A null mutation in the rhodopsin gene causes rod photoreceptor dysfunction and autosomal recessive retinitis pigmentosa.

Mutations within the rhodopsin gene are known to give rise to autosomal dominant retinitis pigmentosa (RP), a common hereditary form of retinal degeneration. We now describe a patient with autosomal recessive RP who is homozygous for a nonsense mutation at codon 249 within exon 4 of the rhodopsin gene. This null mutation, the first gene defect identified in autosomal recessive retinitis pigmentosa, should result in a functionally inactive rhodopsin protein that is missing the sixth and seventh transmembrane domains including the 11-cis-retinal attachment site. We also found a different null mutation carried heterozygously by an unrelated unaffected individual. Heterozygous carriers of either mutation had normal ophthalmologic examinations but their electroretinograms revealed an abnormality in rod photoreceptor function.

Altered Blood Flow in the Ophthalmic and Internal Carotid Arteries in Patients with Age-Related Macular Degeneration Measured Using Noncontrast MR Angiography at 7T.

BACKGROUND AND PURPOSE: Age-related macular degeneration is associated with reduced perfusion of the eye; however, the role of altered blood flow in the upstream ophthalmic or internal carotid arteries is unclear. We used ultra-high-field MR imaging to investigate whether the diameter of and blood flow in the ophthalmic artery and/or the ICA are altered in age-related macular degeneration and whether any blood flow changes are associated with disease progression. MATERIALS AND METHODS: Twenty-four patients with age-related macular degeneration and 13 similarly-aged healthy controls participated. TOF and high-resolution dynamic 2D phase-contrast MRA (0.26 × 0.26 × 2mm3, 100-ms effective sampling rate) was acquired at 7T. Vessel diameters were calculated from cross-sectional areas in phase-contrast acquisitions. Blood flow time-series were measured across the cardiac cycle. RESULTS: The ophthalmic artery vessel diameter was found to be significantly smaller in patients with age-related macular degeneration than in controls. Volumetric flow through the ophthalmic artery was significantly lower in patients with late age-related macular degeneration, with a significant trend of decreasing volumetric ophthalmic artery flow rates with increasing disease severity. The resistance index was significantly greater in patients with age-related macular degeneration than in controls in the ophthalmic artery. Flow velocity through the ophthalmic artery and ICA was significantly higher in patients with age-related macular degeneration. Ophthalmic artery blood flow as a percentage of ipsilateral ICA blood flow was nearly double in controls than in patients with age-related macular degeneration. CONCLUSIONS: These findings support the hypothesis that vascular changes upstream to the eye are associated with the severity of age-related macular degeneration. Additional investigation into the potential causality of this relationship and whether treatments that improve ocular circulation slow disease progression is warranted.

Sequence-specific interactions between cellular DNA-binding proteins and the adenovirus origin of DNA replication.

The adenovirus origin of DNA replication contains three functionally distinct sequence domains (A, B, and C) that are essential for initiation of DNA synthesis. Previous studies have shown that domain B contains the recognition site for nuclear factor I (NF-I), a cellular protein that is required for optimal initiation. In the studies reported here, we used highly purified NF-I, prepared by DNA recognition site affinity chromatography (P. J. Rosenfeld and T. J. Kelly, Jr., J. Biol. Chem. 261:1398-1408, 1986), to investigate the cellular protein requirements for initiation of viral DNA replication. Our data demonstrate that while NF-I is essential for efficient initiation in vitro, other cellular factors are required as well. A fraction derived from HeLa cell nuclear extract (BR-FT fraction) was shown to contain all the additional cellular proteins required for the complete reconstitution of the initiation reaction. Analysis of this complementing fraction by a gel electrophoresis DNA-binding assay revealed the presence of two site-specific DNA-binding proteins, ORP-A and ORP-C, that recognized sequences in domains A and C, respectively, of the viral origin. Both proteins were purified by DNA recognition site affinity chromatography, and the boundaries of their binding sites were defined by DNase I footprint analysis. Additional characterization of the recognition sequences of ORP-A, NF-I, and ORP-C was accomplished by determining the affinity of the proteins for viral origins containing deletion and base substitution mutations. ORP-C recognized a sequence between nucleotides 41 and 51 of the adenovirus genome, and analysis of mutant origins indicated that efficient initiation of replication is dependent on the presence of a high-affinity ORP-C-binding site. The ORP-A recognition site was localized to the first 12 base pairs of the viral genome within the minimal origin of replication. These data provide evidence that the initiation of adenovirus DNA replication involves multiple protein-DNA interactions at the origin.

The International Intravitreal Bevacizumab Safety Survey: using the internet to assess drug safety worldwide.

AIM: Off-label intravitreal injections of bevacizumab (Avastin) have been given for the treatment of neovascular and exudative ocular diseases since May 2005. Since then, the use of intravitreal bevacizumab has spread worldwide, but the drug-related adverse events associated with its use have been reported only in a few retrospective reviews. The International Intravitreal Bevacizumab Safety Survey was initiated to gather timely information regarding adverse events from doctors around the world via the internet. METHODS: An internet-based survey was designed to identify adverse events associated with intravitreal bevacizumab treatment. The survey web address was disseminated to the international vitreoretinal community via email. Rates of adverse events were calculated from participant responses. RESULTS: 70 centres from 12 countries reported on 7113 injections given to 5228 patients. Doctor-reported adverse events included corneal abrasion, lens injury, endophthalmitis, retinal detachment, inflammation or uveitis, cataract progression, acute vision loss, central retinal artery occlusion, subretinal haemorrhage, retinal pigment epithelium tears, blood pressure elevation, transient ischaemic attack, cerebrovascular accident and death. None of the adverse event rates exceeded 0.21%. CONCLUSION: Intravitreal bevacizumab is being used globally for ocular diseases. Self-reporting of adverse events after intravitreal bevacizumab injections did not show an increased rate of potential drug-related ocular or systemic events. These short-term results suggest that intravitreal bevacizumab seems to be safe.

Long-term outcomes of neovascular glaucoma treated with and without intravitreal bevacizumab.

AIMS: To compare the outcomes of neovascular glaucoma (NVG) treated with and without intravitreal bevacizumab in a large case comparison study. METHODS: The study is a retrospective, comparative, case series of 163 eyes of 151 patients with NVG, including 99 treated without and 64 treated with intravitreal bevacizumab. Medical and surgical treatments for NVG were assessed. The main outcome measures were visual acuity (VA) and intraocular pressure (IOP). RESULTS: At the time of NVG diagnosis, the median VA was count fingers (CF) in the non-bevacizumab group and 2/300 in the bevacizumab group. IOP (mean±SD) was 43.1±13.0 mm Hg in the non-bevacizumab group and 40.8±11.5 mm Hg in the bevacizumab group. IOP (mean±SD) decreased to 18.3±13.8 mm Hg in the non-bevacizumab group and 15.3±8.0 mm Hg in the bevacizumab group, and the median VA was CF in both treatment groups at a mean follow-up of 12 months. Panretinal photocoagulation (PRP) substantially reduced the need for glaucoma surgery (P<0.001) in bevacizumab treated NVG eyes. CONCLUSIONS: Although bevacizumab delayed the need for glaucoma surgery, PRP was the most important factor that reduced the need for surgery. Vision and IOP in eyes with NVG treated with bevacizumab showed no long-term differences when compared with eyes that were not treated with bevacizumab. Thus, intravitreal bevacizumab serves as an effective temporizing treatment, but is not a replacement for close monitoring and definitive treatment of NVG. PRP remains the treatment modality that affects the course of NVG in terms of decreasing the need for surgery to control IOP.

Low incidence of retinitis pigmentosa among heterozygous carriers of a specific rhodopsin splice site mutation.

PURPOSE: To determine whether a rhodopsin splice donor site mutation at the 5' end of intron 4 is a cause of autosomal dominant retinitis pigmentosa. METHODS: Heterozygous carriers of the same rhodopsin splice site mutation in two pedigrees were identified using single-strand conformation polymorphism analysis. Twelve heterozygous carriers were evaluated by ophthalmoscopy. Goldmann kinetic visual fields, dark adaptation thresholds, and full-field electroretinograms including rod intensity-response functions. Clinical findings from the heterozygous carriers of the splice site mutation were compared with those from heterozygous carriers from a separate family with a known recessive rhodopsin null mutation, Glu249X. RESULTS: Analysis of DNA from 48 members of two pedigrees revealed 25 heterozygous carriers of the splice site mutation, ranging in age from 14 to 82 years. There were no homozygotes with the rhodopsin splice site mutation. Of the 25 heterozygous carriers, 24 were asymptomatic. Eleven asymptomatic heterozygotes were examined, including four older than 65 years of age. They were found to have normal fundi, full visual fields, and slightly elevated final rod dark adaptation thresholds. Their rod electroretinographic b-wave amplitudes were slightly diminished over the full range of blue light intensities. Rod a-wave implicit times were slightly but significantly prolonged in response to the brightest blue flash of light. These subtle abnormalities in rod function were similar to those found in asymptomatic heterozygous carriers of the recessive Glu249X mutation. Only one of the 25 heterozygous carriers of the splice site mutation had symptoms and signs of retinitis pigmentosa. CONCLUSIONS: Because 96% of these heterozygous carriers do not have retinitis pigmentosa, it is unlikely that this mutation in intron 4 is a dominant allele. The subtle abnormalities of rod function found in asymptomatic carriers are similar to those found in heterozygous carriers of a recessive rhodopsin allele. The one heterozygous carrier with retinitis pigmentosa probably has a second mutation in the rhodopsin gene or has a defect or defects in another gene that causes his disease.

Mutations in ABCR (ABCA4) in patients with Stargardt macular degeneration or cone-rod degeneration.

PURPOSE: To determine the spectrum of ABCR mutations associated with Stargardt macular degeneration and cone-rod degeneration (CRD). METHODS: One hundred eighteen unrelated patients with recessive Stargardt macular degeneration and eight with recessive CRD were screened for mutations in ABCR (ABCA4) by single-strand conformation polymorphism analysis. Variants were characterized by direct genomic sequencing. Segregation analysis was performed on the families of 20 patients in whom at least two or more likely pathogenic sequence changes were identified. RESULTS: The authors found 77 sequence changes likely to be pathogenic: 21 null mutations (15 novel), 55 missense changes (26 novel), and one deletion of a consensus glycosylation site (also novel). Fifty-two patients with Stargardt macular degeneration (44% of those screened) and five with CRD each had two of these sequence changes or were homozygous for one of them. Segregation analyses in the families of 19 of these patients were informative and revealed that the index cases and all available affected siblings were compound heterozygotes or homozygotes. The authors found one instance of an apparently de novo mutation, Ile824Thr, in a patient. Thirty-seven (31%) of the 118 patients with Stargardt disease and one with CRD had only one likely pathogenic sequence change. Twenty-nine patients with Stargardt disease (25%) and two with CRD had no identified sequence changes. CONCLUSIONS: This report of 42 novel mutations brings the growing number of identified likely pathogenic sequence changes in ABCR to approximately 250.

Purtscher-like retinopathy associated with pancreatic adenocarcinoma.

PURPOSE: To investigate a case of Purtscher-like retinopathy that occurred in association with pancreatic adenocarcinoma. METHOD: Case report. RESULTS: A 63-year-old woman presented with multiple gray patches in the central vision of both eyes. Visual acuity was 20/20 in both eyes. Funduscopy showed large peripapillary yellow-white patches within the superficial retina and small superficial retinal hemorrhages in both eyes. The patient subsequently had abdominal pain. Computed tomography of the abdomen demonstrated a large pancreatic mass with extension into the liver. Histologic examination of a percutaneous needle biopsy specimen showed mucinous pancreatic adenocarcinoma. CONCLUSION: Pancreatic adenocarcinoma should be added to the list of systemic diseases that can be associated with Purtscher-like retinopathy.

Age-related maculopathy: an expanded genome-wide scan with evidence of susceptibility loci within the 1q31 and 17q25 regions.

PURPOSE: We seek to identify genetic loci that contribute to age-related maculopathy susceptibility. METHODS: Families consisting of at least two siblings affected by age-related maculopathy were ascertained using eye care records and fundus photographs. Additional family members were used to increase the power to detect linkage. Microsatellite genotyping was conducted by the National Heart, Lung and Blood Institute Mammalian Genotyping Service and the National Institutes of Health Center for Inherited Disease Research. Linkage analyses were conducted with parametric (autosomal dominant; heterogeneity lod score) and nonparametric methods (S(all) statistic) using three diagnostic models. False-positive rates were determined from simulations using actual pedigrees and genotyping data. RESULTS: Under our least stringent diagnostic model, model C, 860 affected individuals from 391 families (452 sib pairs) were genotyped. Sixty-five percent of the affected individuals had evidence of exudative disease. Four regions, 1q31, 9p13, 10q26, and 17q25, showed multipoint heterogeneity lod scores or S(all) scores of 2.0 or greater (under at least one model). Under our most stringent diagnostic model, model A, the 1q31 heterogeneity lod score was 2.46 between D1S1660 and D1S1647. Under model C, the 17q25 heterogeneity lod score at D17S928 was 3.16. Using a threshold of 1.5, additional loci on chromosomes 2 and 12 were identified. CONCLUSIONS: The locus on chromosome 1q31 independently confirms a report by Klein and associates mapping an age-related maculopathy susceptibility gene to this region. Simulations indicate that the 1q31 and 17q25 loci are unlikely to be false positives. There was no evidence that other known macular or retinal dystrophy candidate gene regions are major contributors to the genetics of age-related maculopathy.

Persistent indocyanine green angiographic findings in multiple evanescent white dot syndrome.

This report describes unique findings of persistent peripapillary and posterior pole hypofluorescence on indocyanine green angiography (ICGA) in multiple evanescent white dot syndrome (MEWDS). A 38-year-old woman experienced a sudden decrease of visual acuity in the left eye. Multiple white lesions were seen on fundus examination. Fluorescein angiography, indocyanine green angiography, and automated perimetry were performed. Fundus appearance and fluorescein angiography were consistent with the diagnosis of MEWDS. Automated perimetry revealed an enlarged blind spot. ICGA revealed a zone of hypofluorescence surrounding the optic disc and throughout the posterior pole. The enlarged blind spot resolved after seven weeks along with the signs and symptoms of MEWDS. Nine months after initial presentation, ICGA revealed persistent peripapillary and posterior pole hypofluorescence. Resolution of the enlarged blind spot and return of vision does not completely correlate with the disappearance of hypofluorescent areas on ICGA. These findings suggest that MEWDS may result in persistent abnormalities in choroidal circulation even after clinical symptoms resolve.

Outcomes of vitreoretinal surgery in patients with X-linked retinoschisis.

BACKGROUND AND OBJECTIVE: To assess the outcomes of vitreoretinal surgery in the treatment of vision-threatening posterior segment complications of X-linked retinoschisis. PATIENTS AND METHODS: The authors performed a retrospective analysis of 16 eyes from 11 patients who underwent vitreoretinal surgery. All the patients had a documented positive family history of X-linked retinoschisis, and all patients had bilateral macular disease. RESULTS: The ages of the patients ranged from 14 months to 37 years (mean age 15.1 years; median age 11.5 years), and postoperative follow-up ranged from 3 months to 10 years (mean 2.8 years; median 1 year). The indications for surgical intervention included rhegmatogenous retinal detachment (12 eyes), vitreous hemorrhage (2 eyes), progression of the schisis cavity through the fovea (2 eyes), cataract associated with a persistent hyperplastic primary vitreous-like condition (2 eyes), and exudative maculopathy (1 eye). The primary surgical intervention included pars plana vitrectomy alone (7 eyes), pars plana vitrectomy and pars plana lensectomy (4 eyes), and a scleral buckle procedure alone (5 eyes). Surgical success (defined as reattachment of the retina, removal of media opacities, or arrest of schisis progression) was achieved in 14 of 16 eyes, after an average of 1.2 procedures per eye. The major reason for reoperations was recurrent retinal detachment due to proliferative vitreoretinopathy. Two eyes were eventually enucleated due to pain associated with neovascular glaucoma resulting from recurrent retinal detachment. Of the remaining 14 eyes, visual acuity improved in 8 eyes and remained unchanged in 6 eyes. CONCLUSION: Vitreoretinal surgery is often helpful in stabilizing or improving visual function in patients with posterior segment complications from X-linked retinoschisis.

Outcomes of treatment of neovascular glaucoma with intravitreal bevacizumab.

BACKGROUND/AIMS: To evaluate the course of treatment and outcomes of neovascular glaucoma (NVG) treated with intravitreal bevacizumab. METHODS: The study is a retrospective, non-comparative, consecutive, interventional case series. Demographic data, past ocular history, cause of NVG and anterior chamber angle status were recorded. Visual acuity (VA), intraocular pressure (IOP), number of IOP-lowering medications and type of treatment administered were recorded at the time of NVG diagnosis and at follow-up intervals. Treatment-related complications and reasons for vision loss were recorded. RESULTS: The study included 56 eyes of 52 patients. At the time of NVG diagnosis, the median VA was count fingers, and the mean IOP (SD) was 40 (11) mm Hg. At 6 months after initial bevacizumab injection, the median VA was 1/200, and the mean IOP (SD) was 18 (15) mm Hg. Seventy-one per cent of eyes underwent panretinal photocoagulation after NVG diagnosis. Sixty-one per cent of eyes received a glaucoma drainage implant (GDI). The Kaplan-Meier cumulative proportion of eyes with open angles receiving a GDI after initial bevacizumab injection was not statistically significantly different from that of eyes with closed angles. Forty-six per cent of eyes received repeat bevacizumab injections. Eleven eyes had hyphaema after both bevacizumab injection and GDI surgery, while three eyes had hyphaema after GDI surgery but prior to initial bevacizumab injection. CONCLUSIONS: Intravitreal bevacizumab is now a frequently used adjunct for the treatment of NVG. Eyes must be monitored closely after initial injection of intravitreal bevacizumab, regardless of initial angle status, as many may still require surgery to lower IOP or repeat injections of intravitreal bevacizumab.

Macular spectral-domain optical coherence tomography in patients with X linked retinoschisis.

AIM: To evaluate macular anatomy in patients with X linked retinoschisis (XLRS) using spectral-domain optical coherence tomography (SD-OCT). METHODS: Consecutive observational case series. Clinical features were obtained through retrospective chart review. Only eyes without prior surgical interventions and those scanned with SD-OCT were included. The OCT images were analysed. RESULTS: Fourteen eyes of seven males with XLRS scanned with SD-OCT, age 5 to 45 years, were identified. On clinical examination, stellate spoke-like cystic maculopathy was present in nine eyes, and an atrophic foveal lesion in five eyes. SD-OCT revealed cystoid spaces accounting for retinal splitting in the inner nuclear layer in 12 eyes, and outer plexiform layer in two eyes of one patient. A few small cysts, not accounting for the foveal splitting, were seen in the outer nuclear layer in four eyes and in the ganglion cell layer and/or nerve fibre layer in six eyes. CONCLUSIONS: SD-OCT localised the foveomacular retinoschisis in XLRS to the retinal layers deeper than the nerve fibre layer. In the present study, the foveomacular schisis was seen most frequently in the inner nuclear layer.

Predicted biological activity of intravitreal VEGF Trap.

AIM: To compare the intravitreal binding activity of VEGF Trap with that of ranibizumab against vascular endothelial growth factor (VEGF) using a time-dependent and dose-dependent mathematical model. METHODS: Intravitreal half-lives and relative equimolar VEGF-binding activities of VEGF Trap and ranibizumab were incorporated into a first-order decay model. Time-dependent VEGF Trap activities (relative to ranibizumab) for different initial doses (0.5, 1.15, 2 and 4 mg) were calculated and plotted. RESULTS: Seventy-nine days after a single VEGF Trap (1.15 mg) injection, the intravitreal VEGF-binding activity would be comparable to ranibizumab at 30 days. After injection of 0.5, 2 and 4 mg VEGF Trap, the intravitreal VEGF-binding activities (comparable to ranibizumab at 30 days) would occur at 73, 83 and 87 days, respectively CONCLUSION: On the basis of this mathematical model, VEGF Trap maintains significant intravitreal VEGF-binding activity for 10-12 weeks after a single injection.

[Treatment of neovascular age-related macular degeneration with Ranibizumab/Lucentis]. / Therapie der neovaskulären altersbedingten Makuladegeneration mit Ranibizumab/Lucentis.

Vascular endothelial growth factor (VEGF) is considered to play an essential role in the pathogenesis of age-related macular degeneration due to its vascular permeability-inducing and angiogenic properties. Ranibizumab, a small antibody fragment designed to competitively bind all VEGF isoforms, passes after intravitreal injection all retinal layers reaching the retinal pigment epithelium-choroid complex. Experimental animal models showed the drug to be safe and effective. Subsequently, Phase I/II clinical trials conducted in patients with neovascular AMD demonstrated a good safety profile, and a significant functional benefit. Ranibizumab therapy repeated every four weeks for the treatment of neovascular AMD is currently in Phase III clinical trials. Combination therapy trials aiming for improved treatment durability and effectiveness are currently ongoing as well as new treatment strategies using intermittent, optical coherence tomography (OCT) guided therapy. Anti-VEGF therapy using Ranibizumab is a promising new treatment option for neovascular AMD.

Purification of nuclear factor I by DNA recognition site affinity chromatography.

Nuclear factor I (NF-I) is a cellular protein that enhances the initiation of adenovirus DNA replication in vitro. The enhancement of initiation correlates with the ability of NF-I to bind a specific nucleotide sequence within the viral origin of replication. We have developed a method for the purification of NF-I which is based upon the high affinity interaction between the protein and its recognition site. This approach may be generally applicable to the purification of other site-specific DNA binding proteins. The essential feature of the method is a two-step column chromatographic procedure in which proteins are first fractionated on an affinity matrix consisting of nonspecific (Escherichia coli) DNA and then on a matrix that is highly enriched in the specific DNA sequence that is recognized by NF-I. During the first step NF-I coelutes with proteins that have similar general affinity for DNA. During the second step NF-I elutes at a much higher ionic strength than the contaminating nonspecific DNA binding proteins. The DNA recognition site affinity matrix used in the second step is prepared from a plasmid (pKB67-88) that contains 88 copies of the NF-I binding site. This plasmid was constructed by means of a novel cloning strategy that generates concatenated NF-I binding sites arranged exclusively in a direct head to tail configuration. Using this purification scheme, we have obtained a 2400-fold purification of NF-I from crude HeLa nuclear extract with a 57% recovery of specific DNA binding activity. Throughout the purification procedure NF-I retained the ability to enhance the efficiency of initiation of adenovirus DNA replication in vitro. Electrophoresis of the purified fraction on sodium dodecyl sulfate-polyacrylamide gels revealed a population of related polypeptides that ranged in apparent molecular weight from 66,000 to 52,000. The native molecular weight of NF-I deduced from gel filtration and glycerol sedimentation studies is 55,000 and the frictional ratio is 1.3. These results suggest that NF-I exists as a globular monomer in solution.

A cellular DNA-binding protein that activates eukaryotic transcription and DNA replication.

Transcription factor CTF, which is responsible for selective recognition of eukaryotic promoters that contain the sequence CCAAT, was purified to apparent homogeneity by sequence-specific DNA affinity chromatography. Binding sites for CTF in the human Ha-ras and alpha-globin promoters were highly homologous to sequences recognized by nuclear factor I (NF-I), a cellular DNA-binding protein that is required for the initiation of adenovirus DNA replication in vitro. To determine the relationship between CTF and NF-I, we compared the biochemical properties of these two proteins. CTF and NF-I were found to be indistinguishable in polypeptide composition, DNA-binding properties, immunological cross-reactivity, and in vitro stimulation of DNA replication and transcription initiation. We conclude that CTF/NF-I can serve both as a transcription selectivity factor for RNA polymerase II and as an initiation factor for adenovirus DNA replication.