The impact of genetic mutations on protein trafficking and anchoring in the canalicular membrane has also been investigated for BSEP, the major transport system for bile salts. Mutations in the ABCB11
gene, which encodes BSEP, results in low γ-glutamyl transpeptidase (γGTP) cholestasis and manifest as either a mild clinical syndrome of recurrent symptoms known as BRIC-2 or a more severe disease known as PFIC-2, akin to the phenotypes described for FIC1 deficiency, without extrahepatic manifestations (). In experiments designed to examine how mutations change the biology of BSEP, mutant proteins encoded by genetic mutations found in patients with PFIC-2 were expressed in cell lines and were demonstrated to be retained in the ER at variable degrees and processed by the ER-associated degradation machinery [14
]. Another study added insight into how different ABCB11
missense mutations and single nucleotide polymorphisms (SNPs) influence BSEP expression. The investigators found that most mutations/SNPs resulted in aberrant pre-mRNA splicing, retention in the ER, increased degradation, and lowered canalicular expression of the protein [15
]. Further dissection of the molecular consequences of disease-causing mutations will enable the development of new therapies based on the genetic makeup of the patient, ultimately aiming at restoring the transport of bile acids.
The recessive mode of inheritance for BSEP deficiency implies that mutations affect both alleles. This biological setting was supported by a comprehensive DNA sequence analysis of 109 families with BSEP deficiency, which identified 82 different biallelic mutations [16••
]. It is notable that seven families carried only a single heterozygous mutation, but it remains unclear how a single allele mutation produces a clinical phenotype. Interestingly, 93% of the mutations produced abnormal or absent BSEP expression on liver biopsies; immunostaining identified a variable pattern of BSEP expression in patients carrying the most common E297G or D482G mutations, thus limiting the use of immunohistochemistry to reliably pinpoint BSEP deficiency. This study also showed how the biologic consequences of BSEP deficiency might be far more reaching than previously recognized, with the development of hepatocellular carcinoma or cholangiocarcinoma in 15% of the patients [16••
]. Earlier reports had described liver tumors in patients with ABCB11
mutations, but the strength of the association was unknown [17
]. In the cohort of 109 families, a higher incidence of malignancy (35%) was linked to patients carrying biallelic protein-truncating mutations (vs 10% with less severe genotypes) [16••
]. These findings emphasize the need to maintain close surveillance for the development of malignancy in subjects with chronic cholestasis due to BSEP deficiency.
The evolution of liver disease in children with BSEP can be rapid, reach end-stage cirrhosis, and require transplantation for long-term survival. Transplantation is curative, but a new report described the recurrence of cholestatic liver disease following transplantation because of the development of autoantibodies against BSEP [19••
]. In this report, the child had three homozygous nonsynonymous nucleotide changes that produced incomplete expression of the protein or improper clearance in the ER-associated degradation pathways. Re-transplantation was required because of progressive cholestasis. After observing recurrence of cholestasis in the second functional graft, the investigators uncovered the presence of autoantibodies in the patient’s serum, which recognized the first extracellular loop of the BSEP protein. This new mechanism of recurrence of disease was reported by a different group of investigators in three patients with recurrence of low γGTP cholestasis and giant cell transformation (with no evidence of cellular rejection) following liver transplantation for BSEP deficiency [20••
]. These patients were also shown to have antibodies that recognized BSEP. Together, the findings provide a rationale for the development of trials that assess whether a change in the type of immunosuppression to decrease the production of antibodies (eg, anti-CD20 antibodies) may be more beneficial to patients than an intensification of standard drugs to modulate T-cell function (eg, calcineurin inhibitors) in transplanted patients.