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C. Rivolta, T. L. McGee, N. M. Wade, C. Iseli, J. S. Beckmann, E. L. Berson, T. Rio Frio; A Single-Base Substitution Within an Intronic Repetitive Element in PRPF31 Causes Dominant Retinitis Pigmentosa With Reduced Penetrance. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2318.
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© ARVO (1962-2015); The Authors (2016-present)
To identify the mutation responsible for autosomal dominant retinitis pigmentosa with reduced penetrance in a large American family, clinically reported for the first time in 1969 (Arch Ophthalmol. 81:226-284, 1969), and showing linkage to the RP11 (PRPF31) locus (Am J Hum Genet 61:1059-1066, 1997).
Since previous screens of PRPF31 exonic sequences have failed to detect causative mutations, we sequenced 4 tiled long-range PCR products encompassing the entire genomic region of this gene in 1 affected family member and 2 asymptomatic obligate carriers. PCR products were analyzed by Ultra High Throughput sequencing (UHTseq) using an Illumina-Solexa instrument, and by classical Sanger sequencing. Assembly of the UHTseq reads was performed by 3 different software packages: Rolexa, Maq, and the CLC Genomics Workbench. mRNA analyses were performed by RT-PCR and Q-PCR. PRPF31 protein was assessed by immunoblotting. Segregation analysis was performed on other family members, including 7 affected individuals, by Sanger sequencing.
Among the variants identified, only a single-base substitution (c.1374+654C>G) located deep within intron 13 and inside a 7-element repetitive DNA sequence was common to all 8 affected and 2 obligate carrier family members. It was absent in 150 control individuals. This variant was identified by Sanger sequencing but not by UHTseq, likely because the size of a single repetitive element was bigger than the average length of an UHTseq read. Analysis of mRNA derived from patient cell lines showed that the mutation creates a cryptic splice donor site that leads to the synthesis of 2 mutant PRPF31 isoforms. Both mutant transcripts encode premature termination codons and were shown to be degraded by nonsense-mediated mRNA decay. No mutant protein was detected and a strong reduction in full length PRPF31 mRNA and protein levels was observed.
Our results indicate that the intronic mutation c.1374+654C>G is pathogenic and, like the majority of reported PRPF31 mutations, that haploinsufficiency is the cause of retinitis pigmentosa in this family. Application of UHTseq technologies with traditional Sanger methods may facilitate the continued identification of pathogenic mutations buried deep within intronic gene sequences.
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