The Effect of the CYP1A1*2A Allele on Colorectal Cancer Susceptibility in a British Population

Introduction

Colorectal cancer (CRC) has been an public health problem for many years because of increasing incidence and high mortality rate despite some overall improvements in diagnosis and treatment. Indeed, it is well known that CRC has a complex etiology that has still not been well defined (Siegel et al., 2012). Individual genetic background has been reported to contribute to CRC as well as several anthropometric, lifestyle, and dietary risk factors such as age, smoking, excessive sugar intake, processed meat, and alcohol consumption (Hsing et al., 1998; Haggar and Boushey, 2009; Huxley et al., 2009). Although some candidate genes for CRC susceptibility have been identified, the syndromes caused by mutations on high-penetrant genes (e.g., Lynch syndrome) contribute to less than 5% of all CRC cases. Thus, genetic polymorphisms affecting carcinogen biotransformation or DNA repair either alone or interacting with dietary and environmental factors could affect the carcinogenesis process (Peters et al., 2015).

CYP1A1, a member of CYP1 enzyme family, plays a crucial role in metabolism of many endogenous and exogenous substances (Yu et al., 2014). The CYP1A1 gene has been suggested to be associated with tobacco-related cancers, including CRC. The gene has been studied extensively because of its key role in the metabolic activation of polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (Guengerich and Shimada, 1998; Hou et al., 2005; Yoshida et al., 2006). The CYP1A1*2A (6235 T/C, rs4646903, MspI) allele has been reported to be associated with increased risk of CRC in some studies, but overall, previous results are conflicting (Sivaraman et al., 1994; Inoue et al., 2000; Landi et al., 2005; Nisa et al., 2010). It has been revealed that the CYP1A1*2A allele frequency displays large differences between ethnic groups such as Asians, Caucasians, and Africans. Therefore, the overall effect of CYP1A1*2A on cancer susceptibility may vary between populations (Garte et al., 2001). In this study, we examined a possible association between the CYP1A1*2A allele and CRC in a British population.

Materials and Methods

This study was conducted as a prospective case-control study with British participants from Newcastle and North Tyneside Hospitals over approximately a 1 year time period. Two hundred cases diagnosed with histologically confirmed adenocarcinoma of the colon or rectum and absence of colitis and known genetic predisposition such as polyposis coli and hereditary nonpolyposis CRC were included. Two-hundred and fifty-four age-sex-matched controls who had no history of CRC or colonic adenomatous polyps were selected randomly from hospital patients. Of the participants, 99% were Caucasian and all participants were provided informed consent. Smokers were categorized into two groups: (i) nonsmokers and those who had quit smoking more than 5 years ago (ii) current smokers and those who had quit smoking less than 5 years ago (Welfare et al., 1997).

Genomic DNA was extracted from venous blood by using standard phenol-chloroform extraction protocols. The DNA concentrations were measured spectrophotometrically. Genotyping of CYP1A1*2A was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Each DNA sample was amplified by the following primer mixture: 5′-CAgTgAAgAggTgTAgCCgCT-3′ (forward) and 5′-TAggAgTCTTgTCTCATgCCT-3′ (reverse). Initial denaturation was performed for 60 s at 94°C followed by annealing for 60 s at 63°C in 35 cycles and final extension for 60 s at 72°C. The PCR products were incubated with the restriction enzyme MspI for >1 h at 37°C, which resulted in digestion fragments at 343, 209, and 134 base-pair lengths. The fragments were run on 2% agarose gels stained with ethidium bromide and observed on a UV transilluminator. In each run, controls of known wild-type, heterozygous, and mutant genotype were included to check the accuracy that yielded 100% concordance.

Hardy-Weinberg equilibrium was tested by using Chi-square (χ²) test in case and control subjects. Genotyping analysis was performed based on a dominant model and the wild type was chosen as reference group. Data comparisons were done by using Fisher's exact test. The odds ratios (ORs) and 95% confidence intervals (CIs) were estimated to evaluate the association between cases and controls. Statistical analysis was implemented using the Statistical Package for Social Sciences (SPSS, version 21.0) and GraphPad Prism (version 5.0a) programs. A two-sided p < 0.05 was considered to indicate a statistically significant difference.

Results

Genotype distribution was found to be consistent with the Hardy-Weinberg equilibrium within both cases and controls (p > 0.05). Genotype distribution and estimated OR with 95% CI are summarized in Table 1. Homozygosity for the CYP1A1*2A variant (CC) was observed in only three cases and was not observed in controls. Genotyping analysis based on a dominant model showed that individuals with C allele had about 1.5-fold risk of developing CRC (OR = 1.57; 95% CI = 0.90-2.73); however, the association did not reach statistical significance (p = 0.12).

We also evaluated a possible genotype-smoking association. Fifty-five of the 200 cases were smokers and 43 of these smokers were homozygous for the CYP1A1*2A wild type (TT). We also evaluated a possible genotype-smoking association and found that C allele-carrying individuals with smoking within the past 5 years had a significant risk of CRC (OR = 2.28; 95% CI = 1.07-4.86; p = 0.04), but no significant risk was detected in the never or former smokers.

Discussion

CYP1A1 is one of the most studied genes because of its pivotal role in bioactivation of various number of exogenous substances to their carcinogenic derivatives (Go et al., 2015). Garte et al. (2001) reviewed a number of previous studies and found that CYP1A1 allele frequencies showed large differences across different populations. The CYP1A1*2A variant is about 10 times more frequent in Japanese than in Caucasians (Kawajiri et al., 1993). So, evaluation of an association between CYP1A1*2A and cancer susceptibility is more difficult and needs large number of participants in Caucasians.

Our results showed that CYP1A1*2A was not associated with CRC development in British population. Similar results were reported in Caucasians, Lebanese, Scottish, British, and mixed population mostly consisted of non-Hispanic whites (Sachse et al., 2002; Ye and Parry, 2002; Slattery et al., 2004; Little et al., 2006; Darazy et al., 2011). In contrast, Özhan et al. (2014) observed that CYP1A1*2A is associated with increased risk of CRC in the Turkish population and found that C allele-carrying patients had 2.5-fold higher risk than T allele-carrying patients. Sivaraman et al. (1994) reported a sevenfold increased risk of CRC with CYP1A1*2A allele carriage in a small group of Japanese people living in Hawaii. Subsequent studies showed that CYP1A1*2A was not associated with CRC in Japanese (Inoue et al., 2000; Yoshida et al., 2006; Nisa et al., 2010). The controversial results on the Japanese populations are possibly caused by number of studied individuals. No other study has been conducted in a Turkish population; therefore, it is difficult to make extensive comparisons. As it has been reported that the CYP1A1*2A allele distribution varies by geographic region in the same ethnic group (Garte et al., 2001), differing results between Turkish population and Western Europeans may be explained by regional factors as well as possibly by dietary habits.

Tobacco-related cancers, including CRC, are mainly caused by reactive metabolites of PAHs, for which biotransformation by the CYP1A1 enzyme is one of the three major metabolic pathways (Moorthy et al., 2015). In our study, the genotype-smoking relationship for CRC susceptibility was also evaluated. It was found that C allele-carrying individuals with smoking had 2.5-fold increased risk of CRC. Similarly, Slattery et al. (2004) observed that C allele-carrying current smokers among men had 2.5-fold increased risk of colon cancer. Conversely, in other studies, smoking was found not to be associated in both case-control comparisons and genotype-smoking evaluation (Inoue et al., 2000; Yoshida et al., 2006; Nisa et al., 2010; Özhan et al., 2014). The data from this new study should be useful for understanding CRC etiology and in identifying individual risk of developing CRC.