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Genetic Variations of the ABC Transporter Gene ABCB11 Encoding the Human Bile Salt Export Pump (BSEP) in a Japanese Population
Drug Metab. Pharmacokinet. 24 (3): 277–281 (2009).
SNP Communication Genetic Variations of the ABC Transporter Gene ABCB11 Encoding the Human Bile Salt Export Pump (BSEP) in a Japanese Population Su-Ryang KIM1, Yoshiro SAITO1,2,*, Masaya ITODA1, Keiko MAEKAWA1,2, Manabu KAWAMOTO3, Naoyuki KAMATANI3,**, Shogo OZAWA1,4,† and Jun-ichi SAWADA1,2 1Project
Team for Pharmacogenetics, National Institute of Health Sciences, Tokyo, Japan of Functional Biochemistry and Genomics, National Institute of Health Sciences, Tokyo, Japan 3Division of Genomic Medicine, Department of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, Tokyo, Japan 4 Division of Pharmacology, National Institute of Health Sciences, Tokyo, Japan
2Division
Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk
Summary: The bile salt export pump (BSEP) encoded by ABCB11 is located in the canalicular membrane of hepatocytes and mediates the secretion of numerous conjugated bile salts into the bile canaliculus. In this study, 28 ABCB11 exons (including non-coding exon 1) and their flanking introns were comprehensively screened for genetic variations in 120 Japanese subjects. Fifty-nine genetic variations, including 19 novel ones, were found: 14 in the coding exons (6 nonsynonymous and 8 synonymous variations), 4 in the 3?-UTR, and 41 in the introns. Three novel nonsynonymous variations, 361CÀA (Gln121Lys), 667CÀT (Arg223Cys), and 1460GÀT (Arg487Leu), were found as heterozygotes and at 0.004 allele frequencies. These data provide fundamental and useful information for genotyping ABCB11 in the Japanese and probably other Asian populations. Keywords: BSEP; ABCB11; direct sequencing; novel genetic variation; amino acid change
cholestasis of pregnancy (ICP) and contraceptive-induced cholestasis (CIC), where hepatocellular accumulation of bile salts is observed.1,3) The ABCB11 gene encoding human BSEP consists of 28 exons (including non-coding exon 1) and spans approximately 108 kb on chromosome 2q24–31. Similar to other ABC transporters, BSEP has 12 transmembrane domains (TMDs) and Walker A and B motifs for binding and hydrolysis of ATP in large cytoplasmic domains.4) Human BSEP is a 1,321-residue protein sharing 82% amino acid identity with its rat homologue (rBsep)5) and 68% identity with the marine skate, Raja erinacea, homologue
Introduction The bile salt export pump (BSEP) is a member of the ATP-binding cassette (ABC) transporter family B. BSEP is predominantly expressed in the liver and located in the canalicular membrane of hepatocytes and mediates the secretion of numerous conjugated bile salts into the bile canaliculus.1,2) Inherited and acquired dysfunction of BSEP can lead to a wide spectrum of liver disease such as progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis type 2 (BRIC2), drug-induced cholestasis (DIC), intrahepatic
Received; February 9, 2009, Accepted; March 18, 2009 *To whom correspondence should be addressed: Yoshiro SAITO, Ph.D., Division of Functional Biochemistry and Genomics, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan. Tel. ＋81-3-5717-3831, Fax. ＋81-3-5717-3832, E-mail: yoshiro＠ nihs.go.jp **Present address: Institute for Data Analysis, StaGen Co. Ltd, Orashion Building 9F, 4-31-10 Kuramae, Taito-ku, Tokyo 111-0051, Japan. † Present address: Department of Pharmacodynamics and Molecular Genetics, Faculty of Pharmaceutical Sciences, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba-cho, Shiwa-gun, Iwate 028-3694, Japan As of February 9, 2009, the novel variations reported here are not found in the database of Japanese Single Nucleotide Polymorphisms (http://snp.ims.u-tokyo.ac.jp/), dbSNP in the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/SNP/), or the PharmGKB Database (http://www.pharmgkb.org/). This study was supported in part by the Program for the Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation, and the Health and Labor Sciences Research Grants from the Ministry of Health, Labor and Welfare.
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(sBsep).6) Recently, several genetic polymorphisms in ABCB11, including 10 nonsynonymous SNPs, have been identified in Caucasians, African-Americans and Japanese.7,8) Among them, the common nonsynonymous SNP, 1331CÀT (Ala444Val), may be associated with increased protein expression.9,10) It has been reported that serum bile acid levels in carriers of the 1331CC genotype are higher than those of the 1331TT genotype.11) The 1331C allele is more frequent in patients with DIC, ICP and CIC than the 1331T allele.11,12) In addition, more than one hundred ABCB11 mutations have been identified in BSEP deficiency syndrome patients.1,3,9,13) In this study, all 28 exons and their surrounding introns were resequenced for comprehensive screening of ABCB11 genetic variations. Sequence analysis detected 59 variations including 3 novel nonsynonymous ones from 120 Japanese subjects.
Materials and Methods Human genomic DNA samples: One hundred twenty Japanese healthy volunteers participated in this study and provided written informed consent. The ethical review boards of the Tokyo Women's Medical University and the National Institute of Health Sciences approved this study. Genomic DNA was extracted from Epstein-Barr virus transformed B lymphocytes from volunteers by standard methods. PCR conditions for DNA sequencing: First, multiplex PCR was performed to amplify 5 fragments including 28 exons of ABCB11 from 200 ng of genomic DNA, using 1.25 units of Z-Taq (Takara Bio. Inc., Shiga, Japan) with 0.25 mM each of the mixed primer designed in the intronic regions as listed in Table 1 (1st PCR). The first PCR conditions consisted of 30 cycles of 989C for 5 sec, 559C for 5 sec, and 729 C for 190 sec. Next, each exon was amplified separately by Ex-Taq (0.625 units, Takara Bio. Inc.) with appropriate primers (0.25 mM) designed in the introns (Table 1, 2nd PCR). The conditions for the second round PCR were 949C for 5 min, followed by 30 cycles of 949 C for 30 sec, 559C for 1 min, and 729 C for 2 min, and then a final extension at 729C for 7 min. Following the PCR, the products were treated with a PCR Product Pre-Sequencing Kit (USB Co., Cleveland, OH, USA) and directly sequenced on both strands using an ABI BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) with the sequencing primers listed in Table 1 (Sequencing). Excess dye was removed by a DyeEx96 kit (Qiagen, Hilden, Germany), and the eluates were analyzed on an ABI Prism 3700 DNA Analyzer (Applied Biosystems). All variations were confirmed by sequence analysis of PCR products generated by new amplification of the original genomic DNA templates. Rare SNPs found in single patients as heterozygotes were confirmed by sequencing PCR fragments
produced by amplification with high fidelity DNA polymerase KOD-Plus- (TOYOBO, Tokyo, Japan). Hardy-Weinberg equilibrium analysis was performed by SNPAlyze software (Ver 5.1, Dynacom Co., Yokohama, Japan).
Results and Discussion Sequence analysis from 120 Japanese subjects resulted in the identification of 59 genetic variations, including 19 novel ones (Table 2). Of these, 14 were located in coding exons (6 nonsynonymous and 8 synonymous variations), 4 in the 3?-UTR, and 41 in introns. All detected variations were in Hardy-Weinberg equilibrium (pÀ0.05), except for IVS12＋122GÀA and IVS14＋ 128delA. Deviation from Hardy-Weinberg equilibrium for both variations resulted from a slightly larger number of homozygotes than expected. Of the 6 nonsynonymous variations, 3 were novel: 361CÀA (Gln121Lys), 667CÀT (Arg223Cys), and 1460GÀT (Arg487Leu). All of these variations were found as heterozygotes with frequencies of 0.004. The nonsynonymous SNPs 361CÀA (Gln121Lys), 667CÀT (Arg223Cys), and 1460GÀT (Arg487Leu) are located in the extracellular loop between TMD1 and TMD2, TMD3 and the large cytoplasmic loop between TMD6 and TMD7, respectively.4) Using the PolyPhen program (http:/ /genetics.bwh.harvard.edu/pph/) to predict functional effects of the three amino acid substitutions, two substitutions, Arg223Cys and Arg487Leu, are expected to alter protein function based on PSIC (position-specific independent count) score differences derived from multiple alignments. The Arg223 residue located in TMD3 is well conserved among BSEP proteins including the marine skate, mouse and rat. The Arg487 residue is located in the first nucleotide binding fold (NBF1) domain of the large cytoplasmic loop between TMD6 and TMD7 and highly conserved among human ABC transporter family B proteins, such as ABCB1 and ABCB4. The two substitutions at the same position, Arg487His and Arg487Pro, were found in patients with severe BSEP deficiency syndrome, PFIC2.13,14) Thus, it is possible that bile acid homeostasis in the subject with Arg223Cys or Arg487Leu may be disturbed. However, we have not determined blood bile acid levels in the volunteers. The functional significance of these 3 novel nonsynonymous variations should be clarified in the future. The three known nonsynonymous variations, 896GÀA (Arg299Lys), 1331CÀT (Ala444Val), and 2594CÀT (Ala865Val), were detected at 0.004, 0.267, and 0.004 frequencies, respectively, which are similar to the previously reported Japanese data.7) One variation, 1331CÀT (Ala444Val), was found 14 residues upstream of the Walker A motif in the NBF1 domain in the large cytoplasmic loop between TMD6 and TMD7. This variation results in increased protein expression.9) Its fre-
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Genetic Variations of ABCB11 in Japanese
Table 1. Primer sequences used in this study Amplified or sequenced region 1st PCR
Exons Exons Exons Exons Exons
2nd PCR
Sequencing
1 to 5 6 to 10 11 to 18 19 to 21 22 to 28
Forward primer (5? to 3?)
Reverse primer (5? to 3?)
CAAGGCTGTTTCAAATGTTC ATTGTCCTGGTGAATGAACT GTGTCTGTATTGTTTTTGGC CCTCTGGGTACTGATTAAGGA TATACCTGCTACAACATGCC
ACCTCCCTAGTGACGTTAAG ATGGCATTCACAGCAACCTG TGTCTGTGAAACCTAATTGG CTTCGACGCCTTCAAGATTC GAGTGCTTTTCTGCCATATT
Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Exon 6 Exon 7 Exon 8 Exon 9 Exon 10 Exon 11 Exon 12 Exon 13 Exon 14 Exon 15 Exon 16 Exon 17 Exon 18 Exon 19 Exons 20 and 21 Exon 22 Exon 23 Exon 24 Exon 25 Exon 26 Exon 27 Exon 28
TGTTTGGGGTTATTGCTCTG GGCTCTTTCAGGGAGTTATT AGAGAGACAATATGAGCAGGA CATCTCTGTGAATCGCTAGT AGATAATTGATGTACCGTGC AGAGACTAAAGTGGACCAAG CCACTTTTAGCAGCAGATGA ATTGGCTATCAGGAAAGAGG TTCTTCCTCCTGTCAATGAT GGCAGCTCTGTTAAGTATCG CAGTGGAAAACTTGGAGATG GCAAACTAAGAGGCACAATA CTTTGGGGGCATACATAAAC TTTATGTTTCCACAATGAAT GTAAACCCTGGAATAAAAGC AGACTAGCTTATGATATTTTTTA GACAAGTCCCGAGTTTTATT ACTCTGAATCTGGGTCCAAC TTCAAGCCACAGCAATAGTT CCACAGCTTACATTAGGGTT GGATACAGAGTTTTTGTTGG AATTGTCTCGTGGTCCAGTT TTGAAAACTGCCAGTCCCTT AGCATGGTTTGAAGGTATCT AGCCTTGGGATTGTTAGTCT ATTTGACATCCTCCATCCTA GCACTGCCATTGAAATCACT
GTAGCGTTGTTTACACTGGA TACGTTACATGGATTCTAGG GAATACAGAGGGGACATTTGA ATGATAACCATGGGCTTAGT AGATAGAAGGAAACTTGAGG CATCTGAGAGAAGAATCCCT CACCTGGACAGCTAAATAAC ATTTGACACTGGGGAGTAAT AGGCTTGGGACTTCTTATAG TCCACAGACAGACTCCATAA AGTGTTGCTGAATTAAGGGC TGGTCTGTTGCCTGAGATAA CTGACTGAAATGTTGCTATG TCAGATGAAAGGAAACACTC TTCAACTGTGAGGAAGATTG TAGAAGCCTTGGTTTTAGTG CATGAATTGGGGAGAGAAAT CTAAGACAATTAACCCTCCG CGAGCCCAAATGAGAGTTCT TGCTTTAGCTGCTGTTTGTC GGCTCATACTCAGAGTCTCG GTCAGAAGAACCGCCCTGAT TGTGTCCATGTGTTCTGTTT ATCTGGAACTGGGGAAAATC TGACTGCATTTTGACTGTGA GCCAATTTCTACTGTTAACG TTTCCCTATCCTTAGCCTTA
Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon
CGTATGTCACTGAACTGTGC CAGGGAGTTATTAACCGATT ACAATATGAGCAGGAAGAAAG CTCTGTGAATCGCTAGTGAA TGATGTACCGTGCCGTTTAT GGCTTCCAACAACTGCTAAT TTAGCAGCAGATGACAGAGC TCAGGAAAGAGGCTTGATAA TTCCTCCTGTCAATGATGTT AGCTCTGTTAAGTATCGCCC TGGAGATGCTTTACAGAACA AGAGATACGCCAAAGATGTT ACACACAGACACCGAGTATC AGAACCACATTTCAAGAGGA ACCCTGGAATAAAAGCATAA AGTGTGTGTTGGGGTTTATG GTCCCGAGTTTTATTTATTG TGAATCTGGGTCCAACACTT ATCCCATAGACATTTGAGGT ACAGCTTACATTAGGGTTCA GATAAAGAGGAACAAAGGTC GAGTTTTTGTTGGACATGAT TGATCTCCAGCCACGTCTTT AACTGCCAGTCCCTTAAACA CATGGTTTGAAGGTATCTCA CTTGGGATTGTTAGTCTGTT GGAGGACTCACTCACTGTTC CAGGGAATACTTGCTTAATC AAAGGAGCCTACTACAAACT
AGCGTTGTTTACACTGGACA CCTACTGTTGCTGATTTTTT AGAAGGGATATTCCAAAAGGT GGCAAAGCCTATGACTGAAA ACTTGAGGCAGAGTAATTTC CTCTCCATTCTCTCATGTCC TGAAAAAGCCATGCCACATA TGCTAACTGTACTCAGGAAA CTTAGCTCCCTCTTGAACAA CTGAAGGCACCAAAGTAATA GAATTAAGGGCCTTGCGATA TGCCTGAGATAAGCCAACAC GCCACATCTGACTGTATCTG AGGAAACACTCATGGTACTT TCAGACTAGATGCATGAACC GCTTCAGTTGTTTCCTTTAC TGTCAATATAGCAACCAAGG ATTAACCCTCCGAAGTCCAG CTCAGCTTTGCCTTCTTACC GGCTATTTCTGAGGTCATCA TTAGCTGCTGTTTGTCAGTG ACTCAGAGTCTCGGAGCTTT CTGATGACCCACAGAATCTT GTTCAACTCCCACTTATGAG TTGTGAGGTAGAATCAGGTG ATTTTGACTGTGACCCATCA GTTAACGAATCAGAAAATTG TTAGCTTGGATTCCGATGTA ATCCTTAGCCTTAGAGATGA
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 28
Amplified length (bp) 18,817 11,729 16,682 14,559 14,122 351 435 412 534 604 610 592 521 453 534 443 464 513 584 559 657 407 594 550 683 635 593 512 519 541 420 957
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Table 2. Summary of ABCB11 variations detected in this study SNP ID This Study MPJ6_AB11001 MPJ6_AB11002 MPJ6_AB11003 MPJ6_AB11004 MPJ6_AB11005 MPJ6_AB11006a MPJ6_AB11007 MPJ6_AB11008 MPJ6_AB11009a MPJ6_AB11010a MPJ6_AB11011a MPJ6_AB11012 MPJ6_AB11013a MPJ6_AB11014a MPJ6_AB11015 MPJ6_AB11016 MPJ6_AB11017 MPJ6_AB11018 MPJ6_AB11019 MPJ6_AB11020 MPJ6_AB11021a MPJ6_AB11022a MPJ6_AB11023a MPJ6_AB11024a MPJ6_AB11025 MPJ6_AB11026a MPJ6_AB11027 MPJ6_AB11028 MPJ6_AB11029 MPJ6_AB11030 MPJ6_AB11031 MPJ6_AB11032a MPJ6_AB11033a MPJ6_AB11034 MPJ6_AB11035 MPJ6_AB11036 MPJ6_AB11037 MPJ6_AB11038 MPJ6_AB11039 MPJ6_AB11040 MPJ6_AB11041 MPJ6_AB11042 MPJ6_AB11043 MPJ6_AB11044 MPJ6_AB11045a MPJ6_AB11046 MPJ6_AB11047a MPJ6_AB11048a MPJ6_AB11049 MPJ6_AB11050 MPJ6_AB11051a MPJ6_AB11052 MPJ6_AB11053a MPJ6_AB11054a MPJ6_AB11055 MPJ6_AB11056 MPJ6_AB11057 MPJ6_AB11058 MPJ6_AB11059 a b c
Position dbSNP (NCBI) rs4148772 rs10199694 rs4148775 rs4148776 rs3815675 rs4148777
Reference
7, 8 8 7, 8 7, 8 7 7, 8
7 rs2287614 rs2287615 rs2287616 rs2287617 rs4148780 rs2287618
8 8 7, 8 7 8 7
rs4148783
8
rs55669065 rs2287622 rs2287623 rs2389605 rs4148786
rs2241340 rs2241341 rs55868238 rs2193831 rs2389612 rs4148795 rs4148796 rs853772 rs853773
7, 8 7, 8 8 7 7, 8 8 8 8 8 8 7, 8 8 7
rs11568379 rs497692
7, 8 7
rs7561903 rs579275 rs473351 rs3732038 rs495714 rs496550
7, 8 8, 15 8 8
Location
NT_005403.16
Intron 1 20084130 Intron 3 20082623 Intron 3 20080475 Intron 3 20080300 Exon 4 20080273 Intron 4 20079560 Intron 4 20079512 Exon 5 20079319 Exon 5 20079310 Exon 5 20079228 Intron 5 20062886 Intron 6 20062500 Intron 7 20059917 Exon 8 20059755 Intron 8 20056962 Intron 8 20056940 Exon 9 20056830 Exon 9 20056741 Intron 9 20056621 Intron 9 20052227 Intron 11 20045737 Intron 11 20045731 Intron 11 20042655 Exon 12 20042565 Intron 12 20042389 Intron 12 20042383 Intron 12 20039861 Exon 13 20039746 Intron 13 20039573 Intron 13 20039469 Intron 13 20038064_ 20038065 Exon 14 20037953 Exon 14 20037907 Exon 14 20037808 Intron 14 20037743 Intron 14 20037695 Intron 14 20037647 Intron 14 20036295 Intron 15 20035603 Intron 18 20030037 Intron 18 20030036 Intron 18 20024073 Intron 19 20023765 Exon 21 20010549 Intron 21 20010502 Intron 21 20002386 Intron 22 20002132 Intron 22 20002091 Exon 24 19998434 Intron 24 19998280 Intron 25 19996478 Intron 26 19992983 Intron 26 19992965 Intron 27 19989910 Intron 27 19989784 3?-UTR 19989314 3?-UTR 19989269 3?-UTR 19989182 3?-UTR 19989130
Novel variations detected in this study. Positions in cDNA (NM _003742.2). Numbers in parentheses indicate positions from the termination codon TGA.
From the translational initiation site or from the end of the nearest exonb
Nucleotide change
IVS1－50 IVS3＋83 IVS3－193 IVS3－18 108 IVS4－122 IVS4－74 270 279 361 IVS5－236 IVS6＋63 IVS7－107 667 IVS8－109 IVS8－87 807 896 IVS9＋108 IVS9－15 IVS11 ＋57 IVS11 ＋63 IVS11 －40 1248 IVS12＋116 IVS12＋122 IVS12 －93 1331 IVS13 ＋70 IVS13＋174 IVS13－87_－86 1460 1506 1605 IVS14 ＋32 IVS14 ＋80 IVS14＋128 IVS14－152 IVS15－124 IVS18 ＋97 IVS18 ＋98 IVS18 －17 IVS19＋127 2594 IVS21 ＋31 IVS21 －25 IVS22 ＋26 IVS22 ＋67 3084 IVS24 ＋25 IVS25＋115 IVS26＋101 IVS26＋119 IVS27－160 IVS27 －34 4202 (*236)c 4247 (*281)c 4334 (*368)c 4386 (*420)c
ACTTTGATTAAAG/AAAGAAAGAAGAG AAGCAGAGAATAC/TTTTCATGCACAT TGAGATTGAGCTA/GTACTGAAATCTC GTCTTTAAATCCT/CTATGTTTTTCTC CAGGTTACAAGAT/CGAGAAGAAAGGT CACTCAATTAAGG/ATGATTCCCATGA TGAGAATCTAGTA/TACTAAATTAAGT GACAGATGTTTTT/CATTGACTACGAC TTTTATTGACTC/TGACGTTGAGTTA AGTTCCCTCAACC/AAGAACATGACAA ATATGCATATTTT/CCTGTGATTGGTA ACTACAATGAGAT/GGCAATGTGTTGC ATCCAAGGGTGAT/CAGGGATAGAGAG CTTTTCATTCAGC/TGCATGACCTCGA GTTACAGTGAGAA/CTCTAATATTGTA GTATTAAACCCAT/AGCCACATGTTAA GTTTACGGACTAT/CGAGCTGAAGGCC GTGGTGAGAAAAG/AAGAGGTTGAAAG TCTGTGGCCTCCA/GGAGGAAGTACTT ATTGACTCAAGCG/ATTTTGTCTTCAC GGGGGTGGGGCAC/AAGAATGAACTCC GGGGCACAGAATG/AAACTCCTGAAGA TTGTGCATCTTAG/CTTTGAGTTTACA GTTGGATCGAATC/AAAGGGTGAAATT GTAATAGGGAATG/AGAGGTGTCTTTC GGGAATGGAGGTG/ATCTTTCTCTGAA CACACAGACACCG/AAGTATCAACACA ACCTCAACATGGC/TCATTAAACCAGG ATATTGATCAAAT/CAGAAAGGTGTAG TAACAGTGTTCAA/GTGAATAACCAGT CTCTATTTTTTC-/CTGCCCATTGGTC GCCATGACATTCG/TCTCTCTTAACAT TGGGATAGTGGAG/ACAAGAGCCAGTT TGCCAAGGAGGCC/TAATGCCTACAAC CCTGGGAGAAACC/TAAGAGGTCATAG TACACATTTCTTT/CTCGTATGATTCC TGTTTTAGTTTCA/-TGCCTGAAAAAG AGACAATAACCCA/GTCTGGGGAAGGG AATGTCTGCACAG/ACCTATTTAAGAA TTTTCTAGGTATA/GTATCTAGCAGTG TTTCTAGGTATAT/CATCTAGCAGTGT TGATTAATATAAA/CCCTCTCTCTGCT ATCTCTAAAGAAC/TGAAAAATTTCCT TTGCTACAGATGC/TTTCCCAAGTTCA AATAGAAGTATAT/GTAACTGCATTGG TGTGTCTGAGACG/AGGTTGATTGCTT TCTAATTTTCCCA/GTTCCTCATGGCT AACTGTTAAAAAC/TGAGTAGTACGAA TGTACTGAGTGCG/AACAGCTCTTGGA ATACTATGCAGCC/AATAAAAAAGGAT CTTGCCTAAGGCA/CCTTACCCCATGC CTGAAAATCCCAA/CATCCAAAATGTT AAATGTTCCAAAA/GTTCAAAAATTTT CACTGATGCATTG/ACATTCAGGGAAT GAGCAATCATGCG/ATCTTTGCATCAA ACTAGGGTCCATG/ATGAGGGAAAACC GCCACCACTCAGT/GGCTTCTCTGTGC AACTCCTCAAGGA/GCAGAGAACTGTC AGAGGCGGGTCTG/ATAACAGGCAATC
Amino acid change
Asp36Asp Phe90Phe Tyr93Tyr Gln121Lys
Arg223Cys Tyr269Tyr Arg299Lys
Ile416Ile
Ala444Val
Arg487Leu Glu502Glu Ala535Ala
Ala865Val
Ala1028Ala
Frequency
0.058 0.058 0.225 0.058 0.225 0.029 0.021 0.058 0.004 0.004 0.004 0.017 0.004 0.004 0.058 0.267 0.267 0.004 0.058 0.217 0.004 0.004 0.004 0.008 0.250 0.062 0.012 0.267 0.237 0.237 0.004 0.004 0.004 0.008 0.237 0.237 0.062 0.237 0.237 0.417 0.417 0.412 0.396 0.004 0.004 0.054 0.004 0.004 0.442 0.004 0.042 0.104 0.004 0.004 0.437 0.054 0.004 0.437 0.437
Genetic Variations of ABCB11 in Japanese
quency in Japanese (0.196–0.267) is slightly lower than that in Caucasians (0.405) and African-Americans (0.344) (described as g.44308TÀC [Val444Ala] in a previous paper).7) Two other known variations, 896GÀA (Arg299Lys) and 2594CÀT (Ala865Val), were detected only in the Japanese population at allele frequencies of 0.010 for 896GÀA and 0.024 for 2594CÀT, respectively.7) Both variations are located in the cytoplasmic loops between TMD4 and TMD5 and between TMD8 and TMD9. The variation 2873GÀA (Arg958Gln) previously reported in Japanese at a frequency of 0.011 was not detected in this study. Six variations previously reported in other ethnic groups were not detected: 616AÀG (Ile206Val; found with 0.011 frequency in African-Americans), 851TÀC (Val284Ala; 0.005 in Caucasians), 1846CÀG (Arg616Gly; 0.022 in AfricanAmericans), 1855AÀG (Thr619Ala; 0.011 in AfricanAmericans), 2029AÀG (Met677Val; 0.042 in Caucasians and 0.14 in African-Americans), and 2093GÀA (Arg698His; 0.005 in Caucasians).7) These variations might be ethnic-specific. In conclusion, 59 variations including 19 novel ones were identified in ABCB11 from 120 Japanese subjects. Three novel nonsynonymous variations result in amino acid substitutions. This information is useful for pharmacogenetic studies that investigate the association of ABCB11 variations with interindividual differences in bile salt processing and drug disposition.
Acknowledgments: The authors thank Ms. Chie Sudo for her secretarial assistance.
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port pump is highly conserved during vertebrate evolution and its expression is inhibited by PFIC type II mutations. Am. J. Physiol. Gastrointest. Liver Physiol., 281: G316–G322 (2001). Lang, T., Haberl, M., Jung, D., Drescher, A., Schlagenhaufer, R., Keil, A., Mornhinweg, E., Stieger, B., Kullak-Ublick, G. A. and Kerb, R.: Genetic variability, haplotype structures, and ethnic diversity of hepatic transporters MDR3 (ABCB4) and bile salt export pump (ABCB11). Drug. Metab. Dispos., 34: 1582–1599 (2006). Saito, S., Iida, A., Sekine, A., Miura, Y., Ogawa, C., Kawauchi, S., Higuchi, S. and Nakamura, Y.: Three hundred twenty-six genetic variations in genes encoding nine members of ATP-binding cassette, subfamily B (ABCB/MDR/TAP), in the Japanese population. J. Hum. Genet., 47: 38–50 (2002). Byrne, J. A., Strautnieks, S. S., Ihrke, G., Pagani, F., Knisely, A. S., Linton, K. J., Mieli-Vergani, G. and Thompson, R. J.: Missense mutations and single nucleotide polymorphisms in ABCB11 impair bile salt export pump processing and function or disrupt pre-messenger RNA splicing. Hepatology, 49: 553–567 (2009). Meier, Y., Pauli-Magnus, C., Zanger, U.M., Klein, K., Schaeffeler, E., Nussler, A. K., Nussler, N., Eichelbaum, M., Meier, P. J. and Stieger, B.: Interindividual variability of canalicular ATPbinding cassette (ABC)-transporter expression in human liver. Hepatology, 44: 62–74 (2006). Meier, Y., Zodan, T., Lang, C., Zimmermann, R., Kullak-Ublick, G. A., Meier, P. J., Stieger, B. and Pauli-Magnus, C.: Increased susceptibility for intrahepatic cholestasis of pregnancy and contraceptive-induced cholestasis in carriers of the 1331TÀC polymorphism in the bile salt export pump. World J. Gastroenterol., 14: 38–45 (2008). Lang, C., Meier, Y., Stieger, B., Beuers, U., Lang, T., Kerb, R., Kullak-Ublick, G. A., Meier, P. J. and Pauli-Magnus, C.: Mutations and polymorphisms in the bile salt export pump and the multidrug resistance protein 3 associated with drug-induced liver injury. Pharmacogenet. Genomics, 17: 47–60 (2007). Strautnieks, S. S., Byrne, J. A., Pawlikowska, L., Cebecauerova, D., Rayner, A., Dutton, L., Meier, Y., Antoniou, A., Stieger, B., Arnell, H., Ozcay, F., Al-Hussaini, H. F., Bassas, A. F., Verkade, H. J., Fischler, B., N áemeth, A., Kotalovaá , R., Shneider, B. L., Cielecka-Kuszyk, J., McClean, P., Whitington, P. F., Sokal, E., Jirsa, M., Wali, S. H., Jankowska, I., Pawlowska, J., Mieli-Vergani, G., Knisely, A. S., Bull, L. N. and Thompson, R. J.: Severe bile salt export pump deficiency: 82 different ABCB11 mutations in 109 families. Gastroenterology, 134: 1203–1214 (2008). Goto, K., Sugiyama, K., Sugiura, T., Ando, T., Mizutani, F., Terabe, K., Ban, K. and Togari, H.: Bile salt export pump gene mutations in two Japanese patients with progressive familial intrahepatic cholestasis. J. Pediatr. Gastroenterol. Nutr., 36: 647–650 (2003). Eloranta, M. L., Hakli, T., Hiltunen, M., Helisalmi, S., Punnonen, K. and Heinonen, S.: Association of single nucleotide polymorphisms of the bile salt export pump gene with intrahepatic cholestasis of pregnancy. Scand. J. Gastroenterol., 38: 648–652 (2003).
SNP Communication Genetic Variations of the ABC Transporter Gene ABCB11 Encoding the Human Bile Salt Export Pump (BSEP) in a Japanese Population Su-Ryang KIM1, Yoshiro SAITO1,2,*, Masaya ITODA1, Keiko MAEKAWA1,2, Manabu KAWAMOTO3, Naoyuki KAMATANI3,**, Shogo OZAWA1,4,† and Jun-ichi SAWADA1,2 1Project
Team for Pharmacogenetics, National Institute of Health Sciences, Tokyo, Japan of Functional Biochemistry and Genomics, National Institute of Health Sciences, Tokyo, Japan 3Division of Genomic Medicine, Department of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, Tokyo, Japan 4 Division of Pharmacology, National Institute of Health Sciences, Tokyo, Japan
2Division
Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk
Summary: The bile salt export pump (BSEP) encoded by ABCB11 is located in the canalicular membrane of hepatocytes and mediates the secretion of numerous conjugated bile salts into the bile canaliculus. In this study, 28 ABCB11 exons (including non-coding exon 1) and their flanking introns were comprehensively screened for genetic variations in 120 Japanese subjects. Fifty-nine genetic variations, including 19 novel ones, were found: 14 in the coding exons (6 nonsynonymous and 8 synonymous variations), 4 in the 3?-UTR, and 41 in the introns. Three novel nonsynonymous variations, 361CÀA (Gln121Lys), 667CÀT (Arg223Cys), and 1460GÀT (Arg487Leu), were found as heterozygotes and at 0.004 allele frequencies. These data provide fundamental and useful information for genotyping ABCB11 in the Japanese and probably other Asian populations. Keywords: BSEP; ABCB11; direct sequencing; novel genetic variation; amino acid change
cholestasis of pregnancy (ICP) and contraceptive-induced cholestasis (CIC), where hepatocellular accumulation of bile salts is observed.1,3) The ABCB11 gene encoding human BSEP consists of 28 exons (including non-coding exon 1) and spans approximately 108 kb on chromosome 2q24–31. Similar to other ABC transporters, BSEP has 12 transmembrane domains (TMDs) and Walker A and B motifs for binding and hydrolysis of ATP in large cytoplasmic domains.4) Human BSEP is a 1,321-residue protein sharing 82% amino acid identity with its rat homologue (rBsep)5) and 68% identity with the marine skate, Raja erinacea, homologue
Introduction The bile salt export pump (BSEP) is a member of the ATP-binding cassette (ABC) transporter family B. BSEP is predominantly expressed in the liver and located in the canalicular membrane of hepatocytes and mediates the secretion of numerous conjugated bile salts into the bile canaliculus.1,2) Inherited and acquired dysfunction of BSEP can lead to a wide spectrum of liver disease such as progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis type 2 (BRIC2), drug-induced cholestasis (DIC), intrahepatic
Received; February 9, 2009, Accepted; March 18, 2009 *To whom correspondence should be addressed: Yoshiro SAITO, Ph.D., Division of Functional Biochemistry and Genomics, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan. Tel. ＋81-3-5717-3831, Fax. ＋81-3-5717-3832, E-mail: yoshiro＠ nihs.go.jp **Present address: Institute for Data Analysis, StaGen Co. Ltd, Orashion Building 9F, 4-31-10 Kuramae, Taito-ku, Tokyo 111-0051, Japan. † Present address: Department of Pharmacodynamics and Molecular Genetics, Faculty of Pharmaceutical Sciences, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba-cho, Shiwa-gun, Iwate 028-3694, Japan As of February 9, 2009, the novel variations reported here are not found in the database of Japanese Single Nucleotide Polymorphisms (http://snp.ims.u-tokyo.ac.jp/), dbSNP in the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/SNP/), or the PharmGKB Database (http://www.pharmgkb.org/). This study was supported in part by the Program for the Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation, and the Health and Labor Sciences Research Grants from the Ministry of Health, Labor and Welfare.
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(sBsep).6) Recently, several genetic polymorphisms in ABCB11, including 10 nonsynonymous SNPs, have been identified in Caucasians, African-Americans and Japanese.7,8) Among them, the common nonsynonymous SNP, 1331CÀT (Ala444Val), may be associated with increased protein expression.9,10) It has been reported that serum bile acid levels in carriers of the 1331CC genotype are higher than those of the 1331TT genotype.11) The 1331C allele is more frequent in patients with DIC, ICP and CIC than the 1331T allele.11,12) In addition, more than one hundred ABCB11 mutations have been identified in BSEP deficiency syndrome patients.1,3,9,13) In this study, all 28 exons and their surrounding introns were resequenced for comprehensive screening of ABCB11 genetic variations. Sequence analysis detected 59 variations including 3 novel nonsynonymous ones from 120 Japanese subjects.
Materials and Methods Human genomic DNA samples: One hundred twenty Japanese healthy volunteers participated in this study and provided written informed consent. The ethical review boards of the Tokyo Women's Medical University and the National Institute of Health Sciences approved this study. Genomic DNA was extracted from Epstein-Barr virus transformed B lymphocytes from volunteers by standard methods. PCR conditions for DNA sequencing: First, multiplex PCR was performed to amplify 5 fragments including 28 exons of ABCB11 from 200 ng of genomic DNA, using 1.25 units of Z-Taq (Takara Bio. Inc., Shiga, Japan) with 0.25 mM each of the mixed primer designed in the intronic regions as listed in Table 1 (1st PCR). The first PCR conditions consisted of 30 cycles of 989C for 5 sec, 559C for 5 sec, and 729 C for 190 sec. Next, each exon was amplified separately by Ex-Taq (0.625 units, Takara Bio. Inc.) with appropriate primers (0.25 mM) designed in the introns (Table 1, 2nd PCR). The conditions for the second round PCR were 949C for 5 min, followed by 30 cycles of 949 C for 30 sec, 559C for 1 min, and 729 C for 2 min, and then a final extension at 729C for 7 min. Following the PCR, the products were treated with a PCR Product Pre-Sequencing Kit (USB Co., Cleveland, OH, USA) and directly sequenced on both strands using an ABI BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) with the sequencing primers listed in Table 1 (Sequencing). Excess dye was removed by a DyeEx96 kit (Qiagen, Hilden, Germany), and the eluates were analyzed on an ABI Prism 3700 DNA Analyzer (Applied Biosystems). All variations were confirmed by sequence analysis of PCR products generated by new amplification of the original genomic DNA templates. Rare SNPs found in single patients as heterozygotes were confirmed by sequencing PCR fragments
produced by amplification with high fidelity DNA polymerase KOD-Plus- (TOYOBO, Tokyo, Japan). Hardy-Weinberg equilibrium analysis was performed by SNPAlyze software (Ver 5.1, Dynacom Co., Yokohama, Japan).
Results and Discussion Sequence analysis from 120 Japanese subjects resulted in the identification of 59 genetic variations, including 19 novel ones (Table 2). Of these, 14 were located in coding exons (6 nonsynonymous and 8 synonymous variations), 4 in the 3?-UTR, and 41 in introns. All detected variations were in Hardy-Weinberg equilibrium (pÀ0.05), except for IVS12＋122GÀA and IVS14＋ 128delA. Deviation from Hardy-Weinberg equilibrium for both variations resulted from a slightly larger number of homozygotes than expected. Of the 6 nonsynonymous variations, 3 were novel: 361CÀA (Gln121Lys), 667CÀT (Arg223Cys), and 1460GÀT (Arg487Leu). All of these variations were found as heterozygotes with frequencies of 0.004. The nonsynonymous SNPs 361CÀA (Gln121Lys), 667CÀT (Arg223Cys), and 1460GÀT (Arg487Leu) are located in the extracellular loop between TMD1 and TMD2, TMD3 and the large cytoplasmic loop between TMD6 and TMD7, respectively.4) Using the PolyPhen program (http:/ /genetics.bwh.harvard.edu/pph/) to predict functional effects of the three amino acid substitutions, two substitutions, Arg223Cys and Arg487Leu, are expected to alter protein function based on PSIC (position-specific independent count) score differences derived from multiple alignments. The Arg223 residue located in TMD3 is well conserved among BSEP proteins including the marine skate, mouse and rat. The Arg487 residue is located in the first nucleotide binding fold (NBF1) domain of the large cytoplasmic loop between TMD6 and TMD7 and highly conserved among human ABC transporter family B proteins, such as ABCB1 and ABCB4. The two substitutions at the same position, Arg487His and Arg487Pro, were found in patients with severe BSEP deficiency syndrome, PFIC2.13,14) Thus, it is possible that bile acid homeostasis in the subject with Arg223Cys or Arg487Leu may be disturbed. However, we have not determined blood bile acid levels in the volunteers. The functional significance of these 3 novel nonsynonymous variations should be clarified in the future. The three known nonsynonymous variations, 896GÀA (Arg299Lys), 1331CÀT (Ala444Val), and 2594CÀT (Ala865Val), were detected at 0.004, 0.267, and 0.004 frequencies, respectively, which are similar to the previously reported Japanese data.7) One variation, 1331CÀT (Ala444Val), was found 14 residues upstream of the Walker A motif in the NBF1 domain in the large cytoplasmic loop between TMD6 and TMD7. This variation results in increased protein expression.9) Its fre-
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Genetic Variations of ABCB11 in Japanese
Table 1. Primer sequences used in this study Amplified or sequenced region 1st PCR
Exons Exons Exons Exons Exons
2nd PCR
Sequencing
1 to 5 6 to 10 11 to 18 19 to 21 22 to 28
Forward primer (5? to 3?)
Reverse primer (5? to 3?)
CAAGGCTGTTTCAAATGTTC ATTGTCCTGGTGAATGAACT GTGTCTGTATTGTTTTTGGC CCTCTGGGTACTGATTAAGGA TATACCTGCTACAACATGCC
ACCTCCCTAGTGACGTTAAG ATGGCATTCACAGCAACCTG TGTCTGTGAAACCTAATTGG CTTCGACGCCTTCAAGATTC GAGTGCTTTTCTGCCATATT
Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Exon 6 Exon 7 Exon 8 Exon 9 Exon 10 Exon 11 Exon 12 Exon 13 Exon 14 Exon 15 Exon 16 Exon 17 Exon 18 Exon 19 Exons 20 and 21 Exon 22 Exon 23 Exon 24 Exon 25 Exon 26 Exon 27 Exon 28
TGTTTGGGGTTATTGCTCTG GGCTCTTTCAGGGAGTTATT AGAGAGACAATATGAGCAGGA CATCTCTGTGAATCGCTAGT AGATAATTGATGTACCGTGC AGAGACTAAAGTGGACCAAG CCACTTTTAGCAGCAGATGA ATTGGCTATCAGGAAAGAGG TTCTTCCTCCTGTCAATGAT GGCAGCTCTGTTAAGTATCG CAGTGGAAAACTTGGAGATG GCAAACTAAGAGGCACAATA CTTTGGGGGCATACATAAAC TTTATGTTTCCACAATGAAT GTAAACCCTGGAATAAAAGC AGACTAGCTTATGATATTTTTTA GACAAGTCCCGAGTTTTATT ACTCTGAATCTGGGTCCAAC TTCAAGCCACAGCAATAGTT CCACAGCTTACATTAGGGTT GGATACAGAGTTTTTGTTGG AATTGTCTCGTGGTCCAGTT TTGAAAACTGCCAGTCCCTT AGCATGGTTTGAAGGTATCT AGCCTTGGGATTGTTAGTCT ATTTGACATCCTCCATCCTA GCACTGCCATTGAAATCACT
GTAGCGTTGTTTACACTGGA TACGTTACATGGATTCTAGG GAATACAGAGGGGACATTTGA ATGATAACCATGGGCTTAGT AGATAGAAGGAAACTTGAGG CATCTGAGAGAAGAATCCCT CACCTGGACAGCTAAATAAC ATTTGACACTGGGGAGTAAT AGGCTTGGGACTTCTTATAG TCCACAGACAGACTCCATAA AGTGTTGCTGAATTAAGGGC TGGTCTGTTGCCTGAGATAA CTGACTGAAATGTTGCTATG TCAGATGAAAGGAAACACTC TTCAACTGTGAGGAAGATTG TAGAAGCCTTGGTTTTAGTG CATGAATTGGGGAGAGAAAT CTAAGACAATTAACCCTCCG CGAGCCCAAATGAGAGTTCT TGCTTTAGCTGCTGTTTGTC GGCTCATACTCAGAGTCTCG GTCAGAAGAACCGCCCTGAT TGTGTCCATGTGTTCTGTTT ATCTGGAACTGGGGAAAATC TGACTGCATTTTGACTGTGA GCCAATTTCTACTGTTAACG TTTCCCTATCCTTAGCCTTA
Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon
CGTATGTCACTGAACTGTGC CAGGGAGTTATTAACCGATT ACAATATGAGCAGGAAGAAAG CTCTGTGAATCGCTAGTGAA TGATGTACCGTGCCGTTTAT GGCTTCCAACAACTGCTAAT TTAGCAGCAGATGACAGAGC TCAGGAAAGAGGCTTGATAA TTCCTCCTGTCAATGATGTT AGCTCTGTTAAGTATCGCCC TGGAGATGCTTTACAGAACA AGAGATACGCCAAAGATGTT ACACACAGACACCGAGTATC AGAACCACATTTCAAGAGGA ACCCTGGAATAAAAGCATAA AGTGTGTGTTGGGGTTTATG GTCCCGAGTTTTATTTATTG TGAATCTGGGTCCAACACTT ATCCCATAGACATTTGAGGT ACAGCTTACATTAGGGTTCA GATAAAGAGGAACAAAGGTC GAGTTTTTGTTGGACATGAT TGATCTCCAGCCACGTCTTT AACTGCCAGTCCCTTAAACA CATGGTTTGAAGGTATCTCA CTTGGGATTGTTAGTCTGTT GGAGGACTCACTCACTGTTC CAGGGAATACTTGCTTAATC AAAGGAGCCTACTACAAACT
AGCGTTGTTTACACTGGACA CCTACTGTTGCTGATTTTTT AGAAGGGATATTCCAAAAGGT GGCAAAGCCTATGACTGAAA ACTTGAGGCAGAGTAATTTC CTCTCCATTCTCTCATGTCC TGAAAAAGCCATGCCACATA TGCTAACTGTACTCAGGAAA CTTAGCTCCCTCTTGAACAA CTGAAGGCACCAAAGTAATA GAATTAAGGGCCTTGCGATA TGCCTGAGATAAGCCAACAC GCCACATCTGACTGTATCTG AGGAAACACTCATGGTACTT TCAGACTAGATGCATGAACC GCTTCAGTTGTTTCCTTTAC TGTCAATATAGCAACCAAGG ATTAACCCTCCGAAGTCCAG CTCAGCTTTGCCTTCTTACC GGCTATTTCTGAGGTCATCA TTAGCTGCTGTTTGTCAGTG ACTCAGAGTCTCGGAGCTTT CTGATGACCCACAGAATCTT GTTCAACTCCCACTTATGAG TTGTGAGGTAGAATCAGGTG ATTTTGACTGTGACCCATCA GTTAACGAATCAGAAAATTG TTAGCTTGGATTCCGATGTA ATCCTTAGCCTTAGAGATGA
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 28
Amplified length (bp) 18,817 11,729 16,682 14,559 14,122 351 435 412 534 604 610 592 521 453 534 443 464 513 584 559 657 407 594 550 683 635 593 512 519 541 420 957
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Table 2. Summary of ABCB11 variations detected in this study SNP ID This Study MPJ6_AB11001 MPJ6_AB11002 MPJ6_AB11003 MPJ6_AB11004 MPJ6_AB11005 MPJ6_AB11006a MPJ6_AB11007 MPJ6_AB11008 MPJ6_AB11009a MPJ6_AB11010a MPJ6_AB11011a MPJ6_AB11012 MPJ6_AB11013a MPJ6_AB11014a MPJ6_AB11015 MPJ6_AB11016 MPJ6_AB11017 MPJ6_AB11018 MPJ6_AB11019 MPJ6_AB11020 MPJ6_AB11021a MPJ6_AB11022a MPJ6_AB11023a MPJ6_AB11024a MPJ6_AB11025 MPJ6_AB11026a MPJ6_AB11027 MPJ6_AB11028 MPJ6_AB11029 MPJ6_AB11030 MPJ6_AB11031 MPJ6_AB11032a MPJ6_AB11033a MPJ6_AB11034 MPJ6_AB11035 MPJ6_AB11036 MPJ6_AB11037 MPJ6_AB11038 MPJ6_AB11039 MPJ6_AB11040 MPJ6_AB11041 MPJ6_AB11042 MPJ6_AB11043 MPJ6_AB11044 MPJ6_AB11045a MPJ6_AB11046 MPJ6_AB11047a MPJ6_AB11048a MPJ6_AB11049 MPJ6_AB11050 MPJ6_AB11051a MPJ6_AB11052 MPJ6_AB11053a MPJ6_AB11054a MPJ6_AB11055 MPJ6_AB11056 MPJ6_AB11057 MPJ6_AB11058 MPJ6_AB11059 a b c
Position dbSNP (NCBI) rs4148772 rs10199694 rs4148775 rs4148776 rs3815675 rs4148777
Reference
7, 8 8 7, 8 7, 8 7 7, 8
7 rs2287614 rs2287615 rs2287616 rs2287617 rs4148780 rs2287618
8 8 7, 8 7 8 7
rs4148783
8
rs55669065 rs2287622 rs2287623 rs2389605 rs4148786
rs2241340 rs2241341 rs55868238 rs2193831 rs2389612 rs4148795 rs4148796 rs853772 rs853773
7, 8 7, 8 8 7 7, 8 8 8 8 8 8 7, 8 8 7
rs11568379 rs497692
7, 8 7
rs7561903 rs579275 rs473351 rs3732038 rs495714 rs496550
7, 8 8, 15 8 8
Location
NT_005403.16
Intron 1 20084130 Intron 3 20082623 Intron 3 20080475 Intron 3 20080300 Exon 4 20080273 Intron 4 20079560 Intron 4 20079512 Exon 5 20079319 Exon 5 20079310 Exon 5 20079228 Intron 5 20062886 Intron 6 20062500 Intron 7 20059917 Exon 8 20059755 Intron 8 20056962 Intron 8 20056940 Exon 9 20056830 Exon 9 20056741 Intron 9 20056621 Intron 9 20052227 Intron 11 20045737 Intron 11 20045731 Intron 11 20042655 Exon 12 20042565 Intron 12 20042389 Intron 12 20042383 Intron 12 20039861 Exon 13 20039746 Intron 13 20039573 Intron 13 20039469 Intron 13 20038064_ 20038065 Exon 14 20037953 Exon 14 20037907 Exon 14 20037808 Intron 14 20037743 Intron 14 20037695 Intron 14 20037647 Intron 14 20036295 Intron 15 20035603 Intron 18 20030037 Intron 18 20030036 Intron 18 20024073 Intron 19 20023765 Exon 21 20010549 Intron 21 20010502 Intron 21 20002386 Intron 22 20002132 Intron 22 20002091 Exon 24 19998434 Intron 24 19998280 Intron 25 19996478 Intron 26 19992983 Intron 26 19992965 Intron 27 19989910 Intron 27 19989784 3?-UTR 19989314 3?-UTR 19989269 3?-UTR 19989182 3?-UTR 19989130
Novel variations detected in this study. Positions in cDNA (NM _003742.2). Numbers in parentheses indicate positions from the termination codon TGA.
From the translational initiation site or from the end of the nearest exonb
Nucleotide change
IVS1－50 IVS3＋83 IVS3－193 IVS3－18 108 IVS4－122 IVS4－74 270 279 361 IVS5－236 IVS6＋63 IVS7－107 667 IVS8－109 IVS8－87 807 896 IVS9＋108 IVS9－15 IVS11 ＋57 IVS11 ＋63 IVS11 －40 1248 IVS12＋116 IVS12＋122 IVS12 －93 1331 IVS13 ＋70 IVS13＋174 IVS13－87_－86 1460 1506 1605 IVS14 ＋32 IVS14 ＋80 IVS14＋128 IVS14－152 IVS15－124 IVS18 ＋97 IVS18 ＋98 IVS18 －17 IVS19＋127 2594 IVS21 ＋31 IVS21 －25 IVS22 ＋26 IVS22 ＋67 3084 IVS24 ＋25 IVS25＋115 IVS26＋101 IVS26＋119 IVS27－160 IVS27 －34 4202 (*236)c 4247 (*281)c 4334 (*368)c 4386 (*420)c
ACTTTGATTAAAG/AAAGAAAGAAGAG AAGCAGAGAATAC/TTTTCATGCACAT TGAGATTGAGCTA/GTACTGAAATCTC GTCTTTAAATCCT/CTATGTTTTTCTC CAGGTTACAAGAT/CGAGAAGAAAGGT CACTCAATTAAGG/ATGATTCCCATGA TGAGAATCTAGTA/TACTAAATTAAGT GACAGATGTTTTT/CATTGACTACGAC TTTTATTGACTC/TGACGTTGAGTTA AGTTCCCTCAACC/AAGAACATGACAA ATATGCATATTTT/CCTGTGATTGGTA ACTACAATGAGAT/GGCAATGTGTTGC ATCCAAGGGTGAT/CAGGGATAGAGAG CTTTTCATTCAGC/TGCATGACCTCGA GTTACAGTGAGAA/CTCTAATATTGTA GTATTAAACCCAT/AGCCACATGTTAA GTTTACGGACTAT/CGAGCTGAAGGCC GTGGTGAGAAAAG/AAGAGGTTGAAAG TCTGTGGCCTCCA/GGAGGAAGTACTT ATTGACTCAAGCG/ATTTTGTCTTCAC GGGGGTGGGGCAC/AAGAATGAACTCC GGGGCACAGAATG/AAACTCCTGAAGA TTGTGCATCTTAG/CTTTGAGTTTACA GTTGGATCGAATC/AAAGGGTGAAATT GTAATAGGGAATG/AGAGGTGTCTTTC GGGAATGGAGGTG/ATCTTTCTCTGAA CACACAGACACCG/AAGTATCAACACA ACCTCAACATGGC/TCATTAAACCAGG ATATTGATCAAAT/CAGAAAGGTGTAG TAACAGTGTTCAA/GTGAATAACCAGT CTCTATTTTTTC-/CTGCCCATTGGTC GCCATGACATTCG/TCTCTCTTAACAT TGGGATAGTGGAG/ACAAGAGCCAGTT TGCCAAGGAGGCC/TAATGCCTACAAC CCTGGGAGAAACC/TAAGAGGTCATAG TACACATTTCTTT/CTCGTATGATTCC TGTTTTAGTTTCA/-TGCCTGAAAAAG AGACAATAACCCA/GTCTGGGGAAGGG AATGTCTGCACAG/ACCTATTTAAGAA TTTTCTAGGTATA/GTATCTAGCAGTG TTTCTAGGTATAT/CATCTAGCAGTGT TGATTAATATAAA/CCCTCTCTCTGCT ATCTCTAAAGAAC/TGAAAAATTTCCT TTGCTACAGATGC/TTTCCCAAGTTCA AATAGAAGTATAT/GTAACTGCATTGG TGTGTCTGAGACG/AGGTTGATTGCTT TCTAATTTTCCCA/GTTCCTCATGGCT AACTGTTAAAAAC/TGAGTAGTACGAA TGTACTGAGTGCG/AACAGCTCTTGGA ATACTATGCAGCC/AATAAAAAAGGAT CTTGCCTAAGGCA/CCTTACCCCATGC CTGAAAATCCCAA/CATCCAAAATGTT AAATGTTCCAAAA/GTTCAAAAATTTT CACTGATGCATTG/ACATTCAGGGAAT GAGCAATCATGCG/ATCTTTGCATCAA ACTAGGGTCCATG/ATGAGGGAAAACC GCCACCACTCAGT/GGCTTCTCTGTGC AACTCCTCAAGGA/GCAGAGAACTGTC AGAGGCGGGTCTG/ATAACAGGCAATC
Amino acid change
Asp36Asp Phe90Phe Tyr93Tyr Gln121Lys
Arg223Cys Tyr269Tyr Arg299Lys
Ile416Ile
Ala444Val
Arg487Leu Glu502Glu Ala535Ala
Ala865Val
Ala1028Ala
Frequency
0.058 0.058 0.225 0.058 0.225 0.029 0.021 0.058 0.004 0.004 0.004 0.017 0.004 0.004 0.058 0.267 0.267 0.004 0.058 0.217 0.004 0.004 0.004 0.008 0.250 0.062 0.012 0.267 0.237 0.237 0.004 0.004 0.004 0.008 0.237 0.237 0.062 0.237 0.237 0.417 0.417 0.412 0.396 0.004 0.004 0.054 0.004 0.004 0.442 0.004 0.042 0.104 0.004 0.004 0.437 0.054 0.004 0.437 0.437
Genetic Variations of ABCB11 in Japanese
quency in Japanese (0.196–0.267) is slightly lower than that in Caucasians (0.405) and African-Americans (0.344) (described as g.44308TÀC [Val444Ala] in a previous paper).7) Two other known variations, 896GÀA (Arg299Lys) and 2594CÀT (Ala865Val), were detected only in the Japanese population at allele frequencies of 0.010 for 896GÀA and 0.024 for 2594CÀT, respectively.7) Both variations are located in the cytoplasmic loops between TMD4 and TMD5 and between TMD8 and TMD9. The variation 2873GÀA (Arg958Gln) previously reported in Japanese at a frequency of 0.011 was not detected in this study. Six variations previously reported in other ethnic groups were not detected: 616AÀG (Ile206Val; found with 0.011 frequency in African-Americans), 851TÀC (Val284Ala; 0.005 in Caucasians), 1846CÀG (Arg616Gly; 0.022 in AfricanAmericans), 1855AÀG (Thr619Ala; 0.011 in AfricanAmericans), 2029AÀG (Met677Val; 0.042 in Caucasians and 0.14 in African-Americans), and 2093GÀA (Arg698His; 0.005 in Caucasians).7) These variations might be ethnic-specific. In conclusion, 59 variations including 19 novel ones were identified in ABCB11 from 120 Japanese subjects. Three novel nonsynonymous variations result in amino acid substitutions. This information is useful for pharmacogenetic studies that investigate the association of ABCB11 variations with interindividual differences in bile salt processing and drug disposition.
Acknowledgments: The authors thank Ms. Chie Sudo for her secretarial assistance.
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