Exon-skipping and mRNA decay in human liver tissue: molecular consequences of pathogenic bile salt export pump mutations

Sci Rep. 2016 Apr 26:6:24827. doi: 10.1038/srep24827.

Abstract

The bile salt export pump BSEP mediates bile formation. Over 150 BSEP mutations are associated with progressive familial intrahepatic cholestasis type 2 (PFIC-2), with few characterised specifically. We examined liver tissues from two PFIC-2 patients compound heterozygous for the splice-site mutation c.150 + 3A > C and either c.2783_2787dup5 resulting in a frameshift with a premature termination codon (child 1) or p.R832C (child 2). Splicing was analysed with a minigene system and mRNA sequencing from patients' livers. Protein expression was shown by immunofluorescence. Using the minigene, c.150 + 3A > C causes complete skipping of exon 3. In liver tissue of child 1, c.2783_2787dup5 was found on DNA but not on mRNA level, implying nonsense-mediated mRNA decay (NMD) when c.2783_2787dup5 is present. Still, BSEP protein as well as mRNA with and without exon 3 were detectable and can be assigned to the c.150 + 3A > C allele. Correctly spliced transcripts despite c.150 + 3A > C were also confirmed in liver of child 2. In conclusion, we provide evidence (1) for effective NMD due to a BSEP frameshift mutation and (2) partial exon-skipping due to c.150 + 3A > C. The results illustrate that the extent of exon-skipping depends on the genomic and cellular context and that regulation of splicing may have therapeutic potential.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 11 / genetics*
  • ATP Binding Cassette Transporter, Subfamily B, Member 11 / metabolism
  • Alleles
  • Base Sequence
  • Child, Preschool
  • Cholestasis, Intrahepatic / genetics*
  • Cholestasis, Intrahepatic / pathology
  • Codon, Nonsense
  • DNA Mutational Analysis
  • Exons
  • Frameshift Mutation
  • Genetic Vectors / genetics
  • Genetic Vectors / metabolism
  • Hep G2 Cells
  • Heterozygote
  • Humans
  • Introns
  • Liver / metabolism*
  • Liver / pathology
  • Male
  • Microscopy, Fluorescence
  • RNA Splice Sites
  • RNA Splicing
  • RNA, Messenger / chemistry
  • RNA, Messenger / metabolism
  • gamma-Glutamyltransferase / metabolism

Substances

  • ATP Binding Cassette Transporter, Subfamily B, Member 11
  • Codon, Nonsense
  • RNA Splice Sites
  • RNA, Messenger
  • gamma-Glutamyltransferase

Supplementary concepts

  • Cholestasis, progressive familial intrahepatic 2