ABCMdb

ABCC4 p.Tyr556Cys

Predicted by SNAP2:	A: D (80%), C: D (66%), D: D (91%), E: D (91%), F: N (72%), G: D (91%), H: D (85%), I: D (66%), K: D (91%), L: D (66%), M: D (75%), N: D (85%), P: D (95%), Q: D (85%), R: D (91%), S: D (85%), T: D (80%), V: N (57%), W: D (80%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D,

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[hide] Pharmacogenomics of MRP transporters (ABCC1-5) and... Drug Metab Rev. 2008;40(2):317-54. Gradhand U, Kim RB
Pharmacogenomics of MRP transporters (ABCC1-5) and BCRP (ABCG2).
Drug Metab Rev. 2008;40(2):317-54., [PMID:18464048]

Abstract [show]
Elucidation of the key mechanisms that confer interindividual differences in drug response remains an important focus of drug disposition and clinical pharmacology research. We now know both environmental and host genetic factors contribute to the apparent variability in drug efficacy or in some cases, toxicity. In addition to the widely studied and recognized genes involved in the metabolism of drugs in clinical use today, we now recognize that membrane-bound proteins, broadly referred to as transporters, may be equally as important to the disposition of a substrate drug, and that genetic variation in drug transporter genes may be a major contributor of the apparent intersubject variation in drug response, both in terms of attained plasma and tissue drug level at target sites of action. Of particular relevance to drug disposition are members of the ATP Binding Cassette (ABC) superfamily of efflux transporters. In this review a comprehensive assessment and annotation of recent findings in relation to genetic variation in the Multidrug Resistance Proteins 1-5 (ABCC1-5) and Breast Cancer Resistance Protein (ABCG2) are described, with particular emphasis on the impact of such transporter genetic variation to drug disposition or efficacy.

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191 MRP4 protein has been detected in human kidney (van Aubel et al., 2002), lung (Torky et al., 2005), liver (Rius et al., 2003), prostate (Lee et al., 2000), brain (Nies et al., 2004), pancreas (König et al., 2005), lymphocytes (Schuetz et al., 1999), and platelets Figure 4 Predicted membrance topology of MRP4 (ABCC4) based on hydrophobicity analysis. Locations of the non-synonymous polymorphisms are indicated with arrows. See Table 4 for allele frequencies and description of funtional consequences. NH2 COOH NBD NBD Val854Phe Ile18Leu Ile866Val Arg531Gln Tyr556Cys Thr1142Met Glu757Lys Val776Ile Gly187Trp Lys304Asn in out Membrane Cys171Gly Pro403Leu Lys498Glu Met744Val Met1272Val MRP4 (ABCC4) COOH NBD NBD Val854Phe Ile866Val Arg531Gln Tyr556Cys Thr1142Met Glu757Lys Val776Ile Gly187Trp Lys304AsnCys171Gly Pro403Leu Lys498Glu Met744Val Met1272Val COOH NBD NBD Val854Phe Ile866Val Arg531Gln Tyr556Cys Thr1142Met Glu757Lys Val776Ile Gly187Trp Lys304AsnCys171Gly Pro403Leu Lys498Glu Met744Val Met1272Val (Jedlitschky et al., 2004). Login to comment
216 Polymorphisms in exons 1, 5, 12, 13, 19, 21, and 28 leading to the following amino acid exchanges Ile18Leu, Gly187Trp, Arg531Gln, Tyr556Cys, Val776Ile, Val854Phe, Ile866Val, and Thr1142Met were analysed in relation to expression and localization of MRP4 in human liver. Login to comment
217 Some of the amino acid substitutions are located within highly conserved regions such as membrane spanning domains (Val776Ile, Val854Phe, Ile866Val) or ATP-binding domains (Tyr556Cys, Thr1142Met), others are located in intracellular regions where they might influence substrate recognition (Gly187Trp). Login to comment
236 Following the discovery and Table 4 MRP4 (ABCC4) Single nucleotide polymorphisms. Location, allele frequency and functional effects. Position in coding sequence Amino acid exchange Location Allele frequency Effect NCBI ID ReferenceAf Ca Jp others 52A>C Ile18Leu Exon 1 - 1.1 [1] 0 [2] - No influence on expression and localization in liver [1] rs11568681 511T>G Cys171Gly Exon 4 - 0 [1] [2] - - rs4148460 559G>T Gly187Trp Exon 5 - 2.2 [1] 0 [2] - No influence on expression and localization in liver [1] rs11568658 912G>T Lys304Asn Exon 8 - 9.9 [1] [2] - No influence on expression and localization in liver [1] rs2274407 1208T>C Pro403Leu Exon 9 - - - - - rs11568705 1492A>G Lys498Glu Exon 11 - - - - - rs11568669 1592G>A Arg531Gln Exon 12 - 0.6 [1] 0 [2] - No influence on expression and localization in liver [1] 1667A>G Tyr556Cys Exon 13 - 0.6 [1] 0 [2] - No influence on expression and localization in liver [1] 2230A>G Met744Val Exon 18 - - - - - rs9282570 2269G>A Glu757Lys Exon 18 - 0.6 [1] [2] - No influence on expression and localization in liver [1] rs3765534 2326G>A Val776Ile Exon 19 - 0.6 [1] 0 [2] - No influence on expression and localization in liver [1] 2560G>T Val854Phe Exon 21 - 1.7 [1] 0 [2] - No influence on expression and localization in liver [1] rs11568694 2596A>G Ile866Val Exon 21 - 2.8 [1] 0 [2] - No influence on expression and localization in liver [1] 3425C>T Thr1142Met Exon 27 - 1.6 [1] 0 [2] - No influence on expression and localization in liver [1] rs11568644 3814A>G Met1272Val Exon 30 - - - - - rs1134217 Reference without frequency means that SNP was detected but no frequency determined. Login to comment

[hide] Pharmacogenetics of drug transporters in the enter... Pharmacogenomics. 2011 May;12(5):611-31. Stieger B, Meier PJ
Pharmacogenetics of drug transporters in the enterohepatic circulation.
Pharmacogenomics. 2011 May;12(5):611-31., [PMID:21619426]

Abstract [show]
This article summarizes the impact of the pharmacogenetics of drug transporters expressed in the enterohepatic circulation on the pharmacokinetics and pharmacodynamics of drugs. The role of pharmacogenetics in the function of drug transporter proteins in vitro is now well established and evidence is rapidly accumulating from in vivo pharmacokinetic studies, which suggests that genetic variants of drug transporter proteins can translate into clinically relevant phenotypes. However, a large amount of conflicting information on the clinical relevance of drug transporter proteins has so far precluded the emergence of a clear picture regarding the role of drug transporter pharmacogenetics in medical practice. This is very well exemplified by the case of P-glycoprotein (MDR1, ABCB1). The challenge is now to develop pharmacogenetic models with sufficient predictive power to allow for translation into drug therapy. This will require a combination of pharmacogenetics of drug transporters, drug metabolism and pharmacodynamics of the respective drugs.

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117 Gene name Transporter SNP Protein Population size (n) In vitro function Ref. Liver efflux transporters (cont.) SLC47A1 (cont.) MATE1 (cont.) c.1490G>C c.149G>T p.C497S p.C497F N/A Reduced, unchanged or increased transport activities (substrate dependent) [170,229] c.1557G>C p.Q519H N/A Unchanged [170] ABCC4 MRP4 c.232C>G p.P78A N/A Increased intracellular drug accumulation (substrate dependent), lower transport protein expression [161] c.559C>T p.G187W N/A Increased intracellular drug accumulation, reduced transport protein expression Slightly reduced function [161] [162] c.877A>G p.K293E N/A Unchanged [161] c.912G>T p.K304N N/A Unchanged Unchanged [161] [162] c.1208C>T p.P403L N/A Increased intracellular drug accumulation [161] c.1460G>A p.G487E N/A Increased intracellular drug accumulation Reduced transport activity (substrate dependent) [161] [162] c.1492A>G p.K498E N/A Unaltered [161] c.1667A>G p.Y556C N/A Increased transport activity [162] c.2269G>A p.E575K N/A Increased transport activity [162] c.2230A>G p.M744V N/A Unchanged [161] c.2326G>A p.V776I N/A Reduced transport activity [162] c.2459G>T p.R820I N/A Reduced transport activity [162] c.2560G>T p.V854F N/A Unchanged [162] c.2596A>G p.I866V N/A Unchanged [162] c.2867G>C p.C956S N/A Reduced intracellular drug accumulation [161] c.3211G>A p.V1071I N/A Unchanged [161] c.3425C>T p.T1142M N/A Increased transport activity [162] For more information on members of the SLC superfamily of transporters please consult [301] and for more information of ABC transporters please consult [302]. Login to comment

[hide] Clinical impact of polymorphisms of transport prot... Transplant Proc. 2009 Jun;41(5):1441-55. Rosso Felipe C, de Sandes TV, Sampaio EL, Park SI, Silva HT Jr, Medina Pestana JO
Clinical impact of polymorphisms of transport proteins and enzymes involved in the metabolism of immunosuppressive drugs.
Transplant Proc. 2009 Jun;41(5):1441-55., [PMID:19545654]

Abstract [show]
Individualization of immunosuppressive therapy after solid organ transplantation is a goal that has been pursued for a long time. Nevertheless, in clinical practice, we are still stratifying patients in subgroups in which risk is assessed using demographic information and population analysis. Then, a combination of immunosuppressive drugs is chosen and doses are individualized to compensate for intra- and interindividual variabilities in drug pharmacokinetics, to obtain similar plasma/blood concentrations that are believed to be therapeutic, again based on data derived from population analysis. One step further in this strategy is to recognize, before initiation of immunotherapy, those patients at higher risk to be either under- or overexposed to currently used immunosuppressive drugs. Several studies have been undertaken to correlate single nucleotide polymorphisms in genes encoding transport proteins and metabolizing enzymes involved in the disposition of immunosuppressive drugs. Overall, the results from these studies have been mixed. The causes of these sometimes conflicting results include methodologic, genetic, or nongenetic factors. The degree of linkage disequilibrium, the measure of nonrandom associations between polymorphisms at different loci, not necessarily on the same chromosome, is perhaps the main genetic factor. The influence of the environment, physiology (such as kidney and liver functions), disease state, use of multidrug regimens, and inherent drug-to-drug interactions are present nongenetic factors. Moreover, it is also important to increase our knowledge of the genetic factors involved in the variabilities observed in drug responses of pharmacodynamics. True individualized therapy, with the ability to improve health outcomes of each transplant recipient, will depend on our knowledge of the genetic factors involved in immunological response and drug pharmacokinetics and pharmacodynamics.

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116 This mutation is relatively common (Ͼ18%) in the Japanese population and is associated with increased sensitivity to thiopurines observed in some Japanese patients.72 In one study, 4 MRP4 missense genetic variants (Y556C, E757K, V7761, and T1142M) exhibited a 20% to 40% reduced expression level compared with the wild type. Login to comment

[hide] 6-mercaptopurine and 9-(2-phosphonyl-methoxyethyl)... Hum Mutat. 2008 May;29(5):659-69. Janke D, Mehralivand S, Strand D, Godtel-Armbrust U, Habermeier A, Gradhand U, Fischer C, Toliat MR, Fritz P, Zanger UM, Schwab M, Fromm MF, Nurnberg P, Wojnowski L, Closs EI, Lang T
6-mercaptopurine and 9-(2-phosphonyl-methoxyethyl) adenine (PMEA) transport altered by two missense mutations in the drug transporter gene ABCC4.
Hum Mutat. 2008 May;29(5):659-69., [PMID:18300232]

Abstract [show]
Multiple drug resistance protein 4 (MRP4, ABCC4) belongs to the C subfamily of the ATP-binding cassette (ABC) transporter superfamily and participates in the transport of diverse antiviral and chemotherapeutic agents such as 6-mercaptopurine (6-MP) and 9-(2-phosphonyl methoxyethyl) adenine (PMEA). We have undertaken a comprehensive functional characterization of protein variants of MRP4 found in Caucasians and other ethnicities. A total of 11 MRP4 missense genetic variants (nonsynonymous SNPs), fused to green fluorescent protein (GFP), were examined in Xenopus laevis oocytes for their effect on expression, localization, and function of the transporter. Radiolabeled 6-MP and PMEA were chosen as transport substrates. All MRP4 protein variants were found to be expressed predominantly in the oocyte membrane. A total of four variants (Y556C, E757 K, V776I, and T1142 M) exhibited a 20% to 40% reduced expression level compared to the wild type. Efflux studies showed that 6-MP is transported by MRP4 in unmodified form. Compared to wild-type MRP4, the transmembrane variant V776I, revealed a significant lower activity in 6-MP transport, while the amino acid exchange Y556C in the Walker(B) motif displayed significantly higher transport of PMEA. The transport properties of the other variants were comparable to wild-type MRP4. Our study shows that Xenopus oocytes are well suited to characterize MRP4 and its protein variants. Carriers of the rare MRP4 variants Y556C and V776I may have altered disposition of MRP4 substrates.

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6 A total of four variants (Y556C, E757K, V776I, and T1142M) exhibited a 20% to 40% reduced expression level compared to the wild type. Login to comment
8 Compared to wild-type MRP4, the transmembrane variant V776I, revealed a significant lower activity in 6-MP transport, while the amino acid exchange Y556C in the WalkerB motif displayed significantly higher transport of PMEA. Login to comment
11 Carriers of the rare MRP4 variants Y556C and V776I may have altered disposition of MRP4 substrates. Login to comment
34 We also sequenced large regions of the MRP4 gene of these individuals and identified 74 genetic variations, among them 10 missense mutations (I18L, G187W, K304N, R531Q, Y556C, E757 K, V776I, V854F, I866 V, and T1142 M). Login to comment
164 A summary of all MRP4 protein mutations examined, their distribution within the protein, their frequencies and their functional prediction scores (SIFT/PolyPhen/Grantham) are listed in Supplementary Table S1 and Supplementary Figure S3. A total of six of them (G187W, G487E, Y556C, R820I, V854F, and T1142 M) are located either within transmembrane regions or near the ATP-binding domain of MRP4 and were predicted to have a functional effect by the computer-based algorithms (Supplementary Table S2). Login to comment
170 However, the mutation Y556C located in the WalkerB motif resulted in significantly increased [3 H] PMEA efflux, while the mutation V776I, located in the transmembrane region, led to a significantly decreased [14 C] 6-MP transport activity compared to wild-type (Supplementary Figs. S5 and S6). Login to comment
175 The MRP4 protein variants E757 K, V776I, and T1142 M showed a 20-30% reduced protein expression and Y556C exhibited a 40% reduced protein expression compared to wild type, respectively. Login to comment
182 Quantification of the fluorescence intensity demonstrated a 20-30% reduction in the surface expression for the variants E757 K, V776I, and T1142 M, and a 40% reduction in the surface expression of Y556C (Fig. 4B). Login to comment
185 To investigate intracellular localization of MRP4 wild-type and variants (Y556C, E757 K, V776I, and T1142 M) with a reduced protein level, we prepared thin cryostat sections (12 mm) from oocytes. Login to comment
188 Comparison ofTransport to Cell Surface Expression Y556C caused a two to three times higher [3 H] PMEA efflux, and V776I, G487E, and R820I caused a 25-30% decrease in [14 C] 6-MP efflux compared to wild-type when transport activity was normalized using the MRP4 expression levels (Fig. 5). Login to comment
201 ÃPo0.05 for MRP4(Y556C).EGFP variant vs. wild-type. Login to comment
209 ÃPo0.05 for MRP4(Y556C) variant vs. wild-type. Login to comment
229 MRP4 proteins variants Y556C, E757 K, V776I, and T1142 M were expressed at lower levels (range 20-40% of wild type) compared to MRP4 wild-type protein. Login to comment
236 With the exception of Y556C, the effects of all other MRP4 variants on PMEA transport ranged from none to moderate (o25% decrease). Login to comment
237 Y556C exhibited the greatest reduction in MRP4 protein expression (40%), and it caused an increase in specific PMEA (two- to three-fold of wild type) and 6-MP (1.5-fold of wild type) transport activity (Fig. 5). Login to comment
238 This may be due to an alteration of a region (nucleotide binding domain 1 [NBD1], WalkerB) affecting ATP-binding caused by the radical chemical change at this position indicated by the high Grantham value (Grantham value was 194) (Supplementary Table S1) and by a different drug binding of 6-MP and PMEA in the transmembrane domains of MRP4 Y556C that can induce differential long-range conformational changes in the NBDs, such that these compounds stimulate ATPase activity by decreasing the distance between the Walker A, B, and LSGGQ motif sequences. Login to comment
246 It is possible that MRP4 mutations like Y556C and V776I may affect tissue accumulation and elimination of nucleoside-based agents, including PMEA, 6-MP, or other antiviral agents. Login to comment
248 In addition, four variants have been found as singletons, including the mutation leading to Y556C and V776I substitution (Supplementary Table S1). Login to comment
251 In contrast, variants of MRP4 having an influence on functional activity (G487E, Y556C, V776I, and R820I) were present at allelic frequencies o1%. Login to comment
253 Only for Y556C (WalkerB, consensus sequence ''YLLDD``), the human MRP4 wild-type residue is highly conserved compared to sequences of MRP4 orthologs and other ABCC homologs. Login to comment
257 When SIFT and PolyPhen were used to evaluate the MRP4 nonsynonymous SNPs, both predicted the same variants as deleterious (G187W, G487E, Y556C, R820I, V854F, and TABLE 1. Login to comment
258 Conservation of the MRP4 Polymorphic AminoAcids Among Di¡erent ABCC Orthologs and HomologsÃ Protein Speciesa G187W K304N G487E Y556C E757K V776I R820I V854F I866V T1142M MRP4 Human G K G Y E V R V I T Mouse G K G Y E V R I V T Rat G K G Y G V R I L S MRP1 Human K Q D Y K ^ N C F S Mouse K Q D Y F A ^ V F S Rat K Q D Y ^ G N V V S MRP2 Human K K G Y S G R L V S Mouse R K G Y S G R L V S Rat K K G Y S G R L I S MRP3 Human R Q C F L G R L V S Rat R Q C F S G R I V S MRP5 Human ^ ^ A Y D L R V S T Mouse ^ ^ A Y D L R V S T Rat ^ ^ A Y D L R V S T MRP6 Human K G T Y G H S V V S Mouse K G T Y H G N G V T Rat K G T Y G N G V V T ÃAligned using ClustalW (www.ebi.ac.uk/clustalw). Login to comment
266 It is noteworthy, that MRP4 protein expression in one liver sample carrying Y556C showed a 40% reduction relative to the control group [Gradhand et al., in press]. Login to comment

[hide] Variability in human hepatic MRP4 expression: infl... Pharmacogenomics J. 2008 Feb;8(1):42-52. Epub 2007 Apr 3. Gradhand U, Lang T, Schaeffeler E, Glaeser H, Tegude H, Klein K, Fritz P, Jedlitschky G, Kroemer HK, Bachmakov I, Anwald B, Kerb R, Zanger UM, Eichelbaum M, Schwab M, Fromm MF
Variability in human hepatic MRP4 expression: influence of cholestasis and genotype.
Pharmacogenomics J. 2008 Feb;8(1):42-52. Epub 2007 Apr 3., [PMID:17404579]

Abstract [show]
The multidrug resistance protein 4 (MRP4) is an efflux transporter involved in the transport of endogenous substrates and xenobiotics. We measured MRP4 mRNA and protein expression in human livers and found a 38- and 45-fold variability, respectively. We sequenced 2 kb of the 5'-flanking region, all exons and intron/exon boundaries of the MRP4 gene in 95 patients and identified 74 genetic variants including 10 non-synonymous variations, seven of them being located in highly conserved regions. None of the detected polymorphisms was significantly associated with changes in the MRP4 mRNA or protein expression. Immunofluorescence microscopy indicated that none of the non-synonymous variations affected the cellular localization of MRP4. However, in cholestatic patients the MRP4 mRNA and protein expression both were significantly upregulated compared to non-cholestatic livers (protein: 299+/-138 vs 100+/-60a.u., P<0.001). Taken together, human hepatic MRP4 expression is highly variable. Genetic variations were not sufficient to explain this variability. In contrast, cholestasis is one major determinant of human hepatic MRP4 expression.

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52 MRP4 protein expression in samples carrying non-synonymous polymorphisms relative to the control group (n ¼ 8) was as follows: G187W 58% (n ¼ 2), K304N 54% (n ¼ 3), R531Q 20% (n ¼ 1), Y556C 60% (n ¼ 1), E757K 86% (n ¼ 1), V776I 161% (n ¼ 1), V854F 96% (n ¼ 1), I866V 78% (n ¼ 4) and T1142M 40% (n ¼ 2). Login to comment
72 Prediction of functional effects of non-synonymous variations In silico analysis of all 10 detected amino acid exchanges revealed that five of them (I18L, K304N, R531Q, E757K, V776I) can be considered benign, whereas others especially near or within transmembrane regions or ATP-binding domains are possibly (G187W, Y556C, V854F, I866V) or in one case (T1142M) even very likely damaging for protein localization and/or function (Figure 3). Login to comment
53 MRP4 protein expression in samples carrying non-synonymous polymorphisms relative to the control group (n &#bc; 8) was as follows: G187W 58% (n &#bc; 2), K304N 54% (n &#bc; 3), R531Q 20% (n &#bc; 1), Y556C 60% (n &#bc; 1), E757K 86% (n &#bc; 1), V776I 161% (n &#bc; 1), V854F 96% (n &#bc; 1), I866V 78% (n &#bc; 4) and T1142M 40% (n &#bc; 2). Login to comment
73 Prediction of functional effects of non-synonymous variations In silico analysis of all 10 detected amino acid exchanges revealed that five of them (I18L, K304N, R531Q, E757K, V776I) can be considered benign, whereas others especially near or within transmembrane regions or ATP-binding domains are possibly (G187W, Y556C, V854F, I866V) or in one case (T1142M) even very likely damaging for protein localization and/or function (Figure 3). Login to comment