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Genetic variants in genes related to lipid metabolism and atherosclerosis, dyslipidemia and atorvastatin response
Clinica Chimica Acta 417 (2013) 8–11
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Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Genetic variants in genes related to lipid metabolism and atherosclerosis, dyslipidemia and atorvastatin response A.C. Rodrigues a,⁎, 1, B. Sobrino b, F.D.V. Genvigir a, M.A.V. Willrich a, S.S. Arazi a, E.L. Dorea c, M.M.S. Bernik c, M. Bertolami d, A.A. Faludi d, M.J. Brion b, e, A. Carracedo b, M.H. Hirata a, R.D.C. Hirata a a
Faculdade de Ciências Farmacêuticas, USP, São Paulo, SP, Brazil Grupo de Medicina Xenomica-USC, CIBERER, Fundacion Publica Galega de Medicina Xenómica, Santiago de Compostela, Galícia, Spain Hospital Universitário, USP, São Paulo, SP, Brazil d Instituto Dante Pazzanese de Cardiologia, São Paulo, SP, Brazil e Complexo Hospitalario de Santiago de Compostela, Galícia, Spain b c
a r t i c l e
i n f o
Article history: Received 9 September 2012 Received in revised form 27 November 2012 Accepted 27 November 2012 Available online 13 December 2012 Keywords: Association studies Dyslipidemia SNPs Statins pharmacogenomics
a b s t r a c t Objective: Using candidate gene approach, we have investigated the effect of single nucleotide polymorphism (SNP) in genes related to lipid metabolism and atherosclerosis on dyslipidemia and atorvastatin response. Methods: The study included 157 patients treated with atorvastatin and 145 controls. Genomic DNA was isolated and genotyped using SNPlex technology. Results: Allele and genotype disease association test revealed that APOB rs693 (OR: 2.2 [1.5–3.2], p=0.0001) and CD36 rs1984112 (OR: 3.7 [1.9–7.0], p=0.0002) SNPs were independent risk factors for hypercholesterolemia. Only APOB rs693 T variant allele was associated with increased LDL cholesterol levels (>160 mg/dL). After atorvastatin treatment (10 mg/day/4 weeks), LIPC −514T allele was positively associated with LDL cholesterol reduction. Conclusion: The current study reinforces the current knowledge that carrying APOB rs693 is an independent risk factor for dyslipidemia and higher LDL levels. Furthermore, we found that a variant of CD36 was associated with dyslipidemia as a risk (rs1984112) factor. Finally, atorvastatin response could be predicted by LIPC −514C>T SNP and physical activity. In conclusion, our data evidences the contribution of genetic markers and their interaction with environmental factor in the variability of statin response. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Atherosclerosis is the major cause of cardiovascular disease and there is consistent and convincing evidence that supports an association between dyslipidemia and cardiovascular disease incidence worldwide. Whereas levels of low density lipoprotein (LDL) cholesterol are directly associated with an increased risk for coronary arterial disease (CAD) [1], higher serum levels of high density lipoprotein (HDL) cholesterol are a protection factor [2]. High serum triglycerides (TG) levels may be an additional factor for cardiovascular diseases [3,4], however there is much controversy. Risk factors for dyslipidemia include sedentary lifestyle, high fat diet (cholesterol and monosatured fatty acids enriched) and smoking [1].
⁎ Corresponding author at: Faculty of Pharmaceutical Sciences, Department of Clinical and Toxicological Analyses, Av. Prof. Lineu Prestes, 580, bl17, Sao Paulo, SP 05508-000, Brazil. Tel.: +55 11 30913660; fax: +55 11 3813 2197. E-mail address: [email protected] (A.C. Rodrigues). 1 Present/permanent address: Institute of Biomedical Sciences. Department of Physiology and Biophysics. Av. Prof. Lineu Prestes, 1524, 05508-000, Brazil. 0009-8981/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cca.2012.11.028
Studies with families in many populations suggest, though, that half of the lipid profile variations are genetically determinate [5,6]. Besides this, LDL cholesterol, HDL cholesterol and TG levels are influenced by the genetic constitution of each individual [7–9]. Specifically to LDL cholesterol, variants in LDL receptor (LDLR), apolipoprotein B (APOB) genes have been associated with its plasma levels [10,11]. Although several genes and genetic variants have been associated with individual variation in lipid concentrations, additional variants influencing these traits remain to be identified. The first intervention in dyslipidemia is to introduce to the patients a low-fat diet, however many of the patients do not respond and are initiated on statin therapy. Statins are HMG-CoA reductase inhibitors used as first choice in the treatment of hypercholesterolemia. Variation in LDL cholesterol response has been associated with many genes involved in lipid metabolism and in statin pharmacokinetics (for reviews [12–14]), however no study has found a conclusive genetic marker, and more studies are necessary to elucidate this. Using candidate gene approach, we have performed a case-control study on single nucleotide polymorphisms (SNPs) in genes related to lipid metabolism and atherosclerosis and with statin pharmacokinetics and pharmacodynamics in a group of unrelated dyslipidemic individuals treated with atorvastatin for four weeks.
A.C. Rodrigues et al. / Clinica Chimica Acta 417 (2013) 8–11
2. Materials and methods 2.1. Patients and research design We have selected 302 individuals (145 normolipidemic and 157 dyslipidemic) from the University Hospital from University of Sao Paulo and Institute Dante Pazzanese of Cardiology. Clinical data for the individuals selected are presented in Table 1. The study protocol was approved by the Ethics Committee of these institutions as well as the Committee of the School of Pharmaceutical Sciences (University of Sao Paulo). Information on age, body mass index (BMI), gender, hypertension, obesity, menopause status, cigarette smoking, physical activity, alcohol consumption and family history of CAD was recorded. Dyslipidemic patients were characterized as total cholesterol ≥200 mg/dL, LDL cholesterol ≥160 mg/dL with or without TG ≥150 mg/dL and low levels of HDL cholesterol (men: b 40 mg/dL and women >50 mg/dL). Individuals with diabetes mellitus, hypo- or hyperthyroidism, triglycerides higher than 400 mg/dL, renal or hepatic disease, subjects under treatment with oral contraceptive or hormone replacement and lipid-lowering drugs or pregnant women were excluded from the study. Dyslipidemic subjects even after a low cholesterol diet during 4 weeks were started on atorvastatin therapy, 10 mg orally once daily for 4 weeks. At the end of the protocol, the patients had a last appointment with the doctor and response to atorvastatin as well as any possible Table 1 Clinical and biochemical data for the studied individuals. Variables Alcohol consumption >7 Doses/week b7 Doses/week Smokingc Hypertensiond Familiar history of CAD Menopause Physical activity Ethnicse White Black and intermediates Gender (female) Age—years (mean ± SD) BMI (kg/m2) Total cholesterol (mg/dL) LDL-C (mg/dL) LDL-C post-10 (mg/dL) HDL-C (mg/dL) HDL-C post-10 (mg/dL) TG (mg/dL) TG post-10 (mg/dL) ApoAI (g/L) ApoAI post-10 (g/L) ApoB (g/L) ApoB post-10 (g/L) ApoAI/ApoB ApoAI/ApoB ratio post-10 CK (U/L) CK post-10 (U/L) ALT (U/L) ALT post-10 (U/L)
Normolipidemics (145)
Dyslipidemics (157)
p
1.6% (4/144f) 30.50% (48/144f) 19.4% (24) 33.1% (47/142f) 44.8% (64/143f) 27.42% (23/90f) 45.8% (68)
1.91% (4) 28.02% (40) 15.29% (22) 56.9% (83/146f) 54.8% (80/146) 84.76% (78) 47.74% (68/147f)
0.279a 0.757a 0.794a b0.0001a 0.112a b0.0001a 0.962a 0.008a
54.48% (79) 45.52% (66) 68.3% (99) 47 ± 7 25.7 ± 4.1 (141f) 175 ± 18 98 ± 18 – 62 ± 11 – 77 ± 24 – 147 ± 27 – 86 ± 21 – 1.76 ± 0.68 – 140 ± 116 – 22 ± 15 –
72.22% (104/144f) 27.78% (44/144f) 67.1% (100) 57 ± 11 27.6 ± 4.2 (146f) 282 ± 37 192 ± 34 117 ± 27 57 ± 14 55 ± 13 160 ± 65 131 ± 53 133 ± 26 138 ± 28 143 ± 21 100 ± 21 1.11 ± 0.32 1.10 ± 0.46 98 ± 78 100 ± 84 21 ± 9 25 ± 15
0.930a b0.0001b b0.0001b b0.0001b b0.0001b – b0.0001b – b0.0001b – 0.0254b – b0.0001b – b0.0001b – 0.0002b – 0.4412b –
Post-10: serum concentrations measured after atorvastatin treatment (10 mg/day/4 weeks). CAD: coronary artery disease; BMI: body mass index; LDL-C: low density lipoprotein cholesterol: HDL-C: high-density lipoprotein cholesterol; TG: triglycerides; ApoAI: apolipoprotein AI; ApoB: apolipoprotein B; CK: creatine kinase; ALT: alanine tranasaminase. a p-Value as indicated by Chi-square test. b p-Value as indicated by “t”test. c Current cigarette smoking was defined as daily intake of one or more cigarettes. d Individuals with systolic/diastolic blood pressure higher than 140/90 mmHg were considered hypertensive. e Each individual declared their ethnic group during the interview, as recommended by Brazilian census. f Indicates that some patients did not inform or the information was missing in the medical promtuary.
9
adverse reactions were evaluated. The dose of 10 mg atorvastatin was chosen because the patients had moderate elevations of LDL cholesterol, and LDL cholesterol goal could be achieved with low doses for these patients. In addition, the NCEP recommends checking the response to drug therapy in about 6 weeks. Response to atorvastatin was evaluated by the reduction of LDL cholesterol after the treatment, and adverse effects were monitored by measuring creatine kinase (CK) and alanine aminotransferase (ALT) enzymes. 2.2. Biochemical profile and genes variants genotyping Blood samples for biochemical profile (lipids, CK, and ALT) measurements and genomic DNA extraction were collected after an overnight fast, one day before and 4 weeks after atorvastatin treatment. All patients followed exactly the same study protocol. Laboratory methods for biochemical parameters are described elsewhere [15]. Biochemical data are shown in Table 1. Genomic DNA was extracted from EDTA-anticoagulated blood by a salting-out procedure. The SNPs in candidate genes were selected using the online tool available at Life Technologies website, SNPlex™ Assay Design, and the pools of compatible probes, normally between 24 and 28, were synthesized by Life Technologies. The genes included in multiplex assays are described in Supplemental Table 1. Genes involved in pharmacodynamics and pharmacokinetics of statins and with pleiotropic effects of statins were included. SNPs were genotyped by SNPlex™ (Life Technologies), which consists of four steps: oligonucleotide ligation assay (OLA), PCR amplification, ZipChute™ hybridization and capillary electrophoresis for the detection of ZipChute™ probes. Genotype calling was performed on GeneMapper v.4 (Life Technologies). All genotyping procedures were realized at Centro Nacional do Genotipado (CeGen), Santiago de Compostela, Galícia, Spain. 2.3. Statistical analyses Hardy–Weinberg equilibrium (HWE) was tested for each SNP. Qualitative and quantitative variables were analyzed by Likelihood ratio test and ANOVA, respectively. Allelic and genotypic association tests were corrected by odds ratio Haldane correction. Allele and genotype disease association tests were performed using different model of inheritance (dominant, co-dominant, and recessive). To exclude the possibility of other risk factors to influence our results, we performed a logistic regression association test adjusting by age, hypertension, and history of coronary artery disease, sex and body mass index (BMI). Multiple testing was corrected by Bonferroni's test. Multiple stepwise logistic regression analysis was performed to identify independent predictors of atorvastatin response measured by percentage of LDL cholesterol reduction. A p-value ≤0.05 was considered statistically significant. Analyses were performed by using SNPator [16] and SPSS17 (SPSS Inc., Chicago/IL EUA). 3. Results 3.1. Dyslipidemia We performed a candidate gene association study for dyslipidemia. All the SNPs were in Hardy–Weinberg equilibrium in the control group (normolipidemics), except for two polymorphisms in MBTPS2 gene. These SNPs were excluded from the study. Allele disease association test revealed that carriers of the common C allele for CD36 rs1527483 SNP had 4 times more risk to have dyslipidemia than non-carriers. On the other hand, carriers of APOB T allele (rs693), ATP-binding Cassette transporter G1 (ABCG1) T allele (rs9975740) and CD36 G allele (rs1984112) had 1.7 to 2.2 higher risk to have dyslipidemia than non-carriers (data not shown). After we correct the multiple testing with Bonferroni's test, only APOB rs693T carriers were in risk of dyslipidemia (Table 2).
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A.C. Rodrigues et al. / Clinica Chimica Acta 417 (2013) 8–11
Genotype disease association test using a dominant (AA + AB versus BB) or a recessive (AA versus AB + BB) or a co-dominant model (AA/AB/BB) of inheritance was performed. For APOB rs693, the presence of the variant allele T is a risk factor as evidenced before by allele association test. The heterozygous for this SNP has two-fold risk to have dyslipidemia, and the presence of this allele in homozygosis increases the risk to 4 times. On the other hand, for CD36 rs1984112 polymorphism, carriers of AG genotype have increased risk of dyslipidemia (O.R. 3.55 [1.88–6.70, p=0.0002]) (Table 2). Finally, we performed an association test between the SNPs and the levels of LDL cholesterol. A logistic regression association test (adjusted by sex, BMI, history of DAC and hypertension) showed that RLDL rs688 T allele (OR: 1.7, 95CI: 1.3–2.3, p = 0.018), APOB rs693 T allele (OR: 1.7, 95CI: 1.3–2.3, p = 0.002), and CD36 rs1527483 C allele (OR: 3.1, 95CI: 1.5–6.6, p = 0.0033) were independent risk factors for LDL cholesterol levels higher or equal to 160 mg/dL. However, when we corrected the p-values with Bonferroni correction, only APOB rs693 was at higher risk of LDL > 160 mg/dL.
Table 3 Univariate logistic regression analysis for LDL cholesterol reduction after atorvastatin treatment.
3.2. Atorvastatin response
4. Discussion
Atorvastatin effectively reduced all lipid parameters (Table 1) measured. Association studies between atorvastatin reductions in lipid levels and polymorphisms in the genes analyzed revealed that most of the associations found were related to apoAI increase and ApoB reduction (Tables S2 and S3). In considering pharmacodynamics genes, polymorphisms in HMGCR, LDLR and LIPC genes were associated with apoAI/apoB ratio or ApoAI and HDL cholesterol increases (Table S2). Association studies between polymorphism in genes related to atherosclerosis and atorvastatin response revealed that three inflammatory markers (IL10, TLR4 and TNF) were significantly associated to the reduction in ApoB and increase in HDL cholesterol (Tables S3). These associations found could be confounded with other variables present in the studied population such as family history of CAD, smoking, physical activity, age, and others as described in Table 1. To better evaluate the influence of the many SNPs analyzed on atorvastatin response we performed a univariate logistic regression and a multivariate using stepwise criterion. We have used LDL cholesterol reduction to evaluate atorvastatin response. A cut-off of 36% of LDL cholesterol reduction was used [17]. Results from the univariate logistic regression analysis showed that lower reductions of LDL cholesterol could be predicted by no physical activity practice and SNPs in genes encoding the interleukins 6 (IL-6) and 10 (IL-10) Insulin Induced gene 2 (INSIG2). On the other hand, polymorphisms in hepatic lipase (LIPC), and interleukin 1β (IL-1β) were protection factors for a better atorvastatin response (Table 3). Next, we performed a multivariate logistic regression using stepwise criteria for the selection of the variables for LDL cholesterol reduction (reference was set as LDL-C reduction ≥36%) (Table 4). After this analysis, an inferior atorvastatin response could be predicted by physical inactivity (O.R.=2.92 [1.37–6.20]) and the common allele (C) for rs1800588 of LIPC gene was associated with a better response. The C allele showed a dominant trait in which the presence of two alleles doubles the risk for better response to atorvastatin (Table 4).
The current study reports one variant not previously associated to dyslipidemia, CD36 rs1984112 gene variant and that atorvastatin response can be predicted by LIPC − 514C> T polymorphism. Genetic predisposition to dyslipidemia has been accounted for half of the lipid profile variations. Specifically to LDL cholesterol, familial hypercholesterolemia, characterized by isolated elevation of LDL cholesterol and premature coronary heart disease, is associated with mutations in three major genes: LDLR, APOB and PCSK9. Our population was mainly constituted by individuals with primary hypercholesterolemia (isolated increase of LDL cholesterol), so it is feasible that the variant APOB T allele (XbaI, rs693) was associated to an ~ 2-fold risk for dyslipidemia than non-carriers. Heterozygous for this SNP has two-fold risk to have dyslipidemia, and the presence of this allele in homozygosis increases the risk to 4 times, this is consistent with a dominant-model of inheritance. In a recent genome-wide association study (GWAS) for LDL cholesterol, a meta-analysis from seven populations to determine the effect of each SNP on variations in circulating LDL cholesterol concentrations revealed that from the top 14 SNPs associated to this variable, 10 were from APOB loci [18]. APOB rs693 was in the fourteenth position and C allele was inversely correlated to LDL cholesterol changes. Polymorphisms on specific receptors for oxidized LDL such as CD36 have been related to have a protector effect on the risk for dyslipidemia [19]. Love-Gregory et al. [20] found that for the rs3211938 polymorphism, which results in CD36 deficiency, homozygous for the variant allele was in lower risk to have metabolic syndrome (OR: 0.61, 95%CI: 0.46–0.82, p = 0.0012). In our study, we have studied one intronic SNP (rs1527483) and two in the promoter (rs1761667 and rs1984112). We found that heterozygous for rs1984112 (AG genotype) was related to dyslipidemia. Different from others studies in the literature, our study do not focus only on the association between LDL cholesterol variability and polymorphisms. Our aim was to associate the frequency of SNPs with the incidence of dyslipidemia as well. We point out that individuals not related were investigated in this study, while the majority of the studies are based on transmission disequilibrium test (TDT). A possible limitation of this investigation is the number of individuals studied. Because many of the SNPs analyzed have a low frequency in the population, a higher number of individuals would be necessary to find significant positive associations. On the other hand, we did achieve to replicate the associations previously described in the literature, such as APOB rs693, which may validate our sample of dyslipidemics. Another aim of our study was to investigate the effect of SNPs related to atorvastatin PK/PD that could genetically predict the response to
Table 2 Significant associations between gene polymorphisms and dyslipidemia. Gene
SNP
Change
Majora
Odds ratio
CI95
pb
APOB CD36
rs693 rs1984112
C/T A/G
T AGc
2.20 3.55
1.48–3.20 1.88–6.70
0.0001 0.0002
CI95: confidence interval [5%–95%]; SNP: single nucleotide polymorphism. a The allele or genotype positively associated with dyslipidemia. b Corrected by Bonferroni's test. c Associations between genotypes using a co-dominant model (AA/AB/BB).
Variable
Category
Odds ratioa
Physical activity
Yes No TT CC GG CG GG CG TT CC CC AA TT CT
Ref. 2.35 Ref. 0.27 Ref. 0.35 Ref. 3.86 Ref. 4.55 Ref. 0.22 Ref. 3.14
LIPC rs1800588 (C/T) IL6 rs1800795 (G/C) IL6 rs1800796 (G/C) IL10 rs1800871 (C/T) IL1B rs1800872 (C/A) INSIG2 rs9308762 (T/C)
CI95
p
1.20–4.60
0.0130
0.08–0.90
0.0324
0.17–0.72
0.0043
1.25–11.93
0.0190
1.06–19.51
0.0414
0.05–0.94
0.0410
1.30–7.59
0.0111
CI95: confidence interval [5%–95%]. Ref.=reference variable, coded as zero. In parenthesis the substitutions are indicated. a Risk ratio for LDL-C reduction lower than 36%.
A.C. Rodrigues et al. / Clinica Chimica Acta 417 (2013) 8–11 Table 4 Multivariate logistic regression analysis for LDL cholesterol reduction after atorvastatin treatment. Variable
Category
Odds ratioa
Physical activity
Yes No TT CT CC
Ref. 2.92 Ref. 0.35 0.21
LIPC rs1800588 (C/T)
CI95
p
1.37–6.20
0.005
0.10–1.21 0.06–0.73
0.097 0.014
11
recipients of fellowships from the FAPESP, Sao Paulo, Brazil. M.H. Hirata and R.D.C. Hirata are recipients of fellowships from CNPq, Brasilia, Brazil.
Appendix A. Supplementary data
CI95: confidence interval [5%–95%]. Ref.: reference variable, coded as zero. In parenthesis the substitution is indicated. a Risk ratio for LDL-C reduction lower than 36%.
this drug. Despite some polymorphisms were significantly associated (Tables S3 and S4) to atorvastatin response, after a multivariate regression correcting for other predisposing factors, only the SNP −514C>T at LIPC was an independent predictor of atorvastatin response. Hepatic lipase (HL) promotes the conversion of large triglyceriderich HDL2 to small dense HDL3 particles. In addition, hepatic lipase catalyzes the hydrolysis of triglycerides and phospholipids of the intermediate density lipoproteins (IDL) and large buoyant LDL to form the more atherogenic small, dense LDL particles. The presence of the C >T substitution at position − 514 in the promoter region of the LIPC reduces in 30% of its activity [21]. The C allele has been associated with higher HL activity, more atherogenic LDL particles, and lower levels of antiatherogenic HDL lipoproteins [22]. The possible effect of LIPC −514C>T variant on the lipid-lowering effect of atorvastatin and bezafibrate has been study, however it was conducted in patients with mixed dyslipidemia, which is very different from our population of hypercholesterolemics. Despite −514C allele was associated with greater decrease in triglycerides and greater increase of HDL cholesterol after bezafibrate therapy, no associations were found with atorvastatin response [23]. In accordance, in our study we did not find an effect of the polymorphisms on total and lipoprotein cholesterol, but we did find on ApoAI, which was not measured by the other study. Statin effect on HL has been assessed. Men with LIPC CC genotype had a decreased HL activity after lovastatin combined to colestipol or niacin therapy compared to T carriers [24]. In addition homozygous CC patients exhibited a greater increase in LDL buoyancy and HDL2 cholesterol with lipid lowering therapy than T carriers. This effect was associated with a better response to lipid lowering therapy for CC carriers. However in a double-blind, randomized, placebocontrolled trial for 30 weeks, despite the fact that atorvastatin (10 or 80 mg) has decreased HL activity, this effect was not influenced by LIPIC −514C>T SNP [25]. Our results confirm that CC carriers have a better clinical outcome with lipid-lowering therapy. 5. Conclusion The current study reinforces the current knowledge that carrying APOB rs693 is an independent risk factor for dyslipidemia and higher LDL cholesterol levels. Furthermore, we found that a variant of CD36 gene was associated with dyslipidemia as a risk (rs1984112). Finally, carriers of the LIPC −514CC genotype might experience a better response to atorvastatin and protection from cardiovascular events. Conflict of interest statement The authors have no conflict of interest. Acknowledgments This work was supported by grants from the FAPESP (2008/06667-9). A. C. Rodrigues, F. D. V. Genvigir, M. A. V. Willrich and Arazi, SS were
Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.cca.2012.11.028.
References [1] Grundy SM, Cleeman JI, Merz CN, et al. Implications of recent clinical trials for the National Cholesterol Education Program Adult Treatment Panel III guidelines. Circulation 2004;110:227–39. [2] Gotto Jr AM, Brinton EA. Assessing low levels of high-density lipoprotein cholesterol as a risk factor in coronary heart disease: a working group report and update. J Am Coll Cardiol 2003;43:717–24. [3] Bansal S, Buring JE, Rifai N, Mora S, Sacks FM, Ridker PM. Fasting compared with nonfasting triglycerides and risk of cardiovascular events in women. JAMA 2007;298: 309–16. [4] Nordestgaard BG, Benn M, Schnohr P, Tybjaerg-Hansen A. Nonfasting triglycerides and risk of myocardial infarction, ischemic heart disease, and death in men and women. J Am Med Assoc 2007;298:299–308. [5] Pilia G, Chen WM, Scuteri A, et al. Heritability of cardiovascular and personality traits in 6,148 Sardinians. PLoS Genet 2006;2:e132. [6] Pollin TI, Hsueh WC, Steinle NI, Snitker S, Shuldiner AR, Mitchell BD. A genome-wide scan of serum lipid levels in the old order Amish. Atherosclerosis 2004;173:89–96. [7] Breslow JL. Genetics of lipoprotein abnormalities associated with coronary artery disease susceptibility. Annu Rev Genet 2000;34:233–54. [8] Kathiresan S, Manning AK, Demissie S, et al. A genome-wide association study for blood lipid phenotypes in the Framingham Heart Study. BMC Med Genet 2007;8:S17. [9] Teslovich TM, Musunuru K, Smith AV, et al. Biological, clinical and population relevance of 95 loci for blood lipids. Nature 2010;466:707–13. [10] Benn M, Stene MC, Nordestgaard BG, Jensen GB, Steffensen R, Tybjaerg-Hansen A. Common and rare alleles in apolipoprotein B contribute to plasma levels of low-density lipoprotein cholesterol in the general population. J Clin Endocrinol Metab 2008;93: 1038–45. [11] Zhu H, Tucker HM, Grear KE, et al. A common polymorphism decreases lowdensity lipoprotein receptor exon 12 splicing efficiency and associates with increased cholesterol. Hum Mol Genet 2007;16:1765–72. [12] Rodrigues AC, Hirata MH, Hirata RD. The genetic determinants of atorvastatin response. Curr Opin Mol Ther 2007;9:545–53. [13] Mangravite LM, Wilke RA, Zhang J, Krauss RM. Pharmacogenomics of statin response. Curr Opin Mol Ther 2008;10:555–61. [14] Maggo SD, Kennedy MA, Clark DW. Clinical implications of pharmacogenetic variation on the effects of statins. Drug Saf 2011;34:1–19. [15] Rodrigues AC, Perin PMS, Purim SG, et al. Pharmacogenetics of OATP transporters reveals that SLCO1B1 c.388A>G variant is determinant of increased atorvastatin response. Int J Mol Sci 2011;12:5815–27. [16] Morcillo-Suarez C, Alegre J, Sangros R, et al. SNP analysis to results (SNPator): a web-based environment oriented to statistical genomics analyses upon SNP data. Bioinformatics 2008;24:2790–1. [17] Edwards JE, Moore RA. Statins in hypercholesterolaemia: a dose-specific meta-analysis of lipid changes in randomised, double blind trials. BMC Fam Pract 2003;4:18. [18] Sandhu MS, Waterworth DM, Debenham SL, et al. LDL-cholesterol concentrations: a genome-wide association study. Lancet 2008;371:483–91. [19] Leprêtre F, Cheyssac C, Amouyel P, Froguel P, Helbecque N. A promoter polymorphism in CD36 is associated with an atherogenic lipid profile in a French general population. Atherosclerosis 2004;173:375–7. [20] Love-Gregory L, Sherva R, Sun L, et al. Variants in the CD36 gene associate with the metabolic syndrome and high-density lipoprotein cholesterol. Hum Mol Genet 2008;17:1695–704. [21] Deeb SS, Peng R. The C-514T polymorphism in the human hepatic lipase gene promoter diminishes its activity. J Lipid Res 2000;41:155–8. [22] Zambon A, Deeb SS, Hokanson JE, Brown BG, Brunzell JD. Common variants in the promoter of the hepatic lipase gene are associated with lower levels of hepatic lipase activity, buoyant LDL, and higher HDL2 cholesterol. Arterioscler Thromb Vasc Biol 1998;18:1723–9. [23] Cenarro A, Artieda M, Gonzalvo C, et al. Genetic variation in the hepatic lipase gene is associated with combined hyperlipidemia, plasma lipid concentrations, and lipid-lowering drug response. Am Heart J 2005;150:1154–62. [24] Zambon A, Deeb SS, Brown BG, et al. Common hepatic lipase gene promoter variant determines clinical response to intensive lipid-lowering treatment. Circulation 2001;103:792–8. [25] Berk-Planken II, Hoogerbrugge N, Stolk RP, Bootsma AH, Jansen H, DALI. Study Group. Atorvastatin dose-dependently decreases hepatic lipase activity in type 2 diabetes: effect of sex and the LIPC promoter variant. Diabetes Care 2003;26: 427–32.
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Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Genetic variants in genes related to lipid metabolism and atherosclerosis, dyslipidemia and atorvastatin response A.C. Rodrigues a,⁎, 1, B. Sobrino b, F.D.V. Genvigir a, M.A.V. Willrich a, S.S. Arazi a, E.L. Dorea c, M.M.S. Bernik c, M. Bertolami d, A.A. Faludi d, M.J. Brion b, e, A. Carracedo b, M.H. Hirata a, R.D.C. Hirata a a
Faculdade de Ciências Farmacêuticas, USP, São Paulo, SP, Brazil Grupo de Medicina Xenomica-USC, CIBERER, Fundacion Publica Galega de Medicina Xenómica, Santiago de Compostela, Galícia, Spain Hospital Universitário, USP, São Paulo, SP, Brazil d Instituto Dante Pazzanese de Cardiologia, São Paulo, SP, Brazil e Complexo Hospitalario de Santiago de Compostela, Galícia, Spain b c
a r t i c l e
i n f o
Article history: Received 9 September 2012 Received in revised form 27 November 2012 Accepted 27 November 2012 Available online 13 December 2012 Keywords: Association studies Dyslipidemia SNPs Statins pharmacogenomics
a b s t r a c t Objective: Using candidate gene approach, we have investigated the effect of single nucleotide polymorphism (SNP) in genes related to lipid metabolism and atherosclerosis on dyslipidemia and atorvastatin response. Methods: The study included 157 patients treated with atorvastatin and 145 controls. Genomic DNA was isolated and genotyped using SNPlex technology. Results: Allele and genotype disease association test revealed that APOB rs693 (OR: 2.2 [1.5–3.2], p=0.0001) and CD36 rs1984112 (OR: 3.7 [1.9–7.0], p=0.0002) SNPs were independent risk factors for hypercholesterolemia. Only APOB rs693 T variant allele was associated with increased LDL cholesterol levels (>160 mg/dL). After atorvastatin treatment (10 mg/day/4 weeks), LIPC −514T allele was positively associated with LDL cholesterol reduction. Conclusion: The current study reinforces the current knowledge that carrying APOB rs693 is an independent risk factor for dyslipidemia and higher LDL levels. Furthermore, we found that a variant of CD36 was associated with dyslipidemia as a risk (rs1984112) factor. Finally, atorvastatin response could be predicted by LIPC −514C>T SNP and physical activity. In conclusion, our data evidences the contribution of genetic markers and their interaction with environmental factor in the variability of statin response. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Atherosclerosis is the major cause of cardiovascular disease and there is consistent and convincing evidence that supports an association between dyslipidemia and cardiovascular disease incidence worldwide. Whereas levels of low density lipoprotein (LDL) cholesterol are directly associated with an increased risk for coronary arterial disease (CAD) [1], higher serum levels of high density lipoprotein (HDL) cholesterol are a protection factor [2]. High serum triglycerides (TG) levels may be an additional factor for cardiovascular diseases [3,4], however there is much controversy. Risk factors for dyslipidemia include sedentary lifestyle, high fat diet (cholesterol and monosatured fatty acids enriched) and smoking [1].
⁎ Corresponding author at: Faculty of Pharmaceutical Sciences, Department of Clinical and Toxicological Analyses, Av. Prof. Lineu Prestes, 580, bl17, Sao Paulo, SP 05508-000, Brazil. Tel.: +55 11 30913660; fax: +55 11 3813 2197. E-mail address: [email protected] (A.C. Rodrigues). 1 Present/permanent address: Institute of Biomedical Sciences. Department of Physiology and Biophysics. Av. Prof. Lineu Prestes, 1524, 05508-000, Brazil. 0009-8981/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cca.2012.11.028
Studies with families in many populations suggest, though, that half of the lipid profile variations are genetically determinate [5,6]. Besides this, LDL cholesterol, HDL cholesterol and TG levels are influenced by the genetic constitution of each individual [7–9]. Specifically to LDL cholesterol, variants in LDL receptor (LDLR), apolipoprotein B (APOB) genes have been associated with its plasma levels [10,11]. Although several genes and genetic variants have been associated with individual variation in lipid concentrations, additional variants influencing these traits remain to be identified. The first intervention in dyslipidemia is to introduce to the patients a low-fat diet, however many of the patients do not respond and are initiated on statin therapy. Statins are HMG-CoA reductase inhibitors used as first choice in the treatment of hypercholesterolemia. Variation in LDL cholesterol response has been associated with many genes involved in lipid metabolism and in statin pharmacokinetics (for reviews [12–14]), however no study has found a conclusive genetic marker, and more studies are necessary to elucidate this. Using candidate gene approach, we have performed a case-control study on single nucleotide polymorphisms (SNPs) in genes related to lipid metabolism and atherosclerosis and with statin pharmacokinetics and pharmacodynamics in a group of unrelated dyslipidemic individuals treated with atorvastatin for four weeks.
A.C. Rodrigues et al. / Clinica Chimica Acta 417 (2013) 8–11
2. Materials and methods 2.1. Patients and research design We have selected 302 individuals (145 normolipidemic and 157 dyslipidemic) from the University Hospital from University of Sao Paulo and Institute Dante Pazzanese of Cardiology. Clinical data for the individuals selected are presented in Table 1. The study protocol was approved by the Ethics Committee of these institutions as well as the Committee of the School of Pharmaceutical Sciences (University of Sao Paulo). Information on age, body mass index (BMI), gender, hypertension, obesity, menopause status, cigarette smoking, physical activity, alcohol consumption and family history of CAD was recorded. Dyslipidemic patients were characterized as total cholesterol ≥200 mg/dL, LDL cholesterol ≥160 mg/dL with or without TG ≥150 mg/dL and low levels of HDL cholesterol (men: b 40 mg/dL and women >50 mg/dL). Individuals with diabetes mellitus, hypo- or hyperthyroidism, triglycerides higher than 400 mg/dL, renal or hepatic disease, subjects under treatment with oral contraceptive or hormone replacement and lipid-lowering drugs or pregnant women were excluded from the study. Dyslipidemic subjects even after a low cholesterol diet during 4 weeks were started on atorvastatin therapy, 10 mg orally once daily for 4 weeks. At the end of the protocol, the patients had a last appointment with the doctor and response to atorvastatin as well as any possible Table 1 Clinical and biochemical data for the studied individuals. Variables Alcohol consumption >7 Doses/week b7 Doses/week Smokingc Hypertensiond Familiar history of CAD Menopause Physical activity Ethnicse White Black and intermediates Gender (female) Age—years (mean ± SD) BMI (kg/m2) Total cholesterol (mg/dL) LDL-C (mg/dL) LDL-C post-10 (mg/dL) HDL-C (mg/dL) HDL-C post-10 (mg/dL) TG (mg/dL) TG post-10 (mg/dL) ApoAI (g/L) ApoAI post-10 (g/L) ApoB (g/L) ApoB post-10 (g/L) ApoAI/ApoB ApoAI/ApoB ratio post-10 CK (U/L) CK post-10 (U/L) ALT (U/L) ALT post-10 (U/L)
Normolipidemics (145)
Dyslipidemics (157)
p
1.6% (4/144f) 30.50% (48/144f) 19.4% (24) 33.1% (47/142f) 44.8% (64/143f) 27.42% (23/90f) 45.8% (68)
1.91% (4) 28.02% (40) 15.29% (22) 56.9% (83/146f) 54.8% (80/146) 84.76% (78) 47.74% (68/147f)
0.279a 0.757a 0.794a b0.0001a 0.112a b0.0001a 0.962a 0.008a
54.48% (79) 45.52% (66) 68.3% (99) 47 ± 7 25.7 ± 4.1 (141f) 175 ± 18 98 ± 18 – 62 ± 11 – 77 ± 24 – 147 ± 27 – 86 ± 21 – 1.76 ± 0.68 – 140 ± 116 – 22 ± 15 –
72.22% (104/144f) 27.78% (44/144f) 67.1% (100) 57 ± 11 27.6 ± 4.2 (146f) 282 ± 37 192 ± 34 117 ± 27 57 ± 14 55 ± 13 160 ± 65 131 ± 53 133 ± 26 138 ± 28 143 ± 21 100 ± 21 1.11 ± 0.32 1.10 ± 0.46 98 ± 78 100 ± 84 21 ± 9 25 ± 15
0.930a b0.0001b b0.0001b b0.0001b b0.0001b – b0.0001b – b0.0001b – 0.0254b – b0.0001b – b0.0001b – 0.0002b – 0.4412b –
Post-10: serum concentrations measured after atorvastatin treatment (10 mg/day/4 weeks). CAD: coronary artery disease; BMI: body mass index; LDL-C: low density lipoprotein cholesterol: HDL-C: high-density lipoprotein cholesterol; TG: triglycerides; ApoAI: apolipoprotein AI; ApoB: apolipoprotein B; CK: creatine kinase; ALT: alanine tranasaminase. a p-Value as indicated by Chi-square test. b p-Value as indicated by “t”test. c Current cigarette smoking was defined as daily intake of one or more cigarettes. d Individuals with systolic/diastolic blood pressure higher than 140/90 mmHg were considered hypertensive. e Each individual declared their ethnic group during the interview, as recommended by Brazilian census. f Indicates that some patients did not inform or the information was missing in the medical promtuary.
9
adverse reactions were evaluated. The dose of 10 mg atorvastatin was chosen because the patients had moderate elevations of LDL cholesterol, and LDL cholesterol goal could be achieved with low doses for these patients. In addition, the NCEP recommends checking the response to drug therapy in about 6 weeks. Response to atorvastatin was evaluated by the reduction of LDL cholesterol after the treatment, and adverse effects were monitored by measuring creatine kinase (CK) and alanine aminotransferase (ALT) enzymes. 2.2. Biochemical profile and genes variants genotyping Blood samples for biochemical profile (lipids, CK, and ALT) measurements and genomic DNA extraction were collected after an overnight fast, one day before and 4 weeks after atorvastatin treatment. All patients followed exactly the same study protocol. Laboratory methods for biochemical parameters are described elsewhere [15]. Biochemical data are shown in Table 1. Genomic DNA was extracted from EDTA-anticoagulated blood by a salting-out procedure. The SNPs in candidate genes were selected using the online tool available at Life Technologies website, SNPlex™ Assay Design, and the pools of compatible probes, normally between 24 and 28, were synthesized by Life Technologies. The genes included in multiplex assays are described in Supplemental Table 1. Genes involved in pharmacodynamics and pharmacokinetics of statins and with pleiotropic effects of statins were included. SNPs were genotyped by SNPlex™ (Life Technologies), which consists of four steps: oligonucleotide ligation assay (OLA), PCR amplification, ZipChute™ hybridization and capillary electrophoresis for the detection of ZipChute™ probes. Genotype calling was performed on GeneMapper v.4 (Life Technologies). All genotyping procedures were realized at Centro Nacional do Genotipado (CeGen), Santiago de Compostela, Galícia, Spain. 2.3. Statistical analyses Hardy–Weinberg equilibrium (HWE) was tested for each SNP. Qualitative and quantitative variables were analyzed by Likelihood ratio test and ANOVA, respectively. Allelic and genotypic association tests were corrected by odds ratio Haldane correction. Allele and genotype disease association tests were performed using different model of inheritance (dominant, co-dominant, and recessive). To exclude the possibility of other risk factors to influence our results, we performed a logistic regression association test adjusting by age, hypertension, and history of coronary artery disease, sex and body mass index (BMI). Multiple testing was corrected by Bonferroni's test. Multiple stepwise logistic regression analysis was performed to identify independent predictors of atorvastatin response measured by percentage of LDL cholesterol reduction. A p-value ≤0.05 was considered statistically significant. Analyses were performed by using SNPator [16] and SPSS17 (SPSS Inc., Chicago/IL EUA). 3. Results 3.1. Dyslipidemia We performed a candidate gene association study for dyslipidemia. All the SNPs were in Hardy–Weinberg equilibrium in the control group (normolipidemics), except for two polymorphisms in MBTPS2 gene. These SNPs were excluded from the study. Allele disease association test revealed that carriers of the common C allele for CD36 rs1527483 SNP had 4 times more risk to have dyslipidemia than non-carriers. On the other hand, carriers of APOB T allele (rs693), ATP-binding Cassette transporter G1 (ABCG1) T allele (rs9975740) and CD36 G allele (rs1984112) had 1.7 to 2.2 higher risk to have dyslipidemia than non-carriers (data not shown). After we correct the multiple testing with Bonferroni's test, only APOB rs693T carriers were in risk of dyslipidemia (Table 2).
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A.C. Rodrigues et al. / Clinica Chimica Acta 417 (2013) 8–11
Genotype disease association test using a dominant (AA + AB versus BB) or a recessive (AA versus AB + BB) or a co-dominant model (AA/AB/BB) of inheritance was performed. For APOB rs693, the presence of the variant allele T is a risk factor as evidenced before by allele association test. The heterozygous for this SNP has two-fold risk to have dyslipidemia, and the presence of this allele in homozygosis increases the risk to 4 times. On the other hand, for CD36 rs1984112 polymorphism, carriers of AG genotype have increased risk of dyslipidemia (O.R. 3.55 [1.88–6.70, p=0.0002]) (Table 2). Finally, we performed an association test between the SNPs and the levels of LDL cholesterol. A logistic regression association test (adjusted by sex, BMI, history of DAC and hypertension) showed that RLDL rs688 T allele (OR: 1.7, 95CI: 1.3–2.3, p = 0.018), APOB rs693 T allele (OR: 1.7, 95CI: 1.3–2.3, p = 0.002), and CD36 rs1527483 C allele (OR: 3.1, 95CI: 1.5–6.6, p = 0.0033) were independent risk factors for LDL cholesterol levels higher or equal to 160 mg/dL. However, when we corrected the p-values with Bonferroni correction, only APOB rs693 was at higher risk of LDL > 160 mg/dL.
Table 3 Univariate logistic regression analysis for LDL cholesterol reduction after atorvastatin treatment.
3.2. Atorvastatin response
4. Discussion
Atorvastatin effectively reduced all lipid parameters (Table 1) measured. Association studies between atorvastatin reductions in lipid levels and polymorphisms in the genes analyzed revealed that most of the associations found were related to apoAI increase and ApoB reduction (Tables S2 and S3). In considering pharmacodynamics genes, polymorphisms in HMGCR, LDLR and LIPC genes were associated with apoAI/apoB ratio or ApoAI and HDL cholesterol increases (Table S2). Association studies between polymorphism in genes related to atherosclerosis and atorvastatin response revealed that three inflammatory markers (IL10, TLR4 and TNF) were significantly associated to the reduction in ApoB and increase in HDL cholesterol (Tables S3). These associations found could be confounded with other variables present in the studied population such as family history of CAD, smoking, physical activity, age, and others as described in Table 1. To better evaluate the influence of the many SNPs analyzed on atorvastatin response we performed a univariate logistic regression and a multivariate using stepwise criterion. We have used LDL cholesterol reduction to evaluate atorvastatin response. A cut-off of 36% of LDL cholesterol reduction was used [17]. Results from the univariate logistic regression analysis showed that lower reductions of LDL cholesterol could be predicted by no physical activity practice and SNPs in genes encoding the interleukins 6 (IL-6) and 10 (IL-10) Insulin Induced gene 2 (INSIG2). On the other hand, polymorphisms in hepatic lipase (LIPC), and interleukin 1β (IL-1β) were protection factors for a better atorvastatin response (Table 3). Next, we performed a multivariate logistic regression using stepwise criteria for the selection of the variables for LDL cholesterol reduction (reference was set as LDL-C reduction ≥36%) (Table 4). After this analysis, an inferior atorvastatin response could be predicted by physical inactivity (O.R.=2.92 [1.37–6.20]) and the common allele (C) for rs1800588 of LIPC gene was associated with a better response. The C allele showed a dominant trait in which the presence of two alleles doubles the risk for better response to atorvastatin (Table 4).
The current study reports one variant not previously associated to dyslipidemia, CD36 rs1984112 gene variant and that atorvastatin response can be predicted by LIPC − 514C> T polymorphism. Genetic predisposition to dyslipidemia has been accounted for half of the lipid profile variations. Specifically to LDL cholesterol, familial hypercholesterolemia, characterized by isolated elevation of LDL cholesterol and premature coronary heart disease, is associated with mutations in three major genes: LDLR, APOB and PCSK9. Our population was mainly constituted by individuals with primary hypercholesterolemia (isolated increase of LDL cholesterol), so it is feasible that the variant APOB T allele (XbaI, rs693) was associated to an ~ 2-fold risk for dyslipidemia than non-carriers. Heterozygous for this SNP has two-fold risk to have dyslipidemia, and the presence of this allele in homozygosis increases the risk to 4 times, this is consistent with a dominant-model of inheritance. In a recent genome-wide association study (GWAS) for LDL cholesterol, a meta-analysis from seven populations to determine the effect of each SNP on variations in circulating LDL cholesterol concentrations revealed that from the top 14 SNPs associated to this variable, 10 were from APOB loci [18]. APOB rs693 was in the fourteenth position and C allele was inversely correlated to LDL cholesterol changes. Polymorphisms on specific receptors for oxidized LDL such as CD36 have been related to have a protector effect on the risk for dyslipidemia [19]. Love-Gregory et al. [20] found that for the rs3211938 polymorphism, which results in CD36 deficiency, homozygous for the variant allele was in lower risk to have metabolic syndrome (OR: 0.61, 95%CI: 0.46–0.82, p = 0.0012). In our study, we have studied one intronic SNP (rs1527483) and two in the promoter (rs1761667 and rs1984112). We found that heterozygous for rs1984112 (AG genotype) was related to dyslipidemia. Different from others studies in the literature, our study do not focus only on the association between LDL cholesterol variability and polymorphisms. Our aim was to associate the frequency of SNPs with the incidence of dyslipidemia as well. We point out that individuals not related were investigated in this study, while the majority of the studies are based on transmission disequilibrium test (TDT). A possible limitation of this investigation is the number of individuals studied. Because many of the SNPs analyzed have a low frequency in the population, a higher number of individuals would be necessary to find significant positive associations. On the other hand, we did achieve to replicate the associations previously described in the literature, such as APOB rs693, which may validate our sample of dyslipidemics. Another aim of our study was to investigate the effect of SNPs related to atorvastatin PK/PD that could genetically predict the response to
Table 2 Significant associations between gene polymorphisms and dyslipidemia. Gene
SNP
Change
Majora
Odds ratio
CI95
pb
APOB CD36
rs693 rs1984112
C/T A/G
T AGc
2.20 3.55
1.48–3.20 1.88–6.70
0.0001 0.0002
CI95: confidence interval [5%–95%]; SNP: single nucleotide polymorphism. a The allele or genotype positively associated with dyslipidemia. b Corrected by Bonferroni's test. c Associations between genotypes using a co-dominant model (AA/AB/BB).
Variable
Category
Odds ratioa
Physical activity
Yes No TT CC GG CG GG CG TT CC CC AA TT CT
Ref. 2.35 Ref. 0.27 Ref. 0.35 Ref. 3.86 Ref. 4.55 Ref. 0.22 Ref. 3.14
LIPC rs1800588 (C/T) IL6 rs1800795 (G/C) IL6 rs1800796 (G/C) IL10 rs1800871 (C/T) IL1B rs1800872 (C/A) INSIG2 rs9308762 (T/C)
CI95
p
1.20–4.60
0.0130
0.08–0.90
0.0324
0.17–0.72
0.0043
1.25–11.93
0.0190
1.06–19.51
0.0414
0.05–0.94
0.0410
1.30–7.59
0.0111
CI95: confidence interval [5%–95%]. Ref.=reference variable, coded as zero. In parenthesis the substitutions are indicated. a Risk ratio for LDL-C reduction lower than 36%.
A.C. Rodrigues et al. / Clinica Chimica Acta 417 (2013) 8–11 Table 4 Multivariate logistic regression analysis for LDL cholesterol reduction after atorvastatin treatment. Variable
Category
Odds ratioa
Physical activity
Yes No TT CT CC
Ref. 2.92 Ref. 0.35 0.21
LIPC rs1800588 (C/T)
CI95
p
1.37–6.20
0.005
0.10–1.21 0.06–0.73
0.097 0.014
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recipients of fellowships from the FAPESP, Sao Paulo, Brazil. M.H. Hirata and R.D.C. Hirata are recipients of fellowships from CNPq, Brasilia, Brazil.
Appendix A. Supplementary data
CI95: confidence interval [5%–95%]. Ref.: reference variable, coded as zero. In parenthesis the substitution is indicated. a Risk ratio for LDL-C reduction lower than 36%.
this drug. Despite some polymorphisms were significantly associated (Tables S3 and S4) to atorvastatin response, after a multivariate regression correcting for other predisposing factors, only the SNP −514C>T at LIPC was an independent predictor of atorvastatin response. Hepatic lipase (HL) promotes the conversion of large triglyceriderich HDL2 to small dense HDL3 particles. In addition, hepatic lipase catalyzes the hydrolysis of triglycerides and phospholipids of the intermediate density lipoproteins (IDL) and large buoyant LDL to form the more atherogenic small, dense LDL particles. The presence of the C >T substitution at position − 514 in the promoter region of the LIPC reduces in 30% of its activity [21]. The C allele has been associated with higher HL activity, more atherogenic LDL particles, and lower levels of antiatherogenic HDL lipoproteins [22]. The possible effect of LIPC −514C>T variant on the lipid-lowering effect of atorvastatin and bezafibrate has been study, however it was conducted in patients with mixed dyslipidemia, which is very different from our population of hypercholesterolemics. Despite −514C allele was associated with greater decrease in triglycerides and greater increase of HDL cholesterol after bezafibrate therapy, no associations were found with atorvastatin response [23]. In accordance, in our study we did not find an effect of the polymorphisms on total and lipoprotein cholesterol, but we did find on ApoAI, which was not measured by the other study. Statin effect on HL has been assessed. Men with LIPC CC genotype had a decreased HL activity after lovastatin combined to colestipol or niacin therapy compared to T carriers [24]. In addition homozygous CC patients exhibited a greater increase in LDL buoyancy and HDL2 cholesterol with lipid lowering therapy than T carriers. This effect was associated with a better response to lipid lowering therapy for CC carriers. However in a double-blind, randomized, placebocontrolled trial for 30 weeks, despite the fact that atorvastatin (10 or 80 mg) has decreased HL activity, this effect was not influenced by LIPIC −514C>T SNP [25]. Our results confirm that CC carriers have a better clinical outcome with lipid-lowering therapy. 5. Conclusion The current study reinforces the current knowledge that carrying APOB rs693 is an independent risk factor for dyslipidemia and higher LDL cholesterol levels. Furthermore, we found that a variant of CD36 gene was associated with dyslipidemia as a risk (rs1984112). Finally, carriers of the LIPC −514CC genotype might experience a better response to atorvastatin and protection from cardiovascular events. Conflict of interest statement The authors have no conflict of interest. Acknowledgments This work was supported by grants from the FAPESP (2008/06667-9). A. C. Rodrigues, F. D. V. Genvigir, M. A. V. Willrich and Arazi, SS were
Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.cca.2012.11.028.
References [1] Grundy SM, Cleeman JI, Merz CN, et al. Implications of recent clinical trials for the National Cholesterol Education Program Adult Treatment Panel III guidelines. Circulation 2004;110:227–39. [2] Gotto Jr AM, Brinton EA. Assessing low levels of high-density lipoprotein cholesterol as a risk factor in coronary heart disease: a working group report and update. J Am Coll Cardiol 2003;43:717–24. [3] Bansal S, Buring JE, Rifai N, Mora S, Sacks FM, Ridker PM. Fasting compared with nonfasting triglycerides and risk of cardiovascular events in women. JAMA 2007;298: 309–16. [4] Nordestgaard BG, Benn M, Schnohr P, Tybjaerg-Hansen A. Nonfasting triglycerides and risk of myocardial infarction, ischemic heart disease, and death in men and women. J Am Med Assoc 2007;298:299–308. [5] Pilia G, Chen WM, Scuteri A, et al. Heritability of cardiovascular and personality traits in 6,148 Sardinians. PLoS Genet 2006;2:e132. [6] Pollin TI, Hsueh WC, Steinle NI, Snitker S, Shuldiner AR, Mitchell BD. A genome-wide scan of serum lipid levels in the old order Amish. Atherosclerosis 2004;173:89–96. [7] Breslow JL. Genetics of lipoprotein abnormalities associated with coronary artery disease susceptibility. Annu Rev Genet 2000;34:233–54. [8] Kathiresan S, Manning AK, Demissie S, et al. A genome-wide association study for blood lipid phenotypes in the Framingham Heart Study. BMC Med Genet 2007;8:S17. [9] Teslovich TM, Musunuru K, Smith AV, et al. Biological, clinical and population relevance of 95 loci for blood lipids. Nature 2010;466:707–13. [10] Benn M, Stene MC, Nordestgaard BG, Jensen GB, Steffensen R, Tybjaerg-Hansen A. Common and rare alleles in apolipoprotein B contribute to plasma levels of low-density lipoprotein cholesterol in the general population. J Clin Endocrinol Metab 2008;93: 1038–45. [11] Zhu H, Tucker HM, Grear KE, et al. A common polymorphism decreases lowdensity lipoprotein receptor exon 12 splicing efficiency and associates with increased cholesterol. Hum Mol Genet 2007;16:1765–72. [12] Rodrigues AC, Hirata MH, Hirata RD. The genetic determinants of atorvastatin response. Curr Opin Mol Ther 2007;9:545–53. [13] Mangravite LM, Wilke RA, Zhang J, Krauss RM. Pharmacogenomics of statin response. Curr Opin Mol Ther 2008;10:555–61. [14] Maggo SD, Kennedy MA, Clark DW. Clinical implications of pharmacogenetic variation on the effects of statins. Drug Saf 2011;34:1–19. [15] Rodrigues AC, Perin PMS, Purim SG, et al. Pharmacogenetics of OATP transporters reveals that SLCO1B1 c.388A>G variant is determinant of increased atorvastatin response. Int J Mol Sci 2011;12:5815–27. [16] Morcillo-Suarez C, Alegre J, Sangros R, et al. SNP analysis to results (SNPator): a web-based environment oriented to statistical genomics analyses upon SNP data. Bioinformatics 2008;24:2790–1. [17] Edwards JE, Moore RA. Statins in hypercholesterolaemia: a dose-specific meta-analysis of lipid changes in randomised, double blind trials. BMC Fam Pract 2003;4:18. [18] Sandhu MS, Waterworth DM, Debenham SL, et al. LDL-cholesterol concentrations: a genome-wide association study. Lancet 2008;371:483–91. [19] Leprêtre F, Cheyssac C, Amouyel P, Froguel P, Helbecque N. A promoter polymorphism in CD36 is associated with an atherogenic lipid profile in a French general population. Atherosclerosis 2004;173:375–7. [20] Love-Gregory L, Sherva R, Sun L, et al. Variants in the CD36 gene associate with the metabolic syndrome and high-density lipoprotein cholesterol. Hum Mol Genet 2008;17:1695–704. [21] Deeb SS, Peng R. The C-514T polymorphism in the human hepatic lipase gene promoter diminishes its activity. J Lipid Res 2000;41:155–8. [22] Zambon A, Deeb SS, Hokanson JE, Brown BG, Brunzell JD. Common variants in the promoter of the hepatic lipase gene are associated with lower levels of hepatic lipase activity, buoyant LDL, and higher HDL2 cholesterol. Arterioscler Thromb Vasc Biol 1998;18:1723–9. [23] Cenarro A, Artieda M, Gonzalvo C, et al. Genetic variation in the hepatic lipase gene is associated with combined hyperlipidemia, plasma lipid concentrations, and lipid-lowering drug response. Am Heart J 2005;150:1154–62. [24] Zambon A, Deeb SS, Brown BG, et al. Common hepatic lipase gene promoter variant determines clinical response to intensive lipid-lowering treatment. Circulation 2001;103:792–8. [25] Berk-Planken II, Hoogerbrugge N, Stolk RP, Bootsma AH, Jansen H, DALI. Study Group. Atorvastatin dose-dependently decreases hepatic lipase activity in type 2 diabetes: effect of sex and the LIPC promoter variant. Diabetes Care 2003;26: 427–32.