Identification of a novel nonsense variant in FYCO1 gene associated with infantile cataract and cortical atrophy.

INTRODUCTION: Cataract is a major condition characterized by ocular lens opacification, resulting from alteration in the lens architecture, lens proteins or both. It is responsible for about one-third of infants' blindness worldwide. Variants in the FYCO1 gene have been associated with autosomal recessive infantile cataract. MATERIAL AND METHODS: We conducted whole exome sequencing (WES) in a nine months old male patient who was referred for genetic investigation because of infantile cataract. WES analysis revealed the presence of a homozygous pathogenic variant (c.2365C>T) in exon 8 of the FYCO1 gene. RESULTS AND DISCUSSION: This is the first report on a Lebanese infant with infantile cataract and cortical atrophy which was not previously reported, resulting from a novel homozygous FYCO1 variant; thus expanding the clinical phenotypic spectrum of FYCO1 involvement.

Crystals deposits in the anterior and posterior lens cortex in Bietti corneo-retinal dystrophy.

BACKGROUND: Whereas crystals deposit in the retina, the cornea and limbus in Bietty corneo-retinal dystrophy (BCD) is now well established and documented, only two published cases report their findings in the lens and no cases deep in the lens cortex. MATERIAL AND METHODS: Four consecutive adult patients from three different unrelated families presenting lens crystals associated with advanced genetically confirmed BCD were enrolled with advanced disease and long follow up (>12 years). Demographics, visual acuity, slit lamp biomicroscopy, lens and posterior pole photography, optical coherence tomography (OCT), autofluorescence, and screening for CYP4V2 type of mutation were performed. The setting was Jules Gonin Eye Hospital, Switzerland, between 1.1 2013 and 1.11. 2019. RESULTS: All patients were European women. The ages ranged from 40 to 81 years. Best Snellen visual acuity ranged from light perception to 1.0. All patients presented with limbus and retinal crystals deposit that disappeared over time and the development of severe chorioretinal atrophy. With long-term follow up, multiple crystal-like deposits appeared in the anterior, posterior lens capsule and cortex. All patients, but one, had homozygous or compound heterozygous mutations in CYP4V2 gene. CONCLUSIONS: To the best of our knowledge, there are no published cases of crystal deposits in the cortex of the lens of patients diagnosed with BCD associated with CYP4V2 gene mutation. This could be a feature of advanced BCD, and their presence in the lens cortex questions the hypothesis of floating deposits from posterior pole although their exact etiology remains to be determined.

Drug-induced lenticular opacity and accumulation of cholesterol-related substances in the lens cortex of dogs.

TP0446131, developed as an antidepressant agent, was found to cause lenticular opacity in a 13-week repeated-dose study in dogs. Histopathologically, the lenticular opacity was observed as a degeneration of the lens fibers, characterized by irregularity in the ordered arrangement of the fibers which is necessary to maintain the transparency of the lens, and was considered to manifest clinically as cataract. To evaluate the development mechanism of the lenticular opacity, the chemical constituents of the lens, which is known to be associated with the development of cataract, were examined. The results of liquid chromatography-tandem mass spectrometry analysis revealed an increase in the amplitudes of 3 unknown peaks in a dose- and time-dependent manner in the lens, with no remarkable changes in the other chemical components tested. In addition, the content of cholesterol, alterations of which have been reported to be associated with cataract, remained unchanged. The mass spectral data and chromatographic behavior of the 3 peaks indicated that these peaks corresponded to sterol-related substances, and that one of them was 7-dehydrocholesterol, a precursor of cholesterol biosynthesis. This finding suggested that TP0446131 exerts some effects on the cholesterol biosynthesis pathway, which could be involved in the development of the cataracts. Furthermore, increases in the levels of these sterol-related substances were also detected in the serum, and were, in fact, noted prior to the onset of the cataract, suggesting the possibility that these substances in the serum could be used as potential safety biomarkers for predicting the onset of cataract induced by TP0446131.

Phacoemulsification in bilateral anterior lenticonus in Alport syndrome: A case report.

RATIONALE: To report the visual status and results of phacoemulsification cataract surgery in a young patient with Alport syndrome associated with bilateral anterior lenticonus. The milestone of this report is the use of anterior segment optical coherence tomography (AS-OCT) to confirm the central protrusion of the anterior surface of the crystalline lens. PATIENT CONCERNS: A 23-year-old young woman presented with severe progressive visual loss in both eyes, which started several years ago. DIAGNOSES: Refractive status was indicative of high myopia with astigmatism and vision was not improved with optimal correction to better than 0.1 in the right eye and 0.2 in the left eye (visual acuities given in decimal notation). Slit-lamp examination showed transparent cornea, anterior lenticonus and posterior sub-capsular cataract in both eyes. The classical appearance of oil droplet was evident using retro-illumination on the slit lamp. INTERVENTIONS: The natural lenses were replaced with intraocular lens (IOL). OUTCOMES: An excellent refractive status achieved associated with an uncorrected distance visual acuity 0.9 and 0.8 in the right and left eye, respectively. LESSONS: AS-OCT is a valuable device for confirming the budging of the anterior crystalline lens surface.

Exposure to subthreshold dose of UVR-B induces apoptosis in the lens epithelial cells and does not in the lens cortical fibre cells.

PURPOSE: The aim of this study was to investigate in which part of the lens in vivo exposure to subthreshold dose of UVR-B radiation induces apoptosis. METHODS: Twenty 6-week-old female albino Sprague-Dawley rats were exposed to subthreshold dose (1 kJ/m2 ) of UVR-B unilaterally and killed at 120 hr after exposure. Lenses were enucleated and dissected on three regions: the lens epithelium, the cortex and the nucleus. The lens nucleus then was removed. Apoptosis markers p53 and caspase 3 were used to study apoptosis in the lens regions. qRT-PCR and Western blot were utilized to analyse the lenses. RESULTS: TP53 and CASP3 mRNA expressions are increased in exposed lenses, both in the lens epithelium and in the cortex regions, in relation to non-exposed lenses. Expression of p53 protein is increased in exposed lens epithelium in relation to non-exposed lens epithelium. Caspase 3 protein is expressed in exposed lens epithelial cells, while it is not expressed in non-exposed lens epithelial cells. p53 and caspase 3 proteins are not expressed in either exposed nor non-exposed lens fibre cells. CONCLUSION: Exposure to UVR-B increases mRNA transcription of apoptosis marker p53 in vivo in both regions of the lens and of apoptosis marker caspase 3 in the lens cortex. Exposure to UVR-B increases p53 and caspase 3 proteins expression just in the lens epithelium. In vivo exposure to subthreshold dose of UVR-B induces apoptosis in the lens epithelial cells and does not in the lens fibre cells.

Relationship Between the Altered Expression and Epigenetics of GSTM3 and Age-Related Cataract.

PURPOSE: Glutathione S-Transferase Mu 3 (GSTM3) protects the lens from oxidative stress that contributes to age-related cataract (ARC) formation. We examined the expression and epigenetics of GSTM3 in lens epithelial cells (LECs) and lens cortex of ARC, and investigated the potential role of molecular changes in ARC pathogenesis. METHODS: This study included 120 ARCs and 40 controls. Expression of GSTM3, DNA methylation, and histone modification were assessed by quantificational real-time PCR, Western blot, bisulfite-sequencing PCR, pyrosequencing, and chromatin immunoprecipitation assay. Human lens epithelial (HLE) cell lines, SRA01/04 and HLEB3, were served as an in vitro model to observe the relationship between epigenetic status and GSTM3 expression. Potential transcription factors binding to GSTM3 promoter were detected by electrophoretic mobility shift assay. RESULTS: Expression of GSTM3 decreased in ARC lens tissues compared to that in the controls, which correlated with the hypermethylation of GSTM3 promoter. Lower level of GSTM3 was detected in HLEB3 than in SRA01/04, while HLEB3 displayed hypermethylation of GSTM3 and SRA01/04 did not. Compared to SRA01/04, HLEB3 displayed lower acetylated H3 and higher trimethylated H3K9 levels. After treatment with DNA methyltransferase inhibitor or histone deacetylase inhibitor, HLEB3 had an increased GSTM3 expression. Methylation of GSTM3 promoter abrogated the potential transcription factor binding. The GSTM3 expression declined in hydrogen peroxide-treated HLE cell lines. CONCLUSIONS: Expression of GSTM3 might be regulated by epigenetic changes in lens tissue. Hypermethylation in GSTM3 promoter and altered histone modification might have a role in the ARC formation. The results provided a potential strategy of ARC management by manipulating epigenetic changes.

Effects of lentiviral short hairpin RNA silencing of Toll-like receptor 4 on the lens epithelial cell line HLEC.

The aim of this study was to observe the proliferation of, and cell-cycle changes in, the human lens epithelial cell line HLEC after Toll-like receptor 4 (TLR4) gene silencing. HLEC cells were transfected with four TLR4-short hairpin RNA (shRNA) lentiviral vectors or the control lentivirus (pGCL-GFP-shRP-1, -2, -3, -4, NC). TLR4 silencing was verified in these cells 96 h post-transfection using real-time polymerase chain reaction and western blot. We also observed the change in number of pGCL-GFP-shRP-4-transfected HLEC cells with silenced TLR4 (multiplicity of infection = 10). Cell proliferation was analyzed 48 h after transfection by a standard Cell Counting Kit-8 (CCK-8) assay, and the cell cycle changes were detected by flow cytometry. The number of cells with silenced TLR4 decreased with time. The decrease in TLR4 expression led to decelerated cell proliferation. Cells with silenced TLR4 (for 48 h) were arrested in the G1 phase; that is, the cell cycle was prolonged and cell division was decelerated. Lentivirus-mediated RNA interference effectively silenced TLR4 expression in HLEC cells, which decelerated their proliferation rate and extended the cell cycle.

Objective Assessment of Nuclear and Cortical Cataracts through Scheimpflug Images: Agreement with the LOCS III Scale.

PURPOSE: To assess nuclear and cortical opacities through the objective analysis of Scheimpflug images, and to check the correlation with the Lens Opacity Classification System III (LOCS III). METHODS: Nuclear and cortical opacities were graded according to the LOCS III rules after pupil dilation. The maximum and average pixel intensity values along an elliptical mask within the lens nucleus were taken to analyse nuclear cataracts. A new metric based on the percentage of opaque pixels within a region of interest was used to analyse cortical cataracts. The percentage of opaque pixels was also calculated for half, third and quarter areas from the region of interest's periphery. RESULTS: The maximum and average intensity values along the nucleus were directly proportional to the LOCS III grade: The larger the LOCS III value, the larger maximum and average intensity ones. These metrics showed a positive and significant correlation with the LOCS grade: The larger the LOCS grade, the higher was percentage of opaque pixels along the cortex within the same mask's size. This metric showed a significant correlation to the LOCS grade. CONCLUSION: The metrics used to assess nuclear opacities showed good correlation with the LOCS III. The percentage of opaque pixels showed to be a useful metric to measure objectively the severity of the cortical opacity. These metrics could be implemented in an algorithm to detect and grade lens opacities automatically and objectively.

Measuring the cataractous lens.

PURPOSE: To evaluate the lens thickness, anterior cortex space, nucleus thickness, and posterior cortex space in cataractous eyes and compare them with those in eyes of younger patients with clear lenses. SETTING: Private practice, Lynwood, California, USA. DESIGN: Retrospective observational study. METHODS: The study evaluated a group of cataractous eyes and compared them with a group of eyes of younger patients with clear lenses. All measurements were performed with a biometer (Lenstar LS 900). RESULTS: The cataractous group (200 eyes) had a greater mean lens thickness (4.65 mm ± 0.41 [SD]) than the control group (80 eyes) (4.09 ± 0.33 mm). The mean measured values for the cataractous groups and control groups were 0.84 ± 0.21 mm and 0.35 ± 0.11 mm for anterior cortex space, 3.31 ± 0.25 mm and 3.27 ± 0.27 mm for mean nucleus thickness, and 0.51 ± 0.16 mm and 0.48 ± 0.13 mm for mean posterior cortex space, respectively. Anterior cortex space, nucleus thickness, and posterior cortex space correlated positively with lens thickness (r = 0.69, r = 0.69, and r = 0.59, respectively). Lens thickness, anterior cortex space, nucleus thickness, and posterior cortex space showed a weak inverse correlation with axial length (r = 0.06, r = 0.08, r = 0.10, and r = 0.10, respectively) and an inverse correlation with anterior chamber depth (r = 0.57, r = 0.43, r = 0.42, and r = 0.22, respectively). Lens thickness showed a positive correlation with age (r = 0.28), as did the anterior cortex space (r = 0.32) and posterior cortex space (r = 0.26), but nucleus thickness did not show a positive correlation (r = 0.02). CONCLUSION: Lens thickness increased with age and with cataract formation and was mostly attributable to an increase in the anterior cortex space. FINANCIAL DISCLOSURE: Neither author has a financial or proprietary interest in any material or method mentioned.

Pars Plana Lensectomy After Femtosecond Laser-Assisted Cataract Surgery.

The use of femtosecond laser during cataract surgery is increasing, as it may potentially improve accuracy, safety and refractive outcomes. However, posterior capsule rupture with retained lens material can occur, necessitating vitreoretinal intervention. The authors report the first videographically documented case of removal of retained lens material after femtosecond laser-assisted cataract surgery.

Hydropolish: a controlled trial on a technique to eradicate residual cortical lens fibers in phacoemulsification cataract surgery.

PURPOSE: To assess the efficacy and safety of a noncontact, fluid-based capsular polishing technique (hydropolish) to remove residual cortical fibers (RCFs) and epithelial cells from the posterior and equatorial capsule in phacoemulsification cataract surgery. METHODS: Hydropolish involved manual irrigation of the posterior and equatorial capsule after irrigation/aspiration, using a 27-G hydrodissection cannula. This prospective, consecutive, single surgeon controlled trial was conducted at a dedicated ophthalmic surgery center in Sydney, Australia, between December 20, 2006, and July 14, 2010. Single eyes of consecutive patients underwent cataract surgery without use of hydropolish (control group), while those on or after July 21, 2010, underwent hydropolish (intervention group). Corrected distance visual acuity (CDVA) up to 1 month postoperatively, surgical complications, and hydropolish time were documented. RESULTS: A total of 1531 eyes were included in this study (hydropolish n = 682; control n = 849). After adjusting for age, sex, and nuclear sclerosis grade, no significant difference was found between hydropolish and control groups when preoperative CDVA was compared against postoperative CDVA at 1 day, 1 week, and 1 month (p>0.05). CONCLUSIONS: Hydropolish is a rapid and safe technique that can remove RCFs from the posterior and equatorial capsule in phacoemulsification cataract surgery. It does not compromise postoperative CDVA.

Altered DNA Methylation and Expression Profiles of 8-Oxoguanine DNA Glycosylase 1 in Lens Tissue from Age-related Cataract Patients.

PURPOSE: Oxidative stress and DNA damage contribute to the pathogenesis of age-related cataract (ARC). Most oxidative DNA lesions are repaired via the base excision repair (BER) proteins including 8-oxoguanine DNA glycosylase 1 (OGG1). This study examined DNA methylation of CpG islands upstream of OGG1 and their relation to the gene expression in lens cortex from ARC patients. METHODS: The clinical case-control study consisted of 15 cortical type of ARC patients and 15 age-matched non-ARC controls who received transparent lens extraction due to vitreoretinal diseases. OGG1 expression in lens cortex was analyzed by qRT-PCR and Western blot. The localization and the proportion of cells positive for OGG1 were determined by immunofluorescence. Bisulfite-sequencing PCR (BSP) was performed to evaluate the methylation status of CpG islands near OGG1 in DNA extracted from lens cortex. To test relationship between the methylation and the expression of the gene of interest, 5-Aza-2'-deoxycytidine (5-Aza-dC) was used to induce demethylation of cultured human lens epithelium B-3 (HLE B-3). To test the role of OGG1 in the repair of cellular damage, HLE B-3 was transfected with OGG1 vector, followed by ultraviolet radiation b (UVB) exposure to induce apoptosis. RESULTS: The mRNA and protein levels of OGG1 were significantly reduced in the lens cortex of ARC. Immunofluorescence showed that the proportion of OGG1-positive cells decreased significantly in ARC cortex in comparison with the control. The CpG island in first exon of OGG1 displayed hypermethylation in the DNA extracted from the lens cortex of ARC. Treatment of HLEB-3 cells with 5-Aza-dC upregulated OGG1 expression. UVB-induced apoptosis was attenuated after transfection with OGG1. CONCLUSION: A reduced OGG1 expression was correlated with hypermethylation of a CpG island of OGG1 in lens cortex of ARC. The role of epigenetic change in OGG1 gene in the susceptibility to oxidative stress induced cortical ARC is warranted to further study.

Refractive changes in nuclear, cortical and posterior subcapsular cataracts. effect of the type and grade.

PURPOSE: To determine the effect of main morphological types and grades of age-related cataracts on refractive error. METHODS: We measured 276 subjects with optical compensation prior to the development of cataract. We evaluated 224 eyes with nuclear cataract, 125 with cortical cataract, and 103 with posterior subcapsular (PSC) cataract classified with LOCSIII. We measured visual acuity (VA) with their spectacles and best-corrected visual acuity (BCVA) with chart in decimal scale to obtain the optimal compensation with cataract. We evaluated the differences between compensations. RESULTS: A significant myopic shift was observed in nuclear cataract from low to mild grade (p=0.031), the same as for PSC cataract from mild to advanced grade (p=0.025). No significant changes were found for cortical cataract (p=0.462). Regarding astigmatism, we observed power changes in cortical cataract from low to mild grade (p=0.03) and axis changes in PSC from low to mild grade (p=0.02) and in nuclear cataract from mild to advanced grade (p=0.02). CONCLUSIONS: Cataract produces changes in patient's compensation which depend on severity and type of cataract. For nuclear and PSC cataract, we observed that the higher the grade of severity, the greater the myopic shift. Power astigmatic changes were found in cortical cataract and axis changes in PSC and nuclear cataract.

Molecular mechanism of formation of cortical opacity in CRYAAN101D transgenic mice.

PURPOSE: The CRYAAN101D transgenic mouse model expressing deamidated αA-crystallin (deamidation at N101 position to D) develops cortical cataract at the age of 7 to 9 months. The present study was carried out to explore the molecular mechanism that leads to the development of cortical opacity in CRYAAN101D lenses. METHODS: RNA sequence analysis was carried out on 2- and 4-month-old αA-N101D and wild type (WT) lenses. To understand the biologic relevance and function of significantly altered genes, Ingenuity Pathway Analysis (IPA) was done. To elucidate terminal differentiation defects, immunohistochemical, and Western blot analyses were carried out. RESULTS: RNA sequence and IPA data suggested that the genes belonging to gene expression, cellular assembly and organization, and cell cycle and apoptosis networks were altered in N101D lenses. In addition, the tight junction signaling and Rho A signaling were among the top three canonical pathways that were affected in N101D mutant. Immunohistochemical analysis identified a series of terminal differentiation defects in N101D lenses, specifically, increased proliferation and decreased differentiation of lens epithelial cells (LEC) and decreased denucleation of lens fiber cells (LFC). The expression of Rho A was reduced in different-aged N101D lenses, and, conversely, Cdc42 and Rac1 expressions were increased in the N101D mutants. Moreover, earlier in development, the expression of major membrane-bound molecular transporter Na,K-ATPase was drastically reduced in N101D lenses. CONCLUSIONS: The results suggest that the terminal differentiation defects, specifically, increased proliferation and decreased denucleation are responsible for the development of lens opacity in N101D lenses.

Simple fixation and storage protocol for preserving the internal structure of intact human donor lenses and extracted human nuclear cataract specimens.

PURPOSE: Increased use of phacoemulsification procedures for cataract surgeries has resulted in a dramatic decrease in the availability of cataractous nuclear specimens for basic research into the mechanism of human cataract formation. To overcome such difficulties, a fixation protocol was developed to provide good initial fixation of human donor lenses and extracted nuclei, when available, and is suitable for storing or shipping cataracts to laboratories where structural studies could be completed. METHODS: Cataractous lens nuclei (n=19, ages 12 to 74 years) were obtained from operating suites after extracapsular extraction. Transparent human donor lenses (n=27, ages 22 to 92 years) were obtained from the Ramayamma International Eye Bank. After the dimensions were measured with a digital caliper, samples were preserved in 10% formalin (neutral buffered) for 24 h and followed by fixation in 4% paraformaldehyde (pH 7.2) for 48 h. Samples were stored cold (4 °C) in buffer until shipped. Samples were photographed and measured before further processing for transmission electron microscopy. RESULTS: The dimensions of the samples varied slightly after short fixation followed by 1 to 5 months' storage before transmission electron microscopy processing. The mean change in the axial thickness of the donor lenses was 0.15±0.21 mm or 3.0±5.4%, while that of the extracted nuclei was 0.05±0.24 mm or 1.8±7.6%. Because the initial concern was whether the nuclear core was preserved, thin sections were examined from the embryonic and fetal nuclear regions. All cellular structures were preserved, including the cytoplasm, complex edge processes, membranes, and junctions. The preservation quality was excellent and nearly equivalent to preservation of fresh lenses even for the lens cortex. Cell damage characteristic of specific nuclear cataract types was easily recognized. CONCLUSIONS: The novel fixation protocol appears effective in preserving whole donor lenses and cataractous nuclei over a wide age range. Dimensions varied only 2%-3%, and fiber cell damage correlated well with standard fixation. These methods enable researchers and clinicians in remote settings to preserve donor lenses and rare examples of extracapsular extractions for detailed examination at later times.

Absence of beta-amyloid in cortical cataracts of donors with and without Alzheimer's disease.

Eye lenses from human donors with and without Alzheimer's disease (AD) were studied to evaluate the presence of amyloid in cortical cataract. We obtained 39 lenses from 21 postmortem donors with AD and 15 lenses from age-matched controls provided by the Banco de Ojos para Tratamientos de la Ceguera (Barcelona, Spain). For 17 donors, AD was clinically diagnosed by general physicians and for 4 donors the AD diagnosis was neuropathologically confirmed. Of the 21 donors with AD, 6 had pronounced bilateral cortical lens opacities and 15 only minor or no cortical opacities. As controls, 7 donors with pronounced cortical opacities and 8 donors with almost transparent lenses were selected. All lenses were photographed in a dark field stereomicroscope. Histological sections were analyzed using a standard and a more sensitive Congo red protocol, thioflavin staining and beta-amyloid immunohistochemistry. Brain tissue from two donors, one with cerebral amyloid angiopathy and another with advanced AD-related changes and one cornea with lattice dystrophy were used as positive controls for the staining techniques. Thioflavin, standard and modified Congo red staining were positive in the control brain tissues and in the dystrophic cornea. Beta-amyloid immunohistochemistry was positive in the brain tissues but not in the cornea sample. Lenses from control and AD donors were, without exception, negative after Congo red, thioflavin, and beta-amyloid immunohistochemical staining. The results of the positive control tissues correspond well with known observations in AD, amyloid angiopathy and corneas with lattice dystrophy. The absence of staining in AD and control lenses with the techniques employed lead us to conclude that there is no beta-amyloid in lenses from donors with AD or in control cortical cataracts. The inconsistency with previous studies of Goldstein et al. (2003) and Moncaster et al. (2010), both of which demonstrated positive Congo red, thioflavin, and beta-amyloid immunohistochemical staining in AD and Down syndrome lenses, is discussed.