Angiotensin I-converting enzyme and angiotensinogen geneinteraction and prediction of essential hypertension

ANNA VASKU, MIROSLAV SOUCEK, VLADIMIR ZNOJIL, IVAN RIHACEK, SVATAVA TSCHOPLOVA,LENKA STRELCOVA, KAREL CIDL, MICHAELA BLAZKOVA, DOBROSLAV HAJEK, LYDIE HOLLA, and JIRI VACHA

Institute of Pathological Physiology, Medical Faculty of Masaryk University, and First Department of Internal Medicine, St. Ann’sFaculty Hospital, Brno, Czech Republic

Angiotensin I-converting enzyme and angiotensinogen gene interactionand prediction of essential hypertension. To prove whether the interac-tion between insertion/deletion (I/D) angiotensin I converting enzyme(ACE) and M235T angiotensinogen (AGT) gene polymorphic allelescould contribute to causing essential hypertension, we examined subjectsfrom the Czech Republic (365 Caucasians total; 202 normotensives and163 hypertensives). Subjects were genotyped for insertion/deletion poly-morphism of ACE (I/D ACE, intron 16) and for M235T polymorphism ofangiotensinogen gene (AGT, exon 2) by means of the polymerase chainreaction (PCR) method. The case-control approach was used. Fisher’sexact test followed by Holmes’s test to overcome the problem of multiplecomparisons were used for the statistical analysis of data. No associationof single gene allelic variants with essential hypertension was found in ourpopulation. Having compared only double homozygote combinations, theassociation of the DDMM genotype with essential hypertension wasproven (P 5 0.0081). To the contrary, IITT (P 5 0.0086) was found morefrequently in normotensive subjects. We conclude that the interaction ofthe I/D ACE and M235T AGT polymorphic alleles can contribute toessential hypertension, despite the absence of single gene associations withthe condition.

The aim of this study was to prove whether the interactionbetween insertion/deletion (I/D) angiotensin I converting enzyme(ACE) and M235T angiotensinogen (AGT) gene polymorphicalleles could contribute to the occurrence of essential hyperten-sion.

Blood pressure is a quantitative, multifactorial trait of hetero-geneous origin, in which the definition of abnormality is arbitrary.It reflects a complex interaction between genetic and environmen-tal factors, with genetic susceptibility accounting for much of thevariation in blood pressure within populations and environmentalfactors determining the variation between populations [1]. Thegenetic background not only determines the actual blood pressureresponse to environmental conditions, but also the susceptibily oftarget tissues (heart, blood vessels and kidney) to stressors such asthe high blood pressure [2]. Hypertension is often a cause ofhuman cardiovascular morbidity and mortality because it consti-tutes a major risk for cardiac and cerebral infarction, cerebralhemorrhage, and cardiac and renal failure.

The renin-angiotensin system (RAS) is a hormonal system inthe classical sense, meaning that it is a circulating hormone actingon a target organ. The system apparently functions independentlyat the tissue level in a paracrine and autocrine manner. Subtlederangements in this system that may arise in childhood, mostlikely from a genetic predisposition, almost certainly form thebasis for hypertensive conditions [3]. Laragh proposed that themain pathophysiological problem of essential hypertension is inkidney with a heterogeneous population of nephrons, somehyperfiltering and other ischemic [4]. The heterogeneity arises inpatients with kidney having an abnormal hemodynamic responseto angiotensin II (AT II).

Angiotensinogen as well as angiotensin I converting enzymevariations are supposed to have a role in predisposing theindividuals to essential hypertension. The functional influences ofthe variability at both genes have not been fully established thusfar. I/D angiotensin converting enzyme (ACE) polymorphism hasbeen associated with the level af ACE in serum [5]. An indepen-dent effect of blood group ABO on the serum level of ACE wasrecently described [6]. An enhanced pressor response to angio-tensin I in normotensive men with a DD genotype for I/D ACEpolymorphism was shown, probably as a consequence of thegeneration of increased levels of angiotensin II [7]. The DDgenotype was associated with a significantly greater commoncarotid artery wall thickness (P 5 0.003) [8], without a simulta-neous association of the I/D ACE polymorphism with bloodpressure values according to monitored 24-hour ambulatory bloodpressure values.

The M235T AGT polymorphism codetermines the plasmalevels of angiotensinogen, with increasing levels from MM to TTgenotypes observed both in hypertensive [9, 10] and healthypopulations [11]. T235 allele was found to be associated with theextent of coronary lesions, which were detected in angiograms inpatients with coronary artery disease with a low-risk status in amultigenic study of genetic polymorphisms [12]. Recently, a newpolymorphism [G(26)A] in the angiotensinogen gene promotorwas found. The polymorphism was associated with essentialhypertension [13] and is supposed to affect the basal transcriptionrate of the gene according to tests of promoter activity and toDNA binding studies in vitro. T variant of M235T AGT polymor-phism is in tight linkage disequilibrium with the mutation in theangiotensinogen promotor G(26)A, which leads to elevatedexpression in vitro. Expression of the T235 allele is significantly

Key words: polymorphism allelic variants, angiotensin converting enzyme,blood pressure, renal failure.

© 1998 by the International Society of Nephrology

Kidney International, Vol. 53 (1998), pp. 1479–1482

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higher in decidual spiral arteries of heterozygous women (MT),suggesting that elevated expression of T235 in decidual spiralarteries may cause vascular changes leading to preeclampsia [14].

METHODS

The case-control method was used to study two genetic poly-morphisms of the RAS system in our normotensive and hyper-tensive populations. A sample of healthy population (306 Cauca-sians, Czech nationality, industrial city of Brno with 400,000inhabitants) was examined by the 24-hour blood pressure moni-toring (SpaceLab 90 207). The sample included the entire cohorts(men 40 to 45 years, women 45 to 50 years) of one generalpractitioner, in which essential hypertension had not been diag-nosed thus far. Two hundred and two subjects were diagnosed asnormotensives (controls defined as having , 20% BP valuesabove 140/90 mm Hg during the daytime and/or , 20% BP valuesabove 120/80 mm Hg during the night). Fifty-one subjects werenewly diagnosed as essential hypertensives according to thecriteria of Zachariah et al for 24-hour BP monitoring (.40%values over 140/90 mm Hg from 6:00 to 22:00 and/or . 40% over120/80 mm Hg from 22:00 to 6:00 the next day) [15]. The other 53subjects had . 20% values above 140/90 mm Hg during thedaytime and/or . 20% values above 120/80 mm Hg during thenight, and these subjects were excluded from the next statisticalanalysis. Cases consisted of 112 outpatients of the internal med-icine clinic who had been diagnosed with and treated for essentialhypertension, and of the 52 subjects newly diagnosed with essen-tial hypertension in which nonpharmacological and/or pharmaco-logical interventions were initiated. The patients with secondaryhypertension and/or diabetes mellitus as defined according toWHO recommendations were excluded.

The study was approved by the Committee for the Ethics ofMedical Experiments on Human Subjects, Medical Faculty,Masaryk University, Brno (No. 64/93, 1993). Informed consent ofthe examined subjects was obtained.

Twenty-four-hour blood pressure monitoring

Each record consisted of 48 to 53 readings of the systolic anddiastolic pressures. The daytime frequency of the measurementswas programmed on a three-measurements-per-hour basis from6:00 to 22:00 hours; night time measurements were performed ona one-measurement-per-hour basis from 22:00 until 6:00 hoursthe next day.

Genotype identification

The genomic DNA was isolated from peripheral blood leuko-cytes by a standard method using proteinase K digestion of cellsaccording to Sambrook et al [16].

The analysis of the insertion/deletion polymorphism in theangiotensin I converting enzyme gene (I/D ACE) was performedaccording to Rigat et al [17] with a subsequent verification of theDD genotype by the method of Shannugam, Sell and Saha [18].Using gel electrophoresis, the PCR products were distinguished asa 190 bp fragment in the absence and a 490 bp fragment in thepresence of the insertion (genotypes described as II-490 bps,ID-4901190 bps, DD-190 bps). Polymorphism M2353T in theangiotensinogen gene (M235T AGT) was detected according toRuss et al [19] (mismatch PCR; restriction analysis with Asp I;length of bands 165, 141 at 24 bps on gel electrophoresis withethidium bromide; genotypes described as MM-165 bps, MT-1651141 bps, and TT-141 bps).

Statistical analyses

A multivariate analysis of variance was used to calculate thedifference in body mass index (BMI) and sex between the twogroups.

Coincidences of double genotypes were calculated from prob-abilities of occurrence of possible haploid combinations of the twogene alleles assuming random combinability of haplotypes (thegenes are located at different chromosomes).

Probability of the more general Hardy-Weinberg equilibrium(comparisons of the observed and calculated coincidences) for thetwo genes as well as the difference in double genotype coincidencein the case-control study were calculated by the chi2 test.

Comparisons of the allelic variant frequencies as well ascomparisons of double homozygotes to the other double homozy-gotes in the case-control study were performed by Fisher’s exacttest.

The Holm’s test for multiple comparisons was used whereappropriate. The software program Statistic, version 3.0 (StatsoftInc., Tulsa, OK, USA) was used in all of the calculations.

RESULTS

Using the multivariate analysis of variance, a highly significantdifference in BMI between cases and controls was found (P 50.000003). This difference was not dependent on gender.

There were no significant differences in allelic frequencies ofthe examined genes between the two groups when they werecalculated separately (Table 1).

Both controls (P 5 0.729778) and cases (P 5 0.062687) werefound to be in equilibrium in double genotype coincidence (Table2). A bordeline difference in double genotype coincidence wasfound (P 5 0.054) in the case-control comparison.

Table 1. I/D ACE and M235T AGT allele frequencies in the case-control study

BMI kg/m2 Age years

I/D ACE M235T AGT

I D M T

Controls (N 5 202) 25.4 6 3.3 46 6 3 0.41 0.59 0.54 0.46M/W 5 86/116

Cases (N 5 163) 27.5 6 4.3 50 6 9 0.38 0.62 0.60 0.40M/W 5 95/68

Probability ofdifference

0.000003 NS 0.223182 0.086607

Abbreviations are: M/W, males/ females; BMI, body mass index; I/D ACE, insertion/deletion polymorphism of angiotensin converting enzyme gene;AGT, angiotensinogen gene. I, D, M and T are alleles.

Vasku et al: Gene interaction in essential hypertension1480

The double homozygote coincidence was different when thecases and controls were compared. Statistically significant differ-ences were found in two of the four possible homozygous combi-nations (Table 3): genotype DDMM was more frequent in thecases (P 5 0.008086), while IITT genotype was more frequent inthe controls (P 5 0.008619). After recalculation of probabilities bythe Holm’s test, the probability remained under 0.05.

DISCUSSION

Isolated I/D ACE gene polymorphism in essential hypertensionhas been studied often [20–22], as has isolated M235T AGTpolymorphism [23–27], with researchers using varied study de-signs and finding disparate, often negative, results.

Having compared allelic frequencies of M235T AGT polymor-phism in the studies, our results show a nonsignificant increase inthe M allelic frequency, especially in the controls. Our controlgroup included 202 normotensive subjects confirmed by 24-hourblood pressure monitoring. Surprisingly, only a few genetic studieshave used 24-hour monitoring to determine blood pressure,although the method is more accurate and gives more preciseinformation than office blood pressure. None of the studies[28–30] evaluated both polymorphisms. Another important factorimproving the differences in allelic frequency in Caucasian pop-ulations is a high homogeneity of Czech population historically.The known shift in the incidence of ABO blood groups inEuropean Caucasians could also contribute to the differences.

If the two polymorphisms mentioned above contribute (thoughslightly) to a genetic background affecting blood pressure andtheir contribution can be considered to be additive or multiplica-tive, then the incidence of double homozygotes compared to theother double homozygotes could be expected to be shiftedbetween normotensives and hypertensives. The genotype MMDDsupposedly can be characterized by combination of a high activityof ACE and a low level of angiotensinogen. The variant wasproven to be associated with hypertension. To the contrary,genotype IITT, phenotypically characterized as combination ofthe lowest activity of ACE and the highest level of angiotensino-gen, was found to be associated with normal blood pressure.Another variant, IIMM (the lowest activity of ACE and the lowestlevel of angiotensinogen), did not differ between normotensivesand hypertensives in occurrence. Getopyte DDTT (the highestactivity of ACE with the highest level of angiotensinogen) seemsto slightly diminish the risk of hypertension.

Further candidate gene analysis can be expected to elucidatethe genetic background of essential hypertension in future.

ACKNOWLEDGMENTS

This study was supported by grant 306/93/2192, the Grant Agency of theCzech Republic. We thank Professor Friedrich C. Luft, Professor DetlevGanten and Professor Hermann Haller for the acceptance of this study asa poster for the ISN Forefronts in Nephrology symposium on “Geneticsand Genes in Hypertension.”

Reprint requests to Anna Vasku, Institute of Pathological Physiology,Medical Faculty, Komenskeho nam 2, 662 43 Brno, Czech Republic.E-mail: avasku@med.muni.cz

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Table 2. Comparison of the observed and calculated coincidence of I/DACE with M235T AGT genotypes

II ID DD

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Controlsa

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Casesb

Observ. 9 15 1 21 34 19 28 29 7Calcul. 8.4 11.4 3.9 27.2 37 12.6 22.2 30.2 10.3

Abbreviations are: Observ, observed coincidence; Calcul, calculatedcoincidence of genotypes.

a Equilibrium criterion 5 7.58362; P 5 0.729778.b Equilibrium criterion 5 11.96797; P 5 0.062687.

Table 3. Coincidence of the two gene variants in combination

Allele MM MT TT ALL

Controls 10 19 11 40Cases (M/F) II 5/4 9/6 1/0 15/10

P , 0.4246 , 0.0086

Controls 25 49 12 86Cases (M/F) ID 13/8 16/18 13/6 42/32

Controls 22 37 17 76Cases (M/F) DD 18/10 14/15 6/1 38/26

P , 0.0081 , 0.09432

Controls 57 105 40 202Cases (M/F) ALL 36/22 39/39 20/7 95/68

Abbreviation M/F is male/female.

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