Abstract
Background
CYP2D6 plays a crucial role in drug metabolism of several drugs. It is known to be highly polymorphic with enzymatic activity ranging from poor to ultrarapid metabolic rates. While the frequencies of CYP2D6 alleles are generally known in different Asian populations, data on frequencies of the copy number variations (CNV) and tandems in CYP2D6 in which they occur are less well studied in these populations.
Methods
A cohort of 800 consecutive, unrelated individuals were referred to Prenetics Limited (Prenetics) iGenes test by physicians in Hong Kong as part of their care with informed consent. These clinical samples were deidentified prior to further analysis. Genotyping and copy number determination of CYP2D6 were performed using target specific TaqMan® SNP genotyping and copy number assays. The phenotypes of CYP2D6 were predicted based on its genotypes and is dependent on the biallelic expression of alleles.
Results
Among the Asian group (n = 735, 92%), the observed frequency of CYP2D6*36‐*10 tandems was 34.1%. We also identified duplication of CYP2D6 alleles in 86 (11.7%) individuals of the study cohort. The frequency of all CYP2D6 duplicated alleles was 154 (10.5%) while only 28 (1.9%) of the duplications were of functional alleles (ie CYP2D6*1 and CYP2D6*2).
Conclusion
The present study provides a comprehensive analysis on the occurrences of CNV and tandems of the CYP2D6 gene in the Hong Kong population. The results contribute to the overall knowledge of pharmacogenomics and may accelerate the implementation of precision medicine in Asia.
Keywords: copy number variations, CYP2D6 genotyping, CYP2D6*36‐*10, drug metabolism, tandem repeats
1. INTRODUCTION
CYP2D6 is a clinically important drug‐metabolizing enzyme within Cytochrome P450 (CYP450) superfamily. Although it only constitutes 2%‐4% of hepatic content out of all CYP450s,1, 2, 3 CYP2D6 metabolizes approximately 20% of commonly prescribed medications currently available on the market, including beta blockers, antiarrhythmics, antidepressants, antipsychotics and opioids.4, 5, 6 The CYP2D6 gene is highly polymorphic with numerous allelic variants identified to date by Pharmacogene Variation Consortium (PharmVar) at www.pharmvar.org (previously known as the Human Cytochrome P450 Allele Nomenclature). These CYP2D6 allelic variants consist of combinations of single‐nucleotide polymorphisms, insertions, and copy number variations (CNVs) for multiplication or deletion of an entire gene, leading to altered CYP2D6 enzymatic activity both in vitro and in vivo.7, 8 Evidences have associated these genetic variations of CYP2D6 with variability in drug responses, including treatment failure and toxicity for drugs that are metabolized by CYP2D6.9, 10, 11 Clinical Pharmacogenetic Implementation Consortium (CPIC) guidelines for CYP2D6 genotypes are available for dosing considerations of numerous therapeutic agents.12, 13, 14, 15, 16, 17
Determining copy number variants for deletions and multiplications in the CYP2D6 gene is important for phenotype prediction.2, 18 CYP2D6 gene deletions are indicated by CYP2D6*5 which constitute a non‐functional variant. On the other hand, duplications and multiplications of the gene have been observed. Individuals with multiple copies of functional CYP2D6 alleles exhibit increased enzyme activity with higher rate of drug metabolism. In addition, multiplications and tandems of reduced or non‐functional variants have also been described. CYP2D6 activity in individuals with multiple copies of non‐functional alleles remains negligible.19 Certain CYP2D6 alleles are found in tandem arrangements. Tandem arrangements occur when alleles carry two or more gene units that are not identical.7 One such example is CYP2D6*36‐*10 tandems, which is common in East Asian populations.20, 21 The CYP2D6*36‐*10 tandems convey activity only from CYP2D6*10 allele.7, 22
The frequency of CYP2D6 genetic polymorphisms varies significantly between different ethnic groups. While the frequencies of CYP2D6 alleles are generally known in Asians, distribution of the copy number variations (CNV) and tandems in CYP2D6 in which they occur are less well studied in these populations.19 The objective of this study is to determine and report the observed CYP2D6 allele and predicted phenotype frequencies, specifically the occurrences of CNV and tandems, in the Hong Kong population.
2. METHODS
2.1. Study subjects
Buccal swab samples were collected from 800 consecutive, unrelated individuals for Prenetics Limited (Prenetics) iGenes pharmacogenomic tests with informed consent. These individuals were referred to pharmacogenomic testing by physicians in Hong Kong as part of their care. The clinical samples were deidentified prior to further analysis. Among the genotyped individuals, 735 (92%) were self‐identified as Asians, 56 (7%) were Caucasians and 9 (1%) were mixed race. The study group consists of 357 (44.6%) male and 391 (48.9%) female while 52 (6.5%) of the tested individuals did not report their gender.
2.2. Genotype and copy number determination
As these tests were done for clinical reasons, they undergo extensive quality control procedures in Prenetics’ HOKLAS 15189:2012 accredited laboratory. Prenetic's genotyping tests (including CYP2D6 genotyping and CNV tests) have been validated by external agencies, such as the Core Facilities‐Genome Sequencing Laboratory (CFGSL) at the Chinese University of Hong Kong with an analytical accuracy of 99.9%.
To determine the genotypes of CYP2D6, genotyping experiments were performed using the TaqMan® SNP Genotyping Assays (FAM™ and VIC® dye‐labeled TaqMan® MGB probes) in ViiA7™ Real Time PCR System (Life Technologies, Life Technologies Corporation, Carlsbad, California, USA). Table 1 provides a summary of CYP2D6 variants and alleles that are detected in the present study. To assess the CYP2D6 copy number, two TaqMan® Copy Number Assays (assay ID: Hs00010001_cn (Exon 9) and Hs04502391_cn (Intron 6); Life Technologies Corporation) are used. CYP2D6*36 contains gene conversion to CYP2D7 in exon 9 and the sequence of *10 and *36 are homologous in other regions. TaqMan® SNP Genotyping Assays alone is not sufficient to discriminate the two alleles (CYP2D6*10 and *36). To address this issue, TaqMan® Copy Number Assays (Hs04502391_cn) is used in additional to Hs00010001_cn. Hs04502391_cn targets CYP2D6 intron 6 sequences which will amplify CYP2D6/CYP2D7 hybrid alleles carrying CYP2D6 intron 6 sequences (including *36). On the other hand, Hs00010001_cn targets CYP2D6 exon 9 sequences and will not amplify CYP2D7, CYP2D8 pseudogenes or CYP2D6/CYP2D7 hybrid alleles carrying CYP2D7 exon 9 sequences. As a result, *36 is detected by Hs04502391_cn but not Hs00010001_cn and can be discriminated from *10. TaqMan® Copy Number Assays are run in the same well with the VIC® dye‐labeled TaqMan® Copy Number Reference Assay (RNase P) (assay ID: 4403326; Life Technologies Corporation) in quadruplicates PCR reactions containing 10 ng of purified DNA and TaqMan® Genotyping Master Mix. Reactions were prepared and run on 384‐well plates on the ViiA7™ Real Time PCR System according to the manufacturer's protocol. RNase P is known with two copies in a diploid genome, regardless of the copy number of the target of interest, and are used to normalize sample input and minimize variation between the test targets and reference assays. Relative quantification is performed following the comparative ∆∆CT method in CopyCaller® Software (Life Technologies Corporation) using reference samples purchase from Coriell Cell Institute as calibrators (for example, result of Hs00010001_cn is 2 copies and result of Hs04502391_cn is 3 copies in NA18564). With these calibrators, the copy number of CYP2D6 gene in samples is calculated. SNP genotype and CNV results, generated using TaqMan® SNP assays and TaqMan® Copy Number Assays, respectively, can be translated to star allele diplotypes using AlleleTyper™ software. (Life Technologies Corporation).
Table 1.
Summary of CYP2D6 variants and alleles detected
| Allele | rsID | Molecular consequence | Protein effect |
|---|---|---|---|
| CYP2D6*2 | rs1135840 | NM_000106.5:c.1457G>C | Ser486Thr |
| rs16947 | NM_000106.5:c.886C>T | Arg296Cys | |
| CYP2D6*3 | rs35742686 | NM_000106.5:c.775delA | Arg259Glyfs |
| CYP2D6*4 | rs3892097 | NM_000106.5:c.506‐1G>A | ‐ |
| CYP2D6*5 | ‐ | Gene deletion | ‐ |
| CYP2D6*6 | rs5030655 | NM_000106.5:c.454delT | Trp152Glyfs |
| CYP2D6*9 | rs5030656 | NM_000106.5:c.841_843delAAG | Lys281del |
| CYP2D6*10 | rs1065852 | NM_000106.5:c.100C>T | Pro34Ser |
| rs1135840 | NM_000106.5:c.1457G>C | Ser486Thr | |
| CYP2D6*14A | rs1065852 | NM_000106.5:c.100C>T | Pro34Ser |
| rs1135840 | NM_000106.5:c.1457G>C | Ser486Thr | |
| rs16947 | NM_000106.5:c.886C>T | Arg296Cys | |
| rs5030865 | NM_000106.5:c.505G>T | Gly169Ter | |
| CYP2D6*14B | rs1135840 | NM_000106.5:c.1457G>C | Ser486Thr |
| rs16947 | NM_000106.5:c.886C>T | Arg296Cys | |
| rs5030865 | NM_000106.5:c.505G>T | Gly169Ter | |
| CYP2D6*21 | rs1135840 | NM_000106.5:c.1457G>C | Ser486Thr |
| rs16947 | NM_000106.5:c.886C>T | Arg296Cys | |
| rs72549352 | NM_000106.5:c.805_806insC | Arg269Profs | |
| CYP2D6*36 | rs1065852 | NM_000106.5:c.100C>T | Pro34Ser |
| rs1135840 | NM_000106.5:c.1457G>C | Ser486Thr | |
| rs2267447 | NM_000106.5:c.666 + 90A>G | ‐ | |
| CYP2D6*41 | rs1135840 | NM_000106.5:c.1457G>C | Ser486Thr |
| rs16947 | NM_000106.5:c.886C>T | Arg296Cys | |
| rs28371725 | NM_000106.5:c.985 + 39G>A | ‐ |
2.3. Allelic ratios determination
For samples that carry CYP2D6 duplications and are heterozygous for key CYP2D6 variants, the allele‐specific copy number is determined by digital PCR (dPCR) using QuantStudio™ 3D Digital PCR System (Life Technologies Corporation). Sample DNA is loaded onto nanofluidic chips at the concentrations which would give one or no copies of the target per dPCR. A count of reactions with and without amplification can be used for target quantification purposes. For the allele‐specific dPCR application, TaqMan® SNP assays of CYP2D6 variants that are associated with specific duplicated alleles is run in dPCRs on samples of known SNP genotype and CNV status. Sample input amounts and thermal cycling conditions are optimized to best amplify and resolve each allele in cluster plot analysis. Reactions positive for each allele, detected by allele‐specific VIC® or FAM™ dye‐labeled probes, are counted and the allele ratios are determined. For samples heterozygous for target variants, 2‐copy samples would be close to 1:1 allele ratios, whereas 3‐copy samples would be close to 1:2 ratios and the duplicated allele is readily identified. For example, if a sample with *1/*4 and a total of 3 CYP2D6 gene copies, it will be determined as *1/*4x2 if the dPCR ratio gives 1:2 on rs1065852 G>A, and vice versa. This method facilitates accurate CYP2D6 allele genotyping and better prediction of drug metabolizer phenotype.
2.4. Phenotype prediction
Various systems have been developed to categorize CYP2D6 activity and predicted phenotypes based on its genotypes and is dependent on the biallelic expression of alleles. The *1 (wild‐type) allele encodes a fully functioning enzyme. Allelic variant *2 is considered to be a normal function allele. The *9, *10, *14B, and *41 variant alleles have partial activity and are referred to as decreased‐function or reduced‐function alleles. Examples of non‐functional alleles include *3 (frameshift), *4 (splice site variant), *5 (gene deletion), *6 (frameshift), *14A and *21 (frameshift). The *36 allele is a non‐functional allele that is commonly found in Asians as tandem arrangement with *10. A gene duplication or multiplication of a functional allele results in increased expression of the active enzyme. The phenotypes of CYP2D6 were predicted using an activity scoring system described by Gaedigk et al8 The CYP2D6 activity score is calculated by the summation of the activity value of each allele reported in the diplotype. Based on the activity score, the predicted phenotypes are determined based on CYP2D6 dosing guidelines as follow12, 13, 14, 15, 22, 23: activity score of 0 as poor metabolizer (ie, individual carrying two non‐functional alleles); whereas activity score ranging from 1.5 to 2.0 as normal metabolizer (ie, individual carrying one functional allele and one decreased function alelle or two functional alleles); Predicted phenotype of intermediate metabolizer is more difficult to assess given the lack of consensus in regard to whether individual with a CYP2D6 activity score of 1.0 should be assigned an normal or intermediate phenotype (ie, individual carrying one functional allele and one non‐functional allele or 2 decreased function alleles).13, 15, 22, 23 Pharmacokinetic studies suggest such genotypes would confer decreased activity as compared to normal metabolizer, and could also be considered intermediate metabolizer.24 Our group considered activity score ranging from 0.5 to 1.0 as intermediate metabolizer, which is consistent with the ACMG guidelines for tamoxifen23; activity score greater than 2.0 as ultrarapid metabolizer (ie, individual carrying more than 2 copies of functional alleles).
3. RESULTS AND DISCUSSION
3.1. CYP2D6 alleles and phenotypes
The present study provides a comprehensive analysis on different CYP2D6 alleles in the Hong Kong population which is the largest study to date in terms of the number of individuals genotyped and the number of CYP2D6 alleles analyzed in this population. It is presumed that the majority of the Asian group is Han Chinese due to the geographic location. Any major differences among the other groups in the population (ie, Caucasians and mixed race) would be difficult to conclude due to the small sample size. Previous studies in Asians, specifically in Han Chinese, only reported distributions of three CYP2D6 allelic variants (CYP2D6*2, *5, and *10) out of the twelve that are genotyped in this study (CYP2D6*2, *3, *4, *5, *6, *9, *10, *14, *14B, *21, *36, and *41).25, 26, 27, 28 The frequencies of CYP2D6 alleles and predicted phenotypes among the Asian group in the study cohort are summarized in Tables 2 and 3.
Table 2.
Frequencies of the CYP2D6 alleles, copy number variations, and tandems in the present study
| CYP2D6 alleles | Asian, n (%) Total of 735 subjects | Overall, n (%) Total of 800 subjects |
|---|---|---|
| *1 | 324 (22.0) | 376 (23.5) |
| *1x2 | 14 (1.0) | 18 (1.1) |
| *1x3 | 2 (0.1) | 2 (0.1) |
| *1 total | 340 (23.1) | 396 (24.8) |
| *2 | 131 (8.9) | 156 (9.8) |
| *2x2 | 10 (0.7) | 12 (0.8) |
| *2x3 | 2 (0.1) | 2 (0.1) |
| *2 total | 143 (9.7) | 170 (10.6) |
| *4 | 7 (0.5) | 26 (1.6) |
| *4x2 | ‐ | 1 (0.1) |
| *4 total | 7 (0.5) | 27 (1.7) |
| *5 | 44 (3.0) | 49 (3.1) |
| *5 total | 44 (3.0) | 49 (3.1) |
| *6 | 3 (0.2) | 5 (0.3) |
| *6 total | 3 (0.2) | 5 (0.3) |
| *9 | ‐ | 2 (0.1) |
| *9 total | ‐ | 2 (0.1) |
| *10 | 288 (19.6) | 291 (18.2) |
| *10x2 | 27 (1.8) | 28 (1.8) |
| *10x3 | 2 (0.1) | 2 (0.1) |
| *10 total | 317 (21.6) | 321 (20.1) |
| *14A | 5 (0.3) | 5 (0.3) |
| *14A total | 5 (0.3) | 5 (0.3) |
| *14B | 20 (1.4) | 20 (1.3) |
| *14B total | 20 (1.4) | 20 (1.3) |
| *36 | 32 (2.2) | 33 (2.1) |
| *36x2 | 15 (1.0) | 15 (0.9) |
| *36x3 | 3 (0.2) | 3 (0.2) |
| *36xN | 3 (0.1) | 3 (0.2) |
| *36 total | 53 (3.6) | 54 (3.4) |
| *36‐*10 | 471 (32.0) | 472 (29.5) |
| (*36‐*10)x2 | 29 (2.0) | 29 (1.8) |
| (*36‐*10)xN | 2 (0.1) | 2 (0.1) |
| *36‐*10 tandems total | 502 (34.1) | 503 (31.4) |
| *41 | 35 (2.4) | 46 (2.9) |
| *41x2 | 1 (0.1) | 2 (0.1) |
| *41 total | 36 (2.4) | 48 (3.0) |
| Total | 1470 (100) | 1600 (100) |
xN denotes the presence of more than 3 copies of CYP2D6 allele.
Table 3.
Frequencies of CYP2D6 predicted phenotypes and clinical interpretations in the present study
| Predicted phenotype | CYP2D6 genotype | Clinical interpretation | Asian, n (%) Total of 735 subjects | Total, n (%) Total of 800 subjects |
|---|---|---|---|---|
| Ultrarapid Metabolizer | Individual carrying more than 2 copies of functional alleles | Increased drug metabolism to its less active metabolite. Lower plasma concentrations of active parent drug will likely increase probability of pharmacotherapy failurea | 24 (3.3) | 28 (3.5) |
| Normal Metabolizer | Individual carrying 1 functional allele plus 1 decreased function allele or 2 functional alleles | Likely normal drug metabolism | 367 (49.9) | 401 (50.1) |
| Intermediate Metabolizer | Individual carrying 1 non‐functional allele plus 1 decreased function allele or 2 decreased function alleles | Reduced drug metabolism to less active metabolites, Higher plasma concentrations of active parent drug will likely increase the probability of side effectsb | 341 (46.4) | 365 (45.6) |
| Poor Metabolizer | Individual carrying 2 non‐functional alleles | Reduced drug metabolism to less active metabolites, Higher plasma concentrations of active parent drug will likely increase the probability of side effectsb | 3 (0.4) | 6 (0.8) |
| Total | 735 (100) | 800 (100) |
In the case of prodrugs, CYP2D6 ultrarapid metabolizer would result in increased drug metabolism to active metabolites. Higher plasma concentrations of active metabolite will likely increase the probability of side effects.
In the case of prodrugs, CYP2D6 intermediate and poor metabolizer would result in reduced drug metabolism to active metabolites. Lower plasma concentrations of active metabolites will likely increase the probability of pharmacotherapy failure.
3.2. CYP2D6*36‐*10 tandems
As expected, CYP2D6*10 is one of the most commonly observed decreased‐function alleles reported in Asians. This study reported an allele frequency of 21.6% in the study population which is much less than the observed frequencies of 60‐70% reported previously.25, 26, 27, 28 We suspected the major reason for these differences is due to the number of CYP2D6 alleles tested. Specifically, these other studies conducted in Chinese did not identify neither CYP2D6*36 nor CYP2D6*36‐*10 tandems. The CYP2D6*10 is located downstream of CYP2D6*36 and both sequences are highly homologous. It is important to specifically identify CYP2D6*36 alleles to avoid assigning them as CYP2D6*10.21 Although only a handful of studies investigated the presence of CYP2D6*36‐*10 tandems, it is believed to be highly frequent in Japanese, Korean, Chinese and other East Asian populations. It is probable that the allele frequency of CYP2D6*10 could be overestimated when CYP2D6*36‐*10 tandems and/or CYP2D6*36 are not reported. In the present study, allele frequency of CYP2D6*10 (21.6%) along with CYP2D6*36‐*10 tandems (34.1%) and CYP2D6*36 (3.6%) summed up to 59.3%.
To our knowledge, no study has previously investigated the distribution of CYP2D6*36‐*10 tandems in Han Chinese. This study provides novel findings on the prevalence of CYP2D6*36‐*10 tandems in the Hong Kong population with an observed frequency of 34.1%. In addition, CYP2D6*1/*10‐*36 tandems (15.1%), followed by CYP2D6*36‐*10 tandems/*10‐*36 tandems (12.9%) and CYP2D6*10/*10‐*36 tandems (12.0%) are the most commonly observed genotypes among the study cohort. Thus is consistent with findings by Soyama and colleagues who observed CYP2D6*36‐*10 tandems in 30% of 223 genotyped individuals of Japanese descent.21 Another study reported 24.2% frequency of tandem arrangements in the Japanese population.20
While most other CYP2D6 alleles have been extensively studied (www.pharmvar.org), the in vivo effect of CYP2D6*36‐*10 tandems on CYP2D6 enzymatic activity is less defined. CYP2D6 phenotyping studies concluded that there is no significant difference between CYP2D6*10 and CYP2D6*36‐*10 tandems20, 29 when debrisoquine was used as study probe.
3.3. Copy number variations
The prevalence of duplicated CYP2D6 genes in Asians is poorly understood.30 We identified multiplication of CYP2D6 alleles in 86 (11.7%) Asian individuals of the study cohort. These individuals were further classified into groups of having zero, one, two, and three or more copies of the CYP2D6 gene (Figure 1). The overall distributions of CNVs demonstrated that only 241 (32.8%) of the study subjects possessed two copies of the CYP2D6 gene. In addition, two individuals (0.3%) among the study cohort had no detectable copies of CYP2D6 (ie, homozygous for CYP2D6*5). The frequency of all CYP2D6 duplicated alleles was 154 (10.5%) while only 28 (1.9%) of the duplications were of functional alleles (ie, CYP2D6*1 and CYP2D6*2). The contribution of each alleles with copy number variants is further evaluated in Figures 2 and 3.
Figure 1.
Distribution of individuals carrying *36‐*10 tandems, zero, one, two, three, or more copies of the CYP2D6 gene in the Asian group among the study cohort (n = 735)
Figure 2.
Frequency of alleles with a single gene copy (n = 1316) and those with copy number variants (n = 154) including gene deletion, duplications, multiplications, and tandems in the Asian group among the study cohort
Figure 3.
Functional status of copy number variants in the Asian group among the study cohort. A, Frequencies of alleles which include 2 or more copie of normal function genes (n = 28). B, Frequencies of alleles which include 2 or more copies of decreased function variants or tandems with decreased function (n = 61). xN denotes the presence of more than 3 copies of CYP2D6 allele. C, Frequencies of alleles which include gene deletions (CYP2D6*5) and 2 or more copies of variants with no function (n = 65). xN denotes the presence of more than 3 copies of CYP2D6 allele
The consequences of CYP2D6 gene duplications, especially on those alleles which produce neither fully active nor inactive enzymes, eg, CYP2D6*10 allele, can be very complex. Given CYP2D6*10 is the most frequently observed decreased function allele in the Asian population, the presence of duplicated CYP2D6*10 would be a critical factor affecting the capacity of CYP2D6 in drug metabolism. Although studies have previously described the occurrence of CYP2D6*10x2,25, 26, 31 conclusion on other copy number variations of CYP2D6*10 and their effects on CYP2D6 phenotypes is lacking. Future studies will be warranted to further investigate the clinical implications of CYP2D6 copy number variations in this population.
4. CONCLUSION
This novel finding of the present study is reflective of the prevalence of CYP2D6*36‐*10 tandems in the Hong Kong population. It provides a comprehensive analysis on the occurrences of CNV and tandems of the CYP2D6 gene in the population. The results contribute to the overall knowledge of pharmacogenomics and may accelerate the implementation of precision medicine in Asia.
Chan W, Li MS, Sundaram SK, Tomlinson B, Cheung PY, Tzang CH. CYP2D6 allele frequencies, copy number variants, and tandems in the population of Hong Kong. J Clin Lab Anal. 2019;33:e22634 10.1002/jcla.22634
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