ABCMdb

PMID: 16041243

Letourneau IJ, Deeley RG, Cole SP
Functional characterization of non-synonymous single nucleotide polymorphisms in the gene encoding human multidrug resistance protein 1 (MRP1/ABCC1).
Pharmacogenet Genomics. 2005 Sep;15(9):647-57., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC1 p.Arg723Gln ABCC1 p.Thr73Ile ABCC1 p.Arg1058Gln ABCC1 p.Cys1047Ser ABCC1 p.Arg633Gln ABCC1 p.Arg230Gln ABCC1 p.Ala989Thr ABCC1 p.Ser1512Leu ABCC1 p.Ser92Phe Variants 218C > T (Thr73Ile), 257C > T (Ser92Phe), 350C > T (Thr117Met), 689G > A (Arg230Gln), 1898G > A (Arg633Gln), 2168G > A (Arg723Gln), 2965G > A (Ala989Thr), 3140G > C (Cys1047Ser), 3173G > A (Arg1058Gln) and 4535C > T (Ser1512Leu) were recreated using site-directed mutagenesis and transfected into human embryonic kidney cells. Login to comment 5 ABCC1 p.Ala989Thr Vesicular transport assays revealed that the Ala989Thr mutation caused a significant decrease in estradiol 17b-glucuronide transport due to a decrease in apparent affinity (Km) for this organic anion. Login to comment 7 ABCC1 p.Gly671Val ABCC1 p.Gly671Val ABCC1 p.Arg433Ser ABCC1 p.Arg230Gln ABCC1 p.Val353Met ABCC1 p.Ser92Phe When the MRP1/ABCC1 non-synonymous SNPs were evaluated by the SIFT algorithm using subsets of homologs and orthologs of MRP1/ABCC1, Arg230Gln, Val353Met, Arg433Ser, Gly671Val and Arg1058 mutations were predicted to be deleterious, whereas the PolyPhen algorithm predicted Ser92Phe and Gly671Val to be potentially damaging. Login to comment 28 ABCC1 p.Cys43Ser ABCC1 p.Cys43Ser ABCC1 p.Arg723Gln ABCC1 p.Arg723Gln ABCC1 p.Thr73Ile ABCC1 p.Arg1058Gln ABCC1 p.Arg1058Gln ABCC1 p.Gly671Val ABCC1 p.Gly671Val ABCC1 p.Arg433Ser ABCC1 p.Arg433Ser ABCC1 p.Cys1047Ser ABCC1 p.Cys1047Ser ABCC1 p.Arg633Gln ABCC1 p.Arg633Gln ABCC1 p.Arg230Gln ABCC1 p.Arg230Gln ABCC1 p.Val353Met ABCC1 p.Val353Met ABCC1 p.Ala989Thr ABCC1 p.Ala989Thr ABCC1 p.Ser1512Leu ABCC1 p.Ser1512Leu ABCC1 p.Ser92Phe ABCC1 p.Ser92Phe Of these mutations, the Fig. 1 128G >C (C43S) 128G >T(T73I) 689G >A (R230Q)1057G >A (V353M) 1299G >T(R433S) 1898G >A (R633Q) 2012G >T(G671V) 2168G >A (R723Q) 3173G >A (R1058Q) 4535C >T(S1512L) 3140G >C (C1047S) 2965G >A (A989T) 350C >T(T117M) 257C >T(S92F) 313029282726252423222120181716151413121110987654321 19 MSD1 MSD1 MSD2 MSD3 MSD2 NBD1 MSD3 NBD2 TM 1 2 3 4 5 6 7 8 Val353Met Ala989Thr Cys1047Ser Arg1058Gln NBD2NBD1 Ser1512Leu Arg633Gln Arg433Ser Arg723Gln Thr73lle Thr117Met Arg230Gln Cys43Ser Ser92Phe Gly671Val 9 10 11 12 13 14 15 16 17 (a) (b) Location of reported non-synonymous single nucleotide polymorphisms (SNPs) in MRP1/ABCC1. Login to comment 33 ABCC1 p.Cys43Ser ABCC1 p.Cys43Ser Thus, the Cys43Ser (128G > C) mutation located in TM1 disrupted trafficking of MRP1 to the plasma membrane of HeLa cells and cells expressing the Cys43Ser MRP1 mutant demonstrated a lower level of resistance to vincristine and arsenite than wild-type MRP1. Login to comment 35 ABCC1 p.Arg433Ser A second mutation, Arg433Ser (1299G > T), located in cytoplasmic loop 4 (CL4) proximal to transmembrane (TM) helix 8 in the second MSD, decreased both LTC4 and estrone-3-sulfate (E13SO4) transport but increased resistance to doxorubicin [19]. Login to comment 36 ABCC1 p.Gly671Val Finally, the Gly671Val (2012G > T) mutation located in NBD1 caused no discernible changes in MRP1 function, even though this Gly residue is highly conserved among members of the human ABCC family and also in other species [20]. Login to comment 45 ABCC1 p.Thr73Ile ABCC1 p.Arg230Gln ABCC1 p.Ser92Phe The template for creating the Thr73Ile, Ser92Phe, Thr117Met and Arg230Gln mutants was prepared by subcloning a 865-bp XbaI/BamHI fragment encoding amino acids 1-840 of MRP1 from pcDNA3.1(-)MRP1k into pGEM-3z (Promega, Madison, Wisconsin, USA) [18,21]. Login to comment 46 ABCC1 p.Cys43Ser ABCC1 p.Arg723Gln ABCC1 p.Arg723Gln ABCC1 p.Thr73Ile ABCC1 p.Arg1058Gln ABCC1 p.Gly671Val ABCC1 p.Arg433Ser ABCC1 p.Cys1047Ser ABCC1 p.Arg633Gln ABCC1 p.Arg633Gln ABCC1 p.Arg230Gln ABCC1 p.Val353Met ABCC1 p.Ala989Thr ABCC1 p.Ser1512Leu ABCC1 p.Ser92Phe The template for generating Table 1 Frequencies of non-synonymous single nucleotide polymorphisms in MRP1/ABCC1 Variant Amino acid substitution Allelic frequency Population References 128G > C Cys43Ser 0% (0/26) Japanese [16] 1% (1/96) Japanese [17] 218C > T Thr73Ile 0% (0/26) Japanese [16] 1% (1/96) Japanese [17] 3.7% (2/54) Chinese [37] 257C > T Ser92Phe 0% (0/220) Caucasian www.pharmGKB.org 0.5% (1/200) African-American 0% (0/60) Japanese 0% (0/14) Pacific-Islander 350C > T Thr117Met 1.6% (1/64) Caucasian [28] 689G > A Arg230Gln 0% (0/220) Caucasian www.pharmGKB.org 0.5% (1/200) African-American 0% (0/60) Japanese 0% (0/14) Pacific-Islander 1057G > A Val353Met 0.5% (1/220) Caucasian www.pharmGKB.org 0% (0/200) African-American 0% (0/60) Japanese 0% (0/14) Pacific-Islander 1299G > T Arg433Ser 1.4% (1/72) Caucasian [20] 0% (0/110) Caucasian [19] 1898G > A Arg633Gln 0.8% (2/234) Caucasian [29] 2012G > T Gly671Val 2.8% (2/72) Caucasian [20] 2.6% (6/234) Caucasian [29] 2168G > A Arg723Gln 3.8% (1/26) Japanese [16] 1% (1/96) Japanese [30] 7.3% (7/96) Japanese [17] 5.6% (3/54) Chinese [37] 2965G > A Ala989Thr 0.5% (1/220) Caucasian www.pharmGKB.org 0% (0/200) African-American 0% (0/60) Japanese 0% (0/14) Pacific-Islander 3140G > C Cys1047Ser 0% (0/220) Caucasian www.pharmGKB.org 4.5% (9/200) African-American 0% (0/60) Japanese 0% (0/14) Pacific-Islander 3173G > A Arg1058Gln 0% (0./26) Japanese [16] 1% (1/96) Japanese [17] 4535C > T Ser1512Leu 3.1% (2/24) Caucasian [28] Characterization of MRP1/ABCC1 variants in vitro Le´tourneau et al. 649 the Arg633Gln and Arg723Gln mutants was created by subcloning a HindIII fragment (1329 bp) encoding amino acids 517-959 into pGEM-3z [20]. Login to comment 47 ABCC1 p.Arg1058Gln ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr The mutagenesis template for the Ala989Thr, Cys1047Ser and Arg1058Gln mutants was created by subcloning a 1986-bp XmaI fragment encoding amino acids 780-1440 from pcDNA3.1( - )MRP1k into pGEM-3z [21]. Login to comment 48 ABCC1 p.Ser1512Leu The template for the Ser1512Leu mutant was created by subcloning a 778-bp EcoRI/KpnI fragment encoding amino acids 1295-1531 into pBluescript II KS( + ) (Stratagene). Login to comment 50 ABCC1 p.Arg723Gln ABCC1 p.Thr73Ile ABCC1 p.Arg1058Gln ABCC1 p.Cys1047Ser ABCC1 p.Arg633Gln ABCC1 p.Arg230Gln ABCC1 p.Ala989Thr ABCC1 p.Ser1512Leu ABCC1 p.Ser92Phe Mutagenesis was performed according to the manufacturer`s instructions with the following sense primers (substituted nucleotides for amino acid mutation are underlined, introduced or disrupted restriction sites are italicized and other silent substitutions are in lower case letters) as follows: Thr73Ile (50 -G ATG ACA CCT CTC AAC AAA ATC AAAACTGCCTTGGG-30 ); Ser92Phe (50 -GG GCA GAC CTG TTC TAC TTT TTC TGG GAA AG-30 ) (EarI); Thr117Met (50 -CTC TTG GGC ATC ACC ATG CTG CTT GCT ACC-30 ); Arg230Gln (50 -GG TTG ATT GTA CAG GGC TAC CGC C-30 ) (BsrGI); Arg633Gln (50 -GAC AGC ATC GAG CGA CAG CCT GTG AAA GAC GGC GG-30 ) (Eam1105I); Arg723Gln (50 -CAG AAT GAC TCT CTC CAA GAA AAt ATC CTT TTT GGA TGT CAG C-30 ) (PleI); Ala989Thr (50 -C ATG TGT AAC CAC GTG TCC ACG CTG GCT TCC-30 ) (PmlI); Cys1047Ser (50 - GCT TCC CGC TCT CTG CAT GTG GAC CTG C-30 ) (PmlI); Arg1058Gln (50 -CTG CTG CAC AGC ATC CTC CAG TCA CCC ATG AGC-30 ) (BstEII); and Ser1512Leu (50 -CAG GAG TAC GGA GCC CCA TTG GAC CTt CTG CAG CAG-30 ) (NarI). Login to comment 51 ABCC1 p.Arg723Gln ABCC1 p.Thr73Ile ABCC1 p.Arg1058Gln ABCC1 p.Cys1047Ser ABCC1 p.Arg633Gln ABCC1 p.Arg230Gln ABCC1 p.Ala989Thr ABCC1 p.Ser1512Leu ABCC1 p.Ser92Phe Following mutagenesis, the desired fragment was subcloned back into pcDNA3.1(-) MRP1k as a XbaI/BamHI fragment (865 bp) for the Thr73Ile, Ser92Phe, Thr117Met and Arg230Gln mutants; a Bsu36I/Esp3I fragment (721 bp) for the Arg633Gln and Arg723Gln mutants; a Esp3I/EcoRI fragment (1313 bp) for the Ala989Thr, Cys1047Ser, Arg1058Gln mutants; and a EcoRI/KpnI fragment (778 bp) for the Ser1512Leu mutant. Login to comment 82 ABCC1 p.Val353Met One of the non-synonymous SNPs, Val353Met (1057G > A), is located in the third extracellular loop of MRP1, whereas the remainder are located in the TM helices or intracellular domains of the transporter. Login to comment 83 ABCC1 p.Arg723Gln ABCC1 p.Thr73Ile ABCC1 p.Arg1058Gln ABCC1 p.Cys1047Ser ABCC1 p.Arg633Gln ABCC1 p.Arg230Gln ABCC1 p.Ser1512Leu ABCC1 p.Ser92Phe Expression levels of MRP1 mutants To investigate the effect of the amino acid substitutions resulting from the non-synonymous SNPs on MRP1 protein expression and function, MRP1 expression vectors containing the mutations responsible for the substitutions (Thr73Ile, Ser92Phe, Thr117Met, Arg230Gln, Arg633Gln, Arg723Gln, Thr989Ala, Cys1047Ser, Arg1058Gln, Ser1512Leu) were generated by site-directed mutagenesis and transfected into HEK293T cells. Login to comment 87 ABCC1 p.Arg723Gln ABCC1 p.Thr73Ile ABCC1 p.Arg1058Gln ABCC1 p.Cys1047Ser ABCC1 p.Arg633Gln ABCC1 p.Arg230Gln ABCC1 p.Ala989Thr ABCC1 p.Ser1512Leu ABCC1 p.Ser92Phe The mutants were considered in four groups based on their location in the transporter: (a) MSD1/CL3 mutants Thr73Ile, Ser92Phe, Thr117Met and Arg230Gln; (b) NBD1 mutants Arg633Gln and Arg723Gln; (c) MSD3 mutants Ala989Thr, Cys1047Ser and Arg1058Gln; and (d) COOH-terminus mutant Ser1512Leu. Login to comment 94 ABCC1 p.Cys43Ser ABCC1 p.Gly671Val ABCC1 p.Arg433Ser Results from previously characterized non-synonymous SNPs, Cys43Ser, Arg433Ser and Gly671Val, were included where available for comparison [18-20]. Login to comment 96 ABCC1 p.Arg723Gln ABCC1 p.Arg633Gln ABCC1 p.Ser1512Leu However, the three SNPs located in or close to the NBDs (Arg633Gln, Arg723Gln and Ser1512Leu) caused a consistent decrease (approximately 25%) in [3 H]E217bG (Fig. 3b) and [3 H]MTX uptake when activity levels were normalized for variation in expression level (Fig. 3c). Login to comment 97 ABCC1 p.Arg723Gln ABCC1 p.Arg633Gln ABCC1 p.Ser1512Leu The Arg723Gln and Ser1512Leu mutations had a similar effect on [3 H]LTC4 uptake, whereas the Arg633Gln mutation did not (Fig. 3a). Login to comment 98 ABCC1 p.Ala989Thr The decrease in [3 H]E217bG uptake of approximately 50% by the Ala989Thr mutant was considered substantial enough to warrant further investigation (Fig. 3b). Login to comment 99 ABCC1 p.Ala989Thr ABCC1 p.Ala989Thr Kinetic parameters of [3 H]E217bG uptake by the MRP1 Ala989Thr mutant To further characterize the decrease in [3 H]E217bG transport by the Ala989Thr mutant protein, kinetic analysis was performed. Login to comment 100 ABCC1 p.Ala989Thr A Michaelis-Menten plot of the data yielded a Km of 7.4 ± 1.6 mM and Vmax of 561 ± 34 pmol/mg protein/min for E217bG uptake by the Ala989Thr mutant protein compared to Km and Vmax values for E217bG uptake by WT-MRP1 of 3.1 ± 0.5 mM and 626 ± 22 pmol/mg protein/min, respectively. Login to comment 101 ABCC1 p.Ala989Thr These results indicate that an increase in apparent Km for E217bG is primarily responsible for the decrease in transport of this conjugated oestrogen by the Ala989Thr mutant Fig. 4. Login to comment 102 ABCC1 p.Ala989Thr Inhibition of [3 H]LTC4 and [3 H]MTX transport by E217bG To further characterize the interaction of E217bG with the Ala989Thr mutant protein, its ability to inhibit the transport of other organic anion substrates of MRP1 was investigated. Login to comment 103 ABCC1 p.Ala989Thr The IC50 (E217bG) of [3 H]LTC4 transport for the Ala989Thr mutant was 31 mM whereas the IC50 (E217bG) for WT-MRP1 was 9 mM. Login to comment 106 ABCC1 p.Ala989Thr Thus, the IC50 (E217bG) values were 3 mM and 2 mM for the Ala989Thr mutant and WT-MRP1, respectively Fig. 5. Login to comment 114 ABCC1 p.Ala989Thr The effect of the SNPs (with the exception of Ala989Thr) on E217bG transport also ranged from none to moderate (< 40% decrease or increase). Login to comment 118 ABCC1 p.Ala989Thr Because the Ala989Thr mutation caused the greatest decrease in E217bG uptake (50%), it was further characterized by kinetic analysis. Login to comment 120 ABCC1 p.Ala989Thr Inhibition studies of LTC4 transport by E217bG yielded differences in IC50 values between the Ala989Thr mutant and wild-type 652 Pharmacogenetics and Genomics 2005, Vol 15 No 9 transporters that were in accordance with the approximate two-fold decrease in uptake affinity of the mutant for E217bG. Login to comment 121 ABCC1 p.Ala989Thr By contrast, the IC50 values of E217bG for MTX uptake by the Ala989Thr mutant and wild-type MRP1 were similar. Login to comment 123 ABCC1 p.Cys43Ser ABCC1 p.Cys43Ser ABCC1 p.Arg723Gln ABCC1 p.Arg723Gln ABCC1 p.Arg723Gln ABCC1 p.Arg1058Gln ABCC1 p.Arg1058Gln ABCC1 p.Arg1058Gln ABCC1 p.Gly671Val ABCC1 p.Gly671Val ABCC1 p.Arg433Ser ABCC1 p.Arg433Ser ABCC1 p.Cys1047Ser ABCC1 p.Cys1047Ser ABCC1 p.Cys1047Ser ABCC1 p.Arg633Gln ABCC1 p.Arg633Gln ABCC1 p.Arg633Gln ABCC1 p.Arg230Gln ABCC1 p.Arg230Gln ABCC1 p.Arg230Gln ABCC1 p.Ala989Thr ABCC1 p.Ala989Thr ABCC1 p.Ala989Thr ABCC1 p.Ser1512Leu ABCC1 p.Ser1512Leu ABCC1 p.Ser1512Leu ABCC1 p.Ser92Phe ABCC1 p.Ser92Phe ABCC1 p.Ser92Phe Previous mutagenesis and inhibition studies have Fig. 3 Cys43Ser Thr73lle Ser92Phe Thr117Met Arg230Gln Arg433Ser Arg633Gln Gly671Val Arg723Gln Ala989Thr Cys1047Ser Arg1058Gln Ser1512Leu Cys43Ser Thr73lle Ser92Phe Thr117Met Arg230Gln Arg433Ser Arg633Gln Gly671Val Arg723Gln Ala989Thr Cys1047Ser Arg1058Gln Ser1512Leu Thr73lle Ser92Phe Thr117Met Arg230Gln Arg633Gln Arg723Gln Ala989Thr Cys1047Ser Arg1058Gln Ser1512Leu LTC4 % WT-MRP1 uptake 0 25 50 75 100 125 E217βG % WT-MRP1 uptake 0 25 50 75 100 125 150 MTX % WT-MRP1 uptake 0 25 50 75 100 125 (b) (c) (a) ATP-dependent vesicular transport of organic anions by mutant MRP1 proteins. Login to comment 128 ABCC1 p.Cys43Ser ABCC1 p.Gly671Val ABCC1 p.Arg433Ser Hatched bars represent previously published data on non-synonymous SNPs, Cys43Ser, Arg433Ser and Gly671Val, using membrane vesicles from stably transfected HeLa cells and are included for comparison [18-20]. Login to comment 134 ABCC1 p.Arg1058Gln ABCC1 p.Gly671Val ABCC1 p.Arg433Ser Thus, when the subset included the human homologs MRP2, MRP3 and MRP6, the SIFT analysis predicted that the Arg433Ser, Gly671Val and Arg1058Gln substitutions would be deleterious for MRP1 function. Login to comment 135 ABCC1 p.Gly671Val ABCC1 p.Arg433Ser ABCC1 p.Arg230Gln ABCC1 p.Val353Met However, when the analysis was expanded to include the six known mammalian orthologs of MRP1, the Arg230Gln, Val353Met, Arg433Ser and Gly671Val mutations were also predicted to be deleterious. Login to comment 136 ABCC1 p.Arg433Ser ABCC1 p.Val353Met The Val353Met mutation has not yet been tested in vitro; however, of the remaining five, only Arg433Ser has been found to adversely affect MRP1 function [19]. Login to comment 137 ABCC1 p.Cys43Ser ABCC1 p.Ala989Thr On the other hand, the Cys43Ser and Ala989Thr mutations were found by analysis in vitro to significantly modify MRP1 function, despite the fact that these effects were not predicted by the SIFT algorithm [18]. Login to comment 140 ABCC1 p.Gly671Val ABCC1 p.Ser92Phe Thus, PolyPhen predicted that the Ser92Phe and Gly671Val mutations would likely be deleterious to MRP1 function which our present and previous experimental data show it is not the case, at least in vitro [20]. Login to comment 141 ABCC1 p.Ala989Thr All the other MRP1/ABCC1 Fig. 4 500 250 0 0 25 50 75 100 [3 H]E217βGuptake(pmol/mg/min) [E217βG] (µM) Kinetic analysis of [3 H]E217bG transport by MRP1 mutant Ala989Thr. Login to comment 142 ABCC1 p.Ala989Thr Membrane vesicles (2 mg protein per point) expressing wild-type (`) and Ala989Thr mutant (~) MRP1 were incubated with concentrations of E217bG ranging from 0.25 mM to 100 mM (40-120 nCi per point) for 1 min at 37 1C. Km and Vmax were determined from a Michaelis-Menten curve fitted by nonlinear regression. Login to comment 145 ABCC1 p.Ala989Thr (a) E217bG-mediated inhibition of [3 H]LTC4 transport by wild-type (`) and Ala989Thr mutant (~) MRP1 was measured by incubating 2 mg of membrane vesicles with 50 nM [3 H]LTC4 (10 nCi) and concentrations of E217bG ranging from 2.5-50 mM for 1 min at 231C. Login to comment 146 ABCC1 p.Ala989Thr (b) E217bG-mediated inhibition of [3 H]MTX transport by wild-type (`) and Ala989Thr mutant (~) MRP1 was measured by incubating 5 mg of membrane vesicle protein with 100 mM [3 H]MTX (125 nCi) and concentrations of E217bG ranging from 2.5-50 mM for 20 min at 371C. Results shown are those of a single experiment; each point represents the mean ± SD of triplicate determinations. Login to comment 157 ABCC1 p.Arg723Gln ABCC1 p.Arg1058Gln ABCC1 p.Arg230Gln It is interesting to note that, in some cases, the human MRP1 polymorphic residue is found in the sequence of an MRP1 ortholog or other ABCC homolog (e.g. Thr117Met, Arg230Gln, Arg723Gln and Arg1058Gln). Login to comment 158 ABCC1 p.Cys43Ser ABCC1 p.Arg723Gln ABCC1 p.Thr73Ile ABCC1 p.Arg1058Gln ABCC1 p.Gly671Val ABCC1 p.Arg433Ser ABCC1 p.Cys1047Ser ABCC1 p.Arg230Gln ABCC1 p.Val353Met ABCC1 p.Ala989Thr ABCC1 p.Ser1512Leu ABCC1 p.Ser92Phe This observation may be construed as being Table 2 Conservation of the amino acids substituted by non-synonymous SNP of human MRP1/ABCC1a Protein Speciesb C43S T73I S92F T117Mc R230Q V353M R433S R633Qc G671V R723Q A989T C1047S R1058Q S1512L MRP1 Human C T S T R V R R G R A C R S Monkey C T S M R V R R G Q A C R S Dog C T S M R V R R G R A R R S Cow C A S M Q V R R G R A R R S Rat C A S M Q V R W G R A R R S Mouse C T S M H V R R G R A R R S MRP2 Human L A V T K A K R G K A I R E Monkey L A V T K A K R G K A I R E Dog L A V T K A K R G K A I Q Q Rat L A A T K V K R G K A A R E Mouse L A A T K V K V G K A T R E Rabbit L A V T K V K R G K A I R E MRP3 Human C L S M Y I R K G Q A V R A Rat C L S M L L R K G Q A L R V MRP4 Human - - - - I F K R G R Y T K Y MRP5 Human - - - - V T R S G R T R R S MRP6 Human P A A M R I R S G V A L R A CFTR Human - - - - R Y K A G K L I Q Q SUR1 Human V L L A T V Q R G E L R L E SUR2 Human V L H T Q V Q R G E I N L P Pgp Human - - - - - E K S G A G R R Q YCF1 Saccharomyces cervisiae A I L V T V K L G K S Y R G Mrp1 Caenorhabditis elegans T L D F L I R T G R G L R K Mrp2 Caenorhabditis elegans T F D I L I K T G R G I R K AtMRP2 Arabidopsis thaliana Q L R W L M S P G R R K R E AtMRP1 Arabidopsis thaliana H T A V L M S P G R R K R E a Aligned using Clustal W (http://pbil.univ-lyon1.fr/). Login to comment 163 ABCC1 p.Ala989Thr In addition, many of the variants have been found as singletons, including the mutation leading to Ala989Thr substitution. Login to comment