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1.
J Clin Invest ; 129(8): 3185-3200, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31264971

RESUMO

T follicular helper cells (Tfh), a subset of CD4+ T cells, provide requisite help to B cells in the germinal centers (GC) of lymphoid tissue. GC Tfh are identified by high expression of the chemokine receptor CXCR5 and the inhibitory molecule PD-1. Although more accessible, blood contains lower frequencies of CXCR5+ and PD-1+ cells that have been termed circulating Tfh (cTfh). However, it remains unclear whether GC Tfh exit lymphoid tissues and populate this cTfh pool. To examine exiting cells, we assessed the phenotype of Tfh present within the major conduit of efferent lymph from lymphoid tissues into blood, the human thoracic duct. Unlike what was found in blood, we consistently identified a CXCR5-bright PD-1-bright (CXCR5BrPD-1Br) Tfh population in thoracic duct lymph (TDL). These CXCR5BrPD-1Br TDL Tfh shared phenotypic and transcriptional similarities with GC Tfh. Moreover, components of the epigenetic profile of GC Tfh could be detected in CXCR5BrPD-1Br TDL Tfh and the transcriptional imprint of this epigenetic signature was enriched in an activated cTfh subset known to contain vaccine-responding cells. Together with data showing shared TCR sequences between the CXCR5BrPD-1Br TDL Tfh and cTfh, these studies identify a population in TDL as a circulatory intermediate connecting the biology of Tfh in blood to Tfh in lymphoid tissue.

2.
Am J Hum Genet ; 105(1): 89-107, 2019 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-31204013

RESUMO

Deciphering the impact of genetic variation on gene regulation is fundamental to understanding common, complex human diseases. Although histone modifications are important markers of gene regulatory elements of the genome, any specific histone modification has not been assayed in more than a few individuals in the human liver. As a result, the effects of genetic variation on histone modification states in the liver are poorly understood. Here, we generate the most comprehensive genome-wide dataset of two epigenetic marks, H3K4me3 and H3K27ac, and annotate thousands of putative regulatory elements in the human liver. We integrate these findings with genome-wide gene expression data collected from the same human liver tissues and high-resolution promoter-focused chromatin interaction maps collected from human liver-derived HepG2 cells. We demonstrate widespread functional consequences of natural genetic variation on putative regulatory element activity and gene expression levels. Leveraging these extensive datasets, we fine-map a total of 74 GWAS loci that have been associated with at least one complex phenotype. Our results reveal a repertoire of genes and regulatory mechanisms governing complex disease development and further the basic understanding of genetic and epigenetic regulation of gene expression in the human liver tissue.

3.
Nat Commun ; 10(1): 1260, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30890710

RESUMO

Osteoporosis is a devastating disease with an essential genetic component. GWAS have discovered genetic signals robustly associated with bone mineral density (BMD), but not the precise localization of effector genes. Here, we carry out physical and direct variant to gene mapping in human mesenchymal progenitor cell-derived osteoblasts employing a massively parallel, high resolution Capture C based method in order to simultaneously characterize the genome-wide interactions of all human promoters. By intersecting our Capture C and ATAC-seq data, we observe consistent contacts between candidate causal variants and putative target gene promoters in open chromatin for ~ 17% of the 273 BMD loci investigated. Knockdown of two novel implicated genes, ING3 at 'CPED1-WNT16' and EPDR1 at 'STARD3NL', inhibits osteoblastogenesis, while promoting adipogenesis. This approach therefore aids target discovery in osteoporosis, here on the example of two relevant genes involved in the fate determination of mesenchymal progenitors, and can be applied to other common genetic diseases.


Assuntos
Densidade Óssea/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Osteoporose/genética , Regiões Promotoras Genéticas/genética , Adipogenia/genética , Adulto , Diferenciação Celular/genética , Mapeamento Cromossômico , Feminino , Técnicas de Silenciamento de Genes , Loci Gênicos/genética , Células Hep G2 , Proteínas de Homeodomínio/genética , Humanos , Masculino , Proteínas de Membrana/genética , Células-Tronco Mesenquimais , Proteínas de Neoplasias/genética , Osteoblastos/fisiologia , Osteogênese/genética , Polimorfismo de Nucleotídeo Único , Cultura Primária de Células , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Wnt/genética , Adulto Jovem
4.
Cell ; 2018 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-30392958

RESUMO

Activated T cells differentiate into functional subsets with distinct metabolic programs. Glutaminase (GLS) converts glutamine to glutamate to support the tricarboxylic acid cycle and redox and epigenetic reactions. Here, we identify a key role for GLS in T cell activation and specification. Though GLS deficiency diminished initial T cell activation and proliferation and impaired differentiation of Th17 cells, loss of GLS also increased Tbet to promote differentiation and effector function of CD4 Th1 and CD8 CTL cells. This was associated with altered chromatin accessibility and gene expression, including decreased PIK3IP1 in Th1 cells that sensitized to IL-2-mediated mTORC1 signaling. In vivo, GLS null T cells failed to drive Th17-inflammatory diseases, and Th1 cells had initially elevated function but exhausted over time. Transient GLS inhibition, however, led to increased Th1 and CTL T cell numbers. Glutamine metabolism thus has distinct roles to promote Th17 but constrain Th1 and CTL effector cell differentiation.

5.
Hum Genet ; 137(5): 413-425, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29797095

RESUMO

Although Genome Wide Association Studies (GWAS) have led to many valuable insights into the genetic bases of common diseases over the past decade, the issue of missing heritability has surfaced, as the discovered main effect genetic variants found to date do not account for much of a trait's predicted genetic component. We present a workflow, integrating epigenomics and topologically associating domain data, aimed at discovering trait-associated SNP pairs from GWAS where neither SNP achieved independent genome-wide significance. Each analyzed SNP pair consists of one SNP in a putative active enhancer and another SNP in a putative physically interacting gene promoter in a trait-relevant tissue. As a proof-of-principle case study, we used this approach to identify focused collections of SNP pairs that we analyzed in three independent Type 2 diabetes (T2D) GWAS. This approach led us to discover 35 significant SNP pairs, encompassing both novel signals and signals for which we have found orthogonal support from other sources. Nine of these pairs are consistent with eQTL results, two are consistent with our own capture C experiments, and seven involve signals supported by recent T2D literature.

6.
J Immunol ; 200(1): 82-91, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29150566

RESUMO

T cell differentiation requires appropriate regulation of DNA methylation. In this article, we demonstrate that the methylcytosine dioxygenase ten-eleven translocation (TET)2 regulates CD8+ T cell differentiation. In a murine model of acute viral infection, TET2 loss promotes early acquisition of a memory CD8+ T cell fate in a cell-intrinsic manner without disrupting Ag-driven cell expansion or effector function. Upon secondary recall, TET2-deficient memory CD8+ T cells demonstrate superior pathogen control. Genome-wide methylation analysis identified a number of differentially methylated regions in TET2-deficient versus wild-type CD8+ T cells. These differentially methylated regions did not occur at the loci of differentially expressed memory markers; rather, several hypermethylated regions were identified in known transcriptional regulators of CD8+ T cell memory fate. Together, these data demonstrate that TET2 is an important regulator of CD8+ T cell fate decisions.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Proteínas de Ligação a DNA/metabolismo , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Subpopulações de Linfócitos T/imunologia , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Metilação de DNA , Proteínas de Ligação a DNA/genética , Memória Imunológica , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas/genética
7.
Int J Radiat Oncol Biol Phys ; 97(4): 722-731, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-28244407

RESUMO

PURPOSE: To assess the long-term quality of life (QoL) outcomes from a phase 3 trial comparing 2 modes of intensity modulated radiation therapy (IMRT): conventional IMRT (CIMRT) versus hypofractionated IMRT (HIMRT) in patients with localized prostate cancer. METHODS AND MATERIALS: Between 2002 and 2006, 303 men with low-risk to high-risk prostate cancer were randomized to 76 Gy in 38 fractions (CIMRT) versus 70.2 Gy in 26 fractions (HIMRT). QoL was compared by use of the Expanded Prostate Cancer Index Composite (EPIC), the International Prostate Symptom Score (IPSS), and EuroQoL (EQ5D) questionnaires. The primary outcome of the QoL analysis was a minimum clinically important difference defined as a 0.5 standard deviation change from baseline for each respective QoL parameter. Treatment effects were evaluated with the use of logistic mixed effects regression models. RESULTS: A total of 286, 299, and 218 patients had baseline EPIC, IPSS, or EQ5D data available and were included in the analysis. Overall, there was no statistically significant difference between the 2 treatment arms in terms of EPIC, IPSS, or EQ5D scores over time, although there was a trend toward lower EPIC urinary incontinence scores in the HIMRT arm. More patients in the HIMRT arm had a lower EPIC urinary incontinence score relative to baseline versus patients in the CIMRT arm with long-term follow-up. On multivariable analysis, there was no association between radiation fractionation scheme and any QoL parameter. When other clinical factors were examined, lymph node radiation was associated with worse EPIC hormonal scores versus patients receiving no lymph node radiation. In general, QoL outcomes were generally stable over time, with the exception of EPIC hormonal and EQ5D scores. CONCLUSIONS: In this randomized prospective study, there were stable QoL changes in patients receiving HIMRT or CIMRT. Our results add to the growing body of literature suggesting that HIMRT may be an acceptable treatment modality in clinically localized prostate cancer.


Assuntos
Neoplasias da Próstata/psicologia , Neoplasias da Próstata/radioterapia , Qualidade de Vida/psicologia , Lesões por Radiação/psicologia , Radioterapia Conformacional/psicologia , Radioterapia Conformacional/estatística & dados numéricos , Incontinência Urinária/psicologia , Idoso , Idoso de 80 Anos ou mais , Causalidade , Comorbidade , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Prevalência , Neoplasias da Próstata/epidemiologia , Hipofracionamento da Dose de Radiação , Lesões por Radiação/diagnóstico , Lesões por Radiação/epidemiologia , Radioterapia Conformacional/métodos , Fatores de Risco , Autorrelato , Taxa de Sobrevida , Resultado do Tratamento , Estados Unidos/epidemiologia , Incontinência Urinária/epidemiologia , Incontinência Urinária/prevenção & controle
8.
Can J Urol ; 23(6): 8535-8545, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27995848

RESUMO

INTRODUCTION: To characterize patient reported outcomes for urinary and sexual function using International Prostate Symptom Score (IPSS) and Sexual Health Inventory for Men (SHIM) comparing intensity modulated radiation therapy (IMRT), low dose rate brachytherapy (LDR), post-prostatectomy IMRT (PPRT), and radical prostatectomy (RP). MATERIALS AND METHODS: Patients treated for prostate cancer from 2001-2012 completed self-reported SHIM and IPSS surveys. Subgroups were created by baseline score. Mean change from baseline was determined at each time point for the cohort and subgroups. Statistical analysis was performed with generalized estimating equation method. Incontinence was not captured in the questionnaires. RESULTS: A total of 14,523 IPSS surveys from 3,515 men were evaluated. Patients treated with IMRT experienced a minimal decrease in IPSS score from baseline. PPRT scores did not differ from IMRT at any time point (range: +/- 3 points from baseline in IPSS score over 50 months). LDR had an initial IPSS rise (between 5-10 points on the IPSS over 1-9 months) versus IMRT but returned to comparable levels at 34 months. RP was associated with a lower IPSS versus IMRT. LDR had the largest rise from baseline, with return toward baseline. A total of 2,624 SHIM surveys from 857 men were evaluated. LDR and PPRT did not differ from IMRT at any time point (range: +/- 5 points from baseline in SHIM score for 36 months). RP experienced the largest decline from baseline (up to -7 points on SHIM score), at 3 to 7 months; RP had a larger early decrease in SHIM score versus IMRT between 3 and 22 months, after which there was no difference. CONCLUSIONS: IPSS and SHIM score patterns differed among treatment modalities. These data can be used to predict changes in urinary and sexual function over time based on modality and baseline score.


Assuntos
Complicações Pós-Operatórias , Prostatectomia/efeitos adversos , Neoplasias da Próstata , Qualidade de Vida , Radioterapia de Intensidade Modulada/efeitos adversos , Incontinência Urinária , Idoso , Humanos , Efeitos Adversos de Longa Duração/etiologia , Efeitos Adversos de Longa Duração/fisiopatologia , Efeitos Adversos de Longa Duração/psicologia , Masculino , Pessoa de Meia-Idade , Medidas de Resultados Relatados pelo Paciente , Complicações Pós-Operatórias/fisiopatologia , Complicações Pós-Operatórias/psicologia , Prostatectomia/métodos , Neoplasias da Próstata/patologia , Neoplasias da Próstata/radioterapia , Neoplasias da Próstata/cirurgia , Projetos de Pesquisa , Disfunções Sexuais Fisiológicas/etiologia , Disfunções Sexuais Fisiológicas/psicologia , Estados Unidos , Incontinência Urinária/etiologia , Incontinência Urinária/fisiopatologia , Incontinência Urinária/psicologia
9.
Diabetologia ; 59(11): 2360-2368, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27539148

RESUMO

AIMS/HYPOTHESIS: One of the most strongly associated type 2 diabetes loci reported to date resides within the TCF7L2 gene. Previous studies point to the T allele of rs7903146 in intron 3 as the causal variant at this locus. We aimed to identify the actual gene(s) under the influence of this variant. METHODS: Using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease, we generated a 1.4 kb deletion of the genomic region harbouring rs7903146 in the HCT116 cell line, followed by global gene expression analysis. We then carried out a combination of circularised chromosome conformation capture (4C) and Capture C in cell lines, HCT116 and NCM460 in order to ascertain which promoters of these perturbed genes made consistent physical contact with this genomic region. RESULTS: We observed 99 genes with significant differential expression (false discovery rate [FDR] cut-off:10%) and an effect size of at least twofold. The subsequent promoter contact analyses revealed just one gene, ACSL5, which resides in the same topologically associating domain as TCF7L2. The generation of additional, smaller deletions (66 bp and 104 bp) comprising rs7903146 showed consistently reduced ACSL5 mRNA levels across all three deletions of up to 30-fold, with commensurate loss of acyl-CoA synthetase long-chain family member 5 (ACSL5) protein. Notably, the deletion of this single-nucleotide polymorphism region abolished significantly detectable chromatin contacts with the ACSL5 promoter. We went on to confirm that contacts between rs7903146 and the ACSL5 promoter regions were conserved in human colon tissue. ACSL5 encodes ACSL5, an enzyme with known roles in fatty acid metabolism. CONCLUSIONS/INTERPRETATION: This 'variant to gene mapping' effort implicates the genomic location harbouring rs7903146 as a regulatory region for ACSL5.


Assuntos
Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Western Blotting , Proteínas Associadas a CRISPR/metabolismo , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Colo/metabolismo , Células HCT116 , Humanos , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único/genética , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa
10.
J Natl Compr Canc Netw ; 13(5): 525-30, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25964638

RESUMO

PURPOSE: Characterize use of postprostatectomy radiation (PPRT) for patients with prostate cancer at an NCI-designated comprehensive cancer center. METHODS: We queried our prospective prostate cancer database for patients treated with 60 to 68 Gy of radiation therapy (RT) to the prostate bed after prostatectomy from 2003 to 2011. Prostatectomy cases were obtained from billing records. Patients with an intact prostate treated with definitive RT served as a control for the change in volume of patients with prostate cancer treated in the department. Chi-square analysis assessed differences between adjuvant and salvage RT cohorts. Spearman correlation assessed yearly trends in prostate-specific antigen (PSA) level at the time of referral for RT. Linear regression models tested trends for number of PPRT cases, prostatectomies, and patients with intact prostate receiving radiation across years. RESULTS: PPRT was used to treat 475 men at Fox Chase Cancer Center from 2003 to 2011 (83 adjuvant and 392 salvage). Over time, an increased proportion of patients receiving RT to the prostate were treated with PPRT. No increase was seen in the proportion of patients treated with adjuvant RT compared with salvage RT (P=.5). Patients receiving adjuvant RT were younger, had higher pathologic Gleason score, pathologic T stage, and rates of positive margins than those receiving salvage RT. Pre-RT PSA values were inversely correlated with year (P=.005). The number of patients referred for salvage RT with a PSA of 0.5 ng/mL or less increased significantly from 7.9% in 2003 to 26.6% in 2011 (P=.002). CONCLUSIONS: A larger proportion of patients treated with RT for localized prostate cancer are now receiving PPRT. No increase was seen in the proportion of patients treated with adjuvant RT. Over time, patients with lower PSAs were referred for salvage RT.


Assuntos
Cuidados Pós-Operatórios , Neoplasias da Próstata/radioterapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Prostatectomia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgia , Radioterapia Adjuvante , Terapia de Salvação , Resultado do Tratamento
11.
Clin Breast Cancer ; 15(5): 381-9, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25861716

RESUMO

BACKGROUND: Randomized data examining adjuvant radiation therapy (RT) alone in elderly women with low-risk, hormone receptor-positive (HR(+)) breast cancer is lacking. We investigated the outcomes for elderly women treated with adjuvant RT alone versus RT plus endocrine therapy (ET) after breast-conserving surgery. PATIENTS AND METHODS: We queried our institutional breast cancer database for the following patients: age > 65 years, stage T1-T2N0, HR(+), and treatment with breast-conserving surgery, including adjuvant RT. The χ(2) analysis identified significant baseline differences between the groups. Cox proportional hazard methods identified predictors of endpoints on multivariate analysis. Kaplan-Meier estimates of survival were compared using the log-rank test. RESULTS: A total of 504 patients were identified, 311 had undergone RT plus ET (62%) and 193, RT alone (38%). The median follow-up time was 88 months. The RT-alone group versus RT plus ET group had different median age (72 vs.71 years, P < .001), different median tumor size (1 vs. 1.3 cm, P < .001), lower grade (40% vs. 29%, P = .05), and fewer close or positive margins (11% vs. 19%, P = .01). The adherence rate to prescribed ET was 70%. Tumor size predicted an increased risk of distant metastasis (DM) (hazard ratio, 1.96; 95% confidence interval [CI], 1.23-3.13) and worse disease-free survival (DFS) (hazard ratio, 1.86; 95% CI, 1.22-2.86). ET nonadherence versus adherence predicted for risk of DM (hazard ratio, 5.03; 95% CI, 1.98-12.66) and DFS (HR, 4.24; 95% CI, 1.9-10.3). Of the women with DM, 83.8% had tumors > 1 cm in size. CONCLUSION: ET nonadherence and tumor size > 1 cm predicted an increased risk of DM and worse DFS, favoring the addition of ET in this group. However, RT alone for women with tumors less than or equal to 1 cm may be appropriate.


Assuntos
Antineoplásicos Hormonais/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/radioterapia , Radioterapia Adjuvante/métodos , Tamoxifeno/administração & dosagem , Idoso , Neoplasias da Mama/cirurgia , Terapia Combinada , Intervalo Livre de Doença , Feminino , Seguimentos , Humanos , Estadiamento de Neoplasias , Análise de Sobrevida , Resultado do Tratamento
12.
Med Dosim ; 40(3): 186-9, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25595491

RESUMO

A small decrease in testosterone level has been documented after prostate irradiation, possibly owing to the incidental dose to the testes. Testicular doses from prostate external beam radiation plans with either intensity-modulated radiation therapy (IMRT) or volumetric-modulated arc therapy (VMAT) were calculated to investigate any difference. Testicles were contoured for 16 patients being treated for localized prostate cancer. For each patient, 2 plans were created: 1 with IMRT and 1 with VMAT. No specific attempt was made to reduce testicular dose. Minimum, maximum, and mean doses to the testicles were recorded for each plan. Of the 16 patients, 4 received a total dose of 7800 cGy to the prostate alone, 7 received 8000 cGy to the prostate alone, and 5 received 8000 cGy to the prostate and pelvic lymph nodes. The mean (range) of testicular dose with an IMRT plan was 54.7 cGy (21.1 to 91.9) and 59.0 cGy (25.1 to 93.4) with a VMAT plan. In 12 cases, the mean VMAT dose was higher than the mean IMRT dose, with a mean difference of 4.3 cGy (p = 0.019). There was a small but statistically significant increase in mean testicular dose delivered by VMAT compared with IMRT. Despite this, it unlikely that there is a clinically meaningful difference in testicular doses from either modality.


Assuntos
Neoplasias da Próstata/radioterapia , Dosagem Radioterapêutica , Planejamento da Radioterapia Assistida por Computador/métodos , Radioterapia de Intensidade Modulada/métodos , Testículo/efeitos da radiação , Absorção de Radiação , Humanos , Masculino , Tratamentos com Preservação do Órgão/métodos , Exposição à Radiação/análise , Proteção Radiológica/métodos , Resultado do Tratamento
13.
Nucleic Acids Res ; 43(2): 1268-82, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25567984

RESUMO

FOXP3 is a lineage-specific transcription factor that is required for regulatory T cell development and function. In this study, we determined the crystal structure of the FOXP3 forkhead domain bound to DNA. The structure reveals that FOXP3 can form a stable domain-swapped dimer to bridge DNA in the absence of cofactors, suggesting that FOXP3 may play a role in long-range gene interactions. To test this hypothesis, we used circular chromosome conformation capture coupled with high throughput sequencing (4C-seq) to analyze FOXP3-dependent genomic contacts around a known FOXP3-bound locus, Ptpn22. Our studies reveal that FOXP3 induces significant changes in the chromatin contacts between the Ptpn22 locus and other Foxp3-regulated genes, reflecting a mechanism by which FOXP3 reorganizes the genome architecture to coordinate the expression of its target genes. Our results suggest that FOXP3 mediates long-range chromatin interactions as part of its mechanisms to regulate specific gene expression in regulatory T cells.


Assuntos
Cromossomos/química , DNA/química , Fatores de Transcrição Forkhead/química , Animais , DNA/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Multimerização Proteica , Estrutura Terciária de Proteína , Proteína Tirosina Fosfatase não Receptora Tipo 22/genética
14.
Eur J Hum Genet ; 23(1): 103-9, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24667787

RESUMO

Resolving the underlying functional mechanism to a given genetic association has proven extremely challenging. However, the strongest associated type 2 diabetes (T2D) locus reported to date, TCF7L2, presents an opportunity for translational analyses, as many studies in multiple ethnicities strongly point to SNP rs7903146 in intron 3 as being the causal variant within this gene. We carried out oligo pull-down combined with mass spectrophotometry (MS) to elucidate the specific transcriptional machinery across this SNP using protein extracts from HCT116 cells. We observed that poly (ADP-ribose) polymerase 1 (PARP-1) is by far the most abundant binding factor. Pursuing the possibility of a feedback mechanism, we observed that PARP-1, along with the next most abundant binding proteins, DNA topoisomerase I and ATP-dependent RNA helicase A, dimerize with the TCF7L2 protein and with each other. We uncovered further evidence of a feedback mechanism using a luciferase reporter approach, including observing expression differences between alleles for rs7903146. We also found that there was an allelic difference in the MS results for proteins with less abundant binding, namely X-ray repair cross-complementing 5 and RPA/p70. Our results point to a protein complex binding across rs7903146 within TCF7L2 and suggests a possible mechanism by which this locus confers its T2D risk.


Assuntos
Alelos , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Mutação , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Transcrição Genética , Sequência de Aminoácidos , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Humanos , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteína 2 Semelhante ao Fator 7 de Transcrição/química , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo
15.
BMJ Open Diabetes Res Care ; 2(1): e000052, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25469308

RESUMO

BACKGROUND: The transcription factor 7-like 2 (TCF7L2) locus is strongly implicated in the pathogenesis of type 2 diabetes (T2D). We previously mapped the genomic regions bound by TCF7L2 using ChIP (chromatin immunoprecipitation)-seq in the colorectal carcinoma cell line, HCT116, revealing an unexpected highly significant over-representation of genome-wide association studies (GWAS) loci associated primarily with endocrine (in particular T2D) and cardiovascular traits. METHODS: In order to further explore if this observed phenomenon occurs in other cell lines, we carried out ChIP-seq in HepG2 cells and leveraged ENCODE data for five additional cell lines. Given that only a minority of the predicted genetic component to most complex traits has been identified to date, plus our GWAS-related observations with respect to TCF7L2 occupancy, we investigated if restricting association analyses to the genes yielded from this approach, in order to reduce the constraints of multiple testing, could reveal novel T2D loci. RESULTS: We found strong evidence for the continued enrichment of endocrine and cardiovascular GWAS categories, with additional support for cancer. When investigating all the known GWAS loci bound by TCF7L2 in the shortest gene list, derived from HCT116, the coronary artery disease-associated variant, rs46522 at the UBE2Z-GIP-ATP5G1-SNF8 locus, yielded significant association with T2D within DIAGRAM. Furthermore, when we analyzed tag-SNPs (single nucleotide polymorphisms) in genes not previously implicated by GWAS but bound by TCF7L2 within 5 kb, we observed a significant association of rs4780476 within CPPED1 in DIAGRAM. CONCLUSIONS: ChIP-seq data generated with this GWAS-implicated transcription factor provided a biologically plausible method to limit multiple testing in the assessment of genome-wide genotyping data to uncover two novel T2D-associated loci.

16.
Cancer ; 120(22): 3569-74, 2014 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-24985911

RESUMO

BACKGROUND: There is conflicting evidence regarding the benefit of postmastectomy radiation therapy (PMRT) for pathologic stage T3N0M0 breast cancers. We analyzed data from the Surveillance, Epidemiology, and End Results (SEER) database to investigate the benefit of PMRT in this patient population. METHODS: We queried the SEER database for T3N0M0 breast cancer patients diagnosed from 2000 to 2010 who underwent modified radical mastectomy. We excluded males, patients with unknown radiation timing/type, other primary tumors, and survival <6 months. A total of 2525 patients were included in the analysis. We performed univariate and multivariate statistical analysis using chi-square tests, log-rank tests, and Cox proportional hazards regression. The primary endpoints were overall survival (OS) and cancer-specific survival (CSS). RESULTS: Of the 2525 patients identified, 1063 received PMRT. The median follow-up was 56 months (range, 6-131 months). On univariate analysis, PMRT improved OS (76.5% vs 61.8%, P<.01) and CSS (85.0% vs 82.4%, P<.01) at 8 years. The use of PMRT remained significant on multivariate analysis: PMRT improved OS (hazard ratio 0.63, P<.001) and CSS (hazard ratio 0.77, P = .045). Low tumor grade (P<.01) and marital status of "married" (P = .01) also was a predictor of improved CSS on multivariate analysis. CONCLUSIONS: PMRT was associated with significant improvements in both CSS and OS in patients with T3N0M0 breast cancers treated with modified radical mastectomy from 2000 to 2010. PMRT should be strongly considered in T3N0M0 patients. Postmastectomy radiation therapy is associated with significant improvements in overall and cause-specific survival in patients with T3N0M0 breast cancers treated with modified radical mastectomy from 2000 to 2010 in the SEER database. Postmastectomy radiation therapy should be strongly considered for patients who have T3N0M0 tumors.


Assuntos
Neoplasias da Mama/terapia , Mastectomia , Adolescente , Adulto , Idoso , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Terapia Combinada , Feminino , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Modelos de Riscos Proporcionais , Programa de SEER , Fatores de Tempo
17.
J Clin Endocrinol Metab ; 99(8): E1580-5, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24878043

RESUMO

BACKGROUND: Forkhead Box A2 (FOXA2) exerts an influence on glucose homeostasis via activity in the liver. In addition, a key genome-wide association study (GWAS) recently demonstrated that genetic variation, namely rs6048205, at the FOXA2 locus is robustly associated with fasting glucose levels. Our hypothesis was that this DNA-binding protein regulates the expression of a set of molecular pathways critical to endocrine traits. METHODS: Drawing on our laboratory and bioinformatic experience with chromatin immunoprecipitation followed by massively parallel sequencing, we analyzed our existing FOXA2 chromatin immunoprecipitation followed by massively parallel sequencing data generated in human liver, using the algorithm hypergeometric optimization of motif enrichment, to gain insight into its global genomic binding pattern from a disease perspective. RESULTS: We performed a pathway analysis of the gene list using the gene set enrichment analysis algorithm, which yielded a number of significant annotations. Motivated by the fact that the FOXA2 locus has been implicated by GWAS, we cross-referenced the occupancy sites with the National Institutes of Health GWAS catalog and found strong evidence for the enrichment of loci implicated in endocrine, neuropsychiatric, cardiovascular, and cancer trait categories, but interestingly there was no evidence for enrichment for inflammation related traits. Intriguingly, a FOXA2 occupancy site coincided with rs6048205, suggesting that this variant confers its effect, at least partially, via a perturbation of a FOXA2 feedback mechanism. CONCLUSION: Our data strongly suggest that FOXA2 is acting as a master regulator of key pathways that are enriched for loci implicated by GWAS for most trait categories, with the clear exception of inflammation, suggesting that this factor exerts its effect in this context via noninflammatory processes.


Assuntos
DNA/metabolismo , Redes Reguladoras de Genes , Fator 3-beta Nuclear de Hepatócito/metabolismo , Fígado/metabolismo , Sítios de Ligação/genética , Doenças Cardiovasculares/genética , Imunoprecipitação da Cromatina , Doenças do Sistema Endócrino/genética , Estudo de Associação Genômica Ampla , Fator 3-beta Nuclear de Hepatócito/genética , Humanos , Transtornos Mentais/genética , Redes e Vias Metabólicas/genética , Neoplasias/genética , Doenças do Sistema Nervoso/genética , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Locos de Características Quantitativas
18.
Int J Endocrinol ; 2014: 769671, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24719615

RESUMO

Elucidating the underlying genetic variations influencing various complex diseases is one of the major challenges currently facing clinical genetic research. Although these variations are often difficult to uncover, approaches such as genome-wide association studies (GWASs) have been successful at finding statistically significant associations between specific genomic loci and disease susceptibility. GWAS has been especially successful in elucidating genetic variants that influence type 2 diabetes (T2D) and obesity/body mass index (BMI). Specifically, several GWASs have confirmed that a variant in transcription factor 7-like 2 (TCF7L2) confers risk for T2D, while a variant in fat mass and obesity-associated protein (FTO) confers risk for obesity/BMI; indeed both of these signals are considered the most statistically associated loci discovered for these respective traits to date. The discovery of these two key loci in this context has been invaluable for providing novel insight into mechanisms of heritability and disease pathogenesis. As follow-up studies of TCF7L2 and FTO have typically lead the way in how to follow up a GWAS discovery, we outline what has been learned from such investigations and how they have implications for the myriad of other loci that have been subsequently reported in this disease context.

19.
J Am Acad Dermatol ; 70(6): 1028-35, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24666998

RESUMO

BACKGROUND: Absolute lymphocyte count (ALC) is a laboratory value commonly obtained during workup of patients with Merkel cell carcinoma (MCC). OBJECTIVE: We report the prognostic impact of ALC as a surrogate of immune status in MCC. METHODS: A complete blood cell count was available for 64 patients with MCC in the month before definitive surgery, chemotherapy, or radiation. Statistical analysis was performed with classification and regression tree analysis, log rank test, and Cox model. RESULTS: Median overall survival (OS) for the cohort was 97 months. Median OS for patients with an ALC less than 1.1 k/mm(3) was 18.8 versus 110.1 months for those with ALC greater than or equal to 1.1 k/mm(3) (P = .002, hazard ratio 0.29). Multivariate analysis of OS controlling for ALC, sex, stage, adjuvant chemotherapy, hematologic malignancy, and immunosuppression demonstrated ALC as a prognostic factor (P = .03). Disease-free survival at 36 months for ALC less than 1.1 k/mm(3) was 26.9% versus 64.4% for those with ALC greater than or equal to 1.1 k/mm(3) (P = .01). ALC was not a significant predictor for disease-free survival on multivariate analysis (P = .12). LIMITATIONS: This is a single-institution retrospective data set. CONCLUSION: ALC is associated with OS but not disease-free survival in MCC using a threshold of less than 1.1 k/mm(3). This test may provide additional prognostic information for patients with MCC.


Assuntos
Carcinoma de Célula de Merkel/sangue , Carcinoma de Célula de Merkel/mortalidade , Contagem de Linfócitos , Neoplasias Cutâneas/sangue , Neoplasias Cutâneas/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Carcinoma de Célula de Merkel/imunologia , Carcinoma de Célula de Merkel/terapia , Estudos de Coortes , Terapia Combinada/métodos , Intervalo Livre de Doença , Feminino , Humanos , Linfócitos do Interstício Tumoral/imunologia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Invasividade Neoplásica/patologia , Estadiamento de Neoplasias , Prognóstico , Modelos de Riscos Proporcionais , Análise de Regressão , Estudos Retrospectivos , Medição de Risco , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/terapia , Análise de Sobrevida
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