April 2011
Volume 52, Issue 14
ARVO Annual Meeting Abstract  |   April 2011
Validation of Known and Novel Variations Identified in Retinal Degeneration Patients Using a High-throughput Screening Tool
Author Affiliations & Notes
  • Jin Song
    National Eye Institute, Bethesda, Maryland
  • Xinjing Wang
    National Eye Institute, Bethesda, Maryland
  • Footnotes
    Commercial Relationships  Jin Song, None; Xinjing Wang, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 3318. doi:
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      Jin Song, Xinjing Wang; Validation of Known and Novel Variations Identified in Retinal Degeneration Patients Using a High-throughput Screening Tool. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3318.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : To identify the genetic causes of retinal degeneration in a group of patients and explore the potential clinical utility of genomic DNA sequence screening for mutations in candidate genes using a high-throughput resequencing chip.

Methods: : A custom 300 kb resequencing chip had been developed to detect sequence alterations of 267,550 bases of both sense and antisense sequence in 1,470 exons spanning 93 genes involved in inherited retinal degenerations. This array was evaluated in 20 patient samples with inherited retinal degenerations provided by the eyeGENE® repository and 4 CEPH reference samples through an established high-throughput experimental approach including an automated PCR assay setup and quantification, efficient post-quantification data processing, optimized pooling and fragmentation, and standardized chip processing. A series of custom screening filters was developed for reliably screening the sequence variations in patient samples. The identified variations were independently validated by Sanger sequencing. A number of bioinformatic tools such as PolyPhen-2 and SIFT were further used to evaluate the potential pathogenic variations.

Results: : Three hundred and four variations were initially identified by the resequencing chips. Of 173 variations selected for validation including 134 (77.5%) missense, 4 (2.3%) nonsense, 21 (12.1%) silent, 13 (7.5%) splice-site, and 1 (0.6%) 5’ untranslated region variation, 123 (71.1%) were confirmed, including 92 missense, 2 nonsense, 21 silent, 7 splice-site, and 1 5’ untranslated region variation. This number included all 54 known SNPs (54/54, 100% confirmed) and 69 novel variations (69/120, 57.5% confirmed), whereas 51 novel variations were determined to be artifacts of chip resequencing. Analysis of patient samples using the resequencing chips resulted in the identification of 24 known mutations and 8 novel variations with high probability of deleterious effects.

Conclusions: : The resequencing chip is a valuable tool to detect disease-causative mutations and explore for disease severity modifiers in a single experiment. It may provide a powerful and feasible approach to study the genetic heterogeneity in retinal diseases.

Keywords: retinal degenerations: hereditary • gene screening • gene microarray 

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