Clinically Focused Molecular Investigation of 1000 Consecutive Families with Inherited Retinal Disease

Abstract

Purpose To devise a comprehensive multi-platform genetic testing strategy for inherited retinal disease and describe its performance in 1,000 consecutive families seen by a single clinician. Methods The clinical records of all patients seen by a single retina specialist between January 2010 and June 2016 were reviewed and all patients who met the clinical criteria for a diagnosis of inherited retinal disease were included in the study. Each patient was assigned to one of 62 diagnostic categories and this clinical diagnosis was used to define the scope and order of the molecular investigations that were performed. The number of nucleotides evaluated in a given subject ranged from two (a multiplex allele-specific assay for the most common mutations in BBS1 and BBS10) to nearly 900,000 (the coding sequences, and splice junctions of 305 genes known to cause inherited retinal disease). Results Disease-causing genotypes were identified in 760 families (76%). These genotypes were distributed across 104 different genes. More than 70% of these 104 genes have coding sequences small enough to be efficiently packaged into an adeno-associated virus. Mutations in ABCA4 were the most common cause of disease in this cohort (173 families) while mutations in 80 genes caused disease in five or fewer families (i.e., 0.5% or less). Disease-causing genotypes were identified in 576 of the families without next generation sequencing (NGS). This included 23 families with mutations in the repetitive region of RPGR exon 15 that would have been missed by NGS. Whole exome sequencing of the remaining 424 families revealed mutations in an additional 182, and whole genome sequencing of four of the remaining 242 families revealed two additional genotypes that were invisible by the other methods. Performing the testing in a clinically-focused tiered fashion would be 6.1% more sensitive, 17.7% less expensive and have a significantly lower average false genotype rate than using whole exome sequencing to assess more than 300 genes in all patients (7.1 vs. 128%; p<0.001). Conclusions Genetic testing for inherited retinal disease is now more than 75% sensitive. A clinically-directed tiered testing strategy can increase sensitivity and improve statistical significance without increasing cost.

A nationwide genetic analysis of inherited retinal diseases in Israel as assessed by the Israeli inherited retinal disease consortium (IIRDC)

“…The two most frequently mutated genes in the Israeli IRD cohort are ABCA4 (14%) and USH2A (7%). These findings are similar to those found in large IRD cohorts from other populations (Carss et al, ; Dockery et al, ; Haer‐Wigman et al, ; Stone et al, ). However, some of our findings are unique.…”

“…Overall, we succeeded to identify the genetic basis of disease in 56% of the recruited families (64% among families recruited up to December 2015). This percentage is within the range of diagnostic rates reported recently in other large cohorts of IRD patients (Carss et al, ; Dockery et al, ; Haer‐Wigman et al, ; Stone et al, ). It should be noted that of the unsolved families, only 17% have undergone WES and only 0.5% have undergone WGS.…”

“…Here, we describe a cohort of 3,413 Israeli IRD patients recruited since the year 1999. Though genetic analyses in large cohorts of IRD patients have been previously reported (ranging from 722 to 1000 patients; Carss et al, ; Dockery et al, ; Stone et al, ), to the best of our knowledge this is by far the largest IRD cohort reported to date. In 2017, Dockery et al reported partial results of genetic analysis among 750 individuals, as part of the Target 5000 project, a national effort aimed to genetically characterize IRDs in the Irish population.…”

Limited time window for retinal gene therapy in a preclinical model of ciliopathy

“…Approximately a quarter of inherited retinal degeneration (IRD) genes are involved in building and maintaining the sensory cilium of photoreceptor cells, called the outer segment (OS) (1)(2)(3)(4) (also see the RetNet database: https://sph.uth.edu/retnet/). Retinal degenerations caused by cilium-related defects, or retinal ciliopathies, are particularly detrimental because degeneration often begins very early in life (e.g.…”

Optimizing Donor Cellular Dissociation and Subretinal Injection Parameters for Stem Cell-Based Treatments

“…We and others have previously demonstrated the ability to (a) identify the specific genetic mutations responsible for causing various inherited eye diseases in our patients ; (b) generate patient‐derived iPSCs and correct their disease causing mutations using the CRISPR‐Cas9 system ; (c) differentiate iPSCs into retinal cells using good manufacturing practices (GMP) ; and (d) transplant allogenic iPSC‐derived photoreceptor or RPE cells into a large animal model using pars plana vitrectomy and subretinal injections . The goals of this study were twofold: first, we sought to optimize our donor cell dissociation methods in an attempt to isolate the maximum number of healthy RPCs from three‐dimensional (3D) retinal organoids that could be delivered for seeding of retinal cell grafts; second, we sought to characterize and optimize the subretinal injection parameters used for creating a subretinal bleb prior to retinal graft transplantation.…”