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1.
Transl Res ; 215: 31-40, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31520587

RESUMO

Precision medicine has generated diagnoses for many patients with challenging undiagnosed disorders. Some individuals remain without a diagnosis despite comprehensive testing, and this impedes their treatment. This report addresses the role of personalized medicine in identifying effective therapy for an undiagnosed disease. A 22-year-old woman presented with chronic severe recurrent trismus, facial pain, progressive multicentric inflammatory and fibrotic masses, and high C-reactive protein. Sites of disease included the pterygomaxillary region, masseter muscles, mandible, lung, pericardium, intrabdominal cavity, and retroperitoneum. A diagnosis was not established after an extensive assessment, including multiple biopsies. The patient was subsequently evaluated under the Undiagnosed Diseases Program at the National Institutes of Health. Large scale genotyping, proteomic studies, and in vitro and gene expression analyses of fibroblasts obtained from a major disease locus were performed. Germline genetic testing did not identify strong candidate genes; proteomic studies of the patient's serum and bronchoalveolar lavage fluid and gene expression analyses of her cells were consistent with dysregulation of the tumor necrosis factor-alpha pathway. The patient's cultured fibroblasts were incubated with selected drugs, and cell proliferation was inhibited by hydroxychloroquine. Treatment of the patient with hydroxychloroquine conferred prolonged beneficial clinical effects, including stabilization of trismus and reduction of corticosteroid dose, C-reactive protein, and size of masses. This case represents an example of precision medicine applied to discover effective treatments for individuals with enigmatic undiagnosed disorders.

2.
Nature ; 571(7764): 270-274, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31207604

RESUMO

Tumour-specific CD8 T cell dysfunction is a differentiation state that is distinct from the functional effector or memory T cell states1-6. Here we identify the nuclear factor TOX as a crucial regulator of the differentiation of tumour-specific T (TST) cells. We show that TOX is highly expressed in dysfunctional TST cells from tumours and in exhausted T cells during chronic viral infection. Expression of TOX is driven by chronic T cell receptor stimulation and NFAT activation. Ectopic expression of TOX in effector T cells in vitro induced a transcriptional program associated with T cell exhaustion. Conversely, deletion of Tox in TST cells in tumours abrogated the exhaustion program: Tox-deleted TST cells did not upregulate genes for inhibitory receptors (such as Pdcd1, Entpd1, Havcr2, Cd244 and Tigit), the chromatin of which remained largely inaccessible, and retained high expression of transcription factors such as TCF-1. Despite their normal, 'non-exhausted' immunophenotype, Tox-deleted TST cells remained dysfunctional, which suggests that the regulation of expression of inhibitory receptors is uncoupled from the loss of effector function. Notably, although Tox-deleted CD8 T cells differentiated normally to effector and memory states in response to acute infection, Tox-deleted TST cells failed to persist in tumours. We hypothesize that the TOX-induced exhaustion program serves to prevent the overstimulation of T cells and activation-induced cell death in settings of chronic antigen stimulation such as cancer.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Diferenciação Celular/imunologia , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Homeodomínio/metabolismo , Neoplasias/imunologia , Animais , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/deficiência , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Homeodomínio/genética , Humanos , Memória Imunológica , Linfócitos do Interstício Tumoral/citologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos do Interstício Tumoral/patologia , Camundongos , Neoplasias/patologia , Fenótipo , Receptores de Antígenos de Linfócitos T/imunologia , Transcrição Genética
3.
Nat Genet ; 51(6): 999-1010, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31110351

RESUMO

Human embryonic stem cells (ESCs) and human induced pluripotent stem cells hold great promise for cell-based therapies and drug discovery. However, homogeneous differentiation remains a major challenge, highlighting the need for understanding developmental mechanisms. We performed genome-scale CRISPR screens to uncover regulators of definitive endoderm (DE) differentiation, which unexpectedly uncovered five Jun N-terminal kinase (JNK)-JUN family genes as key barriers of DE differentiation. The JNK-JUN pathway does not act through directly inhibiting the DE enhancers. Instead, JUN co-occupies ESC enhancers with OCT4, NANOG, SMAD2 and SMAD3, and specifically inhibits the exit from the pluripotent state by impeding the decommissioning of ESC enhancers and inhibiting the reconfiguration of SMAD2 and SMAD3 chromatin binding from ESC to DE enhancers. Therefore, the JNK-JUN pathway safeguards pluripotency from precocious DE differentiation. Direct pharmacological inhibition of JNK significantly improves the efficiencies of generating DE and DE-derived pancreatic and lung progenitor cells, highlighting the potential of harnessing the knowledge from developmental studies for regenerative medicine.


Assuntos
Diferenciação Celular/genética , Endoderma/embriologia , Endoderma/metabolismo , Genoma , Genômica , Sistema de Sinalização das MAP Quinases , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Expressão Gênica , Técnicas de Inativação de Genes , Genes Reporter , Genômica/métodos , Humanos , Células-Tronco Pluripotentes Induzidas , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Modelos Biológicos , Reprodutibilidade dos Testes , Proteínas Smad
4.
MBio ; 7(2): e00235, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26980833

RESUMO

UNLABELLED: Respiratory paramyxoviruses, including the highly prevalent human parainfluenza viruses, cause the majority of childhood croup, bronchiolitis, and pneumonia, yet there are currently no vaccines or effective treatments. Paramyxovirus research has relied on the study of laboratory-adapted strains of virus in immortalized cultured cell lines. We show that findings made in such systems about the receptor interaction and viral fusion requirements for entry and fitness-mediated by the receptor binding protein and the fusion protein-can be drastically different from the requirements for infection in vivo. Here we carried out whole-genome sequencing and genomic analysis of circulating human parainfluenza virus field strains to define functional and structural properties of proteins of circulating strains and to identify the genetic basis for properties that confer fitness in the field. The analysis of clinical strains suggests that the receptor binding-fusion molecule pairs of circulating viruses maintain a balance of properties that result in an inverse correlation between fusion in cultured cells and growth in vivo. Future analysis of entry mechanisms and inhibitory strategies for paramyxoviruses will benefit from considering the properties of viruses that are fit to infect humans, since a focus on viruses that have adapted to laboratory work provides a distinctly different picture of the requirements for the entry step of infection. IMPORTANCE: Mechanistic information about viral infection-information that impacts antiviral and vaccine development-is generally derived from viral strains grown under laboratory conditions in immortalized cells. This study uses whole-genome sequencing of clinical strains of human parainfluenza virus 3-a globally important respiratory paramyxovirus-in cell systems that mimic the natural human host and in animal models. By examining the differences between clinical isolates and laboratory-adapted strains, the sequence differences are correlated to mechanistic differences in viral entry. For this ubiquitous and pathogenic respiratory virus to infect the human lung, modulation of the processes of receptor engagement and fusion activation occur in a manner quite different from that carried out by the entry glycoprotein-expressing pair of laboratory strains. These marked contrasts in the viral properties necessary for infection in cultured immortalized cells and in natural host tissues and animals will influence future basic and clinical studies.


Assuntos
Sistema Respiratório/virologia , Respirovirus/fisiologia , Internalização do Vírus , Animais , Genoma Viral , Humanos , Respirovirus/isolamento & purificação , Respirovirus/patogenicidade , Respirovirus/ultraestrutura , Infecções por Respirovirus/virologia , Análise de Sequência de DNA , Sigmodontinae , Virulência
6.
Cell Syst ; 1(1): 72-87, 2015 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-26594662

RESUMO

The panoply of microorganisms and other species present in our environment influence human health and disease, especially in cities, but have not been profiled with metagenomics at a city-wide scale. We sequenced DNA from surfaces across the entire New York City (NYC) subway system, the Gowanus Canal, and public parks. Nearly half of the DNA (48%) does not match any known organism; identified organisms spanned 1,688 bacterial, viral, archaeal, and eukaryotic taxa, which were enriched for harmless genera associated with skin (e.g., Acinetobacter). Predicted ancestry of human DNA left on subway surfaces can recapitulate U.S. Census demographic data, and bacterial signatures can reveal a station's history, such as marine-associated bacteria in a hurricane-flooded station. Some evidence of pathogens was found (Bacillus anthracis), but a lack of reported cases in NYC suggests that the pathogens represent a normal, urban microbiome. This baseline metagenomic map of NYC could help long-term disease surveillance, bioterrorism threat mitigation, and health management in the built environment of cities.

7.
Toxicol Sci ; 148(2): 460-72, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26361796

RESUMO

Formalin-fixed paraffin-embedded (FFPE) tissue samples represent a potentially invaluable resource for transcriptomic research. However, use of FFPE samples in genomic studies has been limited by technical challenges resulting from nucleic acid degradation. Here we evaluated gene expression profiles derived from fresh-frozen (FRO) and FFPE mouse liver tissues preserved in formalin for different amounts of time using 2 DNA microarray protocols and 2 whole-transcriptome sequencing (RNA-seq) library preparation methodologies. The ribo-depletion protocol outperformed the other methods by having the highest correlations of differentially expressed genes (DEGs), and best overlap of pathways, between FRO and FFPE groups. The effect of sample time in formalin (18 h or 3 weeks) on gene expression profiles indicated that test article treatment, not preservation method, was the main driver of gene expression profiles. Meta- and pathway analyses indicated that biological responses were generally consistent for 18 h and 3 week FFPE samples compared with FRO samples. However, clear erosion of signal intensity with time in formalin was evident, and DEG numbers differed by platform and preservation method. Lastly, we investigated the effect of time in paraffin on genomic profiles. Ribo-depletion RNA-seq analysis of 8-, 19-, and 26-year-old control blocks resulted in comparable quality metrics, including expected distributions of mapped reads to exonic, untranslated region, intronic, and ribosomal fractions of the transcriptome. Overall, our results indicate that FFPE samples are appropriate for use in genomic studies in which frozen samples are not available, and that ribo-depletion RNA-seq is the preferred method for this type of analysis in archival and long-aged FFPE samples.


Assuntos
Fixadores , Formaldeído , Secções Congeladas , Furanos/toxicidade , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Inclusão em Parafina , RNA Mensageiro/genética , Fixação de Tecidos/métodos , Animais , Biologia Computacional , Bases de Dados Genéticas , Feminino , Redes Reguladoras de Genes , Fígado/metabolismo , Camundongos , Estabilidade de RNA , Ratos , Reprodutibilidade dos Testes , Análise de Sequência de DNA , Análise de Sequência de RNA , Fatores de Tempo
10.
Nat Biotechnol ; 32(9): 915-925, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25150835

RESUMO

High-throughput RNA sequencing (RNA-seq) greatly expands the potential for genomics discoveries, but the wide variety of platforms, protocols and performance capabilitites has created the need for comprehensive reference data. Here we describe the Association of Biomolecular Resource Facilities next-generation sequencing (ABRF-NGS) study on RNA-seq. We carried out replicate experiments across 15 laboratory sites using reference RNA standards to test four protocols (poly-A-selected, ribo-depleted, size-selected and degraded) on five sequencing platforms (Illumina HiSeq, Life Technologies PGM and Proton, Pacific Biosciences RS and Roche 454). The results show high intraplatform (Spearman rank R > 0.86) and inter-platform (R > 0.83) concordance for expression measures across the deep-count platforms, but highly variable efficiency and cost for splice junction and variant detection between all platforms. For intact RNA, gene expression profiles from rRNA-depletion and poly-A enrichment are similar. In addition, rRNA depletion enables effective analysis of degraded RNA samples. This study provides a broad foundation for cross-platform standardization, evaluation and improvement of RNA-seq.


Assuntos
Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Transcriptoma
11.
Nat Biotechnol ; 32(9): 888-95, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25150837

RESUMO

High-throughput RNA sequencing (RNA-seq) enables comprehensive scans of entire transcriptomes, but best practices for analyzing RNA-seq data have not been fully defined, particularly for data collected with multiple sequencing platforms or at multiple sites. Here we used standardized RNA samples with built-in controls to examine sources of error in large-scale RNA-seq studies and their impact on the detection of differentially expressed genes (DEGs). Analysis of variations in guanine-cytosine content, gene coverage, sequencing error rate and insert size allowed identification of decreased reproducibility across sites. Moreover, commonly used methods for normalization (cqn, EDASeq, RUV2, sva, PEER) varied in their ability to remove these systematic biases, depending on sample complexity and initial data quality. Normalization methods that combine data from genes across sites are strongly recommended to identify and remove site-specific effects and can substantially improve RNA-seq studies.


Assuntos
Análise de Sequência de RNA/métodos , Controle de Qualidade , Reprodutibilidade dos Testes
12.
Genome Biol ; 14(9): R95, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24020486

RESUMO

A large number of computational methods have been developed for analyzing differential gene expression in RNA-seq data. We describe a comprehensive evaluation of common methods using the SEQC benchmark dataset and ENCODE data. We consider a number of key features, including normalization, accuracy of differential expression detection and differential expression analysis when one condition has no detectable expression. We find significant differences among the methods, but note that array-based methods adapted to RNA-seq data perform comparably to methods designed for RNA-seq. Our results demonstrate that increasing the number of replicate samples significantly improves detection power over increased sequencing depth.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/estatística & dados numéricos , Proteínas do Tecido Nervoso/genética , RNA/genética , Análise de Sequência de RNA/estatística & dados numéricos , Software , Química Encefálica , Linhagem Celular , Conjuntos de Dados como Assunto , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Sequência de RNA/métodos , Razão Sinal-Ruído
13.
Cell Rep ; 4(3): 578-88, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23911289

RESUMO

The BCL6 transcriptional repressor is required for the development of germinal center (GC) B cells and diffuse large B cell lymphomas (DLBCLs). Although BCL6 can recruit multiple corepressors, its transcriptional repression mechanism of action in normal and malignant B cells is unknown. We find that in B cells, BCL6 mostly functions through two independent mechanisms that are collectively essential to GC formation and DLBCL, both mediated through its N-terminal BTB domain. These are (1) the formation of a unique ternary BCOR-SMRT complex at promoters, with each corepressor binding to symmetrical sites on BCL6 homodimers linked to specific epigenetic chromatin features, and (2) the "toggling" of active enhancers to a poised but not erased conformation through SMRT-dependent H3K27 deacetylation, which is mediated by HDAC3 and opposed by p300 histone acetyltransferase. Dynamic toggling of enhancers provides a basis for B cells to undergo rapid transcriptional and phenotypic changes in response to signaling or environmental cues.


Assuntos
Linfócitos B/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Animais , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Xenoenxertos , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Camundongos , Modelos Moleculares , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-bcl-6 , Transdução de Sinais
14.
BMC Bioinformatics ; 14 Suppl 5: S10, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23735126

RESUMO

BACKGROUND: DNA methylation profiling reveals important differentially methylated regions (DMRs) of the genome that are altered during development or that are perturbed by disease. To date, few programs exist for regional analysis of enriched or whole-genome bisulfate conversion sequencing data, even though such data are increasingly common. Here, we describe an open-source, optimized method for determining empirically based DMRs (eDMR) from high-throughput sequence data that is applicable to enriched whole-genome methylation profiling datasets, as well as other globally enriched epigenetic modification data. RESULTS: Here we show that our bimodal distribution model and weighted cost function for optimized regional methylation analysis provides accurate boundaries of regions harboring significant epigenetic modifications. Our algorithm takes the spatial distribution of CpGs into account for the enrichment assay, allowing for optimization of the definition of empirical regions for differential methylation. Combined with the dependent adjustment for regional p-value combination and DMR annotation, we provide a method that may be applied to a variety of datasets for rapid DMR analysis. Our method classifies both the directionality of DMRs and their genome-wide distribution, and we have observed that shows clinical relevance through correct stratification of two Acute Myeloid Leukemia (AML) tumor sub-types. CONCLUSIONS: Our weighted optimization algorithm eDMR for calling DMRs extends an established DMR R pipeline (methylKit) and provides a needed resource in epigenomics. Our method enables an accurate and scalable way of finding DMRs in high-throughput methylation sequencing experiments. eDMR is available for download at http://code.google.com/p/edmr/.


Assuntos
Algoritmos , Metilação de DNA , Anotação de Sequência Molecular/métodos , Ilhas de CpG , Epigenômica/métodos , Genoma , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Leucemia/genética , Análise de Sequência de DNA
15.
Nat Genet ; 45(3): 290-4, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23377183

RESUMO

Relapsed childhood acute lymphoblastic leukemia (ALL) carries a poor prognosis, despite intensive retreatment, owing to intrinsic drug resistance. The biological pathways that mediate resistance are unknown. Here, we report the transcriptome profiles of matched diagnosis and relapse bone marrow specimens from ten individuals with pediatric B-lymphoblastic leukemia using RNA sequencing. Transcriptome sequencing identified 20 newly acquired, novel nonsynonymous mutations not present at initial diagnosis, with 2 individuals harboring relapse-specific mutations in the same gene, NT5C2, encoding a 5'-nucleotidase. Full-exon sequencing of NT5C2 was completed in 61 further relapse specimens, identifying additional mutations in 5 cases. Enzymatic analysis of mutant proteins showed that base substitutions conferred increased enzymatic activity and resistance to treatment with nucleoside analog therapies. Clinically, all individuals who harbored NT5C2 mutations relapsed early, within 36 months of initial diagnosis (P = 0.03). These results suggest that mutations in NT5C2 are associated with the outgrowth of drug-resistant clones in ALL.


Assuntos
5'-Nucleotidase/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , 5'-Nucleotidase/metabolismo , Sequência de Bases , Criança , Resistencia a Medicamentos Antineoplásicos , Éxons , Humanos , Dados de Sequência Molecular , Mutação , Conformação Proteica , Recidiva
16.
Nucleic Acids Res ; 41(Database issue): D906-14, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23203872

RESUMO

RNA-based next-generation sequencing (RNA-Seq) provides a tremendous amount of new information regarding gene and transcript structure, expression and regulation. This is particularly true for non-coding RNAs where whole transcriptome analyses have revealed that the much of the genome is transcribed and that many non-coding transcripts have widespread functionality. However, uniform resources for raw, cleaned and processed RNA-Seq data are sparse for most organisms and this is especially true for non-human primates (NHPs). Here, we describe a large-scale RNA-Seq data and analysis infrastructure, the NHP reference transcriptome resource (http://nhprtr.org); it presently hosts data from12 species of primates, to be expanded to 15 species/subspecies spanning great apes, old world monkeys, new world monkeys and prosimians. Data are collected for each species using pools of RNA from comparable tissues. We provide data access in advance of its deposition at NCBI, as well as browsable tracks of alignments against the human genome using the UCSC genome browser. This resource will continue to host additional RNA-Seq data, alignments and assemblies as they are generated over the coming years and provide a key resource for the annotation of NHP genomes as well as informing primate studies on evolution, reproduction, infection, immunity and pharmacology.


Assuntos
Bases de Dados de Ácidos Nucleicos , Genômica , Primatas/genética , Transcriptoma , Animais , Genoma Humano , Humanos , Internet , Primatas/metabolismo , Alinhamento de Sequência , Análise de Sequência de RNA
17.
Cell ; 149(7): 1635-46, 2012 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-22608085

RESUMO

Methylation of the N(6) position of adenosine (m(6)A) is a posttranscriptional modification of RNA with poorly understood prevalence and physiological relevance. The recent discovery that FTO, an obesity risk gene, encodes an m(6)A demethylase implicates m(6)A as an important regulator of physiological processes. Here, we present a method for transcriptome-wide m(6)A localization, which combines m(6)A-specific methylated RNA immunoprecipitation with next-generation sequencing (MeRIP-Seq). We use this method to identify mRNAs of 7,676 mammalian genes that contain m(6)A, indicating that m(6)A is a common base modification of mRNA. The m(6)A modification exhibits tissue-specific regulation and is markedly increased throughout brain development. We find that m(6)A sites are enriched near stop codons and in 3' UTRs, and we uncover an association between m(6)A residues and microRNA-binding sites within 3' UTRs. These findings provide a resource for identifying transcripts that are substrates for adenosine methylation and reveal insights into the epigenetic regulation of the mammalian transcriptome.


Assuntos
Regiões 3' não Traduzidas , Códon de Terminação , Processamento Pós-Transcricional do RNA , Transcriptoma , Adenosina/metabolismo , Metilação , RNA Mensageiro/metabolismo , RNA não Traduzido/metabolismo
18.
Adv Exp Med Biol ; 680: 693-700, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20865556

RESUMO

Next Generation Sequencing technologies are limited by the lack of standard bioinformatics infrastructures that can reduce data storage, increase data processing performance, and integrate diverse information. HDF technologies address these requirements and have a long history of use in data-intensive science communities. They include general data file formats, libraries, and tools for working with the data. Compared to emerging standards, such as the SAM/BAM formats, HDF5-based systems demonstrate significantly better scalability, can support multiple indexes, store multiple data types, and are self-describing. For these reasons, HDF5 and its BioHDF extension are well suited for implementing data models to support the next generation of bioinformatics applications.


Assuntos
Alinhamento de Sequência/estatística & dados numéricos , Análise de Sequência/estatística & dados numéricos , Biologia Computacional , Simulação por Computador , Sistemas de Gerenciamento de Base de Dados , Bases de Dados Genéticas , Alinhamento de Sequência/normas , Alinhamento de Sequência/tendências , Análise de Sequência/normas , Análise de Sequência/tendências , Software/normas , Software/tendências , Desenho de Programas de Computador , Interface Usuário-Computador
19.
Genome Res ; 20(2): 180-9, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20009012

RESUMO

Comparative studies of gene regulation suggest an important role for natural selection in shaping gene expression patterns within and between species. Most of these studies, however, estimated gene expression levels using microarray probes designed to hybridize to only a small proportion of each gene. Here, we used recently developed RNA sequencing protocols, which sidestep this limitation, to assess intra- and interspecies variation in gene regulatory processes in considerably more detail than was previously possible. Specifically, we used RNA-seq to study transcript levels in humans, chimpanzees, and rhesus macaques, using liver RNA samples from three males and three females from each species. Our approach allowed us to identify a large number of genes whose expression levels likely evolve under natural selection in primates. These include a subset of genes with conserved sexually dimorphic expression patterns across the three species, which we found to be enriched for genes involved in lipid metabolism. Our data also suggest that while alternative splicing is tightly regulated within and between species, sex-specific and lineage-specific changes in the expression of different splice forms are also frequent. Intriguingly, among genes in which a change in exon usage occurred exclusively in the human lineage, we found an enrichment of genes involved in anatomical structure and morphogenesis, raising the possibility that differences in the regulation of alternative splicing have been an important force in human evolution.


Assuntos
Processamento Alternativo/genética , Genoma Humano , Macaca mulatta/genética , Pan troglodytes/genética , Seleção Genética , Adulto , Animais , Éxons/genética , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Fígado/metabolismo , Masculino , RNA Mensageiro/genética , Fatores Sexuais , Especificidade da Espécie
20.
Proc Natl Acad Sci U S A ; 106(45): 19096-101, 2009 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-19861545

RESUMO

Protein coding genes constitute only approximately 1% of the human genome but harbor 85% of the mutations with large effects on disease-related traits. Therefore, efficient strategies for selectively sequencing complete coding regions (i.e., "whole exome") have the potential to contribute to the understanding of rare and common human diseases. Here we report a method for whole-exome sequencing coupling Roche/NimbleGen whole exome arrays to the Illumina DNA sequencing platform. We demonstrate the ability to capture approximately 95% of the targeted coding sequences with high sensitivity and specificity for detection of homozygous and heterozygous variants. We illustrate the utility of this approach by making an unanticipated genetic diagnosis of congenital chloride diarrhea in a patient referred with a suspected diagnosis of Bartter syndrome, a renal salt-wasting disease. The molecular diagnosis was based on the finding of a homozygous missense D652N mutation at a position in SLC26A3 (the known congenital chloride diarrhea locus) that is virtually completely conserved in orthologues and paralogues from invertebrates to humans, and clinical follow-up confirmed the diagnosis. To our knowledge, whole-exome (or genome) sequencing has not previously been used to make a genetic diagnosis. Five additional patients suspected to have Bartter syndrome but who did not have mutations in known genes for this disease had homozygous deleterious mutations in SLC26A3. These results demonstrate the clinical utility of whole-exome sequencing and have implications for disease gene discovery and clinical diagnosis.


Assuntos
Algoritmos , Doenças Genéticas Inatas/genética , Técnicas de Diagnóstico Molecular/métodos , Fases de Leitura Aberta/genética , Análise de Sequência de DNA/métodos , Antiporters/genética , Sequência de Bases , Antiportadores de Cloreto-Bicarbonato , Cloretos , Biologia Computacional , Diarreia/genética , Genômica/métodos , Humanos , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genética , Transportadores de Sulfato
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