Towards a Long-Read Sequencing Approach for the Molecular Diagnosis of RPGRORF15 Genetic Variants.

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 RPGRORF15, aiding in the correct annotation of genetic variants in online databases.

Pathogenicity of new BEST1 variants identified in Italian patients with best vitelliform macular dystrophy assessed by computational structural biology.

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.

Macular function and morphologic features in juvenile stargardt disease: longitudinal study.

PURPOSE: To evaluate disease progression in a cohort of patients with a clinical and genetic diagnosis of Stargardt disease. DESIGN: Longitudinal cohort study. PARTICIPANTS: A total of 56 selected patients with a clinical and molecular diagnosis of Stargardt disease, an early age of onset, and a median follow-up length of 2 years. METHODS: Patients underwent routine examination, including full-field electroretinography, microperimetry, and optical coherence tomography. MAIN OUTCOME MEASURES: Best-corrected visual acuity (BCVA), mean retinal sensitivity, fixation stability, preferred retinal locus, inner segment/outer segment (IS/OS) junction loss, and atrophic lesion area. RESULTS: A total of 56 patients with a mean age at disease onset of 15.3 years (range, 3-28 years), a mean disease duration of 12.1 years, and a mean age at baseline of 27.4 years were analyzed. The median BCVA was 20/200 in both eyes. Optical coherence tomography parameters (IS/OS alteration and retinal pigment epithelium lesion area) were obtained in only 49 patients because the signal quality was poor in the remaining 7 patients. Optical coherence tomography revealed a mean retinal pigment epithelium lesion area of 2.6 mm(2), preserved foveal IS/OS in 4.1% of patients, loss of foveal IS/OS in 59.2% of patients, and extensive loss of macular IS/OS in 36.7% of patients. Microperimetric findings showed a reduced macular sensitivity (mean, 10 decibels [dB]) and an unstable fixation in half of the patient cohort. The longitudinal analysis showed a significant progressive reduction of BCVA and macular sensitivity (at an estimated rate of 0.04 decimals and 1.19 dB/year, respectively) associated with a significant enlargement of retinal pigment epithelium lesion area (0.282 mm(2)/year). No significant changes in ophthalmoscopic findings and electroretinographic responses were detected. CONCLUSIONS: This study highlights the importance of microperimetry and optical coherence tomography in monitoring patients with Stargardt disease. Quantifying the decline of visual functionality and detecting morphologic macular changes prove useful in evaluating disease progression over a short-term follow-up and should be taken into account for the design of future clinical trials of gene therapy to treat retinal dystrophy.

SWEPT-SOURCE OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY FINDINGS IN A CASE OF PACHYCHOROID NEOVASCULOPATHY IN RETINITIS PIGMENTOSA.

PURPOSE: To report a rare case of pachychoroid neovasculopathy in retinitis pigmentosa (RP) and to describe its features and changes after treatment with bevacizumab as documented by swept-source optical coherence angiography. METHODS: A case report of a 69-year-old man with a history of RP who presented at our clinic with an acute decrease in visual acuity (Best corrected visual acuity) in his left eye. RESULTS: Upon the first examination, best-corrected visual acuity in the left eye was 20/50. Fundus examination and fundus autofluorescence showed bilaterally typical features of RP, and enhanced depth imaging-optical coherence tomography revealed a pachychoroid pattern. Fluorescein angiography and indocyanine green angiography suggested the presence of a choroidal neovascularization in the left eye, although no clear evidence of any neovascular network could be identified. Swept-source optical coherence angiography was performed, confirming the presence of a choroidal neovascularization network. The patient was treated with 2 monthly intravitreal injections of bevacizumab. After treatment, BVCA improved to 20/25, and no evidence of the vascular network was detectable on swept-source optical coherence angiography. CONCLUSION: Our case reports the uncommon association of RP with pachychoroid neovasculopathy. Swept-source optical coherence angiography proved to be a useful imaging technique for the diagnosis and follow-up of this condition. Intravitreal bevacizumab represents an effective treatment for choroidal neovascularization associated with RP.

Intravitreal Injections for Macular Edema Secondary to Retinal Vein Occlusion: Long-Term Functional and Anatomic Outcomes.

PURPOSE: To report the long-term visual and anatomic outcomes of intravitreal injections for macular edema (ME) secondary to retinal vein occlusion (RVO) in a real-life clinical setting. DESIGN: Retrospective interventional case series. METHODS: A total of 223 consecutive eyes with ME secondary to RVO, treated with the first three intravitreal Ranibizumab or dexamethasone injections between August 2008 and September 2018, were enrolled in the study. Subsequent retreatment was guided by best-corrected visual acuity (BCVA) and central macular thickness (CMT) measurements, aimed at achieving macular fluid regression and BCVA stability. BCVA and CMT were recorded at baseline and at subsequent annual time points. The mean number of injections administered each year and the incidence of adverse events were recorded. RESULTS: The mean BCVA and CMT at baseline were 0.79 logMar (SD 0.71) and 615.7 µm (SD 257.5), respectively. The mean follow-up (FU) period was 47.8 months (min 12-max 120). At 12 months, the mean BCVA and CMT had significantly improved to 0.62 logMar (SD 0.68; p < 0.0001) and 401.04 µm (SD 257.5), respectively. The mean follow-up (FU) period was 47.8 months (min 12-max 120). At 12 months, the mean BCVA and CMT had significantly improved to 0.62 logMar (SD 0.68; p < 0.0001) and 401.04 . CONCLUSION: Intravitreal Ranibizumab and/or dexamethasone injections were found to be effective at inducing a long-lasting improvement of BCVA and CMT in a real-life clinical setting. A safety profile similar to that already well-established in Ranibizumab and dexamethasone treatment was observed, as well as a steady decrease in the number of intraocular injections required. The results support intravitreal treatments for BRVO and CRVO in patient populations with similar characteristics in similar settings.

Anti-Vascular Endothelial Growth Factor Treatment for Diabetic Macular Edema in a Real-World Clinical Setting.

PURPOSE: To report the long-term outcomes of intravitreal anti-vascular endothelial growth factor (VEGF) treatment for diabetic macular edema (DME) in a real-world clinical setting and to assess the efficacy of subsequent alternative treatments in eyes with suboptimal response to anti-VEGF. DESIGN: Retrospective interventional case series. METHODS: The medical records of consecutive eyes with center-involving DME, treated between August 2008 and June 2015 with 3 monthly intravitreal anti-VEGF injections-with or without prompt or deferred laser-followed by pro re nata re-treatment, were reviewed. A subgroup of eyes that were unresponsive to the treatment received subsequent alternative therapeutic options, including switching to another anti-VEGF drug, intravitreal injection of dexamethasone, and vitrectomy. RESULTS: A total of 170 eyes of 129 patients were included in the study. The mean follow-up (FU) was 45.6 months (SD 18; minimum 12-maximum 81). The change in mean best-corrected visual acuity (BCVA) at 1-year FU was +5 ETDRS letters (P < .0001). Improvement in BCVA was statistically significant up to 5 years. Improvement in central macular thickness (CMT) was statistically significant up to the last FU visit. In eyes with suboptimal response, no significant visual improvement was found by switching to another anti-VEGF (P =.4347). Twenty-four eyes treated with intravitreal dexamethasone and 14 with vitrectomy exhibited a significant reduction in CMT with variable functional responses. In these eyes, better BCVA gain was found in cases with an early change of the treatment strategy. CONCLUSION: The results support treatment with intravitreal anti-VEGF for DME in real-world clinical settings and suggest that an early change of the therapeutic strategy should be considered for eyes unresponsive to the treatment.

Clinical Characterization of CNGB1-Related Autosomal Recessive Retinitis Pigmentosa.

IMPORTANCE: There are limited published data on the phenotype of retinitis pigmentosa (RP) related to CNGB1 variants. These data are needed both for prognostic counseling of patients and for understanding potential treatment windows. OBJECTIVE: To describe the detailed clinical and molecular genetic findings in a series of patients with RP with likely pathogenic variants in CNGB1. DESIGN, SETTING, AND PARTICIPANTS: In this case series, 10 patients from 9 families underwent full ophthalmologic examination. Molecular investigations included whole-exome analysis in 6 patients. The study was conducted from April 17, 2013, to March 3, 2016, with final follow-up completed on March 2, 2016, and data were analyzed from October 27, 2014, to March 29, 2016. MAIN OUTCOMES AND MEASURES: Results of ophthalmologic examination and molecular genetic analysis of CNGB1. RESULTS: In this case series, 7 women and 3 men from 9 families with a mean (SD) age of 47.4 (13.2) years identified as having CNGB1 variants were included in this study; there was a mean (SD) follow-up length of 3.7 (2.8) years. The first clinical presentation was with nyctalopia in childhood with visual field loss documented later at a mean (SD) age of 33.2 (8.0) years. All patients had preserved best-corrected visual acuity into adulthood, with a mean of 0.1 logMAR (Snellen equivalent, 20/25) in each eye (logMAR range, 0.0 to 0.3 [Snellen 20/20 to 20/40] in the right eye and -0.1 to 0.3 [Snellen 20/16 to 20/40] in the left eye). Fundus examination revealed midperipheral retinal pigment epithelial atrophy and intraretinal pigment migration. Optical coherence tomography of the macula demonstrated complete preservation of the inner segment ellipsoid band in 1 patient, with variable lateral extent in the other patients corresponding to the diameter of a paracentral ring of increased fundus autofluorescence. Electrophysiologic testing in 6 patients confirmed a rod-cone dystrophy phenotype. Molecular investigations identified a previously reported missense variant (p.[N986I]) and 7 variants not previously reported in disease including 4 nonsense (p.[(Q88*], p.[Q222*], p.[Q318*], and p.[R729*]), 2 frameshift (p.[A1048fs*13], p.[L849Afs*3]), and a splice site variant (c.761 + 2T>A). CONCLUSIONS AND RELEVANCE: The data from this study suggest that visual acuity and foveal structure in patients with RP are preserved into adult life such that a lengthy window of opportunity should exist for intervention with novel therapies.

Photorefractive keratectomy on purely refractive accommodative esotropia.

CONTEXT: Results on refractive keratectomy of the treatment of young patients with purely refractive accommodative esotropia. AIMS: Evaluating the results of refractive keratectomy (PRK) on the treatment of young patients with purely refractive accommodative esotropia. METHODS AND MATERIAL: This prospective study comprised patients with purely accommodative hyperopic esotropia. Patients underwent a complete ophthalmologic examination that included pre-operative and post-operative best corrected visual acuity (BCVA) and uncorrected visual acuity (UCVA) (at 3, 12, and 24 months), alignment and sensory outcomes; keratometry, pachymetry, and corneal topography. STATISTICAL ANALYSIS USED: paired T-student. RESULTS: Twenty eyes of 10 patients (mean age 21.5 years) were treated. The pre-operative mean UCVA was 0.70 logMAR (SD ± 0.177) and 0.02 logMAR (SD ± 0.029) two years later. The pre-operative BCVA was 0.02 logMAR (SD ± 0.037) and 0.01 (SD ± 0.026) two years later. The mean spherical equivalent (SE) in cyclopegia was + 3.92 D (range: +2.75 to +5.00 D; SD = 0.62) pre-operatively, -0.69 D (range -0.5 to -1) at three months and 0 D (range: -0.25 to 0.25) at one and two years later. After surgery, all patients were orthophoric without correction and stereopsis was unaffected by PRK. CONCLUSIONS: A two-year follow-up showed that photorefractive keratectomy was an effective treatment for esotropia associated with mild to moderate hyperopia in young adults with purely refractive accommodative esotropia.

Automatic prediction of cardiovascular and cerebrovascular events using heart rate variability analysis.

BACKGROUND: There is consensus that Heart Rate Variability is associated with the risk of vascular events. However, Heart Rate Variability predictive value for vascular events is not completely clear. The aim of this study is to develop novel predictive models based on data-mining algorithms to provide an automatic risk stratification tool for hypertensive patients. METHODS: A database of 139 Holter recordings with clinical data of hypertensive patients followed up for at least 12 months were collected ad hoc. Subjects who experienced a vascular event (i.e., myocardial infarction, stroke, syncopal event) were considered as high-risk subjects. Several data-mining algorithms (such as support vector machine, tree-based classifier, artificial neural network) were used to develop automatic classifiers and their accuracy was tested by assessing the receiver-operator characteristics curve. Moreover, we tested the echographic parameters, which have been showed as powerful predictors of future vascular events. RESULTS: The best predictive model was based on random forest and enabled to identify high-risk hypertensive patients with sensitivity and specificity rates of 71.4% and 87.8%, respectively. The Heart Rate Variability based classifier showed higher predictive values than the conventional echographic parameters, which are considered as significant cardiovascular risk factors. CONCLUSIONS: Combination of Heart Rate Variability measures, analyzed with data-mining algorithm, could be a reliable tool for identifying hypertensive patients at high risk to develop future vascular events.

Subretinal Fibrosis in Stargardt's Disease with Fundus Flavimaculatus and ABCA4 Gene Mutation.

PURPOSE: To report on 4 patients affected by Stargardt's disease (STGD) with fundus flavimaculatus (FFM) and ABCA4 gene mutation associated with subretinal fibrosis. METHODS: Four patients with a diagnosis of STGD were clinically examined. All 4 cases underwent a full ophthalmologic evaluation, including best-corrected visual acuity measured by the Snellen visual chart, biomicroscopic examination, fundus examination, fundus photography, electroretinogram, microperimetry, optical coherence tomography and fundus autofluorescence. All patients were subsequently screened for ABCA4 gene mutations, identified by microarray genotyping and confirmed by conventional DNA sequencing of the relevant exons. RESULTS: In all 4 patients, ophthalmologic exam showed areas of subretinal fibrosis in different retinal sectors. In only 1 case, these lesions were correlated to an ocular trauma as confirmed by biomicroscopic examination of the anterior segment that showed a nuclear cataract dislocated to the superior site and vitreous opacities along the lens capsule. The other patients reported a lifestyle characterized by competitive sport activities. The performed instrumental diagnostic investigations confirmed the diagnosis of STGD with FFM in all patients. Moreover, in all 4 affected individuals, mutations in the ABCA4 gene were found. CONCLUSIONS: Patients with the diagnosis of STGD associated with FFM can show atypical fundus findings. We report on 4 patients affected by STGD with ABCA4 gene mutation associated with subretinal fibrosis. Our findings suggest that this phenomenon can be accelerated by ocular trauma and also by ocular microtrauma caused by sport activities, highlighting that lifestyle can play a role in the onset of these lesions.