To date only a fraction of the genetic footprint of thyroid function has been clarified. We report a genome-wide association study meta-analysis of thyroid function in up to 271,040 individuals of... Show moreTo date only a fraction of the genetic footprint of thyroid function has been clarified. We report a genome-wide association study meta-analysis of thyroid function in up to 271,040 individuals of European ancestry, including reference range thyrotropin (TSH), free thyroxine (FT4), free and total triiodothyronine (T3), proxies for metabolism (T3/FT4 ratio) as well as dichotomized high and low TSH levels. We revealed 259 independent significant associations for TSH (61% novel), 85 for FT4 (67% novel), and 62 novel signals for the T3 related traits. The loci explained 14.1%, 6.0%, 9.5% and 1.1% of the total variation in TSH, FT4, total T3 and free T3 concentrations, respectively. Genetic correlations indicate that TSH associated loci reflect the thyroid function determined by free T3, whereas the FT4 associations represent the thyroid hormone metabolism. Polygenic risk score and Mendelian randomization analyses showed the effects of genetically determined variation in thyroid function on various clinical outcomes, including cardiovascular risk factors and diseases, autoimmune diseases, and cancer. In conclusion, our results improve the understanding of thyroid hormone physiology and highlight the pleiotropic effects of thyroid function on various diseases. Show less
Kissel, T.; Derksen, V.F.A.M.; Bentlage, A.E.H.; Koeleman, C.; Hafkenscheid, L.; Woude, D. van der; ... ; Toes, R.E.M. 2024
IgG secreted by B cells carry asparagine N(297)-linked glycans in the fragment crystallizable (Fc) region. Changes in Fc glycosylation are related to health or disease and are functionally relevant... Show moreIgG secreted by B cells carry asparagine N(297)-linked glycans in the fragment crystallizable (Fc) region. Changes in Fc glycosylation are related to health or disease and are functionally relevant, as IgG without Fc glycans cannot bind to Fcɣ receptors or complement factors. However, it is currently unknown whether ɣ-heavy chain (ɣHC) glycans also influence the function of membrane-bound IgG-B-cell receptors (BCR) and thus the outcome of the B-cell immune response. Here, we show in a germinal center (GC)-derived human B-cell line that ɣHC glycans do not affect membrane expression of IgG-BCRs. Furthermore, antigen binding or other BCR-facilitated mechanisms appear unaffected, including BCR downmodulation or BCR-mediated signaling. As expected, secreted IgG lacking Fc glycosylation is unable to carry out effector functions. Together, these observations indicate that IgG-Fc glycosylation serves as a mechanism to control the effector functions of antibodies, but does not regulate the activation of IgG-switched B cells, as its absence had no apparent impact on BCR function. Show less
Bacillus Calmette-Guèrin - vaccination induces not only protection in infants and young children against severe forms of tuberculosis, but also against nontuberculosis related all-cause mortality.... Show moreBacillus Calmette-Guèrin - vaccination induces not only protection in infants and young children against severe forms of tuberculosis, but also against nontuberculosis related all-cause mortality. To delineate different factors influencing mycobacterial growth control, here we first investigate the effects of BCG-vaccination in healthy Dutch adults. About a quarter of individuals already control BCG-growth prior to vaccination, whereas a quarter of the vaccinees acquires the capacity to control BCG upon vaccination. This leaves half of the population incapable to control BCG-growth. Single cell RNA sequencing identifies multiple processes associated with mycobacterial growth control. These data suggest (i) that already controllers employ different mechanisms to control BCG-growth than acquired controllers, and (ii) that half of the individuals fail to develop measurable growth control irrespective of BCG-vaccination. These results shed important new light on the variable immune responses to mycobacteria in humans and may impact on improved vaccination against tuberculosis and other diseases. Show less
G protein-coupled receptors (GPCRs) mediate responses to various extracellular and intracellular cues. However, the large number of GPCR genes and their substantial functional redundancy make it... Show moreG protein-coupled receptors (GPCRs) mediate responses to various extracellular and intracellular cues. However, the large number of GPCR genes and their substantial functional redundancy make it challenging to systematically dissect GPCR functions in vivo. Here, we employ a CRISPR/Cas9-based approach, disrupting 1654 GPCR-encoding genes in 284 strains and mutating 152 neuropeptide-encoding genes in 38 strains in C. elegans. These two mutant libraries enable effective deorphanization of chemoreceptors, and characterization of receptors for neuropeptides in various cellular processes. Mutating a set of closely related GPCRs in a single strain permits the assignment of functions to GPCRs with functional redundancy. Our analyses identify a neuropeptide that interacts with three receptors in hypoxia-evoked locomotory responses, unveil a collection of regulators in pathogen-induced immune responses, and define receptors for the volatile food-related odorants. These results establish our GPCR and neuropeptide mutant libraries as valuable resources for the C. elegans community to expedite studies of GPCR signaling in multiple contexts. Show less
Counteracting the overactivation of glucocorticoid receptors (GR) is an important therapeutic goal in stress-related psychiatry and beyond. The only clinically approved GR antagonist lacks... Show moreCounteracting the overactivation of glucocorticoid receptors (GR) is an important therapeutic goal in stress-related psychiatry and beyond. The only clinically approved GR antagonist lacks selectivity and induces unwanted side effects. To complement existing tools of small-molecule-based inhibitors, we present a highly potent, catalytically-driven GR degrader, KH-103, based on proteolysis-targeting chimera technology. This selective degrader enables immediate and reversible GR depletion that is independent of genetic manipulation and circumvents transcriptional adaptations to inhibition. KH-103 achieves passive inhibition, preventing agonistic induction of gene expression, and significantly averts the GR's genomic effects compared to two currently available inhibitors. Application in primary-neuron cultures revealed the dependency of a glucocorticoid-induced increase in spontaneous calcium activity on GR. Finally, we present a proof of concept for application in vivo. KH-103 opens opportunities for a more lucid interpretation of GR functions with translational potential.Stress-hormone receptors are important therapeutic targets for many diseases but the currently clinically approved inhibitor lacks specificity. Here the authors present a stress hormone receptor depletion tool that differs in its mode of action making it specific in counteracting the effects of stress. Show less
Jiang, M.; Huizenga, M.C.W.; Wirth, J.L.; Paloczi, J.; Amedi, A.; Berg, R.J.B.H.N. van den; ... ; Stelt, M. van der 2023
Monoacylglycerol lipase (MAGL) regulates endocannabinoid 2-arachidonoylglycerol (2-AG) and eicosanoid signalling. MAGL inhibition provides therapeutic opportunities but clinical potential is... Show moreMonoacylglycerol lipase (MAGL) regulates endocannabinoid 2-arachidonoylglycerol (2-AG) and eicosanoid signalling. MAGL inhibition provides therapeutic opportunities but clinical potential is limited by central nervous system (CNS)-mediated side effects. Here, we report the discovery of LEI-515, a peripherally restricted, reversible MAGL inhibitor, using high throughput screening and a medicinal chemistry programme. LEI-515 increased 2-AG levels in peripheral organs, but not mouse brain. LEI-515 attenuated liver necrosis, oxidative stress and inflammation in a CCl4-induced acute liver injury model. LEI-515 suppressed chemotherapy-induced neuropathic nociception in mice without inducing cardinal signs of CB1 activation. Antinociceptive efficacy of LEI-515 was blocked by CB2, but not CB1, antagonists. The CB1 antagonist rimonabant precipitated signs of physical dependence in mice treated chronically with a global MAGL inhibitor (JZL184), and an orthosteric cannabinoid agonist (WIN55,212-2), but not with LEI-515. Our data support targeting peripheral MAGL as a promising therapeutic strategy for developing safe and effective anti-inflammatory and analgesic agents. Show less
Hundreds of E3 ligases play a critical role in recognizing specific substrates for modification by ubiquitin (Ub). Separating genuine targets of E3s from E3-interactors remains a challenge. We... Show moreHundreds of E3 ligases play a critical role in recognizing specific substrates for modification by ubiquitin (Ub). Separating genuine targets of E3s from E3-interactors remains a challenge. We present BioE3, a powerful approach for matching substrates to Ub E3 ligases of interest. Using BirA-E3 ligase fusions and bioUb, site-specific biotinylation of Ub-modified substrates of particular E3s facilitates proteomic identification. We show that BioE3 identifies both known and new targets of two RING-type E3 ligases: RNF4 (DNA damage response, PML bodies), and MIB1 (endocytosis, autophagy, centrosome dynamics). Versatile BioE3 identifies targets of an organelle-specific E3 (MARCH5) and a relatively uncharacterized E3 (RNF214). Furthermore, BioE3 works with NEDD4, a HECT-type E3, identifying new targets linked to vesicular trafficking. BioE3 detects altered specificity in response to chemicals, opening avenues for targeted protein degradation, and may be applicable for other Ub-likes (UbLs, e.g., SUMO) and E3 types. BioE3 applications shed light on cellular regulation by the complex UbL network. Show less
Infections and vaccines can induce enhanced long-term responses in innate immune cells, establishing an innate immunological memory termed trained immunity. Here, we show that monocytes with a... Show moreInfections and vaccines can induce enhanced long-term responses in innate immune cells, establishing an innate immunological memory termed trained immunity. Here, we show that monocytes with a trained immunity phenotype, due to exposure to the Bacillus Calmette-Guerin (BCG) vaccine, are characterized by an increased biosynthesis of different lipid mediators (LM) derived from long-chain polyunsaturated fatty acids (PUFA). Pharmacological and genetic approaches show that long-chain PUFA synthesis and lipoxygenase-derived LM are essential for the BCG-induced trained immunity responses of human monocytes. Furthermore, products of 12-lipoxygenase activity increase in monocytes of healthy individuals after BCG vaccination. Grasping the underscoring lipid metabolic pathways contributes to our understanding of trained immunity and may help to identify therapeutic tools and targets for the modulation of innate immune responses.Cellular functional states are supported by metabolic pathways, including lipid metabolism. Here, authors examine the contribution of differential biosynthesis of lipid mediators to innate immune memory (or trained immunity), in human monocytes following Bacillus Calmette-Guerin (BCG) vaccination. Show less
Stallen, M.; Snijder, L.L.; Gross, J.; Hilbert, L.P.; Dreu, C.K.W. de 2023
Cooperation is more likely when individuals can choose their interaction partner. However, partner choice may be detrimental in unequal societies, in which individuals differ in available resources... Show moreCooperation is more likely when individuals can choose their interaction partner. However, partner choice may be detrimental in unequal societies, in which individuals differ in available resources and productivity, and thus in their attractiveness as interaction partners. Here we experimentally examine this conjecture in a repeated public goods game. Individuals (n = 336), participating in groups of eight participants, are assigned a high or low endowment and a high or low productivity factor (the value that their cooperation generates), creating four unique participant types. On each round, individuals are either assigned a partner (assigned partner condition) or paired based on their self-indicated preference for a partner type (partner choice condition). Results show that under partner choice, individuals who were assigned a high endowment and high productivity almost exclusively interact with each other, forcing other individuals into less valuable pairs. Consequently, pre-existing resource differences between individuals increase. These findings show how partner choice in social dilemmas can amplify resource inequality. Show less
The interplay between 3D chromatin architecture and gene silencing is incompletely understood. Here, we report a novel point mutation in the non-canonical SMC protein SMCHD1 that enhances its... Show moreThe interplay between 3D chromatin architecture and gene silencing is incompletely understood. Here, we report a novel point mutation in the non-canonical SMC protein SMCHD1 that enhances its silencing capacity at endogenous developmental targets. Moreover, it also results in enhanced silencing at the facioscapulohumeral muscular dystrophy associated macrosatellite-array, D4Z4, resulting in enhanced repression of DUX4 encoded by this repeat. Heightened SMCHD1 silencing perturbs developmental Hox gene activation, causing a homeotic transformation in mice. Paradoxically, the mutant SMCHD1 appears to enhance insulation against other epigenetic regulators, including PRC2 and CTCF, while depleting long range chromatin interactions akin to what is observed in the absence of SMCHD1. These data suggest that SMCHD1's role in long range chromatin interactions is not directly linked to gene silencing or insulating the chromatin, refining the model for how the different levels of SMCHD1-mediated chromatin regulation interact to bring about gene silencing in normal development and disease.Here the authors reveal that a neomorphic mutation in chromatin protein SMCHD1 enhances SMCHD1-mediated gene silencing, including at the FSHD disease-relevant locus, while depleting SMCHD1-mediated chromatin interactions, suggesting these SMCHD1 functions are unlinked. Show less
The SUMO protease SENP6 maintains genomic stability, but mechanistic understanding of this process remains limited. We find that SENP6 deconjugates SUMO2/3 polymers on a group of DNA damage... Show moreThe SUMO protease SENP6 maintains genomic stability, but mechanistic understanding of this process remains limited. We find that SENP6 deconjugates SUMO2/3 polymers on a group of DNA damage response proteins, including BRCA1-BARD1, 53BP1, BLM and ERCC1-XPF. SENP6 maintains these proteins in a hypo-SUMOylated state under unstressed conditions and counteracts their polySUMOylation after hydroxyurea-induced stress. Co-depletion of RNF4 leads to a further increase in SUMOylation of BRCA1, BARD1 and BLM, suggesting that SENP6 antagonizes targeting of these proteins by RNF4. Functionally, depletion of SENP6 results in uncoordinated recruitment and persistence of SUMO2/3 at UVA laser and ionizing radiation induced DNA damage sites. Additionally, SUMO2/3 and DNA damage response proteins accumulate in nuclear bodies, in a PML-independent manner driven by multivalent SUMO-SIM interactions. These data illustrate coordinated regulation of SUMOylated DNA damage response proteins by SENP6, governing their timely localization at DNA damage sites and nuclear condensation state. Show less
Ecker, J.; Ladoux, B.; Giomi, L.; Schmidt, T. 2023
Tissue-resident macrophage populations constitute a mosaic of phenotypes, yet how their metabolic states link to the range of phenotypes and functions in vivo is still poorly defined. Here, using... Show moreTissue-resident macrophage populations constitute a mosaic of phenotypes, yet how their metabolic states link to the range of phenotypes and functions in vivo is still poorly defined. Here, using high-dimensional spectral flow cytometry, we observe distinct metabolic profiles between different organs and functionally link acetyl CoA carboxylase activity to efferocytotic capacity. Additionally, differences in metabolism are evident within populations from a specific site, corresponding to relative stages of macrophage maturity. Immune perturbation with intestinal helminth infection increases alternative activation and metabolic rewiring of monocyte-derived macrophage populations, while resident TIM4+ intestinal macrophages remain immunologically and metabolically hyporesponsive. Similar metabolic signatures in alternatively-activated macrophages are seen from different tissues using additional helminth models, but to different magnitudes, indicating further tissue-specific contributions to metabolic states. Thus, our high-dimensional, flow-based metabolic analyses indicates complex metabolic heterogeneity and dynamics of tissue-resident macrophage populations at homeostasis and during helminth infection.Gauging the in vivo metabolism of immune cells at the single-cell level has proven challenging. Here the authors use spectral flow cytometry to investigate metabolic profiles in tissue-resident macrophages from several organs and changes in response to helminth infection. Show less
We evaluate the shared genetic regulation of mRNA molecules, proteins and metabolites derived from whole blood from 3029 human donors. We find abundant allelic heterogeneity, where multiple... Show moreWe evaluate the shared genetic regulation of mRNA molecules, proteins and metabolites derived from whole blood from 3029 human donors. We find abundant allelic heterogeneity, where multiple variants regulate a particular molecular phenotype, and pleiotropy, where a single variant associates with multiple molecular phenotypes over multiple genomic regions. The highest proportion of share genetic regulation is detected between gene expression and proteins (66.6%), with a further median shared genetic associations across 49 different tissues of 78.3% and 62.4% between plasma proteins and gene expression. We represent the genetic and molecular associations in networks including 2828 known GWAS variants, showing that GWAS variants are more often connected to gene expression in trans than other molecular phenotypes in the network. Our work provides a roadmap to understanding molecular networks and deriving the underlying mechanism of action of GWAS variants using different molecular phenotypes in an accessible tissue. Show less
Heezen, L.G.M.; Abdelaal, T.; Putten, M. van; Aartsma-Rus, A.; Mahfouz, A.; Spitali, P. 2023
Duchenne muscular dystrophy is caused by mutations in the DMD gene, leading to lack of dystrophin. Chronic muscle damage eventually leads to histological alterations in skeletal muscles. The... Show moreDuchenne muscular dystrophy is caused by mutations in the DMD gene, leading to lack of dystrophin. Chronic muscle damage eventually leads to histological alterations in skeletal muscles. The identification of genes and cell types driving tissue remodeling is a key step to developing effective therapies. Here we use spatial transcriptomics in two Duchenne muscular dystrophy mouse models differing in disease severity to identify gene expression signatures underlying skeletal muscle pathology and to directly link gene expression to muscle histology. We perform deconvolution analysis to identify cell types contributing to histological alterations. We show increased expression of specific genes in areas of muscle regeneration (Myl4, Sparc, Hspg2), fibrosis (Vim, Fn1, Thbs4) and calcification (Bgn, Ctsk, Spp1). These findings are confirmed by smFISH. Finally, we use differentiation dynamic analysis in the D2-mdx muscle to identify muscle fibers in the present state that are predicted to become affected in the future state. Show less
Vegte, Y. van de; Eppinga, R.N.; Ende, M.Y. van der; Hagemeijer, Y.; Mahendran, Y.V.; Salfati, E.Y.; ... ; DCCT EDIC Res Grp 2023
The genetics and clinical consequences of resting heart rate (RHR) remain incompletely understood. Here, the authors discover new genetic variants associated with RHR and find that higher... Show moreThe genetics and clinical consequences of resting heart rate (RHR) remain incompletely understood. Here, the authors discover new genetic variants associated with RHR and find that higher genetically predicted RHR decreases risk of atrial fibrillation and ischemic stroke.Resting heart rate is associated with cardiovascular diseases and mortality in observational and Mendelian randomization studies. The aims of this study are to extend the number of resting heart rate associated genetic variants and to obtain further insights in resting heart rate biology and its clinical consequences. A genome-wide meta-analysis of 100 studies in up to 835,465 individuals reveals 493 independent genetic variants in 352 loci, including 68 genetic variants outside previously identified resting heart rate associated loci. We prioritize 670 genes and in silico annotations point to their enrichment in cardiomyocytes and provide insights in their ECG signature. Two-sample Mendelian randomization analyses indicate that higher genetically predicted resting heart rate increases risk of dilated cardiomyopathy, but decreases risk of developing atrial fibrillation, ischemic stroke, and cardio-embolic stroke. We do not find evidence for a linear or non-linear genetic association between resting heart rate and all-cause mortality in contrast to our previous Mendelian randomization study. Systematic alteration of key differences between the current and previous Mendelian randomization study indicates that the most likely cause of the discrepancy between these studies arises from false positive findings in previous one-sample MR analyses caused by weak-instrument bias at lower P-value thresholds. The results extend our understanding of resting heart rate biology and give additional insights in its role in cardiovascular disease development. Show less
