Comprehensive cDNA study and quantitative transcript analysis of mutantOPA1transcripts containing premature termination codons

Abstract

Autosomal dominant optic atrophy (adOA) is most commonly caused by mutations in the OPA1 gene. There is a considerable allelic heterogeneity among adOA-associated OPA1 mutations, however these mutations have mostly been identified and studied only at the genomic DNA level. Here we report the identification of 22 novel OPA1 mutations and their analysis at the cDNA level along with 15 already known OPA1 mutations. We found that 18 of these mutations cause splice defects that involve either skipping of the adjacent exon or the activation of a cryptic splice site. We also observed a reduced level of the mutant transcript in several adOA subjects. Allele-specific quantification of the transcript steady-state level was performed for 13 different OPA1 mutations applying pyrosequencing to a RT-PCR amplified cSNP (c.2109C>T) in OPA1. Using this new assay we could demonstrate that the majority of OPA1 mutations that lead to a premature termination codon (PTC) undergo nonsense-mediated mRNA decay (NMD). Mutant transcript levels were reduced between 1.25- and 2.5-fold and varied between PTC containing mutations, and between subjects. Our results emphasize the value of cDNA analysis in the characterization of OPA1 mutations and further strengthen the model of haploinsufficiency as a major pathomechanism in OPA1-associated adOA.

Quantitative sequence analysis ofFBN1premature termination codons provides evidence for incomplete NMD in leukocytes

“…This conclusion is in accordance with a previous study which showed that NMD of COL10A1 nonsense mutations causing Schmid-type metaphyseal chondrodysplasia is complete in cartilage chondrocytes but incomplete in noncartilage lymphoblastoid cell lines and bone-derived osteoblast cultures [Bateman et al, 2003]. A difference in NMD has also been previously observed between blood samples and lymphoblastoid cell lines of the same patients, demonstrating high levels of PTCbearing OPA1 transcripts in RNA obtained from blood (40-50%) in comparison with that from lymphoblastoid cell lines (20-30%) [Schimpf et al, 2008]. In addition, intertissue (CVS-stromal cells, amniocytes, and lymphocytes) and interindividual variation in NMD efficiency has been reported for PTCs in different genes, such as DMD and ESCO2 [Kerr et al, 2001;Resta et al, 2006].…”

“…Our finding that PTCbearing transcripts are clearly detectable in PAXgene blood samples seems to be neither restricted to FBN1 nor universally valid (Table 3). This may also be true for lymphoblastoid cell lines, albeit with lower PTC transcript levels as discussed above, because incomplete NMD has also been observed in such cells investigating PTC-containing transcripts of the genes OPA1, BRCA1, and APC [Gismondi et al, 1998;Perrin-Vidoz et al, 2002;Schimpf et al, 2008]. However, it remains unknown whether incomplete NMD occurs with comparable levels of PTC transcripts in all types of leukocytes.…”

OPA1 mutations in Japanese patients suspected to have autosomal dominant optic atrophy

“…NMD is a widespread cellular process that proofreads nascent mRNA transcripts and destroys those with premature termination codons before they are actually translated in truncated and potentially harmful proteins. In a recent study, Schimpf et al [27] demonstrated that the majority of nonsense OPA1 mutations underwent NMD. They found that mutant transcript levels were reduced between 1.25-and 2.5-fold, and varied among premature termination codons containing mutations.…”

Loss of exon identity is a common mechanism of human inherited disease

“…We searched this list of disease-causing mutations for sequences that were amenable for cloning into splicing reporter constructs and were present in exons of near average size and splice-site strength . Of the three different disease-causing mutations we selected-OPA1, PYGM, and TFR2-there was no a priori evidence for any effect on splicing from in vitro analysis (Bruno et al 1999;Camaschella et al 2000;Schimpf et al 2008). However, aberrant splicing of OPA1, PYGM, and TFR2 is observed in patients carrying coding and non-coding mutations at other positions in these genes (Schimpf et al 2006;Biasiotto et al 2008;Nogales-Gadea et al 2008).…”