A catalog of associations between rare coding variants and COVID-19 outcomes.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes coronavirus disease-19 (COVID-19), a respiratory illness that can result in hospitalization or death. We investigated associations between rare genetic variants and seven COVID-19 outcomes in 543,213 individuals, including 8,248 with COVID-19. After accounting for multiple testing, we did not identify any clear associations with rare variants either exome-wide or when specifically focusing on (i) 14 interferon pathway genes in which rare deleterious variants have been reported in severe COVID-19 patients; (ii) 167 genes located in COVID-19 GWAS risk loci; or (iii) 32 additional genes of immunologic relevance and/or therapeutic potential. Our analyses indicate there are no significant associations with rare protein-coding variants with detectable effect sizes at our current sample sizes. Analyses will be updated as additional data become available, with results publicly browsable at https://rgc-covid19.regeneron.com.

Genome-wide analysis in 756,646 individuals provides first genetic evidence that ACE2 expression influences COVID-19 risk and yields genetic risk scores predictive of severe disease.

SARS-CoV-2 enters host cells by binding angiotensin-converting enzyme 2 (ACE2). Through a genome-wide association study, we show that a rare variant (MAF = 0.3%, odds ratio 0.60, P=4.5×10-13) that down-regulates ACE2 expression reduces risk of COVID-19 disease, providing human genetics support for the hypothesis that ACE2 levels influence COVID-19 risk. Further, we show that common genetic variants define a risk score that predicts severe disease among COVID-19 cases.

Aldosterone signaling modifies capillary formation by human bone marrow endothelial cells.

We investigated the regulation of the epithelial sodium channel (ENaC) in human bone marrow endothelial cells (HBMEC) responding to mineralocorticoid hormones and other accessory effectors. The message for both the mineralocorticoid receptor (MCR) and the alpha subunit of ENaC was expressed in HBMEC as predicted bands of 838 and 521 bp, respectively. In Western blots, the MCR of about 107 kDa was localized primarily in the cytoplasmic compartment but migrated to the nucleus when cell cultures were exposed to exogenous aldosterone. On the other hand, the alphaENaC was revealed as a membrane-bound protein of approximately 82 kDa, whose abundance increased after aldosterone treatment. Confocal microscopy confirmed the presence of both the MCR and ENaC as nucleocytoplasmic and membrane-bound proteins, respectively, and both colocalized with tubulin in situ. On Matrigel, the mineralocorticoid aldosterone, by itself, did not influence capillary formation by HBMEC, but the diuretic amiloride reduced the organization of HBMEC into capillary-like networks; curiously, aldosterone further exacerbated this inhibitory effect of amiloride. On the fibrin matrix, aldosterone had no influence at all on the length of the newly formed capillaries, but the capillary diameter was highly increased over the control. Aldosterone-mediated capillary swelling was totally reversed by amiloride, which, by itself, also inhibited capillary elongation by HBMEC. Thus, cell signaling by mineralocorticoid hormones in HBMEC appears to proceed in a manner very similar to that in the epithelial cell, thereby leading to an increase in the endothelial cell volume, which may underline the hypertensive state and which may also modify angiogenesis.

Adaptive immune responses of legumin nanoparticles.

Legumin is one of the main storage proteins in the pea seeds (Pisum sativum L.) and the molecules of this protein have the capacity of binding together to form nanoparticles after aggregation and chemical cross-linkage with glutaraldehyde. The aim of this work was to study the adaptive immune response of legumin nanoparticles in rats. Following intradermal immunisation with the native protein legumin and legumin nanoparticles of about 250 nm, the humoral and cell-mediated immune responses were analysed in rats. The humoral responses against legumin and legumin nanoparticles were examined by western blot and ELISA analysis. Both techniques clearly showed that sera from rats immunised with legumin strongly expressed antibodies against this protein. On the contrary, serum samples from rats inoculated with legumin nanoparticles did not contain detectable amounts of antibodies. These results may be explained by a reduction on the antigenic epitopes of the protein induced by the glutaraldehyde used during the cross-linking step. Concerning the cell-mediated response, neither legumin nor legumin nanoparticles stimulated an immunogenic response. This absence of response of spleen lymphocytes for legumin and legumin nanoparticles may be explained by a cytostatic effect of legumin which was corroborated by the evaluation of the middle phase of cell apoptose. In fact, both legumin and legumin nanoparticles are potent inductors of a cytostatic phenomenon and showed a significant increase of the chromatin condensation (p < 0.05) as compared with control.

Ethanol inhibition of N-methyl-D-aspartate receptors is reduced by site-directed mutagenesis of a transmembrane domain phenylalanine residue.

N-Methyl-D-aspartate (NMDA) receptors (NRs) are ionotropic receptors activated by glutamate and the co-agonist glycine. Ethanol inhibits NMDA receptor function, although its site of action is undefined. We hypothesized that ethanol acts at specific amino acids contained within the transmembrane (TM) domains of the receptor. In this study, NR1 and NR2A subunits were altered by mutagenesis and tested for sensitivity to ethanol. Three NR1 mutants (W636A, F817A, and L819A) and one NR2A mutant (F637A) failed to generate functional receptors. Pre-TM1 (I546A, L551A, F554A, and F558A), TM1 (W563A), and TM2 (W611A) NR1 mutations did not affect ethanol sensitivity of heteromeric receptors. In contrast, altering a TM3 phenylalanine to alanine (F639A) reduced the ethanol inhibition of NMDA receptors expressed in oocytes and human embryonic kidney 293 cells. Mutation of the nearby methionine (M641) to alanine did not affect ethanol sensitivity, whereas changing Phe(639) to tryptophan slightly enhanced ethanol inhibition. NR1(F639A) did not alter the agonist potency of glutamate but did produce a leftward shift in the glycine concentration response for receptors containing NR2A and NR2B subunits. NR1(F639A) also reduced the potency of the competitive glycine antagonist 5,7-dichlorokynurenic acid and increased the efficacy of the glycine partial agonist 3-amino-1-hydroxy-2-pyrrolidinone ((+)-HA-966). These results suggest that ethanol may interact with amino acids contained in the TM3 domain of NMDA subunits that are involved in transducing agonist binding to channel opening.

Receptor-mediated hydrolysis of plasma membrane messenger PIP2 leads to K+-current desensitization.

Phosphatidylinositol bisphosphate (PIP2) directly regulates functions as diverse as the organization of the cytoskeleton, vesicular transport and ion channel activity. It is not known, however, whether dynamic changes in PIP2 levels have a regulatory role of physiological importance in such functions. Here, we show in both native cardiac cells and heterologous expression systems that receptor-regulated PIP2 hydrolysis results in desensitization of a GTP-binding protein-stimulated potassium current. Two receptor-regulated pathways in the plasma membrane cross-talk at the level of these channels to modulate potassium currents. One pathway signals through the betagamma subunits of G proteins, which bind directly to the channel. Gbetagamma subunits stabilize interactions with PIP2 and lead to persistent channel activation. The second pathway activates phospholipase C (PLC) which hydrolyses PIP2 and limits Gbetagamma-stimulated activity. Our results provide evidence that PIP2 itself is a receptor-regulated second messenger, downregulation of which accounts for a new form of desensitization.

Development of a sensitive method for the determination of ganciclovir by reversed-phase high-performance liquid chromatography.

Ganciclovir is a nucleoside analogue widely used in the treatment of cytomegalovirus infections, which affects mainly immunocompromised patients. Recently, new pharmaceutical dosage forms based on the use of albumin nanoparticles have been developed for improving the efficacy of this drug. The aim of this study was to develop an analytical HPLC method for the determination of ganciclovir in both pharmaceuticals (i.e. albumin nanoparticles) and biological medium samples. The chromatography was performed on a reversed-phase encapped column (LiChrospher Select B C8) with a mobile phase consisting of acetonitrile in 0.05 M ammonium acetate (pH 6.5; 2: 98, v/v). Acyclovir was used as internal standard and the detection wavelength was 254 nm. The limit of quantitation of ganciclovir was 50 ng/ml and the average recoveries over a concentration range of 0.05-10 microg/ml ranged from 98 to 102%. Precision did not exceed 5%. In summary, this assay is a selective, sensitive and reproducible method for the determination of the ganciclovir in albumin nanoparticles. It can be successfully applied to the estimation of the ganciclovir uptake by cultured human corneal fibroblasts.

Activation of inwardly rectifying K+ channels by distinct PtdIns(4,5)P2 interactions.

Direct interactions of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) with inwardly rectifying potassium channels are stronger with channels rendered constitutively active by binding to PtdIns(4,5)P2, such as IRK1, than with G-protein-gated channels (GIRKs). As a result, PtdIns(4,5)P2 alone can activate IRK1 but not GIRKs, which require extra gating molecules such as the beta gamma subunits of G proteins or sodium ions. Here we identify two conserved residues near the inner-membrane interface of these channels that are critical in interactions with PtdIns(4,5)P2. Between these two arginines, a conservative change of isoleucine residue 229 in GIRK4 to the corresponding leucine found in IRK1 strengthens GIRK4-PtdIns(4,5)P2 interactions, eliminating the need for extra gating molecules. A negatively charged GIRK4 residue, two positions away from the most strongly interacting arginine, mediates stimulation of channel activity by sodium by strengthening channel-PtdIns(4,5)P2 interactions. Our results provide a mechanistic framework for understanding how distinct gating mechanisms of inwardly rectifying potassium channels allow these channels to subserve their physiological roles.

Identification of a potassium channel site that interacts with G protein betagamma subunits to mediate agonist-induced signaling.

Activation of heterotrimeric GTP-binding (G) proteins by their coupled receptors, causes dissociation of the G protein alpha and betagamma subunits. Gbetagamma subunits interact directly with G protein-gated inwardly rectifying K+ (GIRK) channels to stimulate their activity. In addition, free Gbetagamma subunits, resulting from agonist-independent dissociation of G protein subunits, can account for a major component of the basal channel activity. Using a series of chimeric constructs between GIRK4 and a Gbetagamma-insensitive K+ channel, IRK1, we have identified a critical site of interaction of GIRK with Gbetagamma. Mutation of Leu339 to Glu within this site impaired agonist-induced sensitivity and decreased binding to Gbetagamma, without removing the Gbetagamma contribution to basal currents. Mutation of the corresponding residue in GIRK1 (Leu333) resulted in a similar phenotype. Both the GIRK1 and GIRK4 subunits contributed equally to the agonist-induced sensitivity of the heteromultimeric channel. Thus, we have identified a channel site that interacts specifically with Gbetagamma subunits released through receptor stimulation.

Intracellular calcium enhances the ethanol sensitivity of NMDA receptors through an interaction with the C0 domain of the NR1 subunit.

Previous studies in this laboratory have shown that the ethanol inhibition of recombinant NMDA receptors expressed in Xenopus oocytes is subunit-dependent, with the NR1/2A receptor being more sensitive than NR1/2C receptors. The ethanol sensitivity of NR1/2A receptors is reduced by substitution of the wild-type NR1-1a (NR1(011)) subunit with the calcium-impermeable NR1 (N616R) subunit. In the present study, the ethanol inhibition of NMDA receptors was determined under different conditions to examine the role that calcium plays in determining the ethanol sensitivity of recombinant NMDA receptors. The ethanol sensitivity of NR1/2B or NR1/2C receptors was not affected by alterations in extracellular calcium levels or by coexpression with calcium-impermeable NR1 mutants. In contrast, the inhibition of NR1/2A receptors by 100 mM ethanol was reduced in divalent-free recording medium and was significantly increased when 10 mM calcium was used as the only charge carrier. The increase in the ethanol sensitivity of NR1/2A receptors under high-calcium conditions was prevented by preinjection of oocytes with the calcium chelator 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) but not by inhibitors of calmodulin or protein kinase C. Ethanol did not alter the channel blocking activity of divalent cations on NMDA-induced currents. The enhanced ethanol sensitivity of NR1/2A receptors in 10 mM calcium persisted when the NR1 subunit was replaced by the alternative splice variant NR1-4a (NR1(000)), which lacks the C1 and C2 cassettes. However, expression of a mutant NR1 subunit that lacked the C0, C1, and C2 domains abolished the calcium-dependent enhancement of ethanol's inhibition of NR1/2A receptors. Finally, the ethanol sensitivity of wild-type NR1/2A receptors measured in transfected HEK 293 cells by whole cell patch-clamp electrophysiology was significantly reduced by expression of the C-terminal truncated NR1 subunit. These results demonstrate that the ethanol sensitivity of certain NMDA receptors is modulated by an intracellular, calcium-dependent process that requires the C0 domain of the NR1 subunit.

Effects of the abused solvent toluene on recombinant N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors expressed in Xenopus oocytes.

Previous studies have shown that toluene, which is commonly abused, depresses neuronal activity and causes behavioral effects in both animals and man similar to those observed for ethanol. In this study, the oocyte expression system was used to test the hypothesis that toluene, like ethanol, inhibits the function of ionotropic glutamate receptors. Oocytes were injected with mRNA for specific N-methyl-D-aspartate (NMDA) or non-NMDA subunits and currents were recorded using conventional two-electrode voltage clamp. To enhance the low water solubility of toluene, drug solutions were prepared by mixing toluene with alkamuls (ethoxylated castor oil) at a 1:1 ratio (v:v) and diluting this mixture to the appropriate concentration with barium-containing normal frog Ringer solution. Alkamuls, up to 0.1%, had no significant effects on membrane leak currents or on NMDA-induced currents. Toluene, up to approximately 9 mM, had only minor effects on membrane leak currents but dose-dependently inhibited NMDA-mediated currents in oocytes. The inhibition of NMDA receptor currents by toluene was rapid, reversible and the potency for toluene's effects was subunit dependent. The NR1/2B subunit combination was the most sensitive with an IC50 value for toluene-induced inhibition of 0.17 mM. The NR1/2A and NR1/2C receptors were 6- and 12-fold less sensitive with IC50 values of 1.4 and 2.1 mM, respectively. In contrast, toluene up to approximately 9 mM did not inhibit kainate-induced currents in oocytes expressing GluR1, GluR1(+)R2 or GluR6 subunits. These results suggest that some of the effects of toluene on neuronal activity and behavior may be mediated by inhibition of NMDA receptors.

Effects of acute and chronic ethanol exposure on heteromeric N-methyl-D-aspartate receptors expressed in HEK 293 cells.

Ion flux through native N-methyl-D-aspartate (NMDA) receptors is inhibited by behaviorally relevant concentrations of ethanol (10-100 mM) in a variety of neuronal preparations. However, in animal tissues, it is often difficult to determine accurately which NMDA receptor subunits are responsible for the observed effect. In this study, human embryonic kidney 293 (HEK 293) cells normally devoid of NMDA receptors were transiently transfected with cDNA expression plasmids coding for specific rat NMDA receptor subunits. Brief application of an NMDA/glycine solution to cells markedly increased intracellular calcium in cells transfected with NR1/NR2A, NR1/NR2B, or NR1/NR2A/NR2B as measured by fura-2 calcium imaging. This increase was both NMDA- and glycine-dependent and was inhibited by competitive and noncompetitive NMDA antagonists, including 2-amino-5-phosphopentanoic acid and MK-801. The NR2B-selective antagonist ifenprodil inhibited responses in cells transfected with NR1/NR2B or NR1/NR2A/NR2B, but not NR1/NR2A subunits. Increasing the transfection ratio of NR2B versus NR2A subunit in NR1/NR2A/NR2B-transfected cells greatly increased their ifenprodil sensitivity. Acute exposure to ethanol (25-100 mM) inhibited the NMDA-mediated increase in intracellular calcium in a dose-dependent manner without affecting basal calcium concentrations. There were no statistically significant differences in ethanol's potency or maximal inhibition between any of the subunit combinations tested. HEK 293 cells transfected with NR1/NR2A/NR2B subunits showed an enhanced sensitivity to ifenprodil following a 24-h exposure to concentrations of ethanol of 50 mM and greater. The enhanced ifenprodil sensitivity following ethanol exposure was not associated with changes in NR1, NR2A, or NR2B immunoreactivity. In contrast to results obtained in transfected HEK 293 cells, no effect of chronic ethanol was observed in oocytes expressing NR1/NR2A/NR2B subunits. These results demonstrate that recombinant NMDA receptors expressed in HEK 293 cells form functional receptors that, like native receptors, are sensitive to modulation by both acute and chronic ethanol treatment.

Pharmacological characterization of BNMPA (alpha-benzyl-N-methylphenethylamine), an impurity of illicit methamphetamine synthesis.

alpha-Benzyl-N-methylphenethylamine (BNMPA), an impurity of illicit methamphetamine synthesis, has previously been reported to produce convulsions in mice without affecting spontaneous locomotor activity or altering methamphetamine-induced increases in spontaneous activity. In this study the in vitro effects of BNMPA on a variety of neuronal receptor types was determined to better characterize the pharmacological actions of this novel compound. BNMPA and N-demethyl-BNMPA fully displaced the dopamine transporter selective ligand [3H]CFT (2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane) from rat striatal membranes with Ki values (mean +/- S.E.M) of 6.05 microM +/- 0.15 and 8.73 microM +/- 1.66, respectively. BNMPA also inhibited [3H]dopamine uptake into striatal synaptosomes with an IC50 value of 5.1 +/- 1.4 microM. The basal efflux of [3H]dopamine from striatal slices was slightly enhanced by BNMPA only at concentrations > or = 100 microM. BNMPA had no effect on [3H]norepinephrine efflux from hippocampal slices. BNMPA displaced tritiated paroxetine and prazosin binding from rat cortical membranes with Ki values of 14.5 microM and 11.7 microM respectively. In electrophysiological studies, BNMPA (100 microM) had no significant effects on either GABAA Cl- currents in cultured neurons or non-NMDA glutamate receptors expressed in oocytes. However, BNMPA significantly inhibited NMDA-stimulated currents in oocytes expressing the NR1/2A or NR1/2C receptor subunit combinations (IC50 values = 24.6 +/- 1.8 and 24.0 +/- 1.5 microM, respectively). This inhibition was rapid, reversible and voltage-dependent. These results indicate that BNMPA has multiple sites of action in the CNS that could be important in modulating a variety of behavioral effects upon exposure to this synthetic byproduct of illicit methamphetamine synthesis.

Increased agonist and antagonist sensitivity of N-methyl-D-aspartate stimulated calcium flux in cultured neurons following chronic ethanol exposure.

Cortical cultures of rat brain neurons were exposed to ethanol (100 mM) for 4 days in order to examine whether the pharmacological characteristics of N-methyl-D-aspartate (NMDA) receptors expressed by these neurons were altered by this treatment. In fura-2 loaded control neurons, NMDA (plus 10 microM glycine) stimulated a dose-dependent increase in intracellular calcium concentrations with an estimated EC50 value of 6.8 microM. NMDA-stimulated increases in intracellular calcium reached a plateau at approximately 30 microM with no further increases observed at 100 microM. The EC50 value for NMDA in ethanol-exposed neurons was reduced to 1.8 microM with no alteration in the maximal response. Similarly, the EC50 value for glycine (tested with 100 microM NMDA) was reduced from 2.3 microM in control cultures to 0.67 microM in ethanol-treated cultures. Ifenprodil inhibited NMDA-stimulated increases in intracellular calcium in control cultures only at concentrations of 3 microM and above, with 100 microM producing approximately a 58% inhibition. In ethanol-treated cultures, 0.3 microM ifenprodil inhibited the NMDA response by approximately 60% with 100 microM ifenprodil producing a 72% inhibition. Over the concentration range of ifenprodil tested, half-maximal inhibition occurred at 1.4 microM and 0.18 microM, respectively, for control and ethanol-treated neurons. Although chronic ethanol treatment appeared to alter the sensitivity of neurons to NMDA agonists and antagonists, the inhibitory effects of 50 mM ethanol on NMDA-stimulated increases in intracellular calcium were not different between control (28% inhibition) and ethanol-treated neurons (27% inhibition). Finally, the changes in NMDA receptor sensitivity observed in ethanol-treated neurons were accompanied by an enhanced sensitivity to the neurotoxic effects of NMDA as measured by propidium iodide staining. These results suggest that chronic exposure of neurons to ethanol may result in an altered expression of agonist-sensitive/ifenprodil selective NMDA receptor subunits.

Ethanol sensitivity of heteromeric NMDA receptors: effects of subunit assembly, glycine and NMDAR1 Mg(2+)-insensitive mutants.

In the current study, dimeric and trimeric combinations of N-methyl-D-aspartate (NMDA) receptor subunits were expressed in Xenopus oocytes and their sensitivity to ethanol was examined using conventional two electrode voltage clamp methods. In oocytes expressing the NR1/2A subunits, ethanol (25, 50 and 100 mM) inhibited NMDA (100 microM)/glycine (10 microM) induced currents by 21, 31 and 47%; respectively. NMDA-stimulated currents in oocytes expressing NR1/2B currents were inhibited by 13, 25 and 45% while NR1/NR2C currents were inhibited by 6, 11 and 24%. Ethanol inhibition of NMDA-stimulated currents in oocytes injected with NR1/2A/2B or NR1/2A/2C was not significantly different from that observed in NR1/2B or NR1/2C injected oocytes, respectively. With all receptor combinations, ethanol inhibition was rapid, reversible and not altered by pre-incubation. In the absence of ethanol, glycine enhanced NMDA-induced currents with an EC50 of 1.42 microM for the NR1/NR2A combination and 0.51 microM for the NR1/NR2C combination. Ethanol inhibited NMDA-induced currents at all glycine concentrations tested (1-100-microM) and did not significantly alter the EC50 value for glycine suggesting that ethanol does not compete for the glycine site on the NMDA receptor. Finally, three NR1 mutants which have been previously shown by others to possess either decreased Mg2+ sensitivity and Ca2+ permeability (N616Q and N616R) or reduced current amplitude (F609L) were tested for their ethanol sensitivity when expressed in combination with the NR2A subunit. Substitution of the wild-type NR1 with F609L did not alter the sensitivity of the receptor to ethanol.(ABSTRACT TRUNCATED AT 250 WORDS)