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
SNPs and of their haplotypes varies largely among ethnically identified populations [19
]. Despite the fact that the described frequencies above for SLCO1B1
are similar to others previously reported [16
], 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
]. For this purpose, we have estimated the ACA mean value for our sample and associated it with the alleles of SLC
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
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 ACA quartiles.
These results are in agreement with previous reports showing a low prevalence of this allele in African Americans and a high prevalence in Caucasians [20
]. 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.
2.3. Effect of SLCO1B1 and SLCO2B1 Polymorphisms on Atorvastatin Response
Results from one-way ANOVA regarding the effect of SLCO1B1
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.
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 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
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 [5
]. Current knowledge has shown that SNPs in SLCO1B1
may result in reduced efficacy and increased risk of systemic exposure, leading to adverse effects [5
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
]. 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
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.
Some studies characterizing the impact of SLCO1B1
polymorphisms on lipid-lowering response have been conducted, however they mainly target pravastatin therapy [15
]. 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.
] 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
c.463C>A polymorphism has been previously associated with fluvastatin response [16
]. 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.
] 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 [8
] and pitavastatin [32
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
]. Lee et al.
] 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 [7