Eyes shut homolog is important for the maintenance of photoreceptor morphology and visual function in zebrafish

<div><p>Mutations in <i>eyes shut homolog</i> (<i>EYS</i>), a gene predominantly expressed in the photoreceptor cells of the retina, are among the most frequent causes of autosomal recessive (ar) retinitis pigmentosa (RP), a progressive retinal disorder. Due to the absence of <i>EYS</i> in several rodent species and its retina-specific expression, still little is known about the exact function of EYS and the pathogenic mechanism underlying <i>EYS</i>-associated RP. We characterized <i>eys</i> in zebrafish, by RT-PCR analysis on zebrafish eye-derived RNA, which led to the identification of a 8,715 nucleotide coding sequence that is divided over 46 exons. The transcript is predicted to encode a 2,905-aa protein that contains 39 EGF-like domains and five laminin A G-like domains, which overall shows 33% identity with human EYS. To study the function of EYS, we generated a stable <i>eys</i>^{<i>rmc101/rmc101</i>} mutant zebrafish model using CRISPR/Cas9 technology. The introduced lesion is predicted to result in premature termination of protein synthesis and lead to loss of Eys function. Immunohistochemistry on retinal sections revealed that Eys localizes at the region of the connecting cilium and that both rhodopsin and cone transducin are mislocalized in the absence of Eys. Electroretinogram recordings showed diminished b-wave amplitudes in <i>eys</i>^{<i>rmc101/rmc101</i>} zebrafish (5 dpf) compared to age- and strain-matched wild-type larvae. In addition, decreased locomotor activity in response to light stimuli was observed in <i>eys</i> mutant larvae. Altogether, our study shows that absence of Eys leads to a disorganized retinal architecture and causes visual dysfunction in zebrafish.</p></div

Characterization of a stable <i>eys</i>^{<i>rmc101/rmc101</i>} zebrafish line.

<p><b>(A)</b> Sanger sequencing identified a five base pair deletion in exon 20 in <i>eys</i>^{<i>rmc101/rmc101</i>} zebrafish. <b>(B)</b> Representative gel image of RT-PCR analysis using RNA from a pool of larvae (n = 15), which shows that <i>eys</i> transcripts are present in both wild-type and <i>eys</i>^{<i>rmc101/rmc101</i>} zebrafish (upper panel). Sanger sequencing confirmed the presence of the five base pair deletion in the <i>eys</i>^{<i>rmc101/rmc101</i>} transcript (lower panel). <b>(C)</b> Protein domain structures of wild-type Eys and the truncated Eys protein that is predicted in <i>eys</i>^{<i>rmc101/rmc101</i>} zebrafish.</p

Visual motor response of zebrafish larvae.

<p><b>(A)</b> Distance moved (mm) of wild-type (blue line) and <i>eys</i>^{<i>rmc101/rmc101</i>} (red line) larvae in response to a light stimulus (dark-to-light transition at t = 50 minutes). <b>(B)</b> Comparison of difference in distance moved between wild-type and <i>eys</i>^{<i>rmc101/rmc101</i>} larvae at the dark to light transition zones. <b>(C)</b> Maximum velocity (Vmax; mm/s) of wild-type (blue line) and <i>eys</i>^{<i>rmc101/rmc101</i>} (red line) larvae in response to a light stimulus (dark-to-light transition at t = 50 minutes). <b>(D)</b> Comparison of difference in Vmax between wild-type and <i>eys</i>^{<i>rmc101/rmc101</i>} larvae at the dark to light transition zones. All experiments were done with larvae at 5 dpf (n = 120). Statistical significance (<i>p</i><0.05) is indicated with an asterisk.</p

Immunohistochemistry on retinal sections of wild-type and <i>eys</i>^{<i>rmc101/rmc101</i>} zebrafish.

<p><b>(A)</b> Retinal sections of wild-type and <i>eys</i>^{<i>rmc101/rmc101</i>} zebrafish at 5 dpf and 5 mpf stained with antibodies against Eys (green) and centrin (red). <b>(B)</b> BODIPY (green) staining showing disorganization of photoreceptor outer segments in <i>eys</i>^{<i>rmc101/rmc101</i>} zebrafish (5 dpf and 5 mpf) compared to age- and strain-matched wild-type zebrafish (arrows). <b>(C)</b> Retinal sections of wild-type and <i>eys</i>^{<i>rmc101/rmc101</i>} zebrafish at 5 dpf (upper panel), 2 mpf (middle panel) and 5 mpf (lower panel) stained with antibodies against rhodopsin (green) and F-actin (red). Asterisks indicate mislocalization of rhodopsin to the inner segments and synapses of photoreceptor cells. <b>(D)</b> Retinal sections of wild-type and <i>eys</i>^{<i>rmc101/rmc101</i>} zebrafish at 5 dpf, 2 mpf and 5 mpf stained with antibodies against GNAT2 (green) and F-actin (red). Arrows indicate dysmorphic outer segments in mutant zebrafish. In all images, nuclei are counterstained with DAPI (blue). INL: inner nuclear layer; ONL: outer nuclear layer; OS: outer segments. Scale: 5 μm.</p

Extending the spectrum of EYS-associated retinal disease to macular dystrophy

PURPOSE. To assess the phenotypic variability and natural course of inherited retinal diseases (IRDs) caused by EYS mutations. METHODS. Multiethnic cohort study (N = 30) with biallelic EYS variants from a clinical IRD database (retinitis pigmentosa [RP], N = 27; cone-rod dystrophy [CRD], N = 1; and macular dystrophy, N = 2). In vitro minigene splice assay was performed to determine the effect on EYS pre-mRNA splicing of the c.1299+5_1299+8del variant in macular dystrophy patients. RESULTS. We found 27 different EYS variants in RP patients and 7 were novel. The rate of visual field loss of the V4e isopter area was -0.84 ± 0.44 ln(deg2) per year, and the rate of visual acuity loss was 0.75 Early Treatment Diabetic Retinopathy Study letters per year. Ellipsoid zone width was correlated with area of the hyperautofluorescent ring, with rs = 0.78 and P A), previously identified in RP patients. Two siblings with macular dystrophy carried compound heterozygous EYS variants: c.1299+5_1299+8del and c.6050G>T. The former was novel and shown to result in skipping of exon 8, and the latter was a known RP variant. CONCLUSIONS. We report on EYS-associated macular dystrophy, extending the spectrum of EYSassociated IRDs. We observed heterogeneity between RP patients in age of onset and disease progression. Identical EYS variants were found in cases with RP, CRD, and macular dystrophy. Screening for EYS variants in CRD and macular dystrophy patients might increase the diagnostic yield in previously unsolved cases

Visual function of wild-type and <i>eys</i>^{<i>rmc101/rmc101</i>} zebrafish larvae.

<p><b>(A)</b> ERG measurements of the b-wave of wild-type (black line) and <i>eys</i>^{<i>rmc101/rmc101</i>} (red line) zebrafish larvae at 5 dpf. <b>(B)</b> ERG measurements of the a-wave of wild-type (black line) and <i>eys</i>^{<i>rmc101/rmc101</i>} (red line) zebrafish larvae at 5 dpf. <b>(C)</b> Quantification of the ERG b-wave amplitude of wild-type and <i>eys</i>^{<i>rmc101/rmc101</i>} zebrafish larvae at 5 dpf (n = 30; <i>p</i> = 0.0004). <b>(D)</b> Quantification of the ERG a-wave amplitude of wild-type and <i>eys</i>^{<i>rmc101/rmc101</i>} zebrafish larvae at 5 dpf (n = 20; <i>p</i> = 0.2324). <b>(E)</b> Optokinetic response measurements of wild-type and <i>eys</i>^{<i>rmc101/rmc101</i>} larvae at 5 dpf (n = 19).</p

Development and validation of an early warning score to identify COVID-19 in the emergency department based on routine laboratory tests: a multicentre case-control study

OBJECTIVES: Identifying patients with a possible SARS-CoV-2 infection in the emergency department (ED) is challenging. Symptoms differ, incidence rates vary and test capacity may be limited. As PCR-testing all ED patients is neither feasible nor effective in most centres, a rapid, objective, low-cost early warning score to triage ED patients for a possible infection is developed. DESIGN: Case-control study. SETTING: Secondary and tertiary hospitals in the Netherlands. PARTICIPANTS: The study included patients presenting to the ED with venous blood sampling from July 2019 to July 2020 (n=10 417, 279 SARS-CoV-2-positive). The temporal validation cohort covered the period from July 2020 to October 2021 (n=14 080, 1093 SARS-CoV-2-positive). The external validation cohort consisted of patients presenting to the ED of three hospitals in the Netherlands (n=12 061, 652 SARS-CoV-2-positive). PRIMARY OUTCOME MEASURES: The primary outcome was one or more positive SARS-CoV-2 PCR test results within 1 day prior to or 1 week after ED presentation. RESULTS: The resulting 'CoLab-score' consists of 10 routine laboratory measurements and age. The score showed good discriminative ability (AUC: 0.930, 95% CI 0.909 to 0.945). The lowest CoLab-score had high sensitivity for COVID-19 (0.984, 95% CI 0.970 to 0.991; specificity: 0.411, 95% CI 0.285 to 0.520). Conversely, the highest score had high specificity (0.978, 95% CI 0.973 to 0.983; sensitivity: 0.608, 95% CI 0.522 to 0.685). The results were confirmed in temporal and external validation. CONCLUSIONS: The CoLab-score is based on routine laboratory measurements and is available within 1 hour after presentation. Depending on the prevalence, COVID-19 may be safely ruled out in over one-third of ED presentations. Highly suspect cases can be identified regardless of presenting symptoms. The CoLab-score is continuous, in contrast to the binary outcome of lateral flow testing, and can guide PCR testing and triage ED patients

Alternating Hemiplegia of Childhood mutations have a differential effect on Na(+),K(+)-ATPase activity and ouabain binding

Contains fulltext : 128411.pdf (publisher's version ) (Closed access)De novo mutations in ATP1A3, the gene encoding the alpha3-subunit of Na(+),K(+)-ATPase, are associated with the neurodevelopmental disorder Alternating Hemiplegia of Childhood (AHC). The aim of this study was to determine the functional consequences of six ATP1A3 mutations (S137Y, D220N, I274N, D801N, E815K, and G947R) associated with AHC. Wild type and mutant Na(+),K(+)-ATPases were expressed in Sf9 insect cells using the baculovirus expression system. Ouabain binding, ATPase activity, and phosphorylation were absent in mutants I274N, E815K and G947R. Mutants S137Y and D801N were able to bind ouabain, although these mutants lacked ATPase activity, phosphorylation, and the K(+)/ouabain antagonism indicative of modifications in the cation binding site. Mutant D220N showed similar ouabain binding, ATPase activity, and phosphorylation to wild type Na(+),K(+)-ATPase. Functional impairment of Na(+),K(+)-ATPase in mutants S137Y, I274N, D801N, E815K, and G947R might explain why patients having these mutations suffer from AHC. Moreover, mutant D801N is able to bind ouabain, whereas mutant E815K shows a complete loss of function, possibly explaining the different phenotypes for these mutations