Exon-skipping and mRNA decay in human liver tissue: molecular consequences of pathogenic bile salt export pump mutations.
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.
Department of Gastroenterology, Hepatology and Infectious Diseases, University Hospital, Heinrich Heine University, Düsseldorf, Germany.
Carola
C
Dröge
Institute of Virology, Heinrich Heine University, Düsseldorf, Germany.
Heiner
H
Schaal
Department of Pediatrics, Lukashospital, Neuss, Germany.
Guido
G
Engelmann
Department of General Pediatrics, University Hospital, Heidelberg, Germany.
Daniel
D
Wenning
Department of Gastroenterology, Hepatology and Infectious Diseases, University Hospital, Heinrich Heine University, Düsseldorf, Germany.
Dieter
D
Häussinger
Department of Gastroenterology, Hepatology and Infectious Diseases, University Hospital, Heinrich Heine University, Düsseldorf, Germany.|Medical Clinic I, Bethanien Hospital, Moers, Germany.
Ralf
R
Kubitz
D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11 / Q000235:genetics* / Q000378:metabolism; D000483:Alleles; D001483:Base Sequence; D002675:Child, Preschool; D002780:Cholestasis, Intrahepatic / Q000235:genetics* / Q000473:pathology; D018389:Codon, Nonsense; D004252:DNA Mutational Analysis; D005091:Exons; D016368:Frameshift Mutation; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D056945:Hep G2 Cells; D006579:Heterozygote; D006801:Humans; D007438:Introns; D008099:Liver / Q000378:metabolism* / Q000473:pathology; D008297:Male; D008856:Microscopy, Fluorescence; D022821:RNA Splice Sites; D012326:RNA Splicing; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D005723:gamma-Glutamyltransferase / Q000378:metabolism
D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018389:Codon, Nonsense; D022821:RNA Splice Sites; D012333:RNA, Messenger; D005723:gamma-Glutamyltransferase