Dubin–Johnson syndrome (DJS, OMIM*237500) is an autosomal recessive genetic disease which is characterized by intermittent, predominantly conjugated hyperbilirubinemia and liver pigmentation. DJS has no other features of hepatobiliary disorder. First described by Dubin and Johnson (1954), It is considered a rare disorder worldwide, although its incidence among Sephardic Jews has been reported as approximately 1/3000 (Shani et al., 1970). Pathogenic mutations in the adenosine triphosphate-binding cassette subfamily C member 2 (ABCC2) gene were first discovered to cause DJS in 1997 (Paulusma et al., 1997). The multidrug resistance-associated protein 2 (MRP2) encoded by the ABCC2 gene is principally located in the hepatocyte canalicular membrane. Loss-of-function variants in the ABCC2 gene disturb the secretion of conjugated bilirubin from hepatocytes. DJS does not affect life expectancy and usually requires no treatment (Memon et al., 2016). To date, 127 mutations in ABCC2 have been described in the Human Gene Mutation Database (HGMD; http://www.hgmd.cf.ac.uk/ac/index. php). In the present study, we reported two patients with DJS and identified three novel disease-causing mutations: c.2980delA, c.1834C>T and c.4465_4473delinsGGCCCACAG, in the ABCC2 gene (NM_000392).

In Family 1, a 29-year-old woman (Patient 1) was born to nonconsanguineous parents although her grandparents were cousins (Figure 1A). She was affected with low-grade predominantly conjugated hyperbilirubinemia for more than 6 years. This patient was tested for hyperbilirubinemia in a health physical examination in 2015. There were no other significant abnormalities in her liver function test. She received no other medical treatment then. During her pregnancy, her jaundice was markedly aggravated. She gave birth on 18 August 2018. The obstetrician used reduced glutathione (1.2 g/d, from 16 August 2018 to 20 August 2018) and ursodeoxycholic acid (500 mg/d, from 20 August 2018 to 31 August 2018) to control her jaundice. Her clinical jaundice gradually disappeared after childbirth in 2018. She took oral contraceptives from January 2021 and presented with recurrent mild jaundice again. The jaundice was in remission after she stopped taking oral contraceptives; she was never affected with pruritus. The patient had a previously free medical history. When she came to us as a medical genetic outpatient in July 2021, the physical examination revealed mild splenomegaly. The timeline with relevant data for Patient 1 is shown in Supplementary Figure S1. During the patient’s pregnancy, her level of total bilirubin (TBIL) ranged from 43.1 μmol/L to 82.8 μmol/L (normal range: 3.4–17.1 μmol/L). Her direct bilirubin (DBIL) level ranged from 25.2 μmol/L to 65.1 μmol/L (normal range: 0–6 μmol/L). The ratio of DBIL/TBIL was always greater than 50%. There were no abnormalities in other liver function indexes. She was serologically negative for hepatitis A, B, C, and E viruses. There were no abnormalities in coagulation function, autoimmune liver disease-associated antibodies, immunoglobulin G, or serum ceruloplasmin testing. The abdominal ultrasound examination revealed her portal vein diameter to be about 10 mm and the portal venous maximum velocity was 18 cm/s. The thickness of her spleen was about 49 mm and its inferior margin was about 20 mm below the costal margin. A hepatic hemangioma was also detected by the ultrasound examination (Supplementary Figure S2; Table 1). The whole-exome sequencing (WES) was conducted in July 2021, revealing that Patient 1 carried both the heterozygous c.2980delA and c.1834C>T ABCC2 mutations. Her son also shared the heterozygous c.1834C>T ABCC2 mutation (Figure 1B; Table 2). Considering her symptoms, clinical examinations, and genetic testing, she was diagnosed with DJS. Intrahepatic cholestasis of pregnancy (ICP) was excluded.

In Family 2, an 18-year-old male (Patient 2) presented with intermittent mild jaundice for more than 9 years. This patient showed intermittent jaundice from the age of nine, which would be aggravated by fatigue. He had a previously free medical history and no special personal and family history (Figure 2A). When he came as a medical genetic outpatient to our hospital, he presented with yellow skin and sclera. In the physical examination, his liver could be palpated at the lower margin of the ribs. Patient 2’s TBIL level was 110.6 μmol/L and his DBIL level was 70.4 μmol/L, with no other abnormalities in the liver function test. There were no abnormalities in autoimmune liver disease-associated antibodies or serum hepatitis B and C virus testing. A CT scan revealed hepatomegaly. The lower margin of the liver exceeded the lower costal margin (Table 1, Supplementary Figure S2). The WES indicated that Patient 2 harbored the heterozygous c.2980delA variant inherited from his mother and the heterozygous c.4465_4473delinsGGCCCACAG mutation inherited from his father (Figure 2B; Table 2). He was also diagnosed with DJS and chose to observe his jaundice (without drug interventions). In the telephone follow-up, he still complained of intermittent jaundice.

However, both patients refused the 99mTc-HIDA cholescintigraphy and liver biopsy. The candidate variants in the ABCC2 gene were validated by Sanger sequencing. The primer sequences are listed in Table A1. The methods of genetic testing are described in the supplementary materials. The family members participating in the study signed written informed consent forms. This study complied with the ethical guidelines of the Declaration of Helsinki and was approved by the ethics committee of the Second Xiangya Hospital of Central South University (2014 ethical approval No. S046).

The two patients suffered from chronic low-grade predominantly conjugated hyperbilirubinemia. There were no other features of hepatobiliary disorder. Patient 1’s jaundice increased during pregnancy and while taking oral contraceptives, which was in line with DJS (Erlinger et al., 2014). In the liver biopsy, specimens of DJS had a grossly black appearance and coarse, deep-brown, pigmented granules (Memon et al., 2016). The expression of MRP2 was absent on the hepatocellular membrane in patients with DJS (Morii and Yamamoto, 2016). The MRP2 transporter mediates the canalicular secretion of several organic anions, including many drugs, toxicants, and endogenous compounds (Keppler, 2011). Pigment accumulation is caused by the retention of anionic metabolites of tyrosine, phenylalanine, and tryptophan, which polymerized to form a similar melanin-like pigment in the liver (Kitamura et al., 1992). Postoperative 99mTc-HIDA cholescintigraphy would reveal delayed visualization of the extrahepatic bile ducts (Park et al., 2018). Most patients with DJS have a normal lifespan. Liu et al. even used a graft from a donor with DJS to conduct living donor liver transplantation. The recipient was followed for 1 year and the results were satisfactory (Liu et al., 2012). However, some drugs, such as methotrexate, vincristine, and cisplatin, are excreted from hepatocytes via the MRP2 transporter (Zhang and Fu, 2011). The mutant MRP2 transporter could affect their sensitivity and toxicity (van der Schoor et al., 2015). Therefore, adverse drug reactions need to be carefully noted for DJS patients.

The c.2980delA was a frameshift variant in the ABCC2 gene. Loss of function is a known mechanism of DJS (PVS1). In addition, the c.2980delA was absent from the controls in the 1000G and EXAC databases (PM2). The use of the MutationTaster prediction software (Schwarz et al., 2014) revealed a deleterious effect from c.2980delA (PP3). Patient 1’s phenotype was highly specific for DJS (PP4). Considering the ACMG guidance (Richards et al., 2015), we classified the c.2980delA variant as a pathogenic mutation.

The c.1834C>T mutation was not found in the 1000G or EXAC database (PM2). In addition, c.1834C>T was detected in trans with the pathogenic variant (c.2980delA) in Patient 1, which was confirmed by testing the offspring of her son (PM3). In addition, MutationTaster, PolyPhen-2 (Adzhubei et al., 2010), and SIFT (Kumar et al., 2009) predicted a disease-causing effect for the mutation (PP3). Patient 1 showed highly specific symptoms for DJS (PP4). Based on the ACMG guidelines, c.1834C>T was a likely pathogenic variant. The term “likely pathogenic” indicates a greater than 90% certainty of a variant being disease-causing. The variant had been recorded by gnomAD (with an allele frequency of 4.065e-06) and InterVar (with Uncertain clinical significance) database. But it was clarified for the first time as a likely pathogenic mutation associated with DJS.

The c.2980delA variant was also found in Patient 2. It was a pathogenic variant as analyzed above. The other mutation, c.4465_4473delinsGGCCCACAG, was not found in the 1000G or EXAC database (PM2). For a recessive disorder, c.4465_4473delinsGGCCCACAG was detected in trans with a pathogenic variant (c.2980delA), which was confirmed in his parents (PM3). The c.2980delA and c.4465_4473delinsGGCCCACAG formed a compound heterozygous mutation in Patient 2. The compound heterozygous variant was found to cosegregate in the family members (PP1). In addition, multiple lines of computational evidence supported a deleterious effect of the gene (PP3). Patient 2’s phenotype was highly specific for DJS (PP4). Therefore, c.4465_4473delinsGGCCCACAG was identified as a likely pathogenic variant according to the ACMG guidelines. By scanning the HGMD and previously published works, we concluded that the three mutations are novel pathogenic variants.

Beside the three ABCC2 mutations identified during the steps in the filtering strategies in the WES-yielded data, we also detected a heterozygous mutation, p. G71R, in the UGT1A1 gene in Patient 1 (Table A2). UGT1A1 is the disease-causing gene of Gilbert syndrome (GS), Criggler–Najjar syndrome I (CNS-I), and Criggler–Najjar syndrome II (CNS-II). However, the three diseases are autosomal recessive diseases and cause predominantly unconjugated hyperbilirubinemia. This is not consistent with the patient’s symptoms. In addition, the minor allele frequency of the variant is greater than 0.01 in the 1000G, ExAC, and gnomAD databases. Therefore, we excluded the variant. DJS and Rotor syndrome have similar symptoms, but no pathogenic variant was detected in the OATP1B1 or OATP1B3 genes, which are causative genes for Rotor syndrome. Therefore, Rotor syndrome was excluded. Furthermore, there were no candidate mutations in the ATP8B1, ABCB4, or ABCB11 genes, which could cause benign recurrent intrahepatic cholestasis (BRIC) or ICP.

MRP2 consists of three membrane spanning domains (MSDs) which include 17 transmembrane helices and two intracellular highly conserved nucleotide binding domains (NBDs). The NBD has an ATP binding site and is important for transporting some organic anions out of the hepatocyte. The transport process by MRP2 relies on the energy generated by the hydrolysis of ATP. There have been studies on genotype-phenotype correlations in DJS (Lee et al., 2006; Togawa et al., 2018; Kim et al., 2020). Mutations located in the NBD domain have been associated with early-onset DJS (Lee et al., 2006). Our two patients carried the same heterozygous c.2980delA/p.I994Lfs*29 mutation in the MSD3 domain. However, Patient 2 was affected by a heterozygous deletion-insertion mutation—c.4465_4473delinsGGCCCACAG/p.I1489_I1491delinsGPQ—in the NBD2 domain. Patient 1 carried a heterozygous missense mutation—c.1834C>T/p.R612W—in the peptide chain linking MSD2 and NBD1 (Wen et al., 2017; Xiang et al., 2017) (Figure 2C). Patient 2 carried the variant in the NBD2 domain. He suffered from early-onset and more serious hyperbilirubinemia than Patient 1, consistent with the above study. In addition, the combination of missense and truncating variants may be essential for the phenotype of neonatal DJS (Togawa et al., 2018). Patient 1 was affected by missense and truncating variants but she did not show neonatal DJS.

Because of the favorable prognosis of DJS, the two patients’ stresses were significantly relieved. Patient 1 could also avoid using oral contraceptives, which might aggravate her jaundice. DJS could not cause splenomegaly. The etiology of splenomegaly requires further analysis and follow-up. Patient 2 believed that the genetic testing could facilitate his search for work, as an unclear diagnosis of his liver disease would restrict the range of his job choices. Both patients were satisfied with the outcome of the genetic testing. However, there were several limitations in our research. The 99mTc-HIDA cholescintigraphy and biopsy could not be conducted because of the patients’ rejections. In addition, functional experiments of the novel pathogenic ABCC2 mutations need further investigation.

In conclusion, this study identified three novel pathogenic ABCC2 mutations in two patients with DJS. These results contribute to the genetic diagnosis and counseling of families with DJS and expand the spectrum of ABCC2 mutations.