Genetic Variations in the Xenobiotic-Metabolizing Enzymes CYP1A1,CYP1A2,CYP2C9,CYP2C19 and Susceptibility to Colorectal Cancer Among Turkish People

Abstract

Cytochrome P450 (CYP) enzymes are genetically polymorphic and play key roles in the metabolism of xenobiotics. Colorectal cancer (CRC) is one of the most common malignant tumors in Turkey as well as in the world. In this study, it was aimed both to evaluate the effects of CYP variants on the susceptibility to CRC and to predict the individual response of the Turkish people to xenobiotics metabolized by CYP enzymes. For that, we assessed the association of CYP1A1, CYP1A2, CYP2C9, and CYP2C19 polymorphisms in patients with CRC in the Turkish population through a case-control study. Distributions of the variants were determined in 104 patients with CRC and 183 healthy volunteers. As results, CYP1A1 6235T/C was significantly associated with CRC risk (odds ratio [OR]=2.53; 95% confidence interval [CI]=0.99-6.45; p=0.046). In a haplotype-based analysis, CYP1A1 haplotype C₆₂₃₅-A₂₄₅₅ might be associated with the development of CRC (OR=2.70; 95% CI=0.58-5.90; p=0.046). We believe that the findings are the first results of CYP allele distributions in the Turkish population and provide an understanding of the epidemiological studies that correlate therapeutic approaches and etiology of CRC especially in Turkish patients.

Introduction

Colorectal cancer (CRC) is one of the most frequent tumors and is the main reason for the high mortality ratio among the different types of cancer sufferers in developed countries. It occurs through an interaction between an individual's genetic background and environmental parameters such as dietary factors and smoking. A number of epidemiological studies indicate that dietary carcinogens, such as heterocyclic amines, N-nitroso compounds, and polycyclic aromatic hydrocarbons, are associated with an increased CRC risk. Procarcinogens enter the body and are activated to carcinogens causing the development of CRC or are eliminated by various enzymes (Bae et al., 2006; Fan et al., 2007). CYP enzymes play key roles in the metabolism of xenobiotics. CYP1A1, CYP1A2, CYP2C9, and CYP2C19 are the most important CYP enzymes in xenobiotic metabolism, especially in the activation of procarcinogens (Bozina et al., 2009). Many genes in CYPs, highly polymorphic, could affect metabolic capabilities, which may result in a differential susceptibility to CRC (Kury et al., 2007). Despite all, the biological processes connecting lifestyle characteristics and colorectal carcinogenesis and the importance of CYP's functions on CRC risk remain still unclear (Petrova et al., 2008; Campa et al., 2012).

The CYP alleles and the relation with CRC have not been studied in the Turkish population even though it has been in different populations. In the present study, the objectives were (1) to determine the CYP variants that could serve as biomarkers for CRC prevention and/or prognosis, regarding their possible influence on the relative risk for developing CRC when interacting with environmental factors and (2) to predict the individual response of Turkish people to xenobiotics metabolized by CYP enzymes. Because of this, we investigated nine SNPs of the CYP gene, which were CYP1A1*2A, CYP1A1*2C, CYP1A2*1C, CYP1A2*1F, CYP2C9*2, CYP2C9*3, CYP2C19*2, CYP2C19*3, and CYP2C19*17 alleles in the Turkish population.

Materials and Methods

To examine the association between CYP1A1, CYP1A2, CYP2C9, and CYP2C19 genotypes and susceptibility to colorectal cancer, we conducted a case-control study in the Turkish population. The study was approved by the ethics committee of Istanbul University and all participants provided written informed consent. Blood samples were collected from 104 patients with colorectal cancer and 183 healthy volunteers admitted to the Hospital of Istanbul University and the Bagcilar Training and Research Hospital during the same period. The criteria for the diagnosis of colorectal cancer were as follows: a clinical history consistent with the disease, positive colonoscopic results, and routine laboratory analysis parameters. The histopathological examinations were evaluated according to the established clinical criteria (Compton and Gleene, 2004). Controls were hospital patients with various diagnoses (eye diseases, pulmonary diseases, cardiovascular diseases, and neurological disorders, etc.) who never had colorectal cancer or other cancer or any gastrointestinal diseases. For all subjects, we recorded the smoking status in addition to gender, age, and BMI (body mass index, kg/cm²). For smoking status, a person who had smoked at least once a day for >1 year in his or her lifetime was regarded as a smoker and data collected by interviewing. The smoking status was categorized into two groups: nonsmokers (never) and smokers (former and current). There was no significant difference regarding gender, age, and BMI between case (41 women and 52 men; mean age 49.8±16.7 years; mean BMI 26.6±5.2 kg/cm²) and control groups (79 women and 71 men; mean age 59.4±13.2 years; mean BMI 23.5±3.9 kg/cm²). Sixty-five percent of cases and 47% control subjects were smokers.

Venous blood was drawn from subjects and genomic DNA was extracted from whole blood using standard phenol-chloroform extraction protocols. The DNA purity and concentration were tested spectrophotometrically. The samples were stored at −20°C until analysis. Genotyping of CYP1A1*2A (6235T/C: rs4646903), CYP1A1*2C (2455A/G: rs1048943), CYP1A2*1C (−3860G/A: rs2069514), CYP1A2*1F (−163C/A: rs762551), CYP2C9*2 (430C/T: rs1799853), CYP2C9*3 (1075A/C: rs1057910), CYP2C19*2 (681G/A: rs4244285), CYP2C19*3 (636G/A: rs4986893), and CYP2C19*17 (806C/T: rs12248560) variants was performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism methods (Table 1). The temperature was controlled by a programmable heat block (Applied Biosystems; Gene Amp PCR System 9700). Restriction enzymes were obtained from New England Biolabs and Fermentas. All other molecular biology chemicals were obtained from Fermentas and Sigma-Aldrich. Genotyping was done blinded to case-control status. A 10% random sample was genotyped twice for quality assurance, which yielded 100% concordance.

Table 1.

Details of Methodology Used in Genotyping

	PCR conditions
SNP	Primer sequence	Annealing temperature (°C)	Restriction enzyme	Fragment length (bp)
CYP1A1^2A* 6235 T/C (rs4646903)	5′-CAGTGAAGAGGTGTAGCCGCT-3′	63	MspI	343, 209, 134
	5′-TAGGAGTCTTGTCTCATGCCT-3′
CYP1A1^2C* 2455A/G (rs1048943)	5′-AGTAAGTTCAGAGATGCAGAG-3′	60	BsrDI, BseMI	612, 290, 253, 69
	5′-AGATAGCAAAACTGCAGCCAG-3′
CYP1A2^1C* 3860G/A (rs2069514)	5′-GCTACACATGATCGAGCTATAC-3′	64	DdeI	598, 466, 132
	5′-CAGGTCTCTTCACTGTAAAGTTA 3′
CYP 1A2^ 1 F* 163C/A (rs762551)	5′-CAACCCTGCCAATCTCAAGCAC-3′	65	ApaI	1012, 713, 299
	5′-CATGGCTCTGGTGGACTTTTCA 3′
CYP2C9^2* 430C/T (rs1799853)	5′-GTATTTTGGCCTGAAACCCATA-3′	57.3	AvaII, Eco471	454, 397, 57
	5′-ACCCTTGGTTTTTCTCAACTC-3′
CYP2C9^3* 1075A/C (rs1057910)	5′-TGCACGAGGTCCAGAGATGC-3′	58	NsiI, Mph1103I	156, 135, 21
	5′-GATACTATGAATTTGGGGACTTC-3′
CYP2C19^2* 681G/A (rs4244285)	5′-AATTACAACCAGAGCTTGGC-3′	55.3	SmaI	168, 118, 50
	5′-TATCACTTTCCATAAAAGCAAG-3′
CYP2C19^3* 636G/A (rs4986893)	5′-CATTTAGCTTCACCCTGTG-3′	50.5	BsaJI, BseDI	251, 124, 94, 33
	5′-GGGCTTGGTCAATATAGAAT-3′
CYP2C19^17* 806C/T (rs12248560)	5′-GTGAAGCCTGTTTTATGAA-3′	57.3	SfaNI, LweI	164, 140, 24
	5′-GTGGCGCATTATCTCTTA-3′

bp, base pair; PCR, polymerase chain reaction.

Hardy-Weinberg equilibrium was tested by using χ². For analyses of genotype frequencies, the wild-type category (chosen either as the most common wild-type frequency or arbitrarily if both alleles showed similar frequencies) was the reference group. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by conditional logistic regression analyses based on the comparison of genotypes between patients with colorectal cancer and healthy controls, adjusting for the potential confounders, age, sex, and BMI. Statistical analysis was implemented in the Statistical Package for Social Sciences program (version 13.0). Haplotype frequencies were estimated from genotype data by PHASE (version 2) (Stephens and Donnelly, 2003). The distribution of haplotypes in the cases and controls was compared by the χ² test. A two-sided p-value<0.05 was considered to be statistically significant.

Results

We performed a genetic association study on nine functional SNPs of CYP1A1 (*2A, *2C), CYP1A2 (*1C, *1F), CYP2C9 (*2, *3), and CYP2C19 (*2, *3, *17) with CRC in the Turkish population. There were no significant differences between patients with CRC and healthy volunteers for age and sex distribution, and this suggested that the matching based on these two variables was adequate. The genotype distributions were consistent with the Hardy-Weinberg equilibrium in cases and controls for any of the examined SNPs (p>0.05). We then analyzed the differences between cases and controls in the distribution of genotypes (Table 2). For CYP1A1 6235CC, CYP1A2 3860AA, CYP2C9 430TT, CYP2C9 1075CC, and CYP2C19 636AA genotypes, there was no observation both in cases and controls. In addition, the CYP2C19 681AA genotype was only in cases.

Table 2.

Allele Frequencies of Nine Polymorphisms in Patients with Colorectal Cancer and Healthy Subjects

		Frequency
Polymorphisms	Allele	Cases (n=104)	Controls (n=183)	OR (95% CI)	p-Value
CYP1A1 ^*2A 6235T/C (rs4646903)	T	0.841	0.929	T vs. C	0.046
	C	0.159	0.071	2.53 (0.99-6.45)
CYP1A1 ^*2C 2 455A/G (rs1048943)	A	0.101	0.082	A vs. G	0.624
	G	0.899	0.918	1.278 (0.48-3.38)
CYP1A2 ^1C* 3860G/A (rs2069514)	G	0.971	0.973	G vs. A	1.0
	A	0.029	0.027	1.000 (0.02-5.08)
CYP1A2 ^1F* 163C/A (rs762551)	C	0.317	0.306	C vs. A	0.888
	A	0.683	0.694	0.955 (0.53-1.73)
CYP2C9 ^*2 430C/T (rs1799853)	C	0.909	0.921	C vs. T	0.807
	T	0.091	0.079	1.137 (0.42-3.08)
CYP2C9 ^3* 1075A/C (rs1057910)	A	0.913	0.934	A vs. C	0.603
	C	0.087	0.066	1.314 (0.47-3.68)
CYP2C19 ^*2 681G/A (rs4244285)	G	0.861	0.899	G vs. A	0.383
	A	0.139	0.101	1.465 (0.62-3.48)
CYP2C19 ^*3 636G/A (rs4986893)	G	0.966	0.934	G vs. A	0.195
	A	0.034	0.066	0.411 (0.10-1.64)
CYP2C19 ^17* 806C/T (rs12248560)	C	0.846	0.820	C vs. T	0.566
	T	0.154	0.180	0.804 (0.38-1.70)

Bold values are statistically significant.

OR, odds ratio; CI, confidence interval.

In this study, it was observed that CYP1A1 6235T/C was statistically significantly associated with CRC risk. The association was seen with CYP1A1 6235T/C (p=0.046); specifically, patients carrying the C allele in comparison to patients carrying the T allele had a significantly higher risk of disease (OR=2.53; 95% CI=0.99-6.45) (Table 2). To confirm the genotyping results for this variant, which has an association with CRC, the selected PCR-amplified DNA samples (n=4, for each genotype in both cases and controls) were examined by DNA sequencing, and the results were also 100% concordant. On the other hand, genotype distribution of the other studied SNPs in cases and controls did not significantly differ, and thus, these polymorphisms were not associated with CRC risk (Table 2). Similarly, it was observed that smoking was associated with neither of the studied polymorphisms nor the risk of CRC in the study population (p>0.05) (data not shown).

Next, we examined the association between CYP1A1, CYP1A2, CYP2C9, and CYP2C19 haplotypes and CRC risk. The results of haplotype-based analysis are shown in Table 3. For CYP1A1, haplotype T₆₂₃₅-A₂₄₅₅ being the most common and haplotype C₆₂₃₅-A₂₄₅₅ might be associated with the development of CRC (OR=2.70; 95% CI=0.58-5.90; p=0.046). In addition, there was an observed significant haplotype effect for all haplotypes in CYP1A1 variants (p=0.046). The other studied SNPs were not associated with CRC risk in haplotype analysis.

Table 3.

Haplotype Frequencies Constructed by PHASE Algorithm and the Association with Colorectal Cancer Risk

		Frequency (standard error)
Gene	Haplotype ^a	Cases (n=104)	Control (n=183)	OR (95% CI)	p-Value
CYP1A1 ^b	TA	0.760 (0.006)	0.876 (0.004)	1 (reference)
	TG	0.081 (0.006)	0.053 (0.004)	1.85 (0.58-5.90)	0.290
	CA	0.139 (0.006)	0.059 (0.004)	2.70 (0.99-7.38)	0.046
CYP1A2	GA	0.655 (0.004)	0.675 (0.003)	1 (reference)
	GC	0.316 (0.004)	0.298 (0.003)	1.10 (0.60-2.01)	0.764
CYP2C9	CA	0.825 (0.004)	0.858 (0.003)	1 (reference)
	TA	0.088 (0.004)	0.076 (0.003)	1.38 (0.46-4.15)	0.566
	CC	0.083 (0.004)	0.062 (0.003)	1.17 (0.43-3.17)	0.764
CYP2C19	GGC	0.675 (0.005)	0.688 (0.005)	1 (reference)
	GGT	0.152 (0.004)	0.152 (0.005)	1.02 (0.46-2.24)	1
	AGC	0.137 (0.005)	0.083 (0.004)	1.78 (0.70-4.51)	0.222

The order of genotypes is the order of SNPs as listed in Table 2; haplotypes with frequency less than 0.005 not shown.

p-Value for testing all of haplotypes in CYP1A1 gene: cases versus controls=0.02.

Bold values are statistically significant.

Discussion

In this case-control study, we assessed the evidence of association between nine SNPs in four carcinogen-metabolizing genes (CYP1A1, CYP1A2, CYP2C9, and CYP2C19) and CRC risk. From the studied gene variations, two genetically linked polymorphisms of CYP1A1, *2A and *2C, conferred at least 3-fold increases in its catalytic activity (Garte, 1998). Two CYP1A2 variants, *1C and *1F, have been examined to associate with reduced enzyme activity (Nakajima et al., 1999; Sachse et al., 2003). Alleles CYP2C9*2 and *3 have amino acid replacement and have been reported to reduce in vitro and/or in vivo catalytic activities (Haining et al., 1996; Yamazaki et al., 1998; Lee et al., 2002; Yin et al., 2008; Chan et al., 2009). CYP2C19*17 has been associated with increased enzyme function, although CYP2C19*2 and *3 variants have been reported as loss-of-function (Sim et al., 2006; Simon et al., 2009).

The relationships between CRC and the CYP gene family were reported in several studies. As summarized in Table 4, the previous studies investigating the relationship between CRC risk and CYP genotypes, including CYP1A1, CYP1A2, CYP2C9, and CYP2C19 reached different conclusions. Whereas some studies pointed out that CYP polymorphism might confer an increased risk of CRC, others showed no significant role for CYP polymorphism in CRC. Similar to the four studies carried out in Asian populations (Inoue et al., 2000; Giovannucci, 2001; Sandhu et al., 2001; Zheng et al., 2012), we found that the carriers of the C allele of CYP1A1*2A have a 2.53-fold increased risk for CRC compared with the T allele carriers. Opposite to some researchers, the other SNPs in cases and controls did not significantly differ in the present study (Tables 2 and 4). In the Turkish population, this is the first study to report the relationship between nine polymorphisms and CRC to our knowledge. There were only two studies about these CYP variants on CRC. They found similar results to our findings. Tamer et al. (2006) found that the CYP2C19*2 heterozygote genotype was high in patients with gastric and CRC, however, there was no the relationship between the CYP2C19 polymorphism and susceptibility to CRC. Similarly, it was reported by Buyukdogan et al. (2009) that CYP2C9*2, CYP2C9*3, CYP2C19*2, and CYP2C19*3 were not associated with CRC risk. In the present study, haplotype-based associations between CYP variations and CRC were also evaluated. CYP1A1 haplotype C₆₂₃₅-A₂₄₅₅ might be associated with the development of CRC (p=0.046) (Table 3).

Table 4.

Previous Studies on the Association Between the Studied Variants and the Susceptibility to Colorectal Cancer

Gene	Variation	Population (region)	Association with CRC	Study
CYP1A1	6235T/C (rs4646903)	Caucasian, Asian	−	Inoue et al., 2000; Giovannucci, 2001; Sandhu et al., 2001; Zheng et al., 2012
		Japan, Asian	+	Sivaraman et al., 1994; Raunio et al., 1995; Nisa et al., 2010
		Caucasian	−	Landi et al., 2005
	2455A/G (rs1048943)	Non-Hispanic, Caucasian, Asian	+	Hou et al., 2005; Cleary et al., 2010; Nisa et al., 2010; Jin et al., 2011
CYP1A2	−3860G/A (rs2069514)	Asian	−	Yoshida et al., 2007
		Asian, Caucasian	+	Macleod et al., 1998; Nakajima et al., 1999; Sachse et al., 1999; Yeh et al., 2009
	−163C/A (rs762551)	Caucasian	−	Sachse et al., 2003; Küry et al., 2007; Shin et al., 2008; Northwood et al., 2010; Eichholzer et al., 2012
		Korean	+	Landi et al., 2005; Moonen et al., 2005; Bae et al., 2006
CYP2C9	430C/T (rs1799853)	Asian	−	Küry et al., 2007; Cotterchio et al., 2008; Liang et al., 2012
		American	+	Martinez et al., 2001; Sandberg et al., 2002
	1075A/C (rs1057910)	Asian	−	Liang et al., 2012
		Asian	+	Liao et al., 2007
CYP2C19	681G/A (rs4244285)	Caucasian	−	De Jong et al., 2001; Sachse et al., 2002
		Asian	+	Sachse et al., 2002
	636G/A (rs4986893)	Caucasian	−	Sainz et al., 2011
			+	—
	806C/T (rs12248560)	Caucasian	−	Sainz et al., 2011
			+	—

CRC, colorectal cancer; “+” association with CRC; “−”, no association with CRC.

We suggest that the findings would contribute to the literature, even if the small number of subjects with the association examined in the present study could have limited the power to detect gene effects on CRC risk. In general, polymorphisms of individual genes in carcinogen metabolism may not be significantly associated with CRC. However, they may play an important role in individuals having exposure to known carcinogens such as environmental pollutants and steroid hormones. Future studies of CRC etiology should take into consideration both environmental exposure and genetic susceptibility (Firozi et al., 2002).

In this study, the frequencies of CYP1A2*1C, CYP1A2*1F, CYP2C9*2, CYP2C9*3, CYP2C19*2, and CYP2C19*3 alleles with reduced activity were 0.972, 0.690, 0.916, 0.927, 0.885, and 0.946, respectively (data not shown). The frequencies of CYP1A1*2A, CYP1A1*2C, and CYP2C19*17 alleles with increased activity were 0.897, 0.911, and 0.819, respectively (data not shown). From the point of activities of CYPs, Turkish people seem to be poor metabolizers when assessing the sensitivity of the population to therapeutic drugs and environmental toxicants regardless of the relationship with diseases. Contrary to its relatively high frequency (18-28%) in the Caucasian and African populations, the CYP2C19*17 variant allele is very rare in Asians (1-5%). Whereas CYP2C19*2 is the most common defective allele in all racial groups, CYP2C19*3 is extremely rare in populations other than Asians (Gumus et al., 2012). The frequencies of CYP1A2*1C in Caucasians were quite different from those in Koreans or other Asians (Bae et al., 2006). CYP1A1*2A and CYP1A1*2C variant alleles are more frequent in Asian (Japanese) than Caucasian populations. Among white populations, CYP2C9*2 and CYP2C9*3 variants are of significance, with allelic frequencies of 0.08-0.14 and 0.04-0.16, respectively. In Africans and Asians, both variants are much less frequent and CYP2C9*2 has not yet been detected in Asians. The CYP2C9*3 allele has been detected in many ethnic populations with a minor allele frequency ranging from 0.5 to 16.2% (Xie et al., 2002; Afsar et al., 2010). As to CYP2C9*2, CYP2C9*3, and CYP2C19*17 alleles, Asian populations possess similar allelic frequencies to the Turkish population. On the other hand, they resemble Caucasians in the other alleles.

In conclusion, we suggest that the CYP1A1*2A allele plays an important role in the development of CRC especially in the Turkish population. The results in this article represent an initial step in our effort to understand the susceptibility to develop CRC and will be of importance in providing an understanding of epidemiological studies that correlate therapeutic approaches and etiology of CRC, especially in Turkish patients.

Footnotes

Acknowledgment

The authors wish to thank all the subjects who volunteered to participate. This work conducted in accordance with the Declaration of Helsinki (1964) was supported by the Research Fund of Istanbul University (15607/23505/24985).

Author Disclosure Statement

The study was approved by the ethics committee of Istanbul University and all participants provided written informed consent.

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