Detection of elusive DNA copy-number variations in hereditary disease and cancer through the use of noncoding and off-target sequencing reads.

Copy-number variants (CNVs) play a substantial role in the molecular pathogenesis of hereditary disease and cancer, as well as in normal human interindividual variation. However, they are still rather difficult to identify in mainstream sequencing projects, especially involving exome sequencing, because they often occur in DNA regions that are not targeted for analysis. To overcome this problem, we developed OFF-PEAK, a user-friendly CNV detection tool that builds on a denoising approach and the use of "off-target" DNA reads, which are usually discarded by sequencing pipelines. We benchmarked OFF-PEAK on data from targeted sequencing of 96 cancer samples, as well as 130 exomes of individuals with inherited retinal disease from three different populations. For both sets of data, OFF-PEAK demonstrated excellent performance (>95% sensitivity and >80% specificity vs. experimental validation) in detecting CNVs from in silico data alone, indicating its immediate applicability to molecular diagnosis and genetic research.

Autologous internal limiting membrane transplantation achieves anatomic closure and functional improvement in the treatment of large, persistent macular holes.

PURPOSE: To evaluate the clinical outcome of subretinal autologous internal limiting membrane (ILM) transplantation during pars-plana vitrectomy for persistent full-thickness macular hole (FTMH) repair. METHODS: Retrospective, consecutive case series of 13 eyes (13 patients) undergoing small-incision vitrectomy with ILM transplantation and air tamponade for large persistent FTMH after prior unsuccessful vitrectomy with posterior hyaloid detachment and ILM peeling. MAIN OUTCOME MEASUREMENTS: For all eyes, high-definition spectral domain optical coherence tomography scans (SD-OCT Spectralis, Heidelberg Engineering GmbH, Germany) of the macula were routinely performed before surgery, 1 and 4 weeks after surgery, and at the final follow-up visit. Additionally, age, gender, axial length, macular hole diameter, biomicroscopic fundus evaluation and best-corrected visual acuity (BCVA) at baseline, 1 and 4 weeks after surgery, and at the final follow-up visit were analyzed. RESULTS: Anatomic closure was achieved in all 13 cases (100% success rate). Closure pattern was classified in accordance with to Rossi et al. (Graefe's Arch Clin Exp Ophthalmol 258(12):2629-2638, 2020). Mean baseline BCVA logMAR was 0.93, mean postoperative BCVA logMAR was 0.66 with a mean postoperative follow-up period of 11.4 months. No re-opening occurred during the observation period. CONCLUSIONS: Placing an autologous ILM-transplant in the subretinal space beneath the margin of the FTMH can support anatomic restauration and functional improvement in large, persistent FTMHs.

Relaxing radial retinal incisions for the treatment of persistent macular holes.

PURPOSE: Various surgical techniques have been described for managing persistent macular holes after an unsuccessful vitrectomy with internal limiting membrane (ILM) peeling. However, the closure and functional improvement rates after these procedures are limited. Therefore, the aim of this study was to evaluate the usefulness of radial retinal incisions (retinotomies) in eyes with persistent large macula holes despite previous vitrectomy with ILM peeling. DESIGN: In a retrospective case series, closure rate and best-corrected visual acuity (BCVA) were evaluated in eyes with persistent macular holes after an unsuccessful vitrectomy that included posterior vitreous detachment and ILM peeling. SUBJECTS: 22 eyes of 22 patients (10 men and 12 women) underwent re-vitrectomy with radial retinal incisions. All the patients had undergone an unsuccessful surgery before. METHODS: Small-incision re-vitrectomy with radial retinal incisions (retinotomies) and air tamponade was performed. MAIN OUTCOME MEASUREMENTS: For all eyes, high-definition SD-OCT scans (SD-OCT Spectralis, Heidelberg Engineering GmbH, Germany) of the macula were routinely performed before surgery; 1 week and 1 month after surgery; and at final follow-up. Additionally, age, gender, axial length, macular hole diameter, biomicroscopic fundus evaluation and best-corrected visual acuity (BCVA) in logMAR and Snellen at baseline, 1 and 4 months after operation, and at the final follow-up visit were analyzed. RESULTS: The mean baseline macular hole diameter was 668.5 ± 226.8 µm. At the final examination, 16 (72.72%) of the 22 macula holes were closed. Visual acuity increased in 17 eyes, was stable in 3 eyes, and decreased in 2 eyes owing to central retinal atrophy in both. The mean BCVA increased from logMAR 1.04 ± 0.29 at baseline to 0.57 ± 0.31 (Snellen 0.11 ± 0.05 to 0.33 ± 0.18). In all successful cases, macula hole closure was attained after 3 days, and none of the eyes showed macula hole recurrence. CONCLUSION: The results of this limited case series suggest that radial retinal incisions of the rim in persistent macula holes after initial surgery with ILM peeling increase the success rate of macula hole closure and results in a relevant increase in BCVA. However, as the number of eyes included in this series is limited, the results must be confirmed in a study with a larger sample size.

Human selection bias drives the linear nature of the more ground truth effect in explainable deep learning optical coherence tomography image segmentation.

Supervised deep learning (DL) algorithms are highly dependent on training data for which human graders are assigned, for example, for optical coherence tomography (OCT) image annotation. Despite the tremendous success of DL, due to human judgment, these ground truth labels can be inaccurate and/or ambiguous and cause a human selection bias. We therefore investigated the impact of the size of the ground truth and variable numbers of graders on the predictive performance of the same DL architecture and repeated each experiment three times. The largest training dataset delivered a prediction performance close to that of human experts. All DL systems utilized were highly consistent. Nevertheless, the DL under-performers could not achieve any further autonomous improvement even after repeated training. Furthermore, a quantifiable linear relationship between ground truth ambiguity and the beneficial effect of having a larger amount of ground truth data was detected and marked as the more-ground-truth effect.

Endpoints for clinical trials in ophthalmology.

With the identification of novel targets, the number of interventional clinical trials in ophthalmology has increased. Visual acuity has for a long time been considered the gold standard endpoint for clinical trials, but in the recent years it became evident that other endpoints are required for many indications including geographic atrophy and inherited retinal disease. In glaucoma the currently available drugs were approved based on their IOP lowering capacity. Some recent findings do, however, indicate that at the same level of IOP reduction, not all drugs have the same effect on visual field progression. For neuroprotection trials in glaucoma, novel surrogate endpoints are required, which may either include functional or structural parameters or a combination of both. A number of potential surrogate endpoints for ophthalmology clinical trials have been identified, but their validation is complicated and requires solid scientific evidence. In this article we summarize candidates for clinical endpoints in ophthalmology with a focus on retinal disease and glaucoma. Functional and structural biomarkers, as well as quality of life measures are discussed, and their potential to serve as endpoints in pivotal trials is critically evaluated.

Multidisciplinary team directed analysis of whole genome sequencing reveals pathogenic non-coding variants in molecularly undiagnosed inherited retinal dystrophies.

The purpose of this paper is to identify likely pathogenic non-coding variants in inherited retinal dystrophy (IRD) genes, using genome sequencing (GS). Patients with IRD were recruited to the study and underwent comprehensive ophthalmological evaluation and GS. The results of GS were investigated through virtual gene panel analysis, and plausible pathogenic variants and clinical phenotype evaluated by the multidisciplinary team (MDT) discussion. For unsolved patients in whom a specific gene was suspected to harbor a missed pathogenic variant, targeted re-analysis of non-coding regions was performed on GS data. Candidate variants were functionally tested by messenger RNA analysis, minigene or luciferase reporter assays. Previously unreported, likely pathogenic, non-coding variants in 7 genes (PRPF31, NDP, IFT140, CRB1, USH2A, BBS10 and GUCY2D), were identified in 11 patients. These were shown to lead to mis-splicing (PRPF31, IFT140, CRB1 and USH2A) or altered transcription levels (BBS10 and GUCY2D). MDT-led, phenotype-driven, non-coding variant re-analysis of GS is effective in identifying the missing causative alleles.

Baseline Levels of Retinol-Binding Protein 4 and Vitamin A in Healthy Subjects, Stargardt Disease, and Geographic Atrophy Patients.

INTRODUCTION: The accumulation of lipofuscin is a hallmark in the pathogenesis of Stargardt disease type 1 (STGD1) and geographic atrophy (GA) secondary to age-related macular degeneration. Limiting lipofuscin accumulation by inhibiting the retinol-binding protein 4 (RBP4) is being explored as a potential treatment target for those diseases. In this study, we aimed to establish the concentration of RBP4 in the systemic circulation in different age cohorts of healthy individuals and to check if patients with STGD1 or GA may show abnormal RBP4 levels. METHODS: Forty healthy subjects of various age-groups, 15 Stargardt patients, and 15 GA patients were included in the study. We measured RBP4 levels, serum retinol (SR) levels, complete blood count, and blood chemistry including liver function tests. RESULTS: Mean RBP4 for all cohorts was 26,911.40 ± 6,198.61 ng/mL, and mean SR 1.75 ± 0.36 µmol/L. Age was not found to significantly impact levels neither of RBP4 and SR nor of the RBP4-to-SR ratio. Also, the 2 patient groups showed similar blood levels to their age-matched controls. CONCLUSION: Serum RBP4 and SR do not appear to be affected by age in healthy individuals and remain within normal limits in both STGD1 and GA.

c.-61G>A in OVOL2 is a Pathogenic 5' Untranslated Region Variant Causing Posterior Polymorphous Corneal Dystrophy 1.

PURPOSE: The purpose of this study was to investigate the clinical and genetic features of a man and his daughter with posterior polymorphous corneal dystrophy (PPCD), referred to our clinic for Descemet membrane endothelial keratoplasty. No other known relatives were affected. METHODS: Ophthalmic examination and histology, including electron microscopy, were performed. Genetic testing was conducted by means of whole exome sequencing, and variant analysis was achieved by using an internal in silico pipeline. Molecular tests included a dual-luciferase assay. RESULTS: Slowly progressive blurred vision was reported from childhood by the daughter. The father's symptoms started at age 55. Best-corrected visual acuity was reduced in both patients (0.2-0.4). Slit-lamp examination in both patients revealed bilateral corneal clouding with gray endothelial lesions; other family members had no ophthalmological signs. Descemet membrane endothelial keratoplasty was performed uneventfully in both patients. Histology showed thickened Descemet membrane and abnormal endothelium resembling epithelial-like cells. Both patients carried the OVOL2 5' untranslated region NM_021220.4.c.-61G>A variant in the heterozygous state. This change was associated with increased promoter activity and was not present in the unaffected members of the family. CONCLUSIONS: The 5' untranslated region mutation c.-61G>A in OVOL2 has been previously found in 1 individual with PPCD1 and reported as a variant of unknown significance because of insufficient evidence supporting its pathogenicity. Identification of the second family with 2 individuals affected by PPCD1 carrying this change, together with functional data, provides further proofs that it is disease-causing.

The Progression of Stargardt Disease Using Volumetric Hill of Vision Analyses Over 24 Months: ProgStar Report No.15.

PURPOSE: To report the yearly rate of change in macular function in patients with Stargardt disease type 1 (STGD1) over 24 months and to establish a new volumetric visual function index for use in clinical trials investigating the efficacy on retinal sensitivity. METHODS: Design: International, multicenter, prospective cohort study with 5 study visits every 6 months over 24 months. PARTICIPANTS: A total of 233 individuals with genetically confirmed STGD1 (≥1 disease-causing ABCA4 variant). MAIN OUTCOME MEASURES: The total volume (VTOT) beneath the sensitivity surface of a 3-D model of the hill of vision and mean sensitivity (MS) derived from mesopic microperimetry performed with a white stimulus. Changes of VTOT over time and its correlation with the ABCA4 genotype and baseline features. RESULTS: At baseline, 440 eyes (233 patients) with a mean (SD) age of 33.7 (15.0) years, mean (SD) visual acuity of 46.08 (16.03) ETDRS letters were analyzed with an average VTOT of 0.91 decibel-steradian (dB-sr) and an MS of 10.73 dB. The overall mean rate of decrease in sensitivity [95% confidence interval] was 0.077 [0.064, 0.090] dB-sr/y for VTOT and 0.87 [0.72, 1.02] dB/year for MS. The progression rate of VTOT depended on baseline visual function (0.029 dB-sr/year for low and 0.120 dB-sr/year for high baseline VTOT; P < .001) and exhibited a difference in the first vs second year of follow-up (0.065 dB-sr/year vs 0.089 dB-sr/year, respectively; P < .001). The absence of pigmentary abnormalities of the retinal pigment epithelium at baseline was found to be associated with a faster progression rate (P < .001), whereas a significant association with the genotype was not detected (P = .7). CONCLUSION: In STGD1, both microperimetric outcomes demonstrate statistically significant and clinically meaningful changes after relatively short follow-up periods. Volumetric modeling may be useful in future interventional clinical trials that aim to improve retinal sensitivity or to slow down its decline and for structure-function correlations.

Strabismus Surgery of Consecutive Exotropia.

PURPOSE: The purpose of this study was to determine the surgical outcome, dose-effect (DE), and degree of binocularity in patients undergoing surgery for consecutive exotropia following initial surgery of esotropia. PATIENTS/METHODS: Twenty-one patients were identified. We analyzed the mean angle of deviation pre- and postoperatively as measured with the alternate prism cover test, DE, and binocularity. RESULTS: All patients had had previous strabismus surgery. The surgery for consecutive exotropia had been performed at a mean age of 35.92 ± 18.26 years. In 19 of these patients, surgery of consecutive exotropia involved at least one previously operated extraocular muscle, and the mean interval to the previous surgery was 25.67 ± 16.14 years. The mean angle of deviation (DE) at distance and in the primary position was - 33.43 ± 12.75 prism diopters (PD) preoperatively, + 0.76 ± 7.91 PD 1 week after surgery, and - 7.24 ± 12.14 PD 3 months after surgery. The mean DE was 3.58 ± 1.53 mm/PD at 1 week and 2.70 ± 1.78 mm/PD at 3 months post-surgery. Postoperatively, 62% patients had a binocularity of at least Bagolini positive, 33% had either a positive TNO or Titmus Test, and 24% were Lang I positive (550″). CONCLUSION: Performing strabismus surgery with consecutive exotropia results in restoration of some binocularity in a large number of patients, even in adults, and should be considered as a possibility. The dose-effect is comparable to conventional surgery of exotropia.