Search

Background\ud\udPalmoplantar pustulosis (PPP) is a severe inflammatory skin disorder, characterised by eruptions of painful, neutrophil-filled pustules on the palms and soles. While PPP has a profound effect on quality of life, it remains poorly understood and notoriously difficult to treat.\udObjective\ud\udWe sought to investigate the immune pathways that underlie the pathogenesis of PPP.\udMethods\ud\udWe applied bulk- and single-cell RNA-sequencing methods to the analysis of skin biopsies and peripheral blood mononuclear cells. We validated our results by flow cytometry and immune fluorescence microscopy\udResults\ud\udBulk RNA-sequencing of patient skin detected an unexpected signature of T-cell activation, with a significant overexpression of several Th2 genes typically upregulated in atopic dermatitis. To further explore these findings, we carried out single-cell RNA-sequencing in peripheral blood mononuclear cells of healthy and affected individuals. We found that the memory CD4+T-cells of PPP patients were skewed towards a Th17 phenotype, a phenomenon that was particularly significant among CLA+ skin-homing cells. We also identified a subset of memory CD4+ T-cells which expressed both Th17 (KLRB1/CD161) and Th2 (GATA3) markers, with pseudo-time analysis suggesting that the population was the result of Th17 to Th2 plasticity. Interestingly, the GATA3+/CD161+ cells were over-represented among the PBMCs of affected individuals, both in the scRNA-seq dataset and in independent flow-cytometry experiments. Dual positive cells were also detected in patient skin by means of immune fluorescence microscopy.\udConclusions\ud\udThese observations demonstrate that PPP is associated with complex T-cell activation patterns and may explain why biologics that target individual T-helper populations have shown limited therapeutic efficacy.\udClinical implications\ud\udThe simultaneous activation of Th17 and Th2 responses in PPP supports the therapeutic use of agents that inhibit multiple T-cell pathways.

The identification of disease alleles underlying human autoinflammatory diseases can provide important insights into the mechanisms that maintain neutrophil homeostasis. Here, we focused our attention on generalized pustular psoriasis (GPP), a potentially life-threatening disorder presenting with cutaneous and systemic neutrophilia. Following the whole-exome sequencing of 19 unrelated affected individuals, we identified a subject harboring a homozygous splice-site mutation (c.2031−2A>C) in MPO. This encodes myeloperoxidase, an essential component of neutrophil azurophil granules. MPO screening in conditions phenotypically related to GPP uncovered further disease alleles in one subject with acral pustular psoriasis (c.2031−2A>C;c.2031−2A>C) and in two individuals with acute generalized exanthematous pustulosis (c.1705C>T;c.2031−2A>C and c.1552_1565del;c.1552_1565del). A subsequent analysis of UK Biobank data demonstrated that the c.2031−2A>C and c.1705C>T (p.Arg569Trp) disease alleles were also associated with increased neutrophil abundance in the general population (p = 5.1 × 10−6 and p = 3.6 × 10−5, respectively). The same applied to three further deleterious variants that had been genotyped in the cohort, with two alleles (c.995C>T [p.Ala332Val] and c.752T>C [p.Met251Thr]) yielding p values < 10−10. Finally, treatment of healthy neutrophils with an MPO inhibitor (4-Aminobenzoic acid hydrazide) increased cell viability and delayed apoptosis, highlighting a mechanism whereby MPO mutations affect granulocyte numbers. These findings identify MPO as a genetic determinant of pustular skin disease and neutrophil abundance. Given the recent interest in the development of MPO antagonists for the treatment of neurodegenerative disease, our results also suggest that the pro-inflammatory effects of these agents should be closely monitored.

We propose a new notion of causal contribution which describes the 'intrinsic' part of the contribution of a node on a target node in a DAG. We show that in some scenarios the existing causal quantification methods failed to capture this notion exactly. By recursively writing each node as a function of the upstream noise terms, we separate the intrinsic information added by each node from the one obtained from its ancestors. To interpret the intrinsic information as a causal contribution, we consider 'structure-preserving interventions' that randomize each node in a way that mimics the usual dependence on the parents and do not perturb the observed joint distribution. To get a measure that is invariant across arbitrary orderings of nodes we propose Shapley based symmetrization. We describe our contribution analysis for variance and entropy, but contributions for other target metrics can be defined analogously.

We describe a formal approach to identify 'root causes' of outliers observed in $n$ variables $X_1,\dots,X_n$ in a scenario where the causal relation between the variables is a known directed acyclic graph (DAG). To this end, we first introduce a systematic way to define outlier scores. Further, we introduce the concept of 'conditional outlier score' which measures whether a value of some variable is unexpected *given the value of its parents* in the DAG, if one were to assume that the causal structure and the corresponding conditional distributions are also valid for the anomaly. Finally, we quantify to what extent the high outlier score of some target variable can be attributed to outliers of its ancestors. This quantification is defined via Shapley values from cooperative game theory., 11 pages, 9 Figures

Background & Aims: Excess liver iron content is common and is linked to the risk of hepatic and extrahepatic diseases. We aimed to identify genetic variants influencing liver iron content and use genetics to understand its link to other traits and diseases. Methods: First, we performed a genome-wide association study (GWAS) in 8,289 individuals from UK Biobank, whose liver iron level had been quantified by magnetic resonance imaging, before validating our findings in an independent cohort (n = 1,513 from IMI DIRECT). Second, we used Mendelian randomisation to test the causal effects of 25 predominantly metabolic traits on liver iron content. Third, we tested phenome-wide associations between liver iron variants and 770 traits and disease outcomes. Results: We identified 3 independent genetic variants (rs1800562 [C282Y] and rs1799945 [H63D] in HFE and rs855791 [V736A] in TMPRSS6) associated with liver iron content that reached the GWAS significance threshold (p

A phenocopy is defined as an environmentally induced phenotype of one individual which is identical to the genotype determined phenotype of another individual. In the present work, the phenocopy phenomenon has been translated to the drug discovery process as phenotypes produced by the treatment of cellular systems with small interfering RNAs (siRNAs) or new chemical entities (NCE) may resemble environmentally induced phenotypic modifications. Various new chemical entities exerting inhibition of the kinase activity of Transforming Growth Factor Beta Receptor I (TGF-betaR1) were ranked by high-throughput RNA expression profiling. This chemical genomics approach was able to unravel both on-target effects (effects, caused by the inhibition of the drug target) and off-target effects (effects, caused by the interaction of the NCE with additional molecules). It resulted in a precise time-dependent insight into the TGF-beta biology (referred to as on-target signature) and allowed furthermore a comprehensive analysis of each NCE's off-target effects (re-ferred to as off-target signatures). Both signature types can support the drug discovery process. The on-target signature helps to characterize the mode of action of the drug target (TGF-betaR1) and thereby supports the target validation as well as the assay development process. Furthermore, the evaluation of off-target effects by the Phenocopy approach allows a more accurate and integrated view on the mode of action of the compounds, supplementing classical biological evaluation parameters such as potency and selectivity. The presented proof of concept study allowed the ranking of NCEs that were before indistinguishable solely based on potency and selectivity. According to the newly introduced criteria, several of the tested NCEs revealed liabilities at e.g. the induction of off-target effects and of induction of gene regulation inverse to the desired TGF-beta inhibition effect, at the induction of cell death, at acting as pro-inflammatory stimuli and as promoting cellular growth and at induction of cancer pathways. Ultimately, this approach has therefore the potential to become a novel method for ranking compounds during various drug discovery phases., Eine Phänokopie ist als ein Individuum definiert, dessen Phänotyp durch einen Umwelteinfluss mit dem eines anderen Individuums identisch ist, dessen Phänotyp durch seinen Genotyp bestimmt ist. In der vorliegenden Arbeit wurde das Phänokopiephänomen auf den Wirkstoffentwicklungsprozess übertragen. Hierbei wurden die speziellen Phänotypen in einem Zellsystem durch die Behandlung mit "new chemical entities" (NCE), potentiellen neuen Wirkstoffkandidaten, oder mittels "small interfering RNAs" (siRNAs) induziert. Somit können beide Molekülklassen als externe Umweltbedingung angesehen werden. Hierbei wurden diverse der potentiellen Wirkstoffkandidaten zur Inhibition der Kinaseaktivität von Transforming Growth Factor Beta Receptor I (TGF-betaR1) mittels Hochdurchsatz-Expressionsprofilierung klassifiziert. Dieser „chemical genomics“ Ansatz war in der Lage, sowohl die On-target Effekte (Effekte, welche durch die Inhibition des Wirkstofftargets aus-gelöst werden), als auch die Off-target Effekte (Effekte, welche durch die Interaktion des NCEs mit zusätzlichen Molekülen ausgelöst werden) zu identifizieren. Weiterhin ermöglichte er präzise, zeitlich aufgelöste Einblicke in die TGF-beta Biologie (im Folgenden als On-target Signatur bezeichnet) und erlaubte eine umfassende Analyse der jeweiligen Off-target Effekte der Wirkstoffkandidaten (im Folgenden als Off-target Signatur bezeichnet). Beide Signaturtypen können zur Unterstützung des Wirkstoffentwicklungsprozesses herangezogen werden. Die On-target Signatur charakterisiert die Wirkungsweise des Wirkstoffziels (target) und unterstützt somit sowohl den "target validation" Prozess, als auch den "assay development" Prozess. Des Weiteren erlaubt das Ermitteln der Off-target Effekte durch den Phänokopieansatz einen präzisen und ganzheitlichen Einblick in die Wirkungsweise der Wirkstoffkandidaten und ergänzt klassische, biologische Bewertungsparameter wie Wirksamkeit und Selektivität. Die hier präsentierte Machbarkeitsstudie ermöglicht die Klassifizierung von Wirkstoffkandidaten, die zuvor auf der Basis von Wirksamkeit und Selektivität nicht zu unterscheiden waren. Entsprechend der neu eingeführten Kriterien zeigen verschiedene der getesteten Wirkstoffkandidaten unterschiedliche Vorbelastungen, wie z.B. das Auslösen von Off-target Effekten und von Genregulation, welche gegenläufig zu dem gewünschten TGF-beta Inhibitionseffekt verlaufen. Des Weiteren konnte gezeigt werden, dass manche NCEs Zelltod induzieren, als proinflammatorische Stimuli fungieren, das Zellwachstum steigern könnten und Krebssignalwege induzieren. Letztendlich hat dieser Ansatz somit das Potential eine neue Methode zur Klassifizierung von neuen Wirkstoffkandidaten, während verschiedenster Phasen des Wirkstoffentwicklungsprozesses, zu werden.