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Paola Benaglio, Leonardo P. Capelli, Shyana Harper, Eliot L. Berson, Carlo Rivolta; Next Generation Sequencing Of Pooled DNA From A Large Cohort Of Patients Reveals New SNRNP200 Mutations Associated With Retinitis Pigmentosa. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3316.
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© ARVO (1962-2015); The Authors (2016-present)
Similar to other genes causing dominant retinitis pigmentosa (RP), SNRNP200 encodes a protein essential for pre-mRNA splicing, the 200 kDa helicase hBrr2. Mutations in thisgene have so far been identified in only two Chinese families. In order to investigate the presence of SNRNP200 mutations at a larger scale, we screened an extended cohort of unrelated North American patients with autosomal dominant RP.
The entire 35-kb SNRNP200 genomic region (45 exons) was analyzed in each of 96 such patients, for whom mutations in most known RP genes had previously been excluded. We performed next generation sequencing (NGS) of pooled, untagged PCR products and verified the presence of the detected DNA changes by Sanger sequencing of individual samples. Confirmed changes were tested on 95 additional patients and 175 controls.
One of the two known mutations (c.3260C>T, p.S1087L) was identified in 3 patients of Mexican, French-Canadian, and English/Irish descent. Four new missense changes affecting highly conserved codons and absent in the controls were identified in 6 unrelated individuals: p.R681C was present in 3 patients and p.R681H, p.V683L and p.Y689C were present in one patient each. Pedigree analysis was performed for p.R681C and p.Y689C, which segregated with the disease in affected members of each family. We also used the data obtained to critically evaluate our screening method, in particular with respect to the generation of false positive and negative results.
By this DNA pooling approach combined with NGS, we identified new mutations in SNRNP200 and confirmed that dominant RP associated with hBrr2 impairment is not limited to the Chinese population. Our discovery of p.S1087L in Caucasian patients indicates that either this mutation represents a relatively early event in human history or that nucleotide c.3260 is a mutational hotspot. With respect to the method used, we conclude that, when appropriate settings are implemented, it can be efficiently used for rapid discovery of new disease-associated variants in a large set of DNA samples.
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