Development of ultrasound-guided gene therapy to the sheep fetus.

Fetal gene therapy may treat genetic diseases before significant organ damage, target stem cell populations and avoid immune sensitisation. Candidate diseases include cystic fibrosis, haemophilia and lysosomal storage disorders. This thesis developed ultrasound-guided delivery of viral vectors to the sheep fetus for treatment of these diseases. For haemophilia B treatment we delivered adenovirus vectors containing the p-galactosidase reporter gene (adlacZ) or the human factor IX gene (adhFIX) by ultrasound guidance to the early gestation sheep fetus, when it is considered to be pre-immune. Intraperitoneal injection allowed the earliest time point for gene delivery, achieved the highest hFIX levels and the most localised p-galactosidase expression. Therapeutic hFIX levels were detected after intramuscular and intra-amniotic delivery suggesting that these are potentially alternative sites for therapeutic gene expression. For each route examined, no humoral immune response was observed to the transgene, although antibodies to the adenovirus vector were identified. We achieved intravascular delivery via umbilical vein injection therapeutic hFIX levels were detected. We developed ultrasound-guided transthoracic injection of the mid-gestation fetal trachea for cystic fibrosis treatment, p-galactosidase expression, measured by ELISA, was low after delivery of adlacZ vector alone, but increased 10 fold when the vector was complexed with DEAE dextran. Pretreatment of the fetal airways with sodium caprate increased expression by 90 fold the effect of the two agents was synergistic. Perflubron instillation following vector injection redistributed transgene expression from the large to the small airways. We developed ultrasound-guided fetal intragastric injection and achieved widespread transgene expression throughout the gastrointestinal epithelia after adlacZ vector delivery. For brain manifestation of lysosomal storage disorders we injected adlacZ vectors to the fetal ventricles under ultrasound guidance. Transduction of the choroid plexus was seen. Future application of integrating vectors such as lentivirus may allow for long term therapeutic correction and induce immune tolerance to the transgene

Spectrum of congenital anomalies among VACTERL cases: a EUROCAT population-based study

Background: The VACTERL (Vertebral anomalies, Anal atresia, Cardiac malformations, Tracheo-Esophageal fistula, Renal anomalies, Limb abnormalities) association is the non-random occurrence of at least three of these congenital anomalies: vertebral, anal, cardiac, tracheo-esophageal, renal, and limb anomalies. Diagnosing VACTERL patients is difficult, as many disorders have multiple features in common with VACTERL. The aims of this study were to clearly outline component features, describe the phenotypic spectrum among the largest group of VACTERL patients thus far reported, and to identify phenotypically similar subtypes. Methods: A case-only study was performed assessing data on 501 cases recorded with VACTERL in the JRC-EUROCAT (Joint Research Centre-European Surveillance of Congenital Anomalies) central database (birth years: 1980–2015). We differentiated between major and minor VACTERL features and anomalies outside the VACTERL spectrum to create a clear definition of VACTERL. Results: In total, 397 cases (79%) fulfilled our VACTERL diagnostic criteria. The most commonly observed major VACTERL features were anorectal malformations and esophageal atresia/tracheo-esophageal fistula (both occurring in 62% of VACTERL cases), followed by cardiac (57%), renal (51%), vertebral (33%), and limb anomalies (25%), in every possible combination. Three VACTERL subtypes were defined: STRICT-VACTERL, VACTERL-LIKE, and VACTERL-PLUS, based on severity and presence of additional congenital anomalies. Conclusion: The clearly defined VACTERL component features and the VACTERL subtypes introduced will improve both clinical practice and etiologic research.acceptedVersio

High quality of SARS-CoV-2 molecular diagnostics in a diverse laboratory landscape through supported benchmark testing and External Quality Assessment

A two-step strategy combining assisted benchmark testing (entry controls) and External Quality Assessments (EQAs) with blinded simulated clinical specimens to enhance and maintain the quality of nucleic acid amplification testing was developed. This strategy was successfully applied to 71 diagnostic laboratories in The Netherlands when upscaling the national diagnostic capacity during the SARS-CoV-2 pandemic. The availability of benchmark testing in combination with advice for improvement substantially enhanced the quality of the laboratory testing procedures for SARS-CoV-2 detection. The three subsequent EQA rounds demonstrated high quality testing with regard to specificity (99.6% correctly identified) and sensitivity (93.3% correctly identified). Even with the implementation of novel assays, changing workflows using diverse equipment and a high degree of assay heterogeneity, the overall high quality was maintained using this two-step strategy. We show that in contrast to the limited value of Cq value for absolute proxies of viral load, these Cq values can, in combination with metadata on strategies and techniques, provide valuable information for laboratories to improve their procedures. In conclusion, our two-step strategy (preparation phase followed by a series of EQAs) is a rapid and flexible system capable of scaling, improving, and maintaining high quality diagnostics even in a rapidly evolving (e.g. pandemic) situation.</p

High quality of SARS-CoV-2 molecular diagnostics in a diverse laboratory landscape through supported benchmark testing and External Quality Assessment

A two-step strategy combining assisted benchmark testing (entry controls) and External Quality Assessments (EQAs) with blinded simulated clinical specimens to enhance and maintain the quality of nucleic acid amplification testing was developed. This strategy was successfully applied to 71 diagnostic laboratories in The Netherlands when upscaling the national diagnostic capacity during the SARS-CoV-2 pandemic. The availability of benchmark testing in combination with advice for improvement substantially enhanced the quality of the laboratory testing procedures for SARS-CoV-2 detection. The three subsequent EQA rounds demonstrated high quality testing with regard to specificity (99.6% correctly identified) and sensitivity (93.3% correctly identified). Even with the implementation of novel assays, changing workflows using diverse equipment and a high degree of assay heterogeneity, the overall high quality was maintained using this two-step strategy. We show that in contrast to the limited value of Cq value for absolute proxies of viral load, these Cq values can, in combination with metadata on strategies and techniques, provide valuable information for laboratories to improve their procedures. In conclusion, our two-step strategy (preparation phase followed by a series of EQAs) is a rapid and flexible system capable of scaling, improving, and maintaining high quality diagnostics even in a rapidly evolving (e.g. pandemic) situation.</p

Justify your alpha

Benjamin et al. proposed changing the conventional “statistical significance” threshold (i.e.,the alpha level) from p ≤ .05 to p ≤ .005 for all novel claims with relatively low prior odds. They provided two arguments for why lowering the significance threshold would “immediately improve the reproducibility of scientific research.” First, a p-value near .05provides weak evidence for the alternative hypothesis. Second, under certain assumptions, an alpha of .05 leads to high false positive report probabilities (FPRP2 ; the probability that a significant finding is a false positive

Genome-wide meta-analysis associates HLA-DQA1/DRB1 and LPA and lifestyle factors with human longevity

Genomic analysis of longevity offers the potential to illuminate the biology of human aging. Here, using genome-wide association meta-analysis of 606,059 parents' survival, we discover two regions associated with longevity (HLA-DQA1/DRB1 and LPA). We also validate previous suggestions that APOE, CHRNA3/5, CDKN2A/B, SH2B3 and FOXO3A influence longevity. Next we show that giving up smoking, educational attainment, openness to new experience and high-density lipoprotein (HDL) cholesterol levels are most positively genetically correlated with lifespan while susceptibility to coronary artery disease (CAD), cigarettes smoked per day, lung cancer, insulin resistance and body fat are most negatively correlated. We suggest that the effect of education on lifespan is principally mediated through smoking while the effect of obesity appears to act via CAD. Using instrumental variables, we suggest that an increase of one body mass index unit reduces lifespan by 7 months while 1 year of education adds 11 months to expected lifespan

Justify your alpha

In response to recommendations to redefine statistical significance to p ≤ .005, we propose that researchers should transparently report and justify all choices they make when designing a study, including the alpha level

Implicating genes, pleiotropy, and sexual dimorphism at blood lipid loci through multi-ancestry meta-analysis

Funding Information: GMP, PN, and CW are supported by NHLBI R01HL127564. GMP and PN are supported by R01HL142711. AG acknowledge support from the Wellcome Trust (201543/B/16/Z), European Union Seventh Framework Programme FP7/2007–2013 under grant agreement no. HEALTH-F2-2013–601456 (CVGenes@Target) & the TriPartite Immunometabolism Consortium [TrIC]-Novo Nordisk Foundation’s Grant number NNF15CC0018486. JMM is supported by American Diabetes Association Innovative and Clinical Translational Award 1–19-ICTS-068. SR was supported by the Academy of Finland Center of Excellence in Complex Disease Genetics (Grant No 312062), the Finnish Foundation for Cardiovascular Research, the Sigrid Juselius Foundation, and University of Helsinki HiLIFE Fellow and Grand Challenge grants. EW was supported by the Finnish innovation fund Sitra (EW) and Finska Läkaresällskapet. CNS was supported by American Heart Association Postdoctoral Fellowships 15POST24470131 and 17POST33650016. Charles N Rotimi is supported by Z01HG200362. Zhe Wang, Michael H Preuss, and Ruth JF Loos are supported by R01HL142302. NJT is a Wellcome Trust Investigator (202802/Z/16/Z), is the PI of the Avon Longitudinal Study of Parents and Children (MRC & WT 217065/Z/19/Z), is supported by the University of Bristol NIHR Biomedical Research Centre (BRC-1215–2001) and the MRC Integrative Epidemiology Unit (MC_UU_00011), and works within the CRUK Integrative Cancer Epidemiology Programme (C18281/A19169). Ruth E Mitchell is a member of the MRC Integrative Epidemiology Unit at the University of Bristol funded by the MRC (MC_UU_00011/1). Simon Haworth is supported by the UK National Institute for Health Research Academic Clinical Fellowship. Paul S. de Vries was supported by American Heart Association grant number 18CDA34110116. Julia Ramierz acknowledges support by the People Programme of the European Union’s Seventh Framework Programme grant n° 608765 and Marie Sklodowska-Curie grant n° 786833. Maria Sabater-Lleal is supported by a Miguel Servet contract from the ISCIII Spanish Health Institute (CP17/00142) and co-financed by the European Social Fund. Jian Yang is funded by the Westlake Education Foundation. Olga Giannakopoulou has received funding from the British Heart Foundation (BHF) (FS/14/66/3129). CHARGE Consortium cohorts were supported by R01HL105756. Study-specific acknowledgements are available in the Additional file : Supplementary Note. The views expressed in this manuscript are those of the authors and do not necessarily represent the views of the National Heart, Lung, and Blood Institute; the National Institutes of Health; or the U.S. Department of Health and Human Services. Publisher Copyright: © 2022, The Author(s).Background: Genetic variants within nearly 1000 loci are known to contribute to modulation of blood lipid levels. However, the biological pathways underlying these associations are frequently unknown, limiting understanding of these findings and hindering downstream translational efforts such as drug target discovery. Results: To expand our understanding of the underlying biological pathways and mechanisms controlling blood lipid levels, we leverage a large multi-ancestry meta-analysis (N = 1,654,960) of blood lipids to prioritize putative causal genes for 2286 lipid associations using six gene prediction approaches. Using phenome-wide association (PheWAS) scans, we identify relationships of genetically predicted lipid levels to other diseases and conditions. We confirm known pleiotropic associations with cardiovascular phenotypes and determine novel associations, notably with cholelithiasis risk. We perform sex-stratified GWAS meta-analysis of lipid levels and show that 3–5% of autosomal lipid-associated loci demonstrate sex-biased effects. Finally, we report 21 novel lipid loci identified on the X chromosome. Many of the sex-biased autosomal and X chromosome lipid loci show pleiotropic associations with sex hormones, emphasizing the role of hormone regulation in lipid metabolism. Conclusions: Taken together, our findings provide insights into the biological mechanisms through which associated variants lead to altered lipid levels and potentially cardiovascular disease risk.Peer reviewe

Constraints on spin-0 dark matter mediators and invisible Higgs decays using ATLAS 13 TeV pp collision data with two top quarks and missing transverse momentum in the final state

This paper presents a statistical combination of searches targeting final states with two top quarks and invisible particles, characterised by the presence of zero, one or two leptons, at least one jet originating from a b-quark and missing transverse momentum. The analyses are searches for phenomena beyond the Standard Model consistent with the direct production of dark matter in pp collisions at the LHC, using 139 fb−1 of data collected with the ATLAS detector at a centre-of-mass energy of 13 TeV. The results are interpreted in terms of simplified dark matter models with a spin-0 scalar or pseudoscalar mediator particle. In addition, the results are interpreted in terms of upper limits on the Higgs boson invisible branching ratio, where the Higgs boson is produced according to the Standard Model in association with a pair of top quarks. For scalar (pseudoscalar) dark matter models, with all couplings set to unity, the statistical combination extends the mass range excluded by the best of the individual channels by 50 (25) GeV, excluding mediator masses up to 370 GeV. In addition, the statistical combination improves the expected coupling exclusion reach by 14% (24%), assuming a scalar (pseudoscalar) mediator mass of 10 GeV. An upper limit on the Higgs boson invisible branching ratio of 0.38 (0.30+0.13−0.09) is observed (expected) at 95% confidence level

Study of Z → llγ decays at √s = 8 TeV with the ATLAS detector

This paper presents a study of Z → llγ decays with the ATLAS detector at the Large Hadron Collider. The analysis uses a proton–proton data sample corresponding to an integrated luminosity of 20.2 fb−1 collected at a centre-ofmass energy √s = 8 TeV. Integrated fiducial cross-sections together with normalised differential fiducial cross-sections, sensitive to the kinematics of final-state QED radiation, are obtained. The results are found to be in agreement with stateof-the-art predictions for final-state QED radiation. First measurements of Z → llγ γ decays are also reported