OP0314 DOCK8 MUTATIONS IN COVID-19 AND MIS-C CYTOKINE STORM SYNDROME

Background:We recently identified DOCK8 as a novel gene associated with cytokine storm syndrome (CSS)1. Heterozygous missense mutations in DOCK8 diminish NK cell lytic function and contribute to increased pro-inflammatory cytokine production (CSS). CSS is a potential complication of COVID-19 with severe consequences2. Children are at risk of a SARS-CoV-2 post-infectious CSS, multisystem inflammatory syndrome in children (MIS-C)3. Host genetic factors associated with COVID-19 CSS and MIS-C CSS are unknown.Objectives:The goals are to identify and functionally study rare mutations in DOCK8 in patients with SARS-CoV-2 COVID-19 and MIS-C.Methods:To date, 16 adult patients enrolled in a COVID-19 CSS clinical trial at UAB had whole genome sequencing. Four (25%) had rare heterozygous DOCK8 mutations (3 missense, 1 intronic). A COVID-19 CSS adult patient in Seattle also had a DOCK8 missense mutation. In addition, DOCK8 missense mutations were identified in five children (UAB & Northwell) hospitalized with MIS-C. DOCK8 mutations, or wild-type (WT) sequence controls, were introduced into human NK-92 cells by FOAMY virus transduction. WT and mutant DOCK8-expressing NK-92 cells were incubated with K562 target cells and compared for cytolysis and degranulation (CD107a).Results:One COVID-19 patient DOCK8 mutation (Gly523Arg) reduced NK cell degranulation by 30% and cytolysis by 23% (n=3) (Figure 1). Similar studies of 3 MIS-C patients with DOCK8 missense mutations (Arg899Trp, Ala2Thr, Pro687Leu) revealed up to 31% reduced NK cell degranulation and 48% reduction in cytolysis by 3 distinct mutations (n=3). Two-way ANOVA analysis revealed statistically significant (p<0.05) differences in NK cell degranulation and lysis for four unique DOCK8 mutations.Conclusion:Heterozygous DOCK8 missense mutations may contribute to severe COVID-19 and MIS-C CSS by partial dominant-negative effects yielding decreased NK cell cytolysis.References:[1]Schulert GS, Cron RQ. The genetics of macrophage activation syndrome. Genes Immun 2020:21:169-181.[2] Cron RQ, Chatham WW. The rheumatologist’s role in COVID-19. J Rheumatol 2020:47:639-642.[3]Reiff D, Mannion ML, Samuy N, Scalici P, Cron RQ. Distinguishing active pediatric COVID-19 from MIS-C. Pediatr Rheumatol Online J, in press.Disclosure of Interests:Randy Cron Consultant of: SOBI, Novartis, Pfizer, Sironax, Grant/research support from: SOBI, Mingce Zhang: None declared, Remy Cron: None declared, Devin Absher: None declared, John Bridges: None declared, Amanda Schnell: None declared, Pavan Bhatraju: None declared, Anshul Vagrecha: None declared, Shannon Lozinsky: None declared, Suchitra Acharya: None declared, Carolyn Levy: None declared, Winn Chatham Grant/research support from: SOBI.

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Drosophila transcription factor AP-2 in proboscis, leg and brain central complex development

Development ◽

10.1242/dev.128.8.1239 ◽

2001 ◽

Vol 128 (8) ◽

pp. 1239-1252 ◽

Cited By ~ 2

Author(s):

I. Monge ◽

R. Krishnamurthy ◽

D. Sims ◽

F. Hirth ◽

M. Spengler ◽

...

Keyword(s):

Transcription Factor ◽

Dominant Negative ◽

Central Complex ◽

Missense Mutations ◽

Leg Length ◽

New Paradigm ◽

Negative Effects ◽

Wing Vein ◽

Normal Length ◽

Hypomorphic Alleles

We report loss- and gain-of-function analyses that identify essential roles in development for Drosophila transcription factor AP-2. A mutagenesis screen yielded 16 lethal point mutant alleles of dAP-2. Null mutants die as adults or late pupae with a reduced proboscis, severely shortened legs (~30% of normal length) lacking tarsal joints, and disruptions in the protocerebral central complex, a brain region critical for locomotion. Seven hypomorphic alleles constitute a phenotypic series yielding hemizygous adults with legs ranging from 40–95% of normal length. Hypomorphic alleles show additive effects with respect to leg length and viability; and several heteroallelic lines were established. Heteroallelic adults have moderately penetrant defects that include necrotic leg joints and ectopic growths (sometimes supernumerary antennae) invading medial eye territory. Several dAP-2 alleles with DNA binding domain missense mutations are null in hemizygotes but have dominant negative effects when paired with hypomorphic alleles. In wild-type leg primordia, dAP-2 is restricted to presumptive joints. Ectopic dAP-2 in leg discs can inhibit but not enhance leg elongation indicating that functions of dAP-2 in leg outgrowth are region restricted. In wing discs, ectopic dAP-2 cell autonomously transforms presumptive wing vein epithelium to ectopic sensory bristles, consistent with an instructive role in sensory organ development. These findings reveal multiple functions for dAP-2 during morphogenesis of feeding and locomotor appendages and their neural circuitry, and provide a new paradigm for understanding AP-2 family transcription factors.

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The Vav–Rac1 Pathway in Cytotoxic Lymphocytes Regulates the Generation of Cell-mediated Killing

Journal of Experimental Medicine ◽

10.1084/jem.188.3.549 ◽

1998 ◽

Vol 188 (3) ◽

pp. 549-559 ◽

Cited By ~ 128

Author(s):

Daniel D. Billadeau ◽

Kathryn M. Brumbaugh ◽

Christopher J. Dick ◽

Renee A. Schoon ◽

Xose R. Bustelo ◽

...

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Dominant Negative ◽

Target Cells ◽

Regulation Of Transcription ◽

Cytotoxic Lymphocytes ◽

Cellular Cytotoxicity ◽

Nucleotide Exchange ◽

Exchange Factor ◽

Downstream Signaling

The Rac1 guanine nucleotide exchange factor, Vav, is activated in hematopoietic cells in response to a large variety of stimuli. The downstream signaling events derived from Vav have been primarily characterized as leading to transcription or transformation. However, we report here that Vav and Rac1 in natural killer (NK) cells regulate the development of cell-mediated killing. There is a rapid increase in Vav tyrosine phosphorylation during the development of antibody-dependent cellular cytotoxicity and natural killing. In addition, overexpression of Vav, but not of a mutant lacking exchange factor activity, enhances both forms of killing by NK cells. Furthermore, dominant-negative Rac1 inhibits the development of NK cell–mediated cytotoxicity by two mechanisms: (a) conjugate formation between NK cells and target cells is decreased; and (b) those NK cells that do form conjugates have decreased ability to polarize their granules toward the target cell. Therefore, our results suggest that in addition to participating in the regulation of transcription, Vav and Rac1 are pivotal regulators of adhesion, granule exocytosis, and cellular cytotoxicity.

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Mechanisms of I Ks suppression in LQT1 mutants

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2000.279.6.h3003 ◽

2000 ◽

Vol 279 (6) ◽

pp. H3003-H3011 ◽

Cited By ~ 44

Author(s):

Laura Bianchi ◽

Silvia G. Priori ◽

Carlo Napolitano ◽

Krystyna A. Surewicz ◽

Adrienne T. Dennis ◽

...

Keyword(s):

Cell Surface ◽

Dominant Negative ◽

Delayed Rectifier ◽

Poor Correlation ◽

Type I ◽

Missense Mutations ◽

Wild Type ◽

Negative Effects ◽

Clinical Phenotypes ◽

First Time

Mutations in the cardiac potassium ion channel gene KCNQ1 (voltage-gated K+ channel subtype KvLQT1) cause LQT1, the most common type of hereditary long Q-T syndrome. KvLQT1 mutations prolong Q-T by reducing the repolarizing cardiac current [slow delayed rectifier K+ current ( I Ks )], but, for reasons that are not well understood, the clinical phenotypes may vary considerably even for carriers of the same mutation, perhaps explaining the mode of inheritance. At present, only currents expressed by LQT1 mutants have been studied, and it is unknown whether abnormal subunits are transported to the cell surface. Here, we have examined for the first time trafficking of KvLQT1 mutations and correlated the results with the I Ks currents that were expressed. Two missense mutations, S225L and A300T, produced abnormal currents, and two others, Y281C and Y315C, produced no currents. However, all four KvLQT1 mutations were detected at the cell surface. S225L, Y281C, and Y315C produced dominant negative effects on wild-type I Ks current, whereas the mutant with the mildest dysfunction, A300T, did not. We examined trafficking of a severe insertion deletion mutant Δ544 and detected this protein at the cell surface as well. We compared the cellular and clinical phenotypes and found a poor correlation for the severely dysfunctional mutations.

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Effects of organochlorine pesticides on interleukin secretion from lymphocytes

Human & Experimental Toxicology ◽

10.1177/0960327106070072 ◽

2006 ◽

Vol 25 (11) ◽

pp. 651-659 ◽

Cited By ~ 11

Author(s):

T M Beach ◽

M M Whalen

Keyword(s):

T Cells ◽

Nk Cells ◽

Organochlorine Pesticides ◽

Cell Function ◽

Nk Cell ◽

Organochlorine Compound ◽

Organochlorine Compounds ◽

Negative Effects ◽

Lytic Function ◽

Baseline Levels

Organochlorine pesticides have been used worldwide primarily as insecticides. Due to their chemical stability, they often persist in the environment long after their use has ceased. In a previous study, we found that six organochlorine compounds (α-chlordane, γ-chlordane, 4,4′-DDT, heptachlor, oxychlordane, and pentachlorophenol (PCP)), at concentrations of 5 μM, were able to significantly decrease the ability of highly purified human natural killer (NK) cells to lyse tumor cells after exposures, ranging from 1 hour to 6 days. However, if T cells were present with the NK cells (T/NK cells), loss of lytic function was seen only with oxychlordane and PCP. The purpose of the current study is to begin to investigate the mechanism by which T cells may be blocking the negative effects of some organochlorine compounds on NK cell function. Here, we investigated the hypothesis that T cells could produce significant levels of NK-stimulatory interleukin(s) (ILs), and that this may account for the decreased inhibition seen with organochlorine exposures when T cells were present. Secretion of four cytokines that have a demonstrated capacity to influence NK function, and/or are secreted by T cells, was measured (IL-2, IL-4, IL-10, IL-12). We measured both the baseline levels of ILs and the effects of organochlorine compound on IL secretion in T/NK cells. The results showed that baseline levels of the NK-stimulatory IL, IL-12, were 898±264 pg/mL at 24 hours and IL-10 levels were 564±337 pg/mL. In contrast, IL-2 levels were 14±10 pg/mL, and IL-4 levels were 3±2 pg/mL at 24 hours. The two compounds that retained their capacity to decrease NK lytic function in T/NK cells, oxychlordane (5 μM) and PCP (5 and 10 μM), were able to either decrease the secretion of NK-stimulatory ILs (IL-2, IL-12 and/or IL-10) and/or increase secretion of the NK-inhibitory cytokine, IL-4, at each length of exposure tested.

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Defining relative mutational difficulty to understand cancer formation and prevention

10.1101/789313 ◽

2019 ◽

Author(s):

Lin Shan ◽

Jiao Yu ◽

Zhengjin He ◽

Shishuang Chen ◽

Mingxian Liu ◽

...

Keyword(s):

Human Cancer ◽

Low Frequency ◽

Dominant Negative ◽

Missense Mutations ◽

Nucleotide Change ◽

P53 Mutations ◽

Loss Of Function ◽

Functional Importance ◽

Negative Effects ◽

Different Types

SummaryMost mutations in human cancer are low-frequency missense mutations, whose functional status remains hard to predict. Here we show that depending on the type of nucleotide change and the surrounding sequences, the tendency to generate each type of nucleotide mutations varies greatly, even by several hundred folds. Therefore, a cancer-promoting mutation may appear only in a small number of cancer cases, if the underlying nucleotide change is too difficult to generate. We propose a method that integrates both the original mutation counts and their relative mutational difficulty. Using this method, we can accurately predict the functionality of hundreds of low-frequency missense mutations in p53, PTEN and INK4A. Many loss-of-function p53 mutations with dominant negative effects were identified, and the functional importance of several regions in p53 structure were highlighted by this analysis. Furthermore, mutational difficulty analysis also points to potential means of cancer prevention. Our study not only established relative mutational difficulties for different types of mutations in human cancer, but also showed that by incorporating such parameter, we can bring new angles to understanding cancer formation and prevention.

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Oral Glucosamine Ameliorates Aggravated Neurological Phenotype in Mucopolysaccharidosis III Type C Mouse Model Expressing Misfolded HGSNAT Variant

10.1101/2021.08.26.457793 ◽

2021 ◽

Author(s):

Xuefang Pan ◽

Mahsa Taherzadeh ◽

Poulomee Bose ◽

Rachel Heon-Roberts ◽

Annie L.A. Nguyen ◽

...

Keyword(s):

Heparan Sulfate ◽

Missense Variant ◽

Dominant Negative ◽

Synaptic Proteins ◽

Missense Mutations ◽

Negative Effects ◽

Object Recognition Test ◽

Y Maze ◽

Neurological Phenotype

Objective: Over 55% of mucopolysaccharidosis IIIC (MPS IIIC) patients have at least one allelic missense variant responsible for misfolding of heparan sulfate acetyl-CoA:α-glucosaminide N-acetyltransferase (HGSNAT). These variants are potentially treatable with pharmacological chaperones, such as a competitive HGSNAT inhibitor, glucosamine. Since the constitutive HGSNAT knockout mice, we generated previously cannot be used to test such strategy in vivo, we generated a novel model, the HgsnatP304L strain, expressing misfolded mutant HGSNAT with human missense mutation Pro311Leu (Pro304Leu in the mouse enzyme). Results: HgsnatP304L mice present deficits in short-term (novel object recognition test) and working/spatial (Y-maze test) memory at 4 months of age, 2-4 months earlier than previously described gene-targeted Hgsnat-Geo mice, which lack HGSNAT protein. HgsnatP304L mice also show increased severity of synaptic deficits in CA1 neurons, and accelerated course of CNS pathology including neuronal storage of heparan sulfate, accumulation of misfolded proteins, increase of simple gangliosides, and neuroinflammation as compared with Hgsnat-Geo mice. Expression of misfolded human Pro311Leu HGSNAT protein in cultured hippocampal Hgsnat-Geo neurons aggravated reduction of synaptic proteins. Memory deficits and majority of pathological changes in the brain were rescued in mice receiving daily doses of oral glucosamine. Interpretation: Altogether, our data for the first time demonstrate dominant-negative effects of the misfolded HGSNAT Pro304Leu variant and show that these effects are treatable by oral administration of glucosamine, suggesting that patients, affected with missense mutations preventing normal folding of the enzyme, could benefit from chaperone therapy.

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Robinow syndrome skeletal phenotypes caused by the WNT5AC83S variant are due to dominant interference with chondrogenesis

Human Molecular Genetics ◽

10.1093/hmg/ddz071 ◽

2019 ◽

Vol 28 (14) ◽

pp. 2395-2414

Author(s):

Sarah J Gignac ◽

Sara Hosseini-Farahabadi ◽

Takashi Akazawa ◽

Nathan J Schuck ◽

Katherine Fu ◽

...

Keyword(s):

Planar Cell Polarity ◽

Dominant Negative ◽

Missense Mutations ◽

Negative Effects ◽

Biochemical Assays ◽

Cartilage Differentiation ◽

Robinow Syndrome ◽

Variant Protein ◽

Bone Dimensions ◽

Human Wild Type

Abstract Heterozygous missense mutations in several genes in the WNT5A signaling pathway cause autosomal dominant Robinow syndrome 1 (DRS1). Our objective was to clarify the functional impact of a missense mutation in WNT5A on the skeleton, one of the main affected tissues in RS. We delivered avian replication competent retroviruses (RCAS) containing human wild-type WNT5A (wtWNT5A), WNT5AC83S variant or GFP/AlkPO4 control genes to the chicken embryo limb. Strikingly, WNT5AC83S consistently caused a delay in ossification and bones were more than 50% shorter and 200% wider than controls. In contrast, bone dimensions in wtWNT5A limbs were slightly affected (20% shorter, 25% wider) but ossification occurred on schedule. The dysmorphology of bones was established during cartilage differentiation. Instead of stereotypical stacking of chondrocytes, the WNT5AC83S-infected cartilage was composed of randomly oriented chondrocytes and that had diffuse, rather than concentrated Prickle staining, both signs of disrupted planar cell polarity (PCP) mechanisms. Biochemical assays revealed that C83S variant was able to activate the Jun N-terminal kinase-PCP pathway similar to wtWNT5A; however, the activity of the variant ligand was influenced by receptor availability. Unexpectedly, the C83S change caused a reduction in the amount of protein being synthesized and secreted, compared to wtWNT5A. Thus, in the chicken and human, RS phenotypes are produced from the C83S mutation, even though the variant protein is less abundant than wtWNT5A. We conclude the variant protein has dominant-negative effects on chondrogenesis leading to limb abnormalities.

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Sialic Acids and Their Influence on Human NK Cell Function

Cells ◽

10.3390/cells10020263 ◽

2021 ◽

Vol 10 (2) ◽

pp. 263

Author(s):

Philip Rosenstock ◽

Thomas Kaufmann

Keyword(s):

Nk Cells ◽

Immune Cells ◽

Cell Function ◽

Nk Cell ◽

Sialic Acids ◽

Innate Immune ◽

Target Cells ◽

Carbon Backbone ◽

Nk Cell Receptors ◽

Cell Inhibition

Sialic acids are sugars with a nine-carbon backbone, present on the surface of all cells in humans, including immune cells and their target cells, with various functions. Natural Killer (NK) cells are cells of the innate immune system, capable of killing virus-infected and tumor cells. Sialic acids can influence the interaction of NK cells with potential targets in several ways. Different NK cell receptors can bind sialic acids, leading to NK cell inhibition or activation. Moreover, NK cells have sialic acids on their surface, which can regulate receptor abundance and activity. This review is focused on how sialic acids on NK cells and their target cells are involved in NK cell function.

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Influenza A Virus Hemagglutinin and Other Pathogen Glycoprotein Interactions with NK Cell Natural Cytotoxicity Receptors NKp46, NKp44, and NKp30

Viruses ◽

10.3390/v13020156 ◽

2021 ◽

Vol 13 (2) ◽

pp. 156

Author(s):

Jasmina M. Luczo ◽

Sydney L. Ronzulli ◽

Stephen M. Tompkins

Keyword(s):

Influenza Virus ◽

Nk Cells ◽

Influenza A Virus ◽

Influenza A ◽

Nk Cell ◽

Influenza Virus Infection ◽

Target Cells ◽

Natural Cytotoxicity ◽

Activating Receptors ◽

Natural Cytotoxicity Receptors

Natural killer (NK) cells are part of the innate immunity repertoire, and function in the recognition and destruction of tumorigenic and pathogen-infected cells. Engagement of NK cell activating receptors can lead to functional activation of NK cells, resulting in lysis of target cells. NK cell activating receptors specific for non-major histocompatibility complex ligands are NKp46, NKp44, NKp30, NKG2D, and CD16 (also known as FcγRIII). The natural cytotoxicity receptors (NCRs), NKp46, NKp44, and NKp30, have been implicated in functional activation of NK cells following influenza virus infection via binding with influenza virus hemagglutinin (HA). In this review we describe NK cell and influenza A virus biology, and the interactions of influenza A virus HA and other pathogen lectins with NK cell natural cytotoxicity receptors (NCRs). We review concepts which intersect viral immunology, traditional virology and glycobiology to provide insights into the interactions between influenza virus HA and the NCRs. Furthermore, we provide expert opinion on future directions that would provide insights into currently unanswered questions.

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GSK-3α Inhibition in Drug-Resistant CML Cells Promotes Susceptibility to NK Cell-Mediated Lysis in an NKG2D- and NKp30-Dependent Manner

Cancers ◽

10.3390/cancers13081802 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1802

Author(s):

Nayoung Kim ◽

Mi Yeon Kim ◽

Woo Seon Choi ◽

Eunbi Yi ◽

Hyo Jung Lee ◽

...

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Negative Regulator ◽

Target Cells ◽

Dependent Manner ◽

Cell Functions ◽

Cell Based Therapy ◽

Activating Receptors ◽

Gsk 3Β

Natural killer (NK) cells are innate cytotoxic lymphocytes that provide early protection against cancer. NK cell cytotoxicity against cancer cells is triggered by multiple activating receptors that recognize specific ligands expressed on target cells. We previously demonstrated that glycogen synthase kinase (GSK)-3β, but not GSK-3α, is a negative regulator of NK cell functions via diverse activating receptors, including NKG2D and NKp30. However, the role of GSK-3 isoforms in the regulation of specific ligands on target cells is poorly understood, which remains a challenge limiting GSK-3 targeting for NK cell-based therapy. Here, we demonstrate that GSK-3α rather than GSK-3β is the primary isoform restraining the expression of NKG2D ligands, particularly ULBP2/5/6, on tumor cells, thereby regulating their susceptibility to NK cells. GSK-3α also regulated the expression of the NKp30 ligand B7-H6, but not the DNAM-1 ligands PVR or nectin-2. This regulation occurred independently of BCR-ABL1 mutation that confers tyrosine kinase inhibitor (TKI) resistance. Mechanistically, an increase in PI3K/Akt signaling in concert with c-Myc was required for ligand upregulation in response to GSK-3α inhibition. Importantly, GSK-3α inhibition improved cancer surveillance by human NK cells in vivo. Collectively, our results highlight the distinct role of GSK-3 isoforms in the regulation of NK cell reactivity against target cells and suggest that GSK-3α modulation could be used to enhance tumor cell susceptibility to NK cells in an NKG2D- and NKp30-dependent manner.

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