Vascular endothelial growth factor (−460 C/T, +405 G/C, and +936 C/T) polymorphisms and endometriosis risk in Tunisian population

Figures & data

Table 1. Restriction fragment length polymorphism conditions for identification of VEGF gene polymorphisms.

VEGF SNPs	PCR Primers	PCR product	Restriction enzymes	Restriction fragment length
−460 C/T (rs833061)	5′-TGTGCGTGTGGGGTTGAGCG-3′ (F) 5′-TACGTGCGGACAGGGCCTGA-3′ (R)	175 bp	Bsh12361 (62°C)	175 bp (T) 155 + 20 bp (C)
+405 G/C (rs2010963)	5′-ATTTATTTTTGCTTGCCATT-3′ (F) 5′-GTCTGTCTGTCTGTCCGTCA-3′ (R)	304 bp	FaqI (57°C)	304 bp (C) 203 + 101 bp (G)
+936 C/T (rs3025039)	5′-AGGGTTCGGGAACCAGATC-3′ (F) 5′-CTCGGTGATTTAGCAGCAAG-3′ (R)	266 bp	Hin1II (66°C)	266 bp (C) 211 + 55 bp (T)

VEGF: vascular endothelial growth factor; SNP: single-nucleotide polymorphism; PCR: polymerase chain reaction; bp: base pairs; F: forward; R: reverse.

Table 2. Distribution of genotypes and alleles frequencies of VEGF polymorphisms in endometriosis patients and controls.

	Population
VEGF polymorphisms	Patients n = 105	Controls n = 150	P value	OR (95% CI)
−460C/T Genotype
CC	15 (14.28%)	29 (19.33%)		1.00^a
CT	53 (50.48%)	67 (44.67%)	0.32	1.52 (0.74–3.14)
TT	37 (35.24%)	54 (36%)	0.58	1.32 (0.62–2.8)
TT + CT	90 (85.72%)	121 (80.67%)	0.37	1.43 (0.72–2.84)
−460C/T Allele
C	83 (39.52%)	125 (41.67%)		1.00^a
T	127 (60.48%)	175 (58.33%)	0.69	1.09 (0.72–1.56)
+405G/C Genotype
GG	37 (35.24%)	61 (40.67%)		1.00^a
GC	44 (41.9%)	68 (45.33%)	0.93	1.06 (0.61–1.86)
CC	24 (22.86%)	21 (14%)	0.11	1.88 (0.92–3.84)
GC + CC	68 (64.76%)	89 (46.47%)	0.45	1.26 (0.75–2.11)
+405G/C Allele
G	118 (56.19%)	190 (63.33%)		1.00^a
C	92 (43.81%)	110 (36.67%)	0.12	1.34 (0.93–1.93)
+936C/T Genotype
CC	62 (59.05%)	117 (78%)		1.00^a
CT	33 (31.43%)	26 (17.33%)	0.006*	2.39 (1.31–4.36)
TT	10 (9.52%)	7 (4.67%)	0.08	2.69 (0.97–7.42)
TT + CT	43 (40.95%)	33 (22%)	0.001*	2.45 (1.42–4.25)
+936C/T Allele
C	157 (74.76%)	260 (86.67%)		1.00^a
T	53 (25.24%)	40 (13.33%)	0.0009*	2.19 (1.39–3.46)

VEGF: vascular endothelial growth factor; OR: odds ratio; CI: confidence interval; *significant (P value) < 0.05; ^agenotype or allele category accepted as reference group.

Table 3. Distribution of genotypes and alleles frequencies of VEGF polymorphisms in stages I-II and stages III-IV endometriosis patients and controls.

VEGF polymorphisms	Endometriosis stages I-II n = 63	Endometriosis stages III-IV n = 42	P^b	OR^b (95% CI)	P^c	OR^c (95% CI)
−460C/T Genotype
CC	9 (14.28%)	6 (14.28%)		1.00^a		1.00^a
CT	29 (46.04%)	24 (57.14%)	0.58	1.39 (0.58–3.31)	0.39	1.73 (0.64–4.68)
TT	25 (39.68%)	12 (28.58%)	0.50	1.49 (0.61–3.61)	0.88	1.07 (0.36–3.15)
CT + TT	54 (85.72%)	36 (85.72%)	0.49	1.43 (0.63–3.24)	0.60	1.43 (0.55–3.73)
−460C/T Allele
C	47 (37.3%)	36 (42.85%)		1.00^a		1.00^a
T	79 (62.7%)	48 (57.15%)	0.46	1.2 (0.78–1.84)	0.94	0.95 (0.58–1.55)
+405 G/C Genotype
GG	21 (33.33%)	16 (38.1%)		1.00^a		1.00^a
GC	29 (46.03%)	15 (35.71%)	0.63	1.23 (0.64–2.39)	0.81	0.84 (0.38–1.84)
CC	13 (20.64%)	11 (26.19%)	0.25	1.79 (0.76–4.21)	0.21	1.99 (0.8–4.98)
GC + CC	42 (66.67%)	26 (61.9%)	0.39	1.37 (0.73–2.54)	0.9	1.11 (0.55–2.24)
+405 G/C Allele
G	71 (56.35%)	47 (55.95%)		1.00^a		1.00^a
C	55 (43.65%)	37 (44.05%)	0.21	1.33 (0.87–2.04)	0.27	1.36 (0.83–2.32)
+936C/T Genotype
CC	40 (63.5%)	22 (52.38%)		1.00^a		1.00^a
CT	20 (31.74%)	13 (30.95%)	0.03*	2.25 (1.13–4.46)	0.02*	2.65 (1.18–5.95)
TT	3 (4.76%)	7 (16.67%)	0.95	1.25 (0.3–5.08)	0.005*	5.31 (1.69–16.67)
CT + TT	23 (36.5%)	20 (47.62%)	0.04*	2.03 (1.07–3.87)	0.002*	3.22 (1.57–6.61)
+936C/T Allele
C	100 (79.37%)	57 (67.85%)		1.00^a		1.00^a
T	26 (20.63%)	27 (32.15%)	0.07	1.69 (0.98–2.91)	0.0001*	3.07 (1.74–5.42)

VEGF: vascular endothelial growth factor; OR: odds ratio; CI: confidence interval; *significant (P value) <0.05; ^agenotype or allele category accepted as reference group; ^bstage I-II endometriosis patients versus controls; ^cstage III-IV endometriosis patients versus controls.

Figure 1. Comparison of VEGF serum levels between endometriosis patients and controls.

Figure 2. Comparison of VEGF serum levels with VEGF genotypes in endometriosis patients and controls.

Table 4. Haplotype frequencies of VEGF −460C/T and +405G/C polymorphisms in endometriosis patients and controls.

VEGF haplotypes		Haplotype frequency
−460	+405	Endometriosis (%)	Control (%)	OR (95% CI)	P value
T	G	34.04	38.24	1.00^a	–
C	G	22.16	25.09	0.96 (0.55–1.68)	0.90
T	C	26.45	20.08	1.39 (0.84–2.27)	0.19
C	C	17.35	16.59	1.16 (0.68–1.98)	0.56

VEGF: vascular endothelial growth factor; OR: odds ratio; CI: confidence interval; Significant (P value) < 0.05; ^areference group.

Table 5. A summary of association studies between VEGF gene polymorphisms and endometriosis risk.

				VEGF SNPs
				−460 C/T	+405 G/C	+936 C/T
Country	Ethnicity	Patients	Controls	rs833061	rs2010963	rs3025039	Reference
China	Asian	122	131	<0.05	–	–	Hsieh et al. [2004]
India	Asian	215	210	NS*	<0.05	–	Bhanoori et al. [2005]
China	Asian	215	219	NS	<0.05	–	Kim et al. [2005]
Japan	Asian	147	181	NS	NS	<0.05	Ikuhashi et al. [2007]
Korea	Asian	105	101	–	–	NS	Kim et al. [2008]
Australia	Caucasian	958	959	NS	NS	NS	Zhao et al. [2008]
Spain	Caucasian	186	180	NS	NS	<0.05	Cosín et al. [2009]
China	Asian	344	360	NS	–	NS	Liu et al. [2009]
Turkey	Caucasian	52	60	NS	<0.05	–	Attar et al. [2010]
Estonia	Caucasian	150	199	–	NS	NS	Lamp et al. [2010]
Turkey	Caucasian	98	94	NS	<0.05	–	Altinkaya et al. [2011]
Iran	Asian	480	600	NS	<0.05	–	Emamifar et al. [2012]
Tunisia	African	105	150	NS	NS	<0.05	Our study

VEGF: vascular endothelial growth factor; SNP: single nucleotide polymorphism; *significant p value <0.05.

NS*: non significant P value.

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