Genetic Polymorphisms of Vascular Endothelial Growth Factor (VEGF) -2549 I/D and +405G/C in the susceptibility to Gastric Cancer

Objective: Vascular Endothelial Growth Factor (VEGF) plays an important role in tumor angiogenesis. Although several studies revealed on association of VEGF polymorphisms with gastric cancer, still the results are inconclusive. The role of (VEGF) -2549 I/D and +405G/C polymorphisms in gastric cancer of Telangana Population is evaluated in the present study. Methods: A case control study was carried out on 540 individuals which comprised of 180 Gastric Cancer patients and 360 healthy control subjects.  The association of functional single nucleotide polymorphisms (SNPs) of the VEGF gene (-2549 I/D and +405G/C) were evaluated by polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP). Results: VEGF -2549 polymorphism with I/D and I/Igenotypeshoweda threefold higher risk (95%CI= 1.75-4.63 and 95%CI= 1.72-5.78) to gastric cancer. A signifi cant association was observed with respect to I allele (AOR= 1.67; 95%CI= 1.29-2.15; P= 7.641e-05) while genotypic and allelic frequencies of VEGF +405G/C polymorphism did not show any signifi cant differences in both the groups. A signifi cant increase in the frequency of I-G haplotype in Gastric cancer cases was observed compared to control with an AOR of 2.23 (95%CI= (1.51-3.27); P= 0.0001). Conclusion: In conclusion VEGF -2549I/D polymorphism and I-G haplotype conferred signifi cant susceptibility towards gastric cancer, whereas, no such association with respect to +405G/C polymorphism was observed. Research Article Genetic Polymorphisms of Vascular Endothelial Growth Factor (VEGF) -2549 I/D and +405G/C in the susceptibility to Gastric Cancer Amar chand Bayal, Shehnaz Sultana, Pratibha Nallari and Venkateshwari Ananthapur* Institute of Genetics and Hospital for Genetic Diseases, Osmania University, Begumpet, Hyderabad-500016, Telangana, India Received: 16 December, 2020 Accepted: 15 January, 2021 Published: 16 January, 2021 *Corresponding author: Dr. Venkateshwari Ananthapur, Institute of Genetics and Hospital for Genetic Diseases, Osmania University, Begumpet, Hyderabad-500016, Telangana, India, Tel: 04


Introduction
Gastric Cancer (GC) of the digestive tract is the third leading cause of carcinoma related deaths worldwide [1]. Gastric cancer is a multifactorial disease associated with various risk factors like tobacco chewing, smoking, alcohol consumption, diet, Helicobacter pylori infection and gastric disorders [2]. Genetic factors play a role in the susceptibility to gastric cancer [3][4][5][6], where in Single Nucleotide Polymorphisms (SNPs) of various genes may be causal in the onset of gastric cancer. Angiogenesis is the prerequisite for the growth and progression of tumours with micro invasions. Vascular Endothelial Growth Factor (VEGF) known to infl uence tumour-related angiogenesis [7,8]. The VEGF gene is located on chromosome 6p12-p21 with eight exons [9,10]. Earlier studies have reported an increase in the expression of VEGF to be associated with the grade of angiogenesis and prognosis of various human cancers [11][12][13][14]. However, studies investigating the role of VEGF gene polymorphisms in the outcome of gastric cancer are inconclusive. The present study is taken up to elucidate therole of -2549 Insertion/Deletion polymorphism in the promoter region and +405G/C polymorphism in 5' UTR of VEGF gene towards the susceptibility of gastric cancer.

Genotyping by PCR -RFLP and ARMS PCR
Total genomic DNA was isolated from peripheral blood leukocytes by the salting out method of Lahiri and Nuernberger [15]. The VEGF -2549I/D polymorphism was genotyped by Polymerase Chain Reaction (PCR) using allele specifi c primers. To evaluate the combined effect of VEGF polymorphisms (-2549I/D and +405G/C) in the suspectability to GC, haplotype analysis was performed using SNPSTAT tool. Four haplotype groups were observed (

Discussion
Gastric cancer, a disease of heterogeneous origin involving several factors such as genetic, environmental, immune, diet, infections and infl ammation may lead to disturbances in the signalling pathways related to growth and regulation of gastric tumours due to the infl uence of gene/s variation [18,19].
Genetic variations and disease susceptibility play an important role in the pathogenesis of gastric cancer [20][21][22]. Tumour growth, relapse and metastasis turns on the "angiogenic switch" to induce tumour growth to a size greater than 1-2 mm which is regulated by angiogenic activators and inhibitors [23].    proangiogenic abilities of gastric cancer cells secrete angiogenic cytokines and stimulate ECs to support their own growth in an autocrine manner. VEGF acts as a key mediator of angiogenesis and micro invasion in cancers and is also involved in cellular processes which can be infl uence by the functional polymorphic variants of the gene [27]. VEGF gene polymorphism may alter VEGF production and activity, thereby causing inter-individual differences in the angiogenesis, lymph and vascular and lymphatic tumor spread. According to Ohta, et al. peripheral blood plasma VEGF-A level was reported to be increased in patients with venous invasion and correlated with lymph node metastasis and reported as a sensitive marker for the progression of gastric cancer [28]. Similarly, VEGF + 1612G > A gene polymorphism was found to be associated with gastric cancer in Chinese and Japanese population [29,30]. VEGF − 2578C > A and -634G > C polymorphisms were associated with larger tumor size, poor differentiation, advanced stage of gastric cancer in Greek population [31]. The insertion/deletion (I/D) polymorphism in the promoter region of VEGF at the position -2549 relative to the translation start site has been linked to an altered transcriptional activity [32].
This is the fi rst study reporting an association of VEGF -2549I/D polymorphism with gastric cancer. The fi ndings of the present study are in concordance with the earlier reports that I allele and I allele carrier genotypes (i.e. I/D, I/I) were associated with breast cancer of North Indian origin [33]. The I/D genotype was also being reported to be at greater risk to prostate cancer in a study from North India [34]. Further VEGF +405G/C polymorphism in the 5' UTR has a signifi cant effect on VEGF expression. Similarly, a study on association of +405C/C genotype with poor tumor differentiation and lymph node metastasis in gastric cancer was reported [21]. VEGF +405C allele reduces binding of the transcription factor myeloid zinc fi nger protein MZF1, which then subsequently reduces the gene expression [35][36][37]. According to a study 405C/C genotype was associatedwith larger tumor size, metastasis and advanced stages of gastric cancer [31]. However, in the present study no such association between +405G/C polymorphism and gastric cancer was observed.
Further haplotype analysis showed increased frequency of I-G haplotype in gastric cancer compared to controls. Study carried out by chae, et al. on VEGF gene polymorphisms (-460T > C, +405G > C, and 936C > T) showed TCT haplotype was associated with decreased susceptibility to gastric cancer [38]. Kim, et al. reported association of CACC haplotype with worse survival compared to TGGC haplotype in patients with surgically resected gastric cancer [22]. Penelope, et al. showed CGC haplotype of combined 460T/C, 405G/C, and 936C/T VEGF polymorphisms associated with reduced overall survival and had a potential prognostic signifi cance in oesophageal cancer [39].

Conclusion
In conclusion VEGF -2549I/D polymorphism and I-G haplotype has shown a signifi cant association and susceptibility towards gastric cancer which throws a light on the molecular mechanism of tumour angiogenesis in gastric cancer and helps in developing a novel antiangiogenic strategy. This is the fi rst report of the VEGF polymorphism refl ecting the synergistic action of two SNPs which seem to exert infl uence on the expression of VEGF. Adjusted for: age, sex and addictions; AOR: Adjusted odds ratio, Adjusted P-value: FDR adjusted P-value. AIC: Akaike Information Criterion; *: P <0.05 at 5% level of signifi cance. Codominant AOR 1: G/C vs. G/G; Codominant AOR 2 : C/C vs. G/G; Dominant AOR: G/C-C/C vs. G/G; Recessive AOR: C/C vs. G/G-G/C; Log-additive AOR: G/G-0, G/C-1, C/C-2. Global haplotype association P-value: 0.00012*