Combining a prioritization strategy and functional studies nominates 5’UTR variants underlying inherited retinal disease

BACKGROUND: 5’ untranslated regions (5’UTRs) are essential modulators of protein translation. Predicting the impact of 5’UTR variants is challenging and rarely performed in routine diagnostics. Here, we present a combined approach of a comprehensive prioritization strategy and functional assays to evaluate 5’UTR variation in two large cohorts of patients with inherited retinal diseases (IRDs). METHODS: We performed an isoform-level re-analysis of retinal RNA-seq data to identify the protein-coding transcripts of 378 IRD genes with highest expression in retina. We evaluated the coverage of their 5’UTRs by different whole exome sequencing (WES) kits. The selected 5’UTRs were analyzed in whole genome sequencing (WGS) and WES data from IRD sub-cohorts from the 100,000 Genomes Project (n = 2397 WGS) and an in-house database (n = 1682 WES), respectively. Identified variants were annotated for 5’UTR-relevant features and classified into seven categories based on their predicted functional consequence. We developed a variant prioritization strategy by integrating population frequency, specific criteria for each category, and family and phenotypic data. A selection of candidate variants underwent functional validation using diverse approaches. RESULTS: Isoform-level re-quantification of retinal gene expression revealed 76 IRD genes with a non-canonical retina-enriched isoform, of which 20 display a fully distinct 5’UTR compared to that of their canonical isoform. Depending on the probe design, 3–20% of IRD genes have 5’UTRs fully captured by WES. After analyzing these regions in both cohorts, we prioritized 11 (likely) pathogenic variants in 10 genes (ARL3, MERTK, NDP, NMNAT1, NPHP4, PAX6, PRPF31, PRPF4, RDH12, RD3), of which 7 were novel. Functional analyses further supported the pathogenicity of three variants. Mis-splicing was demonstrated for the PRPF31:c.-9+1G>T variant. The MERTK:c.-125G>A variant, overlapping a transcriptional start site, was shown to significantly reduce both luciferase mRNA levels and activity. The RDH12:c.-123C>T variant was found in cis with the hypomorphic RDH12:c.701G>A (p.Arg234His) variant in 11 patients. This 5’UTR variant, predicted to introduce an upstream open reading frame, was shown to result in reduced RDH12 protein but unaltered mRNA levels. CONCLUSIONS: This study demonstrates the importance of 5’UTR variants implicated in IRDs and provides a systematic approach for 5’UTR annotation and validation that is applicable to other inherited diseases

Impact of biological agents on postsurgical complications in inflammatory bowel disease: A multicentre study of Geteccu

Background: The impact of biologics on the risk of postoperative complications (PC) in inflammatory bowel disease (IBD) is still an ongoing debate. This lack of evidence is more relevant for ustekinumab and vedolizumab. Aims: To evaluate the impact of biologics on the risk of PC. Methods: A retrospective study was performed in 37 centres. Patients treated with biologics within 12 weeks before surgery were considered “exposed”. The impact of the exposure on the risk of 30-day PC and the risk of infections was assessed by logistic regression and propensity score-matched analysis. Results: A total of 1535 surgeries were performed on 1370 patients. Of them, 711 surgeries were conducted in the exposed cohort (584 anti-TNF, 58 vedolizumab and 69 ustekinumab). In the multivariate analysis, male gender (OR: 1.5; 95% CI: 1.2–2.0), urgent surgery (OR: 1.6; 95% CI: 1.2–2.2), laparotomy approach (OR: 1.5; 95% CI: 1.1–1.9) and severe anaemia (OR: 1.8; 95% CI: 1.3–2.6) had higher risk of PC, while academic hospitals had significantly lower risk. Exposure to biologics (either anti-TNF, vedolizumab or ustekinumab) did not increase the risk of PC (OR: 1.2; 95% CI: 0.97–1.58), although it could be a risk factor for postoperative infections (OR 1.5; 95% CI: 1.03–2.27). Conclusions: Preoperative administration of biologics does not seem to be a risk factor for overall PC, although it may be so for postoperative infections

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

Integrated Coastal Management in Cuba: Progress and Challenges in the 2009-2019 Stage

Hace casi una década comenzaron esfuerzos regionales conjuntos por sintetizar la situación del Manejo Costero Integrado y las Políticas Públicas en Iberoamérica. Este capítulo pretende analizar, diez años después, cuánto se ha avanzado en Cuba en la implementación de la gestión costera integrada. En Cuba, un archipiélago del Caribe donde todo está estrechamente ligado a su carácter costero y marino, los asuntos de gestión integrada de zonas costeras, han ganado en atención y se encuentran hoy en una máxima prioridad en las políticas y marcos legales del país. El presente reporte pone en evidencia que en esta etapa transcurrida se ha perfeccionado el marco político-normativo, se ha elevado el número de instituciones que se concentran en evaluar temas de gestión costera, y ha crecido el número de proyectos relacionados con esta temática, así como la actividad de formación de postgrados y la educación comunitaria. Para el país han sido muy relevantes la adopción e implementación del Plan Nacional de Desarrollo Económico y Social hasta el 2030 (PNDES, 2030) y Plan de Estado para el Enfrentamiento al Cambio Climático (Tarea Vida). Ambos planes promueven el análisis de los problemas típicos de las zonas costeras, a la vez que se enfocan en la búsqueda de soluciones. Así mismo, este capítulo identifica los nuevos retos de la gestión de la zona costera en Cuba y esboza las posibles nuevas acciones a emprender y los asuntos que requieren análisis y tratamientos más profundos.Regional efforts jointly done for synthesizing the situation about Integrated Coastal Management and Public Politics in Iberoamerica started almost a decade ago. Ten years later, the present Chapter is aimed to analyze advances regarding implementation of integrated coastal management in Cuba. Due to its condition of being a Caribbean archipelago, everything in Cuba is strongly linked to its coastal and marine characteristics; issues about integrated coastal zone management have received greater attention, becoming, at present, an item of maximum priority among the political and legal frameworks of the country. Improvement to the political-normative framework regarding integrated coastal management, increase in the number of institutions devoted to assess themes related to that topic, as well as in the number of projects, activities for Postgraduate formation and communitarian education regarding the topic, are all widely shown in the present Chapter. Approval and implementation of the National Plan for Social and Economic Development up to 2030 (NPSED, 2030), and of the State Plan to face the Climate Change (“Life Task” in Sp. “Tarea Vida”) have been irrelevant for the country. Both Plans are aimed to analyze typical problems of the coastal zone, focusing on the search of solutions. New challenges for coastal zone management in Cuba are also described in the present Chapter, where possibilities of putting into practice new actions, as well as items, which require deeper analysis, are also given

Multi-omics approach dissects cis-regulatory mechanisms underlying North Carolina macular dystrophy, a retinal enhanceropathy

North Carolina macular dystrophy (NCMD) is a rare autosomal-dominant disease affecting macular development. The disease is caused by non-coding single-nucleotide variants (SNVs) in two hotspot regions near PRDM13 and by duplications in two distinct chromosomal loci, overlapping DNase I hypersensitive sites near either PRDM13 or IRX1. To unravel the mechanisms by which these variants cause disease, we first established a genome-wide multi-omics retinal database, RegRet. Integration of UMI-4C profiles we generated on adult human retina then allowed fine-mapping of the interactions of the PRDM13 and IRX1 promoters and the identification of eighteen candidate cis-regulatory elements (cCREs), the activity of which was investigated by luciferase and Xenopus enhancer assays. Next, luciferase assays showed that the non-coding SNVs located in the two hotspot regions of PRDM13 affect cCRE activity, including two NCMD-associated non-coding SNVs that we identified herein. Interestingly, the cCRE containing one of these SNVs was shown to interact with the PRDM13 promoter, demonstrated in vivo activity in Xenopus, and is active at the developmental stage when progenitor cells of the central retina exit mitosis, suggesting that this region is a PRDM13 enhancer. Finally, mining of single-cell transcriptional data of embryonic and adult retina revealed the highest expression of PRDM13 and IRX1 when amacrine cells start to synapse with retinal ganglion cells, supporting the hypothesis that altered PRDM13 or IRX1 expression impairs interactions between these cells during retinogenesis. Overall, this study provides insight into the cis-regulatory mechanisms of NCMD and supports that this condition is a retinal enhanceropathy

Cooperative Segmental Motions in Ethyl Acrylate/Triethylene Glycol Dimethacrylate Copolymer Networks Studied by Dielectric Techniques

The molecular dynamics of ethyl acrylate/triethylene glycol dimethacrylate (EA/TrEGDMA) copolymer networks on the overall composition range were investigated thoroughly by employing dielectric techniques: thermally stimulated depolarization currents (TSDC) and dielectric relaxation spectroscopy (DRS). Furthermore, di¿erential scanning calorimetry (DSC) was performed in order to investigate the thermal glass transition of these copolymers. The results show that, for low content of the TrEGDMA component, the overall dielectric behavior of the copolymers is dictated by the dynamics of PEA component, exhibiting the ¿cop relaxation process which is strongly a¿ected by the TrEGDMA moieties becoming broader and shifting to higher temperatures with increasing cross-linker (TrEGDMA) content. For the copolymer networks rich in TrEGDMA (w TE g 40 wt %), the overall dielectric behavior indicates that the dynamics of the dimethacrylate component dominates, resembling that of poly(n-alkyl methacrylates) in the sense that the temperature dependence of molecular relaxation processes presents a merging region where the local relaxation process of TrEGDMA,ßPTE, merges with the more cooperative¿cop process of the copolymers into an¿ßcop locally cooperative relaxation process. By increasing the EA content, the strength of ¿cop relaxation process enhances and simultaneously the merging region shifts to lower temperatures. The cooperative motions are signi¿cantly suppressed in the copolymers with TrEGDMA contents higher that 80 wt %. The copolymers present enhanced spatial heterogeneity the more TrEGDMA content, whereas no dynamic heterogeneity was detected.A.T.S. thanks the Department of Physics, National Technical University of Athens, for the financial support of this work.Stathopoulos, AT.; Kyritsis, A.; Gallego Ferrer, G.; Gómez Ribelles, JL.; Christodoulides, C.; Pissis, P. (2011). Cooperative Segmental Motions in Ethyl Acrylate/Triethylene Glycol Dimethacrylate Copolymer Networks Studied by Dielectric Techniques. Macromolecules. 44:8233-8244. doi:10.1021/ma201755yS823382444

Multi-omics profiling, in vitro and in vivo enhancer assays dissect the cis-regulatory mechanisms underlying North Carolina macular dystrophy, a retinal enhanceropathy

North Carolina macular dystrophy (NCMD) is a rare autosomal dominant disease affecting macular development. With the identification of non-coding single nucleotide variants (SNVs) near PRDM13 and duplications overlapping a DNase I hypersensitive site (DHS) near PRDM13 or IRX1 as its underlying genetic cause, we hypothesize that NCMD is a retinal enhanceropathy. Here we aim to provide insight into the cis-regulatory mechanisms of NCMD by integrating multi-omics profiling of human retina with in vitro and in vivo enhancer assays. First, we established RegRet (http://genome.ucsc.edu/s/stvdsomp/RegRet), a genome-wide multi-omics retinal database. Next, UMI-4C profiling was performed on adult human retina to fine-map chromatin interactions of cis-regulatory elements (CREs) with the PRDM13 and IRX1 promoters. Multi-omics analysis including the UMI-4C data revealed sixteen candidate CREs (cCREs), seven for the PRDM13 and nine for the IRX1 region. Subsequently, the activity of cCREs was investigated by in vitro luciferase assays and by in vivo enhancer assays in Xenopus laevis and tropicalis. Four cCREs showed in vivo eye- and brain-specific activity in Xenopus. Furthermore, we expanded the genetic architecture of NCMD with two novel non-coding SNVs (V15, V16) with a likely effect on PRDM13 regulation. Luciferase assays showed that the non-coding SNVs that are located in the two hotspot regions of PRDM13 have an effect on cCRE activity. Interestingly, cCRE4 in which V16 is located was shown to interact with the PRDM13 promoter and demonstrated in vivo activity in Xenopus. This cCRE is active at a specific developmental stage (d103) compatible with the timepoint when retinal progenitor cells of the central retina exit mitosis. Mining of single-cell (sc) transcriptional data of embryonic and adult retina revealed the highest expression of PRDM13 and IRX1 when amacrine cells start to emerge and begin to synapse with retinal ganglion cells. This supports the hypothesis that altered PRDM13 or IRX1 expression impairs synaptic interactions between amacrine and ganglion cells during retinogenesis. Overall, this study gained insight into the cis-regulatory mechanisms of NCMD and supports that NCMD is a retinal enhanceropathy