Angiotensin I-converting enzyme ACE 2350G and ACE-240T-related genotypes and alleles are associated with higher susceptibility to endometriosis

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Angiotensin I-converting enzyme ACE 2350*G and

ACE-240*T-related genotypes and alleles are associated

with higher susceptibility to endometriosis

Yao-Yuan Hsieh

1,4

, Chi-Chen Chang

1

, Fuu-Jen Tsai

2,6

, Chin-Moo Hsu

2

, Cheng-Chieh Lin

3

and Chang-Hai Tsai

2,5

1

_{Department of Obstetrics and Gynecology,}

2

_{Department of Pediatrics and Medical Genetics,}

3

_{Department of Family Medicine, China}

Medical University Hospital, Taichung, Taiwan,

4

Department of Biological Science and Technology, National Chiao Tung University,

Hsinchu, Taiwan and

5

Taichung Health Care and Management University, Taichung, Taiwan, Republic of China

6

_{To whom correspondence should be addressed at: Department of Pediatrics and Medical Genetics, China Medical University}

Hospital, No.2 Yuh-Der Road, Taichung, Taiwan. E-mail: [email protected]

Endometriosis displays features similar to malignancy, ranging from neovascularization to local invasion and aggressive spread

to distant organs. The altered vascular-related genes might be related to the development of endometriosis. This study

investi-gates whether angiotensin I-converting enzyme (ACE) *A2350G and A-240T gene polymorphisms could be used as markers of

susceptibility in endometriosis. Women were divided into two groups: (1) endometriosis group (n 5 150) and (2)

non-endome-triosis group (n 5 159). Genomic DNA was obtained from peripheral leukocytes. ACE A2350G and A-240T gene

polymorph-isms were amplified by PCR and detected after restriction enzyme digestion with Bst UI and Xba I. Genotypes and allelic

frequencies in both groups were compared. We observed that genotype distribution and allele frequency of ACE 2350 and

ACE-240 gene polymorphisms in both groups were significantly different. Proportions of ACE 2350*A

homozygote/heterozygo-te/G homozygote in both groups were: (1) 66.7/29.3/4% and (2) 96.2/3.1/0.7%. Proportions of ACE-240*A

homozygote/hetero-zygote/T homozygote in both groups were: (1) 43.3/46/10.7% and (2) 62.9/35.8/1.3%. We concluded that ACE 2350*G and

ACE-240*T-related genotypes and alleles are associated with higher susceptibility to endometriosis. ACE A2350G and A-240T

gene polymorphisms might be associated with endometriosis development.

Key words: angiotensin I-converting enzyme/ACE/endometriosis/polymorphism/SNP

Introduction

Endometriosis, a polygenic/multi-factorial disease, is associated

with complex interactions between hormone and cytokine activation,

immunoinflammatory processes and genetic factors (Vigano et al.,

1998). Endometriosis displays some features of malignancy,

includ-ing local invasion and aggressive spread to distant organs. Similar

to tumour metastases, endometriotic implants require

neovasculari-zation to become established, grow and invade tissues. Neovascular

processes are prominent in the endometric tissue. Heritable genetic

factors may contribute to the initiation and progression of

endome-triosis (Treloar et al., 1999).

Cardiovascular genes play a role in the regulation and growth of

tumour and altered vascular-related genes might be related to the

development of endometriosis. The renin – angiotensin system

(RAS) regulates blood pressure through its effects on vascular tone,

renal haemodynamics and fluid – electrolyte balance (Fornage et al.,

1998). Angiotensin I-converting enzyme (ACE) regulates systemic

circulation through angiotensin II formation and kinin metabolism.

ACE cleaves angiotensin I to angiotensin II, which is the key

com-ponent in RAS (Berge et al., 1994). The ACE and RAS genes are

related to the regulatory pathway in cardiovascular disease

(Zhu et al., 2001), while the ACE gene is implicated as a risk

factor in coronary artery disease and myocardial infarction (Zhu

et al., 2001).

Gene polymorphisms are useful tools in the study of

multi-factor-ial disorders (Anderson et al., 1994). The analyses of single

nucleo-tide polymorphism (SNP) can be implemented to determine the

mechanisms of complex genetic disorders. Numerous chronic

dis-orders, such as endometriosis, osteoporosis, hypertension, diabetes

and asthma, have been attributed to genetic susceptibility. Most

studies on ACE gene polymorphisms have been focused on their

associations with cardiovascular diseases, serum ACE level and

blood pressure (Zhu et al., 2001).

Angiogenesis and vascular remodelling play critical roles in the

growth, invasion and regression of endometriosis (Donnez et al.,

1998). The presence of angiotensin receptors has been demonstrated

in the endometrial tissue. Angiotensin II in endometrial stromal

cells was mediated via angiotensin I receptors (Braileanu et al.,

2002). Angiotensin II could increase the intracellular calcium

con-centration by interaction with angiotensin receptor in endometrial

stromal cells (Braileanu et al., 2001). Vasopressin also stimulates

phospholipase C activity in endometrial explants (Braileanu et al.,

2001). These findings suggest an underlying contribution of ACE

for the development of endometrium and endometriosis. In this

study, we aimed primarily to evaluate whether ACE A2350G and

A-2240T gene polymorphisms are attractive markers for moderate/

severe endometriosis susceptibility. To the best of our knowledge,

this is the first survey in this field.

Molecular Human Reproduction Vol.11, No.1 pp. 11–14, 2004

Advance Access publication November 5, 2004 doi:10.1093/molehr/gah131

11

at National Chiao Tung University Library on April 26, 2014

http://molehr.oxfordjournals.org/

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Materials and methods

Premenopausal Taiwan Chinese women with surgically diagnosed endome-triosis and non-endomeendome-triosis were included. All patients were divided into two groups: (1) endometriosis stage III/IV (n ¼ 150); (2) non-endometriosis group (n ¼ 159). All individuals with endometriosis accepted laparoscopy or laparotomy management and endometriosis was confirmed pathologically. The non-endometriosis status in the control group was confirmed by sonogra-phy and clinical evaluation. All patients had normal blood pressure without obvious cardiovascular disease. There were non-significant differences between both groups in age (34.5 ^ 4.2 versus 36.2 ^ 5.1 years), weight (51.4 ^ 3.5 versus 53.8 ^ 4.2 kg) and height (159.4 ^ 2.5 versus 157.4 ^ 3.8 cm). In clinical practice, most women with minimal/mild endo-metriosis accept conservative medication instead of invasive management. Therefore, we only recruited the moderate/severe endometriosis women for the survey. All women had consented to peripheral blood sampling for geno-type analyses. The studies were approved by the ethical committee and insti-tutional review board of the China Medical University Hospital. Informed consents were signed by all women who donated their blood.

The ACE A2350G genotypes (intron 17) were determined as previously described (Zhu et al., 2001). The genomic DNA was prepared from periph-eral blood leukocytes by the use of a genomic DNA isolation kit (Blossom, Taipei, Taiwan). A total of 50 ng genomic DNA was mixed with 20 pmol of each PCR primer in a total volume of 25 ml containing 10 mM Tris – HCl pH 8.3, 50 mM potassium chloride, 2.0 mM magnesium chloride, 0.2 mM each deoxyribonucleotide triphosphate and 1 U DNA polymerase (Amplitag; Perkin – Elmer, Foster City, CA, USA). A total of two gene polymorphisms was surveyed, including ACE A2350G and ACE A-240T. The SNP infor-mation for the genes involved was obtained via the Internet (http://www. ncbi.nlm.nih.gov/LocusLink/).

The PCR primer sequences and conditions for each primer are listed in Table I. The PCR amplification was performed in a programmable thermal cycler GenAmp PCR system 2400 (Perkin – Elmer Applied Biosystems, Foster City, CA, USA). After PCR amplification, the individual gene poly-morphisms were analysed by restriction digestion with restriction enzyme (New England Biolabs, Inc., Beverly, MA). The base pairs for their wild, heterozygote and SNP types are listed in Table I.

The PCR products were mixed together and 10 ml of this solution was loaded into 3% agarose gel containing ethidium bromide for electrophoresis. Genotypes and allelic frequencies for ACE A2350G and A-240T gene poly-morphisms in both groups were compared. Correlation between the ACE A2350G genotype and moderate/severe endometriosis was evaluated. Allelic frequencies are expressed as a percentage of the total number of alleles. The SAS system with x2 and Fisher’s exact tests were utilized for statistical analyses. A P-value of , 0.05 was considered statistically significant.

Results

Genotype distribution and allele frequency of ACE 2350 and

ACE-240 gene polymorphisms in both groups were significantly different

(Tables II and III). Proportions of ACE 2350*A

homozygote/hetero-zygote/G homozygote in endometriosis and non-endometriosis

populations were (1) 66.7/29.3/4% and (2) 96.2/3.1/0.7%,

respec-tively ( P , 0.0001; Table II). The percentage of ACE 2350*A/G

alleles in both groups were (1) 81.3/18.7% and (2) 97.8/2.2%,

respectively ( P , 0.0001; Table II). Proportions of ACE-240*A

homozygote/heterozygote/T

homozygote in both groups were

(1)

43.3/46/10.7%

and

(2)

62.9/35.8/1.3%,

respectively

( P , 0.0001; Table III). The percentage of ACE-240*A/T alleles in

both groups were (1) 66.3/33.7% and (2) 81.8/18.2%, respectively

( P , 0.0001; Table III).

The most common genotype and allele for ACE 2350 and

ACE-240 gene polymorphisms in both groups were A homozygote and

allele. However, the ACE 2350*G-related genotypes (A/G

hetero-zygote, G homohetero-zygote, G allele) and ACE-240*T-related genotypes

(A/T heterozygote, T homozygote, T allele) were strikingly higher

among the endometriosis population (Tables II and III). These

find-ings indicated that ACE 2350G and ACE-240T-related genotype

and

allele

were

associated

with

higher

susceptibility

to

endometriosis.

Discussion

The endometrium, which has prominent blood vessels and blood

flow, is one of the few adult tissues that exhibits regular periods of

rapid growth. Angiogenesis, therefore, is an important component

for the growth and function of these tissues. Endometriosis is a

disease of endometrial tissue shedding outside the uterus during

Table I. The primer sequences and PCR conditions for ACE A2350G and A-240T gene polymorphisms Polymorphisms Primer sequences (50_{! 3}0_)* _{PCR conditions}

(8C/s)

Restriction enzyme digestion

Allele DNA fragment size (bp) Denature Annealing Extension

ACE A2350G F-CTGACGAATGTGATGGCCGC 94/30 62/30 72/45 BstUI at 608C for 30 min A 122

R-TGATGAGTTCCACGTATTTCG G 103+19

ACE A-240T F-TCGGGCTGGGAAGATCGAGC 95/30 58/30 72/45 XbaI at 378C for 30 min A 137

R-GAGAAAGGGCCTCCTCTCTCT T 114+23

a_{*F and R indicate forward and reverse primers}

Table II. Genotype and allelic frequencies for ACE A2350G gene polymorphism in individuals with and without endometriosis Genotype Endometriosis (n ¼ 150) Non-endometriosis (n ¼ 159) P A/A 100 (66.7) 153 (96.2) , 0.0001a A/G 44 (29.3) 5 (3.1) G/G 6 (4.0) 1 (0.7) Allelic frequency A 244 (81.3) 311 (97.8) , 0.0001b G 56 (18.7) 7 (2.2) a

P-value was calculated by Fisher’s exact test.

b

P-value was calculated by x2- test.

Table III. Genotype and allelic frequencies for ACE A-240T gene polymorphism in individuals with and without endometriosis Genotype Endometriosis (n ¼ 150) Non-endometriosis (n ¼ 159) Pa A/A 65 (43.3) 100 (62.9) , 0.0001 A/T 69 (46) 57 (35.8) T/T 16 (10.7) 2 (1.3) Allelic frequency A 199 (66.3) 257 (81.8) , 0.0001 T 101 (33.7) 61 (18.2) a

P-values were calculated by x2- test.

Y.-Y.Hsieh et al.

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menstruation. These explants require a rich blood supply which

enables them to survive and grow. The activation of angiogenesis,

therefore, might be a key factor in pathogenesis of endometriosis

(Inan et al., 2003).

ACE activity is associated with angiogenesis. ACE inhibition by

perindopril improves myocardial angiogenesis (Toblli et al., 2004).

Angiotensin II, a key regulator of blood pressure and body fluid

homeostasis, exerts mitogenic effects on endothelial cells.

Angioten-sin II is also a humoral regulator of peripheral angiogenesis

(Walther et al., 2003). ACE catalyses angiotensin II formation;

therefore, the ACE activity is positively correlated with angiotensin

II production.

Recently, ACE inhibitors were found to significantly inhibit

tumour growth and angiogenesis along with suppression of the

vascular endothelial growth factor (VEGF) level (Yasumatsu et al.,

2004). Angiotensin II might have an important role in

carcinogen-esis and the anti-angiogenic activity is partly mediated by

angioten-sin II and ACE inhibition. ACE and angiotenangioten-sin II inhibitors might

be considered as useful anti-tumour agents. However, the

mechan-isms of suppression of the VEGF level are still unclear.

Further-more, in our previous survey, we also observed a correlation

between VEGF and endometriosis (Hsieh et al., 2004). Considering

all these observations ACE appears to have angiogenic and

tumori-genic effects upon endometriosis.

The ACE gene, which is located on chromosome 17q23, contains

some gene polymorphisms and candidate markers for hypertension

and related diseases (Doria et al., 1994). The ACE gene

polymor-phism located on intron 17 of the ACE gene might be in linkage

disequilibrium with other important gene variants. ACE gene

polymorphisms are related to numerous diseases, including carotid

artery wall thickness (Sayed-Tabatabaei et al., 2003), post-transplant

erythrocytosis (Yildiz et al., 2003), diabetic nephropathy (Chang

et al., 2003), Alzheimer’s disease (Kehoe et al., 2003), ischwemic

cerebrovascular disease (Um et al., 2003), dementia (Choi et al.,

2003), segmental glomerulosclerosis (Dixit et al., 2002), cystic

fibrosis (Arkwright et al., 2003), etc. ACE insertion/deletion (I/D)

polymorphism affects the uteroplacental and umbilical flow as well

as the recurrence of an adverse pregnancy outcome in women with

pre-eclampsia (Mello et al., 2003). The ACE I/D and M235T

polymorphisms are associated with an increased risk of developing

coronary heart disease, hypertension and ventricular hypertrophy

(Alvarez et al., 2000; Sethi et al., 2003). Three ACE gene

poly-morphisms [Alu insertion/deletion, 23949 (CT), 10698 (G)] might

influence the development of systemic lupus erythematosus and

nephritis (Parsa et al., 2002).

In contrast, some investigators have demonstrated the

non-associ-ation between the ACE gene polymorphisms with individual

dis-eases, including hypertension (Harrap et al., 1993), left ventricular

hypertrophy (West et al., 1997), pregnancy outcomes,

pregnancy-induced hypertension (Tamura et al., 1996) and nephronophthisis

(Omran et al., 1999). ACE-5466C and 4656 gene polymorphisms

are not directly related to the occurrence of sarcoidosis (Schurmann

et al., 2001). However, the genotype frequencies of the insertion/

deletion polymorphisms of the ACE gene and the M235T

polymorphism for the angiotensin gene might not contribute to

hypertension. This discrepancy might be due to different illness

classifications, or racial and disease variations. In fact, different

eth-nic groups might influence the ACE gene distributions (McKenzie

et al., 2001). Ethnic variation plays a major role in the genetic

regulation of serum ACE activity and ACE gene polymorphism for

cardiovascular disease (Bloem et al., 1996).

Zhu et al. (2001) demonstrated that ACE A2350G and A-240T

polymorphisms are significantly associated with blood pressure

and ACE concentration. The G allele for the ACE 2350 gene

poly-morphism is significantly associated with higher blood pressure and

ACE concentration (Zhu et al., 2001). They suggested that allelic

interaction of these gene polymorphisms might play an important

role in the dissection of complex traits such as blood pressure. They

also indicated that these associations were more obvious in female

individuals than in male patients. Therefore, the gender-specific

influence of these gene polymorphisms should be addressed in these

studies.

In this study, we noted that the genotype distributions for ACE

A2350G and A-240T gene polymorphisms were significantly

differ-ent between the individuals with and without endometriosis. This

finding is the first indicating that ACE gene polymorphisms might

predispose to endometriosis development. We also observed that the

ACE 2350*G-related genotypes and G allele appeared in a higher

percentage among the moderate/severe endometriosis populations

than the controls. This finding is compatible with the result of Zhu

et al. (2001), who suggested that the ACE 2350*G allele might be

associated with a higher risk of vascular lesion (higher systolic

blood pressure) and higher ACE concentrations. Therefore, our data

strongly suggest that the ACE gene polymorphisms might

substan-tially contribute to the pathogenesis of endometriosis. The data also

suggest that RAS might be involved in the pathogenesis of

endometriosis.

The mechanisms of SNPs on individual disease remain uncertain.

Despite SNPs not altering transcript levels, some investigators have

demonstrated that the disequilibrium effects of certain genotypes

might influence the related three-dimensional structure and

effi-ciency of the transcripts (Shintani et al., 1999; Kennon et al., 2004;

Shirasawa et al., 2004). Presumably, the distinct biological

con-dition caused by ACE is among numerous contributions that

influence endometriosis development. These contributions include

genetic, dietary and environmental factors regulating hormonal and

non-hormonal conditions. Furthermore, the ACE polymorphisms

might be in linkage disequilibrium with other unidentified functional

polymorphisms, which co-operatively influence the susceptibility to

endometriosis.

In conclusion, associations of moderate/severe endometriosis

with ACE*A2350G and A-240T gene polymorphisms exist. ACE

2350G and ACE-240T-related genotypes and alleles increase

the susceptibility to endometriosis. The ACE gene polymorphisms

likely contribute to the pathogenesis of endometriosis. Although the

real role and mechanism of ACE gene polymorphisms has not yet

been clarified, this polymorphism deserves more attention for

realiz-ing its importance to endometriosis development. Furthermore, this

study could be extended to determine whether the RAS and its

related gene polymorphism also affect the endometriosis formation.

After the clarification of its role in endometriosis, ACE gene

polymorphism may become a useful marker to predict the future

development of endometriosis and to permit early therapeutic

intervention in women at high risk of endometriosis.

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Submitted on September 19, 2004; resubmitted on October 18, 2004; accepted on October 22, 2004