Polymorphisms of xenobiotic-metabolizing and transporter genes and the risk of gastric and colorectal cancer in an admixed population of Brazil.

Colorectal (CRC) and Gastric (GC) cancers are responsible for considerable morbidity and mortality worldwide. In the North region of Brazil, these neoplasms are among the three most incident and aggressive types of cancer, constituting a severe problem of public health. Single Nucleotide Polymorphisms (SNPs) of xenobiotic metabolism and transporter genes may play a role in individual response to exposure to some of the compounds implicated in the cancer susceptibility. However, few studies have demonstrated the role of polymorphisms of xenobiotic metabolism and transporter genes in the susceptibility to CRC or GC in admixed populations. In this context, the study of variation in the activation and detoxification processes of xenobiotics may help to clarify the development of either GC or CRC in substructured populations, providing new insights about predictive diagnostic criteria in oncological investigations. We performed an association study using 31 SNPs in 15 xenobiotic metabolism and transporter genes. The study was carried out in 121 CRC and 95 GC cases and 140 control individuals from Belém, a city which comprises a population with high levels of miscegenation, located in the Brazilian Amazon. Samples were genotyped using the QuantStudio™12K Flex Real-Time PCR System. Due to the high level of genetic admixture in the studied population, we applied a panel of 61 Ancestry Informative Marker standardized by our research group in an earlier study. Statistical analyses were performed in SPSS v.20.0 and Structure v.2.3.4 The results revealed a significant association between the increased CRC or GC risk and polymorphisms of ABCG2 (rs2231142) and DPYD genes (rs17116806, rs1801159). Our data suggest that polymorphisms in xenobiotic-metabolizing and transporter genes may be relevant to the susceptibility to both CRC and GC.


Conclusions
Our data suggest that polymorphisms in xenobiotic-metabolizing and transporter genes may be relevant to the susceptibility to both CRC and GC.

Introduction
Gastric and colorectal cancers are the main causes of cancer deaths worldwide [1] [2]. In northern Brazil, the incidence of these types of neoplasm is relatively high in comparison with the average rate observed in other regions of the country [2]. The carcinogenesis of colorectal (CRC) and gastric (GC) cancers is still unclear. A number of studies have indicated that the exposure to several environmental or lifestyle risk factors have been identified for CRC and GC, such as obesity, sedentary behavior, high intake of hypercaloric foods, diets rich in fat and poor in fiber, tobacco smoking and alcohol consumption [3] [4] [5] [6]. In addition, the genetic makeup is hypothesized to play an important role in the sporadic form of these neoplasms. Single nucleotide polymorphisms (SNPs) are one of the most common types of genetic variations in the human genome, and from a clinical perspective, are potential diagnostic and therapeutic biomarkers in many cancer types.
SNPs in xenobiotic metabolizing and transporter genes are known to modify the activity of their encoded enzymes, resulting in an increase or decrease predisposition (depending on the pathway of each enzyme) for the development of gastric or colorectal neoplasms [6] [7][8] [9]. However, few studies have demonstrated the role of polymorphisms in xenobiotic metabolism and transporter genes in the susceptibility to CRC or GC in admixed populations, such as the Brazilian one. This factor is highly relevant because if subjects of a genetic association study are sampled from two or more subpopulations for which the frequencies of alleles and traits differ; spurious associations may arise due to the population substructure [10].
Given this, the goal of this work was to investigate the role of 31 polymorphisms in 15 xenobiotic metabolizing and transporter genes and the influence of genetic ancestry in CRC and GC susceptibility in an population from the Brazilian Amazon region with a high degree of interethnic admixture.

Study participants
In this study, we included three groups: (1) 95 individuals with GC diagnosis; (2) 121 individuals with CRC diagnosis; and (3) 140 cancer-free individuals. The cancer-free individuals did not have personal or familial histories of any type of cancer and they did not show any symptoms or signs of cancer. All individuals resided in Belém, which is a city located in the northern region of Brazil. All patients were treated at two local hospitals: Ophir Loyola Hospital and University Hospital João de Barros Barreto. All selected participants were from the same geographical area and groups of similar socioeconomic status.

Ethics, consent and permissions
All participants gave written informed consent to participate and publish this investigation. The protocol used in the study was approved by the Ethics Committee of the

Analysis of genetic ancestry
To avoid misinterpretations caused by a high level of genetic admixture in the studied population, we applied a panel of 61 ancestry-informative markers as described previously [11]. The ancestral populations included representatives of three major ethnic groups, including Amerindian from the Brazilian Amazon, African and European populations. More details on these populations can be found in [12]. The genomic ancestry was performed in

Demographic characteristics
A total of 356 individuals were analyzed in the present study. Demographic data about case and control groups are presented in Table 1 and Table 2, for GC and CRC, respectively. Regarding sex, the CG case group was predominantly composed of men.
Differently, the CRC case group had a predominance of women. Significant differences were found between the two groups (GC and CRC) related to age and sex, therefore, these variables were controlled for in the logistic regression. Genomic ancestry analysis for the CG group showed 45% of European, 22% African and 33% Amerindian ancestries.
The same analysis for CRC patients demonstrated 50% European, 30% Amerindian, and 20% African ancestries. No significant association was found in the genomic ancestry analysis for GC or CRC.

Quality control and Genotyping
A total of 31 polymorphisms were investigated in this study (Table S1). Six variants were excluded from our analysis: nine polymorphisms were out of Hardy-Weinberg Equilibrium and three marker was above the limit of 15% of missing data in Minor Allele Frequency (MAF) analysis. The remaining 19 SNPs were analyzed. Table S2 shows the HWE and MAF data of the 31 polymorphisms and their study status (included or excluded).

Analysis of associations with susceptibility to GC and CRC
Significancy associations were found with the investigated polymorphisms in relation to an increased GC risk ( Table 3 (Table S3 and Table S4).

Discussion
Colorectal and gastric cancers are among the leading causes of death worldwide [1]. In Brazil, estimates for 2018-2019 from the National Cancer Institute (INCA) indicate more than 417000 new cases of these malignant neoplasms [2]. In the northern region of the country, where our target population resides, GC is the second most prevalent type of neoplasia in men and the fourth in women, whereas the CRC is the fourth most incident in men and the third in women [2].
Although the causes of cancer have not yet been completely elucidated, studies have shown that a large group of mutagen-carcinogenic agents require metabolic activation to allow them to bind to DNA, RNA and proteins; therefore, several environmental components are strong risk factors for GC and CRC development [13] [14]. Tomasetti (2017) demonstrated that the percentage of influence of external factors on GC and CRC susceptibility is 55% and 26%, respectively [15], thus, association studies in genetic pathways related to the metabolism and transport of environmental risk factors have aided to better understand the carcinogenesis process in several organs [16][17] [18].
Most genetic association studies on cancer are investigations about tumor suppressors or oncogenes. Our analysis, however, proposes the study of xenobiotic-metabolizing and transporter genes, which can also modulate the susceptibility to different types of cancer.
Additionally, few of these studies have been performed in admixed populations such as the Brazilian one. The Brazilian population is composed, mainly, by the admixture of Amerindian, European, and African ancestral populations [11] [12]. Case-control studies in such admixed populations may be influenced by the variation of allelic frequencies of markers found in each different ethnic groups, which may create biases in the outcomes, especially in investigations of susceptibility to complex diseases, such as cancer [19] [20].
In this case, estimates of ethnic admixture must be taken into consideration. Our investigation of genomic ancestry analysis was based on the set of 61 AIMs used in previous genetic studies of complex human diseases [11]. Our research group have performed some studies that demonstrated the influence of population substructure present in the northern region of Brazil with several types of cancer, for example childhood B-cell Leukemia [20] [21]and breast and gastric cancer [7]. In the present study, however, no significant difference was found in the ethnic profiles between the case and control groups.
In this study, the polymorphisms shown to be associated with colorectal or gastric cancers are related to xenobiotic metabolism. A major group of mutagenic-carcinogenic agents requires metabolic activation to enable them to bind to DNA, RNA, and proteins.
Therefore, genetic polymorphisms in these xenobiotic-metabolizing and transporter genes may account for the individual variation observed in the individual response to exposure [18][19] [20]. where this gene is hypo-expressed [26]. Supporting this finding, the study by Liu et al.
(2010) demonstrated a differential expression of the BCRP at each carcinogenesis stage [27]. For the promotion of carcinogenesis, the expression of BCRP would be decreased to allow the accumulation of genotoxins and nitric oxide, but in the more advanced stages, BRCP can be positively expressed to efficiently transport chemotherapeutic drugs out of the cancerous cells, protecting them. Therefore, the assessment of differential expressions of BRCP may lead the scientific community to create a new method to evaluate progression, metastasis and to predict therapeutic response of colorectal cancer.
The results also corroborate with the findings of Wang and colleagues, who, through an in vitro experiment, demonstrated that the ABCG2 gene shows dysregulated expression in GC tissues and cells [28]. In this study, the high expression of ABCG2/BRCP was correlated with advanced stages and poor prognosis of GC. Deregulated expression of the ABCG2 gene has further been pointed as a promoter factor to GC that affects cell proliferation and induces resistance to cellular apoptosis. These results corroborate with our analyses, confirming the role of SNPs of the ABCG2 gene in the initiation and promotion of GC.
Polymorphisms in the DPYD gene have also been shown to play a role in gastric or colorectal carcinogenesis. Our data demonstrated that the rs1801159 and rs17116806 polymorphisms are associated with a higher risk of GC susceptibility. Additionally, the rs17116806 has been also associated to an increased susceptibility to colorectal carcinogenesis, demonstrating that the same polymorphism can act on tumorigenesis in different tissues. International regulatory agencies, such as the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA), strongly recommend the monitoring of polymorphisms on the DPYD gene for evaluation of therapeutic response in fluoropyrimidine-based treatment [29] [30]. Nevertheless, few investigations have studied genetic variations in the DPYD gene regarding cancer susceptibility, thus, the variety of clinical manifestations resulting by mutations in DPYD is still not well understood [31]. To date, there are no genetic association studies with the rs1801159 and rs17116806 polymorphisms and the susceptibility to the neoplasms investigated hereby. Therefore, this study is the first to investigate the correlation between DPYD polymorphisms and the susceptibility to GC and CRC in the Brazilian Amazon population.
Previous studies have also shown that modifications of the pyrimidines homeostasis and the products of their degradation can result in a number of phenotypic manifestations, including neurological disturbances [32] and gastrointestinal disorders [31]. Tanaka