Abstract
Purpose :
Genetic testing is essential in the diagnosis of inherited eye diseases (IEDs), due to their genetic heterogeneity and phenotypic overlap, and the need to establish a molecular diagnosis for targeted therapy selection. However, IED diagnostics can be compromised by tests which are poorly validated or do not capture the full spectrum of causative genetic variation. We aimed to develop and validate a high quality whole exome sequencing (WES) based platform, with boosted clinical content tailored to IEDs, and to assess diagnostic yield in a large IED cohort.
Methods :
Performance of an xGEN Exome Research Panel assay, with bespoke clinical content, was tested through next generation sequencing (NGS) of reference samples on the Illumina NovaSeq platform. The assay was designed to cover clinically-relevant intronic and difficult-to-sequence regions, and detect copy number variants (CNVs). Based on this assay, 23 ophthalmology gene panels were curated, covering 438 unique genes, and their diagnostic performance evaluated.
Results :
In clinically-relevant genes, the assay showed high average sequencing depth (174x) and coverage (99.4% regions covered >20x). Analytic validation showed excellent sensitivity for SNVs (0.997), INDELs 2-50 base pairs (0.988), 1-exon CNVs (0.92), and 5-exon CNVs (0.99). The assay showed uniform coverage over difficult-to-sequence regions, including RPGR ORF15 and GC-rich 1st exons. The Usher Syndrome Panel gave the highest diagnostic yield (>80%) and the Glaucoma Panel the lowest (11%). A 266-gene Retinal Dystrophy Panel, the largest and most frequently ordered panel, yielded a diagnosis in 58% of (1587) cases. Diagnoses were made in 111 genes in total, the most frequently implicated genes being ABCA4, USH2A, RPGR, RHO, and PRPH2. Diagnoses were also made in genes newly implicated in IEDs such as CTNN1A, TTLL5, and RAB28. Approximately 3% of cases had a diagnostic CNV. Disease-causing variants were detected in RPGR ORF15 and in custom targeted deep intronic regions.
Conclusions :
A WES assay with boosted clinical content provides high sequencing coverage, depth, sensitivity and specificity, and provides high diagnostic yields for IED patients. The genetic variability of diagnoses in our cohort supports the use of comprehensive NGS panels in IED diagnostics, to optimize diagnostic yield and clinical care.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.