Sep 22, 2010 - du CHUM 1560 Sherbrooke Est, Montrå¢al, Quå¢bec, Canada, H2L 4M1 .... 41 unrelated whites canadians from Quebec, Canada. ..... 1833 Gé¦.
Drug Metab. Pharmacokinet. 25 (5): 508–515 (2010).
Short Communication Organic Anion Transporting Polypeptide 1B1 (OATP1B1) and OATP1B3: Genetic Variability and Haplotype Analysis in White Canadians Andree-Anne á BOIVIN1, Helo á ƒä se CARDINAL2, Azemi BARAMA3, 2 and Michel ROGER1,* Á Vincent PICHETTE4, Marie-Josee á HEBERT 1Laboratoire
d'Immunog áen áetique, Centre de Recherche du Centre Hospitalier de l'universit áe de Montr áeal et D áepartement de Microbiologie et Immunologie de l'universit áe de Montr áeal, Montr áeal, Canada 2D áepartement de M áedecine, Service de n áephrologie, Centre Hospitalier de l'universit áe de Montr áeal, Montr áeal, Canada 3D áepartement de Chirurgie, Service de chirurgie greffe r áenale et pancr áeatique, Centre Hospitalier de l'universit áe de Montr áeal, Montr áeal, Canada 4 D áepartement de M áedecine, Service de n áephrologie, H âopital Maisonneuve-Rosemont, Montr áeal, Canada Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk
Summary: Organic anion transporting polypeptide 1B1 (OATP1B1) and OATP1B3 are human hepatocyte transporters that mediate the uptake of various endogenous and exogenous substances. Genetic variations in solute carrier transporter 1B1 (SLCO1B1) and SLCO1B3 genes, which encode OATP1B1 and OATP1B3 proteins, could affect the pharmacokinetics of drugs leading to interindividual differences in drug responses. The full extent of SLCO1B1 and SLCO1B3 polymorphisms in white Canadians was analyzed using DNA sequencing procedures. We identified 49 and 41 nucleotide sequence variants leading to 10 and 9 major haplotypes in SLCO1B1 and SLCO1B3 genes, respectively. We report several novel mutations within regulatory and coding regions that could affect gene transcription, translation and function. Comparison with other studies revealed that the distribution of SLCO1B1 and SLCO1B3 polymorphisms and haplotypes differs widely across populations. Data from this survey will ultimately contribute to the design of pharmacogenetic studies in the Canadian population. Keywords: hepatic transport; ADME; organic anion transport; ethnic differences; genomics; human genetics; single nucleotide polymorphism; OATP; SLC transporters; solute carrier family
SLCO1B1 and SLCO1B3 nucleotide sequence variations have been identified, with frequencies varying between different ethnic groups.4–9) Some of them were associated with altered in vitro transport activity of OATP1B14,10–12) and OATP1B3.13,14) Moreover, studies in humans have shown that plasma concentrations of the OATP1B1 substrate pravastatin were increased in subjects carrying the OATP1B1 Val174Ala variant.15,16) As the significance of SLCO1B1 and SLCO1B3 polymorphisms on drug pharmacokinetics is becoming increasingly evident and inter-ethnic differences exist in the allelic frequency of these variants, there is a need to characterize the full extent of SLCO1B1 and SLCO1B3 genetic variability in any given population. We have
Introduction Organic anion transporting polypeptide 1B1 (OATP1B1) and OATP1B3 are human hepatocyte transporters that mediate the uptake of various endogenous and exogenous substances.1,2) As such, they are both thought to influence overall drug distribution and elimination, and consequently to influence systemic and intracellular levels of many drugs.3) Genetic polymorphisms of drug transporters could affect the pharmacokinetics of drugs, leading to interindividual differences in drug responses. OATP1B1 and OATP1B3 are encoded by solute carrier transporter 1B1 (SLCO1B1) and SLCO1B3 genes, respectively. Several
Received; May 14, 2010, Accepted; June 20, 2010, J-STAGE Advance Published Date: September 22, 2010 *To whom correspondence should be addressed: Michel ROGER M.D., Ph.D, D áepartement de microbiologie et infectiologie, H âopital Notre-Dame du CHUM 1560 Sherbrooke Est, Montr áeal, Qu áebec, Canada, H2L 4M1. Tel. ＋1-514-890-8000 (ext: 25802), Fax. ＋1-514-412-7512, E-mail: michel.roger.chum ＠ssss.gouv.qc.ca This work was supported by Astellas Pharma Canada Inc. The opinions expressed in this paper are those of the authors and Astellas Pharma Inc. had no role in the design of the study, data analysis and interpretation, and in the manuscript preparation. AA Boivin holds a Student Research award from Fonds de la Recherche en Sante du Quebec (FRSQ). V Pichette, MJ Hebert and M Roger are supported by career awards from FRSQ.
508
509
SLCO1B1 and SLCO1B3 Genetic Diversity in White Canadians
therefore investigated the entire nucleotide sequence of SLCO1B1 and SLCO1B3 genes and report the distribution of variants and haplotypes in white Canadians.
Materials and Methods Our samples consisted of stored DNA extracts from 41 unrelated whites canadians from Quebec, Canada.
The use of these samples for the present study was approved by the ethic committee of the Centre Hospitalier de l'universit áe de Montreá al (CHUM). DNA was extracted from whole peripheral blood using standard phenolchloroform procedures. Primers required for PCR (polymerase chain reaction) amplification and sequencing were designed according to wild type SLCO1B1 and
Table 1. Primer sequences and annealing temperatures for amplifying the SLCO1B1 gene for sequencing Annealing temperature (9 C)
Region
Forward primer (5? to 3?)
Reverse primer (5? to 3?)
Promoter
TAACAGGCATAATCTTTGGTCT
CTGAAATAAAGTACAGACCCT
59
498
Promoter
TATGTGAGAGAAGGGTCTGTA
CTACAGGTTACATTGGCATTT
59
389
Exon 1
AAATGCCAATGTAACCTGTAG
AAGGGCTCAGAATGTAAGCG
54
533
Exon 2
TCCTTAGGCTAGAATTTGTGT
CAAAGTGAGTCTCAAGACATT
57
724
Exon 3
TGGCTGAGTAGTAGTACCTG
ATCCTCACTATCAACATTTTCA
53
568
Exon 4
TGAGTGGTCTAATGTAGGTGA
AGGTGTAAGTGTTGAGGTCTT
59
623
Exon 5
ATCTTTCTTGCTGGACACTTC
TATTAAGGAATTTGTTACAGGG
54
674
Exon 6
TTAAGAGTTTACAAGTAGTTAAA
AAGCAATTTTACTAGATGCCAA
53
413
Exon 7
CTTCTTTGTATTTAGGTAATGTA
ATAGTATAAATAGGAGCTGGAT
58
500
Exon 8
TTCCTAGACAGTATCTGTTGC
CTTCCACTTGTTATGTGCTCA
59
632
Fragment size (base pairs)
Exon 9
AGTTACAAAACAGCACTTACG
TCAGGAACTCATCTAAAATAAG
57
643
Exon 10
TTGATAGGTGCAGCAAACCAC
GGAATAAAAGAATGTGTTTGAG
55
581
Exon 11
TCTTTTTGATATATGTCTATCAT
AGTCAAATGAGGTGCTTCTTA
57
514
Exon 12
TTGTCCAAAAGAGTATGTGCT
CAGCCTTGAGAGTTCATAGT
48
708
Exon 13
GTTCTAACCACTTCCTCATAG
TTTTTTTTTTTTCATCATACCTAGT
57
365
Exon 14
TCCTTTTTACCATTCAGGCTTA
ACTAAAATGAGATACGAGATTG
48
573
Exon 15 3?UTR
GATGGCTTAACAGGGCTTGA
AGGCTTATTTATACTTCCACC
53
617
CACATCTTTTATGGTGGAAGT
TGCGGCAAATGATCTAGGAA
57
641
UTR, untranslated region
Table 2. Primer sequences and annealing temperatures for amplifying the SLCO1B3 gene for sequencing Region
Forward primer (5? to 3?)
Reverse primer (5? to 3?)
Annealing temperature (9 C)
Fragment size (base pairs)
Promoter
GCTGCAACTGTATCAACAAC
TCCCTACATGTTACATTAGC
55
549
Promoter
ACAGCCATGTGCCTGAGATA
CTAGGAGTTCACCCCAGAAGC
60
583
Exon 1
TAGGCTTCTGGGGTGAACTC
GTGTGGAGAAGACACAGAGC
65
327
Exon 2
GGTCAGGAAATAGCAGGCCC
CCAAATCATCTCATGTACCCC
60
585
Exon 3
CAACTTGTATAGGGAAAAATGGG
AAAGCATGCATGAACAGTTTG
58
355
Exon 4
CCCAAGTGCTTTCTTTGCAT
CCTCTCAAAAGGTAACTGCC
60
457
Exon 5
TTTGGAGAAGACAGCGGTTC
AAATGCCCTTGGTATACAATAA
58
617
Exon 6
TGTTTTCTTTGTGCCCTTCC
AAACAGAGATCCCAGTGCAAA
60
578 632
Exon 7
TTCCCTGGATCTACCCTTGA
CTTTCGCAAAGCAAACATCA
60
Exon 8
CAATTGCAAGATGTCATCAACC
CTCACTTCTCCGTTTCATTGAG
60
500
Exon 9
TTATTTTGAGCAAAGGTCGC
CAAATGCAGAACAACGATGA
53
480
Exon 10
GAATGGGTGAATTTGGTTGA
CAACCGATGTTGCTTTTCCT
57
506
Exon 11
GCCATTTTGACATCAGCAAA
CACTTCACAGCTATCACGAGGAC
61
514
Exon 12
CAGGGAGCTATTTTGCCTTCACTA
GGCTCATTAGGATGCAACGCAA
56
681
Exon 13
CCAGGGAGAGGAATGATGCTGAT
GACCACTCAATTTTCCCGTTCC
56
330
Exon 14
CGCTCAGTTACATTTGAAGCA
CTGCAGCTGGTCATTCTGTG
60
543
Exon 15 3?UTR
CCTCCCAAAGGAAGGCTAGA
CTAACCATGGGTACTCCCA
56
626
GGGAGTACCCATGGTTAGGA
CCACTTCCATGGGTCCAGTA
62
502
510
Andr áee-Anne BOIVIN, et al.
SLCO1B3 sequences reported in GenBank (NC_000012 regions 21173880 to 21284400 and 20853800 to 20961400, respectively). Tables 1 and 2 describe the various annealing temperatures and primer pairs used to amplify the promoters, the 5?- and 3?-untranslated regions (UTR), the entire coding regions as well as the intron-exon junctions of the SLCO1B1 and SLCO1B3 genes. Amplicons were generated in a total volume of 30 mL using 200 ng of genomic DNA, 2.5 units of AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA), 250 mM each deoxynucleoside-5?-triphosphate multiply symbol (Invitrogen, Carlsbad, CA), 0.5 mM each primer, 1× PCR Buffer (Applied Biosystems) and 1.5 mM MgCl2 (Applied Biosystems); exceptions to the above protocol were SLCO1B1 exon 9 and SLCO1B3 exon 1 amplifications, which both required 2.5 mM MgCl2. PCR cycling conditions included a 10 min initial denaturation step at 959C followed by 40 cycles of the following: 50 s at 959C, 50 s at an appropriate annealing temperature, 50 s at 729C, and a final extension step at 729C for 10 min. The nucleotide sequences of the PCR products were determined by direct sequencing using BigDye terminator cycle sequencing reactions (Applied Biosystems). The primers employed for sequencing were the same as those for the initial PCR amplification. The reaction products were run in an automated DNA sequencing ABI PRISM 3100 capillary sequencer (Applied Biosystems). All PCR products were sequenced in both directions. Sequences were analyzed using Lasergene software (DNA Stars, Madison, WI) and polymorphisms were identified as compared with the reference sequences of SLCO1B1 and SLCO1B3 mentioned above. DNA sequences of the promoter regions were analyzed with the TESS interface (http://www.cbil.upenn.edu/tess) for putative transcription factor binding sites using the TRANSFAC database. Genotypic frequencies were compared with HardyWeinberg expectations using the chi-squared test. Haplotype reconstruction was performed by use of the Bayesian statistical method implemented in PHASE, version 2.1.1,17,18) using polymorphisms with a minimum allele frequency of 5% in the promoter, 5?-, and 3?-UTR regions; coding regions (non-synonymous); and SLCO1B3 intron 4: －3 CÀT. Exceptionally, the SLCO1B1 －11187 GÀA variant, observed in 3.7% of white canadians, was also included for analysis. We applied the algorithm five times using different randomly generated seeds, and consistent results were obtained across runs.
Results and Discussion SLCO1B1 polymorphism and haplotype analysis: The DNA sequence analysis of the SLCO1B1 gene in 41 white Canadians revealed the presence of 49 polymorphisms, including 18 novel variants, with allelic frequencies ranging from 1.2% to 78% (Table 3). Among these variants, 3 were found in the promoter region, 2 in
5?-UTR, 27 in introns, 9 in coding regions and finally 8 polymorphisms were detected in 3?-UTR. The novel variants were found mainly within regulatory regions such as the promoter, 5?- and 3?-UTR, as well as in introns. The promoter nucleotide －10499 A is located within the putative consensus binding site of the nuclear factor 1 (NF-1) regulatory transcription factor. Interestingly, the －10499 C variant, found in 6.1% of the population, predicted the creation of a new binding site for the human ubiquitous transcription factor E12, as suggested by a search in the TRANSFAC database. Among the 27 intron polymorphisms, none were found within splice acceptor sites. Six nucleotide substitutions in coding regions are nonsynonymous mutations. The relatively frequent amino acid substitutions Asn130Asp (50%), Pro155Thr (20.7%) and Val174Ala (18.3%) are situated in the 2nd extracellular loop of the protein. Gly488Ala (1.2%) and Leu643Phe (6.1%) are located in the 5th extracellular loop and 12th transmembrane-spanning domain, respectively. Finally, the substitution from an arginine residue to a termination codon at position 580 leads to a truncated protein missing half of its 11th transmembrane domain and its subsequent components and was observed in 1.2% of individuals. The genotypic distribution of SLCO1B1 variants was in Hardy-Weinberg equilibrium. The allelic frequency of these variants in white Canadians was similar to that reported previously in white Europeans.4,6,15) The Asn130Asp substitution was found at a lower frequency (50%) than that previously observed in Japanese (53.7 to 67%), Chinese (79.5%) and African American (74%) subjects.4,5,8,9) The Pro155Thr variant observed in 20.7% of white Canadians was found in only 2% of African Americans4) and was absent in Japanese and Chinese populations.8,9) The Val174Ala substitution was found at a slightly greater frequency (18.3%) than that previously reported in Japanese subjects (11 to 17.5%) and much greater frequency than reported in African-Americans (2%).4,5,8,15) The Gly488Ala variant was previously reported in 9% of AfricanAmericans,4) and the Arg580stop mutant was found in 0.8% of Japanese subjects.8) The allelic composition of SLCO1B1 haplotypes and their distribution in white Canadians is shown in Table 5. In total, we identified 10 different haplotypes. The wild type H1 was the most frequent haplotype observed (42.1%). Haplotypes 5, 6, and 9 contain the Asn130Asp and Val174Ala variants that in combination are associated with reduced OATP1B1 activity11,12,15,16) and accounted for 18.4% of the variability in the white Canadian population. Nozawa and co-workers5) have also identified a haplotype containing Asn130Asp and Val174Ala variants in 10.3% of Japanese subjects. Interestingly, this haplotype exhibited decreased transport activities for irinotecan as well as for pravastatin, estrone-3-sulfate, and estradiol-17b-glucuronide.12) In addition to these 2nd
511
SLCO1B1 and SLCO1B3 Genetic Diversity in White Canadians
Table 3. Distribution of SLCO1B1 polymorphisms in white Canadians and other ethnic groups Allelic frequencies (%) Location
Nucleotide substitution
Promoter
－11259 AÀGa －11187 GÀA －10499 AÀC
Promoter Promoter 5?UTR 5?UTR Intron 2 Intron 2 Intron 2 Intron 2 Intron 3 Intron 3 Intron 4 Exon 5 Exon 5 Exon 5 Intron 5
Amino acid variation
rs4149021
3.0
rs12812795
＋97 CÀA 388 AÀG 411 GÀA 463 CÀA ＋160 CÀT
7.3
Intron 13 Intron 13
rs2291073 rs2291074
25.6 Asn 130 Asp
50.0
Ser 137 Ser
22.0
13.1
20.7
23.0
53.7–67.0
74.0
rs2306283
0
2.0
rs11045819
rs11045818
22.0
rs11045820
25.6
rs4149044
26.8
rs4149045
40.2
rs4149046
61.0
56.0
rs4149057
Phe 199 Phe
46.3
46.4
rs2291075
39.0
47.0
rs2291076
9.0
rs59502379
13.4
12.0
rs4149070
13.4
12.0
rs4149071
－75 AÀdel －68 GÀA
18.3
28.0
rs4149100
6.1
4.0
rs4149072
－173 TÀAa －171 GÀCa
22.0
0.8
rs71581941
－75 AÀGa 1738 CÀT －198 GÀAa
1.2
rs4149056
2.4
22.0 1.2 Arg 580 STOP
1.2 1.2 22.0 18.3
Intron 14 Exon 15 3?UTR
1929 AÀC 2122 TÀGa
3?UTR
2503 GÀTa
1.2
3?UTR 3?UTR
2515 TÀG 2525 AÀCa
48.8
rs4149080
22.0 1.2 Leu 643 Phe
6.1
4.0–9.0
13.0
rs34671512
1.2 48.8
rs4149087
23.2
3?UTR
2539 AÀG
48.8
3?UTR 3?UTR
2651 GÀAa 2747 AÀGa
2.4 23.2
3?UTR
2772 TÀC
7.3
del, deletion polymorphism; novel variant
2.0
1.2 Gly 488 Ala
－159 CÀAa －97 GÀC
a
11–17.5
1.2
－95 CÀTa －64 AÀGa
Intron 14
rs4149036 51.4
Pro 155 Thr
Intron 11 －323 GGGAATGGAAAA －312Àdela －170 CÀG Intron 11 －89 TÀC Intron 11
Intron 13
8.5 2.4
Leu 191 Leu
1463 GÀC
Exon 13
rs12303784
1.2
14.0–22.2
Exon 11
Intron 12
rs2010668
18.3
Intron 10
Intron 12
rs50710386
2.0
＋23 AÀGa ＋28 AÀGa
Intron 12
rs4149015
8.0
4.9
571 TÀC 597 CÀT ＋33 CÀT
Intron 11
4.0
6.1
1.2
Exon 6
Intron 11
3.7
＋203 AÀT ＋248 AÀG －106 TÀCa ＋89 TÀG ＋224 AÀG
Val 174 Ala
Intron 9
Identity to dbSNP
1.2
521 TÀC
Intron 7
AfricanAmericans4,15)
78.0
Exon 6 Exon 6
Japanese5,8,22)
－155 TÀG －119 TÀCa ＋193 AÀC
Intron 5
Intron 5
White Europeans4,6,15,21)
1.2
＋165 AÀT ＋189 GÀA ＋191 GÀA
Intron 5
White Canadians
rs4149088
rs11045893
512
Andr áee-Anne BOIVIN, et al.
Table 4. Distribution of SLCO1B3 polymorphisms in white Canadians and other ethnic groups Allelic frequencies (%) Amino acid variation
Identity to dbSNP
Location
Nucleotide substitution
Promoter
－5838 CATACACA －5831Àdela －5828 AÀCa －5538 TÀCa
19.5
－28 ATATTCACTTGGTATCTG －11Àdel －7 TTTA－4Àdel ＋72 CÀTa ＋226 TÀCa －138 TÀC
24.4
19.6
rs4149157
24.4
19.6
rs4149158
86.6
rs4149114
－77 AÀG －63 TÀC
86.6
rs4149115
Promoter Promoter 5?UTR 5?UTR Intron 2 Intron 2 Intron 3 Intron 3 Intron 3 Exon 4 Intron 4 Intron 4 Intron 5 Intron 5 Intron 5 Intron 5 Intron 5 Intron 5 Intron 5 Intron 5 Intron 6 Intron 6 Exon 7 Intron 7 Intron 7 Intron 7 Exon 8 Intron 8 Intron 11 Intron 11 Exon 12 Exon 12 Intron 12 Intron 12 Intron 13 Exon 14 Intron 14
White Canadians
White Europeans14,19)
Japanese7,22)
AfricanAmericans19)
25.6 9.8
92.7 26.8
86.6
334 TÀG ＋76 GÀA
Ser 112 Ala
－3 T À C ＋67 CÀG
86.6
rs4149116 81.0–89.0
72.8
41.1
rs4149117
72.0
rs4149118
86.6
rs3764009
86.6
rs3764008
＋91 TÀA ＋106 AÀGa
86.6
rs3764009
＋197 GÀA ＋214/＋215 ins T ＋271 CÀTa
86.6
－141 CÀAa －103 CÀGa ＋34 CÀTa －80 AÀGa
86.6
14.6
86.6 86.6 1.2 86.6
699 GÀA ＋27 TÀGa ＋76 CÀGa
Met 233 Ile
Gly 256 Ala
41.1
rs7311358
12.2
rs60140950
1.2
rs4149139
1.2
rs4149144
86.6
rs2053097
Ala 519 Ala
86.6
rs2053098
Thr 550 Ala
1.2 86.6
＋250 TTGA ＋253Àdela ＋55 CÀT
rs2053099
86.6
1833 GÀA ＋263 CÀTa
Gly 611 Gly
1980 GÀA
Ser 659 Ser
2824 AÀGa
72.8
1.2
1648 AÀGa ＋147 AÀC
3?UTR
81.0–89.0
13.4
＋63 AÀT －82 TÀG 1557 AÀG
2464–2465 ins Aa
86.6 1.2
＋118 CÀGa 767 GÀC －34 AÀT
Exon 15 3?UTR
rs4149119
86.6
86.6
rs4149153
87.8
rs3764006
12.2 2.4
rs60571683
24.4 1.2 a
del, deletion polymorphism; ins, insertion polymorphism; novel variant
extracellular loop variants, haplotypes 6 and 9 contain an other polymorphism in the promoter that is predicted to modify transcription factor binding sites, and 3?-UTR variants that may affect mRNA expression. Further in vitro studies will be needed to assess the possible effects of these promoter and 3?-UTR variants on SLCO1B1 tran-
scriptional and translational activities. SLCO1B3 polymorphism and haplotype analysis: We observed the presence of 41 SLCO1B3 polymorphisms, including 19 novel variants, with allelic frequencies ranging from 1.2% to 92.7% in the white Canadian population. As with SLCO1B1, the SLCO1B3 variants were
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SLCO1B1 and SLCO1B3 Genetic Diversity in White Canadians
G
H7
G
A
H8
G
H9
G
A
H10
A
G
C
C
C
G G
C
G
G
9.2% 15.8%
G
G
C
G
C
5.3%
C
2.6%
C
Other
G
G
G
A
T
T
C
G
H1
G
T
A
G
48.7%
2.6% 2.6%
H2
G
T
A
C
—
—
G
T
A
H4
C
A
2.6%
H3
—
—
G
T
A
C
C
—
—
G
T
A
A
6.6%
H5
C
H6
—
—
T
A
—
A
6.6%
—
H9
T
A
H8
G
C
5.3%
—
C
G
H7
C
6.6% 2.6%
A
5.3%
H, haplotype; UTR, untranslated region
found mainly within regulatory regions such as the promoter, 5?-UTR and 3?-UTR, and in introns. Three relatively frequent new variants in the promoter at position －5538 T/C (9.8%), －5828 A/C (25.6%) and a cluster of 8 nucleotide deletions from position －5838 to －5831 (19.5%) were identified in our sample population, but none of these promoter variants are located within putative binding sites for transcription factors and their significance remains unknown. Two linked clusters of deletions at positions －7 to －4 and －28 to －11 in the 5?-UTR region, previously observed in 19.6% of Japanese subjects7) were found at a frequency of 24.4% in white Canadians. These deletions, located immediately upstream of the translation initiation site, may likely interfere with the mRNA stability and/or the translation initiation since the －7 to －4 deletion is partly positioned within the Kozak consensus sequence, which must be recognized by the ribosomes for the initiation of translation to occur. The intron 4 (－3 CÀT) variant, in proximity to the splicing site of exon 5, is linked with exon 4 Ser112Ala and exon 7 Met233Ile variants located in the 3rd transmembrane-spanning domain and the 3rd extracellular loop of the protein, respectively. The frequencies for the Ser112Ala and Met233Ile linked mutations in white Canadians (86.6%) were similar to those previously reported in white Americans (88%) and Europeans (81%), but greater than those observed in Japanese subjects (72.8%), African Americans (41.1%) and Ghanaians (37.3%).7,19) The amino acid substitution Gly256Ala, located in the 3rd extracellular loop, was observed in 12.2% of our sample population, whereas the novel Thr550Ala variant in the 10th transmembrane-spanning domain was
Wild Type
Other
Allelic frequency
3? UTR: 2464–2465
G
Gly 256 Ala
G
G
C
G
G
H6
T 42.1% 5.3% 5.3%
G
Met 233 lle
A
Intron 4: －3
A
S er 112 Ala
H5
A
5?UTR: －7–4Àdel
H4
T
G
5?UTR: －28 –11Àdel
Promoter: －5538
Promoter: －5828
H3
Promoter: －5838–5831Àdel
A
Table 6. Distribution of SLCO1B3 haplotypes in white Canadians
Allelic frequency
Leu 643 Phe
T
3? UTR: 2772
Val 174 Ala
C
3? UTR: 2747
Pro 155 Thr
A
3? UTR: 2539
Asn 130 Asp
H2
A
3? UTR: 2525
Promoter: －10499
Wild type G H1
3? UTR: 2515
Promoter: －11187
Table 5. Distribution of SLCO1B1 haplotypes in white Canadians
7.9% 3.9% 7.9%
5.3%
Promoter －5838 CATACACA －5831Àdel 5?-UTR －28 ATATTCACTTGGTATCTG －11Àdel 5?UTR －7 TTTA －4Àdel 3?UTR 2464–2465; insertion polymorphism H, haplotype; UTR, untranslated region; —, Deletion polymorphisms
found in only 1.2% of individuals. The genotypic distribution of SLCO1B3 variants was in Hardy-Weinberg equilibrium. Haplotype reconstruction of SLCO1B3 demonstrated similar genetic diversity as observed with SLCO1B1 (Table 6). The haplotype equivalent to the reference wild-type sequence has not been detected in our population. H1 contained a single cluster of linked Ser112Ala, intron 4 (－3 CÀT) and Met233Ile variants and was the most frequent haplotype (48.7%) in white Canadians. OATP1B3 is not only expressed on hepatocytes but it has also been identified on human tumor cells derived from brain, lung, colon, stomach, pancreas, gallbladder20), and more recently in prostatic cancer.14) The Ser112Ala and Met233Ile polymorphisms were recently associated with an improved survival rate in patients with prostatic cancer due to impaired testosterone transport in cancer cells featuring these mutations. Intriguingly, both variants had to be present, as was found in many haplotypes (H1-H7), for the effect to be observed, probably because of an additive effect.14) Until now, the effect of the Gly256Ala variant on OATP1B3 activity remained unknown, but it can be postulated that it might, like many other variations, influence the substrate spectrum of the transporter. One limitation of the present study is the relatively small number of subjects analyzed, which may have
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resulted in underestimation of the prevalence of genetic variants. Nevertheless, our data suggest that SLCO1B1 and SLCO1B3 genes are highly polymorphic in the white Canadian population. Comparison with other studies revealed that the distribution of SLCO1B1 and SLCO1B3 polymorphisms and haplotypes differs widely according to the population. Further studies will be needed to assess the possible effects of the novel mutations on OATP1B1 and OATP1B3 transcription, translation, and function. Data from this survey provide invaluable information for the design of future studies to investigate the contribution of SLCO1B1 and SLCO1B3 polymorphisms to the interindividual variability of OATP1B1 and OATP1B3 substrate drug responses in the Canadian population.
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