The PON1192RR genotype is associated with a higher prevalence of arterial hypertension

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Original article 1293

The PON1192RR genotype is associated with a higherprevalence of arterial hypertensionMaurizio Marraa, Francesca Marchegianib, Roberto Antonicellic,Cristina Sirollad, Liana Spazzafumod, Fabiola Olivierib, Claudio Franceschib,g,h,Roberto Testaa, Giuseppe Paolissoe, Richard W. Jamesf, Massimo Boemia andGianfranco Paratii

Objective To investigate whether genetic polymorphism of

paraoxonase (PON1192), an enzyme which protects low

density lipoprotein from oxidation, is related to the

prevalence of arterial hypertension.

Methods Two groups of carefully selected subjects of both

sexes were enrolled and compared. The first group

comprised 219 healthy controls (mean age

46.5 W 14.7 years) whereas the second comprised 119

hypertensive patients (mean age 47.9 W 10.5 years) with

untreated essential arterial hypertension. Anthropometric

and biochemical parameters were within the normal range

in both groups. The PON1192 polymorphism was

determined by a polymerase chain reaction-restriction

fragment length polymorphism approach.

Results In hypertensive patients, a significant increase of

the frequency of PON1192RR genotype with respect to

healthy controls (14.3 versus 5.0%, P U 0.003) was found.

Logistic regression analyses also showed that the

PON1192RR genotype was independently associated with a

four-fold increase in susceptibility to arterial hypertension

(odds ratio U 4.31; 95% confidence interval U 1.63–11.43,

P U 0.003).

Conclusions The finding that PON1192RR genotype is

associated with a higher prevalence of arterial hypertension

may contribute to improving the stratification of

opyright © Lippincott Williams & Wilkins. Unauth

0263-6352 � 2006 Lippincott Williams & Wilkins

cardiovascular risk within a population aged 30–60 years.

Determination of the PON1192 polymorphism may help to

identify those individuals who are prone to developing

cardiovascular diseases at an early stage, suggesting the

need for close monitoring of cardiovascular risk factors

before the onset of cardiovascular disease. J Hypertens

24:1293–1298 Q 2006 Lippincott Williams & Wilkins.

Journal of Hypertension 2006, 24:1293–1298

Keywords: arterial hypertension, paraoxonase activity, paraoxonase1192gene polymorphism

aDiabetology Unit, INRCA, Ancona, bCenter of Molecular Biology and Genetics,INRCA, Ancona, cCardiology Unit, INRCA, Ancona, dCenter of Epidemiologyand Biostatistic, INRCA, Ancona, eDepartment of Geriatrics and MetabolicDiseases, Second University of Naples, Italy, fUniversity Hospital, Geneva,Switzerland, gDepartment of Experimental Pathology, Bologna University,hInterdepartmental Center ‘Galvani’ (CIG), Bologna University, Bologna andiDepartment of Clinical Medicine, Prevention and Applied Biotechnologies,University of Milano-Bicocca, S-Luca Hospital, Istituto Auxologico Italiano,Milan, Italy

Correspondence and requests for reprints to Dr Maurizio Marra, Department ofGerontological Research, Diabetology Unit-INRCA, via della Montagnola 81,I-60131 Ancona, ItalyTel: +39 0718003854; fax: +39 0718003556; e-mail: [email protected]

Sponsorship: This work was subsidised in part by a grant from the Ministry ofHealth ‘Progetto finalizzato 1998: Interazioni tra fattori di rischio cardiovascolaree fattori genetici dell’aterosclerosi e della trombosi: un approccio integratogenetico-funzionale in casistiche selezionate’. Richard W. James was supportedby the Swiss National Research Foundation.

Received 19 July 2005 Revised 28 December 2005Accepted 27 February 2006

IntroductionThe paraoxonase gene family contains at least three

members, PON1, PON2 and PON3, which are located

on chromosome 7q21.3–22.1 [1]. The PON1 gene pro-

duct in blood circulation is carried by a subfraction of

high-density lipoproteins (HDL), and appears to act on

oxidized phospholipids carried by low-density lipo-

protein (LDL) particles as a physiological substrate

[2,3]. The PON1 gene polymorphism is due to an amino

acid substitution (Gln to Arg) at position 192 in the

coding region of the gene, with low (Q phenotype)

and high (R phenotype) activity towards the artificial

substrate paraoxon [4]. The prevalence of a number of

risk factors for cardiovascular disease (CVD), such as

arterial hypertension, hyperlipidemia and diabetes [5],

may be determined, at least in part, by genetic

factors.

Among these genetic factors, the paraoxonase1192 gene

polymorphism is claimed to play a central role, as

suggested by several studies [6–11].

First, Ruiz et al. [7] reported an association between the

presence of the polymorphism in the heterozygous (QRgenotype) or homozygous (RR genotype) states and cor-

onary heart disease in French Caucasian type 2 diabetic

patients. In particular, the authors suggested a gene–dose

response of the Gln-Arg 192 polymorphism because the

RR homozygotes exhibited a stronger association with

vascular disease than heterozygotes.

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1294 Journal of Hypertension 2006, Vol 24 No 7

In addition, Phfol et al. [9] reported a similar result in a

population of type 2 diabetic patients with vascular

disease. The PON1192 Arg allele was found to be a risk

factor for coronary artery disease. Their study empha-

sized that the association between the PON1192 Arg

allele and coronary artery disease was even stronger after

correction for possible confounders (i.e. age, sex, smoking

history and hypertension).

Finally, Voetsch et al. [11] found increased frequencies of

the PON1192RR genotype and the R allele among young

adults with non-fatal arterial ischemic stroke compared

to controls. In addition, the PON1192RR genotype remai-

ned independently associated with a four-fold increase in

the risk of arterial ischemic stroke after adjustment for

conventional vascular and prothrombotic risk factors

(i.e. hypertension).

The apparent contradiction between the high PON

activity linked to PON1192R allele and the epidemiolo-

gical evidence of its linkage to CVD may be explained by

the study of Mackness et al. [12]. The authors proposed

that this might be due to the different ability of the two

alloenzymes to prevent the accumulation of lipid-pero-

xides on LDL. Oxidized LDLs play an important role in

endothelial dysfunction. It is well known that paraoxo-

nase is a potent inhibitor of LDL and HDL oxidation,

and also protects against the toxic effects of reactive

oxygen species (ROS) produced during the atherogenic

process. It is also well documented that an enhanced

production of ROS is associated with endothelial dys-

function in adult subjects with essential hypertension

[13,14].

Recently, Uzun et al. [15] found that increased oxidative

stress is associated with a consistent decrease in PON1

activity in white-coat hypertension.

In a recent paper [16], we demonstrated that healthy

subjects with the PON1192RR genotype had an impaired

brachial reactivity, with respect to those inidviduals shar-

ing the QQ or QR genotype after infusion of triglycerides

alone or in addition to buccal administration of nitro-

glycerine, suggesting that subjects sharing a different

PON1 genotype might have different risks for endothelial

dysfunction [16].

The present study aimed to assess whether an associa-

tion between the PON1192 polymorphism and arterial

hypertension, a major cardiovascular risk factor, exists in

absence of CVD. In particular, we aimed to assess

whether subjects carrying the PON1192RR genotype

have an increased risk of developing arterial hypertension

by comparing the frequency distribution of PON1192RRversus QQ þ QR genotypes in uncomplicated and

untreated patients with essential hypertension and in a

control group of healthy individuals.

opyright © Lippincott Williams & Wilkins. Unautho

MethodsPatientsTwo hundred and nineteen unrelated healthy controls

and one hundred and nineteen untreated hypertensive

patients were enrolled in our study, which was approved

by the Italian National Research Center on Aging

(INRCA) Ethical Committee. All evaluated subjects

underwent a routine medical check-up, which included

a complete clinical and laboratory examination and an

electrocardiogram (ECG). Subjects with clinical history

of ischemic heart disease (myocardial infarction, angina

pectoris), stroke and peripheral vascular diseases were

excluded from the study, as were subjects with previous

liver or renal disease, or with diabetes mellitus. Enrolled

subjects did not use any drugs. Furthermore, these

subjects were Caucasians, consumed a Mediterranean

diet, and none had a history of alcohol abuse. In particu-

lar, in all subjects, the presence of the classical CVD risk

factors, such as type 2 diabetes mellitus [17], arterial

hypertension [18], a history of CVD, low HDL choles-

terol and smoking history, was assessed. Hypertensive

patients were untreated because they were enrolled at

the first time of diagnosis. In particular, patients with

arterial hypertension were recruited if their clinic systolic

and diastolic blood pressure was repeatedly found to be

> 140 and 90 mmHg, respectively, over three visits

carried out within 3 months, without any pharmacological

treatment. Blood pressure was measured by the physician

with the patients seated after a 10-min resting period,

using a mercury sphygmomanometer (three measure-

ments, one every 5 min). Secondary hypertension was

excluded by standard procedures. The occurrence of

hypertension-related complications was excluded by per-

forming recommended clinical and laboratory examin-

ations in all patients. In particular, all hypertensive

subjects underwent an ECG recording. An ECG was also

performed to exclude the presence of left ventricular

hypertrophy. The presence of clinically relevant hyper-

tensive retinopathy (� II degree of Keith and Wagener

classification) was excluded by examination of the fundus

oculi. Patients with diabetes mellitus or with impaired

fasting glucose were also excluded. It was decided to

include only untreated subjects with arterial hyperten-

sion at the time of their first diagnosis, who were free

of any clinically manifest cardiovascular complications

and who were not receiving any drug treatment. Blood

samples were collected in plain and ethylenediaminete-

traacetic acid-containing tubes (Venoject, Terumo

Europe, Leuven, Belgium) after overnight fasting, and

analyzed immediately or stored at �808C for no more

than 10 days.

Biochemical analysisThe serum concentrations of the main parameters of

lipometabolic balance (fasting glucose, total cholesterol

and triglycerides) were measured by standard laboratory

procedures.

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PON1192 polymorphism and hypertension Marra et al. 1295

Table 1 General patient characteristics

Healthy controls (n ¼ 219) Hypertensive patients (n ¼ 119) P

Age (years) 46.5 � 14.7 47.9 � 10.5 0.30Sex, male (%) 54.6% 65.2% 0.06Smoking status, current þ exsmokers (%) 51.2% 54.6% 0.58Body mass index (kg/m2) 24.7 � 2.7 26.3 � 3.1 < 0.001Fasting glucose (mmol/l) 5.11 � 0.42 5.30 � 0.44 < 0.001Total cholesterol (mmol/l) 5.37 � 1.23 5.88 � 0.94 < 0.001Triglycerides (mmol/l) 0.90 �/� 0.02 1.11 �/� 0.02 < 0.001Paraoxonase (U/ml) 306.8 �/� 1.7 293.5 �/� 1.8 0.47

Values are the arithmetic mean � SD, geometric means �/� antilog SD for skewed data. P ¼ 0.01 was considered to be statistically significanct.

Fig. 1

Frequency (%) of the PON1192 polymorphism (QQ þ QR and RR) inhealthy controls and hypertensive patients.

Paraoxonase PON1192 genotypeDNA was extracted from blood lymphocytes by the

salting-out method [19]. Polymerase chain reactions

were performed using primer sequences derived from

published data and specific for the amplification of the

region surrounding codon 192. The amplification reac-

tions and methodologies have been described previously

[20].

Paraoxonase activityParaoxonase activity was measured by adding serum to a

buffer containing paraoxon (O,O-diethyl-Op-nitrophe-

nylphosphate; Sigma-Aldrich, Milan, Spain). The rate

of generation of p-nitrophenol was measured at 405 nm

using a spectrophotometer (Beckman Du-640; Beckman

Coulter, Fullerton, California, USA) [21].

Statistical analysisData were analyzed using SPSS statistical software for

Windows (version 10.0; SPSS Inc., Chicago, Illinois,

USA). Triglycerides and paraoxonase activity were

natural log-transformed before statistical analyses to

achieve a normal distribution. The corresponding results

are shown as geometric means �/� antilog SD. Differ-

ences between healthy controls and hypertensive pa-

tients were initially assessed by univariate analysis using

Student’s t-test for continuous variables and the chi-

squared test for categorical variables. The distributions

of the PON1192RR genotype versus the 192QQ þ QRgenotype frequencies in the two groups were compared

by the chi-squared test, and odds ratios (OR) and 95%

confidence intervals (CI) were calculated. Deviation of

genotype frequencies from Hardy–Weinberg Equi-

librium was analyzed by chi-squared analysis. A multiple

logistic regression model was used to evaluate the asso-

ciation of paraoxonase activity and the PON1192 poly-

morphism with the presence of arterial hypertension.

The PON1192RR genotype was analyzed using of the

192QQ þ QR genotypes as the reference group. Potential

confounding covariates, such as age, sex, smoking

status, body mass index (BMI), fasting glucose, total

cholesterol and triglycerides, were included in the

model. P < 0.01 was considered to be statistically signifi-

cant.

opyright © Lippincott Williams & Wilkins. Unauth

ResultsGeneral characteristics of hypertensive patients andhealthy controlsAnthropometric, clinical, and laboratory parameters are

reported in Table 1. There were no differences in age and

sex distribution between hypertensive patients and

healthy controls. Hypertensive patients had higher

BMI, fasting serum glucose, total cholesterol and trigly-

ceride levels with respect to healthy controls, whereas

smoking frequency and paraoxonase activity were similar

between the two groups.

PON1192 genotypes and activityThe distribution of the PON1 codon 192 polymorphism

revealed no significant differences between hypertensive

patients and healthy controls either for genotypes or for

alleles at the polymorphic site PON1192 (data not shown).

The distribution respected the Hardy–Weinberg equi-

librium. A significant increase of the PON1192RR geno-

type frequency in hypertensive patients with respect to

healthy controls (14.3 versus 5.0%, x2 ¼ 8.71, d.f. ¼ 1,

P ¼ 0.003) was found when a comparison between RRsubjects and (QQ þ QR) subjects was performed (Fig. 1).

In particular, RR subjects had a prevalence of arterial

hypertension more than three-fold higher than subjects

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1296 Journal of Hypertension 2006, Vol 24 No 7

Fig. 2

1000

900

800

700

600

500

400

300

200

100

0

P < 0.01

Healthy controls Hypertensive patients

Par

aoxo

nase

act

ivit

y (U

/ml)

RRQQ + QR

Paraoxonase activity (U/ml) and the PON1192 polymorphism(QQ þ QR and RR) in healthy controls (~) and hypertensive patients(*). Each symbol represent one individual; the ‘bar’ indicates thegeometric mean per group.

Table 2 Multiple logistic regression model for paraoxonase activityand the PON1192 polymorphism with the presence of hypertensionas the dependent variable

Odds ratio 95% confidence interval P

Age (years) 0.99 0.97–1.01 0.54Sex (male) 0.89 0.51–1.55 0.68Smoking status, current þ

exsmokers1.02 0.61–1.71 0.94

Body mass index (kg/m2) 1.13 1.03–1.24 0.009Fasting glucose (mmol/l) 1.03 0.99–1.07 0.08Total cholesterol (mmol/l) 1.00 0.99–1.01 0.15Triglycerides (mmol/l) 2.15 0.55–8.34 0.27Paraoxonase (U/ml) 0.31 0.09–0.93 0.038PON1192RR genotypea 4.31 1.63–11.43 0.003

aReference groups: QQ þ QR genotypes. Covariates such as age, sex, smokingstatus, body mass index, fasting glucose, total cholesterol and triglycerides wereincluded in the model. P ¼ 0.01 was chosen as level of statistical significance.

characterized by the other genotypes (QQ or QR)

(OR ¼ 3.2; 95% CI ¼ 1.4–7.0, P ¼ 0.003). The effects

of the PON1192 polymorphism on paraoxonase activity

are shown in Fig. 2. As expected, higher paraoxonase

activity was found in RR subjects with respect to

QQ þ QR subjects (F ¼ 62.7, d.f. ¼ 1, P < 0.001). No

differences in paraoxonase activity among hypertensive

patients and healthy controls were found (F ¼ 1.5,

d.f. ¼ 1, P ¼ 0.23). An analysis of variance showed no

interaction between the presence of hypertension and RRgenotype on paraoxonase activity (F ¼ 0.02, d.f. ¼ 1,

P ¼ 0.90). To further investigate the findings obtained

from the univariate analysis, a multiple logistic regression

model was employed to analyse the association between

hypertension and PON1192RR genotype, adjusting for

age, sex, smoking status, BMI, fasting glucose, total

cholesterol, triglycerides and paraoxonase activity. The

results of this analysis are shown in Table 2. The

PON1192RR genotype was independently associated

with a four-fold increase in the prevalence of hyperten-

sion (OR, 4.31; 95% CI, 1.63–11.43, P ¼ 0.003). Among

the covariates, only BMI showed an association with

hypertension (OR, 1.13; 95% CI, 1.03–1.24, P ¼ 0.009).

When multiple logistic regression analysis was performed

by assuming QQ genotype as a reference group in com-

parison with QR or RR genotypes separately considered,

the results showed that the OR associated with the RRgenotype increased up to 6.99 (95% CI, 2.01–24.23;

P ¼ 0.002), whereas the QR genotype showed no associ-

ation with the prevalence of hypertension (OR, 1.60; 955

CI, 0.77–3.32; P ¼ 0.21).

opyright © Lippincott Williams & Wilkins. Unautho

DiscussionThere is an increasing interest in the mechanisms, either

of environmental or of genetic nature, that may be

involved in explaining the prevalence of overt CVD in

a population [22]. In particular, the possible role of

genetic factors is more and more frequently addressed

in this field, in the hope of identifying subjects who are at

higher risk of cardiovascular complications at a very early

stage before the onset of CVD. A number of these studies

have focused on the role of the PON1192 polymorphism

in human vascular disorders. In spite of some contrasting

results, the overall suggestion that can be derived from

these investigations is that this polymorphism may be one

of the many factors contributing towards predicting vas-

cular disease. In particular, the PON1192R allele or the

PON1192RR genotype have been associated with cardio-

vascular disease in several but not in all studies [23]. A

possible reason for these partly heterogeneous results

might be the confounding effect due to the associated

presence of other conventional cardiovascular risk factors

in the populations studied, when considering control

subjects. The need for a careful selection of the control

group, when assessing the relevance of a genetic poly-

morphism in the PON1192 gene, is emphasized by the

demonstration that the R allele may represent a ‘genetic’

risk factor for peripheral and coronary arterial disease, for

other types of cardiovascular diseases and for vascular

dementia. Lack of careful selection of the populations

studied therefore may lead to an overestimation of

R allele frequency [7–11,24]. An increasing interest

towards the PON1192RR genotype has been promoted

by Voetsch et al. [11]. They found an increase in the

frequency of the PON1192RR genotype in patients with

non-fatal arterial ischemic stroke (albeit selected with

less strict criteria than in the present study) compared to

controls. They also reported that the PON1192RR geno-

type increased the susceptibility of developing arterial

ischemic stroke by four-fold, regardless of hypertension.

The present study provides specific additional informa-

tion in this context, by offering, for the first time, exper-

imental support for the involvement of the PON1192

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PON1192 polymorphism and hypertension Marra et al. 1297

polymorphism in an early phase of the process that might

lead to the development of CVD (i.e. in the predisposi-

tion towards one of the most important cardiovascular risk

factors, such as arterial hypertension, in as yet asympto-

matic patients). In particular, in the present study, a

higher frequency of the PON1192RR genotype in

patients with a recent onset of hypertension, who were

still free from any clinically manifest cardiovascular com-

plications, was found compared to a control group of

healthy subjects, who were enrolled on the basis of

rigorous selection criteria to exclude other conventional

risk factors for CVD. To the best of our knowledge, this

demonstration has not yet been reported previously in

such carefully selected populations. An additional merit

of the present study is that it included only healthy

untreated control subjects and untreated hypertensive

patients at the time of their first diagnosis, which also

allowed the avoidance of any interference by drug treat-

ment that could have biased the results. It is probably

due to such careful selection of subjects that our study

was able to provide significant results despite the rela-

tively small sample size. The finding that a homozygous

condition for PON1192RR is associated with a higher

prevalence of hypertension was confirmed by multiple

logistic regression analysis (i.e. after accounting for other

factors that might have independently been predisposed

to the development of arterial hypertension, such as

BMI).

The present study did not aim to explore the mechanisms

responsible for a link between the PON1192 polymorph-

ism and a hypertensive state, for which it cannot provide

an explanation. However, it may offer an explanation for

the discrepancies observed in the results of previous

studies investigating the association between the

PON1192 polymorphism and CVD, suggesting that these

discrepancies might depend on the inclusion of hetero-

geneous populations characterized by a different degree

of CVD progression, and by a different frequency of other

cardiovascular risk factors.

In summary, the present study clearly defines a role for

the PON1192RR genotype in association with a higher

prevalence of arterial hypertension in subjects who are

free from any clinical manifestation of CVD. These

findings might help in the early identification of subjects

with a particularly high predisposition of developing

CVD, because they are characterized by the PON1192RRgenotype. However, it should be acknowledged that our

observations are of a cross-sectional nature. This implies

that our data cannot be used to prove the existence of a

cause–effect relationship between the PON1192 poly-

morphism and the development of arterial hypertension.

To more properly assess whether the PON1192 poly-

morphism does have prognostic value in predicting the

future development of arterial hypertension, and thus

CVD, additional studies with a longitudinal design and an

opyright © Lippincott Williams & Wilkins. Unauth

adequate follow-up duration are needed. The present

study offers a rationale and paves the way for such

investigations.

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