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1.
Nature ; 485(7397): 242-5, 2012 Apr 04.
Artículo en Inglés | MEDLINE | ID: mdl-22495311

RESUMEN

Autism spectrum disorders (ASD) are believed to have genetic and environmental origins, yet in only a modest fraction of individuals can specific causes be identified. To identify further genetic risk factors, here we assess the role of de novo mutations in ASD by sequencing the exomes of ASD cases and their parents (n = 175 trios). Fewer than half of the cases (46.3%) carry a missense or nonsense de novo variant, and the overall rate of mutation is only modestly higher than the expected rate. In contrast, the proteins encoded by genes that harboured de novo missense or nonsense mutations showed a higher degree of connectivity among themselves and to previous ASD genes as indexed by protein-protein interaction screens. The small increase in the rate of de novo events, when taken together with the protein interaction results, are consistent with an important but limited role for de novo point mutations in ASD, similar to that documented for de novo copy number variants. Genetic models incorporating these data indicate that most of the observed de novo events are unconnected to ASD; those that do confer risk are distributed across many genes and are incompletely penetrant (that is, not necessarily sufficient for disease). Our results support polygenic models in which spontaneous coding mutations in any of a large number of genes increases risk by 5- to 20-fold. Despite the challenge posed by such models, results from de novo events and a large parallel case-control study provide strong evidence in favour of CHD8 and KATNAL2 as genuine autism risk factors.


Asunto(s)
Trastorno Autístico/genética , Proteínas de Unión al ADN/genética , Exones/genética , Predisposición Genética a la Enfermedad/genética , Mutación/genética , Factores de Transcripción/genética , Estudios de Casos y Controles , Exoma/genética , Salud de la Familia , Humanos , Modelos Genéticos , Herencia Multifactorial/genética , Fenotipo , Distribución de Poisson , Mapas de Interacción de Proteínas
2.
Nature ; 482(7384): 173-8, 2012 Feb 08.
Artículo en Inglés | MEDLINE | ID: mdl-22318601

RESUMEN

A major challenge of biology is understanding the relationship between molecular genetic variation and variation in quantitative traits, including fitness. This relationship determines our ability to predict phenotypes from genotypes and to understand how evolutionary forces shape variation within and between species. Previous efforts to dissect the genotype-phenotype map were based on incomplete genotypic information. Here, we describe the Drosophila melanogaster Genetic Reference Panel (DGRP), a community resource for analysis of population genomics and quantitative traits. The DGRP consists of fully sequenced inbred lines derived from a natural population. Population genomic analyses reveal reduced polymorphism in centromeric autosomal regions and the X chromosome, evidence for positive and negative selection, and rapid evolution of the X chromosome. Many variants in novel genes, most at low frequency, are associated with quantitative traits and explain a large fraction of the phenotypic variance. The DGRP facilitates genotype-phenotype mapping using the power of Drosophila genetics.


Asunto(s)
Drosophila melanogaster/genética , Estudio de Asociación del Genoma Completo , Genómica , Sitios de Carácter Cuantitativo/genética , Alelos , Animales , Centrómero/genética , Cromosomas de Insectos/genética , Genotipo , Fenotipo , Polimorfismo de Nucleótido Simple/genética , Selección Genética/genética , Inanición/genética , Telómero/genética , Cromosoma X/genética
3.
Proc Natl Acad Sci U S A ; 112(27): 8403-8, 2015 Jul 07.
Artículo en Inglés | MEDLINE | ID: mdl-26080435

RESUMEN

Prostate cancer antigen 3 (PCA3) is the most specific prostate cancer biomarker but its function remains unknown. Here we identify PRUNE2, a target protein-coding gene variant, which harbors the PCA3 locus, thereby classifying PCA3 as an antisense intronic long noncoding (lnc)RNA. We show that PCA3 controls PRUNE2 levels via a unique regulatory mechanism involving formation of a PRUNE2/PCA3 double-stranded RNA that undergoes adenosine deaminase acting on RNA (ADAR)-dependent adenosine-to-inosine RNA editing. PRUNE2 expression or silencing in prostate cancer cells decreased and increased cell proliferation, respectively. Moreover, PRUNE2 and PCA3 elicited opposite effects on tumor growth in immunodeficient tumor-bearing mice. Coregulation and RNA editing of PRUNE2 and PCA3 were confirmed in human prostate cancer specimens, supporting the medical relevance of our findings. These results establish PCA3 as a dominant-negative oncogene and PRUNE2 as an unrecognized tumor suppressor gene in human prostate cancer, and their regulatory axis represents a unique molecular target for diagnostic and therapeutic intervention.


Asunto(s)
Antígenos de Neoplasias/genética , Intrones/genética , Proteínas de Neoplasias/genética , Neoplasias de la Próstata/genética , ARN Largo no Codificante/genética , Proteínas Supresoras de Tumor/genética , Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , Animales , Antígenos de Neoplasias/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Células MCF-7 , Masculino , Ratones SCID , Datos de Secuencia Molecular , Proteínas de Neoplasias/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Unión Proteica , Interferencia de ARN , Precursores del ARN/genética , Precursores del ARN/metabolismo , ARN Largo no Codificante/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Supresoras de Tumor/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto/métodos
4.
PLoS Genet ; 9(4): e1003443, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23593035

RESUMEN

We report on results from whole-exome sequencing (WES) of 1,039 subjects diagnosed with autism spectrum disorders (ASD) and 870 controls selected from the NIMH repository to be of similar ancestry to cases. The WES data came from two centers using different methods to produce sequence and to call variants from it. Therefore, an initial goal was to ensure the distribution of rare variation was similar for data from different centers. This proved straightforward by filtering called variants by fraction of missing data, read depth, and balance of alternative to reference reads. Results were evaluated using seven samples sequenced at both centers and by results from the association study. Next we addressed how the data and/or results from the centers should be combined. Gene-based analyses of association was an obvious choice, but should statistics for association be combined across centers (meta-analysis) or should data be combined and then analyzed (mega-analysis)? Because of the nature of many gene-based tests, we showed by theory and simulations that mega-analysis has better power than meta-analysis. Finally, before analyzing the data for association, we explored the impact of population structure on rare variant analysis in these data. Like other recent studies, we found evidence that population structure can confound case-control studies by the clustering of rare variants in ancestry space; yet, unlike some recent studies, for these data we found that principal component-based analyses were sufficient to control for ancestry and produce test statistics with appropriate distributions. After using a variety of gene-based tests and both meta- and mega-analysis, we found no new risk genes for ASD in this sample. Our results suggest that standard gene-based tests will require much larger samples of cases and controls before being effective for gene discovery, even for a disorder like ASD.


Asunto(s)
Trastornos Generalizados del Desarrollo Infantil/genética , Exoma , Estudio de Asociación del Genoma Completo , Estudios de Casos y Controles , Niño , Trastornos Generalizados del Desarrollo Infantil/fisiopatología , Predisposición Genética a la Enfermedad , Variación Genética , Humanos , Regulación de la Población , Análisis de Secuencia de ADN , Programas Informáticos
5.
BMC Genomics ; 15: 86, 2014 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-24479613

RESUMEN

BACKGROUND: The first generation of genome sequence assemblies and annotations have had a significant impact upon our understanding of the biology of the sequenced species, the phylogenetic relationships among species, the study of populations within and across species, and have informed the biology of humans. As only a few Metazoan genomes are approaching finished quality (human, mouse, fly and worm), there is room for improvement of most genome assemblies. The honey bee (Apis mellifera) genome, published in 2006, was noted for its bimodal GC content distribution that affected the quality of the assembly in some regions and for fewer genes in the initial gene set (OGSv1.0) compared to what would be expected based on other sequenced insect genomes. RESULTS: Here, we report an improved honey bee genome assembly (Amel_4.5) with a new gene annotation set (OGSv3.2), and show that the honey bee genome contains a number of genes similar to that of other insect genomes, contrary to what was suggested in OGSv1.0. The new genome assembly is more contiguous and complete and the new gene set includes ~5000 more protein-coding genes, 50% more than previously reported. About 1/6 of the additional genes were due to improvements to the assembly, and the remaining were inferred based on new RNAseq and protein data. CONCLUSIONS: Lessons learned from this genome upgrade have important implications for future genome sequencing projects. Furthermore, the improvements significantly enhance genomic resources for the honey bee, a key model for social behavior and essential to global ecology through pollination.


Asunto(s)
Abejas/genética , Genes de Insecto , Animales , Composición de Base , Bases de Datos Genéticas , Secuencias Repetitivas Esparcidas/genética , Anotación de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Péptidos/análisis , Análisis de Secuencia de ARN , Homología de Secuencia de Aminoácido
6.
Am J Med Genet A ; 155A(9): 2071-7, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21834044

RESUMEN

Polymicrogyria is a disorder of neuronal development resulting in structurally abnormal cerebral hemispheres characterized by over-folding and abnormal lamination of the cerebral cortex. Polymicrogyria is frequently associated with severe neurologic deficits including intellectual disability, motor problems, and epilepsy. There are acquired and genetic causes of polymicrogyria, but most patients with a presumed genetic etiology lack a specific diagnosis. Here we report using whole-exome sequencing to identify compound heterozygous mutations in the WD repeat domain 62 (WDR62) gene as the cause of recurrent polymicrogyria in a sibling pair. Sanger sequencing confirmed that the siblings both inherited 1-bp (maternal allele) and 2-bp (paternal allele) frameshift deletions, which predict premature truncation of WDR62, a protein that has a role in early cortical development. The probands are from a non-consanguineous family of Northern European descent, suggesting that autosomal recessive PMG due to compound heterozygous mutation of WDR62 might be a relatively common cause of PMG in the population. Further studies to identify mutation frequency in the population are needed.


Asunto(s)
Anomalías Múltiples/genética , Exoma , Malformaciones del Desarrollo Cortical/genética , Proteínas del Tejido Nervioso/genética , Adulto , Secuencia de Bases , Proteínas de Ciclo Celular , Niño , Anomalías Craneofaciales/genética , Femenino , Mutación del Sistema de Lectura , Pruebas Genéticas , Heterocigoto , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Imagen por Resonancia Magnética , Masculino , Mutación , Análisis de Secuencia de ADN , Eliminación de Secuencia , Hermanos
7.
Biochem Pharmacol ; 74(7): 981-91, 2007 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-17692290

RESUMEN

JG-03-14, a substituted pyrrole that inhibits microtubule polymerization, was screened against MCF-7 (p53 wild type), MDA-MB231 (p53 mutant), MCF-7/caspase 3 and MCF-7/ADR (multidrug resistant) breast tumor cell lines. Cell viability and growth inhibition were assessed by the crystal violet dye assay. Apoptosis was evaluated by the TUNEL assay, cell cycle distribution by flow cytometry, autophagy by acridine orange staining of vesicle formation, and senescence based on beta-galactosidase staining and cell morphology. Our studies indicate that exposure to JG-03-14, at a concentration of 500 nM, induces time-dependent cell death in the MCF-7 and MDA-MB231 cell lines. In MCF-7 cells, a residual surviving cell population was found to be senescent; in contrast, there was no surviving senescent population in treated MDA-MB231 cells. No proliferative recovery was detected over a period of 15 days post-treatment in either cell line. Both the TUNEL assay and FLOW cytometry indicated a relatively limited degree of apoptosis (<10%) in response to drug treatment in MCF-7 cells with more extensive apoptosis (but <20%) in MDA-MB231 cells; acidic vacuole formation indicative of autophagic cell death was relatively extensive in both MCF-7 and MDA-MB231 cells. In addition, JG-03-14 induced the formation of a large hyperdiploid cell population in MDA-MB231 cells. JG-03-14 also demonstrated pronounced anti-proliferative activity in MCF-7/caspase 3 cells and in the MCF-7/ADR cell line. The observation that JG-03-14 promotes autophagic cell death and also retains activity in tumor cells expressing the multidrug resistance pump indicates that novel microtubule poisons of the substituted pyrroles class may hold promise in the treatment of breast cancer.


Asunto(s)
Antineoplásicos/farmacología , Autofagia/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Poliploidía , Pirroles/farmacología , Envejecimiento/efectos de los fármacos , Caspasa 3/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Aberraciones Cromosómicas , Resistencia a Antineoplásicos , Humanos , Microtúbulos/efectos de los fármacos , Estructura Molecular , Pirroles/química , Factores de Tiempo
8.
Mol Cancer Ther ; 5(11): 2786-97, 2006 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17121925

RESUMEN

1,25-Dihydroxyvitamin D(3) and vitamin D(3) analogues, such as EB 1089, potentiate the response to ionizing radiation in breast tumor cells. The current studies address the basis for this interaction by evaluating DNA damage and repair, the effect of interference with reactive oxygen generation, the involvement of p53 and caspase-3, signaling through c-myc, as well as the induction of senescence and multiple modes of cell death. EB 1089 failed to increase the extent of radiation-induced DNA damage or to attenuate the rate of DNA repair. The reactive oxygen scavengers N-acetyl-l-cysteine and reduced glutathione failed to protect the cells from the promotion of cell death by EB 1089 and radiation. Whereas MCF-7 cells expressing caspase-3 showed significant apoptosis with radiation alone as well as with EB 1089 followed by radiation, EB 1089 maintained its ability to confer susceptibility to radiation-induced cell killing, in large part by interference with proliferative recovery. In contrast, in breast tumor cells lacking p53, where radiation promoted extensive apoptosis and the cells failed to recover after radiation treatment, EB 1089 failed to influence the effect of radiation. EB 1089 treatment interfered with radiation-induced suppression of c-myc; however, induction of c-myc did not prevent senescence by radiation alone or radiation-induced cell death promoted by EB 1089. EB 1089 did not increase the extent of micronucleation, indicative of mitotic catastrophe, induced by radiation alone. However, EB 1089 did promote extensive autophagic cell death in the irradiated cells. Taken together, these studies suggest that the effect of EB 1089 treatment on the radiation response is related in part to enhanced promotion of autophagic cell death and in part to interference with the proliferative recovery that occurs with radiation alone in p53 wild-type breast tumor cells.


Asunto(s)
Antineoplásicos/farmacología , Autofagia/efectos de los fármacos , Autofagia/efectos de la radiación , Neoplasias de la Mama/metabolismo , Calcitriol/análogos & derivados , Tolerancia a Radiación , Neoplasias de la Mama/patología , Calcitriol/farmacología , Caspasa 3/metabolismo , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Senescencia Celular/efectos de la radiación , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de los fármacos , Reparación del ADN/efectos de la radiación , Femenino , Radicales Libres/metabolismo , Humanos , Especies Reactivas de Oxígeno/metabolismo , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/metabolismo
9.
Oncogene ; 24(43): 6502-15, 2005 Sep 29.
Artículo en Inglés | MEDLINE | ID: mdl-16007173

RESUMEN

4.1B is a member of the protein 4.1 superfamily of proteins that link transmembrane proteins to the actin cytoskeleton. The 4.1B gene localizes to chromosome 18p11.3, which undergoes loss of heterozygosity in mammary tumors. Here, we examine the expression of 4.1B in murine mammary epithelium and find that 4.1B is dramatically upregulated in mammary epithelial cells during pregnancy when there is extensive cell proliferation. In contrast, 4.1B is not expressed in virgin, lactating, or involuting mammary epithelium. To examine the consequence of 4.1B loss on mammary epithelial cell proliferation, we analysed mammary glands in 4.1B-null mice. 4.1B loss results in a significant increase in mammary epithelial cell proliferation during pregnancy, but has no effect on mammary epithelial cell proliferation, in virgin or involuting mice. Furthermore, we show that 4.1B inhibits the proliferation of mammary epithelial cell lines by inducing a G1 cell cycle arrest, characterized by decreased cyclin A expression and reduced Rb phosphorylation, and accompanied by reduced erbB2 phosphorylation. This cell cycle arrest does not involve alterations in the activities of MAPK, JNK, or Akt. Collectively, our findings demonstrate that 4.1B regulates mammary epithelial cell proliferation during pregnancy and suggest that its loss may influence mammary carcinoma pathogenesis in multiparous women.


Asunto(s)
Glándulas Mamarias Animales/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular , Proteínas de Unión al ADN/metabolismo , Células Epiteliales/citología , Femenino , Fase G1/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Lactancia , Glándulas Mamarias Animales/citología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Proteínas de Microfilamentos , Fosforilación , Embarazo , Preñez/genética , Receptor ErbB-2/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas
10.
Mol Cancer ; 5: 4, 2006 Jan 18.
Artículo en Inglés | MEDLINE | ID: mdl-16420693

RESUMEN

BACKGROUND: DAL-1 (Differentially Expressed in Adenocarcinoma of the Lung)/4.1B is a member of the protein 4.1 superfamily that has been shown to suppress growth in lung, breast and brain tumor cells. In the case of the caspase-3 deficient MCF-7 breast cancer cells, this growth suppression has been shown to be partially mediated by the induction of apoptosis. However the exact mechanism of action of DAL-1/4.1B is unknown. Recently, protein arginine N-methyltransferase 3 (PRMT3) was identified as a DAL-1/4.1B interacting protein. Protein arginine methyltransferases (PRMTs) posttranslationally methylate the arginine residues of proteins, a modification which has been implicated in the regulation of multiple cellular processes including nuclear-cytoplasmic transport, signal transduction, and transcription. RESULTS: To investigate the role of protein methylation in cell death induced by DAL-1/4.1B, DAL-1/4.1B-inducible MCF-7 cells were examined for apoptosis and caspase activation in the absence and presence of the protein methylation inhibitor adenosine dialdehyde (AdOX). Flow cytometry analysis revealed that apoptosis was primarily associated with the activation of caspase 8, and inhibition of this activation blocked the ability of DAL-1/4.1B to induce cell death. CONCLUSION: These results suggest that protein methylation cooperates with DAL-1/4.1B-associated caspase 8-specific activation to induce apoptosis in breast cancer cells.


Asunto(s)
Apoptosis , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas de la Membrana/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Western Blotting , Neoplasias de la Mama/enzimología , Caspasa 3/metabolismo , Caspasa 8/metabolismo , Línea Celular Tumoral , Activación Enzimática , Humanos , Metilación , Proteínas de Microfilamentos
11.
Oncogene ; 23(47): 7761-71, 2004 Oct 14.
Artículo en Inglés | MEDLINE | ID: mdl-15334060

RESUMEN

DAL-1 (differentially expressed in adenocarcinoma of the lung)/4.1B is a tumor suppressor gene on human chromosome 18p11.3 whose expression is lost in >50% of primary non-small-cell lung carcinomas. Based on sequence similarity, DAL-1/4.1B has been assigned to the Protein 4.1 superfamily whose members interact with plasma membrane proteins through their N-terminal FERM (4.1/Ezrin/Radixin/Moesin) domain, and cytoskeletal components via their C-terminal SAB (spectrin-actin binding) region. Using the DAL-1/4.1B FERM domain as bait for yeast two-hybrid interaction cloning, we identified protein arginine N-methyltransferase 3 (PRMT3) as a specific DAL-1/4.1B-interacting protein. PRMT3 catalyses the post-translational transfer of methyl groups from S-adenosyl-L-methionine to arginine residues of proteins. Coimmunoprecipitation experiments using lung and breast cancer cell lines confirmed this interaction in mammalian cells in vivo. In vitro binding assays demonstrated that this was an interaction occurring via the C-terminal catalytic core domain of PRMT3. DAL-1/4.1B was determined not to be a substrate for PRMT3-mediated methylation but its presence inhibits the in vitro methylation of a glycine-rich and arginine-rich methyl-accepting protein, GST (glutathione-S-transferase-GAR (glycine- and arginine-rich), which contains 14 'RGG' consensus methylation sites. In addition, induced expression of DAL-1/4.1B in MCF-7 breast cancer cells showed that the DAL-1/4.1B protein significantly inhibits PRMT3 methylation of cellular substrates. These findings suggest that modulation of post-translational methylation may be an important mechanism through which DAL-1/4.1B affects tumor cell growth.


Asunto(s)
Proteínas de la Membrana/metabolismo , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteínas Supresoras de Tumor/metabolismo , Neoplasias de la Mama , Carcinoma de Pulmón de Células no Pequeñas , Línea Celular Tumoral , Clonación Molecular , Humanos , Neoplasias Pulmonares , Metilación , Proteínas de Microfilamentos , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/metabolismo , Saccharomyces cerevisiae/metabolismo
12.
Clin Cancer Res ; 9(12): 4435-42, 2003 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-14555516

RESUMEN

PURPOSE: Loss of heterozygosity (LOH) of alleles on chromosome 10 has been reported in many cancers, leading to the identification of tumor suppressor genes on this chromosome. Several reports implicate LOH of chromosome 10 alleles in meningioma progression, but the frequency and complexity of the loss have not been well characterized. Furthermore, the location and identity of the putative tumor suppressor genes on this chromosome that contribute to meningioma progression are unknown because the currently characterized tumor suppressor genes do not appear to be involved. Therefore, this study was undertaken to (a) assess the frequency and complexity of LOH in meningioma progression, (b) map the LOH patterns of individual meningiomas to define the smallest regions of shared chromosomal deletion, and (c) compare the identified regions with chromosome 10 deletions in other cancers, and thereby initiate the localization of the putative tumor suppressor genes. EXPERIMENTAL DESIGN: We examined 11 microsatellite dinucleotide repeat loci in 208 meningiomas of all grades using laser capture microdissection and fluorescence-based detection of PCR products. RESULTS: For all markers examined, the incidence of LOH was much higher in all grades than that previously reported, with incidence and complexity of LOH increasing with tumor grade. LOH mapping identified four regions of chromosomal deletion: 10pter-D10S89, D10S109-D10S215, D10S187-D10S209, and D10S169-10qter. These deletions on chromosome 10 are shared with other cancer types. CONCLUSIONS: These results delineate chromosomal locations of putative tumor suppressor genes on chromosome 10 that likely play an early role in meningioma tumorigenesis as well as tumor progression.


Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 10/genética , Genes Supresores de Tumor , Pérdida de Heterocigocidad , Neoplasias Meníngeas/genética , Meningioma/genética , Encéfalo/patología , ADN de Neoplasias/genética , Repeticiones de Dinucleótido , Progresión de la Enfermedad , Frecuencia de los Genes , Humanos , Rayos Láser , Linfocitos/patología , Neoplasias Meníngeas/patología , Neoplasias Meníngeas/cirugía , Meningioma/patología , Meningioma/cirugía , Repeticiones de Microsatélite , Neoplasias/genética , Reacción en Cadena de la Polimerasa
13.
Clin Cancer Res ; 9(12): 4443-51, 2003 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-14555517

RESUMEN

PURPOSE: In a study of 208 meningiomas, we found a high incidence of loss of heterozygosity (LOH) on chromosome 10 in benign (73.4%), atypical (80.0%), and malignant (86.7%) tumors. A large percentage of the benign and atypical tumors and an increasing percentage of malignant tumors had LOH on multiple loci (43.9%, 45%, and 66.7%, respectively). The high incidence of LOH occurring early in meningioma progression suggests that LOH at individual alleles may serve as a marker of clinically relevant alterations useful for patient diagnosis, the subclassification of tumors, and/or the treatment of patients. EXPERIMENTAL DESIGN: To test this, we examined 208 sporadic and recurrent meningiomas of all grades for correlations between LOH at 11 markers on chromosome 10 and tumor location, histology, and grade and patient race, gender, age, recurrence, and survival. RESULTS: Several significant correlations were found. The data indicate that genetic differences occur not only between tumors of different grade, but also between tumors of the same grade, and therefore may be useful to define genetic subsets with clinical implications. LOH at D10S179 (P = 0.001) or D10S169 (P = 0.004) is most likely present in higher-grade meningiomas and, when present in benign tumors, may signify sampling error or a morphologically benign but biologically aggressive tumor. Furthermore, LOH at D10S209 (P = 0.06) and D10S169 (P = 0.01) may predict shorter survival and/or higher rates of recurrence, respectively, in tumors with benign or malignant histology. CONCLUSIONS: We conclude that these chromosome 10 markers deserve further testing as unfavorable prognostic indicators for meningioma patients.


Asunto(s)
Cromosomas Humanos Par 10/genética , Marcadores Genéticos , Pérdida de Heterocigocidad , Neoplasias Meníngeas/genética , Meningioma/genética , Recurrencia Local de Neoplasia/genética , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Transicionales/diagnóstico , Carcinoma de Células Transicionales/genética , ADN de Neoplasias/genética , Progresión de la Enfermedad , Etnicidad/genética , Femenino , Frecuencia de los Genes , Genes Supresores de Tumor , Humanos , Linfocitos/patología , Masculino , Neoplasias Meníngeas/diagnóstico , Neoplasias Meníngeas/mortalidad , Meningioma/diagnóstico , Meningioma/mortalidad , Repeticiones de Microsatélite , Persona de Mediana Edad , Recurrencia Local de Neoplasia/diagnóstico , Estadificación de Neoplasias , Reacción en Cadena de la Polimerasa , Pronóstico , Tasa de Supervivencia
14.
Biochem Pharmacol ; 68(9): 1699-708, 2004 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-15450935

RESUMEN

The influence of p53 function and caspase 3 activity on the capacity of the antifolate, methotrexate, to promote senescence arrest and apoptotic cell death was investigated in breast tumor cells. In p53 wild-type, but caspase 3 deficient MCF-7 breast tumor cells, death of approximately 40% of the cell population was observed immediately after acute exposure to 10 microM methotrexate (the IC80 value for a 2 h drug exposure). There was no evidence of either DNA fragmentation, a sub G0 population or morphological alterations indicative of apoptosis; however, PARP cleavage was detected. Cell death was succeeded by growth arrest for at least 72 h--where arrest was characterized by expression of the senescence marker, beta-galactosidase. The response to methotrexate in MCF-7/E6 cells with attenuated p53 function was also primarily growth arrest--but lacking characteristics of senescence. In contrast, MCF-7 cells which expressed caspase 3 demonstrated a gradual and continuous loss of cell viability and unequivocal morphological evidence of apoptosis. DNA fragmentation indicative of apoptosis was also detected after exposure to methotrexate in p53 mutant MDA-MB231 breast tumor cells which also express caspase 3. Methotrexate-induced both p53 and p21waf1/cip1 in MCF-7 cells within 6 h; however, no significant DNA strand breakage was evident before 18 h, suggesting that the induction of p53 reflects a response to cellular stress other than DNA damage, such as nucleotide depletion. Overall, these studies suggest that the nature of the cellular response to methotrexate depends, in large part, on p53 and caspase function. p53 appears to be required for methotrexate-induced senescence, but not apoptosis, caspase 3 is required for DNA fragmentation and the morphological changes associated with apoptosis, while neither p53 nor caspase 3 are required for methotrexate-induced growth arrest. Furthermore, the senescence phenotype may occur in the absence of direct DNA damage.


Asunto(s)
Apoptosis/efectos de los fármacos , Caspasas/fisiología , Senescencia Celular/efectos de los fármacos , Metotrexato/farmacología , Proteína p53 Supresora de Tumor/fisiología , Apoptosis/fisiología , Neoplasias de la Mama/patología , Caspasa 3 , Senescencia Celular/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/biosíntesis , Daño del ADN/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Células Tumorales Cultivadas
15.
Circ Cardiovasc Genet ; 7(3): 335-43, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24951659

RESUMEN

BACKGROUND: Genome-wide association studies have identified thousands of genetic variants that influence a variety of diseases and health-related quantitative traits. However, the causal variants underlying the majority of genetic associations remain unknown. Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium Targeted Sequencing Study aims to follow up genome-wide association study signals and identify novel associations of the allelic spectrum of identified variants with cardiovascular-related traits. METHODS AND RESULTS: The study included 4231 participants from 3 CHARGE cohorts: the Atherosclerosis Risk in Communities Study, the Cardiovascular Health Study, and the Framingham Heart Study. We used a case-cohort design in which we selected both a random sample of participants and participants with extreme phenotypes for each of 14 traits. We sequenced and analyzed 77 genomic loci, which had previously been associated with ≥1 of 14 phenotypes. A total of 52 736 variants were characterized by sequencing and passed our stringent quality control criteria. For common variants (minor allele frequency ≥1%), we performed unweighted regression analyses to obtain P values for associations and weighted regression analyses to obtain effect estimates that accounted for the sampling design. For rare variants, we applied 2 approaches: collapsed aggregate statistics and joint analysis of variants using the sequence kernel association test. CONCLUSIONS: We sequenced 77 genomic loci in participants from 3 cohorts. We established a set of filters to identify high-quality variants and implemented statistical and bioinformatics strategies to analyze the sequence data and identify potentially functional variants within genome-wide association study loci.


Asunto(s)
Envejecimiento/genética , Estudio de Asociación del Genoma Completo , Cardiopatías/genética , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Femenino , Variación Genética , Genómica , Cardiopatías/epidemiología , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Proyectos de Investigación , Análisis de Secuencia de ADN
16.
Neuron ; 77(2): 235-42, 2013 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-23352160

RESUMEN

To characterize the role of rare complete human knockouts in autism spectrum disorders (ASDs), we identify genes with homozygous or compound heterozygous loss-of-function (LoF) variants (defined as nonsense and essential splice sites) from exome sequencing of 933 cases and 869 controls. We identify a 2-fold increase in complete knockouts of autosomal genes with low rates of LoF variation (≤ 5% frequency) in cases and estimate a 3% contribution to ASD risk by these events, confirming this observation in an independent set of 563 probands and 4,605 controls. Outside the pseudoautosomal regions on the X chromosome, we similarly observe a significant 1.5-fold increase in rare hemizygous knockouts in males, contributing to another 2% of ASDs in males. Taken together, these results provide compelling evidence that rare autosomal and X chromosome complete gene knockouts are important inherited risk factors for ASD.


Asunto(s)
Trastornos Generalizados del Desarrollo Infantil/diagnóstico , Trastornos Generalizados del Desarrollo Infantil/genética , Demografía/métodos , Eliminación de Gen , Pérdida de Heterocigocidad/genética , Estudios de Casos y Controles , Trastornos Generalizados del Desarrollo Infantil/epidemiología , Preescolar , Cromosomas Humanos X/genética , Femenino , Variación Genética/genética , Homocigoto , Humanos , Desequilibrio de Ligamiento/genética , Masculino , Factores de Riesgo
17.
Genome Biol ; 12(7): R68, 2011 Jul 25.
Artículo en Inglés | MEDLINE | ID: mdl-21787409

RESUMEN

BACKGROUND: Enrichment of loci by DNA hybridization-capture, followed by high-throughput sequencing, is an important tool in modern genetics. Currently, the most common targets for enrichment are the protein coding exons represented by the consensus coding DNA sequence (CCDS). The CCDS, however, excludes many actual or computationally predicted coding exons present in other databases, such as RefSeq and Vega, and non-coding functional elements such as untranslated and regulatory regions. The number of variants per base pair (variant density) and our ability to interrogate regions outside of the CCDS regions is consequently less well understood. RESULTS: We examine capture sequence data from outside of the CCDS regions and find that extremes of GC content that are present in different subregions of the genome can reduce the local capture sequence coverage to less than 50% relative to the CCDS. This effect is due to biases inherent in both the Illumina and SOLiD sequencing platforms that are exacerbated by the capture process. Interestingly, for two subregion types, microRNA and predicted exons, the capture process yields higher than expected coverage when compared to whole genome sequencing. Lastly, we examine the variation present in non-CCDS regions and find that predicted exons, as well as exonic regions specific to RefSeq and Vega, show much higher variant densities than the CCDS. CONCLUSIONS: We show that regions outside of the CCDS perform less efficiently in capture sequence experiments. Further, we show that the variant density in computationally predicted exons is more than 2.5-times higher than that observed in the CCDS.


Asunto(s)
Secuencia de Consenso , Exoma , Exones , Sistemas de Lectura Abierta/genética , Análisis de Secuencia de ADN , Alelos , Biología Computacional , Frecuencia de los Genes , Genoma Humano , Humanos , Intrones , Tasa de Mutación , Polimorfismo de Nucleótido Simple
18.
Sci Transl Med ; 3(87): 87re3, 2011 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-21677200

RESUMEN

Whole-genome sequencing of patient DNA can facilitate diagnosis of a disease, but its potential for guiding treatment has been under-realized. We interrogated the complete genome sequences of a 14-year-old fraternal twin pair diagnosed with dopa (3,4-dihydroxyphenylalanine)-responsive dystonia (DRD; Mendelian Inheritance in Man #128230). DRD is a genetically heterogeneous and clinically complex movement disorder that is usually treated with l-dopa, a precursor of the neurotransmitter dopamine. Whole-genome sequencing identified compound heterozygous mutations in the SPR gene encoding sepiapterin reductase. Disruption of SPR causes a decrease in tetrahydrobiopterin, a cofactor required for the hydroxylase enzymes that synthesize the neurotransmitters dopamine and serotonin. Supplementation of l-dopa therapy with 5-hydroxytryptophan, a serotonin precursor, resulted in clinical improvements in both twins.


Asunto(s)
Trastornos Distónicos , Genoma Humano , Atención al Paciente , Análisis de Secuencia de ADN , Adolescente , Toma de Decisiones , Trastornos Distónicos/tratamiento farmacológico , Trastornos Distónicos/genética , Femenino , Humanos , Levodopa/uso terapéutico , Masculino , Linaje , Resultado del Tratamiento , Gemelos Dicigóticos/genética
19.
Nat Genet ; 44(2): 165-9, 2011 Dec 25.
Artículo en Inglés | MEDLINE | ID: mdl-22197930

RESUMEN

We sequenced eight melanoma exomes to identify new somatic mutations in metastatic melanoma. Focusing on the mitogen-activated protein (MAP) kinase kinase kinase (MAP3K) family, we found that 24% of melanoma cell lines have mutations in the protein-coding regions of either MAP3K5 or MAP3K9. Structural modeling predicted that mutations in the kinase domain may affect the activity and regulation of these protein kinases. The position of the mutations and the loss of heterozygosity of MAP3K5 and MAP3K9 in 85% and 67% of melanoma samples, respectively, together suggest that the mutations are likely to be inactivating. In in vitro kinase assays, MAP3K5 I780F and MAP3K9 W333* variants had reduced kinase activity. Overexpression of MAP3K5 or MAP3K9 mutants in HEK293T cells reduced the phosphorylation of downstream MAP kinases. Attenuation of MAP3K9 function in melanoma cells using siRNA led to increased cell viability after temozolomide treatment, suggesting that decreased MAP3K pathway activity can lead to chemoresistance in melanoma.


Asunto(s)
MAP Quinasa Quinasa Quinasa 5/genética , Quinasas Quinasa Quinasa PAM/genética , Melanoma/genética , Mutación , Neoplasias Cutáneas/genética , Antineoplásicos/farmacología , Secuencia de Bases , Línea Celular Tumoral , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Exoma , Humanos , Pérdida de Heterocigocidad , Melanoma/tratamiento farmacológico , Melanoma/secundario , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/patología , Temozolomida , Células Tumorales Cultivadas
20.
Genome Biol ; 11(6): R62, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-20565776

RESUMEN

We have developed a solution-based method for targeted DNA capture-sequencing that is directed to the complete human exome. Using this approach allows the discovery of greater than 95% of all expected heterozygous singe base variants, requires as little as 3 Gbp of raw sequence data and constitutes an effective tool for identifying rare coding alleles in large scale genomic studies.


Asunto(s)
Emparejamiento Base/genética , Bases de Datos de Ácidos Nucleicos , Exones/genética , Análisis de Secuencia de ADN/métodos , Biblioteca de Genes , Haplotipos/genética , Humanos , Polimorfismo de Nucleótido Simple/genética , Reproducibilidad de los Resultados , Alineación de Secuencia , Soluciones
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