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

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.

Partial external biliary diversion in bile salt export pump deficiency: Association between outcome and mutation

“…It has been shown in earlier studies that absence of BSEP from the canalicular membrane is a diagnostic feature of PFIC-2/BSEP-disease[ 18 , 25 , 26 ], which is found in up to 72% of affected patients[ 25 ]. Reduced or absent canalicular BSEP expression can for example be caused by splicing defects[ 7 , 27 , 28 ], premature stop codons or by impaired targeting to the canalicular membrane[ 5 , 16 ]. In our patients canalicular BSEP expression was well detectable in liver biopsies and targeting was apparently normal as shown by transient expression of mutated BSEP in HEK293 cells (Figure 2 ).…”

Splicing analysis of rare/novel synonymous or intronic variants identified in ABCB11 heterozygotes presenting as progressive intrahepatic cholestasis with low γ‐glutamyltransferase

“…Progressive familial intrahepatic cholestasis type 2 is an autosomal recessive disease caused by severe BSEP deficiency. At least 150 pathogenic variants have been identified in PFIC2, but only a few are synonymous or intronic variants . In this study, we identified one rare/novel synonymous or intronic variant and one pathogenic variant in each of five progressive intrahepatic cholestatic children with low GGT.…”

“…The majority of pathogenic variants identified in PFIC2 are missense, nonsense, frameshift, or canonical splice site (ss) variants; less than 10, to our best knowledge, are synonymous or intronic variants . Of them, three have been confirmed to cause severe abnormal pre‐RNA splicing by minigene splicing or liver transcripts analysis . Liver transcripts analysis and minigene splicing assay are reliable methods to reveal the effects on pre‐RNA splicing.…”

“…But liver tissue for liver transcripts analysis from the specific patient is not always available. Minigene splicing assay is more frequently used . Cloning three consecutive exons, the affected exon and two flanking exons, into the minigene system may better mimic the procedure of pre‐RNA splicing in vivo , because it can retain the original ss sequences of upstream and downstream introns of the affected exon .…”

“…Cloning three consecutive exons, the affected exon and two flanking exons, into the minigene system may better mimic the procedure of pre‐RNA splicing in vivo , because it can retain the original ss sequences of upstream and downstream introns of the affected exon . The effects on pre‐RNA splicing can also be predicted by in silico tools, including MaxEntScan (MES), Splice‐Site Prediction by Neural Network (NNSplice), Human Splicing Finder (HSF), Splice‐Port, Exonic Splicing Enhancers (ESE) Finder, and RESCUE‐ESE, but they are insufficient. Functional assay is needed for pathogenicity assessment .…”