Whole Genome Sequencing Increases Molecular Diagnostic Yield Compared with Current Diagnostic Testing for Inherited Retinal Disease

Abstract

Purpose: To compare the efficacy of whole genome sequencing (WGS) with targeted next-generation sequencing (NGS) in the diagnosis of inherited retinal disease (IRD).

Design: Case series.

Participants: A total of 562 patients diagnosed with IRD.

Methods: We performed a direct comparative analysis of current molecular diagnostics with WGS. We retrospectively reviewed the findings from a diagnostic NGS DNA test for 562 patients with IRD. A subset of 46 of 562 patients (encompassing potential clinical outcomes of diagnostic analysis) also underwent WGS, and we compared mutation detection rates and molecular diagnostic yields. In addition, we compared the sensitivity and specificity of the 2 techniques to identify known single nucleotide variants (SNVs) using 6 control samples with publically available genotype data.

Main outcome measures: Diagnostic yield of genomic testing.

Results: Across known disease-causing genes, targeted NGS and WGS achieved similar levels of sensitivity and specificity for SNV detection. However, WGS also identified 14 clinically relevant genetic variants through WGS that had not been identified by NGS diagnostic testing for the 46 individuals with IRD. These variants included large deletions and variants in noncoding regions of the genome. Identification of these variants confirmed a molecular diagnosis of IRD for 11 of the 33 individuals referred for WGS who had not obtained a molecular diagnosis through targeted NGS testing. Weighted estimates, accounting for population structure, suggest that WGS methods could result in an overall 29% (95% confidence interval, 15-45) uplift in diagnostic yield.

Conclusions: We show that WGS methods can detect disease-causing genetic variants missed by current NGS diagnostic methodologies for IRD and thereby demonstrate the clinical utility and additional value of WGS.

Figure 1

Data analysis and study design summary. Overview of targeted next-generation sequencing (NGS) diagnostic testing. *Single carrier variant defined as an individual with a pathogenic heterozygous variant found in a gene relevant to their clinical indication of inherited retinal disease (IRD) that is known to cause recessively inherited disease. WGS = whole genome sequencing.

Figure 2

A, Clinical outcome of diagnostic next-generation sequencing (NGS) testing for 562 individuals. Diagnosed, individuals with a molecular diagnosis. Undiagnosed, individuals without a molecular diagnosis. Single carrier variant, individuals who are heterozygous for a “clearly pathogenic” or “likely pathogenic” variant in a gene associated with recessive retinal dystrophy; no second variant was identified. Other variants in additional genes may or may not be determined to cause the phenotype, this refers to diagnosed and single carrier variant and undiagnosed and single carrier variant, respectively. B, Projected impact of whole genome sequencing (WGS) on clinical molecular diagnostics. A weighted biased estimate with 95% confidence intervals (CIs) of the projected increase in molecular diagnostic yield: targeted NGS, number of individuals with a molecular diagnosis through targeted NGS diagnostics; bioinformatics adjustments, the number of individuals expected to receive a molecular diagnosis after alterations to the bioinformatics pipeline for targeted NGS diagnostics; WGS, the number of individuals expected to have a molecular diagnosis if WGS were to be applied to the 562 referred patients.

Figure 3

Sanger sequencing chromatogram showing the breakpoints of a heterozygous deletion removing a single exon from the reading frame of the GPR98 gene.