Reliable and sensitive detection of premature termination mutations using a protein truncation test designed to overcome problems of nonsense-mediated mRNA instability

The protein truncation test (PTT) is a mutation-detection method used to sc an 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 res ultant products can be transcribed and translated in vitro to identify the smaller truncated protein products. mRNA is commonly used as the source mat erial, but success of the PTT and other RNA-based mutation detection method s can be severely compromised by nonsense mutation-induced mRNA decay, a we ll-documented process that is often overlooked in mutation detection strate gies. In this study, we develop an RNA-based PTT that overcomes the problem of mRNA decay by preincubating cells with cycloheximide to stabilise the m utant mRNA. The effectiveness of this method for mutation detection in abun dant mRNAs was demonstrated in osteogenesis imperfecta fibroblasts by the p rotection of type I collagen (COL1A1) mRNA containing nonsense mutations th at normally resulted in mutant mRNA degradation. Stabilisation of mutant mi smatch repair gene (MLH1) mRNA was also observed in transformed lymphocytes from patients with hereditary nonpolyposis colorectal cancer (HNPCC), Impo rtantly, our strategy also stabilised very low-level (or illegitimate) nons ense containing transcripts in lymphoblasts from patients with Bethlem myop athy (COL6A1), familial adenomatous polyposis (APC), and breast cancer (BRC A1), The greatly increased sensitivity and reliability of this RT-PCR/PTT p rotocol has broad applicability to the many genetic diseases in which only blood-derived cells may be readily available for analysis. Hum Mutat 13:311 -317, 1999, (C) 1999 Wiley Liss, Inc.