Exploring the regulation and activation of ALT, the Alternative Lengthening of Telomeres

Cancers and cell lines that utilise the Alternative Lengthening of Telomeres (ALT) to maintain telomere length usually display complex karyotype rearrangements consistent with genome instability that may arise at crisis through dysfunctional telomeres. The absence of the ATRX chromatin remodelling factor is the predominant marker in these cancers and cell lines. It has been shown that specific orphan nuclear receptors (ORs) can bind to (TCAGGG)n repeats in telomeres of some ALT+ cell lines and this binding results in changes to the telomere chromatin that may facilitate the ALT. In this project, the binding site and regulatory roles of ORs were investigated in a panel of five ALT+ cell lines. No relationship was identified between the expression of NR2F2 or NR2C2 proteins and the localisation of these proteins at telomeres in ALT+ cell lines. The frequency of cells with NR2F2 or NR2C2 foci at telomeres varies considerably between the ALT+ cell lines (7- 91% for NR2F2; 1-42% for NR2C2) with only the WI38VA13_2RA ALT+ cell line showing a high frequency for both, consistent with previous work. PCR amplification of XpYp, 12q and 17p telomeres followed by hybridisation to (TTAGGG)n, (TCAGGG)n or (TGAGGG)n variant repeat probes was used to measure the frequency of telomere molecules that contain binding sites for these ORs. This revealed that some telomere alleles in ALT+ cell lines lack binding sites for these ORs, indicating that binding of these ORs is not essential for ALT at some telomeres. Downregulation of NR2F2 in three ALT+ cells resulted in significant changes in the expression of genes involved in DNA replication and repair that resulted in cell cycle arrest in the U2OS, but not in the WV or WI38VA13_2RA ALT+ cell lines. Integrated analyses of whole exome sequence (WES) and RNA-seq analyses were used to uncover the mutation and the gene expression changes associated with ALT activation after ATRX knockout, using CRISPR-Cas9, in SV40- transformed pre-crisis cells. Differential gene expression analysis at early post-crisis time-points in emerging clonal populations showed that downregulation of the JAK-STAT signalling pathway, the ALT-suppressor SP100 and reduced expression of non-homologous end joining (NHEJ) related genes NR4A1 and XLF were the most remarkable events. These gene expression changes were combined with copy number changes in chromosomes 8, 11 and 18 and remarkable level of genome instability. This could suggest that, following the loss of ATRX in SV40-transformed cells, the changes in the expression of the genes required for double strand break (DSB) repair contributed to the activation of ALT

Whole-genome analysis as a diagnostic tool for patients referred for diagnosis of Silver-Russell syndrome: a real-world study

Background Silver-Russell syndrome (SRS) is an imprinting disorder characterised by prenatal and postnatal growth restriction, but its clinical features are non-specific and its differential diagnosis is broad. Known molecular causes of SRS include imprinting disturbance, single nucleotide variant (SNV), CNV or UPD affecting several genes; however, up to 40% of individuals with a clinical diagnosis of SRS currently receive no positive molecular diagnosis.Methods To determine whether whole-genome sequencing (WGS) could uncover pathogenic variants missed by current molecular testing, we analysed data of 72 participants recruited to the 100,000 Genomes Project within the clinical category of SRS.Results In 20 participants (27% of the cohort) we identified genetic variants plausibly accounting for SRS. Coding SNVs were identified in genes including CDKN1C, IGF2, IGF1R and ORC1. Maternal-effect variants were found in mothers of five participants, including two participants with imprinting disturbance and one with multilocus imprinting disorder. Two regions of homozygosity were suggestive of UPD involving imprinted regions implicated in SRS and Temple syndrome, and three plausibly pathogenic CNVs were found, including a paternal deletion of PLAGL1. In 48 participants with no plausible pathogenic variant, unbiased analysis of SNVs detected a potential association with STX4.Conclusion WGS analysis can detect UPD, CNV and SNV and is potentially a valuable addition to diagnosis of SRS and related growth-restricting disorders

Angiotensin II Exaggerates SARS-CoV-2 Specific T-Cell Response in Convalescent Individuals following COVID-19

Dysregulation of renin−angiotensin systems during coronavirus disease 2019 (COVID-19) infection worsens the symptoms and contributes to COVID-19 severity and mortality. This study sought to investigate the effect of exogenous angiotensin II (Ang-II) on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T-cells response in recovered COVID-19 patients. Human peripheral blood mononuclear cells (PBMCs) were treated with Ang II and then stimulated with a SARS-CoV-2 peptide pool. T-cell responses were measured using flow cytometry, while enzyme-linked immunosorbent assay (ELISA) and intracellular cytokine staining (ICS) assays determined functional capability and polarization. Additionally, the relative level of protein phosphorylation was measured using a phosphokinase array. Our results showed that Ang II treatment significantly increased the magnitude of SARS-CoV-2-specific T-cell response in stimulated PBMCs with a SARS-CoV-2 peptide pool. Moreover, the phosphorylation levels of numerous proteins implicated in cardiovascular diseases, inflammation, and viral infection showed significant increases in the presence of Ang II. The mitogenic stimulation of PBMCs after Ang II and SARS-CoV-2 peptide pool stimulation showed functional polarization of T-cells toward Th1/Th17 and Th17 phenotypes, respectively. Meanwhile, ELISA showed increased productions of IL-1β and IL-6 in Ang II-stimulated PBMCs without affecting the IL-10 level. To our knowledge, this study is the first to demonstrate that Ang II exaggerates SARS-CoV-2-specific T-cells response. Therefore, during COVID-19 infection, Ang II may aggravate the inflammatory response and change the immune response toward a more inflammatory profile against SARS-CoV-2 infection

A case of mosaic deletion of paternally‐inherited PLAGL1 and two cases of upd(6)mat add to evidence for PLAGL1 under‐expression as a cause of growth restriction

PLAGL1 is one of a group of imprinted genes, whose altered expression causes imprinting disorders impacting growth, development, metabolism, and behavior. PLAGL1 over-expression causes transient neonatal diabetes mellitus (TNDM type 1) and, based on murine models, under-expression would be expected to cause growth restriction. However, only some reported individuals with upd(6)mat have growth restriction, giving rise to uncertainty about the role of PLAGL1 in human growth. Here we report three individuals investigated for growth restriction, two with upd(6)mat and one with a mosaic deletion of the paternally-inherited allele of PLAGL1. These cases add to evidence of its involvement in pre- and early post-natal human growth