Development and application of loop-mediated isothermal amplification for detection of the F167Y mutation of carbendazim-resistant isolates in Fusarium graminearum

PubMed Central

Duan, Yabing; Zhang, Xiaoke; Ge, Changyan; Wang, Yong; Cao, Junhong; Jia, Xiaojing; Wang, Jianxin; Zhou, Mingguo

2014-01-01

Resistance of Fusarium graminearum to carbendazim is caused by point mutations in the β2-tubulin gene. The point mutation at codon 167 (TTT → TAT, F167Y) occurs in more than 90% of field resistant isolates in China. To establish a suitable method for rapid detection of the F167Y mutation in F. graminearum, an efficient and simple method with high specificity was developed based on loop-mediated isothermal amplification (LAMP). A set of four primers was designed and optimized to specially distinguish the F167Y mutation genotype. The LAMP reaction was optimal at 63°C for 60 min. When hydroxynaphthol blue dye (HNB) was added prior to amplification, samples with DNA of the F167Y mutation developed a characteristic sky blue color after the reaction but those without DNA or with different DNA did not. Results of HNB staining method were reconfirmed by gel electrophoresis. The developed LAMP had good specificity, stability and repeatability and was suitable for monitoring carbendazim-resistance populations of F. graminearum in agricultural production. PMID:25403277

Early-onset severe hereditary sensory and autonomic neuropathy type 1 with S331F SPTLC1 mutation.

PubMed

Suh, Bum Chun; Hong, Young Bin; Nakhro, Khriezhanuo; Nam, Soo Hyun; Chung, Ki Wha; Choi, Byung-Ok

2014-02-01

Hereditary sensory and autonomic neuropathy type I (HSAN I) is an autosomal dominant disease characterized by prominent sensory impairment, resulting in foot ulcers or amputations and has a juvenile to adult onset. The major underlying causes of HSAN I are mutations in SPTLC1, which encodes the first subunit of serine palmitoyltransferase (SPT). To date, there have been no reports with regard to an HSAN patient of Korean origin. In this report we discussed an HSAN I patient with a missense mutation in SPTLC1 (c.992C>T: p.S331F). The patient had noticed frequent falls, lower leg weakness and hand tremors at age five. The patient also presented with foot ulcers, muscle hypotrophy, cataracts, hoarseness, vocal cord palsy and respiratory difficulties and succumbed to the condition at the age of 28 years. In accordance with previous reports, a mutation in Ser331 in the present patient was associated with early-onset and a severe phenotype. Therefore, Ser331 in SPTLC1 is a crucial amino acid, which characterizes the HSAN I phenotype.

A mouse model for the cystic fibrosis delta F508 mutation.

PubMed Central

van Doorninck, J H; French, P J; Verbeek, E; Peters, R H; Morreau, H; Bijman, J; Scholte, B J

1995-01-01

Most cystic fibrosis (CF) patients produce a mutant form (delta F508) of the cystic fibrosis transmembrane conductance regulator (CFTR), which is not properly processed in normal cells but is active as a chloride channel in several experimental systems. We used a double homologous recombination ('Hit and Run') procedure to generate a mouse model for the delta F508 mutation. Targeted embryonic stem (ES) cells (Hit clones) were found; of these either 80 or 20% of the clones had lost the delta F508 mutation, depending on the distance between the linearization site in the targeting construct and the delta F508 mutation. Correctly targeted clones underwent a second selection step resulting in ES cell clones (Run clones) heterozygous for the delta F508 mutation with an efficiency of 2-7%. Chimeric mice were generated and offspring homozygous for the delta F508 mutation showed electrophysiological abnormalities in nasal epithelium, gallbladder and in the intestine, and histological abnormalities in the intestine, typical of CF. Our data suggest that the delta F508 mice have residual delta F508 CFTR activity which would explain the mild pathology of the delta F508 mice. The delta F508 mouse may provide a useful model for the study of the processing defect of delta F508 CFTR and for the development of novel therapeutic approaches based on circumvention of the processing block. Images PMID:7556083

Actomyosin kinetics and in vitro motility of wild-type Drosophila actin and the effects of two mutations in the Act88F gene.

PubMed Central

Anson, M; Drummond, D R; Geeves, M A; Hennessey, E S; Ritchie, M D; Sparrow, J C

1995-01-01

Two missense mutations of the flight muscle-specific actin gene of Drosophila melanogaster, Act88F, assemble into normally structured myofibrils but affect the flight ability of flies and the mechanical kinetics of isolated muscle fibers. We describe the isolation of actin from different homozygous Act88F strains, including wild-type, an Act88F null mutant (KM88), and two Act88F single point mutations (E316K and G368E), their biochemical interactions with rabbit myosin subfragment 1 (S1), and behavior with rabbit myosin and heavy meromyosin in in vitro motility assays. The rabbit and wild-type Drosophila actins have different association rate constants with S1 (2.64 and 1.77 microM-1 s-1, respectively) and in vitro motilities (2.51, 1.60 microns s-1) clearly demonstrating an isoform-specific difference. The G368E mutation shows a reduced affinity for rabbit S1 compared with the wild type (increasing from 0.11 to 0.17 microM) and a reduced velocity in vitro (reduced by 19%). The E316K mutant actin has no change in affinity for myosin S1 or in vitro motility with heavy meromyosin but does have a reduced in vitro motility (15%) with myosin. These results are discussed with respect to the recently published atomic models for the actomyosin structure and our findings that G368E fibers show a reduced rate constant for delayed tension development and increased fiber stiffness. We interpret these results as possibly caused either by effects on A1 myosin light chain binding or conformational changes within the subdomain 1 of actin, which contains the myosin binding site. E316K is discussed with respect to its likely position within the tropomyosin binding site of actin. Images FIGURE 1 FIGURE 9 PMID:7612841

A pathogenic S250F missense mutation results in a mouse model of mild aromatic l-amino acid decarboxylase (AADC) deficiency.

PubMed

Caine, Charlotte; Shohat, Meytal; Kim, Jeong-Ki; Nakanishi, Koki; Homma, Shunichi; Mosharov, Eugene V; Monani, Umrao R

2017-11-15

Homozygous mutations in the aromatic l-amino acid decarboxylase (AADC) gene result in a severe depletion of its namesake protein, triggering a debilitating and often fatal form of infantile Parkinsonism known as AADC deficiency. AADC deficient patients fail to produce normal levels of the monoamine neurotransmitters dopamine and serotonin, and suffer a multi-systemic disorder characterized by movement abnormalities, developmental delay and autonomic dysfunction; an absolute loss of dopamine is generally considered incompatible with life. There is no optimal treatment for AADC deficiency and few truly good models in which to investigate disease mechanisms or develop and refine therapeutic strategies. In this study, we introduced a relatively frequently reported but mildly pathogenic S250F missense mutation into the murine Aadc gene. We show that mutants homozygous for the mutation are viable and express a stable but minimally active form of the AADC protein. Although the low enzymatic activity of the protein resulted in only modestly reduced concentrations of brain dopamine, serotonin levels were markedly diminished, and this perturbed behavior as well as autonomic function in mutant mice. Still, we found no evidence of morphologic abnormalities of the dopaminergic cells in mutant brains. The striatum as well as substantia nigra appeared normal and no loss of dopamine expressing cells in the latter was detected. We conclude that even minute levels of active AADC are sufficient to allow for substantial amounts of dopamine to be produced in model mice harboring the S250F mutation. Such mutants represent a novel, mild model of human AADC deficiency. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Memory Performance and fMRI Signal in Presymptomatic Familial Alzheimer’s Disease

PubMed Central

Braskie, Meredith N.; Medina, Luis D.; Rodriguez-Agudelo, Yaneth; Geschwind, Daniel H.; Macias-Islas, Miguel Angel; Thompson, Paul M.; Cummings, Jeffrey L.; Bookheimer, Susan Y.; Ringman, John M.

2013-01-01

Rare autosomal dominant mutations result in familial Alzheimer’s disease (FAD) with a relatively consistent age of onset within families. This provides an estimate of years until disease onset (relative age) in mutation carriers. Increased AD risk has been associated with differences in functional magnetic resonance imaging (fMRI) activity during memory tasks, but most of these studies have focused on possession of apolipoprotein E allele 4 (APOE4), a risk factor, but not causative variant, of late-onset AD. Evaluation of fMRI activity in presymptomatic FAD mutation carriers versus noncarriers provides insight into preclinical changes in those who will certainly develop AD in a prescribed period of time. Adults from FAD mutation-carrying families (nine mutation carriers, eight noncarriers) underwent fMRI scanning while performing a memory task. We examined fMRI signal differences between carriers and noncarriers, and how signal related to fMRI task performance within mutation status group, controlling for relative age and education. Mutation noncarriers had greater retrieval period activity than carriers in several AD-relevant regions, including the left hippocampus. Better performing noncarriers showed greater encoding period activity including in the parahippocampal gyrus. Poorer performing carriers showed greater retrieval period signal, including in the frontal and temporal lobes, suggesting underlying pathological processes. PMID:22806961

Short-Rib Polydactyly and Jeune Syndromes Are Caused by Mutations in WDR60

PubMed Central

McInerney-Leo, Aideen M.; Schmidts, Miriam; Cortés, Claudio R.; Leo, Paul J.; Gener, Blanca; Courtney, Andrew D.; Gardiner, Brooke; Harris, Jessica A.; Lu, Yeping; Marshall, Mhairi; Scambler, Peter J.; Beales, Philip L.; Brown, Matthew A.; Zankl, Andreas; Mitchison, Hannah M.; Duncan, Emma L.; Wicking, Carol

2013-01-01

Short-rib polydactyly syndromes (SRPS I–V) are a group of lethal congenital disorders characterized by shortening of the ribs and long bones, polydactyly, and a range of extraskeletal phenotypes. A number of other disorders in this grouping, including Jeune and Ellis-van Creveld syndromes, have an overlapping but generally milder phenotype. Collectively, these short-rib dysplasias (with or without polydactyly) share a common underlying defect in primary cilium function and form a subset of the ciliopathy disease spectrum. By using whole-exome capture and massive parallel sequencing of DNA from an affected Australian individual with SRPS type III, we detected two novel heterozygous mutations in WDR60, a relatively uncharacterized gene. These mutations segregated appropriately in the unaffected parents and another affected family member, confirming compound heterozygosity, and both were predicted to have a damaging effect on the protein. Analysis of an additional 54 skeletal ciliopathy exomes identified compound heterozygous mutations in WDR60 in a Spanish individual with Jeune syndrome of relatively mild presentation. Of note, these two families share one novel WDR60 missense mutation, although haplotype analysis suggested no shared ancestry. We further show that WDR60 localizes at the base of the primary cilium in wild-type human chondrocytes, and analysis of fibroblasts from affected individuals revealed a defect in ciliogenesis and aberrant accumulation of the GLI2 transcription factor at the centrosome or basal body in the absence of an obvious axoneme. These findings show that WDR60 mutations can cause skeletal ciliopathies and suggest a role for WDR60 in ciliogenesis. PMID:23910462

F104S c-Mpl responds to a transmembrane domain-binding thrombopoietin receptor agonist: proof of concept that selected receptor mutations in congenital amegakaryocytic thrombocytopenia can be stimulated with alternative thrombopoietic agents.

PubMed

Fox, Norma E; Lim, Jihyang; Chen, Rose; Geddis, Amy E

2010-05-01

To determine whether specific c-Mpl mutations might respond to thrombopoietin receptor agonists. We created cell line models of type II c-Mpl mutations identified in congenital amegakaryocytic thrombocytopenia. We selected F104S c-Mpl for further study because it exhibited surface expression of the receptor. We measured proliferation of cell lines expressing wild-type or F104S c-Mpl in response to thrombopoietin receptor agonists targeting the extracellular (m-AMP4) or transmembrane (LGD-4665) domains of the receptor by 1-methyltetrazole-5-thiol assay. We measured thrombopoietin binding to the mutant receptor using an in vitro thrombopoietin uptake assay and identified F104 as a potentially critical residue for the interaction between the receptor and its ligand by aligning thrombopoietin and erythropoietin receptors from multiple species. Cells expressing F104S c-Mpl proliferated in response to LGD-4665, but not thrombopoietin or m-AMP4. Compared to thrombopoietin, LGD-4665 stimulates signaling with delayed kinetics in both wild-type and F104S c-Mpl-expressing cells. Although F104S c-Mpl is expressed on the cell surface in our BaF3 cell line model, the mutant receptor does not bind thrombopoietin. Comparison to the erythropoietin receptor suggests that F104 engages in hydrogen-bonding interactions that are critical for binding to thrombopoietin. These findings suggest that a small subset of patients with congenital amegakaryocytic thrombocytopenia might respond to treatment with thrombopoietin receptor agonists, but that responsiveness will depend on the type of mutation and agonist used. We postulate that F104 is critical for thrombopoietin binding. The kinetics of signaling in response to a transmembrane domain-binding agonist are delayed in comparison to thrombopoietin. 2010 ISEH Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

S737F is a new CFTR mutation typical of patients originally from the Tuscany region in Italy.

PubMed

Terlizzi, Vito; Di Lullo, Antonella Miriam; Comegna, Marika; Centrone, Claudia; Pelo, Elisabetta; Castaldo, Giuseppe; Raia, Valeria; Braggion, Cesare

2018-01-03

An increasing number of patients have been described as having a number of Cystic Fibrosis Transmembrane conductance Regulator (CFTR) variants for which it lacks a clear genotype-phenotype correlation. We assesses the clinical features of patients bearing the S737F (p.Ser737Phe) CFTR missense variant and evaluated the residual function of CFTR protein on nasal epithelial cells (NEC). A retrospective database was performed from individuals homozygous or compound heterozygous for the S737F variant followed in the Cystic Fibrosis (CF) Centre of Florence. We performed a nasal brushing in cooperating patients and compared the results with those of patients followed in the pediatric CF Centre of Naples. 9/295 (3%) subjects carrying at least S737F CFTR variant on one allele were identified. Patients were diagnosed in 7/9 cases by newborn screening and in two cases for dehydration with hypochloremic metabolic alkalosis; at diagnosis sweat chloride levels (SCL) were in the pathological range in only one case. After a mean follow up of 8,6 years (range 0,5-15,8), SCL were in the pathological range in 8/9 cases (mean age at CF diagnosis: 1,5 years), all patients were pancreatic sufficiency and respiratory function was normal. The gating activity on NEC was 15.6% and 12.7% in two patients compound heterozygous for W1282X and DelE22_24, while it was ranged between 6,2% and 9,8% in CF patients. S737F is a CFTR mutation associated to hypochloremic alkalosis in childhood, mild CF phenotype in teenage years and a residual function of CFTR protein.

Short-rib polydactyly and Jeune syndromes are caused by mutations in WDR60.

PubMed

McInerney-Leo, Aideen M; Schmidts, Miriam; Cortés, Claudio R; Leo, Paul J; Gener, Blanca; Courtney, Andrew D; Gardiner, Brooke; Harris, Jessica A; Lu, Yeping; Marshall, Mhairi; Scambler, Peter J; Beales, Philip L; Brown, Matthew A; Zankl, Andreas; Mitchison, Hannah M; Duncan, Emma L; Wicking, Carol

2013-09-05

Short-rib polydactyly syndromes (SRPS I-V) are a group of lethal congenital disorders characterized by shortening of the ribs and long bones, polydactyly, and a range of extraskeletal phenotypes. A number of other disorders in this grouping, including Jeune and Ellis-van Creveld syndromes, have an overlapping but generally milder phenotype. Collectively, these short-rib dysplasias (with or without polydactyly) share a common underlying defect in primary cilium function and form a subset of the ciliopathy disease spectrum. By using whole-exome capture and massive parallel sequencing of DNA from an affected Australian individual with SRPS type III, we detected two novel heterozygous mutations in WDR60, a relatively uncharacterized gene. These mutations segregated appropriately in the unaffected parents and another affected family member, confirming compound heterozygosity, and both were predicted to have a damaging effect on the protein. Analysis of an additional 54 skeletal ciliopathy exomes identified compound heterozygous mutations in WDR60 in a Spanish individual with Jeune syndrome of relatively mild presentation. Of note, these two families share one novel WDR60 missense mutation, although haplotype analysis suggested no shared ancestry. We further show that WDR60 localizes at the base of the primary cilium in wild-type human chondrocytes, and analysis of fibroblasts from affected individuals revealed a defect in ciliogenesis and aberrant accumulation of the GLI2 transcription factor at the centrosome or basal body in the absence of an obvious axoneme. These findings show that WDR60 mutations can cause skeletal ciliopathies and suggest a role for WDR60 in ciliogenesis. Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Further Insights into the Allan-Herndon-Dudley Syndrome: Clinical and Functional Characterization of a Novel MCT8 Mutation.

PubMed

Armour, Christine M; Kersseboom, Simone; Yoon, Grace; Visser, Theo J

2015-01-01

Mutations in the thyroid hormone (TH) transporter MCT8 have been identified as the cause for Allan-Herndon-Dudley Syndrome (AHDS), characterized by severe psychomotor retardation and altered TH serum levels. Here we report a novel MCT8 mutation identified in 4 generations of one family, and its functional characterization. Proband and family members were screened for 60 genes involved in X-linked cognitive impairment and the MCT8 mutation was confirmed. Functional consequences of MCT8 mutations were studied by analysis of [125I]TH transport in fibroblasts and transiently transfected JEG3 and COS1 cells, and by subcellular localization of the transporter. The proband and a male cousin demonstrated clinical findings characteristic of AHDS. Serum analysis showed high T3, low rT3, and normal T4 and TSH levels in the proband. A MCT8 mutation (c.869C>T; p.S290F) was identified in the proband, his cousin, and several female carriers. Functional analysis of the S290F mutant showed decreased TH transport, metabolism and protein expression in the three cell types, whereas the S290A mutation had no effect. Interestingly, both uptake and efflux of T3 and T4 was impaired in fibroblasts of the proband, compared to his healthy brother. However, no effect of the S290F mutation was observed on TH efflux from COS1 and JEG3 cells. Immunocytochemistry showed plasma membrane localization of wild-type MCT8 and the S290A and S290F mutants in JEG3 cells. We describe a novel MCT8 mutation (S290F) in 4 generations of a family with Allan-Herndon-Dudley Syndrome. Functional analysis demonstrates loss-of-function of the MCT8 transporter. Furthermore, our results indicate that the function of the S290F mutant is dependent on cell context. Comparison of the S290F and S290A mutants indicates that it is not the loss of Ser but its substitution with Phe, which leads to S290F dysfunction.

IDH mutation is paradoxically associated with higher 18F-FDOPA PET uptake in diffuse grade II and grade III gliomas.

PubMed

Verger, A; Metellus, Ph; Sala, Q; Colin, C; Bialecki, E; Taieb, D; Chinot, O; Figarella-Branger, D; Guedj, E

2017-08-01

The World Health Organization Classification of Tumors of the Central Nervous System has recently been updated by the integration of diagnostic and prognostic molecular parameters, giving pivotal attention to IDH mutation as a favourable factor. Amino acid PET is increasingly used in the management of gliomas, but its prognostic value is a matter of debate. The aim of this study was to assess the relationship between IDH mutation and 18 F-FDOPA uptake on PET in newly diagnosed gliomas. A total of 43 patients, presenting with diffuse astrocytic and oligodendroglial grade II and III gliomas, reclassified according to the 2016 WHO classification of tumours of the CNS, were retrospectively included. They had all undergone 18 F-FDOPA PET at an initial stage before surgery and histological diagnosis. 18 F-FDOPA uptake values were compared between patients with and without IDH mutation in terms of maximum standardized uptake value (SUVmax) ratios between tumour and normal contralateral brain (T/N), and between tumour and striatum (T/S). Patients with IDH mutation showed higher 18 F-FDOPA T/N SUVmax ratios (1.6 vs. 1.2) and T/S SUVmax ratios (0.9 vs. 0.6) than patients without IDH mutation (p < 0.05). This study showed paradoxically higher 18 F-FDOPA uptake in diffuse grade II and III gliomas with IDH mutation. Despite evident interest in the management of gliomas, and especially in relation to posttherapy evaluation, our findings raise the question of the prognostic value of 18 F-FDOPA uptake on PET uptake in this group of patients. This may be related to differences in amino acid integration, metabolism, or cell differentiation.

F4-related mutation and expression analysis of the aminopeptidase N gene in pigs.

PubMed

Goetstouwers, T; Van Poucke, M; Nguyen, V U; Melkebeek, V; Coddens, A; Deforce, D; Cox, E; Peelman, L J

2014-05-01

Intestinal infections with F4 enterotoxigenic Escherichia coli (ETEC) are worldwide an important cause of diarrhea in neonatal and recently weaned pigs. Adherence of F4 ETEC to the small intestine by binding to specific receptors is mediated by F4 fimbriae. Porcine aminopeptidase N (ANPEP) was recently identified as a new F4 receptor. In this study, 7 coding mutations and 1 mutation in the 3' untranslated region (3' UTR)were identified in ANPEP by reverse transcriptase (RT-) PCR and sequencing using 3 F4 receptor-positive (F4R+) and 2 F4 receptor-negative (F4R-) pigs, which were F4 phenotyped based on the MUC4 TaqMan, oral immunization, and the in vitro villous adhesion assay. Three potential differential mutations (g.2615C > T, g.8214A > G, and g.16875C > G) identified by comparative analysis between the 3 F4R+ and 2 F4R- pigs were genotyped in 41 additional F4 phenotyped pigs. However, none of these 3 mutations could be associated with F4 ETEC susceptibility. In addition, the RT-PCR experiments did not reveal any differential expression or alternative splicing in the small intestine of F4R+ and F4R- pigs. In conclusion, we hypothesize that the difference in F4 binding to ANPEP is due to modifications in its carbohydrate moieties.

Resensitization to Crizotinib by the Lorlatinib ALK Resistance Mutation L1198F

PubMed Central

Shaw, Alice T.; Friboulet, Luc; Leshchiner, Ignaty; Gainor, Justin F.; Bergqvist, Simon; Brooun, Alexei; Burke, Benjamin J.; Deng, Ya-Li; Liu, Wei; Dardaei, Leila; Frias, Rosa L.; Schultz, Kate R.; Logan, Jennifer; James, Leonard P.; Smeal, Tod; Timofeevski, Sergei; Katayama, Ryohei; Iafrate, A. John; Le, Long; McTigue, Michele; Getz, Gad

2016-01-01

Summary In a patient who had metastatic anaplastic lymphoma kinase (ALK)-rearranged lung cancer, resistance to crizotinib developed because of a mutation in the ALK kinase domain. This mutation is predicted to result in a substitution of cysteine by tyrosine at amino acid residue 1156 (C1156Y). Her tumor did not respond to a second-generation ALK inhibitor, but it did respond to lorlatinib (PF-06463922), a third-generation inhibitor. When her tumor relapsed, sequencing of the resistant tumor revealed an ALK L1198F mutation in addition to the C1156Y mutation. The L1198F substitution confers resistance to lorlatinib through steric interference with drug binding. However, L1198F paradoxically enhances binding to crizotinib, negating the effect of C1156Y and resensitizing resistant cancers to crizotinib. The patient received crizotinib again, and her cancer-related symptoms and liver failure resolved. PMID:26698910

JAK2 (V617F) mutation is not associated with thrombosis in Behcet syndrome.

PubMed

Ar, M Cem; Hatemi, Gülen; Ekizoğlu, Seda; Bilgen, Hülya; Saçli, Sevgi; Buyru, A Nur; Soysal, Teoman; Ülkü, Birsen; Yazici, Hasan

2012-07-01

The Janus kinase 2(V617F) (JAK2 (V617F)) mutation is an acquired genetic defect that is considered to enhance thrombosis in Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). Thrombosis is also a well-defined component of Behcet syndrome (BS). The aim of this study was to determine the frequency of JAK2 ( V617F ) mutation in BS-associated thrombosis. A total of 152 patients with BS (62 with thrombosis and 90 without thrombosis) were enrolled. An additional 186 patients with MPNs and 107 healthy blood donors were included to serve as diseased and healthy controls, respectively. None of the patients with BS and healthy controls carried the JAK2 (V617F) mutation, whereas 67% of patients with MPNs were positive for JAK2 ( V617F ). The frequency of thrombosis in patients with MPNs was not statistically different between carriers and non-carriers of JAK2 ( V617F ) mutation. Our data suggest that JAK2 (V617F) is not directly related to thrombosis in MPNs and in other thrombotic entities, such as BS.

The association of JAK2V617F mutation and leukocytosis with thrombotic events in essential thrombocythemia.

PubMed

Hsiao, Hui-Hua; Yang, Ming-Yu; Liu, Yi-Chang; Lee, Ching-Ping; Yang, Wen-Chi; Liu, Ta-Chih; Chang, Chao-Sung; Lin, Sheng-Fung

2007-11-01

The Janus kinase 2 mutation, JAK2 (V617F), and megakaryocytic mutations, MPL (W515L/K), have been identified and correlated with a subtype of essential thrombocythemia (ET) patients. We investigated the frequency of mutations in ET patients and analyzed the relationship with their clinical features. Fifty-three ET patients were enrolled in the study. The amplification refractory mutation system was applied for the mutation survey of the JAK2V617F, while the polymerase chain reaction with sequencing was used for the mutation survey of MPLW515L/K. Thirty-five (66%) patients harboring the JAK2 (V617F) mutation, including 3 homozygous and 32 heterozygous changes, but no MPLW515L/K mutation, were found. During follow-up, 17 (32.1%) patients suffered from documented thrombotic events, with 15 having JAK2V617F mutations. Statistical analysis showed that patients with the JAK2 mutation had significantly higher leukocytes, hemoglobin level, and thrombotic event (p = 0.043, p = 0.001, and p = 0.029, respectively). Thrombotic events were also significantly correlated with leukocytosis and older age. The JAK2V617F mutation was noted in a certain population of ET patients and correlated with leukocytosis, high hemoglobin level, and thrombosis. Therefore, detection of the JAK2V617F mutation can affect not only the diagnosis, but also the management of ET patients.

ALK F1174V mutation confers sensitivity while ALK I1171 mutation confers resistance to alectinib. The importance of serial biopsy post progression.

PubMed

Ou, Sai-Hong; Milliken, Jeffrey C; Azada, Michele C; Miller, Vincent A; Ali, Siraj M; Klempner, Samuel J

2016-01-01

Many acquired resistant mutations to the anaplastic lymphoma kinase (ALK) gene have been identified during treatment of ALK-rearranged non-small cell lung cancer (NSCLC) patients with crizotinib, ceritinib, and alectinib. These various acquired resistant ALK mutations confer differential sensitivities to various ALK inhibitors and may provide guidance on how to sequence the use of many of the second generation ALK inhibitors. We described a patient who developed an acquired ALK F1174V resistant mutation on progression from crizotinib that responded to alectinib for 18 months but then developed an acquired ALK I1171S mutation to alectinib. Both tumor samples had essentially the same genomic profile by comprehensive genomic profiling otherwise. This is the first patient report that demonstrates ALK F1174V mutation is sensitive to alectinib and further confirms missense acquired ALK I1171 mutation is resistant to alectinib. Sequential tumor re-biopsy for comprehensive genomic profiling (CGP) is important to appreciate the selective pressure during treatment with various ALK inhibitors underpinning the evolution of the disease course of ALK+NSCLC patients while on treatment with the various ALK inhibitors. This approach will likely help inform the optimal sequencing strategy as more ALK inhibitors become available. This case report also validates the importance of developing structurally distinct ALK inhibitors for clinical use to overcome non-cross resistant ALK mutations. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Further Insights into the Allan-Herndon-Dudley Syndrome: Clinical and Functional Characterization of a Novel MCT8 Mutation

PubMed Central

Yoon, Grace; Visser, Theo J.

2015-01-01

Background Mutations in the thyroid hormone (TH) transporter MCT8 have been identified as the cause for Allan-Herndon-Dudley Syndrome (AHDS), characterized by severe psychomotor retardation and altered TH serum levels. Here we report a novel MCT8 mutation identified in 4 generations of one family, and its functional characterization. Methods Proband and family members were screened for 60 genes involved in X-linked cognitive impairment and the MCT8 mutation was confirmed. Functional consequences of MCT8 mutations were studied by analysis of [125I]TH transport in fibroblasts and transiently transfected JEG3 and COS1 cells, and by subcellular localization of the transporter. Results The proband and a male cousin demonstrated clinical findings characteristic of AHDS. Serum analysis showed high T3, low rT3, and normal T4 and TSH levels in the proband. A MCT8 mutation (c.869C>T; p.S290F) was identified in the proband, his cousin, and several female carriers. Functional analysis of the S290F mutant showed decreased TH transport, metabolism and protein expression in the three cell types, whereas the S290A mutation had no effect. Interestingly, both uptake and efflux of T3 and T4 was impaired in fibroblasts of the proband, compared to his healthy brother. However, no effect of the S290F mutation was observed on TH efflux from COS1 and JEG3 cells. Immunocytochemistry showed plasma membrane localization of wild-type MCT8 and the S290A and S290F mutants in JEG3 cells. Conclusions We describe a novel MCT8 mutation (S290F) in 4 generations of a family with Allan-Herndon-Dudley Syndrome. Functional analysis demonstrates loss-of-function of the MCT8 transporter. Furthermore, our results indicate that the function of the S290F mutant is dependent on cell context. Comparison of the S290F and S290A mutants indicates that it is not the loss of Ser but its substitution with Phe, which leads to S290F dysfunction. PMID:26426690

Frequency of the S65C mutation in the hemochromatosis gene in Brazil.

PubMed

Oliveira, V C; Caxito, F A; Gomes, K B; Castro, A M; Pardini, V C; Ferreira, A C S

2009-07-14

Development of hereditary hemochromatosis is associated with the C282Y, H63D or S65C mutations in the hemochromatosis gene. Though there is extensive knowledge about the former two, there is little information on the mechanism of action and the allelic frequency of the S65C mutation. We examined the prevalence of the S65C mutation of the hemochromatosis gene in Brazilians with clinical suspicion of hereditary hemochromatosis. Genotyping for this mutation was carried out in 633 individuals with clinical suspicion of hereditary hemochromatosis, using the polymerase chain reaction, followed by enzymatic digestion. The sample comprised 77.1% men and 22.9% women, giving a ratio of approximately 3:1; the mean age was 48.8 +/- 13.8 years. More than half (57.3%) of the individuals in the sample were 41 to 60 years old. The frequency of heterozygotes for this mutation was 0.016; no homozygous mutant patients were found. This is the first analysis of the S65C mutation in individuals suspected of having hereditary hemochromatosis in Brazil.

Missense variations in the cystic fibrosis gene: Heteroduplex formation in the F508C mutation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Macek, M. Jr.; Ladanyi, L.; Buerger, J.

1992-11-01

Kobayashi et al. (1990) have described missense variations in the conserved region of exon 10 of the cystic fibrosis (CF) transmembrane conductance regulator gene. In their paper, two [Delta]F508/F508C compound heterozygous individuals were reported. Clinical and epithelial physiological studies in both cases were normal, suggesting that the substitution of cysteine for phenylalanine at position 508, the F508C mutation, is benign. However, Kerem et al. reported a patient with this substitution who had typical symptoms of CF. In routine [Delta]F508 mutation screening by visualization of the 3-bp deletion on a 12% polyacrylamide gel the authors detected an abnormal heteroduplex in themore » father of a CF patient of German origin. Subsequent direct sequencing of the PCR product confirmed that this clinically normal father is a compound heterozygote for the [Delta]F508/F508C mutations. This heteroduplex is slightly different from the usual heteroduplex in [Delta]F508/F508C heteroduplex was not published, it is likely that similar cases can be overseen during the widely performed [Delta]F508 mutation screening by PAGE. Detection of more cases, such as the one presented here, together with careful, standardized clinical examination of the proband, would be valuable to verify the nature of this mutation. 4 refs., 1 fig.« less

JAK2V617F mutation is associated with special alleles in essential thrombocythemia.

PubMed

Hsiao, Hui-Hua; Liu, Yi-Chang; Tsai, Hui-Jen; Lee, Ching-Ping; Hsu, Jui-Feng; Lin, Sheng-Fung

2011-03-01

Janus kinase 2 mutation (JAK2V617F) has been identified in myeloproliferative neoplasms. Furthermore, special single nucleoside polymorphisms (SNPs) have been found to be associated with the JAK2V617F mutation. Therefore, the associations among JAK2V617F and special SNPs and the allelic location between them were investigated in patients with essential thrombocythemia (ET). A total of 61 patients with ET and 106 healthy individuals were enrolled. The PCR-RFLP method was applied to investigate the pattern of three SNPs, rs10974944, rs12343867, and rs12340895. Allele-specific PCR was used to examine the allelic location between rs10974944 and JAK2V617F. Among the patients with ET, 34 (55.7%, 34/61) were JAK2V617F positive (heterozygous) while the other 27 (44.3%, 27/61) were negative, and there were no MPLW515L/K mutations noted. The pattern of special SNPs in JAK2V617F(+) was significantly different from that in normal individuals (p <0.05), while there was no difference between JAK2V617F(-) patients and normal individuals. Allele-specific PCR showed high association of a cis-location between the special G-allele of rs10974944 and JAK2V617F(+). Based on this small numbered study, the results show the association between special SNPs and JAK2V617F mutation and a cis-location between the special G-allelic form of rs10974944 and the JAK2V617F mutation. These data highlight a close relationship between them in patients with ET.

Relationship between JAK2V617F mutation, allele burden and coagulation function in Ph-negative myeloproliferative neoplasms.

PubMed

Hu, Linhui; Pu, Lianfang; Ding, Yangyang; Li, Manman; Cabanero, Michael; Xie, Jingxin; Zhou, Dejun; Yang, Dongdong; Zhang, Cui; Wang, Huiping; Zhai, Zhimin; Ru, Xiang; Li, Jingrong; Xiong, Shudao

2017-07-01

Our aim was to explore the relationship between JAK2V617F mutation allele burden and hematological parameters especially in coagulation function in Chinese population. This study included 133 Ph-negative myeloproliferative neoplasms (MPNs) patients between 2013 and 2016. All the clinical and experimental data of patients were collected at the time of the diagnosis without any prior treatment, including blood parameters, coagulation function, splenomegaly, vascular events and chromosome karyotype. PCR and qPCR were used to detect JAK2V617F mutation and JAK2V617F mutation allele burden. In polycythemia vera patients, a positive correlation between the allele burden of JAK2V617F mutation and PLT counts was found; in essential thrombocythemia (ET) patients, WBC counts, RBC counts, HB, and HCT were higher in mutated patients than in wild-type patients. Furthermore, PT-INR was higher in ET and PMF mutated patients. In addition, a positive correlation between the allele burden of JAK2V617F mutation and activated partial thromboplastin time (APTT) was observed in JAK2V617F mutated ET patients. Higher hematologic parameters including counts of WBC, RBC, and PLT are closely associated with JAK2V617F mutation and its burden in Ph-negative MPNs; importantly, PT-INR, APTT are also related to JAK2V617F mutation and allele burden. Thus, our data indicate that JAK2V617F mutation allele burden might not only represent the burden of MPN but also alter the coagulation function.

Altered fast- and slow-twitch muscle fibre characteristics in female mice with a (S248F) knock-in mutation of the brain neuronal nicotinic acetylcholine receptor.

PubMed

Cannata, David J; Finkelstein, David I; Gantois, Ilse; Teper, Yaroslav; Drago, John; West, Jan M

2009-01-01

We generated a mouse line with a missense mutation (S248F) in the gene (CHRNA4) encoding the alpha4 subunit of neuronal nicotinic acetylcholine receptor (nAChR). Mutant mice demonstrate brief nicotine induced dystonia that resembles the clinical events seen in patients with the same mutation. Drug-induced dystonia is more pronounced in female mice, thus our aim was to determine if the S248F mutation changed the properties of fast- and slow-twitch muscle fibres from female mutant mice. Reverse transcriptase-PCR confirmed CHRNA4 gene expression in the brain but not skeletal muscles in normal and mutant mice. Ca(2+) and Sr(2+) force activation curves were obtained using skinned muscle fibres prepared from slow-twitch (soleus) and fast-twitch (EDL) muscles. Two significant results were found: (1) the (pCa(50) - pSr(50)) value from EDL fibres was smaller in mutant mice than in wild type (1.01 vs. 1.30), (2) the percentage force produced at pSr 5.5 was larger in mutants than in wild type (5.76 vs. 0.24%). Both results indicate a shift to slow-twitch characteristics in the mutant. This conclusion is supported by the identification of the myosin heavy chain (MHC) isoforms. Mutant EDL fibres expressed MHC I (usually only found in slow-twitch fibres) as well as MHC IIa. Despite the lack of spontaneous dystonic events, our findings suggest that mutant mice may be having subclinical events or the mutation results in a chronic alteration to muscle neural input.

A novel F11 mutation in a Korean pediatric patient with recurrent epistaxis.

PubMed

Kim, Juwon; Kim, Yoonjung; Shin, Seam; Lyu, Chuhl Joo; Choi, Jong Rak; Lee, Kyung-A

2013-06-01

Congenital FXI deficiency (hemophilia C) is a rare bleeding disorder that has been documented mostly in Ashkenazi Jews. Unlike other hemophilias, bleeding tendency varies considerably among individuals, and FXI deficiency rarely manifests as spontaneous bleeding. FXI deficiency is caused primarily by mutations in the F11 gene. Herein, we report a case of a 10-year-old boy with recurrent nose bleeding due to FXI deficiency who was confirmed to have a novel mutation in F11. A molecular analysis of DNA extracted from peripheral blood collected from the patient [FXI clotting activity (FXI:C): 11%] revealed compound heterozygous mutations, Q226X and L424F, in F11, consistent with the severe disease phenotype of the patient. Pedigree analysis showed that the patient received L424F from his father (FXI:C = 49%) and Q226X from the mother (FXI:C = 48%). The sister (FXI:C = 47%) of the patient only had L424F, presumably inherited from her father. Multiple sequence alignment demonstrated that L424 is highly conserved across mammals, indicating that it is important for the function of FXI. In-silico analysis indicated that replacement of L424 by phenylalanine had a detrimental influence on FXI, consistent with the severe phenotype of the patient. Compilation of FXI deficiency cases in east Asian populations would be of great value because different populations appear to have different F11 mutation spectra.

First report of L1014F-kdr mutation in Culex pipiens complex from Morocco.

PubMed

Bkhache, Meriem; Tmimi, Fatim-Zohra; Charafeddine, Omar; Faraj, Chafika; Failloux, Anna-Bella; Sarih, M'hammed

2016-12-16

Mosquitoes of the Culex pipiens complex, competent vectors for West Nile virus (WNV) and Rift Valley fever virus (RVFV) are widely targeted by insecticide treatments. The intensive application of chemical insecticides led to the development of resistance in many insects including Culex pipiens mosquitoes. The absence of data on resistance mechanisms in Morocco allow us to assess the levels of lambda-cyhalothrin resistance and the frequency of the mutated gene L1014F kdr in different forms of Cx. pipiens complex from three regions of Morocco. Mosquito adults were reared from immature stages collected in three different regions in Morocco (Tangier, Casablanca and Marrakech). Standard WHO insecticide susceptibility tests were conducted on adults emerged from collected larvae. Specimens were identified as belonging to the Culex pipiens complex using a multiplex PCR assay with diagnostic primers designed from the flanking region of microsatellite CQ11. Identified mosquitoes were then tested for the presence of the L1014F kdr mutation using PCR assay. Our results showed that 21% of the tested population has a resistance to lambda-cyhalothrin. The molecular identification of survivors shows that 43% belonged to the Cx. pipiens pipiens and only 9.5% to the Cx. pipiens molestus form. On the other hand, 416 specimens were screened for the L1014F kdr mutation. L1014F mutation was detected in different forms of Cx. pipiens in different sites. The frequency of L1014F mutation was similar between the Cx. pipiens pipiens form and hybrid form, while it was lower in the Cx. pipiens molestus form. The presence of the L1014F kdr allele was significantly associated with resistance to lambda-cyhalothrin in Cx. pipiens pipiens (P < 0.0001) and hybrid form (P < 0.0001). Resistance to lambda-cyhalothrin of Cx. pipiens populations appears to be largely due to the L1014F kdr mutation. To our knowledge, the frequencies of L1014F kdr mutation are examined for the first time in

Low frequency of c-MPL gene mutations in Iranian patients with Philadelphia-negative myeloproliferative disorders.

PubMed

Ghotaslou, A; Nadali, F; Chahardouli, B; Alizad Ghandforosh, N; Rostami, S H; Alimoghaddam, K; Ghavamzadeh, A

2015-01-01

Myeloproliferative disorders are a group of diseases characterized by increased proliferation of myeloid lineage. In addition to JAK2V617F mutation, several mutations in the c-MPL gene have been reported in patients with philadelphia-negative chronic myeloproliferative disorders that could be important in the pathogenesis of diseases. The aim of the present study was to investigate the frequency of c-MPL and JAK2V617F mutations in Iranian patients with Philadelphia-negativemyeloproliferative disorders. Peripheral blood samples were collected from 60 patients with Philadelphia-negative MPD) Subgroups ET and PMF) and 25 healthy subjects as control group. The mutation status of c-MPL and Jak2V617F were investigated by using Amplification-refractory mutation system (ARMS) and Allele-Specific PCR (AS-PCR), respectively. The results were confirmed by sequencing. Among 60 patients, 34 (56.6%) and 1(1.7%) had Jak2V617F and c-MPL mutation, respectively. Patients with Jak2V617F mutation had higher WBC counts and hemoglobin concentration than those without the mutation (p= 0.005, p=0.003). In addition, for all healthy subjects in control group, mutations were negative. The present study revealed that the c-MPL mutations unlike the Jak2V617F mutations are rare in Iranian patients with Ph-negative MPNs and the low mutation rate should be considered in the design of screening strategies of MPD patients.

Low frequency of c-MPL gene mutations in Iranian patients with Philadelphia-negative myeloproliferative disorders

PubMed Central

Ghotaslou, A; Nadali, F; Chahardouli, B; Alizad Ghandforosh, N; Rostami, SH; Alimoghaddam, K; Ghavamzadeh, A

2015-01-01

Background Myeloproliferative disorders are a group of diseases characterized by increased proliferation of myeloid lineage. In addition to JAK2V617F mutation, several mutations in the c-MPL gene have been reported in patients with philadelphia-negative chronic myeloproliferative disorders that could be important in the pathogenesis of diseases. The aim of the present study was to investigate the frequency of c-MPL and JAK2V617F mutations in Iranian patients with Philadelphia-negativemyeloproliferative disorders. Material and Methods Peripheral blood samples were collected from 60 patients with Philadelphia-negative MPD) Subgroups ET and PMF) and 25 healthy subjects as control group. The mutation status of c-MPL and Jak2V617F were investigated by using Amplification-refractory mutation system (ARMS) and Allele-Specific PCR (AS-PCR), respectively. The results were confirmed by sequencing. Results Among 60 patients, 34 (56.6%) and 1(1.7%) had Jak2V617F and c-MPL mutation, respectively. Patients with Jak2V617F mutation had higher WBC counts and hemoglobin concentration than those without the mutation (p= 0.005, p=0.003). In addition, for all healthy subjects in control group, mutations were negative. Conclusions The present study revealed that the c-MPL mutations unlike the Jak2V617F mutations are rare in Iranian patients with Ph-negative MPNs and the low mutation rate should be considered in the design of screening strategies of MPD patients. PMID:25914801

Association between Three Mutations, F1565C, V1023G and S996P, in the Voltage-Sensitive Sodium Channel Gene and Knockdown Resistance in Aedes aegypti from Yogyakarta, Indonesia

PubMed Central

Rochmijati Wuliandari, Juli; Lee, Siu Fai; White, Vanessa Linley; Tantowijoyo, Warsito; Hoffmann, Ary Anthony; Endersby-Harshman, Nancy Margaret

2015-01-01

Mutations in the voltage-sensitive sodium channel gene (Vssc) have been identified in Aedes aegypti and some have been associated with pyrethroid insecticide resistance. Whether these mutations cause resistance, alone or in combination with other alleles, remains unclear, but must be understood if mutations are to become markers for resistance monitoring. We describe High Resolution Melt (HRM) genotyping assays for assessing mutations found in Ae. aegypti in Indonesia (F1565C, V1023G, S996P) and use them to test for associations with pyrethroid resistance in mosquitoes from Yogyakarta, a city where insecticide use is widespread. Such knowledge is important because Yogyakarta is a target area for releases of Wolbachia-infected mosquitoes with virus-blocking traits for dengue suppression. We identify three alleles across Yogyakarta putatively linked to resistance in previous research. By comparing resistant and susceptible mosquitoes from bioassays, we show that the 1023G allele is associated with resistance to type I and type II pyrethroids. In contrast, F1565C homozygotes were rare and there was only a weak association between individuals heterozygous for the mutation and resistance to a type I pyrethroid. As the heterozygote is expected to be incompletely recessive, it is likely that this association was due to a different resistance mechanism being present. A resistance advantage conferred to V1023G homozygotes through addition of the S996P allele in the homozygous form was suggested for the Type II pyrethroid, deltamethrin. Screening of V1023G and S996P should assist resistance monitoring in Ae. aegypti from Yogyakarta, and these mutations should be maintained in Wolbachia strains destined for release in this city to ensure that these virus-blocking strains of mosquitoes are not disadvantaged, relative to resident populations. PMID:26463408

Association between Three Mutations, F1565C, V1023G and S996P, in the Voltage-Sensitive Sodium Channel Gene and Knockdown Resistance in Aedes aegypti from Yogyakarta, Indonesia.

PubMed

Wuliandari, Juli Rochmijati; Lee, Siu Fai; White, Vanessa Linley; Tantowijoyo, Warsito; Hoffmann, Ary Anthony; Endersby-Harshman, Nancy Margaret

2015-07-23

Mutations in the voltage-sensitive sodium channel gene (Vssc) have been identified in Aedes aegypti and some have been associated with pyrethroid insecticide resistance. Whether these mutations cause resistance, alone or in combination with other alleles, remains unclear, but must be understood if mutations are to become markers for resistance monitoring. We describe High Resolution Melt (HRM) genotyping assays for assessing mutations found in Ae. aegypti in Indonesia (F1565C, V1023G, S996P) and use them to test for associations with pyrethroid resistance in mosquitoes from Yogyakarta, a city where insecticide use is widespread. Such knowledge is important because Yogyakarta is a target area for releases of Wolbachia-infected mosquitoes with virus-blocking traits for dengue suppression. We identify three alleles across Yogyakarta putatively linked to resistance in previous research. By comparing resistant and susceptible mosquitoes from bioassays, we show that the 1023G allele is associated with resistance to type I and type II pyrethroids. In contrast, F1565C homozygotes were rare and there was only a weak association between individuals heterozygous for the mutation and resistance to a type I pyrethroid. As the heterozygote is expected to be incompletely recessive, it is likely that this association was due to a different resistance mechanism being present. A resistance advantage conferred to V1023G homozygotes through addition of the S996P allele in the homozygous form was suggested for the Type II pyrethroid, deltamethrin. Screening of V1023G and S996P should assist resistance monitoring in Ae. aegypti from Yogyakarta, and these mutations should be maintained in Wolbachia strains destined for release in this city to ensure that these virus-blocking strains of mosquitoes are not disadvantaged, relative to resident populations.

Cystic fibrosis Δf508 mutation screening in Brazilian women with altered fertility.

PubMed

Brunoro, G V F; Wolfgramm, E V; Louro, I D; Degasperi, I I; Busatto, V C W; Perrone, A M S; Batitucci, M C P

2011-10-01

Cystic Fibrosis (CF) is an autosomal recessive disease, caused by mutations in the Cystic Fibrosis Transmembrane Regulator gene (CFTR). The most frequent mutation in CF is ΔF508. The disease is clinically characterized by elevated concentrations of sweat chlorides and abnormally thick mucus. It affects organs such as lung, pancreas, gastrointestinal and reproductive tract. Women with CF commonly present delayed puberty and amenorrhea due to malnutrition. Our objective was to screen the presence of ΔF508 mutation in 24 women with altered fertility. Nine of these women presented reduced fertility without a known cause, four showed polycystic ovaries and two had early menopause. One woman with early menopause was a carrier of the ΔF508 mutation. Our study demonstrates that it is possible that the frequency of CF mutations among patients with altered fertility may be higher than expected. Previous data showed that fibrocystic women can show reduced fertility, maternal mortality associated with pregnancy and increased incidence of spontaneous abortion. We therefore recommend that women with reduced fertility undertake genetic tests for a better evaluation of pregnancy risks and clinical monitoring.

POPDC1S201F causes muscular dystrophy and arrhythmia by affecting protein trafficking

PubMed Central

Schindler, Roland F.R.; Scotton, Chiara; Zhang, Jianguo; Passarelli, Chiara; Ortiz-Bonnin, Beatriz; Simrick, Subreena; Schwerte, Thorsten; Poon, Kar-Lai; Fang, Mingyan; Rinné, Susanne; Froese, Alexander; Nikolaev, Viacheslav O.; Grunert, Christiane; Müller, Thomas; Tasca, Giorgio; Sarathchandra, Padmini; Drago, Fabrizio; Dallapiccola, Bruno; Rapezzi, Claudio; Arbustini, Eloisa; Di Raimo, Francesca Romana; Neri, Marcella; Selvatici, Rita; Gualandi, Francesca; Fattori, Fabiana; Pietrangelo, Antonello; Li, Wenyan; Jiang, Hui; Xu, Xun; Bertini, Enrico; Decher, Niels; Wang, Jun; Brand, Thomas; Ferlini, Alessandra

2015-01-01

The Popeye domain–containing 1 (POPDC1) gene encodes a plasma membrane–localized cAMP-binding protein that is abundantly expressed in striated muscle. In animal models, POPDC1 is an essential regulator of structure and function of cardiac and skeletal muscle; however, POPDC1 mutations have not been associated with human cardiac and muscular diseases. Here, we have described a homozygous missense variant (c.602C>T, p.S201F) in POPDC1, identified by whole-exome sequencing, in a family of 4 with cardiac arrhythmia and limb-girdle muscular dystrophy (LGMD). This allele was absent in known databases and segregated with the pathological phenotype in this family. We did not find the allele in a further screen of 104 patients with a similar phenotype, suggesting this mutation to be family specific. Compared with WT protein, POPDC1S201F displayed a 50% reduction in cAMP affinity, and in skeletal muscle from patients, both POPDC1S201F and WT POPDC2 displayed impaired membrane trafficking. Forced expression of POPDC1S201F in a murine cardiac muscle cell line (HL-1) increased hyperpolarization and upstroke velocity of the action potential. In zebrafish, expression of the homologous mutation (popdc1S191F) caused heart and skeletal muscle phenotypes that resembled those observed in patients. Our study therefore identifies POPDC1 as a disease gene causing a very rare autosomal recessive cardiac arrhythmia and LGMD, expanding the genetic causes of this heterogeneous group of inherited rare diseases. PMID:26642364

JAK2V617F somatic mutation in the general population: myeloproliferative neoplasm development and progression rate

PubMed Central

Nielsen, Camilla; Bojesen, Stig E.; Nordestgaard, Børge G.; Kofoed, Klaus F.; Birgens, Henrik S.

2014-01-01

Clinical significance of the JAK2V617F mutation in patients with a myeloproliferative neoplasm has been the target of intensive research in recent years. However, there is considerably uncertainty about prognosis in JAK2V617F positive individuals without overt signs of myeloproliferative disease. In this study, we tested the hypothesis that increased JAK2V617F somatic mutation burden is associated with myeloproliferative neoplasm progression rate in the general population. Among 49,488 individuals from the Copenhagen General Population Study, 63 (0.1%) tested positive for the JAK2V617F mutation in the time period 2003–2008. Of these, 48 were available for re-examination in 2012. Level of JAK2V617F mutation burden was associated with myeloproliferative neoplasm progression rate, consistent with a biological continuum of increasing JAK2V617F mutation burden across increasing severity of myeloproliferative neoplasm from no disease (n=8 at re-examination) through essential thrombocythemia (n=20) and polycythemia vera (n=13) to primary myelofibrosis (n=7). Among those diagnosed with a myeloproliferative neoplasm only at re-examination in 2012, in the preceding years JAK2V617F mutation burden increased by 0.55% per year, erythrocyte volume fraction increased by 1.19% per year, and erythrocyte mean corpuscular volume increased by 1.25% per year, while there was no change in platelet count or erythropoietin levels. Furthermore, we established a JAK2V617F mutation burden cut-off point of 2% indicative of disease versus no disease; however, individuals with a mutation burden below 2% may suffer from a latent form of myeloproliferative disease revealed by a slightly larger spleen and/or slightly higher lactic acid dehydrogenase concentration compared to controls. Of all 63 JAK2V617F positive individuals, 48 were eventually diagnosed with a myeloproliferative neoplasm. PMID:24907356

MPL mutation profile in JAK2 mutation-negative patients with myeloproliferative disorders.

PubMed

Ma, Wanlong; Zhang, Xi; Wang, Xiuqiang; Zhang, Zhong; Yeh, Chen-Hsiung; Uyeji, Jennifer; Albitar, Maher

2011-03-01

Mutations in the thrombopoietin receptor gene (myeloproliferative leukemia, MPL) have been reported in patients with JAK2 V617F-negative chronic myeloproliferative disorders (MPDs). We evaluated the prevalence of MPL mutations relative to JAK2 mutations in patients with suspected MPDs. A total of 2790 patient samples submitted for JAK2 mutation analysis were tested using real-time polymerase chain reaction and bidirectional sequencing of plasma RNA. JAK2 V617F-negative samples were tested for JAK2 exons 12 to 14 mutations, and those with negative results were then tested for mutations in MPL exons 10 and 11. Of the 2790 patients, 529 (18.96%) had V617F, 12 (0.43%) had small insertions or deletions in exon 12, and 7 (0.25%) had other JAK2 mutations in exons 12 to 14. Of the 2242 JAK2 mutation-negative patients, 68 (3.03%) had MPL mutations. W515L was the predominant MPL mutation (n=46; 68%), and 10 (15%) patients had other W515 variants. The remaining MPL mutations (n=12, 17%) were detected at other locations in exons 10 and 11 and included 3 insertion/deletion mutations. The S505N mutation, associated with familial MPD, was detected in 3 patients. Overall, for every 100 V617F mutations in patients with suspected MPDs, there were 12.9 MPL mutations, 2.3 JAK2 exon 12 mutations, and 1.3 JAK2 exons 13 to 14 mutations. These findings suggest that MPL mutation screening should be performed before JAK2 exons 12 to 14 testing in JAK2 V617F-negative patients with suspected MPDs.

18F-AV-1451 tau PET imaging correlates strongly with tau neuropathology in MAPT mutation carriers

PubMed Central

Puschmann, Andreas; Schöll, Michael; Ohlsson, Tomas; van Swieten, John; Honer, Michael; Englund, Elisabet

2016-01-01

Abstract Tau positron emission tomography ligands provide the novel possibility to image tau pathology in vivo. However, little is known about how in vivo brain uptake of tau positron emission tomography ligands relates to tau aggregates observed post-mortem. We performed tau positron emission tomography imaging with 18F-AV-1451 in three patients harbouring a p.R406W mutation in the MAPT gene, encoding tau. This mutation results in 3- and 4-repeat tau aggregates similar to those in Alzheimer’s disease, and many of the mutation carriers initially suffer from memory impairment and temporal lobe atrophy. Two patients with short disease duration and isolated memory impairment exhibited 18F-AV-1451 uptake mainly in the hippocampus and adjacent temporal lobe regions, correlating with glucose hypometabolism in corresponding regions. One patient died after 26 years of disease duration with dementia and behavioural deficits. Pre-mortem, there was 18F-AV-1451 uptake in the temporal and frontal lobes, as well as in the basal ganglia, which strongly correlated with the regional extent and amount of tau pathology in post-mortem brain sections. Amyloid-β (18F-flutemetamol) positron emission tomography scans were negative in all cases, as were stainings of brain sections for amyloid. This provides strong evidence that 18F-AV-1451 positron emission tomography can be used to accurately quantify in vivo the regional distribution of hyperphosphorylated tau protein. PMID:27357347

Frequency and clinical features of the JAK2 V617F mutation in pediatric patients with sporadic essential thrombocythemia.

PubMed

Nakatani, Takuya; Imamura, Toshihiko; Ishida, Hiroyuki; Wakaizumi, Katsuji; Yamamoto, Tohru; Otabe, Osamu; Ishigami, Tsuyoshi; Adachi, Souichi; Morimoto, Akira

2008-12-01

Pediatric essential thrombocythemia (ET) is a rare and heterogenous disease entity. While several recent studies have focused on the role of the JAK2 V617F mutation in pediatric ET, the frequency of pediatric ET cases with this mutation and the associated clinical features remain unclear. We examined six childhood cases who had been diagnosed with ET according to WHO criteria (onset age: 0.2-14 years) for the presence of the JAK2 V617F mutation, MPLW515L mutation and JAK2 exon 12 mutations. Two sensitive PCR-based methods were used for the JAK2 V617F genotyping. We also examined the expression of polycythemia rubra vera-1 (PRV-1), which is a diagnostic marker for clonal ET. We found that three of the six cases had the JAK2 V617F mutation and that all six cases expressed PRV-1 in their peripheral granulocytes. Neither MPL W515L mutation nor JAK2 exon 12 mutations was detected in the patients without JAK2 V617F mutation. The two patients who developed thrombocythemia during infancy were JAK2 V617F-negative. These findings suggest that the JAK2 V617F mutation is not rare in childhood sporadic ET cases, and that these cases might be older and myeloproliferative features.

Roles of germline JAK2 activation mutation JAK2 V625F in the pathology of myeloproliferative neoplasms.

PubMed

Wu, Qing-Yun; Ma, Meng-Meng; Fu, Lin; Zhu, Yuan-Yuan; Liu, Yang; Cao, Jiang; Zhou, Ping; Li, Zhen-Yu; Zeng, Ling-Yu; Li, Feng; Wang, Xiao-Yun; Xu, Kai-Lin

2018-05-18

Janus tyrosine kinase 2 (JAK2) mediates downstream signaling of cytokine receptors in all hematological lineages, constitutively active somatic JAK2 mutations play key roles in the pathology of myeloproliferative neoplasms (MPNs). Recently, germline JAK2 mutations are also associated with triple-negative MPNs. A novel germline mutation JAK2 V625F is reported to be involved in a subset of MPNs patients. However, the pathogenesis of this mutation caused MPN is still unclear. In this study, the homology models of JAK2 V625F showed that the newly formed interaction between F625 and Y613 disrupted the JAK2 JH1-JH2 domain interactions was responsible for its activation, when F625 and Y613 interaction was disrupted, its activity significantly decreased. While, when this interaction was repaired whether by forming hydrogen bond or salt bond, it would cause JAK2 activation. Biochemical studies also demonstrated that JAK2 V625F mutation led to JAK2-STAT5 pathway activation and promoted the proliferation of BaF3 cells. Thus, our results herein provide clues to understand the mechanism JAK2 V625F mutation caused MPNs and give information for the development of JAK2 mutation specific inhibitors. Copyright © 2018 Elsevier B.V. All rights reserved.

A novel heterozygous intronic mutation in POU1F1 is associated with combined pituitary hormone deficiency.

PubMed

Takagi, Masaki; Kamasaki, Hotaka; Yagi, Hiroko; Fukuzawa, Ryuji; Narumi, Satoshi; Hasegawa, Tomonobu

2017-02-27

POU class 1 homeobox 1 (POU1F1) regulates pituitary cell-specific gene expression of somatotropes, lactotropes, and thyrotropes. In humans, two POU1F1 isoforms (long and short isoform), which are generated by the alternative use of the splice acceptor site for exon 2, have been identified. To date, more than 30 POU1F1 mutations in patients with combined pituitary hormone deficiency (CPHD) have been described. All POU1F1 variants reported to date affect both the short and long isoforms of the POU1F1 protein; therefore, it is unclear at present whether a decrease in the function of only one of these two isoforms is sufficient for disease onset in humans. Here, we described a sibling case of CPHD carrying a heterozygous mutation in intron 1 of POU1F1. In vitro experiments showed that this mutation resulted in exon 2-skipping of only in the short isoform of POU1F1, while the long isoform remained intact. This result strongly suggests the possibility, for the first time, that isolated mutations in the short isoform of POU1F1 could be sufficient for induction of POU1F1-related CPHD. This finding improves our understanding of the molecular mechanisms, and developmental course associated with mutations in POU1F1.

Frequency of JAK2 V617F mutation in patients with Philadelphia positive Chronic Myeloid Leukemia in Pakistan.

PubMed

Tabassum, Najia; Saboor, Mohammed; Ghani, Rubina; Moinuddin, Moinuddin

2014-01-01

Co-existence of myeloproliferative disorders (MPD) and Janus associated kinase 2 mutation (JAK2 V617F) is a well-established fact. Only few case reports are available showing presence of JAK2 V617F mutation in chronic myeloid leukemia (CML). Purpose of this study was to determine the frequency of JAK2 V617F mutation in Philadelphia Chromosome positive (Ph (+)) CML patients in Pakistan. The study was conducted from August 2009 to July 2010 at Civil Hospital and Baqai Institute of Hematology (BIH) Karachi. Blood samples from 25 patients with CML were collected. Multiplex reverse transcription polymerase chain reaction (RT-PCR) was performed for Breakpoint Cluster Region - Abelson (BCR-ABL) rearrangement. Conventional PCR was performed for JAK2 V617F mutation on BCR-ABL positive samples. All 25 samples showed BCR-ABL rearrangement. Out of these 11 samples (44%) had JAK2 V617F mutation; the remaining 14 (56%) cases showed JAK2 617V wild type. It is concluded that the co-existence of Ph (+)CML and JAK2 V617F mutation is possible.

Altered epidermal lipid processing and calcium distribution in the KID syndrome mouse model Cx26S17F.

PubMed

Bosen, Felicitas; Celli, Anna; Crumrine, Debra; vom Dorp, Katharina; Ebel, Philipp; Jastrow, Holger; Dörmann, Peter; Winterhager, Elke; Mauro, Theodora; Willecke, Klaus

2015-07-08

The keratitis-ichthyosis-deafness (KID) syndrome is caused by mutations in the gap junctional channel protein connexin 26 (Cx26), among them the mutation Cx26S17F. Heterozygous Cx26S17F mice resemble the human KID syndrome, i.e. exhibiting epidermal hyperplasia and hearing impairments. Newborn Cx26S17F mice show a defective epidermal water barrier as well as altered epidermal lipid secretion and location. Linoleoyl ω-esterified ceramides are strongly decreased on the skin surface of Cx26S17F mice. Moreover, the epidermal calcium gradient is altered in the mutant mice. These alterations may be caused by an abnormal Cx26S17F channel function that leads to a defective epidermal water barrier, which in turn may trigger the hyperproliferation seen in the KID syndrome. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

First report of L1014F kdr mutation in Culex quinquefasciatus in Mexico.

PubMed

Ponce, Gustavo; Sanchez, Iram P; García, Selene M; Torrado, Jose M; Lozano-Fuentes, Saúl; Lopez-Monroy, Beatriz; Flores, Adriana E

2016-12-01

The L1014F mutation in the voltage-sodium channel gene has been associated with resistance to DDT and pyrethroids in various arthropod species including mosquitoes. We determined the frequency of the L1014F kdr mutation in 16 field populations of Culex quinquefasciatus from Northeastern Mexico collected between 2008 and 2013. The L1014F was present in all populations analyzed with the lowest frequency (3.33%) corresponding to the population from Monclova collected in 2012, and the highest frequency (63.63%) from the Monterrey population collected in 2012. The presence of a kdr mutation in populations of Cx. quinquefasciatus from northeastern Mexico provides evidence of pyrethroid resistance. This requires a special attention, considering that pyrethroid-based insecticides are commonly used in vector-control campaigns, especially against Aedes aegypti (L.). © 2015 Institute of Zoology, Chinese Academy of Sciences.

Detection of CALR and MPL Mutations in Low Allelic Burden JAK2 V617F Essential Thrombocythemia.

PubMed

Usseglio, Fabrice; Beaufils, Nathalie; Calleja, Anne; Raynaud, Sophie; Gabert, Jean

2017-01-01

Myeloproliferative neoplasms are clonal hematopoietic stem cell disorders characterized by aberrant proliferation and an increased tendency toward leukemic transformation. The genes JAK2, MPL, and CALR are frequently altered in these syndromes, and their mutations are often a strong argument for diagnosis. We analyzed the mutational profiles of these three genes in a cohort of 164 suspected myeloproliferative neoplasms. JAK2 V617F mutation was detected by real-time PCR, whereas high-resolution melting analysis followed by Sanger sequencing were used for searching for mutations in JAK2 exon 12, CALR, and MPL. JAK2 V617F mutation was associated with CALR (n = 4) and MPL (n = 4) mutations in 8 of 103 essential thrombocytosis patients. These cases were harboring a JAK2 V617F allelic burden of <4% and a significantly higher platelet count compared with JAK2 V617F (P < 0.001) and CALR (P = 0.001) single-mutation patients. The findings from this study support the possibility of coexisting mutations of the JAK2, CALR, and MPL genes in myeloproliferative neoplasms and suggest that CALR and MPL should be analyzed not only in JAK2-negative patients but also in low V617F mutation patients. Follow-up of these double-mutation cases will be important for determining whether this group of patients presents particular evolution or complications. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

E2F1 somatic mutation within miRNA target site impairs gene regulation in colorectal cancer.

PubMed

Lopes-Ramos, Camila M; Barros, Bruna P; Koyama, Fernanda C; Carpinetti, Paola A; Pezuk, Julia; Doimo, Nayara T S; Habr-Gama, Angelita; Perez, Rodrigo O; Parmigiani, Raphael B

2017-01-01

Genetic studies have largely concentrated on the impact of somatic mutations found in coding regions, and have neglected mutations outside of these. However, 3' untranslated regions (3' UTR) mutations can also disrupt or create miRNA target sites, and trigger oncogene activation or tumor suppressor inactivation. We used next-generation sequencing to widely screen for genetic alterations within predicted miRNA target sites of oncogenes associated with colorectal cancer, and evaluated the functional impact of a new somatic mutation. Target sequencing of 47 genes was performed for 29 primary colorectal tumor samples. For 71 independent samples, Sanger methodology was used to screen for E2F1 mutations in miRNA predicted target sites, and the functional impact of these mutations was evaluated by luciferase reporter assays. We identified germline and somatic alterations in E2F1. Of the 100 samples evaluated, 3 had germline alterations at the MIR205-5p target site, while one had a somatic mutation at MIR136-5p target site. E2F1 gene expression was similar between normal and tumor tissues bearing the germline alteration; however, expression was increased 4-fold in tumor tissue that harbored a somatic mutation compared to that in normal tissue. Luciferase reporter assays revealed both germline and somatic alterations increased E2F1 activity relative to wild-type E2F1. We demonstrated that somatic mutation within E2F1:MIR136-5p target site impairs miRNA-mediated regulation and leads to increased gene activity. We conclude that somatic mutations that disrupt miRNA target sites have the potential to impact gene regulation, highlighting an important mechanism of oncogene activation.

Mechanisms of mutations in myeloproliferative neoplasms.

PubMed

Levine, Ross L

2009-12-01

In recent years, a series of studies have provided genetic insight into the pathogenesis of myeloproliferative neoplasms (MPNs). It is now known that JAK2V617F mutations are present in 90% of patients with polycythaemia vera (PV), 60% of patients with essential thrombocytosis (ET) and 50% of patients with myelofibrosis (MF). Despite the high prevalence of JAK2V617F mutations in these three myeloid malignancies, several questions remain. For example, how does one mutation contribute to the pathogenesis of three clinically distinct diseases, and how do some patients develop these diseases in the absence of a JAK2V617F mutation? Single nucleotide polymorphisms at various loci and somatic mutations, such as those in MPLW515L/K, TET2 and in exon 12 of JAK2, may also contribute to the pathogenesis of these MPNs. There are likely additional germline and somatic genetic factors important to the MPN phenotype. Additional studies of large MPN and control cohorts with new techniques will help identify these factors.

Genotoxicity testing of sodium formononetin-3'-sulphonate (Sul-F) by assessing bacterial reverse mutation, chromosomal aberrations and micronucleus tests.

PubMed

Li, Chunmei; Gao, Yonglin; Wang, Yunzhi; Li, Guisheng; Fan, Xiaochen; Li, Yanshen; Guo, Chenghua; Tao, Jun

2017-06-01

As part of a safety evaluation, we evaluated the potential genotoxicity of sodium formononetin-3'-sulphonate (Sul-F) using bacterial reverse mutation assay, chromosomal aberrations detection, and mouse micronucleus test. In bacterial reverse mutation assay using five strains of Salmonella typhimurium (TA97, TA98, TA100, TA102 and TA1535), Sul-F (250, 500, 1000, 2000, 4000 μg/plate) did not increase the number of revertant colonies in any tester strain with or without S9 mix. In a chromosomal assay using Chinese hamster lung fibroblast (CHL) cells, there were no increases in either kind of aberration at any dose of Sul-F (400, 800, and 1600 μg/mL) treatment groups with or without S9 metabolic activation. In an in vivo bone marrow micronucleus test in ICR mice, Sul-F at up to 2000 mg/kg (intravenous injection) showed no significant increases in the incidence of micronucleated polychromatic erythrocytes, and the proportion of immature erythrocytes to total erythrocytes. The results demonstrated that Sul-F does not show mutagenic or genotoxic potential under these test conditions. Copyright © 2017. Published by Elsevier Inc.

Risk Profile of the RET A883F Germline Mutation: An International Collaborative Study.

PubMed

Mathiesen, Jes Sloth; Habra, Mouhammed Amir; Bassett, John Howard Duncan; Choudhury, Sirazum Mubin; Balasubramanian, Sabapathy Prakash; Howlett, Trevor A; Robinson, Bruce G; Gimenez-Roqueplo, Anne-Paule; Castinetti, Frederic; Vestergaard, Peter; Frank-Raue, Karin

2017-06-01

The A883F germline mutation of the rearranged during transfection (RET) proto-oncogene causes multiple endocrine neoplasia 2B. In the revised American Thyroid Association (ATA) guidelines for the management of medullary thyroid carcinoma (MTC), the A883F mutation has been reclassified from the highest to the high-risk level, although no well-defined risk profile for this mutation exists. To create a risk profile for the A883F mutation for appropriate classification among the ATA risk levels. Retrospective analysis. International collaboration. Included were 13 A883F carriers. The intervention was thyroidectomy. Earliest age of MTC, regional lymph node metastases, distant metastases, age-related penetrance of MTC and pheochromocytoma (PHEO), overall and disease-specific survival, and biochemical cure rate. One and three carriers were diagnosed at age 7 to 9 years (median, 7.5 years) with a normal thyroid and C-cell hyperplasia, respectively. Nine carriers were diagnosed with MTC at age 10 to 39 years (median, 19 years). The earliest age of MTC, regional lymph node metastasis, and distant metastasis was 10, 20, and 20 years, respectively. Fifty percent penetrance of MTC and PHEO was achieved by age 19 and 34 years, respectively. Five- and 10-year survival rates (both overall and disease specific) were 88% and 88%, respectively. Biochemical cure for MTC at latest follow-up was achieved in 63% (five of eight carriers) with pertinent data. MTC of A883F carriers seems to have a more indolent natural course compared with that of M918T carriers. Our results support the classification of the A883F mutation in the ATA high-risk level. Copyright © 2017 Endocrine Society

Familial Mediterranean fever with a single MEFV mutation: comparison of rare and common mutations in a Turkish paediatric cohort.

PubMed

Soylemezoglu, Oguz; Kandur, Yasar; Duzova, Ali; Ozkaya, Ozan; Kasapcopur, Ozgür; Baskin, Esra; Fidan, Kibriya; Yalcinkaya, Fatos

2015-01-01

Presence of common MEFV gene mutations strengthened the diagnosis of FMF in addition to the typical clinical characteristics of FMF. However, there are also rare mutations. P369S, A744S, R761H, K695R, F479L are the main rare mutations in Turkish population. We aimed to evaluate FMF patients with a single allele MEFV mutation and to compare patients with common and rare mutations. We retrospectively reviewed the medical records of FMF patients with a single allele mutation who were followed up between 2008 and 2013 in six centres. We compared the patients with rare and common mutations for disease severity score, frequent exacerbations ( >1 attack per month), long attack period (>3 day), symptoms, age at the onset of symptoms, gender, consanguinity, and family history. Two hundred and seventeen patients (M/F=101/116) with the diagnosis of FMF and single mutation were included. Heterozygote mutations were defined as common (M694V, V726A, M68OI) and rare mutations (A744S, P369S, K695R, R761H, F479L). Sixty-seven patients (27 males, 40 females) had one single rare mutation and 150 (74 males, 76 females) had one single common mutation. No difference was found between the rare and common mutations with respect to the disease severity score. There was no significant difference between common and rare heterozygote form of mutations in terms of disease severity. Patients with typical characteristics of FMF, with some rare mutations (A744S, P369S) should be treated in the same manner as patients with a common mutation.

Highly preferential association of NonF508del CF mutations with the M470 allele.

PubMed

Ciminelli, B M; Bonizzato, A; Bombieri, C; Pompei, F; Gabaldo, M; Ciccacci, C; Begnini, A; Holubova, A; Zorzi, P; Piskackova, T; Macek, M; Castellani, C; Modiano, G; Pignatti, P F

2007-01-01

On the basis of previous findings on random individuals, we hypothesized a preferential association of CF causing mutations with the M allele of the M470V polymorphic site of the CFTR gene. We have determined the M/V-CF mutation haplotype in a series of 201 North East Italian and 73 Czech CF patients who were not F508del homozygotes, as F508del was already known to be fully associated with the M allele. Out of 358 not F508del CF genes, 84 carried the V allele and 274 the less common M allele. In the N-E Italian population, MM subjects have a risk of carrying a CF causing mutation 6.9x greater than VV subjects when F508del is excluded and 15.4x when F508del is included. In the Czech population a similar, although less pronounced, association is observed. Besides the possible biological significance of this association, the possibility of exploiting it for a pilot screening program has been explored in a local North East Italian population for which CF patients were characterized for their CF mutation. General M470V genotyping followed by common CF mutation screening limited to couples in which each partner carries at least one M allele would need testing only 39% of the couples, which contribute 89% of the total risk, with a cost benefit.

Frequency of JAK2 V617F mutation in patients with Philadelphia positive Chronic Myeloid Leukemia in Pakistan

PubMed Central

Tabassum, Najia; Saboor, Mohammed; Ghani, Rubina; Moinuddin, Moinuddin

2014-01-01

Background and Objective: Co-existence of myeloproliferative disorders (MPD) and Janus associated kinase 2 mutation (JAK2 V617F) is a well-established fact. Only few case reports are available showing presence of JAK2 V617F mutation in chronic myeloid leukemia (CML). Purpose of this study was to determine the frequency of JAK2 V617F mutation in Philadelphia Chromosome positive (Ph +) CML patients in Pakistan. Methods: The study was conducted from August 2009 to July 2010 at Civil Hospital and Baqai Institute of Hematology (BIH) Karachi. Blood samples from 25 patients with CML were collected. Multiplex reverse transcription polymerase chain reaction (RT-PCR) was performed for Breakpoint Cluster Region – Abelson (BCR-ABL) rearrangement. Conventional PCR was performed for JAK2 V617F mutation on BCR-ABL positive samples. Results: All 25 samples showed BCR-ABL rearrangement. Out of these 11 samples (44%) had JAK2 V617F mutation; the remaining 14 (56%) cases showed JAK2 617V wild type. Conclusion: It is concluded that the co-existence of Ph +CML and JAK2 V617F mutation is possible. PMID:24639858

JAK2 V617F, MPL W515L and JAK2 Exon 12 Mutations in Chinese Patients with Primary Myelofibrosis.

PubMed

Xia, Jun; Lu, Mi-Ze; Jiang, Yuan-Qiang; Yang, Guo-Hua; Zhuang, Yun; Sun, Hong-Li; Shen, Yun-Feng

2012-03-01

JAK2 V617F, MPL W515L and JAK2 exon 12 mutations are novel acquired mutations that induce constitutive cytokine-independent activation of the JAK-STAT pathway in myeloproliferative disorders (MPD). The discovery of these mutations provides novel mechanism for activation of signal transduction in hematopoietic malignancies. This research was to investigate their prevalence in Chinese patients with primary myelofibrosis (PMF). We introduced allele-specific PCR (AS-PCR) combined with sequence analysis to simultaneously screen JAK2 V617F, MPL W515L and JAK2 exon 12 mutations in 30 patients with PMF. Fifteen PMF patients (50.0%) carried JAK2 V617F mutation, and only two JAK2 V617F-negative patients (6.7%) harbored MPL W515L mutation. None had JAK2 exon 12 mutations. Furthermore, these three mutations were not detected in 50 healthy controls. MPL W515L and JAK2 V617F mutations existed in PMF patients but JAK2 exon 12 mutations not. JAK2 V617F and MPL W515L and mutations might contribute to the primary molecular pathogenesis in patients with PMF.

F1174V mutation alters the ALK active conformation in response to Crizotinib in NSCLC: Insight from molecular simulations.

PubMed

Dehghanian, Fariba; Kay, Maryam; Vallian, Sadeq

2017-08-01

Crizotinib is an efficient antineoplastic drug for treatment of non-small cell lung carcinoma (NSCLC), which is identified as an anaplastic lymphoma kinase (ALK) inhibitor. F1174V is a recently identified acquired point mutation relating to the Crizotinib resistance in NSCLC patients. The mechanism of Crizotinib resistance relating to F1174V mutation as a non-active site mutation remains unclear. In this study, the molecular dynamic simulation was used to investigate the possible mechanisms by which F1174V mutation may affect the structure and activity of ALK kinase domain. The results suggested that F1174V mutation could cause two important secondary structure alterations, which led to the local conformational change in ALK kinase domain. This causes more positive free energy in the mutant complex in comparison with the wild-type one. In addition, our structural analyses illustrated that F1174V mutation could result in some important interactions, which represent the key characteristics of the ALK active conformation. This study provided a molecular mechanism for ALK Crizotinib resistance caused by F1174V mutation,which could facilitate designing more efficient drugs. Copyright © 2017 Elsevier Inc. All rights reserved.

First report of the presence of L1014S Knockdown-resistance mutation in Anopheles gambiae s.s and Anopheles coluzzii from Togo, West Africa.

PubMed

Djègbè, Innocent; Akoton, Romaric; Tchigossou, Genevieve; Ahadji-Dabla, Koffi Mensah; Atoyebi, Seun Michael; Adéoti, Razack; Zeukeng, Francis; Ketoh, Guillaume Koffivi; Djouaka, Rousseau

2018-01-01

Background: To optimize the success of insecticide-based malaria control intervention, knowledge of the distribution of Anopheles gambiae species and insecticide resistance mechanisms is necessary. This paper reported an updated data on pyrethroids/DDT resistance in the An. gambiae s.l population from Togo.  Methods: From December 2013 to April 2015, females of indoor-resting An. gambiae s.l were captured in three locations belonging to three different ecological zones. Resistance to DDT, permethrin and deltamethrin was screened in F1 progeny of collected mosquitoes using WHO susceptibility tests. The identification of species of An. gambiae complex and the detection of kdr and ace.1 R allele were carried out using DNA-based molecular techniques. Results: An. gambiae from Kovié and Nangbéto were highly resistant to DDT and permethrin with mortalities rate ranging from 0.83% to 1.58% for DDT and zero to 8.54% for permethrin. Mosquitoes collected in Nangbéto displayed 81.53% mortality with deltamethrin. An. coluzzii and An. gambiae s.s were found in sympatry in Nangbéto and Mango . The allelic frequency of L1014F was high, ranging from 66 to 100% in both An. coluzzii and An. gambiae s.s . For the first time we detected the L1014S allele in both An. coluzzii and An. gambiae s.s. from Togo at the frequency ranging from 5% to 13% in all the sites. The kdr N1575Y was present at various frequencies in both species ranging from 10% to 45%. Both An. gambiae s.s. and An. coluzzii shared the ace1 R mutation in all investigated sites with allelic frequency ranging from 4% to 16%. Conclusion: These results showed that multiple mutations are involved in insecticides resistance in An. gambiae populations from Togo including the kdr L1014F, L1014S, and N1575Y and ace.1 R G119S mutations.

First report of the presence of L1014S Knockdown-resistance mutation in Anopheles gambiae s.s and Anopheles coluzzii from Togo, West Africa

PubMed Central

Djègbè, Innocent; Akoton, Romaric; Tchigossou, Genevieve; Ahadji-Dabla, Koffi Mensah; Atoyebi, Seun Michael; Adéoti, Razack; Zeukeng, Francis; Ketoh, Guillaume Koffivi; Djouaka, Rousseau

2018-01-01

Background: To optimize the success of insecticide-based malaria control intervention, knowledge of the distribution of Anopheles gambiae species and insecticide resistance mechanisms is necessary. This paper reported an updated data on pyrethroids/DDT resistance in the An. gambiae s.l population from Togo.  Methods: From December 2013 to April 2015, females of indoor-resting An. gambiae s.l were captured in three locations belonging to three different ecological zones. Resistance to DDT, permethrin and deltamethrin was screened in F1 progeny of collected mosquitoes using WHO susceptibility tests. The identification of species of An. gambiae complex and the detection of kdr and ace.1 R allele were carried out using DNA-based molecular techniques. Results: An. gambiae from Kovié and Nangbéto were highly resistant to DDT and permethrin with mortalities rate ranging from 0.83% to 1.58% for DDT and zero to 8.54% for permethrin. Mosquitoes collected in Nangbéto displayed 81.53% mortality with deltamethrin. An. coluzzii and An. gambiae s.s were found in sympatry in Nangbéto and Mango . The allelic frequency of L1014F was high, ranging from 66 to 100% in both An. coluzzii and An. gambiae s.s. For the first time we detected the L1014S allele in both An. coluzzii and An. gambiae s.s. from Togo at the frequency ranging from 5% to 13% in all the sites. The kdr N1575Y was present at various frequencies in both species ranging from 10% to 45%. Both An. gambiae s.s. and An. coluzzii shared the ace1 R mutation in all investigated sites with allelic frequency ranging from 4% to 16%. Conclusion: These results showed that multiple mutations are involved in insecticides resistance in An. gambiae populations from Togo including the kdr L1014F, L1014S, and N1575Y and ace.1 R G119S mutations. PMID:29707654

F8 and F9 mutations in US haemophilia patients: correlation with history of inhibitor and race/ethnicity.

PubMed

Miller, C H; Benson, J; Ellingsen, D; Driggers, J; Payne, A; Kelly, F M; Soucie, J M; Craig Hooper, W

2012-05-01

Both genetic and treatment-related risk factors contribute to the development of inhibitors in haemophilia. An inhibitor surveillance system piloted at 12 US sites has the goal of assessing risk factors through prospective data collection. This report examines the relationship of genotype and race/ethnicity to history of inhibitor in a large cohort of US haemophilia patients. Mutation analysis was performed on 676 haemophilia A (HA) and 153 haemophilia B (HB) patients by sequencing, Multiplex Ligation-dependent Probe Amplification, and PCR for inversions in F8 introns 22 (inv22) and 1 (inv1). Two HB patients with deletions had history of inhibitor. In severe HA, frequency of history of inhibitor was: large deletion 57.1%, splice site 35.7%, inv22 26.8%, nonsense 24.5%, frameshift 12.9%, inv1 11.1% and missense 9.5%. In HA, 19.6% of 321 White non-Hispanics (Whites), 37.1% of 35 Black non-Hispanics (Blacks) and 46.9% of 32 Hispanics had history of inhibitor (P = 0.0003). Mutation types and novel mutation rates were similar across ethnicities. When F8 haplotypes were constructed, Whites and Hispanics showed only H1 and H2. Within H1, history of inhibitor was 12.4% in Whites, 40.0% in Blacks (P = 0.009) and 32.4% in Hispanics (P = 0.002). Inhibitor frequency is confirmed to vary by mutation type and race in a large US population. White patients with history of inhibitor did not exhibit rare F8 haplotypes. F8 gene analysis did not reveal a cause for the higher inhibitor frequencies in Black and Hispanic patients. © 2011 Blackwell Publishing Ltd.

Advanced cell-based modeling of the royal disease: characterization of the mutated F9 mRNA.

PubMed

Martorell, L; Luce, E; Vazquez, J L; Richaud-Patin, Y; Jimenez-Delgado, S; Corrales, I; Borras, N; Casacuberta-Serra, S; Weber, A; Parra, R; Altisent, C; Follenzi, A; Dubart-Kupperschmitt, A; Raya, A; Vidal, F; Barquinero, J

2017-11-01

Essentials The Royal disease (RD) is a form of hemophilia B predicted to be caused by a splicing mutation. We generated an iPSC-based model of the disease allowing mechanistic studies at the RNA level. F9 mRNA analysis in iPSC-derived hepatocyte-like cells showed the predicted abnormal splicing. Mutated F9 mRNA level was very low but we also found traces of wild type transcripts. Background The royal disease is a form of hemophilia B (HB) that affected many descendants of Queen Victoria in the 19th and 20th centuries. It was found to be caused by the mutation F9 c.278-3A>G. Objective To generate a physiological cell model of the disease and to study F9 expression at the RNA level. Methods Using fibroblasts from skin biopsies of a previously identified hemophilic patient bearing the F9 c.278-3A>G mutation and his mother, we generated induced pluripotent stem cells (iPSCs). Both the patient's and mother's iPSCs were differentiated into hepatocyte-like cells (HLCs) and their F9 mRNA was analyzed using next-generation sequencing (NGS). Results and Conclusion We demonstrated the previously predicted aberrant splicing of the F9 transcript as a result of an intronic nucleotide substitution leading to a frameshift and the generation of a premature termination codon (PTC). The F9 mRNA level in the patient's HLCs was significantly reduced compared with that of his mother, suggesting that mutated transcripts undergo nonsense-mediated decay (NMD), a cellular mechanism that degrades PTC-containing mRNAs. We also detected small proportions of correctly spliced transcripts in the patient's HLCs, which, combined with genetic variability in splicing and NMD machineries, could partially explain some clinical variability among affected members of the European royal families who had lifespans above the average. This work allowed the demonstration of the pathologic consequences of an intronic mutation in the F9 gene and represents the first bona fide cellular model of HB allowing the

Lower cognitive performance in healthy G2019S LRRK2 mutation carriers

PubMed Central

Thaler, Avner; Mirelman, Anat; Gurevich, Tanya; Simon, Ely; Orr-Urtreger, Avi; Marder, Karen; Bressman, Susan

2012-01-01

Objective: To assess cognitive abilities of healthy first-degree relatives of Ashkenazi patients with Parkinson disease (PD), carriers of the G2019S mutation in the LRRK2 gene. Methods: In this observational study, 60 consecutive healthy first-degree relatives (aged 50.9 ± 6.2 years; 48% male; 30 G2019S carriers) were assessed using a computerized cognitive program, the Montreal Cognitive Assessment questionnaire, the Unified Parkinson's Disease Rating Scale Part III, and the Geriatric Depression Scale. Results: G2019S carriers scored significantly lower on the computerized executive function index (p = 0.04) and on specific executive function tasks (Stroop test, p = 0.007). Conclusion: Carrying the LRRK2 G2019S mutation was associated with lower executive performance in a population at risk for PD. PMID:22914834

First somatic mutation of E2F1 in a critical DNA binding residue discovered in well-differentiated papillary mesothelioma of the peritoneum

PubMed Central

2011-01-01

Background Well differentiated papillary mesothelioma of the peritoneum (WDPMP) is a rare variant of epithelial mesothelioma of low malignancy potential, usually found in women with no history of asbestos exposure. In this study, we perform the first exome sequencing of WDPMP. Results WDPMP exome sequencing reveals the first somatic mutation of E2F1, R166H, to be identified in human cancer. The location is in the evolutionarily conserved DNA binding domain and computationally predicted to be mutated in the critical contact point between E2F1 and its DNA target. We show that the R166H mutation abrogates E2F1's DNA binding ability and is associated with reduced activation of E2F1 downstream target genes. Mutant E2F1 proteins are also observed in higher quantities when compared with wild-type E2F1 protein levels and the mutant protein's resistance to degradation was found to be the cause of its accumulation within mutant over-expressing cells. Cells over-expressing wild-type E2F1 show decreased proliferation compared to mutant over-expressing cells, but cell proliferation rates of mutant over-expressing cells were comparable to cells over-expressing the empty vector. Conclusions The R166H mutation in E2F1 is shown to have a deleterious effect on its DNA binding ability as well as increasing its stability and subsequent accumulation in R166H mutant cells. Based on the results, two compatible theories can be formed: R166H mutation appears to allow for protein over-expression while minimizing the apoptotic consequence and the R166H mutation may behave similarly to SV40 large T antigen, inhibiting tumor suppressive functions of retinoblastoma protein 1. PMID:21955916

Greater risk of parkinsonism associated with non-N370S GBA1 mutations

PubMed Central

Giraldo, P.; Capablo, J. L.; Alfonso, P.; Irun, P.; Garcia-Rodriguez, B.; Pocovi, M.; Pastores, G. M.

2014-01-01

Mutations in β-glucosidase (GBA1) are the most common genetic risk factor for Parkinson disease (PD). There is evidence to suggest that PD risk is greater (1) in GBA1 heterozygotes with non-N370S GBA1 mutations compared to N370S mutations and (2) in GD type 1 (GD1) patients compared to GBA1 heterozygotes. This study aimed to determine the comparative risk of parkin-sonism in individuals who are affected or carriers of Gaucher disease (GD) and to ascertain the influence of different GBA1 mutations on risk/clinical expression. We conducted a secondary analysis of cross-sectional data assessing the prevalence of parkinsonism in a population of GD1 patients and their heterozygote and non-carrier family members. Two logistic regression models, both employing a family-specific random effect, were used to assess (1) the association between GBA1 mutation (N370S or non-N370S) and parkinsonism among GBA1 heterozygotes and (2) the association between GBA1 genotype and parkinsonism. Parkinsonism was present in 8.6 % of GD1 (7/81), 8.7 % of GBA1 heterozygotes (18/207), and 2.2 % of non-carriers (1/45). For those greater than 60 years old, parkinsonism was present in 38.5 % (5/13) of GD1 (5/13), 15.3 % of GBA1 heterozygotes (13/85), and 7.1 % of non-carriers (1/14). Among GBA1 heterozygotes, non-N370S mutations were associated with a significantly increased risk of parkinsonism compared to N370S (OR=22.5; p=0.035; 95%CI: 1.24, 411). In this population, each additional GBA1 mutation was associated with a non-significant two-fold increased risk of parkinsonism. GBA1 heterozygotes with non-N370S mutations associated with Gaucher disease have an increased risk of parkinsonism compared to those with N370S mutations. PMID:22968580

JAK2 V617F, MPL, and CALR mutations in essential thrombocythaemia and major thrombotic complications: a single-institute retrospective analysis.

PubMed

Pósfai, Éva; Marton, Imelda; Király, Péter Attila; Kotosz, Balázs; Kiss-László, Zsuzsanna; Széll, Márta; Borbényi, Zita

2015-07-01

Thrombo-haemorrhagic events are the main cause of morbidity and mortality in essential thrombocythemia. The aim of this study was to estimate the incidence of thrombotic events and the impact of the JAK2V617F, MPL (W515L, W515K, W515R, W515A and S505N) and CALR (type-1, type-2) mutations on 101 essential thrombocythaemia patients (72 females and 29 males with a mean age of 61 years) diagnosed in a Southern Hungarian regional academic centre. The incidence of major thrombosis was 13.86 %. Sixty percent of the patients carried the JAK2V617F mutation. The MPL mutations were analysed by sequencing and the W515L was the only one we could identify with an incidence of 3.96 %. Type-2 CALR mutation could be identified in 3 cases among the patients who had JAK2/MPL-unmutated ET. Statistical analyses revealed that the JAK2V617F mutation was associated with significantly increased levels of platelet (p = 0.042), haemoglobin (p = 0.000), red blood cell (p = 0.000) and haematocrit (p = 0.000) and hepatomegaly (p = 0.045) at diagnosis compared to JAK2V617F negative counterparts, however there was no significant association between the JAK2V617F mutation status (relative risk: 1.297, 95 % CI 0.395-4.258; p = 0.668) and subsequent thrombotic complications. The impact of JAK2V617F, MPL W515L and CALR mutations on the clinical findings at the diagnosis of ET was obvious, but their statistically significant role in the prediction of thrombotic events could not be proven in this study. Our results indirectly support the concept that, besides the quantitative and qualitative changes in the platelets, the mechanisms leading to thrombosis are more complex and multifactorial.

STM study of C60F18 high dipole moment molecules on Au(111)

NASA Astrophysics Data System (ADS)

Bairagi, K.; Bellec, A.; Chumakov, R. G.; Menshikov, K. A.; Lagoute, J.; Chacon, C.; Girard, Y.; Rousset, S.; Repain, V.; Lebedev, A. M.; Sukhanov, L. P.; Svechnikov, N. Yu.; Stankevich, V. G.

2015-11-01

Scanning tunneling microscopy and spectroscopy studies of C60F18 molecules deposited on Au(111) are reported and compared to C60 molecules both at liquid helium temperature and room temperature (RT). Whereas adsorption and electronic properties of C60F18 single molecules were studied at low temperature (LT), self-assemblies were investigated at RT. In both cases, the fluorine atoms of the C60F18 molecules are pointed towards the surface. Individual C60F18 molecules on Au(111) have a HOMO-LUMO gap of 2.9 eV. The self-assembled islands exhibit a close-packed hexagonal lattice with amorphous borders. The comparison with C60 molecules clearly demonstrates the influence of the C60F18 electric dipole moment (EDM) on the electronic properties of single molecules and on the thermodynamics of self-assembled islands. Besides, the apparent height value of a separate molecule increases in a self-assembly environment as a result of a depolarization phenomenon.

Implications of fALS Mutations on Sod1 Function and Oligomerization in Cell Models.

PubMed

Brasil, Aline A; Magalhães, Rayne S S; De Carvalho, Mariana D C; Paiva, Isabel; Gerhardt, Ellen; Pereira, Marcos D; Outeiro, Tiago F; Eleutherio, Elis C A

2018-06-01

Among the familial forms of amyotrophic lateral sclerosis (fALS), 20% are associated with the Cu,Zn-superoxide dismutase (Sod1). fALS is characterized by the accumulation of aggregated proteins and the increase in oxidative stress markers. Here, we used the non-invasive bimolecular fluorescence complementation (BiFC) assay in human H4 cells to investigate the kinetics of aggregation and subcellular localization of Sod1 mutants. We also studied the effect of the different Sod1 mutants to respond against oxidative stress by following the levels of reactive oxygen species (ROS) after treatment with hydrogen peroxide. Our results showed that only 30% of cells transfected with A4VSod1 showed no inclusions while for the other Sod1 mutants tested (L38V, G93A and G93C), this percentage was at least 70%. In addition, we found that 10% of cells transfected with A4VSod1 displayed more than five inclusions per cell and that A4V and G93A Sod1 formed inclusions more rapidly than L38V and G93C Sod1. Expression of WTSod1 significantly decreased the intracellular oxidation levels in comparison with expression of fALS Sod1 mutants, suggesting the mutations induce a functional impairment. All fALS mutations impaired nuclear localization of Sod1, which is important for maintaining genomic stability. Consistently, expression of WTSod1, but not of fALS Sod1 mutants, reduced DNA damage, as measured by the comet assay. Altogether, our study sheds light into the effects of fALS Sod1 mutations on inclusion formation, dynamics, and localization as well as on antioxidant response, opening novel avenues for investigating the role of fALS Sod1 mutations in pathogenesis.

Molecular genetic analysis of the F11 gene in 14 Turkish patients with factor XI deficiency: identification of novel and recurrent mutations and their inheritance within families

PubMed Central

Colakoglu, Seyma; Bayhan, Turan; Tavil, Betül; Keskin, Ebru Yılmaz; Cakir, Volkan; Gümrük, Fatma; Çetin, Mualla; Aytaç, Selin; Berber, Ergul

2018-01-01

Background Factor XI (FXI) deficiency is an autosomal bleeding disease associated with genetic defects in the F11 gene which cause decreased FXI levels or impaired FXI function. An increasing number of mutations has been reported in the FXI mutation database, most of which affect the serine protease domain of the protein. FXI is a heterogeneous disorder associated with a variable bleeding tendency and a variety of causative F11 gene mutations. The molecular basis of FXI deficiency in 14 patients from ten unrelated families in Turkey was analysed to establish genotype-phenotype correlations and inheritance of the mutations in the patients’ families. Material and methods Fourteen index cases with a diagnosis of FXI deficiency and family members of these patients were enrolled into the study. The patients’ F11 genes were amplified by polymerase chain reaction and subjected to direct DNA sequencing analysis. The findings were analysed statistically using bivariate correlations, Pearson’s correlation coefficient and the nonparametric Mann-Whitney test. Results Direct DNA sequencing analysis of the F11 genes revealed that all of the 14 patients had a F11 gene mutation. Eight different mutations were identified in the apple 1, apple 2 or serine protease domains, except one which was a splice site mutation. Six of the mutations were recurrent. Two of the mutations were novel missense mutations, p.Val522Gly and p.Cys581Arg, within the catalytic domain. The p.Trp519Stop mutation was observed in two families whereas all the other mutations were specific to a single family. Discussion Identification of mutations confirmed the genetic heterogeneity of FXI deficiency. Most of the patients with mutations did not have any bleeding complications, whereas some had severe bleeding symptoms. Genetic screening for F11 gene mutations is important to decrease the mortality and morbidity rate associated with FXI deficiency, which can be life-threatening if bleeding occurs in tissues

Detection of MPL exon10 mutations in 103 Chinese patients with JAK2V617F-negative myeloproliferative neoplasms.

PubMed

Chen, Xiuhua; Qi, Xiling; Tan, Yanhong; Xu, Zhifang; Xu, Aining; Zhang, Linlin; Wang, Hongwei

2011-06-15

JAK2V617F mutation has been reported in 90% of patients with polycythemia vera (PV) and about 50% of patients with essential thromobocythemia (ET) and primary myelofibrosis (PMF). Recently, acquired mutations in the transmembrane-juxtamembrane region of MPL (MPLW515 mutations) have been reported in approximately 5% of JAK2V617F-negative PMF and about 1% of all cases of ET. MPL is the receptor for thrombopoietin that regulates the production of platelets by bone marrow. It is likely that some mutations more closely related to ET in MPL exon10 may have been missed by current assays. We inferred that there might be other mutations in MPL exon10 for MPN patients in addition to MPLW515 mutations. To investigate its mutation types and prevalence in Chinese patients with myeloproliferative neoplasms (MPN), we performed mutation detection on MPL exon10 in 103 JAK2V617F-negative MPN patients by single strand conformation polymorphism (SSCP) and allele-specific PCR (AS-PCR) combined with sequencing. As a result, one previously unrecognized MPL mutation (12-bp in-frame insertion) was identified in one patient with ET in addition to an MPLW515K mutation identified in one PMF patient. This confirms our hypothesis that BCR/ABL negative and JAK2V617F-negative MPN patients have other mutations besides W515 mutation in MPL exon10 and mutations other than single nucleotide exchange also exist. In addition, MPL mutation was associated with Chinese MPN patients. Copyright © 2011 Elsevier Inc. All rights reserved.

A mutation (L1014F) in the voltage-gated sodium channel of the grain aphid, Sitobion avenae, is associated with resistance to pyrethroid insecticides.

PubMed

Foster, Stephen P; Paul, Verity L; Slater, Russell; Warren, Anne; Denholm, Ian; Field, Linda M; Williamson, Martin S

2014-08-01

The grain aphid, Sitobion avenae Fabricius (Hemiptera: Aphididae), is an important pest of cereal crops. Pesticides are the main method for control but carry the risk of selecting for resistance. In response to reports of reduced efficacy of pyrethroid sprays applied to S. avenae, field samples were collected and screened for mutations in the voltage-gated sodium channel, the primary target site for pyrethroids. Aphid mobility and mortality to lambda-cyhalothrin were measured in coated glass vial bioassays. A single amino acid substitution (L1014F) was identified in the domain IIS6 segment of the sodium channel from the S. avenae samples exhibiting reduced pyrethroid efficacy. Bioassays on aphids heterozygous for the kdr mutation (SR) or homozygous for the wild-type allele (SS) showed that those carrying the mutation had significantly lower susceptibility to lambda-cyhalothrin. The L1014F (kdr) mutation, known to confer pyrethroid resistance in many insect pests, has been identified for the first time in S. avenae. Clonal lines heterozygous for the mutation showed 35-40-fold resistance to lambda-cyhalothrin in laboratory bioassays, consistent with the reported effect of this mutation on pyrethroid sensitivity in other aphid species. © 2013 Society of Chemical Industry.

Impact of JAK2V617F Mutation Burden on Disease Phenotype in Chinese Patients with JAK2V617F-positive Polycythemia Vera (PV) and Essential thrombocythemia (ET).

PubMed

Zhao, Shixiang; Zhang, Xiang; Xu, Yang; Feng, Yufeng; Sheng, Wenhong; Cen, Jiannong; Wu, Depei; Han, Yue

2016-01-01

Most patients with polycythemia vera (PV) and half of essential thrombocythemia (ET) possess an activating JAK2V617F mutation. The objective of this study was to better define the effect of JAK2V617F mutant allele burden on clinical phenotypes in Chinese patients, especially thrombosis. By real-time polymerase chain reaction (RT-PCR), the JAK2V617F mutation burden was detected in 170 JAK2V617F-positive patients, including 54 PV and 116 ET. The results showed that JAK2V617F allele burden was higher in PV than in ET (P< 0.001). Higher percentage of patients had JAK2V617F allele burden over 20% in PV than in ET (68.5% VS 26.7%) (P< 0.001). In PV patients, higher JAK2V617F allele burden was observed in female (P< 0.05) and leukocytosis patients (WBC above 10 × 10(9)/L) (P< 0.001). Meanwhile, ET patients showed increased JAK2V617F allele burden in the group with higher hemoglobin (HGB above 150 g/L) (P< 0.05), leukocytosis (WBC above 10 × 10(9)/L) (P< 0.001), splenomegaly (P< 0.05) and thrombosis (P< 0.05). In conclusion, the JAK2V617F mutation allele burden is higher in Chinese patients with PV than ET. In PV patients, JAK2V617F mutation burden had influence on WBC counts. And the clinical characteristics of ET patients, such as WBC counts, hemoglobin level, splenomegaly and thrombosis, were influenced by JAK2V617F mutation burden. Male, high hemoglobin (HGB above 150 g/L), and increased JAK2V617F mutation burden (JAK2V617F allele burden ≥ 16.5%) were risks of thrombosis (P< 0.05) for ET patients by Logistic Regression.

Krüppel-like factor 1 mutations and expression of hemoglobins F and A2 in homozygous hemoglobin E syndrome.

PubMed

Tepakhan, Wanicha; Yamsri, Supawadee; Fucharoen, Goonnapa; Sanchaisuriya, Kanokwan; Fucharoen, Supan

2015-07-01

The basis for variability of hemoglobin (Hb) F in homozygous Hb E disease is not well understood. We have examined multiple mutations of the Krüppel-like factor 1 (KLF1) gene; an erythroid specific transcription factor and determined their associations with Hbs F and A2 expression in homozygous Hb E. Four KLF1 mutations including G176AfsX179, T334R, R238H, and -154 (C-T) were screened using specific PCR assays on 461 subjects with homozygous Hb E and 100 normal controls. None of these four mutations were observed in 100 normal controls. Among 461 subjects with homozygous Hb E, 306 had high (≥5 %) and 155 had low (<5 %) Hb F. DNA analysis identified the KLF1 mutations in 35 cases of the former group with high Hb F, including the G176AfsX179 mutation (17/306 = 5.6 %), T334R mutation (9/306 = 2.9 %), -154 (C-T) mutation (7/306 = 2.3 %), and R328H mutation (2/306 = 0.7 %). Only two subjects in the latter group with low Hb F carried the G176AfsX179 and -154 (C-T) mutations. Significant higher Hb A2 level was observed in those of homozygous Hb E with the G176AfsX179 mutation as compared to those without KLF1 mutations. These results indicate that KLF1 is among the genetic factors associated with increased Hbs F and A2, and in combination with other factors could explain the variabilities of these Hb expression in Hb E syndrome.

Characterization and Prognosis Significance of JAK2 (V617F), MPL, and CALR Mutations in Philadelphia-Negative Myeloproliferative Neoplasms

PubMed

Singdong, Roongrudee; Siriboonpiputtana, Teerapong; Chareonsirisuthigul, Takol; Kongruang, Adcharee; Limsuwanachot, Nittaya; Sirirat, Tanasan; Chuncharunee, Suporn; Rerkamnuaychoke, Budsaba

2016-10-01

Background: The discovery of somatic acquired mutations of JAK2 (V617F) in Philadelphia-negative myeloproliferative neoplasms (Ph-negative MPNs) including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) has not only improved rational disease classification and prognostication but also brings new understanding insight into the pathogenesis of diseases. Dosage effects of the JAK2 (V617F) allelic burden in Ph-negative MPNs may partially influence clinical presentation, disease progression, and treatment outcome. Material and Methods: Pyrosequencing was performed to detect JAK2 (V617F) and MPL (W515K/L) and capillary electrophoresis to identify CALR exon 9.0 mutations in 100.0 samples of Ph-negative MPNs (38.0 PV, 55 ET, 4 PMF, and 3 MPN-U). Results: The results showed somatic mutations of JAK2 (V617F) in 94.7% of PV, 74.5% of ET, 25.0% of PMF, and all MPN-U. A high proportion of JAK2 (V617F) mutant allele burden (mutational load > 50.0%) was predominantly observed in PV when compared with ET. Although a high level of JAK2 (V617F) allele burden was strongly associated with high WBC counts in both PV and ET, several hematological parameters (hemoglobin, hematocrit, and platelet count) were independent of JAK2 (V617F) mutational load. MPL (W515K/L) mutations could not be detected whereas CALR exon 9.0 mutations were identified in 35.7% of patients with JAK2 negative ET and 33.3% with JAK2 negative PMF. Conclusions: The JAK2 (V617F) allele burden may be involved in progression of MPNs. Furthermore, a high level of JAK2 (V617F) mutant allele appears strongly associated with leukocytosis in both PV and ET. Creative Commons Attribution License

Characterization and Prognosis Significance of JAK2 (V617F), MPL, and CALR Mutations in Philadelphia-Negative Myeloproliferative Neoplasms

PubMed Central

Singdong, Roongrudee; Siriboonpiputtana, Teerapong; Chareonsirisuthigul, Takol; Kongruang, Adcharee; Limsuwanachot, Nittaya; Sirirat, Tanasan; Chuncharunee, Suporn; Rerkamnuaychoke, Budsaba

2016-01-01

Background: The discovery of somatic acquired mutations of JAK2 (V617F) in Philadelphia-negative myeloproliferative neoplasms (Ph-negative MPNs) including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) has not only improved rational disease classification and prognostication but also brings new understanding insight into the pathogenesis of diseases. Dosage effects of the JAK2 (V617F) allelic burden in Ph-negative MPNs may partially influence clinical presentation, disease progression, and treatment outcome. Material and Methods: Pyrosequencing was performed to detect JAK2 (V617F) and MPL (W515K/L) and capillary electrophoresis to identify CALR exon 9 mutations in 100 samples of Ph-negative MPNs (38.0 PV, 55 ET, 4 PMF, and 3 MPN-U). Results: The results showed somatic mutations of JAK2 (V617F) in 94.7% of PV, 74.5% of ET, 25.0% of PMF, and all MPN-U. A high proportion of JAK2 (V617F) mutant allele burden (mutational load > 50.0%) was predominantly observed in PV when compared with ET. Although a high level of JAK2 (V617F) allele burden was strongly associated with high WBC counts in both PV and ET, several hematological parameters (hemoglobin, hematocrit, and platelet count) were independent of JAK2 (V617F) mutational load. MPL (W515K/L) mutations could not be detected whereas CALR exon 9 mutations were identified in 35.7% of patients with JAK2 negative ET and 33.3% with JAK2 negative PMF. Conclusions: The JAK2 (V617F) allele burden may be involved in progression of MPNs. Furthermore, a high level of JAK2 (V617F) mutant allele appears strongly associated with leukocytosis in both PV and ET. PMID:27892678

Developmental and transcriptional consequences of mutations in Drosophila TAF(II)60.

PubMed

Aoyagi, N; Wassarman, D A

2001-10-01

In vitro, the TAF(II)60 component of the TFIID complex contributes to RNA polymerase II transcription initiation by serving as a coactivator that interacts with specific activator proteins and possibly as a promoter selectivity factor that interacts with the downstream promoter element. In vivo roles for TAF(II)60 in metazoan transcription are not as clear. Here we have investigated the developmental and transcriptional requirements for TAF(II)60 by analyzing four independent Drosophila melanogaster TAF(II)60 mutants. Loss-of-function mutations in Drosophila TAF(II)60 result in lethality, indicating that TAF(II)60 provides a nonredundant function in vivo. Molecular analysis of TAF(II)60 alleles revealed that essential TAF(II)60 functions are provided by two evolutionarily conserved regions located in the N-terminal half of the protein. TAF(II)60 is required at all stages of Drosophila development, in both germ cells and somatic cells. Expression of TAF(II)60 from a transgene rescued the lethality of TAF(II)60 mutants and exposed requirements for TAF(II)60 during imaginal development, spermatogenesis, and oogenesis. Phenotypes of rescued TAF(II)60 mutant flies implicate TAF(II)60 in transcriptional mechanisms that regulate cell growth and cell fate specification and suggest that TAF(II)60 is a limiting component of the machinery that regulates the transcription of dosage-sensitive genes. Finally, TAF(II)60 plays roles in developmental regulation of gene expression that are distinct from those of other TAF(II) proteins.

Molecular Dynamic Simulation Reveals Damaging Impact of RAC1 F28L Mutation in the Switch I Region

PubMed Central

Sethumadhavan, Rao; Purohit, Rituraj

2013-01-01

Ras-related C3 botulinum toxin substrate 1 (RAC1) is a plasma membrane-associated small GTPase which cycles between the active GTP-bound and inactive GDP-bound states. There is wide range of evidences indicating its active participation in inducing cancer-associated phenotypes. RAC1 F28L mutation (RACF28L) is a fast recycling mutation which has been implicated in several cancer associated cases. In this work we have performed molecular docking and molecular dynamics simulation (~0.3 μs) to investigate the conformational changes occurring in the mutant protein. The RMSD, RMSF and NHbonds results strongly suggested that the loss of native conformation in the Switch I region in RAC1 mutant protein could be the reason behind its oncogenic transformation. The overall results suggested that the mutant protein attained compact conformation as compared to the native. The major impact of mutation was observed in the Switch I region which might be the crucial reason behind the loss of interaction between the guanine ring and F28 residue. PMID:24146998

Effect of 60-Hz magnetic fields on ultraviolet light-induced mutation and mitotic recombination in Saccharomyces cerevisiae.

PubMed

Ager, D D; Radul, J A

1992-12-01

The purpose of this study was to examine the effect of extremely low frequency (ELF) magnetic fields on the induction of genetic damage. In general, mutational studies involving ELF magnetic fields have proven negative. However, studies examining sister-chromatid exchange and chromosome aberrations have yielded conflicting results. In this study, we have examined whether 60-Hz magnetic fields are capable of inducing mutation or mitotic recombination in the yeast Saccharomyces cerevisiae. In addition we determined whether magnetic fields were capable of altering the genetic response of S. cerevisiae to UV (254 nm). We measured the frequencies of induced mutation, gene conversion and reciprocal mitotic crossing-over for exposures to magnetic fields alone (1 mT) or in combination with various UV exposures (2-50 J/m2). These experiments were performed using a repair-proficient strain (RAD+), as well as a strain of yeast (rad3) which is incapable of excising UV-induced thymine dimers. Magnetic field exposures did not induce mutation, gene conversion or reciprocal mitotic crossing-over in either of these strains, nor did the fields influence the frequencies of UV-induced genetic events.

Molecular dynamics simulations reveal the allosteric effect of F1174C resistance mutation to ceritinib in ALK-associated lung cancer.

PubMed

Ni, Zhong; Wang, Xiting; Zhang, Tianchen; Jin, Rong Zhong

2016-12-01

Anaplastic lymphoma kinase (ALK) has become as an important target for the treatment of various human cancers, especially non-small-cell lung cancer. A mutation, F1174C, suited in the C-terminal helix αC of ALK and distal from the small-molecule inhibitor ceritinib bound to the ATP-binding site, causes the emergence of drug resistance to ceritinib. However, the detailed mechanism for the allosteric effect of F1174C resistance mutation to ceritinib remains unclear. Here, molecular dynamics (MD) simulations and binding free energy calculations [Molecular Mechanics/Generalized Born Surface Area (MM/GBSA)] were carried out to explore the advent of drug resistance mutation in ALK. MD simulations observed that the exquisite aromatic-aromatic network formed by residues F1098, F1174, F1245, and F1271 in the wild-type ALK-ceritinib complex was disrupted by the F1174C mutation. The resulting mutation allosterically affected the conformational dynamic of P-loop and caused the upward movement of the P-loop from the ATP-binding site, thereby weakening the interaction between ceritinib and the P-loop. The subsequent MM/GBSA binding free energy calculations and decomposition analysis of binding free energy validated this prediction. This study provides mechanistic insight into the allosteric effect of F1174C resistance mutation to ceritinib in ALK and is expected to contribute to design the next-generation of ALK inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

Epilepsy in hemiplegic migraine: Genetic mutations and clinical implications.

PubMed

Prontera, P; Sarchielli, P; Caproni, S; Bedetti, C; Cupini, L M; Calabresi, P; Costa, C

2018-02-01

Objective We performed a systematic review on the comorbidities of familial/sporadic hemiplegic migraine (F/SHM) with seizure/epilepsy in patients with CACNA1A, ATP1A2 or SCN1A mutations, to identify the genotypes associated and investigate for the presence of mutational hot spots. Methods We performed a search in MEDLINE and in the Human Gene Mutation and Leiden Open Variation Databases for mutations in the CACNA1A, ATP1A2 and SCN1A genes. After having examined the clinical characteristics of the patients, we selected those having HM and seizures, febrile seizures or epilepsy. For each gene, we determined both the frequency and the positions at protein levels of these mutations, as well as the penetrance of epilepsy within families. Results Concerning F/SHM-Epilepsy1 (F/SHME1) and F/SHME2 endophenotypes, we observed a prevalent involvement of the transmembrane domains, and a strong correlation in F/SHME1 when the positively charged amino acids were involved. The penetrance of epilepsy within the families was highest for patients carrying mutation in the CACNA1A gene (60%), and lower in those having SCN1A (33.3%) and ATP1A2 (30.9%) mutations. Conclusion Among the HM cases with seizure/epilepsy, we observed mutational hot spots in the transmembrane domains of CACNA1A and ATP1A2 proteins. These findings could lead to a better understanding of the pathological mechanisms underlying migraine and epilepsy, therein guaranteeing the most appropriate therapeutic approach.

Increased risk of recurrent thrombosis in patients with essential thrombocythemia carrying the homozygous JAK2 V617F mutation.

PubMed

De Stefano, Valerio; Za, Tommaso; Rossi, Elena; Vannucchi, Alessandro M; Ruggeri, Marco; Elli, Elena; Micò, Caterina; Tieghi, Alessia; Cacciola, Rossella R; Santoro, Cristina; Vianelli, Nicola; Guglielmelli, Paola; Pieri, Lisa; Scognamiglio, Francesca; Cacciola, Emma; Rodeghiero, Francesco; Pogliani, Enrico M; Finazzi, Guido; Gugliotta, Luigi; Leone, Giuseppe; Barbui, Tiziano

2010-02-01

Evidence suggests that the JAK2 V617F mutation is associated with an increased risk of first thrombosis in patients with essential thrombocythemia (ET). Whether this mutation is also a risk factor for recurrent thrombosis is currently unknown. To investigate the impact of the JAK2 V617F mutation on the risk of recurrent thrombosis in patients with ET, we carried out a multicentre retrospective cohort study. We recruited 143 patients with previous arterial (64.4%) or venous major thrombosis (34.8%) or both (0.8%); 98 of them (68.5%) carried the mutation. Thrombosis recurred in 43 of the patients (30%); overall, after adjustment for sex, age, presence of vascular risk factors, and treatment after the first thrombosis, the presence of the JAK2 mutation did not predict recurrence (multivariable hazard ratio, HR, 0.88, 95% CI 0.46-1.68). Indeed, the individuals homozygous for the JAK2 V617F (allele burden >50%) mutation had an increased risk of recurrence in comparison with wild-type patients (HR 6.15, 95% CI 1.51-24.92). In conclusion, a homozygous JAK2 V617F mutation is an independent risk factor for recurrent thrombosis in patients with ET.

Mutations in the tacF gene of clinical strains and laboratory transformants of Streptococcus pneumoniae: impact on choline auxotrophy and growth rate.

PubMed

González, Ana; Llull, Daniel; Morales, María; García, Pedro; García, Ernesto

2008-06-01

The nutritional requirement that Streptococcus pneumoniae has for the aminoalcohol choline as a component of teichoic and lipoteichoic acids appears to be exclusive to this prokaryote. A mutation in the tacF gene, which putatively encodes an integral membrane protein (possibly, a teichoic acid repeat unit transporter), has been recently identified as responsible for generating a choline-independent phenotype of S. pneumoniae (M. Damjanovic, A. S. Kharat, A. Eberhardt, A. Tomasz, and W. Vollmer, J. Bacteriol. 189:7105-7111, 2007). We now report that Streptococcus mitis can grow in choline-free medium, as previously illustrated for Streptococcus oralis. While we confirmed the finding by Damjanovic et al. of the involvement of TacF in the choline dependence of the pneumococcus, the genetic transformation of S. pneumoniae R6 by using S. mitis SK598 DNA and several PCR-amplified tacF fragments suggested that a minimum of two mutations were required to confer improved fitness to choline-independent S. pneumoniae mutants. This conclusion is supported by sequencing results also reported here that indicate that a spontaneous mutant of S. pneumoniae (strain JY2190) able to proliferate in the absence of choline (or analogs) is also a double mutant for the tacF gene. Microscopic observations and competition experiments during the cocultivation of choline-independent strains confirmed that a minimum of two amino acid changes were required to confer improved fitness to choline-independent pneumococcal strains when growing in medium lacking any aminoalcohol. Our results suggest complex relationships among the different regions of the TacF teichoic acid repeat unit transporter.

[Expression of JAK2V617F and MPLW515L/K mutation in 30 suspected cases of early myeloproliferative disorders].

PubMed

Fan, Zheng; Zhang, Ri; Shen, Yi-Min; Fei, Hai-Rong; Zhu, Zi-Ling; Cen, Jian-Nong

2008-09-01

To investigate the prevalence of JAK2V617F and MPLW515L/K mutation in patients with slightly elevated platelets (BPC) or hemoglobin (Hb) not meeting the criteria of polycythemia vera (PV) or essential thrombocythemia (ET). Genomic DNA from bone marrow or blood mononuclear cells was screened with allele specific polymerase chain reaction (AS-PCR) for JAK2V617F and MPLW515L/K mutation. The history of thrombosis was assessed retrospectively by patients files. Of 30 patients, 14 (46.7%) were positive for the JAK2V617F mutation, none of them had the MPLW515L/ K. Five of these 14 patients had a history of thrombosis. Follow-up results were available in 22 patients. Among them, 12 patients with JAK2V617F mutation turned out to be MPD in 6-24 months; only 2 out of 10 patients without this mutation evolved to MPD. JAK2V617F mutation could be one of the diagnosis criteria of early MPD. No MPLW515L/K expression was found in early MPD.

Significance of combined detection of JAK2V617F, MPL and CALR gene mutations in patients with essential thrombocythemia.

PubMed

Ji, Liying; Qian, Mengyao; Wu, Nana; Wu, Jianmin

2017-03-01

The aim of this study was to analyze the mutation rate of JAK2V617F, MPLW515L/K and CALR genes in adult patients with essential thrombocythemia (ET) and the accuracy of the combined detection by the receiver operating curve. Three hundred and forty-two cases with high-platelets (≥300×10 9 /l) were consecutively selected. The patients were analyzed for routine blood examination, bone marrow biopsy and genetic testing. One hundred and fifty-four cases (45.03%) were diagnosed with ET and 188 cases of secondary thrombocythemia according to the hematopoietic and lymphoid tissue tumor classification standards of 2008. It was found that the mutant type of three genes showed three bands, whereas only one band for wild-type. The JAK2V617F and MPL mutations did not cause a change in the open reading frame and the CALR mutation resulted in its change. The mutation rate of JAK2V617F and CALR in ET group was significantly higher than that in the secondary thrombocythemia group (p<0.05). The positive mutation rate of MPL was only 4.55%. JAK2V617F-positive mutation alone was used to diagnose with ET. The area under the curve (AUC) was 0.721. The sensitivity was 72.4%, the specificity was 79.5% and the cut-off value was 0.25. When CALR-positive mutation alone was used to diagnose ET, the AUC, sensitivity, specificity and cut-off value were 0.664, 68.4, 82.4 and 0.09%, respectively. JAK2V617F combined with CALR mutation were used for diagnosis of ET. The AUC was 0.862, the sensitivity was 85.9%, the specificity was 87.8%, and the cut-off values were 0.21 and 0.07. In conclusion, the positive mutation rate of JAK2V617F and CALR in ET was higher, and the sensitivity, specificity and accuracy of the diagnosis of ET were significantly improved using the detection of JAK2V617F and CALR.

Loss-of-function and gain-of-function phenotypes of stomatocytosis mutant RhAG F65S

PubMed Central

Stewart, Andrew K.; Shmukler, Boris E.; Vandorpe, David H.; Rivera, Alicia; Heneghan, John F.; Li, Xiaojin; Hsu, Ann; Karpatkin, Margaret; O'Neill, Allison F.; Bauer, Daniel E.; Heeney, Matthew M.; John, Kathryn; Kuypers, Frans A.; Gallagher, Patrick G.; Lux, Samuel E.; Brugnara, Carlo; Westhoff, Connie M.

2011-01-01

Four patients with overhydrated cation leak stomatocytosis (OHSt) exhibited the heterozygous RhAG missense mutation F65S. OHSt erythrocytes were osmotically fragile, with elevated Na and decreased K contents and increased cation channel-like activity. Xenopus oocytes expressing wild-type RhAG and RhAG F65S exhibited increased ouabain and bumetanide-resistant uptake of Li+ and 86Rb+, with secondarily increased 86Rb+ influx sensitive to ouabain and to bumetanide. Increased RhAG-associated 14C-methylammonium (MA) influx was severely reduced in RhAG F65S-expressing oocytes. RhAG-associated influxes of Li+, 86Rb+, and 14C-MA were pharmacologically distinct, and Li+ uptakes associated with RhAG and RhAG F65S were differentially inhibited by NH4+ and Gd3+. RhAG-expressing oocytes were acidified and depolarized by 5 mM bath NH3/NH4+, but alkalinized and depolarized by subsequent bath exposure to 5 mM methylammonium chloride (MA/MA+). RhAG F65S-expressing oocytes exhibited near-wild-type responses to NH4Cl, but MA/MA+ elicited attenuated alkalinization and strong hyperpolarization. Expression of RhAG or RhAG F65S increased steady-state cation currents unaltered by bath Li+ substitution or bath addition of 5 mM NH4Cl or MA/MA+. These oocyte studies suggest that 1) RhAG expression increases oocyte transport of NH3/NH4+ and MA/MA+; 2) RhAG F65S exhibits gain-of-function phenotypes of increased cation conductance/permeability, and loss-of-function phenotypes of decreased and modified MA/MA+ transport, and decreased NH3/NH4+-associated depolarization; and 3) RhAG transports NH3/NH4+ and MA/MA+ by distinct mechanisms, and/or the substrates elicit distinct cellular responses. Thus, RhAG F65S is a loss-of-function mutation for amine transport. The altered oocyte intracellular pH, membrane potential, and currents associated with RhAG or RhAG F65S expression may reflect distinct transport mechanisms. PMID:21849667

Inferior vena cava thrombosis and its relationship with the JAK2V617F mutation and chronic myeloproliferative disease.

PubMed

Linnemann, Birgit; Kraft, Christiane; Roskos, Martin; Zgouras, Dimitrios; Lindhoff-Last, Edelgard

2012-06-01

Splanchnic vein thrombosis (SVT) is a typical manifestation of polycythaemia vera (PV) or essential thrombocythaemia (ET). The recently discovered JAK2V617F somatic mutation is closely associated with chronic myeloproliferative disease (CMD). We investigated whether thrombosis involving the inferior vena cava (IVC) is also related to the JAK2V617F mutation or CMD. Blood samples were obtained from 40 IVC thrombosis patients. Fifty-three patients with isolated lower extremity deep vein thrombosis (LE-DVT) and 20 SVT patients served as controls. The presence of the JAK2V617F mutation was assessed by real-time polymerase chain reaction (RT-PCR). The JAK2V617F allele was not detected in any of the IVC thrombosis patients but was detected in one patient (2%) with isolated LE-DVT. However, the mutation-carrying patient did not exhibit symptoms of CMD. Even after an observation period of 30months, the patient's complete blood cell count did not exhibit any pathology. In contrast, the JAK2V617F allele was detected in four patients with SVT (20%) and CMD. According to our data, there is no evidence that IVC thrombosis is associated with the JAK2V617F mutation or the presence of chronic myeloproliferative disease. Copyright © 2011 Elsevier Ltd. All rights reserved.

Extending Jak2V617F and MplW515 mutation analysis to single hematopoietic colonies and B and T lymphocytes.

PubMed

Pardanani, Animesh; Lasho, Terra L; Finke, Christy; Mesa, Ruben A; Hogan, William J; Ketterling, Rhett P; Gilliland, Dwight Gary; Tefferi, Ayalew

2007-09-01

JAK2V617F and MPLW515L/K are myeloproliferative disorder (MPD)-associated mutations. We genotyped 552 individual hematopoietic colonies obtained by CD34+ cell culture from 16 affected patients (13 JAK2V617F and 3 MPLW515L/K) to determine (a) the proportion of colonies harboring a particular mutation in the presence or absence of cytokines, (b) the lineage distribution of endogenous colonies for each mutation, and (c) the differences (if any) in the pattern of mutation among the various MPDs, as established by genotyping of individual colonies. Genotyping analysis revealed cohabitation of mutation-negative and mutation-positive endogenous colonies in polycythemia vera as well as other MPDs. Culture of progenitor cells harboring MPLW515L/K yielded virtually no endogenous erythroid colonies in contrast to JAK2V617F-harboring progenitor cells. The mutation pattern (i.e., relative distribution of homozygous, heterozygous, or wild-type colonies) was not a distinguishing feature among the MPDs, and MPLW515 mutations were detected in B and/or T lymphocytes in all three patients tested. These observations suggest that clonal myelopoiesis antedates acquisition of JAK2V617F or MPLW515L/K mutations and that the latter is acquired in a lympho-myeloid progenitor cell.

Kras, Egfr, and Tp53 Mutations in B6C3F1/N Mouse and F344/NTac Rat Alveolar/Bronchiolar Carcinomas Resulting from Chronic Inhalation Exposure to Cobalt Metal

PubMed Central

Hong, Hue-Hua L.; Hoenerhoff, Mark J.; Ton, Thai-Vu; Herbert, Ronald A.; Kissling, Grace E.; Hooth, Michelle J.; Behl, Mamta; Witt, Kristine L.; Smith-Roe, Stephanie L.; Sills, Robert C.; Pandiri, Arun R.

2015-01-01

Rodent lung tumors are morphologically similar to a subtype of human lung adenocarcinomas. The objective of this study was to evaluate Kras, Egfr and Tp53 mutations, which are relevant to human lung cancer, in cobalt metal dust (CMD) induced alveolar/bronchiolar tumors of B6C3F1/N mice and F344/NTac rats. Kras mutations were detected in 67% (mice) and 31% (rats) of CMD-induced lung tumors, and were predominantly exon 1 codon 12 G to T transversions (80% in mice and 57% in rats). Egfr mutations were detected in 17% (both mice and rats) of CMD-induced lung tumors, and were predominantly in exon 20 with 50% G to A transitions (mice and rats). Tp53 mutations were detected in 19% (mice) and 23% (rats) of CMD-induced lung tumors and were predominantly in exon 5 (mice, 69% transversions) and exon 6 (rats, all transitions). No mutations were observed for these genes in spontaneous lung tumors or normal lungs from untreated controls. Ames assays indicated that CMD is mutagenic in the absence but not in the presence of S9 mix. Thus, the mutation data (G to T transversions) and Ames assay results suggest that oxidative damage to DNA may be a contributing factor in CMD-induced pulmonary carcinogenesis in rodents. PMID:26059825

Polymorphism of intron-1 in the voltage-gated sodium channel gene of Anopheles gambiae s.s. populations from Cameroon with emphasis on insecticide knockdown resistance mutations.

PubMed

Etang, Josiane; Vicente, Jose L; Nwane, Philippe; Chouaibou, Mouhamadou; Morlais, Isabelle; Do Rosario, Virgilio E; Simard, Frederic; Awono-Ambene, Parfait; Toto, Jean Claude; Pinto, Joao

2009-07-01

Sequence variation at the intron-1 of the voltage-gated sodium channel gene in Anopheles gambiae M- and S-forms from Cameroon was assessed to explore the number of mutational events originating knockdown resistance (kdr) alleles. Mosquitoes were sampled between December 2005 and June 2006 from three geographical areas: (i) Magba in the western region; (ii) Loum, Tiko, Douala, Kribi, and Campo along the Atlantic coast; and (iii) Bertoua, in the eastern continental plateau. Both 1014S and 1014F kdr alleles were found in the S-form with overall frequencies of 14% and 42% respectively. Only the 1014F allele was found in the M-form at lower frequency (11%). Analysis of a 455 bp region of intron-1 upstream the kdr locus revealed four independent mutation events originating kdr alleles, here named MS1 -1014F, S1-1014S and S2-1014S kdr-intron-1 haplotypes in S-form and MS3-1014F kdr-intron-1 haplotype in the M-form. Furthermore, there was evidence for mutual introgression of kdr 1014F allele between the two molecular forms, MS1 and MS3 being widely shared by them. Although no M/S hybrid was observed in analysed samples, this wide distribution of haplotypes MS1 and MS3 suggests inter-form hybridizing at significant level and emphasizes the rapid diffusion of the kdr alleles in Africa. The mosaic of genetic events found in Cameroon is representative of the situation in the West-Central African region and highlights the importance of evaluating the spatial and temporal evolution of kdr alleles for a better management of insecticide resistance.

Impact of mutations on the midpoint potential of the [4Fe-4S]+1,+2 cluster and on catalytic activity in electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO).

PubMed

Usselman, Robert J; Fielding, Alistair J; Frerman, Frank E; Watmough, Nicholas J; Eaton, Gareth R; Eaton, Sandra S

2008-01-08

Electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is an iron-sulfur flavoprotein that accepts electrons from electron-transfer flavoprotein (ETF) and reduces ubiquinone from the Q-pool. ETF-QO contains a single [4Fe-4S]2+,1+ cluster and one equivalent of FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. Mutations were introduced by site-directed mutagenesis of amino acids in the vicinity of the iron-sulfur cluster of Rhodobacter sphaeroides ETF-QO. Y501 and T525 are equivalent to Y533 and T558 in the porcine ETF-QO. In the porcine protein, these residues are within hydrogen-bonding distance of the Sgamma of the cysteine ligands to the iron-sulfur cluster. Y501F, T525A, and Y501F/T525A substitutions were made to determine the effects on midpoint potential, activity, and EPR spectral properties of the cluster. The integrity of the mutated proteins was confirmed by optical spectra, EPR g-values, and spin-lattice relaxation rates, and the cluster to flavin point-dipole distance was determined by relaxation enhancement. Potentiometric titrations were monitored by changes in the CW EPR signals of the cluster and semiquinone. Single mutations decreased the midpoint potentials of the iron-sulfur cluster from +37 mV for wild type to -60 mV for Y501F and T525A and to -128 mV for Y501F/T525A. Lowering the midpoint potential resulted in a decrease in steady-state ubiquinone reductase activity and in ETF semiquinone disproportionation. The decrease in activity demonstrates that reduction of the iron-sulfur cluster is required for activity. There was no detectable effect of the mutations on the flavin midpoint potentials.

S locus-linked F-box genes expressed in anthers of Hordeum bulbosum.

PubMed

Kakeda, Katsuyuki

2009-09-01

Diploid Hordeum bulbosum (a wild relative of cultivated barley) exhibits a two-locus self-incompatibility (SI) system gametophytically controlled by the unlinked multiallelic loci S and Z. This unique SI system is observed in the grasses (Poaceae) including the tribe Triticeae. This paper describes the identification and characterization of two F-box genes cosegregating with the S locus in H. bulbosum, named Hordeum S locus-linked F-box 1 (HSLF1) and HSLF2, which were derived from an S (3) haplotype-specific clone (HAS175) obtained by previous AMF (AFLP-based mRNA fingerprinting) analysis. Sequence analysis showed that both genes encode similar F-box proteins with a C-terminal leucine-rich repeat (LRR) domain, which are distinct from S locus (or S haplotype-specific) F-box protein (SLF/SFB), a class of F-box proteins identified as the pollen S determinant in S-RNase-based gametophytic SI systems. A number of homologous F-box genes with an LRR domain were found in the rice genome, although the functions of the gene family are unknown. One allele of the HSLF1 gene (HSLF1-S (3)) was expressed specifically in mature anthers, whereas no expression was detected from the other two alleles examined. Although the degree of sequence polymorphism among the three HSLF1 alleles was low, a frameshift mutation was found in one of the unexpressed alleles. The HSLF2 gene showed a low level of expression with no tissue specificity as well as little sequence polymorphism among the three alleles. The multiplicity of S locus-linked F-box genes is discussed in comparison with those found in the S-RNase-based SI system.

Significance of combined detection of JAK2V617F, MPL and CALR gene mutations in patients with essential thrombocythemia

PubMed Central

Ji, Liying; Qian, Mengyao; Wu, Nana; Wu, Jianmin

2017-01-01

The aim of this study was to analyze the mutation rate of JAK2V617F, MPLW515L/K and CALR genes in adult patients with essential thrombocythemia (ET) and the accuracy of the combined detection by the receiver operating curve. Three hundred and forty-two cases with high-platelets (≥300×109/l) were consecutively selected. The patients were analyzed for routine blood examination, bone marrow biopsy and genetic testing. One hundred and fifty-four cases (45.03%) were diagnosed with ET and 188 cases of secondary thrombocythemia according to the hematopoietic and lymphoid tissue tumor classification standards of 2008. It was found that the mutant type of three genes showed three bands, whereas only one band for wild-type. The JAK2V617F and MPL mutations did not cause a change in the open reading frame and the CALR mutation resulted in its change. The mutation rate of JAK2V617F and CALR in ET group was significantly higher than that in the secondary thrombocythemia group (p<0.05). The positive mutation rate of MPL was only 4.55%. JAK2V617F-positive mutation alone was used to diagnose with ET. The area under the curve (AUC) was 0.721. The sensitivity was 72.4%, the specificity was 79.5% and the cut-off value was 0.25. When CALR-positive mutation alone was used to diagnose ET, the AUC, sensitivity, specificity and cut-off value were 0.664, 68.4, 82.4 and 0.09%, respectively. JAK2V617F combined with CALR mutation were used for diagnosis of ET. The AUC was 0.862, the sensitivity was 85.9%, the specificity was 87.8%, and the cut-off values were 0.21 and 0.07. In conclusion, the positive mutation rate of JAK2V617F and CALR in ET was higher, and the sensitivity, specificity and accuracy of the diagnosis of ET were significantly improved using the detection of JAK2V617F and CALR. PMID:28450924

Increased frequency of co-existing JAK2 exon-12 or MPL exon-10 mutations in patients with low JAK2(V617F) allelic burden.

PubMed

Nussenzveig, Roberto H; Pham, Ha T; Perkins, Sherrie L; Prchal, Josef T; Agarwal, Archana M; Salama, Mohamed E

2016-01-01

The frequency of co-existing JAK2(V617F)/MPL and JAK2(V617F)/JAK2 exon-12 mutations has not been previously investigated in MPNs. Poor survival was reported in primary myelofibrosis with low JAK2(V617F) allelic burden. However, mutational status of JAK2 exon-12 or MPL were not reported in these patients. This study developed a cost-effective multiplex high resolution melt assay that screens for mutations in JAK2 gene exons-12 and -14 ((V617F)) and MPL gene exon-10. Co-existing mutations with JAK2(V617F) were detected in 2.9% (6/208; two JAK2 exon-12 and four MPL exon-10) patient specimens with known JAK2(V617F) (allelic-burden range: 0.1-96.8%). Co-existing mutations were detected in specimens with < 12% JAK2(V617F) allelic burden. Current WHO guidelines do not recommend further testing once JAK2(V617F) mutation is detected in MPNs. The findings, however, indicate that quantification of JAK2(V617F) allele burden may be clinically relevant in MPNs and in those with low allelic burden additional testing for JAK2 exon-12 and MPL exon-10 mutation should be pursued.

A novel mutation in the alpha-helix 1 of the C subunit of the F(1)/F(0) ATPase responsible for optochin resistance of a Streptococcus pneumoniae clinical isolate.

PubMed

Cogné, N; Claverys, J; Denis, F; Martin, C

2000-10-01

Previously reported mutations involved in optochin resistance of Streptococcus pneumoniae clinical isolates changed residues 48, 49 or 50, in the transmembrane alpha-helix 2 of the F(1)/F(0) ATPase subunit. We report here an unusual mutation which changes the sequence of the transmembrane alpha-helix 1 of the AtpC subunit. This mutation involves a Gly to Ser substitution resulting from a G to A transition at codon 14 of the atpC gene.

Impact of Mutations on the Midpoint Potential of the [4Fe-4S]+1,+2 Cluster and on Catalytic Activity in Electron Transfer Flavoprotein-ubiquinone Oxidoreductase (ETF-QO)†

PubMed Central

Usselman, Robert J.; Fielding, Alistair J.; Frerman, Frank E.; Watmough, Nicholas J.; Eaton, Gareth R.; Eaton, Sandra S.

2011-01-01

Electron transfer flavoprotein - ubiquinone oxidoreductase (ETF-QO) is an iron-sulfur flavoprotein that accepts electrons from electron-transfer flavoprotein (ETF) and reduces ubiquinone from the Q-pool. ETF-QO contains a single [4Fe-4S]2+,1+ cluster and one equivalent of FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. Mutations were introduced by site-directed mutagenesis of amino acids in the vicinity of the iron-sulfur cluster of Rhodobacter sphaeroides ETF-QO. Y501 and T525 are equivalent to Y533 and T558 in the porcine ETF-QO. In the porcine protein, these residues are within hydrogen bonding distance of the Sγ of the cysteine ligands to the iron-sulfur cluster. Y501F, T525A, and Y501F/T525A substitutions were made to determine the effects on midpoint potential, activity, and EPR spectral properties of the cluster. The integrity of the mutated proteins was confirmed by optical spectra, EPR g-values, and spin-lattice relaxation rates, and the cluster to flavin point-dipole distance was determined by relaxation enhancement. Potentiometric titrations were monitored by changes in the CW EPR signals of the cluster and semiquinone. Single mutations decreased the mid-point potentials of the iron-sulfur cluster from +37 mV for wild type to −60 mV for Y501F and T525A and to −128 mV for Y501F/T525A. Lowering the midpoint potential resulted in a decrease in steady-state ubiquinone reductase activity and in ETF semiquinone disproportionation. The decrease in activity demonstrates that reduction of the iron-sulfur cluster is required for activity. There was no detectable effect of the mutations on the flavin midpoint potentials. PMID:18069858

Cystic fibrosis in the Basque country: high frequency of mutation delta F508 in patients of Basque origin.

PubMed Central

Casals, T; Vázquez, C; Lázaro, C; Girbau, E; Giménez, F J; Estivill, X

1992-01-01

The Basque population is one of the oldest populations of Europe. It has been suggested that the Basques arose from a population established in western Europe during the late Paleolithic Age. The Basque language (Euskera) is a supposedly pre-Indo-European language that originates from the first settlers of Europe. The variable distribution of the major cystic fibrosis (CF) mutation (delta F508 deletion) in Europe, with higher frequencies of the mutation in northern Europe and lower frequencies in southern Europe, has suggested that the delta F508 mutation was spread by early farmers migrating from the Middle East during the Neolithic period. We have studied 45 CF families from the Basque Country, where the incidence of CF is approximately 1/4,500. The birthplaces of the parents and grandparents have been traced and are distributed according to their origin as Basque or Mixed Basque. The frequency of the delta F508 mutation in the chromosomes of Basque origin is 87%, compared with 58% in those of Mixed Basque origin. The analysis of haplotypes, both with markers closely linked to the CF gene and with intragenic markers, suggests that the delta F508 mutation was not spread by the Indo-European invasions but was already present in Europe more than 10,000 years ago, during the Paleolithic period. PMID:1370875

Accurate, simple, and inexpensive assays to diagnose F8 gene inversion mutations in hemophilia A patients and carriers.

PubMed

Dutta, Debargh; Gunasekera, Devi; Ragni, Margaret V; Pratt, Kathleen P

2016-12-27

The most frequent mutations resulting in hemophilia A are an intron 22 or intron 1 gene inversion, which together cause ∼50% of severe hemophilia A cases. We report a simple and accurate RNA-based assay to detect these mutations in patients and heterozygous carriers. The assays do not require specialized equipment or expensive reagents; therefore, they may provide useful and economic protocols that could be standardized for central laboratory testing. RNA is purified from a blood sample, and reverse transcription nested polymerase chain reaction (RT-NPCR) reactions amplify DNA fragments with the F8 sequence spanning the exon 22 to 23 splice site (intron 22 inversion test) or the exon 1 to 2 splice site (intron 1 inversion test). These sequences will be amplified only from F8 RNA without an intron 22 or intron 1 inversion mutation, respectively. Additional RT-NPCR reactions are then carried out to amplify the inverted sequences extending from F8 exon 19 to the first in-frame stop codon within intron 22 or a chimeric transcript containing F8 exon 1 and the VBP1 gene. These latter 2 products are produced only by individuals with an intron 22 or intron 1 inversion mutation, respectively. The intron 22 inversion mutations may be further classified (eg, as type 1 or type 2, reflecting the specific homologous recombination sites) by the standard DNA-based "inverse-shifting" PCR assay if desired. Efficient Bcl I and T4 DNA ligase enzymes that cleave and ligate DNA in minutes were used, which is a substantial improvement over previous protocols that required overnight incubations. These protocols can accurately detect F8 inversion mutations via same-day testing of patient samples.

Correlation of EGFR or KRAS mutation status with 18F-FDG uptake on PET-CT scan in lung adenocarcinoma.

PubMed

Takamochi, Kazuya; Mogushi, Kaoru; Kawaji, Hideya; Imashimizu, Kota; Fukui, Mariko; Oh, Shiaki; Itoh, Masayoshi; Hayashizaki, Yoshihide; Ko, Weijey; Akeboshi, Masao; Suzuki, Kenji

2017-01-01

18F-fluoro-2-deoxy-glucose (18F-FDG) positron emission tomography (PET) is a functional imaging modality based on glucose metabolism. The correlation between EGFR or KRAS mutation status and the standardized uptake value (SUV) of 18F-FDG PET scanning has not been fully elucidated. Correlations between EGFR or KRAS mutation status and clinicopathological factors including SUVmax were statistically analyzed in 734 surgically resected lung adenocarcinoma patients. Molecular causal relationships between EGFR or KRAS mutation status and glucose metabolism were then elucidated in 62 lung adenocarcinomas using cap analysis of gene expression (CAGE), a method to determine and quantify the transcription initiation activities of mRNA across the genome. EGFR and KRAS mutations were detected in 334 (46%) and 83 (11%) of the 734 lung adenocarcinomas, respectively. The remaining 317 (43%) patients had wild-type tumors for both genes. EGFR mutations were more frequent in tumors with lower SUVmax. In contrast, no relationship was noted between KRAS mutation status and SUVmax. CAGE revealed that 4 genes associated with glucose metabolism (GPI, G6PD, PKM2, and GAPDH) and 5 associated with the cell cycle (ANLN, PTTG1, CIT, KPNA2, and CDC25A) were positively correlated with SUVmax, although expression levels were lower in EGFR-mutated than in wild-type tumors. No similar relationships were noted with KRAS mutations. EGFR-mutated adenocarcinomas are biologically indolent with potentially lower levels of glucose metabolism than wild-type tumors. Several genes associated with glucose metabolism and the cell cycle were specifically down-regulated in EGFR-mutated adenocarcinomas.

Identification of Novel PROP1 and POU1F1 Mutations in Patients with Combined Pituitary Hormone Deficiency.

PubMed

Birla, S; Khadgawat, R; Jyotsna, V P; Jain, V; Garg, M K; Bhalla, A S; Sharma, A

2016-12-01

Growth hormone deficiency (GHD) results from variations affecting the production and release of growth hormone (GH) and is of 2 types: isolated growth hormone deficiency (IGHD) and combined pituitary hormone deficiency (CPHD). IGHD results from mutations in GH1 and GHRHR while CPHD is associated with defects in transcription factor genes PROP1 , POU1F1 , and HESX1. The present study reports on screening of POU1F1 , PROP1 , and HESX1 in CPHD patients and the novel variations identified. Fifty-one CPHD patients from 49 unrelated families clinically diagnosed on the basis of biochemical and imaging investigations along with 100 controls were enrolled. Detailed family history was noted from all participants and 5 ml blood samples drawn were processed for DNA isolation followed by direct sequencing of POU1F1 , PROP1 , and HESX1 genes. Of the 51 patients, 8 were females and 43 were males. Mean height standard deviation score (SDS) and weight SDS were -5.50 and -2.76, respectively. Thirty-six of the 51 patients underwent MRI of which 9 (25%) had normal pituitary structure and morphology while 27 (75%) showed abnormalities. Molecular analysis revealed 10 (20%) patients to have POU1F1 and PROP1 mutations/variations of which 5 were novel and 2 previously reported. No mutations were identified in HESX1. The novel variations identified were absent in the 100 healthy individuals screened and the control database Exome Aggregation Consortium (ExAC). Reported POU1F1 and PROP1 mutation hotspots were absent in our patients. Instead, novel POU1F1 changes were identified suggesting existence of a distinct mutation spectrum in our population. © Georg Thieme Verlag KG Stuttgart · New York.

Characterization of an apparently synonymous F5 mutation causing aberrant splicing and factor V deficiency.

PubMed

Nuzzo, F; Bulato, C; Nielsen, B I; Lee, K; Wielders, S J; Simioni, P; Key, N S; Castoldi, E

2015-03-01

Coagulation factor V (FV) deficiency is a rare autosomal recessive bleeding disorder. We investigated a patient with severe FV deficiency (FV:C < 3%) and moderate bleeding symptoms. Thrombin generation experiments showed residual FV expression in the patient's plasma, which was quantified as 0.7 ± 0.3% by a sensitive prothrombinase-based assay. F5 gene sequencing identified a novel missense mutation in exon 4 (c.578G>C, p.Cys193Ser), predicting the abolition of a conserved disulphide bridge, and an apparently synonymous variant in exon 8 (c.1281C>G). The observation that half of the patient's F5 mRNA lacked the last 18 nucleotides of exon 8 prompted us to re-evaluate the c.1281C>G variant for its possible effects on splicing. Bioinformatics sequence analysis predicted that this transversion would activate a cryptic donor splice site and abolish an exonic splicing enhancer. Characterization in a F5 minigene model confirmed that the c.1281C>G variant was responsible for the patient's splicing defect, which could be partially corrected by a mutation-specific morpholino antisense oligonucleotide. The aberrantly spliced F5 mRNA, whose stability was similar to that of the normal mRNA, encoded a putative FV mutant lacking amino acids 427-432. Expression in COS-1 cells indicated that the mutant protein is poorly secreted and not functional. In conclusion, the c.1281C>G mutation, which was predicted to be translationally silent and hence neutral, causes FV deficiency by impairing pre-mRNA splicing. This finding underscores the importance of cDNA analysis for the correct assessment of exonic mutations. © 2014 John Wiley & Sons Ltd.

High penetrance of pheochromocytoma associated with the novel C634Y/Y791F double germline mutation in the RET protooncogene.

PubMed

Toledo, Rodrigo A; Wagner, Simona M; Coutinho, Flavia L; Lourenço, Delmar M; Azevedo, Juliana A; Longuini, Viviane C; Reis, Mariana T A; Siqueira, Sheila A C; Lucon, Antonio M; Tavares, Marcos R; Fragoso, Maria C B V; Pereira, Adelaide A; Dahia, Patricia L M; Mulligan, Lois M; Toledo, Sergio P A

2010-03-01

Previous studies have shown that double RET mutations may be associated with unusual multiple endocrine neoplasia type 2 (MEN 2) phenotypes. Our objective was to report the clinical features of patients harboring a previously unreported double mutation of the RET gene and to characterize this mutation in vitro. Sixteen patients from four unrelated families and harboring the C634Y/Y791F double RET germline mutation were included in the study. Large pheochromocytomas measuring 6.0-14 cm and weighing up to 640 g were identified in the four index cases. Three of the four tumors were bilateral. High penetrance of pheochromocytoma was also seen in the C634Y/Y791F-mutation-positive relatives (seven of nine, 77.7%). Of these, two cases had bilateral tumors, one presented with multifocal tumors, two cases had large tumors (>5 cm), and one case, which was diagnosed with a large (5.5 x 4.5 x 4.0 cm) pheochromocytoma, reported early onset of symptoms of the disease (14 yr old). The overall penetrance of pheochromocytoma was 84.6% (11 of 13). Development of medullary thyroid carcinoma in our patients seemed similar to that observed in patients with codon 634 mutations. Haplotype analysis demonstrated that the mutation did not arise from a common ancestor. In vitro studies showed the double C634Y/Y791F RET receptor was significantly more phosphorylated than either activated wild-type receptor or single C634Y and Y791F RET mutants. Our data suggest that the natural history of the novel C634Y/Y791F double mutation carries a codon 634-like pattern of medullary thyroid carcinoma development, is associated with increased susceptibility to unusually large bilateral pheochromocytomas, and is likely more biologically active than each individual mutation.

Important Mutations Contributing to High-Level Penicillin Resistance in Taiwan19F-14, Taiwan23F-15, and Spain23F-1 of Streptococcus pneumoniae Isolated from Taiwan.

PubMed

Liu, Esther Yip-Mei; Chang, Jen-Chang; Lin, Jung-Chung; Chang, Feng-Yee; Fung, Chang-Phone

2016-12-01

Penicillin-resistant Streptococcus pneumoniae is a serious concern worldwide. In this study, we analyzed the cause of β-lactam resistance in pandemic multidrug-resistant clones. A total of 41 penicillin-nonsusceptible clinical isolates were collected from 1996 to 2012. Sero- and molecular typing confirmed that these isolates were clonal types of Taiwan 19F -14, Taiwan 23F -15, and Spain 23F -1. Sero-switching was found in four isolates. All isolates were multidrug resistant. Sequencing analysis of the penicillin binding proteins (PBPs) was performed on PBP1a, 2b, and 2x, and a large number of mutations were identified in comparing to clinical penicillin-susceptible isolates and the recipient strain R6 used for homologous recombination. The T 451 A substitution was the key amino acid in PBP2b that contributed to penicillin resistance. T 338 A in PBP2x played a role in resistance and reached the highest level of resistance when combined with other mutations in PBP2x. High-level penicillin resistance could not be obtained without the combination of mutations in PBP1a with PBP2b and 2x. The amino acid substitutions in PBP1a, 2b, and 2x were the crucial factors for β-lactam resistance.

Molecular mechanistic explanation for the spectrum of cholestatic disease caused by the S320F variant of ABCB4.

PubMed

Andress, Edward J; Nicolaou, Michael; Romero, Marta R; Naik, Sandhia; Dixon, Peter H; Williamson, Catherine; Linton, Kenneth J

2014-05-01

ABCB4 flops phosphatidylcholine into the bile canaliculus to protect the biliary tree from the detergent activity of bile salts. Homozygous-null ABCB4 mutations cause the childhood liver disease, progressive familial intrahepatic cholestasis, but cause and effect is less clear, with many missense mutations linked to less severe cholestatic diseases. ABCB4(S320F), in particular, is described in 13 patients, including in heterozygosity with ABCB4(A286V), ABCB4(A953D), and null mutants, whose symptoms cover the spectrum of cholestatic disease. We sought to define the impact of these mutations on the floppase, explain the link with multiple conditions at the molecular level, and investigate the potential for reversal. ABCB4(S320F), ABCB4(A286V), and ABCB4(A953D) expression was engineered in naïve cultured cells. Floppase expression, localization, and activity were measured by western blot, confocal microscopy, and lipid transport assays, respectively. ABCB4(S320F) was fully active for floppase activity but expression at the plasma membrane was reduced to 50%. ABCB4(A286V) expressed and trafficked efficiently but could not flop lipid, and ABCB4(A953D) expressed poorly and was impaired in floppase activity. Proteasome inhibition stabilized nascent ABCB4(S320F) and ABCB4(A953D) but did not improve plasma membrane localization. Cyclosporin-A improved plasma membrane localization of both ABCB4(S320F) and ABCB4(A953D), but inhibited floppase activity. The level of ABCB4 functionality correlates with, and is the primary determinant of, cholestatic disease severity in these patients. ABCB4(S320F) homozygosity, with half the normal level of ABCB4, is the tipping point between more benign and potentially fatal cholestasis and makes these patients more acutely sensitive to environmental effects. Cyclosporin-A increased expression of ABCB4(S320F) and ABCB4(A953D), suggesting that chemical chaperones could be exploited for therapeutic benefit to usher in a new era of personalized

A genetic screen of the mutations in the Korean patients with early-onset Alzheimer’s disease

PubMed Central

An, Seong Soo; Park, Sun Ah; Bagyinszky, Eva; Bae, Sun Oh; Kim, Yoon-Jeong; Im, Ji Young; Park, Kyung Won; Park, Kee Hyung; Kim, Eun-Joo; Jeong, Jee Hyang; Kim, Jong Hun; Han, Hyun Jeong; Choi, Seong Hye; Kim, SangYun

2016-01-01

Early-onset Alzheimer’s disease (EOAD) has distinct clinical characteristics in comparison to late-onset Alzheimer’s disease (LOAD). The genetic contribution is suggested to be more potent in EOAD. However, the frequency of causative mutations in EOAD could be variable depending on studies. Moreover, no mutation screening study has been performed yet employing large population in Korea. Previously, we reported that the rate of family history of dementia in EOAD patients was 18.7% in a nationwide hospital-based cohort study, the Clinical Research Center for Dementia of South Korea (CREDOS) study. This rate is much lower than in other countries and is even comparable to the frequency of LOAD patients in our country. To understand the genetic characteristics of EOAD in Korea, we screened the common Alzheimer’s disease (AD) mutations in the consecutive EOAD subjects from the CREDOS study from April 2012 to February 2014. We checked the sequence of APP (exons 16–17), PSEN1 (exons 3–12), and PSEN2 (exons 3–12) genes. We identified different causative or probable pathogenic AD mutations, PSEN1 T116I, PSEN1 L226F, and PSEN2 V214L, employing 24 EOAD subjects with a family history and 80 without a family history of dementia. PSEN1 T116I case demonstrated autosomal dominant trait of inheritance, with at least 11 affected individuals over 2 generations. However, there was no family history of dementia within first-degree relation in PSEN1 L226F and PSEN2 V214L cases. Approximately, 55.7% of the EOAD subjects had APOE ε4 allele, while none of the mutation-carrying subjects had the allele. The frequency of genetic mutation in this study is lower compared to the studies from other countries. The study design that was based on nationwide cohort, which minimizes selection bias, is thought to be one of the contributors to the lower frequency of genetic mutation. However, the possibility of the greater likeliness of earlier onset of sporadic AD in Korea cannot be

Co-occurrence and distribution of East (L1014S) and West (L1014F) African knock-down resistance in Anopheles gambiae sensu lato population of Tanzania

PubMed Central

Kabula, Bilali; Kisinza, William; Tungu, Patrick; Ndege, Chacha; Batengana, Benard; Kollo, Douglas; Malima, Robert; Kafuko, Jessica; Mohamed, Mahdi; Magesa, Stephen

2014-01-01

Objective Insecticide resistance molecular markers can provide sensitive indicators of resistance development in Anopheles vector populations. Assaying these makers is of paramount importance in the resistance monitoring programme. We investigated the presence and distribution of knock-down resistance (kdr) mutations in Anopheles gambiae s.l. in Tanzania. Methods Indoor-resting Anopheles mosquitoes were collected from 10 sites and tested for insecticide resistance using the standard WHO protocol. Polymerase chain reaction-based molecular diagnostics were used to genotype mosquitoes and detect kdr mutations. Results The An. gambiae tested were resistance to lambdacyhalothrin in Muheza, Arumeru and Muleba. Out of 350 An. gambiae s.l. genotyped, 35% were An. gambiae s.s. and 65% An. arabiensis. L1014S and L1014F mutations were detected in both An. gambiae s.s. and An. arabiensis. L1014S point mutation was found at the allelic frequency of 4–33%, while L1014F was at the allelic frequency 6–41%. The L1014S mutation was much associated with An. gambiae s.s. (χ2 = 23.41; P < 0.0001) and L1014F associated with An. arabiensis (χ2 = 11.21; P = 0.0008). The occurrence of the L1014S allele was significantly associated with lambdacyhalothrin resistance mosquitoes (Fisher exact P < 0.001). Conclusion The observed co-occurrence of L1014S and L1014F mutations coupled with reports of insecticide resistance in the country suggest that pyrethroid resistance is becoming a widespread phenomenon among our malaria vector populations. The presence of L1014F mutation in this East African mosquito population indicates the spreading of this gene across Africa. The potential operational implications of these findings on malaria control need further exploration. Objectif Les marqueurs moléculaires de la résistance aux insecticides peuvent fournir des indicateurs sensibles du développement de la résistance dans les populations de vecteurs Anopheles. Le test de ces

Establishment and rapid detection of a heterozygous missense mutation in the CACNA1F gene by ARMS technique with double-base mismatched primers.

PubMed

Yang, W C; Zhu, L; Zhou, B X; Tania, S; Zhou, Q; Khan, M A; Fu, X L; Cheng, J L; Lv, H B; Fu, J J

2015-09-25

Retinitis pigmentosa (RP) is a retinal degenerative disorder that often causes complete blindness. Mutations of more than 50 genes have been identified as associated with RP, including the CACNA1F gene. In a recent study, by employing next-generation sequencing, we identified a novel mutation in the CACNA1F gene. In this study, we used the amplification refractory mutation system (ARMS) and identified a single nucleotide change c.1555C>T in exon 13 of the CACNA1F gene, leading to the substitution of arginine by tryptophan (p.R519W) in a Chinese individual affected by RP. This study actually confirms this novel mutation, and establishes the ARMS technique for the detection of mutations in RP.

Autosomal dominant deficiency of the interleukin-17F in recurrent aphthous stomatitis: Possible novel mutation in a new entity.

PubMed

Zare Bidoki, Alireza; Massoud, Ahmad; Najafi, Shamsolmoulouk; Mohammadzadeh, Mahsa; Rezaei, Nima

2018-05-15

Recurrent Aphthous Stomatitis (RAS) is a common oral inflammatory disease with unknown pathogenesis. Although the immune system alterations could be involved in predisposition of individuals to oral candidiasis, precise etiologies of RAS have not been understood yet. A recent study showed that autosomal dominant IL17F deficiency could cause chronic mucocutaneous candidiasis. Considering the inflammatory nature of interleukin (IL)-17F and RAS, this study was performed to check any disease-associated mutation in a number of patients with RAS. Sixty-two Iranian individuals with RAS were investigated in this study. After DNA extraction using a phenol-chloroform method from the whole blood, amplification was accomplished by polymerase chain reaction and the products were sequenced using a 3730 ABI sequencer. The results of sequencing revealed a missense, heterozygous mutation of IL17F, converting a threonine to proline in a patient with RAS (T79P). The Poly-phen software suggested a damaging probability predicting this substitution to have a harmful effect on IL-17F protein function. This mutation was checked in fifty healthy individuals, and was not detected in any of them. This is the first study showing that a mutation in IL-17F is associated with susceptibility to RAS. However, functional studies and further studies on more patients with RAS are required to confirm such association. Copyright © 2018 Elsevier B.V. All rights reserved.

Mutational Signature Mark Cancer’s Smoking Gun

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alexandrov, Ludmil

A broad computational study of cancer genome sequences by Los Alamos National Laboratory with the UK’s Wellcome Trust Sanger Institute and other collaborators identifies telltale mutational signatures associated with smoking tobacco. The research demonstrates, for the first time, that smoking increases cancer risk by causing somatic mutations in tissues directly and indirectly exposed to tobacco smoke. The international study was published in the November 4 issue of Science. The analysis shows that tobacco smoking causes mutations leading to cancer by multiple distinct mechanisms, including by damaging DNA in organs and by speeding up a mutational cellular clock.

Fabry Disease: prevalence of affected males and heterozygotes with pathogenic GLA mutations identified by screening renal, cardiac and stroke clinics, 1995-2017.

PubMed

Doheny, Dana; Srinivasan, Ram; Pagant, Silvere; Chen, Brenden; Yasuda, Makiko; Desnick, Robert J

2018-04-01

Fabry Disease (FD), an X linked lysosomal storage disease due to pathogenic α-galactosidase A ( GLA ) mutations, results in two major subtypes, the early-onset Type 1 'Classic' and the Type 2 'Later-Onset' phenotypes. To identify previously unrecognised patients, investigators screened cardiac, renal and stroke clinics by enzyme assays. However, some screening studies did not perform confirmatory GLA mutation analyses, and many included recently recognised 'benign/likely-benign' variants, thereby inflating prevalence estimates. Online databases were searched for all FD screening studies in high-risk clinics (1995-2017). Studies reporting GLA mutations were re-analysed for pathogenic mutations, sex and phenotype. Phenotype-specific and sex-specific prevalence rates were determined. Of 67 studies, 63 that screened 51363patients (33943M and 17420F) and provided GLA mutations were reanalysed for disease-causing mutations. Of reported GLA mutations, benign variants occurred in 47.9% of males and 74.1% of females. The following were the revised prevalence estimates: among 36820 (23954M and 12866F) haemodialysis screenees, 0.21% males and 0.15% females; among 3074 (2031M and 1043F) renal transplant screenees, 0.25% males and no females; among 5491 (4054M and 1437F) cardiac screenees, 0.94% males and 0.90% females; and among 5978 (3904M and 2074F) stroke screenees, 0.13% males and 0.14% females. Among male and female screenees with pathogenic mutations, the type 1 Classic phenotype was predominant (~60%), except more male cardiac patients (75%) had type 2 Later-Onset phenotype. Compared with previous findings, reanalysis of 63 studies increased the screenee numbers (~3.4-fold), eliminated 20 benign/likely benign variants, and provided more accurate sex-specific and phenotype-specific prevalence estimates, ranging from ~0.13% of stroke to ~0.9% of cardiac male or female screenees. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article

Polymorphisms and resistance mutations in the protease and reverse transcriptase genes of HIV-1 F subtype Romanian strains.

PubMed

Paraschiv, Simona; Otelea, Dan; Dinu, Magdalena; Maxim, Daniela; Tinischi, Mihaela

2007-03-01

To evaluate the prevalence of resistance mutations in the genome of HIV-1 F subtype strains isolated from Romanian antiretroviral (ARV) treatment-naïve patients and to assess the phylogenetic relatedness of these strains with other HIV-1 strains. Twenty-nine HIV-1 strains isolated from treatment-naïve adolescents (n=15) and adults (n=14) were included in this study. Resistance genotyping was performed by using Big Dye Terminator chemistry provided by the ViroSeq Genotyping System. The sequences of the protease and reverse transcriptase genes were aligned (ClustalW) and a phylogenetic tree was built (MEGA 3 software). For subtyping purposes, all the nucleotide sequences were submitted to the Stanford database. All the studied strains were found to harbor accessory mutations in the protease gene. The most frequent mutation was M36I (29 of 29 strains), followed by L63T, K20R, and L10V. The number of polymorphisms associated with protease inhibitor resistance was different for the two age groups. Intraphylogenetic divergence was greater for adults than for adolescents infected in childhood. All the strains were found to belong to the F1 subtype. The phylogenetic analysis revealed that Romanian strains clustered together, but distinctly from F1 HIV-1 strains isolated in other parts of the world (Brazil, Finland, and Belgium). Protease secondary mutations are present with high frequency in the HIV-1 F subtype strains isolated from Romanian ARV treatment-naïve patients, but no major resistance mutations were found.

An intronic mutation c.6430-3C>G in the F8 gene causes splicing efficiency and premature termination in hemophilia A.

PubMed

Xia, Zunjing; Lin, Jie; Lu, Lingping; Kim, Chol; Yu, Ping; Qi, Ming

2018-06-01

: Hemophilia A is a bleeding disorder caused by coagulation factor VIII protein deficiency or dysfunction, which is classified into severe, moderate, and mild according to factor clotting activity. An overwhelming majority of missense and nonsense mutations occur in exons of F8 gene, whereas mutations in introns can also be pathogenic. This study aimed to investigate the effect of an intronic mutation, c.6430-3C>G (IVS22-3C>G), on pre-mRNA splicing of the F8 gene. We applied DNA and cDNA sequencing in a Chinese boy with hemophilia A to search if any pathogenic mutation in the F8 gene. Functional analysis was performed to investigate the effect of an intronic mutation at the transcriptional level. Human Splicing Finder and PyMol were also used to predict its effect. We found the mutation c.6430-3C>G (IVS22-3C>G) in the F8 gene in the affected boy, with his mother being a carrier. cDNA from the mother and pSPL3 splicing assay showed that the mutation IVS22-3C>G results in a two-nucleotide AG inclusion at the 3' end of intron 22 and leads to a truncated coagulation factor VIII protein, with partial loss of the C1 domain and complete loss of the C2 domain. The in-silico tool predicted that the mutation induces altered pre-mRNA splicing by using a cryptic acceptor site in intron 22. The IVS22-3C>G mutation was confirmed to affect pre-mRNA splicing and produce a truncated protein, which reduces the stability of binding between the F8 protein and von Willebrand factor carrier protein due to the loss of an interaction domain.

Novel mutations of CHST6 in Iranian patients with macular corneal dystrophy

PubMed Central

Salehi, Zivar; Houshmand, Masoud; Mohamadi, Mohamad Javad; Promehr, Leila Azizade; Mozafarzadeh, Zahra

2009-01-01

Purpose To characterize mutations within the carbohydrate sulfotransferase 6 (CHST6) gene in Iranian subjects from 12 families with macular corneal dystrophy (MCD). Methods Genomic DNA was extracted from peripheral blood of 20 affected patients and 60 healthy volunteers followed by polymerase chain reaction (PCR) and direct sequencing of the CHST6 coding region. The observed nucleotide sequences were then compared with those found by investigators in other populations with MCD and in the controls. Results Analysis of CHST6 revealed 11 different mutations. These mutations were comprised of six novel missense mutations (p.F55L, p.P132L, p.S136G, p.C149Y, p.D203Y, and p.H249R), one novel nonsense mutation (p.S48X), one novel frame shift (after P297), and three previously reported missense mutations (p.P31L, p.C165Y, and p.R127C). The majority of the detected MCD mutations are located in the binding sites or the binding pocket, except the p.P31L and p.H249R mutations. Conclusions Nucleotide changes within the coding region of CHST6 are predicted to significantly alter the encoded sulfotransferase within the evolutionary conserved sequences. Our findings show that CHST6 mutations are responsible for the pathogenesis of MCD in Iranian patients. Moreover, the observation that some cases of MCD cannot be explained by mutations in the coding region of CHST6 suggests that MCD may result from possible upstream rearrangements in the CHST6 genomic region. PMID:19223992

Novel mutations of CHST6 in Iranian patients with macular corneal dystrophy.

PubMed

Birgani, Shiva Akbari; Salehi, Zivar; Houshmand, Masoud; Mohamadi, Mohamad Javad; Promehr, Leila Azizade; Mozafarzadeh, Zahra

2009-01-01

To characterize mutations within the carbohydrate sulfotransferase 6 (CHST6) gene in Iranian subjects from 12 families with macular corneal dystrophy (MCD). Genomic DNA was extracted from peripheral blood of 20 affected patients and 60 healthy volunteers followed by polymerase chain reaction (PCR) and direct sequencing of the CHST6 coding region. The observed nucleotide sequences were then compared with those found by investigators in other populations with MCD and in the controls. Analysis of CHST6 revealed 11 different mutations. These mutations were comprised of six novel missense mutations (p.F55L, p.P132L, p.S136G, p.C149Y, p.D203Y, and p.H249R), one novel nonsense mutation (p.S48X), one novel frame shift (after P297), and three previously reported missense mutations (p.P31L, p.C165Y, and p.R127C). The majority of the detected MCD mutations are located in the binding sites or the binding pocket, except the p.P31L and p.H249R mutations. Nucleotide changes within the coding region of CHST6 are predicted to significantly alter the encoded sulfotransferase within the evolutionary conserved sequences. Our findings show that CHST6 mutations are responsible for the pathogenesis of MCD in Iranian patients. Moreover, the observation that some cases of MCD cannot be explained by mutations in the coding region of CHST6 suggests that MCD may result from possible upstream rearrangements in the CHST6 genomic region.

Evaluation of Daptomycin Non-Susceptible Staphylococcus aureus for Stability, Population Profiles, mprF Mutations, and Daptomycin Activity.

PubMed

Steed, Molly E; Hall, Ashley D; Salimnia, Hossein; Kaatz, Glenn W; Kaye, Keith S; Rybak, Michael J

2013-12-01

Despite studies examining daptomycin non-susceptible (DNS) Staphylococcus aureus, examination of the stability and population profiles is limited. The objective was to evaluate the stability, population profiles, and daptomycin activity against DNS isolates. The stability of 12 consecutive clinical DNS strains was evaluated by minimum inhibitory concentration (MICs) and population analysis profiles before and after 5 days of serial passage. Two pairs of DNS S. aureus having the same daptomycin MIC but different daptomycin population profiles were evaluated via an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model of simulated endocardial vegetations for 96 h against daptomycin 6 and 10 mg/kg/day. The sequence of mprF was determined for these isolates before and after 96 h of daptomycin exposure in the in vitro PK/PD model. Daptomycin MIC values were 2-4 mg/L (via Microscan) for the 12 clinical isolates; 9 were confirmed DNS and 3 were within 1 tube dilution of Microscan (daptomycin MIC 1 mg/L). All were stable to serial passage. There was variation in the isolates susceptibility to daptomycin on population analysis (daptomycin population AUC 14.01-26.85). The killing patterns of daptomycin 6 and 10 mg/kg/day differed between isolates with a left-shift and right-shift population profile to daptomycin. Two strains developed additional mprF mutations during daptomycin exposure in the in vitro PK/PD model resulting in P314L, L826F, S337L and a novel Q326Stop mutation. The collection of DNS isolates was stable and displayed variation in susceptibility to daptomycin on population profile. Further research examining this clinical relevance is warranted.

11. Historic American Buildings Survey S.F. Chronicle Library Collection, S.F. ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

11. Historic American Buildings Survey S.F. Chronicle Library Collection, S.F. WALLS AND TOWER AFTER DISASTER OF 1906 - St. Mary's Church, 660 California Street, San Francisco, San Francisco County, CA

Novel domain-specific POU3F4 mutations are associated with X-linked deafness: examples from different populations.

PubMed

Bademci, Guney; Lasisi, Akeem; Yariz, Kemal O; Montenegro, Paola; Menendez, Ibis; Vinueza, Rodrigo; Paredes, Rosario; Moreta, Germania; Subasioglu, Asli; Blanton, Susan; Fitoz, Suat; Incesulu, Armagan; Sennaroglu, Levent; Tekin, Mustafa

2015-02-25

Mutations in the POU3F4 gene cause X-linked deafness type 3 (DFN3), which is characterized by inner ear anomalies. Three Turkish, one Ecuadorian, and one Nigerian families were included based on either inner ear anomalies detected in probands or X-linked family histories. Exome sequencing and/or Sanger sequencing were performed in order to identify the causative DNA variants in these families. Four novel, c.707A>C (p.(Glu236Ala)), c.772delG (p.(Glu258ArgfsX30)), c.902C>T (p.(Pro301Leu)), c.987T>C (p.(Ile308Thr)), and one previously reported mutation c.346delG (p.(Ala116ProfsX26)) in POU3F4, were identified. All mutations identified are predicted to affect the POU-specific or POU homeo domains of the protein and co-segregated with deafness in all families. Expanding the spectrum of POU3F4 mutations in different populations along with their associated phenotypes provides better understanding of their clinical importance and will be helpful in clinical evaluation and counseling of the affected individuals.

Expression of G(alpha)(s) proteins and TSH receptor signalling in hyperfunctioning thyroid nodules with TSH receptor mutations.

PubMed

Holzapfel, Hans-Peter; Bergner, Beate; Wonerow, Peter; Paschke, Ralf

2002-07-01

Constitutively activating mutations of the thyrotrophin receptor (TSHR) are the main molecular cause of hyperfunctioning thyroid nodules (HTNs). The G protein coupling is an important and critical step in the TSHR signalling which mainly includes G(alpha)(s), G(alpha)(i) and G(alpha)(q)/11 proteins. We investigated the in vitro consequences of overexpressing G(alpha) proteins on signalling of the wild-type (WT) or mutated TSHR. Moreover, we investigated whether changes in G(alpha) protein expression are pathophysiologically relevant in HTNs or cold thyroid nodules (CTNs). Wild-type TSH receptor and mutated TSH receptors were coexpressed with G(alpha)(s), G(alpha)(i) or G(alpha)(q)/11, and cAMP and inositol phosphate (IP) production was measured after stimulation with TSH. The expression of G(alpha)(s), G(alpha)(i) and G(alpha)(q)/11 proteins was examined by Western blotting in 28 HTNs and 14 CTNs. Coexpression of G(alpha)(s) with the WT TSH receptor in COS 7 cells significantly increased the basal and TSH-stimulated cAMP accumulation while coexpression of the G(alpha)(q) or G(alpha)11 protein significantly increased the production of cAMP and inositol triphosphate (IP(3)). The coexpression of the TSH receptor mutants (I486F, DEL613-621), known to couple constitutively to G(alpha)(s) and G(alpha)(q) with G(alpha)(s) and G(alpha)(q)/11, significantly increased the basal and stimulated cAMP and IP(3) accumulation. Coexpression of the TSH receptor mutant V556F with G(alpha)(s) only increased the basal and stimulated cAMP production while its coexpression with G(alpha)(q)/11 increased the basal and stimulated IP(3) signalling. The expression of G(alpha)(s) protein subunits determined by Western blotting was significantly decreased in 14 HTNs with a constitutively activating TSH receptor mutation in comparison with the corresponding surrounding tissue, while in 14 HTNs without TSH receptor or G(alpha)(s) protein mutation and in 14 CTNs the expression of G(alpha)(s

[High resolution melting analysis for detecting of JAK2V617F mutation in patients with myeloproliferative neoplasms].

PubMed

Chen, Hai-Hua; Yang, Ji-Long; Lu, Hui-Fang; Zhou, Wei-Jun; Yao, Fei; Deng, Lan

2014-02-01

This study was purposed to investigate the feasibility of high resolution melting (HRM) in the detection of JAK2V617F mutation in patients with myeloproliferative neoplasm (MPN). The 29 marrow samples randomly selected from patients with clinically diagnosed MPN from January 2008 to January 2011 were detected by HRM method. The results of HRM analysis were compared with that detected by allele specific polymerase chain reaction (AS-PCR) and DNA direct sequencing. The results showed that the JAK2V617F mutations were detected in 11 (37.9%, 11/29) cases by HRM, and its comparability with the direct sequencing result was 100%. While the consistency of AS-PCR with the direct sequencing was moderate (Kappa = 0.179, P = 0.316). It is concluded that the HRM analysis may be an optimal method for clinical screening of JAK2V617F mutation due to its simplicity and promptness with a high specificity.

Mutations in the novel protocadherin PCDH15 cause Usher syndrome type 1F.

PubMed

Alagramam, K N; Yuan, H; Kuehn, M H; Murcia, C L; Wayne, S; Srisailpathy, C R; Lowry, R B; Knaus, R; Van Laer, L; Bernier, F P; Schwartz, S; Lee, C; Morton, C C; Mullins, R F; Ramesh, A; Van Camp, G; Hageman, G S; Woychik, R P; Smith, R J; Hagemen, G S

2001-08-01

We have determined the molecular basis for Usher syndrome type 1F (USH1F) in two families segregating for this type of syndromic deafness. By fluorescence in situ hybridization, we placed the human homolog of the mouse protocadherin Pcdh15 in the linkage interval defined by the USH1F locus. We determined the genomic structure of this novel protocadherin, and found a single-base deletion in exon 10 in one USH1F family and a nonsense mutation in exon 2 in the second. Consistent with the phenotypes observed in these families, we demonstrated expression of PCDH15 in the retina and cochlea by RT-PCR and immunohistochemistry. This report shows that protocadherins are essential for maintenance of normal retinal and cochlear function.

Screening mosaic F1 females for mutations affecting zebrafish heart induction and patterning.

PubMed

Alexander, J; Stainier, D Y; Yelon, D

1998-01-01

The genetic pathways underlying the induction and anterior-posterior patterning of the heart are poorly understood. The recent emergence of the zebrafish model system now allows a classical genetic approach to such challenging problems in vertebrate development. Two large-scale screens for mutations affecting zebrafish embryonic development have recently been completed; among the hundreds of mutations identified were several that affect specific aspects of cardiac morphogenesis, differentiation, and function. However, very few mutations affecting induction and/or anterior-posterior patterning of the heart were identified. We hypothesize that a directed approach utilizing molecular markers to examine these particular steps of heart development will uncover additional such mutations. To test this hypothesis, we are conducting two parallel screens for mutations that affect either the induction or the anterior-posterior patterning of the zebrafish heart. As an indicator of cardiac induction, we examine expression of nkx2.5, the earliest known marker of precardiac mesoderm; to assess anterior-posterior patterning, we distinguish ventricle from atrium with antibodies that recognize different myosin heavy chain isoforms. In order to expedite the examination of a large number of mutations, we are screening the haploid progeny of mosaic F1 females. In these ongoing screens, we have identified four mutations that affect nkx2.5 expression as well as 21 that disrupt either ventricular or atrial development and thus far have recovered several of these mutations, demonstrating the value of our approach. Future analysis of these and other cardiac mutations will provide further insight into the processes of induction and anterior-posterior patterning of the heart.

Mapping the binding domain of the F18 fimbrial adhesin.

PubMed

Smeds, A; Pertovaara, M; Timonen, T; Pohjanvirta, T; Pelkonen, S; Palva, A

2003-04-01

F18 fimbrial Esherichia coli strains are associated with porcine postweaning diarrhea and pig edema disease. Recently, the FedF subunit was identified as the adhesin of the F18 fimbriae. In this study, adhesion domains of FedF were further studied by constructing deletions within the fedF gene and expressing FedF proteins with deletions either together with the other F18 fimbrial subunits or as fusion proteins tagged with maltose binding protein. The region essential for adhesion to porcine intestinal epithelial cells was mapped between amino acid residues 60 and 109 of FedF. To map the binding domain even more closely, all eight charged amino acid residues within this region were independently replaced by alanine. Three of these single point mutants expressing F18 fimbriae exhibited significantly diminished capabilities to adhere to porcine epithelial cells in vitro. In addition, a triple point mutation and a double point mutation completely abolished receptor adhesiveness. The result further confirmed that the region between amino acid residues 60 and 109 is essential for the binding of F18 fimbriae to their receptor. In addition, the adhesion capability of the binding domain was eliminated after treatment with iodoacetamide, suggesting the formation of a disulfide bridge between Cys-63 and Cys-83, whereas Cys-111 and Cys-116 could be deleted without affecting the binding ability of FedF.

Mutation detection by mismatch binding protein, MutS, in amplified DNA: Application to the cystic fibrosis gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lishanski, A.; Ostrander, E.A.; Rine, J.

1994-03-29

An experimental strategy for detecting heterozygosity in genomic DNA has been developed based on preferential binding of Escherichia coli MutS protein to DNA molecules containing mismatched bases. The binding was detected by a gel mobility-shift assay. This approach was tested by using as a model the most commonly occurring mutations within the cystic fibrosis (CFTR) gene. Genomic DNA samples were amplified with 5{prime}-end-labeled primers that bracket the site of the {Delta}F508 3-bp deletion in exon 10 of the CFTR gene. The renatured PCR products from homozygotes produced homoduplexes; the PCR products from heterozygotes produced heteroduplexes and homoduplexes (1:1). MutS proteinmore » bound more strongly to heteroduplexes that correspond to heterozygous carriers of {Delta}F508 and contain a CTT or a GAA loop in one of the strands than to homoduplexes corresponding to homozygotes. The ability of MutS protein to detect heteroduplexes in PCR-amplified DNA extended to fragments {approximately} 500 bp long. The method was also able to detect carriers of the point mutations in exon 11 of the CFTR gene by a preferential binding of MutS to single-base mismatches in PCR-amplified DNA.« less

Dark Matter Search Results from the PICO - 60 C 3 F 8 Bubble Chamber

DOE Office of Scientific and Technical Information (OSTI.GOV)

Amole, C.; Ardid, M.; Arnquist, I. J.

2017-06-01

New results are reported from the operation of the PICO-60 dark matter detector, a bubble chamber filled with 52 kg of C 3F 8 located in the SNOLAB underground laboratory. As in previous PICO bubble chambers, PICO-60C 3F 8 exhibits excellent electron recoil and alpha decay rejection, and the observed multiple-scattering neutron rate indicates a single-scatter neutron background of less than one event per month.

HBV subgenotypes F1b and F4 replication induces an incomplete autophagic process in hepatocytes: Role of BCP and preCore mutations.

PubMed

Elizalde, María Mercedes; Pérez, Paula Soledad; Sevic, Ina; Grasso, Daniel; Ropolo, Alejandro; Barbini, Luciana; Campos, Rodolfo Héctor; Vaccaro, María Inés; Flichman, Diego Martín

2018-01-01

Hepatitis B virus (HBV) genotypes and mutants have been associated with differences in clinical and virological characteristics. Autophagy is a cellular process that degrades long-lived proteins and damaged organelles. Viruses have evolved mechanisms to alter this process to survive in host cells. In this work, we studied the modulation of autophagy by the replication of HBV subgenotypes F1b and F4, and the naturally occurring mutants BCP and preCore. HBV subgenotypes F1b and F4 replication induced accumulation of autophagosomes in hepatoma cells. However, no autophagic protein degradation was observed, indicating a blockage of autophagic flux at later stages. This inhibition of autophagy flux might be due to an impairment of lysosomal acidification in hepatoma cells. Moreover, HBV-mediated autophagy modulation was independent of the viral subgenotypes and enhanced in viruses with BCP and preCore naturally occurring mutations. These results contribute to understand the mechanisms by which different HBV variants contribute to the pathogenesis of HBV infections. In addition, this study is the first to describe the role that two highly prevalent naturally occurring mutations exert on the modulation of HBV-induced autophagy.

HBV subgenotypes F1b and F4 replication induces an incomplete autophagic process in hepatocytes: Role of BCP and preCore mutations

PubMed Central

Pérez, Paula Soledad; Sevic, Ina; Ropolo, Alejandro; Barbini, Luciana; Campos, Rodolfo Héctor; Vaccaro, María Inés; Flichman, Diego Martín

2018-01-01

Hepatitis B virus (HBV) genotypes and mutants have been associated with differences in clinical and virological characteristics. Autophagy is a cellular process that degrades long-lived proteins and damaged organelles. Viruses have evolved mechanisms to alter this process to survive in host cells. In this work, we studied the modulation of autophagy by the replication of HBV subgenotypes F1b and F4, and the naturally occurring mutants BCP and preCore. HBV subgenotypes F1b and F4 replication induced accumulation of autophagosomes in hepatoma cells. However, no autophagic protein degradation was observed, indicating a blockage of autophagic flux at later stages. This inhibition of autophagy flux might be due to an impairment of lysosomal acidification in hepatoma cells. Moreover, HBV-mediated autophagy modulation was independent of the viral subgenotypes and enhanced in viruses with BCP and preCore naturally occurring mutations. These results contribute to understand the mechanisms by which different HBV variants contribute to the pathogenesis of HBV infections. In addition, this study is the first to describe the role that two highly prevalent naturally occurring mutations exert on the modulation of HBV-induced autophagy. PMID:29738548

Measurement of Small Molecular Dopant F4TCNQ and C 60F 36 Diffusion in Organic Bilayer Architectures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Jun; Rochester, Chris W.; Jacobs, Ian E.

2015-12-03

The diffusion of molecules through and between organic layers is a serious stability concern in organic electronic devices. In this paper, the temperature-dependent diffusion of molecular dopants through small molecule hole transport layers is observed. Specifically we investigate bilayer stacks of small molecules used for hole transport (MeO-TPD) and p-type dopants (F4TCNQ and C 60F 36) used in hole injection layers for organic light emitting diodes and hole collection electrodes for organic photovoltaics. With the use of absorbance spectroscopy, photoluminescence spectroscopy, neutron reflectometry, and near-edge X-ray absorption fine structure spectroscopy, we are able to obtain a comprehensive picture of themore » diffusion of fluorinated small molecules through MeO-TPD layers. F4TCNQ spontaneously diffuses into the MeO-TPD material even at room temperature, while C 60F 36, a much bulkier molecule, is shown to have a substantially higher morphological stability. Finally, this study highlights that the differences in size/geometry and thermal properties of small molecular dopants can have a significant impact on their diffusion in organic device architectures.« less

24. "GAFFTC 29 SEP 60, F106B STATIC TEST 1." Test ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

24. "G-AFFTC 29 SEP 60, F-106B STATIC TEST 1." Test of the Convair sled escape system at static test site east of Station "50". File no. 11,988-60. - Edwards Air Force Base, South Base Sled Track, Edwards Air Force Base, North of Avenue B, between 100th & 140th Streets East, Lancaster, Los Angeles County, CA

Mutation Spectrum and Phenotypic Features in Noonan Syndrome with PTPN11 Mutations: Definition of Two Novel Mutations.

PubMed

Atik, Tahir; Aykut, Ayca; Hazan, Filiz; Onay, Huseyin; Goksen, Damla; Darcan, Sukran; Tukun, Ajlan; Ozkinay, Ferda

2016-06-01

To evaluate the spectrum of PTPN11 gene mutations in Noonan syndrome patients and to study the genotype-phenotype associations. In this study, twenty Noonan syndrome patients with PTPN11 mutations were included. The patients underwent a detailed clinical and physical evaluation. To identify inherited cases, parents of all mutation positive patients were analyzed. Thirteen different PTPN11 mutations, two of them being novel, were detected in the study group. These mutations included eleven missense mutations: p.G60A, p.D61N, p.Y62D, p.Y63C, p.E69Q, p.Q79R, p.Y279C,p.N308D, p.N308S, p.M504V, p.Q510R and two novel missense mutations: p.I56V and p.I282M. The frequency of cardiac abnormalities and short stature were found to be 80 % and 80 %, respectively. Mental retardation was not observed in patients having exon 8 mutations. No significant correlations were detected between other phenotypic features and genotypes. By identifying genotype-phenotype correlations, this study provides information on phenotypes observed in NS patients with different PTPN11 mutations.

Molecular genetic analysis of the F11 gene in 14 Turkish patients with factor XI deficiency: identification of novel and recurrent mutations and their inheritance within families.

PubMed

Colakoglu, Seyma; Bayhan, Turan; Tavil, Betül; Keskin, Ebru Yılmaz; Cakir, Volkan; Gümrük, Fatma; Çetin, Mualla; Aytaç, Selin; Berber, Ergul

2018-01-01

Factor XI (FXI) deficiency is an autosomal bleeding disease associated with genetic defects in the F11 gene which cause decreased FXI levels or impaired FXI function. An increasing number of mutations has been reported in the FXI mutation database, most of which affect the serine protease domain of the protein. FXI is a heterogeneous disorder associated with a variable bleeding tendency and a variety of causative F11 gene mutations. The molecular basis of FXI deficiency in 14 patients from ten unrelated families in Turkey was analysed to establish genotype-phenotype correlations and inheritance of the mutations in the patients' families. Fourteen index cases with a diagnosis of FXI deficiency and family members of these patients were enrolled into the study. The patients' F11 genes were amplified by polymerase chain reaction and subjected to direct DNA sequencing analysis. The findings were analysed statistically using bivariate correlations, Pearson's correlation coefficient and the nonparametric Mann-Whitney test. Direct DNA sequencing analysis of the F11 genes revealed that all of the 14 patients had a F11 gene mutation. Eight different mutations were identified in the apple 1, apple 2 or serine protease domains, except one which was a splice site mutation. Six of the mutations were recurrent. Two of the mutations were novel missense mutations, p.Val522Gly and p.Cys581Arg, within the catalytic domain. The p.Trp519Stop mutation was observed in two families whereas all the other mutations were specific to a single family. Identification of mutations confirmed the genetic heterogeneity of FXI deficiency. Most of the patients with mutations did not have any bleeding complications, whereas some had severe bleeding symptoms. Genetic screening for F11 gene mutations is important to decrease the mortality and morbidity rate associated with FXI deficiency, which can be life-threatening if bleeding occurs in tissues with high fibrinolytic activity.

Two novel mutations in the POU1F1 gene generate null alleles through different mechanisms leading to combined pituitary hormone deficiency.

PubMed

Turton, J P; Strom, M; Langham, S; Dattani, M T; Le Tissier, P

2012-03-01

  Mutations in the POU1F1 gene severely affect the development and function of the anterior pituitary gland and lead to combined pituitary hormone deficiency (CPHD).   The clinical and genetic analysis of a patient presenting with CPHD and functional characterization of identified mutations.   We describe a male patient with extreme short stature, learning difficulties, anterior pituitary hypoplasia, secondary hypothyroidism and undetectable prolactin, growth hormone (GH) and insulin-like growth factor 1 (IGF1), with normal random cortisol.   The POU1F1 coding region was amplified by PCR and sequenced; the functional consequence of the mutations was analysed by cell transfection and in vitro assays.   Genetic analysis revealed compound heterozygosity for two novel putative loss of function mutations in POU1F1: a transition at position +3 of intron 1 [IVS1+3nt(A>G)] and a point mutation in exon 6 resulting in a substitution of arginine by tryptophan (R265W). Functional analysis revealed that IVS1+3nt(A>G) results in a reduction in the correctly spliced POU1F1 mRNA, which could be corrected by mutations of the +4, +5 and +6 nucleotides. Analysis of POU1F1(R265W) revealed complete loss of function resulting from severely reduced protein stability.   Combined pituitary hormone deficiency in this patient is caused by loss of POU1F1 function by two novel mechanisms, namely aberrant splicing (IVS1+3nt (A>G) and protein instability (R265W). Identification of the genetic basis of CPHD enabled the cessation of hydrocortisone therapy without the need for further assessment for evolving endocrinopathy. © 2012 Blackwell Publishing Ltd.

Cardiomyopathy mutation (F88L) in troponin T abolishes length dependency of myofilament Ca2+ sensitivity.

PubMed

Reda, Sherif M; Chandra, Murali

2018-05-18

Recent clinical studies have revealed a new hypertrophic cardiomyopathy-associated mutation (F87L) in the central region of human cardiac troponin T (TnT). However, despite its implication in several incidences of sudden cardiac death in young and old adults, whether F87L is associated with cardiac contractile dysfunction is unknown. Because the central region of TnT is important for modulating the muscle length-mediated recruitment of new force-bearing cross-bridges (XBs), we hypothesize that the F87L mutation causes molecular changes that are linked to the length-dependent activation of cardiac myofilaments. Length-dependent activation is important because it contributes significantly to the Frank-Starling mechanism, which enables the heart to vary stroke volume as a function of changes in venous return. We measured steady-state and dynamic contractile parameters in detergent-skinned guinea pig cardiac muscle fibers reconstituted with recombinant guinea pig wild-type TnT (TnT WT ) or the guinea pig analogue (TnT F88L ) of the human mutation at two different sarcomere lengths (SLs): short (1.9 µm) and long (2.3 µm). TnT F88L increases pCa 50 (-log [Ca 2+ ] free required for half-maximal activation) to a greater extent at short SL than at long SL; for example, pCa 50 increases by 0.25 pCa units at short SL and 0.17 pCa units at long SL. The greater increase in pCa 50 at short SL leads to the abolishment of the SL-dependent increase in myofilament Ca 2+ sensitivity (ΔpCa 50 ) in TnT F88L fibers, ΔpCa 50 being 0.10 units in TnT WT fibers but only 0.02 units in TnT F88L fibers. Furthermore, at short SL, TnT F88L attenuates the negative impact of strained XBs on force-bearing XBs and augments the magnitude of muscle length-mediated recruitment of new force-bearing XBs. Our findings suggest that the TnT F88L -mediated effects on cardiac thin filaments may lead to a negative impact on the Frank-Starling mechanism. © 2018 Reda and Chandra.

Emergence of CTNNB1 mutation at acquired resistance to KIT inhibitor in metastatic melanoma.

PubMed

Cho, J; Kim, S Y; Kim, Y J; Sim, M H; Kim, S T; Kim, N K D; Kim, K; Park, W; Kim, J H; Jang, K-T; Lee, J

2017-10-01

The KIT inhibitor, imatinib, has shown promising efficacy in patients with KIT-mutated melanoma; however, acquisition of resistance to imatinib occurs rapidly in the majority of patients. The mechanisms of acquired resistance to imatinib in melanoma remain unclear. We analyzed biopsy samples from paired baseline and post-treatment tumor lesions in one patient with KIT-mutated melanoma who had had an initial objective tumor regression in response to imatinib treatment followed by disease progression 8 months later. Targeted deep sequencing from post-treatment biopsy samples detected an additional mutation in CTNNB1 (S33C) with original KIT L576P mutation. We examined the functional role of the additional CTNNB1 S33C mutation in resistance to imatinib indirectly using the Ba/F3 cell model. Ba/F3 cell lines transfected with both the L576P KIT mutation and the CTNNB1 S33C mutation demonstrated no growth inhibition despite imatinib treatment, whereas growth inhibition was observed in the Ba/F3 cell line transfected with the L576 KIT mutation alone. We report the first identification of the emergence of a CTNNB1 mutation that can confer acquired resistance to imatinib. Further investigation into the causes of acquired resistance to imatinib will be essential to improve the prognosis for patients with KIT-mutated melanoma.

Impact of the F508del mutation on ovine CFTR, a Cl− channel with enhanced conductance and ATP-dependent gating

PubMed Central

Cai, Zhiwei; Palmai-Pallag, Timea; Khuituan, Pissared; Mutolo, Michael J; Boinot, Clément; Liu, Beihui; Scott-Ward, Toby S; Callebaut, Isabelle; Harris, Ann; Sheppard, David N

2015-01-01

Cross-species comparative studies are a powerful approach to understanding the epithelial Cl− channel cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in the genetic disease cystic fibrosis (CF). Here, we investigate the single-channel behaviour of ovine CFTR and the impact of the most common CF mutation, F508del-CFTR, using excised inside-out membrane patches from transiently transfected CHO cells. Like human CFTR, ovine CFTR formed a weakly inwardly rectifying Cl− channel regulated by PKA-dependent phosphorylation, inhibited by the open-channel blocker glibenclamide. However, for three reasons, ovine CFTR was noticeably more active than human CFTR. First, single-channel conductance was increased. Second, open probability was augmented because the frequency and duration of channel openings were increased. Third, with enhanced affinity and efficacy, ATP more strongly stimulated ovine CFTR channel gating. Consistent with these data, the CFTR modulator phloxine B failed to potentiate ovine CFTR Cl− currents. Similar to its impact on human CFTR, the F508del mutation caused a temperature-sensitive folding defect, which disrupted ovine CFTR protein processing and reduced membrane stability. However, the F508del mutation had reduced impact on ovine CFTR channel gating in contrast to its marked effects on human CFTR. We conclude that ovine CFTR forms a regulated Cl− channel with enhanced conductance and ATP-dependent channel gating. This phylogenetic analysis of CFTR structure and function demonstrates that subtle changes in structure have pronounced effects on channel function and the consequences of the CF mutation F508del. Key points Malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR), a gated pathway for chloride movement, causes the common life-shortening genetic disease cystic fibrosis (CF). Towards the development of a sheep model of CF, we have investigated the function of sheep CFTR. We found that

Reliable high-power injection locked 6kHz 60W laser for ArF immersion lithography

NASA Astrophysics Data System (ADS)

Watanabe, Hidenori; Komae, Shigeo; Tanaka, Satoshi; Nohdomi, Ryoichi; Yamazaki, Taku; Nakarai, Hiroaki; Fujimoto, Junichi; Matsunaga, Takashi; Saito, Takashi; Kakizaki, Kouji; Mizoguchi, Hakaru

2007-03-01

Reliable high power 193nm ArF light source is desired for the successive growth of ArF-immersion technology for 45nm node generation. In 2006, Gigaphoton released GT60A, high power injection locked 6kHz/60W/0.5pm (E95) laser system, to meet the demands of semiconductor markets. In this paper, we report key technologies for reliable mass production GT laser systems and GT60A high durability performance test results up to 20 billion pulses.

Reduction in hepatic drug metabolizing CYP3A4 activities caused by P450 oxidoreductase mutations identified in patients with disordered steroid metabolism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Flueck, Christa E.; Mullis, Primus E.; Pandey, Amit V., E-mail: amit@pandeylab.org

2010-10-08

Research highlights: {yields} Cytochrome P450 3A4 (CYP3A4), metabolizes 50% of drugs in clinical use and requires NADPH-P450 reductase (POR). {yields} Mutations in human POR cause congenital adrenal hyperplasia from diminished activities of steroid metabolizing P450s. {yields} We are reporting that mutations in POR may reduce CYP3A4 activity. {yields} POR mutants Y181D, A457H, Y459H, V492E and R616X lost 99%, while A287P, C569Y and V608F lost 60-85% CYP3A4 activity. {yields} Reduction of CYP3A4 activity may cause increased risk of drug toxicities/adverse drug reactions in patients with POR mutations. -- Abstract: Cytochrome P450 3A4 (CYP3A4), the major P450 present in human liver metabolizesmore » approximately half the drugs in clinical use and requires electrons supplied from NADPH through NADPH-P450 reductase (POR, CPR). Mutations in human POR cause a rare form of congenital adrenal hyperplasia from diminished activities of steroid metabolizing P450s. In this study we examined the effect of mutations in POR on CYP3A4 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified CYP3A4 to perform kinetic studies. We are reporting that mutations in POR identified in patients with disordered steroidogenesis/Antley-Bixler syndrome (ABS) may reduce CYP3A4 activity, potentially affecting drug metabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had more than 99% loss of CYP3A4 activity, while POR mutations A287P, C569Y and V608F lost 60-85% activity. Loss of CYP3A4 activity may result in increased risk of drug toxicities and adverse drug reactions in patients with POR mutations.« less

Impact of Homologous Resistance Mutations from Pathogenic Yeast on Saccharomyces cerevisiae Lanosterol 14α-Demethylase.

PubMed

Sagatova, Alia A; Keniya, Mikhail V; Tyndall, Joel D A; Monk, Brian C

2018-03-01

Fungal infections frequently affect immunodeficient individuals and are estimated to kill 1.35 million people per annum. Azole antifungals target the membrane-bound cytochrome P450 monooxygenase lanosterol 14α-demethylase (CYP51; Erg11p). Mutations in CYP51 can render the widely used triazole drugs less effective. The Candida albicans CYP51 mutation G464S and the double mutation Y132F G464S (Y140F and G464S by Saccharomyces cerevisiae numbering) as well as the CYP51A G54E/R/W mutations of Aspergillus fumigatus (G73E/R/W by S. cerevisiae numbering) have been reproduced in a recombinant C-terminal hexahistidine-tagged version of S. cerevisiae CYP51 (ScErg11p6×His). Phenotypes and X-ray crystal structures were determined for the mutant enzymes. Liquid microdilution assays showed that the G464S mutation in ScErg11p6×His conferred no difference in the susceptibility of yeast to triazole drugs but in combination with the Y140F mutation gave a 4-fold reduction in susceptibility to the short-tailed triazole fluconazole. The ScErg11p6×His Y140F G464S mutant was unstable during purification and was not crystallized. The ScErg11p6×His G73E/R/W mutations conferred increased susceptibly to all triazoles tested in liquid microdilution assays. High-resolution X-ray crystal structures reveal two different conformations of the ligand itraconazole, including a previously unseen conformation, as well as interactions between the tail of this triazole and the E/W73 residue. This study shows that S. cerevisiae CYP51 adequately represents some but not all mutations in CYP51s of pathogenic fungi. Insight into the molecular mechanisms of resistance mutations in CYP51 will assist the development of inhibitors that will overcome antifungal resistance. Copyright © 2018 American Society for Microbiology.

Analysis of point mutations in an ultraviolet-irradiated shuttle vector plasmid propagated in cells from Japanese xeroderma pigmentosum patients in complementation groups A and F

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yagi, T.; Tatsumi-Miyajima, J.; Sato, M.

1991-06-15

To assess the contribution to mutagenesis by human DNA repair defects, a UV-treated shuttle vector plasmid, pZ189, was passed through fibroblasts derived from Japanese xeroderma pigmentosum (XP) patients in two different DNA repair complementation groups (A and F). Patients with XP have clinical and cellular UV hypersensitivity, increased frequency of skin cancer, and defects in DNA repair. The XP DNA repair defects represented by complementation groups A (XP-A) and F (XP-F) are more common in Japan than in Europe or the United States. In comparison to results with DNA repair-proficient human cells (W138-VA13), UV-treated pZ189 passed through the XP-A (XP2OS(SV))more » or XP-F (XP2YO(SV)) cells showed fewer surviving plasmids (XP-A less than XP-F) and a higher frequency of mutated plasmids (XP-A greater than XP-F). Base sequence analysis of more than 200 mutated plasmids showed the major type of base substitution mutation to be the G:C----A:T transition with all three cell lines. The XP-A and XP-F cells revealed a higher frequency of G:C----A:T transitions and a lower frequency of transversions among plasmids with single or tandem mutations and a lower frequency of plasmids with multiple point mutations compared to the normal line. The spectrum of mutations in pZ189 with the XP-A cells was similar to that with the XP-F cells. Seventy-six to 91% of the single base substitution mutations occurred at G:C base pairs in which the 5{prime}-neighboring base of the cytosine was thymine or cytosine. These studies indicate that the DNA repair defects in Japanese XP patients in complementation groups A and F result in different frequencies of plasmid survival and mutagenesis but in similar types of mutagenic abnormalities despite marked differences in clinical features.« less

AURKA F31I Polymorphism and Breast Cancer Risk in BRCA1 and BRCA2 Mutation Carriers: A CIMBA study

PubMed Central

Couch, Fergus J.; Sinilnikova, Olga; Vierkant, Robert A; Pankratz, V. Shane; Fredericksen, Zachary S.; Stoppa-Lyonnet, Dominique; Coupier, Isabelle; Hughes, David; Hardouin, Agnès; Berthet, Pascaline; Peock, Susan; Cook, Margaret; Baynes, Caroline; Hodgson, Shirley; Morrison, Patrick J.; Porteous, Mary E.; Jakubowska, Anna; Lubinski, Jan; Gronwald, Jacek; Spurdle, Amanda B.; Schmutzler, Rita; Versmold, Beatrix; Engel, Christoph; Meindl, Alfons; Sutter, Christian; Horst, Jurgen; Schaefer, Dieter; Offit, Kenneth; Kirchhoff, Tomas; Andrulis, Irene L.; Ilyushik, Eduard; Glendon, Gordon; Devilee, Peter; Vreeswijk, Maaike P.G.; Vasen, Hans F.A.; Borg, Ake; Backenhorn, Katja; Struewing, Jeffery P.; Greene, Mark H.; Neuhausen, Susan L.; Rebbeck, Timothy R.; Nathanson, Katherine; Domchek, Susan; Wagner, Theresa; Garber, Judy E.; Szabo, Csilla; Zikan, Michal; Foretova, Lenka; Olson, Janet E.; Sellers, Thomas A.; Lindor, Noralane; Nevanlinna, Heli; Tommiska, Johanna; Aittomaki, Kristiina; Hamann, Ute; Rashid, Muhammad U.; Torres, Diana; Simard, Jacques; Durocher, Francine; Guenard, Frederic; Lynch, Henry T.; Isaacs, Claudine; Weitzel, Jeffrey; Olopade, Olufunmilayo I.; Narod, Steven; Daly, Mary B.; Godwin, Andrew K.; Tomlinson, Gail; Easton, Douglas F.; Chenevix-Trench, Georgia; Antoniouon, Antonis C.

2009-01-01

The AURKA oncogene is associated with abnormal chromosome segregation and aneuploidy and predisposition to cancer. Amplification of AURKA has been detected at higher frequency in tumors from BRCA1 and BRCA2 mutation carriers than in sporadic breast tumors, suggesting that overexpression of AURKA and inactivation of BRCA1 and BRCA2 co-operate during tumor development and progression. The F31I polymorphism in AURKA has been associated with breast cancer risk in the homozygous state in prior studies. We evaluated whether the AURKA F31I polymorphism modifies breast cancer risk in BRCA1 and BRCA2 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). CIMBA was established to provide sufficient statistical power through increased numbers of mutation carriers to identify polymorphisms that act as modifiers of cancer risk and can refine breast cancer risk estimates in BRCA1 and BRCA2 mutation carriers. A total of 4935 BRCA1 and 2241 BRCA2 mutation carriers and 11 individuals carrying both BRCA1 and BRCA2 mutations were genotyped for F31I. Overall, homozygosity for the 31I allele was not significantly associated with breast cancer risk in BRCA1 and BRCA2 carriers combined (HR = 0.91; 95% CI 0.77-1.06). Similarly, no significant association was seen in BRCA1 (HR = 0.90; 95% CI 0.75-1.08) or BRCA2 carriers (HR = 0.93; 95% CI 0.67-1.29) or when assessing the modifying effects of either bilateral prophylactic oophorectomy or menopausal status of BRCA1 and BRCA2 carriers. In summary, the F31I polymorphism in AURKA is not associated with a modified risk of breast cancer in BRCA1 and BRCA2 carriers. PMID:17627006

Estimation of diagnosis and prognosis in ET by assessment of CALR and JAK2V617F mutations and laboratory findings: a meta-analysis.

PubMed

Saki, N; Shirzad, R; Rahim, F; Saki Malehi, A

2017-07-01

Essential thrombocythemia (ET) is a benign disease with slow progress in which thrombosis is a cause of mortality. JAK2 V617F and calreticulin (CALR) are the most frequent mutations in this disease. In this systematic review and meta-analysis, we compared the prevalence of JAK2 V617F and CALR mutations in ET and examined the incidence of thrombosis and other hematologic indices. After choosing MeSH keywords, including essential thrombocythemia, JAK2 V617F , calreticulin, prognosis, and diagnosis, as well as searching Medline/PubMed and Scopus, 12 papers were selected. Data were pooled, and summary prevalence and OR were estimated using either a random-effects model or a fixed-effects model. The frequency of JAK2 V617F and CALR shows heterogeneity in Caucasian population [JAK2 V617 I 2 % = 84.3, P < 0.001, 95% CI 0.56 (0.51-0.61)], [CALR I 2 % = 96.1, P < 0.001, 95% CI 0.23 (0.15-0.31)]. The prevalence of JAK2 V617F and CALR was 0.57 (95% CI 0.53-0.61), I 2 % = 79.3 and 0.22 (95% CI 0.16-0.27), I 2 % = 94, respectively. JAK2 V617F positive ET was associated with increasing odds of thrombosis [OR 2.35 (95% CI 1.83-3.02), P < 0.001]. The incidence of splenomegaly was not statistically different between these two mutations. Hemoglobin, platelet, and WBC count did not affect the risk of thrombosis. Detection of CALR mutation is helpful for molecular diagnosis of ET patients as well as JAK2 V617F . Due to reduction of thrombosis in CALR-positive patients, it can be stated that such patients have less thrombotic disorders and better prognosis relative to patients bearing JAK2 V617F mutation. Therefore, detection of mutation in CALR and JAK2 V617F may contribute to diagnosis and prognosis of ET patients.

Development and inter-laboratory validation of unlabeled probe melting curve analysis for detection of JAK2 V617F mutation in polycythemia vera.

PubMed

Wu, Zhiyuan; Yuan, Hong; Zhang, Xinju; Liu, Weiwei; Xu, Jinhua; Zhang, Wei; Guan, Ming

2011-01-01

JAK2 V617F, a somatic point mutation that leads to constitutive JAK2 phosphorylation and kinase activation, has been incorporated into the WHO classification and diagnostic criteria of myeloid neoplasms. Although various approaches such as restriction fragment length polymorphism, amplification refractory mutation system and real-time PCR have been developed for its detection, a generic rapid closed-tube method, which can be utilized on routine genetic testing instruments with stability and cost-efficiency, has not been described. Asymmetric PCR for detection of JAK2 V617F with a 3'-blocked unlabeled probe, saturate dye and subsequent melting curve analysis was performed on a Rotor-Gene® Q real-time cycler to establish the methodology. We compared this method to the existing amplification refractory mutation systems and direct sequencing. Hereafter, the broad applicability of this unlabeled probe melting method was also validated on three diverse real-time systems (Roche LightCycler® 480, Applied Biosystems ABI® 7500 and Eppendorf Mastercycler® ep realplex) in two different laboratories. The unlabeled probe melting analysis could genotype JAK2 V617F mutation explicitly with a 3% mutation load detecting sensitivity. At level of 5% mutation load, the intra- and inter-assay CVs of probe-DNA heteroduplex (mutation/wild type) covered 3.14%/3.55% and 1.72%/1.29% respectively. The method could equally discriminate mutant from wild type samples on the other three real-time instruments. With a high detecting sensitivity, unlabeled probe melting curve analysis is more applicable to disclose JAK2 V617F mutation than conventional methodologies. Verified with the favorable inter- and intra-assay reproducibility, unlabeled probe melting analysis provided a generic mutation detecting alternative for real-time instruments.

A Disease Mutation Causing Episodic Ataxia Type I in the S1 Links Directly to the Voltage Sensor and the Selectivity Filter in Kv Channels.

PubMed

Petitjean, Dimitri; Kalstrup, Tanja; Zhao, Juan; Blunck, Rikard

2015-09-02

The mutation F184C in Kv1.1 leads to development of episodic ataxia type I (EA1). Although the mutation has been said to alter activation kinetics and to lower expression, we show here that the underlying molecular mechanisms may be more complex. Although F184 is positioned in the "peripheral" S1 helix, it occupies a central position in the 3D fold. We show in cut-open oocyte voltage-clamp recordings of gating and ionic currents of the Shaker Kv channel expressed in Xenopus oocytes that F184 not only interacts directly with the gating charges of the S4, but also creates a functional link to the selectivity filter of the neighboring subunit. This link leads to impaired fast and slow inactivation. The effect on fast inactivation is of an allosteric nature considering that fast inactivation is caused by a linked cytosolic ball peptide. The extensive effects of F184C provide a new mechanism underlying EA. Episodic ataxia (EA) is an inherited disease that leads to occasional loss of motor control in combination with variable other symptoms such as vertigo or migraine. EA type I (EA1), studied here, is caused by mutations in a voltage-gated potassium channel that contributes to the generation of electrical signals in the brain. The mechanism by which mutations in voltage-gated potassium channels lead to EA is still unknown and there is no consistent pharmacological treatment. By studying in detail one disease-causing mutation in Kv1.1, we describe a novel molecular mechanism distinct from mechanisms described previously. This mechanism contributes to the understanding of potassium channel function in general and might lead to a better understanding of how EA develops. Copyright © 2015 the authors 0270-6474/15/3512198-09$15.00/0.

Severe coagulation factor VII deficiency caused by a novel homozygous mutation (p. Trp284Gly) in loop 140s.

PubMed

Hao, Xiuping; Cheng, XiaoLi; Ye, Jiajia; Wang, Yingyu; Yang, LiHong; Wang, Mingshan; Jin, Yanhui

2016-06-01

Congenital coagulation factor VII (FVII) deficiency is a rare disorder caused by mutation in F7 gene. Herein, we reported a patient who had unexplained hematuria and vertigo with consanguineous parents. He has been diagnosed as having FVII deficiency based on the results of reduced FVII activity (2.0%) and antigen (12.8%). The thrombin generation tests verified that the proband has obstacles in producing thrombin. Direct sequencing analysis revealed a novel homozygous missense mutation p.Trp284Gly. Also noteworthy is the fact that the mutational residue belongs to structurally conserved loop 140s, which majorly undergo rearrangement after FVII activation. Model analysis indicated that the substitution disrupts these native hydrophobic interactions, which are of great importance to the conformation in the activation domain of FVIIa.

The O-GlcNAc Transferase Intellectual Disability Mutation L254F Distorts the TPR Helix.

PubMed

Gundogdu, Mehmet; Llabrés, Salomé; Gorelik, Andrii; Ferenbach, Andrew T; Zachariae, Ulrich; van Aalten, Daan M F

2018-05-17

O-linked β-N-acetyl- D -glucosamine (O-GlcNAc) transferase (OGT) regulates protein O-GlcNAcylation, an essential post-translational modification that is abundant in the brain. Recently, OGT mutations have been associated with intellectual disability, although it is not understood how they affect OGT structure and function. Using a multi-disciplinary approach we show that the L254F OGT mutation leads to conformational changes of the tetratricopeptide repeats and reduced activity, revealing the molecular mechanisms contributing to pathogenesis. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

L1198F Mutation Resensitizes Crizotinib to ALK by Altering the Conformation of Inhibitor and ATP Binding Sites

PubMed Central

Li, Jian; Sun, Rong; Wu, Yuehong; Song, Mingzhu; Li, Jia; Yang, Qianye; Chen, Xiaoyi; Bao, Jinku; Zhao, Qi

2017-01-01

The efficacy of anaplastic lymphoma kinase (ALK) positive non-small-cell lung cancer (NSCLC) treatment with small molecule inhibitors is greatly challenged by acquired resistance. A recent study reported the newest generation inhibitor resistant mutation L1198F led to the resensitization to crizotinib, which is the first Food and Drug Administration (FDA) approved drug for the treatment of ALK-positive NSCLC. It is of great importance to understand how this extremely rare event occurred for the purpose of overcoming the acquired resistance of such inhibitors. In this study, we exploited molecular dynamics (MD) simulation to dissect the molecular mechanisms. Our MD results revealed that L1198F mutation of ALK resulted in the conformational change at the inhibitor site and altered the binding affinity of ALK to crizotinib and lorlatinib. L1198F mutation also affected the autoactivation of ALK as supported by the identification of His1124 and Tyr1278 as critical amino acids involved in ATP binding and phosphorylation. Our findings are valuable for designing more specific and potent inhibitors for the treatment of ALK-positive NSCLC and other types of cancer. PMID:28245558

Molecular characterization of the Serratia marcescens OmpF porin, and analysis of S. marcescens OmpF and OmpC osmoregulation.

PubMed

Hutsul, J A; Worobec, E

1997-08-01

Serratia marcescens is a nosocomial pathogen with a high incidence of beta-lactam resistance. Reduced amounts of outer-membrane porins have been correlated with increased resistance to beta-lactams but only one porin, OmpC, has been characterized at the molecular level. In this study we present the molecular characterization of a second porin, OmpF, and an analysis of the expression of S. marcescens porins in response to various environmental changes. Two porins were isolated from the outer membrane using urea-SDS-PAGE and the relative amounts were shown to be influenced by the osmolarity of the medium and the presence of salicylate. From a S. marcescens genomic DNA library an 8 kb EcoRI fragment was isolated that hybridized with an oligonucleotide encoding the published N-terminal amino acid sequence of the S. marcescens 41 kDa porin. A 41 kDa protein was detected in the outer membrane of Escherichia coli NM522 carrying the cloned S. marcescens DNA. The cloned gene was sequenced and shown to code for a protein that shared 60-70% identity with other known OmpF and OmpC sequences. The upstream DNA sequence of the S. marcescens gene was similar to the corresponding E. coli ompF sequence; however, a regulatory element important in repression of E. coli ompF at high osmolarity was absent. The cloned S. marcescens OmpF in E. coli increased in expression in conditions of high osmolarity. The potential involvement of micF in the observed osmoregulation of S. marcescens porins is discussed.

Low frequency of V617F mutation in JAK2 gene in Indian patients with hepatic venous outflow obstruction and extrahepatic portal venous obstruction.

PubMed

Rai, Praveer; Kumar, Pankaj; Mishra, Swapnil; Aggarwal, Rakesh

2016-09-01

Hepatic venous outflow tract obstruction (HVOTO) and extrahepatic portal venous obstruction (EHPVO) are important causes of portal hypertension and related complications in India. Both these conditions result from splanchnic venous thrombosis. In recent years, a V617F somatic mutation in Janus kinase 2 (JAK2) gene which is highly specific for myeloproliferative disorders has been detected in 40 % to 50 % and 30 % to 35 % of Western patients with HVOTO and EHPVO, respectively. However, data on this mutation in these conditions from Asian countries are limited. We looked for JAK2 V617F mutation in Indian patients with HVOTO (n = 40, median age 31 [range 17-51] years, 21 female) and EHPVO (n = 50, median age 23 [15-70] years, 25 female) by using two separate methods. Both the methods involved polymerase chain reaction using allele-specific primers. Positive results on one or both of these techniques were confirmed using DNA sequencing. None of the 40 patients with HVOTO and only 1 of 50 patients with EHPVO was found to have JAK2 V617F mutation. In the one patient who was found to have this mutation, both the PCR methods and DNA sequencing showed positive results. Hypercoagulability associated with JAK2 V617F mutation and associated chronic myeloproliferative disorders was not a major cause of HVOTO and EHPVO in this population.

Mucopolysaccharidosis IVA: Identification of a common missense mutation I113F in the N-Acetylgalactosamine-6-sulfate sulfatase gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tomatsu, Shunji; Fukuda, Seiji; Rezvi, Maruf

1995-09-01

Mucopolysaccharidosis IVA is an autosomal recessive lysosomal storage disorder caused by a deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). The recent isolation and characterization of cDNA and genomic sequences encoding GALNS has facilitated identification of the molecular lesions that cause MPS IVA. We identified a common missense mutation among Caucasian MPS IVA patients. The mutation was originally detected by SSCP, and successive sequencing revealed an A{yields}T transversion at nt 393. This substitution altered the isoleucine at position 113 to phenylalanine (I113F) in the 622 amino acid GALNS protein and was associated with a severe phenotype in a homozygote. Compound heterogzygotes with onemore » I113F-allele mutation have a wide range of clinical phenotypes. Transfection experiments in GALNS-deficient fibroblasts revealed that the mutation drastically reduces the enzyme activity of GALNS. Allele-specific oligonucleotide or SSCP analysis indicated that this mutation accounted for 22.5% (9/40) of unrelated MPS IVA chromosomes from 23 Caucasian patients, including 6 consanguineous cases. Of interest, the I1e 113{yields}Phe substitution occurred in only Caucasian MPS IVA patients and in none of the GALNS alleles of 20 Japanese patients. These findings identify a frequent missense mutation among MPS IVA patients of Caucasian ancestry that results in severe MPS IVA when homoallelic, and will facilitate molecular diagnosis of most such patients and identification of heterozygous carriers. In addition to this common mutation, 10 different point mutations and 2 small deletions were detected, suggesting allelic heterogeneity in GALNS gene. 32 refs., 2 figs., 3 tabs.« less

Late manifestation of subclinical hyperthyroidism after goitrogenesis in an index patient with a N670S TSH receptor germline mutation masquerading as TSH receptor antibody negative Graves' disease.

PubMed

Schaarschmidt, J; Paschke, S; Özerden, M; Jäschke, H; Huth, S; Eszlinger, M; Meller, J; Paschke, R

2012-12-01

In 27 families with familial non-autoimmune hyperthyroidism (FNAH) reported up to date, the onset of hyperthyroidism varies from 18 months to 60 years. Also the manifestation of goitres is variable in these families. A 74-year-old woman first presented at the age of 69 years with tachyarrhythmia and hypertension. After initial treatment of her hypertension and oral anticoagulation for her intermittent atrial fibrillation, a thyroid workup revealed a suppressed TSH and normal fT3 and fT4. TPO, TSH receptor (TSHR), and thyroglobulin antibodies were negative. Thyroid ultrasound revealed a thyroid volume of 102 ml with several nodules with diameters of up to 2.6 cm right and up to 1.8 cm left. Scintigraphy showed a homogeneous Technetium-99 m ((99 m)Tc) uptake of 1.27%. She was subsequently treated with 1 GBq radioiodine ((131)I). At the age of 74, her thyroid function was normal and her thyroid volume decreased to 90 ml. Because of the diffuse (99 m)Tc uptake and the negative TPO, TSHR, and thyroglobulin antibodies, genetic analysis of her TSHR gene was performed, in spite of her negative family history for hyperthyroidism. Sequencing revealed a N670S TSHR germline mutation. Previous in vitro characterisation of this TSHR mutation suggests a weak constitutive activity, yet the experimental data are ambiguous. This case illustrates the necessity to analyse patients with hyperthyroidism accompanied by diffuse (99 m)Tc uptake and negative TPO, TSHR, and thyroglobulin antibodies for TSHR germline mutations. Moreover, it demonstrates that TSHR germline mutations may first lead to longstanding nodular goitrogenesis before the late manifestation of subclinical hyperthyroidism. © Georg Thieme Verlag KG Stuttgart · New York.

A novel p.Gly603Arg mutation in CACNA1F causes Åland island eye disease and incomplete congenital stationary night blindness phenotypes in a family

PubMed Central

Vincent, Ajoy; Wright, Tom; Day, Megan A.; Westall, Carol A.

2011-01-01

Purpose To report, for the first time, that X-linked incomplete congenital stationary night blindness (CSNB2A) and Åland island eye disease (AIED) phenotypes coexist in a molecularly confirmed pedigree and to present novel phenotypic characteristics of calcium channel alpha-1F subunit gene (CACNA1F)-related disease. Methods Two affected subjects (the proband and his maternal grandfather) and an unaffected obligate carrier (the proband’s mother) underwent detailed ophthalmological evaluation, fundus autofluorescence imaging, and spectral-domain optical coherence tomography. Goldmann visual field assessment and full-field electroretinogram (ERG) were performed in the two affected subjects, and multichannel flash visual evoked potential was performed on the proband. Scotopic 15 Hz flicker ERG series were performed in both affected subjects to evaluate the function of the slow and fast rod pathways. Haplotype analysis using polymorphic microsatellite markers flanking CACNA1F was performed in all three family members. The proband’s DNA was sequenced for mutations in the coding sequence of CACNA1F and nyctalopin (NYX) genes. Segregation analysis was performed in the family. Results Both affected subjects had symptoms of nonprogressive nyctalopia since childhood, while the proband also had photophobia. Both cases had a distance visual acuity of 20/50 or better in each eye, normal contrast sensitivity, and an incomplete type of Schubert-Bornschein ERGs. The proband also had high myopia, a mild red-green color deficit, hypopigmented fundus, and foveal hypoplasia with no evidence of chiasmal misrouting. Spectral-domain optical coherence tomography confirmed the presence of foveal hypoplasia in the proband. The clinical phenotype of the proband and his maternal grandfather fit the clinical description of AIED and CSNB2A, respectively. The fundus autofluorescence and the visual fields were normal in both cases; the scotopic 15 Hz flicker ERG demonstrated only fast rod

Variation in baseline factor VIII concentration in a retrospective cohort of mild/moderate hemophilia A patients carrying identical F8 mutations.

PubMed

Loomans, J I; van Velzen, A S; Eckhardt, C L; Peters, M; Mäkipernaa, A; Holmstrom, M; Brons, P P; Dors, N; Haya, S; Voorberg, J; van der Bom, J G; Fijnvandraat, K

2017-02-01

Essentials Factor VIII levels vary in mild and moderate hemophilia A (MHA) patients with the same mutation. We aimed to estimate the variation and determinants of factor VIII levels among MHA patients. Age and genotype explain 59% of the observed inter-individual variation in factor VIII levels. Intra-individual variation accounted for 45% of the variation in the three largest mutation groups. Background The bleeding phenotype in patients with mild/moderate hemophilia A (MHA) is inversely associated with the residual plasma concentration of factor VIII (FVIII:C). Within a group of patients with the same F8 missense mutation, baseline FVIII:C may vary, because, in healthy individuals, von Willebrand factor (VWF) levels, ABO blood group and age are also known to influence baseline FVIII:C. Our understanding of the pathophysiologic process of the causative genetic event leading to reduced baseline FVIII:C in MHA patients is still limited. Objectives To estimate the variation and determinants of baseline FVIII:C among MHA patients with the same F8 missense mutation. Methods Three hundred and forty-six patients carrying mutations that were present in at least 10 patients in the cohort were selected from the INSIGHT and the RISE studies, which are cohort studies including data of 3534 MHA patients from Europe, Canada, and Australia. Baseline FVIII:C was measured with a one-stage clotting assay. We used Levene's test, univariate and multivariate linear regression, and mixed-model analyses. Results For 59% of patients, the observed variation in baseline FVIII:C was explained by age and genotype. Compared to FVIII:C in patients with Arg612Cys, FVIII:C was significantly different in patients with eight other F8 missense mutations. Intra-individual variation explained 45% of the observed variance in baseline FVIII:C among patients with the same mutation. Conclusion Our results indicate that baseline FVIII:C levels are not exclusively determined by F8 genotype in MHA patients

An accurate, simple prognostic model consisting of age, JAK2, CALR, and MPL mutation status for patients with primary myelofibrosis.

PubMed

Rozovski, Uri; Verstovsek, Srdan; Manshouri, Taghi; Dembitz, Vilma; Bozinovic, Ksenija; Newberry, Kate; Zhang, Ying; Bove, Joseph E; Pierce, Sherry; Kantarjian, Hagop; Estrov, Zeev

2017-01-01

In most patients with primary myelofibrosis, one of three mutually exclusive somatic mutations is detected. In approximately 60% of patients, the Janus kinase 2 gene is mutated, in 20%, the calreticulin gene is mutated, and in 5%, the myeloproliferative leukemia virus gene is mutated. Although patients with mutated calreticulin or myeloproliferative leukemia genes have a favorable outcome, and those with none of these mutations have an unfavorable outcome, prognostication based on mutation status is challenging due to the heterogeneous survival of patients with mutated Janus kinase 2. To develop a prognostic model based on mutation status, we screened primary myelofibrosis patients seen at the MD Anderson Cancer Center, Houston, USA, between 2000 and 2013 for the presence of Janus kinase 2, calreticulin, and myeloproliferative leukemia mutations. Of 344 primary myelofibrosis patients, Janus kinase 2 V617F was detected in 226 (66%), calreticulin mutation in 43 (12%), and myeloproliferative leukemia mutation in 16 (5%); 59 patients (17%) were triple-negatives. A 50% cut-off dichotomized Janus kinase 2-mutated patients into those with high Janus kinase 2 V617F allele burden and favorable survival and those with low Janus kinase 2 V617F allele burden and unfavorable survival. Patients with a favorable mutation status (high Janus kinase 2 V617F allele burden/myeloproliferative leukemia/calreticulin mutation) and aged 65 years or under had a median survival of 126 months. Patients with one risk factor (low Janus kinase 2 V617F allele burden/triple-negative or age >65 years) had an intermediate survival duration, and patients aged over 65 years with an adverse mutation status (low Janus kinase 2 V617F allele burden or triple-negative) had a median survival of only 35 months. Our simple and easily applied age- and mutation status-based scoring system accurately predicted the survival of patients with primary myelofibrosis. Copyright© Ferrata Storti Foundation.

HER2 activating mutations are targets for colorectal cancer treatment

PubMed Central

Kavuri, Shyam M.; Jain, Naveen; Galimi, Francesco; Cottino, Francesca; Leto, Simonetta M.; Migliardi, Giorgia; Searleman, Adam C.; Shen, Wei; Monsey, John; Trusolino, Livio; Jacobs, Samuel A.; Bertotti, Andrea; Bose, Ron

2015-01-01

The Cancer Genome Atlas project identified HER2 somatic mutations and gene amplification in 7% of colorectal cancer patients. Introduction of the HER2 mutations, S310F, L755S, V777L, V842I, and L866M, into colon epithelial cells increased signaling pathways and anchorage-independent cell growth, indicating that they are activating mutations. Introduction of these HER2 activating mutations into colorectal cancer cell lines produced resistance to cetuximab and panitumumab by sustaining MAPK phosphorylation. HER2 mutations are potently inhibited by low nanomolar doses of the irreversible tyrosine kinase inhibitors, neratinib and afatinib. HER2 gene sequencing of 48 cetuximab resistant, quadruple (KRAS, NRAS, BRAF, and PIK3CA) WT colorectal cancer patient-derived xenografts (PDX’s) identified 4 PDX’s with HER2 mutations. HER2 targeted therapies were tested on two PDX’s. Treatment with a single HER2 targeted drug (trastuzumab, neratinib, or lapatinib) delayed tumor growth, but dual HER2 targeted therapy with trastuzumab plus tyrosine kinase inhibitors produced regression of these HER2 mutated PDX’s. PMID:26243863

Spectrum of CFTR gene mutations in Ecuadorian cystic fibrosis patients: the second report of the p.H609R mutation.

PubMed

Ortiz, Sofía C; Aguirre, Santiago J; Flores, Sofía; Maldonado, Claudio; Mejía, Juan; Salinas, Lilian

2017-11-01

High heterogeneity in the CFTR gene mutations disturbs the molecular diagnosis of cystic fibrosis (CF). In order to improve the diagnosis of CF in our country, the present study aims to define a panel of common CFTR gene mutations by sequencing 27 exons of the gene in Ecuadorian Cystic Fibrosis patients. Forty-eight Ecuadorian individuals with suspected/confirmed CF diagnosis were included. Twenty-seven exons of CFTR gene were sequenced to find sequence variations. Prevalence of pathogenic variations were determined and compared with other countries' data. We found 70 sequence variations. Eight of these are CF-causing mutations: p.F508del, p.G85E, p.G330E, p.A455E, p.G970S, W1098X, R1162X, and N1303K. Also this study is the second report of p.H609R in Ecuadorian population. Mutation prevalence differences between Ecuadorian population and other Latin America countries were found. The panel of mutations suggested as an initial screening for the Ecuadorian population with cystic fibrosis should contain the mutations: p.F508del, p.G85E, p.G330E, p.A455E, p.G970S, W1098X, R1162X, and N1303K. © 2017 NETLAB Laboratorios Especializados. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.

[The quantitative testing of V617F mutation in gen JAK2 using pyrosequencing technique].

PubMed

Dunaeva, E A; Mironov, K O; Dribnokhodova, T E; Subbotina, E E; Bashmakova; Ol'hovskiĭ, I A; Shipulin, G A

2014-11-01

The somatic mutation V617F in gen JAK2 is a frequent cause of chronic myeloprolific diseases not conditioned by BCR/ABL mutation. The quantitative testing of relative percentage of mutant allele can be used in establishing severity of disease and its prognosis and in prescription of remedy inhibiting activity of JAK2. To quantitatively test mutation the pyrosequencing technique was applied. The developed technique permits detecting and quantitatively, testing percentage of mutation fraction since 7%. The "gray zone" is presented by samples with percentage of mutant allele from 4% to 7%. The dependence of expected percentage of mutant fraction in analyzed sample from observed value of signal is described by equation of line with regression coefficients y = - 0.97, x = -1.32 and at that measurement uncertainty consists ± 0.7. The developed technique is approved officially on clinical material from 192 patients with main forms of myeloprolific diseases not conditioned by BCR/ABL mutation. It was detected 64 samples with mautant fraction percentage from 13% to 91%. The developed technique permits implementing monitoring of therapy of myeloprolific diseases and facilitates to optimize tactics of treatment.

The development of gastric cancer in a patient with polycythemia Vera, 3P deletion, and JAK2 V617F mutation.

PubMed

Ayvaz, Ozlem; Yavasoglu, Irfan; Kadikoylu, Gurhan; Meydan, Nezih; Barutca, Sabri; Bolaman, Zahit

2010-12-01

3p deletion which is frequently associated with solitary tumors and hematological malignancies is a chromosomal abnormality. Recently, Janus kinase-2 (JAK2) V617F mutation has an important role in the diagnosis of myeloproliferative disorders, especially in polycythemia vera (PV). We reported the development of gastric cancer in a 75-year-old patient with PV, 3p 12-14 deletion and JAK2 V617F mutation. PV was diagnosed according to the classification of World Health Organization. JAK2 V617F mutation with polymerase chain reaction and 3p12-14 deletion with cytogenetic examination of the bone marrow were detected. We investigated solitary tumors in the patient using computed tomographies of thorax, neck, ear, nose, and throat. However, they were normal. After 2 years, gastric cancer appeared in the patient. In conclusion, cytogenetic examination may be important in both the development and the diagnosis of hematological malignancies and solitary tumors. So the patients should be followed closely.

The CDC Hemophilia B mutation project mutation list: a new online resource.

PubMed

Li, Tengguo; Miller, Connie H; Payne, Amanda B; Craig Hooper, W

2013-11-01

Hemophilia B (HB) is caused by mutations in the human gene F9. The mutation type plays a pivotal role in genetic counseling and prediction of inhibitor development. To help the HB community understand the molecular etiology of HB, we have developed a listing of all F9 mutations that are reported to cause HB based on the literature and existing databases. The Centers for Disease Control and Prevention (CDC) Hemophilia B Mutation Project (CHBMP) mutation list is compiled in an easily accessible format of Microsoft Excel and contains 1083 unique mutations that are reported to cause HB. Each mutation is identified using Human Genome Variation Society (HGVS) nomenclature standards. The mutation types and the predicted changes in amino acids, if applicable, are also provided. Related information including the location of mutation, severity of HB, the presence of inhibitor, and original publication reference are listed as well. Therefore, our mutation list provides an easily accessible resource for genetic counselors and HB researchers to predict inhibitors. The CHBMP mutation list is freely accessible at http://www.cdc.gov/hemophiliamutations.

ZnO:Zn/6LiF scintillator-A low afterglow alternative to ZnS:Ag/6LiF for thermal neutron detection

NASA Astrophysics Data System (ADS)

Sykora, G. Jeff; Schooneveld, Erik M.; Rhodes, Nigel J.

2018-03-01

Current ZnS:Ag/6LiF based scintillation detectors are often count rate limited by the long lifetime afterglow in the scintillator. Despite this drawback, new instruments at neutron scattering facilities, like ISIS in the UK, would still like to use ZnS:Ag/6LiF detectors due to their low gamma sensitivity, high light output, simplicity of detector design and relatively inexpensive production. One particular advantage of ZnS:Ag/6LiF detectors is their ability to provide strong pulse shape discrimination between neutrons and gammas. Despite the advantages of these detectors, it is becoming clear that new and upgraded instruments will be limited by the count rate capability of ZnS:Ag/6LiF, so an alternative scintillator technology with equivalent simplicity is being sought. ZnO:Zn/6LiF is investigated here as a low afterglow alternative to ZnS:Ag/6LiF. Basic scintillation properties of ZnO:Zn are studied and are discussed. Pulse shape discrimination between neutrons and gammas is explored and taken advantage of through simple single photon counting methods. A further step toward a realistic detector for neutron scattering is also taken by fiber coupling the ZnO:Zn/6LiF to a PMT. In an initial study of this fiber coupled configuration, 60Co gamma sensitivity of ∼ 7 × 10-6 is shown and improvements in count rate capability of at least a factor of 6 over ZnS:Ag/6LiF based neutron detectors are demonstrated.

Clinical features of Japanese polycythemia vera and essential thrombocythemia patients harboring CALR, JAK2V617F, JAK2Ex12del, and MPLW515L/K mutations.

PubMed

Okabe, Masahiro; Yamaguchi, Hiroki; Usuki, Kensuke; Kobayashi, Yutaka; Kawata, Eri; Kuroda, Junya; Kimura, Shinya; Tajika, Kenji; Gomi, Seiji; Arima, Nobuyoshi; Mori, Sinichiro; Ito, Shigeki; Koizumi, Masayuki; Ito, Yoshikazu; Wakita, Satoshi; Arai, Kunihito; Kitano, Tomoaki; Kosaka, Fumiko; Dan, Kazuo; Inokuchi, Koiti

2016-01-01

The risk of complication of polycythemia vera (PV) and essential thrombocythemia (ET) by thrombosis in Japanese patients is clearly lower than in western populations, suggesting that genetic background such as race may influence the clinical features. This study aimed to clarify the relationship between genetic mutations and haplotypes and clinical features in Japanese patients with PV and ET. Clinical features were assessed prospectively among 74 PV and 303 ET patients. There were no clinical differences, including JAK2V617F allele burden, between PV patients harboring the various genetic mutations. However, CALR mutation-positive ET patients had a significantly lower WBC count, Hb value, Ht value, and neutrophil alkaline phosphatase score (NAP), and significantly more platelets, relative to JAK2V617F-positive ET patients and ET patients with no mutations. Compared to normal controls, the frequency of the JAK246/1 haplotype was significantly higher among patients with JAK2V617F, JAK2Ex12del, or MPL mutations, whereas no significant difference was found among CALR mutation-positive patients. CALR mutation-positive patients had a lower incidence of thrombosis relative to JAK2V617F-positive patients. Our findings suggest that JAK2V617F-positive ET patients and CALR mutation-positive patients have different mechanisms of occurrence and clinical features of ET, suggesting the potential need for therapy stratification in the future. Copyright © 2015 Elsevier Ltd. All rights reserved.

The CFTR trafficking mutation F508del inhibits the constitutive activity of SLC26A9.

PubMed

Bertrand, Carol A; Mitra, Shalini; Mishra, Sanjay K; Wang, Xiaohui; Zhao, Yu; Pilewski, Joseph M; Madden, Dean R; Frizzell, Raymond A

2017-06-01

Several members of the SLC26A family of anion transporters associate with CFTR, forming complexes in which CFTR and SLC26A functions are reciprocally regulated. These associations are thought to be facilitated by PDZ scaffolding interactions. CFTR has been shown to be positively regulated by NHERF-1, and negatively regulated by CAL in airway epithelia. However, it is unclear which PDZ-domain protein(s) interact with SLC26A9, a SLC26A family member found in airway epithelia. We have previously shown that primary, human bronchial epithelia (HBE) from non-CF donors exhibit constitutive anion secretion attributable to SLC26A9. However, constitutive anion secretion is absent in HBE from CF donors. We examined whether changes in SLC26A9 constitutive activity could be attributed to a loss of CFTR trafficking, and what role PDZ interactions played. HEK293 coexpressing SLC26A9 with the trafficking mutant F508del CFTR exhibited a significant reduction in constitutive current compared with cells coexpressing SLC26A9 and wt CFTR. We found that SLC26A9 exhibits complex glycosylation when coexpressed with F508del CFTR, but its expression at the plasma membrane is decreased. SLC26A9 interacted with both NHERF-1 and CAL, and its interaction with both significantly increased with coexpression of wt CFTR. However, coexpression with F508del CFTR only increased SLC26A9's interaction with CAL. Mutation of SLC26A9's PDZ motif decreased this association with CAL, and restored its constitutive activity. Correcting aberrant F508del CFTR trafficking in CF HBE with corrector VX-809 also restored SLC26A9 activity. We conclude that when SLC26A9 is coexpressed with F508del CFTR, its trafficking defect leads to a PDZ motif-sensitive intracellular retention of SLC26A9. Copyright © 2017 the American Physiological Society.

Somatic mutations of GUCY2F, EPHA3, and NTRK3 in human cancers.

PubMed

Wood, Laura D; Calhoun, Eric S; Silliman, Natalie; Ptak, Janine; Szabo, Steve; Powell, Steve M; Riggins, Gregory J; Wang, Tian-Li; Yan, Hai; Gazdar, Adi; Kern, Scott E; Pennacchio, Len; Kinzler, Kenneth W; Vogelstein, Bert; Velculescu, Victor E

2006-10-01

Tyrosine kinases are major regulators of signal transduction cascades involved in cellular proliferation and have important roles in tumorigenesis. We have recently analyzed the tyrosine kinase gene family for alterations in human colorectal cancers and identified somatic mutations in seven members of this gene family. In this study we have used high-throughput sequencing approaches to further evaluate this subset of genes for genetic alterations in other human tumors. We identified somatic mutations in GUCY2F, EPHA3, and NTRK3 in breast, lung, and pancreatic cancers. Our results implicate these tyrosine kinase genes in the pathogenesis of other tumor types and suggest that they may be useful targets for diagnostic and therapeutic intervention in selected patients.

Amylin S20G mutation in Mexican population.

PubMed

Garcia-Gonzalez, Claudia Lorena; Montoya-Fuentes, Hector; Padilla-Rosas, Miguel; Sanchez-Corona, Jose

2007-04-01

Diabetes Mellitus type 2 (DM2) is a group of metabolic disorders characterized by defective insulin action or secretion or both with a 10.6% incidence in Mexican Mestizo population, DM2 is also classified within the localized misfolding diseases due to the amyloid pancreatic deposits found in 90% of the DM2 necropsies. The pancreatic amyloid main component is a protein known as human islet amyloid polypeptide (hIAPP) or amylin, the most common mutation is the S20G in Asian population with a polymorphic frequency in DM2 Asian patients. The aim of this study was to search this mutation in Mexican Mestizo general population (104) and DM2 patients (100). This is the first molecular study of hIAPP gene in Mexican population and in which we developed an alternative more effective antisense primer for the analysis of the NFGAILSS region in hIAPP exon 3 critical for the amyloid beta structure formation. We did not find the mutation in any of the 204 analyzed samples, thus the findings show that S20G is not a common mutation in Mexican Mestizo population.

Using CRISPR/Cas9-mediated gene editing to further explore growth and trade-off effects in myostatin-mutated F4 medaka (Oryzias latipes).

PubMed

Yeh, Ying-Chun; Kinoshita, Masato; Ng, Tze Hann; Chang, Yu-Hsuan; Maekawa, Shun; Chiang, Yi-An; Aoki, Takashi; Wang, Han-Ching

2017-09-12

Myostatin (MSTN) suppresses skeletal muscle development and growth in mammals, but its role in fish is less well understood. Here we used CRISPR/Cas9 to mutate the MSTN gene in medaka (Oryzias latipes) and evaluate subsequent growth performance. We produced mutant F0 fish that carried different frameshifts in the OlMSTN coding sequence and confirmed the heritability of the mutant genotypes to the F1 generation. Two F1 fish with the same heterozygous frame-shifted genomic mutations (a 22 bp insertion in one allele; a 32 bp insertion in the other) were then crossbred to produce subsequent generations (F2~F5). Body length and weight of the MSTN -/- F4 medaka were significantly higher than in the wild type fish, and muscle fiber density in the inner and outer compartments of the epaxial muscles was decreased, suggesting that MSTN null mutation induces muscle hypertrophy. From 3~4 weeks post hatching (wph), the expression of three major myogenic related factors (MRFs), MyoD, Myf5 and Myogenin, was also significantly upregulated. Some medaka had a spinal deformity, and we also observed a trade-off between growth and immunity in MSTN -/- F4 medaka. Reproduction was unimpaired in the fast-growth phenotypes.

Mapping Polymerization and Allostery of Hemoglobin S Using Point Mutations

PubMed Central

Weinkam, Patrick; Sali, Andrej

2014-01-01

Hemoglobin is a complex system that undergoes conformational changes in response to oxygen, allosteric effectors, mutations, and environmental changes. Here, we study allostery and polymerization of hemoglobin and its variants by application of two previously described methods: (i) AllosMod for simulating allostery dynamics given two allosterically related input structures and (ii) a machine-learning method for dynamics- and structure-based prediction of the mutation impact on allostery (Weinkam et al. J. Mol. Biol. 2013), now applicable to systems with multiple coupled binding sites such as hemoglobin. First, we predict the relative stabilities of substates and microstates of hemoglobin, which are determined primarily by entropy within our model. Next, we predict the impact of 866 annotated mutations on hemoglobin’s oxygen binding equilibrium. We then discuss a subset of 30 mutations that occur in the presence of the sickle cell mutation and whose effects on polymerization have been measured. Seven of these HbS mutations occur in three predicted druggable binding pockets that might be exploited to directly inhibit polymerization; one of these binding pockets is not apparent in the crystal structure but only in structures generated by AllosMod. For the 30 mutations, we predict that mutation-induced conformational changes within a single tetramer tend not to significantly impact polymerization; instead, these mutations more likely impact polymerization by directly perturbing a polymerization interface. Finally, our analysis of allostery allows us to hypothesize why hemoglobin evolved to have multiple subunits and a persistent low frequency sickle cell mutation. PMID:23957820

F.S.V.B. Volleyball

DOE Office of Scientific and Technical Information (OSTI.GOV)

None

2006-06-07

22me assemblée des délégués de la Fédération Suisse de Volley Ball (F.S.V.B.), en présence entre autres de Claude Delay, représentant de la société fédérale de gymnastique et Hans Gauer, représentant de l'association suisse de gymnastique féminine. Annonce d'une démonstration d'un mini match de volley ball à Meyrin dans l'après-midi.

Functional characterization of c-Mpl ectodomain mutations that underlie congenital amegakaryocytic thrombocytopenia.

PubMed

Varghese, Leila N; Zhang, Jian-Guo; Young, Samuel N; Willson, Tracy A; Alexander, Warren S; Nicola, Nicos A; Babon, Jeffrey J; Murphy, James M

2014-02-01

Activation of the cell surface receptor, c-Mpl, by the cytokine, thrombopoietin (TPO), underpins megakaryocyte and platelet production in mammals. In humans, mutations in c-Mpl have been identified as the molecular basis of Congenital Amegakaryocytic Thrombocytopenia (CAMT). Here, we show that CAMT-associated mutations in c-Mpl principally lead to defective receptor presentation on the cell surface. In contrast, one CAMT mutant c-Mpl, F104S, was expressed on the cell surface, but showed defective TPO binding and receptor activation. Using mutational analyses, we examined which residues adjacent to F104 within the membrane-distal cytokine receptor homology module (CRM) of c-Mpl comprise the TPO-binding epitope, revealing residues within the predicted Domain 1 E-F and A-B loops and Domain 2 F'-G' loop as key TPO-binding determinants. These studies underscore the importance of the c-Mpl membrane-distal CRM to TPO-binding and suggest that mutations within this CRM that perturb TPO binding could give rise to CAMT.

Drug-resistant molecular mechanism of CRF01_AE HIV-1 protease due to V82F mutation

NASA Astrophysics Data System (ADS)

Liu, Xiaoqing; Xiu, Zhilong; Hao, Ce

2009-05-01

Human immunodeficiency virus type 1 protease (HIV-1 PR) is one of the major targets of anti-AIDS drug discovery. The circulating recombinant form 01 A/E (CRF01_AE, abbreviated AE) subtype is one of the most common HIV-1 subtypes, which is infecting more humans and is expanding rapidly throughout the world. It is, therefore, necessary to develop inhibitors against subtype AE HIV-1 PR. In this work, we have performed computer simulation of subtype AE HIV-1 PR with the drugs lopinavir (LPV) and nelfinavir (NFV), and examined the mechanism of resistance of the V82F mutation of this protease against LPV both structurally and energetically. The V82F mutation at the active site results in a conformational change of 79's loop region and displacement of LPV from its proper binding site, and these changes lead to rotation of the side-chains of residues D25 and I50'. Consequently, the conformation of the binding cavity is deformed asymmetrically and some interactions between PR and LPV are destroyed. Additionally, by comparing the interactive mechanisms of LPV and NFV with HIV-1 PR we discovered that the presence of a dodecahydroisoquinoline ring at the P1' subsite, a [2-(2,6-dimethylphenoxy)acetyl]amino group at the P2' subsite, and an N2 atom at the P2 subsite could improve the binding affinity of the drug with AE HIV-1 PR. These findings are helpful for promising drug design.

40 CFR 60.1245 - Am I exempt from any appendix B or appendix F requirements to evaluate continuous emission...

Code of Federal Regulations, 2011 CFR

2011-07-01

... appendix F requirements to evaluate continuous emission monitoring systems? 60.1245 Section 60.1245... appendix F requirements to evaluate continuous emission monitoring systems? Yes, the accuracy tests for your sulfur dioxide continuous emission monitoring system require you to also evaluate your oxygen (or...

40 CFR 60.1245 - Am I exempt from any appendix B or appendix F requirements to evaluate continuous emission...

Code of Federal Regulations, 2010 CFR

2010-07-01

... appendix F requirements to evaluate continuous emission monitoring systems? 60.1245 Section 60.1245... appendix F requirements to evaluate continuous emission monitoring systems? Yes, the accuracy tests for your sulfur dioxide continuous emission monitoring system require you to also evaluate your oxygen (or...

Identification of Mutation Accumulation as Resistance Mechanism Emerging in First-Line Osimertinib Treatment.

PubMed

Uchibori, Ken; Inase, Naohiko; Nishio, Makoto; Fujita, Naoya; Katayama, Ryohei

2018-04-24

The survival of patients with EGFR mutation-positive lung cancer has dramatically improved since the introduction of EGFR tyrosine kinase inhibitors (EGFR-TKIs). Recently, osimertinib showed significantly prolonged progression-free survival than first-generation EGFR-TKI in first-line treatment, suggesting that a paradigm change that would move osimetinib to first-line treatment is indicated. We performed N-ethyl-N-nitrosourea (ENU) mutagenesis screening to uncover the resistant mechanism in first- and second-line osimertinib treatment. Ba/F3 cells harboring EGFR activating-mutation with or without secondary resistant mutation were exposed to ENU for 24 hours to introduce random mutations and selected with gefitinib, afatinib, or osimertinib. Mutations of emerging resistant cells were assessed. The resistance of T790M and C797S to gefitinib and osimertinib, respectively, was prevalent in the mutagenesis screening with the Ba/F3 cells harboring activating-mutation alone. From C797S/activating-mutation expressing Ba/F3, the additional T790M was a major resistant mechanism in gefitinib and afatinib selection and the additional T854A and L792H were minor resistance mechanisms only in afatinib selection. However, the additional T854A or L792H mediated resistance to all classes of EGFR-TKI. Surprisingly, no resistant clone due to secondary mutation emerged from activating-mutation alone in the gefitinib + osimertinib selection. We showed the resistance mechanism to EGFR-TKI focusing on first- and second-line osimertinib using ENU mutagenesis screening. Additional T854A and L792H on C797S/activating-mutation were found as afatinib resistance and not as gefitinib resistance. Thus, compared to afatinib, the first-generation EGFR-TKI might be preferable as second-line treatment to C797S/activating-mutation emerging after first-line osimertinib treatment. Copyright © 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights

Acute Myeloid Leukemia with MYC Rearrangement and JAK2 V617F Mutation

PubMed Central

Ohanian, Maro; Bueso-Ramos, Carlos; Ok, Chi Young; Lin, Pei; Patel, Keyur; Alattar, Mona Lisa; Khoury, Joseph D.; Rozovski, Uri; Estrov, Zeev; Huh, Yang O.; Cortes, Jorge; Abruzzo, Lynne V.

2016-01-01

Little is known about MYC dysregulation in myeloid malignancies, and we can find no published studies that have evaluated MYC protein expression in primary cases of myelodysplastic syndromes (MDS) or acute myeloid leukemias (AML). We describe the clinical, morphologic, immunophenotypic, cytogenetic, and molecular genetic findings in two MDS/AML cases that contained both MYC rearrangement and JAK2-V617F mutation. We demonstrate MYC protein expression by immunohistochemistry in both patients. PMID:26382622

Removal of copper (II) from aqueous solutions by flotation using polyaluminum chloride silicate (PAX-XL60 S) as coagulant and carbonate ion as activator.

PubMed

Ghazy, S E; Mahmoud, I A; Ragab, A H

2006-01-01

Flotation is a separation technology for removing toxic heavy metal ions from aqueous solutions. Here a simple and rapid flotation procedure is presented for the removal of copper(II) from aqueous solutions. It is based on the use of polyaluminum chloride silicate (PAX-XL60 S) as coagulant and flocculent, carbonate ion as activator and oleic acid (HOL) as surfactant. Both ion and precipitate flotation are included depending on the solution pH. Ion and precipitate flotation in the aqueous HOL-PAX-XL60 S-Cu2+-CO3(2-) system gave powerful preferential removal of Cu2+ (F -100%) over the HOL-PAX-XL60 S-Cu2+ system containing no CO3(2+) ion (F approximately 86%). The role of CO3(2-) ion is also evident from decreasing the dose of PAX-XL60 S from 700 mg l(-1) to 200 mg l(-1). The other parameters, influencing the flotation process, namely: metal ion, surfactant and PAX-XL60 S concentrations, ionic strength, temperature and foreign ions were examined. Moreover, the procedure was successfully applied to recover Cu2+ ions from different volumes up to 11 and from natural water samples.

Suppression of the UV-sensitive phenotype of Escherichia coli recF mutants by recA(Srf) and recA(Tif) mutations requires recJ+.

PubMed Central

Thoms, B; Wackernagel, W

1988-01-01

Mutations in recA, such as recA801(Srf) (suppressor of RecF) or recA441(Tif) (temperature-induced filamentation) partially suppress the deficiency in postreplication repair of UV damage conferred by recF mutations. We observed that spontaneous recA(Srf) mutants accumulated in cultures of recB recC sbcB sulA::Mu dX(Ap lac) lexA51 recF cells because they grew faster than the parental strain. We show that in a uvrA recB+ recC+ genetic background there are two prerequisites for the suppression by recA(Srf) of the UV-sensitive phenotype of recF mutants. (i) The recA(Srf) protein must be provided in increased amounts either by SOS derepression or by a recA operator-constitutive mutation in a lexA(Ind) (no induction of SOS functions) genetic background. (ii) The gene recJ, which has been shown previously to be involved in the recF pathway of recombination and repair, must be functional. The level of expression of recJ in a lexA(Ind) strain suffices for full suppression. Suppression by recA441 at 30 degrees C also depends on recJ+. The hampered induction by UV of the SOS gene uvrA seen in a recF mutant was improved by a recA(Srf) mutation. This improvement did not require recJ+. We suggest that recA(Srf) and recA(Tif) mutant proteins can operate in postreplication repair independent of recF by using the recJ+ function. PMID:2841294

Restoration of CFTR function in patients with cystic fibrosis carrying the F508del-CFTR mutation

PubMed Central

Stefano, Daniela De; Villella, Valeria R; Esposito, Speranza; Tosco, Antonella; Sepe, Angela; Gregorio, Fabiola De; Salvadori, Laura; Grassia, Rosa; Leone, Carlo A; Rosa, Giuseppe De; Maiuri, Maria C; Pettoello-Mantovani, Massimo; Guido, Stefano; Bossi, Anna; Zolin, Anna; Venerando, Andrea; Pinna, Lorenzo A; Mehta, Anil; Bona, Gianni; Kroemer, Guido; Maiuri, Luigi; Raia, Valeria

2014-01-01

Restoration of BECN1/Beclin 1-dependent autophagy and depletion of SQSTM1/p62 by genetic manipulation or autophagy-stimulatory proteostasis regulators, such as cystamine, have positive effects on mouse models of human cystic fibrosis (CF). These measures rescue the functional expression of the most frequent pathogenic CFTR mutant, F508del, at the respiratory epithelial surface and reduce lung inflammation in CftrF508del homozygous mice. Cysteamine, the reduced form of cystamine, is an FDA-approved drug. Here, we report that oral treatment with cysteamine greatly reduces the mortality rate and improves the phenotype of newborn mice bearing the F508del-CFTR mutation. Cysteamine was also able to increase the plasma membrane expression of the F508del-CFTR protein in nasal epithelial cells from F508del homozygous CF patients, and these effects persisted for 24 h after cysteamine withdrawal. Importantly, this cysteamine effect after washout was further sustained by the sequential administration of epigallocatechin gallate (EGCG), a green tea flavonoid, both in vivo, in mice, and in vitro, in primary epithelial cells from CF patients. In a pilot clinical trial involving 10 F508del-CFTR homozygous CF patients, the combination of cysteamine and EGCG restored BECN1, reduced SQSTM1 levels and improved CFTR function from nasal epithelial cells in vivo, correlating with a decrease of chloride concentrations in sweat, as well as with a reduction of the abundance of TNF/TNF-alpha (tumor necrosis factor) and CXCL8 (chemokine [C-X-C motif] ligand 8) transcripts in nasal brushing and TNF and CXCL8 protein levels in the sputum. Altogether, these results suggest that optimal schedules of cysteamine plus EGCG might be used for the treatment of CF caused by the F508del-CFTR mutation. PMID:25350163

Restoration of CFTR function in patients with cystic fibrosis carrying the F508del-CFTR mutation.

PubMed

De Stefano, Daniela; Villella, Valeria R; Esposito, Speranza; Tosco, Antonella; Sepe, Angela; De Gregorio, Fabiola; Salvadori, Laura; Grassia, Rosa; Leone, Carlo A; De Rosa, Giuseppe; Maiuri, Maria C; Pettoello-Mantovani, Massimo; Guido, Stefano; Bossi, Anna; Zolin, Anna; Venerando, Andrea; Pinna, Lorenzo A; Mehta, Anil; Bona, Gianni; Kroemer, Guido; Maiuri, Luigi; Raia, Valeria

2014-01-01

Restoration of BECN1/Beclin 1-dependent autophagy and depletion of SQSTM1/p62 by genetic manipulation or autophagy-stimulatory proteostasis regulators, such as cystamine, have positive effects on mouse models of human cystic fibrosis (CF). These measures rescue the functional expression of the most frequent pathogenic CFTR mutant, F508del, at the respiratory epithelial surface and reduce lung inflammation in Cftr(F508del) homozygous mice. Cysteamine, the reduced form of cystamine, is an FDA-approved drug. Here, we report that oral treatment with cysteamine greatly reduces the mortality rate and improves the phenotype of newborn mice bearing the F508del-CFTR mutation. Cysteamine was also able to increase the plasma membrane expression of the F508del-CFTR protein in nasal epithelial cells from F508del homozygous CF patients, and these effects persisted for 24 h after cysteamine withdrawal. Importantly, this cysteamine effect after washout was further sustained by the sequential administration of epigallocatechin gallate (EGCG), a green tea flavonoid, both in vivo, in mice, and in vitro, in primary epithelial cells from CF patients. In a pilot clinical trial involving 10 F508del-CFTR homozygous CF patients, the combination of cysteamine and EGCG restored BECN1, reduced SQSTM1 levels and improved CFTR function from nasal epithelial cells in vivo, correlating with a decrease of chloride concentrations in sweat, as well as with a reduction of the abundance of TNF/TNF-alpha (tumor necrosis factor) and CXCL8 (chemokine [C-X-C motif] ligand 8) transcripts in nasal brushing and TNF and CXCL8 protein levels in the sputum. Altogether, these results suggest that optimal schedules of cysteamine plus EGCG might be used for the treatment of CF caused by the F508del-CFTR mutation.

Visualizing Mutation-Specific Differences in the Trafficking-Deficient Phenotype of Kv11.1 Proteins Linked to Long QT Syndrome Type 2.

PubMed

Hall, Allison R; Anderson, Corey L; Smith, Jennifer L; Mirshahi, Tooraj; Elayi, Claude S; January, Craig T; Delisle, Brian P

2018-01-01

KCNH2 encodes the Kv11.1 α-subunit that underlies the rapidly activating delayed-rectifier K + current in the heart. Loss-of-function KCNH2 mutations cause long QT syndrome type 2 (LQT2), and most LQT2-linked missense mutations inhibit the trafficking of Kv11.1 channel protein to the cell surface membrane. Several trafficking-deficient LQT2 mutations (e.g., G601S) generate Kv11.1 proteins that are sequestered in a microtubule-dependent quality control (QC) compartment in the transitional endoplasmic reticulum (ER). We tested the hypothesis that the QC mechanisms that regulate LQT2-linked Kv11.1 protein trafficking are mutation-specific. Confocal imaging analyses of HEK293 cells stably expressing the trafficking-deficient LQT2 mutation F805C showed that, unlike G601S-Kv11.1 protein, F805C-Kv11.1 protein was concentrated in several transitional ER subcompartments. The microtubule depolymerizing drug nocodazole differentially affected G601S- and F805C-Kv11.1 protein immunostaining. Nocodazole caused G601S-Kv11.1 protein to distribute into peripheral reticular structures, and it increased the diffuse immunostaining of F805C-Kv11.1 protein around the transitional ER subcompartments. Proteasome inhibition also affected the immunostaining of G601S- and F805C-Kv11.1 protein differently. Incubating cells in MG132 minimally impacted G601S-Kv11.1 immunostaining, but it dramatically increased the diffuse immunostaining of F805C-Kv11.1 protein in the transitional ER. Similar results were seen after incubating cells in the proteasome inhibitor lactacystin. Differences in the cellular distribution of G601S-Kv11.1 and F805C-Kv11.1 protein persisted in transfected human inducible pluripotent stem cell derived cardiomyocytes. These are the first data to visually demonstrate mutation-specific differences in the trafficking-deficient LQT2 phenotype, and this study has identified a novel way to categorize trafficking-deficient LQT2 mutations based on differences in intracellular

Substituent effects in a series of 1,7-C[subscript 60](R[subscript F])[subscript 2] compounds (R[subscript F] = CF[subscript 3], C[subscript 2]F[subscript 5], n-C[subscrip 3]F[subscript 7], i-C[subscript 3]F[subscript 7], n-C[subscript 4]F[subscript 9], s-C[subscript 4]F[subscript 9], n-C[subscript 8]F[subscript 17]): electron affinities, reduction potentials and E(LUMO) values are not always correlated

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuvychko, Igor V.; Whitaker, James B.; Larson, Bryon W.

2013-04-08

A series of seven structurally-similar compounds with different pairs of R{sub F} groups were prepared, characterized spectroscopically, and studied by electrochemical methods (cyclic and square-wave voltammetry), low-temperature anion photoelectron spectroscopy, and DFT calculations (five of the compounds are reported here for the first time). This is the first time that a set of seven R{sub F} groups have been compared with respect to their relative effects on E{sub 1/2}(0/-), electron affinity (EA), and the DFT-calculated LUMO energy. The compounds, 1,7-C{sub 60}(R{sub F}){sub 2} (R{sub F} = CF{sub 3}, C{sub 2}F{sub 5}, i-C{sub 3}F{sub 7}, n-C{sub 3}F{sub 7}, s-C{sub 4}F{sub 9},more » n-C{sub 4}F{sub 9} and n-C{sub 8}F{sub 21}), were found to have statistically different electron affinities (EA), at the {+-}10 meV level of uncertainty, but virtually identical first reduction potentials, at the {+-}10 mV level of uncertainty. The lack of a correlation between EA and E{sub 1/2}(0/-), and between E(LUMO) and E{sub 1/2}(0/-), for such similar compounds is unprecedented and suggests that explanations for differences in figures of merit for materials and/or devices that are based on equating easily measurable E{sub 1/2}(0/-) values with EAs or E(LUMO) values should be viewed with caution. The solubilities of the seven compounds in toluene varied by nearly a factor of six, but in an unpredictable way, with the C{sub 2}F{sub 5} and s-C{sub 4}F{sub 9} compounds being the most soluble and the i-C{sub 3}F{sub 7} compound being the least soluble. The effects of the different R{sub F} groups on EAs, E(LUMO) values, and solubilities should help fluorine chemists choose the right R{sub F} group to design new materials with improved morphological, electronic, optical, and/or magnetic properties.« less

Mutations in the S gene region of hepatitis B virus genotype D in Turkish patients.

PubMed

Ozaslan, Mehmet; Ozaslan, Ersan; Barsgan, Arzu; Koruk, Mehmet

2007-12-01

The S gene region of the hepatitis B virus (HBV) is responsible for the expression of surface antigens and includes the 'a'-determinant region. Thus, mutation(s) in this region would afford HBV variants a distinct survival advantage, permitting the mutant virus to escape from the immune system. The aim of this study was to search for mutations of the S gene region in different patient groups infected with genotype D variants of HBV, and to analyse the biological significance of these mutations. Moreover, we investigated S gene mutation inductance among family members. Forty HBV-DNA-positive patients were determined among 132 hepatitis B surface antigen (HbsAg) carriers by the first stage of seminested PCR. Genotypes and subtypes were established by sequencing of the amplified S gene regions. Variants were compared with original sequences of these serotypes, and mutations were identified. All variants were designated as genotype D and subtype ayw3. Ten kinds of point mutations were identified within the S region. The highest rates of mutation were found in chronic hepatitis patients and their family members. The amino acid mutations 125 (M -> T) and 127 (T -> P) were found on the first loop of 'a'-determinant. The other consequence was mutation inductance in a family member. We found some mutations in the S gene region known to be stable and observed that some of these mutations affected S gene expression.

Prevalence of Mitochondrial 12S rRNA Mutations Associated with Aminoglycoside Ototoxicity

ERIC Educational Resources Information Center

Guan, Min-Xin

2005-01-01

The mitochondrial DNA (mtDNA) 12S rRNA is a hot spot for mutations associated with both aminoglycoside-induced and nonsyndromic hearing loss. Of those, the homoplasmic A1555G and C1494T mutations at a highly conserved decoding region of the 12S rRNA have been associated with hearing loss. These two mutations account for a significant number of…

Frequency of 8 CFTR gene mutations in cystic fibrosis patients in Minas Gerais, Brazil, diagnosed by neonatal screening.

PubMed

Perone, C; Medeiros, G S; del Castillo, D M; de Aguiar, M J B; Januário, J N

2010-02-01

The nature and frequency of cystic fibrosis mutations in Brazil is not uniform due to the highly varied ethnic composition of the population. The average frequency of the F508del mutation has been reported to be 48.6%. Other common mutations in Brazil are G542X, R1162X, and N1303K. The aim of this study was to analyze the frequency of 8 mutations (F508del, G542X, R1162X, N1303K, W1282X, G85E, 3120+1G>A, and 711+1G>T) in a sample of 111 newborn patients with cystic fibrosis diagnosed by the Cystic Fibrosis Neonatal Screening Program of Minas Gerais State. The mutations were tested by allele-specific oligonucleotide PCR with specially designed primers. An allele frequency of 48.2% was observed for the F508del mutation, and allele frequencies of 5.41, 4.50, 4.05, and 3.60% were found for the R1162X, G542X, 3120+1G>A, and G85E mutations, respectively. The genotypes obtained were in Hardy-Weinberg equilibrium. These data demonstrate that the 8-mutation panel studied here has extensive coverage (68%) for the cystic fibrosis mutations in Minas Gerais. These data improve our knowledge of cystic fibrosis in Brazil, particularly in this region. In addition, this investigation contributed to the establishment of a sensitive and population-specific mutation panel, which can be helpful for molecular diagnosis of cystic fibrosis.

Temporal frequency of knockdown resistance mutations, F1534C and V1016G, in Aedes aegypti in Chiang Mai city, Thailand and the impact of the mutations on the efficiency of thermal fogging spray with pyrethroids.

PubMed

Plernsub, Suriya; Saingamsook, Jassada; Yanola, Jintana; Lumjuan, Nongkran; Tippawangkosol, Pongsri; Walton, Catherine; Somboon, Pradya

2016-10-01

In Thailand, control of dengue outbreaks is currently attained by the use of space sprays, particularly thermal fogging using pyrethroids, with the aim of killing infected Aedes mosquito vectors in epidemic areas. However, the principal dengue vector, Aedes aegypti, is resistant to pyrethroids conferred mainly by mutations in the voltage-gated sodium channel gene, F1534C and V1016G, termed knockdown resistance (kdr). The objectives of this study were to determine the temporal frequencies of F1534C and V1016G in Ae. aegypti populations in relation to pyrethroid resistance in Chiang Mai city, and to evaluate the impact of the mutations on the efficacy of thermal fogging with the pyrethroid deltamethrin. Larvae and pupae were collected from several areas around Chiang Mai city during 2011-2015 and reared to adulthood for bioassays for deltamethrin susceptibility. These revealed no trend of increasing deltamethrin resistance during the study period (mortality 58.0-69.5%, average 62.8%). This corresponded to no overall change in the frequencies of the C1534 allele (0.55-0.66, average 0.62) and G1016 allele (0.34-0.45, average 0.38), determined using allele specific amplification. Only three genotypes of kdr mutations were detected: C1534 homozygous (VV/CC); G1016/C1534 double heterozygous (VG/FC); and G1016 homozygous (GG/FF) indicating that the F1534C and V1016G mutations occurred on separate haplotypic backgrounds and a lack of recombination between them to date. The F1 progeny females were used to evaluate the efficacy of thermal fogging spray with Damthrin-SP(®) (deltamethrin+S-bioallethrin+piperonyl butoxide) using a caged mosquito bioassay. The thermal fogging spray killed 100% and 61.3% of caged mosquito bioassay placed indoors and outdoors, respectively. The outdoor spray had greater killing effect on C1534 homozygous and had partially effect on double heterozygous mosquitoes, but did not kill any G1016 homozygous mutants living outdoors. As this selection

Correlated motion and the effect of distal mutations in dihydrofolate reductase

PubMed Central

Rod, Thomas H.; Radkiewicz, Jennifer L.; Brooks, Charles L.

2003-01-01

Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate to tetrahydrofolate. The catalytic rate in this system has been found to be significantly affected by mutations far from the site of chemical activity in the enzyme [Rajagopalan, P. T. R, Lutz, S., and Benkovic, S. J. (2002) Biochemistry 41, 12618–12628]. On the basis of extensive computer simulations for wild-type DHFR from Escherichia coli and four mutants (G121S, G121V, M42F, and M42F/G121S), we show that key parameters for catalysis are changed. The parameters we study are relative populations of different conformations sampled and hydrogen bonds. We find that the mutations result in long-range structural perturbations, rationalizing the effects that the mutations have on the kinetics of the enzyme. Such perturbations also provide a rationalization for the reported nonadditivity effect for double mutations. We finally examine the role a structural perturbation will have on the hydride transfer step. On the basis of our new findings, we discuss the role of coupled motions between distant regions in the enzyme, which previously was reported by Radkiewicz and Brooks. PMID:12756296

Calreticulin mutation analysis in non-mutated Janus kinase 2 essential thrombocythemia patients in Chiang Mai University: analysis of three methods and clinical correlations.

PubMed

Rattarittamrong, Ekarat; Tantiworawit, Adisak; Kumpunya, Noppamas; Wongtagan, Ornkamon; Tongphung, Ratchanoo; Phusua, Arunee; Chai-Adisaksopha, Chatree; Hantrakool, Sasinee; Rattanathammethee, Thanawat; Norasetthada, Lalita; Charoenkwan, Pimlak; Lekawanvijit, Suree

2018-03-09

The primary objective was to determine the prevalence of calreticulin (CALR) mutation in patients with non-JAK2V617F mutated essential thrombocythemia (ET). The secondary objectives were to evaluate the accuracy of CALR mutation analysis by high-resolution melting (HRM) analysis and real-time polymerase chain reaction (PCR) compared with DNA sequencing and to compare clinical characteristics of CALR mutated and JAK2V617F mutated ET. This was a prospective cohort study involving ET patients registered at Chiang Mai University in the period September 2015-September 2017 who were aged more than 2 years, and did not harbor JAK2V617F mutation. The presence of CALR mutation was established by DNA sequencing, HRM, and real-time PCR for type 1 and type 2 mutation. Clinical data were compared with that from ET patients with mutated JAK2V617F. Twenty-eight patients were enrolled onto the study. CALR mutations were found in 10 patients (35.7%). Three patients had type 1 mutation, 5 patients had type 2 mutation, 1 patient had type 18 mutation, and 1 patients had novel mutations (c.1093 C-G, c.1098_1131 del, c.1135 G-A). HRM could differentiate between the types of mutation in complete agreement with DNA sequencing. Patients with a CALR mutation showed a significantly greater male predominance and had a higher platelet count when compared with 42 JAK2V617F patients. The prevalence of CALR mutation in JAK2V617F-negative ET in this study is 35.7%. HRM is an effective method of detecting CALR mutation and is a more advantageous method of screening for CALR mutation.

Pharmacogenetics and pathophysiology of CACNA1S mutations in malignant hyperthermia.

PubMed

Beam, Teresa A; Loudermilk, Emily F; Kisor, David F

2017-02-01

A review of the pharmacogenetics (PGt) and pathophysiology of calcium voltage-gated channel subunit alpha1 S (CACNA1S) mutations in malignant hyperthermia susceptibility type 5 (MHS5; MIM #60188) is presented. Malignant hyperthermia (MH) is a life-threatening hypermetabolic state of skeletal muscle usually induced by volatile, halogenated anesthetics and/or the depolarizing neuromuscular blocker succinylcholine. In addition to ryanodine receptor 1 (RYR1) mutations, several CACNA1S mutations are known to be risk factors for increased susceptibility to MH (MHS). However, the presence of these pathogenic CACNA1S gene variations cannot be used to positively predict MH since the condition is genetically heterogeneous with variable expression and incomplete penetrance. At present, one or at most six CACNA1S mutations display significant linkage or association either to clinically diagnosed MH or to MHS as determined by contracture testing. Additional pathogenic variants in CACNA1S, either alone or in combination with genes affecting Ca 2+ homeostasis, are likely to be discovered in association to MH as whole exome sequencing becomes more commonplace. Copyright © 2017 the American Physiological Society.

F.S.V.B. Volleyball

ScienceCinema

None

2017-12-09

22me assemblée des délégués de la Fédération Suisse de Volley Ball (F.S.V.B.), en présence entre autres de Claude Delay, représentant de la société fédérale de gymnastique et Hans Gauer, représentant de l'association suisse de gymnastique féminine. Annonce d'une démonstration d'un mini match de volley ball à Meyrin dans l'après-midi.

HSAN1 mutations in serine palmitoyltransferase reveal a close structure-function-phenotype relationship.

PubMed

Bode, Heiko; Bourquin, Florence; Suriyanarayanan, Saranya; Wei, Yu; Alecu, Irina; Othman, Alaa; Von Eckardstein, Arnold; Hornemann, Thorsten

2016-03-01

Hereditary sensory and autonomic neuropathy type 1 (HSAN1) is a rare autosomal dominant inherited peripheral neuropathy caused by mutations in the SPTLC1 and SPTLC2 subunits of serine palmitoyltransferase (SPT). The mutations induce a permanent shift in the substrate preference from L-serine to L-alanine, which results in the pathological formation of atypical and neurotoxic 1-deoxy-sphingolipids (1-deoxySL). Here we compared the enzymatic properties of 11 SPTLC1 and six SPTLC2 mutants using a uniform isotope labelling approach. In total, eight SPT mutants (STPLC1p.C133W, p.C133Y, p.S331F, p.S331Y and SPTLC2p.A182P, p.G382V, p.S384F, p.I504F) were associated with increased 1-deoxySL synthesis. Despite earlier reports, canonical activity with l-serine was not reduced in any of the investigated SPT mutants. Three variants (SPTLC1p.S331F/Y and SPTLC2p.I505Y) showed an increased canonical activity and increased formation of C20 sphingoid bases. These three mutations are associated with an exceptionally severe HSAN1 phenotype, and increased C20 sphingosine levels were also confirmed in plasma of patients. A principal component analysis of the analysed sphingoid bases clustered the mutations into three separate entities. Each cluster was related to a distinct clinical outcome (no, mild and severe HSAN1 phenotype). A homology model based on the protein structure of the prokaryotic SPT recapitulated the same grouping on a structural level. Mutations associated with the mild form clustered around the active site, whereas mutations associated with the severe form were located on the surface of the protein. In conclusion, we showed that HSAN1 mutations in SPT have distinct biochemical properties, which allowed for the prediction of the clinical symptoms on the basis of the plasma sphingoid base profile. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

An MRPS12 mutation modifies aminoglycoside sensitivity caused by 12S rRNA mutations

PubMed Central

Emperador, Sonia; Pacheu-Grau, David; Bayona-Bafaluy, M. Pilar; Garrido-Pérez, Nuria; Martín-Navarro, Antonio; López-Pérez, Manuel J.; Montoya, Julio; Ruiz-Pesini, Eduardo

2015-01-01

Several homoplasmic pathologic mutations in mitochondrial DNA, such as those causing Leber hereditary optic neuropathy or non-syndromic hearing loss, show incomplete penetrance. Therefore, other elements must modify their pathogenicity. Discovery of these modifying factors is not an easy task because in multifactorial diseases conventional genetic approaches may not always be informative. Here, we have taken an evolutionary approach to unmask putative modifying factors for a particular homoplasmic pathologic mutation causing aminoglycoside-induced and non-syndromic hearing loss, the m.1494C>T transition in the mitochondrial DNA. The mutation is located in the decoding site of the mitochondrial ribosomal RNA. We first looked at mammalian species that had fixed the human pathologic mutation. These mutations are called compensated pathogenic deviations because an organism carrying one must also have another that suppresses the deleterious effect of the first. We found that species from the primate family Cercopithecidae (old world monkeys) harbor the m.1494T allele even if their auditory function is normal. In humans the m.1494T allele increases the susceptibility to aminoglycosides. However, in primary fibroblasts from a Cercopithecidae species, aminoglycosides do not impair cell growth, respiratory complex IV activity and quantity or the mitochondrial protein synthesis. Interestingly, this species also carries a fixed mutation in the mitochondrial ribosomal protein S12. We show that the expression of this variant in a human m.1494T cell line reduces its susceptibility to aminoglycosides. Because several mutations in this human protein have been described, they may possibly explain the absence of pathologic phenotype in some pedigree members with the most frequent pathologic mutations in mitochondrial ribosomal RNA. PMID:25642242

Analysis of mutational resistance to trimethoprim in Staphylococcus aureus by genetic and structural modelling techniques.

PubMed

Vickers, Anna A; Potter, Nicola J; Fishwick, Colin W G; Chopra, Ian; O'Neill, Alex J

2009-06-01

This study sought to expand knowledge on the molecular mechanisms of mutational resistance to trimethoprim in Staphylococcus aureus, and the fitness costs associated with resistance. Spontaneous trimethoprim-resistant mutants of S. aureus SH1000 were recovered in vitro, resistance genotypes characterized by DNA sequencing of the gene encoding the drug target (dfrA) and the fitness of mutants determined by pair-wise growth competition assays with SH1000. Novel resistance genotypes were confirmed by ectopic expression of dfrA alleles in a trimethoprim-sensitive S. aureus strain. Molecular models of S. aureus dihydrofolate reductase (DHFR) were constructed to explore the structural basis of trimethoprim resistance, and to rationalize the observed in vitro fitness of trimethoprim-resistant mutants. In addition to known amino acid substitutions in DHFR mediating trimethoprim resistance (F(99)Y and H(150)R), two novel resistance polymorphisms (L(41)F and F(99)S) were identified among the trimethoprim-resistant mutants selected in vitro. Molecular modelling of mutated DHFR enzymes provided insight into the structural basis of trimethoprim resistance. Calculated binding energies of the substrate (dihydrofolate) for the mutant and wild-type enzymes were similar, consistent with apparent lack of fitness costs for the resistance mutations in vitro. Reduced susceptibility to trimethoprim of DHFR enzymes carrying substitutions L(41)F, F(99)S, F(99)Y and H(150)R appears to result from structural changes that reduce trimethoprim binding to the enzyme. However, the mutations conferring trimethoprim resistance are not associated with fitness costs in vitro, suggesting that the survival of trimethoprim-resistant strains emerging in the clinic may not be subject to a fitness disadvantage.

Oncogenetic mutations combined with MRD improve outcome prediction in pediatric T-cell acute lymphoblastic leukemia.

PubMed

Petit, Arnaud; Trinquand, Amélie; Chevret, Sylvie; Ballerini, Paola; Cayuela, Jean-Michel; Grardel, Nathalie; Touzart, Aurore; Brethon, Benoit; Lapillonne, Hélène; Schmitt, Claudine; Thouvenin, Sandrine; Michel, Gerard; Preudhomme, Claude; Soulier, Jean; Landman-Parker, Judith; Leverger, Guy; Macintyre, Elizabeth; Baruchel, André; Asnafi, Vahid

2018-01-18

Risk stratification in childhood T-cell acute lymphoblastic leukemia (T-ALL) is mainly based on minimal residual disease (MRD) quantification. Whether oncogenetic mutation profiles can improve the discrimination of MRD-defined risk categories was unknown. Two hundred and twenty FRALLE2000T-treated patients were tested retrospectively for NOTCH1/FBXW7/RAS and PTEN alterations. Patients with NOTCH1/FBXW7 ( N/F ) mutations and RAS/PTEN ( R/P ) germ line (GL) were classified as oncogenetic low risk (gLoR; n = 111), whereas those with N/F GL and R/P GL mutations or N/F and R/P mutations were classified as high risk (gHiR; n = 109). Day 35 MRD status was available for 191 patients. Five-year cumulative incidence of relapse (CIR) and disease-free survival were 36% and 60% for gHiR patients and 11% and 89% for gLoR patients, respectively. Importantly, among the 60% of patients with MRD <10 -4 , 5-year CIR was 29% for gHiR patients and 4% for gLoR patients. Based on multivariable Cox models and stepwise selection, the 3 most discriminating variables were the oncogenetic classifier, MRD, and white blood cell (WBC) count. Patients harboring a WBC count ≥200 × 10 9 /L, gHiR classifier, and MRD ≥10 -4 demonstrated a 5-year CIR of 46%, whereas the 58 patients (30%) with a WBC count <200 × 10 9 /L, gLoR classifier, and MRD <10 -4 had a very low risk of relapse, with a 5-year CIR of only 2%. In childhood T-ALL, the N/F/R/P mutation profile is an independent predictor of relapse. When combined with MRD and a WBC count ≥200 × 10 9 /L, it identifies a significant subgroup of patients with a low risk of relapse. © 2018 by The American Society of Hematology.

Stabilizing and destabilizing effects of phenylalanine --> F5-phenylalanine mutations on the folding of a small protein.

PubMed

Woll, Matthew G; Hadley, Erik B; Mecozzi, Sandro; Gellman, Samuel H

2006-12-20

We report a systematic evaluation of phenylalanine-to-pentafluorophenylalanine (Phe --> F5-Phe) mutants for the 35-residue chicken villin headpiece subdomain (c-VHP), the hydrophobic core of which features a cluster of three Phe side chains (residues 6, 10, and 17). Phe --> F5-Phe mutations are interesting because aryl-perfluoroaryl interactions of optimal geometry are intrinsically more favorable than aryl-aryl interactions and because perfluoroaryl units are more hydrophobic than are analogous aryl units. One mutant, Phe-10 --> F5-Phe, provides enhanced tertiary structural stability relative to the native sequence. The other six mutants analyzed caused a decrease in stability.

Whole-exome sequencing identifies novel MPL and JAK2 mutations in triple-negative myeloproliferative neoplasms

PubMed Central

Milosevic Feenstra, Jelena D.; Nivarthi, Harini; Gisslinger, Heinz; Leroy, Emilie; Rumi, Elisa; Chachoua, Ilyas; Bagienski, Klaudia; Kubesova, Blanka; Pietra, Daniela; Gisslinger, Bettina; Milanesi, Chiara; Jäger, Roland; Chen, Doris; Berg, Tiina; Schalling, Martin; Schuster, Michael; Bock, Christoph; Constantinescu, Stefan N.; Cazzola, Mario

2016-01-01

Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are chronic diseases characterized by clonal hematopoiesis and hyperproliferation of terminally differentiated myeloid cells. The disease is driven by somatic mutations in exon 9 of CALR or exon 10 of MPL or JAK2-V617F in >90% of the cases, whereas the remaining cases are termed “triple negative.” We aimed to identify the disease-causing mutations in the triple-negative cases of ET and PMF by applying whole-exome sequencing (WES) on paired tumor and control samples from 8 patients. We found evidence of clonal hematopoiesis in 5 of 8 studied cases based on clonality analysis and presence of somatic genetic aberrations. WES identified somatic mutations in 3 of 8 cases. We did not detect any novel recurrent somatic mutations. In 3 patients with clonal hematopoiesis analyzed by WES, we identified a somatic MPL-S204P, a germline MPL-V285E mutation, and a germline JAK2-G571S variant. We performed Sanger sequencing of the entire coding region of MPL in 62, and of JAK2 in 49 additional triple-negative cases of ET or PMF. New somatic (T119I, S204F, E230G, Y591D) and 1 germline (R321W) MPL mutation were detected. All of the identified MPL mutations were gain-of-function when analyzed in functional assays. JAK2 variants were identified in 5 of 57 triple-negative cases analyzed by WES and Sanger sequencing combined. We could demonstrate that JAK2-V625F and JAK2-F556V are gain-of-function mutations. Our results suggest that triple-negative cases of ET and PMF do not represent a homogenous disease entity. Cases with polyclonal hematopoiesis might represent hereditary disorders. PMID:26423830

Whole-exome sequencing identifies novel MPL and JAK2 mutations in triple-negative myeloproliferative neoplasms.

PubMed

Milosevic Feenstra, Jelena D; Nivarthi, Harini; Gisslinger, Heinz; Leroy, Emilie; Rumi, Elisa; Chachoua, Ilyas; Bagienski, Klaudia; Kubesova, Blanka; Pietra, Daniela; Gisslinger, Bettina; Milanesi, Chiara; Jäger, Roland; Chen, Doris; Berg, Tiina; Schalling, Martin; Schuster, Michael; Bock, Christoph; Constantinescu, Stefan N; Cazzola, Mario; Kralovics, Robert

2016-01-21

Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are chronic diseases characterized by clonal hematopoiesis and hyperproliferation of terminally differentiated myeloid cells. The disease is driven by somatic mutations in exon 9 of CALR or exon 10 of MPL or JAK2-V617F in >90% of the cases, whereas the remaining cases are termed "triple negative." We aimed to identify the disease-causing mutations in the triple-negative cases of ET and PMF by applying whole-exome sequencing (WES) on paired tumor and control samples from 8 patients. We found evidence of clonal hematopoiesis in 5 of 8 studied cases based on clonality analysis and presence of somatic genetic aberrations. WES identified somatic mutations in 3 of 8 cases. We did not detect any novel recurrent somatic mutations. In 3 patients with clonal hematopoiesis analyzed by WES, we identified a somatic MPL-S204P, a germline MPL-V285E mutation, and a germline JAK2-G571S variant. We performed Sanger sequencing of the entire coding region of MPL in 62, and of JAK2 in 49 additional triple-negative cases of ET or PMF. New somatic (T119I, S204F, E230G, Y591D) and 1 germline (R321W) MPL mutation were detected. All of the identified MPL mutations were gain-of-function when analyzed in functional assays. JAK2 variants were identified in 5 of 57 triple-negative cases analyzed by WES and Sanger sequencing combined. We could demonstrate that JAK2-V625F and JAK2-F556V are gain-of-function mutations. Our results suggest that triple-negative cases of ET and PMF do not represent a homogenous disease entity. Cases with polyclonal hematopoiesis might represent hereditary disorders. © 2016 by The American Society of Hematology.

The de novo Q167K mutation in the POU1F1 gene leads to combined pituitary hormone deficiency in an Italian patient.

PubMed

Malvagia, Sabrina; Poggi, Giovanni Maria; Pasquini, Elisabetta; Donati, Maria Alice; Pela, Ivana; Morrone, Amelia; Zammarchi, Enrico

2003-11-01

The POU1F1 gene encodes a transcription factor that is important for the development and differentiation of the cells producing GH, prolactin, and TSH in the anterior pituitary gland. Patients with POU1F1 mutations show a combined pituitary hormone deficiency with low or absent levels of GH, prolactin, and TSH. Fourteen mutations have been reported in the POU1F1 gene up to now. These genetic lesions can be inherited either in an autosomal dominant or an autosomal recessive mode. We report on the first Italian patient, a girl, affected by combined pituitary hormone deficiency. The patient was found to be positive for congenital hypothyroidism (with low TSH levels) at neonatal screening. Substitutive therapy was started, but subsequent growth was very poor, although psychomotor development was substantially normal. Hospitalized at 10 mo she showed hypotonic crises, growth retardation, delayed bone age, and facial dysmorphism. In addition to congenital hypothyroidism, GH and prolactin deficiencies were found. Mutation DNA analysis of the patient's POU1F1 gene identified the novel Q167K amino acid change at the heterozygous level. The highly conserved Q167 residue is located in the POU-specific domain. No mutation was detected in the other allele. DNA analysis in the proband's parents did not identify this amino acid substitution, suggesting a de novo genetic lesion. From these data it can be hypothesized that the Q167K mutation has a dominant negative effect.

A KCNQ1 mutation causes age-dependant bradycardia and persistent atrial fibrillation.

PubMed

Ki, Chang-Seok; Jung, Chae Lim; Kim, Hyun-ji; Baek, Kwan-Hyuck; Park, Seung Jung; On, Young Keun; Kim, Ki-Suk; Noh, Su Jin; Youm, Jae Boum; Kim, June Soo; Cho, Hana

2014-03-01

Atrial fibrillation (AF) is the most common arrhythmia. Gain-of-function mutations in KCNQ1, the pore-forming α-subunit of the slow delayed rectifier K current (IKs) channel, have been associated with AF. The purpose of this study was functional assessment of a mutation in KCNQ1 identified in a family with persistent AF and sinus bradycardia. We investigated whether this KCNQ1 missense mutation could form the genetic basis for AF and bradycardia simultaneously in this family. Sanger sequencing in a family with hereditary persistent AF identified a novel KCNQ1 variant (V241F) in a highly conserved region of S4 domain. The proband and her son developed bradycardia and persistent AF in an age-dependent fashion. The other son was a mutation carrier but he showed sinus bradycardia and not AF. Whole-cell patch clamp electrophysiology showed that V241F mutation in KCNQ1 shifted the activation curve to the left and dramatically slowed deactivation, leading to a constitutively open-like phenotype. Computer modeling showed that V241F would slow pacemaker activity. Also, simulations of atrial excitation predicted that V241F results in extreme shortening of action potential duration, possibly resulting in AF. Our study indicates that V241F might cause sinus bradycardia by increasing IKs. Additionally, V241F likely shortens atrial refractoriness to promote a substrate for reentry. KCNQ1 mutations have previously been described in AF, yet this is the first time a mutation in KCNQ1 is associated with age-dependent bradycardia and persistent AF. This finding further supports the hypothesis that sinus node dysfunction contributes to the development of AF.

The novel c.247_249delTTC (p.F83del) GJB2 mutation in a family with prelingual sensorineural deafness.

PubMed

Petersen, Michael B; Grigoriadou, Maria; Koutroumpe, Maria; Kokotas, Haris

2012-07-01

Non-syndromic hearing loss is one of the most common hereditary determined diseases in human, and the disease is a genetically heterogeneous disorder. Mutations in the GJB2 gene, encoding connexin 26 (Cx26), are a major cause of non-syndromic recessive hearing impairment in many countries and are largely dependent on ethnic groups. Due to the high frequency of the c.35delG GJB2 mutation in the Greek population, we have previously suggested that Greek patients with sensorineural, non-syndromic deafness should be tested for the c.35delG mutation and the coding region of the GJB2 gene should be sequenced in c.35delG heterozygotes. Here we present on the clinical and molecular genetic evaluation of a family suffering from prelingual, sensorineural, non-syndromic deafness. A novel c.247_249delTTC (p.F83del) GJB2 mutation was detected in compound heterozygosity with the c.35delG GJB2 mutation in the proband and was later confirmed in the father, while the mother was homozygous for the c.35delG GJB2 mutation. We conclude that compound heterozygosity of the novel c.247_249delTTC (p.F83del) and the c.35delG mutations in the GJB2 gene was the cause of deafness in the proband and his father. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

The CDC Hemophilia A Mutation Project (CHAMP) Mutation List: a New Online Resource

PubMed Central

Payne, Amanda B.; Miller, Connie H.; Kelly, Fiona M.; Soucie, J. Michael; Hooper, W. Craig

2015-01-01

Genotyping efforts in hemophilia A (HA) populations in many countries have identified large numbers of unique mutations in the Factor VIII gene (F8). To assist HA researchers conducting genotyping analyses, we have developed a listing of F8 mutations including those listed in existing locus-specific databases as well as those identified in patient populations and reported in the literature. Each mutation was reviewed and uniquely identified using Human Genome Variation Society (HGVS) nomenclature standards for coding DNA and predicted protein changes as well as traditional nomenclature based on the mature, processed protein. Listings also include the associated hemophilia severity classified by International Society of Thrombosis and Haemostasis (ISTH) criteria, associations of the mutations with inhibitors, and reference information. The mutation list currently contains 2,537 unique mutations known to cause HA. HA severity caused by the mutation is available for 2,022 mutations (80%) and information on inhibitors is available for 1,816 mutations (72%). The CDC Hemophilia A Mutation Project (CHAMP) Mutation List is available at http://www.cdc.gov/hemophiliamutations for download and search and will be updated quarterly based on periodic literature reviews and submitted reports. PMID:23280990

Mutational analysis of the major soybean UreF paralogue involved in urease activation.

PubMed

Polacco, Joe C; Hyten, David L; Medeiros-Silva, Mônica; Sleper, David A; Bilyeu, Kristin D

2011-06-01

The soybean genome duplicated ∼14 and 45 million years ago and has many paralogous genes, including those in urease activation (emplacement of Ni and CO(2) in the active site). Activation requires the UreD and UreF proteins, each encoded by two paralogues. UreG, a third essential activation protein, is encoded by the single-copy Eu3, and eu3 mutants lack activity of both urease isozymes. eu2 has the same urease-negative phenotype, consistent with Eu2 being a single-copy gene, possibly encoding a Ni carrier. Unexpectedly, two eu2 alleles co-segregated with missense mutations in the chromosome 2 UreF paralogue (Ch02UreF), suggesting lack of expression/function of Ch14UreF. However, Ch02UreF and Ch14UreF transcripts accumulate at the same level. Further, it had been shown that expression of the Ch14UreF ORF complemented a fungal ureF mutant. A third, nonsense (Q2*) allelic mutant, eu2-c, exhibited 5- to 10-fold more residual urease activity than missense eu2-a or eu2-b, though eu2-c should lack all Ch02UreF protein. It is hypothesized that low-level activation by Ch14UreF is 'spoiled' by the altered missense Ch02UreF proteins ('epistatic dominant-negative'). In agreement with active 'spoiling' by eu2-b-encoded Ch02UreF (G31D), eu2-b/eu2-c heterozygotes had less than half the urease activity of eu2-c/eu2-c siblings. Ch02UreF (G31D) could spoil activation by Chr14UreF because of higher affinity for the activation complex, or because Ch02UreF (G31D) is more abundant than Ch14UreF. Here, the latter is favoured, consistent with a reported in-frame AUG in the 5' leader of Chr14UreF transcript. Translational inhibition could represent a form of 'functional divergence' of duplicated genes.

TG101209, a small molecule JAK2-selective kinase inhibitor potently inhibits myeloproliferative disorder-associated JAK2V617F and MPLW515L/K mutations.

PubMed

Pardanani, A; Hood, J; Lasho, T; Levine, R L; Martin, M B; Noronha, G; Finke, C; Mak, C C; Mesa, R; Zhu, H; Soll, R; Gilliland, D G; Tefferi, A

2007-08-01

JAK2V617F and MPLW515L/K represent recently identified mutations in myeloproliferative disorders (MPD) that cause dysregulated JAK-STAT signaling, which is implicated in MPD pathogenesis. We developed TG101209, an orally bioavailable small molecule that potently inhibits JAK2 (IC(50)=6 nM), FLT3 (IC(50)=25 nM) and RET (IC(50)=17 nM) kinases, with significantly less activity against other tyrosine kinases including JAK3 (IC(50)=169 nM). TG101209 inhibited growth of Ba/F3 cells expressing JAK2V617F or MPLW515L mutations with an IC(50) of approximately 200 nM. In a human JAK2V617F-expressing acute myeloid leukemia cell line, TG101209-induced cell cycle arrest and apoptosis, and inhibited phosphorylation of JAK2V617F, STAT5 and STAT3. Therapeutic efficacy of TG101209 was demonstrated in a nude mouse model. Furthermore, TG101209 suppressed growth of hematopoietic colonies from primary progenitor cells harboring JAK2V617F or MPL515 mutations.

FlaF is a β-sandwich protein that anchors the archaellum in the archaeal cell envelope by binding the S-layer protein

DOE PAGES

Banerjee, Ankan; Tsai, Chi -Lin; Chaudhury, Paushali; ...

2015-05-01

Archaea employ the archaellum, a type IV pilus-like nanomachine, for swimming motility. In the crenarchaeon Sulfolobus acidocaldarius, the archaellum consists of seven proteins: FlaB/X/G/F/H/I/J. FlaF is conserved and essential for archaellum assembly but no FlaF structures exist. Here, we truncated the FlaF N terminus and solved 1.5-Å and 1.65-Å resolution crystal structures of this monotopic membrane protein. Structures revealed an N-terminal α-helix and an eight-strand β-sandwich, immunoglobulin-like fold with striking similarity to S-layer proteins. Crystal structures, X-ray scattering, and mutational analyses suggest dimer assembly is needed for in vivo function. The sole cell envelope component of S. acidocaldarius is amore » paracrystalline S-layer, and FlaF specifically bound to S-layer protein, suggesting that its interaction domain is located in the pseudoperiplasm with its N-terminal helix in the membrane. From these data, FlaF may act as the previously unknown archaellum stator protein that anchors the rotating archaellum to the archaeal cell envelope.« less

Mutational Signature Mark Cancer’s Smoking Gun

ScienceCinema

Alexandrov, Ludmil

2018-06-13

A broad computational study of cancer genome sequences by Los Alamos National Laboratory with the UK’s Wellcome Trust Sanger Institute and other collaborators identifies telltale mutational signatures associated with smoking tobacco. The research demonstrates, for the first time, that smoking increases cancer risk by causing somatic mutations in tissues directly and indirectly exposed to tobacco smoke. The international study was published in the November 4 issue of Science. The analysis shows that tobacco smoking causes mutations leading to cancer by multiple distinct mechanisms, including by damaging DNA in organs and by speeding up a mutational cellular clock.

RADIATION INDUCED VIABILITY MUTATIONS IN THE HONEY BEE. Progress Report for 1961 and Renewal Proposal of Contract for 1962

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, W.R.

The spectrum of viability mutations ranging from dominant lethals to detrimentals in haploids that resulted from irradiating semen from a single haploid male was studied in the honey bee. From the decrease in viability of diploid progeny following irradiation of the spermatheca of the parental queen, it was calculated that one or more dominant lethals were induced in 60.8% of the sperm cells. In a separate test using the same dosage on an unrelated queen 60.9% dominant lethals were found. Recessive mutations and mutants with incomplete dominance were detected in haploid progeny of F-1 queens. (M.C.G.)

Association of a nonsense mutation (W1282X), the most common mutation in the Ashkenazi Jewish cystic fibrosis patients in Israel, with presentation of severe disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shoshani, T.; Bashan, N.; Seret, H.

1992-01-01

Only about 30% of the cystic fibrosis chromosomes in the Israeli cystic fibrosis patient populations carry the major CF mutation ({Delta}F508). Since different Jewish ethnic groups tended to live as closed isolates until recent times, high frequencies of specific mutations are expected among the remainder cystic fibrosis chromosomes of these ethnic groups. Genetic factors appear to influence the severity of the disease. It is therefore expected that different mutations will be associated with either severe or mild phenotype. Direct genomic sequencing of exons included in the two nucleotide-binding folds of the putative CFTR protein was performed on 119 Israeli cysticmore » fibrosis patients from 97 families. One sequence alteration which is expected to create a termination at residue 1282 (W1282X) was found in 63 chromosomes. Of 95 chromosomes, 57(60%) are of Ashkenazi origin. In conclusion, the W1282X mutation is the most common cystic fibrosis mutation in the Ashkenazi Jewish patient population in Israel. This nonsense mutation is associated with presentation of severe disease.« less

Sequential folding of UmuC by the Hsp70 and Hsp60 chaperone complexes of Escherichia coli.

PubMed

Petit, M A; Bedale, W; Osipiuk, J; Lu, C; Rajagopalan, M; McInerney, P; Goodman, M F; Echols, H

1994-09-23

Replication-blocking lesions generate a signal in Escherichia coli that leads to the induction of the multigene SOS response. Among the SOS-induced genes are umuD and umuC, whose products are necessary for the increased mutation rate in induced bacteria. The mutations are likely to result from replication across the DNA lesion, and such a bypass event has been reconstituted in vitro (Rajagopalan, M., L, C., Woodgate, R., O'Donnel, M., Goodman, M. F., Echols, H. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 10777-10781). In this work, we show that the chaperone proteins promote the proper folding of UmuC protein in vitro. We treated purified and inactive UmuC with Hsp70 and Hsp60. After Hsp70 treatment, the DNA binding activity of UmuC was recovered, but the ability to promote replication across DNA lesions was not. However, lesion bypass activity was recovered upon further treatment with Hsp60. The biological significance of such a folding pathway for UmuC protein is strengthened by in vivo evidence for a role of DnaK in UV-induced mutagenesis.

BG60S dissolution interferes with osteoblast calcium signals.

PubMed

Valério, P; Pereira, M M; Goes, A M; Leite, M F

2007-02-01

We investigated the influence of extracellular calcium concentration, caused by the dissolution of a bioactive glass with 60% of silicon (BG60S), on intracellular calcium (Ca(i) (2 +)) signals and expression of inositol 1, 4, 5-triphosphate receptors (InsP(3)R) in primary culture of osteoblasts. We found that BG60S caused an increase in Ca(i) (2 +) signals in this cell type. Additionally, osteoblasts pre-incubated in the presence of BG60S showed an increase in Ca(i) (2 +) when cells were stimulated with vasopressin. On the other hand, a decrease in Ca(i) (2 +) signals were observed in osteoblasts pre-treated with BG60S and stimulated with KCl. We furher found that in osteoblasts, the type I InsP(3)R is preferentially distributed in the nucleus while the type II InsP(3)R in the cytoplasm. Preincubation of osteoblasts with BG60S altered the receptor expression level, increasing the type I InsP(3)R in the nucleus and decreasing type II InsP(3)R in the cytosol. Together, our results showed that in osteoblasts, BG60S increased Ca(i) (2 +)signals and altered Ca(i) (2 +) machinery.

Protein F, a fibronectin-binding protein, is an adhesin of the group A streptococcus Streptococcus pyogenes.

PubMed

Hanski, E; Caparon, M

1992-07-01

Binding to fibronectin has been suggested to play an important role in adherence of the group A streptococcus Streptococcus pyrogenes to host epithelial cells; however, the identity of the streptococcal fibronectin receptor has been elusive. Here we demonstrate that the fibronectin-binding property of S. pyogenes is mediated by protein F, a bacterial surface protein that binds fibronectin at high affinity. The gene encoding protein F (prtF) produced a functional fibronectin-binding protein in Escherichia coli. Insertional mutagenesis of the cloned gene generated a mutation that resulted in the loss of fibronectin-binding activity. When this mutation was introduced into the S. pyrogenes chromosome by homologous recombination with the wild-type allele, the resulting strains no longer produced protein F and lost their ability to bind fibronectin. The mutation could be complemented by prtF introduced on a plasmid. Mutants lacking protein F had a much lower capacity to adhere to respiratory epithelial cells. These results demonstrate that protein F is an important adhesin of S. pyogenes.

Mutations close to a hub residue affect the distant active site of a GH1 β-glucosidase.

PubMed

Souza, Valquiria P; Ikegami, Cecília M; Arantes, Guilherme M; Marana, Sandro R

2018-01-01

The tertiary structure of proteins has been represented as a network, in which residues are nodes and their contacts are edges. Protein structure networks contain residues, called hubs or central, which are essential to form short connection pathways between any pair of nodes. Hence hub residues may effectively spread structural perturbations through the protein. To test whether modifications nearby to hub residues could affect the enzyme active site, mutations were introduced in the β-glycosidase Sfβgly (PDB-ID: 5CG0) directed to residues that form an α-helix (260-265) and a β-strand (335-337) close to one of its main hub residues, F251, which is approximately 14 Å from the Sfβgly active site. Replacement of residues A263 and A264, which side-chains project from the α-helix towards F251, decreased the rate of substrate hydrolysis. Mutation A263F was shown to weaken noncovalent interactions involved in transition state stabilization within the Sfβgly active site. Mutations placed on the opposite side of the same α-helix did not show these effects. Consistently, replacement of V336, which side-chain protrudes from a β-strand face towards F251, inactivated Sfβgly. Next to V336, mutation S337F also caused a decrease in noncovalent interactions involved in transition state stabilization. Therefore, we suggest that mutations A263F, A264F, V336F and S337F may directly perturb the position of the hub F251, which could propagate these perturbations into the Sfβgly active site through short connection pathways along the protein network.

Assessment of the interlaboratory variability and robustness of JAK2V617F mutation assays: A study involving a consortium of 19 Italian laboratories

PubMed Central

Perricone, Margherita; Palandri, Francesca; Ottaviani, Emanuela; Angelini, Mario; Bagli, Laura; Bellesia, Enrica; Donati, Meris; Gemmati, Donato; Zucchini, Patrizia; Mancini, Stefania; Marchica, Valentina; Trubini, Serena; Matteis, Giovanna De; Zacomo, Silvia Di; Favarato, Mosè; Fioroni, Annamaria; Bolzonella, Caterina; Maccari, Giorgia; Navaglia, Filippo; Gatti, Daniela; Toffolatti, Luisa; Orlandi, Linda; Laloux, Vèronique; Manfrini, Marco; Galieni, Piero; Giannini, Barbara; Tieghi, Alessia; Barulli, Sara; Serino, Maria Luisa; Maccaferri, Monica; Scortechini, Anna Rita; Giuliani, Nicola; Vallisa, Daniele; Bonifacio, Massimiliano; Accorsi, Patrizia; Salbe, Cristina; Fazio, Vinicio; Gusella, Milena; Toffoletti, Eleonora; Salvucci, Marzia; Svaldi, Mirija; Gherlinzoni, Filippo; Cassavia, Francesca; Orsini, Francesco; Martinelli, Giovanni

2017-01-01

To date, a plenty of techniques for the detection of JAK2V617F is used over different laboratories, with substantial differences in specificity and sensitivity. Therefore, to provide reliable and comparable results, the standardization of molecular techniques is mandatory. A network of 19 centers was established to 1) evaluate the inter- and intra-laboratory variability in JAK2V617F quantification, 2) identify the most robust assay for the standardization of the molecular test and 3) allow consistent interpretation of individual patient analysis results. The study was conceived in 3 different rounds, in which all centers had to blindly test DNA samples with different JAK2V617F allele burden (AB) using both quantitative and qualitative assays. The positivity of samples with an AB < 1% was not detected by qualitative assays. Conversely, laboratories performing the quantitative approach were able to determine the expected JAK2V617F AB. Quantitative results were reliable across all mutation loads with moderate variability at low AB (0.1 and 1%; CV = 0.46 and 0.77, respectively). Remarkably, all laboratories clearly distinguished between the 0.1 and 1% mutated samples. In conclusion, a qualitative approach is not sensitive enough to detect the JAK2V617F mutation, especially at low AB. On the contrary, the ipsogen JAK2 MutaQuant CE-IVD kit resulted in a high, efficient and sensitive quantification detection of all mutation loads. This study sets the basis for the standardization of molecular techniques for JAK2V617F determination, which will require the employment of approved operating procedures and the use of certificated standards, such as the recent WHO 1st International Reference Panel for Genomic JAK2V617F. PMID:28427233

Assessment of the interlaboratory variability and robustness of JAK2V617F mutation assays: A study involving a consortium of 19 Italian laboratories.

PubMed

Perricone, Margherita; Palandri, Francesca; Ottaviani, Emanuela; Angelini, Mario; Bagli, Laura; Bellesia, Enrica; Donati, Meris; Gemmati, Donato; Zucchini, Patrizia; Mancini, Stefania; Marchica, Valentina; Trubini, Serena; De Matteis, Giovanna; Di Zacomo, Silvia; Favarato, Mosè; Fioroni, Annamaria; Bolzonella, Caterina; Maccari, Giorgia; Navaglia, Filippo; Gatti, Daniela; Toffolatti, Luisa; Orlandi, Linda; Laloux, Vèronique; Manfrini, Marco; Galieni, Piero; Giannini, Barbara; Tieghi, Alessia; Barulli, Sara; Serino, Maria Luisa; Maccaferri, Monica; Scortechini, Anna Rita; Giuliani, Nicola; Vallisa, Daniele; Bonifacio, Massimiliano; Accorsi, Patrizia; Salbe, Cristina; Fazio, Vinicio; Gusella, Milena; Toffoletti, Eleonora; Salvucci, Marzia; Svaldi, Mirija; Gherlinzoni, Filippo; Cassavia, Francesca; Orsini, Francesco; Martinelli, Giovanni

2017-05-16

To date, a plenty of techniques for the detection of JAK2V617F is used over different laboratories, with substantial differences in specificity and sensitivity. Therefore, to provide reliable and comparable results, the standardization of molecular techniques is mandatory.A network of 19 centers was established to 1) evaluate the inter- and intra-laboratory variability in JAK2V617F quantification, 2) identify the most robust assay for the standardization of the molecular test and 3) allow consistent interpretation of individual patient analysis results. The study was conceived in 3 different rounds, in which all centers had to blindly test DNA samples with different JAK2V617F allele burden (AB) using both quantitative and qualitative assays.The positivity of samples with an AB < 1% was not detected by qualitative assays. Conversely, laboratories performing the quantitative approach were able to determine the expected JAK2V617F AB. Quantitative results were reliable across all mutation loads with moderate variability at low AB (0.1 and 1%; CV = 0.46 and 0.77, respectively). Remarkably, all laboratories clearly distinguished between the 0.1 and 1% mutated samples.In conclusion, a qualitative approach is not sensitive enough to detect the JAK2V617F mutation, especially at low AB. On the contrary, the ipsogen JAK2 MutaQuant CE-IVD kit resulted in a high, efficient and sensitive quantification detection of all mutation loads. This study sets the basis for the standardization of molecular techniques for JAK2V617F determination, which will require the employment of approved operating procedures and the use of certificated standards, such as the recent WHO 1st International Reference Panel for Genomic JAK2V617F.

The Cerebro-oculo-facio-skeletal Syndrome Point Mutation F231L in the ERCC1 DNA Repair Protein Causes Dissociation of the ERCC1-XPF Complex*

PubMed Central

Faridounnia, Maryam; Wienk, Hans; Kovačič, Lidija; Folkers, Gert E.; Jaspers, Nicolaas G. J.; Kaptein, Robert; Hoeijmakers, Jan H. J.; Boelens, Rolf

2015-01-01

The ERCC1-XPF heterodimer, a structure-specific DNA endonuclease, is best known for its function in the nucleotide excision repair (NER) pathway. The ERCC1 point mutation F231L, located at the hydrophobic interaction interface of ERCC1 (excision repair cross-complementation group 1) and XPF (xeroderma pigmentosum complementation group F), leads to severe NER pathway deficiencies. Here, we analyze biophysical properties and report the NMR structure of the complex of the C-terminal tandem helix-hairpin-helix domains of ERCC1-XPF that contains this mutation. The structures of wild type and the F231L mutant are very similar. The F231L mutation results in only a small disturbance of the ERCC1-XPF interface, where, in contrast to Phe231, Leu231 lacks interactions stabilizing the ERCC1-XPF complex. One of the two anchor points is severely distorted, and this results in a more dynamic complex, causing reduced stability and an increased dissociation rate of the mutant complex as compared with wild type. These data provide a biophysical explanation for the severe NER deficiencies caused by this mutation. PMID:26085086

The Cerebro-oculo-facio-skeletal Syndrome Point Mutation F231L in the ERCC1 DNA Repair Protein Causes Dissociation of the ERCC1-XPF Complex.

PubMed

Faridounnia, Maryam; Wienk, Hans; Kovačič, Lidija; Folkers, Gert E; Jaspers, Nicolaas G J; Kaptein, Robert; Hoeijmakers, Jan H J; Boelens, Rolf

2015-08-14

The ERCC1-XPF heterodimer, a structure-specific DNA endonuclease, is best known for its function in the nucleotide excision repair (NER) pathway. The ERCC1 point mutation F231L, located at the hydrophobic interaction interface of ERCC1 (excision repair cross-complementation group 1) and XPF (xeroderma pigmentosum complementation group F), leads to severe NER pathway deficiencies. Here, we analyze biophysical properties and report the NMR structure of the complex of the C-terminal tandem helix-hairpin-helix domains of ERCC1-XPF that contains this mutation. The structures of wild type and the F231L mutant are very similar. The F231L mutation results in only a small disturbance of the ERCC1-XPF interface, where, in contrast to Phe(231), Leu(231) lacks interactions stabilizing the ERCC1-XPF complex. One of the two anchor points is severely distorted, and this results in a more dynamic complex, causing reduced stability and an increased dissociation rate of the mutant complex as compared with wild type. These data provide a biophysical explanation for the severe NER deficiencies caused by this mutation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Bacterial rep- mutations that block development of small DNA bacteriophages late in infection.

PubMed Central

Tessman, E S; Peterson, P K

1976-01-01

Several related mutants of Escherichia coli C have been isolated that block the growth of the small icosahedral DNA phages phiX174 and S13 late in infection. Phage G6 is also blocked, at a stage not yet known. Growth of the filamentous phage M13, though not blocked, is affected in these strains. These host mutations co-transduce with ilv at high frequency, as do rep- mutations. However, the new mutants, designated groL-, differ from previously studied rep- mutants in that they permit synthesis of progeny replicative-form DNA. The groL- mutants are blocked in synthesis of stable single-stranded DNA of phiX174 and related phages. They are gro+ for P2. Evidence that groL- mutations and rep- mutations are in the same gene is presented. Spontaneous mutants (ogr) of phiX174, S13, and the G phages can grow on groL- strains. The ogr mutations are located in the phage's major capsid gene, F, as determined by complementation tests. There are numerous sites for mutation to ogr. Some mutations in genes A and F interfere with the ogr property when combined with an ogr mutation on the same genome. The ogr mutations are cis acting in a groL- cell; i.e., an ogr mutant gives very poor rescue of a non-ogr mutant. The wild-type form of each G phage appears to be naturally in the ogr mutant state for one or more groL- strains. It is suggested that a complex between F and rep proteins is involved in phage maturation. The A protein appears to interact with this complex. PMID:789914

Driver mutations (JAK2V617F, MPLW515L/K or CALR), pentraxin-3 and C-reactive protein in essential thrombocythemia and polycythemia vera.

PubMed

Lussana, Federico; Carobbio, Alessandra; Salmoiraghi, Silvia; Guglielmelli, Paola; Vannucchi, Alessandro Maria; Bottazzi, Barbara; Leone, Roberto; Mantovani, Alberto; Barbui, Tiziano; Rambaldi, Alessandro

2017-02-22

The driver mutations JAK2V617F, MPLW515L/K and CALR influence disease phenotype of myeloproliferative neoplasms (MPNs) and might sustain a condition of chronic inflammation. Pentraxin 3 (PTX3) and high-sensitivity C-reactive protein (hs-CRP) are inflammatory biomarkers potentially useful for refining prognostic classification of MPNs. We evaluated 305 with essential thrombocythemia (ET) and 172 polycythemia vera (PV) patients diagnosed according to the 2016 WHO criteria and with full molecular characterization for driver mutations. PTX3 levels were significantly increased in carriers of homozygous JAK2V617F mutation compared to all the other genotypes and triple negative ET patients, while hs-CRP levels were independent of the mutational profile. The risk of haematological evolution and death from any cause was about 2- and 1.5-fold increased in individuals with high PTX-3 levels, while the thrombosis rate tended to be lower. High hs-CRP levels were associated with risk of haematological evolution, death and also major thrombosis. After sequential adjustment for potential confounders (age, gender, diagnosis and treatments) and the presence of JAK2V617F homozygous status, high hs-CRP levels remained significant for all outcomes, while JAK2V617F homozygous status as well as treatments were the factors independently accounting for adverse outcomes among patients with high PTX3 levels. These results provide evidence that JAK2V617F mutation influences MPN-associated inflammation with a strong correlation between allele burden and PTX3 levels. Plasma levels of hs-CRP and PTX3 might be of prognostic value for patients with ET and PV, but their validation in future prospective studies is needed.

Activation of K-ras by codon 13 mutations in C57BL/6 X C3H F1 mouse tumors induced by exposure to 1,3-butadiene.

PubMed

Goodrow, T; Reynolds, S; Maronpot, R; Anderson, M

1990-08-01

1,3-Butadiene has been detected in urban air, gasoline vapors, and cigarette smoke. It has been estimated that 65,000 workers are exposed to this chemical in occupational settings in the United States. Lymphomas, lung, and liver tumors were induced in female and male C57BL/6 X C3H F1 (hereafter called B6C3F1) mice by inhalation of 6.25 to 625 ppm 1,3-butadiene for 1 to 2 years. The objective of this study was to examine these tumors for the presence of activated protooncogenes by the NIH 3T3 transfection and nude mouse tumorigenicity assays. Transfection of DNA isolated from 7 of 9 lung tumors and 7 of 12 liver tumors induced morphological transformation of NIH 3T3 cells. Southern blot analysis indicated that the transformation induced by 6 lung and 3 liver tumor DNA samples was due to transfer of a K-ras oncogene. Four of the 7 liver tumors that were positive upon transfection contained an activated H-ras gene. The identity of the transforming gene in one of the lung tumors has not been determined but was not a member of the ras family or a met or raf gene. Eleven 1,3-butadiene-induced lymphomas were examined for transforming genes using the nude mouse tumorigenicity assay. Activated K-ras genes were detected in 2 of the 11 lymphomas assayed. DNA sequencing of polymerase chain reaction-amplified ras gene exons revealed that 9 of 11 of the activating K-ras mutations were G to C transversions in codon 13. One liver tumor contained an activated K-ras gene with mutations in both codons 60 and 61. The activating mutation in one of the K-ras genes from a lymphoma was not identified but DNA sequence analysis of amplified regions in proximity to codons 12, 13, and 61 demonstrated that the mutation was not located in or near these codons. Activation of K-ras genes by codon 13 mutations has not been found in any lung or liver tumors or lymphomas from untreated B6C3F1 mice. Thus, the K-ras activation found in 1,3-butadiene-induced B6C3F1 mouse tumors probably occurred as a

Wear Mechanism Maps for Magnesium Alloy AM60 and Composite AM60-9% (Al2O3)f

NASA Astrophysics Data System (ADS)

Ullah Khan, Muhammad Zafar

The purpose of this work was to study the tribological behaviour of squeeze cast Mg alloy AM60 and its composite AM60-9% (Al2O3) f. Dry sliding wear tests were performed on specimens of these materials using a block-on-ring tribometer which was equipped with a COF and temperature measurement system. Wear, COF and temperature maps were constructed to illustrate the effect of temperature and COF on the wear behaviour of the Mg alloy and it's composite. Four wear regimes namely low, mild, transient and severe wear were identified. The transition from mild to severe wear regime was found to be dependent on the bulk temperature of the specimen. Oxidational wear prevailed in low and mild wear whereas plastic deformation induced wear and melt wear controlled the wear rates in transient and severe wear regimes, respectively. This study shows that the incorporation of Al2O3 fibres in AM60 alloy improved the wear resistance of the resulting composite by delaying the transition from mild to severe wear.

Insights into the Functional Architecture of the Catalytic Center of a Maize β-Glucosidase Zm-p60.11

PubMed Central

Zouhar, Jan; Vévodová, Jitka; Marek, Jaromír; Damborský, Jir̆í; Su, Xiao-Dong; Brzobohatý, Br̆etislav

2001-01-01

The maize (Zea mays) β-glucosidase Zm-p60.1 has been implicated in regulation of plant development by the targeted release of free cytokinins from cytokinin-O-glucosides, their inactive storage forms. The crystal structure of the wild-type enzyme was solved at 2.05-Å resolution, allowing molecular docking analysis to be conducted. This indicated that the enzyme specificity toward substrates with aryl aglycones is determined by aglycone aromatic system stacking with W373, and interactions with edges of F193, F200, and F461 located opposite W373 in a slot-like aglycone-binding site. These aglycone-active site interactions recently were hypothesized to determine substrate specificity in inactive enzyme substrate complexes of ZM-Glu1, an allozyme of Zm-p60.1. Here, we test this hypothesis by kinetic analysis of F193I/Y/W mutants. The decreased Km of all mutants confirmed the involvement of F193 in determining enzyme affinity toward substrates with an aromatic aglycone. It was unexpected that a 30-fold decrease in kcat was found in F193I mutant compared with the wild type. Kinetic analysis and computer modeling demonstrated that the F193-aglycone-W373 interaction not only contributes to aglycone recognition as hypothesized previously but also codetermines catalytic rate by fixing the glucosidic bond in an orientation favorable for attack by the catalytic pair, E186 and E401. The catalytic pair, assigned initially by their location in the structure, was confirmed by kinetic analysis of E186D/Q and E401D/Q mutants. It was unexpected that the E401D as well as C205S and C211S mutations dramatically impaired the assembly of a catalysis-competent homodimer, suggesting novel links between the active site structure and dimer formation. PMID:11706179

Identification of Genome-Wide Mutations in Ciprofloxacin-Resistant F. tularensis LVS Using Whole Genome Tiling Arrays and Next Generation Sequencing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jaing, Crystal J.; McLoughlin, Kevin S.; Thissen, James B.

Francisella tularensis is classified as a Class A bioterrorism agent by the U.S. government due to its high virulence and the ease with which it can be spread as an aerosol. It is a facultative intracellular pathogen and the causative agent of tularemia. Ciprofloxacin (Cipro) is a broad spectrum antibiotic effective against Gram-positive and Gram-negative bacteria. Increased Cipro resistance in pathogenic microbes is of serious concern when considering options for medical treatment of bacterial infections. Identification of genes and loci that are associated with Ciprofloxacin resistance will help advance the understanding of resistance mechanisms and may, in the future, providemore » better treatment options for patients. It may also provide information for development of assays that can rapidly identify Cipro-resistant isolates of this pathogen. In this study, we then selected a large number of F. tularensis live vaccine strain (LVS) isolates that survived in progressively higher Ciprofloxacin concentrations, screened the isolates using a whole genome F. tularensis LVS tiling microarray and Illumina sequencing, and identified both known and novel mutations associated with resistance. For genes containing mutations encode DNA gyrase subunit A, a hypothetical protein, an asparagine synthase, a sugar transamine/perosamine synthetase and others. Finally, structural modeling performed on these proteins provides insights into the potential function of these proteins and how they might contribute to Cipro resistance mechanisms.« less

Identification of Genome-Wide Mutations in Ciprofloxacin-Resistant F. tularensis LVS Using Whole Genome Tiling Arrays and Next Generation Sequencing

DOE PAGES

Jaing, Crystal J.; McLoughlin, Kevin S.; Thissen, James B.; ...

2016-09-26

Francisella tularensis is classified as a Class A bioterrorism agent by the U.S. government due to its high virulence and the ease with which it can be spread as an aerosol. It is a facultative intracellular pathogen and the causative agent of tularemia. Ciprofloxacin (Cipro) is a broad spectrum antibiotic effective against Gram-positive and Gram-negative bacteria. Increased Cipro resistance in pathogenic microbes is of serious concern when considering options for medical treatment of bacterial infections. Identification of genes and loci that are associated with Ciprofloxacin resistance will help advance the understanding of resistance mechanisms and may, in the future, providemore » better treatment options for patients. It may also provide information for development of assays that can rapidly identify Cipro-resistant isolates of this pathogen. In this study, we then selected a large number of F. tularensis live vaccine strain (LVS) isolates that survived in progressively higher Ciprofloxacin concentrations, screened the isolates using a whole genome F. tularensis LVS tiling microarray and Illumina sequencing, and identified both known and novel mutations associated with resistance. For genes containing mutations encode DNA gyrase subunit A, a hypothetical protein, an asparagine synthase, a sugar transamine/perosamine synthetase and others. Finally, structural modeling performed on these proteins provides insights into the potential function of these proteins and how they might contribute to Cipro resistance mechanisms.« less

Distinct Brca1 Mutations Differentially Reduce Hematopoietic Stem Cell Function.

PubMed

Mgbemena, Victoria E; Signer, Robert A J; Wijayatunge, Ranjula; Laxson, Travis; Morrison, Sean J; Ross, Theodora S

2017-01-24

BRCA1 is a well-known DNA repair pathway component and a tissue-specific tumor suppressor. However, its role in hematopoiesis is uncertain. Here, we report that a cohort of patients heterozygous for BRCA1 mutations experienced more hematopoietic toxicity from chemotherapy than those with BRCA2 mutations. To test whether this reflects a requirement for BRCA1 in hematopoiesis, we generated mice with Brca1 mutations in hematopoietic cells. Mice homozygous for a null Brca1 mutation in the embryonic hematopoietic system (Vav1-iCre;Brca1 F22-24/F22-24 ) developed hematopoietic defects in early adulthood that included reduced hematopoietic stem cells (HSCs). Although mice homozygous for a huBRCA1 knockin allele (Brca1 BRCA1/BRCA1 ) were normal, mice with a mutant huBRCA1/5382insC allele and a null allele (Mx1-Cre;Brca1 F22-24/5382insC ) had severe hematopoietic defects marked by a complete loss of hematopoietic stem and progenitor cells. Our data show that Brca1 is necessary for HSC maintenance and normal hematopoiesis and that distinct mutations lead to different degrees of hematopoietic dysfunction. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Mutational analysis of the major soybean UreF paralogue involved in urease activation

PubMed Central

Polacco, Joe C.; Hyten, David L.; Medeiros-Silva, Mônica; Sleper, David A.; Bilyeu, Kristin D.

2011-01-01

The soybean genome duplicated ∼14 and 45 million years ago and has many paralogous genes, including those in urease activation (emplacement of Ni and CO2 in the active site). Activation requires the UreD and UreF proteins, each encoded by two paralogues. UreG, a third essential activation protein, is encoded by the single-copy Eu3, and eu3 mutants lack activity of both urease isozymes. eu2 has the same urease-negative phenotype, consistent with Eu2 being a single-copy gene, possibly encoding a Ni carrier. Unexpectedly, two eu2 alleles co-segregated with missense mutations in the chromosome 2 UreF paralogue (Ch02UreF), suggesting lack of expression/function of Ch14UreF. However, Ch02UreF and Ch14UreF transcripts accumulate at the same level. Further, it had been shown that expression of the Ch14UreF ORF complemented a fungal ureF mutant. A third, nonsense (Q2*) allelic mutant, eu2-c, exhibited 5- to 10-fold more residual urease activity than missense eu2-a or eu2-b, though eu2-c should lack all Ch02UreF protein. It is hypothesized that low-level activation by Ch14UreF is ‘spoiled’ by the altered missense Ch02UreF proteins (‘epistatic dominant-negative’). In agreement with active ‘spoiling’ by eu2-b-encoded Ch02UreF (G31D), eu2-b/eu2-c heterozygotes had less than half the urease activity of eu2-c/eu2-c siblings. Ch02UreF (G31D) could spoil activation by Chr14UreF because of higher affinity for the activation complex, or because Ch02UreF (G31D) is more abundant than Ch14UreF. Here, the latter is favoured, consistent with a reported in-frame AUG in the 5' leader of Chr14UreF transcript. Translational inhibition could represent a form of ‘functional divergence’ of duplicated genes. PMID:21430294

Precipitating factors of porphyria cutanea tarda in Brazil with emphasis on hemochromatosis gene (HFE) mutations. Study of 60 patients*

PubMed Central

Vieira, Fatima Mendonça Jorge; Nakhle, Maria Cristina; Abrantes-Lemos, Clarice Pires; Cançado, Eduardo Luiz Rachid; dos Reis, Vitor Manoel Silva

2013-01-01

BACKGROUND Porphyria cutanea tarda is the most common form of porphyria, characterized by the decreased activity of the uroporphyrinogen decarboxylase enzyme. Several reports associated HFE gene mutations of hereditary hemochromatosis with porphyria cutanea tarda worldwide, although up to date only one study has been conducted in Brazil. OBJECTIVES Investigation of porphyria cutanea tarda association with C282Y and H63D mutations in the HFE gene. Identification of precipitating factors (hepatitis C, HIV, alcoholism and estrogen) and their link with HFE mutations. METHODS An ambispective study of 60 patients with PCT was conducted during the period from 2003 to 2012. Serological tests for hepatitis C and HIV were performed and histories of alcohol abuse and estrogen intake were investigated. HFE mutations were identified with real-time PCR. RESULTS Porphyria cutanea tarda predominated in males and alcohol abuse was the main precipitating factor. Estrogen intake was the sole precipitating factor present in 25% of female patients. Hepatitis C was present in 41.7%. All HIV-positive patients (15.3%) had a history of alcohol abuse. Allele frequency for HFE mutations, i.e., C282Y (p = 0.0001) and H63D (p = 0.0004), were significantly higher in porphyria cutanea tarda patients, compared to control group. HFE mutations had no association with the other precipitating factors. CONCLUSIONS Alcohol abuse, hepatitis C and estrogen intake are prevalent precipitating factors in our porphyria cutanea tarda population; however, hemochromatosis in itself can also contribute to the outbreak of porphyria cutanea tarda, which makes the research for HFE mutations necessary in these patients PMID:24068123

Precipitating factors of porphyria cutanea tarda in Brazil with emphasis on hemochromatosis gene (HFE) mutations. Study of 60 patients.

PubMed

Vieira, Fatima Mendonça Jorge; Nakhle, Maria Cristina; Abrantes-Lemos, Clarice Pires; Cançado, Eduardo Luiz Rachid; Reis, Vitor Manoel Silva dos

2013-01-01

Porphyria cutanea tarda is the most common form of porphyria, characterized by the decreased activity of the uroporphyrinogen decarboxylase enzyme. Several reports associated HFE gene mutations of hereditary hemochromatosis with porphyria cutanea tarda worldwide, although up to date only one study has been conducted in Brazil. Investigation of porphyria cutanea tarda association with C282Y and H63D mutations in the HFE gene. Identification of precipitating factors (hepatitis C, HIV, alcoholism and estrogen) and their link with HFE mutations. An ambispective study of 60 patients with PCT was conducted during the period from 2003 to 2012. Serological tests for hepatitis C and HIV were performed and histories of alcohol abuse and estrogen intake were investigated. HFE mutations were identified with real-time PCR. Porphyria cutanea tarda predominated in males and alcohol abuse was the main precipitating factor. Estrogen intake was the sole precipitating factor present in 25% of female patients. Hepatitis C was present in 41.7%. All HIV-positive patients (15.3%) had a history of alcohol abuse. Allele frequency for HFE mutations, i.e., C282Y (p = 0.0001) and H63D (p = 0.0004), were significantly higher in porphyria cutanea tarda patients, compared to control group. HFE mutations had no association with the other precipitating factors. Alcohol abuse, hepatitis C and estrogen intake are prevalent precipitating factors in our porphyria cutanea tarda population; however, hemochromatosis in itself can also contribute to the outbreak of porphyria cutanea tarda, which makes the research for HFE mutations necessary in these patients.

Electron-impact-ionization dynamics of S F6

NASA Astrophysics Data System (ADS)

Bull, James N.; Lee, Jason W. L.; Vallance, Claire

2017-10-01

A detailed understanding of the dissociative electron ionization dynamics of S F6 is important in the modeling and tuning of dry-etching plasmas used in the semiconductor manufacture industry. This paper reports a crossed-beam electron ionization velocity-map imaging study on the dissociative ionization of cold S F6 molecules, providing complete, unbiased kinetic energy distributions for all significant product ions. Analysis of these distributions suggests that fragmentation following single ionization proceeds via formation of S F5 + or S F3 + ions that then dissociate in a statistical manner through loss of F atoms or F2, until most internal energy has been liberated. Similarly, formation of stable dications is consistent with initial formation of S F4 2 + ions, which then dissociate on a longer time scale. These data allow a comparison between electron ionization and photoionization dynamics, revealing similar dynamical behavior. In parallel with the ion kinetic energy distributions, the velocity-map imaging approach provides a set of partial ionization cross sections for all detected ionic fragments over an electron energy range of 50-100 eV, providing partial cross sections for S2 +, and enables the cross sections for S F4 2 + from S F+ to be resolved.

Occult HBV among Anti-HBc Alone: Mutation Analysis of an HBV Surface Gene and Pre-S Gene.

PubMed

Kim, Myeong Hee; Kang, So Young; Lee, Woo In

2017-05-01

The aim of this study is to investigate the molecular characteristics of occult hepatitis B virus (HBV) infection in 'anti-HBc alone' subjects. Twenty-four patients with 'anti-HBc alone' and 20 control patients diagnosed with HBV were analyzed regarding S and pre-S gene mutations. All specimens were analyzed for HBs Ag, anti-HBc, and anti-HBs. For specimens with an anti-HBc alone, quantitative analysis of HBV DNA, as well as sequencing and mutation analysis of S and pre-S genes, were performed. A total 24 were analyzed for the S gene, and 14 were analyzed for the pre-S gene through sequencing. A total of 20 control patients were analyzed for S and pre-S gene simultaneously. Nineteen point mutations of the major hydrophilic region were found in six of 24 patients. Among them, three mutations, S114T, P127S/T, M133T, were detected in common. Only one mutation was found in five subjects of the control group; this mutation was not found in the occult HBV infection group, however. Pre-S mutations were detected in 10 patients, and mutations of site aa58-aa100 were detected in 9 patients. A mutation on D114E was simultaneously detected. Although five mutations from the control group were found at the same location (aa58-aa100), no mutations of occult HBV infection were detected. The prevalence of occult HBV infection is not low among 'anti-HBc alone' subjects. Variable mutations in the S gene and pre-S gene were associated with the occurrence of occult HBV infection. Further larger scale studies are required to determine the significance of newly detected mutations. © Copyright: Yonsei University College of Medicine 2017

Crizotinib-Resistant ROS1 Mutations Reveal a Predictive Kinase Inhibitor Sensitivity Model for ROS1- and ALK-Rearranged Lung Cancers.

PubMed

Facchinetti, Francesco; Loriot, Yohann; Kuo, Mei-Shiue; Mahjoubi, Linda; Lacroix, Ludovic; Planchard, David; Besse, Benjamin; Farace, Françoise; Auger, Nathalie; Remon, Jordi; Scoazec, Jean-Yves; André, Fabrice; Soria, Jean-Charles; Friboulet, Luc

2016-12-15

The identification of molecular mechanisms conferring resistance to tyrosine kinase inhibitor (TKI) is a key step to improve therapeutic results for patients with oncogene addiction. Several alterations leading to EGFR and anaplastic lymphoma kinase (ALK) resistance to TKI therapy have been described in non-small cell lung cancer (NSCLC). Only two mutations in the ROS1 kinase domain responsible for crizotinib resistance have been described in patients thus far. A patient suffering from a metastatic NSCLC harboring an ezrin (EZR)-ROS1 fusion gene developed acquired resistance to the ALK/ROS1 inhibitor crizotinib. Molecular analysis (whole-exome sequencing, CGH) and functional studies were undertaken to elucidate the mechanism of resistance. Based on this case, we took advantage of the structural homology of ROS1 and ALK to build a predictive model for drug sensitivity regarding future ROS1 mutations. Sequencing revealed a dual mutation, S1986Y and S1986F, in the ROS1 kinase domain. Functional in vitro studies demonstrated that ROS1 harboring either the S1986Y or the S1986F mutation, while conferring resistance to crizotinib and ceritinib, was inhibited by lorlatinib (PF-06463922). The patient's clinical response confirmed the potency of lorlatinib against S1986Y/F mutations. The ROS1 S1986Y/F and ALK C1156Y mutations are homologous and displayed similar sensitivity patterns to ALK/ROS1 TKIs. We extended this analogy to build a model predicting TKI efficacy against potential ROS1 mutations. Clinical evidence, in vitro validation, and homology-based prediction provide guidance for treatment decision making for patients with ROS1-rearranged NSCLC who progressed on crizotinib. Clin Cancer Res; 22(24); 5983-91. ©2016 AACR. ©2016 American Association for Cancer Research.

F/FB-111 Avionic Systems Career Ladder, AFSC 452X3

DTIC Science & Technology

1990-07-01

Computer products upon which this report is based are available for use by operations and training officials. The survey instrument was developed by Chief...AND BLOWER 60 64 51 GAS TURBINE GENERATOR/COMPRESSOR 57 64 60 AIRCRAFT RADIO 56 59 62 NITROGEN SERVICING EQUIPMENT 30 34 25 TABLE 13B EQUIPMENT USED OR...F-nf W&#60 &#60 W O / O CD S-c Cr ZC F ODf -C CD ;Z D r- =D CO M1 wD >) C U CCD4F ~-jF-ca- Cr. F- C).FW C) - En z_; > X F-F - : > - &#60 xJC&#60C -)I-CrF- W.JDLU

Effect of the F610A mutation on substrate extrusion in the AcrB transporter: explanation and rationale by molecular dynamics simulations.

PubMed

Vargiu, Attilio V; Collu, Francesca; Schulz, Robert; Pos, Klaas M; Zacharias, Martin; Kleinekathöfer, Ulrich; Ruggerone, Paolo

2011-07-20

The tripartite efflux pump AcrAB-TolC is responsible for the intrinsic and acquired multidrug resistance in Escherichia coli. Its active part, the homotrimeric transporter AcrB, is in charge of the selective binding of substrates and energy transduction. The mutation F610A has been shown to significantly reduce the minimum inhibitory concentration of doxorubicin and many other substrates, although F610 does not appear to interact strongly with them. Biochemical study of transport kinetics in AcrB is not yet possible, except for some β-lactams, and other techniques should supply this important information. Therefore, in this work, we assess the impact of the F610A mutation on the functionality of AcrB by means of computational techniques, using doxorubicin as substrate. We found that the compound slides deeply inside the binding pocket after mutation, increasing the strength of the interaction. During subsequent conformational alterations of the transporter, doxorubicin was either not extruded from the binding site or displaced along a direction other than the one associated with extrusion. Our study indicates how subtle interactions determine the functionality of multidrug transporters, since decreased transport might not be simplistically correlated to decreased substrate binding affinity.

Comparison of ZetaPlus 60S and nitrocellulose membrane filters for the simultaneous concentration of F-RNA coliphages, porcine teschovirus and porcine adenovirus from river water.

PubMed

Jones, T H; Muehlhauser, V; Thériault, G

2014-09-01

Increasing attention is being paid to the impact of agricultural activities on water quality to understand the impact on public health. F-RNA coliphages have been proposed as viral indicators of fecal contamination while porcine teschovirus (PTV) and porcine adenovirus (PAdV) are proposed indicators of fecal contamination of swine origin. Viruses and coliphages are present in water in very low concentrations and must be concentrated to permit their detection. There is little information comparing the effectiveness of the methods for concentrating F-RNA coliphages with concentration methods for other viruses and vice versa. The objective of this study was to compare 5 current published methods for recovering F-RNA coliphages, PTV and PAdV from river water samples concentrated by electronegative nitrocellulose membrane filters (methods A and B) or electropositive Zeta Plus 60S filters (methods C-E). Method A is used routinely for the detection of coliphages (Méndez et al., 2004) and method C (Brassard et al., 2005) is the official method in Health Canada's compendium for the detection of viruses in bottled mineral or spring water. When river water was inoculated with stocks of F-RNA MS2, PAdV, and PTV to final concentrations of 1×10(6) PFU/100 mL, 1×10(5) gc/100 mL and 3×10(5) gc/100 mL, respectively, a significantly higher recovery for each virus was consistently obtained for method A with recoveries of 52% for MS2, 95% for PAdV, and 1.5% for PTV. When method A was compared with method C for the detection of F-coliphages, PAdV and PTV in river water samples, viruses were detected with higher frequencies and at higher mean numbers with method A than with method C. With method A, F-coliphages were detected in 11/12 samples (5-154 PFU/100 mL), PTV in 12/12 samples (397-10,951 gc/100 mL), PAdV in 1/12 samples (15 gc/100 mL), and F-RNA GIII in 1/12 samples (750 gc/100 mL) while F-RNA genotypes I, II, and IV were not detected by qRT-PCR. Crown Copyright © 2014

Mutational analysis of the active site flap (20s loop) of mandelate racemase.

PubMed

Bourque, Jennifer R; Bearne, Stephen L

2008-01-15

Mandelate racemase from Pseudomonas putida catalyzes the Mg2+-dependent 1,1-proton transfer that interconverts the enantiomers of mandelate. Residues of the 20s and 50s loops determine, in part, the topology and polarity of the active site and hence the substrate specificity. Previously, we proposed that, during racemization, the phenyl ring of mandelate moves between an S-pocket comprised of residues from the 50s loop and an R-pocket comprised of residues from the 20s loop [Siddiqi, F., Bourque, J. R., Jiang, H., Gardner, M., St. Maurice, M., Blouin, C., and Bearne, S. L. (2005) Biochemistry 44, 9013-9021]. The 20s loop constitutes a mobile beta-meander flap that covers the active site cavity shielding it from solvent and controlling entry and egress of ligands. To understand the role of the 20s loop in catalysis and substrate specificity, we constructed a series of mutants (V22A, V22I, V22F, T24S, A25V, V26A, V26L, V26F, V29A, V29L, V29F, V26A/V29L, and V22I/V29L) in which the sizes of hydrophobic side chains of the loop residues were varied. Catalytic efficiencies (kcat/Km) for all mutants were reduced between 6- and 40-fold with the exception of those of V22I, V26A, V29L, and V22I/V29L which had near wild-type efficiencies with mandelate. Thr 24 and Ala 25, located at the tip of the 20s loop, were particularly sensitive to minor alterations in the size of their hydrophobic side chains; however, most mutations were tolerated quite well, suggesting that flap mobility could compensate for increases in the steric bulk of hydrophobic side chains. With the exception of V29L, with mandelate as the substrate, and V22F and V26A/V29L, with 2-naphthylglycolate (2-NG) as the substrate, the values of kcat and Km were not altered in a manner consistent with steric obstruction of the R-pocket, perhaps due to flap mobility compensating for the increased size of the hydrophobic side chains. Surprisingly, V22I and V29L catalyzed the racemization of the bulkier substrate 2-NG

Co-occurrence of Point Mutations in the Voltage-Gated Sodium Channel of Pyrethroid-Resistant Aedes aegypti Populations in Myanmar

PubMed Central

Kawada, Hitoshi; Oo, Sai Zaw Min; Thaung, Sein; Kawashima, Emiko; Maung, Yan Naung Maung; Thu, Hlaing Myat; Thant, Kyaw Zin; Minakawa, Noboru

2014-01-01

Background Single amino acid substitutions in the voltage-gated sodium channel associated with pyrethroid resistance constitute one of the main causative factors of knockdown resistance in insects. The kdr gene has been observed in several mosquito species; however, point mutations in the para gene of Aedes aegypti populations in Myanmar have not been fully characterized. The aim of the present study was to determine the types and frequencies of mutations in the para gene of Aedes aegypti collected from used tires in Yangon City, Myanmar. Methodology/Principal Findings We determined high pyrethroid resistance in Aedes aegypti larvae at all collection sites in Yangon City, by using a simplified knockdown bioassay. We showed that V1016G and S989P mutations were widely distributed, with high frequencies (84.4% and 78.8%, respectively). By contrast, we were unable to detect I1011M (or I1011V) or L1014F mutations. F1534C mutations were also widely distributed, but with a lower frequency than the V1016G mutation (21.2%). High percentage of co-occurrence of the homozygous V1016G/S989P mutations was detected (65.7%). Additionally, co-occurrence of homozygous V1016G/F1534C mutations (2.9%) and homozygous V1016G/F1534C/S989P mutations (0.98%) were detected in the present study. Conclusions/Significance Pyrethroid insecticides were first used for malaria control in 1992, and have since been constantly used in Myanmar. This intensive use may explain the strong selection pressure toward Aedes aegypti, because this mosquito is generally a domestic and endophagic species with a preference for indoor breeding. Extensive use of DDT for malaria control before the use of this chemical was banned may also explain the development of pyrethroid resistance in Aedes aegypti. PMID:25077956

Co-occurrence of point mutations in the voltage-gated sodium channel of pyrethroid-resistant Aedes aegypti populations in Myanmar.

PubMed

Kawada, Hitoshi; Oo, Sai Zaw Min; Thaung, Sein; Kawashima, Emiko; Maung, Yan Naung Maung; Thu, Hlaing Myat; Thant, Kyaw Zin; Minakawa, Noboru

2014-01-01

Single amino acid substitutions in the voltage-gated sodium channel associated with pyrethroid resistance constitute one of the main causative factors of knockdown resistance in insects. The kdr gene has been observed in several mosquito species; however, point mutations in the para gene of Aedes aegypti populations in Myanmar have not been fully characterized. The aim of the present study was to determine the types and frequencies of mutations in the para gene of Aedes aegypti collected from used tires in Yangon City, Myanmar. We determined high pyrethroid resistance in Aedes aegypti larvae at all collection sites in Yangon City, by using a simplified knockdown bioassay. We showed that V1016G and S989P mutations were widely distributed, with high frequencies (84.4% and 78.8%, respectively). By contrast, we were unable to detect I1011M (or I1011V) or L1014F mutations. F1534C mutations were also widely distributed, but with a lower frequency than the V1016G mutation (21.2%). High percentage of co-occurrence of the homozygous V1016G/S989P mutations was detected (65.7%). Additionally, co-occurrence of homozygous V1016G/F1534C mutations (2.9%) and homozygous V1016G/F1534C/S989P mutations (0.98%) were detected in the present study. Pyrethroid insecticides were first used for malaria control in 1992, and have since been constantly used in Myanmar. This intensive use may explain the strong selection pressure toward Aedes aegypti, because this mosquito is generally a domestic and endophagic species with a preference for indoor breeding. Extensive use of DDT for malaria control before the use of this chemical was banned may also explain the development of pyrethroid resistance in Aedes aegypti.

Dent’s disease: Identification of seven new pathogenic mutations in the CLCN5 gene

PubMed Central

Ramos-Trujillo, Elena; Claverie-Martin, Felix; Garcia-Nieto, Victor; Ariceta, Gema; Vara, Julia; Gonzalez-Acosta, Hilaria; Garcia-Ramirez, Marta; Fons, Jaime; Cordoba-Lanus, Elizabeth; Gonzalez-Paredes, Javier; Valenciano, Blanca; Ramos, Leticia; Muley, Rafael; Caggiani, Marina; Alvarez-Estrada, Pilar; Madrid, Alvaro

2013-01-01

Dent’s disease is an X-linked proximal tubulopathy characterized by low-molecular-weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis and progressive renal failure. This disorder is frequently caused by mutations in the CLCN5 gene encoding the electrogenic chloride/proton exchanger ClC-5. Occasionally, Dent’s disease has been associated to atypical cases of asymptomatic proteinuria with focal glomerulosclerosis. Twelve unrelated patients with Dent’s disease, including two who presented with asymptomatic proteinuria and developed glomerulosclerosis, were studied. Mutational analysis of the CLCN5 gene was performed by DNA sequencing. We identified thirteen distinct CLCN5 mutations in the twelve patients. Seven of these mutations, p.P416fsX*17, p.[H107P, V108fs*27], p.G466D, p.G65R, p.G462S, p.Y164* and c.723+1G >T, were novel and possibly pathogenic. In one family, the patient’s mother was not a carrier of the respective mutation. Our results increased the spectrum of CLCN5 disease causing defects with seven new pathogenic mutations and established a de novo origin in one of them. Remarkably, three new missense mutations, p.G466D, p.G65R and p.G462S, affect highly conserved glycine residues located in transmembrane α-helix GxxxG packing motifs. The two atypical cases further support that the diagnosis of Dent’s disease should be considered in children with asymptomatic proteinuria and focal glomerulosclerosis and without evidence of primary glomerular disease. PMID:27625851

MPL W515L/K Mutations in Chronic Myeloproliferative Neoplasms.

PubMed

Akpınar, Timur Selçuk; Hançer, Veysel Sabri; Nalçacı, Meliha; Diz-Küçükkaya, Reyhan

2013-03-01

The MPL gene encodes the thrombopoietin receptor. Recently MPL mutations (MPL W515L or MPL W515K) were described in patients with essential thrombocythemia (ET) and primary (idiopathic) myelofibrosis (PMF). The prevalence and the clinical importance of these mutations are not clear. In the present study, we aimed to investigate the frequency and clinical significance of MPL W515L/K mutations in our patients with ET and PMF. A total of 77 patients (66 were diagnosed with ET and 11 with PMF) and 42 healthy controls were included in the study. Using peripheral blood samples, the presence of MPL W515L/K mutations and JAK-2 V617F mutation were analyzed by real-time polymerase chain reaction. In our study, MPL W515L/K or JAK-2 V617F mutations were not observed in healthy controls. JAK-2 V617F mutation was present in 35 patients, of whom 29 had ET (43.9%, 29/66) and 6 had PMF (54.5%, 6/11). In the patient group, MPL W515L/K mutations were found in only 2 PMF cases, and these cases were negative for JAK-2 V617F mutation. The prevalence of MPL W515L/K mutations in the patient group was 2.6%, and the prevalence of MPL W515L/K mutations among the cases negative for the JAK-2 V617F mutation was found to be 4.8%. The 2 cases with MPL W515L/K mutations had long follow-up times (124 months and 71 months, respectively), had no thrombotic or hemorrhagic complications, and had no additional cytogenetic anomalies. MPL W515L/K mutations may be helpful for identifying clonal disease in MPN patients with no established Ph chromosome or JAK-2 V617F mutation. None declared.

Occupancy of Norepinephrine Transporter by Duloxetine in Human Brains Measured by Positron Emission Tomography with (S,S)-[18F]FMeNER-D2.

PubMed

Moriguchi, Sho; Takano, Harumasa; Kimura, Yasuyuki; Nagashima, Tomohisa; Takahata, Keisuke; Kubota, Manabu; Kitamura, Soichiro; Ishii, Tatsuya; Ichise, Masanori; Zhang, Ming-Rong; Shimada, Hitoshi; Mimura, Masaru; Meyer, Jeffrey H; Higuchi, Makoto; Suhara, Tetsuya

2017-12-01

The norepinephrine transporter in the brain has been targeted in the treatment of psychiatric disorders. Duloxetine is a serotonin and norepinephrine reuptake inhibitor that has been widely used for the treatment of depression. However, the relationship between dose and plasma concentration of duloxetine and norepinephrine transporter occupancy in the human brain has not been determined. In this study, we examined norepinephrine transporter occupancy by different doses of duloxetine. We calculated norepinephrine transporter occupancies from 2 positron emission tomography scans using (S,S)-[18F]FMeNER-D2 before and after a single oral dose of duloxetine (20 mg, n = 3; 40 mg, n = 3; 60 mg, n =2). Positron emission tomography scans were performed from 120 to 180 minutes after an i.v. bolus injection of (S,S)-[18F]FMeNER-D2. Venous blood samples were taken to measure the plasma concentration of duloxetine just before and after the second positron emission tomography scan. Norepinephrine transporter occupancy by duloxetine was 29.7% at 20 mg, 30.5% at 40 mg, and 40.0% at 60 mg. The estimated dose of duloxetine inducing 50% norepinephrine transporter occupancy was 76.8 mg, and the estimated plasma drug concentration inducing 50% norepinephrine transporter occupancy was 58.0 ng/mL. Norepinephrine transporter occupancy by clinical doses of duloxetine was approximately 30% to 40% in human brain as estimated using positron emission tomography with (S,S)-[18F]FMeNER-D2. © The Author 2017. Published by Oxford University Press on behalf of CINP.

40 CFR 60.1735 - Am I exempt from any appendix B or appendix F requirements to evaluate continuous emission...

Code of Federal Regulations, 2012 CFR

2012-07-01

... appendix F requirements to evaluate continuous emission monitoring systems? 60.1735 Section 60.1735... Combustion Units Constructed on or Before August 30, 1999 Model Rule-Continuous Emission Monitoring § 60.1735... to also evaluate your oxygen (or carbon dioxide) continuous emission monitoring system. Therefore...

40 CFR 60.1245 - Am I exempt from any appendix B or appendix F requirements to evaluate continuous emission...

Code of Federal Regulations, 2012 CFR

2012-07-01

... appendix F requirements to evaluate continuous emission monitoring systems? 60.1245 Section 60.1245... Commenced After June 6, 2001 Continuous Emission Monitoring § 60.1245 Am I exempt from any appendix B or... carbon dioxide) continuous emission monitoring system. Therefore, your oxygen (or carbon dioxide...

Molecular genetic analysis of some mutations in the cystic fibrosis gene in Moldova: Characterization of molecular markers and their linkage to various mutations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gimbovskaya, S.D.; Kalinin, V.N.; Ivashchenko, T.E.

1994-12-01

Sixty-one patients with cystic fibrosis (CF) from Moldova were tested for mutations {Delta}F508, G551D, and R553X. Frequencies of various alleles of the repeated GATT sequence in intron 6B of the GFTR gene, their linkage to other polymorphic markers, and various mutations were determined. The frequency of occurrence of mutation {Delta}F508 was only 25%. An absolute majority of CF patients (80%) had pancreatic insufficiency. Mutations G551D and R553X were not found in our sample. Each of 31 chromosomes with mutation {Delta}F508 carry the 6-GATT allele. Most {open_quotes}non {Delta}F508{close_quotes} (78%) and normal (80%) chromosomes were marked by the 7-GATT allele. Twenty-seven {Delta}F508more » chromosomes (96.4%) belong to haplotype B6, and only one to D6. Most chromosomes with {open_quotes}non {Delta}F508{close_quotes} mutations are associated with haplotypes D7 (26.3%) and C7 (21%). In addition, a significant portion of chromosomes from this subgroup were associated with haplotypes A7 (23.7%), A6 (10.5%), and C6 (2.7%), which are not yet described for mutant chromosomes. The results obtained demonstrate that CF in Moldova is mainly associated with mutations other than {Delta}F508, G551D, and R553X. Severe forms of the disease, with pancreatic insufficiency, are more frequently caused by these mutations; moreover, our data provides strong evidence for the presence of at least seven additional CF mutations in Moldova, apart from {Delta}F508, G551D, and R553X. Some of these are probably not described.« less

Parkinsonism Associated with Glucocerebrosidase Mutation

PubMed Central

Sunwoo, Mun-Kyung; Kim, Seung-Min; Lee, Sarah

2011-01-01

Background Gaucher's disease is an autosomal recessive, lysosomal storage disease caused by mutations of the β-glucocerebrosidase gene (GBA). There is increasing evidence that GBA mutations are a genetic risk factor for the development of Parkinson's disease (PD). We report herein a family of Koreans exhibiting parkinsonism-associated GBA mutations. Case Report A 44-year-old woman suffering from slowness and paresthesia of the left arm for the previous 1.5years, visited our hospital to manage known invasive ductal carcinoma. During a preoperative evaluation, she was diagnosed with Gaucher's disease and double mutations of S271G and R359X in GBA. Parkinsonian features including low amplitude postural tremors, rigidity, bradykinesia and shuffling gait were observed. Genetic analysis also revealed that her older sister, who had also been diagnosed with PD and had been taking dopaminergic drugs for 8-years, also possessed a heterozygote R359X mutation in GBA. 18F-fluoropropylcarbomethoxyiodophenylnortropane positron-emission tomography in these patients revealed decreased uptake of dopamine transporter in the posterior portion of the bilateral putamen. Conclusions This case study demonstrates Korean familial cases of PD with heterozygote mutation of GBA, further supporting the association between PD and GBA mutation. PMID:21779299

Mutations of Vasopressin Receptor 2 Including Novel L312S Have Differential Effects on Trafficking.

PubMed

Tiulpakov, Anatoly; White, Carl W; Abhayawardana, Rekhati S; See, Heng B; Chan, Audrey S; Seeber, Ruth M; Heng, Julian I; Dedov, Ivan; Pavlos, Nathan J; Pfleger, Kevin D G

2016-08-01

Nephrogenic syndrome of inappropriate antidiuresis (NSIAD) is a genetic disease first described in 2 unrelated male infants with severe symptomatic hyponatremia. Despite undetectable arginine vasopressin levels, patients have inappropriately concentrated urine resulting in hyponatremia, hypoosmolality, and natriuresis. Here, we describe and functionally characterize a novel vasopressin type 2 receptor (V2R) gain-of-function mutation. An L312S substitution in the seventh transmembrane domain was identified in a boy presenting with water-induced hyponatremic seizures at the age of 5.8 years. We show that, compared with wild-type V2R, the L312S mutation results in the constitutive production of cAMP, indicative of the gain-of-function NSIAD profile. Interestingly, like the previously described F229V and I130N NSIAD-causing mutants, this appears to both occur in the absence of notable constitutive β-arrestin2 recruitment and can be reduced by the inverse agonist Tolvaptan. In addition, to understand the effect of various V2R substitutions on the full receptor "life-cycle," we have used and further developed a bioluminescence resonance energy transfer intracellular localization assay using multiple localization markers validated with confocal microscopy. This allowed us to characterize differences in the constitutive and ligand-induced localization and trafficking profiles of the novel L312S mutation as well as for previously described V2R gain-of-function mutants (NSIAD; R137C and R137L), loss-of-function mutants (nephrogenic diabetes insipidus; R137H, R181C, and M311V), and a putative silent V266A V2R polymorphism. In doing so, we describe differences in trafficking between unique V2R substitutions, even at the same amino acid position, therefore highlighting the value of full and thorough characterization of receptor function beyond simple signaling pathway analysis.

Mutations, associated with early-onset Alzheimer’s disease, discovered in Asian countries

PubMed Central

Bagyinszky, Eva; Youn, Young Chul; An, Seong Soo A; Kim, SangYun

2016-01-01

Alzheimer’s disease (AD), the most common form of senile dementia, is a genetically complex disorder. In most Asian countries, the population and the number of AD patients are growing rapidly, and the genetics of AD has been extensively studied, except in Japan. However, recent studies have been started to investigate the genes and mutations associated with AD in Korea, the People’s Republic of China, and Malaysia. This review describes all of the known mutations in three early-onset AD (EOAD) causative genes (APP, PSEN1, and PSEN2) that were discovered in Asian countries. Most of the EOAD-associated mutations have been detected in PSEN1, and several novel PSEN1 mutations were recently identified in patients from various parts of the world, including Asia. Until 2014, no PSEN2 mutations were found in Asian patients; however, emerging studies from Korea and the People’s Republic of China discovered probably pathogenic PSEN2 mutations. Since several novel mutations were discovered in these three genes, we also discuss the predictions on their pathogenic nature. This review briefly summarizes genome-wide association studies of late-onset AD and the genes that might be associated with AD in Asian countries. Standard sequencing is a widely used method, but it has limitations in terms of time, cost, and efficacy. Next-generation sequencing strategies could facilitate genetic analysis and association studies. Genetic testing is important for the accurate diagnosis and for understanding disease-associated pathways and might also improve disease therapy and prevention. PMID:27799753

Dark Matter Search Results from the PICO-60 C$$_3$$F$$_8$$ Bubble Chamber

DOE Office of Scientific and Technical Information (OSTI.GOV)

Amole, C.; et al.

2017-02-24

New results are reported from the operation of the PICO-60 dark matter detector, a bubble chamber filled with 52 kg of Cmore » $$_3$$F$$_8$$ located in the SNOLAB underground laboratory. As in previous PICO bubble chambers, PICO-60 C$$_3$$F$$_8$$ exhibits excellent electron recoil and alpha decay rejection, and the observed multiple-scattering neutron rate indicates a single-scatter neutron background of less than 1 event per month. A blind analysis of an efficiency-corrected 1167-kg-day exposure at a 3.3-keV thermodynamic threshold reveals no single-scattering nuclear recoil candidates, consistent with the predicted background. These results set the most stringent direct-detection constraint to date on the WIMP-proton spin-dependent cross section at 3.4 $$\\times$$ 10$$^{-41}$$ cm$^2$ for a 30-GeV$$\\thinspace$$c$$^{-2}$$ WIMP, more than one order of magnitude improvement from previous PICO results.« less

Dark Matter Search Results from the PICO -60 C 3F8 Bubble Chamber

NASA Astrophysics Data System (ADS)

Amole, C.; Ardid, M.; Arnquist, I. J.; Asner, D. M.; Baxter, D.; Behnke, E.; Bhattacharjee, P.; Borsodi, H.; Bou-Cabo, M.; Campion, P.; Cao, G.; Chen, C. J.; Chowdhury, U.; Clark, K.; Collar, J. I.; Cooper, P. S.; Crisler, M.; Crowder, G.; Dahl, C. E.; Das, M.; Fallows, S.; Farine, J.; Felis, I.; Filgas, R.; Girard, F.; Giroux, G.; Hall, J.; Harris, O.; Hoppe, E. W.; Jin, M.; Krauss, C. B.; Laurin, M.; Lawson, I.; Leblanc, A.; Levine, I.; Lippincott, W. H.; Mamedov, F.; Maurya, D.; Mitra, P.; Nania, T.; Neilson, R.; Noble, A. J.; Olson, S.; Ortega, A.; Plante, A.; Podviyanuk, R.; Priya, S.; Robinson, A. E.; Roeder, A.; Rucinski, R.; Scallon, O.; Seth, S.; Sonnenschein, A.; Starinski, N.; Štekl, I.; Tardif, F.; Vázquez-Jáuregui, E.; Wells, J.; Wichoski, U.; Yan, Y.; Zacek, V.; Zhang, J.; PICO Collaboration

2017-06-01

New results are reported from the operation of the PICO-60 dark matter detector, a bubble chamber filled with 52 kg of C3 F8 located in the SNOLAB underground laboratory. As in previous PICO bubble chambers, PICO -60 C 3F8 exhibits excellent electron recoil and alpha decay rejection, and the observed multiple-scattering neutron rate indicates a single-scatter neutron background of less than one event per month. A blind analysis of an efficiency-corrected 1167-kg day exposure at a 3.3-keV thermodynamic threshold reveals no single-scattering nuclear recoil candidates, consistent with the predicted background. These results set the most stringent direct-detection constraint to date on the weakly interacting massive particle (WIMP)-proton spin-dependent cross section at 3.4 ×10-41 cm2 for a 30 -GeV c-2 WIMP, more than 1 order of magnitude improvement from previous PICO results.

Pathogenic mutation in the ALS/FTD gene, CCNF, causes elevated Lys48-linked ubiquitylation and defective autophagy.

PubMed

Lee, Albert; Rayner, Stephanie L; Gwee, Serene S L; De Luca, Alana; Shahheydari, Hamideh; Sundaramoorthy, Vinod; Ragagnin, Audrey; Morsch, Marco; Radford, Rowan; Galper, Jasmin; Freckleton, Sarah; Shi, Bingyang; Walker, Adam K; Don, Emily K; Cole, Nicholas J; Yang, Shu; Williams, Kelly L; Yerbury, Justin J; Blair, Ian P; Atkin, Julie D; Molloy, Mark P; Chung, Roger S

2018-01-01

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that have common molecular and pathogenic characteristics, such as aberrant accumulation and ubiquitylation of TDP-43; however, the mechanisms that drive this process remain poorly understood. We have recently identified CCNF mutations in familial and sporadic ALS and FTD patients. CCNF encodes cyclin F, a component of an E3 ubiquitin-protein ligase (SCF cyclin F ) complex that is responsible for ubiquitylating proteins for degradation by the ubiquitin-proteasome system. In this study, we examined the ALS/FTD-causing p.Ser621Gly (p.S621G) mutation in cyclin F and its effect upon downstream Lys48-specific ubiquitylation in transfected Neuro-2A and SH-SY5Y cells. Expression of mutant cyclin F S621G caused increased Lys48-specific ubiquitylation of proteins in neuronal cells compared to cyclin F WT . Proteomic analysis of immunoprecipitated Lys48-ubiquitylated proteins from mutant cyclin F S621G -expressing cells identified proteins that clustered within the autophagy pathway, including sequestosome-1 (p62/SQSTM1), heat shock proteins, and chaperonin complex components. Examination of autophagy markers p62, LC3, and lysosome-associated membrane protein 2 (Lamp2) in cells expressing mutant cyclin F S621G revealed defects in the autophagy pathway specifically resulting in impairment in autophagosomal-lysosome fusion. This finding highlights a potential mechanism by which cyclin F interacts with p62, the receptor responsible for transporting ubiquitylated substrates for autophagic degradation. These findings demonstrate that ALS/FTD-causing mutant cyclin F S621G disrupts Lys48-specific ubiquitylation, leading to accumulation of substrates and defects in the autophagic machinery. This study also demonstrates that a single missense mutation in cyclin F causes hyper-ubiquitylation of proteins that can indirectly impair the autophagy degradation pathway, which is

JAK and MPL mutations in myeloid malignancies.

PubMed

Tefferi, Ayalew

2008-03-01

The Janus family of non-receptor tyrosine kinases (JAK1, JAK2, JAK3 and tyrosine kinase 2) transduces signals downstream of type I and II cytokine receptors via signal transducers and activators of transcription (STATs). JAK3 is important in lymphoid and JAK2 in myeloid cell proliferation and differentiation. The thrombopoietin receptor MPL is one of several JAK2 cognate receptors and is essential for myelopoiesis in general and megakaryopoiesis in particular. Germline loss-of-function (LOF) JAK3 and MPL mutations cause severe combined immunodeficiency and congenital amegakaryocytic thrombocytopenia, respectively. Germline gain-of-function (GOF) MPL mutation (MPLS505N) causes familial thrombocytosis. Somatic JAK3 (e.g. JAK3A572V, JAK3V722I, JAK3P132T) and fusion JAK2 (e.g. ETV6-JAK2, PCM1-JAK2, BCR-JAK2) mutations have respectively been described in acute megakaryocytic leukemia and acute leukemia/chronic myeloid malignancies. However, current attention is focused on JAK2 (e.g. JAK2V617F, JAK2 exon 12 mutations) and MPL (e.g. MPLW515L/K/S, MPLS505N) mutations associated with myeloproliferative neoplasms (MPNs). A JAK2 mutation, primarily JAK2V617F, is invariably associated with polycythemia vera (PV). The latter mutation also occurs in the majority of patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF). MPL mutational frequency in MPNs is substantially less (<10%). In general, despite a certain degree of genotype - phenotype correlations, the prognostic relevance of harbouring one of these mutations, or their allele burden when present, remains dubious. Regardless, based on the logical assumption that amplified JAK-STAT signalling is central to the pathogenesis of PV, ET and PMF, several anti-JAK2 tyrosine kinase inhibitors have been developed and are currently being tested in humans with these disorders.

Spectrum of mutations in RARS-T patients includes TET2 and ASXL1 mutations.

PubMed

Szpurka, Hadrian; Jankowska, Anna M; Makishima, Hideki; Bodo, Juraj; Bejanyan, Nelli; Hsi, Eric D; Sekeres, Mikkael A; Maciejewski, Jaroslaw P

2010-08-01

While a majority of patients with refractory anemia with ring sideroblasts and thrombocytosis harbor JAK2V617F and rarely MPLW515L, JAK2/MPL-negative cases constitute a diagnostic problem. 23 RARS-T cases were investigated applying immunohistochemical phospho-STAT5, sequencing and SNP-A-based karyotyping. Based on the association of TET2/ASXL1 mutations with MDS/MPN we studied molecular pattern of these genes. Two patients harbored ASXL1 and another 2 TET2 mutations. Phospho-STAT5 activation was present in one mutated TET2 and ASXL1 case. JAK2V617F/MPLW515L mutations were absent in TET2/ASXL1 mutants, indicating that similar clinical phenotype can be produced by various MPN-associated mutations and that additional unifying lesions may be present in RARS-T. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Special cascade LMS equalization scheme suitable for 60-GHz RoF transmission system.

PubMed

Liu, Siming; Shen, Guansheng; Kou, Yanbin; Tian, Huiping

2016-05-16

We design a specific cascade least mean square (LMS) equalizer and to the best of our knowledge, it is the first time this kind of equalizer has been employed for 60-GHz millimeter-wave (mm-wave) radio over fiber (RoF) system. The proposed cascade LMS equalizer consists of two sub-equalizers which are designated for optical and wireless channel compensations, respectively. We control the linear and nonlinear factors originated from optical link and wireless link separately. The cascade equalization scheme can keep the nonlinear distortions of the RoF system in a low degree. We theoretically and experimentally investigate the parameters of the two sub-equalizers to reach their best performances. The experiment results show that the cascade equalization scheme has a faster convergence speed. It needs a training sequence with a length of 10000 to reach its stable status, which is only half as long as the traditional LMS equalizer needs. With the utility of a proposed equalizer, the 60-GHz RoF system can successfully transmit 5-Gbps BPSK signal over 10-km fiber and 1.2-m wireless link under forward error correction (FEC) limit 10^-3. An improvement of 4dBm and 1dBm in power sensitivity at BER 10^-3 over traditional LMS equalizer can be observed when the signals are transmitted through Back-to-Back (BTB) and 10-km fiber 1.2-m wireless links, respectively.

Painful Charcot-Marie-Tooth neuropathy type 2E/1F due to a novel NEFL mutation.

PubMed

Doppler, Kathrin; Kunstmann, Erdmute; Krüger, Stefan; Sommer, Claudia

2017-05-01

Charcot-Marie-Tooth neuropathy (CMT) 2E/1F is caused by mutations in the neurofilament light-chain polypeptide (NEFL) gene. Giant axons are a histological hallmark frequently seen in nerves of patients with CMT2E. We describe the case of a 43-year-old patient with a painful, predominantly sensory neuropathy. The patient's sural nerve biopsy showed multiple giant axons. Genetic sequencing of the NEFL gene revealed that the patient was heterozygous for an altered sequence of the gene, c.816C>G, p.Asn272Lys, which has not yet been described in CMT2E/1F. In contrast to other cases of CMT2E/1F, where motor symptoms are predominant, pain was the most disabling symptom in this patient. Muscle Nerve 55: 752-755, 2017. © 2016 Wiley Periodicals, Inc.

Disease-Causing Mutations in the G Protein Gαs Subvert the Roles of GDP and GTP.

PubMed

Hu, Qi; Shokat, Kevan M

2018-05-17

The single most frequent cancer-causing mutation across all heterotrimeric G proteins is R201C in Gαs. The current model explaining the gain-of-function activity of the R201 mutations is through the loss of GTPase activity and resulting inability to switch off to the GDP state. Here, we find that the R201C mutation can bypass the need for GTP binding by directly activating GDP-bound Gαs through stabilization of an intramolecular hydrogen bond network. Having found that a gain-of-function mutation can convert GDP into an activator, we postulated that a reciprocal mutation might disrupt the normal role of GTP. Indeed, we found R228C, a loss-of-function mutation in Gαs that causes pseudohypoparathyroidism type 1a (PHP-Ia), compromised the adenylyl cyclase-activating activity of Gαs bound to a non-hydrolyzable GTP analog. These findings show that disease-causing mutations in Gαs can subvert the canonical roles of GDP and GTP, providing new insights into the regulation mechanism of G proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

Rates of spontaneous mutation in an archaeon from geothermal environments.

PubMed Central

Jacobs, K L; Grogan, D W

1997-01-01

To estimate the efficacy of mechanisms which may prevent or repair thermal damage to DNA in thermophilic archaea, a quantitative assay of forward mutation at extremely high temperature was developed for Sulfolobus acidocaldarius, based on the selection of pyrimidine-requiring mutants resistant to 5-fluoro-orotic acid. Maximum-likelihood analysis of spontaneous mutant distributions in wild-type cultures yielded maximal estimates of (2.8 +/- 0.7) x 10(-7) and (1.5 +/- 0.6) x 10(-7) mutational events per cell per division cycle for the pyrE and pyrF loci, respectively. To our knowledge, these results provide the first accurate measurement of the genetic fidelity maintained by archaea that populate geothermal environments. The measured rates of forward mutation at the pyrE and pyrF loci in S. acidocaldarius are close to corresponding rates reported for protein-encoding genes of Escherichia coli. The normal rate of spontaneous mutation in E. coli at 37 degrees C is known to require the functioning of several enzyme systems that repair spontaneous damage in DNA. Our results provide indirect evidence that S. acidocaldarius has cellular mechanisms, as yet unidentified, which effectively compensate for the higher chemical instability of DNA at the temperatures and pHs that prevail within growing Sulfolobus cells. PMID:9150227

Two novel mutations of pfdhps K540T and I588F, affecting sulphadoxine-pyrimethamine-resistant response in uncomplicated falciparum malaria at Banjar district, South Kalimantan Province, Indonesia

PubMed Central

2014-01-01

Background Mutations in pfdhfr and pfdhps genes have been shown to associate with sulphadoxine-pyrimethamine (SP) resistance of Plasmodium falciparum parasites. However, pfdhfr, pfdhps genotypes and the correlations to SP treatment outcome in Indonesia has not yet been well analysed. Methods After obtaining informed consent, 61 uncomplicated falciparum malaria patients were recruited in Banjar district, South Kalimantan Province, Indonesia, from October 2009 to August 2010. They were treated by a single oral dose of SP and its effects on clinical and parasitological status were followed until day 28 after treatment. Occasionally, a thick smear blood film for microscopy observation and blood spot on a filter paper for pfdhfr and pfdhps genotype analysis were collected. Results Pfdhfr and pfdhps genotypes from 24 P. falciparum-infected patients consisting of adequate clinical parasitological response (ACPR) (n = 6; 25.0%) and early treatment failure (ETF) (n = 10; 41.7%) or late parasitological failure (LPF) (n = 8; 33.3%) were obtained by sequencing. Two novel mutations of pfdhps gene, K540T and I588F, were determined in ten and five isolates, respectively. These mutations were present in the pfdhfr/pfdhps combined haplotypes of ANRNI/SGTGA (n = 6), ANRNL/SGTGA (n = 4), and ANRNI/SGEAA(588F) (n = 5), (mutation codons are bold typed); these haplotypes were mostly belonging to parasitological failure (ETF or LPF). The parasites acquiring five mutations in pfdhfr/pfdhps haplotypes and four mutations with additional I588F did not respond adequately to SP treatment. Conclusion Many of Plasmodium falciparum infected patients in Banjar district, South Kalimantan, Indonesia did not respond adequately to SP treatment and these low ineffectiveness of SP in this area was associated with two novel mutations of pfdhps, K540T and I588F. PMID:25187019

Two novel mutations of pfdhps K540T and I588F, affecting sulphadoxine-pyrimethamine-resistant response in uncomplicated falciparum malaria at Banjar district, South Kalimantan Province, Indonesia.

PubMed

Basuki, Sukmawati; Fitriah; Riyanto, Sugeng; Budiono; Dachlan, Yoes P; Uemura, Haruki

2014-04-04

Mutations in pfdhfr and pfdhps genes have been shown to associate with sulphadoxine-pyrimethamine (SP) resistance of Plasmodium falciparum parasites. However, pfdhfr, pfdhps genotypes and the correlations to SP treatment outcome in Indonesia has not yet been well analysed. After obtaining informed consent, 61 uncomplicated falciparum malaria patients were recruited in Banjar district, South Kalimantan Province, Indonesia, from October 2009 to August 2010. They were treated by a single oral dose of SP and its effects on clinical and parasitological status were followed until day 28 after treatment. Occasionally, a thick smear blood film for microscopy observation and blood spot on a filter paper for pfdhfr and pfdhps genotype analysis were collected. Pfdhfr and pfdhps genotypes from 24 P. falciparum-infected patients consisting of adequate clinical parasitological response (ACPR) (n = 6; 25.0%) and early treatment failure (ETF) (n = 10; 41.7%) or late parasitological failure (LPF) (n = 8; 33.3%) were obtained by sequencing. Two novel mutations of pfdhps gene, K540T and I588F, were determined in ten and five isolates, respectively. These mutations were present in the pfdhfr/pfdhps combined haplotypes of ANRNI/SGTGA (n = 6), ANRNL/SGTGA (n = 4), and ANRNI/SGEAA(588F) (n = 5), (mutation codons are bold typed); these haplotypes were mostly belonging to parasitological failure (ETF or LPF). The parasites acquiring five mutations in pfdhfr/pfdhps haplotypes and four mutations with additional I588F did not respond adequately to SP treatment. Many of Plasmodium falciparum infected patients in Banjar district, South Kalimantan, Indonesia did not respond adequately to SP treatment and these low ineffectiveness of SP in this area was associated with two novel mutations of pfdhps, K540T and I588F.

The dual exo/endo-type mode and the effect of ionic strength on the mode of catalysis of chitinase 60 (CHI60) from Serratia sp. TU09 and its mutants.

PubMed

Kuttiyawong, K; Nakapong, S; Pichyangkura, R

2008-11-03

Mutations of the tryptophan residues in the tryptophan-track of the N-terminal domain (W33F/Y and W69F/Y) and in the catalytic domain (W245F/Y) of Serratia sp. TU09 Chitinase 60 (CHI60) were constructed, as single and double point substitutions to either phenylalanine or tyrosine. The enzyme-substrate interaction and mode of catalysis, exo/endo-type, of wild type CHI60 and mutant enzymes on soluble (partially N-acetylated chitin), amorphous (colloidal chitin), and crystalline (β-chitin) substrates were studied. All CHI60 mutants exhibited a reduced substrate binding activity on colloidal chitin. CHI60 possesses a dual mode of catalysis with both exo- and endo-type activities allowing the enzyme to work efficiently on various substrate types. CHI60 preferentially uses the endo-type mode on soluble and amorphous substrates and the exo-type mode on crystalline substrate. However, the prevalent mode of hydrolysis mediated by CHI60 is regulated by ionic strength. Slightly elevated ionic strength, 0.1-0.2M NaCl, which promotes enzyme-substrate interactions, enhances CHI60 hydrolytic activity on amorphous substrate and, interestingly, on partially N-acetylated chitin. High ionic strength, 0.5-2.0M NaCl, prevents the enzyme from dissociating from amorphous substrate, occupying the enzyme in an enzyme-substrate non-productive complex. However, on crystalline substrates, the activity of CHI60 was only inhibited approximately 50% at high ionic strength, suggesting that the enzyme hydrolyzes crystalline substrates with an exo-type mode processively while remaining tightly bound to the substrate. Moreover, substitution of Trp-33 to either phenylalanine or tyrosine reduced the activity of the enzyme at high ionic strength, suggesting an important role of Trp-33 on enzyme processivity.

JAK2 Exon 12 Mutations in Polycythemia Vera and Idiopathic Erythrocytosis

PubMed Central

Scott, Linda M.; Tong, Wei; Levine, Ross L.; Scott, Mike A.; Beer, Philip A.; Stratton, Michael R.; Futreal, P. Andrew; Erber, Wendy N.; McMullin, Mary Frances; Harrison, Claire N.; Warren, Alan J.; Gilliland, D. Gary; Lodish, Harvey F.; Green, Anthony R.

2010-01-01

BACKGROUND The V617F mutation, which causes the substitution of phenylalanine for valine at position 617 of the Janus kinase (JAK) 2 gene (JAK2), is often present in patients with polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. However, the molecular basis of these myeloproliferative disorders in patients without the V617F mutation is unclear. METHODS We searched for new mutations in members of the JAK and signal transducer and activator of transcription (STAT) gene families in patients with V617F-negative polycythemia vera or idiopathic erythrocytosis. The mutations were characterized biochemically and in a murine model of bone marrow transplantation. RESULTS We identified four somatic gain-of-function mutations affecting JAK2 exon 12 in 10 V617F-negative patients. Those with a JAK2 exon 12 mutation presented with an isolated erythrocytosis and distinctive bone marrow morphology, and several also had reduced serum erythropoietin levels. Erythroid colonies could be grown from their blood samples in the absence of exogenous erythropoietin. All such erythroid colonies were heterozygous for the mutation, whereas colonies homozygous for the mutation occur in most patients with V617F-positive polycythemia vera. BaF3 cells expressing the murine erythropoietin receptor and also carrying exon 12 mutations could proliferate without added interleukin-3. They also exhibited increased phosphorylation of JAK2 and extracellular regulated kinase 1 and 2, as compared with cells transduced by wild-type JAK2 or V617F JAK2. Three of the exon 12 mutations included a substitution of leucine for lysine at position 539 of JAK2. This mutation resulted in a myeloproliferative phenotype, including erythrocytosis, in a murine model of retroviral bone marrow transplantation. CONCLUSIONS JAK2 exon 12 mutations define a distinctive myeloproliferative syndrome that affects patients who currently receive a diagnosis of polycythemia vera or idiopathic erythrocytosis

[Study of gene mutation in 62 hemophilia A children].

PubMed

Hu, Q; Liu, A G; Zhang, L Q; Zhang, A; Wang, Y Q; Wang, S M; Lu, Y J; Wang, X

2017-11-02

Objective: To analyze the mutation type of FⅧ gene in children with hemophilia A and to explore the relationship among hemophilia gene mutation spectrum, gene mutation and clinical phenotype. Method: Sixty-two children with hemophilia A from Department of Pediatric Hematology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology between January 2015 and March 2017 were enrolled. All patients were male, aged from 4 months to 7 years and F Ⅷ activity ranged 0.2%-11.0%. Fifty cases had severe, 10 cases had moderate and 2 cases had mild hemophilia A. DNA was isolated from peripheral blood in hemophilia A children and the target gene fragment was amplified by PCR, in combination with the second generation sequencing, 22 and 1 introns were detected. Negative cases were detected by the second generation sequencing and results were compared with those of the international FⅧ gene mutation database. Result: There were 20 cases (32%) of intron 22 inversion, 2 cases (3%) of intron 1 inversion, 18 cases (29%) of missense mutation, 5 cases (8%) of nonsense mutation, 7 cases (11%) of deletion mutation, 1 case(2%)of splice site mutation, 2 cases (3%) of large fragment deletion and 1 case of insertion mutation (2%). No mutation was detected in 2 cases (3%), and 4 cases (7%) failed to amplify. The correlation between phenotype and genotype showed that the most common gene mutation in severe hemophilia A was intron 22 inversion (20 cases), accounting for 40% of severe patients, followed by 11 cases of missense mutation (22%). The most common mutation in moderate hemophilia A was missense mutation (6 cases), accounting for 60% of moderate patients. Conclusion: The most frequent mutation type in hemophilia A was intron 22 inversion, followed by missense mutation, again for missing mutation. The relationship between phenotype and genotype: the most frequent gene mutation in severe hemophilia A is intron 22 inversion, followed by missense

The ΔF508 Mutation in the Cystic Fibrosis Transmembrane Conductance Regulator Is Associated With Progressive Insulin Resistance and Decreased Functional β-Cell Mass in Mice.

PubMed

Fontés, Ghislaine; Ghislain, Julien; Benterki, Isma; Zarrouki, Bader; Trudel, Dominique; Berthiaume, Yves; Poitout, Vincent

2015-12-01

Cystic fibrosis (CF) is the result of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). CF-related diabetes affects 50% of adult CF patients. How CFTR deficiency predisposes to diabetes is unknown. Herein, we examined the impact of the most frequent cftr mutation in humans, deletion of phenylalanine at position 508 (ΔF508), on glucose homeostasis in mice. We compared ΔF508 mutant mice with wild-type (WT) littermates. Twelve-week-old male ΔF508 mutants had lower body weight, improved oral glucose tolerance, and a trend toward higher insulin tolerance. Glucose-induced insulin secretion was slightly diminished in ΔF508 mutant islets, due to reduced insulin content, but ΔF508 mutant islets were not more sensitive to proinflammatory cytokines than WT islets. Hyperglycemic clamps confirmed an increase in insulin sensitivity with normal β-cell function in 12- and 18-week-old ΔF508 mutants. In contrast, 24-week-old ΔF508 mutants exhibited insulin resistance and reduced β-cell function. β-Cell mass was unaffected at 11 weeks of age but was significantly lower in ΔF508 mutants versus controls at 24 weeks. This was not associated with gross pancreatic pathology. We conclude that the ΔF508 CFTR mutation does not lead to an intrinsic β-cell secretory defect but is associated with insulin resistance and a β-cell mass deficit in aging mutants. © 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

23. "GAFFTC 20 APR 60, H65A F106A; ESCAPE SYSTEM RUN ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

23. "G-AFFTC 20 APR 60, H-6-5A F-106A; ESCAPE SYSTEM RUN 5A." Testing the ejection system on a Convair sled. - Edwards Air Force Base, South Base Sled Track, Edwards Air Force Base, North of Avenue B, between 100th & 140th Streets East, Lancaster, Los Angeles County, CA

Confirmation of Pig-a mutation in flow cytometry-identified CD48-deficient T-lymphocytes from F344 rats.

PubMed

Revollo, Javier; Pearce, Mason G; Petibone, Dayton M; Mittelstaedt, Roberta A; Dobrovolsky, Vasily N

2015-05-01

The Pig-a assay is used for monitoring somatic cell mutation in laboratory animals and humans. The assay detects haematopoietic cells deficient in glycosylphosphatidylinositol (GPI)-anchored protein surface markers using flow cytometry. However, given that synthesis of the protein markers (and the expression of their genes) is independent of the expression of the X-linked Pig-a gene and the function of its enzyme product, the deficiency of markers at the surface of the cells may be caused by a number of events (e.g. by mutation or epigenetic silencing in the marker gene itself or in any of about two dozen autosomal genes involved in the synthesis of GPI). Here we provide direct evidence that the deficiency of the GPI-anchored surface marker CD48 in rat T-cells is accompanied by mutation in the endogenous X-linked Pig-a gene. We treated male F344 rats with N-ethyl-N-nitrosourea (ENU), and established colonies from flow cytometry-identified and sorted CD48-deficient spleen T-lymphocytes. Molecular analysis confirmed that the expanded sorted cells have mutations in the Pig-a gene. The spectrum of Pig-a mutation in our model was consistent with the spectrum of ENU-induced mutation determined in other in vivo models, mostly base-pair substitutions at A:T with the mutated T on the non-transcribed strand of Pig-a genomic DNA. We also used next generation sequencing to derive a similar mutational spectrum from a pool of 64 clones developed from flow-sorted CD48-deficient lymphocytes. Our findings confirm that Pig-a assays detect what they are designed to detect-gene mutation in the Pig-a gene. © The Author 2015. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Telomere length is severely and similarly reduced in JAK2V617F-positive and -negative myeloproliferative neoplasms

PubMed Central

Bernard, L; Belisle, C; Mollica, L; Provost, S; Roy, D-C; Gilliland, DG; Levine, RL; Busque, L

2015-01-01

Myeloproliferative neoplasms (MPNs) are clonal stem cell disorders characterized by chronic proliferation of hematopoietic progenitors. We studied the telomere length (TL) of 335 MPN patients and 93 gender- and age-matched controls using a quantitative PCR method (relative TL calculated as the ratio of the amount of telomere DNA vs single-copy DNA: T/S ratio). TL was markedly reduced in MPN patients compared with controls (T/S 0.561 vs 0.990, P<0.001). In JAK2V617F MPN patients, TL correlated inversely with allelic burden (P<0.001). Patients homozygous for the mutation (allelic burden 90–100%) had the shortest TL, even when compared with patients with lower allele burdens consistent with a dominant heterozygous population (allelic burden 55–65%) (T/S 0.367 vs 0.497, P = 0.037). This suggests that the high degree of proliferation of the MPN clone reduces TL and suggests the possibility that TL shortening may be indicative of progressive genomic instability during MPN progression. The TL of JAK2V617F-negative MPN patients was similar to JAK2V617F-positive counterparts (T/S 0.527 vs 0.507, P = 0.603), suggesting that the yet-to-be-discovered causative mutation(s) impact the mutated stem cell similarly to JAK2V617F, and that TL measurement may prove useful in the diagnostic workup of JAK2V617F-negative MPN. PMID:19005480

Meta-analysis of neuroblastomas reveals a skewed ALK mutation spectrum in tumors with MYCN amplification.

PubMed

De Brouwer, Sara; De Preter, Katleen; Kumps, Candy; Zabrocki, Piotr; Porcu, Michaël; Westerhout, Ellen M; Lakeman, Arjan; Vandesompele, Jo; Hoebeeck, Jasmien; Van Maerken, Tom; De Paepe, Anne; Laureys, Geneviève; Schulte, Johannes H; Schramm, Alexander; Van Den Broecke, Caroline; Vermeulen, Joëlle; Van Roy, Nadine; Beiske, Klaus; Renard, Marleen; Noguera, Rosa; Delattre, Olivier; Janoueix-Lerosey, Isabelle; Kogner, Per; Martinsson, Tommy; Nakagawara, Akira; Ohira, Miki; Caron, Huib; Eggert, Angelika; Cools, Jan; Versteeg, Rogier; Speleman, Frank

2010-09-01

Activating mutations of the anaplastic lymphoma kinase (ALK) were recently described in neuroblastoma. We carried out a meta-analysis of 709 neuroblastoma tumors to determine their frequency and mutation spectrum in relation to genomic and clinical parameters, and studied the prognostic significance of ALK copy number and expression. The frequency and type of ALK mutations, copy number gain, and expression were analyzed in a new series of 254 neuroblastoma tumors. Data from 455 published cases were used for further in-depth analysis. ALK mutations were present in 6.9% of 709 investigated tumors, and mutations were found in similar frequencies in favorable [International Neuroblastoma Staging System (INSS) 1, 2, and 4S; 5.7%] and unfavorable (INSS 3 and 4; 7.5%) neuroblastomas (P = 0.087). Two hotspot mutations, at positions R1275 and F1174, were observed (49% and 34.7% of the mutated cases, respectively). Interestingly, the F1174 mutations occurred in a high proportion of MYCN-amplified cases (P = 0.001), and this combined occurrence was associated with a particular poor outcome, suggesting a positive cooperative effect between both aberrations. Furthermore, the F1174L mutant was characterized by a higher degree of autophosphorylation and a more potent transforming capacity as compared with the R1275Q mutant. Chromosome 2p gains, including the ALK locus (91.8%), were associated with a significantly increased ALK expression, which was also correlated with poor survival. ALK mutations occur in equal frequencies across all genomic subtypes, but F1174L mutants are observed in a higher frequency of MYCN-amplified tumors and show increased transforming capacity as compared with the R1275Q mutants.

Single d-metal atoms on F(s) and F(s+) defects of MgO(001): a theoretical study across the periodic table.

PubMed

Neyman, Konstantin M; Inntam, Chan; Matveev, Alexei V; Nasluzov, Vladimir A; Rösch, Notker

2005-08-24

Single d-metal atoms on oxygen defects F(s) and F(s+) of the MgO(001) surface were studied theoretically. We employed an accurate density functional method combined with cluster models, embedded in an elastic polarizable environment, and we applied two gradient-corrected exchange-correlation functionals. In this way, we quantified how 17 metal atoms from groups 6-11 of the periodic table (Cu, Ag, Au; Ni, Pd, Pt; Co, Rh, Ir; Fe, Ru, Os; Mn, Re; and Cr, Mo, W) interact with terrace sites of MgO. We found bonding with F(s) and F(s+) defects to be in general stronger than that with O2- sites, except for Mn-, Re-, and Fe/F(s) complexes. In M/F(s) systems, electron density is accumulated on the metal center in a notable fashion. The binding energy on both kinds of O defects increases from 3d- to 4d- to 5d-atoms of a given group, at variance with the binding energy trend established earlier for the M/O2- complexes, 4d < 3d < 5d. Regarding the evolution of the binding energy along a period, group 7 atoms are slightly destabilized compared to their group 6 congeners in both the F(s) and F(s+) complexes; for later transition elements, the binding energy increases gradually up to group 10 and finally decreases again in group 11, most strongly on the F(s) site. This trend is governed by the negative charge on the adsorbed atoms. We discuss implications for an experimental detection of metal atoms on oxide supports based on computed core-level energies.

40 CFR 60.1735 - Am I exempt from any appendix B or appendix F requirements to evaluate continuous emission...

Code of Federal Regulations, 2011 CFR

2011-07-01

... appendix F requirements to evaluate continuous emission monitoring systems? 60.1735 Section 60.1735... systems? Yes, the accuracy tests for your sulfur dioxide continuous emission monitoring system require you to also evaluate your oxygen (or carbon dioxide) continuous emission monitoring system. Therefore...

40 CFR 60.1735 - Am I exempt from any appendix B or appendix F requirements to evaluate continuous emission...

Code of Federal Regulations, 2010 CFR

2010-07-01

... appendix F requirements to evaluate continuous emission monitoring systems? 60.1735 Section 60.1735... systems? Yes, the accuracy tests for your sulfur dioxide continuous emission monitoring system require you to also evaluate your oxygen (or carbon dioxide) continuous emission monitoring system. Therefore...

The Role of U2AF1 Mutations in the Pathogenesis of Myelodysplastic Syndromes

DTIC Science & Technology

2015-10-01

mutation, U2AF1(S34F), on hematopoiesis and pre-mRNA splicing in vivo, we created doxycycline-inducible U2AF1(WT) and U2AF1(S34F) transgenic mice...U2AF1(S34F) versus U2AF1(WT). Together, these results suggest that mutant U2AF1 expression contributes to the altered hematopoiesis and pre-mRNA...Spliceosome, Mouse Model, Hematopoiesis , RNA-seq, U2AF1 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME

Structural changes induced by L50P and I61T single mutations of ubiquitin affect cell cycle progression while impairing its regulatory and degradative functions in Saccharomyces cerevisiae.

PubMed

Doshi, Ankita; Sharma, Mrinal; Prabha, C Ratna

2017-06-01

Posttranslational conjugation of ubiquitin to proteins either regulates their function directly or concentration through ubiquitination dependent degradation. High degree of conservation of ubiquitin's sequence implies structural and functional importance of the conserved residues. Ubiquitin gene of Saccharomyces cerevisiae was evolved in vitro by us to study the significance of conserved residues. Present study investigates the structural changes in the protein resulting from the single mutations UbS20F, UbA46S, UbL50P, UbI61T and their functional consequences in the SUB60 strain of S. cerevisiae. Expression of UbL50P and UbI61T decreased Cdc28 protein kinase, enhanced Fus3 levels, caused dosage dependent lethality and at sublethal level produced drastic effects on stress tolerance, protein sorting, protein degradation by ubiquitin fusion degradation pathway and by lysosomes. UbS20F and UbA46S produced insignificant effects over the cells. All four mutations of ubiquitin were incorporated into polyubiquitin. However, polyubiquitination with K63 linkage decreased significantly in cells expressing UbL50P and UbI61T. Structural studies on UbL50P and UbI61T revealed distorted structure with greatly reduced α-helical and elevated β-sheet contents, while UbS20F and UbA46S show mild structural alterations. Our results on functional efficacy of ubiquitin in relation to structural integrity may be useful for designing inhibitors to investigate and modulate eukaryotic cellular dynamics. Copyright © 2017 Elsevier B.V. All rights reserved.

HFE Gene Mutations and Iron Status in 100 Healthy Polish Children.

PubMed

Kaczorowska-Hac, Barbara; Luszczyk, Marcin; Antosiewicz, Jedrzej; Ziolkowski, Wieslaw; Adamkiewicz-Drozynska, Elzbieta; Mysliwiec, Malgorzata; Milosz, Ewa; Kaczor, Jan J

2017-07-01

Iron participates in oxygen transport, energetic, metabolic, and immunologic processes. There are 2 main causes of iron overload: hereditary hemochromatosis which is a primary cause, is a metabolic disorder caused by mutations of genes that control iron metabolism and secondary hemochromatosis caused by multitransfusions, chronic hemolysis, and intake of iron rich food. The most common type of hereditary hemochromatosis is caused by HFE gene mutation. In this study, we analyzed iron metabolism in 100 healthy Polish children in relation to their HFE gene status. The wild-type HFE gene was predominant being observed in 60 children (60%). Twenty-five children (25%), presented with heterozygotic H63D mutation, and 15 children (15%), presented with other mutations (heterozygotic C282Y and S65C mutation, compound heterozygotes C282Y/S65C, C282Y/H63D, H63D homozygote). The mean concentration of iron, the level of ferritin, and transferrin saturation were statistically higher in the group of HFE variants compared with the wild-type group. H63D carriers presented with higher mean concentration of iron, ferritin levels, and transferrin saturation compared with the wild-type group. Male HFE carriers presented with higher iron concentration, transferrin saturation, and ferritin levels than females. This preliminary investigation demonstrates allelic impact on potential disease progression from childhood.

Nine known and five novel mutations in the erythroid transcription factor KLF1 gene and phenotypic expression of fetal hemoglobin in hemoglobin E disorder.

PubMed

Tepakhan, Wanicha; Yamsri, Supawadee; Sanchaisuriya, Kanokwan; Fucharoen, Goonnapa; Xu, Xiangmin; Fucharoen, Supan

2016-07-01

Hemoglobin E is the most common Hb variant found in South East Asia. Variation of Hb F expression in Hb E syndrome is associated with several genetic modifiers. We report several single nucleotide polymorphisms (SNPs), including nine known and five novel mutations of the Krüppel-like factor 1 (KLF1; an erythroid specific transcription factor) gene and determine their associations with phenotypic expression of Hb F in Hb E disorders. KLF1 mutations were examined using high resolution melting (HRM) assay and DNA sequencing in 575 homozygous Hb E, 278 heterozygous Hb E and 100 normal subjects. Fourteen mutations were mostly observed in subjects with elevated Hb F, including nine known mutations (G176AfsX179, T334R, R238H, -154 (C>T), A298P, S270W, R301H, -148 (G>A) and G335R and five novel mutations (Q217X, Q223X, Y290_S293del, K307N, and M358I). None of them, but the -148 (G>A), were observed in normal controls to have Hb F <1%. Combined KLF1 mutations with other SNPs including (G)γ-XmnI, BCL11A and HBS1L-MYB were associated with higher Hb F levels. KLF1 is therefore an important genetic factor associated with increased Hb F and in combination with other modifying factors could explain the phenotypic variation of Hb F expression in this common hemoglobinopathy. Copyright © 2016 Elsevier Inc. All rights reserved.

Effect of Mutation Order on Myeloproliferative Neoplasms

PubMed Central

Nangalia, Jyoti; Silber, Yvonne; Wedge, David C.; Grinfeld, Jacob; Baxter, E. Joanna; Massie, Charles E.; Papaemmanuil, Elli; Menon, Suraj; Godfrey, Anna L.; Dimitropoulou, Danai; Guglielmelli, Paola; Bellosillo, Beatriz; Besses, Carles; Döhner, Konstanze; Harrison, Claire N.; Vassiliou, George S.; Vannucchi, Alessandro; Campbell, Peter J.; Green, Anthony R.

2015-01-01

BACKGROUND Cancers result from the accumulation of somatic mutations, and their properties are thought to reflect the sum of these mutations. However, little is known about the effect of the order in which mutations are acquired. METHODS We determined mutation order in patients with myeloproliferative neoplasms by genotyping hematopoietic colonies or by means of next-generation sequencing. Stem cells and progenitor cells were isolated to study the effect of mutation order on mature and immature hematopoietic cells. RESULTS The age at which a patient presented with a myeloproliferative neoplasm, acquisition of JAK2 V617F homozygosity, and the balance of immature progenitors were all influenced by mutation order. As compared with patients in whom the TET2 mutation was acquired first (hereafter referred to as “TET2-first patients”), patients in whom the Janus kinase 2 (JAK2) mutation was acquired first (“JAK2-first patients”) had a greater likelihood of presenting with polycythemia vera than with essential thrombocythemia, an increased risk of thrombosis, and an increased sensitivity of JAK2-mutant progenitors to ruxolitinib in vitro. Mutation order influenced the proliferative response to JAK2 V617F and the capacity of double-mutant hematopoietic cells and progenitor cells to generate colony-forming cells. Moreover, the hematopoietic stem-and-progenitor-cell compartment was dominated by TET2 single-mutant cells in TET2-first patients but by JAK2–TET2 double-mutant cells in JAK2-first patients. Prior mutation of TET2 altered the transcriptional consequences of JAK2 V617F in a cell-intrinsic manner and prevented JAK2 V617F from up-regulating genes associated with proliferation. CONCLUSIONS The order in which JAK2 and TET2 mutations were acquired influenced clinical features, the response to targeted therapy, the biology of stem and progenitor cells, and clonal evolution in patients with myeloproliferative neoplasms. (Funded by Leukemia and Lymphoma Research

Analysis of trafficking, stability and function of human connexin 26 gap junction channels with deafness-causing mutations in the fourth transmembrane helix.

PubMed

Ambrosi, Cinzia; Walker, Amy E; Depriest, Adam D; Cone, Angela C; Lu, Connie; Badger, John; Skerrett, I Martha; Sosinsky, Gina E

2013-01-01

Human Connexin26 gene mutations cause hearing loss. These hereditary mutations are the leading cause of childhood deafness worldwide. Mutations in gap junction proteins (connexins) can impair intercellular communication by eliminating protein synthesis, mis-trafficking, or inducing channels that fail to dock or have aberrant function. We previously identified a new class of mutants that form non-functional gap junction channels and hemichannels (connexons) by disrupting packing and inter-helix interactions. Here we analyzed fourteen point mutations in the fourth transmembrane helix of connexin26 (Cx26) that cause non-syndromic hearing loss. Eight mutations caused mis-trafficking (K188R, F191L, V198M, S199F, G200R, I203K, L205P, T208P). Of the remaining six that formed gap junctions in mammalian cells, M195T and A197S formed stable hemichannels after isolation with a baculovirus/Sf9 protein purification system, while C202F, I203T, L205V and N206S formed hemichannels with varying degrees of instability. The function of all six gap junction-forming mutants was further assessed through measurement of dye coupling in mammalian cells and junctional conductance in paired Xenopus oocytes. Dye coupling between cell pairs was reduced by varying degrees for all six mutants. In homotypic oocyte pairings, only A197S induced measurable conductance. In heterotypic pairings with wild-type Cx26, five of the six mutants formed functional gap junction channels, albeit with reduced efficiency. None of the mutants displayed significant alterations in sensitivity to transjunctional voltage or induced conductive hemichannels in single oocytes. Intra-hemichannel interactions between mutant and wild-type proteins were assessed in rescue experiments using baculovirus expression in Sf9 insect cells. Of the four unstable mutations (C202F, I203T, L205V, N206S) only C202F and N206S formed stable hemichannels when co-expressed with wild-type Cx26. Stable M195T hemichannels displayed an increased

Cardiomyopathy mutations reveal variable region of myosin converter as major element of cross-bridge compliance.

PubMed

Seebohm, B; Matinmehr, F; Köhler, J; Francino, A; Navarro-Lopéz, F; Perrot, A; Ozcelik, C; McKenna, W J; Brenner, B; Kraft, T

2009-08-05

The ability of myosin to generate motile forces is based on elastic distortion of a structural element of the actomyosin complex (cross-bridge) that allows strain to develop before filament sliding. Addressing the question, which part of the actomyosin complex experiences main elastic distortion, we suggested previously that the converter domain might be the most compliant region of the myosin head domain. Here we test this proposal by studying functional effects of naturally occurring missense mutations in the beta-myosin heavy chain, 723Arg --> Gly (R723G) and 736Ile --> Thr (I736T), in comparison to 719Arg --> Trp (R719W). All three mutations are associated with hypertrophic cardiomyopathy and are located in the converter region of the myosin head domain. We determined several mechanical parameters of single skinned slow fibers isolated from Musculus soleus biopsies of hypertrophic cardiomyopathy patients and healthy controls. Major findings of this study for mutation R723G were i), a >40% increase in fiber stiffness in rigor with a 2.9-fold increase in stiffness per myosin head (S( *)(rigor R723G) = 0.84 pN/nm S( *)(rigor WT) = 0.29 pN/nm); and ii), a significant increase in force per head (F( *)(10 degrees C), 1.99 pN vs. 1.49 pN = 1.3-fold increase; F( *)(20 degrees C), 2.56 pN vs. 1.92 pN = 1.3-fold increase) as well as stiffness per head during isometric steady-state contraction (S( *)(active10 degrees C), 0.52 pN/nm vs. 0.28 pN/nm = 1.9-fold increase). Similar changes were found for mutation R719W (2.6-fold increase in S( *)(rigor); 1.8-fold increase in F( *)(10 degrees C), 1.6-fold in F( *)(20 degrees C); twofold increase in S( *)(active10 degrees C)). Changes in active cross-bridge cycling kinetics could not account for the increase in force and active stiffness. For the above estimates the previously determined fraction of mutated myosin in the biopsies was taken into account. Data for wild-type myosin of slow soleus muscle fibers support previous

Gradual Loss of ACTH Due to a Novel Mutation in LHX4: Comprehensive Mutation Screening in Japanese Patients with Congenital Hypopituitarism

PubMed Central

Takagi, Masaki; Ishii, Tomohiro; Inokuchi, Mikako; Amano, Naoko; Narumi, Satoshi; Asakura, Yumi; Muroya, Koji; Hasegawa, Yukihiro; Adachi, Masanori; Hasegawa, Tomonobu

2012-01-01

Mutations in transcription factors genes, which are well regulated spatially and temporally in the pituitary gland, result in congenital hypopituitarism (CH) in humans. The prevalence of CH attributable to transcription factor mutations appears to be rare and varies among populations. This study aimed to define the prevalence of CH in terms of nine CH-associated genes among Japanese patients. We enrolled 91 Japanese CH patients for DNA sequencing of POU1F1, PROP1, HESX1, LHX3, LHX4, SOX2, SOX3, OTX2, and GLI2. Additionally, gene copy numbers for POU1F1, PROP1, HESX1, LHX3, and LHX4 were examined by multiplex ligation-dependent probe amplification. The gene regulatory properties of mutant LHX4 proteins were characterized in vitro. We identified two novel heterozygous LHX4 mutations, namely c.249-1G>A, p.V75I, and one common POU1F1 mutation, p.R271W. The patient harboring the c.249-1G>A mutation exhibited isolated growth hormone deficiency at diagnosis and a gradual loss of ACTH, whereas the patient with the p.V75I mutation exhibited multiple pituitary hormone deficiency. In vitro experiments showed that both LHX4 mutations were associated with an impairment of the transactivation capacities of POU1F1 andαGSU, without any dominant-negative effects. The total mutation prevalence in Japanese CH patients was 3.3%. This study is the first to describe, a gradual loss of ACTH in a patient carrying an LHX4 mutation. Careful monitoring of hypothalamic–pituitary -adrenal function is recommended for CH patients with LHX4 mutations. PMID:23029363

Fabrication of artificially stacked ultrathin ZnS/MgF2 multilayer dielectric optical filters.

PubMed

Kedawat, Garima; Srivastava, Subodh; Jain, Vipin Kumar; Kumar, Pawan; Kataria, Vanjula; Agrawal, Yogyata; Gupta, Bipin Kumar; Vijay, Yogesh K

2013-06-12

We report a design and fabrication strategy for creating an artificially stacked multilayered optical filters using a thermal evaporation technique. We have selectively chosen a zinc sulphide (ZnS) lattice for the high refractive index (n = 2.35) layer and a magnesium fluoride (MgF2) lattice as the low refractive index (n = 1.38) layer. Furthermore, the microstructures of the ZnS/MgF2 multilayer films are also investigated through TEM and HRTEM imaging. The fabricated filters consist of high and low refractive 7 and 13 alternating layers, which exhibit a reflectance of 89.60% and 99%, respectively. The optical microcavity achieved an average transmittance of 85.13% within the visible range. The obtained results suggest that these filters could be an exceptional choice for next-generation antireflection coatings, high-reflection mirrors, and polarized interference filters.

SU-E-T-102: Determination of Dose Distributions and Water-Equivalence of MAGIC-F Polymer Gel for 60Co and 192Ir Brachytherapy Sources

DOE Office of Scientific and Technical Information (OSTI.GOV)

Quevedo, A; Nicolucci, P

2014-06-01

Purpose: Analyse the water-equivalence of MAGIC-f polymer gel for {sup 60}Co and {sup 192}Ir clinical brachytherapy sources, through dose distributions simulated with PENELOPE Monte Carlo code. Methods: The real geometry of {sup 60} (BEBIG, modelo Co0.A86) and {sup 192}192Ir (Varian, model GammaMed Plus) clinical brachytherapy sources were modelled on PENELOPE Monte Carlo simulation code. The most probable emission lines of photons were used for both sources: 17 emission lines for {sup 192}Ir and 12 lines for {sup 60}. The dose distributions were obtained in a cubic water or gel homogeneous phantom (30 × 30 × 30 cm{sup 3}), with themore » source positioned in the middle of the phantom. In all cases the number of simulation showers remained constant at 10{sup 9} particles. A specific material for gel was constructed in PENELOPE using weight fraction components of MAGIC-f: wH = 0,1062, wC = 0,0751, wN = 0,0139, wO = 0,8021, wS = 2,58×10{sup −6} e wCu = 5,08 × 10{sup −6}. The voxel size in the dose distributions was 0.6 mm. Dose distribution maps on the longitudinal and radial direction through the centre of the source were used to analyse the water-equivalence of MAGIC-f. Results: For the {sup 60} source, the maximum diferences in relative doses obtained in the gel and water were 0,65% and 1,90%, for radial and longitudinal direction, respectively. For {sup 192}Ir, the maximum difereces in relative doses were 0,30% and 1,05%, for radial and longitudinal direction, respectively. The materials equivalence can also be verified through the effective atomic number and density of each material: Zef-MAGIC-f = 7,07 e .MAGIC-f = 1,060 g/cm{sup 3} and Zef-water = 7,22. Conclusion: The results showed that MAGIC-f is water equivalent, consequently being suitable to simulate soft tissue, for Cobalt and Iridium energies. Hence, gel can be used as a dosimeter in clinical applications. Further investigation to its use in a clinical protocol is needed.« less

Distinct Viral and Mutational Spectrum of Endemic Burkitt Lymphoma.

PubMed

Abate, Francesco; Ambrosio, Maria Raffaella; Mundo, Lucia; Laginestra, Maria Antonella; Fuligni, Fabio; Rossi, Maura; Zairis, Sakellarios; Gazaneo, Sara; De Falco, Giulia; Lazzi, Stefano; Bellan, Cristiana; Rocca, Bruno Jim; Amato, Teresa; Marasco, Elena; Etebari, Maryam; Ogwang, Martin; Calbi, Valeria; Ndede, Isaac; Patel, Kirtika; Chumba, David; Piccaluga, Pier Paolo; Pileri, Stefano; Leoncini, Lorenzo; Rabadan, Raul

2015-10-01

Endemic Burkitt lymphoma (eBL) is primarily found in children in equatorial regions and represents the first historical example of a virus-associated human malignancy. Although Epstein-Barr virus (EBV) infection and MYC translocations are hallmarks of the disease, it is unclear whether other factors may contribute to its development. We performed RNA-Seq on 20 eBL cases from Uganda and showed that the mutational and viral landscape of eBL is more complex than previously reported. First, we found the presence of other herpesviridae family members in 8 cases (40%), in particular human herpesvirus 5 and human herpesvirus 8 and confirmed their presence by immunohistochemistry in the adjacent non-neoplastic tissue. Second, we identified a distinct latency program in EBV involving lytic genes in association with TCF3 activity. Third, by comparing the eBL mutational landscape with published data on sporadic Burkitt lymphoma (sBL), we detected lower frequencies of mutations in MYC, ID3, TCF3 and TP53, and a higher frequency of mutation in ARID1A in eBL samples. Recurrent mutations in two genes not previously associated with eBL were identified in 20% of tumors: RHOA and cyclin F (CCNF). We also observed that polyviral samples showed lower numbers of somatic mutations in common altered genes in comparison to sBL specimens, suggesting dual mechanisms of transformation, mutation versus virus driven in sBL and eBL respectively.

Distinct Viral and Mutational Spectrum of Endemic Burkitt Lymphoma

PubMed Central

Mundo, Lucia; Laginestra, Maria Antonella; Fuligni, Fabio; Rossi, Maura; Zairis, Sakellarios; Gazaneo, Sara; De Falco, Giulia; Lazzi, Stefano; Bellan, Cristiana; Rocca, Bruno Jim; Amato, Teresa; Marasco, Elena; Etebari, Maryam; Ogwang, Martin; Calbi, Valeria; Ndede, Isaac; Patel, Kirtika; Chumba, David; Piccaluga, Pier Paolo; Pileri, Stefano; Leoncini, Lorenzo; Rabadan, Raul

2015-01-01

Endemic Burkitt lymphoma (eBL) is primarily found in children in equatorial regions and represents the first historical example of a virus-associated human malignancy. Although Epstein-Barr virus (EBV) infection and MYC translocations are hallmarks of the disease, it is unclear whether other factors may contribute to its development. We performed RNA-Seq on 20 eBL cases from Uganda and showed that the mutational and viral landscape of eBL is more complex than previously reported. First, we found the presence of other herpesviridae family members in 8 cases (40%), in particular human herpesvirus 5 and human herpesvirus 8 and confirmed their presence by immunohistochemistry in the adjacent non-neoplastic tissue. Second, we identified a distinct latency program in EBV involving lytic genes in association with TCF3 activity. Third, by comparing the eBL mutational landscape with published data on sporadic Burkitt lymphoma (sBL), we detected lower frequencies of mutations in MYC, ID3, TCF3 and TP53, and a higher frequency of mutation in ARID1A in eBL samples. Recurrent mutations in two genes not previously associated with eBL were identified in 20% of tumors: RHOA and cyclin F (CCNF). We also observed that polyviral samples showed lower numbers of somatic mutations in common altered genes in comparison to sBL specimens, suggesting dual mechanisms of transformation, mutation versus virus driven in sBL and eBL respectively. PMID:26468873

CRISPR/Cas9-mediated knockout of PiSSK1 reveals essential role of S-locus F-box protein-containing SCF complexes in recognition of non-self S-RNases during cross-compatible pollination in self-incompatible Petunia inflata.

PubMed

Sun, Linhan; Kao, Teh-Hui

2018-06-01

Function of Petunia PiSSK1. Self-incompatibility (SI), an inbreeding-preventing mechanism, is regulated in Petunia inflata by the polymorphic S-locus, which houses multiple pollen-specific S-locus F-box (SLF) genes and a single pistil-specific S-RNase gene. S 2 -haplotype and S 3 -haplotype possess the same 17 polymorphic SLF genes (named SLF1 to SLF17), and each SLF protein produced in pollen is assembled into an SCF (Skp1-Cullin1-F-box) E3 ubiquitin ligase complex. A complete suite of SLF proteins is thought to collectively interact with all non-self S-RNases to mediate their ubiquitination and degradation by the 26S proteasome, allowing cross-compatible pollination. For each SCF SLF complex, the Cullin1 subunit (named PiCUL1-P) and Skp1 subunit (named PiSSK1), like the F-box protein subunits (SLFs), are pollen-specific, raising the possibility that they also evolved specifically to function in SI. Here we used CRISPR/Cas9-meditated genome editing to generate frame-shift indel mutations in PiSSK1 and examined the SI behavior of a T 0 plant (S 2 S 3 ) with biallelic mutations in the pollen genome and two progeny plants (S 2 S 2 ) each homozygous for one of the indel alleles and not carrying the Cas9-containing T-DNA. Their pollen was completely incompatible with pistils of seven otherwise-compatible S-genotypes, but fully compatible with pistils of an S 3 S 3 transgenic plant in which production of S 3 -RNase was completely suppressed by an antisense S 3 -RNase gene, and with pistils of immature flower buds, which produce little S-RNase. These results suggest that PiSSK1 specifically functions in SI and support the hypothesis that SLF-containing SCF complexes are essential for compatible pollination.

A molecular description of mutations affecting the pollen component of the Nicotiana alata S locus.

PubMed Central

Golz, J F; Su, V; Clarke, A E; Newbigin, E

1999-01-01

Mutations affecting the self-incompatibility response of Nicotiana alata were generated by irradiation. Mutants in the M1 generation were selected on the basis of pollen tube growth through an otherwise incompatible pistil. Twelve of the 18 M1 plants obtained from the mutagenesis screen were self-compatible. Eleven self-compatible plants had mutations affecting only the pollen function of the S locus (pollen-part mutants). The remaining self-compatible plant had a mutation affecting only the style function of the S locus (style-part mutant). Cytological examination of the pollen-part mutant plants revealed that 8 had an extra chromosome (2n + 1) and 3 did not. The pollen-part mutation in 7 M1 plants was followed in a series of crosses. DNA blot analysis using probes for S-RNase genes (encoding the style function of the S locus) indicated that the pollen-part mutation was associated with an extra S allele in 4 M1 plants. In 3 of these plants, the extra S allele was located on the additional chromosome. There was no evidence of an extra S allele in the 3 remaining M1 plants. The breakdown of self-incompatibility in plants with an extra S allele is discussed with reference to current models of the molecular basis of self-incompatibility. PMID:10388830

Identification of a novel functional JAK1 S646P mutation in acute lymphoblastic leukemia

PubMed Central

Hu, Liangding; Ning, Hongmei; Jiang, Min; Wang, Danhong; Liu, Tingting; Zhang, Bin; Chen, Hu

2017-01-01

The survival rate of childhood acute lymphoblastic leukemia (ALL) is approaching 90%, while the prognosis of adults remains poor due to the limited therapeutic approaches. In order to identify new targets for ALL, we performed whole-exome sequencing on four adults with B-ALL and discovered a somatic JAK1 S646P mutation. Sanger sequencing of JAK1 was conducted on 53 ALL patients, and two cases exhibited A639G and P960S mutations separately. Functional studies demonstrated that only JAK1 S646P mutation could activate multiple signaling pathways, drive cytokine-independent cell growth, and promote proliferation of malignant cells in nude mice. Moreover, a high sensitivity to the JAK1/2 inhibitor ruxolitinib was observed in S646P mutant model. Exploration in a total of 209 ALL cases showed that JAK1 mutations occur at a frequency of 10.5% in T-ALL (2/19) and 1.6% in B-ALL (3/190). Collectively, our results suggested that JAK1 S646P is an activating mutation in vitro and in vivo. JAK-STAT pathway might represent a promising therapeutic target for ALL. PMID:28410228

Identification of a novel functional JAK1 S646P mutation in acute lymphoblastic leukemia.

PubMed

Li, Qian; Li, Botao; Hu, Liangding; Ning, Hongmei; Jiang, Min; Wang, Danhong; Liu, Tingting; Zhang, Bin; Chen, Hu

2017-05-23

The survival rate of childhood acute lymphoblastic leukemia (ALL) is approaching 90%, while the prognosis of adults remains poor due to the limited therapeutic approaches. In order to identify new targets for ALL, we performed whole-exome sequencing on four adults with B-ALL and discovered a somatic JAK1 S646P mutation. Sanger sequencing of JAK1 was conducted on 53 ALL patients, and two cases exhibited A639G and P960S mutations separately. Functional studies demonstrated that only JAK1 S646P mutation could activate multiple signaling pathways, drive cytokine-independent cell growth, and promote proliferation of malignant cells in nude mice. Moreover, a high sensitivity to the JAK1/2 inhibitor ruxolitinib was observed in S646P mutant model. Exploration in a total of 209 ALL cases showed that JAK1 mutations occur at a frequency of 10.5% in T-ALL (2/19) and 1.6% in B-ALL (3/190). Collectively, our results suggested that JAK1 S646P is an activating mutation in vitro and in vivo. JAK-STAT pathway might represent a promising therapeutic target for ALL.

Analysis of human MutS homolog 2 missense mutations in patients with colorectal cancer.

PubMed

Zhang, Xiaomei; Chen, Senqing; Yu, Jun; Zhang, Yuanying; Lv, Min; Zhu, Ming

2018-05-01

Germline mutations of DNA mismatch repair gene human MutS homolog 2 ( hMSH2 ) are associated with hereditary nonpolyposis colorectal cancer (HNPCC). A total of one-third of these mutations are missense mutations. Several hMSH2 missense mutations have been identified in patients in East Asia, although their function has not been evaluated. In the present study, the role of ten hMSH2 missense mutations in the pathogenesis of colorectal cancer was examined. The hMSH2/hMSH6 protein interaction system was established using yeast two-hybrid screening. Next, the missense mutations were analyzed for their ability to affect the protein interaction of hMSH2 with its partner hMSH6. Additionally, the Sorting Intolerant from Tolerant tool was applied to predict the effects of different amino acid substitutions. The results demonstrated that certain hMSH2 mutations (L173R and C199R) caused a significant functional change in the human hMutSα complex and were identified to be pathological mutations. The Y408C, D603Y, P696L and S703Y mutations partially affected interaction and partly affected the function of hMSH2. The remaining four variants, T8M, I169V, A370T and Q419K, may be non-functional polymorphisms or could affect protein function through other molecular mechanisms. The present study evaluated the functional consequences of previously unknown missense mutations in hMSH2 , and may contribute to improved clinical diagnosis and mutation screening of HNPCC.

Group B Streptococcal Toxic Shock Syndrome and covR/S Mutations Revisited.

PubMed

Sendi, Parham; El Hay, Muad Abd; Brandt, Claudia M; Spellerberg, Barbara

2017-01-01

Gene mutations in the virulence regulator CovR/S of group A Streptococcus play a substantial role in the pathogenesis of streptococcal toxic shock syndrome. We screened 25 group B Streptococcus (GBS) isolates obtained from patients with streptococcal toxic shock syndrome and found only 1 GBS clone harboring this kind of mutation.

34 CFR 403.60 - What are the basic programs?

Code of Federal Regulations, 2012 CFR

2012-07-01

... 34 Education 3 2012-07-01 2012-07-01 false What are the basic programs? 403.60 Section 403.60... ADULT EDUCATION, DEPARTMENT OF EDUCATION STATE VOCATIONAL AND APPLIED TECHNOLOGY EDUCATION PROGRAM What... Vocational Education Programs. (f) Postsecondary and Adult Vocational Education Programs. (Authority: 20 U.S...

34 CFR 403.60 - What are the basic programs?

Code of Federal Regulations, 2013 CFR

2013-07-01

... 34 Education 3 2013-07-01 2013-07-01 false What are the basic programs? 403.60 Section 403.60... ADULT EDUCATION, DEPARTMENT OF EDUCATION STATE VOCATIONAL AND APPLIED TECHNOLOGY EDUCATION PROGRAM What... Vocational Education Programs. (f) Postsecondary and Adult Vocational Education Programs. (Authority: 20 U.S...

34 CFR 403.60 - What are the basic programs?

Code of Federal Regulations, 2011 CFR

2011-07-01

... 34 Education 3 2011-07-01 2011-07-01 false What are the basic programs? 403.60 Section 403.60... ADULT EDUCATION, DEPARTMENT OF EDUCATION STATE VOCATIONAL AND APPLIED TECHNOLOGY EDUCATION PROGRAM What... Vocational Education Programs. (f) Postsecondary and Adult Vocational Education Programs. (Authority: 20 U.S...

34 CFR 403.60 - What are the basic programs?

Code of Federal Regulations, 2014 CFR

2014-07-01

... 34 Education 3 2014-07-01 2014-07-01 false What are the basic programs? 403.60 Section 403.60... ADULT EDUCATION, DEPARTMENT OF EDUCATION STATE VOCATIONAL AND APPLIED TECHNOLOGY EDUCATION PROGRAM What... Vocational Education Programs. (f) Postsecondary and Adult Vocational Education Programs. (Authority: 20 U.S...

A Missense Mutation in the Capza3 Gene and Disruption of F-actin Organization in Spermatids of repro32 Infertile Male Mice

PubMed Central

Geyer, Christopher B.; Inselman, Amy L.; Sunman, Jeffrey A.; Bornstein, Sheila; Handel, Mary Ann; Eddy, Edward M.

2009-01-01

Males homozygous for the repro32 ENU-induced mutation produced by the Reproductive Genomics program at The Jackson Laboratory are infertile, have low epididymal sperm concentrations, and produce sperm with abnormally shaped heads and poor motility. The purpose of the present study was to identify the mutated gene in repro32 mice and to define the structural and functional changes causing infertility and the aberrant sperm phenotype. In repro32/repro32 mice, we discovered a failure to shed excess cytoplasm and disorganization of the middle piece of the flagellum at spermiation, resulting in the outer dense fibers being wrapped around the sperm head within a bag of cytoplasm. Using a candidate-gene approach, a mutation was identified in the spermatid-specific “capping protein (actin filament) muscle Z-line, alpha 3” gene (Capza3). CAPZA3 protein localization was altered in spermatids concurrent with altered localization of a unique CAPZB variant isoform and disruption of the filamentous actin (F-actin) network. These observations strongly suggest the missense mutation in Capza3 is responsible for the mutant phenotype of repro32/repro32 sperm and regulation of F-actin dynamics by a spermatogenic cell-specific CAPZ heterodimer is essential for removal of the cytoplasm and maintenance of midpiece integrity during spermiation in the mouse. PMID:19341723

Punctual mutations in 23S rRNA gene of clarithromycin-resistant Helicobacter pylori in Colombian populations

PubMed Central

Matta, Andrés Jenuer; Zambrano, Diana Carolina; Pazos, Alvaro Jairo

2018-01-01

AIM To characterize punctual mutations in 23S rRNA gene of clarithromycin-resistant Helicobacter pylori (H. pylori) and determine their association with therapeutic failure. METHODS PCR products of 23S rRNA gene V domain of 74 H. pylori isolates; 34 resistant to clarithromycin (29 from a low-risk gastric cancer (GC) population: Tumaco-Colombia, and 5 from a high-risk population: Tuquerres-Colombia) and 40 from a susceptible population (28 from Tumaco and 12 from Túquerres) were sequenced using capillary electrophoresis. The concordance between mutations of V domain 23S rRNA gene of H. pylori and therapeutic failure was determined using the Kappa coefficient and McNemar’s test was performed to determine the relationship between H. pylori mutations and clarithromycin resistance. RESULTS 23S rRNA gene from H. pylori was amplified in 56/74 isolates, of which 25 were resistant to clarithromycin (20 from Tumaco and 5 from Túquerres, respectively). In 17 resistant isolates (13 from Tumaco and 4 from Túquerres) the following mutations were found: A1593T1, A1653G2, C1770T, C1954T1, and G1827C in isolates from Tumaco, and A2144G from Túquerres. The mutations T2183C, A2144G and C2196T in H. pylori isolates resistant to clarithromycin from Colombia are reported for the first time. No association between the H. pylori mutations and in vitro clarithromycin resistance was found. However, therapeutic failure of eradication treatment was associated with mutations of 23S rRNA gene in clarithromycin-resistant H. pylori (κ = 0.71). CONCLUSION The therapeutic failure of eradication treatment in the two populations from Colombia was associated with mutations of the 23S rRNA gene in clarithromycin-resistant H. pylori. PMID:29662291

Group B Streptococcal Toxic Shock Syndrome and covR/S Mutations Revisited

PubMed Central

Sendi, Parham; el Hay, Muad Abd; Brandt, Claudia M.

2017-01-01

Gene mutations in the virulence regulator CovR/S of group A Streptococcus play a substantial role in the pathogenesis of streptococcal toxic shock syndrome. We screened 25 group B Streptococcus (GBS) isolates obtained from patients with streptococcal toxic shock syndrome and found only 1 GBS clone harboring this kind of mutation. PMID:27983484

Discovery of Point Mutations in the Voltage-Gated Sodium Channel from African Aedes aegypti Populations: Potential Phylogenetic Reasons for Gene Introgression

PubMed Central

Muranami, Yuto; Kawashima, Emiko; Osei, Joseph H. N.; Sakyi, Kojo Yirenkyi; Dadzie, Samuel; de Souza, Dziedzom K.; Appawu, Maxwell; Ohta, Nobuo; Minakawa, Noboru

2016-01-01

Background Yellow fever is endemic in some countries in Africa, and Aedes aegpyti is one of the most important vectors implicated in the outbreak. The mapping of the nation-wide distribution and the detection of insecticide resistance of vector mosquitoes will provide the beneficial information for forecasting of dengue and yellow fever outbreaks and effective control measures. Methodology/Principal Findings High resistance to DDT was observed in all mosquito colonies collected in Ghana. The resistance and the possible existence of resistance or tolerance to permethrin were suspected in some colonies. High frequencies of point mutations at the voltage-gated sodium channel (F1534C) and one heterozygote of the other mutation (V1016I) were detected, and this is the first detection on the African continent. The frequency of F1534C allele and the ratio of F1534C homozygotes in Ae. aegypti aegypti (Aaa) were significantly higher than those in Ae. aegypti formosus (Aaf). We could detect the two types of introns between exon 20 and 21, and the F1534C mutations were strongly linked with one type of intron, which was commonly found in South East Asian and South and Central American countries, suggesting the possibility that this mutation was introduced from other continents or convergently selected after the introgression of Aaa genes from the above area. Conclusions/Significance The worldwide eradication programs in 1940s and 1950s might have caused high selection pressure on the mosquito populations and expanded the distribution of insecticide-resistant Ae. aegypti populations. Selection of the F1534C point mutation could be hypothesized to have taken place during this period. The selection of the resistant population of Ae. aegypti with the point mutation of F1534C, and the worldwide transportation of vector mosquitoes in accordance with human activity such as trading of used tires, might result in the widespread distribution of F1534C point mutation in tropical countries

Discovery of Point Mutations in the Voltage-Gated Sodium Channel from African Aedes aegypti Populations: Potential Phylogenetic Reasons for Gene Introgression.

PubMed

Kawada, Hitoshi; Higa, Yukiko; Futami, Kyoko; Muranami, Yuto; Kawashima, Emiko; Osei, Joseph H N; Sakyi, Kojo Yirenkyi; Dadzie, Samuel; de Souza, Dziedzom K; Appawu, Maxwell; Ohta, Nobuo; Suzuki, Takashi; Minakawa, Noboru

2016-06-01

Yellow fever is endemic in some countries in Africa, and Aedes aegpyti is one of the most important vectors implicated in the outbreak. The mapping of the nation-wide distribution and the detection of insecticide resistance of vector mosquitoes will provide the beneficial information for forecasting of dengue and yellow fever outbreaks and effective control measures. High resistance to DDT was observed in all mosquito colonies collected in Ghana. The resistance and the possible existence of resistance or tolerance to permethrin were suspected in some colonies. High frequencies of point mutations at the voltage-gated sodium channel (F1534C) and one heterozygote of the other mutation (V1016I) were detected, and this is the first detection on the African continent. The frequency of F1534C allele and the ratio of F1534C homozygotes in Ae. aegypti aegypti (Aaa) were significantly higher than those in Ae. aegypti formosus (Aaf). We could detect the two types of introns between exon 20 and 21, and the F1534C mutations were strongly linked with one type of intron, which was commonly found in South East Asian and South and Central American countries, suggesting the possibility that this mutation was introduced from other continents or convergently selected after the introgression of Aaa genes from the above area. The worldwide eradication programs in 1940s and 1950s might have caused high selection pressure on the mosquito populations and expanded the distribution of insecticide-resistant Ae. aegypti populations. Selection of the F1534C point mutation could be hypothesized to have taken place during this period. The selection of the resistant population of Ae. aegypti with the point mutation of F1534C, and the worldwide transportation of vector mosquitoes in accordance with human activity such as trading of used tires, might result in the widespread distribution of F1534C point mutation in tropical countries.

[MPLW515L point mutation in patients with myeloproliferative disease].

PubMed

Xia, Jun; Xu, Wei; Zhang, Su-Jiang; Fan, Lei; Qiao, Chun; Li, Jian-Yong

2008-12-01

In order to investigate the frequency of MPLW515L and JAK2V617F point mutations of the patients with myeloproliferative disease (MPD) in Nanjing area, MPLW515L and JAK2V617F point mutations were simultaneously detected by alleles specific polymerase chain reaction (AS-PCR) and sequencing in 190 MPD patients. The results showed that MPLW515L point mutation was detected in 1 out of 102 essential thrombocythemia (ET) patients (1.0%) and was not detected in 32 polycythemia vera (PV) patients, 13 idiopathic myelofibrosis (IMF) patients, 43 chronic myelogenous leukemia (CML) patients. JAK2V617F point mutation was detected in 20 out of 32 PV patients (62.5%), 43 out of 102 ET patients (42.2%), 5 out of 13 IMF patients (38.5%), and was not detected in 43 CML patients. It is concluded that MPLW515L point mutation exists in ET patient, but is not found in PV, IMF and CML. JAK2V617F point mutation exists in PV, ET and IMF, but not in CML.

Role of Polymerase Gamma Mutations in Breast Tumorigenesis

DTIC Science & Technology

2010-08-01

2005;309:481–484. [PubMed: 16020738] 47. Alonso A, Martin P, Albarran C, Aquilera B, Garcia O, Guzman A, et al. Detection of somatic mutations in the...Hum. Genet 2005;76:1081–1086. [PubMed: 15877282] 30. Spinazzola A, Viscomi C, Fernandez -Vizarra E, Carrara F, D’Adamo P, Calvo S, et al. MPV17

Chromobacterium violaceum ω-transaminase variant Trp60Cys shows increased specificity for (S)-1-phenylethylamine and 4'-substituted acetophenones, and follows Swain-Lupton parameterisation.

PubMed

Cassimjee, Karim Engelmark; Humble, Maria Svedendahl; Land, Henrik; Abedi, Vahak; Berglund, Per

2012-07-28

For biocatalytic production of pharmaceutically important chiral amines the ω-transaminase enzymes have proven useful. Engineering of these enzymes has to some extent been accomplished by rational design, but mostly by directed evolution. By use of a homology model a key point mutation in Chromobacterium violaceum ω-transaminase was found upon comparison with engineered variants from homologous enzymes. The variant Trp60Cys gave increased specificity for (S)-1-phenylethylamine (29-fold) and 4'-substituted acetophenones (∼5-fold). To further study the effect of the mutation the reaction rates were Swain-Lupton parameterised. On comparison with the wild type, reactions of the variant showed increased resonance dependence; this observation together with changed pH optimum and cofactor dependence suggests an altered reaction mechanism.

Jack Michael's Musings on the 60th Anniversary of Skinner's "Verbal Behavior"

ERIC Educational Resources Information Center

Esch, Barbara E.; Esch, John W.; Palmer, David C.

2017-01-01

When the B. F. Skinner Foundation reprinted Skinner's "Verbal Behavior" in 1992, Jack Michael wrote one of its two forewords, a detailed outline of the book's purpose and scope. On the 60th anniversary of the first publication (1957) of "Verbal Behavior", Jack reflects on the book's impact and its importance to the…

Identification of a novel heterozygous missense mutation in the CACNA1F gene in a chinese family with retinitis pigmentosa by next generation sequencing.

PubMed

Zhou, Qi; Cheng, Jingliang; Yang, Weichan; Tania, Mousumi; Wang, Hui; Khan, Md Asaduzzaman; Duan, Chengxia; Zhu, Li; Chen, Rui; Lv, Hongbin; Fu, Junjiang

2015-01-01

Retinitis pigmentosa (RP) is an inherited retinal degenerative disease, which is clinically and genetically heterogeneous, and the inheritance pattern is complex. In this study, we have intended to study the possible association of certain genes with X-linked RP (XLRP) in a Chinese family. A Chinese family with RP was recruited, and a total of seven individuals were enrolled in this genetic study. Genomic DNA was isolated from peripheral leukocytes, and used for the next generation sequencing (NGS). The affected individual presented the clinical signs of XLRP. A heterozygous missense mutation (c.1555C>T, p.R519W) was identified by NGS in exon 13 of the CACNA1F gene on X chromosome, and was confirmed by Sanger sequencing. It showed perfect cosegregation with the disease in the family. The mutation at this position in the CACNA1F gene of RP was found novel by database searching. By using NGS, we have found a novel heterozygous missense mutation (c.1555C>T, p.R519W) in CACNA1F gene, which is probably associated with XLRP. The findings might provide new insights into the cause and diagnosis of RP, and have implications for genetic counseling and clinical management in this family.

Identification of a Novel Heterozygous Missense Mutation in the CACNA1F Gene in a Chinese Family with Retinitis Pigmentosa by Next Generation Sequencing

PubMed Central

Tania, Mousumi; Wang, Hui; Khan, Md. Asaduzzaman; Duan, Chengxia; Zhu, Li; Chen, Rui; Lv, Hongbin

2015-01-01

Background. Retinitis pigmentosa (RP) is an inherited retinal degenerative disease, which is clinically and genetically heterogeneous, and the inheritance pattern is complex. In this study, we have intended to study the possible association of certain genes with X-linked RP (XLRP) in a Chinese family. Methods. A Chinese family with RP was recruited, and a total of seven individuals were enrolled in this genetic study. Genomic DNA was isolated from peripheral leukocytes, and used for the next generation sequencing (NGS). Results. The affected individual presented the clinical signs of XLRP. A heterozygous missense mutation (c.1555C>T, p.R519W) was identified by NGS in exon 13 of the CACNA1F gene on X chromosome, and was confirmed by Sanger sequencing. It showed perfect cosegregation with the disease in the family. The mutation at this position in the CACNA1F gene of RP was found novel by database searching. Conclusion. By using NGS, we have found a novel heterozygous missense mutation (c.1555C>T, p.R519W) in CACNA1F gene, which is probably associated with XLRP. The findings might provide new insights into the cause and diagnosis of RP, and have implications for genetic counseling and clinical management in this family. PMID:26075273

Detection of MPL mutations by a novel allele-specific PCR-based strategy.

PubMed

Furtado, Larissa V; Weigelin, Helmut C; Elenitoba-Johnson, Kojo S J; Betz, Bryan L

2013-11-01

MPL mutation testing is recommended in patients with suspected primary myelofibrosis or essential thrombocythemia who lack the JAK2 V617F mutation. MPL mutations can occur at allelic levels below 15%, which may escape detection by commonly used mutation screening methods such as Sanger sequencing. We developed a novel multiplexed allele-specific PCR assay capable of detecting most recurrent MPL exon 10 mutations associated with primary myelofibrosis and essential thrombocythemia (W515L, W515K, W515A, and S505N) down to a sensitivity of 2.5% mutant allele. Test results were reviewed from 15 reference cases and 1380 consecutive specimens referred to our laboratory for testing. Assay performance was compared to Sanger sequencing across a series of 58 specimens with MPL mutations. Positive cases consisted of 45 with W515L, 6 with S505N, 5 with W515K, 1 with W515A, and 1 with both W515L and S505N. Seven cases had mutations below 5% that were undetected by Sanger sequencing. Ten additional cases had mutation levels between 5% and 15% that were not consistently detected by sequencing. All results were easily interpreted in the allele-specific test. This assay offers a sensitive and reliable solution for MPL mutation testing. Sanger sequencing appears insufficiently sensitive for robust MPL mutation detection. Our data also suggest the relative frequency of S505N mutations may be underestimated, highlighting the necessity for inclusion of this mutation in MPL test platforms. Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Recent progress in the genetics of motor neuron disease.

PubMed

Finsterer, Josef; Burgunder, Jean-Marc

2014-02-01

Genetic background and pathogenesis of motor neuron diseases (MNDs) have been increasingly elucidated over recent years. To give an overview about publications during the last year concerning the genetic background and phenotypic manifestations of MNDs, such as familial or sporadic amyotrophic lateral sclerosis (fALS, sALS), spinal muscular atrophies (SMA), bulbospinal muscular atrophy (BSMA), and unclassified MNDs. Pubmed search for literature about ALS, SMA, and BSMA for the period 10/2012 to 9/2013. An increasing number of mutated genes is recognised in fALS but also sALS patients. Genes mutated in sALS include C9orf72, SOD1, TARDBP, FUS, UBQL2, SQSTM1, DCTN1, and UNC13A. Juvenile (onset <20y) and adult ALS (early onset 20-60y, late onset >60y) are differentiated. Juvenile fALS is most frequently caused by mutations in ALS2, SETX, spatacsin, or Sigmar1 and adult fALS by mutations in C9orf72, SOD1, TARDBP, and FUS. Onset, phenotype, progression, and outcome of ALS are variable between different mutations, different genes, and different countries. Differentiation between sALS and fALS cases becomes artificial. Further progress has been made over the last year in the clarification and understanding of the aetiology and pathogenesis of MNDs. However, further effort is needed to answer the many remaining questions. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Prevalence of etravirine-associated mutations in clinical samples with resistance to nevirapine and efavirenz.

PubMed

Llibre, J M; Santos, J R; Puig, T; Moltó, J; Ruiz, L; Paredes, R; Clotet, B

2008-11-01

To evaluate the expected activity of etravirine in clinical samples, according to mutational patterns associated with decreased virological response (VR). We identified 1586 routine clinical samples with resistance-associated mutations (RAMs) to nevirapine and efavirenz (K103N 60%, Y181C 37%, G190A 27%, V108I 13%). Concerning in vitro identified etravirine mutations, samples with F227C, Y181I, M230L or L100I plus K103N plus Y181C were considered highly resistant. Samples with two RAMs plus Y181C or V179D or K101E or Y188L were considered intermediate. The prevalence of 13 RAMs recently associated with decreased VR to etravirine in the DUET clinical trials was also investigated. Most samples (69%) harboured more than one IAS-USA RAM to first-generation non-nucleoside reverse transcriptase inhibitors (NNRTIs): 42% harboured two RAMs, 21% three RAMs and 6% four or more RAMs. The prevalence of 13 specific etravirine RAMs was V179F 0.12%, G190S 3.9%, Y181V 0.1%, V106I 2.6%, V179D 1.6%, K101P 2.0%, K101E 10.1%, Y181C 36.9%, A98G 5.9%, V90I 6.9%, Y181I 3.6%, G190A 27% and L100I 9.1%. The five RAMs with the most impact on VR (V179F/D, G190S, Y181V and V106I) occurred less often. Overall, 8.2% of the samples had three or more etravirine RAMs and only 1.1% had four or more. In addition, patterns of RAMs previously associated with intermediate etravirine resistance were present in 26.2% of the samples, whereas 4.85% displayed patterns of high-degree resistance. For RAMs associated with decreased VR, etravirine resistance in routine clinical samples was lower than previously reported. High-degree resistance was uncommon, even in patients with resistance to first-generation NNRTIs, whereas low-to-intermediate etravirine resistance was more common.

[Genetic analysis of the mutations in HIV-1 infected population in Ecuador].

PubMed

González-González, Manuel; Correa-Sierra, Consuelo; Hermida-Álava, Katherine; Machado-Díaz, Ana; Gómez-Andrade, L Fernando; Castillo-Segovia, Martha; Pérez-Santos, C Lissette; Kourí-Cardellá, Vivian

Background The international recommendations of antiretroviral treatment include resistance tests to guide the treatment regimen in each patient, which is not available on a regular basis in Ecuador. Aim To describe mutations that confer resistance to antiretrovirals in a population of Ecuadorian patients. Methods Plasma samples from 101 HIV-1 patients with failure to antiretroviral therapy, divided into 15 children and 86 adults, were studied with the GS Junior (Roche) and the sequences were analyzed with the DeepChek program. Results The most frequent mutations were M184V/I, K101E/P/H, K103N/S, D30N, M46L/I, I54L/M, V82T/F/A/S/L and L90M in adults and F77L, K103N/S, M46L/I, V82T/F/A/S/L and L90M in children. High resistance to non-nucleoside reverse transcriptase (RT) inhibitors in minority viral populations of adults and children (34.9% and 70%) was detected; in children both viral populations (majority and minority viral populations) (> 45%) were protease inhibitor resistant. Patients who had a greater number of therapeutic regimens had higher levels of resistance to antiretrovirals. Most of the samples were subtype B in the TR and protease region, and CRF25_cpx in integrase. Conclusions Mutations and resistance to antiretrovirals are shown in a population of Ecuadorian patients with HIV-1. These results will make it possible to issue a warning to health authorities about the need for resistance studies.

46 CFR 111.105-31 - Flammable or combustible cargo with a flashpoint below 60 °C (140 °F), carriers of liquid-sulphur...

Code of Federal Regulations, 2014 CFR

2014-10-01

... 60 °C (140 °F), carriers of liquid-sulphur or inorganic acid. 111.105-31 Section 111.105-31... below 60 °C (140 °F), carriers of liquid-sulphur or inorganic acid. (a) Applicability. Each vessel that...) or liquid sulphur cargo, or inorganic acid cargo must meet the requirements of this section, except...

46 CFR 111.105-31 - Flammable or combustible cargo with a flashpoint below 60 °C (140 °F), carriers of liquid-sulphur...

Code of Federal Regulations, 2013 CFR

2013-10-01

... 60 °C (140 °F), carriers of liquid-sulphur or inorganic acid. 111.105-31 Section 111.105-31... below 60 °C (140 °F), carriers of liquid-sulphur or inorganic acid. (a) Applicability. Each vessel that...) or liquid sulphur cargo, or inorganic acid cargo must meet the requirements of this section, except...

The novel complex allele [A238V;F508del] of the CFTR gene: clinical phenotype and possible implications for cystic fibrosis etiological therapies.

PubMed

Diana, Anna; Polizzi, Angela Maria; Santostasi, Teresa; Ratclif, Luigi; Pantaleo, Maria Giuseppina; Leonetti, Giuseppina; Iusco, Danila Rosa; Gallo, Crescenzio; Conese, Massimo; Manca, Antonio

2016-06-01

Few mutations in cis have been annotated for F508del homozygous patients. Southern Italy patients who at a first analysis appeared homozygous for the F508del mutation (n=63) or compound heterozygous for the F508del and another mutation in the cystic fibrosis transmembrane conductance regulator gene (n=155) were searched for the A238V mutation in exon 6. The allelic frequency of the complex allele [A238V;F508del] was 0.04. When the whole data set was used (comprised also of 56 F508del/F508del and 34 F508del/other mutation controls), no differences reached the statistical significance in the clinical parameters, except chloride concentrations which were lower in [A238V;F508del]/other mutation compared with F508del/other mutation (P=0.03). The two study groups presented less complications than the control groups. Within the minimal data set (34 F508del/F508del, 27 F508del/other mutation, 4 [A238V;F508del]/F508del cases and 5 [A238V;F508del]/other mutation cases); that is, presenting all the variables in each patient, forced expiratory volume in 1 s and forced vital capacity presented a trend to lower levels in the study groups in comparison with the F508del/F508del group, and C-reactive protein approximated statistically significant higher levels in the [A238V;F508del]/other mutation as compared with F508del/F508del patients (P=0.09). The analysis of statistical dependence among the variables showed a significant anticorrelation between chloride and body mass index in the [A238V;F508del]/other mutation group. In conclusion, the complex allele [A238V;F508del] seems to be associated with less general complications than in the control groups, on the other hand possibly giving a worse pulmonary phenotype and higher systemic/local inflammatory response. These findings have implications for the correct recruitment and clinical response of F508del patients in the clinical trials testing the new etiological drugs for cystic fibrosis.

5-Lipoxygenase-activating protein rescues activity of 5-lipoxygenase mutations that delay nuclear membrane association and disrupt product formation.

PubMed

Gerstmeier, Jana; Newcomer, Marcia E; Dennhardt, Sophie; Romp, Erik; Fischer, Jana; Werz, Oliver; Garscha, Ulrike

2016-05-01

Leukotrienes (LTs) are proinflammatory lipid mediators formed from arachidonic acid in a 2-step reaction catalyzed by 5-lipoxygenase (5-LOX) requiring the formation of 5-HPETE [5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid] and its subsequent transformation to LTA4 5-LOX is thought to receive arachidonic acid from the nuclear membrane-embedded 5-LOX-activating protein (FLAP). The crystal structure of 5-LOX revealed an active site concealed by F177 and Y181 (FY cork). We examined the influence of the FY cork on 5-LOX activity and membrane binding in HEK293 cells in the absence and presence of FLAP. Uncapping the 5-LOX active site by mutation of F177 and/or Y181 to alanine (5-LOX-F177A, 5-LOX-Y181A, 5-LOX-F177/Y181A) resulted in delayed and diminished 5-LOX membrane association in A23187-stimulated cells. For 5-LOX-F177A and 5-LOX-F177/Y181A, formation of 5-LOX products was dramatically reduced relative to 5-LOX-wild type (wt). Strikingly, coexpression of FLAP in A23187-activated HEK293 cells effectively restored formation of 5-H(p)ETE (5-hydroxy- and 5-peroxy-6-trans-8,11,14-cis-eicosatetraenoic acid) by these same 5-LOX mutants (≈60-70% 5-LOX-wt levels) but not of LTA4 hydrolysis products. Yet 5-LOX-Y181A generated 5-H(p)ETE at levels comparable to 5-LOX-wt but reduced LTA4 hydrolysis products. Coexpression of FLAP partially restored LTA4 hydrolysis product formation by 5-LOX-Y181A. Together, the data suggest that the concealed FY cork impacts membrane association and that FLAP may help shield an uncapped active site.-Gerstmeier, J., Newcomer, M. E., Dennhardt, S., Romp, E., Fischer, J., Werz, O., Garscha, U. 5-Lipoxygenase-activating protein rescues activity of 5-lipoxygenase mutations that delay nuclear membrane association and disrupt product formation. © FASEB.

Synthesis and Monkey-PET Study of (R)- and (S)-18F-Labeled 2-Arylbenzoheterocyclic Derivatives as Amyloid Probes with Distinctive in Vivo Kinetics.

PubMed

Yang, Yanping; Wang, Xuedan; Yang, Hui; Fu, Hualong; Zhang, Jinming; Zhang, Xiaojun; Dai, Jiapei; Zhang, Zhiyong; Lin, Chunping; Guo, Yuzhi; Cui, Mengchao

2016-11-07

This study describes an effective strategy to improve pharmacokinetics of Aβ imaging agents, offering a novel class of (R)- and (S)- 18 F-labeled 2-arylbenzoheterocyclic derivatives which bear an additional chiral hydroxyl group on the side chain. These ligands displayed binding abilities toward Aβ aggregates with K i values ranging from 3.2 to 195.6 nM. Chirality-related discrepancy was observed in biodistribution, and (S)-2-phenylbenzoxazole enantiomers exhibited vastly improved brain clearance with washout ratios higher than 20. Notably, (S)-[ 18 F]28 possessed high binding potency (K i = 7.6 nM) and exceptional brain kinetics (9.46% ID/g at 2 min, brain 2min /brain 60min = 27.8) that is superior to well-established [ 18 F]AV45. The excellent pharmacokinetics and low nonspecific binding of (S)-[ 18 F]28 were testified by dynamic PET/CT scans in monkey brains. In addition, (S)-[ 18 F]28 clearly labeled Aβ plaques both in vitro and ex vivo. These results might qualify (S)-[ 18 F]28 to detect Aβ plaques with high signal-to-noise ratio.

Punctual mutations in 23S rRNA gene of clarithromycin-resistant Helicobacter pylori in Colombian populations.

PubMed

Matta, Andrés Jenuer; Zambrano, Diana Carolina; Pazos, Alvaro Jairo

2018-04-14

To characterize punctual mutations in 23S rRNA gene of clarithromycin-resistant Helicobacter pylori ( H. pylori ) and determine their association with therapeutic failure. PCR products of 23S rRNA gene V domain of 74 H. pylori isolates; 34 resistant to clarithromycin (29 from a low-risk gastric cancer (GC) population: Tumaco-Colombia, and 5 from a high-risk population: Tuquerres-Colombia) and 40 from a susceptible population (28 from Tumaco and 12 from Túquerres) were sequenced using capillary electrophoresis. The concordance between mutations of V domain 23S rRNA gene of H. pylori and therapeutic failure was determined using the Kappa coefficient and McNemar's test was performed to determine the relationship between H. pylori mutations and clarithromycin resistance. 23S rRNA gene from H. pylori was amplified in 56/74 isolates, of which 25 were resistant to clarithromycin (20 from Tumaco and 5 from Túquerres, respectively). In 17 resistant isolates (13 from Tumaco and 4 from Túquerres) the following mutations were found: A1593T1, A1653G2, C1770T, C1954T1, and G1827C in isolates from Tumaco, and A2144G from Túquerres. The mutations T2183C, A2144G and C2196T in H. pylori isolates resistant to clarithromycin from Colombia are reported for the first time. No association between the H. pylori mutations and in vitro clarithromycin resistance was found. However, therapeutic failure of eradication treatment was associated with mutations of 23S rRNA gene in clarithromycin-resistant H. pylori ( κ = 0.71). The therapeutic failure of eradication treatment in the two populations from Colombia was associated with mutations of the 23S rRNA gene in clarithromycin-resistant H. pylori .

Identification and functional analysis of CBLB mutations in type 1 diabetes.

PubMed

Yokoi, Norihide; Fujiwara, Yuuka; Wang, He-Yao; Kitao, Mai; Hayashi, Chihiro; Someya, Tomohiro; Kanamori, Masao; Oiso, Yutaka; Tajima, Naoko; Yamada, Yuichiro; Seino, Yutaka; Ikegami, Hiroshi; Seino, Susumu

2008-03-28

Casitas B-lineage lymphoma b (Cblb) is a negative regulator of T-cell activation and dysfunction of Cblb in rats and mice results in autoimmunity. In particular, a nonsense mutation in Cblb has been identified in a rat model of autoimmune type 1 diabetes. To clarify the possible involvement of CBLB mutation in type 1 diabetes in humans, we performed mutation screening of CBLB and characterized functional properties of the mutations in Japanese subjects. Six missense mutations (A155V, F328L, N466D, K837R, T882A, and R968L) were identified in one diabetic subject each, excepting N466D. Of these mutations, F328L showed impaired suppression of T-cell activation and was a loss-of-function mutation. These data suggest that the F328L mutation is involved in the development of autoimmune diseases including type 1 diabetes, and also provide insight into the structure-function relationship of CBLB protein.

Biophysical Properties of 9 KCNQ1 Mutations Associated with Long QT Syndrome (LQTS)

PubMed Central

Yang, Tao; Chung, Seo-Kyung; Zhang, Wei; Mullins, Jonathan G.L.; McCulley, Caroline H.; Crawford, Jackie; MacCormick, Judith; Eddy, Carey-Anne; Shelling, Andrew N.; French, John K.; Yang, Ping; Skinner, Jonathan R.; Roden, Dan M.; Rees, Mark I.

2009-01-01

Background Inherited long QT syndrome (LQTS) is characterized by prolonged QT interval on the EKG, syncope and sudden death due to ventricular arrhythmia. Causative mutations occur mostly in cardiac potassium and sodium channel subunit genes. Confidence in mutation pathogenicity is usually reached through family genotype-phenotype tracking, control population studies, molecular modelling and phylogenetic alignments, however, biophysical testing offers a higher degree of validating evidence. Methods and Results By using in-vitro electrophysiological testing of transfected mutant and wild-type LQTS constructs into Chinese Hamster Ovary cells, we investigated the biophysical properties of 9 KCNQ1 missense mutations (A46T, T265I, F269S, A302V, G316E, F339S, R360G, H455Y, and S546L) identified in a New Zealand based LQTS screening programme. We demonstrate through electrophysiology and molecular modeling that seven of the missense mutations have profound pathological dominant negative loss-of-function properties confirming their likely disease-causing nature. This supports the use of these mutations in diagnostic family screening. Two mutations (A46T, T265I) show suggestive evidence of pathogenicity within the experimental limits of biophysical testing, indicating that these variants are disease-causing via delayed or fast activation kinetics. Further investigation of the A46T family has revealed an inconsistent co-segregation of the variant with the clinical phenotype. Conclusions Electrophysiological characterisation should be used to validate LQTS pathogenicity of novel missense channelopathies. When such results are inconclusive, great care should be taken with genetic counselling and screening of such families, and alternative disease causing mechanisms should be considered. PMID:19808498

Structure and function of human Naa60 (NatF), a Golgi-localized bi-functional acetyltransferase

DOE PAGES

Chen, Ji-Yun; Liu, Liang; Cao, Chun-Ling; ...

2016-08-23

N-terminal acetylation (Nt-acetylation), carried out by N-terminal acetyltransferases (NATs), is a conserved and primary modification of nascent peptide chains. Naa60 (also named NatF) is a recently identified NAT found only in multicellular eukaryotes. This protein was shown to locate on the Golgi apparatus and mainly catalyze the Nt-acetylation of transmembrane proteins, and it also harbors lysine Nε -acetyltransferase (KAT) activity to catalyze the acetylation of lysine ε-amine. Here, we report the crystal structures of human Naa60 (hNaa60) in complex with Acetyl-Coenzyme A (Ac-CoA) or Coenzyme A (CoA). The hNaa60 protein contains an amphipathic helix following its GNAT domain that maymore » contribute to Golgi localization of hNaa60, and the β7-β8 hairpin adopted different conformations in the hNaa60(1-242) and hNaa60(1-199) crystal structures. Remarkably, we found that the side-chain of Phe 34 can influence the position of the coenzyme, indicating a new regulatory mechanism involving enzyme, co-factor and substrates interactions. Moreover, structural comparison and biochemical studies indicated that Tyr 97 and His 138 are key residues for catalytic reaction and that a non-conserved β3-β4 long loop participates in the regulation of hNaa60 activity.« less

Two novel CHN1 mutations in two families with Duane’s retraction syndrome

PubMed Central

Chan, Wai-Man; Miyake, Noriko; Zhu-Tam, Lily; Andrews, Caroline; Engle, Elizabeth C.

2012-01-01

Objective To determine the genetic cause of Duane’s retraction syndrome (DRS) in two families segregating DRS as an autosomal dominant trait. Method Members of two unrelated pedigrees were enrolled in an ongoing genetic study. Linkage analysis was performed using fluorescent microsatellite markers flanking the CHN1 locus. Probands and family members were screened for CHN1 mutations. Results Of the six clinically affected individuals in the two pedigrees, three have bilateral and three have unilateral DRS. Both pedigrees are consistent with linkage to the DURS2 locus, one with complete and one with incomplete penetrance. Sequence analysis revealed the pedigrees segregate novel heterozygous missense CHN1 mutations, c.422C>T and c.754C>T, predicted to result in α2-chimaerin amino acid substitutions P141L and P252S, respectively. Conclusion Genetic analysis of two pedigrees segregating nonsyndromic DRS reveals two novel mutations in CHN1, bringing the number of DRS pedigrees know to harbor CHN1 mutations, and the number of unique CHN1 mutations, from seven to nine. Both mutations identified in this study alter residues that participate in intramolecular interactions that stabilize the inactive, closed conformation of α2-chimerin, and thus are predicted to result in its hyper-activation. Moreover, amino acid residue P252 was altered to a different residue in a previously reported DRS pedigree; thus, this is the first report of two CHN1 mutations altering the same residue, further supporting a gain-of-function etiology. Clinical Relevance Members of families segregating DRS as an autosomal dominant trait should be screened for mutations in the CHN1 gene, enhancing genetic counseling and permitting earlier diagnosis. PMID:21555619

Knockdown resistance (kdr)-like mutations in the voltage-gated sodium channel of a malaria vector Anopheles stephensi and PCR assays for their detection.

PubMed

Singh, Om P; Dykes, Cherry L; Lather, Manila; Agrawal, Om P; Adak, Tridibes

2011-03-14

Knockdown resistance (kdr) in insects, resulting from mutation(s) in the voltage-gated sodium channel (vgsc) gene is one of the mechanisms of resistance against DDT and pyrethroid-group of insecticides. The most common mutation(s) associated with knockdown resistance in insects, including anophelines, has been reported to be present at residue Leu1014 in the IIS6 transmembrane segment of the vgsc gene. This study reports the presence of two alternative kdr-like mutations, L1014S and L1014F, at this residue in a major malaria vector Anopheles stephensi and describes new PCR assays for their detection. Part of the vgsc (IIS4-S5 linker-to-IIS6 transmembrane segment) of An. stephensi collected from Alwar (Rajasthan, India) was PCR-amplified from genomic DNA, sequenced and analysed for the presence of deduced amino acid substitution(s). Analysis of DNA sequences revealed the presence of two alternative non-synonymous point mutations at L1014 residue in the IIS6 transmembrane segment of vgsc, i.e., T>C mutation on the second position and A>T mutation on the third position of the codon, leading to Leu (TTA)-to-Ser (TCA) and -Phe (TTT) amino acid substitutions, respectively. Polymerase chain reaction (PCR) assays were developed for identification of each of these two point mutations. Genotyping of An. stephensi mosquitoes from Alwar by PCR assays revealed the presence of both mutations, with a high frequency of L1014S. The PCR assays developed for detection of the kdr mutations were specific as confirmed by DNA sequencing of PCR-genotyped samples. Two alternative kdr-like mutations, L1014S and L1014F, were detected in An. stephensi with a high allelic frequency of L1014S. The occurrence of L1014S is being reported for the first time in An. stephensi. Two specific PCR assays were developed for detection of two kdr-like mutations in An. stephensi.

Screening for Novel Germline Rare Mutations Associated with Aggressive Prostate Cancer

DTIC Science & Technology

2014-10-01

Schmidt S., Peshkin L., et al. A method and server for predicting damaging missense mutations. Nat Methods. 7, 248-249 (2010). Akbari MR, Trachtenberg...Inst. 2012 Aug 1;104(16):1260-2. Epub 2012 Jul 9. Castro E. G.C.L., Olmos D., et al. Correlation of germ-line BRCA2 mutations with aggressive...prostate cancer. Clin Cancer Res. 16, 2115-2121 (2010). 20    Hammer GE, Gonzalez F, Champsaur M, Cado D, Shastri N. The aminopeptidase ERAAP shapes the

Consequences of the pathogenic T9176C mutation of human mitochondrial DNA on yeast mitochondrial ATP synthase

PubMed Central

Kucharczyk, Roza; Ezkurdia, Nahia; Couplan, Elodie; Procaccio, Vincent; Ackerman, Sharon H.; Blondel, Marc; di Rago, Jean-Paul

2010-01-01

Summary Several human neurological disorders have been associated with various mutations affecting mitochondrial enzymes involved in cellular ATP production. One of these mutations, T9176C in the mitochondrial DNA (mtDNA), changes a highly conserved leucine residue into proline at position 217 of the mitochondrially encoded Atp6p (or a) subunit of the F1FO-ATP synthase. The consequences of this mutation on the mitochondrial ATP synthase are still poorly defined. To gain insight into the primary pathogenic mechanisms induced by T9176C, we have investigated the consequences of this mutation on the ATP synthase of yeast where Atp6p is also encoded by the mtDNA. In vitro, yeast atp6-T9176C mitochondria showed a 30% decrease in the rate of ATP synthesis. When forcing the F1FO complex to work in the reverse mode, i.e. F1-catalyzed hydrolysis of ATP coupled to proton transport out of the mitochondrial matrix, the mutant showed a normal proton-pumping activity and this activity was fully sensitive to oligomycin, an inhibitor of the ATP synthase proton channel. However, under conditions of maximal ATP hydrolytic activity, using non-osmotically protected mitochondria, the mutant ATPase activity was less efficiently inhibited by oligomycin (60% inhibition versus 85% for the wild type control). BN-PAGE analyses revealed that atp6-T9176C yeast accumulated rather good levels of fully assembled ATP synthase complexes. However, a number of subcomplexes (F1, Atp9p-ring, unassembled α-F1 subunits) could be detected as well, presumably because of a decreased stability of Atp6p within the ATP synthase. Although the oxidative phosphorylation capacity was reduced in atp6-T9176C yeast, the number of ATP molecules synthesized per electron transferred to oxygen was similar compared with wild type yeast. It can therefore be inferred that the coupling efficiency within the ATP synthase was mostly unaffected and that the T9176C mutation did not increase the proton permeability of the

The novel Tau mutation G335S: clinical, neuropathological and molecular characterization.

PubMed

Spina, Salvatore; Murrell, Jill R; Yoshida, Hirotaka; Ghetti, Bernardino; Bermingham, Niamh; Sweeney, Brian; Dlouhy, Stephen R; Crowther, R Anthony; Goedert, Michel; Keohane, Catherine

2007-04-01

Mutations in Tau cause the inherited neurodegenerative disease, frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17). Known coding region mutations cluster in the microtubule-binding region, where they alter the ability of tau to promote microtubule assembly. Depending on the tau isoforms, this region consists of three or four imperfect repeats of 31 or 32 amino acids, each of which contains a characteristic and invariant PGGG motif. Here, we report the novel G335S mutation, which changes the PGGG motif of the third tau repeat to PGGS, in an individual who developed social withdrawal, emotional bluntness and stereotypic behavior at age 22, followed by disinhibition, hyperorality and ideomotor apraxia. Abundant tau-positive inclusions were present in neurons and glia in the frontotemporal cortex, hippocampus and brainstem. Sarkosyl-insoluble tau showed paired helical and straight filaments, as well as more irregular rope-like filaments. The pattern of pathological tau bands was like that of Alzheimer disease. Experimentally, the G335S mutation resulted in a greatly reduced ability of tau to promote microtubule assembly, while having no significant effect on heparin-induced assembly of recombinant tau into filaments.

Antisense-based RNA therapy of factor V deficiency: in vitro and ex vivo rescue of a F5 deep-intronic splicing mutation.

PubMed

Nuzzo, Francesca; Radu, Claudia; Baralle, Marco; Spiezia, Luca; Hackeng, Tilman M; Simioni, Paolo; Castoldi, Elisabetta

2013-11-28

Antisense molecules are emerging as a powerful tool to correct splicing defects. Recently, we identified a homozygous deep-intronic mutation (F5 c.1296+268A>G) activating a cryptic donor splice site in a patient with severe coagulation factor V (FV) deficiency and life-threatening bleeding episodes. Here, we assessed the ability of 2 mutation-specific antisense molecules (a morpholino oligonucleotide [MO] and an engineered U7 small nuclear RNA [snRNA]) to correct this splicing defect. COS-1 and HepG2 cells transfected with a F5 minigene construct containing the patient's mutation expressed aberrant messenger RNA (mRNA) in excess of normal mRNA. Treatment with mutation-specific antisense MO (1-5 µM) or a construct expressing antisense U7snRNA (0.25-2 µg) dose-dependently increased the relative amount of correctly spliced mRNA by 1 to 2 orders of magnitude, whereas control MO and U7snRNA were ineffective. Patient-derived megakaryocytes obtained by differentiation of circulating progenitor cells did not express FV, but became positive for FV at immunofluorescence staining after administration of antisense MO or U7snRNA. However, treatment adversely affected cell viability, mainly because of the transfection reagents used to deliver the antisense molecules. Our data provide in vitro and ex vivo proof of principle for the efficacy of RNA therapy in severe FV deficiency, but additional cytotoxicity studies are warranted.

HER2 activating mutations are targets for colorectal cancer treatment.

PubMed

Kavuri, Shyam M; Jain, Naveen; Galimi, Francesco; Cottino, Francesca; Leto, Simonetta M; Migliardi, Giorgia; Searleman, Adam C; Shen, Wei; Monsey, John; Trusolino, Livio; Jacobs, Samuel A; Bertotti, Andrea; Bose, Ron

2015-08-01

The Cancer Genome Atlas project identified HER2 somatic mutations and gene amplification in 7% of patients with colorectal cancer. Introduction of the HER2 mutations S310F, L755S, V777L, V842I, and L866M into colon epithelial cells increased signaling pathways and anchorage-independent cell growth, indicating that they are activating mutations. Introduction of these HER2 activating mutations into colorectal cancer cell lines produced resistance to cetuximab and panitumumab by sustaining MAPK phosphorylation. HER2 mutants are potently inhibited by low nanomolar doses of the irreversible tyrosine kinase inhibitors neratinib and afatinib. HER2 gene sequencing of 48 cetuximab-resistant, quadruple (KRAS, NRAS, BRAF, and PIK3CA) wild-type (WT) colorectal cancer patient-derived xenografts (PDX) identified 4 PDXs with HER2 mutations. HER2-targeted therapies were tested on two PDXs. Treatment with a single HER2-targeted drug (trastuzumab, neratinib, or lapatinib) delayed tumor growth, but dual HER2-targeted therapy with trastuzumab plus tyrosine kinase inhibitors produced regression of these HER2-mutated PDXs. HER2 activating mutations cause EGFR antibody resistance in colorectal cell lines, and PDXs with HER2 mutations show durable tumor regression when treated with dual HER2-targeted therapy. These data provide a strong preclinical rationale for clinical trials targeting HER2 activating mutations in metastatic colorectal cancer. ©2015 American Association for Cancer Research.

Mutation and polymorphism analysis of the human homogentisate 1, 2-dioxygenase gene in alkaptonuria patients.

PubMed Central

Beltrán-Valero de Bernabé, D; Granadino, B; Chiarelli, I; Porfirio, B; Mayatepek, E; Aquaron, R; Moore, M M; Festen, J J; Sanmartí, R; Peñalva, M A; de Córdoba, S R

1998-01-01

Alkaptonuria (AKU), a rare hereditary disorder of phenylalanine and tyrosine catabolism, was the first disease to be interpreted as an inborn error of metabolism. AKU patients are deficient for homogentisate 1,2 dioxygenase (HGO); this deficiency causes homogentisic aciduria, ochronosis, and arthritis. We cloned the human HGO gene and characterized two loss-of-function mutations, P230S and V300G, in the HGO gene in AKU patients. Here we report haplotype and mutational analysis of the HGO gene in 29 novel AKU chromosomes. We identified 12 novel mutations: 8 (E42A, W97G, D153G, S189I, I216T, R225H, F227S, and M368V) missense mutations that result in amino acid substitutions at positions conserved in HGO in different species, 1 (F10fs) frameshift mutation, 2 intronic mutations (IVS9-56G-->A, IVS9-17G-->A), and 1 splice-site mutation (IVS5+1G-->T). We also report characterization of five polymorphic sites in HGO and describe the haplotypic associations of alleles at these sites in normal and AKU chromosomes. One of these sites, HGO-3, is a variable dinucleotide repeat; IVS2+35T/A, IVS5+25T/C, and IVS6+46C/A are intronic sites at which single nucleotide substitutions (dimorphisms) have been detected; and c407T/A is a relatively frequent nucleotide substitution in the coding sequence, exon 4, resulting in an amino acid change (H80Q). These data provide insight into the origin and evolution of the various AKU alleles. PMID:9529363

Broadband Two-Photon Absorption Characteristics of Highly Photostable Fluorenyl-Dicyanoethylenylated [60] Fullerene Dyads (Postprint)

DTIC Science & Technology

2016-05-14

practical use. It is crucial since a i 1. ( ) i it il tauto eri resonances at the bridging C61Hα- [ ( ) ] t t f - j t - ( ) f ll r l s - r ri e si i l... i , lea i t t e f r ati f a f ll -c j ate f r f 6 acce t r ( ) a r ( ), as arke in purple. This resulted in an extended A–D conjugation length a d...absorbing properties. Scheme 1. Synthesis of C60(>CPAF-Cn) 1-Cn (n = 4, 9, 12, or 18) dyads. Reagents and conditions: i . C60, DBU, toluene, rt, 5.0 h; ii

The Role of Polymerase Gamma Mutations in Breast Tumorigenesis