August F. Deutman

Mutations in a human homologue of Drosophila crumbs cause retinitis pigmentosa (RP12)

Retinitis pigmentosa (RP) comprises a clinically and genetically heterogeneous group of diseases that afflicts approximately 1.5 million people worldwide. Affected individuals suffer from a progressive degeneration of the photoreceptors, eventually resulting in severe visual impairment. To isolate candidate genes for chorioretinal diseases, we cloned cDNAs specifically or preferentially expressed in the human retina and the retinal pigment epithelium (RPE) through a novel suppression subtractive hybridization (SSH) method. One of these cDNAs (RET3C11) mapped to chromosome 1q31–q32.1, a region harbouring a gene involved in a severe form of autosomal recessive RP characterized by a typical preservation of the para-arteriolar RPE (RP12; ref. 3). The full-length cDNA encodes an extracellular protein with 19 EGF-like domains, 3 laminin A G-like domains and a C-type lectin domain. This protein is homologous to the Drosophila melanogaster protein crumbs (CRB), and denoted CRB1 (crumbs homologue 1). In ten unrelated RP patients with preserved para-arteriolar RPE, we identified a homozygous AluY insertion disrupting the ORF, five homozygous missense mutations and four compound heterozygous mutations in CRB1. The similarity to CRB suggests a role for CRB1 in cell-cell interaction and possibly in the maintenance of cell polarity in the retina. The distinct RPE abnormalities observed in RP12 patients suggest that CRB1 mutations trigger a novel mechanism of photoreceptor degeneration.

Retinoic acid delays transcription of human retinal pigment neuroepithelium marker genes in ARPE-19 cells

&NA; The effect of retinoic acid on the differentiation of a human retinal pigment epithelium‐derived cell line ARPE‐19 was studied. Differentiation of ARPE‐19 cells is delayed by retinoic acid. The minimum all‐trans‐retinoic acid concentration needed for delay of ARPE‐19 differentiation is 1 μM. A delay of differentiation was also observed using 1 μM 9‐cis or 13‐cis‐retinoic acid. Differentiation at the molecular level was studied by analyzing transcription of two RPE‐marker genes, RPE65 and peropsin. In the presence of 1 μM retinoic acid the onset of transcription of both genes was delayed by 2–3 weeks. We conclude that alltrans‐, 9‐cis‐, and 13‐cis‐retinoic acid delay differentiation of ARPE‐19 cells into cells that phenotypically resemble cells from the human retinal pigment epithelium.

Disruption of the 11-cis-Retinol Dehydrogenase Gene Leads to Accumulation of cis-Retinols and cis-Retinyl Esters

ABSTRACT To elucidate the possible role of 11-cis-retinol dehydrogenase in the visual cycle and/or 9-cis-retinoic acid biosynthesis, we generated mice carrying a targeted disruption of the 11-cis-retinol dehydrogenase gene. Homozygous 11-cis-retinol dehydrogenase mutants developed normally, including their retinas. There was no appreciable loss of photoreceptors. Recently, mutations in the 11-cis-retinol dehydrogenase gene in humans have been associated with fundus albipunctatus. In 11-cis-retinol dehydrogenase knockout mice, the appearance of the fundus was normal and punctata typical of this human hereditary ocular disease were not present. A second typical symptom associated with this disease is delayed dark adaptation. Homozygous 11-cis-retinol dehydrogenase mutants showed normal rod and cone responses. 11-cis-Retinol dehydrogenase knockout mice were capable of dark adaptation. At bleaching levels under which patients suffering from fundus albipunctatus could be detected unequivocally, 11-cis-retinol dehydrogenase knockout animals displayed normal dark adaptation kinetics. However, at high bleaching levels, delayed dark adaptation in 11-cis-retinol dehydrogenase knockout mice was noticed. Reduced 11-cis-retinol oxidation capacity resulted in 11-cis-retinol/13-cis-retinol and 11-cis-retinyl/13-cis-retinyl ester accumulation. Compared with wild-type mice, a large increase in the 11-cis-retinyl ester concentration was noticed in 11-cis-retinol dehydrogenase knockout mice. In the murine retinal pigment epithelium, there has to be an additional mechanism for the biosynthesis of 11-cis-retinal which partially compensates for the loss of the 11-cis-retinol dehydrogenase activity. 11-cis-Retinyl ester formation is an important part of this adaptation process. Functional consequences of the loss of 11-cis-retinol dehydrogenase activity illustrate important differences in the compensation mechanisms between mice and humans. We furthermore demonstrate that upon 11-cis-retinol accumulation, the 13-cis-retinol concentration also increases. This retinoid is inapplicable to the visual processes, and we therefore speculate that it could be an important catabolic metabolite and its biosynthesis could be part of a process involved in regulating 11-cis-retinol concentrations within the retinal pigment epithelium of 11-cis-retinol dehydrogenase knockout mice.

A randomized controlled clinical trial on the efficacy of radiation therapy in the control of subfoveal choroidal neovascularization in age-related macular degeneration : Radiation versus observation

Abstract · Background: The results of several pilot studies concerning radiation therapy for age-related subfoveal choroidal neovascularization (CNV) have been published recently. Although positive treatment results have been described, it is not known whether this therapy alters the natural course of eyes with neovascular age-related macular degeneration (AMD). A randomized controlled clinical trial was conducted in which radiation therapy was compared with observation in patients with subfoveal neovascular AMD. · Methods: Seventy-four patients with a recent drop in central vision due to subfoveal age-related CNV were randomized to either radiation treatment or observation. Patients with either classic, occult or mixed type CNV were included. Eyes in the treatment group received a radiation dose of 24 Gy in four fractions of 6 Gy. Evaluation of data concerning visual acuity (VA) and fluorescein angiography occurred at 3, 6 and 12 months after inclusion. · Results: At 12 months of follow-up 52.2% of the observation group versus 32.0% of the irradiation group had lost 3 or more lines of VA (P=0.03, log rank test). More severe visual decline, 6 lines or more, was observed in 40.9% of the observation versus 8.8% in the irradiation group (P=0.002 using log rank test). At 12 months 39.6% of the observation group and 20.0% of the treatment group had VA of less than 0.1 (P=0.08, log rank test). The size of the CNV membrane doubled in 25.2% of eyes in the observation group versus 20.0% in the treatment group at 12 months (P=0.5, log rank test). No side effects were observed. · Conclusion: Preservation of VA was significantly better in the treatment group compared with the control group at 12 months. Nevertheless we noted a drop in central vision of 3 or more lines in a substantial proportion of the treatment group. Radiation therapy does not prevent visual loss in all patients with age-related subfoveal CNV, and whether the treatment benefit at 12 months will persist has to be awaited.

The spectrum of retinal phenotypes caused by mutations in the ABCA4 gene.

BackgroundThe majority of studies on the retina-specific ATP-binding cassette transporter (ABCA4) gene have focussed on molecular genetic analysis; comparatively few studies have described the clinical aspects of ABCA4-associated retinal disorders. In this study, we demonstrate the spectrum of retinal dystrophies associated with ABCA4 gene mutations.MethodsNine well-documented patients representing distinct phenotypes in the continuum of ABCA4-related disorders were selected. All patients received an extensive ophthalmologic evaluation, including kinetic perimetry, fluorescein angiography, and electroretinography (ERG). Mutation analysis had been performed previously with the genotyping microarray (ABCR400 chip) and/or single-strand conformation polymorphism analysis in combination with direct DNA sequencing.ResultsIn all patients, at least one pathologic ABCA4 mutation was identified. Patient 10034 represented the mild end of the phenotypic spectrum, demonstrating exudative age-related macular degeneration (AMD). Patient 24481 received the diagnosis of late-onset fundus flavimaculatus (FFM), patient 15168 demonstrated the typical FFM phenotype, and patient 19504 had autosomal recessive Stargardt disease (STGD1). Patients 11302 and 7608 exhibited progression from FFM/STGD1 to cone–rod dystrophy (CRD). A more typical CRD phenotype was found in patients 15680 and 12608. Finally, the most severe ABCA4-associated phenotype was retinitis pigmentosa (RP) in patient 11366. This phenotype was characterised by extensive atrophy with almost complete loss of peripheral and central retinal functions.ConclusionWe describe nine patients during different stages of disease progression; together, these patients form a continuum of ABCA4-associated phenotypes. Besides characteristic disorders such as FFM/STGD1, CRD and RP, intermediate phenotypes may be encountered. Moreover, as the disease progresses, marked differences may be observed between initially comparable phenotypes. In contrast, distinctly different phenotypes may converge to a similar final stage, characterised by extensive chorioretinal atrophy and very low visual functions. The identified ABCA4 mutations in most, but not all, patients were compatible with the resulting phenotypes, as predicted by the genotype–phenotype model for ABCA4-associated disorders. With the advent of therapeutic options, recognition by the general ophthalmologist of the various retinal phenotypes associated with ABCA4 mutations is becoming increasingly important.

Hyperhomocysteinemia in Retinal Artery and Retinal Vein Occlusion

In 19 patients who had retinal vein occlusion or retinal artery occlusion before the age of 50 years, the incidence of hyperhomocysteinemia, as observed in heterozygosity for homocystinuria, was studied by the performance of a standardized, oral methionine-loading test. In four of the 19 patients (21%), two with retinal artery occlusion and two with central retinal vein occlusion, the after-load peak levels of homocysteine exceeded the mean level, established in normal control subjects, by more than two standard deviations and were as well within the ranges established in obligate heterozygotes for homocystinuria. Because the frequency of heterozygosity for homocystinuria in the normal population is one in 70 (1.4%) at the most, we conclude that hyperhomocysteinemia predisposes to the development of premature retinal artery and retinal vein occlusion (P < .01; chi 2 test).

Radiation therapy for age-related subfoveal choroidal neovascular membranes

In this pilot study the effect of radiation therapy on subfoveal CNV membranes associated with age-related macular degeneration was evaluated. Four groups of 10 patients were treated with external beam radiotherapy (16 MV photons) on an area of 1 cm2 (macular region) using a lens-sparing technique and total dose of 8 to 24 Gy. The first group received 8 Gy in a single fraction. In this group only 30% had a stable visual acuity and a stable FA after 18 months follow-up. In 50% of patients in group 2 (12 Gy) and 40% of patients in group 3 (18 Gy) the visual acuity and FA appearance remained stable after 18 months of follow-up. In the last group (24 Gy) 80% of patients had a stable visual acuity and FA appearance after 12 months follow-up. Comparison of these findings with the natural history data of subfoveal age-related CNV, suggests a beneficial effect of radiation therapy with a total dose of 12 Gy or more on the progression of CNV membranes.

Radiation therapy for subfoveal choroidal neovascular membranes in age-related macular degeneration

Abstract• Background: The natural course of the visual acuity of age-related subfoveal choroidal neovascularisation (CNV) membranes is poor. Laser photocoagulation of subfoveal CNV is recommended if the patient is willing to accept a large decrease in visual acuity immediately after treatment. A large proportion of patients with subfoveal CNV do not meet the Macular Photocoagulation Study Group (MPS) guidelines for laser photocoagulation. The fact that so few patients meet these criteria makes further research into new treatment techniques warranted. Ionising radiation may prevent the proliferation of endothelial cells of newly formed subretinal capillaries and may induce obliteration of the aberrant new vessels. • Methods: In this study, the effect of radiation therapy on subfoveal CNV membranes was evaluated. Four groups of ten patients were treated with external beam radiotherapy (16-MV photons) on an area of 1 cm2 (macular region) using a lens-sparing technique and total doses of 8–24 Gy. The first group received 8 Gy in one fraction. The second, third and fourth groups received 12 Gy in 2 fractions, 18 Gy in three fractions and 24 Gy in four fractions respectively. The studied parameters included best-corrected visual acuity and membrane size and leakage on the fluorescein angiogram. we included 17 occult and 23 classic CNV membranes as defined by the MPS, with a duration of less than 5 weeks at presentation. Complete ophthalmic examination including fluorescein angiography was performed before and 3, 12 and 18 months after radiation treatment. We analysed the angiogram using a standard over-projection sheet. The results concerning the visual acuity and fluorescein angiography (FA) were compared with the extensively published, natural course data. • Results: The first group (including three cases of occult CNV) received 8 Gy in a single fraction. In this group only four of ten patients had stable visual acuity and stable FA appearance after 21 months follow-up. The visual acuity and FA remained stable after 13.6 months follow-up in seven of the patients in group 2 (12 Gy in two fractions, four occult CNV). The third group (18 Gy in three fractions, seven occult CNV) contained six patients with stable visual acuity, although two of them had CNV deterioration on the FA (11.1 months follow-up). In the last group (24 Gy in four fractions, three occult CNV), with a short follow-up of 5.6 months, eight patients had stable visual acuity and FA appearance. We did not note any regression of the CNV membrane on the angiogram. The visual acuity in groups 2, 3 and 4 decreased to 0.1 or worse in only three cases, three cases and one case respectively after at least 6 months follow-up. • Conclusion: Comparison of these findings with the natural history data of subfoveal age-related CNV suggests a beneficial effect of radiation therapy with a total dose of 12 Gy or more on the progression of CNV To date no negative side effects have been observed.

Juvenile macular dystrophy associated with deficient activity of fatty aldehyde dehydrogenase in Sjögren-Larsson syndrome.

PURPOSE To report the ocular manifestations associated with the Sjögren-Larsson syndrome in a series of patients with proven fatty aldehyde dehydrogenase deficiency. To emphasize the clinical importance of the ophthalmological features of the Sjögren-Larsson syndrome. To discuss the metabolic disturbances that might give rise to the ophthalmological picture. METHODS Fifteen patients with Sjögren-Larsson syndrome underwent a standardized ophthalmological examination. In patients of appropriate age, and who were able to cooperate, additional investigations were performed. RESULTS All patients exhibited bilateral, glistening yellow-white crystalline deposits that were located in the innermost retinal layers and appeared during the first 2 years of life. Repeated fundus photography in individual patients showed that the dots became more numerous as the patients got older. Photophobia, subnormal visual acuity, myopia, and astigmatism were found in most of the patients. Fluorescein angiography was performed in three patients and showed a mottled hyperfluorescence of the retinal pigment epithelium, without leakage. Color vision, electroretinography, and electro-oculography could be performed in only a small number of patients and showed no abnormalities. Visual evoked potentials were found to be abnormal in six of eight patients. CONCLUSIONS In Sjögren-Larsson syndrome, patients exhibit highly characteristic bilateral, glistening yellow-white retinal dots from the age of 1 to 2 years onward. The number of dots increases with age. The extent of the macular abnormality does not correlate with the severity of the ichthyosis or with the severity of the neurological abnormalities. A high percentage of patients shows additional ocular signs and symptoms, notably marked photophobia.

Hereditary retinal dystrophies and choroidal neovascularization

Abstract Background: Choroidal neovascularization infrequently occurs in patients affected by hereditary retinal dystrophies. Methods: We studied eight patients suffering from different hereditary retinal dystrophies (Best’s disease, reticular dystrophy, butterfly-shaped dystrophy, gyrate atrophy, and retinitis pigmentosa) who developed choroidal neovascularization. All patients underwent complete ophthalmic evaluation, electrophysiology, colour vision testing, and fluorescein angiography. In some patients, ICG video-angio- graphy was also performed. Laser treatment was carried out in only one patient. Results: The mean duration of follow-up was 41.7 months (range 6–148 months). At CNV diagnosis, the mean VA was 0.23 (range 0.02–0.6). At the last follow-up, mean VA was 0.34 (range HM to 0.9). At the last follow-up, fluorescein angiography showed a focal, atrophic scar in seven eyes, a fibrotic membrane in two eyes and a still active membrane in two cases. Conclusion: We emphasize the relatively favourable visual prognosis in patients suffering from inherited retinal dystrophies complicated with choroidal neovascularization. Therapeutic approaches other than laser treatment could be attempted in these patients.