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Results for “Loss of Function Mutation”

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PubMed2004

Nitric oxide signaling in colon cancer chemoprevention.

Nitric oxide (NO) is a pleiotrophic regulator, pivotal to numerous biological processes, including vasodilation, neurotransmission, and macrophage-mediated immunity. The highly reactive free radicals, produced by NO synthases (NOS) have been implicated in the modulation of carcinogenesis. Over-expression of inducible NOS (iNOS), a common phenomenon during chronic inflammatory conditions, generates sustainable amounts of NO, that its reactive intermediates are mutagenic, causing DNA damage or impairment of DNA repair, has been well established in carcinogenesis. Recent studies also implicate NO as having a key signaling molecule that regulates processes of tumorigenesis. Increased expression of iNOS has been observed in tumors of the colon, lung, oropharynx, reproductive organs, breast, and central nervous system besides its occurrence in chronic inflammatory diseases. Progression of a large majority of human and experimental colon tumors appears to progress by NO resulting from stimulation of proinflammatory cytokines, and inactivation (nitrosylation) of p53 mediated caspase activities in the tumors, whereas in some cases it associated with induction of apoptosis and tumor regression. This dichotomy is largely explained by the complexity of signaling pathways in tumor cells, that respond to NO very differently depending on its concentration. p53 mutation, functional loss, activation, and inactivation of apoptotic proteins all have been linked with NO resistance and dependence. Evidence from both in vitro and in vivo experiments support that NO and its reactive metabolite peroxynitrite stimulate COX-2 activity leading generation of tumor growth enhancing prostaglandins. Thus, NO mediated signaling can augment the tumor growth and metastasis by promoting invasive and angiogenic properties of tumor cells, which includes triggering and activation of COX-2. Thus, developing selective inhibitors of iNOS and NO-releasing agents may lead to important strategies for chemoprevention of colon cancer. Chemoprevention studies at preclinical level with several selective inhibitors of iNOS in both chemically and transgenic models of colon cancer are encouraging.

Animals↗

PubMed1995

Introduction of a loss-of-function point mutation from the SH3 region of the Caenorhabditis elegans sem-5 gene activates the transforming ability of c-abl in vivo and abolishes binding of proline-rich ligands in vitro.

We have introduced two loss-of-function point mutations from highly conserved regions of the src homology 3 (SH3) domains of the Caenorhabditis elegans sem-5 gene into the SH3 domain of the murine type IV c-abl tyrosine kinase proto-oncogene. One of the mutations, P131L, activated abl to transform fibroblasts while the other, G128R, did not. When combined with independent activating mutations in the c-abl kinase domain or NH2-terminus, the G128R mutation blocked transformation by the double mutant, suggesting that the G128R mutant was unable to transform cells for trivial reasons. The c-Abl G128R mutant, like wild type c-Abl protein, was localized to the nucleus and actin cytoskeleton and had normal tyrosine kinase activity in vitro, while the transforming c-Abl P131L protein was localized exclusively to the cytoplasm and exhibited decreased in vitro kinase activity. By real-time biospecific interaction analysis, the wild type Abl SH3 domain bound to two proteins containing proline-rich motifs with dissociation constants of 0.2 and 17 microM; the G128R mutant bound with 50-fold lower affinity, and no binding was detected by the P131L mutant. Both mutations completely abolished binding of the Abl SH3 domain to proline-rich target proteins in a filter-binding assay. These results suggest that the transforming activity of Abl is regulated in vivo by an inhibitor protein which associates with the SH3 domain via a proline-rich sequence.

3T3 Cells↗

PubMed2005

Loss of lysosomal association of cystatin B proteins representing progressive myoclonus epilepsy, EPM1, mutations.

Loss-of-function mutations in the cystatin B (CSTB), a cysteine protease inhibitor, gene underlie progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1), characterized by myoclonic and tonic-clonic seizures, ataxia and a progressive course. A minisatellite repeat expansion in the promoter region of the CSTB gene is the most common mutation in EPM1 patients and leads to reduced mRNA levels. Seven other mutations altering the structure of CSTB, or predicting altered splicing, have been described. Using a novel monoclonal CSTB antibody and organelle-specific markers in human primary myoblasts, we show here that endogenous CSTB localizes not only to the nucleus and cytoplasm but also associates with lysosomes. Upon differentiation to myotubes, CSTB becomes excluded from the nucleus and lysosomes, suggesting that the subcellular distribution of CSTB is dependent on the differentiation status of the cell. Four patient mutations altering the CSTB polypeptide were transiently expressed in BHK-21 cells. The p.Lys73fsX2-truncated mutant protein shows diffuse cytoplasmic and nuclear distribution, whereas p.Arg68X is rapidly degraded. Two missense mutations, the previously described p.Gly4Arg affecting the highly conserved glycine, critical for cathepsin binding, and a novel mutation, p.Gln71Pro, fail to associate with lysosomes. These data imply an important lysosome-associated physiological function for CSTB and suggest that loss of this association contributes to the molecular pathogenesis of EPM1.

Animals↗

PubMed1996

The Drosophila secreted protein Argos regulates signal transduction in the Ras/MAPK pathway.

The Drosophila argos gene encodes a secreted protein with an EGF motif which acts as an inhibitor of cellular differentiation in multiple developmental processes. To investigate the cellular pathways regulated by Argos, we screened for mutations which could modify the phenotype caused by overexpression of argos. We show that the effects of argos overexpression on the eye and wing vein development are suppressed by gain-of-function mutations of the MAPKK/D-MEK gene (Dsor1/D-mek) and the MAPK/ERK-A gene (rolled) and were enhanced by loss-of-function mutations of Star. Loss-of-function mutations in components of the Ras/MAPK signaling cascade act as dominant suppressors of the phenotype caused by the argos null mutations. A loss-of-function argos mutation enhanced the overproduction of R7 neurons caused by gain-of-function alleles of Son of sevenless and Dsor1. Conversely, overexpression of argos inhibited formation of the extra R7 cells that was caused by high-level MAPK/ERK-A activity. A phenotype of the sev; argos double mutants revealed that sev is epistatic to argos. These results provide evidence that Argos negatively regulates signal transduction events in the Ras/MAPK cascade.

Animals↗

PubMed1998

Pseudohypoparathyroidism type Ia from maternal but not paternal transmission of a Gsalpha gene mutation.

While loss-of-function mutations in Gsalpha are invariably associated with the short stature and brachydactyly of Albright hereditary osteodystrophy (AHO), the association with hormone resistance (to parathyroid hormone and thyrotropin) typical of pseudohypoparathyroidism type Ia (PHP-Ia) is much more variable. Observational studies and DNA polymorphism analysis suggest that maternal transmission of the Gsalpha mutation may be required for full expression of clinical hormone resistance. To test this hypothesis, we studied transmission of a frameshift mutation in Gsalpha through three generations of a pedigree affected by AHO and PHP-Ia. While all family members carrying this loss-of-function mutation in one Gsalpha allele had AHO, neither the presence of the mutation nor the degree of reduction of erythrocyte Gsalpha bioactivity allowed prediction of phenotype (AHO alone versus AHO and PHP-Ia). Paternal transmission of the mutation (from the patriarch of the first generation to three members of the second generation) was not associated with concurrent PHP-Ia, but maternal transmission (from two women in the second generation to four children in the third generation) was invariably associated with PHP-Ia. No expansion of an upstream short CCG nucleotide repeat region was detected, nor was there evidence of uniparental disomy by polymorphism analysis. This report, the first to document the effects across three generations of both paternal and maternal transmission of a specific Gsalpha mutation, strongly supports the hypothesis that a maternal factor determines full expression of Gsalpha dysfunction as PHP-Ia.

DNA Probes↗

PubMedDate not supplied

Molecular pathophysiology and clinical manifestations of gonadotropin receptor defects.

The gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) bind specific receptors, members of the G protein-coupled receptor superfamily. Mutations of gonadotropin receptors are classified into activating (constitutively active or gain-of-function mutations) and inactivating (loss-of-function mutations). Activating mutations of the LH receptor have been described in familial and sporadic forms of male-limited pseudoprecocious puberty, whereas they do not appear to have any particular phenotype in females. The only activating mutation of the FSH receptor described to date was found in a hypophysectomized man who was fertile despite undetectable serum gonadotropin levels; the effects of constitutive FSH receptor activity in the context of normal pituitary function are not known. Homozygous inactivating mutations of the LH and FSH receptor invariably lead to amenorrhea in genotypical female subjects. In males, inactivation of the LH receptor in its more severe form results in a clinical picture similar to the syndrome of complete androgen resistance, but milder forms of hypoandrogenization have been described as well. In males, homozygous inactivation of the FSH receptor can also be associated with infertility. Finally, polymorphic variants of the FSH receptor are present in the normal population.

Animals↗

PubMed1998

Molecular biology of the MEN2 gene.

Cancer is a genetic disease caused by 'gain of function' mutations of oncogenes and 'loss of function' mutations of tumour suppressors and of genes involved in DNA repair mechanisms. The RET gene encodes a tyrosine kinase receptor for molecules belonging to the glial cell line-derived neurotrophic factor (GDNF) family. RET is a paradigmatic example of how different mutations of a single gene can lead to different neoplastic phenotypes. Indeed, gene rearrangements, often caused by chromosomal inversions, activate the oncogenic potential of RET in a fraction of human thyroid papillary carcinomas. On the other hand, different point mutations activate RET in familial multiple endocrine neoplasia syndromes familial medullary thyroid carcinoma (FMTC), MEN-2A and MEN-2B. Little information is so far available on the biochemical mechanisms by which the potent transforming and mitogenic signals of RET are delivered to the nucleus. However, recent data indicate coupling to the Shc-Ras-MAPK pathway as a necessary step in RET signal transduction.

Carcinoma, Papillary↗

PubMed2001

Review: molecular thyroidology.

Novel disorders involving aberrations of the hypothalamic-pituitary-thyroid gland-thyroid hormone axis have been described in the last 5 to 10 years. The following topics are addressed: molecular mutations causing central hypothyroidism (isolated autosomal recessive TRH deficiency; autosomal recessive TRH-receptor inactivating mutations; TSH beta-subunit bio-inactivating mutations; Pit-1 mutations; Prop1 mutations; high molecular weight bio-inactive TSH); defects in response to TSH (mutations in the TSH receptor: TSH receptor gain-of-function mutations; TSH receptor loss-of-function mutations); defects in thyroid gland formation: transcription factor mutations (TTF-2 and Pax8); defects in peripheral thyroid hormone metabolism (defective intrapituitary conversion of T4 to T3; hemangioma consumption of thyroid hormone); and defects in tissue response to thyroid hormone (generalized thyroid hormone resistance, selective pituitary thyroid hormone resistance). While molecular diagnosis of such conditions is rarely indicated for clinical management, knowledge of the molecular mechanisms of these diseases can greatly enhance the clinical laboratory scientist's ability to advise clinicians about appropriate thyroid testing and to interpret the complex and sometimes confusing results of thyroid function tests.

Humans↗

PubMed1998

Molecular definition of an allelic series of mutations disrupting the myostatin function and causing double-muscling in cattle.

We have determined the entire myostatin coding sequence for 32 double-muscled cattle sampled from ten European cattle breeds. Seven DNA sequence polymorphisms were identified, of which five would be predicted to disrupt the function of the protein, one is a conservative amino acid substitution, and one a silent DNA sequence variant. Four additional DNA sequence polymorphisms were identified in myostatin intronic sequences. In all but two breeds, all double-muscled animals were either homozygous or compound heterozygotes for one of the five loss-of-function mutations. The absence of obvious loss-of-function mutations in the coding sequence of the two remaining breeds points either towards additional mutations in unexplored segments of the gene, or towards locus heterogeneity of double-muscling.

Alleles↗

PubMed2000

Effects of loss-of-function and gain-of-function mutations of c-kit on the gastrointestinal tract.

Protooncogene c-kit encodes a receptor tyrosine kinase, KIT. Interstitial cells of Cajal (ICCs) that are important for the autonomous movement of the gastrointestinal tract essentially require the normal function of the KIT for their development. Therefore, germline loss-of-function mutations of the c-kit gene cause deficiency of ICCs that results in disturbed gastrointestinal movement. On the other hand, somatic gain-of-function mutations of the c-kit gene induce gastrointestinal stromal tumors (GIST) that are considered to originate from ICCs. Moreover, germline gain-of-function mutations of the c-kit gene are a cause of familial development of multiple GISTs.

Animals↗

PubMed1999

minifly, a Drosophila gene required for ribosome biogenesis.

We report here the genetic, molecular, and functional characterization of the Drosophila melanogaster minifly (mfl) gene. Genetic analysis shows that mfl is essential for Drosophila viability and fertility. While P-element induced total loss-of-function mutations cause lethality, mfl partial loss-of-function mutations cause pleiotropic defects, such as extreme reduction of body size, developmental delay, hatched abdominal cuticle, and reduced female fertility. Morphological abnormalities characteristic of apoptosis are found in the ovaries, and a proportion of eggs laid by mfl mutant females degenerates during embryogenesis. We show that mfl encodes an ubiquitous nucleolar protein that plays a central role in ribosomal RNA processing and pseudouridylation, whose known eukaryotic homologues are yeast Cfb5p, rat NAP57 and human dyskerin, encoded by the gene responsible for the X-linked dyskeratosis congenita disease. mfl genetic analysis represents the first in vivo functional characterization of a member of this highly conserved gene family from higher eukaryotes. In addition, we report that mfl hosts an intron encoded box H/ACA snoRNA gene, the first member of this class of snoRNAs identified so far from Drosophila.

Amino Acid Sequence↗

PubMed2002

A loss-of-function polymorphic mutation in the cytolytic P2X7 receptor gene and chronic lymphocytic leukaemia: a molecular study.

BACKGROUND: Chronic lymphocytic leukaemia (CLL) has a familial incidence nearly three times higher than expected for the general population and one predisposing factor might be an inherited failure of mechanisms involved in apoptosis of lymphocytes. Our aim was to ascertain whether or not a defect in a proapoptotic pathway, caused by a single nucleotide polymorphism that results in loss-of-function of P2X7 in healthy individuals, was present in leukaemic B lymphocytes of patients with CLL. METHODS: We extracted genomic DNA from the peripheral blood leucocytes of 36 unrelated individuals with CLL, four individuals with familial CLL, and 46 age-matched controls. We sequenced a PCR product to detect mutations in exon 13 of P2X7. In most patients with CLL, we measured expression and function of the P2X7 receptor by flow cytometry in B lymphocytes and T lymphocytes. FINDINGS: The prevalence of the polymorphic mutation and the frequency of the mutant allele were three-fold greater in individuals with CLL than in white, elderly controls. Individuals homozygous for the polymorphic allele had no P2X7 receptor function and heterozygotes had half the mean function of that seen in individuals homozygous for the wildtype allele; amounts of ATP-induced apoptosis varied accordingly. In two families, in which we studied a father-son pair and a sister-sister pair with CLL, loss of P2X7 function arose because of inheritance of one or two 1513A-->C alleles for P2X7. INTERPRETATION: Activation of the P2X7 receptor leads to apoptosis of lymphocytes in individuals with CLL, and reduced function of this receptor has an anti-apoptotic effect, resulting in an increase in B-cell numbers. Thus, inheritance of a loss-of-function polymorphic mutation at position 1513 in the P2X7 gene could contribute to the pathogenesis of CLL.

Aged↗

PubMed1996

Both loss-of-function and gain-of-function mutations in snf define a role for snRNP proteins in regulating Sex-lethal pre-mRNA splicing in Drosophila development.

The Drosophila snf gene encodes a protein with functional homology to the mammalian U1A and U2B" snRNP proteins. Studies, based on the analysis of three viable alleles, have suggested a role for snf in establishing the female-specific splicing pattern of the sex determination switch gene, sex-lethal. Here, we show that the non-sex-specific lethal null allele is required for female sex determination, arguing against the formal possibility that the viable alleles disrupt a function unrelated to snf's wild-type function. Moreover, we find snf is required for normal cell growth and/or survival, as expected for a protein involved in a cell-vital process such as RNA splicing. We also show that of the three viable alleles only one, snfJA2, is a partial loss-of-function mutation. The other two viable alleles, snf1621 and snfe8H, encode antimorphic proteins. We find the antimorphic proteins are mislocalized and correlate their mislocalization with their molecular lesions and mutant phenotypes. Finally, we provide genetic evidence that the antimorphic alleles interfere with the autoregulatory splicing function of the Sex-lethal protein. Based on these studies we suggest a model in which the snRNP protein, Snf, functions with Sex-lethal to block recognition of the regulated male-specific exon.

Alleles↗

PubMed2026

Loss of Function Dnmt3a Mutation Leads to Aberrant Neutrophil Migration.

Clonal hematopoiesis (CH), an age-related expansion of somatically mutated hematopoietic clones, is associated with increased risk of severe infections including coronavirus disease (COVID)-19, yet the underlying mechanisms remain unclear. Here, we investigated the impact of Dnmt3a deficiency in a murine model of influenza A virus (IAV) pneumonia. Dnmt3a-deficient mice exhibited increased pulmonary viral burden and reduced neutrophil accumulation in IAV-infected lungs despite comparable circulating neutrophil numbers. Functional analyses of neutrophils showed impaired chemotactic migration in vitro, whereas maturation, antimicrobial enzyme content, and metabolic capacity were unchanged. Transcriptomic profiling revealed downregulation of pathways involved in chemotaxis, cytokine signaling, and cellular activation, including reduced expression of Cxcr1. Supporting the translational relevance of these findings, proteomic analysis of plasma from individuals with germline DNMT3A mutations (Tatton-Brown-Rahman syndrome) revealed alterations in proteins associated with cell migration and cytoskeletal dynamics. Collectively, our findings demonstrate that Dnmt3a loss compromises innate immune defense by impairing neutrophil migration in a cell-intrinsic manner, leading to ineffective pathogen clearance. This work provides mechanistic insight into how CH-associated mutations contribute to age-associated susceptibility to infection and highlights altered leukocyte trafficking as a potential therapeutic target in aging populations with CH.

Animals↗

PubMed1998

Genetic and molecular characterization of the caenorhabditis elegans gene, mel-26, a postmeiotic negative regulator of mei-1, a meiotic-specific spindle component.

We have previously described the gene mei-1, which encodes an essential component of the Caenorhabditis elegans meiotic spindle. When ectopically expressed after the completion of meiosis, mei-1 protein disrupts the function of the mitotic cleavage spindles. In this article, we describe the cloning and the further genetic characterization of mel-26, a postmeiotic negative regulator of mei-1. mel-26 was originally identified by a gain-of-function mutation. We have reverted this mutation to a loss-of-function allele, which has recessive phenotypes identical to the dominant defects of its gain-of-function parent. Both the dominant and recessive mutations of mel-26 result in mei-1 protein ectopically localized in mitotic spindles and centrosomes, leading to small and misoriented cleavage spindles. The loss-of-function mutation was used to clone mel-26 by transformation rescue. As suggested by genetic results indicating that mel-26 is required only maternally, mel-26 mRNA was expressed predominantly in the female germline. The gene encodes a protein that includes the BTB motif, which is thought to play a role in protein-protein interactions.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Inherited diseases involving g proteins and g protein-coupled receptors.

Heterotrimeric G proteins couple seven-transmembrane receptors for diverse extracellular signals to effectors that generate intracellular signals altering cell function. Mutations in the gene encoding the alpha subunit of the G protein-coupling receptors to stimulation of adenylyl cyclase cause developmental abnormalities of bone, as well as hormone resistance (pseudohypoparathyroidism caused by loss-of-function mutations) and hormone hypersecretion (McCune-Albright syndrome caused by gain-of-function mutations). Loss- and gain-of-function mutations in genes encoding G protein-coupled receptors (GPCRs) have been identified as the cause of an increasing number of retinal, endocrine, metabolic, and developmental disorders. GPCRs comprise an evolutionarily conserved gene superfamily ( 1 ). By coupling to heterotrimeric G proteins, GPCRs transduce a wide variety of extracellular signals including monoamine, amino acid, and nucleoside neurotransmitters, as well as photons, chemical odorants, divalent cations, hormones, lipids, peptides and proteins. Following a brief overview of G protein-coupled signal transduction, we review the growing body of evidence that mutations in genes encoding GPCRs and G proteins are an important cause of human disease.

GTP-Binding Proteins↗

PubMed2001

Loss-of-function EA2 mutations are associated with impaired neuromuscular transmission.

OBJECTIVE: To examine the functional consequences of episodic ataxia type 2 (EA2)-causing nonsense and missense mutations in vitro and to characterize the basis of fluctuating weakness in patients with E2A. BACKGROUND: Mutations in CACNA1A encoding the Ca(v)2.1 calcium channel subunit cause EA2 through incompletely understood mechanisms. Although the Ca(v)2.1 subunit is important for neurotransmission at the neuromuscular junction, weakness has not been considered a feature of EA2. METHODS: The disease-causing mutations in three unrelated patients with EA2 and fluctuating weakness were identified by mutation screening and sequencing. Mutant constructs harboring mutations R1281X, F1406C, R1549X were transfected into COS7 cells and expressed for patch clamp studies. Single-fiber electromyography (SFEMG) was performed in patients to examine synaptic transmission at the neuromuscular junction. RESULTS: Functional studies in COS7 cells of nonsense and missense EA2 mutants demonstrated markedly decreased current densities compared with wild type. SFEMG demonstrated jitter and blocking in these patients with EA2, compared with normal subjects and three patients with SCA-6. CONCLUSION: EA2-causing missense and nonsense mutations in CACNA1A produced mutant channels with diminished whole cell calcium channel activity in vitro due to loss of function. Altered biophysical properties or reduced efficiency of plasma membrane targeting of mutant channels may contribute to abnormal neuromuscular transmission, manifesting as myasthenic syndrome.

Calcium Channels↗

PubMed2024

Proteome-scale prediction of molecular mechanisms underlying dominant genetic diseases.

Many dominant genetic disorders result from protein-altering mutations, acting primarily through dominant-negative (DN), gain-of-function (GOF), and loss-of-function (LOF) mechanisms. Deciphering the mechanisms by which dominant diseases exert their effects is often experimentally challenging and resource intensive, but is essential for developing appropriate therapeutic approaches. Diseases that arise via a LOF mechanism are more amenable to be treated by conventional gene therapy, whereas DN and GOF mechanisms may require gene editing or targeting by small molecules. Moreover, pathogenic missense mutations that act via DN and GOF mechanisms are more difficult to identify than those that act via LOF using nearly all currently available variant effect predictors. Here, we introduce a tripartite statistical model made up of support vector machine binary classifiers trained to predict whether human protein coding genes are likely to be associated with DN, GOF, or LOF molecular disease mechanisms. We test the utility of the predictions by examining biologically and clinically meaningful properties known to be associated with the mechanisms. Our results strongly support that the models are able to generalise on unseen data and offer insight into the functional attributes of proteins associated with different mechanisms. We hope that our predictions will serve as a springboard for researchers studying novel variants and those of uncertain clinical significance, guiding variant interpretation strategies and experimental characterisation. Predictions for the human UniProt reference proteome are available at https://osf.io/z4dcp/.

Humans↗