Association of Epidermal Growth Factor +61 A/G Polymorphism in Chinese Patients with Colon Cancer

Abstract

Epidermal growth factor (EGF) plays an important role in tumorigenesis. The association between the +61 A/G polymorphism of the EGF gene and colon cancer risk remains controversial and unclear. The objective of this study was to investigate the association between EGF +61 A/G polymorphism and colon cancer risk in a Chinese population. A hospital-based case-control study was conducted to assess the possible association between EGF +61 A/G polymorphism and colon cancer risk. A total of 180 colon cancer patients and 180 cancer-free healthy controls were recruited in the Chinese population. Genomic DNA was isolated from peripheral blood, and gene polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Colon cancer patients had a significantly higher frequency of +61 GG genotype (odds ratio [OR]=1.93, 95% confidence interval [CI]=1.07, 3.50; p=0.03) than that of controls. When stratified by the tumor location, tumor size, growth pattern, differentiation, and tumor-node-metastasis (TNM) stage of colon cancer, no statistically significant results were observed. Our study revealed that EGF +61 GG genotype was associated with a higher risk of colon cancer in Chinese population.

Introduction

Colorectal cancer is the third most commonly diagnosed cancer in men and is the second in women, with over 1.2 million new cancer cases and 608,700 deaths estimated to have occurred in 2008 (Jemal et al., 2011). In 2012, there were 143,460 new colorectal cancer cases and 51,690 deaths from colorectal cancer in the United States (Siegel et al., 2012). Colorectal carcinogenesis is a multistep process, where genetic changes occur with mutations of several genes that accumulate with the progression from a normal epithelium to an adenoma to invasive cancer (Vogelstein et al., 1988). As the most common sources of human genetic variation, single-nucleotide polymorphisms (SNPs) may contribute to an individual's susceptibility to cancer.

Epidermal growth factor (EGF) has many biological functions involving stimulation of proliferation, differentiation, and tumorigenesis of epidermal and epithelial tissues (Fisher and Lakshmanan, 1990). EGF was first isolated in 1962 (Cohen, 1962), and is encoded by a single gene on chromosome 4q25-q27 (Morton et al., 1986). Shahbazi et al. (2002) first reported that the +61 A/G SNP in the 5′-untranslated region of the EGF gene was associated with increased EGF production and risk of malignant melanoma.

In recent years, several case-control studies have investigated the association between EGF +61 A/G polymorphism and colon cancer risk (Zhang et al., 2006; Spindler et al., 2007; Kovar et al., 2009; Wu et al., 2009), but results have been inconsistent. The objective of this study was to investigate the association between EGF +61 A/G polymorphism and colon cancer risk in a Chinese population.

Materials and Methods

Subjects

Between January 2010 and January 2012, a total of 180 colon cancer patients were recruited at the Surgical Department of the Hebei Medical University. The control group comprised of 180 cancer-free healthy individuals undergoing colonoscopy as a preventive measure and who had a normal colonoscopy. The healthy controls were selected from a pool of healthy volunteers who visited the hospital during the same period. All patients and healthy controls were Chinese from the same geographic region. None of the patients had received neoadjuvant treatment. Patients with metacronic carcinoma, familial polyposis, hereditary nonpolyposis colon cancer, and inflammatory bowel disease were excluded from the study. The project was approved by the clinical research and ethics committee of the Hebei Medical University. An informed consent was obtained from each subject. Tumor pathology stages were classified according to the tumor-node-metastasis (TNM) classification of the Union Internationale Contra le Cancer (UICC). Pathology grades were determined according to the criteria of World Health Organization (WHO).

Genotyping analysis

For genetic analyses, genomic DNA was isolated from peripheral blood using Blood Kit (Macherey-Nagel). The EGF +61 A/G genotype was analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Based on the GenBank reference sequence, the PCR primers were as follows: forward 5′-GAG AAA CTG TTG GGA GAG GAA TC-3′ and reverse 5′-TCA CAG AGT TTA ACA GCC CTG C-3′. The polymorphism was identified following digestion of amplified DNA with the endonuclease AluI (2 U at 37°C for 4 h). The digested DNA materials were then analyzed by 2% agarose gel electrophoresis, and genotypes were determined distinguishing between digested and undigested PCR products.

Statistical analysis

Statistical analyses were carried out using the STATA program, version 9.0, (StataCorp LP) for Windows^®. Allele and genotype frequencies between the case and control groups were compared by the χ² test. The Hardy-Weinberg equilibrium of alleles at individual loci was tested to compare the observed genotype frequencies with the expected genotype frequencies among the subjects. Stratification analysis was used to study subgroups by tumor location, tumor size, growth pattern, differentiation, and TNM stage. Statistical significance was set at p<0.05.

Results

Clinical characteristics of patients and healthy controls are described in Table 1. The age range was 35-84 years, with a mean of 60.8 years for the patients (85 women and 95 men). The age range was 55-65 years, with a mean of 61.0 years for the healthy controls (87 women and 93 men). The tumor location, tumor size, growth pattern, differentiation, and TNM stage of colon cancer are described in Table 1.

Table 1.

Clinical Characteristics of Colon Cancer Patients and Healthy Controls

Characteristics	Patients	Controls	p-Value
Number of subjects	180	180
Gender: male/female	95/85	93/87	0.83
Age (years), mean±standard deviation	60.8±7.9	61.0±8.0	0.81
Tumor location	180
Left colon	75 (41.7)
Right colon	105 (58.3)
Tumor size	180
<4 cm	104 (57.8)
>4 cm	76 (42.2)
Growth pattern	180
Ulcerative	98 (54.4)
Protruding	82 (45.6)
Differentiation	180
Good	74 (41.1)
Moderate	59 (32.8)
Poor	47 (26.1)
TNM stage	180
I	77 (42.8)
II	63 (35.0)
III	23 (12.8)
IV	17 (9.4)

TNM, tumor-node-metastasis.

The functional EGF +61 A/G polymorphism genotype distribution in patients and controls is described in Table 2. EGF +61 A/G genotype frequencies in both patient and control groups were distributed in accordance with Hardy-Weinberg equilibrium. Colon cancer patients had a significantly higher frequency of +61 GG genotype (odds ratio [OR]=1.93, 95% confidence interval [CI]=1.07, 3.50; p=0.03) than that of controls (Table 2). When stratified by the tumor location, tumor size, growth pattern, differentiation, and TNM stage of colon cancer, no statistically significant results were observed (Table 3).

Table 2.

Genotype Frequencies of EGF +61 A/G Polymorphism in Colon Cancer Patients and Healthy Controls

Genotype	Patients (%)	Controls (%)	OR (95% CI)	p-Value
AA	75 (41.7)	82 (45.6)	0.85 (0.56, 1.29)	0.46
AG	70 (38.9)	78 (43.3)	0.83 (0.55, 1.27)	0.39
GG	35 (19.4)	20 (11.1)	1.93 (1.07, 3.50)	0.03
A allele frequency	220 (61.1)	242 (67.2)	0.77 (0.56, 1.04)	0.09
G allele frequency	140 (38.9)	118 (32.8)	1.30 (0.96, 1.77)	0.09

EGF, epidermal growth factor; OR, odds ratio; CI, confidence interval.

Table 3.

Stratification Analysis of EGF +61 A/G Genotype Frequency in Colon Cancer Patients

		AA			AG			GG
Variable	Cases	n (%)	OR (95% CI)	p-Value	n (%)	OR (95% CI)	p-Value	n (%)	OR (95% CI)	p-Value
Tumor location	180	75 (41.7)	1 (Reference)		70 (38.9)	1 (Reference)		35 (19.4)	1 (Reference)
Left colon	75	32 (42.7)	1.02 (0.63, 1.68)	0.93	30 (40.0)	1.03 (0.62, 1.71)	0.91	13 (17.3)	0.89 (0.45, 1.78)	0.75
Right colon	105	43 (40.9)	0.98 (0.63, 1.54)	0.94	40 (38.1)	0.98 (0.62, 1.55)	0.93	22 (21.0)	1.08 (0.60, 1.93)	0.80
Tumor size	180	75 (41.7)	1 (Reference)		70 (38.9)	1 (Reference)		35 (19.4)	1 (Reference)
<4 cm	104	42 (40.4)	0.97 (0.62, 1.52)	0.89	41 (39.4)	1.01 (0.64, 1.60)	0.95	21 (20.2)	1.04 (0.57, 1.88)	0.90
>4 cm	76	33 (43.4)	1.04 (0.64, 1.70)	0.87	29 (38.2)	0.98 (0.59, 1.63)	0.94	14 (18.4)	0.95 (0.48, 1.86)	0.88
Growth pattern	180	75 (41.7)	1 (Reference)		70 (38.9)	1 (Reference)		35 (19.4)	1 (Reference)
Ulcerative	98	42 (42.8)	1.03 (0.66, 1.61)	0.90	38 (38.8)	0.99 (0.63, 1.59)	0.99	18 (18.4)	0.95 (0.51, 1.76)	0.86
Protruding	82	33 (40.3)	0.97 (0.59, 1.57)	0.89	32 (39.0)	1.00 (0.61, 1.64)	0.99	17 (20.7)	1.07 (0.57, 2.01)	0.84
Differentiation	180	75 (41.7)	1 (Reference)		70 (38.9)	1 (Reference)		35 (19.4)	1 (Reference)
Good	74	33 (44.6)	1.07 (0.66, 1.75)	0.79	27 (36.5)	0.94 (0.56, 1.58)	0.81	14 (18.9)	0.97 (0.49, 1.91)	0.94
Moderate	59	26 (44.1)	1.06 (0.62, 1.80)	0.84	21 (35.6)	0.92 (0.52, 1.62)	0.76	12 (20.3)	1.05 (0.51, 2.15)	0.90
Poor	47	16 (34.0)	0.82 (0.44, 1.53)	0.53	22 (46.8)	1.20 (0.68, 2.14)	0.53	9 (19.2)	0.99 (0.44, 2.19)	0.97
TNM stage	180	75 (41.7)	1 (Reference)		70 (38.9)	1 (Reference)		35 (19.4)	1 (Reference)
I	77	32 (41.6)	0.99 (0.61, 1.63)	0.99	30 (38.9)	1.00 (0.61, 1.66)	0.99	15 (19.5)	1.00 (0.52, 1.94)	0.99
II	63	27 (42.8)	1.03 (0.61, 1.74)	0.92	24 (38.1)	0.98 (0.57, 1.69)	0.94	12 (19.1)	0.98 (0.48, 2.00)	0.96
III	23	10 (43.5)	1.04 (0.47, 2.30)	0.92	9 (39.1)	1.01 (0.44, 2.28)	0.99	4 (17.4)	0.89 (0.29, 2.75)	0.85
IV	17	6 (35.3)	0.85 (0.32, 2.32)	0.74	7 (41.2)	1.06 (0.42, 2.66)	0.90	4 (23.5)	1.21 (0.38, 3.81)	0.75

Discussion

In this study, we found that colon cancer patients had a significantly higher frequency of +61 GG genotype than that of controls. When stratified by the tumor location, tumor size, growth pattern, differentiation, and TNM stage of colon cancer, no statistically significant results were observed. However, a further study involving a larger patient collective is required. Our study revealed that EGF +61 GG genotype was associated with a higher risk of colon cancer in Chinese population.

The mechanism by which the EGF +61 GG genotype increases the risk of colon cancer remains to be defined. Shahbazi et al. (2002) first reported that the +61 A/G SNP in the 5′-untranslated region of the EGF gene was associated with increased EGF production and risk of malignant melanoma. So, we could propose that a higher EGF production is associated with an increased risk of colorectal cancer. Three possible ways by which the EGF +61 GG genotype increases the EGF production could be as follows: (1) the polymorphism itself is functional, (2) the G to A substitution might affect the DNA folding or processing of the mRNA transcript, and (3) the allelic variation at position 61 could be closely linked to a functional polymorphism elsewhere in the gene (Wu et al., 2009).

The possible association of EGF +61 A/G polymorphism with many other cancer risks has been studied. A hospital-based case-control study by Chen et al. (2011) found that EGF +61 GG genotype was associated with a higher risk of chronic hepatitis B virus (HBV)-infected hepatocellular carcinoma in the Chinese population. A prospective study found that EGF +61 A/G polymorphism was associated with risk for hepatocellular carcinoma (Abu Dayyeh et al., 2011). A case-control study involving 180 patients with glioma and 360 controls suggested that the EGF +61 GG genotype was associated with a higher glioma risk in a Chinese population (Li et al., 2012). A meta-analysis of 23 case-control studies, including 5578 cases and 7306 controls, suggested that the EGF 61G allele is a risk factor of cancer, especially for gastric cancer and glioma (Zhang et al., 2010). A case-control study suggested that the EGF +61 A/G polymorphism may alter esophageal adenocarcinoma susceptibility through an interaction with gastroesophageal reflux disease (Cheung et al., 2009). A case-control study found that the frequencies of the EGF +61 GG genotype and G allele were significantly increased among patients with pancreatic head cancer (Wu et al., 2010).

Evidence for a genetic basis for progression of colorectal cancer has been found. A case-control study provided evidence diagnosing that the Leu/Leu genotype of exonuclease 1 showed an inverse association with colorectal cancer (Haghighi et al., 2010). A case-control study found that the GSTM1 gene polymorphism was a significant risk factor for colorectal cancer (Darazy et al., 2011). A case-control study demonstrated that tumor necrosis factor alpha-308AA genotype was associated with a higher risk of colon cancer in the Chinese population (Li et al., 2011). A case-control study suggested that vascular EGF −2578AA genotype was associated with a higher risk of colon cancer in Chinese population (Zhang et al., 2011). A meta-analysis strongly suggested that the two polymorphisms of TGFBR1 may confer low-penetrance susceptibility of colorectal cancer risk (Zhang et al., 2012). A meta-analysis of 3347 colorectal cancer cases and 3102 controls from eight published case-control studies suggested that the MDM2 SNP309 polymorphism is a low-penetrance risk factor for the development of colorectal cancer, particularly among Asians (Cao et al., 2012). A meta-analysis of nine studies, including 7954 colorectal cancer cases and 7369 controls, suggested that E-cadherin -C160A polymorphism provides a possible protection against colorectal cancer, which is especially evident in Caucasian and hospital populations (Geng et al., 2012).

In conclusion, our study revealed that EGF +61 GG genotype was associated with a higher risk of colon cancer in Chinese population. However, further study involving a larger patient collective is required.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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