ABCC2p.R393W variant contributes to Dubin-Johnson syndrome by targeting MRP2 to proteasome degradation

Dubin-Johnson syndrome (DJS), a rare autosomal recessive liver condition, is caused by biallelic loss-of-function mutations of theABCC2gene. This study aimed to investigate genetic variations in the drug efflux transporterABCC2(MRP2) gene in patients with DJS and to characterise the expression and mechanism of theABCC2gene variant.Trio whole exome sequencing was performed in the family to identify the genetic causes. Bioinformatics analysis was performed to assess pathogenicity. Inin vitroexperiments, site-directed mutagenesis was used to introduceABCC2variants in constructs then expressed in HEK293T, HuH-7 and HepG2 cell lines. The expression of total and cell membrane MRP2 was quantified in cells expressing the wild-type or variant forms. Chloroquine and MG132 were used to evaluate the effects of p.R393W on lysosomal and/or proteasomal degradation.The twin probands carry DJS-associated variants c.1177C>T (rs777902199) in theABCC2gene inherited from the father and the c.3632T>C mutation in the other allele inherited from the mother. TheABCC2variant, c.1177C>T, results in a p.R393W substitution in MRP2 that is highly conserved among vertebrates, drastically decreasing the expression of mutant protein by promoting proteasomal degradation. Another variant c.3632T>C results in a p.L1211P substitution in MRP2, decreasing the expression of membrane MRP2 but not changing the expression of total protein.These results strongly suggest that the p.R393W variant affects the stability of the MRP2 protein and decreases its expression by ubiquitin-mediated proteasomal degradation, and the p.L1211P decreases the expression of membrane MRP2, indicating that these two variants, respectively, cause a loss-of-function of the MRP2 protein and membrane MRP2 ultimately leading to DJS development.