RESUMO
This study determined the prevalence of non-Ashkenazi Jewish BRCA1/2 mutations in the Ashkenazi Jewish population in the state of Michigan, current provider testing practices, and the use of mutation probability models in determining which Ashkenazi Jewish individuals should be offered further analysis following negative BRCA1/2 founder testing. Testing patterns, mutation probabilities, and testing results were assessed for 327 Ashkenazi Jewish individuals seen for BRCA1/2 counseling in the state of Michigan who underwent testing for the Ashkenazi Jewish founder mutations. Only one (0.6 %) Ashkenazi Jewish individual with sequencing after negative founder analysis was found to have a non-founder mutation; no rearrangements were identified. Testing patterns varied by clinic, with the proportion of Ashkenazi Jewish individuals undergoing additional sequencing ranging from 22.2 to 92.9 %. In Ashkenazi Jewish individuals with a pre-test BRCAPRO risk calculation, the mean risk was significantly higher in those with follow-up sequencing compared to those who did not pursue additional testing. The low prevalence of non-founder BRCA1/2 mutations in Ashkenazi Jewish individuals does not warrant automatically reflexing to full analysis after negative mutation testing. Increased use of mutation probability models may aid in determining which cases warrant additional testing.
Assuntos
Neoplasias da Mama Masculina/genética , Neoplasias da Mama/genética , Efeito Fundador , Genes BRCA1 , Judeus/genética , Neoplasias Ovarianas/genética , Adulto , Feminino , Testes Genéticos , Humanos , Masculino , Michigan , Mutação , Reflexo , RiscoRESUMO
Human eosinophils spontaneously adhere to various substrates in the absence of exogenously added activators. In the present study a method was developed for characterizing eosinophil adhesion by measuring changes in impedance. Impedance measurements were performed in HCO(3)-buffered HybriCare medium maintained in a humidified 5% CO(2) incubator at 37 degrees C. Impedance increased by more than 1 kOmega within minutes after eosinophils made contact with the substrate, reaching a peak within 20 min. Blocking mobilization of intracellular [Ca(2+)] that precedes adhesion with BAPTA-AM (10 microM) completely inhibited the rise in impedance as well as the changes in cell shape typically observed in adherent cells. However, lowering the extracellular [Ca(2+)] with 2.5 mM EGTA did not inhibit the increase in impedance. Pretreatment with anti-CD18 antibody to block substrate interactions with beta(2)-integrins, or jasplakinolide (2 microM) to block actin reorganization, abolished the increase in impedance and adherent morphology of the cells. Exposure of eosinophils to the phosphatidylinositol 3 kinase inhibitor LY294002 (5 microM) or treatment with protein kinase C zeta pseudosubstrate to competitively inhibit activity of the enzyme significantly reduced the increase in impedance and inhibited the cell spreading associated with adhesion. These results demonstrate a novel method for measuring eosinophil adhesion and showed that, following formation of a tethered attachment, a rapid increase in intracellular [Ca(2+)] precedes the cytoskeletal rearrangements required for cell shape changes and plasma membrane-substrate interactions associated with adhesion.
Assuntos
Antígenos CD18/fisiologia , Cálcio/metabolismo , Adesão Celular/fisiologia , Eosinófilos/citologia , Eosinófilos/metabolismo , Antígenos CD18/metabolismo , Células Cultivadas , Humanos , Imuno-Histoquímica , Microscopia ConfocalRESUMO
We describe the development and validation of a new instrument, the Classroom Discourse Observation Protocol (CDOP), which quantifies teacher discourse moves (TDMs) from observational data in undergraduate STEM classrooms. TDMs can be conceptualized as epistemic tools that can mediate classroom discussions. Through an inductive-deductive coding process, we identified commonly occurring TDMs among a group of biology instructors (n = 13, 37 class session) teaching in Active Learning Environments. We describe the CDOP coding scheme and its associated matrix that allows observers to reliably characterize TDMs in 2-min time intervals over the course of a class period. We present the protocol, discuss how it differs from existing classroom observation protocols, and describe the process by which it was developed and validated. Also, we show how this protocol is able to discriminate the discursive practices of instructors teaching in undergraduate STEM learning environments with sample qualitative and quantitative results that illustrate its utility for assessing and improving STEM instructional practices.
Assuntos
Aprendizagem Baseada em Problemas/métodos , Ensino , Engenharia/educação , Humanos , Matemática/educação , Aprendizagem Baseada em Problemas/estatística & dados numéricos , Reprodutibilidade dos Testes , Ciência/educação , Estudantes , Tecnologia/educação , Estados UnidosRESUMO
Li-Fraumeni syndrome (LFS) is a rare cancer predisposition syndrome usually associated with TP53 germline alterations. Its genetic basis in TP53 wild-type pedigrees is less understood. Using whole-genome sequencing, we identified a germline hemizygous deletion ablating CDKN2A-CDKN2B in a TP53 wild-type patient presenting with high-grade sarcoma, laryngeal squamous cell carcinoma and a family history suggestive of LFS. Patient-derived cells demonstrated reduced basal gene and protein expression of the CDKN2A-encoded tumour suppressors p14ARF and p16INK4A with concomitant decrease in p21 and faster cell proliferation, implying potential deregulation of p53-mediated cell cycle control. Review of 13 additional patients with pathogenic CDKN2A variants suggested associations of germline CDKN2A mutations with an expanded spectrum of non-melanoma familial cancers. To our knowledge, this is the first report of a germline gross deletion of the CDKN2A-CDKN2B locus in an LFS family. These findings highlight the potential contribution of germline CDKN2A deletions to cancer predisposition and the importance of interrogating the full extent of CDKN2A locus in clinical testing gene panels.
RESUMO
Common variable immunodeficiency (CVID) is a primary immunodeficiency disorder that can present with multiple phenotypes, all of which are characterized by hypogammaglobulinemia, in a person at any age. A specific genetic defect that accounts for all CVID phenotypes has not been identified, and it is likely that several distinct genetic disorders with similar clinical presentations are responsible for the observed variation. In this review, we summarize the known genetic mutations that give rise to hypogammaglobulinemia and how these gene products affect normal or abnormal B-cell development and function, with particular emphasis on CVID. Additionally, we describe specific phenotypic and genetic laboratory tests that can be used to diagnose CVID and provide guidelines for test interpretation and subsequent therapeutic intervention.
Assuntos
Linfócitos B/fisiologia , Imunodeficiência de Variável Comum/diagnóstico , Imunodeficiência de Variável Comum/genética , Mutação/genética , Imunodeficiência de Variável Comum/terapia , Testes Genéticos , Humanos , FenótipoRESUMO
The effects of platelet-activating factor (PAF) and IL-5 on intracellular pH were investigated in human eosinophils. Purified peripheral blood eosinophils were loaded with the ratiometric fluorescent pH indicator BCECF-AM ester. Stimulation of eosinophils with PAF produced time-dependent alkalinization of the cytoplasm from an initial pH of 7.1+/-0.04 to 7.5+/-0.05. A similar alkalinization response was produced by the calcium ionophore, ionomycin and by the calcium ATPase inhibitor, thapsigargin. These compounds as well as PAF produce significant increases in cytoplasmic calcium ([Ca2+]i). In contrast, IL-5 and the protein kinase C (PKC) activator phorbol myristate acetate (PMA) did not produce cytoplasmic alkalinization and had no effect on [Ca2+]i in eosinophils. PAF-stimulated alkalinization was not inhibited under conditions that blocked plasma membrane Na+-H+ exchange, proton channel or plasma membrane H+-ATPase activities. Measurements of intragranule pH with a cell permeant pH indicator (LysoSensor Yellow/Blue DND-160), which partitions into intracellular acidic compartments, revealed that PAF-stimulated cytosolic alkalinization correlated with intragranule acidification. These results suggest that the increase in [Ca2+]i after PAF stimulation activates a H+-ATPase present in the granule membranes, leading to enhanced granule acidification and cytoplasmic alkalinization. We propose that granule acidification is an important step in solubilization of major basic protein crystals, which are stored within the granule core, in preparation for degranulation and release of these proteins.
Assuntos
Citoplasma/metabolismo , Grânulos Citoplasmáticos/metabolismo , Proteínas Granulares de Eosinófilos/fisiologia , Eosinófilos/metabolismo , Fator de Ativação de Plaquetas/fisiologia , Adenosina Trifosfatases/química , Western Blotting , Cálcio/metabolismo , Adesão Celular , Membrana Celular/metabolismo , Células Cultivadas , Citosol/metabolismo , Proteína Básica Maior de Eosinófilos/química , Eosinófilos/citologia , Ésteres , Fluoresceínas/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Imuno-Histoquímica , Interleucina-5/metabolismo , Microscopia de Fluorescência , Análise de Sequência com Séries de Oligonucleotídeos , Fator de Ativação de Plaquetas/química , Proteína Quinase C/metabolismo , Prótons , RNA Mensageiro/metabolismo , Acetato de Tetradecanoilforbol , Tapsigargina/farmacologia , Fatores de Tempo , ATPases Vacuolares Próton-Translocadoras/química , Zinco/químicaRESUMO
Protein kinase C (PKC) activation in human eosinophils increases NADPH oxidase activity, which is associated with plasma membrane depolarization. In this study, membrane potential measurements of eosinophils stimulated with phorbol ester (phorbol 12-myristate 13-acetate; PMA) were made using a cell-permeable oxonol membrane potential indicator, diBAC4(3). Within 10 minutes after PMA stimulation, eosinophils depolarized from -32.9+/-5.7 mV to +17.3+/-1.8 mV. The time courses of depolarization and proton channel activation were virtually identical. Blocking the proton conductance with 250 microM ZnCl2 (+43.0+/-4.2 mV) or increasing the proton channel activation threshold by reducing the extracellular pH to 6.5 (+44.4+/-1.4 mV) increased depolarization compared with PMA alone. Additionally, the protein kinase C (PKC) delta-selective blocker, rottlerin, inhibited PMA-stimulated depolarization, indicating that PKCdelta was involved in regulating depolarization associated with eosinophil NADPH oxidase activity. Thus, the membrane depolarization that is associated with NADPH oxidase activation in eosinophils is sufficient to produce marked proton channel activation under physiological conditions.