Novel mutations in the Dubin–Johnson syndrome gene ABCC2/MRP2 and associated biochemical changes

Abstract

A patient with sepsis and jaundice was admitted for diagnosis and treatment. Associated biochemical changes included increased C-reactive protein, conjugated bilirubin and gamma-glutamyltransferase, the duration of which was protracted. High urine coproporphyrin isomer-1 and immunostaining of liver tissue suggested Dubin–Johnson syndrome. DNA sequencing using polymerase chain reaction amplification of the ABCC2 gene revealed the patient to have a compound heterozygous variant of MRP2, a molecule involved in canalicular transport of bilirubin. There was a history of jaundice since infancy.

Case report

A 54-year-old man was referred with a history of vomiting, acute abdominal pains, stress and yellow pigmentation of skin and sclerae. He was a non-smoker; his stated alcohol intake was negligible; he was not prescribed analgesics or other medications; he denied over-the-counter, herbal or homeopathic products; he had not travelled abroad; and he had no history of allergy. The abdomen was soft with no pain, bowel sounds were present, there was no palpable mass, no pruritus, the pulse was normal and there was no history of rectal bleeding. The body mass index was 26 kg/m², blood pressure 150/100 mmHg and body temperature 37.5°C. The respiratory and neurological examination was normal. X-rays, ultrasound and computed tomography were performed. Laboratory tests included blood biochemistry and haematology, urinalysis and histopathological staining of the liver tissue.

Plasma bile acids and urine cholanoids (bile acids and alcohols) were measured by electrospray ionization tandem mass spectrometry. After informed consent, blood from the proband and his mother was obtained for molecular genetic studies of the ABCC2/MRP2 gene. MRP2 is a canalicular multi-specific transporter, expressed on apical membranes, that carries organic anions and bilirubin out of cells. It is a member of the ABCC family located on chromosome 10q24. ¹ Genomic DNA was harvested from whole blood using a Gentra Puregene Kit (Qiagen, Courtaboeuf Cedex, France). MRP2 gene screening was performed using polymerase chain reaction (PCR) amplification, and DNA sequencing of all 32 coding exons and all splice junctions with Big Dye Terminator Chemistry (Applied Biosystems, Applera S.A., Courtaboeuf Cedex, France) in a 3130xl Genetic Analyzer (Applied Biosystems). Identification of alterations was assessed by sequence comparisons with SeqScape software (version 2.5; Applied Biosystems).

Results

On admission, abdominal X-rays and ultrasound showed no focal pathology of the common bile duct, kidney, spleen or pancreas. Urine appeared slightly dark, with a pH 6.0, protein ++, ketone ++, glucose, blood and leukocytes negative and nitrite positive. A urinary tract infection was suspected. Antibiotics were withheld awaiting culture. C-reactive protein (CRP) was 114 mg/L (reference interval <6.0), bilirubin 110 μmol/L (reference interval <22), gamma-glutamyltransferase (GGT) 117 U/L (reference interval <90), prothrombin time 15.1 s (reference interval 11.5–14.5) and Clauss fibrinogen 6.46 g/L (reference interval 1.50–4.50). White blood cell count was 3.44 × 10⁹/L (reference interval 3.60–11.00), and lymphocytes (15%) and monocytes (3.6%) were slightly reduced. Serum B12, folate, other haematology, biochemistry and lipids were within appropriate reference intervals. Serum tests for cytomegalovirus, Epstein–Barr virus and hepatitis viruses were negative. Four days after admission, he was clinically well and discharged home with a diagnosis of sepsis-induced jaundice. However, because of persistent jaundice, he was referred to the gastroenterology clinic.

When seen at the clinic, he was clinically well and symptom-free. Bilirubin remained increased at 59 μmol/L (Figure 1), of which conjugated bilirubin was 40 μmol/L (reference interval 0–8 μmol/L). Mitochondrial, smooth muscle, gastric cell, parietal cell, liver, kidney and microsomal antibodies were negative. Blood film was normal and serum haptoglobin 0.85 g/L (reference interval 0.13–1.63).

Figure 1

Dubin–Johnson syndrome and biochemical changes during and after sepsis (y-axis). Ursodeoxycholic acid when given between 2.3 y and 2.7 y (x-axis) was associated with a reduction in serum GGT but not in TBIL or CBIL. Total bilirubin ( TBIL, μmol/L), gamma-glutamyl transferase ( GGT, U/L), conjugated bilirubin ( CBIL, μmol/L), C-reactive protein ( CRP, mg/L)

Liver biopsy showed no evidence of fibrosis, inflammation, excess iron or copper-associated protein, and immunostaining excluded alpha-1-antitrypsin deficiency. Extensive brown pigmentation was present in perivenular hepatocytes. ² Immunolabelling of the tissue was positive for MRP3 (a basolateral transport protein), indicating the possibility of Rotor's syndrome (RS) or Dubin–Johnson syndrome (DJS). Because of insufficient tissue, MRP2 immunolabelling could not be performed. The patient was started on ursodeoxycholic acid (UDCA) 250 mg twice daily for a trial period ^3,4 and monitored biochemically. Urine cholanoids were negative (i.e. no evidence of an inborn error of bile acid synthesis). Likewise, plasma bile acids were not increased (i.e. no evidence of an inborn error of bile acid metabolism or of a peroxisomal disorder).

The half-lives of the following were: CRP 9 h (reference <20 h), GGT 246 days (reference <26 days) and bilirubin 158 days (reference <20 d) (Figure 1). The calculations were performed during the decreasing phase using the equation C = C ₀e ^−κt , where C ₀ is the initial concentration, C the concentration at time t and κ the elimination rate constant. No change occurred in total or conjugated bilirubin, or alkaline phosphatase while the patient was prescribed UDCA. Serum total bile acids increased from 13 to 28 μmol/L, and decreased to 17 μmol/L on cessation of therapy (Figure 1). GGT decreased from 51 to 22 U/L while alanine transaminase decreased from 26 to 19 U/L on UDCA. Both enzymes reverted to their original activities off therapy. Immediately postsepsis, total bilirubin was 54–64 μmol/L and conjugated 22–27 μmol/L (68–70%), the latter decreasing to 39–45% (mean = 41 μmol/L) at all other times. Serum iron was 29–35 μmol/L (reference interval 12–31). Urine porphobilinogen was <3.1 μmol/L (reference interval <10.2). Spectrophotometry of acidified urine revealed a porphyrin peak (404 nm) with another pigment band at 490 nm. Total porphyrin/creatinine ratio was 24–34 nmol/mmol (reference interval <34). High-performance liquid chromatography analysis showed the stereospecific carboxylated coproporphyrin isomer-I at 80–85% (UCP-I, RR ≤ 30%) and isomer-III at 7–18% (UCP-III, RR ≥ 70%) and UCP-III/UCP-I ratio 0.09 (RR = 1.0–3.6). ^5–8

DJS and RS are inherited conjugated hyperbilirubinaemias; in DJS, the non-enzymatically derived UCP-I (>80%) is higher than in RS (40–65%). Furthermore, the liver lacks brown pigment in RS and has normal MRP2 gene expression. Immunostaining of this patient's liver tissue excluded MRP3 transporter defect and therefore of progressive familial intrahepatic cholestasis. MRP3 is a basolateral transport protein associated with efflux of amphiphilic anion conjugates including bile acids into the blood.

Table 1 shows the forward and reverse primer sequences used for PCR amplifications of all coding exons and splice junctions of the ABCC2 gene (NG_011798.1; NM_000392.3; NP_000383.1). This is the first report of the use of these primer sequences. Our patient was found to have two compound heterozygous variants: NM_000392.3:c.821_822del in exon 7 and NM_000392.3:c.1135C>A in exon 9. The first variant leads to a reading frameshift of the protein MRP2 (NP_000383.1:p.Pro274ArgfsX19) and the mRNA produced might be targeted for nonsense-mediated decay. The second leads to a missense variant of the protein MRP2 (NP_000383.1:p.Gln379Lys). This variant is located in the trans-membrane region TM7 of the MRP2; according to protein prediction software, it may be associated with damage. Finally, the patient had altered MRP2 protein that we predicted should not be seen by immunochemistry of paraffin-embedded liver biopsy slides. The proband's mother presented only the heterozygous missense variant of the MRP2 protein (NM_000392.3:c.1135C>A in exon 9; thus leading to NP_000383.1:p.Gln379Lys, located in the TM7 trans-membrane domain). Her MRP2 protein might be seen by immunochemistry of paraffin-embedded slides of liver because of a possible tolerance of the change of glutamine resulting in a functional protein; all routine serum and urinary biochemistry were normal.

Table 1

Identification of Dubin–Johnson syndrome variants using forward and reverse primer sequences in the polymerase chain reaction amplifications of all coding exons and splice junctions of ABCC2/MRP2 gene (NG_011798.1; NM_000392.3; NP_000383.1)

Forward primers	Reverse primers
ABCC2E1SF 5′ATGGTTGGGATGAAAGGTCA3′	ABCC2E1SR 5′AAGGCAATTTTGCGACTAGC3′
ABCC2E2SF 5′GTGCTGCAGGGTGGTTTT3′	ABCC2E2SR 5′TGCCCTTTGTGTTTAGGGAA3′
ABCC2E3SF 5′TATCCATCACCGGAAACCAT3′	ABCC2E3SR 5′ATTTGTTTCACCCCATTCCA3′
ABCC2E4F 5′TTCAACCTGGCATCTGTGTG3′	ABCC2E4R 5′GCTCAACTTGATGCCATTTA3′
ABCC2E5SF 5′TACGGGCCATGTAGACTTCC3′	ABCC2E5SR 5′ACCTTATTCTGGGCTTGTGG3′
ABCC2E6F 5′GGGTCTCCAAATAAACACAT3′	ABCC2E6R 5′AGCCAAGATCACACCATTGC3′
ABCC2E7SF 5′AAAACAAAGATGCCACAGGG3′	ABCC2E7SR 5′TGCACTGAGAAGTATGAAGTG3′
ABCC2E8SF 5′TTCAACATAGCTTTTGGGGG3′	ABCC2E8SR 5′CCTGCTTGCATACACAGTG3′
ABCC2E9SF 5′TGGCTGTGCATGAGGAGA3′	ABCC2E9SR 5′CCTCTCCACCCATTGTCTGT3′
ABCC2E10SF 5′GGAGCACATCCTTCCATTGT3′	ABCC2E10SR 5′GATGGTAGAAAGTCTTCCACC3′
ABCC2E11SF 5′GATGTCTGCAGCAAACCTGA3′	ABCC2E11SR 5′AGGGACAGGAGGACATGAA3′
ABCC2E12F 5′ATCTTAAAACATGGGTGGATCA3′	ABCC2E12R 5′GCCTAGAGATGCCAGCTAGTCT3′
ABCC2E13SF 5′TAGCACAATGCTGCTTGGTC3′	ABCC2E13SR 5′ACTGGGGCTCAGAAAACTCA3′
ABCC2E14SF 5′AATAAGAGAAAGCCAGGGGC3′	ABCC2E14SR 5′CTTCCCAATCCCACACACTT3′
ABCC2E15SF 5′TAGGAGCCAGCACTTAGCA3′	ABCC2E15SR 5′TGCACTACCTCCATCCACAA3′
ABCC2E16SF 5′AGGGCTCATTAGGGCTCATT3′	ABCC2E16SR 5′ATTCCCAAATCTTACCCCCAG3′
ABCC2E17SF 5′TGGGGCTTTTAATGGTGAAG3′	ABCC2E17SR 5′GTGTAGTTCTTCACCACCATC3′
ABCC2E18SF 5′TTCTTCCTTTTACCCCTCCC3′	ABCC2E18SR 5′CCCACCCCACCTTTATATC3′
ABCC2E19SF 5′GGTGGACATATGGTAATCAACA3′	ABCC2E19SR 5′TCTCTGGGTAGTTTGCTTCCA3′
ABCC2E20SF 5′CAGCAAGATCAGAGGAGGCT3′	ABCC2E20SR 5′CTGACACAACCCCGATCTTT3′
ABCC2E21SF 5′TGCAAGAAGACCCTTGGGA3′	ABCC2E21SR 5′CTTGGTAGAAAGCCCACAGC3′
ABCC2E22SF 5′CTGGCTTGTCCCAATCTGTT3′	ABCC2E22SR 5′CTGAGTCCAGGAAAAGTACA3′
ABCC2E23SF 5′GGGATTCCTGTGCATGGT3′	ABCC2E23SR 5′CTCCCTGGCTTGCTTCTATG3′
ABCC2E24SF 5′TGGGAACACACAGAATCCAA3′	ABCC2E24SR 5′TCCAGAGCCAATCTCAAAGTA3′
ABCC2E25SF 5′CCTTTTGGGGACCTTTTCTC3′	ABCC2E25SR 5′CCCAAAGTACACACATGGG3′
ABCC2E26F 5′ATGTTCTGTGAACGCCAAGG3′	ABCC2E26R 5′GGGTTTAAGAAGGAATCAAG3′
ABCC2E27SF 5′CCAGCTTTAGTCCCAAACCA3′	ABCC2E27SR 5′GTATGTTGCAAGACCCGAC3′
ABCC2E28SF 5′GTGGACTGTTCGGCTGAGTT3′	ABCC2E28SR 5′CCTTCCCTCTGATACTGTG3′
ABCC2E29F 5′TGCCTCTTACCTCCTGTGAC3′	ABCC2E29R 5′CCCGAGTAGACCGTGGAAT3′
ABCC2E30SF 5′AGGTCCTTTTCTGGCATGAG3′	ABCC2E30SR 5′TCACATCCTCTCATTGCCTG3′
ABCC2E31SF 5′ACGGCAACCAAATACAAAGC3′	ABCC2E31SR 5′CACTGTTACTGTTGAGCAAGG3′
ABCC2E32SF 5′CCTAGACTTGAGATGCTGC3′	ABCC2E32SR 5′GGTGGGTAGCAAGGAAGTA3′

Discussion

This patient presented with acute symptoms of jaundice. Associated biochemical changes included increased bilirubin and liver enzymes. Gilbert's syndrome was suspected. ⁹ Normal serum haptoglobin and blood-film excluded haemolysis; clinical and imaging investigations did not show biliary obstruction. Low-grade conjugated hyperbilirubinaemia persisted. This can occur in hepatitis, malignancy, cirrhosis and in rare inherited causes, RS and DJS. RS was excluded in our patient based on liver histology and UCP-I isomer >80%. ⁸ Sepsis and DJS in our patient resulted in the slow clearance of conjugated bilirubin. The reason for this is uncertain, although mutant MRP2 transporter was probably the rate-limiting step in conjugation. (Serum conjugated bilirubin was not available during the acute phase of illness; therefore, its percentage increase during this period is unknown.) For around one year after acute illness, it remained higher compared with the basal levels. Even the presence of albumin-bound delta-bilirubin does not explain this observation. An upregulated MRP3 promotes efflux of conjugated substances back into the circulation. Imbalance between the MRP2 and MRP3 (HGNC:54) transporters may contribute to slow clearance of conjugated bilirubin. Excess pigment deposited in the hepatocytes could be due to this stagnant period of shunt hyperbilirubinaemia. Increase in UCP-I stereoisomer is perhaps a protective mechanism that buffers the liver from the enzymatically derived intermediary tetrapyrrole metabolites at the expense of tissue deposits. Also, compound heterozygote MRP2 that includes a frameshift mutation may have played a role in the slow rate of formation or expression of enzymes involved in the cyclization of hydroxymethylbilane, including slow clearance of conjugated bilirubin.

DJS patients show a wide range of serum total bilirubin concentration. ¹⁰ UDCA induces MRP2 expression in children and reduces the degree of hyperbilirubinaemia. ⁵ However, this effect was not apparent in our patient, possibly due to loss-of-function of MRP2 transporter protein. Furthermore, there was a reduction in GGT and ALT while the patient was on UDCA, reduction to a level not observed at any other time.

This is the first case of DJS in the UK describing the biochemical measurements and the genotype analysis (MIM:601107). The presence of missense variant p.Glu379Lys in the maternal blood did not result in the phenotype. However, the same missense variant together with p.Pro274ArgfsX19 frameshift variant of the ABCC2 gene resulted in the DJS phenotype (MIM: 237500) in our proband. Since the original DJS report in 1954, there have been sporadic case reports, and its prevalence in consanguinous marriage in some Jewish and Japanese groups is high. ^11,12 Isomers of coproporphyrin in urine are readily distinguishable, and their ratio can be used as a biomarker of MRP2/ABCC2 activity. ⁶ Liver biopsy and radionuclide scintiscanning are employed to diagnose DJS phenotype. ¹³ However, a non-invasive UCP isomer ratio method may also help.

Over 45 MRP2 mutations are reported; however, only 15 are known to result in the DJS phenotype. We used a novel set of primers for PCR amplification to identify our proband as compound heterozygote. DJS should be considered when commoner causes of persistent hyperbilirubinaemia have been excluded. In our patient, it resulted in repeated hospitalization, costly clinical examinations, and patient and family distress. The condition appears to be benign and does not require medication when the patient is asymptomatic.

DECLARATIONS

Competing interests: None.

Funding: None.

Ethical approval: Not applicable.

Guarantor: MSD.

Contributorship: MSD was involved in conception and study design, biochemical data analysis and interpretation and writing the manuscript. AME-N was involved in conception and study design, clinical measurements, data analysis and interpretation, and critical revision of the manuscript. GJO'D performed liver biopsy examinations, data interpretation and provided manuscript advice. VB carried out genetic study design, data analysis and interpretation and critical revision of the manuscript. RP was involved in drug trial design, data interpretation and critical revision of the manuscript. All authors approved the manuscript.

Acknowledgements: For help, support, advice and confirmatory tests performed during this study, our thanks to: Dr S Mathie, Wishaw General Hospital; Professor A D Burt, Newcastle University; Dr A Knisely, King's College Hospital, London; Professor P T Clayton, Institute of Child Health, London; Dr M Badminton, Cardiff; and the Lanarkshire Acute Hospitals NHS Trust.