Study population
From the China Registry of Genetic/Metabolic Liver Diseases (ClinicalTrials.gov identifier, NCT03131427), a total of 14 patients suspected with DJS, who had biochemical evidence of fluctuating predominantly conjugated hyperbilirubinemia with or without family history, were initially included in the present study between June 2015 and December 2017. Whole blood samples from the 14 patients were collected and stored at -20˚C for Sanger sequencing. 7 out of the 14 patients had ABCC2 gene mutations, and 2 of the 7 patients had ABCC2 p.G693R mutation [7].
The study was conducted in accordance with the Declaration of Helsinki. The Clinical Research Ethics Committee of Beijing Friendship Hospital, Capital Medical University approved the study protocol (No. 2019-P2-217-02). All patients provided written informed consent.
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.
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.
Analysis of mutation in other known hyperbilirubinemia genes by whole exome sequencing
Approximately 1 μg of genomic DNA was used to construct a whole exome library with an insert size of 150–200 bp by an exome capture strategy using a GenCap custom exome enrichment kit (MyGenostics, Beijing, China). Paired-end 100 bp raw reads from each enriched library were generated with an Illumina HiSeq 2000 platform (Illumina, San Diego, USA) according to the manufacturer’s protocol. The paired-end reads were aligned against NCBI build 37 of the human genome using Burrows Wheeler Aligner. With the GenomeAnalysis Toolkit (GATK4.1.2.0, https://software.broadinstitute.org/gatk/download/), duplicate reads were marked; local indel realignment was performed, and base quality scores were recalibrated for each sample. The identified potential pathogenic variants were confirmed by Sanger sequencing.
Polyphen-2 (http://genetics.bwh.harvard.edu/pph2), SIFT (https://sift.bii.a-star.edu.sg/) and MutationTaster (http://www.mutationtaster.org/) were used to predict the biofunctional consequence of the identified variants.
Functional analysis of ABCC2 p.G693R mutant
Construction of the ABCC2 p.G693R mutant
To create the ABCC2 wild-type plasmid, we amplified ABCC2 from human cDNA and cloned it into Hind III/Not I sites of the pcDNA3.1 vector along with the N-terminal flag tag [8]. The ABCC2 p.G693R constructs were generated using the Gene Tailor Site-Directed Mutagenesis System (Invitrogen, Waltham, MA, USA).
Cell culture and transfection
Human embryonic kidney (HEK) 293A cells and human liver cancer cell lines Huh-7 and HepG2 were obtained from the Cell Resource Center of the Chinese Academy of Medical Science (Beijing, China). The Cell lines were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100 units/ml streptomycin. Then the cells were transfected with plasmids expressing ABCC2 wild-type or ABCC2 p.G693R by Lipofectamine 3000 (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions; the culture medium was changed at 6 h after transfection. Finally, the cells were harvested 24 or 48 h after transfection.
Measurement of MRP2 expression by Western blotting
The cells were lysed in RIPA buffer with proteinase inhibitor. After centrifugation, the supernatant was used for western blot analysis. Proteins were separated by SDS-PAGE (8%) and transferred to nitrocellulose membranes; the membranes were incubated with anti-MRP2 monoclonal antibodies M2III-6 (1:200; sc-59608; Santa Cruz Biotechnology, Dallas, TX; a mouse monoclonal antibody raised against a C-terminal region of MRP2 of human origin) or anti-GAPDH antibodies (1:5000; Santa Cruz Biotechnology) overnight at 4°C, followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (1:5000 dilution; Santa Cruz Biotechnology) for 1 h at 37°C. Immunocomplexes on the membrane were visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, USA) and Image Lab Software (BIO-RAD, Hercules, USA).
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) and mouse anti-flag (#8146; Cell Signaling technology) at 4°C overnight. After three washes with phosphate-buffered saline for 5 min each, the cells were incubated with anti-rabbit Alex 647 and anti-mouse Alex 488-conjugated secondary antibodies (1:200; Invitrogen) for 1 h at room temperature. For F-actin staining, cells were incubated with FITC-conjugated phalloidin at 50 µg/ml (P5282; Sigma) for 40 minutes. Then cells were mounted on a slide in mounting medium (Molecular Probes). Finally, 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 two-sided P value of < 0.05 was considered statistically significant.