The landscape of submicroscopic structural variants at the OPN1LW/OPN1MW gene cluster on Xq28 underlying blue cone monochromacy.

Blue cone monochromacy (BCM) is an X-linked retinal disorder characterized by low vision, photoaversion, and poor color discrimination. BCM is due to the lack of long-wavelength-sensitive and middle-wavelength-sensitive cone photoreceptor function and caused by mutations in the OPN1LW/OPN1MW gene cluster on Xq28. Here, we investigated the prevalence and the landscape of submicroscopic structural variants (SVs) at single-base resolution in BCM patients. We found that about one-third (n = 73) of the 213 molecularly confirmed BCM families carry an SV, most commonly deletions restricted to the OPN1LW/OPN1MW gene cluster. The structure and precise breakpoints of the SVs were resolved in all but one of the 73 families. Twenty-two families-all from the United States-showed the same SV, and we confirmed a common ancestry of this mutation. In total, 42 distinct SVs were identified, including 40 previously unreported SVs, thereby quadrupling the number of precisely mapped SVs underlying BCM. Notably, there was no "region of overlap" among these SVs. However, 90% of SVs encompass the upstream locus control region, an essential enhancer element. Its minimal functional extent based on deletion mapping in patients was refined to 358 bp. Breakpoint analyses suggest diverse mechanisms underlying SV formation as well as in one case the gene conversion-based exchange of a 142-bp deletion between opsin genes. Using parsimonious assumptions, we reconstructed the composition and copy number of the OPN1LW/OPN1MW gene cluster prior to the mutation event and found evidence that large gene arrays may be predisposed to the occurrence of SVs at this locus.

Comprehensive variant spectrum of the CNGA3 gene in patients affected by achromatopsia.

Achromatopsia (ACHM) is a congenital cone photoreceptor disorder characterized by impaired color discrimination, low visual acuity, photosensitivity, and nystagmus. To date, six genes have been associated with ACHM (CNGA3, CNGB3, GNAT2, PDE6C, PDE6H, and ATF6), the majority of these being implicated in the cone phototransduction cascade. CNGA3 encodes the CNGA3 subunit of the cyclic nucleotide-gated ion channel in cone photoreceptors and is one of the major disease-associated genes for ACHM. Herein, we provide a comprehensive overview of the CNGA3 variant spectrum in a cohort of 1060 genetically confirmed ACHM patients, 385 (36.3%) of these carrying "likely disease-causing" variants in CNGA3. Compiling our own genetic data with those reported in the literature and in public databases, we further extend the CNGA3 variant spectrum to a total of 316 variants, 244 of which we interpreted as "likely disease-causing" according to ACMG/AMP criteria. We report 48 novel "likely disease-causing" variants, 24 of which are missense substitutions underlining the predominant role of this mutation class in the CNGA3 variant spectrum. In addition, we provide extensive in silico analyses and summarize reported functional data of previously analyzed missense, nonsense and splicing variants to further advance the pathogenicity assessment of the identified variants.

Network-based visualisation reveals new insights into transposable element diversity.

Transposable elements (TEs) are widespread across eukaryotic genomes, yet their content varies widely between different species. Factors shaping the diversity of TEs are poorly understood. Understanding the evolution of TEs is difficult because their sequences diversify rapidly and TEs are often transferred through non-conventional means such as horizontal gene transfer. We developed a method to track TE evolution using network analysis to visualise TE sequence and TE content across different genomes. We illustrate our method by first using a monopartite network to study the sequence evolution of Tc1/mariner elements across focal species. We identify a connection between two subfamilies associated with convergent acquisition of a domain from a protein-coding gene. Second, we use a bipartite network to study how TE content across species is shaped by epigenetic silencing mechanisms. We show that the presence of Piwi-interacting RNAs is associated with differences in network topology after controlling for phylogenetic effects. Together, our method demonstrates how a network-based approach can identify hitherto unknown properties of TE evolution across species.

VALIDATION OF A DEEP LEARNING-BASED ALGORITHM FOR SEGMENTATION OF THE ELLIPSOID ZONE ON OPTICAL COHERENCE TOMOGRAPHY IMAGES OF AN USH2A-RELATED RETINAL DEGENERATION CLINICAL TRIAL.

PURPOSE: To assess the generalizability of a deep learning-based algorithm to segment the ellipsoid zone (EZ). METHODS: The dataset consisted of 127 spectral-domain optical coherence tomography volumes from eyes of participants with USH2A-related retinal degeneration enrolled in the RUSH2A clinical trial (NCT03146078). The EZ was segmented manually by trained readers and automatically by deep OCT atrophy detection, a deep learning-based algorithm originally developed for macular telangiectasia Type 2. Performance was evaluated using the Dice similarity coefficient between the segmentations, and the absolute difference and Pearson's correlation of measurements of interest obtained from the segmentations. RESULTS: With deep OCT atrophy detection, the average (mean ± SD, median) Dice similarity coefficient was 0.79 ± 0.27, 0.90. The average absolute difference in total EZ area was 0.62 ± 1.41, 0.22 mm2 with a correlation of 0.97. The average absolute difference in the maximum EZ length was 222 ± 288, 126 µm with a correlation of 0.97. CONCLUSION: Deep OCT atrophy detection segmented EZ in USH2A-related retinal degeneration with good performance. The algorithm is potentially generalizable to other diseases and other biomarkers of interest as well, which is an important aspect of clinical applicability.

Establishing the prevalence of common tissue-specific autoantibodies following severe acute respiratory syndrome coronavirus 2 infection.

Coronavirus 19 (COVID-19) has been associated with both transient and persistent systemic symptoms that do not appear to be a direct consequence of viral infection. The generation of autoantibodies has been proposed as a mechanism to explain these symptoms. To understand the prevalence of autoantibodies associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we investigated the frequency and specificity of clinically relevant autoantibodies in 84 individuals previously infected with SARS-CoV-2, suffering from COVID-19 of varying severity in both the acute and convalescent setting. These were compared with results from 32 individuals who were on the intensive therapy unit (ITU) for non-COVID reasons. We demonstrate a higher frequency of autoantibodies in the COVID-19 ITU group compared with non-COVID-19 ITU disease control patients and that autoantibodies were also found in the serum 3-5 months post-COVID-19 infection. Non-COVID patients displayed a diverse pattern of autoantibodies; in contrast, the COVID-19 groups had a more restricted panel of autoantibodies including skin, skeletal muscle and cardiac antibodies. Our results demonstrate that respiratory viral infection with SARS-CoV-2 is associated with the detection of a limited profile of tissue-specific autoantibodies, detectable using routine clinical immunology assays. Further studies are required to determine whether these autoantibodies are specific to SARS-CoV-2 or a phenomenon arising from severe viral infections and to determine the clinical significance of these autoantibodies.

Detecting beta-amyloid glycation by intrinsic fluorescence - Understanding the link between diabetes and Alzheimer's disease.

We monitor early stages of beta-amyloid (Aß1-40) aggregation, one of the key processes leading to Alzheimer's disease (AD), in the presence of high glucose concentrations by measuring Aß1- 40 intrinsic fluorescence. The multiple peaks and their shifts observed in the time-resolved emission spectra (TRES) reveal the impact of glycation on Aß1- 40 oligomerisation. The results show that formation of the advanced glycation end products (AGEs) alters the aggregation pathway. These changes are highly relevant to our understanding of the pathophysiology of AD and the implication of AGE and diabetes in these pathways.

LEBER CONGENITAL AMAUROSIS DUE TO CEP290 MUTATIONS-SEVERE VISION IMPAIRMENT WITH A HIGH UNMET MEDICAL NEED: A Review.

PURPOSE: Leber congenital amaurosis due to CEP290 mutations (LCA10) is an inherited retinal disease that often results in severe visual impairment or blindness in early childhood. Currently, there are no approved treatments, highlighting the considerable unmet medical need associated with LCA10. We aimed to review the clinical characteristics of LCA10, its impact on patients and society, and the investigational treatment strategies currently in development. METHODS: Review of the current literature. RESULTS: LCA10 is an autosomal recessive ciliopathy, for which the CEP290 intronic variant c.2991+1655A>G (p.Cys998X) is the most common mutation. Usually diagnosed in early childhood, most patients with LCA10 have severe visual impairment during their first decade of life, which significantly affects the quality of life and development. LCA10 also has a significant societal burden (direct and indirect costs). RNA editing using antisense oligonucleotides or Staphylococcus aureus CRISPR-associated protein-9 nuclease is currently under investigation for treatment of p.Cys998X LCA10. Specifically, the antisense oligonucleotide therapy QR-110 (sepofarsen) has demonstrated encouraging safety and efficacy data in a first-in-human trial; a phase 3 clinical trial is ongoing. CONCLUSION: Interventions that can preserve or improve vision in patients with LCA10 have considerable potential to improve the patient quality of life and reduce burden of disease.

Influence of local microenvironment on the double hydrogen transfer in porphycene.

We performed time-resolved transient absorption and fluorescence anisotropy measurements in order to study tautomerization of porphycene in rigid polymer matrices at cryogenic temperatures. Studies were carried out in poly(methyl methacrylate) (PMMA), poly(vinyl butyral) (PVB), and poly(vinyl alcohol) (PVA). The results prove that in all studied media hydrogen tunnelling plays a significant role in the double hydrogen transfer which becomes very sensitive to properties of the environment below approx. 150 K. We also demonstrate that there exist two populations of porphycene molecules in rigid media: "hydrogen-transferring" molecules, in which tautomerization occurs on time scales below 1 ns and "frozen" molecules in which double hydrogen transfer is too slow to be monitored with nanosecond techniques. The number of "frozen" molecules increases when the sample is cooled. We explain this effect by interactions of guest molecules with a rigid host matrix which disturbs symmetry of porphycene and hinders tunnelling. Temperature dependence of the number of hydrogen-transferring molecules suggests that the factor which restores the symmetry of the double-minimum potential well in porphycene are intermolecular vibrations localized in separated regions of the amorphous polymer.

Cu2+ Effects on Beta-Amyloid Oligomerisation Monitored by the Fluorescence of Intrinsic Tyrosine.

A non-invasive intrinsic fluorescence sensing of the early stages of Alzheimer's beta amyloid peptide aggregation in the presence of copper ions is reported. By using time-resolved fluorescence techniques the formation of beta amyloid-copper complexes and the accelerated peptide aggregation are demonstrated. The shifts in the emission spectral peaks indicate that the peptides exhibit different aggregation pathways than in the absence of copper.

Adeno-Associated Viral Gene Therapy for Inherited Retinal Disease.

Inherited retinal diseases (IRDs) are a group of rare, heterogenous eye disorders caused by gene mutations that result in degeneration of the retina. There are currently limited treatment options for IRDs; however, retinal gene therapy holds great promise for the treatment of different forms of inherited blindness. One such IRD for which gene therapy has shown positive initial results is choroideremia, a rare, X-linked degenerative disorder of the retina and choroid. Mutation of the CHM gene leads to an absence of functional Rab escort protein 1 (REP1), which causes retinal pigment epithelium cell death and photoreceptor degeneration. The condition presents in childhood as night blindness, followed by progressive constriction of visual fields, generally leading to vision loss in early adulthood and total blindness thereafter. A recently developed adeno-associated virus-2 (AAV2) vector construct encoding REP1 (AAV2-REP1) has been shown to deliver a functional version of the CHM gene into the retinal pigment epithelium and photoreceptor cells. Phase 1 and 2 studies of AAV2-REP1 in patients with choroideremia have produced encouraging results, suggesting that it is possible not only to slow or stop the decline in vision following treatment with AAV2-REP1, but also to improve visual acuity in some patients.

CHOROIDEREMIA: Retinal Degeneration With an Unmet Need.

PURPOSE: Choroideremia is an incurable, X-linked, recessive retinal dystrophy caused by loss of function mutations in the CHM gene. It is estimated to affect approximately 1 in 50,000 male patients. It is characterized by progressive degeneration of the retinal pigment epithelium, choroid, and photoreceptors, resulting in visual impairment and blindness. There is an unmet need in choroideremia, because currently, there are no approved treatments available for patients with the disease. METHODS: We review the patient journey, societal impact, and emerging treatments for patients with choroideremia. RESULTS: Its relative rarity and similarities with other retinal diseases in early years mean that diagnosis of choroideremia can often be delayed. Furthermore, its impact on affected individuals, and wider society, is also likely underestimated. AAV2-mediated gene therapy is an investigational treatment that aims to replace the faulty CHM gene. Early-phase studies reported potentially important visual acuity gains and maintenance of vision in some patients, and a large Phase 3 program is now underway. CONCLUSION: Choroideremia is a disease with a significant unmet need. Interventions that can treat progression of the disease and improve visual and functional outcomes have the potential to reduce health care costs and enhance patient quality of life.

Scotopic Microperimetric Assessment of Rod Function in Stargardt Disease (SMART) Study: Design and Baseline Characteristics (Report No. 1).

PURPOSE: To describe the study design and characteristics at first visit of participants in the longitudinal Scotopic Microperimetric Assessment of Rod Function in Stargardt Disease (SMART) study. METHODS: Scotopic microperimetry (sMP) was performed in one designated study eye in a subset of participants with molecularly proven ABCA4-associated Stargardt disease (STGD1) enrolled in a multicenter natural history study (ProgStar). Study visits were every 6 months over a period ranging from 6 to 24 months, and also included fundus autofluorescence (FAF). RESULTS: SMART enrolled 118 participants (118 eyes). At the first visit of SMART, the mean sensitivity in mesopic microperimetry was 11.48 (±5.05; range 0.00-19.88) dB and in sMP 11.25 (±5.26; 0-19.25) dB. For FAF, all eyes had a lesion of decreased autofluorescence (mean lesion size 3.62 [±3.48; 0.10-21.46] mm2), and a total of 76 eyes (65.5%) had a lesion of definitely decreased autofluorescence with a mean lesion size of 3.46 (±3.60; 0.21-21.46) mm2. CONCLUSIONS: Rod function is impaired in STGD1 and can be assessed by sMP. Testing rod function may serve as a potential outcome measure for future clinical treatment trials. This is evaluated in the SMART study.

Public Health and School Health Education: Aligning Forces for Change.

The interdependent relationship between health and education has long been documented by leading health and education scholars. Children who are not physically, mentally, socially, or emotionally healthy will not be ready to learn and thus hampered to achieve their full potential as productive members of society. Despite this evidence, the United States has yet to bridge the divide between the health and education systems. This perspective introduces three manuscripts in this Special School Health Education Collection on the future of school health education in the United States, and provides a context for the challenges and recommendations each article outlines to improve the quantity and quality of school health education for preK-12 youth. Although some of the challenges and recommendations are not novel, what is exciting is the opportunity to move the agenda forward given the Whole School, Whole Community, Whole Child model and the Every Student Succeeds Act of 2015. Aligning the forces of public health and school health educators is essential to make school health education a societal imperative.

Quality Assurance in Teaching K-12 Health Education: Paving a New Path Forward.

To be effective, school health instruction should be taught by health educators who have graduated from accredited health education teacher education programs and are certified in health education. Unfortunately, the nation has failed to ensure that all those who teach health in schools are well prepared. States vary in the required coursework for health teachers in terms of initial licensure and continuing education for licensure renewal; most elementary teachers are not required to receive preparation in health education; health education and physical education are often viewed as synonymous disciplines; support for in-service education of health teachers is often lacking; and more research is needed in professional preparation and development of school health educators. This article provides a call to action in five areas to strengthen both the professional preparation and professional development of school health educators. Given that education is a social determinant of health, public health educators must become stronger allies in supporting school health to promote health equity. Public health practitioners can advocate to state and community school decision makers for comprehensive school health education taught by teachers with appropriate professional preparation and certification in health education. Public health faculty can educate their students about the Whole School, Whole Community, Whole Child framework and effective strategies for its implementation, and seek rigorous professional preparation and certification and accreditation standards for their school teacher preparation programs. National health and education organizations can call for new leadership and investments in health education teacher preparation and development for a brighter future.

An early nonsense mutation facilitates the expression of a short isoform of CNGA3 by alternative translation initiation.

The cyclic nucleotide-gated (CNG) channel - composed of CNGA3 and CNGB3 subunits - mediates the influx of cations in cone photoreceptors after light stimulation and thus is a key element in cone phototransduction. Mutations in CNGA3 and CNGB3 are associated with achromatopsia, a rare autosomal recessive retinal disorder. Here, we demonstrate that the presence of an early nonsense mutation in CNGA3 induces the usage of a downstream alternative translation initiation site giving rise to a short CNGA3 isoform. The expression of this short isoform was verified by Western blot analysis and DAB staining of HEK293 cells and cone photoreceptor-like 661W cells expressing CNGA3-GST fusion constructs. Functionality of the short isoform was confirmed by a cellular calcium influx assay. Furthermore, patients carrying an early nonsense mutation were analyzed for residual cone photoreceptor function in order to identify a potential role of the short isoform to modify the clinical outcome in achromatopsia patients. Yet the results suggest that the short isoform is not able to compensate for the loss of the long isoform leaving the biological role of this variant unclear.

EVALUATION OF FULL-FIELD ELECTRORETINOGRAM REDUCTIONS AFTER OCRIPLASMIN TREATMENT: Results of the OASIS Trial ERG Substudy.

PURPOSE: To explore a possible association between full-field electroretinograms with vitreomacular adhesion resolution and best-corrected visual acuity as part of the prospective, randomized, double-masked, sham-controlled Ocriplasmin for Treatment for Symptomatic Vitreomacular Adhesion Including Macular Hole (OASIS) trial studying ocriplasmin. METHODS: The ERG substudy enrolled 62 of 220 OASIS subjects (randomized 2:1) and analyzed full-field electroretinograms and their association with both vitreomacular adhesion resolution and best-corrected visual acuity from baseline through Month 24. Electroretinogram reductions were defined as acute full-field electroretinogram reductions in amplitude of ≥40% from baseline occurring at postinjection Day 7 or Day 28. RESULTS: In the ocriplasmin group, 16/40 (40%) subjects developed ERG reductions, compared to 1/21 (4.8%) in the sham group; 13/16 (81.3%) and 1/1 (100%) resolved by study end, respectively. A total of 11/16 (68.8%) ocriplasmin-treated subjects with ERG reductions achieved vitreomacular adhesion resolution, compared to those without (9/24, 37.5%). The ocriplasmin-treated subjects with ERG reductions also gained more letters on average (11.3 vs. 9.3 letters) from baseline and had a difference of 6.7 letters in mean best-corrected visual acuity by study end compared to those without ERG reductions. CONCLUSION: Ocriplasmin-treated subjects with ERG reductions had a higher rate of vitreomacular adhesion resolution and showed better visual improvement than their counterparts without ERG reductions or sham subjects by study end.

The Progression of the Stargardt Disease Type 4 (ProgStar-4) Study: Design and Baseline Characteristics (ProgStar-4 Report No. 1).

BACKGROUND/AIMS: To describe the design and baseline characteristics of patients enrolled in the multicenter, prospective natural history study of Stargardt disease type 4. METHODS: Fifteen eligible patients aged 6 years and older at baseline, harboring disease-causing variants in the PROM1 gene, and with specified ocular lesions were enrolled. They were examined at baseline using a standard protocol, with 6 monthly follow-up visits for a 2-year period including best-corrected ETDRS visual acuity, spectral-domain optical coherence tomography, fundus autofluorescence (FAF), mesopic and scotopic microperimetry (MP). Areas of definitely decreased FAF (DDAF) and questionably decreased FAF were outlined and quantified on FAF images. RESULTS: Amongst the 15 patients (29 eyes) that were enrolled at 5 centers in the USA and Europe, 10 eyes (34.5%) had areas of DDAF with an average lesion area of 3.2 ± 3.5 mm2 (range 0.36-10.39 mm2) at baseline. The mean retinal sensitivity of the posterior pole derived from mesopic MP was 8.8 ± 5.8 dB. CONCLUSIONS: Data on disease progression in PROM1-related retinopathy from this study will contribute to the characterization of the natural history of disease and the exploration of the utility of several modalities to track progression and therefore to potentially be used in future interventional clinical trials.

Molecular Findings in Families with an Initial Diagnose of Autosomal Dominant Retinitis Pigmentosa (adRP).

Genetic testing of probands in families with an initial diagnosis of autosomal dominant retinitis pigmentosa (adRP) usually confirms the diagnosis, but there are exceptions. We report results of genetic testing in a large cohort of adRP families with an emphasis on exceptional cases including X-linked RP with affected females; homozygous affected individuals in families with heterozygous, dominant disease; and independently segregating mutations in the same family. Genetic testing was conducted in more than 700 families with a provisional or probable diagnosis of adRP. Exceptions to the proposed mode of inheritance were extracted from our comprehensive patient and family database. In a subset of 300 well-characterized families with a probable diagnosis of adRP, 195 (70%) have dominant mutations in known adRP genes but 25 (8%) have X-linked mutations, 3 (1%) have multiple segregating mutations, and 3 (1%) have dominant-acting mutations in genes previously associated with recessive disease. It is currently possible to determine the underlying disease-causing gene and mutation in approximately 80% of families with an initial diagnosis of adRP, but 10% of "adRP" families have a variant mode of inheritance. Informed genetic diagnosis requires close collaboration between clinicians, genetic counselors, and laboratory scientists.

Transcription factor PRDM8 is required for rod bipolar and type 2 OFF-cone bipolar cell survival and amacrine subtype identity.

Retinal bipolar (BP) cells mediate the earliest steps in image processing in the visual system, but the genetic pathways that regulate their development and function are incompletely known. We identified PRDI-BF1 and RIZ homology domain containing 8 (PRDM8) as a highly conserved transcription factor that is abundantly expressed in mouse retina. During development and in maturity, PRDM8 is expressed strongly in BP cells and a fraction of amacrine and ganglion cells. To determine whether Prdm8 is essential to BP cell development or physiology, we targeted the gene in mice. Prdm8(EGFP/EGFP) mice showed nonprogressive b-wave deficits on electroretinograms, consistent with compromised BP cell function or circuitry resembling the incomplete form of human congenital stationary night blindness (CSNB). BP cell specification was normal in Prdm8(EGFP/EGFP) retina as determined by VSX2(+) cell numbers and retinal morphology at postnatal day 6. BP subtype differentiation was impaired, however, as indicated by absent or diminished expression of BP subtype-specific markers, including the putative PRDM8 regulatory target PKCα (Prkca) and its protein. By adulthood, rod bipolar (RB) and type 2 OFF-cone bipolar (CB) cells were nearly absent from Prdm8-null mice. Although no change was detected in total amacrine cell (AC) numbers, increased PRKCA(+) and cholinergic ACs and decreased GABAergic ACs were seen, suggesting an alteration in amacrine subtype identity. These findings establish that PRDM8 is required for RB and type 2 OFF-CB cell survival and amacrine subtype identity, and they present PRDM8 as a candidate gene for human CSNB.

Single-base substitutions in the CHM promoter as a cause of choroideremia.

Although over 150 unique mutations affecting the coding sequence of CHM have been identified in patients with the X-linked chorioretinal disease choroideremia (CHM), no regulatory mutations have been reported, and indeed the promoter has not been defined. Here, we describe two independent families affected by CHM bearing a mutation outside the gene's coding region at position c.-98: C>A and C>T, which segregated with the disease. The male proband of family 1 was found to lack CHM mRNA and its gene product Rab escort protein 1, whereas whole-genome sequencing of an affected male in family 2 excluded the involvement of any other known retinal genes. Both mutations abrogated luciferase activity when inserted into a reporter construct, and by further employing the luciferase reporter system to assay sequences 5' to the gene, we identified the CHM promoter as the region encompassing nucleotides c.-119 to c.-76. These findings suggest that the CHM promoter region should be examined in patients with CHM who lack coding sequence mutations, and reveals, for the first time, features of the gene's regulation.