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Results for “Loss of Function Mutation”

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PubMed2003

Na+ channel mutation leading to loss of function and non-progressive cardiac conduction defects.

BACKGROUND: We previously described a Dutch family in which congenital cardiac conduction disorder has clinically been identified. The ECG of the index patient showed a first-degree AV block associated with extensive ventricular conduction delay. Sequencing of the SCN5A locus coding for the human cardiac Na+ channel revealed a single nucleotide deletion at position 5280, resulting in a frame-shift in the sequence coding for the pore region of domain IV and a premature stop codon at the C-terminus. METHODS AND RESULTS: Wild type and mutant Na+ channel proteins were expressed in Xenopus laevis oocytes and in mammalian cells. Voltage clamp experiments demonstrated the presence of fast activating and inactivating inward currents in cells expressing the wild type channel alone or in combination with the beta1 subinut (SCN1B). In contrast, cells expressing the mutant channels did not show any activation of inward current with or without the beta1 subunit. Culturing transfected cells at 25 degrees C did not restore the Na+ channel activity of the mutant protein. Transient expression of WT and mutant Na+ channels in the form of GFP fusion proteins in COS-7 cells indicated protein expression in the cytosol. But in contrast to WT channels were not associated with the plasma membrane. CONCLUSIONS: The SCN5A/5280delG mutation results in the translation into non-function channel proteins that do not reach the plasma membrane. This could explain the cardiac conduction defects in patients carrying the mutation.

Animals↗
PubMed1997

Identification and characterization of genes that interact with lin-12 in Caenorhabditis elegans.

We identified and characterized 14 extragenic mutations that suppressed the dominant egg-laying defect of certain lin-12 gain-of-function mutations. These suppressors defined seven genes: sup-17, lag-2, sel-4, sel-5, sel-6, sel-7 and sel-8. Mutations in six of the genes are recessive suppressors, whereas the two mutations that define the seventh gene, lag-2, are semi-dominant suppressors. These suppressor mutations were able to suppress other lin-12 gain-of-function mutations. The suppressor mutations arose at a very low frequency per gene, 10-50 times below the typical loss-of-function mutation frequency. The suppressor mutations in sup-17 and lag-2 were shown to be rare non-null alleles, and we present evidence that null mutations in these two genes cause lethality. Temperature-shift studies for two suppressor genes, sup-17 and lag-2, suggest that both genes act at approximately the same time as lin-12 in specifying a cell fate. Suppressor alleles of six of these genes enhanced a temperature-sensitive loss-of-function allele of glp-1, a gene related to lin-12 in structure and function. Our analysis of these suppressors suggests that the majority of these genes are part of a shared lin-12/glp-1 signal transduction pathway, or act to regulate the expression or stability of lin-12 and glp-1.

Animals↗
PubMed2005

Genetic and genomic studies of Drosophila parkin mutants implicate oxidative stress and innate immune responses in pathogenesis.

Loss-of-function mutations of the parkin gene, which encodes a ubiquitin-protein ligase, are a common cause of autosomal recessive juvenile parkinsonism (ARJP). Previous work has led to the identification of a number of Parkin substrates that implicate specific pathways in ARJP pathogenesis, including endoplasmic reticulum (ER) stress and cell cycle activation. To test the involvement of previously implicated pathways, as well as to identify novel pathways in ARJP pathogenesis, we are using genetic and genomic approaches to study Parkin function in the fruit fly Drosophila melanogaster. In previous work, we demonstrated that Drosophila parkin null mutants exhibit mitochondrial pathology and flight muscle degeneration. To further explore the mechanisms responsible for pathology in parkin mutants, we analyzed the transcriptional alterations that occur during muscle degeneration and performed a genetic screen for parkin modifiers. Results of these studies indicate that oxidative stress response components are induced in parkin mutants and that loss-of-function mutations in oxidative stress components enhance the parkin mutant phenotypes. Genes involved in the innate immune response are also induced in parkin mutants. In contrast, our studies did not reveal evidence for cell cycle or ER stress pathway induction in parkin mutants. These results suggest that oxidative stress and/or inflammation may play a fundamental role in the etiology of ARJP.

Animals↗
PubMed1996

Fusion of the EWS1 and WT1 genes as a result of the t(11;22)(p13;q12) translocation in desmoplastic small round cell tumors.

The isolation and molecular analysis of genes which cause and/or predispose to Wilms' tumor have yielded fascinating insights into the role of tissue-specific gene regulation in both development and disease processes. Analysis of the WT1 transcription factor has clearly established its role in Wilms' tumorigenesis and a broader role in both urogenital organogenesis and mesenchymal cell differentiation events. Clearly, loss of function mutations in WT1 is correlated with aberrant function as a regulator of gene expression, ultimately resulting in neoplastic transformation in the developing kidney. A question we have pursued is whether alterations of WT1 structure and/or function can be associated with other types of malignancies, possibly reflecting WT1's broader role in mesenchymal differentiation. To this end, we have analyzed a rare solid tumor designated Intra-Abdominal Desmoplastic Small Round Cell Sarcoma (IADSRCT) which often displays a recurrent chromosomal translocation t(11;22)(p13;q12) involving the WT1 genomic locus. We have shown that the EWS1 gene fron chromosome 22q12 is fused to the WT1 gene in IADSRCT and that a fusion protein is produced which functions as a potent activator of transcription. Our results suggest that WT1 has sustained a gain-of function alteration as a results of this fusion and that the fusion gene functions as a dominant oncogene in this disease. Thus, the WT1 locus may be the target for both gain- and loss-of-function mutations resulting in different disease outcomes. A summary of our ongoing analysis of the EWS-WT1 fusion gene is presented.

Adult↗
PubMed1994

The function of argos in regulating cell fate decisions during Drosophila eye and wing vein development.

The Drosophila argos gene, which encodes a secreted protein with an EGF motif, is involved in several developmental processes regulating cell-cell interactions such as eye morphogenesis. Loss-of-function mutations in the argos gene cause an increase in the number of photoreceptor cells and cone cells, impaired retinal projections to the optic lobe, and the formation of extra veins. We show here that ubiquitously expressed argos product restored all these loss-of-function phenotypes. Overexpression of argos in the wild-type background resulted in the reduced number of photoreceptor cells, cone cells, and pigment cells, which are phenotypes opposite to those of the loss-of-function mutants. The argos gene is expressed in developing wing veins. Ubiquitous argos expression caused loss of veins in a dose-dependent manner. This phenotype was enhanced by the loss-of-function rhomboid mutation, implying the possibility that argos and rhomboid play key roles in a common pathway for normal wing vein formation. We propose that argos acts as an inhibitory signal for cellular differentiation in the developing eye and wing.

Animals↗
PubMed2000

The Cdc42p GTPase and its regulators Nrf1p and Scd1p are involved in endocytic trafficking in the fission yeast Schizosaccharomyces pombe.

Nrf1p was first identified in a screen for negative regulators of the Cdc42p GTPase. Overexpression of Nrf1p resulted in dose-dependent lethality, with cells exhibiting an ellipsoidal morphology and abnormal vacuolar phenotypes including an increase in vacuolar fusion. Green fluorescent protein (GFP)-Cdc42p and GFP-Nrf1p colocalized to vacuolar membranes and GFP-Nrf1p vacuolar localization depended on Scd1p, the Schizosaccharomyces pombe homolog of the Cdc24p guanine nucleotide exchange factor. In this study, site-directed mutagenesis was conducted on Nrf1p to determine its functional domains. Mutations in the three putative transmembrane domains resulted in mislocalization of GFP-Nrf1p and an inability to induce lethality, suggesting a loss of function. Mutations in the second extramembranous loop of Nrf1p also resulted in a loss of function and altered the ability of GFP-Nrf1p to localize to vacuolar membranes. Analysis of Deltanrf1 and Deltascd1 mutants revealed defects in endocytosis. In addition, overexpression of constitutively active Cdc42(G12V)p resulted in an increase in endocytosis and an ability to rescue the endocytic defects in Deltanrf1 and Deltascd1 cells. These data are consistent with Nrf1p and Scd1p being necessary for efficient endocytosis, possibly through the regulation of Cdc42p.

Amino Acid Sequence↗
PubMed2003

An extended bipartite nuclear localization signal in Smad4 is required for its nuclear import and transcriptional activity.

Smad proteins are a class of tumor suppressors that play critical roles in inhibiting the proliferation of a variety of cell types by modulating the transcriptions of target genes. Despite recent advances, the mechanism of their nuclear import is not completely understood. Smad proteins contain a conserved basic motif in their N-terminal MH1 domains that resembles a nuclear localization signal (NLS). Previous studies indicate that in receptor-regulated Smads such as Smad1 and Smad3 this motif determines their interactions with nuclear import receptors and mediates their ligand-induced nuclear translocation. Common-Smads such as Smad4 display constant nucleocytoplasmic shuttling and are capable of autonomous nuclear import and export. Mutations of the basic motif in Smad4 disrupted its nuclear accumulation. However, this motif is not sufficient to confer nuclear translocation to a fused heterologous protein, suggesting that it is only part of the bona fide Smad4 NLS. We mapped the Smad4 NLS by fusing various segments of Smad4 sequence covering the basic motif to GFP and tested the localization of the fusion proteins. We identified an extended NLS, starting from the basic motif and extending into the DNA-binding region (AA 45-110), that is sufficient to confer nuclear localization to GFP. Among the 14 basic residues in the NLS, only four (K45, K46, K48 and R81) are critical for import. This NLS is critical not only for autonomous nuclear import of Smad4, but also for its nuclear translocation in the presence of activated R-Smads, further confirming the functional relevance of the Smad4 NLS in TGF-beta signal transduction. Structural modeling demonstrated that the four critical basic residues are all solvent exposed and map to a single localized segment on one surface of the Smad4 MH1 domain. Their distribution and spacing resemble a classical bipartite NLS. Smad4 displays specific binding to importin alpha through its MH1 domain, which was abrogated by loss-of-function mutations in Smad4 NLS. Finally, the Smad4 NLS is essential for its transcriptional activity since loss-of-function NLS mutants are also transcriptionally inactive.

Active Transport, Cell Nucleus↗
PubMed2005

Voltage-dependent electrogenic chloride/proton exchange by endosomal CLC proteins.

Eukaryotic members of the CLC gene family function as plasma membrane chloride channels, or may provide neutralizing anion currents for V-type H(+)-ATPases that acidify compartments of the endosomal/lysosomal pathway. Loss-of-function mutations in the endosomal protein ClC-5 impair renal endocytosis and lead to kidney stones, whereas loss of function of the endosomal/lysosomal protein ClC-7 entails osteopetrosis and lysosomal storage disease. Vesicular CLCs have been thought to be Cl- channels, in particular because ClC-4 and ClC-5 mediate plasma membrane Cl- currents upon heterologous expression. Here we show that these two mainly endosomal CLC proteins instead function as electrogenic Cl-/H+ exchangers (also called antiporters), resembling the transport activity of the bacterial protein ClC-e1, the crystal structure of which has already been determined. Neutralization of a critical glutamate residue not only abolished the steep voltage-dependence of transport, but also eliminated the coupling of anion flux to proton counter-transport. ClC-4 and ClC-5 may still compensate the charge accumulation by endosomal proton pumps, but are expected to couple directly vesicular pH gradients to Cl- gradients.

Animals↗
PubMed2005

Early-onset Parkinson's disease caused by a novel parkin mutation in a genetic isolate from north-eastern Brazil.

We describe clinical and molecular findings in a genetic isolate from north-eastern Brazil with early-onset Parkinson's disease (PD) and a novel mutation in the parkin gene. Genealogical studies could connect 255 individuals, of whom 15 had PD. Geographic isolation and multiple consanguineous marriages initially suggested an autosomal recessive inheritance for PD in these patients. The available individuals were personally examined, and DNA was obtained from 26 members: ten early-onset PD patients, one case with likely neuroleptic-induced parkinsonism and 15 unaffected relatives. The average age at onset of PD symptoms was 30.8 years (range 12-46). Haplotype analysis revealed homozygosity in the PD patients for markers across the PARK2 locus. Genomic sequencing identified a novel homozygous splice-site parkin mutation (IVS1 + 1G/T), which completely co-segregated with the early-onset PD phenotype. cDNA analysis confirmed the total loss of parkin transcript in homozygous mutation carriers, delineating this as a loss-of-function mutation. The case with neuroleptic-induced parkinsonism and 13 of 15 healthy relatives were heterozygous carriers of the mutation. The absence of PD in heterozygous carriers indicates a genuinely recessive nature of this mutation, suggesting that parkin haploinsufficiency is not a relevant risk factor for early- or late-onset PD. However, parkin haploinsufficiency could facilitate the emergence of neuroleptic-induced parkinsonism. The cluster reported here, which to our knowledge is the largest described to date with early-onset PD and parkin mutations, also offers a unique opportunity for the search of modifiers of the parkin-related disease.

Adolescent↗
PubMed2004

Genetic changes associated with floral adaptation restrict future evolutionary potential.

A commonly accepted evolutionary principle is that adaptive change constrains the potential directions of future evolutionary change. One manifestation of this is Dollo's law, which states that character elimination is irreversible. Although the common occurrence of irreversibility has been documented by phylogenetic analyses of phenotypic transitions, little is known about the underlying causes of this phenomenon. One explanation for evolutionary irreversibility relies on the fact that many characteristics result from interactions between multiple gene products. Such characteristics may often be eliminated by inactivation of just one gene in the network. If they serve no other functions, other genes of the network are then free to accumulate mutations or evolve new functions. Evolutionary change after character loss results in the accumulation of redundant loss-of-function mutations. Such pathway degeneration makes it very unlikely that the characteristic will re-evolve, because multiple simultaneous mutations would be required. Here we describe what appear to be the initial stages of such degeneration in the anthyocyanin pigment pathway associated with an adaptive change from blue to red flowers in the morning glory Ipomoea quamoclit.

Adaptation, Physiological↗
PubMed2000

Activation of beta-catenin in epithelial and mesenchymal hepatoblastomas.

Wnt/beta-catenin signaling is frequently activated in cancer cells by stabilizing mutations of beta-catenin or loss-of-function mutations of the APC tumor suppressor gene. We have analysed the role of beta-catenin in the pathogenesis of hepatoblastoma (HB), an embryonic liver tumor occurring mainly in children under 2 years of age. Sequence analysis of the beta-catenin NH2-terminal domain in 18 epithelial and mixed HBs revealed missense mutations in the GSK3beta phosphorylation motif or interstitial deletions in 12 tumors (67%). In the remaining cases, no truncating mutation of APC could be evidenced. Immunohistochemical analysis of beta-catenin in 11 HBs demonstrated nuclear/cytoplasmic accumulation of the protein in all tumors analysed, with predominant nuclear beta-catenin immunostaining in undifferentiated cells. Membranous beta-catenin localization was preserved only in fetal-type tumoral hepatocytes and was associated with E-cadherin expression. Moreover, we show that beta-catenin is aberrantly overexpressed in a large spectrum of tumor components, including hepatocyte-like cells at various differentiation stages and heterologous elements such as squamous, osteoid and chrondroid tissues, and in occasional other mesenchymally-derived cells. These data strongly suggest that activation of beta-catenin signaling is an obligatory step in HB pathogenesis, and raise the possibility that it interferes with developmental signals that specify different tissue types at early stages of hepatic differentiation.

Biological Transport↗
PubMed2000

Seizures induce widespread upregulation of cystatin B, the gene mutated in progressive myoclonus epilepsy, in rat forebrain neurons.

Loss of function mutations in the gene encoding the cysteine protease inhibitor, cystatin B (CSTB), are responsible for the primary defect in human progressive myoclonus epilepsy (EPM1). CSTB inhibits the cathepsins B, H, L and S by tight reversible binding, but little is known regarding its localization and physiological function in the brain and the relation between the depletion of the CSTB protein and the clinical symptoms in EPM1. We have analysed the expression of mRNA and protein for CSTB in the adult rat brain using in situ hybridization and immunocytochemistry. In the control brains, the CSTB gene was differentially expressed with the highest levels in the hippocampal formation and reticular thalamic nucleus, and moderate levels in amygdala, thalamus, hypothalamus and cortical areas. Detectable levels of CSTB were found in virtually all forebrain neurons but not in glial cells. Following 40 rapidly recurring seizures evoked by hippocampal kindling stimulations, CSTB mRNA expression showed marked bilateral increases in the dentate granule cell layer, CA1 and CA4 pyramidal layers, amygdala, and piriform and parietal cortices. Maximum levels were detected at 6 or 24 h, and expression had reached control values at 1 week post-seizures. The changes of mRNA expression were accompanied by transient elevations (at 6-24 h) of CSTB protein in the same brain areas. These findings demonstrate that seizure activity leads to rapid and widespread increases of the synthesis of CSTB in forebrain neurons. We propose that the upregulation of CSTB following seizures may counteract apoptosis by binding cysteine proteases.

Animals↗
PubMed2004

Mild overexpression of MeCP2 causes a progressive neurological disorder in mice.

Mutations in the X-linked methyl-CpG-binding protein 2 (MECP2), encoding a transcriptional repressor, cause Rett syndrome and a variety of related neurodevelopmental disorders. The vast majority of mutations associated with human disease are loss-of-function mutations, but precisely what aspect of MeCP2 function is responsible for these phenotypes remains unknown. We overexpressed wild-type human protein in transgenic mice using a large genomic clone containing the entire human MECP2 locus. Detailed neurobehavioral and electrophysiological studies in transgenic line MeCP2(Tg1), which expresses MeCP2 at approximately 2-fold wild-type levels, demonstrated onset of phenotypes around 10 weeks of age. Surprisingly, these mice displayed enhanced motor and contextual learning and enhanced synaptic plasticity in the hippocampus. After 20 weeks of age, however, these mice developed seizures, became hypoactive and approximately 30% of them died by 1 year of age. These data demonstrate that MeCP2 levels must be tightly regulated in vivo, and that even mild overexpression of this protein is detrimental. Furthermore, these results support the possibility that duplications or gain-of-function mutations in MECP2 might underlie some cases of X-linked delayed-onset neurobehavioral disorders.

Animals↗
PubMed2005

Ankyrin-based cardiac arrhythmias: a new class of channelopathies due to loss of cellular targeting.

PURPOSE OF REVIEW: This review addresses a new mechanism for arrhythmia due to abnormal cellular localization of membrane ion channels and transporters. The focus is on ankyrins, a family of proteins that localize diverse membrane ion channels and transporters, and recent evidence that mutations affecting functions of ankyrins result in cardiac arrhythmia. RECENT FINDINGS: A loss-of-function mutation of ankyrin-B in humans and a null mutation in mice result in a dominantly-inherited fatal cardiac arrhythmia initially classified as type 4 long QT syndrome. Characterization of additional probands suggests ankyrin-B mutations cause a new cardiac arrhythmia syndrome associated with sinus node dysfunction that is distinct from long QT syndrome. Ankyrin-B mutation results in elevated calcium transients in cardiomyocytes accompanied by loss of cellular targeting of Na/K ATPase, Na/Ca exchanger, and InsP3 receptor (all ankyrin-binding proteins) to cardiomyocyte membrane domains. The principal voltage-gated Na channel in heart, Nav1.5, is directly associated with ankyrin-G, which is encoded by a distinct gene from ankyrin-B. Mutation of Nav1.5 causing loss of binding to ankyrin-G results in Brugada syndrome and loss of targeting of Nav1.5 to the cell surface of cardiomyocytes. SUMMARY: Ankyrin-B and ankyrin-G are recently recognized constituents of the heart that target diverse ion channels/pumps/transporters to physiologic sites of action in cardiomyocytes. Mutations of ankyrin-B cause a newly defined cardiac arrhythmia syndrome associated with abnormal calcium homeostasis in a mouse model. Ankyrin-G associates with the principal voltage-gated Na channel in the heart, and loss of this interaction due to mutation of Nav1.5 results in Brugada syndrome.

Animals↗
PubMed2006

The novel brain-specific tryptophan hydroxylase-2 gene in panic disorder.

Panic disorder is a common psychiatric disorder characterized by recurrent anxiety attacks and anticipatory anxiety. Due to the severity of the symptoms of the panic attacks and the frequent additional occurrence of agoraphobia, panic disorder is an often debilitating disease. Elevation of central serotonin levels by drugs such as clomipramine represents one of the most effective treatment options for panic disorder. This points to an important role of dysregulation of the serotonergic system in the genetic etiology of panic disorder. The novel brain-specific 5-HT synthesizing enzyme, tryptophan hydroxylase-2 (TPH2), which represents the rate-limiting enzyme of 5-HT production in the brain, may therefore be of particular importance in panic disorder. We focused on the putative transcriptional control region of TPH2 and identified two novel common single nucleotide polymorphisms (SNPs) of TPH2 in and close to this region. Moreover, a recently described loss-of-function mutation of TPH2 which results in an 80% reduction of serotonin production, was assessed. In an analysis of the putative transcriptional control region SNPs in a sample of panic disorder patients and controls no association of the disorder with the TPH2 SNPs or haplotypes was found. Moreover, the loss-of-function R441H mutation of TPH2 was not present in the panic disorder patients. The results of this first study of TPH2 in panic disorder argue against an importance of allelic variation of TPH2 in the pathogenesis of panic disorder with or without agoraphobia.

Adult↗
PubMed1999

The Aspergillus nidulans sfaD gene encodes a G protein beta subunit that is required for normal growth and repression of sporulation.

flbA encodes an Aspergillus nidulans RGS (regulator of G protein signaling) domain protein that antagonizes FadA (G(i)alpha-subunit of heterotrimeric G protein)-mediated growth signaling to allow asexual development. We previously defined and characterized five suppressors of flbA (sfa) loss-of-function mutations and showed that one suppressor (sfaB) resulted from a novel dominant-negative allele of fadA. In this report we show that a second suppressor gene (sfaD) is predicted to encode the beta subunit of a heterotrimeric G protein. Deletion of sfaD suppressed all defects resulting from complete loss-of-flbA function mutations, caused a hyperactive sporulation phenotype and severely reduced vegetative growth. However, the sfaD deletion could not suppress the growth activation caused by dominant-activating fadA alleles, indicating that constitutively active FadA can cause proliferative growth in the absence of Gbetagamma signaling. We propose that SfaD and FadA are both positive growth regulators with partially overlapping functions and that FlbA has an important role in controlling the activities of both proteins. Inactivation of signaling events stimulated by both components of the heterotrimeric G protein is essential for both sexual and asexual sporulation.

Amino Acid Sequence↗
PubMed1993

Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene.

Estrogen receptor and its ligand, estradiol, have long been thought to be essential for survival, fertility, and female sexual differentiation and development. Consistent with this proposed crucial role, no human estrogen receptor gene mutations are known, unlike the androgen receptor, where many loss of function mutations have been found. We have generated mutant mice lacking responsiveness to estradiol by disrupting the estrogen receptor gene by gene targeting. Both male and female animals survive to adulthood with normal gross external phenotypes. Females are infertile; males have a decreased fertility. Females have hypoplastic uteri and hyperemic ovaries with no detectable corpora lutea. In adult wild-type and heterozygous females, 3-day estradiol treatment at 40 micrograms/kg stimulates a 3- to 4-fold increase in uterine wet weight and alters vaginal cornification, but the uteri and vagina do not respond in the animals with the estrogen receptor gene disruption. Prenatal male and female reproductive tract development can therefore occur in the absence of estradiol receptor-mediated responsiveness.

Animals↗