Genetic Testing of Patients with Inherited Retinal Diseases in the European Countries. An International Survey by the European Vision Institute.

INTRODUCTION: The purpose of this project was to explore the current standards of clinical care genetic testing and counseling for patients with inherited retinal diseases (IRDs) from the perspective of leading experts in selected European countries. Also, to gather opinions on current bottlenecks and future solutions to improve patient care. METHODS: On the initiative of the European Vision Institute, a survey questionnaire with 41 questions was designed and sent to experts in the field from ten European countries. Each participant was asked to answer with reference to the situation in their own country. RESULTS: Sixteen questionnaires were collected by November 2023. IRD genetic tests are performed in clinical care settings for 80% or more of tested patients in 9 countries, and the costs of genetic tests in clinical care are covered by the public health service to the extent of 90% or more in 8 countries. The median proportion of patients who are genetically tested, the median rate of genetically solved patients among those who are tested, and the median proportion of patients receiving counseling are 51-70%, 61-80% and 61-80%, respectively. Improving the education of healthcare professionals who facilitate patient referrals to specialised centres, improving access of patients to more thorough genotyping, and increasing the number of available counselors were the most advocated solutions. CONCLUSION: There is a significant proportion of IRD patients who are not genetically tested, whose genetic testing is inconclusive, or who do not receive counseling. Educational programs, greater availability of state-of-the-art genotyping and genetic counselors could improve healthcare for IRD patients.

The Influence of Various Crosslinking Conditions of EDC/NHS on the Properties of Fish Collagen Film.

The process of crosslinking improves the physicochemical properties of biopolymer-based composites, making them valuable for biomedical applications. EDC/NHS-crosslinked collagen materials have a significant potential for tissue engineering applications, due to their enhanced properties and biocompatibility. Chemical crosslinking of samples can be carried out in several ways, which is crucial and has a direct effect on the final properties of the obtained material. In this study, the effect of crosslinking conditions on the properties of collagen films using EDC and NHS was investigated. Studies included FTIR spectroscopy, AFM, swelling and degradation tests, mechanical testing and contact angle measurements. Evaluation of prepared collagen films indicated that both crosslinking agents and crosslinking conditions influenced film properties. Notable alternations were observed in the infrared spectrum of the sample, to which EDC was added directly to the fish collagen solution. The same sample indicated the lowest Young modulus, tensile strength and breaking force parameters and the highest elongation at break. All samples reached the maximum swelling degree two hours after immersion in PBS solution; however, the immersion-crosslinked samples exhibited a significantly lower degree of swelling and were highly durable. The highest roughness was observed for the collagen film crosslinked with EDC, whereas the lowest was observed for the specimen crosslinked with EDC with NHS addition. The crosslinking agents increased the surface roughness of the collagen film, except for the sample modified with the addition of EDC and NHS mixture. All films were characterized by hydrophilic character. The films' modification resulted in a decrease in their hydrophilicity and wettability. Our research allows for a comparison of proposed EDC/NHS crosslinking conditions and their influence on the physicochemical properties of fish collagen thin films. EDC and NHS are promising crosslinking agents for the modification of fish collagen used in biomedical applications.

Dialdehyde Starch as a Cross-Linking Agent Modifying Fish Collagen Film Properties.

The aim of this research was the modification of fish collagen films with various amounts of dialdehyde starch (DAS). Film properties were examined before and after the cross-linking process by DAS. Prepared biopolymer materials were characterized by Fourier Transform Infrared Spectroscopy and Atomic Force Microscopy. Moreover, the mechanical, thermal and swelling properties of the films were evaluated and the contact angle was measured. Research has shown that dialdehyde starch applied as a cross-linking agent influences collagen film properties. Mechanical testing indicated a decrease in Young's Modulus and an increase in breaking force, elongation at break, and tensile strength parameters. Results for contact angle were significantly higher for collagen films cross-linked with DAS; thus, the hydrophilicity of samples decreased. Modified samples presented a lower swelling degree in PBS than native collagen films. However, the highest values for the degree of swelling among the modified specimens were obtained from the 1% DAS samples, which were 717% and 702% for 1% and 2% collagen, respectively. Based on AFM images and roughness values, it was noticed that DAS influenced collagen film surface morphology. The lowest value of Rq was observed for 2%Coll_2%DAS and was approximately 10 nm. Analyzing thermograms for collagen samples, it was observed that pure collagen samples were less thermally stable than cross-linked ones. Dialdehyde starch is a promising cross-linking agent for collagen extracted from fish skin and may increase its applicability.

Negotiating Safety: Facilitation of Return to Work for Individuals Employed in High-Risk Occupations.

PURPOSE: Return-to-work (RTW) after absence due to a mental illness is a largely understudied area, especially in industries already struggling with retention like those posing unique and high risks for public or personal safety (i.e., pilots, police officers, and health professionals), otherwise known as safety-sensitive sectors. The goal of this paper is to examine how RTW coordinators work with individuals who took a leave of absence for mental illness in safety-sensitive occupations and navigate the RTW process. METHODS: Qualitative methodology was utilized to explore the experiences of 47 RTW coordinators who had worked with individuals employed in safety-sensitive industries. The participants were recruited across Canada using convenience sampling to participate in semi-structured interviews. The interviews were transcribed, anonymized, uploaded to NVIVO 11, and coded using inductive thematic analysis. RESULTS: Our analysis shows that despite the presumed rigidity of occupational health and safety standards for safety-sensitive positions, the notion of "safety" becomes ambiguous in navigating RTW processes, and concerns about safety are often interpreted as the potential risk workers may pose to themselves, other individuals, or the workplace image. Institutional constraints of safety-sensitive jobs shape the ability of RTW coordinators to advocate on behalf of the workers, ultimately placing the workers at a disadvantage by prioritizing safety concerns for organizations over employees' needs. CONCLUSION: It is important to consider how to protect workers in safety-sensitive occupations during the RTW process after absence due to a mental illness to ensure effective integration to the workplace.

Detection of elusive DNA copy-number variations in hereditary disease and cancer through the use of noncoding and off-target sequencing reads.

Copy-number variants (CNVs) play a substantial role in the molecular pathogenesis of hereditary disease and cancer, as well as in normal human interindividual variation. However, they are still rather difficult to identify in mainstream sequencing projects, especially involving exome sequencing, because they often occur in DNA regions that are not targeted for analysis. To overcome this problem, we developed OFF-PEAK, a user-friendly CNV detection tool that builds on a denoising approach and the use of "off-target" DNA reads, which are usually discarded by sequencing pipelines. We benchmarked OFF-PEAK on data from targeted sequencing of 96 cancer samples, as well as 130 exomes of individuals with inherited retinal disease from three different populations. For both sets of data, OFF-PEAK demonstrated excellent performance (>95% sensitivity and >80% specificity vs. experimental validation) in detecting CNVs from in silico data alone, indicating its immediate applicability to molecular diagnosis and genetic research.

GNB1-Related Rod-Cone Dystrophy: A Case Report.

Introduction: The GNB1 (guanine nucleotide-binding protein, ß1) gene encodes for the ubiquitous ß1 subunit of heterotrimeric G proteins, which are associated with G-protein-coupled receptors (GPCRs). GNB1 mutations cause a neurodevelopmental disorder characterized by a broad clinical spectrum. A novel variant has recently been confirmed in a case of rod-cone dystrophy. Case Presentation: We describe the second confirmed case of a classical rod-cone dystrophy associated with a mutation located in exon 6 of GNB1 [NM_002074.5:c.217G>C, p.(Ala73Pro)] in a 56-year-old patient also presenting mild intellectual disability, attention deficit/hyperactivity disorder, and truncal obesity. Conclusion: This paper confirms the role of GNB1 in the pathogenesis of a classic rod-cone dystrophy and highlights the importance of including this gene in the genetic analysis panel for inherited retinal diseases.

Loss-of-function variants in UBAP1L cause autosomal recessive retinal degeneration.

PURPOSE: Inherited retinal diseases (IRDs) are a group of monogenic conditions that can lead to progressive blindness. Their missing heritability is still considerable, due in part to the presence of disease genes that await molecular identification. The purpose of this work was to identify novel genetic associations with IRDs. METHODS: Patients underwent a comprehensive ophthalmological evaluation using standard-of-care tests, such as detailed retinal imaging (macular optical coherence tomography and short-wavelength fundus autofluorescence) and electrophysiological testing. Exome and genome sequencing, as well as computer-assisted data analysis were used for genotyping and detection of DNA variants. A minigene-driven splicing assay was performed to validate the deleterious effects of 1 of such variants. RESULTS: We identified 8 unrelated families from Hungary, the United States, Israel, and The Netherlands with members presenting with a form of autosomal recessive and nonsyndromic retinal degeneration, predominantly described as rod-cone dystrophy but also including cases of cone/cone-rod dystrophy. Age of disease onset was very variable, with some patients experiencing first symptoms during their fourth decade of life or later. Myopia greater than 5 diopters was present in 5 of 7 cases with available refractive data, and retinal detachment was reported in 2 cases. All ascertained patients carried biallelic loss-of-function variants in UBAP1L (HGNC: 40028), a gene with unknown function and with homologies to UBAP1, encoding a protein involved in ubiquitin metabolism. One of these pathogenic variants, the intronic NM_001163692.2:c.910-7G>A substitution, was identified in 5 unrelated families. Minigene-driven splicing assays in HEK293T cells confirmed that this DNA change is responsible for the creation of a new acceptor splice site, resulting in aberrant splicing. CONCLUSION: We identified UBAP1L as a novel IRD gene. Although its function is currently unknown, UBAP1L is almost exclusively expressed in photoreceptors and the retinal pigment epithelium, hence possibly explaining the link between pathogenic variants in this gene and an ocular phenotype.

Genetic profile of syndromic retinitis pigmentosa in Portugal.

PURPOSE: Retinitis pigmentosa (RP) comprises a genetically and clinically heterogeneous group of inherited retinal degenerations, where 20-30% of patients exhibit extra-ocular manifestations (syndromic RP). Understanding the genetic profile of RP has important implications for disease prognosis and genetic counseling. This study aimed to characterize the genetic profile of syndromic RP in Portugal. METHODS: Multicenter, retrospective cohort study. Six Portuguese healthcare providers identified patients with a clinical diagnosis of syndromic RP and available genetic testing results. All patients had been previously subjected to a detailed ophthalmologic examination and clinically oriented genetic testing. Genetic variants were classified according to the American College of Medical Genetics and Genomics; only likely pathogenic or pathogenic variants were considered relevant for disease etiology. RESULTS: One hundred and twenty-two patients (53.3% males) from 100 families were included. Usher syndrome was the most frequent diagnosis (62.0%), followed by Bardet-Biedl (19.0%) and Senior-Løken syndromes (7.0%). Deleterious variants were identified in 86/100 families for a diagnostic yield of 86.0% (87.1% for Usher and 94.7% for Bardet-Biedl). A total of 81 genetic variants were identified in 25 different genes, 22 of which are novel. USH2A and MYO7A were responsible for most type II and type I Usher syndrome cases, respectively. BBS1 variants were the cause of Bardet-Biedl syndrome in 52.6% of families. Best-corrected visual acuity (BCVA) records were available at baseline and last visit for 99 patients (198 eyes), with a median follow-up of 62.0 months. The mean BCVA was 56.5 ETDRS letters at baseline (Snellen equivalent ~ 20/80), declining to 44.9 ETDRS letters (Snellen equivalent ~ 20/125) at the last available follow-up (p < 0.001). CONCLUSION: This is the first multicenter study depicting the genetic profile of syndromic RP in Portugal, thus contributing toward a better understanding of this heterogeneous disease group. Usher and Bardet-Biedl syndromes were found to be the most common types of syndromic RP in this large Portuguese cohort. A high diagnostic yield was obtained, highlighting current genetic testing capabilities in providing a molecular diagnosis to most affected individuals. This has major implications in determining disease-related prognosis and providing targeted genetic counseling for syndromic RP patients in Portugal.

Navigating interprofessional boundaries: Midwifery students in Canada.

The literature on professional socialization focuses on how students adopt and internalize professional identities and values, and assumes that boundary work is essential to learning how best to practice their profession. However, a focus on boundary work in the context of midwifery training - which is embedded in the gendered and hierarchical landscape of maternity care - is lacking. Thus, this article examines how Canadian student-midwives learn to navigate and negotiate interprofessional boundaries. Grounded in a symbolic interactionist approach, it draws on 31 semi-structured qualitative interviews from a mixed-methods national study on midwifery retention, explores how midwifery students make sense of the tensions among midwives, physicians, and nurses, and describes what strategies they utilize when navigating boundaries. Our analysis, based in constructivist grounded theory, revealed that participants learned about interprofessional tensions in clinical placement encounters via direct or indirect interactions with other healthcare professionals, and that strategies to navigate these tensions included educating others about midwifery training and adopting a learner identity. This article proposes that the process of professional socialization enables to reshape professional boundaries and that students are not only learners but also agents of change. These findings may yield practical applications in health education by highlighting opportunities for improving interprofessional collaborations.

Rearrangement of thiazolidine derivatives - a synthesis of a chiral fused oxathiane-γ-lactam bicyclic system.

Reaction of L-cysteine with carbonyl compounds leads to thiazolidine derivatives which undergo a stereoselective conversion to two types of chiral bicyclic products bearing two or three stereogenic centers, including the first fused oxathiane-γ-lactam system.

TBC1D32 variants disrupt retinal ciliogenesis and cause retinitis pigmentosa.

Retinitis pigmentosa (RP) is the most common inherited retinal disease (IRD) and is characterized by photoreceptor degeneration and progressive vision loss. We report 4 patients presenting with RP from 3 unrelated families with variants in TBC1D32, which to date has never been associated with an IRD. To validate TBC1D32 as a putative RP causative gene, we combined Xenopus in vivo approaches and human induced pluripotent stem cell-derived (iPSC-derived) retinal models. Our data showed that TBC1D32 was expressed during retinal development and that it played an important role in retinal pigment epithelium (RPE) differentiation. Furthermore, we identified a role for TBC1D32 in ciliogenesis of the RPE. We demonstrated elongated ciliary defects that resulted in disrupted apical tight junctions, loss of functionality (delayed retinoid cycling and altered secretion balance), and the onset of an epithelial-mesenchymal transition-like phenotype. Last, our results suggested photoreceptor differentiation defects, including connecting cilium anomalies, that resulted in impaired trafficking to the outer segment in cones and rods in TBC1D32 iPSC-derived retinal organoids. Overall, our data highlight a critical role for TBC1D32 in the retina and demonstrate that TBC1D32 mutations lead to RP. We thus identify TBC1D32 as an IRD-causative gene.

Benefits of Treadmill Training for Patients with Down Syndrome: A Systematic Review.

BACKGROUND: The objective of this study was to evaluate the effectiveness of various results of treadmill training in children and adults with Down syndrome (DS). METHODS: To provide an overview of this effectiveness, we conducted a systematic literature review of studies in which participants with DS from all age groups received treadmill training, alone or combined with physiotherapy. We also looked for comparisons with control groups of patients with DS who did not undergo treadmill training. The search was performed in medical databases: PubMed, PEDro, Science Direct, Scopus, and Web of Science, and included trials published until February 2023. Following PRISMA criteria, the risk of bias assessment was conducted using a tool developed by the Cochrane Collaboration for RCT. The selected studies presented multiple outcomes with differences in methodology; therefore, we were not able to conduct any sort of data synthesis, so we present measures of treatment effect as mean differences and corresponding 95% confidence intervals. RESULTS: We selected 25 studies for the analysis with a total number of 687 participants, and identified 25 different outcomes which are presented in a narrative manner. In all outcomes we observed positive results favoring the treadmill training. DISCUSSION: Introducing treadmill exercise into typical physiotherapy generates improvement in mental and physical health of people with DS.

Novel inhibitors of HSV-1 protease effective in vitro and in vivo.

Herpes simplex virus type 1 (HSV-1) is a widespread human pathogen known to cause infections of diverse severity, ranging from mild ulceration of mucosal and dermal tissues to life-threatening viral encephalitis. In most cases, standard treatment with acyclovir is sufficient to manage the disease progression. However, the emergence of ACV-resistant strains drives the need for new therapeutics and molecular targets. HSV-1 VP24 is a protease indispensable for the assembly of mature virions and, as such, constitutes an interesting target for the therapy. In this study, we present novel compounds, KI207M and EWDI/39/55BF, that block the activity of VP24 protease and consequently inhibit HSV-1 infection in vitro and in vivo. The inhibitors were shown to prevent the egress of viral capsids from the cell nucleus and suppress the cell-to-cell spread of the infection. They were also proven effective against ACV-resistant HSV-1 strains. Considering their low toxicity and high antiviral potency, the novel VP24 inhibitors could provide an alternative for treating ACV-resistant infections or a drug to be used in combined, highly effective therapy.

The first genetic landscape of inherited retinal dystrophies in Portuguese patients identifies recurrent homozygous mutations as a frequent cause of pathogenesis.

Inherited retinal diseases (IRDs) are a group of ocular conditions characterized by an elevated genetic and clinical heterogeneity. They are transmitted almost invariantly as monogenic traits. However, with more than 280 disease genes identified so far, association of clinical phenotypes with genotypes can be very challenging, and molecular diagnosis is essential for genetic counseling and correct management of the disease. In addition, the prevalence and the assortment of IRD mutations are often population-specific. In this work, we examined 230 families from Portugal, with individuals suffering from a variety of IRD diagnostic classes (270 subjects in total). Overall, we identified 157 unique mutations (34 previously unreported) in 57 distinct genes, with a diagnostic rate of 76%. The IRD mutational landscape was, to some extent, different from those reported in other European populations, including Spanish cohorts. For instance, the EYS gene appeared to be the most frequently mutated, with a prevalence of 10% among all IRD cases. This was, in part, due to the presence of a recurrent and seemingly founder mutation involving the deletion of exons 13 and 14 of this gene. Moreover, our analysis highlighted that as many as 51% of our cases had mutations in a homozygous state. To our knowledge, this is the first study assessing a cross-sectional genotype-phenotype landscape of IRDs in Portugal. Our data reveal a rather unique distribution of mutations, possibly shaped by a small number of rare ancestral events that have now become prevalent alleles in patients.

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

The purpose of this paper is to identify likely pathogenic non-coding variants in inherited retinal dystrophy (IRD) genes, using genome sequencing (GS). 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 the multidisciplinary team (MDT) discussion. For unsolved patients in whom a specific gene was suspected to harbor a missed pathogenic variant, targeted re-analysis of non-coding regions was performed on GS data. Candidate variants were functionally tested by messenger RNA analysis, minigene or luciferase reporter assays. 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 and USH2A) or altered transcription levels (BBS10 and GUCY2D). MDT-led, phenotype-driven, non-coding variant re-analysis of GS is effective in identifying the missing causative alleles.

The diversity and utility of arylthiazoline and aryloxazoline siderophores: challenges of total synthesis.

Siderophores are unique ferric ion chelators produced and secreted by some organisms like bacteria, fungi and plants under iron deficiency conditions. These molecules possess immense affinity and specificity for Fe3+ and other metal ions, which attracts great interest due to the numerous possibilities of application, including antibiotics delivery to resistant bacteria strains. Total synthesis of siderophores is a must since the compounds are present in natural sources at extremely small concentrations. These molecules are extremely diverse in terms of molecular structure and physical and chemical properties. This review is focused on achievements and developments in the total synthesis strategies of naturally occurring siderophores bearing arylthiazoline and aryloxazoline units.

Chiral sulfonamides with various N-heterocyclic and aromatic units - Synthesis and antiviral activity evaluation.

Chiral sulfonamides with aromatic fragments are important chemical building blocks found widely in many natural products, catalysts, and molecules of biological importance. In this report, we describe the efficient synthesis of a series of chiral sulfonamides which, in addition to the aromatic part (phenyl, biphenyl, and dansyl units), possess N-heterocyclic systems. The described compounds were obtained by nucleophilic substitution of chiral N-heterocyclic amines and commercially available aromatic sulfonyl chlorides under mild conditions. All derivatives were examined in antiviral assay against AdV5, HSV-1, HPIV-3, HCMV, and EMCV viruses.

c.-61G>A in OVOL2 is a Pathogenic 5' Untranslated Region Variant Causing Posterior Polymorphous Corneal Dystrophy 1.

PURPOSE: The purpose of this study was to investigate the clinical and genetic features of a man and his daughter with posterior polymorphous corneal dystrophy (PPCD), referred to our clinic for Descemet membrane endothelial keratoplasty. No other known relatives were affected. METHODS: Ophthalmic examination and histology, including electron microscopy, were performed. Genetic testing was conducted by means of whole exome sequencing, and variant analysis was achieved by using an internal in silico pipeline. Molecular tests included a dual-luciferase assay. RESULTS: Slowly progressive blurred vision was reported from childhood by the daughter. The father's symptoms started at age 55. Best-corrected visual acuity was reduced in both patients (0.2-0.4). Slit-lamp examination in both patients revealed bilateral corneal clouding with gray endothelial lesions; other family members had no ophthalmological signs. Descemet membrane endothelial keratoplasty was performed uneventfully in both patients. Histology showed thickened Descemet membrane and abnormal endothelium resembling epithelial-like cells. Both patients carried the OVOL2 5' untranslated region NM_021220.4.c.-61G>A variant in the heterozygous state. This change was associated with increased promoter activity and was not present in the unaffected members of the family. CONCLUSIONS: The 5' untranslated region mutation c.-61G>A in OVOL2 has been previously found in 1 individual with PPCD1 and reported as a variant of unknown significance because of insufficient evidence supporting its pathogenicity. Identification of the second family with 2 individuals affected by PPCD1 carrying this change, together with functional data, provides further proofs that it is disease-causing.