Clinical and Genetic Findings in a Cohort of Patients with PRPF31-associated Retinal Dystrophy.

PURPOSE: The purpose of our study was to assess the phenotypic and genotypic spectrum in a large cohort of patients with PRPF31-associated retinal dystrophy. DESIGN: Retrospective cohort study METHODS: In this retrospective chart review study, we collected cross-sectional data on the phenotype and genotype of patients with PRPF31-associated retinal dystrophy from the clinics for inherited retinal dystrophies at the University of Tuebingen and the local RetDis database and biobank. Patients underwent thorough ophthalmological examinations and genetic testing. RESULTS: Eighty-six patients from 61 families were available for clinical assessment, while genomic DNA was available for 111 individuals (index patients and family members). Fifty-three different disease-associated variants were observed in our cohort. Point mutations were the most common class. All but two patients exhibited features of a typical Retinitis pigmentosa (RP). One patient showed a cone-rod-dystrophy pattern. One mutation carrier revealed no signs of a retinal dystrophy. There was a statistically significant better visual acuity for patients with large deletions in the 20-39 age group. Cystoid macular edema was common in those with preserved central retina and showed an association with female sex. CONCLUSION: Our study confirms high phenotypic variability in disease onset and age at which legal blindness is reached in PRPF31-linked RP. Non-penetrance is commonly documented in family history, although poorly represented in our study, possibly indicating that true asymptomatic mutation carriers are rare if followed-up over lifetime with thorough ophthalmologic workup.

Genetic and Clinical Profile of Retinopathies Due to Disease-Causing Variants in Leber Congenital Amaurosis (LCA)-Associated Genes in a Large German Cohort.

To report the spectrum of Leber congenital amaurosis (LCA) associated genes in a large German cohort and to delineate their associated phenotype. Local databases were screened for patients with a clinical diagnosis of LCA and for patients with disease-causing variants in known LCA-associated genes independent of their clinical diagnosis. Patients with a mere clinical diagnosis were invited for genetic testing. Genomic DNA was either analyzed in a diagnostic-genetic or research setup using various capture panels for syndromic and non-syndromic IRD (inherited retinal dystrophy) genes. Clinical data was obtained mainly retrospectively. Patients with genetic and phenotypic information were eventually included. Descriptive statistical data analysis was performed. A total of 105 patients (53 female, 52 male, age 3-76 years at the time of data collection) with disease-causing variants in 16 LCA-associated genes were included. The genetic spectrum displayed variants in the following genes: CEP290 (21%), CRB1 (21%), RPE65 (14%), RDH12 (13%), AIPL1 (6%), TULP1 (6%), and IQCB1 (5%), and few cases harbored pathogenic variants in LRAT, CABP4, NMNAT1, RPGRIP1, SPATA7, CRX, IFT140, LCA5, and RD3 (altogether accounting for 14%). The most common clinical diagnosis was LCA (53%, 56/105) followed by retinitis pigmentosa (RP, 40%, 42/105), but also other IRDs were seen (cone-rod dystrophy, 5%; congenital stationary night blindness, 2%). Among LCA patients, 50% were caused by variants in CEP290 (29%) and RPE65 (21%), whereas variants in other genes were much less frequent (CRB1 11%, AIPL1 11%, IQCB1 9%, and RDH12 7%, and sporadically LRAT, NMNAT1, CRX, RD3, and RPGRIP1). In general, the patients showed a severe phenotype hallmarked by severely reduced visual acuity, concentric narrowing of the visual field, and extinguished electroretinograms. However, there were also exceptional cases with best corrected visual acuity as high as 0.8 (Snellen), well-preserved visual fields, and preserved photoreceptors in spectral domain optical coherence tomography. Phenotypic variability was seen between and within genetic subgroups. The study we are presenting pertains to a considerable LCA group, furnishing valuable comprehension of the genetic and phenotypic spectrum. This knowledge holds significance for impending gene therapeutic trials. In this German cohort, CEP290 and CRB1 are the most frequently mutated genes. However, LCA is genetically highly heterogeneous and exhibits clinical variability, showing overlap with other IRDs. For any therapeutic gene intervention, the disease-causing genotype is the primary criterion for treatment access, but the clinical diagnosis, state of the retina, number of to be treated target cells, and the time point of treatment will be crucial.

The Usher syndrome 1C protein harmonin regulates canonical Wnt signaling.

Human Usher syndrome (USH) is the most common form of hereditary combined deaf-blindness. USH is a complex genetic disorder, and the pathomechanisms underlying the disease are far from being understood, especially in the eye and retina. The USH1C gene encodes the scaffold protein harmonin which organizes protein networks due to binary interactions with other proteins, such as all USH proteins. Interestingly, only the retina and inner ear show a disease-related phenotype, although USH1C/harmonin is almost ubiquitously expressed in the human body and upregulated in colorectal cancer. We show that harmonin binds to ß-catenin, the key effector of the canonical Wnt (cWnt) signaling pathway. We also demonstrate the interaction of the scaffold protein USH1C/harmonin with the stabilized acetylated ß-catenin, especially in nuclei. In HEK293T cells, overexpression of USH1C/harmonin significantly reduced cWnt signaling, but a USH1C-R31* mutated form did not. Concordantly, we observed an increase in cWnt signaling in dermal fibroblasts derived from an USH1C R31*/R80Pfs*69 patient compared with healthy donor cells. RNAseq analysis reveals that both the expression of genes related to the cWnt signaling pathway and cWnt target genes were significantly altered in USH1C patient-derived fibroblasts compared to healthy donor cells. Finally, we show that the altered cWnt signaling was reverted in USH1C patient fibroblast cells by the application of Ataluren, a small molecule suitable to induce translational read-through of nonsense mutations, hereby restoring some USH1C expression. Our results demonstrate a cWnt signaling phenotype in USH establishing USH1C/harmonin as a suppressor of the cWnt/ß-catenin pathway.

Development of retinal atrophy after subretinal gene therapy with voretigene neparvovec.

BACKGROUND/AIMS: Voretigene neparvovec (VN) is the first and only subretinal gene therapy approved by the Food and Drug Administration and European Medicines Agency. Real-world application has started in 2018 in patients with vision impairment due to biallelic retinal pigment epithelium (RPE) 65 mutation-associated inherited retinal degenerations. Herein, we evaluated the development of retinal atrophy within in a single-centre patient cohort treated with VN. METHODS: 13 eyes of eight patients treated with VN were retrospectively analysed for areas of retinal atrophy over a period of 6-24 months following surgery. Ultrawide field images were used to measure the area of atrophy. Fundus autofluorescence imaging is presented as an instrument for early detection of signs of retinal atrophy in these patients. RESULTS: Atrophic changes beyond the retinotomy site were observed in all eyes. Areas of atrophy developed within the area of detachment (bleb) in all eight patients and outside the bleb in three patients. Changes in autofluorescence preceded the development of retinal atrophy and were already evident 2 weeks after surgery in the majority of patients. The areas of atrophy increase with time and progression continued over year 1. Functional outcomes remained stable (VA, FST, visual field). CONCLUSION: Subretinal injection of VN can lead to RPE atrophy with consequent photoreceptor loss in and outside of the bleb area. Fundus autofluorescence is an important tool to monitor atrophic changes in patients after gene therapy. Interestingly, while areas of atrophy also included central areas, the functional benefits of the treatment did not appear to be affected and remained stable.

Novel OPN1LW/OPN1MW Exon 3 Haplotype-Associated Splicing Defect in Patients with X-Linked Cone Dysfunction.

Certain combinations of common variants in exon 3 of OPN1LW and OPN1MW, the genes encoding the apo-protein of the long- and middle-wavelength sensitive cone photoreceptor visual pigments in humans, induce splicing defects and have been associated with dyschromatopsia and cone dysfunction syndromes. Here we report the identification of a novel exon 3 haplotype, G-C-G-A-T-T-G-G (referring to nucleotide variants at cDNA positions c.453, c.457, c.465, c.511, c.513, c.521, c.532, and c.538) deduced to encode a pigment with the amino acid residues L-I-V-V-A at positions p.153, p.171, p.174, p.178, and p.180, in OPN1LW or OPN1MW or both in a series of seven patients from four families with cone dysfunction. Applying minigene assays for all observed exon 3 haplotypes in the patients, we demonstrated that the novel exon 3 haplotype L-I-V-V-A induces a strong but incomplete splicing defect with 3-5% of residual correctly spliced transcripts. Minigene splicing outcomes were similar in HEK293 cells and the human retinoblastoma cell line WERI-Rb1, the latter retaining a cone photoreceptor expression profile including endogenous OPN1LW and OPN1MW gene expression. Patients carrying the novel L-I-V-V-A haplotype presented with a mild form of Blue Cone Monochromacy or Bornholm Eye Disease-like phenotype with reduced visual acuity, reduced cone electroretinography responses, red-green color vision defects, and frequently with severe myopia.

Three-year results of phase I retinal gene therapy trial for CNGA3-mutated achromatopsia: results of a non randomised controlled trial.

AIMS: To determine long-term safety and efficacy outcomes of a subretinal gene therapy for CNGA3-associated achromatopsia. We present data from an open-label, nonrandomised controlled trial (NCT02610582). METHODS: Details of the study design have been previously described. Briefly, nine patients were treated in three escalating dose groups with subretinal AAV8.CNGA3 gene therapy between November 2015 and October 2016. After the first year, patients were seen on a yearly basis. Safety assessment constituted the primary endpoint. On a secondary level, multiple functional tests were carried out to determine efficacy of the therapy. RESULTS: No adverse or serious adverse events deemed related to the study drug occurred after year 1. Safety of the therapy, as the primary endpoint of this trial, can, therefore, be confirmed. The functional benefits that were noted in the treated eye at year 1 were persistent throughout the following visits at years 2 and 3. While functional improvement in the treated eye reached statistical significance for some secondary endpoints, for most endpoints, this was not the case when the treated eye was compared with the untreated fellow eye. CONCLUSION: The results demonstrate a very good safety profile of the therapy even at the highest dose administered. The small sample size limits the statistical power of efficacy analyses. However, trial results inform on the most promising design and endpoints for future clinical trials. Such trials have to determine whether treatment of younger patients results in greater functional gains by avoiding amblyopia as a potential limiting factor.

Short term morphological rescue of the fovea after gene therapy with voretigene neparvovec.

PURPOSE: Leber congenital amaurosis type 2 (LCA2) and early-onset severe retinal dystrophy (EOSRD) are linked to visual impairment with nyctalopia and visual acuity reduction in early childhood. In 2017, the first gene therapy voretigene neparvovec (Luxturna™) for patients with LCA and EOSRD cause by bi-allelic mutations in the RPE65 gene has been approved. Here we report on an example of short-term change in the foveal morphology after functionally successful gene therapy with voretigene neparvovec in a 15-year old patient. METHODS: The clinical examinations included best corrected visual acuity (BCVA), spectral domain optical coherence tomography (OCT) and adaptive optics retinal imaging. RESULTS: During follow-up over a period of 3 months after the treatment, an improvement of the central foveal morphology could be observed in OCT, with a clear demarcation of the external limiting membrane and changes in the photoreceptor mosaic on adaptive optics retinal imaging. These morphological rescue parameters correlated in part with the improvement in foveal-mediated vision after the treatment and adaptive optics imaging. Although the visual acuity improved only slightly at month 3, objective central cone evaluation with chromatic pupil campimetry showed an increase in the central sensitivity. In daily life, the patient reported her visional experience after the treatment as 'brighter'. CONCLUSION: Rapid changes in the correlates of photoreceptor morphology after successful gene therapy in patients with LCA/EORD can be quantifiable on individual level.

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.

Safety and Vision Outcomes of Subretinal Gene Therapy Targeting Cone Photoreceptors in Achromatopsia: A Nonrandomized Controlled Trial.

Importance: Achromatopsia linked to variations in the CNGA3 gene is associated with day blindness, poor visual acuity, photophobia, and involuntary eye movements owing to lack of cone photoreceptor function. No treatment is currently available. Objective: To assess safety and vision outcomes of supplemental gene therapy with adeno-associated virus (AAV) encoding CNGA3 (AAV8.CNGA3) in patients with CNGA3-linked achromatopsia. Design, Setting, and Participants: This open-label, exploratory nonrandomized controlled trial tested safety and vision outcomes of gene therapy vector AAV8.CNGA3 administered by subretinal injection at a single center. Nine patients (3 per dose group) with a clinical diagnosis of achromatopsia and confirmed biallelic disease-linked variants in CNGA3 were enrolled between November 5, 2015, and September 22, 2016. Data analysis was performed from June 6, 2017, to March 12, 2018. Intervention: Patients received a single unilateral injection of 1.0 × 1010, 5.0 × 1010, or 1.0 × 1011 total vector genomes of AAV8.CNGA3 and were followed up for a period of 12 months (November 11, 2015, to October 10, 2017). Main Outcomes and Measures: Safety as the primary end point was assessed by clinical examination of ocular inflammation. Systemic safety was assessed by vital signs, routine clinical chemistry testing, and full and differential blood cell counts. Secondary outcomes were change in visual function from baseline in terms of spatial and temporal resolution and chromatic, luminance, and contrast sensitivity throughout a period of 12 months after treatment. Results: Nine patients (mean [SD] age, 39.6 [11.9] years; age range, 24-59 years; 8 [89%] male) were included in the study. Baseline visual acuity letter score (approximate Snellen equivalent) ranged from 34 (20/200) to 49 (20/100), whereas baseline contrast sensitivity log scores ranged from 0.1 to 0.9. All 9 patients underwent surgery and subretinal injection of AAV8.CNGA3 without complications. No substantial safety problems were observed during the 12-month follow-up period. Despite the congenital deprivation of cone photoreceptor-mediated vision in achromatopsia, all 9 treated eyes demonstrated some level of improvement in secondary end points regarding cone function, including mean change in visual acuity of 2.9 letters (95% CI, 1.65-4.13; P = .006, 2-sided t test paired samples). Contrast sensitivity improved by a mean of 0.33 log (95% CI, 0.14-0.51 log; P = .003, 2-sided t test paired samples). Conclusions and Relevance: Subretinal gene therapy with AAV8.CNGA3 was not associated with substantial safety problems and was associated with cone photoreceptor activation in adult patients, as reflected by visual acuity and contrast sensitivity gains. Trial Registration: ClinicalTrials.gov Identifier: NCT02610582.

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.

Molecular and clinical analysis of 27 German patients with Leber congenital amaurosis.

Leber congenital amaurosis (LCA) is the earliest and most severe form of all inherited retinal dystrophies (IRD) and the most frequent cause of inherited blindness in children. The phenotypic overlap with other early-onset and severe IRDs as well as difficulties associated with the ophthalmic examination of infants can complicate the clinical diagnosis. To date, 25 genes have been implicated in the pathogenesis of LCA. The disorder is usually inherited in an autosomal recessive fashion, although rare dominant cases have been reported. We report the mutation spectra and frequency of genes in 27 German index patients initially diagnosed with LCA. A total of 108 LCA- and other genes implicated in IRD were analysed using a cost-effective targeted next-generation sequencing procedure based on molecular inversion probes (MIPs). Sequencing and variant filtering led to the identification of putative pathogenic variants in 25 cases, thereby leading to a detection rate of 93%. The mutation spectrum comprises 34 different alleles, 17 of which are novel. In line with previous studies, the genetic results led to a revision of the initial clinical diagnosis in a substantial proportion of cases, demonstrating the importance of genetic testing in IRD. In addition, our detection rate of 93% shows that MIPs are a cost-efficient and sensitive tool for targeted next-generation sequencing in IRD.

Development of Methodology and Study Protocol: Safety and Efficacy of a Single Subretinal Injection of rAAV.hCNGA3 in Patients with CNGA3-Linked Achromatopsia Investigated in an Exploratory Dose-Escalation Trial.

Achromatopsia is an autosomal recessively inherited congenital defect characterized by a lack of cone photoreceptor function, leading to severely impaired vision. In this clinical study, achromatopsia patients were treated with a single subretinal injection of rAAV.hCNGA3 to restore cone function. The focus of this trial was on the safety of the treatment. After surgery, patients were monitored in eight extensive visits during the first year, followed by a 4-year follow-up period with annual visits. For essential complementation of the standard ophthalmological and systemic examinations, disease-specific methods were developed to assess the safety, efficacy, and patient-reported outcomes in this trial.

Development of a Chromatic Pupillography Protocol for the First Gene Therapy Trial in Patients With CNGA3-Linked Achromatopsia.

Purpose: To establish a feasible and sensitive pupillographic protocol to assess outer and inner retinal function for the first gene therapy trial in achromatopsia patients (ACHM) with mutations in CNGA3. Methods: Twenty-seven CNGA3-ACHM patients and 22 age-matched control subjects were tested using chromatic pupillography. Three different protocols were established to assess the pupillary light reflex parameters and to create the final protocol. In the individual protocols, various stimulus parameters (i.e., intensity, duration, wavelength, adaptation states) were applied to evaluate the impact of these stimuli on the pupillary response in untreated ACHM patients. Results: In the light-adapted conditions, CNGA3-ACHM patients showed significantly reduced maximal amplitudes compared with the control group when using a 1-second high intensity (28-lux corneal illumination) blue or red stimulus (P < 0.005). In the dark-adapted conditions, CNGA3-ACHM patients unexpectedly revealed significantly increased maximal amplitudes when stimulating with red (1 second) or blue (4 ms and 1 second) stimuli of low intensity (0.01-lux corneal illumination; P < 0.05). Pupil responses of CNGA3-ACHM patients after high intensity (28 lux) red and blue 1-second stimuli were within the normal range. Conclusions: Chromatic pupillography demonstrated significant reduced pupil responses to stimuli addressing primarily cone function, an increased sensitivity to rod-favoring stimuli and evidence for disinhibition of intrinsically photosensitive retinal ganglion cells in CNGA3-ACHM patients. A final protocol was established based on these findings. These conclusions may be useful for the objective assessment of efficacy gained by gene therapy or other innovative interventions in this hereditary retinal disorder.

In Vivo Analysis of Disease-Associated Point Mutations Unveils Profound Differences in mRNA Splicing of Peripherin-2 in Rod and Cone Photoreceptors.

Point mutations in peripherin-2 (PRPH2) are associated with severe retinal degenerative disorders affecting rod and/or cone photoreceptors. Various disease-causing mutations have been identified, but the exact contribution of a given mutation to the clinical phenotype remains unclear. Exonic point mutations are usually assumed to alter single amino acids, thereby influencing specific protein characteristics; however, they can also affect mRNA splicing. To examine the effects of distinct PRPH2 point mutations on mRNA splicing and protein expression in vivo, we designed PRPH2 minigenes containing the three coding exons and relevant intronic regions of human PRPH2. Minigenes carrying wild type PRPH2 or PRPH2 exon 2 mutations associated with rod or cone disorders were expressed in murine photoreceptors using recombinant adeno-associated virus (rAAV) vectors. We detect three PRPH2 splice isoforms in rods and cones: correctly spliced, intron 1 retention, and unspliced. In addition, we show that only the correctly spliced isoform results in detectable protein expression. Surprisingly, compared to rods, differential splicing leads to lower expression of correctly spliced and higher expression of unspliced PRPH2 in cones. These results were confirmed in qRT-PCR experiments from FAC-sorted murine rods and cones. Strikingly, three out of five cone disease-causing PRPH2 mutations profoundly enhanced correct splicing of PRPH2, which correlated with strong upregulation of mutant PRPH2 protein expression in cones. By contrast, four out of six PRPH2 mutants associated with rod disorders gave rise to a reduced PRPH2 protein expression via different mechanisms. These mechanisms include aberrant mRNA splicing, protein mislocalization, and protein degradation. Our data suggest that upregulation of PRPH2 levels in combination with defects in the PRPH2 function caused by the mutation might be an important mechanism leading to cone degeneration. By contrast, the pathology of rod-specific PRPH2 mutations is rather characterized by PRPH2 downregulation and impaired protein localization.

Homozygosity mapping and whole-genome sequencing reveals a deep intronic PROM1 mutation causing cone-rod dystrophy by pseudoexon activation.

Several genes have been implicated in the autosomal recessive form of cone-rod dystrophy (CRD), but the majority of cases remain unsolved. We identified a homozygous interval comprising two known genes associated with the autosomal recessive form of CRD, namely RAB28 and PROM1, in a consanguineous family with clinical evidence of CRD. Both genes proved to be mutation negative upon sequencing of exons and canonical splice sites but whole-genome sequencing revealed a private variant located deep in intron 18 of PROM1. In silico and functional analyses of this variant using minigenes as splicing reporters revealed the integration of a pseudoexon in the mutant transcript, thereby leading to a premature termination codon and presumably resulting in a functional null allele. This is the first report of a deep intronic variant that acts as a splicing mutation in PROM1. The detection of such variants escapes the exon-focused techniques typically used in genetic analyses. Sequencing the entire genomic regions of known disease genes might identify more causal mutations in the autosomal recessive form of CRD.

Blue cone monochromacy: visual function and efficacy outcome measures for clinical trials.

BACKGROUND: Blue Cone Monochromacy (BCM) is an X-linked retinopathy caused by mutations in the OPN1LW / OPN1MW gene cluster, encoding long (L)- and middle (M)-wavelength sensitive cone opsins. Recent evidence shows sufficient structural integrity of cone photoreceptors in BCM to warrant consideration of a gene therapy approach to the disease. In the present study, the vision in BCM is examined, specifically seeking clinically-feasible outcomes for a future clinical trial. METHODS: BCM patients (n = 25, ages 5-72) were studied with kinetic and static chromatic perimetry, full-field sensitivity testing, and eye movement recordings. Vision at the fovea and parafovea was probed with chromatic microperimetry. RESULTS: Kinetic fields with a Goldmann size V target were generally full. Short-wavelength (S-) sensitive cone function was normal or near normal in most patients. Light-adapted perimetry results on conventional background lights were abnormally reduced; 600-nm stimuli were seen by rods whereas white stimuli were seen by both rods and S-cones. Under dark-adapted conditions, 500-nm stimuli were seen by rods in both BCM and normals. Spectral sensitivity functions in the superior retina showed retained rod and S-cone functions in BCM under dark-adapted and light-adapted conditions. In the fovea, normal subjects showed L/M-cone mediation using a 650-nm stimulus under dark-adapted conditions, whereas BCM patients had reduced sensitivity driven by rod vision. Full-field red stimuli on bright blue backgrounds were seen by L/M-cones in normal subjects whereas BCM patients had abnormally reduced and rod-mediated sensitivities. Fixation location could vary from fovea to parafovea. Chromatic microperimetry demonstrated a large loss of sensitivity to red stimuli presented on a cyan adapting background at the anatomical fovea and surrounding parafovea. CONCLUSIONS: BCM rods continue to signal vision under conditions normally associated with daylight vision. Localized and retina-wide outcome measures were examined to evaluate possible improvement of L/M-cone-based vision in a clinical trial.

Novel compound heterozygous NMNAT1 variants associated with Leber congenital amaurosis.

PURPOSE: The gene encoding nicotinamide nucleotide adenylyltransferase 1 (NMNAT1) was recently found to be mutated in a subset of patients with Leber congenital amaurosis (LCA) with macular atrophy. The aim of this study was to determine the occurrence and frequency of NMNAT1 mutations and associated phenotypes in different types of inherited retinal dystrophies. METHODS: DNA samples of 161 patients with LCA without genetic diagnosis were analyzed for variants in NMNAT1 using Sanger sequencing. Variants in exon 5 of NMNAT1, which harbors the majority of the previously identified mutations, were screened in 532 additional patients with retinal dystrophies. This cohort encompassed 108 persons with isolated or autosomal recessive cone-rod dystrophy (CRD), 271 with isolated or autosomal recessive retinitis pigmentosa (RP), and 49 with autosomal dominant RP, as well as 104 persons with LCA in whom the causative mutation was previously identified. RESULTS: Compound heterozygous alterations were found in six patients with LCA and in one person with early-onset RP. All except one carried the common p.E257K variant on one allele. Macular atrophy was absent in one patient, who carried this variant in combination with a truncating mutation on the other allele. The p.E257K alteration was also found in a heterozygous state in five individuals with LCA and one with RP while no mutation was detected on the other allele. Two individuals with LCA carried other NMNAT1 variants in a heterozygous state, whereas no NMNAT1 variants in exon 5 were identified in individuals with CRD. The p.E257K variant was found to be enriched in a heterozygous state in individuals with LCA (0.94%) compared to Caucasian controls (0.18%), although the difference was statistically insignificant (p=0.12). CONCLUSIONS: Although macular atrophy can occur in LCA and CRD, no NMNAT1 mutations were found in the latter cohort. NMNAT1 variants were also not found in a large group of patients with sporadic or autosomal recessive RP. The enrichment of p.E257K in a heterozygous state in patients with LCA versus controls suggests that this allele could act as a modifier in other genetic subtypes of LCA.

Union makes strength: a worldwide collaborative genetic and clinical study to provide a comprehensive survey of RD3 mutations and delineate the associated phenotype.

Leber congenital amaurosis (LCA) is the earliest and most severe retinal degeneration (RD), and the most common cause of incurable blindness diagnosed in children. It is occasionally the presenting symptom of multisystemic ciliopathies which diagnosis will require a specific care of patients. Nineteen LCA genes are currently identified and three of them account for both non-syndromic and syndromic forms of the disease. RD3 (LCA12) was implicated as a LCA gene based on the identification of homozygous truncating mutations in two LCA families despite the screening of large cohorts of patients. Here we provide a comprehensive survey of RD3 mutations and of their clinical expression through the screening of a cohort of 852 patients originating worldwide affected with LCA or early-onset and severe RD. We identified three RD3 mutations in seven unrelated consanguineous LCA families - i.e., a 2 bp deletion and two nonsense mutations - predicted to cause complete loss of function. Five families originating from the Southern Shores of the Mediterranean segregated a similar mutation (c.112C>T, p.R38*) suggesting that this change may have resulted from an ancient founder effect. Considering the low frequency of RD3 carriers, the recurrence risk for LCA in non-consanguineous unions is negligible for both heterozygote and homozygote RD3 individuals. The LCA12 phenotype in our patients is highly similar to those of patients with mutant photoreceptor-specific guanylate cyclase (GUCY2D/LCA1). This observation is consistent with the report of the role of RD3 in trafficking of GUCYs and gives further support to a common mechanism of photoreceptor degeneration in LCA12 and LCA1, i.e., inability to increase cytoplasmic cGMP concentration in outer segments and thus to recover the dark-state. Similar to LCA1, LCA12 patients have no extraocular symptoms despite complete inactivation of both RD3 alleles, supporting the view that extraocular investigations in LCA infants with RD3 mutations should be avoided.

Cone-rod dystrophy associated with amelogenesis imperfecta in a child with neurofibromatosis type 1.

PURPOSE: To report a case of a 9-year-old child with neurofibromatosis type 1 (NF1) and Jalili syndrome, the latter denoting a rare combination of cone-rod dystrophy and amelogenesis imperfecta. METHODS: Detailed ophthalmological and electrophysiological examinations were carried out and blood samples were taken from the patient and her father for molecular genetic analysis by direct DNA sequencing of the NF1 and the ancient conserved domain protein 4 (CNNM4) gene. RESULTS: The diagnosis of neurofibromatosis type 1 (NF1) could be confirmed clinically and genetically. Furthermore, cone-rod dystrophy and amelogenesis imperfecta could be observed as typical features of a rare condition, acknowledged as Jalili syndrome. The diagnosis was assured on the basis of clinical examinations and molecular genetic analysis of the CNNM4 gene, which was previously shown to cause Jalili syndrome. CONCLUSION: Our case shows a unique combination of NF1 and Jalili syndrome. The random association of two diseases is unusual and deserves attention. This case highlights the importance not only of detailed clinical examination, but also of molecular genetic analysis, which together provide a precise diagnosis.