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Results for “Gain of Function Mutation”

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PubMed1996

Enhancement of lymphocyte responsiveness by a gain-of-function mutation of ZAP-70.

The protein tyrosine kinase ZAP-70 plays an essential role in T-cell activation and development. After T-cell receptor stimulation, ZAP-70 is associated with the receptor and is phosphorylated on many tyrosine residues, including tyrosine 292 (Y-292), in the region between the C-terminal SH2 domain and the kinase domain (interdomain B). Here we show that a mutation of Y-292 (292F) or deletion of interdomain B enhanced the ability of ZAP-70 to reconstitute B-cell receptor stimulation-dependent NF-AT induction in a B-cell line deficient in Syk. In contrast, in a T-cell line, expression of 292F led to basal NF-AT induction independent of T-cell receptor stimulation. These results demonstrate that the role of Y-292 is to negatively regulate the function of ZAP-70 in lymphocytes. This appears to be a dominant function of interdomain B because deletion of most of interdomain B also resulted in a mutant of ZAP-70 with enhanced ability to reconstitute Syk-deficient DT-40 B cells. Since our biochemical studies did not reveal an effect of the 292F mutation on either the kinase activity of ZAP-70 or on the ability of ZAP-70 to bind to the receptor, we propose a model in which Y-292 interacts with an inhibitory protein to negatively regulate ZAP-70 function.

Gene Expression Regulation↗
PubMed2005

No GIST-type c-kit gain of function mutations in neuroblastic tumours.

AIMS: Neuroblastic tumours (NTs) have been shown to respond to imatinib treatment in vivo and in vitro, possibly via inactivating the c-kit receptor. The purpose of this study was to identify gastrointestinal stromal tumour (GIST)-type c-kit gene associated mutations in exons 9, 11, 13, and 17 in NTs to recognise a subset of tumours that would probably respond to imatinib treatment. METHODS: Expression of the c-kit protein was detected immunohistochemically in a total of 37 archival paraffin wax embedded NTs using polyclonal rabbit antihuman c-kit antibody. After immunohistochemistry, c-kit gene associated chromosomal mutations in all cases of NT were detected with denaturing high performance liquid chromatography (HPLC). RESULTS: Denaturing HLPC analysis did not reveal GIST-type mutations in four immunohistochemically detected c-kit positive or in 33 c-kit negative NTs. CONCLUSIONS: c-kit receptor expression and GIST-type c-kit gene mutations are rare events in NTs. Oncogenic activation of c-kit in NTs presumably differs from that of GISTs, which may influence their responsiveness to imatinib treatment. Whether c-kit has an essential role in the pathogenesis of NTs remains to be investigated.

Antineoplastic Agents↗
PubMed2002

A family of autosomal dominant hypocalcemia with a positive correlation between serum calcium and magnesium: identification of a novel gain of function mutation (Ser(820)Phe) in the calcium-sensing receptor.

To date about 20 activating mutations in the calcium-sensing receptor (CaR) gene have been identified to cause autosomal dominant hypocalcemia (ADH) or sporadic hypoparathyroidism. We report a novel activating mutation in the CaR gene in a Japanese family with ADH. The proband, a 15-yr-old boy, and 5 other patients in 3 generations were asymptomatic, except for the proband's grandmother who had a history of seizures. They showed mild hypocalcemia (1.68-1.98 mmol/liter) with normal urinary calcium excretion and low normal serum PTH levels. Their serum magnesium concentrations were below normal in 3 adults and within the normal range in 3 teenagers. There was a significant positive correlation (r = 0.90; P < 0.05) between the serum calcium and magnesium concentrations of 6 affected members. Nucleotide sequencing revealed that the proband had a known polymorphism (Gly(990)Arg) and a novel heterozygous mutation substituting phenylalanine for serine at codon 820 (Ser(820)Phe) in the sixth transmembrane helix of the CaR. In other family members, the Ser(820)Phe mutation cosegregated with hypocalcemia. The mutation was not detected in 50 control subjects. The Gly(990)Arg polymorphism was observed in 8 of 9 family members with or without hypocalcemia and in 36 of 50 controls. The sensitivity of the Ser(820)Phe mutant CaR to calcium was assessed using transiently transfected HEK293 cells and measuring the increases in intracellular Ca(2+) concentrations in response to the changes in extracellular Ca(2+). The concentration-response curve of the mutant receptor was left-shifted, and its EC(50) (2.5 mM) was significantly (P < 0.05) lower than that of the wild-type CaR (3.3 mM). We conclude that the Ser(820)Phe mutation in the CaR caused ADH in this family. The positive correlation between serum calcium and magnesium levels observed in this family may support the concept that renal CaR acts as a magnesium sensor as well as a calcium sensor.

Adolescent↗
PubMed2000

Loss- and gain-of-function mutations show a polycomb group function for Ring1A in mice.

The products of the Polycomb group (PcG) of genes act as transcriptional repressors involved in the maintenance of homeotic gene expression patterns throughout development, from flies to mice. Biochemical and molecular evidence suggests that the mouse Ring1A gene is a member of the PcG of genes. However, genetic evidence is needed to establish PcG function for Ring1A, since contrary to all other murine PcG genes, there is no known Drosophila PcG gene encoding a homolog of the Ring1A protein. To study Ring1A function we have generated a mouse line lacking Ring1A and mouse lines overexpressing Ring1A. Both Ring1A(-/-)and Ring1A(+/-) mice show anterior transformations and other abnormalities of the axial skeleton, which indicates an unusual sensitivity of axial skeleton patterning to Ring1A gene dosage. Ectopic expression of Ring1A also results in dose-dependent anterior transformations of vertebral identity, many of which, interestingly, are shared by Ring1A(-/-) mice. In contrast, the alterations of Hox gene expression observed in both type of mutant mice are subtle and involve a reduced number of Hox genes. Taken together, these results provide genetic evidence for a PcG function of the mouse Ring1A gene.

Animals↗
PubMed2006

A gain-of-function mutation in synaptotagmin-1 reveals a critical role of Ca2+-dependent soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex binding in synaptic exocytosis.

Synaptotagmin-1, the Ca2+ sensor for fast neurotransmitter release, was proposed to function by Ca2+-dependent phospholipid binding and/or by Ca2+-dependent soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex binding. Extensive in vivo data support the first hypothesis, but testing the second hypothesis has been difficult because no synaptotagmin-1 mutation is known that selectively interferes with SNARE complex binding. Using knock-in mice that carry aspartate-to-asparagine substitutions in a Ca2+-binding site of synaptotagmin-1 (the D232N or D238N substitutions), we now show that the D232N mutation dramatically increases Ca2+-dependent SNARE complex binding by native synaptotagmin-1, but leaves phospholipid binding unchanged. In contrast, the adjacent D238N mutation does not significantly affect SNARE complex binding, but decreases phospholipid binding. Electrophysiological recordings revealed that the D232N mutation increased Ca2+-triggered release, whereas the D238N mutation decreased release. These data establish that fast vesicle exocytosis is driven by a dual Ca2+-dependent activity of synaptotagmin-1, namely Ca2+-dependent binding both to SNARE complexes and to phospholipids.

Animals↗
PubMed2025

Gain-of-function PPM1D mutations attenuate ischemic stroke.

Identification of genetic aberrations in stroke, the second leading cause of death worldwide, is of paramount importance for understanding the disease pathogenesis and generating new therapies. Whole-genome sequencing from 10,241 ischemic stroke patients identified eight patients carrying gain-of-function mutations on coding variants in the protein phosphatase magnesium-dependent 1 &#x3b4; (PPM1D) gene. Patients carrying PPM1D mutations exhibit better stroke-related clinical phenotypes, including improvements in peripheral inflammation, fibrinogen, low-density lipoprotein, cholesterol&#xa0;and plateletcrit level. Experimental brain ischemia in Ppm1d-deficient (Ppm1d-/-) mice resulted in enlarged lesions and pronounced neurological impairments. Spatial transcriptomics revealed a distinct Ppm1d-associated gene expression pattern, indicating disrupted endothelial homeostasis during ischemic brain injury. Proteomic analysis demonstrated that differentially expressed proteins in primary brain endothelial cells from Ppm1d-/- mice were significantly enriched in the peroxisome proliferator-activated receptors (PPARs)-mediated metabolic signaling. Mechanistically, Ppm1d deficiency promoted aberrant fatty acid &#x3b2;-oxidation and increased oxidative stress, which impaired endothelial cell function through the PPAR&#x3b1; pathway. A small molecule, T2755, was identified to engage Trp427 and stabilize PPM1D, thereby mitigating ischemic brain injury in mice. Collectively, we find that PPM1D protects against ischemic brain injury and validates its pharmacological stabilizer T2755 as a promising therapy for ischemic stroke. Gain-of-function PPM1D mutations attenuate ischemic cerebral injury. Whole-genome sequencing data of 10,241 ischemic stroke patients from the Third Chinese National Stroke Registry (CNSR-III) identified eight patients with gain-of-function mutations in the protein phosphatase magnesium-dependent 1 &#x3b4; (PPM1D) gene (17q23.2). These mutation carriers displayed improved peripheral inflammation,&#xa0;decreased&#xa0;fibrinogen, low-density lipoprotein, cholesterol&#xa0;and plateletcrit level. Ppm1d-deficient (Ppm1d-/-) mice exhibited exacerbated stroke outcomes, characterized by enlarged infarct volumes, disrupted cerebrovascular architecture, and enhanced neuro-inflammation. Mechanistically, Ppm1d deficiency induced the disturbance of endothelial fatty acid metabolism involving the PPAR&#x3b1; pathway. Through integrated computational modeling, virtual screening, and in vitro validation, T2755 was identified as a small molecule PPM1D stabilizer. Pharmacological PPM1D stabilization with T2755 significantly attenuated ischemic brain injury in murine models.

Aged↗
PubMed2000

Functional characterization of five constitutively activating thyrotrophin receptor mutations.

OBJECTIVE: Gain of function mutations of the thyrotrophin receptor (TSHR) affect several functional characteristics, such as cAMP and inositol phosphate (IP) accumulation, cell surface expression and TSH affinity. In this study we compared five constitutively activating TSHR mutations, four receptors with a point mutation (S505N, L629F, I630L, V656F) and a nine amino acid (aa) deletion mutant (aa positions 613-621) for these functional parameters in parallel transfection experiments. METHODS: The wild-type TSHR (wt) and TSHRs containing the mutations S505N, L629F, I630L, V656F and the deletion 613-621 (all cloned in the expression vector pSVL) were transiently expressed in COS-7 cells in parallel experiments. Forty-eight hours after transfection the basal and stimulated cAMP and inositol phosphate accumulation as well as the cell surface expression (by FACS and ELISA), KD-values and TSHR down regulation by different stimuli were determined. RESULTS: In contrast to the very different values for specific constitutive activity (sca) (ranging from 7.5 to 100.3-fold wt) and very different levels of receptor cell surface expression (11-94% wt level) the basal cAMP accumulation determined in transfected COS-7 cells was surprisingly uniform (6.5-8.0 over wt basal). None of the point mutated receptors constitutively activates the phospholipase C cascade. In contrast the deletion 613-621 mutant showed constitutive activity for the IP pathway with a twofold increase in basal IP accumulation compared to the wild type TSHR. All investigated TSHR-mutants showed a TSH-stimulated receptor down-regulation, which seems to be independent of the phospholipase C pathway. CONCLUSIONS: The uniform basal cAMP values in spite of the large variation in specific constitutive activity values suggest that the COS-7 cell overexpression system used for the in vitro characterization is partly regulated. This regulation is most likely due to receptor down regulation. The TSHR deletion mutant (613-621) showed a constitutive activity for both the Galphas and the Galphaq/11 pathways. The TSH-mediated IP-stimulation by this mutant contrasts with its unresponsiveness to TSH for cAMP accumulation and therefore supports the model of different active conformations of the TSHR.

Animals↗
PubMed1992

Disorganization is a completely dominant gain-of-function mouse mutation causing sporadic developmental defects.

Disorganization (Ds) is an exceptional mutation because of its diverse and profound developmental effects. Although other mouse mutations produce similar congenital defects, extreme pleiotropism, random occurrence, developmental independence of multiple defects, and type of anomaly make Ds unique. Examples of developmental defects include cranioschisis, rachischisis, thoracoschisis, exencephaly, hamartomas, and anomalies of appendages, digestive, genital and urinary tracts, sense organs, limbs and girdles, tail and pharynx. No other mutation in the mouse has such broad effects. Ds is therefore an important model for studying not only the genetic control of lineage determination and pattern formation, but also the occurrence of sporadic congenital defects. To characterize the effects of gene dosage, we examined the viability and phenotype of Ds homozygotes and the phenotype of +/+/Ds trisomic fetuses. Occurrence of homozygotes was tested by intercrossing Ds/+ heterozygotes, typing genetic markers that flank Ds, and examining homozygotes for morphological abnormalities. Not only were Ds homozygotes found in their expected frequency, homozygotes were not more severely affected than heterozygotes. Trisomies provide a direct test for determining whether Ds is a gain-of-function mutation. Trisomic fetuses were derived by crossing Ds/Ds homozygous mice to hybrid mice that were heterozygous for two related Robertsonian translocations. Two trisomic fetuses had developmental defects characteristic of Ds mice. Together these results demonstrate that Ds is a completely dominant, gain-of-function mutation.

Abnormalities, Multiple↗
PubMed2005

Epilepsy and sodium channel gene mutations: gain or loss of function?

Mutations in voltage-gated sodium channel genes (SCN1A, SCN2A, SCN1B) have been reported to be responsible for some epilepsies. Although studying such mutations to elucidate the disease mechanisms would be indispensable for the development of effective therapies, the functional consequences of these mutations remain controversial. Here, I propose a novel hypothesis for an epileptic disease mechanism which could drive the design of further studies to understand the molecular pathology of these diseases.

Epilepsy↗
PubMed2004

Oropharyngeal skeletal disease accompanying high bone mass and novel LRP5 mutation.

UNLABELLED: Gain-of-function mutation in the gene encoding LRP5 causes high bone mass. A 59-year-old woman carrying a novel LRP5 missense mutation, Arg154Met, manifested skeletal disease affecting her oropharynx as well as dense bones, showing that exuberant LRP5 effects are not always benign. INTRODUCTION: Gain-of-function mutation (Gly171Val) of LDL receptor-related protein 5 (LRP5) was discovered in 2002 in two American kindreds with high bone mass and benign phenotypes. In 2003, however, skeletal disease was reported for individuals from the Americas and Europe carrying any of six novel LRP5 missense mutations affecting the same LRP5 protein domain. Furthermore, in 2004, we described a patient with neurologic complications from dense bones and extensive oropharyngeal exostoses caused by the Gly171Val defect. MATERIALS AND METHODS: A 59-year-old woman was referred for dense bones. Three years before, mandibular buccal and lingual exostoses (osseous "tori") were removed because of infections from food trapping between the teeth and exostoses. Maxillary buccal and palatal exostoses were asymptomatic. Radiographic skeletal survey showed marked thickening of the skull base and diaphyses of long bones (endosteal hyperostosis). BMD Z scores assessed by DXA were +8.5 and +8.7 in the total hip and L(1)-L(4) spine (both approximately 195% average control), respectively. LRP5 mutation analysis was carried out for the LRP5 domain known to cause high bone mass. RESULTS: Biochemical evaluation excluded most secondary causes of dense bones, and male-to-male transmission in her family indicated autosomal dominant inheritance. PCR amplification and sequencing of LRP5 exons 2-4 and adjacent splice sites revealed heterozygosity for a new LRP5 missense mutation, Arg154Met. CONCLUSIONS: LRP5 Arg154Met is a novel defect that changes the same first "beta-propeller" module as the eight previously reported LRP5 gain-of-function missense mutations. Arg154Met alters a region important for LRP5 antagonism by dickkopf (Dkk). Therefore, our patient's extensive oropharyngeal exostoses and endosteal hyperostosis likely reflect increased Wnt signaling and show that exuberant LRP5 effects are not always benign.

Alleles↗
PubMed2000

Gain of function mutants: ion channels and G protein-coupled receptors.

Many ion channels and receptors display striking phenotypes for gain-of-function mutations but milder phenotypes for null mutations. Gain of molecular function can have several mechanistic bases: selectivity changes, gating changes including constitutive activation and slowed inactivation, elimination of a subunit that enhances inactivation, decreased drug sensitivity, changes in regulation or trafficking of the channel, or induction of apoptosis. Decreased firing frequency can occur via increased function of K+ or Cl- channels. Channel mutants also cause gain-of-function syndromes at the cellular and circuit level; of these syndromes, the cardiac long-QT syndromes are explained in a more straightforward way than are the epilepsies. G protein-coupled receptors are also affected by activating mutations.

Animals↗
PubMed1992

cDNA cloning of MEV, a mutant protein that facilitates cellular uptake of mevalonate, and identification of the point mutation responsible for its gain of function.

We report the expression cloning of pMev, a cDNA that facilitates cellular uptake of mevalonate. pMev was isolated from the met-18b-2 clone of Chinese hamster ovary (CHO) cells, which were selected for growth in low concentrations of mevalonate when synthesis is blocked by compactin (Faust, J. R., and Krieger, M. (1987) J. Biol. Chem. 262, 1996-2004). pMev encodes a 494-residue protein, Mev, that is predicted to have 12 membrane-spanning regions, consistent with a membrane transporter. Surprisingly, levels of Mev mRNA and protein are similar in CHO and met-18b-2 cells. The Mev gene differs from the wild-type gene by a single base change that substitutes a cysteine for phenylalanine in the 10th membrane-spanning region. met-18b-2 cells are heterozygous for this dominant gain-of-function mutation. Transfection of a cDNA encoding pMev, but not the wild-type cDNA, elicited a marked increase in [3H]mevalonate uptake and incorporation into cellular lipids in stably and transiently transfected cells. The availability of pMev will facilitate studies of [3H]mevalonate incorporation into trace products, including p21ras and other prenylated proteins.

Amino Acid Sequence↗
PubMed2004

Distribution pattern of Notch3 mutations suggests a gain-of-function mechanism for CADASIL.

Mutations in Notch3 cause the syndrome CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy). The mechanism by which these mutations result in a CADASIL phenotype has been widely speculated upon. A first step toward understanding a disease mechanism is to learn whether the mutations result in the loss of Notch3 function, in particular, its role in signaling or in the gain of a novel function. Notch3 genomic sequences were analyzed for sites of conservation across species. We present here a bioinformatic analysis of the Notch paralogs and orthologs that suggest that CADASIL mutations result in a gain of function. This finding diminishes the likelihood that a Notch3 signaling deficit is responsible for the phenotype and increases the likelihood that CADASIL joins the growing list of neurological diseases with protein deposits due to misfolding and aggregation.

Animals↗
PubMed2006

Gain-of-function SOS1 mutations cause a distinctive form of Noonan syndrome.

Noonan syndrome is a developmental disorder characterized by short stature, facial dysmorphia, congenital heart defects and skeletal anomalies. Increased RAS-mitogen-activated protein kinase (MAPK) signaling due to PTPN11 and KRAS mutations causes 50% of cases of Noonan syndrome. Here, we report that 22 of 129 individuals with Noonan syndrome without PTPN11 or KRAS mutation have missense mutations in SOS1, which encodes a RAS-specific guanine nucleotide exchange factor. SOS1 mutations cluster at codons encoding residues implicated in the maintenance of SOS1 in its autoinhibited form. In addition, ectopic expression of two Noonan syndrome-associated mutants induces enhanced RAS and ERK activation. The phenotype associated with SOS1 defects lies within the Noonan syndrome spectrum but is distinctive, with a high prevalence of ectodermal abnormalities but generally normal development and linear growth. Our findings implicate gain-of-function mutations in a RAS guanine nucleotide exchange factor in disease for the first time and define a new mechanism by which upregulation of the RAS pathway can profoundly change human development.

Animals↗