SPACR Encoded by IMPG1 Is Essential for Photoreceptor Survival by Interplaying between the Interphotoreceptor Matrix and the Retinal Pigment Epithelium.

Several pathogenic variants have been reported in the IMPG1 gene associated with the inherited retinal disorders vitelliform macular dystrophy (VMD) and retinitis pigmentosa (RP). IMPG1 and its paralog IMPG2 encode for two proteoglycans, SPACR and SPACRCAN, respectively, which are the main components of the interphotoreceptor matrix (IPM), the extracellular matrix surrounding the photoreceptor cells. To determine the role of SPACR in the pathological mechanisms leading to RP and VMD, we generated a knockout mouse model lacking Impg1, the mouse ortholog. Impg1-deficient mice show abnormal accumulation of autofluorescent deposits visible by fundus imaging and spectral-domain optical coherence tomography (SD-OCT) and attenuated electroretinogram responses from 9 months of age. Furthermore, SD-OCT of Impg1-/- mice shows a degeneration of the photoreceptor layer, and transmission electron microscopy shows a disruption of the IPM and the retinal pigment epithelial cells. The decrease in the concentration of the chromophore 11-cis-retinal supports this loss of photoreceptors. In conclusion, our results demonstrate the essential role of SPACR in maintaining photoreceptors. Impg1-/- mice provide a novel model for mechanistic investigations and the development of therapies for VMD and RP caused by IMPG1 pathogenic variants.

Optic neuropathy linked to ACAD9 pathogenic variants: A potentially riboflavin-responsive disorder?

Mitochondrial complex I (CI) deficiencies (OMIM 252010) are the commonest inherited mitochondrial disorders in children. Acyl-CoA dehydrogenase 9 (ACAD9) is a flavoenzyme involved chiefly in CI assembly and possibly in fatty acid oxidation. Biallelic pathogenic variants result in CI dysfunction, with a phenotype ranging from early onset and sometimes fatal mitochondrial encephalopathy with lactic acidosis to late-onset exercise intolerance. Cardiomyopathy is often associated. We report a patient with childhood-onset optic and peripheral neuropathy without cardiac involvement, related to CI deficiency. Genetic analysis revealed compound heterozygous pathogenic variants in ACAD9, expanding the clinical spectrum associated to ACAD9 mutations. Importantly, riboflavin treatment (15 mg/kg/day) improved long-distance visual acuity and demonstrated significant rescue of CI activity in vitro.

A ROD-CONE DYSTROPHY IS SYSTEMATICALLY ASSOCIATED TO THE RTN4IP1 RECESSIVE OPTIC ATROPHY.

PURPOSE: RTN4IP1 biallelic mutations cause a recessive optic atrophy, sometimes associated to more severe neurological syndromes, but so far, no retinal phenotype has been reported in RTN4IP1 patients, justifying their reappraisal. METHODS: Seven patients from four families carrying biallelic RTN4IP1 variants were retrospectively reviewed, with emphasis on their age of onset, visual acuity, multimodal imaging including color and autofluorescence frames, spectral-domain optical coherence tomography with RNFL and macular analyses. RESULTS: Seven patients from four RTN4IP1 families developed in their first decade of life a bilateral recessive optic atrophy with severe central visual loss, and primary nystagmus developed in 5 of 7 patients. Six patients were legally blind. In a second stage, the seven individuals developed a rod-cone dystrophy, sparing the macular zone and the far periphery. This retinal damage was identified by 55° field fundus autofluorescence frames and also by spectral-domain optical coherence tomography scans of the temporal part of the macular zone in five of the seven patients. Full-field electroretinography measurements disclosed reduced b-wave amplitude of the rod responses in all patients but two. Family 4 with the p.R103H and c.601A > T (p.K201*) truncating mutation had further combined neurological signs with cerebellar ataxia, seizures, and intellectual disability. CONCLUSION: RTN4IP1 recessive optic atrophy is systematically associated to a rod-cone dystrophy, which suggests that both the retinal ganglion cells and the rods are affected as a result of a deficit in the mitochondrial respiratory chain. Thus, systematic widefield autofluorescence frames and temporal macular scans are recommended for the evaluation of patients with optic neuropathies.

Pathogenic variants in IMPG1 cause autosomal dominant and autosomal recessive retinitis pigmentosa.

BACKGROUND: Inherited retinal disorders are a clinically and genetically heterogeneous group of conditions and a major cause of visual impairment. Common disease subtypes include vitelliform macular dystrophy (VMD) and retinitis pigmentosa (RP). Despite the identification of over 90 genes associated with RP, conventional genetic testing fails to detect a molecular diagnosis in about one third of patients with RP. METHODS: Exome sequencing was carried out for identifying the disease-causing gene in a family with autosomal dominant RP. Gene panel testing and exome sequencing were performed in 596 RP and VMD families to identified additional IMPG1 variants. In vivo analysis in the medaka fish system by knockdown assays was performed to screen IMPG1 possible pathogenic role. RESULTS: Exome sequencing of a family with RP revealed a splice variant in IMPG1. Subsequently, the same variant was identified in individuals from two families with either RP or VMD. A retrospective study of patients with RP or VMD revealed eight additional families with different missense or nonsense variants in IMPG1. In addition, the clinical diagnosis of the IMPG1 retinopathy-associated variant, originally described as benign concentric annular macular dystrophy, was also revised to RP with early macular involvement. Using morpholino-mediated ablation of Impg1 and its paralog Impg2 in medaka fish, we confirmed a phenotype consistent with that observed in the families, including a decreased length of rod and cone photoreceptor outer segments. CONCLUSION: This study discusses a previously unreported association between monoallelic or biallelic IMPG1 variants and RP. Notably, similar observations have been reported for IMPG2.

Dominant mutations in mtDNA maintenance gene SSBP1 cause optic atrophy and foveopathy.

Mutations in genes encoding components of the mitochondrial DNA (mtDNA) replication machinery cause mtDNA depletion syndromes (MDSs), which associate ocular features with severe neurological syndromes. Here, we identified heterozygous missense mutations in single-strand binding protein 1 (SSBP1) in 5 unrelated families, leading to the R38Q and R107Q amino acid changes in the mitochondrial single-stranded DNA-binding protein, a crucial protein involved in mtDNA replication. All affected individuals presented optic atrophy, associated with foveopathy in half of the cases. To uncover the structural features underlying SSBP1 mutations, we determined a revised SSBP1 crystal structure. Structural analysis suggested that both mutations affect dimer interactions and presumably distort the DNA-binding region. Using patient fibroblasts, we validated that the R38Q variant destabilizes SSBP1 dimer/tetramer formation, affects mtDNA replication, and induces mtDNA depletion. Our study showing that mutations in SSBP1 cause a form of dominant optic atrophy frequently accompanied with foveopathy brings insights into mtDNA maintenance disorders.

Hereditary spastic paraplegia and prominent sensorial involvement: think MAG mutations!

Homozygous mutations in MAG, encoding the myelin-associated glycoprotein, a transmembrane component of the myelin sheath, have been associated with SPG 75 recessive spastic paraplegia. Here, we report the first patient with two compound heterozygous novel MAG mutations (p.A151V and p.S373R) and early developmental delay with a progressive complex phenotype characterized by spastic paraplegia, peripheral sensorimotor neuropathy, intellectual disability, and sensorial dysfunctions with severe optic atrophy and hearing involvement. Brain imaging showed progressive global cerebellar atrophy. We propose that complex hereditary spastic paraplegia, with axonal and demyelinating polyneuropathy, sensorial impairment and intellectual disability might suggest MAG mutations.

Generation of a human iPSC line, INMi003-A, with a missense mutation in CRX associated with autosomal dominant cone-rod dystrophy.

We generated an induced pluripotent stem cell (iPSC) line using dermal fibroblasts from a 53 year-old patient with autosomal dominant cone-rod dystrophy (CRD) caused by a missense mutation, c.121C > T, in the CRX gene. Patient fibroblasts were reprogrammed using the non-integrative Sendai virus reprogramming system and the human OSKM transcription factor cocktail. The generated iPSCs contained the congenital mutation in exon 3 of CRX and were pluripotent and genetically stable. This iPSC line will be an important tool for retinal differentiation studies to better understand the CRD phenotype caused by the mutant p.Arg41Trp CRX protein.

Generation of a human iPSC line, INMi004-A, with a point mutation in CRX associated with autosomal dominant Leber congenital amaurosis.

The human induced pluripotent stem cell (iPSC) line, INMi004-A, was generated using dermal fibroblasts from a 6 year-old patient with autosomal dominant Leber Congenital Amaurosis (LCA) caused by the point mutation c.695delC (p.Pro232Argfs*139) in the CRX gene. We used non-integrative Sendai virus vectors containing the human OSKM transcription factor cocktail to reprogram patient fibroblasts. The generated iPSC line contained the congenital deletion c.695delC in exon 4 of CRX, had a normal karyotype, and was capable of differentiation into all three germ layers. This cell line represents an important tool to study the pathophysiology of CRX-associated LCA.

Genome Editing as a Treatment for the Most Prevalent Causative Genes of Autosomal Dominant Retinitis Pigmentosa.

: Inherited retinal dystrophies (IRDs) are a clinically and genetically heterogeneous group of diseases with more than 250 causative genes. The most common form is retinitis pigmentosa. IRDs lead to vision impairment for which there is no universal cure. Encouragingly, a first gene supplementation therapy has been approved for an autosomal recessive IRD. However, for autosomal dominant IRDs, gene supplementation therapy is not always pertinent because haploinsufficiency is not the only cause. Disease-causing mechanisms are often gain-of-function or dominant-negative, which usually require alternative therapeutic approaches. In such cases, genome-editing technology has raised hopes for treatment. Genome editing could be used to i) invalidate both alleles, followed by supplementation of the wild type gene, ii) specifically invalidate the mutant allele, with or without gene supplementation, or iii) to correct the mutant allele. We review here the most prevalent genes causing autosomal dominant retinitis pigmentosa and the most appropriate genome-editing strategy that could be used to target their different causative mutations.

Natural models for retinitis pigmentosa: progressive retinal atrophy in dog breeds.

Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal disorders eventually leading to blindness with different ages of onset, progression and severity. Human RP, first characterized by the progressive degeneration of rod photoreceptor cells, shows high genetic heterogeneity with more than 90 genes identified. However, about one-third of patients have no known genetic causes. Interestingly, dogs are also severely affected by similar diseases, called progressive retinal atrophy (PRA). Indeed, RP and PRA have comparable clinical signs, physiopathology and outcomes, similar diagnosis methods and most often, orthologous genes are involved. The many different dog PRAs often segregate in specific breeds. Indeed, undesired alleles have been selected and amplified through drastic selection and excessive use of inbreeding. Out of the 400 breeds, nearly 100 have an inherited form of PRA, which are natural animal models that can be used to investigate the genetics, disease progression and therapies in dogs for the benefit of both dogs and humans. Recent knowledge on the canine genome and access to new genotyping and sequencing technologies now efficiently allows the identification of mutations involved in canine genetic diseases. To date, PRA genes identified in dog breeds correspond to the same genes in humans and represent relevant RP models, and new genes found in dogs represent good candidate for still unknown human RP. We present here a review of the main advantages of the dog models for human RP with the genes already identified and an X-linked PRA in the Border collie as a model for orphan X-linked RPs in human.

Generation of a human iPSC line, INMi002-A, carrying the most prevalent USH2A variant associated with Usher syndrome type 2.

We generated an induced pluripotent stem cell (iPSC) line using dermal fibroblasts from a patient with Usher syndrome type 2 (USH2). This individual was homozygous for the most prevalent variant reported in the USH2A gene, c.2299delG localized in exon 13. Reprogramming was performed using the non-integrative Sendai virus reprogramming method and the human OSKM transcription factor cocktail under feeder-free culture conditions. This iPSC line will be an invaluable tool for studying the pathophysiology of USH2 and for testing the efficacy of novel treatments.

Generation of an iPSC line, INMi001-A, carrying the two most common USH2A mutations from a compound heterozygote with non-syndromic retinitis pigmentosa.

We generated an induced pluripotent stem cell (iPSC) line from a patient with non-syndromic retinitis pigmentosa who is a compound heterozygote for the two most frequent USH2A variants, c.2276G > T and c.2299delG localized in exon 13. Patient fibroblasts were reprogrammed using the non-integrative Sendai virus reprogramming method and the human OSKM transcription factor cocktail. The generated cells were pluripotent and genetically stable. This iPSC line will be an important tool for studying the pathogenesis of these USH2A mutations and for developing treatments that, due their high prevalence, will target a large patient population.

AP4 deficiency: A novel form of neurodegeneration with brain iron accumulation?

OBJECTIVE: To describe the clinico-radiological phenotype of 3 patients harboring a homozygous novel AP4M1 pathogenic mutation. METHODS: The 3 patients from an inbred family who exhibited early-onset developmental delay, tetraparesis, juvenile motor function deterioration, and intellectual deficiency were investigated by magnetic brain imaging using T1-weighted, T2-weighted, T2*-weighted, fluid-attenuated inversion recovery, susceptibility weighted imaging (SWI) sequences. Whole-exome sequencing was performed on the 3 patients. RESULTS: In the 3 patients, brain imaging identified the same pattern of bilateral SWI hyposignal of the globus pallidus, concordant with iron accumulation. A novel homozygous nonsense mutation was identified in AP4M1, segregating with the disease and leading to truncation of half of the adap domain of the protein. CONCLUSIONS: Our results suggest that AP4M1 represents a new candidate gene that should be considered in the neurodegeneration with brain iron accumulation (NBIA) spectrum of disorders and highlight the intersections between hereditary spastic paraplegia and NBIA clinical presentations.

Identification of Inherited Retinal Disease-Associated Genetic Variants in 11 Candidate Genes.

Inherited retinal diseases (IRDs) display an enormous genetic heterogeneity. Whole exome sequencing (WES) recently identified genes that were mutated in a small proportion of IRD cases. Consequently, finding a second case or family carrying pathogenic variants in the same candidate gene often is challenging. In this study, we searched for novel candidate IRD gene-associated variants in isolated IRD families, assessed their causality, and searched for novel genotype-phenotype correlations. Whole exome sequencing was performed in 11 probands affected with IRDs. Homozygosity mapping data was available for five cases. Variants with minor allele frequencies ≤ 0.5% in public databases were selected as candidate disease-causing variants. These variants were ranked based on their: (a) presence in a gene that was previously implicated in IRD; (b) minor allele frequency in the Exome Aggregation Consortium database (ExAC); (c) in silico pathogenicity assessment using the combined annotation dependent depletion (CADD) score; and (d) interaction of the corresponding protein with known IRD-associated proteins. Twelve unique variants were found in 11 different genes in 11 IRD probands. Novel autosomal recessive and dominant inheritance patterns were found for variants in Small Nuclear Ribonucleoprotein U5 Subunit 200 (SNRNP200) and Zinc Finger Protein 513 (ZNF513), respectively. Using our pathogenicity assessment, a variant in DEAH-Box Helicase 32 (DHX32) was the top ranked novel candidate gene to be associated with IRDs, followed by eight medium and lower ranked candidate genes. The identification of candidate disease-associated sequence variants in 11 single families underscores the notion that the previously identified IRD-associated genes collectively carry > 90% of the defects implicated in IRDs. To identify multiple patients or families with variants in the same gene and thereby provide extra proof for pathogenicity, worldwide data sharing is needed.

A novel duplication of PRMD13 causes North Carolina macular dystrophy: overexpression of PRDM13 orthologue in drosophila eye reproduces the human phenotype.

In this study, we report a novel duplication causing North Carolina macular dystrophy (NCMD) identified applying whole genome sequencing performed on eight affected members of two presumed unrelated families mapping to the MCDR1 locus. In our families, the NCMD phenotype was associated with a 98.4 kb tandem duplication encompassing the entire CCNC and PRDM13 genes and a common DNase 1 hypersensitivity site. To study the impact of PRDM13 or CCNC dysregulation, we used the Drosophila eye development as a model. Knock-down and overexpression of CycC and CG13296, Drosophila orthologues of CCNC and PRDM13, respectively, were induced separately during eye development. In flies, eye development was not affected, while knocking down either CycC or CG13296 mutant models. Overexpression of CycC also had no effect. Strikingly, overexpression of CG13296 in Drosophila leads to a severe loss of the imaginal eye-antennal disc. This study demonstrated for the first time in an animal model that overexpression of PRDM13 alone causes a severe abnormal retinal development. It is noteworthy that mutations associated with this autosomal dominant foveal developmental disorder are frequently duplications always including an entire copy of PRDM13, or variants in one DNase 1 hypersensitivity site at this locus.

Cone dystrophy or macular dystrophy associated with novel autosomal dominant GUCA1A mutations.

PURPOSE: Sixteen different mutations in the guanylate cyclase activator 1A gene (GUCA1A), have been previously identified to cause autosomal dominant cone dystrophy (adCOD), cone-rod dystrophy (adCORD), macular dystrophy (adMD), and in an isolated patient, retinitis pigmentosa (RP). The purpose of this study is to report on two novel mutations and the patients' clinical features. METHODS: Clinical investigations included visual acuity and visual field testing, fundus examination, high-resolution spectral-domain optical coherence tomography (OCT), fundus autofluorescence imaging, and full-field and multifocal electroretinogram (ERG) recordings. GUCA1A was screened by Sanger sequencing in a cohort of 12 French families with adCOD, adCORD, and adMD. RESULTS: We found two novel GUCA1A mutations-one amino acid deletion, c.302_304delTAG (p.Val101del), and one missense mutation, c.444T>A (p.Asp148Glu)-each of which was found in one family. The p.Asp148Glu mutation affected one of the Ca2+-binding amino acids of the EF4 hand, while the p.Val101del mutation resulted in the in-frame deletion of Valine-101, localized between two Ca2+-binding aspartic acid residues at positions 100 and 102 of the EF3 hand. Both families complained of visual acuity loss worsening with age. However, the p.Asp148Glu mutation was present in one family with adCOD involving abnormal cone function and an absence of macular atrophy, whereas p.Val101del mutation was encountered in another family with adMD without a generalized cone defect. CONCLUSIONS: The two novel mutations described in this study are associated with distinct phenotypes, MD for p.Val101del and COD for p.Asp148Glu, with no intrafamilial phenotypic heterogeneity.

A dominant mutation in MAPKAPK3, an actor of p38 signaling pathway, causes a new retinal dystrophy involving Bruch's membrane and retinal pigment epithelium.

Inherited retinal dystrophies are clinically and genetically heterogeneous with significant number of cases remaining genetically unresolved. We studied a large family from the West Indies islands with a peculiar retinal disease, the Martinique crinkled retinal pigment epitheliopathy that begins around the age of 30 with retinal pigment epithelium (RPE) and Bruch's membrane changes resembling a dry desert land and ends with a retinitis pigmentosa. Whole-exome sequencing identified a heterozygous c.518T>C (p.Leu173Pro) mutation in MAPKAPK3 that segregates with the disease in 14 affected and 28 unaffected siblings from three generations. This unknown variant is predicted to be damaging by bioinformatic predictive tools and the mutated protein to be non-functional by crystal structure analysis. MAPKAPK3 is a serine/threonine protein kinase of the p38 signaling pathway that is activated by a variety of stress stimuli and is implicated in cellular responses and gene regulation. In contrast to other tissues, MAPKAPK3 is highly expressed in the RPE, suggesting a crucial role for retinal physiology. Expression of the mutated allele in HEK cells revealed a mislocalization of the protein in the cytoplasm, leading to cytoskeleton alteration and cytodieresis inhibition. In Mapkapk3-/- mice, Bruch's membrane is irregular with both abnormal thickened and thinned portions. In conclusion, we identified the first pathogenic mutation in MAPKAPK3 associated with a retinal disease. These findings shed new lights on Bruch's membrane/RPE pathophysiology and will open studies of this signaling pathway in diseases with RPE and Bruch's membrane alterations, such as age-related macular degeneration.

Recessive Mutations in RTN4IP1 Cause Isolated and Syndromic Optic Neuropathies.

Autosomal-recessive optic neuropathies are rare blinding conditions related to retinal ganglion cell (RGC) and optic-nerve degeneration, for which only mutations in TMEM126A and ACO2 are known. In four families with early-onset recessive optic neuropathy, we identified mutations in RTN4IP1, which encodes a mitochondrial ubiquinol oxydo-reductase. RTN4IP1 is a partner of RTN4 (also known as NOGO), and its ortholog Rad8 in C. elegans is involved in UV light response. Analysis of fibroblasts from affected individuals with a RTN4IP1 mutation showed loss of the altered protein, a deficit of mitochondrial respiratory complex I and IV activities, and increased susceptibility to UV light. Silencing of RTN4IP1 altered the number and morphogenesis of mouse RGC dendrites in vitro and the eye size, neuro-retinal development, and swimming behavior in zebrafish in vivo. Altogether, these data point to a pathophysiological mechanism responsible for RGC early degeneration and optic neuropathy and linking RTN4IP1 functions to mitochondrial physiology, response to UV light, and dendrite growth during eye maturation.

Mutation in NDUFA13/GRIM19 leads to early onset hypotonia, dyskinesia and sensorial deficiencies, and mitochondrial complex I instability.

Mitochondrial complex I (CI) deficiencies are causing debilitating neurological diseases, among which, the Leber Hereditary Optic Neuropathy and Leigh Syndrome are the most frequent. Here, we describe the first germinal pathogenic mutation in the NDUFA13/GRIM19 gene encoding a CI subunit, in two sisters with early onset hypotonia, dyskinesia and sensorial deficiencies, including a severe optic neuropathy. Biochemical analysis revealed a drastic decrease in CI enzymatic activity in patient muscle biopsies, and reduction of CI-driven respiration in fibroblasts, while the activities of complex II, III and IV were hardly affected. Western blots disclosed that the abundances of NDUFA13 protein, CI holoenzyme and super complexes were drastically reduced in mitochondrial fractions, a situation that was reproduced by silencing NDUFA13 in control cells. Thus, we established here a correlation between the first mutation yet identified in the NDUFA13 gene, which induces CI instability and a severe but slowly evolving clinical presentation affecting the central nervous system.

Neuroradiological findings expand the phenotype of OPA1-related mitochondrial dysfunction.

OBJECTIVE: OPA1 mutations are responsible for more than half of autosomal dominant optic atrophy (ADOA), a blinding disease affecting the retinal ganglion neurons. In most patients the clinical presentation is restricted to the optic nerve degeneration, albeit in 20% of them, additional neuro-sensorial symptoms might be associated to the loss of vision, as frequently encountered in mitochondrial diseases. This study describes clinical and neuroradiological features of OPA1 patients. METHODS: Twenty two patients from 17 families with decreased visual acuity related to optic atrophy and carrying an OPA1 mutation were enrolled. Patients underwent neuro-ophthalmological examinations. Brain magnetic resonance imaging (T1, T2 and flair sequences) was performed on a 1.5-Tesla MR Unit. Twenty patients underwent 2-D proton spectroscopic imaging. RESULTS: Brain imaging disclosed abnormalities in 12 patients. Cerebellar atrophy mainly involving the vermis was observed in almost a quarter of the patients; other abnormalities included unspecific white matter hypersignal, hemispheric cortical atrophy, and lactate peak. Neurological examination disclosed one patient with a transient right hand motor deficit and ENT examination revealed hearing impairment in 6 patients. Patients with abnormal MRI were characterized by: (i) an older age (ii) more severe visual impairment with chronic visual acuity deterioration, and (iii) more frequent associated deafness. CONCLUSIONS: Our results demonstrate that brain imaging abnormalities are common in OPA1 patients, even in those with normal neurological examination. Lactate peak, cerebellar and cortical atrophies are consistent with the mitochondrial dysfunction related to OPA1 mutations and might result from widespread neuronal degeneration.