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.

Cataract progression following lens-sparing pars plana vitrectomy for rhegmatogenous retinal detachment.

Lens-sparing pars plana vitrectomy (PPV) is often followed by cataract development. However, there have been few prospective studies evaluating the timing of cataract progression and potential associated factors. This was an observational study conducted at the Ophthalmology Unit of the University Hospital of Parma (Parma, Italy). Patients presenting with rhegmatogenous retinal detachment (RRD), who underwent PPV with preservation of the lens, were examined according to a scheduled follow-up (3, 6 and 12 months after PPV) and then preoperatively when cataract extraction surgery (CES) was indicated, or at the end of the study follow-up period (May 2021). The primary outcome was the interval between PPV and CES indication (based on predefined refractive criteria). A total of 36 eyes of 36 patients (mean age: 52 ± 10 years) were included in the study. Nineteen eyes (53%) were indicated for CES a median of 14.5 months (IQR: 12.0-24.8) after PPV. The nuclear and posterior subcapsular forms of cataract progressed significantly starting at 6 months after PPV. Older age at the time of PPV, silicone oil tamponade and RRD without macular involvement were significantly and independently associated with an earlier indication for CES. Patient age and the use of silicone oil tamponade must be taken into consideration when evaluating the risk of cataract development after PPV.

Tilt and decentration of posterior and anterior iris-claw intraocular lenses: a pilot study using anterior segment optical coherence tomography.

BACKGROUND: Information on the centration and tilt of iris-claw intraocular lenses (IC-IOLs) is limited. In this study, we tested the capacity of an anterior segment optical coherence tomography (AS-OCT) instrument to measure decentration and tilt of anterior and posterior IC-IOLs through an integrated software. METHODS: The present observational, cross-sectional study was conducted at University Eye Clinic of Parma (Parma, Italy). The CASIA2 swept-source AS-OCT (Tomey Corp.) was used to measure the tilt and decentration of posterior and anterior IC-IOLs in patients implanted at least 6 months in advance. After failure with full-automation, semi-manual IOL tracing was applied. In-the-bag (IB) contralateral IOLs, when present, were measured automatically. The Bland-Altman method was used to evaluate the agreement between repeated measurements (2 images for each study eye). The amount and direction of tilt and decentration were recorded and plotted into polar charts for evaluation. RESULTS: A total of 21 patients were included: 14 with posterior and 7 with anterior IC-IOL fixation. In 17 eyes (81%), the AS-OCT provided a repeatable measurement of tilt and decentration. All contralateral eyes with IB IOL were automatically measured. The median decentration was 0.67 mm, 0.24 mm, and 0.24 mm in posterior IC-IOLs, anterior IC-IOLs, and IB IOLs group, respectively. The median tilt was 5.0°, 5.6°, and 5.6° for posterior IC-IOLs, anterior IC-IOLs, and IB IOLs, respectively. Tilt direction was mainly temporal, while decentration was inferior-temporal with posterior IC-IOLs and scattered with anterior IC-IOLs and IB IOLs. CONCLUSIONS: The semi-manual tracing function of the CASIA2 AS-OCT provides repeatable and affordable measurements of the decentration and tilt of IC-IOLs in both the anterior and posterior chamber. Data from the former group were similar to the IB group.

Short-term changes in retinal and choroidal relative flow volume after anti-VEGF treatment for neovascular age-related macular degeneration.

The effects of anti-vascular endothelial growth factor (anti-VEGF) agents on the native ocular vasculature are poorly understood. This pilot study aimed to assess short-term changes in retinal and choroidal perfusion after anti-VEGF treatment for neovascular exudative age-related macular degeneration (nAMD) using the relative flow volume (RFV) parameter derived from laser speckle flowgraphy. Ten treatment-naïve nAMD patients underwent measurements of mean, maximum, minimum, and differential RFV within a retinal arteriolar segment and a choroidal vessel segment outside the neovascular area. Measurement of retinal RFV (rRFV), choroidal RFV (cRFV), and subfoveal choroidal thickness (SCT) was repeated 9 and 35 days after a single anti-VEGF injection. The treatment caused a statistically significant decrease in the mean rRFV, mean cRFV, and SCT during the follow-up (p < 0.05). At the intermediate visit, the mean cRFV and SCT were - 17.6% and - 6.4% compared to baseline, respectively. However, at the final measurement, the mean cRFV was not different from the baseline value, which indicated waning of the anti-VEGF effect. In conclusion, a single anti-VEGF injection in treatment-naïve nAMD resulted in a decrease in retinal arteriolar and choroidal perfusion, according to the RFV parameter, which is a promising tool to simultaneously assess retinal and choroidal perfusion changes in response to anti-VEGF therapy.

Parsplana vitrectomy alone versus parsplana vitrectomy combined with phacoemulsification for the treatment of rhegmatogenous retinal detachment: a randomized study.

BACKGROUND: To compare parsplana vitrectomy (PPV) with and without phacoemulsification to treat rhegmatogenous retinal detachment (RRD). METHODS: Subjects aged 48-65 years with RRD in a phakic eye due to superior retinal tears with an overall extension of retinal breaks < 90° underwent to PPV alone (group A); or PPV plus phacoemulsification (phacovitrectomy, PCV, group B). Post-operative follow-up visits occurred at 1 week, 1 month (m1), 3 months (m3), and 6 months (m6) after surgery. The main outcome was the rate of retinal reattachment. Secondary outcomes included best-corrected visual acuity (BCVA), intraocular pressure (IOP), central macular thickness (CMT), and cataract progression (in the lens-sparing [PPV-alone] group). RESULTS: In this initial phase of the study a total of 59 patients (mean age: 55 years, 59 eyes) were enrolled: 29 eyes in group A and 30 eyes in group B. Both groups had similar gas tamponade. During the follow-up there were three cases of RRD recurrence in group A and one in group B. The relative risk of recurrence in group A was 3.22 times higher but the difference was not significant (p = 0.3). The two groups were also similar in terms of BCVA and IOP variation. At m3, CMT was significantly higher in group B (p = 0.014). In group A, cataract progression was significant at m6 (p = 0.003). CONCLUSIONS: In a cohort of RRD patients selected according to their preoperative clinical characteristics, PPV was comparable to PCV in terms of the rate of retinal reattachment after 6 months. TRIAL REGISTRATION: ISRCTN15940019 . Date registered: 15/01/2021 (retrospectively registered).

Assessment of Choroidal Neovascularization Perfusion: A Pilot Study With Laser Speckle Flowgraphy.

Purpose: The purpose of this study was to quantify perfusion in the area of choroidal neovascularization (CNV) using laser speckle flowgraphy (LSFG) before and after intravitreal anti-vascular endothelial growth factor (VEGF) injection. Methods: Retrospective case series. Fifteen eyes of 15 patients with treatment-naïve CNV due to age-related macular degeneration (AMD) and with available LSFG images were included. The main outcome was the mean blur rate (MBR) quantified as a measure of perfusion within the CNV area observed on indocyanine green angiography. Twelve patients had available longitudinal data until one month after the injection, used to evaluate changes in perfusion, central macular thickness (CMT), visual acuity, and ocular perfusion pressure. Reproducibility of LSFG measurements was investigated at each time point from two images taken within five minutes. Results: Intraclass correlation coefficients for LSFG measurements were higher than 0.8 indicating excellent reproducibility. There was a significant decrease in perfusion after one week (-26.4 ± 14.4%; P = 0.027), whereas, after one month, perfusion was no longer significantly different from baseline (P = 0.121). CMT showed a progressive decrease over the follow-up period. Changes in perfusion were strongly correlated with changes in CMT after one week, but not thereafter. Conclusions: This pilot study suggests a method to select a region in the CNV area to quantify perfusion using LSFG. MBR could represent a parameter possibly related to regrowth of the CNV after anti-VEGF treatment. Large-scale studies are needed to assess the usefulness of LSFG in defining re-treatment criteria for neovascular AMD. Translational Relevance: LSFG technology to quantify perfusion could provide useful biomarkers for therapeutic management of CNV.

A case of morgagnian cataract mimicking an iris tumor.

PURPOSE: To report, with the aid of original imaging, an unusual differential diagnosis of an iris tumor. OBSERVATIONS: A 60-year-old Caucasian man visited our clinic with a large amelanotic iris mass in the left eye in the absence of concomitant extraocular inflammation or neoplastic evidence. The patient reported an ocular trauma caused by a wire 5 years prior to his visit, which resolved after a short course of antibiotic eye drops. Orbital magnetic resonance imaging with contrast enhancement was consistent with an iris tumor; ultrasound biomicroscopy indicated a semi-solid, mid-stromal iris formation continuous with the lens. Surgical inspection was performed. Surgery showed the presence of a hypermature cataract with a fine break in the anterior capsule of the lens covered by the iris surface. The liquefied cortex infiltrated the iris without diffusing into the aqueous humor. CONCLUSIONS AND IMPORTANCE: A so-called morgagnian cataract developed, likely following a penetrating ocular wound. The progressive, slow infiltration of the iris stroma by the crystalline matrix mimicked the appearance of an amelanotic iris tumor.

Comparative Analysis of the Safety and Functional Outcomes of Anterior versus Retropupillary Iris-Claw IOL Fixation.

PURPOSE: To compare the functional and clinical outcomes of the iris-claw intraocular lens (IOL) placed on the anterior versus posterior surface of the iris. PATIENTS AND METHODS: A multicenter, retrospective study. Data on eyes that underwent anterior or retropupillary iris-claw IOL implantation because of inadequate capsular support secondary to complicated cataract surgery, trauma, and dislocated/opacified IOLs since January 2015 were analyzed. For study inclusion, evaluation results had to be available in the medical records both preoperatively and at 1 and 12 months after implantation. The following parameters were compared between the groups: best-corrected distance visual acuity (BCDVA), spherical and cylindrical refractive error, endothelial cell density (ECD), central macular thickness (CMT), and percentage and type of postoperative complications. RESULTS: In total, 60 eyes of 60 patients aged 73 ± 13 years were included: 28 eyes (47%) involved anterior, and 32 eyes (53%) retropupillary, iris-claw IOL fixations. Preoperatively, the groups were similar in all parameters except for a significantly higher proportion of retropupillary fixations in patients who had previously experienced a closed-globe trauma (p=0.03). The groups showed comparable improvements in BCDVA after surgery (final BCDVA: 0.34 ± 0.45 vs. 0.37 ± 0.50 logMAR in the anterior and retropupillary placement groups, respectively). During follow-up, no group difference was observed in refractive error or CMT. Both groups experienced similarly marked ECD loss and showed similar incidence of postoperative complications, with cystoid macular edema being the most common complication. Multivariable linear regression showed that BCDVA at 1 month was the best predictor of the final BCDVA. CONCLUSIONS: Anterior chamber and posterior chamber iris-claw IOL fixations proved equally effective and safe for aphakic correction in eyes with inadequate capsular support.

Assessment of choroidal blood flow using laser speckle flowgraphy.

BACKGROUND/AIMS: There is considerable interest in novel techniques to quantify choroidal blood flow (CBF) in humans. In the present study, we investigated a novel technique to measure CBF based on laser speckle flowgraphy (LSFG) in healthy subjects. METHODS: This study included 31 eyes of 31 healthy, non-smoking subjects aged between 19 and 74 years. A commercial LSFG instrument was used to measure choroidal vessel diameter (CVD) and relative flow volume (RFV) in choroidal vessels that were identified on fundus photos, an approach that was used previously only for retinal vessels. The reproducibility and the effect of isometric exercise on these parameters were investigated. The latter was compared with measurement of subfoveal CBF using laser Doppler flowmetry (LDF). RESULTS: Intraclass correlation coefficients for CVD and RFV were higher than 0.8 indicating excellent reproducibility. During isometric exercise, we observed an increase in ocular perfusion pressure of approximately 60% (P<0.001). The increase in RFV and CBF was lower, but also highly significant versus baseline (at minute 6 of isometric exercise: RFV 10.5%±4.2%, CBF 8.3%±3.6%; P<0.001 each) indicating choroidal autoregulation. CONCLUSION: LSFG may be a novel approach to study blood flow in choroidal vessels. Data are reproducible and show good agreement with LDF data. TRIAL REGISTRATION NUMBER: NCT02102880, Results.