Vascular Endothelial Growth Factor Single Nucleotide Polymorphism +405 G/C (rs2010963) is associated with Levels, Infection Severity, and Amputation among South Indian Diabetic Foot Ulcer Patients

Background The regulation of vascular endothelial growth factor (VEGF) by genetic factors in T2DM and DFU still requires thorough investigation. Hence, the present study aimed to investigate the association of VEGF +405 G/C in DFU subjects and correlate it with its circulatory levels, infection severity, and amputation rate. Materials and Methods This study registered a total of 754 participants of which group I: healthy controls (n = 297), group II: T2DM subjects (n = 242), and group III: DFU subjects (n = 215). Genotyping and levels of rs2010963 were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and ELISA, respectively. Results Results of the current study showed a clear decline in circulatory VEGF-A levels in DFU subjects. VEGF-A was decreased in DFU subjects with the mutant “CC” genotype. The mutant “CC” of VEGF +405G/C was also found to be more susceptible to ulcer grade (III and IV) and major amputations. Conclusion VEGF +405G/C SNP is associated with levels, infection severity, and amputation amongst South Indian DFU patients.


Introduction
Te global spread of Type 2 diabetes mellitus (T2DM) and its late complications increases with a serious impact on public health as well as health care systems. It has been well estimated by the International Diabetes Federation (IDF) that, by the year 2017 about 425 million people were afected with T2DM and this fgure is assessed to rise up to 628 million by the year 2045 [1]. Amongst various other T2DM complications, diabetic foot ulcer (DFU) is the most distressing one which frequently ends in nontraumatic foot amputation and with an increased mortality rate [2]. Indeed, earlier studies have well reported that the incidence of amputation among the diabetic population is 10 times higher than that of the nondiabetic population [3]. Although the etiology of a foot ulcer is multifactorial, one prime prognostic factor for the consequence of ulceration is the infection which may cause septic gangrene and thereby leads to foot amputation [4]. Tus, managing DFU entails a complete multidisciplinary method such as glycemia management, debridement, adequate antibiotic therapy, of-loading and surgical revascularization [5,6]. Furthermore, when associated with various other T2DM complications, the genetics of DFU is not yet fully investigated. An earlier report from our laboratory on the South Indian race investigated the association of various single nucleotide polymorphisms (SNPs) in chemokines and cytokines genes with the severity and treatment outcomes of DFU [7]. Apart from the earlier report, there are hardly any other studies available to link SNPs in association with microbial infection load, ulcer severity, and treatment regimen for DFU. Indeed, another study by our team had already shed light on the genetic association of interleukin-6, tumor necrosis factor-α, and stromal-derived factor-1 SNPs in association with its circulatory cytokine levels among South Indian subjects with DFU [8].
Among several other growth factors that are intricated in the progression of late T2DM complications, VEGF has established mounting attention [9][10][11]. It is an efective proangiogenic growth factor that surges vascular permeability in vivo and triggers endothelial cells in vitro [12]. Te gene encoding VEGF is situated on chromosome 6p21.3 and is highly polymorphic and has 8 exons and seven introns [13]. Earlier studies have well documented that VEGF is intricated in the pathogenesis of various T2DM complications [14,15]. Studies had also indicated that the efect on SNPs has functional signifcance afecting the level of mRNA expression [16]. Further, circulatory levels were found elevated among T2DM and its late complications like DFU, diabetic retinopathy, and diabetic nephropathy [17][18][19]. To date, various variants have been identifed in the VEGF gene, in which +405 G/C (rs2010963) SNP has a direct correlation with its circulatory levels. Despite many debatable reports discussing the association of VEGF gene +405 G/C (rs2010963) SNP and its circulatory levels, however, nil reports are seen to assess the association between the VEGF gene and its circulatory efect in subjects with DFU, particularly from our South Indian population.
Tus, the current study was designed to investigate the association of VEGF +405 G/C (rs2010963) in DFU subjects and correlate it with its circulatory levels, infection severity, and amputation rate.

Recruitment of Study Subjects.
In this study, we registered a total of 754 patients that included 3 diferent groups, namely, Group I healthy controls (n � 297; M/F: 150/147), Group II consisting of T2DM patients (n � 242; M/F: 123/ 119), Group III consists of DFU patients (n � 215; M/F: 117/ 98). Further, Group III subjects were segregated into subgroups based on their grades (G1 (n � 84), G2(n � 60), G3(n � 45), and G4 (n � 26)), as per IDSA-IWGDF classifcation of foot infections [20]. All the study participants were unconnected participants of the South Indian race, after obtaining informed consent in agreement with the principles of the Declaration of Helsinki the study was initiated as per the institutional ethical clearance (Ref. No: IHEC/N-002/05/2020). Te exclusion criteria were subjects with type 1 DM, gestational DM, autoimmune, and rheumatic disorders. Diagnosis of healthy controls and T2DM patients was well defned in our earlier laboratory publications and also from other reports [21][22][23]. Diagnosis of DFU was performed by a well-trained team of podiatrists who scored the gradings of infection as per the distinct criteria. Patients with DFU were investigated for the occurrence of osteomyelitis by the probe-to-bone test, microbiologic culture, and imaging procedures. 5 ml venous blood samples were collected in fasting condition to evaluate various clinical and genotypic analyses. Details on the anthropometric, demographic, and surgical procedures/interventions were recorded from the eligible participants. Further, the abbreviations for the acronyms used in the manuscript are detailed in Supplementary (S. Table 1).

Analysis of the Power of the Study.
Tis study was undertaken with 75 subjects in each group. Te sample size for this study was derived from the initial results with an estimated P < 0.05, with a power of 80% and a confdence interval (CI) of 95%.

Genotyping VEGF+405G/C Ser338Phe (rs2010963) SNP.
Te phenol-chloroform method was adopted for DNA extraction. PCR-RFLP was used to genotype the nonsynonymous SNP of VEGF+405G/C (rs2010963) [8]. Te 304 bp segment containing SNP rs2010963 (p.Ser338Phe) in the VEGF gene was amplifed by using the following primers; Forward: 5′-TGTGGATTTTGGAAACCAGCA GA-3″; Reverse: 5′-CGGTGTCTGTCTGTCTGTCCG-3″. As a next step, the amplicon was kept for restriction digestion using the BsmFI enzyme (New England Biolabs, MA). 193 and 111 bands were observed in the existence of a wild genotype (GG) and only one band of 304 bp was observed in the existence of a mutant genotype (CC). Te heterozygous genotype (G/C) was confrmed in the presence of three bands 304, 193, and 111 bps. Plasma VEGF-A levels were quantitatively analyzed using Sandwich ELISA (R&D Systems, MN, USA) and expressed as pg/mL.

Statistical
Analysis. SPSS-20 was used for all statistical purposes. Clinical data are represented as mean ± SD. Genotype distribution and allele frequency were compared across groups using an independence χ2 test with 2 × 2 contingency statistics [24]. Te odds ratio (OR) was calculated with 95% CI as appropriate. Te genetic association of genotypes with diferent stages of ulcer grade and the treatment regimens were carried out using backward logistic regression analysis. Initially, the odds ratios were calculated without including the confounding variables and then the signifcance of its association between the genotypes was tested by adding the confounding variables. P value <0.05 was considered to be statistically signifcant.

Biochemical Investigations on the Recruited Participants.
Te details of the biochemical investigations were illustrated in Table 1. All the clinical investigations (except age) displayed a signifcant diference in T2DM patients when compared to healthy controls. On the other hand, fasting plasma glucose (FPG), postprandial plasma glucose (PPG), HbA1c, triglycerides (TGL), LDL-cholesterol (LDL-c), and urea were signifcantly elevated in DFU patients when compared to T2DM patients, whereas the other biochemical parameters did not show any statistical diferences in the DFU subjects. With respect to other diabetic complications in DFU subjects, 37.2% had diabetic nephropathy, 20.9% had diabetic retinopathy, 16.7% had both retinopathy and nephropathy, 11.2% had myocardial ischemia, 8.4% had coronary artery disease, and 5.6% had cardiovascular disease.

Genetic Association of VEGF +405G/C Ser338-Phe(rs2010963) with the Disease Phenotype.
A total of 754 subjects were genotyped for the VEGF +405G/C (rs2010963) SNP using the PCR-RFLP approach which is depicted in Te allele and genotype frequencies of VEGF SNP were shown in Table 2. Te genotypic distribution of these SNPs was found to be in Hardy-Weinberg equilibrium. Te frequency of minor CC genotypes was increased in DFU subjects (14.88%), followed by T2DM (7.85%), compared to control participants (3.03%) (P < 0.0001). Te frequency of the minor "C" allele showed signifcantly higher among DFU 24.88% and 17.36% in T2DM, respectively. Further, the DFU subjects were subdivided based on their ulcer grades and it was observed that the frequencies of the "C" allele of VEGF SNP were found to be signifcantly increased among Grade-IV infection (57.69%), followed by Grade-III subjects (32.22%) and Grade-II (22.50%), respectively, when compared to grade-I subjects (Table 3).
All data are reported as mean ± SD for continuous variables. BMI-Body mass index; SBP-systolic blood pressure; DBP-diastolic blood pressure; FPG-fasting plasma glucose; PPG-postprandial plasma glucose; HbA1c-glycated hemoglobin; HDL-high-density lipoprotein; LDL-low-density lipoprotein. * P < 0.05; * * P < 0.01; * * * P < 0.001. Ϯindicates comparison was made between T2DM and healthy controls subjects. ϮϮ indicates comparison was made between T2DM and DFU subjects. Evidence-Based Complementary and Alternative Medicine 3 In addition, to reconfrm, the association of VEGF +405G/C in both T2DM and DFU patients was found by calculating an Odds Ratio (OR). In this logistic regression analysis, the "GG" genotype was taken as the reference genotype, and when the healthy control group was taken as a reference and compared with T2DM and DFU subjects, the results of the study showed that the "CC" genotype conferred signifcant risk towards DFU (OR: 6.0; 95% CI: 2.4-8.3; P � 0.0001) andT2DM (OR: 2.2; 95% CI: 1.1-5.2; P � 0.001) over the GG genotype, even after adjusting for its potential confounders like BMI, age, and gender. Te unadjusted OR for the "C" allele in the DFU group was observed as 2.25 (95% CI: 1.63-3.1; P � 0.0001), for the T2DM group it showed 1.43 (95% CI: 1.0-2.0; P � 0.03), as compared to "C" allele of controls (Table 4). Next, the odds ratio was generated using the T2DM group as the reference type and compared it with DFU subjects. Te study fndings showed that the "CC" genotype confers signifcant susceptibility for DFU subjects with OR as 2.7 (95% CI: 1.0-3.5; P � 0.001) after adjusting for various confounding factors like BMI, age, and gender. Te unadjusted OR for the "C" allele in DFU subjects was observed as 1.5 (95% CI: 1.1-2.1; P � 0.004), in comparison to "G" allele of T2DM subjects. As an overall result, the mutant "CC" genotype of VEGF +405G/C Ser338Phe (rs2010963) SNP exhibited a risk for both T2DM and DFU (Table 4). Table 5 shows the genetic association of the VEGF genotypes with the ulcer grades and treatment regimen in DFU subjects. Using backward logistic regression analysis, it was found that the recessive model genotype CC vs GG + GC conferred signifcant risk for Grades III and IV ulcer (OR: 4.3; 95% CI: 1.5-78.8; P � 0.0001) after adjusting for confounding variables like age, sex, and BMI. Figure 2 shows the percentage of various surgical treatment regimens performed on the recruited. Out of a total of 215 DFU, debridement (DB) was the most common form of treatments-115 (53%) followed by minor amputation: toe amputation-52 (25%) and trans metatarsal amputation-28 (15%) and major amputation: below knee amputation-15 (7%) and above knee amputations-5 (2%). Te genetic association of VEGF genotypes was computed with respect to the treatment procedures for DFU subjects using backward logistic regression analysis, it was found that subjects with recessive model genotype CC vs GG + GC conferred signifcant risk for major amputation procedures (OR: 1.9; 95% CI: 1.1-2.7; P � 0.004), after adjusting for various confounding factors (Table 5). Tus, our overall results showed that the mutant genotype "CC" was found to be more susceptible to ulcer grade (III and IV) and major amputations (below and above knee amputations).

Circulatory Plasma Levels of VEGF-A in the Study
Participants. As can be seen in Figure 3 the median circulatory VEGF-A levels were found to be signifcantly decreased among DFU subjects (76.3 (56.3-81.8); P � 0.007), as compared to that of healthy group participants (118.6 (98.8-141.1)). Although the circulatory levels of VEGF-A were found to be increased in T2DM (135.6 (128.0-156.3); P � 0.006), we found a loss in statistical signifcance as in comparison with healthy controls. As a next step, the comparison was done between T2DM and DFU groups, and the results displayed a signifcant decrease in the levels of DFU (P � 0.004). Figure 4 illustrates an association between VEGF +405G/C Ser338-Phe (rs2010963) SNP and circulatory VEGF-A levels. To fnd this association, we segregated mean values of VEGF-A based on their genotype identifed. In the case of the mutant "CC" genotype, our results explored the median VEGF-A levels to be signifcantly reduced in DFU subjects (60.

Discussion
Earlier fndings from our laboratory had reported an increased infammatory cytokine that was associated with the circulation of DFU patients and thus impair the proper wound-healing process [25]. Tus, chronic infammation which is coupled with infection delays wound healing and thereby results in amputations. On the other hand, VEGF efectively participates in vascular and angiogenic functions that are usually associated with the development of diabetic complications [26]. Hence, this present study explored the association of VEGF +405 G/C (rs2010963) in subjects with DFU which may provide a chance to measure the impact of a candidate gene on the progression of DFU and also, we have correlated the efect of SNP with its phenotypic impact on the levels, infection severity, and amputation.    Although numerous SNPs in the VEGF gene had been reported, the SNP at +405 G/C (rs2010963) in the 5′ untranslated region has a direct efect on the release of VEGF. Further, the GG genotype was also found to be associated with an elevated VEGF production among healthy persons [27]. To the best of our knowledge, the present study is the frst one to explore the association of VEGF +405 G/C SNP genotype (rs2010963) and DFU along with infection severity and amputation rates. To date, there are hardly three case control studies available in the literature on VEGF SNP, particularly among DFU subjects. Te frst study by Amoli et al. [28] from Iranian DFU patients (but not with the same genotype chosen as per the present study), reported that the "C" allele of VEGF -278C/A was associated with the progression of DFU, however, one limitation of the study is that the VEGF levels in DFU patients were not measured and correlated with the clinical parameters of DFU patients. Te second study by Li et al. [29] explored two diferent SNPS of VEGF (rs699947 andrs13207351) among Chinese Han DFU patients and reported that −2578 C/A (rs699947) conferred signifcant protection for the development of DFU. Te apparent disparity between the aforementioned study and our study could be due to diferences in race. Te third study by Li et al. [17] reported that the GG genotype of VEGF-634G/C SNP conferred signifcant risk for DFU patients.
Te results of the current study had shown that the CC genotype of VEGF +405 G/C SNP conferred signifcant risk for both T2DM and DFU patients. Our results are in agreement with a study that explored diferent disease conditions. Farhangi et al. [30] documented an association between the CC genotype of VEGF +405 G/C SNP and dietary diversity score among patients with metabolic syndrome. In contrast to the present study, Nikzamir et al. reported that the GG genotype of VEGF +405 G/C SNP is independently related to the progression of diabetic nephropathy [31]. Two controversial fndings on diferent disease conditions in the same variant of VEGF +405G/C    [32]. Another study by Guruvaiah et al. [33] had also shown that the +405G/C in which the "G" allele conferred signifcant risk among South Indian polycystic ovary syndrome. Possible reasons for these discrepancies could be due to diferent disease subjects adopted for the study. Another reason for this disparity is the sample size in both the aforementioned studies was comparatively lesser than in the present study. Another important aim is to estimate the plasma VEGF-A levels in DFU patients. Te results showed that circulatory levels are signifcantly decreased among DFU subjects when compared to T2DM. Tis fnding is in agreement with another study by Li et al., who had shown a decreased expression of VEGF levels in DFU patients when compared with the T2DM population from the Han Chinese population [17]. On the other hand, a study from a similar race to ours, but under a diferent disease condition had shown no association between VEGF +405 G/C SNP (rs2010963) and its levels among subjects with psoriasis [15].
Further, the results of the present study showed that the CC genotype of DFU patients was associated with a decrease in circulatory levels. Tis result was in accordance with an earlier study by Watson et al., who had conducted an in vitro study and identifed a signifcant correlation between lipopolysaccharide-stimulated peripheral blood mononuclear cell VEGF protein production and VEGF +405 genotypes. It was also found that +405 CC showed decreased protein than +405 GG genotypes [34].
In addition, the present study was designed to observe the relationship between the VEGF +405 G/C SNP genotypes and correlate it with the outcome of treatment procedures for DFU. Te present study results had shown that the mutant genotype "CC" was found to be more susceptible to develop higher DFU grades and amputations. Although reports on the association of VEGF (rs2010963) genotypes and treatment outcomes are less inconsistent, our results agree with an earlier study conducted by Viswanathan et al. on DFU from the South Indian race [7]. Tey showed that increased risk for developing higher grades of DFU and amputations among, IL-6 (-174G/C), SDF-1 (+801G/A), and TNF-α (-308G/A) genotypes. Tus, our present study on VEGF SNP has emerged as a strong risk factor for developing DFU.

Conclusion
To the best of our knowledge, this is the frst study on VEGF +405 G/C SNP to steadily compare the genetic association with the outcome of ulcer severity and treatment procedures in an alarming disease condition such as DFU. However, the major limitation of our study is its cross-sectional nature. Going forward, our team will focus on assessing the physiological validity and relevance of the circulatory levels observed among the diferent grades of DFU. Terefore, the study concludes that VEGF +405 G/C SNP has a genetic susceptibility factor, which would assist in preventing amputation.

Data Availability
Te data that support the fndings of this study are available from the corresponding authors upon reasonable request.

Additional Points
Highlights of the Study . (1) Te mutant "CC" genotype of VEGF +405G/C Ser338Phe (rs2010963) SNP exhibited a signifcant risk towards T2DM and DFU. (2) VEGF-A levels were signifcantly decreased among DFU subjects when compared to T2DM. Further, VEGF-A was declined in DFU patients with mutant "CC" than the wild "GG" genotype. (3) Te mutant genotype "CC" of VEGF +405G/C was found to be more susceptible to ulcer grade (III and IV) and major amputations (below and above knee amputations).

Conflicts of Interest
Te authors declare that they have no conficts of interest.