Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 14 de 14
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
2.
Arthritis Rheumatol ; 69(4): 774-784, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28002888

RESUMO

OBJECTIVE: Ankylosing spondylitis (AS), a chronic inflammatory disorder, has a notable association with HLA-B27. One hypothesis suggests that a common antigen that binds to HLA-B27 is important for AS disease pathogenesis. This study was undertaken to determine sequences and motifs that are shared among HLA-B27-positive AS patients, using T cell repertoire next-generation sequencing. METHODS: To identify motifs enriched among B27-positive AS patients, we performed T cell receptor ß (TCRß) repertoire sequencing on samples from 191 B27-positive AS patients, 43 B27-negative AS patients, and 227 controls, and we obtained >77 million TCRß clonotype sequences. First, we assessed whether any of 50 previously published sequences were enriched in B27-positive AS patients. We then used training and test cohorts to identify discovered motifs that were enriched in B27-positive AS patients versus controls. RESULTS: Six previously published and 11 discovered motifs were enriched in the B27-positive AS samples as compared to controls. After combining motifs related by sequence, we identified a total of 15 independent motifs. Both the full set of 15 motifs and a set of 6 published motifs were enriched in the B27-positive AS patients as compared to B27-positive healthy individuals (P = 0.049 and P = 0.001, respectively). Using an independent cohort, we validated that at least some of these motifs were associated with AS, and not simply with B27-positive status. CONCLUSION: We identified TCRß motifs that are enriched in B27-positive AS patients as compared to B27-positive healthy controls. This suggests that a common antigen, presented by HLA-B27 and detected by CD8+ T cells, may be associated with AS disease pathogenesis.


Assuntos
Antígeno HLA-B27/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Espondilite Anquilosante/imunologia , Adolescente , Adulto , Motivos de Aminoácidos , Sequência de Aminoácidos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência , Adulto Jovem
3.
PLoS One ; 10(10): e0141561, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26509579

RESUMO

Monitoring antigen-specific T cells is critical for the study of immune responses and development of biomarkers and immunotherapeutics. We developed a novel multiplex assay that combines conventional immune monitoring techniques and immune receptor repertoire sequencing to enable identification of T cells specific to large numbers of antigens simultaneously. We multiplexed 30 different antigens and identified 427 antigen-specific clonotypes from 5 individuals with frequencies as low as 1 per million T cells. The clonotypes identified were validated several ways including repeatability, concordance with published clonotypes, and high correlation with ELISPOT. Applying this technology we have shown that the vast majority of shared antigen-specific clonotypes identified in different individuals display the same specificity. We also showed that shared antigen-specific clonotypes are simpler sequences and are present at higher frequencies compared to non-shared clonotypes specific to the same antigen. In conclusion this technology enables sensitive and quantitative monitoring of T cells specific for hundreds or thousands of antigens simultaneously allowing the study of T cell responses with an unprecedented resolution and scale.


Assuntos
ELISPOT , Epitopos de Linfócito T/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Receptores de Antígenos de Linfócitos T/genética , Receptores Imunológicos/genética , Especificidade do Receptor de Antígeno de Linfócitos T/genética , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Evolução Clonal/genética , Evolução Clonal/imunologia , ELISPOT/métodos , ELISPOT/normas , Humanos , Reprodutibilidade dos Testes
4.
Lancet Oncol ; 16(5): 541-9, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25842160

RESUMO

BACKGROUND: Diffuse large-B-cell lymphoma is curable, but when treatment fails, outcome is poor. Although imaging can help to identify patients at risk of treatment failure, they are often imprecise, and radiation exposure is a potential health risk. We aimed to assess whether circulating tumour DNA encoding the clonal immunoglobulin gene sequence could be detected in the serum of patients with diffuse large-B-cell lymphoma and used to predict clinical disease recurrence after frontline treatment. METHODS: We used next-generation DNA sequencing to retrospectively analyse cell-free circulating tumour DNA in patients assigned to one of three treatment protocols between May 8, 1993, and June 6, 2013. Eligible patients had diffuse large-B-cell lymphoma, no evidence of indolent lymphoma, and were previously untreated. We obtained serial serum samples and concurrent CT scans at specified times during most treatment cycles and up to 5 years of follow-up. VDJ gene segments of the rearranged immunoglobulin receptor genes were amplified and sequenced from pretreatment specimens and serum circulating tumour DNA encoding the VDJ rearrangements was quantitated. FINDINGS: Tumour clonotypes were identified in pretreatment specimens from 126 patients who were followed up for a median of 11 years (IQR 6·8-14·2). Interim monitoring of circulating tumour DNA at the end of two treatment cycles in 108 patients showed a 5-year time to progression of 41·7% (95% CI 22·2-60·1) in patients with detectable circulating tumour DNA and 80·2% (69·6-87·3) in those without detectable circulating tumour DNA (p<0·0001). Detectable interim circulating tumour DNA had a positive predictive value of 62·5% (95% CI 40·6-81·2) and a negative predictive value of 79·8% (69·6-87·8). Surveillance monitoring of circulating tumour DNA was done in 107 patients who achieved complete remission. A Cox proportional hazards model showed that the hazard ratio for clinical disease progression was 228 (95% CI 51-1022) for patients who developed detectable circulating tumour DNA during surveillance compared with patients with undetectable circulating tumour DNA (p<0·0001). Surveillance circulating tumour DNA had a positive predictive value of 88·2% (95% CI 63·6-98·5) and a negative predictive value of 97·8% (92·2-99·7) and identified risk of recurrence at a median of 3·5 months (range 0-200) before evidence of clinical disease. INTERPRETATION: Surveillance circulating tumour DNA identifies patients at risk of recurrence before clinical evidence of disease in most patients and results in a reduced disease burden at relapse. Interim circulating tumour DNA is a promising biomarker to identify patients at high risk of treatment failure. FUNDING: National Cancer Institute and Adaptive Biotechnologies.


Assuntos
DNA de Neoplasias/genética , Sequenciamento de Nucleotídeos em Larga Escala , Linfoma Difuso de Grandes Células B/diagnóstico por imagem , Linfoma Difuso de Grandes Células B/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Criança , DNA de Neoplasias/isolamento & purificação , Feminino , Humanos , Linfoma Difuso de Grandes Células B/patologia , Masculino , Pessoa de Meia-Idade , Células Neoplásicas Circulantes , Tomografia Computadorizada por Raios X
5.
Biol Blood Marrow Transplant ; 20(9): 1307-13, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24769317

RESUMO

Minimal residual disease (MRD) quantification is an important predictor of outcome after treatment for acute lymphoblastic leukemia (ALL). Bone marrow ALL burden ≥ 10(-4) after induction predicts subsequent relapse. Likewise, MRD ≥ 10(-4) in bone marrow before initiation of conditioning for allogeneic (allo) hematopoietic cell transplantation (HCT) predicts transplantation failure. Current methods for MRD quantification in ALL are not sufficiently sensitive for use with peripheral blood specimens and have not been broadly implemented in the management of adults with ALL. Consensus-primed immunoglobulin (Ig), T cell receptor (TCR) amplification and high-throughput sequencing (HTS) permit use of a standardized algorithm for all patients and can detect leukemia at 10(-6) or lower. We applied the LymphoSIGHT HTS platform (Sequenta Inc., South San Francisco, CA) to quantification of MRD in 237 samples from 29 adult B cell ALL patients before and after allo-HCT. Using primers for the IGH-VDJ, IGH-DJ, IGK, TCRB, TCRD, and TCRG loci, MRD could be quantified in 93% of patients. Leukemia-associated clonotypes at these loci were identified in 52%, 28%, 10%, 35%, 28%, and 41% of patients, respectively. MRD ≥ 10(-4) before HCT conditioning predicted post-HCT relapse (hazard ratio [HR], 7.7; 95% confidence interval [CI], 2.0 to 30; P = .003). In post-HCT blood samples, MRD ≥10(-6) had 100% positive predictive value for relapse with median lead time of 89 days (HR, 14; 95% CI, 4.7 to 44, P < .0001). The use of HTS-based MRD quantification in adults with ALL offers a standardized approach with sufficient sensitivity to quantify leukemia MRD in peripheral blood. Use of this approach may identify a window for clinical intervention before overt relapse.


Assuntos
Genes Codificadores dos Receptores de Linfócitos T/genética , Transplante de Células-Tronco Hematopoéticas/métodos , Imunoglobulinas/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Condicionamento Pré-Transplante/métodos , Transplante Homólogo/métodos , Adolescente , Adulto , Idoso , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Neoplasia Residual , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Prognóstico , Estudos Retrospectivos , Análise de Sobrevida , Adulto Jovem
6.
PLoS One ; 8(9): e74231, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24069285

RESUMO

In this study, we combined a novel sequencing method, which can identify individual clonotypes based on their unique T cell receptor (TCR) rearrangement, with existing immune assays to characterize antigen-specific T cell responses. We validated this approach using three types of assays routinely used to measure antigen-specific responses: pentamers which enable identification of T cells bearing specific TCRs, activation marker expression following antigen stimulation and antigen-induced proliferation to identify cytomegalovirus (CMV) specific clonotypes. In one individual, 8 clonotypes were identified using a pentamer reagent derived from the CMV pp65 protein. The same 8 clonotypes were also identified following sequencing of cells that upregulated an activation marker following incubation with an identical peptide derived from pp65. These 8 and an additional 8 clonotypes were identified using a more sensitive CFSE-based proliferation assay. We found clear sequence homology among some of the clonotypes identified, and the CDR3 region in one clonotype was identical to a previously published pp65-specific clonotype sequence. Many of these CMV-specific clonotypes were present at frequencies below 10(-5) which are undetectable using standard flow-cytometric methods. These studies suggest that an immune response is comprised of a diverse set of clones, many of which are present at very low frequencies. Thus, the combination of immune assays and sequencing depicts the richness and diversity of an immune response at a level that is not possible using standard immune assays alone. The methods articulated in this work provide an enhanced understanding of T cell-mediated immune responses at the clonal level.


Assuntos
Epitopos de Linfócito T/imunologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Imunoensaio/métodos , Subpopulações de Linfócitos T/imunologia , Sequência de Aminoácidos , Antígenos/imunologia , Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/metabolismo , Citomegalovirus/imunologia , Citometria de Fluxo/métodos , Humanos , Imunofenotipagem , Ativação Linfocitária/imunologia , Fosfoproteínas/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Subpopulações de Linfócitos T/metabolismo , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Proteínas da Matriz Viral/imunologia
7.
Blood ; 120(26): 5173-80, 2012 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-23074282

RESUMO

The persistence of minimal residual disease (MRD) during therapy is the strongest adverse prognostic factor in acute lymphoblastic leukemia (ALL). We developed a high-throughput sequencing method that universally amplifies antigen-receptor gene segments and identifies all clonal gene rearrangements (ie, leukemia-specific sequences) at diagnosis, allowing monitoring of disease progression and clonal evolution during therapy. In the present study, the assay specifically detected 1 leukemic cell among greater than 1 million leukocytes in spike-in experiments. We compared this method with the gold-standard MRD assays multiparameter flow cytometry and allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) using diagnostic and follow-up samples from 106 patients with ALL. Sequencing detected MRD in all 28 samples shown to be positive by flow cytometry and in 35 of the 36 shown to be positive by ASO-PCR and revealed MRD in 10 and 3 additional samples that were negative by flow cytometry and ASO-PCR, respectively. We conclude that this new method allows monitoring of treatment response in ALL and other lymphoid malignancies with great sensitivity and precision. The www.clinicaltrials.gov identifier number for the Total XV study is NCT00137111.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Sequência de Bases , Criança , Evolução Clonal/genética , Evolução Clonal/fisiologia , Genes de Cadeia Pesada de Imunoglobulina/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Modelos Biológicos , Técnicas de Diagnóstico Molecular/métodos , Dados de Sequência Molecular , Neoplasia Residual , Reação em Cadeia da Polimerase/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Prognóstico , Sensibilidade e Especificidade
8.
Nature ; 466(7308): 869-73, 2010 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-20668451

RESUMO

The systematic characterization of somatic mutations in cancer genomes is essential for understanding the disease and for developing targeted therapeutics. Here we report the identification of 2,576 somatic mutations across approximately 1,800 megabases of DNA representing 1,507 coding genes from 441 tumours comprising breast, lung, ovarian and prostate cancer types and subtypes. We found that mutation rates and the sets of mutated genes varied substantially across tumour types and subtypes. Statistical analysis identified 77 significantly mutated genes including protein kinases, G-protein-coupled receptors such as GRM8, BAI3, AGTRL1 (also called APLNR) and LPHN3, and other druggable targets. Integrated analysis of somatic mutations and copy number alterations identified another 35 significantly altered genes including GNAS, indicating an expanded role for galpha subunits in multiple cancer types. Furthermore, our experimental analyses demonstrate the functional roles of mutant GNAO1 (a Galpha subunit) and mutant MAP2K4 (a member of the JNK signalling pathway) in oncogenesis. Our study provides an overview of the mutational spectra across major human cancers and identifies several potential therapeutic targets.


Assuntos
Genes Neoplásicos/genética , Mutação/genética , Neoplasias/genética , Neoplasias/metabolismo , Transdução de Sinais/genética , Neoplasias da Mama/classificação , Neoplasias da Mama/genética , Variações do Número de Cópias de DNA/genética , Análise Mutacional de DNA , Feminino , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Humanos , Neoplasias Pulmonares/classificação , Neoplasias Pulmonares/genética , MAP Quinase Quinase 4/genética , Masculino , Neoplasias/enzimologia , Neoplasias/patologia , Neoplasias Ovarianas/classificação , Neoplasias Ovarianas/genética , Neoplasias da Próstata/classificação , Neoplasias da Próstata/genética , Proteínas Quinases/genética , Receptores Acoplados a Proteínas-G/genética
9.
Proc Natl Acad Sci U S A ; 106(16): 6712-7, 2009 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-19342489

RESUMO

Although genomewide association studies have successfully identified associations of many common single-nucleotide polymorphisms (SNPs) with common diseases, the SNPs implicated so far account for only a small proportion of the genetic variability of tested diseases. It has been suggested that common diseases may often be caused by rare alleles missed by genomewide association studies. To identify these rare alleles we need high-throughput, high-accuracy resequencing technologies. Although array-based genotyping has allowed genomewide association studies of common SNPs in tens of thousands of samples, array-based resequencing has been limited for 2 main reasons: the lack of a fully multiplexed pipeline for high-throughput sample processing, and failure to achieve sufficient performance. We have recently solved both of these problems and created a fully multiplexed high-throughput pipeline that results in high-quality data. The pipeline consists of target amplification from genomic DNA, followed by allele enrichment to generate pools of purified variant (or nonvariant) DNA and ends with interrogation of purified DNA on resequencing arrays. We have used this pipeline to resequence approximately 5 Mb of DNA (on 3 arrays) corresponding to the exons of 1,500 genes in >473 samples; in total >2,350 Mb were sequenced. In the context of this large-scale study we obtained a false positive rate of approximately 1 in 500,000 bp and a false negative rate of approximately 10%.


Assuntos
Análise de Sequência com Séries de Oligonucleotídeos , Análise de Sequência de DNA/métodos , Alelos , Automação , Pareamento Incorreto de Bases , Genoma Humano/genética , Humanos , Mutação/genética , Curva ROC , Análise de Sequência de DNA/normas
10.
Proc Natl Acad Sci U S A ; 105(27): 9296-301, 2008 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-18599465

RESUMO

We developed a robust and reproducible methodology to amplify human sequences in parallel for use in downstream multiplexed sequence analyses. We call the methodology SMART (Spacer Multiplex Amplification Reaction), and it is based, in part, on padlock probe technology. As a proof of principle, we used SMART technology to simultaneously amplify 485 human exons ranging from 100 to 500 bp from human genomic DNA. In multiple repetitions, >90% of the targets were successfully amplified with a high degree of uniformity, with 70% of targets falling within a 10-fold range and all products falling within a 100-fold range of each other in abundance. We used long padlock probes (LPPs) >300 bases in length for the assay, and the increased length of these probes allowed for the capture of human sequences up to 500 bp in length, which is optimal for capturing most human exons. To engineer the LPPs, we developed a method that generates ssDNA molecules with precise ends, using an appropriately designed dsDNA template. The template has appropriate restriction sites engineered into it that can be digested to generate nucleotide overhangs that are suitable for lambda exonuclease digestion, producing a single-stranded probe from dsDNA. The SMART technology is flexible and can be easily adapted to multiplex tens of thousands of target sequences in a single reaction.


Assuntos
Sequência de Bases/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Sondas de DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Éxons/genética , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase
11.
Hum Mutat ; 29(3): 441-50, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18186519

RESUMO

Mismatch repair detection (MRD) was used to screen 93 matched tumor-normal sample pairs and 22 cell lines for somatic mutations in 30 cancer relevant genes. Using a starting amount of only 150 ng of genomic DNA, we screened 102 kb of sequence for somatic mutations in colon and breast cancer. A total of 152 somatic mutations were discovered, encompassing previously reported mutations, such as BRAF V600E and KRAS G12S, G12V, and G13D, as well as novel mutations, including some in genes in which somatic mutations have not previously been reported, such as MAP2K1 and MAP2K2. The distribution of mutations ranged widely within and across tumor types. The functional significance of many of these mutations is not understood, with patterns of selection only evident in KRAS and BRAF in colon cancer. These results present a novel approach to high-throughput mutation screening using small amounts of starting material and reveal a mutation spectrum across 30 genes in a large cohort of breast and colorectal cancers.


Assuntos
Neoplasias da Mama/genética , Neoplasias Colorretais/genética , Reparo de Erro de Pareamento de DNA , Análise Mutacional de DNA/métodos , Mutação , Sequência de Bases , Linhagem Celular Tumoral , DNA de Neoplasias/genética , Feminino , Humanos , Masculino
12.
Nat Methods ; 4(9): 713-5, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17704782

RESUMO

The discovery of somatic mutations in cancer tissue is extremely laborious, time-consuming and costly. In an evaluation comparing mismatch repair detection (MRD) against Sanger sequencing for somatic-mutation detection, we found that MRD had a specificity of 96% and a sensitivity of 92%. Our results showed that MRD is a robust and cost-effective alternative to Sanger sequencing for identifying somatic mutations in human tumors.


Assuntos
Pareamento Incorreto de Bases/genética , DNA de Neoplasias/genética , Escherichia coli/genética , Mutação , Neoplasias/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Clonagem Molecular , Humanos , Neoplasias Pulmonares/genética , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade
13.
Proc Natl Acad Sci U S A ; 102(41): 14717-22, 2005 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-16203980

RESUMO

Identification of the genetic basis of common disease may require comprehensive sequence analysis of coding regions and regulatory elements in patients and controls to find genetic effects caused by rare or heterogeneous mutations. In this study, we demonstrate how mismatch repair detection on tag arrays can be applied in a case-control study. Mismatch repair detection allows >1,000 amplicons to be screened for variations in a single laboratory reaction. Variation scanning in 939 amplicons, mostly in coding regions within a linkage peak, was done for 372 patients and 404 controls. In total, >180 Mb of DNA was scanned. Several variants more prevalent in patients than in controls were identified. This study demonstrates an approach to the discovery of susceptibility genes for common disease: large-scale direct sequence comparison between patients and controls. We believe this approach can be scaled up to allow sequence comparison in the whole-genome coding regions among large sets of cases and controls at a reasonable cost in the near future.


Assuntos
Transtorno Autístico/genética , Pareamento Incorreto de Bases/genética , Cromossomos Humanos Par 2/genética , Predisposição Genética para Doença/genética , Testes Genéticos/métodos , Análise por Conglomerados , Éxons/genética , Humanos , Mutação/genética
14.
Genome Res ; 14(7): 1404-12, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15231755

RESUMO

A targeted discovery effort is required to identify low frequency single nucleotide polymorphisms (SNPs) in human coding and regulatory regions. We here describe combining mismatch repair detection (MRD) with dideoxy terminator sequencing to detect SNPs in pooled DNA samples. MRD enriches for variant alleles in the pooled sample, and sequencing determines the nature of the variants. By using a genomic DNA pool as a template, approximately 100 fragments were amplified and subsequently combined and subjected en masse to the MRD procedure. The variant-enriched pool from this one MRD reaction is enriched for the population variants of all the tested fragments. Each fragment was amplified from the variant-enriched pool and sequenced, allowing the discovery of alleles with frequencies as low as 1% in the initial population. Our results support that MRD-based SNP discovery can be used for large-scale discovery of SNPs at low frequencies in a population.


Assuntos
Pareamento Incorreto de Bases/genética , Reparo do DNA/genética , Polimorfismo de Nucleotídeo Único/genética , Animais , Mapeamento Cromossômico/métodos , Cromossomos Humanos Par 21/genética , Clonagem Molecular/métodos , Reações Falso-Negativas , Reações Falso-Positivas , Frequência do Gene/genética , Genes BRCA1 , Genes BRCA2 , Variação Genética/genética , Genética Populacional , Humanos , Células Híbridas , Camundongos , Técnicas de Amplificação de Ácido Nucleico/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA