A novel ABCC2 p.G693R mutation resulting in loss of function of MRP2: a recurrent cause of hyperbilirubinemia in Dubin-Johnson syndrome in China

Background Dubin-Johnson syndrome (DJS) is a rare autosomal recessive disorder characterized by predominantly conjugated hyperbilirubinemia that is caused by pathogenic mutations in the adenosine triphosphate-binding cassette subfamily C member 2 ( ABCC2 ) gene, which encodes multidrug resistance-associated protein 2 (MRP2). However, little is known about the causative mutation of DJS in China. Recently, we have reported a novel ABCC2 p.G693R mutation in two unrelated cases. In the present study, we investigated the pathogenicity of the ABCC2 p.G693R mutation in DJS in China. Methods Clinical and genetic analysis was conducted for the two patients with the ABCC2 p.G693R mutation. Whole exome sequencing for mutations in other known hyperbilirubinemia-related genes was conducted for the cases with ABCC2 p.G693R. Expression and cellular localization of the mutant MRP2 p.G693R were analyzed by Western blotting and immunofluorescence assay, respectively. Organic anion transport activity was evaluated by the analysis of glutathione-conjugated-monochlorobimane. Results The two DJS patients with ABCC2 p.G693R mutation, which was conserved among different species, showed typical hyperbilirubinemia phenotype. No pathogenic mutation was identified in the other known hyperbilirubinemia related genes. Functional studies in three cell lines showed that the expression, localization and the organic anion transport activity were significantly compromised by MRP2 p.G693R mutation compared with wild-type MRP2. Conclusions The ABCC2 p.G693R mutation is associated with loss of function of the MRP2 protein, which may represent one of the major etiological factors of The clinical manifestation of the patients with the ABCC2 p.G693R, including age of onset, duration of jaundice, aggravating or relieving factors was recorded. Past medical history including drug or toxin exposure, alcohol intake, and family history of jaundice or other liver diseases were collected. Relevant laboratory data of the two patients with the ABCC2 p.G693R were analyzed, including complete blood count, liver function tests, renal function tests and electrolytes, coagulation profile. Abdominal ultrasonography was done to exclude obstruction or dilation of the hepatobiliary tract exist and transient elastography (FibroScan) was conducted to evaluate the liver stiffness. Conservative analysis was performed by http://genome.ucsc.edu/. Aligned amino acid sequences of human, rhesus, mouse, dog, elephant, chicken, xenopus tropicalis, zebrafish, and lamprey MRP2 with mutation p.G693R loci were analyzed.


Abstract
Background Dubin-Johnson syndrome (DJS) is a rare autosomal recessive disorder characterized by predominantly conjugated hyperbilirubinemia that is caused by pathogenic mutations in the adenosine triphosphate-binding cassette subfamily C member 2 ( ABCC2 ) gene, which encodes multidrug resistance-associated protein 2 (MRP2). However, little is known about the causative mutation of DJS in China.
Recently, we have reported a novel ABCC2 p.G693R mutation in two unrelated cases. In the present study, we investigated the pathogenicity of the ABCC2 p.G693R mutation in DJS in China.
Methods Clinical and genetic analysis was conducted for the two patients with the ABCC2 p.G693R mutation. Whole exome sequencing for mutations in other known hyperbilirubinemia-related genes was conducted for the cases with ABCC2 p.G693R.
Expression and cellular localization of the mutant MRP2 p.G693R were analyzed by Western blotting and immunofluorescence assay, respectively. Organic anion transport activity was evaluated by the analysis of glutathione-conjugatedmonochlorobimane.

Results
The two DJS patients with ABCC2 p.G693R mutation, which was conserved among different species, showed typical hyperbilirubinemia phenotype. No pathogenic mutation was identified in the other known hyperbilirubinemia related genes. Functional studies in three cell lines showed that the expression, localization and the organic anion transport activity were significantly compromised by MRP2 p.G693R mutation compared with wild-type MRP2.
Conclusions The ABCC2 p.G693R mutation is associated with loss of function of the MRP2 protein, which may represent one of the major etiological factors of hyperbilirubinemia in DJS in China.

Background
Dubin-Johnson syndrome (DJS) is an autosomal recessive disorder which was first described in 1954 [1]. As a rare disorder affecting both genders, DJS has been identified in all nationalities and races. Its incidence in Sephardic Jews is approximately 1 in 3,000 [2]. The syndrome is characterized by predominantly conjugated hyperbilirubinemia, which is caused by impairment in the transfer of non-bile acid organic anions from hepatocytes into canaliculi [3].
The adenosine triphosphate-binding cassette subfamily C member 2 (ABCC2) gene, located on chromosome 10q24, encodes the multidrug resistance-associated protein 2 (MRP2). Comprised of 1,545 amino acids, this protein belongs to an integral membrane glycoprotein family [4]. MRP2 traffic from the endoplasmic reticulum to the canalicular membrane of hepatocytes where it functions and then relocalizes back to endosomal vesicles for recycling 5 . This protein is a non-bile acid organic anion transporter and mediates the active transport of conjugate compounds with glutathione or glucuronate from the cytoplasm of hepatocytes into the canaliculi [5].
According to Human Gene Mutation Database (HGMD; www.hgmd.cf.ac.uk), a total of 67 pathogenic genetic mutations in the ABCC2 gene including missense, nonsense, deletions and splice site mutations have been identified in DJS patients.
However, no hotspot mutations have been identified in the ABCC2 gene. The majority of the DJS-causing mutations in ABCC2 are related to defects in MRP2 protein synthesis, localization or secretion activities. Some mutations may cause rapid degradation of the mRNA, mislocalization of protein or decreased organic anion transport activity [6]. ABCC2 mutations have been identified in DJS patients worldwide. However, less is known about the causative mutation of DJS in China.
Recently, we identified a novel ABCC2 p.G693R mutation in our previous study [7].
Therefore, in the present study, we investigated the frequency of ABCC2 p.G693R in Chinese DJS patients and examined the pattern and biological consequences of the ABCC2 p.G693R mutation, focusing on their effects on protein maturation, localization and transport activity. Clinical and genetic analysis of the two patients with the ABCC2 p.G693R The clinical manifestation of the patients with the ABCC2 p.G693R, including age of onset, duration of jaundice, aggravating or relieving factors was recorded. Past medical history including drug or toxin exposure, alcohol intake, and family history of jaundice or other liver diseases were collected.
Relevant laboratory data of the two patients with the ABCC2 p.G693R were analyzed, including complete blood count, liver function tests, renal function tests and electrolytes, coagulation profile. Abdominal ultrasonography was done to exclude obstruction or dilation of the hepatobiliary tract exist and transient elastography (FibroScan) was conducted to evaluate the liver stiffness.

Measurement of subcellular localization by indirect immunofluorescence staining
Immunofluorescence analysis was performed as described previously [9]. The cells were incubated with a primary antibody directed against rabbit anti-MRP2 (ab172630; Abcam) at 4C overnight. After three washes with phosphate-buffered saline for 5 min each, the cells were incubated with anti-rabbit Alex 647-conjugated secondary antibodies (1:200; Invitrogen) for 1 h at room temperature. Then the cells were visualized and photographed using an FV 300 confocal microscope (Olympus, Tokyo, Japan).
Measurement of organic anion transport activity by export of glutathione conjugated monochlorobimane (GS-MCLB) assay MCLB is an organic anion transport substrate for MRP2 and has absorption/emission maximal ~394/490 nm. MCLB (M1381MP, Thermo, USA) transport study was conducted as described by Terlouw et al [10]. The cells were pre-incubated with 0.2 mmol/L MCLB in medium for 30 min on ice. The medium was replaced with fresh Hank's medium and incubated at 37°C. At different time points, medium was collected and the GS-MCLB in the medium was measured by the fluorescence method with a spectrophotometer.

Statistical analysis
All experiments were carried out at least three times. Statistical analyses were performed using SPSS V12.0 software. Results were expressed as mean ± standard deviation (SD). Continuous variables were analyzed using the Student's test. A twosided P value of < 0.05 was considered statistically significant.

Results
Clinical and genetic profiles of the DJS patients with ABCC2 p.G693R mutation  (Fig. 1a). Sequence comparison showed that amino acid 693 of MRP2 was conserved among different species (Fig. 1b). The two patients harbored the variant of p.G808V or p.R529Q in another allele respectively.
The clinical features of these two patients are shown in Table 1 Table 1).

MRP2 expression was decreased in cell lines stably expressing MRP2 p.G693R
Western blotting for MRP2 in HEK293, Huh-7 and HepG2 cell lines transfected with vector expressing ABCC2 with the p.G693R mutation demonstrated a decreased expression of theMRP2 p.G693R mutant, probably due to the degradation of the mutant protein, compared with wild-type MRP2 (Fig. 2a). MRP2 p.G693R mutant was predominantly retained in the cytoplasm rather than the cell surface Indirect immunofluorescence staining in HEK293A, Huh-7 and HepG2 cell lines showed that wild-type MRP2 predominantly localized to the cell surface and cytoplasm (Fig. 2b). In contrast, we observed mislocalization of the MRP2 p.G693R mutant, which was predominantly retained in the cytoplasm rather than the cell surface.
MRP2 p.G693R mutant exhibited decreased organic anion transport activity Following pre-incubation with MCLB, which was conjugated with glutathione to produce GS-MCLB, the GS-MCLB efflux in HEK293A, Huh-7 and HepG2 cells lines which stably expressed wild-type MRP2 was markedly increased compared with negative control cells (p < 0.05) (Fig. 3). However, the efflux of GS-MCLB in cells expressing MRP2 p.G693R was similar to the negative control. These results indicated that the p.G693R mutant exhibited significantly decreased organic anion transport activity compared with the wild-type MRP2.

DISCUSSION
In the present study, we reported on the clinical manifestations and biological functional consequences of the ABCC2 p.G693R mutation, which was identified with a high frequency of 28.6% (2/7) in a small cohort of Chinese patients with DJS.
Functional studies indicated significantly decreased expression, mislocalization, and decreased organic anion transport activity of mutant MRP2 compared with wild-type MRP2, indicating that this variant resulted in loss of function of the MRP2 protein.
DJS is characterized by predominantly conjugated hyperbilirubinemia in liver function tests and black liver in liver biopsies [11]. Urinary coproporphyrin levels, bromsulphalein excretion test and hepatobiliary scintigraphy with iodopanoic acid provide effective methods for identification and diagnosis on DJS [12,13] In the present study, whole exome sequencing revealed additional HSD3B7 p.R110Q mutation in a case with ABCC2 p.G693R mutation, but no mutations in the other known hyperbilirubinemia-related genes in the other case with ABCC2 p.G693R mutation. Gene HSD3B7, located on chromosome 16p11.2, encodes an enzyme which is a member of the short-chain dehydrogenase/reductase superfamily and involved in the initial stages of the synthesis of bile acids from cholesterol [14].
Pathogenic mutations in HSD3B7 are associated with congenital bile acid deficiency type I, which is a life-threatening liver disease and manifests as hyperbilirubinemia and neonatal cholestasis [15,16]. However, in silico analysis showed this variant was benign, indicating it might be common variant but not a pathogenic mutation.
Thus, the two cases with ABCC2 p.G693R heterozygous mutation showed that the ABCC2 mutation might be the main genetic factor of DJS in China.
In the present study, the p.G693R mutation was observed in 2 out of 7 patients, indicating that the p.G693R mutation might be a potential hotspot mutation in Chinese DJS patients. ABCC2 p.G693R is a missense mutation located in the first ATP-binding domain of MRP2 protein [17]. We demonstrated that while wild-type MRP2 predominantly localized to the cell surface and cytoplasm, the MRP2 p.G693R mutant was predominantly retained in the cytoplasm rather than the cell surface.
The mislocalization of the p.G693R mutant may likely be due to deficient maturation and sorting, causing impaired insertion trafficking from the endoplasmic reticulum to the canalicular membrane in hepatocytes, similar to the p.R768W and p.W709R mutants reported in previous studies [18,19].
Furthermore, efflux of GS-MCLB uptake into plasma from cells expressing the p.G693R mutant was markedly reduced compared with cells expressing wild-type MRP2, suggesting that the organic anion transport activity of p.G693R MRP2 on the canalicular membrane was defective [13]. Similarly, Hashimoto et al. reported that p.Q1382R MRP2 impairs substrate-induced ATP hydrolysis, which lead to the defect of organic anion transport activity [6].
We understand that the number of cases analyzed in the current study was limited, and thus more cases with DJS are required to confirm these conclusions. Using the China Registry of Genetic/Metabolic Liver Diseases, we will conduct further genetic and functional studies of DJS to strengthen the relationship between the genotype and phenotype of DJS.
In conclusion, here we show that the ABCC2 p. Availability of data and materials All the data were collected from the hospital information system and can be available from the corresponding author upon reasonable request.

Competing interests
The authors declare that they have no competing interests.   Figure 1 Mutation analysis of the two DJS cases with ABCC2 p.G693R. a Sequencing of the heterozygo Figure 2 The MRP2 p.G693R mutant showed decreased expression and mislocalization in three cell lin