Scrutinizing pathogenicity of the USH2A c.2276 G > T; p.(Cys759Phe) variant.

The USH2A variant c.2276 G > T (p.(Cys759Phe)) has been described by many authors as a frequent cause of autosomal recessive retinitis pigmentosa (arRP). However, this is in contrast with the description of two asymptomatic individuals homozygous for this variant. We therefore assessed pathogenicity of the USH2A c.2276 G > T variant using extensive genetic and functional analyses. Whole genome sequencing and optical genome mapping were performed for three arRP cases homozygous for USH2A c.2276 G > T to exclude alternative genetic causes. A minigene splice assay was designed to investigate the effect of c.2276 G > T on pre-mRNA splicing, in presence or absence of the nearby c.2256 T > C variant. Moreover, an ush2ap.(Cys771Phe) zebrafish knock-in model mimicking human p.(Cys759Phe) was generated and characterized using functional and immunohistochemical analyses. Besides the homozygous c.2276 G > T USH2A variant, no alternative genetic causes were identified. Evaluation of the ush2ap.(Cys771Phe) zebrafish model revealed strongly reduced levels of usherin expression at the photoreceptor periciliary membrane, increased levels of rhodopsin localization in the photoreceptor cell body and decreased electroretinogram (ERG) b-wave amplitudes compared to wildtype controls. In conclusion, we confirmed pathogenicity of USH2A c.2276 G > T (p.(Cys759Phe)). Consequently, cases homozygous for c.2276 G > T can now receive a definite genetic diagnosis and can be considered eligible for receiving future QR-421a-mediated exon 13 skipping therapy.

Comparing the Efficacy of Bevacizumab and Ranibizumab in Patients with Diabetic Macular Edema (BRDME): The BRDME Study, a Randomized Trial.

PURPOSE: To generate conclusive evidence regarding the noninferiority of intravitreal bevacizumab compared with ranibizumab in patients with diabetic macular edema (DME). DESIGN: Comparative, randomized, double-masked, multicenter, noninferiority clinical trial. PARTICIPANTS: Eligible patients were older than 18 years, diagnosed with type 1 or type 2 diabetes mellitus, with glycosylated hemoglobin of less than 12%, central area thickness of more than 325 µm, and visual impairment from DME with a best-corrected visual acuity (BCVA) between 24 letters and 78 letters. METHODS: From June 2012 through February 2018, a total of 170 participants were randomized to receive 6 monthly injections of either 1.25 mg bevacizumab (n = 86) or 0.5 mg ranibizumab (n = 84). MAIN OUTCOME MEASURES: Primary outcome was change in BCVA from baseline to month 6 compared between the 2 treatment arms. The noninferiority margin was 3.5 letters. RESULTS: The difference in mean BCVA between treatment arms was 1.8 letters in favor of ranibizumab after 6 months of follow-up; BCVA improved by 4.9±6.7 letters in the bevacizumab group and 6.7±8.7 letters in the ranibizumab group. The lower bound of the 2-sided 90% confidence interval (CI) was -3.626 letters, exceeding the noninferiority margin of 3.5 letters. Central area thickness decreased more with ranibizumab (138.2±114.3 µm) compared with bevacizumab (64.2±104.2 µm). In a post hoc subgroup analysis, participants with a worse BCVA at baseline (≤69 letters) improved by 6.7±7.0 letters with bevacizumab and 10.4±10.0 letters with ranibizumab, and central area thickness decreased significantly more in the ranibizumab arm of this subgroup compared with the bevacizumab arm. Participants with an initially better BCVA at baseline (≥70 letters) did not demonstrate differences in BCVA or OCT outcomes between treatment arms. CONCLUSIONS: Based on change in BCVA from baseline to month 6, the noninferiority of 1.25 mg bevacizumab to 0.5 mg ranibizumab was not confirmed. Only the subgroup of patients with a lower BCVA at baseline showed better visual acuity and anatomic outcomes with ranibizumab. Our study confirmed the potential differential efficacy of anti-vascular endothelial growth factor agents in the treatment of DME as well as the difference in response between patient groups with different baseline visual acuities.

Comparing the Efficacy of Bevacizumab and Ranibizumab in Patients with Retinal Vein Occlusion: The Bevacizumab to Ranibizumab in Retinal Vein Occlusions (BRVO) study, a Randomized Trial.

PURPOSE: Comparing the efficacy of intravitreal injections of bevacizumab to ranibizumab in the treatment of macular edema (ME) resulting from retinal vein occlusion (RVO). DESIGN: Comparative, randomized, double-masked, multicenter, noninferiority clinical trial. The noninferiority margin was 4 letters. PARTICIPANTS: Patients with vision loss resulting from ME secondary to a branch or (hemi) central RVO who might benefit from anti-vascular endothelial growth factor treatment were eligible for participation. METHODS: From June 2012 through February 2018, 277 participants were randomized to receive injections of 1.25 mg bevacizumab (n = 139) or 0.5 mg ranibizumab (n = 138). The follow-up was 6 months with a monthly dosing interval. MAIN OUTCOME MEASURES: The primary outcome was a change in visual acuity from baseline at 6 months. Changes in the central area thickness and safety were studied as secondary outcomes. RESULTS: The mean visual acuity (±standard deviation) improved, with 15.3±13.0 letters for bevacizumab and 15.5±13.3 letters for ranibizumab after 6 months of monthly treatment. The lower limit of the 2-sided 90% confidence interval was -1.724 letters, which is within the noninferiority margin of 4 letters. Even in the branch and (hemi-)central RVO subgroups, minimal differences were found in visual acuity outcomes between treatment arms. Changes in central area thickness on OCT at 6 months did not differ significantly between treatment groups, with a decrease of 287.0±231.3 µm in the bevacizumab group and 300.8±224.8 µm in the ranibizumab group. Severe adverse events (SAEs) were also distributed equally over both treatment groups: 10 participants (7.1%) in the bevacizumab group and 13 participants (9.2%) in the ranibizumab group experienced SAEs. CONCLUSIONS: This study showed, based on the change in visual acuity, that bevacizumab is noninferior to ranibizumab for patients with ME resulting from RVO of either subtype when receiving monthly injections for a period of 6 months. In addition, anatomic and safety outcomes did not differ between treatment groups. Based on our findings, bevacizumab may be an effective alternative to ranibizumab.

Mutations in MFSD8, encoding a lysosomal membrane protein, are associated with nonsyndromic autosomal recessive macular dystrophy.

PURPOSE: This study aimed to identify the genetic defects in 2 families with autosomal recessive macular dystrophy with central cone involvement. DESIGN: Case series. PARTICIPANTS: Two families and a cohort of 244 individuals with various inherited maculopathies and cone disorders. METHODS: Genome-wide linkage analysis and exome sequencing were performed in 1 large family with 5 affected individuals. In addition, exome sequencing was performed in the proband of a second family. Subsequent analysis of the identified mutations in 244 patients was performed by Sanger sequencing or restriction enzyme digestion. The medical history of individuals carrying the MFSD8 variants was reviewed and additional ophthalmic examinations were performed, including electroretinography (ERG), multifocal ERG (mfERG), perimetry, optical coherence tomography (OCT), fundus autofluorescence, and fundus photography. MAIN OUTCOME MEASURES: MFSD8 variants, age at diagnosis, visual acuity, fundus appearance, color vision defects, visual field, ERG, mfERG, fundus autofluorescence, and OCT findings. RESULTS: Compound heterozygous variants in MFSD8, a gene encoding a lysosomal transmembrane protein, were identified in 2 families with macular dystrophy with a normal or subnormal ERG, but reduced mfERG. In both families, a heterozygous missense variant p.Glu336Gln was identified, which was predicted to have a mild effect on the protein. In the first family, a protein-truncating variant (p.Glu381*) was identified on the other allele, and in the second family, a variant (c.1102G>C) was identified that results in a splicing defect leading to skipping of exon 11 (p.Lys333Lysfs*3). The p.Glu336Gln allele was found to be significantly enriched in patients with maculopathies and cone disorders (6/488) compared with ethnically matched controls (35/18 682; P < 0.0001), suggesting that it may act as a genetic modifier. CONCLUSIONS: In this study, we identified variants in MFSD8 as a novel cause of nonsyndromic autosomal recessive macular dystrophy with central cone involvement. Affected individuals showed no neurologic features typical for variant late-infantile neuronal ceroid lipofuscinosis (vLINCL), a severe and devastating multisystem lysosomal storage disease previously associated with mutations in MFSD8. We propose a genotype-phenotype model in which a combination of a severe and a mild variant cause nonsyndromic macular dystrophy with central cone involvement, and 2 severe mutations cause vLINCL.

A prospective, observational, open-label, multicentre study to investigate the daily treatment practice of ranibizumab in patients with neovascular age-related macular degeneration.

PURPOSE: The HELIOS (Health Economics with Lucentis in Observational Settings) study was designed on request of the Dutch Health Authority for an observational study to assess the effectiveness and safety of ranibizumab for neovascular age-related macular degeneration (wet AMD) in daily practice. METHODS: The HELIOS study was a 2-year prospective, observational, open-label, multicentre study involving 14 sites. Patients with wet AMD were enrolled and observed for a period of 24 months. The data were collected at baseline and at the visits closest around the time-points 3, 6, 12, 18 and 24 months after inclusion. RESULTS: Treatment with ranibizumab resulted in prevention of vision loss. The mean ETDRS score increased from 45.1 letters at baseline to 48.5 letters at 24 months. This was achieved with a mean of 7.8 injections over 24 months. Stabilization of visual acuity was also reflected by the scores on the quality of life EQ-5D questionnaire, which did not significantly change over the study period. The more subjective EQ-VAS questionnaire showed an overall improvement. The VFQ-25 questionnaire was also mostly stable over time. After 24 months, 32.2% of the patients gained ≥1 letter and 17.1% gained >15 letters. Patients completing the loading phase were better responders, as demonstrated by increased long-term visual acuity. In addition, ranibizumab was well tolerated and had a safety profile commonly seen in routine clinical practice. CONCLUSION: This study demonstrates that also in daily practice ranibizumab was effective in preventing vision loss over a period of 24 months. No new safety findings were identified.

Dominant cystoid macular dystrophy.

OBJECTIVE: To describe the clinical characteristics and long-term follow-up in patients with autosomal dominant cystoid macular dystrophy (DCMD). DESIGN: Retrospective case series. PARTICIPANTS: Ninety-seven patients with DCMD. METHODS: Extensive ophthalmic examination, including visual acuity (VA), fundus photography, fluorescein angiography (FA), fundus autofluorescence (FAF) imaging, optical coherence tomography (OCT), color vision testing, dark adaptation testing, full-field electroretinography (ERG), and electro-oculography (EOG). Blood samples were obtained for DNA extraction and subsequent haplotype analysis. MAIN OUTCOME MEASURES: Age at onset, VA, fundus appearance, and characteristics on FA, FAF, OCT, ERG, and EOG. RESULTS: Cystoid fluid collections (CFCs) were the first retinal abnormalities detectable in DCMD, developing during childhood. At long-term follow-up, the CFCs decreased in size and number, and eventually disappeared with concurrent development of progressive chorioretinal atrophy and hyperpigmented deposits in the posterior pole. Dominant cystoid macular dystrophy could be classified into 3 stages, based on characteristics on ophthalmoscopy, FAF, FA, and OCT, as well as on results of electrophysiologic analysis. The staging system correlated with age and VA. In stage 1 DCMD (20 patients; 22%), patients generally were younger than 20 years and had CFCs with fine folding of the internal limiting membrane and mild pigment changes. In stage 2 DCMD (48 patients; 52%), the CFCs tended to decrease in size, and moderate macular chorioretinal atrophy developed. Patients with stage 3 DCMD (24 patients; 26%) generally were older than 50 years and showed profound chorioretinal atrophy, as well as coarse hyperpigmented deposits in the posterior pole. Most patients were (highly) hyperopic (72 patients; 92%). All DCMD patients shared the disease haplotype at the DCMD locus at 7p15.3. CONCLUSIONS: Dominant cystoid macular dystrophy is a progressive retinal dystrophy, characterized primarily by early-onset cystoid fluid collections in the neuroretina, which distinguishes this disorder from other retinal dystrophies. The phenotypic range of DCMD can be classified into 3 stages. The genetic locus for this retinal dystrophy has been mapped to 7p15.3, but the involved gene is currently unknown.

Comprehensive analysis of the achromatopsia genes CNGA3 and CNGB3 in progressive cone dystrophy.

OBJECTIVE: To investigate whether the major achromatopsia genes (CNGA3 and CNGB3) play a role in the cause of progressive cone dystrophy (CD). DESIGN: Prospective multicenter study. PARTICIPANTS: Probands (N = 60) with autosomal recessive (ar) CD from various ophthalmogenetic clinics in The Netherlands. METHODS: All available ophthalmologic data from the arCD probands were registered from medical charts and updated by an additional ophthalmologic examination. Mutations in the CNGA3 and CNGB3 genes were analyzed by direct sequencing. MAIN OUTCOME MEASURES: CNGA3 and CNGB3 mutations and clinical course in arCD probands. RESULTS: In 3 arCD probands (3/60; 5%) we found 2 mutations in the CNGB3 gene. Two of these probands had compound heterozygous mutations (p.R296YfsX9/p.R274VfsX12 and p.R296YfsX9/c.991-3T>g). The third proband revealed homozygous missense mutations (p.R403Q) with 2 additional variants in the CNGA3 gene (p.E228K and p.V266M). These probands did not have a congenital nystagmus, but had a progressive deterioration of visual acuity, color vision, and photopic electroretinogram, with onset in the second decade. In 6 other unrelated probands, we found 6 different heterozygous amino acid changes in the CNGA3 (N = 4) and CNGB3 (N = 2) gene. CONCLUSIONS: The CNGB3 gene accounts for a small fraction of the later onset progressive form of cone photoreceptor disorders, and CNGA3 may have an additive causative effect. Our data indicate that these genes are involved in a broader spectrum of cone dysfunction, and it remains intriguing why initial cone function can be spared despite similar gene defects. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Homozygosity mapping reveals PDE6C mutations in patients with early-onset cone photoreceptor disorders.

Cone photoreceptor disorders form a clinical spectrum of diseases that include progressive cone dystrophy (CD) and complete and incomplete achromatopsia (ACHM). The underlying disease mechanisms of autosomal recessive (ar)CD are largely unknown. Our aim was to identify causative genes for these disorders by genome-wide homozygosity mapping. We investigated 75 ACHM, 97 arCD, and 20 early-onset arCD probands and excluded the involvement of known genes for ACHM and arCD. Subsequently, we performed high-resolution SNP analysis and identified large homozygous regions spanning the PDE6C gene in one sibling pair with early-onset arCD and one sibling pair with incomplete ACHM. The PDE6C gene encodes the cone alpha subunit of cyclic guanosine monophosphate (cGMP) phosphodiesterase, which converts cGMP to 5'-GMP, and thereby plays an essential role in cone phototransduction. Sequence analysis of the coding region of PDE6C revealed homozygous missense mutations (p.R29W, p.Y323N) in both sibling pairs. Sequence analysis of 104 probands with arCD and 10 probands with ACHM revealed compound heterozygous PDE6C mutations in three complete ACHM patients from two families. One patient had a frameshift mutation and a splice defect; the other two had a splice defect and a missense variant (p.M455V). Cross-sectional retinal imaging via optical coherence tomography revealed a more pronounced absence of cone photoreceptors in patients with ACHM compared to patients with early-onset arCD. Our findings identify PDE6C as a gene for cone photoreceptor disorders and show that arCD and ACHM constitute genetically and clinically overlapping phenotypes.

Genetic etiology and clinical consequences of complete and incomplete achromatopsia.

OBJECTIVE: To investigate the genetic causes of complete and incomplete achromatopsia (ACHM) and assess the association between disease-causing mutations, phenotype at diagnosis, and visual prognosis. DESIGN: Clinic-based, longitudinal, multicenter study. PARTICIPANTS: Probands with complete ACHM (n = 35), incomplete ACHM (n = 26), or nonspecific ACHM (n = 2) and their affected relatives (n = 18) from various ophthalmogenetic clinics in The Netherlands. METHODS: Ophthalmologic clinical data were assessed over a life time and were registered from medical charts and updated by ophthalmologic examination. Mutations in the CNGB3, CNGA3, and GNAT2 genes were analyzed by direct sequencing. MAIN OUTCOME MEASURES: Genetic mutations and clinical course of ACHM. RESULTS: CNGB3 mutations were identified in 55 of 63 (87%) of probands and all caused premature truncation of the protein. The most common mutation was p.T383IfsX13 (80%); among the 4 other mutations was the novel frameshift mutation p.G548VfsX35. CNGA3 mutations were detected in 3 of 63 (5%) probands; all caused an amino acid change of the protein. No mutations were found in the GNAT2 gene. The ACHM subtype, visual acuity, color vision, and macular appearance were equally distributed among the CNGB3 genotypes, but were more severely affected among CNGA3 genotypes. Visual acuity deteriorated from infancy to adulthood in 12% of patients, leading to 0.10 in 61%, and even lower than 0.10 in 20% of patients. CONCLUSIONS: In this well-defined cohort of ACHM patients, the disease seemed much more genetically homogeneous than previously described. The CNGB3 gene was by far the most important causal gene, and T383IfsX13 the most frequent mutation. The ACHM subtype did not associate with a distinct genetic etiology, nor were any other genotype-phenotype correlations apparent. The distinction between complete and incomplete subtypes of ACHM has no clinical value, and the assumption of a stationary nature is misleading.

Novel compound heterozygous TULP1 mutations in a family with severe early-onset retinitis pigmentosa.

OBJECTIVE: To describe the clinical characteristics and determine the genetic defect in a Surinamese family with autosomal recessive retinitis pigmentosa. METHODS: Family members underwent blood sampling and ophthalmologic examinations. After exclusion of all known mutations in all genes involved in autosomal recessive retinitis pigmentosa, a genome-wide linkage scan was performed using 11,555 single-nucleotide polymorphisms spread throughout the genome. Mutation analysis of the TULP1 gene was performed by direct sequencing. RESULTS: All affected family members had a severe retinal dystrophy with a history of nystagmus, low visual acuity, and nyctalopia since infancy. The scotopic and photopic responses were nonrecordable on electroretinography. A genome-wide scan suggested linkage to the chromosomal region containing the TULP1 gene. Mutation analysis of TULP1 identified novel compound heterozygous mutations (p.Arg482Trp and p.Leu504fsX140) in all affected family members. CONCLUSIONS: The affected members of the Surinamese family have a severe early-onset form of autosomal recessive retinitis pigmentosa, which is caused by compound heterozygous mutations in the TULP1 gene. Clinical Relevance Clinical and molecular genetic characterization of autosomal recessive retinitis pigmentosa may help to provide a more accurate prognosis in individual patients. This study confirms that TULP1 mutations cause a severe early-onset form of autosomal recessive retinitis pigmentosa.

Mutations in the peripherin/RDS gene are an important cause of multifocal pattern dystrophy simulating STGD1/fundus flavimaculatus.

AIM: To describe the phenotype and to analyse the peripherin/RDS gene in 10 unrelated families with multifocal pattern dystrophy simulating Stargardt disease (STGD1). METHODS: The probands of 10 families and 20 affected family members underwent an ophthalmic examination including dilated fundus examination, fundus autofluorescence imaging and optical coherence tomography (OCT). In all probands and in selected family members, fluorescein angiography, electrophysiological testing and visual field analysis were performed. Blood samples were obtained from affected and unaffected family members for analysis of the peripherin/RDS gene. RESULTS: All 10 probands carried mutations in the peripherin/RDS gene. Nine different mutations were identified, including six mutations that were not described previously. All probands showed a pattern dystrophy with yellow-white flecks in the posterior pole that strongly resembled the flecks seen in STGD1, on ophthalmoscopy as well as on autofluorescence and OCT. Clinical findings in the family members carrying the same mutation as the proband were highly variable, ranging from no visible abnormalities to retinitis pigmentosa. CONCLUSIONS: Mutations in the peripherin/RDS gene are the major cause of multifocal pattern dystrophy simulating STGD1/fundus flavimaculatus. This autosomal dominant disorder should be distinguished from autosomal recessive STGD1, in view of the different inheritance pattern and the overall better visual prognosis.

The benign concentric annular macular dystrophy locus maps to 6p12.3-q16.

PURPOSE: To describe the clinical findings and to identify the genetic locus in a Dutch family with autosomal dominant benign concentric annular macular dystrophy (BCAMD). METHODS: All family members underwent ophthalmic examination. Linkage analysis of candidate retinal dystrophy loci and a whole genome scan were performed. Five candidate genes from the linked locus were analyzed for mutations by direct sequencing. RESULTS: The BCAMD phenotype is initially characterized by parafoveal hypopigmentation and good visual acuity, but progresses to a retinitis pigmentosa-like phenotype. Linkage analysis established complete segregation of the BCAMD phenotype (maximum multipoint LOD score, 3.8) with DNA markers at chromosome 6, region p12.3-q16. Recombination events defined a critical interval spanning 30.7 cM at the long arm of chromosome 6 between markers D6S269 and D6S300. This interval encompasses several retinal dystrophy loci, including the ELOVL4 gene, mutated in autosomal dominant Stargardt disease, and the RIM1 gene, mutated in autosomal dominant cone-rod dystrophy, as well as the retinally expressed GABRR1 and -2 genes. Mutation screening of these four genes revealed no mutations. Sequence analysis of the interphotoreceptor matrix proteoglycan 1 gene IMPG1, also residing in the BCAMD locus, revealed a single base-pair change (T-->C) of nucleotide 1866 in exon 13, resulting in a Leu579Pro amino acid substitution. This mutation was absent in 190 control individuals. CONCLUSIONS: Significant linkage was found for the BCAMD defect with chromosomal 6, region p12.3-q16. A Leu579Pro mutation in the IMPG1 gene may play a causal role.

A peculiar autosomal dominant macular dystrophy caused by an asparagine deletion at codon 169 in the peripherin/RDS gene.

OBJECTIVE: To describe the clinical and genetic findings in a family with a peculiar autosomal dominant macular dystrophy with peripheral deposits. METHODS: All family members underwent an ophthalmic examination, and their genomic DNA was screened for mutations in the human retinal degeneration slow (peripherin/RDS) and rhodopsin genes. In selected cases, fluorescein angiography and electrophysiologic testing were performed. RESULTS: The age at onset of the disease was between the third and fourth decades of life, starting with mild visual acuity loss and periods of metamorphopsia. Clinical signs included subretinal yellowish macular deposits evolving into geographic atrophy and retinal hypopigmentation and hyperpigmentation. Electroretinography demonstrated rod dysfunction, and electro-oculograms were mildly to severely disturbed. All affected members were found to carry a 3-base pair deletion affecting codon 169 of the peripherin/RDS gene. This mutation resulted in an asparagine (Asn) deletion in the peripherin/RDS protein and was not found in 155 control individuals. CONCLUSION: A deletion of Asn169 in the peripherin/RDS protein causes a peculiar form of autosomal dominant macular dystrophy in a large family from the Netherlands. CLINICAL RELEVANCE: Characterizing the phenotype and genotype in this family may, in the long term, result in a better understanding of the precise mechanism underlying this retinal degeneration.

Genetic heterogeneity of butterfly-shaped pigment dystrophy of the fovea.

PURPOSE: Butterfly-shaped macular dystrophy (BSMD) has so far only been associated with mutations in the peripherin/RDS gene. The initial aim of our study was to investigate the peripherin/RDS gene as the causative gene in a family with BSMD. Subsequently the putative involvement of the ROM-1 gene, 4 genes expressed in cone photoreceptors, all known non-syndromic macular, retinal pigment epithelium and choroidal dystrophy loci, all known Leber congenital amaurosis loci and all known non-syndromic congenital and stationary retinal disease loci was examined. METHODS: Thirteen members from the original family with autosomal dominant BSMD were examined. The protein coding exons of the peripherin/RDS gene were screened for mutations by sequence analysis. Linkage analysis was performed using markers flanking the peripherin/RDS gene to rule out the presence of a heterozygous deletion. Likewise, involvement of the ROM-1 gene, four cone genes, 41 non-syndromic retinal disease loci and one syndromic retinal disease locus was investigated. RESULTS: Sequence analysis of the peripherin/RDS gene revealed no mutations. In addition, the BSMD phenotype could not be genetically linked to the peripherin/RDS gene, the ROM-1 gene and the four cone genes nor to any of the 42 retinal disease loci. CONCLUSIONS: This study reveals genetic heterogeneity for BSMD by the identification of a BSMD family, which is not associated with a mutation in the peripherin/RDS gene nor with any other known non-syndromic retinal disease gene.