Viewing Ageing Eyes: Diverse Sites of Amyloid Beta Accumulation in the Ageing Mouse Retina and the Up-Regulation of Macrophages

Amyloid beta (Aβ) accumulates in the ageing central nervous system and is associated with a number of age-related diseases, including age-related macular degeneration (AMD) in the eye. AMD is characterised by accumulation of extracellular deposits called drusen in which Aβ is a key constituent. Aβ activates the complement cascade and its deposition is associated with activated macrophages. So far, little is known about the quantitative measurements of Aβ accumulation and definitions of its relative sites of ocular deposition in the normal ageing mouse. METHODOLOGY/PRINCIPAL FINDINGS: We have traced Aβ accumulation quantitatively in the ageing mouse retina using immunohistochemistry and Western blot analysis. We reveal that it is not only deposited at Bruch's membrane and along blood vessels, but unexpectedly, it also coats photoreceptor outer segments. While Aβ is present at all sites of deposition from 3 months of age, it increases markedly from 6 months onward. Progressive accumulation of deposits on outer segments was confirmed with scanning electron microscopy, revealing age-related changes in their morphology. Such progress of accumulation of Aβ on photoreceptor outer segments with age was also confirmed in human retinae using immunohistochemistry. We also chart the macrophage response to increases in Aβ showing up-regulation in their numbers using both confocal laser imaging of the eye in vivo followed by in vitro immunostaining. With age macrophages become bloated with cellular debris including Aβ, however, their increasing numbers fail to stop Aβ accumulation. CONCLUSIONS: Increasing Aβ deposition in blood vessels and Bruch's membrane will impact upon retinal perfusion and clearance of cellular waste products from the outer retina, a region of very high metabolic activity. This accumulation of Aβ may contribute to the 30% reduction of photoreceptors found throughout life and the shortening of those that remain. The coating of Aβ on outer segments may also have an impact upon visual function with ag

Biallelic Variants in TTLL5, Encoding a Tubulin Glutamylase, Cause Retinal Dystrophy

In a subset of inherited retinal degenerations (including cone, cone-rod, and macular dystrophies), cone photoreceptors are more severely affected than rods; ABCA4 mutations are the most common cause of this heterogeneous class of disorders. To identify retinal-disease-associated genes, we performed exome sequencing in 28 individuals with “cone-first” retinal disease and clinical features atypical for ABCA4 retinopathy. We then conducted a gene-based case-control association study with an internal exome data set as the control group. TTLL5, encoding a tubulin glutamylase, was highlighted as the most likely disease-associated gene; 2 of 28 affected subjects harbored presumed loss-of-function variants: c.[1586_1589delAGAG];[1586_1589delAGAG], p.[Glu529Valfs∗2];[Glu529Valfs∗2], and c.[401delT(;)3354G>A], p.[Leu134Argfs∗45(;)Trp1118∗]. We then inspected previously collected exome sequence data from individuals with related phenotypes and found two siblings with homozygous nonsense variant c.1627G>T (p.Glu543∗) in TTLL5. Subsequently, we tested a panel of 55 probands with retinal dystrophy for TTLL5 mutations; one proband had a homozygous missense change (c.1627G>A [p.Glu543Lys]). The retinal phenotype was highly similar in three of four families; the sibling pair had a more severe, early-onset disease. In human and murine retinae, TTLL5 localized to the centrioles at the base of the connecting cilium. TTLL5 has been previously reported to be essential for the correct function of sperm flagella in mice and play a role in polyglutamylation of primary cilia in vitro. Notably, genes involved in the polyglutamylation and deglutamylation of tubulin have been associated with photoreceptor degeneration in mice. The electrophysiological and fundus autofluorescence imaging presented here should facilitate the molecular diagnosis in further families

EyeG2P: an automated variant filtering approach improves efficiency of diagnostic genomic testing for inherited ophthalmic disorders

BACKGROUND: Genomic variant prioritisation is one of the most significant bottlenecks to mainstream genomic testing in healthcare. Tools to improve precision while ensuring high recall are critical to successful mainstream clinical genomic testing, in particular for whole genome sequencing where millions of variants must be considered for each patient. METHODS: We developed EyeG2P, a publicly available database and web application using the Ensembl Variant Effect Predictor. EyeG2P is tailored for efficient variant prioritisation for individuals with inherited ophthalmic conditions. We assessed the sensitivity of EyeG2P in 1234 individuals with a broad range of eye conditions who had previously received a confirmed molecular diagnosis through routine genomic diagnostic approaches. For a prospective cohort of 83 individuals, we assessed the precision of EyeG2P in comparison with routine diagnostic approaches. For 10 additional individuals, we assessed the utility of EyeG2P for whole genome analysis. RESULTS: EyeG2P had 99.5% sensitivity for genomic variants previously identified as clinically relevant through routine diagnostic analysis (n=1234 individuals). Prospectively, EyeG2P enabled a significant increase in precision (35% on average) in comparison with routine testing strategies (p<0.001). We demonstrate that incorporation of EyeG2P into whole genome sequencing analysis strategies can reduce the number of variants for analysis to six variants, on average, while maintaining high diagnostic yield. CONCLUSION: Automated filtering of genomic variants through EyeG2P can increase the efficiency of diagnostic testing for individuals with a broad range of inherited ophthalmic disorders

A detailed clinical and molecular survey of subjects with nonsyndromic USH2A retinopathy reveals an allelic hierarchy of disease-causing variants.

Defects in USH2A cause both isolated retinal disease and Usher syndrome (ie, retinal disease and deafness). To gain insights into isolated/nonsyndromic USH2A retinopathy, we screened USH2A in 186 probands with recessive retinal disease and no hearing complaint in childhood (discovery cohort) and in 84 probands with recessive retinal disease (replication cohort). Detailed phenotyping, including retinal imaging and audiological assessment, was performed in individuals with two likely disease-causing USH2A variants. Further genetic testing, including screening for a deep-intronic disease-causing variant and large deletions/duplications, was performed in those with one likely disease-causing change. Overall, 23 of 186 probands (discovery cohort) were found to harbour two likely disease-causing variants in USH2A. Some of these variants were predominantly associated with nonsyndromic retinal degeneration ('retinal disease-specific'); these included the common c.2276 G>T, p.(Cys759Phe) mutation and five additional variants: c.2802 T>G, p.(Cys934Trp); c.10073 G>A, p.(Cys3358Tyr); c.11156 G>A, p.(Arg3719His); c.12295-3 T>A; and c.12575 G>A, p.(Arg4192His). An allelic hierarchy was observed in the discovery cohort and confirmed in the replication cohort. In nonsyndromic USH2A disease, retinopathy was consistent with retinitis pigmentosa and the audiological phenotype was variable. USH2A retinopathy is a common cause of nonsyndromic recessive retinal degeneration and has a different mutational spectrum to that observed in Usher syndrome. The following model is proposed: the presence of at least one 'retinal disease-specific' USH2A allele in a patient with USH2A-related disease results in the preservation of normal hearing. Careful genotype-phenotype studies such as this will become increasingly important, especially now that high-throughput sequencing is widely used in the clinical setting.European Journal of Human Genetics advance online publication, 4 February 2015; doi:10.1038/ejhg.2014.283

Multi-disciplinary team directed analysis of whole genome sequencing reveals pathogenic non-coding variants in molecularly undiagnosed inherited retinal dystrophies

PURPOSE: To identify, using genome sequencing (GS), likely pathogenic non-coding variants in inherited retinal dystrophy (IRD) genes Methods: Patients with IRD were recruited to the study and underwent comprehensive ophthalmological evaluation and GS. The results of GS were investigated through virtual gene panel analysis and plausible pathogenic variants and clinical phenotype evaluated by multi-disciplinary team (MDT) discussion. For unsolved patients in whom a specific gene was suspected to harbour a missed pathogenic variant, targeted re-analysis of non-coding regions was performed on GS data. Candidate variants were functionally tested including by mRNA analysis, minigene and luciferase reporter assays. RESULTS: Previously unreported, likely pathogenic, non-coding variants, in 7 genes (PRPF31, NDP, IFT140, CRB1, USH2A, BBS10, and GUCY2D), were identified in 11 patients. These were shown to lead to mis-splicing (PRPF31, IFT140, CRB1, USH2A) or altered transcription levels (BBS10, GUCY2D). CONCLUSION: MDT-led, phenotype driven, non-coding variant re-analysis of GS is effective in identifying missing causative alleles

Clinical utility of genetic testing in 201 preschool children with inherited eye disorders

From Springer Nature via Jisc Publications RouterHistory: received 2019-09-23, registration 2019-11-25, accepted 2019-11-25, pub-electronic 2019-12-18, online 2019-12-18, pub-print 2020-04-01Publication status: PublishedAbstract: Purpose: A key property to consider in all genetic tests is clinical utility, the ability of the test to influence patient management and health outcomes. Here we assess the current clinical utility of genetic testing in diverse pediatric inherited eye disorders (IEDs). Methods: Two hundred one unrelated children (0–5 years old) with IEDs were ascertained through the database of the North West Genomic Laboratory Hub, Manchester, UK. The cohort was collected over a 7-year period (2011–2018) and included 74 children with bilateral cataracts, 8 with bilateral ectopia lentis, 28 with bilateral anterior segment dysgenesis, 32 with albinism, and 59 with inherited retinal disorders. All participants underwent panel-based genetic testing. Results: The diagnostic yield of genetic testing for the cohort was 64% (ranging from 39% to 91% depending on the condition). The test result led to altered management (including preventing additional investigations or resulting in the introduction of personalized surveillance measures) in 33% of probands (75% for ectopia lentis, 50% for cataracts, 33% for inherited retinal disorders, 7% for anterior segment dysgenesis, 3% for albinism). Conclusion: Genetic testing helped identify an etiological diagnosis in the majority of preschool children with IEDs. This prevented additional unnecessary testing and provided the opportunity for anticipatory guidance in significant subsets of patients

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

© 2024 The Authors. Journal of Extracellular Vesicles, published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY), https://creativecommons.org/licenses/by/4.0/Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.Peer reviewe

Minimal information for studies of extracellular vesicles 2018 (MISEV2018):a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points