ABCMdb

ABCC2 p.Arg1150His

Predicted by SNAP2:	A: D (91%), C: D (91%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: N (57%), I: D (95%), K: D (91%), L: D (95%), M: D (91%), N: D (95%), P: D (95%), Q: D (91%), S: D (95%), T: D (91%), V: D (91%), W: D (95%), Y: D (95%),
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, S: D, T: D, V: D, W: D, Y: 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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101 Several molecular defects in MRP2 have been suggested to result in DJS including those which produce deficient protein maturation (Hashimoto et al., 2002; Keitel et al., 2003), proteasomal degradation (Keitel, 2003), impaired membrane sorting (Hashimoto et al., 2002; Mor-Cohen et al., 2001), loss in transport activity (Mor-Cohen et al., 2001), Figure 2 Predicted membrance topology of MRP2 (ABCC2) based on hydrophobicity analysis. Locations of the non-synonymous polymorphisms are indicated with arrows. See Table 2 for allele frequencies and description of funtional consequences. NH2 COOH NBD NBD in out Membrane Pro19Leu Phe39Tyr Arg100* Arg100Gln Ser281Asn Ser325* Asp333Gly Arg353His Arg412Gly Val417Ile Lys430Arg Thr486Ile Gly676Arg Trp709Arg Asn718Ser Ser789Phe Arg768Trp Asp833Asn Glu893Gln Leu927Arg Lys961Arg Tyr967* Phe981Leu Gln1019His Arg1066* Arg1150His Arg1100Cys Arg1100His Ile1137Phe Ile1173Phe Val1188Glu Arg1174His Arg1181Leu Asn1244Lys Thr1273Ala Pro1291Leu Lys1299Gln Arg1310* Ser1367Cys Gln1382Arg Arg1392del Met1393del Ala1450Thr Thr1476Met Cys1515Tyr MRP2 (ABCC2) NBD NBD Asp833Asn Glu893Gln Leu927Arg Lys961Arg Tyr967* NBD NBDNBD Asp833Asn Glu893Gln Leu927Arg Lys961Arg Tyr967* 325 Table2MRP2(ABCC2)singlenucleotidepolymorphisms.Location,allelefrequencyandfunctionaleffects. Positionin codingsequence Amino acidexchangeLocation Allelefrequency EffectNCBIIDReferenceAfCaJpothers 56C>TPro19LeuExon2--1[1]b -- 116T>APhe39TyrExon2--0[2]--rs927344 298C>TArg100*Exon3--[3]-DJS[3] 299G>AArg100GlnExon3--1[1]b -- 842G>ASer281AsnExon7-0[4]1[1]b -- 974C>GSer325*Exon8---Malayan[5]DJS[5] 998A>GAsp333GlyExon8--0[2]--rs17222674 1058G>AArg353HisExon9--0[2]--rs7080681 1271A>GArg412GlyExon10-[6]0[2]-DJS;Decreaseinmethotrexateelimination[6] 1249G>AVal417IleExon10-22[7]13[9]-lowermRNAand(protein)expressioninpreterm placenta[11] rs2273697 26[8]16[4]noeffectonRNAandproteinininduodenum[12] 19[10]noeffectonproteininliver[8] noeffectonconjugatedbilirubinlevelinserum[13] changesinlocalizationinneuroepithelialtumors[14] possibleassociationwithtenofovir-inducedrenal proximaltubulopathy[15] 1289A>GLys430ArgExon10-4[16]0[2]-- 1457C>TThr486IleExon10-0[4]3[1]b -- 2026G>CGly676Arg--0[2]-DJS[17] 2125T>CTrp709Arg--0[2]-DJS[17] 2153A>GAsn718SerExon17-0[4]0[2]--rs3740072 2302C>TArg768TrpExon18-0[18]1[9]-DJS;deficientmaturationandimpairedsorting[19] 2366C>TSer789PheExon18-0[18]1[9]-lowerexpressionandmembranelocalization[20] noeffectonconjugatedbilirubinlevelinserum[13]/ heterozygous 2647G>AAsp883AsnExon20--1[1]b -- 2677G>CGlu893GlnExon20--0[2]--rs3740071 2780T>GLeu927ArgExon21-1[10]0[2]-- (Continued) Table2(Continued) Positionin codingsequence Aminoacid exchangeLocation Allelefrequency EffectNCBIIDReferenceAfCaJpothers 2882A>GLys961ArgExon21--1[1]b --- 2901C>ATyr967*Exon22--0[2]--rs17222547 2943C>GPhe981LeuExon22-2[21]0[2]-Noinfluenceonpravastatinkinetics[21] 3057G>TGln1019HisExon22--1[1]b -- 3196C>TArg1066*Exon23-[22]0[2]-DJS;truncatedprotein[22][23] 3298C>TArg1100CysExon24-1[10]0[2]-- 3299G>AArg1100HisExon24-1[10]0[2]-- 3449G>AArg1150HisExon25--0[2]Israeli[24]DJS;impairedtransportactivityintransfectedcells althoughnormalexpressionandlocalization[24] 3517A>TIle1173PheExon25--0[2]Israeli[24]DJS;impairedproteinmaturationandproteasomal degradation[25] lowexpression,mislocation,andimpairedtransport activityintransfectedcells[24] 3521G>AArg1174HisExon25-0[4]1[1]b -- 3542G>TArg1181LeuExon25-0[4]0[2]--rs8187692 3563T>AVal1188GluExon25-7[4]1[1]b -noeffectonnelfinaviraccumulationinPBMC[4],rs17222723 4[16]associatedwithanthracycline-induced cardiotoxicity[26] 6[8] 3732C>TAsn1244LysExon26--0[1]b -- 0[2] 3817A>GThr1273AlaExon27--0[2]--rs8187699 3872C>TPro1291LeuExon28--0[2]--rs17216317 3897A>CLys1299GlnExon28--0[2]--rs4148400 3928C>TArg1310*Exon28--0[2]-DJS[17,27] 4100C>GSer1367CysExon29--1[1]b -- 4145A>GGln1382ArgExon29--[28]-DJS;noeffectonmaturationorsorting,impaired substrate-inducedATPhydrolysis[19] 4175-80delArg1392delExon30--0[2]-DJS;deficientMRP2maturationandimpaired sortingtoapicalmembraneintransfectedcells[29] 327 4348G>AAla11450ThrExon31-0[18]1[9]-lowerexperssionandmembracelocalizationin transfectedcells[20] 4461C>TThr1476MetExon31-[30]1[2]-- 4544G>ACys1515TyrExon32-9[4]1[1]b -noeffectonnelfinaviraccumulationinPBMC[4]rs8187710 5[10]associatedwithanthracycline-induced cardiotoxicity[26] 4[16] 6[8] ReferencewithoutfrequencymeansthatSNPwasdetectedbutnofrequencydetermined. Login to comment

[hide] Pharmacogenetics of intestinal absorption. Curr Drug Deliv. 2008 Jul;5(3):153-69. Nakamura T, Yamamori M, Sakaeda T
Pharmacogenetics of intestinal absorption.
Curr Drug Deliv. 2008 Jul;5(3):153-69., [PMID:18673259]

Abstract [show]
The small intestine is the primary site of absorption for many drugs administered orally and so is the target tissue for pharmacotherapeutic strategies to control the oral absorption of drugs. Drug transporters, including the ATP-binding cassette (ABC) superfamily and the solute carrier (SLC) superfamily, have been considered to play a physiological role in regulating the absorption of xenobiotics, and variations in their expression level and function in the small intestine cause intra- and inter-individual variation in the oral absorption of drugs. Recent advances in molecular biology have suggested that genetic polymorphisms are associated with the expression level and function, and thereby inter-individual variation. In this review, the pharmacogenetics of these transporters is summarized, and their future significance in the clinical setting is discussed.

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33 Two mutations 3517A>T and 3449G>A, predicting the substitution Ile1173Phe and Arg1150His, respectively, impaired the transport of carboxyfluorescein by ABCC2, while the 3517A>T mutation induced weaker ABCC2 expression and mislocation to the ER [39, 41]. Login to comment

[hide] The MRP-related and BCRP/ABCG2 multidrug resistanc... Curr Drug Metab. 2004 Feb;5(1):21-53. Haimeur A, Conseil G, Deeley RG, Cole SP
The MRP-related and BCRP/ABCG2 multidrug resistance proteins: biology, substrate specificity and regulation.
Curr Drug Metab. 2004 Feb;5(1):21-53., [PMID:14965249]

Abstract [show]
Several members of different families of the ATP-binding cassette (ABC) superfamily of transport proteins are capable of transporting an extraordinarily structurally diverse array of endo- and xenobiotics and their metabolites across cell membranes. Together, these transporters play an important role in the absorption, disposition and elimination of these chemicals in the body. In tumor cells, increased expression of these drug transporters is associated with resistance to multiple chemotherapeutic agents. In this review, current knowledge of the biochemical, physiological and pharmacological properties of nine members of the multidrug resistance protein (MRP)-related ABCC family (MRP1, MRP2, MRP3, MRP4, MRP5, MRP6, MRP7, ABCC11 and ABCC12) as well as the G family member, ABCG2/BCRP, are summarized. A focus is placed on the structural similarities and differences of these drug transporters as well as the molecular determinants of their substrate specificities and transport activities. Factors that regulate expression of the MRP-related proteins and ABCG2/BCRP are also reviewed.

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380 Two other DJS mutations that are not in the NBDs are Arg1150His and Ile1173Phe [276, 277]. Login to comment
381 When recreated in vitro, MRP2-Arg1150His correctly localized to the apical membrane, but its transport activity was impaired. Login to comment
383 The Ile1173Phe and Arg1150His mutations are both in MSD3 but their precise location varies according to different topological models of the protein. Login to comment

[hide] Polymorphisms of MRP1 (ABCC1) and related ATP-depe... Pharmacogenet Genomics. 2005 Aug;15(8):523-33. Conseil G, Deeley RG, Cole SP
Polymorphisms of MRP1 (ABCC1) and related ATP-dependent drug transporters.
Pharmacogenet Genomics. 2005 Aug;15(8):523-33., [PMID:16006996]

Abstract [show]
Genetic variations in drug metabolizing enzymes and targets are established determinants of adverse drug reactions and interactions, but less is known about the role of genetic polymorphisms in membrane transport proteins. MRP1 (ABCC1) is one of 13 polytopic membrane proteins that comprise the 'C' subfamily of the ATP-binding cassette (ABC) superfamily of transport proteins. MRP1 and related ABCC family members, including MRP2, 3, 4 and 5 (ABCC2, 3, 4 and 5), each have a distinctive pattern of tissue expression and substrate specificity. Together, these five transporters play important roles in the disposition and elimination of drugs and other organic anions, and in maintenance of blood-tissue barriers, as confirmed by enhanced chemosensitivity of respective knockout mice. Moreover, Mrp2 (Abcc2) deficient animals display mild conjugated hyperbilirubinemia, corresponding to a human condition known as Dubin-Johnson syndrome (DJS). Naturally occurring mutations in MRP/ABCC-related drug transporters have been reported, some of which are non-synonymous single nucleotide polymorphisms. The consequences of the resulting amino acid changes can sometimes be predicted from in vitro site-directed mutagenesis studies or from knowledge of mutations of analogous (conserved) residues in ABCC proteins that cause DJS, Pseudoxanthoma elasticum (ABCC6), cystic fibrosis (CFTR/ABCC7) or persistent hyperinsulinemic hypoglycemia of infancy (SUR1/ABCC8). Continual updating of databases of sequence variants and haplotype analysis, together with in vitro biochemical validation assays and pharmacological studies in knockout animals, should make it possible to determine how genetic variation in the MRP-related transporters contributes to the range of responses to drugs and chemicals observed in different human populations.

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167 The G3449A mutation (which results in a substitution of His for Arg1150 ) is found in Moroccan- Jewish DJS patients whereas the MRP2-Ile1173Phe (A3517T) mutation is mainly found in Iranian-Jewish patients. Login to comment
168 Although both the Arg1150His and Ile1173Phe mutations eliminate MRP2 transport activity in vitro, the Ile1173Phe variant protein also exists predominantly in an underglycosylated form that is retained in the endoplasmic reticulum [47,48]. Login to comment

[hide] Influence of ABCB1, ABCC1, ABCC2, and ABCG2 haplot... Pharmacogenet Genomics. 2005 Sep;15(9):599-608. Colombo S, Soranzo N, Rotger M, Sprenger R, Bleiber G, Furrer H, Buclin T, Goldstein D, Decosterd L, Telenti A
Influence of ABCB1, ABCC1, ABCC2, and ABCG2 haplotypes on the cellular exposure of nelfinavir in vivo.
Pharmacogenet Genomics. 2005 Sep;15(9):599-608., [PMID:16041239]

Abstract [show]
OBJECTIVES: The human immunodeficiency virus protease inhibitor nelfinavir is substrate of polyspecific drug transporters encoded by ABCB1 (P-glycoprotein), ABCC1 (MRP1) and ABCC2 (MRP2), and an inhibitor of BCRP, encoded by ABCG2. Genetic polymorphism in these genes may be associated with changes in transport function. METHODS: A comprehensive evaluation of single nucleotide polymorphisms (39 SNPs in ABCB1, 7 in ABCC1, 27 in ABCC2, and 16 in ABCG2), and inferred haplotypes was done to assess possible associations of genetic variants with cellular exposure of nelfinavir in vivo. Analysis used peripheral mononuclear cells from individuals receiving nelfinavir (n=28). Key results were re-examined in a larger sample size (n=129) contributing data on plasma drug levels. RESULTS AND CONCLUSIONS: There was no significant association between cellular nelfinavir area under the curve (AUC) and SNPs or haplotypes at ABCC1, ABCC2, ABCG2. There was an association with cellular exposure for two loci in strong linkage disequilibrium: ABCB1 3435C>T; AUCTT>AUCCT>AUCCC (ratio 2.1, 1.4, 1, Ptrend=0.01), and intron 26 +80T>C; AUCCC> AUCCT > AUCTT (ratio 2.4, 1.3, 1, Ptrend=0.006). Haplotypic analysis using tagging SNPs did not improve the single SNP association values.

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71 - 24C > T exon 1 Itoda et al., 2002 mp-v-004 rs717620 IVS 6-30 G > T intron 6 Epidauros mp-v-051 rs8187666 c.842G > A exon 7 p.S281N Epidauros mp-v-115 c.998G > A intron 7 Epidauros mp-v-083 c.1219C > T exon 10 synonymous (p.L407L) Epidauros mp-v-007 rs8187669 c.1249G > A exon 10 p.V417I Itoda et al., 2002 mp-v-008 rs2273697 c.1346C > G exon 10 synonymous (p.T482T) Epidauros mp-v-114 c.1457C > T exon 10 p.T486I Epidauros mp-v-055 rs8187670 IVS 16 - 47 G > A intron 16 Epidauros mp-v-118 IVS 16 - 30 T > A intron 16 Epidauros mp-v-119 c.2153A > G exon 17 p.N718S Epidauros mp-v-093 rs3740072 c.2216T > C exon 17 p.L739P Epidauros mp-v-108 c.3449G > A exon 25 p.R1150H Mor-Cohen et al., 2001 mp-v-085 c.3517A > T exon 25 p.I1173F Keitel et al., 2003 mp-v-096 c.3521G > A exon 25 p.R1174H Epidauros mp-v-068 c.3542G > T exon 25 p.R1181L Epidauros mp-v-069 rs8187692 c.3563T > A exon 25 p.V1188E Epidauros mp-v-025 rs8187694 IVS 30 - 53 C > T intron 30 Epidauros mp-v-105 rs3824610 c.4348G > A exon 31 p.A1450T Suzuki et al. 2002 mp-v-106 c.4410G > A exon 31 synonymous (p.E1470E) Epidauros mp-v-077 rs8187706 c.4488C > T exon 31 synonymous (p.H1496H) Epidauros mp-v-038 rs8187707 IVS 31 + 12 G > A intron 31 Epidauros mp-v-039 rs8187708 IVS 31 + 74 C > T intron 31 Epidauros mp-v-040 IVS 31 - 9 T > C intron 31 Epidauros mp-v-042 c 4527C > T exon 32 synonymous (p.A1509A) Epidauros mp-v-048 rs8187709 c.4544G > A exon 32 p.C1515Y Epidauros mp-v-043 rs8187710 + 259 G > T 30 flanking Epidauros mp-v-120 Transporter polymorphisms and HIV treatment Colombo et al. 601 BCRP (ABCG2) g. Login to comment

[hide] Functional importance of three basic residues clus... J Biol Chem. 2006 Jan 6;281(1):43-50. Epub 2005 Oct 17. Conseil G, Deeley RG, Cole SP
Functional importance of three basic residues clustered at the cytosolic interface of transmembrane helix 15 in the multidrug and organic anion transporter MRP1 (ABCC1).
J Biol Chem. 2006 Jan 6;281(1):43-50. Epub 2005 Oct 17., 2006-01-06 [PMID:16230346]

Abstract [show]
The multidrug resistance protein 1 (MRP1) mediates drug and organic anion efflux across the plasma membrane. The 17 transmembrane (TM) helices of MRP1 are linked by extracellular and cytoplasmic (CL) loops of various lengths and two cytoplasmic nucleotide binding domains. In this study, three basic residues clustered at the predicted TM15/CL7 interface were investigated for their role in MRP1 expression and activity. Thus, Arg1138, Lys1141, and Arg1142 were replaced with residues of the same or opposite charge, expressed in human embryonic kidney cells, and the properties of the mutant proteins were assessed. Neither Glu nor Lys substitutions of Arg1138 and Arg1142 affected MRP1 expression; however, all four mutants showed a decrease in organic anion transport with a relatively greater decrease in leukotriene C4 and glutathione transport. These mutations also modulated MRP1 ATPase activity as reflected by a decreased vanadate-induced trapping of 8-azido-[32P]ADP. Mutation of Lys1141 to either Glu or Arg reduced MRP1 expression, and routing to the plasma membrane was impaired. However, only the Glu-substituted Lys1141 mutant showed a decrease in organic anion transport, and this was associated with decreased substrate binding and vanadate-induced trapping of 8-azido-ADP. These studies identified a cluster of basic amino acids likely at the TM15/CL7 interface as a region important for both MRP1 expression and activity and demonstrated that each of the three residues plays a distinct role in the substrate specificity and catalytic activity of the transporter.

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174 In addition, a His substitution of Arg1150 in MRP2 (also analogous to MRP1-Arg1142 ) has been reported in a group of Moroccan Jewish individuals affected by Dubin-Johnson syndrome (38). Login to comment

[hide] Insight in eukaryotic ABC transporter function by ... FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19. Frelet A, Klein M
Insight in eukaryotic ABC transporter function by mutation analysis.
FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19., 2006-02-13 [PMID:16442101]

Abstract [show]
With regard to structure-function relations of ATP-binding cassette (ABC) transporters several intriguing questions are in the spotlight of active research: Why do functional ABC transporters possess two ATP binding and hydrolysis domains together with two ABC signatures and to what extent are the individual nucleotide-binding domains independent or interacting? Where is the substrate-binding site and how is ATP hydrolysis functionally coupled to the transport process itself? Although much progress has been made in the elucidation of the three-dimensional structures of ABC transporters in the last years by several crystallographic studies including novel models for the nucleotide hydrolysis and translocation catalysis, site-directed mutagenesis as well as the identification of natural mutations is still a major tool to evaluate effects of individual amino acids on the overall function of ABC transporters. Apart from alterations in characteristic sequence such as Walker A, Walker B and the ABC signature other parts of ABC proteins were subject to detailed mutagenesis studies including the substrate-binding site or the regulatory domain of CFTR. In this review, we will give a detailed overview of the mutation analysis reported for selected ABC transporters of the ABCB and ABCC subfamilies, namely HsCFTR/ABCC7, HsSUR/ABCC8,9, HsMRP1/ABCC1, HsMRP2/ABCC2, ScYCF1 and P-glycoprotein (Pgp)/MDR1/ABCB1 and their effects on the function of each protein.

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496 R1150H and I1173F mutations eliminated transport activity in vitro. Login to comment

[hide] Multidrug resistance associated proteins as determ... Curr Drug Metab. 2007 Dec;8(8):787-802. Yu XQ, Xue CC, Wang G, Zhou SF
Multidrug resistance associated proteins as determining factors of pharmacokinetics and pharmacodynamics of drugs.
Curr Drug Metab. 2007 Dec;8(8):787-802., [PMID:18220559]

Abstract [show]
The multidrug resistance associated proteins (MRP1, MRP2, MRP3, MRP4, MRP5, MRP6, MRP7, MRP8 and MRP9) belong to the ATP-binding cassette superfamily (ABCC family) of transporters. They are expressed differentially in the liver, kidney, intestine, brain and other tissues. These transporters are localized to the apical and/or basolateral membrane of the hepatocytes, enterocytes, renal proximal tubule cells and endothelial cells of the blood-brain barrier. Several MRPs (mainly MRP1-3) are associated with tumor resistance which is often caused by an increased efflux and decreased intracellular accumulation of natural product anticancer drugs and other anticancer agents. MRPs transport a structurally diverse array of important endogenous substances and xenobiotics and their metabolites (in particular conjugates) with different substrate specificity and transport kinetics. Most MRPs are subject to induction and inhibition by a variety of compounds. Several nuclear receptors, including pregnane X receptor (PXR), liver X receptor (LXR), and farnesoid receptor (FXR) participate in the regulation of MRPs. MRPs play an important role in the absorption, distribution and elimination of various drugs in the body and thus may affect their efficacy and toxicity and cause drug-drug interactions. MRPs located in the blood-brain barrier can restrict the penetration of compounds into the central nervous system. Mutation of MRP2 causes Dubin-Johnson syndrome, while mutations in MRP6 are responsible for pseudoxanthoma elasticum. More recently, mutations in mouse Mrp6/Abcc6 gene is associated with dystrophic cardiac calcification (DCC), a disease characterized by hydroxyapatite deposition in necrotic myocytes. A single nucleotide polymorphism, 538G>A in the MRP8/ABCC11 gene, is responsible for determination of earwax type. A better understanding of the function and regulating mechanism of MRPs can help minimize and avoid drug toxicity, unfavourable drug-drug interactions, and to overcome drug resistance.

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405 Important Single Nucleotide Polymorphisms (SNPs) of MRP Genes MRP Chromosomal location Amino acid variation Nucleotide variation Location References Cys43Ser Thr73Ile G128C C218T Exon2 Exon2 [239] Arg433Ser G1299T Exon10 [258] Gly671Val G2012T Exon16 [259] Arg723Gln G2168A Exon17 [239] MRP1 16p13.11-p13.12 Arg1058Gln G3173A Exon23 [239] C-24T Promoter [100, 239] Val417Ile G1249A Exon10 [100, 238, 239] Gly676Arg G2026C Exon16 [237] Try709Arg T2125C Exon17 [236] Arg768Trp Ser789Phe C2302T C2366T Exon18 Exon18 [100, 238, 239] I1173F R1150H A3517T G3449A Exon25 Exon25 [240] Ile1324Ile C3972T Exon28 [100, 239] MRP2 10q23-24 Ala1450Thr G4348A Exon31 [100, 238, 239] (Table 2) contd…. Login to comment

[hide] ABCC6/MRP6 mutations: further insight into the mol... Eur J Hum Genet. 2003 Mar;11(3):215-24. Hu X, Plomp A, Wijnholds J, Ten Brink J, van Soest S, van den Born LI, Leys A, Peek R, de Jong PT, Bergen AA
ABCC6/MRP6 mutations: further insight into the molecular pathology of pseudoxanthoma elasticum.
Eur J Hum Genet. 2003 Mar;11(3):215-24., [PMID:12673275]

Abstract [show]
Pseudoxanthoma elasticum (PXE) is a hereditary disease characterized by progressive dystrophic mineralization of the elastic fibres. PXE patients frequently present with skin lesions and visual acuity loss. Recently, we and others showed that PXE is caused by mutations in the ABCC6/MRP6 gene. However, the molecular pathology of PXE is complicated by yet unknown factors causing the variable clinical expression of the disease. In addition, the presence of ABCC6/MRP6 pseudogenes and multiple ABCC6/MRP6-associated deletions complicate interpretation of molecular genetic studies. In this study, we present the mutation spectrum of ABCC6/MRP6 in 59 PXE patients from the Netherlands. We detected 17 different mutations in 65 alleles. The majority of mutations occurred in the NBF1 (nucleotide binding fold) domain, in the eighth cytoplasmatic loop between the 15th and 16th transmembrane regions, and in NBF2 of the predicted ABCC6/MRP6 protein. The R1141X mutation was by far the most common mutation identified in 19 (32.2%) patients. The second most frequent mutation, an intragenic deletion from exon 23 to exon 29 in ABCC6/MRP6, was detected in 11 (18.6%) of the patients. Our data include 11 novel ABCC6/MRP6 mutations, as well as additional segregation data relevant to the molecular pathology of PXE in a limited number of patients and families. The consequences of our data for the molecular pathology of PXE are discussed.

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128 Further alignment showed that the R765Q mutation in ABCC6/MRP6 is the positional equivalent of both the R560T mutation in ABCC7,28 and the R842G mutation in ABCC8.29 Similarly, additional possible positional equivalent clusters of conserved and mutated residues were found between ABCC6/ MRP6 and ABCC2 (R1114H and R1150H),30 ABCC6/MRP6 and ABCC7 (3775 del T and W1204X),31 ABCC6/MRP6 and ABCR (R1459C and H2128R, 4220InsAGAA and R2077W, R1141X and L1631P).32,33 Interestingly, for both ABCC7 and ABCR, models were postulated in which the severity of the disease shows an inverse correlation with the predicted transport activity of the ABC protein. Login to comment

[hide] Molecular genetics of pseudoxanthoma elasticum: ty... Hum Mutat. 2005 Sep;26(3):235-48. Miksch S, Lumsden A, Guenther UP, Foernzler D, Christen-Zach S, Daugherty C, Ramesar RK, Lebwohl M, Hohl D, Neldner KH, Lindpaintner K, Richards RI, Struk B
Molecular genetics of pseudoxanthoma elasticum: type and frequency of mutations in ABCC6.
Hum Mutat. 2005 Sep;26(3):235-48., [PMID:16086317]

Abstract [show]
Pseudoxanthoma elasticum (PXE) is a systemic heritable disorder that affects the elastic tissue in the skin, eye, and cardiovascular system. Mutations in the ABCC6 gene cause PXE. We performed a mutation screen in ABCC6 using haplotype analysis in conjunction with direct sequencing to achieve a mutation detection rate of 97%. This screen consisted of 170 PXE chromosomes in 81 families, and detected 59 distinct mutations (32 missense, eight nonsense, and six likely splice-site point mutations; one small insertion; and seven small and five large deletions). Forty-three of these mutations are novel variants, which increases the total number of PXE mutations to 121. While most mutations are rare, three nonsense mutations, a splice donor site mutation, and the large deletion comprising exons 23-29 (c.2996_4208del) were identified as relatively frequent PXE mutations at 26%, 5%, 3.5%, 3%, and 11%, respectively. Chromosomal haplotyping with two proximal and two distal polymorphic markers flanking ABCC6 demonstrated that most chromosomes that carry these relatively frequent PXE mutations have related haplotypes specific for these mutations, which suggests that these chromosomes originate from single founder mutations. The types of mutations found support loss-of-function as the molecular mechanism for the PXE phenotype. In 76 of the 81 families, the affected individuals were either homozygous for the same mutation or compound heterozygous for two mutations. In the remaining five families with one uncovered mutation, affected showed allelic compound heterozygosity for the cosegregating PXE haplotype. This demonstrates pseudo-dominance as the relevant inheritance mechanism, since disease transmission to the next generation always requires one mutant allelic variant from each parent. In contrast to other previous clinical and molecular claims, our results show evidence only for recessive PXE. This has profound consequences for the genetic counseling of families with PXE.

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293 R1150H in ABCC2 aligns with R1114H in ABCC6 within the second cytosolic loop of TMD2. Login to comment

[hide] Genetic association analysis of transporters ident... Pharmacogenet Genomics. 2012 Jun;22(6):447-65. Grover S, Gourie-Devi M, Bala K, Sharma S, Kukreti R
Genetic association analysis of transporters identifies ABCC2 loci for seizure control in women with epilepsy on first-line antiepileptic drugs.
Pharmacogenet Genomics. 2012 Jun;22(6):447-65., [PMID:22565165]

Abstract [show]
OBJECTIVE: The ATP-binding cassette (ABC) superfamily of transporters is known to efflux antiepileptic drugs (AEDs) primarily in the brain, gastrointestinal tract, liver, and kidneys. In addition, they are also known to be involved in estrogen disposition and may modulate seizure susceptibility and drug response. The objective of the present study was to investigate the role of genetic variants from ABC transporters in seizure control in epilepsy patients treated with monotherapy of first-line AEDs for 12 months. METHODS: On the basis of gene coverage and functional significance, a total of 98 single nucleotide polymorphisms from ABCB1, ABCC1, and ABCC2 were genotyped in 400 patients from North India. Of these, 216 patients were eligible for therapeutic assessment. Genetic variants were compared between the 'no-seizures' and the 'recurrent-seizures' groups. Bonferroni corrections for multiple comparisons and adjustment for covariates were performed before assessment of associations. RESULTS: Functionally relevant promoter polymorphisms from ABCC2: c.-1549G>A and c.-1019A>G either considered alone or in haplotype and diplotype combinations were observed for a significant association with seizure control in women (odds ratio>3.5, P<10, power>95%). Further, low protein-expressing CGT and TGT (c.-24C>T, c.1249G>A, c.3972C>T) haplotypes were always observed to be present in combination with the AG (c.-1549G>A, c.-1019A>G) haplotype that was over-represented in women with 'no seizures'. CONCLUSION: The distribution of the associated variants supports the involvement of ABCC2 in controlling seizures in women possibly by lowering of its expression. The biological basis of this finding could be an altered interaction of ABCC2 with AEDs and estrogens. These results necessitate replication in a larger pool of patients.

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89 - 24C > T 50 UTR k Promoter activity [haplotype containing (- 1549A)-(- 24T)] [33] k mRNA expression [29] m Expression and activity [35] m Expression [haplotype containing (- 24C)-1249A- 3972C] [36] k Expression [haplotype containing (- 24C)- 1249G- 3972T, (-24T)-1249G- 3972C or (-24T)-1249G- 3972T] [36] k Clearance of mycophenolic acid [35] k Clearance of methotrexate [37] k Clearance of irinotecan (ABCC2*2 containing the wild-type C allele) [29] 0.137 5 rs4919395 chr10:101542963 c.33 + 329G > A Intron 1 0.384 6 rs2756104 chr10:101544026 c.34 - 339C > T Intron 1 0.392 7 rs927344 chr10:101544447 c.116T > A Exon 2 (Phe39Tyr) 0.000 8 rs4148385 chr10:101548177 c.207+ 3639C > A Intron 2 0.382 9 rs2180990 chr10:101548974 c.208 - 3017C > G Intron 2 0.392 10 rs35191126 chr10:101549533:34 c.208 - 2458_208 - 2457G > delG Intron 2 0.384 11 rs4148389 chr10:101549911 c.208 - 2080A > G Intron 2 0.396 12 rs2804400 chr10:101553259 c.334 - 49C > T Intron 3 0.394 13 rs2756109 chr10:101558746 c.868 - 218T > G Intron 7 0.364 14 rs7080681 chr10:101560169 c.1058G > A Exon 9 (Arg353His) 0.000 15 rs2273697 chr10:101563815 c.1249G > A Exon 10 (Val417Ile) m mRNA expression [38] m Expression [haplotype containing (- 24C)-1249A- 3972C] [36] k Expression [haplotype containing (- 24C)- 1249G- 3972T, (-24T)-1249G- 3972C or (-24T)-1249G- 3972T] [36] k Clearance of irinotecan (ABCC*2 containing G allele) [34] 0.271 16 rs113646094 chr10:101564012 c.1446C > G Exon 10 (Thr482Thr) m mRNA expression [39] 0.002 17 rs2073337 chr10:101567426 c.1668 + 148A > G Intron 12 0.388 18 rs2756114 chr10:101569483 c.1816 - 408T > C Intron 13 0.393 19 rs3740074 chr10:101571528 c.1967+ 169T > C Intron 15 0.368 20 rs4148394 chr10:101572343 c.1968 - 432A > C Intron 15 0.206 21 rs3740072 chr10:101577123 c.2153A > G Exon 17 (Asn718Ser) 0.000 22 rs56199535 chr10:101578577 c.2302C > T Exon 18 (Arg768Trp) 0.000 23 rs56220353 chr10:101578641 c.2366C > G/T Exon 18 (Ser789Cys/Phe) k Activity, k expression and impaired membrane localization [40] 0.000 24 rs2002042 chr10:101587931 c.2621 - 2133C > T Intron 19 0.204 25 rs11442349 chr10:101589215:16 c.2621 - 849_2621 - 848T > delT Intron 19 0.375 26 rs3740071 chr10:101590120 c.2677C > G Exon 20 (Gln893Glu) 0.000 27 rs7898096 chr10:101593385 c.3259 -752G > A Intron 23 0.000 28 rs17216345 chr10:101594274 c.3396T > C Exon 24 (Ile1132Ile) 0.027 29 rs72558200 chr10:101595882 c.3449 G > A Exon 25 (Arg1150His) 0.000 30 rs72558201 chr10:101595950 c.3517 A > T Exon 25 (Ile1173Phe) 0.000 31 rs8187692 chr10:101595975 c.3542G > T Exon 25 (Leu1181Arg) 0.000 32 rs17222723 chr10:101595996 c.3563T > A Exon 25 (Val1188Glu) m Expression [41] 0.016 ABCC2 polymorphism and response to AEDs Grover et al. 451 or liver functioning. Login to comment

[hide] Functional characterization of protein variants of... Hum Mutat. 2012 Apr;33(4):750-62. doi: 10.1002/humu.22041. Epub 2012 Feb 28. Arlanov R, Porter A, Strand D, Brough R, Karpova D, Kerb R, Wojnowski L, Schwab M, Lang T
Functional characterization of protein variants of the human multidrug transporter ABCC2 by a novel targeted expression system in fibrosarcoma cells.
Hum Mutat. 2012 Apr;33(4):750-62. doi: 10.1002/humu.22041. Epub 2012 Feb 28., [PMID:22290738]

Abstract [show]
The multidrug resistance-associated protein 2 (MRP2/ABCC2) is involved in the efflux of endogenous and xenobiotic substrates, including several anticancer and antiviral drugs. The functional consequences of ABCC2 protein variants remain inconsistent, which may be due to shortcomings of the in vitro assays used. To study systematically the functional consequences of nonsynonymous ABCC2 variants, we used a novel "Screen and Insert" (ScIn) technology to achieve stable and highly reproducible expression of 13 ABCC2 variants in HT1080 cells. Western blotting revealed lower (30-65%) ABCC2 expression for D333G, R1174H, and R1181L as compared with wild type (WT; 100%), whereas the linked variant V1188E/C1515Y resulted in higher expression (150%). R1174H caused mislocalization of ABCC2 to the cytoplasm with an endoplasmic reticulum-like distribution. Variants N1244K and R1174H decreased transport of glutathione-methylfluorescein (GS-MF) and glutathione-monochlorobimane (GS-MCB) by 80% and 50%, respectively, whereas R1181L and P1291L reduced only GS-MCB transport by 50% as compared with WT. Contrary to protein data, the double variant V1188E/C1515Y decreased specific transport activity for GS-MF and GS-MCB by 40%. The ScIn approach is a feasible and reliable method to functionally characterize systematically ABCC2 variants. D333G, R1174H, R1181L, N1244K, P1291L, and double variant V1188E/C1515Y have been identified as most promising for further clinical evaluation.

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169 Computational comparative genomic studies using PolyPhen 2 predicted that nine of these ABCC2 variants (F39Y, D333G, I670T, I1036T, R1174H, R1181L, V1188E, N1244K, and P1291L) would be located within the transmembrane regions, close to the ATP-binding domain of ABCC2 or close to two missense variants (I1173F, R1150H) causing Dubin-Johnson syndrome [Keitel et al., 2003; Mor-Cohen et al., 2001]. Login to comment
272 The selected variants R1174H, R1181L, and V1188E (linked to C1515Y) from the present study, in addition to two recently identified variants that cause Dubin-Johnson syndrome (R1150H, I1173F), are located at the same gene region between the transmembrane helices TM15 and TM16. Login to comment

[hide] ABCC2/Abcc2: a multispecific transporter with domi... Drug Metab Rev. 2010 Aug;42(3):402-36. Jemnitz K, Heredi-Szabo K, Janossy J, Ioja E, Vereczkey L, Krajcsi P
ABCC2/Abcc2: a multispecific transporter with dominant excretory functions.
Drug Metab Rev. 2010 Aug;42(3):402-36., [PMID:20082599]

Abstract [show]
ABCC2/Abcc2 (MRP2/Mrp2) is expressed at major physiological barriers, such as the canalicular membrane of liver cells, kidney proximal tubule epithelial cells, enterocytes of the small and large intestine, and syncytiotrophoblast of the placenta. ABCC2/Abcc2 always localizes in the apical membranes. Although ABCC2/Abcc2 transports a variety of amphiphilic anions that belong to different classes of molecules, such as endogenous compounds (e.g., bilirubin-glucuronides), drugs, toxic chemicals, nutraceuticals, and their conjugates, it displays a preference for phase II conjugates. Phenotypically, the most obvious consequence of mutations in ABCC2 that lead to Dubin-Johnson syndrome is conjugate hyperbilirubinemia. ABCC2/Abcc2 harbors multiple binding sites and displays complex transport kinetics.

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113 Among these mutations recently compiled by Nies and Keppler (2007), of the five amino acids affected that are located outside the nucleotide-binding domains, four are basic (R100X, R393W, R1066X, and R1150H) and one is neutral (I1173F). Login to comment
114 R1150H is normally processed, but nonfunctional (Mor-Cohen et al., 2001). Login to comment

[hide] Age estimates of ancestral mutations causing facto... Blood Coagul Fibrinolysis. 2007 Mar;18(2):139-44. Mor-Cohen R, Zivelin A, Fromovich-Amit Y, Kovalski V, Rosenberg N, Seligsohn U
Age estimates of ancestral mutations causing factor VII deficiency and Dubin-Johnson syndrome in Iranian and Moroccan Jews are consistent with ancient Jewish migrations.
Blood Coagul Fibrinolysis. 2007 Mar;18(2):139-44., [PMID:17287630]

Abstract [show]
Factor VII (FVII) deficiency and Dubin-Johnson syndrome (DJS) are rare autosomal recessive disorders caused by mutations in F7 and MRP2 genes, respectively. Both disorders are relatively frequent among Iranian and Moroccan Jews. FVII deficiency in both populations is caused by a founder A244V mutation in the F7 gene and DJS is caused by two founder mutations, I1173F and R1150H in the MRP2 gene that are specific for Iranian and Moroccan Jewish patients, respectively. We estimated the age of FVII A244V and MRP2 I1173F by analysis of microsatellite markers flanking F7 and MRP2 genes, respectively, in 13 Iranian Jewish homozygotes for the I1173F mutation and 21 Iranian and Moroccan Jewish homozygotes for the A244V mutation. Dating of the mutations was estimated by the DMLE+2.0 program employing observed linkage disequilibria of multiple genetic markers. The estimated age of the I1173F mutation was approximately 1500 years, and the age of the A244V mutation was approximately 2600 years. These estimates suggest that I1173F causing DJS in Iranian Jews occurred after the separation of Iranian Jews from Moroccan Jews 2000-2600 years ago, while A244V causing FVII deficiency in Iranian and Moroccan Jews occurred prior to the divergence of these two populations.

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2 FVII deficiency in both populations is caused by a founder A244V mutation in the F7 gene and DJS is caused by two founder mutations, I1173F and R1150H in the MRP2 gene that are specific for Iranian and Moroccan Jewish patients, respectively. Login to comment
19 We previously reported two distinct missense mutations, I1173F and R1150H, causing DJS in Iranian and Moroccan Jewish patients, respectively. Login to comment
67 We also showed that DJS is prevalent in both populations and is caused by distinct mutations, I1173F in Iranian Jews and R1150H in Moroccan Jews [17]. Login to comment
74 An age estimate was not performed for the R1150H mutation due to the small number of available patients (five patients from four unrelated families). Login to comment
75 The fact that R1150H is a founder mutation unique for Moroccan Jews, however, indicates that it probably occurred after the separation of the two populations. Login to comment

[hide] The apical conjugate efflux pump ABCC2 (MRP2). Pflugers Arch. 2007 Feb;453(5):643-59. Epub 2006 Jul 18. Nies AT, Keppler D
The apical conjugate efflux pump ABCC2 (MRP2).
Pflugers Arch. 2007 Feb;453(5):643-59. Epub 2006 Jul 18., [PMID:16847695]

Abstract [show]
ABCC2 is a member of the multidrug resistance protein subfamily localized exclusively to the apical membrane domain of polarized cells, such as hepatocytes, renal proximal tubule epithelia, and intestinal epithelia. This localization supports the function of ABCC2 in the terminal excretion and detoxification of endogenous and xenobiotic organic anions, particularly in the unidirectional efflux of substances conjugated with glutathione, glucuronate, or sulfate, as exemplified by leukotriene C(4), bilirubin glucuronosides, and some steroid sulfates. The hepatic ABCC2 pump contributes to the driving forces of bile flow. Acquired or hereditary deficiency of ABCC2, the latter known as Dubin-Johnson syndrome in humans, causes an increased concentration of bilirubin glucuronosides in blood because of their efflux from hepatocytes via the basolateral ABCC3, which compensates for the deficiency in ABCC2-mediated apical efflux. In this article we provide an overview on the molecular characteristics of ABCC2 and its expression in various tissues and species. We discuss the transcriptional and posttranscriptional regulation of ABCC2 and review approaches to the functional analysis providing information on its substrate specificity. A comprehensive list of sequence variants in the human ABCC2 gene summarizes predicted and proven functional consequences, including variants leading to Dubin-Johnson syndrome.

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139 Although all sequence variants associated with Dubin-Johnson syndrome result in the absence of a Table 3 Nucleotide sequence variants in the human ABCC2 gene (NM_000392) leading to amino acid changes in the ABCC2/MRP2 protein (NP_000383) Location Nucleotide changea Deduced effect on proteina Causing Dubin-Johnson syndromeb Predicted effect by PolyPhen databasec Experimentally proven functional consequence Average frequency of indicated nucleotide exchange in population NCBI SNP IDd and/or references Exon 2 c.56 C>Te p.P19L Probably damaging T: 0.007 [63] Exon 2 c.116 T>A p.F39Y Benign A: 0.010 rs927344 A: 0.008 rs17222603 Exon 3 c.298 C>T p.R100Xf DJS [154] Exon 3 c.299 G>Ae p.R100Q Possibly damaging A: 0.007 [63] Exon 7 c.736 A>C p.M246L Benign C: 0.002 rs8187667 C: 0.002 rs17222744 Exon 7 c.842 G>A p.S281N Benign A: 0.0060.056 [117] Exon 8 c.998 A>G p.D333G Possibly damaging G: 0.002 rs8187668 G: 0.004 rs17222674 Exon 9 c.1058 G>A p.R353H Benign A: 0.009 rs7080681 A: 0.014 rs17216205 Exon 9 c.1177 C>T p.R393W DJS Probably damaging [104, 112] Exon 10 c.1234 A>G p.R412G Probably damaging Deficient methotrexate transport function [56] Exon 10 c.1249 G>A p.V417I Benign None apparent [50] A: 0.163 rs2273697, [146] A: 0.158 rs17216184 A: 0.125 [62] A: 0.1830.312 [117] Exon 10 c.1457 C>T p.T486I Benign T: 0.002 rs8187670 T: 0.002 rs17222589 Exon 11 c.1483 A>G p.K495E Possibly damaging G: 0.002 rs8187672 G: 0.002 rs17222561 Exon 13 c.1686 T>G p.F562L Benign G: 0.002 rs8187673 G: 0.002 rs17216233 Exon 16 c.2009 T>C p.I670T Benign rs8187676 C: 0.006 rs17222632 Exon 16 c.2026 G>C p.G676R DJS Probably damaging [181] Exon 17 c.2125 T>C p.W709R DJS Probably damaging [111] Exon 17 c.2153 A>G p.N718S Possibly damaging rs3740072 Exon 17 c.2215 C>T p.L739F Probably damaging T: 0.006 [51] Exon 18 c.2302 C>T p.R768W DJS Probably damaging Deficient maturation and impaired sorting [47] T: 0.010 [62] [168, 180] Exon 18 c.2366 C>T p.S789F Probably damaging Reduced protein levels [50] T: 0.010 [62] Exon 19 c.2546 T>G p.L849R Benign G: 0.002 rs8187689 G: 0.006 rs17222617 Exon 20 c.2647 G>Ae p.D883N Benign A: 0.007 [63] Exon 20 c.2677 G>C p.E893Q Benign rs3740071 Exon 21 c.2882 A>Ge p.K961R Benign G: 0.007 [63] Exon 22 c.2901 C>A p.Y967Xf A: 0.002 rs8187683 A: 0.002 rs17222547 Exon 22 c.2944 A>G p.I982V Benign G: 0.002 rs8187684 G: 0.002 rs17222554 Exon 22 c.3057 G>Te p.Q1019H Benign T: 0.007 [63] Exon 23 c.3107 T>C p.I1036T Possibly damaging C: 0.002 rs8187685 C: 0.004 rs17216149 Exon 23 c.3188 A>G p.N1063S Benign G: 0.002 rs8187686 G: 0.002 rs17222540 Exon 23 c.3196 C>T p.R1066Xf DJS No ABCC2 protein in liver [134] Exon 25 c.3449 G>A p.R1150H DJS Probably damaging Deficient transport function A: 00.009 [117] Exon 25 c.3517 A>T p.I1173F DJS Probably damaging Deficient maturation and impaired sorting, deficient transport function T: 00.029 [117] [80, 117] Exon 25 c.3521 G>Ae p.R1174H Probably damaging A: 0.007 [63] Exon 25 c.3542 G>T p.R1181L Possibly damaging T: 0.039 rs8187692 T: 0.034 rs17222702 Exon 25 c.3563 T>A p.V1188E Benign A: 0.059 rs8187694 A: 0.059 rs17222723 Exon 26 c.3732 T>Ge p.N1244K Possibly damaging G: 0.014 [63] Exon 27 c.3817 A>G p.T1273A Benign G: 0.002 rs8187699 G: 0.004 rs17222582 Exon 27 c.3825 C>G p.Y1275Xf DJS No ABCC2 protein in liver [104] Exon 28 c.3872 C>T p.P1291L Possibly damaging T: 0.012 rs8187700 T: 0.010 rs17216317 Exon 28 c.3895 A>C p.K1299Q Benign rs4148400, [146] Exon 28 c.3928 C>T p.R1310Xf DJS [166] Exon 29 c.4100 C>Ge p.S1367C Possibly damaging G: 0.007 [63] Exon 29 c.4145 A>G p.Q1382R DJS Probably Deficient [47, 168] Table 3 (continued) Location Nucleotide changea Deduced effect on proteina Causing Dubin-Johnson syndromeb Predicted effect by PolyPhen databasec Experimentally proven functional consequence Average frequency of indicated nucleotide exchange in population NCBI SNP IDd and/or references functionally active ABCC2 protein from the canalicular membrane, their effects on the synthesis and function of the ABCC2 protein differ. Login to comment
140 Although all sequence variants associated with Dubin-Johnson syndrome result in the absence of a Table 3 Nucleotide sequence variants in the human ABCC2 gene (NM_000392) leading to amino acid changes in the ABCC2/MRP2 protein (NP_000383) Location Nucleotide changea Deduced effect on proteina Causing Dubin-Johnson syndromeb Predicted effect by PolyPhen databasec Experimentally proven functional consequence Average frequency of indicated nucleotide exchange in population NCBI SNP IDd and/or references Exon 2 c.56 C>Te p.P19L Probably damaging T: 0.007 [63] Exon 2 c.116 T>A p.F39Y Benign A: 0.010 rs927344 A: 0.008 rs17222603 Exon 3 c.298 C>T p.R100Xf DJS [154] Exon 3 c.299 G>Ae p.R100Q Possibly damaging A: 0.007 [63] Exon 7 c.736 A>C p.M246L Benign C: 0.002 rs8187667 C: 0.002 rs17222744 Exon 7 c.842 G>A p.S281N Benign A: 0.0060.056 [117] Exon 8 c.998 A>G p.D333G Possibly damaging G: 0.002 rs8187668 G: 0.004 rs17222674 Exon 9 c.1058 G>A p.R353H Benign A: 0.009 rs7080681 A: 0.014 rs17216205 Exon 9 c.1177 C>T p.R393W DJS Probably damaging [104, 112] Exon 10 c.1234 A>G p.R412G Probably damaging Deficient methotrexate transport function [56] Exon 10 c.1249 G>A p.V417I Benign None apparent [50] A: 0.163 rs2273697, [146] A: 0.158 rs17216184 A: 0.125 [62] A: 0.1830.312 [117] Exon 10 c.1457 C>T p.T486I Benign T: 0.002 rs8187670 T: 0.002 rs17222589 Exon 11 c.1483 A>G p.K495E Possibly damaging G: 0.002 rs8187672 G: 0.002 rs17222561 Exon 13 c.1686 T>G p.F562L Benign G: 0.002 rs8187673 G: 0.002 rs17216233 Exon 16 c.2009 T>C p.I670T Benign rs8187676 C: 0.006 rs17222632 Exon 16 c.2026 G>C p.G676R DJS Probably damaging [181] Exon 17 c.2125 T>C p.W709R DJS Probably damaging [111] Exon 17 c.2153 A>G p.N718S Possibly damaging rs3740072 Exon 17 c.2215 C>T p.L739F Probably damaging T: 0.006 [51] Exon 18 c.2302 C>T p.R768W DJS Probably damaging Deficient maturation and impaired sorting [47] T: 0.010 [62] [168, 180] Exon 18 c.2366 C>T p.S789F Probably damaging Reduced protein levels [50] T: 0.010 [62] Exon 19 c.2546 T>G p.L849R Benign G: 0.002 rs8187689 G: 0.006 rs17222617 Exon 20 c.2647 G>Ae p.D883N Benign A: 0.007 [63] Exon 20 c.2677 G>C p.E893Q Benign rs3740071 Exon 21 c.2882 A>Ge p.K961R Benign G: 0.007 [63] Exon 22 c.2901 C>A p.Y967Xf A: 0.002 rs8187683 A: 0.002 rs17222547 Exon 22 c.2944 A>G p.I982V Benign G: 0.002 rs8187684 G: 0.002 rs17222554 Exon 22 c.3057 G>Te p.Q1019H Benign T: 0.007 [63] Exon 23 c.3107 T>C p.I1036T Possibly damaging C: 0.002 rs8187685 C: 0.004 rs17216149 Exon 23 c.3188 A>G p.N1063S Benign G: 0.002 rs8187686 G: 0.002 rs17222540 Exon 23 c.3196 C>T p.R1066Xf DJS No ABCC2 protein in liver [134] Exon 25 c.3449 G>A p.R1150H DJS Probably damaging Deficient transport function A: 00.009 [117] Exon 25 c.3517 A>T p.I1173F DJS Probably damaging Deficient maturation and impaired sorting, deficient transport function T: 00.029 [117] [80, 117] Exon 25 c.3521 G>Ae p.R1174H Probably damaging A: 0.007 [63] Exon 25 c.3542 G>T p.R1181L Possibly damaging T: 0.039 rs8187692 T: 0.034 rs17222702 Exon 25 c.3563 T>A p.V1188E Benign A: 0.059 rs8187694 A: 0.059 rs17222723 Exon 26 c.3732 T>Ge p.N1244K Possibly damaging G: 0.014 [63] Exon 27 c.3817 A>G p.T1273A Benign G: 0.002 rs8187699 G: 0.004 rs17222582 Exon 27 c.3825 C>G p.Y1275Xf DJS No ABCC2 protein in liver [104] Exon 28 c.3872 C>T p.P1291L Possibly damaging T: 0.012 rs8187700 T: 0.010 rs17216317 Exon 28 c.3895 A>C p.K1299Q Benign rs4148400, [146] Exon 28 c.3928 C>T p.R1310Xf DJS [166] Exon 29 c.4100 C>Ge p.S1367C Possibly damaging G: 0.007 [63] Exon 29 c.4145 A>G p.Q1382R DJS Probably Deficient [47, 168] Table 3 (continued) Location Nucleotide changea Deduced effect on proteina Causing Dubin-Johnson syndromeb Predicted effect by PolyPhen databasec Experimentally proven functional consequence Average frequency of indicated nucleotide exchange in population NCBI SNP IDd and/or references functionally active ABCC2 protein from the canalicular membrane, their effects on the synthesis and function of the ABCC2 protein differ. Login to comment

[hide] Identification of a novel 974C-->G nonsense mutati... Am J Gastroenterol. 2006 Oct;101(10):2427-32. Epub 2006 Sep 4. Corpechot C, Ping C, Wendum D, Matsuda F, Barbu V, Poupon R
Identification of a novel 974C-->G nonsense mutation of the MRP2/ABCC2 gene in a patient with Dubin-Johnson syndrome and analysis of the effects of rifampicin and ursodeoxycholic acid on serum bilirubin and bile acids.
Am J Gastroenterol. 2006 Oct;101(10):2427-32. Epub 2006 Sep 4., [PMID:16952291]

Abstract [show]
Rifampicin (RIF) and ursodeoxycholic acid (UDCA) therapies have beneficial effects in chronic cholestatic diseases. These may result in part from the induction of multidrug-resistance protein 2 (MRP2/ABCC2) expression in the liver and kidney. However, the precise mechanisms by which RIF and UDCA act in cholestasis remain unclear. In the present study, we report the effects of chronic administration of both drugs in a patient with Dubin-Johnson syndrome (DJS), an inherited autosomal recessive disorder characterized by the absence of functional MRP2 protein at the canalicular hepatocyte membrane. A novel 974C-->G nonsense mutation was identified in the MRP2 gene sequence from this patient. RIF induced further increase in conjugated bilirubinemia, whereas concomitant administration of RIF and UDCA led to a dramatic rise in serum bile acid concentrations. These biochemical effects, which are in marked contrast to those observed in cholestatic settings, were concomitant with an increased MRP3, but not MRP4, expression on basolateral hepatocyte membrane. Such findings highlight the key role of MRP2 in the pharmacological properties of RIF and UDCA and suggest that both drugs should be used with caution in pathologic settings in which MRP2 expression may be downregulated, as in advanced stage of cholestatic diseases.

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78 Mutations in the MRP2/ABCC2 Gene Associated with DJS Nucleotide Mutation Exon Predicted Effect Reference 298C→T 3 R100X 27 974C→G 8 S325X This article IVS8 + 4A→G Intron 8 Aberrant splicing 28 1177C→T 9 R393W 29 1256insCT/ delAAACAG TGAACCT- GATG 10 Frameshift 30 1271A→G 10 R412G 31 1815 + 2T→A 13 Skipped exon 32, 33 1967 + 2T→C 15 Skipped exon 34, 35 2026G→C 16 G676R 35 2125T→C 17 W709R 36 2302C→T 18 R768W 32, 37, 38 2439 + 2T→C 18 Skipped exon 32, 35, 37 3196C→T 23 R1066X 39, 40 3449Gș2;A 25 R1150H 41 3517A→T 25 I1173F 41 3928C→T 28 R1310X 27, 33 4145A→G 29 Q1382R 37 4175delGGATGA 30 R1392 + M1393 deletion 40 4292delCA 30 Frameshift 30 DISCUSSION Identification of a Novel Nonsense Mutation of the MRP2/ABCC2 Gene Up to now, 18 mutations in the sequence of the MRP2/ABCC2 gene have been reported in DJS, including nonsense mutations, deletions, splicing junction mutations, and missense mutations (Table 1). Login to comment

[hide] Single nucleotide polymorphisms in ABCC2 and ABCB1... Cancer Lett. 2006 Mar 8;234(1):40-50. Epub 2005 Dec 27. Wada M
Single nucleotide polymorphisms in ABCC2 and ABCB1 genes and their clinical impact in physiology and drug response.
Cancer Lett. 2006 Mar 8;234(1):40-50. Epub 2005 Dec 27., [PMID:16377077]

Abstract [show]
Among the ABC proteins, some members including ABCB1, ABCC1, ABCC2 and ABCG2 are believed to contribute to multidrug resistance of cancer chemotherapy. In addition, the broad substrate-specificity and apical localization of the ABCB1 and ABCC2 in mucosal epithelium of intestine and hepatocyte give them a protective role against xenobiotics. The inter-individual variations in activity and expression levels of ABCB1 and ABCC2, thus, might affect on drug response and response to toxic substrates. In this review, I focus on (1) physiological and toxicological relevance of ABCB1 and ABCC2, and on (2) genetic variations of ABCB1 and ABCC2 genes and their association with biochemical function, expression level and tumor incidence.

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41 In Japan, the expected number of Table 1 Summary of mutations identified in Dubin-Johnson syndrome (DJS) Mutation Exon IVS Amino acid alteration Reference 298COT 3 R100X a,b 1815C 2TOA 13 Exon13 skip [38] 1967C 2TOC 15 Exon15 skip [62] 2026GOC 16 G676R [92] 2302COT 18 R768W [49,91]c 2439C 2TOC 18 Exon18 skip [38]a,c 3196COT 23 R1066X [47] 3449GOA 25 R1150H [52] 3517AOT 25 I1173F [52] 3928COT 28 R1310X [50] 4145AOG 29 Q1382R [38] 4175- 4180del 30 RM1392-1393del [48] a Adachi and Wada, unpublished data. b Houkibara and Wada, unpublished data. Login to comment
59 [42,49,53,91]c 18 2366COT S789F NBD1 (Transport activity) Not reported 0.9 [42]a 25 3449GOA R1150H MSD3 DJS (transport activity) 0.3 Not reported [52] 25 3517AOT I1173F MSD3 DJS (protein maturation) 1.4 Not reported [52] 28 3895AOC K1299Q NBD2 Unkown Not reported 1? Login to comment
66 Immunoblot analysis and immunocytochemistry showed that one mutant ABCC2 (R1150H) matured properly and localized at the plasma membrane of transfected cells. Login to comment

[hide] Apical/basolateral surface expression of drug tran... Pharm Res. 2005 Oct;22(10):1559-77. Epub 2005 Sep 22. Ito K, Suzuki H, Horie T, Sugiyama Y
Apical/basolateral surface expression of drug transporters and its role in vectorial drug transport.
Pharm Res. 2005 Oct;22(10):1559-77. Epub 2005 Sep 22., [PMID:16180115]

Abstract [show]
It is well known that transporter proteins play a key role in governing drug absorption, distribution, and elimination in the body, and, accordingly, they are now considered as causes of drug-drug interactions and interindividual differences in pharmacokinetic profiles. Polarized tissues directly involved in drug disposition (intestine, kidney, and liver) and restricted distribution to naive sanctuaries (blood-tissue barriers) asymmetrically express a variety of drug transporters on the apical and basolateral sides, resulting in vectorial drug transport. For example, the organic anion transporting polypeptide (OATP) family on the sinusoidal (basolateral) membrane and multidrug resistance-associated protein 2 (MRP2/ABCC2) on the apical bile canalicular membrane of hepatocytes take up and excrete organic anionic compounds from blood to bile. Such vectorial transcellular transport is fundamentally attributable to the asymmetrical distribution of transporter molecules in polarized cells. Besides the apical/basolateral sorting direction, distribution of the transporter protein between the membrane surface (active site) and the intracellular fraction (inactive site) is of practical importance for the quantitative evaluation of drug transport processes. The most characterized drug transporter associated with this issue is MRP2 on the hepatocyte canalicular (apical) membrane, and it is linked to a genetic disease. Dubin-Johnson syndrome is sometimes caused by impaired canalicular surface expression of MRP2 by a single amino acid substitution. Moreover, single nucleotide polymorphisms in OATP-C/SLC21A6 (SLCO1B1) also affect membrane surface expression, and actually lead to the altered pharmacokinetic profile of pravastatin in healthy subjects. In this review article, the asymmetrical transporter distribution and altered surface expression in polarized tissues are discussed.

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239 Similarly, R1150H was also found as the mature glycosylated form on the membrane surface of transfected HEK293 cells but was not functional (123). Login to comment

[hide] Dual hereditary jaundice: simultaneous occurrence ... Gastroenterology. 2005 Jul;129(1):315-20. Cebecauerova D, Jirasek T, Budisova L, Mandys V, Volf V, Novotna Z, Subhanova I, Hrebicek M, Elleder M, Jirsa M
Dual hereditary jaundice: simultaneous occurrence of mutations causing Gilbert's and Dubin-Johnson syndrome.
Gastroenterology. 2005 Jul;129(1):315-20., [PMID:16012956]

Abstract [show]
BACKGROUND & AIMS: Dubin-Johnson syndrome is recessively inherited, conjugated hyperbilirubinemia induced by mutations in the ABCC2/MRP2 gene encoding the canalicular transporter for conjugated bilirubin. Gilbert's syndrome is recessively inherited, unconjugated hyperbilirubinemia caused by decreased conjugation rate of bilirubin associated mostly with homozygous A(TA) 7 TAA variant of the TATAA-box in the UGT1A1 gene promoter. Our aim was to establish the molecular diagnosis in a 3-year-old male with atypical, intermittent, predominantly unconjugated, hyperbilirubinemia. METHODS: 99m Tc-HIDA cholescintigraphy was used for imaging the biliary tree. Expression of ABCC2/MRP2 protein in hepatocytes was investigated immunohistochemically. UGT1A1 and ABCC2/MRP2 genes were sequenced from genomic DNA, and the mutations were verified by fragment analysis, sequencing the cloned exons, and restriction fragment length polymorphism. RESULTS: Cholescintigraphy revealed delayed visualization of the gallbladder. A brown granular lipopigment differing from melanin-like pigment reported in Dubin-Johnson syndrome was present in hepatocytes, but, otherwise, liver histology was normal. ABCC2/MRP2 protein was not detected on the canalicular membrane of hepatocytes, and 2 novel mutations were found in the ABCC2/MRP2 gene: a heterozygous in-frame insertion-deletion mutation 1256insCT/delAAACAGTGAACCTGATG in exon 10 inherited from the father and a heterozygous deletion 4292delCA in exon 30 inherited from the mother. In addition, the patient was homozygous for -3279T>G and A(TA) 7 TAA mutations in the UGT1A1 gene promoter. CONCLUSIONS: Our patient represents a case of digenic mixed hyperbilirubinemia-a distinct type of constitutive jaundice resulting from coinherited defects in ABCC2/MRP2 and UGT1A1 genes.

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60 3449GϾA 25 R1150H Mor-Cohen R et al, J Biol Chem 2001;276:36923-36930. Login to comment

[hide] Identification of a novel 2026G-->C mutation of th... J Hum Genet. 2003;48(8):425-9. Epub 2003 Jul 22. Wakusawa S, Machida I, Suzuki S, Hayashi H, Yano M, Yoshioka K
Identification of a novel 2026G-->C mutation of the MRP2 gene in a Japanese patient with Dubin-Johnson syndrome.
J Hum Genet. 2003;48(8):425-9. Epub 2003 Jul 22., [PMID:12884082]

Abstract [show]
Dubin-Johnson syndrome is a recessive inherited disorder with conjugated hyperbilirubinemia caused by a dysfunction of multidrug resistance protein 2 (MRP2) on the canalicular membrane of hepatocytes. A mutational analysis of the MRP2 gene was carried out in three Japanese patients and their family members. In two patients, the homozygous mutations c.1901del67 and c,2272del168 were found. In the third patient, a -24C-->T polymorphism and the two mutations c.1901del67 and 2026G-->C were detected. The 2026G-->C mutation was a novel mutation in exon 16 affecting the conversion of Gly(676) to Arg(676) (G676R) in the MRP2 protein, and was not detected in fifty healthy volunteers. The G676R mutation was located in the Walker A motif of the first nucleotide binding domain in the MRP2 protein, and it was suggested that the mutation induced the dysfunction of the MRP2 protein. It was concluded that the compound heterozygosity of the two mutations of the MRP2 gene in the third patient contributed to the induction of hyperbilirubinemia in this case.

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43 With respect to the polymorphism )24C fi T, we also employed a restriction enzyme assay with BbsI; the results showed that his spouse and one daughter were heterozygous for this SNP, but other Table 1 Mutations of MRP2 in Dubin-Johnson syndrome (DJS) Nucleotide mutation Predicted effects References Splice site mutation 1815+2T fi A 1669del147 (exon13 skipping) Wada et al. 1998 1967+2T fi C 1901del67 (exon15 skipping) Kajihara et al. 1998 2439+2T fi C 2272del168 (exon18 skipping) Toh et al. 1999 Deletion mutation Del4170-5 Del R1392 , M1393 Tsujii et al. 1999 Missense mutation 2302C fi T R768 W Wada et al. 1998 3449G fi A R1150H Mor-Cohen et al. 2001 3517A fi T I1173F Mor-Cohen et al. 2001 4145A fi G Q1382R Toh et al. 1999 Nonesense mutation 3196C fi T R1066X Paulusma et al. 1997 3928C fi T R1310X Tate et al. 2002 family members did not possess it (Fig. 4). Login to comment

[hide] Single nucleotide polymorphisms in multidrug resis... Adv Drug Deliv Rev. 2002 Nov 18;54(10):1311-31. Suzuki H, Sugiyama Y
Single nucleotide polymorphisms in multidrug resistance associated protein 2 (MRP2/ABCC2): its impact on drug disposition.
Adv Drug Deliv Rev. 2002 Nov 18;54(10):1311-31., [PMID:12406647]

Abstract [show]
Multidrug resistance associated protein 2 (MRP2/ABCC2), expressed on the bile canalicular membrane, plays an important role in the biliary excretion of various kinds of substrates. In addition, MRP2 is also expressed on the apical membrane of epithelial cells such as enterocytes. It is possible that the inter-individual difference in the function of MRP2 affects the drug disposition. In the present article, we will summarize the physiological and pharmacological role of MRP2, particularly focusing on the factors affecting its transport function such as single nucleotide polymorphisms and/or the induction/down regulation of this transporter. Mutations found in patients suffering from the Dubin-Johnson syndrome, along with the amino acid residues which are involved in supporting the transport activity of MRP2, are also summarized.

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109 mutation, and deletion mutation have all been re- Moreover, I1173F and R1150H have been found ported. Login to comment
114 In addition, Western blot and immunohistochemical analysis indicated that, Asp1208Asn lost their ability to transport these although R1150H was mature and targeted to the conjugates (Fig. 2) [109]. Login to comment
116 The mechanism for Met transport monovalent bile salts, which are not the impaired function of R1150H remains to be transported by wt MRP2 (Fig. 2) [110]. Login to comment

[hide] Regulation of expression of the multidrug resistan... J Pharmacol Exp Ther. 2002 Aug;302(2):407-15. Gerk PM, Vore M
Regulation of expression of the multidrug resistance-associated protein 2 (MRP2) and its role in drug disposition.
J Pharmacol Exp Ther. 2002 Aug;302(2):407-15., [PMID:12130697]

Abstract [show]
The multidrug resistance protein 2 (MRP2; ABCC2) is an ATP-binding cassette transporter accepting a diverse range of substrates, including glutathione, glucuronide, and sulfate conjugates of many endo- and xenobiotics. MRP2 generally performs excretory or protective roles, and it is expressed on the apical domain of hepatocytes, enterocytes of the proximal small intestine, and proximal renal tubular cells, as well as in the brain and the placenta. MRP2 is regulated at several levels, including membrane retrieval and reinsertion, translation, and transcription. In addition to transport of conjugates, MRP2 transports cancer chemotherapeutics, uricosurics, antibiotics, leukotrienes, glutathione, toxins, and heavy metals. Several mutagenesis studies have described critical residues for substrate binding and various naturally occurring mutations that eliminate MRP2 expression or function. MRP2 is important clinically as it modulates the pharmacokinetics of many drugs, and its expression and activity are also altered by certain drugs and disease states.

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38 Unlike other mutations, R1150H mutants of the MRP2 protein mature and are properly localized, but transport activity is impaired (Mor-Cohen et al., 2001). Login to comment
53 Site-directed mutagenesis studies substi- TABLE 1 Mutations in human MRP2 Mutation Location Translation Domain Phenotype Reference -24(C-T) Promoter 5Ј-UTR Not reported (Ito et al., 2001e) 1249(G-A)/wt Exon 10 V4171 MSD2 Not reported (Ito et al., 2001e) 1815ϩ2(T-A)/1815ϩ2(T-A) Exon 13 147-bp deletion MSD2 DJS (Wada et al., 1998; Toh et al., 1999) 2002del67/2002del67 Exon 16 Premature termination codon NBD1 DJS (Konig et al., 1999a) 2302(C-T)/2302(C-T) Exon 18 R768W/R768W NBD1 DJS (Wada et al., 1998; Toh et al., 1999; Ito et al., 2001e) 2302(C-T)/2439ϩ2(T-C) Exon 18 R768W/168-bp deletion NBD1 DJS (Toh et al., 1999) 2302(C-T)/wt Exon 18 R768W/wt NBD1 Increased UCP1 (Toh et al., 1999) 2366(C-T)/wt Exon 18 S789F/wt NBD1 Not reported (Ito et al., 2001e) 2439ϩ2(T-C)/2439ϩ2(T-C) Exon 18 168-bp deletion/168-bp deletion NBD1 DJS (Wada et al., 1998; Toh et al., 1999) 2439ϩ2(T-C)/4145(A-G) Exon 18/29 168-bp deletion/Q1328R NBD1/2 DJS (Toh et al., 1999) 2439ϩ2(T-C)/wt Exon 18 168-bp deletion/wt NBD1 Increased UCP1 (Toh et al., 1999) 3196(C-T)/3196(C-T) Exon 23 Premature termination codon MSD3 DJS (Paulusma et al., 1997; Tsujii et al., 1999) 3449(G-A)/3449(G-A) Exon 25 R1150H/R1150H MSD3 DJS (Mor-Cohen et al., 2001) 3517(A-T)/3517(A-T) Exon 25 I1173F/I1173F MSD3 DJS (Mor-Cohen et al., 2001) 3972(C-T)/wt Exon 28 I1324I/wt near NBD2 None (Ito et al., 2001e) 4175del6 Exon 30 Loss of R1392 and M1393 NBD2 DJS (Tsujii et al., 1999) 4348(G-A)/wt Exon 31 A1450T/wt NBD2 Not reported (Ito et al., 2001e) UTR, untranslated region; wt, wild type; bp, base pair; UCP1, urinary coproporphyrin fraction 1. tuting the cationic amino acid Arg586 and Arg1096 in rat Mrp2 with neutral amino acids (R586L, R586I, R1096L, and R1096M) or a cationic amino acid (R1096K) led to acquisition of taurocholate transport and retention of glucuronide and glutathione conjugate transport by Mrp2 (Ito et al., 2001d). Login to comment
186 Since MRP2 seems to act as a protective barrier in the TABLE 2 Factors altering MRP2/Mrp2 mRNA or protein expression and function in vitro or in vivo, as discussed in the text Regulation of expression Renal failure Pregnancy and lactation Cholestasis MRP2 mutations Sepsis (lipopolysaccharide) Hypo-or hyperosmolarity Phalloidin Bile duct ligation Acute-phase response Rifampin FXR, CAR, and PXR ligands Dibutyryl cAMP Inhibition or stimulation of activity ATP depletion Estrogen glucuronides Cholestasis MRP2 mutation: R1150H Cancer chemotherapeutics: irinotecan, methotrexate, and vinblastine Uricosurics: sulfinpyrazone, probenecid, and benzbromarone brain, intestine, and placenta, MRP2 alterations may also affect the absorption and distribution of these compounds, thus affecting therapeutics or toxicology. Login to comment

[hide] Identification and functional analysis of two nove... J Biol Chem. 2001 Oct 5;276(40):36923-30. Epub 2001 Jul 26. Mor-Cohen R, Zivelin A, Rosenberg N, Shani M, Muallem S, Seligsohn U
Identification and functional analysis of two novel mutations in the multidrug resistance protein 2 gene in Israeli patients with Dubin-Johnson syndrome.
J Biol Chem. 2001 Oct 5;276(40):36923-30. Epub 2001 Jul 26., [PMID:11477083]

Abstract [show]
Dubin-Johnson syndrome (DJS) is an inherited disorder characterized by conjugated hyperbilirubinemia and is caused by a deficiency of the multidrug resistance protein 2 (MRP2) located in the apical membrane of hepatocytes. The aim of this study was to identify the mutations in two previously characterized clusters of patients with Dubin-Johnson syndrome among Iranian and Moroccan Jews and determine the consequence of the mutations on MRP2 expression and function by expression studies. All 32 exons and adjacent regions of the MRP2 gene were screened by polymerase chain reaction and DNA sequencing. Two novel mutations were identified in exon 25. One mutation, 3517A-->T, predicting a I1173F substitution, was found in 22 homozygous Iranian Jewish DJS patients from 13 unrelated families and a second mutation, 3449G-->A, predicting a R1150H substitution, was found in 5 homozygous Moroccan Jewish DJS patients from 4 unrelated families. Use of four intragenic dimorphisms and haplotype analyses disclosed a specific founder effect for each mutation. The mutations were introduced into an MRP2 expression vector by site-directed mutagenesis, transfected into HEK-293 cells, and analyzed by a fluorescence transport assay, immunoblot, and immunocytochemistry. Continuous measurement of probenecid-sensitive carboxyfluorescein efflux revealed that both mutations impaired the transport activity of MRP2. Immunoblot analysis and immunocytochemistry showed that MRP2 (R1150H) matured properly and localized at the plasma membrane of transfected cells. In contrast, expression of MRP2 (I1173F) was low and mislocated to the endoplasmic reticulum of the transfected cells. These findings provide an explanation for the DJS phenotype in these two patient groups. Furthermore, the close localization of the two mutations identify this region of MRP2 as important for both activity and processing of the protein.

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3 One mutation, 3517A3T, predicting a I1173F substitution, was found in 22 homozygous Iranian Jewish DJS patients from 13 unrelated families and a second mutation, 3449G3A, predicting a R1150H substitution, was found in 5 homozygous Moroccan Jewish DJS patients from 4 unrelated families. Login to comment
7 Immunoblot analysis and immunocytochemistry showed that MRP2 (R1150H) matured properly and localized at the plasma membrane of transfected cells. Login to comment
65 The primers used to introduce the R1150H mutation are the forward primer (5Ј-GCCAGCTGAG- GCATCTGGACTCTGTCACCAG-3Ј) and the reverse primer (5Ј-CTG- GTGACAGAGTCCAGATGCCTCAGCTGGC-3Ј). Login to comment
105 The mutation predicts an R1150H substitution and results in loss of a BsaHI restriction site. Login to comment
157 Carboxyfluorescein Transport of Mutants MRP2-Transfected HEK-293 cells expressing the I1173F- and R1150H-MRP2 mutants were used to study the effect of the mutations on CF transport. Login to comment
160 Cells transfected with either I1173F-MRP2 (Fig. 5C) or R1150H-MRP2 (Fig. 5D) showed a slow efflux of CF, which was similar to that measured in control cells (Figs. Login to comment
164 The average rate constant of CF efflux (in s-1 ) by WT-MRP2 (562 Ϯ 56, n ϭ 4) was significantly higher than that measured in control cells (151 Ϯ 45, n ϭ 5), R1150H-MRP2 cells (90 Ϯ 40, n ϭ 5) and I1173F-MRP2 cells (104 Ϯ 66, n ϭ 3). Login to comment
165 No significant difference was found between the average rate constant of control, R1150H-MRP2, and I1173F-MRP2 cells. These data indicate TABLE III Frequency of haplotypes in Iranian Jewish and Moroccan Jewish controls and DJS patients Haplotype 5Ј-Untranslated region -24C3T Exon 7 842G3A Exon 10 1249G3A IVS29-35 G3A Frequency in Iranian Jewsa Frequency in Moroccan Jewsa Controls (n ϭ 144) Patients (n ϭ 26) Controls (n ϭ 118) Patients (n ϭ 8) % % 1 C G A A 0 100 0 0 2 C A G G 0.7 0 1.7 100 3 C G G G 63.2 0 61 0 4 C G A G 23.6 0 14.4 0 5 T G G G 11.1 0 18.6 0 Seven additional haplotypes 2.1 0 4.3 0 a n indicates number of informative alleles of unrelated individuals. Login to comment
178 Panel E shows the irreversible inhibition of efflux by cyclosporine A. that both I1173F and R1150H mutations impair the transport activity of MRP2. Login to comment
186 The R1150H-MRP2 behaved similarly to WT-MRP2, showing a very intense band at 190 kDa and a very weak band at 175 kDa when 20 ␮g of protein was used, and no 175-kDa band when 13 ␮g of protein was used. Login to comment
188 At 20 ␮g of I1173F-MRP2 protein, staining was much weaker than that observed with either WT or R1150H-MRP2. Login to comment
189 When 55 ␮g of protein from cells expressing I1173F-MRP2 was used, the overall amount of protein was comparable to that measured with 13 ␮g of WT or R1150H-MRP2. Login to comment
194 In cells transfected with WT-MRP2 and in R1150H-MRP2, the protein was localized at the plasma membrane (Fig. 7, A and B) and a Golgi-like structure. Login to comment
195 This suggested that processing and targeting of the R1150H-MRP2 mutant was normal. Login to comment
199 Both mutations are located in exon 25 of the MRP2 gene, with 3517A3T predicting I1173F substitution found in a cluster of Iranian Jewish DJS patients, and 3449G3A predicting R1150H substitution found in a cluster of Moroccan Jewish patients. Login to comment
218 Cells were transfected with GFP only (control, A), WT-MRP2 (B), I1173F-MRP2 (C), or R1150H-MRP2 (D). Login to comment
233 With this assay we showed that both I1173F and R1150H mutations impaired the MRP2 activity (Fig. 5). Login to comment
237 The R1150H mutation had no apparent effect on the relative amount of each MRP2 forms. Login to comment
240 Indeed, immunolocalization of the different constructs showed that WT and R1150H-MRP2 were properly targeted to the plasma membrane, whereas I1173F-MRP2 remained largely confined to the ER. Login to comment
242 Our expression studies suggest that the I1173F mutation causes impaired maturation of MRP2, mislocalization probably to the ER, and augmented degradation, whereas the R1150H mutation does not affect the maturation and localization, but impairs the MRP2 transport activity. Login to comment
252 HEK 293 cells transfected with WT-MRP2 (A), R1150H-MRP2 (B), or I1173F-MRP2 (C) were detected by the monoclonal antibody M2III-6 and were examined by confocal laser scanning microscopy. Login to comment

[hide] Structure, function, expression, genomic organizat... Int J Toxicol. 2006 Jul-Aug;25(4):231-59. Choudhuri S, Klaassen CD
Structure, function, expression, genomic organization, and single nucleotide polymorphisms of human ABCB1 (MDR1), ABCC (MRP), and ABCG2 (BCRP) efflux transporters.
Int J Toxicol. 2006 Jul-Aug;25(4):231-59., [PMID:16815813]

Abstract [show]
The ATP-binding cassette (ABC) transporters constitute a large family of membrane proteins, which transport a variety of compounds through the membrane against a concentration gradient at the cost of ATP hydrolysis. Substrates of the ABC transporters include lipids, bile acids, xenobiotics, and peptides for antigen presentation. As they transport exogenous and endogenous compounds, they reduce the body load of potentially harmful substances. One by-product of such protective function is that they also eliminate various useful drugs from the body, causing drug resistance. This review is a brief summary of the structure, function, and expression of the important drug resistance-conferring members belonging to three subfamilies of the human ABC family; these are ABCB1 (MDR1/P-glycoprotein of subfamily ABCB), subfamily ABCC (MRPs), and ABCG2 (BCRP of subfamily ABCG), which are expressed in various organs. In the text, the transporter symbol that carries the subfamily name (such as ABCB1, ABCC1, etc.) is used interchangeably with the corresponding original names, such as MDR1P-glycoprotein, MRP1, etc., respectively. Both nomenclatures are maintained in the text because both are still used in the transporter literature. This helps readers relate various names that they encounter in the literature. It now appears that P-glycoprotein, MRP1, MRP2, and BCRP can explain the phenomenon of multidrug resistance in all cell lines analyzed thus far. Also discussed are the gene structure, regulation of expression, and various polymorphisms in these genes. Because genetic polymorphism is thought to underlie interindividual differences, including their response to drugs and other xenobiotics, the importance of polymorphism in these genes is also discussed.

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340 Additional missense mutations Ile1173Phe and Arg1150His have been found in Iranian Jewish and Moroccan Jewish DJS patients, respectively. Login to comment