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Mitomycin C as well as other antiproliferative drugs are off-label agents widely used to prevent the failure of glaucoma surgery due to activation of Tenon's fibroblasts and the ensuing excessive subconjunctival scarring. Though efficacious, these treatments are associated with some severe long-term complications, so it is crucial to investigate less cytotoxic compounds as adjuvant therapy in glaucoma surgery. The aim of this study was to evaluate the effect and potential cytotoxicity of Myriocin, a natural sphingolipid synthesis inhibitor, on TGF-β1-induced myofibroblasts transformation of human dermal fibroblasts. We found that myriocin significantly attenuated the transcript levels of αSMA, CTGF, and MMP9 which are involved in the fibrosis process. Mitomycin C poorly affects the same pro-fibrotic markers while reducing fibroblasts motility as much as myriocin. At similar doses, five minutes of mitomycin C treatment consistently affects human dermal fibroblast viability and proliferation compared to prolonged myriocin application, strengthening already published data on the good tolerability of this natural compound. Our results draw attention to the use of myriocin as an adjuvant in glaucoma surgery due to the effectiveness in reducing fibroblasts to myofibroblasts transformation and the low cytotoxicity., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests Anna Caretti, Luca Rossetti, Leonardo Colombo and Dario Romano have patent pending to assignee. The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Introduction: Concomitant manifestation of PPRCA in one eye and RP-like retinopathy in the fellow eye is a rare clinical entity, with limited published descriptions to date. The aim of this study is to describe comprehensive clinical evaluations and long-term follow-up of three patients affected by this clinical picture., Methods: Three patients with concurrent PPRCA and RP-like retinopathy were prospectively re-evaluated and comprehensive assessments were performed. The progression of disease was assessed by comparing best corrected visual acuity (BCVA) and ellipsoid zone (EZ) width with data available from each patient's medical charts and previous SD-OCT scans. Blood samples were collected and tested to rule out autoimmune or infectious ocular diseases, for testing anti-retinal autoantibodies (ARAs) and for genetic analysis., Results: Reduction in BCVA and a progressive concentric loss of the EZ band over time were detected in the eye with RP-like phenotype in all three patients, while in the eye with PPRCA none of the patients showed significant changes in BCVA and only one patient showed a progressive reduction of the EZ width. No clear etiology has been identified. Two or more ARAs subtypes were detected in two out of three patients., Conclusions: PPRCA was demonstrated to be a non-progressive or slowly progressive disease, instead the eye with RP-like phenotype showed a progressive visual impairment, highlighting the importance of shedding light on this condition. Its etiology remains unclear: a genetic trait with different phenotype between the two eyes is conceivable, as well as a potential role of ARAs., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Purpose: To compare within-subject efficacy and safety of intravitreal dexamethasone implant and topical carbonic anhydrase inhibitors in the treatment of retinitis pigmentosa-related cystoid macular edema., Methods: Patients with bilateral retinitis pigmentosa-related cystoid macular edema were treated with intravitreal dexamethasone implant in one eye and topical carbonic anhydrase inhibitors in the contralateral eye. The primary endpoint was a change in central macular thickness. Secondary endpoints were changes in best-corrected visual acuity and microperimetric central retinal sensitivity. Intraocular pressure and other ocular complications were evaluated for safety assessment., Results: Nine patients were recruited for this 12-month follow-up study. Central macular thickness was significantly lower in intravitreal dexamethasone implant-treated eyes than in topical carbonic anhydrase inhibitors-treated eyes at Months 1 and 7, whereas mean best-corrected visual acuity was better in eyes treated with topical carbonic anhydrase inhibitors at Month 12 (borderline significant P = 0.0510). There was no difference in microperimetric sensitivity between the two treatments. Three patients developed ocular hypertension after intravitreal dexamethasone implant. Intravitreal dexamethasone implant showed an effect on the contralateral eye in five of nine patients., Conclusion: Intravitreal dexamethasone implant was more effective than topical carbonic anhydrase inhibitors in reducing retinitis pigmentosa-related cystoid macular edema 1 month after treatment. Corticosteroids can play a key role in the management of retinitis pigmentosa-related cystoid macular edema; however, their routes, timing, and modes of administration should be further explored., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the Opthalmic Communications Society, Inc.)

Introduction: Retinitis pigmentosa (RP), a heterogeneous inherited retinal disorder causing gradual vision loss, affects over 1 million people worldwide. Pathogenic variants in CNGA1 and CNGB1 genes, respectively, accounting for 1% and 4% of cases, impact the cyclic nucleotide-gated channel in rod photoreceptor cells. The aim of this study was to describe and compare genotypic and clinical characteristics of a cohort of patients with CNGA1- or CNGB1-related RP and to explore potential genotype-phenotype correlations., Methods: The following data from patients with CNGA1- or CNGB1-related RP, followed in five Italian inherited retinal degenerations services, were retrospectively collected: genetic variants in CNGA1 and CNGB1, best-corrected visual acuity (BCVA), ellipsoid zone (EZ) width, fundus photographs, and short-wavelength fundus autofluorescence (SW-AF) images. Comparisons and correlation analyses were performed by first dividing the cohort in two groups according to the gene responsible for the disease (CNGA1 and CNGB1 groups). In parallel, the whole cohort of RP patients was divided into two other groups, according to the expected impact of the variants at protein level (low and high group)., Results: In total, 29 patients were recruited, 11 with CNGA1- and 18 with CNGB1-related RP. In both CNGA1 and CNGB1, 5 novel variants in CNGA1 and 5 in CNGB1 were found. BCVA was comparable between CNGA1 and CNGB1 groups, as well as between low and high groups. CNGA1 group had a larger mean EZ width compared to CNGB1 group, albeit not statistically significant, while EZ width did not differ between low and high groups A statistically significant correlation between EZ width and BCVA as well as between EZ width and age were observed in the whole cohort of RP patients. Fundus photographs of all patients in the cohort showed classic RP pattern, and in SW-AF images an hyperautofluorescent ring was observed in 14/21 patients., Conclusion: Rod CNG channel-associated RP was demonstrated to be a slowly progressive disease in both CNGA1- and CNGB1-related forms, making it an ideal candidate for gene augmentation therapies., (© 2024 The Author(s). Published by S. Karger AG, Basel.)

Sequencing of the low-complexity ORF15 exon of RPGR, a gene correlated with retinitis pigmentosa and cone dystrophy, is difficult to achieve with NGS and Sanger sequencing. False results could lead to the inaccurate annotation of genetic variants in dbSNP and ClinVar databases, tools on which HGMD and Ensembl rely, finally resulting in incorrect genetic variants interpretation. This paper aims to propose PacBio sequencing as a feasible method to correctly detect genetic variants in low-complexity regions, such as the ORF15 exon of RPGR, and interpret their pathogenicity by structural studies. Biological samples from 75 patients affected by retinitis pigmentosa or cone dystrophy were analyzed with NGS and repeated with PacBio. The results showed that NGS has a low coverage of the ORF15 region, while PacBio was able to sequence the region of interest and detect eight genetic variants, of which four are likely pathogenic. Furthermore, molecular modeling and dynamics of the RPGR Glu-Gly repeats binding to TTLL5 allowed for the structural evaluation of the variants, providing a way to predict their pathogenicity. Therefore, we propose PacBio sequencing as a standard procedure in diagnostic research for sequencing low-complexity regions such as RPGR ORF15 , aiding in the correct annotation of genetic variants in online databases.

Purpose: To describe a case of extensive macular atrophy with pseudodrusen-like appearance complicated by Bruch membrane rupture and choroidal neovascularization, treated with intravitreal injection of an antivascular endothelial growth factor drug., Methods: A 54-year-old woman, affected by extensive macular atrophy with pseudodrusen-like appearance, has developed Bruch membrane rupture and choroidal neovascularization. This report discusses the development of the disease and the improvement after a single intravitreal injection of Avastin., Results: After treatment, the visual acuity increased from 20/400 to 20/200; clinical and anatomical improvement remained stable with a 2-month follow-up., Conclusion: Extensive macular atrophy with pseudodrusen-like appearance could be complicated by Bruch membrane rupture and subsequent choroidal neovascularization. Antivascular endothelial growth factor intravitreal injection could have beneficial effects on this entity, but concern could raise in respect of possible increase of chorioretinal atrophy., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the Opthalmic Communications Society, Inc.)

Third generation sequencing methods, like PacBio, provide information about structural variants, introns, enhancers and promoters. We developed an automated pipeline, called PacMAGI, including quality control, alignment, SNV, INDELs, structural variant calling, phasing, annotation and variant interpretation, for the analysis of PacBio data for any target region. Bi-allelic mutations in the RPE65 gene are associated with different inherited retinal dystrophies, such as Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP). Diagnostic panel-based NGS analysis is performed on coding regions and intron/exon junctions of genes. To obtain a more conclusive diagnosis, we applied PacMAGI to obtain a second hit on RPE65 in LCA or RP patients who showed a single heterozygous variant by NGS. We used PacBio to sequence the full gene and identify putative second-hits in intronic, problematic and promoter regions. All variants identified in the diagnostic setting with NGS were correctly detected by the pipeline, and thanks to our custom algorithm for INDELs, a previously undetected 'Pathogenic' frameshift variant was found in a RP patient already identified to carry a 'Likely Pathogenic' variant., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: Describing the clinical and genetic features of an ethnically heterogeneous group of (inherited retinal diseases) IRD patients from different underrepresented countries, referring to specialized Italian Hospitals, and expanding the epidemiological spectrum of the IRD in understudied populations. Methods: The patients' phenotypes underwent were characterized by exhaustive ophthalmological examinations, including morpho-functional testing. Genetic testing was performed using next-generation sequencing (NGS) and gene sequencing panels targeting a specific set of genes, Sanger sequencing and-when necessary-multiplex ligation-dependent probe amplification (MLPA) to better identify the genotype. When possible, segregation analysis was performed in order to confirm unsolved cases. Results: The article reports the results of the phenotypes and genotypes of 123 IRD probands, 69 males and 54 females, mean age 41 (IQR, 54-30) years, disease onset at 13 (IQR, 27.25-5) years. Thirty-three patients out of 123 (26.8%) were Africans (North/Northwest Africa), 21 (17.1%) Asians, 19 (15.4%) Americans (South/Central America) and 50 (40.7%) Europeans (Eastern Europe). Retinitis pigmentosa was the most represented phenotype (56%), followed by cone dystrophy (11%) and Leber congenital amaurosis (7%), while ABCA4 was the most frequently mutated gene (18%), followed by USH2A (9%) and RPGR (5%). About ABCA4 variants found in Stargardt disease, macular and cone dystrophies were predominant in Asian (42%) and European (21%) patients. The most represented inheritance pattern was autosomal recessive, while a higher frequency of homozygous patients versus compound heterozygotes as compared to previous studies on Italian IRD patients was evidenced, reflecting a possible higher frequency of inbreeding marriages. Conclusion: Though limited by the relatively low number of patients, the present paper paints a picture of the clinical and genetic features of IRD patients from understudied ethnic groups referred to Italian specialized hospitals and extended the epidemiological studies on underrepresented world regional areas., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Maltese, Colombo, Martella, Rossetti, El Shamieh, Sinibaldi, Passarelli, Coppè, Buzzonetti, Falsini, Chiurazzi, Placidi, Tanzi, Bertelli and Iarossi.)

Purpose: To investigate the course of inherited retinal degenerations (IRD) due to mutations in the RPE65 gene., Methods: This longitudinal multicentric retrospective chart-review study was designed to collect best corrected visual acuity (BCVA), Goldman visual field, optical coherence tomography (OCT), and electroretinography (ERG) measurements. The data, including imaging, were collected using an electronic clinical research form and were reviewed at a single center to improve consistency., Results: From an overall cohort of 60 Italian patients with RPE65-associated IRD, 43 patients (mean age, 27.8 ± 19.7 years) were included and showed a mean BCVA of 2.0 ± 1.0 logMAR. Time-to-event analysis revealed a median age of 33.8 years and 41.4 years to reach low vision and blindness based on BCVA, respectively. ERG (available for 34 patients) showed undetectable responses in most patients (26; 76.5%). OCT (available for 31 patients) revealed epiretinal membranes in five patients (16.1%). Central foveal thickness significantly decreased with age at a mean annual rate of -0.6%/y (P = 0.044). We identified 43 different variants in the RPE65 gene in the entire cohort. Nine variants were novel. Finally, to assess genotype-phenotype correlations, patients were stratified according to the number of RPE65 loss-of-function (LoF) alleles. Patients without LoF variants showed significantly (P < 0.05) better BCVA compared to patients with one or two LoF alleles., Conclusions: We described the natural course of RPE65-associated IRD in an Italian cohort showing for the first time a specific genotype-phenotype association. Our findings can contribute to a better management of RPE65-associated IRD patients.

Introduction: This study aimed to analyze macular structure by using spectral-domain optical coherence tomography (SD-OCT) in a cohort of patients affected by autosomal recessive retinitis pigmentosa and Usher syndrome, due to genetic variants in USH2A gene, and to correlate optical coherence tomography (OCT) parameters with functional and genetic data., Methods: The subjects of this study were 92 patients, 46 syndromic (Usher syndrome type IIa [Ush2]) and 46 nonsyndromic (autosomal recessive RP [arRP]), with clinical and genetic diagnosis of USH2A-related retinal dystrophy, who underwent a complete ophthalmic examination and spectral-domain OCT analysis. The study focused on evaluating the differences between the 2 groups in the following parameters: best-corrected visual acuity (BCVA), ellipsoid zone (EZ) width, presence of epiretinal membrane (ERM), and cystic macular lesions (CMLs). Variants in USH2A gene were divided into 3 categories, according to the expected impact (low/high) at protein level of the different variants on each allele., Results: BCVA and EZ width were significantly lower in Ush2 than in arRP patients (p < 0.0001 and p = 0.001). ERM was detected in 34.8% (16/46) of arRP patients and in 65.2% (30/46) of Ush2 patients (p = 0.003). CML was detected in 17.4% (8/46) of arRP patients and 30.4% (14/46) of Ush2 patients (p = 0.14). The allelic distribution was statistically different (p = 0.0003) by dividing the 2 diseases: for Ush2 patients it was 45.7% (high/high), 39.1% (low/high) and 15.2% (low/low); for arRP patients it was 8.7% (high/high), 56.5% (low/high), and 34.8% (low/low). The severity class of the variants significantly affected visual acuity and EZ width parameters (p = 0.004 and p = 0.002, respectively)., Conclusion: Retinal disease, as evaluated by means of SD-OCT, shows more advanced degeneration signs in the syndromic than the nonsyndromic form of retinal dystrophy related to USH2A gene. Variant types and allelic profiles are determining factors for the onset of syndromic features. However, since the 3 allelic profiles can be found in both Usher and RP patients, other factors must necessarily play a determining role., (© 2021 The Author(s). Published by S. Karger AG, Basel.)

Guanylate cyclase-activating protein 1 (GCAP1), encoded by the GUCA1A gene, is a neuronal calcium sensor protein involved in shaping the photoresponse kinetics in cones and rods. GCAP1 accelerates or slows the cGMP synthesis operated by retinal guanylate cyclase (GC) based on the light-dependent levels of intracellular Ca 2+ , thereby ensuring a timely regulation of the phototransduction cascade. We found a novel variant of GUCA1A in a patient affected by autosomal dominant cone dystrophy (adCOD), leading to the Asn104His (N104H) amino acid substitution at the protein level. While biochemical analysis of the recombinant protein showed impaired Ca 2+ sensitivity of the variant, structural properties investigated by circular dichroism and limited proteolysis excluded major structural rearrangements induced by the mutation. Analytical gel filtration profiles and dynamic light scattering were compatible with a dimeric protein both in the presence of Mg 2+ alone and Mg 2+ and Ca 2+ . Enzymatic assays showed that N104H-GCAP1 strongly interacts with the GC, with an affinity that doubles that of the WT. The doubled IC 50 value of the novel variant (520 nM for N104H vs. 260 nM for the WT) is compatible with a constitutive activity of GC at physiological levels of Ca 2+ . The structural region at the interface with the GC may acquire enhanced flexibility under high Ca 2+ conditions, as suggested by 2 μs molecular dynamics simulations. The altered interaction with GC would cause hyper-activity of the enzyme at both low and high Ca 2+ levels, which would ultimately lead to toxic accumulation of cGMP and Ca 2+ in the photoreceptor outer segment, thus triggering cell death.

Electrical stimulation (ES) of the eye represents a therapeutic approach in various clinical applications ranging from retinal dystrophies, age-related macular degeneration, retinal artery occlusion and nonarteritic ischemic optic neuropathy. In clinical practice, ES of the eye is mainly performed with a transcorneal or transpalpebral approach. These procedures are non-invasive and well-tolerated by the patients, reporting only minimal and transient adverse events, while serious adverse effects were not observed. Despite the growing literature on animal models, only clinical parameters have been investigated in humans and few data are available about biochemical changes induced by ES of the eye. The purpose of this study is to investigate the possible mechanism that regulates the beneficial effects of ES on retinal cells function and survival in humans. 28 patients undergoing pars plana vitrectomy (PPV) for idiopathic epiretinal membrane (iERM) were randomly divided in two groups: 13 patients were treated with transpalpebral ES before surgery and 15 underwent surgery with no prior treatment. Vitreous samples were collected for biochemical analysis during PPV. ES treatment leads to a reduction in the vitreous expression of both proinflammatory cytokines, namely IL-6 and IL-8, and proinflammatory lipid mediators, such as lysophosphatidylcholine. Indeed, we observed a 70% decrease of lysophosphatidylcholine 18:0, which has been proven to exert the greatest proinflammatory activities among the lysophosphatidylcholine class. The content of triglycerides is also affected and significantly decreased following ES application. The vitreous composition of patients undergoing PPV for iERM displays significant changes following ES treatment. Proinflammatory cytokines and bioactive lipid mediators expression decreases, suggesting an overall anti-inflammatory potential of ES. The investigation of the mechanism by which this treatment alters the retinal neurons leading to good outcomes is essential for supporting ES therapeutic application in various types of retinal diseases., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: To describe the molecular epidemiology of nonsyndromic retinitis pigmentosa (RP) and Usher syndrome (US) in Italian patients., Methods: A total of 591 probands (315 with family history and 276 sporadics) were analyzed. For 155 of them, we performed a family segregation study, considering a total of 382 relatives. Probands were analyzed by a customized multigene panel approach. Sanger sequencing was used to validate all genetic variants and to perform family segregation studies. Copy number variants of selected genes were analyzed by multiplex ligation-dependent probe amplification. Four patients who tested negative to targeted next-generation sequencing analysis underwent clinical exome sequencing., Results: The mean diagnostic yield of molecular testing among patients with a family history of retinal disorders was 55.2% while the diagnostic yield including sporadic cases was 37.4%. We found 468 potentially pathogenic variants, 147 of which were unpublished, in 308 probands and 66 relatives. Mean ages of onset of the different classes of RP were autosomal dominant RP, 19.3 ± 12.6 years; autosomal recessive RP, 23.2 ± 16.6 years; X-linked RP, 13.9 ± 9.9 years; and Usher syndrome, 18.9 ± 9.5 years. We reported potential new genotype-phenotype correlations in three probands, two revealed by TruSight One testing. All three probands showed isolated RP caused by biallelic variants in genes usually associated with syndromes such as PERCHING and Senior-Loken or with retinal dystrophy, iris coloboma, and comedogenic acne syndrome., Conclusions: This is the largest molecular study of Italian patients with RP in the literature, thus reflecting the epidemiology of the disease in Italy with reasonable accuracy.

The small Ras-related GTPase Rab-28 is highly expressed in photoreceptor cells, where it possibly participates in membrane trafficking. To date, six alterations in the RAB28 gene have been associated with autosomal recessive cone-rod dystrophies. Confirmed variants include splicing variants, missense and nonsense mutations. Here, we present a thorough phenotypical and genotypical characterization of five individuals belonging to four Italian families, constituting the largest cohort of RAB28 patients reported in literature to date. All probands displayed similar clinical phenotype consisting of photophobia, decreased visual acuity, central outer retinal thinning, and impaired color vision. By sequencing the four probands, we identified: a novel homozygous splicing variant; two novel nonsense variants in homozygosis; a novel missense variant in compound heterozygous state with a previously reported nonsense variant. Exhaustive molecular dynamics simulations of the missense variant p.(Thr26Asn) in both its active and inactive states revealed an allosteric structural mechanism that impairs the binding of Mg 2+ , thus decreasing the affinity for GTP. The impaired GTP-GDP exchange ultimately locks Rab-28 in a GDP-bound inactive state. The loss-of-function mutation p.(Thr26Asn) was present in a compound heterozygosis with the nonsense variant p.(Arg137*), which does not cause mRNA-mediated decay, but is rather likely degraded due to its incomplete folding. The frameshift p.(Thr26Valfs4*) and nonsense p.(Leu13*) and p.(Trp107*) variants, if translated, would lack several key structural components necessary for the correct functioning of the encoded protein.

Purpose: 1) To investigate morphologic and histochemical characteristics of an epiretinal fibrosis removed in an Argus II-implanted eye; 2) to evaluate the Argus II function before and after the fibrosis removal, and 3) to compare morphologic and functional data., Methods: Fibrosis, which developed between the Argus II prosthesis and the retina two years after implant, was surgically removed. Its morphologic and histochemical characteristics were evaluated both in light and transmission electron microscopy, with special stains and immunohistochemistry. The Argus II function was evaluated during the follow-up before surgical removal and 1 month later., Results: Fibrosis was successfully removed. It was composed of a fibrotic tissue with spindle cells arranged in nodular aggregates with a symmetric distribution, mixed with an inflammatory infiltrate. Extra- and intracellular, irregular, small iron particles were found and confirmed ultrastructural characterization with degenerative cellular changes. The repositioned Argus II restored, and its function was partially nearly to normal values 1 month after surgery., Conclusion: Fibrosis can develop between the Argus II and the retina with increasing reduced function. Morphologic characteristics of the removed fibrosis suggested a pathogenesis based on an inflammatory process involved in a foreign body reaction with progressing connective tissue deposition leading to sclerosis. Adequate clinical follow-up is critical to successful removal of the fibrosis with reactivation of the Argus II function.

Background: Alström syndrome is a rare recessively inherited disorder caused by variants in the ALMS1 gene. It is characterized by multiple organ dysfunction, including cone-rod retinal dystrophy, dilated cardiomyopathy, hearing loss, obesity, insulin resistance, hyperinsulinemia, type 2 diabetes mellitus and systemic fibrosis. Heterogeneity and age-dependent development of clinical manifestations make it difficult to obtain a clear diagnosis, especially in pediatric patients., Case Presentation: Here we report the case of a girl with Alström syndrome. Genetic examination was proposed at age 22 months when suspected macular degeneration was the only major finding. Next generation sequencing of a panel of genes linked to eye-related pathologies revealed two compound heterozygous variants in the ALMS1 gene. Frameshift variants c.1196&#95;1202del, p.(Thr399Lysfs*11), rs761292021 and c.11310&#95;11313del, (p.Glu3771Trpfs*18), rs747272625 were detected in exons 5 and 16, respectively. Both variants cause frameshifts and generation of a premature stop-codon that probably leads to mRNA nonsense-mediated decay. Validation and segregation of ALMS1 variants were confirmed by Sanger sequencing., Conclusions: Genetic testing makes it possible, even in childhood, to increase the number of correct diagnoses of patients who have ambiguous phenotypes caused by rare genetic variants. The development of high-throughput sequencing technologies offers an exceptionally valuable screening tool for clear genetic diagnoses and ensures early multidisciplinary management and treatment of the emerging symptoms.

Background: Best vitelliform macular dystrophy (BVMD) is an autosomal dominant macular degeneration. The typical central yellowish yolk-like lesion usually appears in childhood and gradually worsens. Most cases are caused by variants in the BEST1 gene which encodes bestrophin-1, an integral membrane protein found primarily in the retinal pigment epithelium., Methods: Here we describe the spectrum of BEST1 variants identified in a cohort of 57 Italian patients analyzed by Sanger sequencing. In 13 cases, the study also included segregation analysis in affected and unaffected relatives. We used molecular mechanics to calculate two quantitative parameters related to calcium-activated chloride channel (CaCC composed of 5 BEST1 subunits) stability and calcium-dependent activation and related them to the potential pathogenicity of individual missense variants detected in the probands., Results: Thirty-six out of 57 probands (63% positivity) and 16 out of 18 relatives proved positive to genetic testing. Family study confirmed the variable penetrance and expressivity of the disease. Six of the 27 genetic variants discovered were novel: p.(Val9Gly), p.(Ser108Arg), p.(Asn179Asp), p.(Trp182Arg), p.(Glu292Gln) and p.(Asn296Lys). All BEST1 variants were assessed in silico for potential pathogenicity. Our computational structural biology approach based on 3D model structure of the CaCC showed that individual amino acid replacements may affect channel shape, stability, activation, gating, selectivity and throughput, and possibly also other features, depending on where the individual mutated amino acid residues are located in the tertiary structure of BEST1. Statistically significant correlations between mean logMAR best-corrected visual acuity (BCVA), age and modulus of computed BEST1 dimerization energies, which reflect variations in the in CaCC stability due to amino acid changes, permitted us to assess the pathogenicity of individual BEST1 variants., Conclusions: Using this computational approach, we designed a method for estimating BCVA progression in patients with BEST1 variants.

Oguchi disease, is a very rare form of night blindness caused by biallelic variations in the SAG or GRK1 genes, both involved in rod restoration after light stimuli. Here we report the clinical and genetic findings of an 8-year old boy with a history of reduced visual acuity, nyctalpia and hemeralopia. Clinical findings, in particular the Mizuo-Nakamura phenomenon, were compatible with a diagnosis of Oguchi disease. Genetic testing revealed a novel missense homozygous variation in the SAG gene. This is the first evidence that the disease can be caused by missense variations in this gene., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Background: Bardet-Biedl syndrome (BBS) is a rare inherited multisystemic disorder with autosomal recessive or complex digenic triallelic inheritance. There is currently no treatment for BBS, but some morbidities can be managed. Accurate molecular diagnosis is often crucial for the definition of appropriate patient management and for the development of a potential personalized therapy., Methods: We developed a next-generation-sequencing (NGS) protocol for the screening of the 18 most frequently mutated genes to define the genotype and clarify the mutation spectrum of a cohort of 20 BBS Italian patients., Results: We defined the causative variants in 60% of patients; four of those are novel. 33% of patients also harboured variants in additional gene/s, suggesting possible oligogenic inheritance. To explore the function of different genes, we looked for correlations between genotype and phenotype in our cohort. Hypogonadism was more frequently detected in patients with variants in BBSome proteins, while renal abnormalities in patients with variations in BBSome chaperonin genes., Conclusions: NGS is a powerful tool that can help understanding BBS patients' phenotype through the identification of mutations that could explain differences in phenotype severity and could provide insights for the development of targeted therapy. Furthermore, our results support the existence of additional BBS loci yet to be identified.

Background: The aim of this study is to analyze and compare the progression of photoreceptor atrophy among siblings affected by retinitis pigmentosa by means of spectral SD-OCT., Methods: Fifty three eyes of 27 patients belonging to 12 family clusters were analyzed. To assess the annual progression rate of photoreceptor atrophy, the ellipsoid zone (EZ) line was measured in OCT sections through the fovea. We used multivariate generalized mixed effects to model the rate of progression and its relation to the initial ellipsoid zone line width., Results: During our 4.84 years (± 1.44) mean follow up time (range 3-7) 53 eyes were examined. The ellipsoid zone line width declined with a yearly average rate of 76.4 μm (4.16% / year) (p-value < 0.0001). Progression rates were poorly correlated within family clusters (p-value = 0.23) and showed statistical difference between affected siblings (p-value = 0.007). There was no correlation between inter-familiar progression rate and mode of inheritance (p-value = 0.98) as well as between age and ellipsoid zone line width among siblings (p-value = 0.91)., Conclusion: RP could be extremely heterogeneous even among siblings: an accurate and sensitive method to follow the progression of the disease is fundamental for future development of clinical trials and therapy strategies.

Obesity phenotype can be manifested as an isolated trait or accompanied by multisystem disorders as part of a syndromic picture. In both situations, same molecular pathways may be involved to different degrees. This evidence is stronger in syndromic obesity, in which phenotypes of different syndromes may overlap. In these cases, genetic testing can unequivocally provide a final diagnosis. Here we describe a patient who met the diagnostic criteria for Alström syndrome only during adolescence. Genetic testing was requested at 25 years of age for a final confirmation of the diagnosis. The genetic diagnosis of Alström syndrome was obtained through a Next Generation Sequencing genetic test approach using a custom-designed gene panel of 47 genes associated with syndromic and non-syndromic obesity. Genetic analysis revealed a novel homozygous frameshift variant p.(Arg1550Lysfs*10) on exon 8 of the ALMS1 gene. This case shows the need for a revision of the diagnostic criteria guidelines, as a consequence of the recent advent of massive parallel sequencing technology. Indications for genetic testing reported in these currently accepted diagnostic criteria for Alström syndrome, were drafted when sequencing was expensive and time consuming. Nowadays, Next Generation Sequencing testing could be considered as first line diagnostic tool not only for Alström syndrome but, more generally, for all those atypical or not clearly distinguishable cases of syndromic obesity, thus avoiding delayed diagnosis and treatments. Early diagnosis permits a better follow-up and pre-symptomatic interventions., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

The main objective of our work is to perform an in depth analysis of the structural features of normal choriocapillaris imaged with OCT Angiography. Specifically, we provide an optimal radius for a circular Region of Interest (ROI) to obtain a stable estimate of the subfoveal choriocapillaris density and characterize its textural properties using Markov Random Fields. On each binarized image of the choriocapillaris OCT Angiography we performed simulated measurements of the subfoveal choriocapillaris densities with circular Regions of Interest (ROIs) of different radii and with small random displacements from the center of the Foveal Avascular Zone (FAZ). We then calculated the variability of the density measure with different ROI radii. We then characterized the textural features of choriocapillaris binary images by estimating the parameters of an Ising model. For each image we calculated the Optimal Radius (OR) as the minimum ROI radius required to obtain a standard deviation in the simulation below 0.01. The density measured with the individual OR was 0.52 ± 0.07 (mean ± STD). Similar density values (0.51 ± 0.07) were obtained using a fixed ROI radius of 450 μm. The Ising model yielded two parameter estimates (β = 0.34 ± 0.03; γ = 0.003 ± 0.012; mean ± STD), characterizing pixel clustering and white pixel density respectively. Using the estimated parameters to synthetize new random textures via simulation we obtained a good reproduction of the original choriocapillaris structural features and density. In conclusion, we developed an extensive characterization of the normal subfoveal choriocapillaris that might be used for flow analysis and applied to the investigation pathological alterations.

Purpose: To assess the correlation between the 5-year risk of developing primary open-angle glaucoma (POAG) [on the basis of the Ocular Hypertension Treatment Study (OHTS)-European Glaucoma Prevention Study (EGPS) model] and optic nerve head and retinal nerve fiber layer (RNFL) indices as evaluated by optical coherence tomography (Stratus OCT), scanning laser polarimetry (GDx ECC), and confocal laser ophthalmoscopy [Heidelberg Retinal Tomograph (HRT) III] in ocular hypertensive (OHT) individuals., Participants and Methods: Sixty-eight OHT individuals with the untreated intraocular pressure in both eyes between 22 and 32 mm Hg, an ophthalmoscopically normal optic disc with a cup/disc ratio asymmetry between the 2 eyes of ≤0.2, and a visual field with glaucoma hemifield test within normal limits, and pattern SD within P<0.05 were recruited. A comprehensive glaucoma workup and examination by means of OCT, GDx ECC, and HRT III were performed in all of the patients. The probability of developing POAG in 5 years was calculated by means of the OHTS-EGPS model (risk calculator score-RS), which has been developed by the OHTS-EGPS Collaborative Study Group by taking into account the age and the mean values of the central corneal thickness, the intraocular pressure, the pattern SD, and the cup/disc ratio collected in the 2 eyes. Correlations between the individual RS and OCT, GDx, and HRT parameters were then assessed by regression analysis (exponential and linear), with a statistically significant level of P<0.01., Results: The RS of the 68 individuals ranged between 1.5% and 28.6%, with a mean of 10.2% (SD 6.4). Only OCT RNFL-related parameters showed a statistically significant correlation with the RS. The average RNFL thickness had an exponential regression r of 0.21 (P<0.0001) and a linear regression r of 0.14 (P=0.0012); the superior average RNFL thickness had an exponential regression r of 0.26 (P<0.0001) and a linear regression r of 0.23 (P<0.0001); the nasal average RNFL thickness had an exponential regression r of only 0.12 (P=0.0031). Neither GDx RNFL-related parameters nor HRT disc-related parameters showed statistically significant correlations with the RC score., Conclusions: This explorative study showed that the individual risk to develop POAG within 5 years in OHT individuals is significantly correlated with OCT RNFL parameters, but not with GDx RNFL parameters or HRT disc parameters. OCT parameters could explain about 20% to 25% of the variability in individual RS. The RS seems to take into account the subclinical RNFL thinning shown by the OCT.

Background: It is known that office-hour measurements might not adequately estimate IOP mean, peaks and fluctuations in healthy subjects. The purpose of the present study is to verify whether office-hour measurements in patients in different body positions can estimate the characteristics of 24-hour intraocular pressure (IOP) in treated POAG patients., Methods: The 24-hour IOP curves of 70 eyes of 70 caucasian patients with treated glaucoma were analyzed. Measurements were taken at 9 AM; 12, 3, 6, and 9 PM; and 12, 3, and 6 AM, both in the supine (TonoPen XL) and sitting (Goldmann tonometer) positions. The ability of five strategies to estimate IOP mean, peak and fluctuation was evaluated. Each method was analyzed both with regression of the estimate error on the real value and with "hit or miss" analysis., Results: The least biased estimate of the Peak IOP was obtained using measurements from both supine and sitting positions, also yielding the highest rate of correct predictions (which was significantly different from 3 of the remaining 4 strategies proposed, p < 0.05). Strategies obtained from the combination of supine, sitting and peak measurements resulted to be least biased for the Mean IOP and the IOP Fluctuation estimate, but all strategies were not found significantly different in terms of correct prediction rate (the only significant difference being between the two strategies based on sitting or supine measurements only, with the former being the one with the highest correct prediction rate)., Conclusions: The results of this study remark the concept that IOP is a dynamic parameter and that intensive measurement is helpful in determining its characteristics. All office-hour strategies showed a very poor performance of in correctly predicting the considered parameters within the thresholds used in this paper, all scoring a correct prediction rate below 52%.