Association of Vascular Endothelial Growth Factor A gene polymorphisms with susceptibility to Systemic lupus erythematosus in Iranian population

Personal non-commercial use only.Rheumatology Research Journal. Copyright © 2019. All rights reserved *Corresponding Author: Mahdi Mahmoudi, Ph.D, and Elham Farhadi, Ph.D; ; Rheumatology Research Center, Tehran University of Medical Sciences, Tehran, Iran, Email: mahmoudim@tums.ac.ir and farhadie@tums.ac.ir, Tel/Fax: (+98) 21-8822-0067 Po Box: 1411713137 Received: 21 October 2019; Accepted: 11 January 2020 Systemic lupus erythematosus (SLE) is an autoimmune, autoinflammatory disorder in which genetic factors have been implicated in the etiopathogenesis. Elevated levels of the vascular endothelial growth factor (VEGF) have been reported in patients with SLE. This study intended to evaluate the association of the VEGFA gene rs833061 and rs2010963 single nucleotide polymorphisms (SNPs) with the risk of SLE susceptibility in an Iranian population. In this case-control study, 400 SLE patients and 400 age-, sex-, and ethnically-matched healthy controls were recruited. Genotyping of VEGFA gene rs833061 and rs2010963 polymorphisms in both SLE and control groups was done using real-time PCR allelic discrimination technique. No significant difference between patient and control groups was detected in the alleles or genotypes of either rs833061 or rs2010963 SNPs. Moreover, the haplotypes were not associated with SLE susceptibility. However, rs833061 and rs2010963 polymorphisms were in linkage disequilibrium according to Dꞌ = 95 % but not according to r2 = 42%. The associations between rs833061 (C vs. T: OR = 0.98, 95% CI = 0.80-1.20, P value = 0.87) and rs2010963 (C vs. G: OR = 0.89, 95% CI = 0.73 1.09, P value = 0.28) with risk of SLE were not significant. The clinical data of the patients, including anti-dsDNA (P value = 0.036), anti-SSA (P value = 0.039), and anti-SSAB (P value = 0.036), were associated with the genotypes of the VEGFA gene rs2010963 SNP. It was recognized that VEGFA gene rs833061 and rs2010963 polymorphisms did not affect SLE susceptibility in the Iranian population.


Introduction
Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disorder in which genetic, epigenetic, and environmental factors interplay determining disease susceptibility [1,2]. The chronic inflammatory responses, immunopathogenic mediators, and several autoantibodies may result in an immune complex-associated vasculitis as well as endothelial damage [3,4]. SLE is seen all over the world [5]. Epidemiological studies have indicated that the global incidence of SLE is between 4 and 7 in 100,000 per year [6,7]. Most SLE patients are affected by the disease at a period of 15 to 40 years, and SLE is predominantly more common in women than men [5]. Despite a decrease in the mortality rate of SLE patients, They still experience an intense physical and psychological burden [8].
According to the suggested hypothesis, the pathogenesis of SLE may be initiated when vascular endothelial cells are damaged and activated [9]. It has been observed that the vascular endothelial growth factor (VEGF) is upregulated in SLE patients [10] and is involved in various biological functions, including endothelial cell proliferation and migration, angiogenesis, and vascular permeability [11]. It has further been established that an increased VEGF level is associated with the disease activity in SLE patients [3,4]. On the other hand, vascular endothelial growth factor receptor 2 (VEGFR2) gene polymorphism has been reported to be involved in the pathogenesis of vascular diseases and may be involved in the endothelial function, and integrity [12]. In addition to the involvement of VEGF level in the disease activity of SLE patients, studies have also indicated that an abnormal VEGF level is associated with the clinical presentations of patients, including higher mean carotid intima media thickness and lupus nephritis [13,14], platelet count [10], and pulmonary hypertension [15]. The chromosome 6p.12 harbors the human VEGFA gene, and genetic single nucleotide polymorphisms (SNPs) in this gene that may cause functional impressions in the coding protein have previously been identified [16,17]. Many studies have implied the role of VEGFA gene polymorphisms in different immunity disorders such as rheumatoid arthritis (RA) and SLE [18,19]. Concerning this, the current study aimed to investigate the possible association of VEGFA gene rs833061 and rs2010963 SNPs with SLE susceptibility in the Iranian population.

Patients and controls
In this association study, 400 SLE patients who fulfilled the American College of Rheumatology (ACR) criteria for the diagnosis of SLE [20] and referred to the rheumatology clinic of Shariati Hospital, Tehran, Iran, were recruited. The baseline characteristics of the 400 SLE patients are provided in Table 1. Moreover, 400 healthy individuals who were age-, sex-, and ethnicity-matched with participants in the SLE group were selected as the control group. Healthy controls had no history of autoimmune or other disorders, either in themselves or in their first-degree relatives. This study gained approval from the Ethical Committee of Tehran University of Medical Sciences. Prior to blood sampling, written informed consent forms was signed by all SLE patients and healthy controls. For genotyping, of peripheral venous blood were collected from all study participants in EDTA-coated tubes using venipuncture.

Genotyping of VEGFA gene polymorphisms
The DNA was extracted from the peripheral blood of the study subjects using the phenol-chloroform DNA extraction approach. All study subjects were genotyped for VEGFA gene rs833061 and rs2010963 polymorphisms using the real-time allelic discrimination TaqMan method (Applied Biosystems, Foster City, USA). The amplification mixture, with a final volume of 10 μl, contained 5 µl TaqMan Master Mix containing Taq DNA polymerase and dNTPs (Applied Biosystems, Foster City, USA), 0.25 µl TaqMan Genotyping Assay mix containing primers and FAM or VIC labeled probes (Applied Biosystems, Foster City, USA), 4.5 μl of genomic DNA (20 -30 ng/μl), and 0.25 μl of H 2 O. Real-time allelic discrimination PCR was performed by StepOnePlus Real-Time PCR system (Applied Biosystems, Foster City, USA), based on the following conditions: initially 60 °C for 30 seconds, then 95 °C for 10 min, then 40 cycles of amplification (95 °C for 15 seconds and 60 °C for 1 min), and finally 60 °C for 30 seconds.

Statistical analysis
The chi-square test was used to survey the association of alleles, genotypes, dominant, and recessive models of inheritance with disease risk. Odds ratios (ORs) with 95% confidence intervals (95% CI) were also measured for each comparison. The Pearson's chi-square or Fisher exact test was used to evaluate the associations between genotype frequency and the clinical data in SLE subjects. The Hardy-Weinberg Equilibrium (HWE) was calculated for the control group in each SNP. For the analysis of allele, genotype, and haplotype frequencies, the SHEsis online tool (http://shesis.bio-x.cn/SHEsis.html) [21] was used. P values less than 0.05 were considered significant in all tests.
With respect to haplotype frequencies, according to the rs833061 (C/T) and rs2010963 (C/G) order of polymor-   phisms, four haplotypes (CC, CG, TC, and TG) were identified, none of which was significantly associated with SLE risk (Table 3). The linkage disequilibrium (LD) analysis indicated that rs833061 and rs2010963 were in LD according to Dꞌ = 95 %, but not according to r 2 = 42% ( Figure 1).
As seen in Table 4, none of the clinical presentations of the SLE subjects was significantly associated with the frequency of the genotypes for the rs833061 SNP. However, the clinical data of the SLE patients, including anti-dsD-NA (P value < 0.001), anti-SSA (P value = 0.001), and anti-SSAB (P value = 0.001) was significantly associated with the genotypes of the rs2010963 SNP.

Discussion
SLE is defined as a multisystemic autoimmune disorder, and autoantibodies developed against several nuclear antigens have been implicated in its pathogenesis [22]. It has been reported that a systemic chronic inflammatory condition in SLE leads to damage in vascular endothelial cells, which in turn culminates in a marked promotion of angiogenic factors synthesis. These factors are involved in vascular permeability, vascular growth, and inflammatory response, resulting in a disruption of vascular network and impairment of several internal organs, which finally represents with complications like kidney and skin dysfunctions [23].
It has been reported that the VEGF gene plays a role in the normal function of lung and kidney and confers a surviving mediator in the neuronal cells [24]. SNPs in angiogenesis-regulating genes may impress the response toward angiogenic factors and, thereupon, modulate the proneness, initiation, and perpetuation of angiogenesis-associated disorders. Multiple SNPs have been discovered in the upstream and 5'-untranslated regions (5'-UTR) of the VEGF gene due to the existence of highly polymorphic sites [16,25]. These polymorphisms may affect protein expression or specific disease status [17]. Moreover, SNPs in the VEGF and VEGFR2 genes have been associated with the development of angiogenesis-dependent diseases [26]. The VEGF level has been reported to be highly heritable [27], and its polymorphisms include at least three SNPs that have been observed to impress the expression of VEGF mRNA. It has been observed that the SNPs at the −2549 (rs35569394) position located in the promoter region and the −634G/C (rs2010963) SNP found in the 5ʹ-UTR are associated with overexpression of VEGF [28]. Furthermore, the 936C/T (rs3025039) SNP located in the 3ʹ-UTR was associated with a significantly higher VEGF level in serum [28]. Tang et al. tried to perform a meta-analysis to disclose the association between the VEGF gene 634G/C (rs2010963) polymorphism and VEGF serum levels with SLE proneness. They found that the VEGF level was associated with increased SLE risk as well as with active SLE risk. They further observed that the VEGF level was associated with the development of lupus nephritis risk in SLE patients [29].
With respect to the studies implying the role of VEGF in the modulation of angiogenesis and endothelial cell proliferation and function, a limited number of studies have surveyed the possible association of VEGF gene polymorphisms with SLE susceptibility as well as its clinical presentations. In line with this, a study tried to investigate the association of the VEGF gene G1612A (rs10434) polymorphism with lupus-related neuropsychiatric manifestations in SLE patients. It was found that the AA genotype of rs10434 was significantly more prevalent in SLE patients with neuropsychiatric manifestations than in the subjects without this manifestation; hence, the AA genotype conferred a susceptibility risk of developing neuropsychiatric manifestations in SLE patients [19]. Moreover, it was found that the C allele VEGF gene rs2010963 SNP was associated with the development of SLE, while the G allele of this polymorphism conferred a protective effect in SLE susceptibility. Moreover, G allele and GG genotypes of rs1570360 were associated with an increased risk of lupus nephritis development [30]. Conversely, rs2010963 SNP was not identified in a recent meta-analysis to be associated with SLE susceptibility [29]. In addition, a meta-analysis of an rs2010963 association with chronic immune-mediated inflammatory diseases resulted in no significant association [31]. In the current study, the association of rs833061 and rs2010963 SNPs with SLE susceptibility were analyzed, and it was observed that none of the alleles, genotypes, or haplotypes of these polymorphisms were involved in the disease risk. This was the first study to evaluate the association of rs833061 SNP with SLE susceptibility.
Nonetheless, neither the rs833061 nor the rs2010963 polymorphism was associated with RA susceptibility in a previous study by the authors in the Iranian population [18]. Moreover, the data testified to a relation between the genotypes of the VEGFA gene rs2010963 SNP and the clinical data of SLE subjects, such as anti-dsDNA, anti-SSA, and anti-SSAB. However, the authors' previous study indicated an association between the genotypes of the VEGFA gene rs833061 SNP and rheumatoid factor (RF) in RA patients.

Conclusion
Considering all the facts, the present study intended to investigate the association of VEGFA gene polymorphisms, including rs833061 and rs2010963, in SLE susceptibility in the Iranian population. It was observed that none of the alleles, genotypes, and haplotypes of these SNPs were associated with the risk of SLE development, despite an LD according to only Dʹ value. Although rs2010963 has not been associated with SLE in previous studies or in other populations, this was the first report of an rs833061 association in SLE that needs to be further investigated in different ethnicities.