Genetic Polymorphisms of CYP2C8 in the Czech Republic

Abstract

Aim: CYP2C8 represents 7% of the hepatic cytochrome system and metabolizes around 5% of drugs in phase I processes. It also plays a significant role in metabolism of endogenous compounds. More than 20 single-nucleotide polymorphisms (SNPs) have been noted, mainly in exons 3, 5, and 8. The most studied SNPs may lead to decreased enzyme activity and may have impact on drug metabolism. Variant alleles are called CYP2C8*2 (I269F), CYP2C8*3 (R139K, K399R), and CYP2C8*4(I264M). Our aim was to investigate the frequency of major functional SNPs among the Czech population. Material and methods: DNA was isolated from whole blood of 161 healthy, young, and unrelated subjects (94 men and 67 women, aged from 23 to 28 years). The genotypes of polymorphic positions CYP2C8*2, CYP2C8*3 (G416A, A1196G), and CYP2C8*4 were determined by polymerase chain reaction–restriction fragment length polymorphism. Results and conclusion: Observed allele frequencies were 10.9%, 5.9%, and 0.3% for the alleles CYP2C8*3, CYP2C8*4, and CYP2C8*2, respectively. Both CYP2C8*3 (G416A, A1196G) alleles have been found in complete linkage disequilibrium. The allele distribution complies well with Hardy–Weinberg equilibrium. Allele frequencies of functionally important CYP2C8 variants in the Czech population are similar to that of other Caucasian populations.

Introduction

Cytochrome P4502C8 (CYP2C8) is a member of the CYP2C subfamily of the human cytochrome P450 enzyme family. The CYP2C subfamily metabolizes about 20% of clinically prescribed drugs. CYP2C8 represents 7% of the hepatic cytochrome system, metabolizes around 5% of drugs in phase I processes, and plays a significant role in the metabolism of endogenous compounds such as arachidonic acids and retinoic acid (Capdevila et al., 1981).

This enzyme is clearly different from the other members of the subfamily (CYP2C9, CYP2C18, and CYP2C19). Compared to the enzymes mentioned above, it has an uncharacteristically large active site, which can recognize a distinct type of compound as substrates (Jiang et al., 2010). The CYP enzymes are found primarily in liver, but are also found in various extrahepatic tissues; for example, CYP2C8 is expressed mainly in kidney, as well as in brain, adrenal gland, mammary gland, ovary, duodenum, etc. (Klose et al., 1999).

CYP2C genes are located at chromosome 10q24 in the following order: 2C18-2C19-2C9-2C8. All CYP2C enzymes are genetically polymorphic and share significant sequence homology—for example, the CYP2C8 gene shares 74% sequence homology with CYP2C9. The CYP2C8 gene consists of 9 exons and >20 single-nucleotide polymorphisms (SNPs) have been noted mainly in exons 3, 5, and 8. Polymorphisms of CYP2C8 have been determined quite recently in 2001 as the last and least known ones of the CYP2C subfamily (Dai et al., 2001; Soyama et al., 2001). The most studied SNPs that may lead to decreased enzyme activity are alleles called CYP2C8*2 (I269F), CYP2C8*3 (R139K, K399R), and CYP2C8*4(I264M).

CYP2C8 has been shown to be the main isoenzyme that metabolizes the anticancer drug paclitaxel to its major metabolite 6α-hydroxy paclitaxel (Rahman et al., 1994). Clinical studies have demonstrated significant interindividual responses of patients to paclitaxel (Evans and Relling, 1989). The hydroxylation activity of this enzyme is mainly reduced in the presence of the variant CYP2C8*3. A recently characterized novel CYP2C8*14 allele among the Japanese population showed decreased affinity of the enzyme toward paclitaxel and influenced clinical response to amiodarone (Hanioka et al., 2010, 2011). In addition, hydroxylation of amiodarone is also significantly lower with the CYP2C8 P404A variant allele than that of the wild-type allele CYP2C8*1 (Soyama et al., 2002). It has been demonstrated that this enzyme plays an important role in the metabolism of many well-known and prescribed drugs such as thiazolidinediones, ibuprofen, and fluvastatin. The main substrates and inhibitors of CYP2C8 are summarized in Table 1 (Daily and Aquilante, 2009). It is also suggested that CYP2C8 may influence important physiological and pathological processes in man. It converts arachidonic acid to biologically active epoxyeicosatrienoic acids, which modulate hepatic glycogenolysis, peptide hormone secretion in pancreas, platelet aggregation, etc. (Zeldin et al., 1996). The CYP2C8*3 allele was also found to be associated with markedly defective metabolism of arachidonic acid.

Table 1.

Substrates and Inhibitors of CYP2C8

Specific drugs	Drug class	Other involved CYP enzymes
CYP2C8 major metabolizing enzyme
All-trans-retinoic acid	Vitamin A derivative	CYP2C9
Amiodarone	Class III antiarrhythmic	CYP3A4, CYP1A2, CYP2C19, CYP2D6
Amodiaquine	4-aminoquinoline	CYP3A4, CYP1A1, CYP1B1
Cerivastatin	HMG-CoA reductase inhibitor	CYP3A4
Chloroquine	4-aminoquinoline	CYP3A4, CYP2D6
Paclitaxel	Taxane	CYP3A4, CYP3A5
Repaglinide	Antidiabetics	CYP3A4
Rosiglitazone Pioglitazone	Thiazolidinedione	CYP2C9, CYP3A4, CYP1A1
Tazarotenic acid	Retinoid derivative
Troglitazone	Thiazolidinedione	CYP3A4
CYP2C8 intermediate or minor metabolizing enzyme
Carbamazepine	Anticonvulsant	CYP3A4 (major)
Cyclophosphamide	Nitrogen mustard alkylating agent	CYP2A6, CYP2B6, CYP2C9,CYP2C19, CYP3A
Dapsone	Antibacterial sulfone	CYP2C9 (major)
Diclofenac	NSAID	CYP2C9 (major), CYP3A4, and CYP2C19
Diltiazem	Calcium channel blocker	CYP3A4, CYP2C9
Fluvastatin	HMG-CoA reductase inhibitor	CYP2C9 (major), CYP3A4
Ibuprofen	NSAID	CYP2C9
Ifosfamide	Nitrogen mustard alkylating agent	CYP2A6, CYP2B6, CYP2C9,CYP2C19, CYP3A
Loperamide	μ-opioid receptor agonist	CYP2B6, CYP2D6, CYP3A4
Methadone	Opioid antagonist	CYP3A4, CYP2D6, CYP2B6
Morphine	Opioid agonist	CYP3A4
Simvastatin	acid HMG-CoA reductase inhibitor	CYP3A4 (major)
Tenoxicam	NSAID	CYP2C9 (major)
Torsemide	Loop diuretic	CYP2C9 (major)
Verapamil	Calcium channel blocker	CYP3A4 (major), CYP3A5
Zopiclone	Nonbenzodiazepine sedative hypnotic	CYP3A4 (major)
Inhibitors of CYP2C8
Amitriptyline	Tricyclic antidepressants	CYP3A4
α-naphthoflavone	—	CYP1A
Gemfibrozil	Fibrates
Ketoconazol	Antimycotics	CYP3A4
Montelukast	Leukotriene receptor antagonist
Quercetin	Plant-derived flavonoid	CYP1A2
Terfenadine	Antihistaminic	CYP3A4
Triazolam	Benzodiazepine	CYP3A4
Trimethoprim	Dihydrofolate reductase inhibitors	–

Table was adapted from previously published articles in Daily and Aquilante (2009) and Totah and Rettie (2005).

CYP2C8*3 has been found primarily in Caucasians (allele frequency 13%), while the frequency among African Americans is 2%. This allele has not been described in Asians. The CYP2C8*3 allele variant appears to be linked to the CYP2C9*2 allele. The variant allele CYP2C8*4 has been only detected in Caucasians with an allele frequency of 7.5% (Dai et al., 2001; Bahadur et al., 2002). The 404A allele was determined only in the Japanese population at a very low frequency (<0.007%).

Although the functional genetic polymorphisms of this cytochrome P450 member have been reported previously, there is no information on the occurrence of CYP2C8 polymorphisms in the Czech population.

Materials and Methods

Subjects

The studied population comprised a randomly selected sample of 161 healthy unrelated Czech subjects (94 men and 67 women, aged from 23 to 28 years, average age was 24.3 years, SD=1.6). Subjects were briefly interviewed regarding their medical history to include only healthy subjects. Each of the subjects gave their written informed consent.

Genotyping

Venous blood with EDTA (7 mL) was obtained and DNA was isolated from a subject's leukocytes using QIAamp DNA Blood Mini Kit (Qiagen).

Polymerase chain reaction (PCR)–restriction fragment length polymorphism was used for analysis. The genotypes of variant CYP2C8 alleles were determined using a modified analysis by Dai et al. (2001); for CYP2C8 P404A allele-specific PCR was used according to Nakajima et al. (2003). DNA amplification was done in a Biorad termocyclerMyCycle. Primer sets and restriction enzymes used are shown in Table 2.

Table 2.

Primers and Enzymes Used for Genotyping of Variant Alleles of CYP2C8

Allele CYP 2C8	Sense primers	Antisense primers	Enzyme	Fragment length	Exon
* 2	5′-AAGATACATATATCTTATACATG-3′	5′-ATCCTTAGTAAATTACAGAAGG-3′	BclI	wt: 214 98 v: 312	5
*3 G416A	5′-AGGCAATTCCCCAATATCTC-3′	5′-ACTCCTCCACAAGGCAGTGA-3′	BseRI	wt: 310 37 v: 347	3
*3 A1196G	5′-CTTCCGTGCTACATGATGACG-3′	5′-CTGCTGAGAAAGGCATGAAG-3′	XmnI	wt: 92 25 v: 117	8
*4	5′-AAAGTAAAAGAACACCAAGC-3′	5′-AAACATCCTTAGTAAATTACA-3′	TaqI	wt: 83, 53, 31 v: 136, 31	5

PCR amplification consisted of initial denaturation for 2 min at 94°C followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 48°C for 80 s, and extension at 72°C for 1 min. Terminal elongation ran at 72°C for 7 min. Restriction produced DNA fragments that were separated on 3.5% agarose gel and visualized after ethidium bromide staining on the ultraviolet transilluminator.

Statistical analysis

Statistical significance (p<0.05) was assessed by the χ² test for 2 degrees of freedom. Expected distribution of genotypes was calculated by Hardy–Weinberg equilibrium from the allele frequencies. Data were compiled according to the genotype and allele frequencies with 95% confidence intervals (CIs).

Results

We have analyzed 161 subjects to detect the SNPs in enzyme CYP2C8.

CYP2C8*3 G416A and *3 A1196G were the most frequent alleles in the Czech population. Both these alleles were in complete linkage disequilibrium. The frequency of the variant allele was 10.9%. The allele frequency of CYP2C8*4 was 5.9%. Only one subject was a homozygous carrier of the variant allele and 17 subjects were heterozygous.

The allele frequencies were all in Hardy–Weinberg equilibrium. All achieved results are summarized in Table 3. Expected allele frequencies fit into the 95% CI in all variants.

Table 3.

Genotype and Allele Frequencies of CYP2C8 in the Czech Population

			Allele		Genotype
Nomenclature CYP2C8	Polymorphism	Effect		f		f	95% CI
*2	A805T	I269F	A	0.997	Wt	0.994	1.0, 0.982
			T	0.003	Het	0.006	0, 0.018
					Var	0	0, 0
*3	G416A	R139K	G	0.891	Wt	0.801	0.863, 0.740
			A	0.109	Het	0.180	0.240, 0.120
					Var	0.019	0.040, −0.001
*3	A1196G	K399R	A	0.891	Wt	0.801	0.863, 0.740
			G	0.109	Het	0.180	0.240, 0.120
					Var	0.019	0.040, −0.001
*4	C792G	I264M	C	0.941	Wt	0.888	0.937, 0.840
			G	0.059	Het	0.106	0.153, 0.058
					Var	0.006	0.018, −0.006
P404A	C1210G		C	0.966	Wt	0.932	0.970, 0.893
			G	0.034	Het	0.068	0.107, 0.029
					Var	0	0, 0

CI, confidence interval; f, frequency; Wt, homozygous wild type carrier; Het, heterozygous carrier; Var, homozygous variant carrier.

Discussion

Recently, the structure of the CYP2C8 isoenzyme was characterized and the polymorphisms within the CYP2C8 gene have also been identified. In this study we assessed frequencies of the most common polymorphisms with possible functional consequences in the Czech population.

The genetic polymorphisms of CYP2C8 have been described in 2001 (Dai et al., 2001). Totally, 20 SNPs have been characterized, including the most common alleles CYP2C8*2, CYP2C8*3, and CYP2C8*4. Subsequently, several studies identified frequencies of relevant SNPs in various ethnic populations (Table 4).

Table 4.

Comparison of CYP2C8 Allele Frequencies in Different Populations

Nomenclature CYP2C8	Population	Number of subjects	Allele frequency	Reference
*2	Czech Republic	161	0.003	—
	Zanzibar	165	0.139	Cavaco et al. (2005)
	US African-Americans	82	0.183	Dai et al. (2001)
	North East England	116	0.004	Bahadur et al. (2002)
	West Germany	122	0.016	Weise et al. (2004)
	Japan	360	0.000	Nakajima et al. (2003)
	Ghanaian	204	0.170	Kudzi et al. (2009)
	Malaysian	123	0.008	Muthiah et al. (2005)
*3	Czech Republic	161	0.109	—
	Zanzibar	165	0.021	Cavaco et al. (2005)
	US African-Americans	82	0.018	Dai, et al. (2001)
	North East England	107	0.150	Bahadur et al. (2002)
	West Germany	122	0.140	Weise et al. (2004)
	Japan	360	0.000	Nakajima et al. (2003)
	Ghanaian	204	0.000	Kudzi et al. (2009)
	Malaysian	123	0.012	Muthiah et al. (2005)
	Faroese	311	0.069	Halling et al. (2005)
	Spanish	130	0.15	Garcia-Martin et al. (2004)
	Sweden	1503	0.095	Yasar et al. (2003)
*4	Czech Republic	161	0.059	—
	Zanzibar	165	0.006	Cavaco et al. (2005)
	North East England	107	0.075	Bahadur et al. (2002)
	West Germany	122	0.074	Weise et al. (2004)
	Japan	360	0.000	Nakajima et al. (2003)
	Ghanaian	204	0.000	Kudzi et al. (2009)
	Malaysian	123	0.000	Muthiah et al. (2005)
P404A	Czech Republic	161	0.034	—
	Japan	200	0.000	Nakajima et al. (2003)

Two substitutions CYP2C8*3 G416A and *3 A1196G occur in complete linkage, while the CYP2C8*3 allele is found in an incomplete linkage disequilibrium with the CYP2C9*2 allele. Furthermore, the CYP2C8*3 variant allele is also associated with altered metabolism of CYP2C8 substrates. It was proven that the CYP2C8*3 allele results in impaired metabolism of the anticancer drug paclitaxel, with 50% reduction of the enzyme activity in paclitaxel metabolism (Bahadur et al., 2002). The CYP2C8*2 variant allele showed a 15% reduction of paclitaxel metabolism. Thus, both these CYP2C8 variant alleles can increase the paclitaxel toxicity and lead to variations in clinical response. Data also suggested lower metabolic activity of CYP2C8*4 than wild-type enzyme for paclitaxel, but it was not statistically significant (Bahadur et al., 2002).

Next, homozygous carriers for both variant alleles CYP2C8*3 and CYP2C9*2 manifested lower clearance of ibuprofen in comparison with carriers of the wild-type allele (p<0.001) (Garcia-Martin et al., 2004).

In our study we investigated three common polymorphisms of CYP2C8 and one rare allele P404A. The results were compared with the results from other published studies concerning the SNP frequencies (Table 4).

CYP2C8*2 homozygous was not found in the Czech population and variant allele frequency was 0.3%. This finding was in good accordance with reported Caucasian populations (0%–2%) because this allele is very rare in Caucasians and absent in Asian populations (Garcia-Martin et al., 2006). The CYP2C8*3 variant allele was observed with a frequency of 10.9%, while 1.2% of subjects should be homozygous carriers of this allele in Caucasian populations (Dai et al., 2001), which is similar to our finding of 1.9%. We have found good consistency of our results with the data from other studies among Caucasian populations with allele frequencies of the variant allele reported between 9.5% and 15% (Table 4).

Distribution frequencies of the CYP2C8*4 variant allele were determined to be between 5.4% and 7.5% (Table 4). Homozygous frequency for the CYP2C8*4 variant allele should occur at 0.3% according to Hardy–Weinberg equilibrium in the Czech population.

CYP2C8*3 and CYP2C8*4 variant alleles have not been detected within Asian populations.

The CYP2C8 P404A allele has been until now described only in the Japanese population at very low frequency. Among the Czech population we have confirmed 11 heterozygous carriers of the P404A allele.

It was shown that CYP2C8*2 and CYP2C8*3 homozygous genotypes can lead to poor metabolic phenotype, which could result in drug toxicity. It was also speculated that the expression of CYP2C8 enzymes in the human intestine and liver is quite low and therefore other metabolic pathways may compensate for the possible genetic deficit of the enzyme activity (Lapple et al., 2003).

Nevertheless, the routine testing for the SNPs is becoming a useful tool for optimizing dosage and therapy. Knowledge of the frequency of functionally important SNPs in the CYP2C8 gene in the Czech population is a substantial asset for designing future pharmacokinetic and pharmacodynamic studies conducted with its substrates. These variant alleles, to our knowledge, are being estimated for the first time among the Czech population.

Acknowledgment

This study has been supported by Grants VZ MSM0021620849 and VZ MSM 0021620820.

Author Disclosure Statement

No competing financial interests exist.