A Case of Dubin-Johnson Syndrome Presenting as Neonatal Cholestasis With Paucity of Interlobular Bile Ducts.

Dubin-Johnson syndrome (DJS) is a rare autosomal recessive disorder that typically manifests in young adulthood as jaundice with conjugated hyperbilirubinemia. We report a case presenting as neonatal cholestasis with the unexpected histologic finding of paucity of interlobular bile ducts, a feature that is not typically seen in DJS. The diagnosis was confirmed by absent canalicular multidrug-resistance-associated protein 2 (MRP2) immunohistochemical staining on liver biopsy tissue and molecular genetic testing that demonstrated heterozygous mutations in the ATP-Binding Cassette Subfamily C Member 2 (ABCC2) gene, including a novel missense mutation. This report describes a case of DJS with atypical clinicopathologic findings and suggests that DJS should be considered in patients with neonatal cholestasis and bile duct paucity.

Inherited disorders of bilirubin transport and conjugation: new insights into molecular mechanisms and consequences.

Inherited disorders of bilirubin metabolism might reduce bilirubin uptake by hepatocytes, bilirubin conjugation, or secretion of bilirubin into bile. Reductions in uptake could increase levels of unconjugated or conjugated bilirubin (Rotor syndrome). Defects in bilirubin conjugation could increase levels of unconjugated bilirubin; the effects can be benign and frequent (Gilbert syndrome) or rare but severe, increasing the risk of bilirubin encephalopathy (Crigler-Najjar syndrome). Impairment of bilirubin secretion leads to accumulation of conjugated bilirubin (Dubin-Johnson syndrome). We review the genetic causes and pathophysiology of disorders of bilirubin transport and conjugation as well as clinical and therapeutic aspects. We also discuss the possible mechanisms by which hyperbilirubinemia protects against cardiovascular disease and the metabolic syndrome and the effects of specific genetic variants on drug metabolism and cancer development.

The roles of MRP2, MRP3, OATP1B1, and OATP1B3 in conjugated hyperbilirubinemia.

Increased concentrations of bilirubin glucuronides in blood plasma indicate hepatocellular dysfunction. Elucidation of the transport processes of bilirubin conjugates across the basolateral (sinusoidal) and the canalicular plasma membrane domains of hepatocytes has decisively contributed to our current understanding of the molecular basis of conjugated hyperbilirubinemia in human liver diseases. Under normal conditions, unconjugated bilirubin is taken up into hepatocytes by transporters of the organic anion-transporting polypeptide (OATP) family, followed by conjugation with glucuronic acid, and ATP-dependent transport into bile. This efflux across the canalicular membrane is mediated by multidrug resistance protein 2 (MRP2 or ABCC2), which is a 190-kDa glycoprotein transporting with high affinity and efficiency monoglucuronosyl bilirubin and bisglucuronosyl bilirubin into bile. MRP2 is hereditarily deficient in human Dubin-Johnson syndrome. Under pathophysiological conditions such as cholestatic liver injury and MRP2 inhibition, the basolateral efflux pump multidrug resistance protein 3 (MRP3 or ABCC3) is responsible for the occurrence of conjugated hyperbilirubinemia. MRP3 is a glycoprotein with a similar molecular mass as MRP2, with 48% amino acid identity, and with overlapping substrate specificity. Human MRP3 is the only basolateral efflux pump shown to transport bilirubin glucuronides. In human and rat hepatocytes, MRP3/Mrp3 is strongly upregulated under conditions of cholestasis and MRP2 deficiency. This is in line with the concept that basolateral efflux pumps of the hepatocyte compensate for impaired canalicular efflux of compounds into bile and contribute to balance the rate of uptake or synthesis of compounds in hepatocytes with the capacity for efflux into bile.

[Neonatal hyperbilirubinemia and molecular mechanisms of jaundice]. / Vrozené hyperbilirubinemie a molekulární mechanizmy zloutenky.

The introductory summarises the classical path of heme degradation and classification of jaundice. Subsequently, a description of neonatal types of jaundice is given, known as Crigler Najjar, Gilberts, DubinJohnson and Rotor syndromes, emphasising the explanation of the molecular mechanisms of these metabolic disorders. Special attention is given to a recently discovered molecular mechanism of the Rotor syndrome. The mechanism is based on the inability of the liver to retrospectively uptake the conjugated bilirubin fraction primarily excreted into the blood, not bile. A reduced ability of the liver to uptake the conjugated bilirubin contributes to the development of hyperbilirubinemia in common disorders of the liver and bile ducts and to the toxicity of xenobiotics and drugs using transport proteins for conjugated bilirubin.

Role of Multidrug-Resistance Protein 2 in coproporphyrin transport: results from experimental studies in bile fistula rat models.

Coproporphyrin (CP) is one of the main by-products of heme biosynthesis and its abnormal accumulation is associated with different forms of porphyria. Indirect data obtained from animal and human models have suggested a possible role for Multidrug Resistance-associated Protein 2 (MRP2) and other MRPs in hepatocyte excretion of CP. Using normal, MRP2-deficient and a cholestatic rat model, we have assessed the role of MRPs in CP disposition. MRP levels were assayed using immunofluorescence. Biliary and urinary excretion patterns of CP and conjugate bilirubin were measured during equimolar infusions of CP isomers with and without phenoldibromopthalein sulfonate (BSP), a well-known MRP2 substrate. Our results suggest a role for the MRP system as a possible regulator of CP traffic and accumulation in normal and pathological conditions. Alteration in this systems (as observed in cholestatic disease) may play an important role in triggering clinical expression of porphyria in individuals with underlying mutations leading to porphyrin accumulation and may help explain the phenotypic heterogeneity in patients affected by different forms of porphyrias.

Structure and function of the MRP2 (ABCC2) protein and its role in drug disposition.

The multi-drug resistance protein 2 (MRP2; ABCC2) is an ATP-binding cassette transporter playing an important role in detoxification and chemoprotection by transporting a wide range of compounds, especially conjugates of lipophilic substances with glutathione, glucuronate and sulfate, which are collectively known as phase II products of biotransformation. In addition, MRP2 can also transport uncharged compounds in cotransport with glutathione, and thus can modulate the pharmacokinetics of many drugs. The other way around, its expression and activity are also altered by certain drugs and disease states. Unlike other members of the MRP/ABCC family, MRP2 is specifically expressed on the apical membrane domain of polarised cells as hepatocytes, renal proximal tubular cells, enterocytes and syncytiotrophoblasts of the placenta. Several naturally occurring mutations leading to the absence of functional MRP2 protein from the apical membrane have been described causing the human Dubin-Johnson syndrome associated with conjugated hyperbilirubinaemia. Experimental mutation studies have revealed critical amino acids for substrate binding in the MRP2 molecule. This review is, therefore, focused on the structure and function of MRP2, the substrates transported and the clinical relevance of MRP2.

Increased hepatic and renal expressions of multidrug resistance-associated protein 3 in Eisai hyperbilirubinuria rats.

BACKGROUND AND AIM: Eisai hyperbilirubinuria rats (EHBR) are animal models of Dubin-Johnson syndrome, which suffer from jaundice due to impaired biliary excretion of bilirubin glucuronides. In EHBR, deficiency of multidrug resistance-associated protein 2 (mrp2) causes defective biliary excretion of numerous organic anions. However, little is known about the expression of other organic anion transporters in this mrp2-deficient model. The aim of the present study was to investigate adaptive expressions of mrp1, mrp3, mrp6, organic anion transporting polypeptide 1 (oatp1) and oatp2 in liver and kidney of EHBR. METHODS: For the present study, EHBR (n = 5) were used. Hepatic and renal mRNA expression of the aforementioned transporters was determined by constructed semiquantitative reverse transcription polymerase chain reaction assay. Their protein expression was determined by western blotting. Localization of hepatic and renal mrp3 was confirmed by immunohistochemistry. Sprague-Dawley (SD) rats (n = 5) were used as normal controls. RESULTS: Deficiency of mrp2 protein was confirmed in EHBR. Hepatic and renal expression of mrp3 mRNA was 53.6% (P < 0.001) and 82.9% (P < 0.001), and its protein expression was 298.9% (P < 0.001) and 245.0% (P = 0.001) higher in EHBR than in SD rats, respectively. Hepatic and renal expression of mrp1 and mrp6 mRNA was not significantly different between EHBR and SD rats. The mrp1 and mrp6 proteins were expressed in very low amounts in the liver and kidney of both EHBR and SD rats. In contrast to mrp3, hepatic expression of oatp1 and oatp2 mRNA was 33.9% (P = 0. 001) and 38.6% (P < 0.001), and their protein expression was 57.4% (P < 0.05) and 51.0% (P < 0.01) lower in EHBR than in SD rats, respectively. Hepatic and renal mrp3 protein was localized at the basolateral membrane. CONCLUSIONS: Mrp3 plays an important role in the compensation of mrp2 deficiency in liver and kidney of EHBR. Hepatic expressions of mrp3, oatp1 and oatp2 changed adaptively in this animal model. This is a compensatory mechanism for reducing injury to hepatocytes from cytotoxic materials that increase in mrp2 deficiency.

A common Dubin-Johnson syndrome mutation impairs protein maturation and transport activity of MRP2 (ABCC2).

Absence of a functional multidrug resistance protein 2 (MRP2; symbol ABCC2) from the hepatocyte canalicular membrane is the molecular basis of Dubin- Johnson syndrome, an inherited disorder associated with conjugated hyperbilirubinemia in humans. In this work, we analyzed a relatively frequent Dubin-Johnson syndrome mutation that leads to an exchange of two hydrophobic amino acids, isoleucine 1173 to phenylalanine (MRP2I1173F), in a predicted extracellular loop of MRP2. HEK-293 cells stably transfected with MRP2I1173F cDNA synthesized a mutant protein that was mainly core-glycosylated, predominantly retained in the endoplasmic reticulum, and degraded by proteasomes. MRP2I1173F did not mediate ATP-dependent transport of leukotriene C(4) (LTC(4)) into vesicles from plasma membrane and endoplasmic reticulum preparations while normal MRP2 was functionally active. Human HepG2 cells were used to study localization of MRP2I1173F in a polarized cell system. Quantitative analysis showed that GFP-tagged MRP2I1173F was localized to the apical membrane in only 5% of transfected, polarized HepG2 cells compared with 80% for normal MRP2-GFP. Impaired protein maturation followed by proteasomal degradation of inactive MRP2I1173F explain the deficient hepatobiliary elimination observed in this group of Dubin-Johnson syndrome patients.

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.

A new mutation of the ATP-binding cassette, sub-family C, member 2 (ABCC2) gene in a Japanese patient with Dubin-Johnson syndrome.

Dubin-Johnson syndrome (DJS) is an inherited disorder characterized by conjugated hyperbilirubinemia and is caused by mutations of the canalicular multispecific organic anion transporter (cMOAT)/ multidrug resistance protein 2 (MRP2)/ ATP-binding cassette, sub-family C, member 2 (ABCC2) gene. The ABCC2 protein is located in the apical membrane of hepatocytes, and known mutations of this gene cause impaired maturation and trafficking of the mutated protein from the endoplasmic reticulum (ER) to the Golgi complex. We have characterized the ABCC2 gene in a Japanese DJS patient by polymerase chain reaction and DNA sequencing, resulting in the identification of two mutations. One mutation, 1815+2 (T>A) in the splice donor site of intron 13, has already been reported. However, we have identified a novel nonsense mutation consisting of a (C>T) transition at nucleotide 3928 in exon 28.

Genetic defects in hepatobiliary transport.

Bile formation, the exocrine function of the liver, represents a process that is unique to the hepatocyte as a polarized epithelial cell. The generation of bile flow is an osmotic process and largely depends on solute secretion by primary active transporters in the apical membrane of the hepatocyte. In recent years an impressive progress has been made in the discovery of these proteins, most of which belong to the family of ABC transporters. The number of identified ABC transporter genes has been exponentially increasing and the mammalian subfamily now counts at least 52. This development has been of crucial importance for the elucidation of the mechanism of bile formation, and it is therefore not surprising that the development in this field has run in parallel with the discovery of the ABC genes. With the identification of these transporter genes, the background of a number of inherited diseases, which are caused by mutations in these solute pumps, has now been elucidated. We now know that at least six primary active transporters are involved in canalicular secretion of biliary components (MDR1, MDR3, BSEP, MRP2, BCRP and FIC1). Four of these transporter genes are associated with inherited diseases. In this minireview we will shortly describe our present understanding of bile formation and the associated inherited defects.

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.

Exon-intron organization of the human multidrug-resistance protein 2 (MRP2) gene mutated in Dubin-Johnson syndrome.

BACKGROUND & AIMS: The Dubin-Johnson syndrome is characterized by conjugated hyperbilirubinemia and by impaired secretion of anionic conjugates from hepatocytes into bile. Absence of the multidrug-resistance protein 2 (MRP2; symbol ABCC2), an adenosine triphosphate-dependent conjugate export pump, from the hepatocyte canalicular membrane is the molecular basis of this syndrome. The aim of this study was the elucidation of all exon-intron boundaries of the MRP2 gene as a prerequisite for the analysis of mutations in patients with Dubin-Johnson syndrome. METHODS: Exon-intron boundaries of MRP2 were determined, and the amplified exons were screened for mutations. Immunofluorescence microscopy served to localize the MRP2 protein in human liver. RESULTS: The human MRP2 gene is approximately 45 kilobases long; it contains 32 exons and a high proportion of class 0 introns. In 2 patients with Dubin-Johnson syndrome, we detected a nonsense mutation at codon 1066 and a 6-nucleotide deletion mutation affecting codons 1392-1394. The MRP2 protein was absent from the canalicular membrane of both patients. CONCLUSIONS: The mutations detected so far show that various mutations in the MRP2 gene can lead to the Dubin-Johnson syndrome. The exon-intron boundaries established in this article will facilitate the analysis of additional mutations in the MRP2 gene.

Melanins: an old problem revisited or the usefulness of infrared spectroscopy.

This mini review remembers the implication, thirty nine years ago, of professor Wegmann in a fascinating adventure on the serendipitous study of melanin, after that a case of Dubin-Johnson's icterus had been submitted to him. To this investigation have been applied, one after the other and often simultaneously, for the first time, the diverse technologies of pathology, histochemistry, histoenzymology, biochemistry and infrared spectrography, having permitted to detect for the first time melanin in a human liver, associated with a melaninuria. But it was thanks to a marvellous collaboration with many colleagues that we came to a good end. It was the beginning of the interdisciplinary science of Cellular and Molecular Biology. To day remains to explain the mechanisms leading to the formation of melanin in liver and spleen.

The canalicular multispecific organic anion transporter and conjugated hyperbilirubinemia in rat and man.

The human Dubin-Johnson syndrome is an autosomal recessive liver disease characterized by a chronic conjugated hyperbilirubinemia. Patients have impaired hepatobiliary transport of many endogenous and xenobiotic compounds. A similar disease phenotype has been described for a naturally occurring mutant Wistar rat strain, the TR- rat, which is defective in the, functionally defined, canalicular multispecific organic anion transporter (cMOAT). The complementary DNA encoding this protein has been cloned from rat and recently from human liver. cMOAT is a new member of the ATP-binding cassette transporter family, and homologous to the multidrug resistance-associated protein 1. A mutation in the cMOAT gene is responsible for the phenotype observed in TR- rats. This information should soon lead tc a complete genetic characterization of the human Dubin-Johnson syndrome.

A mutation in the human canalicular multispecific organic anion transporter gene causes the Dubin-Johnson syndrome.

The human Dubin-Johnson syndrome (DJS) is a rare autosomal recessive liver disorder characterized by chronic conjugated hyperbilirubinemia. Patients have impaired hepatobiliary transport of non-bile salt organic anions. A highly similar phenotype has been described for a mutant Wistar rat strain, the transport-deficient (TR-) rat, which is defective in the canalicular multispecific organic anion transporter (cmoat). This protein mediates adenosine triphosphate-dependent transport of a broad range of endogenous and xenobiotic compounds across the (apical) canalicular membrane of the hepatocyte. The complementary DNA (cDNA) encoding rat cmoat has recently been cloned, and the mutation underlying the defect in TR- rats has been identified. In the present study, we have isolated the human homologue of rat cmoat, human cMOAT, and analyzed the corresponding cDNA from fibroblasts of a DJS patient for mutations. Our results show that a mutation in this gene is the cause of DJS.

Absence of the canalicular isoform of the MRP gene-encoded conjugate export pump from the hepatocytes in Dubin-Johnson syndrome.

The Dubin-Johnson syndrome is characterized by an inherited defect in the secretion of amphiphilic anionic conjugates from hepatocytes into the bile. We have recently identified the membrane protein mediating the adenosine triphosphate (ATP)-dependent transport of glutathione and glucuronate conjugates as a multidrug-resistance protein (MRP) and localized it to the canalicular as well as to the lateral hepatocyte plasma membrane. In the present study we show the selective absence of the canalicular isoform of MRP (cMRP) from the hepatocytes in a patient with Dubin-Johnson syndrome by double-label immunofluorescence and confocal laser scanning microscopy using antibodies directed against MRP and dipeptidyl-peptidase IV (DPPIV). Another isoform of MRP was detected, however, in the lateral hepatocyte membrane of the patient. Moreover, MRP was present on immunoblots of erythrocyte membranes from Dubin-Johnson syndrome and normal humans. These findings are analogous to our recent observations on the localization of the rat homolog of MRP and its canalicular isoform, cMrp, in normal and transport-deficient GY/TR- Wistar rat liver. The elucidation of the selective absence of an isoform of MRP and from the canalicular membrane domain in conjunction with the defined substrate specificity of the MRP and cMRP gene-encoded conjugate export pumps contributes to the molecular definition of the transport defect in Dubin-Johnson syndrome.