A novel deep intronic COL2A1 mutation in a family with early-onset high myopia/ocular-only Stickler syndrome.

PURPOSE: To identify the genetic defect causing early-onset high myopia (eoHM)/ocular-only Stickler syndrome (ocular-STL) in a large Chinese family. METHODS: Genomic DNA and clinical data from a four-generation family with eoHM/ocular-STL were collected. Whole-exome sequencing was performed on one affected member in initial screening. Linkage scan based on microsatellite markers was carried out initially from candidate loci associated with autosomal dominant eoHM and Stickler syndrome. Sanger sequencing was used to detect potential variants. The pathogenicity of candidate variants was evaluated using mini genes ex vivo. RESULTS: Eight patients and five unaffected members in the family participated in the study, in which the patients had high myopia with other variable ocular phenotypes but without extraocular abnormalities. Whole exome sequencing did not detect any potential pathogenic variant in all genes known to associate with the disease. The eoHM/ocular-STL in the family was mapped to markers around COL2A1 by candidate loci linkage scan, with a maximum lod score of 3.31 for D12S1590 at θ = 0. A novel deep intronic variant, c.86-50C > G in intron 1 of COL2A1, was detected by Sanger sequencing and co-segregated with eoHM/ocular-STL in the family. Ex vivo splicing test using mini genes confirmed that the variant created a new splicing acceptor 49 bp before the canonical splicing site of exon 2, resulted in addition of 49 bp fragment in the transcript (from c.86-49 to c.86-1) and premature termination. CONCLUSIONS: Linkage study, bioinformatics prediction, and ex vivo transcript analysis suggest a novel deep intronic variant adjacent to 5-prime of exon 2 of COL2A1, affecting exon 2 splicing, as a potential cause of ocular-STL in a large family. To our knowledge, this is the first report of an intronic variant around exon 2 as a cause of ocular-STL while a series of variants in the coding region of exon 2, a dispensable alternative-splicing exon for extraocular tissues, in COL2A1 have been reported to cause Stickler syndrome-related ocular phenotype alone.

Iron is neurotoxic in retinal detachment and transferrin confers neuroprotection.

In retinal detachment (RD), photoreceptor death and permanent vision loss are caused by neurosensory retina separating from the retinal pigment epithelium because of subretinal fluid (SRF), and successful surgical reattachment is not predictive of total visual recovery. As retinal iron overload exacerbates cell death in retinal diseases, we assessed iron as a predictive marker and therapeutic target for RD. In the vitreous and SRF from patients with RD, we measured increased iron and transferrin (TF) saturation that is correlated with poor visual recovery. In ex vivo and in vivo RD models, iron induces immediate necrosis and delayed apoptosis. We demonstrate that TF decreases both apoptosis and necroptosis induced by RD, and using RNA sequencing, pathways mediating the neuroprotective effects of TF are identified. Since toxic iron accumulates in RD, we propose TF supplementation as an adjunctive therapy to surgery for improving the visual outcomes of patients with RD.

Endoplasmic Reticulum Stress and Unfolded Protein Response in Cartilage Pathophysiology; Contributing Factors to Apoptosis and Osteoarthritis.

Chondrocytes of the growth plate undergo apoptosis during the process of endochondral ossification, as well as during the progression of osteoarthritis. Although the regulation of this process is not completely understood, alterations in the precisely orchestrated programmed cell death during development can have catastrophic results, as exemplified by several chondrodystrophies which are frequently accompanied by early onset osteoarthritis. Understanding the mechanisms that underlie chondrocyte apoptosis during endochondral ossification in the growth plate has the potential to impact the development of therapeutic applications for chondrodystrophies and associated early onset osteoarthritis. In recent years, several chondrodysplasias and collagenopathies have been recognized as protein-folding diseases that lead to endoplasmic reticulum stress, endoplasmic reticulum associated degradation, and the unfolded protein response. Under conditions of prolonged endoplasmic reticulum stress in which the protein folding load outweighs the folding capacity of the endoplasmic reticulum, cellular dysfunction and death often occur. However, unfolded protein response (UPR) signaling is also required for the normal maturation of chondrocytes and osteoblasts. Understanding how UPR signaling may contribute to cartilage pathophysiology is an essential step toward therapeutic modulation of skeletal disorders that lead to osteoarthritis.

THE PATHOGENY OF PROLIFERATIVE VITREORETINOPATHY.

Proliferative vitreoretinopathy (PVR) is the most important complication of rhegmatogenous retinal detachment (RRD) and the main cause of RRD surgery failure. This is a review of recent literature data, which concerns PVR pathogeny and risk factors. The occurrence of pre- and subretinal membranes is a consequence of retinal pigment epithelial cells activation and migration, with concomitant participation of inflammatory cells. The newly synthesized extracellular matrix interacts with cells promoting membrane contraction. Photoreceptor apoptosis limits functional recovery--but there is ongoing research for neuroprotective mechanisms. A lot of evidence has been accumulated about the role of growth factors (PDGF, VEGF, HGF, EGF, TGF α and ß, G-CSF, FGF, IGF-1,CTGF), cytokines (interleukins IL-1, -6, -8, -10 and interferon γ), matrix metalloproteinases and chemokines, by measuring their concentrations in the vitreous or the subretinal fluid of PVR patients. A list of risk factors (common or more controversial) may help the surgeon make the best approach for the management of individual cases. Adjuvant therapies tested for PVR prevention (steroids, heparin, 5 fluorouracil, daunomycin, colchicine and 13-cis retinoic acid) did not enter current practice, but there are numerous research directions currently being developed.

Hearing impairment in Stickler syndrome: a systematic review.

BACKGROUND: Stickler syndrome is a connective tissue disorder characterized by ocular, skeletal, orofacial and auditory defects. It is caused by mutations in different collagen genes, namely COL2A1, COL11A1 and COL11A2 (autosomal dominant inheritance), and COL9A1 and COL9A2 (autosomal recessive inheritance). The auditory phenotype in Stickler syndrome is inconsistently reported. Therefore we performed a systematic review of the literature to give an up-to-date overview of hearing loss in Stickler syndrome, and correlated it with the genotype. METHODS: English-language literature was reviewed through searches of PubMed and Web of Science, in order to find relevant articles describing auditory features in Stickler patients, along with genotype. Prevalences of hearing loss are calculated and correlated with the different affected genes and type of mutation. RESULTS: 313 patients (102 families) individually described in 46 articles were included. Hearing loss was found in 62.9%, mostly mild to moderate when reported. Hearing impairment was predominantly sensorineural (67.8%). Conductive (14.1%) and mixed (18.1%) hearing loss was primarily found in young patients or patients with a palatal defect. Overall, mutations in COL11A1 (82.5%) and COL11A2 (94.1%) seem to be more frequently associated with hearing impairment than mutations in COL2A1 (52.2%). CONCLUSIONS: Hearing impairment in patients with Stickler syndrome is common. Sensorineural hearing loss predominates, but also conductive hearing loss, especially in children and patients with a palatal defect, may occur. The distinct disease-causing collagen genes are associated with a different prevalence of hearing impairment, but still large phenotypic variation exists. Regular auditory follow-up is strongly advised, particularly because many Stickler patients are visually impaired.

Oral vitamin E absorption in English Cocker Spaniels with familial vitamin E deficiency and retinal pigment epithelial dystrophy.

BACKGROUND: Retinal Pigment Epithelial Dystrophy (RPED) with neuroaxonal degeneration in English Cocker Spaniels (ECS) is associated with systemic vitamin E deficiency in the absence of dietary insufficiency. OBJECTIVE: To evaluate the ability of ECS with RPED to absorb orally administered vitamin E and establish a basis for vitamin E supplementation in affected dogs. ANIMALS STUDIED: 8 RPED-affected ECS and five clinically normal dogs. PROCEDURES: An oral vitamin E tolerance test (OVETT) was conducted in each dog. Blood samples were obtained prior to and at 3, 6, 9, 12, 24, 120, and 240 h following oral administration of 90 iu/kg of RRR-α-tocopherol. Plasma alpha tocopherol (αTOC) content was measured by normal phase, high-performance liquid chromatography, and indices of vitamin E absorption calculated. RESULTS: There was marked variation in OVETT results between individuals. In RPED-affected ECS, mean peak plasma αTOC concentration (17.87 ± 13.21 µg/mL), attained after administration of a large oral dose of the vitamin, was significantly lower than the mean peak plasma αTOC concentration attained in normal dogs (47.61 ± 17.17 µg/mL; P < 0.005). However, the plasma concentrations achieved in 7/8 RPED-affected dogs remained within the normal reference range for plasma αTOC in vitamin E-replete dogs, for at least 12 h postdose. CONCLUSIONS: Vitamin E-deficient ECS with RPED are capable of absorbing orally administered vitamin E. Twice daily administration of 600-900 iu tocopherol is likely to restore plasma vitamin E concentrations to the normal range in most affected dogs.

Ceramide production associated with retinal apoptosis after retinal detachment.

BACKGROUND: During retinal detachment, premature apoptosis of photoreceptors and a loss of optimally corrected visual acuity occur. We hypothesized that retinal cell death and generation of ceramide, a pro-apoptotic lipid, would progress as a function of time following experimental retinal detachment, and undertook to define the appropriate temporal window. METHODS: Unilateral retinal detachment was induced in white New Zealand rabbits by subretinal injection of sodium hyaluronate. In experimental animals, we injected sphingosine-1-P into the vitreous 2 hours before retinal detachment. Both eyes were removed on days 1, 3 and 6 for histological and biochemical examination. The number of photoreceptors was counted in section, the level of apoptosis was assessed using the TUNEL assay, and the production of ceramide was analyzed in situ with immunohistochemistry. The concentration of ceramide was also determined on retinal homogenates using a diacylglycerol kinase assay. RESULTS: We confirmed that the average number of live photoreceptors decreased gradually after retinal detachment. In eyes pre-treated with sphingosine-1-P the number of apoptotic photoreceptors was significantly lower. The proportion of apoptotic photoreceptors (14%) remained constant as a function of time in the window studied. As compared to controls, the detached retina showed intense ceramide immunostaining that was prominent in the photoreceptors, but also present to a lesser extent in other retinal layers. The total concentration of intra-retinal ceramide increased by 40% on the first day and continued augmenting through the sixth day after retinal detachment. CONCLUSIONS: Retinal apoptosis during experimental retinal detachment is associated with in vivo production of ceramide.

[The use of flavonoid antioxidants in the complex treatment of patients with peripheral vitreo-chorioretinal dystrophies and dystrophic retinal detachment]. / Primenenie flavonoidnykh antioksidantov v kompleksnom lechenii bol'nykh s perifericheskimi vitreokhorioretinal'nymi distrofiiami i distroficheskoi otsloikoi setchatki.

Parameters of lipid peroxidation (LP) and antioxidant activity (AOA) of tear and blood plasma were examined in 22 healthy subjects (44 eyes) as well as in 33 patients with peripheral vitreochoreoretinal dystrophies (PVCRD--60 eyes), in 32 patients with non-operated dystrophic retinal detachment (DRD--34 eyes) and in 135 patients with operated retinal detachment, stable visual functions and with the postoperative period ranging from 4 months to 10 years (137 eyes). The results denoted a lower tear AOA (on the average by 35%) in patients with DRD and PVCRD, whereas, the blood plasma AOA or LP products' content remained unchanged. It is indicative of a local nature of metabolic impairments, specifically, of impairments in the system of antioxidant protection of the eye. Flavonoid antioxidants (dikvertin and ginkgo biloba) reduced the content of LP products, and induced the AOA in tear and blood plasma in patients with PVCRD and retinal detachment; they also improved the visual functions in patients with operated retinal detachment. Therefore, the flavonoid antioxidants can be recommended for adding to the complex treatment of PVCRD and DRD for the purpose of improving and stabilizing the visual functions and for neuroprotection.

Laser and proton radiation to reduce uveal melanoma-associated exudative retinal detachments.

PURPOSE: To assess whether laser-induced hyperthermia in conjunction with proton irradiation of choroidal melanoma may more rapidly decrease exudative retinal detachments. DESIGN: Case-control study. METHODS: This was a single-center prospective phase 1 study of choroidal melanoma patients with exudative retinal detachments. These tumors did not overhang the optic disc, involve the fovea, or have greater than 40% involvement of the ciliary body. Patients were treated with laser-induced hyperthermia and proton radiation; results were compared with those of similar patients treated at the same institution with only proton radiation. Patients were followed up in an identical manner for loss of subretinal fluid, visual acuity change, and visual field alterations. RESULTS: All 11 patients treated with combined laser and proton therapy had resorption of subretinal fluid with a mean duration of retinal detachment of 193 days, compared with 263 days in the group treated with only proton therapy (P<.04). At 1 year, visual acuity was similar in both groups. CONCLUSIONS: Combined laser-induced hyperthermia and proton radiation may dissipate exudative detachments more rapidly than radiation alone.

Gene therapy for detached retina by adeno-associated virus vector expressing glial cell line-derived neurotrophic factor.

PURPOSE: To examine the protective effect of glial cell line-derived neurotrophic factor (GDNF) on retinal detachment (RD)-induced photoreceptor damage by using gene delivery. METHODS: Gene delivery to photoreceptors was achieved by subretinal injection of recombinant adeno-associated virus expressing GDNF (rAAV-GDNF) in the right eyes and AAV expressing Escherichia coli LacZ (rAAV-LacZ) in the left eyes of Lewis rats. RD in bilateral eyes was induced with subretinal injection of high-density vitreous substitute in the temporal retina 3 weeks after gene delivery. The synthesis and accumulation of GDNF within the retina was monitored 3 weeks after RD by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA), respectively. The rescue of photoreceptors was evaluated by monitoring the preservation of the thickness of photoreceptor outer segment (OS) and outer nuclear layer (ONL). Apoptosis in the photoreceptors was studied using the TdT-dUTP terminal nick-end labeling (TUNEL) method 2 days after RD. Müller cell activity was checked using the immunohistochemistry with glial fibrillary acidic protein (GFAP) antibody 28 days after RD. RESULTS: Gene delivery was demonstrated by immunohistochemical study. The results of ELISA confirmed that high levels of neurotrophic factors were produced in retinas. Photoreceptor OS degeneration and the gradual shortening of the ONL were noted after RD in all the eyes. However, rAAV-GDNF-treated eyes retained longer OS than rAAV-LacZ-treated eyes 7 (P = 0.012) and 28 days (P = 0.008) after RD. ONL was also longer in rAAV-GDNF-treated eyes than in rAAV-LacZ-treated eyes 7 (P = 0.012) and 28 days (P = 0.008) after RD. GDNF-treated eyes had statistically less apoptotic cells than control eyes in photoreceptor layer (P = 0.043). Subretinal proliferation of Müller cells was suppressed in the GDNF-treated group, indicating less scar formation. CONCLUSIONS: GDNF is a potential factor that can protect photoreceptors from degeneration. In addition to preserving the OS and ONL structures, GDNF may exert its protective action by preventing the apoptosis of photoreceptors after RD. GDNF gene therapy may be a valuable adjuvant to current treatments in certain complicated forms of RD.

Limiting the proliferation and reactivity of retinal Müller cells during experimental retinal detachment: the value of oxygen supplementation.

PURPOSE: To assess the role of hypoxia in inducing the proliferation, hypertrophy, and dysfunction of Muller cells in detached retina and the effectiveness of supplemental oxygen in limiting these reactions. METHODS: Retinal detachments were produced in the right eye of each of 13 cats; the cats survived surgery for 3 days, during which six were kept in normoxia (room air, 21%) and seven in hyperoxia (70% oxygen). Retinas were labeled for proliferation with an antibody (MIB-1) to a cell cycle protein (Ki-67), for evidence of hypertrophy employing antibodies to the intermediate filament protein glial fibrillary acidic protein (GFAP) and to beta-tubulin and for disturbance of glutamate neurochemistry employing antibodies to glutamate to a glutamate receptor (GluR-2) and to glutamine synthetase. RESULTS: Results from the two animals kept in normoxia after retinal detachment confirmed previous reports that detachment caused the proliferation of Muller cells, the hypertrophy of Muller cell processes, and the disruption of glutamate recycling by Muller cells. Oxygen supplementation during detachment reduced Muller cell proliferation and hypertrophy and reduced the abnormalities in the distributions of glutamate, GluR-2, and glutamine synthetase. CONCLUSIONS: Oxygen supplementation reduced the reaction of retinal Muller cells to retinal detachment, limiting their proliferation and helping to maintain their normal structure and function. In the clinical setting, oxygen supplementation between diagnosis and reattachment surgery may reduce the incidence and severity of glial-based complications, such as proliferative vitreoretinopathy.