Molecular diagnosis of Stickler syndrome: A COL2A1 stop codon mutation screening strategy that is not compromised by mutant mRNA instability

We have developed a novel strategy for screening families with type 1 Stick ler syndrome due to COL2AI nonsense mutations, using a modified RNA-based p rotein truncation test. To overcome the problem of the unavailability of co llagen II-producing cartilage cells, reverse transcription polymerase chain reaction (RT-PCR) was performed on the illegitimate transcripts of accessi ble cells (lymphoblasts and fibroblasts), which were pre-incubated with cyc loheximide to prevent nonsense mutation-induced mRNA decay, The five overla pping RT-PCR fragments covering the COL2AI coding region were then transcri bed and translated in vitro to identify smaller truncated protein products which result from a premature stop codon, This method was used to screen a 4-generation Stickler family and a protein truncating mutation was identifi ed, which was present in all affected individuals, Targeted sequencing iden tified the mutation as a G(+1) to A substitution at the 5' splice donor sit e of intron 25, which led to the activation of a cryptic splice site 8-bp u pstream causing aberrant mRNA splicing and a translational frameshift that introduced a premature stop codon, Mutant mRNA was undetectable without cyc loheximide protection, demonstrating that the mutant mRNA was subjected to nonsense-mediated mRNA decay, As well as providing further evidence that ty pe 1 Stickler syndrome results from COL2AI premature stop codon mutations, this study suggests mutant mRNA instability leading to haploinsufficiency m ay also be an important, but previously unrecognized, molecular basis of St ickler syndrome. This rapid new test for COL2AI nonsense mutations is of pa rticular clinical importance to Stickler syndrome families, where the ident ification of individuals who are at risk of this potentially preventable fo rm of blindness will allow them to undergo regular ophthalmological surveil lance and preventative or early ameliorative treatment. (C) 2000 Wiley-Liss , Inc.