Targeted deletion of an NRL- and CRX-regulated alternative promoter specifically silences FERM and PDZ domain containing 1 (Frmpd1) in rod photoreceptors.

Regulation of cell type-specific gene expression is critical for generating neuronal diversity. Transcriptome analyses have unraveled extensive heterogeneity of transcribed sequences in retinal photoreceptors because of alternate splicing and/or promoter usage. Here we show that Frmpd1 (FERM and PDZ domain containing 1) is transcribed from an alternative promoter specifically in the retina. Electroporation of Frmpd1 promoter region, -505 to +382 bp, activated reporter gene expression in mouse retina in vivo. A proximal promoter sequence (-8 to +33 bp) of Frmpd1 binds to neural retina leucine zipper (NRL) and cone-rod homeobox protein (CRX), two rod-specific differentiation factors, and is necessary for activating reporter gene expression in vitro and in vivo. Clustered regularly interspaced short palindromic repeats/Cas9-mediated deletion of the genomic region, including NRL and CRX binding sites, in vivo completely eliminated Frmpd1 expression in rods and dramatically reduced expression in rod bipolar cells, thereby overcoming embryonic lethality caused by germline Frmpd1 deletion. Our studies demonstrate that a cell type-specific regulatory control region is a credible target for creating loss-of-function alleles of widely expressed genes.

Targeted next generation sequencing and family survey enable correct genetic diagnosis in CRX associated macular dystrophy - a case report.

BACKGROUND: We present 3 members of a family with macular dystrophy, originally diagnosed as Stargardt disease, with a significantly variable age at onset, caused by a heterozygous mutation in CRX. CASE PRESENTATION: A 43-year-old female with bull's eye maculopathy, whose sister was diagnosed with Stargardt disease previously at another centre, was found to have a single ABCA4 variant. Further examination of the family revealed that the asymptomatic father was also affected, indicating a dominant pattern of inheritance. In addition, the ABCA4 variant was not identified in the sister originally diagnosed with Stargardt disease. Next generation sequencing identified a heterozygous c.121C > T, p.R41W missense mutation in CRX in all 3 affected members. CONCLUSIONS: We describe a common phenotype, but with variable age at onset, with autosomal dominant inheritance and reduced penetrance in a family found to have a pathogenic sequence variant in CRX. This illustrates the importance of panel based molecular genetic testing accompanied by family studies to establish a definitive diagnosis.

Phenopolis: an open platform for harmonization and analysis of genetic and phenotypic data.

SUMMARY: Phenopolis is an open-source web server providing an intuitive interface to genetic and phenotypic databases. It integrates analysis tools such as variant filtering and gene prioritization based on phenotype. The Phenopolis platform will accelerate clinical diagnosis, gene discovery and encourage wider adoption of the Human Phenotype Ontology in the study of rare genetic diseases. AVAILABILITY AND IMPLEMENTATION: A demo of the website is available at https://phenopolis.github.io . If you wish to install a local copy, source code and installation instruction are available at https://github.com/phenopolis . The software is implemented using Python, MongoDB, HTML/Javascript and various bash shell scripts. CONTACT: n.pontikos@ucl.ac.uk. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Novel homozygous splicing mutations in ARL2BP cause autosomal recessive retinitis pigmentosa.

Purpose: Mutations in ARL2BP, encoding ADP-ribosylation factor-like 2 binding protein, have recently been implicated as a cause of autosomal recessive retinitis pigmentosa (arRP), with three homozygous variants identified to date. In this study, we performed next-generation sequencing to reveal additional arRP cases associated with ARL2BP variants. Methods: Whole-genome sequencing (WGS) or whole-exome sequencing (WES) was performed in 1,051 unrelated individuals recruited for the UK Inherited Retinal Disease Consortium and NIHR-BioResource Rare Diseases research studies. Sanger sequencing was used to validate the next-generation sequencing data, and reverse transcriptase (RT)-PCR analysis was performed on RNA extracted from blood from affected individuals to test for altered splicing of ARL2BP. Detailed phenotyping was performed, including clinical evaluation, electroretinography, fundus photography, fundus autofluorescence imaging, and spectral-domain optical coherence tomography. Results: Homozygous variants in ARL2BP (NM_012106.3) were identified in two unrelated individuals with RP. The variants, c.207+1G>A and c.390+5G>A, at conserved splice donor sites for intron 3 and intron 5, respectively, were predicted to alter the pre-mRNA splicing of ARL2BP. RT-PCR spanning the affected introns revealed that both variants caused abnormal splicing of ARL2BP in samples from affected individuals. Conclusions: This study identified two homozygous variants in ARL2BP as a rare cause of arRP. Further studies are required to define the underlying disease mechanism causing retinal degeneration as a result of mutations in ARL2BP and any phenotype-genotype correlation associated with residual levels of the wild-type transcript.

The hypothalamic photoreceptors regulating seasonal reproduction in birds: a prime role for VA opsin.

Extraretinal photoreceptors located within the medio-basal hypothalamus regulate the photoperiodic control of seasonal reproduction in birds. An action spectrum for this response describes an opsin photopigment with a λmax of ∼ 492 nm. Beyond this however, the specific identity of the photopigment remains unresolved. Several candidates have emerged including rod-opsin; melanopsin (OPN4); neuropsin (OPN5); and vertebrate ancient (VA) opsin. These contenders are evaluated against key criteria used routinely in photobiology to link orphan photopigments to specific biological responses. To date, only VA opsin can easily satisfy all criteria and we propose that this photopigment represents the prime candidate for encoding daylength and driving seasonal breeding in birds. We also show that VA opsin is co-expressed with both gonadotropin-releasing hormone (GnRH) and arginine-vasotocin (AVT) neurons. These new data suggest that GnRH and AVT neurosecretory pathways are endogenously photosensitive and that our current understanding of how these systems are regulated will require substantial revision.

Identification of rod- and cone-specific expression signatures to identify candidate genes for retinal disease.

Recent advances in technology have greatly increased our ability to identify genetic variants in individuals with retinal disease. However, determining which are likely to be pathogenic remains a challenging task. Using a transgenic coneless (cl) mouse model, together with rodless (rd/rd) and rodless/coneless (rd/rd cl) mice, we have characterised patterns of gene expression in the rod and cone photoreceptors at a genome-wide level. We examined the expression of >27,000 genes in the mice lacking rods, cones or both and compared them with wild type animals. We identified a list of 418 genes with highly significant changes in expression in one or more of the transgenic strains. Pathway analysis confirmed that expected Gene Ontology terms such as phototransduction were over-represented amongst these genes. However, many of these genes have no previously known function in the retina. Gene set enrichment analysis further demonstrated that the mouse orthologues of known human retinal disease genes were significantly enriched amongst those genes with decreased expression. Comparison of our data to human disease loci with no known causal genetic changes has highlighted genes with significant changes in expression making these strong candidates for further screening. These data add to the current literature through the utilisation of the specific cl and rd/rd cl models. Moreover, this study identifies genes that appear to be implicated in photoreceptor function thereby providing a valuable filter for variants identified by high-throughput sequencing in individuals with retinal disease.

Detailed phenotypic and genotypic characterization of bietti crystalline dystrophy.

OBJECTIVE: To provide a detailed phenotype/genotype characterization of Bietti crystalline dystrophy (BCD). DESIGN: Observational case series. PARTICIPANTS: Twenty patients from 17 families recruited from a multiethnic British population. METHODS: Patients underwent color fundus photography, near-infrared (NIR) imaging, fundus autofluorescence (FAF) imaging, spectral domain optical coherence tomography (SD-OCT), and electroretinogram (ERG) assessment. The gene CYP4V2 was sequenced. MAIN OUTCOME MEASURES: Clinical, imaging, electrophysiologic, and molecular genetics findings. RESULTS: Patients ranged in age from 19 to 72 years (median, 40 years), with a visual acuity of 6/5 to perception of light (median, 6/12). There was wide intrafamilial and interfamilial variability in clinical severity. The FAF imaging showed well-defined areas of retinal pigment epithelium (RPE) loss that corresponded on SD-OCT to well-demarcated areas of outer retinal atrophy. Retinal crystals were not evident on FAF imaging and were best visualized with NIR imaging. Spectral domain OCT showed them to be principally located on or in the RPE/Bruch's membrane complex. Disappearance of the crystals, revealed by serial recording, was associated with severe disruption and thinning of the RPE/Bruch's membrane complex. Cases with extensive RPE degeneration (N = 5) had ERGs consistent with generalized rod and cone dysfunction, but those with more focal RPE atrophy showed amplitude reduction without delay (N = 3), consistent with restricted loss of function, or that was normal (N = 2). Likely disease-causing variants were identified in 34 chromosomes from 17 families. Seven were novel, including p.Met66Arg, found in all 11 patients from 8 families of South Asian descent. This mutation appears to be associated with earlier onset (median age, 30 years) compared with other substitutions (median age, 41 years). Deletions of exon 7 were associated with more severe disease. CONCLUSIONS: The phenotype is highly variable. Several novel variants are reported, including a highly prevalent substitution in patients of South Asian descent that is associated with earlier-onset disease. Autofluorescence showed sharply demarcated areas of RPE loss that coincided with abrupt edges of outer retinal atrophy on SD-OCT; crystals were generally situated on or in the RPE/Bruch's complex but could disappear over time with associated RPE disruption. These results support a role for the RPE in disease pathogenesis.

Intraretinal Microvascular Abnormalities and Venous Beading Have Different Genetic Profiles in Caucasian Patients with Non-Proliferative Diabetic Retinopathy.

Diabetic Retinopathy (DR) is a leading cause of preventable visual impairment in the working age population. Despite the increasing prevalence of DR, there remain gaps in our understanding of its pathophysiology. This is a prospective case-control study comparing the genetic profiles of patients with no DR vs. non-proliferative DR (NPDR) focusing on intraretinal microvascular abnormalities (IRMA) and venous beading (VB) in Caucasians. A total of 596 participants were recruited to the study; 199 with moderate/severe NPDR and 397 with diabetes for at least 5 years without DR. Sixty-four patients were excluded due to technical issues. In total, 532 were analysed; 181 and 351 were in the NPDR group and no DR group, respectively. Those with severe IRMA and VB had distinctly different genetic profiles from each other and from the no DR group, which further supports the theory that these two features of DR might have different etiologies. This also suggests that IRMA and VB are independent risk factors for the development of PDR and may have different pathophysiologies. If these findings are confirmed in larger studies, this could pave the way for personalised treatment options for those more at risk of developing different features of NPDR.

A Carrier Female Manifesting an Unusual X-Linked Retinoschisis Phenotype Associated with the Pathogenic Variant c.266delA, p.(Tyr89LeufsTer37) in RS1, and Skewed X-Inactivation.

X-linked retinoschisis (XLRS) is the most common juvenile macular degeneration in males. Unlike most other X-linked retinal dystrophies, carrier heterozygous females are very rarely reported to show clinical features of the disease. Herein, we describe unusual retinal features in a 2-year-old female infant with family history and genetic testing consistent with XLRS.

Vertebrate ancient opsin photopigment spectra and the avian photoperiodic response.

In mammals, photoreception is restricted to cones, rods and a subset of retinal ganglion cells. By contrast, non-mammalian vertebrates possess many extraocular photoreceptors but in many cases the role of these photoreceptors and their underlying photopigments is unknown. In birds, deep brain photoreceptors have been shown to sense photic changes in daylength (photoperiod) and mediate seasonal reproduction. Nonetheless, the specific identity of the opsin photopigment 'sensor' involved has remained elusive. Previously, we showed that vertebrate ancient (VA) opsin is expressed in avian hypothalamic neurons and forms a photosensitive molecule. However, a direct functional link between VA opsin and the regulation of seasonal biology was absent. Here, we report the in vivo and in vitro absorption spectra (λ(max) = ~490 nm) for chicken VA photopigments. Furthermore, the spectral sensitivity of these photopigments match the peak absorbance of the avian photoperiodic response (λ(max) = 492 nm) and permits maximum photon capture within the restricted light environment of the hypothalamus. Such a correspondence argues strongly that VA opsin plays a key role in regulating seasonal reproduction in birds.

Functional diversity of melanopsins and their global expression in the teleost retina.

Melanopsin (OPN4) is an opsin photopigment that, in mammals, confers photosensitivity to retinal ganglion cells and regulates circadian entrainment and pupil constriction. In non-mammalian species, two forms of opn4 exist, and are classified into mammalian-like (m) and non-mammalian-like (x) clades. However, far less is understood of the function of this photopigment family. Here we identify in zebrafish five melanopsins (opn4m-1, opn4m-2, opn4m-3, opn4x-1 and opn4x-2), each encoding a full-length opsin G protein. All five genes are expressed in the adult retina in a largely non-overlapping pattern, as revealed by RNA in situ hybridisation and immunocytochemistry, with at least one melanopsin form present in all neuronal cell types, including cone photoreceptors. This raises the possibility that the teleost retina is globally light sensitive. Electrophysiological and spectrophotometric studies demonstrate that all five zebrafish melanopsins encode a functional photopigment with peak spectral sensitivities that range from 470 to 484 nm, with opn4m-1 and opn4m-3 displaying invertebrate-like bistability, where the retinal chromophore interchanges between cis- and trans-isomers in a light-dependent manner and remains within the opsin binding pocket. In contrast, opn4m-2, opn4x-1 and opn4x-2 are monostable and function more like classical vertebrate-like photopigments, where the chromophore is converted from 11-cis to all-trans retinal upon absorption of a photon, hydrolysed and exits from the binding pocket of the opsin. It is thought that all melanopsins exhibit an invertebrate-like bistability biochemistry. Our novel findings, however, reveal the presence of both invertebrate-like and vertebrate-like forms of melanopsin in the teleost retina, and indicate that photopigment bistability is not a universal property of the melanopsin family. The functional diversity of these teleost melanopsins, together with their widespread expression pattern within the retina, suggests that melanopsins confer global photosensitivity to the teleost retina and might allow for direct "fine-tuning" of retinal circuitry and physiology in the dynamic light environments found in aquatic habitats.

Whole genome sequencing in a Knobloch syndrome family confirms the molecular diagnosis.

BACKGROUND: To establish the molecular diagnosis in two brothers presenting with the ocular features of Knobloch Syndrome using whole genome sequencing (WGS). METHODS: Clinical examination and ophthalmological phenotyping were completed under general anaesthesia. DNA samples were tested on a targeted retinal dystrophy next-generation sequencing panel. Subsequently, WGS was performed to identify additional variants. RESULTS: Clinical examination confirmed the diagnosis of Knobloch Syndrome. Targeted sequencing identified a novel heterozygous frameshift pathogenic variant in COL18A1, c.2864dupC; p.(Gly956ArgfsX20), inherited from their mother. A second paternally inherited heterozygous missense variant was identified in both brothers, c.5014 G > A; p.(Asp1672Asn), which was initially considered to have too high frequency to be pathogenic (MAF 8.8%). This led to an in-depth analysis of the COL18A1 locus using WGS data, which confirmed that Asp1672Asn is a likely pathogenic hypomorphic allele. CONCLUSION: To date, all confirmed genetic diagnoses of Knobloch syndrome are attributable to variants in COL18A1. The family described here has a heterozygous novel loss of function variant. Detailed analysis of WGS data combined with family segregation studies concluded that although Asp1672Asn has a high population frequency, it is the most likely second pathogenic variant in our family. This supports the hypothesis that this is a hypomorphic allele, which, in combination with a loss of function pathogenic variant, leads to Knobloch syndrome.To our knowledge, this is the first time that WGS has been used to confirm a molecular diagnosis of Knobloch syndrome in this way and has provided further insight into the molecular mechanisms in this rare disorder.

Phenotypic and Genetic Characteristics in a Cohort of Patients with Usher Genes.

Background: This study aimed to compare phenotype−genotype correlation in patients with Usher syndrome (USH) to those with autosomal recessive retinitis pigmentosa (NS-ARRP) caused by genes associated with Usher syndrome. Methods: Case notes of patients with USH or NS-ARRP and a molecularly confirmed diagnosis in genes associated with Usher syndrome were reviewed. Phenotypic information, including the age of ocular symptoms, hearing impairment, visual acuity, Goldmann visual fields, fundus autofluorescence (FAF) imaging and spectral domain optical coherence tomography (OCT) imaging, was reviewed. The patients were divided into three genotype groups based on variant severity for genotype-phenotype correlations. Results: 39 patients with Usher syndrome and 33 patients with NS-ARRP and a molecular diagnosis in an Usher syndrome-related gene were identified. In the 39 patients diagnosed with Usher syndrome, a molecular diagnosis was confirmed as follows: USH2A (28), MYO7A (4), CDH23 (2), USH1C (2), GPR98/VLGR1 (2) and PCDH15 (1). All 33 patients with NS-ARRP had variants in USH2A. Further analysis was performed on the patients with USH2A variants. USH2A patients with syndromic features had an earlier mean age of symptom onset (17.9 vs. 31.7 years, p < 0.001), had more advanced changes on FAF imaging (p = 0.040) and were more likely to have cystoid macular oedema (p = 0.021) when compared to USH2A patients presenting with non-syndromic NS-ARRP. Self-reported late-onset hearing loss was identified in 33.3% of patients with NS-ARRP. Having a syndromic phenotype was associated with more severe USH2A variants (p < 0.001). Eighteen novel variants in genes associated with Usher syndrome were identified in this cohort. Conclusions: Patients with Usher syndrome, whatever the associated gene in this cohort, tended to have an earlier onset of retinal disease (other than GPR98/VLGR1) when compared to patients presenting with NS-ARRP. Analysis of genetic variants in USH2A, the commonest gene in our cohort, showed that patients with a more severe genotype were more likely to be diagnosed with USH compared to NS-ARRP. USH2A patients with syndromic features have an earlier onset of symptoms and more severe features on FAF and OCT imaging. However, a third of patients diagnosed with NS-ARRP developed later onset hearing loss. Eighteen novel variants in genes associated with Usher syndrome were identified in this cohort, thus expanding the genetic spectrum of known pathogenic variants. An accurate molecular diagnosis is important for diagnosis and prognosis and has become particularly relevant with the advent of potential therapies for Usher-related gene

An Overview of the Genetics of ABCA4 Retinopathies, an Evolving Story.

Stargardt disease (STGD1) and ABCA4 retinopathies (ABCA4R) are caused by pathogenic variants in the ABCA4 gene inherited in an autosomal recessive manner. The gene encodes an importer flippase protein that prevents the build-up of vitamin A derivatives that are toxic to the RPE. Diagnosing ABCA4R is complex due to its phenotypic variability and the presence of other inherited retinal dystrophy phenocopies. ABCA4 is a large gene, comprising 50 exons; to date > 2000 variants have been described. These include missense, nonsense, splicing, structural, and deep intronic variants. Missense variants account for the majority of variants in ABCA4. However, in a significant proportion of patients with an ABCA4R phenotype, a second variant in ABCA4 is not identified. This could be due to the presence of yet unknown variants, or hypomorphic alleles being incorrectly classified as benign, or the possibility that the disease is caused by a variant in another gene. This underlines the importance of accurate genetic testing. The pathogenicity of novel variants can be predicted using in silico programs, but these rely on databases that are not ethnically diverse, thus highlighting the need for studies in differing populations. Functional studies in vitro are useful towards assessing protein function but do not directly measure the flippase activity. Obtaining an accurate molecular diagnosis is becoming increasingly more important as targeted therapeutic options become available; these include pharmacological, gene-based, and cell replacement-based therapies. The aim of this review is to provide an update on the current status of genotyping in ABCA4 and the status of the therapeutic approaches being investigated.

The role of multimodal imaging and vision function testing in ABCA4-related retinopathies and their relevance to future therapeutic interventions.

The aim of this review article is to describe the specific features of Stargardt disease and ABCA4 retinopathies (ABCA4R) using multimodal imaging and functional testing and to highlight their relevance to potential therapeutic interventions. Standardised measures of tissue loss, tissue function and rate of change over time using formal structured deep phenotyping in Stargardt disease and ABCA4R are key in diagnosis, and prognosis as well as when selecting cohorts for therapeutic intervention. In addition, a meticulous documentation of natural history will be invaluable in the future to compare treated with untreated retinas. Despite the familiarity with the term Stargardt disease, this eponymous classification alone is unhelpful when evaluating ABCA4R, as the ABCA4 gene is associated with a number of phenotypes, and a range of severity. Multimodal imaging, psychophysical and electrophysiologic measurements are necessary in diagnosing and characterising these differing retinopathies. A wide range of retinal dystrophy phenotypes are seen in association with ABCA4 mutations. In this article, these will be referred to as ABCA4R. These different phenotypes and the existence of phenocopies present a significant challenge to the clinician. Careful phenotypic characterisation coupled with the genotype enables the clinician to provide an accurate diagnosis, associated inheritance pattern and information regarding prognosis and management. This is particularly relevant now for recruiting to therapeutic trials, and in the future when therapies become available. The importance of accurate genotype-phenotype correlation studies cannot be overemphasised. This approach together with segregation studies can be vital in the identification of causal mutations when variants in more than one gene are being considered as possible. In this article, we give an overview of the current imaging, psychophysical and electrophysiological investigations, as well as current therapeutic research trials for retinopathies associated with the ABCA4 gene.

"Genetic and clinical findings in an ethnically diverse retinitis pigmentosa cohort associated with pathogenic variants in EYS".

BACKGROUND AND OBJECTIVES: The EYS gene is an important cause of autosomal recessive retinitis pigmentosa (arRP). The objective of this study is to report on novel pathogenic variants in EYS and the range of associated phenotypes. SUBJECTS AND METHODS: This retrospective case series at a tertiary referral centre for inherited retinal diseases describes patients with an IRD and at least two variants in the EYS gene. Phenotyping included multimodal retinal imaging; genotyping molecular genetic analysis using targeted next generation sequencing. Sanger sequencing verification and analysis of novel variants using in silico approaches to determine their predicted pathogenicity. RESULTS: Eight male and four female patients were included. Age at onset ranged from 11 to 62 years with variable symptom presentation; ten patients showed classical features of retinitis pigmentosa, albeit with great variation in disease severity and extent. Two patients had atypical phenotypes: one with localised inferior sector pigmentation and a mild RP phenotype with changes predominantly at the posterior pole. Eighteen variants in EYS were identified, located across the gene: six were novel. Eight variants were missense, two altered splicing, one was a whole exon duplication and the remainder were predicted to result in premature truncation of the protein. CONCLUSION: The marked variability in severity and age of onset in most patients in this ethnically diverse cohort adds to growing evidence that that mild phenotypes are associated with EYS variants. Similarly, the two atypical cases add to the growing diversity of EYS disease as do the six novel pathogenic variants described.

Differential expression of two distinct functional isoforms of melanopsin (Opn4) in the mammalian retina.

Melanopsin is the photopigment that confers photosensitivity to a subset of retinal ganglion cells (pRGCs) that regulate many non-image-forming tasks such as the detection of light for circadian entrainment. Recent studies have begun to subdivide the pRGCs on the basis of morphology and function, but the origin of these differences is not yet fully understood. Here we report the identification of two isoforms of melanopsin from the mouse Opn4 locus, a previously described long isoform (Opn4L) and a novel short isoform (Opn4S) that more closely resembles the sequence and structure of rat and human melanopsins. Both isoforms, Opn4L and Opn4S, are expressed in the ganglion cell layer of the retina, traffic to the plasma membrane and form a functional photopigment in vitro. Quantitative PCR revealed that Opn4S is 40 times more abundant than Opn4L. The two variants encode predicted proteins of 521 and 466 aa and only differ in the length of their C-terminal tails. Antibodies raised to isoform-specific epitopes identified two discrete populations of melanopsin-expressing RGCs, those that coexpress Opn4L and Opn4S and those that express Opn4L only. Recent evidence suggests that pRGCs show a range of anatomical subtypes, which may reflect the functional diversity reported for mouse Opn4-mediated light responses. The distinct isoforms of Opn4 described in this study provide a potential molecular basis for generating this diversity, and it seems likely that their differential expression plays a role in generating the variety of pRGC light responses found in the mammalian retina.

Novel Pathogenic Sequence Variants in NR2E3 and Clinical Findings in Three Patients.

A retrospective review of the clinical records of patients seen at the Oxford Eye Hospital identified as having NR2E3 mutations was performed. The data included symptoms, best-corrected visual acuity, multimodal retinal imaging, visual fields and electrophysiology testing. Three participants were identified with biallelic NR2E3 pathogenic sequence variants detected using a targeted NGS gene panel, two of which were novel. Participant I was a Nepalese male aged 68 years, and participants II and III were white Caucasian females aged 69 and 10 years old, respectively. All three had childhood onset nyctalopia, a progressive decrease in central vision, and visual field loss. Patients I and III had photopsia, patient II had photosensitivity and patient III also had photophobia. Visual acuities in patients I and II were preserved even into the seventh decade, with the worst visual acuity measured at 6/36. Visual field constriction was severe in participant I, less so in II, and fields were full to bright targets targets in participant III. Electrophysiology testing in all three demonstrated loss of rod function. The three patients share some of the typical distinctive features of NR2E3 retinopathies, as well as a novel clinical observation of foveal ellipsoid thickening.

Association of Clinical and Genetic Heterogeneity With BEST1 Sequence Variations.

Importance: Detailed phenotypic information on the spectrum of fundus abnormalities and clinical variability of all phenotypes associated with sequence variations in BEST1 is limited. Objective: To report a detailed phenotypic and genetic analysis of a patient cohort with sequence variations in BEST1. Design, Setting, and Participants: This retrospective case series took place at the Oxford Eye Hospital in Oxford, UK. Thirty-six patients from a single center with disease-causing sequence variations in BEST1 from 25 different families were analyzed. Data were collected from November 2017 to June 2018, and analysis began April 2018. Main Outcomes and Measures: Results of ocular phenotyping and genetic testing using targeted next-generation sequencing to identify BEST1 sequence variations. Results: Thirty-six patients from 25 families with disease-causing sequence variations in BEST1 were included. Of 36 patients, 20 (55.6%) were female. Three distinct clinical phenotypes were identified: autosomal recessive bestrophinopathy (ARB), best vitelliform macular dystrophy (BVMD), and adult-onset vitelliform macular dystrophy. The ARB phenotype group comprised 18 patients from 9 families with age in years at symptom onset ranging from less than 10 to 40s. All patients showed a common phenotype of fundus autofluorescence abnormalities, and spectral-domain optical coherence tomography features were similar in all patients with schitic and cystoid changes. A phenotype of a beaten metallic retinal appearance extending from the mid periphery to the far periphery was identified in 8 patients. Four patients from 1 family with ARB were previously reported to have autosomal recessive retinitis pigmentosa but were reclassified as having ARB as part of this study. The BVMD phenotype group comprised 16 patients from 14 families with age at symptom onset ranging from less than 10 to 70s. Fundus features were localized to the macula and consistent with the stage of BVMD. In the adult-onset vitelliform macular dystrophy phenotype group, the age in years at symptom onset varied from 50s to 70s in 2 patients from 2 families. Fundus features included small vitelliform lesions. Where available, electro-oculogram results demonstrated a reduced or absent light rise in all patients with ARB and BVMD. Genetic testing identified 22 variants in BEST1. Conclusions and Relevance: These findings support the notion that ARB, BVMD, and adult-onset vitelliform macular dystrophy are clinically distinct and recognizable phenotypes and suggest that the association of autosomal recessive retinitis pigmentosa with sequence variations in BEST1 should be rereviewed.