PFIC2 and ethnicity‐specific bile salt export pump (BSEP, ABCB11) mutations: where do we go from here?

Bile formation is a major function performed by the liver and bile secretion is critical for the absorption of fats and fat-soluble vitamins (1). Bile flow consists of bile derived from bile acid (BA)-dependent and BA-independent secretory fractions. BA-dependent fraction of bile flow is influenced significantly by the function of the basolateral (NTCP, sodium taurocholate cotransporting polypeptide, SLC10A1) and canalicular [bile salt export pump (BSEP) , ABCB11] BA transporters, which determine the intracellular BA concentrations in concert with BA synthetic enzymes (2). Therefore, it is imperative that both transporters operate normally to maintain normal bile secretion and subsequent digestion of fats. Any disruption in the function of these transporters because of changes in transcriptional and post-transcriptional mechanisms of regulation can result in abnormal bile flow, which is seen in experimental and human cholestatic diseases (3). Work from multiple laboratories including ours have shown the role played by Farnesoid X receptor (FXR, NRH14) in transcriptional regulation of BSEP and NTCP (4, 5). Post-transcriptional mechanisms that result in increased insertion/retrieval of transporters and/or by post-translational mechanisms (such as phosphorylation) have also been demonstrated (6). Progressive familial intrahepatic cholestasis (PFIC) refers to multiple syndromes in which patients suffer from cholestatic episodes because of inherited defects in a few genes some of which have been discovered during the last decade. PFIC1 (normal GGT) and PFIC3 (high GGT) are because of mutations in the genes encoding ATP8B1 (a P4 type ATPase, that flips phosphatidyl serine from the outer to inner hemileaflet) and MDR3 (a multidrug resistant transporter) that flips phosphatidyl choline from the inner to the outer leaflet respectively. In contrast, PFIC2 (normal to low GGT) is caused by mutations in BSEP, an ATP-binding cassette (ABC) transporter that secretes conjugated divalent bile salts into bile utilizing the energy derived from the hydrolysis of ATP. Analysis of mutations in Caucasians, Africans, Arab and Taiwan Chinese patients suffering from PFIC2 have been conducted over the last several years. These studies revealed mutations that included missense, nonsense and single nucleotide polymorphisms (SNPs) suggesting multiple genetic defects in ABCB11 gene can cause liver disease. However, to date very few studies have been conducted in other ethnic groups (such as Chinese from China) leaving open the possibility that further unknown mutations could be discovered. It is exactly that possibility that has been explored in the report appearing in this issue by Li-yan Liu et al. (7). These studies reveal that PFIC2 patients in China (thus representing this Asian ethnic subgroup) are carriers of ABCB11 mutations that are entirely different from those discovered in other populations. This interesting report has thus raised the question as to what is the significance of such ethnicity-specific mutations and its implications for treatment of PFIC2 cholestasis. In their study, the authors found a total of 12 mutations in ABCB11 gene. Among them were three nonsense, six missense, two splicing and one intronic mutation. In a recent study, Byrne et al. (8) analysed a total of 42 variants (37 missense and five SNPs) encoding mutant BSEP proteins where altered amino acids were putatively incorporated. Protein expression abundance was examined using insect cells and functional analysis was conducted with the help of transport studies using radioactively labelled BA tracers. It is imperative that such studies need to be conducted by expressing the mutant proteins produced by the mutations reported in this study and what their effect is on the protein as well as the BA transport function mediated by the mutant BSEPs. In addition, splicing mutations also need to be experimentally studied using the in vitro minigene vector constructs containing additional domains such as those described by Byrne et al. (8) to prove that these mutations do indeed affect the nature of the BSEP mRNA. A note also may be added about splice prediction programs (such as ESE finder and SPLICE prediction by Neural Network) that the authors have used in this study. Although these programs can to some extent predict what type of splicing (alterations) ‘may’ occur, further testing using in vitro mutagenesis, minigene vector construction and identification of spliced products as demonstrated by Byrne and colleagues are essential. Even with such experimentation, it still remains plausible that these in vitro splicing events may not truly reflect what happens in vivo in the native liver. Without such further analysis, it is impossible to predict how the mutations in these patients lead to cholestasis as evidenced by clinical findings. The only previous study that reported mutations in BSEP from Southeast Asians are those of Chen et al. (9) in Taiwan Chinese. Mutations reported in this study except one (R303K) were unique to this population