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

doi:10.1016/S0016-5085(99)70459-2

Gastroenterology

Volume 117, Issue 3, September 1999, Pages 653-660

https://doi.org/10.1016/S0016-5085(99)70459-2 Get rights and content

Abstract

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 ~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.

GASTROENTEROLOGY 1999;117:653-660

Section snippets

Patients and tissue samples

Patient H1 with Dubin–Johnson syndrome has been described in detail recently.⁴ Patient H2 was a white woman, 25 years of age, who had a 10-year history of intermittent jaundice. Hyperbilirubinemia was moderate with serum levels of 47 and 27 μmol/L for conjugated and unconjugated bilirubin, respectively. Serum activities of alanine aminotransferase, γ-glutamyltransferase, and alkaline phosphatase were in the normal range. Patient H2 gave written informed consent to the needle biopsy that was

Exon-intron organization of the human MRP2 gene

As a prerequisite for a detailed analysis of mutations in the coding region of the human MRP2 gene, we determined the exon-intron boundaries of this gene. The MRP2 gene spans ~45 kilobases (kb). The analysis of the amplified fragments indicated that the human MRP2 gene contains 32 exons ranging in size from 56 to 255 base pairs (bp; Fig. 1, Fig. 1). Intron sizes varied from 145 bp to ~3.6 kb. Of the 31 introns, 21 were class 0 (where the splice occurs between codons), 3 were class 1 (where the

Discussion

Knowledge of the exon-intron organization of the human MRP2 gene provides the basis for an efficient screening for mutations located in the coding region. Our analysis indicates that the human MRP2 gene comprises 32 exons and spans ~45 kb (Figure 1A). The genomic structure of the human MRP2 gene exhibits a remarkable similarity to that of the human MRP1 gene reported by Grant et al.,²⁰ as indicated by the number and size of exons and by the high proportion of class 0 introns (Figure 5).

Acknowledgements

The authors thank Marion Pfannschmidt for excellent technical assistance.

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2023, MacSween's Pathology of the Liver, Eighth Edition
A section on the approach to diagnostic histological interpretation is the overture to this chapter on inherited and developmental disorders. This initial section is split chronologically into the early neonatal and infantile period and later childhood and adulthood, with the intention of reflecting clinical practice as closely and succinctly as possible. Disorders of the biliary tree, bile formation and secretion and hepatocyte metabolism are the core of this chapter, a merger of Chapters 3 and 4 of previous editions. Considerations on the pathogenetic and/or clinical overlap among developmental, genetic and metabolic disorders were the rationale behind this change. The complexity of hepatocyte metabolism is reflected into the myriad of related pathological conditions. Two short new paragraphs on disorders of manganese metabolism and DNA repair and nuclear envelope have been added. Recent technological advances, particularly in genomics in the last 5 years, have resulted in a plethora of new entities and changes in terminology, challenging the authors to balance detail and application to clinical practice. Tables and figures from the previous edition have been largely kept due to their quality and contemporary relevance, and updated where necessary. Liver involvement in immunodeficiency and miscellaneous disorders precede the final section on anatomical anomalies. Vascular anomalies are now included in the chapters on vascular disorders.
In silico screening and analysis of single-nucleotide polymorphic variants of the ABCC2 gene affecting Dubin–Johnson syndrome
2022, Arab Journal of Gastroenterology
Citation Excerpt :
Studies have identified that SNPs in the TMDs and NBDs of ABCC2 are harmful. SNPs result in rapid mRNA degradation and impaired protein maturation, leading to the production of non-functional MRP2 [20,21]. According to previous reports, the deleterious effects of SNPs in the ABCC2 gene have not been studied using in silico methods.
Dubin–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.
The 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.
In 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.
In 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.
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Bilirubin is an endogenous substance derived from heme catabolism. In this study, we aimed to assess the anti-inflammatory activity of bilirubin, and to determine the mechanism thereof. The anti-inflammatory activity of bilirubin was evaluated using lipopolysaccharide (LPS)-treated peritoneal macrophages (PMs) and Raw264.7 cells, and mice with alum-induced peritonitis. The mRNA and proteins of NOD-like receptor family pyrin domain containing 3 (Nlrp3) and inflammatory cytokines were determined using qPCR and Western blotting, respectively. Distribution of phosphorylated (p) p65 [a NF-κB (nuclear factor-κB) subunit] in the cytoplasm and nucleus were evaluated by immunofluorescence analysis and electrophoretic mobility shift assay. Bilirubin prior to LPS treatment decreased protein expressions of Nlrp3, pro-interleukin (IL)-1β and mature IL-1β in PMs, whereas bilirubin post LPS treatment showed no effects. Bilirubin prior to LPS treatment dose-dependently repressed expressions of Nlrp3 and IL-1β, and inhibited translocation of p-p65 to nucleus in Raw264.7 cells. Bilirubin treatment decreased myeloperoxidase activity and reduced the levels of inflammatory cytokines (i.e., IL-1β, TNFα and IL-6) in lavage fluid in mice with alum-induced peritonitis. This was accompanied by a lower mortality rate. In addition, the mRNAs of Nlrp3 and IL-1β in peritoneal exudates cells were decreased, and the levels of p-p65 and mature IL-1β proteins were reduced. In conclusion, bilirubin acted on inflammation and alleviated alum–induced peritonitis through inactivation of Nlrp3 inflammasome.
Bile pigment metabolism and its disorders
2019, Emery and Rimoin's Principles and Practice of Medical Genetics and Genomics: Cardiovascular, Respiratory, and Gastrointestinal Disorders
Bilirubin is the breakdown product of the heme moiety of hemoproteins, including hemoglobin, myoglobin, and several hepatic hemoproteins. About 80% of the 200–300 mg bilirubin is produced daily in reticuloendothelial cells from hemoglobin of senescent erythrocytes, the remainder being derived mostly from hepatic hemoproteins. Microsomal heme oxygenases cleave heme forming biliverdin, which is reduced to bilirubin by biliverdin reductase. At low concentrations, bilirubin is cytoprotective and provides metabolic benefits. However, bilirubin-induced neurological damage (BIND) caused by severe unconjugated hyperbilirubinemia is a major clinical concern. Physiologically, BIND is prevented by binding to plasma albumin, rapid uptake by hepatocytes, UGT1A1-mediated glucuronidation, and active canalicular excretion. A fraction of the bilirubin glucuronides is pumped out by ABCC3 through the sinusoidal surface of hepatocytes and undergoes reuptake by hepatocytes located downstream to sinusoidal blood flow. A small portion of bilirubin excreted in bile undergoes enterohepatic recycling, the remainder being degraded by bacteria to urobilinogen. Unconjugated hyperbilirubinemia can be caused by excessive bilirubin production, reduced hepatocellular uptake, or defective glucuronidation, whereas conjugated hyperbilirubinemia results from defective canalicular excretion or abnormal reuptake. Hyperbilirubinemia, which is universal in neonates, normally resolves in 1–2 weeks, but can be exacerbated or prolonged by breastfeeding or delayed UGT1A1 maturation (Lucey–Driscoll syndrome). In addition to acquired hepatobiliary diseases and hemolysis, a number of inherited liver disorders can cause lifelong hyperbilirubinemia. Coding region mutations of UGT1A1 can cause complete or partial loss of bilirubin glucuronidation (Crigler–Najjar syndrome, type 1 and type 2, respectively). A variant UGT1A1 promoter can reduce UGT1A1 expression, causing mild and innocuous unconjugated hyperbilirubinemia (Gilbert syndrome). Conjugated hyperbilirubinemia can result from mutations of the ABCC2 that reduce canalicular secretion of bilirubin glucuronides and many other organic anions causing Dubin–Johnson syndrome. Concurrent mutations of ABCB1 and ABCB3 disable reuptake of bilirubin glucuronides, causing mild conjugated hyperbilirubinemia (Rotor syndrome). Several other inherited defects of bile canalicular proteins cause four types of progressive familial intrahepatic cholestasis that cause mixed hyperbilirubinemia as well as liver injury.
Developmental and Inherited Liver Disease
2018, MacSween's Pathology of the Liver
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Visualization of liver uptake function using the uptake contrast-enhanced ratio in hepatobiliary phase imaging
2014, Magnetic Resonance Imaging
To visualize liver uptake function using the uptake contrast-enhanced ratio in hepatobiliary phase (uptake CERH) magnetic resonance imaging.
Thirty-seven patients with hepatocellular carcinoma (HCC) and 23 with metastatic liver cancer were evaluated. Hepatobiliary phase images were acquired 20 min after an intravenous bolus injection of gadoxetic acid disodium. We assumed that the contrast-enhanced ratio in the hepatobiliary phase (CERH) in the spleen was similar to the contrast-enhanced ratio in the extracellular matrix (CEREM). The Uptake CERH value was defined as the percentage signal gain between the precontrast and hepatobiliary phase images (without CEREM). The Uptake CERH value measured the tumor-free liver parenchyma. The association of the uptake CERH value with the biochemical liver function test results, and hepatocellular density in the liver parenchyma was assessed. Correlations were examined using Pearson correlation coefficient and the Mann–Whitney test.
The uptake CERH value was correlated with albumin, bilirubin, indocyanine green retention rate at 15 min, prothrombin activity(%), platelet count, and cellular density in the liver parenchyma (p < 0.01).
Uptake CERH images are useful for visualizing liver uptake function.

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^☆: Address requests for reprints to: Dietrich Keppler, M.D., Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. e-mail: [email protected]; fax: (49) 6221-422402.

^☆☆: Supported in part by the Alexander von Humboldt Foundation and the Deutsche Forschungsgemeinschaft through Sonderforschungsbereich 601 and 352 and through Forschungsschwerpunkt Transplantation Heidelberg.

^★: This article is dedicated to Professor emeritus Tadasu Tsujii, Nara Medical University, Japan, on the occasion of his 70th birthday.

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Gastroenterology

Abstract

Section snippets

Patients and tissue samples

Exon-intron organization of the human MRP2 gene

Discussion

Acknowledgements

References (24)

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

Hepatology

cDNA cloning of the hepatocyte canalicular isoform of the multidrug resistance protein, cMRP, reveals a novel conjugate export pump deficient in hyperbilirubinemic mutant rats

J Biol Chem

The sister of P-glycoprotein represents the canalicular bile salt export pump of mammalian liver

J Biol Chem

Analysis of the intronexon organization of the human multidrug-resistance protein gene (MRP) and alternative splicing of its mRNA

Genomics

Genomic structure of the canalicular multispecific organic anion-transporter gene (MRP2/cMOAT) and mutations in the ATP-binding-cassette region in Dubin–Johnson syndrome

Am J Hum Genet

Chronic idiopathic jaundice with unidentified pigment in liver cells; a new clinicopathologic entity with report of 12 cases

Medicine

Persistent nonhemolytic hyperbilirubinemia associated with lipochrome-like pigment in liver cells; report of four cases

Ann Intern Med

Heme and bile pigment metabolism

The canalicular conjugate export pump encoded by the cmrp/cmoat gene

Expression of the MRP gene–encoded conjugate export pump in liver and its selective absence from the canalicular membrane in transport-deficient mutant hepatocytes

J Cell Biol

Congenital jaundice in rats with a mutation in a multidrug resistance–associated protein gene

Science

Molecular cloning of canalicular multispecific organic anion transporter defective in Eisai hyperbilirubinemic rats

Am J Physiol

Cited by (140)

Developmental and Inherited Liver Disease

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

Bilirubin alleviates alum–induced peritonitis through inactivation of NLRP3 inflammasome

Bile pigment metabolism and its disorders

Developmental and Inherited Liver Disease

Visualization of liver uptake function using the uptake contrast-enhanced ratio in hepatobiliary phase imaging

Liver, Pancreas, and Biliary TractExon-intron organization of the human multidrug-resistance protein 2 (MRP2) gene mutated in Dubin–Johnson syndrome☆,☆☆,★

Abstract

Section snippets

Patients and tissue samples

Exon-intron organization of the human MRP2 gene

Discussion

Acknowledgements

Hepatology

J Biol Chem

J Biol Chem

Genomics

Am J Hum Genet

Chronic idiopathic jaundice with unidentified pigment in liver cells; a new clinicopathologic entity with report of 12 cases

Medicine

Persistent nonhemolytic hyperbilirubinemia associated with lipochrome-like pigment in liver cells; report of four cases

Ann Intern Med

Heme and bile pigment metabolism

The canalicular conjugate export pump encoded by the cmrp/cmoat gene

Expression of the MRP gene–encoded conjugate export pump in liver and its selective absence from the canalicular membrane in transport-deficient mutant hepatocytes

J Cell Biol

Congenital jaundice in rats with a mutation in a multidrug resistance–associated protein gene

Science

Molecular cloning of canalicular multispecific organic anion transporter defective in Eisai hyperbilirubinemic rats

Am J Physiol

Liver, Pancreas, and Biliary Tract
Exon-intron organization of the human multidrug-resistance protein 2 (MRP2) gene mutated in Dubin–Johnson syndrome☆,☆☆,★