Fragment length analysis screening for detection of CEBPA mutations in intermediate-risk karyotype acute myeloid leukemia.

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Fuster, Oscar; Barragán, Eva; Bolufer, Pascual; Such, Esperanza; Valencia, Ana; Ibáñez, Mariam; Dolz, Sandra; de Juan, Inmaculada; Jiménez, Antonio; Gómez, Maria Teresa; Buño, Ismael; Martínez, Joaquín; Cervera, José; Montesinos, Pau; Moscardó, Federico; Sanz, Miguel Ángel

2012-01-01

During last years, molecular markers have been increased as prognostic factors routinely screened in acute myeloid leukemia (AML). Recently, an increasing interest has been reported in introducing to clinical practice screening for mutations in the CCAAT/enhancer-binding protein α (CEBPA) gene in AML, as it seems to be a good prognostic factor. However, there is no reliable established method for assessing CEBPA mutations during the diagnostic work-up of AMLs. We describe here a straightforward and reliable fragment analysis method based in PCR capillary electrophoresis (PCR-CE) for screening of CEBPA mutations; moreover, we present the results obtained in 151 intermediate-risk karyotype AML patients (aged 16-80 years). The method gave a specificity of 100% and sensitivity of 93% with a lower detection limit of 1-5% for CEBPA mutations. The series found 19 mutations and four polymorphisms in 12 patients, seven of whom (58%) presented two mutations. The overall frequency of CEBPA mutations in AML was 8% (n = 12). CEBPA mutations showed no coincidence with FLT3-ITD or NPM1 mutations. CEBPA mutation predicted better disease-free survival in the group of patients without FLT3-ITD, NPM, or both genes mutated (HR 3.6, IC 95%; 1.0-13.2, p = 0.05) and better overall survival in patients younger than 65 of this group without molecular markers (HR 4.0, IC 95%; 1.0-17.4, p = 0.05). In conclusion, the fragment analysis method based in PCR-CE is a rapid, specific, and sensitive method for CEBPA mutation screening and our results confirm that CEBPA mutations can identify a subgroup of patients with favorable prognosis in AML with intermediate-risk karyotype.

Is there new public health management (NPM) in Nepal? Arguments for and against NPM in Nepal.

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Paudel, Mohan

2013-01-01

This article is a reflection about whether new public management (NPM) styles of reforms seen in other developing countries are also seen in Nepal, and to substantiate these facts with the available evidence and findings. The author saw the emergence of NPM ideas in Western industrialized countries like the United Kingdom and New Zealand. Now it exists in several developing countries of Africa and Asia; but it is very hard to generalize the degree and scope of NPM elements' existence. In Southeast Asia, there is still a mix of the old bureaucratic system with new NPM-oriented reform initiatives. Series of administrative reforms, donor conditionality, and the reestablishment of democracy in the country after 1991 have influenced an orientation toward an efficient, people-oriented, mixed-economy model with increasing partnership of private agencies and nongovernmental organizations in Nepal. The political movement of the last 15 years in the country has strongly called for a new, efficient, and performance-oriented administration and management culture in the country. There are several initiatives already introduced (public-private partnership, decentralization, good governance, accountability/public auditing, performance-based outcome/results-oriented financing and reporting systems). However, to take this momentum up, it still requires strong willingness of political leaders and senior administrators. At the moment, peace and stability of turmoil, political stability, state-of-the-art management skills, and supportive organizational culture are the fundamental requirements for increasing the realization of, and sustaining the NPM-oriented reforms in Nepal.

Oncogenic kinase NPM/ALK induces through STAT3 expression of immunosuppressive protein CD274 (PD-L1, B7-H1)

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Marzec, Michal; Zhang, Qian; Goradia, Ami

2008-01-01

The mechanisms of malignant cell transformation caused by the oncogenic, chimeric nucleophosmin (NPM)/anaplastic lymphoma kinase (ALK) remain only partially understood, with most of the previous studies focusing mainly on the impact of NPM/ALK on cell survival and proliferation. Here we report th...

Structural investigation of nucleophosmin interaction with the tumor suppressor Fbw7γ.

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Di Matteo, A; Franceschini, M; Paiardini, A; Grottesi, A; Chiarella, S; Rocchio, S; Di Natale, C; Marasco, D; Vitagliano, L; Travaglini-Allocatelli, C; Federici, L

2017-09-18

Nucleophosmin (NPM1) is a multifunctional nucleolar protein implicated in ribogenesis, centrosome duplication, cell cycle control, regulation of DNA repair and apoptotic response to stress stimuli. The majority of these functions are played through the interactions with a variety of protein partners. NPM1 is frequently overexpressed in solid tumors of different histological origin. Furthermore NPM1 is the most frequently mutated protein in acute myeloid leukemia (AML) patients. Mutations map to the C-terminal domain and lead to the aberrant and stable localization of the protein in the cytoplasm of leukemic blasts. Among NPM1 protein partners, a pivotal role is played by the tumor suppressor Fbw7γ, an E3-ubiquitin ligase that degrades oncoproteins like c-MYC, cyclin E, Notch and c-jun. In AML with NPM1 mutations, Fbw7γ is degraded following its abnormal cytosolic delocalization by mutated NPM1. This mechanism also applies to other tumor suppressors and it has been suggested that it may play a key role in leukemogenesis. Here we analyse the interaction between NPM1 and Fbw7γ, by identifying the protein surfaces implicated in recognition and key aminoacids involved. Based on the results of computational methods, we propose a structural model for the interaction, which is substantiated by experimental findings on several site-directed mutants. We also extend the analysis to two other NPM1 partners (HIV Tat and CENP-W) and conclude that NPM1 uses the same molecular surface as a platform for recognizing different protein partners. We suggest that this region of NPM1 may be targeted for cancer treatment.

Oncogenic tyrosine kinase NPM-ALK induces expression of the growth-promoting receptor ICOS

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Zhang, Qian; Wang, HongYi; Kantekure, Kanchan

2011-01-01

Here we report that T-cell lymphoma cells carrying the NPM-ALK fusion protein (ALK(+) TCL) frequently express the cell-stimulatory receptor ICOS. ICOS expression in ALK(+) TCL is moderate and strictly dependent on the expression and enzymatic activity of NPM-ALK. NPM-ALK induces ICOS expression v...

Polo-like kinase 2-dependent phosphorylation of NPM/B23 on serine 4 triggers centriole duplication.

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Annekatrin Krause

Full Text Available Duplication of the centrosome is well controlled during faithful cell division while deregulation of this process leads to supernumary centrosomes, chromosome missegregation and aneuploidy, a hallmark of many cancer cells. We previously reported that Polo-like kinase 2 (Plk2 is activated near the G1/S phase transition, and regulates the reproduction of centrosomes. In search for Plk2 interacting proteins we have identified NPM/B23 (Nucleophosmin as a novel Plk2 binding partner. We find that Plk2 and NPM/B23 interact in vitro in a Polo-box dependent manner. An association between both proteins was also observed in vivo. Moreover, we show that Plk2 phosphorylates NPM/B23 on serine 4 in vivo in S-phase. Notably, expression of a non-phosphorylatable NPM/B23 S4A mutant interferes with centriole reduplication in S-phase arrested cells and leads to a dilution of centriole numbers in unperturbed U2OS cells. The corresponding phospho-mimicking mutants have the opposite effect and their expression leads to the accumulation of centrioles. These findings suggest that NPM/B23 is a direct target of Plk2 in the regulation of centriole duplication and that phosphorylation on serine 4 can trigger this process.

From Autonomy to Quality Management: NPM Impacts on School Governance in Switzerland

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Hangartner, Judith; Svaton, Carla Jana

2013-01-01

This article reviews the impact of discourses on "New Public Management" (NPM) on compulsory schooling in Switzerland during the last two decades and traces its implementation in the Canton of Bern. The analysis suggests that while NPM reformers initially promoted increased school autonomy, the introduction of market elements and school…

The historical development of management accounting discursive role in NPM reforms

DEFF Research Database (Denmark)

Malmmose, Margit

Much research has focused on the role and limitations that management accounting techniques have had in the development of New Public Management (NPM) reforms during the past 30 years. However, research of the origin of NPM, the reason for NPM reforms consumed with the role of management accounting...... from a historical perspective is limited. This paper investigates the historical and discursive development of management accounting in health care through general history, organisational and national documents along with academic literature. The framework is informed by the governmentality concept...... described by Miller and O’Leary (1987) and this is used to reflect on developments in the past century including the role of discursive formation and practice. The intention is to explain and put into perspective how the current role of management accounting developed. The aim is to guide future research...

Homozygous and compound heterozygous mutations in the FBN1 gene: unexpected findings in molecular diagnosis of Marfan syndrome.

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Arnaud, Pauline; Hanna, Nadine; Aubart, Mélodie; Leheup, Bruno; Dupuis-Girod, Sophie; Naudion, Sophie; Lacombe, Didier; Milleron, Olivier; Odent, Sylvie; Faivre, Laurence; Bal, Laurence; Edouard, Thomas; Collod-Beroud, Gwenaëlle; Langeois, Maud; Spentchian, Myrtille; Gouya, Laurent; Jondeau, Guillaume; Boileau, Catherine

2017-02-01

Marfan syndrome (MFS) is an autosomal-dominant connective tissue disorder usually associated with heterozygous mutations in the gene encoding fibrillin-1 (FBN1). Homozygous and compound heterozygous cases are rare events and have been associated with a clinical severe presentation. Report unexpected findings of homozygosity and compound heterozygosity in the course of molecular diagnosis of heterozygous MFS and compare the findings with published cases. In the context of molecular diagnosis of heterozygous MFS, systematic sequencing of the FBN1 gene was performed in 2500 probands referred nationwide. 1400 probands carried a heterozygous mutation in this gene. Unexpectedly, among them four homozygous cases (0.29%) and five compound heterozygous cases (0.36%) were identified (total: 0.64%). Interestingly, none of these cases carried two premature termination codon mutations in the FBN1 gene. Clinical features for these carriers and their families were gathered and compared. There was a large spectrum of severity of the disease in probands carrying two mutated FBN1 alleles, but none of them presented extremely severe manifestations of MFS in any system compared with carriers of only one mutated FBN1 allele. This observation is not in line with the severe clinical features reported in the literature for four homozygous and three compound heterozygous probands. Homozygotes and compound heterozygotes were unexpectedly identified in the course of molecular diagnosis of MFS. Contrary to previous reports, the presence of two mutated alleles was not associated with severe forms of MFS. Although homozygosity and compound heterozygosity are rarely found in molecular diagnosis, they should not be overlooked, especially among consanguineous families. However, no predictive evaluation of severity should be provided. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Molecular Clock of Neutral Mutations in a Fitness-Increasing Evolutionary Process.

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Kishimoto, Toshihiko; Ying, Bei-Wen; Tsuru, Saburo; Iijima, Leo; Suzuki, Shingo; Hashimoto, Tomomi; Oyake, Ayana; Kobayashi, Hisaka; Someya, Yuki; Narisawa, Dai; Yomo, Tetsuya

2015-07-01

The molecular clock of neutral mutations, which represents linear mutation fixation over generations, is theoretically explained by genetic drift in fitness-steady evolution or hitchhiking in adaptive evolution. The present study is the first experimental demonstration for the molecular clock of neutral mutations in a fitness-increasing evolutionary process. The dynamics of genome mutation fixation in the thermal adaptive evolution of Escherichia coli were evaluated in a prolonged evolution experiment in duplicated lineages. The cells from the continuously fitness-increasing evolutionary process were subjected to genome sequencing and analyzed at both the population and single-colony levels. Although the dynamics of genome mutation fixation were complicated by the combination of the stochastic appearance of adaptive mutations and clonal interference, the mutation fixation in the population was simply linear over generations. Each genome in the population accumulated 1.6 synonymous and 3.1 non-synonymous neutral mutations, on average, by the spontaneous mutation accumulation rate, while only a single genome in the population occasionally acquired an adaptive mutation. The neutral mutations that preexisted on the single genome hitchhiked on the domination of the adaptive mutation. The successive fixation processes of the 128 mutations demonstrated that hitchhiking and not genetic drift were responsible for the coincidence of the spontaneous mutation accumulation rate in the genome with the fixation rate of neutral mutations in the population. The molecular clock of neutral mutations to the fitness-increasing evolution suggests that the numerous neutral mutations observed in molecular phylogenetic trees may not always have been fixed in fitness-steady evolution but in adaptive evolution.

Molecular Clock of Neutral Mutations in a Fitness-Increasing Evolutionary Process.

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Toshihiko Kishimoto

2015-07-01

Full Text Available The molecular clock of neutral mutations, which represents linear mutation fixation over generations, is theoretically explained by genetic drift in fitness-steady evolution or hitchhiking in adaptive evolution. The present study is the first experimental demonstration for the molecular clock of neutral mutations in a fitness-increasing evolutionary process. The dynamics of genome mutation fixation in the thermal adaptive evolution of Escherichia coli were evaluated in a prolonged evolution experiment in duplicated lineages. The cells from the continuously fitness-increasing evolutionary process were subjected to genome sequencing and analyzed at both the population and single-colony levels. Although the dynamics of genome mutation fixation were complicated by the combination of the stochastic appearance of adaptive mutations and clonal interference, the mutation fixation in the population was simply linear over generations. Each genome in the population accumulated 1.6 synonymous and 3.1 non-synonymous neutral mutations, on average, by the spontaneous mutation accumulation rate, while only a single genome in the population occasionally acquired an adaptive mutation. The neutral mutations that preexisted on the single genome hitchhiked on the domination of the adaptive mutation. The successive fixation processes of the 128 mutations demonstrated that hitchhiking and not genetic drift were responsible for the coincidence of the spontaneous mutation accumulation rate in the genome with the fixation rate of neutral mutations in the population. The molecular clock of neutral mutations to the fitness-increasing evolution suggests that the numerous neutral mutations observed in molecular phylogenetic trees may not always have been fixed in fitness-steady evolution but in adaptive evolution.

Benefit of high-dose daunorubicin in AML induction extends across cytogenetic and molecular groups.

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Luskin, Marlise R; Lee, Ju-Whei; Fernandez, Hugo F; Abdel-Wahab, Omar; Bennett, John M; Ketterling, Rhett P; Lazarus, Hillard M; Levine, Ross L; Litzow, Mark R; Paietta, Elisabeth M; Patel, Jay P; Racevskis, Janis; Rowe, Jacob M; Tallman, Martin S; Sun, Zhuoxin; Luger, Selina M

2016-03-24

The initial report of the Eastern Cooperative Oncology Group-American College of Radiology Imaging Network Cancer Research Group trial E1900 (#NCT00049517) showed that induction therapy with high-dose (HD) daunorubicin (90 mg/m(2)) improved overall survival in adults cytogenetics or aFLT3-ITD mutation. Here, we update the results of E1900 after longer follow-up (median, 80.1 months among survivors), focusing on the benefit of HD daunorubicin on common genetic subgroups. Compared with standard-dose daunorubicin (45 mg/m(2)), HD daunorubicin is associated with a hazard ratio (HR) for death of 0.74 (P= .001). Younger patients (cytogenetics (HR, 0.51;P= .03 and HR, 0.68;P= .01, respectively). Patients with unfavorable cytogenetics were shown to benefit from HD daunorubicin on multivariable analysis (adjusted HR, 0.66;P= .04). Patients with FLT3-ITD (24%),DNMT3A(24%), and NPM1(26%) mutant AML all benefited from HD daunorubicin (HR, 0.61,P= .009; HR, 0.62,P= .02; and HR, 0.50,P= .002; respectively). HD benefit was seen in the subgroup of older patients (50-60 years) with the FLT3-ITD or NPM1 mutation. Additionally, the presence of an NPM1 mutation confers a favorable prognosis only for patients receiving anthracycline dose intensification during induction. © 2016 by The American Society of Hematology.

Insight on Mutation-Induced Resistance from Molecular Dynamics Simulations of the Native and Mutated CSF-1R and KIT.

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Priscila Da Silva Figueiredo Celestino Gomes

Full Text Available The receptors tyrosine kinases (RTKs for the colony stimulating factor-1, CSF-1R, and for the stem cell factor, SCFR or KIT, are important mediators of signal transduction. The abnormal function of these receptors, promoted by gain-of-function mutations, leads to their constitutive activation, associated with cancer or other proliferative diseases. A secondary effect of the mutations is the alteration of receptors' sensitivity to tyrosine kinase inhibitors, compromising effectiveness of these molecules in clinical treatment. In particular, the mutation V560G in KIT increases its sensitivity to Imatinib, while the D816V in KIT, and D802V in CSF-1R, triggers resistance to the drug. We analyzed the Imatinib binding affinity to the native and mutated KIT (mutations V560G, S628N and D816V and CSF-1R (mutation D802V by using molecular dynamics simulations and energy calculations of Imatinib•target complexes. Further, we evaluated the sensitivity of the studied KIT receptors to Imatinib by measuring the inhibition of KIT phosphorylation. Our study showed that (i the binding free energy of Imatinib to the targets is highly correlated with their experimentally measured sensitivity; (ii the electrostatic interactions are a decisive factor affecting the binding energy; (iii the most deleterious impact to the Imatinib sensitivity is promoted by D802V (CSF-1R and D816V (KIT mutations; (iv the role of the juxtamembrane region, JMR, in the imatinib binding is accessory. These findings contribute to a better description of the mutation-induced effects alternating the targets sensitivity to Imatinib.

High-risk Long QT Syndrome Mutations in the Kv7.1 (KCNQ1) Pore Disrupt the Molecular Basis for Rapid K+ Permeation

Science.gov (United States)

Burgess, Don E.; Bartos, Daniel C.; Reloj, Allison R.; Campbell, Kenneth S.; Johnson, Jonathan N.; Tester, David J.; Ackerman, Michael J.; Fressart, Véronique; Denjoy, Isabelle; Guicheney, Pascale; Moss, Arthur J.; Ohno, Seiko; Horie, Minoru; Delisle, Brian P.

2012-01-01

Type 1 long QT syndrome (LQT1) syndrome is caused by loss-of-function mutations in the KCNQ1, which encodes the K+ channel (Kv7.1) that underlies the slowly activating delayed rectifier K+ current in the heart. Intragenic risk stratification suggests LQT1 mutations that disrupt conserved amino acid residues in the pore are an independent risk factor for LQT1-related cardiac events. The purpose of this study is to determine possible molecular mechanisms that underlie the loss-of-function for these high-risk mutations. Extensive genotype-phenotype analyses of LQT1 patients showed that T322M-, T322A-, or G325R-Kv7.1 confer a high risk for LQT1-related cardiac events. Heterologous expression of these mutations with KCNE1 revealed they generated non-functional channels and caused dominant negative suppression of WT-Kv7.1 current. Molecular dynamic simulations (MDS) of analogous mutations in KcsA (T85M-, T85A-, and G88R-KcsA) demonstrated that they disrupted the symmetrical distribution of the carbonyl oxygen atoms in the selectivity filter, which upset the balance between the strong attractive and K+-K+ repulsive forces required for rapid K+ permeation. We conclude high-risk LQT1 mutations in the pore likely disrupt the architectural and physical properties of the K+ channel selectivity filter. PMID:23092362

High-risk long QT syndrome mutations in the Kv7.1 (KCNQ1) pore disrupt the molecular basis for rapid K(+) permeation.

Science.gov (United States)

Burgess, Don E; Bartos, Daniel C; Reloj, Allison R; Campbell, Kenneth S; Johnson, Jonathan N; Tester, David J; Ackerman, Michael J; Fressart, Véronique; Denjoy, Isabelle; Guicheney, Pascale; Moss, Arthur J; Ohno, Seiko; Horie, Minoru; Delisle, Brian P

2012-11-13

Type 1 long QT syndrome (LQT1) is caused by loss-of-function mutations in the KCNQ1 gene, which encodes the K(+) channel (Kv7.1) that underlies the slowly activating delayed rectifier K(+) current in the heart. Intragenic risk stratification suggests LQT1 mutations that disrupt conserved amino acid residues in the pore are an independent risk factor for LQT1-related cardiac events. The purpose of this study is to determine possible molecular mechanisms that underlie the loss of function for these high-risk mutations. Extensive genotype-phenotype analyses of LQT1 patients showed that T322M-, T322A-, or G325R-Kv7.1 confers a high risk for LQT1-related cardiac events. Heterologous expression of these mutations with KCNE1 revealed they generated nonfunctional channels and caused dominant negative suppression of WT-Kv7.1 current. Molecular dynamics simulations of analogous mutations in KcsA (T85M-, T85A-, and G88R-KcsA) demonstrated that they disrupted the symmetrical distribution of the carbonyl oxygen atoms in the selectivity filter, which upset the balance between the strong attractive and K(+)-K(+) repulsive forces required for rapid K(+) permeation. We conclude high-risk LQT1 mutations in the pore likely disrupt the architectural and physical properties of the K(+) channel selectivity filter.

Molecular screening of 980 cases of suspected hereditary optic neuropathy with a report on 77 novel OPA1 mutations

DEFF Research Database (Denmark)

Ferré, Marc; Bonneau, Dominique; Milea, Dan

2009-01-01

We report the results of molecular screening in 980 patients carried out as part of their work-up for suspected hereditary optic neuropathies. All the patients were investigated for Leber's hereditary optic neuropathy (LHON) and autosomal dominant optic atrophy (ADOA), by searching for the ten...... and OPA3 mutations in cases of suspected hereditary optic neuropathy, even in absence of a family history of the disease....... novel OPA1 mutations reported here. The statistical analysis of this large set of mutations has led us to propose a diagnostic strategy that should help with the molecular work-up of optic neuropathies. Our results highlight the importance of investigating LHON-causing mtDNA mutations as well as OPA1...

The tyrosine phosphatase Shp2 interacts with NPM-ALK and regulates anaplastic lymphoma cell growth and migration

DEFF Research Database (Denmark)

Voena, Claudia; Conte, Chiara; Ambrogio, Chiara

2007-01-01

Anaplastic large cell lymphomas (ALCL) are mainly characterized by the reciprocal translocation t(2;5)(p23;q35) that involves the anaplastic lymphoma kinase (ALK) gene and generates the fusion protein NPM-ALK with intrinsic tyrosine kinase activity. NPM-ALK triggers several signaling cascades......, leading to increased cell growth, resistance to apoptosis, and changes in morphology and migration of transformed cells. To search for new NPM-ALK interacting molecules, we developed a mass spectrometry-based proteomic approach in HEK293 cells expressing an inducible NPM-ALK and identified the tyrosine...... phosphatase Shp2 as a candidate substrate. We found that NPM-ALK was able to bind Shp2 in coprecipitation experiments and to induce its phosphorylation in the tyrosine residues Y542 and Y580 both in HEK293 cells and ALCL cell lines. In primary lymphomas, antibodies against the phosphorylated tyrosine Y542...

The interplay of management accounting research and NPM health initiatives

DEFF Research Database (Denmark)

Malmmose, Margit

This paper investigates the development of management accounting research in the context of New Public Management (NPM) initiatives in health care. Drawing on concepts from diffusion theory and earlier literature reviews, the paper examines the interplay between management accounting research...... and health care reforms in relation to country of origin, development, theoretical approach, research method and topic. The study thus establishes a different focus; namely the interrelationship between the development of management accounting research and practical socio-political NPM innovations. The study...... shows that management accounting techniques are increasingly adopted in governmental health reforms and diffused across nations, themes and initiatives through time with the result that wider social practices become more and more integrated in management accounting research themes...

Activity of second-generation ALK inhibitors against crizotinib-resistant mutants in an NPM-ALK model compared to EML4-ALK

International Nuclear Information System (INIS)

Fontana, Diletta; Ceccon, Monica; Gambacorti-Passerini, Carlo; Mologni, Luca

2015-01-01

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor involved in both solid and hematological tumors. About 80% of ALK-positive anaplastic large-cell lymphoma (ALCL) cases are characterized by the t(2;5)(p23;q35) translocation, encoding for the aberrant fusion protein nucleophosmin (NPM)-ALK, whereas 5% of non-small-cell lung cancer (NSCLC) patients carry the inv(2)(p21;p23) rearrangement, encoding for the echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion. The ALK/c-MET/ROS inhibitor crizotinib successfully improved the treatment of ALK-driven diseases. However, several cases of resistance appeared in NSCLC patients, and ALK amino acid substitutions were identified as a leading cause of resistance to crizotinib. Second-generation ALK inhibitors have been developed in order to overcome crizotinib resistance. In this work, we profiled in vitro the activity of crizotinib, AP26113, ASP3026, alectinib, and ceritinib against six mutated forms of ALK associated with clinical resistance to crizotinib (C1156Y, L1196M, L1152R, G1202R, G1269A, and S1206Y) and provide a classification of mutants according to their level of sensitivity/resistance to the drugs. Since the biological activity of ALK mutations extends beyond the specific type of fusion, both NPM-ALK- and EML4-ALK-positive cellular models were used. Our data revealed that most mutants may be targeted by using different inhibitors. One relevant exception is represented by the G1202R substitution, which was highly resistant to all drugs (>10-fold increased IC 50 compared to wild type) and may represent the most challenging mutation to overcome. These results provide a prediction of cross-resistance of known crizotinib-resistant mutations against all second-generation tyrosine kinase inhibitors (TKIs) clinically available, and therefore could be a useful tool to help clinicians in the management of crizotinib-resistance cases

Whole-exome sequencing reveals the spectrum of gene mutations and the clonal evolution patterns in paediatric acute myeloid leukaemia.

Science.gov (United States)

Shiba, Norio; Yoshida, Kenichi; Shiraishi, Yuichi; Okuno, Yusuke; Yamato, Genki; Hara, Yusuke; Nagata, Yasunobu; Chiba, Kenichi; Tanaka, Hiroko; Terui, Kiminori; Kato, Motohiro; Park, Myoung-Ja; Ohki, Kentaro; Shimada, Akira; Takita, Junko; Tomizawa, Daisuke; Kudo, Kazuko; Arakawa, Hirokazu; Adachi, Souichi; Taga, Takashi; Tawa, Akio; Ito, Etsuro; Horibe, Keizo; Sanada, Masashi; Miyano, Satoru; Ogawa, Seishi; Hayashi, Yasuhide

2016-11-01

Acute myeloid leukaemia (AML) is a molecularly and clinically heterogeneous disease. Targeted sequencing efforts have identified several mutations with diagnostic and prognostic values in KIT, NPM1, CEBPA and FLT3 in both adult and paediatric AML. In addition, massively parallel sequencing enabled the discovery of recurrent mutations (i.e. IDH1/2 and DNMT3A) in adult AML. In this study, whole-exome sequencing (WES) of 22 paediatric AML patients revealed mutations in components of the cohesin complex (RAD21 and SMC3), BCORL1 and ASXL2 in addition to previously known gene mutations. We also revealed intratumoural heterogeneities in many patients, implicating multiple clonal evolution events in the development of AML. Furthermore, targeted deep sequencing in 182 paediatric AML patients identified three major categories of recurrently mutated genes: cohesion complex genes [STAG2, RAD21 and SMC3 in 17 patients (8·3%)], epigenetic regulators [ASXL1/ASXL2 in 17 patients (8·3%), BCOR/BCORL1 in 7 patients (3·4%)] and signalling molecules. We also performed WES in four patients with relapsed AML. Relapsed AML evolved from one of the subclones at the initial phase and was accompanied by many additional mutations, including common driver mutations that were absent or existed only with lower allele frequency in the diagnostic samples, indicating a multistep process causing leukaemia recurrence. © 2016 John Wiley & Sons Ltd.

The heat shock protein-90 co-chaperone, Cyclophilin 40, promotes ALK-positive, anaplastic large cell lymphoma viability and its expression is regulated by the NPM-ALK oncoprotein

International Nuclear Information System (INIS)

Pearson, Joel D; Mohammed, Zubair; Bacani, Julinor T C; Lai, Raymond; Ingham, Robert J

2012-01-01

Anaplastic lymphoma kinase-positive, anaplastic large cell lymphoma (ALK+ ALCL) is a T cell lymphoma defined by the presence of chromosomal translocations involving the ALK tyrosine kinase gene. These translocations generate fusion proteins (e.g. NPM-ALK) with constitutive tyrosine kinase activity, which activate numerous signalling pathways important for ALK+ ALCL pathogenesis. The molecular chaperone heat shock protein-90 (Hsp90) plays a critical role in allowing NPM-ALK and other signalling proteins to function in this lymphoma. Co-chaperone proteins are important for helping Hsp90 fold proteins and for directing Hsp90 to specific clients; however the importance of co-chaperone proteins in ALK+ ALCL has not been investigated. Our preliminary findings suggested that expression of the immunophilin co-chaperone, Cyclophilin 40 (Cyp40), is up-regulated in ALK+ ALCL by JunB, a transcription factor activated by NPM-ALK signalling. In this study we examined the regulation of the immunophilin family of co-chaperones by NPM-ALK and JunB, and investigated whether the immunophilin co-chaperones promote the viability of ALK+ ALCL cell lines. NPM-ALK and JunB were knocked-down in ALK+ ALCL cell lines with siRNA, and the effect on the expression of the three immunophilin co-chaperones: Cyp40, FK506-binding protein (FKBP) 51, and FKBP52 examined. Furthermore, the effect of knock-down of the immunophilin co-chaperones, either individually or in combination, on the viability of ALK+ ALCL cell lines and NPM-ALK levels and activity was also examined. We found that NPM-ALK promoted the transcription of Cyp40 and FKBP52, but only Cyp40 transcription was promoted by JunB. We also observed reduced viability of ALK+ ALCL cell lines treated with Cyp40 siRNA, but not with siRNAs directed against FKBP52 or FKBP51. Finally, we demonstrate that the decrease in the viability of ALK+ ALCL cell lines treated with Cyp40 siRNA does not appear to be due to a decrease in NPM-ALK levels or the

Molecular dynamic simulation reveals damaging impact of RAC1 F28L mutation in the switch I region.

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Ambuj Kumar

Full Text Available 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 (RAC(F28L 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.

Spectrum of novel mutations found in Waardenburg syndrome types 1 and 2: implications for molecular genetic diagnostics.

Science.gov (United States)

Wildhardt, Gabriele; Zirn, Birgit; Graul-Neumann, Luitgard M; Wechtenbruch, Juliane; Suckfüll, Markus; Buske, Annegret; Bohring, Axel; Kubisch, Christian; Vogt, Stefanie; Strobl-Wildemann, Gertrud; Greally, Marie; Bartsch, Oliver; Steinberger, Daniela

2013-03-18

Till date, mutations in the genes PAX3 and MITF have been described in Waardenburg syndrome (WS), which is clinically characterised by congenital hearing loss and pigmentation anomalies. Our study intended to determine the frequency of mutations and deletions in these genes, to assess the clinical phenotype in detail and to identify rational priorities for molecular genetic diagnostics procedures. Prospective analysis. 19 Caucasian patients with typical features of WS underwent stepwise investigation of PAX3 and MITF. When point mutations and small insertions/deletions were excluded by direct sequencing, copy number analysis by multiplex ligation-dependent probe amplification was performed to detect larger deletions and duplications. Clinical data and photographs were collected to facilitate genotype-phenotype analyses. All analyses were performed in a large German laboratory specialised in genetic diagnostics. 15 novel and 4 previously published heterozygous mutations in PAX3 and MITF were identified. Of these, six were large deletions or duplications that were only detectable by copy number analysis. All patients with PAX3 mutations had typical phenotype of WS with dystopia canthorum (WS1), whereas patients with MITF gene mutations presented without dystopia canthorum (WS2). In addition, one patient with bilateral hearing loss and blue eyes with iris stroma dysplasia had a de novo missense mutation (p.Arg217Ile) in MITF. MITF 3-bp deletions at amino acid position 217 have previously been described in patients with Tietz syndrome (TS), a clinical entity with hearing loss and generalised hypopigmentation. On the basis of these findings, we conclude that sequencing and copy number analysis of both PAX3 and MITF have to be recommended in the routine molecular diagnostic setting for patients, WS1 and WS2. Furthermore, our genotype-phenotype analyses indicate that WS2 and TS correspond to a clinical spectrum that is influenced by MITF mutation type and position.

SETBP1 mutations drive leukemic transformation in ASXL1-mutated MDS.

Science.gov (United States)

Inoue, D; Kitaura, J; Matsui, H; Hou, H-A; Chou, W-C; Nagamachi, A; Kawabata, K C; Togami, K; Nagase, R; Horikawa, S; Saika, M; Micol, J-B; Hayashi, Y; Harada, Y; Harada, H; Inaba, T; Tien, H-F; Abdel-Wahab, O; Kitamura, T

2015-04-01

Mutations in ASXL1 are frequent in patients with myelodysplastic syndrome (MDS) and are associated with adverse survival, yet the molecular pathogenesis of ASXL1 mutations (ASXL1-MT) is not fully understood. Recently, it has been found that deletion of Asxl1 or expression of C-terminal-truncating ASXL1-MTs inhibit myeloid differentiation and induce MDS-like disease in mice. Here, we find that SET-binding protein 1 (SETBP1) mutations (SETBP1-MT) are enriched among ASXL1-mutated MDS patients and associated with increased incidence of leukemic transformation, as well as shorter survival, suggesting that SETBP1-MT play a critical role in leukemic transformation of MDS. We identify that SETBP1-MT inhibit ubiquitination and subsequent degradation of SETBP1, resulting in increased expression. Expression of SETBP1-MT, in turn, inhibited protein phosphatase 2A activity, leading to Akt activation and enhanced expression of posterior Hoxa genes in ASXL1-mutant cells. Biologically, SETBP1-MT augmented ASXL1-MT-induced differentiation block, inhibited apoptosis and enhanced myeloid colony output. SETBP1-MT collaborated with ASXL1-MT in inducing acute myeloid leukemia in vivo. The combination of ASXL1-MT and SETBP1-MT activated a stem cell signature and repressed the tumor growth factor-β signaling pathway, in contrast to the ASXL1-MT-induced MDS model. These data reveal that SETBP1-MT are critical drivers of ASXL1-mutated MDS and identify several deregulated pathways as potential therapeutic targets in high-risk MDS.

N-(1-Pyrenyl Maleimide Induces Bak Oligomerization and Mitochondrial Dysfunction in Jurkat Cells

Directory of Open Access Journals (Sweden)

Pei-Rong Huang

2015-01-01

Full Text Available N-(1-pyrenyl maleimide (NPM is a fluorescent reagent that is frequently used as a derivatization agent for the detection of thio-containing compounds. NPM has been shown to display a great differential cytotoxicity against hematopoietic cancer cells. In this study, the molecular mechanism by which NPM induces apoptosis was examined. Here, we show that treatment of Jurkat cells with NPM leads to Bak oligomerization, loss of mitochondrial membrane potential (Δψm, and release of cytochrome C from mitochondria to cytosol. Induction of Bak oligomerization appears to play a critical role in NPM-induced apoptosis, as downregulation of Bak by shRNA significantly prevented NPM-induced apoptosis. Inhibition of caspase 8 by Z-IETD-FMK and/or depletion of Bid did not affect NPM-induced oligomerization of Bak. Taken together, these results suggest that NPM-induced apoptosis is mediated through a pathway that is independent of caspase-8 activation.

Molecular methods for the detection of mutations.

Science.gov (United States)

Monteiro, C; Marcelino, L A; Conde, A R; Saraiva, C; Giphart-Gassler, M; De Nooij-van Dalen, A G; Van Buuren-van Seggelen, V; Van der Keur, M; May, C A; Cole, J; Lehmann, A R; Steinsgrimsdottir, H; Beare, D; Capulas, E; Armour, J A

2000-01-01

We report the results of a collaborative study aimed at developing reliable, direct assays for mutation in human cells. The project used common lymphoblastoid cell lines, both with and without mutagen treatment, as a shared resource to validate the development of new molecular methods for the detection of low-level mutations in the presence of a large excess of normal alleles. As the "gold standard, " hprt mutation frequencies were also measured on the same samples. The methods under development included i) the restriction site mutation (RSM) assay, in which mutations lead to the destruction of a restriction site; ii) minisatellite length-change mutation, in which mutations lead to alleles containing new numbers of tandem repeat units; iii) loss of heterozygosity for HLA epitopes, in which antibodies can be used to direct selection for mutant cells; iv) multiple fluorescence-based long linker arm nucleotides assay (mf-LLA) technology, for the detection of substitutional mutations; v) detection of alterations in the TP53 locus using a (CA) array as the target for the screening; and vi) PCR analysis of lymphocytes for the presence of the BCL2 t(14:18) translocation. The relative merits of these molecular methods are discussed, and a comparison made with more "traditional" methods.

Control and Autonomy—The SAIs in Norway, Denmark, and Germany as Watchdogs in an NPM-Era?

DEFF Research Database (Denmark)

Jantz, Bastian; Reichborn-Kjennerud, Kristin; Vrangbæk, Karsten

2015-01-01

on an evaluative role when judging NPM instruments. At the same time their emphasis on legality and compliance can be at odds with some of the operating principles in NPM. All in all the German SAI seems to be the most radical critic. This may be linked to the lack of openness of the German SAIs results that makes...... it more internally focused within a mindset of administrative accountability....

Molecular basis of hereditary C1q deficiency-revisited: identification of several novel disease-causing mutations

DEFF Research Database (Denmark)

Schejbel, L; Skattum, L; Hagelberg, S

2011-01-01

C1q is the central pattern-recognition molecule in the classical pathway of the complement system and is known to have a key role in the crossroads between adaptive and innate immunity. Hereditary C1q deficiency is a rare genetic condition strongly associated with systemic lupus erythematosus...... and increased susceptibility to bacterial infections. However, the clinical symptoms may vary. For long, the molecular basis of C1q deficiency was ascribed to only six different mutations. In the present report, we describe five new patients with C1q deficiency, present the 12 causative mutations described till...... now and review the clinical spectrum of symptoms found in patients with C1q deficiency. With the results presented here, confirmed C1q deficiency is reported in 64 patients from at least 38 families....

Spectrum of novel mutations found in Waardenburg syndrome types 1 and 2: implications for molecular genetic diagnostics

Science.gov (United States)

Wildhardt, Gabriele; Zirn, Birgit; Graul-Neumann, Luitgard M; Wechtenbruch, Juliane; Suckfüll, Markus; Buske, Annegret; Bohring, Axel; Kubisch, Christian; Vogt, Stefanie; Strobl-Wildemann, Gertrud; Greally, Marie; Bartsch, Oliver; Steinberger, Daniela

2013-01-01

Objectives Till date, mutations in the genes PAX3 and MITF have been described in Waardenburg syndrome (WS), which is clinically characterised by congenital hearing loss and pigmentation anomalies. Our study intended to determine the frequency of mutations and deletions in these genes, to assess the clinical phenotype in detail and to identify rational priorities for molecular genetic diagnostics procedures. Design Prospective analysis. Patients 19 Caucasian patients with typical features of WS underwent stepwise investigation of PAX3 and MITF. When point mutations and small insertions/deletions were excluded by direct sequencing, copy number analysis by multiplex ligation-dependent probe amplification was performed to detect larger deletions and duplications. Clinical data and photographs were collected to facilitate genotype–phenotype analyses. Setting All analyses were performed in a large German laboratory specialised in genetic diagnostics. Results 15 novel and 4 previously published heterozygous mutations in PAX3 and MITF were identified. Of these, six were large deletions or duplications that were only detectable by copy number analysis. All patients with PAX3 mutations had typical phenotype of WS with dystopia canthorum (WS1), whereas patients with MITF gene mutations presented without dystopia canthorum (WS2). In addition, one patient with bilateral hearing loss and blue eyes with iris stroma dysplasia had a de novo missense mutation (p.Arg217Ile) in MITF. MITF 3-bp deletions at amino acid position 217 have previously been described in patients with Tietz syndrome (TS), a clinical entity with hearing loss and generalised hypopigmentation. Conclusions On the basis of these findings, we conclude that sequencing and copy number analysis of both PAX3 and MITF have to be recommended in the routine molecular diagnostic setting for patients, WS1 and WS2. Furthermore, our genotype–phenotype analyses indicate that WS2 and TS correspond to a clinical spectrum

Molecular characteristics of the KCNJ5 mutated aldosterone-producing adenomas.

Science.gov (United States)

Murakami, Masanori; Yoshimoto, Takanobu; Nakabayashi, Kazuhiko; Nakano, Yujiro; Fukaishi, Takahiro; Tsuchiya, Kyoichiro; Minami, Isao; Bouchi, Ryotaro; Okamura, Kohji; Fujii, Yasuhisa; Hashimoto, Koshi; Hata, Ken-Ichiro; Kihara, Kazunori; Ogawa, Yoshihiro

2017-10-01

The pathophysiology of aldosterone-producing adenomas (APAs) has been investigated via genetic approaches and the pathogenic significance of a series of somatic mutations, including KCNJ5 , has been uncovered. However, how the mutational status of an APA is associated with its molecular characteristics, including its transcriptome and methylome, has not been fully understood. This study was undertaken to explore the molecular characteristics of APAs, specifically focusing on APAs with KCNJ5 mutations as opposed to those without KCNJ5 mutations, by comparing their transcriptome and methylome status. Cortisol-producing adenomas (CPAs) were used as reference. We conducted transcriptome and methylome analyses of 29 APAs with KCNJ5 mutations, 8 APAs without KCNJ5 mutations and 5 CPAs. Genome-wide gene expression and CpG methylation profiles were obtained from RNA and DNA samples extracted from these 42 adrenal tumors. Cluster analysis of the transcriptome and methylome revealed molecular heterogeneity in APAs depending on their mutational status. DNA hypomethylation and gene expression changes in Wnt signaling and inflammatory response pathways were characteristic of APAs with KCNJ5 mutations. Comparisons between transcriptome data from our APAs and that from normal adrenal cortex obtained from the Gene Expression Omnibus suggested similarities between APAs with KCNJ5 mutations and zona glomerulosa. The present study, which is based on transcriptome and methylome analyses, indicates the molecular heterogeneity of APAs depends on their mutational status. Here, we report the unique characteristics of APAs with KCNJ5 mutations. © 2017 Society for Endocrinology.

Molecular evaluation of a novel missense mutation & an insertional truncating mutation in SUMF1 gene

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Udhaya H Kotecha

2014-01-01

Full Text Available Background & objectives: Multiple suphphatase deficiency (MSD is an autosomal recessive disorder affecting the post translational activation of all enzymes of the sulphatase family. To date, approximately 30 different mutations have been identified in the causative gene, sulfatase modifying factor 1 (SUMF1. We describe here the mutation analysis of a case of MSD. Methods: The proband was a four year old boy with developmental delay followed by neuroregression. He had coarse facies, appendicular hypertonia, truncal ataxia and ichthyosis limited to both lower limbs. Radiographs showed dysostosis multiplex. Clinical suspicion of MSD was confirmed by enzyme analysis of four enzymes of the sulphatase group. Results: The patient was compound heterozygote for a c.451A>G (p.K151E substitution in exon 3 and a single base insertion mutation (c.690_691 InsT in exon 5 in the SUMF1 gene. The bioinformatic analysis of the missense mutation revealed no apparent effect on the overall structure. However, the mutated 151-amino acid residue was found to be adjacent to the substrate binding and the active site residues, thereby affecting the substrate binding and/or catalytic activity, resulting in almost complete loss of enzyme function. Conclusions: The two mutations identified in the present case were novel. This is perhaps the first report of an insertion mutation in SUMF1 causing premature truncation of the protein.

NPM Can Work: An optimistic review of the impact of New Public Management reforms in central and eastern Europe

NARCIS (Netherlands)

S. Dan (Sorin); Ch. Pollitt (Christopher)

2014-01-01

textabstractThis article reviews the New Public Management (NPM) literature in central and eastern Europe (CEE) with the aim of assessing whether reforms have 'worked'. Increasingly, academics have tended to argue against the suitability of NPM instruments in this region. To understand the impact of

TSC1/2 mutations define a molecular subset of HCC with aggressive behaviour and treatment implication.

Science.gov (United States)

Ho, Daniel W H; Chan, Lo K; Chiu, Yung T; Xu, Iris M J; Poon, Ronnie T P; Cheung, Tan T; Tang, Chung N; Tang, Victor W L; Lo, Irene L O; Lam, Polly W Y; Yau, Derek T W; Li, Miao X; Wong, Chun M; Ng, Irene O L

2017-08-01

We investigated the mutational landscape of mammalian target of rapamycin (mTOR) signalling cascade in hepatocellular carcinomas (HCCs) with chronic HBV background, aiming to evaluate and delineate mutation-dependent mechanism of mTOR hyperactivation in hepatocarcinogenesis. We performed next-generation sequencing on human HCC samples and cell line panel. Systematic mutational screening of mTOR pathway-related genes was undertaken and mutant genes were evaluated based on their recurrence. Protein expressions of tuberous sclerosis complex (TSC)1, TSC2 and pRPS6 were assessed by immunohistochemistry in human HCC samples. Rapamycin sensitivity was estimated by colony-formation assay in HCC cell lines and the treatment was further tested using our patient-derived tumour xenograft (PDTX) models. We identified and confirmed multiple mTOR components as recurrently mutated in HBV-associated HCCs. Of significance, we detected frequent (16.2%, n=18/111) mutations of TSC1 and TSC2 genes in the HCC samples. The spectrum of TSC1/2 mutations likely disrupts the endogenous gene functions in suppressing the downstream mTOR activity through different mechanisms and leads to more aggressive tumour behaviour. Mutational disruption of TSC1 and TSC2 was also observed in HCC cell lines and our PDTX models. TSC -mutant cells exhibited reduced colony-forming ability on rapamycin treatment. With the use of biologically relevant TSC2 -mutant PDTXs, we demonstrated the therapeutic benefits of the hypersensitivity towards rapamycin treatment. Taken together, our findings suggest the significance of previously undocumented mutation-dependent mTOR hyperactivation and frequent TSC1/2 mutations in HBV-associated HCCs. They define a molecular subset of HCC having genetic aberrations in mTOR signalling, with potential significance of effective specific drug therapy. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Molecular Dynamics and Bioactivity of a Novel Mutated Human ...

African Journals Online (AJOL)

Keywords: Parathyroid hormone, Mutation prediction, Molecular dynamics, RANKL/OPG, UAMS-32P cell. Tropical .... PTH1R were used as MD simulation starting points. A full-atom ... Values of RMSD, Rg, and potential energy evaluation ...

PENGARUH NPM, FDR, KOMITE AUDIT, PERTUMBUHAN USAHA, LEVERAGE DAN SIZE TERHADAP MANAJEMEN LABA

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Mahfudzotun Nahar

2017-04-01

Full Text Available The purpose of this study was to know the influence of NPM , FDR, Audit Committee, the sales growth (growth, leverage and size of the company earnings management practices of Islamic banking in Indonesia. The dependent variable used in this study was calculated using the earnings management of discretionary accruals. The independent variables used in this study is the net profit margin ratio, the ratio of Financing to Deposit Ratio, the Audit Committee, Sales Growth (Growth, Leverage and Firm Size.             The sample in the study of Islamic banking, comprising both Sharia Bank or Sharia in commercial banks by the Financial Services Authority statistics as of June 2015. The sample was selected using purposive sampling was then obtained 6 Islamic Banks and 12 Sharia sampled in this study ,             The results of this study indicate that there is significant influence between NPM ratio to earnings management of Islamic banking. As for the ratio of FDR, the Audit Committee, Growth, Leverage and Size (size of the company had no significant effect on earnings management practices in Islamic banking. Keywords: earnings management, NPM, FDR,audit committee, Growth, Leverage, Company Size

Metastatic Breast Cancer With ESR1 Mutation: Clinical Management Considerations From the Molecular and Precision Medicine (MAP) Tumor Board at Massachusetts General Hospital.

Science.gov (United States)

Bardia, Aditya; Iafrate, John A; Sundaresan, Tilak; Younger, Jerry; Nardi, Valentina

2016-09-01

: The last decade in oncology has witnessed impressive response rates with targeted therapies, largely because of collaborative efforts at understanding tumor biology and careful patient selection based on molecular fingerprinting of the tumor. Consequently, there has been a push toward routine molecular genotyping of tumors, and large precision medicine-based clinical trials have been launched to match therapy to the molecular alteration seen in a tumor. However, selecting the "right drug" for an individual patient in clinic is a complex decision-making process, including analytical interpretation of the report, consideration of the importance of the molecular alteration in driving growth of the tumor, tumor heterogeneity, the availability of a matched targeted therapy, efficacy and toxicity considerations of the targeted therapy (compared with standard therapy), and reimbursement issues. In this article, we review the key considerations involved in clinical decision making while reviewing a molecular genotyping report. We present the case of a 67-year-old postmenopausal female with metastatic estrogen receptor-positive (ER+) breast cancer, whose tumor progressed on multiple endocrine therapies. Molecular genotyping of the metastatic lesion revealed the presence of an ESR1 mutation (encoding p.Tyr537Asn), which was absent in the primary tumor. The same ESR1 mutation was also detected in circulating tumor DNA (ctDNA) extracted from her blood. The general approach for interpretation of genotyping results, the clinical significance of the specific mutation in the particular cancer, potential strategies to target the pathway, and implications for clinical practice are reviewed in this article. ER+ breast tumors are known to undergo genomic evolution during treatment with the acquisition of new mutations that confer resistance to treatment.ESR1 mutations in the ligand-binding domain of ER can lead to a ligand-independent, constitutively active form of ER and mediate

Molecular characterization of WFS1 in an Iranian family with Wolfram syndrome reveals a novel frameshift mutation associated with early symptoms.

Science.gov (United States)

Sobhani, Maryam; Tabatabaiefar, Mohammad Amin; Rajab, Asadollah; Kajbafzadeh, Abdol-Mohammad; Noori-Daloii, Mohammad Reza

2013-10-10

Wolfram syndrome (WS) is a rare autosomal recessive neurodegenerative disorder that represents a likely source of childhood diabetes especially among countries in the consanguinity belt. The main responsible gene is WFS1 for which over one hundred mutations have been reported from different ethnic groups. The aim of this study was to identify the molecular etiology of WS and to perform a possible genotype-phenotype correlation in Iranian kindred. An Iranian family with two patients was clinically studied and WS was suspected. Genetic linkage analysis via 5 STR markers was carried out. For identification of mutations, DNA sequencing of WFS1 including all the exons, exon-intron boundaries and the promoter was performed. Linkage analysis indicated linkage to the WFS1 region. After DNA sequencing of WFS1, one novel pathogenic mutation, which causes frameshift alteration c.2177_2178insTCTTC (or c.2173_2177dupTCTTC) in exon eight, was found. The genotype-phenotype correlation analysis suggests that the presence of the homozygous mutation may be associated with early onset of disease symptoms. This study stresses the necessity of considering the molecular analysis of WFS1 in childhood diabetes with some symptoms of WS. © 2013 Elsevier B.V. All rights reserved.

Molecular Clock of Neutral Mutations in a Fitness-Increasing Evolutionary Process

OpenAIRE

Kishimoto, Toshihiko; Ying, Bei-Wen; Tsuru, Saburo; Iijima, Leo; Suzuki, Shingo; Hashimoto, Tomomi; Oyake, Ayana; Kobayashi, Hisaka; Someya, Yuki; Narisawa, Dai; Yomo, Tetsuya

2015-01-01

The molecular clock of neutral mutations, which represents linear mutation fixation over generations, is theoretically explained by genetic drift in fitness-steady evolution or hitchhiking in adaptive evolution. The present study is the first experimental demonstration for the molecular clock of neutral mutations in a fitness-increasing evolutionary process. The dynamics of genome mutation fixation in the thermal adaptive evolution of Escherichia coli were evaluated in a prolonged evolution e...

IDH1/2 mutations target a key hallmark of cancer by deregulating cellular metabolism in glioma.

Science.gov (United States)

Zhang, Chunzhi; Moore, Lynette M; Li, Xia; Yung, W K Alfred; Zhang, Wei

2013-09-01

Isocitrate dehydrogenase (IDH) enzymes have recently become a focal point for research aimed at understanding the biology of glioma. IDH1 and IDH2 are mutated in 50%-80% of astrocytomas, oligodendrogliomas, oligoastrocytomas, and secondary glioblastomas but are seldom mutated in primary glioblastomas. Gliomas with IDH1/2 mutations always harbor other molecular aberrations, such as TP53 mutation or 1p/19q loss. IDH1 and IDH2 mutations may serve as prognostic factors because patients with an IDH-mutated glioma survive significantly longer than those with an IDH-wild-type tumor. However, the molecular pathogenic role of IDH1/2 mutations in the development of gliomas is unclear. The production of 2-hydroxyglutarate and enhanced NADP+ levels in tumor cells with mutant IDH1/2 suggest mechanisms through which these mutations contribute to tumorigenesis. Elucidating the pathogenesis of IDH mutations will improve understanding of the molecular mechanisms of gliomagenesis and may lead to development of a new molecular classification system and novel therapies.

Highly sensitive detection of ESR1 mutations in cell-free DNA from patients with metastatic breast cancer using molecular barcode sequencing.

Science.gov (United States)

Masunaga, Nanae; Kagara, Naofumi; Motooka, Daisuke; Nakamura, Shota; Miyake, Tomohiro; Tanei, Tomonori; Naoi, Yasuto; Shimoda, Masafumi; Shimazu, Kenzo; Kim, Seung Jin; Noguchi, Shinzaburo

2018-01-01

We aimed to develop a highly sensitive method to detect ESR1 mutations in cell-free DNA (cfDNA) using next-generation sequencing with molecular barcode (MB-NGS) targeting the hotspot segment (c.1600-1713). The sensitivity of MB-NGS was tested using serially diluted ESR1 mutant DNA and then cfDNA samples from 34 patients with metastatic breast cancer were analyzed with MB-NGS. The results of MB-NGS were validated in comparison with conventional NGS and droplet digital PCR (ddPCR). MB-NGS showed a higher sensitivity (0.1%) than NGS without barcode (1%) by reducing background errors. Of the cfDNA samples from 34 patients with metastatic breast cancer, NGS without barcode revealed seven mutations in six patients (17.6%) and MB-NGS revealed six additional mutations including three mutations not reported in the COSMIC database of breast cancer, resulting in total 13 ESR1 mutations in ten patients (29.4%). Regarding the three hotspot mutations, all the patients with mutations detected by MB-NGS had identical mutations detected by droplet digital PCR (ddPCR), and mutant allele frequency correlated very well between both (r = 0.850, p < 0.01). Moreover, all the patients without these mutations by MB-NGS were found to have no mutations by ddPCR. In conclusion, MB-NGS could successfully detect ESR1 mutations in cfDNA with a higher sensitivity of 0.1% than conventional NGS and was considered as clinically useful as ddPCR.

Preleukemic and second-hit mutational events in an acute myeloid leukemia patient with a novel germline RUNX1 mutation.

Science.gov (United States)

Ng, Isaac Ks; Lee, Joanne; Ng, Christopher; Kosmo, Bustamin; Chiu, Lily; Seah, Elaine; Mok, Michelle Meng Huang; Tan, Karen; Osato, Motomi; Chng, Wee-Joo; Yan, Benedict; Tan, Lip Kun

2018-01-01

Germline mutations in the RUNX1 transcription factor give rise to a rare autosomal dominant genetic condition classified under the entity: Familial Platelet Disorders with predisposition to Acute Myeloid Leukaemia (FPD/AML). While several studies have identified a myriad of germline RUNX1 mutations implicated in this disorder, second-hit mutational events are necessary for patients with hereditary thrombocytopenia to develop full-blown AML. The molecular picture behind this process remains unclear. We describe a patient of Malay descent with an unreported 7-bp germline RUNX1 frameshift deletion, who developed second-hit mutations that could have brought about the leukaemic transformation from a pre-leukaemic state. These mutations were charted through the course of the treatment and stem cell transplant, showing a clear correlation between her clinical presentation and the mutations present. The patient was a 27-year-old Malay woman who presented with AML on the background of hereditary thrombocytopenia affecting her father and 3 brothers. Initial molecular testing revealed the same novel RUNX1 mutation in all 5 individuals. The patient received standard induction, consolidation chemotherapy, and a haploidentical stem cell transplant from her mother with normal RUNX1 profile. Comprehensive genomic analyses were performed at diagnosis, post-chemotherapy and post-transplant. A total of 8 mutations ( RUNX1 , GATA2 , DNMT3A , BCORL1 , BCOR , 2 PHF6 and CDKN2A ) were identified in the pre-induction sample, of which 5 remained ( RUNX1 , DNMT3A , BCORL1 , BCOR and 1 out of 2 PHF6 ) in the post-treatment sample and none were present post-transplant. In brief, the 3 mutations which were lost along with the leukemic cells at complete morphological remission were most likely acquired leukemic driver mutations that were responsible for the AML transformation from a pre-leukemic germline RUNX1 -mutated state. On the contrary, the 5 mutations that persisted post

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

Science.gov (United States)

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.

PENERAPAN NEW PUBLIC MANAGEMENT (NPM DI INDONESIA (Reformasi Birokrasi, Desentralisasi, Kerjasama Pemerintah dan Swasta Dalam Meningkatkan Pelayanan Publik

Directory of Open Access Journals (Sweden)

ANI AGUS PUSPAWATI

2016-04-01

Full Text Available New Public management (NPM is a new paradigm in public sector management. And was first developed in the 1980s, especially in New Zealand, the UK and the U.S. as a result of the emergence of welfare state crisis. This paper aims to look at how the application of NPM in Indonesia, particularly in the form of bureaucratic reform through decentralization and relationship between the government (public and private, to improve public services.

Molecular analysis of carbon ion-induced mutations in Arabidopsis thaliana

International Nuclear Information System (INIS)

Shikazono, Naoya; Tanaka, Atsushi; Watanabe, Hiroshi; Tano, Shigemitsu; Yokota, Yukihiko

1998-01-01

In order to elucidate the characteristics of the mutations induced by ion particles at the molecular level in plants, mutated loci in carbon ion-induced mutants of Arabidopsis were investigated by PCR and Southern blot analyses. In the present study, two lines of gl1 mutant and two lines of tt4 mutant were isolated after carbon ion-irradiation. Out of four mutants, one had a deletion, other two contained rearrangements, and one had a point-like mutation. From the present result, it was suggested that ion particles induced different kinds of alterations of the DNA and therefore they could produce various types of mutant alleles in plants. (author)

NPM-ALK mediates phosphorylation of MSH2 at tyrosine 238, creating a functional deficiency in MSH2 and the loss of mismatch repair

International Nuclear Information System (INIS)

Bone, K M; Wang, P; Wu, F; Wu, C; Li, L; Bacani, J T; Andrew, S E; Lai, R

2015-01-01

The vast majority of anaplastic lymphoma kinase-positive anaplastic large cell lymphoma (ALK+ALCL) tumors express the characteristic oncogenic fusion protein NPM-ALK, which mediates tumorigenesis by exerting its constitutive tyrosine kinase activity on various substrates. We recently identified MSH2, a protein central to DNA mismatch repair (MMR), as a novel binding partner and phosphorylation substrate of NPM-ALK. Here, using liquid chromatography–mass spectrometry, we report for the first time that MSH2 is phosphorylated by NPM-ALK at a specific residue, tyrosine 238. Using GP293 cells transfected with NPM-ALK, we confirmed that the MSH2 Y238F mutant is not tyrosine phosphorylated. Furthermore, transfection of MSH2 Y238F into these cells substantially decreased the tyrosine phosphorylation of endogenous MSH2. Importantly, gene transfection of MSH2 Y238F abrogated the binding of NPM-ALK with endogenous MSH2, re-established the dimerization of MSH2:MSH6 and restored the sensitivity to DNA mismatch-inducing drugs, indicative of MMR return. Parallel findings were observed in two ALK+ALCL cell lines, Karpas 299 and SUP-M2. In addition, we found that enforced expression of MSH2 Y238F into ALK+ALCL cells alone was sufficient to induce spontaneous apoptosis. In conclusion, our findings have identified NPM-ALK-induced phosphorylation of MSH2 at Y238 as a crucial event in suppressing MMR. Our studies have provided novel insights into the mechanism by which oncogenic tyrosine kinases disrupt MMR

Investigating the structural impacts of I64T and P311S mutations in APE1-DNA complex: a molecular dynamics approach.

Directory of Open Access Journals (Sweden)

C George Priya Doss

Full Text Available Elucidating the molecular dynamic behavior of Protein-DNA complex upon mutation is crucial in current genomics. Molecular dynamics approach reveals the changes on incorporation of variants that dictate the structure and function of Protein-DNA complexes. Deleterious mutations in APE1 protein modify the physicochemical property of amino acids that affect the protein stability and dynamic behavior. Further, these mutations disrupt the binding sites and prohibit the protein to form complexes with its interacting DNA.In this study, we developed a rapid and cost-effective method to analyze variants in APE1 gene that are associated with disease susceptibility and evaluated their impacts on APE1-DNA complex dynamic behavior. Initially, two different in silico approaches were used to identify deleterious variants in APE1 gene. Deleterious scores that overlap in these approaches were taken in concern and based on it, two nsSNPs with IDs rs61730854 (I64T and rs1803120 (P311S were taken further for structural analysis.Different parameters such as RMSD, RMSF, salt bridge, H-bonds and SASA applied in Molecular dynamic study reveals that predicted deleterious variants I64T and P311S alters the structure as well as affect the stability of APE1-DNA interacting functions. This study addresses such new methods for validating functional polymorphisms of human APE1 which is critically involved in causing deficit in repair capacity, which in turn leads to genetic instability and carcinogenesis.

Homozygous EDNRB mutation in a patient with Waardenburg syndrome type 1.

Science.gov (United States)

Morimoto, Noriko; Mutai, Hideki; Namba, Kazunori; Kaneko, Hiroki; Kosaki, Rika; Matsunaga, Tatsuo

2018-04-01

To examine and expand the genetic spectrum of Waardenburg syndrome type 1 (WS1). Clinical features related to Waardenburg syndrome (WS) were examined in a five-year old patient. Mutation analysis of genes related to WS was performed in the proband and her parents. Molecular modeling of EDNRB and the p.R319W mutant was conducted to predict the pathogenicity of the mutation. The proband showed sensorineural hearing loss, heterochromia iridis, and dystopia canthorum, fulfilling the clinical criteria of WS1. Genetic analyses revealed that the proband had no mutation in PAX3 which has been known as the cause of WS1, but had a homozygous missense mutation (p.R319W) in endothelin receptor type B (EDNRB) gene. The asymptomatic parents had the mutation in a heterozygote state. This mutation has been previously reported in a heterozygous state in a patient with Hirschsprung's disease unaccompanied by WS, but the patient and her parents did not show any symptoms in gastrointestinal tract. Molecular modeling of EDNRB with the p.R319W mutation demonstrated reduction of the positively charged surface area in this region, which might reduce binding ability of EDNRB to G protein and lead to abnormal signal transduction underlying the WS phenotype. Our findings suggested that autosomal recessive mutation in EDNRB may underlie a part of WS1 with the current diagnostic criteria, and supported that Hirschsprung's disease is a multifactorial genetic disease which requires additional factors. Further molecular analysis is necessary to elucidate the gene interaction and to reappraise the current WS classification. Copyright © 2017 Elsevier B.V. All rights reserved.

Integrating or disintegrating effects of customised care: the role of professions beyond NPM.

Science.gov (United States)

Liff, Roy; Andersson, Thomas

2011-01-01

This paper aims to describe the integrating and disintegrating effects of professional actions in customised care. Using a qualitative case study, the authors examine the work practices and cultures of three Swedish child and adolescent psychiatric care units (CAP) charged with providing customised care in collaboration with other organisations. The authors conducted 62 interviews, made 11 half-day observations, and shadowed employees for two days. The social embeddedness of action is crucial to understanding the professions' integrating/disintegrating activities. In the internal social context of CAP, the professions adapt to productivity-enhancing new public management (NPM) principles, resulting in integrating effects between the different professions and administrative management in the CAP units. However, CAP exercises professional dominance over the cooperating organisations. Thus, in the external social context, CAP's resistance to customised care principles exacerbates the disintegration problems among the different organisations. The study concludes that, contrary to findings in many other studies, neither the professional logic nor NPM/customised care reforms determine the actions of professionals. In this case, the institutionalisation of some NPM methods blocks the adoption of customised care practices. Contrary to the widely accepted idea that resource restriction is a main source of conflict between management and the professions, the professions accept and adapt to resource restrictions, even at the expense of de-emphasising the practices of customised care. Thus, since professionals choose different operational strategies depending on the social context, the success of a normative reform measure may depend in part on its social context.

Molecular analysis on germline mutation caused by low-dose irradiation

International Nuclear Information System (INIS)

Uchiyama, R.; Fujikawa, K.; Nishimura, M.; Adzuma, H.; Shimada, Y.; Yamauchi, M.

2003-01-01

Full text: Genetic heterogeneity and a low frequency of germline mutation at single-copy gene loci have limited the direct measurement of germline mutation in human populations. Two conflicting results have been reported for the effect of ionizing radiation on germline mutation in human populations. A study conducted on the first-generation progeny of the survivors of the atomic bombs at Hiroshima and Nagasaki found no significant increase in germline mutations. On the other hand, a significant increase in germline mutation was reported among the human population in the Belarus area after the Chernobyl accident in 1986. We investigated the germline mutation at the molecular level using experimental mouse strains with different genetic backgrounds to assess the risk of ionizing radiation on human populations. The C3H male parents were exposed to X ray (0, 0.3, 1, and 3Gy) and mated with unexposed C57BL females after two weeks interval, so as to detect the germline mutation occurred at the spermatid stage. Genomic DNA samples were prepared from the both parents and F1s, and the genomic DNA sequences were compared between parents and offspring at the specific genomic gene loci, such as adenine phosphoribosyl transferase (aprt) gene and cytidine triphosphate synthetase (ctps) gene, using the automated DNA sequencer. Also hypervariable Pc-1 (Ms6-hm) minisatellite repeat locus was analyzed by using Southern blot hybridization technique. Our preliminary results indicated that the changes of the restriction DNA fragment length in offspring did not reflect the occurrence of the mutation, such as point mutation, insertion, and deletion, in the genomic gene loci including the intervening sequence (intron)

Identification of NPM and DDX5 as Therapeutic Targets in TSC

Science.gov (United States)

2017-12-01

Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012 DISTRIBUTION STATEMENT: Approved for Public Release; Distribution Unlimited...9. SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S) U.S. Army Medical Research and Materiel Command Fort...complexes in TSC cells. 15. SUBJECT TERMS NPM, DDX5, TSC, chemical library , split-luciferase 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT

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

OpenAIRE

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

2010-01-01

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 ...

Molecular analysis of lipoid proteinosis: identification of a novel nonsense mutation in the ECM1 gene in a Pakistani family

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Naeem Muhammad

2011-07-01

Full Text Available Abstract Lipoid proteinosis is a rare autosomal recessive disease characterized by cutaneous and mucosal lesions and hoarseness appearing in early childhood that is caused by homozygous or compound heterozygous mutations in the ECM1 gene located on chromosome 1q21. The aim of the study was to investigate the molecular genetic defect underlying lipoid proteinosis in a consanguineous Pakistani family. Methods Genotyping of seven members of the family was performed by amplifying microsatellite markers, tightly linked to the ECM1 gene. To screen for mutations in the ECM1 gene, all of its exons and splice junctions were PCR amplified from genomic DNA and analyzed by SSCP and sequenced directly in an ABI 3130 genetic analyzer. Results The results revealed linkage of the LP family to the ECM1 locus. Sequence analysis of the coding exons and splice junctions of the ECM1 gene revealed a novel homozygous mutation (c.616C > T in exon 6, predicted to replace glutamine with stop codon (p.Q206X at amino acid position 206. Conclusions The finding of a novel mutation in Pakistani family extends the body of evidence that supports the importance of ECM1 gene for the development of lipoid proteinosis.

CFTR mutations spectrum and the efficiency of molecular diagnostics in Polish cystic fibrosis patients.

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Ewa Ziętkiewicz

Full Text Available Cystic fibrosis (CF is caused by mutations in the cystic fibrosis transmembrane regulator gene (CFTR. In light of the strong allelic heterogeneity and regional specificity of the mutation spectrum, the strategy of molecular diagnostics and counseling in CF requires genetic tests to reflect the frequency profile characteristic for a given population. The goal of the study was to provide an updated comprehensive estimation of the distribution of CFTR mutations in Polish CF patients and to assess the effectiveness of INNOLiPA_CFTR tests in Polish population. The analyzed cohort consisted of 738 patients with the clinically confirmed CF diagnosis, prescreened for molecular defects using INNOLiPA_CFTR panels from Innogenetics. A combined efficiency of INNOLiPA CFTR_19 and CFTR_17_TnUpdate tests was 75.5%; both mutations were detected in 68.2%, and one mutation in 14.8% of the affected individuals. The group composed of all the patients with only one or with no mutation detected (109 and 126 individuals, respectively was analyzed further using a mutation screening approach, i.e. SSCP/HD (single strand conformational polymorphism/heteroduplex analysis of PCR products followed by sequencing of the coding sequence. As a result, 53 more mutations were found in 97 patients. The overall efficiency of the CF allele detection was 82.5% (7.0% increase compared to INNOLiPA tests alone. The distribution of the most frequent mutations in Poland was assessed. Most of the mutations repetitively found in Polish patients had been previously described in other European populations. The most frequent mutated allele, F508del, represented 54.5% of Polish CF chromosomes. Another eight mutations had frequencies over 1%, 24 had frequencies between 1 and 0.1%; c.2052-2053insA and c.3468+2_3468+3insT were the most frequent non-INNOLiPA mutations. Mutation distribution described herein is also relevant to the Polish diaspora. Our study also demonstrates that the reported

Targeting Nucleophosmin 1 Represents a Rational Strategy for Radiation Sensitization

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Sekhar, Konjeti R. [Department of Radiation Oncology, Vanderbilt University School of Medicine, Nashville, Tennessee (United States); Benamar, Mouadh [Department of Radiation Oncology, University of Virginia School of Medicine, Charlottesville, Virginia (United States); Venkateswaran, Amudhan; Sasi, Soumya [Department of Radiation Oncology, Vanderbilt University School of Medicine, Nashville, Tennessee (United States); Penthala, Narsimha R.; Crooks, Peter A. [Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, Arkansas (United States); Hann, Stephen R. [Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee (United States); Geng, Ling [Department of Radiation Oncology, Vanderbilt University School of Medicine, Nashville, Tennessee (United States); Balusu, Ramesh [Department of Internal Medicine, University of Kansas Medical Center, Kansas City, Kansas (United States); Abbas, Tarek [Department of Radiation Oncology, University of Virginia School of Medicine, Charlottesville, Virginia (United States); Freeman, Michael L., E-mail: michael.freeman@vanderbilt.edu [Department of Radiation Oncology, Vanderbilt University School of Medicine, Nashville, Tennessee (United States)

2014-08-01

Purpose: To test the hypothesis that small molecule targeting of nucleophosmin 1 (NPM1) represents a rational approach for radiosensitization. Methods and Materials: Wilde-type and NPM1-deficient mouse embryo fibroblasts (MEFs) were used to determine whether radiosensitization produced by the small molecule YTR107 was NPM1 dependent. The stress response to ionizing radiation was assessed by quantifying pNPM1, γH2AX, and Rad51 foci, neutral comet tail moment, and colony formation. NPM1 levels in a human-derived non-small-cell lung cancer (NSCLC) tissue microarray (TMA) were determined by immunohistochemistry. YTR107-mediated radiosensitization was assessed in NSCLC cell lines and xenografts. Results: Use of NPM1-null MEFs demonstrated that NPM1 is critical for DNA double- strand break (DSB) repair, that loss of NPM1 increases radiation sensitivity, and that YTR107-mediated radiosensitization is NPM1 dependent. YTR107 was shown to inhibit NPM1 oligomerization and impair formation of pNPM1 irradiation-induced foci that colocalized with γH2AX foci. Analysis of the TMA demonstrated that NPM1 is overexpressed in subsets of NSCLC. YTR107 inhibited DNA DSB repair and radiosensitized NSCLC lines and xenografts. Conclusions: These data demonstrate that YTR107-mediated targeting of NPM1 impairs DNA DSB repair, an event that increases radiation sensitivity.

Targeting Nucleophosmin 1 Represents a Rational Strategy for Radiation Sensitization

International Nuclear Information System (INIS)

Sekhar, Konjeti R.; Benamar, Mouadh; Venkateswaran, Amudhan; Sasi, Soumya; Penthala, Narsimha R.; Crooks, Peter A.; Hann, Stephen R.; Geng, Ling; Balusu, Ramesh; Abbas, Tarek; Freeman, Michael L.

2014-01-01

Purpose: To test the hypothesis that small molecule targeting of nucleophosmin 1 (NPM1) represents a rational approach for radiosensitization. Methods and Materials: Wilde-type and NPM1-deficient mouse embryo fibroblasts (MEFs) were used to determine whether radiosensitization produced by the small molecule YTR107 was NPM1 dependent. The stress response to ionizing radiation was assessed by quantifying pNPM1, γH2AX, and Rad51 foci, neutral comet tail moment, and colony formation. NPM1 levels in a human-derived non-small-cell lung cancer (NSCLC) tissue microarray (TMA) were determined by immunohistochemistry. YTR107-mediated radiosensitization was assessed in NSCLC cell lines and xenografts. Results: Use of NPM1-null MEFs demonstrated that NPM1 is critical for DNA double- strand break (DSB) repair, that loss of NPM1 increases radiation sensitivity, and that YTR107-mediated radiosensitization is NPM1 dependent. YTR107 was shown to inhibit NPM1 oligomerization and impair formation of pNPM1 irradiation-induced foci that colocalized with γH2AX foci. Analysis of the TMA demonstrated that NPM1 is overexpressed in subsets of NSCLC. YTR107 inhibited DNA DSB repair and radiosensitized NSCLC lines and xenografts. Conclusions: These data demonstrate that YTR107-mediated targeting of NPM1 impairs DNA DSB repair, an event that increases radiation sensitivity

Wild-type and mutated IDH1/2 enzymes and therapy responses.

Science.gov (United States)

Molenaar, Remco J; Maciejewski, Jaroslaw P; Wilmink, Johanna W; van Noorden, Cornelis J F

2018-04-01

Isocitrate dehydrogenase 1 and 2 (IDH1/2) are key enzymes in cellular metabolism, epigenetic regulation, redox states, and DNA repair. IDH1/2 mutations are causal in the development and/or progression of various types of cancer due to supraphysiological production of D-2-hydroxyglutarate. In various tumor types, IDH1/2-mutated cancers predict for improved responses to treatment with irradiation or chemotherapy. The present review discusses the molecular basis of the sensitivity of IDH1/2-mutated cancers with respect to the function of mutated IDH1/2 in cellular processes and their interactions with novel IDH1/2-mutant inhibitors. Finally, lessons learned from IDH1/2 mutations for future clinical applications in IDH1/2 wild-type cancers are discussed.

Mesothelioma patient derived tumor xenografts with defined BAP1 mutations that mimic the molecular characteristics of human malignant mesothelioma

International Nuclear Information System (INIS)

Kalra, Neetu; Zhang, Jingli; Thomas, Anish; Xi, Liqiang; Cheung, Mitchell; Talarchek, Jacqueline; Burkett, Sandra; Tsokos, Maria G; Chen, Yuanbin; Raffeld, Mark; Miettinen, Markku; Pastan, Ira; Testa, Joseph R; Hassan, Raffit

2015-01-01

The development and evaluation of new therapeutic approaches for malignant mesothelioma has been sparse due, in part, to lack of suitable tumor models. We established primary mesothelioma cultures from pleural and ascitic fluids of five patients with advanced mesothelioma. Electron microscopy and immunohistochemistry (IHC) confirmed their mesothelial origin. Patient derived xenografts were generated by injecting the cells in nude or SCID mice, and malignant potential of the cells was analyzed by soft agar colony assay. Molecular profiles of the primary patient tumors, early passage cell cultures, and patient derived xenografts were assessed using mutational analysis, fluorescence in situ hybridization (FISH) analysis and IHC. Primary cultures from all five tumors exhibited morphologic and IHC features consistent to those of mesothelioma cells. Mutations of BAP1 and CDKN2A were each detected in four tumors. BAP1 mutation was associated with the lack of expression of BAP1 protein. Three cell cultures, all of which were derived from BAP1 mutant primary tumors, exhibited anchorage independent growth and also formed tumors in mice, suggesting that BAP1 loss may enhance tumor growth in vivo. Both early passage cell cultures and mouse xenograft tumors harbored BAP1 mutations and CDKN2A deletions identical to those found in the corresponding primary patient tumors. The mesothelioma patient derived tumor xenografts with mutational alterations that mimic those observed in patient tumors which we established can be used for preclinical development of novel drug regimens and for studying the functional aspects of BAP1 biology in mesothelioma. The online version of this article (doi:10.1186/s12885-015-1362-2) contains supplementary material, which is available to authorized users

New mutations and an updated database for the patched-1 (PTCH1) gene.

Science.gov (United States)

Reinders, Marie G; van Hout, Antonius F; Cosgun, Betûl; Paulussen, Aimée D; Leter, Edward M; Steijlen, Peter M; Mosterd, Klara; van Geel, Michel; Gille, Johan J

2018-05-01

Basal cell nevus syndrome (BCNS) is an autosomal dominant disorder characterized by multiple basal cell carcinomas (BCCs), maxillary keratocysts, and cerebral calcifications. BCNS most commonly is caused by a germline mutation in the patched-1 (PTCH1) gene. PTCH1 mutations are also described in patients with holoprosencephaly. We have established a locus-specific database for the PTCH1 gene using the Leiden Open Variation Database (LOVD). We included 117 new PTCH1 variations, in addition to 331 previously published unique PTCH1 mutations. These new mutations were found in 141 patients who had a positive PTCH1 mutation analysis in either the VU University Medical Centre (VUMC) or Maastricht University Medical Centre (MUMC) between 1995 and 2015. The database contains 331 previously published unique PTCH1 mutations and 117 new PTCH1 variations. We have established a locus-specific database for the PTCH1 gene using the Leiden Open Variation Database (LOVD). The database provides an open collection for both clinicians and researchers and is accessible online at http://www.lovd.nl/PTCH1. © 2018 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.

The germacranolide sesquiterpene lactone neurolenin B of the medicinal plant Neurolaena lobata (L.) R.Br. ex Cass inhibits NPM/ALK-driven cell expansion and NF-κB-driven tumour intravasation.

Science.gov (United States)

Unger, Christine; Kiss, Izabella; Vasas, Andrea; Lajter, Ildikó; Kramer, Nina; Atanasov, Atanas Georgiev; Nguyen, Chi Huu; Chatuphonprasert, Waranya; Brenner, Stefan; Krieger, Sigurd; McKinnon, Ruxandra; Peschel, Andrea; Kain, Renate; Saiko, Philipp; Szekeres, Thomas; Kenner, Lukas; Hassler, Melanie R; Diaz, Rene; Frisch, Richard; Dirsch, Verena M; Jäger, Walter; de Martin, Rainer; Bochkov, Valery N; Passreiter, Claus M; Peter-Vörösmarty, Barbara; Mader, Robert M; Grusch, Michael; Dolznig, Helmut; Kopp, Brigitte; Zupko, Istvan; Hohmann, Judit; Krupitza, Georg

2015-08-15

The t(2;5)(p23;q35) chromosomal translocation results in the expression of the fusion protein NPM/ALK that when expressed in T-lymphocytes gives rise to anaplastic large cell lymphomas (ALCL). In search of new therapy options the dichloromethane extract of the ethnomedicinal plant Neurolaena lobata (L.) R.Br. ex Cass was shown to inhibit NPM/ALK expression. Therefore, we analysed whether the active principles that were recently isolated and found to inhibit inflammatory responses specifically inhibit growth of NPM/ALK+ ALCL, leukaemia and breast cancer cells, but not of normal cells, and the intravasation through the lymphendothelial barrier. ALCL, leukaemia and breast cancer cells, and normal peripheral blood mononuclear cells (PBMCs) were treated with isolated sesquiterpene lactones and analysed for cell cycle progression, proliferation, mitochondrial activity, apoptosis, protein and mRNA expression, NF-κB and cytochrome P450 activity, 12(S)-HETE production and lymphendothelial intravasation. In vitro treatment of ALCL by neurolenin B suppressed NPM/ALK, JunB and PDGF-Rβ expression, inhibited the growth of ALCL cells late in M phase, and induced apoptosis via caspase 3 without compromising mitochondrial activity (as a measure of general exogenic toxicity). Moreover, neurolenin B attenuated tumour spheroid intravasation probably through inhibition of NF-κB and CYP1A1. Neurolenin B specifically decreased pro-carcinogenic NPM/ALK expression in ALK+ ALCL cells and, via the inhibition of NF-kB signalling, attenuated tumour intra/extravasation into the lymphatics. Hence, neurolenin B may open new options to treat ALCL and to manage early metastatic processes to which no other therapies exist. Copyright © 2015 Elsevier GmbH. All rights reserved.

Acquired copy-neutral loss of heterozygosity of chromosome 1p as a molecular event associated with marrow fibrosis in MPL-mutated myeloproliferative neoplasms.

Science.gov (United States)

Rumi, Elisa; Pietra, Daniela; Guglielmelli, Paola; Bordoni, Roberta; Casetti, Ilaria; Milanesi, Chiara; Sant'Antonio, Emanuela; Ferretti, Virginia; Pancrazzi, Alessandro; Rotunno, Giada; Severgnini, Marco; Pietrelli, Alessandro; Astori, Cesare; Fugazza, Elena; Pascutto, Cristiana; Boveri, Emanuela; Passamonti, Francesco; De Bellis, Gianluca; Vannucchi, Alessandro; Cazzola, Mario

2013-05-23

We studied mutations of MPL exon 10 in patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF), first investigating a cohort of 892 consecutive patients. MPL mutation scanning was performed on granulocyte genomic DNA by using a high-resolution melt assay, and the mutant allele burden was evaluated by using deep sequencing. Somatic mutations of MPL, all but one involving codon W515, were detected in 26/661 (4%) patients with ET, 10/187 (5%) with PMF, and 7/44 (16%) patients with post-ET myelofibrosis. Comparison of JAK2 (V617F)-mutated and MPL-mutated patients showed only minor phenotypic differences. In an extended group of 62 MPL-mutated patients, the granulocyte mutant allele burden ranged from 1% to 95% and was significantly higher in patients with PMF or post-ET myelofibrosis compared with those with ET. Patients with higher mutation burdens had evidence of acquired copy-neutral loss of heterozygosity (CN-LOH) of chromosome 1p in granulocytes, consistent with a transition from heterozygosity to homozygosity for the MPL mutation in clonal cells. A significant association was found between MPL-mutant allele burden greater than 50% and marrow fibrosis. These observations suggest that acquired CN-LOH of chromosome 1p involving the MPL location may represent a molecular mechanism of fibrotic transformation in MPL-mutated myeloproliferative neoplasms.

Noonan syndrome-causing genes: Molecular update and an assessment of the mutation rate

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Ihssane El Bouchikhi

2016-12-01

Full Text Available Noonan syndrome is a common autosomal dominant disorder characterized by short stature, congenital heart disease and facial dysmorphia with an incidence of 1/1000 to 2500 live births. Up to now, several genes have been proven to be involved in the disturbance of the transduction signal through the RAS-MAP Kinase pathway and the manifestation of Noonan syndrome. The first gene described was PTPN11, followed by SOS1, RAF1, KRAS, BRAF, NRAS, MAP2K1, and RIT1, and recently SOS2, LZTR1, and A2ML1, among others. Progressively, the physiopathology and molecular etiology of most signs of Noonan syndrome have been demonstrated, and inheritance patterns as well as genetic counseling have been established. In this review, we summarize the data concerning clinical features frequently observed in Noonan syndrome, and then, we describe the molecular etiology as well as the physiopathology of most Noonan syndrome-causing genes. In the second part of this review, we assess the mutational rate of Noonan syndrome-causing genes reported up to now in most screening studies. This review should give clinicians as well as geneticists a full view of the molecular aspects of Noonan syndrome and the authentic prevalence of the mutational events of its causing-genes. It will also facilitate laying the groundwork for future molecular diagnosis research, and the development of novel treatment strategies.

FANCA Gene Mutations with 8 Novel Molecular Changes in Indian Fanconi Anemia Patients.

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Avani Solanki

Full Text Available Fanconi anemia (FA, a rare heterogeneous genetic disorder, is known to be associated with 19 genes and a spectrum of clinical features. We studied FANCA molecular changes in 34 unrelated and 2 siblings of Indian patients with FA and have identified 26 different molecular changes of FANCA gene, of which 8 were novel mutations (a small deletion c.2500delC, 4 non-sense mutations c.2182C>T, c.2630C>G, c.3677C>G, c.3189G>A; and 3 missense mutations; c.1273G>C, c.3679 G>C, and c.3992 T>C. Among these only 16 patients could be assigned FA-A complementation group, because we could not confirm single exon deletions detected by MLPA or cDNA amplification by secondary confirmation method and due to presence of heterozygous non-pathogenic variations or heterozygous pathogenic mutations. An effective molecular screening strategy should be developed for confirmation of these mutations and determining the breakpoints for single exon deletions.

FANCA Gene Mutations with 8 Novel Molecular Changes in Indian Fanconi Anemia Patients.

Science.gov (United States)

Solanki, Avani; Mohanty, Purvi; Shukla, Pallavi; Rao, Anita; Ghosh, Kanjaksha; Vundinti, Babu Rao

2016-01-01

Fanconi anemia (FA), a rare heterogeneous genetic disorder, is known to be associated with 19 genes and a spectrum of clinical features. We studied FANCA molecular changes in 34 unrelated and 2 siblings of Indian patients with FA and have identified 26 different molecular changes of FANCA gene, of which 8 were novel mutations (a small deletion c.2500delC, 4 non-sense mutations c.2182C>T, c.2630C>G, c.3677C>G, c.3189G>A; and 3 missense mutations; c.1273G>C, c.3679 G>C, and c.3992 T>C). Among these only 16 patients could be assigned FA-A complementation group, because we could not confirm single exon deletions detected by MLPA or cDNA amplification by secondary confirmation method and due to presence of heterozygous non-pathogenic variations or heterozygous pathogenic mutations. An effective molecular screening strategy should be developed for confirmation of these mutations and determining the breakpoints for single exon deletions.

From molecular genetics to phylodynamics: evolutionary relevance of mutation rates across viruses.

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Rafael Sanjuán

Full Text Available Although evolution is a multifactorial process, theory posits that the speed of molecular evolution should be directly determined by the rate at which spontaneous mutations appear. To what extent these two biochemical and population-scale processes are related in nature, however, is largely unknown. Viruses are an ideal system for addressing this question because their evolution is fast enough to be observed in real time, and experimentally-determined mutation rates are abundant. This article provides statistically supported evidence that the mutation rate determines molecular evolution across all types of viruses. Properties of the viral genome such as its size and chemical composition are identified as major determinants of these rates. Furthermore, a quantitative analysis reveals that, as expected, evolution rates increase linearly with mutation rates for slowly mutating viruses. However, this relationship plateaus for fast mutating viruses. A model is proposed in which deleterious mutations impose an evolutionary speed limit and set an extinction threshold in nature. The model is consistent with data from replication kinetics, selection strength and chemical mutagenesis studies.

Exploring the common molecular basis for the universal DNA mutation bias: Revival of Loewdin mutation model

International Nuclear Information System (INIS)

Fu, Liang-Yu; Wang, Guang-Zhong; Ma, Bin-Guang; Zhang, Hong-Yu

2011-01-01

Highlights: → There exists a universal G:C → A:T mutation bias in three domains of life. → This universal mutation bias has not been sufficiently explained. → A DNA mutation model proposed by Loewdin 40 years ago offers a common explanation. -- Abstract: Recently, numerous genome analyses revealed the existence of a universal G:C → A:T mutation bias in bacteria, fungi, plants and animals. To explore the molecular basis for this mutation bias, we examined the three well-known DNA mutation models, i.e., oxidative damage model, UV-radiation damage model and CpG hypermutation model. It was revealed that these models cannot provide a sufficient explanation to the universal mutation bias. Therefore, we resorted to a DNA mutation model proposed by Loewdin 40 years ago, which was based on inter-base double proton transfers (DPT). Since DPT is a fundamental and spontaneous chemical process and occurs much more frequently within GC pairs than AT pairs, Loewdin model offers a common explanation for the observed universal mutation bias and thus has broad biological implications.

Contribution of CYP1B1 mutations and founder effect to primary congenital glaucoma in Mexico.

Science.gov (United States)

Zenteno, Juan Carlos; Hernandez-Merino, Elena; Mejia-Lopez, Herlinda; Matías-Florentino, Margarita; Michel, Norma; Elizondo-Olascoaga, Celia; Korder-Ortega, Vincent; Casab-Rueda, Homero; Garcia-Ortiz, Jose Elias

2008-01-01

The frequency of primary congenital glaucoma (PCG)-causing CYP1B1 mutations varies importantly among distinct populations, ranging from 20% in Indonesians and Japanese to about 100% among the Saudi Arabians and Slovakian Gypsies. Thus, the molecular characterization of large groups of PCG from different ethnic backgrounds is important to establish the actual CYP1B1 contribution in specific populations. In this work, the molecular analysis of the CYP1B1 gene in a group of Mexican PCG patients is reported. Thirty unrelated Mexican patients fulfilling the clinical criteria for PCG were included. Two cases were familial and with proven consanguinity, originating from distinct regions of the country. Polymerase chain reaction amplification and direct automated sequencing of the CYP1B1 coding region was performed in each participating subject. An identical pathogenic CYP1B1 mutation was demonstrated in 2 unrelated PCG subjects. The mutation consisted of a homozygous G to A transition at nucleotide position 1505 in exon 3, which predicted a substitution of glutamic acid for lysine at residue 387 of the protein (E387K). In the remaining 28 PCG subjects, no deleterious mutations were identified. Both subjects with the E387K mutation shared a same haplotype for 5 CYP1B1 intragenic single nucleotide polymorphisms, indicating a common origin of the allele. Mexican patients with PCG are rarely (less than 10%) due to CYP1B1 mutations. Available data indicate that most of the non-Brazilian Latin American PCG patients investigated to date are not due to CYP1B1 defects. Populations with low incidence of CYP1B1 mutations are appropriate candidates for the identification of novel PCG-causing genes.

Clinical and molecular characterization of limb-girdle muscular dystrophy due to LAMA2 mutations

DEFF Research Database (Denmark)

Gavassini, Bruno F; Carboni, Nicola; Nielsen, Jørgen E

2011-01-01

In this study we describe the clinical and molecular characteristics of limb-girdle muscular dystrophy (LGMD) due to LAMA2 mutations.......In this study we describe the clinical and molecular characteristics of limb-girdle muscular dystrophy (LGMD) due to LAMA2 mutations....

Aromatase expression is increased in BRCA1 mutation carriers

International Nuclear Information System (INIS)

Chand, Ashwini L; KConFab; Simpson, Evan R; Clyne, Colin D

2009-01-01

Until recently, the molecular mechanisms explaining increased incidence of ovarian and breast cancers in carriers of BRCA1 gene mutations had not been clearly understood. Of significance is the finding that BRCA1 negatively regulates aromatase expression in vitro. Our objective was to characterise aromatase gene (CYP19A1) and its promoter expression in breast adipose and ovarian tissue in BRCA1 mutation carriers and unaffected controls. We measured aromatase transcripts, total and promoter-specific (PII, PI.3, PI.4) in prophylactic oophorectomy or mastectomy, therapeutic mastectomy, ovarian and breast tissue from unaffected women. We demonstrate that the lack of functional BRCA1 protein correlates to higher aromatase levels in 85% of BRCA1 mutation carriers. This increase is mediated by aberrant transcriptional regulation of aromatase; in breast adipose by increases in promoter II/I.3 and I.4-specific transcripts; and in the ovary with elevation in promoter I.3 and II-specific transcripts. Understanding the link between BRCA1 and aromatase is significant in terms of understanding why carcinogenesis is restricted to estrogen-producing tissues in BRCA1 mutation carriers

Adenoviral protein V promotes a process of viral assembly through nucleophosmin 1

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Ugai, Hideyo; Dobbins, George C.; Wang, Minghui [Division of Human Gene Therapy, Departments of Medicine, Obstetrics and Gynecology, Pathology, and Surgery, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Le, Long P. [Massachusetts General Hospital, Pathology Service, 55 Fruit St.-GRJ 249, Boston, MA 02114 (United States); Matthews, David A. [School of Cellular and Molecular Medicine, Medical Sciences Building, University of Bristol, Bristol BS8 1TD (United Kingdom); Curiel, David T., E-mail: dcuriel@radonc.wustl.edu [Division of Human Gene Therapy, Departments of Medicine, Obstetrics and Gynecology, Pathology, and Surgery, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); The Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL 35294 (United States)

2012-10-25

Adenoviral infection induces nucleoplasmic redistribution of a nucleolar nucleophosmin 1/NPM1/B23.1. NPM1 is preferentially localized in the nucleoli of normal cells, whereas it is also present at the nuclear matrix in cancer cells. However, the biological roles of NPM1 during infection are unknown. Here, by analyzing a pV-deletion mutant, Ad5-dV/TSB, we demonstrate that pV promotes the NPM1 translocation from the nucleoli to the nucleoplasm in normal cells, and the NPM1 translocation is correlated with adenoviral replication. Lack of pV causes a dramatic reduction of adenoviral replication in normal cells, but not cancer cells, and Ad5-dV/TSB was defective in viral assembly in normal cells. NPM1 knockdown inhibits adenoviral replication, suggesting an involvement of NPM1 in adenoviral biology. Further, we show that NPM1 interacts with empty adenovirus particles which are an intermediate during virion maturation by immunoelectron microscopy. Collectively, these data implicate that pV participates in a process of viral assembly through NPM1.

Research progress of IDH1 and IDH2 mutations in gliomas

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Shan-shan ZHANG

2015-11-01

Full Text Available The gene mutations of isocitrate dehydrogenase 1 and 2 (IDH1/2 mainly occur in astrocytoma, anaplastic astrocytoma, oligodendroglioma, anaplastic oligodendroglioma, oligoastrocytoma, anaplastic oligoastrocytoma and secondary glioblastoma. The IDH1/2 gene mutation can alter proteinase function, consume α-ketoglutarate and nicotinamide adenine dinucleotide phosphate-reduced (NADPH and thus produce carcinogenic metabolite, 2-hydroxyglutarate. The intracellular accumulation of 2-hydroxyglutarate will induce a series of downstream effects which may result in the development of gliomas mentioned above. Both IDH1/2 mutations and other concomitant hereditary variations are biomarkers for differential diagnosis and IDH1/2 mutations are also independent factors for the prognosis of gliomas. The molecular targeting therapy for IDH1/2 mutations has become the research focus of glioma treatment. This review summarizes the recent progress of this field. DOI: 10.3969/j.issn.1672-6731.2015.11.017

Seeking new mutation clues from Bacillus licheniformis amylase by molecular dynamics simulations

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Lu, Tao

2009-07-01

Amylase is one of the most important industrial enzymes in the world. Researchers have been searching for a highly thermal stable mutant for many years, but most focus on point mutations of one or few nitrogenous bases. According to this molecular dynamic simulation of amylase from Bacillus licheniformis (BLA), the deletion of some nitrogenous bases would be more efficacious than point mutations. The simulation reveals strong fluctuation of the BLA structure at optimum temperature. The fluctuation of the outer domains of BLA is stronger than that of the core domain. Molecular simulation provides a clue to design thermal stable amylases through deletion mutations in the outer domain.

Vibrational, DFT, thermal and dielectric studies on 3-nitrophenol-1,3,5-triazine-2,4,6-triamine (2/1)

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Sangeetha, V.; Govindarajan, M.; Kanagathara, N.; Marchewka, M. K.; Gunasekaran, S.; Anbalagan, G.

2014-01-01

A new organic-organic salt, 3-nitrophenol-1,3,5-triazine-2,4,6-triamine (2/1) (3-NPM) has been synthesized by slow evaporation technique at room temperature. Single crystal X-ray diffraction analysis reveals that 3-NPM crystallizes in orthorhombic system with centrosymmetric space group Pbca and the lattice parameters are a = 15.5150(6) Å, b = 12.9137(6) Å, c = 17.8323(6) Å, α = β = γ = 90° and V = 3572.8(2) (Å)3. The geometry, fundamental vibrational frequencies are interpreted with the aid of structure optimization and normal coordinate force field calculations based on density functional theory (DFT) B3LYP/6-311G(d,p) method. IR and Raman spectra of 3-NPM have been recorded and analyzed. The complete vibrational assignments are made on the basis of potential energy distribution (PED). The electric dipole moment, polarizability and the first order hyperpolarizability values of the 3-NPM have been calculated. 1H and 13C NMR chemical shifts are calculated by using the gauge independent atomic orbital (GIAO) method with B3LYP method with 6-311G (d,p) basis set. Moreover, molecular electrostatic potential (MEP) and thermodynamic properties are performed. Mulliken and Natural charges of the title molecule are also calculated and interpreted. Thermal decomposition behavior of 3-NPM has been studied by means of thermogravimetric analysis. The dielectric measurements on the powdered sample have been carried out and the variation of dielectric constant and dielectric loss at different frequencies of the applied field has been studied and the results are discussed in detail.

Insights into amyloid-like aggregation of H2 region of the C-terminal domain of nucleophosmin.

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Russo, Anna; Diaferia, Carlo; La Manna, Sara; Giannini, Cinzia; Sibillano, Teresa; Accardo, Antonella; Morelli, Giancarlo; Novellino, Ettore; Marasco, Daniela

2017-02-01

Nucleophosmin (NPM1) is a multifunctional protein involved in a variety of biological processes including the pathogenesis of several human malignancies and is the most frequently mutated gene in Acute Myeloid Leukemia (AML). To deepen the role of protein regions in its biological activities, lately we reported on the structural behavior of dissected C-terminal domain (CTD) helical fragments. Unexpectedly the H2 (residues 264-277) and H3 AML-mutated regions showed a remarkable tendency to form amyloid-like assemblies with fibrillar morphology and β-sheet structure that resulted as toxic when exposed to human neuroblastoma cells. More recently NPM1 was found to be highly expressed and toxic in neurons of mouse models of Huntington's disease (HD). Here we investigate the role of each residue in the β-strand aggregation process of H2 region of NPM1 by performing a systematic alanine scan of its sequence and structural and kinetic analyses of aggregation of derived peptides by means of Circular Dichorism (CD) and Thioflavin T (Th-T) assay. These solution state investigations pointed out the crucial role exerted by the basic amyloidogenic stretch of H2 (264-271) and to shed light on the initial and main interactions involved in fibril formation we performed studies on fibrils deriving from the related Ala peptides through the analysis of fibrils with birefringence of polarized optical microscopy and wide-angle X-ray scattering (WAXS). This analysis suggested that the presence of branched Ile 269 conferred preferential packing patterns that, instead, appeared geometrically hampered by the aromatic side-chain of Phe 268 . Present investigations could be useful to deepen the knowledge of AML molecular mechanisms and the role of cytoplasmatic aggregates of NPM1c+. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular Diagnostics of Copper-Transporting Protein Mutations Allows Early Onset Individual Therapy of Menkes Disease.

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Králík, L; Flachsová, E; Hansíková, H; Saudek, V; Zeman, J; Martásek, P

2017-01-01

Menkes disease is a severe X-linked recessive disorder caused by a defect in the ATP7A gene, which encodes a membrane copper-transporting ATPase. Deficient activity of the ATP7A protein results in decreased intestinal absorption of copper, low copper level in serum and defective distribution of copper in tissues. The clinical symptoms are caused by decreased activities of copper-dependent enzymes and include neurodegeneration, connective tissue disorders, arterial changes and hair abnormalities. Without therapy, the disease is fatal in early infancy. Rapid diagnosis of Menkes disease and early start of copper therapy is critical for the effectiveness of treatment. We report a molecular biology-based strategy that allows early diagnosis of copper transport defects and implementation of individual therapies before the full development of pathological symptoms. Low serum copper and decreased activity of copperdependent mitochondrial cytochrome c oxidase in isolated platelets found in three patients indicated a possibility of functional defects in copper-transporting proteins, especially in the ATPA7 protein, a copper- transporting P-type ATPase. Rapid mutational screening of the ATP7A gene using high-resolution melting analysis of DNA indicated presence of mutations in the patients. Molecular investigation for mutations in the ATP7A gene revealed three nonsense mutations: c.2170C>T (p.Gln724Ter); c.3745G>T (p.Glu1249Ter); and c.3862C>T (p.Gln1288Ter). The mutation c.3745G>T (p.Glu1249Ter) has not been identified previously. Molecular analysis of the ATOX1 gene as a possible modulating factor of Menkes disease did not reveal presence of pathogenic mutations. Molecular diagnostics allowed early onset of individual therapies, adequate genetic counselling and prenatal diagnosis in the affected families.

HAEdb: a novel interactive, locus-specific mutation database for the C1 inhibitor gene.

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Kalmár, Lajos; Hegedüs, Tamás; Farkas, Henriette; Nagy, Melinda; Tordai, Attila

2005-01-01

Hereditary angioneurotic edema (HAE) is an autosomal dominant disorder characterized by episodic local subcutaneous and submucosal edema and is caused by the deficiency of the activated C1 esterase inhibitor protein (C1-INH or C1INH; approved gene symbol SERPING1). Published C1-INH mutations are represented in large universal databases (e.g., OMIM, HGMD), but these databases update their data rather infrequently, they are not interactive, and they do not allow searches according to different criteria. The HAEdb, a C1-INH gene mutation database (http://hae.biomembrane.hu) was created to contribute to the following expectations: 1) help the comprehensive collection of information on genetic alterations of the C1-INH gene; 2) create a database in which data can be searched and compared according to several flexible criteria; and 3) provide additional help in new mutation identification. The website uses MySQL, an open-source, multithreaded, relational database management system. The user-friendly graphical interface was written in the PHP web programming language. The website consists of two main parts, the freely browsable search function, and the password-protected data deposition function. Mutations of the C1-INH gene are divided in two parts: gross mutations involving DNA fragments >1 kb, and micro mutations encompassing all non-gross mutations. Several attributes (e.g., affected exon, molecular consequence, family history) are collected for each mutation in a standardized form. This database may facilitate future comprehensive analyses of C1-INH mutations and also provide regular help for molecular diagnostic testing of HAE patients in different centers.

Effects of Mutations on Structure-Function Relationships of Matrix Metalloproteinase-1.

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Singh, Warispreet; Fields, Gregg B; Christov, Christo Z; Karabencheva-Christova, Tatyana G

2016-10-14

Matrix metalloproteinase-1 (MMP-1) is one of the most widely studied enzymes involved in collagen degradation. Mutations of specific residues in the MMP-1 hemopexin-like (HPX) domain have been shown to modulate activity of the MMP-1 catalytic (CAT) domain. In order to reveal the structural and conformational effects of such mutations, a molecular dynamics (MD) study was performed of in silico mutated residues in the X-ray crystallographic structure of MMP-1 complexed with a collagen-model triple-helical peptide (THP). The results indicate an important role of the mutated residues in MMP-1 interactions with the THP and communication between the CAT and the HPX domains. Each mutation has a distinct impact on the correlated motions in the MMP-1•THP. An increased collagenase activity corresponded to the appearance of a unique anti-correlated motion and decreased correlated motions, while decreased collagenase activity corresponded both to increased and decreased anti-correlated motions.

Effects of Mutations on Structure–Function Relationships of Matrix Metalloproteinase-1

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Warispreet Singh

2016-10-01

Full Text Available Matrix metalloproteinase-1 (MMP-1 is one of the most widely studied enzymes involved in collagen degradation. Mutations of specific residues in the MMP-1 hemopexin-like (HPX domain have been shown to modulate activity of the MMP-1 catalytic (CAT domain. In order to reveal the structural and conformational effects of such mutations, a molecular dynamics (MD study was performed of in silico mutated residues in the X-ray crystallographic structure of MMP-1 complexed with a collagen-model triple-helical peptide (THP. The results indicate an important role of the mutated residues in MMP-1 interactions with the THP and communication between the CAT and the HPX domains. Each mutation has a distinct impact on the correlated motions in the MMP-1•THP. An increased collagenase activity corresponded to the appearance of a unique anti-correlated motion and decreased correlated motions, while decreased collagenase activity corresponded both to increased and decreased anti-correlated motions.

Pitfalls in genetic testing: the story of missed SCN1A mutations.

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Djémié, Tania; Weckhuysen, Sarah; von Spiczak, Sarah; Carvill, Gemma L; Jaehn, Johanna; Anttonen, Anna-Kaisa; Brilstra, Eva; Caglayan, Hande S; de Kovel, Carolien G; Depienne, Christel; Gaily, Eija; Gennaro, Elena; Giraldez, Beatriz G; Gormley, Padhraig; Guerrero-López, Rosa; Guerrini, Renzo; Hämäläinen, Eija; Hartmann, Corinna; Hernandez-Hernandez, Laura; Hjalgrim, Helle; Koeleman, Bobby P C; Leguern, Eric; Lehesjoki, Anna-Elina; Lemke, Johannes R; Leu, Costin; Marini, Carla; McMahon, Jacinta M; Mei, Davide; Møller, Rikke S; Muhle, Hiltrud; Myers, Candace T; Nava, Caroline; Serratosa, Jose M; Sisodiya, Sanjay M; Stephani, Ulrich; Striano, Pasquale; van Kempen, Marjan J A; Verbeek, Nienke E; Usluer, Sunay; Zara, Federico; Palotie, Aarno; Mefford, Heather C; Scheffer, Ingrid E; De Jonghe, Peter; Helbig, Ingo; Suls, Arvid

2016-07-01

Sanger sequencing, still the standard technique for genetic testing in most diagnostic laboratories and until recently widely used in research, is gradually being complemented by next-generation sequencing (NGS). No single mutation detection technique is however perfect in identifying all mutations. Therefore, we wondered to what extent inconsistencies between Sanger sequencing and NGS affect the molecular diagnosis of patients. Since mutations in SCN1A, the major gene implicated in epilepsy, are found in the majority of Dravet syndrome (DS) patients, we focused on missed SCN1A mutations. We sent out a survey to 16 genetic centers performing SCN1A testing. We collected data on 28 mutations initially missed using Sanger sequencing. All patients were falsely reported as SCN1A mutation-negative, both due to technical limitations and human errors. We illustrate the pitfalls of Sanger sequencing and most importantly provide evidence that SCN1A mutations are an even more frequent cause of DS than already anticipated.

Molecular Mechanisms of Glutamine Synthetase Mutations that Lead to Clinically Relevant Pathologies.

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Benedikt Frieg

2016-02-01

Full Text Available Glutamine synthetase (GS catalyzes ATP-dependent ligation of ammonia and glutamate to glutamine. Two mutations of human GS (R324C and R341C were connected to congenital glutamine deficiency with severe brain malformations resulting in neonatal death. Another GS mutation (R324S was identified in a neurologically compromised patient. However, the molecular mechanisms underlying the impairment of GS activity by these mutations have remained elusive. Molecular dynamics simulations, free energy calculations, and rigidity analyses suggest that all three mutations influence the first step of GS catalytic cycle. The R324S and R324C mutations deteriorate GS catalytic activity due to loss of direct interactions with ATP. As to R324S, indirect, water-mediated interactions reduce this effect, which may explain the suggested higher GS residual activity. The R341C mutation weakens ATP binding by destabilizing the interacting residue R340 in the apo state of GS. Additionally, the mutation is predicted to result in a significant destabilization of helix H8, which should negatively affect glutamate binding. This prediction was tested in HEK293 cells overexpressing GS by dot-blot analysis: Structural stability of H8 was impaired through mutation of amino acids interacting with R341, as indicated by a loss of masking of an epitope in the glutamate binding pocket for a monoclonal anti-GS antibody by L-methionine-S-sulfoximine; in contrast, cells transfected with wild type GS showed the masking. Our analyses reveal complex molecular effects underlying impaired GS catalytic activity in three clinically relevant mutants. Our findings could stimulate the development of ATP binding-enhancing molecules by which the R324S mutant can be repaired extrinsically.

FANCA Gene Mutations with 8 Novel Molecular Changes in Indian Fanconi Anemia Patients

OpenAIRE

Solanki, Avani; Mohanty, Purvi; Shukla, Pallavi; Rao, Anita; Ghosh, Kanjaksha; Vundinti, Babu Rao

2016-01-01

Fanconi anemia (FA), a rare heterogeneous genetic disorder, is known to be associated with 19 genes and a spectrum of clinical features. We studied FANCA molecular changes in 34 unrelated and 2 siblings of Indian patients with FA and have identified 26 different molecular changes of FANCA gene, of which 8 were novel mutations (a small deletion c.2500delC, 4 non-sense mutations c.2182C>T, c.2630C>G, c.3677C>G, c.3189G>A; and 3 missense mutations; c.1273G>C, c.3679 G>C, and c.3992 T>C). Among t...

Pitfalls in genetic testing: the story of missed SCN1A mutations

OpenAIRE

Djémié, T.; Weckhuysen, S.; von Spiczak, S.; Carvill, G. L.; Jaehn, J.; Anttonen, A-K; Brilstra, E.; Caglayan, H. S.; de Kovel, C. G.; Depienne, C.; Gaily, E.; Gennaro, E.; Giraldez, B. G.; Gormley, P.; Guerrero-López, R.

2016-01-01

BACKGROUND: Sanger sequencing, still the standard technique for genetic testing in most diagnostic laboratories and until recently widely used in research, is gradually being complemented by next-generation sequencing (NGS). No single mutation detection technique is however perfect in identifying all mutations. Therefore, we wondered to what extent inconsistencies between Sanger sequencing and NGS affect the molecular diagnosis of patients. Since mutations in SCN1A, the major gene implicated ...

Pitfalls in genetic testing : the story of missed SCN1A mutations

OpenAIRE

Djémié, Tania; Weckhuysen, Sarah; von Spiczak, Sarah; Carvill, Gemma L; Jaehn, Johanna; Anttonen, Anna-Kaisa; Brilstra, Eva; Caglayan, Hande S; de Kovel, Carolien G; Depienne, Christel; Gaily, Eija; Gennaro, Elena; Giraldez, Beatriz G; Gormley, Padhraig; Guerrero-López, Rosa

2016-01-01

BACKGROUND: Sanger sequencing, still the standard technique for genetic testing in most diagnostic laboratories and until recently widely used in research, is gradually being complemented by next-generation sequencing (NGS). No single mutation detection technique is however perfect in identifying all mutations. Therefore, we wondered to what extent inconsistencies between Sanger sequencing and NGS affect the molecular diagnosis of patients. Since mutations in SCN1A, the major gene implicated ...

The Human Gene Mutation Database: building a comprehensive mutation repository for clinical and molecular genetics, diagnostic testing and personalized genomic medicine.

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Stenson, Peter D; Mort, Matthew; Ball, Edward V; Shaw, Katy; Phillips, Andrew; Cooper, David N

2014-01-01

The Human Gene Mutation Database (HGMD®) is a comprehensive collection of germline mutations in nuclear genes that underlie, or are associated with, human inherited disease. By June 2013, the database contained over 141,000 different lesions detected in over 5,700 different genes, with new mutation entries currently accumulating at a rate exceeding 10,000 per annum. HGMD was originally established in 1996 for the scientific study of mutational mechanisms in human genes. However, it has since acquired a much broader utility as a central unified disease-oriented mutation repository utilized by human molecular geneticists, genome scientists, molecular biologists, clinicians and genetic counsellors as well as by those specializing in biopharmaceuticals, bioinformatics and personalized genomics. The public version of HGMD (http://www.hgmd.org) is freely available to registered users from academic institutions/non-profit organizations whilst the subscription version (HGMD Professional) is available to academic, clinical and commercial users under license via BIOBASE GmbH.

Identification of four novel mutations of the WFS1 gene in Iranian Wolfram syndrome pedigrees.

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Ghahraman, Martha; Abbaszadegan, Mohammad Reza; Vakili, Rahim; Hosseini, Sousan; Fardi Golyan, Fatemeh; Ghaemi, Nosrat; Forghanifard, Mohammad Mahdi

2016-12-01

Wolfram syndrome is a rare neurodegenerative disorder with an autosomal recessive pattern of inheritance characterized by various clinical manifestations. The related gene, WFS1, encodes a transmembrane glycoprotein, named wolframin. Genetic analyses demonstrated that mutations in this gene are associated with WS type 1. Our aim in this study was to sequence WFS1 coding region in Iranian Wolfram syndrome pedigrees. Genomic DNA was extracted from peripheral blood of 12 WS patients and their healthy parents. Exons 2-8 and the exon-intron junctions of WFS1 were sequenced. DNA sequences were compared to the reference using Sequencher software. Molecular analysis of WFS1 revealed six different mutations. Four novel and two previously reported mutations were identified. One novel mutation, c.1379_1381del, is predicted to produce an aberrant protein. A second novel mutation, c.1384G > T, encodes a truncated protein. Novel mutation, c.1097-1107dup (11 bp), causes a frameshift which results in a premature stop codon. We screened for the novel missense mutation, c.1010C > T, in 100 control alleles. This mutation was not found in any of the healthy controls. Our study increased the spectrum of WFS1 mutations and supported the role of WFS1 in susceptibility to WS. We hope that these findings open new horizons to future molecular investigations which may help to prevent and treat this devastating disease.

Mutation Analysis of COL1A1 and COL1A2 in Fetuses with Osteogenesis Imperfecta Type II/III.

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Wang, Wenbo; Wu, Qichang; Cao, Lin; Sun, Li; Xu, Yasong; Guo, Qiwei

2015-01-27

Aim: To analyze COL1A1/2 mutations in prenatal-onset OI for determine the proportion of mutations in type I collagen genes among prenatal onset OI and to provide additional data for genotype-phenotype analyses. Material and Methods: Ten cases of severe fetal short-limb dwarfism detected by antenatal ultrasonography were referred to our center. Before the termination of pregnancy, cordocentesis was performed for fetal karyotype and COL1A1/2 gene sequencing analysis. Postmortem radiographic examination was performed at all instances for definitive diagnosis. Results: COL1A1 and COL1A2 SNP and mutations were identified in all the cases. Among these, one synonymous SNP and four synonymous SNPs were recognized in COL1A1/2, respectively, seven cases have distinct heterozygous mutations and six new COL1A1/2 gene mutations were identified. Conclusion: There has been substantial progress in the identification of the molecular defects responsible for skeletal dysplasias. With the constant increase in the number of identified mutations in COL1A1 and COL1A2, genotype-phenotype correlation is becoming increasingly pertinent. © 2015 S. Karger AG, Basel.

Two families with normosmic congenital hypogonadotropic hypogonadism and biallelic mutations in KISS1R (KISS1 receptor: clinical evaluation and molecular characterization of a novel mutation.

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Frédéric Brioude

Full Text Available CONTEXT: KISS1R mutations have been reported in few patients with normosmic congenital hypogonadotropic hypogonadism (nCHH (OMIM #146110. OBJECTIVE: To describe in detail nCHH patients with biallelic KISS1R mutations belonging to 2 unrelated families, and to functionally characterize a novel KISS1R mutation. RESULTS: An original mutant, p.Tyr313His, was found in the homozygous state in 3 affected kindred (2 females and 1 male from a consanguineous Portuguese family. This mutation, located in the seventh transmembrane domain, affects a highly conserved amino acid, perturbs the conformation of the transmembrane segment, and impairs MAP kinase signaling and intracellular calcium release. In the second family, a French Caucasian male patient with nCHH was found to carry two recurrent mutations in the compound heterozygous state (p.Leu102Pro/Stop399Arg. In this man, pulsatile GnRH (Gonadotropin Releasing Hormone administration restored pulsatile LH (Luteinizing Hormone secretion and testicular hormone secretion. Later, long-term combined gonadotropin therapy induced spermatogenesis, enabling 3 successive pregnancies that resulted in 2 miscarriages and the birth of a healthy boy. CONCLUSION: We show that a novel loss-of-function mutation (p.Tyr313His in the KISS1R gene can cause familial nCHH, revealing the crucial role of this amino acid in KISS1R function. The observed restoration of gonadotropin secretion by exogenous GnRH administration further supports, in humans, the hypothalamic origin of the gonadotropin deficiency in this genetic form of nCHH.

Pitfalls in molecular analysis for mismatch repair deficiency in a family with biallelic pms2 germline mutations.

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Leenen, C H M; Geurts-Giele, W R R; Dubbink, H J; Reddingius, R; van den Ouweland, A M; Tops, C M J; van de Klift, H M; Kuipers, E J; van Leerdam, M E; Dinjens, W N M; Wagner, A

2011-12-01

Heterozygous germline mutations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2 cause Lynch syndrome. Biallelic mutations in the MMR genes are associated with a childhood cancer syndrome [constitutional mismatch repair deficiency (CMMR-D)]. This is predominantly characterized by hematological malignancies and tumors of the bowel and brain, often associated with signs of neurofibromatosis type 1 (NF1). Diagnostic strategies for selection of patients for MMR gene analysis include analysis of microsatellite instability (MSI) and immunohistochemical (IHC) analysis of MMR proteins in tumor tissue. We report the clinical characterization and molecular analyses of tumor specimens from a family with biallelic PMS2 germline mutations. This illustrates the pitfalls of present molecular screening strategies. Tumor tissues of five family members were analyzed for MSI and IHC. MSI was observed in only one of the analyzed tissues. However, IHC analysis of brain tumor tissue of the index patient and his sister showed absence of PMS2 expression, and germline mutation analyses showed biallelic mutations in PMS2: p.Ser46IIe and p.Pro246fs. The same heterozygous mutations were confirmed in the father and mother, respectively. These data support the conclusion that in case of a clinical phenotype of CMMR-D, it is advisable to routinely combine MSI analysis with IHC analysis for the expression of MMR proteins. With inconclusive or conflicting results, germline mutation analysis of the MMR genes should be considered after thorough counselling of the patients and/or their relatives. © 2011 John Wiley & Sons A/S.

Novel autosomal dominant TNNT1 mutation causing nemaline myopathy.

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Konersman, Chamindra G; Freyermuth, Fernande; Winder, Thomas L; Lawlor, Michael W; Lagier-Tourenne, Clotilde; Patel, Shailendra B

2017-11-01

Nemaline myopathy (NEM) is one of the three major forms of congenital myopathy and is characterized by diffuse muscle weakness, hypotonia, respiratory insufficiency, and the presence of nemaline rod structures on muscle biopsy. Mutations in troponin T1 (TNNT1) is 1 of 10 genes known to cause NEM. To date, only homozygous nonsense mutations or compound heterozygous truncating or internal deletion mutations in TNNT1 gene have been identified in NEM. This extended family is of historical importance as some members were reported in the 1960s as initial evidence that NEM is a hereditary disorder. Proband and extended family underwent Sanger sequencing for TNNT1. We performed RT-PCR and immunoblot on muscle to assess TNNT1 RNA expression and protein levels in proband and father. We report a novel heterozygous missense mutation of TNNT1 c.311A>T (p.E104V) that segregated in an autosomal dominant fashion in a large family residing in the United States. Extensive sequencing of the other known genes for NEM failed to identify any other mutant alleles. Muscle biopsies revealed a characteristic pattern of nemaline rods and severe myofiber hypotrophy that was almost entirely restricted to the type 1 fiber population. This novel mutation alters a residue that is highly conserved among vertebrates. This report highlights not only a family with autosomal dominant inheritance of NEM, but that this novel mutation likely acts via a dominant negative mechanism. © 2017 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.

Molecular profiling of appendiceal epithelial tumors using massively parallel sequencing to identify somatic mutations.

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Liu, Xiaoying; Mody, Kabir; de Abreu, Francine B; Pipas, J Marc; Peterson, Jason D; Gallagher, Torrey L; Suriawinata, Arief A; Ripple, Gregory H; Hourdequin, Kathryn C; Smith, Kerrington D; Barth, Richard J; Colacchio, Thomas A; Tsapakos, Michael J; Zaki, Bassem I; Gardner, Timothy B; Gordon, Stuart R; Amos, Christopher I; Wells, Wendy A; Tsongalis, Gregory J

2014-07-01

Some epithelial neoplasms of the appendix, including low-grade appendiceal mucinous neoplasm and adenocarcinoma, can result in pseudomyxoma peritonei (PMP). Little is known about the mutational spectra of these tumor types and whether mutations may be of clinical significance with respect to therapeutic selection. In this study, we identified somatic mutations using the Ion Torrent AmpliSeq Cancer Hotspot Panel v2. Specimens consisted of 3 nonneoplastic retention cysts/mucocele, 15 low-grade mucinous neoplasms (LAMNs), 8 low-grade/well-differentiated mucinous adenocarcinomas with pseudomyxoma peritonei, and 12 adenocarcinomas with/without goblet cell/signet ring cell features. Barcoded libraries were prepared from up to 10 ng of extracted DNA and multiplexed on single 318 chips for sequencing. Data analysis was performed using Golden Helix SVS. Variants that remained after the analysis pipeline were individually interrogated using the Integrative Genomics Viewer. A single Janus kinase 3 (JAK3) mutation was detected in the mucocele group. Eight mutations were identified in the V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and GNAS complex locus (GNAS) genes among LAMN samples. Additional gene mutations were identified in the AKT1 (v-akt murine thymoma viral oncogene homolog 1), APC (adenomatous polyposis coli), JAK3, MET (met proto-oncogene), phosphatidylinositol-4,5-bisphosphate 3-kinase (PIK3CA), RB1 (retinoblastoma 1), STK11 (serine/threonine kinase 11), and tumor protein p53 (TP53) genes. Among the PMPs, 6 mutations were detected in the KRAS gene and also in the GNAS, TP53, and RB1 genes. Appendiceal cancers showed mutations in the APC, ATM (ataxia telangiectasia mutated), KRAS, IDH1 [isocitrate dehydrogenase 1 (NADP+)], NRAS [neuroblastoma RAS viral (v-ras) oncogene homolog], PIK3CA, SMAD4 (SMAD family member 4), and TP53 genes. Our results suggest molecular heterogeneity among epithelial tumors of the appendix. Next generation sequencing efforts

657del5 mutation of the NBS1 gene in myelodysplastic syndrome

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Bunjevacki Vera

2014-01-01

Full Text Available Myelodysplastic syndromes (MDS are clonal hematologic stem cell disorders with an as yet unknown molecular pathology. Genetic instability has been proposed as a cause of MDS. Mutations in the NBS1 gene, whose product nibrin (p95 is involved in DNA damage repair and cell-cycle control, might be associated with an elevated predisposition to the development of MDS. The aim of the study was to examine truncating 5 bp deletion (657del5, the most frequent NBS1 gene mutation in Slavic populations, in MDS patients. Among 71 MDS patients, we found one case that was heterozygous for the NBS1 657del5 mutation. To the best of our knowledge, this is the first report of a NBS1 mutation in MDS. [Projekat Ministarstva nauke Republike Srbije, br. 175091

Bone marrow miR-10a overexpression is associated with genetic events but not affects clinical outcome in acute myeloid leukemia.

Science.gov (United States)

Zhang, Ting-Juan; Guo, Hong; Zhou, Jing-Dong; Li, Xi-Xi; Zhang, Wei; Ma, Ji-Chun; Wen, Xiang-Mei; Yao, Xin-Yu; Lin, Jiang; Qian, Jun

2018-01-01

Accumulating studies have linked the disruptions of microRNA-10 (miR-10) to acute myeloid leukemia (AML) with NPM1 mutation. However, miR-10 expression and its clinical implication in AML remain poorly defined. Although a recent report showed high serum level of miR-10a was associated with adverse prognosis in AML, herein, we found bone marrow (BM) miR-10 overexpression was not a prognostic biomarker in AML. BM miR-10 expression was examined by real-time quantitative PCR in BM mononuclear cells in 115 de novo AML patients and 45 controls. BM miR-10 (miR-10a/b) expression was significantly up-regulated in AML patients, and was positively correlated with each other. Overexpression of miR-10a was associated with lower percentage of BM blasts, whereas miR-10b overexpression tended to correlate with higher percentage of BM blasts. Importantly, miR-10a overexpression was significantly associated with FAB-M3/t(15;17) subtypes and NPM1 mutation, meanwhile, overexpression of miR-10b was correlated with NPM1 and DNMT3A mutations. However, miR-10a/b overexpression was not associated with complete remission rate, and did not have an impact on both leukemia free survival and overall survival time in non-M3 AML patients without NPM1 mutation. BM miR-10 overexpression is associated with genetic events but not affects clinical outcome in AML. Copyright © 2017 Elsevier GmbH. All rights reserved.

Molecular analysis of radiation-induced mutations in vitro

International Nuclear Information System (INIS)

Kronenberg, A.

1996-01-01

This review will focus on the nature of specific locus mutations detected in mammalian cells exposed in vitro to different types of ionizing radiations. Ionizing radiation has been shown to produce a wide variety of heritable alterations in DNA. These range from single base pair substitutions to stable loss or translocation of large portions of whole chromosomes. Data will be reviewed for certain test systems that reveal different mutation spectra. Techniques for the analysis of molecular alterations include applications of the polymerase chain reaction, some of which may be coupled with DNA sequence analysis, and a variety of hybridization-based techniques. The complexity of large scale rearrangements is approached with cytogenetic techniques including high resolution banding and various applications of the fluorescence in situ hybridization (FISH) technique. Radiation-induced mutant frequencies and mutation spectra are a function of the linkage constraints on the recovery of viable mutants for a given locus and test system. 44 refs

[Schinzel-Giedion syndrome: a new mutation in SETBP1].

Science.gov (United States)

López-González, V; Domingo-Jiménez, M R; Burglen, L; Ballesta-Martínez, M J; Whalen, S; Piñero-Fernández, J A; Guillén-Navarro, E

2015-01-01

Schinzel-Giedion syndrome (SGS) (#MIM 269150) is a rare genetic disorder characterized by very marked craniofacial dysmorphism, multiple congenital anomalies and severe intellectual disability. Most affected patients die in early childhood. SETBP1 was identified as the causative gene, but a limited number of patients with molecular confirmation have been reported to date. The case is reported of a 4 and a half year-old male patient, affected by SGS. SETBP1 sequencing analysis revealed the presence of a non-previously described mutation: c.2608G>T (p.Gly870Cys). The clinical features and differential diagnosis of this rare condition are reviewed. Dysmorphic features are strongly suggestive of SGS. Its clinical recognition is essential to enable an early diagnosis, a proper follow-up, and to provide the family with genetic counseling. To date, this is the seventeenth SGS patient published with SETBP1 mutation, and the first in Spain, helping to widen clinical and molecular knowledge of the disease. Copyright © 2014 Asociación Española de Pediatría. Published by Elsevier Espana. All rights reserved.

Molecular analysis of formaldehyde-induced mutations in human lymphoblasts and E. coli

International Nuclear Information System (INIS)

Crosby, R.M.; Richardson, K.K.; Craft, T.R.; Benforado, K.B.; Liber, H.L.; Skopek, T.R.

1988-01-01

The molecular nature of formaldehyde (HCHO)-induced mutations was studied in both human lymphoblasts and E. coli. Thirty HPRT - human lymphoblast colonies induced by eight repetitive 150 μM HCHO treatments were characterized by Southern blot analysis. Fourteen of these mutants (47%) had visible deletions of some or all of the X-linked HPRT bands, indicating that HCHO can induce large losses of DNA in human lymphoblasts. In E. coli., DNA alterations induced by HCHO were characterized with use of the xanthine guanine phosphoribosyl transferase (gpt) gene as the genetic target. Exposure of E. coli to 4 mM HCHO for 1 hr induced large insertions (41%), large deletions (18%), and point mutations (41%). Dideoxy DNA sequencing revealed that most of the point mutations were transversions at GC base pairs. In contrast, exposure of E. coli to 40 mM HCHO for 1 hr produced 92% point mutations, 62% of which were transitions at a single AT base pair in the gene. Therefore, HCHO is capable of producing different genetic alterations in E. coli at different concentrations, suggesting fundamental differences in the mutagenic mechanisms operating at the two concentrations used. Naked pSV2gpt plasmid DNA was exposed to 3.3 or 10 mM HCHO and transformed into E. coli. Most of the resulting mutations were frameshifts, again suggesting a different mutagenic mechanism

Introduction of Molecular Diagnosis of Hemochromatosis Type 1 in Cuba

Directory of Open Access Journals (Sweden)

Ismael Aramís Cervera García

2013-06-01

Full Text Available Background: hemochromatosis type 1 is an autosomal recessive genetic disorder, which should be diagnosed during its preclinical phase in order to prevent severe organ damage. Objective: to establish the diagnosis of hemochromatosis type 1 in Cuba, and calculate its frequencies in patients with hepatopathies. Methods: an analytic cross-sectional study was conducted including 65 patients with liver disease, who were referred to the laboratory of Molecular Biology of the National Medical Genetics Center by clinical geneticists. A PCR-RFLP analysis was used for detecting the C282Y and H63D mutations in the HFE gene. Results: PCR-RFLP analysis was standardized for the detection of C282Y and H63D mutations. Frequencies of C282Y and H63D mutations in the HFE gene in patients with hepatopathies were 6.3% and 18.2% respectively. Conclusions: molecular diagnosis of C282Y and H63D mutations in the HFE gene causing hemochromatosis type 1 contributed to the identification of 28 carriers in the 65 patients who were studied, as well as a homozygous individual for the H63D mutation, which shows the high prevalence of these mutations in Cuban patients with liver disease.

Haplotype analysis suggest that the MLH1 c.2059C > T mutation is a Swedish founder mutation.

Science.gov (United States)

von Salomé, Jenny; Liu, Tao; Keihäs, Markku; Morak, Moni; Holinski-Feder, Elke; Berry, Ian R; Moilanen, Jukka S; Baert-Desurmont, Stéphanie; Lindblom, Annika; Lagerstedt-Robinson, Kristina

2017-12-29

Lynch syndrome (LS) predisposes to a spectrum of cancers and increases the lifetime risk of developing colorectal- or endometrial cancer to over 50%. Lynch syndrome is dominantly inherited and is caused by defects in DNA mismatch-repair genes MLH1, MSH2, MSH6 or PMS2, with the vast majority detected in MLH1 and MSH2. Recurrent LS-associated variants observed in apparently unrelated individuals, have either arisen de novo in different families due to mutation hotspots, or are inherited from a founder (a common ancestor) that lived several generations back. There are variants that recur in some populations while also acting as founders in other ethnic groups. Testing for founder mutations can facilitate molecular diagnosis of Lynch Syndrome more efficiently and more cost effective than screening for all possible mutations. Here we report a study of the missense mutation MLH1 c.2059C > T (p.Arg687Trp), a potential founder mutation identified in eight Swedish families and one Finnish family with Swedish ancestors. Haplotype analysis confirmed that the Finnish and Swedish families shared a haplotype of between 0.9 and 2.8 Mb. While MLH1 c.2059C > T exists worldwide, the Swedish haplotype was not found among mutation carriers from Germany or France, which indicates a common founder in the Swedish population. The geographic distribution of MLH1 c.2059C > T in Sweden suggests a single, ancient mutational event in the northern part of Sweden.

Mutation spectrum of RB1 mutations in retinoblastoma cases from Singapore with implications for genetic management and counselling.

Directory of Open Access Journals (Sweden)

Swati Tomar

Full Text Available Retinoblastoma (RB is a rare childhood malignant disorder caused by the biallelic inactivation of RB1 gene. Early diagnosis and identification of carriers of heritable RB1 mutations can improve disease outcome and management. In this study, mutational analysis was conducted on fifty-nine matched tumor and peripheral blood samples from 18 bilateral and 41 unilateral unrelated RB cases by a combinatorial approach of Multiplex Ligation-dependent Probe Amplification (MLPA assay, deletion screening, direct sequencing, copy number gene dosage analysis and methylation assays. Screening of both blood and tumor samples yielded a mutation detection rate of 94.9% (56/59 while only 42.4% (25/59 of mutations were detected if blood samples alone were analyzed. Biallelic mutations were observed in 43/59 (72.9% of tumors screened. There were 3 cases (5.1% in which no mutations could be detected and germline mutations were detected in 19.5% (8/41 of unilateral cases. A total of 61 point mutations were identified, of which 10 were novel. There was a high incidence of previously reported recurrent mutations, occurring at 38.98% (23/59 of all cases. Of interest were three cases of mosaic RB1 mutations detected in the blood from patients with unilateral retinoblastoma. Additionally, two germline mutations previously reported to be associated with low-penetrance phenotypes: missense-c.1981C>T and splice variant-c.607+1G>T, were observed in a bilateral and a unilateral proband, respectively. These findings have implications for genetic counselling and risk prediction for the affected families. This is the first published report on the spectrum of mutations in RB patients from Singapore and shows that further improved mutation screening strategies are required in order to provide a definitive molecular diagnosis for every case of RB. Our findings also underscore the importance of genetic testing in supporting individualized disease management plans for patients and

Nucleophosmin is required for DNA integrity and p19Arf protein stability

DEFF Research Database (Denmark)

Colombo, Emanuela; Bonetti, Paola; Lazzerini Denchi, Eros

2005-01-01

, such as mutated Ras or overexpressed Myc. In the absence of NPM, Arf protein is excluded from nucleoli and is markedly less stable. Our data demonstrate that NPM regulates DNA integrity and, through Arf, inhibits cell proliferation and are consistent with a putative tumor-suppressive function of NPM....

Molecular genetic mutation analysis in Menkes-disease with prenatal diagnosis

DEFF Research Database (Denmark)

László, Aranka; Endreffy, Emoke; Tümer, Zeynep

2010-01-01

Menkes disease (MD) is an X-linked recessive multisystemic lethal, heredodegenerative disorder. Progressive neurodegeneration and connective tissue disturbances with microscopically kinky hair are the main symptoms. Molecular genetic mutation analysis was made at a Hungarian male infant suffering...... from MD and prenatal diagnosis was done in this MD loaded family. METHOD: The 12th exon of ATP7A gene has been analyzed by dideoxy-finger printing (DDF), polymerase chain reaction (PCR), direct sequencing of exon 12. The specific mutation was screened from chorionic villi of the maternal aunt at the 14......th gestational week. RESULTS: In the exon 12th a basic pair substitution with Arg 844 His change was detected leading to very severe fatal missense mutation....

Novel mutations in the homogentisate 1,2 dioxygenase gene identified in Jordanian patients with alkaptonuria.

Science.gov (United States)

Al-sbou, Mohammed

2012-06-01

This study was conducted to identify mutations in the homogentisate 1,2 dioxygenase gene (HGD) in alkaptonuria patients among Jordanian population. Blood samples were collected from four alkaptonuria patients, four carriers, and two healthy volunteers. DNA was isolated from peripheral blood. All 14 exons of the HGD gene were amplified using the polymerase chain reaction (PCR) technique. The PCR products were then purified and analyzed by sequencing. Five mutations were identified in our samples. Four of them were novel C1273A, T1046G, 551-552insG, T533G and had not been previously reported, and one mutation T847C has been described before. The types of mutations identified were two missense mutations, one splice site mutation, one frameshift mutation, and one polymorphism. We present the first molecular study of the HGD gene in Jordanian alkaptonuria patients. This study provides valuable information about the molecular basis of alkaptonuria in Jordanian population.

NSD1 mutations generate a genome-wide DNA methylation signature.

LENUS (Irish Health Repository)

Choufani, S

2015-12-22

Sotos syndrome (SS) represents an important human model system for the study of epigenetic regulation; it is an overgrowth\\/intellectual disability syndrome caused by mutations in a histone methyltransferase, NSD1. As layered epigenetic modifications are often interdependent, we propose that pathogenic NSD1 mutations have a genome-wide impact on the most stable epigenetic mark, DNA methylation (DNAm). By interrogating DNAm in SS patients, we identify a genome-wide, highly significant NSD1(+\\/-)-specific signature that differentiates pathogenic NSD1 mutations from controls, benign NSD1 variants and the clinically overlapping Weaver syndrome. Validation studies of independent cohorts of SS and controls assigned 100% of these samples correctly. This highly specific and sensitive NSD1(+\\/-) signature encompasses genes that function in cellular morphogenesis and neuronal differentiation, reflecting cardinal features of the SS phenotype. The identification of SS-specific genome-wide DNAm alterations will facilitate both the elucidation of the molecular pathophysiology of SS and the development of improved diagnostic testing.

PIK3CA activating mutation in colorectal carcinoma: associations with molecular features and survival.

Directory of Open Access Journals (Sweden)

Christophe Rosty

Full Text Available Mutations in PIK3CA are present in 10 to 15% of colorectal carcinomas. We aimed to examine how PIK3CA mutations relate to other molecular alterations in colorectal carcinoma, to pathologic phenotype and survival. PIK3CA mutation testing was carried out using direct sequencing on 757 incident tumors from the Melbourne Collaborative Cohort Study. The status of O-6-methylguanine-DNA methyltransferase (MGMT was assessed using both immunohistochemistry and methyLight techniques. Microsatellite instability, CpG island phenotype (CIMP, KRAS and BRAF V600E mutation status, and pathology review features were derived from previous reports. PIK3CA mutation was observed in 105 of 757 (14% of carcinomas, characterized by location in the proximal colon (54% vs. 34%; P<0.001 and an increased frequency of KRAS mutation (48% vs. 25%; P<0.001. High-levels of CIMP were more frequently found in PIK3CA-mutated tumors compared with PIK3CA wild-type tumors (22% vs. 11%; P = 0.004. There was no difference in the prevalence of BRAF V600E mutation between these two tumor groups. PIK3CA-mutated tumors were associated with loss of MGMT expression (35% vs. 20%; P = 0.001 and the presence of tumor mucinous differentiation (54% vs. 32%; P<0.001. In patients with wild-type BRAF tumors, PIK3CA mutation was associated with poor survival (HR 1.51 95% CI 1.04-2.19, P = 0.03. In summary, PIK3CA-mutated colorectal carcinomas are more likely to develop in the proximal colon, to demonstrate high levels of CIMP, KRAS mutation and loss of MGMT expression. PIK3CA mutation also contributes to significantly decreased survival for patients with wild-type BRAF tumors.

A novel nonsense mutation in the WFS1 gene causes the Wolfram syndrome.

Science.gov (United States)

Noorian, Shahab; Savad, Shahram; Mohammadi, Davood Shah

2016-05-01

Wolfram syndrome is a rare autosomal recessive neurodegenerative disorder, which is mostly caused by mutations in the WFS1 gene. The WFS1 gene product, which is called wolframin, is thought to regulate the function of endoplasmic reticulum. The endoplasmic reticulum has a critical role in protein folding and material transportation within the cell or to the surface of the cell. Identification of new mutations in WFS1 gene will unravel the molecular pathology of WS. The aim of this case report study is to describe a novel mutation in exon 4 of the WFS1 gene (c.330C>A) in a 9-year-old boy with WS.

DNA Variations in Oculocutaneous Albinism: An Updated Mutation List and Current Outstanding Issues in Molecular Diagnostics

Science.gov (United States)

Simeonov, Dimitre R.; Wang, Xinjing; Wang, Chen; Sergeev, Yuri; Dolinska, Monika; Bower, Matthew; Fischer, Roxanne; Winer, David; Dubrovsky, Genia; Balog, Joan Z.; Huizing, Marjan; Hart, Rachel; Zein, Wadih M.; Gahl, William A.; Brooks, Brian P.; Adams, David R.

2014-01-01

Oculocutaneous albinism (OCA) is a rare genetic disorder of melanin synthesis that results in hypopigmented hair, skin, and eyes. There are four types of OCA, caused by mutations in TYR (OCA-1), OCA2 (OCA-2), TYRP1 (OCA-3), or SLC45A2 (OCA-4). Here we report 22 novel mutations; 14 from a cohort of 61 patients seen as part of the NIH OCA Natural History Study and 8 from a prior study at the University of Minnesota. We also include a comprehensive list of almost 600 previously reported OCA mutations, along with ethnicity information, carrier frequencies, and in silico pathogenicity predictions. In addition to discussing the clinical and molecular features of OCA, we address the cases of apparent missing heritability. In our cohort, 25% of patients did not have two mutations in a single OCA gene. We demonstrate the utility of multiple detection methods to reveal mutations missed by Sanger sequencing. Finally, we review the TYR p.R402Q temperature sensitive variant and confirm its association with cases of albinism with only one identifiable TYR mutation. PMID:23504663

Use of Wilms Tumor 1 Gene Expression as a Reliable Marker for Prognosis and Minimal Residual Disease Monitoring in Acute Myeloid Leukemia With Normal Karyotype Patients.

Science.gov (United States)

Marjanovic, Irena; Karan-Djurasevic, Teodora; Ugrin, Milena; Virijevic, Marijana; Vidovic, Ana; Tomin, Dragica; Suvajdzic Vukovic, Nada; Pavlovic, Sonja; Tosic, Natasa

2017-05-01

Acute myeloid leukemia with normal karyotype (AML-NK) represents the largest group of AML patients classified with an intermediate prognosis. A constant need exists to introduce new molecular markers for more precise risk stratification and for minimal residual disease (MRD) monitoring. Quantitative assessment of Wilms tumor 1 (WT1) gene transcripts was performed using real-time polymerase chain reaction. The bone marrow samples were collected at the diagnosis from 104 AML-NK patients and from 34 of these patients during follow-up or disease relapse. We found that overexpression of the WT1 gene (WT1 high status), present in 25.5% of patients, was an independent unfavorable factor for achieving complete remission. WT1 high status was also associated with resistance to therapy and shorter disease-free survival and overall survival. Assessment of the log reduction value of WT1 expression, measured in paired diagnosis/complete remission samples, revealed that patients with a log reduction of < 2 had a tendency toward shorter disease-free survival and overall survival and a greater incidence of disease relapse. Combining WT1 gene expression status with NPM1 and FLT3-ITD mutational status, we found that the tumor behavior of intermediate patients (FLT3-ITD - /NPM1 - double negative) with WT1 high status is almost the same as the tumor behavior of the adverse risk group. WT1 expression status represents a good molecular marker of prognosis, response to treatment, and MRD monitoring. Above all, the usage of the WT1 expression level as an additional marker for more precise risk stratification of AML-NK patients could lead to more adapted, personalized treatment protocols. Copyright © 2017 Elsevier Inc. All rights reserved.

KRAS and BRAF Mutation Detection: Is Immunohistochemistry a Possible Alternative to Molecular Biology in Colorectal Cancer?

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Nicolas Piton

2015-01-01

Full Text Available KRAS genotyping is mandatory in metastatic colorectal cancer treatment prior to undertaking antiepidermal growth factor receptor (EGFR monoclonal antibody therapy. BRAF V600E mutation is often present in colorectal carcinoma with CpG island methylator phenotype and microsatellite instability. Currently, KRAS and BRAF evaluation is based on molecular biology techniques such as SNaPshot or Sanger sequencing. As molecular testing is performed on formalin-fixed paraffin-embedded (FFPE samples, immunodetection would appear to be an attractive alternative for detecting mutations. Thus, our objective was to assess the validity of KRAS and BRAF immunodetection of mutations compared with the genotyping reference method in colorectal adenocarcinoma. KRAS and BRAF genotyping was assessed by SNaPshot. A rabbit anti-human KRAS polyclonal antibody was tested on 33 FFPE colorectal tumor samples with known KRAS status. Additionally, a mouse anti-human BRAF monoclonal antibody was tested on 30 FFPE tumor samples with known BRAF status. KRAS immunostaining demonstrated both poor sensitivity (27% and specificity (64% in detecting KRAS mutation. Conversely, BRAF immunohistochemistry showed perfect sensitivity (100% and specificity (100% in detecting V600E mutation. Although molecular biology remains the reference method for detecting KRAS mutation, immunohistochemistry could be an attractive method for detecting BRAF V600E mutation in colorectal cancer.

KRAS and BRAF Mutation Detection: Is Immunohistochemistry a Possible Alternative to Molecular Biology in Colorectal Cancer?

Science.gov (United States)

Borrini, Francesco; Bolognese, Antonio; Lamy, Aude; Sabourin, Jean-Christophe

2015-01-01

KRAS genotyping is mandatory in metastatic colorectal cancer treatment prior to undertaking antiepidermal growth factor receptor (EGFR) monoclonal antibody therapy. BRAF V600E mutation is often present in colorectal carcinoma with CpG island methylator phenotype and microsatellite instability. Currently, KRAS and BRAF evaluation is based on molecular biology techniques such as SNaPshot or Sanger sequencing. As molecular testing is performed on formalin-fixed paraffin-embedded (FFPE) samples, immunodetection would appear to be an attractive alternative for detecting mutations. Thus, our objective was to assess the validity of KRAS and BRAF immunodetection of mutations compared with the genotyping reference method in colorectal adenocarcinoma. KRAS and BRAF genotyping was assessed by SNaPshot. A rabbit anti-human KRAS polyclonal antibody was tested on 33 FFPE colorectal tumor samples with known KRAS status. Additionally, a mouse anti-human BRAF monoclonal antibody was tested on 30 FFPE tumor samples with known BRAF status. KRAS immunostaining demonstrated both poor sensitivity (27%) and specificity (64%) in detecting KRAS mutation. Conversely, BRAF immunohistochemistry showed perfect sensitivity (100%) and specificity (100%) in detecting V600E mutation. Although molecular biology remains the reference method for detecting KRAS mutation, immunohistochemistry could be an attractive method for detecting BRAF V600E mutation in colorectal cancer. PMID:25983749

TBC1D24 Mutations in a Sibship with Multifocal Polymyoclonus

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Adeline Ngoh

2017-04-01

Full Text Available Background: Advances in molecular genetic technologies have improved our understanding of genetic causes of rare neurological disorders with features of myoclonus.Case Report: A family with two affected siblings, presenting with multifocal polymyoclonus and neurodevelopmental delay, was recruited for whole-exome sequencing following unyielding diagnostic neurometabolic investigations. Compound heterozygous mutations in TBC1D24, a gene previously associated with various epilepsy phenotypes and hearing loss, were identified in both siblings. The mutations included a missense change c.457G>A (p.Glu157Lys, and a novel frameshift mutation c.545del (p.Thr182Serfs*6.Discussion: We propose that TBC1D24-related diseases should be in the differential diagnosis for children with polymyoclonus. 

Mutational analysis in patients with Autosomal Dominant Polycystic Kidney Disease (ADPKD): Identification of five mutations in the PKD1 gene.

Science.gov (United States)

Abdelwahed, Mayssa; Hilbert, Pascale; Ahmed, Asma; Mahfoudh, Hichem; Bouomrani, Salem; Dey, Mouna; Hachicha, Jamil; Kamoun, Hassen; Keskes-Ammar, Leila; Belguith, Neïla

2018-05-31

Autosomal Dominant Polycystic Kidney Disease (ADPKD), the most frequent genetic disorder of the kidneys, is characterized by a typical presenting symptoms include cysts development in different organs and a non-cysts manifestations. ADPKD is caused by mutations in PKD1 or PKD2 genes. In this study, we aimed to search for molecular causative defects among PKD1 and PKD2 genes. Eighteen patients were diagnosed based on renal ultrasonography and renal/extra-renal manifestations. Then, Sanger sequencing was performed for PKD1 and PKD2 genes. Multiplex Ligation dependent Probe Amplification method (MLPA) methods was performed for both PKD genes. Mutational analysis of the PKD2 gene revealed the absence of variants and no deletions or duplications of both PKD genes were detected. But three novels mutations i.e. p.S463C exon 7; c. c.11156+2T>C IVS38 and c.8161-1G>A IVS22 and two previously reported c.1522T>C exon 7 and c.412C>T exon 4 mutations in the PKD1 gene were detected. Bioinformatics tools predicted that the novel variants have a pathogenic effects on splicing machinery, pre-mRNA secondary structure and stability and protein stability. Our results highlighted molecular features of Tunisian patients with ADPKD and revealed novel variations that can be utilized in clinical diagnosis and in the evaluation of living kidney donor. To the best of our knowledge, this is the first report of Autosomal Polycystic Kidney Disease in Tunisia. Copyright © 2017. Published by Elsevier B.V.

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

Science.gov (United States)

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.

Loss of stability and hydrophobicity of presenilin 1 mutations causing Alzheimer's Disease

DEFF Research Database (Denmark)

Somavarapu, Arun Kumar; Kepp, Kasper Planeta

2016-01-01

Nearly 200 mutations in the gene coding for presenilin 1 (PSEN1) cause early-onset Alzheimer's Disease, yet the molecular mechanism remains obscure. As a meta-analysis, we compiled available clinical and biochemical data for PSEN1 variants and correlated these to chemical properties of the mutant...

The c.IVS1+1G>A mutation inthe GJB2 gene is prevalent and large ...

Indian Academy of Sciences (India)

IVS1+1G>A mutation inthe GJB2 gene is prevalent and large deletions involving the GJB6 gene are not present in the Turkish population. ASLI SIRMACI, DUYGU AKCAYOZ-DUMAN and MUSTAFA TEKIN∗. Division of Pediatric Molecular Genetics, Ankara University School of Medicine, Ankara 06100, Turkey. Introduction.

UBIAD1 mutation alters a mitochondrial prenyltransferase to cause Schnyder corneal dystrophy.

Directory of Open Access Journals (Sweden)

Michael L Nickerson

2010-05-01

Full Text Available Mutations in a novel gene, UBIAD1, were recently found to cause the autosomal dominant eye disease Schnyder corneal dystrophy (SCD. SCD is characterized by an abnormal deposition of cholesterol and phospholipids in the cornea resulting in progressive corneal opacification and visual loss. We characterized lesions in the UBIAD1 gene in new SCD families and examined protein homology, localization, and structure.We characterized five novel mutations in the UBIAD1 gene in ten SCD families, including a first SCD family of Native American ethnicity. Examination of protein homology revealed that SCD altered amino acids which were highly conserved across species. Cell lines were established from patients including keratocytes obtained after corneal transplant surgery and lymphoblastoid cell lines from Epstein-Barr virus immortalized peripheral blood mononuclear cells. These were used to determine the subcellular localization of mutant and wild type protein, and to examine cholesterol metabolite ratios. Immunohistochemistry using antibodies specific for UBIAD1 protein in keratocytes revealed that both wild type and N102S protein were localized sub-cellularly to mitochondria. Analysis of cholesterol metabolites in patient cell line extracts showed no significant alteration in the presence of mutant protein indicating a potentially novel function of the UBIAD1 protein in cholesterol biochemistry. Molecular modeling was used to develop a model of human UBIAD1 protein in a membrane and revealed potentially critical roles for amino acids mutated in SCD. Potential primary and secondary substrate binding sites were identified and docking simulations indicated likely substrates including prenyl and phenolic molecules.Accumulating evidence from the SCD familial mutation spectrum, protein homology across species, and molecular modeling suggest that protein function is likely down-regulated by SCD mutations. Mitochondrial UBIAD1 protein appears to have a highly

Molecular characterization of Als1, an acetohydroxyacid synthase mutation conferring resistance to sulfonylurea herbicides in soybean.

Science.gov (United States)

Ghio, Cecilia; Ramos, María Laura; Altieri, Emiliano; Bulos, Mariano; Sala, Carlos A

2013-12-01

The AHAS gene family in soybean was characterized. The locus Als1 for sulfonylurea resistance was mapped and the resistant allele was characterized at the molecular level. Sulfonylurea (SU) resistance in soybean is controlled by Als1, a semi-dominant allele obtained by EMS mutagenesis over the cultivar Williams 82. The overall objective of this research was to map Als1 in the soybean genome and to determine the nucleotidic changes conferring resistance to SU. Four nucleotide sequences (GmAhas1-4) showing high homology with the Arabidopsis thaliana acetohydroxyacid synthase (AHAS, EC 4.1.3.18) gene sequence were identified by in silico analysis, PCR-amplified from the SU-resistant line BTK323STS and sequenced. Expression analysis showed that GmAhas1, located on chromosome 4 by in silico analysis, is the most expressed sequence in true leaves. F2:3 families derived from the cross between susceptible and resistant lines were evaluated for SU resistance. Mapping results indicate that the locus als1 is located on chromosome 4. Sequence comparison of GmAhas1 between BTK323STS and Williams 82 showed a single nucleotide change from cytosine to thymine at position 532. This transversion generates an amino acid change from proline to serine at position 197 (A. thaliana nomenclature) of the AHAS catalytic subunit. An allele-specific marker developed for the GmAhas1 mutant sequence cosegregated with SU resistance in the F2 population. Taking together, the mapping, expression and sequencing results indicate that the GmAhas1 sequence corresponds to the Als1 gene sequence controlling SU resistance in soybean. The molecular breeding tools described herein create the basis to speed up the identification of new mutations in soybean AHAS leading to enhanced levels of resistance to SU or to other families of AHAS inhibitor herbicides.

[Wolfram syndrome: clinical features, molecular genetics of WFS1 gene].

Science.gov (United States)

Tanabe, Katsuya; Matsunaga, Kimie; Hatanaka, Masayuki; Akiyama, Masaru; Tanizawa, Yukio

2015-02-01

Wolfram syndrome(WFS: OMIM 222300) is a rare recessive neuro-endocrine degenerative disorder, known as DIDMOAD(Diabetes Insipidus, early-onset Diabetes Mellitus, Optic Atrophy and Deafness) syndrome. Most affected individuals carry recessive mutations in the Wolfram syndrome 1 gene(WFS1). The WFS1 protein is an endoplasmic reticulum(ER) embedded protein, which functions in ER calcium homeostasis and unfolded protein responses. Dysregulation of these cellular processes results in the development of ER stress, leading to apoptosis. In addition, abundantly present WFS1 protein in insulin secretory granules plays a role in the intra-granular acidification. However, the phenotypic pleiomorphism and molecular complexity of this disease limit the understanding of WFS. Here we review clinical features, molecular mechanisms and mutations of WFS1 gene that relate to this syndrome.

High Mutation Levels are Compatible with Normal Embryonic Development in Mlh1-Deficient Mice.

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Fan, Xiaoyan; Li, Yan; Zhang, Yulong; Sang, Meixiang; Cai, Jianhui; Li, Qiaoxia; Ozaki, Toshinori; Ono, Tetsuya; He, Dongwei

2016-10-01

To elucidate the role of the mismatch repair gene Mlh1 in genome instability during the fetal stage, spontaneous mutations were studied in Mlh1-deficient lacZ-transgenic mouse fetuses. Mutation levels were high at 9.5 days post coitum (dpc) and gradually increased during the embryonic stage, after which they remained unchanged. In addition, mutations that were found in brain, liver, spleen, small intestine and thymus showed similar levels and no statistically significant difference was found. The molecular nature of mutations at 12.5 dpc in fetuses of Mlh1 +/+ and Mlh1 -/- mice showed their own unique spectra, suggesting that deletion mutations were the main causes in the deficiency of the Mlh1 gene. Of note, fetuses of irradiated mice exhibited marked differences such as post-implantation loss and Mendelian distribution. Collectively, these results strongly suggest that high mutation ofMlh1 -/- -deficient fetuses has little effect on the fetuses during their early developmental stages, whereas Mlh1 -/- -deficient fetuses from X-ray irradiated mothers are clearly effected.

Comparison of risk assessment models of BRCA1 and BRCA2 mutation carrier in patients with breast cancer

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Rybchenko L.A.

2013-12-01

Full Text Available Analysis of efficiency of the algorithm BOADICEA using and Manchester scoring system to predict the carrier of BRCA1 and BRCA2 mutations in Ukranian patients with breast cancer was performed. Materials for this study were the results of clinical, imunogistological, pathogistological, genealogical, molecular genetic researches of 146 patients with breast cancer. Calculations of mutations risk were performed using BOADICEA algorithm and Manchester scoring system. In the total group of patients the area under the curve while predicting BRCA1 mutations with algorithm BOADICEA was 0.86, with Manchester scoring system - 0.84, and in calculation of the combined risk of BRCA mutations - 0.83 and 0.84, respectively. However, statistical difference between the areas of algorithms has not been established (p> 0.05, it indicates to the same discriminatory power of the test models. Better sensitivity, specificity, positive and negative predictive value of results of BOADICEA algorithm was reached in 6% of BRCA1 probability and in 8% threshold of BRCA1/2 mutations. The Manchester scoring system has showed the best operating characteristics with 6 and 13-point probability of BRCA1 and BRCA1/2 mutations respectively. Patients with probability of mutations with such thresholds may be offered molecular study of pathogenic alleles.

Developmentally Regulated Post-translational Modification of Nucleoplasmin Controls Histone Sequestration and Deposition

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Takashi Onikubo

2015-03-01

Full Text Available Nucleoplasmin (Npm is an abundant histone chaperone in vertebrate oocytes and embryos. During embryogenesis, regulation of Npm histone binding is critical for its function in storing and releasing maternal histones to establish and maintain the zygotic epigenome. Here, we demonstrate that Xenopus laevis Npm post-translational modifications (PTMs specific to the oocyte and egg promote either histone deposition or sequestration, respectively. Mass spectrometry and Npm phosphomimetic mutations used in chromatin assembly assays identified hyperphosphorylation on the N-terminal tail as a critical regulator for sequestration. C-terminal tail phosphorylation and PRMT5-catalyzed arginine methylation enhance nucleosome assembly by promoting histone interaction with the second acidic tract of Npm. Electron microscopy reconstructions of Npm and TTLL4 activity toward the C-terminal tail demonstrate that oocyte- and egg-specific PTMs cause Npm conformational changes. Our results reveal that PTMs regulate Npm chaperoning activity by modulating Npm conformation and Npm-histone interaction, leading to histone sequestration in the egg.

Identification of a novel WFS1 homozygous nonsense mutation in Jordanian children with Wolfram syndrome.

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Bodoor, Khaldon; Batiha, Osama; Abu-Awad, Ayman; Al-Sarihin, Khaldon; Ziad, Haya; Jarun, Yousef; Abu-Sheikha, Aya; Abu Jalboush, Sara; Alibrahim, Khoulod S

2016-09-01

Wolfram syndrome (WS) is a rare autosomal recessive neurodegenerative disorder characterized by the presentation of early onset type I diabetes mellitus and optic atrophy with later onset diabetes insipidus and deafness. WFS1 gene was identified on chromosome 4p16.1 as the gene responsible for WS disease given that most of the WS patients were found to carry mutations in this gene. This study was carried out to investigate the molecular spectrum of WFS1 gene in Jordanian families. Molecular and clinical characterization was performed on five WS patients from two unrelated Jordanian families. Our data indicated that WS patients of the first family harbored two deletion mutations (V415del and F247fs) located in exon 8 and exon 7 respectively, with a compound heterozygous pattern of inheritance; while in the second family, we identified a novel nonsense mutation (W185X) located in exon 5 in the N-terminal cytoplasmic domain with a homozygous pattern of inheritance. This mutation can be considered as loss of function mutation since the resulting truncated protein lost both the transmembrane domain and the C-terminal domain. Additionally, the W185X mutation lies within the CaM binding domain in wolframin protein which is thought to have a role in the regulation of wolframin function in response to calcium levels.

Clinical spectrum and molecular diagnosis of Angelman and Prader-Willi syndrome patients with an imprinting mutation

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Saitoh, S.; Cassidy, S.B.; Conroy, J.M. [Univ. of Hospitals of Cleveland, OH (United States)] [and others

1997-01-20

Recent studies have identified a new class of Prader-Willi syndrome (PWS) and Angelman syndrome (AS) patients who have biparental inheritance, but neither the typical deletion nor uniparental disomy (UPD) or translocation. However, these patients have uniparental DNA methylation throughout 15q11-q13, and thus appear to have a mutation in the imprinting process for this region. Here we describe detailed clinical findings of five AS imprinting mutation patients (three families) and two PWS imprinting mutation patients (one new family). All these patients have essentially the classical clinical phenotype for the respective syndrome, except that the incidence of microcephaly is lower in imprinting mutation AS patients than in deletion AS patients. Furthermore, imprinting mutation AS and PWS patients do not typically have hypopigmentation, which is commonly found in patients with the usual large deletion. Molecular diagnosis of these cases is initially achieved by DNA methylation analyses of the DN34/ZNF127, PW71 (D15S63), and SNRPN loci. The latter two probes have clear advantages in the simple molecular diagnostic analysis of PWS and AS patients with an imprinting mutation, as has been found for typical deletion or UPD PWS and AS cases. With the recent finding of inherited microdeletions in PWS and AS imprinting mutation families, our studies define a new class of these two syndromes. The clinical and molecular identification of these PWS and AS patients has important genetic counseling consequences. 49 refs., 4 figs., 3 tabs.

Dominant mutations in S. cerevisiae PMS1 identify the Mlh1-Pms1 endonuclease active site and an exonuclease 1-independent mismatch repair pathway.

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Smith, Catherine E; Mendillo, Marc L; Bowen, Nikki; Hombauer, Hans; Campbell, Christopher S; Desai, Arshad; Putnam, Christopher D; Kolodner, Richard D

2013-10-01

Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC) is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR) caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. To better understand the function of Mlh1-Pms1 in MMR, we used Saccharomyces cerevisiae to identify six pms1 mutations (pms1-G683E, pms1-C817R, pms1-C848S, pms1-H850R, pms1-H703A and pms1-E707A) that were weakly dominant in wild-type cells, which surprisingly caused a strong MMR defect when present on low copy plasmids in an exo1Δ mutant. Molecular modeling showed these mutations caused amino acid substitutions in the metal coordination pocket of the Pms1 endonuclease active site and biochemical studies showed that they inactivated the endonuclease activity. This model of Mlh1-Pms1 suggested that the Mlh1-FERC motif contributes to the endonuclease active site. Consistent with this, the mlh1-E767stp mutation caused both MMR and endonuclease defects similar to those caused by the dominant pms1 mutations whereas mutations affecting the predicted metal coordinating residue Mlh1-C769 had no effect. These studies establish that the Mlh1-Pms1 endonuclease is required for MMR in a previously uncharacterized Exo1-independent MMR pathway.

Dominant mutations in S. cerevisiae PMS1 identify the Mlh1-Pms1 endonuclease active site and an exonuclease 1-independent mismatch repair pathway.

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Catherine E Smith

2013-10-01

Full Text Available Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. To better understand the function of Mlh1-Pms1 in MMR, we used Saccharomyces cerevisiae to identify six pms1 mutations (pms1-G683E, pms1-C817R, pms1-C848S, pms1-H850R, pms1-H703A and pms1-E707A that were weakly dominant in wild-type cells, which surprisingly caused a strong MMR defect when present on low copy plasmids in an exo1Δ mutant. Molecular modeling showed these mutations caused amino acid substitutions in the metal coordination pocket of the Pms1 endonuclease active site and biochemical studies showed that they inactivated the endonuclease activity. This model of Mlh1-Pms1 suggested that the Mlh1-FERC motif contributes to the endonuclease active site. Consistent with this, the mlh1-E767stp mutation caused both MMR and endonuclease defects similar to those caused by the dominant pms1 mutations whereas mutations affecting the predicted metal coordinating residue Mlh1-C769 had no effect. These studies establish that the Mlh1-Pms1 endonuclease is required for MMR in a previously uncharacterized Exo1-independent MMR pathway.

Screening of mutations affecting protein stability and dynamics of FGFR1—A simulation analysis

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C. George Priya Doss

2012-12-01

Full Text Available Single amino acid substitutions in Fibroblast Growth Factor Receptor 1 (FGFR1 destabilize protein and have been implicated in several genetic disorders like various forms of cancer, Kallamann syndrome, Pfeiffer syndrome, Jackson Weiss syndrome, etc. In order to gain functional insight into mutation caused by amino acid substitution to protein function and expression, special emphasis was laid on molecular dynamics simulation techniques in combination with in silico tools such as SIFT, PolyPhen 2.0, I-Mutant 3.0 and SNAP. It has been estimated that 68% nsSNPs were predicted to be deleterious by I-Mutant, slightly higher than SIFT (37%, PolyPhen 2.0 (61% and SNAP (58%. From the observed results, P722S mutation was found to be most deleterious by comparing results of all in silico tools. By molecular dynamics approach, we have shown that P722S mutation leads to increase in flexibility, and deviated more from the native structure which was supported by the decrease in the number of hydrogen bonds. In addition, biophysical analysis revealed a clear insight of stability loss due to P722S mutation in FGFR1 protein. Majority of mutations predicted by these in silico tools were in good concordance with the experimental results.

Novel mutation in forkhead box G1 (FOXG1) gene in an Indian patient with Rett syndrome.

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Das, Dhanjit Kumar; Jadhav, Vaishali; Ghattargi, Vikas C; Udani, Vrajesh

2014-03-15

Rett syndrome (RTT) is a severe neurodevelopmental disorder characterized by the progressive loss of intellectual functioning, fine and gross motor skills and communicative abilities, deceleration of head growth, and the development of stereotypic hand movements, occurring after a period of normal development. The classic form of RTT involves mutation in MECP2 while the involvement of CDKL5 and FOXG1 genes has been identified in atypical RTT phenotype. FOXG1 gene encodes for a fork-head box protein G1, a transcription factor acting primarily as transcriptional repressor through DNA binding in the embryonic telencephalon as well as a number of other neurodevelopmental processes. In this report we have described the molecular analysis of FOXG1 gene in Indian patients with Rett syndrome. FOXG1 gene mutation analysis was done in a cohort of 34 MECP2/CDKL5 mutation negative RTT patients. We have identified a novel mutation (p. D263VfsX190) in FOXG1 gene in a patient with congenital variant of Rett syndrome. This mutation resulted into a frameshift, thereby causing an alteration in the reading frames of the entire coding sequence downstream of the mutation. The start position of the frameshift (Asp263) and amino acid towards the carboxyl terminal end of the protein was found to be well conserved across species using multiple sequence alignment. Since the mutation is located at forkhead binding domain, the resultant mutation disrupts the secondary structure of the protein making it non-functional. This is the first report from India showing mutation in FOXG1 gene in Rett syndrome. Copyright © 2014 Elsevier B.V. All rights reserved.

New splice site acceptor mutation in AIRE gene in autoimmune polyendocrine syndrome type 1.

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Mireia Mora

Full Text Available Autoimmune polyglandular syndrome type 1 (APS-1, OMIM 240300 is a rare autosomal recessive disorder, characterized by the presence of at least two of three major diseases: hypoparathyroidism, Addison's disease, and chronic mucocutaneous candidiasis. We aim to identify the molecular defects and investigate the clinical and mutational characteristics in an index case and other members of a consanguineous family. We identified a novel homozygous mutation in the splice site acceptor (SSA of intron 5 (c.653-1G>A in two siblings with different clinical outcomes of APS-1. Coding DNA sequencing revealed that this AIRE mutation potentially compromised the recognition of the constitutive SSA of intron 5, splicing upstream onto a nearby cryptic SSA in intron 5. Surprisingly, the use of an alternative SSA entails the uncovering of a cryptic donor splice site in exon 5. This new transcript generates a truncated protein (p.A214fs67X containing the first 213 amino acids and followed by 68 aberrant amino acids. The mutation affects the proper splicing, not only at the acceptor but also at the donor splice site, highlighting the complexity of recognizing suitable splicing sites and the importance of sequencing the intron-exon junctions for a more precise molecular diagnosis and correct genetic counseling. As both siblings were carrying the same mutation but exhibited a different APS-1 onset, and one of the brothers was not clinically diagnosed, our finding highlights the possibility to suspect mutations in the AIRE gene in cases of childhood chronic candidiasis and/or hypoparathyroidism otherwise unexplained, especially when the phenotype is associated with other autoimmune diseases.

DNMT1 mutations found in HSANIE patients affect interaction with UHRF1 and neuronal differentiation.

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Smets, Martha; Link, Stephanie; Wolf, Patricia; Schneider, Katrin; Solis, Veronica; Ryan, Joel; Meilinger, Daniela; Qin, Weihua; Leonhardt, Heinrich

2017-04-15

DNMT1 is recruited to substrate sites by PCNA and UHRF1 to maintain DNA methylation after replication. The cell cycle dependent recruitment of DNMT1 is mediated by the PCNA-binding domain (PBD) and the targeting sequence (TS) within the N-terminal regulatory domain. The TS domain was found to be mutated in patients suffering from hereditary sensory and autonomic neuropathies with dementia and hearing loss (HSANIE) and autosomal dominant cerebellar ataxia deafness and narcolepsy (ADCA-DN) and is associated with global hypomethylation and site specific hypermethylation. With functional complementation assays in mouse embryonic stem cells, we showed that DNMT1 mutations P496Y and Y500C identified in HSANIE patients not only impair DNMT1 heterochromatin association, but also UHRF1 interaction resulting in hypomethylation. Similar DNA methylation defects were observed when DNMT1 interacting domains in UHRF1, the UBL and the SRA domain, were deleted. With cell-based assays, we could show that HSANIE associated mutations perturb DNMT1 heterochromatin association and catalytic complex formation at methylation sites and decrease protein stability in late S and G2 phase. To investigate the neuronal phenotype of HSANIE mutations, we performed DNMT1 rescue assays and could show that cells expressing mutated DNMT1 were prone to apoptosis and failed to differentiate into neuronal lineage. Our results provide insights into the molecular basis of DNMT1 dysfunction in HSANIE patients and emphasize the importance of the TS domain in the regulation of DNA methylation in pluripotent and differentiating cells. © The Author 2017. Published by Oxford University Press.

Genotype-phenotype correlation of Coffin-Siris syndrome caused by mutations in SMARCB1, SMARCA4, SMARCE1, and ARID1A.

Science.gov (United States)

Kosho, Tomoki; Okamoto, Nobuhiko

2014-09-01

Coffin-Siris syndrome (CSS) is a rare congenital malformation syndrome, recently found to be caused by mutations in several genes encoding components of the BAF complex. To date, 109 patients have been reported with their mutations: SMARCB1 (12%), SMARCA4 (11%), SMARCE1 (2%), ARID1A (7%), ARID1B (65%), and PHF6 (2%). We review genotype-phenotype correlation of all previously reported patients with mutations in SMARCB1, SMARCA4, SMARCE1, and ARID1A through reassessment of their clinical and molecular findings. Cardinal features of CSS included variable degrees of intellectual disability (ID) predominantly affecting speech, sucking/feeding difficulty, and craniofacial (thick eyebrows, long eyelashes), digital (hypoplastic 5th fingers or toes, hypoplastic 5th fingernails or toenails), and other characteristics (hypertrichosis). In addition, patients with SMARCB1 mutations had severe neurodevelopmental deficits including severe ID, seizures, CNS structural abnormalities, and no expressive words as well as scoliosis. Especially, those with a recurrent mutation "p.Lys364del" represented strikingly similar phenotypes including characteristic facial coarseness. Patients with SMARCA4 mutations had less coarse craniofacial appearances and behavioral abnormalities. Patients with SMARCE1 mutations had a wide spectrum of manifestations from severe to moderate ID. Patients with ARID1A also had a wide spectrum of manifestations from severe ID and serous internal complications that could result in early death to mild ID. Mutations in SMARCB1, SMARCA4, and SMARCE1 are expected to exert dominant-negative or gain-of-function effects, whereas those in ARID1A are expected to exert loss-of-function effects. © 2014 Wiley Periodicals, Inc.

Classifications within molecular subtypes enables identification of BRCA1/BRCA2 mutation carriers by RNA tumor profiling

DEFF Research Database (Denmark)

Larsen, Martin J; Kruse, Torben A; Tan, Qihua

2013-01-01

Pathogenic germline mutations in BRCA1 or BRCA2 are detected in less than one third of families with a strong history of breast cancer. It is therefore expected that mutations still remain undetected by currently used screening methods. In addition, a growing number of BRCA1/2 sequence variants...... of unclear pathogen significance are found in the families, constituting an increasing clinical challenge. New methods are therefore needed to improve the detection rate and aid the interpretation of the clinically uncertain variants. In this study we analyzed a series of 33 BRCA1, 22 BRCA2, and 128 sporadic...... tumors by RNA profiling to investigate the classification potential of RNA profiles to predict BRCA1/2 mutation status. We found that breast tumors from BRCA1 and BRCA2 mutation carriers display characteristic RNA expression patterns, allowing them to be distinguished from sporadic tumors. The majority...

[Molecular genetics of pigmentary retinopathies: identification of mutations in CHM, RDS, RHO, RPE65, USH2A and XLRS1 genes].

Science.gov (United States)

Hamel, C P; Griffoin, J M; Bazalgette, C; Lasquellec, L; Duval, P A; Bareil, C; Beaufrère, L; Bonnet, S; Eliaou, C; Marlhens, F; Schmitt-Bernard, C F; Tuffery, S; Claustres, M; Arnaud, B

2000-12-01

To evaluate the occurrence and inheritance of various types of pigmentary retinopathy in patients followed at the outpatient clinic in the university hospital, Montpellier, France. To characterize genes and mutations causing these conditions. Ophthalmic examination and various visual tests were performed. Mutations were sought from genomic DNA by PCR amplification of exons associated with single-strand conformation analysis and/or direct sequencing. Among 315 patients over an 8-year period, cases of retinitis pigmentosa (63.2%), Usher's syndrome (10.2%), Stargardt's disease (5.4%), choroideremia (3.2%), Leber's congenital amaurosis (3.2%), congenital stationary night blindness (2.9%), cone dystrophy (2.5%), dominant optic atrophy (1.9%), X-linked juvenile retinoschisis (1.6%), Best's disease (1.6%), and others (4.3%) were diagnosed. In retinitis pigmentosa, inheritance could be determined in 54.2% of the cases including dominant autosomic (26.6%), recessive autosomic (22.6%), and X-linked cases (5%) while it could not be confirmed in 45.7% of the cases (simplex cases in the majority). For the 6 examined genes, mutations were found in 22 out of 182 propositus (12.1%). Analysis of phenotype-genotype correlations indicates that in retinitis pigmentosa, RDS is more frequently associated with macular involvement and retinal flecks, RHO with regional disease, and RPE65 with the great severity of the disease with some cases of Leber's congenital amaurosis. Identification of genes may help in diagnosis and in genetic counseling, especially in simplex cases with retinitis pigmentosa. In this latter condition, molecular diagnosis will be necessary to rationalize future treatments.

Molecular characterization of three novel Fanconi anemia mutations in Israeli Arabs.

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Tamary, Hannah; Dgany, Orly; Toledano, Helen; Shalev, Zvi; Krasnov, Tatyana; Shalmon, Lea; Schechter, Tali; Bercovich, Dani; Attias, Dina; Laor, Ruth; Koren, Ariel; Yaniv, Isaac

2004-05-01

In a previous study, we investigated the molecular basis of Fanconi anemia (FA) in 13 unrelated Israeli Jewish FA patients and identified four ethnicity specific mutations. In the present study we extended our study to Israeli Arab patients. We studied three consanguineous families with nine FA patients and an additional unrelated patient. DNA single-strand conformation polymorphism of each exon of the FANCA and FANCG genes was followed by sequence analysis of the aberrantly migrating fragments and by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of the splice-site mutations identified. Three unique disease-causing mutations were identified: (i) FANCA gross deletion of exons 6-31; (ii) FANCA splice-site mutation IVS 42-2A>C; (iii) FANCG splice-site mutation IVS4+3A>G. Sequence analysis of the FANCA gross deletion revealed recombination between two highly homologous Alu elements. cDNA analysis of the two splice mutations suggested intron 42 retention in FANCA IVS 42-2A>C and exon 4 skipping in FANCG IVS4+3A>G. The clinical condition of eight patients with FANCA mutations was severe. Two unique FANCA mutations and one FANCG mutation were identified in Israeli Arab FA patients. Deletion of FANCA exon 6-31 as in previously described gross deletions was within introns rich in Alu repeats. To the best of our knowledge, the FANCA IVS 42-2A>C mutation is the first in this gene to result in intron retention. Further analysis of FA mutations will enable prenatal diagnosis and a rational therapeutic approach including frequent monitoring and early bone marrow transplantation. Copyright Blackwell Munksgaard 2004.

A recurrent deletion mutation in OPA1 causes autosomal dominant optic atrophy in a Chinese family

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Zhang, Liping; Shi, Wei; Song, Liming; Zhang, Xiao; Cheng, Lulu; Wang, Yanfang; Ge, Xianglian; Li, Wei; Zhang, Wei; Min, Qingjie; Jin, Zi-Bing; Qu, Jia; Gu, Feng

2014-11-01

Autosomal dominant optic atrophy (ADOA) is the most frequent form of hereditary optic neuropathy and occurs due to the degeneration of the retinal ganglion cells. To identify the genetic defect in a family with putative ADOA, we performed capture next generation sequencing (CNGS) to screen known retinal disease genes. However, six exons failed to be sequenced by CNGS in optic atrophy 1 gene (OPA1). Sequencing of those exons identified a 4 bp deletion mutation (c.2983-1_2985del) in OPA1. Furthermore, we sequenced the transcripts of OPA1 from the patient skin fibroblasts and found there is six-nucleotide deletion (c.2984-c.2989, AGAAAG). Quantitative-PCR and Western blotting showed that OPA1 mRNA and its protein expression have no obvious difference between patient skin fibroblast and control. The analysis of protein structure by molecular modeling suggests that the mutation may change the structure of OPA1 by formation of an alpha helix protruding into an existing pocket. Taken together, we identified an OPA1 mutation in a family with ADOA by filling the missing CNGS data. We also showed that this mutation affects the structural intactness of OPA1. It provides molecular insights for clinical genetic diagnosis and treatment of optic atrophy.

Molecular grading of tumors of the upper urothelial tract using FGFR3 mutation status identifies patients with favorable prognosis

OpenAIRE

Fernandez, Cecilia; Lyle,Stephen; Hsieh,; Shuber,Anthony

2012-01-01

Stephen R Lyle,1 Chung-Cheng Hsieh,1 Cecilia A Fernandez,2 Anthony P Shuber21University of Massachusetts, Worcester, MA, 2Predictive Biosciences Inc., Lexington, MA, USABackground: Mutations in FGFR3 have been shown to occur in tumors of the upper urothelial tract and may be indicative of a good prognosis. In bladder tumors, the combination of FGFR3 mutation status and Ki-67 level has been used to define a tumor's molecular grade and predict survival. Pathological evaluation of upper ...

ENPP1 Mutation Causes Recessive Cole Disease by Altering Melanogenesis.

Science.gov (United States)

Chourabi, Marwa; Liew, Mei Shan; Lim, Shawn; H'mida-Ben Brahim, Dorra; Boussofara, Lobna; Dai, Liang; Wong, Pui Mun; Foo, Jia Nee; Sriha, Badreddine; Robinson, Kim Samirah; Denil, Simon; Common, John Ea; Mamaï, Ons; Ben Khalifa, Youcef; Bollen, Mathieu; Liu, Jianjun; Denguezli, Mohamed; Bonnard, Carine; Saad, Ali; Reversade, Bruno

2018-02-01

Cole disease is a genodermatosis of pigmentation following a strict dominant mode of inheritance. In this study, we investigated eight patients affected with an overlapping genodermatosis after recessive inheritance. The patients presented with hypo- and hyperpigmented macules over the body, resembling dyschromatosis universalis hereditaria in addition to punctuate palmoplantar keratosis. By homozygosity mapping and whole-exome sequencing, a biallelic p.Cys120Arg mutation in ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) was identified in all patients. We found that this mutation, like those causing dominant Cole disease, impairs homodimerization of the ENPP1 enzyme that is mediated by its two somatomedin-B-like domains. Histological analysis revealed structural and molecular changes in affected skin that were likely to originate from defective melanocytes because keratinocytes do not express ENPP1. Consistently, RNA-sequencing analysis of patient-derived primary melanocytes revealed alterations in melanocyte development and in pigmentation signaling pathways. We therefore conclude that germline ENPP1 cysteine-specific mutations, primarily affecting the melanocyte lineage, cause a clinical spectrum of dyschromatosis, in which the p.Cys120Arg allele represents a recessive and more severe form of Cole disease. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Two Finnish USH1B patients with three novel mutations in myosin VIIA.

Science.gov (United States)

Vastinsalo, Hanna; Isosomppi, Juha; Aittakorpi, Anne; Sankila, Eeva-Marja

2006-09-21

Usher syndrome (USH) is an autosomal recessive disorder resulting in retinal degeneration and sensorineural deafness caused by mutations in at least 10 gene loci. USH is divided into three main clinical types: USH1 (33-44%), USH2 (56-67%), and USH3. Worldwide, USH1 and USH2 account for most of the Usher syndrome cases with rare occurrence of USH3. In Finland, however, USH3 is the most common type (40%), explained by genetic and geographical isolation accompanied with a founder mutation, while USH1 is estimated to comprise 34% and USH2 12% of all USH cases. We examined two unrelated Finnish USH1 patients by sequencing. We found three new myosin VIIA (MYO7A) mutations: p.K923AfsX8, p.Q1896X, and p.E1349K. The p.K923AfsX8 mutation was present in both patients as well as in one of 200 Finnish control chromosomes. This is the first molecular genetic study of USH1 in Finland. We have found three new pathological mutations causing either premature termination of translation or replacement of an evolutionary conserved MYO7A amino acid.

Clinical and molecular response to interferon-α therapy in essential thrombocythemia patients with CALR mutations.

Science.gov (United States)

Verger, Emmanuelle; Cassinat, Bruno; Chauveau, Aurélie; Dosquet, Christine; Giraudier, Stephane; Schlageter, Marie-Hélène; Ianotto, Jean-Christophe; Yassin, Mohammed A; Al-Dewik, Nader; Carillo, Serge; Legouffe, Eric; Ugo, Valerie; Chomienne, Christine; Kiladjian, Jean-Jacques

2015-12-10

Myeloproliferative neoplasms are clonal disorders characterized by the presence of several gene mutations associated with particular hematologic parameters, clinical evolution, and prognosis. Few therapeutic options are available, among which interferon α (IFNα) presents interesting properties like the ability to induce hematologic responses (HRs) and molecular responses (MRs) in patients with JAK2 mutation. We report on the response to IFNα therapy in a cohort of 31 essential thrombocythemia (ET) patients with CALR mutations (mean follow-up of 11.8 years). HR was achieved in all patients. Median CALR mutant allelic burden (%CALR) significantly decreased from 41% at baseline to 26% after treatment, and 2 patients even achieved complete MR. In contrast, %CALR was not significantly modified in ET patients treated with hydroxyurea or aspirin only. Next-generation sequencing identified additional mutations in 6 patients (affecting TET2, ASXL1, IDH2, and TP53 genes). The presence of additional mutations was associated with poorer MR on CALR mutant clones, with only minor or no MRs in this subset of patients. Analysis of the evolution of the different variant allele frequencies showed that the mutated clones had a differential sensitivity to IFNα in a given patient, but no new mutation emerged during treatment. In all, this study shows that IFNα induces high rates of HRs and MRs in CALR-mutated ET, and that the presence of additional nondriver mutations may influence the MR to therapy. © 2015 by The American Society of Hematology.

Splicing mutation in Sbf1 causes nonsyndromic male infertility in the rat

Czech Academy of Sciences Publication Activity Database

Liška, F.; Chylíková, B.; Janků, M.; Šeda, Ondřej; Vernerová, Z.; Pravenec, Michal; Křen, Vladimír

2016-01-01

Roč. 152, č. 3 (2016), s. 215-223 ISSN 1470-1626 R&D Projects: GA CR(CZ) GA16-06548S Institutional support: RVO:67985823 ; RVO:68378050 Keywords : Sbf1 (SET binding factor 1) gene mutation * male infertility * spontaneously hypertensive rat Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.100, year: 2016

Understanding the cross-resistance of oseltamivir to H1N1 and H5N1 influenza A neuraminidase mutations using multidimensional computational analyses

Directory of Open Access Journals (Sweden)

Singh A

2015-07-01

Full Text Available Ashona Singh, Mahmoud E Soliman School of Health Sciences, University of KwaZulu-Natal, Westville, Durban, South Africa Abstract: This study embarks on a comprehensive description of the conformational contributions to resistance of neuraminidase (N1 in H1N1 and H5N1 to oseltamivir, using comparative multiple molecular dynamic simulations. The available data with regard to elucidation of the mechanism of resistance as a result of mutations in H1N1 and H5N1 neuraminidases is not well established. Enhanced post-dynamic analysis, such as principal component analysis, solvent accessible surface area, free binding energy calculations, and radius of gyration were performed to gain a precise insight into the binding mode and origin of resistance of oseltamivir in H1N1 and H5N1 mutants. Three significant features reflecting resistance in the presence of mutations H274Y and I222K, of the protein complexed with the inhibitor are: reduced flexibility of the a-carbon backbone; an improved ΔEele of ~15 (kcal/mol for H1N1 coupled with an increase in ΔGsol­ (~13 kcal/mol from wild-type to mutation; a low binding affinity in comparison with the wild-type of ~2 (kcal/mol and ~7 (kcal/mol with respect to each mutation for the H5N1 systems; and reduced hydrophobicity of the overall surface structure due to an impaired hydrogen bonding network. We believe the results of this study will ultimately provide a useful insight into the structural landscape of neuraminidase-associated binding of oseltamivir. Furthermore, the results can be used in the design and development of potent inhibitors of neuraminidases. Keywords: neuraminidase, molecular dynamics, resistance, mutation, binding free energy

SQSTM1 Mutations and Glaucoma.

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Todd E Scheetz

Full Text Available Glaucoma is the most common cause of irreversible blindness worldwide. One subset of glaucoma, normal tension glaucoma (NTG occurs in the absence of high intraocular pressure. Mutations in two genes, optineurin (OPTN and TANK binding kinase 1 (TBK1, cause familial NTG and have known roles in the catabolic cellular process autophagy. TKB1 encodes a kinase that phosphorylates OPTN, an autophagy receptor, which ultimately activates autophagy. The sequestosome (SQSTM1 gene also encodes an autophagy receptor and also is a target of TBK1 phosphorylation. Consequently, we hypothesized that mutations in SQSTM1 may also cause NTG. We tested this hypothesis by searching for glaucoma-causing mutations in a cohort of NTG patients (n = 308 and matched controls (n = 157 using Sanger sequencing. An additional 1098 population control samples were also analyzed using whole exome sequencing. A total of 17 non-synonymous mutations were detected which were not significantly skewed between cases and controls when analyzed separately, or as a group (p > 0.05. These data suggest that SQSTM1 mutations are not a common cause of NTG.

Consistency and reproducibility of next-generation sequencing and other multigene mutational assays: A worldwide ring trial study on quantitative cytological molecular reference specimens.

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Malapelle, Umberto; Mayo-de-Las-Casas, Clara; Molina-Vila, Miguel A; Rosell, Rafael; Savic, Spasenija; Bihl, Michel; Bubendorf, Lukas; Salto-Tellez, Manuel; de Biase, Dario; Tallini, Giovanni; Hwang, David H; Sholl, Lynette M; Luthra, Rajyalakshmi; Weynand, Birgit; Vander Borght, Sara; Missiaglia, Edoardo; Bongiovanni, Massimo; Stieber, Daniel; Vielh, Philippe; Schmitt, Fernando; Rappa, Alessandra; Barberis, Massimo; Pepe, Francesco; Pisapia, Pasquale; Serra, Nicola; Vigliar, Elena; Bellevicine, Claudio; Fassan, Matteo; Rugge, Massimo; de Andrea, Carlos E; Lozano, Maria D; Basolo, Fulvio; Fontanini, Gabriella; Nikiforov, Yuri E; Kamel-Reid, Suzanne; da Cunha Santos, Gilda; Nikiforova, Marina N; Roy-Chowdhuri, Sinchita; Troncone, Giancarlo

2017-08-01

Molecular testing of cytological lung cancer specimens includes, beyond epidermal growth factor receptor (EGFR), emerging predictive/prognostic genomic biomarkers such as Kirsten rat sarcoma viral oncogene homolog (KRAS), neuroblastoma RAS viral [v-ras] oncogene homolog (NRAS), B-Raf proto-oncogene, serine/threonine kinase (BRAF), and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA). Next-generation sequencing (NGS) and other multigene mutational assays are suitable for cytological specimens, including smears. However, the current literature reflects single-institution studies rather than multicenter experiences. Quantitative cytological molecular reference slides were produced with cell lines designed to harbor concurrent mutations in the EGFR, KRAS, NRAS, BRAF, and PIK3CA genes at various allelic ratios, including low allele frequencies (AFs; 1%). This interlaboratory ring trial study included 14 institutions across the world that performed multigene mutational assays, from tissue extraction to data analysis, on these reference slides, with each laboratory using its own mutation analysis platform and methodology. All laboratories using NGS (n = 11) successfully detected the study's set of mutations with minimal variations in the means and standard errors of variant fractions at dilution points of 10% (P = .171) and 5% (P = .063) despite the use of different sequencing platforms (Illumina, Ion Torrent/Proton, and Roche). However, when mutations at a low AF of 1% were analyzed, the concordance of the NGS results was low, and this reflected the use of different thresholds for variant calling among the institutions. In contrast, laboratories using matrix-assisted laser desorption/ionization-time of flight (n = 2) showed lower concordance in terms of mutation detection and mutant AF quantification. Quantitative molecular reference slides are a useful tool for monitoring the performance of different multigene mutational

Comprehensive molecular diagnosis of 67 Chinese Usher syndrome probands: high rate of ethnicity specific mutations in Chinese USH patients.

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Jiang, Lichun; Liang, Xiaofang; Li, Yumei; Wang, Jing; Zaneveld, Jacques Eric; Wang, Hui; Xu, Shan; Wang, Keqing; Wang, Binbin; Chen, Rui; Sui, Ruifang

2015-09-04

Usher syndrome (USH) is the most common disease causing combined deafness and blindness. It is predominantly an autosomal recessive genetic disorder with occasionally digenic cases. Molecular diagnosis of USH patients is important for disease management. Few studies have tried to find the genetic cause of USH in Chinese patients. This study was designed to determine the mutation spectrum of Chinese USH patients. We applied next generation sequencing to characterize the mutation spectrum in 67 independent Chinese families with at least one member diagnosed with USH. Blood was collected at Peking Union Medical College Hospital. This cohort is one of the largest USH cohorts reported. We utilized customized panel and whole exome sequencing, variant analysis, Sanger validation and segregation tests to find disease causing mutations in these families. We identified biallelic disease causing mutations in known USH genes in 70 % (49) of our patients. As has been previously reported, MYO7A is the most frequently mutated gene in our USH type I patients while USH2A is the most mutated gene in our USH type II patients. In addition, we identify mutations in CLRN1, DFNB31, GPR98 and PCDH15 for the first time in Chinese USH patients. Together, mutations in CLRN1, DNFB31, GPR98 and PCDH15 account for 11.4 % of disease in our cohort. Interestingly, although the spectrum of disease genes is quite similar between our Chinese patient cohort and other patient cohorts from different (and primarily Caucasian) ethnic backgrounds, the mutations themselves are dramatically different. In particular, 76 % (52/68) of alleles found in this study have never been previously reported. Interestingly, we observed a strong enrichment for severe protein truncating mutations expected to have severe functional consequence on the protein in USH II patients compared to the reported mutation spectrum in RP patients, who often carry partial protein truncating mutations. Our study provides the first

Molecular analysis of the eighteen most frequent mutations in the BRCA1 gene in 63 Chilean breast cancer families

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LILIAN JARA

2004-01-01

Full Text Available BRCA1 gene mutations account for nearly all families with multiple cases of both early onset breast and/or ovarian cancer and about 30% of hereditary breast cancer. Although to date more than 1,237 distinct mutations, polymorphisms, and variants have been described, several mutations have been found to be recurrent in this gene. We have analyzed 63 Chilean breast/ovarian cancer families for eighteen frequent BRCA1 mutations. The analysis of the five exons and two introns in which these mutations are located was made using mismatch PCR assay, ASO hybridization assay, restriction fragment analysis, allele specific PCR assay and direct sequentiation techniques. Two BRCA1 mutations (185delAG and C61G and one variant of unknown significance (E1250K were found in four of these families. Also, a new mutation (4185delCAAG and one previously described polymorphism (E1038G were found in two other families. The 185delAG was found in a 3.17 % of the families and the others were present only in one of the families of this cohort. Therefore these mutations are not prominent in the Chilean population. The variant of unknown significance and the polymorphism detected could represent a founder effect of Spanish origin

Overexpression of Wilms Tumor 1 Gene as a Negative Prognostic Indicator in Acute Myeloid Leukemia

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Mi, Ruihua; Ding, Jing; Wang, Xianwei; Hu, Jieying; Fan, Ruihua; Wei, Xudong; Song, Yongping; Zhao, Richard Y.

2014-01-01

Chromosomal aberrations are useful in assessing treatment options and clinical outcomes of acute myeloid leukemia (AML) patients. However, 40∼50% of the AML patients showed no chromosomal abnormalities, i.e., with normal cytogenetics aka the CN-AML patients. Testing of molecular aberrations such as FLT3 or NPM1 can help to define clinical outcomes in the CN-AML patients but with various successes. Goal of this study was to test the possibility of Wilms’ tumor 1 (WT1) gene overexpression as an additional molecular biomarker. A total of 103 CN-AML patients, among which 28% had overexpressed WT1, were studied over a period of 38 months. Patient’s response to induction chemotherapy as measured by the complete remission (CR) rate, disease-free survival (DFS) and overall survival (OS) were measured. Our data suggested that WT1 overexpression correlated negatively with the CR rate, DFS and OS. Consistent with previous reports, CN-AML patients can be divided into three different risk subgroups based on the status of known molecular abnormalities, i.e., the favorable (NPM1mt/no FLT3ITD), the unfavorable (FLT3ITD) and the intermediate risk subgroups. The WT1 overexpression significantly reduced the CR, DFS and OS in both the favorable and unfavorable groups. As the results, patients with normal WT1 gene expression in the favorable risk group showed the best clinical outcomes and all survived with complete remission and disease-free survival over the 37 month study period; in contrast, patients with WT1 overexpression in the unfavorable risk group displayed the worst clinical outcomes. WT1 overexpression by itself is an independent and negative indicator for predicting CR rate, DFS and OS of the CN-AML patients; moreover, it increases the statistical power of predicting the same clinical outcomes when it is combined with the NPM1 mt or the FLT3 ITD genotypes that are the good or poor prognostic markers of CN-AML. PMID:24667279

Identification of a mutation in ADD1/SREBP-1 in the spontaneously hypertensive rat

Czech Academy of Sciences Publication Activity Database

Pravenec, Michal; Jansa, Petr; Kostka, Vlastimil; Zídek, Václav; Křen, Vladimír; Forejt, Jiří; Kurtz, T. W.

2001-01-01

Roč. 12, č. 4 (2001), s. 295-298 ISSN 0938-8990 R&D Projects: GA ČR(CZ) GA305/00/1646; GA MŠk(CZ) LN00A079; GA ČR(CZ) GV204/98/K015 Grant - others:HHMI(US) 55000331 Institutional research plan: CEZ:AV0Z5011922 Keywords : mutations in genes * ADD1/SREBP-1c * spontaneously hypertensive rat Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.318, year: 2001

FBXW7 mutations in patients with advanced cancers: clinical and molecular characteristics and outcomes with mTOR inhibitors.

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Denis L Jardim

Full Text Available FBXW7 is a tumor suppressor gene responsible for the degradation of several proto-oncogenes. Preclinical data suggest that FBXW7 mutations sensitize cells to mTOR inhibitors. Clinicopathologic characteristics of cancer patients with FBXW7 mutations and their responses to mTOR inhibitors remain unknown.Using multiplex gene panels we evaluated how the FBXW7 mutation affected the cancer phenotype of patients referred to a phase I clinic starting January 2012. Whenever possible patients positive for FBXW7 mutation were treated with regimens containing an mTOR inhibitors and their outcomes were reviewed.FBXW7 mutations were detected in 17 of 418 patients (4.0%. Among tumor types with more than 10 patients tested, FBXW7 mutations occurred in colorectal cancer (7/49; 14.3%, squamous cell cancer of head and neck (2/18; 11.1%, liver (1/13; 7.7%, and ovarian cancers (1/40; 2.5%. No one clinical, pathological or demographic feature was characteristic of the FBXW7-mutated patient population. The mutation occurred in isolation in only 2/17 (12% patients, and KRAS was frequently found as a concomitant mutation, especially in patients with colorectal cancer (6/7; 86%. Ten patients were treated on a protocol containing an mTOR inhibitor, with a median time to treatment failure of 2.8 months (range, 1.3-6.8. One patient with liver cancer (fibrolamellar subtype continues to have a prolonged stable disease for 6.8+ months.In patients with advanced cancers, somatic mutations in FBXW7 usually occur with other simultaneous molecular aberrations, which can contribute to limited therapeutic efficacy of mTOR inhibitors.

Osteogenesis imperfecta type I: second-trimester diagnosis and incidental identification of a dominant COL1A1 deletion mutation in the paucisymptomatic father.

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Chen, Chih-Ping; Su, Yi-Ning; Chang, Tung-Yao; Chern, Schu-Rern; Chen, Chen-Yu; Su, Jun-Wei; Wang, Wayseen

2012-06-01

To present second-trimester ultrasound and molecular diagnosis for osteogenesis imperfecta (OI) type I in a female fetus and incidental identification of a dominant COL1A1 deletion mutation in her paucisymptomatic father. A 30-year-old, primigravid woman was referred for genetic counseling in the second trimester because of bowing of the fetal lower limbs. She and her husband were non-consanguineous, and there was no family history of skeletal dysplasias. Prenatal ultrasound at 22 weeks of gestation revealed short and curved right femur and left tibia, and a short left fibula. The lengths of other long bones were normal. The husband was 158 cm tall, had blue sclerae, a history of habitual subluxation and dislocation of bilateral elbows and left knee, and an episode of left ulna fracture, and was not aware of his being affected with OI type I. The woman underwent amniocentesis. Cytogenetic analysis revealed a karyotype of 46,XX. Molecular analysis of the amniocytes revealed a heterozygous deletion mutation of c.1064_1068delCTGGT in exon 17 of the COL1A1 gene. By genetic testing the husband was found to carry the same mutation. Despite counseling of favorable outcome for OI type I with the parents, the woman elected to terminate the pregnancy. Postnatal skeletal X-ray findings were consistent with OI type I. Prenatal ultrasound diagnosis of mild forms of OI should include molecular analysis of type I collagen genes in both fetus and parents. Molecular genetic analysis of the family may incidentally identify a collagen gene mutation in the paucisymptomatic affected parent. Copyright © 2012. Published by Elsevier B.V.

A mutational analysis and molecular dynamics simulation of quinolone resistance proteins QnrA1 and QnrC from Proteus mirabilis

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Ye Xinyu

2010-10-01

Full Text Available Abstract Background The first report on the transferable, plasmid-mediated quinolone-resistance determinant qnrA1 was in 1998. Since then, qnr alleles have been discovered worldwide in clinical strains of Gram-negative bacilli. Qnr proteins confer quinolone resistance, and belong to the pentapeptide repeat protein (PRP family. Several PRP crystal structures have been solved, but little is known about the functional significance of their structural arrangement. Results We conducted random and site-directed mutagenesis on qnrA1 and on qnrC, a newly identified quinolone-resistance gene from Proteus mirabilis. Many of the Qnr mutants lost their quinolone resistance function. The highly conserved hydrophobic Leu or Phe residues at the center of the pentapeptide repeats are known as i sites, and loss-of-function mutations included replacement of the i site hydrophobic residues with charged residues, replacing the i-2 site, N-terminal to the i residues, with bulky side-chain residues, introducing Pro into the β-helix coil, deletion of the N- and C-termini, and excision of a central coil. Molecular dynamics simulations and homology modeling demonstrated that QnrC overall adopts a stable β-helix fold and shares more similarities with MfpA than with other PRP structures. Based on homology modeling and molecular dynamics simulation, the dysfunctional point mutations introduced structural deformations into the quadrilateral β-helix structure of PRPs. Of the pentapeptides of QnrC, two-thirds adopted a type II β-turn, while the rest adopted type IV turns. A gap exists between coil 2 and coil 3 in the QnrC model structure, introducing a structural flexibility that is similar to that seen in MfpA. Conclusion The hydrophobic core and the β-helix backbone conformation are important for maintaining the quinolone resistance property of Qnr proteins. QnrC may share structural similarity with MfpA.

Genetic and molecular analyses of UV radiation-induced mutations in the fem-3 gene of Caenorhabditis elegans

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Hartman, P S; De Wilde, D; Dwarakanath, V N [Texas Christian Univ., Fort Worth, TX (United States). Dept. of Biology

1995-06-01

The utility of a new target gene (fem-3) is described for investigating the molecular nature of mutagenesis in the nematode Caenorhabditis elegans. As a principal attribute, this system allows for the selection, maintenance and molecular analysis of any type of mutation that disrupts the gene, including deletions. In this study, 86 mutant strains were isolated, of which 79 proved to have mutations in fem-3. Twenty of these originally tested as homozygous inviable. Homozygous inviability was expected, as Stewart and coworkers had previously observed that, unlike in other organisms, most UV radiation-induced mutations in C. elegans are chromosomal rearrangements of deficiencies (Mutat. Res 249, 37-54, 1991). However, additional data, including Southern blot analyses on 49 of the strains, indicated that most of the UV radiation-induced fem-3 mutations were not deficiencies, as originally inferred from their homozygous inviability. Instead, the lethals were most likely ``coincident mutations`` in linked, essential genes that were concomitantly induced. As such, they were lost owing to genetic recombination during stock maintenance. As in mammalian cells, yeast and bacteria, the frequency of coincident mutations was much higher than would be predicted by chance. (Author).

Genaesthics : Breast Surgery in BRCA1/2 Gene Mutation Carriers

OpenAIRE

Verschuer, Victorien

2017-01-01

markdownabstractThe present thesis focuses on breast surgery in BRCA1/2 gene mutation carriers. The topics that are studied vary broadly, representing the multiple disciplines that are involved in the diagnostic work-up and treatment of BRCA1/2-associated breast cancer. The first part contains studies on molecular and prognostic tumor characteristics in breast cancer. The thesis continues with an anatomical study on safety of prophylactic mastectomy, and finishes with studies on aesthetics an...

Gene expression profiling of acute myeloid leukemia samples from adult patients with AML-M1 and -M2 through boutique microarrays, real-time PCR and droplet digital PCR.

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Handschuh, Luiza; Kaźmierczak, Maciej; Milewski, Marek C; Góralski, Michał; Łuczak, Magdalena; Wojtaszewska, Marzena; Uszczyńska-Ratajczak, Barbara; Lewandowski, Krzysztof; Komarnicki, Mieczysław; Figlerowicz, Marek

2018-03-01

Acute myeloid leukemia (AML) is the most common and severe form of acute leukemia diagnosed in adults. Owing to its heterogeneity, AML is divided into classes associated with different treatment outcomes and specific gene expression profiles. Based on previous studies on AML, in this study, we designed and generated an AML-array containing 900 oligonucleotide probes complementary to human genes implicated in hematopoietic cell differentiation and maturation, proliferation, apoptosis and leukemic transformation. The AML-array was used to hybridize 118 samples from 33 patients with AML of the M1 and M2 subtypes of the French-American‑British (FAB) classification and 15 healthy volunteers (HV). Rigorous analysis of the microarray data revealed that 83 genes were differentially expressed between the patients with AML and the HV, including genes not yet discussed in the context of AML pathogenesis. The most overexpressed genes in AML were STMN1, KITLG, CDK6, MCM5, KRAS, CEBPA, MYC, ANGPT1, SRGN, RPLP0, ENO1 and SET, whereas the most underexpressed genes were IFITM1, LTB, FCN1, BIRC3, LYZ, ADD3, S100A9, FCER1G, PTRPE, CD74 and TMSB4X. The overexpression of the CPA3 gene was specific for AML with mutated NPM1 and FLT3. Although the microarray-based method was insufficient to differentiate between any other AML subgroups, quantitative PCR approaches enabled us to identify 3 genes (ANXA3, S100A9 and WT1) whose expression can be used to discriminate between the 2 studied AML FAB subtypes. The expression levels of the ANXA3 and S100A9 genes were increased, whereas those of WT1 were decreased in the AML-M2 compared to the AML-M1 group. We also examined the association between the STMN1, CAT and ABL1 genes, and the FLT3 and NPM1 mutation status. FLT3+/NPM1- AML was associated with the highest expression of STMN1, and ABL1 was upregulated in FLT3+ AML and CAT in FLT3- AML, irrespectively of the NPM1 mutation status. Moreover, our results indicated that CAT and WT1

Lobatin B inhibits NPM/ALK and NF-κB attenuating anaplastic-large-cell-lymphomagenesis and lymphendothelial tumour intravasation.

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Kiss, Izabella; Unger, Christine; Huu, Chi Nguyen; Atanasov, Atanas Georgiev; Kramer, Nina; Chatruphonprasert, Waranya; Brenner, Stefan; McKinnon, Ruxandra; Peschel, Andrea; Vasas, Andrea; Lajter, Ildiko; Kain, Renate; Saiko, Philipp; Szekeres, Thomas; Kenner, Lukas; Hassler, Melanie R; Diaz, Rene; Frisch, Richard; Dirsch, Verena M; Jäger, Walter; de Martin, Rainer; Bochkov, Valery N; Passreiter, Claus M; Peter-Vörösmarty, Barbara; Mader, Robert M; Grusch, Michael; Dolznig, Helmut; Kopp, Brigitte; Zupko, Istvan; Hohmann, Judit; Krupitza, Georg

2015-01-28

An apolar extract of the traditional medicinal plant Neurolaena lobata inhibited the expression of the NPM/ALK chimera, which is causal for the majority of anaplastic large cell lymphomas (ALCLs). Therefore, an active principle of the extract, the furanoheliangolide sesquiterpene lactone lobatin B, was isolated and tested regarding the inhibition of ALCL expansion and tumour cell intravasation through the lymphendothelium. ALCL cell lines, HL-60 cells and PBMCs were treated with plant compounds and the ALK inhibitor TAE-684 to measure mitochondrial activity, proliferation and cell cycle progression and to correlate the results with protein- and mRNA-expression of selected gene products. Several endpoints indicative for cell death were analysed after lobatin B treatment. Tumour cell intravasation through lymphendothelial monolayers was measured and potential causal mechanisms were investigated analysing NF-κB- and cytochrome P450 activity, and 12(S)-HETE production. Lobatin B inhibited the expression of NPM/ALK, JunB and PDGF-Rβ, and attenuated proliferation of ALCL cells by arresting them in late M phase. Mitochondrial activity remained largely unaffected upon lobatin B treatment. Nevertheless, caspase 3 became activated in ALCL cells. Also HL-60 cell proliferation was attenuated whereas PBMCs of healthy donors were not affected by lobatin B. Additionally, tumour cell intravasation, which partly depends on NF-κB, was significantly suppressed by lobatin B most likely due to its NF-κB-inhibitory property. Lobatin B, which was isolated from a plant used in ethnomedicine, targets malignant cells by at least two properties: I) inhibition of NPM/ALK, thereby providing high specificity in combating this most prevalent fusion protein occurring in ALCL; II) inhibition of NF-κB, thereby not affecting normal cells with low constitutive NF-κB activity. This property also inhibits tumour cell intravasation into the lymphatic system and may provide an option to manage this

Effects on survival of BAP1 and PBRM1 mutations in sporadic clear-cell renal-cell carcinoma: a retrospective analysis with independent validation.

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Kapur, Payal; Peña-Llopis, Samuel; Christie, Alana; Zhrebker, Leah; Pavía-Jiménez, Andrea; Rathmell, W Kimryn; Xie, Xian-Jin; Brugarolas, James

2013-02-01

Clear-cell renal-cell carcinomas display divergent clinical behaviours. However, the molecular genetic events driving these behaviours are unknown. We discovered that BAP1 is mutated in about 15% of clear-cell renal-cell carcinoma, and that BAP1 and PBRM1 mutations are largely mutually exclusive. The aim of this study was to investigate the clinicopathological significance of these molecular subtypes and to determine whether patients with BAP1-mutant and PBRM1-mutant tumours had different overall survival. In this retrospective analysis, we assessed 145 patients with primary clear-cell renal-cell carcinoma and defined PBRM1 and BAP1 mutation status from the University of Texas Southwestern Medical Center (UTSW), TX, USA, between 1998 and 2011. We classified patients into those with BAP1-mutant tumours and those with tumours exclusively mutated for PBRM1 (PBRM1-mutant). We used a second independent cohort (n=327) from The Cancer Genome Atlas (TCGA) for validation. In both cohorts, more than 80% of patients had localised or locoregional disease at presentation. Overall both cohorts were similar, although the TCGA had more patients with metastatic and higher-grade disease, and more TCGA patients presented before molecularly targeted therapies became available. The median overall survival in the UTSW cohort was significantly shorter for patients with BAP1-mutant tumours (4·6 years; 95% CI 2·1-7·2), than for patients with PBRM1-mutant tumours (10·6 years; 9·8-11·5), corresponding to a HR of 2·7 (95% CI 0·99-7·6, p=0·044). Median overall survival in the TCGA cohort was 1·9 years (95% CI 0·6-3·3) for patients with BAP1-mutant tumours and 5·4 years (4·0-6·8) for those with PBRM1-mutant tumours. A HR similar to the UTSW cohort was noted in the TCGA cohort (2·8; 95% CI 1·4-5·9; p=0·004). Patients with mutations in both BAP1 and PBRM1, although a minority (three in UTSW cohort and four in TCGA cohort), had the worst overall survival (median 2·1 years, 95

Base substitution mutations in uridinediphosphate-dependent glycosyltransferase 76G1 gene of Stevia rebaudiana causes the low levels of rebaudioside A: mutations in UGT76G1, a key gene of steviol glycosides synthesis.

Science.gov (United States)

Yang, Yong-Heng; Huang, Su-Zhen; Han, Yu-Lin; Yuan, Hai-Yan; Gu, Chun-Sun; Zhao, Yan-Hai

2014-07-01

Steviol glycosides, extracted from the leaves of Stevia rebaudiana (Bert) Bertoni, are calorie-free sugar substitute of natural origin with intensely sweet (Boileau et al., 2012). Stevioside and rebaudioside A are the two main kinds of the diterpenic glycosides. We analyzed the concentration of stevioside and rebaudioside A in Stevia leaves of about 500 samples (hybrid progenies) and discovered a mutation plant "Z05" with very low levels of rebaudioside A. Because UGT76G1, a uridinediphosphate-dependent glycosyltransferases, is responsible for the conversion from stevioside to rebaudioside A (Richman et al., 2005), so mutation identification was done by sequencing the candidate gene, UGT76G1. In this study molecular analysis of two strains revealed a heterozygotic nonsense mutation of c.389T > G (p.L121X) in UGT76G1. Meanwhile, we found some amino acid substitutions significant change the protein structure. And the difference of enzyme activity between two strains proved the lack of functionality of UGT76G1 of the mutation "Z05". So the nonsense mutation and amino acid substitution mutation resulted in the low levels of rebaudioside A. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Somatic mutations in ATP1A1 and ATP2B3 lead to aldosterone-producing adenomas and secondary hypertension

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Beuschlein, Felix; Boulkroun, Sheerazed; Osswald, Andrea

2013-01-01

Primary aldosteronism is the most prevalent form of secondary hypertension. To explore molecular mechanisms of autonomous aldosterone secretion, we performed exome sequencing of aldosterone-producing adenomas (APAs). We identified somatic hotspot mutations in the ATP1A1 (encoding an Na+/K+ ATPase α...

Molecular Genetic Analysis of the PLP1 Gene in 38 Families with PLP1-related disorders: Identification and Functional Characterization of 11 Novel PLP1 Mutations

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Marchiani Valentina

2011-06-01

Full Text Available Abstract Background The breadth of the clinical spectrum underlying Pelizaeus-Merzbacher disease and spastic paraplegia type 2 is due to the extensive allelic heterogeneity in the X-linked PLP1 gene encoding myelin proteolipid protein (PLP. PLP1 mutations range from gene duplications of variable size found in 60-70% of patients to intragenic lesions present in 15-20% of patients. Methods Forty-eight male patients from 38 unrelated families with a PLP1-related disorder were studied. All DNA samples were screened for PLP1 gene duplications using real-time PCR. PLP1 gene sequencing analysis was performed on patients negative for the duplication. The mutational status of all 14 potential carrier mothers of the familial PLP1 gene mutation was determined as well as 15/24 potential carrier mothers of the PLP1 duplication. Results and Conclusions PLP1 gene duplications were identified in 24 of the unrelated patients whereas a variety of intragenic PLP1 mutations were found in the remaining 14 patients. Of the 14 different intragenic lesions, 11 were novel; these included one nonsense and 7 missense mutations, a 657-bp deletion, a microdeletion and a microduplication. The functional significance of the novel PLP1 missense mutations, all occurring at evolutionarily conserved residues, was analysed by the MutPred tool whereas their potential effect on splicing was ascertained using the Skippy algorithm and a neural network. Although MutPred predicted that all 7 novel missense mutations would be likely to be deleterious, in silico analysis indicated that four of them (p.Leu146Val, p.Leu159Pro, p.Thr230Ile, p.Ala247Asp might cause exon skipping by altering exonic splicing elements. These predictions were then investigated in vitro for both p.Leu146Val and p.Thr230Ile by means of RNA or minigene studies and were subsequently confirmed in the case of p.Leu146Val. Peripheral neuropathy was noted in four patients harbouring intragenic mutations that altered RNA

Novel Mutations in Sandhoff Disease: A Molecular Analysis among Iranian Cohort of Infantile Patients

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Aryan, H; Aryani, O; Banihashemi, K; Zaman, T; Houshmand, M

2012-01-01

Background Sandhoff disease is an autosomal recessive disorder caused by β-hexosaminidase deficiency and accumulation of GM2 ganglioside resulting in progressive motor neuron manifestations and death from respiratory failure and infections in infantiles. Pathogenic mutations in HEXB gene were observed which leads to enzyme activity reduction and interruption of normal metabolic cycle of GM2 ganglioside in sandhoff patients. Methods: Six infantile index patients with typical biochemical and clinical picture of the disease were studied at the molecular level. After DNA extraction and amplification, probands and their parents, were evaluated by direct sequencing of amplicons. Results: We identified 7 different mutations among which 4 were novel. The most prevalent finding (50%) among our population was a 16 kb deletion including the promoter and exons 1–5. The other findings included c.1552delG and c.410G>A, c.362 A>G, c.550delT, c.1597C>T, c.1752delTG. Conclusion: We conclude that Cys137Tyr and R533C mutations may be pathogenic because of changing amino acid and locating at the conserved region and also they have not been observed in hundred controls. Besides, four mutations including: Cys137Tyr, c.1552delG, c.1597C>T and c.550delT fulfilled almost criteria for pathogenic mutation. PMID:23113155

Molecular dynamics simulations suggest that electrostatic funnel directs binding of Tamiflu to influenza N1 neuraminidases.

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Ly Le

2010-09-01

Full Text Available Oseltamivir (Tamiflu is currently the frontline antiviral drug employed to fight the flu virus in infected individuals by inhibiting neuraminidase, a flu protein responsible for the release of newly synthesized virions. However, oseltamivir resistance has become a critical problem due to rapid mutation of the flu virus. Unfortunately, how mutations actually confer drug resistance is not well understood. In this study, we employ molecular dynamics (MD and steered molecular dynamics (SMD simulations, as well as graphics processing unit (GPU-accelerated electrostatic mapping, to uncover the mechanism behind point mutation induced oseltamivir-resistance in both H5N1 "avian" and H1N1pdm "swine" flu N1-subtype neuraminidases. The simulations reveal an electrostatic binding funnel that plays a key role in directing oseltamivir into and out of its binding site on N1 neuraminidase. The binding pathway for oseltamivir suggests how mutations disrupt drug binding and how new drugs may circumvent the resistance mechanisms.

Studies of human mutation rates, December 1, 1985--November 30, 1986

International Nuclear Information System (INIS)

Neel, J.V.

1985-01-01

This program seeks to quantify native human mutation rates and to determine how man's activities may affect these rates. The program is divided into six tasks, i.e. The American Indian mutation rate, monitoring populations for frequency of mutation by electrophoresis of blood proteins, application of molecular biological approaches to the detection and study of mutational events in human populations, development of two-dimensional electrophoresis for identification of mutant proteins, co-operative program with the Radiation Effects Research Foundation in Hiroshima and Nagasaki, Japan, and statistical problems associated with the estimation of mutation rates. Progress of each of the above tasks is related in detail. (DT)

Mutation inactivation of Nijmegen breakage syndrome gene (NBS1 in hepatocellular carcinoma and intrahepatic cholangiocarcinoma.

Directory of Open Access Journals (Sweden)

Yan Wang

Full Text Available Nijmegen breakage syndrome (NBS with NBS1 germ-line mutation is a human autosomal recessive disease characterized by genomic instability and enhanced cancer predisposition. The NBS1 gene codes for a protein, Nbs1(p95/Nibrin, involved in the processing/repair of DNA double-strand breaks. Hepatocellular carcinoma (HCC is a complex and heterogeneous tumor with several genomic alterations. Recent studies have shown that heterozygous NBS1 mice exhibited a higher incidence of HCC than did wild-type mice. The objective of the present study is to assess whether NBS1 mutations play a role in the pathogenesis of human primary liver cancer, including HBV-associated HCC and intrahepatic cholangiocarcinoma (ICC. Eight missense NBS1 mutations were identified in six of 64 (9.4% HCCs and two of 18 (11.1% ICCs, whereas only one synonymous mutation was found in 89 control cases of cirrhosis and chronic hepatitis B. Analysis of the functional consequences of the identified NBS1 mutations in Mre11-binding domain showed loss of nuclear localization of Nbs1 partner Mre11, one of the hallmarks for Nbs1 deficiency, in one HCC and two ICCs with NBS1 mutations. Moreover, seven of the eight tumors with NBS1 mutations had at least one genetic alteration in the TP53 pathway, including TP53 mutation, MDM2 amplification, p14ARF homozygous deletion and promoter methylation, implying a synergistic effect of Nbs1 disruption and p53 inactivation. Our findings provide novel insight on the molecular pathogenesis of primary liver cancer characterized by mutation inactivation of NBS1, a DNA repair associated gene.

Mutations in LPL, APOC2, APOA5, GPIHBP1 and LMF1 in patients with severe hypertriglyceridaemia

NARCIS (Netherlands)

Surendran, R P; Visser, M E; Heemelaar, S; Wang, J; Peter, J; Defesche, J C; Kuivenhoven, J A; Hosseini, M; Péterfy, M; Kastelein, J J P; Johansen, C T; Hegele, R A; Stroes, E S G; Dallinga-Thie, G M

OBJECTIVES: The severe forms of hypertriglyceridaemia (HTG) are caused by mutations in genes that lead to the loss of function of lipoprotein lipase (LPL). In most patients with severe HTG (TG > 10 mmol L(-1) ), it is a challenge to define the underlying cause. We investigated the molecular basis of

The rad2 mutation affects the molecular nature of UV and acridine-mustard-induced mutations in the ADE2 gene of Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Ivanov, E.L.; Kovaltzova, S.V.; Kassinova, G.V.; Gracheva, L.M.; Korolev, V.G.; Zakharov, I.A.

1986-01-01

The authors have studied the molecular nature of ade2 mutations induced by UV light and bifunctional acridine-mustard (BAM) in wild-type (RAD) and in excision-deficient (rad2) strains of the yeast, Saccharomyces cerevisiae. In the RAD strain, UV causes 45% GC → AT transitions among all mutations; in the rad2 strain this value is 77%. BAM was shown to be highly specific for frameshift mutagenesis: 60% frameshifts in the RAD strain, and as many as 84% frameshifts in the rad2 strain were induced. Therefore, the rad2 mutation affects the specificity of UV- and BAM-induced mutagenesis in yeast. Experimental data agree with the view that the majority of mutations in the RAD strain are induced by a prereplicative mechanism, whereas mutations in the rad2 strain are predominantly postreplicative events. (Auth.)

POLG1 mutations and stroke like episodes: a distinct clinical entity rather than an atypical MELAS syndrome.

Science.gov (United States)

Cheldi, Antonella; Ronchi, Dario; Bordoni, Andreina; Bordo, Bianca; Lanfranconi, Silvia; Bellotti, Maria Grazia; Corti, Stefania; Lucchini, Valeria; Sciacco, Monica; Moggio, Maurizio; Baron, Pierluigi; Comi, Giacomo Pietro; Colombo, Antonio; Bersano, Anna

2013-01-15

POLG1 mutations have been associated with MELAS-like phenotypes. However given several clinical differences it is unknown whether POLG1 mutations are possible causes of MELAS or give raise to a distinct clinical and genetic entity, named POLG1-associated encephalopathy. We describe a 74 years old man carrying POLG1 mutations presenting with strokes, myopathy and ragged red fibers with some atypical aspects for MELAS such as late onset, lack of cerebral calcification and presence of frontal and occipital MRI lesions better consistent with the POLG associated-encephalopathy spectrum. The lack of available data hampers a definite diagnosis in our patient as well as makes it difficult to compare MELAS, which is a clearly defined clinical syndrome, with POLG1-associated encephalopathy, which is so far a purely molecularly defined syndrome with a quite heterogeneous clinical picture. However, the present report contributes to expand the phenotypic spectrum of POLG1 mutations underlining the importance of searching POLG1 mutations in patients with mitochondrial signs and MELAS like phenotypes but negative for common mtDNA mutations.

Identification of a novel LMF1 nonsense mutation responsible for severe hypertriglyceridemia by targeted next-generation sequencing.

Science.gov (United States)

Cefalù, Angelo B; Spina, Rossella; Noto, Davide; Ingrassia, Valeria; Valenti, Vincenza; Giammanco, Antonina; Fayer, Francesca; Misiano, Gabriella; Cocorullo, Gianfranco; Scrimali, Chiara; Palesano, Ornella; Altieri, Grazia I; Ganci, Antonina; Barbagallo, Carlo M; Averna, Maurizio R

Severe hypertriglyceridemia (HTG) may result from mutations in genes affecting the intravascular lipolysis of triglyceride (TG)-rich lipoproteins. The aim of this study was to develop a targeted next-generation sequencing panel for the molecular diagnosis of disorders characterized by severe HTG. We developed a targeted customized panel for next-generation sequencing Ion Torrent Personal Genome Machine to capture the coding exons and intron/exon boundaries of 18 genes affecting the main pathways of TG synthesis and metabolism. We sequenced 11 samples of patients with severe HTG (TG>885 mg/dL-10 mmol/L): 4 positive controls in whom pathogenic mutations had previously been identified by Sanger sequencing and 7 patients in whom the molecular defect was still unknown. The customized panel was accurate, and it allowed to confirm genetic variants previously identified in all positive controls with primary severe HTG. Only 1 patient of 7 with HTG was found to be carrier of a homozygous pathogenic mutation of the third novel mutation of LMF1 gene (c.1380C>G-p.Y460X). The clinical and molecular familial cascade screening allowed the identification of 2 additional affected siblings and 7 heterozygous carriers of the mutation. We showed that our targeted resequencing approach for genetic diagnosis of severe HTG appears to be accurate, less time consuming, and more economical compared with traditional Sanger resequencing. The identification of pathogenic mutations in candidate genes remains challenging and clinical resequencing should mainly intended for patients with strong clinical criteria for monogenic severe HTG. Copyright © 2017 National Lipid Association. Published by Elsevier Inc. All rights reserved.

BRCA1/2 mutation analysis in 41 ovarian cell lines reveals only one functionally deleterious BRCA1 mutation.

LENUS (Irish Health Repository)

Stordal, Britta

2013-06-01

Mutations in BRCA1\\/2 increase the risk of developing breast and ovarian cancer. Germline BRCA1\\/2 mutations occur in 8.6-13.7% of unselected epithelial ovarian cancers, somatic mutations are also frequent. BRCA1\\/2 mutated or dysfunctional cells may be sensitive to PARP inhibition by synthetic lethality. The aim of this study is to comprehensively characterise the BRCA1\\/2 status of a large panel of ovarian cancer cell lines available to the research community to assist in biomarker studies of novel drugs and in particular of PARP inhibitors. The BRCA1\\/2 genes were sequenced in 41 ovarian cell lines, mRNA expression of BRCA1\\/2 and gene methylation status of BRCA1 was also examined. The cytotoxicity of PARP inhibitors olaparib and veliparib was examined in 20 cell lines. The cell line SNU-251 has a deleterious BRCA1 mutation at 5564G > A, and is the only deleterious BRCA1\\/2 mutant in the panel. Two cell lines (UPN-251 and PEO1) had deleterious mutations as well as additional reversion mutations that restored the protein functionality. Heterozygous mutations in BRCA1\\/2 were relatively common, found in 14.6% of cell lines. BRCA1 was methylated in two cell lines (OVCAR8, A1847) and there was a corresponding decrease in gene expression. The BRCA1 methylated cell lines were more sensitive to PARP inhibition than wild-type cells. The SNU-251 deleterious mutant was more sensitive to PARP inhibition, but only in a long-term exposure to correct for its slow growth rate. Cell lines derived from metastatic disease are significantly more resistant to veliparib (2.0 fold p = 0.03) compared to those derived from primary tumours. Resistance to olaparib and veliparib was correlated Pearsons-R 0.5393, p = 0.0311. The incidence of BRCA1\\/2 deleterious mutations 1\\/41 cell lines derived from 33 different patients (3.0%) is much lower than the population incidence. The reversion mutations and high frequency of heterozygous mutations suggest that there is a selective

Splicing of phenylalanine hydroxylase (PAH) exon 11 is vulnerable - Molecular pathology of mutations in PAH exon 11

DEFF Research Database (Denmark)

Heintz, Caroline; Dobrowolski, Steven F.; Andersen, Henriette Skovgaard

2012-01-01

as a vulnerable exon and used patient derived lymphoblast cell lines and PAH minigenes to study the molecular defect that impacted pre-mRNA processing. We showed that the c.1144T>C and c.1066-3C>T mutations cause exon 11 skipping, while the c.1139C>T mutation is neutral or slightly beneficial. The c.1144T......In about 20-30% of phenylketonuria (PKU) patients, phenylalanine (Phe) levels can be controlled by cofactor 6R-tetrahydrobiopterin (BH(4)) administration. The phenylalanine hydroxylase (PAH) genotype has a predictive value concerning BH(4)-response and therefore a correct assessment of the mutation...... molecular pathology is important. Mutations that disturb the splicing of exons (e.g. interplay between splice site strength and regulatory sequences like exon splicing enhancers (ESEs)/exon splicing silencers (ESSs)) may cause different severity of PKU. In this study, we identified PAH exon 11...

Olaparib in Treating Patients With Metastatic or Advanced Urothelial Cancer With DNA-Repair Defects

Science.gov (United States)

2018-06-14

Abnormal DNA Repair; ATM Gene Mutation; ATR Gene Mutation; BAP1 Gene Mutation; BARD1 Gene Mutation; BLM Gene Mutation; BRCA1 Gene Mutation; BRCA2 Gene Mutation; BRIP1 Gene Mutation; CHEK1 Gene Mutation; CHEK2 Gene Mutation; FANCC Gene Mutation; FANCD2 Gene Mutation; FANCE Gene Mutation; FANCF Gene Mutation; MEN1 Gene Mutation; Metastatic Urothelial Carcinoma; MLH1 Gene Mutation; MSH2 Gene Mutation; MSH6 Gene Mutation; MUTYH Gene Mutation; NPM1 Gene Mutation; PALB2 Gene Mutation; PMS2 Gene Mutation; POLD1 Gene Mutation; POLE Gene Mutation; PRKDC Gene Mutation; RAD50 Gene Mutation; RAD51 Gene Mutation; SMARCB1 Gene Mutation; Stage III Bladder Urothelial Carcinoma AJCC v6 and v7; Stage IV Bladder Urothelial Carcinoma AJCC v7; STK11 Gene Mutation; Urothelial Carcinoma

The molecular landscape of pediatric acute myeloid leukemia reveals recurrent structural alterations and age-specific mutational interactions | Office of Cancer Genomics

Science.gov (United States)

We present the molecular landscape of pediatric acute myeloid leukemia (AML) and characterize nearly 1,000 participants in Children’s Oncology Group (COG) AML trials. The COG–National Cancer Institute (NCI) TARGET AML initiative assessed cases by whole-genome, targeted DNA, mRNA and microRNA sequencing and CpG methylation profiling. Validated DNA variants corresponded to diverse, infrequent mutations, with fewer than 40 genes mutated in >2% of cases.

Engineered mutations in fibrillin-1 leading to Marfan syndrome act at the protein, cellular and organismal levels.

Science.gov (United States)

Zeyer, Karina A; Reinhardt, Dieter P

2015-01-01

Fibrillins are the major components of microfibrils in the extracellular matrix of elastic and non-elastic tissues. They are multi-domain proteins, containing primarily calcium binding epidermal growth factor-like (cbEGF) domains and 8-cysteine/transforming growth factor-beta binding protein-like (TB) domains. Mutations in the fibrillin-1 gene give rise to Marfan syndrome, a connective tissue disorder with clinical complications in the cardiovascular, skeletal, ocular and other organ systems. Here, we review the consequences of engineered Marfan syndrome mutations in fibrillin-1 at the protein, cellular and organismal levels. Representative point mutations associated with Marfan syndrome in affected individuals have been introduced and analyzed in recombinant fibrillin-1 fragments. Those mutations affect fibrillin-1 on a structural and functional level. Mutations which impair folding of cbEGF domains can affect protein trafficking. Protein folding disrupted by some mutations can lead to defective secretion in mutant fibrillin-1 fragments, whereas fragments with other Marfan mutations are secreted normally. Many Marfan mutations render fibrillin-1 more susceptible to proteolysis. There is also evidence that some mutations affect heparin binding. Few mutations have been further analyzed in mouse models. An extensively studied mouse model of Marfan syndrome expresses mouse fibrillin-1 with a missense mutation (p.C1039G). The mice display similar characteristics to human patients with Marfan syndrome. Overall, the analyses of engineered mutations leading to Marfan syndrome provide important insights into the pathogenic molecular mechanisms exerted by mutated fibrillin-1. Copyright © 2015 Elsevier B.V. All rights reserved.

Molecular mechanisms of induced-mutations

International Nuclear Information System (INIS)

Kato, Takeshi

1985-01-01

The outcome of recent studies on mechanisms of induced-mutations is outlined with particular emphasis on the dependence of recA gene function in Escherichia coli. Genes involved in spontaneous mutation and x-ray- and chemical-induced mutation and genes involved in adaptive response are presented. As for SOS mutagenesis, SOS-induced regulation mechanisms and mutagenic routes are described. Furthermore, specificity of mutagens themselves are discussed in relation to mechanisms of base substitution, frameshift, and deletion mutagenesis. (Namekawa, K.)

Molecular epidemiology of DFNB1 deafness in France

Directory of Open Access Journals (Sweden)

Molinari Nicolas

2004-03-01

Full Text Available Abstract Background Mutations in the GJB2 gene have been established as a major cause of inherited non syndromic deafness in different populations. A high number of sequence variations have been described in the GJB2 gene and the associated pathogenic effects are not always clearly established. The prevalence of a number of mutations is known to be population specific, and therefore population specific testing should be a prerequisite step when molecular diagnosis is offered. Moreover, population studies are needed to determine the contribution of GJB2 variants to deafness. We present our findings from the molecular diagnostic screening of the GJB2 and GJB6 genes over a three year period, together with a population-based study of GJB2 variants. Methods and results Molecular studies were performed using denaturing High Performance Liquid Chromatograghy (DHPLC and sequencing of the GJB2 gene. Over the last 3 years we have studied 159 families presenting sensorineural hearing loss, including 84 with non syndromic, stable, bilateral deafness. Thirty families were genotyped with causative mutations. In parallel, we have performed a molecular epidemiology study on more than 3000 dried blood spots and established the frequency of the GJB2 variants in our population. Finally, we have compared the prevalence of the variants in the hearing impaired population with the general population. Conclusion Although a high heterogeneity of sequence variation was observed in patients and controls, the 35delG mutation remains the most common pathogenic mutation in our population. Genetic counseling is dependent on the knowledge of the pathogenicity of the mutations and remains difficult in a number of cases. By comparing the sequence variations observed in hearing impaired patients with those sequence variants observed in general population, from the same ethnic background, we show that the M34T, V37I and R127H variants can not be responsible for profound or severe

Neonatal Marfan syndrome caused by an exon 25 mutation of the fibrillin-1 gene.

Science.gov (United States)

Elçioglu, N H; Akalin, F; Elçioglu, M; Comeglio, P; Child, A H

2004-01-01

Neonatal Marfan syndrome caused by an exon 25 mutation of the Fibrillin-1 gene: We describe a male infant with severe arachnodactyly, hypermobility of the fingers, flexion contractures of elbows, wrists, hips, and knees, microretrognathia, crumpled ears, rockerbottom feet, loose redundant skin, and lens dislocations. Cardiac valve insufficiency and aortic dilatation resulted in cardiac failure, decompensated with digitalisation and death occurred at the age of 4 months. This case represents the severe end of the clinical spectrum of Marfan syndrome, namely neonatal Marfan syndrome. Molecular diagnostic analyses confirmed a de novo exon 25 mutation in the FBN1 gene.

Genetic heterogeneity and minor CYP1B1 involvement in the molecular basis of primary congenital glaucoma in Gypsies.

Science.gov (United States)

Sivadorai, P; Cherninkova, S; Bouwer, S; Kamenarova, K; Angelicheva, D; Seeman, P; Hollingsworth, K; Mihaylova, V; Oscar, A; Dimitrova, G; Kaneva, R; Tournev, I; Kalaydjieva, L

2008-07-01

Primary congenital glaucoma (PCG) is a genetically heterogeneous disorder of autosomal recessive inheritance, with mutations in the cytochrome P450 1B1 (CYP1B1) gene detected in an average of approximately 50% of cases worldwide. The Roma/Gypsies are considered to be a rare example of a single founder CYP1B1 mutation, E387K (identified in the Slovak Roma), accounting for 100% of disease alleles. Contrary to this concept, unusual genetic heterogeneity was revealed in this study of 21 Gypsy PCG patients from Bulgaria and 715 controls from the general Gypsy population. In our small sample of affected subjects, we identified five different CYP1B1 mutations - four known (E229K, R368H, E387K and R390C) and one novel and potentially pathogenic (F445I), which together accounted for approximately 30% of disease alleles. E387K was rare in both the patient and the control group, indicating that its high frequency in the Slovak Roma is the product of local founder effect not representative of the overall molecular pattern of PCG in the Gypsy population. Data on other Mendelian disorders and on the population genetics of the Gypsies suggest that a true founder mutation is likely to exist and has remained undetected. Our analysis of another candidate gene, MYOC, and the GLC3B and GLC3C loci did not provide support for their involvement. The molecular basis of PCG in the Gypsies is thus unresolved, and diagnostic analyses should be extended beyond the E387K mutation.

Standard Mutation Nomenclature in Molecular Diagnostics

Science.gov (United States)

Ogino, Shuji; Gulley, Margaret L.; den Dunnen, Johan T.; Wilson, Robert B.

2007-01-01

To translate basic research findings into clinical practice, it is essential that information about mutations and variations in the human genome are communicated easily and unequivocally. Unfortunately, there has been much confusion regarding the description of genetic sequence variants. This is largely because research articles that first report novel sequence variants do not often use standard nomenclature, and the final genomic sequence is compiled over many separate entries. In this article, we discuss issues crucial to clear communication, using examples of genes that are commonly assayed in clinical laboratories. Although molecular diagnostics is a dynamic field, this should not inhibit the need for and movement toward consensus nomenclature for accurate reporting among laboratories. Our aim is to alert laboratory scientists and other health care professionals to the important issues and provide a foundation for further discussions that will ultimately lead to solutions. PMID:17251329

Investigating the Impact of Asp181 Point Mutations on Interactions between PTP1B and Phosphotyrosine Substrate

Science.gov (United States)

Liu, Mengyuan; Wang, Lushan; Sun, Xun; Zhao, Xian

2014-05-01

Protein tyrosine phosphatase 1B (PTP1B) is a key negative regulator of insulin and leptin signaling, which suggests that it is an attractive therapeutic target in type II diabetes and obesity. The aim of this research is to explore residues which interact with phosphotyrosine substrate can be affected by D181 point mutations and lead to increased substrate binding. To achieve this goal, molecular dynamics simulations were performed on wild type (WT) and two mutated PTP1B/substrate complexes. The cross-correlation and principal component analyses show that point mutations can affect the motions of some residues in the active site of PTP1B. Moreover, the hydrogen bond and energy decomposition analyses indicate that apart from residue 181, point mutations have influence on the interactions of substrate with several residues in the active site of PTP1B.

HFE gene mutations in patients with primary iron overload: is there a significant improvement in molecular diagnosis yield with HFE sequencing?

Science.gov (United States)

Santos, Paulo C J L; Pereira, Alexandre C; Cançado, Rodolfo D; Schettert, Isolmar T; Sobreira, Tiago J P; Oliveira, Paulo S L; Hirata, Rosario D C; Hirata, Mario H; Figueiredo, Maria Stella; Chiattone, Carlos S; Krieger, Jose E; Guerra-Shinohara, Elvira M

2010-12-15

Rare HFE variants have been shown to be associated with hereditary hemochromatosis (HH), an iron overload disease. The low frequency of the HFE p.C282Y mutation in HH-affected Brazilian patients may suggest that other HFE-related mutations may also be implicated in the pathogenesis of HH in this population. The main aim was to screen for new HFE mutations in Brazilian individuals with primary iron overload and to investigate their relationship with HH. Fifty Brazilian patients with primary iron overload (transferrin saturation>50% in females and 60% in males) were selected. Subsequent bidirectional sequencing for each HFE exon was performed. The effect of HFE mutations on protein structure were analyzed by molecular dynamics simulation and free binding energy calculations. p.C282Y in homozygosis or in heterozygosis with p.H63D were the most frequent genotypic combinations associated with HH in our sample population (present in 17 individuals, 34%). Thirty-six (72.0%) out of the 50 individuals presented at least one HFE mutation. The most frequent genotype associated with HH was the homozygous p.C282Y mutation (n=11, 22.0%). One novel mutation (p.V256I) was indentified in heterozygosis with the p.H63D mutation. In silico modeling analysis of protein behavior indicated that the p.V256I mutation does not reduce the binding affinity between HFE and β2-microglobulin (β2M) in the same way the p.C282Y mutation does compared with the native HFE protein. In conclusion, screening of HFE through direct sequencing, as compared to p.C282Y/p.H63D genotyping, was not able to increase the molecular diagnosis yield of HH. The novel p.V256I mutation could not be implicated in the molecular basis of the HH phenotype, although its role cannot be completely excluded in HH-phenotype development. Our molecular modeling analysis can help in the analysis of novel, previously undescribed, HFE mutations. Copyright © 2010 Elsevier Inc. All rights reserved.

Investigation of mutations in the HBB gene using the 1,000 genomes database.

Science.gov (United States)

Carlice-Dos-Reis, Tânia; Viana, Jaime; Moreira, Fabiano Cordeiro; Cardoso, Greice de Lemos; Guerreiro, João; Santos, Sidney; Ribeiro-Dos-Santos, Ândrea

2017-01-01

Mutations in the HBB gene are responsible for several serious hemoglobinopathies, such as sickle cell anemia and β-thalassemia. Sickle cell anemia is one of the most common monogenic diseases worldwide. Due to its prevalence, diverse strategies have been developed for a better understanding of its molecular mechanisms. In silico analysis has been increasingly used to investigate the genotype-phenotype relationship of many diseases, and the sequences of healthy individuals deposited in the 1,000 Genomes database appear to be an excellent tool for such analysis. The objective of this study is to analyze the variations in the HBB gene in the 1,000 Genomes database, to describe the mutation frequencies in the different population groups, and to investigate the pattern of pathogenicity. The computational tool SNPEFF was used to align the data from 2,504 samples of the 1,000 Genomes database with the HG19 genome reference. The pathogenicity of each amino acid change was investigated using the databases CLINVAR, dbSNP and HbVar and five different predictors. Twenty different mutations were found in 209 healthy individuals. The African group had the highest number of individuals with mutations, and the European group had the lowest number. Thus, it is concluded that approximately 8.3% of phenotypically healthy individuals from the 1,000 Genomes database have some mutation in the HBB gene. The frequency of mutated genes was estimated at 0.042, so that the expected frequency of being homozygous or compound heterozygous for these variants in the next generation is approximately 0.002. In total, 193 subjects had a non-synonymous mutation, which 186 (7.4%) have a deleterious mutation. Considering that the 1,000 Genomes database is representative of the world's population, it can be estimated that fourteen out of every 10,000 individuals in the world will have a hemoglobinopathy in the next generation.

Molecular screening of deafness in Algeria: high genetic heterogeneity involving DFNB1 and the Usher loci, DFNB2/USH1B, DFNB12/USH1D and DFNB23/USH1F.

Science.gov (United States)

Ammar-Khodja, Fatima; Faugère, Valérie; Baux, David; Giannesini, Claire; Léonard, Susana; Makrelouf, Mohamed; Malek, Rahia; Djennaoui, Djamel; Zenati, Akila; Claustres, Mireille; Roux, Anne-Françoise

2009-01-01

A systematic approach, involving haplotyping and genotyping, to the molecular diagnosis of non-syndromic deafness within 50 families and 9 sporadic cases from Algeria is described. Mutations at the DFNB1 locus (encompassing the GJB2 and GJB6 genes) are responsible for more than half of autosomal recessive prelingual non-syndromic deafness in various populations. A c.35delG mutation can account for up to 85% of GJB2 mutations and two large deletions del(GJB6-D13S1830) and del(GJB6-D13S1854) have also been reported in several population groups. In view of the genetic heterogeneity a strategy was developed which involved direct analysis of DFNB1. In negative familial cases, haplotype analysis was carried out, where possible, to exclude DFNB1 mutations. Following this, haplotype analysis of five Usher syndrome loci, sometimes involved in autosomal non-syndromic hearing loss, was carried out to identify cases in which Usher gene sequencing was indicated. When homozygosity was observed at a locus in a consanguineous family, the corresponding gene was exhaustively sequenced. Pathogenic DFNB1 genotypes were identified in 40% of the cases. Of the 21 cases identified with 2 pathogenic mutations, c.35delG represented 76% of the mutated alleles. The additional mutations were one nonsense, two missense and one splicing mutation. Four additional patients were identified with a single DFNB1 mutation. None carried the large deletions. Three families with non-syndromic deafness carried novel unclassified variants (UVs) in MYO7A (1 family) and CDH23 (2 families) of unknown pathogenic effect. Additionally, molecular diagnosis was carried out on two Usher type I families and pathogenic mutations in MYO7A and PCDH15 were found.

Somatic mutations of isocitrate dehydrogenases 1 and 2 are prognostic and follow-up markers in patients with acute myeloid leukaemia with normal karyotype

Directory of Open Access Journals (Sweden)

Virijevic Marijana

2016-12-01

Full Text Available Mutations in the isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2 genes are frequent molecular lesions in acute myeloid leukaemia with normal karyotype (AML-NK. The effects of IDH mutations on clinical features and treatment outcome in AML-NK have been widely investigated, but only a few studies monitored these mutations during follow-up.

Molecular pathogenesis of megalencephalic leukoencephalopathy with subcortical cysts: mutations in MLC1 cause folding defects

NARCIS (Netherlands)

Duarri, A.; Teijido, O.; Lopez-Hernandez, T.; Scheper, G.C.; Barriere, H.; Boor, P.K.I.; Aguado, F.; Zorzano, A.; Palacin, M.; Martinez, A; Lukacs, G.L.; van der Knaap, M.S.; Nunes, V.; Estevez, R.

2008-01-01

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy, most often caused by mutations in the MLC1 gene. MLC1 is an oligomeric plasma membrane (PM) protein of unknown function expressed mainly in glial cells and neurons. Most disease-causing missense

Clinical and molecular diagnosis of a Costa Rican family with autosomal recessive myotonia congenita (Becker disease carrying a new mutation in the CLCN1 gene

Directory of Open Access Journals (Sweden)

Fernando Morales

2008-03-01

Full Text Available Myotonia congenita is a muscular disease characterized by myotonia, hypertrophy, and stiffness. It is inherited as either autosomal dominant or recessive known as Thomsen and Becker diseases, respectively. Here we confirm the clinical diagnosis of a family diagnosed with a myotonic condition many years ago and report a new mutation in the CLCN1 gene. The clinical diagnosis was established using ocular, cardiac, neurological and electrophysiological tests and the molecular diagnosis was done by PCR, SSCP and sequencing of the CLCN1 gene. The proband and the other affected individuals exhibited proximal and distal muscle weakness but no hypertrophy or muscular pain was found. The myotatic reflexes were lessened and sensibility was normal. Electrical and clinical myotonia was found only in the sufferers. Slit lamp and electrocardiogram tests were normal. Two affected probands presented diminution of the sensitive conduction velocities and prolonged sensory distal latencies. The clinical spectrum for this family is in agreement with a clinical diagnosis of Becker myotonia. This was confirmed by molecular diagnosis where a new disease-causing mutation (Q412P was found in the family and absent in 200 unaffected chromosomes. No latent myotonia was found in this family; therefore the ability to cause this subclinical sign might be intrinsic to each mutation. Implications of the structure-function-genotype relationship for this and other mutations are discussed. Adequate clinical diagnosis of a neuromuscular disorder would allow focusing the molecular studies toward the confirmation of the initial diagnosis, leading to a proper clinical management, genetic counseling and improving in the quality of life of the patients and relatives. Rev. Biol. Trop. 56 (1: 1-11. Epub 2008 March 31.La miotonía congénita es una enfermedad muscular caracterizada por miotonía, hipertrofia y rigidez. Se presenta con dos patrones de herencia, autosómica dominante en cuyo

Laboratory practice guidelines for detecting and reporting JAK2 and MPL mutations in myeloproliferative neoplasms: a report of the Association for Molecular Pathology.

Science.gov (United States)

Gong, Jerald Z; Cook, James R; Greiner, Timothy C; Hedvat, Cyrus; Hill, Charles E; Lim, Megan S; Longtine, Janina A; Sabath, Daniel; Wang, Y Lynn

2013-11-01

Recurrent mutations in JAK2 and MPL genes are genetic hallmarks of BCR-ABL1-negative myeloproliferative neoplasms. Detection of JAK2 and MPL mutations has been incorporated into routine diagnostic algorithms for these diseases. This Special Article summarizes results from a nationwide laboratory survey of JAK2 and MPL mutation analysis. Based on the current practice pattern and the literature, this Special Article provides recommendations and guidelines for laboratory practice for detection of mutations in the JAK2 and MPL genes, including clinical manifestations for prompting the mutation analysis, current and recommended methodologies for testing the mutations, and standardization for reporting the test results. This Special Article also points to future directions for genomic testing in BCR-ABL1-negative myeloproliferative neoplasms. Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

A novel mutation in TFL1 homolog affecting determinacy in cowpea (Vigna unguiculata).

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Dhanasekar, P; Reddy, K S

2015-02-01

Mutations in the widely conserved Arabidopsis Terminal Flower 1 (TFL1) gene and its homologs have been demonstrated to result in determinacy across genera, the knowledge of which is lacking in cowpea. Understanding the molecular events leading to determinacy of apical meristems could hasten development of cowpea varieties with suitable ideotypes. Isolation and characterization of a novel mutation in cowpea TFL1 homolog (VuTFL1) affecting determinacy is reported here for the first time. Cowpea TFL1 homolog was amplified using primers designed based on conserved sequences in related genera and sequence variation was analysed in three gamma ray-induced determinate mutants, their indeterminate parent "EC394763" and two indeterminate varieties. The analyses of sequence variation exposed a novel SNP distinguishing the determinate mutants from the indeterminate types. The non-synonymous point mutation in exon 4 at position 1,176 resulted from transversion of cytosine (C) to adenine (A) leading to an amino acid change (Pro-136 to His) in determinate mutants. The effect of the mutation on protein function and stability was predicted to be detrimental using different bioinformatics/computational tools. The functionally significant novel substitution mutation is hypothesized to affect determinacy in the cowpea mutants. Development of suitable regeneration protocols in this hitherto recalcitrant crop and subsequent complementation assay in mutants or over-expressing assay in parents could decisively conclude the role of the SNP in regulating determinacy in these cowpea mutants.

A Mononucleotide Markers Panel to Identify hMLH1/hMSH2 Germline Mutations

Directory of Open Access Journals (Sweden)

M. Pedroni

2007-01-01

Full Text Available Hereditary NonPolyposis Colorectal Cancer (Lynch syndrome is an autosomal dominant disease caused by germline mutations in a class of genes deputed to maintain genomic integrity during cell replication, mutations result in a generalized genomic instability, particularly evident at microsatellite loci (Microsatellite Instability, MSI. MSI is present in 85–90% of colorectal cancers that occur in Lynch Syndrome. To standardize the molecular diagnosis of MSI, a panel of 5 microsatellite markers was proposed (known as the “Bethesda panel”. Aim of our study is to evaluate if MSI testing with two mononucleotide markers, such as BAT25 and BAT26, was sufficient to identify patients with hMLH1/hMSH2 germline mutations. We tested 105 tumours for MSI using both the Bethesda markers and the two mononucleotide markers BAT25 and BAT26. Moreover, immunohistochemical evaluation of MLH1 and MSH2 proteins was executed on the tumours with at least one unstable microsatellite, whereas germline hMLH1/hMSH2 mutations were searched for all cases showing two or more unstable microsatellites.

A novel mutation in the albumin gene (c.1A>C) resulting in analbuminemia.

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Caridi, Gianluca; Dagnino, Monica; Lugani, Francesca; Shalev, Stavit A; Campagnoli, Monica; Galliano, Monica; Spiegel, Ronen; Minchiotti, Lorenzo

2013-01-01

Analbuminemia (OMIM # 103600) is a rare autosomal recessive disorder manifested by the absence or severe reduction of circulating serum albumin in homozygous or compound heterozygous subjects. The trait is caused by a variety of mutations within the albumin gene. We report here the clinical and molecular characterisation of two new cases of congenital analbuminemia diagnosed in two members of the Druze population living in a Galilean village (Northern Israel) on the basis of their low level of circulating albumin. The albumin gene was screened by single-strand conformation polymorphism and heteroduplex analysis, and the mutated region was submitted to DNA sequencing. Both the analbuminemic subjects resulted homozygous for a previously unreported c.1 A>C transversion, for which we suggest the name Afula from the hospital where the two cases were investigated. This mutation causes the loss of the primary start codon ATG for Met1, which is replaced by a - then untranslated - triplet CTG for Leu. (p.Met1Leu). The use of an alternative downstream ATG codon would probably give rise to a completely aberrant polypeptide chain, leading to a misrouted intracellular transport and a premature degradation. The discovery of this new ALB mutation, probably inherited from a common ancestor, sheds light on the molecular mechanism underlying the analbuminemic trait and may serve in the development of a rapid genetic test for the identification of a-symptomatic heterozygous carriers in the Druze population in the Galilee. © 2012 The Authors. European Journal of Clinical Investigation © 2012 Stichting European Society for Clinical Investigation Journal Foundation.

Molecular defects of the growth hormone receptor gene, including a new mutation, in Laron syndrome patients in Israel: relationship between defects and ethnic groups.

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Shevah, Orit; Rubinstein, Menachem; Laron, Zvi

2004-10-01

Laron Syndrome, first described in Israel, is a form of dwarfism similar to isolated growth hormone deficiency caused by molecular defects in the GH receptor gene. To characterize the molecular defects of the GH-R in Laron syndrome patients followed in our clinic. Of the 63 patients in the cohort, we investigated 31 patients and 32 relatives belonging to several ethnic origins. Molecular analysis of the GH-R gene was performed using the single strand conformation polymorphism and DNA sequencing techniques. Eleven molecular defects including a novel mutation were found. Twenty-two patients carried mutations in the extracellular domain, one in the transmembrane domain, and 3 siblings with typical Laron syndrome presented a normal GH-R. Of interest are, on one hand, different mutations within the same ethnic groups: W-15X and 5, 6 exon deletion in Jewish-Iraqis, and E180 splice and 5, 6 exon deletion in Jewish-Moroccans; and on the other hand, identical findings in patients from distinct regions: the 785-1 G to T mutation in an Israeli-Druze and a Peruvian patient. A polymorphism in exon 6, Gly168Gly, was found in 15 probands. One typical Laron patient from Greece was heterozygous for R43X in exon 4 and heterozygous for Gly168Gly. In addition, a novel mutation in exon 5: substitution of T to G replacing tyrosine 86 for aspartic acid (Y86D) is described. This study demonstrates: a) an increased focal incidence of Laron syndrome in different ethnic groups from our area with a high incidence of consanguinity; and b) a relationship between molecular defects of the GH-R, ethnic group and geographic area.

Bi-allelic Mutations in PKD1L1 Are Associated with Laterality Defects in Humans.

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Vetrini, Francesco; D'Alessandro, Lisa C A; Akdemir, Zeynep C; Braxton, Alicia; Azamian, Mahshid S; Eldomery, Mohammad K; Miller, Kathryn; Kois, Chelsea; Sack, Virginia; Shur, Natasha; Rijhsinghani, Asha; Chandarana, Jignesh; Ding, Yan; Holtzman, Judy; Jhangiani, Shalini N; Muzny, Donna M; Gibbs, Richard A; Eng, Christine M; Hanchard, Neil A; Harel, Tamar; Rosenfeld, Jill A; Belmont, John W; Lupski, James R; Yang, Yaping

2016-10-06

Disruption of the establishment of left-right (L-R) asymmetry leads to situs anomalies ranging from situs inversus totalis (SIT) to situs ambiguus (heterotaxy). The genetic causes of laterality defects in humans are highly heterogeneous. Via whole-exome sequencing (WES), we identified homozygous mutations in PKD1L1 from three affected individuals in two unrelated families. PKD1L1 encodes a polycystin-1-like protein and its loss of function is known to cause laterality defects in mouse and medaka fish models. Family 1 had one fetus and one deceased child with heterotaxy and complex congenital heart malformations. WES identified a homozygous splicing mutation, c.6473+2_6473+3delTG, which disrupts the invariant splice donor site in intron 42, in both affected individuals. In the second family, a homozygous c.5072G>C (p.Cys1691Ser) missense mutation was detected in an individual with SIT and congenital heart disease. The p.Cys1691Ser substitution affects a highly conserved cysteine residue and is predicted by molecular modeling to disrupt a disulfide bridge essential for the proper folding of the G protein-coupled receptor proteolytic site (GPS) motif. Damaging effects associated with substitutions of this conserved cysteine residue in the GPS motif have also been reported in other genes, namely GPR56, BAI3, and PKD1 in human and lat-1 in C. elegans, further supporting the likely pathogenicity of p.Cys1691Ser in PKD1L1. The identification of bi-allelic PKD1L1 mutations recapitulates previous findings regarding phenotypic consequences of loss of function of the orthologous genes in mice and medaka fish and further expands our understanding of genetic contributions to laterality defects in humans. Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Molecular alterations underlying the spontaneous and γ-ray-induced point mutations at the white locus of Drosophila Melanogaster

International Nuclear Information System (INIS)

Aleksandrova, M.V.; Lapidus, I.L.; Aleksandrov, I.D.; Karpovskij, A.L.

1996-01-01

The white locus in D.Melanogaster was selected as a target gene for the study of the mutational spectra of spontaneously arising and radiation-induced gene mutations in a whole organism. Analysis of 6 spontaneous and 73 γ-ray-induced white mutations by a combination of cytological, genetic and molecular techniques revealed that on the chromosomal and genetic levels all spontaneous mutations showed themselves to be point mutants. The share of such mutants among all heritable radiation-induced gene mutations is about 40%, whereas the rest ones are due to exchange breaks (8%) as well as multilocus, single-locus or partial-locus (intragenic) deletions (52%). The DNAs from 4 spontaneous and 17 γ-ray-induced point mutants were analysed by Southern blot-hybridization. The three spontaneous and 7 radiation mutants showed an altered DNA sequence at the left (distal) half of the white gene due to insertion or DNA rearrangement. The rest (58%) of the radiation-induced point mutations did not indicate any alternations in this part of the gene as detected by this technique and probes employed. 15 refs., 3 figs., 1 tab

Mosaicism in segmental darier disease: an in-depth molecular analysis quantifying proportions of mutated alleles in various tissues

DEFF Research Database (Denmark)

Harboe, Theresa Larriba; Willems, Patrick; Jespersgaard, Cathrine

2011-01-01

Darier disease is an autosomal dominant genodermatosis caused by germline mutations in the ATP2A2 gene. Clinical expression is variable, including rare segmental phenotypes thought to be caused by postzygotic mosaicism. Genetic counseling of segmental Darier patients is complex, as risk of transm......Darier disease is an autosomal dominant genodermatosis caused by germline mutations in the ATP2A2 gene. Clinical expression is variable, including rare segmental phenotypes thought to be caused by postzygotic mosaicism. Genetic counseling of segmental Darier patients is complex, as risk...... of transmitting a nonsegmental phenotype to offspring is of unknown magnitude. We present the first in-depth molecular analysis of a mosaic patient with segmental disease, quantifying proportions of mutated and normal alleles in various tissues. Pyrosequence analysis of DNA from semen, affected and normal skin......, peripheral leukocytes and hair revealed an uneven distribution of the mutated allele, from 14% in semen to 37% in affected skin. We suggest a model for segmental manifestation expression where a threshold number of mutated cells is needed for manifestation development. We further recommend molecular analysis...

Leber's hereditary optic neuropathy is associated with mitochondrial ND1 T3394C mutation

International Nuclear Information System (INIS)

Liang, Min; Guan, Minqiang; Zhao, Fuxing; Zhou, Xiangtian; Yuan, Meixia; Tong, Yi; Yang, Li; Wei, Qi-Ping; Sun, Yan-Hong; Lu, Fan; Qu, Jia

2009-01-01

We report here the clinical, genetic and molecular characterization of four Chinese families with Leber's hereditary optic neuropathy (LHON). There were variable severity and age-of-onset in visual impairment among these families. Strikingly, there were extremely low penetrances of visual impairment in these Chinese families. Sequence analysis of complete mitochondrial genomes in these pedigrees showed the homoplasmic T3394C (Y30H) mutation, which localized at a highly conserved tyrosine at position 30 of ND1, and distinct sets of mtDNA polymorphisms belonging to haplogroups D4b and M9a. The occurrence of T3394C mutation in these several genetically unrelated subjects affected by visual impairment strongly indicates that this mutation is involved in the pathogenesis of visual impairment. However, there was the absence of functionally significant mtDNA mutations in these four Chinese pedigrees carrying the T3394C mutation. Therefore, nuclear modifier gene(s) or environmental factor(s) may play a role in the phenotypic expression of the LHON-associated T3394C mutation.

Molecular imaging with (99m)Tc-MIBI and molecular testing for mutations in differentiating benign from malignant follicular neoplasm: a prospective comparison.

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Giovanella, L; Campenni, A; Treglia, G; Verburg, F A; Trimboli, P; Ceriani, L; Bongiovanni, M

2016-06-01

To compare mutation analysis of cytology specimens and (99m)Tc-MIBI thyroid scintigraphy for differentiating benign from malignant thyroid nodules in patients with a cytological reading of follicular neoplasm. Patients ≥18 years of age with a solitary hypofunctioning thyroid nodule (≥10 mm), normal thyrotropin and calcitonin levels, and a cytological diagnosis of follicular neoplasm were prospectively enrolled. Mutation analysis and (99m)Tc-MIBI scintigraphy were performed and patients were subsequently operated on to confirm or exclude a malignant lesion. Mutations for KRAS, HRAS and NRAS and for BRAF and translocations of PAX8/PPARγ, RET/PTC1 and RET/PTC3 were investigated. Static thyroid scintigraphic images were acquired 10 and 60 min after intravenous injection of 200 MBq of (99m)Tc-MIBI and visually assessed. Additionally, the MIBI washout index was calculated using a semiquantitative method. In our series, 26 % of nodules with a follicular pattern on cytology were malignant with a prevalence of follicular carcinomas. (99m)Tc-MIBI scintigraphy was found to be significantly more accurate (positive likelihood ratio 4.56 for visual assessment and 12.35 for semiquantitative assessment) than mutation analysis (positive likelihood ratio 1.74). A negative (99m)Tc-MIBI scan reliably excluded malignancy. In patients with a thyroid nodule cytologically diagnosed as a follicular proliferation, semiquantitative analysis of (99m)Tc-MIBI scintigraphy should be the preferred method for differentiating benign from malignant nodules. It is superior to molecular testing for the presence of differentiated thyroid cancer-associated mutations in fine-needle aspiration cytology sample material.

MLH1-deficient Colorectal Carcinoma With Wild-type BRAF and MLH1 Promoter Hypermethylation Harbor KRAS Mutations and Arise From Conventional Adenomas.

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Farchoukh, Lama; Kuan, Shih-Fan; Dudley, Beth; Brand, Randall; Nikiforova, Marina; Pai, Reetesh K

2016-10-01

Between 10% and 15% of colorectal carcinomas demonstrate sporadic DNA mismatch-repair protein deficiency as a result of MLH1 promoter methylation and are thought to arise from sessile serrated adenomas, termed the serrated neoplasia pathway. Although the presence of the BRAF V600E mutation is indicative of a sporadic cancer, up to 30% to 50% of colorectal carcinomas with MLH1 promoter hypermethylation will lack a BRAF mutation. We report the clinicopathologic and molecular features of MLH1-deficient colorectal carcinoma with wild-type BRAF and MLH1 promoter hypermethylation (referred to as MLH1-hypermethylated BRAF wild-type colorectal carcinoma, n=36) in comparison with MLH1-deficient BRAF-mutated colorectal carcinoma (n=113) and Lynch syndrome-associated colorectal carcinoma (n=36). KRAS mutations were identified in 31% of MLH1-hypermethylated BRAF wild-type colorectal carcinomas compared with 0% of MLH1-deficient BRAF-mutated colorectal carcinomas and 37% of Lynch syndrome-associated colorectal carcinomas. When a precursor polyp was identified, MLH1-hypermethylated BRAF wild-type colorectal carcinomas arose from precursor polyps resembling conventional tubular/tubulovillous adenomas in contrast to MLH1-deficient BRAF-mutated colorectal carcinomas, which arose from precursor sessile serrated adenomas (PMLH1-hypermethylated BRAF wild-type colorectal carcinoma and MLH1-deficient BRAF-mutated colorectal carcinoma had a predilection for the right colon compared with Lynch syndrome-associated colorectal carcinoma (86% vs. 92% vs. 49%, P0.05). In conclusion, our results indicate that MLH1-hypermethylated BRAF wild-type colorectal carcinomas can harbor KRAS mutations and arise from precursor polyps resembling conventional tubular/tubulovillous adenomas.

Clinical and molecular characterization of 112 single-center patients with Neurofibromatosis type 1.

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Corsello, Giovanni; Antona, Vincenzo; Serra, Gregorio; Zara, Federico; Giambrone, Clara; Lagalla, Luca; Piccione, Maria; Piro, Ettore

2018-04-04

The aim of this retrospective study was to define clinical and molecular characteristics of a large sample of neurofibromatosis type 1 (NF1) patients, as well as to evaluate mutational spectrum and genotype-phenotype correlation. NF1 is a relatively common neurogenetic disorder (1:2500-1:3000 individuals). It is caused by mutations of the NF1 gene on chromosome 17ql1.2, with autosomal dominant pattern of inheritance and wide phenotypical variability. Café-au-lait spots (CALs), cutaneous and/or subcutaneous neurofibromas (CNFs/SCNFs), skinfold freckling, skeletal abnormalities, Lisch nodules of the iris and increased risk of learning and intellectual disabilities, as well as tumors of the nervous system and other organs are its main clinical features. The preliminary group collected 168 subjects with clinical suspicion of NF1. They were evaluated following the National Institutes of Health (NIH) criteria for NF1, revised by Gutmann et al. 1997, integrated for 67 of them by molecular testing. According to these references, 112 of 168 patients were diagnosed as NF1. The sample was characterized by an equal sex ratio (57 males, 55 females) and age distribution ranging from 10 days to 60 years of age (mean age, 13 years). A wide spectrum of clinical features has been observed in our patients. Mutational analysis resulted positive in 51 cases (76%). Twenty-four mutations detected in our cohort have not been reported to date. This study may contribute to a better definition of genotypic and phenotypic features of NF1 patients, with respect to further insights into the clinical characterization of the disease. In addition, an amplification of the spectrum of mutations in the NF1 gene has been documented.

The enzymatic activity of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC) is enhanced by NPM-ALK

DEFF Research Database (Denmark)

Boccalatte, Francesco E; Voena, Claudia; Riganti, Chiara

2009-01-01

. A well-defined set of ALK-associated tyrosine phospho-peptides, including metabolic enzymes, kinases, ribosomal and cytoskeletal proteins was identified. Validation studies confirmed that VASP and ATIC associated with NPM-ALK and their phosphorylation required ALK activity. ATIC phosphorylation was also...... documented in cell lines and primary tumors carrying ALK proteins and other tyrosine kinases, including TPR-Met and wild type c-Met. Functional analyses revealed that ALK-mediated ATIC phosphorylation enhanced its enzymatic activity, dampering the methotrexate-mediated transformylase activity inhibition...

RAI1 gene mutations: mechanisms of Smith–Magenis Syndrome

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Falco M

2017-11-01

Full Text Available Mariateresa Falco,1,* Sonia Amabile,1,* Fabio Acquaviva2 1Department of Molecular Medicine and Medical Biotechnology, University of Naples “Federico II”, Naples, Italy; 2Department of Translational Medical Sciences (DISMET, Section of Pediatric Clinical Genetics, University of Naples “Federico II”, Naples, Italy *These authors contributed equally to this work Abstract: Smith–Magenis syndrome (SMS; OMIM #182290 is a complex genetic disorder characterized by distinctive physical features, developmental delay, cognitive impairment, and a typical behavioral phenotype. SMS is caused by interstitial 17p11.2 deletions, encompassing multiple genes and including the retinoic acid-induced 1 gene (RAI1, or by mutations in RAI1 itself. About 10% of all the SMS patients, in fact, carry an RAI1 mutation responsible for the phenotype. RAI1 (OMIM *607642 is a dosage-sensitive gene expressed in many tissues and highly conserved among species. Over the years, several studies have demonstrated that RAI1 (or its homologs in animal models acts as a transcriptional factor implicated in embryonic neurodevelopment, neuronal differentiation, cell growth and cell cycle regulation, bone and skeletal development, lipid and glucose metabolisms, behavioral functions, and circadian activity. Patients with RAI1 pathogenic variants show some phenotypic differences when compared to those carrying the typical deletion. They usually have lower incidence of hypotonia and less cognitive impairment than those with 17p11.2 deletions but more frequently show the behavioral characteristics of the syndrome and overeating issues. These differences reflect the primary pathogenetic role of RAI1 without the pathogenetic contribution of the other genes included in the typical 17p11.2 deletion. The better comprehension of physiological roles of RAI1, its molecular co-workers and interactors, and its contribution in determining the typical SMS phenotype will certainly open a new path

Structural insights into transient receptor potential vanilloid type 1 (TRPV1) from homology modeling, flexible docking, and mutational studies.

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Lee, Jin Hee; Lee, Yoonji; Ryu, HyungChul; Kang, Dong Wook; Lee, Jeewoo; Lazar, Jozsef; Pearce, Larry V; Pavlyukovets, Vladimir A; Blumberg, Peter M; Choi, Sun

2011-04-01

The transient receptor potential vanilloid subtype 1 (TRPV1) is a non-selective cation channel composed of four monomers with six transmembrane helices (TM1-TM6). TRPV1 is found in the central and peripheral nervous system, and it is an important therapeutic target for pain relief. We describe here the construction of a tetrameric homology model of rat TRPV1 (rTRPV1). We experimentally evaluated by mutational analysis the contribution of residues of rTRPV1 contributing to ligand binding by the prototypical TRPV1 agonists, capsaicin and resiniferatoxin (RTX). We then performed docking analysis using our homology model. The docking results with capsaicin and RTX showed that our homology model was reliable, affording good agreement with our mutation data. Additionally, the binding mode of a simplified RTX (sRTX) ligand as predicted by the modeling agreed well with those of capsaicin and RTX, accounting for the high binding affinity of the sRTX ligand for TRPV1. Through the homology modeling, docking and mutational studies, we obtained important insights into the ligand-receptor interactions at the molecular level which should prove of value in the design of novel TRPV1 ligands.

The infiltration, and prognostic importance, of Th1 lymphocytes vary in molecular subgroups of colorectal cancer.

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Ling, Agnes; Lundberg, Ida V; Eklöf, Vincy; Wikberg, Maria L; Öberg, Åke; Edin, Sofia; Palmqvist, Richard

2016-01-01

Giving strong prognostic information, T-cell infiltration is on the verge of becoming an additional component in the routine clinical setting for classification of colorectal cancer (CRC). With a view to further improving the tools for prognostic evaluation, we have studied how Th1 lymphocyte infiltration correlates with prognosis not only by quantity, but also by subsite, within CRCs with different molecular characteristics (microsatellite instability, CpG island methylator phenotype status, and BRAF and KRAS mutational status). We evaluated the Th1 marker T-bet by immunohistochemistry in 418 archival tumour tissue samples from patients who underwent surgical resection for CRC. We found that a high number of infiltrating Th1 lymphocytes is strongly associated with an improved prognosis in patients with CRC, irrespective of intratumoural subsite, and that both extent of infiltration and patient outcome differ according to molecular subgroup. In brief, microsatellite instability, CpG island methylator phenotype-high and BRAF mutated tumours showed increased infiltration of Th1 lymphocytes, and the most pronounced prognostic effect of Th1 infiltration was found in these tumours. Interestingly, BRAF mutated tumours were found to be more highly infiltrated by Th1 lymphocytes than BRAF wild-type tumours whereas the opposite was seen for KRAS mutated tumours. These differences could be explained at least partly by our finding that BRAF mutated, in contrast to KRAS mutated, CRC cell lines and tumour specimens expressed higher levels of the Th1-attracting chemokine CXCL10, and reduced levels of CCL22 and TGFB1, stimulating Th2/Treg recruitment and polarisation. In conclusion, the strong prognostic importance of Th1 lymphocyte infiltration in CRC was found at all subsites evaluated, and it remained significant in multivariable analyses, indicating that T-bet may be a valuable marker in the clinical setting. Our results also indicate that T-bet is of value when analysed in

Proceedings of the FNCA workshop on plant mutation breeding 2001. Molecular biological techniques

International Nuclear Information System (INIS)

Kume, Tamikazu; Watanabe, Kazuo; Tano, Shigemitsu

2002-02-01

The FNCA (Forum for Nuclear Cooperation in Asia) Workshop on Plant Mutation Breeding was held on 20-24 August 2001 in Bangkok, Thailand. The Workshop was sponsored by the Ministry of Education, Culture, Sports, Science and Technology (MEXT). The Kasetsart University (KU), the Office of Atomic Energy for Peace (OAEP) and Department of Agriculture (DOA) acted as local host and the organizer with the cooperation of the Ministry of Agriculture, Forestry and Fisheries (MAFF) of Japan, the Japan Atomic Industrial Forum (JAIF) and Japan Atomic Energy Research Institute (JAERI). The Workshop was attended by two participants, a Project Leader and an expert on molecular biological techniques for plant mutation breeding, from each of the participating countries, i.e. China, Indonesia, Malaysia, the Philippines and Vietnam. One participant from the Republic of Korea, nine participants from Japan and thirteen participants from Thailand including three invited speakers attended the Workshop. Eleven papers including three invited papers on the current status of molecular biological techniques for plant mutation breeding were presented. Discussions were focused to further regional cooperation, to review and discuss results of past activities. The Medium-Term Plan of the project on the application of radiation and radioisotopes for agriculture in participating countries of Regional Nuclear Cooperation Activities (RNCA) was formulated and agreed. This proceeding compiles the invited and contributed papers that were submitted from the speakers. (author)

Transcriptome analysis of skin fibroblasts with dominant negative COL3A1 mutations provides molecular insights into the etiopathology of vascular Ehlers-Danlos syndrome.

Science.gov (United States)

Chiarelli, Nicola; Carini, Giulia; Zoppi, Nicoletta; Ritelli, Marco; Colombi, Marina

2018-01-01

Vascular Ehlers-Danlos syndrome (vEDS) is a dominantly inherited connective tissue disorder caused by mutations in the COL3A1 gene that encodes type III collagen (COLLIII), which is the major expressed collagen in blood vessels and hollow organs. The majority of disease-causing variants in COL3A1 are glycine substitutions and in-frame splice mutations in the triple helix domain that through a dominant negative effect are associated with the severe clinical spectrum potentially lethal of vEDS, characterized by fragility of soft connective tissues with arterial and organ ruptures. To shed lights into molecular mechanisms underlying vEDS, we performed gene expression profiling in cultured skin fibroblasts from three patients with different structural COL3A1 mutations. Transcriptome analysis revealed significant changes in the expression levels of several genes involved in maintenance of cell redox and endoplasmic reticulum (ER) homeostasis, COLLs folding and extracellular matrix (ECM) organization, formation of the proteasome complex, and cell cycle regulation. Protein analyses showed that aberrant COLLIII expression is associated with the disassembly of many structural ECM constituents, such as fibrillins, EMILINs, and elastin, as well as with the reduction of the proteoglycans perlecan, decorin, and versican, all playing an important role in the vascular system. Furthermore, the altered distribution of the ER marker protein disulfide isomerase PDI and the strong reduction of the COLLs-modifying enzyme FKBP22 are consistent with the disturbance of ER-related homeostasis and COLLs biosynthesis and post-translational modifications, indicated by microarray analysis. Our findings add new insights into the pathophysiology of this severe vascular disorder, since they provide a picture of the gene expression changes in vEDS skin fibroblasts and highlight that dominant negative mutations in COL3A1 also affect post-translational modifications and deposition into the ECM of

Transcriptome analysis of skin fibroblasts with dominant negative COL3A1 mutations provides molecular insights into the etiopathology of vascular Ehlers-Danlos syndrome.

Directory of Open Access Journals (Sweden)

Nicola Chiarelli

Full Text Available Vascular Ehlers-Danlos syndrome (vEDS is a dominantly inherited connective tissue disorder caused by mutations in the COL3A1 gene that encodes type III collagen (COLLIII, which is the major expressed collagen in blood vessels and hollow organs. The majority of disease-causing variants in COL3A1 are glycine substitutions and in-frame splice mutations in the triple helix domain that through a dominant negative effect are associated with the severe clinical spectrum potentially lethal of vEDS, characterized by fragility of soft connective tissues with arterial and organ ruptures. To shed lights into molecular mechanisms underlying vEDS, we performed gene expression profiling in cultured skin fibroblasts from three patients with different structural COL3A1 mutations. Transcriptome analysis revealed significant changes in the expression levels of several genes involved in maintenance of cell redox and endoplasmic reticulum (ER homeostasis, COLLs folding and extracellular matrix (ECM organization, formation of the proteasome complex, and cell cycle regulation. Protein analyses showed that aberrant COLLIII expression is associated with the disassembly of many structural ECM constituents, such as fibrillins, EMILINs, and elastin, as well as with the reduction of the proteoglycans perlecan, decorin, and versican, all playing an important role in the vascular system. Furthermore, the altered distribution of the ER marker protein disulfide isomerase PDI and the strong reduction of the COLLs-modifying enzyme FKBP22 are consistent with the disturbance of ER-related homeostasis and COLLs biosynthesis and post-translational modifications, indicated by microarray analysis. Our findings add new insights into the pathophysiology of this severe vascular disorder, since they provide a picture of the gene expression changes in vEDS skin fibroblasts and highlight that dominant negative mutations in COL3A1 also affect post-translational modifications and deposition

A recessive founder mutation in regulator of telomere elongation helicase 1, RTEL1, underlies severe immunodeficiency and features of Hoyeraal Hreidarsson syndrome.

Science.gov (United States)

Ballew, Bari J; Joseph, Vijai; De, Saurav; Sarek, Grzegorz; Vannier, Jean-Baptiste; Stracker, Travis; Schrader, Kasmintan A; Small, Trudy N; O'Reilly, Richard; Manschreck, Chris; Harlan Fleischut, Megan M; Zhang, Liying; Sullivan, John; Stratton, Kelly; Yeager, Meredith; Jacobs, Kevin; Giri, Neelam; Alter, Blanche P; Boland, Joseph; Burdett, Laurie; Offit, Kenneth; Boulton, Simon J; Savage, Sharon A; Petrini, John H J

2013-08-01

Dyskeratosis congenita (DC) is a heterogeneous inherited bone marrow failure and cancer predisposition syndrome in which germline mutations in telomere biology genes account for approximately one-half of known families. Hoyeraal Hreidarsson syndrome (HH) is a clinically severe variant of DC in which patients also have cerebellar hypoplasia and may present with severe immunodeficiency and enteropathy. We discovered a germline autosomal recessive mutation in RTEL1, a helicase with critical telomeric functions, in two unrelated families of Ashkenazi Jewish (AJ) ancestry. The affected individuals in these families are homozygous for the same mutation, R1264H, which affects three isoforms of RTEL1. Each parent was a heterozygous carrier of one mutant allele. Patient-derived cell lines revealed evidence of telomere dysfunction, including significantly decreased telomere length, telomere length heterogeneity, and the presence of extra-chromosomal circular telomeric DNA. In addition, RTEL1 mutant cells exhibited enhanced sensitivity to the interstrand cross-linking agent mitomycin C. The molecular data and the patterns of inheritance are consistent with a hypomorphic mutation in RTEL1 as the underlying basis of the clinical and cellular phenotypes. This study further implicates RTEL1 in the etiology of DC/HH and immunodeficiency, and identifies the first known homozygous autosomal recessive disease-associated mutation in RTEL1.

A recessive founder mutation in regulator of telomere elongation helicase 1, RTEL1, underlies severe immunodeficiency and features of Hoyeraal Hreidarsson syndrome.

Directory of Open Access Journals (Sweden)

Bari J Ballew

2013-08-01

Full Text Available Dyskeratosis congenita (DC is a heterogeneous inherited bone marrow failure and cancer predisposition syndrome in which germline mutations in telomere biology genes account for approximately one-half of known families. Hoyeraal Hreidarsson syndrome (HH is a clinically severe variant of DC in which patients also have cerebellar hypoplasia and may present with severe immunodeficiency and enteropathy. We discovered a germline autosomal recessive mutation in RTEL1, a helicase with critical telomeric functions, in two unrelated families of Ashkenazi Jewish (AJ ancestry. The affected individuals in these families are homozygous for the same mutation, R1264H, which affects three isoforms of RTEL1. Each parent was a heterozygous carrier of one mutant allele. Patient-derived cell lines revealed evidence of telomere dysfunction, including significantly decreased telomere length, telomere length heterogeneity, and the presence of extra-chromosomal circular telomeric DNA. In addition, RTEL1 mutant cells exhibited enhanced sensitivity to the interstrand cross-linking agent mitomycin C. The molecular data and the patterns of inheritance are consistent with a hypomorphic mutation in RTEL1 as the underlying basis of the clinical and cellular phenotypes. This study further implicates RTEL1 in the etiology of DC/HH and immunodeficiency, and identifies the first known homozygous autosomal recessive disease-associated mutation in RTEL1.

Novel POC1A mutation in primordial dwarfism reveals new insights for centriole biogenesis.

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Koparir, Asuman; Karatas, Omer F; Yuceturk, Betul; Yuksel, Bayram; Bayrak, Ali O; Gerdan, Omer F; Sagiroglu, Mahmut S; Gezdirici, Alper; Kirimtay, Koray; Selcuk, Ece; Karabay, Arzu; Creighton, Chad J; Yuksel, Adnan; Ozen, Mustafa

2015-10-01

POC1A encodes a WD repeat protein localizing to centrioles and spindle poles and is associated with short stature, onychodysplasia, facial dysmorphism and hypotrichosis (SOFT) syndrome. These main features are related to the defect in cell proliferation of chondrocytes in growth plate. In the current study, we aimed at identifying the molecular basis of two patients with primordial dwarfism (PD) in a single family through utilization of whole-exome sequencing. A novel homozygous p.T120A missense mutation was detected in POC1A in both patients, a known causative gene of SOFT syndrome, and confirmed using Sanger sequencing. To test the pathogenicity of the detected mutation, primary fibroblast cultures obtained from the patients and a control individual were used. For evaluating the global gene expression profile of cells carrying p.T120A mutation in POC1A, we performed the gene expression array and compared their expression profiles to those of control fibroblast cells. The gene expression array analysis showed that 4800 transcript probes were significantly deregulated in cells with p.T120A mutation in comparison to the control. GO term association results showed that deregulated genes are mostly involved in the extracellular matrix and cytoskeleton. Furthermore, the p.T120A missense mutation in POC1A caused the formation of abnormal mitotic spindle structure, including supernumerary centrosomes, and changes in POC1A were accompanied by alterations in another centrosome-associated WD repeat protein p80-katanin. As a result, we identified a novel mutation in POC1A of patients with PD and showed that this mutation causes the formation of multiple numbers of centrioles and multipolar spindles with abnormal chromosome arrangement. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Unique mutation portraits and frequent COL2A1 gene alteration in chondrosarcoma

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Totoki, Yasushi; Yoshida, Akihiko; Hosoda, Fumie; Nakamura, Hiromi; Hama, Natsuko; Ogura, Koichi; Yoshida, Aki; Fujiwara, Tomohiro; Arai, Yasuhito; Toguchida, Junya; Tsuda, Hitoshi; Miyano, Satoru; Kawai, Akira

2014-01-01

Chondrosarcoma is the second most frequent malignant bone tumor. However, the etiological background of chondrosarcomagenesis remains largely unknown, along with details on molecular alterations and potential therapeutic targets. Massively parallel paired-end sequencing of whole genomes of 10 primary chondrosarcomas revealed that the process of accumulation of somatic mutations is homogeneous irrespective of the pathological subtype or the presence of IDH1 mutations, is unique among a range of cancer types, and shares significant commonalities with that of prostate cancer. Clusters of structural alterations localized within a single chromosome were observed in four cases. Combined with targeted resequencing of additional cartilaginous tumor cohorts, we identified somatic alterations of the COL2A1 gene, which encodes an essential extracellular matrix protein in chondroskeletal development, in 19.3% of chondrosarcoma and 31.7% of enchondroma cases. Epigenetic regulators (IDH1 and YEATS2) and an activin/BMP signal component (ACVR2A) were recurrently altered. Furthermore, a novel FN1-ACVR2A fusion transcript was observed in both chondrosarcoma and osteochondromatosis cases. With the characteristic accumulative process of somatic changes as a background, molecular defects in chondrogenesis and aberrant epigenetic control are primarily causative of both benign and malignant cartilaginous tumors. PMID:25024164

Quality control in mutation analysis: the European Molecular Genetics Quality Network (EMQN).

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Müller, C R

2001-08-01

The demand for clinical molecular genetics testing has steadily grown since its introduction in the 1980s. In order to reach and maintain the agreed quality standards of laboratory medicine, the same internal and external quality assurance (IQA/EQA) criteria have to be applied as for "conventional" clinical chemistry or pathology. In 1996 the European Molecular Genetics Quality Network (EMQN) was established in order to spread QA standards across Europe and to harmonise the existing national activities. EMQN is operated by a central co-ordinator and 17 national partners from 15 EU countries; since 1998 it is being funded by the EU commission for a 3-year period. EMQN promotes QA by two tools: by providing disease-specific best practice meetings (BPM) and EQA schemes. A typical BPM is focussed on one disease or group of related disorders. International experts report on the latest news of gene characterisation and function and the state-of-the-art techniques for mutation detection. Disease-specific EQA schemes are provided by experts in the field. DNA samples are sent out together with mock clinical referrals and a diagnostic question is asked. Written reports must be returned which are marked for genotyping and interpretation. So far, three BPMs have been held and six EQA schemes are in operation at various stages. Although mutation types and diagnostic techniques varied considerably between schemes, the overall technical performance showed a high diagnostic standard. Nevertheless, serious genotyping errors have been occurred in some schemes which underline the necessity of quality assurance efforts. The European Molecular Genetics Quality Network provides a necessary platform for the internal and external quality assurance of molecular genetic testing.

Molecular imaging with {sup 99m}Tc-MIBI and molecular testing for mutations in differentiating benign from malignant follicular neoplasm: a prospective comparison

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Giovanella, L.; Treglia, G.; Ceriani, L. [Oncology Institute of Southern Switzerland, Department of Nuclear Medicine and Thyroid Centre, Bellinzona (Switzerland); Campenni, A. [Policlinico Universitario, Istituto di Medicina Nucleare, Messina (Italy); Verburg, F.A. [RWTH University Hospital Aachen, Department of Nuclear Medicine, Aachen (Germany); Trimboli, P. [Oncology Institute of Southern Switzerland, Department of Nuclear Medicine and Thyroid Centre, Bellinzona (Switzerland); Ospedale Israelitico, Sezione di Endocrinologia e Diabetologia, Roma (Italy); Bongiovanni, M. [Centre Hopitalier Universitaire Vaudouise, Institut de Pathologie, Lausanne (Switzerland)

2016-06-15

To compare mutation analysis of cytology specimens and {sup 99m}Tc-MIBI thyroid scintigraphy for differentiating benign from malignant thyroid nodules in patients with a cytological reading of follicular neoplasm. Patients ≥18 years of age with a solitary hypofunctioning thyroid nodule (≥10 mm), normal thyrotropin and calcitonin levels, and a cytological diagnosis of follicular neoplasm were prospectively enrolled. Mutation analysis and {sup 99m}Tc-MIBI scintigraphy were performed and patients were subsequently operated on to confirm or exclude a malignant lesion. Mutations for KRAS, HRAS and NRAS and for BRAF and translocations of PAX8/PPARγ, RET/PTC1 and RET/PTC3 were investigated. Static thyroid scintigraphic images were acquired 10 and 60 min after intravenous injection of 200 MBq of {sup 99m}Tc-MIBI and visually assessed. Additionally, the MIBI washout index was calculated using a semiquantitative method. In our series, 26 % of nodules with a follicular pattern on cytology were malignant with a prevalence of follicular carcinomas. {sup 99m}Tc-MIBI scintigraphy was found to be significantly more accurate (positive likelihood ratio 4.56 for visual assessment and 12.35 for semiquantitative assessment) than mutation analysis (positive likelihood ratio 1.74). A negative {sup 99m}Tc-MIBI scan reliably excluded malignancy. In patients with a thyroid nodule cytologically diagnosed as a follicular proliferation, semiquantitative analysis of {sup 99m}Tc-MIBI scintigraphy should be the preferred method for differentiating benign from malignant nodules. It is superior to molecular testing for the presence of differentiated thyroid cancer-associated mutations in fine-needle aspiration cytology sample material. (orig.)

Insight into resistance mechanisms of AZD4547 and E3810 to FGFR1 gatekeeper mutation via theoretical study

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Liang D

2017-02-01

Full Text Available Donglou Liang,1,* Qiaowan Chen,2,* Yujin Guo,1 Ting Zhang,3 Wentao Guo4 1Pharmacy Department, Jining First People’s Hospital, 2Department of Obstetrics, Affiliated Hospital of Jining Medical University, Jining, Shandong, 3Department of Rheumatology, The First Affiliated Hospital of Wenzhou Medical University, 4School of Pharmacy, Wenzhou Medical University, Wenzhou, Zhejiang, People’s Republic of China *These authors contributed equally to this work Abstract: Inhibitors targeting the amplification of the fibroblast growth factor receptor 1 (FGFR1 have found success in the treatment of FGFR1-positive squamous cell lung and breast cancers. A secondary mutation of gatekeeper residue (V561M in the binding site has been linked to the acquired resistance. Recently, two well-known small molecule inhibitors of FGFR1, AZD4547 and E3810, reported that the V561M mutation confers significant resistance to E3810, while retaining affinity for AZD4547. FGFR1 is widely investigated as potential therapeutic target, while there are few computational studies made to understand the resistance mechanisms about FGFR1 V561M gatekeeper mutation. In this study, molecular docking, classical molecular dynamics simulations, molecular mechanics/generalized born surface area (MM/GBSA free energy calculations, and umbrella sampling (US simulations were carried out to make clear the principle of the binding preference of AZD4547 and E3810 toward FGFR1 V561M gatekeeper mutation. The results provided by MM/GBSA reveal that AZD4547 has similar binding affinity to both FGFR1WT and FGFR1V561M, whereas E3810 has much higher binding affinity to FGFR1WT than to FGFR1V561M. Comparison of individual energy terms indicates that the major variation of E3810 between FGFR1WT and FGFR1V561M are van der Waals interactions. In addition, US simulations prove that the potential of mean force (PMF profile of AZD4547 toward FGFR1WT and FGFR1V561M has similar PMF depth. However, the PMF profile

A novel fully automated molecular diagnostic system (AMDS for colorectal cancer mutation detection.

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Shiro Kitano

Full Text Available BACKGROUND: KRAS, BRAF and PIK3CA mutations are frequently observed in colorectal cancer (CRC. In particular, KRAS mutations are strong predictors for clinical outcomes of EGFR-targeted treatments such as cetuximab and panitumumab in metastatic colorectal cancer (mCRC. For mutation analysis, the current methods are time-consuming, and not readily available to all oncologists and pathologists. We have developed a novel, simple, sensitive and fully automated molecular diagnostic system (AMDS for point of care testing (POCT. Here we report the results of a comparison study between AMDS and direct sequencing (DS in the detection of KRAS, BRAF and PI3KCA somatic mutations. METHODOLOGY/PRINCIPAL FINDING: DNA was extracted from a slice of either frozen (n = 89 or formalin-fixed and paraffin-embedded (FFPE CRC tissue (n = 70, and then used for mutation analysis by AMDS and DS. All mutations (n = 41 among frozen and 27 among FFPE samples detected by DS were also successfully (100% detected by the AMDS. However, 8 frozen and 6 FFPE samples detected as wild-type in the DS analysis were shown as mutants in the AMDS analysis. By cloning-sequencing assays, these discordant samples were confirmed as true mutants. One sample had simultaneous "hot spot" mutations of KRAS and PIK3CA, and cloning assay comfirmed that E542K and E545K were not on the same allele. Genotyping call rates for DS were 100.0% (89/89 and 74.3% (52/70 in frozen and FFPE samples, respectively, for the first attempt; whereas that of AMDS was 100.0% for both sample sets. For automated DNA extraction and mutation detection by AMDS, frozen tissues (n = 41 were successfully detected all mutations within 70 minutes. CONCLUSIONS/SIGNIFICANCE: AMDS has superior sensitivity and accuracy over DS, and is much easier to execute than conventional labor intensive manual mutation analysis. AMDS has great potential for POCT equipment for mutation analysis.

Aarskog-Scott syndrome: clinical update and report of nine novel mutations of the FGD1 gene.

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Orrico, A; Galli, L; Faivre, L; Clayton-Smith, J; Azzarello-Burri, S M; Hertz, J M; Jacquemont, S; Taurisano, R; Arroyo Carrera, I; Tarantino, E; Devriendt, K; Melis, D; Thelle, T; Meinhardt, U; Sorrentino, V

2010-02-01

Mutations in the FGD1 gene have been shown to cause Aarskog-Scott syndrome (AAS), or facio-digito-genital dysplasia (OMIM#305400), an X-linked disorder characterized by distinctive genital and skeletal developmental abnormalities with a broad spectrum of clinical phenotypes. To date, 20 distinct mutations have been reported, but little phenotypic data are available on patients with molecularly confirmed AAS. In the present study, we report on our experience of screening for mutations in the FGD1 gene in a cohort of 60 European patients with a clinically suspected diagnosis of AAS. We identified nine novel mutations in 11 patients (detection rate of 18.33%), including three missense mutations (p.R402Q; p.S558W; p.K748E), four truncating mutations (p.Y530X; p.R656X; c.806delC; c.1620delC), one in-frame deletion (c.2020_2022delGAG) and the first reported splice site mutation (c.1935+3A>C). A recurrent mutation (p.R656X) was detected in three independent families. We did not find any evidence for phenotype-genotype correlations between type and position of mutations and clinical features. In addition to the well-established phenotypic features of AAS, other clinical features are also reported and discussed. Copyright 2010 Wiley-Liss, Inc.

Phenotypes in siblings with homozygous mutations of TRAPPC9 and/or MCPH1 support a bifunctional model of MCPH1.

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Duerinckx, Sarah; Meuwissen, Marije; Perazzolo, Camille; Desmyter, Laurence; Pirson, Isabelle; Abramowicz, Marc

2018-04-24

Autosomal recessive intellectual disability (ARID) is vastly heterogeneous. Truncating mutations of TRAPPC9 were reported in 8 ARID families. Autosomal recessive primary microcephaly (MCPH) represents another subgroup of ARID, itself very heterogeneous, where the size of the brain is very small since birth. MCPH1 plays a role at the centrosome via a BRCT1 domain, and in DNA Damage Repair (DDR) via BRCT2 and BRCT3, and it is not clear which of these two mechanisms causes MCPH in man. We studied the phenotype and sequenced the exome in two siblings with MCPH and their unaffected sister. Homozygous mutations of TRAPPC9 (p.Leu178Pro) and of MCPH1 (p.Arg741X) were found in both affected siblings. Brain MRI showed anomalies previously associated with TRAPPC9 defects, supporting the implication of TRAPPC9 in the phenotype. Importantly, the asymptomatic sister with normal head size was homozygous for the MCPH1 truncating mutation and heterozygous for the TRAPPC9 mutation. The affected siblings represent the first ARID cases with a TRAPPC9 missense mutation and with microcephaly of prenatal onset of. Furthermore, their unaffected sister represents strong evidence that the lack of MCPH1 BRCT3 domain does not cause MCPH in man, supporting a bifunctional model of MCPH1 where the centrosomal function is involved in brain volumic development and not the DDR function. © 2018 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.

Lung cancer with concurrent EGFR mutation and ROS1 rearrangement: a case report and review of the literature

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Zhu YC

2016-07-01

Full Text Available You-cai Zhu,1,2,* Chun-wei Xu,3,* Xiao-qian Ye,4 Man-xiang Yin,4 Jin-xian Zhang,2 Kai-qi Du,2 Zhi-hao Zhang,2 Jian Hu1 1Department of Thoracic Surgery, The First Affiliated Hospital of Medical School of Zhejiang University, Hangzhou, 2Department of Thoracic Surgery, Chinese People’s Armed Police Force, Zhejiang Corps Hospital, Jiaxing, Zhejiang, 3Department of Pathology, Affiliated Hospital of Academy of Military Medical Sciences, Beijing, 4Department of Pathology, Chinese People’s Armed Police Force, Zhejiang Corps Hospital, Jiaxing, Zhejiang, People’s Republic of China *These authors contributed equally to this work Abstract: ROS1 rearrangement has recently emerged as a new molecular subtype in non-small cell lung cancer, and is predominantly found in lung adenocarcinomas compared with other oncogenes such as EGFR, KRAS, or ALK. Patients who have both mutations are extremely rare. Here we report a 50-year-old female diagnosed with adenocarcinoma with sarcomatoid differentiation, who was shown to have EGFR and ROS1 mutations. The patient was treated surgically and received three cycles of adjuvant postoperative chemotherapy. In addition, we reviewed the previously reported cases and related literature. This presentation will provide further understanding of the underlying molecular biology and optimal treatment for non-small cell lung cancer patients with more than one driver mutation. Keywords: non-small cell lung cancer, EGFR gene mutation, ROS1 fusion gene

A Novel WT1 Gene Mutation in a Three-Generation Family with Progressive Isolated Focal Segmental Glomerulosclerosis

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Caridi, Gianluca; Malaventura, Cristina; Dagnino, Monica; Leonardi, Emanuela; Artifoni, Lina; Ghiggeri, Gian Marco; Tosatto, Silvio C.E.; Murer, Luisa

2010-01-01

Background and objectives: Wilms tumor-suppressor gene-1 (WT1) plays a key role in kidney development and function. WT1 mutations usually occur in exons 8 and 9 and are associated with Denys-Drash, or in intron 9 and are associated with Frasier syndrome. However, overlapping clinical and molecular features have been reported. Few familial cases have been described, with intrafamilial variability. Sporadic cases of WT1 mutations in isolated diffuse mesangial sclerosis or focal segmental glomerulosclerosis have also been reported. Design, setting, participants, & measurements: Molecular analysis of WT1 exons 8 and 9 was carried out in five members on three generations of a family with late-onset isolated proteinuria. The effect of the detected amino acid substitution on WT1 protein's structure was studied by bioinformatics tools. Results: Three family members reached end-stage renal disease in full adulthood. None had genital abnormalities or Wilms tumor. Histologic analysis in two subjects revealed focal segmental glomerulosclerosis. The novel sequence variant c.1208G>A in WT1 exon 9 was identified in all of the affected members of the family. Conclusions: The lack of Wilms tumor or other related phenotypes suggests the expansion of WT1 gene analysis in patients with focal segmental glomerulosclerosis, regardless of age or presence of typical Denys-Drash or Frasier syndrome clinical features. Structural analysis of the mutated protein revealed that the mutation hampers zinc finger-DNA interactions, impairing target gene transcription. This finding opens up new issues about WT1 function in the maintenance of the complex gene network that regulates normal podocyte function. PMID:20150449

The L444P Gba1 mutation enhances alpha-synuclein induced loss of nigral dopaminergic neurons in mice

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Migdalska-Richards, Anna; Wegrzynowicz, Michal; Rusconi, Raffaella; Deangeli, Giulio; Di Monte, Donato A; Spillantini, Maria G; Schapira, Anthony H V

2017-01-01

Abstract Mutations in glucocerebrosidase 1 (GBA1) represent the most prevalent risk factor for Parkinson’s disease. The molecular mechanisms underlying the link between GBA1 mutations and Parkinson’s disease are incompletely understood. We analysed two aged (24-month-old) Gba1 mouse models, one carrying a knock-out mutation and the other a L444P knock-in mutation. A significant reduction of glucocerebrosidase activity was associated with increased total alpha-synuclein accumulation in both these models. Gba1 mutations alone did not alter the number of nigral dopaminergic neurons nor striatal dopamine levels. We then investigated the effect of overexpression of human alpha-synuclein in the substantia nigra of aged (18 to 21-month-old) L444P Gba1 mice. Following intraparenchymal injections of human alpha-synuclein carrying viral vectors, pathological accumulation of phosphorylated alpha-synuclein occurred within the transduced neurons. Stereological counts of nigral dopaminergic neurons revealed a significantly greater cell loss in Gba1-mutant than wild-type mice. These results indicate that Gba1 deficiency enhances neuronal vulnerability to neurodegenerative processes triggered by increased alpha-synuclein expression. PMID:28969384

Molecular nature of mutations induced by high-LET irradiation with argon and carbon ions in Arabidopsis thaliana

International Nuclear Information System (INIS)

Hirano, Tomonari; Kazama, Yusuke; Ohbu, Sumie; Shirakawa, Yuki; Liu Yang; Kambara, Tadashi; Fukunishi, Nobuhisa; Abe, Tomoko

2012-01-01

Linear energy transfer (LET) is an important parameter to be considered in heavy-ion mutagenesis. However, in plants, no quantitative data are available on the molecular nature of the mutations induced with high-LET radiation above 101–124 keV μm −1 . In this study, we irradiated dry seeds of Arabidopsis thaliana with Ar and C ions with an LET of 290 keV μm −1 . We analyzed the DNA alterations caused by the higher-LET radiation. Mutants were identified from the M 2 pools. In total, 14 and 13 mutated genes, including bin2, egy1, gl1, gl2, hy1, hy3–5, ttg1, and var2, were identified in the plants derived from Ar- and C-ions irradiation, respectively. In the mutants from both irradiations, deletion was the most frequent type of mutation; 13 of the 14 mutated genes from the Ar ion-irradiated plants and 11 of the 13 mutated genes from the C ion-irradiated plants harbored deletions. Analysis of junction regions generated by the 2 types of irradiation suggested that alternative non-homologous end-joining was the predominant pathway of repair of break points. Among the deletions, the proportion of large deletions (>100 bp) was about 54% for Ar-ion irradiation and about 64% for C-ion irradiation. Both current results and previously reported data revealed that the proportions of the large deletions induced by 290-keV μm −1 radiations were higher than those of the large deletions induced by lower-LET radiations (6% for 22.5–30.0 keV μm −1 and 27% for 101–124 keV μm −1 ). Therefore, the 290 keV μm −1 heavy-ion beams can effectively induce large deletions and will prove useful as novel mutagens for plant breeding and analysis of gene functions, particularly tandemly arrayed genes.

Molecular nature of mutations induced by high-LET irradiation with argon and carbon ions in Arabidopsis thaliana

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Hirano, Tomonari; Kazama, Yusuke [Nishina Center for Accelerator-Based Science, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); Innovation Center, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); Ohbu, Sumie; Shirakawa, Yuki; Liu Yang; Kambara, Tadashi; Fukunishi, Nobuhisa [Nishina Center for Accelerator-Based Science, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); Abe, Tomoko, E-mail: tomoabe@riken.jp [Nishina Center for Accelerator-Based Science, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); Innovation Center, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan)

2012-07-01

Linear energy transfer (LET) is an important parameter to be considered in heavy-ion mutagenesis. However, in plants, no quantitative data are available on the molecular nature of the mutations induced with high-LET radiation above 101-124 keV {mu}m{sup -1}. In this study, we irradiated dry seeds of Arabidopsis thaliana with Ar and C ions with an LET of 290 keV {mu}m{sup -1}. We analyzed the DNA alterations caused by the higher-LET radiation. Mutants were identified from the M{sub 2} pools. In total, 14 and 13 mutated genes, including bin2, egy1, gl1, gl2, hy1, hy3-5, ttg1, and var2, were identified in the plants derived from Ar- and C-ions irradiation, respectively. In the mutants from both irradiations, deletion was the most frequent type of mutation; 13 of the 14 mutated genes from the Ar ion-irradiated plants and 11 of the 13 mutated genes from the C ion-irradiated plants harbored deletions. Analysis of junction regions generated by the 2 types of irradiation suggested that alternative non-homologous end-joining was the predominant pathway of repair of break points. Among the deletions, the proportion of large deletions (>100 bp) was about 54% for Ar-ion irradiation and about 64% for C-ion irradiation. Both current results and previously reported data revealed that the proportions of the large deletions induced by 290-keV {mu}m{sup -1} radiations were higher than those of the large deletions induced by lower-LET radiations (6% for 22.5-30.0 keV {mu}m{sup -1} and 27% for 101-124 keV {mu}m{sup -1}). Therefore, the 290 keV {mu}m{sup -1} heavy-ion beams can effectively induce large deletions and will prove useful as novel mutagens for plant breeding and analysis of gene functions, particularly tandemly arrayed genes.

Insights into the mutation-induced HHH syndrome from modeling human mitochondrial ornithine transporter-1.

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Jing-Fang Wang

Full Text Available Human mitochondrial ornithine transporter-1 is reported in coupling with the hyperornithinemia-hyperammonemia-homocitrullinuria (HHH syndrome, which is a rare autosomal recessive disorder. For in-depth understanding of the molecular mechanism of the disease, it is crucially important to acquire the 3D structure of human mitochondrial ornithine transporter-1. Since no such structure is available in the current protein structure database, we have developed it via computational approaches based on the recent NMR structure of human mitochondrial uncoupling protein (Berardi MJ, Chou JJ, et al. Nature 2011, 476:109-113. Subsequently, we docked the ligand L-ornithine into the computational structure to search for the favorable binding mode. It was observed that the binding interaction for the most favorable binding mode is featured by six remarkable hydrogen bonds between the receptor and ligand, and that the most favorable binding mode shared the same ligand-binding site with most of the homologous mitochondrial carriers from different organisms, implying that the ligand-binding sites are quite conservative in the mitochondrial carriers family although their sequences similarity is very low with 20% or so. Moreover, according to our structural analysis, the relationship between the disease-causing mutations of human mitochondrial ornithine transporter-1 and the HHH syndrome can be classified into the following three categories: (i the mutation occurs in the pseudo-repeat regions so as to change the region of the protein closer to the mitochondrial matrix; (ii the mutation is directly affecting the substrate binding pocket so as to reduce the substrate binding affinity; (iii the mutation is located in the structural region closer to the intermembrane space that can significantly break the salt bridge networks of the protein. These findings may provide useful insights for in-depth understanding of the molecular mechanism of the HHH syndrome and

Presymptomatic breast cancer in Egypt: role of BRCA1 and BRCA2 tumor suppressor genes mutations detection

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Hashishe Mervat M

2010-06-01

Full Text Available Abstract Background Breast cancer is one of the most common diseases affecting women. Inherited susceptibility genes, BRCA1 and BRCA2, are considered in breast, ovarian and other common cancers etiology. BRCA1 and BRCA2 genes have been identified that confer a high degree of breast cancer risk. Objective Our study was performed to identify germline mutations in some exons of BRCA1 and BRCA2 genes for the early detection of presymptomatic breast cancer in females. Methods This study was applied on Egyptian healthy females who first degree relatives to those, with or without a family history, infected with breast cancer. Sixty breast cancer patients, derived from 60 families, were selected for molecular genetic testing of BRCA1 and BRCA2 genes. The study also included 120 healthy first degree female relatives of the patients, either sisters and/or daughters, for early detection of presymptomatic breast cancer mutation carriers. Genomic DNA was extracted from peripheral blood lymphocytes of all the studied subjects. Universal primers were used to amplify four regions of the BRCA1 gene (exons 2,8,13 and 22 and one region (exon 9 of BRCA2 gene using specific PCR. The polymerase chain reaction was carried out. Single strand conformation polymorphism assay and heteroduplex analysis were used to screen for mutations in the studied exons. In addition, DNA sequencing of the normal and mutated exons were performed. Results Mutations in both BRCA1 and BRCA2 genes were detected in 86.7% of the families. Current study indicates that 60% of these families were attributable to BRCA1 mutations, while 26.7% of them were attributable to BRCA2 mutations. Results showed that four mutations were detected in the BRCA1 gene, while one mutation was detected in the BRCA2 gene. Asymptomatic relatives, 80(67% out of total 120, were mutation carriers. Conclusions BRCA1 and BRCA2 genes mutations are responsible for a significant proportion of breast cancer. BRCA mutations

Exome sequencing finds a novel PCSK1 mutation in a child with generalized malabsorptive diarrhea and diabetes insipidus.

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Yourshaw, Michael; Solorzano-Vargas, R Sergio; Pickett, Lindsay A; Lindberg, Iris; Wang, Jiafang; Cortina, Galen; Pawlikowska-Haddal, Anna; Baron, Howard; Venick, Robert S; Nelson, Stanley F; Martín, Martín G

2013-12-01

Congenital diarrhea disorders are a group of genetically diverse and typically autosomal recessive disorders that have yet to be well characterized phenotypically or molecularly. Diagnostic assessments are generally limited to nutritional challenges and histologic evaluation, and many subjects eventually require a prolonged course of intravenous nutrition. Here we describe next-generation sequencing techniques to investigate a child with perplexing congenital malabsorptive diarrhea and other presumably unrelated clinical problems; this method provides an alternative approach to molecular diagnosis. We screened the diploid genome of an affected individual, using exome sequencing, for uncommon variants that have observed protein-coding consequences. We assessed the functional activity of the mutant protein, as well as its lack of expression using immunohistochemistry. Among several rare variants detected was a homozygous nonsense mutation in the catalytic domain of the proprotein convertase subtilisin/kexin type 1 gene. The mutation abolishes prohormone convertase 1/3 endoprotease activity as well as expression in the intestine. These primary genetic findings prompted a careful endocrine reevaluation of the child at 4.5 years of age, and multiple significant problems were subsequently identified consistent with the known phenotypic consequences of proprotein convertase subtilisin/kexin type 1 (PCSK1) gene mutations. Based on the molecular diagnosis, alternate medical and dietary management was implemented for diabetes insipidus, polyphagia, and micropenis. Whole-exome sequencing provides a powerful diagnostic tool to clinicians managing rare genetic disorders with multiple perplexing clinical manifestations.

Molecular nature of X-ray-induced mutations compared with that of spontaneous ones in human c-hprt gene integrated into mammalian chromosomal DNA

International Nuclear Information System (INIS)

Kimura, Hiroshi; Kato, Takesi.

1992-01-01

X-ray-induced mutations were analysed at molecular levels in comparison with spontaneous mutations. Altered sequences were determined tentatively of 30 independent X-ray-induced mutations in a cDNA of the human hprt gene which was integrated into mammalian chromosome as a part of a shuttle vector. Mutations consisted of base substitutions (37 %), frameshifts (27 %), deletions (27 %) and others (10 %). All these mutational events were distributed randomly over the gene without there being hot spots. The spectrum and distribution of X-ray-induced mutations resembled those of spontaneous mutations. Among base substitutions, transversions were predominant and base substitution mutations occurred more at A:T sites than at G:C sites, which is also the case in spontaneous mutations. Most of the frameshift and deletion mutations induced by X-rays, as well as those spontaneously arising, were characterized by the existence of short direct repeats of several identical bases in a row at the sites of the mutations. A slippage misalignment mechanism in replication well accounts for the generation of these classes of mutations. Judging from the data accumulated so far, it can be concluded that X-ray-induced mutations at molecular levels are similar to those spontaneously occurring. (author)

Preappointment testing for BRAF/KIT mutation in advanced melanoma: a model in molecular data delivery for individualized medicine.

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Mounajjed, Taofic; Brown, Char L; Stern, Therese K; Bjorheim, Annette M; Bridgeman, Andrew J; Rumilla, Kandelaria M; McWilliams, Robert R; Flotte, Thomas J

2014-11-01

The emergence of individualized medicine is driven by developments in precision diagnostics, epitomized by molecular testing. Because treatment decisions are being made based on such molecular data, data management is gaining major importance. Among data management challenges, creating workflow solutions for timely delivery of molecular data has become pivotal. This study aims to design and implement a scalable process that permits preappointment BRAF/KIT mutation analysis in melanoma patients, allowing molecular results necessary for treatment plans to be available before the patient's appointment. Process implementation aims to provide a model for efficient molecular data delivery for individualized medicine. We examined the existing process of BRAF/KIT testing in melanoma patients visiting our institution for oncology consultation. We created 5 working groups, each designing a specific segment of an alternative process that would allow preappointment BRAF/KIT testing and delivery of results. Data were captured and analyzed to evaluate the success of the alternative process. For 1 year, 35 (59%) of 55 patients had prior BRAF/KIT testing. The remaining 20 patients went through the new process of preappointment testing; results were available at the time of appointment for 12 patients (overall preappointment results availability, 85.5%). The overall process averaged 13.4 ± 4.7 days. In conclusion, we describe the successful implementation of a scalable workflow solution that permits preappointment BRAF/KIT mutation analysis and result delivery in melanoma patients. This sets the stage for further applications of this model to other conditions, answering an increasing demand for robust delivery of molecular data for individualized medicine. Copyright © 2014 Elsevier Inc. All rights reserved.

Exome sequencing links mutations in PARN and RTEL1 with familial pulmonary fibrosis and telomere shortening.

Science.gov (United States)

Stuart, Bridget D; Choi, Jungmin; Zaidi, Samir; Xing, Chao; Holohan, Brody; Chen, Rui; Choi, Mihwa; Dharwadkar, Pooja; Torres, Fernando; Girod, Carlos E; Weissler, Jonathan; Fitzgerald, John; Kershaw, Corey; Klesney-Tait, Julia; Mageto, Yolanda; Shay, Jerry W; Ji, Weizhen; Bilguvar, Kaya; Mane, Shrikant; Lifton, Richard P; Garcia, Christine Kim

2015-05-01

Idiopathic pulmonary fibrosis (IPF) is an age-related disease featuring progressive lung scarring. To elucidate the molecular basis of IPF, we performed exome sequencing of familial kindreds with pulmonary fibrosis. Gene burden analysis comparing 78 European cases and 2,816 controls implicated PARN, an exoribonuclease with no previous connection to telomere biology or disease, with five new heterozygous damaging mutations in unrelated cases and none in controls (P = 1.3 × 10(-8)); mutations were shared by all affected relatives (odds in favor of linkage = 4,096:1). RTEL1, an established locus for dyskeratosis congenita, harbored significantly more new damaging and missense variants at conserved residues in cases than in controls (P = 1.6 × 10(-6)). PARN and RTEL1 mutation carriers had shortened leukocyte telomere lengths, and we observed epigenetic inheritance of short telomeres in family members. Together, these genes explain ~7% of familial pulmonary fibrosis and strengthen the link between lung fibrosis and telomere dysfunction.

The ter mutation in the rat Dnd1 gene initiates gonadal teratomas and infertility in both genders.

Science.gov (United States)

Northrup, Emily; Zschemisch, Nils-Holger; Eisenblätter, Regina; Glage, Silke; Wedekind, Dirk; Cuppen, Edwin; Dorsch, Martina; Hedrich, Hans-Jürgen

2012-01-01

A spontaneous mutation leading to the formation of congenital ovarian and testicular tumors was detected in the WKY/Ztm rat strain. The histological evaluation revealed derivatives from all three germ layers, thereby identifying these tumors as teratomas. Teratocarcinogenesis was accompanied by infertility and the underlying mutation was termed ter. Linkage analysis of 58 (WKY-ter×SPRD-Cu3) F2 rats associated the ter mutation with RNO18 (LOD = 3.25). Sequencing of candidate genes detected a point mutation in exon 4 of the dead-end homolog 1 gene (Dnd1), which introduces a premature stop codon assumed to cause a truncation of the Dnd1 protein. Genotyping of the recessive ter mutation revealed a complete penetrance of teratocarcinogenesis and infertility in homozygous ter rats of both genders. Morphologically non-tumorous testes of homozygous ter males were reduced in both size and weight. This testicular malformation was linked to a lack of spermatogenesis using immunohistochemical and histological staining. Our WKY-Dnd1(ter)/Ztm rat is a novel animal model to investigate gonadal teratocarcinogenesis and the molecular mechanisms involved in germ cell development of both genders.

Extended mutation spectrum of Usher syndrome in Finland.

Science.gov (United States)

Västinsalo, Hanna; Jalkanen, Reetta; Bergmann, Carsten; Neuhaus, Christine; Kleemola, Leenamaija; Jauhola, Liisa; Bolz, Hanno Jörn; Sankila, Eeva-Marja

2013-06-01

The Finnish distribution of clinical Usher syndrome (USH) types is 40% USH3, 34% USH1 and 12% USH2. All patients with USH3 carry the founder mutation in clarin 1 (CLRN1), whereas we recently reported three novel myosin VIIA (MYO7A) mutations in two unrelated patients with USH1. This study was carried out to further investigate the USH mutation spectrum in Finnish patients. We analysed samples from nine unrelated USH patients/families without known mutations and two USH3 families with atypically severe phenotype. The Asper Ophthalmics USH mutation chip was used to screen for known mutations and to evaluate the chip in molecular diagnostics of Finnish patients. The chip revealed a heterozygous usherin (USH2A) mutation, p.N346H, in one patient. Sequencing of MYO7A and/or USH2A in three index patients revealed two novel heterozygous mutations, p.R873W in MYO7A and c.14343+2T>C in USH2A. We did not identify definite pathogenic second mutations in the patients, but identified several probably nonpathogenic variations that may modify the disease phenotype. Possible digenism could not be excluded in two families segregating genomic variations in both MYO7A and USH2A, and two families with CLRN1 and USH2A. We conclude that there is considerable genetic heterogeneity of USH1 and USH2 in Finland, making molecular diagnostics and genetic counselling of patients and families challenging. © 2012 The Authors. Acta Ophthalmologica © 2012 Acta Ophthalmologica Scandinavica Foundation.

Quantitative and molecular analyses of mutation in a pSV2gpt transformed CHO cell line

International Nuclear Information System (INIS)

Stankowski, L.F. Jr.; Tindall, K.R.; Hsie, A.W.

1983-01-01

Following NDA-mediated gene transfer we have isolated a cell line useful for studying gene mutation at the molecular level. This line, AS52, derived from a hypoxanthine-guanine phosphoribosyl transferase (HGPRT) deficient Chinese hamster ovary (CHO) cell line, carries a single copy of the E. coli xanthine-guanine phosphoribosyl transferase (XGPRT) gene (gpt) and exhibits a spontaneous mutant frequency of 20 TG/sup r/ mutants/10 6 clonable cells. As with HGPRT - mutants, XGPRT - mutants can be selected in 6-thioguanine. AS52 (XGPRT + ) and wild type CHO (HGPRT + ) cell exhibit almost identical cytotoxic responses to various agents. We observed significant differences in mutation induction by UV light and ethyl methanesulfonate (EMS). Ratios of XGPRT - to HGPRT - mutants induced per unit dose (J/m 2 for UV light and μg/ml for EMS) are 1.4 and 0.70, respectively. Preliminary Southern blot hybridization analyses has been performed on 30 XGPRT - AS52 mutants. A majority of spontaneous mutants have deletions ranging in size from 1 to 4 kilobases (9/19) to complete loss of gpt sequences (4/19); the remainder have no detectable (5/19) or only minor (1/19) alterations. 5/5 UV-induced and 5/6 EMS-induced mutants do not show a detectable change. Similar analyses are underway for mutations induced by x-irradiation and ICR 191 treatment

Mutation induction in γ-irradiated primary human bronchial epithelial cells and molecular analysis of the HPRT- mutants

International Nuclear Information System (INIS)

Suzuki, Keiji; Hei, Tom K.

1996-01-01

We have examined various radiobiological parameters using commercially-available primary normal human bronchial epithelial (NHBE) cells, which can be subcultured more than 20 population doublings, and have established the mutation system in order to characterize the molecular changes in γ-irradiated primary cells. The survival curve, obtained after irradiation of cells with 137 Cs γ-rays, indicates that the D 0 , D q , and n values are 1.34 Gy, 1.12 Gy, and 2.3, respectively. The induction of HPRT - mutation was dose-dependent and the mutant fraction increased in a non-linear fashion. Since the doubling number of NHBE cells is limited, DNA was extracted directly from the single mutant colonies and alteration in the HPRT gene locus was analyzed using multiplex PCR technique. Among spontaneous mutants, the proportion with total and partial deletions of the gene was 10.0% (2/20) and 60.0% (12/20), respectively, while 30.0% (6/20) did not have any detectable changes in the nine exons examined. On the other hand, the fraction of total deletion increased by more than 2-fold among mutants induced by γ-rays in that 26.3% (10/38) of them showed the total gene deletions. Twenty-five out of 38 γ-induced mutants (65.8%) had partial deletions and 3 mutants (7.9%) had no detectable alteration. The present results showed that γ-irradiation efficiently induced HPRT gene mutation in primary human epithelial cells and that most of the induced mutants suffered larger deletions compared to that observed in spontaneous mutants. This system provides a useful tool for determination of mutagenicity and understanding the molecular mechanisms of environmental carcinogens in primary human bronchial cells

MEN1 redefined, a clinical comparison of mutation-positive and mutation-negative patients

NARCIS (Netherlands)

de Laat, Joanne M; van der Luijt, Rob B; Pieterman, Carolina R C; Oostveen, Maria P; Hermus, Ad R; Dekkers, Olaf M; de Herder, Wouter W; van der Horst-Schrivers, Anouk N; Drent, Madeleine L; Bisschop, Peter H; Havekes, Bas; Vriens, Menno R; Valk, Gerlof D

2016-01-01

BACKGROUND: Multiple Endocrine Neoplasia type 1 (MEN1) is diagnosed when two out of the three primary MEN1-associated endocrine tumors occur in a patient. Up to 10-30 % of those patients have no mutation in the MEN1 gene. It is unclear if the phenotype and course of the disease of mutation-negative

MEN1 redefined, a clinical comparison of mutation-positive and mutation-negative patients

NARCIS (Netherlands)

de Laat, Joanne M.; van der Luijt, Rob B.; Pieterman, Carolina R. C.; Oostveen, Maria P.; Hermus, Ad R.; Dekkers, Olaf M.; de Herder, Wouter W.; van der Horst-Schrivers, Anouk N.; Drent, Madeleine L.; Bisschop, Peter H.; Havekes, Bas; Vriens, Menno R.; Valk, Gerlof D.

2016-01-01

Background: Multiple Endocrine Neoplasia type 1 (MEN1) is diagnosed when two out of the three primary MEN1-associated endocrine tumors occur in a patient. Up to 10-30 % of those patients have no mutation in the MEN1 gene. It is unclear if the phenotype and course of the disease of mutation-negative

SOS1 and PTPN11 mutations in five cases of Noonan syndrome with multiple giant cell lesions.

Science.gov (United States)

Beneteau, Claire; Cavé, Hélène; Moncla, Anne; Dorison, Nathalie; Munnich, Arnold; Verloes, Alain; Leheup, Bruno

2009-10-01

We report five cases of multiple giant cell lesions in patients with typical Noonan syndrome. Such association has frequently been referred to as Noonan-like/multiple giant cell (NL/MGCL) syndrome before the molecular definition of Noonan syndrome. Two patients show mutations in PTPN11 (p.Tyr62Asp and p.Asn308Asp) and three in SOS1 (p.Arg552Ser and p.Arg552Thr). The latter are the first SOS1 mutations reported outside PTPN11 in NL/MGCL syndrome. MGCL lesions were observed in jaws ('cherubism') and joints ('pigmented villonodular synovitis'). We show through those patients that both types of MGCL are not PTPN11-specific, but rather represent a low penetrant (or perhaps overlooked) complication of the dysregulated RAS/MAPK signaling pathway. We recommend discarding NL/MGCL syndrome from the nosology, as this presentation is neither gene-nor allele-specific of Noonan syndrome; these patients should be described as Noonan syndrome with MGCL (of the mandible, the long bone...). The term cherubism should be used only when multiple giant cell lesions occur without any other clinical and molecular evidence of Noonan syndrome, with or without mutations of the SH3BP2 gene.

MEN1 redefined, a clinical comparison of mutation-positive and mutation-negative patients

NARCIS (Netherlands)

de Laat, Joanne M.; van der Luijt, Rob B.; Pieterman, Carolina R. C.; Oostveen, Maria P.; Hermus, Ad R.; Dekkers, Olaf M.; de Herder, Wouter W.; van der Horst-Schrivers, Anouk N.; Drent, Madeleine L.; Bisschop, Peter H.; Havekes, Bas; Vriens, Menno R.; Valk, Gerlof D.

2016-01-01

Multiple Endocrine Neoplasia type 1 (MEN1) is diagnosed when two out of the three primary MEN1-associated endocrine tumors occur in a patient. Up to 10-30 % of those patients have no mutation in the MEN1 gene. It is unclear if the phenotype and course of the disease of mutation-negative patients is

Autosomal dominant cutis laxa with progeroid features due to a novel, de novo mutation in ALDH18A1.

Science.gov (United States)

Bhola, Priya T; Hartley, Taila; Bareke, Eric; Boycott, Kym M; Nikkel, Sarah M; Dyment, David A

2017-06-01

De novo dominant mutations in the aldehyde dehydrogenase 18 family member A1 (ALDH18A1) gene have recently been shown to cause autosomal dominant cutis laxa with progeroid features (MIM 616603). To date, all de novo dominant mutations have been found in a single highly conserved amino acid residue at position p.Arg138. We report an 8-year-old male with a clinical diagnosis of autosomal dominant cutis laxa (ADCL) with progeroid features and a novel de novo missense mutation in ALDH18A1 (NM_002860.3: c.377G>A (p.Arg126His)). This is the first report of an individual with ALDH18A1-ADCL due to a substitution at a residue other than p.Arg138. Knowledge of the complete spectrum of dominant-acting mutations that cause this rare syndrome will have implications for molecular diagnosis and genetic counselling of these families.

BCR-ABL1 mutation development during first-line treatment with dasatinib or imatinib for chronic myeloid leukemia in chronic phase.

Science.gov (United States)

Hughes, T P; Saglio, G; Quintás-Cardama, A; Mauro, M J; Kim, D-W; Lipton, J H; Bradley-Garelik, M B; Ukropec, J; Hochhaus, A

2015-09-01

BCR-ABL1 mutations are a common, well-characterized mechanism of resistance to imatinib as first-line treatment of chronic myeloid leukemia in chronic phase (CML-CP). Less is known about mutation development during first-line treatment with dasatinib and nilotinib, despite increased use because of higher response rates compared with imatinib. Retrospective analyses were conducted to characterize mutation development in patients with newly diagnosed CML-CP treated with dasatinib (n=259) or imatinib (n=260) in DASISION (Dasatinib versus Imatinib Study in Treatment-Naive CML-CP), with 3-year minimum follow-up. Mutation screening, including patients who discontinued treatment and patients who had a clinically relevant on-treatment event (no confirmed complete cytogenetic response (cCCyR) and no major molecular response (MMR) within 12 months; fivefold increase in BCR-ABL1 with loss of MMR; loss of CCyR), yielded a small number of patients with mutations (dasatinib, n=17; imatinib, n=18). Dasatinib patients had a narrower spectrum of mutations (4 vs 12 sites for dasatinib vs imatinib), fewer phosphate-binding loop mutations (1 vs 9 mutations), fewer multiple mutations (1 vs 6 patients) and greater occurrence of T315I (11 vs 0 patients). This trial was registered at www.clinicaltrials.gov as NCT00481247.

ORAI1 mutations abolishing store-operated Ca2+ entry cause anhidrotic ectodermal dysplasia with immunodeficiency.

Science.gov (United States)

Lian, Jayson; Cuk, Mario; Kahlfuss, Sascha; Kozhaya, Lina; Vaeth, Martin; Rieux-Laucat, Frédéric; Picard, Capucine; Benson, Melina J; Jakovcevic, Antonia; Bilic, Karmen; Martinac, Iva; Stathopulos, Peter; Kacskovics, Imre; Vraetz, Thomas; Speckmann, Carsten; Ehl, Stephan; Issekutz, Thomas; Unutmaz, Derya; Feske, Stefan

2017-11-16

Store-operated Ca 2+ entry (SOCE) through Ca 2+ release-activated Ca 2+ channels is an essential signaling pathway in many cell types. Ca 2+ release-activated Ca 2+ channels are formed by ORAI1, ORAI2, and ORAI3 proteins and activated by stromal interaction molecule (STIM) 1 and STIM2. Mutations in the ORAI1 and STIM1 genes that abolish SOCE cause a combined immunodeficiency (CID) syndrome that is accompanied by autoimmunity and nonimmunologic symptoms. We performed molecular and immunologic analysis of patients with CID, anhidrosis, and ectodermal dysplasia of unknown etiology. We performed DNA sequencing of the ORAI1 gene, modeling of mutations on ORAI1 crystal structure, analysis of ORAI1 mRNA and protein expression, SOCE measurements, immunologic analysis of peripheral blood lymphocyte populations by using flow cytometry, and histologic and ultrastructural analysis of patient tissues. We identified 3 novel autosomal recessive mutations in ORAI1 in unrelated kindreds with CID, autoimmunity, ectodermal dysplasia with anhidrosis, and muscular dysplasia. The patients were homozygous for p.V181SfsX8, p.L194P, and p.G98R mutations in the ORAI1 gene that suppressed ORAI1 protein expression and SOCE in the patients' lymphocytes and fibroblasts. In addition to impaired T-cell cytokine production, ORAI1 mutations were associated with strongly reduced numbers of invariant natural killer T and regulatory T (Treg) cells and altered composition of γδ T-cell and natural killer cell subsets. ORAI1 null mutations are associated with reduced numbers of invariant natural killer T and Treg cells that likely contribute to the patients' immunodeficiency and autoimmunity. ORAI1-deficient patients have dental enamel defects and anhidrosis, representing a new form of anhidrotic ectodermal dysplasia with immunodeficiency that is distinct from previously reported patients with anhidrotic ectodermal dysplasia with immunodeficiency caused by mutations in the nuclear factor κB signaling

Single nucleotide polymorphism array lesions, TET2, DNMT3A, ASXL1 and CBL mutations are present in systemic mastocytosis.

Directory of Open Access Journals (Sweden)

Fabiola Traina

Full Text Available We hypothesized that analysis of single nucleotide polymorphism arrays (SNP-A and new molecular defects may provide new insight in the pathogenesis of systemic mastocytosis (SM. SNP-A karyotyping was applied to identify recurrent areas of loss of heterozygosity and bidirectional sequencing was performed to evaluate the mutational status of TET2, DNMT3A, ASXL1, EZH2, IDH1/IDH2 and the CBL gene family. Overall survival (OS was analyzed using the Kaplan-Meier method. We studied a total of 26 patients with SM. In 67% of SM patients, SNP-A karyotyping showed new chromosomal abnormalities including uniparental disomy of 4q and 2p spanning TET2/KIT and DNMT3A. Mutations in TET2, DNMT3A, ASXL1 and CBL were found in 23%, 12%, 12%, and 4% of SM patients, respectively. No mutations were observed in EZH2 and IDH1/IDH2. Significant differences in OS were observed for SM mutated patients grouped based on the presence of combined TET2/DNMT3A/ASXL1 mutations independent of KIT (P = 0.04 and sole TET2 mutations (P<0.001. In conclusion, TET2, DNMT3A and ASXL1 mutations are also present in mastocytosis and these mutations may affect prognosis, as demonstrated by worse OS in mutated patients.

Mutational spectrum in a worldwide study of 29,700 families with BRCA1 or BRCA2 mutations.

Science.gov (United States)

Rebbeck, Timothy R; Friebel, Tara M; Friedman, Eitan; Hamann, Ute; Huo, Dezheng; Kwong, Ava; Olah, Edith; Olopade, Olufunmilayo I; Solano, Angela R; Teo, Soo-Hwang; Thomassen, Mads; Weitzel, Jeffrey N; Chan, T L; Couch, Fergus J; Goldgar, David E; Kruse, Torben A; Palmero, Edenir Inêz; Park, Sue Kyung; Torres, Diana; van Rensburg, Elizabeth J; McGuffog, Lesley; Parsons, Michael T; Leslie, Goska; Aalfs, Cora M; Abugattas, Julio; Adlard, Julian; Agata, Simona; Aittomäki, Kristiina; Andrews, Lesley; Andrulis, Irene L; Arason, Adalgeir; Arnold, Norbert; Arun, Banu K; Asseryanis, Ella; Auerbach, Leo; Azzollini, Jacopo; Balmaña, Judith; Barile, Monica; Barkardottir, Rosa B; Barrowdale, Daniel; Benitez, Javier; Berger, Andreas; Berger, Raanan; Blanco, Amie M; Blazer, Kathleen R; Blok, Marinus J; Bonadona, Valérie; Bonanni, Bernardo; Bradbury, Angela R; Brewer, Carole; Buecher, Bruno; Buys, Saundra S; Caldes, Trinidad; Caliebe, Almuth; Caligo, Maria A; Campbell, Ian; Caputo, Sandrine M; Chiquette, Jocelyne; Chung, Wendy K; Claes, Kathleen B M; Collée, J Margriet; Cook, Jackie; Davidson, Rosemarie; de la Hoya, Miguel; De Leeneer, Kim; de Pauw, Antoine; Delnatte, Capucine; Diez, Orland; Ding, Yuan Chun; Ditsch, Nina; Domchek, Susan M; Dorfling, Cecilia M; Velazquez, Carolina; Dworniczak, Bernd; Eason, Jacqueline; Easton, Douglas F; Eeles, Ros; Ehrencrona, Hans; Ejlertsen, Bent; Engel, Christoph; Engert, Stefanie; Evans, D Gareth; Faivre, Laurence; Feliubadaló, Lidia; Ferrer, Sandra Fert; Foretova, Lenka; Fowler, Jeffrey; Frost, Debra; Galvão, Henrique C R; Ganz, Patricia A; Garber, Judy; Gauthier-Villars, Marion; Gehrig, Andrea; Gerdes, Anne-Marie; Gesta, Paul; Giannini, Giuseppe; Giraud, Sophie; Glendon, Gord; Godwin, Andrew K; Greene, Mark H; Gronwald, Jacek; Gutierrez-Barrera, Angelica; Hahnen, Eric; Hauke, Jan; Henderson, Alex; Hentschel, Julia; Hogervorst, Frans B L; Honisch, Ellen; Imyanitov, Evgeny N; Isaacs, Claudine; Izatt, Louise; Izquierdo, Angel; Jakubowska, Anna; James, Paul; Janavicius, Ramunas; Jensen, Uffe Birk; John, Esther M; Vijai, Joseph; Kaczmarek, Katarzyna; Karlan, Beth Y; Kast, Karin; Investigators, KConFab; Kim, Sung-Won; Konstantopoulou, Irene; Korach, Jacob; Laitman, Yael; Lasa, Adriana; Lasset, Christine; Lázaro, Conxi; Lee, Annette; Lee, Min Hyuk; Lester, Jenny; Lesueur, Fabienne; Liljegren, Annelie; Lindor, Noralane M; Longy, Michel; Loud, Jennifer T; Lu, Karen H; Lubinski, Jan; Machackova, Eva; Manoukian, Siranoush; Mari, Véronique; Martínez-Bouzas, Cristina; Matrai, Zoltan; Mebirouk, Noura; Meijers-Heijboer, Hanne E J; Meindl, Alfons; Mensenkamp, Arjen R; Mickys, Ugnius; Miller, Austin; Montagna, Marco; Moysich, Kirsten B; Mulligan, Anna Marie; Musinsky, Jacob; Neuhausen, Susan L; Nevanlinna, Heli; Ngeow, Joanne; Nguyen, Huu Phuc; Niederacher, Dieter; Nielsen, Henriette Roed; Nielsen, Finn Cilius; Nussbaum, Robert L; Offit, Kenneth; Öfverholm, Anna; Ong, Kai-Ren; Osorio, Ana; Papi, Laura; Papp, Janos; Pasini, Barbara; Pedersen, Inge Sokilde; Peixoto, Ana; Peruga, Nina; Peterlongo, Paolo; Pohl, Esther; Pradhan, Nisha; Prajzendanc, Karolina; Prieur, Fabienne; Pujol, Pascal; Radice, Paolo; Ramus, Susan J; Rantala, Johanna; Rashid, Muhammad Usman; Rhiem, Kerstin; Robson, Mark; Rodriguez, Gustavo C; Rogers, Mark T; Rudaitis, Vilius; Schmidt, Ane Y; Schmutzler, Rita Katharina; Senter, Leigha; Shah, Payal D; Sharma, Priyanka; Side, Lucy E; Simard, Jacques; Singer, Christian F; Skytte, Anne-Bine; Slavin, Thomas P; Snape, Katie; Sobol, Hagay; Southey, Melissa; Steele, Linda; Steinemann, Doris; Sukiennicki, Grzegorz; Sutter, Christian; Szabo, Csilla I; Tan, Yen Y; Teixeira, Manuel R; Terry, Mary Beth; Teulé, Alex; Thomas, Abigail; Thull, Darcy L; Tischkowitz, Marc; Tognazzo, Silvia; Toland, Amanda Ewart; Topka, Sabine; Trainer, Alison H; Tung, Nadine; van Asperen, Christi J; van der Hout, Annemieke H; van der Kolk, Lizet E; van der Luijt, Rob B; Van Heetvelde, Mattias; Varesco, Liliana; Varon-Mateeva, Raymonda; Vega, Ana; Villarreal-Garza, Cynthia; von Wachenfeldt, Anna; Walker, Lisa; Wang-Gohrke, Shan; Wappenschmidt, Barbara; Weber, Bernhard H F; Yannoukakos, Drakoulis; Yoon, Sook-Yee; Zanzottera, Cristina; Zidan, Jamal; Zorn, Kristin K; Hutten Selkirk, Christina G; Hulick, Peter J; Chenevix-Trench, Georgia; Spurdle, Amanda B; Antoniou, Antonis C; Nathanson, Katherine L

2018-05-01

The prevalence and spectrum of germline mutations in BRCA1 and BRCA2 have been reported in single populations, with the majority of reports focused on White in Europe and North America. The Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) has assembled data on 18,435 families with BRCA1 mutations and 11,351 families with BRCA2 mutations ascertained from 69 centers in 49 countries on six continents. This study comprehensively describes the characteristics of the 1,650 unique BRCA1 and 1,731 unique BRCA2 deleterious (disease-associated) mutations identified in the CIMBA database. We observed substantial variation in mutation type and frequency by geographical region and race/ethnicity. In addition to known founder mutations, mutations of relatively high frequency were identified in specific racial/ethnic or geographic groups that may reflect founder mutations and which could be used in targeted (panel) first pass genotyping for specific populations. Knowledge of the population-specific mutational spectrum in BRCA1 and BRCA2 could inform efficient strategies for genetic testing and may justify a more broad-based oncogenetic testing in some populations. © 2018 Wiley Periodicals, Inc.

Emergence of MPLW515 mutation in a patient with CALR deletion: Evidence of secondary acquisition of MPL mutation in the CALR clone.

Science.gov (United States)

Partouche, Nicolas; Conejero, Carole; Barathon, Quentin; Moroch, Julien; Tulliez, Michel; Cordonnier, Catherine; Giraudier, Stephane

2018-02-01

Myeloproliferative neoplasms are characterized by transduction pathway recognized as mutually exclusive molecular abnormalities such as BCR-ABL translocation, JAK2V617F or JAK2 exon 12 mutations, MPL w515, and CALR mutations. However, in some rare cases, associations of such mutations are found in 1 patient. This can be related to 2 pathologies (at least 2 different clones harboring 2 mutations) or associated mutations in 1 clone. We describe here such an association of CALR and MPL mutations in a patient harboring the second mutation in a subclone during the phenotypic evolution of the myeloproliferative neoplasms. Copyright © 2017 John Wiley & Sons, Ltd.

RANK ligand as a potential target for breast cancer prevention in BRCA1-mutation carriers.

Science.gov (United States)

Nolan, Emma; Vaillant, François; Branstetter, Daniel; Pal, Bhupinder; Giner, Göknur; Whitehead, Lachlan; Lok, Sheau W; Mann, Gregory B; Rohrbach, Kathy; Huang, Li-Ya; Soriano, Rosalia; Smyth, Gordon K; Dougall, William C; Visvader, Jane E; Lindeman, Geoffrey J

2016-08-01

Individuals who have mutations in the breast-cancer-susceptibility gene BRCA1 (hereafter referred to as BRCA1-mutation carriers) frequently undergo prophylactic mastectomy to minimize their risk of breast cancer. The identification of an effective prevention therapy therefore remains a 'holy grail' for the field. Precancerous BRCA1(mut/+) tissue harbors an aberrant population of luminal progenitor cells, and deregulated progesterone signaling has been implicated in BRCA1-associated oncogenesis. Coupled with the findings that tumor necrosis factor superfamily member 11 (TNFSF11; also known as RANKL) is a key paracrine effector of progesterone signaling and that RANKL and its receptor TNFRSF11A (also known as RANK) contribute to mammary tumorigenesis, we investigated a role for this pathway in the pre-neoplastic phase of BRCA1-mutation carriers. We identified two subsets of luminal progenitors (RANK(+) and RANK(-)) in histologically normal tissue of BRCA1-mutation carriers and showed that RANK(+) cells are highly proliferative, have grossly aberrant DNA repair and bear a molecular signature similar to that of basal-like breast cancer. These data suggest that RANK(+) and not RANK(-) progenitors are a key target population in these women. Inhibition of RANKL signaling by treatment with denosumab in three-dimensional breast organoids derived from pre-neoplastic BRCA1(mut/+) tissue attenuated progesterone-induced proliferation. Notably, proliferation was markedly reduced in breast biopsies from BRCA1-mutation carriers who were treated with denosumab. Furthermore, inhibition of RANKL in a Brca1-deficient mouse model substantially curtailed mammary tumorigenesis. Taken together, these findings identify a targetable pathway in a putative cell-of-origin population in BRCA1-mutation carriers and implicate RANKL blockade as a promising strategy in the prevention of breast cancer.

Leber's hereditary optic neuropathy is associated with mitochondrial ND1 T3394C mutation

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Liang, Min [School of Ophthalmology and Optometry, Wenzhou Medical College, Wenzhou, Zhejiang 325003 (China); Zhejiang Provincial Key Laboratory of Medical Genetics, School of Life Sciences, Wenzhou Medical College, Wenzhou, Zhejiang 325003 (China); Guan, Minqiang [Zhejiang Provincial Key Laboratory of Medical Genetics, School of Life Sciences, Wenzhou Medical College, Wenzhou, Zhejiang 325003 (China); Zhao, Fuxing; Zhou, Xiangtian [School of Ophthalmology and Optometry, Wenzhou Medical College, Wenzhou, Zhejiang 325003 (China); Yuan, Meixia [School of Ophthalmology and Optometry, Wenzhou Medical College, Wenzhou, Zhejiang 325003 (China); Zhejiang Provincial Key Laboratory of Medical Genetics, School of Life Sciences, Wenzhou Medical College, Wenzhou, Zhejiang 325003 (China); Tong, Yi [School of Ophthalmology and Optometry, Wenzhou Medical College, Wenzhou, Zhejiang 325003 (China); The First Affiliated Hospital, Fujian Medical University, Fuzhou, Fujian 350005 (China); Yang, Li [Division of Human Genetics, Cincinnati Children' s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229 (United States); Wei, Qi-Ping; Sun, Yan-Hong [Department of Ophthalmology, Dongfang Hospital, Beijing University of Chinese Medicine and Pharmacology, Beijing 100078 (China); Lu, Fan [School of Ophthalmology and Optometry, Wenzhou Medical College, Wenzhou, Zhejiang 325003 (China); Qu, Jia, E-mail: jqu@wzmc.net [School of Ophthalmology and Optometry, Wenzhou Medical College, Wenzhou, Zhejiang 325003 (China); Zhejiang Provincial Key Laboratory of Medical Genetics, School of Life Sciences, Wenzhou Medical College, Wenzhou, Zhejiang 325003 (China); and others

2009-06-05

We report here the clinical, genetic and molecular characterization of four Chinese families with Leber's hereditary optic neuropathy (LHON). There were variable severity and age-of-onset in visual impairment among these families. Strikingly, there were extremely low penetrances of visual impairment in these Chinese families. Sequence analysis of complete mitochondrial genomes in these pedigrees showed the homoplasmic T3394C (Y30H) mutation, which localized at a highly conserved tyrosine at position 30 of ND1, and distinct sets of mtDNA polymorphisms belonging to haplogroups D4b and M9a. The occurrence of T3394C mutation in these several genetically unrelated subjects affected by visual impairment strongly indicates that this mutation is involved in the pathogenesis of visual impairment. However, there was the absence of functionally significant mtDNA mutations in these four Chinese pedigrees carrying the T3394C mutation. Therefore, nuclear modifier gene(s) or environmental factor(s) may play a role in the phenotypic expression of the LHON-associated T3394C mutation.

Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia

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Puente, Xose S.; Pinyol, Magda; Quesada, Víctor; Conde, Laura; Ordóñez, Gonzalo R.; Villamor, Neus; Escaramis, Georgia; Jares, Pedro; Beà, Sílvia; González-Díaz, Marcos; Bassaganyas, Laia; Baumann, Tycho; Juan, Manel; López-Guerra, Mónica; Colomer, Dolors; Tubío, José M. C.; López, Cristina; Navarro, Alba; Tornador, Cristian; Aymerich, Marta; Rozman, María; Hernández, Jesús M.; Puente, Diana A.; Freije, José M. P.; Velasco, Gloria; Gutiérrez-Fernández, Ana; Costa, Dolors; Carrió, Anna; Guijarro, Sara; Enjuanes, Anna; Hernández, Lluís; Yagüe, Jordi; Nicolás, Pilar; Romeo-Casabona, Carlos M.; Himmelbauer, Heinz; Castillo, Ester; Dohm, Juliane C.; de Sanjosé, Silvia; Piris, Miguel A.; de Alava, Enrique; Miguel, Jesús San; Royo, Romina; Gelpí, Josep L.; Torrents, David; Orozco, Modesto; Pisano, David G.; Valencia, Alfonso; Guigó, Roderic; Bayés, Mónica; Heath, Simon; Gut, Marta; Klatt, Peter; Marshall, John; Raine, Keiran; Stebbings, Lucy A.; Futreal, P. Andrew; Stratton, Michael R.; Campbell, Peter J.; Gut, Ivo; López-Guillermo, Armando; Estivill, Xavier; Montserrat, Emili; López-Otín, Carlos; Campo, Elías

2012-01-01

Chronic lymphocytic leukaemia (CLL), the most frequent leukaemia in adults in Western countries, is a heterogeneous disease with variable clinical presentation and evolution1,2. Two major molecular subtypes can be distinguished, characterized respectively by a high or low number of somatic hypermutations in the variable region of immunoglobulin genes3,4. The molecular changes leading to the pathogenesis of the disease are still poorly understood. Here we performed whole-genome sequencing of four cases of CLL and identified 46 somatic mutations that potentially affect gene function. Further analysis of these mutations in 363 patients with CLL identified four genes that are recurrently mutated: notch 1 (NOTCH1), exportin 1 (XPO1), myeloid differentiation primary response gene 88 (MYD88) and kelch-like 6 (KLHL6). Mutations in MYD88 and KLHL6 are predominant in cases of CLL with mutated immunoglobulin genes, whereas NOTCH1 and XPO1 mutations are mainly detected in patients with unmutated immunoglobulins. The patterns of somatic mutation, supported by functional and clinical analyses, strongly indicate that the recurrent NOTCH1, MYD88 and XPO1 mutations are oncogenic changes that contribute to the clinical evolution of the disease. To our knowledge, this is the first comprehensive analysis of CLL combining whole-genome sequencing with clinical characteristics and clinical outcomes. It highlights the usefulness of this approach for the identification of clinically relevant mutations in cancer. PMID:21642962

[Clinical relevance of ESR1 circulating mutations detection in hormone receptor positive metastatic breast cancer].

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Clatot, Florian; Perdrix, Anne; Sefrioui, David; Sarafan-Vasseur, Nasrin; Di Fiore, Frédéric

2018-01-01

If hormone therapy is a key treatment for hormone receptor positive advanced breast cancers, secondary resistance occurs as a rule. Recently, acquired alterations of the ESR1 gene have been identified as a mechanism of resistance on aromatase inhibitor (AI) treatment. The selective pressure by AI exposure during the metastatic setting triggers the emergence of ESR1 activating mutations. In that context, the "liquid biopsy" concept has been used to detect this molecular resistance before progression. Thus, the ESR1 circulating mutation detection will soon be used in daily practice to help monitoring patients on AI treatment and provide an early change for specific therapies that still have to be determined in prospective clinical trials. This review will present the acquired ESR1 mutations, as well as the methods used for their detection in blood and the potential clinical impact of this approach for hormone receptor positive breast cancer management. Copyright © 2017 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.

Disease-associated extracellular loop mutations in the adhesion G protein-coupled receptor G1 (ADGRG1; GPR56) differentially regulate downstream signaling.

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Kishore, Ayush; Hall, Randy A

2017-06-09

Mutations to the adhesion G protein-coupled receptor ADGRG1 (G1; also known as GPR56) underlie the neurological disorder bilateral frontoparietal polymicrogyria. Disease-associated mutations in G1 studied to date are believed to induce complete loss of receptor function through disruption of either receptor trafficking or signaling activity. Given that N-terminal truncation of G1 and other adhesion G protein-coupled receptors has been shown to significantly increase the receptors' constitutive signaling, we examined two different bilateral frontoparietal polymicrogyria-inducing extracellular loop mutations (R565W and L640R) in the context of both full-length and N-terminally truncated (ΔNT) G1. Interestingly, we found that these mutations reduced surface expression of full-length G1 but not G1-ΔNT in HEK-293 cells. Moreover, the mutations ablated receptor-mediated activation of serum response factor luciferase, a classic measure of Gα 12/13 -mediated signaling, but had no effect on G1-mediated signaling to nuclear factor of activated T cells (NFAT) luciferase. Given these differential signaling results, we sought to further elucidate the pathway by which G1 can activate NFAT luciferase. We found no evidence that ΔNT activation of NFAT is dependent on Gα q/11 -mediated or β-arrestin-mediated signaling but rather involves liberation of Gβγ subunits and activation of calcium channels. These findings reveal that disease-associated mutations to the extracellular loops of G1 differentially alter receptor trafficking, depending on the presence of the N terminus, and differentially alter signaling to distinct downstream pathways. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Genetic and epigenetic similarities and differences between childhood and adult AML

DEFF Research Database (Denmark)

Juhl-Christensen, Caroline; Ommen, Hans Beier; Aggerholm, Anni

2012-01-01

The biology of acute myeloid leukemia (AML) is complex and includes both genetic and epigenetic aberrations. We addressed the combined consequences of promoter hypermethylation of p15, CDH1, ER, MDR1, and RARB2 and mutation of NPM1, CEBPA, FLT3, and WT1 in a Danish cohort of 70 pediatric and 383...

The molecular anatomy of spontaneous germline mutations in human testes.

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Jian Qin

2007-09-01

Full Text Available The frequency of the most common sporadic Apert syndrome mutation (C755G in the human fibroblast growth factor receptor 2 gene (FGFR2 is 100-1,000 times higher than expected from average nucleotide substitution rates based on evolutionary studies and the incidence of human genetic diseases. To determine if this increased frequency was due to the nucleotide site having the properties of a mutation hot spot, or some other explanation, we developed a new experimental approach. We examined the spatial distribution of the frequency of the C755G mutation in the germline by dividing four testes from two normal individuals each into several hundred pieces, and, using a highly sensitive PCR assay, we measured the mutation frequency of each piece. We discovered that each testis was characterized by rare foci with mutation frequencies 10(3 to >10(4 times higher than the rest of the testis regions. Using a model based on what is known about human germline development forced us to reject (p < 10(-6 the idea that the C755G mutation arises more frequently because this nucleotide simply has a higher than average mutation rate (hot spot model. This is true regardless of whether mutation is dependent or independent of cell division. An alternate model was examined where positive selection acts on adult self-renewing Ap spermatogonial cells (SrAp carrying this mutation such that, instead of only replacing themselves, they occasionally produce two SrAp cells. This model could not be rejected given our observed data. Unlike the disease site, similar analysis of C-to-G mutations at a control nucleotide site in one testis pair failed to find any foci with high mutation frequencies. The rejection of the hot spot model and lack of rejection of a selection model for the C755G mutation, along with other data, provides strong support for the proposal that positive selection in the testis can act to increase the frequency of premeiotic germ cells carrying a mutation

Mutational Analysis of Oculocutaneous Albinism: A Compact Review

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Kamaraj, Balu

2014-01-01

Oculocutaneous albinism (OCA) is an autosomal recessive disorder caused by either complete lack of or a reduction of melanin biosynthesis in the melanocytes. The OCA1A is the most severe type with a complete lack of melanin production throughout life, while the milder forms OCA1B, OCA2, OCA3, and OCA4 show some pigment accumulation over time. Mutations in TYR, OCA2, TYRP1, and SLC45A2 are mainly responsible for causing oculocutaneous albinism. Recently, two new genes SLC24A5 and C10orf11 are identified that are responsible to cause OCA6 and OCA7, respectively. Also a locus has been mapped to the human chromosome 4q24 region which is responsible for genetic cause of OCA5. In this paper, we summarized the clinical and molecular features of OCA genes. Further, we reviewed the screening of pathological mutations of OCA genes and its molecular mechanism of the protein upon mutation by in silico approach. We also reviewed TYR (T373K, N371Y, M370T, and P313R), OCA2 (R305W), TYRP1 (R326H and R356Q) mutations and their structural consequences at molecular level. It is observed that the pathological genetic mutations and their structural and functional significance of OCA genes will aid in development of personalized medicine for albinism patients. PMID:25093188

Molecular analysis of the most prevalent mutations of the FANCA and FANCC genes in Brazilian patients with Fanconi anaemia

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David Enrique Aguilar Rodriguez

2005-01-01

Full Text Available Fanconi anaemia (FA is a recessive autosomal disease determined by mutations in genes of at least eleven complementation groups, with distinct distributions in different populations. As far as we know, there are no reports regarding the molecular characterisation of the disease in unselected FA patients in Brazil. OBECTIVE: This study aimed to investigate the most prevalent mutations of FANCA and FANCC genes in Brazilian patients with FA. METHODS: Genomic DNA obtained from 22 racially and ethnically diverse unrelated FA patients (mean age ± SD: 14.0 ± 7.8 years; 10 male, 12 female; 14 white, 8 black was analysed by polymerase chain reaction and restriction site assays for identification of FANCA (delta3788-3790 and FANCC (delta322G, IVS4+4A -> T, W22X, L496R, R548X, Q13X, R185X, and L554P gene mutations. RESULTS: Mutations in FANCA and FANCC genes were identified in 6 (27.3% and 14 (63.6% out of 22 patients, respectively. The disease could not be attributed to the tested mutations in the two remaining patients enrolled in the study (9.1%. The registry of the two most prevalent gene abnormalities (delta3788-3790 and IVS4 + 4 -> T revealed that they were present in 18.2% and 15.9% of the FA alleles, respectively. Additional FANCC gene mutations were found in the study, with the following prevalence: delta322G (11.4%, W22X (9.1%, Q13X (2.3%, L554P (2.3%, and R548X (2.3% of total FA alleles. CONCLUSION: These results suggest that mutations of FANCA and FANCC genes are the most prevalent mutations among FA patients in Brazil.

Induced mutations and molecular techniques for crop improvement. Proceedings of an international symposium

International Nuclear Information System (INIS)

1995-01-01

The symposium was aimed at reviewing current aspects of mutation and molecular biology techniques for use in crop improvement and to bridge the gap between practical plant breeding and molecular laboratory techniques. Over the past few years, many transgenic plants have been developed in important crops such as rice, wheat, maize, soybean, banana, cassava and cotton, as well as in many food, industrial and pharmaceutical plant species. More than 180 participants from 48 countries of which 31 were from developing countries, attended which provided a forum for the discussion of problems related to crop improvement world wide, and their possible solutions. Refs, figs and tabs

Plant Mutation Reports, Vol. 3, No. 1, July 2013

International Nuclear Information System (INIS)

2013-07-01

your submissions of officially released mutant varieties to our Mutant Varieties Database (MVD). This database is unique and functions as a witness for the useful application of nuclear technology in food and agriculture. Currently the database lists more than 3200 released mutant varieties in more than 200 crop species and the number of plant species subject to mutagenesis also continues to rise. The MVD is currently being improved to facilitate submission of information and to provide more advanced search and data analysis tools. As to the current and final regular volume of PMR, I am happy that we can present an interesting mixture of two short notes on the development of new mutant varieties of rice in India, a review on the current status and trends in cassava mutation breeding and four research articles dealing with various topics in plant mutation. One of the articles addresses the characterization of various types of mutations in wheat as a resource for functional genomics, thus giving an example of the trend in broadening the use of mutation induction. Another article reports on the nature of molecular variation induced by gamma irradiation of barley as analysed by Amplified Fragment Length Polymorphisms (AFLPs) and Single Sequence Repeats (SSRs). This issue of PMR also gives a forum for results produced by a recently completed CRP on ''Molecular Tools for Quality Improvement in Vegetatively Propagated Crops Including Banana and Cassava''. Reports are included on the production of haploid tissue of the diploid Musa species M. acuminata cv. 'Matti' and on the analysis of carotenoid-protein content variation in pigmented cassava storage roots and its implication for traditional breeding strategies and use of induced mutations

Determining the role of missense mutations in the POU domain of HNF1A that reduce the DNA-binding affinity: A computational approach.

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Sneha P

Full Text Available Maturity-onset diabetes of the young type 3 (MODY3 is a non-ketotic form of diabetes associated with poor insulin secretion. Over the past years, several studies have reported the association of missense mutations in the Hepatocyte Nuclear Factor 1 Alpha (HNF1A with MODY3. Missense mutations in the POU homeodomain (POUH of HNF1A hinder binding to the DNA, thereby leading to a dysfunctional protein. Missense mutations of the HNF1A were retrieved from public databases and subjected to a three-step computational mutational analysis to identify the underlying mechanism. First, the pathogenicity and stability of the mutations were analyzed to determine whether they alter protein structure and function. Second, the sequence conservation and DNA-binding sites of the mutant positions were assessed; as HNF1A protein is a transcription factor. Finally, the biochemical properties of the biological system were validated using molecular dynamic simulations in Gromacs 4.6.3 package. Two arginine residues (131 and 203 in the HNF1A protein are highly conserved residues and contribute to the function of the protein. Furthermore, the R131W, R131Q, and R203C mutations were predicted to be highly deleterious by in silico tools and showed lower binding affinity with DNA when compared to the native protein using the molecular docking analysis. Triplicate runs of molecular dynamic (MD simulations (50ns revealed smaller changes in patterns of deviation, fluctuation, and compactness, in complexes containing the R131Q and R131W mutations, compared to complexes containing the R203C mutant complex. We observed reduction in the number of intermolecular hydrogen bonds, compactness, and electrostatic potential, as well as the loss of salt bridges, in the R203C mutant complex. Substitution of arginine with cysteine at position 203 decreases the affinity of the protein for DNA, thereby destabilizing the protein. Based on our current findings, the MD approach is an important

Mutations in the SRY, DAX1, SF1 and WNT4 genes in Brazilian sex-reversed patients

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S. Domenice

2004-01-01

Full Text Available In most mammals, male development is triggered by the transient expression of the SRY gene, which initiates a cascade of gene interactions ultimately leading to the formation of a testis from the indifferent fetal gonad. Mutation studies have identified several genes essential for early gonadal development. We report here a molecular study of the SRY, DAX1, SF1 and WNT4 genes, mainly involved in sexual determination, in Brazilian 46,XX and 46,XY sex-reversed patients. The group of 46,XX sex-reversed patients consisted of thirteen 46,XX true hermaphrodites and four 46,XX males, and was examined for the presence of the SRY gene and for the loss of function (inactivating mutations and deletions of DAX1 and WNT4 genes. In the second group consisting of thirty-three 46,XY sex-reversed patients we investigated the presence of inactivating mutations in the SRY and SF1 genes as well as the overexpression (duplication of the DAX1 and WNT4 genes. The SRY gene was present in two 46,XX male patients and in none of the true hermaphrodites. Only one mutation, located outside homeobox domain of the 5' region of the HMG box of SRY (S18N, was identified in a patient with 46,XY sex reversal. A novel 8-bp microdeletion of the SF1 gene was identified in a 46,XY sex-reversed patient without adrenal insufficiency. The dosage of DAX1 and WNT4 was normal in the sex-reversed patients studied. We conclude that these genes are rarely involved in the etiology of male gonadal development in sex-reversed patients, a fact suggesting the presence of other genes in the sex determination cascade.

Mutations in polymerase genes enhanced the virulence of 2009 pandemic H1N1 influenza virus in mice.

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Wenfei Zhu

Full Text Available Influenza A virus can infect a wide variety of animal species with illness ranging from mild to severe, and is a continual cause for concern. Genetic mutations that occur either naturally or during viral adaptation in a poorly susceptible host are key mechanisms underlying the evolution and virulence of influenza A virus. Here, the variants containing PA-A36T or PB2-H357N observed in the mouse-adapted descendants of 2009 pandemic H1N1 virus (pH1N1, A/Sichuan/1/2009 (SC, were characterized. Both mutations enhanced polymerase activity in mammalian cells. These effects were confirmed using recombinant SC virus containing polymerase genes with wild type (WT or mutant PA or PB2. The PA-A36T mutant showed enhanced growth property compared to the WT in both human A549 cells and porcine PK15 cells in vitro, without significant effect on viral propagation in murine LA-4 cells and pathogenicity in mice; however, it did enhance the lung virus titer. PB2-H357N variant demonstrated growth ability comparable to the WT in A549 cells, but replicated well in PK15, LA-4 cells and in mice with an enhanced pathogenic phenotype. Despite such mutations are rare in nature, they could be observed in avian H5 and H7 subtype viruses which were currently recognized to pose potential threat to human. Our findings indicated that pH1N1 may adapt well in mammals when acquiring these mutations. Therefore, future molecular epidemiological surveillance should include scrutiny of both markers because of their potential impact on pathogenesis.

Autosomal recessive deafness 1A (DFNB1A) in Yakut population isolate in Eastern Siberia: extensive accumulation of the splice site mutation IVS1+1G>A in GJB2 gene as a result of founder effect.

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Barashkov, Nikolay A; Dzhemileva, Lilya U; Fedorova, Sardana A; Teryutin, Fedor M; Posukh, Olga L; Fedotova, Elvira E; Lobov, Simeon L; Khusnutdinova, Elza K

2011-09-01

Hereditary forms of hearing impairment (HI) caused by GJB2 (Cx26) mutations are the frequent sensory disorders registered among newborns in various human populations. In this study, we present data on the molecular, audiological and population features of autosomal recessive deafness 1A (DFNB1A) associated with the donor splicing site IVS1+1G>A mutation of GJB2 gene in Yakut population isolate of the Sakha Republic (Yakutia) located in Eastern Siberia (Russian Federation). The Yakut population exhibits high frequency of some Mendelian disorders, which are rare in other populations worldwide. Mutational analysis of GJB2 gene in 86 unrelated Yakut patients with congenital HI without other clinical features has been performed. In this study, we registered a large cohort of Yakut patients homozygous for the IVS1+1G>A mutation (70 unrelated deaf subjects in total). Detailed audiological analysis of 40 deaf subjects with genotype IVS1+1G>A/IVS1+1G>A revealed significant association of this genotype with mostly symmetrical bilateral severe to profound HI (85% severe-to-profound HI versus 15% mild-to-moderate HI, PA mutation (11.7%) has been found in Yakut population. Reconstruction of 140 haplotypes with IVS1+1G>A mutation demonstrates the common origin of all mutant chromosomes found in Yakuts. The age of mutation was estimated to be approximately 800 years. These findings characterize Eastern Siberia as the region with the most extensive accumulation of the IVS1+1G>A mutation in the world as a result of founder effect.

Performance of Lynch syndrome predictive models in quantifying the likelihood of germline mutations in patients with abnormal MLH1 immunoexpression.

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Cabreira, Verónica; Pinto, Carla; Pinheiro, Manuela; Lopes, Paula; Peixoto, Ana; Santos, Catarina; Veiga, Isabel; Rocha, Patrícia; Pinto, Pedro; Henrique, Rui; Teixeira, Manuel R

2017-01-01

Lynch syndrome (LS) accounts for up to 4 % of all colorectal cancers (CRC). Detection of a pathogenic germline mutation in one of the mismatch repair genes is the definitive criterion for LS diagnosis, but it is time-consuming and expensive. Immunohistochemistry is the most sensitive prescreening test and its predictive value is very high for loss of expression of MSH2, MSH6, and (isolated) PMS2, but not for MLH1. We evaluated if LS predictive models have a role to improve the molecular testing algorithm in this specific setting by studying 38 individuals referred for molecular testing and who were subsequently shown to have loss of MLH1 immunoexpression in their tumors. For each proband we calculated a risk score, which represents the probability that the patient with CRC carries a pathogenic MLH1 germline mutation, using the PREMM 1,2,6 and MMRpro predictive models. Of the 38 individuals, 18.4 % had a pathogenic MLH1 germline mutation. MMRpro performed better for the purpose of this study, presenting a AUC of 0.83 (95 % CI 0.67-0.9; P < 0.001) compared with a AUC of 0.68 (95 % CI 0.51-0.82, P = 0.09) for PREMM 1,2,6 . Considering a threshold of 5 %, MMRpro would eliminate unnecessary germline mutation analysis in a significant proportion of cases while keeping very high sensitivity. We conclude that MMRpro is useful to correctly predict who should be screened for a germline MLH1 gene mutation and propose an algorithm to improve the cost-effectiveness of LS diagnosis.

BESTROPHINOPATHY: A Spectrum of Ocular Abnormalities Caused by the c.614T>C Mutation in the BEST1 Gene

NARCIS (Netherlands)

Toto, L.; Boon, C.J.F.; Antonio, L. Di; Parodi, M. Battaglia; Mastropasqua, R.; Antonucci, I.; Stuppia, L.; Mastropasqua, L.

2016-01-01

PURPOSE: To describe the variable ocular phenotype associated with a heterozygous mutation in the BEST1 gene. METHODS: Clinical and genetic assessment was performed in five members of the same family. Molecular genetic analysis of the BEST1 gene was performed by direct sequencing. Extensive

Two novel mutations in the homogentisate-1,2-dioxygenase gene identified in Chinese Han Child with Alkaptonuria.

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Li, Hongying; Zhang, Kaihui; Xu, Qun; Ma, Lixia; Lv, Xin; Sun, Ruopeng

2015-03-01

Alkaptonuria (AKU) is an autosomal recessive disorder of tyrosine metabolism, which is caused by a defect in the enzyme homogentisate 1,2-dioxygenase (HGD) with subsequent accumulation of homogentisic acid. Presently, more than 100 HGD mutations have been identified as the cause of the inborn error of metabolism across different populations worldwide. However, the HGD mutation is very rarely reported in Asia, especially China. In this study, we present mutational analyses of HGD gene in one Chinese Han child with AKU, which had been identified by gas chromatography-mass spectrometry detection of organic acids in urine samples. PCR and DNA sequencing of the entire coding region as well as exon-intron boundaries of HGD have been performed. Two novel mutations were identified in the HGD gene in this AKU case, a frameshift mutation of c.115delG in exon 3 and the splicing mutation of IVS5+3 A>C, a donor splice site of the exon 5 and exon-intron junction. The identification of these mutations in this study further expands the spectrum of known HGD gene mutations and contributes to prenatal molecular diagnosis of AKU.

Molecular pathology and thyroid FNA.

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Poller, D N; Glaysher, S

2017-12-01

This review summarises molecular pathological techniques applicable to thyroid FNA. The molecular pathology of thyroid tumours is now fairly well understood. Molecular methods may be used as a rule-in test for diagnosis of malignancy in thyroid nodules, eg BRAF V600E point mutation, use of a seven-gene mutational panel (BRAF V600E, RAS genes, RET/PTC or PAX8/PPARG rearrangement), or as a comprehensive multigene next-generation sequencing panel, eg ThyroSeq v2. Molecular methods can also be applied as rule-out tests for malignancy in thyroid nodules, eg Afirma or ThyroSeq v2 or as markers of prognosis, eg TERT promoter mutation or other gene mutations including BRAF V600E, TP53 and AKT1, and as tests for newly defined tumour entities such as non-invasive follicular thyroid neoplasm with papillary like nuclei, or as a molecular marker(s) for targeted therapies. This review describes practical examples of molecular techniques as applied to thyroid FNA in routine clinical practice and the value of molecular diagnostics in thyroid FNA. It describes the range of molecular abnormalities identified in thyroid nodules and thyroid cancers with some practical applications of molecular methods to diagnosis and prognosis of thyroid nodules and thyroid cancer. © 2017 John Wiley & Sons Ltd.

Germline CDKN1B/p27Kip1 mutation in multiple endocrine neoplasia

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Georgitsi, Marianthi; Raitila, Anniina; Karhu, Auli; van der Luijt, Rob B.; Aalfs, Cora M.; Sane, Timo; Vierimaa, Outi; Mäkinen, Markus J.; Tuppurainen, Karoliina; Paschke, Ralph; Gimm, Oliver; Koch, Christian A.; Gündogdu, Sadi; Lucassen, Anneke; Tischkowitz, Marc; Izatt, Louise; Aylwin, Simon; Bano, Gul; Hodgson, Shirley; de Menis, Ernesto; Launonen, Virpi; Vahteristo, Pia; Aaltonen, Lauri A.

2007-01-01

Germline mutations in the MEN1 gene predispose to multiple endocrine neoplasia type 1 (MEN1) syndrome, but in up to 20-25% of clinical MEN1 cases, no MEN1 mutations can be found. Recently, a germline mutation in the CDKN1B gene, encoding p27(Kip1), was reported in one suspected MEN1 family with two

Insights into the binding specificity of wild type and mutated wheat germ agglutinin towards Neu5Acα(2-3)Gal: a study by in silico mutations and molecular dynamics simulations.

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Parasuraman, Ponnusamy; Murugan, Veeramani; Selvin, Jeyasigamani F A; Gromiha, M Michael; Fukui, Kazuhiko; Veluraja, Kasinadar

2014-08-01

Wheat germ agglutinin (WGA) is a plant lectin, which specifically recognizes the sugars NeuNAc and GlcNAc. Mutated WGA with enhanced binding specificity can be used as biomarkers for cancer. In silico mutations are performed at the active site of WGA to enhance the binding specificity towards sialylglycans, and molecular dynamics simulations of 20 ns are carried out for wild type and mutated WGAs (WGA1, WGA2, and WGA3) in complex with sialylgalactose to examine the change in binding specificity. MD simulations reveal the change in binding specificity of wild type and mutated WGAs towards sialylgalactose and bound conformational flexibility of sialylgalactose. The mutated polar amino acid residues Asn114 (S114N), Lys118 (G118K), and Arg118 (G118R) make direct and water mediated hydrogen bonds and hydrophobic interactions with sialylgalactose. An analysis of possible hydrogen bonds, hydrophobic interactions, total pair wise interaction energy between active site residues and sialylgalactose and MM-PBSA free energy calculation reveals the plausible binding modes and the role of water in stabilizing different binding modes. An interesting observation is that the binding specificity of mutated WGAs (cyborg lectin) towards sialylgalactose is found to be higher in double point mutation (WGA3). One of the substituted residues Arg118 plays a crucial role in sugar binding. Based on the interactions and energy calculations, it is concluded that the order of binding specificity of WGAs towards sialylgalactose is WGA3 > WGA1 > WGA2 > WGA. On comparing with the wild type, double point mutated WGA (WGA3) exhibits increased specificity towards sialylgalactose, and thus, it can be effectively used in targeted drug delivery and as biological cell marker in cancer therapeutics. Copyright © 2014 John Wiley & Sons, Ltd.

A Molecular Modeling Study of the Hydroxyflutamide Resistance Mechanism Induced by Androgen Receptor Mutations

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Hong-Li Liu

2017-08-01

Full Text Available Hydroxyflutamide (HF, an active metabolite of the first generation antiandrogen flutamide, was used in clinic to treat prostate cancer targeting androgen receptor (AR. However, a drug resistance problem appears after about one year’s treatment. AR T877A is the first mutation that was found to cause a resistance problem. Then W741C_T877A and F876L_T877A mutations were also reported to cause resistance to HF, while W741C and F876L single mutations cannot. In this study, molecular dynamics (MD simulations combined with the molecular mechanics generalized Born surface area (MM-GBSA method have been carried out to analyze the interaction mechanism between HF and wild-type (WT/mutant ARs. The obtained results indicate that AR helix 12 (H12 plays a pivotal role in the resistance of HF. It can affect the coactivator binding site at the activation function 2 domain (AF2, surrounded by H3, H4, and H12. When H12 closes to the AR ligand-binding domain (LBD like a lid, the coactivator binding site can be formed to promote transcription. However, once H12 is opened to expose LBD, the coactivator binding site will be distorted, leading to invalid transcription. Moreover, per-residue free energy decomposition analyses indicate that N705, T877, and M895 are vital residues in the agonist/antagonist mechanism of HF.

Dysmorphic Facial Features and Other Clinical Characteristics in Two Patients with PEX1 Gene Mutations

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Gunduz, Mehmet

2016-01-01

Peroxisomal disorders are a group of genetically heterogeneous metabolic diseases related to dysfunction of peroxisomes. Dysmorphic features, neurological abnormalities, and hepatic dysfunction can be presenting signs of peroxisomal disorders. Here we presented dysmorphic facial features and other clinical characteristics in two patients with PEX1 gene mutation. Follow-up periods were 3.5 years and 1 year in the patients. Case I was one-year-old girl that presented with neurodevelopmental delay, hepatomegaly, bilateral hearing loss, and visual problems. Ophthalmologic examination suggested septooptic dysplasia. Cranial magnetic resonance imaging (MRI) showed nonspecific gliosis at subcortical and periventricular deep white matter. Case II was 2.5-year-old girl referred for investigation of global developmental delay and elevated liver enzymes. Ophthalmologic examination findings were consistent with bilateral nystagmus and retinitis pigmentosa. Cranial MRI was normal. Dysmorphic facial features including broad nasal root, low set ears, downward slanting eyes, downward slanting eyebrows, and epichantal folds were common findings in two patients. Molecular genetic analysis indicated homozygous novel IVS1-2A>G mutation in Case I and homozygous p.G843D (c.2528G>A) mutation in Case II in the PEX1 gene. Clinical findings and developmental prognosis vary in PEX1 gene mutation. Kabuki-like phenotype associated with liver pathology may indicate Zellweger spectrum disorders (ZSD). PMID:27882258

Triplet-Based Codon Organization Optimizes the Impact of Synonymous Mutation on Nucleic Acid Molecular Dynamics.

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Babbitt, Gregory A; Coppola, Erin E; Mortensen, Jamie S; Ekeren, Patrick X; Viola, Cosmo; Goldblatt, Dallan; Hudson, André O

2018-02-01

Since the elucidation of the genetic code almost 50 years ago, many nonrandom aspects of its codon organization remain only partly resolved. Here, we investigate the recent hypothesis of 'dual-use' codons which proposes that in addition to allowing adjustment of codon optimization to tRNA abundance, the degeneracy in the triplet-based genetic code also multiplexes information regarding DNA's helical shape and protein-binding dynamics while avoiding interference with other protein-level characteristics determined by amino acid properties. How such structural optimization of the code within eukaryotic chromatin could have arisen from an RNA world is a mystery, but would imply some preadaptation in an RNA context. We analyzed synonymous (protein-silent) and nonsynonymous (protein-altering) mutational impacts on molecular dynamics in 13823 identically degenerate alternative codon reorganizations, defined by codon transitions in 7680 GPU-accelerated molecular dynamic simulations of implicitly and explicitly solvated double-stranded aRNA and bDNA structures. When compared to all possible alternative codon assignments, the standard genetic code minimized the impact of synonymous mutations on the random atomic fluctuations and correlations of carbon backbone vector trajectories while facilitating the specific movements that contribute to DNA polymer flexibility. This trend was notably stronger in the context of RNA supporting the idea that dual-use codon optimization and informational multiplexing in DNA resulted from the preadaptation of the RNA duplex to resist changes to thermostability. The nonrandom and divergent molecular dynamics of synonymous mutations also imply that the triplet-based code may have resulted from adaptive functional expansion enabling a primordial doublet code to multiplex gene regulatory information via the shape and charge of the minor groove.

Report of Chinese family with severe dermatitis, multiple allergies and metabolic wasting syndrome caused by novel homozygous desmoglein-1 gene mutation.

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Cheng, Ruhong; Yan, Ming; Ni, Cheng; Zhang, Jia; Li, Ming; Yao, Zhirong

2016-10-01

Recently, homozygous mutations in the desmoglein-1 (DSG1) gene and heterozygous mutation in the desmoplakin (DSP) gene have been demonstrated to be associated with severe dermatitis, multiple allergies and metabolic wasting (SAM) syndrome (Mendelian Inheritance in Man no. 615508). We aim to identify the molecular basis for a Chinese pedigree of SAM syndrome. A Chinese pedigree of SAM syndrome was subjected to mutation detection in the DSG1 gene. Sequence analysis of the DSG1 gene and quantitative reverse transcriptase polymerase chain reaction analysis for gene expression of DSG1 using cDNA derived from the epidermis of patients and controls were both performed. Skin biopsies were also taken from patients for pathological study and transmission electron microscopy observation. Novel homozygous splicing mutation c.1892-1delG in the exon-intron border of the DSG1 gene has been demonstrated to be associated with SAM syndrome. We report a new family of SAM syndrome of Asian decent and expand the spectrum of mutations in the DSG1 gene. © 2016 Japanese Dermatological Association.

Concurrent IDH1 and SMARCB1 Mutations in Pediatric Medulloblastoma: A Case Report

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Moatasem El-Ayadi

2018-06-01

Full Text Available Isocitrate Dehydrogenase-1 (IDH1 is a driver gene in several cancers including brain tumors such as low-grade and high-grade gliomas. Mutations of SMARCB1 were described in atypical teratoid rhabdoid tumors and to date have not been associated with the pathogenesis of medulloblastoma. We report concurrent IDH1 and SMARCB1 mutations in a medulloblastoma patient. We searched the catalog of somatic mutations in cancer (COSMIC database and other mutation databases and -to our knowledge- this is the first reported case of medulloblastoma harboring both mutations together. Our patient is a 13-year-old male presenting with headache and vomiting at diagnosis. MRI revealed left cerebellar expansive lesion with no evidence of metastasis. A histopathological diagnosis of desmoplastic/nodular medulloblastoma was made after complete resection of the tumor. Immunophenotypic characterization and methylation profiling suggested a medulloblastoma with SHH activation. Next generation sequencing of a panel of 400 genes revealed heterozygous somatic IDH1(p.R132C, SMARCB1(p.R201Q, and CDH11(p.L625T mutations. The patient was treated according to the HIT-SIOP PNET 4 protocol. He is in complete remission more than 2 years after diagnosis. In conclusion, increasing use of high throughput sequencing will certainly increase the frequency with which rare mutations or mutation combinations are identified. The exact frequency of this mutation combination and whether it has any particular therapeutic implications or prognostic relevance requires further investigation.

The driver and passenger effects of isocitrate dehydrogenase 1 and 2 mutations in oncogenesis and survival prolongation.

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Molenaar, Remco J; Radivoyevitch, Tomas; Maciejewski, Jaroslaw P; van Noorden, Cornelis J F; Bleeker, Fonnet E

2014-12-01

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are key events in the development of glioma, acute myeloid leukemia (AML), chondrosarcoma, intrahepatic cholangiocarcinoma (ICC), and angioimmunoblastic T-cell lymphoma. They also cause D-2-hydroxyglutaric aciduria and Ollier and Maffucci syndromes. IDH1/2 mutations are associated with prolonged survival in glioma and in ICC, but not in AML. The reason for this is unknown. In their wild-type forms, IDH1 and IDH2 convert isocitrate and NADP(+) to α-ketoglutarate (αKG) and NADPH. Missense mutations in the active sites of these enzymes induce a neo-enzymatic reaction wherein NADPH reduces αKG to D-2-hydroxyglutarate (D-2HG). The resulting D-2HG accumulation leads to hypoxia-inducible factor 1α degradation, and changes in epigenetics and extracellular matrix homeostasis. Such mutations also imply less NADPH production capacity. Each of these effects could play a role in cancer formation. Here, we provide an overview of the literature and discuss which downstream molecular effects are likely to be the drivers of the oncogenic and survival-prolonging properties of IDH1/2 mutations. We discuss interactions between mutant IDH1/2 inhibitors and conventional therapies. Understanding of the biochemical consequences of IDH1/2 mutations in oncogenesis and survival prolongation will yield valuable information for rational therapy design: it will tell us which oncogenic processes should be blocked and which "survivalogenic" effects should be retained. Copyright © 2014 Elsevier B.V. All rights reserved.

Autosomal recessive posterior column ataxia with retinitis pigmentosa caused by novel mutations in the FLVCR1 gene.

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Shaibani, Aziz; Wong, Lee-Jun; Wei Zhang, Victor; Lewis, Richard Alan; Shinawi, Marwan

2015-01-01

Posterior column ataxia with retinitis pigmentosa (PCARP) is an autosomal recessive disorder characterized by severe sensory ataxia, muscle weakness and atrophy, and progressive pigmentary retinopathy. Recently, mutations in the FLVCR1 gene were described in four families with this condition. We investigated the molecular basis and studied the phenotype of PCARP in a new family. The proband is a 33-year-old woman presented with sensory polyneuropathy and retinitis pigmentosa (RP). The constellation of clinical findings with normal metabolic and genetic evaluation, including mitochondrial DNA (mtDNA) analysis and normal levels of phytanic acid and vitamin E, prompted us to seek other causes of our patient's condition. Sequencing of FLVCR1 in the proband and targeted mutation testing in her two affected siblings revealed two novel variants, c.1547G > A (p.R516Q) and c.1593+5_+8delGTAA predicted, respectively, to be highly conserved throughout evolution and affecting the normal splicing, therefore, deleterious. This study supports the pathogenic role of FLVCR1 in PCARP and expands the molecular and clinical spectra of PCARP. We show for the first time that nontransmembrane domain (TMD) mutations in the FLVCR1 can cause PCARP, suggesting different mechanisms for pathogenicity. Our clinical data reveal that impaired sensation can be part of the phenotypic spectrum of PCARP. This study along with previously reported cases suggests that targeted sequencing of the FLVCR1 gene should be considered in patients with severe sensory ataxia, RP, and peripheral sensory neuropathy.

PIK3CA Mutation in Colorectal Cancer: Relationship with Genetic and Epigenetic Alterations

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Katsuhiko Nosho

2008-06-01

Full Text Available Somatic PIK3CA mutations are often present in colorectal cancer. Mutant PIK3CA activates AKT signaling, which up-regulates fatty acid synthase (FASN. Microsatellite instability (MSI and CpG island methylator phenotype (CIMP are important molecular classifiers in colorectal cancer. However, the relationship between PIK3CA mutation, MSI and CIMP remains uncertain. Using Pyrosequencing technology, we detected PIK3CA mutations in 91 (15% of 590 population-based colorectal cancers. To determine CIMP status, we quantified DNA methylation in eight CIMP-specific promoters [CACNA1G, CDKN2A (p16, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1] by real-time polymerase chain reaction (MethyLight. PIK3CA mutation was significantly associated with mucinous tumors [P = .0002; odds ratio (OR = 2.44], KRAS mutation (P < .0001; OR = 2.68, CIMP-high (P = .03; OR = 2.08, phospho–ribosomal protein S6 expression (P = .002; OR = 2.19, and FASN expression (P = .02; OR = 1.85 and inversely with p53 expression (P = .01; OR = 0.54 and β-catenin (CTNNB1 alteration (P = .004; OR = 0.43. In addition, PIK3CA G-to-A mutations were associated with MGMT loss (P = .001; OR = 3.24 but not with MGMT promoter methylation. In conclusion, PIK3CA mutation is significantly associated with other key molecular events in colorectal cancer, and MGMT loss likely contributes to the development of PIK3CA G>A mutation. In addition, Pyrosequencing is useful in detecting PIK3CA mutation in archival paraffin tumor tissue. PIK3CA mutational data further emphasize heterogeneity of colorectal cancer at the molecular level.

Oncogene mutational profile in nasopharyngeal carcinoma

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Zhang ZC

2014-03-01

Full Text Available Zi-Chen Zhang,1,* Sha Fu,1,* Fang Wang,1 Hai-Yun Wang,1 Yi-Xin Zeng,2 Jian-Yong Shao11Department of Molecular Diagnostics, 2Department of Experimental Research, Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou, People's Republic of China *These authors contributed equally to this work Abstract: Nasopharyngeal carcinoma (NPC is a common tumor in Southern China, but the oncogene mutational status of NPC patients has not been clarified. Using time-of-flight mass spectrometry, 238 mutation hotspots in 19 oncogenes were examined in 123 NPC patients. The relationships between mutational status and clinical data were assessed with a χ2 or Fisher's exact test. Survival analysis was performed using the Kaplan–Meier method with the log-rank test. In 123 patients, 21 (17.1% NPC tumors were positive for mutations in eight oncogenes: six patients had PIK3CA mutations (4.9%, five NRAS mutations (4.1%, four KIT mutations (3.3%, two PDGFRA mutations (1.6%, two ABL mutations (1.6%, and one with simultaneous mutations in HRAS, EGFR, and BRAF (1%. Patients with mutations were more likely to relapse or develop metastasis than those with wild-type alleles (P=0.019. No differences or correlations were found in other clinical characteristics or in patient survival. No mutations were detected in oncogenes AKT1, AKT2, CDK, ERBB2, FGFR1, FGFR3, FLT3, JAK2, KRAS, MET, and RET. These results demonstrate an association between NPC and mutations in NRAS, KIT, PIK3CA, PDGFRA, and ABL, which are associated with patient relapse and metastasis. Keywords: NPC, oncogene, mutation

Novel compound heterozygous mutations of ALDH1A3 contribute to anophthalmia in a non-consanguineous Chinese family

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Yunqiang Liu

2017-06-01

Full Text Available Abstract Anophthalmia is a rare eye development anomaly resulting in absent ocular globes or tissue in the orbit since birth. Here, we investigated a newborn with bilateral anophthalmia in a Chinese family. Exome sequencing revealed that compound heterozygous mutations c.287G > A (p.(Arg96His and c.709G > A (p.(Gly237Arg of the ALDH1A3 gene were present in the affected newborn. Both mutations were absent in all of the searched databases, including 10,000 in-house Chinese exome sequences, and these mutations were confirmed as having been transmitted from the parents. Comparative amino acid sequence analysis across distantly related species revealed that the residues at positions 96 and 234 were evolutionarily highly conserved. In silico analysis predicted these changes to be damaging, and in vitro expression analysis revealed that the mutated alleles were associated with decreased protein production and impaired tetrameric protein formation. This study firstly reported that compound heterozygous mutations of the ALDH1A3 gene can result in anophthalmia in humans, thus highlighting those heterozygous mutations in ALDH1A3 should be considered for molecular screening in anophthalmia, particularly in cases from families without consanguineous relationships.

Multiple spinal nerve enlargement and SOS1 mutation: Further evidence of overlap between neurofibromatosis type 1 and Noonan phenotype.

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Santoro, C; Giugliano, T; Melone, M A B; Cirillo, M; Schettino, C; Bernardo, P; Cirillo, G; Perrotta, S; Piluso, G

2018-01-01

Neurofibromatosis type 1 (NF1) has long been considered a well-defined, recognizable monogenic disorder, with neurofibromas constituting a pathognomonic sign. This dogma has been challenged by recent descriptions of patients with enlarged nerves or paraspinal tumors, suggesting that neurogenic tumors and hypertrophic neuropathy may be a complication of Noonan syndrome with multiple lentigines (NSML) or RASopathy phenotype. We describe a 15-year-old boy, whose mother previously received clinical diagnosis of NF1 due to presence of bilateral cervical and lumbar spinal lesions resembling plexiform neurofibromas and features suggestive of NS. NF1 molecular analysis was negative in the mother. The boy presented with Noonan features, multiple lentigines and pectus excavatum. Next-generation sequencing analysis of all RASopathy genes identified p.Ser548Arg missense mutation in SOS1 in the boy, confirmed in his mother. Brain and spinal magnetic resonance imaging scans were negative in the boy. No heart involvement or deafness was observed in proband or mother. This is the first report of a SOS1 mutation associated with hypertrophic neuropathy resembling plexiform neurofibromas, a rare complication in Noonan phenotypes with mutations in RASopathy genes. Our results highlight the overlap between RASopathies, suggesting that NF1 diagnostic criteria need rethinking. Genetic analysis of RASopathy genes should be considered when diagnosis is uncertain. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Two novel mutations in the SLC40A1 and HFE genes implicated in iron overload in a Spanish man.

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Del-Castillo-Rueda, Alejandro; Moreno-Carralero, María-Isabel; Alvarez-Sala-Walther, Luis-Antonio; Cuadrado-Grande, Nuria; Enríquez-de-Salamanca, Rafael; Méndez, Manuel; Morán-Jiménez, María-Josefa

2011-03-01

The most common form of hemochromatosis is caused by mutations in the HFE gene. Rare forms of the disease are caused by mutations in other genes. We present a patient with hyperferritinemia and iron overload, and facial flushing. Magnetic resonance imaging was performed to measure hepatic iron overload, and a molecular study of the genes involved in iron metabolism was undertaken. The iron overload was similar to that observed in HFE hemochromatosis, and the patient was double heterozygous for two novel mutations, c.-20G>A and c.718A>G (p.K240E), in the HFE and ferroportin (FPN1 or SLC40A1) genes, respectively. Hyperferritinemia and facial flushing improved after phlebotomy. Two of the patient's children were also studied, and the daughter was heterozygous for the mutation in the SLC40A1 gene, although she did not have hyperferritinemia. The patient presented a mild iron overload phenotype probably because of the two novel mutations in the HFE and SLC40A1 genes. © 2011 John Wiley & Sons A/S.

Molecular perspectives in differentiated thyroid cancer.

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Buffet, C; Groussin, L

2015-02-01

Progress in understanding the molecular genetics of thyroid cancer in the last 20 years has accelerated recently with the advent of high-throughput sequencing technologies known as Next-Generation Sequencing. Besides classical molecular abnormalities involving the MAPK (Mitogen Activated Protein Kinase) and PI3K (PhosphoInositide 3-Kinase) pathways that play a key role in follicular-derived thyroid tumorigenesis, new molecular abnormalities have been discovered. The major advances in recent years have been the discovery of new somatic driver gene point mutations (such as RASAL1 [RAS protein activator Like 1] mutations in follicular cancer) and/or mutations that have prognostic value (such as TERT [Telomerase reverse transcriptase] promoter mutations); new chromosomal rearrangements, usually having close connection with exposure to ionizing radiation (such as ALK [Anaplastic Lymphoma Kinase] rearrangements); and deregulation of some gene or microRNA expression representing a molecular signature. Progress made in understanding the molecular mechanisms of thyroid cancer offers new perspectives for the diagnosis of the benign or malignant status of a thyroid nodule, to refine prognosis and offer new perspectives of targeted therapy for radioiodine-refractory cancers. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Adult Diffuse Astrocytoma in the Medulla Oblongata: Molecular Biological Analyses Including H3F3A Mutation of Histone H3.3.

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Uekawa, Ken; Nakamura, Hideo; Shinojima, Naoki; Takezaki, Tatsuya; Yano, Shigetoshi; Kuratsu, Jun-Ichi

2016-04-01

Unlike in children, brain stem gliomas in adult are rare and still poorly understood. In addition, most adult brain stem gliomas result predominantly in the pons and are less often found in the medulla oblongata. Here, we report a case of an adult glioma in the medulla oblongata and its molecular biological features. A 46-year-old male presented with gait disturbance, paresthesia, and dysphagia. Magnetic resonance imaging (MRI) showed a diffuse hyper-intensive lesion in the medulla oblongata on a T 2 -weighted image without gadolinium contrast enhancement. We performed an open biopsy and the lesion was pathologically diagnosed as a diffuse astrocytoma. Molecular biological analyses revealed the absence of histone H3.3 mutation (H3F3A K27M), and presence of methylation of O-6-methylguanine-DNA methyltransferase (MGMT) promoter and a mutation in isocitrate dehydrogenase 1 (IDH-1). The patient received local radiotherapy and temozolomide chemotherapy. The patient's symptoms were ameliorated, and MRI showed no tumor growth at 6 months after the initial treatment. Biopsy for brain stem lesions is generally thought to have risk of complications, but if performed minimally, it is useful to diagnose and determine treatment strategy. Obtaining patient characteristics and molecular biological features will provide insight towards therapeutic treatment for adult brain stem gliomas.

Status of biotechnology with emphasis on molecular techniques for mutation breeding in the Philippines

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Lapade, A.G.; Nazarea, T.Y.; Veluz, A.M.S.; Marbella, L.J.; Nato, A.Q.; Coloma, C.B. Jr.; Asencion, A.B. [Philippine Nuclear Research Institute, Commonwealth Avenue, Quezon (Philippines)

2002-02-01

This paper summarizes the status of biotechnology with emphasis on molecular techniques for plant breeding in the Philippines. Several molecular and in-vitro culture techniques are integrated in plant breeding for crop improvement at PNRI, UPLB, IRRI and PhilRice. At IRRI, PCR techniques, RFLP and RAPD, PCR techniques, RFLP and RAPD were developed to establish high density molecular maps, determine breadth and diversity of germplasm and characterize alien introgression. The molecular maps have identified DNA sequence of resistance genes of HYVs and NPTs to abiotic and biotic stresses, the major achievement is the development of high density molecular maps in rice with at least 2000 markers. The biotechnology program at PhilRice for varietal improvement includes: (1) utilization of molecular marker technology such gene mapping of desired traits in rice, analysis of genetic relationships of germplasm materials and breeding lines through DNA fingerprinting and genetic diversity studies and development and application of marker aided selection for disease resistance (RTD and BLB); (2) application of in-vitro techniques in the development of lines with tolerance to adverse conditions; (3) molecular cloning of important genes for RTD resistance; (4) genetic transformation for male sterility and resistance to sheath blight and stem borers; and (5) transfer of disease resistance from wild species to cultivated varieties. In IPB, molecular markers:microsatellites or SSR, AFLP and RGA are being used for mapping and diversity studies in coconut, mango, banana, mungbean, corn and tomato. Mutation breeding at PNRI using gamma radiation has resulted in the development of crop varieties with desirable traits. The use of AFLP coupled to PCR is being used to study polymorphism in plant variants of radiation-induced mutants of rice, pineapple and ornamentals. (author)

Mutational analysis of COL1A1 and COL1A2 genes among Estonian osteogenesis imperfecta patients.

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Zhytnik, Lidiia; Maasalu, Katre; Reimann, Ene; Prans, Ele; Kõks, Sulev; Märtson, Aare

2017-08-15

Osteogenesis imperfecta (OI) is a rare bone disorder. In 90% of cases, OI is caused by mutations in the COL1A1/2 genes, which code procollagen α1 and α2 chains. The main aim of the current research was to identify the mutational spectrum of COL1A1/2 genes in Estonian patients. The small population size of Estonia provides a unique chance to explore the collagen I mutational profile of 100% of OI families in the country. We performed mutational analysis of peripheral blood gDNA of 30 unrelated Estonian OI patients using Sanger sequencing of COL1A1 and COL1A2 genes, including all intron-exon junctions and 5'UTR and 3'UTR regions, to identify causative OI mutations. We identified COL1A1/2 mutations in 86.67% of patients (26/30). 76.92% of discovered mutations were located in the COL1A1 (n = 20) and 23.08% in the COL1A2 (n = 6) gene. Half of the COL1A1/2 mutations appeared to be novel. The percentage of quantitative COL1A1/2 mutations was 69.23%. Glycine substitution with serine was the most prevalent among missense mutations. All qualitative mutations were situated in the chain domain of pro-α1/2 chains. Our study shows that among the Estonian OI population, the range of collagen I mutations is quite high, which agrees with other described OI cohorts of Northern Europe. The Estonian OI cohort differs due to the high number of quantitative variants and simple missense variants, which are mostly Gly to Ser substitutions and do not extend the chain domain of COL1A1/2 products.

Characterization and molecular profiling of PSEN1 familial Alzheimer's disease iPSC-derived neural progenitors.

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Andrew A Sproul

Full Text Available Presenilin 1 (PSEN1 encodes the catalytic subunit of γ-secretase, and PSEN1 mutations are the most common cause of early onset familial Alzheimer's disease (FAD. In order to elucidate pathways downstream of PSEN1, we characterized neural progenitor cells (NPCs derived from FAD mutant PSEN1 subjects. Thus, we generated induced pluripotent stem cells (iPSCs from affected and unaffected individuals from two families carrying PSEN1 mutations. PSEN1 mutant fibroblasts, and NPCs produced greater ratios of Aβ42 to Aβ40 relative to their control counterparts, with the elevated ratio even more apparent in PSEN1 NPCs than in fibroblasts. Molecular profiling identified 14 genes differentially-regulated in PSEN1 NPCs relative to control NPCs. Five of these targets showed differential expression in late onset AD/Intermediate AD pathology brains. Therefore, in our PSEN1 iPSC model, we have reconstituted an essential feature in the molecular pathogenesis of FAD, increased generation of Aβ42/40, and have characterized novel expression changes.

Ocular Phenotype Analysis of a Family With Biallelic Mutations in the BEST1 Gene

DEFF Research Database (Denmark)

Sharon, Dror; Al-Hamdani, Sermed; Engelsberg, Karl

2014-01-01

in the inner nuclear layer, no light rise in the electro-oculography, and a reduced central but preserved peripheral retinal function by multifocal electroretinography. Full-field electroretinography demonstrated a reduced rod response and inner retina dysfunction. Retinal structure was normal in all 3 family......PURPOSE: To investigate the genetic cause and perform a comprehensive clinical analysis of a Danish family with autosomal recessive bestrophinopathy; to investigate whether Bestrophin may be expressed in normal human retina. DESIGN: Retrospective clinical and molecular genetic analysis...... and immunohistochemical observational study. METHODS: setting: National referral center. participants: A family with 5 individuals and biallelic BEST1 mutations, and enucleated eyes from 2 individuals with nonaffected retinas. observation procedures: Molecular genetic analysis included sequencing of BEST1 and co...

Novel mutations in the SCNN1A gene causing Pseudohypoaldosteronism type 1.

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Jian Wang

Full Text Available Pseudohypoaldosteronism type 1 (PHA1 is a rare inherited disease characterized by resistance to the actions of aldosterone. Mutations in the subunit genes (SCNN1A, SCNN1B, SCNN1G of the epithelial sodium channel (ENaC and the NR3C2 gene encoding the mineralocorticoid receptor, result in systemic PHA1 and renal PHA1 respectively. Common clinical manifestations of PHA1 include salt wasting, hyperkalaemia, metabolic acidosis and elevated plasma aldosterone levels in the neonatal period. In this study, we describe the clinical and biochemical manifestations in two Chinese patients with systemic PHA1. Sequence analysis of the SCNN1A gene revealed a compound heterozygous mutation (c.1311delG and c.1439+1G>C in one patient and a homozygous mutation (c.814_815insG in another patient, all three variants are novel. Further analysis of the splicing pattern in a minigene construct showed that the c.1439+1G>C mutation can lead to the retainment of intron 9 as the 5'-donor splice site disappears during post-transcriptional processing of mRNA. In conclusion, our study identified three novel SCNN1A gene mutations in two Chinese patients with systemic PHA1.

A novel COL11A1 missense mutation in siblings with non-ocular Stickler syndrome.

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Kohmoto, Tomohiro; Tsuji, Atsumi; Morita, Kei-Ichi; Naruto, Takuya; Masuda, Kiyoshi; Kashimada, Kenichi; Enomoto, Keisuke; Morio, Tomohiro; Harada, Hiroyuki; Imoto, Issei

2016-01-01

Stickler syndrome (STL) is an autosomal, dominantly inherited, clinically variable and genetically heterogeneous connective tissue disorder characterized by ocular, auditory, orofacial and skeletal abnormalities. We conducted targeted resequencing using a next-generation sequencer for molecular diagnosis of a 2-year-old girl who was clinically suspected of having STL with Pierre Robin sequence. We detected a novel heterozygous missense mutation, NM_001854.3:n.4838G>A [NM_001854.3 (COL11A1_v001):c.4520G>A], in COL11A1, resulting in a Gly to Asp substitution at position 1507 [NM_001854.3(COL11A1_i001)] within one of the collagen-like domains of the triple helical region. The same mutation was detected in her 4-year-old brother with cleft palate and high-frequency sensorineural hearing loss.

Amyotrophic Lateral Sclerosis Type 20 - In Silico Analysis and Molecular Dynamics Simulation of hnRNPA1.

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Bruna Baumgarten Krebs

Full Text Available Amyotrophic Lateral Sclerosis (ALS is a fatal neurodegenerative disease that affects the upper and lower motor neurons. 5-10% of cases are genetically inherited, including ALS type 20, which is caused by mutations in the hnRNPA1 gene. The goals of this work are to analyze the effects of non-synonymous single nucleotide polymorphisms (nsSNPs on hnRNPA1 protein function, to model the complete tridimensional structure of the protein using computational methods and to assess structural and functional differences between the wild type and its variants through Molecular Dynamics simulations. nsSNP, PhD-SNP, Polyphen2, SIFT, SNAP, SNPs&GO, SNPeffect and PROVEAN were used to predict the functional effects of nsSNPs. Ab initio modeling of hnRNPA1 was made using Rosetta and refined using KoBaMIN. The structure was validated by PROCHECK, Rampage, ERRAT, Verify3D, ProSA and Qmean. TM-align was used for the structural alignment. FoldIndex, DICHOT, ELM, D2P2, Disopred and DisEMBL were used to predict disordered regions within the protein. Amino acid conservation analysis was assessed by Consurf, and the molecular dynamics simulations were performed using GROMACS. Mutations D314V and D314N were predicted to increase amyloid propensity, and predicted as deleterious by at least three algorithms, while mutation N73S was predicted as neutral by all the algorithms. D314N and D314V occur in a highly conserved amino acid. The Molecular Dynamics results indicate that all mutations increase protein stability when compared to the wild type. Mutants D314N and N319S showed higher overall dimensions and accessible surface when compared to the wild type. The flexibility level of the C-terminal residues of hnRNPA1 is affected by all mutations, which may affect protein function, especially regarding the protein ability to interact with other proteins.

BCOR and BCORL1 mutations in myelodysplastic syndromes and related disorders.

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Damm, Frederik; Chesnais, Virginie; Nagata, Yasunobu; Yoshida, Kenichi; Scourzic, Laurianne; Okuno, Yusuke; Itzykson, Raphael; Sanada, Masashi; Shiraishi, Yuichi; Gelsi-Boyer, Véronique; Renneville, Aline; Miyano, Satoru; Mori, Hiraku; Shih, Lee-Yung; Park, Sophie; Dreyfus, François; Guerci-Bresler, Agnes; Solary, Eric; Rose, Christian; Cheze, Stéphane; Prébet, Thomas; Vey, Norbert; Legentil, Marion; Duffourd, Yannis; de Botton, Stéphane; Preudhomme, Claude; Birnbaum, Daniel; Bernard, Olivier A; Ogawa, Seishi; Fontenay, Michaela; Kosmider, Olivier

2013-10-31

Patients with low-risk myelodysplastic syndromes (MDS) that rapidly progress to acute myeloid leukemia (AML) remain a challenge in disease management. Using whole-exome sequencing of an MDS patient, we identified a somatic mutation in the BCOR gene also mutated in AML. Sequencing of BCOR and related BCORL1 genes in a cohort of 354 MDS patients identified 4.2% and 0.8% of mutations respectively. BCOR mutations were associated with RUNX1 (P = .002) and DNMT3A mutations (P = .015). BCOR is also mutated in chronic myelomonocytic leukemia patients (7.4%) and BCORL1 in AML patients with myelodysplasia-related changes (9.1%). Using deep sequencing, we show that BCOR mutations arise after mutations affecting genes involved in splicing machinery or epigenetic regulation. In univariate analysis, BCOR mutations were associated with poor prognosis in MDS (overall survival [OS]: P = .013; cumulative incidence of AML transformation: P = .005). Multivariate analysis including age, International Prognostic Scoring System, transfusion dependency, and mutational status confirmed a significant inferior OS to patients with a BCOR mutation (hazard ratio, 3.3; 95% confidence interval, 1.4-8.1; P = .008). These data suggest that BCOR mutations define the clinical course rather than disease initiation. Despite infrequent mutations, BCOR analyses should be considered in risk stratification.

Molecular nature of forvard gene mutations induced by γ- and UV-irradiation ip the yeast Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Ivanov, E.L.; Koval'tsova, S.V.; Korolev, V.G.

1983-01-01

Gamma and UV-radiation induce the following mutation spectra in the ADE2 gene of Saccharomyces cerevisial yeast respectively: 27 and 41% of GTs→AT transitions, 8 and 11% of AT→GTs transitions, 59 and 40% transversions, 6 and 8% mutations of the reading fame shift type. The results obtained prove the presence of specific nature of UV rays in respect to induction of GTs→AT transitions. The experimental data are discussed from the point of view of studying molecular mechanisms of radiation mutagenesis

BRCA2, EGFR, and NTRK mutations in mismatch repair-deficient colorectal cancers with MSH2 or MLH1 mutations.

Science.gov (United States)

Deihimi, Safoora; Lev, Avital; Slifker, Michael; Shagisultanova, Elena; Xu, Qifang; Jung, Kyungsuk; Vijayvergia, Namrata; Ross, Eric A; Xiu, Joanne; Swensen, Jeffrey; Gatalica, Zoran; Andrake, Mark; Dunbrack, Roland L; El-Deiry, Wafik S

2017-06-20

Deficient mismatch repair (MMR) and microsatellite instability (MSI) contribute to ~15% of colorectal cancer (CRCs). We hypothesized MSI leads to mutations in DNA repair proteins including BRCA2 and cancer drivers including EGFR. We analyzed mutations among a discovery cohort of 26 MSI-High (MSI-H) and 558 non-MSI-H CRCs profiled at Caris Life Sciences. Caris-profiled MSI-H CRCs had high mutation rates (50% vs 14% in non-MSI-H, P MLH1-mutant CRCs showed higher mutation rates in BRCA2 compared to non-MSH2/MLH1-mutant tumors (38% vs 6%, P MLH1-mutant CRCs included 75 unique mutations not known to occur in breast or pancreatic cancer per COSMIC v73. Only 5 deleterious BRCA2 mutations in CRC were previously reported in the BIC database as germ-line mutations in breast cancer. Some BRCA2 mutations were predicted to disrupt interactions with partner proteins DSS1 and RAD51. Some CRCs harbored multiple BRCA2 mutations. EGFR was mutated in 45.5% of MSH2/MLH1-mutant and 6.5% of non-MSH2/MLH1-mutant tumors (P MLH1-mutant CRC including NTRK1 I699V, NTRK2 P716S, and NTRK3 R745L. Our findings have clinical relevance regarding therapeutic targeting of BRCA2 vulnerabilities, EGFR mutations or other identified oncogenic drivers such as NTRK in MSH2/MLH1-mutant CRCs or other tumors with mismatch repair deficiency.

Ovarian carcinomas with genetic and epigenetic BRCA1 loss havedistinct molecular abnormalities

Energy Technology Data Exchange (ETDEWEB)

Press, Joshua Z.; De Luca, Alessandro; Boyd, Niki; Young, Sean; Troussard, Armelle; Ridge, Yolanda; Kaurah, Pardeep; Kalloger, Steve E.; Blood, Katherine A.; Smith, Margaret; Spellman, Paul T.; Wang, Yuker; Miller, Dianne M.; Horsman, Doug; Faham, Malek; Gilks, C. Blake; Gray,Joe; Huntsman, David G.

2007-07-23

Subclassification of ovarian carcinomas can be used to guide treatment and determine prognosis. Germline and somatic mutations, loss of heterozygosity (LOH), and epigenetic events such as promoter hypermethylation can lead to decreased expression of BRCA1/2 in ovarian cancers. The mechanism of BRCA1/2 loss is a potential method of subclassifying high grade serous carcinomas. A consecutive series of 49 ovarian cancers was assessed for mutations status of BRCA1 and BRCA2, LOH at the BRCA1 and BRCA2 loci, methylation of the BRCA1 promoter, BRCA1, BRCA2, PTEN, and PIK3CA transcript levels, PIK3CA gene copy number, and BRCA1, p21, p53, and WT-1 immunohistochemistry. Eighteen (37%) of the ovarian carcinomas had germline or somatic BRCA1 mutations, or epigenetic loss of BRCA1. All of these tumors were high-grade serous or undifferentiated type. None of the endometrioid (n = 5), clear cell (n = 4), or low grade serous (n = 2) carcinomas showed loss of BRCA1, whereas 47% of the 38 high-grade serous or undifferentiated carcinomas had loss of BRCA1. It was possible to distinguish high grade serous carcinomas with BRCA1 mutations from those with epigenetic BRCA1 loss: tumors with BRCA1 mutations typically had decreased PTEN mRNA levels while those with epigenetic loss of BRCA1 had copy number gain of PIK3CA. Overexpression of p53 with loss of p21 expression occurred significantly more frequently in high grade serous carcinomas with epigenetic loss of BRCA1, compared to high grade serous tumors without loss of BRCA1. High grade serous carcinomas can be subclassified into three groups: BRCA1 loss (genetic), BRCA1 loss (epigenetic), and no BRCA1 loss. Tumors in these groups show distinct molecular alterations involving the PI3K/AKT and p53 pathways.

Minimal Residual Disease Diagnostics and Chimerism in the Post-Transplant Period in Acute Myeloid Leukemia

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Ulrike Bacher

2011-01-01

Full Text Available In acute myeloid leukemia (AML, the selection of poor-risk patients for allogeneic hematopoietic stem cell transplantation (HSCT is associated with rather high post-transplant relapse rates. As immunotherapeutic intervention is considered to be more effective before the cytomorphologic manifestation of relapse, post-transplant monitoring gains increasing attention in stem cell recipients with a previous diagnosis of AML. Different methods for detection of chimerism (e.g., microsatellite analysis or quantitative real-time PCR are available to quantify the ratio of donor and recipient cells in the post-transplant period. Various studies demonstrated the potential use of mixed chimerism kinetics to predict relapse of the AML. CD34+-specific chimerism is associated with a higher specificity of chimerism analysis. Nevertheless, a decrease of donor cells can have other causes as well. Therefore, efforts continue to introduce minimal residual disease (MRD monitoring based on molecular mutations in the post-transplant period. The NPM1 (nucleophosmin mutations can be monitored by sensitive quantitative real-time PCR in subsets of stem cell recipients with AML, but for approximately 20% of patients, suitable molecular mutations for post-transplant MRD monitoring are not available so far. This emphasizes the need for an expansion of the panel of MRD markers in the transplant setting.

Whole-exome re-sequencing in a family quartet identifies POP1 mutations as the cause of a novel skeletal dysplasia.

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Evgeny A Glazov

2011-03-01

Full Text Available Recent advances in DNA sequencing have enabled mapping of genes for monogenic traits in families with small pedigrees and even in unrelated cases. We report the identification of disease-causing mutations in a rare, severe, skeletal dysplasia, studying a family of two healthy unrelated parents and two affected children using whole-exome sequencing. The two affected daughters have clinical and radiographic features suggestive of anauxetic dysplasia (OMIM 607095, a rare form of dwarfism caused by mutations of RMRP. However, mutations of RMRP were excluded in this family by direct sequencing. Our studies identified two novel compound heterozygous loss-of-function mutations in POP1, which encodes a core component of the RNase mitochondrial RNA processing (RNase MRP complex that directly interacts with the RMRP RNA domains that are affected in anauxetic dysplasia. We demonstrate that these mutations impair the integrity and activity of this complex and that they impair cell proliferation, providing likely molecular and cellular mechanisms by which POP1 mutations cause this severe skeletal dysplasia.

HEXIM1, a New Player in the p53 Pathway

Energy Technology Data Exchange (ETDEWEB)

Lew, Qiao Jing; Chu, Kai Ling; Chia, Yi Ling; Cheong, Nge [Expression Engineering Group, Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01, Singapore 138668 (Singapore); Chao, Sheng-Hao, E-mail: jimmy_chao@bti.a-star.edu.sg [Expression Engineering Group, Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01, Singapore 138668 (Singapore); Department of Microbiology, National University of Singapore, Singapore 117597 (Singapore)

2013-07-04

Hexamethylene bisacetamide-inducible protein 1 (HEXIM1) is best known as the inhibitor of positive transcription elongation factor b (P-TEFb), which controls transcription elongation of RNA polymerase II and Tat transactivation of human immunodeficiency virus. Besides P-TEFb, several proteins have been identified as HEXIM1 binding proteins. It is noteworthy that more than half of the HEXIM1 binding partners are involved in cancers. P53 and two key regulators of the p53 pathway, nucleophosmin (NPM) and human double minute-2 protein (HDM2), are among the factors identified. This review will focus on the functional importance of the interactions between HEXIM1 and p53/NPM/HDM2. NPM and the cytoplasmic mutant of NPM, NPMc+, were found to regulate P-TEFb activity and RNA polymerase II transcription through the interaction with HEXIM1. Importantly, more than one-third of acute myeloid leukemia (AML) patients carry NPMc+, suggesting the involvement of HEXIM1 in tumorigenesis of AML. HDM2 was found to ubiquitinate HEXIM1. The HDM2-mediated ubiquitination of HEXIM1 did not lead to protein degradation of HEXIM1 but enhanced its inhibitory activity on P-TEFb. Recently, HEXIM1 was identified as a novel positive regulator of p53. HEXIM1 prevented p53 ubiquitination by competing with HDM2 in binding to p53. Taken together, the new evidence suggests a role of HEXIM1 in regulating the p53 pathway and tumorigenesis.

Repair-resistant mutation in Neurospora

International Nuclear Information System (INIS)

Stadler, D.; Macleod, H.; Loo, M.

1987-01-01

Chronic UV treatment produces severalfold fewer mutations in Neurospora conidia than does the same total dose of acute UV. Experiments were designed to determine the conditions required for chronic UV mutagenesis. Measurement of the coincidence frequency for two independent mutations revealed the existence of a subset of cells which are mutable by chronic UV. Analysis of forward mutation at the mtr locus showed that the genetic alterations produced by chronic UV were virtually all point mutants, even though the assay system could detect alterations or deletions extending into neighboring genes. A significant fraction of the mutants produced by acute UV were multigenic deletions. The size of the dose-rate effect (acute UV mutation frequency divided by chronic UV mutation frequency) was compared for several different mutation assay systems. Forward mutations (recessive lethals and mtr) gave values ranging from four to nine. For events which were restricted to specific molecular sites (specific reversions and nonsense suppressor mutations), there was a wider range of dose-rate ratios. This suggests that chronic UV mutation may be restricted to certain molecular sequences or configurations

The effect of point mutations on structure and mechanical properties of collagen-like fibril: A molecular dynamics study

International Nuclear Information System (INIS)

Marlowe, Ashley E.; Singh, Abhishek; Yingling, Yaroslava G.

2012-01-01

Understanding sequence dependent mechanical and structural properties of collagen fibrils is important for the development of artificial biomaterials for medical and nanotechnological applications. Moreover, point mutations are behind many collagen associated diseases, including Osteogenesis Imperfecta (OI). We conducted a combination of classical and steered atomistic molecular dynamics simulations to examine the effect of point mutations on structure and mechanical properties of short collagen fibrils which include mutations of glycine to alanine, aspartic acid, cysteine, and serine or mutations of hydroxyproline to arginine, asparagine, glutamine, and lysine. We found that all mutations disrupt structure and reduce strength of the collagen fibrils, which may affect the hierarchical packing of the fibrils. The glycine mutations were more detrimental to mechanical strength of the fibrils (WT > Ala > Ser > Cys > Asp) than that of hydroxyproline (WT > Arg > Gln > Asn > Lys). The clinical outcome for glycine mutations agrees well with the trend in reduction of fibril's tensile strength predicted by our simulations. Overall, our results suggest that the reduction in mechanical properties of collagen fibrils may be used to predict the clinical outcome of mutations. Highlights: ► All mutations disrupt structure and bonding pattern and reduce strength of the collagen fibrils. ► Gly based mutations are worst to mechanical integrity of fibrils than that of Hyp. ► Lys and Arg mutations most dramatically destabilize collagen fibril properties. ► Clinical outcome of mutations may be related to the reduced mechanical properties of fibrils.

Establishment of novel monoclonal antibodies KMab-1 and MMab-1 specific for IDH2 mutations

Energy Technology Data Exchange (ETDEWEB)

Kaneko, Mika Kato [Regional Innovation Strategy Support Program, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575 (Japan); Molecular Tumor Marker Research Team, Global COE Program, Yamagata University Faculty of Medicine, 2-2-2 Iida-nishi, Yamagata 990-9585 (Japan); Morita, Shunpei; Tsujimoto, Yuta; Yanagiya, Ryo; Nasu, Kana; Sasaki, Hiroko [Molecular Tumor Marker Research Team, Global COE Program, Yamagata University Faculty of Medicine, 2-2-2 Iida-nishi, Yamagata 990-9585 (Japan); Hozumi, Yasukazu; Goto, Kaoru [Department of Anatomy and Cell Biology, Yamagata University School of Medicine, 2-2-2 Iida-nishi, Yamagata 990-9585 (Japan); Natsume, Atsushi [Department of Neurosurgery, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan); Watanabe, Mika [Department of Pathology and Histotechnology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575 (Japan); Kumabe, Toshihiro [Department of Neurosurgery, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8574 (Japan); Takano, Shingo [Department of Neurosurgery, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8575 (Japan); Kato, Yukinari, E-mail: yukinari-k@bea.hi-ho.ne.jp [Regional Innovation Strategy Support Program, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575 (Japan); Molecular Tumor Marker Research Team, Global COE Program, Yamagata University Faculty of Medicine, 2-2-2 Iida-nishi, Yamagata 990-9585 (Japan)

2013-03-01

Highlights: ► IDH1/2 mutations are early and frequent genetic alterations in gliomas. ► We established anti-mutated IDH2-specific mAbs KMab-1 and MMab-1. ► KMab-1 or MMab-1 specifically reacted with mutated IDH2 in ELISA. ► MMab-1 specifically stained IDH2-R172M-expressing CHO cells in ICC. ► MMab-1 specifically stained IDH2-R172M-expressing gliomas in IHC. - Abstract: Isocitrate dehydrogenase 1/2 (IDH1/2) mutations have been detected in gliomas, cartilaginous tumors, and leukemias. IDH1/2 mutations are early and frequent genetic alterations, are specific to a single codon in the conserved and functionally important Arginine 132 (R132) in IDH1 and Arginine 172 (R172) in IDH2. We previously established several monoclonal antibodies (mAbs), which are specific for IDH1 mutations: clones IMab-1 or HMab-1 against IDH1-R132H or clone SMab-1 against IDH1-R132S. However, specific mAbs against IDH2 mutations have not been reported. To establish IDH2-mutation-specific mAbs, we immunized mice or rats with each mutation-containing IDH2 peptides including IDH2-R172K and IDH2-R172M. After cell fusion, IDH2 mutation-specific mAbs were screened in Enzyme-Linked Immunosorbent Assay (ELISA). Established mAbs KMab-1 and MMab-1 reacted with the IDH2-R172K and IDH2-R172M peptides, respectively, but not with IDH2-wild type (WT) in ELISA. Western-blot analysis also showed that KMab-1 and MMab-1 reacted with the IDH2-R172K and IDH2-R172M recombinant proteins, respectively, not with IDH2-WT or other IDH2 mutants, indicating that KMab-1 and MMab-1 are IDH2-mutation-specific. Furthermore, MMab-1 specifically stained the IDH2-R172M-expressing cells in immunocytochemistry, but did not stain IDH2-WT and other IDH2-mutation-containing cells. In immunohistochemical analysis, MMab-1 specifically stained IDH2-R172M-expressing glioma. This is the first report to establish anti-IDH2-mutation-specific mAbs, which could be useful in diagnosis of mutation-bearing tumors.

Establishment of novel monoclonal antibodies KMab-1 and MMab-1 specific for IDH2 mutations

International Nuclear Information System (INIS)

Kaneko, Mika Kato; Morita, Shunpei; Tsujimoto, Yuta; Yanagiya, Ryo; Nasu, Kana; Sasaki, Hiroko; Hozumi, Yasukazu; Goto, Kaoru; Natsume, Atsushi; Watanabe, Mika; Kumabe, Toshihiro; Takano, Shingo; Kato, Yukinari

2013-01-01

Highlights: ► IDH1/2 mutations are early and frequent genetic alterations in gliomas. ► We established anti-mutated IDH2-specific mAbs KMab-1 and MMab-1. ► KMab-1 or MMab-1 specifically reacted with mutated IDH2 in ELISA. ► MMab-1 specifically stained IDH2-R172M-expressing CHO cells in ICC. ► MMab-1 specifically stained IDH2-R172M-expressing gliomas in IHC. - Abstract: Isocitrate dehydrogenase 1/2 (IDH1/2) mutations have been detected in gliomas, cartilaginous tumors, and leukemias. IDH1/2 mutations are early and frequent genetic alterations, are specific to a single codon in the conserved and functionally important Arginine 132 (R132) in IDH1 and Arginine 172 (R172) in IDH2. We previously established several monoclonal antibodies (mAbs), which are specific for IDH1 mutations: clones IMab-1 or HMab-1 against IDH1-R132H or clone SMab-1 against IDH1-R132S. However, specific mAbs against IDH2 mutations have not been reported. To establish IDH2-mutation-specific mAbs, we immunized mice or rats with each mutation-containing IDH2 peptides including IDH2-R172K and IDH2-R172M. After cell fusion, IDH2 mutation-specific mAbs were screened in Enzyme-Linked Immunosorbent Assay (ELISA). Established mAbs KMab-1 and MMab-1 reacted with the IDH2-R172K and IDH2-R172M peptides, respectively, but not with IDH2-wild type (WT) in ELISA. Western-blot analysis also showed that KMab-1 and MMab-1 reacted with the IDH2-R172K and IDH2-R172M recombinant proteins, respectively, not with IDH2-WT or other IDH2 mutants, indicating that KMab-1 and MMab-1 are IDH2-mutation-specific. Furthermore, MMab-1 specifically stained the IDH2-R172M-expressing cells in immunocytochemistry, but did not stain IDH2-WT and other IDH2-mutation-containing cells. In immunohistochemical analysis, MMab-1 specifically stained IDH2-R172M-expressing glioma. This is the first report to establish anti-IDH2-mutation-specific mAbs, which could be useful in diagnosis of mutation-bearing tumors

Arrestin gene mutations in autosomal recessive retinitis pigmentosa.

Science.gov (United States)

Nakazawa, M; Wada, Y; Tamai, M

1998-04-01

To assess the clinical and molecular genetic studies of patients with autosomal recessive retinitis pigmentosa associated with a mutation in the arrestin gene. Results of molecular genetic screening and case reports with DNA analysis and clinical features. University medical center. One hundred twenty anamnestically unrelated patients with autosomal recessive retinitis pigmentosa. DNA analysis was performed by single strand conformation polymorphism followed by nucleotide sequencing to search for a mutation in exon 11 of the arrestin gene. Clinical features were characterized by visual acuity slitlamp biomicroscopy, fundus examinations, fluorescein angiography, kinetic visual field testing, and electroretinography. We identified 3 unrelated patients with retinitis pigmentosa associated with a homozygous 1-base-pair deletion mutation in codon 309 of the arrestin gene designated as 1147delA. All 3 patients showed pigmentary retinal degeneration in the midperipheral area with or without macular involvement. Patient 1 had a sibling with Oguchi disease associated with the same mutation. Patient 2 demonstrated pigmentary retinal degeneration associated with a golden-yellow reflex in the peripheral fundus. Patients 1 and 3 showed features of retinitis pigmentosa without the golden-yellow fundus reflex. Although the arrestin 1147delA has been known as a frequent cause of Oguchi disease, this mutation also may be related to the pathogenesis of autosomal recessive retinitis pigmentosa. This phenomenon may provide evidence of variable expressivity of the mutation in the arrestin gene.

Mutations in KCNT1 cause a spectrum of focal epilepsies

Science.gov (United States)

Møller, Rikke S.; Heron, Sarah E.; Larsen, Line H. G.; Lim, Chiao Xin; Ricos, Michael G.; Bayly, Marta A.; van Kempen, Marjan J. A.; Klinkenberg, Sylvia; Andrews, Ian; Kelley, Kent; Ronen, Gabriel M.; Callen, David; McMahon, Jacinta M.; Yendle, Simone C.; Carvill, Gemma L.; Mefford, Heather C.; Nabbout, Rima; Poduri, Annapurna; Striano, Pasquale; Baglietto, Maria G.; Zara, Federico; Smith, Nicholas J.; Pridmore, Clair; Gardella, Elena; Nikanorova, Marina; Dahl, Hans Atli; Gellert, Pia; Scheffer, Ingrid E.; Gunning, Boudewijn; Kragh-Olsen, Bente; Dibbens, Leanne M.

2018-01-01

Summary Autosomal dominant mutations in the sodium-gated potassium channel subunit gene KCNT1 have been associated with two distinct seizure syndromes, nocturnal frontal lobe epilepsy (NFLE) and malignant migrating focal seizures of infancy (MMFSI). To further explore the phenotypic spectrum associated with KCNT1, we examined individuals affected with focal epilepsy or an epileptic encephalopathy for mutations in the gene. We identified KCNT1 mutations in 12 previously unreported patients with focal epilepsy, multifocal epilepsy, cardiac arrhythmia, and in a family with sudden unexpected death in epilepsy (SUDEP), in addition to patients with NFLE and MMFSI. In contrast to the 100% penetrance so far reported for KCNT1 mutations, we observed incomplete penetrance. It is notable that we report that the one KCNT1 mutation, p.Arg398Gln, can lead to either of the two distinct phenotypes, ADNFLE or MMFSI, even within the same family. This indicates that genotype–phenotype relationships for KCNT1 mutations are not straightforward. We demonstrate that KCNT1 mutations are highly pleiotropic and are associated with phenotypes other than ADNFLE and MMFSI. KCNT1 mutations are now associated with Ohtahara syndrome, MMFSI, and nocturnal focal epilepsy. They may also be associated with multifocal epilepsy and cardiac disturbances. PMID:26122718

Mutation induction by ion beams in arabidopsis

International Nuclear Information System (INIS)

Tanaka, Atsushi

1999-01-01

An investigation was made on characteristics of ion beams for the biological effects and the induction of mutation using Arabidopsis plant as a model plant for the molecular genetics. Here, the characteristics of mutation at the molecular level as well as new mutants induced by ion beams were described. The ast and sep1 were obtained from the offspring of 1488 carbon ion-irradiated seeds respectively. The uvi1-uvi4 mutants were also induced from 1280 M 1 lines. Thus, ion beams can induce not only known mutants such as tt, gl and hy but also novel mutants with high frequency. Even in the tt phenotype, two new mutant loci other than known loci were found. In chrysanthemum, several kinds of single, complex or stripped flower-color mutants that have been never induced by γirradiation, indicating that ion beams could produce a variety of mutants with the same phenotype. In conclusion, ion beams for the mutation induction are characterized by 1) to induce mutants with high frequency, 2) to show broad mutation spectrum and 3) to produce novel mutants. For these reasons, chemical mutagens such as EMS and low LET ionizing radiation such as X-rays and γ-rays will predominantly induce many but small modifications or DNA damages on the DNA strands. As the result, several point mutations will be produced on the genome. On the contrary, ion beams as a high LET ionizing radiation will not cause so many but large and irreparable DNA damage locally, resulting in production of limited number of null mutation. (M.N.)

Mutation induction by ion beams in arabidopsis

Energy Technology Data Exchange (ETDEWEB)

Tanaka, Atsushi [Japan Atomic Energy Research Inst., Takasaki, Gunma (Japan). Takasaki Radiation Chemistry Research Establishment

1999-07-01

An investigation was made on characteristics of ion beams for the biological effects and the induction of mutation using Arabidopsis plant as a model plant for the molecular genetics. Here, the characteristics of mutation at the molecular level as well as new mutants induced by ion beams were described. The ast and sep1 were obtained from the offspring of 1488 carbon ion-irradiated seeds respectively. The uvi1-uvi4 mutants were also induced from 1280 M{sub 1} lines. Thus, ion beams can induce not only known mutants such as tt, gl and hy but also novel mutants with high frequency. Even in the tt phenotype, two new mutant loci other than known loci were found. In chrysanthemum, several kinds of single, complex or stripped flower-color mutants that have been never induced by {gamma}irradiation, indicating that ion beams could produce a variety of mutants with the same phenotype. In conclusion, ion beams for the mutation induction are characterized by 1) to induce mutants with high frequency, 2) to show broad mutation spectrum and 3) to produce novel mutants. For these reasons, chemical mutagens such as EMS and low LET ionizing radiation such as X-rays and {gamma}-rays will predominantly induce many but small modifications or DNA damages on the DNA strands. As the result, several point mutations will be produced on the genome. On the contrary, ion beams as a high LET ionizing radiation will not cause so many but large and irreparable DNA damage locally, resulting in production of limited number of null mutation. (M.N.)

SPOP Mutations in Prostate Cancer across Demographically Diverse Patient Cohorts

Directory of Open Access Journals (Sweden)