Home About us Contact
|
Home About us Contact | ||
|
|
||
Gain-of-function Mutations (gain-of-function + mutation)
Selected AbstractsOropharyngeal Skeletal Disease Accompanying High Bone Mass and Novel LRP5 Mutation,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 5 2005Michael R Rickels Abstract 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 L1 -L4 spine (both ,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 ",-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. [source] RET receptor signaling: Dysfunction in thyroid cancer and Hirschsprung's diseasePATHOLOGY INTERNATIONAL, Issue 4 2006Naoya Asai Gain-of-function mutations within the receptor tyrosine kinase gene RET cause inherited and non-inherited thyroid cancer. Somatic gene rearrangements of RET have been found in papillary thyroid carcinoma and germline point mutations in multiple endocrine neoplasia (MEN) types 2A and 2B and familial medullary thyroid carcinoma (FMTC). Conversely, loss-of-function mutations are responsible for the development of Hirschsprung's disease, a congenital malformation of the enteric nervous system. Comparison between normal RET signaling activated by the RET ligand glial cell line-derived neurotrophic factor (GDNF) and abnormal RET signaling caused by various mutations has led to a deeper understanding of disease mechanisms. The focus of the present review is on recent progress in the study of RET signaling dysfunction in human diseases. [source] Activity of triptolide against human mast cells harboring the kinase domain mutant KITCANCER SCIENCE, Issue 7 2009Yanli Jin Gain-of-function mutations of the receptor tyrosine kinase KIT can cause systemic mastocytosis (SM) and gastrointestinal stromal tumors. Most of the constitutively active KIT can be inhibited by imatinib; D816V KIT cannot. In this study, we investigated the activity of triptolide, a diterpenoid isolated from the Chinese herb Tripterygium wilfordii Hook. f., in cells expressing mutant KIT, including D816V KIT. Imatinib-sensitive HMC-1.1 cells harboring the mutation V560G in the juxtamembrane domain of KIT, imatinib-resistant HMC-1.2 cells harboring both V560G and D816V mutations, and murine P815 cells, were treated with triptolide, and analyzed in terms of growth, apoptosis, and signal transduction. The in vivo antitumor activity was evaluated by using the nude mouse xenograft model. Our results demonstrated that triptolide potently inhibits the growth of both human and murine mast cells harboring not only imatinib-sensitive KIT mutation but also imatinib-resistant D816V KIT. Triptolide markedly inhibited KIT mRNA levels and strikingly reduced the levels of phosphorylated and total Stat3, Akt, and Erk1/2, downstream targets of KIT. Triptolide triggered apoptosis by inducing depolarization of mitochondrial potential and release of cytochrome c, downregulation of Mcl-1 and XIAP. Furthermore, triptolide significantly abrogated the growth of imatinib-resistant HMC-1.2 cell xenografts in nude mice and decreased KIT expression in xenografts. Our data demonstrate that triptolide inhibits imatinib-resistant mast cells harboring D816V KIT. Further investigation of triptolide for treatment of human neoplasms driven by gain-of-function KIT mutations is warranted. (Cancer Sci 2009; 100: 1335,1343) [source] Dynamic morphological changes in the skulls of mice mimicking human Apert syndrome resulting from gain-of-function mutation of FGFR2 (P253R)JOURNAL OF ANATOMY, Issue 2 2010Xiaolan Du Abstract Apert syndrome is caused mainly by gain-of-function mutations of fibroblast growth factor receptor 2. We have generated a mouse model (Fgfr2+/P253R) mimicking human Apert syndrome resulting from fibroblast growth factor receptor 2 Pro253Arg mutation using the knock-in approach. This mouse model in general has the characteristic skull morphology similar to that in humans with Apert syndrome. To characterize the detailed changes of form in the overall skull and its major anatomic structures, euclidean distance matrix analysis was used to quantitatively compare the form and growth difference between the skulls of mutants and their wild-type controls. There were substantial morphological differences between the skulls of mutants and their controls at 4 and 8 weeks of age (P < 0.01). The mutants showed shortened skull dimensions along the rostrocaudal axis, especially in their face. The width of the frontal bone and the distance between the two orbits were broadened mediolaterally. The neurocrania were significantly increased along the dorsoventral axis and slightly increased along the mediolateral axis, and also had anteriorly displayed opisthion along the rostrocaudal axis. Compared with wild-type, the mutant mandible had an anteriorly displaced coronoid process and mandibular condyle along the rostrocaudal axis. We further found that there was catch-up growth in the nasal bone, maxilla, zygomatic bone and some regions of the mandible of the mutant skulls during the 4,8-week interval. The above-mentioned findings further validate the Fgfr2+/P253R mouse strain as a good model for human Apert syndrome. The changes in form characterized in this study will help to elucidate the mechanisms through which the Pro253Arg mutation in fibroblast growth factor receptor 2 affects craniofacial development and causes Apert syndrome. [source] Amino acids involved in conformational dynamics and G protein coupling of an odorant receptor: targeting gain-of-function mutationJOURNAL OF NEUROCHEMISTRY, Issue 5 2008Aya Kato Abstract Thousands of different odorants are recognized and discriminated by odorant receptors (ORs) in the guanine nucleotide-binding protein (G protein)-coupled seven-transmembrane receptor family. Odorant-bound ORs stimulate Gs-type G proteins, G,olf, which in turn activates cAMP-mediated signaling pathway in olfactory sensory neurons. To better understand the molecular basis for OR activation and G protein coupling, we analyzed the effects of a series of site-directed mutations of mouse ORs, on function. Mutations of conserved amino acid residues in an intracellular loop or the C-terminus resulted in loss of activity without impairing ligand-binding activity, indicating that these residues are involved in G,s/olf coupling. Moreover, mutation of the serine in KAFSTC, the OR-specific sequence motif, resulted in a dramatic increase in odorant responsiveness, suggesting that the motif is involved in a conformational change of the receptor that regulates G protein coupling efficiency. Our results provide insights into how ORs switch from an inactive to an active state, as well as where and how activated ORs interact with G proteins. [source] Purified A2 domain of von Willebrand factor binds to the active conformation of von Willebrand factor and blocks the interaction with platelet glycoprotein Ib,JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 7 2007C. MARTIN Summary.,Background:,von Willebrand factor (VWF) does not interact with circulating platelets unless it is induced to expose the binding site for platelet glycoprotein (GP)Ib, in the A1 domain by high shear stress, immobilization, and/or a modulator. Previous studies have implied indirectly that the A2 domain may be involved in regulating A1,GPIb, binding. Objective and methods:,Because the relationship between the A1 and A2 domains has not been defined, we have investigated the effect of the A2 domain on the binding activity of the A1 domain using recombinant A domain polypeptides, multimeric VWF, and monoclonal antibodies (mAb). Results:,The A2 domain polypeptide bound specifically to the immobilized A1 domain polypeptide or full-length VWF, with half-maximal binding being obtained at 60 or 168 nm, respectively. This A1,A2 interaction was inhibited by mAb against the A2 or A1 domain and by the A1 domain polypeptide. The A2 domain polypeptide effectively blocked GPIb,-mediated platelet adhesion under high flow conditions. The A2 domain polypeptide specifically recognizes the GPIb,-binding conformation in the A1 domain, as it only interacted with VWF activated by the modulator ristocetin or immobilized VWF. Furthermore, in contrast to plasma VWF, the ultra-large (UL)VWF multimers or a recombinant VWF,A1A2A3 polypeptide containing a gain-of-function mutation (R1308 L) of type 2B von Willebrand disease bound to the A2 domain polypeptide without the need for ristocetin. Conclusions:,The recombinant A2 domain polypeptide specifically binds to the active conformation of the A1 domain in VWF and effectively blocks the interaction with platelet GPIb, under high-flow conditions. [source] Gene therapy flexes muscleTHE JOURNAL OF GENE MEDICINE, Issue 9 2005A European Society of Gene Therapy commentary on progress in gene therapy for Duchenne muscular dystrophy, amyotrophic lateral sclerosis Abstract This commentary highlights the promising results of recent studies in animal models of Duchenne muscular dystrophy and amyotrophic lateral sclerosis that have clearly demonstrated the potential of gene therapy for tackling these diseases. In the absence of effective drugs or other treatments, these advances in gene therapy technology represent the best hope for those patients and families that are blighted by these diseases. Background Diseases characterized by progressive muscle degeneration are often incurable and affect a relatively large number of individuals. The progressive deterioration of muscle function is like the sword of Damocles that constantly reminds patients suffering from these diseases of their tragic fate, since most of them will eventually die from cardiac or pulmonary dysfunction. Some of these disorders are due to mutations in genes that directly influence the integrity of muscle fibers, such as in Duchenne muscular dystrophy (DMD), a recessive X-linked genetic disease. Others result from a progressive neurodegeneration of the motoneurons that are essential for maintaining muscle function, such as in amyotrophic lateral sclerosis (ALS), also commonly known as Lou Gehrig's disease. The genetic basis of DMD is relatively well understood as it is due to mutations in the dystrophin gene that encodes the cognate sarcolemmal protein. In contrast, the cause of ALS is poorly defined, with the exception of some dominantly inherited familial cases of ALS that are due to gain-of-function mutations in the gene encoding superoxide dismutase (SODG93A). Gene therapy for these disorders has been hampered by the inability to achieve widespread gene transfer. Moreover, since familial ALS is due to a dominant gain-of-function mutation, inhibition of gene expression (rather than gene augmentation) would be required to correct the phenotype, which is particularly challenging. Copyright © 2005 John Wiley & Sons, Ltd. [source] Gibberellin controls the nodulation signaling pathway in Lotus japonicusTHE PLANT JOURNAL, Issue 2 2009Takaki Maekawa Summary Root nodule formation is regulated by several plant hormones, but the details of the regulation of the nodulation signaling pathway are largely unknown. In this study, the role of gibberellin (GA) in the control of root nodule symbiosis was investigated at the physiological and genetic levels in Lotus japonicus. Exogenous application of biologically active GA, GA3, inhibited the formation of infection threads and nodules, which was counteracted by the application of a biosynthesis inhibitor of GA, Uniconazole P. Nod factor-induced root hair deformation was severely blocked in the presence of GA, which was phenocopied by nsp2 mutants. The number of spontaneous nodules triggered by the gain-of-function mutation of calcium/calmodulin-dependent kinase (CCaMK) or the lotus histidine kinase 1 (LHK1) was decreased upon the addition of GA; moreover, the overexpression of the gain-of-function mutation of L. japonicus, SLEEPY1, a positive regulator of GA signaling, resulted in a reduced nodule number, without other aspects of root development being affected. These results indicate that higher GA signaling levels specifically inhibit the nodulation signaling pathway. Nod factor-dependent induction of NSP2 and NIN was inhibited by exogenous GA. Furthermore, the cytokinin-dependent induction of NIN was suppressed by GA. From these results, we conclude that GA inhibits the nodulation signaling pathway downstream of cytokinin, possibly at NSP2, which is required for Nod factor-dependent NIN expression. These results clarify the roles of GA in the nodulation signaling pathway, and in relation to the cytokinin signaling pathway for nodulation in L. japonicus. [source] Maize mutants affected at distinct stages of the arbuscular mycorrhizal symbiosisTHE PLANT JOURNAL, Issue 2 2006Uta Paszkowski Summary Maize mutants affected in the symbiotic interaction with the arbuscular mycorrhizal fungus Glomus mosseae have been found by a visual, macroscopic screen in a Mutator -tagged population of maize. Seven mutants have been identified, falling into three phenotypic classes. For each class one mutant has been characterized in more detail. The nope1 (noperception 1) mutant does not support appressoria formation of G. mosseae, suggesting the absence of a plant-encoded function necessary for early recognition prior to contact. The phenotype segregated as a monogenic recessive trait, indicating that a mutation in a single locus abolished compatibility of maize to G. mosseae. On a second mutant termed taci1 (taciturn 1), appressoria form at reduced frequency but their morphology is normal and leads to penetration of the rhizodermis. However, intraradically, the majority of hyphae are septate, resulting in terminated fungal spreading. This phenotype suggests that the mutation of taci1 has an effect on recognition and on cortex invasion. Segregation analysis indicates taci1 to carry a recessive mutation. In contrast, wild-type fungal morphology has been recorded in the Pram1 (Precocious arbuscular mycorrhiza 1) mutant, which displays enhanced and earlier fungal invasion. This trait segregates in a dominant fashion indicative of a gain-of-function mutation affecting the plant's control over restricting fungal colonization. [source] High humidity suppresses ssi4 -mediated cell death and disease resistance upstream of MAP kinase activation, H2O2 production and defense gene expressionTHE PLANT JOURNAL, Issue 6 2004Fasong Zhou Summary The Arabidopsis ssi4 mutant, which exhibits spontaneous lesion formation, constitutive expression of pathogenesis-related (PR) genes and enhanced resistance to virulent bacterial and oomycete pathogens, contains a gain-of-function mutation in a TIR-NBS-LRR type R gene. Epistatic analyses revealed that both PR gene expression and disease resistance are activated via a salicylic acid (SA)- and EDS1 -dependent, but NPR1 - and NDR1 -independent signaling pathway. In this study, we demonstrate that in moderate relative humidity (RH; 60%), the ssi4 mutant accumulates H2O2 and SA prior to lesion formation and displays constitutive activation of the MAP kinases AtMPK6 and AtMPK3. It also constitutively expresses a variety of defense-associated genes, including those encoding the WRKY transcription factors AtWRKY29 and AtWRKY6, the MAP kinases AtMPK6 and AtMPK3, the powdery mildew R proteins RPW8.1 and RPW8.2, EDS1 and PR proteins. All of these ssi4 -induced responses, as well as the chlorotic, stunted morphology and enhanced disease resistance phenotype, are suppressed by high RH (95%) growth conditions. Thus, a humidity sensitive factor (HSF) appears to function at an early point in the ssi4 signaling pathway. All ssi4 phenotypes, except for MAP kinase activation, also were suppressed by the eds1-1 mutation. Thus, ssi4 -induced MAP kinase activation occurs downstream of the HSF but either upstream of EDS1 or on a separate branch of the ssi4 signaling pathway. SA is a critical signaling component in ssi4 -mediated defense responses. However, exogenously supplied SA failed to restore lesion formation in high RH-grown ssi4 plants, although it induced defense gene expression. Thus, additional signals also are involved. [source] Sporadic onset of erythermalgia: A gain-of-function mutation in Nav1.7ANNALS OF NEUROLOGY, Issue 3 2006Chongyang Han BS Objective Inherited erythermalgia (erythromelalgia) is an autosomal dominant disorder in which patients experience severe burning pain in the extremities, in response to mild thermal stimuli and exercise. Although mutations in sodium channel Nav1.7 have been shown to underlie erythermalgia in several multigeneration families with the disease that have been investigated to date, the molecular basis of erythermalgia in sporadic cases is enigmatic. We investigated the role of Nav1.7 in a sporadic case of erythermalgia in a Chinese family. Methods Genomic DNA from patients and their asymptomatic family members were sequenced to identify mutations in Nav1.7. Whole-cell patch clamp analysis was used to characterize biophysical properties of wild-type and mutant Nav1.7 channels in mammalian cells. Results A single amino acid substitution in the DIIS4-S5 linker of Nav1.7 was present in two children whose parents were asymptomatic. The asymptomatic father was genetically mosaic for the mutation. This mutation produces a hyperpolarizing shift in channel activation and an increase in amplitude of the response to slow, small depolarizations. Interpretation Founder mutations in Nav1.7, which can confer hyperexcitability on peripheral sensory neurons, can underlie sporadic erythermalgia. Ann Neurol 2006 [source] Towards characterization of palmoplantar keratoderma caused by gain-of-function mutation in loricrin: analysis of a family and review of the literatureBRITISH JOURNAL OF DERMATOLOGY, Issue 1 2006M.M. Gedicke Summary Loricrin keratoderma is an autosomal dominant palmoplantar keratoderma heterogeneous in clinical appearance. We report a family with diffuse ichthyosis and honeycomb palmoplantar keratoderma but no occurrence of pseudoainhums or autoamputations. All patients were born as collodion babies and displayed prominent knuckle pads. We identified the previously reported mutation 730insG in LOR, which elongates loricrin by 22 amino acids because of delayed termination. As pseudoainhums are missing in all patients of the family reported, we propose two compulsory features of loricrin keratoderma: (i) honeycomb palmoplantar keratoderma and (ii) diffuse ichthyosiform dermatosis. Therefore we suggest that the condition should be described clinically as ,honeycomb palmoplantar keratoderma with ichthyosis'. Furthermore, we have assessed the amounts of transcript of LOR using pyrosequencing. This revealed an equal expression of mutant and wild-type alleles of LOR in an affected individual. These findings further underline the gain-of-function theory for mutant LOR in loricrin keratoderma. [source] Alterations of the c-kit gene in testicular germ cell tumorsCANCER SCIENCE, Issue 6 2003Yuji Sakuma Expression and gain-of-function mutation of the c-kit gene, that encodes a receptor tyrosine kinase (KIT), have been reported in mast cell tumors and gastrointestinal stromal tumors (GISTs). Among human testicular germ cell tumors (GCTs), seminomas and seminoma components of mixed GCTs have also been shown to express KIT, but only one study has found the c-kit gene mutation at exon 17 in seminoma. To elucidate the frequency and location of the c-kit gene mutation of testicular GCTs, we analyzed the whole coding region of the c-kit complementary DNA along with 4 mutational hot spots (exons 9, 11, 13 and 17) of the c-kit genomic DNA by polymerase chain reaction and direct sequencing. Somatic mutations were found in 4 pure seminomas of 34 testicular GCTs (11.8%). One mutation was found in exon 11 (W557R) and the others were observed in exon 17 (D816H and D816V). These types of mutations were reported in GISTs (W557R), seminoma (D816H) and mastocytosis (D816V) and were considered to be gain-of-function mutations, although there were no differences of any clinicopathological factors or outcome between patients with and without mutations. Additionally, we also demonstrated coexpression of Gly-Asn-Asn-Lys510,513 (GNNK)+ and GNNK- isoforms of the c-kit gene with dominance of the GNNK- transcript in all testicular GCTs. The mutations and/or preferential expression of GNNK- isoform of the c-kit gene might play an important role in the development of testicular GCTs, and these tumors may also be targets for STI571, which is a promising drug for advanced and metastatic GISTs. [source] Drosophila RSK negatively regulates bouton number at the neuromuscular junctionDEVELOPMENTAL NEUROBIOLOGY, Issue 4 2009Matthias Fischer Abstract Ribosomal S6 kinases (RSKs) are growth factor-regulated serine-threonine kinases participating in the RAS-ERK signaling pathway. RSKs have been implicated in memory formation in mammals and flies. To characterize the function of RSK at the synapse level, we investigated the effect of mutations in the rsk gene on the neuromuscular junction (NMJ) in Drosophila larvae. Immunostaining revealed transgenic expressed RSK in presynaptic regions. In mutants with a full deletion or an N-terminal partial deletion of rsk, an increased bouton number was found. Restoring the wild-type rsk function in the null mutant with a genomic rescue construct reverted the synaptic phenotype, and overexpression of the rsk -cDNA in motoneurons reduced bouton numbers. Based on previous observations that RSK interacts with the Drosophila ERK homologue Rolled, genetic epistasis experiments were performed with loss- and gain-of-function mutations in Rolled. These experiments provided evidence that RSK mediates its negative effect on bouton formation at the Drosophila NMJ by inhibition of ERK signaling. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009 [source] Loss-of-function variants of the human melanocortin-1 receptor gene in melanoma cells define structural determinants of receptor functionFEBS JOURNAL, Issue 24 2002Jesús Sánchez Más The ,-melanocyte-stimulating hormone (,MSH) receptor (MC1R) is a major determinant of mammalian skin and hair pigmentation. Binding of ,MSH to MC1R in human melanocytes stimulates cell proliferation and synthesis of photoprotective eumelanin pigments. Certain MC1R alleles have been associated with increased risk of melanoma. This can be theoretically considered on two grounds. First, gain-of-function mutations may stimulate proliferation, thus promoting dysplastic lesions. Second, and opposite, loss-of-function mutations may decrease eumelanin contents, and impair protection against the carcinogenic effects of UV light, thus predisposing to skin cancers. To test these possibilities, we sequenced the MC1R gene from seven human melanoma cell (HMC) lines and three giant congenital nevus cell (GCNC) cultures. Four HMC lines and two GCNC cultures contained MC1R allelic variants. These were the known loss-of-function Arg142His and Arg151Cys alleles and a new variant, Leu93Arg. Moreover, impaired response to a superpotent ,MSH analog was demonstrated for the cell line carrying the Leu93Arg allele and for a HMC line homozygous for wild-type MC1R. Functional analysis in heterologous cells stably or transiently expressing this variant demonstrated that Leu93Arg is a loss-of-function mutation abolishing agonist binding. These results, together with site-directed mutagenesis of the vicinal Glu94, demonstrate that the MC1R second transmembrane fragment is critical for agonist binding and maintenance of a resting conformation, whereas the second intracellular loop is essential for coupling to the cAMP system. Therefore, loss-of-function, but not activating MC1R mutations are common in HMC. Their study provides important clues to understand MC1R structure-function relationships. [source] Novel missense mutations in the FOXC2 gene alter transcriptional activity,HUMAN MUTATION, Issue 12 2009M.A.M. van Steensel Abstract Mutations in the FOXC2 gene that codes for a forkhead transcription factor are associated with primary lymphedema that usually develops around puberty. Associated abnormalities include distichiasis and, very frequently, superficial and deep venous insufficiency. Most mutations reported so far either truncate the protein or are missense mutations in the forkhead domain causing a loss of function. The haplo-insufficient state is associated with lymphatic hyperplasia in mice as well as in humans. We analyzed the FOXC2 gene in 288 patients with primary lymphedema and found 11 pathogenic mutations, of which 9 are novel. Of those, 5 were novel missense mutations of which 4 were located outside of the forkhead domain. To examine their pathogenic potential we performed a transactivation assay using a luciferase reporter construct driven by FOXC1 response elements. We found that the mutations outside the forkhead domain cause a gain of function as measured by luciferase activity. Patient characteristics conform to previous reports with the exception of distichiasis, which was found in only 2 patients out of 11. FOXC2 mutations causing lymphedema-distichiasis syndrome reported thus far result in haplo-insufficiency and lead to lymphatic hyperplasia. Our results suggest that gain-of-function mutations may also cause lymphedema. One would expect that in this case, lymphatic hypoplasia would be the underlying abnormality. Patients with activating mutations might present with Meige disease. © 2009 Wiley-Liss, Inc. [source] Dynamic morphological changes in the skulls of mice mimicking human Apert syndrome resulting from gain-of-function mutation of FGFR2 (P253R)JOURNAL OF ANATOMY, Issue 2 2010Xiaolan Du Abstract Apert syndrome is caused mainly by gain-of-function mutations of fibroblast growth factor receptor 2. We have generated a mouse model (Fgfr2+/P253R) mimicking human Apert syndrome resulting from fibroblast growth factor receptor 2 Pro253Arg mutation using the knock-in approach. This mouse model in general has the characteristic skull morphology similar to that in humans with Apert syndrome. To characterize the detailed changes of form in the overall skull and its major anatomic structures, euclidean distance matrix analysis was used to quantitatively compare the form and growth difference between the skulls of mutants and their wild-type controls. There were substantial morphological differences between the skulls of mutants and their controls at 4 and 8 weeks of age (P < 0.01). The mutants showed shortened skull dimensions along the rostrocaudal axis, especially in their face. The width of the frontal bone and the distance between the two orbits were broadened mediolaterally. The neurocrania were significantly increased along the dorsoventral axis and slightly increased along the mediolateral axis, and also had anteriorly displayed opisthion along the rostrocaudal axis. Compared with wild-type, the mutant mandible had an anteriorly displaced coronoid process and mandibular condyle along the rostrocaudal axis. We further found that there was catch-up growth in the nasal bone, maxilla, zygomatic bone and some regions of the mandible of the mutant skulls during the 4,8-week interval. The above-mentioned findings further validate the Fgfr2+/P253R mouse strain as a good model for human Apert syndrome. The changes in form characterized in this study will help to elucidate the mechanisms through which the Pro253Arg mutation in fibroblast growth factor receptor 2 affects craniofacial development and causes Apert syndrome. [source] Calcium Channel Antagonism Reduces Exercise-Induced Ventricular Arrhythmias in Catecholaminergic Polymorphic Ventricular Tachycardia Patients with RyR2 MutationsJOURNAL OF CARDIOVASCULAR ELECTROPHYSIOLOGY, Issue 2 2005HEIKKI SWAN M.D. Introduction: Recently, gain-of-function mutations of cardiac ryanodine receptor RyR2 gene have been identified as a cause of familial or catecholaminergic polymorphic ventricular tachycardia. We examined the influence of the calcium channel blockers, verapamil and magnesium, on exercise-induced ventricular arrhythmias in patients with RyR2 mutations. Methods and Results: Six molecularly defined catecholaminergic polymorphic ventricular tachycardia patients, all carrying a RyR2 mutation and on ,-adrenergic blocker therapy, underwent exercise stress test four times: at baseline, after verapamil and magnesium sulphate infusions, and finally, without interventions. The number of isolated and successive premature ventricular complexes during exercise ranged from 40 to 374 beats (mean 165 beats) at baseline, and was reduced during verapamil by 76 ± 17% (P < 0.05). Premature ventricular complexes appeared later and at higher heart rate during verapamil than at baseline (119 ± 21 vs. 127 ± 27 min,1, P < 0.05). Magnesium did not inhibit the arrhythmias. Results in the fourth exercise stress test without interventions were similar to those in the first baseline study. Conclusions: This study provides the first in vivo demonstration that a calcium channel antagonist, verapamil, can suppress premature ventricular complexes and nonsustained ventricular salvoes in catecholaminergic polymorphic ventricular tachycardia caused by RyR2 mutations. Modifying the abnormal calcium handling by calcium antagonists might have therapeutic value. [source] Syndromic craniosynostosis: from history to hydrogen bondsORTHODONTICS & CRANIOFACIAL RESEARCH, Issue 2 2007ML Cunningham Structured Abstract Authors,,, Cunningham ML, Seto ML, Ratisoontorn C, Heike CL, Hing AV The syndromic craniosynostoses, usually involving multiple sutures, are hereditary forms of craniosynostosis associated with extracranial phenotypes such as limb, cardiac, CNS and tracheal malformations. The genetic etiology of syndromic craniosynostosis in humans is only partially understood. Syndromic synostosis has been found to be associated with mutations of the fibroblast growth factor receptor family (FGFR1, -R2, -R3), TWIST1, MSX2, and EFNB1. Apert, Pfeiffer, Crouzon, and Jackson-Weiss syndromes are due to gain-of-function mutations of FGFR2 in either the Ig II,III linker region (Apert) or Ig III domain. Loss of function mutations of TWIST1 and gain-of-function mutations of MSX2 lead to Saethre,Chotzen and Boston-type syndromes, respectively. The mutations in Pfeiffer (FGFR1), Muenke (FGFR3), and Apert syndrome (FGFR2) are caused by the same amino acid substitution in a highly conserved region of the Ig II,III linker region of these proteins, which suggests that these receptor tyrosine kinases have an overlapping function in suture biology. In this review we will discuss the historical descriptions, current phenotypes and molecular causes of the more common forms of syndromic craniosynostosis. [source] Voltage-Gated Sodium Channels: Therapeutic Targets for PainPAIN MEDICINE, Issue 7 2009Sulayman D. Dib-Hajj PhD ABSTRACT Objective., To provide an overview of the role of voltage-gated sodium channels in pathophysiology of acquired and inherited pain states, and of recent developments that validate these channels as therapeutic targets for treating chronic pain. Background., Neuropathic and inflammatory pain conditions are major medical needs worldwide with only partial or low efficacy treatment options currently available. An important role of voltage-gated sodium channels in many different pain states has been established in animal models and, empirically, in humans, where sodium channel blockers partially ameliorate pain. Animal studies have causally linked changes in sodium channel expression and modulation that alter channel gating properties or current density in nociceptor neurons to different pain states. Biophysical and pharmacological studies have identified the sodium channel isoforms Nav1.3, Nav1.7, Nav1.8, and Nav1.9 as particularly important in the pathophysiology of different pain syndromes. Recently, gain-of-function mutations in SCN9A, the gene which encodes Nav1.7, have been linked to two human-inherited pain syndromes, inherited erythromelalgia and paroxysmal extreme pain disorder, while loss-of-function mutations in SCN9A have been linked to complete insensitivity to pain. Studies on firing properties of sensory neurons of dorsal root ganglia demonstrate that the effects of gain-of-function mutations in Nav1.7 on the excitability of these neurons depend on the presence of Nav1.8, which suggests a similar physiological interaction of these two channels in humans carrying the Nav1.7 pain mutation. Conclusions., These studies suggest that isoform-specific blockers of these channels or targeting of their modulators may provide novel approaches to treatment of pain. [source] Pathology of gastrointestinal stromal tumorsPATHOLOGY INTERNATIONAL, Issue 1 2006Seiichi Hirota Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors in the gastrointestinal tract. It was found that most GIST expressed KIT, a receptor tyrosine kinase encoded by protooncogene c- kit. In normal gastrointestinal wall, KIT is expressed by interstitial cells of Cajal (ICC), which are a pacemaker for autonomous gastrointestinal movement. Because both GIST and ICC are double-positive for KIT and CD34, and because familial and multiple GIST appear to develop from diffuse hyperplasia of ICC, GIST are considered to originate from ICC or their precursor cells. It was also found that approximately 90% of the sporadic GIST have somatic gain-of-function mutations of the c- kit gene, and that the patients with familial and multiple GIST have germline gain-of-function mutations of the c- kit gene. These facts strongly suggest that the c- kit gene mutations are a cause of GIST. Approximately half of the sporadic GIST without c- kit gene mutations were demonstrated to have gain-of-function mutations in platelet-derived growth factor receptor-, (PDGFRA) gene that encodes another receptor tyrosine kinase. Because KIT is immunohistochemically negative in a minority of GIST, especially in PDGFRA gene mutation-harboring GIST, mutational analyses of c- kit and PDGFRA genes may be required to diagnose such GIST definitely. Imatinib mesylate was developed as a selective tyrosine kinase inhibitor. It inhibits constitutive activation of mutated KIT and PDGFRA, and is now being used for KIT-positive metastatic or unresectable GIST as a molecular target drug. Confirmation of KIT expression by immunohistochemistry is necessary for application of the drug. The effect of imatinib mesylate is different in various types of c- kit and PDGFRA gene mutations, and the secondary resistance against imatinib mesylate is often acquired by the second mutation of the identical genes. Mutational analyses of c- kit and PDGFRA genes are also significant for prediction of effectiveness of drugs including newly developed agents. [source] PKC theta, a novel immunohistochemical marker for gastrointestinal stromal tumors (GIST), especially useful for identifying KIT-negative tumorsPATHOLOGY INTERNATIONAL, Issue 3 2005Atsushi Motegi Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor in the digestive tract and the majority of GIST has characteristic gain-of-function mutations of the c-kit gene, which encodes the KIT receptor for stem cell factor. The present study aimed to establish the usefulness of protein kinase C theta (PKC ,) as an immunohistochemical marker for GIST in comparison with KIT immunohistochemistry. PKC , immunohistochemistry was carried out not only on 48 cases of GIST and another 40 cases of gastrointestinal mesenchymal tumors, but also on 24 cases of various tumors known to be immunohistochemically positive for KIT. Immunohistochemically, 41 out of 48 cases (85%) of GIST were positive for PKC ,, and its expression was confirmed by Western blot analysis using six cases of surgically resected GIST. In the present study there were six GIST immunohistochemically negative for KIT, which histologically revealed a myxoid epithelioid appearance characteristic to that of GIST with platelet-derived growth factor receptor alpha mutation. All six GIST were immunohistochemically positive for PKC ,. No PKC , immunoreactivity was observed in other gastrointestinal mesenchymal tumors and various KIT-positive tumors except for three cases (14%) of gastrointestinal schwannomas. The present study revealed that PKC , is an immunohistochemically novel and useful marker for GIST, especially for GIST negative for KIT. [source] Gene therapy flexes muscleTHE JOURNAL OF GENE MEDICINE, Issue 9 2005A European Society of Gene Therapy commentary on progress in gene therapy for Duchenne muscular dystrophy, amyotrophic lateral sclerosis Abstract This commentary highlights the promising results of recent studies in animal models of Duchenne muscular dystrophy and amyotrophic lateral sclerosis that have clearly demonstrated the potential of gene therapy for tackling these diseases. In the absence of effective drugs or other treatments, these advances in gene therapy technology represent the best hope for those patients and families that are blighted by these diseases. Background Diseases characterized by progressive muscle degeneration are often incurable and affect a relatively large number of individuals. The progressive deterioration of muscle function is like the sword of Damocles that constantly reminds patients suffering from these diseases of their tragic fate, since most of them will eventually die from cardiac or pulmonary dysfunction. Some of these disorders are due to mutations in genes that directly influence the integrity of muscle fibers, such as in Duchenne muscular dystrophy (DMD), a recessive X-linked genetic disease. Others result from a progressive neurodegeneration of the motoneurons that are essential for maintaining muscle function, such as in amyotrophic lateral sclerosis (ALS), also commonly known as Lou Gehrig's disease. The genetic basis of DMD is relatively well understood as it is due to mutations in the dystrophin gene that encodes the cognate sarcolemmal protein. In contrast, the cause of ALS is poorly defined, with the exception of some dominantly inherited familial cases of ALS that are due to gain-of-function mutations in the gene encoding superoxide dismutase (SODG93A). Gene therapy for these disorders has been hampered by the inability to achieve widespread gene transfer. Moreover, since familial ALS is due to a dominant gain-of-function mutation, inhibition of gene expression (rather than gene augmentation) would be required to correct the phenotype, which is particularly challenging. Copyright © 2005 John Wiley & Sons, Ltd. [source] Aldosterone responsiveness of the epithelial sodium channel (ENaC) in colon is increased in a mouse model for Liddle's syndromeTHE JOURNAL OF PHYSIOLOGY, Issue 2 2008Marko Bertog Liddle's syndrome is an autosomal dominant form of human hypertension, caused by gain-of-function mutations of the epithelial sodium channel (ENaC) which is expressed in aldosterone target tissues including the distal colon. We used a mouse model for Liddle's syndrome to investigate ENaC-mediated Na+ transport in late distal colon by measuring the amiloride-sensitive transepithelial short circuit current (,ISC-Ami) ex vivo. In Liddle mice maintained on a standard salt diet, ,ISC-Ami was only slightly increased but plasma aldosterone (PAldo) was severely suppressed. Liddle mice responded to a low or a high salt diet by increasing or decreasing, respectively, their PAldo and ,ISC-Ami. However, less aldosterone was required in Liddle animals to achieve similar or even higher Na+ transport rates than wild-type animals. Indeed, the ability of aldosterone to stimulate ,ISC-Ami was about threefold higher in Liddle animals than in the wild-type controls. Application of aldosterone to colon tissue in vitro confirmed that ENaC stimulation by aldosterone was not only preserved but enhanced in Liddle mice. Aldosterone-induced transcriptional up-regulation of the channel's ,- and ,-subunit (,ENaC and ,ENaC) and of the serum- and glucocorticoid-inducible kinase 1 (SGK1) was similar in colon tissue from Liddle and wild-type animals, while aldosterone had no transcriptional effect on the ,-subunit (,ENaC). Moreover, Na+ feedback regulation was largely preserved in colon tissue of Liddle animals. In conclusion, we have demonstrated that in the colon of Liddle mice, ENaC-mediated Na+ transport is enhanced with an increased responsiveness to aldosterone. This may be pathophysiologically relevant in patients with Liddle's syndrome, in particular on a high salt diet, when suppression of PAldo is likely to be insufficient to reduce Na+ absorption to an appropriate level. [source] A novel Nav1.7 mutation producing carbamazepine-responsive erythromelalgia,ANNALS OF NEUROLOGY, Issue 6 2009Tanya Z. Fischer MD Objective Human and animal studies have shown that Nav1.7 sodium channels, which are preferentially expressed within nociceptors and sympathetic neurons, play a major role in inflammatory and neuropathic pain. Inherited erythromelalgia (IEM) has been linked to gain-of-function mutations of Nav1.7. We now report a novel mutation (V400M) in a three-generation Canadian family in which pain is relieved by carbamazepine (CBZ). Methods We extracted genomic DNA from blood samples of eight members of the family, and the sequence of SCN9A coding exons was compared with the reference Nav1.7 complementary DNA. Wild-type Nav1.7 and V400M cell lines were then analyzed using whole-cell patch-clamp recording for changes in activation, deactivation, steady-state inactivation, and ramp currents. Results Whole-cell patch-clamp studies of V400M demonstrate changes in activation, deactivation, steady-state inactivation, and ramp currents that can produce dorsal root ganglia neuron hyperexcitability that underlies pain in these patients. We show that CBZ, at concentrations in the human therapeutic range, normalizes the voltage dependence of activation and inactivation of this inherited erythromelalgia mutation in Nav1.7 but does not affect these parameters in wild-type Nav1.7. Interpretation Our results demonstrate a normalizing effect of CBZ on mutant Nav1.7 channels in this kindred with CBZ-responsive inherited erythromelalgia. The selective effect of CBZ on the mutant Nav1.7 channel appears to explain the ameliorative response to treatment in this kindred. Our results suggest that functional expression and pharmacological studies may provide mechanistic insights into hereditary painful disorders. Ann Neurol 2009;65:733,741 [source] An unusual class of PITX2 mutations in Axenfeld-Rieger syndromeBIRTH DEFECTS RESEARCH, Issue 3 2006Irfan Saadi Abstract BACKGROUND Mutations in the PITX2 homeobox gene are known to contribute to Axenfeld-Rieger syndrome (ARS), an autosomal-dominant developmental disorder. Although most mutations are in the homeodomain and result in a loss of function, there is a growing subset in the C-terminal domain that has not yet been characterized. These mutations are of particular interest because the C-terminus has both inhibitory and stimulatory activities. METHODS In this study we used a combination of in vitro DNA binding and transfection reporter assays to investigate the fundamental issue of whether C-terminal mutations result in gain or loss of function at a cellular level. RESULTS We report a new frameshift mutation in the PITX2 allele that predicts a truncated protein lacking most of the C-terminal domain (D122FS). This newly reported mutant and another ARS C-terminal mutant (W133Stop) both have greater binding than wild-type to the bicoid element. Of interest, the mutants yielded ,5-fold greater activation of the prolactin promoter in CHO cells, even though the truncated proteins were expressed at lower levels than the wild-type protein. The truncated proteins also had greater than wild-type activity in 2 other cell lines, including the LS8 oral epithelial line that expresses the endogenous Pitx2 gene. CONCLUSIONS The results indicate that the PITX2 C-terminal domain has inhibitory activity and support the notion that ARS may also be caused by gain-of-function mutations. Birth Defects Research (Part A), 2006. © 2006 Wiley-Liss, Inc. [source] Alterations of the c-kit gene in testicular germ cell tumorsCANCER SCIENCE, Issue 6 2003Yuji Sakuma Expression and gain-of-function mutation of the c-kit gene, that encodes a receptor tyrosine kinase (KIT), have been reported in mast cell tumors and gastrointestinal stromal tumors (GISTs). Among human testicular germ cell tumors (GCTs), seminomas and seminoma components of mixed GCTs have also been shown to express KIT, but only one study has found the c-kit gene mutation at exon 17 in seminoma. To elucidate the frequency and location of the c-kit gene mutation of testicular GCTs, we analyzed the whole coding region of the c-kit complementary DNA along with 4 mutational hot spots (exons 9, 11, 13 and 17) of the c-kit genomic DNA by polymerase chain reaction and direct sequencing. Somatic mutations were found in 4 pure seminomas of 34 testicular GCTs (11.8%). One mutation was found in exon 11 (W557R) and the others were observed in exon 17 (D816H and D816V). These types of mutations were reported in GISTs (W557R), seminoma (D816H) and mastocytosis (D816V) and were considered to be gain-of-function mutations, although there were no differences of any clinicopathological factors or outcome between patients with and without mutations. Additionally, we also demonstrated coexpression of Gly-Asn-Asn-Lys510,513 (GNNK)+ and GNNK- isoforms of the c-kit gene with dominance of the GNNK- transcript in all testicular GCTs. The mutations and/or preferential expression of GNNK- isoform of the c-kit gene might play an important role in the development of testicular GCTs, and these tumors may also be targets for STI571, which is a promising drug for advanced and metastatic GISTs. [source] Gastrointestinal stromal tumors (GIST): A model for molecule-based diagnosis and treatment of solid tumorsCANCER SCIENCE, Issue 4 2003Yukihiko Kitamura Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the human gastrointestinal (GI) tract. The c-kit receptor tyrosine kinase (KIT) is expressed by practically all GISTs, and gain-of-function mutations of KIT are present in most GISTs. Interstitial cells of Cajal (ICC) are the pacemaker of the peristaltic movement of the GI tract. Since signals through KIT are essential for development of ICC and since multiple GISTs develop from the hyperplastic lesion of ICCs in familial GIST patients with germ-line mutations of KIT, GISTs are considered to originate from ICC. Imatinib mesylate, which was developed for treatment of chronic myeloid leukemia (CML), was found to be useful for treatment of GISTs. Imatinib mesylate inhibits BCR-ABL fused tyrosine kinase that causes CML. Imatinib mesylate also inhibits the mutated KIT observed in most GISTs, and this explains the effectiveness of Imatinib mesylate on GISTs. GISTs appear to serve as a model for molecule-based diagnosis and treatment of solid tumors. (Cancer Sci 2003; 94: 315,320) [source] | |||||||||||||