Search

Abstract: The G haplotype is a group of co-inherited single nucleotide variants in the F5 gene that reduce venous thromboembolism (VTE) risk. Although 7% of the population is homozygous for the G haplotype (F5-G/G), the underlying mechanism of VTE protection is poorly understood. Using RNA sequencing data from 4651 blood donors in the INTERVAL study, we detected a rare excision event at the factor V (FV)-short splice sites in 5% of F5-G/Gs carriers as compared with 2.16% of homozygotes for the F5 reference sequence (F5-ref; P = .003). Highly elevated (∼10-fold) FV-short, a FV isoform that lacks most of the B-domain, has been linked with increased tissue factor inhibitor α (TFPIα) levels in rare hemorrhagic diathesis, including East Texas bleeding disorder. To ascertain whether the enhanced FV-short splicing seen in F5-G/G INTERVAL participants translated to increased plasma FV-short levels, we analyzed plasma samples from 7 F5-G/G and 13 F5-ref individuals in a recall-by-genotype study. A ∼2.2-fold higher amount of FV-short was found in a plasma pool from F5-G/G participants when compared with the pool of F5-refs (P = .029), but there was no difference in the total FV levels. Although no significant difference in TFPI levels were found, F5-G/Gs showed a ∼1.4-fold TFPI-dependent increase in lag time to thrombin generation than F5-refs (P = .0085). Finally, in an analysis of 117 699 UK Biobank participants, we discovered that, although being protective against VTE, the G haplotype also confers an increase in bleeding episodes (P = .011). Our study provides evidence that the effect of the common G haplotype is mediated by the FV-short/TFPI pathway., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Gene misexpression is the aberrant transcription of a gene in a context where it is usually inactive. Despite its known pathological consequences in specific rare diseases, we have a limited understanding of its wider prevalence and mechanisms in humans. To address this, we analyzed gene misexpression in 4,568 whole-blood bulk RNA sequencing samples from INTERVAL study blood donors. We found that while individual misexpression events occur rarely, in aggregate they were found in almost all samples and a third of inactive protein-coding genes. Using 2,821 paired whole-genome and RNA sequencing samples, we identified that misexpression events are enriched in cis for rare structural variants. We established putative mechanisms through which a subset of SVs lead to gene misexpression, including transcriptional readthrough, transcript fusions, and gene inversion. Overall, we develop misexpression as a type of transcriptomic outlier analysis and extend our understanding of the variety of mechanisms by which genetic variants can influence gene expression., Competing Interests: Declaration of interests The authors declare the following interests: T.V. has received PhD studentship funding from AstraZeneca. J.M. completed this work while employed by the University of Cambridge but is now an employee of Genomics plc. S.P. is a current employee and stockholder of AstraZeneca. D.J.R. is an employee of NHS Blood and Transplant. A.B. is currently an employee of Bayer AG, Research and Early Development Precision Medicine, Research & Development, Pharmaceutical Division, Wuppertal, DE. A.P. is a current employee and stockholder of AstraZeneca. D.S.P. is a current employee and stockholder of AstraZeneca. K.K. is a current employee and stockholder of AstraZeneca. J.D. serves on scientific advisory boards for AstraZeneca, Novartis, and UK Biobank and has received multiple grants from academic, charitable, and industry sources outside of the submitted work. M.I. is a trustee of the Public Health Genomics (PHG) Foundation, is a member of the Scientific Advisory Board of Open Targets, and has a research collaboration with AstraZeneca, which is unrelated to this study., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The ithomiine butterflies (Nymphalidae: Danainae) represent the largest known radiation of Müllerian mimetic butterflies. They dominate by number the mimetic butterfly communities, which include species such as the iconic neotropical Heliconius genus. Recent studies on the ecology and genetics of speciation in Ithomiini have suggested that sexual pheromones, colour pattern and perhaps hostplant could drive reproductive isolation. However, no reference genome was available for Ithomiini, which has hindered further exploration on the genetic architecture of these candidate traits, and more generally on the genomic patterns of divergence. Here, we generated high-quality, chromosome-scale genome assemblies for two Melinaea species, M. marsaeus and M. menophilus, and a draft genome of the species Ithomia salapia. We obtained genomes with a size ranging from 396 to 503 Mb across the three species and scaffold N50 of 40.5 and 23.2 Mb for the two chromosome-scale assemblies. Using collinearity analyses we identified massive rearrangements between the two closely related Melinaea species. An annotation of transposable elements and gene content was performed, as well as a specialist annotation to target chemosensory genes, which is crucial for host plant detection and mate recognition in mimetic species. A comparative genomic approach revealed independent gene expansions in ithomiines and particularly in gustatory receptor genes. These first three genomes of ithomiine mimetic butterflies constitute a valuable addition and a welcome comparison to existing biological models such as Heliconius, and will enable further understanding of the mechanisms of adaptation in butterflies., (© 2023 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

The use of omic modalities to dissect the molecular underpinnings of common diseases and traits is becoming increasingly common. But multi-omic traits can be genetically predicted, which enables highly cost-effective and powerful analyses for studies that do not have multi-omics 1 . Here we examine a large cohort (the INTERVAL study 2 ; n = 50,000 participants) with extensive multi-omic data for plasma proteomics (SomaScan, n = 3,175; Olink, n = 4,822), plasma metabolomics (Metabolon HD4, n = 8,153), serum metabolomics (Nightingale, n = 37,359) and whole-blood Illumina RNA sequencing (n = 4,136), and use machine learning to train genetic scores for 17,227 molecular traits, including 10,521 that reach Bonferroni-adjusted significance. We evaluate the performance of genetic scores through external validation across cohorts of individuals of European, Asian and African American ancestries. In addition, we show the utility of these multi-omic genetic scores by quantifying the genetic control of biological pathways and by generating a synthetic multi-omic dataset of the UK Biobank 3 to identify disease associations using a phenome-wide scan. We highlight a series of biological insights with regard to genetic mechanisms in metabolism and canonical pathway associations with disease; for example, JAK-STAT signalling and coronary atherosclerosis. Finally, we develop a portal ( https://www.omicspred.org/ ) to facilitate public access to all genetic scores and validation results, as well as to serve as a platform for future extensions and enhancements of multi-omic genetic scores., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Natural killer (NK) cells are cytotoxic and cytokine-producing lymphocytes that play an important role in the first line of defense against malignant or virus-infected cells. A better understanding of the transcriptional regulation of human NK cell differentiation is crucial to improve the efficacy of NK cell-mediated immunotherapy for cancer treatment. Here, we studied the role of the transcription factor interferon regulatory factor (IRF) 2 in human NK cell differentiation by stable knockdown or overexpression in cord blood hematopoietic stem cells and investigated its effect on development and function of the NK cell progeny. IRF2 overexpression had limited effects in these processes, indicating that endogenous IRF2 expression levels are sufficient. However, IRF2 knockdown greatly reduced the cell numbers of all early differentiation stages, resulting in decimated NK cell numbers. This was not caused by increased apoptosis, but by decreased proliferation. Expression of IRF2 is also required for functional maturation of NK cells, as the remaining NK cells after silencing of IRF2 had a less mature phenotype and showed decreased cytotoxic potential, as well as a greatly reduced cytokine secretion. Thus, IRF2 plays an important role during development and functional maturation of human NK cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Persyn, Wahlen, Kiekens, Van Loocke, Siwe, Van Ammel, De Vos, Van Nieuwerburgh, Matthys, Taghon, Vandekerckhove, Van Vlierberghe and Leclercq.)

Understanding how genetic variants influence disease risk and complex traits (variant-to-function) is one of the major challenges in human genetics. Here we present a model-driven framework to leverage human genome-scale metabolic networks to define how genetic variants affect biochemical reaction fluxes across major human tissues, including skeletal muscle, adipose, liver, brain and heart. As proof of concept, we build personalised organ-specific metabolic flux models for 524,615 individuals of the INTERVAL and UK Biobank cohorts and perform a fluxome-wide association study (FWAS) to identify 4312 associations between personalised flux values and the concentration of metabolites in blood. Furthermore, we apply FWAS to identify 92 metabolic fluxes associated with the risk of developing coronary artery disease, many of which are linked to processes previously described to play in role in the disease. Our work demonstrates that genetically personalised metabolic models can elucidate the downstream effects of genetic variants on biochemical reactions involved in common human diseases., (© 2022. The Author(s).)

Type 3 innate lymphoid cells (ILC3s) are characterized by RORγt expression and they produce IL-22 upon activation. ILC3s play a role in maintenance of barrier integrity in the intestine. Under inflammatory conditions, the ILC composition of the mucosal tissues is altered due to a high degree of plasticity. It has been extensively demonstrated that both murine and human ILC3s convert into ILC1s to mediate appropriate immune responses. However, plasticity between human ILC3s and NK cells is less well documented. As T-BET and EOMES are key transcription factors in NK cell differentiation, we investigated whether ectopic T-BET or EOMES expression converts human ILC3s into NK cells. ILC3s with ectopic T-BET and EOMES expression downregulate RORγt expression, while T-BET-overexpressing ILC3s additionally upregulate EOMES expression. High E ctopic T-BET expression in ILC3s results in transdifferentiation towards CD94 + NK cells, whereas ectopic EOMES overexpression results in dedifferentiation of ILC3s into CD94-CD117 -/low cells but is ineffective in NK cell generation. Dedifferentiating ILC3s from both T-BET and EOMES overexpression cultures upregulate NK cell receptors, perforin and granzyme B. Finally, IL-22 secretion is completely blocked in transdifferentiating ILC3s with both T-BET and EOMES ectopic expression, whereas only T-BET overexpression increases IFN-γ secretion and cytotoxicity. Altogether, these findings demonstrate that human ILC3s can convert into functional NK cells, wherein T-BET, and not EOMES, is the main driver., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kiekens, Wahlen, Persyn, De Vos, Taghon, Vandekerckhove and Leclercq.)

Stroke is the second leading cause of death with substantial unmet therapeutic needs. To identify potential stroke therapeutic targets, we estimate the causal effects of 308 plasma proteins on stroke outcomes in a two-sample Mendelian randomization framework and assess mediation effects by stroke risk factors. We find associations between genetically predicted plasma levels of six proteins and stroke (P ≤ 1.62 × 10 -4 ). The genetic associations with stroke colocalize (Posterior Probability >0.7) with the genetic associations of four proteins (TFPI, TMPRSS5, CD6, CD40). Mendelian randomization supports atrial fibrillation, body mass index, smoking, blood pressure, white matter hyperintensities and type 2 diabetes as stroke risk factors (P ≤ 0.0071). Body mass index, white matter hyperintensity and atrial fibrillation appear to mediate the TFPI, IL6RA, TMPRSS5 associations with stroke. Furthermore, thirty-six proteins are associated with one or more of these risk factors using Mendelian randomization. Our results highlight causal pathways and potential therapeutic targets for stroke., (© 2022. The Author(s).)

The ability of natural killer (NK) cells to kill tumor cells without prior sensitization makes them a rising player in immunotherapy. Increased understanding of the development and functioning of NK cells will improve their clinical utilization. As opposed to murine NK cell development, human NK cell development is still less understood. Here, we studied the role of thioredoxin-interacting protein (TXNIP) in human NK cell differentiation by stable TXNIP knockdown or overexpression in cord blood hematopoietic stem cells, followed by in vitro NK cell differentiation. TXNIP overexpression only had marginal effects, indicating that endogenous TXNIP levels are sufficient in this process. TXNIP knockdown, however, reduced proliferation of early differentiation stages and greatly decreased NK cell numbers. Transcriptome analysis and experimental confirmation showed that reduced protein synthesis upon TXNIP knockdown likely caused this low proliferation. Contrary to its profound effects on the early differentiation stages, TXNIP knockdown led to limited alterations in NK cell phenotype, and it had no effect on NK cell cytotoxicity or cytokine production. Thus, TXNIP promotes human NK cell differentiation by affecting protein synthesis and proliferation of early NK cell differentiation stages, but it is redundant for functional NK cell maturation.

We investigated the in vitro susceptibility of ceftazidime-avibactam (CZA) resistant Stenotrophomonas maltophilia to the associations aztreonam/amoxicillin-clavulanate (ATM-AMC) and ATM-CZA. Forty clinical isolates of S. maltophilia recovered from sputum samples of 40 cystic fibrosis people were selected from the collection of the Nantes University Hospital, based on their resistance to CZA. Minimum inhibitory concentrations (MICs) of ATM-CZA and ATM-AMC were determined for each isolate by an Etest strip superposition method, and by Etest for each individual antibiotic. MICs of CZA, ATM, and AMC ranged from 12 to ≥256, ≥256, and 16 to ≥256 mg/L, respectively. Synergistic effects were observed with the ATM-CZA combination for all isolates (fractional inhibitory concentration index range of 0.01 to 0.27), with combination MICs ranging from 0.75 to 16 mg/L (MIC 50/90  = 3/12 mg/L), corresponding to a decrease of at least 16-folds in the MIC of ATM. In 23 (57.5%) S. maltophilia isolates, the association of AMC to ATM was also synergistic and combination MICs were ≤16 mg/L (EUCAST breakpoint for ATM resistance in Pseudomonas aeruginosa ). Our results show that ATM-CZA or ATM-AMC could be alternative therapeutic options against some highly resistant S. maltophilia . This encourages further experimental studies, in particular time-kill analyses, and clinical trials to delineate conditions required for use of these combinations in practice.

The bitter taste, triggered via gustatory receptors, serves as an important natural defense against the ingestion of poisonous foods in animals, and the increased host breadth is usually linked to an increase in the number of gustatory receptor genes. This has been especially observed in polyphagous insect species, such as noctuid species from the Spodoptera genus. However, the dynamic and physical mechanisms leading to these gene expansions and the evolutionary pressures behind them remain elusive. Among major drivers of genome dynamics are the transposable elements but, surprisingly, their potential role in insect gustatory receptor expansion has not been considered yet. In this work, we hypothesized that transposable elements and possibly positive selection would be involved in the highly dynamic evolution of gustatory receptor in Spodoptera spp. We first sequenced de novo the full 465 Mb genome of S. littoralis, and manually annotated the main chemosensory genes, including a large repertoire of 373 gustatory receptor genes (including 19 pseudogenes). We also improved the completeness of S. frugiperda and S. litura gustatory receptor gene repertoires. Then, we annotated transposable elements and revealed that a particular category of class I retrotransposons, the SINE transposons, was significantly enriched in the vicinity of gustatory receptor gene clusters, suggesting a transposon-mediated mechanism for the formation of these clusters. Selection pressure analyses indicated that positive selection within the gustatory receptor gene family is cryptic, only 7 receptors being identified as positively selected. Altogether, our data provide a new good quality Spodoptera genome, pinpoint interesting gustatory receptor candidates for further functional studies and bring valuable genomic information on the mechanisms of gustatory receptor expansions in polyphagous insect species., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)

Natural killer (NK) cells are innate lymphocytes that eliminate virus-infected and cancer cells by cytotoxicity and cytokine secretion. In addition to circulating NK cells, distinct tissue-resident NK subsets have been identified in various organs. Although transcription factors regulating NK cell development and function have been extensively studied in mice, the role of RUNX2 in these processes has not been investigated, neither in mice nor in human. Here, by manipulating RUNX2 expression with either knockdown or overexpression in human haematopoietic stem cell-based NK cell differentiation cultures, combined with transcriptomic and ChIP-sequencing analyses, we established that RUNX2 drives the generation of NK cells, possibly through induction of IL-2Rβ expression in NK progenitor cells. Importantly, RUNX2 promotes tissue residency in human NK cells. Our findings have the potential to improve existing NK cell-based cancer therapies and can impact research fields beyond NK cell biology, since tissue-resident subsets have also been described in other lymphocyte subpopulations., Competing Interests: SW, FM, WV, ST, LK, EP, EV, ZD, SD, PM, FV, TT, BV, PV, GL No competing interests declared, (© 2022, Wahlen et al.)

Previous single-center, cross-sectional studies have reported a steep increase in the prevalence and severity of fibrosis through 10 to 15 years after pediatric liver transplantation. We report a multicenter study of paired surveillance biopsies in a contemporary cohort. Children who underwent liver transplant when younger than 6 years old and had paired surveillance liver biopsies were enrolled (n = 78, 35% girls, median 1.2 years old at transplant). A central pathologist graded inflammation, assessed rejection activity index, and staged fibrosis in the portal, sinusoidal, and perivenular compartments, allowing for calculation of the Liver Allograft Fibrosis Score (LAFSc). Analysis of variance tested associations between fibrosis progression and clinical parameters. The first biopsy, at a median 8.2 years (interquartile range, 5.9-11.6 years) after transplantation, showed absent to mild fibrosis (LAFSc 0-2) in 29%, moderate (LAFSc 3-5) in 56%, and severe (LAFSc 6-7) in 14% of patients. The second biopsy, at a median 4.7 years (IQR, 4.3-5.1 years) later, showed fibrosis progression (LAFSc increased by ≥3) in 10 (13%) and regression (LAFSc decreased by ≥3) in 4 (5%) patients. After adjusting for baseline LAFSc, younger age at transplant was the only risk factor for fibrosis progression. Although fibrosis prevalence and severity 6 to 12 years after transplant was similar to previous reports, fibrosis trajectory during the next 4 to 5 years was stable. Our data may be reassuring for children with consistently normal liver tests. A comprehensive understanding of factors determining allograft health during the very long term is essential to optimizing allograft and patient health., (© 2022 by the American Association for the Study of Liver Diseases.)

Background: Cardiorespiratory fitness (CRF) and physical activity (PA) are well-established predictors of morbidity and all-cause mortality. However, CRF is not routinely measured and PA not routinely prescribed as part of standard healthcare. The American Heart Association (AHA) recently presented a scientific case for the inclusion of CRF as a clinical vital sign based on epidemiological and clinical observation. Here, we leverage genetic data in the UK Biobank (UKB) to strengthen the case for CRF as a vital sign and make a case for the prescription of PA., Methods: We derived two CRF measures from the heart rate data collected during a submaximal cycle ramp test: CRF-vo2max, an estimate of the participants' maximum volume of oxygen uptake, per kilogram of body weight, per minute; and CRF-slope, an estimate of the rate of increase of heart rate during exercise. Average PA over a 7-day period was derived from a wrist-worn activity tracker. After quality control, 70,783 participants had data on the two derived CRF measures, and 89,683 had PA data. We performed genome-wide association study (GWAS) analyses by sex, and post-GWAS techniques to understand genetic architecture of the traits and prioritise functional genes for follow-up., Results: We found strong evidence that genetic variants associated with CRF and PA influenced genetic expression in a relatively small set of genes in the heart, artery, lung, skeletal muscle and adipose tissue. These functionally relevant genes were enriched among genes known to be associated with coronary artery disease (CAD), type 2 diabetes (T2D) and Alzheimer's disease (three of the top 10 causes of death in high-income countries) as well as Parkinson's disease, pulmonary fibrosis, and blood pressure, heart rate, and respiratory phenotypes. Genetic variation associated with lower CRF and PA was also correlated with several disease risk factors (including greater body mass index, body fat and multiple obesity phenotypes); a typical T2D profile (including higher insulin resistance, higher fasting glucose, impaired beta-cell function, hyperglycaemia, hypertriglyceridemia); increased risk for CAD and T2D; and a shorter lifespan., Conclusions: Genetics supports three decades of evidence for the inclusion of CRF as a clinical vital sign. Given the genetic, clinical and epidemiological evidence linking CRF and PA to increased morbidity and mortality, regular measurement of CRF as a marker of health and routine prescription of PA could be a prudent strategy to support public health., (© 2021. The Author(s).)

Ecological speciation entails divergent selection on specific traits and ultimately on the developmental pathways responsible for these traits. Selection can act on gene sequences but also on regulatory regions responsible for gene expression. Mimetic butterflies are a relevant system for speciation studies because wing colour pattern (WCP) often diverges between closely related taxa and is thought to drive speciation through assortative mating and increased predation on hybrids. Here, we generate the first transcriptomic resources for a mimetic butterfly of the tribe Ithomiini, Melinaea marsaeus, to examine patterns of differential expression between two subspecies and between tissues that express traits that likely drive reproductive isolation; WCP and chemosensory genes. We sequenced whole transcriptomes of three life stages to cover a large catalogue of transcripts, and we investigated differential expression between subspecies in pupal wing discs and antennae. Eighteen known WCP genes were expressed in wing discs and 115 chemosensory genes were expressed in antennae, with a remarkable diversity of chemosensory protein genes. Many transcripts were differentially expressed between subspecies, including two WCP genes and one odorant receptor. Our results suggest that in M. marsaeus the same genes as in other mimetic butterflies are involved in traits causing reproductive isolation, and point at possible candidates for the differences in those traits between subspecies. Differential expression analyses of other developmental stages and body organs and functional studies are needed to confirm and expand these results. Our work provides key resources for comparative genomics in mimetic butterflies, and more generally in Lepidoptera., (© 2021 European Society for Evolutionary Biology.)

T-bet and Eomes are transcription factors that are known to be important in maturation and function of murine natural killer (NK) cells. Reduced T-BET and EOMES expression results in dysfunctional NK cells and failure to control tumor growth. In contrast to mice, the current knowledge on the role of T-BET and EOMES in human NK cells is rudimentary. Here, we ectopically expressed either T-BET or EOMES in human hematopoietic progenitor cells. Combined transcriptome, chromatin accessibility and protein expression analyses revealed that T-BET or EOMES epigenetically represses hematopoietic stem cell quiescence and non-NK lineage differentiation genes, while activating an NK cell-specific transcriptome and thereby drastically accelerating NK cell differentiation. In this model, the effects of T-BET and EOMES are largely overlapping, yet EOMES shows a superior role in early NK cell maturation and induces faster NK receptor and enhanced CD16 expression. T-BET particularly controls transcription of terminal maturation markers and epigenetically controls strong induction of KIR expression. Finally, NK cells generated upon T-BET or EOMES overexpression display improved functionality, including increased IFN-γ production and killing, and especially EOMES overexpression NK cells have enhanced antibody-dependent cellular cytotoxicity. Our findings reveal novel insights on the regulatory role of T-BET and EOMES in human NK cell maturation and function, which is essential to further understand human NK cell biology and to optimize adoptive NK cell therapies., Competing Interests: An international patent application WO2020070070 was filed by Ghent University (Ghent, Belgium) on 30/09/2019, with title ‘Accelerated human hematopoietic stem cell differentiation towards mature natural killer cells with enhanced antibody-dependent cytotoxic activity’, with LK and GL as inventors. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kiekens, Van Loocke, Taveirne, Wahlen, Persyn, Van Ammel, De Vos, Matthys, Van Nieuwerburgh, Taghon, Van Vlierberghe, Vandekerckhove and Leclercq.)

Achromobacter spp. are increasingly reported among cystic fibrosis patients. Genotyping requires time-consuming methods such as multilocus sequence typing or pulsed-field gel electrophoresis. Therefore, data on the prevalence of multiresistant epidemic clones, especially A. xylosoxidans ST137 (AxST137) and the Danish epidemic strain A. ruhlandii (DES), are lacking. We recently developed and published a database for Achromobacter species identification by matrix-assisted laser desorption-ionization-time of flight mass spectrometry (MALDI-TOF MS; Bruker Daltonics). The aim of this study was to evaluate the ability of the MALDI-TOF MS to distinguish these multiresistant epidemic clones within Achromobacter species. All the spectra of A. xylosoxidans ( n  = 1,571) and A. ruhlandii ( n  = 174) used to build the local database were analyzed by ClinProTools, MALDI Biotyper PCA, MALDI Biotyper dendrogram, and flexAnalysis software for biomarker peak detection. Two hundred two isolates (including 48 isolates of AxST137 and 7 of DES) were tested. Specific biomarker peaks were identified: absent peak at m/z 6,651 for AxST137 isolates and present peak at m/z 9,438 for DES isolates. All tested isolates were well typed by our local database and clustered within distinct groups (ST137 or non-ST137 and DES or non-DES) no matter the MALDI-TOF software or only by simple visual inspection of the spectra by any user. The use of MALDI-TOF MS allowed us to identify isolates of A. xylosoxidans belonging to the AxST137 clone that spread in France and Belgium (the Belgian epidemic clone) and of A. ruhlandii belonging to the DES clone. This tool will help the implementation of segregation measures to avoid interpatient transmission of these resistant clones.

Background: Automated molecular panels are attractive tools for improving early meningitis diagnosis. This study assessed the Eazyplex ® CSF direct M panel (EP), a multiplex real-time Loop-Mediated Isothermal Amplification assay. Methods: From December 2016 to December 2019, cerebrospinal fluid (CSF) samples were routinely tested with the EP V1.0. CSF parameters and microbiological and clinical data were retrospectively collected. Results: Out of 230 CSF samples, the EP yielded positive, negative, and invalid results for 32 (13.9%) (16 N. meningitidis , nine S. pneumoniae , two S. agalactiae , two E. coli , two H. influenzae , one L. monocytogenes ), 182 (79.1%), and 16 (7%) samples, respectively. Among the positive samples, 14 (44%) remained negative in culture (antibiotic therapy before lumbar puncture ( n = 11), meningococcal meningitis ( n = 3)). High CSF protein concentrations and cellularity were associated with LAMP inhibition, counteracted by centrifugation. The automated software yielded 13 false positive and five false negative results. Amplification curve analysis was necessary and enabled the attainment of positive (PPA) and negative percentage agreement and positive and negative predictive values of 91.4%, 100%, 100%, and 98.3%. Three false negative results remained (two E. coli and one N. meningitidis ). E. coli presented the poorest PPA (50%). Conclusion : This work confirms the strong performance of the EP, of particular interest in cases of antibiotic therapy before lumbar puncture.

Background: The genetic basis of lacunar stroke is poorly understood, with a single locus on 16q24 identified to date. We sought to identify novel associations and provide mechanistic insights into the disease., Methods: We did a pooled analysis of data from newly recruited patients with an MRI-confirmed diagnosis of lacunar stroke and existing genome-wide association studies (GWAS). Patients were recruited from hospitals in the UK as part of the UK DNA Lacunar Stroke studies 1 and 2 and from collaborators within the International Stroke Genetics Consortium. Cases and controls were stratified by ancestry and two meta-analyses were done: a European ancestry analysis, and a transethnic analysis that included all ancestry groups. We also did a multi-trait analysis of GWAS, in a joint analysis with a study of cerebral white matter hyperintensities (an aetiologically related radiological trait), to find additional genetic associations. We did a transcriptome-wide association study (TWAS) to detect genes for which expression is associated with lacunar stroke; identified significantly enriched pathways using multi-marker analysis of genomic annotation; and evaluated cardiovascular risk factors causally associated with the disease using mendelian randomisation., Findings: Our meta-analysis comprised studies from Europe, the USA, and Australia, including 7338 cases and 254 798 controls, of which 2987 cases (matched with 29 540 controls) were confirmed using MRI. Five loci (ICA1L-WDR12-CARF-NBEAL1, ULK4, SPI1-SLC39A13-PSMC3-RAPSN, ZCCHC14, ZBTB14-EPB41L3) were found to be associated with lacunar stroke in the European or transethnic meta-analyses. A further seven loci (SLC25A44-PMF1-BGLAP, LOX-ZNF474-LOC100505841, FOXF2-FOXQ1, VTA1-GPR126, SH3PXD2A, HTRA1-ARMS2, COL4A2) were found to be associated in the multi-trait analysis with cerebral white matter hyperintensities (n=42 310). Two of the identified loci contain genes (COL4A2 and HTRA1) that are involved in monogenic lacunar stroke. The TWAS identified associations between the expression of six genes (SCL25A44, ULK4, CARF, FAM117B, ICA1L, NBEAL1) and lacunar stroke. Pathway analyses implicated disruption of the extracellular matrix, phosphatidylinositol 5 phosphate binding, and roundabout binding (false discovery rate <0·05). Mendelian randomisation analyses identified positive associations of elevated blood pressure, history of smoking, and type 2 diabetes with lacunar stroke., Interpretation: Lacunar stroke has a substantial heritable component, with 12 loci now identified that could represent future treatment targets. These loci provide insights into lacunar stroke pathogenesis, highlighting disruption of the vascular extracellular matrix (COL4A2, LOX, SH3PXD2A, GPR126, HTRA1), pericyte differentiation (FOXF2, GPR126), TGF-β signalling (HTRA1), and myelination (ULK4, GPR126) in disease risk., Funding: British Heart Foundation., (Copyright © 2021 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Endogenous viruses form an important proportion of eukaryote genomes and a source of novel functions. How large DNA viruses integrated into a genome evolve when they confer a benefit to their host, however, remains unknown. Bracoviruses are essential for the parasitism success of parasitoid wasps, into whose genomes they integrated ~103 million years ago. Here we show, from the assembly of a parasitoid wasp genome at a chromosomal scale, that bracovirus genes colonized all ten chromosomes of Cotesia congregata. Most form clusters of genes involved in particle production or parasitism success. Genomic comparison with another wasp, Microplitis demolitor, revealed that these clusters were already established ~53 mya and thus belong to remarkably stable genomic structures, the architectures of which are evolutionary constrained. Transcriptomic analyses highlight temporal synchronization of viral gene expression without resulting in immune gene induction, suggesting that no conflicts remain between ancient symbiotic partners when benefits to them converge.

The FilmArray ® Pneumonia plus Panel (FAPP) is a new multiplex molecular test for hospital-acquired pneumonia (HAP), which can rapidly detect 18 bacteria, 9 viruses, and 7 resistance genes. We aimed to compare the diagnosis performance of FAPP with conventional testing in 100 intensive care unit (ICU) patients who required mechanical ventilation, with clinically suspected HAP. A total of 237 samples [76 bronchoalveolar lavages (BAL DS ) and 82 endotracheal aspirates (ETA DS ) obtained at HAP diagnosis, and 79 ETA obtained during follow-up (ETA TT )], were analyzed independently by routine microbiology testing and FAPP. 58 patients had paired BAL DS and ETA DS . The positivity thresholds of semi-quantified bacteria were 10 3 -10 4 CFUs/mL or 10 4 copies/mL for BAL, and 10 5 CFUs/mL or copies/mL for ETA. Respiratory commensals ( H. influenzae , S. aureus , E. coli , S. pneumoniae ) were the most common pathogens. Discordant results for bacterial identification were observed in 33/76 (43.4%) BAL DS and 36/82 (43.9%) ETA DS , and in most cases, FAPP identified one supplemental bacteria (23/33 BAL DS and 21/36 ETA DS ). An absence of growth, or polybacterial cultures, explained almost equally the majority of the non-detections in culture. No linear relationship was observed between bin and CFUs/mL variables. Concordant results between paired BAL DS and ETA DS were obtained in 46/58 (79.3%) patients with FAPP. One of the 17 resistance genes detected with FAPP ( mecA/C and MREJ) was not confirmed by conventional testing. Overall, FAPP enhanced the positivity rate of diagnostic testing, with increased recognition of coinfections. Implementing this strategy may allow clinicians to make more timely and informed decisions., (Copyright © 2020 Crémet, Gaborit, Bouras, Drumel, Guillotin, Poulain, Persyn, Lakhal, Rozec, Vibet, Roquilly and Gibaud.)

Natural killer (NK) cells are important in the immune defense against tumor cells and pathogens, and they regulate other immune cells by cytokine secretion. Although murine NK cell biology has been extensively studied, knowledge about transcriptional circuitries controlling human NK cell development and maturation is limited. By generating ETS1-deficient human embryonic stem cells and by expressing the dominant-negative ETS1 p27 isoform in cord blood hematopoietic progenitor cells, we show that the transcription factor ETS1 is critically required for human NK cell differentiation. Genome-wide transcriptome analysis determined by RNA-sequencing combined with chromatin immunoprecipitation-sequencing analysis reveals that human ETS1 directly induces expression of key transcription factors that control NK cell differentiation (ie, E4BP4, TXNIP, TBET, GATA3, HOBIT, BLIMP1). In addition, ETS1 regulates expression of genes involved in apoptosis and NK cell activation. Our study provides important molecular insights into the role of ETS1 as an important regulator of human NK cell development and terminal differentiation., (© 2020 by The American Society of Hematology.)

The tremendous diversity of Hymenoptera is commonly attributed to the evolution of parasitoidism in the last common ancestor of parasitoid sawflies (Orussidae) and wasp-waisted Hymenoptera (Apocrita). However, Apocrita and Orussidae differ dramatically in their species richness, indicating that the diversification of Apocrita was promoted by additional traits. These traits have remained elusive due to a paucity of sawfly genome sequences, in particular those of parasitoid sawflies. Here, we present comparative analyses of draft genomes of the primarily phytophagous sawfly Athalia rosae and the parasitoid sawfly Orussus abietinus. Our analyses revealed that the ancestral hymenopteran genome exhibited traits that were previously considered unique to eusocial Apocrita (e.g., low transposable element content and activity) and a wider gene repertoire than previously thought (e.g., genes for CO2 detection). Moreover, we discovered that Apocrita evolved a significantly larger array of odorant receptors than sawflies, which could be relevant to the remarkable diversification of Apocrita by enabling efficient detection and reliable identification of hosts., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Cerebral small vessel disease is a major cause of stroke and dementia, but its genetic basis is incompletely understood. We perform a genetic study of three MRI markers of the disease in UK Biobank imaging data and other sources: white matter hyperintensities (N = 42,310), fractional anisotropy (N = 17,663) and mean diffusivity (N = 17,467). Our aim is to better understand the disease pathophysiology. Across the three traits, we identify 31 loci, of which 21 were previously unreported. We perform a transcriptome-wide association study to identify associations with gene expression in relevant tissues, identifying 66 associated genes across the three traits. This genetic study provides insights into the understanding of the biological mechanisms underlying small vessel disease.

Biofilms produced by Pseudomonas aeruginosa present a serious threat to cystic fibrosis patients. Here, we report the draft genome sequences of four cystic fibrosis isolates displaying various mucoid and biofilm phenotypes. The estimated average genome size was about 6,255,986 ± 50,202 bp with a mean G+C content of 66.52 ± 0.06%., (Copyright © 2020 Boukerb et al.)

Treatment with antibiotics leads to the selection of isolates with increased resistance. We investigated if evolution towards resistance was associated with virulence changes, in the context of P. aeruginosa ventilator-associated pneumonia (VAP). Four patients were selected because they had multiple VAP episodes during short periods (12 days to 5 weeks), with emergence of resistance. We performed whole-genome sequencing of 12 P. aeruginosa from bronchoalveolar lavages or blood culture (3 isolates per patient). Production of quorum sensing-dependent virulence factors, serum resistance, cytotoxicity against A549 cells, biofilm production, and twitching motility were studied. Each patient was infected with a unique strain. For all patients, resistance development was explained by genetic events in ampD, mexR or oprD. Additional variations were detected in virulence- and/or fitness-associated genes (algB, gacA, groEL, lasR, mpl, pilE, pilM, rhlR) depending on the strain. We noticed a convergence towards quorum sensing deficiency, correlated with a decrease of pyocyanin and protease production, survival in serum, twitching motility and cytotoxicity. In one patient, changes in pilM and pilE were related to enhanced twitching. We show that the emergence of resistance in P. aeruginosa is associated with virulence modification, even in acute infections. The consequences of this short-term pathoadaptation need to be explored.

Population stratification is a well-known confounding factor in both common and rare variant association analyses. Rare variants tend to be more geographically clustered than common variants, because of their more recent origin. However, it is not yet clear if population stratification at a very fine scale (neighboring administrative regions within a country) would lead to statistical bias in rare variant analyses. As the inclusion of convenience controls from external studies is indeed a common procedure, in order to increase the power to detect genetic associations, this problem is important. We studied through simulation the impact of a fine scale population structure on different rare variant association strategies, assessing type I error and power. We showed that principal component analysis (PCA) based methods of adjustment for population stratification adequately corrected type I error inflation at the largest geographical scales, but not at finest scales. We also showed in our simulations that adding controls obviously increased power, but at a considerably lower level when controls were drawn from another population., Competing Interests: The authors have declared that no competing interests exist.

Next-generation sequencing technologies made it possible to assay the effect of rare variants on complex diseases. As an extension of the "common disease-common variant" paradigm, rare variant studies are necessary to get a more complete insight into the genetic architecture of human traits. Association studies of these rare variations show new challenges in terms of statistical analysis. Due to their low frequency, rare variants must be tested by groups. This approach is then hindered by the fact that an unknown proportion of the variants could be neutral. The risk level of a rare variation may be determined by its impact but also by its position in the protein sequence. More generally, the molecular mechanisms underlying the disease architecture may involve specific protein domains or inter-genic regulatory regions. While a large variety of methods are optimizing functionality weights for each single marker, few evaluate variant position differences between cases and controls. Here, we propose a test called DoEstRare, which aims to simultaneously detect clusters of disease risk variants and global allele frequency differences in genomic regions. This test estimates, for cases and controls, variant position densities in the genetic region by a kernel method, weighted by a function of allele frequencies. We compared DoEstRare with previously published strategies through simulation studies as well as re-analysis of real datasets. Based on simulation under various scenarios, DoEstRare was the sole to consistently show highest performance, in terms of type I error and power both when variants were clustered or not. DoEstRare was also applied to Brugada syndrome and early-onset Alzheimer's disease data and provided complementary results to other existing tests. DoEstRare, by integrating variant position information, gives new opportunities to explain disease susceptibility. DoEstRare is implemented in a user-friendly R package.

The Brugada syndrome (BrS) is a rare heritable cardiac arrhythmia disorder associated with ventricular fibrillation and sudden cardiac death. Mutations in the SCN5A gene have been causally related to BrS in 20-30% of cases. Twenty other genes have been described as involved in BrS, but their overall contribution to disease prevalence is still unclear. This study aims to estimate the burden of rare coding variation in arrhythmia-susceptibility genes among a large group of patients with BrS. We have developed a custom kit to capture and sequence the coding regions of 45 previously reported arrhythmia-susceptibility genes and applied this kit to 167 index cases presenting with a Brugada pattern on the electrocardiogram as well as 167 individuals aged over 65-year old and showing no history of cardiac arrhythmia. By applying burden tests, a significant enrichment in rare coding variation (with a minor allele frequency below 0.1%) was observed only for SCN5A, with rare coding variants carried by 20.4% of cases with BrS versus 2.4% of control individuals (P = 1.4 × 10(-7)). No significant enrichment was observed for any other arrhythmia-susceptibility gene, including SCN10A and CACNA1C. These results indicate that, except for SCN5A, rare coding variation in previously reported arrhythmia-susceptibility genes do not contribute significantly to the occurrence of BrS in a population with European ancestry. Extreme caution should thus be taken when interpreting genetic variation in molecular diagnostic setting, since rare coding variants were observed in a similar extent among cases versus controls, for most previously reported BrS-susceptibility genes., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

1-Arylmethyl-2-(cyanomethyl)aziridines were prepared in high yields from the corresponding 2-(bromomethyl)aziridines upon treatment with potassium cyanide in DMSO. Ring opening of the aziridine moiety with N-chlorosuccinimide in CCl4 and subsequent treatment of the thus formed 4-chloro-3-(N-chloro-N-(alpha,alpha-dichlorobenzyl)amino)butanenitriles with sodium methoxide in methanol resulted in novel methyl N-(2-chloro-1-(cyanomethyl)ethyl)benzimidates, although in low yields. The latter gamma-chloro nitriles were smoothly converted into methyl N-(2-cyanocyclopropyl)benzimidates as precursors of biologically relevant beta-ACC derivatives through a 1,3-cyclization protocol by reaction with potassium tert-butoxide in THF.