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Die hereditaren Netzhautdystrophien (IRD; inherited retinal diseases) sind eine heterogene Gruppe von Krankheiten – unter anderem Retinitis pigmentosa (RP), Lebersche kongenitale Amaurose (LCA) und Netzhautdystrophie mit fruhem Krankheitsbeginn (EO[S]RD; early-onset [severe] retinal dystrophy) –, die sich hinsichtlich der Schwere der Krankheit und des Alters bei Krankheitsbeginn unterscheiden. IRD konnen durch Mutationen in >250 verschiedenen Genen hervorgerufen werden. Varianten des RPE65-Gens sind die Ursache fur 0,6–6% aller Falle von RP und fur 3–16% der Falle von LCA/EORD. Voretigen Neparvovec ist eine Gentherapie, die zur Behandlung von Patienten mit autosomal-rezessiver Retinadystrophie infolge biallelischer RPE65-Varianten ( RPE65-IRD) zugelassen ist. Die korrekte molekulare Diagnostik ist daher bei RPE65-IRD von entscheidender Bedeutung, um therapeutisch nutzbare, d. h. potenziell von der Therapie profitierende Genotypen zu identifizieren, und sie ist eine tragende Saule des Patientenmanagements. Es sind bereits Hunderte von RPE65-Varianten bekannt, und wahrend ein Teil als pathogen oder wahrscheinlich pathogen identifiziert werden konnte, ist die Signifikanz anderer Varianten noch unklar. In dieser Ubersichtsarbeit beschreiben wir das erforderliche gendiagnostische Vorgehen, um diejenigen Patienten auszuwahlen, die die Voraussetzungen fur die Behandlung mit Voretigen Neparvovec erfullen. Die sorgfaltige klinische Charakterisierung der Patienten durch interdisziplinare Expertenteams in Kombination mit der Verfugbarkeit von Next-Genera­tion-Sequenzierung kann den Zugang der Patienten zu verfugbaren Therapieoptionen beschleunigen.

Purpose: To present visual outcomes of the first two Italian patients with RPE65-related inherited retinal dystrophy (RPE65-IRD) treated with voretigene neparvovec (VN). Methods: Two pediatric patients with RPE65-IRD were treated with VN in both eyes. Patients were evaluated by best-corrected visual acuity (BCVA), full-field stimulus threshold (FST) test, semiautomated kinetic visual field (SKVF), microperimetry, and chromatic pupillometry over 6 months. Results: No complications occurred in the first patient, whereas in the second a subreti-nal hemorrhage was observed in the first treated eye, and excessive resistance to drug injection occurred during treatment of the second eye. BCVA improved by at least one Early Treatment Diabetic Retinopathy Study line in all treated eyes. The FST test and SKVF showed clinically significant improvements in all eyes (i.e., change of light sensitivity > 10 decibels; area enlargement of at least 20%). Moreover, microperimetry showed better fixation stability. Finally, chromatic pupillometry showed increases in pupillary constric-tion that ranged from 10% to 20%. All visual changes remained stable during follow-up. Conclusions: The first VN treatments in two pediatric Italian patients in clinical practice showed significant improvements in visual outcomes, even in the case of surgical complications, which spontaneously recovered without sequelae. Translational Relevance: These findings with VN in patients with RPE65-IRD confirm the results of clinical trials.

PURPOSE. The purpose of this study was to perform a detailed longitudinal phenotyping and genetic characterization of 32 Italian patients with a nonsyndromic retinal dystrophy and mutations in the CEP290 gene. METHODS. We reviewed the clinical history and examinations of 32 patients with a nonsyndromic retinal dystrophy due to mutations in the CEP290 gene, followed up (mean followup: 5.9 years) at 3 Italian centers. The clinical examinations included: best corrected visual acuity (BCVA), optical coherence tomography (OCT), and full-field electroretinogram (ERG). RESULTS. Patients (mean age = 19.0 ± 3.4 years) had a mean BCVA of 1.73 ± 0.20 logMAR. Longitudinal analysis of BCVA showed a nonsignificant decline. Central retinal thickness (CRT) declined significantly with age at an exponential rate of 1.0%/year (P = 0.001). At disease onset, most patients (19/32; 49.4%) had nystagmus. The absence of nystagmus was significantly associated with better BCVA and more preserved CRT (P < 0.05). ERG showed undetectable responses in most patients (64.0%), whereas reduced scotopic and photopic responses were observed in four patients (16.0%) who had no nystagmus. We identified 35 different variants, among which 12 were novel. Our genotype-phenotype correlation analysis shows a significantly worse BCVA in patients harboring a loss-of-function mutation and the deep-intronic variant c.2991+1655A>G. CONCLUSIONS. Our study highlights a mild phenotype of the disease, characterized by absence of nystagmus, good visual acuity, considerably preserved retinal morphology, and recordable ERG, confirming the wide spectrum of CEP290-related retinal dystrophies. Finally, in our cohort, the deep intronic variant c.2991+1655A>G was associated with a more severe phenotype.

PURPOSE: We studied microRNAs as potential biomarkers for Pompe disease. METHODS: We analyzed microRNA expression by small RNA-seq in tissues from the disease murine model at two different ages (3 and 9 months), and in plasma from Pompe patients. RESULTS: In the mouse model we found 211 microRNAs that were differentially expressed in gastrocnemii and 66 in heart, with a different pattern of expression at different ages. In a preliminary analysis in plasma from six patients 55 microRNAs were differentially expressed. Sixteen of these microRNAs were common to those dysregulated in mouse tissues. These microRNAs are known to modulate the expression of genes involved in relevant pathways for Pompe disease pathophysiology (autophagy, muscle regeneration, muscle atrophy). One of these microRNAs, miR-133a, was selected for further quantitative real-time polymerase chain reaction analysis in plasma samples from 52 patients, obtained from seven Italian and Dutch biobanks. miR-133a levels were significantly higher in Pompe disease patients than in controls and correlated with phenotype severity, with higher levels in infantile compared with late-onset patients. In three infantile patients miR-133a decreased after start of enzyme replacement therapy and evidence of clinical improvement. CONCLUSION: Circulating microRNAs may represent additional biomarkers of Pompe disease severity and of response to therapy.

Pathogenic variants in the MKS1 gene are responsible for a ciliopathy with a wide spectrum of clinical manifestations ranging from Meckel and Joubert syndrome (JBTS) to Bardet-Biedl syndrome, and involving the central nervous system, liver, kidney, skeleton, and retina. We report a 39-year-old male individual presenting with isolated Retinitis Pigmentosa (RP), as assessed by full ophthalmological evaluation including Best-Corrected Visual Acuity measurements, fundus examination, Goldmann Visual Field test, and full-field Electroretinography. A clinical exome identified biallelic nonsense variants in MKS1 that prompted post-genotyping investigations for systemic abnormalities of ciliopathy. Brain magnetic resonance imaging revealed malformations of the posterior cranial fossa with the ‘molar tooth sign’ and cerebellar folia dysplasia, which are both distinctive features of JBTS. No other organ or skeletal abnormalities were detected. This case illustrates the power of clinical exome for the identification of the mildest forms of a disease spectrum, such as a mild JBTS with RP in the presented case of an individual carrying biallelic truncating variants in MKS1.

International audience; Purpose: The purpose of this study was to perform a detailed longitudinal phenotyping of X-linked retinitis pigmentosa (RP) caused by mutations in the RPGR gene during a long follow-up period.Methods: An Italian cohort of 48 male patients (from 31 unrelated families) with RPGR-associated RP was clinically assessed at a single center (mean follow-up = 6.5 years), including measurements of best-corrected visual acuity (BCVA), Goldmann visual field (GVF), optical coherence tomography (OCT), fundus autofluorescence (FAF), microperimetry, and full-field electroretinography (ERG).Results: Patients (29.6 ± 15.2 years) showed a mean BCVA of 0.6 ± 0.7 logMAR, mostly with myopic refraction (79.2%). Thirty patients (62.5%) presented a typical RP fundus, while the remaining sine pigmento RP. Over the follow-up, BCVA significantly declined at a mean rate of 0.025 logMAR/year. Typical RP and high myopia were associated with a significantly faster decline of BCVA. Blindness was driven primarily by GVF loss. ERG responses with a rod-cone pattern of dysfunction were detectable in patients (50%) that were significantly younger and more frequently presented sine pigmento RP. Thirteen patients (27.1%) had macular abnormalities without cystoid macular edema. Patients (50%) with a perimacular hyper-FAF ring were significantly younger, had a higher BCVA and a better-preserved ellipsoid zone band than those with markedly decreased FAF. Patients harboring pathogenic variants in exons 1 to 14 showed a milder phenotype compared to those with ORF15 mutations.Conclusions: Our monocentric, longitudinal retrospective study revealed a spectrum disease progression in male patients with RPGR-associated RP. Slow disease progression correlated with sine pigmento RP, absence of high myopia, and mutations in RPGR exons 1 to 14.

Achromatopsia (ACHM) is a rare genetic disorder of infantile onset affecting cone photoreceptors. To determine the extent of progressive retinal changes in achromatopsia, we performed a detailed longitudinal phenotyping and genetic characterization of an Italian cohort comprising 21 ACHM patients (17 unrelated families). Molecular genetic testing identified biallelic pathogenic mutations in known ACHM genes, including four novel variants. At baseline, the patients presented a reduced best corrected visual acuity (BCVA), reduced macular sensitivity (MS), normal dark-adapted electroretinogram (ERG) responses and undetectable or severely reduced light-adapted ERG. The longitudinal analysis of 16 patients (mean follow-up: 5.4 ± 1.0 years) showed a significant decline of BCVA (0.012 logMAR/year) and MS (−0.16 dB/year). Light-adapted and flicker ERG responses decreased below noise level in three and two patients, respectively. Only two patients (12.5%) progressed to a worst OCT grading during the follow-up. Our findings corroborate the notion that ACHM is a progressive disease in terms of BCVA, MS and ERG responses, and affects slowly the structural integrity of the retina. These observations can serve towards the development of guidelines for patient selection and intervention timing in forthcoming gene replacement therapies.

Purpose: Missing heritability in human diseases represents a major challenge, and this is particularly true for ABCA4-associated Stargardt disease (STGD1). We aimed to elucidate the genomic and transcriptomic variation in 1054 unsolved STGD and STGD-like probands. Methods: Sequencing of the complete 128-kb ABCA4 gene was performed using single-molecule molecular inversion probes (smMIPs), based on a semiautomated and cost-effective method. Structural variants (SVs) were identified using relative read coverage analyses and putative splice defects were studied using in vitro assays. Results: In 448 biallelic probands 14 known and 13 novel deep-intronic variants were found, resulting in pseudoexon (PE) insertions or exon elongations in 105 alleles. Intriguingly, intron 13 variants c.1938-621G>A and c.1938-514G>A resulted in dual PE insertions consisting of the same upstream, but different downstream PEs. The intron 44 variant c.6148-84A>T resulted in two PE insertions and flanking exon deletions. Eleven distinct large deletions were found, two of which contained small inverted segments. Uniparental isodisomy of chromosome 1 was identified in one proband. Conclusion: Deep sequencing of ABCA4 and midigene-based splice assays allowed the identification of SVs and causal deep-intronic variants in 25% of biallelic STGD1 cases, which represents a model study that can be applied to other inherited diseases. Fil: Khan, Mubeen. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Cornelis, Stéphanie S.. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Del Pozo Valero, Marta. Hospital Universitario Fundación Jiménez Díaz; España. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Whelan, Laura. Trinity College; Estados Unidos Fil: Runhart, Esmee H.. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Mishra, Ketan. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Bults, Femke. Radboud University Nijmegen Medical Centre; Países Bajos Fil: AlSwaiti, Yahya. St John of Jerusalem Eye Hospital Group; Palestina (ANP) Fil: AlTalbishi, Alaa. St John of Jerusalem Eye Hospital Group; Palestina (ANP) Fil: De Baere, Elfride. University of Ghent; Bélgica Fil: Banfi, Sandro. Seconda Universita Degli Studi Di Napoli; Italia Fil: Banin, Eyal. The Hebrew University of Jerusalem; Israel Fil: Bauwens, Miriam. University of Ghent; Bélgica Fil: Ben Yosef, Tamar. The Ruth And Bruce Rappaport Faculty Of Medicine; Israel Fil: Boon, Camiel J. F.. Leiden University. Leiden University Medical Center; Países Bajos Fil: van den Born, L. Ingeborgh. Rotterdam Ophthalmic Institute; Países Bajos Fil: Defoort, Sabine. Universite Lille; Francia Fil: Devos, Aurore. Universite Lille; Francia Fil: Dockery, Adrian. Trinity College; Estados Unidos Fil: Dudakova, Lubica. Charles University and General University Hospital; República Checa Fil: Fakin, Ana. Charles University and General University Hospital; República Checa Fil: Farrar, G. Jane. Trinity College; Estados Unidos Fil: Ferraz Sallum, Juliana Maria. Universidade Federal de Sao Paulo; Brasil Fil: Fujinami, Kaoru. UCL Institute of Ophthalmology; Reino Unido Fil: Gilissen, Christian. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Glavac, Damjan. University of Ljubljana; Eslovenia Fil: Gorin, Michael B.. University of California at Los Angeles. School of Medicine; Estados Unidos Fil: Greenberg, Jacquie. University of Cape Town; Sudáfrica Fil: Hayashi, Takaaki. The Jikei University School of Medicine; Japón Fil: Hettinga, Ymkje M.. Bartiméus Diagnostic Center for Complex Visual Disorders; Países Bajos Fil: Hoischen, Alexander. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Hoyng, Carel B.. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Hufendiek, Karsten. University Eye Hospital Hannover Medical School; Alemania Fil: Jägle, Herbert. University Regensburg; Alemania Fil: Kamakari, Smaragda. OMMA Ophthalmological Institute of Athens; Grecia Fil: Karali, Marianthi. Seconda Universita Degli Studi Di Napoli; Italia Fil: Kellner, Ulrich. No especifíca; Fil: Klaver, Caroline C. W.. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Kousal, Bohdan. Charles University and General University Hospital; República Checa Fil: Lamey, Tina M.. University of Western Australia; Australia Fil: MacDonald, Ian M.. University of Alberta; Canadá Fil: Matynia, Anna. University of California at Los Angeles. School of Medicine; Estados Unidos Fil: McLaren, Terri L.. University of Western Australia; Australia Fil: Mena, Marcela D.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Meunier, Isabelle. Université Montpellier II; Francia Fil: Miller, Rianne. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Newman, Hadas. Universitat Tel Aviv; Israel Fil: Ntozini, Buhle. University of Cape Town; Sudáfrica Fil: Oldak, Monika. No especifíca; Fil: Pieterse, Marc. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Podhajcer, Osvaldo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Puech, Bernard. Universite Lille; Francia Fil: Ramesar, Raj. University of Cape Town; Sudáfrica Fil: Rüther, Klaus. No especifíca; Fil: Salameh, Manar. No especifíca; Fil: Salles, Mariana Vallim. Universidade de Sao Paulo; Brasil Fil: Sharon, Dror. The Hebrew University of Jerusalem; Israel Fil: Simonelli, Francesca. Seconda Universita Degli Studi Di Napoli; Italia Fil: Spital, Georg. No especifíca; Fil: Steehouwer, Marloes. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Szaflik, Jacek P.. No especifíca; Fil: Thompson, Jennifer A.. No especifíca; Fil: Thuillier, Caroline. Universite Lille; Francia Fil: Tracewska, Anna M.. No especifíca; Fil: van Zweeden, Martine. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Vincent, Andrea L.. University of Auckland; Nueva Zelanda Fil: Zanlonghi, Xavier. No especifíca; Fil: Liskova, Petra. Charles University and General University Hospital; República Checa Fil: Stöhr, Heidi. Universitat Regensburg; Alemania Fil: De Roach, John N.. University of Western Australia; Australia Fil: Ayuso, Carmen. Hospital Universitario Fundación Jiménez Díaz; España Fil: Roberts, Lisa. University of Cape Town; Sudáfrica Fil: Weber, Bernhard H. F.. Universitat Regensburg; Alemania Fil: Dhaenens, Claire Marie. Universite Lille; Francia Fil: Cremers, Frans P. M.. Radboud University Nijmegen Medical Centre; Países Bajos

Retinitis pigmentosa (RP) is a clinically heterogenous disease that comprises a wide range of phenotypic and genetic subtypes. Pericentral RP is an atypical form of RP characterized by bone-spicule pigmentation and/or atrophy confined in the near mid-periphery of the retina. In contrast to classic RP, the far periphery is better preserved in pericentral RP. The aim of this study was to perform the first detailed clinical and genetic analysis of a cohort of European subjects with pericentral RP to determine the phenotypic features and the genetic bases of the disease. A total of 54 subjects from 48 independent families with pericentral RP, non-syndromic and syndromic, were evaluated through a full ophthalmological examination and underwent clinical exome or retinopathy gene panel sequencing. Disease-causative variants were identified in 22 of the 35 families (63%) in 10 different genes, four of which are also responsible for syndromic RP. Thirteen of the 34 likely pathogenic variants were novel. Intra-familiar variability was also observed. The current study confirms the mild phenotype of pericentral RP and extends the spectrum of genes associated with this condition. Retinitis pigmentosa (RP) is a clinically heterogenous disease that comprises a wide range of phenotypic and genetic subtypes. Pericentral RP is an atypical form of RP characterized by bone-spicule pigmentation and/or atrophy confined in the near mid-periphery of the retina. In contrast to classic RP, the far periphery is better preserved in pericentral RP. The aim of this study was to perform the first detailed clinical and genetic analysis of a cohort of European subjects with pericentral RP to determine the phenotypic features and the genetic bases of the disease. A total of 54 subjects from 48 independent families with pericentral RP, non-syndromic and syndromic, were evaluated through a full ophthalmological examination and underwent clinical exome or retinopathy gene panel sequencing. Disease-causative variants were identified in 22 of the 35 families (63%) in 10 different genes, four of which are also responsible for syndromic RP. Thirteen of the 34 likely pathogenic variants were novel. Intra-familiar variability was also observed. The current study confirms the mild phenotype of pericentral RP and extends the spectrum of genes associated with this condition.

Missing heritability in human diseases represents a major challenge. Although whole-genome sequencing enables the analysis of coding and non-coding sequences, substantial costs and data storage requirements hamper its large-scale use to (re)sequence genes in genetically unsolved cases. The ABCA4 gene implicated in Stargardt disease (STGD1) has been studied extensively for 22 years, but thousands of cases remained unsolved. Therefore, single molecule molecular inversion probes were designed that enabled an automated and cost-effective sequence analysis of the complete 128-kb ABCA4 gene. Analysis of 1,054 unsolved STGD and STGD-like probands resulted in bi-allelic variations in 448 probands. Twenty-seven different causal deep-intronic variants were identified in 117 alleles. Based on in vitro splice assays, the 13 novel causal deep-intronic variants were found to result in pseudo-exon (PE) insertions (n=10) or exon elongations (n=3). Intriguingly, intron 13 variants c.1938-621G>A and c.1938-514G>A resulted in dual PE insertions consisting of the same upstream, but different downstream PEs. The intron 44 variant c.6148-84A>T resulted in two PE insertions that were accompanied by flanking exon deletions. Structural variant analysis revealed 11 distinct deletions, two of which contained small inverted segments. Uniparental isodisomy of chromosome 1 was identified in one proband. Integrated complete gene sequencing combined with transcript analysis, identified pathogenic deep-intronic and structural variants in 26% of bi-allelic cases not solved previously by sequencing of coding regions. This strategy serves as a model study that can be applied to other inherited diseases in which only one or a few genes are involved in the majority of cases.

Purpose RAX2 encodes a homeobox-containing transcription factor, in which four monoallelic pathogenic variants have been described in autosomal dominant cone-dominated retinal disease. Methods Exome sequencing in a European cohort with inherited retinal disease (IRD) (n = 2086) was combined with protein structure modeling of RAX2 missense variants, bioinformatics analysis of deletion breakpoints, haplotyping of RAX2 variant c.335dup, and clinical assessment of biallelic RAX2-positive cases and carrier family members. Results Biallelic RAX2 sequence and structural variants were found in five unrelated European index cases, displaying nonsyndromic autosomal recessive retinitis pigmentosa (ARRP) with an age of onset ranging from childhood to the mid-40s (average mid-30s). Protein structure modeling points to loss of function of the novel recessive missense variants and to a dominant-negative effect of the reported dominant RAX2 alleles. Structural variants were fine-mapped to disentangle their underlying mechanisms. Haplotyping of c.335dup in two cases suggests a common ancestry. Conclusion This study supports a role for RAX2 as a novel disease gene for recessive IRD, broadening the mutation spectrum from sequence to structural variants and revealing a founder effect. The identification of biallelic RAX2 pathogenic variants in five unrelated families shows that RAX2 loss of function may be a nonnegligible cause of IRD in unsolved ARRP cases.

Inherited retinal diseases (IRDs) represent a frequent cause of genetic blindness. Their high genetic heterogeneity hinders the application of gene-specific therapies to the vast majority of patients. We recently demonstrated that the microRNA miR-204 is essential for retinal function, although the underlying molecular mechanisms remain poorly understood. Here, we investigated the therapeutic potential of miR-204 in IRDs. We subretinally delivered an adeno-associated viral (AAV) vector carrying the miR-204 precursor to two genetically different IRD mouse models. The administration of AAV-miR-204 preserved retinal function in a mouse model for a dominant form of retinitis pigmentosa (RHO-P347S). This was associated with a reduction of apoptotic photoreceptor cells and with a better preservation of photoreceptor marker expression. Transcriptome analysis showed that miR-204 shifts expression profiles of transgenic retinas toward those of healthy retinas by the downregulation of microglia activation and photoreceptor cell death. Delivery of miR-204 exerted neuroprotective effects also in a mouse model of Leber congenital amaurosis, due to mutations of the Aipl1 gene. Our study highlights the mutation-independent therapeutic potential of AAV-miR204 in slowing down retinal degeneration in IRDs and unveils the previously unreported role of this miRNA in attenuating microglia activation and photoreceptor cell death.

We identified a 14q21.2 microdeletion in a 16-year-old boy with autism spectrum disorder (ASD), IQ in the lower part of normal range but high-functioning memory skills. The deletion affects a gene desert, and the non-deletedgene closest to the microdeletion boundaries is LRFN5, which encodes a protein involved in synaptic plasticity and implicated in neuro-psychiatric disorders. LRFN5 expression was significantly decreased in the proband’s skin fibroblasts. The deleted region includes the pseudogene chr14.232.a, which is transcribed into a long non-coding RNA (lncLRFN5-10), whose levels were also significantly reduced in the proband’s fibroblasts compared to controls. Transfection of the patient’s fibroblasts with a plasmid expressing chr14.232.a significantly increased LRFN5 expression, while siRNA targeting chr14.232.a-derived lncLRFN5-10 reduced LRFN5 levels. In summary, we report on an individual with ASD carrying a microdeletion encompassing the pseudogene chr14.232.a encoding for lncLRFN5-10, which was found to affect the expression levels of the nearby, non-deleted LRFN5. This case illustrates the potential role of long non-coding RNAs in regulating expression of neighbouring genes with a functional role in ASD pathogenesis.

Purpose: RAX2 encodes a homeobox-containing transcription factor, in which four monoallelic pathogenic variants have been described in autosomal dominant cone-dominated retinal disease. Methods: Exome sequencing in a European cohort with inherited retinal disease (IRD) (n = 2086) was combined with protein structure modeling of RAX2 missense variants, bioinformatics analysis of deletion breakpoints, haplotyping of RAX2 variant c.335dup, and clinical assessment of biallelic RAX2-positive cases and carrier family members. Results: Biallelic RAX2 sequence and structural variants were found in five unrelated European index cases, displaying nonsyndromic autosomal recessive retinitis pigmentosa (ARRP) with an age of onset ranging from childhood to the mid-40s (average mid-30s). Protein structure modeling points to loss of function of the novel recessive missense variants and to a dominant-negative effect of the reported dominant RAX2 alleles. Structural variants were fine-mapped to disentangle their underlying mechanisms. Haplotyping of c.335dup in two cases suggests a common ancestry. Conclusion: This study supports a role for RAX2 as a novel disease gene for recessive IRD, broadening the mutation spectrum from sequence to structural variants and revealing a founder effect. The identification of biallelic RAX2 pathogenic variants in five unrelated families shows that RAX2 loss of function may be a nonnegligible cause of IRD in unsolved ARRP cases. Purpose: RAX2 encodes a homeobox-containing transcription factor, in which four monoallelic pathogenic variants have been described in autosomal dominant cone-dominated retinal disease. Methods: Exome sequencing in a European cohort with inherited retinal disease (IRD) (n = 2086) was combined with protein structure modeling of RAX2 missense variants, bioinformatics analysis of deletion breakpoints, haplotyping of RAX2 variant c.335dup, and clinical assessment of biallelic RAX2-positive cases and carrier family members. Results: Biallelic RAX2 sequence and structural variants were found in five unrelated European index cases, displaying nonsyndromic autosomal recessive retinitis pigmentosa (ARRP) with an age of onset ranging from childhood to the mid-40s (average mid-30s). Protein structure modeling points to loss of function of the novel recessive missense variants and to a dominant-negative effect of the reported dominant RAX2 alleles. Structural variants were fine-mapped to disentangle their underlying mechanisms. Haplotyping of c.335dup in two cases suggests a common ancestry. Conclusion: This study supports a role for RAX2 as a novel disease gene for recessive IRD, broadening the mutation spectrum from sequence to structural variants and revealing a founder effect. The identification of biallelic RAX2 pathogenic variants in five unrelated families shows that RAX2 loss of function may be a nonnegligible cause of IRD in unsolved ARRP cases.

Accumulating evidence on the role of non-protein-coding RNA sequences in the regulation of gene expression is greatly expanding our understanding of the flow of genetic information within biological systems. The interplay between protein-coding and non-coding RNAs (ncRNAs) is essential for tissue development, homeostasis, and function. NcRNAs can be divided in short ncRNAs, whose main subtype is represented by microRNAs, and long ncRNAs, which constitute a more heterogeneous class. The retina is a light-sensitive tissue consisting of highly interconnected cell types and is the primary target of many genetic diseases. Among these, the genetically heterogeneous group of inherited retinal diseases (IRDs) represents the most frequent monogenic cause of visual impairment that can ultimately lead to blindness. Here, we provide an overview on the role of ncRNAs in retinal development and function with an emphasis on microRNAs and on different types of long ncRNAs. We also review how sequence variations in ncRNAs can play a pathogenic role in IRDs as well as in multifactorial ocular disorders. These data indicate that a comprehensive study of the contribution of ncRNAs to the mutation repertoire associated with retinal disease can shed light on previously unknown pathophysiological mechanisms and open new therapeutic avenues. We conclude that a more comprehensive dissection of the pathogenic role of non-coding RNAs in retinal function and disease will not only improve our diagnostic ability, but will allow the development of novel targeted therapies for ocular disease.

MicroRNAs play a fundamental role in retinal development and function. To characterise the miRNome of the human retina, we carried out deep sequencing analysis on sixteen individuals. We established the catalogue of retina-expressed miRNAs, determined their relative abundance and found that a small number of miRNAs accounts for almost 90% of the retina miRNome. We discovered more than 3000 miRNA variants (isomiRs), encompassing a wide range of sequence variations, which include seed modifications that are predicted to have an impact on miRNA action. We demonstrated that a seed-modifying isomiR of the retina-enriched miR-124-3p was endowed with different targeting properties with respect to the corresponding canonical form. Moreover, we identified 51 putative novel, retina-specific miRNAs and experimentally validated the expression for nine of them. Finally, a parallel analysis of the human Retinal Pigment Epithelium (RPE)/choroid, two tissues that are known to be crucial for retina homeostasis, yielded notably distinct miRNA enrichment patterns compared to the retina. The generated data are accessible through an ad hoc database. This study is the first to reveal the complexity of the human retina miRNome at nucleotide resolution and constitutes a unique resource to assess the contribution of miRNAs to the pathophysiology of the human retina.

We performed a clinical and genetic characterization of a pediatric cohort of patients with inherited retinal dystrophy (IRD) to identify the most suitable cases for gene therapy. The cohort comprised 43 patients, aged between 2 and 18 years, with severe isolated IRD at the time of presentation. The ophthalmological characterization also included assessment of the photoreceptor layer integrity in the macular region (ellipsoid zone (EZ) band). In parallel, we carried out a targeted, next-generation sequencing (NGS)-based analysis using a panel that covers over 150 genes with either an established or a candidate role in IRD pathogenesis. Based on the ophthalmological assessment, the cohort was composed of 24 Leber congenital amaurosis, 14 early onset retinitis pigmentosa, and 5 achromatopsia patients. We identified causative mutations in 58.1% of the cases. We also found novel genotype-phenotype correlations in patients harboring mutations in the CEP290 and CNGB3 genes. The EZ band was detectable in 40%of the analyzed cases, also in patients with genotypes usually associated with severe clinical manifestations. This study provides the first detailed clinical-genetic assessment of severe IRDs with infantile onset and lays the foundation of a standardized protocol for the selection of patients that are more likely to benefit from gene replacement therapeutic approaches. We performed a clinical and genetic characterization of a pediatric cohort of patients with inherited retinal dystrophy (IRD) to identify the most suitable cases for gene therapy. The cohort comprised 43 patients, aged between 2 and 18 years, with severe isolated IRD at the time of presentation. The ophthalmological characterization also included assessment of the photoreceptor layer integrity in the macular region (ellipsoid zone (EZ) band). In parallel, we carried out a targeted, next-generation sequencing (NGS)-based analysis using a panel that covers over 150 genes with either an established or a candidate role in IRD pathogenesis. Based on the ophthalmological assessment, the cohort was composed of 24 Leber congenital amaurosis, 14 early onset retinitis pigmentosa, and 5 achromatopsia patients. We identified causative mutations in 58.1% of the cases. We also found novel genotype-phenotype correlations in patients harboring mutations in the CEP290 and CNGB3 genes. The EZ band was detectable in 40% of the analyzed cases, also in patients with genotypes usually associated with severe clinical manifestations. This study provides the first detailed clinical-genetic assessment of severe IRDs with infantile onset and lays the foundation of a standardized protocol for the selection of patients that are more likely to benefit from gene replacement therapeutic approaches.

MicroRNAs (miRNAs) are small noncoding RNAs that have important roles in the regulation of gene expression. The roles of individual miRNAs in controlling vertebrate eye development remain, however, largely unexplored. Here, we show that a single miRNA, miR-204, regulates multiple aspects of eye development in the medaka fish ( Oryzias latipes ). Morpholino-mediated ablation of miR-204 expression resulted in an eye phenotype characterized by microphthalmia, abnormal lens formation, and altered dorsoventral (D-V) patterning of the retina, which is associated with optic fissure coloboma. Using a variety of in vivo and in vitro approaches, we identified the transcription factor Meis2 as one of the main targets of miR-204 function. We show that, together with altered regulation of the Pax6 pathway, the abnormally elevated levels of Meis2 resulting from miR-204 inactivation are largely responsible for the observed phenotype. These data provide an example of how a specific miRNA can regulate multiple events in eye formation; at the same time, they uncover an as yet unreported function of Meis2 in the specification of D-V patterning of the retina.

Inherited Retinal Dystrophies (IRDs) are a clinically and genetically heterogeneous group of rare disorders characterized by a significant impairment in retinal function and vision. More than 150 genes have been associated with retinal dystrophies and the genetic overlap among different IRDs renders diagnosis and prognosis challenging. In this In Focus article, we give a summary on the pathogenic role of gene expression regulators in IRDs. Emphasis is given on key transcription factors that participate to regulatory gene networks controlling photoreceptor specification and maintenance, and their possible relevance as therapeutic targets. The increasing knowledge on the composition and function of these transcriptional regulatory networks indicates that intervening on transcription factors may be instrumental for a more effective treatment of some forms of IRDs, although the development of appropriate molecular tools to target them remains a formidable challenge.

Ocular developmental disorders, including the group classified as microphthalmia, anophthalmia, and coloboma (MAC) and inherited retinal dystrophies, collectively represent leading causes of hereditary blindness. Characterized by extreme genetic and clinical heterogeneity, the separate groups share many common genetic causes, in particular relating to pathways controlling retinal and retinal pigment epithelial maintenance. To understand these shared pathways and delineate the overlap between these groups, we investigated the genetic cause of an autosomal dominantly inherited condition of retinal dystrophy and bilateral coloboma, present in varying degrees in a large, five-generation family. By linkage analysis and exome sequencing, we identified a previously undescribed heterozygous mutation, n.37C > T, in the seed region of microRNA-204 (miR-204), which segregates with the disease in all affected individuals. We demonstrated that this mutation determines significant alterations of miR-204 targeting capabilities via in vitro assays, including transcriptome analysis. In vivo injection, in medaka fish (Oryzias latipes), of the mutated miR-204 caused a phenotype consistent with that observed in the family, including photoreceptor alterations with reduced numbers of both cones and rods as a result of increased apoptosis, thereby confirming the pathogenic effect of the n.37C > T mutation. Finally, knockdown assays in medaka fish demonstrated that miR-204 is necessary for normal photoreceptor function. Overall, these data highlight the importance of miR-204 in the regulation of ocular development and maintenance and provide the first evidence, to our knowledge, of its contribution to eye disease, likely through a gain-of-function mechanism.

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the retrieval of ocular tissues, especially cornea, by cutting it separate from the ocular globe. Evisceration is the process of removing the internal organs referred here as retina. The ocular globe consists of the cornea, the sclera, the vitreous body, the lens, the iris, the retina, the choroid, muscles etc (Suppl. Figure 1). When a patient is suffering from corneal damage, the cornea needs to be removed and a healthy one must be transplanted by keratoplastic surgeries. Genetic disorders or defects in retinal function can compromise vision. Human ocular globes can be used for various surgical procedures such as eye banking, transplantation of human cornea or sclera and research on ocular tissues. However, there is little information available on human corneal and retinal excision, probably due to the limited accessibility to human tissues. Most of the studies describing similar procedures are performed on animal models. Research scientists rely on the availability of properly dissected and well-conserved ocular tissues in order to extend the knowledge on human eye development, homeostasis and function. As we receive high amount of ocular globes out of which approximately 40% (Table 1) of them are used for research purposes, we are able to perform huge amount of experiments on these tissues, defining techniques to excise and preserve them regularly. The cornea is an avascular tissue which enables the transmission of light onto the retina and for this purpose should always maintain a good degree of transparency. Within the cornea, the limbus region, which is a reservoir of the stem cells, helps the reconstruction of epithelial cells and restricts the overgrowth of the conjunctiva maintaining corneal transparency and clarity. The size and thickness of the cornea are critical for clear vision, as changes in either of them could lead to distracted, unclear vision. The cornea comprises of 5 layers; a) epithelium, b) Bowman's layer, c) stroma, d) Descemet's membrane and e) endothelium. All layers should function properly to ensure clear vision4,5,6. The choroid is the intermediate tunic between the sclera and retina, bounded on the interior by the Bruch's membrane and is responsible for blood flow in the eye. The choroid also helps to regulate the temperature and supplies nourishment to the outer layers of the retina5,6. The retina is a layer of nervous tissue that covers the back of the ocular globe (Suppl. Figure 1) and consists of two parts: a photoreceptive part and a non-receptive part. The retina helps to receive the light from the cornea and lens and converts it into the chemical energy eventually transmitted to the brain with help of the optic nerve5,6. The aim of this paper is to provide a protocol for the dissection of corneal and retinal tissues from human ocular globes. Avoiding cross-contamination with adjacent tissues and preserving RNA integrity is of fundamental importance as such tissues are indispensable for research purposes aimed at (i) characterizing the transcriptome of the ocular tissues, (ii) isolating stem cells for regenerative medicine projects, and (iii) evaluating histological differences between tissues from normal/affected subjects. In this paper we describe the technique we currently use to remove the cornea, the choroid and retinal tissues from an ocular globe. Here we provide a detailed protocol for the dissection of the human ocular globe and the excision of corneal and retinal tissues. The accompanying video will help researchers to learn an appropriate technique for the retrieval of precious human tissues which are difficult to find regularly.

Background: Gene transfer using adeno-associated viral (AAV) vectors has been successfully applied in the retina for the treatment of inherited retinal dystrophies. Recently, microRNAs have been exploited to fine-tune transgene expression improving therapeutic outcomes. Here we evaluated the ability of retinal-expressed microRNAs to restrict AAV-mediated transgene expression to specific retinal cell types that represent the main targets of common inherited blinding conditions. Methodology/Principal Findings: To this end, we generated AAV2/5 vectors expressing EGFP and containing four tandem copies of miR-124 or miR-204 complementary sequences in the 3′UTR of the transgene expression cassette. These vectors were administered subretinally to adult C57BL/6 mice and Large White pigs. Our results demonstrate that miR-124 and miR-204 target sequences can efficiently restrict AAV2/5-mediated transgene expression to retinal pigment epithelium and photoreceptors, respectively, in mice and pigs. Interestingly, transgene restriction was observed at low vector doses relevant to therapy. Conclusions: We conclude that microRNA-mediated regulation of transgene expression can be applied in the retina to either restrict to a specific cell type the robust expression obtained using ubiquitous promoters or to provide an additional layer of gene expression regulation when using cell-specific promoters. © 2011 Karali et al.

Background MicroRNAs (miRNAs) are key regulators of biological processes. To define miRNA function in the eye, it is essential to determine a high-resolution profile of their spatial and temporal distribution. Results In this report, we present the first comprehensive survey of miRNA expression in ocular tissues, using both microarray and RNA in situ hybridization (ISH) procedures. We initially determined the expression profiles of miRNAs in the retina, lens, cornea and retinal pigment epithelium of the adult mouse eye by microarray. Each tissue exhibited notably distinct miRNA enrichment patterns and cluster analysis identified groups of miRNAs that showed predominant expression in specific ocular tissues or combinations of them. Next, we performed RNA ISH for over 220 miRNAs, including those showing the highest expression levels by microarray, and generated a high-resolution expression atlas of miRNAs in the developing and adult wild-type mouse eye, which is accessible in the form of a publicly available web database. We found that 122 miRNAs displayed restricted expression domains in the eye at different developmental stages, with the majority of them expressed in one or more cell layers of the neural retina. Conclusions This analysis revealed miRNAs with differential expression in ocular tissues and provided a detailed atlas of their tissue-specific distribution during development of the murine eye. The combination of the two approaches offers a valuable resource to decipher the contributions of specific miRNAs and miRNA clusters to the development of distinct ocular structures.

PURPOSE. MicroRNAs (miRNAs) are a class of small, endogenous RNAs that negatively regulate gene expression post-transcriptionally by binding to target sites in the 3' untranslated region (UTR) of messenger RNAs. Although they have been found to regulate developmental and physiological processes in several organs and tissues, their role in the eye transcriptome is completely unknown. This study was conducted to gain understanding of their eye-related function in mammals, by looking for miRNAs significantly expressed in the mouse eye by means of high-resolution expression analysis. METHODS. The spatiotemporal localization of miRNAs was analyzed in the murine embryonic and postnatal eye by RNA in situ hybridization (ISH) using LNA-modified oligonucleotide probes. RESULTS. Seven miRNAs were expressed in the eye with diverse and partially overlapping patterns, which may reflect their role in controlling cell differentiation of the retina as well as of other ocular structures. Most eye-expressed miRNAs overlap with or are in the near vicinity of transcripts derived predominantly from eye cDNA libraries. We found that these transcripts share very similar cellular distribution with their corresponding miRNAs, suggesting that miRNAs may share common expression regulatory elements with their host genes. CONCLUSIONS. The data provide a detailed characterization of expression of eye-enriched miRNAs. Knowledge of the spatiotemporal distribution of miRNAs is an essential step toward the identification of their targets and eventually the elucidation of their biological role in eye development and function.

International audience; HP1 proteins are essential components of heterochromatin and contribute to the transcriptional repression of euchromatic genes via the recruitment to specific promoters by corepressor proteins including TIF1 and Rb. The Caenorhabditis elegans HP1 homologue HPL-2 acts in the "synMuv" (synthetic multivulval) pathway, which defines redundant negative regulators of a Ras signaling cascade required for vulval induction. Several synMuv genes encode for chromatin-associated proteins involved in transcriptional regulation, including Rb and components of the Mi-2/NuRD and TIP60/NuA4 chromatin remodeling complexes. Here, we show that HPL-2 physically interacts in vitro and in vivo with the multiple zinc finger protein LIN-13, another member of the synMuv pathway. A variant of the conserved PXVXL motif found in many HP1-interacting proteins mediates LIN-13 binding to the CSD of HPL-2. We further show by in vivo localization studies that LIN-13 is required for HPL-2 recruitment in nuclear foci. Our data suggest that the LIN-13/HPL-2 complex may physically link a subset of the Rb related synMuv proteins to chromatin.

Liver-directed gene transfer is being investigated for the treatment of systemic or liver-specific diseases. Recombinant vectors based on adeno-associated virus serotype 8 (AAV2/8) efficiently transduce liver cells allowing long term transgene expression after a single administration in animal models and in patients. We evaluated the impact on AAV2/8-mediated rat liver transduction of the following variables: i) age at vector administration, ii) presence of lysosomal storage in liver cells, and iii) regulatory elements included in the transgene expression cassette. We found that systemic administration of AAV2/8 to newborn rats results in vector genome dilution and reduced transduction efficacy when compared to adult injected animals, presumably due to hepatocyte proliferation. Accumulation of glycosaminoglycans in lysosomes does not impact on levels and distribution of AAV2/8-mediated liver transduction. Transgene expression occurs in hepatocytes but not in Kupffer or liver endothelial cells when the liver-specific thyroxine-binding-globulin promoter is used. However, extra-hepatic transduction is observed in the spleen and kidney of animals injected at birth. The use of target sequences for the hematopoietic-specific microRNA miR142-3p does not improve liver transduction efficacy neither reduce immune responses to the lysosomal enzyme arylsulfatase B. The inclusion of a variant of the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE-m) decreases AAV2/8-mediated liver transduction levels. As AAV2/8-mediated liver gene transfer is entering in the clinical arena, these data will provide relevant information to the design of efficient AAV2/8-based therapeutic strategies.