Association of CHEK2 I157T and SULT1A1 R213H genetic variants with risk of sporadic colorectal cancer in a sample of Egyptian patients

Recent research proposed an association between functional defects involving CHEK2 I157T and SULT1A1 R213H variants and increased incidence of several types of cancer. A total of 86 unrelated colorectal cancer patients attending the Surgical Oncology Department were recruited in the study. The second group of 152 healthy age- and sex-matched volunteers were included as controls. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) was applied for genotyping. Chi-square test was applied to compare genotype and allele frequencies in the studied groups. The purpose of the present study was to evaluate the association between CHEK2 I157T and SULT1A1 R213H polymorphisms and colorectal cancer. No significant differences in genotypes were detected between cases and controls in the present study for both CHEK2 I157T and SULT1A1 R213H polymorphisms (χ2 = 1.839; P = 0.399/χ2 = 2.831; P = 0.243), respectively. Likewise, discrepancies in allele frequency for the wild-type or mutant alleles were non-statistically significant in CHEK2 I157T and SULT1A1 R213H (χ2 = 1.231; P = 0.267/χ2 = 0.180; P = 0.671), respectively. Results of the current study propose that CHEK2 I157T and SULT1A1 R213H polymorphisms are not associated with CRC development in Egyptian population. Further future studies on the functional implications of these polymorphisms are strongly recommended.


Background
Colorectal cancer (CRC) ranks third among the most frequent cancers and the fourth most common etiology of global cancer fatalities. CRC has become a leading health problem based on the fact that new CRC cases diagnosed yearly exceed a million worldwide and death represents the outcome in more than 30% of them [1]. Nowadays, CRC is considered in many countries as a major community health burden. Therefore, understanding the etiologies of this cancer is an area of extreme importance.
CRC development is a complex process involving the interplay between many factors. Both gene mutation and environmental factors have a crucial role in CRC development [2].
A multitude of evidence highlights the crucial genetic role in CRC risk [2]. Multiple reports concluded that inherited factors affect DNA repairing capacity which may result in cancer development [3][4][5]. Hence, subjects with hereditarily impaired DNA repairing capability are usually related to increased cancer risks [6,7].
The Checkpoint kinase 2 (CHEK2) gene is recognized as a breast cancer susceptibility gene [8], and multiple germ-line variants may be associated with an increased risk of colorectal, prostate, thyroid, and renal cancer in certain populations [9,10]. CHEK2 encodes the human homologue of the CDP-diacylglycerol synthase 1 (Cds1) and RADiation sensitive (RAD53) checkpoint kinases and serves a crucial role in DNA damage checkpoint pathway. Following DNA damage exacerbated by ionizing radiation, CHEK2 activation is triggered by the ataxia-telangiectasia mutated (ATM) protein and thereafter, phosphorylates multiple substrates, including p53, Breast cancer type 1 susceptibility protein (BRCA1), Mouse Double Minute 2(Mdm2), Cell division cycle 25 A (Cdc25A), and Cell division cycle 25 C (Cdc25C), causing activation of DNA repairing pathways, cell cycle arrest, or apoptosis. Activated Chk2 was detected in early precursor specimens of urinary bladder, lung, breast, and colorectal carcinoma (but not in normal specimens) before genomic instability occurs and hence malignant transformation [11], raising a suggestion that DNA damage checkpoints are activated early in tumorigenesis stages. Hence, CHEK2 mutations, or other genes involved in the ATM-CHEK2-p53 pathway, may permit tumorigenic cells evasion of normal cell cycle checkpoints, causing aberrant cell proliferation and survival, decreased genomic stability, and eventually, tumor progression [12].
Heterozygosity of I157T (rs17879961) in CHEK2 gene that results in the substitution of an isoleucine (Ilu) for a threonine (Thr) is shown to reduce the functional pool of CHEK2 protein by forming heterodimers with the wild type [13] leading to impaired binding to BRCA1, CDC25A, and p53. As I157T is localized in a functionally important domain of CHEK2, and the protein with this mutation has been proven deficient in its ability to bind p53 and BRCA1 and to bind and phosphorylate Cdc25A [14]. A functionally defective CHEK2 variant I157T was suggested to be associated with increased breast cancer risk, together with prostate cancer and a number of other cancers [9,13,15]. Sulfotransferases (SULTs) serve a crucial function in the normal physiological processes in addition to malignant transformation [16]. In humans, three members of the phenol sulfotransferase family exist (SULT1A1, SULT1A2, and SULT1A3). SULT1A1 is expressed in the liver together with multiple extrahepatic sites like colon mucosa and plays a role in various xenobiotic detoxication pathways [17]. It serves a vital function in the metabolism and bioactivation of numerous environmental and dietary mutagenic factors, including heterocyclic amines involved in colorectal carcinogenesis together with various cancers [18]. Consequently, SULT1A1 gene may represent a suitable candidate for genetic CRC studies. The SULT1A1 gene resides on chromosome 16p12.1-p11.2 [19]. A polymorphism R213H(rs1042028) in the SULT1A1 gene was recognized in the coding sector at nucleotide 638 (a G to A transition). This base replacement leads to amino acid sequential change from arginine to histidine (Arg213His), causing a reduction in enzyme activity [20].
The association between the genetic variants CHEK2 I157T and SULT1A1 R213H and colorectal cancer susceptibility still needs to be further explored. The aim of the present study was to evaluate the association between CHEK2 I157T (rs17879961) and SULT1A1 R213H (rs1042028) polymorphisms and CRC in an Egyptian population.

Population studied
The study was conducted on 86 sporadic unrelated (nonconsanguineous) CRC patients attending the Surgical Oncology Department, from March 2018 to January 2019. The diagnosis and confirmation of colorectal cancer were done based on endoscopic and histopathological results. 152 healthy age-and sex-matched volunteers were recruited as controls. The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics of research committee. Informed written consents were obtained from all participants after explanation of the purpose of the study.

History, clinical evaluation, and blood sampling
All participants were subjected to careful medical and family history taking. Data collected from CRC patients included tumor site, Dukes stage (stage A: limited to muscularis propria, stage B: extending beyond muscularis propria, stage C: nodes involved and stage D: distant metastatic spread) [21,22] and tumor grade (well differentiated (low grade), moderately differentiated (intermediate grade) and poorly differentiated (high grade)). Venous blood samples for the molecular analysis were collected in EDTA tubes from all patients and controls.

Molecular study
Genomic DNA was extracted from peripheral blood leukocytes by salting out technique [23]. TheCHEK2 gene polymorphism I157T (rs17879961)was investigated by Polymerase Chain Reaction (PCR) amplification of genomic DNA followed by Restriction Fragment Length Polymorphism (RFLP); according to the method previously reported by Cybulski et al. [24].
Amplification via Veriti Thermal Cycler (Applied Biosystems) was performed using the following primer sequences: 5'-ACC CAT GTA TCT AGG AGA GCTG-3' (forward) and 5'-CCA CTG TGA TCT TCT ATG TCT GCA -3' (reverse). The PCR reaction was performed in a total volume of 50 ul including 25 ul 2X PCR master mix (0.05 U/μL Taq DNA polymerase, reaction buffer, 4 mM MgCl2, 0.4 mM of each dNTP) (Thermo-scientific), 1uM each primer, 5 ug DNA and nuclease free water up to 50 ul. The PCR program included: An initial denaturing step of 4 min at 95 °C followed by 30 cycles of 94 °C for 30 s, 57 °C for 1 min for annealing, and 1-min elongation at 72 °C, with a final elongation step of 72 °C for 7 min. The PCR products were digested with pst1 restriction enzyme (fast digest) (Thermoscientific)according to the manufacturer's protocol. For the SULT1A1 gene polymorphism R213H(rs1042028) genotyping, the same method was applied except for an annealing temperature of 55C with the following primers sequences: 5'-GGG TCT CTA GGA GAG GTG GC-3' (forward) and 5'-GCT GTG GTC CAT GAA CTC CT-3' (reverse) [25]. The amplified segments were digested with Hha1 restriction enzyme (fast digest) (Thermoscientific) according to the manufacturer's instructions.

Analysis of rs17879961 and rs1042028 polymorphisms
The digested PCR products were resolved by electrophoresis on 3% agarose gel stained with ethidium bromide for 20 min at 200 V and were sized with reference to a 50-bp DNA ladder.

Statistical analysis
Data were analysed using IBM SPSS software package version 20.0. (Armonk, NY: IBM Corp). The Kolmogorov-Smirnov, Shapiro and D'agstino tests were used to verify the normality of distribution of variables, Comparisons between groups for categorical variables were assessed using Chi-square test (Fisher or Monte Carlo). Student t-test was used to compare two groups for normally distributed quantitative variables. Odd ratio (OR) was used to calculate the ratio of the odds and 95% Confidence Interval of an event occurring in one risk group to the odds of it occurring in the non-risk group. Regression analysis was applied to detect the most independent/ affecting factor for affecting cases. Significance of the obtained results was judged at the 5% level.

Results
A total of 86 sporadic unrelated CRC patients and 152 controls were recruited in this study. There was statistical difference between cases and controls regarding the smoking status ( MC p < 0.001*). Among the CRC cases, 74.4% of patients suffered from colon cancer, 21.1% from rectal cancer and 3.5% with rectosigmoid cancer. Regarding histologic differentiation, 19.8%, 68.6%, and 11.6% of CRCs were classified as low grade, intermediate grade, and high grade, respectively. The Dukes A, B, C, and D stages were 0%, 1.2%, 2.3%, and 96.5%, respectively (Table1).
The allelic distribution of rs17879961 and rs1042028 polymorphisms were in Hardy-Weinberg equilibrium (HWE) among the cases and the control groups (Tables 2, 3). The genotype and allele frequencies for the two single nucleotide polymorphisms (SNPs) between the cases and the control group are shown in Table 4 and Figs. 1 and 2. No significant differences in genotypes were detected  between the cases and control in the present study for both rs17879961 and rs1042028 polymorphisms (χ 2 = 1.839; P = 0.399/χ 2 = 2.831; P = 0.243), respectively. Likewise, discrepancies in allele frequency for the wild-type or mutant alleles were non-statistically significant in rs17879961 and rs1042028 (χ 2 = 1.231; P = 0.267/χ 2 = 0.180; P = 0.671) respectively. No significant association was found between rs17879961 and rs1042028 and CRC susceptibility in different models (Table 5). Logistic regression analysis showed that age, gender and smoking are risk factors for colorectal cancer (Table 6). No significant association was detected between rs17879961 and rs1042028 polymorphisms and various clinicopathologic parameters of CRC (Table 7).

Discussion
The CHEK2 is a serine/threonine protein kinase that plays a crucial role in DNA damage response and the regulation of cell cycle. CHEK2 protein consists of three functional domains: SQ/TQ cluster domain (SCD), forkhead associated (FHA) domain, and kinase domain. The SCD is a target for (auto)phosphorylation and is thus crucial for activation and regulation of CHEK2 functions. The FHA domain is in charge of substrate specificity of CHEK2 through phosphorylation dependent proteinprotein interactions and plays a role in the activation process of CHEK2 [26]. The I157T (c.470T > C) (rs17879961) variant resides in a phosphopeptide recognition domain (FHA domain), whose normal function is to enable protein formation of homodimers as well as substrate binding. Protein FHA domain mutation has been shown to interfere with complex formation with key substrates-p53 and Cdc25A. Since its ability of dimer formation remains intact, CHEK2 I157T has been postulated to reduce the pool of wild-type CHEK2 protein via a dominant-negative interaction [13]. CHEK2 gene is evolutionarily conserved with few germline variants described. CHEK2 I157T, together with 1100delC, were originally recognized in Li-Fraumeni syndrome families and speculated to be the etiological factor of the disease. Other studies showed that such variants may exert their effects through low-penetrant, rather than high-penetrance multiorgan tumor-susceptibility alleles [27]. The distribution of CHEK2 I157T among European populations appears quite heterogeneous which may be attributed to migration and founder effects. Its frequency is highest in Russia 7.6% [28] and Finland 5.3% [29] followed  by the Czech Republic 2.5% [28,30]. In Italy it was not detected in any of 365 studied unrelated males [31]. In Germany, it was detected in 0.6% of controls and in Bella Russia in 1.3% [32]. Despite marked discrepancy in allele frequency, the variant has shown consistent association with CRC risk in various reports [9,29,33,34]. In the present study, no association was detected between CHEK2 I157T polymorphism and CRC risk, which is consistent Konstantinova et al. [35] who published his study on Bulgarian population, and was the first to report Fig. 1 The genotype distribution of rs17879961 polymorphisms among cases and control a population in which CHEK2 I157T does not increase CRC risk, although a very low penetrance effect could not be excluded. The variation between the different studies may be attributed to ethnic discrepancies, differences in sample size, variability in the inclusion criteria, and the study techniques.
One study correlated CHEK2 I157T with colon cancer tumor characteristics [29], but prevalence in any of the grade or stage subclassification was not detected. In the study conducted by Konstantinova et al. [35], no such prevalence was detectable. Instead, they reported a relation to two other tumor criteria-histological type   (p = 0.26) and multiple polyps presence (p = 0.28), however, the corresponding patient groups were limited yielding non statistically significant results. This goes in agreement with the present study, where no significant association was detected between rs17879961 and rs1042028 polymorphisms and various clinicopathologic parameters of CRC. Numerous lines of evidence suggest a crucial genetic role in cancer risk determination, and association studies are important in the search for susceptibility genes related to cancer [36]. In the current study, no significant association between the SULT1A1 R213H (rs1042028) polymorphism and CRC was detected among cases and control groups.
Results of the present work suggest that SULT1A1 R213H polymorphism shows no association with CRC development. SULT1A1 is related to activation and detoxification of various carcinogens, as well as different hormones regulation [18]. G to A transition at nucleotide 638 in SULT1A1 gene has been shown to induce an Arg to His swap associated with reduced enzyme activity [20]. Multiple reports concluded that SULT1A1 HH genotype was related with an increase in cancer risks, namely breast, lung and esophageal cancers37-39. These study results support the hypothesis that low SULT1A1*H allozyme activity reduces protection against environmental and or dietary carcinogens. However, our study did not confirm the association between SULT1A1 and CRC risk. Raftogianis et al. study [20] suggested that this polymorphism is related to low enzymatic activity. Enzyme activity was recorded using platelet preparations. However, the use of platelets in the determination of a specific enzymatic activity can be misleading due to methodological inability to distinguish which enzyme is responsible for the studied activity [40]. In addition, initial modelling reports showed that such polymorphism has no direct effect on the binding plot of the substrate or the universal sulphonate donor 39phosphoadenosine-59-phosphosulphate (PAPS) [41]. Supplemental future research on the functional consequences of SULT1A1 R213H polymorphism are strongly recommended.
The results of the present study are consistent with a meta-analysis demonstrating lack of association between the SULT1A1 R213H polymorphism and CRC, specifically in Caucasian population [42]. However, such metaanalysis results should be cautiously interpreted because SULT1A1 R213H polymorphism prevalence may vary with various CRC subtypes; therefore, analysis classified by variable CRC subtypes may yield more accurate results.
The present study also goes in concordance with Chung Fai Won et al. [40] research on Australian population, reporting insignificant correlation between the SULT1A1 R213H polymorphism and CRC development. Moreover, substrate assays research reported no functional difference between R213 SULT1A1 and H213 SULT1A1 in sulphonating the model substrate p-nitrophenol, the sulphonate donor PAPS or the drug substrate paracetamol.

Conclusion
We conclude that CHEK2 I127T (rs17879961) and SULT1A1 R213H (rs1042028) polymorphisms might not be associated with CRC development in Egyptian population; however, larger scale studies are recommended.