Pharmacogenetics of OATP Transporters Reveals That SLCO1B1 c.388A>G Variant Is Determinant of Increased Atorvastatin Response.
ABSTRACT The relationship between variants in SLCO1B1 and SLCO2B1 genes and lipid-lowering response to atorvastatin was investigated.
One-hundred-thirty-six unrelated individuals with hypercholesterolemia were selected and treated with atorvastatin (10 mg/day/4 weeks). They were genotyped with a panel of ancestry informative markers for individual African component of ancestry (ACA) estimation by SNaPshot(®) and SLCO1B1 (c.388A>G, c.463C>A and c.521T>C) and SLCO2B1 (-71T>C) gene polymorphisms were identified by TaqMan(®) Real-time PCR.
Subjects carrying SLCO1B1 c.388GG genotype exhibited significantly high low-density lipoprotein (LDL) cholesterol reduction relative to c.388AA+c.388AG carriers (41 vs. 37%, p = 0.034). Haplotype analysis revealed that homozygous of SLCO1B1*15 (c.521C and c.388G) variant had similar response to statin relative to heterozygous and non-carriers. A multivariate logistic regression analysis confirmed that c.388GG genotype was associated with higher LDL cholesterol reduction in the study population (OR: 3.2, CI95%:1.3-8.0, p < 0.05).
SLCO1B1 c.388A>G polymorphism causes significant increase in atorvastatin response and may be an important marker for predicting efficacy of lipid-lowering therapy.
- [show abstract] [hide abstract]
ABSTRACT: Recent pharmacogenomic/pharmacogenetic studies have disclosed important roles of drug transporters in the pharmacokinetic/pharmacodynamic (PK/PD) profiles of some clinically relevant drugs. It has concurrently been explained that variations in the drug transporter genes are associated with not only inter-individual but also inter-ethnic differences in PK/PD profiles of these drugs. This review focuses on two uptake and two efflux transporters. Organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 are uptake transporters, specifically expressed in the liver, and considered important for drugs, particularly as their pharmacological target organ is the liver. Two ATP-binding cassette transporters, multi-drug resistance-associated protein 2 and breast cancer resistance protein, are efflux transporters, expressed in various human tissues, and considered particularly important for intestinal drug absorption and hepatic drug elimination. All 3-hydroxyl-3-methylglutaryl-CoA reductase inhibitors (statins) except fluvastatin are substrates for OATP1B1, but hepatobiliary (canalicular) efflux transporters differ among statins. In this review, we update the pharmacogenomic/pharmacogenetic properties of these transporters and their effects on PK/PD profiles of statins and other clinically relevant drugs. In addition, we describe a physiologically-based pharmacokinetic model for predicting the effects of changes in transporter activities on systemic and hepatic exposure to pravastatin.Expert Opinion on Drug Metabolism & Toxicology 06/2009; 5(7):703-29. · 2.94 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: This 6-week, double-blind clinical trial evaluated lipid parameter responses to different dosages of atorvastatin in patients with primary hypercholesterolemia. Atorvastatin is a new 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor under development. After completing an 8-week placebo-baseline dietary phase, 81 patients were randomly assigned to receive either placebo or 2.5, 5, 10, 20, 40, or 80 mg atorvastatin once daily for 6 weeks. Plasma LDL cholesterol reductions from baseline were dose related, with 25% to 61% reduction from the minimum dose to the maximum dose of 80 mg atorvastatin once a day. Plasma total cholesterol and apo B reductions were also dose related. Previously, reductions in LDL cholesterol of the magnitude observed in this study have been seen only with combination drug therapy. In this study, atorvastatin was well tolerated by hyperlipidemic patients, had an acceptable safety profile, and provided greater reduction in cholesterol than other previously reported HMG-CoA reductase inhibitors.Arteriosclerosis Thrombosis and Vascular Biology 06/1995; 15(5):678-82. · 6.34 Impact Factor
Article: Update on statins: 2003.Circulation 09/2004; 110(7):886-92. · 15.20 Impact Factor
Int. J. Mol. Sci. 2011, 12, 5815-5827; doi:10.3390/ijms12095815
International Journal of
Pharmacogenetics of OATP Transporters Reveals That
SLCO1B1 c.388A>G Variant Is Determinant of Increased
Alice C. Rodrigues 1,*, Paula M. S. Perin 1, Sheila G. Purim 2, Vivian N. Silbiger 1,
Fabiana D. V. Genvigir 1, Maria Alice V. Willrich 1, Simone S. Arazi 1, Andre D. Luchessi 1,
Mario H. Hirata 1, Marcia M. S. Bernik 3, Egidio L. Dorea 3, Carla Santos 4,5, Andre A. Faludi 6,
Marcelo C. Bertolami 6, Antonio Salas 5, Ana Freire 5, Maria V. Lareu 5, Christopher Phillips 5,
Liliana Porras-Hurtado 5, Manuel Fondevila 5, Angel Carracedo 5 and Rosario D. C. Hirata 1
1 Faculty of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo 05508-900, Brazil;
E-Mails: firstname.lastname@example.org (P.M.S.P.); email@example.com (V.N.S.);
firstname.lastname@example.org (F.D.V.G.); email@example.com (M.A.V.W.); firstname.lastname@example.org (S.S.A.);
email@example.com (A.D.L.); firstname.lastname@example.org (M.H.H.); email@example.com (R.D.C.H.)
2 Life Technologies, Sao Paulo 04311-000, Brazil; E-Mail: firstname.lastname@example.org
3 University Hospital, University of Sao Paulo, Sao Paulo 05508-000, Brazil;
E-Mails: email@example.com (M.M.S.B.); firstname.lastname@example.org (E.L.D.)
4 Department of Biology, University of Aveiro, Aveiro 3810-193, Portugal;
5 Forensic Genetics Unit, Institute of Legal Medicine, University of Santiago de Compostela, Galicia
15705, Spain; E-Mails: email@example.com (A.S.); firstname.lastname@example.org (A.F.);
email@example.com (M.V.L.); firstname.lastname@example.org (C.P.);
email@example.com (L.P.-H.); firstname.lastname@example.org (M.F.); email@example.com (A.C.)
6 Dante Pazzanese Institute, Sao Paulo 04012-909, Brazil; E-Mail: firstname.lastname@example.org (A.A.F.);
* Author to whom correspondence should be addressed; E-Mail: email@example.com;
Tel.: +55-11-3091-3660; Fax: +55-11-3813-2197.
Received: 29 July 2011; in revised form: 29 August 2011 / Accepted: 30 August 2011 /
Published: 9 September 2011
Abstract: Aims: The relationship between variants in SLCO1B1 and SLCO2B1 genes and
lipid-lowering response to atorvastatin was investigated. Material and Methods:
One-hundred-thirty-six unrelated individuals with hypercholesterolemia were selected and
Int. J. Mol. Sci. 2011, 12
treated with atorvastatin (10 mg/day/4 weeks). They were genotyped with a panel of
ancestry informative markers for individual African component of ancestry (ACA)
estimation by SNaPshot® and SLCO1B1 (c.388A>G, c.463C>A and c.521T>C) and
SLCO2B1 (−71T>C) gene polymorphisms were identified by TaqMan® Real-time PCR.
Results: Subjects carrying SLCO1B1 c.388GG genotype exhibited significantly high
low-density lipoprotein (LDL) cholesterol reduction relative to c.388AA+c.388AG carriers
(41 vs. 37%, p = 0.034). Haplotype analysis revealed that homozygous of SLCO1B1*15
(c.521C and c.388G) variant had similar response to statin relative to heterozygous and
non-carriers. A multivariate logistic regression analysis confirmed that c.388GG genotype
was associated with higher LDL cholesterol reduction in the study population (OR: 3.2,
CI95%:1.3–8.0, p < 0.05). Conclusion: SLCO1B1 c.388A>G polymorphism causes
significant increase in atorvastatin response and may be an important marker for predicting
efficacy of lipid-lowering therapy.
Keywords: OATP; atorvastatin; single nucleotide polymorphisms; pharmacogenetics
Organic anion transporting polypeptides (OATPs) are plasma membrane transport proteins that
mediate the active cellular influx of a variety of amphipathic compounds. OATP1B1, OATP2B1 and
OATP1B3 are expressed in the sinusoidal membrane of hepatocytes and transport a large number of
therapeutic drugs, such as statins . The uptake of statins by OATPs not only represents the first step
of hepatic drug elimination, but is also a delivery system to the liver as the target organ. Such transport
therefore potentially influences the efficacy of the therapy of this drug class, as differences in OATP
activity may result in variability of statins plasma levels and consequently variability in drug response.
Atorvastatin is a potent inhibitor of the 3-hydroxy-3-methlyglutaryl-coenzyme A reductase
(HMGCR), the rate-limiting enzyme in the cholesterol biosynthesis pathway . It plays an important
role in reducing plasma low-density lipoprotein (LDL) cholesterol and in preventing the risk of
coronary artery disease (CAD) [3,4]. Hepatic uptake of atorvastatin has been demonstrated to be
mediated in an OATP-dependent manner .
OATP1B1 and 2B1 are codified by solute carrier organic anion transporter family genes, member
1B1 (SLCO1B1) and 2B1 (SLCO2B1), respectively. SLCO1B1 has several common polymorphisms
and its relation with statin efficacy remains uncertain. The single nucleotide polymorphism (SNP)
SLCO1B1 c.521T>C has been associated with markedly increased plasma concentrations of
simvastatin, rosuvastatin, pravastatin, and atorvastatin [6–12]. These studies have shown that
homozygous for c.521C allele presented the highest plasma concentration as compared to TC
heterozygote or TT homozygote. The increase in plasma concentration of statins may increase the
exposure of the drug and lead to adverse drug reactions. Indeed, SLCO1B1*5 (c.521C) was associated
with increased risk of statin-induced myopathy in a genome-wide association study in patients taking
simvastatin 80 mg . The SNP c.388A>G (SLCO1B1*1b) has been also associated with higher
activity of OATP1B1 resulting in lower oral bioavailability of pravastatin .
Int. J. Mol. Sci. 2011, 12
Many studies have focused only on the pharmacokinetics of statin, whereas the impact of
SLCO1B1 genotypes on lipid-lowering response to statins remains unsure. In one study, in Japanese
hypercholesterolemic patients treated with pravastatin for eight weeks, heterozygous carriers of
SLCO1B1*15 allele (c.388G and c.521C alleles) had poor LDL cholesterol reduction as compared
with non-carriers (reduction: −14.1 vs. −28.9%) . On the other hand, in a cohort of elderly
hypercholesterolemic patients treated with fluvastatin extended-release, the SLCO1B1 c.463C>A SNP
was significantly associated with enhanced fluvastatin response .
The potential contribution of genetic variations in SLCO2B1 in statins efficacy is not known. Until
now, only one study has accessed the impact of variants of SLCO2B1; however, no differences were
found . OATP2B1, differently from OATP1B1 is localized not only in hepatocytes, but at
membranes of enterocytes, human skeletal muscle and heart. Recent studies have suggested that
OATP2B1 may play a role in statin-induced myopathy, since the presence of OATP2B1 in primary
muscle myoblast cells caused a significant increase in intracellular retention of statins .
The uptake and delivery of atorvastatin into hepatocytes by OATP is essential for its action.
Because some studies have previously associated OATP variants with altered pharmacokinetic profile
of atorvastatin, the aim of this study was to describe the influence of SLCO1B1 and SLCO2B1
genotypes on the pharmacological efficacy of atorvastatin.
2. Results and Discussion
2.1. Characteristics of the Hypercholesterolemic Individuals
Clinical and laboratory data of the HC subjects were previously described by
Rebecchi et al. (2009) . Atorvastatin treatment significantly reduced total LDL cholesterol and
triglycerides values (Table 1). Concomitant ingestion of CYP3A4 substrates or inhibitors did not affect
atorvastatin response (p > 0.05), as evaluated by Chi-square test (data not shown). We did not observe
an increase in high-density lipoprotein (HDL) cholesterol levels as it has been described for this drug.
In addition, atorvastatin treatment did not cause a significant increase in CK levels. There was no
report of intolerance or adverse effects related to atorvastatin therapy. We have observed an increase of
ALT levels after treatment, but this increase did not translate into hepatotoxicity for the patients that
have undergone atorvastatin treatment.
Table 1. Biochemical profile of hypercholesterolemic individuals in response to atorvastatin
(10 mg/day/4 weeks).
281 ± 38
193 ± 55
56 ± 14
160 ± 66
102 ± 80
22 ± 10
130 ± 25
140 ± 22
198 ± 30
118 ± 27
54 ± 13
132 ± 52
104 ± 88
25 ± 15
136 ± 27
102 ± 22
−28.9 ± 9.5
−38.3 ± 12.4
−2.5 ± 10.5
−26.9 ± 52.5
4.9 ± 36.5
23.0 ± 63.2
4.9 ± 15.4
−28 ± 46.1
TC: total cholesterol, LDL-C: low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol; TG: triglyceride;
CK: Creatine kinase; ALT: Alanine aminotransferase; ApoAI: Apolipoprotein AI; ApoB: Apolipoprotein B. * 10mg/daily for 4 weeks.
Int. J. Mol. Sci. 2011, 12
2.2. SLCO1B1 and SLCO2B1 Polymorphisms
Genotype and allele frequencies for SLCO1B1 and SLCO2B1 polymorphisms were calculated for
this sample of the Brazilian population. As expected, allele frequencies of these variants were in
Hardy-Weinberg Equilibrium confirming the random selection of the individuals. The frequencies of
the three variants (c.388A>G, c.463C>A and c.521T>C) for SLCO1B1 gene in Brazilian individuals
were 32%, 16% and 12%, respectively. Minor allele frequency for SLCO2B1 −71C allele was 53%.
Linkage disequilibrium was tested for SLCO1B1 variants. Association was found between
c.388A>G and c.521T>C polymorphisms (D' = 0.84; χ2 = 9.56, p = 0.049) and c.388A>G and
c.463C>A SNPs were also consistently associated (D' = 1.0; χ2 = 69.94, p < 0.0001). Nevertheless,
c.521T>C and c.463C>A were not associated (χ2 = 2.32, p = 0.677). Therefore, six SLCO1B1
haplotypes were found in our study group: *1a (39.3%), *1b (33.3%), *14 (16.0%), *15 (10.3%),
and *4 (1.1%).
The frequency of SLCO1B1 and SLCO2B1 SNPs and of their haplotypes varies largely among
ethnically identified populations [19–21]. Despite the fact that the described frequencies above for
SLCO1B1 are similar to others previously reported [16,20], Brazilians are a highly admixed population
with Amerindian, European and African ancestral roots and estimation of the genetic ancestry
provided by AIMs may allow more realistic representations of such diversity [22–25]. For this
purpose, we have estimated the ACA mean value for our sample and associated it with the alleles of
The individual ACA values across the study population ranged from 0.003 to 0.989. ACA mean
values between ancestral and variant allele of each SNP are presented in Figure S1. We observed that
,only for SLCO2B1 −71T>C polymorphism, the ACA mean value was significantly higher in subjects
carrying −71T allele compared to −71C allele carriers [0.461 (0.010–0.687) vs. 0.112 (0.037–0.243),
p = 0.023].
Categorization of ACA values in four quartiles (<0.25; 0.25–0.50; 0.50–0.75; >0.75) revealed that
frequency of the SLCO2B1 −71C allele decreased progressively from the lowest (<0.25 ACA) to the
highest (>0.75 ACA) quartile, showing its higher prevalence in people with minor African influence
(Supplemental Table 1). For SLCO1B1 gene, the frequencies of the SNPs were not different among the
SLCO1B1 c.463C>A SNP showed a trend for decreasing the frequency of c.463A variant from low
ACA values (<25%) to high ACA values (<75%) (Supplemental Figure 1, Supplemental Table 1).
These results are in agreement with previous reports showing a low prevalence of this allele in
African Americans and a high prevalence in Caucasians . For SLCO2B1 variant, a significant
association between −71C allele and ACA values was found. There is no study reporting this
relationship, but we may conclude that −71C allele varies among ethically identified populations and
presents a low frequency in people with high African background.
The variables’ age, BMI, gender, hypertension, obesity, menopause, cigarette smoking, alcohol
consumption, physical activity, and baseline mean plasma lipid parameters were not different among
the genotypes or haplotypes for all the polymorphisms studied (data not shown). These results suggest
that SLCO1B1 and SLCO2B1 variants were not associated with these variables in this sample.
Int. J. Mol. Sci. 2011, 12
2.3. Effect of SLCO1B1 and SLCO2B1 Polymorphisms on Atorvastatin Response
Results from one-way ANOVA regarding the effect of SLCO1B1 and SLCO2B1 SNPs on total and
LDL cholesterol are presented in Table 2. For SLCO1B1 c.388A>G polymorphism, homozygous for
c.388G allele presented higher mean percentage of LDL cholesterol reduction than carriers of c.388A
allele (41.3 ± 12.4% for GG vs. 36.6 ± 12.1% for AA + AG, p = 0.034), in a dominant model. For
SLCO2B1 polymorphism there was no association between lipid parameters and the genotypes.
Table 2. Association of SLCO1B1 and SLCO2B1 variants with total and LDL cholesterol
in individuals treated with atorvastatin.
199 ± 29
192 ± 32
193 ± 29
200 ± 31
199 ± 32
194 ± 27
200 ± 34
198 ± 29
28.7 ± 9.1
31.8 ± 9.3
30.6 ± 9.8
28.0 ± 9.2
29.4 ± 9.3
27.9 ± 9.9
28.6 ± 8.6
29.4 ± 9.4
192 ± 34
193 ± 31
193 ± 36
191 ± 28
196 ± 35
184 ± 27
194 ± 37
198 ± 29
118 ± 26
114 ± 28
111 ± 25
121 ± 27
120 ± 27
113 ± 26
120 ± 28
116 ± 27
38.1 ± 12.4
40.9 ± 11.6
41.3 ± 12.4
36.6 ± 12.1
38.4 ± 11.5
38.0 ± 14.4
37.6 ± 10.6
39.0 ± 12.9
TC + CC (28)
AA + AG (82)
CA + AA (41)
TC + CC (94)
Number of individuals is given in parenthesis. Values are mean ± standard deviation. P: p-values as evaluated by
one-way analysis of variance, significant p-values are indicated in bold. TC: total cholesterol (mg/dL); LDL-C: low-density
lipoprotein cholesterol (mg/dL).
281 ± 37
282 ± 35
279 ± 32
280 ± 40
283 ± 38
271 ± 31
281 ± 43
282 ± 35
In addition, the effect of SLCO1B1 haplotypes on total and LDL cholesterol before and after
atorvastatin treatment was investigated. We have compared the effect of *15 homozygous (*15/*15),
*15 heterozygous (*1a/*15 and *1b/*15) and *15 non-carriers. (*1a, *1b, and *1a/*1b). Despite the
fact that *15/*15 subjects presented lower total and LDL cholesterol reductions than *15 heterozygous
and *15 non-carriers, this association lacked statistical significance (Figure 1). There was no effect of
*14 allele on atorvastatin response.
After atorvastatin treatment, LDL cholesterol serum concentrations varied largely from reduction of
61.7% to 6.4%. Therefore, individuals with LDL cholesterol in the first quartile (reduction higher than
48%) were compared with those with lower response. First, a stepwise forward multiple regression
analysis including all parameters (age, BMI, gender, basal LDL cholesterol, and c.388A>G genotypes)
was performed. After this analysis we concluded that BMI and gender were not related to atorvastatin
Int. J. Mol. Sci. 2011, 12
response. Then, a multivariate logistic regression including all the remaining parameters was
performed. Results from logistic regression showed that SLCO1B1 c.388GG and higher LDL basal
levels were the most significant factors positively related to atorvastatin response (Table 3).
Figure 1. Influence of the SLCO1B1 *15 variant on reduction of total (TC) and
low-density lipoprotein (LDL-C) cholesterol
(10 mg/day/4 weeks). P > 0.05, as compared by One-Way Analysis of Variance followed
by Hom-Sidak test. Number of individuals in parenthesis.
in response to atorvastatin
Table 3. Multiple logistic regression analysis for reduction of LDL cholesterol after
Basal LDL cholesterol (≥208 mg/dL)
Age (<60 years)
SLCO1B1 c.388G allele (dominant)
CI: Confidence interval; Significant values are highlighted in bold. LDL cholesterol reduction was considered higher than
48% of the basal level.
SLCO1B1 and SLCO2B1 polymorphisms may have particularly important consequences for
cholesterol-lowering therapy with HMGCR inhibitors, as OATPs (1A2, 1B1, 1B3, and 2B1) are
involved in the hepatic uptake of statins . Current knowledge has shown that SNPs in SLCO1B1
may result in reduced efficacy and increased risk of systemic exposure, leading to adverse effects .
Studies of SLCO1B1 SNPs have focused mainly on c.521T>C polymorphism. They have shown
that c.521C allele causes reduced OATP1B1 activity, thus increasing plasma concentrations of all
statins except fluvastatin [6–12]. The area under the curve (AUC) of atorvastatin was 1.5–2.0-fold
higher in subjects with the 521C/C genotype than in those with the 521T/T [6,7,9].
The effect of c.521T>C polymorphism on atorvastatin therapy has been investigated in this study.
We have found no association between c.521C allele carriers and changes in lipid parameters after
4 weeks of atorvastatin treatment. One reason for that lack of association may be due to a limited
number of subjects with 521C/C genotype. Because only two individuals in our sample were
homozygous for the variant allele they were pooled with the 521C/T genotype, then we could not
effectively analyze the effect of 521C/C genotype.
Int. J. Mol. Sci. 2011, 12
Some studies characterizing the impact of SLCO1B1 polymorphisms on lipid-lowering response
have been conducted, however they mainly target pravastatin therapy [15,26–30]. Because these
studies were very heterogeneous among the study population (healthy, hypercholesterolemic or elderly
subjects), duration of treatment (single dose, 3 or 8 weeks, 1 year) and daily dose (20, 40 or
9.4 mg/day), divergent findings have been reported. For instance, Zhang et al. (2007)  reported an
attenuated pravastatin (20 mg/day for 30 days) pharmacodynamic effect on total cholesterol in patients
with 521TC heterozygous compared to 521TT homozygous. On the other hand, treatment with 40 mg
pravastatin for 3 weeks caused no difference in lipid-lowering efficacy between c.521C carriers
(i.e., SLCO1B1*15 and *17) and non-carriers (SLCO1B1*1a).
The SLCO1B1 c.463C>A polymorphism has been previously associated with fluvastatin
response . Carriers of *14 allele had better response to fluvastatin as compared to *1a/*1a or
*1a/*14 genotypes. We have found no association between c.463C>A variant and atorvastatin
response. In fact, this is not the first study to describe a lack of association between c.463C>A SNP
and atorvastatin response. Thompson et al. (2005)  using a much larger sample (n = 1902) also did
not find any association between this polymorphism and response to atorvastatin. The lack of effect of
this polymorphism on atorvastatin response may be due to a substrate-specific effect of this OATP1B1
variant. This substrate-specific effect has been clearly shown for SLCO1B1 c.521T>C SNP. It has been
associated with a markedly reduced uptake of all statins except fluvastatin, as discussed before. Then,
it is possible that SLCO1B1 c.463C>A variant has a high affinity for fluvastatin, however it needs to
be verified by transporter function analyses.
Significantly high reduction of LDL cholesterol in response to atorvastatin treatment was found in
individuals homozygous for SLCO1B1 c.388G allele when compared to c.388A allele carriers
(−41.3 vs. −36.6%). This finding is consistent with previous in vivo studies reporting a higher transport
function for OATP1B1 in subjects carrying *1b variant, resulting in lower oral bioavailability of
pravastatin  and pitavastatin .
There is some evidence that SLCO1B1*15 variant (c.388G and c.521C) exhibits reduced
transport function and play an important role in pravastatin and atorvastatin systemic exposure and
elimination [6,33–35]. Lee et al. (2010)  have shown that the AUC of atorvastatin was 1.8 higher
in *15/*15 subjects than in 1a/*15 and *1b/*15 and 2.2-fold than for *1a/*1a, *1a/*1b and *1b/*1b.
Haplotype analysis revealed that mean percentage reduction in total and LDL cholesterol values at
4 weeks post-treatment with atorvastatin were lower in *15/*15 than in *15 heterozygous and
*15 non-carries. The allele frequency of SLCO1B1*15 was 10.3% in our population, then the sample
size was not enough to find many subjects carrying *15/*15 genotype, so the association lacks
statistical significance. Multiple regression analysis in the study population revealed that only
c.388GG was correlated with statin response.
With respect to SLCO2B1 polymorphism we have not found significant differences between the
different genotypes and atorvastatin response. A previous study also failed to find relationship between
polymorphisms of SLCO2B1 and pharmacokinetics of pravastatin .
Int. J. Mol. Sci. 2011, 12
3. Material and Methods
3.1. Subjects and Study Protocol
The characteristics of study design have been previously reported . Briefly, 136
hypercholesterolemic (HC) individuals were selected randomly among the outpatients evaluated for
the presence of risk factors for coronary artery disease (CAD) at the University Hospital of the Sao
Paulo University (Sao Paulo City, Brazil). The study protocol was approved by the Ethics Committee
of this institution as well the Committee of the Faculty of Pharmaceutical Sciences (University of Sao
Paulo). Individuals diagnosed with thyroid, liver and kidney diseases, diabetes, and triglycerides
higher than 400 mg/dL or subjects under treatment with lipid-lowering drugs, hormone replacement or
oral contraceptives were not included. Pregnant women or patients with heart disease known
previously were not included too.
Information on age, body mass index (BMI), gender, hypertension, obesity, menopause status,
cigarette smoking, physical activity, alcohol consumption and family history of CAD were recorded.
HC patients with (LDL) cholesterol higher than 160 mg/dL, 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 adverse reactions was evaluated. The study design was based on the recommendations of the
National Cholesterol Education Program (NCEP) for treatment of high blood cholesterol in adults .
The dose of 10 mg atorvastatin was chosen because the patients had moderate elevations of LDL
cholesterol, and LDL cholesterol goal will be achieved with low doses for these patients. In addition,
the NCEP recommend checking the response to drug therapy in about 6 weeks.
Response to atorvastatin was evaluated by reduction of LDL cholesterol after the treatment, and
adverse effects were monitored by measuring creatine kinase (CK) and alanine aminotransferase
3.2. Biochemical Profile and SLCO 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 .
Genomic DNA was extracted from EDTA-anticoagulated blood by a salting-out procedure
optimized in our laboratory . Polymorphisms of SLCO1B1 [c.521T>C (Val174Ala, rs4149056),
c.388A>G (Asp130Asn, rs2306283), c.463C>A (Pro155Thr, rs11045819)], and SLCO2B1 [−71T>C
(rs2851069)] were detected by TaqMan® Real time PCR. TaqMan Drug Metabolism Genotyping
Assay (20X) were obtained from Life Technologies (Foster City, CA, USA).
PCR assays contained 4 μL of Universal Master Mix (2X) (Life Technologies), 0.4 μL of TaqMan
Drug Metabolism Genotyping Assay (20X) and 3.6 μL de DNA (20 ng) diluted in nuclease-free water.
The thermal cycling protocol consisted of initial cycle at 10 min a 95 °C followed by 40 cycles at 92 °C
for 15 s, 60 °C for 1 min, using standard 7500 conditions. For SLCO2B1 polymorphisms the cycles
were increased to 50, and the time for extension was 90 s. The amplification was carried out in a
Int. J. Mol. Sci. 2011, 12
7500 fast real-time system (Life Technologies). Genotype calling was performed using the SDS
software (Life Technologies).
3.3. Ancestry Informative Markers (AIMs)
The ancestral origin and African component ancestry (ACA) of the individuals was explored using
a 34-plex AIM-SNPs assay. SNPs were genotyped by multiplex-PCR followed by 34-plex SNaPshot®
primer extension reactions (Life Technologies, Foster City, USA). Extension products were separated
by capillary electrophoresis (3130 Analyzer, Life Technologies) and POP6™ polymer (details in ).
The ACA of the samples was estimated and categorized into four ancestral categories (<0.25;
0.25–0.50; 0.50–0.75; >0.75) according to the relative contribution of a variable number of African
3.4. Statistical Analysis
For SLCO1B1, as previously described by Tirona and colleagues (2002) , haplotypes
were defined based on the presence of c.388A>G, c.463C>A and c.521T>C polymorphisms alone
or in combination, as follows: SLCO1B1*1a (wild type), *1b (c.388G), *4 (c.463A), *5 (c.521C),
*14 (c.388G and c.463A) or *15 (c.388G and c.521C). The agreement of genotypes frequencies with
Hardy-Weinberg equilibrium expectations was tested by χ2 test using Haploview software. Relationships
between the genotypes or haplotypes and categorical variables were evaluated by the Chi-square or
Exact Fisher test.
Continuous variables are presented as mean ± SD. Those without normal distribution were analyzed
by a non parametric test, and they are presented as median (25%–75%). Numerical variables were
compared by t test (two variables) and One-way ANOVA (three or more variables) and Holm-Sidak
method was used for multiple comparisons. Logistic regression analysis was used to evaluate the
relationships between reduction of serum LDL cholesterol and other variables after treatment with
atorvastatin. Statistical tests were performed using the Sigma Stat version 3.5 (SPSS Inc., Chicago, IL,
USA). Significance was considered P < 0.05.
The lack of association between lipid response to atorvastatin and SLCO1B1 c.521T>C polymorphism
may be due to the size of our sample since we could not find many individuals homozygous to the rare
allele. This caused the statistical power of the test performed to be below the desired level. In addition,
the positive association between c.388GG carriers and higher LDL cholesterol reductions would be
greatly strengthened if the sample were larger.
SLCO1B1 c.388A>G polymorphism causes significant increase in atorvastatin response and may be
an important marker for predicting efficacy of lipid-lowering therapy. However, others factors, such as
the drug given to the patient, duration of the treatment, daily dose, basal LDL cholesterol, may
influence the efficacy of the therapy and needs to be taken into consideration.
Int. J. Mol. Sci. 2011, 12
This work was supported by grants from FAPESP (2008/06667-9). A.C. Rodrigues, F.D.V.
Genvigir and M.A.V. Willrich are recipients of fellowships from FAPESP, Sao Paulo, Brazil. M.H.
Hirata and R.D.C. Hirata are recipients of fellowships from CNPq, Brasilia, Brazil.
This work was partially supported by Life Technologies, Sao Paulo, SP, Brazil. The sponsor of this
study had no role in the study design, data collection, data analysis, data interpretation or writing of the
report. S.G. Purim was salaried personnel of Life Technologies. The authors have no other relevant
affiliation or financial involvement with any organization or entity with a financial interest in or
financial conflict with the subject matter or materials in the manuscript apart from those disclosed.
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