Pharmacological EZH2 inhibition combined with retinoic acid treatment promotes differentiation and apoptosis in rhabdomyosarcoma cells.

BACKGROUND: Rhabdomyosarcomas (RMS) are predominantly paediatric sarcomas thought to originate from muscle precursor cells due to impaired myogenic differentiation. Despite intensive treatment, 5-year survival for patients with advanced disease remains low (< 30%), highlighting a need for novel therapies to improve outcomes. Differentiation therapeutics are agents that induce differentiation of cancer cells from malignant to benign. The histone methyltransferase, Enhancer of Zeste Homolog 2 (EZH2) suppresses normal skeletal muscle differentiation and is highly expressed in RMS tumours. RESULTS: We demonstrate combining inhibition of the epigenetic modulator EZH2 with the differentiating agent retinoic acid (RA) is more effective at reducing cell proliferation in RMS cell lines than single agents alone. In PAX3-FOXO1 positive RMS cells this is due to an RA-driven induction of the interferon pathway resulting in apoptosis. In fusion negative RMS, combination therapy led to an EZH2i-driven upregulation of myogenic signalling resulting in differentiation. In both subtypes, EZH2 is significantly associated with enrichment of trimethylated lysine 27 on histone 3 (H3K27me3) in genes that are downregulated in untreated RMS cells and upregulated with EZH2 inhibitor treatment. These results provide insight into the mechanism that drives the anti-cancer effect of the EZH2/RA single agent and combination treatment and indicate that the reduction of EZH2 activity combined with the induction of RA signalling represents a potential novel therapeutic strategy to treat both subtypes of RMS. CONCLUSIONS: The results of this study demonstrate the potential utility of combining EZH2 inhibitors with differentiation agents for the treatment of paediatric rhabdomyosarcomas. As EZH2 inhibitors are currently undergoing clinical trials for adult and paediatric solid tumours and retinoic acid differentiation agents are already in clinical use this presents a readily translatable potential therapeutic strategy. Moreover, as inhibition of EZH2 in the poor prognosis FPRMS subtype results in an inflammatory response, it is conceivable that this strategy may also synergise with immunotherapies for a more effective treatment in these patients.

Review: Targeting EZH2 in neuroblastoma.

Neuroblastoma is one of the commonest extra-cranial pediatric tumors, and accounts for over 15% of all childhood cancer mortality. Risk stratification for children with neuroblastoma is based on age, stage, histology, and tumor cytogenetics. The majority of patients are considered to have high-risk neuroblastoma, for which the long-term survival is less than 50%. Current treatments combine surgical resection, chemotherapy, stem cell transplantation, radiotherapy, anti-GD2 based immunotherapy as well as the differentiating agent isotretinoin. Despite the intensive multimodal therapies applied, there are high relapse rates, and recurrent disease is often resistant to further therapy. Enhancer of Zeste Homolog 2 (EZH2), a catalytic subunit of Polycomb Repressive Complex 2 (PRC2), is a histone methyltransferase that represses transcription through trimethylation of lysine residue K27 on histone H3 (H3K27me3). It is responsible for epigenetic repression of transcription, making EZH2 an essential regulator for cell differentiation. Overexpression of EZH2 has been shown to promote tumorigenesis, cancer cell proliferation and prevent tumor cells from differentiating in a number of cancers. Therefore, research has been ongoing for the past decade, developing treatments that target EZH2 in neuroblastoma. This review summarises the role of EZH2 in neuroblastoma and evaluates the latest research findings on the therapeutic potential of targeting EZH2 in the treatment of neuroblastoma.

Whole-genome sequencing of chronic lymphocytic leukemia identifies subgroups with distinct biological and clinical features.

The value of genome-wide over targeted driver analyses for predicting clinical outcomes of cancer patients is debated. Here, we report the whole-genome sequencing of 485 chronic lymphocytic leukemia patients enrolled in clinical trials as part of the United Kingdom's 100,000 Genomes Project. We identify an extended catalog of recurrent coding and noncoding genetic mutations that represents a source for future studies and provide the most complete high-resolution map of structural variants, copy number changes and global genome features including telomere length, mutational signatures and genomic complexity. We demonstrate the relationship of these features with clinical outcome and show that integration of 186 distinct recurrent genomic alterations defines five genomic subgroups that associate with response to therapy, refining conventional outcome prediction. While requiring independent validation, our findings highlight the potential of whole-genome sequencing to inform future risk stratification in chronic lymphocytic leukemia.

Pathogenic KDM5B variants in the context of developmental disorders.

Histone modifying enzymes are involved in the posttranslational modification of histones and the epigenetic control of gene expression. They play a critical role in normal development, and there is increasing evidence of their role in developmental disorders (DDs). DDs are a group of chronic, severe conditions that impact the physical, intellectual, language and/or behavioral development of an individual. There are very few treatment options available for DDs such that these are conditions with significant unmet clinical need. Recessive variants in the gene encoding histone modifying enzyme KDM5B are associated with a DD characterized by developmental delay, facial dysmorphism and camptodactyly. KDM5B is responsible for the demethylation of lysine 4 on the amino tail of histone 3 and plays a vital role in normal development and regulating cell differentiation. This review explores the literature on KDM5B and what is currently known about its roles in development and developmental disorders.

Considerations of the built environment for autistic individuals: A review of the literature.

LAY ABSTRACT: Factors related to the interiors of buildings, including the layout of rooms, colours, smells, noises, temperature, ventilation, colour and clutter, among other things, can change the way we interact with our environment and the people around us. Autistic individuals can have differences in processing sensory information and may find aspects of the built environment (BE) over-whelming and difficult to navigate. We reviewed the existing literature exploring the BE and autism. This study found that it is possible to make changes to the BE to create more inclusive and friendly environments for everyone, including autistic individuals. Findings from this study provide clear recommendations that can be used by interior designers, architects, builders, and clinical practitioners to make a positive difference. Key recommendations include using simple spatial layouts, compartmentalising and zoning spaces into specific activity sections and providing retreat spaces. The thoughtful placement of windows and blinds and the installation of dimmable lights, for example, will allow users to manage or reduce sensory over-stimulation caused by lights. Similarly, we recommend creating soundproofing and sound absorbent materials to reduce background noise and sound levels. We also recommend using neutral or simple colour palettes and restrained use of patterns. Finally, and most importantly, the BE needs to be flexible and adaptable to meet the unique needs of each person. This study provides a starting point for design guidelines and recommendations towards making a difference to the everyday experiences of the interiors of buildings.

Phosphodiesterase type 5 inhibitors enhance chemotherapy in preclinical models of esophageal adenocarcinoma by targeting cancer-associated fibroblasts.

The chemotherapy resistance of esophageal adenocarcinomas (EACs) is underpinned by cancer cell extrinsic mechanisms of the tumor microenvironment (TME). We demonstrate that, by targeting the tumor-promoting functions of the predominant TME cell type, cancer-associated fibroblasts (CAFs) with phosphodiesterase type 5 inhibitors (PDE5i), we can enhance the efficacy of standard-of-care chemotherapy. In ex vivo conditions, PDE5i prevent the transdifferentiation of normal fibroblasts to CAF and abolish the tumor-promoting function of established EAC CAFs. Using shotgun proteomics and single-cell RNA-seq, we reveal PDE5i-specific regulation of pathways related to fibroblast activation and tumor promotion. Finally, we confirm the efficacy of PDE5i in combination with chemotherapy in close-to-patient and in vivo PDX-based model systems. These findings demonstrate that CAFs drive chemotherapy resistance in EACs and can be targeted by repurposing PDE5i, a safe and well-tolerated class of drug administered to millions of patients world-wide to treat erectile dysfunction.

Genomic Analysis of Response to Neoadjuvant Chemotherapy in Esophageal Adenocarcinoma.

Neoadjuvant therapy followed by surgery is the standard of care for locally advanced esophageal adenocarcinoma (EAC). Unfortunately, response to neoadjuvant chemotherapy (NAC) is poor (20-37%), as is the overall survival benefit at five years (9%). The EAC genome is complex and heterogeneous between patients, and it is not yet understood whether specific mutational patterns may result in chemotherapy sensitivity or resistance. To identify associations between genomic events and response to NAC in EAC, a comparative genomic analysis was performed in 65 patients with extensive clinical and pathological annotation using whole-genome sequencing (WGS). We defined response using Mandard Tumor Regression Grade (TRG), with responders classified as TRG1-2 (n = 27) and non-responders classified as TRG4-5 (n =38). We report a higher non-synonymous mutation burden in responders (median 2.08/Mb vs. 1.70/Mb, p = 0.036) and elevated copy number variation in non-responders (282 vs. 136/patient, p < 0.001). We identified copy number variants unique to each group in our cohort, with cell cycle (CDKN2A, CCND1), c-Myc (MYC), RTK/PIK3 (KRAS, EGFR) and gastrointestinal differentiation (GATA6) pathway genes being specifically altered in non-responders. Of note, NAV3 mutations were exclusively present in the non-responder group with a frequency of 22%. Thus, lower mutation burden, higher chromosomal instability and specific copy number alterations are associated with resistance to NAC.

Protein over-expression in Escherichia coli triggers adaptation analogous to antimicrobial resistance.

BACKGROUND: The E. coli pET system is the most widely used protein over-expression system worldwide. It relies on the assumption that all cells produce target protein and it is generally believed that integral membrane protein (IMP) over-expression is more toxic than their soluble counterparts. RESULTS: Using GFP-tagged proteins, high level over-expression of either soluble or IMP targets results in > 99.9% cell loss with survival rate of only < 0.03%. Selective pressure generates three phenotypes: large green, large white and small colony variants. As a result, in overnight cultures, ~ 50% of the overall cell mass produces no protein. Genome sequencing of the phenotypes revealed genomic mutations that causes either the loss of T7 RNAP activity or its transcriptional downregulation. The over-expression process is bactericidal and is observed for both soluble and membrane proteins. CONCLUSIONS: We demonstrate that it is the act of high-level over-expression of exogenous proteins in E. coli that sets in motion a chain of events leading to > 99.9% cell death. These results redefine our understanding of protein over-production and link it to the adaptive survival response seen in the development of antimicrobial resistance.

A small gene sequencing panel realises a high diagnostic rate in patients with congenital nystagmus following basic phenotyping.

Nystagmus is a disorder of uncontrolled eye movement and can occur as an isolated trait (idiopathic INS, IINS) or as part of multisystem disorders such as albinism, significant visual disorders or neurological disease. Eighty-one unrelated patients with nystagmus underwent routine ocular phenotyping using commonly available phenotyping methods and were grouped into four sub-cohorts according to the level of phenotyping information gained and their findings. DNA was extracted and sequenced using a broad utility next generation sequencing (NGS) gene panel. A clinical subpanel of genes for nystagmus/albinism was utilised and likely causal variants were prioritised according to methods currently employed by clinical diagnostic laboratories. We determine the likely underlying genetic cause for 43.2% of participants with similar yields regardless of prior phenotyping. This study demonstrates that a diagnostic workflow combining basic ocular phenotyping and a clinically available targeted NGS panel, can provide a high diagnostic yield for patients with infantile nystagmus, enabling access to disease specific management at a young age and reducing the need for multiple costly, often invasive tests. By describing diagnostic yield for groups of patients with incomplete phenotyping data, it also permits the subsequent design of 'real-world' diagnostic workflows and illustrates the changing role of genetic testing in modern diagnostic workflows for heterogeneous ophthalmic disorders.

Systematic Review of Somatic Mutations in Splenic Marginal Zone Lymphoma.

The aims of this systematic review are to refine the catalogue of somatic variants in splenic marginal zone lymphoma (SMZL) and to provide a well-annotated, manually curated database of high-confidence somatic mutations to facilitate variant interpretation for further biological studies and future clinical implementation. Two independent reviewers systematically searched PubMed and Ovid in January 2019 and included studies that sequenced SMZL cases with confirmed diagnosis. The database included fourteen studies, comprising 2817 variants in over 1000 genes from 475 cases. We confirmed the high prevalence of NOTCH2, KLF2 and TP53 mutations and analysis of targeted genes further implicated TNFAIP3, KMT2D, and TRAF3 as recurrent targets of somatic mutation based on their high incidence across studies. The major limitations we encountered were the low number of patients with whole-genome, unbiased analysis and the relative sensitivities of differing sequencing approaches. Overall, we showed that there is little concordance between whole exome sequencing studies of SMZL. We strongly support the continuing unbiased analysis of the SMZL genome for mutations in all protein-coding genes and provide a valuable database resource to facilitate this endeavour that will ultimately improve our understanding of SMZL pathobiology.

Eggplant Domestication: Pervasive Gene Flow, Feralization, and Transcriptomic Divergence.

In the context of food security, examining the genomics of domestication will help identify genes underlying adaptive and economically important phenotypes, for example, larger fruit, improved taste, and loss of agronomically inferior phenotypes. Examination of genome-scale single nucleotide polymorphisms demonstrates the relationships between wild ancestors of eggplant (Solanum melongena L.), confirming that Solanum insanum L. is the wild progenitor. This species is split roughly into an Eastern (Malaysian, Thai, and Vietnamese) and Western (Indian, Madagascan, and Sri Lankan) group, with domesticates derived from the former. Additional "wild" accessions from India appear to be feral escapes, derived multiple times from domesticated varieties through admixture. Accessions with small egg-shaped fruit are generally found intermixed with East Asian Solanum insanum confirming they are primitive relative to the large-fruited domesticates. Comparative transcriptomics was used to track the loci under selection. Sequence analysis revealed a genetic bottleneck reducing variation by almost 50% in the primitive accessions relative to the wild species and a further 10% in the landraces. We also show evidence for selection on genes with a role in response to wounding and apoptosis. Genes showing a significant difference in expression between wild and primitive or between primitive and landrace genepools were mostly (>75%) downregulated in the derived populations and enriched for gene ontologies related to defense, flowering, signaling, and response to biotic and abiotic stimuli. This work reveals genomic changes involved in crop domestication and improvement, and the population genetics work explains why defining the eggplant domestication trajectory has been so challenging.

Comprehensive sequencing of the myocilin gene in a selected cohort of severe primary open-angle glaucoma patients.

Primary open-angle glaucoma (POAG) is the most common form of glaucoma, prevalent in approximately 1-2% of Caucasians in the UK over the age of 40. It is characterised by an open anterior chamber angle, raised intraocular pressure (IOP) and optic nerve damage leading to loss of sight. The myocilin gene (MYOC) is the most common glaucoma-causing gene, accounting for ~2% of British POAG cases. 358 patients were selected for next generation sequencing (NGS) with the following selection criteria: Caucasian ethnicity, intraocular pressure (IOP) 21-40 mm Hg, cup:disc ratio ≥0.6 and visual field mean deviation ≤-3. The entire MYOC gene (17,321 bp) was captured including the promoter, introns, UTRs and coding exons. We identify 12 exonic variants (one stop-gain, five missense and six synonymous variants), two promoter variants, 133 intronic variants, two 3' UTR variants and 23 intergenic variants. Four known or predicted pathogenic exonic variants (p.R126W, p.K216K, p.Q368* and p.T419A) were identified across 11 patients, which accounts for 3.07% of this POAG cohort. This is the first time that the entire region of MYOC has been sequenced and variants reported for a cohort of POAG patients.

A Review of the Genomic Analysis of Children Presenting with Developmental Delay/Intellectual Disability and Associated Dysmorphic Features.

This review describes the clinical criteria of developmental delay (DD)/intellectual disability (ID) as well as the various techniques that are currently implemented to diagnose neurodevelopmental disorders that typically present with associated dysmorphic features such as Angelman syndrome, Prader-Willi syndrome, and DiGeorge syndrome. These analyses include various forms of chromosomal microarray (CMA), which have proven to be superior to previously implemented techniques such as G-banded karyotyping and fluorescent in situ hybridization (FISH) analysis, as well as whole exome sequencing (WES), which is implemented as a secondary examination when CMA analysis is unrevealing. The clinical significance of identified variants and how it relates to facilitating the management of specific genetic disorders such as the above mentioned is also discussed. In addition, the importance of genomic databases and bioinformatics technologies as they relate to variant classification is also considered. Essentially, the discovery of pathogenic variants allows for enhanced management of a patient's clinical phenotype, whereas the identification of variants of uncertain significance (VUS) has proven to have an increase in the number of associated conflicts as they typically generate more ambiguity in regard to the clinical manifestations present within the child. As a result, additional procedures need to be implemented to mitigate the issues that surround their identification. The concluding remarks are in regard to both the ethical and legal considerations of genetic testing as they relate to informed consent, testing of minors, how to handle secondary findings, as well as the anticipated future direction of genomic analysis.

Accurate PCR Detection of Influenza A/B and Respiratory Syncytial Viruses by Use of Cepheid Xpert Flu+RSV Xpress Assay in Point-of-Care Settings: Comparison to Prodesse ProFlu.

The Xpert Flu+RSV Xpress Assay is a fast, automated in vitro diagnostic test for qualitative detection and differentiation of influenza A and B viruses and respiratory syncytial virus (RSV) performed on the Cepheid GeneXpert Xpress System. The objective of this study was to establish performance characteristics of the Xpert Flu+RSV Xpress Assay compared to those of the Prodesse ProFlu+ real-time reverse transcription-PCR (RT-PCR) assay (ProFlu+) for the detection of influenza A and B viruses as well as RSV in a Clinical Laboratory Improvement Amendments (CLIA)-waived (CW) setting. Overall, the assay, using fresh and frozen nasopharyngeal (NP) swabs, demonstrated high concordance with results of the ProFlu+ assay in the combined CW and non-CW settings with positive percent agreements (PPA) (100%, 100%, and 97.1%) and negative percent agreements (NPA) (95.2%, 99.5%, and 99.6%) for influenza A and B viruses and RSV, respectively. In conclusion, this multicenter study using the Cepheid Xpert Flu+RSV Xpress Assay demonstrated high sensitivities and specificities for influenza A and B viruses and RSV in ∼60 min for use at the point-of-care in the CW setting.

Multi-center evaluation of the cobas® Liat® Influenza A/B & RSV assay for rapid point of care diagnosis.

Point of Care Testing (POCT) provides the capability for rapid laboratory test results in patient care environments where a traditional clinical laboratory is not available. POCTs have shorter turn-around times (TATs), they may be performed by non-laboratory personnel, and the need for transport time is eliminated. The Food and Drug Administration (FDA) recently granted Clinical Laboratory Improvements Amendment (CLIA) waiver status to the cobas® Influenza A/B & RSV assay, a rapid, accurate point-of-care test for Influenza and respiratory syncytial virus (RSV) performed on the Liat® System. The performance characteristics of this test were determined though a multi-site study consisting of different point of care testing environments. Prospectively collected Nasopharyngeal (NP) swabs from 1361 patients seen at 8 primary care clinics and 4 emergency departments (EDs) and 295 retrospectively identified specimens were tested for Influenza A/B and RSV on the cobas® Liat® platform. Performance characteristics were determined through comparison to ProFlu+, a laboratory-based PCR test for Influenza A/B and RSV (reference test). Discordant specimens were adjudicated following bi-directional sequencing. The cobas® Influenza A/B and RSV assay showed sensitivities of 99.6%, 99.3%, and 96.8% for Influenza A, Influenza B, and RSV, respectively as determined from percent positive agreement (PPA) following comparison to the reference test. Sequencing confirmed cobas® Influenza A/B and RSV results in 49.2% of reference test discordant specimens, while crossing threshold data suggest increased sensitivity compared to the reference test. The cobas® Influenza A/B and RSV assay was found to be a rapid, sensitive POCT for the detection of these viruses, and provides laboratory-quality PCR-based diagnostic results in point of care settings.

Identification of a functionally significant tri-allelic genotype in the Tyrosinase gene (TYR) causing hypomorphic oculocutaneous albinism (OCA1B).

Oculocutaneous albinism (OCA) and ocular albinism (OA) are inherited disorders of melanin biosynthesis, resulting in loss of pigment and severe visual deficits. OCA encompasses a range of subtypes with overlapping, often hypomorphic phenotypes. OCA1 is the most common cause of albinism in European populations and is inherited through autosomal recessive mutations in the Tyrosinase (TYR) gene. However, there is a high level of reported missing heritability, where only a single heterozygous mutation is found in TYR. This is also the case for other OCA subtypes including OCA2 caused by mutations in the OCA2 gene. Here we have interrogated the genetic cause of albinism in a well phenotyped, hypomorphic albinism population by sequencing a broad gene panel and performing segregation studies on phenotyped family members. Of eighteen probands we can confidently diagnose three with OA and OCA2, and one with a PAX6 mutation. Of six probands with only a single heterozygous mutation in TYR, all were found to have the two common variants S192Y and R402Q. Our results suggest that a combination of R402Q and S192Y with a deleterious mutation in a 'tri-allelic genotype' can account for missing heritability in some hypomorphic OCA1 albinism phenotypes.