Bilirubin is the end product of heme catabolism, which is produced primarily from the breakdown of erythrocyte hemoglobin in the reticuloendothelial system. There are several major steps in the hepatic clearance of bilirubin, including hepatocytes ingesting and storing unconjugated bilirubin, the conjugation of bilirubin to bilirubin glucuronides, the excretion of conjugated bilirubin into bile, and hepatocytes resorbing the conjugated bilirubin (1). Hyperbilirubinemia is not only induced by increased bilirubin synthesis, but it can also be caused by decreased bilirubin clearance (2). There are several inherited disorders, which can contribute to hyperbilirubinemia (3), including Dubin-Johnson syndrome (DJS), Crigler-Najjar syndrome, Gilbert syndrome and Lucey-Driscoll syndrome. As a rare autosomal recessive disorder, DJS is characterized by predominantly conjugated hyperbilirubinemia without progression to end-stage liver disease (4–6).

To date, there are several genes, which have been identified to contribute to hyperbilirubinemia, including UGT1A1, SLCO1B1/OATP1B1, SLCO1B3/OATP1B3, MRP2/ABCC2 and ABCG2/BCRP (1,7,8). At present, at least 24 ABCC2/MRP2 point mutations have been reported in DJS, a number of which are predicted to result in truncated proteins. In the present study, the possible causative gene was investigated in a patient with hyperbilirubinemia. The results revealed two novel mutations (c.1303A>C/p.T435P and c.1326G>A/p.W442X) in exon 10 of ABCC2. To the best of our knowledge, these mutations have not been reported in previous studies, neither have they been presented in the single nucleotide polymorphism (dbSNP) databases (https://www.ncbi.nlm.nih.gov/projects/SNP/) and Exome Variant Server databases (http://evs.gs.washington.edu/EVS/).

The present study reported on a patient with hyperbilirubinemia with a total bilirubin level of 30.6 µmol/l and direct bilirubin of 10.5 µmol/l, whereas the reference standard values are 5.1–17.1 and 0–6.0 µmol/l, respectively. The possibility of the induction of hyperbilirubinemia by known genes was investigated. Using Sanger sequencing, a missense mutation (c.1303A>C/p.T435P) and a nonsense mutation (c.1326G>A/p.W442X) in ABCC2 were identified and co-segregated with the affected members. (Fig. 1B and C). The allelic segregation analysis revealed that the missense mutation was carried by the mother, whereas the nonsense mutation was inherited from the father. These newly identified missense mutations c.1303A>C and c.1326G>A were not found in a cohort of 200 controls, as described in our previous study (9). In addition, these two mutations were not present in the dbSNP and Exome Variant Server databases. In humans, macaques, cats, mice and zebrafish, the amino acid sequences of ABCC2 were found to be aligned, which revealed that the affected amino acids were evolutionarily conserved (Fig. 2A and B). Three programs were used for analyzing the protein functions of ABCC2; polyphen2, SIFT and Mutation Taster, predicted that the two variants were likely to be damaging, deleterious and disease-causing, respectively. The consistent findings of the detrimental effects of the variants by all these bioinformatics programs suggested that these mutations are important in the function of ABCC2.

The present study presented a case of hyperbilirubinemia associated with a compound heterozygous mutation (c.1303A>C/p.T435P and c.1326G>A/p.W442X) in exon 10 of ABCC2. A previous study found that mutations in ABCC2 may cause DJS, and this syndrome was characterized by biphasic, predominantly conjugated, hyperbilirubinemia. At present, ~17 point mutations of ABCC2 have been reported in patients with DJS (Fig. 3). The outcome of the molecular genetic investigations performed in the present study was consistent with and confirmed the clinical diagnosis of DJS.

Under normal conditions, hepatocytes take up unconjugated bilirubin by transporters of the organic anion-transporting polypeptide family, followed by conjugation with glucuronic acid and ATP-dependent transport into bile. This efflux across the canalicular membrane is mediated by ABCC2/MRP2, which has a high affinity and efficiency for monoglucuronosyl and bisglucuronosyl bilirubin into bile. Therefore, mutations in ABCC2 may lead to DJS (13,14).

In the present study, the missense and nonsense mutations were located in a conserved membrane-spanning domain (MSD), namely MSD2, of the ABCC2 protein (15). The nonsense mutation can also lead to the absence of the complete nucleotide-binding domain (NBD)-1, MSD3 and NBD2. Mutations in MSD2 may affect the subcellular localization of ABCC2, and the truncated mutation may cause the functional defect of ABCC2. These two point mutations were present in patients, and this compound heterozygous mutation was associated with DJS recessive hereditary mode.

Among the compound heterozygous mutations in DJS, 13 cases have been reported, including that identified in the present study (5,16). At present, a total of 24 MRP2/ABCC2 point mutations have been reported, 17 of which are associated with DJS (17–19) (Fig. 3). In the last 25 years, different types of viral vectors have been used in clinical trials for the treatment of a variety of monogenetic disorders. It has been suggested that this technique may be used to treat hereditary hyperbilirubinemia (20,21), however, further investigation and improvements are required (22).

In conclusion, the present study identified an ABCC2 compound heterozygous mutation (c.1303A>C/p.T435P and c.1326G>A/p.W442X) in a patient with DJS. To the best of our knowledge, this may be the first report of these two mutations worldwide. The results of the present study offer further support for the significant involvement of ABCC2 in DJS. The results also expand on the spectrum of ABCC2 mutations, and contribute to the genetic diagnosis and counseling of families with DJS.