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

Abstract

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.

Dominance and gene dosage balance in health and disease: why levels matter!

“…In the same vein, the most common vitreoretinal degeneration, known as Stickler syndrome, can be due to mutations leading to haploinsufficiency of collagen 2A1 (OMIM 108300). Studies on this collagen have shown that mutant mRNA molecules containing truncating mutations undergo nonsense-mediated mRNA decay [13]. Such mRNA instability leads to haploinsufficiency.…”

A type II collagen mutation also results in oto-spondylo-megaepiphyseal dysplasia

“…EB virus-transformed lymphoblastoid cells were established from the blood of the patient and a control individual as described previously (Fukushima et al 1992). The lymphoblastoid cells were cultured with or without cycloheximide as described previously (Freddi et al 2000) to examine the effects of mRNA decay. mRNAs were isolated from lymphoblastoid cells and a cartilage specimen from the normal individual using the FastTrack 2.0 Kit (Invitrogen, Carlsbad, CA, USA).…”

The Stickler syndrome: Genotype/phenotype correlation in 10 families with Stickler syndrome resulting from seven mutations in the type II collagen gene locus COL2A1

“…19,20 A variety of mutations spread throughout the entire COL2A1 gene have been identified in families with the Stickler syndrome. [21][22][23][24][25][26][27][28][29][30][31] Most of the Stickler COL2A1 mutations noted to date result in the introduction of a premature stop codon, suggesting that the phenotype usually results from a quantitative defect in type II procollagen biosynthesis either because of the nonsense-mediated mRNA decay pathway or failure of chain association. COL2A1 mutations associated with phenotypes more severe than the Stickler syndrome such as achondrogenesis type II, hypochondrogenesis, spondyloepiphyseal dysplasia congenita, and Kniest dysplasia result from missense mutations that cause defects in the structure of type II collagen protein.…”