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Revital Bronstein, Sudeep Mehrotra, Elizabeth E Capowski, David M Gamm, Eric A Pierce; A novel genetic diagnostic approach combines NGS and RNA-seq methodologies for detection of non-coding mutations in IRD patients. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5412. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Despite substantial progress in sequencing and bioinformatics tool development, current genetic diagnostic strategies can solve only about 60% of inherited retinal degeneration (IRD) cases. Some of these elusive disease causalities may be attributed to non-coding variants whose effect on gene structure and function cannot be readily assessed using current methodologies. The purpose of this study was to establish a new diagnostic pipeline that allows the identification of non-coding disease causality.
The patients included in the study were recruited and clinically examined at Massachusetts Eye and Ear. Patients underwent full ophthalmic examinations. Blood samples were taken from affected and unaffected family members and used for the isolation of DNA and generation of induced pluripotent stem cells (iPSC). DNA samples were studied by whole genome (WGS) sequencing. WGS sequence reads were aligned, and variants identified using the GATK pipeline. RNA samples from iPSC-derived optical vesicles (OV) were sequenced using standard methods. Differential splicing was assessed using the CASH algorithm and results were verified by RT-PCR.
A pilot study was conducted on a five member family with two siblings affected by cone dysfunction syndrome. WGS of all family members identified 8,191 pairs of rare variants in single genes as potential causes of recessive disease. RNA-seq analysis of 160 day OV identified 291 alternative splicing events present in OV from the affected siblings, but not in their unaffected sibling. Combined analysis of the WGS and RNA-seq data determined that disease is caused by a frame shift mutation and a novel deep intronic mutation in the CNGB3 gene. The intronic mutation creates a novel splice donor site, leading to a splice gain, which results in a premature stop codon in the mutant transcript. Mutations in CNGB3 are a common cause of cone dysfunction.
WGS and RNA-seq analyses alone identified a long list of plausible disease causing variants or events in this family. Their combined analysis allowed direct identification of disease causality, including a novel non-coding variant, even in a small family. The use of iPSC-derived OV was successful in providing a relevant transcriptome for these analyses, as readily accessible tissues such as blood and skin express only a fraction of genes expressed in the retina.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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