Background. Human genetic polymorphisms in metabolic activation and detoxification pathways are a major source of inter-individual variation in susceptibility to environmentally induced disease. The group has developed genotyping assays for the at-risk variants of enzymes that protect against carcinogens in cigarette smoke, diet, industrial processes and environmental pollution. Population studies indicate that for these candidate susceptibility genes, the frequency of the at-risk genotypes for glutathione transferase M1 (GSTM1), theta 1 (GSTT1), Pi (GSTP1) and N-acetyltransferase (NAT1 and NAT2), XRCC1, XPD, vary significantly between ethnic groups. Some differences in cancer incidence among groups may be due to genetic metabolic differences as well as exposure differences. Mission: Our long-term goal is to understanding how genes and environment interact to influence risk of environmentally induced disease. To this end we are engaged in Environmental Genomics. This encompasses: 1) identification of candidate environmental response genes, 2) discovery and functional characterization of genetic, epigenetic and phenotypic variation in these genes, and; 3) the analysis in population studies of environmental disease susceptibility associated with functional polymorphisms, acquired susceptibility factors such as epigenetic changes and exposures; and the interactions between these factors. Eventually we hope these genomic approaches may identify biomarkers of exposure and effect, that will be predictive of future risk. A current primary focus is to look at methylation levels of CpG sites in the human genome in relationship to exposures. This information will allow us to more carefully determine the bounds of human variability to guide risk assessment and may be useful in developing prevention strategies to reduce disease incidence. In the Genetic Susceptibility Project we take the candidate susceptibility factors from the laboratory genotype/phenotype studies and test them in population studies. We are collaborating with numerous NIH, and university-based epidemiology groups to design and carryout appropriate tests of these factors in population-based epidemiology studies. Progress/accomplishments: 1) Chronic cigarette smoking exposes airway epithelial cells to thousands of carcinogens, oxidants and DNA damaging agents, creating a field of molecular injury in the airway and altering gene expression of a number of p53-regulated genes. The ability of p53 to regulate transcription is critical for tumor suppression and this suggests that single nucleotide polymorphisms (SNPs) in functional p53-binding sites (p53REs) that affect gene expression could influence susceptibility to cancer. To connect p53RE SNP genotype, gene expression, and cancer risk, we identified a set of 204 SNPs in putative p53REs, and performed cis eQTL (expression quantitative trait loci) analysis, assessing associations between genotypes and mRNA levels of adjacent genes in bronchial epithelial cells obtained from 44 cigarette smokers. To further validate these genotype-expression associations, we searched published eQTL studies from independent populations and determined that 53% (39/74) of the bronchial epithelial eQTLs were observed in at least one other study. SNPs in p53REs were also evaluated for effects on p53-DNA binding using a quantitative in vitro protein-DNA binding assay. Last, based on linkage disequilibrium, we found 6 p53RE eQTL SNPs were identified as cancer risk SNPs. We provide an approach for identifying and evaluating potentially functional SNPs that may modulate the airway gene expression response to smoking and may influence susceptibility to cancers. (Wang et al Human Genetics, 2014). 2) The ability of p53 to regulate transcription is crucial for tumor suppression and implies that inherited polymorphisms in functional p53 binding sites could influence cancer. Here, we identify a polymorphic p53 responsive element, and demonstrate its influence on cancer risk using genome-wide datasets of cancer susceptibility loci, genetic variation, p53 occupancy and p53 binding sites. We uncovered a single nucleotide polymorphism (SNP) in a functional p53 binding site and establish its influence on the ability of p53 to bind to and regulate transcription of the KITLG gene. The SNP resides in KITLG and associates with one of the largest risks identified among cancer genome-wide association studies (Zeron-Medina et al). Decades of research has proven that mutations in the p53 stress response pathway affect the incidence of diverse cancers more than mutations in other pathways. However, most evidence is limited to rare inherited, and somatic mutations. Using newly abundant genomic data, we demonstrate that commonly inherited genetic variants in the p53 pathway also affect the incidence of a broad range of cancers more than variants in other pathways. The p53 pathway cancer-associated polymorphisms have strikingly similar characteristics to well-studied p53 pathway mutations. Our results enable insights into p53-mediated tumor suppression in humans and into p53 pathway-based surveillance and treatment strategies. (Stracquadanio et al submitted, 2015). 3) As part of a team led by Dr. Stephanie London, EB, NIEHS, we have examined CpG methylation in cord blood in relation to maternal smoking. Epigenetic modifications due to in utero exposures may play a critical role in early programming for childhood and adult illness. Using Illumina 450K array, we previously identified differential DNA methylation related to maternal smoking during pregnancy at 26 CpG sites (CpGs) in 10 genes in newborn cord bloods from the Norwegian Mother and Child Cohort Study (MoBa). We evaluated the impact of the timing of mothers smoking (before or during pregnancy using cotinine measured at 18 weeks gestation), the fathers smoking before conception, and the grandmothers smoking during her pregnancy with the mother on methylation at these 26 CpGs in 1,042 MoBa newborns. We used robust linear regression, adjusting for covariates, applying Bonferroni correction. The strongest, and only statistically significant associations were observed for sustained smoking by the mother during pregnancy through at least 18 weeks gestation (p<1.6x10-5 for all 26 CpGs). We observed no statistically significant differential methylation due to smoking by mother prior to pregnancy or that ceased by week 18, fathers smoking before conception, or grandmothers smoking while pregnant with the mother. Differential methylation at these CpGs in newborns appears to reflect sustained in utero exposure rather than epigenetic inheritance. Impact: Smoking cessation in early pregnancy may negate effects on methylation (Joubert et al, 2014). Examining adult smoking we found that DNA Methylation of the Aryl Hydrocarbon Receptor Repressor Links Cigarette Smoking to Subclinical Atherosclerosis. Functionally, we found methylation of cg05575921 was linked with up-regulated Aryl-Hydrocarbon Receptor Repressor (AHRR) (p = 1.4x10-17), and significantly mediated (p = 3.1x10-4) the associations between cumulative smoking exposure (pack-years) and higher carotid plaque score in current and former smokers. We replicated the association of cg05575921 methylation with smoking (p=0.002) and arterial extended fatty streaks (p=0.002) in biopsies of 141 males. These findings suggest AHRR methylation may be mechanistically linked to AHRR expression in monocytes, and represents a potential biomarker of subclinical atherosclerosis in smokers.
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