Lack of Influence of the SMAD 7 Gene rs 2337107 Polymorphism on Risk of Colorectal Cancer in an Iranian Population

Colorectal cancer (CRC) as the third most common cancer brings about the fourth cause of worldwide cancer mortality (Jemal et al., 2011; Shemirani et al., 2011). According on World Health Organization (WHO) reports, CRC is one of the common diseases in an Asian population (Moghimi-Dehkordi et al., 2012; Haerian et al., 2014). It is noticeable that incidence of this cancer has been rising in an Iranian population during recent decades (Azadeh et al., 2007; Milanizadeh et al., 2013). Thereby, the importance of CRC as a major worldwide health issue is also increasing in our population (Haghighi et al., 2009). Although numerous risk factors and causes have been taken into account for CRC, genetic component has a great contribution on CRC development. To emphasize this fact, nearly 35% of total cases have been reflected by twinand family based studies (Lichtenstein et al., 2000).


Introduction
Colorectal cancer (CRC) as the third most common cancer brings about the fourth cause of worldwide cancer mortality (Jemal et al., 2011;Shemirani et al., 2011).According on World Health Organization (WHO) reports, CRC is one of the common diseases in an Asian population (Moghimi-Dehkordi et al., 2012;Haerian et al., 2014).It is noticeable that incidence of this cancer has been rising in an Iranian population during recent decades (Azadeh et al., 2007;Milanizadeh et al., 2013).Thereby, the importance of CRC as a major worldwide health issue is also increasing in our population (Haghighi et al., 2009).Although numerous risk factors and causes have been taken into account for CRC, genetic component has a great contribution on CRC development.To emphasize this fact, nearly 35% of total cases have been reflected by twin-and family based studies (Lichtenstein et al., 2000).
SMAD7 was initially identified as an inhibitor of TGF-β because of its ability to bind TGF-β receptor typeI and prevent TGF-β-associated SMAD signaling (Yan et al., 2011).SMAD7 as a negative regulator of the TGF-b signaling pathway promotes the anti-inflammatory effects of TGF-b signaling, which performed by binding to TAB2 and TAB3 and inhibiting TAK1 (Hong et al., 2007).Two GWAS introduced a risk locus for CRC at 18q21 which maps to SMAD7, a functional candidate gene for CRC (Broderick et al., 2007;Tenesa et al., 2008).Despite of SMAD7 role in hepatic metastasis in CRC (Halder et al., 2008), its role in cancer development, particularly colorectal cancer, has not been utterly investigated.
Although it has been shown that SMAD7 has a compounding role as an intracellular mediator of TGF-b typeI receptor in cancer development, the relevance of several genetic variants within SMAD7 with CRC has been proved by several studies (Thompson et al., 2009;Slattery et al., 2010;Li et al., 2011;Nassiri et al., Singlenucleotide polymorphisms (SNPs) were determined as the most frequent and subtle genetic variation in the human genome and has great potential for application to association studies of multifactorial disorder (Kirk et al., 2002).
In this end, we explored one polymorphism of SMAD7, rs2337107; based on hypothesis that there may be the association between that SNP and CRC in an Iranian population.Additionally, we used the clinical features to investigate the correlation between them and mentioned SNP.Factors evaluated including an age, sex, tumor site, tumor grade and stage.

Study population
Our sample study was comprised of one hundred and five controls and one hundred and five patients.All patients were diagnosed with CRC and histologically confirmed by positive colonoscopy and pathology results for colon or rectum malignant tumor.Patients for the present study were recruited from October 2007 until January 2009 in cancer registry unit of the Gastroenterology and Liver Diseases Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.All participants signed an informed consent prior to participation in the study, and all healthy subjects and patients completed a selfadministrated questionnaire.Besides, all procedures were approved by Ethic committee of the Gastroenterology and Liver Diseases Research Center.Clinical data, pathological grade and clinical stage, were abstracted from medical reports.The histological classification and pathological staging were determined on the criteria of the UICC Tumor-Node-Metastasis classification of Malignant Tumors (TNM) 6 th edition, 2002, colon and rectum (ICD-O C18-C 20).The controls were randomly selected in the same time.Our control population was individuals who showed no colonoscopy report for malignancy inflammatory ulcers or polyps, and they also have no families with the history of gastrointestinal defects.

DNA isolation and genotyping
Genomic DNA was extracted from peripheral blood using a phenol-chloroform standard protocol (Green et al., 2012).Agarose gel-electrophoresis was used to assay the quality of genomic DNA and then DNA concentration determined by NanoDrop spectrophotometry 1000.Determination of samples genotyping for the one SMAD7 SNP (rs2337107) was performed via predesigned TaqMan probes (C-26177715-20; Applied Biosystems, Foster City, CA) via an ABI 7500Real Time PCR System (Applied Biosystems).The polymerase chain reaction was done with these conditions: 95 °C for10 minutes and 40 cycles of 92 °C for 15 seconds and 60 °C for 1 minute.SDS software version 1.3 (Applied Biosystems) was our analytical tool to identify individual genotypes.

Statistical analysis
SPSS software version 13 was applied to calculate statistical analysis.Distribution of the allele and genotype frequencies and also clinicopathological characteristics were compared by using of χ 2 test.Logistic regression analysis which used for the adjustment of confounding factors such as age, gender and smoking, was carried out to calculate Odds ratio (OR) and its 95% confidence intervals (95% CI).T-test or χ 2 test were used to determine differences in demographic factors.Data were considered significant if they had p values less than 0.05 in all comparisons.

Baseline characteristics
The frequency distributions of baseline characteristics and histological parameters in one hundred and five colorectal cancer cases and one hundred and five healthy subjects are presented in Table 1.Baseline characteristics of patients and controls were all well matched.
The mean age of CRC patients and controls were 51.27 (Standard deviation, SD±15.05) and 42.74 (SD±15.24)years respectively.Logistic regression method was applied to adjust the effect of age, gender and smoking status as confusing variations.
The prevalence of colon cancer in the patients was noticeably more than rectal cancer (70.5% and 29.5%, respectively).According to histological differentiation of tumor grades, 5.6%, 27.6%, and 46.7%, patients were classified in three grades, poor, moderate, or well grade, respectively.Besides, patients were grouped into four classes from I to IV regarding tumor node metastasis (TNM) at the time of diagnosis, 14.3%, 33.3%, 44.8%, and 7.6%, respectively.

Genotypic and allelic frequencies
The genotypic and allelic frequencies of the one SNP of SMAD7 in the whole study groups are summarized in Table 2. Furthermore, Figure 1, 2 illustrates the patterns of genotypes of SMAD7 gene.Genotype frequencies of the SNP for each group separately were in accordance with Hardy-Weinberg equilibrium (for cases: p value =0.955 and for controls p value =0.145).Genotypes and allelic distributions of this genetic polymorphism of SMAD7 were not significantly different between CRC patients and control groups.
The results of association between the SMAD7 genotypes and clinicopathological characteristics in colorectal cancer patients are shown in Table 3.There was no significant association between SMAD7 genotypes of rs12337107 and these features.Although there was not any significant association between genotypes and disorder, CT was the most common genotype in this population.This genotype prevalence was also higher in the patients with well grade and colon tumors.

Discussion
SMAD7 acts as an intracellular antagonist of TGF-b signaling by binding stably to the receptor complex and blocking activation of downstream signaling events (Tenesa et al., 2009).Perturbation of SMAD7 expression has been documented to influence CRC progression (Levy et al., 2006), and loss of chromosome 18q21 including SMAD7 is common in CRC (Gaasenbeek et al., 2006).In this study, one polymorphism of this gene, rs2337107, was investigated to determine the correlation between SMAD7 gene and CRC in an Iranian population.We also pursued the association of this polymorphism with clinicopathological factors such as tumor location, TNM stage and tumor grade.Our analysis demonstrated that variant genotypes of this polymorphism had no association with the risk of colorectal cancer in our population.
The noticeable point is that data emerging from experimental studies indicate that SMAD7 may be differently regulated in various tumors depending on the context analyzed (Stolfi et al., 2013).Since SMAD7 is considered as an inhibitor of TGF-β signaling, it's opposite (pro-and anti-tumorigenic) effects could originate from the different functionality of TGF-b1 pathway among distinct cancer types (Stolfi et al., 2013).Although the potential importance of SMAD7 in the etiology of CRC is supported by several researches and the GWAS (Tomlinson et al., 2008;Slattery et al., 2010;Song et al., 2012), underlying mechanism has not been fully elucidated.Boulay et al. (2003) found that CRC patients with deletion of SMAD7 had a favorable clinical outcome compared with patients with SMAD7 amplification (Boulay et al., 2003), and Halder et al reported that SMAD7-overexpressing FET cells show aggressive colony formation on soft agar and increased tumorigenicity in vivo compared with control FET cells (Halder et al., 2005).In contrast above finding, the opposing role of SMAD7 in the control of sporadic and colitis-associated CRC has been reported by one study in which they showed that over-expression of SMAD7 in T cells associates with severe colitis and reduces the growth of colitis-associated CRC (Rizzo et al., 2011).Therefore, further research on the functionality of SMAD7 variants would be needed to better understand the observed associations.

Table 2. Distribution of SMAD7 Genotypes among Colorectal Cancer Patients and Controls
Our results showed no significant genetic association between this intronic SNP, rs2337107 C>T, with CRC.Although genotype and allele distributions of this polymorphism did not show any significant disease association, our results confirmed that the heterozygote genotype of this SNP (CT) is the most common ones like Asian, European and some of Sub-Saharan African populations, according on NCBI information (www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi.rs=2337107).Furthermore, there were also no significant differences between this polymorphism and clinicopathological factors.Despite more frequency of CT genotype in patients with colon location and well grade tumors than others, the results indicated that we cannot recommend this polymorphism as a biomarker for CRC in an Iranian population.
There have been some earlier studies on the association SMAD7 gene variants and CRC.Alemn et al. interrogated all polymorphisms within the 17-kb region of the 18q21 locus, which on the basis of LD (linkage disequilibrium), protects the disease-causing variants responsible for the SMAD7-18q21 association with CRC.Their results showed that rs2337107, not alone but together with 24 another SNPs, had an association with the developing of CRC at the 5% statistical threshold (Pittman et al., 2009).There is two another studies which implemented by Martah et al. and Xuejuan et al. separately, that their results demonstrated no association between rs2337107 and CRC in their population (Slattery et al., 2010;Jiang et al., 2013).Our result also appears consistent with these results.
This study was conducted in a well-defined homogenous sample with detailed clinical data.However, one of our limitations was sample size.Since we were in sanction and the price of probes were so high, we could only provide the small size of predesigned probes (S: 188uL; 40X).It seems logical that the genotype differences may be strictly attributed to chance due to the modest sample size.Therefore, larger population is needed to elucidate the exact conclusion of this SNP frequency in our population.Another point is that only one variant of the SMAD7 gene was genotyped, and it was insufficient to conclude about the effect of whole gene.Since it has been documented that this SNP has a high linkage disequilibrium with three common genetic variations of SMAD7 which strongly associated to CRC, it may be more informative to study the more number of SMAD7 polymorphisms along with this SNP to throw light on the role of CRC progression in an Iranian population.
In conclusion, there is the first case-control study which has explored the influence of rs2337107 of SMAD7 gene on clinicopathological features and CRC risk in Iranian population.Furthermore, the results showed no evidence of association between this SNP and the risk of initiation and development of CRC, also there was no significant effect of this SNP on clinicopathological features.To regard this point that this study had some inherent limitations such as small sample size; further studies in various populations should be implemented to clarify the association of this SNP with colorectal cancer.
distribution of patients and controls were in agreement with the Hardy-Weinberg equilibrium; b Additive Genetic model; c Adjusted for age, gender and smoking status

Figure 1 .Figure 2 .
Figure 1.Allelic Discrimination Plot Represents Genotypes with Four Symbols (Squares, Diamonds, Triangles and Circles) for NTC Sample, CC, CT and TT Genotypes Respectively.The x-axis is amount of emission for flourophore channels (FAM) and on the y-axis represents emission for flourophore channels (VIC) DOI:http://dx.doi.org/10.7314/APJCP.2014.15.11.4437Lack of Influence of SMAD7 Gene rs2337107 Polymorphism on the Risk of Colorectal Cancer in an Iranian Population 0

Table 3 . Association between SMAD7 Genotypes and Clinic Pathological Characteristics
*TNM, Tumor Node Metastasis