Abstract
BackgroundWhole 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.ResultsSubsequent 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, 353-bp 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.ConclusionsNegative 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.
Highlights
Whole exome sequencing (WES) allows for an unbiased search of the genetic cause of a disease
Copy number variations (CNVs) within an exon are covered by WES chemistry but likely to be missed in the reporting when the size exceeds 50 bp based on the analysis pipeline
Despite the high diagnostic yield of WES, there are inherent technical limitations that lead to missed variant callings
Summary
Whole exome sequencing (WES) allows for an unbiased search of the genetic cause of a disease. Genome-wide association studies (GWAS) have revealed that a significant proportion of variants within the noncoding genome are clinically relevant; mutations in the regulatory DNA sequences are either pathogenic themselves or they affect complex interactions between individual genetic features that lead to disease [11]. Such findings accentuate the inherent limitation of WES, as its coverage of exons and immediately adjacent introns fails to identify variants in the remaining 98% of the genome. When clinical indications are prominent, a negative WES analysis should be re-evaluated, as it can be insufficient to exclude disorders in the differential diagnoses [13]
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