Detection of Variants in 15 Genes in 87 Unrelated Chinese Patients with Leber Congenital Amaurosis

BACKGROUND: Leber congenital amaurosis (LCA) is the earliest onset and most severe form of hereditary retinal dystrophy. So far, full spectrum of variations in the 15 genes known to cause LCA has not been systemically evaluated in East Asians. Therefore, we performed comprehensive detection of variants in these 15 genes in 87 unrelated Han Chinese patients with LCA. METHODOLOGY/PRINCIPAL FINDINGS: The 51 most frequently mutated exons and introns in the 15 genes were selected for an initial scan using cycle sequencing. All the remaining exons in 11 of the 15 genes were subsequently sequenced. Fifty-three different variants were identified in 44 of the 87 patients (50.6%), involving 78 of the 88 alleles (11 homozygous and 56 heterozygous variants). Of the 53 variants, 35 (66%) were novel pathogenic mutations. In these Chinese patients, variants in GUCY2D are the most common cause of LCA (16.1% cases), followed by CRB1 (11.5%), RPGRIP1 (8%), RPE65 (5.7%), SPATA7 (4.6%), CEP290 (4.6%), CRX (3.4%), LCA5 (2.3%), MERTK (2.3%), AIPL1 (1.1%), and RDH12 (1.1%). This differs from the variation spectrum described in other populations. An initial scan of 55 of 215 PCR amplicons, including 214 exons and 1 intron, detected 83.3% (65/78) of the mutant alleles ultimately found in these 87 patients. In addition, sequencing only 9 exons would detect over 50% of the identified variants and require less than 5% of the labor and cost of comprehensive sequencing for all exons. CONCLUSIONS/SIGNIFICANCE: Our results suggest that specific difference in the variation spectrum found in LCA patients from the Han Chinese and other populations are related by ethnicity. Sequencing exons in order of decreasing risk is a cost-effective way to identify causative mutations responsible for LCA, especially in the context of genetic counseling for individual patients in a clinical setting

Electrospun nanofibrous meshes cultured with Wharton’s Jelly Stem Cell: an alternative for cartilage regeneration, without the need of growth factors

Many efforts are being directed worldwide to the treatment of OA-focal lesions. The majority of those efforts comprise either the refinement of surgical techniques or combinations of biomaterials with various autologous cells. Herein, we tested electrospun polycaprolactone (PCL) nanofibrous meshes for cartilage tissue engineering. For that, articular chondrocytes (hACs) isolated from human osteoarthritic joints and Whartonâ s Jelly Stem Cells (hWJSCs) are cultured on electrospun nanofiber meshes, without adding external growth factors. We observed higher glycosaminoglycans production and higher overexpression of cartilage-related genes from hWJSCs cultured with basal medium, when compared to hACs isolated from osteoarthritic joints. Moreover, the presence of sulfated proteoglycans and collagen type II is observed on both types of cell cultures. We believe that this effect is due to either the electrospun nanofibers topography or the intrinsic chondrogenic differentiation potential of hWJSCs. Therefore, we propose the electrospun nanofibrous scaffolds in combination with hWJSCs as a viable alternative to the commercial membranes used in autologous chondrogenic regeneration approaches.The authors thank the Portuguese Foundation for Science and Technology for the Post-Doc grant of Marta Alves da Silva (SFRH/BPD/73322/2010, financed by POPH QREN Tipologia 4.1 – Advanced Formation, co-financed by Fundo Social Europeu and MEC national funds). This work was supported by the project SPARTAN (PTDC/CTM-BIO/4388/2014) FCT/MEC with PIDDAC funds. It was also partly supported by the POLARIS (FP7-REGPOT-2012-2013-1) and the Project “New methodologies for the isolation and control of stem cells differentiation using advanced culturing conditions and/or nanomaterials” (RL2 SCN NORTE-01-0124-FEDER-000018), co-financed by North Portugal Regional Operational Programme (ON.2 – O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF).info:eu-repo/semantics/publishedVersio

661W photoreceptor cell line as a cell model for studying retinal ciliopathies

Copyright © 2019 Wheway, Nazlamova, Turner and Cross. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. The retina contains several ciliated cell types, including the retinal pigment epithelium (RPE) and photoreceptor cells. The photoreceptor cilium is one of the most highly modified sensory cilia in the human body. The outer segment of the photoreceptor is a highly elaborate primary cilium, containing stacks or folds of membrane where the photopigment molecules are located. Perhaps unsurprisingly, defects in cilia often lead to retinal phenotypes, either as part of syndromic conditions involving other organs, or in isolation in the so-called retinal ciliopathies. The study of retinal ciliopathies has been limited by a lack of retinal cell lines. RPE1 retinal pigment epithelial cell line is commonly used in such studies, but the existence of a photoreceptor cell line has largely been neglected in the retinal ciliopathy field. 661W cone photoreceptor cells, derived from mouse, have been widely used as a model for studying macular degeneration, but not described as a model for studying retinal ciliopathies such as retinitis pigmentosa. Here, we characterize the 661W cell line as a model for studying retinal ciliopathies. We fully characterize the expression profile of these cells, using whole transcriptome RNA sequencing, and provide this data on Gene Expression Omnibus for the advantage of the scientific community. We show that these cells express the majority of markers of cone cell origin. Using immunostaining and confocal microscopy, alongside scanning electron microscopy, we show that these cells grow long primary cilia, reminiscent of photoreceptor outer segments, and localize many cilium proteins to the axoneme, membrane and transition zone. We show that siRNA knockdown of cilia genes Ift88 results in loss of cilia, and that this can be assayed by high-throughput screening. We present evidence that the 661W cell line is a useful cell model for studying retinal ciliopathies

A clinical and molecular characterisation of CRB1-associated maculopathy

To date, over 150 disease-associated variants in CRB1 have been described, resulting in a range of retinal disease phenotypes including Leber congenital amaurosis and retinitis pigmentosa. Despite this, no genotype–phenotype correlations are currently recognised. We performed a retrospective review of electronic patient records to identify patients with macular dystrophy due to bi-allelic variants in CRB1. In total, seven unrelated individuals were identified. The median age at presentation was 21 years, with a median acuity of 0.55 decimalised Snellen units (IQR = 0.43). The follow-up period ranged from 0 to 19 years (median = 2.0 years), with a median final decimalised Snellen acuity of 0.65 (IQR = 0.70). Fundoscopy revealed only a subtly altered foveal reflex, which evolved into a bull’s-eye pattern of outer retinal atrophy. Optical coherence tomography identified structural changes—intraretinal cysts in the early stages of disease, and later outer retinal atrophy. Genetic testing revealed that one rare allele (c.498_506del, p.(Ile167_Gly169del)) was present in all patients, with one patient being homozygous for the variant and six being heterozygous. In trans with this, one variant recurred twice (p.(Cys896Ter)), while the four remaining alleles were each observed once (p.(Pro1381Thr), p.(Ser478ProfsTer24), p.(Cys195Phe) and p.(Arg764Cys)). These findings show that the rare CRB1 variant, c.498_506del, is strongly associated with localised retinal dysfunction. The clinical findings are much milder than those observed with bi-allelic, loss-of-function variants in CRB1, suggesting this in-frame deletion acts as a hypomorphic allele. This is the most prevalent disease-causing CRB1 variant identified in the non-Asian population to date

Phylogenetic relationships of cone snails endemic to Cabo Verde based on mitochondrial genomes

Background: Due to their great species and ecological diversity as well as their capacity to produce hundreds of different toxins, cone snails are of interest to evolutionary biologists, pharmacologists and amateur naturalists alike. Taxonomic identification of cone snails still relies mostly on the shape, color, and banding patterns of the shell. However, these phenotypic traits are prone to homoplasy. Therefore, the consistent use of genetic data for species delimitation and phylogenetic inference in this apparently hyperdiverse group is largely wanting. Here, we reconstruct the phylogeny of the cones endemic to Cabo Verde archipelago, a well-known radiation of the group, using mitochondrial (mt) genomes. Results: The reconstructed phylogeny grouped the analyzed species into two main clades, one including Kalloconus from West Africa sister to Trovaoconus from Cabo Verde and the other with a paraphyletic Lautoconus due to the sister group relationship of Africonus from Cabo Verde and Lautoconus ventricosus from Mediterranean Sea and neighboring Atlantic Ocean to the exclusion of Lautoconus endemic to Senegal (plus Lautoconus guanche from Mauritania, Morocco, and Canary Islands). Within Trovaoconus, up to three main lineages could be distinguished. The clade of Africonus included four main lineages (named I to IV), each further subdivided into two monophyletic groups. The reconstructed phylogeny allowed inferring the evolution of the radula in the studied lineages as well as biogeographic patterns. The number of cone species endemic to Cabo Verde was revised under the light of sequence divergence data and the inferred phylogenetic relationships. Conclusions: The sequence divergence between continental members of the genus Kalloconus and island endemics ascribed to the genus Trovaoconus is low, prompting for synonymization of the latter. The genus Lautoconus is paraphyletic. Lautoconus ventricosus is the closest living sister group of genus Africonus. Diversification of Africonus was in allopatry due to the direct development nature of their larvae and mainly triggered by eustatic sea level changes during the Miocene-Pliocene. Our study confirms the diversity of cone endemic to Cabo Verde but significantly reduces the number of valid species. Applying a sequence divergence threshold, the number of valid species within the sampled Africonus is reduced to half.Spanish Ministry of Science and Innovation [CGL2013-45211-C2-2-P, CGL2016-75255-C2-1-P, BES-2011-051469, BES-2014-069575, Doctorado Nacional-567]info:eu-repo/semantics/publishedVersio

Kinome rewiring reveals AURKA limits PI3K-pathway inhibitor efficacy in breast cancer.

Dysregulation of the PI3K-AKT-mTOR signaling network is a prominent feature of breast cancers. However, clinical responses to drugs targeting this pathway have been modest, possibly because of dynamic changes in cellular signaling that drive resistance and limit drug efficacy. Using a quantitative chemoproteomics approach, we mapped kinome dynamics in response to inhibitors of this pathway and identified signaling changes that correlate with drug sensitivity. Maintenance of AURKA after drug treatment was associated with resistance in breast cancer models. Incomplete inhibition of AURKA was a common source of therapy failure, and combinations of PI3K, AKT or mTOR inhibitors with the AURKA inhibitor MLN8237 were highly synergistic and durably suppressed mTOR signaling, resulting in apoptosis and tumor regression in vivo. This signaling map identifies survival factors whose presence limits the efficacy of targeted therapies and reveals new drug combinations that may unlock the full potential of PI3K-AKT-mTOR pathway inhibitors in breast cancer

Mutation screening of retinal dystrophy patients by targeted capture from tagged pooled DNAs and next generation sequencing.

Purpose: Retinal dystrophies are genetically heterogeneous, resulting from mutations in over 200 genes. Prior to the development of massively parallel sequencing, comprehensive genetic screening was unobtainable for most patients. Identifying the causative genetic mutation facilitates genetic counselling, carrier testing and prenatal/pre-implantation diagnosis, and often leads to a clearer prognosis. In addition, in a proportion of cases, when the mutation is known treatment can be optimised and patients are eligible for enrolment into clinical trials for gene-specific therapies. Methods: Patient genomic DNA was sheared, tagged and pooled in batches of four samples, prior to targeted capture and next generation sequencing. The enrichment reagent was designed against genes listed on the RetNet database (July 2010). Sequence data were aligned to the human genome and variants were filtered to identify potential pathogenic mutations. These were confirmed by Sanger sequencing. Results: Molecular analysis of 20 DNAs from retinal dystrophy patients identified likely pathogenic mutations in 12 cases, many of them known and/or confirmed by segregation. These included previously described mutations in ABCA4 (c.6088C>T,p.R2030*; c.5882G>A,p.G1961E), BBS2 (c.1895G>C,p.R632P), GUCY2D (c.2512C>T,p.R838C), PROM1 (c.1117C>T,p.R373C), RDH12 (c.601T>C,p.C201R; c.506G>A,p.R169Q), RPGRIP1 (c.3565C>T,p.R1189*) and SPATA7 (c.253C>T,p.R85*) and new mutations in ABCA4 (c.3328+1G>C), CRB1 (c.2832_2842+23del), RP2 (c.884-1G>T) and USH2A (c.12874A>G,p.N4292D). Conclusions: Tagging and pooling DNA prior to targeted capture of known retinal dystrophy genes identified mutations in 60% of cases. This relatively high success rate may reflect enrichment for consanguineous cases in the local Yorkshire population, and the use of multiplex families. Nevertheless this is a promising high throughput approach to retinal dystrophy diagnostics

Area-level deprivation and overall and cause-specific mortality: 12 years' observation on British women and systematic review of prospective studies.

BACKGROUND: Prospective studies have suggested a negative impact of area deprivation on overall mortality, but its effect on cause-specific mortality and the mechanisms that account for this association remain unclear. We investigate the association of area deprivation, using Index of Multiple deprivation (IMD), with overall and cause-specific mortality, contextualising findings within a systematic review. METHODS AND FINDINGS: We used data from 4,286 women from the British Women's Heart Health Study (BWHHS) recruited at 1999-2001 to examine the association of IMD with overall and cause-specific mortality using Cox regression models. One standard deviation (SD) increase in the IMD score had a hazard ratio (HR) of 1.21 (95% CI: 1.13-1.30) for overall mortality after adjustment for age and lifecourse individual deprivation, which was attenuated to 1.15 (95% CI: 1.04-1.26) after further inclusion of mediators (health behaviours, biological factors and use of statins and blood pressure-lowering medications). A more pronounced association was observed for respiratory disease and vascular deaths. The meta-analysis, based on 20 published studies plus the BWHHS (n=21), yielded a summary relative risk (RR) of 1.15 (95% CI: 1.11-1.19) for area deprivation (top [least deprived; reference] vs. bottom tertile) with overall mortality in an age and sex adjusted model, which reduced to 1.06 (95% CI: 1.04-1.08) in a fully adjusted model. CONCLUSIONS: Health behaviours mediate the association between area deprivation and cause-specific mortality. Efforts to modify health behaviours may be more successful if they are combined with measures that tackle area deprivation