Diagnostic genome sequencing improves diagnostic yield: a prospective single-centre study in 1000 patients with inherited eye diseases.

PURPOSE: Genome sequencing (GS) is expected to reduce the diagnostic gap in rare disease genetics. We aimed to evaluate a scalable framework for genome-based analyses 'beyond the exome' in regular care of patients with inherited retinal degeneration (IRD) or inherited optic neuropathy (ION). METHODS: PCR-free short-read GS was performed on 1000 consecutive probands with IRD/ION in routine diagnostics. Complementary whole-blood RNA-sequencing (RNA-seq) was done in a subset of 74 patients. An open-source bioinformatics analysis pipeline was optimised for structural variant (SV) calling and combined RNA/DNA variation interpretation. RESULTS: A definite genetic diagnosis was established in 57.4% of cases. For another 16.7%, variants of uncertain significance were identified in known IRD/ION genes, while the underlying genetic cause remained unresolved in 25.9%. SVs or alterations in non-coding genomic regions made up for 12.7% of the observed variants. The RNA-seq studies supported the classification of two unclear variants. CONCLUSION: GS is feasible in clinical practice and reliably identifies causal variants in a substantial proportion of individuals. GS extends the diagnostic yield to rare non-coding variants and enables precise determination of SVs. The added diagnostic value of RNA-seq is limited by low expression levels of the major IRD disease genes in blood.

Age-dependencies of the electroretinogram in healthy subjects.

OBJECTIVE: The purpose of this study was to evaluate the age-dependency of amplitudes and implicit times in the electroretinograms (ERGs) of healthy individuals and provide clinicians and researchers with a reference for a variety of stimulus paradigms. DESIGN AND METHODS: Full-field electroretinography was conducted on 73 healthy participants aged 14-73 using an extended ISCEV standard protocol that included an additional 9 Hz flicker stimulus for assessing rod function and special paradigms for isolated On-Off and S-cone responses. Correlation coefficients and best-fit regression models for each parameter's age-dependency were calculated. RESULTS: Dark-adapted ERGs, in particular, displayed notable age-related alterations. The attenuation and delay of the b-wave with higher age were most significant in the dark-adapted, rod-driven 0.001 cd s/m2 flash ERG. The age-dependent reduction of the a-wave amplitude was strongest in the standard dark-adapted 3 cd s/m2 flash condition. Cone-driven, light-adapted responses to either flash or flicker stimuli displayed comparatively small alterations at higher age. S-cone function tended to diminish at an early age, but the effect was not significant in the whole population. CONCLUSION: The results suggest that rod and cone function decline at different rates with age, with rods being generally more affected by aging. Nonetheless, response amplitudes displayed a wide variability across the whole sample.

Elaborate Evaluation of Farnsworth Dichotomous D-15 Panel Test Can Help Differentiate between Best Vitelliform Macular Dystrophy and Autosomal Recessive Bestrophinopathy.

INTRODUCTION: The colour vision in bestrophinopathies has not been assessed in detail so far. The aim of this study was to explore the extent to which distinct types of bestrophinopathies differ in regard to colour vision deficiencies using Farnsworth Dichotomous D-15 and Lanthony Desaturated D-15 panel tests. METHODS: Both D-15 tests were performed in 52 eyes of 26 patients with Best vitelliform macular dystrophy (BVMD) and 10 eyes of 5 patients with autosomal recessive bestrophinopathy (ARB). Two methods were used for a quantitative assessment of the colour vision deficiencies: moment of inertia method and Bowman method. The following parameters were calculated: confusion angle, confusion index (C-index), selectivity index (S-index), total error score (TES), and colour confusion index (CCI). RESULTS: The median value of confusion angle for all stages of BVMD fell into a narrow range around 62, indicating normal results. The median confusion angle value was 57 in ARB patients within a very wide range down to -82, indicating non-specific deficits. These differences were statistically significant. Significantly abnormal C-index and CCI values were found only in ARB patients, being 2.0 and 1.49, respectively. The majority of parameters of D-15 tests were independent of the visual acuity in both bestrophinopathies. CONCLUSIONS: Elaborate evaluation of the D-15 panel tests might help establish a differential diagnosis between different bestrophinopathies, as the pattern of the colour vision loss is different between BVMD and ARB. The quantitative parameters of colour vision tests in bestrophinopathies are independent of the visual acuity.

CNGB1-related rod-cone dystrophy: A mutation review and update.

Cyclic nucleotide-gated channel ß1 (CNGB1) encodes the 240-kDa ß subunit of the rod photoreceptor cyclic nucleotide-gated ion channel. Disease-causing sequence variants in CNGB1 lead to autosomal recessive rod-cone dystrophy/retinitis pigmentosa (RP). We herein present a comprehensive review and analysis of all previously reported CNGB1 sequence variants, and add 22 novel variants, thereby enlarging the spectrum to 84 variants in total, including 24 missense variants (two of which may also affect splicing), 21 nonsense, 19 splicing defects (7 at noncanonical positions), 10 small deletions, 1 small insertion, 1 small insertion-deletion, 7 small duplications, and 1 gross deletion. According to the American College of Medical Genetics and Genomics classification criteria, 59 variants were considered pathogenic or likely pathogenic and 25 were variants of uncertain significance. In addition, we provide further phenotypic data from 34 CNGB1-related RP cases, which, overall, are in line with previous findings suggesting that this form of RP has long-term retention of useful central vision despite the early onset of night blindness, which is valuable for patient counseling, but also has implications for it being considered a priority target for gene therapy trials.

Clinical Phenotype of PDE6B-Associated Retinitis Pigmentosa.

In this retrospective, longitudinal, observational cohort study, we investigated the phenotypic and genotypic features of retinitis pigmentosa associated with variants in the PDE6B gene. Patients underwent clinical examination and genetic testing at a single tertiary referral center, including best-corrected visual acuity (BCVA), kinetic visual field (VF), full-field electroretinography, full-field stimulus threshold, spectral domain optical coherence tomography, and fundus autofluorescence imaging. The genetic testing comprised candidate gene sequencing, inherited retinal disease gene panel sequencing, whole-genome sequencing, and testing for familial variants by Sanger sequencing. Twenty-four patients with mutations in PDE6B from 21 families were included in the study (mean age at the first visit: 32.1 ± 13.5 years). The majority of variants were putative splicing defects (8/23) and missense (7/23) mutations. Seventy-nine percent (38/48) of eyes had no visual acuity impairment at the first visit. Visual acuity impairment was mild in 4% (2/48), moderate in 13% (6/48), and severe in 4% (2/48). BCVA was symmetrical in the right and left eyes. The kinetic VF measurements were highly symmetrical in the right and left eyes, as was the horizontal ellipsoid zone (EZ) width. Regarding the genetic findings, 43% of the PDE6B variants found in our patients were novel. Thus, this study contributed substantially to the PDE6B mutation spectrum. The visual acuity impairment was mild in 83% of eyes, providing a window of opportunity for investigational new drugs. The EZ width was reduced in all patients and was highly symmetric between the eyes, making it a promising outcome measure. We expect these findings to have implications on the design of future PDE6B-related retinitis pigmentosa (RP) clinical trials.

Genetic architecture of inherited retinal degeneration in Germany: A large cohort study from a single diagnostic center over a 9-year period.

We aimed to unravel the molecular genetic basis of inherited retinal degeneration (IRD) in a comprehensive cohort of patients diagnosed in the largest center for IRD in Germany. A cohort of 2,158 affected patients from 1,785 families diagnosed with IRD was analyzed by targeted next-generation sequencing (NGS). Patients with single-gene disorders (i.e., choroideremia and retinoschisis) were analyzed by Sanger sequencing and multiplex ligation-dependent probe amplification. Our study cohort accounts for ∼7% of the estimated 30,000 patients with IRD in Germany, thereby providing representative data for both the prevalence of IRDs and the mutation spectrum of IRD genes for the population in Germany. We achieved a molecular diagnostic rate of 35-95%, depending on the clinical entities, with a high detection rate for achromatopsia, retinoschisis, and choroideremia, and a low detection rate for central areolar choroidal dystrophy and macular dystrophy. A total of 1,161 distinct variants were identified, including 501 novel variants, reaffirming the known vast genetic heterogeneity of IRD in a mainly outbred European population. This study demonstrates the clinical utility of panel-based NGS in a large and highly heterogeneous cohort from an outbred population and for the first time gives a comprehensive representation of the genetic landscape of IRDs in Germany. The data are valuable and crucial for the scientific community and healthcare providers, but also for the pharmaceutical industry in the progressing field of personalized medicine and gene therapy.

Correlating Adaptive Optics Images to Clinical Findings in Juvenile Macular Dystrophy with Hypotrichosis in Siblings with Homozygous CDH3 Pathogenic Variation.

OBJECTIVE: We report on two German siblings diagnosed with congenital hypotrichosis and juvenile macular dystrophy, an extremely rare syndrome affecting both hair growth and visual functions. METHODS: A detailed ophthalmological examination was carried out including fundus examination, visual acuity assessment, visual field determination, color vision testing, and electrophysiology (electroretinography [ERG]). Additionally, fundus photography and autofluorescence imaging (FAF) was performed, along with optical coherence tomography (OCT) and adaptive optics (AO) fundus imaging. Targeted Sanger sequencing and next-generation gene panel sequencing were carried out. RESULTS: Macular dystrophy was evident in the fundus of both patients, as was a central scotoma in the static visual field. The kinetic visual field was normal. The ERG recordings were also normal, but the amplitudes of the multifocal ERG were reduced in the central 4-5° of the retina. The FAF images revealed a large central hypofluorescent area surrounded by a hyperfluorescent ring. The OCT images showed atrophy in the outer layers and tubulations. The AO images depicted a loss of central photoreceptors, as well as severe central atrophy in patient 1. A cone mosaic was observable in the peripheral AO fundus images of both patients. The disrupted cone mosaic on the AO images correlated with the hypofluorescent areas on autofluorescence. DNA testing identified the homozygous, likely pathogenic variant c.1508G>A/p.(Arg503His) (chr16:68719191) in the CDH3 gene. CONCLUSIONS: The two siblings revealed hypotrichosis and macular dystrophy in both eyes. The identification of a homozygous CDH3 mutation in each patient confirms the syndromic entity of hypotrichosis with juvenile macular degeneration.

Objective assessment of visual acuity: a refined model for analyzing the sweep VEP.

PURPOSE: The aim of this study was to develop a simple and reliable method for the objective assessment of visual acuity by optimizing the stimulus used in commercially available systems and by improving the methods of evaluation using a nonlinear function, the modified Ricker model. METHODS: Subjective visual acuity in the normal subjects was measured with Snellen targets, best-corrected, and in some cases also uncorrected and with plus lenses (+ 1 D, + 2 D, + 3 D). In patients, subjective visual acuity was measured best-corrected using the Freiburg Visual Acuity Test. Sweep VEP recordings to 11 spatial frequencies, with check sizes in logarithmically equidistant steps (0.6, 0.9, 1.4, 2.1, 3.3, 4.9, 7.3, 10.4, 18.2, 24.4, and 36.5 cpd), were obtained from 56 healthy subjects aged between 17 and 69 years (mean 42.5 ± 15.3 SD years) and 20 patients with diseases of the lens (n = 6), retina (n = 8) or optic nerve (n = 6). The results were fit by a multiple linear regression (2nd-order polynomial) or a nonlinear regression (modified Ricker model) and parameters compared (limiting spatial frequency (sflimiting) and the spatial frequency of the vertex (sfvertex) of the parabola for the 2nd-order polynomial fitting, and the maximal spatial frequency (sfmax), and the spatial frequency where the amplitude is 2 dB higher than the level of noise (sfthreshold) for the modified Ricker model. RESULTS: Recording with 11 spatial frequencies allows a more accurate determination of acuities above 1.0 logMAR. Tuning curves fitted to the results show that compared to the normal 2nd-order polynomial analysis, the modified Ricker model is able to describe closely the amplitudes of the sweep VEP in relation to the spatial frequencies of the presented checkerboards. In patients with a visual acuity better than about 0.5 (decimal), the predicted acuities based on the different parameters show a good match of the predicted visual acuities based on the models established in healthy volunteers to the subjective visual acuities. However, for lower visual acuities, both models tend to overestimate the visual acuity (up to ~ 0.4 logMAR), especially in patients suffering from AMD. CONCLUSIONS: Both models, the 2nd-order polynomial and the modified Ricker model performed equally well in the prediction of the visual acuity based on the amplitudes recorded using the sweep VEP. However, the modified Ricker model does not require the exclusion of data points from the fit, as necessary when fitting the 2nd-order polynomial model making it more reliable and robust against outliers, and, in addition, provides a measure for the noise of the recorded results.

A 73,128 bp de novo deletion encompassing the OPN1LW/OPN1MW gene cluster in sporadic Blue Cone Monochromacy: a case report.

BACKGROUND: Blue Cone Monochromacy (BCM) is a rare congenital cone dysfunction disorder with X-linked recessive mode of inheritance. BCM is caused by mutations at the OPN1LW/MW cone opsin gene cluster including deletions of the locus control region (LCR) and/or parts of the gene cluster. We aimed at investigating the clinical presentation, genetic cause and inheritance underlying a sporadic case of BCM. CASE PRESENTATION: We report a 24-year-old male presenting with congenital photophobia, nystagmus and colour vision abnormalities. There was no history of retinal dystrophy in the family. Clinical diagnosis of BCM was supported by genetic investigations of the patient and his family members. Molecular genetic analysis of the OPN1LW/OPN1MW gene cluster revealed a novel deletion of about 73 kb in the patient encompassing the LCR. The deletion was absent in the X-chromosomes of both the mother and transmitting grandfather. CONCLUSIONS: The present report provides the clinical findings and the genetic basis underlying a sporadic BCM case which is caused by a de novo deletion within the OPN1LW/MW gene cluster originating from the mother's germline due to Alu-repeat mediated recombination. This is the first report of a de novo deletion resulting in BCM, highlighting the importance to consider BCM and perform genetic testing for this condition in male patients with cone dysfunction also in the absence of a positive family history.

Adaptive optics retinal imaging in patients with usher syndrome.

Purpose: To determine the structure of the cone photoreceptor mosaic in the macula in eyes with retinitis pigmentosa related to Usher syndrome using adaptive optics fundus (AO) imaging and to correlate these findings with those of the standard clinical diagnostics. Methods: Ten patients with a genetically confirmed retinitis pigmentosa in Usher syndrome due to biallelic variants in MYO7A or USH2A were enrolled in the study. All patients underwent a complete ophthalmological examination including best corrected visual acuity (BCVA), spectral-domain optical coherence tomography (SD-OCT) with fundus autofluorescence photography (FAF), full-field (ffERG) and multifocal electroretinography (mfERG) and Adaptive Optics Flood Illuminated Ophthalmoscopy (AO, rtx1™, Imagine Eyes, Orsay, France). The cone density was assessed centrally and at each 0.5 degree horizontally and vertically from 1-4 degree of eccentricity. Results: In the AO images, photoreceptor cell death was visualized as a disruption of the cone mosaic and low cone density. In the early stage of the disease, cones were still visible in the fovea, whereas outside the fovea a loss of cones was recognizable by blurry, dark patches. The blurry patches corresponded to the parafoveal hypofluorescent ring in the FAF images and the beginning loss of the IS/OS line and external limiting membrane in the SD-OCT images. FfERGs were non-recordable in 7 patients and reduced in 3. The mfERG was reduced in all patients and correlated significantly (p <0.001) with the cone density. The kinetic visual field area, measured with III4e and I4e, did not correlate with the cone density. Conclusion: The structure of the photoreceptors in Usher syndrome patients were detectable by AO fundus imaging. The approach of using high-resolution technique to assess the photoreceptor structure complements the established clinical examinations and allows a more sensitive monitoring of early stages of retinitis pigmentosa in Usher syndrome.

KCNV2-associated retinopathy: genotype-phenotype correlations - KCNV2 study group report 3.

BACKGROUND/AIMS: To investigate genotype-phenotype associations in patients with KCNV2 retinopathy. METHODS: Review of clinical notes, best-corrected visual acuity (BCVA), molecular variants, electroretinography (ERG) and retinal imaging. Subjects were grouped according to the combination of KCNV2 variants-two loss-of-function (TLOF), two missense (TM) or one of each (MLOF)-and parameters were compared. RESULTS: Ninety-two patients were included. The mean age of onset (mean±SD) in TLOF (n=55), TM (n=23) and MLOF (n=14) groups was 3.51±0.58, 4.07±2.76 and 5.54±3.38 years, respectively. The mean LogMAR BCVA (±SD) at baseline in TLOF, TM and MLOF groups was 0.89±0.25, 0.67±0.38 and 0.81±0.35 for right, and 0.88±0.26, 0.69±0.33 and 0.78±0.33 for left eyes, respectively. The difference in BCVA between groups at baseline was significant in right (p=0.03) and left eyes (p=0.035). Mean outer nuclear layer thickness (±SD) at baseline in TLOF, MLOF and TM groups was 37.07±15.20 µm, 40.67±12.53 and 40.38±18.67, respectively, which was not significantly different (p=0.85). The mean ellipsoid zone width (EZW) loss (±SD) was 2051 µm (±1318) for patients in the TLOF, and 1314 µm (±965) for MLOF. Only one patient in the TM group had EZW loss at presentation. There was considerable overlap in ERG findings, although the largest DA 10 ERG b-waves were associated with TLOF and the smallest with TM variants. CONCLUSIONS: Patients with missense alterations had better BCVA and greater structural integrity. This is important for patient prognostication and counselling, as well as stratification for future gene therapy trials.

Ophthalmic and Genetic Features of Bardet Biedl Syndrome in a German Cohort.

The aim of this study was to characterize the ophthalmic and genetic features of Bardet Biedl (BBS) syndrome in a cohort of patients from a German specialized ophthalmic care center. Sixty-one patients, aged 5−56 years, underwent a detailed ophthalmic examination including visual acuity and color vision testing, electroretinography (ERG), visually evoked potential recording (VEP), fundus examination, and spectral domain optical coherence tomography (SD-OCT). Adaptive optics flood illumination ophthalmoscopy was performed in five patients. All patients had received diagnostic genetic testing and were selected upon the presence of apparent biallelic variants in known BBS-associated genes. All patients had retinal dystrophy with morphologic changes of the retina. Visual acuity decreased from ~0.2 (decimal) at age 5 to blindness 0 at 50 years. Visual field examination could be performed in only half of the patients and showed a concentric constriction with remaining islands of function in the periphery. ERG recordings were mostly extinguished whereas VEP recordings were reduced in about half of the patients. The cohort of patients showed 51 different likely biallelic mutations­of which 11 are novel­in 12 different BBS-associated genes. The most common associated genes were BBS10 (32.8%) and BBS1 (24.6%), and by far the most commonly observed variants were BBS10 c.271dup;p.C91Lfs*5 (21 alleles) and BBS1 c.1169T>G;p.M390R (18 alleles). The phenotype associated with the different BBS-associated genes and genotypes in our cohort is heterogeneous, with diverse features without genotype−phenotype correlation. The results confirm and expand our knowledge of this rare disease.

Disease expression caused by different variants in the BEST1 gene: genotype and phenotype findings in bestrophinopathies.

PURPOSE: To analyse the spectrum of clinical features and molecular genetic data in a series of patients carrying likely disease-associated variants in the BEST1 gene. METHODS: Retrospective observational analysis of clinical data extracted from the medical records of visual function, multimodal imaging and electrophysiology of 62 eyes of 31 patients. Molecular genetic analysis was performed by means of panel-based NGS or Sanger sequencing. RESULTS: The spectrum of variants in the BEST1 gene comprised 19 different variants and three of which are novel. Fundus photographs and OCT images allowed categorization of 52 eyes as Best vitelliform macular dystrophy (BVMD) with stages 1 to 5 and 10 eyes with autosomal recessive bestrophinopathy (ARB), with more severe phenotype. One patient was shown to be heterozygous for a variant, which has so far been described only in ARB, but this patient had the BVMD phenotype. There was no significant progression of the visual acuity during the follow-up period of 5 years both in BVMD and ARB. The most prevalent pattern of fundus autofluorescence (FAF) in BVMD was 'patchy'. There were diverse visual field defects in static automated perimetry (SAP) depending on the stage. The Arden ratio was significantly lower in ARB patients and in eyes with stage 5 of BVMD. CONCLUSIONS: The genotype does not always predict the phenotype in patients with BVMD and ARB; however, having two mutations in the BEST1 gene causes a more severe phenotype. FAF helped to distinguish ARB from BVMD. Most of the observed eyes did not progress functionally during the follow-up. ARB and the atrophic stage of BVMD as the disease end-stage had the worst visual functions and EOG results.

Spatial and temporal resolution of the photoreceptors rescue dynamics after treatment with voretigene neparvovec.

BACKGROUND: Voretigene neparvovec is a gene therapeutic agent for treatment of retinal dystrophies caused by bi-allelic RPE65 mutations. In this study, we report on a novel and objective evaluation of a retinotopic photoreceptor rescue. METHODS: Seven eyes of five patients (14, 21, 23, 24, 36 years, 1 male, 4 females) with bi-allelic RPE65 mutations have been treated with voretigene neparvovec. The clinical examinations included visual acuity testing, dark-adapted full-field stimulus threshold (FST), dark-adapted chromatic perimeter (DAC) with a 30-degree grid, and a 30 degrees grid scotopic and photopic chromatic pupil campimetry (CPC). All evaluations and spectral domain optical coherence tomography were performed at baseline, 1 month and 3 months. RESULTS: All except the oldest patient had a measurable improvement of the rod function assessed via FST, DAC or scotopic CPC at 1 month. The visual acuity improved slightly or remained stable in all eyes. A cone function improvement as measured by photopic CPC was observed in three eyes. The gain of the dark-adapted threshold with blue FST and the DAC stimuli (cyan) average correlated strongly with age (R2>0.7). The pupil response improvement in the scotopic CPC correlated with the baseline local retinal volume (R2=0.5). CONCLUSIONS: The presented protocols allow evaluating the individual spatial and temporal effects of gene therapy effects. Additionally, we explored parameters that correlated with the success of the therapy. CPC and DAC present new and fast ways to assess functional changes in retinotopic maps of rod and cone function, measuring complementary aspects of retinal function.

KCNV2-Associated Retinopathy: Detailed Retinal Phenotype and Structural Endpoints-KCNV2 Study Group Report 2.

PURPOSE: To describe the detailed retinal phenotype of KCNV2-associated retinopathy. STUDY DESIGN: Multicenter international retrospective case series. METHODS: Review of retinal imaging including fundus autofluorescence (FAF) and optical coherence tomography (OCT), including qualitative and quantitative analyses. RESULTS: Three distinct macular FAF features were identified: (1) centrally increased signal (n = 35, 41.7%), (2) decreased autofluorescence (n = 27, 31.1%), and (3) ring of increased signal (n = 37, 44.0%). Five distinct FAF groups were identified based on combinations of those features, with 23.5% of patients changing the FAF group over a mean (range) follow-up of 5.9 years (1.9-13.1 years). Qualitative assessment was performed by grading OCT into 5 grades: (1) continuous ellipsoid zone (EZ) (20.5%); (2) EZ disruption (26.1%); (3) EZ absence, without optical gap and with preserved retinal pigment epithelium complex (21.6%); (4) loss of EZ and a hyporeflective zone at the foveola (6.8%); and (5) outer retina and retinal pigment epithelium complex loss (25.0%). Eighty-six patients had scans available from both eyes, with 83 (96.5%) having the same grade in both eyes, and 36.1% changed OCT grade over a mean follow-up of 5.5 years. The annual rate of outer nuclear layer thickness change was similar for right and left eyes. CONCLUSIONS: KCNV2-associated retinopathy is a slowly progressive disease with early retinal changes, which are predominantly symmetric between eyes. The identification of a single OCT or FAF measurement as an endpoint to determine progression that applies to all patients may be challenging, although outer nuclear layer thickness is a potential biomarker. Findings suggest a potential window for intervention until 40 years of age.

KCNV2-Associated Retinopathy: Genetics, Electrophysiology, and Clinical Course-KCNV2 Study Group Report 1.

PURPOSE: To investigate genetics, electrophysiology, and clinical course of KCNV2-associated retinopathy in a cohort of children and adults. STUDY DESIGN: This was a multicenter international clinical cohort study. METHODS: Review of clinical notes and molecular genetic testing. Full-field electroretinography (ERG) recordings, incorporating the international standards, were reviewed and quantified and compared with age and recordings from control subjects. RESULTS: In total, 230 disease-associated alleles were identified from 117 patients, corresponding to 75 different KCNV2 variants, with 28 being novel. The mean age of onset was 3.9 years old. All patients were symptomatic before 12 years of age (range, 0-11 years). Decreased visual acuity was present in all patients, and 4 other symptoms were common: reduced color vision (78.6%), photophobia (53.5%), nyctalopia (43.6%), and nystagmus (38.6%). After a mean follow-up of 8.4 years, the mean best-corrected visual acuity (BCVA ± SD) decreased from 0.81 ± 0.27 to 0.90 ± 0.31 logarithm of minimal angle of resolution. Full-field ERGs showed pathognomonic waveform features. Quantitative assessment revealed a wide range of ERG amplitudes and peak times, with a mean rate of age-associated reduction indistinguishable from the control group. Mean amplitude reductions for the dark-adapted 0.01 ERG, dark-adapted 10 ERG a-wave, and LA 3.0 30 Hz and LA3 ERG b-waves were 55%, 21%, 48%, and 74%, respectively compared with control values. Peak times showed stability across 6 decades. CONCLUSION: In KCNV2-associated retinopathy, full-field ERGs are diagnostic and consistent with largely stable peripheral retinal dysfunction. Report 1 highlights the severity of the clinical phenotype and established a large cohort of patients, emphasizing the unmet need for trials of novel therapeutics.

Ophthalmic features of retinitis pigmentosa in Cohen syndrome caused by pathogenic variants in the VPS13B gene.

PURPOSE: The aim of this study is to report on the phenotype and genotype of five patients diagnosed with Cohen syndrome, an extremely rare autosomal recessive disorder manifesting with mental and physiological defects. METHODS: Five patients from three German families and one Syrian family underwent a comprehensive ophthalmological examination. The scheduled visual acuity measurements, fundus ophthalmoscopy, spectral domain optical coherence tomography (OCT), full-field electrophysiological recordings of scotopic and photopic electroretinograms (ERGs) and colour vision testing could not be carried out in all subjects, because of the mental and physical retardation. The genetic diagnosis was achieved by next-generation sequencing. RESULTS: The ophthalmic and systemic phenotype of the patients is typical for Cohen syndrome including myopia, night blindness, photophobia, fundus pigmentary changes and bull's eye maculopathy. Electroretinograms (ERGs) were extinguished in the four patients, whose recording was possible. Genetic testing revealed homozygous or two heterozygous bi-allelic mutations in the VPS13B (COH1) gene in all five patients, with five different allelic variants observed. The homozygous mutation c.6055_6056delGA; p.Asp2019Glnfs*15 in two sibling patients as well as the homozygous nonsense mutation c.8112C>G;p.Tyr2704* have not previously been reported. CONCLUSIONS: The phenotype of the five patients reported here is typical for Cohen syndrome; however, their genotype is heterogeneous. Two new allelic variants were found to be the causative mutation.

CEP290 Mutation Spectrum and Delineation of the Associated Phenotype in a Large German Cohort: A Monocentric Study.

PURPOSE: Gene therapy for Leber congenital amaurosis (LCA) is becoming available, and therefore it is crucial to identify eligible candidates. We report the spectrum and associated phenotype of CEP290 mutations in the largest German cohort observed by a single clinical site. DESIGN: Prospective cohort study. METHODS: Twenty-three patients with mutations in CEP290 were included. Genomic DNA was analyzed by Sanger sequencing or high-throughput sequencing for all retinitis pigmentosa-associated genes in patients, and segregation analysis was done in family members. Patients underwent functional and morphologic examinations, including fundus autofluorescence and spectral-domain optical coherence tomography. RESULTS: The most frequent mutation was c.2991+1655A>G, found in 87% of patients (20/23). Thirty percent of patients (7/23) carried the mutation in an apparent homozygous state and 57% (13/23) in a likely compound heterozygous state. The most common clinical diagnosis was LCA and/or early onset severe retinal dystrophy in 82% (19/23), followed by retinitis pigmentosa in 14% (3/23) and cone-rod dystrophy (4%, 1/23). Best-corrected visual acuity was severely reduced to residual light perception and hand motion vision, with the exception of 3 patients with best-corrected visual acuity of 0.8 (Snellen). The visual field was severely decreased and electroretinogram was undetectable in most cases; however, retinal layers at the fovea appeared to be relatively well preserved. Systemic disorders were not noticed. CONCLUSIONS: c.2991+1655A>G is by far the most important CEP290 mutation, contributing to 87% of patients with the CEP290 mutation in Germany. In our cohort, a homozygous c.2991+1655A>G genotype presented with a more severe phenotype. National studies and further detailed phenotype analysis seem to be important to assess the need for and promise of specific gene therapies.

Clinical Phenotype and Course of PDE6A-Associated Retinitis Pigmentosa Disease, Characterized in Preparation for a Gene Supplementation Trial.

Importance: Treatment trials require sound knowledge on the natural course of disease. Objective: To assess clinical features, genetic findings, and genotype-phenotype correlations in patients with retinitis pigmentosa (RP) associated with biallelic sequence variations in the PDE6A gene in preparation for a gene supplementation trial. Design, Setting, and Participants: This prospective, longitudinal, observational cohort study was conducted from January 2001 to December 2019 in a single center (Centre for Ophthalmology of the University of Tübingen, Germany) with patients recruited multinationally from 12 collaborating European tertiary referral centers. Patients with retinitis pigmentosa, sequence variants in PDE6A, and the ability to provide informed consent were included. Exposures: Comprehensive ophthalmological examinations; validation of compound heterozygosity and biallelism by familial segregation analysis, allelic cloning, or assessment of next-generation sequencing-read data, where possible. Main Outcomes and Measures: Genetic findings and clinical features describing the entire cohort and comparing patients harboring the 2 most common disease-causing variants in a homozygous state (c.304C>A;p.(R102S) and c.998 + 1G>A;p.?). Results: Fifty-seven patients (32 female patients [56%]; mean [SD], 40 [14] years) from 44 families were included. All patients completed the study. Thirty patients were homozygous for disease-causing alleles. Twenty-seven patients were heterozygous for 2 different PDE6A variants each. The most frequently observed alleles were c.304C>A;p.(R102S), c.998 + 1G>A;p.?, and c.2053G>A;p.(V685M). The mean (SD) best-corrected visual acuity was 0.43 (0.48) logMAR (Snellen equivalent, 20/50). The median visual field area with object III4e was 660 square degrees (5th and 95th percentiles, 76 and 11 019 square degrees; 25th and 75th percentiles, 255 and 3923 square degrees). Dark-adapted and light-adapted full-field electroretinography showed no responses in 88 of 108 eyes (81.5%). Sixty-nine of 108 eyes (62.9%) showed additional findings on optical coherence tomography imaging (eg, cystoid macular edema or macular atrophy). The variant c.998 + 1G>A;p.? led to a more severe phenotype when compared with the variant c.304C>A;p.(R102S). Conclusions and Relevance: Seventeen of the PDE6A variants found in these patients appeared to be novel. Regarding the clinical findings, disease was highly symmetrical between the right and left eyes and visual impairment was mild or moderate in 90% of patients, providing a window of opportunity for gene therapy.

Ophthalmic features of cone-rod dystrophy caused by pathogenic variants in the ALMS1 gene.

PURPOSE: We aim to describe ophthalmic characteristics and systemic findings in a cohort of seven patients with cone-rod retinal dystrophy (CORD) caused by pathogenic variants in the ALMS1 gene. METHODS: Seven patients with Alström syndrome (ALMS) were included in the study. A comprehensive ophthalmological examination was performed, including best-corrected visual acuity (BCVA), a semiautomated kinetic visual field exam, colour vision testing, full-field electroretinography testing according to International Society for Clinical Electrophysiology of Vision (ISCEV) standards, spectral domain optical coherence tomography (SD-OCT) and fundus autofluorescence (FAF) imaging, and slit lamp and dilated fundus examination. DNA samples were analysed using Sanger sequencing or exome sequencing. RESULTS: In our cohort, the ocular phenotype presented with a wide variability in retinal function and disease severity. However, age of symptom onset (i.e. nystagmus and photophobia) was at 6-9 months in all patients. These symptoms mostly mislead to the diagnosis of congenital achromatopsia (ACHM), Leber congenital amaurosis (LCA), isolated CORD or Bardet-Biedl syndrome. The systemic manifestations in our cohort were highly variable. CONCLUSION: In summary, we can report that most of our ALMS patients primarily presented with nystagmus and severe photophobia since early childhood interestingly without night blindness in the absence of systemic symptoms. Only genetic testing analysing both nonsyndromic retinal disease (RD) genes and syndromic ciliopathy genes by comprehensive panel sequencing can result in the correct diagnosis, genetically and clinically, with important implication for the physical health of the individual.