Frequency of Certain Single-Nucleotide Polymorphisms and Duplication of CYP2D6 in the Jordanian Population

Abstract

The CYP2D6 isozymes are responsible for metabolism of 7-10% of clinically available drugs. Genetic polymorphism in CYP2D6 may have an impact on drug efficacy and toxicity. The aim of this study was to determine the allelic frequency of CYP2D6*4, *10, and *17 and CYP2D6*2×N duplication allele in 192 healthy unrelated male and female Jordanian volunteers. Polymerase chain reaction (PCR)-restriction fragment length polymorphism-based methods were used to identify the CYP2D6*4, *10, and *17 genotypes; and allele-specific long PCR was used to determine the CYP2D6*2×N allelic frequency. The CYP2D6*10 allele was the most frequent mutant allele in Jordanians (14.8%) followed by CYP2D6*4 and *17 at 12.8%, and 8.3%, respectively. The duplication allele was found in 13.5% of the studied sample. The CYP2D6*4 G-A heterozygote genotype frequency was 20.3%, and the homozygous mutant genotype was 2.6%. In case of CYP2D6*10 C-T and CYP2D6*17 G-C heterozygote genotypes, the frequencies were 21.4% and 12.5%, respectively, while the homozygous mutant genotype frequencies of T-T and C-C were 4.2% and 2.1%, respectively. In conclusion, the allelic distributions of the CYP2D6 gene among Jordanians are different from other Mediterranean groups, especially the *10 and *17 single-nucleotide polymorphisms, and more importantly the CYP2D6*2×N duplication allele, which seems to follow a gradient reduction in prevalence from Ethiopia to Northern Europe.

Introduction

Genetic polymorphisms in drug-metabolizing enzymes are a major cause of inherited differences in drug metabolism that may influence drug efficacy and toxicity (Benny and Abraham, 2001). The highest impact among drug-metabolizing enzymes is exerted by the cytochromes P450s (Bernard et al., 2006; Risch et al., 2007). Among these, CYP2D6 is thought to be involved in the metabolism of up to 10% of all prescription drugs, including the selective serotonin-reuptake inhibitors, tricylic antidepressants, beta-blockers, opiates, neuroleptics, antiarrhythmics, cardiovascular drugs, and a variety of other drugs (Ingelman-Sundberg and Evans, 2001).

CYP2D6 is highly polymorphic, with over 90 known allelic variants (Ma et al., 2002; Teh and Bertilsson, 2012). Some of the alleles are fully functional, sometimes with multiple copies, whereas others have reduced or null function, resulting in a wide interindividual variability in drug metabolism (Kitada, 2003). Depending on the individual's ability to metabolize CYP2D6 substrates, a population can be divided into four categories: poor metabolizers (PMs), intermediate metabolizers (IMs), extensive metabolizers (EMs), and ultrarapid metabolizers (UMs) (Jann and Cohen, 2000).

The majority of the population expresses the EM phenotype (Evans and Relling, 2004). In case of PMs, two CYP2D6 alleles are inactive resulting in a lower substrate metabolism rate, leading to high levels of unmetabolized drugs (Ma et al., 2002). CYP2D6*4 is the most common polymorphism that is associated with the PM phenotype (Scordo et al., 2004). The IM has three possibilities: two reduced function alleles, one active and one inactive allele, and one decreased activity and one inactive allele (Ingelman-Sundberg et al., 1999). The most common alleles that result in reduced CYP2D6 activity are the CYP2D6*10 and the CYP2D6*17 alleles (Tod et al., 2011). The CYP2D6*10, which is most common in the Asian population, is a mutation that causes an amino acid substitution for proline by serine at the 34th amino acid. This alteration results in a reduction in the enzyme substrate affinity through abolishing the polyglycoprotein sequence necessary for folding of the enzyme (Tod et al., 2001; Zuo et al., 2012). The CYP2D6*17 is most common in the African population. It is associated with reduced enzymatic activity as a result of two mis-sense mutations that alter the active site structure (Ingelman-Sundberg et al., 1999).

The UM phenotype is caused by multiple functional copies of the active CYP2D6 genes, including primarily the CYP2D6*2 allele, but also involves CYP2D6*1 and others. Individuals with the UM phenotype metabolize drugs at a very high rate, leading to a loss of therapeutic efficacy at standard doses (Ingelman-Sundberg et al., 1999). There is evidence to suggest that the frequency of the polymorphism in CYP2D6 varies among ethnic groups. The UMs have a low prevalence of 0.8% in some European white populations, whereas the prevalence is much higher in Ethiopians and Saudi Arabs scoring 29% and 21%, respectively (Oscarson et al., 1997).

The aim of this study was to determine the allelic frequency of the most common CYP2D6 single-nucleotide polymorphisms (SNPs) in the Jordanian population. In particular, the allelic frequency of CYP2D6*4, *10, and *17 and CYP2D6*2×N duplication allele will be assessed.

Materials and Methods

Sample collection

A total of 192 healthy unrelated Jordanian volunteers (77 women and 115 men) with an average age of 27 years volunteered to participate in the study and signed an informed consent. The Faculty of Medicine Ethics Committee approved the study. From each volunteer, 3-5 mL venous blood was collected in ethylenediaminetetraacetic acid tubes.

DNA extraction

DNA was extracted using the Wizard DNA extraction kit (Promega) according to the manufacturer's protocol.

Genotyping method

Genotypes were determined for CYP2D6*4, CYP2D6*10, and CYP2D6*17 using the polymerase chain reaction (PCR)-restriction fragment length polymorphism method as described by Theophilus et al. (2006), with minor modifications. Briefly, the PCR mixture (50 μL) was prepared using 200 ng of genomic DNA, 10 μL of Taq polymerase buffer, 0.2 mM of dNTPs, 2.0, 1.5, and 2.5 mM of MgCl₂ for the *4, *10, and *17 polymorphism, respectively, and 0.2 pM each of the forward and reverse primers (Table 1) (Invitrogen) and 2.5 U of Taq DNA polymerase.

Table 1.

Primer Sequences Used for Amplification of CYP2D6^*4, CYP2D6^*10, and CYP2D6^*17, and Long Polymerase Chain Reaction Assay

Primers	Sequence (5′-3′)
CYP2D6^4-F*	TGC CGC CTT CGC CAA CCA CT
CYP2D6^4-R*	TCG CCC TGC AGA GAC TCC TC
CYP2D6^10-F*	GTG CTG AGA GTG TCC TGC C
CYP2D6^10-R*	CAC CCA CCA TCC ATG TTT GC
CYP2D6^17-F*	TTT TGC ACT GTG GTG CCT CGC
CYP2D6^17-R*	CCC GGG TTC CAC GGA AAT CT
CYP-17F^*	TCC CCC ACT GAC CCA ACT CT
CYP-32R^*	CAC GTG CAG GCG ACC TAG AT
CYP-207F^*	CCC TCA GCC TCG TCA CCT CAC

These primers were used to detect the duplicated allele.

F, forward; R, reverse.

After an initial denaturation at 94°C for 8 min, 30 cycles were performed consisting of a denaturation step at 94°C for 30 s, an annealing step at 58°C, 56°C, and 60°C for 30 s for *4, *10, and *17 polymorphisms, respectively, and an elongation step at 72°C for 30 s and a final elongation step at 72°C for 5 min.

To identify individuals carrying duplicate CYP2D6 genes, a long-PCR-based assay was performed according to the method adopted from Lovlie et al. (1996) with some modifications. Long PCR was carried out using the Gene Amp XL PCR kit (Applied Biosystem) in 50-mL reaction volumes containing 1× PCR reaction buffer, 400 ng of genomic DNA, 200 mM of each dNTP, 0.35 mM of each primer (Table 1), 4 mM Mg(OAc)₂, and 1 U of rTth DNA polymerase. For the internal control of the PCR reaction, the primer combination cyp-17f and cyp-32r was used to amplify the 5.2-kb fragment from a CYP2D7-CYP2D6 intergenic region, which should appear in all samples (see Fig. 1A). To detect the CYP2D6-CYP2D6 intergenic sequence, cyp-207f and cyp-32r primers were used. Only subjects carrying two CYP2D6 gene copies on the same allele show a 3.2-kb fragment, which indicates a CYP2D6-CYP2D6 intergenic sequence, whereas no amplification is seen in individuals without duplicate genes (see Fig. 1B).

FIG. 1.

Long-polymerase chain reaction assays for detection of alleles with duplicate CYP2D6 genes. (A) Lane 1 is the ladder. Lanes 1-3 show the results of DNA amplification using the primer pair cyp-17f-cyp-32r in samples 11, 12, and 13, respectively. Patient in lane 1 has CYP2D6 gene duplication; patients in lanes 2 and 3 do not carry duplicate genes. (B) Lane 1 is the ladder. The results of amplification by the primer pair cyp-207f-cyp-32r are seen in lanes 1-3 in samples 11, 12, and 13, respectively. Patient in lane 1 has CYP2D6 gene duplication, whereas patients in lanes 2 and 3 do not carry duplicate genes.

The conditions for amplification with the primer pairs cyp-17f/cyp-32r and cyp-207f/cyp-32r were as follows: an initial denaturing step at 93°C for 1 min, followed by 37 cycles of 93°C for 1 min, 60°C for 30 s, and 68°C for 6 min, and a final elongation step of 72°C for 10 min. The resulting long-PCR products were separated and detected in an ethidium bromide-containing 0.8% agarose gel.

Restriction enzyme analysis

The PCR products for CYP2D6*4, CYP2D6*10, and CYP2D6*17 were digested with BstNI, HphI, and BtsC1, respectively, following the manufacturer's instructions. DNA fragments were separated on 3% agarose and visualized by ethidium bromide.

Statistical analysis

Expected genotype frequencies were calculated using the Hardy-Weinberg equilibrium from the observed allelic frequencies. The 95% confidence interval was calculated using Microsoft Excel 8.0 (Microsoft).

Results

Table 2 shows the allele and genotype frequencies of CYP2D6 distribution among 192 Jordanian volunteers. The frequencies of CYP2D6*4, *10, and *17 variant alleles were 12.8%, 14.8%, and 8.3%, respectively. For the genotype frequency, CYP2D6*4 scored 77.1% for the G-G wild type, 20.3% for the heterozygote G-A, and 2.6% for the mutant A-A genotype. In case of CYP2D6*10, the genotype frequencies were 74.5%, 21.4%, and 4.2% for wild-type C-C, C-T, and the mutant T-T, respectively. For CYP2D6*17, the genotype frequencies were 85.4%, 12.5%, and 2.1% for wild-type G-G, G-A, and mutant A-A, respectively. Taken together, these findings point out that most of the polymorphisms were heterozygous, in which case the volunteers only carry one non- or reduced functional allele, and only a low percentage of the volunteers carries homozygous mutant alleles. For the CYP2D6 duplication, 26 of the 192 volunteers (13.5%) were found to carry a duplicated functional CYP2D6 gene.

Table 2.

CYP2D6 Genotype and Allele Frequencies in Samples of Jordanian Population

Alleles	Number (n=192)	Genotype frequency (%)	Allele frequency (%) n=384	95% CI
CYP2D6^4*
G-G	148	77.1
G-A	39	20.3	12.8 (A)	(0.096-0.1636)
A-A	5	2.6
CYP2D6^10*
C-C	143	74.5
C-T	41	21.4	14.8 (T)	(0.1143-0.1857)
T-T	8	4.2
CYP2D6^17*
G-G	164	85.4
G-C	24	12.5	8.3 (C)	(0.053-0.107)
C-C	4	2.1
Ultrarapid	26	13.5	—

CI, confidence interval.

Depending on the classifications that have been set by Ingelman-Sundberg and Evans (2001), our subjects have been divided into four metabolic groups, namely, PMs, IMs, Ems, and UMs (see Table 3). Interestingly, the frequency of the genotypes encoding intermediate metabolism was 21.1%, which is higher than the 2.6% genotypes encoding poor metabolism.

Table 3.

Frequencies of the CYP2D6 Genotypes and Predicted Phenotypes

Predicted phenotype	CYP2D6 alleles/genotype	Count (192)	Frequency (%)
Poor metabolism	4(A)/4(A)	5	2.6
Intermediate metabolism	4(A)/4(G), 4(A)/10(T), 4(A)/17(C), 10(T)/10(T), 17(C)/17(C), 10(T)/17(C)	41	21.1
Extensive metabolism	4(G)/4(G)	120	62.5
Ultrarapid metabolism		26	13.5

Discussion

Interethnic variation in the CYP2D6 polymorphism has been reported (Ingelman-Sundberg 2005; Ingelman-Sundberg et al., 2007). Therefore, it is important to determine the frequency of the most common CYP2D6 SNPs in the Jordanian population. We studied four polymorphisms: CYP2D6*4, *10, and *17, and 2×N duplication alleles. Our results on the frequency of the CYP2D6*4 alleles in the Jordanian population was 12.8%, which is in accordance with the values reported in the Jordanian population by Hadidi et al. (1994). It is also close to the CYP2D6*4 allelic frequencies in other Mediterranean populations such as Turkish (13.9%), Southern Spaniards (17.7%), Sardinians (12.5%), Syrians (9.8%), and Italians (15.3%) (see Table 4) (Fuselli et al., 2004; Scordo et al., 2004; Koseler et al., 2007). At the genotyping level, 5 of the 192 volunteers (2.6%) were found to carry two nonfunctional alleles, which classifies them as PMs, while 39 of the 192 volunteers (20.31%) were carrying only one nonfunctional allele. Again, this finding is compatible with those of the previous Jordanian and Mediterranean studies (Hadidi et al., 1994; Fuselli et al., 2004; Scordo et al., 2004; Koseler et al., 2007).

Table 4.

Comparison of CYP2D6 Alleles in Jordanians with Major Ethnic Groups

Major variants alleles (%)
Population	CYP2D6^4*	CYP2D6^10*	CYP2D6^17*	Ultrarapid	References
Caucasians	12-21	1-2	0.0	1-5	Ingelman-Sundberg et al. (1999)
Turkish	15	—	—	8.7	Koseler et al. (2007)
Asians	1	51	0.0	0-2	Ingelman-Sundberg et al. (1999)
Spanish	17.7	1.0	1.0	10	Fuselli et al. (2004)
Ethiopians and Saudi Arabians	1-4	3-9	3-9	10-16	Ingelman-Sundberg et al. (1999)
Sardinians	12.5	4.2	0.00	2.1	Fuselli et al. (2004)
Syrians	9.8	2.9	0.00	3.9	Fuselli et al. (2004)
Jordanians (our study)	12.8	14.8	8.3	13.5

Surprisingly, the determined allelic frequency of CYP2D6*10, which has a 50% allelic frequency in the Asian population (Scordo et al., 2004; Tateishi et al., 1999), was higher than the expectations; it scored 14.8%, which is higher than what has been reported in many Mediterranean populations such as the Southern Spaniards (0.98%), Basques (1.3%), Sardinians (4.2%), Central Italians (8.1%), and Syrians (2.9%) (see Table 4) (Fuselli et al., 2004; Scordo et al., 2004). This indicates that the Jordanian population is among those populations that exert high frequency of this reduced function allele. Similarly, the allelic frequency of CYP2D6*17, which is a rare SNP in the Caucasian and Asians, was relatively high, scoring 8.3%; a value that seems to be higher than those frequencies in other Mediterranean populations such as Southern Spaniards (0.98%), Italians (0.00%), and Syrians (0.00%) (see Table 4) (Fuselli et al., 2004; Scordo et al., 2004; Koseler et al., 2007). Looking into the genotypes of the CYP2D6*10 and *17 shows that most of these reduced function alleles are heterozygous in origin. The percentage of homozygous reduced function alleles is only 2.18% and 4.16% for CYP2D6*17 and CYP2D6*10, respectively.

Taking together, those findings for CYP2D6*4, *10, and *17 indicate that the prevalence of the PMs in the Jordanian population is relatively low (2.6), whereas that of the IMs reaches 21.1%. This high percentage value is due to the high frequency of the heterozygous CYP2D6*4 and also to the relatively high heterozygous frequency of the CYP2D6*10 and *17 alleles. Therefore, attention should be given when prescribing drugs with a narrow therapeutic index and which are substrate for the CYP2D6 enzyme. This recommendation is supported by other studies that have shown that the side effect of drugs increased in CYP2D6 IMs. Laika et al. (2009) have shown that IMs have increased side effects of psychoactive drugs (Laika et al., 2009; Zhou, 2009). Similarly, Mulder et al. (2006) demonstrated that the antidepressant risk of a plasma concentration above the therapeutic range can be increased in the IMs, although to a lesser extent than found in PMs.

Concerning CYP2D6 duplication, we found that 26 of the 192 volunteers (13.5%) carry a duplicated functional CYP2D6 gene and were thus classified as UMs. Importantly, this percentage is higher from the values reported for northern Europeans (1-2%) (Bradford, 2002) and slightly higher than the CYP2D6*2×N allelic frequency in a Spanish population (10%) (Bernal et al., 1999). However, it is lower than frequencies reported from more southern geographical areas (29% in an Ethiopian population) (Aklillu et al., 1996). This finding supports the hypothesis that there is a gradient of decreasing prevalence of CYP2D6 gene duplications from Ethiopia to northern Europe (Bernal et al., 1999; Aklillu et al., 1996; Bradford, 2002). Perhaps, this high frequency of UMs in a Jordanian population should be carefully considered, because the UM patients generally may have therapeutic failure with drugs on account of increased enzymatic activity. On the other hand, the UMs rapidly may exhibit exaggerated toxicity to agents that are activated by CYP2D6, such as the analgesic codeine. The importance of determining the CYP2D6*2×N frequency is such population clearly can be strengthened after the 2007 Food and Drug Administration Codeine Warning for Breastfeeding Mothers, which was based on the idea that ultrarapid codeine metabolizers change codeine to morphine faster and more completely than in other mothers, resulting in high and unsafe levels of morphine in blood and breast milk (Madadi et al., 2007; Crews et al., 2012).

In conclusion, the allelic frequencies of the most common CYP2D6 SNPs in the Jordanian population show some similarities and some differences from those of the other populations. The CYP2D6*4 showed a close frequency to the frequencies of the Caucasian and Mediterranean populations. In the cases of CYP2D6*10 and CYP2D6*17, the frequencies were relatively higher than those reported in the Caucasian and Mediterranean populations. Interestingly, the predicted phenotype revealed a relatively high frequency of the IMs reaching 21.1% that may be due to the high frequency of heterozygous CYP2D6*4, *10, and *17 alleles. Importantly, the frequency of the CYP2D6*2×N duplication allele was relatively high and seems to follow a gradient reduction in prevalence from the Mediterranean countries to northern Europe.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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