The farnesoid X receptor -1G>T polymorphism influences the lipid response to rosuvastatin.

The bile acid-activated nuclear receptor farnesoid X receptor (FXR) plays an important role in lipid and glucose metabolism, and in addition, it regulates multiple drug transporters involved in statin disposition. We examined whether a functional single nucleotide polymorphism (SNP) in FXR (-1G>T) influenced the lipid-lowering effect of rosuvastatin. In 385 Chinese patients with hyperlipidemia who had been treated with rosuvastatin 10 mg daily for at least 4 weeks, the association between the FXR -1G>T SNP and lipid response to rosuvastatin was analyzed. The FXR -1G>T SNP was not associated with baseline lipids but was significantly associated with the LDL cholesterol (LDL-C) and total cholesterol response to rosuvastatin. Carriers of the T-variant allele (GT+TT = 68+3) had 4.4% (95% CI: 1.2, 7.5%, P = 0.006) and 2.6% (95% CI: 0.3, 5.0%; P < 0.05) greater reductions in LDL-C and total cholesterol, respectively, compared with those with homozygous wild-type alleles. The association between the FXR polymorphism and the LDL-C response to rosuvastatin remained significant after adjusting for other covariants. This association of the variant allele of the FXR -1G>T polymorphism with a greater LDL-C response to rosuvastatin may suggest that this polymorphism influences the expression of the hepatic efflux transporters involved in biliary excretion of rosuvastatin.

The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily, which is activated by bile acids. Activation of FXR leads to altered expression of many genes responsible for bile acid and lipid and glucose metabolism and transport, resulting in decreased intracellular bile acid concentrations and reduced plasma glucose and triglyceride levels ( 1,2 ). Current evidence suggests All patients were treated with a single lipid-lowering therapy of rosuvastatin 10 mg per day for at least four weeks (median duration of treatment was six weeks), and drug compliance was assessed at study visit by tablet counting. Subjects with poor adherence (reported or calculated taking <80% of the prescribed number of tablets) were excluded from the analysis. The study was approved by the local Clinical Research Ethics Committee and was performed in accordance with the Declaration of Helsinki. All participants gave written informed consent.

Laboratory tests
Fasting blood samples before and after at least four weeks of treatment with rosuvastatin 10 mg daily were collected for the measurement of lipid profi les and laboratory safety data, including serum alanine aminotransferase, creatine kinase, and creatinine. LDL-C concentrations were calculated according to the Friedewald formula or directly measured if the triglyceride level was greater than 4.5 mmol/l ( 23 ). All biochemistry tests were performed by standard methods in the Chemical Pathology Laboratory, Prince of Wales Hospital, which has international laboratory accreditation.

Genotyping
The FXR -1G>T polymorphism (rs56163822) was genotyped by using Taqman genotyping assay (C_25598386_10) on the ABI GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA) with reactions performed according to manufacturer's instructions. The SNPs 421C>A in ABCG2 and 521T>C in SLCO1B1 were genotyped as described previously ( 19,20 ).

Statistical analysis
Differences in baseline characteristics and lipid responses to rosuvastatin between the FXR -1G>T genotype groups were assessed using Student t -test for normally distributed variables or Mann-Whitney test for skewed variables. The degree of skewness of the variables was assessed by the Kolmogorov-Smirnov normality test and the Normal Q-Q plot. 2 tests were used to test Hardy-Weinberg equilibrium and comparisons for categorical variables. ANCOVA was performed to determine the effect of the FXR -1G>T polymorphism on the lipid responses to rosuvastatin after adjusting for confounding factors. The effects of the FXR and ABCG2 genotypes on LDL-C response to rosuvastatin were analyzed by ANOVA, followed by the posthoc Fisher's least signifi cant difference test. P < 0.05 was considered to be statistically signifi cant. All statistical analyses were performed using SPSS Version 17.0 (SPSS Inc., Chicago, IL).

RESULTS
In 386 patients with good adherence to rosuvastatin, genotyping of FXR -1G>T SNP was successful in 385 subjects. Of these patients, 314 had GG genotype, 68 were heterozygous, and 3 were homozygous for the T-variant alleles. The frequency of the T-variant allele was 9.9%, and the genotype distribution was in Hardy-Weinberg equilibrium ( P > 0.05). The data of the GT and TT genotype groups were combined due to the small number of the homozygous TT subjects for comparison with those who were homozygous for the G wild-type allele.
The baseline characteristics of the study subjects stratifi ed by the FXR -1G>T genotype groups are shown in Table 1 . There were no signifi cant differences in the baseline characteristics, except for body mass index (BMI, certain effl ux transporters ( 4,8,(11)(12)(13). It has been reported that a common noncoding SNP resulting in a G-1T (*1B) substitution in the nucleotide adjacent to the translation initiation site of FXR was associated with signifi cantly reduced function in vitro ( 14 ) and probably resulted from decreased translational effi ciency ( 15 ). Gene expression analysis showed that livers carrying one copy of the variant allele had signifi cantly decreased expression of hepatic FXR target genes, including small heterodimer partner (SHP), SLCO1B1 , and SLCO1B3 ( 14 ). This polymorphism is relatively common among some populations, with a prevalence of 2.5% in Europeans, 3.2% in Africans, and 12.1% in Chinese. However, there is no study to report whether the FXR -1G>T polymorphism relates to interindividual or interethnic differences in the pharmacokinetics and pharmacodynamics of the substrate drugs of these transporters.
Rosuvastatin undergoes little metabolism, but it is a substrate of a number of drug transporters, including the uptake transporters SLCO1B1, SLCO1B3, SLCO2B1, SLCO1A2, and NTCP and the effl ux transporters ABCG2 and ABCC2 (16)(17)(18). Early studies showed that loss-of-function SNPs in SLCO1B1 and ABCG2 were signifi cantly associated with increased systemic drug exposure to rosuvastatin ( 6 ). Our previous study in Chinese patients with hypercholesterolemia also showed that the ABCG2 421C>A polymorphism was signifi cantly associated with the lipid-lowering effect of rosuvastatin ( 19,20 ). Given the importance of the FXR in lipid metabolism and in regulating the expression of the drug transporter genes that are involved in rosuvastatin disposition, we examined the association between the functional SNP FXR -1G>T and the lipid response to rosuvastatin in Chinese patients with hypercholesterolemia.

Study population and design
Patients included in this analysis were Hong Kong Han Chinese patients with hypercholesterolemia, who had been involved in the pharmacogenetic analysis of the lipid response to rosuvastatin as described previously ( 19,20 ). In brief, patients ages у 18 years with baseline LDL cholesterol (LDL-C) > 2.6 mmol/l were included if they were considered at increased risk of cardiovascular disease because of a history of coronary heart disease (CHD), other clinical evidence of atherosclerosis, diabetes mellitus, calculated 10-year CHD risk score > 20% ( 21 ), or clinically diagnosed with familial hypercholesterolemia (FH) ( 22 ). A small group of patients with rheumatoid arthritis ( n = 36) who had participated in a double-blind, placebo-controlled, randomized study to assess the effect of rosuvastatin 10 mg on carotid intimamedia thickness and pulse wave velocity (ClinicalTrials.gov NCT00555230) were also included.
Individuals with poorly controlled hypo/hyperthyroidism, diabetes, hypertension, a history of statin intolerance, signifi cant renal impairment, hepatic dysfunction, or unexplained high (>3 ULN) serum creatine kinase, or who had experienced a cardiovascular event within the three months before recruitment, or who were taking other drugs known to modify plasma lipids or to have an interaction with rosuvastatin (corticosteroids, cyclosporine, etc.) were excluded from this study. of patients as shown previously ( 19,20 ), further analyses were carried out to adjust for its effect. There were significant differences ( P < 2 × 10 Ϫ 6 ) in LDL-C response among the four groups stratifi ed according to the FXR -1G>T and the ABCG2 421C>A polymorphisms ( Fig. 2 ). Subjects homozygous for the wild-type alleles of the FXR -1G>T and the ABCG2 421C>A polymorphisms ( FXR -1GG / ABCG2 421CC) had the smallest reduction in LDL-C, those with variant alleles of these two SNPs had the greatest LDL-C reduction, and those with mutations in one of the two SNPs had intermediate values. The difference in LDL-C response to rosuvastatin between subjects with FXR -1GG versus -1GT/TT was signifi cant in subjects with the ABCG2 421CC genotype but not for those with one or two copies of the ABCG2 421A variant allele ( Fig. 2 ).

DISCUSSION
It has been proposed that variants in regulatory protein coding genes, such as FXR , may contribute to the overall variation of drug transporter expression in tissues and thereby infl uence drug disposition and responses ( 4,24 ). This study is the fi rst to report that the common polymorphism (-1G>T) in FXR resulting in signifi cantly reduced function of the gene was associated with a greater LDL-C response to rosuvastatin in Chinese patients with hypercholesterolemia and that this association remained significant after adjusting for the potential confounding factors. P = 0.023) between the two genotype groups ( Table 1 ). Subjects with one or two copies of the T-variant allele had a higher BMI than those with GG genotype, but the differences in waist circumference and body fat were not statistically signifi cant between the two groups. Further subgroup analysis showed that the signifi cant association between the FXR -1G>T genotype and BMI was found only in females and not in males (supplementary Table I).
There were signifi cant differences in the percentage reductions in LDL-C ( P = 0.006) and total cholesterol ( P = 0.026) between the FXR -1G>T genotype groups ( Table 2 ). Subjects with one or two copies of the T-variant allele (n = 71) had 4.4% (95% CI: 1.2, 7.5%) and 2.6% (95% CI: 0.3, 5.0%) greater reductions in LDL-C and total cholesterol, respectively, compared with those with homozygous wildtype alleles. The three subjects who were homozygous for TT appeared to have the greatest LDL-C response to rosuvastatin, whereas the heterozygotes had intermediate responses, which were signifi cantly different from those in homozygotes for the wild-type alleles ( Fig. 1 ). The differences in LDL-C response to rosuvastatin between the genotype groups remained signifi cant after adjustment for age, gender, BMI, having FH, and the SLCO1B1 521T>C and ABCG2 421C>A polymorphisms, which may potentially confound the results [GG versus GT+TT = Ϫ 51.3% ( Ϫ 52.6, Ϫ 49.9%) versus Ϫ 55.0% ( Ϫ 57.8, Ϫ 52.2%), P = 0.021].
As the ABCG2 421C>A polymorphism had a signifi cant effect on the LDL-C response to rosuvastatin in this group between the SLCO1B1 polymorphism and the lipid response to rosuvastatin, as liver exposure would determine the lipid-lowering effect of statins. Rosuvastatin is excreted mostly unchanged into the bile; therefore, interactions with hepatic apical effl ux transporters may infl uence its lipid-lowering effi cacy in the liver. There is convincing evidence showing that human ABCG2 is the major determinant of the pharmacokinetics and lipid-lowering effect of rosuvastatin ( 5 ). In addition, it has been suggested that another effl ux transporter, ABCC2, is involved in the biliary excretion of rosuvastatin in humans ( 18 ), whereas animal studies in rats identifi ed a possible role of bile acid export pump (Bsep) in the biliary clearance of rosuvastatin ( 29 ). It is known that ABCC2 ( 11 ) and BSEP ( 30 ) are both regulated by FXR. The mutation -1G>T in FXR may result in reduced function of these effl ux transporters and thus reduced biliary clearance of rosuvastatin, which is in line with the greater LDL-C response in subjects with the variant allele of -1G>T in this study.
To our knowledge, there is no study reporting whether the gene for ABCG2, the major effl ux transporter of rosuvastatin, is a target of FXR, but indirect evidence suggests that hepatic ABCG2 expression is regulated by some nuclear receptors, such as constitutive androstane receptor (CAR) and pregnane X receptor (PXR) ( 31 ), and the latter is a target of FXR ( 32 ). Therefore, it is possible that the reduced FXR activity due to the -1G>T mutation may reduce the ABCG2 expression via effects on PXR. Given the strong association between the ABCG2 421C>A polymorphism and the LDL-C response to rosuvastatin, we further evaluated the effect of the FXR -1G>T polymorphism in subgroups of patients stratifi ed by the ABCG2 421C>A genotype. It appears that the association between the FXR -1G>T polymorphism and the LDL-C response to rosuvastatin is more evident in subjects homozygous for the wildtype alleles than in those with one or two copies of the variant allele of the ABCG2 421C>A polymorphism. This result may suggest that the effect of the FXR polymorphism It has been shown that the T-variant allele of the FXR -1G>T was associated with reduced mRNA expression of SLCO1B1 ( 14 ), but the result of the present study suggests that the association of the FXR polymorphism and statin response is mediated by transporters other than SLCO1B1, as reduced expression of SLCO1B1 is expected to be associated with a reduced LDL-C response to statins as shown previously ( 25 ). The functional SNP 521T>C in SLCO1B1 is associated with decreased transport activity of SLCO1B1 and is consequently associated with increased systemic exposure to rosuvastatin ( 26 ), but it did not infl uence the lipid response to rosuvastatin signifi cantly in our study subjects (supplementary Table II) ( 19 ). It has been proposed that the SLCO1B1 polymorphism may affect the systemic exposure to substrate drugs but may only have a minor infl uence on liver exposure, as observed with rifampin ( 27,28 ). This probably explains the lack of association  on the lipid-lowering effect of rosuvastatin is mediated through a regulatory effect on ABCG2 expression, but this idea needs to be further investigated.
FXR plays an important role in maintaining bile acid, lipid, and glucose homeostasis, and it is a key regulator of whole-body energy metabolism ( 1,2 ). Defi ciency of FXR in mice results in increases in systemic and hepatic HDL cholesterol (HDL-C) and triglyceride concentrations ( 33 ), whereas activation of FXR by synthetic FXR agonists reduces plasma triglyceride and HDL-C levels (34)(35)(36). In this study, the FXR -1G>T polymorphism was not associated with baseline lipid levels, but a potential infl uence of this polymorphism on the lipid metabolism pathways, which may infl uence statin pharmacodynamics and thereby the lipid-lowering effect of rosuvastatin, cannot be excluded. In this study, female but not male subjects with the FXR -1T-variant allele had higher BMI than those homozygous for the wild-type alleles. The association between the FXR -1G>T polymorphism and obesity in females is probably related to the reduced energy metabolism induced by the reduced function of FXR in subjects with the variant allele ( 2 ).
It is also noteworthy that a recent in vitro study suggested that statins could activate some nuclear receptors, including PXR, CAR and FXR, in a dose-dependent and chemical-specifi c manner ( 37 ). Rosuvastatin signifi cantly activated the expression of CAR and FXR at high concentrations (10 M) by about 2-fold. Whether statins can infl uence the expression of these nuclear receptors in vivo and whether polymorphisms in nuclear receptor genes could infl uence this interaction remain unknown and warrant further evaluation.
In conclusion, this study showed that the variant allele of the common FXR -1G>T polymorphism was signifi cantly associated with a greater LDL-C response to rosuvastatin in Chinese patients with hypercholesterolemia. The association is probably through the infl uence of the FXR -1G>T polymorphism on the expression of the effl ux transporters that determine the hepatic exposure to rosuvastatin, but a potential effect of this polymorphism on lipid metabolism/pharmacodynamic pathways of rosuvastatin cannot be excluded.