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IntroductionMyopia is a major cause of degenerative eye disease and increases the risk of secondary visual impairment. Mitigating its progression therefore has great potential of clinically relevant benefit as shown by using highly diluted atropine eye drops in children of Asian origin. However, limited evidence is available regarding the efficacy and safety of low-dose atropine therapy in non-Asian populations. Hence, the Low-dose AtropIne for Myopia Control in Children (AIM) study will test the efficacy and safety of 0.02% atropine vs placebo in a German population.Methods and analysisAIM is a national, multicentre, prospective, randomised, placebo-controlled, double-blind trial with two parallel arms. The primary objective is to assess the efficacy of atropine 0.02% eyedrops for myopia control in children of Caucasian origin. The primary outcome is the change in cycloplegic refraction after 1 year of treatment (D/year). Secondary and tertiary outcome measures comprise the change in axial length (mm/year) in children treated with 0.02% atropine compared with placebo, the myopic progression of participants treated with 0.01% compared with 0.02% atropine (D/year and mm/year), and the safety profile of both 0.02% and 0.01% atropine. Furthermore, the myopic progression 1 year after cessation of therapy with 0.02% atropine will be evaluated. Inclusion criteria are an age of 8–12 years and myopia of −1 D to −6 D with an estimated annual myopia progression of ≥0.5 D. After randomisation, patients will receive either atropine 0.02% (arm A) or placebo eye drops (arm B) in the first year of treatment. In the second year, they will continue to receive atropine 0.02% (arm A) or switch to atropine 0.01% (arm B). In the third year, they will switch to placebo (arm A) or continue with atropine 0.01% (arm B). To achieve a statistical power of 80%, the calculated sample size is 300. The trial has started in October 2021 with a planned recruitment period of 18 months.Ethics and disseminationAIM has been approved by the Central Ethics Committee of the University Medical Center Freiburg (21-1106), local ethics committees of each participating centre and the German Federal Institute for Drugs and Medical Devices (61-3910-4044659). It complies with the Declaration of Helsinki, local laws and ICH-GCP. Results and underlying data from this trial will be disseminated through peer-reviewed publications and conference presentations.Trial registration numberNCT03865160.

Leber congenital amaurosis caused by mutations in the RPE65 gene belongs to the most severe early-onset hereditary childhood retinopathies naturally progressing to legal blindness. The novel gene therapy voretigene neparvovec is the first approved causative treatment option for this devastating eye disease and is specifically designed to treat RPE65-mediated retinal dystrophies. Herein, we present a follow-up of the youngest treated patients in Germany so far, including four pre-school children who received treatment with voretigene neparvovec at a single treatment center between January 2020 and May 2022. All patients underwent pars plana vitrectomy with circumferential peeling of the internal limiting membrane at the injection site and subretinal injection of voretigene neparvovec. Pre- and postoperative diagnostics included imaging (spectral domain optical coherence tomography, fundus autofluorescence, fundus wide-angle imaging), electrophysiologic examination (ERG), retinal light sensitivity measurements (FST) and visual acuity testing. Behavioral changes were assessed using a questionnaire and by observing the children’s vision-guided behavior in different levels of illumination. All children showed marked increase in vision-guided behavior shortly after therapy, as well as marked increase in visual acuity in the postoperative course up to full visual acuity in one child. Two eyes showed partial electrophysiological recovery of an ERG that was undetectable before treatment—a finding that has not been described in humans before.

Zusammenfassung Hintergrund Das Retina.net ROP-Register erhebt Daten von Kindern, die eine seltene behandlungsbedürftige Frühgeborenenretinopathie (Retinopathy of Prematurity, ROP) entwickeln. Ziel dieser Auswertung ist die Untersuchung der Daten zur behandlungsbedürftigen ROP, Epidemiologie, Therapie und deren Änderungen über einen Zeitraum von 15 Jahren an der Medizinischen Hochschule Hannover. Methoden Analyse der Daten der therapiebedürftigen Fälle der ROP eines Zentrums für die Geburtsjahre 2001 bis 2016 (Therapie in 2002 bis 2017) als Gesamtzeitraum und in 5 Abschnitten. Ergebnisse Es wurden 65 Kinder behandelt (23 weiblich), davon wurden 11 (16,9 %) extern auf ROP gescreent und zur ROP-Behandlung zugewiesen. Für den Zeitraum 2006 bis 2016 lag die Inzidenz der behandlungsbedürftigen ROP unter den gescreenten Kindern bei 4,1 %. Das mittlere Gestationsalter betrug 25,7 Schwangerschaftswochen (SSW) (Standardabweichung [SA] = 1,8), das Geburtsgewicht 763 g (SA = 235), das postmenstruelle Alter bei Behandlung 38,2 Wochen (SA = 3,2), das postnatale Alter 12,4 Wochen (SA = 3,2). Über die Zeit zeigte sich kein signifikanter Unterschied der demografischen Parameter. Am häufigsten (57 Augen bei 31 Kindern) wurde eine Zone II, 3+-Erkrankung behandelt; 58 Kinder erhielten eine Laserkoagulation (N = 114 Augen), 7 Kinder bilateral eine Anti-VEGF-Therapie (Bevacizumab) (N = 14 Augen), welche ab 2014 eingesetzt wurde. Eine Wiederbehandlung bei Wiederaufflammen der behandlungsbedürftigen ROP war in einem Fall nach initialer Laserbehandlung notwendig. Kinder mit behandlungsbedürftiger ROP zeigten häufig neonatologische Komorbiditäten, in mehr als 90 % Beatmung, bronchopulmonale Dysplasie und erhielten Transfusionen. Schlussfolgerung Dies ist die erste monozentrische Auswertung über 15 Jahre im Rahmen des Retina.net ROP-Registers. Ab 2014 sehen wir einen Wechsel von der Laserkoagulation zur Anti-VEGF-Therapie (Bevacizumab), während im betrachteten Kollektiv die demografischen Daten und Behandlungsparameter weitgehend konstant waren.

Inherited retinal diseases can result from various genetic defects and are one of the leading causes for blindness in the working-age population. The present study aims to provide a comprehensive description of changes in retinal structure associated with phenotypic disease entities and underlying genetic mutations. Full macular spectral domain optical coherence tomography scans were obtained and manually segmented in 16 patients with retinitis pigmentosa, 7 patients with cone–rod dystrophy, and 7 patients with Stargardt disease, as well as 23 age- and sex-matched controls without retinal disease, to assess retinal layer thicknesses. As indicated by generalized least squares models, all IRDs were associated with retinal thinning (p < 0.001), especially of the outer nuclear layer (ONL, p < 0.001). Except for the retinal nerve fiber layer, such thinning was associated with a reduced visual acuity (p < 0.001). These advances in our understanding of ultrastructural retinal changes are important for the development of gene-, cell-, and optogenetic therapy. Longitudinal studies are warranted to describe the temporal component of those changes.

Proper treatment of the two-wall fractured orbit is still controversial. Specifically, there is no consensus on the issue of the necessity of medial orbital wall repair. With anatomically critical structures at risk during the surgical approach, surgeons’ view on the necessity of medial orbital wall repair often is restricted and an aesthetically disturbing enophthalmos is more likely to be accepted. Therefore, treatment options range from leaving the medial wall without repair to reconstruction with autogenous tissue or alloplastic materials, which can lead to moderate to severe side effects. However, emerging technologies such as patient-specific implants (PSI) offer a reliable and anatomically correct reconstruction of the bony orbit. This study aimed to evaluate the outcome of full orbital reconstruction using PSIs compared to only orbital floor repair using PDS (bioresorbable polydioxanone) foils leaving the medial orbital wall untouched in traumatic two-wall orbital fractures. Of all patients treated at the University Hospital of Düsseldorf between 2017 and 2019 who suffered from traumatic orbital fracture, only patients with a two-wall orbital fracture involving both the orbital floor and the medial wall (n = 68) were included. Patients were treated either with a PSI (n = 35) or a PDS foil (n = 33). Primary outcome parameters were ophthalmological disturbances analyzed via clinical investigation and intra-orbital angles, volumes and implant position analyzed with radiological 3D-datasets. While a two-wall reconstruction using PSIs led to a significant improvement of the enophthalmos, the rate of postoperative enophthalmos was significantly increased in cases of only orbital floor repair with PDS foils. Radiologically, a significant reconstruction of the three-dimensional bony orbit succeeded with the simple use of PSIs leading to a significant reduction in the traumatically enlarged orbital volume. PSI also led to a significant reduction in the traumatically enlarged medial angle of the orbit. This was not the case for single-floor repair with PDS foil. The results of this study suggest that complex orbital fractures can be reconstructed at an even higher degree of accuracy with selective laser-melted PSIs than PDS foils. In order to achieve a true to original reconstruction of the bony orbit, surgical treatment of the medial orbital wall can be advocated for in the long term depending on the indication.

Contemporary advances in technology have allowed the transfer of knowledge from industrial laser melting systems to surgery; such an approach could increase the degree of accuracy in orbital restoration. The aim of this study was to examine the accuracy of selective laser melted PSIs (patient-specific implants) and navigation in primary orbital reconstruction. Ninety-six patients with orbital fractures were included in this study. Planned vs. achieved orbital volumes (a) and angles (b) were compared to the unaffected side (n = 96). The analysis included the overlay of post-treatment on planned images (iPlan 3.0.5, Brainlab®, Feldkirchen, Germany). The mean difference in orbital volume between the digitally planned orbit and the postoperative orbit was 29.16 cm3 (SD 3.54, presurgical) to 28.33 cm3 (SD 3.64, postsurgical, t = 5.00, df = 95.00; p < 0.001), resulting in a mean volume difference (planned vs. postop) of less than 1 cm3. A 3D analysis of the color mapping showed minor deviations compared to the mirrored unaffected side. The results suggested that primary reconstruction in complex orbital wall fractures can be routinely achieved with a high degree of accuracy by using selective laser melted orbital PSIs.

Study Design: This study presents a case-control study of 33 patients who underwent secondary orbital reconstruction, evaluating techniques and outcome. Objective: Adequate functional and aesthetical appearance are main goals for secondary orbital reconstruction. Insufficient premorbid orbital reconstruction can result in hypoglobus, enophthalmos, and diplopia. Computer-assisted surgery and the use of patient-specific implants (PSIs) is widely described in the literature. The authors evaluate the use of selective laser-melted PSIs and hypothesize that PSIs are an excellent option for secondary orbital reconstruction. Methods: The sample was composed of 33 patients, previously treated with primary orbital reconstruction, presenting themselves with indications for secondary reconstruction (i.e. enophthalmos, diplopia, or limited eye motility). Computed tomography and/or cone beam data sets were assessed before and after secondary reconstruction comparing intraorbital volumes, infraorbital angles, and clinical symptoms. Clinical outcomes were assessed using a standardized protocol. Results: Results show a significant change in intraorbital volumes and a reduction of clinical symptoms after secondary reconstruction. Conclusions: Outcomes of this study suggest that secondary orbital reconstruction can be performed routinely using selective laser-melted PSIs and titanium spacers.

Vorstellung und Anwendung eines pachymetrischen Verfahrens, das auf einem definierten optischen Schnitt an der Spaltlampe beruht. An insgesamt 103 Augen (68 mit Glaukom und 35 bei Patienten mit Keratokonus) wurde die sichtbare Breite eines unter 45° einfallenden optischen Schnittes an der Spaltlampe bestimmt. Die gemessenen Werte wurden mit externen Pachymetriewerten dieser Augen verglichen, um einen Proportionalitatsfaktor zur pachymetrischen Abschatzung aus den Spaltlampendaten zu ermitteln. Aus einer linearen Regression zwischen externer Pachymetrie und sichtbarer Breite des optischen Schnitts an der Spaltlampe ergab sich ein Proportionalitatsfaktor von 1,71. Wendet man diesen Faktor auf die einzelnen Messungen an, weichen die an der Spaltlampe bestimmten Pachymetriewerte bei 82 von 103 der Augen hochstens 40 µm von den externen Werten ab. Mithilfe eines definierten optischen Schnittes lassen sich an einer Spaltlampe klinisch verwertbare Aussagen zur zentralen Hornhautdicke bei Krankheitsbildern wie Glaukom und Keratokonus machen. Die Abweichung von extern gemessenen Werten liegt zu 80 % im Bereich der bekannten Schwankungsbreite der Hornhautdicke im Tagesverlauf. Ahnlich wie bei anderen pachymetrischen Verfahren steigert sich die Verlasslichkeit der Bestimmung der Hornhautdicke an der Spaltlampe bei Anwendung im Seitenvergleich oder Zeitverlauf am selben Auge.

Purpose Transforming growth factor-beta (TGFB)-mediated epithelial-mesenchymal transition (EMT) plays a crucial role in the pathogenesis of retinal fibrosis, which is one of the leading causes of impaired vision. Current approaches to treating retinal fibrosis focus, among other things, on inhibiting the TGFB signaling pathway. Transient expression of microRNAs (miRNAs) is one way to inhibit the TGFB pathway post-transcriptionally. Our previous study identified the miRNA miR-302d as a regulator of multiple TGFB-related genes in ARPE-19 cells. To further explore its effect on primary cells, the effect of miR-302d on TGFB-induced EMT in primary human retinal pigment epithelium (hRPE) was investigated in vitro. Methods hRPE cells were extracted from patients receiving enucleation. Transfection of hRPE cells with miR-302d was performed before or after TGFB1 stimulation. Live-cell imaging, immunocytochemistry staining, Western blot, and ELISA assays were utilized to identify the alterations of cellular morphology and EMT-related factors expressions in hRPE cells. Results hRPE cells underwent EMT by TGFB1 exposure. The transfection of miR-302d inhibited the transition with decreased production of mesenchymal markers and increased epithelial factors. Meanwhile, the phosphorylation of SMAD2 activated by TGFB1 was suppressed. Moreover, miR-302d expression promoted TGFB1-induced fibroblast-like hRPE cells to revert towards an epithelial stage. As confirmed by ELISA, miR-302d reduced TGFB receptor 2 (TGFBR2) and vascular endothelial growth factor A (VEGFA) levels 48 hours after transfection. Conclusions The protective effect of miR-302d might be a promising approach for ameliorating retinal fibrosis and neovascularization. MiR-302d suppresses TGFB-induced EMT in hRPE cells via downregulation of TGFBR2, even reversing the process. Furthermore, miR-302d reduces the constitutive secretion of VEGFA from hRPE cells.

Summary Background The human cytokine erythropoietin conveys neuroprotection in animal models but has shown ambiguous results in phase 2 clinical trials in patients with optic neuritis. We assessed the safety and efficacy of erythropoietin in patients with optic neuritis as a clinically isolated syndrome in a multicentre, prospective, randomised clinical trial. Methods This randomised, placebo-controlled, double-blind phase 3 trial, conducted at 12 tertiary referral centres in Germany, included participants aged 18–50 years, within 10 days of onset of unilateral optic neuritis, with visual acuity of 0·5 or less, and without a previous diagnosis of multiple sclerosis. Participants were randomly assigned (1:1) to receive either 33 000 IU erythropoietin or placebo intravenously for 3 days as an adjunct to high-dose intravenous methylprednisolone (1000 mg per day). Block randomisation was performed by the trial statistician using an SAS code that generated randomly varying block sizes, stratified by study site and distributed using sealed envelopes. All trial participants and all study staff were masked to treatment assignment, except the trial pharmacist. The first primary outcome was atrophy of the peripapillary retinal nerve fibre layer (pRNFL), measured by optic coherence tomography (OCT) as the difference in pRNFL thickness between the affected eye at week 26 and the unaffected eye at baseline. The second primary outcome was low contrast letter acuity at week 26, measured as the 2·5% Sloan chart score of the affected eye. Analysis was performed in the full analysis set of all randomised participants for whom treatment was started and at least one follow-up OCT measurement was available. Safety was analysed in all patients who received at least one dose of the trial medication. This trial is registered at ClinicalTrials.gov, NCT01962571. Findings 108 participants were enrolled between Nov 25, 2014, and Oct 9, 2017, of whom 55 were assigned to erythropoietin and 53 to placebo. Five patients were excluded from the primary analysis due to not receiving the allocated medication, withdrawn consent, revised diagnosis, or loss to follow-up, yielding a full analysis set of 52 patients in the erythropoietin group and 51 in the placebo group. Mean pRNFL atrophy was 15·93 μm (SD 14·91) in the erythropoietin group and 14·65 μm (15·60) in the placebo group (adjusted mean treatment difference 1·02 μm; 95% CI −5·51 to 7·55; p=0·76). Mean low contrast letter acuity scores were 49·60 (21·31) in the erythropoietin group and 49·06 (21·93) in the placebo group (adjusted mean treatment difference −4·03; −13·06 to 5·01). Adverse events occurred in 43 (81%) participants in the erythropoietin group and in 42 (81%) in the placebo group. The most common adverse event was headache, occuring in 15 (28%) patients in the erythropoietin group and 13 (25%) patients in the placebo group. Serious adverse events occurred in eight (15%) participants in the erythropoietin and in four (8%) in the placebo group. One patient (2%) in the erythropoietin group developed a venous sinus thrombosis, which was treated with anticoagulants and resolved without sequelae. Interpretation Erythropoietin as an adjunct to corticosteroids conveyed neither functional nor structural neuroprotection in the visual pathways after optic neuritis. Future research could focus on modified erythropoietin administration, assess its efficacy independent of corticosteroids, and investigate whether it affects the conversion of optic neuritis to multiple sclerosis. Funding German Federal Ministry of Education and Research (BMBF).

Autosomal recessive bestrophinopathy (ARB) has been reported as clinically heterogeneous. Eighteen patients (mean age: 22.5 years, 15 unrelated families) underwent ophthalmological examination, fundus photography, fundus autofluorescence, and optical coherence tomography (OCT). Molecular genetic testing of the BEST1 gene was conducted by the chain-terminating dideoxynucleotide Sanger methodology. Onset of symptoms (3 to 50 years of age) and best-corrected visual acuity (0.02&ndash, 1.0) were highly variable. Ophthalmoscopic and retinal imaging defined five phenotypes. Phenotype I presented with single or confluent yellow lesions at the posterior pole and midperiphery, serous retinal detachment, and intraretinal cystoid spaces. In phenotype II fleck-like lesions were smaller and extended to the far periphery. Phenotype III showed a widespread continuous lesion with sharp peripheral demarcation. Single (phenotype IV) or multifocal (phenotype V) vitelliform macular dystrophy-like lesions were observed as well. Phenotypes varied within families and in two eyes of one patient. In addition, OCT detected hyperreflective foci (13/36 eyes) and choroidal excavation (11/36). Biallelic mutations were identified in each patient, six of which have not been reported so far [c.454C&gt, T/p.(Pro152Ser), c.620T&gt, A/p.(Leu207His), c.287_298del/p.(Gln96_Asn99del), c.199_200del/p.(Leu67Valfs*164), c.524del/p.(Ser175Thrfs*19), c.590_615del/p.(Leu197Profs*26)]. BEST1-associated ARB presents with a variable age of onset and clinical findings, that can be categorized in 5 clinical phenotypes. Hyperreflective foci and choroidal excavation frequently develop as secondary manifestations.

Objective: We report the case of a young woman with postinfectious onset of myasthenia gravis after COVID-19 with mild respiratory symptoms and anosmia/ageusia 1 month before admission to our neurological department. Methods: Patient data were derived from medical records of Hannover Medical School, Germany. Written informed consent was obtained from the patient. Results: The 21-year-old female patient presented with subacute, vertically shifted double vision evoked by right sided partial oculomotor paresis and ptosis. About 4 weeks earlier she had suffered from mild respiratory symptoms, aching limbs and head without fever, accompanied by anosmia/ageusia. During the persistence of the latter symptoms for around 10 days the patient had already noticed “tired eyes” and fluctuating double vision. Clinical assessment including a positive test with edrophonium chloride and increased acetylcholine receptor antibodies related the ocular manifestation etiologically to myasthenia gravis. Antibodies (IgA/IgG) against SARS-CoV-2 using three different serological tests (Abbott, DiaSorin, Euroimmun) were detected in serum suggesting this specific coronavirus as previously infectious agent in our patient. The myasthenic syndrome was treated successfully with intravenous immunoglobulins and oral pyridostigmine. Conclusion: This is the first case presentation of postinfectious myasthenia gravis as neurological complication in a COVID-19 patient.

Presentation and application of a method for pachymetry with the slit lamp based on the concept of a defined optical section.The visible width of an optical section on slit lamp examination with a 45° angle between the slit beam and the central corneal surface was determined in 103 eyes (68 with glaucoma and 35 in patients with keratoconus). The measurements were compared to the pachymetric values previously determined elsewhere for these eyes in order to establish a proportionality factor for calculation of corneal thickness from slit lamp measurements.From a linear regression between the external pachymetry and visible width of the optical section in the split lamp, 1.71 was determined to be the factor of proportionality. Applying this factor to single measurements, 82 out of 103 eyes had a maximum difference of 40 µm compared to the external pachymetric values.Based on a defined optical section at the slit lamp, clinically relevant estimates can be made for central corneal thickness in diseases, such as glaucoma or keratoconus. The difference to the externally measured pachymetric values was within the range of diurnal changes in corneal thickness for 80% of the eyes. Similar to other pachymetric procedures, the reliability of determination of the corneal thickness with the slit lamp also increases when applied intraindividually or for the same eye in the course of time.HINTERGRUND UND ZIEL: Vorstellung und Anwendung eines pachymetrischen Verfahrens, das auf einem definierten optischen Schnitt an der Spaltlampe beruht.An insgesamt 103 Augen (68 mit Glaukom und 35 bei Patienten mit Keratokonus) wurde die sichtbare Breite eines unter 45° einfallenden optischen Schnittes an der Spaltlampe bestimmt. Die gemessenen Werte wurden mit externen Pachymetriewerten dieser Augen verglichen, um einen Proportionalitätsfaktor zur pachymetrischen Abschätzung aus den Spaltlampendaten zu ermitteln.Aus einer linearen Regression zwischen externer Pachymetrie und sichtbarer Breite des optischen Schnitts an der Spaltlampe ergab sich ein Proportionalitätsfaktor von 1,71. Wendet man diesen Faktor auf die einzelnen Messungen an, weichen die an der Spaltlampe bestimmten Pachymetriewerte bei 82 von 103 der Augen höchstens 40 µm von den externen Werten ab.Mithilfe eines definierten optischen Schnittes lassen sich an einer Spaltlampe klinisch verwertbare Aussagen zur zentralen Hornhautdicke bei Krankheitsbildern wie Glaukom und Keratokonus machen. Die Abweichung von extern gemessenen Werten liegt zu 80 % im Bereich der bekannten Schwankungsbreite der Hornhautdicke im Tagesverlauf. Ähnlich wie bei anderen pachymetrischen Verfahren steigert sich die Verlässlichkeit der Bestimmung der Hornhautdicke an der Spaltlampe bei Anwendung im Seitenvergleich oder Zeitverlauf am selben Auge.

Purpose: Missing heritability in human diseases represents a major challenge, and this is particularly true for ABCA4-associated Stargardt disease (STGD1). We aimed to elucidate the genomic and transcriptomic variation in 1054 unsolved STGD and STGD-like probands. Methods: Sequencing of the complete 128-kb ABCA4 gene was performed using single-molecule molecular inversion probes (smMIPs), based on a semiautomated and cost-effective method. Structural variants (SVs) were identified using relative read coverage analyses and putative splice defects were studied using in vitro assays. Results: In 448 biallelic probands 14 known and 13 novel deep-intronic variants were found, resulting in pseudoexon (PE) insertions or exon elongations in 105 alleles. Intriguingly, intron 13 variants c.1938-621G>A and c.1938-514G>A resulted in dual PE insertions consisting of the same upstream, but different downstream PEs. The intron 44 variant c.6148-84A>T resulted in two PE insertions and flanking exon deletions. Eleven distinct large deletions were found, two of which contained small inverted segments. Uniparental isodisomy of chromosome 1 was identified in one proband. Conclusion: Deep sequencing of ABCA4 and midigene-based splice assays allowed the identification of SVs and causal deep-intronic variants in 25% of biallelic STGD1 cases, which represents a model study that can be applied to other inherited diseases. Fil: Khan, Mubeen. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Cornelis, Stéphanie S.. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Del Pozo Valero, Marta. Hospital Universitario Fundación Jiménez Díaz; España. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Whelan, Laura. Trinity College; Estados Unidos Fil: Runhart, Esmee H.. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Mishra, Ketan. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Bults, Femke. Radboud University Nijmegen Medical Centre; Países Bajos Fil: AlSwaiti, Yahya. St John of Jerusalem Eye Hospital Group; Palestina (ANP) Fil: AlTalbishi, Alaa. St John of Jerusalem Eye Hospital Group; Palestina (ANP) Fil: De Baere, Elfride. University of Ghent; Bélgica Fil: Banfi, Sandro. Seconda Universita Degli Studi Di Napoli; Italia Fil: Banin, Eyal. The Hebrew University of Jerusalem; Israel Fil: Bauwens, Miriam. University of Ghent; Bélgica Fil: Ben Yosef, Tamar. The Ruth And Bruce Rappaport Faculty Of Medicine; Israel Fil: Boon, Camiel J. F.. Leiden University. Leiden University Medical Center; Países Bajos Fil: van den Born, L. Ingeborgh. Rotterdam Ophthalmic Institute; Países Bajos Fil: Defoort, Sabine. Universite Lille; Francia Fil: Devos, Aurore. Universite Lille; Francia Fil: Dockery, Adrian. Trinity College; Estados Unidos Fil: Dudakova, Lubica. Charles University and General University Hospital; República Checa Fil: Fakin, Ana. Charles University and General University Hospital; República Checa Fil: Farrar, G. Jane. Trinity College; Estados Unidos Fil: Ferraz Sallum, Juliana Maria. Universidade Federal de Sao Paulo; Brasil Fil: Fujinami, Kaoru. UCL Institute of Ophthalmology; Reino Unido Fil: Gilissen, Christian. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Glavac, Damjan. University of Ljubljana; Eslovenia Fil: Gorin, Michael B.. University of California at Los Angeles. School of Medicine; Estados Unidos Fil: Greenberg, Jacquie. University of Cape Town; Sudáfrica Fil: Hayashi, Takaaki. The Jikei University School of Medicine; Japón Fil: Hettinga, Ymkje M.. Bartiméus Diagnostic Center for Complex Visual Disorders; Países Bajos Fil: Hoischen, Alexander. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Hoyng, Carel B.. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Hufendiek, Karsten. University Eye Hospital Hannover Medical School; Alemania Fil: Jägle, Herbert. University Regensburg; Alemania Fil: Kamakari, Smaragda. OMMA Ophthalmological Institute of Athens; Grecia Fil: Karali, Marianthi. Seconda Universita Degli Studi Di Napoli; Italia Fil: Kellner, Ulrich. No especifíca; Fil: Klaver, Caroline C. W.. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Kousal, Bohdan. Charles University and General University Hospital; República Checa Fil: Lamey, Tina M.. University of Western Australia; Australia Fil: MacDonald, Ian M.. University of Alberta; Canadá Fil: Matynia, Anna. University of California at Los Angeles. School of Medicine; Estados Unidos Fil: McLaren, Terri L.. University of Western Australia; Australia Fil: Mena, Marcela D.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Meunier, Isabelle. Université Montpellier II; Francia Fil: Miller, Rianne. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Newman, Hadas. Universitat Tel Aviv; Israel Fil: Ntozini, Buhle. University of Cape Town; Sudáfrica Fil: Oldak, Monika. No especifíca; Fil: Pieterse, Marc. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Podhajcer, Osvaldo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Puech, Bernard. Universite Lille; Francia Fil: Ramesar, Raj. University of Cape Town; Sudáfrica Fil: Rüther, Klaus. No especifíca; Fil: Salameh, Manar. No especifíca; Fil: Salles, Mariana Vallim. Universidade de Sao Paulo; Brasil Fil: Sharon, Dror. The Hebrew University of Jerusalem; Israel Fil: Simonelli, Francesca. Seconda Universita Degli Studi Di Napoli; Italia Fil: Spital, Georg. No especifíca; Fil: Steehouwer, Marloes. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Szaflik, Jacek P.. No especifíca; Fil: Thompson, Jennifer A.. No especifíca; Fil: Thuillier, Caroline. Universite Lille; Francia Fil: Tracewska, Anna M.. No especifíca; Fil: van Zweeden, Martine. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Vincent, Andrea L.. University of Auckland; Nueva Zelanda Fil: Zanlonghi, Xavier. No especifíca; Fil: Liskova, Petra. Charles University and General University Hospital; República Checa Fil: Stöhr, Heidi. Universitat Regensburg; Alemania Fil: De Roach, John N.. University of Western Australia; Australia Fil: Ayuso, Carmen. Hospital Universitario Fundación Jiménez Díaz; España Fil: Roberts, Lisa. University of Cape Town; Sudáfrica Fil: Weber, Bernhard H. F.. Universitat Regensburg; Alemania Fil: Dhaenens, Claire Marie. Universite Lille; Francia Fil: Cremers, Frans P. M.. Radboud University Nijmegen Medical Centre; Países Bajos

Purpose Stargardt disease (STGD1) is an autosomal recessive retinopathy, caused by mutations in the retina-specific ATP-binding cassette transporter (ABCA4) gene. To establish the mutational spectrum and to assess effects of selected deep intronic and common genetic variants on disease, we performed a comprehensive sequence analysis in a large cohort of German STGD1 patients. Methods DNA samples of 335 STGD1 patients were analyzed for ABCA4 mutations in its 50 coding exons and adjacent intronic sequences by resequencing array technology or next generation sequencing (NGS). Parts of intron 30 and 36 were screened by Sanger chain-terminating dideoxynucleotide sequencing. An in vitro splicing assay was used to test selected variants for their splicing behavior. By logistic regression analysis we assessed the association of common ABCA4 alleles while a multivariate logistic regression model calculated a genetic risk score (GRS). Results Our analysis identified 148 pathogenic or likely pathogenic mutations, of which 48 constitute so far unpublished ABCA4-associated disease alleles. Four rare deep intronic variants were found once in 472 alleles analyzed. In addition, we identified six risk-modulating common variants. Genetic risk score estimates suggest that defined common ABCA4 variants influence disease risk in carriers of a single pathogenic ABCA4 allele. Conclusions Our study adds to the mutational spectrum of the ABCA4 gene. Moreover, in our cohort, deep intronic variants in intron 30 and 36 likely play no or only a minor role in disease pathology. Of note, our findings demonstrate a possible modifying effect of common sequence variants on ABCA4-associated disease.

Missing heritability in human diseases represents a major challenge. Although whole-genome sequencing enables the analysis of coding and non-coding sequences, substantial costs and data storage requirements hamper its large-scale use to (re)sequence genes in genetically unsolved cases. The ABCA4 gene implicated in Stargardt disease (STGD1) has been studied extensively for 22 years, but thousands of cases remained unsolved. Therefore, single molecule molecular inversion probes were designed that enabled an automated and cost-effective sequence analysis of the complete 128-kb ABCA4 gene. Analysis of 1,054 unsolved STGD and STGD-like probands resulted in bi-allelic variations in 448 probands. Twenty-seven different causal deep-intronic variants were identified in 117 alleles. Based on in vitro splice assays, the 13 novel causal deep-intronic variants were found to result in pseudo-exon (PE) insertions (n=10) or exon elongations (n=3). Intriguingly, intron 13 variants c.1938-621G>A and c.1938-514G>A resulted in dual PE insertions consisting of the same upstream, but different downstream PEs. The intron 44 variant c.6148-84A>T resulted in two PE insertions that were accompanied by flanking exon deletions. Structural variant analysis revealed 11 distinct deletions, two of which contained small inverted segments. Uniparental isodisomy of chromosome 1 was identified in one proband. Integrated complete gene sequencing combined with transcript analysis, identified pathogenic deep-intronic and structural variants in 26% of bi-allelic cases not solved previously by sequencing of coding regions. This strategy serves as a model study that can be applied to other inherited diseases in which only one or a few genes are involved in the majority of cases.

A primary skeletal muscle cell culture, in which myoblasts derived from newborn rabbit hindlimb muscles grow on gelatin bead microcarriers in suspension and differentiate into myotubes, has been established previously. In the course of differentiation and beginning spontaneous contractions, these multinucleated myotubes do not detach from their support. Here, we describe the development of the primary myotubes with respect to their ultrastructural differentiation. Scanning electron microscopy reveals that myotubes not only grow around the surface of one carrier bead but also attach themselves to neighboring carriers, forming bridges between carriers. Transmission electron microscopy demonstrates highly ordered myofibrils, T-tubules, and sarcoplasmic reticulum. The functionality of the contractile apparatus is evidenced by contractile activity that occurs spontaneously or can be elicited by electrostimulation. Creatine kinase activity increases steadily until day 20 of culture. Regarding the expression of isoforms of myosin heavy chains (MHC), we could demonstrate that from day 16 on, no non-adult MHC isoform mRNAs are present. Instead, on day 28 the myotubes express predominantly adult fast MHCIId/x mRNA and protein. This MHC pattern resembles that of fast muscles of adult rabbits. In contrast, primary myotubes grown on matrigel-covered culture dishes express substantial amounts of non-adult MHC protein even on day 21. To conclude, primary myotubes grown on microcarriers in their later stages exhibit many features of adult skeletal muscle and characteristics of fast type II fibers. Thus, the culture represents an excellent model of adult fast skeletal muscle, for example, when investigating molecular mechanisms of fast-to-slow fiber-type transformation.

Purpose To evaluate the cleavage plane, corneal cytoarchitecture, and cell vitality of separated corneal epithelial sheets created with 3 commonly used microkeratomes. Setting Laboratories of the Regensburg University Medical Center, Regensburg, Germany. Methods Mechanical separation of the epithelial layer in 10 porcine eyes and 2 human eyes was performed with 3 different microkeratomes (Amadeus II, Zyoptix XP, Epivision). Five of 10 porcine corneas and the 2 human corneas treated with each microkeratome were processed for histology, electron microscopy, and immunohistochemistry. In 5 of 10 porcine corneas, the corneal epithelial sheets were separated from the globe and cell vitality was assessed with the trypan blue dye vitality test. Results A reproducible epithelial separation with a smooth surface was achieved in all eyes. The cleavage plane was located between the lamina lucida and the lamina densa. Damage to epithelial cells was mainly limited to the cut margins. Conclusions Mechanical separation of the epithelial sheet in epithelial laser in situ keratomileusis (epi-LASIK) was safe and reproducible with all evaluated microkeratomes. Immunohistochemistry and electron microscopy showed the cleavage plane in epi-LASIK was between the basal epithelium and the basement membrane at the level of the lamina lucida.