Publications by authors named "Marianthi Karali"

27 Publications

  • Page 1 of 1

Clinical and Molecular Characterization of Achromatopsia Patients: A Longitudinal Study.

Int J Mol Sci 2021 Feb 7;22(4). Epub 2021 Feb 7.

Eye Clinic, Multidisciplinary Department of Medical, Surgical and Dental Sciences, Università degli Studi della Campania "Luigi Vanvitelli", via Pansini 5, 80131 Naples, Italy.

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.
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http://dx.doi.org/10.3390/ijms22041681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7914547PMC
February 2021

Sophisticated Gene Regulation for a Complex Physiological System: The Role of Non-coding RNAs in Photoreceptor Cells.

Front Cell Dev Biol 2020 18;8:629158. Epub 2021 Jan 18.

Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy.

Photoreceptors (PRs) are specialized neuroepithelial cells of the retina responsible for sensory transduction of light stimuli. In the highly structured vertebrate retina, PRs have a highly polarized modular structure to accommodate the demanding processes of phototransduction and the visual cycle. Because of their function, PRs are exposed to continuous cellular stress. PRs are therefore under pressure to maintain their function in defiance of constant environmental perturbation, besides being part of a highly sophisticated developmental process. All this translates into the need for tightly regulated and responsive molecular mechanisms that can reinforce transcriptional programs. It is commonly accepted that regulatory non-coding RNAs (ncRNAs), and in particular microRNAs (miRNAs), are not only involved but indeed central in conferring robustness and accuracy to developmental and physiological processes. Here we integrate recent findings on the role of regulatory ncRNAs (e.g., miRNAs, lncRNAs, circular RNAs, and antisense RNAs), and of their contribution to PR pathophysiology. We also outline the therapeutic implications of translational studies that harness ncRNAs to prevent PR degeneration and promote their survival and function.
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http://dx.doi.org/10.3389/fcell.2020.629158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7848107PMC
January 2021

Spectrum of Disease Severity in Patients With X-Linked Retinitis Pigmentosa Due to RPGR Mutations.

Invest Ophthalmol Vis Sci 2020 Dec;61(14):36

Eye Clinic, Multidisciplinary Department of Medical, Surgical and Dental Sciences, Università degli Studi della Campania "Luigi Vanvitelli," Naples, Italy.

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.
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http://dx.doi.org/10.1167/iovs.61.14.36DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7774109PMC
December 2020

Mild form of Zellweger Spectrum Disorders (ZSD) due to variants in : Detailed clinical investigation in a 9-years-old female.

Mol Genet Metab Rep 2020 Sep 20;24:100615. Epub 2020 Jun 20.

Eye Clinic, Multidisciplinary Department of Medical, Surgical and Dental Sciences, University of Campania Luigi Vanvitelli, Via Pansini 5, 80131 Naples, Italy.

Peroxisomal biogenesis disorders (PBD) are rare autosomal recessive disorders with various degrees of severity caused by hypomorphic mutations in 13 different peroxin (PEX) genes. In this study, we report the clinical and molecular characterization of a 9-years-old female presenting an apparently isolated pre-lingual sensorineural hearing loss (SNHL) and early onset Retinitis Pigmentosa (RP) that may clinically overlap with Usher syndrome. Genetic testing by clinical exome sequencing identified two variants in : the missense variant c.274G > C; p.(Val92Leu) that was already reported in a PBD patient, and the variant c.2140_2145dup; p.(Ser714_Gln715dup) which is a novel, non-frameshift variant, absent in control databases. On the basis of the molecular analysis, a thorough clinical examination revealed nail and dental abnormalities, a mild cognitive impairment, learning disabilities and poor feeding, apart from the retinal and audiological features initially identified. The clinical and molecular findings led us to the diagnosis of a mild form of PBD. This study further emphasizes that mild forms of PBD can be a differential diagnosis of Usher syndrome and suggests that patients with mild cognitive impairment associated to visual and hearing loss should perform a comprehensive mutation screening that includes genes.
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http://dx.doi.org/10.1016/j.ymgmr.2020.100615DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7306489PMC
September 2020

Resolving the dark matter of ABCA4 for 1054 Stargardt disease probands through integrated genomics and transcriptomics.

Genet Med 2020 Jul 20;22(7):1235-1246. Epub 2020 Apr 20.

Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands.

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.
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http://dx.doi.org/10.1038/s41436-020-0787-4DOI Listing
July 2020

Clinical and Genetic Analysis of a European Cohort with Pericentral Retinitis Pigmentosa.

Int J Mol Sci 2019 Dec 20;21(1). Epub 2019 Dec 20.

Eye Clinic, Multidisciplinary Department of Medical, Surgical and Dental Sciences, Università degli Studi della Campania 'Luigi Vanvitelli', via Pansini 5, 80131 Naples, Italy.

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.
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http://dx.doi.org/10.3390/ijms21010086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6982348PMC
December 2019

AAV-miR-204 Protects from Retinal Degeneration by Attenuation of Microglia Activation and Photoreceptor Cell Death.

Mol Ther Nucleic Acids 2020 Mar 18;19:144-156. Epub 2019 Nov 18.

Telethon Institute of Genetics and Medicine, via Campi Flegrei 34, 80078 Pozzuoli (NA), Italy; Department of Precision Medicine, University of Campania 'Luigi Vanvitelli,' via Luigi De Crecchio 7, 80138 Naples (NA), Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.omtn.2019.11.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6920266PMC
March 2020

Microdeletion of pseudogene chr14.232.a affects LRFN5 expression in cells of a patient with autism spectrum disorder.

Eur J Hum Genet 2019 09 31;27(9):1475-1480. Epub 2019 May 31.

Department of Translational Medicine, Section of Paediatrics, Federico II University, Naples, Italy.

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-deleted gene 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.
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http://dx.doi.org/10.1038/s41431-019-0430-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6777536PMC
September 2019

Correction: Biallelic sequence and structural variants in RAX2 are a novel cause for autosomal recessive inherited retinal disease.

Genet Med 2019 04;21(4):1028

Center for Medical Genetics, Ghent University and Ghent University Hospital, Ghent, Belgium.

The original version of this Article contained an incorrect version of Fig. 3, which included two variants initially shown in black text in Fig. 3a that the authors removed from the final manuscript. The correct version of Fig. 3 without the two variants now appears in the PDF and HTML versions of the Article.
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http://dx.doi.org/10.1038/s41436-018-0392-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6752296PMC
April 2019

Biallelic sequence and structural variants in RAX2 are a novel cause for autosomal recessive inherited retinal disease.

Genet Med 2019 06 31;21(6):1319-1329. Epub 2018 Oct 31.

Center for Medical Genetics, Ghent University and Ghent University Hospital, Ghent, Belgium.

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.
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http://dx.doi.org/10.1038/s41436-018-0345-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6752271PMC
June 2019

Non-coding RNAs in retinal development and function.

Hum Genet 2019 Sep 5;138(8-9):957-971. Epub 2018 Sep 5.

Medical Genetics, Department of Precision Medicine, Università degli Studi della Campania "Luigi Vanvitelli", Via Luigi De Crecchio 7, 80138, Naples, Italy.

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.
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http://dx.doi.org/10.1007/s00439-018-1931-yDOI Listing
September 2019

microRNAs as biomarkers in Pompe disease.

Genet Med 2019 03 12;21(3):591-600. Epub 2018 Jul 12.

Department of Translational Medical Sciences, Federico II University, Naples, Italy.

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.
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http://dx.doi.org/10.1038/s41436-018-0103-8DOI Listing
March 2019

MiR-211 is essential for adult cone photoreceptor maintenance and visual function.

Sci Rep 2017 12 5;7(1):17004. Epub 2017 Dec 5.

Telethon Institute of Genetics and Medicine, Via Campi Flegrei 34, Pozzuoli (Naples), 80078, Italy.

MicroRNAs (miRNAs) are key post-transcriptional regulators of gene expression that play an important role in the control of fundamental biological processes in both physiological and pathological conditions. Their function in retinal cells is just beginning to be elucidated, and a few have been found to play a role in photoreceptor maintenance and function. MiR-211 is one of the most abundant miRNAs in the developing and adult eye. However, its role in controlling vertebrate visual system development, maintenance and function so far remain incompletely unexplored. Here, by targeted inactivation in a mouse model, we identify a critical role of miR-211 in cone photoreceptor function and survival. MiR-211 knockout (-/-) mice exhibited a progressive cone dystrophy accompanied by significant alterations in visual function. Transcriptome analysis of the retina from miR-211-/- mice during cone degeneration revealed significant alteration of pathways related to cell metabolism. Collectively, this study highlights for the first time the impact of miR-211 function in the retina and significantly contributes to unravelling the role of specific miRNAs in cone photoreceptor function and survival.
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http://dx.doi.org/10.1038/s41598-017-17331-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5717140PMC
December 2017

Clinical and Genetic Evaluation of a Cohort of Pediatric Patients with Severe Inherited Retinal Dystrophies.

Genes (Basel) 2017 Oct 20;8(10). Epub 2017 Oct 20.

Eye Clinic, Multidisciplinary Department of Medical, Surgical and Dental Sciences, Università degli Studi della Campania Luigi Vanvitelli, via Pansini 5, Naples 80131, Italy.

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 and 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.
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http://dx.doi.org/10.3390/genes8100280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5664130PMC
October 2017

An atlas of gene expression and gene co-regulation in the human retina.

Nucleic Acids Res 2016 07 27;44(12):5773-84. Epub 2016 May 27.

Telethon Institute of Genetics and Medicine (TIGEM), Via Campi Flegrei 34, 80078 Pozzuoli, Italy Dept. Of Chemical, Materials and Industrial Production Engineering, University of Naples 'Federico II', Piazzale Tecchio 80, 80125 Naples, Italy

The human retina is a specialized tissue involved in light stimulus transduction. Despite its unique biology, an accurate reference transcriptome is still missing. Here, we performed gene expression analysis (RNA-seq) of 50 retinal samples from non-visually impaired post-mortem donors. We identified novel transcripts with high confidence (Observed Transcriptome (ObsT)) and quantified the expression level of known transcripts (Reference Transcriptome (RefT)). The ObsT included 77 623 transcripts (23 960 genes) covering 137 Mb (35 Mb new transcribed genome). Most of the transcripts (92%) were multi-exonic: 81% with known isoforms, 16% with new isoforms and 3% belonging to new genes. The RefT included 13 792 genes across 94 521 known transcripts. Mitochondrial genes were among the most highly expressed, accounting for about 10% of the reads. Of all the protein-coding genes in Gencode, 65% are expressed in the retina. We exploited inter-individual variability in gene expression to infer a gene co-expression network and to identify genes specifically expressed in photoreceptor cells. We experimentally validated the photoreceptors localization of three genes in human retina that had not been previously reported. RNA-seq data and the gene co-expression network are available online (http://retina.tigem.it).
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http://dx.doi.org/10.1093/nar/gkw486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4937338PMC
July 2016

High-resolution analysis of the human retina miRNome reveals isomiR variations and novel microRNAs.

Nucleic Acids Res 2016 Feb 26;44(4):1525-40. Epub 2016 Jan 26.

Telethon Institute of Genetics and Medicine, via Campi Flegrei 34, 80078 Pozzuoli (NA), Italy Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, via Luigi De Crecchio 7, 80138 Naples (NA), Italy

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.
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http://dx.doi.org/10.1093/nar/gkw039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4770244PMC
February 2016

MiR-204 is responsible for inherited retinal dystrophy associated with ocular coloboma.

Proc Natl Acad Sci U S A 2015 Jun 8;112(25):E3236-45. Epub 2015 Jun 8.

Manchester Centre for Genomic Medicine, Institute of Human Development, Faculty of Medical and Human Sciences, University of Manchester, Manchester M13 9WL, United Kingdom; Manchester Centre for Genomic Medicine, St. Mary's Hospital, Central Manchester University Hospitals National Health Service Foundation Trust, Manchester Academic Health Sciences Centre, Manchester M13 9WL, United Kingdom; European Retinal Dystrophy Consortium; United Kingdom Inherited Retinal Disease Consortium, Manchester Centre for Genomic Medicine, St. Mary's Hospital, Central Manchester University Hospitals National Health Service Foundation Trust, Manchester Academic Health Sciences Centre, Manchester M13 9WL, United Kingdom

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.37 C > 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.37 C > 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.
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http://dx.doi.org/10.1073/pnas.1401464112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4485104PMC
June 2015

Inherited Retinal Dystrophies: the role of gene expression regulators.

Int J Biochem Cell Biol 2015 Apr 16;61:115-9. Epub 2015 Feb 16.

Telethon Institute for Genetics and Medicine, via Campi Flegrei 34, 80078 Pozzuoli, NA, Italy; Medical Genetics, Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, via Luigi De Crecchio 7, 80138 Naples, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.biocel.2015.02.007DOI Listing
April 2015

A simplified technique for in situ excision of cornea and evisceration of retinal tissue from human ocular globe.

J Vis Exp 2012 Jun 12(64):e3765. Epub 2012 Jun 12.

Fondazione Banca Degli Occhi del Veneto ONLUS.

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 vision(4,5,6). 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 retina(5,6). 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 nerve(5,6). 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.
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http://dx.doi.org/10.3791/3765DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3471310PMC
June 2012

Impact of age at administration, lysosomal storage, and transgene regulatory elements on AAV2/8-mediated rat liver transduction.

PLoS One 2012 13;7(3):e33286. Epub 2012 Mar 13.

Telethon Institute of Genetics and Medicine, Naples, Italy.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0033286PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3302848PMC
August 2012

MicroRNA-restricted transgene expression in the retina.

PLoS One 2011 26;6(7):e22166. Epub 2011 Jul 26.

Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0022166PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3144214PMC
December 2011

miRNeye: a microRNA expression atlas of the mouse eye.

BMC Genomics 2010 Dec 20;11:715. Epub 2010 Dec 20.

Telethon Institute for Genetics and Medicine, Via P. Castellino 111, Naples, Italy.

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.
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http://dx.doi.org/10.1186/1471-2164-11-715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3018480PMC
December 2010

miR-204 is required for lens and retinal development via Meis2 targeting.

Proc Natl Acad Sci U S A 2010 Aug 16;107(35):15491-6. Epub 2010 Aug 16.

Telethon Institute of Genetics and Medicine, 80131 Naples, Italy.

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.
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http://dx.doi.org/10.1073/pnas.0914785107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2932609PMC
August 2010

Inducible gene expression systems and plant biotechnology.

Biotechnol Adv 2009 Nov-Dec;27(6):733-743. Epub 2009 May 19.

Centre for Plant Science, University of Leeds, Leeds, LS29JT, United Kingdom.

Plant biotechnology relies heavily on the genetic manipulation of crops. Almost invariantly, the gene of interest is expressed in a constitutive fashion, although this may not be strictly necessary for several applications. Currently, there are several regulatable expression systems for the temporal, spatial and quantitative control of transgene activity. These molecular switches are based on components derived from different organisms, which range from viruses to higher eukaryotes. Many inducible systems have been designed for fundamental and applied research and since their initial development, they have become increasingly popular in plant molecular biology. This review covers a broad number of inducible expression systems examining their properties and relevance for plant biotechnology in its various guises, from molecular breeding to pharmaceutical and industrial applications. For each system, we examine some advantages and limitations, also in relation to the strategy on which they rely. Besides being necessary to control useful genes that may negatively affect crop yield and quality, we discuss that inducible systems can be also used to increase public acceptance of GMOs, reducing some of the most common concerns. Finally, we suggest some directions and future developments for their further diffusion in agriculture and biotechnology.
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http://dx.doi.org/10.1016/j.biotechadv.2009.05.006DOI Listing
January 2010

A high-resolution RNA expression atlas of retinitis pigmentosa genes in human and mouse retinas.

Invest Ophthalmol Vis Sci 2008 Jun 15;49(6):2330-6. Epub 2008 Feb 15.

Telethon Institute of Genetics and Medicine, Naples, Italy.

Purpose: Retinitis pigmentosa (RP) is one of the leading causes of visual handicap in the world population and is characterized by high genetic heterogeneity. The study of the disease mechanisms and the development of efficient therapeutic approaches have mostly relied on the availability of animal models for this condition, so far. Nevertheless, little information is available about the RNA expression profiles of RP genes in the human retina. An expression atlas of 34 known RP genes in human and murine retinas was generated to overcome this lack of information.

Methods: Appropriate templates were retrieved for 34 RP genes that were used to perform RNA in situ hybridization studies on human and murine adult eyes.

Results: Most of the genes displayed similar patterns between human and mouse retina. Different expression patterns were observed for the CNGB1, USH2A, and FSCN2 genes, compared with those in previously reported profiles. In addition, different expression profiles were detected for the RPGR, CA4, PAP1, RGR, and RLBP1 genes in human and mouse retinas.

Conclusions: The first gene expression atlas has been generated of RP genes in human and murine retinas. Differences observed in the expression patterns of some genes in humans and mice, will open new perspectives on the function of these genes and their putative roles in disease pathogenesis.
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http://dx.doi.org/10.1167/iovs.07-1513DOI Listing
June 2008

Identification and characterization of microRNAs expressed in the mouse eye.

Invest Ophthalmol Vis Sci 2007 Feb;48(2):509-15

Telethon Institute for Genetics and Medicine, Naples, Italy.

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.
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http://dx.doi.org/10.1167/iovs.06-0866DOI Listing
February 2007

The C. elegans HP1 homologue HPL-2 and the LIN-13 zinc finger protein form a complex implicated in vulval development.

Dev Biol 2006 Sep 23;297(2):308-22. Epub 2006 May 23.

Laboratoire de Biologie Moleculaire de la Cellule, Ecole Normale Supérieure de Lyon, 69007 Lyon, France.

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.
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http://dx.doi.org/10.1016/j.ydbio.2006.04.474DOI Listing
September 2006