Abstract
Abstract: :
Purpose: Autosomal dominant optic atrophy is the most common form of inherited optic neuropathy. In the majority of cases adOA is caused by mutations in the OPA1 gene. Because of the complex structure of the OPA1 gene within 30 coding exons, commonly used mutation detection techniques (e.g. exon by exon sequencing) are laborious and expensive. Moreover putative splice site mutations cannot be functionally characterised. To overcome these problems we have established a cDNA–based screening analysis for the OPA1 gene. Methods: Total RNA was extracted from patients blood samples or cell lines. RNA samples were reverse transcribed and the entire coding region of the OPA1 gene was amplified as four overlapping cDNA fragments. Mutation screening was done by direct sequencing of RT–PCR products. For allele specific cDNA quantification we applied the Pyrosequencing technology. Results: Using cDNA analysis we have identified 36 different OPA1 mutations including 6 frameshift, 10 missense, 3 nonsense and 17 splicing mutations. Twelve of the splicing mutations lead to skipping of the flanking exon and five of them to the activation of a cryptic splice site. We noticed that some mutations which lead to a premature stop codon are underrepresented at the cDNA level, which most likely results from nonsense mediated mRNA decay. Allele specific cDNA quantification applying pyrosequencing was used to confirm and quantify the reduced levels of the mutant transcripts. Conclusions: We have established an efficient cDNA–based screening method for the OPA1 gene. Our analyses also underline the value of the cDNA analysis for the assessment of splicing mutations. The results of the allele specific cDNA quantification provide additional evidence that haploinsufficiency may represent a mechanism for the development of adOA.
Keywords: gene screening • mutations • neuro-ophthalmology: optic nerve