title: Nonsense-mediated decay of human LDL receptor mRNA.
abstract: OBJECTIVE
  The objective of this project was to determine whether nonsense mutation in the low density lipoprotein receptor (LDLR) induces nonsense-mediated mRNA decay (NMD).
  
  
  METHODS
  Four known nonsense mutations (W23X, S78X, E207X and W541X) in the LDLR gene, which are found in Norwegian familial hypercholesterolaemia (FH) patients, were investigated. Epstein-Barr virus (EBV) transformed lymphocytes from patients heterozygous for these mutations in the LDLR gene were analysed. Flow cytometric analysis was used to determine the amount and function of the cell surface LDLRs. The expression of LDLR mRNA in lymphocytes was quantified by real-time polymerase chain reaction (PCR). The presence of NMD was tested using the inhibitors gentamicin, emetine or cycloheximide.
  
  
  RESULTS
  Cells from heterozygous FH patients with nonsense mutations in the LDLR gene contained significantly less LDLR protein (p<0.05). In addition, flow cytometric analysis revealed that these patients had a reduced LDL-uptake compared to controls (p<0.005). Cells from heterozygous FH patients with nonsense mutations W23X, S78X or W541X in the LDLR gene showed significantly decreased levels of LDLR mRNA (p<0.005). LDLR mRNA was reduced in the mutant lymphocyte S78X prior to treatment with pharmacological inhibitors, and after treatment the level of LDLR mRNA increased to the same level as that of normal cells.
  
  
  CONCLUSIONS
  In the present study, NMD was confirmed in the LDLR gene. Translation inhibitors showed reduced NMD caused by nonsense mutated LDLR transcripts. Knowledge of NMD might have an important impact in clinical medicine as genetic intervention develops.
authors.affiliation: Department of Medical Genetics, Rikshospitalet University Hospital, Oslo, Norway.
authors.forename: Øystein Lunde
authors.initials: ØL
authors.lastname: Holla
authors.forename: Mari Ann
authors.initials: MA
authors.lastname: Kulseth
authors.forename: Knut Erik
authors.initials: KE
authors.lastname: Berge
authors.forename: Trond Paul
authors.initials: TP
authors.lastname: Leren
authors.forename: Trine
authors.initials: T
authors.lastname: Ranheim
mesh_terms: D015153:Blotting, Western; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D018389:Codon, Nonsense; D003513:Cycloheximide / Q000494:pharmacology; D005434:Flow Cytometry; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D006938:Hyperlipoproteinemia Type II / Q000235:genetics / Q000378:metabolism; D008077:Lipoproteins, LDL / Q000378:metabolism; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis / Q000187:drug effects; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D020871:RNA Stability / Q000187:drug effects / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism*; D011973:Receptors, LDL / Q000235:genetics* / Q000378:metabolism
chemical_list: D018389:Codon, Nonsense; D008077:Lipoproteins, LDL; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D011973:Receptors, LDL; D003513:Cycloheximide