Faculty Opinions recommendation of A dominant-negative effect drives selection of TP53 missense mutations in myeloid malignancies.

TP53, which encodes the tumor suppressor p53, is the most frequently mutated gene in human cancer. The selective pressures shaping its mutational spectrum, dominated by missense mutations, are enigmatic, and neomorphic gain-of-function (GOF) activities have been implicated. We used CRISPR-Cas9 to generate isogenic human leukemia cell lines of the most common TP53 missense mutations. Functional, DNA-binding, and transcriptional analyses revealed loss of function but no GOF effects. Comprehensive mutational scanning of p53 single–amino acid variants demonstrated that missense variants in the DNA-binding domain exert a dominant-negative effect (DNE). In mice, the DNE of p53 missense variants confers a selective advantage to hematopoietic cells on DNA damage. Analysis of clinical outcomes in patients with acute myeloid leukemia showed no evidence of GOF for TP53 missense mutations. Thus, a DNE is the primary unit of selection for TP53 missense mutations in myeloid malignancies.

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Four novel mutations in the ALPL gene in Chinese patients with odonto, childhood, and adult hypophosphatasia

Bioscience Reports ◽

10.1042/bsr20171377 ◽

2018 ◽

Vol 38 (4) ◽

Cited By ~ 2

Author(s):

Lijun Xu ◽

Qianqian Pang ◽

Yan Jiang ◽

Ou Wang ◽

Mei Li ◽

...

Keyword(s):

Serum Alkaline Phosphatase ◽

Gene Mutations ◽

Dominant Negative ◽

Chinese Patients ◽

Dominant Negative Effect ◽

Healthy Controls ◽

Missense Mutations ◽

Genetic Characteristics ◽

Chinese Families ◽

Negative Effect

Hypophosphatasia (HPP) is a rare inherited disorder characterized by defective bone and/or dental mineralization, and decreased serum alkaline phosphatase (ALP) activity. ALPL, the only gene related with HPP, encodes tissue non-specific ALP (TNSALP). Few studies were carried out in ALPL gene mutations in the Chinese population with HPP. The purpose of the present study is to elucidate the clinical and genetic characteristics of HPP in five unrelated Chinese families and two sporadic patients. Ten clinically diagnosed HPP patients from five unrelated Chinese families and two sporadic patients and fifty healthy controls were genetically investigated. All 12 exons and exon–intron boundaries of the ALPL gene were amplified by PCR and directly sequenced. The laboratory and radiological investigations were conducted simultaneously in these HPP ten patients. A 3D model of the TNSALP was used to predict the dominant negative effect of identified missense mutations. Three odonto, three childhood, and four adult types of HPP were clinically diagnosed. Ten mutations were identified in five unrelated Chinese families and two sporadic patients, including eight missense mutations and two frameshift mutations. Of which, four were novel: one frameshift mutation (p.R138Pfsx45); three missense mutations (p.C201R, p.V459A, p.C497S). No identical mutations and any other new ALPL mutations were found in unrelated 50 healthy controls. Our study demonstrated that the ALPL gene mutations are responsible for HPP in these Chinese families. These findings will be useful for clinicians to improve understanding of this heritable bone disorder.

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The Human Glucocorticoid Receptor (GR) Isoform β Differentially Suppresses GRα-Induced Transactivation Stimulated by Synthetic Glucocorticoids

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2004-1646 ◽

2005 ◽

Vol 90 (6) ◽

pp. 3505-3509 ◽

Cited By ~ 26

Author(s):

Oren Fruchter ◽

Tomoshige Kino ◽

Emmanouil Zoumakis ◽

Salvatore Alesci ◽

Massimo De Martino ◽

...

Keyword(s):

Glucocorticoid Receptor ◽

Suppressive Effect ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Compound A ◽

Pathological Conditions ◽

Dominant Negative Inhibitor ◽

Negative Effect ◽

Synthetic Glucocorticoids ◽

Selection Of

The β-isoform of human glucocorticoid receptor β (hGRβ) acts as a natural dominant negative inhibitor of hGRα-induced transactivation of glucocorticoid-responsive genes. We determined hGRβ ability to suppress hGRα transactivation that was induced by commonly used synthetic glucocorticoids. HepG2/C3A cells were transiently cotransfected with GR cDNA and a glucocorticoid-responsive promoter, luciferase (MMTV-luc). Transfected cells were incubated for 16 h with glucocorticoid and luciferase. For each compound, a dose-response curve was constructed, and half-maximal effective concentrations and maximal transcriptional activities were compared. hGRβ, at a 1:1 ratio to hGRα, differentially suppressed hGRα-induced maximal transcriptional activity stimulated by triamcinolone, dexamethasone, hydrocortisone, and betamethasone (by 96, 68, 62, and 49%, respectively) but not by methylprednisolone. The suppressive effect of hGRβ on hGRα-induced transactivation was stronger at lower concentrations of all tested glucocorticoids, whereas it was blunted at higher concentrations. We conclude that the potency of the dominant negative effect of hGRβ on hGRα-induced transactivation depends on both the type and the dose of the synthetic glucocorticoids in use. These results may provide helpful information concerning the selection of synthetic glucocorticoids for treatment of pathological conditions in which hGRβ modulates the sensitivity of tissues to glucocorticoids.

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Characterization of ANGPT2 mutations associated with primary lymphedema

Science Translational Medicine ◽

10.1126/scitranslmed.aax8013 ◽

2020 ◽

Vol 12 (560) ◽

pp. eaax8013 ◽

Cited By ~ 1

Author(s):

Veli-Matti Leppänen ◽

Pascal Brouillard ◽

Emilia A. Korhonen ◽

Tuomas Sipilä ◽

Sawan Kumar Jha ◽

...

Keyword(s):

De Novo ◽

Vascular System ◽

Lymphatic Vessels ◽

Mouse Skin ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Missense Mutations ◽

Activation Mechanisms ◽

Primary Lymphedema ◽

Negative Effect

Primary lymphedema is caused by developmental and functional defects of the lymphatic vascular system that result in accumulation of protein-rich fluid in tissues, resulting in edema. The 28 currently known genes causing primary lymphedema can explain <30% of cases. Angiopoietin 1 (ANGPT1) and ANGPT2 function via the TIE1-TIE2 (tyrosine kinase with immunoglobulin-like and epidermal growth factor–like domains 1 and 2) receptor complex and α5β1 integrin to form an endothelial cell signaling pathway that is critical for blood and lymphatic vessel formation and remodeling during embryonic development, as well as for homeostasis of the mature vasculature. By screening a cohort of 543 individuals affected by primary lymphedema, we identified one heterozygous de novo ANGPT2 whole-gene deletion and four heterozygous ANGPT2 missense mutations. Functional analyses revealed three missense mutations that resulted in decreased ANGPT2 secretion and inhibited the secretion of wild-type (WT)–ANGPT2, suggesting that they have a dominant-negative effect on ANGPT2 signaling. WT-ANGPT2 and soluble mutants T299M and N304K activated TIE1 and TIE2 in an autocrine assay in human lymphatic endothelial cells. Molecular modeling and biophysical studies showed that amino-terminally truncated ANGPT subunits formed asymmetrical homodimers that bound TIE2 in a 2:1 ratio. The T299M mutant, located in the dimerization interphase, showed reduced integrin α5 binding, and its expression in mouse skin promoted hyperplasia and dilation of cutaneous lymphatic vessels. These results demonstrate that primary lymphedema can be associated with ANGPT2 mutations and provide insights into TIE1 and TIE2 activation mechanisms.

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Selective functional inhibition of a tumor-derived p53 mutant by cytosolic chaperones identified using split-YFP in budding yeast

10.1101/2021.04.02.438278 ◽

2021 ◽

Author(s):

Ashley S. Denney ◽

Andrew D. Weems ◽

Michael A. McMurray

Keyword(s):

Three Dimensional ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Temperature Sensitive ◽

Missense Mutations ◽

Tumor Suppressor P53 ◽

Wild Type ◽

Type Function ◽

Negative Effect ◽

Chaperone Interactions

ABSTRACTLife requires the oligomerization of individual proteins into higher-order assemblies. In order to form functional oligomers, monomers must adopt appropriate three-dimensional structures. Molecular chaperones transiently bind nascent or misfolded proteins to promote proper folding. Single missense mutations frequently cause disease by perturbing folding despite chaperone engagement. A misfolded mutant capable of oligomerizing with wild-type proteins can dominantly poison oligomer function. We previously found evidence that human-disease-linked mutations in Saccharomyces cerevisiae septin proteins slow folding and attract chaperones, resulting in a kinetic delay in oligomerization that prevents the mutant from interfering with wild-type function. Here we build upon our septin studies to develop a new approach to identifying chaperone interactions in living cells, and use it to expand our understanding of chaperone involvement, kinetic folding delays, and oligomerization in the recessive behavior of tumor-derived mutants of the tumor suppressor p53. We find evidence of increased binding of several cytosolic chaperones to a recessive, misfolding-prone mutant, p53(V272M). Similar to our septin results, chaperone overexpression inhibits the function of p53(V272M) with minimal effect on the wild type. Unlike mutant septins, p53(V272M) is not kinetically delayed under conditions in which it is functional. Instead, it interacts with wild-type p53 but this interaction is temperature sensitive. At high temperatures or upon chaperone overexpression, p53(V272M) is excluded from the nucleus and cannot function or perturb wild-type function. Chaperone inhibition liberates the mutant to enter the nucleus where it has a slight dominant-negative effect. These findings provide new insights into the effects of missense mutations.

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Role of the novel endoribonuclease SLFN14 and its disease causing mutations in ribosomal degradation

10.1101/267633 ◽

2018 ◽

Author(s):

Sarah J. Fletcher ◽

Vera P. Pisareva ◽

Abdullah Khan ◽

Andrew Tcherepanov ◽

Neil V. Morgan ◽

...

Keyword(s):

In Vitro Models ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Heterozygous Mutation ◽

Missense Mutations ◽

The Novel ◽

Excessive Bleeding ◽

Negative Effect ◽

Inherited Thrombocytopenia

ABSTRACTPlatelets are anucleate and mostly ribosome-free cells within the bloodstream, derived from megakaryocytes within bone marrow and crucial for cessation of bleeding at sites of injury. Inherited thrombocytopenias are a group of disorders characterized by alow platelet count and are frequently associated with excessive bleeding. SLFN14 is one of the most recently discovered genes linked to inherited thrombocytopenia where several heterozygous missense mutations in SLFN14 were identified to cause defective megakaryocyte maturation and platelet dysfunction. Yet, SLFN14 was recently described as a ribosome-associated protein resulting in rRNA and ribosome-bound mRNA degradation in rabbit reticulocytes. To unveil the cellular function of SLFN14 and the link between SLFN14 and thrombocytopenia, we examined SLFN14 (WT/mutants) in in vitro models. Here, we show that all SLFN14 variants co-localize with ribosomes and mediate rRNA endonucleolytic degradation and ribosome clearance. Compare dto SLFN14 WT, expression of mutants is dramatically reduced as a result of post-translational degradation due to partial misfolding of the protein. Moreover, all SLFN14 variants tend to form oligomers. These findings could explain the dominant negative effect of heterozygous mutation on SLFN14 expression in patients’ platelets. Overall we suggest that SLFN14 could be involved in ribosome degradation during platelet formation and maturation.

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The Impact of Different DNMT3A Mutations on Outcome in Younger Adults with Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v124.21.67.67 ◽

2014 ◽

Vol 124 (21) ◽

pp. 67-67

Author(s):

Rosemary E Gale ◽

Katarina Lamb ◽

Christopher Allen ◽

Dima El-Sharkawi ◽

Cassandra Stowe ◽

...

Keyword(s):

Conflicts Of Interest ◽

Multivariable Analysis ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Heterozygous Mutation ◽

Prognostic Impact ◽

Missense Mutations ◽

Limited Information ◽

Negative Effect ◽

The Impact

Abstract DNMT3A mutations (DNMT3AMUT) are recurrent in AML. They predominate in patients with intermediate-risk (IR) cytogenetics and are often co-incident with FLT3ITD and NPM1MUT. Their prognostic impact is unclear. Most reports suggest they are associated with a worse outcome, but a large study including 1060 younger adult IR patients found that DNMT3AMUT had no significant impact on survival endpoints. Variable results have also been reported for different FLT3/NPM1 subgroups. Missense mutations at R882 in exon 23 occur in ≈65% of patients, but other missense and truncation mutations occur throughout the gene, mainly in exons 13-23. There is limited information on the prognostic impact of the different mutations, although they may have differing functional consequences. We therefore screened exons 13-23 in DNA samples from 914 younger adult AML patients (median age 43 years) with IR cytogenetics treated on UK MRC trials and evaluated outcome according to type of DNMT3Amutation. Overall, 278 mutations were detected in 272 (30%) patients; 175 (64%) had R882 missense mutations, 59 (22%) other missense mutations, 35 (13%) truncations or in-frame deletions; 3 (1%) had 2 mutations of differing types. Median R882 mutant level in 172 mutated cases was 47% (range 15-85%), consistent with a heterozygous mutation in most cells. Patients with DNMT3AMUT were significantly older than those with DNMT3A wild-type (DNMT3AWT) (P<.0001), more likely to be female (P=.004), have a higher presenting WBC (P<.0001), and a normal rather than abnormal karyotype (P<.0001). The presence of DNMT3AMUT positively correlated with FLT3ITD (P=.0003) and NPM1MUT (P<.0001) and negatively correlated with CEBPAMUT (P<.0001). Patients with R882 mutations had significantly higher WBC (P=.005) and correlation with NPM1MUT (P=.01) than non-R882 mutated patients; non-R882 missense mutated patients had higher WBC (P=.05) and non-significant higher co-incidence with FLT3ITD than those with truncations. Presence of DNMT3AMUT was associated with a poorer prognosis, but this difference was only seen if the results were analyzed separately according to NPM1 genotype, where DNMT3AMUT was associated with higher cumulative incidence of relapse (CIR) than DNMT3AWT in cases with NPM1MUT (49% vs 40%, P=.01) and NPM1WT (61% vs 58%, P=.5) genotype. Similarly, DNMT3AMUT patients had worse overall survival (OS) than DNMT3AWT patients with NPM1MUT (38% vs 50%, P=.008) and NPM1WT (15% vs 25%, P=.09) genotype. This statistical anomaly is an example of Simpson’s paradox. It results from the strong co-incidence of DNMT3A and NPM1 mutations with opposing prognostic associations that mask the effect seen separately when the groups are combined. Although the differences were smaller for NPM1WT cases, tests for heterogeneity showed that the impact of a DNMT3A mutation did not differ between NPM1MUT and NPM1WT for either CIR or OS, nor between the 4 genotypes defined by the combination of NPM1 and FLT3ITD genotypes. In multivariable analysis including age, WBC, NPM1 and FLT3ITD, DNMT3AMUT was a significant adverse risk factor for CIR (HR=1.27, CI=1.01-1.61; P=.04), and showed a trend for being adverse for OS (HR=1.19, CI=.98-1.45; P=.08). When outcome was considered according to the type of mutation (R882, other missense or truncations), for the NPM1MUT genotype cases CIR was highest in R882 and other missense cases (51%, 50%) and truncation cases were similar to DNMT3AWT (35%, 40%). For NPM1WT, CIR was highest in R882 cases (76%), similar in other missense and DNMT3AWT cases (55%, 58%) and lowest in truncation cases (40%). Consistent with this data, for NPM1MUT genotype, OS was lowest in R882 and other missense cases (35%, 38%), better in DNMT3AWT (50%) and highest in truncation cases (57%). For NPM1WT, OS was lowest in R882 cases (11%), and similar in DNMT3AWT, other missense and truncation cases (25%, 21%, 18% respectively). These data suggest that screening cannot be limited to the hotspot R882 mutations and that cases with missense mutations should be treated as poor risk, including those patients currently considered as favorable risk such as NPM1MUTFLT3WT. Conversely, truncation mutations have a different functional impact from missense mutations, more likely to result in haploinsufficiency than a dominant-negative effect, and these cases should be considered as equivalent to DNMT3AWT for prognostication and selection of therapy in 1st remission. Disclosures No relevant conflicts of interest to declare.

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A Dominant Negative Effect of eth-1r, a Mutant Allele of the Neurospora crassa S-Adenosylmethionine Synthetase-Encoding Gene Conferring Resistance to the Methionine Toxic Analogue Ethionine

Genetics ◽

10.1093/genetics/144.4.1455 ◽

1996 ◽

Vol 144 (4) ◽

pp. 1455-1462

Author(s):

José L Barra ◽

Mario R Mautino ◽

Alberto L Rosa

Keyword(s):

Neurospora Crassa ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Resistant Phenotype ◽

Negative Effect ◽

Encoding Gene ◽

Adenosylmethionine Synthetase ◽

Adomet Synthetase ◽

Identical Gene

eth-1r a thermosensitive allele of the Neurospora crassa S-adenosylmethionine (AdoMet) synthetase gene that confers ethionine resistance, has been cloned and sequenced. Replacement of an aspartic amino acid residue (D48 → N48), perfectly conserved in prokaryotic, fungal and higher eukaryotic AdoMet synthetases, was found responsible for both thermosensitivity and ethionine resistance conferred by eth-1r. Gene fusion constructs, designed to overexpress eth-1r in vivo, render transformant cells resistant to ethionine. Dominance of ethionine resistance was further demonstrated in eth-1 +/eth-1r partial diploids carrying identical gene doses of both alleles. Heterozygous eth-1 +/eth-1r cells have, at the same time, both the thermotolerance conferred by eth-1 + and the ethionine-resistant phenotype conferred by eth-1r. AdoMet levels and AdoMet synthetase activities were dramatically decreased in heterozygous eth-1 +/eth-1r cells. We propose that this negative effect exerted by eth-1r results from the in vivo formation of heteromeric eth-1 +/eth-1r AdoMet synthetase molecules.

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Evidence for a dominant‐negative effect of a missense mutation in the SERPING1 gene responsible for hereditary angioedema type I

The Journal of Dermatology ◽

10.1111/1346-8138.15930 ◽

2021 ◽

Author(s):

Shuichiro Yasuno ◽

Osamu Ansai ◽

Ryota Hayashi ◽

Sawako Nakamura ◽

Yutaka Shimomura

Keyword(s):

Missense Mutation ◽

Hereditary Angioedema ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Type I ◽

Negative Effect ◽

Serping1 Gene

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Restoration of Silencing in Saccharomyces cerevisiae by Tethering of a Novel Sir2-Interacting Protein, Esc8

Genetics ◽

10.1093/genetics/162.2.633 ◽

2002 ◽

Vol 162 (2) ◽

pp. 633-645 ◽

Cited By ~ 2

Author(s):

Guido Cuperus ◽

David Shore

Keyword(s):

Protein A ◽

Dominant Negative ◽

Class I ◽

Dominant Negative Effect ◽

Rna Pol Ii ◽

Interacting Protein ◽

Nucleosome Remodeling ◽

Negative Effect ◽

Close Homolog

Abstract We previously described two classes of SIR2 mutations specifically defective in either telomeric/HM silencing (class I) or rDNA silencing (class II) in S. cerevisiae. Here we report the identification of genes whose protein products, when either overexpressed or directly tethered to the locus in question, can establish silencing in SIR2 class I mutants. Elevated dosage of SCS2, previously implicated as a regulator of both inositol biosynthesis and telomeric silencing, suppressed the dominant-negative effect of a SIR2-143 mutation. In a genetic screen for proteins that restore silencing when tethered to a telomere, we isolated ESC2 and an uncharacterized gene, (YOL017w), which we call ESC8. Both Esc2p and Esc8p interact with Sir2p in two-hybrid assays, and the Esc8p-Sir2 interaction is detected in vitro. Interestingly, Esc8p has a single close homolog in yeast, the ISW1-complex factor Ioc3p, and has also been copurified with Isw1p, raising the possibility that Esc8p is a component of an Isw1p-containing nucleosome remodeling complex. Whereas esc2 and esc8 deletion mutants alone have only marginal silencing defects, cells lacking Isw1p show a strong silencing defect at HMR but not at telomeres. Finally, we show that Esc8p interacts with the Gal11 protein, a component of the RNA pol II mediator complex.

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