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Background and Purpose— Transorbital sonography may help establish diagnosis of central retinal artery occlusion (CRAO). Next to Doppler sonographic proof of CRAO, an intra-arterial spot sign can be detected in some cases. We hypothesized that it reflects calcified components. It may be associated with embolization from atherosclerotic plaques and may negatively influence thrombolysis. Methods— Prospective monocenter study of 46 patients with ophthalmologically confirmed CRAO. Systemic tissue-type plasminogen activator thrombolysis was performed when appropriate. All patients received etiologic workup. Results— CRAO was confirmed by Doppler in all patients. Fifty-nine percent of patients with arterio-arterial embolization were spot sign–positive compared with 20% from cardiac source ( P P Conclusions— Transbulbar ultrasound is valuable for initial diagnosis and diagnostic workup of CRAO. In the light of inconsistent results of previous thrombolysis trials, ultrasound may identify patients more likely to benefit from thrombolytic treatment.

Most retinal neovascular disorders are caused by upregulation of vascular endothelial growth factor (VEGF) expression. These disorders are treated with repeated injections of anti-VEGF molecules, which may have severe side effects. The expression of anti-VEGF molecules by the retina itself in a controlled manner following adeno-associated viral (AAV) gene transfer could be a replacement of this therapy.The open reading frames (orf) of the light and the heavy chain of ranibizumab were cloned into an expression plasmid separated by an internal ribosomal entry site (IRES). The construct was mutated to generate ranibizumab single-chain variable fragments (scFv). Expression was verified by western blotting and the concentrations were measured with a custom-made ranibizumab ELISA. Biological activity, VEGF-binding properties, and the doxycycline-dependent induction of anti-VEGF expression were tested. An AAV2/5 vector was generated containing the optimal variant Ra02.Ra01-Ra05 molecules were detected in the cell culture medium. While the VEGF-binding affinity was significantly lower for Ra01 and Ra02 compared to Lucentis(®), the inhibition of cell migration was comparable and the maximum inhibition of Ra01 and Ra02 was reached at lower doses. The expression of Ra01 and Ra02 was shown to be regulable with the TetOn-system(®) as plasmid (Ra01, Ra02) and AAV vector construct (Ra02).Ra01 and Ra02 can be produced in eukaryotic cells after AAV-mediated gene transfer in a regulable manner in vitro and display comparable biological activity as Lucentis. These results are the basis for in vivo studies in human VEGF-overexpressing mice, a model for human neovascular disorders.

We report on the clinical, molecular and biochemical findings of a patient with the rare event (4.02 x 10(-9) per generation) of coinciding de novo mutations in the nuclear PAX6 (c.1252-1267del16) and the mitochondrial mt.RNA (Lys) (8347A--G) genes. The boy suffers from exercise intolerance, ptosis, nystagmus, macular hypoplasia and anterior segment abnormalities evocative of Axenfeld-Rieger anomaly. The PAX6 mutation is predicted to cause haploinsufficiency. The novel mt.RNA (Lys) mutation is located close to the classic myoclonic epilepsy with ragged-red-fibers mutation, but the patient exhibits neither myoclonic epilepsy nor ragged-red-fibers. The degree of mutant mtDNA heteroplasmy, as determined by a very accurate pyrosequencing assay, varies between 31% (muscle) and 38% (fibroblasts). We discuss a potential effect of the PAX6 mutation on the mtDNA mutation rate.

To report the nature of the contents of a cyst associated with microphthalmia and optic nerve coloboma in a male infant.Clinicopathologic report.A male infant in good general health presented with a massively swollen, protruded left lower eyelid since birth. The globe was not identifiable. MRI disclosed a microphthalmic globe in the upper nasal orbit and an orbital cystic mass consisting of various compartments. Examination of the fellow eye showed a large optic nerve coloboma. As the protrusion of the left lower eyelid was progressive, aspiration of the cyst was performed twice. Finally, the cyst was removed surgically due to repeated filling.Examination of the aspirated fluid showed resemblance to cerebrospinal fluid (CSF). During surgery, the cyst was not found to communicate with the cerebrospinal fluid system. However, tissue resembling choroidal plexus lined the wall of the cyst that was adherent to the optic nerve. Histological evaluation of the tissue revealed ependymal cells.The presence of CSF in microphthalmic cysts does not necessarily imply a connection to the CNS, but may be due to local CSF production by ectopic ependymal cells.

Oculo-auriculo-vertebral spectrum (OMIM164210) is a phenotypically and probably also a genetically heterogeneous disorder, characterized by anomalies of the ear (mostly microtia), hemifacial microsomia, and defects of the vertebral column. Associated clinical findings include anomalies of the eye and brain, and developmental delay. We have evaluated the clinical data and photographs of 53 unrelated patients with OAVS, all presenting with either isolated microtia or preauricular tags in association with hemifacial microsomia as minimal diagnostic criteria; five had a positive family history for OAVS. Based on the main clinical findings and unilateral or bilateral involvement, we have developed a new classification system for OAVS, consisting of six subgroups. There is a statistically significant correlation between the subgroup and number of associated clinical findings, and a statistically significant difference regarding prognosis in uni- and bilaterally affected patients, suggesting that this classification is clinically relevant to the categorization of patients with OAVS. The newly developed scoring system (two points for each main clinical finding and one for each associated clinical finding) presented here, also aids prognosis, especially for delay of motor development and brain anomalies, and statistical analysis revealed significant clustering between different clinical findings of OAVS confirming the clinical impression previously published by several authors.

Hintergrund: Das Alagille-Syndrom ist eine autosomal-dominante Erkrankung, die durch neonatale Cholestase, typische Facies und kardiale Anomalien gekennzeichnet ist. Charakteristische okuläre Anomalien sind Embryotoxon posterius, Irisstroma-Hypoplasie mit Mosaikmuster, Mikrocornea, papilläre Drusen und Retinopathia pigmentosa. Wir legen hier den zweiten Bericht zur okulären Pathologie in zwei Fällen des Alagille-Syndroms vor. Methoden: Makroskopische und histologische Untersuchung von Präparaten von vier Augen zweier Patienten. Ergebnisse: In beiden Fällen liegt ein Embryotoxon posterius vor; in Fall 1 mit Iris-Prozessbildung bis zum Embryotoxon. Fall 1 wies ein deutlich anomales Irisstroma mit ausgeprägter Spaltbildung zwischen anteriorem und posteriorem Stroma auf. In Fall 2 war Vakuolenbildung mit Spitzenmuster zu beobachten. Schlussfolgerungen: Das Alagille-Syndrom ist primär eine hepatische Erkrankung, weist jedoch auch mehrere charakterische okuläre Krankheitsmerkmale auf, insbesondere Embryotoxon posterius. Dies und die Anomalien des Irisstromas könnten auf die fehlerhafte Migration von Neuralleistenzellen infolge der Mutation des Jagged-1-Gens zurückzuführen sein, die das Alagille-Syndrom verursacht. Patienten mit Alagille-Syndrom werden selten einer okulären Autopsie unterzogen. Die Ergebnisse dieser Untersuchung könnten zum besseren Verständnis der Pathophysiologie der okulären Anomalien bei dieser Erkrankung beitragen.

Ziel: Beurteilung der Auswirkung einer einmaligen intravitrealen Injektion mit Bevacizumab (IVB) auf verschiedene Parameter des Blutflusses in der Arteria ophthalmica (AO) und Arteria cerebri media (ACM) bei Säuglingen mit Retinopathia praematurorum (RPM). Material und Methoden: In diese prospektive, interventionelle Studie wurden 15 Säuglinge mit RPM eingeschlossen, die mit IVB behandelt wurden. Untersucht wurden die systolische Spitzengeschwindigkeit (PSV), die enddiastolische Geschwindigkeit, die mittlere Geschwindigkeit (MV) und der Resistivitätsindex, gemessen mittels Pulswellen-Dopplersonographie (Philips En Visor C, Amsterdam, Niederlande) in der AO und ACM vor der Injektion mit IVB sowie einen Tag, eine Woche und einen Monat nach der IVB-Injektion. Ergebnisse: Die gemessenen PSV-AO-, MV-AO- und PSV-ACM-Werte zeigten signifikante Veränderungen nach der IVB-Behandlung (p = 0,01, p = 0,02, bzw. p = 0,02). Die PSV-AO-Werte nach einer Woche und einem Monat waren signifikant niedriger als der PSV-AO-Ausgangswert vor der Behandlung (p = 0,03, bzw. p = 0,01). Der MV-AO-Wert war einen Monat nach der IVB-Behandlung signifikant niedriger als der Ausgangswert (p = 0,03). Der PSV-ACM-Wert zeigte einen signifikanten Rückgang einen Tag nach der Injektion (p = 0,03). Schlussfolgerungen: Die Studie belegt, dass IVB bei Säuglingen mit RPM signifikante Veränderungen bei Blutfluss-Parametern der AO und ACM bewirkt, wie die Dopplersonographie zeigt.

Isolated functional methionine synthase deficiency occurs in the cblE and cblG defects of methylcobalamin metabolism and is one of a number of causes of severely elevated plasma homocysteine. Clinical features are predominantly of a neurological nature but also include functional restriction of the visual system manifesting as loss of visual acuity and nystagmus. As yet, the origin and pathogenesis of impaired vision have not been explained.We investigated a patient who was proven by complementation analysis in cultured fibroblasts to belong to the cblG complementation group. Ganzfeld electroretinograms (ERG) and flash visual evoked potentials (VEP) were recorded over a period of 4 years.Amplitudes of all International Society for Clinical Electrophysiology of Vision (ISCEV) standard responses were below normal. The greatest reductions were of rod response to 24 microV, of standard combined response (SC) b-wave to 120 microV, of oscillatory potentials (OP) to 5 microV, of cone response b-wave to 35 microV, and of 30 Hz flicker response to 8 microV. Except for SC and cone a-waves at age 2.5 and 3.5 years, as well as cone b-wave at 3.5 years, amplitudes remained at a subnormal level at follow-up examinations. Implicit times were slightly prolonged (SC b-wave 6 ms, OPs 2 ms, cone b-wave 2 ms, 30 Hz flicker 4 ms) or fell within the normal range. Responses of the flash VEP were severely deformed but reproducible.This is the first report of detailed investigations of the visual system in a patient with isolated methionine synthase deficiency. Reduced oscillatory potentials suggest microvascular damage to the retina through homocysteine. Decreased photoreceptor function as well as ganglion cell loss as indicated by pathological flash VEPs may reflect a cytotoxic impact of homocysteine on neurons of the visual pathway.

Background Duane retraction syndrome (DURS) accounts for 1 - 4 % of all cases of strabismus. Approximately 90 % of the cases are sporadic with a preponderance for females and the left eye. Many associated ocular and systemic findings have been described. Recently, mutations of SALL4 have been found in patients with autosomal-dominantly inherited Okihiro syndrome (DURS associated with forearm malformations). The aim of this study was the clinical examination of patients with isolated sporadic DURS and the molecular genetic analysis of SALL4 in these patients. Subjects and methods Twenty-five patients with non-familial DURS (aged 1 - 75 years, 16 female, 9male) were examined clinically and were interviewed concerning associated pathologies. DNA was prepared from peripheral lymphocytes, and the complete coding region of SALL4 was sequenced. Results In 18 patients DURS affected the left eye, in four the right eye, and was bilateral in three patients. One patient had fused vertebrae, one had a cone-rod-dystrophy. No hearing impairments or malformation of the upper limbs were observed. No mutation in the coding region of SALL4 could be detected. Discussion Associated conditions in DURS patients most commonly involve the ear, the spinal column, the kidneys and the heart and the upper limbs. No mutations in SALL4 could be detected in patients with isolated sporadic DURS as opposed to findings in familial Okihiro syndrome. However, Okihiro syndrome shows marked intra- and interfamilial variability, suggesting that in rare cases of isolated DURS a causative SALL4 mutation may be found.

Adult malignant optic nerve gliomas are rare and rapidly fatal visual pathway tumours. They represent a clinical entity different from the more common childhood benign optic nerve gliomas, which are frequently associated with neurofibromatosis I. A 61-year-old woman presented with rapidly progressing right vision loss, lower altitudinal visual field defect and papilloedema. MRI showed intraorbital and intracranial swelling of the right optic nerve. Resection of the intracranial part of the right optic nerve up to the chiasm revealed anaplastic astrocytoma grade III. Within 1 year, the patient died of leptomeningeal metastasis despite radiotherapy. Clinical and MRI evaluation of the left eye and optic nerve were normal at all times. Unilateral adult malignant glioma of the optic nerve is exceptional. The final diagnosis was only confirmed by optic nerve biopsy. In the literature, only one patient has been reported with a unilateral tumour manifestation; he was lost to follow-up 3 months later. All other cases were bilateral. To date, 44 case reports of adult malignant optic nerve glioma have been published, either malignant astrocytoma or glioblastoma. These tumours can mimic optic neuritis in their initial presentation. The diagnosis is seldom made before craniotomy. On MRI images, malignant glioma cannot be distinguished from optic nerve enlargement due to other causes. Although radiotherapy appears to prolong life expectancy, all presently available treatment options (radiation, surgery, radio-chemotherapy) are of limited value. Most patients go blind and die within 1 or 2 years.

PURPOSE Craniosynostoses are premature ossifications of cranial sutures. They occur isolated and syndromic. Syndromic craniosynostoses are mainly associated with mutations of the Fibroblast Growth Factor Receptors (FGFR) 1 - 3. This paper gives an overview of the etiology and pathophysiology of isolated and syndromic craniosynostoses and discusses the molecular genetic results in 21 index cases (19 seemingly isolated craniosynostoses, 2 cases with a clinical diagnosis of Crouzon's syndrome). METHOD Mutation analysis in exons of the FGFR 1 - 3 known to be preferentially affected in craniosynostoses was performed on DNA samples from peripheral blood and bone specimen excised at the time of surgery to correct the craniosynostosis. RESULTS In a girl with seemingly isolated plagiocephaly we identified a P250L (749C-->T) mutation in FGFR3. Her mother showed minor signs of craniosynostosis when the family was re-evaluated. She was shown to carry the same mutation. In two patients with suspected Crouzon's syndrome 2 different mutations were detected at the same nucleotide (1025G-->A or C) and confirmed the clinical diagnosis. No mutation was found in 18/19 seemingly isolated craniosynostosis cases. CONCLUSION In contrast to syndromic forms isolated craniosynostoses are rarely associated with mutations in FGFR. The affection of further family members is a strong indication of involvement of FGFR mutations. Because of variable expressivity, parents should be examined carefully in isolated craniosynostoses to identify minor signs.

Aim: To determine the anatomical site and patterns of cause of blindness in one of the biggest schools for the blind in Indonesia with a view to determining potentially preventable and treatable causes. Methods: 165 students in one school for the blind in Bandung, Indonesia, were examined clinically and data reported using the WHO/PBL childhood blindness assessment form. Results: Most of the students (96.4%) were blind (BL); 3% were severely visually impaired (SVI). The major causes of SVI/BL in this study were: (1) corneal staphyloma, corneal scar, and phthisis bulbi (mainly attributed to infection) in 29.7%; (2) retinal dystrophies (mainly Leber congenital amaurosis, early onset retinitis pigmentosa) in 20.6%; (3) congenital and familial cataract (13.3%); (4) microphthalmus, anophthalmus (10.9%). The whole globe was the major anatomical site of visual loss (32.7%), followed by the retina (26.0%), cornea (17.6%), lens (13.3%), optic nerve (6.1%), and uvea (4.3%). Conclusions: This is a small study in a selected population and the results should be interpreted with caution. This blind school study, adopting the WHO/PBL eye form for data analysing, is the first reported for Indonesia. Hereditary disease and infective causes of blindness are the predominant causes of blindness, accounting for 42.4% and 29.7%, respectively. This pattern of causes is a mixed pattern which lies in an intermediate position between the patterns seen in developing countries and those seen in developed countries. The importance both of preventive public health strategies and of specialist paediatric ophthalmic and optical services in the management of childhood blindness in Indonesia are therefore strongly suggested to cover the problems that exist.

Blepharophimosis syndrome (BPES), an autosomal dominant syndrome in which an eyelid malformation is associated (type I) or not (type II) with premature ovarian failure (POF), has recently been ascribed to mutations in FOXL2, a putative forkhead transcription factor gene. We previously reported 22 FOXL2 mutations and suggested a preliminary genotype-phenotype correlation. Here, we describe 21 new FOXL2 mutations (16 novel ones) through sequencing of open reading frame, 5′ untranslated region, putative core promoter, and fluorescence in situ hybridization analysis. Our study shows the existence of two mutational hotspots: 30% of FOXL2 mutations lead to polyalanine (poly-Ala) expansions, and 13% are a novel out-of-frame duplication. In addition, this is the first study to demonstrate intra- and interfamilial phenotypic variability (both BPES types caused by the same mutation). Furthermore, the present study allows a revision of the current genotype-phenotype correlation, since we found exceptions to it. We assume that for predicted proteins with a truncation before the poly-Ala tract, the risk for development of POF is high. For mutations leading to a truncated or extended protein containing an intact forkhead and poly-Ala tract, no predictions are possible, since some of these mutations lead to both types of BPES, even within the same family. Poly-Ala expansions may lead to BPES type II. For missense mutations, no correlations can be made yet. Microdeletions are associated with mental retardation. We conclude that molecular testing may be carefully used as a predictor for POF risk in a limited number of mutations.

To describe the phenotype of three patients and two carriers from two families with mutations in the RPGR gene. The genotypes (a 75-kb deletion on the X chromosome spanning the RPGR gene and the first exon of the SRPX gene, and a stop mutation (G52X) in the RPGR gene) have been reported previously.A clinical examination including Goldmann perimetry, full-field electroretinography (ERG), dark adaptometry, and dark- and light-adapted two-color threshold (500-nm cut-off, 600-nm cut-on filter) perimetry was performed in all patients and one carrier. The second carrier was only examined clinically.All affected males presented with a marked decrease in visual acuity of 0.3 to 0.5 at the age of 17-22.5 years, and a typical fundus appearance. The stop mutation (G52X) appeared to be associated with a more pronounced bone spicule formation compared to the deletion of the entire RPR gene and the first exon of the SRPX gene. The kinetic visual fields were constricted to20 degrees eccentricity, in part with a residual island in the temporal field. Using two-color dark-adapted threshold perimetry, rod function was more reduced than cone function. The ERG was extinguished. The carrier with the stop mutation showed sectorial peripheral bone spicules and ERG changes typical of carriers of XLRP. The carrier with the deletion had no visual complaints, full visual acuity, and only minimal peripheral retinal changes. Goldmann perimetry showed minor peripheral defects with small targets. ERG amplitudes were reduced below the 10th percentile of normals, without selective loss in rods or cones. The scotopic (rod) sensitivity loss at 500 nm was more pronounced than the photopic (cone) sensitivity loss at 600 nm. Neither of the two carriers showed a tapetal reflex.The affected males of the two families with RPGR mutations already exhibited retinitis pigmentosa with severe impairment of the rod and cone system during their second decade of life. The degree of bone spicules differed between the two families. Psychophysics detected a slightly more pronounced affection of the rod system compared to the cone system in both the hemizygous males and the carrier with the deletion of the RPGR gene and the first exon of the SRPX gene. Psychophysics disclosed mild progression of the disease in the carrier underlining the potential of the method in monitoring the disease course. As in most other reported phenotypes of RPGR mutations, no tapetal reflex was found in the carriers.

Cleidocranial dysplasia (CCD) and the Rubinstein- Taybi syndrome (RTS) are two rare congenital syndromes that have many clinical signs in common. We present an 18-year- old-patient with untypical CCD expression who was misdiag- nosed with RTS at the age of 2 years. An extensive craniofacial examination was carried out with respect to morphological and dental aspects. The molecular-genetic analysis of two under- lying genes (CBFA1 and CBP) for CCD and RTS was per- formed using SSCP, direct sequencing and FISH. While the clinical examination showed uncharacteristic CCD symptoms with some findings common for RTS, the molecular- genetic analysis revealed a missense mutation in the CBFA1 gene, which is considered to be the etiological factor for CCD. Our findings with this patient presented clear evidence for the wide morphologic variety that can be related to a certain gene such as CBFA1. The diagnosis of rare diseases is currently based on the clinical phenomenology of small groups or single cases. The use of molecular-genetic biology extends the hori- zon of diagnostic and scientific possibilities. In this patient, it allowed us to compare the clinically diagnosis to molecular- genetic data. We conclude that molecular-genetic analysis may be a helpful tool in the differential diagnosis of many congenital diseases such as CCD and RTS.

Purpose. To determine the phenotypic variability in patients with compound heterozygous or homozygous ABCA4 mutations, and to correlate the phenotypes with the functional properties of the altered protein. Methods. Sixteen patients from 13 families with signs of Stargardt macular dystrophy/fundus flavimaculatus and known mutations on both alleles of the ABCA4 gene (15 compound heterozygous, one homozygous) were characterized by clinical examination, fundus autofluorescence, psychophysics (color vision, kinetic and two-color dark- and light-adapted static threshold perimetry), and electrophysiology (Ganzfeld, multifocal ERG, EOG). Results. The homozygous 5917delG mutation resulted in the earliest disease manifestation (at 5 years) and a general cone–rod dysfunction, whereas the compound heterozygous mother (5917delG, G1961E) exhibited a very mild phenotype. Compound heterozygotes for the IVS40+5G→A and the C1488Y or Y362X mutation showed also an early age of onset but only a central dysfunction. The effect of the 2588G→C mutation, the G1961E mutation, and the complex mutation L541P-A1038V depended on the mutation in the second allele. Genotype–phenotype correlation appeared possible in most instances. Psychophysics revealed a simultaneous yet not necessarily congruent cone and rod dysfunction. Conclusions. The type and combination of ABCA4 mutations in compound heterozygous patients determined were compatible with the severity of the phenotype as to age of onset and the functional consequences in the majority of patients. Unexplained phenotypic differences indicate the influence of other factors. ABCA4 mutations result in cone and rod dysfunction. Different disease durations limit the power of presently available genotype–phenotype correlations.

Hintergrund. Die pra- und postoperativen Befunde bei Patienten mit ein- und beidseitiger Abduzensparalyse werden vorgestellt.

Family studies have demonstrated the genetic etiology of concomitant strabismus (prevalence in different populations 1-5%, positive family history in 37% on average), as have twin studies (mean concordance in monozygous twins 73% compared to 35% in dizygous twins). In Duane's syndrome, three chromosomal loci have been identified to date: 2q31, 8q13, and 22q11; loci have also been identified in Moebius syndrome (various inheritance patterns): 13q12.2-q13, 3q21-q22, and 10q21.3-q22, as well as in congenital fibrosis of the extraocular muscles (CFEOM; 3 loci, 1 gene).(1) Already identified are the genes for a number of rarer muscular dystrophy syndromes with ocular involvement, various forms of congenital myasthenia, and mitochondrial diseases (with the primary defect located either in the mtDNA, resulting in a mitochondrial inheritance pattern, or in the nuclear DNA with Mendelian inheritance). A number of hereditary retinal diseases (Mendelian inheritance) may also be associated with strabismus. More than one gene is likely to be involved in the frequently occurring concomitant strabismus, making the analysis more difficult. Patients with chromosomal rearrangements, large families and isolated populations (see also the contributions by Preising and Zitzlsperger et al. in this issue)(2,3) will be instrumental in gene identification. In view of the high prevalence of concomitant strabismus, the disclosure of its etiology will have considerable medical, psychosocial and health-cost impact.

This study was intended to identify communities with an increased prevalence rate of inherited eye diseases (different forms of strabismus, juvenile maculopathy, AMD, myopia magna, etc.) in the districts Oberpfalz (Opf.) and Niederbayern (Ndb.). The data obtained in this study will serve to identify larger families and inbred populations for genetic testing to identify the cause of eye diseases with autosomal recessive and multifactorial inheritance.In cooperation with the local ophthalmologists in two Bavarian districts anonymized lists were compiled correlating diagnosis, postal code, and municipality of strabismus patients. The office software used in the participating practices was a limiting factor as well as the hand-written records used in most of these. The collected data were correlated with the published prevalence rates in the general population.Of the 104 local ophthalmologists who were invited to participate, only 17 provided information. The results indicate an increased prevalence rate of strabismus and microstrabismus patients from Neumarkt (Opf.) / Postbauer-Heng, Amberg, Hauzenberg and Pocking.Documentation as it is currently done - local ophthalmologist generally have written records and computer systems that are unsuitable for detailed surveys - restricts the possibility of completing the survey in the whole area. Nevertheless, regional accumulations of strabismus/microstrabismus could be described, providing populations for further investigations to uncover the genetic causes.

Purpose Alternating oxygen levels during delivery and supplemental oxygen therapies can cause pathological vascularization of the retina (retinopathy of prematurity, ROP) and/or loss of oligodendrocytes in the periventricular area of the brain with subsequent white matter damage (periventricular leukomalacia (PVL). The aim of this study was to examine retinal vessel pathologies in the standard PVL rat model in order to identify uniform damage pathways. Methods Ischemia was induced in post natal day (P) 6 Long Evans rats with unilateral (left side) carotid ligation (UCL) and exposure to different oxygen concentrations. In total, four different groups were examined: (A) hypoxia/ischemia (UCL + 6% O2, 1 h), (B) hyperoxia (80%O2, 24 h), (C) hypoxia/ischemia + hyperoxia (UCL + 6% O2, 1 h + 80% O2, 24h), (D) normoxia. Morphological analysis of vessel development was performed at P11 and P21. Quantitative PCR was performed at P7, P11, and P21 (VEGF, HIF1a, EPOR, TNFa, iNOS and BMP-9). Results Distinct retardation in deeper vascular plexus development was observed most prominent in left eyes of groups A and C. Diameters of big retinal arteries were significantly increased in central retinae at P21 and showed retardation in vascular remodeling. Left retinae of groups A and C at P11 displayed reduced capillary free zones (CFZ) and an increase in the number of branching points (BP). Quantitative gene expression analysis showed down regulation of angiogenic factors at P7. Conclusion This is the first report on concurring damage to the retina that was evaluated in a model of white matter injury in the neonatal brain. We observed mild damage to the vessel development and maturation, which is the basis for a model for moderate forms of ROP.

The aim of this study was to provide fundus-controlled two-color adaptometry with an existing device. A quick and easy approach extends the application possibilities of a commercial fundus-controlled perimeter. An external filter holder was placed in front the objective lens of the MP1 (Nidek, Italy) and fitted with filters to modify background, stimulus intensity, and color. Prior to dark adaptometry, the subject’s visual sensitivity profile was measured for red and blue stimuli to determine whether rods or cones or both mediated the absolute threshold. After light adaptation, 20 healthy subjects were investigated with a pattern covering six spots at the posterior pole of the retina up to 45 min of dark adaptation. Thresholds were determined using a 200 ms red Goldmann IV and a blue Goldmann II stimulus. The pre-test sensitivity showed a typical distribution of values along the meridian, with high peripheral light increment sensitivity (LIS) and low central LIS for rods and the reverse for cones. After bleach, threshold recovery had a classic biphasic shape. The absolute threshold was reached after approximately 10 min for the red and 15 min for the blue stimulus. Two-color fundus-controlled adaptometry with a commercial MP1 without internal changes to the device provides a quick and easy examination of rod and cone function during dark adaptation at defined retinal loci of the posterior pole. This innovative method will be helpful to measure rod vs. cone function at known loci of the posterior pole in early stages of retinal degenerations.

Optic atrophy type 1 (OPA1, MIM 165500) is a dominantly inherited optic neuropathy that features low visual acuity leading in many cases to legal blindness. We have recently shown, with others, that mutations in the OPA1 gene encoding a dynamin-related mitochondrial protein, underlie the dominant form of optic atrophy. Here we report that OPA1 has eight mRNA isoforms as a result of the alternative splicing of exon 4 and two novel exons named 4b and 5b. In addition, we screened a cohort of 19 unrelated patients with dominant optic atrophy by direct sequencing of the 30 OPA1 exons (including exons 4b and 5b) and found mutations in 17 (89%) of them of which 8 were novel. A majority of these mutations were truncative (65%) and located in exons 8 to 28, but a number of them were amino acid changes predominantly found in the GTPase domain (exons 8 to 15). We hypothesize that at least two modifications of OPA1 may lead to dominant optic atrophy, that is alteration in GTPase activity and loss of the last seven C-terminal amino acids that putatively interact with other proteins.

We recently showed that mutations in the CNGA3 gene encoding the alpha -subunit of the cone photoreceptor cGMP-gated channel cause autosomal recessive complete achromatopsia linked to chromosome 2q11. We now report the results of a first comprehensive screening for CNGA3 mutations in a cohort of 258 additional independent families with hereditary cone photoreceptor disorders. CNGA3 mutations were detected not only in patients with the complete form of achromatopsia but also in incomplete achromats with residual cone photoreceptor function and (rarely) in patients with evidence for severe progressive cone dystrophy. In total, mutations were identified in 53 independent families comprising 38 new CNGA3 mutations, in addition to the 8 mutations reported elsewhere. Apparently, both mutant alleles were identified in 47 families, including 16 families with presumed homozygous mutations and 31 families with two heterozygous mutations. Single heterozygous mutations were identified in six additional families. The majority of all known CNGA3 mutations (39/46) are amino acid substitutions compared with only four stop-codon mutations, two 1-bp insertions and one 3-bp in-frame deletion. The missense mutations mostly affect amino acids conserved among the members of the cyclic nucleotide gated (CNG) channel family and cluster at the cytoplasmic face of transmembrane domains (TM) S1 and S2, in TM S4, and in the cGMP-binding domain. Several mutations were identified recurrently (e.g., R277C, R283W, R436W, and F547L). These four mutations account for 41.8% of all detected mutant CNGA3 alleles. Haplotype analysis suggests that the R436W and F547L mutant alleles have multiple origins, whereas we found evidence that the R283W alleles, which are particularly frequent among patients from Scandinavia and northern Italy, have a common origin.

Ocular albinism type 1 (OA1) is an X-linked disorder mainly characterized by congenital nystagmus and photodysphoria, moderate to severe reduction of visual acuity, hypopigmentation of the retina, and the presence of macromelanosomes in the skin and eyes. We have previously isolated the gene for OA1 and characterized its protein product as melanosomal membrane glycoprotein displaying structural and functional features of G protein-coupled receptors. We and others have identified mutations of various types within the OA1 gene in patients with this disorder, including deletions and splice site, frameshift, nonsense, and missense mutations. However, different prevalences of large intragenic deletions have been reported, ranging from 10% to 50% in independent studies. To determine whether these differences might be related to the geographic origin of the OA1 families tested, we performed a further extensive mutation analysis study leading to the identification of pathogenic mutations in 30 unrelated OA1 patients mainly from Europe and North America. These results, together with our earlier mutation reports on OA1, allow us to resolve the apparent discrepancies between previous studies and point to a substantial difference in the frequency of large intragenic deletions in European ( 50%) OA1 families. These observations and our overall refinement of point mutation distribution within the OA1 gene have important implications for the molecular diagnosis of OA1 and for the establishment of any mutation detection program for this disorder

Aim: Analysis of the diseases underlying congenital nystagmus in a series of patients registered during 6 years as a prerequisite for adequate counselling of the families. Patients and Methods: Retrospective study of all patients that presented between 1992 and 1998 with congenital nystagmus not related to visual deprivation or acquired pathologies of the visual pathways. The patients were examined clinically and in dependence on the findings also by electrophysiological (Ganzfeld ERG and VEP, Albino-flash-VEP), psychophysical (colour vision, dark adaptation, spectral sensitivity), and molecular genetic methods. When estimated necessary, family members affected by history and unaffected family members were also examined. In cases of complex neuroophthalmological diseases a neuropaediatric examination including neuroimaging was initiated. Results: In total, 180 patients could be analysed. A sensory defect nystagmus (SDN) was present in 142 patients (79%). The diagnoses were as follows: albinism (any form) in 56 patients (30%), progressive photoreceptor dystrophy in 20 patients (11%), stationary cone dysfunction in 18 patients (10%), bilateral optic nerve hypoplasia in 15 patients (8%), chorioretinal or optic nerve colobomata in 10 patients (6%), aniridia and its variants in 10 patients (6%), familial isolated nystagmus in 8 patients (5%), and congenital stationary night blindness in 5 patients (3%). 38 patients (21 %) could not (yet) be classified. Discussion: The prevalence of SDN as the manifesting symptom of a variety of well defined diseases in the present series of at least 79% is similar to that of 90% reported earlier. The precise diagnosis is a prerequisite for counselling the families as to functional prognosis and recurrence risk. Unnecessary neurological examinations including neuroimaging can be avoided.

The filter settings of the amplifier section of ERG recording systems have large impact on the waveforms of the recorded responses. In this study, the effects of a 50 Hz notch filter were assessed for both First-Order-Kernel- (FOK) and Second-Order-Kernel-responses (SOK 1st slice) from multifocal ERGs recorded with a VERIS III system. Amplitude and phase responses of the amplifier section were recorded for typical filter settings with the notch filter active and inactive. Multifocal ERGs (MERGs) from a group of 11 normal subjects were recorded for both amplifier settings, the waveforms resulted from those recordings were compared in the time and frequency domain. To verify the results, the recordings without the notch filter were digitally filtered with a simulated notch filter and compared to the responses recorded with an active analog notch filter. The line filter has the biggest attenuation at 50 Hz with an additional phase jump of 180°. The FOK responses of MERGs are assembled by frequencies below 65 Hz with main spectral components between 19 and 47 Hz. The 1st slice of the SOK consists of frequencies up to 100 Hz with main components between 19 and 84 Hz. Thus, if FOK recordings are to be analyzed, the notch filter of the amplifier can be used in order to cope with noise problems caused by the line frequency of 50 Hz. However, one must be aware that the attenuation of higher frequency components will lead to changes of the waveforms. For SOK analysis, main spectral components are attenuated and/or shifted in phase, which leads to completely different waveforms and severe distortion of the recording results.

The RS1 gene is the causative gene in X-linked juvenile retinoschisis (RS). We have screened this gene for mutations in 13 patients with RS and in 7 probands with senile retinoschisis, a sporadic, later-onset form of retinoschisis. Mutations were detected in all RS patients. Of the 11 different mutations identified, six have been reported previously and five are novel. We did not find mutations in any of the senile retinoschisis patients and conclude that senile retinoschisis is not the result of germline mutations in the RS1 gene.

Hintergrund: Die Frühgeborenen-Retinopathie (ROP; Retinopathia praematurorum) ist eine potenziell zur Erblindung führende Krankheit, die bei extrem verfrüht geborenen Säuglingen auftritt. Die Einführung neuer ROP-Vorsorgeuntersuchungssysteme mit höherer Sensitivität und Spezifität als bisherige Leitlinien für das ROP-Screening hat das Potenzial, die Zahl belastender Augenuntersuchung bei diesen Säuglingen zu verringern. Ziele: Erhöhung der Spezifizität des WINROP-Überwachungssystems (Weight, Insulin-like growth factor-I, Neonatal, ROP) zur Identifikation extrem frühgeborener Säuglinge mit behandlungsbedürftiger ROP. Methoden: Zwei Kohorten, die zuvor WINROP-Analysen unterzogen worden waren, wurden in die Studie eingeschlossen und erneut evaluiert. Das Körpergewicht bei WINROP-Alarm für extrem frühgeborene Säuglinge (Entbindung vor Schwangerschaftswoche 27) wurde reevaluiert, und durch Setzung «sicherer» WINROP-Alarmgrenzwerte für das Körpergewicht erfolgte eine Neubeurteilung der Stichproben nach WINROP-Alarm. Die folgenden beiden Kohorten wurden untersucht: (1) die Kohorte der EXPRESS-Studie (Extremely Preterm Infants in Sweden Study) mit 2004-2007 in Schweden geborenen Säuglingen (n = 407) und (2) die extrem frühgeborenen Säuglinge einer nordamerikanischen Kohorte, Geburtsjahrgänge 2006-2009 (n = 566). Ergebnisse: In der EXPRESS-Kohorte lag bei 12,5% (40/319) der Säuglinge, bei denen zuvor ein WINROP-Alarm erfolgt war, nun kein Alarm mehr vor; die Spezifität von WINROP in der EXPRESS-Kohorte stieg damit von 23,9% (86/360) auf 35,0% (126/360). In der nordamerikanischen Kohorte hatten nach der erneuten Beurteilung 15,4% (81/526) keinen Alarm mehr; die Spezifität stieg von 8,5% (38/447) auf 26,6% (119/447). Die Sensitivität betrug unverändert 97,5% in der EXPRESS-Kohorte (45/47) und 98,3% (117/119) in der nordamerikanischen Kohorte. Schlussfolgerungen: Die Spezifität des WINROP-Überwachungssystems für extrem frühgeborene Säuglinge kann durch erneute Evaluierung anhand des Gewichts bei WINROP-Alarm signifikant verbessert werden. Neonatology 2015;108:152-156 (DOI:10.1159/000435770)

Background Complex posttraumatic malpositions of the orbital walls require repositioning osteotomy. Computer tomography, stereolithography models and tele-X-rays are used in planning. However, the precision achieved in the planning phase could not so far be translated to patients (1). The Surgical Segment Navigator SSN is the first highly precise computer-assisted system to transfer laboratory planning data concerning the repositioning osteotomy of orbital walls to a surgical site. Materials and methods The SSN is based on infrared technology such as the Surgical Tool Navigator STN and the Surgical Microscope Navigator SMN manufactured by Carl Zeiss. Laboratory planning data are transferred to the surgical site by measurements with infrared transmitters which are checked by an infrared camera. Results A surgical planning can be carried out exactly using the Surgical Segment Navigator. Moreover, the SSN displays hidden levels of an extensive bone segment which are not visible via a bicoronary approach (e.g. orbital floor and facial wall of the maxillary sinus) clearly on monitor and helps to navigate the complete segment. Conclusions The Surgical Segment Navigator is the first computer-assisted system for highly precise repositioning osteotomy of the orbital walls.

The First Order Kernel responses (FOK) from a group of 15 normal subjects aged 21–36 yrs were recorded under clinical conditions with the multifocal ERG (MERG) systems VERIS and RETiscan using system-specific software and hardware under various parameter settings. First, the multifocal ERG's of normal subjects were recorded with the standard-parameters of VERIS and RETIscan. Thereafter the VERIS system parameters were set as close as possible to the RETIscan-setup, and the recordings were repeated. The VERIS parameters changed were the luminance- and contrast-settings of the stimulus-monitor and m-sequence-timing. In addition, the effects of different sampling rates were also examined. The consequences of the parameter changes were analyzed by displaying the peak times of the First-Order-Kernel (P1). The parameter-combinations used for recording lead to system-specific results. By varying the luminance/contrast settings and/or the m-sequence-timing the results can be changed. The amplitude of the recorded results can be influenced by the luminance-settings of the stimulus-monitor, and the topography of the P1 peak times is affected by the m-/f-frame-combination of the recording stimulus. With their standard parameters, the two systems give different measures of the P1 peak times. By adjusting the recording parameters, these differences can be reduced significantly. The main parameter here is the m-sequence-timing, although the sampling rate must also be taken into account with regard to the accuracy of the recording results.

PURPOSE To develop and test an algorithm to segment intraretinal layers irrespectively of the actual Optical Coherence Tomography (OCT) device used. METHODS The developed algorithm is based on the graph theory optimization. The algorithm's performance was evaluated against that of three expert graders for unsigned boundary position difference and thickness measurement of a retinal layer group in 50 and 41 B-scans, respectively. Reproducibility of the algorithm was tested in 30 C-scans of 10 healthy subjects each with the Spectralis and the Stratus OCT. Comparability between different devices was evaluated in 84 C-scans (volume or radial scans) obtained from 21 healthy subjects, two scans per subject with the Spectralis OCT, and one scan per subject each with the Stratus OCT and the RTVue-100 OCT. Each C-scan was segmented and the mean thickness for each retinal layer in sections of the early treatment of diabetic retinopathy study (ETDRS) grid was measured. RESULTS The algorithm was able to segment up to 11 intraretinal layers. Measurements with the algorithm were within the 95% confidence interval of a single grader and the difference was smaller than the interindividual difference between the expert graders themselves. The cross-device examination of ETDRS-grid related layer thicknesses highly agreed between the three OCT devices. The algorithm correctly segmented a C-scan of a patient with X-linked retinitis pigmentosa. CONCLUSIONS The segmentation software provides device-independent, reliable, and reproducible analysis of intraretinal layers, similar to what is obtained from expert graders. TRANSLATIONAL RELEVANCE Potential application of the software includes routine clinical practice and multicenter clinical trials.

SummaryIn most patients with isolated unilateral retinoblastoma, tumor development is initiated by somatic inactivation of both alleles of the RB1 gene. However, some of these patients can transmit retinoblastoma predisposition to their offspring. To determine the frequency and nature of constitutional RB1-gene mutations in patients with isolated unilateral retinoblastoma, we analyzed DNA from peripheral blood and from tumor tissue. The analysis of tumors from 54 (71%) of 76 informative patients showed loss of constitutional heterozygosity (LOH) at intragenic loci. Three of 13 uninformative patients had constitutional deletions. For 39 randomly selected tumors, SSCP, hetero-duplex analysis, sequencing, and Southern blot analysis were used to identify mutations. Mutations were detected in 21 (91%) of 23 tumors with LOH. In 6 (38%) of 16 tumors without LOH, one mutation was detected, and in 9 (56%) of the tumors without LOH, both mutations were found. Thus, a total of 45 mutations were identified in tumors of 36 patients. Thirty-nine of the mutations—including 34 small mutations, 2 large structural alterations, and hypermethylation in 3 tumors—were not detected in the corresponding peripheral blood DNA. In 6 (17%) of the 36 patients, a mutation was detected in constitutional DNA, and 1 of these mutations is known to be associated with reduced expressivity. The presence of a constitutional mutation was not associated with an early age at treatment. In 1 patient, somatic mosaicism was demonstrated by molecular analysis of DNA and RNA from peripheral blood. In 2 patients without a detectable mutation in peripheral blood, mosaicism was suggested because 1 of the patients showed multifocal tumors and the other later developed bilateral retinoblastoma. In conclusion, our results emphasize that the manifestation and transmissibility of retinoblastoma depend on the nature of the first mutation, its time in development, and the number and types of cells that are affected.

To evaluate the range of peripheral retinal thickness (PRT) in young healthy human subjects by Spectralis HRA + OCT, and to analyze a potential association between the peripheral location, spherical equivalent (SE), axial length (AL), and gender. After pupil dilation, the peripheral retina was scanned by means of six volume protocols (9 × 7.5 mm), each consisting of 31 B-scans. PRT was determined at 4,500, 5,500, 6,500 and 7,500 μm eccentricity from the fovea and the optic nerve head (ONH). Data points were collected every 22.5°. Six additional data points at a distance of 9,000 μm were included. In 11 subjects, OCT measurements were performed twice to evaluate reproducibility. Coefficients of variation (COV) were calculated. Randomly selected eyes of 50 subjects (19–30 years) with AL of 21–27 mm (SE: −5.75 to +5.25 dpt) were included in the study. Mean PRT decreased significantly (p ≤ 0.001, r = −0.99) towards the periphery, reaching a minimum at 9,000 μm eccentricity (mean PRT: 187.7 ± 8.9 μm). Multiple regression analysis revealed a significant association of PRT with AL at nasal and temporal locations as well as gender for temporal locations. COVs ranged from 0.44 to 2.90 %, with highest COVs found nasal to the fovea. This is the first study to report normative data of PRT outside the ETDRS grid and to show a significant continuous almost linear decrease of the RT from the center into the periphery. The data will be valuable to detect peripheral pathologies of the retina in early stages of peripheral retinal dystrophies.

PURPOSE To develop and evaluate a method for automated segmentation and quantitative analysis of pathological cavities in the retina visualized by spectral-domain optical coherence tomography (SD-OCT) scans. METHODS The algorithm is based on the segmentation of the gray-level intensities within a B-scan by a k-means cluster analysis and subsequent classification by a k-nearest neighbor algorithm. Accuracy was evaluated against three clinical experts using 130 bullous cavities identified on eight SD-OCT B-scans of three patients with wet age-related macular degeneration (AMD) and five patients with X-linked retinoschisis, as well as on one volume scan of a patient with X-linked retinoschisis. The algorithm calculated the surface area of the cavities for the B-scans and the volume of all cavities for the volume scan. In order to validate the applicability of the algorithm in clinical use, we analyzed 31 volume scans taken over the course of 4 years for one AMD patient with a serous retinal detachment. RESULTS Discrepancies in area measurements between the segmentation results of the algorithm and the experts were within the range of the area deviations among the experts. Volumes interpolated from the B-scan series of the volume scan were comparable among experts and algorithm (0.249 mm³ for the algorithm, 0.271 mm³ for expert 1, 0.239 mm³ for expert 2, and 0.262 mm³ for expert 3). Volume changes of the serous retinal detachment were quantifiable. CONCLUSIONS The segmentation algorithm represents a method for the automated analysis of large numbers of volume scans during routine diagnostics and in clinical trials.

PURPOSE With "standard" stimuli (white, Goldmann size III, 200 ms), the Nidek Microperimeter MP1 underestimates retinal light increment sensitivity (LIS). We thoroughly analyze this problem, suggest alternative settings to improve sensitivity to detect dysfunction, and provide true normal values. METHODS LIS was tested at 55 positions in the macular region using a 4-2-1 staircase strategy with 200 ms white or red stimuli on a 1.3 cd m⁻² background. Stimulus size was Goldmann III and I, and additionally II in the healthy subjects. All participants underwent a complete ophthalmologic examination, spectral domain optical coherence tomography (OCT), and fundus autofluorescence (FAF). RESULTS In normals, distributions of LIS for white Goldmann sizes II and III within the central 6° to 10° were clipped off at 20 dB--the MP1 cannot attenuate them any further. When the stimulus size was reduced to Goldmann I or the color changed to red, median LIS in the fovea (∼15 dB) was approximately 5 dB higher than at 10° eccentricity. Estimated from these results, central LIS for white Goldmann sizes II and III stimuli were 21 and 27 dB, respectively. In four patients with either focal or diffuse macular pathology, as confirmed by funduscopy, OCT, or FAF, reduced LIS was detected clearly with Goldmann size I stimuli, but not III. CONCLUSIONS In all subjects reported here, standard central LIS was above the technical limit of the MP1. To measure true thresholds in healthy subjects, either smaller (Goldmann size I) or dimmer stimuli (red) must be used.

The aim of the present population-based cross-sectional study was to examine the prevalence and diagnostic spectrum of generalized retinal dystrophy in Danish children.The Danish Registry for the Blind and Partially Sighted Children comprises all visually impaired children residing in Denmark aged 0-17 years. Among registered children, the primary diagnosis of generalized retinal dystrophy was assessed by chart review, including fundus photographs and electroretinograms. Age-specific data for live children in Denmark were retrieved from Statistics Denmark.Of the 1,204,235 Danish children aged 0-17 years on 1 October 2011, 2017 children were registered as visually impaired. Of these, 153 cases were attributed to generalized retinal dystrophy, corresponding to a prevalence of 13 per 100,000 children. The age-specific prevalence increased prominently with increasing age. In 43% of the children the eye condition was part of a syndrome, while the remaining 57% had eye disease only. The most common hereditary pattern was autosomal recessive (99 children, 66%).This epidemiological survey demonstrates that the prevalence of generalized retinal dystrophy in Danish children is 13 per 100,000, which is a considerable increase compared to the 9.8 per 100,000 reported by Rosenberg in 1988. The prevalence of Leber congenital amaurosis, Usher syndrome, and Bardet-Biedl syndrome doubled, which may be explained by a documented history of consanguinity in more than one third of the children. Many of the dystrophies are the subject of clinical intervention trials, and nation-wide epidemiological data can help assess the future need for treatment.

Background Classifying congenital nystagmus in the absence of biomicroscopically detectable abnormalities of the eye, and in an otherwise healthy child is difficult, especially early in life. At that age, nystagmus and visual loss may be the predominant symptoms of congenital stationary night-blindness. Unless night-blindness is specifically asked for or an ERG performed the correct diagnosis may be missed. Patients and methods We present the clinical data of two families with X-linked incomplete CSNB previously undiagnosed. ERG recordings in both families were suggestive of CSNB. The ERG of the obligate carrier was normal. In an attempt to distinguish between the complete and the incomplete type, and to identify further carrier signs, scotopic perimetry and dark adaptation were performed in both affected males and carriers. Scotopic perimetry allows to test the rod-mediated visual pathway in its spatial distribution. Results In affected males with non-recordable ERGs scotopic perimetry and dark adaptation disclosed residual rod function indicating an incomplete type. In carriers, there was a sensitivity loss at 600 nm, which may be a new carrier sign. Conclusions The correct diagnosis of the different forms of CSNB together with the identification of carriers is important for (1) ) genetic counselling and (2) linkage studies to identify the gene(s) for CSNB.

Mutations in CACNA1F encoding the α1-subunit of the retinal Cav1.4 L-type calcium channel have been linked to Cav1.4 channelopathies including incomplete congenital stationary night blindness type 2A (CSNB2), Åland Island eye disease (AIED) and cone-rod dystrophy type 3 (CORDX3). Since CACNA1F is located on the X chromosome, Cav1.4 channelopathies are typically affecting male patients via X-chromosomal recessive inheritance. Occasionally, clinical symptoms have been observed in female carriers, too. It is currently unknown how these mutations lead to symptoms in carriers and how the retinal network in these females is affected. To investigate these clinically important issues, we compared retinal phenotypes in Cav1.4-deficient and Cav1.4 heterozygous mice and in human female carrier patients. Heterozygous Cacna1f carrier mice have a retinal mosaic consistent with differential X-chromosomal inactivation, characterized by adjacent vertical columns of affected and non-affected wild-type-like retinal network. Vertical columns in heterozygous mice are well comparable to either the wild-type retinal network of normal mice or to the retina of homozygous mice. Affected retinal columns display pronounced rod and cone photoreceptor synaptopathy and cone degeneration. These changes lead to vastly impaired vision-guided navigation under dark and normal light conditions and reduced retinal electroretinography (ERG) responses in Cacna1f carrier mice. Similar abnormal ERG responses were found in five human CACNA1F carriers, four of which had novel mutations. In conclusion, our data on Cav1.4 deficient mice and human female carriers of mutations in CACNA1F are consistent with a phenotype of mosaic CSNB2.

Retinopathy of prematurity (ROP) is an eye disease that affects prematurely-born babies. It may even lead to blindness in serious cases. Among others, ROP can be recognized by evaluating the tortuosity of retinal vessels. An automatic detection and evaluation of tortuous vessels can support ophthalmologists when judging retinal images. There already exist various methods for computing vessel tortuosity. However, they do not always cover the medical properties and requirements. Thus, we propose an efficient measure, which is based to the total curvature and the number of curvature sign changes (twists) of the vessel. In order to achieve a stable detection of twists we represent the extracted vessels by curves composed of circular arcs and line segments: The vessels center pixels are approximated by smoothly joined segments of constant curvature. For any maximum tolerance we obtain the minimum number of segments. This way vessels are represented by preferably long curve segments of constant curvature. Together with the proposed measure this representation enables an effective and stable method for evaluating vessel tortuosity. Its performance is examined by means of both synthetic data and vessels of real retinal images.

Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene cause severe X-linked retinitis pigmentosa (XLRP). More than 80% of the mutations are located in the terminal exon ORF15 of the RPGR gene. Genome editing, which represents a novel approach to treat monogenic disorders, is based on highly specific nucleases that cleave or nick at a chosen position within the complex genome followed by the repair of the double or single strand break (DSB or SSB) by endogenous repair mechanisms. The major pathways include error prone non-homologous end joining (NHEJ), microhomology-mediated end-joining (MMEJ), and homologous recombination (HR), the latter two with the help of a donor template. Currently, endonucleases for inducing the DSB are based on the CRISPR-Cas9 system or TALE proteins fused to the non-specific FokI nuclease (TALEN). However, specificity and toxicity of both endonuclease types raise concerns about their use for therapeutic in vivo applications. In order to study in vivo genome editing for the treatment of XLRP, our lab has generated a mouse model containing a point mutation in the ORF15 exon. In the present study, we characterize advanced variants of both endonuclease types (Cas9-FokI and TALE-MutH) for their activity and toxicity at the murine Rpgr-ORF15 locus for later usage in the mouse model. In total, ten sequences within or near the ORF15 exon have been targeted for the induction of DSB or SSB. Nine target sites for CRISPR/Cas9-FokI were chosen: three before, within, and behind the exon, respectively, and one target site for TALE-MutH within the exon. These sequences have been cloned into the traffic light reporter (TLR) gene expression system at the homing endonuclease I-SceI site. The TLR system has been modified to express either GFP in case of successful HR or BFP in case of NHEJ. Plasmids containing substrate, nucleases and template DNA were transfected into HEK293T cells. Efficiency of DNA modification was measured by FACS analysis and T7 surveyor assay, and toxicity was assessed by cell survival assay. In addition to the episomal TLR system within a human cell line (HEK293T), the genome of murine C2C12 cells was targeted by all endonuclease variants and toxicity was analysed via the T7 surveyor assay. Toxicity of Cas9-FokI and TALE-MutH are comparable to the golden standard ISceI while standard Cas9 nucleases showed slightly increased toxicity in HEK293T cells. Two different concentrations of the nucleases were used in a toxicity assay and were equally tolerated. Cas9-FokI showed preferences in its activity at the nine target sites with activities well above ISce-I level, while the one target site of TALE-MutH was as efficient as ISceI. Activity results were confirmed in the murine cell line C2C12. Off target toxicity in C2C12 cells was non-detectable. The characterization of the activity and toxicity of the tested endonucleases helped us to identify the most promising tailored nuclease and its target sequence in our gene targeting approach to treat XLRP. With the help of mouse retinal explants and subsequently in vivo experiments, we will confirm the efficacy of endonuclease mediated genome editing in photoreceptors.

X-linked retinitis pigmentosa (XLRP) is characterized by retinal degeneration with night blindness and progressive reduction of the visual fields. By linkage and deletion analysis a gene locus (RP3) has been mapped to the short arm of the X chromosome between the genes CYBB and OTC. Analysis of transcript in this region has revealed a gene which is abundantly expressed in human retina and encodes a putative membrane protein with significant homologies to short consensus repeat (SCR/sushi) domains known from selections and complement proteins. The gene termed SRPX (sushi-repeat-containing protein, x chromosome) is deleted in an RP patient who also suffers from chronic granulomatous disease and McLeod syndrome. A 75 kb deletion removing exon 1 of the gene was also found in two brothers of a second XLRP family. However, no further functionally significant mutations were detected by SSCP screening of all 10 exons in 34 unrelated XLRP patients nor by full length RT-PCR sequencing in two RP3 families. The role of this highly conserved retinal gene in the pathogenesis of RP therefore remains to be determined.

Purpose Objective methods for the characterization of photosensitive cells are highly needed in patients with retinal dystrophies. The pupil light reflex is driven by rod, cone, and intrinsic photosensitive retinal ganglion cell (ipRGC) input. The aim of this study is to analyze two protocols of chromatic pupillometry with differing stimulus paradigms to gather isolated functional information of the three cell populations in patients with different forms of retinal dystrophies. Methods The study group comprised 60 patients with different forms of retinal dystrophies, and 32 healthy probands. Patients were grouped according to their electroretinography (ERG) data. A custom made binocular chromatic pupillometer (Bino I, AMTech) connected to the Colordome Ganzfeld stimulator (Diagnosys LLC) was used to assess changes in pupil diameter in response to red (640nm) and blue (462nm) light stimuli. Results Patients who had no measurable scotopic ERG also had the most reduced yet often still measurable rod-weighted pupil responses (PR), while patients with abnormal or normal scotopic ERG had reduced or normal rod-weighted PR. Similarly, patients with no measurable photopic ERG had reduced but still measurable cone-weighted PR, while patients with abnormal of normal photopic ERG had mostly normal cone-weighted PR. Reduced ipRGC-weighted PR could not be correlated with the ERG classification. Conclusion In the absence of a measurable ERG, chromatic pupillometry generates important functional and quantifiable data on residual rod and cone activity. These data show that this technique should be added to the panel of examination tools for the characterisation of retinal dystrophies and for quantifying the clinical benefit of experimental treatment protocols.

PURPOSE The aim of the study was to objectively characterize the function of rods, cones, and intrinsic photosensitive retinal ganglion cells (ipRGCs) in patients with RPE65 mutations by using two published protocols for chromatic pupillometry, and to correlate the data with the clinical phenotype. METHODS The study group comprised 11 patients with RPE65 mutations, and for control purposes, 32 healthy probands and 2 achromats. A custom-made binocular chromatic pupillometer (Bino I) connected to a ColorDome Ganzfeld stimulator was used to assess changes in pupil diameter in response to red (640 nm) and blue (462 nm) light stimuli. Light intensities, stimulus duration, and background varied depending on the protocol used. Results were compared to the clinical phenotype, that is, visual field (Goldmann perimetry), best corrected visual acuity, and full-field stimulus testing (FST). RESULTS No significant differences in any of the pupil response parameters were observed in intraday or intervisit variability tests. Pupil responses to rod-weighted stimulation were significantly diminished in all RPE65 patients. Pupil responses to cone-weighted stimuli differed among RPE65 patients and did not always correlate with residual visual field and cone sensitivity loss in FST. Pupil responses to ipRGC-weighted answers were slightly but significantly diminished, and the postillumination pupil response was significantly increased. CONCLUSIONS Chromatic pupillometry represents a highly sensitive and objective test to quantify the function of rods, cones, and ipRGCs in patients with RPE65 mutations.

To identify the underlying mutation and describe the phenotype in a consanguineous Kurdish family with Leber's congenital amaurosis (LCA)/early onset severe retinal dystrophy (EOSRD).Members of the index family were followed up to 22 years by ophthalmological examinations, including best corrected visual acuity (BCVA), Goldmann visual field (GVF), two-color-threshold perimetry (2CTP) and Ganzfeld electroretinogram (ERG), fundus photographs, fundus autofluorescence (FAF), and optical coherence tomography (OCT). After excluding seven of nine known LCA/EOSRD genes in the index patient, linkage analysis was performed in the family using a microarray followed by microsatellite fine mapping and direct sequencing of candidate genes. RD3 was screened by direct sequencing of 85 independent patients with LCA/EOSRD presenting with a BCVA ≥ 1.0 LogMAR before the age of 2 years to assess the prevalence of RD3 mutations in LCA/EOSRD. Since RD3 and RetGC1 have a functional relation, study authors screened for a modifying effect of RD3 mutations in 17 independent patients with mutations in GUCY2D.BCVA was severely reduced from the earliest examinations (as early as 3 months), never exceeding 1.3 LogMAR. The disease presented as cone-rod dystrophy with dystrophic changes in the macula and bone spicules in the periphery on progression. Linkage analysis narrowed the region of interest towards the LCA12 locus. Direct sequencing of RD3 revealed a homozygous nonsense mutation (c.180CA) in all affected members tested. Screening of additional unrelated LCA/EOSRD patients revealed only polymorphisms in RD3.This is the second family reported so far with mutations in RD3. Mutations in RD3 are a very rare cause of LCA associated with an extremely severe form of retinal dystrophy.

The correct segmentation of blood vessels in optical coherence tomography (OCT) images may be an important requirement for the analysis of intra-retinal layer thickness in human retinal diseases. We developed a shape model based procedure for the automatic segmentation of retinal blood vessels in spectral domain (SD)-OCT scans acquired with the Spectralis OCT system. The segmentation procedure is based on a statistical shape model that has been created through manual segmentation of vessels in a training phase. The actual segmentation procedure is performed after the approximate vessel position has been defined by a shadowgraph that assigns the lateral vessel positions. The active shape model method is subsequently used to segment blood vessel contours in axial direction. The automated segmentation results were validated against the manual segmentation of the same vessels by three expert readers. Manual and automated segmentations of 168 blood vessels from 34 B-scans were analyzed with respect to the deviations in the mean Euclidean distance and surface area. The mean Euclidean distance between the automatically and manually segmented contours (on average 4.0 pixels respectively 20 µm against all three experts) was within the range of the manually marked contours among the three readers (approximately 3.8 pixels respectively 18 µm for all experts). The area deviations between the automated and manual segmentation also lie within the range of the area deviations among the 3 clinical experts. Intra reader variability for the experts was between 0.9 and 0.94. We conclude that the automated segmentation approach is able to segment blood vessels with comparable accuracy as expert readers and will provide a useful tool in vessel analysis of whole C-scans, and in particular in multicenter trials.