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PubMed2003

Serotonin transporter missense mutation associated with a complex neuropsychiatric phenotype.

Two common serotonin transporter (SERT) untranslated region gene variants have been intensively studied, but remain inconclusively linked to depression and other neuropsychiatric disorders. We now report an uncommon coding region SERT mutation, Ile425Val, in two unrelated families with OCD and other serotonin-related disorders. Six of the seven family members with this mutation had OCD (n=5) or obsessive-compulsive personality disorder (n=1) and some also met diagnostic criteria for multiple other disorders (Asperger's syndrome, social phobia, anorexia nervosa, tic disorder and alcohol and other substance abuse/dependence). The four most clinically affected individuals--the two probands and their two slbs--had the I425V SERT gene gain-of-function mutation and were also homozygous for 5'-UTR SERT gene variant with greater transcriptional efficacy.

Amino Acid Sequence↗
PubMed1999

Altered substrate selectivity in a mutant of an intrahelical salt bridge in UhpT, the sugar phosphate carrier of Escherichia coli.

Site-directed and second site suppressor mutagenesis identify an intrahelical salt bridge in the eleventh transmembrane segment of UhpT, the sugar phosphate carrier of Escherichia coli. Glucose 6-phosphate (G6P) transport by UhpT is inactivated if cysteine replaces either Asp388 or Lys391 but not if both are replaced. This suggests that Asp388 and Lys391 are involved in an intrahelical salt bridge and that neither is required for normal UhpT function. This interpretation is strengthened by the finding that mutations at Lys391 (K391N, K391Q, and K391T) are recovered as revertants of the inactive D388C variant. Further work shows that although the D388C variant is null for G6P transport, movement of 32Pi by homologous Pi/Pi exchange is unaffected. This raises the possibility that this derivative may have latent function, a possibility confirmed by showing that D388C is a gain-of-function mutation in which phosphoenolpyruvate (PEP) is the preferred substrate. Added study of the Pi/Pi exchange shows that in wild type UhpT this partial reaction is readily blocked by G6P but not PEP. By contrast, in the D388C variant, Pi/Pi exchange is unaffected by G6P but is inhibited by both PEP and 3-phosphoglycerate. These latter substrates are used by PgtP, a related Pi-linked antiporter, which lacks the Asp388-Lys391 salt bridge but has instead an uncompensated arginine at position 391. For this reason, we conclude that in both UhpT and PgtP position 391 can serve as a determinant of substrate selectivity by acting as a receptor for the anionic carboxyl brought into the translocation pathway by PEP.

Amino Acid Sequence↗
PubMed2004

Activating mutations of the noonan syndrome-associated SHP2/PTPN11 gene in human solid tumors and adult acute myelogenous leukemia.

The SH2 domain-containing protein-tyrosine phosphatase PTPN11 (Shp2) is required for normal development and is an essential component of signaling pathways initiated by growth factors, cytokines, and extracellular matrix. In many of these pathways, Shp2 acts upstream of Ras. About 50% of patients with Noonan syndrome have germ-line PTPN11 gain of function mutations. Associations between Noonan syndrome and an increased risk of some malignancies, notably leukemia and neuroblastoma, have been reported, and recent data indicate that somatic PTPN11 mutations occur in children with sporadic juvenile myelomonocytic leukemia, myelodysplasic syndrome, B-cell acute lymphoblastic leukemia, and acute myelogenous leukemia (AML). Juvenile myelomonocytic leukemia patients without PTPN11 mutations have either homozygotic NF-1 deletion or activating RAS mutations. Given the role of Shp2 in Ras activation and the frequent mutation of RAS in human tumors, these data raise the possibility that PTPN11 mutations play a broader role in cancer. We asked whether PTPN11 mutations occur in other malignancies in which activating RAS mutations occur at low but significant frequency. Sequencing of PTPN11 from 13 different human neoplasms including breast, lung, gastric, and neuroblastoma tumors and adult AML and acute lymphoblastic leukemia revealed 11 missense mutations. Five are known mutations predicted to result in an activated form of Shp2, whereas six are new mutations. Biochemical analysis confirmed that several of the new mutations result in increased Shp2 activity. Our data demonstrate that mutations in PTPN11 occur at low frequency in several human cancers, especially neuroblastoma and AML, and suggest that Shp2 may be a novel target for antineoplastic therapy.

Adult↗
PubMed1993

Deregulation of Pax-2 expression in transgenic mice generates severe kidney abnormalities.

The Pax genes comprise a family of transcription factors active in specific tissues during embryonic development and are associated with at least three developmental mutations in mouse and man. In the developing kidney, Pax-2 is expressed in the induced mesenchyme, in the ureter epithelium, and in early epithelial structures derived from the mesenchyme. Pax-2 expression is repressed upon terminal differentiation of the renal tubule epithelium, but persists in the undifferentiated epithelium of human Wilms' tumours. We have produced a dominant gain-of-function mutation in transgenic mice by deregulating the expression of the mouse Pax-2 gene. The data obtained with four independently derived transgenic embryos and with one transgenic line demonstrate that deregulated Pax-2 expression results in histologically abnormal and dysfunctional renal epithelium with properties similar to congenital nephrotic syndrome. Thus, repression of Pax-2 is required for normal kidney development and persistent expression of Pax-2 may restrict the differentiation potential of renal epithelial cells.

Animals↗
PubMed2005

Identification of an acquired JAK2 mutation in polycythemia vera.

Polycythemia vera (PV) is a human clonal hematological disorder. The molecular etiology of the disease has not been identified. PV hematopoietic progenitor cells exhibit hypersensitivity to growth factors and cytokines, suggesting possible abnormalities in protein-tyrosine kinases and phosphatases. By sequencing the entire coding regions of cDNAs of candidate enzymes, we identified a G:C--> T:A point mutation of the JAK2 tyrosine kinase in 20 of 24 PV blood samples but none in 12 normal samples. The mutation has varying degrees of heterozygosity and is apparently acquired. It changes conserved Val(617) to Phe in the pseudokinase domain of JAK2 that is known to have an inhibitory role. The mutant JAK2 has enhanced kinase activity, and when overexpressed together with the erythropoietin receptor in cells, it caused hyperactivation of erythropoietin-induced cell signaling. This gain-of-function mutation of JAK may explain the hypersensitivity of PV progenitor cells to growth factors and cytokines. Our study thus defines a molecular defect of PV.

Amino Acid Sequence↗
PubMed2005

Gain-of-function amino acid substitutions drive positive selection of FGFR2 mutations in human spermatogonia.

Despite the importance of mutation in genetics, there are virtually no experimental data on the occurrence of specific nucleotide substitutions in human gametes. C>G transversions at position 755 of FGF receptor 2 (FGFR2) cause Apert syndrome; this mutation, encoding the gain-of-function substitution Ser252Trp, occurs with a birth rate elevated 200- to 800-fold above background and originates exclusively from the unaffected father. We previously demonstrated high levels of both 755C>G and 755C>T FGFR2 mutations in human sperm and proposed that these particular mutations are enriched because the encoded proteins confer a selective advantage to spermatogonial cells. Here, we examine three corollaries of this hypothesis. First, we show that mutation levels at the adjacent FGFR2 nucleotides 752-754 are low, excluding any general increase in local mutation rate. Second, we present three instances of double-nucleotide changes involving 755C, expected to be extremely rare as chance events. Two of these double-nucleotide substitutions are shown, either by assessment of the pedigree or by direct analysis of sperm, to have arisen in sequential steps; the third (encoding Ser252Tyr) was predicted from structural considerations. Finally, we demonstrate that both major alternative spliceforms of FGFR2 (Fgfr2b and Fgfr2c) are expressed in rat spermatogonial stem cell lines. Taken together, these observations show that specific FGFR2 mutations attain high levels in sperm because they encode proteins with gain-of-function properties, favoring clonal expansion of mutant spermatogonial cells. Among FGFR2 mutations, those causing Apert syndrome may be especially prevalent because they enhance signaling by FGF ligands specific for each of the major expressed isoforms.

Acrocephalosyndactylia↗
PubMed1997

Mutations in the alpha1 subunit of an L-type voltage-activated Ca2+ channel cause myotonia in Caenorhabditis elegans.

The control of excitable cell action potentials is central to animal behavior. We show that the egl-19 gene plays a pivotal role in regulating muscle excitation and contraction in the nematode Caenorhabditis elegans and encodes the alphal subunit of a homologue of vertebrate L-type voltage-activated Ca2+ channels. Semi-dominant, gain-of-function mutations in egl-19 cause myotonia: mutant muscle action potentials are prolonged and the relaxation delayed. Partial loss-of-function mutations cause slow muscle depolarization and feeble contraction. The most severe loss-of-function mutants lack muscle contraction and die as embryos. We localized two myotonic mutations in the sixth membrane-spanning domain of the first repeat (IS6) region, which has been shown to be responsible for voltage-dependent inactivation. A third myotonic mutation implicates IIIS4, a region involved in sensing plasma-membrane voltage change, in the inactivation process.

Action Potentials↗
PubMed2001

Muscular degeneration in the absence of dystrophin is a calcium-dependent process.

Duchenne muscular dystrophy (DMD) is a progressive degenerative muscular disease that is due to mutations in the dystrophin gene. Neither the function of dystrophin nor the physiopathology of the disease have been clearly established yet. Several groups have reported elevated calcium concentrations in the mdx mouse model of DMD, but the effect of calcium levels on the progression of the disease continues to be a matter of debate. Here, we show that, in Caenorhabditis elegans, a gain-of-function mutation in the egl-19 calcium channel gene dramatically increases muscle degeneration in dystrophin mutants. Conversely, RNAi-mediated inhibition of egl-19 function reduces muscle degeneration by half. Therefore, our results demonstrate that calcium channel activity is a critical factor in the progression of dystrophin-dependent muscle degeneration.

Animals↗
PubMed1991

A functional and structural analysis of the Sex combs reduced locus of Drosophila melanogaster.

We have undertaken a developmental genetic analysis of the homeotic gene Sex combs reduced (Scr) of Drosophila melanogaster by examining embryonic and adult phenotypes of mutations affecting Scr gene function. Molecular mapping of Scr breakpoint lesions has defined a segment of greater than 70 kb of DNA necessary for proper Scr gene function. This region is split by the fushi tarazu (ftz) gene, with lesions affecting embryonic Scr function molecularly mapping to the region proximal (5') to ftz and those exhibiting polyphasic semilethality predominantly mapping distal (3') to ftz. Gain-of-function mutations are associated with genomic rearrangements and map throughout the Scr locus. Our analysis has revealed that the Scr locus encompasses genetic elements that are responsible for functions in both the embryonic and larval to adult periods of development. From these studies, we conclude that Scr is a complex genetic locus with an extensive regulatory region that directs functions required for normal head and thoracic development in both the embryo and the adult and that the regulation of Scr during these two periods is distinct.

Alleles↗
PubMed1992

Unraveling the knots in plant development.

Homeobox genes, first discovered from studies of homeotic mutations in Drosophila, have recently been found in plants. The proteins encoded by homeobox genes thus join the ranks of other animal transcription factors that have plant developmental counterparts, suggesting that even though plant and animal development are very different, regulatory mechanisms that direct development may be shared among all higher eukaryotes. The role of homeobox genes in plants remains elusive; nonetheless, gain-of-function mutations of one homeobox gene, Knotted, profoundly affect development. The phenotype suggests that ectopic expression of Knotted in leaves causes cells to take on alternative fates.

Animals↗
PubMed2001

A point mutation in the cysteine-rich domain of glycoprotein (GP) IIIa results in the expression of a GPIIb-IIIa (alphaIIbbeta3) integrin receptor locked in a high-affinity state and a Glanzmann thrombasthenia-like phenotype.

This article reports a Glanzmann thrombasthenia (GT) patient, N.M., with a point mutation in the third cysteine-rich repeat of beta3-integrin or platelet glycoprotein (GP) IIIa, leading to the expression of a constitutively activated fibrinogen receptor. The diagnosis of GT was based on a severely reduced platelet-aggregation response to a series of agonists and approximately 20% of surface-expressed GPIIb-IIIa. The patient's GPIIb-IIIa constitutively expressed epitopes recognized by antibodies to ligand-induced binding sites (LIBS) and also spontaneously bound the fibrinogen-mimetic antibody, PAC-1. Furthermore, significant amounts of bound fibrinogen were detected on his platelets ex vivo. No signs of platelet activation were observed on sections of unstimulated platelets from N.M. by electron microscopy. Immunogold labeling highlighted the presence of surface-bound fibrinogen but revealed platelet heterogeneity with regard to the surface density. When the patient's platelets were stimulated by thrombin-receptor activating peptide, amounts of surface-expressed GPIIb-IIIa increased and the aggregation response improved, although it failed to normalize. Platelets from N.M. were able to adhere and spread on immobilized fibrinogen. Sequence analysis of genomic DNA from N.M. revealed a homozygous g1776T>C mutation in GPIIIa, leading to a Cys560Arg amino acid substitution. A stable Chinese hamster ovary (CHO) cell line was prepared expressing surface GPIIb-Arg560IIIa. Like platelets from the patient, GPIIb-Arg560IIIa-transfected CHO cells constitutively bound LIBS antibodies and PAC-1. They also showed an enhanced ability to adhere on surface-bound fibrinogen. Overall, these data demonstrate that a gain-of-function mutation can still be associated with a thrombasthenic phenotype even though platelets show spontaneous fibrinogen binding.

Adult↗
PubMed1998

Severe B cell deficiency and disrupted splenic architecture in transgenic mice expressing the E41K mutated form of Bruton's tyrosine kinase.

To identify B-cell signaling pathways activated by Bruton's tyrosine kinase (Btk) in vivo, we generated transgenic mice in which Btk expression is driven by the MHC class II Ea gene locus control region. Btk overexpression did not have significant adverse effects on B cell function, and essentially corrected the X-linked immunodeficiency (xid) phenotype in Btk- mice. In contrast, expression of a constitutively activated form of Btk carrying the E41K gain-of-function mutation resulted in a B cell defect that was more severe than xid. The mice showed a marked reduction of the B cell compartment in spleen, lymph nodes, peripheral blood and peritoneal cavity. The levels in the serum of most immunoglobulin subclasses decreased with age, and B cell responses to both T cell-independent type II and T cell-dependent antigens were essentially absent. Expression of the E41K Btk mutant enhanced blast formation of splenic B cells in vitro in response to anti-IgM stimulation. Furthermore, the mice manifested a disorganization of B cell areas and marginal zones in the spleen. Our findings demonstrate that expression of constitutively activated Btk blocks the development of follicular recirculating B cells.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1998

Myelin-associated glycoprotein is a myelin signal that modulates the caliber of myelinated axons.

Myelination increases neuronal conduction velocity through its insulating properties and an unidentified extrinsic effect that increases axonal caliber. Although it is well established that demyelination can cause axonal atrophy, the myelin molecule that regulates axonal caliber is not known. Loss of the structural proteins of compact peripheral nervous system (PNS) myelin, P0 protein, and myelin basic protein does not lead to axonal atrophy. This study demonstrates that mice with a null mutation of the myelin-associated glycoprotein (MAG) gene have a chronic atrophy of myelinated PNS axons that results in paranodal myelin tomaculi and axonal degeneration. Absence of MAG was correlated with reduced axonal calibers, decreased neurofilament spacing, and reduced neurofilament phosphorylation. Because axonal atrophy and degeneration in MAG-deficient mice occur in the absence of inflammation, hypomyelination, significant demyelination-remyelination, or gain of function mutations, these data support a functional role for MAG in modulating the maturation and viability of myelinated axons.

Animals↗
PubMed2006

Molecular and biochemical analyses of platelet-derived growth factor receptor (PDGFR) B, PDGFRA, and KIT receptors in chordomas.

PURPOSE: We have previously shown the presence of an activated platelet-derived growth factor (PDGF) receptor (PDGFR) B and its ligand PDGFB in a limited number of patients with clinical and radiological responses to imatinib mesylate treatment. This article describes the results of comprehensive molecular/biochemical analyses of the three receptors targeted by the drug (PDGFRB, PDGFRA, and KIT) in a series of 31 chordoma patients. EXPERIMENTAL DESIGN: The presence and activation status of PDGFRB, PDGFRA, and KIT receptors were investigated by means of immunoprecipitation and Western blot analyses complemented by immunohistochemistry, their expression level was analyzed by means of real-time PCR, and the occurrence of activating point mutations was investigated by means of cDNA sequencing. The PDGFB, PDGFA, and stem cell factor cognate ligands were investigated by reverse transcription-PCR, and gene status was assessed by fluorescence in situ hybridization. RESULTS: The results show that PDGFRB was highly expressed and phosphorylated, whereas PDGFRA and KIT were less expressed but phosphorylated and thus activated. These findings, together with the absence of gain-of-function mutations and the presence of the cognate ligands, strongly support the hypothesis that the activation mechanism is the autocrine/paracrine loop. No role seems to be played by gene amplification. CONCLUSIONS: In the light of our findings, the clinical benefit observed in chordoma patients treated with imatinib seems to be attributable to the switching off of all three receptors.

Adult↗
PubMed2004

Mutational analysis of ABCG2: role of the GXXXG motif.

ABCG2 (BCRP/MXR/ABCP) is a half-transporter associated with multidrug resistance that presumably homodimerizes for function. It has a conserved GXXXG motif in its first transmembrane segment, a motif that has been linked with dimerization in other proteins, e.g., glycophorin A. We substituted either or both glycines of this GXXXG motif with leucines to evaluate the impact on drug transport, ATP hydrolysis, cross-linking, and susceptibility to degradation. All mutants also carried the R482G gain-of-function mutation, and all migrated to the cell surface. The mutations resulted in lost transport for rhodamine 123 and impaired mitoxantrone, pheophorbide a, and BODIPY-prazosin transport, particularly in the double leucine mutant (G406L/G410L). Basal ATPase activity of the G406L/G410L mutant was comparable to the empty vector transfected cells with no substrate induction. Despite impaired function, the mutants retained susceptibility to cross-linking using either disuccinimidyl suberate (DSS) or the reducible dithiobis(succinimidyl propionate) (DSP) and demonstrated a high molecular weight complex under nonreducing conditions. Mutations to alanine at the same positions yielded fully functional transporters. Finally, we exposed cells to mitoxantrone to promote folding and processing of the mutant proteins, which in the leucine mutants resulted in increased amounts detected on immunoblot and by immunofluorescence. These studies support a hypothesis that the GXXXG motif promotes proper packing of the transmembrane segments in the functional ABCG2 homodimer, although it does not solely arbitrate dimerization.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2004

Crystal structure of the wild-type von Willebrand factor A1-glycoprotein Ibalpha complex reveals conformation differences with a complex bearing von Willebrand disease mutations.

The adhesion of platelets to the subendothelium of blood vessels at sites of vascular injury under high shear conditions is mediated by a direct interaction between the platelet receptor glycoprotein Ibalpha (GpIbalpha) and the A1 domain of the von Willebrand factor (VWF). Here we report the 2.6-A crystal structure of a complex comprised of the extracellular domain of GpIbalpha and the wild-type A1 domain of VWF. A direct comparison of this structure to a GpIbalpha-A1 complex containing "gain-of-function" mutations, A1-R543Q and GpIbalpha-M239V, reveals specific structural differences between these complexes at sites near the two GpIbalpha-A1 binding interfaces. At the smaller interface, differences in interaction show that the alpha1-beta2 loop of A1 serves as a conformational switch, alternating between an open alpha1-beta2 isomer that allows faster dissociation of GpIbalpha-A1, as observed in the wild-type complex, and an extended isomer that favors tight association as seen in the complex containing A1 with a type 2B von Willebrand Disease (VWD) mutation associated with spontaneous binding to GpIbalpha. At the larger interface, differences in interaction associated with the GpIbalpha-M239V platelet-type VWD mutation are minor and localized but feature discrete gamma-turn conformers at the loop end of the beta-hairpin structure. The beta-hairpin, stabilized by a strong classic gamma-turn as seen in the mutant complex, relates to the increased affinity of A1 binding, and the beta-hairpin with a weak inverse gamma-turn observed in the wild-type complex corresponds to the lower affinity state of GpIbalpha. These findings provide important details that add to our understanding of how both type 2B and platelet-type VWD mutations affect GpIbalpha-A1 binding affinity.

Binding Sites↗
PubMed1999

A mutation in the C. elegans EXP-2 potassium channel that alters feeding behavior.

The nematode pharynx has a potassium channel with unusual properties, which allows the muscles to repolarize quickly and with the proper delay. Here, the Caenorhabditis elegans exp-2 gene is shown to encode this channel. EXP-2 is a Kv-type (voltage-activated) potassium channel that has inward-rectifying properties resembling those of the structurally dissimilar human ether-à-go-go-related gene (HERG) channel. Null and gain-of-function mutations affect pharyngeal muscle excitability in ways that are consistent with the electrophysiological behavior of the channel, and thereby demonstrate a direct link between the kinetics of this unusual channel and behavior.

Action Potentials↗
PubMed1997

Clustered amino acid substitutions in the yeast transcription regulator Pdr3p increase pleiotropic drug resistance and identify a new central regulatory domain.

In the yeast Saccharomyces cerevisiae mutations in the genes encoding the transcription factors Pdr1p and Pdr3p are known to be associated with pleiotropic drug resistance mediated by the overexpression of the efflux pumps Pdr5p, Snq2p, and Yor1p. Mutagenesis of PDR3 was used to induce multidrug resistance phenotypes and independent pdr3 mutants were isolated and characterized. DNA sequence analysis revealed seven different pdr3 alleles with mutations in the N-terminal region of PDR3. The pdr3 mutants were semidominant and conferred different drug resistance patterns on host strains deleted either for PDR3 or for PDR3 and PDR1. Transactivation experiments proved that the mutated forms of Pdr3p induced increased activation of the PDR3, PDR5, and SNQ2 promoters. The amino acid changes encoded by five pdr3 mutant alleles were found to occur in a short protein segment (amino acids 252-280), thus revealing a regulatory domain. This region may play an important role in protein-DNA or protein-protein interactions during activation by Pdr3p. Moreover, this hot spot for gain-of-function mutations overlaps two structural motifs, MI and MII, recently proposed to be conserved in the large family of Zn2Cys6 transcription factors.

ATP-Binding Cassette Transporters↗