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1.
Diabetes ; 69(3): 465-476, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32029481

RESUMO

Children at increased genetic risk for type 1 diabetes (T1D) after environmental exposures may develop pancreatic islet autoantibodies (IA) at a very young age. Metabolic profile changes over time may imply responses to exposures and signal development of the first IA. Our present research in The Environmental Determinants of Diabetes in the Young (TEDDY) study aimed to identify metabolome-wide signals preceding the first IA against GAD (GADA-first) or against insulin (IAA-first). We profiled metabolomes by mass spectrometry from children's plasma at 3-month intervals after birth until appearance of the first IA. A trajectory analysis discovered each first IA preceded by reduced amino acid proline and branched-chain amino acids (BCAAs), respectively. With independent time point analysis following birth, we discovered dehydroascorbic acid (DHAA) contributing to the risk of each first IA, and γ-aminobutyric acid (GABAs) associated with the first autoantibody against insulin (IAA-first). Methionine and alanine, compounds produced in BCAA metabolism and fatty acids, also preceded IA at different time points. Unsaturated triglycerides and phosphatidylethanolamines decreased in abundance before appearance of either autoantibody. Our findings suggest that IAA-first and GADA-first are heralded by different patterns of DHAA, GABA, multiple amino acids, and fatty acids, which may be important to primary prevention of T1D.

2.
Diabetologia ; 63(2): 278-286, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31728565

RESUMO

AIMS/HYPOTHESIS: We studied the association of plasma ascorbic acid with the risk of developing islet autoimmunity and type 1 diabetes and examined whether SNPs in vitamin C transport genes modify these associations. Furthermore, we aimed to determine whether the SNPs themselves are associated with the risk of islet autoimmunity or type 1 diabetes. METHODS: We used a risk set sampled nested case-control design within an ongoing international multicentre observational study: The Environmental Determinants of Diabetes in the Young (TEDDY). The TEDDY study followed children with increased genetic risk from birth to endpoints of islet autoantibodies (350 cases, 974 controls) and type 1 diabetes (102 cases, 282 controls) in six clinical centres. Control participants were matched for family history of type 1 diabetes, clinical centre and sex. Plasma ascorbic acid concentration was measured at ages 6 and 12 months and then annually up to age 6 years. SNPs in vitamin C transport genes were genotyped using the ImmunoChip custom microarray. Comparisons were adjusted for HLA genotypes and for background population stratification. RESULTS: Childhood plasma ascorbic acid (mean ± SD 10.76 ± 3.54 mg/l in controls) was inversely associated with islet autoimmunity risk (adjusted OR 0.96 [95% CI 0.92, 0.99] per +1 mg/l), particularly islet autoimmunity, starting with insulin autoantibodies (OR 0.94 [95% CI 0.88, 0.99]), but not with type 1 diabetes risk (OR 0.93 [95% Cl 0.86, 1.02]). The SLC2A2 rs5400 SNP was associated with increased risk of type 1 diabetes (OR 1.77 [95% CI 1.12, 2.80]), independent of plasma ascorbic acid (OR 0.92 [95% CI 0.84, 1.00]). CONCLUSIONS/INTERPRETATION: Higher plasma ascorbic acid levels may protect against islet autoimmunity in children genetically at risk for type 1 diabetes. Further studies are warranted to confirm these findings. DATA AVAILABILITY: The datasets generated and analysed during the current study will be made available in the NIDDK Central Repository at https://www.niddkrepository.org/studies/teddy.

3.
Nat Biotechnol ; 37(5): 561-566, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30936564

RESUMO

Benchmark small variant calls are required for developing, optimizing and assessing the performance of sequencing and bioinformatics methods. Here, as part of the Genome in a Bottle (GIAB) Consortium, we apply a reproducible, cloud-based pipeline to integrate multiple short- and linked-read sequencing datasets and provide benchmark calls for human genomes. We generate benchmark calls for one previously analyzed GIAB sample, as well as six genomes from the Personal Genome Project. These new genomes have broad, open consent, making this a 'first of its kind' resource that is available to the community for multiple downstream applications. We produce 17% more benchmark single nucleotide variations, 176% more indels and 12% larger benchmark regions than previously published GIAB benchmarks. We demonstrate that this benchmark reliably identifies errors in existing callsets and highlight challenges in interpreting performance metrics when using benchmarks that are not perfect or comprehensive. Finally, we identify strengths and weaknesses of callsets by stratifying performance according to variant type and genome context.


Assuntos
Benchmarking , Biologia Computacional/tendências , Genoma Humano/genética , Genômica/tendências , Variação Genética/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação INDEL/genética , Polimorfismo de Nucleotídeo Único , Software/tendências
4.
Steroids ; 140: 159-166, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30352204

RESUMO

Glucocorticoids are standard of care for many chronic inflammatory conditions, including juvenile dermatomyositis (JDM) and anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). We sought to define pharmacodynamic biomarkers of therapeutic efficacy and safety concerns of glucocorticoid treatment for these two disorders. Previous proteomic profiling of patients with Duchenne muscular dystrophy (DMD) and inflammatory bowel disease (IBD) treated with glucocorticoids identified candidate biomarkers for efficacy and safety concerns of glucocorticoids. Serial serum samples from patients with AAV (n = 30) and JDM (n = 12) were obtained during active disease, and after treatment with glucocorticoids. For AAV, 8 of 11 biomarkers of the anti-inflammatory response to glucocorticoids were validated (P-value ≤0.05; CD23, macrophage-derived cytokine, interleukin-22 binding protein, matrix metalloproteinase-12, T lymphocyte surface antigen Ly9, fibrinogen gamma chain, angiopoietin-2 [all decreased], and protein C [increased]), as were 5 of 7 safety biomarkers (P-value ≤0.05; afamin, matrix metalloproteinase-3, insulin growth factor binding protein-5, angiotensinogen, leptin [all increased]). For JDM, 10 of 11 efficacy biomarkers were validated (P-value ≤0.05; all proteins except fibrinogen gamma chain) and 6 of 7 safety biomarkers (P-value ≤0.05; AAV proteins plus growth hormone binding protein). The identified efficacy biomarkers may be useful as objective outcome measures for early phase proof-of-concept studies when assessing novel anti-inflammatory drugs in JDM and AAV, and likely in other inflammatory disorders. Similarly, safety biomarkers may also be helpful assessing toxicity of alternatives to glucocorticoids.


Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/sangue , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/tratamento farmacológico , Dermatomiosite/sangue , Dermatomiosite/tratamento farmacológico , Glucocorticoides/efeitos adversos , Glucocorticoides/farmacologia , Segurança , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Criança , Pré-Escolar , Feminino , Glucocorticoides/uso terapêutico , Humanos , Masculino , Pessoa de Meia-Idade , Proteômica , Resultado do Tratamento , Adulto Jovem
5.
Ann Rheum Dis ; 77(8): 1226-1233, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29724730

RESUMO

OBJECTIVES: To characterise renal tissue metabolic pathway gene expression in different forms of glomerulonephritis. METHODS: Patients with nephrotic syndrome (NS), antineutrophil cytoplasmic antibody-associated vasculitis (AAV), systemic lupus erythematosus (SLE) and healthy living donors (LD) were studied. Clinically indicated renal biopsies were obtained at time of diagnosis and microdissected into glomerular and tubulointerstitial compartments. Microarray-derived differential gene expression of 88 genes representing critical enzymes of metabolic pathways and 25 genes related to immune cell markers was compared between disease groups. Correlation analyses measured relationships between metabolic pathways, kidney function and cytokine production. RESULTS: Reduced steady state levels of mRNA species were enriched in pathways of oxidative phosphorylation and increased in the pentose phosphate pathway (PPP) with maximal perturbation in AAV and SLE followed by NS, and least in LD. Transcript regulation was isozymes specific with robust regulation in hexokinases, enolases and glucose transporters. Intercorrelation networks were observed between enzymes of the PPP (eg, transketolase) and macrophage markers (eg, CD68) (r=0.49, p<0.01). Increased PPP transcript levels were associated with reduced glomerular filtration rate in the glomerular (r=-0.49, p<0.01) and tubulointerstitial (r=-0.41, p<0.01) compartments. PPP expression and tumour necrosis factor activation were tightly co-expressed (r=0.70, p<0.01). CONCLUSION: This study demonstrated concordant alterations of the renal transcriptome consistent with metabolic reprogramming across different forms of glomerulonephritis. Activation of the PPP was tightly linked with intrarenal macrophage marker expression, reduced kidney function and increased production of cytokines. Modulation of glucose metabolism may offer novel immune-modulatory therapeutic approaches in rare kidney diseases.


Assuntos
Glomerulonefrite/metabolismo , Redes e Vias Metabólicas/genética , Adulto , Idoso , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/genética , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/metabolismo , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/patologia , Biópsia , Citocinas/biossíntese , Feminino , Regulação da Expressão Gênica , Glomerulonefrite/genética , Glomerulonefrite/patologia , Humanos , Isoenzimas/metabolismo , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/metabolismo , Lúpus Eritematoso Sistêmico/patologia , Masculino , Redes e Vias Metabólicas/imunologia , Pessoa de Meia-Idade , Síndrome Nefrótica/genética , Síndrome Nefrótica/metabolismo , Síndrome Nefrótica/patologia , Via de Pentose Fosfato/genética , RNA Mensageiro/genética , Transcriptoma , Adulto Jovem
6.
Gut ; 67(3): 521-533, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28634199

RESUMO

OBJECTIVE: To elucidate the genetic architecture of gene expression in pancreatic tissues. DESIGN: We performed expression quantitative trait locus (eQTL) analysis in histologically normal pancreatic tissue samples (n=95) using RNA sequencing and the corresponding 1000 genomes imputed germline genotypes. Data from pancreatic tumour-derived tissue samples (n=115) from The Cancer Genome Atlas were included for comparison. RESULTS: We identified 38 615 cis-eQTLs (in 484 genes) in histologically normal tissues and 39 713 cis-eQTL (in 237 genes) in tumour-derived tissues (false discovery rate <0.1), with the strongest effects seen near transcriptional start sites. Approximately 23% and 42% of genes with significant cis-eQTLs appeared to be specific for tumour-derived and normal-derived tissues, respectively. Significant enrichment of cis-eQTL variants was noted in non-coding regulatory regions, in particular for pancreatic tissues (1.53-fold to 3.12-fold, p≤0.0001), indicating tissue-specific functional relevance. A common pancreatic cancer risk locus on 9q34.2 (rs687289) was associated with ABO expression in histologically normal (p=5.8×10-8) and tumour-derived (p=8.3×10-5) tissues. The high linkage disequilibrium between this variant and the O blood group generating deletion variant in ABO (exon 6) suggested that nonsense-mediated decay (NMD) of the 'O' mRNA might explain this finding. However, knockdown of crucial NMD regulators did not influence decay of the ABO 'O' mRNA, indicating that a gene regulatory element influenced by pancreatic cancer risk alleles may underlie the eQTL. CONCLUSIONS: We have identified cis-eQTLs representing potential functional regulatory variants in the pancreas and generated a rich data set for further studies on gene expression and its regulation in pancreatic tissues.


Assuntos
Sistema do Grupo Sanguíneo ABO/genética , Expressão Gênica , Pâncreas , Neoplasias Pancreáticas/genética , Locos de Características Quantitativas , RNA Neoplásico/análise , Transcriptoma , Alelos , Cromossomos Humanos Par 9 , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Degradação do RNAm Mediada por Códon sem Sentido , Polimorfismo de Nucleotídeo Único , Sequências Reguladoras de Ácido Nucleico , Análise de Sequência de RNA
7.
Nat Genet ; 49(9): 1326-1335, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28759004

RESUMO

Previous genome-wide association studies have identified a melanoma-associated locus at 1q42.1 that encompasses a ∼100-kb region spanning the PARP1 gene. Expression quantitative trait locus (eQTL) analysis in multiple cell types of the melanocytic lineage consistently demonstrated that the 1q42.1 melanoma risk allele (rs3219090[G]) is correlated with higher PARP1 levels. In silico fine-mapping and functional validation identified a common intronic indel, rs144361550 (-/GGGCCC; r2 = 0.947 with rs3219090), as displaying allele-specific transcriptional activity. A proteomic screen identified RECQL as binding to rs144361550 in an allele-preferential manner. In human primary melanocytes, PARP1 promoted cell proliferation and rescued BRAFV600E-induced senescence phenotypes in a PARylation-independent manner. PARP1 also transformed TERT-immortalized melanocytes expressing BRAFV600E. PARP1-mediated senescence rescue was accompanied by transcriptional activation of the melanocyte-lineage survival oncogene MITF, highlighting a new role for PARP1 in melanomagenesis.


Assuntos
Proliferação de Células/genética , Íntrons/genética , Melanócitos/metabolismo , Fator de Transcrição Associado à Microftalmia/genética , Poli(ADP-Ribose) Polimerase-1/genética , Polimorfismo de Nucleotídeo Único , Sequência de Bases , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Células Cultivadas , Senescência Celular/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação INDEL , Immunoblotting , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Fator de Transcrição Associado à Microftalmia/metabolismo , Microscopia Confocal , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Fatores de Risco , Telomerase/genética , Telomerase/metabolismo
8.
BMC Genomics ; 17: 64, 2016 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-26772178

RESUMO

BACKGROUND: The human genome contains variants ranging in size from small single nucleotide polymorphisms (SNPs) to large structural variants (SVs). High-quality benchmark small variant calls for the pilot National Institute of Standards and Technology (NIST) Reference Material (NA12878) have been developed by the Genome in a Bottle Consortium, but no similar high-quality benchmark SV calls exist for this genome. Since SV callers output highly discordant results, we developed methods to combine multiple forms of evidence from multiple sequencing technologies to classify candidate SVs into likely true or false positives. Our method (svclassify) calculates annotations from one or more aligned bam files from many high-throughput sequencing technologies, and then builds a one-class model using these annotations to classify candidate SVs as likely true or false positives. RESULTS: We first used pedigree analysis to develop a set of high-confidence breakpoint-resolved large deletions. We then used svclassify to cluster and classify these deletions as well as a set of high-confidence deletions from the 1000 Genomes Project and a set of breakpoint-resolved complex insertions from Spiral Genetics. We find that likely SVs cluster separately from likely non-SVs based on our annotations, and that the SVs cluster into different types of deletions. We then developed a supervised one-class classification method that uses a training set of random non-SV regions to determine whether candidate SVs have abnormal annotations different from most of the genome. To test this classification method, we use our pedigree-based breakpoint-resolved SVs, SVs validated by the 1000 Genomes Project, and assembly-based breakpoint-resolved insertions, along with semi-automated visualization using svviz. CONCLUSIONS: We find that candidate SVs with high scores from multiple technologies have high concordance with PCR validation and an orthogonal consensus method MetaSV (99.7 % concordant), and candidate SVs with low scores are questionable. We distribute a set of 2676 high-confidence deletions and 68 high-confidence insertions with high svclassify scores from these call sets for benchmarking SV callers. We expect these methods to be particularly useful for establishing high-confidence SV calls for benchmark samples that have been characterized by multiple technologies.


Assuntos
Genoma Humano , Variação Estrutural do Genoma , Software , Benchmarking , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Anotação de Sequência Molecular , Linhagem , Polimorfismo de Nucleotídeo Único/genética
9.
Hum Mol Genet ; 25(21): 4726-4738, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28172817

RESUMO

Genome-wide association studies (GWAS) have identified multiple common susceptibility loci for pancreatic cancer. Here we report fine-mapping and functional analysis of one such locus residing in a 610 kb gene desert on chr13q22.1 (marked by rs9543325). The closest candidate genes, KLF5, KLF12, PIBF1, DIS3 and BORA, range in distance from 265-586 kb. Sequencing three sub-regions containing the top ranked SNPs by imputation P-value revealed a 30 bp insertion/deletion (indel) variant that was significantly associated with pancreatic cancer risk (rs386772267, P = 2.30 × 10-11, OR = 1.22, 95% CI 1.15-1.28) and highly correlated to rs9543325 (r2 = 0.97 in the 1000 Genomes EUR population). This indel was the most significant cis-eQTL variant in a set of 222 histologically normal pancreatic tissue samples (ß = 0.26, P = 0.004), with the insertion (risk-increasing) allele associated with reduced DIS3 expression. DIS3 encodes a catalytic subunit of the nuclear RNA exosome complex that mediates RNA processing and decay, and is mutated in several cancers. Chromosome conformation capture revealed a long range (570 kb) physical interaction between a sub-region of the risk locus, containing rs386772267, and a region ∼6 kb upstream of DIS3 Finally, repressor regulatory activity and allele-specific protein binding by transcription factors of the TCF/LEF family were observed for the risk-increasing allele of rs386772267, indicating that expression regulation at this risk locus may be influenced by the Wnt signaling pathway. In conclusion, we have identified a putative functional indel variant at chr13q22.1 that associates with decreased DIS3 expression in carriers of pancreatic cancer risk-increasing alleles, and could therefore affect nuclear RNA processing and/or decay.


Assuntos
Cromossomos Humanos Par 13 , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Neoplasias Pancreáticas/genética , Alelos , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Mapeamento Cromossômico/métodos , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Loci Gênicos , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Mutação INDEL , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/metabolismo , Análise de Sequência de DNA , Fatores de Transcrição/genética
10.
Urol Oncol ; 33(2): 69.e11-8, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25035170

RESUMO

OBJECTIVE: To identify prognostic biomarkers in clear cell renal cell carcinoma (ccRCC) using a proteomic approach. MATERIAL AND METHODS: We performed a comparative proteomic profiling of ccRCC and normal renal tissues from 9 different human specimens. We assessed differential protein expression by iTRAQ (isobaric tagging reagent for absolute quantify) labeling with regard to tumor aggressiveness according to the stage, size, grade, and necrosis (SSIGN) score and confirmed our results using Western blot (9 patients) and immunohistochemistry (135 patients) analysis. RESULTS: After proteomic analysis, 928 constitutive proteins were identified. Among these proteins, 346 had a modified expression in tumor compared with that of normal tissue. Pathway and integrated analyses indicated the presence of an up-regulation of the pentose phosphate pathway in aggressive tumors. In total, 14 proteins were excreted and could potentially become biomarkers. Overexpression of transforming growth factor, beta-induced (TGFBI) in ccRCC was confirmed using Western blot and immunohistochemistry analysis. A significant association was found between the presence of TGFBI expression with tumor category T3-4 (P<0.0001), Fuhrman grades III and IV (P<0.0001), tumor size>4cm (P<0.0001), presence of tumor necrosis (P<0.0001), nodal involvement (n = 0.009), metastasis (P = 0.012), SSIGN score≥5 (P<0.0001), cancer progression (P<0.0001), and cancer-specific death (P<0.0001). Cancer-specific survival was significantly better for patients with no cytoplasmic TGFBI expression (1-, 3-, 5-y cancer-specific survival of 94.7%, 87.8%, and 73.4% vs. 92.9%, 71.2%, and 49.8%, respectively; P<0.0001). CONCLUSION: We identified 346 proteins involved in renal carcinogenesis and confirmed the presence of a metabolic shift in aggressive tumors. TGFBI was overexpressed in tumors with high SSIGN scores and was significantly associated with oncologic outcomes.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Renais/metabolismo , Neoplasias Renais/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Biomarcadores Tumorais/genética , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Feminino , Humanos , Neoplasias Renais/genética , Masculino , Espectrometria de Massas , Prognóstico , Proteômica , Fator de Crescimento Transformador beta1/genética
11.
Carcinogenesis ; 35(12): 2670-8, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25233928

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is driven by the accumulation of somatic mutations, epigenetic modifications and changes in the micro-environment. New approaches to investigating disruptions of gene expression networks promise to uncover key regulators and pathways in carcinogenesis. We performed messenger RNA-sequencing in pancreatic normal (n = 10) and tumor (n = 8) derived tissue samples, as well as in pancreatic cancer cell lines (n = 9), to determine differential gene expression (DE) patterns. Sub-network enrichment analyses identified HNF1A as the regulator of the most significantly and consistently dysregulated expression sub-network in pancreatic tumor tissues and cells (median P = 7.56×10(-7), median rank = 1, range = 1-25). To explore the effects of HNF1A expression in pancreatic tumor-derived cells, we generated stable HNF1A-inducible clones in two pancreatic cancer cell lines (PANC-1 and MIA PaCa-2) and observed growth inhibition (5.3-fold, P = 4.5×10(-5) for MIA PaCa-2 clones; 7.2-fold, P = 2.2×10(-5) for PANC-1 clones), and a G0/G1 cell cycle arrest and apoptosis upon induction. These effects correlated with HNF1A-induced down-regulation of 51 of 84 cell cycle genes (e.g. E2F1, CDK2, CDK4, MCM2/3/4/5, SKP2 and CCND1), decreased expression of anti-apoptotic genes (e.g. BIRC2/5/6 and AKT) and increased expression of pro-apoptotic genes (e.g. CASP4/9/10 and APAF1). In light of the established role of HNF1A in the regulation of pancreatic development and homeostasis, our data suggest that it also functions as an important tumor suppressor in the pancreas.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma Ductal Pancreático/genética , Perfilação da Expressão Gênica , Genes Supressores de Tumor , Fator 1-alfa Nuclear de Hepatócito/genética , Neoplasias Pancreáticas/genética , Apoptose , Biomarcadores Tumorais/metabolismo , Western Blotting , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Ciclo Celular , Proliferação de Células , Células Cultivadas , Citometria de Fluxo , Redes Reguladoras de Genes , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Humanos , Técnicas Imunoenzimáticas , Pâncreas/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
12.
Thyroid ; 24(10): 1524-32, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25135760

RESUMO

BACKGROUND: Cigarette smoking is a risk factor for the development of Graves' ophthalmopathy (GO). In a previous study of gene expression in intraorbital fat, adipocyte-related immediate early genes (IEGs) were overexpressed in patients with GO compared to controls. We investigated whether IEGs are upregulated by smoking, and examined other pathways that may be affected by smoking. METHODS: Gene expression in intraorbital fat was studied in smokers (n=8) and nonsmokers (n=8) with severe active GO, as well as in subcutaneous fat in thyroid-healthy smokers (n=5) and nonsmokers (n=5) using microarray and real-time polymerase chain reaction (PCR). RESULTS: With microarray, eight IEGs were upregulated more than 1.5-fold in smokers compared to nonsmokers with GO. Five were chosen for confirmation and were also overexpressed with real-time PCR. Interleukin-1 beta/IL-1B/(2.3-fold) and interleukin-6/IL-6/(2.4-fold) were upregulated both with microarray and with real-time PCR in smokers with GO compared to nonsmokers. Major histocompatibility complex, class II, DR beta 1/HLA-DRB1/was upregulated with microarray (2.1-fold) and with borderline significance with real-time PCR. None of these genes were upregulated in smokers compared to nonsmokers in subcutaneous fat. CONCLUSIONS: IEGs, IL-1B, and IL-6 were overexpressed in smokers with severe active GO compared to nonsmokers, suggesting that smoking activates pathways associated with adipogenesis and inflammation. This study underlines the importance of IEGs in the pathogenesis of GO, and provides evidence for possible novel therapeutic interventions in GO. The mechanisms activated by smoking may be shared with other conditions such as rheumatoid arthritis.


Assuntos
Tecido Adiposo/química , Genes Precoces , Oftalmopatia de Graves/genética , Fumar/efeitos adversos , Fumar/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Perfilação da Expressão Gênica/métodos , Marcadores Genéticos , Oftalmopatia de Graves/diagnóstico , Oftalmopatia de Graves/terapia , Humanos , Interleucina-1beta/genética , Interleucina-6/genética , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Risco , Índice de Gravidade de Doença , Regulação para Cima
13.
Hum Mol Genet ; 23(24): 6616-33, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-25027329

RESUMO

Genome-wide association studies (GWAS) have mapped risk alleles for at least 10 distinct cancers to a small region of 63 000 bp on chromosome 5p15.33. This region harbors the TERT and CLPTM1L genes; the former encodes the catalytic subunit of telomerase reverse transcriptase and the latter may play a role in apoptosis. To investigate further the genetic architecture of common susceptibility alleles in this region, we conducted an agnostic subset-based meta-analysis (association analysis based on subsets) across six distinct cancers in 34 248 cases and 45 036 controls. Based on sequential conditional analysis, we identified as many as six independent risk loci marked by common single-nucleotide polymorphisms: five in the TERT gene (Region 1: rs7726159, P = 2.10 × 10(-39); Region 3: rs2853677, P = 3.30 × 10(-36) and PConditional = 2.36 × 10(-8); Region 4: rs2736098, P = 3.87 × 10(-12) and PConditional = 5.19 × 10(-6), Region 5: rs13172201, P = 0.041 and PConditional = 2.04 × 10(-6); and Region 6: rs10069690, P = 7.49 × 10(-15) and PConditional = 5.35 × 10(-7)) and one in the neighboring CLPTM1L gene (Region 2: rs451360; P = 1.90 × 10(-18) and PConditional = 7.06 × 10(-16)). Between three and five cancers mapped to each independent locus with both risk-enhancing and protective effects. Allele-specific effects on DNA methylation were seen for a subset of risk loci, indicating that methylation and subsequent effects on gene expression may contribute to the biology of risk variants on 5p15.33. Our results provide strong support for extensive pleiotropy across this region of 5p15.33, to an extent not previously observed in other cancer susceptibility loci.


Assuntos
Cromossomos Humanos Par 5/química , Regulação Neoplásica da Expressão Gênica , Loci Gênicos , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Telomerase/genética , Alelos , Biologia Computacional , Metilação de DNA , Epigênese Genética , Feminino , Frequência do Gene , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Masculino , Neoplasias/patologia , Razão de Chances , Polimorfismo de Nucleotídeo Único , Risco
14.
Cancer Res ; 74(10): 2785-95, 2014 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-24648346

RESUMO

Genome-wide association studies (GWAS) of 10 different cancers have identified pleiotropic cancer predisposition loci across a region of chromosome 5p15.33 that includes the TERT and CLPTM1L genes. Of these, susceptibility alleles for pancreatic cancer have mapped to the CLPTM1L gene, thus prompting an investigation of the function of CLPTM1L in the pancreas. Immunofluorescence analysis indicated that CLPTM1L localized to the endoplasmic reticulum where it is likely embedded in the membrane, in accord with multiple predicted transmembrane domains. Overexpression of CLPTM1L enhanced growth of pancreatic cancer cells in vitro (1.3-1.5-fold; PDAY7 < 0.003) and in vivo (3.46-fold; PDAY68 = 0.039), suggesting a role in tumor growth; this effect was abrogated by deletion of two hydrophilic domains. Affinity purification followed by mass spectrometry identified an interaction between CLPTM1L and non-muscle myosin II (NMM-II), a protein involved in maintaining cell shape, migration, and cytokinesis. The two proteins colocalized in the cytoplasm and, after treatment with a DNA-damaging agent, at the centrosomes. Overexpression of CLPTM1L and depletion of NMM-II induced aneuploidy, indicating that CLPTM1L may interfere with normal NMM-II function in regulating cytokinesis. Immunohistochemical analysis revealed enhanced staining of CLPTM1L in human pancreatic ductal adenocarcinoma (n = 378) as compared with normal pancreatic tissue samples (n = 17; P = 1.7 × 10(-4)). Our results suggest that CLPTM1L functions as a growth-promoting gene in the pancreas and that overexpression may lead to an abrogation of normal cytokinesis, indicating that it should be considered as a plausible candidate gene that could explain the effect of pancreatic cancer susceptibility alleles on chr5p15.33.


Assuntos
Carcinoma Ductal Pancreático/patologia , Proteínas de Membrana/biossíntese , Proteínas de Neoplasias/biossíntese , Neoplasias Pancreáticas/patologia , Aneuploidia , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Feminino , Células HEK293 , Xenoenxertos , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Nus , Miosina Tipo II/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Frações Subcelulares/metabolismo
15.
BMC Med Genomics ; 6: 33, 2013 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-24053169

RESUMO

BACKGROUND: Pancreatic cancer is a highly lethal cancer with limited diagnostic and therapeutic modalities. METHODS: To begin to explore the genomic landscape of pancreatic cancer, we used massively parallel sequencing to catalog and compare transcribed regions and potential regulatory elements in two human cell lines derived from normal and cancerous pancreas. RESULTS: By RNA-sequencing, we identified 2,146 differentially expressed genes in these cell lines that were enriched in cancer related pathways and biological processes that include cell adhesion, growth factor and receptor activity, signaling, transcription and differentiation. Our high throughput Chromatin immunoprecipitation (ChIP) sequence analysis furthermore identified over 100,000 regions enriched in epigenetic marks, showing either positive (H3K4me1, H3K4me3, RNA Pol II) or negative (H3K27me3) correlation with gene expression. Notably, an overall enrichment of RNA Pol II binding and depletion of H3K27me3 binding were seen in the cancer derived cell line as compared to the normal derived cell line. By selecting genes for further assessment based on this difference, we confirmed enhanced expression of aldehyde dehydrogenase 1A3 (ALDH1A3) in two larger sets of pancreatic cancer cell lines and in tumor tissues as compared to normal derived tissues. CONCLUSIONS: As aldehyde dehydrogenase (ALDH) activity is a key feature of cancer stem cells, our results indicate that a member of the ALDH superfamily, ALDH1A3, may be upregulated in pancreatic cancer, where it could mark pancreatic cancer stem cells.


Assuntos
Aldeído Oxirredutases/genética , Epigenômica , Perfilação da Expressão Gênica , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/genética , Linhagem Celular Tumoral , Humanos , MicroRNAs/genética , Neoplasias Pancreáticas/patologia , RNA Mensageiro/genética , Análise de Sequência de RNA
16.
PLoS One ; 8(4): e60083, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23573233

RESUMO

BACKGROUND: Genome-wide association studies (GWAS) have identified multiple SNPs associated with prostate cancer (PrCa). Population isolates may have different sets of risk alleles for PrCa constituting unique population and individual risk profiles. METHODS: To test this hypothesis, associations between 31 GWAS SNPs of PrCa were examined among 979 PrCa cases and 1,251 controls of Ashkenazic descent using logistic regression. We also investigated risks by age at diagnosis, pathological features of PrCa, and family history of cancer. Moreover, we examined associations between cumulative number of risk alleles and PrCa and assessed the utility of risk alleles in PrCa risk prediction by comparing the area under the curve (AUC) for different logistic models. RESULTS: Of the 31 genotyped SNPs, 8 were associated with PrCa at p ≤ 0.002 (corrected p-value threshold) with odds ratios (ORs) ranging from 1.22 to 1.42 per risk allele. Four SNPs were associated with aggressive PrCa, while three other SNPs showed potential interactions for PrCa by family history of PrCa (rs8102476; 19q13), lung cancer (rs17021918; 4q22), and breast cancer (rs10896449; 11q13). Men in the highest vs. lowest quartile of cumulative number of risk alleles had ORs of 3.70 (95% CI 2.76-4.97); 3.76 (95% CI 2.57-5.50), and 5.20 (95% CI 2.94-9.19) for overall PrCa, aggressive cancer and younger age at diagnosis, respectively. The addition of cumulative risk alleles to the model containing age at diagnosis and family history of PrCa yielded a slightly higher AUC (0.69 vs. 0.64). CONCLUSION: These data define a set of risk alleles associated with PrCa in men of Ashkenazic descent and indicate possible genetic differences for PrCa between populations of European and Ashkenazic ancestry. Use of genetic markers might provide an opportunity to identify men at highest risk for younger age of onset PrCa; however, their clinical utility in identifying men at highest risk for aggressive cancer remains limited.


Assuntos
Polimorfismo de Nucleotídeo Único , Neoplasias da Próstata/genética , Alelos , Área Sob a Curva , Estudos de Casos e Controles , Cromossomos Humanos , Frequência do Gene , Estudo de Associação Genômica Ampla , Humanos , Judeus/genética , Masculino , Pessoa de Meia-Idade , Razão de Chances , Linhagem , Curva ROC , Risco , Análise de Sequência de DNA
17.
Pancreas ; 42(2): 209-15, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23295781

RESUMO

OBJECTIVE: The objective of this study was to fine-map common pancreatic cancer susceptibility regions. METHODS: We conducted targeted Roche-454 resequencing across 428 kb in 3 genomic regions identified in genome-wide association studies (GWAS) of pancreatic cancer, on chromosomes 1q32.1, 5p15.33, and 13q22.1. RESULTS: An analytical pipeline for calling genotypes was developed using HapMap samples sequenced on chr5p15.33. Concordance to 1000 Genomes data for chr5p15.33 was greater than 96%. The concordance for chr1q32.1 and chr13q22.1 with pancreatic cancer GWAS data was greater than 99%. Between 9.2% and 19.0% of variants detected were not present in 1000 Genomes for the respective continental population. The majority of completely novel single-nucleotide polymorphisms (SNPs) were less common (minor allele frequency [MAF], ≤5%) or rare (MAF, ≤2%), illustrating the value of enlarging test sets for discovery of less common variants. Using the data set, we examined haplotype blocks across each region using a tag SNP analysis (r² > 0.8 for MAF of ≥5%) and determined that at least 196, 243, and 63 SNPs are required for fine-mapping chr1q32.1, chr5p15.33, and chr13q22.1, respectively, in European populations. CONCLUSIONS: We have characterized germline variation in 3 regions associated with pancreatic cancer risk and show that targeted resequencing leads to the discovery of novel variants and improves the completeness of germline sequence variants for fine-mapping GWAS susceptibility loci.


Assuntos
Cromossomos Humanos Par 13 , Cromossomos Humanos Par 1 , Cromossomos Humanos Par 5 , Loci Gênicos , Neoplasias Pancreáticas/genética , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , Grupos de Populações Continentais/genética , Bases de Dados Genéticas , Frequência do Gene , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Haplótipos , Humanos , Desequilíbrio de Ligação , Neoplasias Pancreáticas/etnologia , Medição de Risco , Fatores de Risco
18.
Diabetes ; 61(12): 3322-32, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23028138

RESUMO

To identify epigenetic patterns, which may predispose to type 2 diabetes (T2D) due to a family history (FH) of the disease, we analyzed DNA methylation genome-wide in skeletal muscle from individuals with (FH(+)) or without (FH(-)) an FH of T2D. We found differential DNA methylation of genes in biological pathways including mitogen-activated protein kinase (MAPK), insulin, and calcium signaling (P ≤ 0.007) and of individual genes with known function in muscle, including MAPK1, MYO18B, HOXC6, and the AMP-activated protein kinase subunit PRKAB1 in skeletal muscle of FH(+) compared with FH(-) men. We further validated our findings from FH(+) men in monozygotic twin pairs discordant for T2D, and 40% of 65 analyzed genes exhibited differential DNA methylation in muscle of both FH(+) men and diabetic twins. We further examined if a 6-month exercise intervention modifies the genome-wide DNA methylation pattern in skeletal muscle of the FH(+) and FH(-) individuals. DNA methylation of genes in retinol metabolism and calcium signaling pathways (P < 3 × 10(-6)) and with known functions in muscle and T2D including MEF2A, RUNX1, NDUFC2, and THADA decreased after exercise. Methylation of these human promoter regions suppressed reporter gene expression in vitro. In addition, both expression and methylation of several genes, i.e., ADIPOR1, BDKRB2, and TRIB1, changed after exercise. These findings provide new insights into how genetic background and environment can alter the human epigenome.


Assuntos
Metilação de DNA/fisiologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/terapia , Exercício/fisiologia , Músculo Esquelético/metabolismo , Adulto , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Metilação de DNA/genética , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Domínio MADS/genética , Fatores de Transcrição MEF2 , Masculino , Fatores de Regulação Miogênica/genética , Proteínas de Neoplasias/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Receptores de Adiponectina/genética
19.
Cell Metab ; 16(1): 122-34, 2012 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-22768844

RESUMO

Close to 50 genetic loci have been associated with type 2 diabetes (T2D), but they explain only 15% of the heritability. In an attempt to identify additional T2D genes, we analyzed global gene expression in human islets from 63 donors. Using 48 genes located near T2D risk variants, we identified gene coexpression and protein-protein interaction networks that were strongly associated with islet insulin secretion and HbA(1c). We integrated our data to form a rank list of putative T2D genes, of which CHL1, LRFN2, RASGRP1, and PPM1K were validated in INS-1 cells to influence insulin secretion, whereas GPR120 affected apoptosis in islets. Expression variation of the top 20 genes explained 24% of the variance in HbA(1c) with no claim of the direction. The data present a global map of genes associated with islet dysfunction and demonstrate the value of systems genetics for the identification of genes potentially involved in T2D.


Assuntos
Diabetes Mellitus Tipo 2/genética , Ilhotas Pancreáticas/metabolismo , Mapas de Interação de Proteínas/genética , Idoso , Animais , Estudos de Casos e Controles , Linhagem Celular , Diabetes Mellitus Tipo 2/patologia , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla , Humanos , Insulina/metabolismo , Secreção de Insulina , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único , Ratos , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo , Biologia de Sistemas
20.
Am J Clin Nutr ; 96(1): 196-207, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22648723

RESUMO

BACKGROUND: Differential gene expression in adipose tissue during diet-induced weight loss followed by a weight stability period is poorly characterized. Markers of these processes may provide a deeper understanding of underlying mechanisms. OBJECTIVE: We aimed to identify differentially expressed genes in human adipose tissue during weight loss and weight maintenance after weight loss. DESIGN: RNA from subcutaneous abdominal adipose tissue from 9 obese subjects was analyzed by using a complementary DNA microarray at baseline after weight loss on a low-calorie diet and after weight maintenance. RESULTS: Subjects lost 18.8 ± 1.8% of weight and maintained this loss during weight maintenance (1.1 ± 2.1%; range: -9.3 to 10.6%). Most differentially expressed genes exhibited a reciprocal regulation and returned to baseline after weight loss (2163 genes) and weight maintenance (3175 genes). CETP and ABCG1, both of which participate in the HDL-mediated reverse cholesterol transport (RCT), were among the most upregulated of the 750 genes that were differentially expressed after both processes. Several genes involved in inflammation were downregulated. The use of real-time polymerase chain reaction confirmed or partially confirmed the previously implicated genes TNMD and MMP9 (both downregulated), PNPLA3 (upregulated), and CIDEA and SCD (both reciprocally regulated). CONCLUSIONS: The beneficial effects of weight loss should be investigated after long-term weight maintenance. The processes of weight loss and weight maintenance should be viewed as biologically distinct. CETP and ABCG1 may be important mediators of these effects through HDL-mediated RCT.


Assuntos
Regulação da Expressão Gênica , Obesidade/metabolismo , Gordura Subcutânea Abdominal/metabolismo , Perda de Peso , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adulto , Biomarcadores/metabolismo , Biópsia , Índice de Massa Corporal , Proteínas de Transferência de Ésteres de Colesterol/genética , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Dieta Redutora , Feminino , Seguimentos , Perfilação da Expressão Gênica , Humanos , Masculino , Obesidade/dietoterapia , Obesidade/patologia , Obesidade/prevenção & controle , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , Prevenção Secundária , Gordura Subcutânea Abdominal/patologia
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