Characterising splicing defects of ABCA4 variants within exons 13-50 in patient-derived fibroblasts.

The ATP-binding cassette subfamily A member 4 gene (ABCA4)-associated retinopathy, Stargardt disease, is the most common monogenic inherited retinal disease. Given the pathogenicity of numerous ABCA4 variants is yet to be examined and a significant proportion (more than 15%) of ABCA4 variants are categorized as splice variants in silico, we therefore established a fibroblast-based splice assay to analyze ABCA4 variants in an Australian Stargardt disease cohort and characterize the pathogenic mechanisms of ABCA4 variants. A cohort of 67 patients clinically diagnosed with Stargardt disease was recruited. Genomic DNA was analysed using a commercial panel for ABCA4 variant detection and the consequences of ABCA4 variants were predicted in silico. Dermal fibroblasts were propagated from skin biopsies, total RNA was extracted and the ABCA4 transcript was amplified by RT-PCR. Our analysis identified a total of 67 unique alleles carrying 74 unique variants. The most prevalent splice-affecting complex allele c.[5461-10T>C; 5603A>T] was carried by 10% of patients in a compound heterozygous state. ABCA4 transcripts from exon 13 to exon 50 were readily detected in fibroblasts. In this region, aberrant splicing was evident in 10 out of 57 variant transcripts (18%), carried by 19 patients (28%). Patient-derived fibroblasts provide a feasible platform for identification of ABCA4 splice variants located within exons 13-50. Experimental evidence of aberrant splicing contributes to the pathogenic classification for ABCA4 variants. Moreover, identification of variants that affect splicing processes provides opportunities for intervention, in particular antisense oligonucleotide-mediated splice correction.

Localised relative scotoma in cuticular drusen.

PURPOSE: To investigate retinal sensitivity changes in eyes with pure cuticular drusen. METHODS: Multimodal imaging and microperimetry (37-loci grid) data were examined retrospectively to evaluate functional changes in eyes with pure cuticular drusen. Mean sensitivity in the cuticular drusen cohort was compared to age-matched normals. An age- and loci-specific normative reference was created to analyse localised sensitivity deviation. RESULTS: The mean number loci with relative scotoma in the cuticular drusen cohort (n = 27, mean [SD] age: 48.5 [12.4] years) referenced to normal eyes (n = 80, 53.5 [14.6] years) was 5.5 (95% confidence interval 3.0 to 8.1). However, mean sensitivity was not statistically different to the age-matched normal cohort (95% CI, - 2.3 to + 3.4 dB). The 37-loci grid was stratified into three rings of the approximately same number of loci, and the percentage of cuticular drusen eyes with pointwise deviation was significantly lower in the inner compared to the middle ring (12.3 [5.3]% vs. 17.3 [5.1]%, p < 0.05). CONCLUSIONS: Eyes with cuticular drusen demonstrated relative scotoma, but mean sensitivity was not affected. Pointwise sensitivity provides a more robust measure of retinal sensitivity than mean sensitivity in cuticular drusen and should be assessed both in the clinic and in future clinical trials.

Determinants of Disease Penetrance in PRPF31-Associated Retinopathy.

Retinitis pigmentosa 11 (RP11) is caused by dominant mutations in PRPF31, however a significant proportion of mutation carriers do not develop retinopathy. Here, we investigated the relationship between CNOT3 polymorphism, MSR1 repeat copy number and disease penetrance in RP11 patients and non-penetrant carriers (NPCs). We further characterized PRPF31 and CNOT3 expression in fibroblasts from eight RP11 patients and one NPC from a family carrying the c.1205C>T variant. Retinal organoids (ROs) and retinal pigment epithelium (RPE) were differentiated from induced pluripotent stem cells derived from RP11 patients, an NPC and a control subject. All RP11 patients were homozygous for the 3-copy MSR1 repeat in the PRPF31 promoter, while 3/5 NPCs carried a 4-copy MSR1 repeat. The CNOT3 rs4806718 genotype did not correlate with disease penetrance. PRFP31 expression declined with age in adult cadaveric retina. PRPF31 and CNOT3 expression was reduced in RP11 fibroblasts, RO and RPE compared with controls. Both RP11 and NPC RPE displayed shortened primary cilia compared with controls, however a subpopulation of cells with normal cilia lengths was present in NPC RPE monolayers. Our results indicate that RP11 non-penetrance is associated with the inheritance of a 4-copy MSR1 repeat, but not with CNOT3 polymorphisms.

Increased interdigitation zone visibility on optical coherence tomography following systemic fibroblast growth factor receptor 1-3 tyrosine kinase inhibitor anticancer therapy.

BACKGROUND: To describe ocular adverse events and retinal changes during fibroblast growth factor receptor (FGFR) inhibitor (AZD4547) anticancer therapy. METHODS: This is a sub-study examining ocular adverse effects from AZD4547 therapy (single-centre, open-label, single arm phase II clinical trial). Comprehensive ocular examinations were performed 3 weekly in 24 patients. Macular optical coherence tomography (OCT) scan (300 × 250 ) was obtained at each visit and OCT parameters [central 1 mm retinal thickness (CRT) and total macular volume in central 6 mm] extracted. OCT scans were subdivided into outer (ELM to RPE) and inner (ELM to ILM) layers to compare outer and inner retinal changes. RESULTS: In 24 patients, AZD4547 was associated with eyelash elongation (n = 5, 21%) and punctate corneal erosion (n = 2, 8%). One patient developed clinically significant posterior capsular opacification during the study. OCT data were available in 23 patients, retinal changes ranged from an asymptomatic increased visibility of the interdigitation zone (IDZ) (n = 10, 43%) to multilobular subretinal fluid pockets (n = 5, 22%), which was associated with mild visual acuity loss. In a subset of patients (n = 9) with pre-AZD4547 dosing OCT baseline, CRT increased by mean (SD) of 9 (4) µm in those with IDZ change only compared with 64 (38) µm in those with other retinal changes. Retinal changes tended to be bilateral, self-limiting and improved over time without medical intervention. CONCLUSIONS: The ocular signs and symptoms did not result in dose cessation. Posteriorly, FGFR inhibition leads to outer retinal changes ranging from increased visibility of IDZ to distinct, multiple fluid pockets.

Exploring microperimetry and autofluorescence endpoints for monitoring disease progression in PRPF31-associated retinopathy.

BACKGROUND: Mutations in the splicing factor pre-messenger RNA processing factor 31 (PRPF31) gene cause autosomal dominant retinitis pigmentosa 11 (RP11) through a haplo-insufficiency mechanism. We describe the phenotype and progression of microperimetry and autofluorescence endpoints in an Indigenous Australian RP11 family. PATIENTS AND METHODS: Ophthalmic examination, optical coherence tomography, fundus autofluorescence and microperimetry were performed at baseline and every 6-12 months. Baseline and annual change in best-corrected visual acuity (BCVA), microperimetry mean sensitivity (MS) and number of scotoma loci, residual ellipsoid zone (EZ) span and hyperautofluorescent ring (HAR) area were reported. Next-generation and Sanger sequencing were performed in available members. RESULTS: 12 affected members from three generations were examined. Mean (SD, range) age at onset of symptoms was 11 (4.5, 4-19) years. MS declined steadily from the third decade and EZ span and HAR area declined rapidly during the second decade. Serial microperimetry showed negligible change in MS over 2-3 years. However, mean EZ span, near-infrared and short-wavelength HAR area reduction was 203 (6.4%) µm/year, 1.8 (8.7%) mm2/year and 1.1 (8.6%) mm2/year, respectively. Genetic testing was performed on 11 affected and 10 asymptomatic members and PRPF31 c.1205 C > A (p.Ser402Ter) mutation was detected in all affected and two asymptomatic members (non-penetrant carriers). CONCLUSIONS: Our findings suggest that in the studied cohort, the optimal window for therapeutic intervention is the second decade of life and residual EZ span and HAR area can be considered as efficacy outcome measures. Further studies on larger samples with different PRPF31 mutations and longer follow-up duration are recommended.

Deep learning segmentation of hyperautofluorescent fleck lesions in Stargardt disease.

Stargardt disease is one of the most common forms of inherited retinal disease and leads to permanent vision loss. A diagnostic feature of the disease is retinal flecks, which appear hyperautofluorescent in fundus autofluorescence (FAF) imaging. The size and number of these flecks increase with disease progression. Manual segmentation of flecks allows monitoring of disease, but is time-consuming. Herein, we have developed and validated a deep learning approach for segmenting these Stargardt flecks (1750 training and 100 validation FAF patches from 37 eyes with Stargardt disease). Testing was done in 10 separate Stargardt FAF images and we observed a good overall agreement between manual and deep learning in both fleck count and fleck area. Longitudinal data were available in both eyes from 6 patients (average total follow-up time 4.2 years), with both manual and deep learning segmentation performed on all (n = 82) images. Both methods detected a similar upward trend in fleck number and area over time. In conclusion, we demonstrated the feasibility of utilizing deep learning to segment and quantify FAF lesions, laying the foundation for future studies using fleck parameters as a trial endpoint.

Edge of Scotoma Sensitivity as a Microperimetry Clinical Trial End Point in USH2A Retinopathy.

Purpose: Microperimetry is commonly used to assess retinal function. We perform cross-sectional and longitudinal analysis on microperimetry parameters in USH2A retinopathy and explore end points suitable for future clinical trials. Methods: Microperimetry was performed using two grids, Grid 1 (18° diameter) and Grid 2 (6° diameter). In Grid 1, four parameters (number of nonscotomatous loci, mean sensitivity [MS], responding point sensitivity [RPS], and edge of scotoma sensitivity [ESS]) were analyzed. In Grid 2, number of nonscotomatous loci and MS were examined. Interocular symmetry was also examined. Longitudinal analysis was conducted in a subset of eyes. Results: Microperimetry could be performed in 16 of 21 patients. In Grid 1 (n = 15; average age, 35.6 years), average number of nonscotomatous loci, MS, RPS, and ESS were 46.6 loci, 10.0 dB, 14.7 and 9.6 dB, respectively. In Grid 2 (n = 13; average age, 37.4 years), 12 eyes had measurable sensitivity across the entire grid. Average MS was 23.8 dB. Interocular analysis revealed large 95% confidence intervals for all parameters. Longitudinally, Grid 1 (n = 12, average follow-up 2.6 years) ESS showed the fastest rate of decline (-1.84 dB/y) compared with MS (-0.34 dB/y) and RPS (-0.90 dB/y). Conclusions: Our data suggest that ESS may be more useful than MS and RPS in test grids that cover a large extent of the macula. We caution the use of contralateral eye as an internal control. Translational Relevance: ESS may decrease the duration or sample size of treatment trials in USH2A retinopathy.

Interpreting MAIA Microperimetry Using Age- and Retinal Loci-Specific Reference Thresholds.

Purpose: Macular Integrity Assessment (MAIA) microperimetry is used widely in clinical trials and routine practice to assess paracentral scotoma. Current interpretation of MAIA is based on an assumed uniform 25 decibel (dB) cutoff for normal function irrespective of subject age and retinal location. We examined this convention by establishing an age- and loci-specific reference in healthy eyes and comparing this to the <25 dB cutoff. Methods: Retrospective MAIA results from healthy eyes were analyzed for prevalence of loci with <25 dB. At each locus, a new reference cutoff was derived from quantile regression of sensitivity against age at the 2.5th percentile. Two clinical cases of serial MAIA testing were analyzed using the new approach and compared to the <25 dB cutoff. Results: Fifty-four and 56 age-matched (range: 16-75 years) healthy eyes underwent small (37 loci) and large (68 loci) grid testing, respectively. Retinal sensitivity <25 dB was found in 5% of the small grid (1998 data points) and 10% of the large grid (3808 data points). These were found predominantly in older subjects and at the central point or in the perifoveal region. Quantile regression at each individual locus showed age-related decline with a median gradient of 0.6 dB/decade. Conclusions: We caution against using <25 dB cutoff in MAIA interpretation and advocate an age- and loci-specific cutoff criterion. Translational Relevance: Our study suggests that MAIA interpretation is influenced by the criterion used for defining abnormal pointwise measurement.

A phase II trial of single oral FGF inhibitor, AZD4547, as second or third line therapy in malignant pleural mesothelioma.

OBJECTIVES: Currently, there is no optimal salvage therapy for patients with malignant pleural mesothelioma (MPM) who relapse after treatment with first-line chemotherapy. In line with the strong preclinical rationale for targeting fibroblast growth factor receptor (FGFR) signalling in malignant mesothelioma, we conducted a phase II study assessing the efficacy of AZD4547, an oral tyrosine multi-kinase FGFR 1-3 inhibitor, as a second or third-line treatment. MATERIALS AND METHODS: We conducted a single-center, open-label, single-arm phase II study of AZD4547 in eligible patients with confirmed, measurable MPM and radiological progression after first or second-line systemic chemotherapy. Patients received continuous, twice-daily oral AZD4547 on a 3-weekly cycle. The primary end point was 6-month progression free survival (PFS6). Response was assessed with CT scan every 6 weeks according to the modified RECIST criteria for mesothelioma (mRECIST) and toxicities were also assessed. The study used a Simon's two-stage design: 26 patients would be recruited to the first stage and more than 7 (27 %) of 26 patients were required to achieve PFS6 to continue to stage two, for a potential total cohort of 55 patients. RESULTS: 3 of 24 patients (12 %) were progression-free at 6 months. Hence, the study fulfilled stopping criteria regardless of further recruitment and warranted discontinuation. The most common toxicities (across all grades) were hyperphosphatemia, xerostomia, mucositis, retinopathy, dysgeusia, and fatigue. Maximum toxicities were grade 2 or below for all patients across all cycles. There was no association between tumour BAP1 protein loss and clinical outcomes. CONCLUSIONS: The FGFR 1-3 inhibitor AZD4547 did not demonstrate efficacy in patients with MPM who had progressed after first line treatment with platinum-based chemotherapy.

Functional results and photic phenomena with new extended-depth-of-focus intraocular Lens.

BACKGROUND: Evaluation of clinical and functional results of a new extended depth of focus intraocular lens (EDOF-IOL). METHODS: Fourteen cataract patients (28 bilateral implantations) were assessed for uncorrected (UDVA) and corrected (CDVA) distance visual acuities; uncorrected (UNVA), distance-corrected (DCNVA) and best corrected (CNVA) near visual acuities; and uncorrected (UIVA) and distance-corrected (DCIVA) intermediate visual acuities - as well as binocular defocus curves. Photopic and mesopic contrast sensitivity was recorded. Reading acuity was evaluated using an electronic reading desk at fixed distances and at the patient's preferred near and intermediate distances. Visual symptoms were assessed with a halo and glare simulator plus a patient questionnaire which also recorded quality of life. RESULTS: Median postoperative monocular UDVA was 0.13logMAR (range - 0.08 to 0.42logMAR), median CDVA was - 0.01logMAR (range - 0.20 to 0.22logMAR), median UIVA at 80 cm was - 0.05logMAR (range - 0.18 to 0.58logMAR) and median UNVA at 40 cm was 0.14logMAR (range - 0.10 to 0.64logMAR). Binocular uncorrected reading acuity was 0.10logMAR at 40 cm and 0.11logMAR at 80 cm. Patients preferred a median intermediate reading distance of 62.8 cm over the predetermined 80 cm, which allowed them to read smaller letter size but did not improve reading acuity. Patients reported a high rate of spectacle independence and satisfaction in everyday life and little to no dysphotopsia. CONCLUSION: The Mini WELL Ready IOL provided good postoperative functional results at far and intermediate distances and improved the visual and reading acuity at reading distance. The lens caused little to no dysphotopsia. TRIAL REGISTRATION: The study protocol was registered at the German Clinical Trials Register: DRKS00007837 (Registered Date: March 9th, 2015).

Clinical Evaluation of an Extended Depth of Focus Intraocular Lens With the Salzburg Reading Desk.

BACKGROUND: Clinical evaluation of an extended depth of focus (EDOF) intraocular lens (IOL) regarding visual performance at various distances, reading performance on an electronic reading desk, and depth of focus on the defocus curve. METHODS: In this prospective study, 30 eyes of 15 patients who received the Tecnis Symfony IOL (Abbott Medical Optics, Inc., Santa Ana, CA) were examined 3.60 ± 1.54 months postoperatively. Uncorrected and corrected distance (UDVA and CDVA), uncorrected and distance-corrected intermediate (UIVA and DCIVA), and uncorrected and distance-corrected near (UNVA and DCNVA) visual acuity were determined. The defocus curve was performed with distance correction. Furthermore, the reading acuity at the preferred near and intermediate distances was measured with consideration of the reading distance, speed, and print size. A subjective questionnaire was also administered. RESULTS: Results showed a median UDVA of 0.03 logMAR or 20/21.43 Snellen (range: 0.44 to -0.18 logMAR or 20/55.08 to 20/13.21 Snellen), UNVA of 0.20 logMAR or 20/31.70 Snellen (range: 0.46 to 0.00 logMAR or 20/57.68 to 20/20 Snellen), and UIVA of -0.03 logMAR or 20/18.67 Snellen (range: 0.14 to -0.18 logMAR or 20/27.61 to 20/13.21 Snellen). The improved performance at intermediate distance was confirmed by an uncorrected reading acuity of 0.09 logMAR or 20/24.61 Snellen (range: 0.36 to 0.00 logMAR or 20/45.82 to 20/20.00 Snellen) at a preferred intermediate distance of 64.10 cm (range: 52.0 to 75.0 cm). The defocus curve showed an extended range of visual acuity of 0.10 logMAR or better between 1.00 and -1.50 diopters. CONCLUSIONS: The EDOF IOL design provided a wide range of improved visual and reading function between far and intermediate distances and also improved near visual acuity. Patients reported a high rate of satisfaction and spectacle independence at the various distances. [J Refract Surg. 2017;33(10):664-669.].

Clinical Evaluation of Reading Performance Using the Salzburg Reading Desk With a Refractive Rotational Asymmetric Multifocal Intraocular Lens.

PURPOSE: To evaluate functional results and reading performance using the Salzburg Reading Desk after implantation of a sector-shaped near-embedded, rotational asymmetrical multifocal intraocular lens (IOL) and a multifocal toric IOL with a +3.00 diopter (D) near addition. METHODS: In a prospective study, the LentisMplus and Mplus toric IOLs (Oculentis GmbH, Berlin, Germany) were implanted in 34 eyes of 18 patients at the University Eye Hospital of Heidelberg. Uncorrected and corrected distance visual acuity (UDVA, CDVA) and uncorrected and corrected near visual acuity (UNVA, CNVA) were evaluated using standardized visual acuity charts (ETDRS). The Salzburg Reading Desk was used to analyze unilateral and bilateral uncorrected and corrected reading acuity, reading distance, reading speed, and the smallest log-scaled print size that could be read effectively at a set (40 cm/80 cm) and subjective chosen near and intermediate distance. RESULTS: Postoperatively, the median UDVA was 0.08 logMAR (20/25 Snellen) and the median CDVA was 0.01 logMAR (20/20 Snellen). The median UNVA was 0.12 logMAR (20/25 Snellen) and the median CNVA was 0.03 logMAR (20/20 Snellen). The median uncorrected reading acuity measured with the Salzburg Reading Desk for near distance at 40 cm was 0.18 logMAR (20/32 Snellen). The subjectively preferred near distance was 39 cm and revealed similar visual acuity results. The best reading acuity for intermediate distance with a median of 0.22 logMAR (20/32 Snellen) was achieved at a median distance of 62 cm. CONCLUSIONS: Reading performance of the multifocal IOL corresponded for near standardized and individual distance, whereas reading function was better at the patient's preferred intermediate distance. [J Refract Surg. 2016;32(8):526-532.].

Near and intermediate visual and reading performance of patients with a multifocal apodized diffractive intraocular lens using an electronic reading desk.

PURPOSE: To functionally evaluate an apodized diffractive multifocal intraocular lens (IOL) using an electronic reading desk (Salzburg Reading Desk). SETTING: University Eye Clinic Heidelberg, Heidelberg, Germany. DESIGN: Prospective case series. METHODS: After implantation of an Acrysof IQ Restor +3.0 diopter (D) multifocal IOL, uncorrected (UDVA) and corrected (CDVA) distance visual acuities, uncorrected (UNVA) and distance-corrected (DCNVA) near visual acuities, and uncorrected (UIVA) and distance-corrected (DCIVA) intermediate visual acuities were determined. Criteria for evaluating reading acuity were the reading speed and print size. Monocular and binocular examinations were performed with the electronic reading desk at fixed distances and at the patient's preferred near and intermediate distances. In addition, a subjective questionnaire was administered. RESULTS: Forty eyes (20 patients) were enrolled. Monocularly, the medians were UDVA, 0.00 logMAR (range 0.26 to -0.14 logMAR); CDVA, -0.08 logMAR (range 0.16 to -0.24 logMAR); 40 cm UNVA, 0.04 logMAR (range 0.24 to -0.10 logMAR); DCNVA, 0.00 logMAR (range 0.18 to -0.22 logMAR); 80 cm UIVA, 0.15 logMAR (range 0.40 to -0.18 logMAR); DCIVA, 0.16 logMAR (range 0.42 to -0.14 logMAR). The monocular reading desk examinations resulted in an uncorrected reading acuity of 0.11 logMAR (range 0.49 to -0.01 logMAR) at 40 cm and 0.30 logMAR (range 0.48 to 0.00 logMAR) at 80 cm. Patients reported a high rate of spectacle independence and satisfaction in everyday life. CONCLUSION: The +3.0 D IOL provided good postoperative functional results at far and near and improved the visual and reading acuity at an intermediate distance. FINANCIAL DISCLOSURES: The International Vision Correction Research Centre has received research grants, travel expenses, and lecture fees from various intraocular lens manufacturers. None of the authors has a financial or proprietary interest in any material or method mentioned.

Near and intermediate reading performance of a diffractive trifocal intraocular lens using a reading desk.

PURPOSE: To evaluate reading performance of a trifocal intra-ocular lens (IOL) at near and intermediate distances using the Salzburg Reading Desk. SETTING: International Vision Correction Research Centre and David J. Apple Laboratory, University Eye Clinic, Heidelberg, Germany. DESIGN: Prospective, nonrandomized clinical study. METHODS: Follow-up examinations at a minimum of 3 months postoperatively included uncorrected (UDVA) and corrected (CDVA) distance visual acuity, uncorrected (UIVA) and distance-corrected (DCIVA)) intermediate as well as uncorrected (UNVA) and distance-corrected (DCNVA) near visual acuity. Uncorrected and distance-corrected reading acuity at 40 cm for near, 80 cm for intermediate distance, and at the patient's preferred near and intermediate distances were evaluated with the reading desk. RESULTS: In this study, 22 eyes of 11 patients having cataract surgery with implantation of the trifocal Finevision IOL were evaluated. Postoperative monocular Snellen median values were 20/21.44 (range 20/52.61 to 20/14.49) for UDVA, 20/20.47 (range 20/38.11 to 20/16.64) for UIVA, and 20/26.39 (range 20/43.76 to 20/18.24) for UNVA. Subjective intermediate distance at the binocular uncorrected examination on the reading desk was 64.2 cm (range 51.9 to 80.0) with a reading acuity of 0.10 logMAR (range 0.32 to 0.00). Subjective near distance at the uncorrected binocular reading desk examination was 36.5 cm (30.8 to 41.2) with a near reading acuity of 0.06 logMAR (range 0.23 to -0.01). The preferred distances differed significantly from the fixed ones of 40 and 80 cm. The preferred intermediate distance was almost consistent, with the intermediate addition of 1.75 D corresponding to 57.1 cm. CONCLUSION: The visual and reading function of the trifocal IOL was better at the patient's preferred near and intermediate distances. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.