A Functional Polymorphism in the Epidermal Growth Factor Gene Is Associated with Risk for Glioma in a Chinese Population

Abstract

Epidermal growth factor (EGF) plays a key role in survival of neural and glial precursor cells. A single nucleotide polymorphism of the EGF gene +61G/A in the 5'-untranslated region has been reported to be associated with susceptibility to glioma. The purpose of this study was to investigate the potential association between EGF +61G/A and brain glioma in a Chinese population. A case-control study involving 180 patients with glioma and 360 controls was done. Polymerase chain reaction restriction fragment-length polymorphism assay was used to analyze the EGF +61G/A genotypes. Patients with glioma had a significantly higher frequency of AA genotype (odds ratio, 2.25; 95% confidence interval, 1.20, 4.21; p=0.01] than controls. When stratified by histologic features and World Health Organization grade of glioma, distribution of each genotype did not significantly differ. Our data suggested that the EGF +61G AA genotype was associated with a higher glioma risk in a Chinese population. This finding is in contrast with previous studies that reported the G allele as a risk factor for glioma in white patients.

Introduction

Gliomas are the most common primary tumors of the central nervous system (Jemal et al., 2009), but despite the marked advances in the characterization of their molecular pathogenesis, these tumors remain incurable. The median survival time of patients with malignant glioma remains only approximately 12 months; less than 3% of patients with glioblastoma survive for 5 years after diagnosis (Ohgaki and Kleihues, 2005; Liu et al., 2009). Several occupations (De Roos et al., 2003; Samkange-Zeeb et al., 2010), environmental carcinogens (Maker et al., 1976), and diet have been reported to be associated with an elevated glioma risk (Groothuis et al., 1992; Bunin et al., 1994; Chen et al., 2002; Thivat et al., 2009; Kabat et al., 2011), as have genetic factors, such as single nucleotide polymorphisms (SNPs) (Ohgaki and Kleihues, 2005).

The epidermal growth factor (EGF) gene, located in 4q25-q27 (Normanno et al., 2006), encodes a ligand for the EGF receptor (EGFR) that activates a cascade of events responsible for promoting cell proliferation, inhibition of apoptosis, and differentiation (Salomon et al., 1995). A SNP involving an A-to-G mutation at position 61 of the 5′ untranslated region of the EGF gene (rs4444903) results in higher EGF levels in persons with EGF genotype G/G and has been associated with an increased risk for several human malignancies (Shahbazi et al., 2002).

Although an association between EGF +61G/A genotype and brain glioma is biologically plausible, epidemiologic studies on this relationship have yielded inconsistent results (Costa et al., 2007; Liu et al., 2009; Bao et al., 2010; Tan et al., 2010; Wang et al., 2010). The purpose of this study was to investigate the potential association between EGF +61G/A genotype and brain glioma in a Chinese population.

Materials and Methods

Study population

From June 2009 to June 2011, 180 primary gliomas surgically resected from the Department of Neurosurgery of the First Affiliated Hospital of Dalian Medical University were analyzed. At the same time, 360 healthy controls were recruited from the same geographic region. Tumor type and stage were determined according to the World Health Organization criteria. Smoking status was defined as nonsmoker (smoked < 100 cigarettes in lifetime) and smoker. Drinking status was defined as nondrinker and drinker (consumed more than one cup [200 mL] per day). Informed consent was obtained according to the Declaration of Helsinki. The institutional review board of the Dalian Medical University approved the study.

DNA extraction and genotyping

Genomic DNA was extracted from blood samples using a commercialized kit (QIAamp, QIAGEN Inc., Valencia, CA). EGF +61 A/G polymorphisms were analyzed with polymerase chain reaction (PCR) amplification followed by restriction fragment-length polymorphism (RFLP). The following primers were used: forward-5′-GAGAAACTGTTGGGAGAGGAATC-3′ and reverse-5′- TCACAGAGTTTAACAGCCCTGC-3′. PCR cycling conditions were 5 min at 94°C, followed by 35 cycles of 30 s at 94°C, 30 s at 58°C, and 30 s at 72°C, with a final elongation step at 72°C for 5 min. For RFLP, the PCR products were digested with AluI (2 U at 37°C for 4 h).

Statistical analysis

Chi-square tests were used to compare the genotype distribution between the 2 groups. Multivariate logistic regression analysis was performed to analyze the association between the genotypes and risk for glioma after stratifying the participants according to age, sex, and histologic grade. The odds ratio (OR) and 95% confidence interval (CI) were calculated by using a logistic regression model. A 2-sided significance level of p<0.05 was used for all analyses. All calculations were performed by using a commercially available program (SPSS for Windows, version 14.0, Chicago, IL).

Results

Characteristics of the 180 patients with glioma and 360 controls are shown in Table 1. The groups did not differ regarding sex (p=0.50) or age (p=0.52). The mean age±standard deviation was 41.5±12.5 years for the glioma group and 40.8±11.7 years for the control group. In addition, the 2 groups did not differ for smoking status, drinking, or family history of cancer (Table 1). Among the 180 patients with glioma, 67 (37.2%) had astrocytomas, 59 (32.8%) had glioblastoma, and 54 (30.0%) had other gliomas. Genotype and allele frequencies were in Hardy-Weinberg equilibrium in both groups.

Table 1.

Characteristics of Patients with Glioma and Controls

Characteristic	Glioma group (n=180)	Control group (n=360)	p value
Mean age±SD (y)	41.5±12.5	40.8±11.7	0.52
Sex, n (%)			0.50
Male	106 (58.9)	201 (55.8)
Female	74 (41.1)	159 (44.2)
Smoking status, n (%)			0.61
Never	123 (68.3)	238 (66.1)
Ever	57 (31.7)	122 (33.9)
Drinking (%)			0.80
Never	112 (62.2)	220 (61.1)
Ever	68 (37.8)	140 (38.9)
Family history of cancer, n (%)			0.28
No	157 (87.2)	325 (90.3)
Yes	23 (12.8)	35 (9.7)
Histologic type, n (%)
Astrocytomas	67 (37.2)
Glioblastoma	59 (32.8)
Other gliomas	54 (30.0)
WHO grade, n (%)
I	16 (8.9)
II	57 (31.7)
III	36 (20.0)
IV	71 (39.4)

SD, standard deviation; WHO, World Health Organization.

Patients with glioma had a significantly higher frequency of AA genotype (OR, 2.25; 95% CI, 1.20, 4.21; p=0.01) than did controls (Table 2). When stratified by histologic and World Health Organization grade of glioma, the distribution of each genotype did not significantly differ between groups (Table 3).

Table 2.

Genotype Frequencies of EGF +61 Polymorphism Among Patients with Glioma and Controls

Genotype	Glioma group (%) (n=180)	Control group (%) (n=360)	Odds ratio (95% CI)	p value
GG	75 (41.7)	163 (45.3)	0.86 (0.60, 1.24)	0.43
GA	83 (46.1)	176 (48.9)	0.90 (0.63, 1.28)	0.54
AA	22 (12.2)	21 (5.8)	2.25 (1.20, 4.21)	0.01
A allele frequency	233 (64.7)	502 (69.7)	0.80 (0.61, 1.04)	0.10
G allele frequency	127 (35.3)	218 (30.3)	1.26 (0.96, 1.64)	0.10

Table 3.

Stratification Analysis of EGF +61 Genotype Frequency in Glioma

		GG			GA			AA
Variable	Glioma Group (n=180)	Patients, n (%)	OR (95% CI)	p value	Patients, n (%)	OR (95% CI)	p value	Patients, n (%)	OR (95% CI)	p value
Histologic type	180	75 (41.7)	1 (Reference)		83 (46.1)	1 (Reference)		22 (12.2)	1 (Reference)
Astrocytomas	67	27 (40.3)	0.95 (0.53, 1.67)	0.85	31 (46.3)	1.01 (0.57, 1.77)	0.98	9 (13.4)	1.11 (0.49, 2.56)	0.80
Glioblastoma	59	24 (40.7)	0.96 (0.53, 1.75)	0.89	27 (45.8)	0.99 (0.55, 1.78)	0.96	8 (13.6)	1.13 (0.47, 2.69)	0.79
Other gliomas	54	24 (44.4)	1.12 (0.61, 2.07)	0.72	25 (46.3)	1.01 (0.55, 1.85)	0.98	5 (9.3)	0.73 (0.26, 2.04)	0.55
WHO grade	180	75 (41.7)	1 (Reference)		83 (46.1)	1 (Reference)		22 (12.2)	1 (Reference)
I	16	6 (37.5)	0.84 (0.29, 2.41)	0.75	7 (43.8)	0.91 (0.32, 2.55)	0.86	3 (18.8)	1.66 (0.44, 6.28)	0.46
II	57	23 (40.4)	0.95 (0.52, 1.74)	0.86	26 (45.6)	0.98 (0.54, 1.78)	0.95	8 (14.0)	1.17 (0.49, 2.80)	0.72
III	36	15 (41.7)	1.00 (0.48, 2.07)	1.00	17 (47.2)	1.05 (0.51, 2.14)	0.90	4 (11.1)	0.90 (0.29, 2.78)	0.85
IV	71	31 (43.7)	1.09 (0.62, 1.89)	0.77	33 (46.5)	1.02 (0.59, 1.76)	0.96	7 (9.8)	0.79 (0.32, 1.93)	0.60

OR, odds ratio.

Discussion

EGF plays a key role in the survival of neural and glial precursor cells. The +61A/G polymorphism of the EGF gene is located in the 5′-untranslated region of EGF messenger RNA and may affect DNA folding or gene transcription, leading to the increase in EGF protein expression (Tan et al., 2010). The association between the +61A/G polymorphism of the EGF gene and glioma risk has been widely reported; however, epidemiologic studies on this relationship have yielded inconsistent results. A meta-analysis of 6 case-control studies found that EGF +61G allele represented a risk factor for glioma in European populations and, conversely, a protective factor in Chinese populations (Tan et al., 2010). Thus, we investigated the potential association between EGF +61G/A genotype and brain glioma in a Chinese population. Our data suggested that EGF +61 AA genotype was associated with a higher glioma risk in this population. This is in contrast with previous studies, which reported that G allele was a risk factor for glioma in white patients.

Studies investigating the association between genetic polymorphisms and glioma risk are increasingly being reported. A case-control study indicated that vascular EGF+936 C/T genotype was associated with a higher glioma risk in a Chinese Han population (Bao et al., 2011). Another case-control study showed that XRCC1 Arg399Gln polymorphism is a significant risk factor and that the 399Gln (G) allele carries a 3.5 times greater risk for glioma, whereas the PARP1 Val/Ala genotype may be protective against it (Yosunkaya et al., 2010). A hospital-based case-control study suggested that CT genotype and the T allele of hypoxia-inducible factor-1α C1772T gene polymorphism were associated with a higher glioma risk in the Han population of China (Xu et al., 2011). From a total of 11 SNPs in 7 cytokine genes within a hospital-based study, Brenner et al. (2007) found that the IL4 (rs2243248, -1098T>G) and IL6 (rs1800795, -174G>C) polymorphisms were significantly associated with risk for glioma in the pooled analysis.

EGF +61G/A polymorphism is associated with many diseases. A meta-analysis of 23 case-control studies that consisted of 5578 case-patients and 7306 controls suggested that the EGF 61G allele was a risk factor for cancer, especially for gastric cancer and glioma (Zhang et al., 2010). Another meta-analysis from 21 studies with 14,609 participants suggested that the EGF +61G/A polymorphism most likely contributed to decreased susceptibility to cancers among Asians and Americans and that the A allele may be a protective factor for gastric cancer, esophageal cancer, hepatoma, and glioma (Tan et al., 2010). A hospital-based case-control study suggested that the EGF +61 GG genotype was associated with a higher risk for hepatocellular carcinoma (Abu Dayyeh et al., 2011; Chen et al., 2011).

Some limitations of our study should be mentioned. First, the sample size of the study is small, and there may not be enough statistical power to explore the real association. Second, the interactions between gene-gene, gene-environment, and even different polymorphic loci of the same gene may modulate glioma risk. Third, these results should be interpreted with caution because the entire population was from China; this reduces the possibility of confounding due to ethnicity but does not permit extrapolation of the results to other ethnic groups.

In conclusion, our data suggested that the EGF +61 AA genotype was associated with a higher glioma risk in a Chinese population. This is in contrast with previous studies, which reported G allele as a risk factor of glioma in white persons. Additional studies are needed to confirm this finding.

Footnotes

Disclosure Statement

The authors state that they have no conflicts of interest.

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