Missense mutations and single nucleotide polymorphisms in ABCB11 impair bile salt export pump processing and function or disrupt pre-messenger RNA splicing

Abstract

The gene encoding the human bile salt export pump (BSEP), ABCB11, is mutated in several forms of intrahepatic cholestasis. Here we classified the majority (63) of known ABCB11 missense mutations and 21 single-nucleotide polymorphisms (SNPs) to determine whether they caused abnormal ABCB11 pre-messenger RNA splicing, abnormal processing of BSEP protein, or alterations in BSEP protein function. Using an in vitro minigene system to analyze splicing events, we found reduced wild-type splicing for 20 mutations/SNPs, with normal mRNA levels reduced to 5% or less in eight cases. The common ABCB11 missense mutation encoding D482G enhanced aberrant splicing, whereas the common SNP A1028A promoted exon skipping. Addition of exogenous splicing factors modulated several splicing defects. Of the mutants expressed in vitro in CHO-K1 cells, most appeared to be retained in the endoplasmic reticulum and degraded. A minority had BSEP levels similar to wild-type. The SNP variant A444 had reduced levels of protein compared with V444. Treatment with glycerol and incubation at reduced temperature overcame processing defects for several mutants, including E297G. Taurocholate transport by two assessed mutants, N490D and A570T, was reduced compared with wild-type. Conclusion: This work is a comprehensive analysis of 80% of ABCB11 missense mutations and singlenucleotide polymorphisms at pre-mRNA splicing and protein processing/functional levels. We show that aberrant pre-mRNA splicing occurs in a considerable number of cases, leading to reduced levels of normal mRNA. Thus, primary defects at either the protein or the mRNA level (or both) contribute significantly to BSEP deficiency. These results will help to develop mutation-specific therapies for children and adults suffering from intrahepatic cholestasis due to BSEP deficiency. (HEPATOLOGY 2009;49:553-567.)

No Contribution of the ABCB11 p.444A Polymorphism in Japanese Patients with Drug-Induced Cholestasis

“…Therefore, individuals with the 1331CC genotype had an ABCB11 expression of 80% compared with those harboring the 1331TT genotype. Our in vitro study reinforced the results of Byrne et al (2009) and Meier et al (2006) and identified decreased protein stability as a cause of reduced protein expression. Although some missense mutations, such as the D482G, enhanced aberrant splicing shown using a minigene system, this SNP (1331T.C) did not alter the pre-mRNA splicing pattern (Byrne et al, 2009).…”

“…Our in vitro study reinforced the results of Byrne et al (2009) and Meier et al (2006) and identified decreased protein stability as a cause of reduced protein expression. Although some missense mutations, such as the D482G, enhanced aberrant splicing shown using a minigene system, this SNP (1331T.C) did not alter the pre-mRNA splicing pattern (Byrne et al, 2009). A major concern is whether a 444A protein with slightly reduced transport activity can cause acquired cholestasis.…”

“…In the mammalian cell line HeLa, Ho et al (2010) did not find any significant differences in bile acid transport activity and protein expression between 444A and 444V. In contrast, 444V was expressed to a greater extent than 444A in CHO-K1 cells (Byrne et al, 2009). Meier et al (2006) studied the hepatic expression of ABCB11 protein in 110 individuals undergoing liver resection and demonstrated a trend toward reduced expression in individuals harboring the 1331C-allele (444A) polymorphism compared with those harboring the 1331T-allele (444V).…”

“…Although we studied two independent cohorts, their sizes were relatively small and might have led to sampling errors. We also did not analyze the ABCB11 mutations that cause PFCI2 and BRIC2 (Pauli-Magnus et al, 2005;Byrne et al, 2009). Heterozygous carriers of these mutations can potentially develop druginduced cholestasis, although such cases have not been reported.…”

In Vitro Inhibition of the Bile Salt Export Pump Correlates with Risk of Cholestatic Drug-Induced Liver Injury in Humans

“…PFIC2 is a consequence of mutations in the gene encoding the human bile salt export pump (BSEP, ABCB11; rodent ortholog Bsep, Abcb11; originally termed "sister of P-glycoprotein," spgp), which mediates efflux of bile salts from hepatocytes into bile and is essential for normal bile formation and flow. In patients with PFIC2, mutations or single nucleotide polymorphisms (SNPs) in the BSEP gene result in either reduced levels of mRNA transcription or translation or reduced protein stability or activity (Strautnieks et al, 2008;Byrne et al, 2009;Ho et al, 2010), and liver injury is considered to be caused by intracellular accumulation of cytotoxic bile constituents (Perez and Briz, 2009). Less functionally severe ABCB11 gene mutations have been shown to result in benign recurrent intrahepatic cholestasis type 2, which is characterized by nonprogressive cholestasis (Noe et al, 2005).…”