ABSTRACT Dubin-Johnson syndrome (DJS) is an autosomal recessive disorder characterized by conjugated hyperbilirubinemia and caused by mutations of the ATP-binding cassette (ABC) transporter

encoding gene_ MRP2/cMOAT/ABCC2_. Previous studies reported on mutations in DJS patients and polymorphisms in healthy human individuals. The genomic DNA sequence of a female Caucasian DJS

patient was analyzed by DNA sequencing and revealed the identification of a homozygous missense mutation C2302T. This DJS-causing alteration results in an amino acid exchange Arg768Trp.

INTRODUCTION Dubin-Johnson syndrome (DJS, MIM 237500) is a rare, benign, and autosomal recessive disorder, which is characterized by a defect in the transfer of endogenous and exogenous

anion transporter (cMOAT) or multidrug resistance protein 2 (MRP2), is an integral membrane glycoprotein of about 190 kDa localized to the apical domain of polarized cells, such as the

canalicular membrane of hepatocytes or the apical membrane of renal proximal tubules (König et al. 1999). In the liver, the ABCC2 protein mediates the multispecific efflux of various types

of organic anions, including glucuronate, sulfate, and glutathione conjugates, across the canalicular hepatocyte membrane into the bile, dependent on ATP-hydrolysis (Suzuki and Sugiyama

2002). So far, several mutations and polymorphisms of the human_ ABCC2_ gene have been reported (Wada et al. 1998; Toh et al. 1999; Ito et al. 2001; Mor-Cohen et al. 2001; Itoda et al. 2002;

Saito et al. 2002). In this study, we report a mutation of the_ ABCC2_ gene in a patient with DJS, which was also described to be a single nucleotide polymorphism (SNP) in healthy specimens

by other authors (Ito et al. 2001; Saito et al. 2002). Additionally, the SNP C-24T (NCBI SNP ID rs717620) of the 5'-untranslated region (5'-UTR) in a set of healthy European

individuals and a panel of human tumor cell lines was analyzed. PATIENT AND METHODS A female Caucasian DJS patient of Turkish origin, aged 37, was examined by a needle biopsy performed for

diagnostic reasons. Anamnesis of the family showed no additional proven case of DJS. The serum total and conjugated bilirubin levels were elevated (2.4 mg/dl and 1.9 mg/dl, respectively).

Histopathologic examination of the liver biopsy revealed golden-brown pigmented granules in the cytoplasm of the liver cells, typical for DJS (Fig. 1). Control subjects were healthy patients

in terms of DJS at the University Hospital Charité (Berlin, Germany). The DJS patient and control subjects gave their informed consent prior to their inclusion in the study. Blood samples

of the DJS patient and 20 control subjects were used for genetic analysis. The following human tumor cell lines were used: the melanoma cell line MeWo (Fogh et al. 1978), the gastric

carcinoma cell line EPG85–257 (Dietel et al. 1990), the pancreatic carcinoma cell line EPP85–181 (Lage und Dietel 2002), the ovarian carcinoma cell line A2780 (Eva et al. 1982), the colon

carcinoma cell line HT-29 (Thomas et al. 1974), and the adrenocortical carcinoma cell line D43/86 (not published). Cell lines were grown using standard procedures as described previously

(Dietel et al. 1990; Lage and Dietel 2002). Genomic DNA was prepared from peripheral blood leukocytes and cultured tumor cell lines by standard methods as described previously (Lage and

Dietel 2002). Polymerase chain reaction (PCR) amplification was performed using 150 ng genomic DNA, 0.2 µM of each oligonucleotide primer (Table 1), 0.2 mM of each dNTP, 50 mM KCl, 10 mM

Tris-HCl, pH 8.3, 1.5 mM MgCl2, 0.001% gelatin, and 1 unit Ampli_Taq-_Gold DNA-polymerase (Perkin Elmer) in a final volume of 25 µl. Cycling conditions: initial denaturation at 94°C for 12 

min followed 36 cycles of amplification (denaturation at 94°C, 1 min; annealing at 55°C, 90 s; extension at 72°C, 90 s). The last cycle contained a 5-min extension step at 72°C. The PCR

products were sequenced directly by using dye terminator sequencing and an ABI-373 sequencer (Perkin Elmer). The sequencing was repeated independently, and mutation analysis was performed by

comparison with the_ ABCC2_ reference sequence NM_000392 (NCBI). RESULTS AND DISCUSSION The genomic DNA of a Caucasian DJS patient was analyzed with PCR methods using primer pairs (Table 1)

that cover the entire ABCC2 cDNA and exon-intron junction regions and screened for mutations. The only alteration found was the missense mutation C2302T causing an amino acid exchange

Arg768Trp indicating to be the molecular basis for DJS in this Turkish patient. This mutation was not found in any of the 26 control specimens tested. The nucleotide exchange C2302T located

in the highly conserved domain, the Walker C motif, was previously described by other authors (Wada et al. 1998; Toh et al. 1999; Ito et al. 2001) in DJS patients as well as in healthy

specimens. Wada et al. (1998) and Toh et al. (1999) reported the homozygous mutation C2302T as genetic alteration causing DJS in patients. Ito et al. (2001) examined a panel of healthy

Japanese specimens to detect mutations in the_ ABCC2_ gene and found the mutation C2302T to be heterozygous in one of 48 specimens. In accordance with our results, Itoda et al. (2002) could

not detect the alteration C2302T in any of 72 cell lines derived from various tissues of healthy Japanese individuals for the purpose of detecting polymorphisms in the_ ABCC2_ gene. It was

reported that the amino acid exchange Arg768Trp causes a deficient maturation of the ABCC2 protein leading to a diminished glycosylation, impaired sorting to the apical membrane, and

degradation via the proteasome pathway (Hashimoto et al. 2002). These results support our findings that the missense mutation Arg768Trp alone is responsible for the defect in transporting

bilirubin conjugates, leading to hyperbilirubinemia in the context of DJS in the examined patient. The observation that this homozygous mutation was found in Japanese patients (Wada et al.

1998; Toh et al. 1999) and in a Caucasian DJS patient with Turkish descent (present study) suggests that this dysfunctional allel is relatively common in the human genetic pool. Further

analyzes with more healthy specimens and DJS patients are needed to determine whether this allel is also present in other ethnic populations, and to compare allel frequencies among them.

With data of other genetic loci, these data could give new insights in population founding, migration, and development. In the_ ABCC2_ gene mutation analysis of the DJS patient we used

genomic DNA of 20 healthy individuals and six human tumor cell lines as control. The DJS-causing base substitution C2302T was not detected in any of the control specimens. In order to

demonstrate that the used control subjects and cell lines are representative for the population, one of the most common polymorphisms of the ABCC2 encoding gene (Ito et al. 2001; Mor-Cohen

et al. 2001; Itoda et al. 2002), SNP C-24T, was analyzed. In four of 20 control individuals the SNP C-24T was detected, whereas the DJS patient did not show this polymorphism. Among the six

human tumor cell lines analyzed, the melanoma cell line MeWo and the pancreatic carcinoma cell line EPP85–181 contained this SNP, whereas the other four cell lines did not (Table 2). All six

control specimen with the SNP C-24T were heterozygous for this polymorphism. The allel frequency of 0.889 for C and 0.111 for T at this locus was similar to other studies, i.e., C=0.813 and

T=0.188 (Ito et al., 2001), or C=0.819 and T=0.181 (Itoda et al. 2002) in Japanese healthy individuals. Mor-Cohen et al. (2001) examined three Jewish populations and reported allel

frequencies between 0.739 and 0.877 for C and between 0.122 and 0.261 for T for the SNP C-24T. These data indicate a comparable allel frequency for the SNP C-24T in all analyzed populations.

CONCLUSIONS The homozygous missense mutation C2302T causing the amino acid exchange Arg768Trp is the molecular reason for DJS in a female Caucasian patient examined. Since this DJS-causing

mutation was reported in a Japanese population previously, the data indicate that C2302T is a common mutation in DJS patients in different populations. Analysis of the_ ABCC2_ gene in DJS

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The authors thank Dr. Christiane Otto (Berlin) for her support and for providing clinical data. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Institute of Pathology, Charité Campus Mitte,

Humboldt University Berlin, Schumannstr. 20/21, 10117, Berlin, Germany Verena Materna & Hermann Lage Authors * Verena Materna View author publications You can also search for this author

inPubMed Google Scholar * Hermann Lage View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Hermann Lage. RIGHTS AND

PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Materna, V., Lage, H. Homozygous mutation Arg768Trp in the ABC-transporter encoding gene_ MRP2/cMOAT/ABCC2_ causes

Dubin-Johnson syndrome in a Caucasian patient. _J Hum Genet_ 48, 484–486 (2003). https://doi.org/10.1007/s10038-003-0057-8 Download citation * Received: 15 May 2003 * Accepted: 01 July 2003

* Published: 27 August 2003 * Issue Date: September 2003 * DOI: https://doi.org/10.1007/s10038-003-0057-8 SHARE THIS ARTICLE Anyone you share the following link with will be able to read

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KEYWORDS * MRP2 * cMOAT * ABCC2 * DJS * Mutation * Polymorphism