BACKGROUND:Genomic variant prioritisation is one of the most significant bottlenecks to mainstream genomic testing in healthcare. Tools to improve precision while ensuring high recall are critical to successful mainstream clinical genomic testing, in particular for whole genome sequencing where millions of variants must be considered for each patient. METHODS:We developed EyeG2P, a publicly available database and web application using the Ensembl Variant Effect Predictor. EyeG2P is tailored for efficient variant prioritisation for individuals with inherited ophthalmic conditions. We assessed the sensitivity of EyeG2P in 1234 individuals with a broad range of eye conditions who had previously received a confirmed molecular diagnosis through routine genomic diagnostic approaches. For a prospective cohort of 83 individuals, we assessed the precision of EyeG2P in comparison with routine diagnostic approaches. For 10 additional individuals, we assessed the utility of EyeG2P for whole genome analysis. RESULTS:EyeG2P had 99.5% sensitivity for genomic variants previously identified as clinically relevant through routine diagnostic analysis (n=1234 individuals). Prospectively, EyeG2P enabled a significant increase in precision (35% on average) in comparison with routine testing strategies (p<0.001). We demonstrate that incorporation of EyeG2P into whole genome sequencing analysis strategies can reduce the number of variants for analysis to six variants, on average, while maintaining high diagnostic yield. CONCLUSION:Automated filtering of genomic variants through EyeG2P can increase the efficiency of diagnostic testing for individuals with a broad range of inherited ophthalmic disorders.

The purpose of this paper is to identify likely pathogenic non-coding variants in inherited retinal dystrophy (IRD) genes, using genome sequencing (GS). Patients with IRD were recruited to the study and underwent comprehensive ophthalmological evaluation and GS. The results of GS were investigated through virtual gene panel analysis, and plausible pathogenic variants and clinical phenotype evaluated by the multidisciplinary team (MDT) discussion. For unsolved patients in whom a specific gene was suspected to harbor a missed pathogenic variant, targeted re-analysis of non-coding regions was performed on GS data. Candidate variants were functionally tested by messenger RNA analysis, minigene or luciferase reporter assays. Previously unreported, likely pathogenic, non-coding variants in 7 genes (PRPF31, NDP, IFT140, CRB1, USH2A, BBS10 and GUCY2D), were identified in 11 patients. These were shown to lead to mis-splicing (PRPF31, IFT140, CRB1 and USH2A) or altered transcription levels (BBS10 and GUCY2D). MDT-led, phenotype-driven, non-coding variant re-analysis of GS is effective in identifying the missing causative alleles.

Purpose:North Carolina macular dystrophy (NCMD) is an autosomal dominant, congenital disorder affecting the central retina. Here, we report clinical and genetic findings in three families segregating NCMD and use epigenomic datasets from human tissues to gain insights into the effect of NCMD-implicated variants.Methods:Clinical assessment and genetic testing were performed. Publicly available transcriptomic and epigenomic datasets were analyzed and the activity-by-contact method for scoring enhancer elements and linking them to target genes was used.Results:A previously described, heterozygous, noncoding variant upstream of the PRDM13 gene was detected in all six affected study participants (chr6:100,040,987G>C [GRCh37/hg19]). Interfamilial and intrafamilial variability were observed; the visual acuity ranged from 0.0 to 1.6 LogMAR and fundoscopic findings ranged from visually insignificant, confluent, drusen-like macular deposits to coloboma-like macular lesions. Variable degrees of peripheral retinal spots (which were easily detected on widefield retinal imaging) were observed in all study subjects. Notably, a 6-year-old patient developed choroidal neovascularization and required treatment with intravitreal bevacizumab injections. Computational analysis of the five single nucleotide variants that have been implicated in NCMD revealed that these noncoding changes lie within two putative enhancer elements; these elements are predicted to interact with PRDM13 in the developing human retina. PRDM13 was found to be expressed in the fetal retina, with greatest expression in the amacrine precursor cell population.Conclusions:We provide further evidence supporting the role of PRDM13 dysregulation in the pathogenesis of NCMD and highlight the usefulness of widefield retinal imaging in individuals suspected to have this condition.

OBJECTIVE:USH2A-related disorders are characterised by genetic and phenotypic heterogeneity, and are associated with a spectrum of sensory deficits, ranging from deaf blindness to blindness with normal hearing. It has been previously proposed that the presence of specific USH2A alleles can be predictive of unaffected hearing. This study reports the clinical and genetic findings in a group of patients with USH2A-related disease and evaluates the validity of the allelic hierarchy model. PATIENTS AND INTERVENTION:USH2A variants from 27 adults with syndromic and nonsyndromic USH2A-related disease were analyzed according to a previously reported model of allelic hierarchy. The analysis was replicated on genotype-phenotype correlation information from 197 individuals previously reported in 2 external datasets. MAIN OUTCOME MEASURE:Genotype-phenotype correlations in USH2A-related disease. RESULTS:A valid allelic hierarchy model was observed in 93% of individuals with nonsyndromic USH2A-retinopathy (n = 14/15) and in 100% of patients with classic Usher syndrome type IIa (n = 8/8). Furthermore, when two large external cohorts of cases were combined, the allelic hierarchy model was valid across 85.7% (n = 78/91) of individuals with nonsyndromic USH2A-retinopathy and 95% (n = 123/129) of individuals with classic Usher syndrome type II (p = 0.012, χ test). Notably, analysis of all three patient datasets revealed that USH2A protein truncating variants were reported most frequently in individuals with hearing loss. CONCLUSION:Genetic testing results in individuals suspected to have an USH2A-related disorder have the potential to facilitate personalized audiological surveillance and rehabilitation pathways.

Purpose A key property to consider in all genetic tests is clinical utility, the ability of the test to influence patient management and health outcomes. Here we assess the current clinical utility of genetic testing in diverse pediatric inherited eye disorders (IEDs). Methods Two hundred one unrelated children (0–5 years old) with IEDs were ascertained through the database of the North West Genomic Laboratory Hub, Manchester, UK. The cohort was collected over a 7-year period (2011–2018) and included 74 children with bilateral cataracts, 8 with bilateral ectopia lentis, 28 with bilateral anterior segment dysgenesis, 32 with albinism, and 59 with inherited retinal disorders. All participants underwent panel-based genetic testing. Results The diagnostic yield of genetic testing for the cohort was 64% (ranging from 39% to 91% depending on the condition). The test result led to altered management (including preventing additional investigations or resulting in the introduction of personalized surveillance measures) in 33% of probands (75% for ectopia lentis, 50% for cataracts, 33% for inherited retinal disorders, 7% for anterior segment dysgenesis, 3% for albinism). Conclusion Genetic testing helped identify an etiological diagnosis in the majority of preschool children with IEDs. This prevented additional unnecessary testing and provided the opportunity for anticipatory guidance in significant subsets of patients.

Macular pattern dystrophies of the retinal pigment epithelium have various causes and effects on vision, with abnormalities particularly evident on short-wavelength autofluorescence imaging. A number of genes have been implicated, frequently PRPH2. In 2015, 3 heterozygous missense variants in CTNNA1 (encoding the widely expressed α-catenin protein) were associated with butterfly-shaped pigment dystrophy: c.953T→C (p.Leu318Ser), c.1293T→G (p.Ile431Met), and c.919G→A (p.Glu307Lys).1 Herein, we describe 6 additional families with CTNNA1 missense variants (affected individuals exhibiting pattern dystrophy), corroborating the previous report.

Next-generation sequencing has revolutionized rare disease diagnostics, but many patients remain without a molecular diagnosis, particularly because many candidate variants usually survive despite strict filtering. Exomiser was launched in 2014 as a Java tool that performs an integrative analysis of patients’ sequencing data and their phenotypes encoded with Human Phenotype Ontology (HPO) terms. It prioritizes variants by leveraging information on variant frequency, predicted pathogenicity, and gene-phenotype associations derived from human diseases, model organisms, and protein–protein interactions. Early published releases of Exomiser were able to prioritize disease-causative variants as top candidates in up to 97% of simulated whole-exomes. The size of the tested real patient datasets published so far are very limited. Here, we present the latest Exomiser version 12.0.1 with many new features. We assessed the performance using a set of 134 whole-exomes from patients with a range of rare retinal diseases and known molecular diagnosis. Using default settings, Exomiser ranked the correct diagnosed variants as the top candidate in 74% of the dataset and top 5 in 94%; not using the patients’ HPO profiles (i.e., variant-only analysis) decreased the performance to 3% and 27%, respectively. In conclusion, Exomiser is an effective support tool for rare Mendelian phenotype-driven variant prioritization.

Thirty percent of all inherited retinal disease (IRD) is accounted for by conditions with extra-ocular features. This study aimed to establish the genetic diagnostic pick-up rate for IRD patients with one or more extra-ocular features undergoing panel-based screening in a clinical setting. One hundred and six participants, tested on a gene panel which contained both isolated and syndromic IRD genes, were retrospectively ascertained from the Manchester Genomic Diagnostics Laboratory database spanning 6 years (2012–2017). Phenotypic features were extracted from the clinical notes and classified according to Human Phenotype Ontology; all identified genetic variants were interpreted in accordance to the American College of Medical Genetics and Genomics guidelines. Overall, 49% ( n = 52) of patients received a probable genetic diagnosis. A further 6% ( n = 6) had a single disease-associated variant in an autosomal recessive disease-relevant gene. Fifty-two percent ( n = 55) of patients had a clinical diagnosis at the time of testing. Of these, 71% ( n = 39) received a probable genetic diagnosis. By contrast, for those without a provisional clinical diagnosis ( n = 51), only 25% ( n = 13) received a probable genetic diagnosis. The clinical diagnosis of Usher ( n = 33) and Bardet–Biedl syndrome ( n = 10) was confirmed in 67% ( n = 22) and 80% ( n = 8), respectively. The testing diagnostic rate in patients with clinically diagnosed multisystemic IRD conditions was significantly higher than those without one (71% versus 25%; p value < 0.001). The lower pick-up rate in patients without a clinical diagnosis suggests that panel-based approaches are unlikely to be the most effective means of achieving a molecular diagnosis for this group. Here, we suggest that genome-wide approaches (whole exome or genome) are more appropriate.

Persistent placoid maculopathy (PPM) is a bilateral inflammatory chorioretinopathy characterized by long-standing plaque-like macular lesions. No systemic manifestations have been reported to date. We describe a case of PPM complicated by cerebral vasculitis, suggesting that neurological symptoms, including headache, should be enquired about in all PPM subjects.

Linear scleroderma is a characteristic form of scleroderma that typically affects children. Ocular manifestations may be present, especially when the frontoparietal area of the head is affected. We present the case of a 5-year-old boy with craniofacial linear scleroderma ("en coup de sabre") who developed exudative retinal detachment. Angiographic and neuroimaging findings are presented, and the importance of regular fundus examination is highlighted.

To gain insights into microgravity-induced ophthalmic changes (microgravity ocular syndrome), and as part of a project investigating effects of future planetary habitats, we investigated the effect of acute hypercapnia following 10-day bed rest and hypoxia on posterior eye structures. Female subjects (N = 7) completed three 10-day experimental interventions: 1) normoxic bed rest [NBR; partial pressure of inspired O2 (PiO2 ) = 132.9 ± 0.3 Torr]; 2) hypoxic ambulatory confinement (HAMB; PiO2 = 90.4 ± 0.3 Torr); and 3) hypoxic bed rest (HBR; n = 12; PiO2 = 90.4 ± 0.3 Torr). Before and on the last day of each intervention, optical coherence tomography (OCT) of the optic disk was performed, and the thicknesses of the retinal nerve fiber layer (RNFL), retina, and choroid were measured. OCT examinations were conducted with the subjects breathing the prevailing normocapnic breathing mixture (either normoxic or hypoxic) and then following a 10-min period of breathing the same gas mixture, but with the addition of 1% CO2 Choroidal thickness was greater during both bed-rest conditions (NBR and HBR) compared with the ambulatory (HAMB) condition (ANOVA, P < 0.001). Increases in RNFL thickness compared with baseline were observed in the hypoxic trials (HBR, P < 0.001; and HAMB, P = 0.021), but not the normoxic trial (NBR). A further increase in RNFL thickness (P = 0.019) was observed after the 10-min hypercapnic trial in the NBR condition only. The fact that choroidal thickness was not affected by Po2 or Pco2, but increased by bed rest, suggests a hydrostatic rather than a vasoactive effect. The increments in RNFL thickness were most likely associated with local hypoxia and hypercapnia-induced dilatation of the retinal blood vessels.

Early onset retinal dystrophy (EORD) is a clinical and genetically heterozygous disorder characterized by progressive photoreceptor degeneration resulting in severe visual impairment from early childhood. Mutations in the CRB1 gene are one of the commonest causes of EORD in European populations (Henderson et al. 2011; Bujakowska et al. 2012) and, to date, over 250 mutations have been reported in this gene. Although Crumbs-homologue-1 (CRB1) retino-pathy exhibits significant clinical variability (Bujakowska et al. 2012; Alves et al. 2014), there is a recognized phenotype that can help guide molecular genetic testing. Common clinical features include increased retinal thickness on optical coherence tomography (OCT), hypermetropic refractive error and deep nummular pigmentation. We present the case of a child who presented during screening for uveitis associated with juvenile idiopathic arthritis with macular oedema and was found to have an EORD and mutations in CRB1. An 18-month-old boy was referred from a rheumatologist for uveitis screening after a diagnosis of juvenile idiopathic arthritis was made. The child was experiencing left ankle pain, but there were no signs of uveitis and was in good health. There was no family history of arthritis or ocular disease. On examination, there were no signs or symptoms of uveitis, and fundoscopy was normal. Two years later in a routine follow-up appointment, although he remained asymptomatic at the time and still had no evidence of uveitis, he was found to have macula oedema in both eyes. A month later, he reported nyctalopia and central visual disturbance. Refraction revealed a +3.50/+0.50 × 86 and +4.50/+0.50 × 84 hypermetropic correction. Examination revealed mottled pigmentary changes in the retinal periphery (Fig. 1A,B) and macular thickening in both eyes. Optical coherence tomography (OCT) showed large hypo-reflective intraretinal spaces in the right eye and smaller cystic spaces in the left eye consistent with macular oedema (Fig. 1C–F). A full field electroretinogram revealed no recordable response in both eyes. Based on the phenotype described above, and in the absence of uveitis, a retinal dystrophy was suspected and the family underwent genetic counselling and panel-based genetic diagnostic testing (105 retinal dystrophy associated genes) (O'Sullivan et al. 2012). Two missense variants were detected in the CRB1 gene: c.584G>T, p.(Cys195Phe) and c.2843G>A, p.(Cys948Tyr). These changes have previously been described as disease causing (den Hollander et al. 1999, 2004) and are likely responsible for the patient's phenotype. The CRB1 gene encodes the CRB1 protein. This protein, localized above the adherens junctions in Müller glial cells, has a role in retinal development (Alves et al. 2014). Individuals with defects in CRB1 are likely to possess abnormal retinal architecture. Typically, there is severe retinal involvement with early macular changes. A previous study has also identified macular oedema in six of 11 cases (age range: 6–39 years; Bujakowska et al. 2012). This case highlights the need to be vigilant in our examination of patients when screening for uveitis for the presence of coexisting ocular disease. Furthermore, our findings add to and support the association between macular oedema and CRB1-related disease in a younger child. Crumbs-homologue-1 (CRB1) retinopathy should be considered as a differential in children presenting with bilateral macular oedema. Although carbonic anhydrase inhibitors have been previously used in such cases, there is no conclusive evidence of their efficacy. This report demonstrates the importance of the utility of a combined approach incorporating the assessment of retinal function (electrophysiology), retinal structure (fundus appearance and OCT) and genomic analysis in cases with early onset visual loss.

Defects in USH2A cause both isolated retinal disease and Usher syndrome (ie, retinal disease and deafness). To gain insights into isolated/nonsyndromic USH2A retinopathy, we screened USH2A in 186 probands with recessive retinal disease and no hearing complaint in childhood (discovery cohort) and in 84 probands with recessive retinal disease (replication cohort). Detailed phenotyping, including retinal imaging and audiological assessment, was performed in individuals with two likely disease-causing USH2A variants. Further genetic testing, including screening for a deep-intronic disease-causing variant and large deletions/duplications, was performed in those with one likely disease-causing change. Overall, 23 of 186 probands (discovery cohort) were found to harbour two likely disease-causing variants in USH2A. Some of these variants were predominantly associated with nonsyndromic retinal degeneration (‘retinal disease-specific’); these included the common c.2276 G>T, p.(Cys759Phe) mutation and five additional variants: c.2802 T>G, p.(Cys934Trp); c.10073 G>A, p.(Cys3358Tyr); c.11156 G>A, p.(Arg3719His); c.12295-3 T>A; and c.12575 G>A, p.(Arg4192His). An allelic hierarchy was observed in the discovery cohort and confirmed in the replication cohort. In nonsyndromic USH2A disease, retinopathy was consistent with retinitis pigmentosa and the audiological phenotype was variable. USH2A retinopathy is a common cause of nonsyndromic recessive retinal degeneration and has a different mutational spectrum to that observed in Usher syndrome. The following model is proposed: the presence of at least one ‘retinal disease-specific’ USH2A allele in a patient with USH2A-related disease results in the preservation of normal hearing. Careful genotype–phenotype studies such as this will become increasingly important, especially now that high-throughput sequencing is widely used in the clinical setting.