Vascular Endothelial Growth Factor Haplotypes Associated with Childhood Obesity

Abstract

Expansion of adipose tissue in obesity is associated with angiogenesis and adipose tissue mass depends on neovascularization. Vascular endothelial growth factor (VEGF) is the main angiogenic factor in the adipose tissue, and VEGF expression is tightly regulated at both transcriptional and translational levels. However, no previous study has tested the hypothesis that genetic polymorphisms in the VEGF gene could affect susceptibility to obesity. To test this hypothesis, we compared the distribution of genotypes and haplotypes including three VEGF genetic polymorphisms in obese children and adolescents with those found in healthy controls. We studied 172 healthy children and adolescents and 113 obese children and adolescents. Genotypes of three clinically relevant VEGF polymorphisms in the promoter region (C−2578A, G−1154A, and G−634C) of the VEGF gene were determined by TaqMan allele discrimination assay and real-time polymerase chain reaction. VEGF haplotypes were inferred using Haplo.stats and PHASE 2.1 programs. We found no differences in the distributions of VEGF genotypes and alleles (p > 0.05). However, the CAG haplotype was more frequent in the obese group than in the control group (4% versus 0%, respectively, in white subjects; p = 0.008; odds ratio = 10.148 (95% confidence interval: 1.098–93.788). Our findings suggest that VEGF haplotypes affect susceptibility to obesity in children and adolescents.

Introduction

O besity is reaching epidemic proportions worldwide, and its prevalence is increasing among children and adolescents, likely causing several cardiovascular complications in adults (Chinn and Rona, 2001). Important features of this condition include expansion of adipose tissue mass with increased size and number of mature adipocytes, extracellular matrix remodeling, and angiogenesis (Lijnen, 2008). The expansion of adipose tissue is associated with angiogenesis and inhibition of angiogenesis prevents its development, thus suggesting that adipose tissue mass depends on neovascularization and therefore could be regulated through the vasculature (Rupnick et al., 2002; Brakenhielm et al., 2004; Kolonin et al., 2004; Kim et al., 2010).

Vascular endothelial growth factor (VEGF) is the main angiogenic factor in adipose tissue. VEGF is a mediator of vascular permeability, and stimulates proliferation and migration of endothelial cells (Fain and Zhang et al., 1997; Madan, 2005; Cao, 2007; Ledoux et al., 2008; Lijnen, 2008). The expression and activity of VEGF are tightly regulated at both transcriptional and translational levels (Loureiro and D'Amore, 2005). In this respect, hypoxia is the most important mechanism for VEGF modulation (Pugh and Ratcliffe, 2003; Roy et al., 2006; Nagy et al., 2007), and there is strong evidence suggesting that hypoxia may cause adipose tissue dysfunction in obesity (Trayhurn et al., 2008; Wood et al., 2009). These findings give support to a major role for VEGF in obesity because VEGF affects angiogenesis in adipose tissue, and weight reduction with adipose tissue loss has been reported in obese mice treated with inhibitors of angiogenesis (Rupnick et al., 2002; Brakenhielm et al., 2004). In addition, clinical studies have shown that VEGF is probably involved in the development of obesity in humans, as suggested by increased VEGF levels in obese subjects (Miyazawa-Hoshimoto et al., 2003; Silha et al., 2005; Garcia de la Torre, et al. 2008; Gomez-Ambrosi et al., 2010), which decrease after weight loss (Garcia de la Torre et al., 2008; Gomez-Ambrosi et al., 2010).

The relevance of VEGF for development of obesity suggests that genetic polymorphism in the VEGF gene could affect the susceptibility to this metabolic disorder. Recent studies have associated three clinically significant single-nucleotide polymorphisms localized in promoter region (C−2578A, G−1154A, and G−634C) with disease (Del Bo et al., 2005; Biselli et al., 2008; Churchill et al., 2008; Douvaras et al., 2009; Sandrim et al., 2009). However, no previous study has examined whether genetic polymorphisms in the VEGF gene affect susceptibility to the development of obesity. To test this hypothesis, we compared the distribution of genotypes and haplotypes of three VEGF genetic polymorphisms in obese children and adolescents with those found in healthy children and adolescents.

Materials and Methods

Subjects

Approval for use of human subjects in this study was obtained from the Institutional Review Board at the Federal University of Juiz de Fora, Brazil. Parents and children were informed about the nature and purpose of this study. Parents gave their written consent and children gave their verbal consent.

The study participants included 113 normotensive obese subjects recruited from the Endocrinology Ambulatory of the Adolescent and Child Institute at Juiz de Fora and from the Childhood Endocrinology Ambulatory of the IMEPEN Foundation at Juiz de Fora. The control group consisted of 172 healthy children and adolescents recruited from the local community.

All children underwent thorough physical examination. Height was measured to the nearest 0.1 cm by using a wall-mounted stadiometer and body weight was measured with a digital scale to the nearest 0.1 kg. Body mass index (BMI) was calculated as the weight in kilograms divided by height in meters squared. Obesity was defined as BMI greater than the 95th percentile, matched according to age and sex (Kuczmarski et al., 2000). Systolic and diastolic blood pressures were measured at least three times and the presence of hypertension was defined as SBP and/or DBP exceeding the 95th percentile. Children with hypertension were not included in this study.

At the time of clinic attendance, venous blood samples were collected from the study participants and genomic DNA was extracted from the cellular component of 1 mL of whole blood by a salting-out method and stored at −20°C until analyzed.

Laboratory analyses

Glucose and lipid (total cholesterol, triglycerides, and high-density lipoprotein [HDL] cholesterol) concentrations were determined in plasma and serum, respectively, with routine enzymatic methods using commercial kits (Labtest Diagnostic, SA, Lagoa Santa, Brazil). Low-density lipoprotein (LDL) concentration was calculated according to the Friedewald formula.

Genotype determination

Three clinically relevant polymorphisms in the VEGF gene were studied: C−2578A (rs699947), G−1154A (rs1570360), and G−634C (rs2010963). Genotypes were determined using the TaqMan^® Allele Discrimination assay (Applied Biosystems, Foster City, CA) (Goncalves et al., 2010). Probes and primers used were designed by Applied Biosystems (Assay ID: C_8311602_10/-2578; C_1647379_10/-1154; C_8311614_10/-634). TaqMan polymerase chain reaction (PCR) was performed in a total volume of 12 μL (3 ng of dried DNA, 1X TaqMan master mix, 900 nM of each primer and 200 nM of each probe) placed in 96-well PCR plates. Fluorescence from PCR amplification was detected using Chromo 4 Detector (Bio-Rad Laboratories, Hercules, CA) and analyzed with manufacturer's software. The PCR assay was carried out following the manufacturer's instructions (Applied Biosystems) that include one step of 10 min at 95°C (Ultra Pure AmpliTaq Gold^® DNA Polymerase Enzyme Activation) followed by 40 cycles of DNA denaturation at 92°C for 15 s and annealing/extension step at 60°C for 1 min.

Statistical analysis

The clinical characteristics of study groups were compared by unpaired Student t-test in case of normally distributed variables. The Mann-Whitney U-test was used to compare non-normally distributed variables.

The distribution of genotypes for each polymorphism was assessed for deviation from the Hardy-Weinberg equilibrium, and differences in genotype frequency and allele frequency between groups were assessed using chi-squared tests. A value of p < 0.05 was considered to be statistically significant.

The Haplo.stats package (version 1.4.0; www.r-project.org) was used to estimate the haplotype frequencies. The function haplo.em computes maximum likelihood estimates of haplotype probabilities using the progressive insertion algorithm, which progressively inserts batches of loci into haplotypes of growing lengths. The function haplo.score was used to compute haplotype-specific score statistics to test for association (Schaid et al., 2002), with the value of p < 0.05 to be statistically significant. Only the haplotypes with frequencies greater than 1% were taken into consideration for the haplotype-specific score analysis. To further confirm haplotype estimates and frequencies comparisons between groups, we also used the Bayesian statistical based program PHASE version 2.1 (www.stat.washington.edu/stephens/software.html), as well as to calculate odds ratio (OR) and 95% confidence intervals (CIs) for each haplotype. The possible haplotypes including genetic variants of three polymorphisms in the VEGF gene studied (C−2578A; G−1154A; and G−634C) were: H1 (CGC); H2 (CGG); H3 (CAG); H4 (CAC); H5 (AGG); H6(AGC); H7 (AAG); and H8 (AAC).

Results

The clinical and laboratorial characteristics of the study groups are presented in Table 1. As expected, subjects in the obese group had higher BMI than controls (p < 0.05; Table 1). In addition, obese subjects had increased SBP, and higher LDL cholesterol and triglycerides levels than controls (all p < 0.05; Table 1). Obese subjects had lower HDL cholesterol levels than controls (p < 0.05; Table 1).

Table 1.

Demographic Characteristics of Study Participants

Parameters	Controls	Obese
N	172	113
Age (years)	11.9 ± 3.1	10.3 ± .2.7^a
Ethnicity (% white)	51	53
BMI (kg/m²)	17.7 ± 2.2	26.1 ± 4.4^a
SBP (mmHg)	105.5 ± 11.7	111.1 ± 10.1^a
DPB (mmHg)	65.6 ± 9.0	70.6 ± 8.8^a
Fasting glucose (mg/dL)	81.4 ± 10.8	85.3 ± 8.8
Total cholesterol (mg/dL)	141.9 ± 42.5	147.9 ± 39.9
LDL cholesterol (mg/dL)	74.6 ± 23.0	89.3 ± 31.5^a
HDL cholesterol (mg/dL)	43.6 ± 8.9	38.7 ± 9.9^a
Triglycerides (mg/dL)	76.8 ± 28.7	88.8 ± 43.4^a

p < 0.05 versus controls.

Values are the mean ± standard deviation.

BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; LDL, low-density lipoprotein; HDL, high-density lipoprotein.

Table 2 shows VEGF genotypes and allele frequencies in the two study groups. The distribution of genotypes for each polymorphism showed no deviation from Hardy-Weinberg equilibrium (all p > 0.05). As significant interethnic differences exist in the distribution of VEGF polymorphisms (Muniz et al., 2009), we carried out two different analyses. The first analysis included black and white children, whereas the second analysis took into consideration only white children, which corresponded to 50%–56% of the subjects. Both analyses showed no significant differences in genotype and allele distributions for the three VEGF polymorphisms when the control group was compared with the obese group (all p > 0.05; Table 2).

Table 2.

Genotypes and Alleles Frequencies in the Control Group and in the Obese Group (White and Black or Only White) Shown as N (%)

	All patients (White and black)			White patients only
Polymorphism	Control (N = 172)	Obese (N = 113)	p-Value ^a	Control (N = 96)	Obese (N = 57)	p-Value ^a
C-2578A
CC	72 (42)	47 (42)		38 (40)	17 (30)
CA	77 (45)	53 (47)	ns	44 (46)	32 (56)	ns
AA	23 (13)	13 (12)		14 (15)	8 (14)
C allele	221 (64)	147 (65)	ns	120 (63)	66 (58)	ns
A allele	123 (36)	79 (35)		72 (37)	48 (42)
G-1154A
GG	103 (60)	67 (59)		55 (57)	31 (54)
GA	61 (35)	35 (31)	ns	35 (36)	19 (33)	ns
AA	8 (5)	11 (10)		6 (6)	7 (12)
G Allele	267 (78)	169 (75)	ns	145 (76)	81 (71)	ns
A Allele	77 (22)	57 (25)		47 (24)	33 (29)
G−634C
GG	72 (42)	46 (41)		42 (44)	25 (44)
GC	82 (48)	52 (46)	ns	44 (46)	24 (42)	ns
CC	18 (10)	15 (13)		10 (10)	8 (14)
G allele	226 (66)	144 (64)	ns	128 (67)	74 (65)	ns
C allele	118 (34)	82 (36)		64 (33)	40 (35)

p-Value for control versus obese.

ns, not significant.

We found significant differences in the distributions of VEGF haplotype frequencies (p < 0.05; Table 3). When white and black subjects were taken into consideration, the H3 haplotype including the variants CAG was more frequent in the obese group than in the control group (Table 3, OR = 2.255, CI = 0.676 to 7.515). When we took into consideration only white subjects, the same CAG haplotype was more frequent in obese group than in the control group (Table 3, OR = 10.148, CI = 1.098 to 93.788). No other significant differences were found in haplotype frequencies distribution when the two groups were compared. Very similar results were found when Haplo.stats and PHASE version 2.1 were used to estimate the haplotype frequencies (data not shown).

Table 3.

Estimated Haplotypes Frequencies in Control Group and Obese Group (White and Black or Only White) Shown as N (%)

				All patients (white and black)				White patients only
Haplotype				Control	Obese	p-Value ^a	OR (CI)	Control	Obese	p-Value ^a	OR (CI)
H1	C	G	C	111 (32)	77 (34)	ns	1.000 (Reference)	19 (10)	15 (13)	0.528	1.357 (0.755–2.440)
H2	C	G	G	107 (31)	62 (28)	ns	0.881 (0.556–1.395)	101 (53)	47 (41)	0.064	1.000 (Reference)
H3	C	A	G	0 (0)	4 (2)	0.032^b	2.255 (0.676–7.515)	0 (0)	4 (4)	0.008^b	10.148^b (1.098–93.788)
H4	C	A	C	2 (1)	4 (2)	ns	2.255 (0.676–7.515)	0 (0)	0 (0)	—	—
H5	A	G	G	48 (14)	30 (13)	ns	0.909 (0.521–1.585)	59 (31)	37 (32)	0.745	1.460 (0.792–2.690)
H6	A	G	C	0 (0)	0 (0)	-	-	1 (0)	1 (1)	0.707	2.149 (0.132–35.130)
H7	A	A	G	70 (21)	48 (21)	ns	0.976 (0.612–1.557)	12 (6)	10 (9)	0.539	1.236 (0.589–2.590)
H8	A	A	C	4 (1)	1 (0)	ns	2.255 (0.676–7.515)	0 (0)	0 (0)	—	—

VEGF haplotypes included genetic variants for three VEGF polymorphisms in promoter region (C−2578A, G−1154A, and G−634C).

p-Value for control versus obese.

p < 0.05.

CI, 95% confidence interval; OR, odds ratio for control versus obese; VEGF, vascular endothelial growth factor.

Discussion

This was the first study to investigate the association of three single-nucleotide polymorphisms localized in the VEGF promotor and obesity. The main finding was that the CAG haplotype was more common in obese children and adolescents than in the control group, and this finding suggests that VEGF haplotypes are associated with variable susceptibility to obesity in children and adolescents.

Obesity requires the formation of new blood vessels to supply increased size and number of mature adipocytes with nutrients and oxygen (Lijnen, 2008). This process involves VEGF, which is the most important angiogenic factor responsible for the expansion of adipose tissue (Lijnen, 2008) and recent reports have attempted to show the role played by VEGF in the pathogenesis of obesity (Rupnick et al., 2002; Miyazawa-Hoshimoto et al., 2003; Brakenhielm et al., 2004; Silha et al., 2005; Garcia de la Torre et al., 2008; Gomez-Ambrosi et al., 2010). Accordingly, the manipulation of VEGF has been suggested as a possible pharmacological target in the therapy of obesity (Gomez-Ambrosi et al., 2010). Consistent with these recent findings, genetic variations in the VEGF gene may affect susceptibility to obesity, as we have found in the present study.

There is evidence for a genetic contribution to differences in VEGF expression levels (Watson et al., 2000; Stevens et al., 2003). While we have not attempted to demonstrate the functionality of the VEGF polymorphisms in the present study, previous findings showed that these polymorphisms are associated with variable circulating VEGF levels. For example, the alleles −1154A and −2578A for the G⁻¹¹⁵⁴A and C−2578A polymorphisms, respectively, were previously associated with lower circulating VEGF levels when compared with the −1154G and −2578C alleles, respectively (Shahbazi et al., 2002; Lambrechts et al., 2003). However, conflicting results have been reported in relation to the G−634C polymorphism. Whereas one study showed higher serum VEGF levels in subjects with −634 CC genotype (Awata et al., 2002), another study showed higher VEGF production by stimulated mononuclear cells in individuals with the −634 GG genotype (Watson et al., 2000). In the present study, we found no significant differences in genotypes and alleles distributions for the three VEGF polymorphisms. Conversely, the CAG haplotype was more common in obese children than in healthy controls, thus supporting the notion that the examination of combinations of polymorphisms (haplotypes) rather than only one polymorphism provides much more accurate genetic information (Crawford and Nickerson, 2005). While we have no precise mechanism that may help to explain this significant association of one VEGF haplotype with obesity, our findings are in line with recent studies suggesting a role for VEGF in the pathogenesis of obesity (Rupnick et al., 2002; Miyazawa-Hoshimoto et al., 2003; Brakenhielm et al., 2004; Silha et al., 2005; Garcia de la Torre et al., 2008; Gomez-Ambrosi et al., 2010). It remains to be determined whether the CAG haplotype (which was found in association with obesity) increases VEGF levels.

Although no previous study has examined whether genetic polymorphisms in the VEGF gene affect the susceptibility to the development of obesity, either in children and adolescents or in adults, recent reports have showed significant association of VEGF polymorphisms and cardiovascular diseases (Biselli et al., 2008; Douvaras et al., 2009; Sandrim et al., 2009). Some differences between studies may have resulted from significant interethnic differences in the distribution of VEGF polymorphisms (Muniz et al., 2009). In the present study, we carried out two different analyses that have lead to very similar results. The first analysis included black and white children, whereas the second analysis took into consideration only white children. Both analyses showed that CAG haplotype was more common in obese children and adolescents than in the control group. These findings are consistent with the suggestion that genetic determinants of complex diseases may be associated with disease conditions independently of ethnicity, as previously reported (Sandrim et al., 2006, 2007, 2008). While our findings suggest that race or ethnicity does not matter, it is clear that obesity is not a consequence of genetic variations in the VEGF gene only, and other genes are clearly involved, as well as environmental factors.

In conclusion, we found significant differences in the distribution of VEGF haplotypes when obese children and adolescents were compared with a control group. Our findings suggest that the CAG haplotype is associated with increased susceptibility to obesity. Further studies examining the possible interactions of VEGF haplotypes with environmental factors and with other genetic markers involved in the development of obesity and its complications are warranted.

Footnotes

Acknowledgments

This study was supported by Fundaçao de Amparo a Pesquisa do Estado de Sao Paulo, Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, and IMEPEN Foundation at Juiz de Fora.

Disclosure Statement

No competing financial interests exist.

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