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J. Biol. Chem., Vol. 276, Issue 40, 36923-36930, October 5, 2001

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Identification and Functional Analysis of Two Novel Mutations in the Multidrug Resistance Protein 2 Gene in Israeli Patients with Dubin-Johnson Syndrome^*

Ronit Mor-Cohen^§, Ariella Zivelin, Nurit Rosenberg, Mordechai Shani, Shmuel Muallem^§¶, and Uri Seligsohn

From the  Institute of Thrombosis and Hemostasis, Department of Hematology, Chaim Sheba Medical Center and Sackler Faculty of Medicine, Tel Aviv University, Tel Hashomer 52621, Israel and the ^§ Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235

Received for publication, June 1, 2001, and in revised form, July 23, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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, 3517AT, predicting a I1173F substitution, was found in 22 homozygous Iranian Jewish DJS patients from 13 unrelated families and a second mutation, 3449GA, 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Dubin-Johnson syndrome (DJS)¹ is an autosomal recessive disorder manifested by chronic conjugated hyperbilirubinemia and accumulation of a dark pigment in liver parenchymal cells (1, 2). The disorder has recently been associated with several mutations in the multidrug resistance protein 2 (MRP2) gene (3-7). MRP2, also known as the canalicular multispecific organic anion transporter, is a 190-kDa integral membrane glycoprotein expressed mainly in the canalicular (apical) membrane of liver cells. It belongs to the superfamily of ATP-binding cassette transporters, and transports endogenous and exogenous anionic conjugates from hepatocytes to the bile (8-13). MRP2 is one of seven known MRPs that are involved in resistance of cancer cells to chemotherapeutic drugs (13-17). The MRP2 consists of 1545 amino acids, and its gene is located on chromosome 10q24 (13, 14, 16, 18). The genomic structure of the MRP2 gene exhibits a remarkable similarity to the MRP1 gene; it contains 32 exons and spans ~45 kilobase pairs (6, 7).

Since the original description of DJS, many cases have been described in different populations (19-22) and a cluster of 101 patients was ascertained in Israel (23). Sixty-three percent of the Israeli patients were of Iranian Jewish origin, and 9% were of Moroccan Jewish origin.

Expression of recombinant MRP2 in mammalian cell lines provides an important tool for functional characterization of this transporter. The activity of MRP2 has been evaluated by uptake of radiolabeled substrates into membrane vesicles prepared from MRP2-transfected cells (24-27), or by measurements of the accumulation of fluorescent compounds in intact cells (28-33). The fluorescent anion 5-carboxyfluorescein (CF; Ref. 34) has been used as a substrate for transport by MRP1 and MRP2 (32, 35, 36). The results obtained by these measurements have a relatively poor temporal resolution and require transfection with high efficiency or development of stable cell lines.

In this study we identified two novel mutations causing DJS in the Iranian and Moroccan Jewish patients, respectively, and obtained evidence for specific founder effects which account for the observed clusters. Both mutations were functionally analyzed by expressing the mutated MRP2 proteins in HEK-293 cells, testing their transport activity by a CF transport assay in single cells with high temporal resolution, and determining their cellular localization.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Patients-- Most patients with DJS were ascertained previously (23) and have been followed for more than 3 decades. The diagnosis was based on the finding of chronic or intermittent conjugated hyperbilirubinemia, and on either finding in liver biopsy material the typical pigment in hepatocytes or a predominance of coproporphyrin I urinary excretion (37). A total of 35 patients of 24 unrelated families were included in the study. Twenty-two were of Iranian Jewish origin (13 families), 5 were of Moroccan Jewish origin (4 families), 2 were offspring of a Moroccan Jewish mother and an Iranian Jewish father, 3 were of Ashkenazi Jewish origin (3 families) and 3 were of Turkish, Kurdish, and Afghani Jewish origins, respectively.

Control Subjects-- Control subjects were patients consecutively admitted to the Sheba Medical Center or healthy individuals who were Sheba Medical Center personnel. Definition of the ethnic origin of each subject was based on the country of birth of the individual's 4 grandparents. The human subject ethics committee of Sheba Medical Center approved the performance of the study.

Materials and Antibodies-- LipofectAMINE and Geneticin (G418) were obtained from Life Technologies, Inc. 5-(and-6)-Carboxyfluorescein diacetate was obtained from Molecular Probes, Inc. (Eugene, OR). Probenecid was from Sigma and cyclosporine A was from Sandoz Research Institute (Hanover, NJ). The monoclonal antibody M₂III-6 (38) was obtained from Kamiya Biomedical Co. (Seattle, WA). PNGase F was obtained from New England Biolabs (Beverly, MA).

Plasmids-- The human MRP2 expression vector, pcDNA3.1/MRP2, was a gift from Professor D. Keppler and has been described previously (27). Green fluorescent protein (GFP)-expressing plasmid was purchased from Life Technologies, Inc.

Amplification of Genomic DNA-- Genomic DNA was extracted from peripheral blood leukocytes by the desalting procedure (39). Polymerase chain reaction (PCR) was used to amplify each of the 32 exons of the MRP2 gene and their intronic-exonic boundaries. Primers were designed according to intronic sequences flanking the exons (6, 7) and are presented in Table I. PCR was performed in a 20-µl PCR buffer containing 1.5 mM MgCl₂, 100 to 200 ng of genomic DNA, 500 nM each primer, 200 µM each dNTP, and 0.125 unit of Taq polymerase (Super Nova; Laboratory Products, Kent, United Kingdom). The reactions were subjected to 35 cycles of 45 s of denaturation at 94 °C, 45 s of annealing at 55 °C, and 1 min of extension at 72 °C.

Identification of Sequence Alterations-- PCR fragments were sequenced either directly or after subcloning into pGEM-T vector using pGEM-T vector System (Promega, Madison, WI) and transformation into JM109-competent cells (Promega). Plasmid DNA was isolated using Wizard^TM Plus Minipreps DNA purification system (Promega). Sequencing was carried out in an automatic sequencer (ABI Prism 377, PerkinElmer Life Sciences).

Restriction Analysis of Mutations and Polymorphisms-- Following identification of mutations and intragenic polymorphisms by sequence alterations, methods for their easy detection were designed using amplified DNA segments and restriction analyses. Amplified DNA segments were digested by 5U of the respective enzymes (EcoRV, BsaHI, BbsI, BsaBI, Psp1406I, and NlaIII) and the products were separated on 3-4% agarose or metaphor gels (FMC-Bioproducts, Rockland, ME). Allele frequencies for identified mutations and polymorphisms were determined in patients with DJS and in control populations.

Haplotype Analysis and Assessment of Founder Effect-- Haplotypes of informative alleles from patients with DJS and controls were determined. Assessments of founder haplotypes were based on ² analysis.

Site-directed Mutagenesis-- Mutations were introduced into the pcDNA3.1/MRP2 vector using the QuikChange^TM site-directed mutagenesis kit (Stratagene, La Jolla, CA). Incorporation of the mutations was verified by DNA sequencing. Two sets of primers were used to introduce the I1173F mutation:1) the forward primer (5'-CAGGTTTGCCAGTTTTCCGTGCCTTTGAGC-3') and the reverse primer (5'-GCTCAAAGGCACGGAAAACTGGCAAACCTG-3'); 2) the forward primer (5'-CCGTATCAGGTTTGCCAGTTTTCCGTGCCTTTGAGC-3') and the reverse primer (5'-GCTCAAAGGCACGGAAAACTGGCAAACCTGATACGG-3'). This was done to ensure that low expression of this construct was not due to unexpected interference in any region of the vector except for the desired mutation. The primers used to introduce the R1150H mutation are the forward primer (5'-GCCAGCTGAGGCATCTGGACTCTGTCACCAG-3') and the reverse primer (5'-CTGGTGACAGAGTCCAGATGCCTCAGCTGGC-3'). All procedures were as suggested by the manufacturer. Positive clones were selected on ampicillin agar plates and all clones were verified by sequencing. At least three clones of each mutant were analyzed for activity, and all six clones of the I1173F mutant were analyzed for expression by Western blot.

Cell Culture and Transfections-- HEK-293 cells were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 10% fetal bovine serum, 100 µg/ml penicillin, and 100 µg/ml streptomycin. The expression vectors containing WT or the mutated MRP2 were transfected into HEK-293 cells using LipofectAMINE reagent according to instructions provided by the manufacturer. Cells were used 48-72 h after transfection. For stable expression, cells were selected with 1000 µg/ml G418.

Carboxyfluorescein Transport Assay-- HEK-293 cells were plated on a sterile 22 × 22-mm coverslips. On the following day, WT or mutant MRP2 plasmids and GFP-expressing plasmid were co-transfected into the cells. 48-72 h after transfection, coverslips with cells attached to them were washed once with a Hepes-buffered solution and assembled to form the bottom of a perfusion chamber. The Hepes-buffered solution contained (in mM) 140 NaCl, 5 KCL, 1 MgCl₂, 1 CaCl₂, 10 glucose, 10 Hepes (pH 7.4 with NaOH, osmolarity 310 with NaCl). GFP-expressing cells were identified by viewing GFP fluorescence and one cell was selected. GFP fluorescence intensity was recorded at an excitation wavelength of 490 nm and used to normalize MRP2 expression in different experiments. Subsequently, cells were loaded with carboxyfluorescein (CF) by a 10-min incubation at room temperature in Hepes-buffered solution containing 100 µM carboxyfluorescein diacetate and 1.5 mM probenecid. CF fluorescence was continuously monitored, and after it exceeded GFP fluorescence by at least 10-fold, the cells were perfused with Hepes-buffered solution containing 1.5 mM probenecid to establish a base line. Since inhibition by probenecid is completely reversible, transport of CF by MRP2 was initiated by removing probenecid from the perfusate. CF fluorescence was measured from single cells at excitation wavelengths of 490 and 440 nm using the recording setup from PTI (PTI Delta Ram, Brunswick, NJ). Since the fluorescence at the excitation wavelength of 440 nm is the isosbestic point for pH sensitivity of CF, fluorescence recorded at 440 nm was used to evaluate CF efflux. The fluorescence recorded at 490 nm was used to follow intracellular pH during the experiment to verify that the observed effect was not secondary to changes in pH_i.

Immunoblot Analysis of MRP2 Expression-- HEK-293 cells, transiently or stably transfected with WT or mutant MRP2, were disrupted in RIPA buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.2 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, 10 µM pepstatin, 10 µM aprotinin) and centrifuged at 15,000 rpm for 15 min. The clarified supernatant was recovered, and protein concentrations were determined with a Bio-Rad protein assay. Between 13 and 55 µg of protein was separated on a 7.5% SDS-polyacrylamide gel and electrophoretically transferred to nitrocellulose membrane (Bio-Rad). The membrane was blocked with 5% skim milk in 20 mM Tris/HCl, pH 7.4, 150 mM NaCl, 4 mM Trizma base, 0.1% Tween 20 and probed with a 1:100 dilution of M₂III-6 antibody in 5% skim milk in 20 mM Tris/HCl, pH 7.4, 150 mM NaCl, 4 mM Trizma base, 0.1% Tween 20. Horseradish peroxidase-conjugated goat anti-mouse IgG (Bio-Rad) at a 1:1000 dilution was used as a secondary antibody, and the signal was detected using ECL detection system (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom). For PNGase F digestion assay, samples (25 µl) were incubated with 1000 units of PNGase F for 2 h at 37 °C before separation and probing as above.

Immunofluorescence and Confocal Laser Scanning Microscopy-- Stable or transiently transfected HEK-293 cells cultured on 12-mm glass coverslips were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.05% Triton X-100 in PBS. After washing with PBS, cells were incubated for 1 h in blocking serum (1% bovine serum albumin, 0.1% gelatinin, 0.01% sodium azide, and 5% normal goat serum in PBS). The cells were then incubated for 2 h at room temperature with the M₂III-6 antibody diluted 1:20 in the blocking serum, washed with PBS, and reincubated for 1 h at room temperature with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Jackson Immunoresearch Laboratories, Inc., West Grove, PA; dilution 1:100 in the blocking serum). The cells were than mounted on glass slides and examined with a confocal laser-scanning microscope (MRC 1024, Bio-Rad).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Identification of Candidate Mutations in DJS Patients-- Screening of all 32 exons of the MRP2 gene in an Iranian Jewish patient disclosed a 3517AT transition in exon 25, predicting an I1173F substitution. The mutation was detectable by restriction analysis of nested PCR performed as follows. First, exon 25 was amplified by the forward and reverse primers depicted in Table I, and then nested PCR was carried out using a mutated forward primer (5'-ACCGTATCAGGTTTGCCAGAT-3') that created with the reverse primer an EcoRV restriction site in the normal sequence of exon 25. The amplified fragment was digested with EcoRV and its products analyzed by 4% metaphor gel electrophoresis (Fig. 1A). All 22 Iranian Jewish patients were homozygous for the 3517AT mutation, 2 affected siblings of mixed Iranian and Moroccan Jewish origin were heterozygous, and 11 non-Iranian Jewish patients did not bear this mutation. In the general Iranian Jewish population, heterozygosity for this mutation was observed in 14/243 subjects examined (5.8%), whereas none of 164 Moroccan Jews and 108 Ashkenazi Jews carried this mutation. The estimated allele frequency of the mutation in the general Iranian Jewish population was 2.9% (95% confidence interval of 1.6-4.8%) (Table II).

View larger version (44K): [in this window] [in a new window]   Fig. 1.   Detection of the 3517AT and the 3449GA mutations in DJS patients and segregation of the mutations in a mixed Iranian and Moroccan Jewish family. Panel A, the 3517AT mutation was detected using the restriction enzyme EcoRV and electrophoretic separation on 4% metaphor gel. The three possible genotypes are illustrated for an Iranian Jewish DJS patient (TT), an obligate carrier (TA), and a normal subject (AA). Panel B, the 3449G A mutation was detected using the restriction enzyme BsaHI and electrophoretic separation on 3% agarose gel. The three possible genotypes are illustrated for a Moroccan Jewish DJS patient (AA), an obligate carrier (GA), and a normal subject (GG). Panel C, 2 out of 4 offspring of the Moroccan Jewish mother and the Iranian Jewish father were affected by DJS, and both were compound heterozygotes for the 3517AT (black) and 3449GA (gray) mutations. The 2 phenotypically normal brothers and the mother were heterozygous for the 3449GA mutation, whereas the father was heterozygous for the 3517AT mutation.

View this table: [in this window] [in a new window]   Table II Allele frequencies of the 3517A T and the 3449GA mutations and of the 4 polymorphisms in the MRP2 gene in Iranian, Moroccan, and Ashkenazi Jews

A different mutation in the MRP2 gene, a 3449GA transition in exon 25, was identified in a Moroccan Jewish patient. The mutation predicts an R1150H substitution and results in loss of a BsaHI restriction site. For detection of this mutation, exon 25 was amplified by the primers depicted in Table I, then digested with BsaHI (New England Biolabs) and the products analyzed by 3% agarose gel electrophoresis (Fig. 1B). All 5 Moroccan Jewish patients were homozygous for the 3449GA mutation, whereas none of the 28 non-Moroccan patients carried it. The 2 affected siblings of the mixed Moroccan and Iranian Jewish origin were heterozygous for the mutation, and were thus compound heterozygotes for the 3449GA and the 3517AT mutations (Fig. 1C). The mother of these siblings of Moroccan Jewish origin was heterozygous for the 3449GA mutation, and the father, of Iranian Jewish origin, was heterozygous for the 3517AT mutation. In the general Moroccan Jewish population, heterozygosity for the 3449GA mutation was observed in 4/226 subjects examined (1.8%), whereas none of 156 Iranian Jews and 156 Ashkenazi Jews carried this mutation. The estimated allele frequency of the mutation in the general Moroccan Jewish population was 0.9% (95% confidence interval, 0.24-2.27%) (Table II).

Identification of Novel Polymorphisms in the MRP2 Gene-- Four dimorphisms in the MRP2 gene were identified: 1) 24CT in the 5'-untranslated region, 2) 842GA in exon 7 predicting a S281N substitution, 3) 1249GA in exon 10 predicting a V417I substitution, and 4) IVS29-35GA. 842GA and IVS29-35G  A are novel polymorphisms, whereas 24CT and 1249GA were recently published (40). Methods were devised for simple detection of these four polymorphisms by PCR and restriction analysis (Fig. 2). Table II shows the allele frequencies of the four dimorphisms in Iranian, Moroccan, and Ashkenazi Jews. A pronounced difference was observed for the rare 842GA dimorphism; in Moroccan Jews, its frequency was 6-fold higher than in Ashkenazi Jews and 9-fold higher than in Iranian Jews.

View larger version (77K): [in this window] [in a new window]   Fig. 2.   Allele-specific restriction analysis of the four dimorphisms in the MRP2 gene. The dimorphisms and the restriction enzymes by which they were detected are depicted. A, DNA fragments were amplified by using primers 1F and 1R (Table I), then digested with BbsI, which cuts the 24C sequence, and analyzed by 3% agarose gel electrophoresis. B, PCR was performed using a mutated forward primer of exon 7 (5'-CCTGGCTTGAACAAGGATCAG-3') that created a BsaBI restriction site in the 842A sequence, and 7R (Table I). PCR fragments were digested by BsaBI and analyzed on 4% agarose gel electrophoresis. C, PCR was performed using primers 10F and 10R followed by nested PCR using 10R (Table I) and a mutated forward primer (5'-TGGCCAGGAAGGAGTACAAC-3'), which created a Psp1406I restriction site in the 1249G sequence. The amplified fragments were digested with Psp1406I and analyzed by 4% metaphor gel electrophoresis. D, PCR fragments using primers 30F and 30R (Table I) were digested with NlaIII, which cuts the IVS29-35G sequence, and then analyzed by 3% agarose gel electrophoresis. The three possible genotypes for each polymorphism are illustrated.

Founder Effect for the Iranian Jewish 3517AT Mutation-- Haplotype analysis disclosed that all 26 alleles of 13 unrelated Iranian Jewish patients carried the same haplotype (24C, 842G, 1249A, IVS29-35A), whereas none of 144 informative alleles (out of 168 examined) of control Iranian Jews carried the same haplotype (Table III). The common haplotype among Iranian Jewish controls was 24C, 842G, 1249G, IVS29-35G; it accounted for 63% of the alleles. Statistical analysis yielded highly significant differences in the haplotype distribution between patients and controls (² = 271, p < 0.0001).

The 3517AT mutation and the four dimorphisms were examined in a large Iranian Jewish family. As can be seen in Fig. 3, all 6 DJS patients examined were homozygous for the 3517AT mutation and the founder haplotype: 24C, 842G, 1249A, IVS29-35A. All 7 obligate carriers examined were heterozygous for the mutation and the founder haplotype. One normal subject, a descendent of an obligatory carrier, bore neither the mutation nor the founder haplotype.

View larger version (35K): [in this window] [in a new window]   Fig. 3.   Distribution of the 3517AT mutation and the four dimorphisms in an Iranian Jewish family. Patients affected by DJS are depicted by filled symbols and obligatory carriers by half-filled symbols. The mutated allele 3517T and the founder haplotype (24C, 842G, 1249A, IVS29-35A) are depicted in bold letters.

Founder Effect for the Moroccan Jewish 3449GA Mutation-- Haplotype analysis disclosed that the 8 alleles of 4 unrelated Moroccan Jewish patients had the same haplotype (24C, 842A, 1249G, IVS29-35G), whereas only 2 out of 118 (1.7%) informative alleles (out of 138 examined) of control Moroccan Jews carried this haplotype (Table III). Similarly to Iranian Jewish controls, the common haplotype among Moroccan Jewish controls was also 24C, 842G, 1249G, IVS29-35G, accounting for 61% of the alleles. Statistical analysis yielded highly significant differences in the haplotype distributions between patients and controls (² = 99, p < 0.0001).

Carboxylfluorescein Transport Assay of MRP2 Activity-- A new procedure was developed to estimate MRP2 activity with high temporal resolution. The procedure is based on measurements of probenecid-sensitive CF efflux. Probenecid is an organic anion transport inhibitor (41) that has been shown to increase CF accumulation in primary human hepatocytes (36), probably by inhibition of MRP-dependent transport. Fig. 4 illustrates the protocols used to establish the technique as a transport assay of MRP2 activity in single cells and reveal several properties of CF transport by MRP2. Cells were perfused with a solution containing 1.5 mM probenecid for 150-200 s to establish a base line. A similar slow reduction in CF fluorescent was seen in control HEK-293 cells and HEK-293 cells expressing WT-MRP2 (Fig. 4, A-E). Upon removal of probenecid, CF efflux from control cells remained slow (Fig. 4A). By contrast, an almost immediate high rate of CF efflux was seen in cells expressing WT-MRP2 (Fig. 4B). MRP2-mediated CF efflux followed a single exponential with a rate constant of ~600 s¹.

View larger version (29K): [in this window] [in a new window]   Fig. 4.   Properties of CF efflux by MRP2. HEK-293 cells transfected with GFP only (control, A) or GFP and MRP2 (B-E) were loaded with CF by incubation in a solution containing 100 µM 5-(and-6)-carboxyfluorescein diacetate and 1.5 mM probenecid. All experiments were initiated by perfusing the cells with a standard bath solution containing 1.5 mM probenecid to establish the rate of nonspecific CF leak. Efflux started by removal of probenecid from the perfusate (A and B). To demonstrate reversibility of inhibition by probenecid, the inhibitor was removed and added back to the perfusate (C). The dose dependence of inhibition by probenecid was evaluated from a stepwise reduction in perfusate probenecid concentration (D). Panel E shows the irreversible inhibition of efflux by cyclosporine A.

Fig. 4C shows that the effect of probenecid was completely reversible. Removal and addition of probenecid resulted in an immediate onset and inhibition of CF transport. Fig. 4D illustrates the dose-dependent effect of probenecid. WT-MRP2-transfected cells were perfused with reducing probenecid concentrations (in mM) (1.5, 0.5, 0.25, 0.15, and 0). CF transport rate indicated by the slope in each probenecid concentration, decreased in a concentration-dependent manner between 1.5 and 0.15 mM probenecid, with 50% inhibition at ~0.2 mM. Another well established inhibitor of MRP2 is cyclosporine A. Fig. 4E shows that 10 µM cyclosporine A inhibited MRP2-dependent CF efflux in an irreversible manner.

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. Experiments were performed with each mutant, and results were always compared with efflux by WT-MRP2 and control, GFP-transfected cells. A typical CF efflux from control, mutant, and WT-MRP2 proteins is illustrated in Fig. 5. 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. 4A and 5A), while cells transfected with WT-MRP2 displayed a robust efflux (Figs. 4B and 5B). The rate constant of CF efflux measured in multiple experiments are summarized in Fig. 5E. The average rate constant of CF efflux (in s¹) by WT-MRP2 (562 ± 56, n = 4) was significantly higher than that measured in control cells (151 ± 45, n = 5), R1150H-MRP2 cells (90 ± 40, n = 5) and I1173F-MRP2 cells (104 ± 66, n = 3). No significant difference was found between the average rate constant of control, R1150H-MRP2, and I1173F-MRP2 cells. These data indicate that both I1173F and R1150H mutations impair the transport activity of MRP2.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   CF transport by MRP2 mutants. Cells were transfected with GFP only (control, A), WT-MRP2 (B), I1173F-MRP2 (C), or R1150H-MRP2 (D). CF efflux was measured as described in Fig. 4. The average rate constant from multiple experiments (n) is summarized in E.

Expression of Mutant MRP2 Proteins-- Reduction in CF transport by the mutants can be due to inhibition of MRP2 activity or misprocessing of the protein. To determine the cause of the reduced activity, we analyzed protein expression and localization. For immunoblot analysis we used the monoclonal antibody M₂III-6, which recognizes the carboxyl terminus of rat and human MRP2. As shown in Fig. 6A, WT-MRP2 was detected mainly as a mature glycoprotein of ~190 kDa. A minor band of ~175 kDa was also observed in all experiments in which at least 20 µg of protein was used. This band probably represents the core, unglycosylated protein since treatment of WT-MRP2 with PNGase F resulted in a lower intensity of the 190-kDa band and an increased intensity in the 175-kDa band (Fig. 6B). In experiments shown in Fig. 6A, different amounts of protein from WT and mutant MRP2 samples were loaded in each lane. 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 µg of protein was used, and no 175-kDa band when 13 µg of protein was used. By contrast, the amount of protein and the ratio between the two bands was different for I1173F-MRP2. At 20 µg of I1173F-MRP2 protein, staining was much weaker than that observed with either WT or R1150H-MRP2. When 55 µg of protein from cells expressing I1173F-MRP2 was used, the overall amount of protein was comparable to that measured with 13 µg of WT or R1150H-MRP2. Notably, the 175-kDa band was more intense than the 190-kDa band for I1173F-MRP2.

View larger version (54K): [in this window] [in a new window]   Fig. 6.   Immunoblot analysis of WT-MRP2 and mutants. Panel A, cells transfected with vector (Mock), WT-MRP2, or the indicated mutants were used to prepare lysates and the proteins were detected by immunoblotting. Note that different amounts of protein were used in different lanes. Panel B, cells transfected with WT-MRP2. Samples (25 µl) were incubated with 1000 units of PNGase F for 2 h at 37 °C. PNGase F-treated (+) and untreated () samples were separated and probed as above. The 190- and 175-kDa bands are marked.

Cellular Localization of WT and Mutants MRP2-- The significant amount of the 175-kDa band found in cells expressing I1173F-MRP2 suggests that processing of MRP2 was affected by this mutation. Consequently, it was of interest to determine the intracellular localization of WT and MRP2 mutants. The results are shown in Fig. 7. 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. This suggested that processing and targeting of the R1150H-MRP2 mutant was normal. In contrast, I1173F-MRP2 showed largely a reticular pattern of staining (Fig. 7C). Examination of several fields in five separate transfection experiments consistently showed a sparse intracellular distribution of this mutant, in agreement with the results obtained in the Western blot.

View larger version (62K): [in this window] [in a new window]   Fig. 7.   Immunolocalization of WT-MRP2 and mutants. 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.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In this study we identified two novel mutations in the MRP2 gene that were associated with DJS. Both mutations are located in exon 25 of the MRP2 gene, with 3517AT predicting I1173F substitution found in a cluster of Iranian Jewish DJS patients, and 3449GA predicting R1150H substitution found in a cluster of Moroccan Jewish patients. None of 544 non-Iranian Jewish alleles carried the 3517AT mutation, and none of 624 non-Moroccan Jewish alleles bore the 3499GA mutation (Table II), indicating that these mutations are unique for these specific populations. Both mutations strongly correlate with DJS as indicated by the following evidence. 1) Twenty-two Iranian Jewish patients were homozygous for the 3517AT mutation and 7 obligatory carriers from one kindred were heterozygous for the mutation (Fig. 3), and all 5 patients of Moroccan Jewish origin were homozygous for the 3449GA mutation; 2) 2 patients of mixed Iranian and Moroccan Jewish origin were compound heterozygotes for both mutations (Fig. 1); 3) sequencing of all 32 exons and exon-intron junctions of the MRP2 gene in an Iranian Jewish patient and a Moroccan Jewish patient did not disclose any alternations except for the respective mutations and polymorphisms.

During the screening of the MRP2 gene for mutations, we identified four polymorphisms. Two were in non-coding regions of the gene, 24CT and IVS29-35GA, and two were in coding regions of the gene, 842GA and 1249GA predicting S281N and V417I substitutions, respectively. The 4 polymorphisms were identified in all three control populations examined (Table II). The 24CT and 1249GA polymorphisms were relatively frequent in Iranian, Moroccan, and Ashkenazi Jews, while the novel 842GA and IVS-35GA polymorphisms were less frequent. Interestingly, the 842GA polymorphism was significantly more frequent in Moroccan Jews than in Iranian or Ashkenazi Jews.

The 4 polymorphisms were used for haplotype analysis in patients and controls. One distinct haplotype (24C, 842G, 1249A, IVS29-35A) was discerned in all 26 alleles bearing the 3517AT mutation, but was absent in 144 alleles of the control Iranian Jewish population (Table III). This finding was consistent with a founder effect for the 3517AT mutation in Iranian Jewish patients with DJS. Another haplotype (24C, 842A, 1249G, IVS29-35G) was associated with the 3449GA mutation in all 8 alleles of 4 unrelated Moroccan Jewish patients and was found only in 2 of 118 (1.7%) normal alleles in the control Moroccan Jewish population. These results are consistent with a founder effect for the 3449GA mutation in Moroccan Jewish patients afflicted by DJS.

In a previous study, we estimated that 1:1300 Iranian Jews residing in Israel are affected by DJS, which implied a 5.4% prevalence of heterozygotes in this population (23). In the present study, screening of 486 control Iranian Jewish alleles for the 3517AT mutation yielded an almost identical result. Thus, the frequency of the mutant allele was 2.9%, which is consistent with a 5.8% prevalence of heterozygotes (Table II). In 452 Moroccan Jewish control alleles, the prevalence of the 3449GA mutation was only 0.9%, which predicted a 1.8% prevalence of heterozygotes in this population (Table II). The significantly lower prevalence of the 3449GA mutation in Moroccan Jews than the prevalence of the 3517AT mutation in Iranian Jews explains the substantially lower number of patients with DJS among Moroccan Jews (23).

Seven mutations in the MRP2 gene have been described previously in DJS patients of Japanese and Caucasian origins (3-7). These mutations include two missense mutations (R768Y, Q1382R), a nonsense mutation (R1066stop), three splice site mutations (1815+2, TA; 1967+2, TC; 2439+2, TC) and a deletion mutation (4175del6). The predicted structure of rat MRP2 (41) places the two missense mutations described in this study in an extracellular loop of the third membrane-spanning domain, between transmembrane helices 12 and 13. However, a prediction of human MRP2 membrane topology places the mutations in an intracellular loop of the third membrane-spanning domain between transmembrane helices 15 and 16 (33). We used several algorithms (TMpred, DAS, TMHMM) for prediction of the localization. Although the exact localization varied somewhat among the algorithms, all indicated that both mutations were localized within an intracellular loop of the third membrane-spanning domain. This site is different from the localizations of previously described mutations that were predicted to be in the ATP binding fold or adjacent regions. Interestingly, I1173 and R1150 are conserved in MRP2 analogues in plants, nematodes, rodents, and canine (42-45), and in human MRP1, MRP3, MRP4, and MRP5 (14). Hence, it was of particular interest to determine the effect of the newly identified mutations on the expression and activity of MRP2. To evaluate MRP2 activity, we established a new method for measurement of MRP2-dependent CF efflux. The method was validated by demonstration of a dose-dependent and reversible inhibition of the efflux by probenecid and irreversible inhibition by cyclosporine A. With this assay we showed that both I1173F and R1150H mutations impaired the MRP2 activity (Fig. 5). However, this was due to a different effect by each of the mutations (see below).

Immunoblot analysis revealed that MRP2 exists in at least two forms: a mature glycosylated form of ~190 kDa apparent molecular mass and an immature unglycosylated form of ~175 kDa apparent molecular mass. Similar observations were made for other ATP-binding cassette transporters such as MRP1 (46), P-glycoprotein (47), and cystic fibrosis transmembrane conductance regulator (48). The R1150H mutation had no apparent effect on the relative amount of each MRP2 forms. In contrast, the I1173F mutation gave rise to a markedly reduced relative amount of the 190-kDa band and an increased relative amount of the 175-kDa form. Furthermore, the total amount of I1173F-MRP2 that was expressed was substantially decreased (Fig. 6), which suggests enhanced intracellular degradation, due to mistargeting and misfolding of the mutant protein. 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.

Expression studies have provided a useful tool for studying the underlying mechanism causing DJS as was shown for the 4175del6 mutation (49). 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. Interestingly, two site-directed mutations, which replaced arginine by alanine in the predicted third transmembrane region of MRP2 (R1210A and R1257A), were shown to cause normal expression but impaired transport activity of MRP2 (33). This may imply that this region is important for MRP2 transport activity and that arginine residues in this region play an important role in its function.

In summary, our results provide strong evidence that two ancestral mutations cause DJS in two clusters of Israeli patients and provide a probable mechanism for their effect. The new transport assay that we devised can be useful in studying additional mutations, in particular those with normal expression. Moreover, the assay may be used with other fluorescent substrates in order to better characterize transport activity of MRP2.

	ACKNOWLEDGEMENT

We thank Dr. Dietrich Keppler (Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, Heidelberg, Germany) for kindly providing us the pcDNA3.1/MRP2 plasmid and for support and encouragement. We are indebted to Dr. Meir Muallem (Department of Medicine, Sheba Medical Center) for allowing us to examine one of his patients with DJS.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence may be addressed: Dept. of Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9040. Tel.: 214-648-2593; Fax: 214-648-8879; E-mail: shmuel.muallem@utsouthwestern.edu.

To whom correspondence may be addressed: Inst. of Thrombosis and Hemostasis, Dept. of Hematology, Sheba Medical Center, Tel Hashomer 52621, Israel. Tel.: 972-3-5302104; Fax: 972-3-5351568; E-mail: zeligson@post.tau.ac.il.

Published, JBC Papers in Press, July 26, 2001, DOI 10.1074/jbc.M105047200

	ABBREVIATIONS

The abbreviations used are: DJS, Dubin-Johnson syndrome; MRP, multidrug resistance protein; CF, 5-carboxyfluorescein; PCR, polymerase chain reaction; PNGase F, peptide N-glycosidase F; GFP, green fluorescent protein; WT, wild type; PBS, phosphate-buffered saline.

	REFERENCES

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