In Utero Gene Therapy (IUGT) Using GLOBE Lentiviral Vector Phenotypically Corrects the Heterozygous Humanised Mouse Model and Its Progress Can Be Monitored Using MRI Techniques

Funder: UK Thalassaemia SocietyAbstract: In utero gene therapy (IUGT) to the fetal hematopoietic compartment could be used to treat congenital blood disorders such as β-thalassemia. A humanised mouse model of β-thalassemia was used, in which heterozygous animals are anaemic with splenomegaly and extramedullary hematopoiesis. Intrahepatic in utero injections of a β globin-expressing lentiviral vector (GLOBE), were performed in fetuses at E13.5 of gestation. We analysed animals at 12 and 32 weeks of age, for vector copy number in bone marrow, peripheral blood liver and spleen and we performed integration site analysis. Compared to noninjected heterozygous animals IUGT normalised blood haemoglobin levels and spleen weight. Integration site analysis showed polyclonality. The left ventricular ejection fraction measured using magnetic resonance imaging (MRI) in treated heterozygous animals was similar to that of normal non-β-thalassemic mice but significantly higher than untreated heterozygous thalassemia mice suggesting that IUGT ameliorated poor cardiac function. GLOBE LV-mediated IUGT normalised the haematological and anatomical phenotype in a heterozygous humanised model of β-thalassemia

Longitudinal associations between circulating interleukin-6 and C-reactive protein in childhood, and eating disorders and disordered eating in adolescence

Objective: Few studies have explored the association between inflammation and eating disorders and none used a longitudinal design. We investigated the association between serum-levels of interleukin 6 (IL-6) and C-reactive protein (CRP) measured in childhood and eating disorders and related behaviours and cognitions in adolescence in a large general population sample. Methods: We used data from the Avon Longitudinal Study of Parents and Children (ALSPAC). Our exposures were thirds of IL6 and CRP derived from serum measurements taken at age nine years, and outcomes were eating disorder diagnoses and self-reported disordered eating behaviours at ages 14, 16, and 18 years. We used univariable and multivariable multilevel logistic regression models adjusting for a number of potential confounders, including sex, fat mass, and pre-existing mental health difficulties. Results: Our sample included 3480 children. Those in the top third of CRP had lower odds of binge eating (odds ratio(OR):0.62, 95% confidence interval (CI):0.39,1.00,p “equals” 0.05) and fasting (OR:0.63, 95% CI:0.38,1.07,p “equals” 0.09) after adjustment for confounders. We also observed weak associations of comparable magnitude for purging, anorexia nervosa, and bulimia nervosa. We did not find any associations between levels of IL6 and any of the outcomes under study. Conclusions: There was little evidence of an association between CRP and IL-6 and adolescent eating disorder outcomes. The inverse association observed between CRP and binge eating was unexpected, so caution is needed when interpreting it. One possible explanation is that higher CRP levels could have a protective role for disordered eating by affecting appetitive traits

Publisher Correction: In Utero Gene Therapy (IUGT) Using GLOBE Lentiviral Vector Phenotypically Corrects the Heterozygous Humanised Mouse Model and Its Progress Can Be Monitored Using MRI Techniques

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Publisher Correction: In Utero Gene Therapy (IUGT) Using GLOBE Lentiviral Vector Phenotypically Corrects the Heterozygous Humanised Mouse Model and Its Progress Can Be Monitored Using MRI Techniques.

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Placenta Imaging Workshop 2018 report:Multiscale and multimodal approaches

The Centre for Medical Image Computing (CMIC) at University College London (UCL) hosted a two-day workshop on placenta imaging on April 12th and 13th 2018. The workshop consisted of 10 invited talks, 3 contributed talks, a poster session, a public interaction session and a panel discussion about the future direction of placental imaging. With approximately 50 placental researchers in attendance, the workshop was a platform for engineers, clinicians and medical experts in the field to network and exchange ideas. Attendees had the chance to explore over 20 posters with subjects ranging from the movement of blood within the placenta to the efficient segmentation of fetal MRI using deep learning tools. UCL public engagement specialists also presented a poster, encouraging attendees to learn more about how to engage patients and the public with their research, creating spaces for mutual learning and dialogue

The BRCA1 Variant p.Ser36Tyr Abrogates BRCA1 Protein Function and Potentially Confers a Moderate Risk of Breast Cancer

<div><p>The identification of variants of unknown clinical significance (VUS) in the <i>BRCA1</i> gene complicates genetic counselling and causes additional anxiety to carriers. <i>In silico</i> approaches currently used for VUS pathogenicity assessment are predictive and often produce conflicting data. Furthermore, functional assays are either domain or function specific, thus they do not examine the entire spectrum of BRCA1 functions and interpretation of individual assay results can be misleading. PolyPhen algorithm predicted that the BRCA1 p.Ser36Tyr VUS identified in the Cypriot population was damaging, whereas Align-GVGD predicted that it was possibly of no significance. In addition the BRCA1 p.Ser36Tyr variant was found to be associated with increased risk (OR = 3.47, 95% CI 1.13-10.67, P = 0.02) in a single case-control series of 1174 cases and 1109 controls. We describe a cellular system for examining the function of exogenous full-length BRCA1 and for classifying VUS. We achieved strong protein expression of full-length BRCA1 in transiently transfected HEK293T cells. The p.Ser36Tyr VUS exhibited low protein expression similar to the known pathogenic variant p.Cys61Gly. Co-precipitation analysis further demonstrated that it has a reduced ability to interact with BARD1. Further, co-precipitation analysis of nuclear and cytosolic extracts as well as immunofluorescence studies showed that a high proportion of the p.Ser36Tyr variant is withheld in the cytoplasm contrary to wild type protein. In addition the ability of p.Ser36Tyr to co-localize with conjugated ubiquitin foci in the nuclei of S-phase synchronized cells following genotoxic stress with hydroxyurea is impaired at more pronounced levels than that of the p.Cys61Gly pathogenic variant. The p.Ser36Tyr variant demonstrates abrogated function, and based on epidemiological, genetic, and clinical data we conclude that the p.Ser36Tyr variant is probably associated with a moderate breast cancer risk.</p></div

Ribbon representation of the BARD1:BRCA1 complex.

<p>A. The RING domains of BRCA1 and BARD1 (PDB 1JM7 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093400#pone.0093400-Brzovic3" target="_blank">[63]</a>) are represented in magenta and blue respectively and the residues isoleucine 26, serine 36 and C61G are indicated in yellow. Orange spheres represent the Zn²⁺ ions. This model demonstrates that serine 36 lies within the second β-sheet of the RING structure. B. and C. represent the complex in a different orientation revealing the position of serine 36 (B.) and its substitution by tyrosine (C.) may distort the structure. The model was modified using PyMOL software.</p

p.Ser36Tyr (S36Y) BRCA1:BARD1 complex is withheld within the cytoplasm following HU treatment.

<p>Lysates of transfected cells were fractionated into nuclear and cytosolic extracts following S-phase synchronization and treatment with HU. BARD1 was co-precipitated with the ectopically expressed BRCA1 in the A. Nuclear and B. Cytosolic extracts with the anti-DYKDDDDK tag antibody which recognizes only the exogenous BRCA1. A. Immunoblot analysis of the pull-downs demonstrated that unlike wild type BRCA1, both the p.Ser36Tyr and p.Cys61Gly (C61G) variants exhibit reduced ability to co-precipitate BARD1 in the nuclear extracts of stressed cells. B. However, in the cytosolic extracts the p.Ser36Tyr variant, but not p.Cys61Gly, co-precipitated a higher proportion of BARD1 in treated cells compared to wild type transfected cells. In untreated cells, both variants, including wild type BRCA1, co-precipitated larger amounts of BARD1 compared to treated cells. Bar charts showing that C. in the nuclei of treated cells, both the p.Ser36Tyr and p.Cys61Gly variants exhibit reduced ability to co-precipitate with BARD1, whereas D. in the cytosol the p.Ser36Tyr variant co-precipitates similar levels of BARD1 compared to wild type BRCA1, but it is not statistically significant. Immunoblot analysis of nuclear and cytoplasmic extracts demonstrating their purity is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093400#pone.0093400.s002" target="_blank">Fig. S2</a>. The results are representative of 2 experiments.</p