Multidisciplinary team directed analysis of whole genome sequencing reveals pathogenic non-coding variants in molecularly undiagnosed inherited retinal dystrophies.

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.

Optic Disc and Retinal Architecture Changes in Patients with Spinocerebellar Ataxia Type 2.

BACKGROUND: ATXN2 is the causative gene of spinocerebellar ataxia type 2 (SCA2) and has been implicated in glaucoma pathogenesis. Therefore, studying ocular changes in SCA2 could uncover clinically relevant changes. OBJECTIVE: The aim was to investigate optic disc and retinal architecture in SCA2. METHODS: We evaluated 14 patients with SCA2 and 26 controls who underwent intraocular pressure measurement, fundoscopy, and macular and peripapillary spectral domain optical coherence tomography (SD-OCT). We compared SD-OCT measurements in SCA2 and controls, and the frequency of glaucomatous changes among SCA2, controls, and 76 patients with other SCAs (types 1, 3, 6, and 7). RESULTS: The macula, peripapillary retinal nerve fiber and inner plexiform layers were thinner in SCA2 than in controls. Increased cup-to-disc ratio was more frequent in SCA2 than in controls and other SCAs. CONCLUSIONS: Ocular changes are part of SCA2 phenotype. Future studies should further investigate retinal and optic nerve architecture in this disorder.

Characterization of Retinal Architecture in Spinocerebellar Ataxia Type 3 and Correlation with Disease Severity.

BACKGROUND: Neurodegeneration affects the brain and peripheral nervous system in spinocerebellar ataxia type 3 (SCA3). As the retina is also involved, studying the retinal architecture in a cohort of patients could reveal clinically relevant biomarkers. OBJECTIVE: The aim is to investigate retinal architecture in SCA3 to identify potential biomarkers. METHODS: We evaluated 38 patients with SCA3 and 25 healthy age-matched controls, who underwent visual acuity assessment, intraocular pressure measurement, and fundoscopy and macular and peripapillary spectral domain optical coherence tomography (SD-OCT). We measured the peripapillary retinal nerve fiber layer (pRNFL) thickness in each quadrant of the temporal-superior-nasal-inferior-temporal chart and the macular layer thicknesses in each sector of the inner circle of the Early Treatment Diabetic Retinopathy Study (IC-ETDRS) grid. Linear regression analysis was employed to test the associations between retinal parameters and age, disease duration, CAG repeats, and SARA (Scale of the Assessment and Rating of Ataxia) and ICARS (International Cooperative Ataxia Rating Scale) scores in SCA3. RESULTS: In all sectors, except for the temporal quadrant, pRNFL was significantly thinner in SCA3 patients than in controls. Average total macular, ganglion cell layer (GCL), and inner plexiform layer (IPL) thicknesses were significantly decreased in SCA3 patients in comparison to controls. The average total macular thickness and the average thicknesses of RNFL, GCL, and IPL negatively correlated with ICARS scores, whereas average GCL and IPL thicknesses negatively correlated with SARA scores. CONCLUSIONS: The retinal ganglion cells, their dendrites, and axons are selectively affected in SCA3 patients. The RNFL, GCL, and IPL thicknesses in SD-OCT correlate with the clinical phenotype and represent potential biomarkers for future clinical trials and natural history studies. © 2021 International Parkinson and Movement Disorder Society.

A Typical Case Presentation with Spontaneous Visual Recovery in Patient Diagnosed with Leber Hereditary Optic Neuropathy due to Rare Point Mutation in MT-ND4 Gene (m.11253T>C) and Literature Review.

Leber hereditary optic neuropathy (LHON) is one of the most common inherited mitochondrial optic neuropathies, caused by mitochondrial DNA (mtDNA) mutations. Three most common mutations, namely m.11778G>A, m.14484T>G and m.3460G>A, account for the majority of LHON cases. These mutations lead to mitochondrial respiratory chain complex I damage. Typically, LHON presents at the 15-35 years of age with male predominance. LHON is associated with severe, subacute, painless bilateral vision loss and account for one of the most common causes of legal blindness in young individuals. Spontaneous visual acuity recovery is rare and has been reported in patients harbouring m.14484T>C mutation. Up to date LHON treatment is limited. Idebenone has been approved by European Medicines Agency (EMA) to treat LHON. However better understanding of disease mechanisms and ongoing treatment trials are promising and brings hope for patients. In this article we report on a patient diagnosed with LHON harbouring rare m.11253T>C mutation in MT-ND4 gene, who experienced spontaneous visual recovery. In addition, we summarise clinical presentation, diagnostic features, and treatment.

Genetic Basis of Inherited Retinal Disease in a Molecularly Characterized Cohort of More Than 3000 Families from the United Kingdom.

PURPOSE: In a large cohort of molecularly characterized inherited retinal disease (IRD) families, we investigated proportions with disease attributable to causative variants in each gene. DESIGN: Retrospective study of electronic patient records. PARTICIPANTS: Patients and relatives managed in the Genetics Service of Moorfields Eye Hospital in whom a molecular diagnosis had been identified. METHODS: Genetic screening used a combination of single-gene testing, gene panel testing, whole exome sequencing, and more recently, whole genome sequencing. For this study, genes listed in the Retinal Information Network online resource (https://sph.uth.edu/retnet/) were included. Transcript length was extracted for each gene (Ensembl, release 94). MAIN OUTCOME MEASURES: We calculated proportions of families with IRD attributable to variants in each gene in the entire cohort, a cohort younger than 18 years, and a current cohort (at least 1 patient encounter between January 1, 2017, and August 2, 2019). Additionally, we explored correlation between numbers of families and gene transcript length. RESULTS: We identified 3195 families with a molecular diagnosis (variants in 135 genes), including 4236 affected individuals. The pediatric cohort comprised 452 individuals from 411 families (66 genes). The current cohort comprised 2614 families (131 genes; 3130 affected individuals). The 20 most frequently implicated genes overall (with prevalence rates per families) were as follows: ABCA4 (20.8%), USH2A (9.1%), RPGR (5.1%), PRPH2 (4.6%), BEST1 (3.9%), RS1 (3.5%), RP1 (3.3%), RHO (3.3%), CHM (2.7%), CRB1 (2.1%), PRPF31 (1.8%), MY07A (1.7%), OPA1 (1.6%), CNGB3 (1.4%), RPE65 (1.2%), EYS (1.2%), GUCY2D (1.2%), PROM1 (1.2%), CNGA3 (1.1%), and RDH12 (1.1%). These accounted for 71.8% of all molecularly diagnosed families. Spearman coefficients for correlation between numbers of families and transcript length were 0.20 (P = 0.025) overall and 0.27 (P = 0.017), -0.17 (P = 0.46), and 0.71 (P = 0.047) for genes in which variants exclusively cause recessive, dominant, or X-linked disease, respectively. CONCLUSIONS: Our findings help to quantify the burden of IRD attributable to each gene. More than 70% of families showed pathogenic variants in 1 of 20 genes. Transcript length (relevant to gene delivery strategies) correlated significantly with numbers of affected families (but not for dominant disease).

SSBP1 mutations in dominant optic atrophy with variable retinal degeneration.

OBJECTIVE: Autosomal dominant optic atrophy (ADOA) starts in early childhood with loss of visual acuity and color vision deficits. OPA1 mutations are responsible for the majority of cases, but in a portion of patients with a clinical diagnosis of ADOA, the cause remains unknown. This study aimed to identify novel ADOA-associated genes and explore their causality. METHODS: Linkage analysis and sequencing were performed in multigeneration families and unrelated patients to identify disease-causing variants. Functional consequences were investigated in silico and confirmed experimentally using the zebrafish model. RESULTS: We defined a new ADOA locus on 7q33-q35 and identified 3 different missense variants in SSBP1 (NM_001256510.1; c.113G>A [p.(Arg38Gln)], c.320G>A [p.(Arg107Gln)] and c.422G>A [p.(Ser141Asn)]) in affected individuals from 2 families and 2 singletons with ADOA and variable retinal degeneration. The mutated arginine residues are part of a basic patch that is essential for single-strand DNA binding. The loss of a positive charge at these positions is very likely to lower the affinity of SSBP1 for single-strand DNA. Antisense-mediated knockdown of endogenous ssbp1 messenger RNA (mRNA) in zebrafish resulted in compromised differentiation of retinal ganglion cells. A similar effect was achieved when mutated mRNAs were administered. These findings point toward an essential role of ssbp1 in retinal development and the dominant-negative nature of the identified human variants, which is consistent with the segregation pattern observed in 2 multigeneration families studied. INTERPRETATION: SSBP1 is an essential protein for mitochondrial DNA replication and maintenance. Our data have established pathogenic variants in SSBP1 as a cause of ADOA and variable retinal degeneration. ANN NEUROL 2019;86:368-383.

Multirater Validation of Peripapillary Hyperreflective Ovoid Mass-like Structures (PHOMS).

Peripapillary hyperreflective ovoid mass-like structures (PHOMS) are a new retinal optical coherence tomography (OCT) finding. The Optic Disc Drusen Studies Consortium had made recommendations to distinguish PHOMS from true optic disc drusen (ODD) in 2018. While publications on PHOMS have increased since then, the accuracy of the definition of PHOMS and reliability of detection is unknown. In this multi-rater study, we demonstrate that the 2018 definition of PHOMS resulted in a poor multi-rater kappa of 0.356. We performed a Delphi consensus process to develop a consistent and refined definition of PHOMS with clear principles around the nature of PHOMS and how they differ from normal anatomy. Fifty explanatory teaching slides, provided as supplementary material, allowed our expert group of raters to achieve a good level of agreement (kappa 0.701, 50 OCT scans, 21 raters). We recommend adopting the refined definition for PHOMS.

Treatment strategies for Leber hereditary optic neuropathy.

PURPOSE OF REVIEW: Leber hereditary optic neuropathy (LHON) is the most common primary mitochondrial DNA (mtDNA) disorder in the population and it carries a poor visual prognosis. In this article, we review the development of treatment strategies for LHON, the evidence base and the areas of unmet clinical need. RECENT FINDINGS: There is accumulating evidence that increasing mitochondrial biogenesis could be an effective strategy for protecting retinal ganglion cells in LHON. A number of clinical trials are currently investigating the efficacy of viral-based gene therapy for patients harbouring the m.11778G>A mtDNA mutation. For female LHON carriers of childbearing age, mitochondrial replacement therapy is being offered to prevent the maternal transmission of pathogenic mtDNA mutations. SUMMARY: Although disease-modifying treatment options remain limited, a better understanding of the underlying disease mechanisms in LHON is paving the way for complementary neuroprotective and gene therapeutic strategies for this mitochondrial optic nerve disorder.

Leber hereditary optic neuropathy: bridging the translational gap.

PURPOSE OF REVIEW: Leber hereditary optic neuropathy (LHON) is the most common primary mitochondrial DNA (mtDNA) genetic disorder in the population. We address the clinical evolution of the disease, the secondary etiological factors that could contribute to visual loss, and the challenging task of developing effective treatments. RECENT FINDINGS: LHON is characterized by a preclinical phase that reflects retinal ganglion cell (RGC) dysfunction before rapid visual deterioration ensues. Children can present atypically with slowly progressive visual loss or an insidious/subclinical onset that frequently results in considerable diagnostic delays. The LHON mtDNA mutation is not sufficient on its own to precipitate RGC loss and the current body of evidence supports a role for smoking and estrogen levels influencing disease conversion. Clinical trials are currently investigating the efficacy of adeno-associated viral vectors-based gene therapy approaches for patients carrying the m.11778G>A mutation. Mitochondrial replacement therapy is being developed as a reproductive option to prevent the maternal transmission of pathogenic mtDNA mutations. SUMMARY: LHON is phenotypically more heterogeneous than previously considered and a complex interplay of genetic, environmental and hormonal factors modulates the risk of a LHON carrier losing vision. Advances in disease modelling, drug screening and genetic engineering offer promising avenues for therapeutic breakthroughs in LHON.

Electrodiagnostic tests of the visual pathway and applications in neuro-ophthalmology.

This article describes the main visual electrodiagnostic tests relevant to neuro-ophthalmology practice, including the visual evoked potential (VEP), and the full-field, pattern and multifocal electroretinograms (ffERG; PERG; mfERG). The principles of electrophysiological interpretation are illustrated with reference to acquired and inherited optic neuropathies, and retinal disorders that may masquerade as optic neuropathy, including ffERG and PERG findings in cone and macular dystrophies, paraneoplastic and vascular retinopathies. Complementary VEP and PERG recordings are illustrated in demyelinating, ischaemic, nutritional (B12), and toxic (mercury, cobalt, and ethambutol-related) optic neuropathies and inherited disorders affecting mitochondrial function such as Leber hereditary optic neuropathy and dominant optic atrophy. The value of comprehensive electrophysiological phenotyping in syndromic diseases is highlighted in cases of SSBP1-related disease and ROSAH (Retinal dystrophy, Optic nerve oedema, Splenomegaly, Anhidrosis and Headache). The review highlights the value of different electrophysiological techniques, for the purposes of differential diagnosis and objective functional phenotyping.

Diagnostic Value of Inter-Eye Difference Metrics on OCT for Myelin Oligodendrocyte Glycoprotein Antibody-Associated Optic Neuritis.

BACKGROUND AND OBJECTIVES: The 2022 International Consortium for Optic Neuritis diagnostic criteria for optic neuritis (ON) include optical coherence tomography (OCT). The diagnostic value of intereye difference (IED) metrics is high for ON in patients with multiple sclerosis and aquaporin-4 antibody seropositive neuromyelitis optica spectrum disorders, but unknown in myelin oligodendrocyte glycoprotein antibody-associated ON (MOG-ON). METHODS: A multicenter validation study was conducted on the published IED cutoff values (>4% or >4 µm in the macular ganglion cell and inner plexiform layer [mGCIP] or >5% or >5 µm in the peripapillary retinal nerve fiber layer [pRNFL]) in individuals with MOG-ON and age-matched and sex-matched healthy controls (HCs). Structural data were acquired with Spectralis spectral-domain OCT >6 months after ON. We calculated sensitivity, specificity, and receiver operating characteristics for both intereye percentage (IEPD) and absolute difference (IEAD). RESULTS: A total of 66 individuals were included (MOG-ON N = 33; HCs N = 33). ON was unilateral in 20 and bilateral in 13 subjects. In the pooled analysis, the mGCIP IEPD was most sensitive (92%), followed by the mGCIP IEAD (88%) and pRNFL (84%). The same pattern was found for the specificity (mGCIP IEPD 82%, IEAD 82%; pRNFL IEPD 82%, IEAD 79%).In subgroup analyses, the diagnostic sensitivity was higher in subjects with unilateral ON (>99% for all metrics) compared with bilateral ON (61%-78%). DISCUSSION: In individuals with MOG-ON, the diagnostic accuracy of OCT-based IED metrics for ON was high, especially of mGCIP IEPD. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that the intereye difference on OCT can distinguish between those with MOG and normal controls.

Investigating Splice Defects in USH2A Using Targeted Long-Read Sequencing.

Biallelic variants in USH2A are associated with retinitis pigmentosa (RP) and Type 2 Usher Syndrome (USH2), leading to impaired vision and, additionally, hearing loss in the latter. Although the introduction of next-generation sequencing into clinical diagnostics has led to a significant uplift in molecular diagnostic rates, many patients remain molecularly unsolved. It is thought that non-coding variants or variants of uncertain significance contribute significantly to this diagnostic gap. This study aims to demonstrate the clinical utility of the reverse transcription-polymerase chain reaction (RT-PCR)-Oxford Nanopore Technology (ONT) sequencing of USH2A mRNA transcripts from nasal epithelial cells to determine the splice-altering effect of candidate variants. Five affected individuals with USH2 or non-syndromic RP who had undergone whole genome sequencing were recruited for further investigation. All individuals had uncertain genotypes in USH2A, including deep intronic rare variants, c.8682-654C>G, c.9055+389G>A, and c.9959-2971C>T; a synonymous variant of uncertain significance, c.2139C>T; p.(Gly713=); and a predicted loss of function duplication spanning an intron/exon boundary, c.3812-3_3837dup p.(Met1280Ter). In silico assessment using SpliceAI provided splice-altering predictions for all candidate variants which were investigated using ONT sequencing. All predictions were found to be accurate; however, in the case of c.3812-3_3837dup, the outcome was a complex cryptic splicing pattern with predominant in-frame exon 18 skipping and a low level of exon 18 inclusion leading to the predicted stop gain. This study detected and functionally characterised simple and complex mis-splicing patterns in USH2A arising from previously unknown deep intronic variants and previously reported variants of uncertain significance, confirming the pathogenicity of the variants.

Spectrum of Genetic Variants in the Most Common Genes Causing Inherited Retinal Disease in a Large Molecularly Characterized United Kingdom Cohort.

PURPOSE: Inherited retinal disease (IRD) is a leading cause of blindness. Recent advances in gene-directed therapies highlight the importance of understanding the genetic basis of these disorders. This study details the molecular spectrum in a large United Kingdom (UK) IRD patient cohort. DESIGN: Retrospective study of electronic patient records. PARTICIPANTS: Patients with IRD who attended the Genetics Service at Moorfields Eye Hospital between 2003 and July 2020, in whom a molecular diagnosis was identified. METHODS: Genetic testing was undertaken via a combination of single-gene testing, gene panel testing, whole exome sequencing, and more recently, whole genome sequencing. Likely disease-causing variants were identified from entries within the genetics module of the hospital electronic patient record (OpenEyes Electronic Medical Record). Analysis was restricted to only genes listed in the Genomics England PanelApp R32 Retinal Disorders panel (version 3.24), which includes 412 genes associated with IRD. Manual curation ensured consistent variant annotation and included only plausible disease-associated variants. MAIN OUTCOME MEASURES: Detailed analysis was performed for variants in the 5 most frequent genes (ABCA4, USH2A, RPGR, PRPH2, and BEST1), as well as for the most common variants encountered in the IRD study cohort. RESULTS: We identified 4415 individuals from 3953 families with molecularly diagnosed IRD (variants in 166 genes). Of the families, 42.7% had variants in 1 of the 5 most common IRD genes. Complex disease alleles contributed to disease in 16.9% of affected families with ABCA4-associated retinopathy. USH2A exon 13 variants were identified in 43% of affected individuals with USH2A-associated IRD. Of the RPGR variants, 71% were clustered in the ORF15 region. PRPH2 and BEST1 variants were associated with a range of dominant and recessive IRD phenotypes. Of the 20 most prevalent variants identified, 5 were not in the most common genes; these included founder variants in CNGB3, BBS1, TIMP3, EFEMP1, and RP1. CONCLUSIONS: We describe the most common pathogenic IRD alleles in a large single-center multiethnic UK cohort and the burden of disease, in terms of families affected, attributable to these variants. Our findings will inform IRD diagnoses in future patients and help delineate the cohort of patients eligible for gene-directed therapies under development. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Therapeutic benefit of idebenone in patients with Leber hereditary optic neuropathy: The LEROS nonrandomized controlled trial.

Leber hereditary optic neuropathy (LHON) is a mitochondrial disease leading to rapid and severe bilateral vision loss. Idebenone has been shown to be effective in stabilizing and restoring vision in patients treated within 1 year of onset of vision loss. The open-label, international, multicenter, natural history-controlled LEROS study (ClinicalTrials.gov NCT02774005) assesses the efficacy and safety of idebenone treatment (900 mg/day) in patients with LHON up to 5 years after symptom onset (N = 199) and over a treatment period of 24 months, compared to an external natural history control cohort (N = 372), matched by time since symptom onset. LEROS meets its primary endpoint and confirms the long-term efficacy of idebenone in the subacute/dynamic and chronic phases; the treatment effect varies depending on disease phase and the causative mtDNA mutation. The findings of the LEROS study will help guide the clinical management of patients with LHON.

Mitochondria and the eye-manifestations of mitochondrial diseases and their management.

Historically, distinct mitochondrial syndromes were recognised clinically by their ocular features. Due to their predilection for metabolically active tissue, mitochondrial diseases frequently involve the eye, resulting in a range of ophthalmic manifestations including progressive external ophthalmoplegia, retinopathy and optic neuropathy, as well as deficiencies of the retrochiasmal visual pathway. With the wider availability of genetic testing in clinical practice, it is now recognised that genotype-phenotype correlations in mitochondrial diseases can be imprecise: many classic syndromes can be associated with multiple genes and genetic variants, and the same genetic variant can have multiple clinical presentations, including subclinical ophthalmic manifestations in individuals who are otherwise asymptomatic. Previously considered rare diseases with no effective treatments, considerable progress has been made in our understanding of mitochondrial diseases with new therapies emerging, in particular, gene therapy for inherited optic neuropathies.