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C. Rivolta, T.L. McGee, M.A. Stillberger, E.L. Berson, T.P. Dryja; RP11 Transcripts With Premature Stop Codons May Escape Nonsense-Mediated Decay; Possible Role in Explaining Reduced Penetrance . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2019.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Mutations in the RP11 (PRPF31) gene have been associated with dominant retinitis pigmentosa with reduced penetrance, possibly mediated by isoalleles of RP11 itself. In this work we are investigating how penetrance is defined at the molecular level. Methods: We ascertained individuals from 11 RP11-linked families, of which 10 had identified heterozygous mutations in the PRPF31 gene. So far we have extracted total leukocyte RNA from 14 members of 4 of these 11 families. The leukocyte RNA from 6 individuals from 2 families was analyzed by reverse transcription-polymerase chain reaction (RT-PCR), and automated DNA sequencing. Results: The PRPF31 mutation in the first family is a deletion of part of exon 9 [Arg293(34-bp del)]. The mutation in the second family (IVS8-2A>G) abolishes the normal splice-acceptor site of exon 9 and, based on our analysis of the mutant mRNA in leukocytes, produces a mutant transcript that incorporates 28 extra nucleotides of IVS8 because of a cryptic splice-acceptor site at IVS8-29. Both of these mutations cause frameshifts and introduce premature stops. If translated, mutant mRNAs from both of them would result in truncated proteins having abnormal sequences at their carboxy-termini. We sequenced RT-PCR products from leukocyte RNA from these 4 patients (2 affected patients and 1 asymptomatic carrier with Arg293(34-bp del) and 1 affected patient with IVS8-2A>G). All 3 affected patients expressed approximately equal amounts of normal and mutant transcript. In the asymptomatic carrier, the mutant mRNA was expressed at a level much lower than the wild-type mRNA (approximate ratio wt:mutant = 4:1). Conclusions: In some patients with dominant RP11 mutations that create premature termination codons, there is little or no nonsense-mediated decay of the resulting mutant mRNA, and possibly this results in the production of a truncated and aberrant protein. The predicted mutant protein might have a role in the pathogenesis of retinitis pigmentosa. At least one asymptomatic carrier appears to have a reduced level of mutant mRNA, suggesting that the ratio of wild-type versus mutant mRNA may have a role in determining the penetrance of RP11 mutations. We are currently extending the mRNA analyses to include additional RP11 patients and asymptomatic carriers to assess how general these findings are.
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