Mutations in RPGR and RP2 account for 15% of males with simplex retinal degenerative disease.

PURPOSE: To determine the proportion of male patients presenting simplex retinal degenerative disease (RD: retinitis pigmentosa [RP] or cone/cone-rod dystrophy [COD/CORD]) with mutations in the X-linked retinal degeneration genes RPGR and RP2. METHODS: Simplex males were defined as patients with no known affected family members. Patients were excluded if they had a family history of parental consanguinity. Blood samples from a total of 214 simplex males with a diagnosis of retinal degeneration were collected for genetic analysis. The patients were screened for mutations in RPGR and RP2 by direct sequencing of PCR-amplified genomic DNA. RESULTS: We identified pathogenic mutations in 32 of the 214 patients screened (15%). Of the 29 patients with a diagnosis of COD/CORD, four mutations were identified in the ORF15 mutational hotspot of the RPGR gene. Of the 185 RP patients, three patients had mutations in RP2 and 25 had RPGR mutations (including 12 in the ORF15 region). CONCLUSIONS: This study represents mutation screening of RPGR and RP2 in the largest cohort, to date, of simplex males affected with RP or COD/CORD. Our results demonstrate a substantial contribution of RPGR mutations to retinal degenerations, and in particular, to simplex RP. Based on our findings, we suggest that RPGR should be considered as a first tier gene for screening isolated males with retinal degeneration.

Behçet disease in children.

PURPOSE: To investigate the incidence and clinical characteristics of Behçet disease in children. METHODS: The authors retrospectively reviewed the charts of 3382 patients with Behçet disease from October 1986 to December 2005 at Ankara University Medical School Behçet Unit and/or Atmaca private clinic. RESULTS: 110 children were diagnosed with Behçet disease. The 110 children represented 3.3% of the total number of Behçet patients that were reviewed. 62.7% were girls and 37.3% were boys. The mean age at initial manifestation was 11.63 ± 3.46 years and at diagnosis was 14.15 ± 2.13 years. Ocular involvement was found in 30.9% of the children. Of these, 61.8% were bilateral and 38.2% were unilateral. Anterior uveitis was found in 18 eyes (32.8%), posterior uveitis in 24 eyes (43.6%), and panuveitis in 13 eyes (23.6%). CONCLUSION: Behçet disease should be considered in the differential diagnosis of childhood uveitis, particularly in endemic areas. The collaboration of pediatricians, rheumatologists, dermatologists, ophthalmologists, and other specialists when necessary is required in the diagnosis and management of children with Behçet disease.

Laser treatment in 341 patients with exudative age-related macular degeneration.

AIM: To document the prognosis of laser treatment in patients with exudative age-related macular degeneration (AMD). METHODS: Efficacy of the intervention was evaluated using a before-after method. RESULTS: A total of 392 eyes of 341 patients with exudative AMD were examined. 77.6% had choroideal neovascularisation (CNV). Before the use of indocyanine green (ICG) angiography, occult CNV was detected in only 1.8% of the eyes, but after the use of ICG angiography, this increased to 19.5% (P<0.001). Of the 349 eyes which were followed up, visual acuity had remained stable in 68.2%. There was a statistically significant relationship between the localization of lesion and visual acuity changes on pre- and post-laser treatment (P<0.001). Also there was a statistically significant relationship between the localization of lesion and recurrence (P<0.05). The recurrence was less in subfoveal lesions than that in juxtafoveal and extrafoveal lesions. CONCLUSION: ICG angiography is highly important in the treatment of occult CNV.

Ocular involvement in sarcoidosis.

PURPOSE: To evaluate ocular involvement in patients with sarcoidosis. METHODS: The prevalence of ocular manifestations was studied in 139 Turkish patients with sarcoidosis as a cross section. Color photography and fluorescein angiography were performed on patients suspected of having ocular involvement. RESULTS: Ocular involvement was seen in 12.9% patients. Of 18 patients with ocular sarcoidosis, 39% had anterior and posterior involvement, 28% only anterior, 11% only posterior, 16.7% conjuctival, and 5.6% eyelid involvement. There was no statistically significant relationship between radiological grading and ocular involvement. CONCLUSIONS: The most common ocular involvement in this study was both anterior and posterior segment involvement.

Endogenous bone marrow derived cells express retinal pigment epithelium cell markers and migrate to focal areas of RPE damage.

PURPOSE: The aim of the present study was to investigate whether bone marrow-derived cells (BMCs) can be induced to express retinal pigment epithelial (RPE) cell markers in vitro and can home to the site of RPE damage after mobilization and express markers of RPE lineage in vivo. METHODS: Adult RPE cells were cocultured with green fluorescence protein (GFP)-labeled stem cell antigen-1 positive (Sca-1(+)) BMCs for 1, 2, and 3 weeks. Cell morphology and expression of RPE-specific markers and markers for other retinal cell types were studied. Using an animal model of sodium iodate (NaIO(3))-induced RPE degeneration, BMCs were mobilized into the peripheral circulation by granulocyte-colony stimulating factor, flt3 ligand, or both. Immunocytochemistry was used to identify and characterize BMCs in the subretinal space in C57BL/6 wild-type (wt) mice and GFP chimeric mice. RESULTS: In vitro, BMCs changed from round to flattened, polygonal cells and expressed cytokeratin, RPE65, and microphthalmia transcription factor (MITF) when cocultured in direct cell-cell contact with RPE. In vivo, BMCs were identified in the subretinal space as Sca-1(+) or c-kit(+) cells. They were also double labeled for GFP and RPE65 or MITF. These cells formed a monolayer on the Bruch membrane in focal areas of RPE damage. CONCLUSIONS: Thus, it appears that BMCs, when mobilized into the peripheral circulation, can home to focal areas of RPE damage and express cell markers of RPE lineage. The use of endogenous BMCs to replace damaged retinal tissue opens new possibilities for cell replacement therapy in ophthalmology.

Cone photoreceptor function loss-3, a novel mouse model of achromatopsia due to a mutation in Gnat2.

PURPOSE: To report a novel mouse model of achromatopsia with a cpfl3 mutation found in the ALS/LtJ strain. METHODS: The effects of a cpfl3 mutation were documented using fundus photography, electroretinography (ERG), and histopathology. Genetic analysis was performed using linkage studies and PCR gene identification. RESULTS: Homozygous cpfl3 mice had poor cone-mediated responses on ERG at 3 weeks that became undetectable by 9 months. Rod-mediated waveforms were initially normal, but declined with age. Microscopy of the retinas revealed progressive vacuolization of the photoreceptor outer segments. Immunocytochemistry with cone-specific markers showed progressive loss of labeling for alpha-transducin, but the cone outer segments in the oldest mice examined remained intact and positive with peanut agglutinin (PNA). The cpfl3 mapped to mouse chromosome 3 at the same location as human GNAT2, known to cause achromatopsia. Sequence analysis revealed a missense mutation due to a single base pair substitution in exon 6 in cpfl3. CONCLUSIONS: The Gnat2(cpfl3) mutation leads to cone dysfunction and the progressive loss of cone alpha-transducin immunolabeling. Despite a poor cone ERG signal and loss of cone alpha-transducin label, the cones survive at 14 weeks as demonstrated by PNA staining. This mouse model of achromatopsia will be useful in the study of the development, pathophysiology, and treatment of achromatopsia and other cone degenerations. The gene symbol for the cpfl3 mutation has been changed to Gnat2(cpfl3).

Systemically transferred hematopoietic stem cells home to the subretinal space and express RPE-65 in a mouse model of retinal pigment epithelium damage.

Stem cell regeneration of damaged tissue has recently been reported in many different organs. Since the loss of retinal pigment epithelium (RPE) in the eye is associated with a major cause of visual loss - specifically, age-related macular degeneration - we investigated whether hematopoietic stem cells (HSC) given systemically can home to the damaged subretinal space and express markers of RPE lineage. Green fluorescent protein (GFP) cells of bone marrow origin were used in a sodium iodate (NaIO(3)) model of RPE damage in the mouse. The optimal time for adoptive transfer of bone marrow-derived stem cells relative to the time of injury and the optimal cell type [whole bone marrow, mobilized peripheral blood, HSC, facilitating cells (FC)] were determined by counting the number of GFP(+) cells in whole eye flat mounts. Immunocytochemistry was performed to identify the bone marrow origin of the cells in the RPE using antibodies for CD45, Sca-1, and c-kit, as well as the expression of the RPE-specific marker, RPE-65. The time at which bone marrow-derived cells were adoptively transferred relative to the time of NaIO(3) injection did not significantly influence the number of cells that homed to the subretinal space. At both one and two weeks after intravenous (i.v.) injection, GFP(+) cells of bone marrow origin were observed in the damaged subretinal space, at sites of RPE loss, but not in the normal subretinal space. The combined transplantation of HSC+FC cells appeared to favor the survival of the homed stem cells at two weeks, and RPE-65 was expressed by adoptively transferred HSC by four weeks. We have shown that systemically injected HSC homed to the subretinal space in the presence of RPE damage and that FC promoted survival of these cells. Furthermore, the RPE-specific marker RPE-65 was expressed on adoptively transferred HSC in the denuded areas.

CFH haplotypes without the Y402H coding variant show strong association with susceptibility to age-related macular degeneration.

In developed countries, age-related macular degeneration is a common cause of blindness in the elderly. A common polymorphism, encoding the sequence variation Y402H in complement factor H (CFH), has been strongly associated with disease susceptibility. Here, we examined 84 polymorphisms in and around CFH in 726 affected individuals (including 544 unrelated individuals) and 268 unrelated controls. In this sample, 20 of these polymorphisms showed stronger association with disease susceptibility than the Y402H variant. Further, no single polymorphism could account for the contribution of the CFH locus to disease susceptibility. Instead, multiple polymorphisms defined a set of four common haplotypes (of which two were associated with disease susceptibility and two seemed to be protective) and multiple rare haplotypes (associated with increased susceptibility in aggregate). Our results suggest that there are multiple disease susceptibility alleles in the region and that noncoding CFH variants play a role in disease susceptibility.

Fluorescein and indocyanine green angiography in ocular toxoplasmosis.

PURPOSE: To document fluorescein and indocyanine green angiographic findings in patients with ocular toxoplasmosis. METHODS: Charts of patients with ocular toxoplasmosis who were evaluated with fluorescein and indocyanine green angiograpy were reviewed. RESULTS: In this study, eight (38%) females and 13 (62%) males with a mean age of 20.3 years were included. Of the 21 patients, five (24%) had bilateral involvement with active or inactive toxoplasmic lesion. There were active lesions in 12 (46%) eyes and inactive lesions in 14 (54%) eyes. Indocyanine green angiograpy showed hypofluorescence of the active and inactive retinochoroiditis lesions at all phases. Hypofluorescent multiple satellite dark dots were observed in 11 (92%) eyes with active retinochoroiditis and in two (14%) eyes with inactive lesions. In two patients with unilateral active toxoplasmic retinochoroiditis, hyperfluorescent plaques were observed in the fellow eyes on indocyanine green angiograpy. The fundus examination and fluorescein angiography of the fellow eyes were normal and had a visual acuity of 10/10. Choroidal neovascularization was observed in two (8%) eyes. In eyes with active inflammation, fluorescein angiography revealed early hypo-fluorescence and late intense hyper-fluorescence with fuzzy margins of the retinochoroiditis lesion (12 eyes), hyperfluorescence of the optic nerve head (four eyes) and leakage from the vessels and/or vascular sheathing (four eyes) and neuroretinitis (one eye). CONCLUSION: Toxoplasmic retinochoroiditis is a more widespread inflammation than visible fundus lesions. Indocyanine green angiography is a useful method for evaluating the amount of inflammatory activity and late complications in patients with ocular toxoplasmosis.

Retinal pigment epithelium damage enhances expression of chemoattractants and migration of bone marrow-derived stem cells.

PURPOSE: To characterize chemoattractants expressed by the retinal pigment epithelium (RPE) after sodium iodate (NaIO3)-induced damage and to investigate whether ocular-committed stem cells preexist in the bone marrow (BM) and migrate in response to the chemoattractive signals expressed by the damaged RPE. METHODS: C57/BL6 mice were treated with a single intravenous injection of NaIO3 (50 mg/kg) to create RPE damage. At different time points real-time RT-PCR, ELISA, and immunohistochemistry were used to identify chemoattractants secreted in the subretinal space. Conditioned medium from NaIO3-treated mouse RPE was used in an in vitro assay to assess chemotaxis of stem cell antigen-1 positive (Sca-1+) BM mononuclear cells (MNCs). The expression of early ocular markers (MITF, Pax-6, Six-3, Otx) in migrated cells and in MNCs isolated from granulocyte colony-stimulating factor (G-CSF) and Flt3 ligand (FL)-mobilized and nonmobilized peripheral blood (PB) was analyzed by real-time RT-PCR. RESULTS: mRNA for stromal cell-derived factor-1 (SDF-1), C3, hepatocyte growth factor (HGF), and leukemia inhibitory factor (LIF) was significantly increased, and higher SDF-1 and C3 protein secretion from the RPE was found after NaIO3 treatment. A higher number of BMMNCs expressing early ocular markers migrated to conditioned medium from damaged retina. There was also increased expression of early ocular markers in PBMNCs after mobilization. CONCLUSIONS: Damaged RPE secretes cytokines that have been shown to serve as chemoattractants for BM-derived stem cells (BMSCs). Retina-committed stem cells appear to reside in the BM and can be mobilized into the PB by G-CSF and FL. These stem cells may have the potential to serve as an endogenous source for tissue regeneration after RPE damage.

A long-term follow-up of Eales' disease.

PURPOSE: To provide long-term follow-up information on Eales' patients. METHODS: Eales' patients, who had been examined at varying periods between the years 1970 and 1991 with a minimum five-year follow-up, were included in the study. RESULTS: A total of 130 patients were followed up for a minimum of five and a maximum of 26.5 years. The retinal lesions found during the first examination included vascular sheathing, disc and/or retinal neovascularization, vitreous hemorrhage, branch retinal vein occlusion, retinitis proliferans, and retinal detachment. Visual acuity improved in 37 (20%) of the 185 treated eyes, was maintained in 79 (43%), and worsened in 69 (37%). The complications were tractional detachment, cataract, rubeosis iridis, neovascular glaucoma, and phthisis bulbi. CONCLUSION: The most important elements in dealing with Eales' disease are periodic follow-up, a good and adequate laser treatment, pars plana vitrectomy combined with procedures for nonclearing vitreous hemorrhage, and retinal vasoproliferative changes to stabilize the retinal lesions and maintain functional levels of vision.