Mutation and functional analysis of ABCC2/multidrug resistance protein 2 in a Japanese patient with Dubin–Johnson syndrome

Dubin–Johnson syndrome (DJS) is a recessive inherited disorder characterized by conjugated hyperbilirubinemia. It is caused by dysfunction of adenosine triphosphate‐binding cassette, sub‐family C, member 2 (ABCC2/MRP2) on the canalicular membrane of hepatocytes. We performed mutational analysis of the ABCC2/MRP2 gene in a Japanese female with DJS. Furthermore, we investigated the effects of the two identified DJS‐associated mutations on MRP2 function. We found a compound heterozygous mutation in the patient: W709R (c.2124T>C), a missense mutation in exon 17, and R1310X (c.3928C>T), a nonsense mutation in exon 28. DJS‐associated mutations have been shown to impair the protein maturation and transport activity of ABCC2/MRP2. We established HEK293 cell lines stably expressing one of the two identified DJS‐associated mutations. Expressed W709R MRP2 was mainly core‐glycosylated, predominantly retained in the endoplasmic reticulum, and exhibited no transport activity, suggesting that this mutation causes deficient maturation and impaired protein sorting. No MRP2 protein was expressed from HEK293 cells transfected with an R1310X‐containing construct. This compound heterozygous mutation of the MRP2 gene causes dysfunction of the MRP2 protein and the hyperbilirubinemia seen in DJS.     

Dubin-Johnson综合征临床及病理特征

目的探讨Dubin-Johnson综合征(DJS)的临床及病理特点。方法分析解放军第三○二医院2006年1月至2016年4月收治的21例DJS患者的临床资料,回顾性分析患者的临床及病理资料。结果男17例,女4例,平均年龄(28.7±8.1)岁,平均TBil(59.2±11.1)μmol/L;平均DBil(40.9±9.3)μmol/L,肝穿病理组织肉眼可见呈黑色、灰褐色、黄绿色、灰黑色等黑肝表现。光镜下主要病理改变:肝细胞内大量较粗大的深棕色颗粒沉积,以中央静脉周围为著,少数肝细胞水样变性,窦周炎不明显,汇管区无或轻度扩大,少量炎细胞浸润,未见明确界面炎。结论 DJS多发于男性,以青少年期发病为主,升高以DBil升高为主,肝穿病理检查为确诊DJS主要手段,黑肝表现及镜下深棕色颗粒沉积为其特异性病理特征。     

Mutational analysis of the MRP2 gene and long-term follow-up of Dubin-Johnson syndrome in Japan

Background Recent studies have indicated that dysfunction or loss of the multidrug resistance protein 2 (MRP2) is the molecular basis of Dubin-Johnson syndrome (DJS). To clarify the genetic basis of the disease and the long-term stability of serum bilirubin levels, we conducted a mutational analysis of the MRP2 gene and followed up serum bilirubin levels in Japanese DJS patients 30 years after they were originally diagnosed, based on traditional criteria.Methods Patients were interviewed by telephone, and blood tests, including a genetic analysis of MRP2, were performed on the patients and family members who gave informed consent.Results Over the 30 years, hyperbilirubinemia remained unchanged in four of the five patients studied, while it worsened in 1 patient whose DJS was complicated by chronic hepatitis C. From an MRP2 gene mutational analysis, six mutations, including the novel mutation 1177C>T, were found. Three patients of a consanguineous family were homozygotes for three mutations (298C>T, 1967+2T>C, and 2439+2T>C). Two patients were compound heterozygotes (1177C>T/2302C>T and 1967+2T>C/2026G>C). A familial study showed no difference in serum bilirubin levels between mutant/wild heterozygotes and wild/wild homozygotes.Conclusions The hyperbilirubinemia of four Japanese patients with DJS, one of whom had a novel mutation, 1177C>T, of the MRP2 gene, had not worsened with aging.     

In silico screening and analysis of single-nucleotide polymorphic variants of the ABCC2 gene affecting Dubin–Johnson syndrome

Background and Study AimsDubin–Johnson syndrome (DJS) is a benevolent genetic disorder of the liver with autosomal inheritance. It is a rare disorder characterized by an increase in conjugated bilirubin and anomaly in coproporphyrin clearance. DJS is caused by deleterious mutations in the ABCC2 gene. A polymorphism in the ABCC2 gene causes malfunctions in its ability to regulate the efflux of different organic anions, such as bilirubin, from hepatocytes to the canaliculi. Multidrug resistance protein 2 (MRP2) encoded by the ABCC2 gene is one of the main regulators of the export of bilirubin to respective sites. ABCC2 gene mutations have widely drawn attention in the pathology of DJS in various populations.Patients and MethodsThe ABCC2 gene was subjected to the National Center for Biotechnology Information (NCBI) database in 2020, and non-synonymous single-nucleotide polymorphisms (nsSNPs) and variants in untranslated regions were studied using different computational servers. SIFT, Protein variation effect analyzer, and PolyPhen-2 were used to retrieve the damaging Single-nucleotide polymorphisms (SNPs); PhD-SNP, SNPs&GO, and Protein Analysis Through Evolutionary Relationships were used to predict the association of nsSNPs with DJS; Mutation3D illustrated the location of variants in the protein; SNAP2, MutPred2, ELASPIC, and HOPE were used to predict the structural and functional effects of these mutations on MRP2; and I-mutant 3.0 and MuPro were used to determine the effects of polymorphism on the function of MRP2.ResultsIn this study, 18,947 SNPs were screened from the NCBI database, followed by a series of refinement of variants using online available servers. We concluded that 41 ABCC2 gene variants are vital etiological candidates for DJS in humans. These 41 variants had highly damaging effects on the MRP2 protein, which may lead to deficient transportation capacity, thereby affecting the efflux of bilirubin across the canalicular membrane.ConclusionIn silico tools are an alternative approach for predicting the target SNPs. Hence, previously unreported variants can be considered strong etiological candidates for diseases related to MRP2.     

Ultrastructure of Kupffer cells and hepatocytes in the Dubin-Johnson syndrome： A case report

Ultrastructure of Kupffer cells and hepatocytes in liverbioptate was evaluated in a 17-year-old boy with Dubin-Johnson syndrome(DJS).The liver tissue obtainedby needle biopsy was fixed in glutaraldehyde andparaformaldehyde and routinely processed for electronmicroscopic analysis.The ultrastructural examinations ofliver bioptate revealed the accumulation of membrane-bound,electron-dense lysosomal granules within thecytoplasm of hepatocytes,characteristic of DJS.Theywere located mainly in the vicinity of the biliary pole,andpreferentially in the centrilobular region that correspondedto the pigment deposits seen under light microscope.Thepresence of the granules was accompanied by dilatedelements of the granular endoplasmic reticulum andparacrystalline mitochondrial inclusions as well as dilationof the bile canaliculi.The changes in hepatocytes co-existed with marked stimulation and enhanced phagocyticactivity of Kupffer cells.This was manifested in theaccumulation of pigment deposits within their cytoplasmthat corresponded to those observed in hepatocytes.Hyperactive pericentral Kupffer cells which are involvedin the response to pigmentary material originating fromdisintegrated hepatocytes may play an essential role inthe development of DJS.     

A novel ancestral splicing mutation in the multidrug resistance protein 2 gene causes Dubin-Johnson syndrome in Ashkenazi Jewish patients.

Dubin-Johnson syndrome (DJS) is an inherited disorder characterized by chronic conjugated hyperbilirubinemia due to the absence or dysfunction of the multidrug resistance protein 2 (MRP2). We previously identified two distinct ancestral mutations causing DJS in 22 unrelated Iranian and five unrelated Moroccan Jewish patients, respectively. In this study we identified and characterized the mutation causing DJS in Ashkenazi Jewish patients and assessed a possible founder effect. Sequencing of all 32 exons of the MRP2 gene identified a novel IVS8+4A-->G mutation in three unrelated homozygotes. Haplotype analysis using four intragenic dimorphisms disclosed a founder effect for the mutation. RT-PCR and real time PCR analysis of mRNA from one patient revealed three splice variants all leading to frameshifts and predicting premature termination codons. The main splice variant was a consequence of the use of a cryptic donor splice site inside exon 8. Liver biopsy in one patient revealed complete absence of MRP2 from the canalicular membrane of hepatocytes. In conclusion, our results provide strong evidence that an ancestral IVS8+4A-->G mutation causes DJS in Ashkenazi Jewish patients by abolishing normal splicing of intron 8 leading to aberrantly spliced products that predict truncation of MRP2.     

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

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.     

Trafficking and functional defects by mutations of the ATP-binding domains in MRP2 in patients with Dubin-Johnson syndrome

Dubin-Johnson syndrome (DJS) is a hereditary disease characterized by hyperbilirubinemia. We investigated the consequences of 2 missense mutations, R768W and Q1382R, of nucleotide-binding domains (NBDs) of the multidrug resistance protein 2 (MRP2; ABCC2) that were previously identified in patients with DJS. Pulse chase analysis revealed that the precursor form of the wild-type and Q1382R MRP2 were converted to the mature form, which is resistant to endoglycosidase H (Endo H) in about 60 minutes. However, the precursor form of the R768W MRP2, which is sensitive to endoglycosidase H, was degraded within 120 minutes and did not mature to the fully glycosylated form. Proteasome inhibitors inhibited the degradation of the precursor form of the R768W MRP2. Unlike the R768W MRP2, the Q1382R MRP2 was mainly localized on the apical membrane in the wild-type form. However, efflux of glutathione monochlorobimane (GS-MCLB) and ATP-dependent leukotriene C(4) (LTC(4)) uptake into plasma membrane vesicles from cells expressing the Q1382R MRP2 were markedly reduced, suggesting that the Q1382R MRP2 on the apical membrane was nonfunctional. Vanadate-induced nucleotide trapping with 8-azido-[alpha-32P]ATP in the wild-type MRP2 was stimulated by estradiol glucuronide (E(2)17betaG) in a concentration-dependent manner but that in the Q1382R MRP2 was not. In conclusion, the R768W mutation causes deficient maturation and impaired sorting, and the Q1382R mutation does not affect maturation or sorting but impairs the substrate-induced ATP hydrolysis.     

Expression of hepatic transporters OATP‐C and MRP2 in primary sclerosing cholangitis

Abstract: Background/Aims: In chronic cholestatic liver diseases, biliary excretion of organic anions from blood into bile is impaired. The aim of this study was to identify the underlying mechanism. Methods: Expression of the basolateral organic anion transporting polypeptide OATP‐C (SLC21A6) and the canalicular multidrug resistance protein 2 (MRP2) was studied in patients with primary sclerosing cholangitis (PSC) (n=4), a chronic cholestatic liver disease, and in non‐cholestatic controls (n=4) (two with chronic hepatitis C, one with idiopathic liver cirrhosis and one with fatty liver). Total RNA was isolated from liver tissue, reverse transcribed and subjected to polymerase chain reaction (PCR) amplification using primers specific for OATP‐C, MRP2 and β‐actin. PCR products were quantified densitometrically. Results: When normalized for β‐actin expression, the level of OATP‐C mRNA in liver tissue of patients with PSC was 49% of controls (OATP‐C/β‐actin 1.60±0.25 vs. 3.24±0.69; p<0.05) and the level of MRP2 mRNA was 27% of controls (MRP2/β‐actin 0.70±0.36 vs. 2.54±0.56; p<0.01). Conclusions: Both OATP‐C and MRP2 are decreased as measured by mRNA level in PSC. Downregulation of OATP‐C might be the consequence of impaired canalicular secretion of organic anions and could serve to reduce the organic anion load of cholestatic hepatocytes.     

Characterization of the human multidrug resistance protein isoform MRP3 localized to the basolateral hepatocyte membrane

Several members of the multidrug resistance protein (MRP) family are expressed in the liver. Adenosine triphosphate (ATP)-dependent transport of glutathione and glucuronoside conjugates across the hepatocyte canalicular membrane is mediated by the apical MRP isoform, MRP2 (APMRP), also known as canalicular multispecific organic anion transporter (cMOAT). We have cloned an additional MRP isoform, MRP3, from human liver and localized it to the basolateral membrane domain of hepatocytes. Basolateral MRP (BLMRP) is composed of 1,527 amino acids and encoded by 4,581 base pairs of complementary DNA. Northern blotting of various human tissues indicated an expression of MRP3 in the liver, colon, pancreas, and, at a lower level, in the kidney. The amino acid identity of MRP3 with MRP1 and MRP2 is 58% and 48%, respectively. These three isoforms, encoded by genes on different chromosomes, have a similar predicted topology of transmembrane segments and ATP-binding domains. Antibodies raised against two peptide sequences of MRP3 that are not shared by other MRP family members detected recombinant MRP3 expressed in polarized MDCK cells. Both antibodies served to localize MRP3 to the basolateral membrane of hepatocytes. Double-label immunofluorescence microscopy confirmed that MRP3 was not detectable in the canalicular membrane domain. A particularly strong expression of the MRP3 protein was observed in the basolateral hepatocyte membrane of two patients with Dubin-Johnson syndrome who are deficient in MRP2. These results indicate that the basolateral MRP isoform, MRP3, may be upregulated when the canalicular secretion of anionic conjugates by MRP2 is impaired.     

Dubin-Johnson综合征合并慢性乙型肝炎患者脂褐素小体的超微结构特点

目的 研究Dubin-Johnson综合征合并慢性乙型肝炎患者肝细胞脂褐素小体的超微结构特点.方法用透射电子显微镜(简称电镜)对11例Dubin-Johnson综合征合并慢性乙型肝炎(轻度)患者、5例Dubin-Johnson综合征患者的肝细胞超微结构进行观察,重点观察脂褐素小体的形态及密度.对数据的统计学分析采用Fisher's精确概率法.结果 11例Dubin-Johnson综合征合并慢性乙型肝炎(轻度)患者的肝细胞脂褐素小体形态呈现多形性,电镜下至少有5种形态:"明亮型"(18.2%,2/11)、"网眼型"(9.1%,1/11)、"点状型"(54.5%,6/11)、"不规则型"(9.1%,1/11)和"基本型"(9.1%,1/11);而未合并慢性乙型肝炎的Dubin-Johnson综合征患者的肝细胞脂褐素小体形态相对较为单一,为"基本型"(100.0%,5/5)."基本型"的差异在两组间比较,P=0.0013.两组患者脂褐素小体的密度也不均等,合并慢性乙型肝炎(轻度)患者的肝细胞脂褐素小体密度低,未合并慢性乙型肝炎(轻度)的Dubin-Johnson综合征患者肝细胞脂褐素小体密度高.结论 Dubin-Johnson综合征合并慢性乙型肝炎(轻度)患者肝细胞脂褐素小体形态呈现多形性,含高电子密度物质的比例少;而Dubin-Johnson综合征患者的肝细胞脂褐素小体形态相对较为单一,以色素沉着较重的"基本型"为主,含高电子密度物质的比例多.二者的差别在诊断上可能有一定价值.

Abstract：

Objective To explore characteristics of the myelin-like bodies in the hepatocytes of patients with Dubin-Johnson syndrome (DJS) complicated with chronic hepatitis B (CHB). Methods 11 cases of DJS complicated with CHB and 5 cases DJS without CHB were studied clinicopathologically. The hepatocyte ultrastructure was observed with transmission electron microscope and taken photos. The data were compared and analyzed using Fisher's Exact Test. Results Deposition of myelin-like bodies can be observed in the hepatocytes of DJS patients with CHB but can not in DJS patients without CHB. The morphology of pigment varys. The electron density and volume of pigment in DJS patients with CHB can be classified into five types: brights (2/11,18.2%), reticulation (1/11, 9.1%), punctiform (6/11, 54.5%), abnormiry (1 / 11, 9.1%) and primary type (1 / 11, 9.1%). The myelin-like bodies in the hepatocytes of patients with DJS are high density and round with membrance (we named it as primary type) (5/5, 100%). Conclusions The myelin-like bodies in the hepatocytes of DJS patients with CHB possess special pleomorphism and may have important diagnostic value.     

Dubin-Johnson syndrome with systemic lupus erythematosus: a case report

Introduction Dubin-Johnson syndrome (DJS) is a rare recessively inherited conjugated hyperbilirubi- nemia caused by deficiency of the canalicular multi-drug resistance/multi-specific organic anionic transporter protein (MDR2/cMOAT). Thus bilirubin is conjugated but inefficiently secreted into bile, which results in accumulation of conjugated and, to some extent, unconjugated bilirubin in blood, leading to hyperbilirubinemia and bilirubinuria. But, the results of liver function tests are no…     

Dual hereditary jaundice: simultaneous occurrence of mutations causing Gilbert's and Dubin-Johnson syndrome

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.     

Dubin-Johnson综合征的临床特点分析

Dubin-Johnson综合征（DJS）是一种少见的常染色体隐性遗传病,是由于毛细胆管上特异性有机阴离子转运蛋白基因缺陷,导致结合胆红素排泄障碍,血液中结合胆红素升高,黑色色素在肝细胞内沉着,大体表现为黑肝。该疾病预后良好,不需特殊治疗,但容易被误诊导致患者反复就诊治疗,承担不必要的精神和经济负担。近年来有报道本病可合并多种疾病,诊断较困难,从而对持续性黄疸难以解释,并且患者可在中老年时期就诊,而不是在青少年好发时期,容易遗漏该疾病导致误诊。本文对DJS的发病机制、临床特点及近年来的诊断方法等作一综述。     

Age estimates of ancestral mutations causing factor VII deficiency and Dubin-Johnson syndrome in Iranian and Moroccan Jews are consistent with ancient Jewish migrations.

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.     

Role of transferrin receptor 2 in hepatic accumulation of iron in patients with chronic hepatitis C

Background and Aim: Iron deposition in the liver is a common finding in patients with chronic hepatitis C (CH‐C). The mechanism of this hepatic accumulation of iron is not completely understood. This study assessed if the protein expression of transferrin receptor 2 (TfR2) is upregulated in the liver of patients with CH‐C and if TfR2 protein mediates iron accumulation during hepatitis C virus (HCV) infection. Method: Liver specimens from patients with CH‐C that underwent interferon (IFN) therapy (n = 23) and from patients with CH‐B (n = 18) were evaluated. Hepatic expression of TfR2 protein was analyzed by immunohistochemistry. Total hepatic iron score (THIS) was evaluated by Prussian blue staining. Results: TfR2 protein was expressed in the cell membrane and cytosol of hepatocytes. Cytosol TfR2 protein was found to co‐localize with Tf. THIS (P = 0.0198) and hepatic TfR2 (P = 0.0047) expression were significantly higher in CH‐C than in CH‐B. The change in THIS values (rho = 0.580, P = 0.0079) and the grade of histological activity (rho = 0.444, P = 0.0373) were significantly correlated with changes in TfR2 expression after IFN therapy. Conclusions: The protein expression of TfR2 is significantly associated with iron deposition in the liver in patients with CH‐C. HCV infection may affect the hepatic expression of TfR2, leading to iron accumulation in the liver.     

多耐药相关蛋白MRP2与核受体RXRα、RARα在人阻塞性胆汁淤积肝组织中的表达变化

目的观察多耐药相关蛋白MRP2（multidrug resistance associated protein2）与核受体RXRα、RARα在人阻塞性胆汁淤积肝组织的表达变化。方法收集人正常及阻塞性胆汁淤积肝组织标本各20例,HE染色验证阻塞性黄疸病变,免疫组化方法检测人多耐药相关蛋白MRP2及核受体RXRα、RARα的表达变化。结果 人阻塞性胆汁淤积肝组织中核受体RXRα、RARα表达减弱,多耐药相关蛋白MRP2表达也减弱。结论与体外培养肝细胞与模型动物一样,人阻塞性胆汁淤积肝组织中MRP2表达减弱也可能由核受体RXRα、RARα表达减弱导致。     

Impaired protein maturation of the conjugate export pump multidrug resistance protein 2 as a consequence of a deletion mutation in Dubin-Johnson syndrome

The Dubin-Johnson syndrome is an inherited disorder characterized by conjugated hyperbilirubinemia. The deficient hepatobiliary transport of anionic conjugates is caused by the absence of a functional multidrug-resistance protein 2 (MRP2, symbol ABCC2) from the apical (canalicular) membrane of hepatocytes. Mechanisms underlying this deficiency may include rapid degradation of mutated MRP2 messenger RNA (mRNA) or impaired MRP2 protein maturation and trafficking. We investigated the consequences of the mutation MRP2Delta(R,M), which leads to the loss of 2 amino acids from the second ATP-binding domain of MRP2. The MRP2Delta(R,M) mutation is associated with the absence of the MRP2 glycoprotein from the apical membrane of hepatocytes. Transfection of mutated MRP2 complementary DNA (cDNA) led to an MRP2Delta(R,M) protein that was only core glycosylated, sensitive to endoglycosidase H digestion, and located in the endoplasmic reticulum (ER) of transfected HEK293 and HepG2 cells. This indicated that deletion of Arg1392 and Met1393 leads to impaired maturation and trafficking of the protein from the ER to the Golgi complex. Inhibition of proteasome function resulted in a paranuclear accumulation of the MRP2Delta(R,M) protein, suggesting that proteasomes are involved in the degradation of the mutant protein. This is the first mutation in Dubin-Johnson syndrome shown to cause deficient MRP2 maturation and impaired sorting of this glycoprotein to the apical membrane.