Klaus Rohrschneider

Mutations in the CEP290 (NPHP6) Gene Are a Frequent Cause of Leber Congenital Amaurosis

Leber congenital amaurosis (LCA) is one of the main causes of childhood blindness. To date, mutations in eight genes have been described, which together account for approximately 45% of LCA cases. We localized the genetic defect in a consanguineous LCA-affected family from Quebec and identified a splice defect in a gene encoding a centrosomal protein (CEP290). The defect is caused by an intronic mutation (c.2991+1655A-->G) that creates a strong splice-donor site and inserts a cryptic exon in the CEP290 messenger RNA. This mutation was detected in 16 (21%) of 76 unrelated patients with LCA, either homozygously or in combination with a second deleterious mutation on the other allele. CEP290 mutations therefore represent one of the most frequent causes of LCA identified so far.

Multilayer amniotic membrane transplantation for reconstruction of deep corneal ulcers

PURPOSE To evaluate the efficacy of multilayer amniotic membrane transplantation for reconstruction of corneal epithelium and stroma in the context of deep corneal ulcers. DESIGN Prospective, noncomparative, interventional case series. PARTICIPANTS Eleven consecutive patients with deep corneal ulcers refractory to conventional treatment; six patients had herpetic keratitis and five had other forms of neurotrophic keratitis. INTERVENTION Multilayer amniotic membrane transplantation with kryopreserved human amniotic membrane. MAIN OUTCOME MEASURES Integrity of corneal epithelium and stroma, opacification, and appearance of grafted membrane during 12 months follow-up. RESULTS Amniotic membrane transplantation markedly reduced ocular inflammation in all patients. Epithelium healed above all corneal ulcers within 4 weeks and remained stable in 9 of 11 patients for 1 year. Two patients with recurrent epithelial defect suffered from severe neurotrophic keratitis. Following transplantation the amniotic membranes gradually dissolved over a period of 12 months, but stromal thickness remained stable. CONCLUSION Amniotic membrane transplantation allows corneal surface reconstruction in patients with persistent epithelial defects. The multilayer technique is useful for treating deep corneal ulcers and even descemetoceles. Because the procedure results in stability of the ocular surface over a period of more than 12 months in most patients, it may be considered an alternative to conventional surgical techniques for ocular surface reconstruction.

Reproducibility of the Optic Nerve Head Topography with a New Laser Tomographic Scanning Device

BACKGROUND Laser scanning tomography has been shown to be an accurate and reliable method for the assessment of the three-dimensional optic disc topography. The authors investigate the reliability of morphometric measurements with the Heidelberg retina tomograph, a new instrument which was designed based on this technology, which simplifies handling and is much smaller than the laser tomographic scanner. METHODS Three independent measurements of the optic disc were performed in 39 eyes of 39 patients which were equally divided into the following three groups: glaucoma, glaucoma suspects, and controls. RESULTS The mean coefficient of variation for measurement in the glaucoma, glaucoma suspect, and control groups was 2.9%, 5.0%, and 3.4%, respectively, for cup area; 4.9%, 4.6%, and 4.6%, respectively, for cup volume; 5.2%, 3.8% and 3.3%, respectively, for mean cup depth; and 5.2%, 4.1%, and 4.0%, respectively, for maximal cup depth. The mean standard deviation for one pixel of the total image was 30 +/- 6 microns, 28 +/- 7 microns, and 22 +/- 6 microns for the three groups, respectively. CONCLUSION The Heidelberg retina tomograph enables fast and reliable measurement of the optic disc topography and therefore may allow exact follow-up of patients.

Cryopreserved human amniotic membrane for ocular surface reconstruction

Abstract · Background: Amniotic membrane transplantation is used for the reconstruction of the ocular surface in the context of, for example, corneal ulcers or conjunctival scarring. The mechanisms by which preserved amniotic membrane grafts promote reepithelialization are unknown. As a first step the viability and proliferative capacity of amnion cells following cryopreservation of membranes in glycerol is investigated. · Methods: Fresh and cryopreserved (in 50% glycerol) amniotic membranes were investigated histologically and by vital stains. Following enzymatic digestion, amniotic cells were stained for viability and cultured in DMEM+10% FBS. In addition, explant cultures were established from fresh and cryopreserved membranes. · Results: Histologicacl examination showed no significant morphological alteration following cryopreservation. While fresh membranes contained predominantly vital cells, no such cells were detected following cryopreservation. Also, cells removed enzymatically from cryopreserved membranes were not viable and did not grow in culture. While both epithelial and fibroblastic cells grew from fresh membranes, no growth was seen from cryopreserved membranes. · Conclusion: The results suggest that the technique for preservation which is most widely used for ophthalmological amniotic membrane transplantation significantly impairs viability and proliferative capacity. This supports the clinical finding that neither immunological reactions nor signs of ingrowth of amniotic cells are observed in patients. Furthermore amniotic membrane grafts seem to function primarily as matrix and not by virtue of transplanted functional cells.

Thalidomide inhibits corneal angiogenesis induced by vascular endothelial growth factor

Abstract · Background: Ocular diseases caused by neovascularization are among the leading causes of blindness. No specific pharmacological treatment is available. Among potential drugs, thalidomide deserves special interest since a wide body of clinical experience exists. However, its antiangiogenic effect is controversial. We therefore investigated the effect of thalidomide on corneal angiogenesis induced by vascular endothelial growth factor (VEGF), which has a special role among angiogenic growth factors. · Methods: Corneal neovascularization was induced in NZW rabbits by an intrastromal pellet loaded with 500 or 750 ng VEGF. Animals received two daily feedings of 200 mg/kg thalidomide. · Results: Significant inhibition of corneal angiogenesis (P<0.0001) was caused by the teratogenic dose of thalidomide after the 5th day of treatment and persisted for more than 16 days. No obvious side effects were recorded. · Conclusions: Thalidomide has a significant antiangiogenic effect against VEGF-induced neovasclar growth. Together with earlier findings this observation indicates that the drug inhibits two angiogenic pathways which are mediated through integrin adhesion molecules.

Use of fundus perimetry (microperimetry) to quantify macular sensitivity

The advances in retinal imaging technologies have led to enormous innovation towards diagnostic in current ophthalmology, enabling the practitioner to detect early retinal changes and to document treatment effects. While, in the past, retinoscopy, visual acuity testing and perimetry played the major role in functional diagnostics, today, laser-based systems like laser scanning imaging systems especially for fluorescein-angiography, optical coherence tomography, electrodiagnostic systems and the analysis of retinal vessels may be used as well. However, the challenge to correlate subjective alterations or clinical changes with visual function, still remains. Micro- or fundus perimetry offers the option to test retinal sensitivity while directly observing the fundus. In this paper, we review the literature encompassing the results of more than 25 years of fundus perimetry, i.e. perimetry under simultaneous visualization of the fundus. During this time, results on known diseases and reproducibility of the technique were published, but a lot of work was also performed on the combination of different examination methods, allowing a synopsis of long-term results and new approaches by combining different methods and improving each of them. The first part of this review attends to improvements of the method. The second part addresses the clinical and diagnostic values. The final part is dedicated to diagnostic and long-term observation of fundus perimetric results beginning with common and rare diseases like age-related macular degeneration, macular holes and diabetic retinopathy, various types of macular dystrophies ending with challenges in conventional perimetry like glaucoma and malingering. Due to the experience and progress in the field of fundus perimetry and retinal imaging, the method has long passed its role of observing and has all the potential for prediction, early detection and treatment-monitoring of macular diseases.

Microarray-based mutation analysis of the ABCA4 (ABCR) gene in autosomal recessive cone-rod dystrophy and retinitis pigmentosa.

Mutations in the ABCA4 gene have been associated with autosomal recessive Stargardt disease (STGD1), cone–rod dystrophy (CRD), and retinitis pigmentosa (RP). We employed a recently developed genotyping microarray, the ABCR400-chip, to search for known ABCA4 mutations in patients with isolated or autosomal recessive CRD (54 cases) or RP (90 cases). We performed detailed ophthalmologic examinations and identified at least one ABCA4 mutation in 18 patients (33%) with CRD and in five patients (5.6%) with RP. Single-strand conformation polymorphism (SSCP) analysis and subsequent DNA sequencing revealed four novel missense mutations (R24C, E161K, P597S, G618E) and a novel 1-bp deletion (5888delG). Ophthalmoscopic abnormalities in CRD patients ranged from minor granular pigmentary changes in the posterior pole to widespread atrophy. In 12 patients with recordable electroretinogram (ERG) tracings, a cone–rod pattern was detected. Three patients demonstrated progression from a retinal dystrophy resembling STGD1 to a more widespread degeneration, and were subsequently diagnosed as CRD. In addition to a variable degree of atrophy, all RP patients displayed ophthalmologic characteristics of classic RP. When detectable, ERG recordings in these patients demonstrated rod–cone patterns of photoreceptor degeneration. In conclusion, in this study, we show that the ABCA4 mutation chip is an efficient first screening tool for arCRD.

IQCB1 Mutations in Patients with Leber Congenital Amaurosis

PURPOSE Leber congenital amaurosis (LCA) is genetically heterogeneous, with 15 genes identified thus far, accounting for ∼70% of LCA patients. The aim of the present study was to identify new genetic causes of LCA. METHODS Homozygosity mapping in >150 LCA patients of worldwide origin was performed with high-density SNP microarrays to identify new disease-causing genes. RESULTS In three isolated LCA patients, the authors identified large homozygous regions on chromosome 3 encompassing the IQCB1 gene, which has been associated with Senior-Loken syndrome (SLSN), characterized by nephronophthisis and retinal degeneration. Mutation analysis of IQCB1 in these three patients and a subsequent cohort of 222 additional LCA patients identified frameshift and nonsense mutations in 11 patients diagnosed with LCA. On re-inspection of the patients disease status, seven were found to have developed SLSN, but four maintained the diagnosis of LCA as the kidney function remained normal. CONCLUSIONS Results show that the onset of renal failure in patients with IQCB1 mutations is highly variable, and that mutations are also found in LCA patients without nephronophthisis, rendering IQCB1 a new gene for LCA. However, these patients are at high risk for developing renal failure, which in early stages is often not recognized and can cause sudden death from fluid and electrolyte imbalance. It is therefore recommended that all LCA patients be screened for IQCB1 mutations, to follow them more closely for kidney disease.

Scanning laser ophthalmoscope fundus perimetry before and after laser photocoagulation for clinically significant diabetic macular edema

PURPOSE To prospectively evaluate functional and funduscopic changes after laser treatment in patients with diabetic retinopathy and clinically significant macular edema by scanning laser ophthalmoscope fundus perimetry. METHODS Thirty eyes of 30 patients with clinically significant macular edema as a result of diabetic retinopathy were prospectively examined before and at least 3 months after focal laser treatment with automatic fundus threshold perimetry using the scanning laser ophthalmoscope. Thresholds of light sensitivity were compared with age-corrected normal values and correlated with corrected visual acuity and subjective appraisal of visual function. RESULTS In 30 eyes, fundus perimetry lasted for 10.5+/-2.7 (mean+/-SD) minutes with 322+/-67 stimulus presentations for each eye. Whereas eight eyes remained stable (< +/-1 dB change), 15 improved concerning mean deviation (MD) (3.1+/-1.7 dB) after focal laser treatment. Stability of fixation remained the same after focal laser treatment (0.75+/-0.57 degree). Laser scars showed marked loss of function (MD > 13 dB). CONCLUSIONS Although light sensitivity was reduced in areas of macular edema, there was no correlation between the amount of edema and visual function. Fundus perimetry allows the creation of exact maps of retinal dysfunction before and after laser treatment. It may help in making management decisions in diabetic and nondiabetic patients by offering a sensitive parameter in addition to visual acuity.

Homozygosity mapping in patients with cone-rod dystrophy: novel mutations and clinical characterizations.

PURPOSE To determine the genetic defect and to describe the clinical characteristics in a cohort of mainly nonconsanguineous cone-rod dystrophy (CRD) patients. METHODS One hundred thirty-nine patients with diagnosed CRD were recruited. Ninety of them were screened for known mutations in ABCA4, and those carrying one or two mutations were excluded from further research. Genome-wide homozygosity mapping was performed in the remaining 108. Known genes associated with autosomal recessive retinal dystrophies located within a homozygous region were screened for mutations. Patients in whom a mutation was detected underwent further ophthalmic examination. RESULTS Homozygous sequence variants were identified in eight CRD families, six of which were nonconsanguineous. The variants were detected in the following six genes: ABCA4, CABP4, CERKL, EYS, KCNV2, and PROM1. Patients carrying mutations in ABCA4, CERKL, and PROM1 had typical CRD symptoms, but a variety of retinal appearances on funduscopy, optical coherence tomography, and autofluorescence imaging. CONCLUSIONS Homozygosity mapping led to the identification of new mutations in consanguineous and nonconsanguineous patients with retinal dystrophy. Detailed clinical characterization revealed a variety of retinal appearances, ranging from nearly normal to extensive retinal remodeling, retinal thinning, and debris accumulation. Although CRD was initially diagnosed in all patients, the molecular findings led to a reappraisal of the diagnosis in patients carrying mutations in EYS, CABP4, and KCNV2.