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

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

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.1865 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 192.1-192

Author(s):

R. Cron ◽

M. Zhang ◽

D. Absher ◽

J. Bridges ◽

A. Schnell ◽

...

Keyword(s):

Nk Cell ◽

Foamy Virus ◽

Dominant Negative ◽

Target Cells ◽

Missense Mutations ◽

Cytokine Storm ◽

Negative Effects ◽

Research Support ◽

Rare Mutations ◽

Lytic Function

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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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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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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Glucocorticoid Receptor β (GRβ): Beyond Its Dominant-Negative Function

International Journal of Molecular Sciences ◽

10.3390/ijms22073649 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3649

Author(s):

Patricia Ramos-Ramírez ◽

Omar Tliba

Keyword(s):

Current Knowledge ◽

Inflammatory Diseases ◽

Cell Communication ◽

Cell Types ◽

The Body ◽

Dominant Negative ◽

Negative Effects ◽

Independent Manner ◽

Distinct Cell ◽

Induced Apoptosis

Glucocorticoids (GCs) act via the GC receptor (GR), a receptor ubiquitously expressed in the body where it drives a broad spectrum of responses within distinct cell types and tissues, which vary in strength and specificity. The variability of GR-mediated cell responses is further extended by the existence of GR isoforms, such as GRα and GRβ, generated through alternative splicing mechanisms. While GRα is the classic receptor responsible for GC actions, GRβ has been implicated in the impairment of GRα-mediated activities. Interestingly, in contrast to the popular belief that GRβ actions are restricted to its dominant-negative effects on GRα-mediated responses, GRβ has been shown to have intrinsic activities and “directly” regulates a plethora of genes related to inflammatory process, cell communication, migration, and malignancy, each in a GRα-independent manner. Furthermore, GRβ has been associated with increased cell migration, growth, and reduced sensitivity to GC-induced apoptosis. We will summarize the current knowledge of GRβ-mediated responses, with a focus on the GRα-independent/intrinsic effects of GRβ and the associated non-canonical signaling pathways. Where appropriate, potential links to airway inflammatory diseases will be highlighted.

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In Caenorhabditis elegans, the RNA-Binding Domains of the Cytoplasmic Polyadenylation Element Binding Protein FOG-1 Are Needed to Regulate Germ Cell Fates

Genetics ◽

10.1093/genetics/159.4.1617 ◽

2001 ◽

Vol 159 (4) ◽

pp. 1617-1630

Author(s):

Suk-Won Jin ◽

Nancy Arno ◽

Adam Cohen ◽

Amy Shah ◽

Qijin Xu ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Germ Cell ◽

Rna Binding ◽

Dominant Negative ◽

Missense Mutations ◽

Biochemical Tests ◽

Cell Fates ◽

Cytoplasmic Polyadenylation ◽

Binding Domains ◽

Rna Binding Domains

Abstract FOG-1 controls germ cell fates in the nematode Caenorhabditis elegans. Sequence analyses revealed that FOG-1 is a cytoplasmic polyadenylation element binding (CPEB) protein; similar proteins from other species have been shown to bind messenger RNAs and regulate their translation. Our analyses of fog-1 mutations indicate that each of the three RNA-binding domains of FOG-1 is essential for activity. In addition, biochemical tests show that FOG-1 is capable of binding RNA sequences in the 3′-untranslated region of its own message. Finally, genetic assays reveal that fog-1 functions zygotically, that the small fog-1 transcript has no detectable function, and that missense mutations in fog-1 cause a dominant negative phenotype. This last observation suggests that FOG-1 acts in a complex, or as a multimer, to regulate translation. On the basis of these data, we propose that FOG-1 binds RNA to regulate germ cell fates and that it does so by controlling the translation of its targets. One of these targets might be the fog-1 transcript itself.

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Mutant VAPB: Culprit or Innocent Bystander of Amyotrophic Lateral Sclerosis?

Contact ◽

10.1177/25152564211022515 ◽

2021 ◽

Vol 4 ◽

pp. 251525642110225

Author(s):

Nica Borgese ◽

Francesca Navone ◽

Nobuyuki Nukina ◽

Tomoyuki Yamanaka

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Motor Neuron ◽

Motor Neurons ◽

Dominant Negative ◽

Negative Effects ◽

Membrane Contact Sites ◽

Familial Form ◽

Main Driver ◽

Mechanistic Basis ◽

Lateral Sclerosis

Nearly twenty years ago a mutation in the VAPB gene, resulting in a proline to serine substitution (p.P56S), was identified as the cause of a rare, slowly progressing, familial form of the motor neuron degenerative disease Amyotrophic Lateral Sclerosis (ALS). Since then, progress in unravelling the mechanistic basis of this mutation has proceeded in parallel with research on the VAP proteins and on their role in establishing membrane contact sites between the ER and other organelles. Analysis of the literature on cellular and animal models reviewed here supports the conclusion that P56S-VAPB, which is aggregation-prone, non-functional and unstable, is expressed at levels that are insufficient to support toxic gain-of-function or dominant negative effects within motor neurons. Instead, insufficient levels of the product of the single wild-type allele appear to be required for pathological effects, and may be the main driver of the disease. In light of the multiple interactions of the VAP proteins, we address the consequences of specific VAPB depletion and highlight various affected processes that could contribute to motor neuron degeneration. In the future, distinction of specific roles of each of the two VAP paralogues should help to further elucidate the basis of p.P56S familial ALS, as well as of other more common forms of the disease.

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Dominant Negative Mutants Implicate STAT5 in Myeloid Cell Proliferation and Neutrophil Differentiation

Blood ◽

10.1182/blood.v93.12.4154 ◽

1999 ◽

Vol 93 (12) ◽

pp. 4154-4166 ◽

Cited By ~ 80

Author(s):

Robert L. Ilaria ◽

Robert G. Hawley ◽

Richard A. Van Etten

Keyword(s):

Dna Binding ◽

Transcriptional Activation ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Cytokine Signaling ◽

Transactivation Domain ◽

Negative Effects ◽

Murine Bone Marrow ◽

Negative Effect

Abstract STAT5 is a member of the signal transducers and activation of transcription (STAT) family of latent transcription factors activated in a variety of cytokine signaling pathways. We introduced alanine substitution mutations in highly conserved regions of murine STAT5A and studied the mutants for dimerization, DNA binding, transactivation, and dominant negative effects on erythropoietin-induced STAT5-dependent transcriptional activation. The mutations included two near the amino-terminus (W255KR→AAA and R290QQ→AAA), two in the DNA-binding domain (E437E→AA and V466VV→AAA), and a carboxy-terminal truncation of STAT5A (STAT5A/▵53C) analogous to a naturally occurring isoform of rat STAT5B. All of the STAT mutant proteins were tyrosine phosphorylated by JAK2 and heterodimerized with STAT5B except for the WKR mutant, suggesting an important role for this region in STAT5 for stabilizing dimerization. The WKR, EE, and VVV mutants had no detectable DNA-binding activity, and the WKR and VVV mutants, but not EE, were defective in transcriptional induction. The VVV mutant had a moderate dominant negative effect on erythropoietin-induced STAT5 transcriptional activation, which was likely due to the formation of heterodimers that are defective in DNA binding. Interestingly, the WKR mutant had a potent dominant negative effect, comparable to the transactivation domain deletion mutant, ▵53C. Stable expression of either the WKR or ▵53C STAT5 mutants in the murine myeloid cytokine-dependent cell line 32D inhibited both interleukin-3–dependent proliferation and granulocyte colony-stimulating factor (G-CSF)–dependent differentiation, without induction of apoptosis. Expression of these mutants in primary murine bone marrow inhibited G-CSF–dependent granulocyte colony formation in vitro. These results demonstrate that mutations in distinct regions of STAT5 exert dominant negative effects on cytokine signaling, likely through different mechanisms, and suggest a role for STAT5 in proliferation and differentiation of myeloid cells.

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Mutation-specific non-canonical pathway of PTEN as a distinct therapeutic target for glioblastoma

Cell Death and Disease ◽

10.1038/s41419-021-03657-0 ◽

2021 ◽

Vol 12 (4) ◽

Author(s):

Seung Won Choi ◽

Yeri Lee ◽

Kayoung Shin ◽

Harim Koo ◽

Donggeon Kim ◽

...

Keyword(s):

Functional Properties ◽

Therapeutic Target ◽

Medical Center ◽

Dominant Negative ◽

The Cancer Genome Atlas ◽

Dominant Negative Effect ◽

Cell Periphery ◽

Missense Mutations ◽

Phosphatase Domain ◽

Mutant Proteins

AbstractPTEN is one of the most frequently altered tumor suppressor genes in malignant tumors. The dominant-negative effect of PTEN alteration suggests that the aberrant function of PTEN mutation might be more disastrous than deletion, the most frequent genomic event in glioblastoma (GBM). This study aimed to understand the functional properties of various PTEN missense mutations and to investigate their clinical relevance. The genomic landscape of PTEN alteration was analyzed using the Samsung Medical Center GBM cohort and validated via The Cancer Genome Atlas dataset. Several hotspot mutations were identified, and their subcellular distributions and phenotypes were evaluated. We established a library of cancer cell lines that overexpress these mutant proteins using the U87MG and patient-derived cell models lacking functional PTEN. PTEN mutations were categorized into two major subsets: missense mutations in the phosphatase domain and truncal mutations in the C2 domain. We determined the subcellular compartmentalization of four mutant proteins (H93Y, C124S, R130Q, and R173C) from the former group and found that they had distinct localizations; those associated with invasive phenotypes (‘edge mutations’) localized to the cell periphery, while the R173C mutant localized to the nucleus. Invasive phenotypes derived from edge substitutions were unaffected by an anti-PI3K/Akt agent but were disrupted by microtubule inhibitors. PTEN mutations exhibit distinct functional properties regarding their subcellular localization. Further, some missense mutations (‘edge mutations’) in the phosphatase domain caused enhanced invasiveness associated with dysfunctional cytoskeletal assembly, thus suggesting it to be a potent therapeutic target.

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