Genome-wide RNA sequencing of ocular fibroblasts from glaucomatous and normal eyes: Implications for glaucoma management.

Primary open angle glaucoma is a leading cause of visual impairment and blindness which is commonly treated with drugs or laser but may require surgery. Tenon's ocular fibroblasts are involved in wound-healing after glaucoma filtration surgery and may compromise a favourable outcome of glaucoma surgery by contributing to fibrosis. To investigate changes in gene expression and key pathways contributing to the glaucomatous state we performed genome-wide RNA sequencing. Human Tenon's ocular fibroblasts were cultured from normal and glaucomatous human donors undergoing eye surgery (n = 12). mRNA was extracted and RNA-Seq performed on the Illumina platform. Differentially expressed genes were identified using a bioinformatics pipeline consisting of FastQC, STAR, FeatureCounts and edgeR. Changes in biological functions and pathways were determined using Enrichr and clustered using Cytoscape. A total of 5817 genes were differentially expressed between Tenon's ocular fibroblasts from normal versus glaucomatous eyes. Enrichment analysis showed 787 significantly different biological functions and pathways which were clustered into 176 clusters. Tenon's ocular fibroblasts from glaucomatous eyes showed signs of fibrosis with fibroblast to myofibroblast transdifferentiation and associated changes in mitochondrial fission, remodeling of the extracellular matrix, proliferation, unfolded protein response, inflammation and apoptosis which may relate to the pathogenesis of glaucoma or the detrimental effects of topical glaucoma therapies. Altered gene expression in glaucomatous Tenon's ocular fibroblasts may contribute to an unfavourable outcome of glaucoma filtration surgery. This work presents a genome-wide transcriptome of glaucomatous versus normal Tenon's ocular fibroblasts which may identify genes or pathways of therapeutic value to improve surgical outcomes.

The TGFß Induced MicroRNAome of the Trabecular Meshwork.

Primary open-angle glaucoma (POAG) is a progressive optic neuropathy with a complex, multifactorial aetiology. Raised intraocular pressure (IOP) is the most important clinically modifiable risk factor for POAG. All current pharmacological agents target aqueous humour dynamics to lower IOP. Newer therapeutic agents are required as some patients with POAG show a limited therapeutic response or develop ocular and systemic side effects to topical medication. Elevated IOP in POAG results from cellular and molecular changes in the trabecular meshwork driven by increased levels of transforming growth factor ß (TGFß) in the anterior segment of the eye. Understanding how TGFß affects both the structural and functional changes in the outflow pathway and IOP is required to develop new glaucoma therapies that target the molecular pathology in the trabecular meshwork. In this study, we evaluated the effects of TGF-ß1 and -ß2 treatment on miRNA expression in cultured human primary trabecular meshwork cells. Our findings are presented in terms of specific miRNAs (miRNA-centric), but given miRNAs work in networks to control cellular pathways and processes, a pathway-centric view of miRNA action is also reported. Evaluating TGFß-responsive miRNA expression in trabecular meshwork cells will further our understanding of the important pathways and changes involved in the pathogenesis of glaucoma and could lead to the development of miRNAs as new therapeutic modalities in glaucoma.

The Suppression of the Epithelial to Mesenchymal Transition in Prostate Cancer through the Targeting of MYO6 Using MiR-145-5p.

Aberrant expression of miR-145-5p has been observed in prostate cancer where is has been suggested to play a tumor suppressor role. In other cancers, miR-145-5p acts as an inhibitor of epithelial-to-mesenchymal transition (EMT), a key molecular process for tumor progression. However, the interaction between miR-145-5p and EMT remains to be elucidated in prostate cancer. In this paper the link between miR-145-5p and EMT in prostate cancer was investigated using a combination of in silico and in vitro analyses. miR-145-5p expression was significantly lower in prostate cancer cell lines compared to normal prostate cells. Bioinformatic analysis of The Cancer Genome Atlas prostate adenocarcinoma (TCGA PRAD) data showed significant downregulation of miR-145-5p in prostate cancer, correlating with disease progression. Functional enrichment analysis significantly associated miR-145-5p and its target genes with EMT. MYO6, an EMT-associated gene, was identified and validated as a novel target of miR-145-5p in prostate cancer cells. In vitro manipulation of miR-145-5p levels significantly altered cell proliferation, clonogenicity, migration and expression of EMT-associated markers. Additional TCGA PRAD analysis suggested miR-145-5p tumor expression may be useful predictor of disease recurrence. In summary, this is the first study to report that miR-145-5p may inhibit EMT by targeting MYO6 in prostate cancer cells. The findings suggest miR-145-5p could be a useful diagnostic and prognostic biomarker for prostate cancer.

Ocular phenotype and therapeutic interventions in keratitis-ichthyosis-deafness (KID) syndrome.

BACKGROUND: To report ocular manifestations, clinical course, and therapeutic management of patients with molecular genetically confirmed keratitis-ichthyosis-deafness syndrome. METHODS: Four patients, aged 19 to 46, with keratitis-ichthyosis-deafness syndrome from across the UK were recruited for a general and ocular examination and GJB2 (Cx26) mutational analysis. The ocular examination included best-corrected visual acuity, slit-lamp bio-microscopy, and ocular surface assessment. Mutational analysis of the coding region of GJB2 (Cx26) was performed by bidirectional Sanger sequencing. RESULTS: All four individuals had the characteristic systemic features of keratitis-ichthyosis-deafness syndrome. Each patient was found to have a missense mutation, resulting in the substitution of aspartic acid with asparagine at codon 50 (p.D50N). Main ophthalmic features were vascularizing keratopathy, ocular surface disease, hyperkeratotic lid lesions, recurrent epithelial defects, and corneal stromal scarring. One patient had multiple surgical procedures, including superficial keratectomies and lamellar keratoplasty, which failed to prevent severe visual loss. In contrast, oral therapy with ketoconazole stabilized the corneal and skin disease in two other patients with keratitis-ichthyosis-deafness syndrome. The patient who underwent intracorneal bevacizumab injection showed a marked reduction in corneal vascularization following a single application. CONCLUSIONS: Keratitis-ichthyosis-deafness syndrome is a rare ectodermal dysplasia caused by heterozygous mutations in GJB2 (Cx26) with a severe, progressive vascularizing keratopathy. Oral ketoconazole therapy may offer benefit in stabilizing the corneal and skin disease.

Targeting of AKT1 by miR-143-3p Suppresses Epithelial-to-Mesenchymal Transition in Prostate Cancer.

An altered expression of miR-143-3p has been previously reported in prostate cancer where it is purported to play a tumor suppressor role. Evidence from other cancers suggests miR-143-3p acts as an inhibitor of epithelial-to-mesenchymal transition (EMT), a key biological process required for metastasis. However, in prostate cancer the interaction between miR-143-3p and EMT-associated mechanisms remains unclear. Therefore, this paper investigated the link between miR-143-3p and EMT in prostate cancer using in vitro and in silico analyses. PCR detected that miR-143-3p expression was significantly decreased in prostate cancer cell lines compared to normal prostate cells. Bioinformatic analysis of The Cancer Genome Atlas Prostate Adenocarcinoma (TCGA PRAD) data showed a significant downregulation of miR-143-3p in prostate cancer, correlating with pathological markers of advanced disease. Functional enrichment analysis confirmed the significant association of miR-143-3p and its target genes with EMT. The EMT-linked gene AKT1 was subsequently shown to be a novel target of miR-143-3p in prostate cancer cells. The in vitro manipulation of miR-143-3p levels significantly altered the cell proliferation, clonogenicity, migration and expression of EMT-associated markers. Further TCGA PRAD analysis suggested miR-143-3p tumor expression may be a useful predictor of disease recurrence. In summary, this is the first study to report that miR-143-3p overexpression in prostate cancer may inhibit EMT by targeting AKT1. The findings suggest miR-143-3p could be a useful diagnostic and prognostic biomarker for prostate cancer.

Dermatological findings in Rubinstein-Taybi Syndrome.

Rubinstein-Taybi Syndrome is a rare congenital multisystem syndrome inherited in an autosomal dominant pattern caused by mutations in CREBBP and EP300 genes in approximately 60% and 10% respectively. These genes encode two highly evolutionarily conserved, ubiquitously expressed, and homologous lysine-acetyltransferases, that are involved in number of basic cellular activities, such as DNA repair, cell proliferation, growth, differentiation, apoptosis of cells, and tumor suppression. It is mainly characterized by global developmental delay, moderate to severe intellectual disability, postnatal retardation, microcephaly, skeletal anomalies including broad/short, angled thumbs and/or large first toes, short stature, and dysmorphic facial features. There is an increased risk to develop tumors mainly meningiomas and pilomatrixomas, without a clear genotype-phenotype correlation. Although not considered as characteristic manifestations, numerous cutaneous anomalies have also been reported in patients with this entity. Both susceptibility to the formation of keloids and pilomatricomas are the most often associated cutaneous features. In this review, we discuss the genetics, diagnosis, and clinical features in Rubinstein-Taybi Syndrome with a review of the major dermatological manifestations.

Next generation sequencing panel target genes: possible diagnostic tool for ectodermal dysplasia related diseases.

BACKGROUND: Ectodermal dysplasias (EDs) are a large and complex group of disorders affecting the ectoderm-derived organs; the clinical and genetic heterogeneity of these conditions renders an accurate diagnosis more challenging. The aim of this study is to demonstrate the clinical utility of a targeted resequencing panel through enhancing the molecular and clinical diagnosis of EDs. Given the recent developments in gene and protein-based therapies for X-linked hypohidrotic ectodermal dysplasia, there is a re-emerging interest in identifying the genetic basis of EDs and the respective phenotypic presentations, in an aim to facilitate potential treatments for affected families. METHODS: We assessed seventeen individuals, from three unrelated families, who presented with diverse phenotypes suggestive of ED. An extensive multidisciplinary clinical evaluation was performed followed by a targeted exome resequencing panel (including genes that are known to cause EDs). MiSeqTM data software was used, variants with Qscore >30 were accepted. RESULTS: Three different previously reported hemizygous EDA mutations were found in the families. However, a complete genotype-phenotype correlation could not be established, neither in our patients nor in the previously reported patients. CONCLUSIONS: Targeted exome resequencing can provide a rapid and accurate diagnosis of EDs, while further contributing to the existing ED genetic data. Moreover, the identification of the disease-causing mutation in an affected family is crucial for proper genetic counseling and the establishment of a genotype-phenotype correlation which will subsequently provide the affected individuals with a more suitable treatment plan.

A Severe Case of Spondylometaphyseal Dysplasia Algerian Type with Two Mutations in COL2A1.

Spondylometaphyseal dysplasia Algerian type (MIM no.: 184253) is an uncommon autosomal dominant skeletal dysplasia caused by heterozygous mutations in the COL2A1 gene (MIM no.: 120140). In this case based review, we reported a 5-year-old boy with short stature, severe dorsolumbar scoliosis, lumbar hyperlordosis, short trunk, and severe genu valgum . Radiological examination showed platyspondyly, irregular metaphyseal radiolucencies intermingled with radiodensities, and corner fractures. The patient has a c.3275G > A; p.Gly1092Asp mutation in exon 47 of the COL2A1 gene and a variant of unknown significance in c.1366-13C > A in intron 21. This latter sequence variant could partially or completely disrupt the natural splice acceptor site of intron 21/exon 22 in the COL2A1 gene leading to a potential modification of the phenotypic severity.

Erratum: Corrigendum: A Severe Case of Spondylometaphyseal Dysplasia Algerian Type with Two Mutations in COL2A1.

[This corrects the article DOI: 10.1055/s-0041-1732474.].

Extended Overview of Ocular Phenotype with Recent Advances in Hypohidrotic Ectodermal Dysplasia.

The term ectodermal dysplasias (EDs) describes a heterogeneous group of inherited developmental disorders that affect several tissues of ectodermal origin. The most common form of EDs is hypohidrotic ectodermal dysplasia (HED), which is characterized by hypodontia, hypotrichosis, and partial or total eccrine sweat gland deficiency. HED is estimated to affect at least 1 in 17,000 people worldwide. Patients with HED have characteristic facies with periorbital hyperpigmentation, depressed nasal bridge, malar hypoplasia, and absent or sparse eyebrows and eyelashes. The common ocular features of HED include madarosis, trichiasis, and ocular chronic surface disease due to dry eye syndrome, which manifests clinically with discomfort, photophobia, and redness. Dry eye is common in HED and results from a combination of ocular surface defects: mucus abnormalities (abnormal conjunctival mucinous glands), aqueous tear deficiency (abnormalities in the lacrimal gland) and lipid deficiency (due to the partial or total absence of the meibomian glands; modified sebaceous glands with the tarsal plate). Sight-threatening complications result from ocular surface disease, including corneal ulceration and perforation with subsequent corneal scarring and neovascularization. Rare ocular features have been reported and include bilateral or unilateral congenital cataracts, bilateral glaucoma, chorioretinal atrophy and atresia of the nasolacrimal duct. Recognition of the ocular manifestations of HED is required to perform clinical surveillance, instigate supportive and preventative treatment, and manage ocular complications.

MiR-18a-5p Targets Connective Tissue Growth Factor Expression and Inhibits Transforming Growth Factor ß2-Induced Trabecular Meshwork Cell Contractility.

Increased trabecular meshwork (TM) cell and tissue contractility is a driver of the reduced outflow facility and elevation of intraocular pressure (IOP) associated with primary open-angle glaucoma (POAG). Connective tissue growth factor (CTGF) is an established mediator of TM cell contractility, and its expression is increased in POAG due to transforming growth factor ß 2 (TGFß2) signalling. Inhibiting CTGF upregulation using microRNA (miRNA) mimetics could represent a new treatment option for POAG. A combination of in silico predictive tools and a literature review identified a panel of putative CTGF-targeting miRNAs. Treatment of primary human TM cells with 5 ng/mL TGFß2 for 24 h identified miR-18a-5p as a consistent responder, being upregulated in cells from five different human donors. Transfection of primary donor TM cells with 20 nM synthetic miR-18a-5p mimic reduced TGFß2-induced CTGF protein expression, and stable lentiviral-mediated overexpression of this miRNA reduced TGFß2-induced contraction of collagen gels. Together, these findings identify miR-18a-5p as a mediator of the TGFß2 response and a candidate therapeutic agent for glaucoma via its ability to inhibit CTGF-associated increased TM contractility.

Genome-wide transcriptome profiling of human trabecular meshwork cells treated with TGF-ß2.

Glaucoma is a complex neurodegenerative disease resulting in progressive optic neuropathy and is a leading cause of irreversible blindness worldwide. Primary open angle glaucoma (POAG) is the predominant form affecting 65.5 million people globally. Despite the prevalence of POAG and the identification of over 120 glaucoma related genetic loci, the underlaying molecular mechanisms are still poorly understood. The transforming growth factor beta (TGF-ß) signalling pathway is implicated in the molecular pathology of POAG. To gain a better understanding of the role TGF-ß2 plays in the glaucomatous changes to the molecular pathology in the trabecular meshwork, we employed RNA-Seq to delineate the TGF-ß2 induced changes in the transcriptome of normal primary human trabecular meshwork cells (HTM). We identified a significant number of differentially expressed genes and associated pathways that contribute to the pathogenesis of POAG. The differentially expressed genes were predominantly enriched in ECM regulation, TGF-ß signalling, proliferation/apoptosis, inflammation/wound healing, MAPK signalling, oxidative stress and RHO signalling. Canonical pathway analysis confirmed the enrichment of RhoA signalling, inflammatory-related processes, ECM and cytoskeletal organisation in HTM cells in response to TGF-ß2. We also identified novel genes and pathways that were affected after TGF-ß2 treatment in the HTM, suggesting additional pathways are activated, including Nrf2, PI3K-Akt, MAPK and HIPPO signalling pathways. The identification and characterisation of TGF-ß2 dependent differentially expressed genes and pathways in HTM cells is essential to understand the patho-physiology of glaucoma and to develop new therapeutic agents.

Corneal Endothelial Cell Loss in Glaucoma and Glaucoma Surgery and the Utility of Management with Descemet Membrane Endothelial Keratoplasty (DMEK).

The corneal endothelium has a crucial role in maintaining a clear and healthy cornea. Corneal endothelial cell loss occurs naturally with age; however, a diagnosis of glaucoma and surgical intervention for glaucoma can exacerbate a decline in cell number and impairment in morphology. In glaucoma, the mechanisms for this are not well understood and this accelerated cell loss can result in corneal decompensation. Given the high prevalence of glaucoma worldwide, this review aims to explore the abnormalities observed in the corneal endothelium in differing glaucoma phenotypes and glaucoma therapies (medical or surgical including with new generation microinvasive glaucoma surgeries). Descemet membrane endothelial keratoplasty (DMEK) is increasingly being used to manage corneal endothelial failure for glaucoma patients and we aim to review the recent literature evaluating the use of this technique in this clinical scenario.

Prognostic value of miR-21 for prostate cancer: a systematic review and meta-analysis.

Elevated levels of miR-21 expression are associated with many cancers, suggesting it may be a promising clinical biomarker. In prostate cancer (PCa), however, there is still no consensus about the usefulness of miR-21 as an indicator of disease progression. This systematic review and meta-analysis was conducted to investigate the value of miR-21 expression as a prognostic measurement in PCa patients. Medline (Ovid), EMBASE, Web of Science, Scopus and Cochrane Library databases were systematically searched for relevant publications between 2010 to 2021. Studies exploring the relationship between miR-21 expression, PCa prognosis and clinicopathological factors were selected for review. Those reporting hazard ratio (HR) and 95% confidence intervals (CIs) were subject to meta-analyses. Fixed-effect models were employed to calculated pooled HRs and 95% CIs. Risk of bias in each study was assessed using QUIPS tool. Certainty of evidence in each meta-analysis was assessed using GRADE guidelines. A total of 64 studies were included in the systematic review. Of these, 11 were eligible for inclusion in meta-analysis. Meta-analyses revealed that high miR-21 expression was associated with poor prognosis: HR = 1.58 (95% CI = 1.19-2.09) for biochemical recurrence, MODERATE certainty; HR = 1.46 (95% CI = 1.06-2.01) for death, VERY LOW certainty; and HR = 1.26 (95% CI = 0.70-2.27) for disease progression, VERY LOW certainty. Qualitative summary revealed elevated miR-21 expression was significantly positively associated with PCa stage, Gleason score and risk groups. This systematic review and meta-analysis suggests that elevated levels of miR-21 are associated with poor prognosis in PCa patients. miR-21 expression may therefore be a useful prognostic biomarker in this disease.

Replacement of the Trabecular Meshwork Cells-A Way Ahead in IOP Control?

Glaucoma is one of the leading causes of vision loss worldwide, characterised with irreversible optic nerve damage and progressive vision loss. Primary open-angle glaucoma (POAG) is a subset of glaucoma, characterised by normal anterior chamber angle and raised intraocular pressure (IOP). Reducing IOP is the main modifiable factor in the treatment of POAG, and the trabecular meshwork (TM) is the primary site of aqueous humour outflow (AH) and the resistance to outflow. The structure and the composition of the TM are key to its function in regulating AH outflow. Dysfunction and loss of the TM cells found in the natural ageing process and more so in POAG can cause abnormal extracellular matrix (ECM) accumulation, increased TM stiffness, and increased IOP. Therefore, repair or regeneration of TM's structure and function is considered as a potential treatment for POAG. Cell transplantation is an attractive option to repopulate the TM cells in POAG, but to develop a cell replacement approach, various challenges are still to be addressed. The choice of cell replacement covers autologous or allogenic approaches, which led to investigations into TM progenitor cells, induced pluripotent stem cells (iPSCs), and mesenchymal stem cells (MSCs) as potential stem cell source candidates. However, the potential plasticity and the lack of definitive cell markers for the progenitor and the TM cell population compound the biological challenge. Morphological and differential gene expression of TM cells located within different regions of the TM may give rise to different cell replacement or regenerative approaches. As such, this review describes the different approaches taken to date investigating different cell sources and their differing cell isolation and differentiation methodologies. In addition, we highlighted how these approaches were evaluated in different animal and ex vivo model systems and the potential of these methods in future POAG treatment.

Chemical Cross-Linking of Corneal Tissue to Reduce Progression of Loss of Sight in Patients With Keratoconus.

Purpose: We aimed to develop a novel chemical cross-linker treatment for keratoconus by reacting dicarboxylic acid spacer molecules and amine functional groups on protein structure of the tissue using carbodi-imide chemistry. We propose this as an alternative to conventional cross-linking treatment for keratoconus. Methods: The study involved optimization of the cross-linker formulation. Mechanical stiffness of ex vivo porcine and human corneas after application of the cross-linker was measured. Histochemical analysis was performed to record changes in gross morphology after cross-linker treatment on ex vivo porcine and human and in vivo rabbit corneas. Terminal deoxynucleotidyl transferase-mediated dUTP-X nick-end-labeling (TUNEL) staining was performed to study apoptotic effects of cross-linker. Cytotoxicity potential of cross-linker was evaluated by studying explant cultures for cellular outgrowth and immunostaining assays on porcine and human corneas after treatment. Results: We demonstrated a clinically relevant increase in stiffness in ex vivo experiments using porcine and human cornea without removal of corneal epithelium. Histological analysis showed no change in gross morphology of cornea and no evidence of apoptosis. In vivo treatment of rabbit eyes demonstrated initial thinning of corneal epithelium that recovered after seven days although with abnormal regularity of cells. Cellular outgrowth from corneal explant cultures after treatment further confirmed cell survival after treatment. Conclusions: This chemical cross-linking of corneal tissue has potential advantages over current therapeutic options including lower cytotoxicity to stromal cells than ultraviolet A treatment. Translational Relevance: The cross-linker has potential to become a treatment for keratoconus because it overcomes the need for procedures using specialized equipment and ensures accessibility to large populations.

An Intraocular Pressure Polygenic Risk Score Stratifies Multiple Primary Open-Angle Glaucoma Parameters Including Treatment Intensity.

PURPOSE: To examine the combined effects of common genetic variants associated with intraocular pressure (IOP) on primary open-angle glaucoma (POAG) phenotype using a polygenic risk score (PRS) stratification. DESIGN: Cross-sectional study. PARTICIPANTS: For the primary analysis, we examined the glaucoma phenotype of 2154 POAG patients enrolled in the Australian and New Zealand Registry of Advanced Glaucoma, including patients recruited from the United Kingdom. For replication, we examined an independent cohort of 624 early POAG patients. METHODS: Using IOP genome-wide association study summary statistics, we developed a PRS derived solely from IOP-associated variants and stratified POAG patients into 3 risk tiers. The lowest and highest quintiles of the score were set as the low- and high-risk groups, respectively, and the other quintiles were set as the intermediate risk group. MAIN OUTCOME MEASURES: Clinical glaucoma phenotype including maximum recorded IOP, age at diagnosis, number of family members affected by glaucoma, cup-to-disc ratio, visual field mean deviation, and treatment intensity. RESULTS: A dose-response relationship was found between the IOP PRS and the maximum recorded IOP, with the high genetic risk group having a higher maximum IOP by 1.7 mmHg (standard deviation [SD], 0.62 mmHg) than the low genetic risk group (P = 0.006). Compared with the low genetic risk group, the high genetic risk group had a younger age of diagnosis by 3.7 years (SD, 1.0 years; P < 0.001), more family members affected by 0.46 members (SD, 0.11 members; P < 0.001), and higher rates of incisional surgery (odds ratio, 1.5; 95% confidence interval, 1.1-2.0; P = 0.007). No statistically significant difference was found in mean deviation. We further replicated the maximum IOP, number of family members affected by glaucoma, and treatment intensity (number of medications) results in the early POAG cohort (P ≤ 0.01). CONCLUSIONS: The IOP PRS was correlated positively with maximum IOP, disease severity, need for surgery, and number of affected family members. Genes acting via IOP-mediated pathways, when considered in aggregate, have clinically important and reproducible implications for glaucoma patients and their close family members.

A Venezuelan Case of Schmid-Type Metaphyseal Chondrodysplasia with a Novel Mutation in COL10A1.

Schmid-type metaphyseal chondrodysplasia (MIM 156500) is an uncommon autosomal dominant skeletal dysplasia caused by heterozygous mutations in the COL10A1 gene (MIM 120110) encoding the α1(X) chains of type X collagen. We report an 8-year-old girl with waddling gait, short stature, mild dorsal scoliosis, coxa vara, short lower limbs, bowing of the femurs, genu varum, and metaphyseal fraying and splaying, who is a carrier of a novel heterozygous 2-bp (c.1894_1895dupTA; p.Leu633Thrfs*45) duplication in exon 3 of the COL10A1 gene.