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Published on: December 13, 2014
Nonsense-mediated mRNA decay efficiency varies in choroideremia providing a target to boost small molecule
Hajrah Sarkar1, Andreas Mitsios1,2, Matthew Smart1
1Development, Ageing and Disease, UCL Institute of Ophthalmology, London, UK.
Abstract:
Choroideremia (CHM) is an x-linked recessive chorioretinal dystrophy, with 30% caused by nonsense mutations in the CHM gene resulting in an in-frame premature termination codon (PTC). Nonsense-mediated mRNA decay (NMD) is the cell's natural surveillance mechanism that detects and destroys PTC-containing transcripts, with UPF1 being the central NMD modulator. NMD efficiency can be variable amongst individuals with some transcripts escaping destruction, leading to the production of a truncated non-functional or partially functional protein. Nonsense suppression drugs, such as ataluren, target these transcripts and read-through the PTC, leading to the production of a full length functional protein. Patients with higher transcript levels are considered to respond better to these drugs, as more substrate is available for read-through. Using Quantitative reverse transcription PCR (RT-qPCR), we show that CHM mRNA expression in blood from nonsense mutation CHM patients is 2.8-fold lower than controls, and varies widely amongst patients, with 40% variation between those carrying the same UGA mutation [c.715 C>T; p.(R239*)]. These results indicate that although NMD machinery is at work, efficiency is highly variable and not wholly dependent on mutation position. No significant difference in CHM mRNA levels was seen between two patients' fibroblasts and their induced pluripotent stem cell-derived retinal pigment epithelium. There was no correlation between CHM mRNA expression and genotype, phenotype or UPF1 transcript levels. NMD inhibition with caffeine was shown to restore CHM mRNA transcripts to near wild-type levels. Baseline mRNA levels may provide a prognostic indicator for response to nonsense suppression therapy, and caffeine may be a useful adjunct to enhance treatment efficacy where indicated.
Insights
Choroideremia (CHM) patients have lower CHM mRNA levels, with significant variation impacting treatment response. Inhibiting nonsense-mediated mRNA decay (NMD) with caffeine can restore CHM mRNA, potentially improving outcomes for nonsense suppression therapies.
Area of Science:
- Genetics and Molecular Biology
- Ophthalmology
- Pharmacology
Background:
- Choroideremia (CHM) is an X-linked chorioretinal dystrophy often caused by nonsense mutations in the CHM gene, leading to premature termination codons (PTCs).
- Nonsense-mediated mRNA decay (NMD) is a cellular process that degrades PTC-containing transcripts, with UPF1 as a key regulator.
- Variability in NMD efficiency can result in truncated proteins, impacting disease severity and response to therapies like ataluren, which aim to suppress nonsense mutations.
Purpose of the Study:
- To investigate CHM mRNA expression levels in patients with nonsense mutations.
- To assess the variability of NMD efficiency in CHM patients and its correlation with clinical factors.
- To evaluate the potential of NMD inhibition to restore CHM mRNA levels and improve treatment efficacy.
Main Methods:
- Quantitative reverse transcription PCR (RT-qPCR) was used to measure CHM mRNA expression in patient blood samples and cell lines (fibroblasts and iPSC-derived RPE).
- NMD was inhibited using caffeine to assess its effect on CHM mRNA levels.
- Correlation analyses were performed between CHM mRNA levels, genotype, phenotype, and UPF1 transcript levels.
Main Results:
- CHM mRNA expression was 2.8-fold lower in patients with nonsense mutations compared to controls, exhibiting significant inter-patient variability (up to 40% variation for the same mutation).
- No significant differences in CHM mRNA levels were observed between fibroblasts and iPSC-derived RPE, nor were correlations found with genotype, phenotype, or UPF1 levels.
- NMD inhibition with caffeine successfully restored CHM mRNA transcripts to near wild-type levels.
Conclusions:
- Baseline CHM mRNA levels are highly variable in patients and may serve as a prognostic indicator for response to nonsense suppression therapy.
- NMD efficiency is variable and not solely dependent on mutation position, suggesting complex regulatory mechanisms.
- Caffeine-induced NMD inhibition shows promise as an adjunct therapy to enhance the efficacy of nonsense suppression treatments for Choroideremia.
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