The first reported case of a deletion of the entire RPGR gene in a family with X-linked retinitis pigmentosa.

Clinical phenotypes of a patient with a deletion of the entire RPGR gene have not been described in the literature yet. We hereby report a new mutation in a family of X-linked retinitis pigmentosa (×lRP), showing the deletion of the entire RPGR gene. Gene therapy for inherited retinal diseases holds great promise; however, so far there has been no approved treatment of RPGR-mediated retinitis pigmentosa. The presented evidence of genotype-phenotype correlation may be useful for genetic diagnosis or even genetic treatment in the near future.

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.

Mutation spectrum of the OPA1 gene in a large cohort of patients with suspected dominant optic atrophy: Identification and classification of 48 novel variants.

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.

Dominant ACO2 mutations are a frequent cause of isolated optic atrophy.

Biallelic mutations in ACO2, encoding the mitochondrial aconitase 2, have been identified in individuals with neurodegenerative syndromes, including infantile cerebellar retinal degeneration and recessive optic neuropathies (locus OPA9). By screening European cohorts of individuals with genetically unsolved inherited optic neuropathies, we identified 61 cases harbouring variants in ACO2, among whom 50 carried dominant mutations, emphasizing for the first time the important contribution of ACO2 monoallelic pathogenic variants to dominant optic atrophy. Analysis of the ophthalmological and clinical data revealed that recessive cases are affected more severely than dominant cases, while not significantly earlier. In addition, 27% of the recessive cases and 11% of the dominant cases manifested with extraocular features in addition to optic atrophy. In silico analyses of ACO2 variants predicted their deleterious impacts on ACO2 biophysical properties. Skin derived fibroblasts from patients harbouring dominant and recessive ACO2 mutations revealed a reduction of ACO2 abundance and enzymatic activity, and the impairment of the mitochondrial respiration using citrate and pyruvate as substrates, while the addition of other Krebs cycle intermediates restored a normal respiration, suggesting a possible short-cut adaptation of the tricarboxylic citric acid cycle. Analysis of the mitochondrial genome abundance disclosed a significant reduction of the mitochondrial DNA amount in all ACO2 fibroblasts. Overall, our data position ACO2 as the third most frequently mutated gene in autosomal inherited optic neuropathies, after OPA1 and WFS1, and emphasize the crucial involvement of the first steps of the Krebs cycle in the maintenance and survival of retinal ganglion cells.

Haploinsufficiency due to a novel ACO2 deletion causes mitochondrial dysfunction in fibroblasts from a patient with dominant optic nerve atrophy.

ACO2 is a mitochondrial protein, which is critically involved in the function of the tricarboxylic acid cycle (TCA), the maintenance of iron homeostasis, oxidative stress defense and the integrity of mitochondrial DNA (mtDNA). Mutations in the ACO2 gene were identified in patients suffering from a broad range of symptoms, including optic nerve atrophy, cortical atrophy, cerebellar atrophy, hypotonia, seizures and intellectual disabilities. In the present study, we identified a heterozygous 51 bp deletion (c.1699_1749del51) in ACO2 in a family with autosomal dominant inherited isolated optic atrophy. A complementation assay using aco1-deficient yeast revealed a growth defect for the mutant ACO2 variant substantiating a pathogenic effect of the deletion. We used patient-derived fibroblasts to characterize cellular phenotypes and found a decrease of ACO2 protein levels, while ACO2 enzyme activity was not affected compared to two age- and gender-matched control lines. Several parameters of mitochondrial function, including mitochondrial morphology, mitochondrial membrane potential or mitochondrial superoxide production, were not changed under baseline conditions. However, basal respiration, maximal respiration, and spare respiratory capacity were reduced in mutant cells. Furthermore, we observed a reduction of mtDNA copy number and reduced mtDNA transcription levels in ACO2-mutant fibroblasts. Inducing oxidative stress led to an increased susceptibility for cell death in ACO2-mutant fibroblasts compared to controls. Our study reveals that a monoallelic mutation in ACO2 is sufficient to promote mitochondrial dysfunction and increased vulnerability to oxidative stress as main drivers of cell death related to optic nerve atrophy.

Novel likely pathogenic variants in TMEM126A identified in non-syndromic autosomal recessive optic atrophy: two case reports.

BACKGROUND: Reports on autosomal recessive optic atrophy (arOA) are sparse and so far, only one gene has been specifically associated with non-syndromic arOA, namely TMEM126A. To date, all reports of pathogenic TMEM126A variants are from affected individuals of Maghrebian origin, who all carry an identical nonsense variant. Here we report two novel variants in the TMEM126A gene from non-Maghreb individuals, both found in affected individuals with an arOA phenotype. CASE PRESENTATION: We report three affected individuals from two families. The proband of family A, a 24-year-old Turkish woman, was diagnosed with visual loss in early childhood but a diagnosis of optic atrophy was only made at 14 years. A diagnostic gene panel revealed a splice donor variant (c.86 + 2 T > C) in homozygous state in the TMEM126A gene. Analysis of this variant based on RNA from whole blood revealed a single aberrant transcript lacking exon 2, presumably representing a functional null allele. Two siblings from family B, a 16-year old Iraqi girl and her 14-year old brother, were diagnosed with optic atrophy in early childhood. A missense variant p.(S36 L) in the TMEM126A gene was identified in homozygous state in a gene panel-based diagnostic setting in both siblings. This missense variant is ultra rare in the general population, affects a highly evolutionarily conserved amino acid and segregates with the disease within the family. The three probands reported in this study had a relatively mild clinical course without any evidence of a syndromic (e.g. neurological) comorbidity, which is in line with previous studies. CONCLUSIONS: We provide additional evidence for the implication of biallelic pathogenic TMEM126A variants in arOA. Our findings extend both the mutational spectrum and geographic presence of TMEM126A in arOA. Screening of the entire gene should be considered in affected individuals presenting with features resembling arOA and also from non-Maghrebian descent.

Allelic Expression Imbalance in the Human Retinal Transcriptome and Potential Impact on Inherited Retinal Diseases.

Inherited retinal diseases (IRDs) are often associated with variable clinical expressivity (VE) and incomplete penetrance (IP). Underlying mechanisms may include environmental, epigenetic, and genetic factors. Cis-acting expression quantitative trait loci (cis-eQTLs) can be implicated in the regulation of genes by favoring or hampering the expression of one allele over the other. Thus, the presence of such loci elicits allelic expression imbalance (AEI) that can be traced by massive parallel sequencing techniques. In this study, we performed an AEI analysis on RNA-sequencing (RNA-seq) data, from 52 healthy retina donors, that identified 194 imbalanced single nucleotide polymorphisms(SNPs) in 67 IRD genes. Focusing on SNPs displaying AEI at a frequency higher than 10%, we found evidence of AEI in several IRD genes regularly associated with IP and VE (BEST1, RP1, PROM1, and PRPH2). Based on these SNPs commonly undergoing AEI, we performed pyrosequencing in an independent sample set of 17 healthy retina donors in order to confirm our findings. Indeed, we were able to validate CDHR1, BEST1, and PROM1 to be subjected to cis-acting regulation. With this work, we aim to shed light on differentially expressed alleles in the human retina transcriptome that, in the context of autosomal dominant IRD cases, could help to explain IP or VE.

A novel heterozygous OPA3 mutation located in the mitochondrial target sequence results in altered steady-state levels and fragmented mitochondrial network.

BACKGROUND: Mutations in OPA3 have been reported in patients with autosomal dominant optic atrophy plus cataract and Costeff syndrome. Here, we report the results of a comprehensive study on OPA3 mutations, including the mutation spectrum and its prevalence in a large cohort of OPA1-negative autosomal dominant optic atrophy (ADOA) patients, the associated clinical phenotype and the functional characterisation of a newly identified OPA3 mutant. METHODS: Mutation analysis was carried out in a patient cohort of 121 independent ADOA patients. To characterise a novel OPA3 mutation, we analysed the mitochondrial import, steady-state levels and the mitochondrial localisation of the mutated protein in patients' fibroblasts. Furthermore, the morphology of mitochondria harbouring the mutated OPA3 was monitored. RESULTS: We identified four independent cases (representing families with multiple affected members) with OPA3 mutations. Besides the known p.Q105E mutation, we observed a novel insertion, c.10_11insCGCCCG/p.V3_G4insAP which is located in the mitochondrial presequence. Detailed functional analysis of mitochondria harbouring this novel mutation demonstrates a fragmented mitochondrial network with a decreased mitochondrial mass in patient fibroblasts. In addition, quantification of the OPA3 protein reveals decreased steady-state levels of the mutant protein compared with the native one. Comparison of the clinical phenotypes suggests that OPA3 mutations can additionally evoke hearing loss and by that extend the clinical manifestation of OPA3-associated optic atrophy. This finding is supported by expression analysis of OPA3 in murine cochlear tissue. CONCLUSIONS: In summary, our study provides new insights into the clinical spectrum and the pathogenesis of dominant optic atrophy caused by mutations in the OPA3 gene.