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Apoliprotein E genotype is associated with apoliprotein B plasma levels but not with coronary calcium score in very elderly individuals in primary care setting
Gene 539 (2014) 275–278
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Gene journal homepage: www.elsevier.com/locate/gene
Short Communication
Apoliprotein E genotype is associated with apoliprotein B plasma levels but not with coronary calcium score in very elderly individuals in primary care setting Adriane D. Henriques a, Audrey C. Tonet-Furioso a, Wilcelly Machado-Silva a, Wladimir M. Freitas a,b, Luiz A. Quaglia b, Simone N. Santos a, Cláudio Córdova c, Andrei C. Sposito a,d, Otávio T. Nóbrega a,⁎, on behalf of the Brazilian Study on Healthy Aging Group a
Universidade de Brasília (UnB), Campus Universitário Darcy Ribeiro, 70.910-900 Brasília, DF, Brazil Instituto de Cardiologia Biocardios, SEPS 709/909 Sul Edifício Biocenter, Asa Sul, 70.390-095 Brasília, DF, Brazil c Universidade Católica de Brasília (UCB-DF), QS 07 Lote 01 EPCT, 71.966-700, Taguatinga, DF, Brazil d Universidade Estadual de Campinas (UNICAMP), Universidade Estadual de Campinas, Rua Tessália Vieira de Camargo 126, 13.083-887, Campinas, SP, Brazil b
a r t i c l e
i n f o
Article history: Received 18 November 2013 Received in revised form 26 December 2013 Accepted 31 January 2014 Available online 12 February 2014 Keywords: Apolipoprotein Lipid metabolism Atherosclerosis Primary prevention Aging
a b s t r a c t Background: Epidemiological surveys indicate the influence of polymorphisms of apolipoprotein (apo) E on plasma lipids and triglyceride-rich lipoprotein levels, with impact on atherosclerotic phenotypes. Aim: We studied the association of classic genotypes of the apoE gene with clinical and biochemical risk factors for atherosclerosis in a segment of the very-old Brazilian individuals, with emphasis on the lipemic profile. Methods: We performed cross-sectional analyses of clinical and laboratory assessments, including cardiac computed tomography, across ε2, ε3 and ε4 carriers of the apoE gene with a convenience sample of 208 participants eligible for prevention against cardiovascular events. Results: When non-ε4 carriers were compared with ε4 carrying subjects, lower levels of ApoB as well as ApoB/ ApoA ratios were observed in the former group. Tests between apoE polymorphisms with other clinical/ biochemical variables and those with arterial calcification showed no significant differences between groups. Conclusion: The study suggests a possible atherogenic role of the ε4 allele attributable to increased ApoB levels and ApoB/ApoA ratios among very-old subjects in primary care setting. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Cardiovascular diseases (CVD) account for 48% of the deaths from noncommunicable diseases worldwide (Finegold et al., 2013). In Brazil, roughly one third of total deaths are represented by CVD (Sociedade Brasileira de Cardiologia et al., 2010), the main cause of deaths in the elderly population (Mansur and Favarato, 2012). Many clinical factors that contribute to CVD mortality such as obesity, hypertension, type-2 diabetes and dyslipidemia bear significant genetic contributors (Glass and Witztum, 2001). An increasing number of genetic Abbreviations: ANCOVA, analysis of covariance; Apo, apolipoprotein; BMI, body mass index; CVD, cardiovascular disease; HDL-c, high density lipoprotein cholesterol; LDL-c, low density lipoprotein cholesterol; MANCOVA, multivariate analysis of covariance; SD, standard deviation; SPSS, Statistical Package For Social Sciences; TC, total cholesterol; TG, triglyceride; TRL, triglyceride-rich lipoprotein. ⁎ Corresponding author at: Universidade de Brasília (UnB), Campus Universitário Darcy Ribeiro, 70.910-900, Brasília, DF, Brazil. Tel.: +55 61 31071913. E-mail addresses: [email protected] (A.D. Henriques), [email protected] (A.C. Tonet-Furioso), [email protected] (W. Machado-Silva), [email protected] (W.M. Freitas), [email protected] (L.A. Quaglia), [email protected] (S.N. Santos), [email protected] (C. Córdova), [email protected] (A.C. Sposito), [email protected] (O.T. Nóbrega). 0378-1119/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2014.01.077
polymorphisms have been associated with susceptibility to cardiovascular disease, mainly atherosclerosis, with emphasis on the important variability of the apolipoprotein (apo) E gene. Epidemiological surveys assessing the role of the apoE polymorphism on plasma lipids and triglyceride-rich lipoproteins (TRL) have shown that the presence of the ε4 allele is associated with elevations in low density lipoprotein cholesterol (LDL-c), while the presence of ε2 has the opposite effect on these particles (Alvim et al., 2010), possibly by virtue of the greater ability of the former and minor ability of the latter to act as ligands for specialized lipoprotein receptors (Demant et al., 1991) and hence an adaptative up-regulation of the receptor in ε2 carriers (Alvim et al., 2010; Davignon et al., 1999). These gene–gene interactions are important because lipoproteins play a central role in the development of the atherosclerotic phenotype (Tiret et al., 1994), and ApoE is a key protein in the modulation of the catabolism of the most atherogenic particles (Alvim et al., 2010). There is evidence that ApoE has a profound impact on the ApoB metabolism (Demant et al., 1991) and on ApoB-related lipids and lipoproteins (Davignon et al., 1999). Recent genome wide association studies confirmed strong statistical association between apoE with CVD risk (Waterworth et al., 2010), primarily due to influences on levels of LDL-c (Smith et al., 2010) and total cholesterol
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(Aulchenko et al., 2009). Despite prospective studies indicating that the ApoB/ApoA-I ratio is a useful predictor of risk for myocardial infarction (MI) and other CVD (Sierra-Johnson et al., 2009), to our knowledge no studies sought to associate apoE genotypes with this aspect of the lipid metabolism of older adults. The very elderly are the age group that is growing the most rapidly in westernized societies such as those in Brazil (Nobrega et al., 2009) with an increasing number of individuals reaching this age stratum devoid of severe circulatory disorders (Freitas et al., 2012), and being eligible for primary prevention against major cardiovascular events. The goal of our work is to study the association of classic genotypes of the apoE gene with the atherosclerotic risk inherent to the lipemic profile of a segment of the very-old Brazilian population in primary care setting. 2. Methods 2.1. Subjects Study subjects are participants in the ongoing Brazilian Study on Healthy Aging, which is a prospective cohort study that was started in December 2008 and designed to identify markers of cardiovascular risk in very elderly individuals eligible for primary prevention as published elsewhere (Freitas et al., 2011). For this proposal, noninstitutionalized consecutive patients aged 80 years or over who have sought the outpatient clinic for preventive care and have never manifested myocardial infarction, stroke or peripheral arterial disease were enrolled. Additional selection criteria were the absence of autoimmune disease (including rheumatic disorders), chronic or recurrent infections, prior or current neoplastic disease, and use of steroidal or nonsteroidal anti-inflammatory drugs in the past 30 days. After baseline measurements, all the participants were referred to the study outpatient clinic for prospective medical follow-up evaluations. Nonetheless, the results described herein derived from crosssectional analyses with data at entry. The study was approved by the institutional research ethics committee and procedures were in accordance with the ethical standards of the Helsinki Declaration, with all participants having signed informed consent before enrollment.
prospective electrocardiography triggering. Coronary artery calcification was defined as a minimum of 3 contiguous pixels with a peak Hounsfield unit density N130. Coronary artery calcifications were scored by a certified radiologist using the Agatston score to express its extent. 2.5. apoE genotyping Whole blood was obtained during sampling for biochemical analysis and stored at −20 °C until use. Genomic DNA was purified according to standard extraction kits (QIAamp DNA Mini Kit, Qiagen, Brazil). To determine the classic genotypes for epsilon allelic variants of the apoE gene (ε2, ε3 and ε4), amplifications based on a refractory mutation system (ARMS) for multiplex polymerase chain reactions were carried out using the method described by Donohoe et al. (1999), followed by electrophoresis of the amplification products in a 1.6% agarose gel. Each sample was run at least twice, and further checked only if the genotypes of the first two productive analyses were in conflict. 2.6. Statistical analysis Violation of the Hardy–Weinberg equilibrium was tested using Fisher's exact test. The Kolmogorov–Smirnov test was used to verify normal distribution of data from continuous variables in a withingroup approach. Whenever appropriate, data are expressed as means ± standard deviation (SD) or frequency (%). The Student's t test was used to compare the means of age and BMI across genotypes. Frequencies (e.g.: gender) were compared using the chi-square test. Analysis of covariance (ANCOVA) was used to analyze the variance in the main dependent variables across genotypes, with adjustment to age, BMI and gender. To test whether the intercorrelated nature of some dependent variables affected the parametric analyses outputs, MANCOVA was run to compare cholesterol-related metabolite values across groups, with the same adjustments. All the analyses were performed employing the Statistical Package for Social Sciences (SPSS) for Windows (version 17.0). A P value b 0.05 was considered significant. 3. Results
2.2. Clinical inspection All the subjects were submitted to assessments of total body mass (kg), body height (m), and blood pressure (mm Hg). Body mass index (BMI; kg/m2) was defined as usual whereas waist circumference (WC; cm) was measured 2 cm above the umbilicus scar. 2.3. Biochemical analysis After 12 h of overnight fasting, blood was collected and centrifuged at 5 °C, 4500 rpm for 15 min to separate plasma that was used exclusively to carry out the measurements of the metabolic variables analyzed herein: fasting glucose, glycated hemoglobin A (HbA) 1c, total cholesterol (TC), triglycerides (TG) and high density lipoprotein cholesterol (HDL-c). All glycemic and lipemic assessments were performed using reagents from Roche Diagnostics (Mannheim, USA) according to manufacturer recommendations and carried out by the same certified clinical laboratory. Low density lipoprotein cholesterol (LDL-c) was determined using the Friedewald equation (Friedewald et al., 1972). Assessments of C-reactive protein (highly sensitive, CardioPhase, Dade Behring, Marburg, USA), ApoA and ApoB (Behring Nephelometer BNII, Dade Behring, Marburg, Germany) were also performed. 2.4. Cardiac computed tomography Computed tomography was performed in a 64-slice scanner (Aquillion 64, Toshiba, Ottawara, Japan). Axial slices of 3 mm thickness with 3 mm table-feed were acquired at 70% of R–R intervals with
During the recruitment of patients, we assessed the clinical and biochemical characteristics of each subject at admission, along with the genotypes of the apoE gene. The allele frequencies of the ε2, ε3 and ε4 in the whole sample of 208 admitted individuals were 1.0%, 91.3% and 7.7%, respectively. Due to a low count of ε2 carriers in the sample, these individuals were merged with the ε3 homozygotes (non-ε4 carries) for the purpose of comparison with ε4 carriers. A single patient with the ε2/ε4 genotype was excluded from the analysis because of the putative opposite effects of these alleles in the phenotypes studied (Tiret et al., 1994), constituting a final sample of 207 subjects. This sample showed a homogeneous pattern of baseline characteristics such as mean values of age and BMI and gender proportion across genotypes. Even so, these features were treated as covariates in the subsequent analyses. Regarding lipemic and clinical variables, ANCOVA revealed no differences in terms of plasma lipids (TC, LDL-c, HDL-c and TG) when non-ε4 carriers were compared with ε4 carrying subjects, albeit a non-significant trend towards lower LDL-c levels (P = 0.08) could be observed among the non-ε4 carriers. Only in terms of serum ApoB levels and ApoB/ApoA ratios, were mean scores rendered significant (P b 0.05). There were no other clinical or laboratory differences observed between the groups (Table 1). MANCOVA for cholesterolrelated metabolites as dependent variables (TC, LDL-c, HDL-c, TG, ApoB, ApoA and ApoB/ApoA) yielded similar results (Hotelling's Trace (7197) = 0.058), with greater serum ApoB levels (F = 4.43, P = 0.036) and ApoB/ApoA ratios (F = 4.88, P = 0.028) among the e4-carriers. The association of this structural polymorphism with the quantitative levels of ApoB, but not with ApoA, is suggestive of
A.D. Henriques et al. / Gene 539 (2014) 275–278 Table 1 Characteristics of the very-old individuals according to the apolipoprotein E genotypic group.
Age, years Male, % Body mass index, kg/m2 Waist circumference, cm SBP, mm Hg DBP, mm Hg Total cholesterol, mg/dL HDL cholesterol, mg/dL LDL cholesterol, mg/dL Triglyceride, mg/dL HbA1c, % ApoA, mg/dL ApoB, mg/dL ApoB/ApoA Agatston score, unit density C-reactive protein, mg/L
Non-ε4 carriers
ε4 carriers
(n = 179)
(n = 28)
84.5 ± 4.2 18.8 26.5 ± 4.8 94.4 ± 12.1 144.8 ± 19.4 75.3 ± 10.8 197.4 ± 41.0 54.9 ± 13.8 112.3 ± 34.9 127.0 ± 55.8 6.19 ± 1.02 150.9 ± 24.8 84.4 ± 23.2 0.57 ± 0.20 262.9 ± 300.6 3.82 ± 6.90
84.1 ± 4.5 35.7 24.9 ± 3.6 93.3 ± 12.1 148.1 ± 27.6 75.2 ± 10.7 204.5 ± 41.9 53.4 ± 14.3 122.6 ± 37.0 128.5 ± 58.2 6.23 ± 1.63 144.7 ± 22.8 94.0 ± 23.0 0.67 ± 0.21 269.1 ± 331.4 4.11 ± 6.75
P⁎
0.66† 0.13§ 0.16† 0.65† 0.43 0.96 0.39 0.59 0.08 0.89 0.84 0.22 0.04 0.03 0.92 0.84
SBP: systolic blood pressure; DBP: diastolic blood pressure; HDL: high density lipoprotein; LDL: low density lipoprotein; HbA1c: glycated hemoglobin type-A 1c; Apo: apolipoprotein. Data expressed as mean ± SD or percentage (%) within genotypic group. ⁎P values for comparison of differences using ANCOVA adjusted for age, BMI and gender, exception for use of the chi-square test§ or the Student's t test†.
a selective interaction between the apoE gene with this other gene product within the family of apolipoproteins. Regarding the assessments of coronary artery calcification, Agatston scores N100 were found in 128 patients (61.8%) of our sample. Notably, 34 (16.4%) subjects were found with roughly null levels of coronary calcification (Agatston scores b 10). The relationship between the apoE polymorphism and these measures showed no significant difference in levels of coronary artery calcification between groups of carriers and non-carriers of ε4. During clinical assessments, 19 (9.2%) of the patients were found to be users of lipid-lowering drugs. Nonetheless, we do not believe that the discrete results we observed were pharmaceutically influenced since chi-square tests failed to reveal any quantitative variances in the distribution of users and non-users of antilipidemic drugs across apolipotrotein genotypes (P N 0.05). 4. Discussion In a whole group approach, it is noteworthy that most of the mean levels of important clinical and biochemical parameters were found within the range of acceptable for that age stratum, with emphasis on average levels of C-reactive protein (3.86 mg/L), diastolic blood pressure (75.3 mm Hg), HbA1c (6.20%), HDL-c (54.7 mg/dL), LDL-c (113.6 mg/dL), TC (198.4 mg/dL) and triglyceride (127.2 mg/dL), and with an exception made for the mean systolic blood pressure (145 mm Hg). These findings are compatible with an overall good health status that is expected from patients eligible for primary prevention against cardiovascular disorders. With this background, our main result suggests that apoE plays a role in modulating the expression of ApoB regardless of causing detectable fluctuation in ApoB-containing lipoproteins. Lower ApoB levels were found among the non-carriers of ε4, whereas the highest average levels were associated with ε4 carriers. In line with this, the study of Boerwinkle and co-workers (Boerwinkle and Utermann, 1988) found that the total ApoB concentration (as the sum of lipoprotein particles with very-low, low and intermediate densities) was markedly decreased in ε2 homozygotes. This very same finding was also observed by Luc et al. (1994) in the case–control study that included four Europe regions and that by Bagger et al. (2007) while investigating a wide array of serum lipids and lipoproteins as surrogate measures of osteoporosis in Danish postmenopausal women. Despite neither the kinetics of ApoE integration on TRL particles nor the
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influence of this multifunctional protein on the metabolism of ApoB being fully understood, Millar et al. observed a negative correlation between the production of ApoE-containing TRL and the production of ApoB-containing LDL-c (Millar et al., 2001). Moreover, it is demonstrated by studies elsewhere (Davignon et al., 1988, 1999; Millar et al., 2001) that a differential expression occurs between apoE alleles, with the ε2 and ε3 alleles bearing a greater expression rate compared to that of ε4. For this reason, the results obtained in our study may at least in part derive from the competitive interaction between gene products, since the inherently higher rate of ε2 and ε3 expression may justify proportionally lower levels of ApoB in TRL particles in the former subjects. Also, this greater expression of ε3 compared to ε4 may justify the tendency towards more elimination of ApoB-containing lipoproteins (e.g.: trend for reduced LDL-c) since greater expression of apoE promotes the enhanced clearance of these particles through hepatic LDL receptors (Shimano et al., 1992). In our conditions, no interaction of apoE with ApoA alone was observed. In studies with younger cohorts, ε2 carriers tend to express lower levels of whole plasma cholesterol and LDL-c than ε3/ε3 homozygotes, with subjects carrying the ε4 allele having higher cholesterol and LDL levels accordingly (Alvim et al., 2010; Davignon et al., 1988, 1999; Yin et al., 2008). However, for very-old adults, as those included herein, the relationship between ApoE isoforms and plasma lipid levels becomes less evident (Davignon et al., 1988), probably due to lifestyle interference (e.g.: malnutrition) and/or age-related deregulation. The works from Davignon et al. as well as those from Pablos-Mendez et al. (1997) observed that ε4 octogenarians had the same mean LDL-c concentrations as those with the ε3/ε3 phenotype, in contrast to findings in the younger population or in younger coronary arterial disease patients. Thereby, the typical LDL-c decreasing gradient from ε4/ε3 to non-ε4 carriers could not be observed in our very elderly sample but the almost significant difference observed among genotypic groups is consistent with such a trend. Having this finding in mind and also that Apo B and Apo A are respectively addressed as surrogates for the assessments of LDL and HDL particles, our results may help in strengthening the assumption that Apo B/A bears more information among healthy very-aged individuals where the mere levels of lipoproteins per se might not reveal the actual metabolic processes taking place in the individual. Regardless of the fact that the association of the apoE classic polymorphism with ApoB levels has long been reported in the literature (Demant et al., 1991), further investigation is still required to reveal how useful this finding would be in the primary care setting, by means of studies with longitudinal design. The present work also suggests that the apoE polymorphism is not related to arterial calcification. As demonstrated elsewhere, a whole body of evidence now indicates that the apoE variability poorly accounts for calcification phenotypes, either ectopic or not (Bagger et al., 2007; Kardia et al., 1999), including those in the general Brazilian population (Alvim et al., 2010). In spite of the well acknowledged fact that apoE impairment leads to severe hypercholesterolemia, atherosclerosis and plaque calcification (Rosenfeld et al., 2000), the presumed role of apoE in the calcification process probably derives from no more than the differential profile of extracellular cholesterol transport and accumulation across genotypes, modulating the contribution of known coronary risk factors but with no major biological role of the gene per se in the onset of abnormal calcium deposition. Currently, the mechanism of vascular calcification is still under investigation, and it is likely that more than one mechanism may arise since atherosclerosis itself occurs by several different mechanisms (Weber and Noels, 2011). Among these possible hypotheses, the osteoblast-like differentiation of human coronary artery smooth muscle triggered by inflammatory mediators (Satomi-Kobayashi et al., 2012) stands as a promising explanation, among others which demand proper clarification. Despite all methodological care, our study has potential limitations. We did not evaluate the usual dietary intake or physical fitness of the very-old adults, which could confound the observed associations.
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Other limitation was that assessing very-low density lipoprotein levels was beyond the original scope of our work. The authors are unaware of previous studies that have observed the relationship of apoE with the ratio between ApoB and ApoA in very-old subjects eligible for primary prevention. 5. Conclusion Our results are suggestive of a competitive interaction between gene products, with ε4 carriers of the apoE gene showing higher levels of the serum apolipoprotein B. Forthcoming studies with prospective design should determine whether the resulting ε4-associated increase in ApoB/ApoA ratio bears clinical relevance in determining cardiovascular events among apparently healthy very-old subjects. Conflict of interest The authors declare no conflict of interest. Author contributions AD Henriques, AC Tonet-Furioso and W Machado-Silva: genotyped all subjects, analyzed and interpreted data used in the study. LA Quaglia, SN Santos: executed the radiological component of the study. WM Freitas: supervised and executed the medical component of the study. C Córdova: advised on the statistical analysis and interpretation of results. AC Sposito, OT Nóbrega: designed and coordinated the study, analyzed and interpreted the results. AD Henriques, AC Tonet-Furioso and OT Nóbrega: participated in the preparation of the original manuscript. Acknowledgments This research was supported by the CNPq with grant # 471872/ 2011-3, with a stipend to A.C. Tonet-Furioso, and with fellowships for productivity in research to A.C. Sposito and to O.T. Nóbrega. References Alvim, R.O., et al., 2010. APOE polymorphism is associated with lipid profile, but not with arterial stiffness in the general population. Lipids Health Dis. 9, 128 (PubMed PMID: 21059196. Pubmed Central PMCID: 2992057). Aulchenko, Y.S., et al., 2009. Loci influencing lipid levels and coronary heart disease risk in 16 European population cohorts. Nat. Genet. 41 (1), 47–55 (Jan, PubMed PMID: 19060911. Pubmed Central PMCID: 2687074). Bagger, Y.Z., et al., 2007. Links between cardiovascular disease and osteoporosis in postmenopausal women: serum lipids or atherosclerosis per se? Osteoporos. Int. 18 (4), 505–512 (Apr, PubMed PMID: 17109061. Pubmed Central PMCID: 1820757). Boerwinkle, E., Utermann, G., 1988. Simultaneous effects of the apolipoprotein E polymorphism on apolipoprotein E, apolipoprotein B, and cholesterol metabolism. Am. J. Hum. Genet. 42 (1), 104–112 (Jan, PubMed PMID: 3337104. Pubmed Central PMCID: 1715322). Davignon, J., Bouthillier, D., Nestruck, A.C., Sing, C.F., 1988. Apolipoprotein E polymorphism and atherosclerosis: insight from a study in octogenarians. Trans. Am. Clin. Climatol. Assoc. 99, 100–110 (PubMed PMID: 3503432. Pubmed Central PMCID: 2376437). Davignon, J., Cohn, J.S., Mabile, L., Bernier, L., 1999. Apolipoprotein E and atherosclerosis: insight from animal and human studies. Clin. Chim. Acta 286 (1–2), 115–143 (Aug, PubMed PMID: 10511288. Epub 1999/10/08. eng). Demant, T., Bedford, D., Packard, C.J., Shepherd, J., 1991. Influence of apolipoprotein E polymorphism on apolipoprotein B-100 metabolism in normolipemic subjects.
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Gene journal homepage: www.elsevier.com/locate/gene
Short Communication
Apoliprotein E genotype is associated with apoliprotein B plasma levels but not with coronary calcium score in very elderly individuals in primary care setting Adriane D. Henriques a, Audrey C. Tonet-Furioso a, Wilcelly Machado-Silva a, Wladimir M. Freitas a,b, Luiz A. Quaglia b, Simone N. Santos a, Cláudio Córdova c, Andrei C. Sposito a,d, Otávio T. Nóbrega a,⁎, on behalf of the Brazilian Study on Healthy Aging Group a
Universidade de Brasília (UnB), Campus Universitário Darcy Ribeiro, 70.910-900 Brasília, DF, Brazil Instituto de Cardiologia Biocardios, SEPS 709/909 Sul Edifício Biocenter, Asa Sul, 70.390-095 Brasília, DF, Brazil c Universidade Católica de Brasília (UCB-DF), QS 07 Lote 01 EPCT, 71.966-700, Taguatinga, DF, Brazil d Universidade Estadual de Campinas (UNICAMP), Universidade Estadual de Campinas, Rua Tessália Vieira de Camargo 126, 13.083-887, Campinas, SP, Brazil b
a r t i c l e
i n f o
Article history: Received 18 November 2013 Received in revised form 26 December 2013 Accepted 31 January 2014 Available online 12 February 2014 Keywords: Apolipoprotein Lipid metabolism Atherosclerosis Primary prevention Aging
a b s t r a c t Background: Epidemiological surveys indicate the influence of polymorphisms of apolipoprotein (apo) E on plasma lipids and triglyceride-rich lipoprotein levels, with impact on atherosclerotic phenotypes. Aim: We studied the association of classic genotypes of the apoE gene with clinical and biochemical risk factors for atherosclerosis in a segment of the very-old Brazilian individuals, with emphasis on the lipemic profile. Methods: We performed cross-sectional analyses of clinical and laboratory assessments, including cardiac computed tomography, across ε2, ε3 and ε4 carriers of the apoE gene with a convenience sample of 208 participants eligible for prevention against cardiovascular events. Results: When non-ε4 carriers were compared with ε4 carrying subjects, lower levels of ApoB as well as ApoB/ ApoA ratios were observed in the former group. Tests between apoE polymorphisms with other clinical/ biochemical variables and those with arterial calcification showed no significant differences between groups. Conclusion: The study suggests a possible atherogenic role of the ε4 allele attributable to increased ApoB levels and ApoB/ApoA ratios among very-old subjects in primary care setting. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Cardiovascular diseases (CVD) account for 48% of the deaths from noncommunicable diseases worldwide (Finegold et al., 2013). In Brazil, roughly one third of total deaths are represented by CVD (Sociedade Brasileira de Cardiologia et al., 2010), the main cause of deaths in the elderly population (Mansur and Favarato, 2012). Many clinical factors that contribute to CVD mortality such as obesity, hypertension, type-2 diabetes and dyslipidemia bear significant genetic contributors (Glass and Witztum, 2001). An increasing number of genetic Abbreviations: ANCOVA, analysis of covariance; Apo, apolipoprotein; BMI, body mass index; CVD, cardiovascular disease; HDL-c, high density lipoprotein cholesterol; LDL-c, low density lipoprotein cholesterol; MANCOVA, multivariate analysis of covariance; SD, standard deviation; SPSS, Statistical Package For Social Sciences; TC, total cholesterol; TG, triglyceride; TRL, triglyceride-rich lipoprotein. ⁎ Corresponding author at: Universidade de Brasília (UnB), Campus Universitário Darcy Ribeiro, 70.910-900, Brasília, DF, Brazil. Tel.: +55 61 31071913. E-mail addresses: [email protected] (A.D. Henriques), [email protected] (A.C. Tonet-Furioso), [email protected] (W. Machado-Silva), [email protected] (W.M. Freitas), [email protected] (L.A. Quaglia), [email protected] (S.N. Santos), [email protected] (C. Córdova), [email protected] (A.C. Sposito), [email protected] (O.T. Nóbrega). 0378-1119/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2014.01.077
polymorphisms have been associated with susceptibility to cardiovascular disease, mainly atherosclerosis, with emphasis on the important variability of the apolipoprotein (apo) E gene. Epidemiological surveys assessing the role of the apoE polymorphism on plasma lipids and triglyceride-rich lipoproteins (TRL) have shown that the presence of the ε4 allele is associated with elevations in low density lipoprotein cholesterol (LDL-c), while the presence of ε2 has the opposite effect on these particles (Alvim et al., 2010), possibly by virtue of the greater ability of the former and minor ability of the latter to act as ligands for specialized lipoprotein receptors (Demant et al., 1991) and hence an adaptative up-regulation of the receptor in ε2 carriers (Alvim et al., 2010; Davignon et al., 1999). These gene–gene interactions are important because lipoproteins play a central role in the development of the atherosclerotic phenotype (Tiret et al., 1994), and ApoE is a key protein in the modulation of the catabolism of the most atherogenic particles (Alvim et al., 2010). There is evidence that ApoE has a profound impact on the ApoB metabolism (Demant et al., 1991) and on ApoB-related lipids and lipoproteins (Davignon et al., 1999). Recent genome wide association studies confirmed strong statistical association between apoE with CVD risk (Waterworth et al., 2010), primarily due to influences on levels of LDL-c (Smith et al., 2010) and total cholesterol
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(Aulchenko et al., 2009). Despite prospective studies indicating that the ApoB/ApoA-I ratio is a useful predictor of risk for myocardial infarction (MI) and other CVD (Sierra-Johnson et al., 2009), to our knowledge no studies sought to associate apoE genotypes with this aspect of the lipid metabolism of older adults. The very elderly are the age group that is growing the most rapidly in westernized societies such as those in Brazil (Nobrega et al., 2009) with an increasing number of individuals reaching this age stratum devoid of severe circulatory disorders (Freitas et al., 2012), and being eligible for primary prevention against major cardiovascular events. The goal of our work is to study the association of classic genotypes of the apoE gene with the atherosclerotic risk inherent to the lipemic profile of a segment of the very-old Brazilian population in primary care setting. 2. Methods 2.1. Subjects Study subjects are participants in the ongoing Brazilian Study on Healthy Aging, which is a prospective cohort study that was started in December 2008 and designed to identify markers of cardiovascular risk in very elderly individuals eligible for primary prevention as published elsewhere (Freitas et al., 2011). For this proposal, noninstitutionalized consecutive patients aged 80 years or over who have sought the outpatient clinic for preventive care and have never manifested myocardial infarction, stroke or peripheral arterial disease were enrolled. Additional selection criteria were the absence of autoimmune disease (including rheumatic disorders), chronic or recurrent infections, prior or current neoplastic disease, and use of steroidal or nonsteroidal anti-inflammatory drugs in the past 30 days. After baseline measurements, all the participants were referred to the study outpatient clinic for prospective medical follow-up evaluations. Nonetheless, the results described herein derived from crosssectional analyses with data at entry. The study was approved by the institutional research ethics committee and procedures were in accordance with the ethical standards of the Helsinki Declaration, with all participants having signed informed consent before enrollment.
prospective electrocardiography triggering. Coronary artery calcification was defined as a minimum of 3 contiguous pixels with a peak Hounsfield unit density N130. Coronary artery calcifications were scored by a certified radiologist using the Agatston score to express its extent. 2.5. apoE genotyping Whole blood was obtained during sampling for biochemical analysis and stored at −20 °C until use. Genomic DNA was purified according to standard extraction kits (QIAamp DNA Mini Kit, Qiagen, Brazil). To determine the classic genotypes for epsilon allelic variants of the apoE gene (ε2, ε3 and ε4), amplifications based on a refractory mutation system (ARMS) for multiplex polymerase chain reactions were carried out using the method described by Donohoe et al. (1999), followed by electrophoresis of the amplification products in a 1.6% agarose gel. Each sample was run at least twice, and further checked only if the genotypes of the first two productive analyses were in conflict. 2.6. Statistical analysis Violation of the Hardy–Weinberg equilibrium was tested using Fisher's exact test. The Kolmogorov–Smirnov test was used to verify normal distribution of data from continuous variables in a withingroup approach. Whenever appropriate, data are expressed as means ± standard deviation (SD) or frequency (%). The Student's t test was used to compare the means of age and BMI across genotypes. Frequencies (e.g.: gender) were compared using the chi-square test. Analysis of covariance (ANCOVA) was used to analyze the variance in the main dependent variables across genotypes, with adjustment to age, BMI and gender. To test whether the intercorrelated nature of some dependent variables affected the parametric analyses outputs, MANCOVA was run to compare cholesterol-related metabolite values across groups, with the same adjustments. All the analyses were performed employing the Statistical Package for Social Sciences (SPSS) for Windows (version 17.0). A P value b 0.05 was considered significant. 3. Results
2.2. Clinical inspection All the subjects were submitted to assessments of total body mass (kg), body height (m), and blood pressure (mm Hg). Body mass index (BMI; kg/m2) was defined as usual whereas waist circumference (WC; cm) was measured 2 cm above the umbilicus scar. 2.3. Biochemical analysis After 12 h of overnight fasting, blood was collected and centrifuged at 5 °C, 4500 rpm for 15 min to separate plasma that was used exclusively to carry out the measurements of the metabolic variables analyzed herein: fasting glucose, glycated hemoglobin A (HbA) 1c, total cholesterol (TC), triglycerides (TG) and high density lipoprotein cholesterol (HDL-c). All glycemic and lipemic assessments were performed using reagents from Roche Diagnostics (Mannheim, USA) according to manufacturer recommendations and carried out by the same certified clinical laboratory. Low density lipoprotein cholesterol (LDL-c) was determined using the Friedewald equation (Friedewald et al., 1972). Assessments of C-reactive protein (highly sensitive, CardioPhase, Dade Behring, Marburg, USA), ApoA and ApoB (Behring Nephelometer BNII, Dade Behring, Marburg, Germany) were also performed. 2.4. Cardiac computed tomography Computed tomography was performed in a 64-slice scanner (Aquillion 64, Toshiba, Ottawara, Japan). Axial slices of 3 mm thickness with 3 mm table-feed were acquired at 70% of R–R intervals with
During the recruitment of patients, we assessed the clinical and biochemical characteristics of each subject at admission, along with the genotypes of the apoE gene. The allele frequencies of the ε2, ε3 and ε4 in the whole sample of 208 admitted individuals were 1.0%, 91.3% and 7.7%, respectively. Due to a low count of ε2 carriers in the sample, these individuals were merged with the ε3 homozygotes (non-ε4 carries) for the purpose of comparison with ε4 carriers. A single patient with the ε2/ε4 genotype was excluded from the analysis because of the putative opposite effects of these alleles in the phenotypes studied (Tiret et al., 1994), constituting a final sample of 207 subjects. This sample showed a homogeneous pattern of baseline characteristics such as mean values of age and BMI and gender proportion across genotypes. Even so, these features were treated as covariates in the subsequent analyses. Regarding lipemic and clinical variables, ANCOVA revealed no differences in terms of plasma lipids (TC, LDL-c, HDL-c and TG) when non-ε4 carriers were compared with ε4 carrying subjects, albeit a non-significant trend towards lower LDL-c levels (P = 0.08) could be observed among the non-ε4 carriers. Only in terms of serum ApoB levels and ApoB/ApoA ratios, were mean scores rendered significant (P b 0.05). There were no other clinical or laboratory differences observed between the groups (Table 1). MANCOVA for cholesterolrelated metabolites as dependent variables (TC, LDL-c, HDL-c, TG, ApoB, ApoA and ApoB/ApoA) yielded similar results (Hotelling's Trace (7197) = 0.058), with greater serum ApoB levels (F = 4.43, P = 0.036) and ApoB/ApoA ratios (F = 4.88, P = 0.028) among the e4-carriers. The association of this structural polymorphism with the quantitative levels of ApoB, but not with ApoA, is suggestive of
A.D. Henriques et al. / Gene 539 (2014) 275–278 Table 1 Characteristics of the very-old individuals according to the apolipoprotein E genotypic group.
Age, years Male, % Body mass index, kg/m2 Waist circumference, cm SBP, mm Hg DBP, mm Hg Total cholesterol, mg/dL HDL cholesterol, mg/dL LDL cholesterol, mg/dL Triglyceride, mg/dL HbA1c, % ApoA, mg/dL ApoB, mg/dL ApoB/ApoA Agatston score, unit density C-reactive protein, mg/L
Non-ε4 carriers
ε4 carriers
(n = 179)
(n = 28)
84.5 ± 4.2 18.8 26.5 ± 4.8 94.4 ± 12.1 144.8 ± 19.4 75.3 ± 10.8 197.4 ± 41.0 54.9 ± 13.8 112.3 ± 34.9 127.0 ± 55.8 6.19 ± 1.02 150.9 ± 24.8 84.4 ± 23.2 0.57 ± 0.20 262.9 ± 300.6 3.82 ± 6.90
84.1 ± 4.5 35.7 24.9 ± 3.6 93.3 ± 12.1 148.1 ± 27.6 75.2 ± 10.7 204.5 ± 41.9 53.4 ± 14.3 122.6 ± 37.0 128.5 ± 58.2 6.23 ± 1.63 144.7 ± 22.8 94.0 ± 23.0 0.67 ± 0.21 269.1 ± 331.4 4.11 ± 6.75
P⁎
0.66† 0.13§ 0.16† 0.65† 0.43 0.96 0.39 0.59 0.08 0.89 0.84 0.22 0.04 0.03 0.92 0.84
SBP: systolic blood pressure; DBP: diastolic blood pressure; HDL: high density lipoprotein; LDL: low density lipoprotein; HbA1c: glycated hemoglobin type-A 1c; Apo: apolipoprotein. Data expressed as mean ± SD or percentage (%) within genotypic group. ⁎P values for comparison of differences using ANCOVA adjusted for age, BMI and gender, exception for use of the chi-square test§ or the Student's t test†.
a selective interaction between the apoE gene with this other gene product within the family of apolipoproteins. Regarding the assessments of coronary artery calcification, Agatston scores N100 were found in 128 patients (61.8%) of our sample. Notably, 34 (16.4%) subjects were found with roughly null levels of coronary calcification (Agatston scores b 10). The relationship between the apoE polymorphism and these measures showed no significant difference in levels of coronary artery calcification between groups of carriers and non-carriers of ε4. During clinical assessments, 19 (9.2%) of the patients were found to be users of lipid-lowering drugs. Nonetheless, we do not believe that the discrete results we observed were pharmaceutically influenced since chi-square tests failed to reveal any quantitative variances in the distribution of users and non-users of antilipidemic drugs across apolipotrotein genotypes (P N 0.05). 4. Discussion In a whole group approach, it is noteworthy that most of the mean levels of important clinical and biochemical parameters were found within the range of acceptable for that age stratum, with emphasis on average levels of C-reactive protein (3.86 mg/L), diastolic blood pressure (75.3 mm Hg), HbA1c (6.20%), HDL-c (54.7 mg/dL), LDL-c (113.6 mg/dL), TC (198.4 mg/dL) and triglyceride (127.2 mg/dL), and with an exception made for the mean systolic blood pressure (145 mm Hg). These findings are compatible with an overall good health status that is expected from patients eligible for primary prevention against cardiovascular disorders. With this background, our main result suggests that apoE plays a role in modulating the expression of ApoB regardless of causing detectable fluctuation in ApoB-containing lipoproteins. Lower ApoB levels were found among the non-carriers of ε4, whereas the highest average levels were associated with ε4 carriers. In line with this, the study of Boerwinkle and co-workers (Boerwinkle and Utermann, 1988) found that the total ApoB concentration (as the sum of lipoprotein particles with very-low, low and intermediate densities) was markedly decreased in ε2 homozygotes. This very same finding was also observed by Luc et al. (1994) in the case–control study that included four Europe regions and that by Bagger et al. (2007) while investigating a wide array of serum lipids and lipoproteins as surrogate measures of osteoporosis in Danish postmenopausal women. Despite neither the kinetics of ApoE integration on TRL particles nor the
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influence of this multifunctional protein on the metabolism of ApoB being fully understood, Millar et al. observed a negative correlation between the production of ApoE-containing TRL and the production of ApoB-containing LDL-c (Millar et al., 2001). Moreover, it is demonstrated by studies elsewhere (Davignon et al., 1988, 1999; Millar et al., 2001) that a differential expression occurs between apoE alleles, with the ε2 and ε3 alleles bearing a greater expression rate compared to that of ε4. For this reason, the results obtained in our study may at least in part derive from the competitive interaction between gene products, since the inherently higher rate of ε2 and ε3 expression may justify proportionally lower levels of ApoB in TRL particles in the former subjects. Also, this greater expression of ε3 compared to ε4 may justify the tendency towards more elimination of ApoB-containing lipoproteins (e.g.: trend for reduced LDL-c) since greater expression of apoE promotes the enhanced clearance of these particles through hepatic LDL receptors (Shimano et al., 1992). In our conditions, no interaction of apoE with ApoA alone was observed. In studies with younger cohorts, ε2 carriers tend to express lower levels of whole plasma cholesterol and LDL-c than ε3/ε3 homozygotes, with subjects carrying the ε4 allele having higher cholesterol and LDL levels accordingly (Alvim et al., 2010; Davignon et al., 1988, 1999; Yin et al., 2008). However, for very-old adults, as those included herein, the relationship between ApoE isoforms and plasma lipid levels becomes less evident (Davignon et al., 1988), probably due to lifestyle interference (e.g.: malnutrition) and/or age-related deregulation. The works from Davignon et al. as well as those from Pablos-Mendez et al. (1997) observed that ε4 octogenarians had the same mean LDL-c concentrations as those with the ε3/ε3 phenotype, in contrast to findings in the younger population or in younger coronary arterial disease patients. Thereby, the typical LDL-c decreasing gradient from ε4/ε3 to non-ε4 carriers could not be observed in our very elderly sample but the almost significant difference observed among genotypic groups is consistent with such a trend. Having this finding in mind and also that Apo B and Apo A are respectively addressed as surrogates for the assessments of LDL and HDL particles, our results may help in strengthening the assumption that Apo B/A bears more information among healthy very-aged individuals where the mere levels of lipoproteins per se might not reveal the actual metabolic processes taking place in the individual. Regardless of the fact that the association of the apoE classic polymorphism with ApoB levels has long been reported in the literature (Demant et al., 1991), further investigation is still required to reveal how useful this finding would be in the primary care setting, by means of studies with longitudinal design. The present work also suggests that the apoE polymorphism is not related to arterial calcification. As demonstrated elsewhere, a whole body of evidence now indicates that the apoE variability poorly accounts for calcification phenotypes, either ectopic or not (Bagger et al., 2007; Kardia et al., 1999), including those in the general Brazilian population (Alvim et al., 2010). In spite of the well acknowledged fact that apoE impairment leads to severe hypercholesterolemia, atherosclerosis and plaque calcification (Rosenfeld et al., 2000), the presumed role of apoE in the calcification process probably derives from no more than the differential profile of extracellular cholesterol transport and accumulation across genotypes, modulating the contribution of known coronary risk factors but with no major biological role of the gene per se in the onset of abnormal calcium deposition. Currently, the mechanism of vascular calcification is still under investigation, and it is likely that more than one mechanism may arise since atherosclerosis itself occurs by several different mechanisms (Weber and Noels, 2011). Among these possible hypotheses, the osteoblast-like differentiation of human coronary artery smooth muscle triggered by inflammatory mediators (Satomi-Kobayashi et al., 2012) stands as a promising explanation, among others which demand proper clarification. Despite all methodological care, our study has potential limitations. We did not evaluate the usual dietary intake or physical fitness of the very-old adults, which could confound the observed associations.
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Other limitation was that assessing very-low density lipoprotein levels was beyond the original scope of our work. The authors are unaware of previous studies that have observed the relationship of apoE with the ratio between ApoB and ApoA in very-old subjects eligible for primary prevention. 5. Conclusion Our results are suggestive of a competitive interaction between gene products, with ε4 carriers of the apoE gene showing higher levels of the serum apolipoprotein B. Forthcoming studies with prospective design should determine whether the resulting ε4-associated increase in ApoB/ApoA ratio bears clinical relevance in determining cardiovascular events among apparently healthy very-old subjects. Conflict of interest The authors declare no conflict of interest. Author contributions AD Henriques, AC Tonet-Furioso and W Machado-Silva: genotyped all subjects, analyzed and interpreted data used in the study. LA Quaglia, SN Santos: executed the radiological component of the study. WM Freitas: supervised and executed the medical component of the study. C Córdova: advised on the statistical analysis and interpretation of results. AC Sposito, OT Nóbrega: designed and coordinated the study, analyzed and interpreted the results. AD Henriques, AC Tonet-Furioso and OT Nóbrega: participated in the preparation of the original manuscript. Acknowledgments This research was supported by the CNPq with grant # 471872/ 2011-3, with a stipend to A.C. Tonet-Furioso, and with fellowships for productivity in research to A.C. Sposito and to O.T. Nóbrega. References Alvim, R.O., et al., 2010. APOE polymorphism is associated with lipid profile, but not with arterial stiffness in the general population. Lipids Health Dis. 9, 128 (PubMed PMID: 21059196. Pubmed Central PMCID: 2992057). Aulchenko, Y.S., et al., 2009. Loci influencing lipid levels and coronary heart disease risk in 16 European population cohorts. Nat. Genet. 41 (1), 47–55 (Jan, PubMed PMID: 19060911. Pubmed Central PMCID: 2687074). Bagger, Y.Z., et al., 2007. Links between cardiovascular disease and osteoporosis in postmenopausal women: serum lipids or atherosclerosis per se? Osteoporos. Int. 18 (4), 505–512 (Apr, PubMed PMID: 17109061. Pubmed Central PMCID: 1820757). Boerwinkle, E., Utermann, G., 1988. Simultaneous effects of the apolipoprotein E polymorphism on apolipoprotein E, apolipoprotein B, and cholesterol metabolism. Am. J. Hum. Genet. 42 (1), 104–112 (Jan, PubMed PMID: 3337104. Pubmed Central PMCID: 1715322). Davignon, J., Bouthillier, D., Nestruck, A.C., Sing, C.F., 1988. Apolipoprotein E polymorphism and atherosclerosis: insight from a study in octogenarians. Trans. Am. Clin. Climatol. Assoc. 99, 100–110 (PubMed PMID: 3503432. Pubmed Central PMCID: 2376437). Davignon, J., Cohn, J.S., Mabile, L., Bernier, L., 1999. Apolipoprotein E and atherosclerosis: insight from animal and human studies. Clin. Chim. Acta 286 (1–2), 115–143 (Aug, PubMed PMID: 10511288. Epub 1999/10/08. eng). Demant, T., Bedford, D., Packard, C.J., Shepherd, J., 1991. Influence of apolipoprotein E polymorphism on apolipoprotein B-100 metabolism in normolipemic subjects.
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