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

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 This study modified the long PCR method reported by Johansson et al. (1996) to screen for multiplication, CYP2D6*5, and wild-type CYP2D6 in post-operative Minangkabau patients receiving tramadol analgesia. The genetic distribution and clinical effects of tramadol were also investigated.

2. Methods

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, D: for CYP2D6*5 detection, and Wt: for Wild-type detection; 10 kb, 6 kb, and 5 kb for multiplication, CYP2D6*5, and wild-type CYP2D6.

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 patient sample for detection of CYP2D6*5, multiplication and wild-type.

The target was shown with the red arrow: M: for multiplication detection, D: for CYP2D6*5 detection, and Wt: for Wild-type detection; 10 kb, 6 kb, and 5 kb for multiplication, CYP2D6*5, and wild-type CYP2D6.

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. 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 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	31
Central Han Chinese	7.17	1.35	31
Malaysia Chinese	2.54	4.24	31
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.

4. Discussion

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 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. 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

5. Conclusion

In conclusion, 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. Furthermore, 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 administration of tramadol (100 mg) to patients led to optimal pain relief, as evidenced by a study analyzed using IBM SPPS 24. The results showed a significant correlation (p = 0.008) with the weight of postoperative patients receiving tramadol analgesia but not with sex or age. This also suggests that in the Minangkabau ethnic group, all genotypes were capable of metabolizing the administered tramadol.

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.