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Adam P DeLuca, Heather T Daggett, Jade S East, Arlene V Drack, Todd E Scheetz, Val Sheffield, Edwin M Stone; Whole genome sequencing to find missing alleles in patients with retinal disease.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2756.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in any of hundreds of genes can cause inherited retinal disease, yet even with an exome-scale tiered genetic testing strategy, about a quarter of all cases go unsolved. While a portion of the remaining cases are likely caused by novel disease genes, others are likely caused by variants in known genes that are not detected with current methods. The identification of common missing alleles would improve the sensitivity of genetic tests. We studied a subset of these unsolved cases in which a combination of genotypic and phenotypic information reduced the hypothesis to a single gene with a single missing allele.
Tiered genetic testing – consisting of focused tests for common alleles followed by whole-exome sequencing – was performed on over 1000 patients clinically diagnosed with inherited retinal disease. In 45 negative cases where a combination of phenotypic and genotypic information yielded a single gene hypothesis, 30x Whole Genome Sequencing (WGS) was obtained. This cohort included 10 cases where we suspected Copy Number Variants (CNVs) based on exome coverage data. Identified variants were confirmed via Sanger sequencing. Segregation and phase were established by sequencing available family members. Unrelated affected individuals were screened using PCR-based allele-specific assays in an attempt to find common missing alleles.
Putative disease-causing variants were identified in 26/45 cases. Of these 26 cases, 4 had exonic single nucleotide variants, 19 had CNVs that removed or added exons (including 10/10 suspected by exome coverage data), and 3 had structural variants disrupting regulatory sequence. Seven of these 26 variants were screened in cohorts of unrelated affected individuals but no recurrent mutations were identified.
Our data show that if sufficient genotypic and phenotypic information exist to narrow a hypothesis to a single gene, WGS can identify the missing allele in the majority of cases that are negative after allele-specific or exome sequencing.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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