title: Reliable and sensitive detection of premature termination mutations using a protein truncation test designed to overcome problems of nonsense-mediated mRNA instability.
abstract: The protein truncation test (PTT) is a mutation-detection method used to scan for premature termination (nonsense) mutations. PCR amplification of the DNA or mRNA source material is performed using forward primers containing a T7-promoter sequence and translation initiation signals such that the resultant products can be transcribed and translated in vitro to identify the smaller truncated protein products. mRNA is commonly used as the source material, but success of the PTT and other RNA-based mutation detection methods can be severely compromised by nonsense mutation-induced mRNA decay, a well-documented process that is often overlooked in mutation detection strategies. In this study, we develop an RNA-based PTT that overcomes the problem of mRNA decay by preincubating cells with cycloheximide to stabilise the mutant mRNA. The effectiveness of this method for mutation detection in abundant mRNAs was demonstrated in osteogenesis imperfecta fibroblasts by the protection of type I collagen (COL1A1) mRNA containing nonsense mutations that normally resulted in mutant mRNA degradation. Stabilisation of mutant mismatch repair gene (MLH1) mRNA was also observed in transformed lymphocytes from patients with hereditary nonpolyposis colorectal cancer (HNPCC). Importantly, our strategy also stabilised very low-level (or illegitimate) nonsense-containing transcripts in lymphoblasts from patients with Bethlem myopathy (COL6A1), familial adenomatous polyposis (APC), and breast cancer (BRCA1). The greatly increased sensitivity and reliability of this RT-PCR/PTT protocol has broad applicability to the many genetic diseases in which only blood-derived cells may be readily available for analysis.
authors.affiliation: Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Parkville, Victoria, Australia. bateman@cryptic.rch.unimelb.edu.au
authors.forename: J F
authors.initials: JF
authors.lastname: Bateman
authors.forename: S
authors.initials: S
authors.lastname: Freddi
authors.forename: S R
authors.initials: SR
authors.lastname: Lamandé
authors.forename: P
authors.initials: P
authors.lastname: Byers
authors.forename: S
authors.initials: S
authors.lastname: Nasioulas
authors.forename: J
authors.initials: J
authors.lastname: Douglas
authors.forename: R
authors.initials: R
authors.lastname: Otway
authors.forename: M
authors.initials: M
authors.lastname: Kohonen-Corish
authors.forename: E
authors.initials: E
authors.lastname: Edkins
authors.forename: S
authors.initials: S
authors.lastname: Forrest
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D011125:Adenomatous Polyposis Coli / Q000235:genetics; D025601:Adenomatous Polyposis Coli Protein; D019313:BRCA1 Protein / Q000235:genetics; D002352:Carrier Proteins; D002478:Cells, Cultured; D003094:Collagen / Q000235:genetics; D003513:Cycloheximide / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D008967:Molecular Biology / Q000379:methods*; D000070957:MutL Protein Homolog 1; D009363:Neoplasm Proteins / Q000235:genetics; D009687:Nuclear Proteins; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013997:Time Factors
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D025601:Adenomatous Polyposis Coli Protein; D019313:BRCA1 Protein; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; C501486:MLH1 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D003094:Collagen; D003513:Cycloheximide; D000070957:MutL Protein Homolog 1