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

Abstract

Although rare, inherited retinal degenerations (IRDs) are the most common reason for blind registration in the working age population. They are highly genetically heterogeneous (>300 known genetic loci), and confirmation of a molecular diagnosis is a prerequisite for many therapeutic clinical trials and approved treatments. First-tier genetic testing of IRDs with panel-based next-generation sequencing (pNGS) has a diagnostic yield of ≈70-80%, leaving the remaining more challenging cases to be resolved by second-tier testing methods. This study describes the phenotypic reassessment of patients with a negative result from first-tier pNGS and the rationale, outcomes, and cost of second-tier genetic testing approaches. Removing non-IRD cases from consideration and utilizing case-appropriate second-tier genetic testing techniques, we genetically resolved 56% of previously unresolved pedigrees, bringing the overall resolve rate to 92% (388/423). At present, pNGS remains the most cost-effective first-tier approach for the molecular assessment of diverse IRD populations Second-tier genetic testing should be guided by clinical (i.e., reassessment, multimodal imaging, electrophysiology), and genetic (i.e., single alleles in autosomal recessive disease) indications to achieve a genetic diagnosis in the most cost-effective manner.

Keywords: genetic testing; inherited retinal degenerations; next generation sequencing; retinal dystrophy; single gene sequencing; unresolved inherited retinal degenerations; whole exome sequencing.

Figure 1

Proposed algorithm for clinical and genetic reassessment of ‘gene-negative’ cases. (A) Algorithm for clinical reassessment of ‘gene-negative’ cases. (B) Algorithm for selecting the most appropriate further genetic testing modalities. * https://blueprintgenetics.com/tests/panels/ophthalmology/retinal-dystrophy-panel/ (accessed on 8 November 2021).

Figure 1

Proposed algorithm for clinical and genetic reassessment of ‘gene-negative’ cases. (A) Algorithm for clinical reassessment of ‘gene-negative’ cases. (B) Algorithm for selecting the most appropriate further genetic testing modalities. * https://blueprintgenetics.com/tests/panels/ophthalmology/retinal-dystrophy-panel/ (accessed on 8 November 2021).

Figure 2

Non-IRD (‘gene negative’) examples with markedly asymmetric disease. Pedigree #2: Colour fundus photographs a 70-year-old man with markedly asymmetrical retinal pigmentation. The right eye (A) has more classic features of retinitis pigmentosa (RP) while the left eye (B) has less pronounced paraarteriolar intraretinal pigment migration only. Fundus autofluorescence delineates a small central island of residual RPE in the right eye (C) while the left eye (D) shows subtle hyperautofluorescent paravascular changes not in keeping with RP. Optical coherence tomography confirms asymmetric disease with relative sparing of the central macular outer retina in the right eye (E) with entirely normal retinal lamination in the left eye (F). No relevant genetic variants were detected on pNGS. This patient had a history of childhood meningitis with no family history, stable visual acuity (6/12 right, 6/6 left), and no progression of visual symptoms. Clinical consensus opinion was reached of asymmetric post-inflammatory pigmentary retinal changes, and no further genetic testing was indicated. Pedigree #3: Colour fundus photographs (G,H) of a 47-year-old woman with asymmetrical macular atrophy. Autofluorescence (I,J) shows hypoautofluorescent areas of macular atrophy with surrounding/intervening areas of hyperautofluorescence. Retinal vasculature and periphery are otherwise normal. OCT (K,L) shows outer retinal atrophy, subretinal fibrosis, and focal choroidal attenuation. The clinical phenotype was reassessed, and a diagnosis of punctate inner choroidopathy was made, with the decision to not pursue further genetic testing. The patient was referred to the uveitis clinic.

Figure 3

Multimodal imaging (MMI) of cases consistent with IRD. Pedigree #18: (A,B): Colour fundus photographs demonstrating symmetrical predominantly midperipheral pigmentary changes. (C,D): Autofluorescence showing patchy midperipheral and focal posterior pole hypoautofluorescence consistent with retinal pigment epithelium (RPE) atrophy. (E,F): OCT demonstrating predominant preservation of photoreceptor/RPE laminae with a focal nasal defect of photoreceptor inner segments (IS) in E and multiple (nasal and temporal) IS/RPE defects in F. A second pathogenic PEX7 variant (OMIM*601757, c.40A > C, p.Thr14Pro) was detected via single gene sequencing, confirming a genetic diagnosis of autosomal recessive (AR) Refsum disease (OMIM#614879). This patient also had systemic features of this condition including ataxia. Pedigree #22: (G,H): Colour fundus photographs showing macular atrophy with surrounding subretinal flecks with sparing outside the vascular arcades. (I,J): Autofluorescence confirms foveal hypoautofluorescence surrounded by hyperautofluorescent flecks mostly confined to the macula with some flecks nasal to the optic nerve head in J. (K,L): OCT showing foveal outer retinal atrophy. These multimodal imaging features are in keeping with AR Stargardt disease (OMIM#248200). Single gene (i.e., ABCA4) testing allowed the detection of a second pathogenic allele (c.4253 + 43G > A p.[=, Ile1377Hisfs*3]), genetically resolving this case.