Fundoscopy-directed genetic testing to re-evaluate negative whole exome sequencing results

Abstract

Background: Whole exome sequencing (WES) allows for an unbiased search of the genetic cause of a disease. Employing it as a first-tier genetic testing can be favored due to the associated lower incremental cost per diagnosis compared to when using it later in the diagnostic pathway. However, there are technical limitations of WES that can lead to inaccurate negative variant callings. Our study presents these limitations through a re-evaluation of negative WES results using subsequent tests primarily driven by fundoscopic findings. These tests included targeted gene testing, inherited retinal gene panels, whole genome sequencing (WGS), and array comparative genomic hybridization. Results: Subsequent genetic testing guided by fundoscopy findings identified the following variant types causing retinitis pigmentosa that were not detected by WES: frameshift deletion and nonsense variants in the RPGR gene, 353bp Alu repeat insertions in the MAK gene, and large exonic deletion variants in the EYS and PRPF31 genes. Deep intronic variants in the ABCA4 gene causing Stargardt disease and the GUCY2D gene causing Leber congenital amaurosis were also identified. Conclusions: Negative WES analyses inconsistent with the phenotype should raise clinical suspicion. Subsequent genetic testing may detect genetic variants missed by WES and can make patients eligible for gene replacement therapy and upcoming clinical trials. When phenotypic findings support a genetic etiology, negative WES results should be followed by targeted gene sequencing, array based approach or whole genome sequencing.

Landscape of pathogenic variants in six pre‐mRNA processing factor genes for retinitis pigmentosa based on large in‐house data sets and database comparisons

“…3 and S9). This was identified in six patients from our cohort for whom SLO images were available and was also observed in other studies (Van Cauwenbergh et al 2017;Vincent et al 2017;Cho et al 2020). Of the 49 patients with RP, an electroretinogram was available for 17 patients, and only text results were available for four patients; the results for the remaining 13 patients are shown in Fig.…”

Frequency of Eye Changes in Patients of Chronic Kidney Disease

“…Generally, if hypertension is well controlled, there is no additional risk caused by anticoagulation done by heparin in patients on hemodialysis. 11 Hypertension in itself can impose additional hazard for progression of diabetic retinopathy if I is not optimized, can lead to complications such as vitreous hemorrhage or retinal hemorrhage [12][13] .…”

Clinical and Genetic Re-Evaluation of Inherited Retinal Degeneration Pedigrees following Initial Negative Findings on Panel-Based Next Generation Sequencing

“…Cloning allowing bidirectional sequence reads of such regions has been demonstrated as a cost-effective way to overcome this issue [29,30]. The RPGR ORF15 region is a crucial locus to assess X-linked RP (XLRP) pedigrees, as ≈60% of variants causing XLRP may be found in this mutational 'hot spot' [31]. Pedigrees that were resolved by the resequencing of RPGR include pedigree #26 (resolved by an expanded pNGS (351 genes) at an accredited laboratory) and pedigree #27 (resolved using trio WES) [32].…”

“…WES is gaining popularity as a primary method for the genetic assessment for IRDs [31]. Benefits include the ability to store entire exomes and apply a 'virtual panel' for genes of interest in specific areas of medicine [37].…”

“…Costs are upfront in terms of DNA sequencing, and data can be securely stored for future research or clinical applications. Published series report a 49-63% genetic resolve rate for their IRD cohorts using WES [22,31,38,39]; thus, the financial and efficiency benefit remains with pNGS as in our pathway [4,5]. Although a panel-based method eliminates the possibility of re-analysis with a broader virtual gene panel, this is less likely to be an issue for conditions whereby the gene associations have been largely already established.…”