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1.
J Vis Exp ; (78): e50405, 2013 Aug 22.
Artículo en Inglés | MEDLINE | ID: mdl-23995536

RESUMEN

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.


Asunto(s)
Bacterias/clasificación , Análisis de Secuencia de ADN/métodos , Bacterias/genética , Secuencia de Bases , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Difosfatos/química , Difosfatos/metabolismo , Datos de Secuencia Molecular , Nucleótidos/química , Nucleótidos/genética , Nucleótidos/metabolismo , Sulfato Adenililtransferasa/química , Sulfato Adenililtransferasa/metabolismo , Moldes Genéticos
2.
Clin Cancer Res ; 14(15): 4726-34, 2008 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-18676741

RESUMEN

PURPOSE: To provide a comprehensive, thorough analysis of somatic mutation and promoter hypermethylation of the von Hippel-Lindau (VHL) gene in the cancer genome, unique to clear cell renal cancer (ccRCC). Identify relationships between the prevalence of VHL gene alterations and alteration subtypes with patient and tumor characteristics. EXPERIMENTAL DESIGN: As part of a large kidney cancer case-control study conducted in Central Europe, we analyzed VHL mutations and promoter methylation in 205 well-characterized, histologically confirmed patient tumor biopsies using a combination of sensitive, high-throughput methods (endonuclease scanning and Sanger sequencing) and analysis of 11 CpG sites in the VHL promoter. RESULTS: We identified mutations in 82.4% of cases, the highest VHL gene mutation prevalence reported to date. Analysis of 11 VHL promoter CpG sites revealed that 8.3% of tumors were hypermethylated and all were mutation negative. In total, 91% of ccRCCs exhibited alteration of the gene through genetic or epigenetic mechanisms. Analysis of patient and tumor characteristics revealed that certain mutation subtypes were significantly associated with Fuhrman nuclear grade, metastasis, node positivity, and self-reported family history of RCC. CONCLUSION: Detection of VHL gene alterations using these accurate, sensitive, and practical methods provides evidence that the vast majority of histologically confirmed ccRCC tumors possess genetic or epigenetic alteration of the VHL gene and support the hypothesis that VHL alteration is an early event in ccRCC carcinogenesis. These findings also indicate that VHL molecular subtypes can provide a sensitive marker of tumor heterogeneity among histologically similar ccRCC cases for etiologic, prognostic, and translational studies.


Asunto(s)
Adenocarcinoma de Células Claras/genética , Carcinoma de Células Renales/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Renales/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Adenocarcinoma de Células Claras/metabolismo , Carcinoma de Células Renales/metabolismo , Estudios de Casos y Controles , Islas de CpG , Metilación de ADN , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Mutación , Neoplasias/metabolismo , Regiones Promotoras Genéticas , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo
3.
Proc Natl Acad Sci U S A ; 103(16): 6224-9, 2006 Apr 18.
Artículo en Inglés | MEDLINE | ID: mdl-16603627

RESUMEN

Although a large proportion of patients with polycythemia vera (PV) harbor a valine-to-phenylalanine mutation at amino acid 617 (V617F) in the JAK2 signaling molecule, the stage of hematopoiesis at which the mutation arises is unknown. Here we isolated and characterized hematopoietic stem cells (HSC) and myeloid progenitors from 16 PV patient samples and 14 normal individuals, testing whether the JAK2 mutation could be found at the level of stem or progenitor cells and whether the JAK2 V617F-positive cells had altered differentiation potential. In all PV samples analyzed, there were increased numbers of cells with a HSC phenotype (CD34+CD38-CD90+Lin-) compared with normal samples. Hematopoietic progenitor assays demonstrated that the differentiation potential of PV was already skewed toward the erythroid lineage at the HSC level. The JAK2 V617F mutation was detectable within HSC and their progeny in PV. Moreover, the aberrant erythroid potential of PV HSC was potently inhibited with a JAK2 inhibitor, AG490.


Asunto(s)
Células Precursoras Eritroides/citología , Hematopoyesis/genética , Células Madre Hematopoyéticas/enzimología , Policitemia Vera/genética , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , ADP-Ribosil Ciclasa 1/análisis , Sustitución de Aminoácidos/genética , Antígenos CD34/análisis , Células Precursoras Eritroides/enzimología , Células Madre Hematopoyéticas/química , Células Madre Hematopoyéticas/citología , Humanos , Janus Quinasa 2 , Fenilalanina/química , Fenilalanina/genética , Mutación Puntual , Policitemia Vera/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Antígenos Thy-1/análisis , Tirfostinos/farmacología , Valina/química , Valina/genética
4.
Blood ; 106(8): 2865-70, 2005 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-15972446

RESUMEN

The majority of patients with systemic mast cell disease express the imatinib-resistant Asp816Val (D816V) mutation in the KIT receptor tyrosine kinase. Limited treatment options exist for aggressive systemic mastocytosis (ASM) and mast cell leukemia (MCL). We evaluated whether PKC412, a small-molecule inhibitor of KIT with a different chemical structure from imatinib, may have therapeutic use in advanced SM with the D816V KIT mutation. We treated a patient with MCL (with an associated myelodysplastic syndrome (MDS)/myeloproliferative disorder [MPD]) based on in vitro studies demonstrating that PKC412 could inhibit D816V KIT-transformed Ba/F3 cell growth with a 50% inhibitory concentration (IC50) of 30 nM to 40 nM. The patient exhibited a partial response with significant resolution of liver function abnormalities. In addition, PKC412 treatment resulted in a significant decline in the percentage of peripheral blood mast cells and serum histamine level and was associated with a decrease in KIT phosphorylation and D816V KIT mutation frequency. The patient died after 3 months of therapy due to progression of her MDS/MPD to acute myeloid leukemia (AML). This case indicates that KIT tyrosine kinase inhibition is a feasible approach in SM, but single-agent clinical efficacy may be limited by clonal evolution in the advanced leukemic phase of this disease.


Asunto(s)
Ácido Aspártico/genética , Leucemia de Mastocitos/tratamiento farmacológico , Leucemia de Mastocitos/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-kit/genética , Estaurosporina/análogos & derivados , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Femenino , Humanos , Inmunofenotipificación , Leucemia de Mastocitos/metabolismo , Leucemia de Mastocitos/patología , Persona de Mediana Edad , Mutación/genética , Fosforilación/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , Estaurosporina/farmacocinética , Estaurosporina/uso terapéutico
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