). The wild-type PCR product was used as the control reaction. The method was applied to screen postoperative patients of Minangkabau ethnicity. A statistical study was conducted using analysis of variance (ANOVA), and a chi-square test. Result The Long PCR was successfully modified with a two-step PCR at 68°C as the annealing and extension temperatures. The screened genotype patients were dominated by the wild-type, followed by CYP2D6*5, and multiplication CYP2D6 with percentages of 89%, 6,3%, and 4,7% respectively. A total of 6.3% of CYP2D6*5 cases were classified as heterozygous and predicted as intermediate metabolizers. Based on ANOVA analysis, there was a significant association between analgesic tramadol and VAS value, as well as between tramadol and molecular screening, with P-values of 0.045 and 0.003, respectively. Conclusion The percentages of CYP2D6*5 and CYP2D6 multiplication genotypes were similar to those observed in another Asian population. Based on statistical analysis, tramadol was effective as an analgesic for postoperative Minangkabau ethnicity patients with wild-type, CYP2D6*5, and multiple CYP2D6 genotypes. 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F1000Research 2025, 13 :1526 ( https://doi.org/10.12688/f1000research.155988.3 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Research Article Revised Distribution of CYP2D6 multiplication, CYP2D6*5, and clinical implications in postoperative patients receiving tramadol analgesia in the Minangkabau ethnic group, Indonesia [version 3; peer review: 2 approved] Rinal Effendi https://orcid.org/0009-0000-6850-4959 1 , Aufa Rizqia Haz 2 , Asrul Muhammad Fuad 2 , Bagus Dermawan https://orcid.org/0009-0008-0521-9884 3 , Nuruliawaty Utami https://orcid.org/0000-0001-7720-2450 2 , . Desriani https://orcid.org/0000-0003-0445-6569 2 Rinal Effendi https://orcid.org/0009-0000-6850-4959 1 , Aufa Rizqia Haz 2 , [...] Asrul Muhammad Fuad 2 , Bagus Dermawan https://orcid.org/0009-0008-0521-9884 3 , Nuruliawaty Utami https://orcid.org/0000-0001-7720-2450 2 , . Desriani https://orcid.org/0000-0003-0445-6569 2 PUBLISHED 23 Sep 2025 Author details Author details 1 Department of Anesthesiology, Faculty of Medicine, Universitas Andalas, Padang, West Sumatra, Indonesia 2 Research Center for Genetic Engineering, Soekarno Sains and Technology Park, National Research and Innovation Agency (BRIN), Jl. Raya Bogor Km, 46 Cibinong, 16911, Indonesia 3 Department of Neurology, Jl dr. Soetomo no 16, Dr Kariadi General Hospital Medical Center, Semarang, Central Java, 50244, Indonesia Rinal Effendi Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Aufa Rizqia Haz Roles: Data Curation, Formal Analysis, Methodology, Validation, Visualization, Writing – Review & Editing Asrul Muhammad Fuad Roles: Formal Analysis, Methodology, Supervision, Validation, Writing – Review & Editing Bagus Dermawan Roles: Data Curation, Formal Analysis, Methodology, Supervision, Validation, Visualization, Writing – Review & Editing Nuruliawaty Utami Roles: Formal Analysis, Methodology, Supervision, Validation, Visualization, Writing – Review & Editing . Desriani Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing OPEN PEER REVIEW DETAILS REVIEWER STATUS This article is included in the Genomics and Genetics gateway. Abstract Background Cytochrome P450 2D6 (CYP2D6) is an important enzyme that metabolizes commonly used drugs, such as tramadol hydrochloride. Genetic polymorphisms of CYP2D6 have been shown to influence the pharmacodynamic properties of administered drugs. This study aimed to screen postoperative patients (wild-type, CYP2D6*5, and CYP2D6 multiplication) of Minangkabau ethnicity in West Sumatera, Indonesia, who received tramadol using a modified long PCR method, and to investigate the clinical impact of tramadol on these patients. This study had a retrospective clinical trial registry number NCT06642480, and a total of 65 patients participated. Methods The Reported Long PCR was modified using different DNA polymerases, optimizing annealing, and the number of step PCR to detect wild-type, CYP2D6*5, and multiplication CYP2D6. To ensure accurate PCR, the size of the PCR product was monitored: wild-type (5kb), CYP2D6*5 (6kb), and CYP2D6 multiplication genotype (10 kb). The wild-type PCR product was used as the control reaction. The method was applied to screen postoperative patients of Minangkabau ethnicity. A statistical study was conducted using analysis of variance (ANOVA), and a chi-square test. Result The Long PCR was successfully modified with a two-step PCR at 68°C as the annealing and extension temperatures. The screened genotype patients were dominated by the wild-type, followed by CYP2D6*5, and multiplication CYP2D6 with percentages of 89%, 6,3%, and 4,7% respectively. A total of 6.3% of CYP2D6*5 cases were classified as heterozygous and predicted as intermediate metabolizers. Based on ANOVA analysis, there was a significant association between analgesic tramadol and VAS value, as well as between tramadol and molecular screening, with P-values of 0.045 and 0.003, respectively. Conclusion The percentages of CYP2D6*5 and CYP2D6 multiplication genotypes were similar to those observed in another Asian population. Based on statistical analysis, tramadol was effective as an analgesic for postoperative Minangkabau ethnicity patients with wild-type, CYP2D6*5, and multiple CYP2D6 genotypes. READ ALL READ LESS Keywords CYP2D6, Long PCR, Opioid Analgesic, Post Operative, Tramadol, West Sumatera Corresponding Author(s) Rinal Effendi ( [email protected] ) . Desriani ( [email protected] ) Close Corresponding authors: Rinal Effendi, . Desriani Competing interests: No competing interests were disclosed. Grant information: Financial support for this research was provided by the Rumah Program Organisasi Riset Ilmu Pengetahuan Teknik, Badan Riset dan Inovasi Nasional 2022 Number 2/III/HK/2022. The funders had no role in the study design, data collection and analysis, decision to publish, or manuscript preparation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2025 Effendi R et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: Effendi R, Rizqia Haz A, Muhammad Fuad A et al. Distribution of CYP2D6 multiplication, CYP2D6*5, and clinical implications in postoperative patients receiving tramadol analgesia in the Minangkabau ethnic group, Indonesia [version 3; peer review: 2 approved] . F1000Research 2025, 13 :1526 ( https://doi.org/10.12688/f1000research.155988.3 ) First published: 17 Dec 2024, 13 :1526 ( https://doi.org/10.12688/f1000research.155988.1 ) Latest published: 23 Sep 2025, 13 :1526 ( https://doi.org/10.12688/f1000research.155988.3 ) Revised Amendments from Version 2 Please kindly find our version 3 revision. We have revised it based on the valuable comments and input from the reviewer. We have rewritten our objective. We also revised the narrative of our statistical study in the Methods section. An analysis of variance (ANOVA) was conducted to analyze the relationship between the amount of tramadol administered and the VAS value, as well as between the amount of tramadol administered and the molecular screening result. Demographic characteristics were analysed using a chi-square test. Statistical analysis was conducted using IBM SPSS software, version 24. P-values below 0.05 were considered statistically significant. We completed the legend for Figures 1 (1-3) and 2 (2-1 and 2-2). Further, we also completed our results and discussion using not only a chi-square test but also ANOVA analysis data. This ANOVA data is already available in the supplementary material. Additional ANOVA data in the manuscript does not change the substance of the discussion; it strengthens it. Detailed information can be found in the manuscript. Please kindly find our version 3 revision. We have revised it based on the valuable comments and input from the reviewer. We have rewritten our objective. We also revised the narrative of our statistical study in the Methods section. An analysis of variance (ANOVA) was conducted to analyze the relationship between the amount of tramadol administered and the VAS value, as well as between the amount of tramadol administered and the molecular screening result. Demographic characteristics were analysed using a chi-square test. Statistical analysis was conducted using IBM SPSS software, version 24. P-values below 0.05 were considered statistically significant. We completed the legend for Figures 1 (1-3) and 2 (2-1 and 2-2). Further, we also completed our results and discussion using not only a chi-square test but also ANOVA analysis data. This ANOVA data is already available in the supplementary material. Additional ANOVA data in the manuscript does not change the substance of the discussion; it strengthens it. Detailed information can be found in the manuscript. See the authors' detailed response to the review by Heri Setiawan See the authors' detailed response to the review by Anom Bowolaksono READ REVIEWER RESPONSES 1. Introduction Individuals vary in their P450 2D6 (CYP2D6) activity levels, ranging from complete lack of metabolism of certain drugs to ultra-rapid metabolism, which can lead to adverse effects during standard treatment. CYP2D6 metabolizes 15-25% of clinically used drugs, including a broad spectrum of medications such as antidepressants (paroxetine, tricyclic antidepressants, etc.), analgesics (codeine, tramadol, oxycodone, etc.), oncology (tamoxifen, etc.), antihypertensives (metoprolol, bisoprolol, etc.), and cardiology drugs. The gene encoding the enzyme is located on chromosome 22q13.1, and contains nine exons. It is positioned alongside two pseudogenes, CYP2D7 and CYP2D8, which are highly homologous to the transcribed active area with a similarity percentage of 94.2% and 89.1%, respectively. These two pseudogenes were also composed of l9 exons. 1 , 2 According to the prevailing metabolic capacity, the CYP2D6 phenotype is classified into four distinct categories: poor metabolizer (PM), intermediate metabolizer (IM), extensive metabolizer (EM), and ultra-metabolizer (UM). 3 , 4 Arneth et al. 2009 clasiffied EM as a characteristic of the normal population with two functional wild type alleles; PM usually results from the deletion or mutation of both alleles and is an autosomal recessive condition, IM those with one functional allele; Rapid metabolism UM is believed to be an autosomal dominant feature that results from either functional gene multiplication or duplication (>30%). 3 Further Darney et al. 2021, reported that extensive metabolism as normal, and it is defined as having two functional wild-type alleles (i.e. *1, *2), or heterozygosity with one decreased-activity and one non-activity allele. PM is associated with CYP2D6 alleles with no activity both alleles (i.e., *3, *4, *5, *6, *7, *8, *11, *14, *15, *18, *19, *21, *29, *40). The IM phenotype has been associated with a decrease in CYP2D6 activity alleles, i.e. *9, *10, *17, *21, *36, *29, *41, *45, and *46, or heterozygosity for one decreased activity allele and one non-activity allele. The UM phenotype has been linked to at least one active CYP2D6 gene duplication. Patients exhibiting the UM phenotype may face an elevated risk of therapeutic failure or an augmented propensity for adverse metabolic toxicities. 4 These phenotypic differences alter the capacity of enzymes to metabolize drugs. As mention above, phenotypic variations in CYP2D6 have been attributed to polymorphisms, deletions, and variations in the number of copies of the enzyme. The Pharmacogene Variation (PharmVar) Consortium classified the gene as highly polymorphic. Further details can be found at https://www.pharmvar.org/gene/CYP2D6 . 5 According to a previous study, the frequencies of CYP2D6 in Hong Kong were UM 3.3%, EM 49.9%, IM 46.4%, and PM 0.4%. 6 The frequency of CYP2D6 alleles, especially CYP2D6*5, varies worldwide. CYP2D6*5 associated loss of function allele varied by 3-6% in African, European, and East Asian populations. 2 CYP2D6*5 heterozygotes with the IM phenotype were 2.4% in Malay, 2.2% in Indian, and 0.5% in Chinese, while the UM phenotype was 11% for Chinese, 5% for Indian and 4.8% for Malay ethnicity in the Singapore population. 7 Tramadol hydrochloride is a popular analgesic opioid that is widely used for the treatment of postoperative, dental, cancer, neuropathic, acute musculoskeletal neuropathic, and acute musculoskeletal pain. It is associated with high clinical efficacy, a low incidence of adverse effects, and low abuse potential. Tramadol can be administered in several ways, including oral, rectal, sustained-release, and parenteral (IV/IM). Tramadol is converted into two active metabolites: M1 (O-desmethyltramadol) and M2 (N-desmethyltramadol). CYP P450 2D6 catalyzes o-demethylation to M1 (the main analgesic effective metabolite), while CYP2B6 and CYP3A4 catalyze n-demethylation to M2. The main route of elimination of tramadol and its metabolites is through the kidneys. The average elimination half-life is approximately 6 h. 8 , 9 The typical dosage of tramadol administered was within the range of 50–100 milligrams. 10 The dosage for pediatric patients was 50 mg, whereas the adult dosage was 100 mg. As mentioned above, CYP2D6 is polymorphic, with different alleles encoding functionally different enzymes. This suggests that different genetics would influence the pharmacokinetics and/or pharmacodynamics of tramadol. 8 , 9 , 11 , 12 West Sumatra is a province in Indonesia, and its population comprises the Minangkabau ethnic group. Previous studies have reported the analgesic effect of endogenous opioids and receptor polymorphisms of OPRM A118G and COMT G158A in the Minangkabau ethnic group. The results showed that gene polymorphisms were not significantly associated with pain sensitivity, and there is still limited information on gene polymorphisms related to opioid analgesics in West Sumatra. 13 CYP2D6 genetic detection technologies have been developed, including TaqMan technology, microarray, Southern blotting RFLP, PCR-RFLP, Long PCR, and single-strand conformation polymorphism (SSCP). 1 , 14 – 16 The methods used to count the number of CYP2D6 copies were Southern blotting, TaqMan technology, HRM qPCR, 17 , 18 and pyrosequencing genotyping. 19 Johansson et al. (1996) reported a method for detecting multiplication and deletion using Long PCR. 20 This method used rTth DNA Polymerase from Perkin Elmar and 0.09U Vent Polymerase (New England Biolabs) in a total volume of 25 μL. PCR products with size of 10kb, 6kb, and 5kb signify multiplication, CYP2D6*5, and wild-type. 20 The objective of this study is to investigate the genetic distribution of multiplication, CYP2D6*5, and wild-type CYP2D6 in post-operative Minangkabau patients using modified Johansson et al. (1996) Long PCR methods and to investigate the clinical effects of tramadol on patients. 20 2. Methods 2.1 Determination of Patients’ Visual Analogue Scale (VAS) “The study was approved on 21 March 2022, by the Ethics Committee of the Faculty of Medicine, University of Andalas West Sumatra, Indonesia (number 653/UN.16.2/KEP-FK/2022). Informed consent was also signed and obtained from the patients for blood collection, relevant clinic data collection, and determination of the patients’ visual analog scale (VAS). These studies have fulfilled WMA declaration of Helsinki-Ethical principles. In addition, this study had a retrospective clinical trial registry number NCT06642480. All clinical trials were registered, allowing information to be shared between clinicians, researchers, and patients. This increases public confidence in the research. A clinical trial registry (or retrospective clinical trial registry) can be enrolled and issued at https://register.clinicaltrials.gov/ . All of the study’s participants were patients who had undergone surgery at M. Djamil Padang Hospital. The inclusion criteria were: Patients with elective surgery at M. Djamil Hospital; proven Minangkabau ethnicity through family history; signed informed consent for blood sampling and pain assessment (VAS); and American Society of Anesthesiologists (ASA) criteria levels 1–2. The exclusion criteria were: Patients who refused to sign the informed consent form for sample collection; patients with ASA criteria levels ≥3 (patients with severe comorbidities e.g. renal failure or severe liver disease) that may interfere with tramadol metabolism, this also included patients who were taking other drugs that could significantly induce or inhibit CYP2D6 (e.g. fluoxetine, paroxetine and rifampicin). The VAS scores of patients were determined by injecting tramadol at a dose of 100 mg 30 min before the operation was completed. This was observed in the recovery room at 30, 60, and 120 min. Tramadol had an onset time of 15-60 minutes, with peak effectiveness at 2-6 hours postoperatively. Pain level was categorized as 0 (no pain), 1-3 (signifying mild), 4-6 (middle), and 7-10 (heavy pain). 2.2 Blood sample collection and DNA extraction A total of 65 patients participated in this study. DNA was extracted using the Purelink Genomic DNA Mini Kit from Thermo Fisher Scientific (# K1820-01). The extracted DNA was further verified using 1% agarose, Ist Base #BIO-1000-500g and its concentration was measured using a spectrophotometer at A260/280. 2.3 Long PCR modification for CYP2D6*5, multiplication, and wild-type CYP2D6 genotyping The long PCR method of Johansson et al. (1996) was modified using Promega GoTaq Long PCR Master Mix #M4021, with the following DNA primers: Lx2F: 5′ GCC ACC ATG GTG TCT TTG CTT TC 3′, Lx2R: 5′ ACC GGA TTC CAG CTG GGA AAT G 3′ for multiplication detection amplification, DF: 5′ GCC ACT CTC GTG TCG TCA GCT TT 3′; DR: 5′ GGC ATG AGC TAA GGC ACC 3′ for detecting CYP2D6*5, LongPCR-CYP2D6-Fw: 5′ CCA GAA GGC TTT GCA GGC TTC A 3′, and LongPCR-CYP2D6-Rev: 5′ ACT GAG CCC TGG GAG GTA GGT A 3′ for wild-type detection amplification and serving the purpose as control reaction. Successful PCR optimization of DNA amplification resulted in 10 kb, 6 kb, and 5 kb fragments for multiplication of the CYP2D6 gene, CYP2D6*5, and wild-type, respectively. Three tubes were required to genotype each patient sample. Each tube contained the DNA primers described above. Two samples with pain level scales of 0 and 8 were selected as DNA templates for Long PCR modifications. DNA samples were selected based on VAS criteria. The Long PCR ingredient of each amplification target contained 10 μl GoTaq ® Long PCR Master Mix Promega #M4021, 0.4 μl FW primer 10 μM, 0.4 μl Rev primer 10 μM, 1 μl DNA template (0.1-0.5 μg), and 8.2 μl nuclease-free water. Each PCR amplification included a Negative Control (NTC) containing the same ingredients, except that the DNA template was substituted with nuclease-free water. The two-step PCR was optimized using the gradient PCR method. Amplification was performed using a Kyratec SuperCycler SC-200 (Kyratec Life Science). The conditions included pre-denaturation at 95°C for 2 min, followed by 30x amplification with denaturation at 94°C for 30 sec, and annealing temperature of 63/63,95/65,2/67,15/68°C for 12 min, followed by post-extension at 72°C for 10 min. 2.4 Statistical analysis An analysis of variance (ANOVA) was conducted to analyze the relationship between the amount of tramadol administered and the VAS value, as well as between the amount of tramadol administered and the molecular screening result. The demographic characteristics data were analyzed with a Chi-Square test. The statistical analysis was conducted with the IBM SPSS software version 24, with p values less than 0.05 being considered statistically significant. 3. Result 3.1 Long PCR modified from Johansson et al. 20 Long PCR was selected for CYP2D6*5 or multiplication and detection of wild-type CYP2D6. Although this method is time-consuming, it is more cost-effective than the other methods. The long PCR method developed by Johansson et al. (1996) 20 was modified in this study. The PCR products of wild-type CYP2D6, CYP2D6*5, and multiplication detection yielded 5 kb, 6 kb, and 10 kb, respectively. In contrast to Johansson et al. (1996), who used PerkinElmer’s rTDNA and New England Biolabs’ 0.09U Vent, the present study used Promega’s GoTaqLong PCR Mastermix #M4021. GoTaq ® Long PCR Master Mix uses a combination of recombinant hot-start Taq DNA polymerase and recombinant proofreading DNA polymerase. The amplification conditions must be further optimized. In a preliminary study, the annealing temperature of 55°C-67°C was optimized using a three-step PCR (denaturation, annealing, and extension) for three DNA primers. No PCR product was detected (data not shown). Further, the annealing temperature across five different temperatures starting from 65 °C to 68°C was optimized using two-step PCR (denaturation, annealing also as an extension step) for each DNA primer. Among the five annealing temperatures tested, 68°C was the best temperature. One patient DNA sample with an 0 VAS scale, produced 6kb and 5kb for the amplified CYP2D6*5 DNA primer and wild-type CYP2D6 DNA primer, respectively, with no PCR product in all NTC. Using this 0 VAS scale patient DNA sample, amplification with a CYP2D6*5 DNA primer produced a 6 kb PCR product that consistently showed up at all annealing temperatures. The PCR product showed a specific amplification product at 68°C annealing temperature. In addition, amplification using a wild-type CYP2D6 DNA primer produced a 5 kb PCR product that consistently showed at all annealing temperatures, and specific PCR product were provide at 67.15°C and 68°C. Furthermore, DNA samples from other patients (second patient) that had 8 VAS scale showed only produced 5kb for the wild-type DNA primer at all annealing temperatures, and specific PCR product showed at 67.15°C and 68°C annealing temperature. The DNA primer used for the detection of CYP2D6 multiplication did not generate a PCR product for either sample ( Figure 1 ). This showed that the two samples used for optimization were CYP2D6*5 for the first sample (0 VAS scale) and the wild-type for the second sample (8 VAS scale). Specifically, CYP2D6*5 was identified as heterozygous CYP2D6*5 as the PCR product was produced using both CYP2D6*5 and wild-type DNA primers. According to Johansson et al., 20 heterozygous CYP2D6*5 led to PCR products with both CYP2D6*5 and wild-type DNA primers, whereas homozygous CYP2D6*5 resulted in no PCR product with wild-type DNA primers. Based on these results, there was an inconsistency between the VAS scale measurement and the genotype detection. This discrepancy was possible since VAS scale measurements are highly subjective and depend on patient pain tolerance. Figure 1. Long PCR annealing temperature optimization. Sample: 1, 2. N: No template control. M: for multiplication detection (10kb), D: for CYP2D6*5 detection (6kb), and Wt: for Wild-type detection (5kb). The red arrow indicates the PCR target of each DNA primer. The optimal PCR setup was used to screen several samples. The screening results showed that one sample produced 10kb which was suspected to be a multiplication sample ( Figure 2 ). This sample was optimized for its optimal PCR setup, and the process was used to confirm and verify the results. Consistency was observed with a 10 kb PCR product as a single band across temperatures ranging from 63 to 68°C. The optimal PCR annealing temperature was determined to be 63.95, 65.2, and 67.15, indicating thick single-band DNA ( Figure 3 ). However, 68°C was selected because it produced a single band and was the most effective temperature for detecting both CYP2D6*5 and wild-type alleles. In addition, all the annealing optimization processes showed clear and clean NTC. The remaining samples were screened using a modified method. Figure 2. Long PCR screening of patient sample for detection of CYP2D6*5, multiplication, and wild-type. The target was shown with the red arrow: M: for multiplication detection (10kb), D: for CYP2D6*5 detection (6kb), and Wt: for Wild-type detection (5kb). Patients Sample: 1-10. Figure 3. Long PCR annealing PCR optimization for multiplication CYP2D6. The target was shown with the red arrow, 10 kb. S: Sample, N: No template control. 3.2 Distribution of CYP2D6*5, or multiplication, and wild-type genotype of Minangkabau ethnicity As previously reported, the modified Long PCR was applied to postoperative patients of the Minangkabau ethnicity who were administered tramadol as an analgesic. A total of 65 patients participated in this study. 62 patients who received tramadol analgesia, plus one patient were tested for genetics, while the other two samples did not undergo genetic testing because they had no detailed information. Out of 63 DNA patient samples, four samples could not be amplified due to poor DNA quality. The distribution of patients with multiplication, wild type, or CYP2D6*5 based on age, sex, and weight is shown in the following graphic ( Figure 4 ). The majority of patients of Minangkabau ethnicity had a wild-type genotype (89%). The frequencies of CYP2D6*5 and multiplication were 6.3% and 4.7%, respectively, for each genotype. CYP2D6*5 is a heterozygous allele, as all samples produced 6 and 5 kb PCR DNA products in tubes containing CYP2D6*5 and wild-type DNA primers, respectively. The distribution of CYP2D6*5 and the multiplication genotypes in some areas are shown in Table 1 . Figure 4. Distribution of multiplication, CYP2D6*5 (Heterozygous) and wild-type CYP2D6 in the Minangkabau ethnicity. A. Based on age, B. gender, and C. weight. Table 1. Comparison of CYP2D6*5, multiplication genotype distribution in some areas. Ethnic groups Genotype (%) Ref. CYP2D6*5 (Deletion) Multiplication Eastern Han Chinese 4.82 0.69 33 Central Han Chinese 7.17 1.35 33 Malaysia Chinese 2.54 4.24 33 Malay 2.4 4.8 7 Minangkabau 6.3 4.7 Present report The modified Long PCR method is straightforward and highly accurate for genotyping. The PCR product sizes reported by Johansson et al. (1996) was monitored for determination of each genotype. 20 The sizes of the wild-type, CYP2D6*5, and CYP2D6 multiplication PCR products were 5 kb, 6 kb, and 10 kb, respectively. 3.3 Patients demographics The administration of tramadol (100 mg) to the patients resulted in optimal pain relief. This was evidenced in the present study, ANOVA analysis showed a significant association between the drug and VAS scores of postoperative patients with p = 0.045, and a significant association also existed between the drug and molecular screening with p = 0.003 (in the Supplementary). In addition, wild-type genotypes, heterozygote CYP2D6*5, and multiplication were observed in all the patients. Chi-square analysis showed that patients’ demographic characteristics, including age, gender, and VAS value, showed no significant correlations (p = 0.052; 0.243; and 0.244, respectively). Meanwhile, the weight of the patients is indicated as biased. Inconsistent p-values are shown between the Pearson chi-square test (p = 0.008), the likelihood ratio test (p = 0.149), and the linear-by-linear association test (p = 0.124). Further, Table 2 shows that there was no correlation between the VAS score and molecular detection. The VAS is a subjective measure that depends on the pain tolerance of patients and the type of surgery. However, recent studies have not differentiated between the various classifications of surgery. Table 2. The characteristic demographic of CYP2D6*5, multiplication and wild-type polymorphism of CYP2D6 (age, sex, and weight as VAS measurement). Characteristics Distribution Molecular screening Total (n) p-value Wild-type CYP2D6*5 (Heterozygous) Multiplication No details Age 15-35 years 23 (85.2%) 1 (3.7%) 0 (0.0%) 3 (11.1%) 27 0.052 36-50 years 13 (76.5%) 0 (0.0%) 3 (17.6%) 1 (5.9%) 17 51-65 years 16 (76.2%) 3 (14.3%) 0 (0.0%) 2 (9.5%) 21 Gender Female 31 (81.6%) 1 (2.6%) 1 (2.6%) 5 (13.2%) 38 0.243 Male 21 (77.8%) 3 (11.1%) 2 (7.4%) 1 (3.7%) 27 Weight 40-55 kg 24 (82.8%) 1 (3.4%) 2 (6.9%) 2 (6.9%) 29 0.008 56-70 kg 21 (80.8%) 2 (7.7%) 1 (3.8%) 2 (7.7%) 26 71-85 kg 7 (87.5%) 1 (12.5%) 0 (0.0%) 0 (0.0%) 8 No information 0 (0.0%) 0 (0.0%) 0 (0.0%) 2 (100.0%) 2 VAS value 0 (no pain) 13 (76.5%) 2 (11.8%) 1 (5.9%) 1 (5.9%) 17 0.244 1-3 (signifying mild) 17 (85.0%) 1 (5.0%) 1 (5.0%) 1 (5.0%) 20 4-6 (middle) 5 (83.3%) 0 (0.0%) 0 (0.0%) 1 (16.7%) 6 7-10 (heavy pain) 16 (84.2%) 1 (5.3%) 1 (5.3%) 1 (5.3%) 19 No information 1 (33.3%) 0 (0.0%) 0 (0.0%) 2 (66.7%) 3 4. Discussion This study aimed to investigate the genetic distribution of CYP2D6*5 and CYP2D6 multiplication in post-operative Minangkabau ethnicity patients receiving tramadol analgesia and further investigate the efficacy of tramadol in these patients. To achieve this, the Long PCR method developed by Johansson et al ., 20 was modified. The Long PCR, reported by Johansson et al . 1996, was successfully modified with the GoTaq Long PCR master mix from Promega #M4021, with two-step PCR at 68°C annealing and extension PCR amplification temperatures. It is important to optimise the annealing temperature, particularly when synthesising long PCR products or using total genomic DNA as the PCR substrate. An annealing temperature that is too high will reduced the PCR product yielded while an annealing temperature that is too low will produce an unspecific PCR product. PCR annealing temperature optimization was one of the key successes for developing PCR methods. 21 GoTaq ® Long PCR Master Mix uses a combination of recombinant hot-start Taq DNA polymerase and recombinant proofreading DNA polymerase. Both enzymes in the GoTaq Long PCR master mix from Promega #M4021 were required to amplify long targets. Taq DNA Polymerase alone cannot correct nucleotide mismatches resulting from misincorporation and is therefore ineffective in amplifying segments longer than 3-5 kb. Products are truncated if nucleotides are misincorporated; further, they cannot be extended in subsequent cycles. Long amplicons can be amplified with a high yield by adding a small amount of proofreading enzyme to fix mismatches and allow extension. To ensure accurate PCR, the size of the product was monitored. The sizes of the wild-type, CYP2D6*5, and CYP2D6 multiplication PCR products were 5kb, 6kb, and 10 kb, respectively. The wild-type genotype PCR product was used as a control reaction. Furthermore, heterozygous CYP2D6*5 produced a DNA PCR product in a tube containing CYP2D6*5 and wild-type DNA primers. In contrast, homozygous CYP2D6*5 produced no PCR products in the tube containing wild-type DNA primers. Although the real-time method offers a simpler and faster alternative to long PCR, it requires costly real-time PCR machines, and pyrosequencing is also costly. The modified Long PCR method was used to generate a distribution report of the Minangkabau ethnicity genotypes. The majority of CYP2D6 genotypes were dominated by the wild-type genotype, with only 6.3% and 4.7% for CYP2D6*5 and multiplication, respectively. Furthermore, 6.3% of CYP2D6*5 patients were classified as heterozygous and predicted as intermediate metabolizers. These percentages are similar to the frequencies observed in other Asian populations for CYP2D6*5 and multiplication ( Table 1 ). Allele frequencies of CYP2D6 5* were 3.08% (European), 5.67% (African), 2.96% (Hispanic), 6.24% (Asian), and 1.30% (Samoan) respectively. 22 In Singaporean populations, ultra metabolizers presented 11, 5, and 4.8% of Chinese, Indian, and Malay ethnicity participants, CYP2D6*5 heterozygotes with IM phenotype were 2.4% for Malay, 2.2% for Indian, and 0.5% for Chinese ethnicity participants. 7 According to Arneth at al. 2009, Individuals with a single functioning CYP2D6 allele are characterized as intermediate metabolism (IM). 3 Several reports have also supported the phenotype prediction, classifying heterozygous CYP2D6*5 as an intermediate metabolizer. 7 , 23 Further, it is important for the next activity to measure tramadol’s enantiomers in plasma, their primary phase I metabolites, and also epinephrine to evaluate the CYP2D6 phenotype as reported by Garcia-Quetglas et al. , 2007. 24 Also following the recommendation of the consensus on translating CYP2D6 genotype to metabolizer phenotype. 23 It was reported that there were discrepancies between clinical genetic testing laboratories and guidelines for translating genotype to metabolizer phenotype. These differences will result in inconsistent therapeutic recommendations. 23 The two enantiomers that make up tramadol each contribute to its analgesic effects in a distinct way in cytochrome P450 system. The highly polymorphic enzyme CYP2D6 catalyzes the formation of M1 from O-desmethyltramadol. N-demethylation, however, was catalyzed by a different mechanism. As previously mentioned, the phenotypic variance of CYP2D6 is explained by its highly polymorphic character (mutation, deletion, and multiplication). Furthermore, depending on CYP2D6 phenotypes and other genetic factors, that the mean elimination half-life is around 6 hours, while the initial distribution half-life is 6 minutes. Three hours after delivery, O-Desmethyltramadol (M1), the main active metabolite of tramadol, reaches Cmax at 18–26% of the tramadol Cmax value. The average elimination half-life is roughly seven hours. 9 , 25 Deletion and multiplication of genes are important for phenotype prediction. Deletion of the entire CYP2D6 gene (*5) results in loss (in the homozygote) or reduction (in the heterozygote) of its protein production. 3 , 26 , 27 Furthermore, it is important to acknowledge that drug metabolism in the multiplication genotype increases the activity of the enzyme acitivity at a high rate as the functional increase. 5 , 22 The UM phenotype may also be at risk of therapeutic failure or increased toxic side effects of metabolites. The ANOVA analysis showed that administered tramadol was significantly associated with the VAS value of postoperative patients receiving analgesia with p = 0.045 (Supplementary). This result supported and indicated that administration of 100 mg of the drug resulted in optimal pain relief. In addition, the typical dosage of tramadol administered is within the range of 50 to 100 milligrams. 10 The dosage for pediatric patients was 50 mg, whereas the adult dosage was 100 mg. Furthermore, a significant association was also shown between tramadol administration and molecular screening, with p = 0.003 (in the Supplementary). This could be interpreted as meaning that all genotypes in this activity individually metabolize the given tramadol is still possible. This also shows that both genotypes (heterozygote CYP2D6 *5 and the multiplication) are clinically functional. In Table 2 , the Chi-square test showed that the distribution of the molecular screening among the demographic factors (Age, Gender, and VAS value) of the West Sumatra patients does not have a significant correlation. While the weight of the patients is indicated as biased. As it showed inconsistent p-values between Pearson Chi-Square (p = 0.008), Likelihood Ratio (p = 0.149) and Linear-by-Linear Association (p = 0.124). According to McHugh 2013, too small sample size will affect to Pearson Chi Square, Likehood Ratio and Linear-by-linear P Value in the Chi-square test. 28 Therefore, for a better understanding of this, further study needs a larger number of samples. In addition, gene polymorphisms are the most common type of genetic variation, defined as variations in gene sequences caused by changes in the DNA sequence that are heritable. 29 Further, the VAS value has a high subjectivity, and open possibilities have no significant correlation with molecular screening, as proved by the Chi-square result ( Table 2 ). The potency of tramadol analgesia is approximately 10% that of morphine when administered intravenously. Tramadol has also been reported to be comparable to morphine. 30 , 31 Tramadol is an efficient and well-tolerated medication for the treatment of chronic pain, whether of malignant or non-malignant origin, especially neuropathic pain. It can also be used to relieve pain related to trauma, renal or biliary colic, and labor. When taken as a non-opioid analgesic, the analgesic effect of the drug can be enhanced. 30 Furthermore, tramadol administration is ideally based on body weight. According to Sidiq et al . (2015), 2 mg/kg body weight of tramadol hydrochloride is an optimal dose for postoperative analgesia when administered epidurally in urological surgery, with no significant increase in side effects. 32 Pang et al . (2005) reported that the recommended intraoperative dose of tramadol is 2.5 mg/kg body weight for effective postoperative analgesia with minimal sedation, when considering the efficacy and side-effect profile. 31 Patients without CYP2D6 activity may not be able to metabolize drugs. Ultra-rapid metabolizers may also experience treatment failure owing to the rapid metabolism of active drugs, resulting in drug levels below the therapeutic range. 5 The data obtained specifically in the multiplication part were not in line with the presented statement. This discrepancy may be attributed to the absence of a subsequent follow-up. To enhance the quality of future data, it is essential to categorize the type of operation, collect a larger number of samples, and conduct longer follow-up periods, and also estimate the pharmacokinetic parameter of tramadol in plasma. Tramadol’s enantiomers, their primary phase I metabolites, and epinephrin were pharmacokinetic parameter of tramadol in plasma in order to evaluate the CYP2D6 phenotype. 23 Also following the recommendation of the consensus on translating CYP2D6 genotype to metabolizer phenotype. 24 5. Conclusion In conclusion, the majority of the CYP2D6 genotypes were dominated by the wild-type genotype, with only 6.3% and 4.7% for CYP2D6*5 (heterozygote) and multiplication, respectively. The modified long PCR method for CYP2D6 genotyping successfully detected CYP2D6*5, multiplication, and wild-type genotypes. The proposed method is simple and highly accurate. To ensure accurate results, the size of the PCR products was monitored. The sizes of the wild-type, CYP2D6*5, and multiplication genotype PCR products were 5 kb, 6 kb, and 10 kb, respectively. The wild-type PCR product was used as control reaction. The administration of tramadol (100 mg) to patients led to optimal pain relief, as evidenced by an ANOVA study analyzed using IBM SPPS 24. The ANOVA analysis showed that administered tramadol was significantly associated with the VAS value of postoperative patients receiving analgesia with p = 0.045. A significant association was also shown between tramadol administration and molecular screening, with p = 0.003. This also suggests that in the Minangkabau ethnic group, all genotypes were capable of metabolizing the administered tramadol. Ethics and consent The study was approved by the Ethics Committee of the Faculty of Medicine, University of Andalas West Sumatra, Indonesia (number 653/UN.16.2/KEP-FK/2022) on 21 March 2022. Informed consent was also signed and obtained from the patients for blood collection and determination of the patients’ Visual Analog Scale (VAS). The participants in this study were 65 post-operative patients from M Djamil Central Hospital of West Sumatra. Among the patients, 62 received tramadol analgesia and 63 were tested for genetics, while the other two samples did not have detailed information. In the case of participants who were minors, consent was obtained and signed by their legal guardian or parent. Author roles Desriani: Conceptualization, Data curation, Formal Analysis, Investigation, Project Administration, Funding Acquisition, Methodology, Validation, Writing-Original Draft Preparation, Writing-Review and Editing, Supervision; Rinal Effendi: Conceptualization, Funding Acquisition, Data curation, Formal Analysis, Investigation, Project Administration, Methodology, Validation, Writing-Original Draft Preparation, Writing-Review and Editing, Supervision; Aufa RH : Data Curation, Methodology, Investigation, Formal Analysis, Validation, Writing-Review and Editing; Asrul MF : Methodology, Formal Analysis, Validation, Writing-Review and Editing, Supervision; Bagus Dermawan : Methodology, Validation, Writing-Original Draft Preparation, Writing-Review and Editing, Supervision: Nuruliawaty Utami: Data Curation, Methodology, investigation, Formal Analysis, Validation, Writing-Review and Editing, Supervision. Data availability Underlying data figshare: Distribution of CYP2D6 multiplication, CYP2D6*5, and clinical implications in postoperative patients receiving tramadol analgesia in the Minangkabau ethnic group, Indonesia https://doi.org/10.6084/m9.figshare.26870995 . The project contains the following data: • Dataset from 65 postoperative Minangkabau ethnic patients Figshare: Desriani, Desriani; Effendi, Rinal; Utami, Nuruliawaty; Dermawan, Bagus; Muhamad Fuad, Asrul; Rizqia Haz, Aufa (2024). Statistic analysis West Sumatera Patients.pdf. figshare. Dataset. https://doi.org/10.6084/m9.figshare.27895956.v1 The project contains the following data: • Statistic analysis West Sumatera Patients.pdf Data is available under CC0 license . Acknowledgment We thank you for the financial support for this research provided by the Rumah Program Organisasi Riset Ilmu Pengetahuan Teknik, Badan Riset dan Inovasi Nasional 2022 Number 2/III/HK/2022, on behalf of Dr. Eng. Desriani, M.Si. We also want to express our gratitude to Oktri Yurika for her technical support. References 1. Meijerman I, Sanderson LM, Smits PHM, et al. : Pharmacogenetic screening of the gene deletion and duplications of CYP2D6. Drug Metab. Rev. 2007; 39 (1): 45–60. PubMed Abstract | Publisher Full Text 2. Taylor C, Crosby I, Yip V, et al. : A review of the important role of cyp2d6 in pharmacogenomics. Genes (Basel). 2020; 11 (11): 1–23. Publisher Full Text 3. Arneth B, Shams M, Hiemke C, et al. : Rapid and reliable genotyping procedure for detection of alleles with mutations, deletion, or/and duplication of the CYP2D6 gene. Clin. Biochem. 2009; 42 (12): 1282–1290. Publisher Full Text 4. Darney K, Lautz LS, Béchaux C, et al. : Human variability in polymorphic CYP2D6 metabolism: Implications for the risk assessment of chemicals in food and emerging designer drugs. Environ. Int. 2021; 156 : 106760. Publisher Full Text 5. Gaedigk A, Turner A, Everts RE, et al. : Characterization of Reference Materials for Genetic Testing of CYP2D6 Alleles: A GeT-RM Collaborative Project. J. Mol. Diagnostics. 2019; 21 (6): 1034–1052. PubMed Abstract | Publisher Full Text | Free Full Text 6. Chan W, Li MS, Sundaram SK, et al. : CYP2D6 allele frequencies, copy number variants, and tandems in the population of Hong Kong.2019; July 2018): 1–7. 7. Goh LL, Lim CW, Sim WC, et al. : Analysis of genetic variation in CYP450 genes for clinical implementation. PLoS One. 2017; 12 (1): e0169233. PubMed Abstract | Publisher Full Text | Free Full Text 8. Vadivelu N, Chang D, Helander EM, et al. : Ketorolac, Oxymorphone, Tapentadol, and Tramadol: A Comprehensive Review. Anesthesiol. Clin. 2017; 35 (2): e1–e20. PubMed Abstract | Publisher Full Text 9. Lassen D, Damkier P, Brøsen K: The Pharmacogenetics of Tramadol. Clin. Pharmacokinet. 2015; 54 (8): 825–836. Publisher Full Text 10. Nakhaee S, Hoyte C, Dart RC, et al. : A review on tramadol toxicity: mechanism of action, clinical presentation, and treatment. Forensic Toxicol. 2021; 39 (2): 293–310. Publisher Full Text 11. Grond S, Sablotzki A: Clinical pharmacology of tramadol. Clin. Pharmacokinet. 2004; 43 (13): 879–923. Publisher Full Text 12. Dean L: Tramadol Therapy and CYP2D6 Genotype. Med. Genet. Summ. 2012; 1–16. (Md). Reference Source 13. Indra B, Lipoeto NI, Tjong DH, et al. : Polymorphism of Gene OPRM A118G and COMT G158A and Pain Sensitivity of the Minangkabau Ethnic, Indonesia. J. Cell Mol. Anesth. 2023; 8 (2): 73–83. 14. Unknown - Stuven 1996 Pharmacogenetics - Rapid detection of CYP2D6 null. http 15. Schaeffeler E, Schwab M, Eichelbaum M, et al. : CYP2D6 Genotyping Strategy Based on Gene Copy Number Determination by TaqMan Real-Time PCR. Hum. Mutat. 2003; 22 (6): 476–485. PubMed Abstract | Publisher Full Text 16. Bodin L, Beaune PH, Loriot MA: Determination of cytochrome P450 2D6 (CYP2D6) gene copy number by real-time quantitative PCR. J. Biomed. Biotechnol. 2005; 2005 (3): 248–253. PubMed Abstract 17. Kisoi M, Imai M, Yamamura M, et al. : Unique genotyping protocol of CYP2D6 allele frequency using real time quantitative PCR from Japanese healthy women. Biol. Pharm. Bull. 2020; 43 (5): 904–907. PubMed Abstract | Publisher Full Text 18. Larsen JB, Jørgensen S: Simple and Robust Detection of CYP2D6 Gene Deletions and Duplications Using CYP2D8P as Reference. Pharmaceuticals. 2022; 15 (2): 1–13. Publisher Full Text 19. Langaee T, Hamadeh I, Chapman AB, et al. : A novel simple method for determining CYP2D6 gene copy number and identifying allele(s) with duplication/multiplication. PLoS One. 2015; 10 (1): 2–12. Publisher Full Text 20. Johansson I, Lundqvist E, Dahl MJISM: 23-johansson1996.pdf. Pharmacogenetics. 1996; 6 : 351–5. PubMed Abstract | Publisher Full Text 21. Rychlik W, Spencer WJ, Rhoads RE: Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Res. 1990; 18 (21): 6409–6412. PubMed Abstract | Publisher Full Text | Free Full Text 22. Kane M: CYP2D6 Overview: Allele and Phenotype Frequencies. Med. Genet. Summ. 2021; 1–32. (Md). Reference Source 23. Caudle KE, Sangkuhl K, Whirl-Carrillo M, et al. : Standardizing CYP2D6 Genotype to Phenotype Translation: Consensus Recommendations from the Clinical Pharmacogenetics Implementation Consortium and Dutch Pharmacogenetics Working Group. Clin. Transl. Sci. 2020; 13 (1): 116–124. PubMed Abstract | Publisher Full Text | Free Full Text 24. García-Quetglas E, Azanza JR, Sádaba B, et al. : Pharmacokinetics of tramadol enantiomers and their respective phase I metabolites in relation to CYP2D6 phenotype. Pharmacol. Res. 2007; 55 (2): 122–130. PubMed Abstract | Publisher Full Text 25. Stamer UM, Lehnen K, Höthker F, et al. : Impact of CYP2D6 genotype on postoperative tramadol analgesia. Pain. 2003; 105 (1–2): 231–238. PubMed Abstract | Publisher Full Text 26. Jarvis JP, Peter AP, Shaman JA: Consequences of CYP2D6Copy-number variation for pharmacogenomics in psychiatry. Front. Psych. 2019; 10 (JUN): 1–14. Publisher Full Text 27. Hersberger M, Marti-Jaun J, Rentsch K, et al. : Rapid detection of the CYP2D6*3, CYP2D6*4, and CYP2D6*6 alleles by tetra-primer PCR and of the CYP2D6*5 allele by multiplex long PCR. Clin. Chem. 2000; 46 (8 I): 1072–1077. Publisher Full Text 28. Mchugh ML: The Chi-square test of independence Lessons in biostatistics. Biochem. Med [Internet]. 2013; 23 (2): 143–149. PubMed Abstract | Publisher Full Text | Free Full Text 29. Chiarella P, Capone P, Sisto R: Contribution of Genetic Polymorphisms in Human Health. Int. J. Environ. Res. Public Health. 2023; 20 (2). PubMed Abstract | Publisher Full Text | Free Full Text 30. Lewis KSHN: Tramadol: A new centrally acting analgesic. Am. J. Heal. Pharm. 1997; 54 : 643–652. Publisher Full Text 31. Pang WW, Wu HS, Tung CC: Tramadol 2.5 mg·kg-1 appears to be the optimal intraoperative loading dose before patient-controlled analgesia. Can. J. Anesth. 2003; 50 (1): 48–51. PubMed Abstract | Publisher Full Text 32. Sidiq S, Bacha UQ, Mir AW, et al. : Optimum Dose of Epidural Tramadol Hydrochloride for Post Operative Analgesia. Indian J. Clin. Anaesth. 2015; 2 (4): 262. Publisher Full Text 33. Sheng HH, Zeng AP, Zhu WX, et al. : Allelic distributions of CYP2D6 gene copy number variation in the Eastern Han Chinese population. Acta Pharmacol. Sin. 2007; 28 (2): 279–286. PubMed Abstract | Publisher Full Text Comments on this article Comments (0) Version 3 VERSION 3 PUBLISHED 17 Dec 2024 ADD YOUR COMMENT Comment Author details Author details 1 Department of Anesthesiology, Faculty of Medicine, Universitas Andalas, Padang, West Sumatra, Indonesia 2 Research Center for Genetic Engineering, Soekarno Sains and Technology Park, National Research and Innovation Agency (BRIN), Jl. Raya Bogor Km, 46 Cibinong, 16911, Indonesia 3 Department of Neurology, Jl dr. Soetomo no 16, Dr Kariadi General Hospital Medical Center, Semarang, Central Java, 50244, Indonesia Rinal Effendi Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Aufa Rizqia Haz Roles: Data Curation, Formal Analysis, Methodology, Validation, Visualization, Writing – Review & Editing Asrul Muhammad Fuad Roles: Formal Analysis, Methodology, Supervision, Validation, Writing – Review & Editing Bagus Dermawan Roles: Data Curation, Formal Analysis, Methodology, Supervision, Validation, Visualization, Writing – Review & Editing Nuruliawaty Utami Roles: Formal Analysis, Methodology, Supervision, Validation, Visualization, Writing – Review & Editing . Desriani Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Competing interests No competing interests were disclosed. Grant information Financial support for this research was provided by the Rumah Program Organisasi Riset Ilmu Pengetahuan Teknik, Badan Riset dan Inovasi Nasional 2022 Number 2/III/HK/2022. The funders had no role in the study design, data collection and analysis, decision to publish, or manuscript preparation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Article Versions (3) version 3 Revised Published: 23 Sep 2025, 13:1526 https://doi.org/10.12688/f1000research.155988.3 version 2 Revised Published: 22 Aug 2025, 13:1526 https://doi.org/10.12688/f1000research.155988.2 version 1 Published: 17 Dec 2024, 13:1526 https://doi.org/10.12688/f1000research.155988.1 Copyright © 2025 Effendi R et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article Effendi R, Rizqia Haz A, Muhammad Fuad A et al. Distribution of CYP2D6 multiplication, CYP2D6*5, and clinical implications in postoperative patients receiving tramadol analgesia in the Minangkabau ethnic group, Indonesia [version 3; peer review: 2 approved] . F1000Research 2025, 13 :1526 ( https://doi.org/10.12688/f1000research.155988.3 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Version 3 VERSION 3 PUBLISHED 23 Sep 2025 Revised Views 0 Cite How to cite this report: Setiawan H. Reviewer Report For: Distribution of CYP2D6 multiplication, CYP2D6*5, and clinical implications in postoperative patients receiving tramadol analgesia in the Minangkabau ethnic group, Indonesia [version 3; peer review: 2 approved] . F1000Research 2025, 13 :1526 ( https://doi.org/10.5256/f1000research.188479.r416788 ) The direct URL for this report is: https://f1000research.com/articles/13-1526/v3#referee-response-416788 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 07 Oct 2025 Heri Setiawan , Universitas Indonesia, Depok, Indonesia Approved VIEWS 0 https://doi.org/10.5256/f1000research.188479.r416788 The manuscript has been revised, and ... Continue reading READ ALL The manuscript has been revised, and all my concerns have been addressed adequately. Competing Interests: No competing interests were disclosed. Reviewer Expertise: Pharmacology and toxicology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Setiawan H. Reviewer Report For: Distribution of CYP2D6 multiplication, CYP2D6*5, and clinical implications in postoperative patients receiving tramadol analgesia in the Minangkabau ethnic group, Indonesia [version 3; peer review: 2 approved] . F1000Research 2025, 13 :1526 ( https://doi.org/10.5256/f1000research.188479.r416788 ) The direct URL for this report is: https://f1000research.com/articles/13-1526/v3#referee-response-416788 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Version 2 VERSION 2 PUBLISHED 22 Aug 2025 Revised Views 0 Cite How to cite this report: Setiawan H. Reviewer Report For: Distribution of CYP2D6 multiplication, CYP2D6*5, and clinical implications in postoperative patients receiving tramadol analgesia in the Minangkabau ethnic group, Indonesia [version 3; peer review: 2 approved] . F1000Research 2025, 13 :1526 ( https://doi.org/10.5256/f1000research.185264.r407882 ) The direct URL for this report is: https://f1000research.com/articles/13-1526/v2#referee-response-407882 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 16 Sep 2025 Heri Setiawan , Universitas Indonesia, Depok, Indonesia Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.185264.r407882 Only one out of the six points of review has been adequately addressed, which is about the explanation of a normal metabolizer. Please revise and explain adequately each point, especially the association between the VAS value and the CYP2D6 polymorphism. ... Continue reading READ ALL Only one out of the six points of review has been adequately addressed, which is about the explanation of a normal metabolizer. Please revise and explain adequately each point, especially the association between the VAS value and the CYP2D6 polymorphism. However, the data showed significant different proportion (p = 0.008) between body weight (not VAS value) and CYP2D6 polymorphism. Competing Interests: No competing interests were disclosed. Reviewer Expertise: Pharmacology and toxicology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Setiawan H. Reviewer Report For: Distribution of CYP2D6 multiplication, CYP2D6*5, and clinical implications in postoperative patients receiving tramadol analgesia in the Minangkabau ethnic group, Indonesia [version 3; peer review: 2 approved] . F1000Research 2025, 13 :1526 ( https://doi.org/10.5256/f1000research.185264.r407882 ) The direct URL for this report is: https://f1000research.com/articles/13-1526/v2#referee-response-407882 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 26 Sep 2025 Desriani Desriani , Research Center for Genetic Engineering, Soekarno Sains and Technology Park, National Research and Innovation Agency (BRIN), Jl. Raya Bogor Km, 16911, Indonesia 26 Sep 2025 Author Response 1. Only one out of the six points of review has been adequately addressed, which is about the explanation of a normal metabolizer. Please revise and explain adequately each point, ... Continue reading 1. Only one out of the six points of review has been adequately addressed, which is about the explanation of a normal metabolizer. Please revise and explain adequately each point, especially the association between the VAS value and the CYP2D6 polymorphism. However, the data showed significant different proportion (p = 0.008) between body weight (not VAS value) and CYP2D6 polymorphism. > Thank you very much for your valuable input and comment. Please find below our explanation, which we have also included in the revised manuscript (in the abstract, methods, results, and discussion parts). In the present study, we used two different statistical methods: Analysis of variance (ANOVA) was conducted to analyze the relationship between the amount of tramadol administered and the VAS value, as well as between the amount of tramadol administered and the molecular screening result. The demographic characteristics data were analyzed with a Chi-Square test. The statistical analysis was conducted with the IBM SPSS software version 24, with p values less than 0.05 being related as statistically significant.” The ANOVA analysis showed that administered tramadol was significantly associated with the VAS value of postoperative patients receiving analgesia with p = 0.045 (Supplementary). This result supported and indicated that administration of 100 mg of the drug resulted in optimal pain relief. In addition, the typical dosage of tramadol administered is within the range of 50 to 100 milligrams. 10 The dosage for pediatric patients was 50 mg, whereas the adult dosage was 100 mg. Furthermore, a significant association was also shown between tramadol administration and molecular screening, with p = 0.003 (in the Supplementary). This could be interpreted as meaning that all genotypes in this activity individually metabolize the given tramadol is still possible. This also shows that both genotypes (heterozygote CYP2D6 *5 and the multiplication) are clinically functional. In Table 2, the Chi-square test showed that the distribution of the molecular screening among the demographic factors (Age, Gender, and VAS value) of the West Sumatra patients does not have a significant correlation. While the weight of the patients is indicated as biased. As it showed inconsistent p-values between Pearson Chi-Square (p = 0.008), Likelihood Ratio (p = 0.149) and Linear-by-Linear Association (p = 0.124). According to McHugh 2013, too small sample size will affect to Pearson Chi Square, Likehood Ratio and Linear-by-linear P Value in the Chi-square test. Therefore, for a better understanding of this, further study needs a larger number of samples. In addition, gene polymorphisms are the most common type of genetic variation, defined as variations in gene sequences caused by changes in the DNA sequence that are heritable. 29 Further, the VAS value has a high subjectivity, and open possibilities have no significant correlation with molecular screening, as proved by the Chi-square result (Table 2). 2. "This study had a retrospective clinical trial registry number NCT06642480." In my opinion, it is not necessary to include the trial registry number in the abstract. "It is our first time using the clinical trial registry because the research already finished, so we applied for a retrospective registry clinical trial". This information may not be of concern to the reader Thank you very much for your valuable input and comment. We delete the sentences. 3. The images of the PCR product in gel electrophoresis are not clear Thank you very much for your valuable comment. We are very sorry that our trays for visualizing PCR products were cracked when we conducted this research in 2022. This explains why the background of our PCR product figure was cracked. But according to us, the images are still readable and analyzable. Furthermore, in Figure 1, since M-D-Wt were not always in the same gel, we used a white line to indicate the same temperature annealing and a black line to indicate different annealing temperatures. This makes the data clearer and easier to read and analyze. In addition, We completed the figure label in Figure 1 (1-3), and also in Figure 2 (2-1 and 2-2). There is an inconsistency about the patient number. Thank you very much for your valuable input and comment. We revised the sentences. A total of 65 patients participated in this study. For detailed information, please kindly see in https://doi.org/10.6084/m9.figshare.26870995, as mentioned in the data availability section of the manuscript. 1. Only one out of the six points of review has been adequately addressed, which is about the explanation of a normal metabolizer. Please revise and explain adequately each point, especially the association between the VAS value and the CYP2D6 polymorphism. However, the data showed significant different proportion (p = 0.008) between body weight (not VAS value) and CYP2D6 polymorphism. > Thank you very much for your valuable input and comment. Please find below our explanation, which we have also included in the revised manuscript (in the abstract, methods, results, and discussion parts). In the present study, we used two different statistical methods: Analysis of variance (ANOVA) was conducted to analyze the relationship between the amount of tramadol administered and the VAS value, as well as between the amount of tramadol administered and the molecular screening result. The demographic characteristics data were analyzed with a Chi-Square test. The statistical analysis was conducted with the IBM SPSS software version 24, with p values less than 0.05 being related as statistically significant.” The ANOVA analysis showed that administered tramadol was significantly associated with the VAS value of postoperative patients receiving analgesia with p = 0.045 (Supplementary). This result supported and indicated that administration of 100 mg of the drug resulted in optimal pain relief. In addition, the typical dosage of tramadol administered is within the range of 50 to 100 milligrams. 10 The dosage for pediatric patients was 50 mg, whereas the adult dosage was 100 mg. Furthermore, a significant association was also shown between tramadol administration and molecular screening, with p = 0.003 (in the Supplementary). This could be interpreted as meaning that all genotypes in this activity individually metabolize the given tramadol is still possible. This also shows that both genotypes (heterozygote CYP2D6 *5 and the multiplication) are clinically functional. In Table 2, the Chi-square test showed that the distribution of the molecular screening among the demographic factors (Age, Gender, and VAS value) of the West Sumatra patients does not have a significant correlation. While the weight of the patients is indicated as biased. As it showed inconsistent p-values between Pearson Chi-Square (p = 0.008), Likelihood Ratio (p = 0.149) and Linear-by-Linear Association (p = 0.124). According to McHugh 2013, too small sample size will affect to Pearson Chi Square, Likehood Ratio and Linear-by-linear P Value in the Chi-square test. Therefore, for a better understanding of this, further study needs a larger number of samples. In addition, gene polymorphisms are the most common type of genetic variation, defined as variations in gene sequences caused by changes in the DNA sequence that are heritable. 29 Further, the VAS value has a high subjectivity, and open possibilities have no significant correlation with molecular screening, as proved by the Chi-square result (Table 2). 2. "This study had a retrospective clinical trial registry number NCT06642480." In my opinion, it is not necessary to include the trial registry number in the abstract. "It is our first time using the clinical trial registry because the research already finished, so we applied for a retrospective registry clinical trial". This information may not be of concern to the reader Thank you very much for your valuable input and comment. We delete the sentences. 3. The images of the PCR product in gel electrophoresis are not clear Thank you very much for your valuable comment. We are very sorry that our trays for visualizing PCR products were cracked when we conducted this research in 2022. This explains why the background of our PCR product figure was cracked. But according to us, the images are still readable and analyzable. Furthermore, in Figure 1, since M-D-Wt were not always in the same gel, we used a white line to indicate the same temperature annealing and a black line to indicate different annealing temperatures. This makes the data clearer and easier to read and analyze. In addition, We completed the figure label in Figure 1 (1-3), and also in Figure 2 (2-1 and 2-2). There is an inconsistency about the patient number. Thank you very much for your valuable input and comment. We revised the sentences. A total of 65 patients participated in this study. For detailed information, please kindly see in https://doi.org/10.6084/m9.figshare.26870995, as mentioned in the data availability section of the manuscript. Competing Interests: No competing interests were disclosed Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 26 Sep 2025 Desriani Desriani , Research Center for Genetic Engineering, Soekarno Sains and Technology Park, National Research and Innovation Agency (BRIN), Jl. Raya Bogor Km, 16911, Indonesia 26 Sep 2025 Author Response 1. Only one out of the six points of review has been adequately addressed, which is about the explanation of a normal metabolizer. Please revise and explain adequately each point, ... Continue reading 1. Only one out of the six points of review has been adequately addressed, which is about the explanation of a normal metabolizer. Please revise and explain adequately each point, especially the association between the VAS value and the CYP2D6 polymorphism. However, the data showed significant different proportion (p = 0.008) between body weight (not VAS value) and CYP2D6 polymorphism. > Thank you very much for your valuable input and comment. Please find below our explanation, which we have also included in the revised manuscript (in the abstract, methods, results, and discussion parts). In the present study, we used two different statistical methods: Analysis of variance (ANOVA) was conducted to analyze the relationship between the amount of tramadol administered and the VAS value, as well as between the amount of tramadol administered and the molecular screening result. The demographic characteristics data were analyzed with a Chi-Square test. The statistical analysis was conducted with the IBM SPSS software version 24, with p values less than 0.05 being related as statistically significant.” The ANOVA analysis showed that administered tramadol was significantly associated with the VAS value of postoperative patients receiving analgesia with p = 0.045 (Supplementary). This result supported and indicated that administration of 100 mg of the drug resulted in optimal pain relief. In addition, the typical dosage of tramadol administered is within the range of 50 to 100 milligrams. 10 The dosage for pediatric patients was 50 mg, whereas the adult dosage was 100 mg. Furthermore, a significant association was also shown between tramadol administration and molecular screening, with p = 0.003 (in the Supplementary). This could be interpreted as meaning that all genotypes in this activity individually metabolize the given tramadol is still possible. This also shows that both genotypes (heterozygote CYP2D6 *5 and the multiplication) are clinically functional. In Table 2, the Chi-square test showed that the distribution of the molecular screening among the demographic factors (Age, Gender, and VAS value) of the West Sumatra patients does not have a significant correlation. While the weight of the patients is indicated as biased. As it showed inconsistent p-values between Pearson Chi-Square (p = 0.008), Likelihood Ratio (p = 0.149) and Linear-by-Linear Association (p = 0.124). According to McHugh 2013, too small sample size will affect to Pearson Chi Square, Likehood Ratio and Linear-by-linear P Value in the Chi-square test. Therefore, for a better understanding of this, further study needs a larger number of samples. In addition, gene polymorphisms are the most common type of genetic variation, defined as variations in gene sequences caused by changes in the DNA sequence that are heritable. 29 Further, the VAS value has a high subjectivity, and open possibilities have no significant correlation with molecular screening, as proved by the Chi-square result (Table 2). 2. "This study had a retrospective clinical trial registry number NCT06642480." In my opinion, it is not necessary to include the trial registry number in the abstract. "It is our first time using the clinical trial registry because the research already finished, so we applied for a retrospective registry clinical trial". This information may not be of concern to the reader Thank you very much for your valuable input and comment. We delete the sentences. 3. The images of the PCR product in gel electrophoresis are not clear Thank you very much for your valuable comment. We are very sorry that our trays for visualizing PCR products were cracked when we conducted this research in 2022. This explains why the background of our PCR product figure was cracked. But according to us, the images are still readable and analyzable. Furthermore, in Figure 1, since M-D-Wt were not always in the same gel, we used a white line to indicate the same temperature annealing and a black line to indicate different annealing temperatures. This makes the data clearer and easier to read and analyze. In addition, We completed the figure label in Figure 1 (1-3), and also in Figure 2 (2-1 and 2-2). There is an inconsistency about the patient number. Thank you very much for your valuable input and comment. We revised the sentences. A total of 65 patients participated in this study. For detailed information, please kindly see in https://doi.org/10.6084/m9.figshare.26870995, as mentioned in the data availability section of the manuscript. 1. Only one out of the six points of review has been adequately addressed, which is about the explanation of a normal metabolizer. Please revise and explain adequately each point, especially the association between the VAS value and the CYP2D6 polymorphism. However, the data showed significant different proportion (p = 0.008) between body weight (not VAS value) and CYP2D6 polymorphism. > Thank you very much for your valuable input and comment. Please find below our explanation, which we have also included in the revised manuscript (in the abstract, methods, results, and discussion parts). In the present study, we used two different statistical methods: Analysis of variance (ANOVA) was conducted to analyze the relationship between the amount of tramadol administered and the VAS value, as well as between the amount of tramadol administered and the molecular screening result. The demographic characteristics data were analyzed with a Chi-Square test. The statistical analysis was conducted with the IBM SPSS software version 24, with p values less than 0.05 being related as statistically significant.” The ANOVA analysis showed that administered tramadol was significantly associated with the VAS value of postoperative patients receiving analgesia with p = 0.045 (Supplementary). This result supported and indicated that administration of 100 mg of the drug resulted in optimal pain relief. In addition, the typical dosage of tramadol administered is within the range of 50 to 100 milligrams. 10 The dosage for pediatric patients was 50 mg, whereas the adult dosage was 100 mg. Furthermore, a significant association was also shown between tramadol administration and molecular screening, with p = 0.003 (in the Supplementary). This could be interpreted as meaning that all genotypes in this activity individually metabolize the given tramadol is still possible. This also shows that both genotypes (heterozygote CYP2D6 *5 and the multiplication) are clinically functional. In Table 2, the Chi-square test showed that the distribution of the molecular screening among the demographic factors (Age, Gender, and VAS value) of the West Sumatra patients does not have a significant correlation. While the weight of the patients is indicated as biased. As it showed inconsistent p-values between Pearson Chi-Square (p = 0.008), Likelihood Ratio (p = 0.149) and Linear-by-Linear Association (p = 0.124). According to McHugh 2013, too small sample size will affect to Pearson Chi Square, Likehood Ratio and Linear-by-linear P Value in the Chi-square test. Therefore, for a better understanding of this, further study needs a larger number of samples. In addition, gene polymorphisms are the most common type of genetic variation, defined as variations in gene sequences caused by changes in the DNA sequence that are heritable. 29 Further, the VAS value has a high subjectivity, and open possibilities have no significant correlation with molecular screening, as proved by the Chi-square result (Table 2). 2. "This study had a retrospective clinical trial registry number NCT06642480." In my opinion, it is not necessary to include the trial registry number in the abstract. "It is our first time using the clinical trial registry because the research already finished, so we applied for a retrospective registry clinical trial". This information may not be of concern to the reader Thank you very much for your valuable input and comment. We delete the sentences. 3. The images of the PCR product in gel electrophoresis are not clear Thank you very much for your valuable comment. We are very sorry that our trays for visualizing PCR products were cracked when we conducted this research in 2022. This explains why the background of our PCR product figure was cracked. But according to us, the images are still readable and analyzable. Furthermore, in Figure 1, since M-D-Wt were not always in the same gel, we used a white line to indicate the same temperature annealing and a black line to indicate different annealing temperatures. This makes the data clearer and easier to read and analyze. In addition, We completed the figure label in Figure 1 (1-3), and also in Figure 2 (2-1 and 2-2). There is an inconsistency about the patient number. Thank you very much for your valuable input and comment. We revised the sentences. A total of 65 patients participated in this study. For detailed information, please kindly see in https://doi.org/10.6084/m9.figshare.26870995, as mentioned in the data availability section of the manuscript. Competing Interests: No competing interests were disclosed Close Report a concern COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Bowolaksono A. Reviewer Report For: Distribution of CYP2D6 multiplication, CYP2D6*5, and clinical implications in postoperative patients receiving tramadol analgesia in the Minangkabau ethnic group, Indonesia [version 3; peer review: 2 approved] . F1000Research 2025, 13 :1526 ( https://doi.org/10.5256/f1000research.185264.r407883 ) The direct URL for this report is: https://f1000research.com/articles/13-1526/v2#referee-response-407883 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 05 Sep 2025 Anom Bowolaksono , Cellular and Molecular Mechanisms in Biological System (CEMBIOS) Research Group, Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Indonesia, Depok, Indonesia Approved VIEWS 0 https://doi.org/10.5256/f1000research.185264.r407883 Most of ... Continue reading READ ALL Most of comments are resolved Competing Interests: No competing interests were disclosed. Reviewer Expertise: Molecular biology in aging and cancer I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Bowolaksono A. Reviewer Report For: Distribution of CYP2D6 multiplication, CYP2D6*5, and clinical implications in postoperative patients receiving tramadol analgesia in the Minangkabau ethnic group, Indonesia [version 3; peer review: 2 approved] . F1000Research 2025, 13 :1526 ( https://doi.org/10.5256/f1000research.185264.r407883 ) The direct URL for this report is: https://f1000research.com/articles/13-1526/v2#referee-response-407883 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Version 1 VERSION 1 PUBLISHED 17 Dec 2024 Views 0 Cite How to cite this report: Bowolaksono A. Reviewer Report For: Distribution of CYP2D6 multiplication, CYP2D6*5, and clinical implications in postoperative patients receiving tramadol analgesia in the Minangkabau ethnic group, Indonesia [version 3; peer review: 2 approved] . F1000Research 2025, 13 :1526 ( https://doi.org/10.5256/f1000research.171234.r395048 ) The direct URL for this report is: https://f1000research.com/articles/13-1526/v1#referee-response-395048 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 22 Jul 2025 Anom Bowolaksono , Cellular and Molecular Mechanisms in Biological System (CEMBIOS) Research Group, Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Indonesia, Depok, Indonesia Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.171234.r395048 This paper would be reviewed per section in points manner as would written below. There are some questions and critics those need to be cleared out in this paper Introduction Here were said ... Continue reading READ ALL This paper would be reviewed per section in points manner as would written below. There are some questions and critics those need to be cleared out in this paper Introduction Here were said about average elimination half-life of the drug. Beforehand, it mentioned tramadol is converted into M1 and M2. So, which one is referred in the average elimination of drugs? Why does CYP2D6*5 alleles need to be studied? how does it significantly contribute to CYP2D6 activity levels? Methods Standardization of VAS in this study is not really explained such as categorization criterions, patient symptoms, and valuation of VAS. Type of ANOVA model that used in this paper, for example Dunnet, Sidaks, Tukey, or else, is not clear. Result It is hard to interpret from Long PCR results because legends each figures have different description. It always needs to recheck before seeing the results. As mentioned in Methods . Bar graph or table need are to be specified in related of statistical analysis beforehand of significancy conclusion. Discussion - Conclusion There are some typos From this study, can it conclude the dose that might be works related to this study? Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly Competing Interests: No competing interests were disclosed. Reviewer Expertise: Molecular biology in aging and cancer I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Bowolaksono A. Reviewer Report For: Distribution of CYP2D6 multiplication, CYP2D6*5, and clinical implications in postoperative patients receiving tramadol analgesia in the Minangkabau ethnic group, Indonesia [version 3; peer review: 2 approved] . F1000Research 2025, 13 :1526 ( https://doi.org/10.5256/f1000research.171234.r395048 ) The direct URL for this report is: https://f1000research.com/articles/13-1526/v1#referee-response-395048 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 05 Sep 2025 Desriani Desriani , Research Center for Genetic Engineering, Soekarno Sains and Technology Park, National Research and Innovation Agency (BRIN), Jl. Raya Bogor Km, 16911, Indonesia 05 Sep 2025 Author Response This paper would be reviewed per section in points manner as would written below. There are some questions and critics those need to be cleared out in this paper >Thank ... Continue reading This paper would be reviewed per section in points manner as would written below. There are some questions and critics those need to be cleared out in this paper >Thank you very much for your valuable input and comment. Introduction Here were said about average elimination half-life of the drug. Beforehand, it mentioned tramadol is converted into M1 and M2. So, which one is referred in the average elimination of drugs? Response: Thank you very much for your comment. Please kindly find the explanation below. We have also included this explanation in the manuscript. The two enantiomers that make up tramadol each contribute to its analgesic effects in a distinct way in cytochrome P450 system. The highly polymorphic enzyme CYP2D6 catalyzes the formation of M1 from O-desmethyltramadol. N-demethylation, however, was catalyzed by a different mechanism. As previously mentioned, the phenotypic variance of CYP2D6 is explained by its highly polymorphic character (mutation, deletion, and multiplication). Furthermore, depending on CYP2D6 phenotypes and other genetic factors, that the mean elimination half-life is around 6 hours, while the initial distribution half-life is 6 minutes. Three hours after delivery, O-Desmethyltramadol (M1), the main active metabolite of tramadol, reaches Cmax at 18–26% of the tramadol Cmax value. The average elimination half-life is roughly seven hours 9,23 . Why does CYP2D6*5 alleles need to be studied? how does it significantly contribute to CYP2D6 activity levels? Response: Thank you very much for your comment. Please kindly find the explanation below. We have also included some of this explanation in the manuscript. To date, prominent pharmacogenomics societies have developed clinical guidelines for at least 48 CYP2D6-substrate drugs. These guidelines contain therapeutic recommendations based on predicted CYP2D6 metaboliser phenotypes. The CYP2D6 pharmacogenomic guidelines use these metaboliser categories to provide clinicians with drug-specific recommendations, aiming to avoid prescribing ineffective or harmful doses to patients with a particular CYP2D6 metaboliser status. Phenotypic differences alter the capacity of enzymes to metabolize drugs. Deletion and multiplication of genes are important for phenotype prediction. Deletion of the entire CYP2D6 gene (*5) results in loss (in the homozygote) or reduction (in the heterozygote) of its protein production. 3, 26, 27 Furthermore, it is important to acknowledge that drug metabolism in the multiplication genotype increases the activity of the enzyme acitivity at a high rate as the functional increase. 5, 22 The UM phenotype may also be at risk of therapeutic failure or increased toxic side effects of metabolites. In this study showed that tramadol was significantly associated with the weight of postoperative patients receiving analgesia (p = 0.008). This indicated that administration of 100 mg of the drug resulted in optimal pain relief. As stated above, the typical dosage of tramadol administered is within the range of 50 to 100 milligrams. 10 The dosage for pediatric patients was 50 mg, whereas the adult dosage was 100 mg. Age and sex showed no significant correlations (p = 0.052 and 0.243, respectively). This could be interpreted as meaning that all genotypes in this activity individually metabolize the given tramadol is still possible. This also shows that both genotypes (heterozygote CYP2D6 *5 and the multiplication) are clinically functional. Methods Standardization of VAS in this study is not really explained such as categorization criterions, patient symptoms, and valuation of VAS. Response: Thank you very much for your valuable comment. Please kindly find the explanation below. We have also included this explanation in the manuscript (in methods part). The VAS is a subjective measure that depends on the pain tolerance of patients. However standarization of VAS scale was categorized as: 0 (no pain), 1-3 (signifying mild), 4-6 (middle), and 7-10 ( heavy pain). We also provide additional information on the exclusion and inclusion criteria. All of the study's participants were patients who had undergone surgery at M. Djamil Padang Hospital. The inclusion criteria were: Patients with elective surgery at M. Djamil Hospital; proven Minangkabau ethnicity through family history; signed informed consent for blood sampling and pain assessment (VAS); and American Society of Anesthesiologists (ASA) criteria levels 1–2. The exclusion criteria were: Patients who refused to sign the informed consent form for sample collection; patients with ASA criteria levels ≥3 (patients with severe comorbidities e.g. renal failure or severe liver disease) that may interfere with tramadol metabolism, this also included patients who were taking other drugs that could significantly induce or inhibit CYP2D6 (e.g. fluoxetine, paroxetine and rifampicin). Type of ANOVA model that used in this paper, for example Dunnet, Sidaks, Tukey, or else, is not clear. Thank you for your valuable comments. The information about statistical analysis that was used ini this study was mentioned in the manuscript on “2.4 Statistical Analysis” section: “ The demographic characteristics data were presented as the mean value ± standard deviation (SD). Subsequently, the data were analyzed with analysis of variance (ANOVA), followed by a chi-square test. The statistical analysis was conducted with the IBM SPSS software version 24, with p values less than 0.05 being related as statistically significant.” Here it is the detailed explanation about statistical analysis using SPSS that has been used on the data consists of: The significancies value for Visual Analogue Scale (VAS), age, sex, body weight, and molecular values, was firstly analyzed with Wild-typeity Test to evaluate the data distribution, after that ANOVA test was conducted and followed by Chi-square test. This was mentioned in 3. Results section, point 3.3 Patients demographics : “The sig value for VAS, age, sex, body weight, and molecular values <0.05 indicates that the data are not wild-typely distributed according to the Wild-typeity Test table. The demographic characteristics data presented as the mean value ± standard deviation (SD) showed no significant difference in VAS value, age, gender, and weight. The mean value range was 1.43-1.91, while the standard deviation ranged from 0.497 to 1.014 (detailed data were not shown). Further, the data was analyzed with analysis of variance (ANOVA), followed by a chi-square test.” The characteristic demographic of deletion, multiplication and wild-type polymorphism of CYP2D6 (age, sex, and weight as VAS measurement) was analyzed using Chi-square test to evaluate whether there is a correlation between the variables. The correlation between the administration of tramadol and the Visual Analogue Scale (VAS) score (which indicates the pain ratin scale on patiens) was analyzed using ANOVA test (the data have been evaluated with Normality (Wild-typeity Test) and Homogenity tests, and the data was distributed normally and homogen). The correlation between the administration of tramadol and the molecular examination was analyzed using ANOVA test (the data have been evaluated with Normality and Homogenity tests, and the data was distibuted normally and homogen). Result It is hard to interpret from Long PCR results because legends each figures have different description. It always needs to recheck before seeing the results. >Thank you very much for your comments and input. We have updated the legend in all figures to be consistent. Please kindly see the updated manuscript. As mentioned in Methods . Bar graph or table need are to be specified in related of statistical analysis beforehand of significancy conclusion. Response: Thank you for your valuable comments. Please kindly find all the detailed statistical analyses that are reported in https://doi.org/10.6084/m9.figshare.27895956.v1, as mentioned in the data availability section of the manuscript. Discussion - Conclusion There are some typos Response: Thank you very much for your comments and input. We have corrected the typos. Please kindly see the updated manuscript. From this study, can it conclude the dose that might be works related to this study? Response: Thank you very much for your valuable comments and input. Based on this study indicates that 100 mg of tramadol is an effective dose. In this study showed that tramadol was significantly associated with the weight of postoperative patients receiving analgesia (p = 0.008). This indicated that administration of 100 mg of the drug resulted in optimal pain relief. As stated above, the typical dosage of tramadol administered is within the range of 50 to 100 milligrams 10 . The dosage for pediatric patients was 50 mg, whereas the adult dosage was 100 mg. Age and sex showed no significant correlations (p = 0.052 and 0.243, respectively). This could be interpreted as meaning that all genotypes in this activity individually metabolize the given tramadol is still possible. This also shows that both genotypes ( heterozygote CYP2D6 *5 and the multiplication) are clinically functional. This paper would be reviewed per section in points manner as would written below. There are some questions and critics those need to be cleared out in this paper >Thank you very much for your valuable input and comment. Introduction Here were said about average elimination half-life of the drug. Beforehand, it mentioned tramadol is converted into M1 and M2. So, which one is referred in the average elimination of drugs? Response: Thank you very much for your comment. Please kindly find the explanation below. We have also included this explanation in the manuscript. The two enantiomers that make up tramadol each contribute to its analgesic effects in a distinct way in cytochrome P450 system. The highly polymorphic enzyme CYP2D6 catalyzes the formation of M1 from O-desmethyltramadol. N-demethylation, however, was catalyzed by a different mechanism. As previously mentioned, the phenotypic variance of CYP2D6 is explained by its highly polymorphic character (mutation, deletion, and multiplication). Furthermore, depending on CYP2D6 phenotypes and other genetic factors, that the mean elimination half-life is around 6 hours, while the initial distribution half-life is 6 minutes. Three hours after delivery, O-Desmethyltramadol (M1), the main active metabolite of tramadol, reaches Cmax at 18–26% of the tramadol Cmax value. The average elimination half-life is roughly seven hours 9,23 . Why does CYP2D6*5 alleles need to be studied? how does it significantly contribute to CYP2D6 activity levels? Response: Thank you very much for your comment. Please kindly find the explanation below. We have also included some of this explanation in the manuscript. To date, prominent pharmacogenomics societies have developed clinical guidelines for at least 48 CYP2D6-substrate drugs. These guidelines contain therapeutic recommendations based on predicted CYP2D6 metaboliser phenotypes. The CYP2D6 pharmacogenomic guidelines use these metaboliser categories to provide clinicians with drug-specific recommendations, aiming to avoid prescribing ineffective or harmful doses to patients with a particular CYP2D6 metaboliser status. Phenotypic differences alter the capacity of enzymes to metabolize drugs. Deletion and multiplication of genes are important for phenotype prediction. Deletion of the entire CYP2D6 gene (*5) results in loss (in the homozygote) or reduction (in the heterozygote) of its protein production. 3, 26, 27 Furthermore, it is important to acknowledge that drug metabolism in the multiplication genotype increases the activity of the enzyme acitivity at a high rate as the functional increase. 5, 22 The UM phenotype may also be at risk of therapeutic failure or increased toxic side effects of metabolites. In this study showed that tramadol was significantly associated with the weight of postoperative patients receiving analgesia (p = 0.008). This indicated that administration of 100 mg of the drug resulted in optimal pain relief. As stated above, the typical dosage of tramadol administered is within the range of 50 to 100 milligrams. 10 The dosage for pediatric patients was 50 mg, whereas the adult dosage was 100 mg. Age and sex showed no significant correlations (p = 0.052 and 0.243, respectively). This could be interpreted as meaning that all genotypes in this activity individually metabolize the given tramadol is still possible. This also shows that both genotypes (heterozygote CYP2D6 *5 and the multiplication) are clinically functional. Methods Standardization of VAS in this study is not really explained such as categorization criterions, patient symptoms, and valuation of VAS. Response: Thank you very much for your valuable comment. Please kindly find the explanation below. We have also included this explanation in the manuscript (in methods part). The VAS is a subjective measure that depends on the pain tolerance of patients. However standarization of VAS scale was categorized as: 0 (no pain), 1-3 (signifying mild), 4-6 (middle), and 7-10 ( heavy pain). We also provide additional information on the exclusion and inclusion criteria. All of the study's participants were patients who had undergone surgery at M. Djamil Padang Hospital. The inclusion criteria were: Patients with elective surgery at M. Djamil Hospital; proven Minangkabau ethnicity through family history; signed informed consent for blood sampling and pain assessment (VAS); and American Society of Anesthesiologists (ASA) criteria levels 1–2. The exclusion criteria were: Patients who refused to sign the informed consent form for sample collection; patients with ASA criteria levels ≥3 (patients with severe comorbidities e.g. renal failure or severe liver disease) that may interfere with tramadol metabolism, this also included patients who were taking other drugs that could significantly induce or inhibit CYP2D6 (e.g. fluoxetine, paroxetine and rifampicin). Type of ANOVA model that used in this paper, for example Dunnet, Sidaks, Tukey, or else, is not clear. Thank you for your valuable comments. The information about statistical analysis that was used ini this study was mentioned in the manuscript on “2.4 Statistical Analysis” section: “ The demographic characteristics data were presented as the mean value ± standard deviation (SD). Subsequently, the data were analyzed with analysis of variance (ANOVA), followed by a chi-square test. The statistical analysis was conducted with the IBM SPSS software version 24, with p values less than 0.05 being related as statistically significant.” Here it is the detailed explanation about statistical analysis using SPSS that has been used on the data consists of: The significancies value for Visual Analogue Scale (VAS), age, sex, body weight, and molecular values, was firstly analyzed with Wild-typeity Test to evaluate the data distribution, after that ANOVA test was conducted and followed by Chi-square test. This was mentioned in 3. Results section, point 3.3 Patients demographics : “The sig value for VAS, age, sex, body weight, and molecular values <0.05 indicates that the data are not wild-typely distributed according to the Wild-typeity Test table. The demographic characteristics data presented as the mean value ± standard deviation (SD) showed no significant difference in VAS value, age, gender, and weight. The mean value range was 1.43-1.91, while the standard deviation ranged from 0.497 to 1.014 (detailed data were not shown). Further, the data was analyzed with analysis of variance (ANOVA), followed by a chi-square test.” The characteristic demographic of deletion, multiplication and wild-type polymorphism of CYP2D6 (age, sex, and weight as VAS measurement) was analyzed using Chi-square test to evaluate whether there is a correlation between the variables. The correlation between the administration of tramadol and the Visual Analogue Scale (VAS) score (which indicates the pain ratin scale on patiens) was analyzed using ANOVA test (the data have been evaluated with Normality (Wild-typeity Test) and Homogenity tests, and the data was distributed normally and homogen). The correlation between the administration of tramadol and the molecular examination was analyzed using ANOVA test (the data have been evaluated with Normality and Homogenity tests, and the data was distibuted normally and homogen). Result It is hard to interpret from Long PCR results because legends each figures have different description. It always needs to recheck before seeing the results. >Thank you very much for your comments and input. We have updated the legend in all figures to be consistent. Please kindly see the updated manuscript. As mentioned in Methods . Bar graph or table need are to be specified in related of statistical analysis beforehand of significancy conclusion. Response: Thank you for your valuable comments. Please kindly find all the detailed statistical analyses that are reported in https://doi.org/10.6084/m9.figshare.27895956.v1, as mentioned in the data availability section of the manuscript. Discussion - Conclusion There are some typos Response: Thank you very much for your comments and input. We have corrected the typos. Please kindly see the updated manuscript. From this study, can it conclude the dose that might be works related to this study? Response: Thank you very much for your valuable comments and input. Based on this study indicates that 100 mg of tramadol is an effective dose. In this study showed that tramadol was significantly associated with the weight of postoperative patients receiving analgesia (p = 0.008). This indicated that administration of 100 mg of the drug resulted in optimal pain relief. As stated above, the typical dosage of tramadol administered is within the range of 50 to 100 milligrams 10 . The dosage for pediatric patients was 50 mg, whereas the adult dosage was 100 mg. Age and sex showed no significant correlations (p = 0.052 and 0.243, respectively). This could be interpreted as meaning that all genotypes in this activity individually metabolize the given tramadol is still possible. This also shows that both genotypes ( heterozygote CYP2D6 *5 and the multiplication) are clinically functional. Competing Interests: No competing interests were disclosed Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 05 Sep 2025 Desriani Desriani , Research Center for Genetic Engineering, Soekarno Sains and Technology Park, National Research and Innovation Agency (BRIN), Jl. Raya Bogor Km, 16911, Indonesia 05 Sep 2025 Author Response This paper would be reviewed per section in points manner as would written below. There are some questions and critics those need to be cleared out in this paper >Thank ... Continue reading This paper would be reviewed per section in points manner as would written below. There are some questions and critics those need to be cleared out in this paper >Thank you very much for your valuable input and comment. Introduction Here were said about average elimination half-life of the drug. Beforehand, it mentioned tramadol is converted into M1 and M2. So, which one is referred in the average elimination of drugs? Response: Thank you very much for your comment. Please kindly find the explanation below. We have also included this explanation in the manuscript. The two enantiomers that make up tramadol each contribute to its analgesic effects in a distinct way in cytochrome P450 system. The highly polymorphic enzyme CYP2D6 catalyzes the formation of M1 from O-desmethyltramadol. N-demethylation, however, was catalyzed by a different mechanism. As previously mentioned, the phenotypic variance of CYP2D6 is explained by its highly polymorphic character (mutation, deletion, and multiplication). Furthermore, depending on CYP2D6 phenotypes and other genetic factors, that the mean elimination half-life is around 6 hours, while the initial distribution half-life is 6 minutes. Three hours after delivery, O-Desmethyltramadol (M1), the main active metabolite of tramadol, reaches Cmax at 18–26% of the tramadol Cmax value. The average elimination half-life is roughly seven hours 9,23 . Why does CYP2D6*5 alleles need to be studied? how does it significantly contribute to CYP2D6 activity levels? Response: Thank you very much for your comment. Please kindly find the explanation below. We have also included some of this explanation in the manuscript. To date, prominent pharmacogenomics societies have developed clinical guidelines for at least 48 CYP2D6-substrate drugs. These guidelines contain therapeutic recommendations based on predicted CYP2D6 metaboliser phenotypes. The CYP2D6 pharmacogenomic guidelines use these metaboliser categories to provide clinicians with drug-specific recommendations, aiming to avoid prescribing ineffective or harmful doses to patients with a particular CYP2D6 metaboliser status. Phenotypic differences alter the capacity of enzymes to metabolize drugs. Deletion and multiplication of genes are important for phenotype prediction. Deletion of the entire CYP2D6 gene (*5) results in loss (in the homozygote) or reduction (in the heterozygote) of its protein production. 3, 26, 27 Furthermore, it is important to acknowledge that drug metabolism in the multiplication genotype increases the activity of the enzyme acitivity at a high rate as the functional increase. 5, 22 The UM phenotype may also be at risk of therapeutic failure or increased toxic side effects of metabolites. In this study showed that tramadol was significantly associated with the weight of postoperative patients receiving analgesia (p = 0.008). This indicated that administration of 100 mg of the drug resulted in optimal pain relief. As stated above, the typical dosage of tramadol administered is within the range of 50 to 100 milligrams. 10 The dosage for pediatric patients was 50 mg, whereas the adult dosage was 100 mg. Age and sex showed no significant correlations (p = 0.052 and 0.243, respectively). This could be interpreted as meaning that all genotypes in this activity individually metabolize the given tramadol is still possible. This also shows that both genotypes (heterozygote CYP2D6 *5 and the multiplication) are clinically functional. Methods Standardization of VAS in this study is not really explained such as categorization criterions, patient symptoms, and valuation of VAS. Response: Thank you very much for your valuable comment. Please kindly find the explanation below. We have also included this explanation in the manuscript (in methods part). The VAS is a subjective measure that depends on the pain tolerance of patients. However standarization of VAS scale was categorized as: 0 (no pain), 1-3 (signifying mild), 4-6 (middle), and 7-10 ( heavy pain). We also provide additional information on the exclusion and inclusion criteria. All of the study's participants were patients who had undergone surgery at M. Djamil Padang Hospital. The inclusion criteria were: Patients with elective surgery at M. Djamil Hospital; proven Minangkabau ethnicity through family history; signed informed consent for blood sampling and pain assessment (VAS); and American Society of Anesthesiologists (ASA) criteria levels 1–2. The exclusion criteria were: Patients who refused to sign the informed consent form for sample collection; patients with ASA criteria levels ≥3 (patients with severe comorbidities e.g. renal failure or severe liver disease) that may interfere with tramadol metabolism, this also included patients who were taking other drugs that could significantly induce or inhibit CYP2D6 (e.g. fluoxetine, paroxetine and rifampicin). Type of ANOVA model that used in this paper, for example Dunnet, Sidaks, Tukey, or else, is not clear. Thank you for your valuable comments. The information about statistical analysis that was used ini this study was mentioned in the manuscript on “2.4 Statistical Analysis” section: “ The demographic characteristics data were presented as the mean value ± standard deviation (SD). Subsequently, the data were analyzed with analysis of variance (ANOVA), followed by a chi-square test. The statistical analysis was conducted with the IBM SPSS software version 24, with p values less than 0.05 being related as statistically significant.” Here it is the detailed explanation about statistical analysis using SPSS that has been used on the data consists of: The significancies value for Visual Analogue Scale (VAS), age, sex, body weight, and molecular values, was firstly analyzed with Wild-typeity Test to evaluate the data distribution, after that ANOVA test was conducted and followed by Chi-square test. This was mentioned in 3. Results section, point 3.3 Patients demographics : “The sig value for VAS, age, sex, body weight, and molecular values <0.05 indicates that the data are not wild-typely distributed according to the Wild-typeity Test table. The demographic characteristics data presented as the mean value ± standard deviation (SD) showed no significant difference in VAS value, age, gender, and weight. The mean value range was 1.43-1.91, while the standard deviation ranged from 0.497 to 1.014 (detailed data were not shown). Further, the data was analyzed with analysis of variance (ANOVA), followed by a chi-square test.” The characteristic demographic of deletion, multiplication and wild-type polymorphism of CYP2D6 (age, sex, and weight as VAS measurement) was analyzed using Chi-square test to evaluate whether there is a correlation between the variables. The correlation between the administration of tramadol and the Visual Analogue Scale (VAS) score (which indicates the pain ratin scale on patiens) was analyzed using ANOVA test (the data have been evaluated with Normality (Wild-typeity Test) and Homogenity tests, and the data was distributed normally and homogen). The correlation between the administration of tramadol and the molecular examination was analyzed using ANOVA test (the data have been evaluated with Normality and Homogenity tests, and the data was distibuted normally and homogen). Result It is hard to interpret from Long PCR results because legends each figures have different description. It always needs to recheck before seeing the results. >Thank you very much for your comments and input. We have updated the legend in all figures to be consistent. Please kindly see the updated manuscript. As mentioned in Methods . Bar graph or table need are to be specified in related of statistical analysis beforehand of significancy conclusion. Response: Thank you for your valuable comments. Please kindly find all the detailed statistical analyses that are reported in https://doi.org/10.6084/m9.figshare.27895956.v1, as mentioned in the data availability section of the manuscript. Discussion - Conclusion There are some typos Response: Thank you very much for your comments and input. We have corrected the typos. Please kindly see the updated manuscript. From this study, can it conclude the dose that might be works related to this study? Response: Thank you very much for your valuable comments and input. Based on this study indicates that 100 mg of tramadol is an effective dose. In this study showed that tramadol was significantly associated with the weight of postoperative patients receiving analgesia (p = 0.008). This indicated that administration of 100 mg of the drug resulted in optimal pain relief. As stated above, the typical dosage of tramadol administered is within the range of 50 to 100 milligrams 10 . The dosage for pediatric patients was 50 mg, whereas the adult dosage was 100 mg. Age and sex showed no significant correlations (p = 0.052 and 0.243, respectively). This could be interpreted as meaning that all genotypes in this activity individually metabolize the given tramadol is still possible. This also shows that both genotypes ( heterozygote CYP2D6 *5 and the multiplication) are clinically functional. This paper would be reviewed per section in points manner as would written below. There are some questions and critics those need to be cleared out in this paper >Thank you very much for your valuable input and comment. Introduction Here were said about average elimination half-life of the drug. Beforehand, it mentioned tramadol is converted into M1 and M2. So, which one is referred in the average elimination of drugs? Response: Thank you very much for your comment. Please kindly find the explanation below. We have also included this explanation in the manuscript. The two enantiomers that make up tramadol each contribute to its analgesic effects in a distinct way in cytochrome P450 system. The highly polymorphic enzyme CYP2D6 catalyzes the formation of M1 from O-desmethyltramadol. N-demethylation, however, was catalyzed by a different mechanism. As previously mentioned, the phenotypic variance of CYP2D6 is explained by its highly polymorphic character (mutation, deletion, and multiplication). Furthermore, depending on CYP2D6 phenotypes and other genetic factors, that the mean elimination half-life is around 6 hours, while the initial distribution half-life is 6 minutes. Three hours after delivery, O-Desmethyltramadol (M1), the main active metabolite of tramadol, reaches Cmax at 18–26% of the tramadol Cmax value. The average elimination half-life is roughly seven hours 9,23 . Why does CYP2D6*5 alleles need to be studied? how does it significantly contribute to CYP2D6 activity levels? Response: Thank you very much for your comment. Please kindly find the explanation below. We have also included some of this explanation in the manuscript. To date, prominent pharmacogenomics societies have developed clinical guidelines for at least 48 CYP2D6-substrate drugs. These guidelines contain therapeutic recommendations based on predicted CYP2D6 metaboliser phenotypes. The CYP2D6 pharmacogenomic guidelines use these metaboliser categories to provide clinicians with drug-specific recommendations, aiming to avoid prescribing ineffective or harmful doses to patients with a particular CYP2D6 metaboliser status. Phenotypic differences alter the capacity of enzymes to metabolize drugs. Deletion and multiplication of genes are important for phenotype prediction. Deletion of the entire CYP2D6 gene (*5) results in loss (in the homozygote) or reduction (in the heterozygote) of its protein production. 3, 26, 27 Furthermore, it is important to acknowledge that drug metabolism in the multiplication genotype increases the activity of the enzyme acitivity at a high rate as the functional increase. 5, 22 The UM phenotype may also be at risk of therapeutic failure or increased toxic side effects of metabolites. In this study showed that tramadol was significantly associated with the weight of postoperative patients receiving analgesia (p = 0.008). This indicated that administration of 100 mg of the drug resulted in optimal pain relief. As stated above, the typical dosage of tramadol administered is within the range of 50 to 100 milligrams. 10 The dosage for pediatric patients was 50 mg, whereas the adult dosage was 100 mg. Age and sex showed no significant correlations (p = 0.052 and 0.243, respectively). This could be interpreted as meaning that all genotypes in this activity individually metabolize the given tramadol is still possible. This also shows that both genotypes (heterozygote CYP2D6 *5 and the multiplication) are clinically functional. Methods Standardization of VAS in this study is not really explained such as categorization criterions, patient symptoms, and valuation of VAS. Response: Thank you very much for your valuable comment. Please kindly find the explanation below. We have also included this explanation in the manuscript (in methods part). The VAS is a subjective measure that depends on the pain tolerance of patients. However standarization of VAS scale was categorized as: 0 (no pain), 1-3 (signifying mild), 4-6 (middle), and 7-10 ( heavy pain). We also provide additional information on the exclusion and inclusion criteria. All of the study's participants were patients who had undergone surgery at M. Djamil Padang Hospital. The inclusion criteria were: Patients with elective surgery at M. Djamil Hospital; proven Minangkabau ethnicity through family history; signed informed consent for blood sampling and pain assessment (VAS); and American Society of Anesthesiologists (ASA) criteria levels 1–2. The exclusion criteria were: Patients who refused to sign the informed consent form for sample collection; patients with ASA criteria levels ≥3 (patients with severe comorbidities e.g. renal failure or severe liver disease) that may interfere with tramadol metabolism, this also included patients who were taking other drugs that could significantly induce or inhibit CYP2D6 (e.g. fluoxetine, paroxetine and rifampicin). Type of ANOVA model that used in this paper, for example Dunnet, Sidaks, Tukey, or else, is not clear. Thank you for your valuable comments. The information about statistical analysis that was used ini this study was mentioned in the manuscript on “2.4 Statistical Analysis” section: “ The demographic characteristics data were presented as the mean value ± standard deviation (SD). Subsequently, the data were analyzed with analysis of variance (ANOVA), followed by a chi-square test. The statistical analysis was conducted with the IBM SPSS software version 24, with p values less than 0.05 being related as statistically significant.” Here it is the detailed explanation about statistical analysis using SPSS that has been used on the data consists of: The significancies value for Visual Analogue Scale (VAS), age, sex, body weight, and molecular values, was firstly analyzed with Wild-typeity Test to evaluate the data distribution, after that ANOVA test was conducted and followed by Chi-square test. This was mentioned in 3. Results section, point 3.3 Patients demographics : “The sig value for VAS, age, sex, body weight, and molecular values <0.05 indicates that the data are not wild-typely distributed according to the Wild-typeity Test table. The demographic characteristics data presented as the mean value ± standard deviation (SD) showed no significant difference in VAS value, age, gender, and weight. The mean value range was 1.43-1.91, while the standard deviation ranged from 0.497 to 1.014 (detailed data were not shown). Further, the data was analyzed with analysis of variance (ANOVA), followed by a chi-square test.” The characteristic demographic of deletion, multiplication and wild-type polymorphism of CYP2D6 (age, sex, and weight as VAS measurement) was analyzed using Chi-square test to evaluate whether there is a correlation between the variables. The correlation between the administration of tramadol and the Visual Analogue Scale (VAS) score (which indicates the pain ratin scale on patiens) was analyzed using ANOVA test (the data have been evaluated with Normality (Wild-typeity Test) and Homogenity tests, and the data was distributed normally and homogen). The correlation between the administration of tramadol and the molecular examination was analyzed using ANOVA test (the data have been evaluated with Normality and Homogenity tests, and the data was distibuted normally and homogen). Result It is hard to interpret from Long PCR results because legends each figures have different description. It always needs to recheck before seeing the results. >Thank you very much for your comments and input. We have updated the legend in all figures to be consistent. Please kindly see the updated manuscript. As mentioned in Methods . Bar graph or table need are to be specified in related of statistical analysis beforehand of significancy conclusion. Response: Thank you for your valuable comments. Please kindly find all the detailed statistical analyses that are reported in https://doi.org/10.6084/m9.figshare.27895956.v1, as mentioned in the data availability section of the manuscript. Discussion - Conclusion There are some typos Response: Thank you very much for your comments and input. We have corrected the typos. Please kindly see the updated manuscript. From this study, can it conclude the dose that might be works related to this study? Response: Thank you very much for your valuable comments and input. Based on this study indicates that 100 mg of tramadol is an effective dose. In this study showed that tramadol was significantly associated with the weight of postoperative patients receiving analgesia (p = 0.008). This indicated that administration of 100 mg of the drug resulted in optimal pain relief. As stated above, the typical dosage of tramadol administered is within the range of 50 to 100 milligrams 10 . The dosage for pediatric patients was 50 mg, whereas the adult dosage was 100 mg. Age and sex showed no significant correlations (p = 0.052 and 0.243, respectively). This could be interpreted as meaning that all genotypes in this activity individually metabolize the given tramadol is still possible. This also shows that both genotypes ( heterozygote CYP2D6 *5 and the multiplication) are clinically functional. Competing Interests: No competing interests were disclosed Close Report a concern COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Setiawan H. Reviewer Report For: Distribution of CYP2D6 multiplication, CYP2D6*5, and clinical implications in postoperative patients receiving tramadol analgesia in the Minangkabau ethnic group, Indonesia [version 3; peer review: 2 approved] . F1000Research 2025, 13 :1526 ( https://doi.org/10.5256/f1000research.171234.r391019 ) The direct URL for this report is: https://f1000research.com/articles/13-1526/v1#referee-response-391019 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 01 Jul 2025 Heri Setiawan , Universitas Indonesia, Depok, Indonesia Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.171234.r391019 This is an interesting study that describes the polymorphism of CYP2D6 in the Minangkabu ethic group. However, there are several aspects of this manuscript that should be revised: 1. Please improve the narration: - "(wild-type, CYP2D6*5, ... Continue reading READ ALL This is an interesting study that describes the polymorphism of CYP2D6 in the Minangkabu ethic group. However, there are several aspects of this manuscript that should be revised: 1. Please improve the narration: - "(wild-type, CYP2D6*5, and CYP2D6 multiplication)" Please explain shortly the metabolic capacity of each of these genotypes? - "This study had a retrospective clinical trial registry number NCT06642480." In my opinion, it is not necessary to include the trial registry number in the abstract. -"It is our first time using the clinical trial registry because the research already finished, so we applied for a retrospective registry clinical trial". This information may not be of concern to the reader -"Based on metabolic capacity, the phenotype of CYP2D6 can be divided into four parts: poor metabolizer (PM), intermediate metabolizer (IM), extensive metabolizer (EM), and ultra-metabolizer (UM)". Is there any phenotype that has a normal function of metabolism? Can the wild type in this study be considered a normal metabolizer? 2. There is considerable explanation about the PCR method and its optimization, which has been published elsewhere. I think the scope of this study is the distribution of CYP26D and its clinical implications in the Minangkabau ethnicity. Therefore, please just show the best result of the PCR method. 3. The images of the PCR product in gel electrophoresis are not clear, and the explanations are also inadequate. 4. There is an inconsistency about the patient number. In the abstract and method, it is written that 63 patients were included. However, in Table 2, the total number of patients of both genders, all body weights, and ages is 65. Furthermore, the total number of patients with all VAS scores is 62. 5. There is no explanation about the patient and its inclusion-exclusion criteria. It is important to control the confounding variables, such as disease severity, comorbidity, and the use of other medications. 6. "This was evidenced in the present study, which showed a significant association between the drug and weight of postoperative patients (p = 0.008)." I think the association established in this analysis is between the class of body weight and genotype, which needs further explanation of its biological plausibility. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly Competing Interests: No competing interests were disclosed. Reviewer Expertise: Pharmacology and toxicology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Setiawan H. Reviewer Report For: Distribution of CYP2D6 multiplication, CYP2D6*5, and clinical implications in postoperative patients receiving tramadol analgesia in the Minangkabau ethnic group, Indonesia [version 3; peer review: 2 approved] . F1000Research 2025, 13 :1526 ( https://doi.org/10.5256/f1000research.171234.r391019 ) The direct URL for this report is: https://f1000research.com/articles/13-1526/v1#referee-response-391019 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 05 Sep 2025 Desriani Desriani , Research Center for Genetic Engineering, Soekarno Sains and Technology Park, National Research and Innovation Agency (BRIN), Jl. Raya Bogor Km, 16911, Indonesia 05 Sep 2025 Author Response This is an interesting study that describes the polymorphism of CYP2D6 in the Minangkabu ethic group. However, there are several aspects of this manuscript that should be revised: ... Continue reading This is an interesting study that describes the polymorphism of CYP2D6 in the Minangkabu ethic group. However, there are several aspects of this manuscript that should be revised: Thank you very much for your kind review and for considering our manuscript. 1. Please improve the narration: - "(wild-type, CYP2D6*5, and CYP2D6 multiplication)" Please explain shortly the metabolic capacity of each of these genotypes? Thank you very much for your kind input. We have revised the manuscript and added additional information. Please find the revised version below or in the first paragraph of the manuscript. …According to the prevailing metabolic capacity, the CYP2D6 phenotype is classified into four distinct categories: poor metabolizer (PM), intermediate metabolizer (IM), extensive metabolizer (EM), and ultra-metabolizer (UM). 3,4 Arneth et al 2009 clasiffied EM as a characteristic of the normal population with two functional wild type alleles; PM usually results from the deletion or mutation of both alleles and is an autosomal recessive condition, IM those with one functional allele; Rapid metabolism UM is believed to be an autosomal dominant feature that results from either functional gene multiplication or duplication (>30%). 3 Further Darney et al 2021, reported that extensive metabolism as normal, and it is defined as having two functional wild-type alleles (i.e. *1, *2), or heterozygosity with one decreased-activity and one non-activity allele. PM is associated with CYP2D6 alleles with no activity both alleles (i.e., *3, *4, *5, *6, *7, *8, *11, *14, *15, *18, *19, *21, *29, *40). The IM phenotype has been associated with a decrease in CYP2D6 activity alleles, i.e. *9, *10, *17, *21, *36, *29, *41, *45, and *46, or heterozygosity for one decreased activity allele and one non-activity allele. The UM phenotype has been linked to at least one active CYP2D6 gene duplication. Patients exhibiting the UM phenotype may face an elevated risk of therapeutic failure or an augmented propensity for adverse metabolic toxicities. 4 - "This study had a retrospective clinical trial registry number NCT06642480." In my opinion, it is not necessary to include the trial registry number in the abstract. "It is our first time using the clinical trial registry because the research already finished, so we applied for a retrospective registry clinical trial". This information may not be of concern to the reader Thank you very much for your valuable comment. We input this information based on Editor F 1000 Research request. -"Based on metabolic capacity, the phenotype of CYP2D6 can be divided into four parts: poor metabolizer (PM), intermediate metabolizer (IM), extensive metabolizer (EM), and ultra-metabolizer (UM)". Is there any phenotype that has a normal function of metabolism? Can the wild type in this study be considered a normal metabolizer? Thank you very much for your comment. As explained in the additional information in the first paragraph, extensive metabolisers are classified as the normal population. Further, in the case of our manuscript, we revised and consistently used the term 'wild type'. Please kindly see the updated manuscript. 2. There is considerable explanation about the PCR method and its optimization, which has been published elsewhere. I think the scope of this study is the distribution of CYP26D and its clinical implications in the Minangkabau ethnicity. Therefore, please just show the best result of the PCR method. Thank you very much for your kind comments and inputs. As we have no positive samples for the optimisation of long PCR for the wild-type, CYP2D6*5 and CYP2D6 multiplication targets, according to our opinion, besides reporting the distribution of CYP26D and its clinical implications in the Minangkabau ethnicity, we also should report how we modified and optimized the Johansson 1996 procedures for Long PCR. The long PCR method developed by Johansson et al. (1996) 20 was modified in this study. The PCR products of wild-type CYP2D6, CYP2D6*5, and multiplication detection yielded 5 kb, 6 kb, and 10 kb, respectively. In contrast to Johansson et al. (1996), who used PerkinElmer's rTDNA and New England Biolabs' 0.09U Vent, the present study used Promega's GoTaqLong PCR Mastermix #M4021. GoTaq ® Long PCR Master Mix uses a combination of recombinant hot-start Taq DNA polymerase and recombinant proofreading DNA polymerase. The amplification conditions must be further optimized. In a preliminary study, the annealing temperature of 55°C-67°C was optimized using a three-step PCR (denaturation, annealing, and extension) for three DNA primers. No PCR product was detected (data not shown). Further, the annealing temperature across five different temperatures starting from 65 °C to 68°C was optimized using two-step PCR (denaturation, annealing also as an extension step) for each DNA primer. Among the five annealing temperatures tested, 68°C was the best temperature. One patient DNA sample with an 0 VAS scale, produced 6kb and 5kb for the amplified CYP2D6*5 DNA primer and wild-type CYP2D6 DNA primer, respectively, with no PCR product in all NTC. Using this 0 VAS scale patient DNA sample, amplification with a CYP2D6*5 DNA primer produced a 6 kb PCR product that consistently showed up at all annealing temperatures. The PCR product showed a specific amplification product at 68°C annealing temperature. In addition, amplification using a wild-type CYP2D6 DNA primer produced a 5 kb PCR product that consistently showed at all annealing temperatures, and specific PCR product were provide at 67.15°C and 68°C. Furthermore, DNA samples from other patients (second patient) that had 8 VAS scale showed only produced 5kb for the wild-type DNA primer at all annealing temperatures, and specific PCR product showed at 67.15°C and 68°C annealing temperature. The DNA primer used for the detection of CYP2D6 multiplication did not generate a PCR product for either sample ( Figure 1). This showed that the two samples used for optimization were CYP2D6*5 for the first sample (0 VAS scale) and the wild-type for the second sample (8 VAS scale). Specifically, CYP2D6*5 was identified as heterozygous CYP2D6*5 as the PCR product was produced using both CYP2D6*5 and wild-type DNA primers. According to Johansson et al., 20 heterozygous CYP2D6*5 led to PCR products with both CYP2D6*5 and wild-type DNA primers, whereas homozygous CYP2D6*5 resulted in no PCR product with wild-type DNA primers. Based on these results, there was an inconsistency between the VAS scale measurement and the genotype detection. This discrepancy was possible since VAS scale measurements are highly subjective and depend on patient pain tolerance. The optimal PCR setup was used to screen several samples. The screening results showed that one sample produced 10kb which was suspected to be a multiplication sample ( Figure 2). This sample was optimized for its optimal PCR setup, and the process was used to confirm and verify the results. Consistency was observed with a 10 kb PCR product as a single band across temperatures ranging from 63 to 68°C. The optimal PCR annealing temperature was determined to be 63.95, 65.2, and 67.15, indicating thick single-band DNA ( Figure 3). However, 68°C was selected because it produced a single band and was the most effective temperature for detecting both CYP2D6*5 and wild-type alleles. In addition, all the annealing optimization processes showed clear and clean NTC. The remaining samples were screened using a modified method. 3. The images of the PCR product in gel electrophoresis are not clear, and the explanations are also inadequate. Thank you very much for your comment and we regret to inform you that, at that moment, our gel doc DNA tray was in a crack condition. We believe that, despite the crack situation, our DNA target was clear and visible. We also indicated the DNA target with an arrow, as shown in the figure in the text. Further We add additional explanation regarding the PCR in order have adequate explanation (highlighted with yellow). Please kindly find below or in the manuscript This study aimed to investigate CYP2D6*5 and CYP2D6 multiplication in post-operative Minangkabau ethnicity patients receiving tramadol analgesia and further investigate the efficacy of tramadol in these patients. To achieve this, the Long PCR method developed by Johansson et al., 20 was modified. The Long PCR, reported by Johansson et al 1996, was successfully modified with the GoTaq Long PCR master mix from Promega #M4021, with two-step PCR at 68°C annealing and extension PCR amplification temperatures. It is important to optimise the annealing temperature, particularly when synthesising long PCR products or using total genomic DNA as the PCR substrate. An annealing temperature that is too high will reduced the PCR product yielded while an annealing temperature that is too low will produce an unspecific PCR product. PCR annealing temperature optimization was one of the key successes for developing PCR methods. 21 . GoTaq ® Long PCR Master Mix uses a combination of recombinant hot-start Taq DNA polymerase and recombinant proofreading DNA polymerase. Both enzymes in the GoTaq Long PCR master mix from Promega #M4021 were required to amplify long targets. Taq DNA Polymerase alone cannot correct nucleotide mismatches resulting from misincorporation and is therefore ineffective in amplifying segments longer than 3-5 kb. Products are truncated if nucleotides are misincorporated; further, they cannot be extended in subsequent cycles. Long amplicons can be amplified with a high yield by adding a small amount of proofreading enzyme to fix mismatches and allow extension. To ensure accurate PCR, the size of the product was monitored. The sizes of the wild-type, CYP2D6*5, and CYP2D6 multiplication PCR products were 5kb, 6kb, and 10 kb, respectively. The wild-type genotype PCR product was used as an control reaction. Furthermore, heterozygous CYP2D6*5 produced a DNA PCR product in a tube containing CYP2D6*5 and wild-type DNA primers. In contrast, homozygous CYP2D6*5 produced no PCR products in the tube containing wild-type DNA primers. Although the real-time method offers a simpler and faster alternative to long PCR, it requires costly real-time PCR machines, and pyrosequencing is also costly. The modified Long PCR method was used to generate a distribution report of the Minangkabau ethnicity genotypes. 4. There is an inconsistency about the patient number. In the abstract and method, it is written that 63 patients were included. However, in Table 2, the total number of patients of both genders, all body weights, and ages is 65. Furthermore, the total number of patients with all VAS scores is 62. Thank you very much for your kind correction. This study included a total of 65 patients. Among the patients, 62 received tramadol analgesia and 63 were tested for genetics, while the other two samples did not have detailed information. Please kindly see detailed information in https://doi.org/10.6084/m9.figshare.26870995, as mentioned in the data availability section of the manuscript. 5. There is no explanation about the patient and its inclusion-exclusion criteria. It is important to control the confounding variables, such as disease severity, comorbidity, and the use of other medications. Thank you very much for your kind input. Please kindly find our manuscript that we have added and completed the inclusion and exclusion criteria, In the methods section. ..All of the study's participants were patients who had undergone surgery at M. Djamil Padang Hospital. The inclusion criteria were: Patients with elective surgery at M. Djamil Hospital; proven Minangkabau ethnicity through family history; signed informed consent for blood sampling and pain assessment (VAS); and American Society of Anesthesiologists (ASA) criteria levels 1–2. The exclusion criteria were: Patients who refused to sign the informed consent form for sample collection; patients with ASA criteria levels ≥3 (patients with severe comorbidities e.g. renal failure or severe liver disease) that may interfere with tramadol metabolism, this also included patients who were taking other drugs that could significantly induce or inhibit CYP2D6 (e.g. fluoxetine, paroxetine and rifampicin)… 6. "This was evidenced in the present study, which showed a significant association between the drug and weight of postoperative patients (p = 0.008)." I think the association established in this analysis is between the class of body weight and genotype, which needs further explanation of its biological plausibility. Thank you very much for your kind comment. Please find the additional explanation below. The two enantiomers that make up tramadol each contribute to its analgesic effects in a distinct way in cytochrome P450 system. The highly polymorphic enzyme CYP2D6 catalyzes the formation of M1 from O-desmethyltramadol. N-demethylation, however, was catalyzed by a different mechanism. As previously mentioned, the phenotypic variance of CYP2D6 is explained by its highly polymorphic character (mutation, deletion, and multiplication). Furthermore, depending on CYP2D6 phenotypes and other genetic factors, that the mean elimination half-life is around 6 hours, while the initial distribution half-life is 6 minutes. Three hours after delivery, O-Desmethyltramadol (M1), the main active metabolite of tramadol, reaches Cmax at 18–26% of the tramadol Cmax value. The average elimination half-life is roughly seven hours. 9 , 25 Deletion and multiplication of genes are important for phenotype prediction. Deletion of the entire CYP2D6 gene (*5) results in loss (in the homozygote) or reduction (in the heterozygote) of its protein production. 3, 26, 27 Furthermore, it is important to acknowledge that drug metabolism in the multiplication genotype increases the activity of the enzyme acitivity at a high rate as the functional increase. 5, 22 The UM phenotype may also be at risk of therapeutic failure or increased toxic side effects of metabolites. In this study showed that tramadol was significantly associated with the weight of postoperative patients receiving analgesia (p = 0.008). This indicated that administration of 100 mg of the drug resulted in optimal pain relief. As stated above, the typical dosage of tramadol administered is within the range of 50 to 100 milligrams. 10 The dosage for pediatric patients was 50 mg, whereas the adult dosage was 100 mg. Age and sex showed no significant correlations (p = 0.052 and 0.243, respectively). This could be interpreted as meaning that all genotypes in this activity individually metabolize the given tramadol is still possible. This also shows that both genotypes (heterozygote CYP2D6 *5 and the multiplication) are clinically functional. The potency of tramadol analgesia is approximately 10% that of morphine when administered intravenously. Tramadol has also been reported to be comparable to morphine. 28, 29 Tramadol is an efficient and well-tolerated medication for the treatment of chronic pain, whether of malignant or non-malignant origin, especially neuropathic pain. It can also be used to relieve pain related to trauma, renal or biliary colic, and labor. When taken as a non-opioid analgesic, the analgesic effect of the drug can be enhanced. 28 Furthermore, tramadol administration is ideally based on body weight. According to Sidiq et al. (2015), 2 mg/kg body weight of tramadol hydrochloride is an optimal dose for postoperative analgesia when administered epidurally in urological surgery, with no significant increase in side effects. 30 Pang et al. (2005) reported that the recommended intraoperative dose of tramadol is 2.5 mg/kg body weight for effective postoperative analgesia with minimal sedation, when considering the efficacy and side-effect profile. 29 Patients without CYP2D6 activity may not be able to metabolize drugs. Ultra-rapid metabolizers may also experience treatment failure owing to the rapid metabolism of active drugs, resulting in drug levels below the therapeutic range. 5 The data obtained specifically in the multiplication part were not in line with the presented statement. This discrepancy may be attributed to the absence of a subsequent follow-up. To enhance the quality of future data, it is essential to categorize the type of operation, collect a larger number of samples, and conduct longer follow-up periods, and also estimate the pharmacokinetic parameter of tramadol in plasma. Tramadol's enantiomers, their primary phase I metabolites, and epinephrin were pharmacokinetic parameter of tramadol in plasma in order to evaluate the CYP2D6 phenotype. 23 Also following the recommendation of the consensus on translating CYP2D6 genotype to metabolizer phenotype. 24 This is an interesting study that describes the polymorphism of CYP2D6 in the Minangkabu ethic group. However, there are several aspects of this manuscript that should be revised: Thank you very much for your kind review and for considering our manuscript. 1. Please improve the narration: - "(wild-type, CYP2D6*5, and CYP2D6 multiplication)" Please explain shortly the metabolic capacity of each of these genotypes? Thank you very much for your kind input. We have revised the manuscript and added additional information. Please find the revised version below or in the first paragraph of the manuscript. …According to the prevailing metabolic capacity, the CYP2D6 phenotype is classified into four distinct categories: poor metabolizer (PM), intermediate metabolizer (IM), extensive metabolizer (EM), and ultra-metabolizer (UM). 3,4 Arneth et al 2009 clasiffied EM as a characteristic of the normal population with two functional wild type alleles; PM usually results from the deletion or mutation of both alleles and is an autosomal recessive condition, IM those with one functional allele; Rapid metabolism UM is believed to be an autosomal dominant feature that results from either functional gene multiplication or duplication (>30%). 3 Further Darney et al 2021, reported that extensive metabolism as normal, and it is defined as having two functional wild-type alleles (i.e. *1, *2), or heterozygosity with one decreased-activity and one non-activity allele. PM is associated with CYP2D6 alleles with no activity both alleles (i.e., *3, *4, *5, *6, *7, *8, *11, *14, *15, *18, *19, *21, *29, *40). The IM phenotype has been associated with a decrease in CYP2D6 activity alleles, i.e. *9, *10, *17, *21, *36, *29, *41, *45, and *46, or heterozygosity for one decreased activity allele and one non-activity allele. The UM phenotype has been linked to at least one active CYP2D6 gene duplication. Patients exhibiting the UM phenotype may face an elevated risk of therapeutic failure or an augmented propensity for adverse metabolic toxicities. 4 - "This study had a retrospective clinical trial registry number NCT06642480." In my opinion, it is not necessary to include the trial registry number in the abstract. "It is our first time using the clinical trial registry because the research already finished, so we applied for a retrospective registry clinical trial". This information may not be of concern to the reader Thank you very much for your valuable comment. We input this information based on Editor F 1000 Research request. -"Based on metabolic capacity, the phenotype of CYP2D6 can be divided into four parts: poor metabolizer (PM), intermediate metabolizer (IM), extensive metabolizer (EM), and ultra-metabolizer (UM)". Is there any phenotype that has a normal function of metabolism? Can the wild type in this study be considered a normal metabolizer? Thank you very much for your comment. As explained in the additional information in the first paragraph, extensive metabolisers are classified as the normal population. Further, in the case of our manuscript, we revised and consistently used the term 'wild type'. Please kindly see the updated manuscript. 2. There is considerable explanation about the PCR method and its optimization, which has been published elsewhere. I think the scope of this study is the distribution of CYP26D and its clinical implications in the Minangkabau ethnicity. Therefore, please just show the best result of the PCR method. Thank you very much for your kind comments and inputs. As we have no positive samples for the optimisation of long PCR for the wild-type, CYP2D6*5 and CYP2D6 multiplication targets, according to our opinion, besides reporting the distribution of CYP26D and its clinical implications in the Minangkabau ethnicity, we also should report how we modified and optimized the Johansson 1996 procedures for Long PCR. The long PCR method developed by Johansson et al. (1996) 20 was modified in this study. The PCR products of wild-type CYP2D6, CYP2D6*5, and multiplication detection yielded 5 kb, 6 kb, and 10 kb, respectively. In contrast to Johansson et al. (1996), who used PerkinElmer's rTDNA and New England Biolabs' 0.09U Vent, the present study used Promega's GoTaqLong PCR Mastermix #M4021. GoTaq ® Long PCR Master Mix uses a combination of recombinant hot-start Taq DNA polymerase and recombinant proofreading DNA polymerase. The amplification conditions must be further optimized. In a preliminary study, the annealing temperature of 55°C-67°C was optimized using a three-step PCR (denaturation, annealing, and extension) for three DNA primers. No PCR product was detected (data not shown). Further, the annealing temperature across five different temperatures starting from 65 °C to 68°C was optimized using two-step PCR (denaturation, annealing also as an extension step) for each DNA primer. Among the five annealing temperatures tested, 68°C was the best temperature. One patient DNA sample with an 0 VAS scale, produced 6kb and 5kb for the amplified CYP2D6*5 DNA primer and wild-type CYP2D6 DNA primer, respectively, with no PCR product in all NTC. Using this 0 VAS scale patient DNA sample, amplification with a CYP2D6*5 DNA primer produced a 6 kb PCR product that consistently showed up at all annealing temperatures. The PCR product showed a specific amplification product at 68°C annealing temperature. In addition, amplification using a wild-type CYP2D6 DNA primer produced a 5 kb PCR product that consistently showed at all annealing temperatures, and specific PCR product were provide at 67.15°C and 68°C. Furthermore, DNA samples from other patients (second patient) that had 8 VAS scale showed only produced 5kb for the wild-type DNA primer at all annealing temperatures, and specific PCR product showed at 67.15°C and 68°C annealing temperature. The DNA primer used for the detection of CYP2D6 multiplication did not generate a PCR product for either sample ( Figure 1). This showed that the two samples used for optimization were CYP2D6*5 for the first sample (0 VAS scale) and the wild-type for the second sample (8 VAS scale). Specifically, CYP2D6*5 was identified as heterozygous CYP2D6*5 as the PCR product was produced using both CYP2D6*5 and wild-type DNA primers. According to Johansson et al., 20 heterozygous CYP2D6*5 led to PCR products with both CYP2D6*5 and wild-type DNA primers, whereas homozygous CYP2D6*5 resulted in no PCR product with wild-type DNA primers. Based on these results, there was an inconsistency between the VAS scale measurement and the genotype detection. This discrepancy was possible since VAS scale measurements are highly subjective and depend on patient pain tolerance. The optimal PCR setup was used to screen several samples. The screening results showed that one sample produced 10kb which was suspected to be a multiplication sample ( Figure 2). This sample was optimized for its optimal PCR setup, and the process was used to confirm and verify the results. Consistency was observed with a 10 kb PCR product as a single band across temperatures ranging from 63 to 68°C. The optimal PCR annealing temperature was determined to be 63.95, 65.2, and 67.15, indicating thick single-band DNA ( Figure 3). However, 68°C was selected because it produced a single band and was the most effective temperature for detecting both CYP2D6*5 and wild-type alleles. In addition, all the annealing optimization processes showed clear and clean NTC. The remaining samples were screened using a modified method. 3. The images of the PCR product in gel electrophoresis are not clear, and the explanations are also inadequate. Thank you very much for your comment and we regret to inform you that, at that moment, our gel doc DNA tray was in a crack condition. We believe that, despite the crack situation, our DNA target was clear and visible. We also indicated the DNA target with an arrow, as shown in the figure in the text. Further We add additional explanation regarding the PCR in order have adequate explanation (highlighted with yellow). Please kindly find below or in the manuscript This study aimed to investigate CYP2D6*5 and CYP2D6 multiplication in post-operative Minangkabau ethnicity patients receiving tramadol analgesia and further investigate the efficacy of tramadol in these patients. To achieve this, the Long PCR method developed by Johansson et al., 20 was modified. The Long PCR, reported by Johansson et al 1996, was successfully modified with the GoTaq Long PCR master mix from Promega #M4021, with two-step PCR at 68°C annealing and extension PCR amplification temperatures. It is important to optimise the annealing temperature, particularly when synthesising long PCR products or using total genomic DNA as the PCR substrate. An annealing temperature that is too high will reduced the PCR product yielded while an annealing temperature that is too low will produce an unspecific PCR product. PCR annealing temperature optimization was one of the key successes for developing PCR methods. 21 . GoTaq ® Long PCR Master Mix uses a combination of recombinant hot-start Taq DNA polymerase and recombinant proofreading DNA polymerase. Both enzymes in the GoTaq Long PCR master mix from Promega #M4021 were required to amplify long targets. Taq DNA Polymerase alone cannot correct nucleotide mismatches resulting from misincorporation and is therefore ineffective in amplifying segments longer than 3-5 kb. Products are truncated if nucleotides are misincorporated; further, they cannot be extended in subsequent cycles. Long amplicons can be amplified with a high yield by adding a small amount of proofreading enzyme to fix mismatches and allow extension. To ensure accurate PCR, the size of the product was monitored. The sizes of the wild-type, CYP2D6*5, and CYP2D6 multiplication PCR products were 5kb, 6kb, and 10 kb, respectively. The wild-type genotype PCR product was used as an control reaction. Furthermore, heterozygous CYP2D6*5 produced a DNA PCR product in a tube containing CYP2D6*5 and wild-type DNA primers. In contrast, homozygous CYP2D6*5 produced no PCR products in the tube containing wild-type DNA primers. Although the real-time method offers a simpler and faster alternative to long PCR, it requires costly real-time PCR machines, and pyrosequencing is also costly. The modified Long PCR method was used to generate a distribution report of the Minangkabau ethnicity genotypes. 4. There is an inconsistency about the patient number. In the abstract and method, it is written that 63 patients were included. However, in Table 2, the total number of patients of both genders, all body weights, and ages is 65. Furthermore, the total number of patients with all VAS scores is 62. Thank you very much for your kind correction. This study included a total of 65 patients. Among the patients, 62 received tramadol analgesia and 63 were tested for genetics, while the other two samples did not have detailed information. Please kindly see detailed information in https://doi.org/10.6084/m9.figshare.26870995, as mentioned in the data availability section of the manuscript. 5. There is no explanation about the patient and its inclusion-exclusion criteria. It is important to control the confounding variables, such as disease severity, comorbidity, and the use of other medications. Thank you very much for your kind input. Please kindly find our manuscript that we have added and completed the inclusion and exclusion criteria, In the methods section. ..All of the study's participants were patients who had undergone surgery at M. Djamil Padang Hospital. The inclusion criteria were: Patients with elective surgery at M. Djamil Hospital; proven Minangkabau ethnicity through family history; signed informed consent for blood sampling and pain assessment (VAS); and American Society of Anesthesiologists (ASA) criteria levels 1–2. The exclusion criteria were: Patients who refused to sign the informed consent form for sample collection; patients with ASA criteria levels ≥3 (patients with severe comorbidities e.g. renal failure or severe liver disease) that may interfere with tramadol metabolism, this also included patients who were taking other drugs that could significantly induce or inhibit CYP2D6 (e.g. fluoxetine, paroxetine and rifampicin)… 6. "This was evidenced in the present study, which showed a significant association between the drug and weight of postoperative patients (p = 0.008)." I think the association established in this analysis is between the class of body weight and genotype, which needs further explanation of its biological plausibility. Thank you very much for your kind comment. Please find the additional explanation below. The two enantiomers that make up tramadol each contribute to its analgesic effects in a distinct way in cytochrome P450 system. The highly polymorphic enzyme CYP2D6 catalyzes the formation of M1 from O-desmethyltramadol. N-demethylation, however, was catalyzed by a different mechanism. As previously mentioned, the phenotypic variance of CYP2D6 is explained by its highly polymorphic character (mutation, deletion, and multiplication). Furthermore, depending on CYP2D6 phenotypes and other genetic factors, that the mean elimination half-life is around 6 hours, while the initial distribution half-life is 6 minutes. Three hours after delivery, O-Desmethyltramadol (M1), the main active metabolite of tramadol, reaches Cmax at 18–26% of the tramadol Cmax value. The average elimination half-life is roughly seven hours. 9 , 25 Deletion and multiplication of genes are important for phenotype prediction. Deletion of the entire CYP2D6 gene (*5) results in loss (in the homozygote) or reduction (in the heterozygote) of its protein production. 3, 26, 27 Furthermore, it is important to acknowledge that drug metabolism in the multiplication genotype increases the activity of the enzyme acitivity at a high rate as the functional increase. 5, 22 The UM phenotype may also be at risk of therapeutic failure or increased toxic side effects of metabolites. In this study showed that tramadol was significantly associated with the weight of postoperative patients receiving analgesia (p = 0.008). This indicated that administration of 100 mg of the drug resulted in optimal pain relief. As stated above, the typical dosage of tramadol administered is within the range of 50 to 100 milligrams. 10 The dosage for pediatric patients was 50 mg, whereas the adult dosage was 100 mg. Age and sex showed no significant correlations (p = 0.052 and 0.243, respectively). This could be interpreted as meaning that all genotypes in this activity individually metabolize the given tramadol is still possible. This also shows that both genotypes (heterozygote CYP2D6 *5 and the multiplication) are clinically functional. The potency of tramadol analgesia is approximately 10% that of morphine when administered intravenously. Tramadol has also been reported to be comparable to morphine. 28, 29 Tramadol is an efficient and well-tolerated medication for the treatment of chronic pain, whether of malignant or non-malignant origin, especially neuropathic pain. It can also be used to relieve pain related to trauma, renal or biliary colic, and labor. When taken as a non-opioid analgesic, the analgesic effect of the drug can be enhanced. 28 Furthermore, tramadol administration is ideally based on body weight. According to Sidiq et al. (2015), 2 mg/kg body weight of tramadol hydrochloride is an optimal dose for postoperative analgesia when administered epidurally in urological surgery, with no significant increase in side effects. 30 Pang et al. (2005) reported that the recommended intraoperative dose of tramadol is 2.5 mg/kg body weight for effective postoperative analgesia with minimal sedation, when considering the efficacy and side-effect profile. 29 Patients without CYP2D6 activity may not be able to metabolize drugs. Ultra-rapid metabolizers may also experience treatment failure owing to the rapid metabolism of active drugs, resulting in drug levels below the therapeutic range. 5 The data obtained specifically in the multiplication part were not in line with the presented statement. This discrepancy may be attributed to the absence of a subsequent follow-up. To enhance the quality of future data, it is essential to categorize the type of operation, collect a larger number of samples, and conduct longer follow-up periods, and also estimate the pharmacokinetic parameter of tramadol in plasma. Tramadol's enantiomers, their primary phase I metabolites, and epinephrin were pharmacokinetic parameter of tramadol in plasma in order to evaluate the CYP2D6 phenotype. 23 Also following the recommendation of the consensus on translating CYP2D6 genotype to metabolizer phenotype. 24 Competing Interests: No competing interests were disclosed Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 05 Sep 2025 Desriani Desriani , Research Center for Genetic Engineering, Soekarno Sains and Technology Park, National Research and Innovation Agency (BRIN), Jl. Raya Bogor Km, 16911, Indonesia 05 Sep 2025 Author Response This is an interesting study that describes the polymorphism of CYP2D6 in the Minangkabu ethic group. However, there are several aspects of this manuscript that should be revised: ... Continue reading This is an interesting study that describes the polymorphism of CYP2D6 in the Minangkabu ethic group. However, there are several aspects of this manuscript that should be revised: Thank you very much for your kind review and for considering our manuscript. 1. Please improve the narration: - "(wild-type, CYP2D6*5, and CYP2D6 multiplication)" Please explain shortly the metabolic capacity of each of these genotypes? Thank you very much for your kind input. We have revised the manuscript and added additional information. Please find the revised version below or in the first paragraph of the manuscript. …According to the prevailing metabolic capacity, the CYP2D6 phenotype is classified into four distinct categories: poor metabolizer (PM), intermediate metabolizer (IM), extensive metabolizer (EM), and ultra-metabolizer (UM). 3,4 Arneth et al 2009 clasiffied EM as a characteristic of the normal population with two functional wild type alleles; PM usually results from the deletion or mutation of both alleles and is an autosomal recessive condition, IM those with one functional allele; Rapid metabolism UM is believed to be an autosomal dominant feature that results from either functional gene multiplication or duplication (>30%). 3 Further Darney et al 2021, reported that extensive metabolism as normal, and it is defined as having two functional wild-type alleles (i.e. *1, *2), or heterozygosity with one decreased-activity and one non-activity allele. PM is associated with CYP2D6 alleles with no activity both alleles (i.e., *3, *4, *5, *6, *7, *8, *11, *14, *15, *18, *19, *21, *29, *40). The IM phenotype has been associated with a decrease in CYP2D6 activity alleles, i.e. *9, *10, *17, *21, *36, *29, *41, *45, and *46, or heterozygosity for one decreased activity allele and one non-activity allele. The UM phenotype has been linked to at least one active CYP2D6 gene duplication. Patients exhibiting the UM phenotype may face an elevated risk of therapeutic failure or an augmented propensity for adverse metabolic toxicities. 4 - "This study had a retrospective clinical trial registry number NCT06642480." In my opinion, it is not necessary to include the trial registry number in the abstract. "It is our first time using the clinical trial registry because the research already finished, so we applied for a retrospective registry clinical trial". This information may not be of concern to the reader Thank you very much for your valuable comment. We input this information based on Editor F 1000 Research request. -"Based on metabolic capacity, the phenotype of CYP2D6 can be divided into four parts: poor metabolizer (PM), intermediate metabolizer (IM), extensive metabolizer (EM), and ultra-metabolizer (UM)". Is there any phenotype that has a normal function of metabolism? Can the wild type in this study be considered a normal metabolizer? Thank you very much for your comment. As explained in the additional information in the first paragraph, extensive metabolisers are classified as the normal population. Further, in the case of our manuscript, we revised and consistently used the term 'wild type'. Please kindly see the updated manuscript. 2. There is considerable explanation about the PCR method and its optimization, which has been published elsewhere. I think the scope of this study is the distribution of CYP26D and its clinical implications in the Minangkabau ethnicity. Therefore, please just show the best result of the PCR method. Thank you very much for your kind comments and inputs. As we have no positive samples for the optimisation of long PCR for the wild-type, CYP2D6*5 and CYP2D6 multiplication targets, according to our opinion, besides reporting the distribution of CYP26D and its clinical implications in the Minangkabau ethnicity, we also should report how we modified and optimized the Johansson 1996 procedures for Long PCR. The long PCR method developed by Johansson et al. (1996) 20 was modified in this study. The PCR products of wild-type CYP2D6, CYP2D6*5, and multiplication detection yielded 5 kb, 6 kb, and 10 kb, respectively. In contrast to Johansson et al. (1996), who used PerkinElmer's rTDNA and New England Biolabs' 0.09U Vent, the present study used Promega's GoTaqLong PCR Mastermix #M4021. GoTaq ® Long PCR Master Mix uses a combination of recombinant hot-start Taq DNA polymerase and recombinant proofreading DNA polymerase. The amplification conditions must be further optimized. In a preliminary study, the annealing temperature of 55°C-67°C was optimized using a three-step PCR (denaturation, annealing, and extension) for three DNA primers. No PCR product was detected (data not shown). Further, the annealing temperature across five different temperatures starting from 65 °C to 68°C was optimized using two-step PCR (denaturation, annealing also as an extension step) for each DNA primer. Among the five annealing temperatures tested, 68°C was the best temperature. One patient DNA sample with an 0 VAS scale, produced 6kb and 5kb for the amplified CYP2D6*5 DNA primer and wild-type CYP2D6 DNA primer, respectively, with no PCR product in all NTC. Using this 0 VAS scale patient DNA sample, amplification with a CYP2D6*5 DNA primer produced a 6 kb PCR product that consistently showed up at all annealing temperatures. The PCR product showed a specific amplification product at 68°C annealing temperature. In addition, amplification using a wild-type CYP2D6 DNA primer produced a 5 kb PCR product that consistently showed at all annealing temperatures, and specific PCR product were provide at 67.15°C and 68°C. Furthermore, DNA samples from other patients (second patient) that had 8 VAS scale showed only produced 5kb for the wild-type DNA primer at all annealing temperatures, and specific PCR product showed at 67.15°C and 68°C annealing temperature. The DNA primer used for the detection of CYP2D6 multiplication did not generate a PCR product for either sample ( Figure 1). This showed that the two samples used for optimization were CYP2D6*5 for the first sample (0 VAS scale) and the wild-type for the second sample (8 VAS scale). Specifically, CYP2D6*5 was identified as heterozygous CYP2D6*5 as the PCR product was produced using both CYP2D6*5 and wild-type DNA primers. According to Johansson et al., 20 heterozygous CYP2D6*5 led to PCR products with both CYP2D6*5 and wild-type DNA primers, whereas homozygous CYP2D6*5 resulted in no PCR product with wild-type DNA primers. Based on these results, there was an inconsistency between the VAS scale measurement and the genotype detection. This discrepancy was possible since VAS scale measurements are highly subjective and depend on patient pain tolerance. The optimal PCR setup was used to screen several samples. The screening results showed that one sample produced 10kb which was suspected to be a multiplication sample ( Figure 2). This sample was optimized for its optimal PCR setup, and the process was used to confirm and verify the results. Consistency was observed with a 10 kb PCR product as a single band across temperatures ranging from 63 to 68°C. The optimal PCR annealing temperature was determined to be 63.95, 65.2, and 67.15, indicating thick single-band DNA ( Figure 3). However, 68°C was selected because it produced a single band and was the most effective temperature for detecting both CYP2D6*5 and wild-type alleles. In addition, all the annealing optimization processes showed clear and clean NTC. The remaining samples were screened using a modified method. 3. The images of the PCR product in gel electrophoresis are not clear, and the explanations are also inadequate. Thank you very much for your comment and we regret to inform you that, at that moment, our gel doc DNA tray was in a crack condition. We believe that, despite the crack situation, our DNA target was clear and visible. We also indicated the DNA target with an arrow, as shown in the figure in the text. Further We add additional explanation regarding the PCR in order have adequate explanation (highlighted with yellow). Please kindly find below or in the manuscript This study aimed to investigate CYP2D6*5 and CYP2D6 multiplication in post-operative Minangkabau ethnicity patients receiving tramadol analgesia and further investigate the efficacy of tramadol in these patients. To achieve this, the Long PCR method developed by Johansson et al., 20 was modified. The Long PCR, reported by Johansson et al 1996, was successfully modified with the GoTaq Long PCR master mix from Promega #M4021, with two-step PCR at 68°C annealing and extension PCR amplification temperatures. It is important to optimise the annealing temperature, particularly when synthesising long PCR products or using total genomic DNA as the PCR substrate. An annealing temperature that is too high will reduced the PCR product yielded while an annealing temperature that is too low will produce an unspecific PCR product. PCR annealing temperature optimization was one of the key successes for developing PCR methods. 21 . GoTaq ® Long PCR Master Mix uses a combination of recombinant hot-start Taq DNA polymerase and recombinant proofreading DNA polymerase. Both enzymes in the GoTaq Long PCR master mix from Promega #M4021 were required to amplify long targets. Taq DNA Polymerase alone cannot correct nucleotide mismatches resulting from misincorporation and is therefore ineffective in amplifying segments longer than 3-5 kb. Products are truncated if nucleotides are misincorporated; further, they cannot be extended in subsequent cycles. Long amplicons can be amplified with a high yield by adding a small amount of proofreading enzyme to fix mismatches and allow extension. To ensure accurate PCR, the size of the product was monitored. The sizes of the wild-type, CYP2D6*5, and CYP2D6 multiplication PCR products were 5kb, 6kb, and 10 kb, respectively. The wild-type genotype PCR product was used as an control reaction. Furthermore, heterozygous CYP2D6*5 produced a DNA PCR product in a tube containing CYP2D6*5 and wild-type DNA primers. In contrast, homozygous CYP2D6*5 produced no PCR products in the tube containing wild-type DNA primers. Although the real-time method offers a simpler and faster alternative to long PCR, it requires costly real-time PCR machines, and pyrosequencing is also costly. The modified Long PCR method was used to generate a distribution report of the Minangkabau ethnicity genotypes. 4. There is an inconsistency about the patient number. In the abstract and method, it is written that 63 patients were included. However, in Table 2, the total number of patients of both genders, all body weights, and ages is 65. Furthermore, the total number of patients with all VAS scores is 62. Thank you very much for your kind correction. This study included a total of 65 patients. Among the patients, 62 received tramadol analgesia and 63 were tested for genetics, while the other two samples did not have detailed information. Please kindly see detailed information in https://doi.org/10.6084/m9.figshare.26870995, as mentioned in the data availability section of the manuscript. 5. There is no explanation about the patient and its inclusion-exclusion criteria. It is important to control the confounding variables, such as disease severity, comorbidity, and the use of other medications. Thank you very much for your kind input. Please kindly find our manuscript that we have added and completed the inclusion and exclusion criteria, In the methods section. ..All of the study's participants were patients who had undergone surgery at M. Djamil Padang Hospital. The inclusion criteria were: Patients with elective surgery at M. Djamil Hospital; proven Minangkabau ethnicity through family history; signed informed consent for blood sampling and pain assessment (VAS); and American Society of Anesthesiologists (ASA) criteria levels 1–2. The exclusion criteria were: Patients who refused to sign the informed consent form for sample collection; patients with ASA criteria levels ≥3 (patients with severe comorbidities e.g. renal failure or severe liver disease) that may interfere with tramadol metabolism, this also included patients who were taking other drugs that could significantly induce or inhibit CYP2D6 (e.g. fluoxetine, paroxetine and rifampicin)… 6. "This was evidenced in the present study, which showed a significant association between the drug and weight of postoperative patients (p = 0.008)." I think the association established in this analysis is between the class of body weight and genotype, which needs further explanation of its biological plausibility. Thank you very much for your kind comment. Please find the additional explanation below. The two enantiomers that make up tramadol each contribute to its analgesic effects in a distinct way in cytochrome P450 system. The highly polymorphic enzyme CYP2D6 catalyzes the formation of M1 from O-desmethyltramadol. N-demethylation, however, was catalyzed by a different mechanism. As previously mentioned, the phenotypic variance of CYP2D6 is explained by its highly polymorphic character (mutation, deletion, and multiplication). Furthermore, depending on CYP2D6 phenotypes and other genetic factors, that the mean elimination half-life is around 6 hours, while the initial distribution half-life is 6 minutes. Three hours after delivery, O-Desmethyltramadol (M1), the main active metabolite of tramadol, reaches Cmax at 18–26% of the tramadol Cmax value. The average elimination half-life is roughly seven hours. 9 , 25 Deletion and multiplication of genes are important for phenotype prediction. Deletion of the entire CYP2D6 gene (*5) results in loss (in the homozygote) or reduction (in the heterozygote) of its protein production. 3, 26, 27 Furthermore, it is important to acknowledge that drug metabolism in the multiplication genotype increases the activity of the enzyme acitivity at a high rate as the functional increase. 5, 22 The UM phenotype may also be at risk of therapeutic failure or increased toxic side effects of metabolites. In this study showed that tramadol was significantly associated with the weight of postoperative patients receiving analgesia (p = 0.008). This indicated that administration of 100 mg of the drug resulted in optimal pain relief. As stated above, the typical dosage of tramadol administered is within the range of 50 to 100 milligrams. 10 The dosage for pediatric patients was 50 mg, whereas the adult dosage was 100 mg. Age and sex showed no significant correlations (p = 0.052 and 0.243, respectively). This could be interpreted as meaning that all genotypes in this activity individually metabolize the given tramadol is still possible. This also shows that both genotypes (heterozygote CYP2D6 *5 and the multiplication) are clinically functional. The potency of tramadol analgesia is approximately 10% that of morphine when administered intravenously. Tramadol has also been reported to be comparable to morphine. 28, 29 Tramadol is an efficient and well-tolerated medication for the treatment of chronic pain, whether of malignant or non-malignant origin, especially neuropathic pain. It can also be used to relieve pain related to trauma, renal or biliary colic, and labor. When taken as a non-opioid analgesic, the analgesic effect of the drug can be enhanced. 28 Furthermore, tramadol administration is ideally based on body weight. According to Sidiq et al. (2015), 2 mg/kg body weight of tramadol hydrochloride is an optimal dose for postoperative analgesia when administered epidurally in urological surgery, with no significant increase in side effects. 30 Pang et al. (2005) reported that the recommended intraoperative dose of tramadol is 2.5 mg/kg body weight for effective postoperative analgesia with minimal sedation, when considering the efficacy and side-effect profile. 29 Patients without CYP2D6 activity may not be able to metabolize drugs. Ultra-rapid metabolizers may also experience treatment failure owing to the rapid metabolism of active drugs, resulting in drug levels below the therapeutic range. 5 The data obtained specifically in the multiplication part were not in line with the presented statement. This discrepancy may be attributed to the absence of a subsequent follow-up. To enhance the quality of future data, it is essential to categorize the type of operation, collect a larger number of samples, and conduct longer follow-up periods, and also estimate the pharmacokinetic parameter of tramadol in plasma. Tramadol's enantiomers, their primary phase I metabolites, and epinephrin were pharmacokinetic parameter of tramadol in plasma in order to evaluate the CYP2D6 phenotype. 23 Also following the recommendation of the consensus on translating CYP2D6 genotype to metabolizer phenotype. 24 This is an interesting study that describes the polymorphism of CYP2D6 in the Minangkabu ethic group. However, there are several aspects of this manuscript that should be revised: Thank you very much for your kind review and for considering our manuscript. 1. Please improve the narration: - "(wild-type, CYP2D6*5, and CYP2D6 multiplication)" Please explain shortly the metabolic capacity of each of these genotypes? Thank you very much for your kind input. We have revised the manuscript and added additional information. Please find the revised version below or in the first paragraph of the manuscript. …According to the prevailing metabolic capacity, the CYP2D6 phenotype is classified into four distinct categories: poor metabolizer (PM), intermediate metabolizer (IM), extensive metabolizer (EM), and ultra-metabolizer (UM). 3,4 Arneth et al 2009 clasiffied EM as a characteristic of the normal population with two functional wild type alleles; PM usually results from the deletion or mutation of both alleles and is an autosomal recessive condition, IM those with one functional allele; Rapid metabolism UM is believed to be an autosomal dominant feature that results from either functional gene multiplication or duplication (>30%). 3 Further Darney et al 2021, reported that extensive metabolism as normal, and it is defined as having two functional wild-type alleles (i.e. *1, *2), or heterozygosity with one decreased-activity and one non-activity allele. PM is associated with CYP2D6 alleles with no activity both alleles (i.e., *3, *4, *5, *6, *7, *8, *11, *14, *15, *18, *19, *21, *29, *40). The IM phenotype has been associated with a decrease in CYP2D6 activity alleles, i.e. *9, *10, *17, *21, *36, *29, *41, *45, and *46, or heterozygosity for one decreased activity allele and one non-activity allele. The UM phenotype has been linked to at least one active CYP2D6 gene duplication. Patients exhibiting the UM phenotype may face an elevated risk of therapeutic failure or an augmented propensity for adverse metabolic toxicities. 4 - "This study had a retrospective clinical trial registry number NCT06642480." In my opinion, it is not necessary to include the trial registry number in the abstract. "It is our first time using the clinical trial registry because the research already finished, so we applied for a retrospective registry clinical trial". This information may not be of concern to the reader Thank you very much for your valuable comment. We input this information based on Editor F 1000 Research request. -"Based on metabolic capacity, the phenotype of CYP2D6 can be divided into four parts: poor metabolizer (PM), intermediate metabolizer (IM), extensive metabolizer (EM), and ultra-metabolizer (UM)". Is there any phenotype that has a normal function of metabolism? Can the wild type in this study be considered a normal metabolizer? Thank you very much for your comment. As explained in the additional information in the first paragraph, extensive metabolisers are classified as the normal population. Further, in the case of our manuscript, we revised and consistently used the term 'wild type'. Please kindly see the updated manuscript. 2. There is considerable explanation about the PCR method and its optimization, which has been published elsewhere. I think the scope of this study is the distribution of CYP26D and its clinical implications in the Minangkabau ethnicity. Therefore, please just show the best result of the PCR method. Thank you very much for your kind comments and inputs. As we have no positive samples for the optimisation of long PCR for the wild-type, CYP2D6*5 and CYP2D6 multiplication targets, according to our opinion, besides reporting the distribution of CYP26D and its clinical implications in the Minangkabau ethnicity, we also should report how we modified and optimized the Johansson 1996 procedures for Long PCR. The long PCR method developed by Johansson et al. (1996) 20 was modified in this study. The PCR products of wild-type CYP2D6, CYP2D6*5, and multiplication detection yielded 5 kb, 6 kb, and 10 kb, respectively. In contrast to Johansson et al. (1996), who used PerkinElmer's rTDNA and New England Biolabs' 0.09U Vent, the present study used Promega's GoTaqLong PCR Mastermix #M4021. GoTaq ® Long PCR Master Mix uses a combination of recombinant hot-start Taq DNA polymerase and recombinant proofreading DNA polymerase. The amplification conditions must be further optimized. In a preliminary study, the annealing temperature of 55°C-67°C was optimized using a three-step PCR (denaturation, annealing, and extension) for three DNA primers. No PCR product was detected (data not shown). Further, the annealing temperature across five different temperatures starting from 65 °C to 68°C was optimized using two-step PCR (denaturation, annealing also as an extension step) for each DNA primer. Among the five annealing temperatures tested, 68°C was the best temperature. One patient DNA sample with an 0 VAS scale, produced 6kb and 5kb for the amplified CYP2D6*5 DNA primer and wild-type CYP2D6 DNA primer, respectively, with no PCR product in all NTC. Using this 0 VAS scale patient DNA sample, amplification with a CYP2D6*5 DNA primer produced a 6 kb PCR product that consistently showed up at all annealing temperatures. The PCR product showed a specific amplification product at 68°C annealing temperature. In addition, amplification using a wild-type CYP2D6 DNA primer produced a 5 kb PCR product that consistently showed at all annealing temperatures, and specific PCR product were provide at 67.15°C and 68°C. Furthermore, DNA samples from other patients (second patient) that had 8 VAS scale showed only produced 5kb for the wild-type DNA primer at all annealing temperatures, and specific PCR product showed at 67.15°C and 68°C annealing temperature. The DNA primer used for the detection of CYP2D6 multiplication did not generate a PCR product for either sample ( Figure 1). This showed that the two samples used for optimization were CYP2D6*5 for the first sample (0 VAS scale) and the wild-type for the second sample (8 VAS scale). Specifically, CYP2D6*5 was identified as heterozygous CYP2D6*5 as the PCR product was produced using both CYP2D6*5 and wild-type DNA primers. According to Johansson et al., 20 heterozygous CYP2D6*5 led to PCR products with both CYP2D6*5 and wild-type DNA primers, whereas homozygous CYP2D6*5 resulted in no PCR product with wild-type DNA primers. Based on these results, there was an inconsistency between the VAS scale measurement and the genotype detection. This discrepancy was possible since VAS scale measurements are highly subjective and depend on patient pain tolerance. The optimal PCR setup was used to screen several samples. The screening results showed that one sample produced 10kb which was suspected to be a multiplication sample ( Figure 2). This sample was optimized for its optimal PCR setup, and the process was used to confirm and verify the results. Consistency was observed with a 10 kb PCR product as a single band across temperatures ranging from 63 to 68°C. The optimal PCR annealing temperature was determined to be 63.95, 65.2, and 67.15, indicating thick single-band DNA ( Figure 3). However, 68°C was selected because it produced a single band and was the most effective temperature for detecting both CYP2D6*5 and wild-type alleles. In addition, all the annealing optimization processes showed clear and clean NTC. The remaining samples were screened using a modified method. 3. The images of the PCR product in gel electrophoresis are not clear, and the explanations are also inadequate. Thank you very much for your comment and we regret to inform you that, at that moment, our gel doc DNA tray was in a crack condition. We believe that, despite the crack situation, our DNA target was clear and visible. We also indicated the DNA target with an arrow, as shown in the figure in the text. Further We add additional explanation regarding the PCR in order have adequate explanation (highlighted with yellow). Please kindly find below or in the manuscript This study aimed to investigate CYP2D6*5 and CYP2D6 multiplication in post-operative Minangkabau ethnicity patients receiving tramadol analgesia and further investigate the efficacy of tramadol in these patients. To achieve this, the Long PCR method developed by Johansson et al., 20 was modified. The Long PCR, reported by Johansson et al 1996, was successfully modified with the GoTaq Long PCR master mix from Promega #M4021, with two-step PCR at 68°C annealing and extension PCR amplification temperatures. It is important to optimise the annealing temperature, particularly when synthesising long PCR products or using total genomic DNA as the PCR substrate. An annealing temperature that is too high will reduced the PCR product yielded while an annealing temperature that is too low will produce an unspecific PCR product. PCR annealing temperature optimization was one of the key successes for developing PCR methods. 21 . GoTaq ® Long PCR Master Mix uses a combination of recombinant hot-start Taq DNA polymerase and recombinant proofreading DNA polymerase. Both enzymes in the GoTaq Long PCR master mix from Promega #M4021 were required to amplify long targets. Taq DNA Polymerase alone cannot correct nucleotide mismatches resulting from misincorporation and is therefore ineffective in amplifying segments longer than 3-5 kb. Products are truncated if nucleotides are misincorporated; further, they cannot be extended in subsequent cycles. Long amplicons can be amplified with a high yield by adding a small amount of proofreading enzyme to fix mismatches and allow extension. To ensure accurate PCR, the size of the product was monitored. The sizes of the wild-type, CYP2D6*5, and CYP2D6 multiplication PCR products were 5kb, 6kb, and 10 kb, respectively. The wild-type genotype PCR product was used as an control reaction. Furthermore, heterozygous CYP2D6*5 produced a DNA PCR product in a tube containing CYP2D6*5 and wild-type DNA primers. In contrast, homozygous CYP2D6*5 produced no PCR products in the tube containing wild-type DNA primers. Although the real-time method offers a simpler and faster alternative to long PCR, it requires costly real-time PCR machines, and pyrosequencing is also costly. The modified Long PCR method was used to generate a distribution report of the Minangkabau ethnicity genotypes. 4. There is an inconsistency about the patient number. In the abstract and method, it is written that 63 patients were included. However, in Table 2, the total number of patients of both genders, all body weights, and ages is 65. Furthermore, the total number of patients with all VAS scores is 62. Thank you very much for your kind correction. This study included a total of 65 patients. Among the patients, 62 received tramadol analgesia and 63 were tested for genetics, while the other two samples did not have detailed information. Please kindly see detailed information in https://doi.org/10.6084/m9.figshare.26870995, as mentioned in the data availability section of the manuscript. 5. There is no explanation about the patient and its inclusion-exclusion criteria. It is important to control the confounding variables, such as disease severity, comorbidity, and the use of other medications. Thank you very much for your kind input. Please kindly find our manuscript that we have added and completed the inclusion and exclusion criteria, In the methods section. ..All of the study's participants were patients who had undergone surgery at M. Djamil Padang Hospital. The inclusion criteria were: Patients with elective surgery at M. Djamil Hospital; proven Minangkabau ethnicity through family history; signed informed consent for blood sampling and pain assessment (VAS); and American Society of Anesthesiologists (ASA) criteria levels 1–2. The exclusion criteria were: Patients who refused to sign the informed consent form for sample collection; patients with ASA criteria levels ≥3 (patients with severe comorbidities e.g. renal failure or severe liver disease) that may interfere with tramadol metabolism, this also included patients who were taking other drugs that could significantly induce or inhibit CYP2D6 (e.g. fluoxetine, paroxetine and rifampicin)… 6. "This was evidenced in the present study, which showed a significant association between the drug and weight of postoperative patients (p = 0.008)." I think the association established in this analysis is between the class of body weight and genotype, which needs further explanation of its biological plausibility. Thank you very much for your kind comment. Please find the additional explanation below. The two enantiomers that make up tramadol each contribute to its analgesic effects in a distinct way in cytochrome P450 system. The highly polymorphic enzyme CYP2D6 catalyzes the formation of M1 from O-desmethyltramadol. N-demethylation, however, was catalyzed by a different mechanism. As previously mentioned, the phenotypic variance of CYP2D6 is explained by its highly polymorphic character (mutation, deletion, and multiplication). Furthermore, depending on CYP2D6 phenotypes and other genetic factors, that the mean elimination half-life is around 6 hours, while the initial distribution half-life is 6 minutes. Three hours after delivery, O-Desmethyltramadol (M1), the main active metabolite of tramadol, reaches Cmax at 18–26% of the tramadol Cmax value. The average elimination half-life is roughly seven hours. 9 , 25 Deletion and multiplication of genes are important for phenotype prediction. Deletion of the entire CYP2D6 gene (*5) results in loss (in the homozygote) or reduction (in the heterozygote) of its protein production. 3, 26, 27 Furthermore, it is important to acknowledge that drug metabolism in the multiplication genotype increases the activity of the enzyme acitivity at a high rate as the functional increase. 5, 22 The UM phenotype may also be at risk of therapeutic failure or increased toxic side effects of metabolites. In this study showed that tramadol was significantly associated with the weight of postoperative patients receiving analgesia (p = 0.008). This indicated that administration of 100 mg of the drug resulted in optimal pain relief. As stated above, the typical dosage of tramadol administered is within the range of 50 to 100 milligrams. 10 The dosage for pediatric patients was 50 mg, whereas the adult dosage was 100 mg. Age and sex showed no significant correlations (p = 0.052 and 0.243, respectively). This could be interpreted as meaning that all genotypes in this activity individually metabolize the given tramadol is still possible. This also shows that both genotypes (heterozygote CYP2D6 *5 and the multiplication) are clinically functional. The potency of tramadol analgesia is approximately 10% that of morphine when administered intravenously. Tramadol has also been reported to be comparable to morphine. 28, 29 Tramadol is an efficient and well-tolerated medication for the treatment of chronic pain, whether of malignant or non-malignant origin, especially neuropathic pain. It can also be used to relieve pain related to trauma, renal or biliary colic, and labor. When taken as a non-opioid analgesic, the analgesic effect of the drug can be enhanced. 28 Furthermore, tramadol administration is ideally based on body weight. According to Sidiq et al. (2015), 2 mg/kg body weight of tramadol hydrochloride is an optimal dose for postoperative analgesia when administered epidurally in urological surgery, with no significant increase in side effects. 30 Pang et al. (2005) reported that the recommended intraoperative dose of tramadol is 2.5 mg/kg body weight for effective postoperative analgesia with minimal sedation, when considering the efficacy and side-effect profile. 29 Patients without CYP2D6 activity may not be able to metabolize drugs. Ultra-rapid metabolizers may also experience treatment failure owing to the rapid metabolism of active drugs, resulting in drug levels below the therapeutic range. 5 The data obtained specifically in the multiplication part were not in line with the presented statement. This discrepancy may be attributed to the absence of a subsequent follow-up. To enhance the quality of future data, it is essential to categorize the type of operation, collect a larger number of samples, and conduct longer follow-up periods, and also estimate the pharmacokinetic parameter of tramadol in plasma. Tramadol's enantiomers, their primary phase I metabolites, and epinephrin were pharmacokinetic parameter of tramadol in plasma in order to evaluate the CYP2D6 phenotype. 23 Also following the recommendation of the consensus on translating CYP2D6 genotype to metabolizer phenotype. 24 Competing Interests: No competing interests were disclosed Close Report a concern COMMENT ON THIS REPORT Comments on this article Comments (0) Version 3 VERSION 3 PUBLISHED 17 Dec 2024 ADD YOUR COMMENT Comment keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 2 Version 3 (revision) 23 Sep 25 read Version 2 (revision) 22 Aug 25 read read Version 1 17 Dec 24 read read Heri Setiawan , Universitas Indonesia, Depok, Indonesia Anom Bowolaksono , Universitas Indonesia, Depok, Indonesia Comments on this article All Comments (0) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Setiawan H. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 07 Oct 2025 | for Version 3 Heri Setiawan , Universitas Indonesia, Depok, Indonesia 0 Views copyright © 2025 Setiawan H. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The manuscript has been revised, and all my concerns have been addressed adequately. Competing Interests No competing interests were disclosed. Reviewer Expertise Pharmacology and toxicology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Setiawan H. Peer Review Report For: Distribution of CYP2D6 multiplication, CYP2D6*5, and clinical implications in postoperative patients receiving tramadol analgesia in the Minangkabau ethnic group, Indonesia [version 3; peer review: 2 approved] . F1000Research 2025, 13 :1526 ( https://doi.org/10.5256/f1000research.188479.r416788) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-1526/v3#referee-response-416788 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Setiawan H. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 16 Sep 2025 | for Version 2 Heri Setiawan , Universitas Indonesia, Depok, Indonesia 0 Views copyright © 2025 Setiawan H. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Only one out of the six points of review has been adequately addressed, which is about the explanation of a normal metabolizer. Please revise and explain adequately each point, especially the association between the VAS value and the CYP2D6 polymorphism. However, the data showed significant different proportion (p = 0.008) between body weight (not VAS value) and CYP2D6 polymorphism. Competing Interests No competing interests were disclosed. Reviewer Expertise Pharmacology and toxicology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (1) Author Response 26 Sep 2025 Desriani Desriani, Research Center for Genetic Engineering, Soekarno Sains and Technology Park, National Research and Innovation Agency (BRIN), Jl. Raya Bogor Km, 16911, Indonesia 1. Only one out of the six points of review has been adequately addressed, which is about the explanation of a normal metabolizer. Please revise and explain adequately each point, especially the association between the VAS value and the CYP2D6 polymorphism. However, the data showed significant different proportion (p = 0.008) between body weight (not VAS value) and CYP2D6 polymorphism. > Thank you very much for your valuable input and comment. Please find below our explanation, which we have also included in the revised manuscript (in the abstract, methods, results, and discussion parts). In the present study, we used two different statistical methods: Analysis of variance (ANOVA) was conducted to analyze the relationship between the amount of tramadol administered and the VAS value, as well as between the amount of tramadol administered and the molecular screening result. The demographic characteristics data were analyzed with a Chi-Square test. The statistical analysis was conducted with the IBM SPSS software version 24, with p values less than 0.05 being related as statistically significant.” The ANOVA analysis showed that administered tramadol was significantly associated with the VAS value of postoperative patients receiving analgesia with p = 0.045 (Supplementary). This result supported and indicated that administration of 100 mg of the drug resulted in optimal pain relief. In addition, the typical dosage of tramadol administered is within the range of 50 to 100 milligrams. 10 The dosage for pediatric patients was 50 mg, whereas the adult dosage was 100 mg. Furthermore, a significant association was also shown between tramadol administration and molecular screening, with p = 0.003 (in the Supplementary). This could be interpreted as meaning that all genotypes in this activity individually metabolize the given tramadol is still possible. This also shows that both genotypes (heterozygote CYP2D6 *5 and the multiplication) are clinically functional. In Table 2, the Chi-square test showed that the distribution of the molecular screening among the demographic factors (Age, Gender, and VAS value) of the West Sumatra patients does not have a significant correlation. While the weight of the patients is indicated as biased. As it showed inconsistent p-values between Pearson Chi-Square (p = 0.008), Likelihood Ratio (p = 0.149) and Linear-by-Linear Association (p = 0.124). According to McHugh 2013, too small sample size will affect to Pearson Chi Square, Likehood Ratio and Linear-by-linear P Value in the Chi-square test. Therefore, for a better understanding of this, further study needs a larger number of samples. In addition, gene polymorphisms are the most common type of genetic variation, defined as variations in gene sequences caused by changes in the DNA sequence that are heritable. 29 Further, the VAS value has a high subjectivity, and open possibilities have no significant correlation with molecular screening, as proved by the Chi-square result (Table 2). 2. "This study had a retrospective clinical trial registry number NCT06642480." In my opinion, it is not necessary to include the trial registry number in the abstract. "It is our first time using the clinical trial registry because the research already finished, so we applied for a retrospective registry clinical trial". This information may not be of concern to the reader Thank you very much for your valuable input and comment. We delete the sentences. 3. The images of the PCR product in gel electrophoresis are not clear Thank you very much for your valuable comment. We are very sorry that our trays for visualizing PCR products were cracked when we conducted this research in 2022. This explains why the background of our PCR product figure was cracked. But according to us, the images are still readable and analyzable. Furthermore, in Figure 1, since M-D-Wt were not always in the same gel, we used a white line to indicate the same temperature annealing and a black line to indicate different annealing temperatures. This makes the data clearer and easier to read and analyze. In addition, We completed the figure label in Figure 1 (1-3), and also in Figure 2 (2-1 and 2-2). There is an inconsistency about the patient number. Thank you very much for your valuable input and comment. We revised the sentences. A total of 65 patients participated in this study. For detailed information, please kindly see in https://doi.org/10.6084/m9.figshare.26870995, as mentioned in the data availability section of the manuscript. View more View less Competing Interests No competing interests were disclosed reply Respond Report a concern Setiawan H. Peer Review Report For: Distribution of CYP2D6 multiplication, CYP2D6*5, and clinical implications in postoperative patients receiving tramadol analgesia in the Minangkabau ethnic group, Indonesia [version 3; peer review: 2 approved] . F1000Research 2025, 13 :1526 ( https://doi.org/10.5256/f1000research.185264.r407882) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-1526/v2#referee-response-407882 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Bowolaksono A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 05 Sep 2025 | for Version 2 Anom Bowolaksono , Cellular and Molecular Mechanisms in Biological System (CEMBIOS) Research Group, Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Indonesia, Depok, Indonesia 0 Views copyright © 2025 Bowolaksono A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Most of comments are resolved Competing Interests No competing interests were disclosed. Reviewer Expertise Molecular biology in aging and cancer I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Bowolaksono A. Peer Review Report For: Distribution of CYP2D6 multiplication, CYP2D6*5, and clinical implications in postoperative patients receiving tramadol analgesia in the Minangkabau ethnic group, Indonesia [version 3; peer review: 2 approved] . F1000Research 2025, 13 :1526 ( https://doi.org/10.5256/f1000research.185264.r407883) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-1526/v2#referee-response-407883 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Bowolaksono A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 22 Jul 2025 | for Version 1 Anom Bowolaksono , Cellular and Molecular Mechanisms in Biological System (CEMBIOS) Research Group, Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Indonesia, Depok, Indonesia 0 Views copyright © 2025 Bowolaksono A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions This paper would be reviewed per section in points manner as would written below. There are some questions and critics those need to be cleared out in this paper Introduction Here were said about average elimination half-life of the drug. Beforehand, it mentioned tramadol is converted into M1 and M2. So, which one is referred in the average elimination of drugs? Why does CYP2D6*5 alleles need to be studied? how does it significantly contribute to CYP2D6 activity levels? Methods Standardization of VAS in this study is not really explained such as categorization criterions, patient symptoms, and valuation of VAS. Type of ANOVA model that used in this paper, for example Dunnet, Sidaks, Tukey, or else, is not clear. Result It is hard to interpret from Long PCR results because legends each figures have different description. It always needs to recheck before seeing the results. As mentioned in Methods . Bar graph or table need are to be specified in related of statistical analysis beforehand of significancy conclusion. Discussion - Conclusion There are some typos From this study, can it conclude the dose that might be works related to this study? Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly Competing Interests No competing interests were disclosed. Reviewer Expertise Molecular biology in aging and cancer I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (1) Author Response 05 Sep 2025 Desriani Desriani, Research Center for Genetic Engineering, Soekarno Sains and Technology Park, National Research and Innovation Agency (BRIN), Jl. Raya Bogor Km, 16911, Indonesia This paper would be reviewed per section in points manner as would written below. There are some questions and critics those need to be cleared out in this paper >Thank you very much for your valuable input and comment. Introduction Here were said about average elimination half-life of the drug. Beforehand, it mentioned tramadol is converted into M1 and M2. So, which one is referred in the average elimination of drugs? Response: Thank you very much for your comment. Please kindly find the explanation below. We have also included this explanation in the manuscript. The two enantiomers that make up tramadol each contribute to its analgesic effects in a distinct way in cytochrome P450 system. The highly polymorphic enzyme CYP2D6 catalyzes the formation of M1 from O-desmethyltramadol. N-demethylation, however, was catalyzed by a different mechanism. As previously mentioned, the phenotypic variance of CYP2D6 is explained by its highly polymorphic character (mutation, deletion, and multiplication). Furthermore, depending on CYP2D6 phenotypes and other genetic factors, that the mean elimination half-life is around 6 hours, while the initial distribution half-life is 6 minutes. Three hours after delivery, O-Desmethyltramadol (M1), the main active metabolite of tramadol, reaches Cmax at 18–26% of the tramadol Cmax value. The average elimination half-life is roughly seven hours 9,23 . Why does CYP2D6*5 alleles need to be studied? how does it significantly contribute to CYP2D6 activity levels? Response: Thank you very much for your comment. Please kindly find the explanation below. We have also included some of this explanation in the manuscript. To date, prominent pharmacogenomics societies have developed clinical guidelines for at least 48 CYP2D6-substrate drugs. These guidelines contain therapeutic recommendations based on predicted CYP2D6 metaboliser phenotypes. The CYP2D6 pharmacogenomic guidelines use these metaboliser categories to provide clinicians with drug-specific recommendations, aiming to avoid prescribing ineffective or harmful doses to patients with a particular CYP2D6 metaboliser status. Phenotypic differences alter the capacity of enzymes to metabolize drugs. Deletion and multiplication of genes are important for phenotype prediction. Deletion of the entire CYP2D6 gene (*5) results in loss (in the homozygote) or reduction (in the heterozygote) of its protein production. 3, 26, 27 Furthermore, it is important to acknowledge that drug metabolism in the multiplication genotype increases the activity of the enzyme acitivity at a high rate as the functional increase. 5, 22 The UM phenotype may also be at risk of therapeutic failure or increased toxic side effects of metabolites. In this study showed that tramadol was significantly associated with the weight of postoperative patients receiving analgesia (p = 0.008). This indicated that administration of 100 mg of the drug resulted in optimal pain relief. As stated above, the typical dosage of tramadol administered is within the range of 50 to 100 milligrams. 10 The dosage for pediatric patients was 50 mg, whereas the adult dosage was 100 mg. Age and sex showed no significant correlations (p = 0.052 and 0.243, respectively). This could be interpreted as meaning that all genotypes in this activity individually metabolize the given tramadol is still possible. This also shows that both genotypes (heterozygote CYP2D6 *5 and the multiplication) are clinically functional. Methods Standardization of VAS in this study is not really explained such as categorization criterions, patient symptoms, and valuation of VAS. Response: Thank you very much for your valuable comment. Please kindly find the explanation below. We have also included this explanation in the manuscript (in methods part). The VAS is a subjective measure that depends on the pain tolerance of patients. However standarization of VAS scale was categorized as: 0 (no pain), 1-3 (signifying mild), 4-6 (middle), and 7-10 ( heavy pain). We also provide additional information on the exclusion and inclusion criteria. All of the study's participants were patients who had undergone surgery at M. Djamil Padang Hospital. The inclusion criteria were: Patients with elective surgery at M. Djamil Hospital; proven Minangkabau ethnicity through family history; signed informed consent for blood sampling and pain assessment (VAS); and American Society of Anesthesiologists (ASA) criteria levels 1–2. The exclusion criteria were: Patients who refused to sign the informed consent form for sample collection; patients with ASA criteria levels ≥3 (patients with severe comorbidities e.g. renal failure or severe liver disease) that may interfere with tramadol metabolism, this also included patients who were taking other drugs that could significantly induce or inhibit CYP2D6 (e.g. fluoxetine, paroxetine and rifampicin). Type of ANOVA model that used in this paper, for example Dunnet, Sidaks, Tukey, or else, is not clear. Thank you for your valuable comments. The information about statistical analysis that was used ini this study was mentioned in the manuscript on “2.4 Statistical Analysis” section: “ The demographic characteristics data were presented as the mean value ± standard deviation (SD). Subsequently, the data were analyzed with analysis of variance (ANOVA), followed by a chi-square test. The statistical analysis was conducted with the IBM SPSS software version 24, with p values less than 0.05 being related as statistically significant.” Here it is the detailed explanation about statistical analysis using SPSS that has been used on the data consists of: The significancies value for Visual Analogue Scale (VAS), age, sex, body weight, and molecular values, was firstly analyzed with Wild-typeity Test to evaluate the data distribution, after that ANOVA test was conducted and followed by Chi-square test. This was mentioned in 3. Results section, point 3.3 Patients demographics : “The sig value for VAS, age, sex, body weight, and molecular values <0.05 indicates that the data are not wild-typely distributed according to the Wild-typeity Test table. The demographic characteristics data presented as the mean value ± standard deviation (SD) showed no significant difference in VAS value, age, gender, and weight. The mean value range was 1.43-1.91, while the standard deviation ranged from 0.497 to 1.014 (detailed data were not shown). Further, the data was analyzed with analysis of variance (ANOVA), followed by a chi-square test.” The characteristic demographic of deletion, multiplication and wild-type polymorphism of CYP2D6 (age, sex, and weight as VAS measurement) was analyzed using Chi-square test to evaluate whether there is a correlation between the variables. The correlation between the administration of tramadol and the Visual Analogue Scale (VAS) score (which indicates the pain ratin scale on patiens) was analyzed using ANOVA test (the data have been evaluated with Normality (Wild-typeity Test) and Homogenity tests, and the data was distributed normally and homogen). The correlation between the administration of tramadol and the molecular examination was analyzed using ANOVA test (the data have been evaluated with Normality and Homogenity tests, and the data was distibuted normally and homogen). Result It is hard to interpret from Long PCR results because legends each figures have different description. It always needs to recheck before seeing the results. >Thank you very much for your comments and input. We have updated the legend in all figures to be consistent. Please kindly see the updated manuscript. As mentioned in Methods . Bar graph or table need are to be specified in related of statistical analysis beforehand of significancy conclusion. Response: Thank you for your valuable comments. Please kindly find all the detailed statistical analyses that are reported in https://doi.org/10.6084/m9.figshare.27895956.v1, as mentioned in the data availability section of the manuscript. Discussion - Conclusion There are some typos Response: Thank you very much for your comments and input. We have corrected the typos. Please kindly see the updated manuscript. From this study, can it conclude the dose that might be works related to this study? Response: Thank you very much for your valuable comments and input. Based on this study indicates that 100 mg of tramadol is an effective dose. In this study showed that tramadol was significantly associated with the weight of postoperative patients receiving analgesia (p = 0.008). This indicated that administration of 100 mg of the drug resulted in optimal pain relief. As stated above, the typical dosage of tramadol administered is within the range of 50 to 100 milligrams 10 . The dosage for pediatric patients was 50 mg, whereas the adult dosage was 100 mg. Age and sex showed no significant correlations (p = 0.052 and 0.243, respectively). This could be interpreted as meaning that all genotypes in this activity individually metabolize the given tramadol is still possible. This also shows that both genotypes ( heterozygote CYP2D6 *5 and the multiplication) are clinically functional. View more View less Competing Interests No competing interests were disclosed reply Respond Report a concern Bowolaksono A. Peer Review Report For: Distribution of CYP2D6 multiplication, CYP2D6*5, and clinical implications in postoperative patients receiving tramadol analgesia in the Minangkabau ethnic group, Indonesia [version 3; peer review: 2 approved] . F1000Research 2025, 13 :1526 ( https://doi.org/10.5256/f1000research.171234.r395048) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-1526/v1#referee-response-395048 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Setiawan H. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 01 Jul 2025 | for Version 1 Heri Setiawan , Universitas Indonesia, Depok, Indonesia 0 Views copyright © 2025 Setiawan H. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions This is an interesting study that describes the polymorphism of CYP2D6 in the Minangkabu ethic group. However, there are several aspects of this manuscript that should be revised: 1. Please improve the narration: - "(wild-type, CYP2D6*5, and CYP2D6 multiplication)" Please explain shortly the metabolic capacity of each of these genotypes? - "This study had a retrospective clinical trial registry number NCT06642480." In my opinion, it is not necessary to include the trial registry number in the abstract. -"It is our first time using the clinical trial registry because the research already finished, so we applied for a retrospective registry clinical trial". This information may not be of concern to the reader -"Based on metabolic capacity, the phenotype of CYP2D6 can be divided into four parts: poor metabolizer (PM), intermediate metabolizer (IM), extensive metabolizer (EM), and ultra-metabolizer (UM)". Is there any phenotype that has a normal function of metabolism? Can the wild type in this study be considered a normal metabolizer? 2. There is considerable explanation about the PCR method and its optimization, which has been published elsewhere. I think the scope of this study is the distribution of CYP26D and its clinical implications in the Minangkabau ethnicity. Therefore, please just show the best result of the PCR method. 3. The images of the PCR product in gel electrophoresis are not clear, and the explanations are also inadequate. 4. There is an inconsistency about the patient number. In the abstract and method, it is written that 63 patients were included. However, in Table 2, the total number of patients of both genders, all body weights, and ages is 65. Furthermore, the total number of patients with all VAS scores is 62. 5. There is no explanation about the patient and its inclusion-exclusion criteria. It is important to control the confounding variables, such as disease severity, comorbidity, and the use of other medications. 6. "This was evidenced in the present study, which showed a significant association between the drug and weight of postoperative patients (p = 0.008)." I think the association established in this analysis is between the class of body weight and genotype, which needs further explanation of its biological plausibility. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly Competing Interests No competing interests were disclosed. Reviewer Expertise Pharmacology and toxicology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (1) Author Response 05 Sep 2025 Desriani Desriani, Research Center for Genetic Engineering, Soekarno Sains and Technology Park, National Research and Innovation Agency (BRIN), Jl. Raya Bogor Km, 16911, Indonesia This is an interesting study that describes the polymorphism of CYP2D6 in the Minangkabu ethic group. However, there are several aspects of this manuscript that should be revised: Thank you very much for your kind review and for considering our manuscript. 1. Please improve the narration: - "(wild-type, CYP2D6*5, and CYP2D6 multiplication)" Please explain shortly the metabolic capacity of each of these genotypes? Thank you very much for your kind input. We have revised the manuscript and added additional information. Please find the revised version below or in the first paragraph of the manuscript. …According to the prevailing metabolic capacity, the CYP2D6 phenotype is classified into four distinct categories: poor metabolizer (PM), intermediate metabolizer (IM), extensive metabolizer (EM), and ultra-metabolizer (UM). 3,4 Arneth et al 2009 clasiffied EM as a characteristic of the normal population with two functional wild type alleles; PM usually results from the deletion or mutation of both alleles and is an autosomal recessive condition, IM those with one functional allele; Rapid metabolism UM is believed to be an autosomal dominant feature that results from either functional gene multiplication or duplication (>30%). 3 Further Darney et al 2021, reported that extensive metabolism as normal, and it is defined as having two functional wild-type alleles (i.e. *1, *2), or heterozygosity with one decreased-activity and one non-activity allele. PM is associated with CYP2D6 alleles with no activity both alleles (i.e., *3, *4, *5, *6, *7, *8, *11, *14, *15, *18, *19, *21, *29, *40). The IM phenotype has been associated with a decrease in CYP2D6 activity alleles, i.e. *9, *10, *17, *21, *36, *29, *41, *45, and *46, or heterozygosity for one decreased activity allele and one non-activity allele. The UM phenotype has been linked to at least one active CYP2D6 gene duplication. Patients exhibiting the UM phenotype may face an elevated risk of therapeutic failure or an augmented propensity for adverse metabolic toxicities. 4 - "This study had a retrospective clinical trial registry number NCT06642480." In my opinion, it is not necessary to include the trial registry number in the abstract. "It is our first time using the clinical trial registry because the research already finished, so we applied for a retrospective registry clinical trial". This information may not be of concern to the reader Thank you very much for your valuable comment. We input this information based on Editor F 1000 Research request. -"Based on metabolic capacity, the phenotype of CYP2D6 can be divided into four parts: poor metabolizer (PM), intermediate metabolizer (IM), extensive metabolizer (EM), and ultra-metabolizer (UM)". Is there any phenotype that has a normal function of metabolism? Can the wild type in this study be considered a normal metabolizer? Thank you very much for your comment. As explained in the additional information in the first paragraph, extensive metabolisers are classified as the normal population. Further, in the case of our manuscript, we revised and consistently used the term 'wild type'. Please kindly see the updated manuscript. 2. There is considerable explanation about the PCR method and its optimization, which has been published elsewhere. I think the scope of this study is the distribution of CYP26D and its clinical implications in the Minangkabau ethnicity. Therefore, please just show the best result of the PCR method. Thank you very much for your kind comments and inputs. As we have no positive samples for the optimisation of long PCR for the wild-type, CYP2D6*5 and CYP2D6 multiplication targets, according to our opinion, besides reporting the distribution of CYP26D and its clinical implications in the Minangkabau ethnicity, we also should report how we modified and optimized the Johansson 1996 procedures for Long PCR. The long PCR method developed by Johansson et al. (1996) 20 was modified in this study. The PCR products of wild-type CYP2D6, CYP2D6*5, and multiplication detection yielded 5 kb, 6 kb, and 10 kb, respectively. In contrast to Johansson et al. (1996), who used PerkinElmer's rTDNA and New England Biolabs' 0.09U Vent, the present study used Promega's GoTaqLong PCR Mastermix #M4021. GoTaq ® Long PCR Master Mix uses a combination of recombinant hot-start Taq DNA polymerase and recombinant proofreading DNA polymerase. The amplification conditions must be further optimized. In a preliminary study, the annealing temperature of 55°C-67°C was optimized using a three-step PCR (denaturation, annealing, and extension) for three DNA primers. No PCR product was detected (data not shown). Further, the annealing temperature across five different temperatures starting from 65 °C to 68°C was optimized using two-step PCR (denaturation, annealing also as an extension step) for each DNA primer. Among the five annealing temperatures tested, 68°C was the best temperature. One patient DNA sample with an 0 VAS scale, produced 6kb and 5kb for the amplified CYP2D6*5 DNA primer and wild-type CYP2D6 DNA primer, respectively, with no PCR product in all NTC. Using this 0 VAS scale patient DNA sample, amplification with a CYP2D6*5 DNA primer produced a 6 kb PCR product that consistently showed up at all annealing temperatures. The PCR product showed a specific amplification product at 68°C annealing temperature. In addition, amplification using a wild-type CYP2D6 DNA primer produced a 5 kb PCR product that consistently showed at all annealing temperatures, and specific PCR product were provide at 67.15°C and 68°C. Furthermore, DNA samples from other patients (second patient) that had 8 VAS scale showed only produced 5kb for the wild-type DNA primer at all annealing temperatures, and specific PCR product showed at 67.15°C and 68°C annealing temperature. The DNA primer used for the detection of CYP2D6 multiplication did not generate a PCR product for either sample ( Figure 1). This showed that the two samples used for optimization were CYP2D6*5 for the first sample (0 VAS scale) and the wild-type for the second sample (8 VAS scale). Specifically, CYP2D6*5 was identified as heterozygous CYP2D6*5 as the PCR product was produced using both CYP2D6*5 and wild-type DNA primers. According to Johansson et al., 20 heterozygous CYP2D6*5 led to PCR products with both CYP2D6*5 and wild-type DNA primers, whereas homozygous CYP2D6*5 resulted in no PCR product with wild-type DNA primers. Based on these results, there was an inconsistency between the VAS scale measurement and the genotype detection. This discrepancy was possible since VAS scale measurements are highly subjective and depend on patient pain tolerance. The optimal PCR setup was used to screen several samples. The screening results showed that one sample produced 10kb which was suspected to be a multiplication sample ( Figure 2). This sample was optimized for its optimal PCR setup, and the process was used to confirm and verify the results. Consistency was observed with a 10 kb PCR product as a single band across temperatures ranging from 63 to 68°C. The optimal PCR annealing temperature was determined to be 63.95, 65.2, and 67.15, indicating thick single-band DNA ( Figure 3). However, 68°C was selected because it produced a single band and was the most effective temperature for detecting both CYP2D6*5 and wild-type alleles. In addition, all the annealing optimization processes showed clear and clean NTC. The remaining samples were screened using a modified method. 3. The images of the PCR product in gel electrophoresis are not clear, and the explanations are also inadequate. Thank you very much for your comment and we regret to inform you that, at that moment, our gel doc DNA tray was in a crack condition. We believe that, despite the crack situation, our DNA target was clear and visible. We also indicated the DNA target with an arrow, as shown in the figure in the text. Further We add additional explanation regarding the PCR in order have adequate explanation (highlighted with yellow). Please kindly find below or in the manuscript This study aimed to investigate CYP2D6*5 and CYP2D6 multiplication in post-operative Minangkabau ethnicity patients receiving tramadol analgesia and further investigate the efficacy of tramadol in these patients. To achieve this, the Long PCR method developed by Johansson et al., 20 was modified. The Long PCR, reported by Johansson et al 1996, was successfully modified with the GoTaq Long PCR master mix from Promega #M4021, with two-step PCR at 68°C annealing and extension PCR amplification temperatures. It is important to optimise the annealing temperature, particularly when synthesising long PCR products or using total genomic DNA as the PCR substrate. An annealing temperature that is too high will reduced the PCR product yielded while an annealing temperature that is too low will produce an unspecific PCR product. PCR annealing temperature optimization was one of the key successes for developing PCR methods. 21 . GoTaq ® Long PCR Master Mix uses a combination of recombinant hot-start Taq DNA polymerase and recombinant proofreading DNA polymerase. Both enzymes in the GoTaq Long PCR master mix from Promega #M4021 were required to amplify long targets. Taq DNA Polymerase alone cannot correct nucleotide mismatches resulting from misincorporation and is therefore ineffective in amplifying segments longer than 3-5 kb. Products are truncated if nucleotides are misincorporated; further, they cannot be extended in subsequent cycles. Long amplicons can be amplified with a high yield by adding a small amount of proofreading enzyme to fix mismatches and allow extension. To ensure accurate PCR, the size of the product was monitored. The sizes of the wild-type, CYP2D6*5, and CYP2D6 multiplication PCR products were 5kb, 6kb, and 10 kb, respectively. The wild-type genotype PCR product was used as an control reaction. Furthermore, heterozygous CYP2D6*5 produced a DNA PCR product in a tube containing CYP2D6*5 and wild-type DNA primers. In contrast, homozygous CYP2D6*5 produced no PCR products in the tube containing wild-type DNA primers. Although the real-time method offers a simpler and faster alternative to long PCR, it requires costly real-time PCR machines, and pyrosequencing is also costly. The modified Long PCR method was used to generate a distribution report of the Minangkabau ethnicity genotypes. 4. There is an inconsistency about the patient number. In the abstract and method, it is written that 63 patients were included. However, in Table 2, the total number of patients of both genders, all body weights, and ages is 65. Furthermore, the total number of patients with all VAS scores is 62. Thank you very much for your kind correction. This study included a total of 65 patients. Among the patients, 62 received tramadol analgesia and 63 were tested for genetics, while the other two samples did not have detailed information. Please kindly see detailed information in https://doi.org/10.6084/m9.figshare.26870995, as mentioned in the data availability section of the manuscript. 5. There is no explanation about the patient and its inclusion-exclusion criteria. It is important to control the confounding variables, such as disease severity, comorbidity, and the use of other medications. Thank you very much for your kind input. Please kindly find our manuscript that we have added and completed the inclusion and exclusion criteria, In the methods section. ..All of the study's participants were patients who had undergone surgery at M. Djamil Padang Hospital. The inclusion criteria were: Patients with elective surgery at M. Djamil Hospital; proven Minangkabau ethnicity through family history; signed informed consent for blood sampling and pain assessment (VAS); and American Society of Anesthesiologists (ASA) criteria levels 1–2. The exclusion criteria were: Patients who refused to sign the informed consent form for sample collection; patients with ASA criteria levels ≥3 (patients with severe comorbidities e.g. renal failure or severe liver disease) that may interfere with tramadol metabolism, this also included patients who were taking other drugs that could significantly induce or inhibit CYP2D6 (e.g. fluoxetine, paroxetine and rifampicin)… 6. "This was evidenced in the present study, which showed a significant association between the drug and weight of postoperative patients (p = 0.008)." I think the association established in this analysis is between the class of body weight and genotype, which needs further explanation of its biological plausibility. Thank you very much for your kind comment. Please find the additional explanation below. The two enantiomers that make up tramadol each contribute to its analgesic effects in a distinct way in cytochrome P450 system. The highly polymorphic enzyme CYP2D6 catalyzes the formation of M1 from O-desmethyltramadol. N-demethylation, however, was catalyzed by a different mechanism. As previously mentioned, the phenotypic variance of CYP2D6 is explained by its highly polymorphic character (mutation, deletion, and multiplication). Furthermore, depending on CYP2D6 phenotypes and other genetic factors, that the mean elimination half-life is around 6 hours, while the initial distribution half-life is 6 minutes. Three hours after delivery, O-Desmethyltramadol (M1), the main active metabolite of tramadol, reaches Cmax at 18–26% of the tramadol Cmax value. The average elimination half-life is roughly seven hours. 9 , 25 Deletion and multiplication of genes are important for phenotype prediction. Deletion of the entire CYP2D6 gene (*5) results in loss (in the homozygote) or reduction (in the heterozygote) of its protein production. 3, 26, 27 Furthermore, it is important to acknowledge that drug metabolism in the multiplication genotype increases the activity of the enzyme acitivity at a high rate as the functional increase. 5, 22 The UM phenotype may also be at risk of therapeutic failure or increased toxic side effects of metabolites. In this study showed that tramadol was significantly associated with the weight of postoperative patients receiving analgesia (p = 0.008). This indicated that administration of 100 mg of the drug resulted in optimal pain relief. As stated above, the typical dosage of tramadol administered is within the range of 50 to 100 milligrams. 10 The dosage for pediatric patients was 50 mg, whereas the adult dosage was 100 mg. Age and sex showed no significant correlations (p = 0.052 and 0.243, respectively). This could be interpreted as meaning that all genotypes in this activity individually metabolize the given tramadol is still possible. This also shows that both genotypes (heterozygote CYP2D6 *5 and the multiplication) are clinically functional. The potency of tramadol analgesia is approximately 10% that of morphine when administered intravenously. Tramadol has also been reported to be comparable to morphine. 28, 29 Tramadol is an efficient and well-tolerated medication for the treatment of chronic pain, whether of malignant or non-malignant origin, especially neuropathic pain. It can also be used to relieve pain related to trauma, renal or biliary colic, and labor. When taken as a non-opioid analgesic, the analgesic effect of the drug can be enhanced. 28 Furthermore, tramadol administration is ideally based on body weight. According to Sidiq et al. (2015), 2 mg/kg body weight of tramadol hydrochloride is an optimal dose for postoperative analgesia when administered epidurally in urological surgery, with no significant increase in side effects. 30 Pang et al. (2005) reported that the recommended intraoperative dose of tramadol is 2.5 mg/kg body weight for effective postoperative analgesia with minimal sedation, when considering the efficacy and side-effect profile. 29 Patients without CYP2D6 activity may not be able to metabolize drugs. Ultra-rapid metabolizers may also experience treatment failure owing to the rapid metabolism of active drugs, resulting in drug levels below the therapeutic range. 5 The data obtained specifically in the multiplication part were not in line with the presented statement. This discrepancy may be attributed to the absence of a subsequent follow-up. To enhance the quality of future data, it is essential to categorize the type of operation, collect a larger number of samples, and conduct longer follow-up periods, and also estimate the pharmacokinetic parameter of tramadol in plasma. Tramadol's enantiomers, their primary phase I metabolites, and epinephrin were pharmacokinetic parameter of tramadol in plasma in order to evaluate the CYP2D6 phenotype. 23 Also following the recommendation of the consensus on translating CYP2D6 genotype to metabolizer phenotype. 24 View more View less Competing Interests No competing interests were disclosed reply Respond Report a concern Setiawan H. Peer Review Report For: Distribution of CYP2D6 multiplication, CYP2D6*5, and clinical implications in postoperative patients receiving tramadol analgesia in the Minangkabau ethnic group, Indonesia [version 3; peer review: 2 approved] . F1000Research 2025, 13 :1526 ( https://doi.org/10.5256/f1000research.171234.r391019) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-1526/v1#referee-response-391019 Alongside their report, reviewers assign a status to the article: Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. 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