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 Stickler syndrome due to COL2A1 nonsense mutations, using a modified RNA-based protein truncation test. To overcome the problem of the unavailability of collagen II-producing cartilage cells, reverse transcription polymerase chain reaction (RT-PCR) was performed on the illegitimate transcripts of accessible cells (lymphoblasts and fibroblasts), which were pre-incubated with cycloheximide to prevent nonsense-mutation-induced mRNA decay. The five overlapping RT-PCR fragments covering the COL2A1 coding region were then transcribed 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 identified, which was present in all affected individuals. Targeted sequencing identified the mutation as a G(+1) to A substitution at the 5' splice donor site of intron 25, which led to the activation of a cryptic splice site 8-bp upstream causing aberrant mRNA splicing and a translational frameshift that introduced a premature stop codon. Mutant mRNA was undetectable without cycloheximide protection, demonstrating that the mutant mRNA was subjected to nonsense-mediated mRNA decay. As well as providing further evidence that type 1 Stickler syndrome results from COL2A1 premature stop codon mutations, this study suggests mutant mRNA instability leading to haploinsufficiency may also be an important, but previously unrecognized, molecular basis of Stickler syndrome. This rapid new test for COL2A1 nonsense mutations is of particular clinical importance to Stickler syndrome families, where the identification of individuals who are at risk of this potentially preventable form of blindness will allow them to undergo regular ophthalmological surveillance and preventative or early ameliorative treatment.