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PubMed2006

Transgenic Drosophila models of Noonan syndrome causing PTPN11 gain-of-function mutations.

Mutations in the PTPN11 gene, which encodes the protein tyrosine phosphatase SHP-2, causes Noonan syndrome (NS), an autosomal dominant disorder with pleomorphic developmental abnormalities. Certain germline and somatic PTPN11 mutations cause leukemias. Mutations have gain-of-function (GOF) effects with the commonest NS allele, N308D, being weaker than the leukemia-causing mutations. To study the effects of disease-associated PTPN11 alleles, we generated transgenic fruitflies with GAL4-inducible expression of wild-type or mutant csw, the Drosophila orthologue of PTPN11. All three transgenic mutant CSWs rescued a hypomorphic csw allele's eye phenotype, documenting activity. Ubiquitous expression of two strong csw mutant alleles were lethal, but did not perturb development from some CSW-dependent receptor tyrosine kinase pathways. Ubiquitous expression of the weaker N308D allele caused ectopic wing veins, identical to the EGFR GOF phenotype. Epistatic analyses established that csw(N308D)'s ectopic wing vein phenotype required intact EGF ligand and receptor, and that this transgene interacted genetically with Notch, DPP and JAK/STAT signaling. Expression of the mutant csw transgenes increased RAS-MAP kinase activation, which was necessary but not sufficient for transducing their phenotypes. The findings from these fly models provided hypotheses testable in mammalian models, in which these signaling cassettes are largely conserved. In addition, these fly models can be used for sensitized screens to identify novel interacting genes as well as for high-throughput screening of therapeutic compounds for NS and PTPN11-related cancers.

Animals↗
PubMed2002

A novel gain-of-function mutation of the integrin alpha2 VWFA domain.

Integrin alpha2beta1 is the major receptor for collagens in human tissues, being involved in cell adhesion and the control of collagen and collagenase gene expression. The collagen binding site of alpha2beta1 has been localized to the alpha2 von Willebrand Factor type A (VWFA) domain (A-domain or I-domain) and the residues responsible for the interaction with collagen have been mapped. We report a study of alpha2 VWFA domain in which residue E318, which lies outside the collagen binding site, is mutated to tryptophan, showing that this is a gain-of-function mutation. Recombinant alpha2-E318W VWFA domain showed elevated and specific binding to collagen I compared with the wild-type. Side chain hydrophobicity was important for the gain-of-function as elevated binding was seen with E318I and E318Y, but not with E318R. The E318W mutation had additional effects on VWFA domain properties as alpha2-E318W VWFA domain differed from the wild-type in its cation preferences for ligand binding and in binding to monoclonal antibody JA203, which bound at a site distal to E318. The gain-of-function effect was not restricted to binding to collagen I as alpha2-E318W also showed elevated binding to collagen IV, collagen I C-propeptide, laminin and E-cadherin. Binding to these ligands was inhibited by collagen peptide containing the GFOGER motif, indicating that these bound to the VWFA domain by a similar mechanism to collagen I. These data indicate that residue E318 plays a novel and important role in modulating alpha2 VWFA domain--ligand binding and may be involved in the conformational changes associated with its regulation.

Amino Acid Sequence↗
PubMed1998

A novel gain-of-function mutation of c-kit gene in gastrointestinal stromal tumors.

BACKGROUND & AIMS: The c-kit gene encodes a receptor tyrosine kinase (KIT). Recently, we found gain-of-function mutations of the c-kit gene in gastrointestinal stromal tumors (GISTs). All mutations were confined within the 11 amino acids (Lys-550 to Val-560) in the juxtamembrane domain, but one GIST showed a novel deletion-type mutation at codon 579 (Asp) in the juxtamembrane domain. The aim of this study was to clarify whether the mutation is activating. METHODS: Mutant c-kit cDNA was transfected into an interleukin 3 (IL-3)-dependent Ba/F3 murine lymphoid cell line, and the magnitude of autophosphorylation of the mutant KIT was examined with or without stem cell factor (SCF), a ligand of KIT. An in vitro kinase assay was also performed. The biological behavior of the transfectant was estimated by both an in vitro proliferation assay and in vivo transplantation to nude mice. RESULTS: The mutant KIT exhibited constitutive phosphorylation and strong kinase activity without SCF. The transfectant grew autonomously without IL-3 and SCF, and it formed tumors in nude mice. CONCLUSIONS: Deletion at codon 579 (Asp) in the juxtamembrane domain of the c-kit gene is a novel gain-of-function mutation other than the region between Lys-550 and Val-560.

Amino Acid Sequence↗
PubMed1997

Sporadic hypoparathyroidism caused by de Novo gain-of-function mutations of the Ca(2+)-sensing receptor.

Activating mutations of the Ca(2+)-sensing receptor (CaR) gene have been identified in families with autosomal dominant hypoparathyroidism and in one patient with sporadic hypoparathyroidism. Here, we describe two additional patients with sporadic hypoparathyroidism. One patient presented with mild symptoms at age 18 yr; the other was severely symptomatic from infancy. A heterozygous missense mutation was identified in each patient. One mutation (L773R) involved the fifth transmembrane domain of the CaR, the other (N118K) affected the amino-terminal, extracellular domain. In both cases, the probands' parents lacked the mutation, indicating that the mutations arose de novo. In expression studies the mutations shifted the concentration-response curve to the left and increased maximal activity. We conclude that 1) sporadic hypoparathyroidism can be caused by de novo gain-of-function mutations of the CaR; 2) the phenotype can vary from mild to life-threatening hypocalcemia; 3) gain-of-function mutations can involve not only extracellular regions, as previously reported, but also transmembrane domains of the CaR; and 4) the mechanism of activation can involve both increased receptor sensitivity to Ca2+ and increased maximal signal transduction.

Adult↗
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↗
PubMed2001

Gain-of-function mutation at the extracellular domain of KIT in gastrointestinal stromal tumours.

Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal tumours of the human gastrointestinal tract. Previous studies of GISTs found gain-of-function mutations of the c-kit gene, which encodes a receptor tyrosine kinase (KIT). All the mutations were confined to exon 11, which encodes the juxtamembrane domain. By further examination of the whole coding region of c-kit complementary DNA in 35 GISTs, two were found to show the identical mutation at exon 9, which encodes the extracellular domain. The aims of the present study were to examine the frequency of the extracellular domain mutation and to determine whether the mutation is a gain-of-function type or not. Genomic DNA was extracted from paraffin-embedded tissues of 133 GISTs and exon 9 of the c-kit gene was amplified by polymerase chain reaction. Screening of the mutation was carried out by single-strand conformation polymorphism analysis and direct sequencing was done. Mutant c-kit cDNA was transfected into 293T human embryonic kidney cells and the magnitude of autophosphorylation of the mutant KIT was examined with or without the ligand of KIT, stem cell factor (SCF). In total, seven GIST cases (approximately 5%) were found with the identical mutation at exon 9. The mutant KIT exhibited constitutive autophosphorylation without SCF stimulation. The prognosis of the patients with the extracellular domain mutation was comparable to that of the patients with the juxtamembrane domain mutation.

Adult↗
PubMed2005

A case of gain-of-function mutation in calcium-sensing receptor: supplemental hydration is required for renal protection.

AIMS: The calcium-sensing receptor (CaSR) regulates the extracellular calcium level, mainly by controlling parathyroid hormon secretion and renal calcium reabsorption. In gain-of-function CaSR mutations, the genetic abnormalities increase CaSR activity leading to the development of such clinical manifestations as hypercalciuric hypocalcemia and hypoparathyroidism. We report a Japanese case of CaSR gain-of-function mutation and represent a therapeutic intervention based on the functional characteristics of CaSR in renal tubule. METHODS AND RESULTS (CASE): DNA sequence analysis revealed a heterozygous G to T mutation identified in a 12-year-old Japanese girl presenting with sporadic onset of hypercalciuric hypocalcemia and hypoparathyroidism. The mutation is located in the N-terminal extracellular domain of the CaSR gene, one of the most important parts for the three-dimensional construction of the receptor, resulting in the substitution of phenylalanine for cysteine at amino acid 131 (C131F) in exon 3. Based on the diagnosis of the gain-of-function mutation in the CaSR, oral hydrochlorothiazide administration and supplemental hydration were started in addition to calcium supplementation. The combination therapy of thiazide and supplemental hydration markedly reduced both renal calcium excretion and urinary calcium concentration from 0.4-0.7 to less than 0.1 mg/mg (urinary calcium/creatinine ratio) and from 10-15 to 3-5 mg/dl (urinary calcium concentration), respectively. This therapy stopped the progression of renal calcification during the follow-up period. CONCLUSION: Supplemental hydration should be considered essential for the following reasons: (1) calcium supplementation activates the CaSR in the kidney and suppresses renal urinary concentrating ability, (2) the thiazide has a diuretic effect, (3) as calcium supplementation increases renal calcium excretion, the supplemental hydration decreases urinary calcium concentration by increasing urinary volume, thereby diminishing the risk of intratubular crystallization of calcium ion.

Administration, Oral↗
PubMed2003

Autonomously functioning thyroid nodules in a former iodine-deficient area commonly harbor gain-of-function mutations in the thyrotropin signaling pathway.

BACKGROUND: Somatic activating mutations of the thyrotropin (thyroid-stimulating hormone (TSH)) receptor (TSHR) and G(alphas) protein have been detected in solitary toxic adenomas and toxic multinodular goiters, but their role in the pathogenesis of autonomous nodules is debated. The frequency of mutations is highly variable among populations and is inversely proportional to iodine intake. DESIGN AND PATIENTS: We screened 28 clinically and histologically heterogeneous autonomous nodules from 24 Greek patients for the presence of TSHR and G(alphas) mutations. RESULTS: By direct sequencing of genomic DNA, we detected 11 somatic heterozygous gain-of-function mutations in TSHR and one in G(alphas). Forty-three percent (12 of 28) of all nodules and 57% (four of seven) of solitary toxic adenomas harbored an activating mutation. Typical adenomas and hyperplastic nodules did not differ in mutation frequency. Substitutions I568T and T632I were detected in both histological types of nodules. CONCLUSIONS: Our findings indicate that activating somatic mutations in the TSH signaling pathway are frequent in autonomous nodules in Greece. This may be due to earlier exposure of the population to iodine deficiency, which was corrected in Greece only over the past two decades. Gain-of-function mutations are shared by nodules with varying histological and clinical presentations. Thus, they may represent a common molecular mechanism underlying the pathogenesis of non-autoimmune thyroid autonomy.

Adenoma↗
PubMed2005

Fibroblast growth factor receptor 2, gain-of-function mutations, and tumourigenesis: investigating a potential link.

Activating germline mutations in the fibroblast growth factor receptor (FGFR) gene family have been identified in several dominantly inherited skeletal disorders; in the case of FGFR3, the same somatically arising mutations have also been isolated from a variety of tumour tissues. Whilst the role of FGFR2 mutations in congenital syndromes has been well documented, their relationship with cancer has not been clearly defined. Based on evidence that gain-of-function mutations in FGFR2 drive positive selection in adult spermatogonia, the present study investigated, by denaturing high-performance liquid chromatography (DHPLC), DNA sequencing, and restriction digestion, the prevalence of FGFR2 mutations in 58 tumour cell lines of various types, and 29 testicular germ cell tumour samples. Although sequence variations and allelic imbalance were identified in FGFR2, none of the previously documented dominant mutations was detected in any of the tumour types examined. This suggests that gain-of-function FGFR2 mutations are not commonly encountered in tumourigenesis and specifically excludes a major contribution in testicular tumours.

Acrocephalosyndactylia↗
PubMed2026

A new MRR1 gain-of-function mutation involved in cross-resistance to antifungal agents in the fungal priority pathogen Candida parapsilosis.

OBJECTIVES: Candida parapsilosis is a leading cause of invasive candidiasis globally, with rising reports of fluconazole resistance threatening its clinical management. Among the mechanisms involved, gain-of-function mutations in the MRR1 gene have emerged as key drivers of antifungal resistance. We aimed to investigate a novel amino acid substitution (G982E) in the Mrr1 zinc cluster transcription factor, identified in a fluconazole-resistant C. parapsilosis isolate from a patient exposed to fluconazole. METHODS: Using CRISPR-Cas9 genome editing, we introduced the G982E variant into two fluconazole-susceptible C. parapsilosis genetic backgrounds. The antifungal susceptibility of the engineered mutants was assessed in vitro against a broad panel of systemic antifungal agents. A Galleria mellonella infection model was also used to evaluate the impact of the G982E variant on antifungal treatment efficacy and virulence in vivo. RESULTS: Acquisition of the G982E substitution dramatically altered the antifungal susceptibility profile, particularly for fluconazole for which the MIC increased to >256 µg/mL. However, the magnitude of the MIC increase varied by azole, with the greatest increase seen for fluconazole (>9-10-fold), followed by voriconazole (5-fold), isavuconazole (3-fold), but also flucytosine (1.5-fold). In contrast, susceptibility to posaconazole remained largely unchanged. In vivo, this new variant conferred fluconazole treatment failure but was associated with a significant reduction in virulence. CONCLUSIONS: The G982E is a novel Mrr1 gain-of-function mutation driving high-level fluconazole resistance in C. parapsilosis. These findings reinforce the central role of Mrr1 in antifungal resistance, underscore the functional diversity of its mutational landscape, with potential implications for fungal fitness and transcriptional regulation.

Candida parapsilosis↗
PubMed2004

A gain-of-function mutation of Fgfr2c demonstrates the roles of this receptor variant in osteogenesis.

The b and c variants of fibroblast growth factor receptor 2 (FGFR2) differ in sequence, binding specificity, and localization. Fgfr2b, expressed in epithelia, is required for limb outgrowth and branching morphogenesis, whereas the mesenchymal Fgfr2c variant is required by the osteocyte lineage for normal skeletogenesis. Gain-of-function mutations in human FGFR2c are associated with craniosynostosis syndromes. To confirm and extend this evidence, we introduced a Cys342Tyr replacement into Fgfr2c to create a gain-of-function mutation equivalent to a mutation in human Crouzon and Pfeiffer syndromes. Fgfr2c(C342Y/)(+) heterozygote mice are viable and fertile with shortened face, protruding eyes, premature fusion of cranial sutures, and enhanced Spp1 expression in the calvaria. Homozygous mutants display multiple joint fusions, cleft palate, and trachea and lung defects, and die shortly after birth. They show enhanced Cbfa1/Runx2 expression without significant change in chondrocyte-specific Ihh, PTHrP, Sox9, Col2a, or Col10a gene expression. Histomorphometric analysis and bone marrow stromal cell culture showed a significant increase of osteoblast progenitors with no change in osteoclastogenic cells. Chondrocyte proliferation was decreased in the skull base at embryonic day 14.5 but not later. These results suggest that long-term aspects of the mutant phenotype, including craniosynostosis, are related to the Fgfr2c regulation of the osteoblast lineage. The effect on early chondrocyte proliferation but not gene expression suggests cooperation of Fgfr2c with Fgfr3 in the formation of the cartilage model for endochondral bone.

Animals↗
PubMed2003

A case for gastrointestinal stromal tumor (GIST) with reference to its ultrastructure and 'gain-of-function' mutation.

A case for primary gastrointestinal stromal tumor (GIST) is described with reference to its ultrastructural characteristics and mutation within the exon 11 of c-kit gene. A forty-seven years old woman complaining of dysphasia was examined by endoscopy, which depicted a submucosal tumor (70 mm in diameter) with ulcerations at the fundus of the stomach. Histopathologically, the tumor cells had large nuclei and eosinophilic cytoplasm and were frequently during mitosis phase. The tumor cells were immunopositive for KIT, CD 34 and vimentin, suggesting their fibroblast-like characteristics. In contrast, desmin and S-100, a smooth muscle and an enteroglial marker, were not immunopositive within the cells. At least 30 % of the tumor cells possessed MIB-I and 20 % of them possessed p53, which are compatible with fast development of the tumor. By electron microscopy, the tumor cells possessed large oval nuclei, abundant mitochondria, caveolae and smooth endoplasmic reticulums, while no gap junctions were seen on the cells: The tumor cells thus possessed interstitial cells-like characteristics at least in part. DNA mutation search for the tumor cells however realized no gain-of-function mutation within the exon 11 of the c-kit gene, suggesting existence of other mechanism for neoplasmic growth of the tumor cells classified as gastrointestinal stromal tumors.

DNA↗
PubMed1994

The sevenmaker gain-of-function mutation in p42 MAP kinase leads to enhanced signalling and reduced sensitivity to dual specificity phosphatase action.

A mammalian mutant MAP kinase, D319N ERK2, analogous to Drosophila melanogaster sevenmaker (rlsem) gain-of-function mutation was shown to have an increased sensitivity to low levels of signalling in vivo. However, the mutation does not lead to an elevated basal kinase activity and still requires activation by MAP kinase kinase (MAPKK) as does wild type ERK2. This increased responsiveness seen in vivo is not due to an increased ability to phosphorylate substrates but appears to reflect a reduced sensitivity to a MAP kinase phosphatase CL100.

3T3 Cells↗
PubMed2006

Germline gain-of-function mutations in SOS1 cause Noonan syndrome.

Noonan syndrome, the most common single-gene cause of congenital heart disease, is characterized by short stature, characteristic facies, learning problems and leukemia predisposition. Gain-of-function mutations in PTPN11, encoding the tyrosine phosphatase SHP2, cause approximately 50% of Noonan syndrome cases. SHP2 is required for RAS-ERK MAP kinase (MAPK) cascade activation, and Noonan syndrome mutants enhance ERK activation ex vivo and in mice. KRAS mutations account for <5% of cases of Noonan syndrome, but the gene(s) responsible for the remainder are unknown. We identified missense mutations in SOS1, which encodes an essential RAS guanine nucleotide-exchange factor (RAS-GEF), in approximately 20% of cases of Noonan syndrome without PTPN11 mutation. The prevalence of specific cardiac defects differs in SOS1 mutation-associated Noonan syndrome. Noonan syndrome-associated SOS1 mutations are hypermorphs encoding products that enhance RAS and ERK activation. Our results identify SOS1 mutants as a major cause of Noonan syndrome, representing the first example of activating GEF mutations associated with human disease and providing new insights into RAS-GEF regulation.

Adolescent↗
PubMed1997

Mechanism of activation of the Caenorhabditis elegans ras homologue let-60 by a novel, temperature-sensitive, gain-of-function mutation.

The Caenorhabditis elegans let-60 gene encodes a Ras protein that mediates induction of the hermaphrodite vulva. To better understand how mutations constitutively activate Ras and cause unregulated cell division, we have characterized ga89, a temperature-sensitive, gain-of-function mutation in let-60 ras. At 25 degrees, ga89 increases let-60 activity resulting in a multivulva phenotype. At 15 degrees, ga89 decreases let-60 activity resulting in a vulvaless phenotype in let-60(ga89)/Df animals. The ga89 mutation causes a leucine (L) to phenylalanine (F) substitution at amino acid 19, a residue conserved in all Ras proteins. We introduced the L19F change into human H-Ras protein and found that the in vitro GTPase activity of H-Ras became temperature-dependent. Genetic experiments suggest that LET-60 (L19F) interacts with GAP and GNEF, since mutations that decrease GAP and GNEF activity affect the multivulva phenotype of let-60(ga89) animals. These results suggest that the L19F mutation primarily affects the intrinsic rate of GTP hydrolysis by Ras, and that this effect may be sufficient to account for the activated-Ras phenotype caused by let-60(ga89). Our results suggest that a mutation in a human ras gene analogous to ga89 might contribute to oncogenic transformation.

Animals↗
PubMed1990

Analysis of gain-of-function mutations of the lin-12 gene of Caenorhabditis elegans.

Certain cell fate decisions are specified by cell-cell interactions during the development of the nematode Caenorhabditis elegans. For example, in a wild-type hermaphrodite gonad, two cells, Z1.ppp and Z4.aaa, have the potential to become the anchor cell (AC). Intercellular communication establishes their fates and ensures that only one cell becomes the AC, while the other becomes a ventral uterine precursor cell (VU). One component of this intercellular communication seems to be the 'AC-to-VU' signal from the presumptive AC that causes the other cell to become a VU. Genetic and developmental studies indicated that the lin-12 gene specifies the fates of Z1.ppp and Z4.aaa. Molecular studies suggest that lin-12 directly participates in their communications, perhaps acting as the receptor for the 'AC-to-VU' signal. Here, we report the molecular lesions associated with lin-12 gain-of-function mutations, cell isolation experiments, and genetic studies of an unusual lin-12 allele. These data suggest that self-association of the putative lin-12-encoded receptor leads to its activation, and that certain gain-of-function mutations result in ligand-independent activation.

Alleles↗
PubMed2004

Gain-of-function mutation in the KCNMB1 potassium channel subunit is associated with low prevalence of diastolic hypertension.

Hypertension is the most prevalent risk factor for cardiovascular diseases, present in almost 30% of adults. A key element in the control of vascular tone is the large-conductance, Ca(2+)-dependent K(+) (BK) channel. The BK channel in vascular smooth muscle is formed by an ion-conducting alpha subunit and a regulatory beta(1) subunit, which couples local increases in intracellular Ca(2+) to augmented channel activity and vascular relaxation. Our large population-based genetic epidemiological study has identified a new single-nucleotide substitution (G352A) in the beta(1) gene (KCNMB1), corresponding to an E65K mutation in the protein. This mutation results in a gain of function of the channel and is associated with low prevalence of moderate and severe diastolic hypertension. BK-beta(1E65K) channels showed increased Ca(2+) sensitivity, compared with wild-type channels, without changes in channel kinetics. In conclusion, the BK-beta(1E65K) channel might offer a more efficient negative-feedback effect on vascular smooth muscle contractility, consistent with a protective effect of the K allele against the severity of diastolic hypertension.

Adult↗
PubMed2000

Novel gain-of-function mutations of platelet glycoprotein IBalpha by valine mutagenesis in the Cys209-Cys248 disulfide loop. Functional analysis under statis and dynamic conditions.

Platelet-type von Willebrand disease is a bleeding disorder resulting from gain-of-function mutations of glycoprotein (GP) Ibalpha that increase its affinity for von Willebrand factor (vWf). The two known naturally occurring mutations, G233V and M239V, both enrich the valine content of an already valine-rich region within the Cys(209)-Cys(248) disulfide loop. We tested the effect of converting other non-valine residues in this region to valine. Of 10 mutants expressed in CHO cells as components of GP Ib-IX complexes, four displayed a gain-of-function phenotype (G233V, D235V, K237V, and M239V) based on (125)I-vWf binding and adhesion to immobilized vWf. The remainder displayed loss-of-function phenotypes. The gain-of-function mutants bound vWf spontaneously and had a heightened response to low concentrations of ristocetin or botrocetin, whereas the loss-of-function mutants bound vWf more poorly than wild-type GP Ibalpha. No distinct gain- or loss-of-function conformations were identified with conformation-sensitive antibodies. Compared with cells expressing wild-type GP Ibalpha, cells expressing the gain-of-function mutants rolled significantly more slowly over immobilized vWf under flow than wild-type cells and were able to adhere to vWf coated at lower densities. In aggregate, these data indicate that the region of GP Ibalpha bounded by Asn(226) and Ala(244) regulates the affinity for vWf.

Amino Acid Sequence↗