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Archives of cardiovascular diseases
Volume 103, n° 1
pages 10-18 (janvier 2010)
Doi : 10.1016/j.acvd.2009.10.001
Received : 30 April 2009 ;  accepted : 8 October 2009
Influence of polymorphisms in candidate genes on early vascular alterations in obese children
Gènes candidats et atteintes vasculaires précoces des enfants obèses
 

Agnès Tounian a, b, Yacine Aggoun c, Jean-Marc Lacorte b, d, Béatrice Dubern a, b, Karine Clément b, Damien Bonnet c, Patrick Tounian a, , b
a Département de gastro-entérologie et nutrition pédiatriques, hôpital Armand-Trousseau, AP–HP, 26, avenue du Dr-A.-Netter, 75012 Paris, France 
b U872 nutriomique, Inserm, université Pierre-et-Marie-Curie–Paris-6, Paris, France 
c Département de cardiologie pédiatrique, hôpital Necker–Enfants-Malades, AP–HP, Paris, France 
d Département de biochimie endocrinienne et oncologique, hôpital Pitié-Salpêtrière, AP–HP, Paris, France 

Corresponding author. Fax: +33 1 44 73 62 28.
Summary
Background

Many studies performed in adults have reported the involvement of genetic determinants in vascular alterations that predispose to cardiovascular diseases later in life. To date, no study has assessed the co-involvement of gene polymorphisms as cardiovascular risk factors in children.

Aims

To search for variants involved in early vascular alterations in obese children.

Methods

In 232 obese children, we performed an association study between variables related to endothelial function or arterial mechanical properties and functional variants reported to predispose towards vascular alterations in adults. Candidate polymorphisms were selected in genes involved in the renin–angiotensin system, vascular endothelial cell remodelling and communication, arterial inflammation, adiponectin production and lipoprotein metabolism. Non-invasive arterial measurements were performed to evaluate the mechanical characteristics of the common carotid artery and the endothelial function of the brachial artery.

Results

We found no association between the polymorphisms studied, taken alone or in combination with the arterial variables measured.

Conclusion

Our hypothesis predicting that the tested genetic variants, which are involved in adult cardiovascular diseases, may influence the susceptibility to early vascular alterations in obese children was not validated. Thus, obesity-associated metabolic complications appear to remain the main predictors of arterial alterations in obese children.

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Résumé
Contexte

De nombreuses études réalisées chez l’adulte ont permis de mettre en évidence l’implication d’une composante génétique dans la survenue et la progression d’altérations vasculaires à l’origine de cardiopathies ultérieures. À ce jour, aucune étude visant à mettre en évidence des marqueurs génétiques précoces du risque cardiovasculaire n’a été réalisée chez l’enfant.

But de l’étude

Rechercher des marqueurs génétiques de lésions vasculaires précoces dans notre cohorte d’enfants obèses.

Méthodes

Une étude d’association entre des paramètres de la fonction endothéliale et de la mécanique artérielle et des variants fonctionnels préalablement associés à des lésions cardiovasculaires chez l’adulte a été menée chez 232 enfants obèses. Les polymorphismes ont été choisis dans des gènes impliqués dans le système rénine–angiotensine, le remodelage vasculaire, les jonctions entre cellules endothéliales, l’inflammation artérielle, la sécrétion d’adiponectine et le métabolisme des lipoprotéines. Au site artériel carotidien, ont été mesurés par échographie vasculaire de haute résolution les caractéristiques de la mécanique artérielle. Au site artériel brachial, la fonction endothéliale a été évaluée par la vasodilatation induite par l’hyperhémie provoquée.

Résultats

Aucune association entre les polymorphismes étudiés, pris individuellement ou en combinaison, et les caractéristiques artérielles phénotypiques des enfants obèses étudiés n’a pu être mise en évidence.

Conclusion

Les variants sélectionnés ne semblent pas intervenir dans la survenue des lésions précoces des enfants obèses de notre cohorte. La réplication de cette étude dans une cohorte plus importante est nécessaire pour confirmer ou infirmer ces données.

The full text of this article is available in PDF format.

Abbreviations : ACE, AGT, AGTR1, APM1, CX37, CX3CR1, dD, DWS, E inc , FMD, GTNMD, I/D, IMT, LCSA, MMP3, NOS3, ΔP , PAI-1, PON1, sD, SNP

Keywords : Gene variants, Vascular alterations, Obesity, Children

Mots clés : Variants géniques, Risque vasculaire, Obésité, Enfant


Introduction

The presence of early arterial alterations has been widely studied in obese children [1, 2, 3]. These vascular alterations lay the foundations for atherosclerosis and favour adverse adult cardiovascular outcome, independent of adult weight [4, 5]. Metabolic disorders (insulin resistance, abnormal plasma lipids) and inflammatory conditions associated with excess fat mass – especially visceral fat – are surely involved in the occurrence of these early arterial lesions [1, 2, 3]. Besides these conventional risk factors, the so-called atherosclerotic “burden” also involves a predisposing genetic background, as suggested by twin-, sibling- and family-based studies [6, 7], as well as candidate-gene analyses, performed in adults [8]. So far, no study has been designed to identify the involvement of several polymorphisms, alone or in combination, in the susceptibility to precocious endothelial dysfunction and arterial mechanical property abnormalities in children. It stands to reason that such a study has to be performed, firstly because the foundations of atherosclerosis are laid as early as childhood, and secondly because factors that act early in life have a major impact on lifetime risk of cardiovascular disease [5, 9, 10]. Furthermore, it is worth investigating whether genetic variants implicated in vascular alterations in adults have an impact in obese children who are already presenting with vascular conditions caused by obesity [1, 2, 3].

The present study was undertaken to assess the contribution of specific genetic variants, known to predispose towards cardiovascular diseases in adults, to vascular alterations in obese children.

Materials and methods
Study population

This association study was performed in a cohort of 232 unrelated severely obese children recruited prospectively over 5 years at our outpatient department. This cohort was extended by 60 more children for the AGTR1 A1166C polymorphism, to further explore the trend of the association between the C allele and several arterial and endothelial parameters revealed by the first statistical analyses. Severe obesity was defined as a body mass index Z-score ≥3 (standard deviation over mean age and sex-specific body mass index values determined in French children by Rolland-Cachera et al. [11]).

The protocol was approved by the institutional review board and written informed consent was obtained from the older children and from both parents of all children.

Design and procedures
Baseline anthropometric and metabolic characteristics of obese subjects

Blood samples were collected from the children after an overnight fast to determine plasma glucose, insulin, lipid profile (total cholesterol, triglycerides, low-density lipoprotein, high-density lipoprotein, and apolipoprotein B and A-1), ferritin and leptin by routine laboratory procedures as described previously [1, 12]. Insulin sensitivity was evaluated using homeostasis model assessment (HOMA), a surrogate measure of insulin sensitivity validated in children [13]. Anthropometric data (Tanner stage, weight, height) were collected. Body fat mass was measured by dual-energy X-ray absorptiometry with a total-body scanner (QDR 2000-HOLOGIC, Waltham, MA, USA). Percentage body fat was calculated as 100×total body fat mass/total body mass. To evaluate the effects of fat distribution, the body was divided into areas corresponding to arms, legs, trunk and head. The trunk region was delineated by an upper horizontal border below the chin, vertical borders lateral to the ribs and a lower border formed by the oblique lines passing through the hip joints. The leg region was defined as the tissue below the oblique lines passing through the hip joints. Body fat distribution was calculated as the ratio of the amount of fat tissue in the trunk region over the amount of fat tissue in the leg region, and was defined as android/gynoid fat mass ratio.

Arterial measurements

A single investigator (YA), who used a real-time B-mode ultrasound imager (Acuson XP 128, Mountain View, CA, USA) performed non-invasive arterial measurements. IMT and lumen diameters during dD and sD were measured at the common carotid artery as described previously [1]. LCSA was calculated as πdD2/4 and wall cross-sectional area (WCSA) as π(dD/2+IMT)2π(dD/2)2. Cross-sectional compliance (CSC) and cross-sectional distensibility (CSD) of the common carotid artery were determined from diameter changes during systole and from simultaneously measured pulse pressure (ΔP ), according to the following formulae: CSC=(π[sD2dD2])/4ΔP (mm2mmHg−1); and CSD=(sD2dD2)/(dD2×ΔP) (mmHg−1×10−2). ΔP was measured using applanation tonometry during arterial measurements. DWS was calculated as mean arterial pressure×dD/2×IMT. Incremental elastic modulus (E inc ) was calculated as 3×[1+LCSA/WCSA])/CSD (mmHg×102). In contrast to compliance, which provides information about elasticity of the artery as a hollow structure, the E inc provides information about the properties of the wall material, independent of the geometry.

After at least 30min of rest in a recumbent position, arterial endothelial function was studied using a high-resolution vascular ultrasound system. The investigator measured FMD, defined as arterial diameter changes in response to reactive hyperaemia (endothelium-dependent vasodilation induced by a flow increase); and GTNMD, defined as arterial diameter changes in response to the endothelium-independent vasodilator glyceryltrinitrate. Reactive hyperaemia was induced by inflating a blood pressure cuff to 300mmHg for 4min; the artery was scanned for 30s before and 90s after cuff deflation. A resting scan was recorded 10m later. Glyceryltrinitrate (400μg spray) was then given sublingually and the artery was scanned 3min after the dose.

Selection of polymorphisms and genotyping

With the use of the public databases PubMed® (query.fcgi%3Fdb=Pubmed) and Online Mendelian Inheritance in Man (query.fcgi%3Fdb=OMIM), we selected variants reported previously to be associated with vascular endothelial dysfunction and artery mechanical property alterations in adults. These polymorphisms are located in genes coding for proteins involved in the renin–angiotensin system (ACE I/D, AGT G-6A and Met235Thr, AGTR1 A1166C), vascular endothelial cell remodelling (MMP3 5A/6A, PAI-1 4G/5G), cell junction and communication (CX37 Pro319Ser), leucocyte adhesion during inflammation (CX3CR1 Val249Ile), arterial vasodilation (NOS3 Glu298Asp), adiponectin secretion (APM1 C-11377G and C-11391G) and lipoprotein metabolism (PON1 Glu192Arg).

All polymorphisms were SNP except for the ACE I/D, which corresponds to the insertion of an Alu-like repetitive sequence into intron 16 [14]. These polymorphisms are located either in the promoter region or in introns or exons and have been shown to cause changes in the function or levels of expression of the encoded protein; the variant allele was reported to occur at least in 5% of the population in previous studies.

The subjects’ DNA was extracted from ethylenediaminetetraacetic acid whole blood samples using the Puregen kit (Gentra Systems, Minneapolis, MN, USA). The SNPs were genotyped with the LightCycler™ (Roche Diagnostics, Basel, Switzerland) based on probes and fluorescence resonance energy transfer between fluorescein and LC Red 640 (Roche Diagnostics). Primers and hybridization probes were designed by TIB MolBiol Syntheselabor (Berlin, Germany). Detailed experimental conditions are available from the authors.

The ACE I/D polymorphism was determined according the technique developed by Marre et al. [15].

Data analyses

The number of subjects included (n =232) was sufficient to detect, with a power of 80%, a 4% variation (threshold based on previous results of our own [1]) of the flow-mediated dilation trait, defined as the more relevant marker of endothelial function, assuming a recessive genetic model for each polymorphism.

The Chi-square test was applied to check compliance with the Hardy-Weinberg equilibrium of each allele frequency and that no difference in genotype distribution of each polymorphism was present between sexes. Quantitative trait data are expressed as mean±standard error of the mean. The Gaussian distribution of each variable was tested with the Shapiro-Wilk test. For distributions skewed positively, a log-transformation was applied before the statistical tests but untransformed values are given in tables.

Associations between genotypes (each allele or haplotype alone and combinations of alleles when possible) and phenotype traits were tested with the general linear model analysis of covariance (least-square mean regression) with adjustments on covariates as follows: age, sex, Tanner stage, homeostasis model assessment, ferritin, android/gynoid fat-mass ratio, leptin and lipid profile. Each genotype was assessed with the use of dominant, recessive and additive genetic models.

All statistical analyses were performed with JMP software (SAS Institute Inc, Cary, NC, USA). Statistical significance was defined from p <0.05.

Results

The baseline demographic, metabolic and haemodynamic characteristics of the 232 severely obese children involved in the study are shown in Table 1, Table 2. The average duration of the obesity was 8.4±3.1 years.

The prevalence of the 12 polymorphisms screened in our population-based study is given in Table 3. As described previously in the literature [16], the A and C alleles of the AGT G-6A and Met235Thr (a substitution of the T nucleotide for a C, which changes the ATG codon coding for methionine into an ACG codon coding for threonine) polymorphisms were in very tight disequilibrium (D=0.96; AC=51%, GT=47%). The genotype frequencies for each polymorphism were in Hardy-Weinberg equilibrium and in keeping with values reported in the National Centre for Biotechnology Information, Entrez Single Nucleotide Polymorphisms Data Bank (query.fcgi%3FCMD=search%26DB=snp). There were no significant differences between different genotypes within the cohort with respect to age, sex, anthropometric or metabolic estimates, or obesity comorbidities.

Multivariable logistic-regression analysis with adjustments for covariates failed to show that the 12 polymorphisms, taken alone or in combination, had any impact either on the geometrical and mechanical characteristics of the common carotid artery or the endothelial function of the brachial artery (Table 4). The trend of association between the AGTR1 1166C allele, in a dominant model, and common carotid artery compliance (CSC, p =0.06) and distensibility (CSD, p =0.06), and E inc (p =0.07), first revealed by the statistical analyses performed in the cohort of 232 children, was not confirmed when 60 extra children were added. It is noteworthy that initial statistical analyses performed without adjustments also led to negative results. Grouping subjects according to age, sex, android or fat mass distribution made no difference (data not shown).

Discussion

We examined, in 232 prospectively recruited obese children, the relationships between 12 polymorphisms located in candidate genes relevant to vascular alterations in adults, and the risk of early endothelial dysfunction and mechanical property changes, which are markers of the arterial damage that precedes plaque formation [2, 3]. We found no association between the arterial variables measured and the polymorphisms studied, taken alone or in combination. Therefore, we cannot support the hypothesis that they confer, in either way, susceptibility to vascular alterations in obese children.

The absence of a positive result provides an informative conclusion, nevertheless. Indeed, the result may be wrongly attributed to statistical pitfalls due to the size of the cohort. However, the statistical differences were so far from significance (range 0.1–0.8) that it is more realistic to conclude an absence of effect of the variants tested, rather than an impossibility to detect a possible effect. It is noteworthy that underpowered epidemiological studies lead more usually to statistical type I errors and to hastily drawn positive susceptibility effects, which are not confirmed in larger populations [17]. This explains why the trend towards a positive association between the AGTR1 1166C allele and CSC, CSD Einc, which was detected in the cohort of 232 children, was lost when we added 60 extra children.

Thus, the positive associations described in previous studies, on which our own study is based, appear more probably to be due to a gene expression and/or a phenotypical expression of the polymorphisms that occur at an older age [18, 19]. For some of them, combination with concomitant expression of other variants [20, 21] and/or other conventional risk factors that are present to a lesser extent in children (hypercholesterolaemia, hypertension, smoking, drinking) [22, 23] is required.

In light of the results of this survey, obesity per se [24], metabolic disorders (insulin resistance, abnormal plasma lipids) associated with adipose tissue (especially visceral fat) [2, 9, 10] and low-grade inflammation [25, 26] remain the main predictors of the occurrence of early morphological and functional vascular changes. Previously, in a study based on a subset of the cohort used in this paper, we reported that lower arterial compliance and distensibility correlated positively with android fat distribution, which was associated with insulin resistance and plasma triglyceride concentration [1]. We also observed that altered endothelium function correlated with low plasma apolipoprotein A-1 and insulin resistance indices [1]. Later, our results were confirmed and completed by others [27, 28]. More recently, we extended the previous cohort, and using three different definitions of metabolic syndrome, we found that if metabolic factors taken individually are associated with vascular dysfunction, no synergistic effect is observed when they are combined to define the metabolic syndrome [29]. The fact that metabolic factors, which are known to be powerful in affecting vascular properties, failed to show a synergistic effect, illustrated for us the difficulty in bringing a genetic effect to the fore. Indeed, frequent polymorphisms, such as those we selected to ensure a sufficient number of each genotype to enable relevant statistical analyses, carry only a modest relative risk, unlike rare polymorphisms [17]. Conceivably, this small effect can be masked by prevailing metabolic abnormalities early in life, and become detectable later due to the progressive pressure on vasculature.

Besides metabolic factors, features of acute-phase activation and low-grade inflammation, demonstrated by a plasma increase in C-reactive protein, develop with weight gain and extend the preatherosclerotic burden. Indeed, adipose tissue – and visceral adiposity in particular – is a key regulator of inflammation, coagulation and fibrinolysis through the secretion of proinflammatory cytokines (leptin, resistin, tumour necrosis factor-⍺, interleukin-6, etc.) and fibrinolytic regulators [30, 31]. Recent studies performed in overweight children have reported a positive association between proinflammatory factors, body mass index, visceral fat and insulin resistance [25]. Obviously, inflammation promotes early arterial changes, as suggested by the positive association between inflammation, endothelial dysfunction and structural arterial disease demonstrated clearly in children [32, 33]. In our cohort, 53% of the children had a high android/gynoid ratio; it is thus conceivable that the associated inflammatory status plays a non-negligible part in the increased stiffness of the common carotid artery and the endothelial dysfunction we brought to light several years ago in this cohort of obese children [1]. As with metabolic factors, it can also be hypothesized that inflammatory variables override the phenotypical expression of polymorphisms.

Further studies are necessary to confirm our results. To date, as genetic predisposition to atherosclerosis does not seem to play a part as early as in childhood, the role of obesity-associated factors in the occurrence of arterial alterations in obese children still predominates. Therefore, the early setup of established treatments for metabolic complications should certainly help to preserve vasculature in obese children.

Conflicts of interest

None.


Acknowledgements

We warmly thank Dominique Pepin for her skilled technical assistance and Pierre-Yves Boelle for in-depth statistical analyses and fruitful discussions.

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