Extract
Note: Please read the complete
full text with Figures and Tables at
Introduction
The members of the organic anion-transporting
polypeptides (OATP) represent a family of important proteins
involved in the membrane transport of endogenous and
xenobiotic compounds. OATP are expressed in a wide
variety of tissues, including the liver, kidney, brain, and small
intestine[1_2]. Human OATP1B1 (also known as OATP-C,
OATP2, SLCO1B1 gene), a sodium-independent bile acid
transporter, is specifically expressed on the basolateral
membrane of hepatocytes and translocate a broad range of
compounds, such as bile acids, bilirubin, sulfate and
glucuronide conjugates, thyroid hormones, peptides, and drugs
like 3-hydroxy-3-methylglutaryl-co-enzymeA (HMG
CoA)-reductase inhibitors (pravastatin, rosuvastatin, pitavastatin)
and methotrexate[3_5]. Recent studies have proven that
OATP1B1 plays an important role in the hepatocellular
uptake and consequently the elimination of numerous chemicals.
A number of single nucleotide polymorphisms (SNP)
have been identified in the encoding and regulating regions
of the OATP1B1 gene among different populations. The
frequency of the SLCO1B1*5
(521T>C, Val174Ala) variant frequency is 14% in European Americans and the
SLCO1B1*9 (1463G>C, Gly488Ala) variant frequency is 9%
in African Americans[6], but these 2 common polymorphisms
are extremely low in Japanese populations, which exhibit
significant ethnic difference. In Orientals,
SLCO1B1*1b (388G>A, Asn130Asp) and
SLCO1B1*15 (a haplotype of
SLCO1B1*1b and
SLCO1B1*5) are 2 common SNP with
relatively high frequencies of 66% and 16%, respectively.
Recent studies have elucidated that both
SLCO1B1*1a and
SLCO1B1*15 exhibit reduced transport function and play
an important role in pravastatin, pitavastatin, rosuvastatin
(a substrate for OATP1B1) systemic exposure and
elimination[7_10]. SLCO1B1*17
(_11187G>A, 388G>A, and
521T>C) was found to be associated with increased plasma
concentrations of pravastatin in humans. However, there
are no published studies concerning the functional
significance of the _11187G>A promoter SNP
in vitro until now[9,10].
Therefore, this study was carried out to determine the
SLCO1B1*1b and *15 polymorphisms in the Chinese
population.
Materials and methods
Chemicals and reagents All primers for the PCR were
synthesized by Bioasia Company (Shanghai, China).
Taq DNA polymerase, dNTP mixture, PCR buffer, GeneRuler 500
bp DNA ladder, and the restriction endonuclease
ClaI were all purchased from MBI Fermentas (Vilnius, Lithuania). All
other chemicals were of highest grade and available from
commercial sources.
Patients One hundred and eleven unrelated, healthy,
Chinese Han male volunteers were recruited in this study.
They were medical students at the Central South University,
Xiangya Medical College (Hunan, China) and their mean age
was 20±2 years. This research was approved by the Ethics
Committee of the Central South University and written
informed consent was obtained from each participant. The
participants were assessed by medical history, physical
examination, and clinical laboratory test of hematological,
liver and kidney function, blood tests for human hepatitis
B or C, and blood glucose. They were non-smokers who
did not take any medication or alcohol in the 14 d before
the study.
Blood acquisition and DNA isolation Five milliliters of
venous blood was collected in a sterile tube containing EDTA
and stored at -80 °C. Genomic DNA was isolated from
leukocytes through a standard manual chloroform_phenol
extraction procedure and stored at 4 °C until use.
PCR_restriction fragment length
polymorphism assay for 388G>A
genotyping The 388G>A genotype was
determined by means of PCR_restriction fragment length
polymorphism (RFLP) analysis according to Torina
et al[6] with some modifications. The PCR reaction was carried out in a
total volume of 25 µL consisting of 2.5 µL 10×PCR buffer
(with MgCl2), 0.2 mmol/L of each dNTP, 60 pmol/L of each
primer, 100 ng of genomic DNA as a template, and 2.5 U
Taq polymerase. The sequences of the primers used for
detection of 388G>A were: forward primer,
5'-GCAAATAAA-GGGGAATATTTCTC-3' and reverse primer,
5'-AGAGATGT-AATTAAA TGTATAC-3'. PCR amplification to detect
388G>A was performed using the Gene Amplification PCR System
2400 (Perkin Elmer, Foster City, CA, USA) with an initial
denaturation at 94 °C for 5 min, followed by 37 cycles of
denaturation at 94 °C for 30 s, annealing at 46 °C for 30 s, and
extension at 72 °C for 30 s. A final 5 min extension at 72 °C
was adopted. After amplification, the PCR products (274 bp)
were digested with the CLaI restriction endonuclease at 30
°C for at least 6 h. Digested products were analyzed by
electrophoresis on a 2.5% agarose gel in the presence of
ethidium bromide.
Amplification refractory mutation system_PCR
assay for 521T>C genotyping The amplification
refractory mutation system (ARMS) was introduced, avoiding the
use of restriction enzymes[10]. The 4 primers used in
ARMS_PCR were according to Torina et
al[6] with slight modifications. PCR amplification was performed in a
Perkin Elmer DNA Model PJ2000 thermal cycler (USA)
in a total volume of 25 µL solution containing 2.5 µL 10×buffer
(with MgCl2), 0.4 mmol/L of each dNTP, 40 pmol/L of each
primer, 2 U LA_Taq enzyme and approximately 200 ng
genomic DNA as a template. The PCR conditions involved an
initial denaturation at 94 °C for 5 min, followed by 35 cycles
of denaturation at 94 °C for 30 s, annealing at 48 °C for 30 s,
and extension at 72 °C for 30 s with a final extension at 72 °C
for 7 min. Because the restriction endonuclease was
unnecessary, the PCR products (totally 260 bp) were
detected by means of 2% agarose gel electrophoresis and were
detected by ethidium bromide staining. Two samples of
521T>C of each genotype were directly sequenced to
confirm our genotyping result. The primers and conditions are
listed in detail in Table 1.
Data analysis Haplotypes were reconstructed on the
basis of the phase-unknown genotype data using PHASE
version 2 software (Stephens, et al, Seattle, Washington,
USA), a computer-assisted statistical analysis based on
Bayesian statistics. Haplotypes, as well as the genotype
frequency deviation from the Hardy_Weinberg equilibrium,
were evaluated by appropriate χ2-test. The SPSS software
package version 11.2 (Chicago, IL, USA) was used to
perform the statistical analysis. A P-value of less than 0.05 was
accepted as significant.
Results
The PCR product in different 388G>A genotypes are
shown in Figure 1. Patients with 388GG produced 155 and
119 bp fragments, whereas those from 388AA
homozygotes generated an additional 274 bp fragment. Heterozygous
388G>A-mutated genotypes produced 3 fragments of 155,
119, and 274 bp.
Four specific primers were used to amplify 3 fragments in
different 521T>C genotypes (Figure 2). The wild-type allele
yielded 2 fragments of 260 and 179 bp in length, while the
variant 521T>C homozygotes resulted in 2 fragments of 260
and 123 bp in length. The heterozygotes resulted in 3
fragments of 260, 123, and 179 bp.
Of the 111 Chinese patients, 39 patients (35.1%) were
heterozygotes and 62 (55.9%) were homozygotes for the 388G
allele and 27 (24.3%) were heterozygotes and 2
(1.8%) were homozygotes for 521T>C mutation. The
frequencies of the alleles and genotypes were calculated and
are listed in Table 2. The distribution of the 3 genotypes of
the 388G>A and 521T>C polymorphisms conformed well to
the predictions of Hardy_Weinberg equilibrium
(P>0.05).
A haplotype comprising of the 2 common SNP was
constructed for these 111 individuals (222 sequences). The
results of the haplotype analysis using a pseudo-Bayesian
algorithm revealed 3 types of haplotypes in our Chinese
population. These haplotypes were A-T (*1a), G-T
(*1b), and G-C (*15). The haplotype frequencies were 26.1%,
59.9%, and 14%, respectively (Table 2). Of the 4 (2×2)
possible combinations of the SNP, haplotype A-C (in order
388_521) was not present in our patients. In the population in our
present study, the diplotypes of the individuals were
determined by PHASE 2.0 software consisting of 6 types in all:
*1a/*1a, *1a/*1b,
*1a/*15, *1b/*1b,
*1b/*15, and *15/*15. The detailed haplotypes and their frequencies
are shown in Table 3.
Discussion
Drug metabolic enzymes and transporters have long been
determined as major determinants of drug metabolism and
disposition. However, accumulating evidence has proved
that membrane transporters are also critical factors to the
drug disposition process both in vitro and
in vivo. Human OATP1B1 belongs to the OATP family of drug uptake
transporters. It is specifically localized at the basolateral
membrane of hepatocytes and is responsible for the hepatic
uptake of a series of structurally-divergent compounds,
including some endogenous chemicals and many
clinically-used drugs.
More than 20 functionally-relevant SNP in the
SLCO1B1 gene have been identified in different
populations[6,12_14]. The common SNP with impaired transport activity appeared to
be 521T>C (Val174Ala) in European Americans and
Japanese and G1463>C (Gly488Ala) in African Americans.
However, the clinical significance of these commonly seen
mutations for large numbers of endogenous and xenobiotic
substrates transported by OATP1B1 remains to be further
studied.
We detected both the SLCO1B1*1b
and SLCO1B1*15 variants in this study to characterize OATP1B1 genetic
polymorphisms in the Chinese population. The frequency of the
SLCO1B1*1b haplotype was 59.9% in our study, which is
similar to previous reports of Japanese
(62.9%)[7] and African Americans (74%), but higher than that of European
Americans (30%)[6]. The frequency of the
SLCO1B1*15 haplotype was 14% in Chinese, similar to that in Japanese
(15.8%), but greater than that of Caucasians (2.4%) and
African Americans (0%)[15].
The allele frequencies of drug transporter polymorphisms
among different ethnic groups may contribute to drug
therapeutic effects as well as toxicity. For example, pravastatin is
one of the HMG-CoA reductase inhibitors (statins) widely
used in the treatment of
hypercholesterolemia[16], which experienced no obvious metabolism by cytochrome P450s, but
could be efficiently taken up from circulation by the liver
through OATP1B1[6]. It has been reported that increased
systemic exposure and decreased non-renal elimination to
pravastatin in vivo was significantly associated with the
521T>C variant in both Europeans and Japanese. The
388G>A site and novel mutation
_11187G>A were also proven to be of pharmacokinetic
significance[7_9]. In a recent study, patients with the
SLCO1B1*15 allele were proven to be associated with higher serum bilirubin
levels[17_19]. Thus, the determination of
OATP1B1 gene polymorphisms in specific ethnic groups is very important for contributing to
individualized drug therapeutics and the pathogenesis of
hereditary diseases. However, the effect of the
521T>C genotype on the pharmacokinetics of rosuvastatin was not
observed significantly in the Chinese, Malay, and
Asian_Indian populations[20]. Our study indicated that the
SLCO1B1 polymorphisms could not fully explain the interindividual
difference of rosuvastatin disposition. The functional
polymorphisms of CYP2C9 and some certain drug transporters,
for instance breast cancer resistance protein (BCRP), may
also contribute to the disposition of
rosuvastatin[21], whereas pravastatin is not obviously metabolized by CYP450.
In summary, the present study has shown that the
SLCO1B1*1b and
SLCO1B1*15 variants represent common
genetic polymorphisms in the Chinese population. There
were remarkable ethnic differences in the frequencies of these
2 haplotypes. Our findings suggest that about 26.1% of the
Chinese population carrying the
SLCO1B1*15 variant might exhibit impaired transport activity of OATP1B1.
References
1 Hagenbuch B, Meier PJ. The superfamily of organic anion
transporting polypeptides. Biochim Biophys Acta 2003;1609(1):1_18.
Review.
2 Kim RB. Organic anion-transporting polypeptide (OATP)
transporter family and drug disposition. Eur J Clin Invest 2003; 33
(Suppl 2): 1_5.
3 Abe T, Kakyo M, Tokui T, Nakagomi R, Nishio T, Nakai D,
et al. Identification of a novel gene family encoding human
liver-specific organic anion transporter LST-1. J Biol Chem 1999;
120: 17159_63.
4 Hsiang B, Zhu Y, Wang Z, Wu Y, Sasseville V, Yang WP,
et al. A novel human hepatic organic anion transporting polypeptide 2
(OATP2): identification of a liver-specific organic anion
transporting polypeptide and identification of rat and human
hydroxymethylglutaryl-CoA-reductase inhibitor transporters. J
Biol Chem 1999; 274: 37161_8.
5 Konig J, Cui Y, Nies AT, Keppler D. A novel human organic
anion transporting polypeptide localized to the basolateral
hepatocyte membrane. Am J Physiol Gastrointest Liver Physiol 2000;
278: G156_64.
6 Tirona RG, Leake BF, Merino G, Kim RB. Polymorphisms in
OATP-C: identification of multiple allelic variants associated
with altered transport activity among European and
African_Americans. J Biol Chem 2001; 276: 35 669_75.
7 Nishizato Y, Ieiri I, Suzuki H, Kimura M, Kawabata K, Hirota T,
et al. Polymorphisms of OATP-C (SLC21A6) and OAT3
(SLC22A8) genes: consequences for pravastatin
pharmaco-kinetics. Clin Pharmacol Ther 2003; 73: 554_65.
8 Mwinyi J, Johne A, Bauer S, Roots I, Gerloff T. Evidence for
inverse effects of OATP-C (SLC21A6)
*5 and *1b haplotypes on pravastatin kinetics. Clin Pharmacol Ther 2004; 75: 415_
21.
9 Niemi M, Schaeffeler E, Langb T, Fromma MF, Neuvonen M,
Kyrklund C, et al. High plasma pravastatin concentrations are
associated with single nucleotide polymorphisms and haplotypes
of organic anion transporting polypeptide-C (OATP-C,
SLCO1B1). Pharmacogenetics 2004, 14: 429_40.
10 Kameyama Y, Yamashita K, Kobayash K, Hosokawa M, Chiba
K. Functional characterization of SLCO1B1 (OATP-C) variants,
SLCO1B1*5, SLCO1B1*15 and SLCO1B1*15+C1007G, by using transient expression systems of HeLa and HEK293 cells.
Pharmacogenet Genomics 2005; 15: 513_22.
11 Ye S, Dhillon S, Ke XY, Collins AR, Day INM. An efficient
procedure for genotyping single nucleotide polymorphisms.
Nucleic Acids Res 2001; 29: E88_8.
12 Kim RB. 3-Hydroxy-3-methylglutaryl_coenzyme A reductase
inhibitors (statins) and genetic variability (single nucleotide
polymorphisms) in a hepatic drug uptake transporter: What's it
all about? Clin Pharmacol Ther 2004; 75: 381_5.
13 Tirona RG, Kim RB. Pharmacogenomics of organic
anion-transporting polypeptides (OATP). Adv Drug Deliv Rev 2002; 54:
1343_52.
14 Morimoto K, Oishi T, Ueda S, Ueda M, Hosokawa M, Chiba K. A
novel variant allele of OATP-C (SLCO1B1) found in a Japanese
patient with pravastatin-induced myopathy. Drug Metab
Pharmacokinet 2004; 19: 453_5.
15 Pasanen MK, Backman JT, Neuvonen PJ, Niemi M. Frequencies
of single nucleotide polymorphisms and haplotypes of organic
anion transporting polypeptide 1B1 SLCO1B1 gene in a Finnish
population. Eur J Clin Pharmacol 2006; 62: 409_15.
16 Hatanaka T. Clinical pharmacokinetics of pravastatin
mechanisms of pharmacokinetic events. Clin Pharmacokinet 2000;
39: 397_412.
17 Cui YH, Konig J, Leier I, Buchholz U, Keppler D. Hepatic
uptake of bilirubin and its conjugates by the human organic anion
transporter SLC21A6*. J Biol Chem 2001; 276: 9626_30.
18 Ieiri I, Suzuki H, Kimura M, Takane H, Nishizato Y, Irie S,
et al. Influence of common variants in the pharmacokinetic genes
(OATP-C, UGT1A1, and MRP2) on serum bilirubin levels in
healthy subjects. Hepatol Res 2004; 30: 91_5.
19 Nozawa T, Nakajima M, Tamai I, Noda K, Nezu JC, Sai Y,
et al. Genetic polymorphisms of human organic anion transporters
OATP-C (SLC21A6) and OATP-B (SLC21A9): allele
frequencies in the Japanese population and functional analysis. J
Pharmacol Exp Ther 2002; 302: 804_13.
20 Lee E, Ryan S, Birmingham B, Zalikowski J, March R, Ambrose
H, et al. Rosuvastatin pharmacokinetics and pharmacogenetics
in white and Asian subjects residing in the same environment.
Clin Pharmacol Ther 2005; 78: 330_41.
21 Zhang W, Yu BN, He YJ, Fan L, Li Q, Liu ZQ,
et al. Role of BCRP 421C>A polymorphism on rosuvastatin
pharmacokinetics in healthy Chinese males. Clin Chim Acta 2006; 373:
99_103.
|
