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ABSTRACT: Acute lung injury (ALI) is a syndrome with significant morbidity and mortality, but its genetic susceptibility is not clearly understood. In the present study, we characterized functional promoter single nucleotide polymorphisms (SNPs) in the phase II antioxidant gene NQO1 (NAD(P)H:quinone oxidoreductase1) to evaluate its role in susceptibility to ALI. Three previously uncharacterized SNPs in the NQO1 promoter were selected for investigation. Luciferase assays were performed using constructs of each promoter polymorphism to evaluate function. Functional SNPs were genotyped in a prospective cohort of major trauma patients (N = 264) and assessed for association with development of ALI. The A/C SNP at -1221 decreased in vitro transcription of NQO1 at baseline and after exposure to hyperoxia and other oxidant stressors. Patients heterozygous for the -1221 C allele were at significantly lesser risk of ALI after major trauma compared with patients with wild-type alleles, even after adjustment for APACHE III score, and mechanism of trauma [OR, 0.46 (95% CI 0.23, 0.90); P = 0.024]. This study demonstrated that the AC genotype at position -1221 in the NQO1 gene caused decreased transcription and was associated with a lower incidence of ALI following major trauma. These novel findings may have important implications in diseases with oxidant stress aetiologies.
Journal of Cellular and Molecular Medicine 12/2008; 13(8B):1784-91. · 4.13 Impact Factor
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ABSTRACT: Respiratory syncytial virus (RSV) is the most frequent cause of significant lower respiratory illness in infants and young children, but its pathogenesis is not fully understood. The transcription factor Nrf2 protects lungs from oxidative injury and inflammation via antioxidant response element (ARE)-mediated gene induction.
The current study was designed to determine the role of Nrf2-mediated cytoprotective mechanisms in murine airway RSV disease.
Nrf2-deficient (Nrf2(-/-)) and wild-type (Nrf2(+/+)) mice were intranasally instilled with RSV or vehicle. In a separate study, Nrf2(+/+) and Nrf2(-/-) mice were treated orally with sulforaphane (an Nrf2-ARE inducer) or phosphate-buffered saline before RSV infection.
RSV-induced bronchopulmonary inflammation, epithelial injury, and mucus cell metaplasia as well as nasal epithelial injury were significantly greater in Nrf2(-/-) mice than in Nrf2(+/+) mice. Compared with Nrf2(+/+) mice, significantly attenuated viral clearance and IFN-gamma, body weight loss, heightened protein/lipid oxidation, and AP-1/NF-kappaB activity along with suppressed antioxidant induction was found in Nrf2(-/-) mice in response to RSV. Sulforaphane pretreatment significantly limited lung RSV replication and virus-induced inflammation in Nrf2(+/+) but not in Nrf2(-/-) mice.
The results of this study support an association of oxidant stress with RSV pathogenesis and a key role for the Nrf2-ARE pathway in host defense against RSV.
American Journal of Respiratory and Critical Care Medicine 11/2008; 179(2):138-50. · 11.08 Impact Factor
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ABSTRACT: The increasing number of population-based and epidemiologic associations between oxidant pollutant exposures and cardiopulmonary disease exacerbation, decrements in pulmonary function, and mortality underscores the important detrimental effects of oxidants on public health. Because inhaled oxidants initiate a number of pathologic processes, including inflammation of the airways, which may contribute to the pathogenesis and/or exacerbation of airways disease, it is critical to understand the mechanisms through which exogenous and endogenous oxidants interact with molecules in the cells, tissues, and epithelial lining fluid of the lung. Furthermore, it is clear that interindividual variation in response to a given exposure also exists across an individual lifetime. Because of the potential impact that oxidant exposures may have on reproductive outcomes and infant, child, and adult health, identification of the intrinsic and extrinsic factors that may influence susceptibility to oxidants remains an important issue. In this review, we discuss mechanisms of oxidant stress in the lung, the role of oxidants in lung disease pathogenesis and exacerbation (eg, asthma, chronic obstructive pulmonary disease, and acute respiratory distress syndrome), and the potential risk factors (eg, age, genetics) for enhanced susceptibility to oxidant-induced disease.
The Journal of allergy and clinical immunology 10/2008; 122(3):456-68; quiz 469-70. · 9.17 Impact Factor
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ABSTRACT: Recent studies have suggested a genetic component to heart rate (HR) and HR variability (HRV). However, a systematic examination of the genetic contribution to the variation in HR and HRV has not been performed. This study investigated the genetic contribution to HR and HRV using a wide range of inbred and recombinant inbred (RI) mouse strains. Electrocardiogram data were recorded from 30 strains of inbred mice and 29 RI strains. Significant differences in mean HR and total power (TP) HRV were identified between inbred strains and RI strains. Multiple significant differences within the strain sets in mean low-frequency (LF) and high-frequency (HF) power were also found. No statistically significant concordance was found between strain distribution patterns for HR and HRV phenotypes. Genomewide interval mapping identified a significant quantitative trait locus (QTL) for HR [LOD (likelihood of the odds) score = 3.763] on chromosome 6 [peak at 53.69 megabases (Mb); designated HR 1 (Hr1)]. Suggestive QTLs for TP were found on chromosomes 2, 4, 5, 6, and 14. A suggestive QTL for LF was found on chromosome 16; for HF, we found one significant QTL on chromosome 5 (LOD score = 3.107) [peak at 53.56 Mb; designated HRV-high-frequency 1 (Hrvhf1)] and three suggestive QTLs on chromosomes 2, 11 and 15. In conclusion, the results demonstrate a strong genetic component in the regulation of resting HR and HRV evidenced by the significant differences between strains. A lack of correlation between HR and HRV phenotypes in some inbred strains suggests that different sets of genes control the phenotypes. Furthermore, QTLs were found that will provide important insight to the genetic regulation of HR and HRV at rest.
AJP Heart and Circulatory Physiology 08/2008; 295(1):H59-68. · 3.71 Impact Factor
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M Inés Klein,
Eduardo Bergel,
Luz Gibbons,
Silvina Coviello,
Gabriela Bauer,
Alicia Benitez,
M Elina Serra,
M Florencia Delgado,
Guillermina A Melendi,
Susana Rodríguez, Steven R Kleeberger,
Fernando P Polack
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ABSTRACT: The protective role of breastfeeding against severe acute lung disease in infants is well established, but its mechanism is unclear. Most hypotheses assume that breastfeeding confers similar passive protection to every infant; however, a few observations have suggested that the benefits of breast milk against severe lung disease may differ according to gender. The objective of this study was to determine whether the effect of breastfeeding on susceptibility to severe acute lung disease among infants at high risk is different for girls and boys.
A cohort was analyzed prospectively by use of 2 different strategies: (1) predictors of first episode of rehospitalization by univariate and multivariate analyses using robust Poisson regression and (2) mean number of rehospitalizations between groups using multiple regression negative binomial models.
A total of 119 high-risk, very low birth weight infants were enrolled. Breast milk protected girls but not boys against severe acute lung disease. The interaction between breastfeeding and gender was clinically and statistically significant, even after adjustment for variables that can affect severity of acute lung disease. Disease was most severe in formula-fed girls (versus formula-fed boys).
Breastfeeding decreased the risk for severe acute lung disease in girls but not in boys. These findings suggest that breast milk protection is not universally conferred by passive transfer of humoral immunity (which should be gender indifferent), show that respiratory symptoms may be amenable to nonspecific modulation, and identify nonbreastfed preterm infant girls as an at-risk group for severe acute lung disease.
PEDIATRICS 06/2008; 121(6):e1510-6. · 4.47 Impact Factor
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Proceedings of the American Thoracic Society 05/2008; 5(3):367.
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Proceedings of the American Thoracic Society 05/2008; 5(3):368.
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ABSTRACT: The genomic locations and identities of the genes that regulate voluntary physical activity are presently unknown. The purpose of this study was to search for quantitative trait loci (QTL) that are linked with daily mouse running wheel distance, duration, and speed of exercise. F(2) animals (n = 310) derived from high active C57L/J and low active C3H/HeJ inbred strains were phenotyped for 21 days. After phenotyping, genotyping with a fully informative single-nucleotide polymorphism panel with an average intermarker interval of 13.7 cM was used. On all three activity indexes, sex and strain were significant factors, with the F(2) animals similar to the high active C57L/J mice in both daily exercise distance and duration of exercise. In the F(2) cohort, female mice ran significantly farther, longer, and faster than male mice. QTL analysis revealed no sex-specific QTL but at the 5% experimentwise significance level did identify one QTL for duration, one QTL for distance, and two QTL for speed. The QTL for duration (DUR13.1) and distance (DIST13.1) colocalized with the QTL for speed (SPD13.1). Each of these QTL accounted for approximately 6% of the phenotypic variance, whereas SPD9.1 (chromosome 9, 7 cM) accounted for 11.3% of the phenotypic variation. DUR13.1, DIST13.1, SPD13.1, and SPD9.1 were subsequently replicated by haplotype association mapping. The results of this study suggest a genetic basis of voluntary activity in mice and provide a foundation for future candidate gene studies.
Physiological Genomics 03/2008; 32(3):401-8. · 2.73 Impact Factor
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ABSTRACT: Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disorder in which excessive deposition of extracellular matrix leads to irreversible scarring of interstitial lung tissue. The etiology of IPF remains unknown, but growing evidence suggests that disequilibrium in oxidant/antioxidant balance contributes significantly. IPF is currently regarded as a fibroproliferative disorder triggered by repeated alveolar epithelial cell injury. Oxidative stress plays a role in many processes involved in alveolar epithelial cell injury and fibrogenesis. Here we review the role of oxidative stress in IPF, and other forms of pulmonary fibrosis, with particular attention to antioxidant defenses regulated by the redox-sensitive transcription factor nuclear factor, erythroid derived 2, like (Nrf2). Nrf2 binds specific antioxidant response elements (AREs) in the promoter of antioxidant enzyme and defense protein genes and regulates their expression in many tissue types. Nrf2 protects from several phenotypes in which enhanced oxidative burden contributes to disease pathogenesis, including cancer, acute lung injury, and pulmonary fibrosis. We suggest that promoter polymorphisms in human NRF2 may contribute to IPF susceptibility, although this hypothesis has not been tested. Pulmonary fibrosis is a highly complex disease and involves multiple genes and processes, and new therapies for cellular and molecular targets involved in pathogenic mechanisms are needed.
Antioxidants and Redox Signaling 03/2008; 10(2):321-32. · 8.46 Impact Factor
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ABSTRACT: Pulmonary fibrosis is a component of many interstitial lung diseases, including idiopathic pulmonary fibrosis, a chronic, progressive disease for which there is currently no effective therapy. Bleomycin has been widely used in rodents to model pulmonary fibrosis for the study of mechanisms involved in fibrogenesis and for evaluation of potential therapies. Bleomycin induces DNA strand breaks, resulting in pulmonary inflammation, injury, and subsequent interstitial fibrosis. This unit describes methods for delivering bleomycin, either directly into the lung or systemically, to create models of pulmonary fibrosis in rodents. Also described is a rapid and easy procedure for measuring lung collagen content to quantify the severity of fibrosis.
Current protocols in pharmacology 03/2008; Chapter 5:Unit 5.46.
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ABSTRACT: Lung diseases such as asthma, chronic obstructive pulmonary disease (COPD), and acute respiratory distress syndrome (ARDS) have complex etiologies. It is generally agreed that genetic background has an important role in susceptibility to these diseases, and the genetic contribution to disease phenotypes varies between populations. Linkage analyses have identified some predisposing genes. However, genetic background cannot account for all of the inter-individual variation in disease susceptibility. Interaction between genetic background and exposures to environmental stimuli, and understanding of the mechanisms through which environmental exposure interact with susceptibility genes, is critical to disease prevention. Use of animal models, particularly inbred mice, has provided important insight to understand human disease etiologies because genetic background and environmental exposures can be controlled. We have utilized a positional cloning approach in inbred mice to identify candidate susceptibility genes for oxidant-induced lung injury. Subsequent investigations with cell models identified functional polymorphisms in human homologues that confer enhanced risk of lung injury in humans. This 'bench to bedside' approach may provide an understanding of gene-environment interactions in complex lung diseases is essential to the development of new strategies for lung disease prevention and treatment.
Novartis Foundation symposium 02/2008; 293:168-78; discussion 178-83.
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ABSTRACT: The beta zipper (bZip) transcription factor, nuclear factor erythroid 2, like 2 (Nrf2), acting via an antioxidant/electrophile response element, regulates the expression of several antioxidant enzymes and maintains cellular redox homeostasis. Nrf2 deficiency diminishes pulmonary expression of several antioxidant enzymes, rendering them highly susceptible to various mouse models of prooxidant-induced lung injury. We recently demonstrated that Nrf2 deficiency impairs primary cultured pulmonary epithelial cell proliferation and greatly enhances sensitivity to prooxidant-induced cell death. Glutathione (GSH) supplementation rescued cells from these defects associated with Nrf2 deficiency. To further delineate the mechanisms by which Nrf2, via redox signaling, regulates cellular protection and proliferation, we compared the global expression profiling of Nrf2-deficient cells with and without GSH supplementation. We found that GSH regulates the expression of various networks of transcriptional programs including 1) several antioxidant enzymes involved in cellular detoxification of reactive oxygen species and recycling of thiol status and 2) several growth factors, growth factor receptors, and integrins that are critical for cell growth and proliferation. We also found that Nrf2 deficiency enhances the expression levels of several genes encoding proinflammatory cytokines; however, GSH supplementation markedly suppressed their expression. Collectively, these findings uncover an important insight into the nature of genes regulated by Nrf2-dependent redox signaling through GSH that are involved in cellular detoxification and proliferation.
Physiological Genomics 01/2008; 32(1):74-81. · 2.73 Impact Factor
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ABSTRACT: Exposure to ozone causes airway inflammation, hyperreactivity, lung hyper-permeability, and epithelial cell injury. An early inflammatory response induced by inhaled O(3) is characterized primarily by release of inflammatory mediators such as cytokines, chemokines, and airway neutrophil accumulation. Matrix metalloproteinases (MMPs) have been implicated in the pathogenesis of oxidative lung disorders including acute lung injury, asthma, and chronic obstructive pulmonary disease.
We hypothesized that MMPs have an important role in the pathogenesis of O(3)-induced airway inflammation.
We compared the lung injury responses in either Mmp7- (Mmp7(-/-)) or Mmp9-deficient (Mmp9(-/-)) mice and their wild-type controls (Mmp7(+/+), Mmp9(+/+)) after exposure to 0.3 ppm O(3) or filtered air.
Relative to air-exposed controls, MMP-9 activity in bronchoalveolar lavage fluid (BALF) was significantly increased by O(3) exposure in Mmp9(+/+) mice. O(3)-induced increases in the concentration of total protein (a marker of lung permeability) and the numbers of neutrophils and epithelial cells in BALF were significantly greater in Mmp9(-/-) mice compared with Mmp9(+/+) mice. Keratinocyte-derived chemokine (KC) and macrophage inflammatory protein (MIP)-2 levels in BALF were also significantly higher in Mmp9(-/-) mice than in Mmp9(+/+) mice after O(3) exposure, although no differences in mRNA expression for these chemokines were found between genotypes. Mean BALF protein concentration and numbers of inflammatory cells were not significantly different between Mmp7(+/+) and Mmp7(-/-) mice after O(3) exposure.
Results demonstrated a protective role of MMP-9 but not of MMP-7, in O(3)-induced lung neutrophilic inflammation and hyperpermeability. The mechanism through which Mmp9 limits O(3)-induced airway injury is not known but may be via posttranscriptional effects on proinflammatory CXC chemokines including KC and MIP-2.
Environmental Health Perspectives 12/2007; 115(11):1557-63. · 7.04 Impact Factor
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ABSTRACT: Cytotoxic T lymphocytes (CTLs) are important for the control of virus replication during respiratory infections. For human metapneumovirus (hMPV), an H-2(d)-restricted CTL epitope in the M2-2 protein has been described. In this study, we screened the hMPV F, G, N, M, M2-1, and M2-2 proteins using three independent algorithms to predict H-2(d) CTL epitopes in BALB/c mice. A dominant epitope (GYIDDNQSI) in positions 81 to 89 of the antitermination factor M2-1 and a subdominant epitope (SPKAGLLSL) in N(307-315) were detected during the anti-hMPV CTL response. Passive transfer of CD8(+) T-cell lines against M2-1(81-89) and N(307-315) protected Rag1(-/-) mice against hMPV challenge. Interestingly, diversification of CTL targets to include multiple epitopes was observed after repetitive infections. A subdominant response against the previously described M2-2 epitope was detected after the third infection. An understanding of the CTL response against hMPV is important for developing preventive and therapeutic strategies against the virus.
Journal of Virology 11/2007; 81(20):11461-7. · 5.40 Impact Factor
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Journal of Allergy and Clinical Immunology 10/2007; 120(3):719-22. · 11.00 Impact Factor
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ABSTRACT: We characterized the T helper cytokine profiles in the respiratory tract of infants infected with influenza virus, human metapneumovirus, and respiratory syncytial virus to examine whether these agents elicit similar cytokine responses and whether T helper type 2 polarization is associated with wheezing and severe disease.
A prospective study of infants who were seeking medical help for acute upper and/or lower respiratory tract infection symptoms for the first time and were found to be infected with influenza, human metapneumovirus, or respiratory syncytial virus was performed. Respiratory viruses were detected in nasal secretions with reverse transcriptase-polymerase chain reaction assays. The study was performed in emergency departments and outpatient clinics in Buenos Aires, Argentina. T cell cytokine responses were determined in nasal secretions with immunoassays and reverse transcriptase-polymerase chain reaction assays.
Influenza elicited higher levels of interferon-gamma, interleukin-4, and interleukin-2 than did the other agents. Human metapneumovirus had the lowest interferon-gamma/interleukin-4 ratio (T helper type 2 bias). However, no association was found between T helper type 2 bias and overall wheezing or hospitalization rates.
These findings show that viral respiratory infections in infants elicit different cytokine responses and that the pathogeneses of these agents should be studied individually.
PEDIATRICS 09/2007; 120(2):e410-5. · 4.47 Impact Factor
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ABSTRACT: Redox imbalance has been implicated in the pathogenesis of many acute and chronic lung diseases. The b-Zip transcription factor Nrf2 acts via an antioxidant/electrophilic response element to regulate antioxidants and maintain cellular redox homeostasis. Our previous studies have shown that Nrf2-deficient mice (Nrf2(-/-)) show reduced pulmonary expression of several antioxidant enzymes, which renders them highly susceptible to hyperoxia-induced lung injury. To better understand the physiologic significance of Nrf2-induced redox signaling, we have used primary cells isolated from the lungs of Nrf2(+/+) and Nrf2(-/-) mice. Our studies were focused on type II cells because these cells are constantly exposed to the oxidant environment and play key roles in host defense, injury, and repair processes. Using this system, we now report that an Nrf2 deficiency leads to defects in type II cell proliferation and greatly enhances the cells' sensitivity to oxidant-induced cell death. These defects were closely associated with high levels of reactive oxygen species (ROS) and redox imbalance in Nrf2(-/-) cells. Glutathione (GSH) supplementation rescued these phenotypic defects associated with the Nrf2 deficiency. Intriguingly, although the antioxidant N-acetyl-cysteine drastically squelched ROS levels, it was unable to counteract growth arrest in Nrf2(-/-) cells. Moreover, despite their elevated levels of ROS, Nrf2(-/-) type II cells were viable and, like their wild-type counterparts, exhibited normal differentiation characteristics. Our data suggest that dysfunctional Nrf2-regulated GSH-induced signaling is associated with deregulation of type II cell proliferation, which contributes to abnormal injury and repair and leads to respiratory impairment.
American Journal of Respiratory Cell and Molecular Biology 08/2007; 37(1):3-8. · 5.13 Impact Factor
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ABSTRACT: The role of genetics in the determination of maximal exercise endurance is unclear. Six- to nine-week-old F2 mice (n = 99; 60 female, 39 male), derived from an intercross of two inbred strains that had previously been phenotyped as having high maximal exercise endurance (Balb/cJ) and low maximal exercise endurance (DBA/2J), were treadmill tested to estimate exercise endurance. Selective genotyping of the F2 cohort (n = 12 high exercise endurance; n = 12 low exercise endurance) identified a significant quantitative trait locus (QTL) on chromosome X (53.7 cM, DXMit121) in the entire cohort and a suggestive QTL on chromosome 8 (36.1 cM, D8Mit359) in the female mice. Fine mapping with the entire F2 cohort and additional informative markers confirmed and narrowed the QTLs. The chromosome 8 QTL (EE8(F)) is homologous with two suggestive human QTLs and one significant rat QTL previously linked with exercise endurance. No effect of sex (P = 0.33) or body weight (P = 0.79) on exercise endurance was found in the F2 cohort. These data indicate that genetic factors in distinct chromosomal regions may affect maximal exercise endurance in the inbred mouse. Whereas multiple genes are located in the identified QTL that could functionally affect exercise endurance, this study serves as a foundation for further investigations delineating the identity of genetic factors influencing maximum exercise endurance.
Journal of Applied Physiology 08/2007; 103(1):105-10. · 3.75 Impact Factor
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Jacqui M Marzec,
Jason D Christie,
Sekhar P Reddy,
Anne E Jedlicka,
Hue Vuong,
Paul N Lanken,
Richard Aplenc,
Tae Yamamoto,
Masayuki Yamamoto,
Hye-Youn Cho, Steven R Kleeberger
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ABSTRACT: We recently used positional cloning to identify the transcription factor Nrf2 (NF-E2 related factor 2) as a susceptibility gene in a murine model of oxidant-induced acute lung injury (ALI). NRF2 binds to antioxidant response elements (ARE) and up-regulates protective detoxifying enzymes in response to oxidative stress. This led us to investigate NRF2 as a candidate susceptibility gene for risk of development of ALI in humans. We identified multiple single nucleotide polymorphisms (SNPs) by resequencing NRF2 in ethnically diverse subjects, and one (-617 C/A) significantly (P<0.001) diminished luciferase activity of promoter constructs containing the SNP and significantly decreased the binding affinity (P<0.001) relative to the wild type at this locus (-617 CC). In a nested case-control study, patients with the -617 A SNP had a significantly higher risk for developing ALI after major trauma (OR 6.44; 95% CI 1.34, 30.8; P=0.021) relative to patients with the wild type (-617 CC). This translational investigation provides novel insight into the molecular mechanisms of susceptibility to ALI and may help to identify patients who are predisposed to develop ALI under at risk conditions, such as trauma and sepsis. Furthermore, these findings may have important implications in other oxidative stress related illnesses.
The FASEB Journal 08/2007; 21(9):2237-46. · 5.71 Impact Factor
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ABSTRACT: Single nucleotide polymorphisms (SNPs) in transcription factor binding sites (TFBSs) may affect the binding of transcription factors, lead to differences in gene expression and phenotypes and therefore affect susceptibility to environmental exposure. We developed an integrated computational system for discovering functional SNPs in TFBSs in the human genome and predicting their impact on the expression of target genes. In this system, we (i) construct a position weight matrix (PWM) from a collection of experimentally discovered TFBSs; (ii) predict TFBSs in SNP sequences using the PWM and map SNPs to the upstream regions of genes; (iii) examine the evolutionary conservation of putative TFBSs by phylogenetic footprinting; (iv) prioritize candidate SNPs based on microarray expression profiles from tissues in which the transcription factor of interest is either deleted or over-expressed and (v) finally, analyze association of SNP genotypes with gene expression phenotypes. The application of our system has been tested to identify functional polymorphisms in the antioxidant response element (ARE), a cis-acting enhancer sequence found in the promoter region of many genes that encode antioxidant and Phase II detoxification enzymes/proteins. In response to oxidative stress, the transcription factor NRF2 (nuclear factor erythroid-derived 2-like 2) binds to AREs, mediating transcriptional activation of its responsive genes and modulating in vivo defense mechanisms against oxidative damage. Using our novel computational tools, we have identified a set of polymorphic AREs with functional evidence, showing the utility of our system to direct further experimental validation of genomic sequence variations that could be useful for identifying high-risk individuals.
Human Molecular Genetics 06/2007; 16(10):1188-200. · 7.64 Impact Factor
