Lack of Association between �460 C / T and 936 C / Tof the Vascular Endothelial Growth Factor

and Angiopoietin-2 Exon 4 G /A Polymorphismsand Ovarian, Cervical, and Endometrial Cancers

ECE KONAC,1 H. ILKE ONEN,1 JALE METINDIR,2 EBRU ALP,1

AYDAN ASYALI BIRI,3 and ABDULLAH EKMEKCI1

ABSTRACT

Tumor growth, which employs a number of regulators, requires the formation of new blood vessels. The mostimportant regulators are vascular endothelial growth factor (VEGF) and angiopoietin-2 (ANGPT-2). DNA

sequence variations in VEGF and ANGPT-2 genes may lead to altered productions and/or activities of these

genes. In this study, we aimed to determine the polymorphic effects of the changes in the VEGF �460 C/T,

VEGF 936 C/T, and ANGPT-2 exon 4 G/A, which we perceive as risk factors in the progress and metasta-

sis of cancer, on the gynecologic cancer patients in the Turkish population. Forty-seven ovarian, 32 cervical,

and 21 endometrial cancer patients and 106 healthy controls were studied. The genomic DNA was extracted

from the whole blood by using DNA extraction techniques. DNA samples were analyzed by polymerase chain

reaction and restriction fragment length polymorphism. There were no significant differences between any ofthe three types of gynecologic cancer patients and controls in terms of the distribution of VEGF� 460, VEGF

936, and ANGPT-2 genotypes and alleles ( p> 0.05). Odds ratios (ORs) were calculated by logistic regression

analysis in comparison with the most common homozygote genotype observed in the studied population. No

evidence of a relationship that would constitute a risk factor ( p> 0.05) was found between genotype and allele

frequencies of patients and controls for VEGF� 460, VEGF 936, and ANGPT-2 genes. A multivariable logistic

regression analysis with the involvement of covariant factors, such as the history of gynecologic cancer and/or

other cancer types in the family, stages of tumor, smoking habits, and existence of other diseases, did not

change the results. The present study is the first case-control study of VEGF and ANGPT-2 polymorphisms inrelation to ovarian, cervical, and endometrial cancers.

INTRODUCTION

O VARIAN, CERVICAL, AND ENDOMETRIAL CANCERS are the most

common types of gynecologic cancers. Parallel to the

increasing female life expectancy, the likelihood of encoun-

tering these cancer types, which are often observed in females

in the age range of 45–80 and constitute roughly one-fifth of all

cancers observed in females, also increases (Repetto et al.,

1998).

Angiogenesis, which would allow better flow of blood to the

ischemic tissues, is the formation of new capillary blood ves-

sels (neovascularization) rooting from pre-existing ones. It

occurs in normal adult tissues during the healing of a wound

and female reproductive cycle, and further activated during the

cancer progression. Tumor growth and the process of metas-

tasis employ a number of regulators and require the formation

of new blood vessels (Saaristo et al., 2000). Various angiogenic

growth factors, such as the vascular endothelial growth factor

1Department of Medical Biology and Genetics, Faculty of Medicine, Gazi University, Besevler, Ankara, Turkey.2Department of Obstetrics and Gynecology, Ankara Oncology Education and Research Hospital, Demetevler, Ankara, Turkey.3Department of Obstetrics and Gynecology, Faculty of Medicine, Gazi University, Besevler, Ankara, Turkey.

DNA AND CELL BIOLOGYVolume 26, Number 7, 2007# Mary Ann Liebert, Inc.Pp. 453–463DOI: 10.1089/dna.2007.0585

453

(VEGF) and angiopoietin (ANGPT), as well as cytokines, in-

duce neovascularization in tumors (Cao, 2004; Tait and Jones,

2004; Tammela et al., 2005).

The gene that encodes VEGF is located on chromosome 6

(6p21.3) and comprises a 14-kb-coding region with eight exons

and seven introns (Vincenti et al., 1996). VEGF is a heparin-

binding homodimeric glycoprotein of 45 kDa, and at least six

VEGF isoforms of variable amino acids are produced through

alternative splicing (Ferrara et al., 2003). It has been shown

that elements in both the 50- and 30-flanking regions are sen-

sitive to hypoxia (Minchenko et al., 1994). Additionally,

VEGF strongly stimulates angiogenesis in hypoxic conditions

via hypoxia-inducible factor (HIF), which is a regulated ele-

ment of the VEGF gene (Pugh and Ratcliffe, 2003). A strong

correlation has been found between VEGF expression and in-

creased tumor microvasculature, malignancy, and metastasis in

breast cancer (Saaristo et al., 2000). There is a considerable

variation between individuals in terms of VEGF expression,

and analysis of the 50-flanking region of the gene has shown the

presence of many polymorphisms (Schultz et al., 1999; Watson

et al., 2000). Polymorphisms within the VEGF gene have been

associated with production of VEGF protein and reported to be

susceptible to several diseases whose progress may be critically

affected by angiogenesis (Renner et al., 2000; Watson et al.,

2000; Awata et al., 2002; Cheng-Chieh et al., 2003; Krippl

et al., 2003; Morohashi et al., 2003; Han et al., 2004; Papa-

zoglou et al., 2004a, 2004b; Ray et al., 2004). The most fre-

quently seen polymorphisms are Bsh1236I (C to T) located at

the� 460th (promoter) and Hin1II (C to T) located at the 936th

(30 untranslated) nucleotides of the gene (Cheng-Chieh et al.,

2003; Krippl et al., 2003). Bsh1236I is a suitable genetic

marker of breast cancer (Krippl et al., 2003), whereas Hin1II is

of prostate cancer (Cheng-Chieh et al., 2003). On the basis of

these facts, VEGF may contribute to the development and

progress of gynecologic cancers. However, there have been no

published studies on any populations so far about the associ-

ation of VEGF� 460 C/T and VEGF 936 C/T polymorphisms

with the ovarian, cervical, and endometrial cancers.

The family of human ANGPTs comprises factors with criti-

cally important roles in vascular development and angiogenesis,

especially in the female reproductive tract (Davis et al., 1996;

Maisonpierre et al., 1997; Valenzuela et al., 1999). Due to their

critical roles in adult angiogenesis, the ANGPTs have natu-

rally become the focus of cancer studies (Tait and Jones, 2004).

Three human ANGPTs have been identified: angiopoietin-1

(ANGPT-1), angiopoietin-2 (ANGPT-2), and angiopoietin-4

(ANGPT-4) (Ward et al., 2001). ANGPT-2, comprising 496

amino acids and consisting of nine exons and eight introns, en-

codes the ANGPT-2 protein (Grosios et al., 1999) on chromo-

some 8 (8p23.1) and is highly expressed only at sites of vascular

remodeling in the adult, notably in the ovary, uterus, and placenta

(Maisonpierre et al., 1997; Grosios et al., 1999; Holash et al.,

1999; Valenzuela et al., 1999; Dunk et al., 2000; Ward et al.,

2001). In addition, ANGPT-2 seems to exert vasodilatative ef-

fects in hypoxic conditions, possibly stimulated by the most

prominent mediator of angiogenesis, VEGF (Dunk et al., 2000).

The ANGPT-2 acts through inhibition of the endothelial cell–

specific receptor tyrosine kinase (Tie-2) signaling and leads to a

loosening of cell–matrix and cell–cell contacts, allowing access

to angiogenic inducers, for example, VEGF. Thus, coexpression

of VEGF and that of ANGPT-2 whose mRNA and protein are

detectable in ovarian cancer specimens lead to angiogenesis

(Hirchenhain et al., 2003; Zhang et al., 2003). In ANGPT-2,

three positions were found to be highly polymorphic between

individuals: at position 759 in exon 2, position 1087 in exon 4, and

position 1233 in exon 5 of the cDNA sequence. All detected

polymorphisms are silent mutations and do not result in amino

acid changes in the encoded protein (Ward et al., 2001). The most

common polymorphism of ANGPT-2 is a G/A polymorphism

in exon 4, which is believed to alter protein expression and pos-

sibly inhibit angiogenesis (Pietrowski et al., 2003). ANGPT-2

gene polymorphism was studied, so far, in idiopathic recurrent

miscarriage (IRM), unexplained intrauterine fetal death (IUFD),

and uterine leiomyoma (Pietrowski et al., 2003; Denschlag

et al., 2005; Huber et al., 2005). The biological actions of the

ANGPTs in tumor growth and metastasis have not been fully

ascertained although they have been implicated in the devel-

opment of the vasculature of a wide variety of tumors (Tait and

Jones, 2004). Therefore, the ANGPT-2 polymorphism has only

been linked to obstetric conditions/diseases so far (Huber et al.,

2005). The capability of angiogenic polymorphism to affect the

growth and invasion rates of the tumor led us to investigate the

relationship between them.

In the present study, we aimed to determine whether the

polymorphic effects of the changes in the VEGF� 460 C/T,

VEGF 936 C/T, and ANGPT-2 exon 4 G/A are risk factors

for ovarian, cervical, and endometrial cancer patients in the

Turkish population.

MATERIALS AND METHODS

Study population

A total of 206 subjects, consisting of 106 healthy volunteers

as controls and 100 gynecologic cancer patients who were

selected from patients admitted to the Department of Obstetrics

and Gynecology, Faculty of Medicine, Gazi University, Be-

sevler, Turkey, and to the same department of the Ankara On-

cology Education and Research Hospital, Demetevler, Turkey,

were enrolled in this study. The research was carried out in

accordance with the Declaration of Helsinki (1989) of the

World Medical Association, and the study was approved by

the Committee of Ethics of the Gazi University. All cases and

controls were of Caucasian origin and provided their written

consents for inclusion in the study. Our study conforms to a

great extent the standards presented by Hegele in the form of a

list of the desirable attributes for genetic case–control studies

(Hegele, 2002). In each case, age of menarche and first diag-

nosis; history of gynecologic cancer and/or other cancer types

in the family; stages (I–IV) of tumor; information on smoking

habits; and existence of other diseases, such as psoriasis, dia-

betic retinopathy, rheumatoid arthritis, and cardiovascular dis-

eases (for which angiogenesis may be critical), were obtained

from the patient files. Out of 100 patients, 47 had epithelial

ovarian cancer, 32 women had cervical cancer, and the remain-

ing 21 women had endometrial cancer. Clinical characteristics

of the participating patients were shown in Table 1. The mean

454 KONAC ET AL.

age of menarche of the control group was 13.0� 0.18 years,

and the mean age of the control group was 47� 0.5 years

(range: 25–65 years). None of them had history of gynecologic

cancer or other cancer types in the family, smoking habits, or

other diseases.

Extraction of genomic DNA and amplification of thegenes� 460 VEGF, 936 VEGF, and ANGPT-2

Genomic DNA was isolated from peripheral blood by using a

DNA extraction kit (Heliosis�, Metis Biotechnology, Ankara,

Turkey) according to the manufacturer’s instructions. Amplifica-

tions of the two regions of the VEGF gene and the gene encod-

ing for ANGPT-2 were carried out by placing in a Mastercycler

gradient (Eppendorf, Hamburg, Germany) thermal cycler a total

volume of 50mL PCR mixture containing 50 ng genomic DNA,

2.5 mM magnesium chloride, 100mM deoxyribonucleotide tri-

phosphate, 50 pmol/mL of each primer, and 1.0 U/mL Taq DNA

polymerase. For the� 460 C/T (rs833061), the following pair

of primers produced a PCR product of 175 bp: forward, 50-TGTGCGTGTGGGGTTGAGCG-30; reverse, 50-TACGTGCG

GACAGGGCCTGA-30 (Gene Bank accession no. AF095785;

nucleotides 1883–2057). For the 936 C/T (rs3025039), the

following pair of primers produced a PCR product of 208 bp:

forward, 50-AAGGAAGAGGAGACTCTGCGCAGAGC-30;reverse, 50-TAAATGTATGTATGTGGGTGGGTGTGTCTA

CAGG-30 (Gene Bank accession no. AF437895; nucleotides

12450–12657). For the ANGPT-2 exon 4 G/A (rs3020221),

the following pair of primers produced a PCR product of 335 bp:

forward, 50-CATTAGAATAGCCTTCAC-30; reverse, 50-GAG

TGTTTTACTGACTAAAGG-30 (Gene Bank accession no.

AC018398; nucleotides 83277–83611). We set the PCR cycling

conditions for the VEGF and ANGPT-2 genes as explained by

Cheng-Chieh et al. (2003), Pietrowski et al. (2003), and Papa-

zoglou et al. (2004a). The VEGF polymorphisms were analyzed

by digestion of the PCR products with restriction endonucle-

ases, Bsh1236I for� 460 C/T and Hin1II for 936 C/T (MBI

Fermantas, Vilnius, Lithuania). The� 460 C allele was cut into

two fragments of 155 bp and 20 bp, while the� 460 T allele re-

mained uncut (175 bp). The 936 C allele remained uncut (208 bp),

while the 936 T was cut into two fragments of 122 bp and 86 bp.

The ANGPT-2 exon 4 G/A polymorphism was analyzed by

digestion of the PCR product with restriction endonuclease

Eco57I (Fermantas). After digestion, the G allele generated two

fragments of 193 bp and 142 bp, whereas the A allele remained

uncut (335 bp). The PCR products and restriction fragments of

VEGF� 460 C/T, VEGF 936 C/T, and ANGPT-2 exon 4 G/A

were loaded directly onto 4%, 3%, and 2% agarose gel (con-

taining 0.5% ethidium bromide), respectively. The products were

separated by electrophoresis and visualized by Gel Logic 100 gel

image system (Kodak, Rochester, NY).

Statistical analysis

We used the goodness-of-fit chi-square (w2) test with one

degree of freedom while testing the Hardy–Weinberg equilib-

rium of alleles at individual loci to compare the observed ge-

notype frequencies with the expected genotype frequencies

among the subjects. We then used Pearson’s two-way w2 test to

analyze the distribution of genotype and allele frequencies

of the VEGF� 460 C/T, VEGF 936 C/T, and ANGPT-2 exon 4

G/A polymorphisms in the patients and control group.

When the assumption of the w2 was violated, Fisher’s exact test

was performed. A power calculation was conducted for each

single nucleotide polymorphism (SNP) to indicate which ap-

proximate odds ratio (OR) levels could achieve significant

association between the three types of gynecologic cancer and

the genotypes, given the sample size used and the observed

allele frequencies in controls. We used the logistic regression

analysis to calculate the ORs at 95% confidence interval (CI).

Adjusted ORs for the epidemiological covariant factors, such

as the history of gynecologic cancer and/or other cancer types

in the family, stages of tumor, smoking habits, and existence of

other diseases, were determined by using a multivariable lo-

gistic regression method. We considered results with p< 0.05

as statistically significant. SPSS V.11.5 was employed to carry

out the required statistical analysis.

RESULTS

There were no significant differences between the cases and

controls in mean age and mean age of menarche, suggesting

that the matching based on these two variables was adequate.

The genotype distributions of the three polymorphisms in the

controls and cases were in Hardy–Weinberg equilibrium.

For VEGF� 460 genotypes, frequencies of C alleles of all

three types of gynecologic cancer patients and controls were

TABLE 1. CLINICAL CHARACTERISTICS OF THE

PARTICIPATING PATIENTS

Ovarian

cancer

Cervical

cancer

Endometrial

cancer

Number of cases 47 (47) 32 (32) 21 (21)

Mean ages of

menarche (years)

13� 0.23 13� 0.18 13� 0.14

Mean ages of

first diagnosis (years)

48� 0.51 49� 0.46 50� 0.52

Range (years) 16–73 25–68 39–81

1a 8 (17.0) 7 (21.9) 9 (42.9)

2b

I 1 (2.1) 5 (15.6) 10 (47.6)

II 5 (10.6) 16 (50.0) 5 (23.8)

III 40 (85.1) 10 (31.3) 5 (23.8)

IV 1 (2.1) 1 (3.1) 1 (4.8)

3c 8 (17.0) 4 (12.5) 1 (4.8)

4d 11 (23.4) 5 (15.6) 5 (23.8)

Values in parentheses are percentages.aHistory of gynecologic cancer and/or other cancer types in the

family.bStages of tumor (staging was performed according to the current

International Federation of Gynecology and Obstetrics [FIGO] classi-

fication).cSmoking habits.dExistence of other diseases.

VEGF AND ANGPT-2 POLYMORPHISMS IN GYNECOLOGIC CANCERS 455

TABLE 2. GENOTYPE AND ALLELE FREQUENCIES (%) OF THE VEGF AND ANGPT-2 POLYMORPHISMS IN

OVARIAN, CERVICAL, AND ENDOMETRIAL CANCERS AND CONTROLS

Cancer

types

Patients

(n)

Controls

(n¼ 106) p-valuesaOR

(95% CI)b p-valuesb

VEGF� 460 C> T (rs833061)

Ovarian n¼ 47

Genotypes 0.383

CC 5 (10) 13 (12) 0.64 (0.20–2.05) 0.452

CT 21 (45) 58 (55) 0.60 (0.29–1.26) 0.177

TT 21 (45) 35 (33) 1

Alleles 0.269

C 31 (33) 84 (40) 0.75 (0.45–1.25) 0.269

T 63 (67) 128 (60) 1

Cervical n¼ 32

Genotypes 0.324

CC 4 (12) 13 (12) 0.72 (0.20–2.56) 0.760

CT 13 (41) 58 (55) 0.52 (0.22–1.23) 0.133

TT 15 (47) 35 (33) 1

Alleles 0.325

C 21 (33) 84 (40) 0.74 (0.41–1.34) 0.325

T 43 (67) 128 (60) 1

Endometrial n¼ 21

Genotypes 0.257

CC 5 (24) 13 (12) 1.68 (0.46–6.09) 0.499

CT 8 (38) 58 (55) 0.60 (0.21–1.75) 0.350

TT 8 (38) 35 (33) 1

Alleles 0.696

C 18 (43) 84 (40) 1.14 (0.58–2.23) 0.696

T 24 (57) 128 (60) 1

VEGF 936 C> T (rs3025039)

Ovarian n¼ 47

Genotypes 0.655

CC 1 (2) 1 (1) 2.15 (0.13–35.47) 0.541

CT 13 (28) 34 (32) 0.82 (0.38–1.76) 0.615

TT 33 (70) 71 (67) 1

Alleles 0.825

C 15 (16) 36 (17) 0.93 (0.48–1.79) 0.825

T 79 (84) 176 (83) 1

Cervical n¼ 32

Genotypes 0.626

CC 0 (0) 1 (1) Not calculatedc 1.000

CT 8 (25) 34 (32) 0.70 (0.28–1.71) 0.428

TT 24 (75) 71 (67) 1

Alleles 0.391

C 8 (12) 36 (17) 0.70 (0.31–1.59) 0.391

T 56 (88) 176 (83) 1

Endometrial n¼ 21

Genotypes 0.225

CC 0 (0) 1 (1) Not calculatedd 1.000

CT 3 (14) 34 (32) 0.35 (0.10–1.26) 0.096

TT 18 (86) 71 (67) 1

456 KONAC ET AL.

smaller than the frequencies of T alleles. We did not find sta-

tistically significant differences in the genotypes and allele

frequencies between the patients and controls of all three types

of gynecologic cancers. The ORs per copy of the C allele were

0.75 (95% CI: 0.45–1.25; p¼ 0.269) for ovarian cancer, 0.74

(95% CI: 0.41–1.34; p¼ 0.325) for cervical cancer, and 1.14

(95% CI: 0.58–2.23; p¼ 0.696) for endometrial cancer (Table

2). In other words, the allelic frequencies of the gene were not

significantly associated with any of the three types of gyne-

cologic cancer patients.

For VEGF 936 genotypes, out of 106 healthy controls, 1 was

type CC, 34 were type CT, and 71 were type TT. In addition, in

the whole group of 100 patients there was only one type CC,

which was encountered in 1 of the ovarian cancer patients. VEGF

936 TT was the most common genotype observed in the Turk-

ish population. Genotype and allele frequencies of VEGF 936

were again not significantly different between the patients and

controls of the three types of gynecologic cancers. The ORs per

copy of the C allele were 0.93 (95% CI: 0.48–1.79; p¼ 0.825) for

ovarian cancer, 0.70 (95% CI: 0.31–1.59; p¼ 0.391) for cervi-

cal cancer, and 0.38 (95% CI: 0.11–1.28; p¼ 0.106) for endo-

metrial cancer (Table 2), again pointing to the fact that the allelic

frequencies of the gene were not significantly associated with

any of the three types of gynecologic cancer patients.

TABLE 2. (Continued)

Cancer

types

Patients

(n)

Controls

(n¼ 106) p-valuesaOR

(95% CI)b p-valuesb

VEGF 936 C> T (rs3025039)

Alleles 0.106

C 3 (7) 36 (17) 0.38 (0.11–1.28) 0.106

T 39 (93) 176 (83) 1

ANGPT-2 exon 4 G>A (rs3020221)

Ovarian n¼ 47

Genotypes 0.989

GG 11 (23) 26 (25) 1.10 (0.41–2.27) 0.892

GA 22 (47) 49 (46) 1.10 (0.45–2.52) 0.893

AA 14 (30) 31 (29) 1

Alleles 0.893

G 44 (47) 101 (48) 1.03 (0.64–1.68) 0.893

A 50 (53) 111 (52) 1

Cervical n¼ 32

Genotypes 0.157

GG 5 (16) 26 (25) 1.01 (0.27–3.68) 0.992

GA 21 (66) 49 (46) 2.23 (0.75–6.60) 0.141

AA 6 (18) 31 (29) 1

Alleles 0.570

G 31 (48) 101 (48) 1.21 (0.62–2.37) 0.570

A 33 (52) 111 (52) 1

Endometrial n¼ 21

Genotypes 0.031

GG 1 (5) 26 (25) 3.35 (0.35–31.91) 0.376

GA 16 (76) 49 (46) 8.49 (1.06–67.60) 0.019

AA 4 (19) 31 (29) 1

Alleles 0.570

G 18 (43) 101 (48) 1.21 (0.62–2.37) 0.570

A 24 (57) 111 (52) 1

Values in parentheses are percentages.aResults of ovarian, cervical, and endometrial cancer groups compared with control group.bCalculations were performed TT vs. CT and CC for VEGF� 460 and 936; AA vs. GA and GG for ANGPT-2.cThere are no CC-genotype patients.dThere are no CC-genotype patients.

OR, odds ratio; CI, confidence interval.

VEGF AND ANGPT-2 POLYMORPHISMS IN GYNECOLOGIC CANCERS 457

Finally, regarding the ANGPT-2 exon 4 G/A genotypes,

forty-eight percent of the control group and 43–48% of the

cancer patients were found to have G allele frequencies. There

were no significant differences in the genotype and allele fre-

quencies of ANGPT-2 between the controls and patients. The

ORs per copy of the G allele were 1.03 (95% CI: 0.64–1.68;

p¼ 0.893) for ovarian cancer, 1.21 (95% CI: 0.62–2.37;

p¼ 0.570) for cervical cancer, and 1.21 (95% CI: 0.62–2.37;

p¼ 0.570) for endometrial cancer (Table 2). In other words, the

allelic frequencies of the gene were again not significantly

associated with the three types of gynecologic cancer patients.

Power calculation was conducted for each SNP (VEGF�460 C/T, VEGF 936 C/T, and ANGPT-2 exon 4 G/A) to

find out approximate OR levels, which would achieve signifi-

cant association between the three types of gynecologic cancer

and the genotypes, given the sample sizes used and the ob-

served allele frequencies in controls. OR levels calculated by

the power analysis were from 2.7 (1.3–5.8) to 15.2 (1.7–132.1)

for ovarian cancer, from 3.05 (1.35–6.89) to 8.7 (1.9–38.8) for

cervical cancer, and from 3.4 (1.3–8.96) to 9.4 (1.9– 47.2) for

endometrial cancer with respect to reference genotypes (power

of 0.80, significance level of 0.05).

Multivariable logistic regression analysis demonstrated that

as far as VEGF� 460 C/T, VEGF 936 C/T, and ANGPT-2

exon 4 G/A polymorphisms were concerned, the occurrences of

ovarian, cervical, and endometrial cancers were not associated

with the history of gynecologic cancer or other cancer types in

the family, smoking habits, stages of tumor, or existence of

other diseases (Table 3).

DISCUSSION

Humans are believed to carry over a million distinct SNPs

(Li et al., 2001). Determination of SNPs is a new means to

study the etiology of polygenetic disorders with complex in-

heritance patterns (Denschlag et al., 2005). For instance, they

affect the sensibility of cancer development and progression

via actions on the pathways of tumor angiogenesis. This may

be achieved by enhanced or reduced transcription, altered

posttranscriptional or posttranslational activity, or changes in

the tertiary structure of the gene product (Liu et al., 1995).

Polymorphisms of VEGF and ANGPT-2 genes may lead to

differences in their expression levels among individuals and

could potentially contribute to a variety of pathological pro-

cesses. Since the implications of VEGF and ANGPT-2 poly-

morphisms for gynecologic cancers have not been studied so

far, we, in this study, investigate the polymorphic effects of the

changes in the VEGF� 460 C/T, VEGF 936 C/T, and

ANGPT-2 exon 4 G/A on the ovarian, cervical, and endometrial

cancer patients in the Turkish population.

VEGF is a major angiogenic factor and is a prime regulator

of endothelial cell proliferation (Ferrara et al., 2003). A 28-bp

element within the human VEGF promoter believed to contain

a consensus binding site for hypoxia-inducible factor-1 (HIF-1)

and is necessary to increase transcription of a chloramphenicol

acetyltransferase (CAT) reporter gene in endothelial cells

cultured under hypoxic conditions (Liu et al., 1995). Many

reports have indicated that VEGF might combine with up-

stream signals or other growth factors to enhance prostate

cancer growth and progression (Ravindranath et al., 2001).

VEGF� 460 C/T polymorphism is associated with prostate

cancer. While the TT homozygotes, the most common geno-

type found in patients with prostate cancer in Taiwan, seemed

to impose a significant risk for the emergence of prostate

cancer, it did not play a role in the cancer progression (Cheng-

Chieh et al., 2003). Another study showed that TT homozy-

gotes and the T allele of the VEGF� 460 gene were associated

with a higher risk of endometriosis. In other words, VEGF

polymorphism is likely to contribute to the pathogenesis of en-

dometriosis and may become a useful marker for predicting

endometriosis susceptibility (Hsieh et al., 2004). However, the

distribution of genotype and allele frequencies of the VEGF�460 C/T polymorphism did not differ significantly between en-

dometriosis patients and controls in South Indian and Korean

populations (Bhanoori et al., 2005; Kim et al., 2005). In another

study in which we had investigated VEGF� 634 G>C poly-

morphism, we reported that VEGF� 634 genetic variants did

not play a relevant role in the pathogenesis of ovarian, cervical,

and endometrial cancers (Konac et al., 2006). However, in our

recent study (Konac et al., 2007), in which we had investigated

HIF-1a gene polymorphisms, results suggested that the C1772T

polymorphism of the HIF-1a might be associated with cervical

and endometrial cancers. In this study, we found that the least

common genotype of VEGF� 460 C/T polymorphism was

CC homozygote in both the gynecologic cancer patients and the

control group. The distribution of genotypes and allele frequen-

cies of the� 460 C/T polymorphism in controls in South

Indian population (Bhanoori et al., 2005) is consistent with the

distribution of genotypes and allele frequencies presented in the

current study. Studies on Korean population have revealed that

the most common genotype was TT in endometriosis and lung

cancer patients (Kim et al., 2005; Lee et al., 2005). We found

that� 460 T allele carriers (CTþTT genotypes) were most

frequent among the cancer patients. However, there was no sta-

tistically significant difference between the patients and the con-

trols in terms of genotypes ( p> 0.05). Multivariable logistic

regression analysis for the VEGF� 460 demonstrated that the

occurrences of ovarian, cervical, and endometrial cancers were

not associated with the history of gynecologic cancer or other

cancer types in the family, stages of tumor, smoking habits, and

existence of other diseases ( p> 0.05) (Table 3).

A C/T polymorphism at position 936 in the 30 untranslated

region of the VEGF gene has been associated with VEGF

plasma levels (Renner et al., 2000; Krippl et al., 2003). Al-

though it is unclear how the polymorphism in the untranslated

region of the gene influences its protein production, several

reports have demonstrated that SNPs of the VEGF gene are

associated with VEGF synthesis (Watson et al., 2000; Awata

et al., 2002). The T variant, which is linked to lower VEGF

levels, has been associated with breast cancer, sarcoidosis, and

spontaneous preterm delivery (Krippl et al., 2003; Morohashi

et al., 2003; Papazoglou et al., 2004a). Individuals with VEGF

936 TT or CT genotype demonstrated a statistically significant

association with preterm delivery compared with those sharing

936 CC genotype in the Greek population (Papazoglou et al.,

2004a). The 936 C allele carries a potential binding site for

activator protein 4 (AP-4), a helix-loop-helix transcription

factor enhancing expression of several viral and cellular genes

by binding to specific enhancer sites, which are abolished in the

458 KONAC ET AL.

TABLE

3.

AD

JUS

TE

DO

DD

SR

AT

IOS

FO

RA

LL

TH

ET

HR

EE

TY

PE

SO

FG

YN

EC

OL

OG

ICC

AN

CE

RA

SS

OC

IAT

ED

WIT

HV

EG

FA

ND

AN

GP

T-2

GE

NO

TY

PE

S

Cancertypes

OR

a,b(95%

CI)

p-values

a,b

OR

a,c(95%

CI)

p-values

a,c

OR

a,d(95%

CI)

p-values

a,d

OR

a,e(95%

CI)

p-values

a,e

VEGF�460C>T(rs833061)

Ovarian

Gen

oty

pes

CC

0.7

5(0

.58

–0

.97

)0

.29

80

.67

(0.0

5–

8.2

0)

1.0

00

0.7

6(0

.60

–0

.97

)0

.54

50

.76

(0.6

0–

0.9

7)

0.5

45

CT

0.2

6(0

.05

–1

.50

)0

.23

81

.58

(0.2

4–

10

.61

)1

.00

00

.53

(0.1

1–

2.5

9)

0.6

97

1.2

8(0

.32

–5

.09

)0

.72

6

TT

11

11

Cervical

Gen

oty

pes

CC

2.1

7(0

.14

–3

2.5

3)

0.5

30

0.6

7(0

.45

–1

.00

)0

.24

50

.77

(0.5

9–

1.0

0)

1.0

00

0.7

3(0

.54

–1

.00

)0

.53

0

CT

2.8

9(0

.43

–1

9.2

8)

0.3

72

0.5

1(0

.11

–2

.40

)0

.39

01

.18

(0.1

4–

9.8

3)

1.0

00

0.2

3(0

.02

–2

.38

)0

.33

3

TT

11

11

Endometrial

Gen

oty

pes

CC

2.0

0(0

.18

–2

2.0

6)

1.0

00

0.4

4(0

.21

–0

.92

)0

.10

5N

ot

calc

ula

ted

f1

.75

(0.0

8–

36

.29

)1

.00

0

CT

5.0

0(0

.58

–4

2.8

0)

0.3

15

0.3

3(0

.04

–2

.77

)0

.60

80

.47

(0.2

7–

0.8

0)

1.0

00

7.0

0(0

.57

–8

6.3

2)

0.2

82

TT

11

11

VEGF936C>T(rs3025039)

Ovarian

Gen

oty

pes

CC

0.9

6(0

.89

–1

.04

)1

.00

01

.04

(0.9

7–

1.1

1)

1.0

00

1.2

5(0

.81

–1

.94

)0

.14

70

.96

(0.8

9–

1.0

4)

1.0

00

CT

0.3

1(0

.03

–2

.81

)0

.40

92

.14

(0.2

3–

20

.35

)0

.65

92

.18

(0.4

1–

11

.45

)0

.38

50

.94

(0.2

1–

4.2

7)

1.0

00

TT

11

11

Cervical

Gen

oty

pes

CC

No

tca

lcu

late

dg

No

tca

lcu

late

dg

No

tca

lcu

late

dg

No

tca

lcu

late

dg

CT

0.4

3(0

.04

–4

.23

)0

.64

60

.56

(0.0

9–

3.3

7)

0.6

81

1.0

0(0

.09

–1

1.2

4)

1.0

00

0.7

1(0

.07

–7

.52

)1

.00

0

TT

11

11

Endometrial

Gen

oty

pes

CC

No

tca

lcu

late

dh

No

tca

lcu

late

dh

No

tca

lcu

late

dh

No

tca

lcu

late

dh

CT

0.7

5(0

.54

–1

.04

)0

.22

91

.30

(0.1

0–

17

.73

)1

.00

00

.10

(0.0

3–

0.3

7)

0.1

43

0.8

0(0

.62

–1

.03

)0

.52

6

TT

11

11

(continued

)

459

TABLE

3.

(Continued

)

Cancertypes

OR

a,b(95%

CI)

p-values

a,b

OR

a,c(95%

CI)

p-values

a,c

OR

a,d(95%

CI)

p-values

a,d

OR

a,e(95%

CI)

p-values

a,e

ANGPT-2

exon4G>A(rs3020221)

Ovarian

Gen

oty

pes

GG

2.7

3(0

.24

–3

0.6

6)

0.6

04

1.3

0(0

.07

–2

3.4

3)

1.0

00

1.8

0(0

.26

–1

2.3

0)

0.6

61

1.0

7(0

.18

–6

.21

)1

.00

0

GA

2.2

2(0

.22

–2

2.7

)0

.64

30

.45

(0.0

4–

4.6

0)

0.6

43

0.4

5(0

.05

–3

.72

)0

.58

60

.59

(0.1

1–

3.2

9)

0.6

61

AA

11

11

Cervical

Gen

oty

pes

GG

0.8

0(0

.04

–1

7.2

0)

1.0

00

0.3

0(0

.02

–4

.91

)0

.54

52

.00

(0.1

3–

31

.98

)1

.00

00

.44

(0.2

1–

0.9

2)

0.4

55

GA

1.2

5(0

.11

–1

3.9

2)

1.0

00

0.9

2(0

.13

–6

.78

)1

.00

00

.20

(0.0

1–

3.9

1)

0.3

54

0.7

8(0

.63

–0

.97

)1

.00

0

AA

11

11

Endometrial

Gen

oty

pes

GG

1.3

3(0

.76

–2

.35

)1

.00

01

.33

(0.7

6–

2.3

5)

1.0

00

No

tca

lcu

late

di

0.6

7(0

.30

–1

.48

)1

.00

0

GA

1.2

0(0

.84

–1

.72

)0

.35

31

.50

(0.6

7–

3.3

4)

0.1

76

0.9

4(0

.83

–1

.06

)1

.00

00

.92

(0.7

9–

1.0

8)

1.0

00

AA

11

11

OR

,o

dd

sra

tio

;C

I,co

nfi

den

cein

terv

al.

aC

alcu

lati

ons

wer

eper

form

edT

Tvs.

CT

and

CC

for

VE

GF�

46

0an

d9

36

;A

Av

s.G

Aan

dG

Gfo

rA

NG

PT

-2.

bO

Rad

just

edfo

rh

isto

ryo

fg

yn

ecolo

gic

can

cer

and

/or

oth

erca

nce

rty

pes

inth

efa

mil

y.

cO

Rad

just

edfo

rst

ages

of

tum

or.

dO

Rad

just

edfo

rsm

ok

ing

hab

its.

eO

Rad

just

edfo

rex

iste

nce

of

oth

erd

isea

ses.

f Th

ere

are

no

CC

-gen

oty

pe

pat

ien

tsw

ho

smo

ke.

gT

her

ear

en

oC

C-g

enoty

pe

pat

ien

ts.

hT

her

ear

en

oC

C-g

enoty

pe

pat

ien

ts.

i Th

ere

are

no

GG

-gen

oty

pe

pat

ien

tsw

ho

smo

ke.

460

936 T allele (Renner et al., 2000). The loss of this potential

binding site could be responsible for decreased VEGF expres-

sion by the T allele (Hu et al., 1990). In our study, for VEGF 936

C/T polymorphism, there were no statistically significant dif-

ferences between the genotype and allele frequencies of the

patients and the control group ( p> 0.05). The reason for this

result is that genotype TT was the most common in both the

control group and the patients (Table 2). Interestingly, we found

only one CC genotype (ovarian) among the whole patient group

and another in the control group. There was no CC genotype in

cervical and endometrial patients. In contrast, a low number of

TT homozygotes was found in patients and controls in Austrian

(Krippl et al., 2003) and Tunisian population (Sfar et al., 2006).

In another study, allelic frequency of 936 T variant in pre-

eclampsia, in which VEGF plays a crucial role, was found to be

20.2% (Papazoglou et al., 2004a). Furthermore, carriers of the

VEGF 936 T allele were less frequent in breast cancer patients

than in healthy subjects, indicating that this genetic variant may

be protective against breast cancer (Krippl et al., 2003). The

936 CT genotype and the combined 936 CT and TT genotype

were also associated with a significantly decreased risk of small-

cell lung cancer compared with the 936 CC genotype (Lee et al.,

2005). The discrepancy might be due to ethnic composition

differences between the studies, sizes of study population, or the

fact that the mechanism by which the 936 C/T polymorphism

affects serum levels of VEGF might differ during the progres-

sion of breast, lung, and gynecologic cancers. No evidence of a

relationship between clinicopathological characteristics, in-

cluding family history, stages and grades of tumor, smoking

habits, and existence of other diseases, and 936 C/T poly-

morphism in ovarian, cervical, and endometrial cancer patients

was observed.

ANGPT-2 appears to be highly polymorphic in a few indi-

viduals. The polymorphism in exon 2 suggests that this position

has been the site of two independent mutations and that the

surrounding structure is conducive to mutations (Ward et al.,

2001). On the other hand, the polymorphism in exon 4 of

ANGPT-2 is a silent mutation and does not result in amino acid

changes in the encoded protein (Ward et al., 2001). However,

silent mutations are often associated with expression differences

in various genes on the protein level (Stanford et al., 2000). It

should be noted that there are just a few studies on ANGPT-2

exon 4 G/A polymorphism, such as those on IUFD and IRM

(Pietrowski et al., 2003; Huber et al., 2005). The G/A poly-

morphism in exon 4 of ANGPT-2 is not associated with IRM in

a Middle European white population (Pietrowski et al., 2003).

Furthermore, the polymorphism does not seem to be a candidate

gene for unexplained IUFD in Caucasian women (Huber et al.,

2005). Denschlag et al. (2005) reported that there was no asso-

ciation between the ANGPT-2 genotype and its allele frequen-

cies and leiomyoma in Caucasian population. Nevertheless, there

are no data published to date with respect to this polymorphism

in gynecologic cancers. In our study, we found that the most

common genotype in all three types of gynecologic cancer pa-

tients and controls was GA heterozygote (Table 2). Therefore,

there was no statistically significant difference between the

patients and the controls in terms of genotypes and allele fre-

quencies ( p> 0.05). In addition, ORs per copy of the G allele

showed that carriers of an ANGPT-2 G allele did not seem to

increase the risk of gynecologic cancers in the Turkish popula-

tion. In addition, there was no statistically significant associa-

tion between the genotypes of patients and family history, stages

of tumor, smoking habits, and existence of other diseases (Table

3). Since the association between the three polymorphisms we

studied and gynecologic cancers has not yet been investigated,

we were unable to compare our results with those of similar

studies covering other ethnic populations.

In conclusion, the results of this study suggest that VEGF�460 C/T, VEGF 936 C/T, and ANGPT-2 exon 4 G/A poly-

morphisms are not associated with any of the three types of

gynecologic cancers. It is unlikely that these three types of

polymorphisms account for a substantial proportion of ovarian,

cervical, and endometrial cancers. The main limitation of this

study is the small sample size due to our criteria for the se-

lection of the cases and controls. Therefore, larger prospective

and multiethnic studies are needed to investigate the functional

relevance of the three polymorphic genes and their receptors to

gynecologic cancer development and progression. Further-

more, the functionality of the polymorphic regions needs to be

analyzed by expression studies. Certainly, further in vivo and in

vitro experimentations are required to demonstrate the specific

role of VEGF and ANGPT-2 in angiogenesis of gynecologic

cancer types and elucidate the mechanisms involved. Our re-

sults, which should be bolstered by future complementary

in vivo, in vitro, and gene expression studies, are expected to

help better understand the bond between the SNPs and the

ovarian, cervical, and endometrial cancers, as well as the pro-

spective diagnosis and treatment phases.

ACKNOWLEDGMENT

This study has been supported by Gazi University Research

Fund (No. 11/2004-13).

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Address reprint requests to:

Ece Konac, Ph.D.

Department of Medical Biology and Genetics

Faculty of Medicine

Gazi University

06500 Besevler, Ankara

Turkey

E-mail: ecemercanoglu@yahoo.com

Received for publication February 8, 2007; received in revised

form March 1, 2007; accepted March 5, 2007.

VEGF AND ANGPT-2 POLYMORPHISMS IN GYNECOLOGIC CANCERS 463