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Lee-Jun C Wong, Victor Zhang, Yanming Feng, Fangyuan Li, Cavatina K Truong, Guoli Wang, Jing Wang, Richard A Lewis; Next Generation Approach to Molecular Diagnosis of Ocular Diseases. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6392.
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Ocular disease is a group of genetically and clinically heterogeneous disorders affecting the eye, including degenerative retinal diseases, glaucoma, age-related macular degeneration, and Leber congenital amaurosis (LCA). Identification of disease-causing mutations in affected individuals is essential for genetic counseling, carrier testing, and future gene-specific therapies. However, current molecular diagnosis analyzes candidate genes one-by-one, which is time consuming and costly. The purpose of this report is to demonstrate the clinical utility of a cost effective, high throughput, comprehensive next generation sequencing (NGS) approach for the molecular diagnosis of ocular diseases.
All coding exons and 20 bp of their flanking intron regions of 207 genes related to ocular diseases, including 794,035 bp of target sequences and 2837 exons, were captured by probe hybridization, followed by massively parallel sequencing (MPS) on Illumina HiSeq2000. The sequencing results of a panel of specific disease genes of interest were validated by Sanger sequencing for clinical application. All mutations identified are confirmed by Sanger sequencing.
This approach achieved a mean sequence depth of 1000X per base. We validated 4 panels; nonsyndromic retinitis pigmentosa (RP), LCA, Usher syndrome (USH) and glaucoma, each containing 66, 19, 9, and 5 genes, respectively, for clinical application. We ensure 100% coverage by PCR/Sanger sequencing to complete the insufficiently sequenced (<20X) exons (about 0-3% per panel). Using this gene panel capture/MPS analysis the diagnostic yields for RP and USH reaches 80% and >92% respectively.
Our data demonstrated the power of MPS-based analysis in modern molecular diagnosis of ocular diseases. The target gene capture followed by deep sequencing allows accurate identification of all types of mutations including point mutations, exonic deletions and large insertions, for an improved diagnostic acumen in a cost- and time-efficient manner.
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