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1.
Nat Genet ; 38(9): 1049-54, 2006 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-16936733

RESUMEN

In developed countries, age-related macular degeneration is a common cause of blindness in the elderly. A common polymorphism, encoding the sequence variation Y402H in complement factor H (CFH), has been strongly associated with disease susceptibility. Here, we examined 84 polymorphisms in and around CFH in 726 affected individuals (including 544 unrelated individuals) and 268 unrelated controls. In this sample, 20 of these polymorphisms showed stronger association with disease susceptibility than the Y402H variant. Further, no single polymorphism could account for the contribution of the CFH locus to disease susceptibility. Instead, multiple polymorphisms defined a set of four common haplotypes (of which two were associated with disease susceptibility and two seemed to be protective) and multiple rare haplotypes (associated with increased susceptibility in aggregate). Our results suggest that there are multiple disease susceptibility alleles in the region and that noncoding CFH variants play a role in disease susceptibility.


Asunto(s)
Factor H de Complemento/genética , Haplotipos , Degeneración Macular/genética , Anciano , Estudios de Casos y Controles , Susceptibilidad a Enfermedades , Frecuencia de los Genes , Humanos , Modelos Logísticos , Degeneración Macular/patología , Polimorfismo de Nucleótido Simple
2.
Blood Cells Mol Dis ; 52(1): 35-45, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23993337

RESUMEN

In congenital Chuvash polycythemia (CP), VHL(R200W) homozygosity leads to elevated hypoxia inducible factor (HIF) levels at normoxia. CP is often treated by phlebotomy resulting in iron deficiency, permitting us to examine the separate and synergistic effects of iron deficiency and HIF signaling on gene expression. We compared peripheral blood mononuclear cell gene expression profiles of eight VHL(R200W) homozygotes with 17 wildtype individuals with normal iron status and found 812 up-regulated and 2120 down-regulated genes at false discovery rate of 0.05. Among differential genes we identified three major gene regulation modules involving induction of innate immune responses, alteration of carbohydrate and lipid metabolism, and down-regulation of cell proliferation, stress-induced apoptosis and T-cell activation. These observations suggest molecular mechanisms for previous observations in CP of lower blood sugar without increased insulin and low oncogenic potential. Studies including 16 additional VHL(R200W) homozygotes with low ferritin indicated that iron deficiency enhanced the induction effect of VHL(R200W) for 50 genes including hemoglobin synthesis loci but suppressed the effect for 107 genes enriched for HIF-2 targets. This pattern is consistent with potentiation of HIF-1α protein stability by iron deficiency but a trend for down-regulation of HIF-2α translation by iron deficiency overriding an increase in HIF-2α protein stability.


Asunto(s)
Anemia Ferropénica/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Hipoxia/genética , Hierro/metabolismo , Policitemia/congénito , Anemia Ferropénica/etiología , Anemia Ferropénica/inmunología , Anemia Ferropénica/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Glucemia/metabolismo , Regulación de la Expresión Génica , Homocigoto , Humanos , Hipoxia/inmunología , Hipoxia/metabolismo , Hipoxia/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Inmunidad Innata , Insulina/sangre , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/patología , Flebotomía/efectos adversos , Policitemia/genética , Policitemia/inmunología , Policitemia/metabolismo , Policitemia/patología , Estabilidad Proteica , Transducción de Señal , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo
3.
Proc Natl Acad Sci U S A ; 108(49): E1321-9, 2011 Dec 06.
Artículo en Inglés | MEDLINE | ID: mdl-22065740

RESUMEN

Thyroid hormone (TH) metabolism, mediated by deiodinase types 1, 2, and 3 (D1, D2, and D3) is profoundly affected by acute illness. We examined the role of TH metabolism during ventilator-induced lung injury (VILI) in mice. Mice exposed to VILI recapitulated the serum TH findings of acute illness, namely a decrease in 3,5,3'-triiodothyronine (T(3)) and thyroid-stimulating hormone and an increase in reverse T(3). Both D2 immunoreactivity and D2 enzymatic activity were increased significantly. D1 and D3 activity did not change. Using D2 knockout (D2KO) mice, we determined whether the increase in D2 was an adaptive response. Although similar changes in serum TH levels were observed in D2KO and WT mice, D2KO mice exhibited greater susceptibility to VILI than WT mice, as evidenced by poorer alveoli integrity and quantified by lung chemokine and cytokine mRNA induction. These data suggest that an increase in lung D2 is protective against VILI. Similar findings of increased inflammatory markers were found in hypothyroid WT mice exposed to VILI compared with euthyroid mice, indicating that the lungs were functionally hypothyroid. Treatment of D2KO mice with T(3) reversed many of the lung chemokine and cytokine profiles seen in response to VILI, demonstrating a role for T(3) in the treatment of lung injury. We conclude that TH metabolism in the lung is linked to the response to inflammatory injury and speculate that D2 exerts its protective effect by making more TH available to the injured lung tissue.


Asunto(s)
Lesión Pulmonar Aguda/enzimología , Yoduro Peroxidasa/metabolismo , Pulmón/enzimología , Lesión Pulmonar Inducida por Ventilación Mecánica/enzimología , Lesión Pulmonar Aguda/sangre , Lesión Pulmonar Aguda/genética , Animales , Quimiocinas/genética , Citocinas/genética , Activación Enzimática/fisiología , Expresión Génica/efectos de los fármacos , Genotipo , Inmunohistoquímica , Yoduro Peroxidasa/genética , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Endogámicos , Ratones Noqueados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad de la Especie , Tirotropina/sangre , Tiroxina/sangre , Triyodotironina/sangre , Triyodotironina/farmacología , Lesión Pulmonar Inducida por Ventilación Mecánica/sangre , Lesión Pulmonar Inducida por Ventilación Mecánica/genética , Yodotironina Deyodinasa Tipo II
4.
Am J Physiol Lung Cell Mol Physiol ; 305(7): L467-77, 2013 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-23911438

RESUMEN

The genetic mechanisms underlying the susceptibility to acute respiratory distress syndrome (ARDS) are poorly understood. We previously demonstrated that sphingosine 1-phosphate (S1P) and the S1P receptor S1PR3 are intimately involved in lung inflammatory responses and vascular barrier regulation. Furthermore, plasma S1PR3 protein levels were shown to serve as a biomarker of severity in critically ill ARDS patients. This study explores the contribution of single nucleotide polymorphisms (SNPs) of the S1PR3 gene to sepsis-associated ARDS. S1PR3 SNPs were identified by sequencing the entire gene and tagging SNPs selected for case-control association analysis in African- and ED samples from Chicago, with independent replication in a European case-control study of Spanish individuals. Electrophoretic mobility shift assays, luciferase activity assays, and protein immunoassays were utilized to assess the functionality of associated SNPs. A total of 80 variants, including 29 novel SNPs, were identified. Because of limited sample size, conclusive findings could not be drawn in African-descent ARDS subjects; however, significant associations were found for two promoter SNPs (rs7022797 -1899T/G; rs11137480 -1785G/C), across two ED samples supporting the association of alleles -1899G and -1785C with decreased risk for sepsis-associated ARDS. In addition, these alleles significantly reduced transcription factor binding to the S1PR3 promoter; reduced S1PR3 promoter activity, a response particularly striking after TNF-α challenge; and were associated with lower plasma S1PR3 protein levels in ARDS patients. These highly functional studies support S1PR3 as a novel ARDS candidate gene and a potential target for individualized therapy.


Asunto(s)
Regiones Promotoras Genéticas , Receptores de Lisoesfingolípidos/genética , Síndrome de Dificultad Respiratoria/genética , Sepsis/complicaciones , Secuencia de Bases , Biomarcadores/sangre , Estudios de Casos y Controles , Ensayo de Cambio de Movilidad Electroforética , Femenino , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Lisofosfolípidos/metabolismo , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Receptores de Lisoesfingolípidos/sangre , Síndrome de Dificultad Respiratoria/sangre , Síndrome de Dificultad Respiratoria/etiología , Análisis de Secuencia de ADN , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato
5.
Am J Respir Crit Care Med ; 186(4): 359-68, 2012 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-22679008

RESUMEN

RATIONALE: An increased tricuspid regurgitation jet velocity (TRV > 2.5 m/s) and pulmonary hypertension defined by right heart catheterization both independently confer increased mortality in sickle cell disease (SCD). OBJECTIVES: We explored the usefulness of peripheral blood mononuclear cell-derived gene signatures as biomarkers for an elevated TRV in SCD. METHODS: Twenty-seven patients with SCD underwent echocardiography and peripheral blood mononuclear cell isolation for expression profiling and 112 patients with SCD were genotyped for single-nucleotide polymorphisms. MEASUREMENTS AND MAIN RESULTS: Genome-wide gene and miRNA expression profiles were correlated against TRV, yielding 631 transcripts and 12 miRNAs. Support vector machine analysis identified a 10-gene signature including GALNT13 (encoding polypeptide N-acetylgalactosaminyltransferase 13) that discriminates patients with and without increased TRV with 100% accuracy. This finding was then validated in a cohort of patients with SCD without (n = 10) and with pulmonary hypertension (n = 10, 90% accuracy). Increased TRV-related miRNAs revealed strong in silico binding predictions of miR-301a to GALNT13 corroborated by microarray analyses demonstrating an inverse correlation between their expression. A genetic association study comparing patients with an elevated (n = 49) versus normal (n = 63) TRV revealed five significant single-nucleotide polymorphisms within GALNT13 (P < 0.005), four trans-acting (P < 2.1 × 10(-7)) and one cis-acting (P = 0.6 × 10(-4)) expression quantitative trait locus upstream of the adenosine-A2B receptor gene (ADORA2B). CONCLUSIONS: These studies validate the clinical usefulness of genomic signatures as potential biomarkers and highlight ADORA2B and GALNT13 as potential candidate genes in SCD-associated elevated TRV.


Asunto(s)
Anemia de Células Falciformes/genética , Anemia de Células Falciformes/fisiopatología , N-Acetilgalactosaminiltransferasas/genética , Receptor de Adenosina A2B/genética , Insuficiencia de la Válvula Tricúspide/genética , Insuficiencia de la Válvula Tricúspide/fisiopatología , Adulto , Anemia de Células Falciformes/diagnóstico por imagen , Cateterismo Cardíaco , Estudios de Cohortes , Ecocardiografía Doppler/métodos , Femenino , Perfilación de la Expresión Génica/métodos , Marcadores Genéticos/genética , Estudio de Asociación del Genoma Completo/métodos , Humanos , Hipertensión Pulmonar/diagnóstico por imagen , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/fisiopatología , Masculino , Análisis por Micromatrices/métodos , Polimorfismo de Nucleótido Simple/genética , Arteria Pulmonar/diagnóstico por imagen , Arteria Pulmonar/fisiopatología , Reproducibilidad de los Resultados , Máquina de Vectores de Soporte , Insuficiencia de la Válvula Tricúspide/diagnóstico por imagen , Polipéptido N-Acetilgalactosaminiltransferasa
6.
Am J Respir Cell Mol Biol ; 47(5): 628-36, 2012 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-22771388

RESUMEN

The inflamed lung exhibits oxidative and nitrative modifications of multiple target proteins, potentially reflecting disease severity and progression. We identified sphingosine-1-phosphate receptor-3 (S1PR3), a critical signaling molecule mediating cell proliferation and vascular permeability, as a nitrated plasma protein in mice with acute lung injury (ALI). We explored S1PR3 as a potential biomarker in murine and human ALI. In vivo nitrated and total S1PR3 concentrations were determined by immunoprecipitation and microarray studies in mice, and by ELISA in human plasma. In vitro nitrated S1PR3 concentrations were evaluated in human lung vascular endothelial cells (ECs) or within microparticles shed from ECs after exposure to barrier-disrupting agonists (LPS, low-molecular-weight hyaluronan, and thrombin). The effects of S1PR3-containing microparticles on EC barrier function were assessed by transendothelial electrical resistance (TER). Nitrated S1PR3 was identified in the plasma of murine ALI and in humans with severe sepsis-induced ALI. Elevated total S1PR3 plasma concentrations (> 251 pg/ml) were linked to sepsis and ALI mortality. In vitro EC exposure to barrier-disrupting agents induced S1PR3 nitration and the shedding of S1PR3-containing microparticles, which significantly reduced TER, consistent with increased permeability. These changes were attenuated by reduced S1PR3 expression (small interfering RNAs). These results suggest that microparticles containing nitrated S1PR3 shed into the circulation during inflammatory lung states, and represent a novel ALI biomarker linked to disease severity and outcome.


Asunto(s)
Lesión Pulmonar Aguda/sangre , Receptores de Lisoesfingolípidos/sangre , Lesión Pulmonar Aguda/inmunología , Lesión Pulmonar Aguda/mortalidad , Adulto , Anciano , Animales , Biomarcadores/sangre , Permeabilidad Capilar , Estudios de Casos y Controles , Micropartículas Derivadas de Células/metabolismo , Células Cultivadas , Impedancia Eléctrica , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Endotelio Vascular/patología , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Estimación de Kaplan-Meier , Lipopolisacáridos/farmacología , Pulmón/patología , Masculino , Ratones , Persona de Mediana Edad , Arteria Pulmonar/patología , Interferencia de ARN , Receptores de Lisoesfingolípidos/genética , Receptores de Esfingosina-1-Fosfato , Tirosina/análogos & derivados , Tirosina/sangre , Lesión Pulmonar Inducida por Ventilación Mecánica/metabolismo
7.
Am J Respir Cell Mol Biol ; 45(6): 1203-11, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21685153

RESUMEN

The role of thyroid hormone metabolism in clinical outcomes of the critically ill remains unclear. Using preclinical models of acute lung injury (ALI), we assessed the gene and protein expression of type 2 deiodinase (DIO2), a key driver for synthesis of biologically active triiodothyronine, and addressed potential association of DIO2 genetic variants with ALI in a multiethnic cohort. DIO2 gene and protein expression levels in murine lung were validated by microarrays and immunoblotting. Lung injury was assessed by levels of bronchoalveolar lavage protein and leukocytes. Single-nucleotide polymorphisms were genotyped and ALI susceptibility association assessed. Significant increases in both DIO2 gene and D2 protein expression were observed in lung tissues from murine ALI models (LPS- and ventilator-induced lung injury), with expression directly increasing with the extent of lung injury. Mice with reduced levels of DIO2 expression (by silencing RNA) demonstrated reduced thyroxine levels in plasma and increased lung injury (increased bronchoalveolar lavage protein and leukocytes), suggesting a protective role for DIO2 in ALI. The G (Ala) allele of the Thr92Ala coding single-nucleotide polymorphism (rs225014) was protective in severe sepsis and severe sepsis-associated ALI after adjustments for age, sex, and genetic ancestry in a logistic regression model in European Americans. Our studies indicate that DIO2 is a novel ALI candidate gene, the nonsynonymous Thr92Ala coding variant of which confers ALI protection. Increased DIO2 expression may dampen the ALI inflammatory response, thereby strengthening the premise that thyroid hormone metabolism is intimately linked to the integrated response to inflammatory injury in critically ill patients.


Asunto(s)
Lesión Pulmonar Aguda , Regulación Enzimológica de la Expresión Génica , Yoduro Peroxidasa , Polimorfismo de Nucleótido Simple , Sepsis , Hormonas Tiroideas/metabolismo , Lesión Pulmonar Aguda/enzimología , Lesión Pulmonar Aguda/etnología , Lesión Pulmonar Aguda/genética , Factores de Edad , Alelos , Animales , Estudios de Cohortes , Enfermedad Crítica , Modelos Animales de Enfermedad , Humanos , Yoduro Peroxidasa/biosíntesis , Yoduro Peroxidasa/genética , Pulmón/enzimología , Ratones , Sepsis/enzimología , Sepsis/etnología , Sepsis/genética , Factores Sexuales , Hormonas Tiroideas/genética , Yodotironina Deyodinasa Tipo II
8.
J Allergy Clin Immunol ; 126(2): 241-9, 249.e1-3, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20624651

RESUMEN

BACKGROUND: The genetic mechanisms underlying asthma remain unclear. Increased permeability of the microvasculature is a feature of asthma, and the sphingosine-1-phosphate receptor (S1PR1) is an essential participant regulating lung vascular integrity and responses to lung inflammation. OBJECTIVE: We explored the contribution of polymorphisms in the S1PR1 gene to asthma susceptibility. METHODS: A combination of gene resequencing for single nucleotide polymorphism (SNP) discovery, case-control association, functional evaluation of associated SNPs, and protein immunochemistry studies was used. RESULTS: Immunohistochemistry studies demonstrated significantly decreased S1PR1 protein expression in pulmonary vessels in lungs of asthmatic patients compared with those of nonasthmatic subjects (P < .05). Direct DNA sequencing of 27 multiethnic samples identified 39 S1PR1 variants (18 novel SNPs). Association studies were performed based on genotyping results from cosmopolitan tagging SNPs in 3 case-control cohorts from Chicago and New York totaling 1,061 subjects (502 cases and 559 control subjects). The promoter SNP rs2038366 (-1557G/T) was found to be associated with asthma (P = .03) in European Americans. In African Americans an association was found for both asthma and severe asthma for intronic SNP rs3753194 (c.-164+170A/G; P = .006 and P = .040, respectively) and for promoter SNP rs59317557 (-532C/G) with severe asthma (P = .028). Consistent with predicted in silico functionality, alleles of the promoter SNPs rs2038366 (-1557G/T) and rs59317557 (-532C/G) influenced the activity of a luciferase S1PR1 reporter vector in transfected endothelial cells exposed to growth factors (epidermal growth factor, platelet-derived growth factor, and vascular endothelial growth factor) known to be increased in asthmatic airways. CONCLUSION: These data provide strong support for a role for S1PR1 gene variants in asthma susceptibility and severity.


Asunto(s)
Alelos , Asma/genética , Predisposición Genética a la Enfermedad/genética , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas/genética , Receptores de Lisoesfingolípidos/genética , Negro o Afroamericano/genética , Asma/epidemiología , Asma/metabolismo , Asma/patología , Estudios de Casos y Controles , Línea Celular , Chicago , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Predisposición Genética a la Enfermedad/etnología , Genotipo , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular/farmacología , Pulmón/metabolismo , Pulmón/patología , Masculino , Ciudad de Nueva York , Receptores de Lisoesfingolípidos/biosíntesis , Índice de Severidad de la Enfermedad , Receptores de Esfingosina-1-Fosfato , Transfección , Población Blanca/genética
9.
Genet Epidemiol ; 32(8): 757-66, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-18521921

RESUMEN

Genomic regions with replicated linkage to asthma-related phenotypes likely harbor multiple susceptibility loci with relatively minor effects on disease susceptibility. The 11q13 chromosomal region has repeatedly been linked to asthma with five genes residing in this region with reported replicated associations. Cortactin, an actin-binding protein encoded by the CTTN gene in 11q13, constitutes a key regulator of cytoskeletal dynamics and contractile cell machinery, events facilitated by interaction with myosin light chain kinase; encoded by MYLK, a gene we recently reported as associated with severe asthma in African Americans. To evaluate potential association of CTTN gene variation with asthma susceptibility, CTTN exons and flanking regions were re-sequenced in 48 non-asthmatic multiethnic samples, leading to selection of nine tagging polymorphisms for case-control association studies in individuals of European and African descent. After ancestry adjustments, an intronic variant (rs3802780) was significantly associated with severe asthma (odds ratio [OR]: 1.71; 95% confidence interval [CI]: 1.20-2.43; p=0.003) in a joint analysis. Further analyses evidenced independent and additive effects of CTTN and MYLK risk variants for severe asthma susceptibility in African Americans (accumulated OR: 2.93, 95% CI: 1.40-6.13, p=0.004). These data suggest that CTTN gene variation may contribute to severe asthma and that the combined effects of CTTN and MYLK risk polymorphisms may further increase susceptibility to severe asthma in African Americans harboring both genetic variants.


Asunto(s)
Asma/etnología , Asma/genética , Cortactina/genética , Predisposición Genética a la Enfermedad , Variación Genética , Polimorfismo de Nucleótido Simple , Población Negra , Proteínas de Unión al Calcio/genética , Estudios de Casos y Controles , Cromosomas Humanos Par 11 , Citoesqueleto/metabolismo , Etnicidad , Humanos , Quinasa de Cadena Ligera de Miosina/genética , Oportunidad Relativa , Polimorfismo Genético , Población Blanca
10.
Pulm Circ ; 6(4): 465-471, 2016 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-28090288

RESUMEN

Pulmonary hypertension (PH), when it complicates sarcoidosis, carries a poor prognosis, in part because it is difficult to detect early in patients with worsening respiratory symptoms. Pathogenesis of sarcoidosis occurs via incompletely characterized mechanisms that are distinct from the mechanisms of pulmonary vascular remodeling well known to occur in conjunction with other chronic lung diseases. To address the need for a biomarker to aid in early detection as well as the gap in knowledge regarding the mechanisms of PH in sarcoidosis, we used genome-wide peripheral blood gene expression analysis and identified an 18-gene signature capable of distinguishing sarcoidosis patients with PH (n = 8), sarcoidosis patients without PH (n = 17), and healthy controls (n = 45). The discriminative accuracy of this 18-gene signature was 100% in separating sarcoidosis patients with PH from those without it. If validated in a large replicate cohort, this signature could potentially be used as a diagnostic molecular biomarker for sarcoidosis-associated PH.

11.
PLoS One ; 9(6): e100169, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24940746

RESUMEN

RATIONALE: Growth arrest DNA damage inducible alpha (GADD45a) is a stress-induced gene we have shown to participate in the pathophysiology of ventilator-induced lung injury (VILI) via regulation of mechanical stress-induced Akt ubiquitination and phosphorylation. The regulation of GADD45a expression by mechanical stress and its relationship with acute lung injury (ALI) susceptibility and severity, however, remains unknown. OBJECTIVES: We examined mechanical stress-dependent regulatory elements (MSRE) in the GADD45a promoter and the contribution of promoter polymorphisms in GADD45a expression and ALI susceptibility. METHODS AND RESULTS: Initial studies in GADD45a knockout and heterozygous mice confirmed the relationship of GADD45a gene dose to VILI severity. Human lung endothelial cells (EC) transfected with a luciferase vector containing the full length GADD45a promoter sequence (-771 to +223) demonstrated a >4 fold increase in GADD45a expression in response to 18% cyclic stretch (CS, 4 h) compared to static controls while specific promoter regions harboring CS-dependent MSRE were identified using vectors containing serial deletion constructs of the GADD45a promoter. In silico analyses of GADD45a promoter region (-371 to -133) revealed a potential binding site for specificity protein 1 (SP1), a finding supported by confirmed SP1 binding with the GADD45a promoter and by the significant attenuation of CS-dependent GADD45a promoter activity in response to SP1 silencing. Separately, case-control association studies revealed a significant association of a GADD45a promoter SNP at -589 (rs581000, G>C) with reduced ALI susceptibility. Subsequently, we found allelic variation of this SNP is associated with both differential GADD45a expression in mechanically stressed EC (18% CS, 4 h) and differential binding site of interferon regulatory factor 7 (IRF7) at this site. CONCLUSION: These results strongly support a functional role for GADD45a in ALI/VILI and identify a specific gene variant that confers risk for ALI.


Asunto(s)
Lesión Pulmonar Aguda/genética , Proteínas de Ciclo Celular/genética , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Lesión Pulmonar Inducida por Ventilación Mecánica/genética , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/patología , Adulto , Anciano , Animales , Sitios de Unión , Proteínas de Ciclo Celular/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/patología , Femenino , Dosificación de Gen , Regulación de la Expresión Génica , Genes Reporteros , Heterocigoto , Humanos , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , Pulmón/metabolismo , Pulmón/patología , Masculino , Mecanotransducción Celular , Ratones , Ratones Noqueados , Persona de Mediana Edad , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleótido Simple , Unión Proteica , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/metabolismo , Estrés Mecánico , Lesión Pulmonar Inducida por Ventilación Mecánica/metabolismo , Lesión Pulmonar Inducida por Ventilación Mecánica/patología
12.
PLoS One ; 9(2): e88001, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24505347

RESUMEN

BACKGROUND: Angiotensin I-converting enzyme (ACE) has two functional N- and C-domain active centers that display differences in the metabolism of biologically-active peptides including the hemoregulatory tetrapeptide, Ac-SDKP, hydrolysed preferentially by the N domain active center. Elevated Ac-SDKP concentrations are associated with reduced tissue fibrosis. RESULTS: We identified a patient of African descent exhibiting unusual blood ACE kinetics with reduced relative hydrolysis of two synthetic ACE substrates (ZPHL/HHL ratio) suggestive of the ACE N domain center inactivation. Inhibition of blood ACE activity by anti-catalytic mAbs and ACE inhibitors and conformational fingerprint of blood ACE suggested overall conformational changes in the ACE molecule and sequencing identified Ser333Trp substitution in the N domain of ACE. In silico analysis demonstrated S333W localized in the S1 pocket of the active site of the N domain with the bulky Trp adversely affecting binding of ACE substrates due to steric hindrance. Expression of mutant ACE (S333W) in CHO cells confirmed altered kinetic properties of mutant ACE and conformational changes in the N domain. Further, the S333W mutant displayed decreased ability (5-fold) to cleave the physiological substrate AcSDKP compared to wild-type ACE. CONCLUSIONS AND SIGNIFICANCE: A novel Ser333Trp ACE mutation results in dramatic changes in ACE kinetic properties and lowered clearance of Ac-SDKP. Individuals with this mutation (likely with significantly increased levels of the hemoregulatory tetrapeptide in blood and tissues), may confer protection against fibrosis.


Asunto(s)
Fibrosis/genética , Mutación/genética , Oligopéptidos/genética , Oligopéptidos/metabolismo , Péptidos/genética , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Línea Celular , Cricetulus , Fibrosis/metabolismo , Humanos , Cinética , Datos de Secuencia Molecular , Péptidos/metabolismo , Alineación de Secuencia
13.
Lancet Respir Med ; 1(4): 309-317, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24429156

RESUMEN

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a devastating disease that probably involves several genetic loci. Several rare genetic variants and one common single nucleotide polymorphism (SNP) of MUC5B have been associated with the disease. Our aim was to identify additional common variants associated with susceptibility and ultimately mortality in IPF. METHODS: First, we did a three-stage genome-wide association study (GWAS): stage one was a discovery GWAS; and stages two and three were independent case-control studies. DNA samples from European-American patients with IPF meeting standard criteria were obtained from several US centres for each stage. Data for European-American control individuals for stage one were gathered from the database of genotypes and phenotypes; additional control individuals were recruited at the University of Pittsburgh to increase the number. For controls in stages two and three, we gathered data for additional sex-matched European-American control individuals who had been recruited in another study. DNA samples from patients and from control individuals were genotyped to identify SNPs associated with IPF. SNPs identified in stage one were carried forward to stage two, and those that achieved genome-wide significance (p<5 × 10(-8)) in a meta-analysis were carried forward to stage three. Three case series with follow-up data were selected from stages one and two of the GWAS using samples with follow-up data. Mortality analyses were done in these case series to assess the SNPs associated with IPF that had achieved genome-wide significance in the meta-analysis of stages one and two. Finally, we obtained gene-expression profiling data for lungs of patients with IPF from the Lung Genomics Research Consortium and analysed correlation with SNP genotypes. FINDINGS: In stage one of the GWAS (542 patients with IPF, 542 control individuals matched one-by-one to cases by genetic ancestry estimates), we identified 20 loci. Six SNPs reached genome-wide significance in stage two (544 patients, 687 control individuals): three TOLLIP SNPs (rs111521887, rs5743894, rs5743890) and one MUC5B SNP (rs35705950) at 11p15.5; one MDGA2 SNP (rs7144383) at 14q21.3; and one SPPL2C SNP (rs17690703) at 17q21.31. Stage three (324 patients, 702 control individuals) confirmed the associations for all these SNPs, except for rs7144383. Linkage disequilibrium between the MUC5B SNP (rs35705950) and TOLLIP SNPs (rs111521887 [r(2)=0·07], rs5743894 [r(2)=0·16], and rs5743890 [r(2)=0·01]) was low. 683 patients from the GWAS were included in the mortality analysis. Individuals who developed IPF despite having the protective TOLLIP minor allele of rs5743890 carried an increased mortality risk (meta-analysis with fixed-effect model: hazard ratio 1·72 [95% CI 1·24-2·38]; p=0·0012). TOLLIP expression was decreased by 20% in individuals carrying the minor allele of rs5743890 (p=0·097), 40% in those with the minor allele of rs111521887 (p=3·0 × 10(-4)), and 50% in those with the minor allele of rs5743894 (p=2·93 × 10(-5)) compared with homozygous carriers of common alleles for these SNPs. INTERPRETATION: Novel variants in TOLLIP and SPPL2C are associated with IPF susceptibility. One novel variant of TOLLIP, rs5743890, is also associated with mortality. These associations and the reduced expression of TOLLIP in patients with IPF who carry TOLLIP SNPs emphasise the importance of this gene in the disease. FUNDING: National Institutes of Health; National Heart, Lung, and Blood Institute; Pulmonary Fibrosis Foundation; Coalition for Pulmonary Fibrosis; and Instituto de Salud Carlos III.


Asunto(s)
ADN/genética , Predisposición Genética a la Enfermedad , Variación Genética , Estudio de Asociación del Genoma Completo/métodos , Fibrosis Pulmonar Idiopática/genética , Adulto , Anciano , Femenino , Genotipo , Humanos , Fibrosis Pulmonar Idiopática/mortalidad , Desequilibrio de Ligamiento , Masculino , Persona de Mediana Edad , Fenotipo , Estudios Retrospectivos , Tasa de Supervivencia/tendencias , Estados Unidos/epidemiología
14.
J Mol Med (Berl) ; 90(3): 299-308, 2012 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-22015949

RESUMEN

Intronic single-nucleotide polymorphisms (SNPs) are commonly associated with complex diseases but exhibit unknown biologic functionality. Myosin light-chain kinase (MLCK), a central cytoskeletal regulator encoded by MYLK, plays a key pathophysiological role in complex diseases including acute lung injury (ALI) and asthma. We studied the potential regulatory roles of two intronic MYLK SNPs (rs936170 and rs820336) previously associated with ALI and asthma. Due to their genomic location at the junction encoding the non-muscle and smooth muscle MLCK (smMLCK) isoforms, we first identified the transcription start site (TSS) of the smMLCK isoform, and isolated a 2,954-bp DNA fragment upstream of the smMLCK TSS. Serial 5' deletion of the fragment revealed a proximal promoter region exhibiting strong promoter activity with potential inhibitory elements in the distal region. Site-directed mutageneses and luciferase reporter assays showed no effect of the distal promoter SNP rs936170 on smMLCK promoter activity. In contrast, SNP rs820336, located in an enhancer/repressor region downstream of TSS, was identified to regulate smMLCK promoter activity in an allelic-dependent manner. The A allele interrupted the binding site for Forkhead box protein N1 (FOXN1), a transcription factor governing expression of immune response genes. Silencing of FOXN1 expression (siRNA) reduced FOXN1 interaction with cis-regulatory elements in proximity to rs820336 and significantly decreased smMLCK expression. These functional insights into the involvement of intronic MYLK SNPs further strengthen the concept that MYLK contributes to inflammatory disease susceptibility and represents an attractive molecular target in complex inflammatory disorders.


Asunto(s)
Lesión Pulmonar Aguda/genética , Asma/genética , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Intrones/genética , Músculo Liso/enzimología , Quinasa de Cadena Ligera de Miosina/genética , Quinasa de Cadena Ligera de Miosina/metabolismo , Polimorfismo de Nucleótido Simple/genética , Lesión Pulmonar Aguda/fisiopatología , Animales , Asma/fisiopatología , Células Cultivadas , Ensayo de Cambio de Movilidad Electroforética , Elementos de Facilitación Genéticos , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Silenciador del Gen , Genes Reporteros , Predisposición Genética a la Enfermedad , Células HeLa , Humanos , Enfermedades Pulmonares/genética , Mutagénesis Sitio-Dirigida , Regiones Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratas
15.
PLoS One ; 7(1): e26807, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22235241

RESUMEN

BACKGROUND: Asthma is a common complex condition with clear racial and ethnic differences in both prevalence and severity. Asthma consultation rates, mortality, and severe symptoms are greatly increased in African descent populations of developed countries. African ancestry has been associated with asthma, total serum IgE and lower pulmonary function in African-admixed populations. To replicate previous findings, here we aimed to examine whether African ancestry was associated with asthma susceptibility in African Americans. In addition, we examined for the first time whether African ancestry was associated with asthma exacerbations. METHODOLOGY/PRINCIPAL FINDINGS: After filtering for self-reported ancestry and genotype data quality, samples from 1,117 self-reported African-American individuals from New York and Baltimore (394 cases, 481 controls), and Chicago (321 cases followed for asthma exacerbations) were analyzed. Genetic ancestry was estimated based on ancestry informative markers (AIMs) selected for being highly divergent among European and West African populations (95 AIMs for New York and Baltimore, and 66 independent AIMs for Chicago). Among case-control samples, the mean African ancestry was significantly higher in asthmatics than in non-asthmatics (82.0±14.0% vs. 77.8±18.1%, mean difference 4.2% [95% confidence interval (CI):2.0-6.4], p<0.0001). This association remained significant after adjusting for potential confounders (odds ratio: 4.55, 95% CI: 1.69-12.29, p = 0.003). African ancestry failed to show an association with asthma exacerbations (p = 0.965) using a model based on longitudinal data of the number of exacerbations followed over 1.5 years. CONCLUSIONS/SIGNIFICANCE: These data replicate previous findings indicating that African ancestry constitutes a risk factor for asthma and suggest that elevated asthma rates in African Americans can be partially attributed to African genetic ancestry.


Asunto(s)
Asma/etnología , Negro o Afroamericano/genética , África/etnología , Asma/epidemiología , Asma/genética , Estudios de Casos y Controles , Susceptibilidad a Enfermedades , Humanos , Modelos Logísticos , Riesgo
16.
PLoS One ; 7(9): e44818, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22984568

RESUMEN

Sarcoidosis, a systemic granulomatous syndrome invariably affecting the lung, typically spontaneously remits but in ~20% of cases progresses with severe lung dysfunction or cardiac and neurologic involvement (complicated sarcoidosis). Unfortunately, current biomarkers fail to distinguish patients with remitting (uncomplicated) sarcoidosis from other fibrotic lung disorders, and fail to identify individuals at risk for complicated sarcoidosis. We utilized genome-wide peripheral blood gene expression analysis to identify a 20-gene sarcoidosis biomarker signature distinguishing sarcoidosis (n = 39) from healthy controls (n = 35, 86% classification accuracy) and which served as a molecular signature for complicated sarcoidosis (n = 17). As aberrancies in T cell receptor (TCR) signaling, JAK-STAT (JS) signaling, and cytokine-cytokine receptor (CCR) signaling are implicated in sarcoidosis pathogenesis, a 31-gene signature comprised of T cell signaling pathway genes associated with sarcoidosis (TCR/JS/CCR) was compared to the unbiased 20-gene biomarker signature but proved inferior in prediction accuracy in distinguishing complicated from uncomplicated sarcoidosis. Additional validation strategies included significant association of single nucleotide polymorphisms (SNPs) in signature genes with sarcoidosis susceptibility and severity (unbiased signature genes - CX3CR1, FKBP1A, NOG, RBM12B, SENS3, TSHZ2; T cell/JAK-STAT pathway genes such as AKT3, CBLB, DLG1, IFNG, IL2RA, IL7R, ITK, JUN, MALT1, NFATC2, PLCG1, SPRED1). In summary, this validated peripheral blood molecular gene signature appears to be a valuable biomarker in identifying cases with sarcoidoisis and predicting risk for complicated sarcoidosis.


Asunto(s)
Biomarcadores/metabolismo , Regulación de la Expresión Génica , Sarcoidosis/sangre , Sarcoidosis/genética , Adulto , Estudios de Casos y Controles , Femenino , Perfilación de la Expresión Génica , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Genómica , Humanos , Masculino , Persona de Mediana Edad , Modelos Biológicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal
17.
Transl Res ; 152(1): 11-7, 2008 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-18593632

RESUMEN

Experimental and clinical studies support the key role of interleukin 6 (IL-6), a potent proinflammatory cytokine, in the development of acute lung injury (ALI). Plasma IL-6 levels are influenced mainly by genetic determinants, and a -174G/C polymorphism of the gene has been recently associated with susceptibility to ALI. Here we aimed to validate the association of the IL6 gene with ALI in a case-control sample from Spain. DNA was isolated from 67 consecutive patients who fulfilled international criteria for severe sepsis and for ALI and 96 population-based controls drawn from the general population. Genotypes of the -174G/C polymorphism along with other 14 tagging variants of the IL6 gene were evaluated. Twenty polymorphisms unlinked to IL6 gene were additionally compared between cases and controls to rule out population stratification. None of the individual single-nucleotide polymorphisms was significantly associated with susceptibility to ALI. However, we found that a common haplotype from -1363 to +4835 from the transcription start site, and spanning the gene, conferred risk for susceptibility to ALI (odds ratio, 2.73; 95% confidence interval, 1.39-5.37; P = 0.003). Adjustment for relevant covariates did not modify this result. These data support the association of the IL6 gene with ALI susceptibility and illustrate the value of haplotype analysis as a robust approach for evaluating IL6 gene effects in association studies.


Asunto(s)
Predisposición Genética a la Enfermedad , Haplotipos , Interleucina-6/genética , Síndrome de Dificultad Respiratoria/genética , APACHE , Anciano , Estudios de Casos y Controles , Estudios de Cohortes , Intervalos de Confianza , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Polimorfismo Genético , Factores de Riesgo , Población Blanca
18.
Cancer Res ; 68(24): 10205-14, 2008 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-19074888

RESUMEN

There are currently no effective therapies for metastatic melanoma and targeted immunotherapy results in the remission of only a very small percentage of tumors. In this study, we show that the noncanonical Wnt ligand, Wnt5A, can increase melanoma metastasis in vivo while down-regulating the expression of tumor-associated antigens important in eliciting CTL responses (e.g., MART-1, GP100, and tyrosinase). Melanosomal antigen expression is governed by MITF, PAX3, and SOX10 and is inhibited upon signal transducers and activators of transcription 3 (STAT3) activation, via decreases in PAX3 and subsequently MITF expression. Increasing Wnt5A in Wnt5A-low cells activated STAT3, and STAT3 was decreased upon Wnt5A knockdown. Downstream targets such as PAX3, MITF, and MART-1 were also affected by Wnt5A treatment or knockdown. Staining of a melanoma tissue array also highlighted the inverse relationship between MART-1 and Wnt5A expression. PKC activation by phorbol ester mimicked Wnt5A effects, and Wnt5A treatment in the presence of STAT3 or PKC inhibitors did not lower MART-1 levels. CTL activation studies showed that increases in Wnt5A correspond to decreased CTL activation and vice versa, suggesting that targeting Wnt5A before immunotherapy may lead to the enhancement of current targeted immunotherapy for patients with metastatic melanoma.


Asunto(s)
Antígenos de Neoplasias/biosíntesis , Melanoma Experimental/metabolismo , Melanoma/metabolismo , Proteínas de Neoplasias/biosíntesis , Factor de Transcripción STAT3/metabolismo , Proteínas Wnt/metabolismo , Animales , Antígenos de Neoplasias/genética , Humanos , Activación de Linfocitos , Antígeno MART-1 , Melanoma/genética , Melanoma/inmunología , Melanoma/patología , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Metástasis de la Neoplasia , Proteínas de Neoplasias/genética , Fosforilación , ARN Interferente Pequeño/genética , Linfocitos T/inmunología , Transcripción Genética , Transfección , Proteínas Wnt/biosíntesis , Proteínas Wnt/genética , Proteína Wnt-5a
19.
Exp Cell Res ; 313(3): 450-61, 2007 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-17188679

RESUMEN

Microarray technology was utilized to isolate disease-specific changes in gene expression by sampling across inferior parietal lobes of patients suffering from late onset AD or non-AD-associated dementia and non-demented controls. Primary focus was placed on understanding how inflammation plays a role in AD pathogenesis. Gene ontology analysis revealed that the most differentially expressed genes related to nervous system development and function and neurological disease followed by genes involved in inflammation and immunological signaling. Pathway analysis also implicated a role for chemokines and their receptors, specifically CXCR4 and CCR3, in AD. Immunohistological analysis revealed that these chemokine receptors are upregulated in AD patients. Western analysis demonstrated an increased activation of PKC, a downstream mediator of chemokine receptor signaling, in the majority of AD patients. A very specific cohort of genes related to amyloid beta accumulation and clearance were found to be significantly altered in AD. The most significantly downregulated gene in this data set was the endothelin converting enzyme 2 (ECE2), implicated in amyloid beta clearance. These data were subsequently confirmed by real-time PCR and Western blot analysis. Together, these findings open up new avenues of investigation and possible therapeutic strategies targeting inflammation and amyloid clearance in AD patients.


Asunto(s)
Enfermedad de Alzheimer/metabolismo , Corteza Cerebelosa/metabolismo , Quimiocinas/genética , Demencia/metabolismo , Neuronas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores de Quimiocina/genética , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/inmunología , Ácido Aspártico Endopeptidasas/metabolismo , Estudios de Casos y Controles , Análisis por Conglomerados , Demencia/inmunología , Regulación hacia Abajo , Enzimas Convertidoras de Endotelina , Femenino , Humanos , Masculino , Metaloendopeptidasas/metabolismo , Persona de Mediana Edad , Modelos Biológicos , Proteína Quinasa C/metabolismo , Receptores CXCR4/metabolismo , Transducción de Señal , Regulación hacia Arriba
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