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Koji M Nishiguchi,
Tetsuhiro R Yasuma,
Daisuke Tomida,
Makoto Nakamura,
Kohei Ishikawa,
Masato Kikuchi,
Yuhsuke Ohmi,
Toshimitsu Niwa,
Nobuyuki Hamajima,
Koichi Furukawa,
Hiroko Terasaki
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ABSTRACT: A non-sense mutation at codon 95 in the gene encoding complement factor C9 (C9-R95X) is found most frequently among Japanese. The authors investigated the association between C9-R95X and Japanese patients with neovascular age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV).
The presence of the C9-R95X polymorphism was assessed by direct sequencing in Japanese patients with either PCV (n = 105) or neovascular AMD (n = 198) and 396 control subjects. Multivariate regression analyses were conducted. Photocoagulation was applied in the eyes of mice with a heterozygous defect in the C3 gene and control wild-type mice. Photocoagulation was also applied to wild-type mice before either anti-C9 antibody or isotype IgG was injected into the eyes. The eyes were collected later for measurement of vascular endothelial growth factor (VEGF) and histological evaluation of choroidal neovascularization (CNV).
The frequency of those with one or two C9-R95X variants was lower in neovascular AMD (2.02%) than in PCV (5.71%) and controls (6.05%). The presence of C9-R95X conferred a 4.7-fold reduction (95% confidence interval, 1.2-18.1; P = 0.021) in the risk for neovascular AMD after adjusting for the major AMD risk factors. A heterozygous defect in the C3 gene was associated with the reduced growth of laser-induced CNV, as was intraocular injection of anti-C9 antibody. This reduced CNV growth was accompanied by a decreased level of secreted VEGF in the intraocular fluid.
These findings support the notion that the haploinsufficiency of C9, a terminal complement complex component, engenders reduced intraocular secretion of VEGF and decreased risk for CNV development.
Investigative ophthalmology & visual science 12/2011; 53(1):508-12. · 3.43 Impact Factor
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ABSTRACT: To study the effect of heparin on the development of laser-induced choroidal neovascularization (CNV) and to assess the underlying molecular mechanisms.
Bone marrow transplantation (BMT) was conducted by intravenous injection of green fluorescence protein (GFP)-labeled bone marrow cells (1 × 10(7) cells) into irradiated (9 Gy) C57BL/6J mice. Laser photocoagulation was applied to induce CNV; subsequently, unfractionated heparin or phosphate-buffered saline was injected into mice that did or did not undergo BMT. The area of CNV, distribution of injected heparin, and quantities of infiltrating cells positive for Griffonia simplicifolia (GS) and GFP inside and outside the CNV were evaluated. Effects of heparin on the secretion of VEGF, CCL2, and TNF-α by ARPE19 cells and on the binding of VEGF, CCL2, TNF-α, and their receptors were analyzed in vitro.
Intravitreal injection of heparin at higher doses reduced the size of the CNV. Heparin localized at the vascular structures and photoreceptor layers adjacent to the laser scar. Only GS-positive cells infiltrating outside the CNV were reduced significantly, but not those inside the CNV or those expressing GFP. Relative decreases in VEGF and CCL2 levels were observed in media of ARPE19 cells at higher heparin concentrations. In vitro binding assays revealed that heparin and porcine ocular fluid, respectively, suppressed the binding of VEGF to VEGFR2 and CCL2 to CCR2.
Intravitreal heparin injection inhibited CNV development. Reduced VEGF and CCL2 secretion by RPE cells and suppression of VEGF-VEGFR2 and CCL2-CCR2 interactions at the laser site mediated by heparin may contribute to the pharmacologic effect.
Investigative ophthalmology & visual science 02/2011; 52(6):3193-9. · 3.43 Impact Factor
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ABSTRACT: To analyze the roles of vitreal macrophages and circulating leukocytes in retinal vascular growth.
Bone marrow (BM) cells from green fluorescent protein (GFP) transgenic mice were transplanted into postnatal day (P)1 mice after irradiation. The mice were exposed to 76% to 78% oxygen (P7-P12), to initiate oxygen-induced retinopathy (OIR). The eyes were collected at P8, P17, and P30, to analyze the engraftment of GFP-positive cells in the retina. GFP-positive peritoneal macrophages, clodronate liposomes, or control liposomes were injected into the eyes at P5 or P12 to examine the effects at P8 or P17. The number of Iba1-positive vitreal macrophages was quantified from histologic sections at P12 and P17.
Few transplanted GFP-positive cells were found in the retina at P8 in both wild-type and OIR mice. However, their number increased at P17 during retinal neovascularization in OIR. Most GFP-positive cells were Iba1-positive microglia, which comprised a minority of the total retinal microglia. Intravitreal injection of peritoneal macrophages showed only incidental migration of these cells into the wild-type retinas (P8), whereas the engraftment was more robust, typically around the neovascularization, in OIR mice (P17). Furthermore, native macrophages in the vitreous cavity became fewer (37.7% reduction) during neovascularization in OIR at P17. The selective depletion of vitreal macrophages by clodronate liposomes at P12 reduced retinal neovascularization in OIR mice by 59.0% at P17.
Vitreal macrophages are attracted to the site of pathologic angiogenesis triggered by retinal ischemia, where they actively participate in vascular development.
Investigative ophthalmology & visual science 11/2010; 52(3):1431-8. · 3.43 Impact Factor
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ABSTRACT: To identify human transient receptor potential cation channel, subfamily M, member 1 (TRPM1) gene mutations in patients with congenital stationary night blindness (CSNB).
We analyzed four different Japanese patients with complete CSNB in whom previous molecular examination revealed no mutation in either nyctalopin (NYX) or glutamate receptor, metabotropic 6 (GRM6). The ophthalmologic examination included best-corrected visual acuity, refraction, biomicroscopy, ophthalmoscopy, fundus photography, Goldmann kinetic perimetry, color vision tests, and electroretinography (ERG). Exons 2 through 27 and the exon-intron junction regions of human TRPM1 were sequenced.
Five different mutations in human TRPM1 were identified. Mutations were present in three unrelated patients with complete CSNB. All three patients were compound heterozygotes. Fundus examination revealed no abnormalities other than myopic changes, and the single bright-flash, mixed rod-cone ERG showed a "negative-type" configuration with a reduced normal a-wave and a significantly reduced b-wave amplitude. Our biochemical and cell biologic analyses suggest that the two identified IVS mutations lead to abnormal TRPM1 protein production, and imply that the two identified missense mutations lead to the mislocalization of the TRPM1 protein in bipolar cells (BCs).
Human TRPM1 mutations are associated with the complete form of CSNB in Japanese patients, suggesting that TRPM1 plays an essential role in mediating the photoresponse in ON BCs in humans as well as in mice.
Molecular vision 01/2010; 16:425-37. · 2.20 Impact Factor
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ABSTRACT: To investigate the association between the serum high sensitivity C-reactive protein (hs-CRP) levels and variants in age-related maculopathy susceptibility 2 (ARMS2)/HtrA serine peptidase 1 (HTRA1) genes in normal subjects with no evidence of age-related macular degeneration (AMD).
After clinical evaluation, information related to medical and social history was collected from 476 Japanese individuals (age range 17-89 years) along with blood samples. These subjects were medical checkup participants recruited at Nagoya University Hospital with no macular disease, as confirmed by fundus photographs. Serum hs-CRP levels were measured using a highly sensitive latex aggregation immunoassay. The genotypes of three polymorphisms in the ARMS2/HTRA1 locus, i.e., *372_815del443ins54 (del/ins), rs10490924, and rs11200638 were determined using direct sequencing and/or PCR-based assays. After the haplotype was constructed and analyzed, the associations between hs-CRP levels and representative del/ins genotypes were studied with and without adjustment for potential confounding factors.
All three polymorphisms in the ARMS2/HTRA1 region were in almost complete linkage disequilibrium. Haplotype analyses showed the existence of only two common haplotypes, together comprising 98.9%. Regression analyses showed that the level of hs-CRP was positively correlated with increasing age. This age-dependent increase of hs-CRP levels was greatest in those with homozygous del/ins alleles and lowest in those with homozygous wild-type alleles, which was significant assuming an additive model for gene-dosage association (univariate analyses: p=0.016, multivariate analyses including smoking status, past medical history, and BMI: p=0.043). Consequently, the level of hs-CRP was greatest in those with homozygous del/ins alleles and lowest in those with homozygous wild-type alleles when subjects older than 60 were analyzed. This was significant assuming an additive model for gene-dosage association (univariate analyses: p=0.032).
An age-dependent elevation of serum hs-CRP levels may be accelerated in normal subjects with one or two risk alleles in the ARMS2/HTRA1 locus compared to those with homozygous wild-type alleles. The results of the current study show that the as-yet undetermined function of variants in the ARMS2/HTRA1 locus might be linked to inflammation, possibly contributing to the development of neovascular AMD.
Molecular vision 01/2010; 16:2923-30. · 2.20 Impact Factor
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ABSTRACT: Development of the retinal vascular network is strictly confined within the neuronal retina, allowing the intraocular media to be optically transparent. However, in retinal ischemia, pro-angiogenic factors (including vascular endothelial growth factor-A, VEGF-A) induce aberrant guidance of retinal vessels into the vitreous. Here, we show that the soluble heparan sulfate level in murine intraocular fluid is high particularly during ocular development. When the eyes of young mice with retinal ischemia were treated with heparan sulfate-degrading enzyme, the subsequent aberrant angiogenesis was greatly enhanced compared to PBS-injected contralateral eyes; however, increased angiogenesis was completely antagonized by simultaneous injection of heparin. Intraocular injection of heparan sulfate or heparin alone in these eyes resulted in reduced neovascularization. In cell cultures, the porcine ocular fluid suppressed the dose-dependent proliferation of human umbilical vein endothelial cells (HUVECs) mediated by VEGF-A. Ocular fluid and heparin also inhibited the migration and tube formation by these cells. The binding of VEGF-A and HUVECs was reduced under a high concentration of heparin or ocular fluid compared to lower concentrations of heparin. In vitro assays demonstrated that the ocular fluid or soluble heparan sulfate or heparin inhibited the binding of VEGF-A and immobilized heparin or VEGF receptor 2 but not VEGF receptor 1. The recognition that the high concentration of soluble heparan sulfate in the ocular fluid allows it to serve as an endogenous inhibitor of aberrant retinal vascular growth provides a platform for modulating heparan sulfate/heparin levels to regulate angiogenesis.
PLoS ONE 01/2010; 5(10):e13493. · 4.09 Impact Factor
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ABSTRACT: To study the differentiation of immature retinal neurons/retinal precursors in the ciliary epithelium after retinal histogenesis in mice with inherited or acquired retinal degeneration.
Immunoreactivity to anti-recoverin, rhodopsin, and Pax6 antibodies and binding to peanut agglutinin were analyzed histologically. The distribution and differentiation of immature retinal neurons/retinal precursors in the ciliary epithelium of mice with inherited (C3H/HeJ) and acquired (C57BL mice injected with 60 mg/kg N-methyl-N-nitrosourea) retinal degeneration were assessed. Proliferating retinal progenitors were labeled with bromodeoxyuridine (BrdU), and they were studied histologically using retinal markers.
Many cells of rod and cone photoreceptor lineage were identified within the ciliary epithelium of the pars plana in adult mice with inherited retinal degeneration. Tracking experiments using BrdU indicated that some of recoverin-positive cells in the pars plana (approximately 3%) were generated after retinal histogenesis, and few were produced at or after postnatal day 24 (P24). The induction of acquired retinal degeneration in adult wild-type mice (P30) increased the number of BrdU-positve cells by roughly fourfold and recoverin-positive cells by approximately 17-fold in the pars plana. Moreover, some (approximately 1.5%) of the recoverin-positive cells were newly generated from dividing retinal progenitors in the adult pars plana.
In response to retinal damage, an increased number of immature retinal neurons/retinal precursors was observed in the pars plana of mice with acquired and inherited retinal degeneration. Some of these cells differentiated from proliferating cells even after retinal histogenesis.
Molecular vision 02/2009; 15:187-99. · 2.20 Impact Factor
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ABSTRACT: To compare the distribution and immunologic characteristics of bone marrow (BM)-derived and resident microglia in the retina.
Mice were irradiated and injected with enhanced green fluorescent protein-positive (EGFP(+)) BM cells. One month to 12 months after BM transplantation, eyes were analyzed histologically for the expression of EGFP and various monocyte/microglia/macrophage markers (Iba-1, F4/80, GS-1, major histocompatibility complex [MHC] class II). N-methyl-N-nitrosourea (MNU) was injected or retinal detachment was created to induce retinal damage.
Many BM-derived EGFP(+) cells were found in the ciliary body and choroid and around the optic nerve in the uninjured eyes. Within the retina, few such cells existed at the retinal margin and juxtapapillary area at 3 to 12 months after BM transplantation. However, after MNU injection, many EGFP(+) cells were found in the retina adjacent to the retinal vessels, optic nerve, and ciliary body that rapidly spread throughout the retina. Most of them showed morphologic and immunohistochemical features of microglia. By 7 days after MNU injection, EGFP(+) BM-derived cells occupied approximately 15% of the total Iba-1(+) retinal microglia. Meanwhile, the proportion of MHC class II(+) cells was larger among BM-derived (EGFP(+)/Iba-1(+)) than resident (EGFP(-)/Iba-1(+)) microglia. In the eyes with retinal detachment, EGFP(+)/F4/80(+) cells engrafted exclusively around the detached retina.
In response to retinal damage, numerous BM-derived cells migrated to the retina from the ciliary body, optic nerve, and retinal vessels and differentiated into microglia. The higher rate of immunologic activation and the increased specificity to the damaged site appeared to be the characteristic features of BM-derived microglia.
Investigative ophthalmology & visual science 06/2008; 49(9):4162-8. · 3.43 Impact Factor
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ABSTRACT: To study the neuroprotective effect of experimental retinal detachment (RD) on photoreceptor degeneration in rd1 mice.
RD was produced in the eyes of rd1 mice at postnatal day (P) 9. These eyes were collected and compared to controls without RD. The effects of RD on retinal degeneration were evaluated by histochemical staining of nuclei in the outer nuclear layer (ONL), rod and cone photoreceptors, and retinal vessels at P30 in retinal sections and flatmounts. Apoptotic photoreceptors were detected by TdT-mediated dUTP nick-end labeling (TUNEL) at P15. Mice with or without RD were also reared in darkness and evaluated immunohistochemically at P30.
The numbers of rhodopsin-positive (rod), peanut agglutinin-positive (cone), and diamino-2-phenyl-indol-stained (rod-plus-cone) cells in the ONL were increased by 2.0-fold, 1.3-fold, and 1.2-fold, respectively, in the rd1 eyes with RD compared to those without RD at P30. In the detached retina, the cone photoreceptor inner/outer segment structures and the deep retinal vessels surrounding the inner nuclear layer and the ONL, but not the ganglion cell layer, were preserved. At P15, TUNEL-positive cell numbers in the ONL were significantly reduced in the eyes with RD. Light exposure had no effect on photoreceptor degeneration in the eyes with or without RD.
RD mediates the preservation of cone and rod photoreceptors in the ONL and surrounding vascular structures by reducing the rate of apoptosis of photoreceptors in rd1 mice. Light deprivation does not appear to be one of the mechanisms of photoreceptor protection in the detached retinas in these mice.
Investigative Ophthalmology & Visual Science 03/2008; 49(2):781-7. · 3.60 Impact Factor
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ABSTRACT: To study the distribution and differentiation of photoreceptor precursors in the ciliary epithelium in mice.
Proliferating cells in flat-mount specimens of the ciliary body and retina were studied by bromodeoxyuridine (BRDU; 150 mg/kg) labeling in young C57Bl mice. Immunoreactivity to anti-recoverin, rhodopsin, and Pax6 antibodies and binding to peanut agglutinin were analyzed histologically to assess the distribution and differentiation of photoreceptor progenitors or precursors. Mice injected intraperitoneally with N-methyl-N-nitrosourea (MNU; 60 mg/kg) were also examined.
Part of the neuroblast layer composed of BrdU-positive retinal progenitor cells was identified within the ciliary epithelium of the pars plana in continuation of the layer of the peripheral retina during ocular development. In both the ciliary epithelium and the retina, the layer size decreased rapidly and disappeared mostly by postnatal day (P)9. Within the ciliary epithelium of the pars plana, numerous postmitotic rod and cone photoreceptor precursors were identified that rapidly differentiated morphologically and decreased in number with ocular development. Rod precursors were no longer seen in the pars plana at P12, whereas rare presumptive cone precursors persisted even at P120. An increase in the number of presumptive cone precursors (approximately 16-fold) was identified in the pars plana of adult mice with MNU-induced photoreceptor degeneration.
Rod and cone precursors were identified in the ciliary epithelium of the murine pars plana during ocular development but nearly disappeared after the completion of histogenesis. However, in response to retinal injury, an increased number of presumptive cone precursors was found even in the adult pars plana.
Investigative Ophthalmology & Visual Science 02/2008; 49(1):422-8. · 3.60 Impact Factor
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ABSTRACT: To analyze the role of VEGF and its receptors, VEGFR2/Flk1 and VEGFR1/Flt1, on retinal progenitor cells (RPCs) in a murine model of inherited retinal degeneration (rd1 mice).
After proliferating RPCs in the retina of rd1 mice were labeled with bromodeoxyuridine (BrdU), expressions of VEGFR2/Flk1 and VEGFR1/Flt1 were immunohistochemically analyzed. To examine its effect on the proliferation of BrdU-positive RPCs in rd1 mice, VEGF was administered into retinal culture medium with or without blocking agents against VEGFR2/Flk1 or VEGFR1/Flt1 in vitro or injected into vitreous cavity in vivo.
BrdU-labeled RPCs in rd1 mice expressed VEGFR2/Flk1 but not VEGFR1/Flt1. These cells later expressed retinal neuronal markers such as Pax6 and rhodopsin. Exposure of the retinas from postnatal day (P) 9 rd1 mice to VEGF increased the number of proliferating RPCs by 61% in vitro. This effect was blocked by concomitant administration of VEGFR2/Flk1 kinase inhibitor. In vivo, a single intravitreal injection of VEGF in rd1 mice at P9 increased by 138% the number of RPCs and cells that developed from RPCs in the peripheral retina at P18.
VEGF stimulates the proliferation of RPCs through VEGFR2/Flk1 in rd1 mice. The observed proliferation of RPCs that have the potential to differentiate into retinal neurons may enhance the regeneration of the degenerating retina.
Investigative Ophthalmology & Visual Science 10/2007; 48(9):4315-20. · 3.60 Impact Factor
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ABSTRACT: The transcription factor neural retina leucine zipper (NRL) is required for rod photoreceptor differentiation during mammalian retinal development. NRL interacts with CRX, NR2E3, and other transcription factors and synergistically regulates the activity of photoreceptor-specific genes. Mutations in the human NRL gene are associated with retinal degenerative diseases. Here we report functional analyses of 17 amino acid variations and/or mutations of NRL. We show that 13 of these lead to changes in NRL phosphorylation. Six mutations at residues p.S50 (c.148T>A, c.148T>C, and c.149C>T) and p.P51 (c.151C>A, c.151C>T, and c.152C>T), identified in patients with autosomal dominant retinitis pigmentosa, result in a major NRL isoform that exhibits reduced phosphorylation but enhanced activation of the rhodopsin promoter. The truncated NRL mutant proteins-p.L75fs (c.224_225insC) and p.L160fs (c.459_477dup)-do not localize to the nucleus because of the absence of bZIP domain. The p.L160P (c.479T>C), p.L160fs, and p.R218fs (c.654delC) mutant proteins do not bind to the NRL-response element, as revealed by electrophoretic mobility shift assays. These three and p.S225N (c.674G>A) mutant show reduced transcriptional activity and may contribute to recessive disease. The p.P67S (c.199C>T) and p.L235F (c.703C>T) variations in NRL do not appear to directly cause retinitis pigmentosa, while p.E63K (c.187G>A), p.A76V (c.227C>T), p.G122E (c.365G>A), and p.H125Q (c.375C>G) are of uncertain significance. Our results support the notion that gain-of-function mutations in the NRL gene cause autosomal dominant retinitis pigmentosa while loss-of-function NRL mutations lead to autosomal recessive retinitis pigmentosa. We propose that differential phosphorylation of NRL fine-tunes its transcriptional regulatory activity, leading to a more precise control of gene expression.
Human Mutation 06/2007; 28(6):589-98. · 5.69 Impact Factor
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ABSTRACT: Unrelated patients with achromatopsia, macular degeneration with onset under age 50 years, cone degeneration or dysfunction, cone-rod degeneration, or macular malfunction were screened for mutations in the three genes known to be associated with achromatopsia: the GNAT2 gene encoding the alpha subunit of cone transducin and the CNGA3 and CNGB3 genes encoding the alpha and beta subunits of the cone cGMP-gated cation channel. We found no examples of patients with GNAT2 mutations. Out of 36 achromats, 12 (33%) had mutations in CNGA3 (13 different mutations including five novel mutations) and 12 (33%) had mutations in CNGB3 (six different mutations including four novel mutations). All achromats with CNG mutations had residual, presumably cone function as determined by computer-averaged 30-Hz electroretinograms (ERGs). There was considerable variability in acuity and color vision, with most patients having acuities of 20/200-20/400 and complete absence of color perception, and others having acuities of 20/25-20/40 and some color vision. Two pseudodominant achromatopsia cases were uncovered, both with CNGA3 mutations, including one family in which some compound heterozygotes with achromatopsia mutations were clinically unaffected. We found two novel CNGB3 changes in three patients with juvenile macular degeneration, a phenotype not previously associated with mutations in the cone channel subunits. These patients had subnormal acuity (20/30-20/60), normal to subnormal color vision, and normal to subnormal full-field cone ERG amplitudes. Our results indicate that some patients with channel protein mutations retain residual foveal cone function. Based on our findings, CNGB3 should be considered as a candidate gene to be evaluated in patients with forms of cone dysfunction, including macular degeneration.
Human Mutation 04/2005; 25(3):248-58. · 5.69 Impact Factor
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ABSTRACT: Mice lacking the transcription factor Nrl have no rod photoreceptors and an increased number of short-wavelength-sensitive cones. Missense mutations in NRL are associated with autosomal dominant retinitis pigmentosa; however, the phenotype associated with the loss of NRL function in humans has not been reported. We identified two siblings who carried two allelic mutations: a predicted null allele (L75fs) and a missense mutation (L160P) altering a highly conserved residue in the domain involved in DNA-binding-site recognition. In vitro luciferase reporter assays demonstrated that the NRL-L160P mutant had severely reduced transcriptional activity compared with the WT NRL protein, consistent with a severe loss of function. The affected patients had night blindness since early childhood, consistent with a severe reduction in rod function. Color vision was normal, suggesting the presence of all cone color types; nevertheless, a comparison of central visual fields evaluated with white-on-white and blue-on-yellow light stimuli was consistent with a relatively enhanced function of short-wavelength-sensitive cones in the macula. The fundi had signs of retinal degeneration (such as vascular attenuation) and clusters of large, clumped, pigment deposits in the peripheral fundus at the level of the retinal pigment epithelium (clumped pigmentary retinal degeneration). Our report presents an unusual clinical phenotype in humans with loss-of-function mutations in NRL.
Proceedings of the National Academy of Sciences 01/2005; 101(51):17819-24. · 9.68 Impact Factor
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ABSTRACT: To identify pathogenic mutations in the guanylate cyclase-activating protein 1 (GCAP1) and GCAP2 genes and to characterize the biochemical effect of mutation on guanylate cyclase (GC) stimulation.
The GCAP1 and GCAP2 genes were screened by direct sequencing for mutations in 216 patients and 421 patients, respectively, with various hereditary retinal diseases. A mutation in GCAP1 segregating with autosomal dominant cone degeneration was further evaluated biochemically by employing recombinant proteins, immunoblotting, Ca2+-dependent stimulation of GC, fluorescence emission spectra, and limited proteolysis in the absence and presence of Ca2+.
A novel GCAP1 mutation, I143NT (substitution of Ile at codon 143 by Asn and Thr), affecting the EF4 Ca2+-binding loop, was identified in a heterozygote father and son with autosomal dominant cone degeneration. Both patients had much greater loss of cone function versus rod function; previous histopathologic evaluation of the father's eyes at autopsy (age 75 years) showed no foveal cones but a few, scattered cones remaining in the peripheral retina. Biochemical analysis showed that the GCAP1-I143NT mutant adopted a conformation susceptible to proteolysis, and the mutant inhibited GC only partially at high Ca2+ concentrations. Individual patients with atypical or recessive retinitis pigmentosa (RP) had additional heterozygous GCAP1-T114I and GCAP2 gene changes (V85M and F150C) of unknown pathogenicity.
A novel GCAP1 mutation, I143NT, caused a form of autosomal dominant cone degeneration that destroys foveal cones by mid-life but spares some cones in the peripheral retina up to 75 years. Properties of the GCAP1-I143NT mutant protein suggested that it is incompletely inactivated by high Ca2+ concentrations as should occur with dark adaptation. The continued activity of the mutant GCAP1 likely results in higher-than-normal scotopic cGMP levels which may, in turn, account for the progressive loss of cones.
Investigative Ophthalmology & Visual Science 12/2004; 45(11):3863-70. · 3.60 Impact Factor
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ABSTRACT: To search for a phenotype associated with mutations in the phosducin gene PDC.
We screened 853 patients with retinitis pigmentosa or an allied disease diseases, including groups of 61 to 212 patients, each with dominant retinitis pigmentosa (RP), recessive RP, Leber congenital amaurosis, or cone-rod degeneration, for mutations in the PDC gene using direct genomic sequencing of the three coding exons and their flanking intron splice sites.
We found one polymorphism in the 5' untranslated region (minor allele frequency of 0.149) and three rare single-base sequence variants (one missense change, one isocoding change, and one in the 3' untranslated region). The rare variants were found in one heterozygous patient each and none was interpreted as pathogenic.
Phosducin mutations are not a major cause of dominant or recessive RP, Leber congenital amaurosis, or cone-rod degeneration. The human phenotype associated with phosducin defects remains unknown.
Molecular vision 02/2004; 10:62-4. · 2.20 Impact Factor
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ABSTRACT: The RGS proteins are GTPase activating proteins that accelerate the deactivation of G proteins in a variety of signalling pathways in eukaryotes. RGS9 deactivates the G proteins (transducins) in the rod and cone phototransduction cascades. It is anchored to photoreceptor membranes by the transmembrane protein R9AP (RGS9 anchor protein), which enhances RGS9 activity up to 70-fold. If RGS9 is absent or unable to interact with R9AP, there is a substantial delay in the recovery from light responses in mice. We identified five unrelated patients with recessive mutations in the genes encoding either RGS9 or R9AP who reported difficulty adapting to sudden changes in luminance levels mediated by cones. Standard visual acuity was normal to moderately subnormal, but the ability to see moving objects, especially with low-contrast, was severely reduced despite full visual fields; we have termed this condition bradyopsia. To our knowledge, these patients represent the first identified humans with a phenotype associated with reduced RGS activity in any organ.
Nature 02/2004; 427(6969):75-8. · 36.28 Impact Factor
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ABSTRACT: To evaluate the molecular genetic defects associated with retinitis punctata albescens (RPA) in 5 patients from 3 families with this disease.
We examined 3 probands and 2 clinically affected relatives with RPA. Clinical examinations included best-corrected visual acuity, visual field testing, electroretinography, dilated fundus examination, and fundus photography. Leukocyte DNA was analyzed for mutations in the exons of the genes encoding cellular retinaldehyde-binding protein 1 (RLBP1), 11-cis-retinol dehydrogenase (RDH5), interphotoreceptor retinoid-binding protein (RBP3), and photoreceptor all-trans-retinol dehydrogenase (RDH8). Not all patients were evaluated for mutations in each gene. The exons were individually amplified and screened for mutations by single-stranded conformational polymorphism analysis or direct genomic sequencing.
The 3 probands had similar clinical findings, including a history of poor night vision, the presence of punctate white deposits in the retina, and substantially reduced or absent rod responses on electroretinogram testing. One of the probands (patient 2:III:2) had 2 novel mutations in the RLBP1 gene (Arg151Trp and Gly31[2-base pair deletion], [GGA-->G-]). Segregation analysis showed that the 2 mutations were allelic and that the patient was a compound heterozygote. Both parents of the proband manifested round white deposits in the retina. The other 2 probands had no detected pathogenic mutations in RLBP1 or in the other 3 genes evaluated.
The identification of novel RLBP1 mutations in 1 of our 3 probands, all with RPA, is further evidence of genetic (nonallelic) heterogeneity in this disease. The presence of round white deposits in the retina may be observed in those heterozygous for RLBP1. Clinical Relevance Patients with a clinical presentation of RPA can have genetically different mutations. Drusen-like lesions may be observed in heterozygotes in families with this disease and a mutation in RLBP1.
Archives of Ophthalmology 02/2004; 122(1):70-5. · 3.71 Impact Factor
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ABSTRACT: The transcription factor neural retina leucine zipper (NRL) is required for rod photoreceptor differentiation during mammalian retinal development. NRL interacts with CRX, NR2E3, and other transcription factors and synergistically regulates the activity of photoreceptor-specific genes. Mutations in the human NRL gene are associated with retinal degenerative diseases. Here we report functional analyses of 17 amino acid variations and/or mutations of NRL. We show that 13 of these lead to changes in NRL phosphorylation. Six mutations at residues p.S50 (c.148T>A, c.148T>C, and c.149C>T) and p.P51 (c.151C>A, c.151C>T, and c.152C>T), identified in patients with autosomal dominant retinitis pigmentosa, result in a major NRL isoform that exhibits reduced phosphorylation but enhanced activation of the rhodopsin promoter. The truncated NRL mutant proteins—p.L75fs (c.224-225insC) and p.L160fs (c.459-477dup)—do not localize to the nucleus because of the absence of bZIP domain. The p.L160P (c.479T>C), p.L160fs, and p.R218fs (c.654delC) mutant proteins do not bind to the NRL-response element, as revealed by electrophoretic mobility shift assays. These three and p.S225N (c.674G>A) mutant show reduced transcriptional activity and may contribute to recessive disease. The p.P67S (c.199C>T) and p.L235F (c.703C>T) variations in NRL do not appear to directly cause retinitis pigmentosa, while p.E63K (c.187G>A), p.A76V (c.227C>T), p.G122E (c.365G>A), and p.H125Q (c.375C>G) are of uncertain significance. Our results support the notion that gain-of-function mutations in the NRL gene cause autosomal dominant retinitis pigmentosa while loss-of-function NRL mutations lead to autosomal recessive retinitis pigmentosa. We propose that differential phosphorylation of NRL fine-tunes its transcriptional regulatory activity, leading to a more precise control of gene expression. Hum Mutat 28(6), 589–598, 2007. © Published 2007 Wiley-Liss, Inc. Peer Reviewed http://deepblue.lib.umich.edu/bitstream/2027.42/56084/1/20488_ftp.pdf
