Carotid Intima-media Thickness, Paraoxonase-1 Gene Polymorphism, Inflammation and Oxidation Status in Children with Family History of Premature Cardiovascular Diseases

Aims: Paraoxonase-1 (PON-1) gene polymorphisms and carotid intima-media thickness (cIMT) in adults have been associated to increased incidence of cardiovascular diseases (CVD). Possible Original Research Article Ioannou et al.; CA, 5(2): 1-8, 2016; Article no.CA.25664 2 relation of cIMT to PON-1 polymorphisms and to markers of inflammation and oxidation, in children with family history of premature CVD, was investigated. Place of the Study: Laboratory for Lipids and Cardiovascular Disease Prevention, 2 Pediatric Department, AHEPA University Hospital, Thessaloniki, Greece. Methodology: Eighty four healthy children of normal BMI, were recruited; 42 with positive family history of premature CVD, median age 10 (7-15) years old (study group) and 42 ageand gendermatched controls. Levels of lipid profile parameters, high sensitivity CRP (hsCRP) and oxidized LDL (oxLDL) were determined. cIMT was measured by ultrasounds and PON-1 gene polymorphisms, Q192R and L55M, were investigated using standard PCR-RFLP. Results: Median cIMT was higher in study group than in controls [0.45 (0.30-0.65) vs 0.4 (0.300.60)]. Only ApoA-I presented significant difference between the three subgroups with different PON-1 SNP of the L55M polymorphism (P=.03). Regression analysis showed that there was no statistically significant dependence of cIMT on age, lipid profile parameters or markers of inflammation and oxidation. Conclusion: Family history of premature CVD and PON-1 gene polymorphism are not related with significant differences in cIMT in children. Inflammation and oxidation do not markedly affect cIMT in children with family history of premature CVD.


INTRODUCTION
Paraoxonase 1 (PON-1) is a calcium-dependent enzyme exhibiting esterase, lactonase and peroxidase activity.PON-1 accepts a broad range of substrates including organophosphates, diverse lactones and lipid peroxides and has been widely studied for its ability to breakdown pesticides and nerve gases [1].
PON-1 is predominantly synthesized by the liver and it is distributed to other tissues, but mainly found in serum [2].In serum, PON-1 is attached to high density lipoprotein (HDL) particles and seems to contribute to the anti-atherogenic and anti-inflammatory properties of HDL, as it degrades lipid peroxides, decreases HDL susceptibility to peroxidation, glycation, and homocysteinylation, and increases cholesterol efflux from macrophages.Therefore, it is thought to protect against the development of atherosclerosis [3].
The human PON-1 gene is located on the long arm of chromosome 7 between q21 and q22 [4].Two common coding region polymorphisms occur: a glutamine to arginine substitution at a.a position 192 (Q192R, rs662) and a leucine to methionine substitution at a.a position 55 (L55M, rs854560) [5].These single nucleotide polymorphisms (SNP) impair PON-1 activity [6]; L55M seems to impair PON-1 bioavailability and Q192R SNP determines PON-1 hydrolytic activity towards paraoxon [7].Studies have shown that carriers of the R and the L allele seem to have a greater risk of developing Cardiovascular Disease (CVD) [4].
The pathogenetic mechanisms of CVD development involve arterial damage, including both media and intima of large-and mediumsized arteries [8].In adults, arteriosclerosis and atherosclerosis are closely related, share common risk factors and they increase arterial stiffening and the risk of atherothrombosis, respectively [9].Among the noninvasive methods for the evaluation of arterial structure, ultrasound imaging of arterial walls allows measurement of intima-media thickness (IMT) [8,9].The standard sites of IMT measurement in adults are the common carotid artery (CCA), carotid bulb and internal carotid artery (ICA).The basic mode includes evaluation in B-mode presentation, and results are usually shown as the average value of IMT measurement from the right and left CCA.Studies in adult populations have shown that there is a steady increase in the diameter of CCA with age, followed by increased IMT, and that increased IMT is in part a physiological reaction in elevated blood pressure [10].In the general pediatric population, IMT also increases with age and is related to blood pressure, even in the normal range [10].Moreover, increased IMT has been observed in CCA of children with metabolic diseases, such as familial hyperlipidemia, and in obese children [11,12].
Low density lipoprotein (LDL) oxidation is considered to play a triggering role in the atherosclerotic plaque formation.This process is possibly inhibited by HDL, by PON-1 in particular [3].Moreover, low-grade systemic and subendothelial inflammation seems to be implicated in this chronic and complex procedure, as well.Markers of inflammation, such as C-reactive protein (CRP), have been related to the presence of active atherosclerotic disease and to the risk for CVD [13].PON

METHODOLOGY
Eighty four healthy children, with normal BMI, participated in the study and they were divided in two groups; 42 children with positive family history of premature CVD, median age 10 (7-15) years old (study group), 23 (55%) boys and 19 (45%) girls, and 42 age-and gender-matched children without family history of premature CVD (controls).The family history of premature CVD was not used for risk estimation, but as a risk factor which might or might not charge each of the two groups negatively, in reference to cardiovascular disease, as long as the hypothesis set in this study aimed in investigating whether family history plays a role in the differences in cIMT among children and its relation to PON-1 gene expression, oxidation and inflammation status.The children were recruited from the Outpatient Clinic for Lipids and Cardiovascular Disease Prevention from Childhood of our Department.Written consent for the participation in the study was obtained from the parents of the children.
Blood was collected after a fasting period of at least 8-hours.Renal, liver and thyroid function, i.e. creatinine, SGOT, SGPT and TSH, as well as blood pressure and fasting blood glucose were determined and used as exclusion criteria.Only healthy children, with normal BMI were included in the study.Blood in EDTA was kept in -80°Celsius for the PON-1 polymorphism study, and serum was isolated and frozen in -80°C.Levels of lipid profile parameters, such as total cholesterol (TC), HDL-cholesterol (HDL), LDLcholesterol (LDL), triglycerides (TG), The presence of the two polymorphisms was assessed by restriction fragment length polymorphism (RFLP).Hinf1 was used to digest 5 µl of the PCR reaction in a 20 µl final reaction.The result of the enzymatic cut with the Hinf1 was visualized, after electrophoresis, in a 3% agarose gel stained with Et-Br.
Carotid IMT (cIMT) in both carotid arteries was measured by Doppler ultrasound.The IMT was measured as the distance between the leading edge of the lumen-intima interface and the media-adventitia interface on the far wall of the artery.A mean IMT (mcIMT) was calculated.

Statistical Analysis
Statistical analysis was performed with ΙΒΜ SPSS Statistics 20, for Windows.In each group of children the normality of distribution of values was investigated with Kolmogorov-Smirnov test.Due to significant difference from normal distribution for most of the variables, median and range of values were used for the description of the sample.For comparison between study group and controls non-parametric Mann-Whitney and median comparison tests of independent samples were performed.Studying the children as a whole (N=84), we divided them in three subgroups, for each one of the genotypes of the PON-1 polymorphisms, i.e.QQ, QR, RR for Q192R polymorphism and MM, ML, LL for L55M polymorphism of PON-1.For comparisons between these subgroups of the children, non-parametric Kruskall-Wallis test for independent samples was used.Finally, multiple regression analysis was performed in order to investigate possible dependence of cIMT to other parameters under study.Statistical significance was set for P < .05.

RESULTS
Values of mcIMT and other biochemical parameters assayed, as well as of hsCRP and oxLDL, in both groups as shown in Table 1.Between the two groups only cIMT presented marginally significant difference between the two groups, with those children with positive family history of premature CVD having higher median cIMT than controls (Fig. 1).
Considering all of the 84 children, values of the parameters assayed in the three subgroups of children created with different PON-1 SNP are shown in Table 2. Apart from ApoA-I, no other parameter presented any difference between these subgroups.Genotype distribution for Q192R and L55M was in agreement with frequencies expected under Hardy-Weinberg equilibrium.The relation between different SNPs of PON-1 and mcIMT is also shown in Table 2.
Results of multiple regression analysis between mcIMT and parameters of lipid profile, markers of inflammation and oxidation are shown in Table 3.The dependency of cIMT on the different variables investigated, i.e. age, lipid parameters, inflammatory and oxidative stress markers, seemed to be non-significant.Family history contributed in mcIMT only 1.7%, and all the other parameters studied contributed below 5%.The least contribution was by TG and Lp(α), with 0.5% and 0.3% respectively.

DISCUSSION
Family history of premature CVD is considered one of the major risk factors for the onset of CVD, even from childhood.The antioxidant capacity of HDL, due to PON-1, is considered one of the causes for the inverse relation of HDL serum levels to cardiovascular risk [14].One of the markers for the estimation of the cardiovascular risk is cIMT, in adults and children as well.Various studies have been conducted in children populations investigating cIMT in children in relation to inherited metabolic diseases, obesity, hypertension, biochemical risk factors for CVD, etc. [15][16][17][18].Nevertheless, results in healthy children population are absent, and in Greece in particular, are absent and, to our knowledge, there are no reports on cIMT in relation to family history of premature CVD.In our study there was no statistical significant difference in cIMT between children with positive family history of premature CVD and controls, as shown in Table 1, with the study group having higher median values than controls (Fig. 1).Nevertheless, there was no child in either group with cIMT > 0.9 mm, which is considered to have negative prognostic value for future CVD.There was no difference in the lipid profile parameters, hsCRP and oxLDL, markers of inflammation and oxidation, respectively, in the two groups of children.Therefore, children with family history of premature CVD do not seem to have any difference in levels of inflammatory or oxidative indices, in the present study, which are considered secondary markers of increased risk for CVD.Those results are in contrast to previous studies [19,20] showing significant elevated lipids and inflammation markers in children with positive family history of premature CVD.
Moreover, the investigation of lipid profile parameters, hsCRP and oxLDL between children with different Q192R SNP and L55M SNP (Table 2), showed significant difference only in ApoA-I in the latter.The M-variant, which has been related to lower PON-1 concentration and bioactivity [21], seemed to be related to lower levels of ApoA-I in the children of this study.Nonsignificant was the difference of mcIMT between the subgroups of children with different genotypes of PON-1 (Table 2).This is in contrast to other studies which have shown that decreased PON-1 activity was related to more severe atherosclerosis in adults [22]

CONCLUSIONS
In the present study, neither positive family history for premature CVD nor the polymorphism of PON-1 gene seems to be related with significant differences in cIMT.Inflammation and oxidation do not markedly affect cIMT in children with family history of premature CVD.This is the first time, to our knowledge, that such results combining cIMT, PON-1 polymorphism, inflammatory and oxidation indices are presented in a population of children.The weakness of our study is the small number of children that participated and the results can only provide indications on the certain hypothesis.Therefore, larger studies are needed to ascertain these results and provide more information on the relation of vascular integrity indices, such as cIMT, and PON-1 polymorphisms, as well as markers of inflammation and oxidation, in order to establish whether the investigation of such parameters can be used as early biomarkers of atherosclerosis progression beginning from childhood.