Revista Română de Medicină de Laborator Vol. 18, Nr. 4/4, Decembrie 2010

Angiotensin-converting enzyme gene polymorphisms inpulmonary arterial hypertension in children

Polimorfismul genei enzimei de conversie a angiotensinei în hipertensiuneaarterial ă pulmonară la copil

Rodica Togănel1,2*, Claudia Bănescu3, Carmen Duicu4, Carmen Şuteu2,Iolanda Muntean1,2, Amalia Făgărăşan1,2, Liliana Gozar2, Sorina Pasc2

1. University of Medicine and Pharmacy Tg. Mureş - Cardiology Clinic Tg. Mureş2. Institute of Cardiovascular Diseases and Transplantation Tg. Mureş - Cardiology Clinic

3. University of Medicine and Pharmacy Tg. Mureş - Genetics Department4. University of Medicine and Pharmacy Tg. Mureş - Pediatric Clinic Tg. Mureş

Abstract

Introduction. Angiotensin converting enzyme (ACE) plays an important role in the pathogenesis of pul-monary arterial hypertension. In this study we determined whether the deletion (D)/insertion (I) polymorphism inthe ACE gene may be associated with pulmonary arterial hypertension in children. Methods and Results. Weperformed a case-control study involving 29 patients with pulmonary arterial hypertension and 26 healthy con-trols. Genomic DNA extracted from peripheral blood was amplified using the polymerase chain reaction to de-tect the polymorphic marker. In patients, ACE genotype distribution of DD, ID, and II was 34.5%, 58.6%, and6.9%, respectively, whereas in controls it was 11.5%, 69.3%, and 19.2%, respectively. Conclusion. The ACE DDgenotype is significantly increased in patients with pulmonary hypertension compared with normal controls, sug-gesting that certain individuals may be genetically predisposed to developing pulmonary hypertension.

Keywords: pulmonary hypertension, gene polymorphisms, angiotensin converting enzyme.

Rezumat

Introducere. Enzima de conversie a angiotensinei (ACE) joacă un rol important în patogeneza hipertensiuniiarteriale pulmonare. În acest studiu am analizat posibila asociere a polimorfismului genei ACE deleţie (D) / inserţie (I) cuhipertensiunea arteriala pulmonară la copii. Metodă şi rezultate. Am efectuat un studiu caz-control cu 29 pacienţi cu hi-pertensiune arterială pulmonară şi 26 martori sănătoşi. ADN-ul genomic extras din sângele periferic a fost amplificatprin reacţia polimerizării în lanţ pentru a detecta markerul polimorfic. Frecvenţa genotipului DD, ID, şi II în gena ACE lapacienţi a fost de 34.5%, 58.6%, respectiv 6.9% , iar la lotul control a fost de 11.5%, 69.3%, respectiv 19.2%. Concluzii.Genotipul DD în gena ACE este semnificativ crescut la pacienţi comparativ cu lotul control, sugerând faptul că anumiţiindivizi au o predispoziţie genetică în dezvoltarea hipertensiunii pulmonare.

Cuvinte cheie : hipertensiune pulmonară, polimorfism genic, enzima de conversie a angiotensinei

*Corresponding author: Rodica Togănel, University of Medicine and Pharmacy Tg. Mureş - Cardiology ClinicTg. Mureş, 38, Gh. Marinescu Str, Tg. Mureş 540042, Romania.Tel: +40 265 210505, Email: rodicatoganel@yahoo.com

31

Revista Română de Medicină de Laborator Vol. 18, Nr. 4/4, Decembrie 2010

Introduction

Pulmonary arterial hypertension (PAH)consists of a group of vascular abnormalitieswith elevated pulmonary arterial pressure andpulmonary vascular resistance. PAH is definedas a mean pulmonary artery pressure of over25mmHg at rest or over 30mmHg with exercisein the absence of increased left heart bloodpressure. PAH is a life-threatening disease char-acterized by a progressive increase of pulmon-ary vascular resistance (PVR), which, if left un-treated, progresses rapidly to right ventricularfailure. Cardiac disorders, pulmonary disorders,or both in combination are the most commoncauses of secondary pulmonary hypertension.The current nosology of pulmonary hyperten-sion (PH) has been provided by consensus ofthe Venice classification in 2003. There are fivegroups of PH arranged according to pathogenet-ic, clinical and therapeutic criteria. The firstgroup (PAH) includes the idiopathic (IPAH),familial (FPAH) forms and PAH associatedwith different conditions such as congenital he-art defects, portal hypertension, connective tis-sue disease and others that share similar clinicaland hemodynamic aspects and comparablepathological changes of the lung microcircula-tion. The other four groups include the PH thatdevelops secondary to left-heart diseases, hyp-oxic lung diseases, pulmonary embolism, andmiscellaneous conditions. Moreover, the func-tional classification of PH is performed inagreement with classification of the New YorkHeart Association according to the WorldHealth Organization (1998).

There are generally four main altera-tions in the walls of the pulmonary vessels.These include vasoconstriction, rarefaction ofvessels, vascular remodeling and the occlusionof vascular lumen by a thrombus with sub-sequent structural remodeling of the vascularand extracellular matrix (1).

A lot of studies suggest that the ren-in-angiotensin system (RAS) has a fundamentalrole in the onset and progression of cardiovascu-

lar diseases and in regulating blood pressure (2).Genes encoding components of RAS are pos-sible candidate genes for hypertension. Amongthe candidate genes of the RAS, the an-giotensinogen (AGT), angiotensin-convertingenzyme (ACE), angiotensinogen II type-1 re-ceptor (AGTR1), and aldosterone synthase(CYP11B2) genes have been widely investigatedas genetic determinants of hypertension (3 - 5).

Angiotensin converting enzyme (ACE)plays an important role in the pathogenesis of pul-monary arterial hypertension. ACE is a key en-zyme in renin-angiotensin system (RAS) andwidely distributed in human tissues including thelung, vascular endothelium, kidney and heart.ACE converts angiotensin I to angiotensin II, andinactivates bradykinin through the kallikre-in-kininogen system. Angiotensin II may play arole in vascular diseases through vascular smoothmuscle cell contraction and proliferation, mono-cyte adhesion, platelet aggregation, mediatedeither directly or via various factors such as en-dothelin, nitric oxide, and prostacyclin (6).

The ACE gene contains a polymorphismbased on the presence (insertion, I) or absence (de-letion, D) within an intron of a 287-bp non-senseDNA domain, resulting in three genotypes (DDand II homozygotes, and DI heterozygotes) (7).

To date, the ACE DD genotype has beenassociated with increased circulating and cardiac tis-sue ACE activity (8), the risk for coronary artery dis-ease and myocardial infarction (9), the risk for eitherischemic or idiopathic dilated cardiomyopathy (10),hypertrophic cardiomyopathy (11), and possibly therisk of left ventricular hypertrophy (2, 12). Sincethese syndromes are associated with abnormal vaso-constriction and vascular smooth muscle prolifera-tion, Abraham et al. postulated a role for the RASand the ACE genotype in the pathophysiology ofpulmonary hypertension (PH) (2).

Also, Tanabe et al. identified this poly-morphism as risk factor for chronic thrombo-em-bolic pulmonary hypertension (13). According toOztürk et al. ACE gene I/D polymorphism D al-lele may modulate the relationship between an-

32

Revista Română de Medicină de Laborator Vol. 18, Nr. 4/4, Decembrie 2010

terior acute myocardial infarction and pulse pres-sure (14). Another study suggests that I/D ACEgene polymorphism is linked to pulmonaryartery pressure in Caucasian patients with chron-ic obstructive pulmonary disease (15).

Although there are some studies whichreported a positive association between alleleand coronary artery disease susceptibility (9),this result was not replicated in a recent studyby Shafiee SM et al. The study showed that DDgenotype does not increase the coronary arterydisease susceptibility in the studied populationand may not be a risk factor (16).

The relation between ACE gene poly-morphism and pulmonary arterial hypertension inchildren has not been previously reported. There-fore, the objective of this study was to investigatethe relation between ACE gene polymorphismand pulmonary arterial hypertension in children.

Methods

SubjectsThe study protocol was approved by

the Ethics Committee of the University ofMedicine and Pharmacy Tg. Mures and Insti-tute of Cardiovascular Diseases Tg. Mures.Written informed consent was obtained fromeach subject’s parent included in the study.

The study included 55 children: 26healthy individuals as a control group and 29 pa-tients with pulmonary hypertension. Among them,2 patients were diagnosed with idiopathic pul-monary arterial hypertension (IPAH) and 27 withPAH associated with CHD. They were included inNYHA functional classes: 8 patients pertained tothe NHYA II class, 16 patients to the NYHA IIIclass, and 5 patients to the NYHA IV class.

The patients were evaluated by echo-cardiography, using an IE33 Philips echocardi-ograph. The echocardiographic technique toevaluate the pulmonary hypertension consistedin: indirect assessment of pulmonary arterialhypertension, measurement of pulmonary arterypressure from tricuspid valve regurgitation andfrom pulmonary regurgitation, the impact of

pulmonary arterial hypertension on the rightventricle. Estimation of right atrial pressure ismost often done by assessing the IVC diameterand its degree of collapse with inspiration. Tri-cuspid annular plane systolic excursion(TAPSE) was used to evaluate right ventricularlongitudinal function. This parameter is meas-ured using an M-mode cursor passed throughthe tricuspid lateral annulus in a four – chamberview. This parameter measures the extent ofsystolic motion of the lateral portion of the tri-cuspid ring towards the apex. The tricuspid in-flow pattern was used to evaluate the rightventricular diastolic function. The tricuspid in-flow pattern is obtained in a four- chamberview by placing a cursor at the tips of the tri-cuspid valve. The Tei index, which measuresglobal ventricular function, has been used to as-sess the impact of increased pulmonary arterypressure. It is calculated as the ratio betweenthe sum of the times of the isovolumetric peri-ods and the ejection time for the right ventricle.

GenotypingGenomic DNA was extracted from peri-

pheral blood as described previously (7). Briefly,genomic DNA was extracted from whole bloodcontaining ethylenediamine-tetraacetic acid(EDTA) as an anticoagulant, by using the Gen-omic DNA Purification Kit (ZymoResearch).The sequences of the forward and reverseprimers used were: 5’-CTGGAGAC-CACTCCCATCCTTTCT-3’ and 5’-GATGTG-GCCATCACATTGGTCAGAT-3’ (Fermentas).DNA amplifications were performed with aMastercycler Gradient Thermal Cycler (Eppen-dorf) with 5 min of denaturation at 94°C, fol-lowed by 30 cycles with denaturation for 1 minat 94°C, annealing for 1 min at 58°C, and exten-sion for 2 min at 72°C, followed by 5 min of ex-tension at 72°C.

Detection of ACE Polymorphism byElectrophoresis

The reaction products were separated byelectrophoresis in 2% agarose gels and stainedwith ethidium bromide. Under ultraviolet light

33

Revista Română de Medicină de Laborator Vol. 18, Nr. 4/4, Decembrie 2010

two bands, insertion (I allele; 490 bp) and deletion(D allele; 190 bp) were visible (Figure 1).

Statistical analysisData analysis was performed using the stat-

istical analysis software system SPSS.15. Chi squaretests according to Pearson were performed to com-pare the frequency of ACE genotype between pa-tients and control groups. A p value less than 0.05was considered significant. Allele frequencies wereestimated by the gene counting method.

Results

Since no reports are available regardingthe association between ACE I/D polymorphismand pulmonary hypertension in children in Ro-mania, the present case control study examinedthe possible association of ACE I/D polymorph-ism in the pulmonary hypertensive patients.

The patient group comprised 15 boys and14 girls. The mean age of the patients was 10.83years. In the control group there were 12 girls and 14boys. The mean age of the group was 12.1 years.

The frequencies of II and ID genotypes ofACE gene polymorphism were not significantly dif-ferent between patients and controls. ACE-DD gen-otype was significantly higher in patients comparedwith controls (0.046). The frequency of I/D hetero-zygotes as compared to homozygotes, was higherboth in the patient and control group (Table 1). Bothgroups were in Hardy-Weinberg equilibrium.

We have also analyzed the associationof all the three genotypes with the phenotypic

variables such as gender and severity of pul-monary hypertension. Genotype frequencieswere not different between NYHA functionalclasses (Table 2) and we did not find a signific-ant difference between boys and girls.

As summarized in Table 2, I allele fre-quencies analysis revealed a significant differ-ence between the two groups (p=0.046).

Discussion

Pulmonary arterial hypertension (PAH) isa debilitating chronic disease of the small pul-monary arteries that is characterized by vasocon-striction and vascular remodeling. Endothelialdysfunction is believed to occur early in diseasepathogenesis, leading to endothelial and smoothmuscle cell proliferation and structural changes orremodeling of the pulmonary vascular bed result-ing in an increase in pulmonary vascular resist-ance (17). The renin-angiotensin system compon-ents appear to play an important role in the patho-genesis of pulmonary hypertension (18).

Angiotensin-converting enzyme (ACE),the key enzyme mainly responsible for the con-version of angiotensin I to angiotensin II, is be-lieved to contribute to the development of pul-monary hypertension, because angiotensin IIstimulates the growth and proliferation of hu-man vascular smooth muscle cells (19). ACEexpression was found to be elevated in patientswith pulmonary hypertension (20).

The expression of ACE and angiotensinII are partially genetically determined, with theD allele of ACE associated with higher bloodACE levels and activity (19).

We determined the prevalence of inser-tion/deletion (I/D) polymorphism of the ACEgene in children with pulmonary hypertension.ACE DD genotype was significantly higher inpatients compared with controls. Our findingsin this study suggest that patients with DD gen-otype may have a greater increase in tissueACE activity than those with the II and ID gen-otypes. The increased local production of ACEcould contribute to elevated pulmonary vascu-

34

Figure 1. Agarose gel electrophoresis of PCR productsof angiotensin-converting enzyme gene. M: marker, lanes1,12: homozygous II cases, lanes 2-5, 7-10 hetrozygous ID

and lane 6,11: homozygous DD case

Revista Română de Medicină de Laborator Vol. 18, Nr. 4/4, Decembrie 2010

lar tone and pulmonary vascular remodeling byincreasing the local production of angiotensin II,because angiotensin II is known to stimulate thegrowth of human vascular smooth muscle cells.The association between the D allele and pul-monary hypertension in patients suggests a po-tential role for renin-angiotensin system in thepathogenesis of PH.

There are a few reports that have foundan association between the D allele and pulmon-ary hypertension. Kanazawa H et al suggestedthat I/D polymorphism in the ACE gene may beassociated with pulmonary hypertension evokedby exercise challenge in patients with chronic ob-structive pulmonary disease (21). Abraham et al.demonstrated an increased incidence of the ACEDD genotype in patients with primary pulmonaryhypertension, and an association of the ACE DDgenotype with preserved right ventricular functionin these patients (2). Solari et al. suggested that D

allele of the ACE gene insertion/deletion poly-morphism may be associated with primary pul-monary hypertension in newborns with congenitaldiaphragmatic hernia (19). In contrast to theaforementioned studies, Hoeper et al. found noevidence for an association between ACE geno-type and ACE serum activity, respectively, andright ventricular performance in patients withprimary pulmonary hypertension (22).

The present investigation providesevidence of a role for the RAS in the pathogen-esis of pulmonary hypertension, by demonstrat-ing a high incidence of the ACE DD genotypein PAH patients. The frequency of the ACE DDgenotype in our patients is smaller (34.5%) thanthat reported by Abraham et al (45%) (2).

In our study, the incidence of I allele andII genotype was higher in controls than in childrenwith pulmonary hypertension. These findings maysuggest that the I allele and II genotype of the

ACE gene could be protectiveagainst PAH in children.

To our knowledge, this isthe first study that evaluates theassociation of ACE I/D poly-morphism with pulmonary hyper-tension in children in Romania.The number of patients in thisstudy was relatively small for agenetic association study, and ourresults should be enlarged uponin wider future studies.

In conclusion, our resultsshowed that pulmonary hyperten-sion is associated with a higherincidence of DD genotype.

Acknowledgements

The authors thank to Konc-ság Előd for statistical analysis.

The study was realized inthe research program projectMAMI no 41-042/2007 financedby the Romanian Ministry ofEducation, Research and Youth.

35

Table 1. Distribution of ACE genotype and allele frequencies inpatients and controls

Patientsn=29 (%)

Controlsn=26 (%)

p value

Genotype frequency II ID DD

2 (6.9)17 (58.6)10 (34.5)

5 (19.2)18 (69.3)3 (11.5)

0.4260.4140.046

Allele frequency I D

19 (39.6)29 (60.4)

23 (52.3)21 (47.7)

0.1710.046

ACE- angiotensin converting enzyme; I- Insertion; D- deletion

Table 2. Frequency of Genotypes of ACE-I/D polymorphismaccording to NYHA functional class in PH

ACE genotype II ID DD

NYHA I n= 0 (%) 0 (0) 0 (0) 0 (0)NYHA IIn= 7 (%) 1 (14.3) 5 (71.4) 1(14.3)NYHA IIIn= 16 (%) 1 (6.3) 7 (43.7) 8 (50)NYHA IV n= 4 (%) 0 (0) 3 (75) 1(25)

NYHA - New York Heart Association functional class

Revista Română de Medicină de Laborator Vol. 18, Nr. 4/4, Decembrie 2010

Abbreviations

ACE= angiotensin converting enzymeI= insertionD= deletionDNA= deoxyribonucleic acidNYHA = New York Heart Association functional

classPCR= polymerase chain reactionPAH= pulmonary arterial hypertensionRAS= renin-angiotensin systembp= base pairs

References1. Götting M, Addicks JP, Uibel S, Jensen AM, Grone-berg DA, Quarcoo D. Pulmonary hypertension-clinical as-pects, pathophysiology, diagnostic and therapy. Versicher-ungsmedizin. 2010, 62(1):9-15.2. Abraham WT, Raynolds MV, Badesch DB, WynneKM, Groves BM, Roden RL et al. Angiotensin-convertingenzyme DD genotype in patients with primary pulmonaryhypertension: increased frequency and association withpreserved haemodynamics. J Renin Angiotensin Aldoster-one Syst. 2003, 4(1):27-303. Nawaz SK, Hasnain S. Pleiotropic effects of ACEpolymorphism. Biochemia Medica 2009;19(1):36-49. 4. Ortlepp JR, Metrikat J, Mevissen V, Schmitz F,Albrecht M, Maya-Pelzer P et al. Relation between the an-giotensinogen (AGT) M235T gene polymorphism andblood pressure in a large, homogeneous study populationJournal of Human Hypertension, 2003, 17:555-559.5. Buraczynska M, Ksiazek P, Drop A, Zaluska W,Spasiewicz D, Ksiazek A. Genetic polymorphisms of therenin-angiotensin system in end-stage renal disease. Neph-rol Dial Transplant, 2006, 21: 979-9836. Sayed-Tabatabaei FA, Oostra BA, Isaacs A, VanDuijn CM, Witteman JCM. ACE Polymorphisms Circ.Res. 2006;98;1123-1133.7. Rigat B, Hubert C, Corvol P, Soubrier F. PCR detec-tion of the insertion/deletion polymorphism of the humanangiotensin converting enzyme gene (DCP1) (dipeptidylcarboxy peptidase 1). Nucl. Acids Res. 1992, 20:1433. 8. Danser AH, Schalekamp MA, Bax WA , van denBrink A M, Saxena PR, Riegge G et al. Angiotensincon-verting enzyme in the human heart: effect of thedeletion/insertion polymorphism. Circulation. 1995,92:1387-1388.9. Cambien F, Poirier O, Lecerf L, Evans A, Cambou JP,Arveiler D et al. Deletion polymorphism in the gene forangiotensin-converting enzyme is a potent risk factor formyocardial infarction. Nature 1992, 359:641-644.10. Raynolds MV, Bristow MR, Bush E, Abraham WT,Lowes BD, Zisman LS et al. The angiotensin converting

enzyme D/D genotype frequency is increased in patientswith ischemic or idiopathic dilated cardiomyopathy. Lan-cet 1993; 342:1073-1075.11. Marian AJ, Yu Q-t, Workman R, Greve G, RobertsR. Angiotensin-converting enzyme polymorphism in hy-pertrophic cardiomyopathy and sudden cardiac death. Lan-cet 1993, 342:1085-1086.12. Iwai N, Ohmichi N, Nakamura Y, Kinoshita M. DDgenotype of the angiotensin-converting enzyme gene is arisk factor for left ventricular hypertrophy. Circulation1994, 90:2622-262813. Tanabe N, Amano S, Tatsumi K, Kominami S, Ig-arashi N, Shimura R, et al. Angiotensin-converting en-zyme gene polymorphisms and prognosis in chronicthromboembolic pulmonary hypertension. Circ J. 2006;70(9):1174-1179.14. Oztürk O, Oztürk U. Relation between angiotensin-con-verting enzyme I/D gene polymorphism and pulse pressure inpatients with a first anterior acute myocardial infarction. Anad-olu Kardiyol Derg. 2009; 9(1):9-14.15. Tkácová R, Joppa P, Stancák B, Salagovic J, Mis-íková S, Kalina I. The link between angiotensin-convert-ing enzyme genotype and pulmonary artery pressure in pa-tients with COPD. Wien Klin Wochenschr. 2005, 117(5-6):210-214.16. Shafiee SM, Firoozrai M, Salimi S, Zand H, HesabiB, Mohebbi A. Angiotensin converting enzyme DD geno-type not associated with increased risk of coronary arterydisease in the Iranian population. Pathophysiology 2010,17:163-167.17. Humbert M, Sitbon O, Simonneau G. Treatment ofpulmonary arterial hypertension. N Engl J Med 2004,351:1425-1436.18. Morrell NW, Morris KG, Stenmark KR. Role of an-giotensin converting enzyme and angiotensin II in the de-velopment of hypoxic pulmonary hypertension. Am JPhysiol. 1995, 269: H1186-H1194.19. Solari V, Puri P. Genetic Polymorphisms of An-giotensin System Genes in Congenital Diaphragmatic Her-nia Associated With Persistent Pulmonary Hypertension.Journal of Pediatric Surgery, 2004, 39(3): 302-306.20. Orte C, Polak JM, Haworth SG. Expression of pul-monary vascular angiotensin-converting enzyme inprimary and secondary plexiform pulmonary hypertension.J Pathol 2000, 192: 379-384.21. Kanazawa H, Okamoto T, Hirata K, Yoshikawa J.Deletion Polymorphisms in the Angiotensin ConvertingEnzyme Gene Are Associated with Pulmonary Hyperten-sion Evoked by Exercise Challenge in Patients withChronic Obstructive Pulmonary Disease. Am J Respir CritCare Med, 2000, 162: 1235-1238.22. Hoeper MM, Tacacs A, Stellmacher U, Lichtingha-gen R. Lack of association between angiotensin convertingenzyme (ACE) genotype, serum ACE activity, and haemo-dynamics in patients with primary pulmonary hyperten-sion Heart 2003, 89: 445-446.

36