Evaluation of a Novel Missense Mutation inABCB4Gene Causing Progressive Familial Intrahepatic Cholestasis Type 3

Abstract

Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a hepatic disorder occurring predominantly in childhood and is difficult to diagnose. PFIC3, being a rare autosomal recessive disease, is caused by genetic mutations in both alleles of ABCB4, resulting in the disruption of the bile secretory pathway. The identification of pathogenic effects resulting from different mutations in ABCB4 is the key to revealing the internal cause of disease. These mutations cause truncation, instability, misfolding, and impaired trafficking of the MDR3 protein. Here, we reported a girl, with a history of intrahepatic cholestasis and progressive liver cirrhosis, with an elevated gamma-glutamyltransferase level. Genetic screening via whole exome sequencing found a novel homozygous missense mutation ABCB4:c.1195G>C:p.V399L, and the patient was diagnosed with PFIC3. Various computational tools predicted the variant to be deleterious and evolutionary conserved. For functional characterization studies, plasmids, encoding ABCB4 wild-type and selected established mutant constructs, were expressed in human embryonic kidney (HEK-293T) and hepatocellular carcinoma (HepG2) cells. In vitro expression analysis observed a reduced expression of mutant protein compared to wild-type protein. We found that ABCB4 wild type was localized at the apical canalicular membrane, while mutant p.V399L showed intracellular retention. Intracellular mistrafficking proteins usually undergo proteasomal or lysosomal degradation. We found that after treatment with proteasomal inhibitor MG132 and lysosomal inhibitor bafilomycin A1, MDR3 expression of V399L was significantly increased. A decrease in MDR3 expression of mutant V399L protein may be a result of proteasomal or lysosomal degradation. Pharmacological modulator cyclosporin A and intracellular low temperature (30°C) treatment significantly rescued both the folding defect and the active maturation of the mutant protein. Our study identified a novel pathogenic mutation which expanded the mutational spectrum of the ABCB4 gene and may contribute to understanding the molecular basis of PFIC3. Therefore, genetic screening plays a conclusive role in the diagnosis of rare heterogenic disorders like PFIC3.

A female case of progressive familial intrahepatic cholestasis type 3 caused by heterozygous mutations of ABCB4 gene and her cirrhosis improved after treatment of ursodeoxycholic acid

“…Preventing the toxic damage of bile salt plays an important role in protecting the liver [20]. ABCB4 gene mutations can cause the truncation, instability, misfoldin of the MDR3 [21] which leads to abnormal transport of phospholipid on different levels. The lack of phospholipids and phosphatidylcholine in bile results in bile salt released from bile ingredients, the increase in the ratio of cholesterol to phosphatidylcholine, easy formation of cholesterol crystals and obstruction of small bile duct [22].…”

Case series of progressive familial intrahepatic cholestasis type 3: Characterization of variants in ABCB4 in China

“…More than 40 different ABCB4 gene mutations have been reported, including missense mutations, senseless mutations, deletion mutations, and insertion of small fragments of bases ( 32 ). Different types of pathogenic mutations in ABCB4 (≥ 70% missense) lead to distinct clinical outcomes and are associated with different cumulative risks ( 33 ). Delaunay et al devised a classification system for the various forms of these mutations.…”

Influencing factors of hyperuricemia and gene analysis of familial gout among the elderly in Shijing Town, Nan'an City, Fujian Province

“…High-risk factors for the disease include age, females, obesity (especially central obesity), blood lipids, type 2 diabetes, etc. [15][16][17]. This variant is a rare variant, which is not included in the gnomAD population database and the local population database.…”