A Roberts Syndrome Individual With Differential Genotoxin Sensitivity and a DNA Damage Response Defect

© 2018 Elsevier. This manuscript version is made available under the CC-BY-NC-ND 4.0 license: http://creativecommons.org/licenses/by-nc-nd/4.0/ This author accepted manuscript is made available following 12 month embargo from date of publication (November 2018) in accordance with the publisher’s archiving policyPurpose Roberts syndrome (RBS) is a rare, recessively transmitted developmental disorder characterized by growth retardation, craniofacial abnormalities, and truncation of limbs. All affected individuals to date have mutations in the ESCO2 (establishment of cohesion 2) gene, a key regulator of the cohesin complex, which is involved in sister chromatid cohesion and DNA double-strand break (DSB) repair. Here we characterize DNA damage responses (DDRs) for the first time in an RBS-affected family. Methods and Materials Lymphoblastoid cell lines were established from an RBS family, including the proband and parents carrying ESCO2 mutations. Various DDR assays were performed on these cells, including cell survival, chromosome break, and apoptosis assays; checkpoint activation indicators; and measures of DNA breakage and repair. Results Cells derived from the RBS-affected individual showed sensitivity to ionizing radiation (IR) and mitomycin C–induced DNA damage. In this ESCO2 compound heterozygote, other DDRs were also defective, including enhanced IR-induced clastogenicity and apoptosis; increased DNA DSB induction; and a reduced capacity for repairing IR-induced DNA DSBs, as measured by γ-H2AX foci and the comet assay. Conclusions In addition to its developmental features, RBS can be, like ataxia telangiectasia, considered a DDR-defective syndrome, which contributes to its cellular, molecular, and clinical phenotype

Randomized-controlled trial of the DIALIVE liver dialysis device vs. standard of care in patients with acute-on-chronic liver failure

BACKGROUND AND AIMS: Acute on chronic liver failure (ACLF) is characterized by severe systemic inflammation, multi-organ failure and high mortality rates. Its treatment is an urgent unmet need. DIALIVE is a novel liver dialysis device that aims to exchange d ysfunctional albumin and remove damage- and pathogen-associated molecular patterns. This first-in-man randomized, controlled clinical trial was performed with the primary aim of assessing its safety in ACLF patients with secondary aims to evaluate its clinical effects, device performance and effect on pathophysiologically-relevant biomarkers. METHODS: 32 alcoholic cirrhosis patients with ACLF were included. Patients were treated with DIALIVE for up to 5-days and end points were assessed at Day-10. Safety was assessed in all patients (n=32). The secondary aims were assessed in a pre-specified subgroup that had at least 3-treatment sessions with DIALIVE (n=30). RESULTS: There were no significant differences in 28-day mortality or occurrence of serious adverse events between the groups. Significant reduction in the severity of endotoxemia and improvement in albumin function was observed in DIALIVE group, which translated into a significant reduction in the CLIF-C (Chronic Liver Failure consortium) organ failure (p=0.018) and CLIF-C ACLF scores (p=0.042) at Day-10. Time to resolution of ACLF was significantly faster in DIALIVE group (p=0.036). Biomarkers of systemic inflammation such as IL-8 (p=0.006), cell death [cytokeratin-18: M30 (p=0.005) and M65 (p=0.029)], endothelial function [asymmetric dimethylarginine (p=0.002)] and, ligands for toll-like receptor 4 (p=0.030) and inflammasome (p=0.002) improved significantly in DIALIVE group. CONCLUSIONS: These data indicate that DIALIVE appears to be safe and impacts positively on prognostic scores and pathophysiologically relevant biomarkers in patients with ACLF. Larger, adequately powered studies are warranted to further confirm its safety and efficacy. LAY SUMMARY: This is the first-in-man clinical trial which tested DIALIVE, a novel liver dialysis device for the treatment of liver cirrhosis and acute on chronic liver failure, a condition associated with severe inflammation, organ failures and a high risk of death. The study met the primary end point confirming DIALIVE system to be safe. Additionally, it reduced inflammation with improved clinical parameters. It did not, however, reduce mortality in this small study and requires further larger clinical trials to re-confirm its safety and evaluate efficacy. CLINICAL TRIAL NUMBER: NCT03065699

Atrasentan and renal events in patients with type 2 diabetes and chronic kidney disease (SONAR): a double-blind, randomised, placebo-controlled trial

Background: Short-term treatment for people with type 2 diabetes using a low dose of the selective endothelin A receptor antagonist atrasentan reduces albuminuria without causing significant sodium retention. We report the long-term effects of treatment with atrasentan on major renal outcomes. Methods: We did this double-blind, randomised, placebo-controlled trial at 689 sites in 41 countries. We enrolled adults aged 18–85 years with type 2 diabetes, estimated glomerular filtration rate (eGFR)25–75 mL/min per 1·73 m 2 of body surface area, and a urine albumin-to-creatinine ratio (UACR)of 300–5000 mg/g who had received maximum labelled or tolerated renin–angiotensin system inhibition for at least 4 weeks. Participants were given atrasentan 0·75 mg orally daily during an enrichment period before random group assignment. Those with a UACR decrease of at least 30% with no substantial fluid retention during the enrichment period (responders)were included in the double-blind treatment period. Responders were randomly assigned to receive either atrasentan 0·75 mg orally daily or placebo. All patients and investigators were masked to treatment assignment. The primary endpoint was a composite of doubling of serum creatinine (sustained for ≥30 days)or end-stage kidney disease (eGFR <15 mL/min per 1·73 m 2 sustained for ≥90 days, chronic dialysis for ≥90 days, kidney transplantation, or death from kidney failure)in the intention-to-treat population of all responders. Safety was assessed in all patients who received at least one dose of their assigned study treatment. The study is registered with ClinicalTrials.gov, number NCT01858532. Findings: Between May 17, 2013, and July 13, 2017, 11 087 patients were screened; 5117 entered the enrichment period, and 4711 completed the enrichment period. Of these, 2648 patients were responders and were randomly assigned to the atrasentan group (n=1325)or placebo group (n=1323). Median follow-up was 2·2 years (IQR 1·4–2·9). 79 (6·0%)of 1325 patients in the atrasentan group and 105 (7·9%)of 1323 in the placebo group had a primary composite renal endpoint event (hazard ratio [HR]0·65 [95% CI 0·49–0·88]; p=0·0047). Fluid retention and anaemia adverse events, which have been previously attributed to endothelin receptor antagonists, were more frequent in the atrasentan group than in the placebo group. Hospital admission for heart failure occurred in 47 (3·5%)of 1325 patients in the atrasentan group and 34 (2·6%)of 1323 patients in the placebo group (HR 1·33 [95% CI 0·85–2·07]; p=0·208). 58 (4·4%)patients in the atrasentan group and 52 (3·9%)in the placebo group died (HR 1·09 [95% CI 0·75–1·59]; p=0·65). Interpretation: Atrasentan reduced the risk of renal events in patients with diabetes and chronic kidney disease who were selected to optimise efficacy and safety. These data support a potential role for selective endothelin receptor antagonists in protecting renal function in patients with type 2 diabetes at high risk of developing end-stage kidney disease. Funding: AbbVie

Investigation of hospital discharge cases and SARS-CoV-2 introduction into Lothian care homes

Background The first epidemic wave of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in Scotland resulted in high case numbers and mortality in care homes. In Lothian, over one-third of care homes reported an outbreak, while there was limited testing of hospital patients discharged to care homes. Aim To investigate patients discharged from hospitals as a source of SARS-CoV-2 introduction into care homes during the first epidemic wave. Methods A clinical review was performed for all patients discharges from hospitals to care homes from 1st March 2020 to 31st May 2020. Episodes were ruled out based on coronavirus disease 2019 (COVID-19) test history, clinical assessment at discharge, whole-genome sequencing (WGS) data and an infectious period of 14 days. Clinical samples were processed for WGS, and consensus genomes generated were used for analysis using Cluster Investigation and Virus Epidemiological Tool software. Patient timelines were obtained using electronic hospital records. Findings In total, 787 patients discharged from hospitals to care homes were identified. Of these, 776 (99%) were ruled out for subsequent introduction of SARS-CoV-2 into care homes. However, for 10 episodes, the results were inconclusive as there was low genomic diversity in consensus genomes or no sequencing data were available. Only one discharge episode had a genomic, time and location link to positive cases during hospital admission, leading to 10 positive cases in their care home. Conclusion The majority of patients discharged from hospitals were ruled out for introduction of SARS-CoV-2 into care homes, highlighting the importance of screening all new admissions when faced with a novel emerging virus and no available vaccine

SARS-CoV-2 Omicron is an immune escape variant with an altered cell entry pathway

Vaccines based on the spike protein of SARS-CoV-2 are a cornerstone of the public health response to COVID-19. The emergence of hypermutated, increasingly transmissible variants of concern (VOCs) threaten this strategy. Omicron (B.1.1.529), the fifth VOC to be described, harbours multiple amino acid mutations in spike, half of which lie within the receptor-binding domain. Here we demonstrate substantial evasion of neutralization by Omicron BA.1 and BA.2 variants in vitro using sera from individuals vaccinated with ChAdOx1, BNT162b2 and mRNA-1273. These data were mirrored by a substantial reduction in real-world vaccine effectiveness that was partially restored by booster vaccination. The Omicron variants BA.1 and BA.2 did not induce cell syncytia in vitro and favoured a TMPRSS2-independent endosomal entry pathway, these phenotypes mapping to distinct regions of the spike protein. Impaired cell fusion was determined by the receptor-binding domain, while endosomal entry mapped to the S2 domain. Such marked changes in antigenicity and replicative biology may underlie the rapid global spread and altered pathogenicity of the Omicron variant

ATM and cellular response to DNA damage

Most human cancers display a myriad of genetic changes, a characteristic often attributed to genome instability. Cytogenetic studies have long identified chromosomal aberrations as a hallmark of human tumours, but the causes and consequences of genomic defects in tumours still remain to be fully understood. In particular, the role of genome instability in the development of human cancers as well as its relevance to treatment paradigms continue to evoke intense debate. To address these critical issues it is clearly important to understand the mechanisms that give rise to genome instability. This book reviews both genetic and biochemical data on the origin of genome instability and its impact on carcinogenesis. Reflecting recent discoveries and ongoing research, it discusses DNA repair mechanisms and hereditary cancer syndromes, as well as checkpoint mechanisms operating to safeguard chromosome integrity during cell cycle progression. Moreover, it summarises our current understanding of the various defects that may allow cancer cells to rapidly accumulate critical mutations and evolve, through processes reminiscent of Darwinian selection, an increasingly aggressive behaviour. Hopefully, this book will stimulate thought, discussion and experimentation, and serve as a rich source of information for a wide audience, including advanced students, researchers and clinical oncologists

Association between sucrase-isomaltase and p53 expression in colorectal cancer

BACKGROUND: Sucrase-isomaltase (SI) is a tissue-based phenotypic marker that is an independent prognostic factor in colorectal cancer (CRC). DF3 and galectin 3 are two other tissue-based markers that are upregulated during neoplastic transformation. Because p53 mutations are acquired during neoplastic progression, we reasoned that alterations in SI and p53 may be associated despite an apparent lack of biological interaction. METHODS: Paraffin sections from 183 patients who underwent surgery at New England Deaconess Hospital (NEDH) between 1965 and 1977 were analyzed first by immunohistochemistry (IHC) for the expression of the markers SI, DF3, and galectin 3, which were scored as absent or present. Paraffin sections from a second group of 59 patients who underwent surgery at NEDH between 1985 and 1992 were analyzed by IHC for the expression of p53 as well as SI, DF3, and galectin 3. p53 nuclear staining was scored as absent or present. Previous work has shown that p53 is mutated in all cells with nuclear staining and in 10% of tumors that are unstained. RESULTS: SI expression was not associated with the expression of either DF3 or galectin 3, and neither DF3 nor galectin 3 were prognostic factors in CRC. None of the phenotypic markers were associated with any of the clinicopathologic variables. However, 21 of 24 p53-positive cases (88%) expressed SI, whereas 15 of 35 p53-negative cases (43%) were also SI negative (p = 0.02, Fisher exact test). p53 expression was not associated with expression of DF3 or galectin 3. CONCLUSIONS: SI expression and p53 mutation are associated significantly in CRC. Although the mechanism underlying such an association in presently unknown, the association may define a subset of patients with a worse prognosis

Involvement of novel autophosphorylation sites in ATM activation

ATM kinase plays a central role in signaling DNA double-strand breaks to cell cycle checkpoints and to the DNA repair machinery. Although the exact mechanism of ATM activation remains unknown, efficient activation requires the Mre11 complex, autophosphorylation on S1981 and the involvement of protein phosphatases and acetylases. We report here the identification of several additional phosphorylation sites on ATM in response to DNA damage, including autophosphorylation on pS367 and pS1893. ATM autophosphorylates all these sites in vitro in response to DNA damage. Antibodies against phosphoserine 1893 revealed rapid and persistent phosphorylation at this site after in vivo activation of ATM kinase by ionizing radiation, paralleling that observed for S1981 phosphorylation. Phosphorylation was dependent on functional ATM and on the Mre11 complex. All three autophosphorylation sites are physiologically important parts of the DNA damage response, as phosphorylation site mutants (S367A, S1893A and S1981A) were each defective in ATM signaling in vivo and each failed to correct radiosensitivity, genome instability and cell cycle checkpoint defects in ataxia-telangiectasia cells. We conclude that there are at least three functionally important radiation-induced autophosphorylation events in ATM

Sucrase-isomaltase is an independent prognostic marker for colorectal carcinoma

PURPOSE: Expression of disaccharidase sucrase-isomaltase (SI) is significantly enhanced during neoplastic transformation of colonic epithelium. Our study was designed to determine whether expression of SI within primary tumors was significantly associated with survival in patients with colorectal carcinoma (CRC). METHODS: SI expression was analyzed by immunohistochemistry in paraffin sections from 182 Stage I to III CRC that had been resected for cure at the New England Deaconess Hospital between 1965 and 1977. Expression was scored as absent or present in 1 to 50 percent or more than 50 percent of tumor cells. Associations were explored among SI expression, other clinical or pathologic variables, and overall survival. The data set is mature, with 91 (56 percent) patients who had died of CRC at a median follow-up of 96 months. RESULTS: Fifty-five percent of primary CRC expressed SI. When the multivariate Cox analysis was performed, nodal status, T stage, primary site, grade, and SI expression were independent covariates. SI expression was not associated with the expression of other clinicopathologic variables but increased the risk of death from colorectal carcinoma by 1.83-fold. DISCUSSION: These results indicate that SI is a prognostic marker for CRC that is independent of stage-related variables in patients who have undergone potentially curative resections