Autosomal dominant optic atrophy is one of the most common inherited optic neuropathies. This disease is genetically heterogeneous, but most cases are due to pathogenic variants in the OPA1 gene: depending on the population studied, 32–90% of cases harbor pathogenic variants in this gene. The aim of this study was to provide a comprehensive overview of the entire spectrum of likely pathogenic variants in the OPA1 gene in a large cohort of patients. Over a period of 20 years, 755 unrelated probands with a diagnosis of bilateral optic atrophy were referred to our laboratory for molecular genetic investigation. Genetic testing of the OPA1 gene was initially performed by a combined analysis using either single-strand conformation polymorphism or denaturing high performance liquid chromatography followed by Sanger sequencing to validate aberrant bands or melting profiles. The presence of copy number variations was assessed using multiplex ligation-dependent probe amplification. Since 2012, genetic testing was based on next-generation sequencing platforms. Genetic screening of the OPA1 gene revealed putatively pathogenic variants in 278 unrelated probands which represent 36.8% of the entire cohort. A total of 156 unique variants were identified, 78% of which can be considered null alleles. Variant c.2708_2711del/p.(V903Gfs*3) was found to constitute 14% of all disease-causing alleles. Special emphasis was placed on the validation of splice variants either by analyzing cDNA derived from patients´ blood samples or by heterologous splice assays using minigenes. Splicing analysis revealed different aberrant splicing events, including exon skipping, activation of exonic or intronic cryptic splice sites, and the inclusion of pseudoexons. Forty-eight variants that we identified were novel. Nine of them were classified as pathogenic, 34 as likely pathogenic and five as variant of uncertain significance. Our study adds a significant number of novel variants to the mutation spectrum of the OPA1 gene and will thereby facilitate genetic diagnostics of patients with suspected dominant optic atrophy.

BackgroundLeber’s hereditary optic neuropathy (LHON) has been considered a prototypical mitochondriopathy and a textbook example for maternal inheritance linked to certain disease-causing variants in the mitochondrial genome. Recently, an autosomal recessive form of LHON (arLHON) has been described, caused by disease-causing variants in the nuclear encoded gene DNAJC30.Methods and resultsIn this study, we screened the DNAJC30 gene in a large Central European cohort of patients with a clinical diagnosis of LHON or other autosomal inherited optic atrophies (OA). We identified likely pathogenic variants in 35/1202 patients, corresponding to a detection rate of 2.9%. The previously described missense variant c.152A>G;p.(Tyr51Cys) accounts for 90% of disease-associated alleles in our cohort and we confirmed a strong founder effect. Furthermore, we identified two novel pathogenic variants in DNAJC30: the nonsense variant c.610G>T;p.(Glu204*) and the in-frame deletion c.230_232del;p.(His77del). Clinical investigation of the patients with arLHON revealed a younger age of onset, a more frequent bilateral onset and an increased clinically relevant recovery compared with LHON associated with disease-causing variants in the mitochondrial DNA.ConclusionThis study expands previous findings on arLHON and emphasises the importance of DNAJC30 in the genetic diagnostics of LHON and OA in European patients.

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.

Background Dominant optic atrophy (DOA) is an inherited optic neuropathy that mainly affects visual acuity, central visual fields and color vision due to a progressive loss of retinal ganglion cells and their axons that form the optic nerve. Approximately 45–90% of affected individuals with DOA harbor pathogenic variants in the OPA1 gene. The mutation spectrum of OPA1 comprises nonsense, canonical and non-canonical splice site, frameshift and missense as well as copy number variants, but intragenic inversions have not been reported so far. Case presentation We report a 33-year-old male with characteristic clinical features of DOA. Whole-genome sequencing identified a structural variant of 2.4 kb comprising an inversion of 937 bp at the OPA1 locus. Fine mapping of the breakpoints to single nucleotide level revealed that the structural variation was an inversion flanked by two deletions. As this rearrangement inverts the entire first exon of OPA1, it was classified as likely pathogenic. Conclusions We report the first DOA case harboring an inversion in the OPA1 gene. Our study demonstrates that copy-neutral genomic rearrangements have to be considered as a possible cause of disease in DOA cases.

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We present a long-term follow-up in autosomal dominant gyrate atrophy-like choroidal dystrophy (adGALCD) and propose a possible genotype/phenotype correlation. Ophthalmic examination of six patients from two families revealed confluent areas of choroidal atrophy resembling gyrate atrophy, starting in the second decade of life. Progression continued centrally, reaching the fovea at about 60 years of age. Subretinal deposits, retinal pigmentation or choroidal neovascularization as seen in late-onset retinal degeneration (LORD) were not observed. Whole genome sequencing revealed a novel missense variant in the C1QTNF5 gene (p.(Q180E)) which was found in heterozygous state in all affected subjects. Haplotype analysis showed that this variant found in both families is identical by descent. Three-dimensional modeling of the possible supramolecular assemblies of C1QTNF5 revealed that the p.(Q180E) variant led to the destabilization of protein tertiary and quaternary structures, affecting both the stability of the single protomer and the entire globular head, thus exerting detrimental effects on the formation of C1QTNF5 trimeric globular domains and their interaction. In conclusion, we propose that the p.(Q180E) variant causes a specific phenotype, adGALCD, that differs in multiple clinical aspects from LORD. Disruption of optimal cell-adhesion mechanisms is expected when analyzing the effects of the point mutation at the protein level.

Mutations in POC1B are a rare cause of inherited retinal degeneration. In this study, we present a thorough phenotypic and genotypic characterization of three individuals harboring putatively pathogenic variants in the POC1B gene. All patients displayed a similar, slowly progressive retinopathy (cone dystrophy or cone-rod dystrophy) with normal funduscopy but disrupted outer retinal layers on optical coherence tomography and variable age of onset. Other symptoms were decreased visual acuity and photophobia. Whole genome sequencing revealed a novel homozygous frameshift variant in one patient. Another patient was shown to harbor a novel deep intronic variant in compound heterozygous state with a previously reported canonical splice site variant. The third patient showed a novel nonsense variant and a novel non-canonical splice site variant. We aimed to validate the effect of the deep intronic variant and the non-canonical splice site variant by means of in vitro splice assays. In addition, direct RNA analysis was performed in one patient. Splicing analysis revealed that the non-canonical splice site variant c.561-3T>C leads to exon skipping while the novel deep intronic variant c.1033-327T>A causes pseudoexon activation. Our data expand the genetic landscape of POC1B mutations and confirm the benefit of genome sequencing in combination with downstream functional validation using minigene assays for the analysis of putative splice variants. In addition, we provide clinical multimodal phenotyping of the affected individuals.