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Summary Tobacco smoke exposure contributes to the global burden of communicable and chronic diseases. To identify the immune cells affected by smoking, we use single-cell RNA sequencing on peripheral blood from smokers and nonsmokers. Transcriptomes reveal a subpopulation of FCGR3A (CD16)-expressing natural killer (NK)-like CD8 T lymphocytes that increase in smokers. Mass cytometry confirms elevated CD16+ CD8 T cells in smokers. Inferred as highly differentiated by pseudotime analysis, NK-like CD8 T cells express markers that are characteristic of effector memory re-expressing CD45RA T (TEMRA) cells. Indicative of immune aging, smokers’ CD8 T cells are biased toward differentiated cells, and smokers have fewer naive cells than nonsmokers. DNA methylation-based models show that smoking dose is associated with accelerated aging and decreased telomere length, a biomarker of T cell senescence. Immune aging accompanies T cell senescence, which can ultimately lead to impaired immune function. This suggests a role for smoking-induced, senescence-associated immune dysregulation in smoking-mediated pathologies., Graphical Abstract, Highlights Smoking shifts the composition of CD8 T cells from naive to differentiated states NK-like CD16+ CD8 TEMRA cells are elevated in smokers and express GZMB and PRF1 DNA methylation links smoking dose with age acceleration and shortened telomeres CD8 T, CD4 T, NKT, NK, and monocytes express senescence-linked genes in smokers, Smoking increases the risk of inflammatory diseases and decreases immunity. Martos et al. characterize immune cells and find that smoking reduces naive and increases late-stage CD8 T cells. They show that smoking dose is associated with age acceleration and shortened telomeres. These changes are consistent with immune aging and senescence.

The Anniston Community Health Survey (ACHS-I) was initially conducted from 2005 to 2007 to assess polychlorinated biphenyl (PCB) exposures in Anniston, Alabama residents. In 2014, a follow-up study (ACHS-II) was conducted to measure the same PCBs as in ACHS-I and additional compounds e.g., polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and dioxin-like non-ortho (cPCBs) substituted PCBs. In this epigenome-wide association study (EWAS), we examined the associations between PCDD, PCDF, and PCB exposures and DNA methylation. Whole blood DNA methylation was measured using Illumina EPIC arrays (n=292). We modeled lipid-adjusted toxic equivalencies (TEQs) for: ΣDioxins (sum of 28 PCDDs, PCDFs, cPCBs, and mPCBs), PCDDs, PCDFs, cPCBs, and mPCBs using robust multivariable linear regression adjusting for age, race, sex, smoking, bisulfite conversion batch, and estimated percentages of six blood cell types. Among all exposures we identified 10 genome-wide (Bonferroni p≤6.74E-08) and 116 FDR (p≤5.00E-02) significant associations representing 10 and 113 unique CpGs, respectively. Of the 10 genome-wide associations, seven (70%) occurred in the PCDDs and four (40%) of these associations had an absolute differential methylation ≥1.00%, based on the methylation difference between the highest and lowest exposure quartiles. Most of the associations (six, 60%) represented hypomethylation changes. Of the 10 unique CpGs, eight (80%) were in genes shown to be associated with dioxins and/or PCBs based on data from the 2019 Comparative Toxicogenomics Database. In this study, we have identified a set of CpGs in blood DNA that may be particularly susceptible to dioxin, furan, and dioxin-like PCB exposures.

SUMMARYTobacco smoke exposure has been found to impact immune response, leukocyte subtypes, DNA methylation, and gene expression in human whole blood. Analysis with single cell technologies will resolve smoking associated (sub)population compositions, gene expression differences, and identification of rare subtypes masked by bulk fraction data. To characterize smoking-related gene expression changes in primary immune cells, we performed single-cell RNA sequencing (scRNAseq) on >45,000 human peripheral blood mononuclear cells (PBMCs) from smokers (n=4) and nonsmokers (n=4). Major cell type population frequencies showed strong correlation between scRNAseq and mass cytometry. Transcriptomes revealed an altered subpopulation of Natural Killer (NK)-like T lymphocytes in smokers, which expressed elevated levels of FCGR3A (gene encoding CD16) compared to other CD8 T cell subpopulations. Relatively rare in nonsmokers (median: 1.8%), the transcriptionally unique subset of CD8 T cells comprised 7.3% of PBMCs in smokers. Mass cytometry confirmed a significant increase (p = 0.03) in the frequency of CD16+ CD8 T cells in smokers. The majority of CD16+ CD8 T cells were CD45RA positive, indicating an effector memory re-expressing CD45RA T cell (TEMRA) phenotype. We expect that cigarette smoke alters CD8 T cell composition by shifting CD8 T cells toward differentiated functional states. Pseudotemporal ordering of CD8 T cell clusters revealed that smokers’ cells were biased toward later pseudotimes, and characterization of established markers in CD8 T cell subsets indicates a higher frequency of terminally differentiated cells in smokers than in nonsmokers, which corresponded with a lower frequency in naïve CD8 T cells. Consistent with an end-stage TEMRA phenotype, FCGR3A-expressing CD8 T cells were inferred as the most differentiated cluster by pseudotime analysis and expressed markers linked to senescence. Examination of differentially expressed genes in other PBMCs uncovered additional senescence-associated genes in CD4 T cells, NKT cells, NK cells, and monocytes. We also observed elevated Tregs, inducers of T cell senescence, in smokers. Taken together, our results suggest smoking-induced, senescence-associated immune cell dysregulation contributes to smoking-mediated pathologies.

Maternal tobacco smoke exposure has been associated with altered DNA methylation. However, previous studies largely used methylation arrays, which cover a small fraction of CpGs, and focused on whole cord blood.The current study examined the impact of in utero exposure to maternal tobacco smoke on the cord blood [Formula: see text] DNA methylome.The methylomes of 20 Hispanic white newborns ([Formula: see text] exposed to any maternal tobacco smoke in pregnancy; [Formula: see text] unexposed) from the Maternal and Child Health Study (MACHS) were profiled by whole-genome bisulfite sequencing (median coverage: [Formula: see text]). Statistical analyses were conducted using the Regression Analysis of Differential Methylation (RADMeth) program because it performs well on low-coverage data (minimizes false positives and negatives).We found that 10,381 CpGs were differentially methylated by tobacco smoke exposure [neighbor-adjusted p-values that are additionally corrected for multiple testing based on the Benjamini-Hochberg method for controlling the false discovery rate (FDR) [Formula: see text]]. From these CpGs, RADMeth identified 557 differentially methylated regions (DMRs) that were overrepresented ([Formula: see text]) in important regulatory regions, including enhancers. Of nine DMRs that could be queried in a reduced representation bisulfite sequencing (RRBS) study of adult [Formula: see text] cells ([Formula: see text] smokers; [Formula: see text] nonsmokers), four replicated ([Formula: see text]). Additionally, a CpG in the promoter of SLC7A8 (percent methylation difference: [Formula: see text] comparing exposed to unexposed) replicated ([Formula: see text]) in an EPIC (Illumina) array study of cord blood [Formula: see text] cells ([Formula: see text] exposed to sustained maternal tobacco smoke; [Formula: see text] unexposed) and in a study of adult [Formula: see text] cells across two platforms (EPIC: [Formula: see text] smokers; [Formula: see text] nonsmokers; 450K: [Formula: see text] smokers; [Formula: see text] nonsmokers).Maternal tobacco smoke exposure in pregnancy is associated with cord blood [Formula: see text] DNA methylation in key regulatory regions, including enhancers. While we used a method that performs well on low-coverage data, we cannot exclude the possibility that some results may be false positives. However, we identified a differentially methylated CpG in amino acid transporter SLC7A8 that is highly reproducible, which may be sensitive to cigarette smoke in both cord blood and adult [Formula: see text] cells. https://doi.org/10.1289/EHP3398.

Background: Maternal tobacco smoke exposure has been associated with altered DNA methylation. However, previous studies largely used methylation arrays, which cover a small fraction of CpGs, and fo...

Background Numerous studies have demonstrated that DNA methylation levels in the aryl hydrocarbon receptor repressor (AHRR) gene measured in cord blood are significantly associated with prenatal tobacco smoke exposure and can be used as a fetal exposure biomarker. The mechanism driving this demethylation has not been determined and it is unclear if all cord blood cell types are impacted. Nucleated red blood cells (nRBCs/CD235a+ cells) are developmentally immature RBCs that display genome-wide hypomethylation and are observed at increased frequency in the cord blood of smoking mothers. We tested if AHRR methylation levels in CD235a+ nRBCs or nRBC counts influenced AHRR methylation in whole cord blood. Methods Cord blood was collected from smoking (n = 34) and nonsmoking (n = 19) mothers and DNA was prepared from whole cord blood, isolated CD235a+ nRBCs, and CD14+ monocytes. AHRR methylation in cord blood DNA was measured using Illumina 850K arrays (cg05575921, chr5:373378). Pyrosequencing was used to compare methylation levels among cord blood, CD235a+, and CD14+ cells. We measured nRBC percentages using conventional complete blood counts and estimated percent nRBCs by a deconvolution model. Results Methylation levels in AHRR were significantly lower in nRBCs relative to whole cord blood and CD14+ monocytes. While AHRR methylation levels in the cell types were significantly correlated across all subjects, methylation values at the chr5:373378 CpG averaged 14.6% lower in nRBCs (range 0.4 to 24.8%; p = 3.8E−13) relative to CD14+, with nonsmokers showing a significantly greater hypomethylation (− 4.1%, p = 1.8E−02). Methylation level at the AHRR chr5:373378 CpG was strongly associated with self-reported smoking in both CD14+ monocytes (t test p = 5.7E−09) and nRBCs (p = 4.8E−08), as well as cotinine levels (regression p = 1.1E−07 and p = 3.6E−04, respectively). For subjects with whole blood 850K data, robust linear regression models adjusting for estimated cell type composition, either including nRBCs counts or estimates, modestly increased the association between smoking and cg05575921 methylation. Conclusions Prenatal smoke exposure was highly significantly associated with AHRR methylation in cord blood, CD14+ monocytes, and CD235a+ nRBCs. AHRR methylation levels in nRBCs and nRBC counts had minimal effect on cord blood methylation measurements. However, regression models using estimated nRBCs or actual nRBC counts outperformed those lacking these covariates. Electronic supplementary material The online version of this article (10.1186/s13148-019-0686-1) contains supplementary material, which is available to authorized users.

Background: Cigarette smoke is a causal factor in cancers and cardiovascular disease. Smoking-associated differentially methylated regions (SM-DMRs) have been observed in disease studies, but the causal link between altered DNA methylation and transcriptional change is obscure. Objective: Our objectives were to finely resolve SM-DMRs and to interrogate the mechanistic link between SM-DMRs and altered transcription of enhancer noncoding RNA (eRNA) and mRNA in human circulating monocytes. Method: We integrated SM-DMRs identified by reduced representation bisulfite sequencing (RRBS) of circulating CD14+ monocyte DNA collected from two independent human studies [n=38 from Clinical Research Unit (CRU) and n=55 from the Multi-Ethnic Study of Atherosclerosis (MESA), about half of whom were active smokers] with gene expression for protein-coding genes and noncoding RNAs measured by RT-PCR or RNA sequencing. Candidate SM-DMRs were compared with RRBS of purified CD4+ T cells, CD8+ T cells, CD15+ granulocytes, CD19+ B cells, and CD56+ NK cells (n=19 females, CRU). DMRs were validated using pyrosequencing or bisulfite amplicon sequencing in up to 85 CRU volunteers, who also provided saliva DNA. Results: RRBS identified monocyte SM-DMRs frequently located in putative gene regulatory regions. The most significant monocyte DMR occurred at a poised enhancer in the aryl-hydrocarbon receptor repressor gene (AHRR) and it was also detected in both granulocytes and saliva DNA. To our knowledge, we identify for the first time that SM-DMRs in or near AHRR, C5orf55-EXOC-AS, and SASH1 were associated with increased noncoding eRNA as well as mRNA in monocytes. Functionally, the AHRR SM-DMR appeared to up-regulate AHRR mRNA through activating the AHRR enhancer, as suggested by increased eRNA in the monocytes, but not granulocytes, from smokers compared with nonsmokers. Conclusions: Our findings suggest that AHRR SM-DMR up-regulates AHRR mRNA in a monocyte-specific manner by activating the AHRR enhancer. Cell type–specific activation of enhancers at SM-DMRs may represent a mechanism driving smoking-related disease. https://doi.org/10.1289/EHP2395

Background— Tobacco smoke contains numerous agonists of the aryl hydrocarbon receptor (AhR) pathway, and activation of the AhR pathway was shown to promote atherosclerosis in mice. Intriguingly, cigarette smoking is most strongly and robustly associated with DNA modifications to an AhR pathway gene, the AhR repressor ( AHRR ). We hypothesized that altered AHRR methylation in monocytes, a cell type sensitive to cigarette smoking and involved in atherogenesis, may be a part of the biological link between cigarette smoking and atherosclerosis. Methods and Results— DNA methylation profiles of AHRR in monocytes (542 CpG sites±150 kb of AHRR , using Illumina 450K array) were integrated with smoking habits and ultrasound-measured carotid plaque scores from 1256 participants of the Multi-Ethnic Study of Atherosclerosis (MESA). Methylation of cg05575921 significantly associated ( P =6.1×10 −134 ) with smoking status (current versus never). Novel associations between cg05575921 methylation and carotid plaque scores ( P =3.1×10 −10 ) were identified, which remained significant in current and former smokers even after adjusting for self-reported smoking habits, urinary cotinine, and well-known cardiovascular disease risk factors. This association replicated in an independent cohort using hepatic DNA (n=141). Functionally, cg05575921 was located in a predicted gene expression regulatory element (enhancer) and had methylation correlated with AHRR mRNA profiles ( P =1.4×10 −17 ) obtained from RNA sequencing conducted on a subset (n=373) of the samples. Conclusions— These findings suggest that AHRR methylation may be functionally related to AHRR expression in monocytes and represents a potential biomarker of subclinical atherosclerosis in smokers.

Tobacco smoke exposure is a risk factor for many human diseases and the global disease burden attributed to smoking remains substantial. In addition to DNA damage, smoking-induced epigenetic changes may contribute to etiology of complex smoking-associated diseases. Smoking alters the epigenome and transcriptome of human blood leukocytes. However, interpretation of bulk genomic approaches is limited because changes could indicate altered distribution of cell (sub)populations or changes in expression within (sub)populations. To characterize smoking-related gene expression changes in primary immune cells, we performed single cell RNA sequencing on human peripheral blood mononuclear cells (PBMCs) from smokers (n=4) and nonsmokers (n=4). Transcriptomes of 45,965 cells (78,183 reads per cell) revealed an altered population of Natural Killer (NK)-like T lymphocytes in smokers. Compared to NK cells, the NK-like T cell cluster had elevated expression of CD8A, CD8B, CD3D, CD3E, CD3G, CD6, and CD2, indicative of CD8+ T lymphocytes. Relatively rare in nonsmokers (2.2%), the transcriptionally unique subset of CD8+ T cells comprised 8.7% of PBMCs in smokers. Among CD8+ T cell subtypes, the increase in NK-like CD8+ T cells (Mann-Whitney p = 0.03) corresponded with a decrease in Naïve CD8+ T cells (p = 0.03). We did not observe changes in the frequencies of two additional CD8+ T cell clusters or in the overall frequency of CD8+ T cells. Mass cytometry of a 26-antibody leukocyte panel confirmed no differences in the frequencies of total CD8+ T (CD3+/CD8+/CD56-), total NKT (CD3+/CD56+) or CD8+ NKT (CD3+/CD8+/CD56+) cells between smokers and nonsmokers. This suggests smoking is associated with an increased number of CD8+ T cells that share characteristics with NK cells, but are not NKT cells. Consistent with an NK-like phenotype, altered effector CD8+ T cells had elevated expression of genes reported to be upregulated in T cells reprogrammed to NK-like cells. Compared to other effector CD8+ T cells, altered effector CD8+ T cells had reduced IL7R and increased FCGR3A (CD16), IFNG (interferon gamma), GZMB (granzyme B), and PRF1 (perforin) expression. In mice, granzyme B and perforin expressing CD8+ T cells contribute to the development of atherosclerotic plaques. Our data highlights a potential link between smoking-induced functional changes in human CD8+ T cells and atherosclerosis and /or immune surveillance in cancer. Citation Format: Suzanne N. Martos, Michelle R. Campbell, Marie A. Iannone, Gary S. Pittman, Ma Wan, Douglas A. Bell. Single cell RNA sequencing reveals altered natural killer-like, effector CD8+ T lymphocytes in smokers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5057.

Prenatal tobacco smoke exposure is associated with numerous adverse health outcomes at birth and in later life. Numerous studies have demonstrated that Aryl Hydrocarbon Receptor Repressor gene (AHRR) hypomethylation in umbilical cord blood is significantly associated with prenatal tobacco smoke exposure and is suitable as a fetal exposure biomarker. The mechanism driving this demethylation and whether all cord blood cell types are impacted is unclear. Nucleated red blood cells (nucleated CD235a+ cells, nRBCs) are developmentally immature RBCs that display genome-wide demethylation and are observed at increased frequency in the cord blood of smoking mothers. We assessed if nRBC counts or methylation level affected smoking-associated DNA methylation in the AHRR gene as measured in whole cord blood. We analyzed methylation in DNA from whole cord blood, CD14+ monocytes, and nRBCs across four AHRR CpGs using pyrosequencing and also with Illumina 850K arrays (cg05575921) from smoking (n=34) and nonsmoking (n=19) mothers. nRBCs present in cord blood were determined by conventional CBCs and also estimated with a Houseman deconvolution model. AHRR methylation levels were significantly lower in nRBCs relative to whole blood and CD14+ monocytes. Methylation values at each AHRR CpG averaged 14.6% lower in nRBCs (range 0.4% to 24%, p=3.9E-13) relative to CD14+ monocytes with nonsmokers displaying significantly greater hypomethylation in nRBCs than smokers. Methylation levels at each CpG across the AHRR DMR were strongly associated with either self-reported smoking or cord blood cotinine levels in both CD14+ monocytes (r2=0.45, p=4.6E-08) and nRBCs (r2=0.26, p=1.2E-04). The most significant prenatal smoking effect on methylation occurred in CD14+ monocytes at the CpG located at chr5:373490 (-16.7%, p=6.7E-09), 112nt downstream of cg05575921. For subjects with 850K data, adjusting a regression analysis model with estimated cell-type composition, including nRBC counts or estimates, marginally increased the magnitude and significance of the prenatal smoke exposure effect on AHRR cg05575921 methylation. Prenatal tobacco smoke exposure strongly affects DNA methylation in the AHRR gene and this prenatal smoke exposure biomarker is largely unaffected by cord blood cell type composition. Citation Format: Matthew A. Bergens, Gary S. Pittman, Isabel J. Thompson, Michelle R. Campbell, Xuting Wang, Ma Wan, Cathrine Hoyo, Douglas A. Bell. Smoking-associated AHRR demethylation in cord blood DNA: Impact of CD235a+ nucleated red blood cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 599.

Tobacco smoke exposure dramatically alters DNA methylation in blood cells and may mediate smoking-associated complex diseases through effects on immune cell function. However, knowledge of smoking effects in specific leukocyte subtypes is limited. To better characterize smoking-associated methylation changes in whole blood and leukocyte subtypes, we used Illumina 450K arrays and Reduced Representation Bisulfite Sequencing (RRBS) to assess genome-wide DNA methylation. Differential methylation analysis in whole blood DNA from 172 smokers and 81 nonsmokers revealed 738 CpGs, including 616 previously unreported CpGs, genome-wide significantly associated with current smoking (p 22 cigarettes/day, n = 86) which might relate to long-term heavy-smoking pathology. In purified leukocyte subtypes from an independent group of 20 smokers and 14 nonsmokers we further examined methylation and gene expression for selected genes among CD14+ monocytes, CD15+ granulocytes, CD19+ B cells, and CD2+ T cells. In 10 smokers and 10 nonsmokers we used RRBS to fine map differential methylation in CD4+ T cells, CD8+ T cells, CD14+, CD15+, CD19+, and CD56+ natural killer cells. Distinct cell-type differences in smoking-associated methylation and gene expression were identified. AHRR (cg05575921), ALPPL2 (cg21566642), GFI1 (cg09935388), IER3 (cg06126421) and F2RL3 (cg03636183) showed a distinct pattern of significant smoking-associated methylation differences across cell types: granulocytes> monocytes>> B cells. In contrast GPR15 (cg19859270) was highly significant in T and B cells and ITGAL (cg09099830) significant only in T cells. Numerous other CpGs displayed distinctive cell-type responses to tobacco smoke exposure that were not apparent in whole blood DNA. Assessing the overlap between these CpG sites and differential methylated regions (DMRs) with RRBS in 6 cell types, we confirmed cell-type specificity in the context of DMRs. We identified new CpGs associated with current smoking, pack-years, duration, and revealed unique profiles of smoking-associated DNA methylation and gene expression among immune cell types, providing potential clues to hematopoietic lineage-specific effects in disease etiology.

Exposure to tobacco smoke dramatically alters DNA methylation in blood cells and this immune cell modulation may mediate smoking-associated complex diseases. To identify cell-type specific, smoking-associated differentially methylated regions (SM-DMRs) in hematopoietic cells from smokers and non-smokers, we used Illumina 450/850K arrays, reduced representation (RRBS) and whole genome bisulfite sequencing (WGBS) to measure methylation on DNA extracted from whole blood and 7 leukocyte subtypes (CD4+, CD8+, CD14+ monocytes, CD15+ granulocytes, B cells, NK cells and CD34+ progenitor cells). SM-DMRs preferentially occurred at enhancers (H3K27ac), and/or CpG island shores and were enriched for transcription factor (TF) binding suggesting crosstalk between TFs, DNA methylation, and tobacco smoke exposure. SM-DMRs in or near genes AHRR, ALPPL2, and GFI1 were highly significant in CD34+, CD14+, CD15+, B cells and NK cells but were absent in CD4+ and CD8+ T cells. We also identified genomic regions with distinct cell-type and lineage-specific differences in smoking-associated methylation and gene expression. AHRR displayed increased levels of both noncoding RNA and mRNA gene expression only in CD14+ monocytes. The SM-DMR in GPR15 was highly significant in CD4+ and CD8+ T cells, B cells, and NK cells but absent in myeloid cells. To explore possible links between smoking, DNA methylation/gene expression and immune cell functions, we carried out pathway analysis on smoking-affected methylomes and transcriptomes in each cell type. The smokers' transcriptome of CD56+ NK cells showed perturbation of genes either altered in leukemias, linked with leukemia GWAS SNPs, or under various types of innate immune stimulation. In particular, the locus containing HLA-DQA1 and HLA-DQB1 is highly significantly linked with chronic lymphocytic leukemia and these genes were strongly down-regulated in NK cells of smokers. The NK cell methylome of smokers also displayed perturbation of genes altered in leukemia cell lines. To identify and characterize rare blood cells among cell-type subsets that have smoking-altered transcriptomes, we are carrying out single cell RNA-seq. These studies may reveal new biological connections between tobacco smoke exposure and smoking-associated disease etiology. Citation Format: Xuting Wang, Ma Wan, Brian D. Bennett, Gary S. Pittman, Michelle Campbell, Dan Su, Isabel J. Thompson, Suzanne N. Martos, Douglas A. Bell. Cell-type specific epigenetic effects of tobacco smoking on the immune cell methylome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4317.

The most common form of genetic variation, single nucleotide polymorphisms or SNPs, can affect the way an individual responds to the environment and modify disease risk. Although most of the millions of SNPs have little or no effect on gene regulation and protein activity, there are many circumstances where base changes can have deleterious effects. Non-synonymous SNPs that result in amino acid changes in proteins have been studied because of their obvious impact on protein activity. It is well known that SNPs within regulatory regions of the genome can result in disregulation of gene transcription. However, the impact of SNPs located in putative regulatory regions, or rSNPs, is harder to predict for two primary reasons. First, the mechanistic roles of non-coding genomic sequence remain poorly defined. Second, experimental validation of the functional consequences of rSNPs is often slow and laborious. In this review, we summarize traditional and novel methodologies for candidate rSNPs selection, in particular in silico techniques that aid in candidate rSNP selection. Additionally we will discuss molecular biological techniques that assess the impact of rSNPs on binding of regulatory machinery, as well as functional consequences on transcription. Standard techniques such as EMSA and luciferase reporter constructs are still widely used to assess effects of rSNPs on binding and gene transcription; however, these protocols are often bottlenecks in the discovery process. Therefore, we highlight novel and developing high-throughput protocols that promise to aid in shortening the process of rSNP validation. Given the large amount of genomic information generated from a multitude of re-sequencing and genome-wide SNP array efforts, future focus should be to develop validation techniques that will allow greater understanding of the impact these polymorphisms have on human health and disease.

Discovery and functional evaluation of biologically significant regulatory single nucleotide polymorphisms (SNP) in carcinogen metabolism genes is a difficult challenge because the phenotypic consequences may be both transient and subtle. We have used a gene expression screening approach to identify a functional regulatory SNP in glutathione S-transferase M3 (GSTM3). Anttila et al. proposed that variation in GSTM3 expression was affected by exposure to cigarette smoke and inheritance of the GSTM1-null genotype. To investigate the mechanism of GSTM3 expression was affected by exposure to cigarette smoke and inheritance of the GSTM1-null genotype. To investigate the mechanism of GSTM3 expression variation, we measured GSTM3 expression in lymphoblast cells from a human Centre d'Etude du Polymorphisme Humain family and observed a low expression phenotype. Promoter sequencing revealed two novel GSTM3 promoter SNPs: A/C and A/G SNPs, 63 and 783 bp upstream of the codon 1 start site, respectively. In this pedigree, the two children homozygous for the −63C/C genotype had 8-fold lower GSTM3 expression relative to the two children with the −63A/A genotype, with no association between A−783G SNP and GSTM3 expression. Further evaluation using genotyped glioma cell lines and with luciferase reporter constructs showed that the −63C allele was associated with lower GSTM3 expression (P < 0.0001 and P < 0.003). RNA pol II chromatin immunoprecipitation was combined with quantitative probed-based allelic discrimination genotyping to provide direct evidence of a 9-fold reduced RNA pol II binding capacity for the −63C allele. These results show that the GSTM3 −63C allele strongly affects gene expression in human cell lines and suggests that individuals who carry the low expression allele may be deficient in glutathione transferase catalyzed biological functions.

Introduction A polymorphism in the manganese superoxide dismutase (MnSOD) gene, Ala-9Val, has been examined in association with breast cancer risk in several epidemiologic studies. Results suggest that the Ala allele increases the risk of breast cancer and modifies the effects of environmental exposures that produce oxidative damage to DNA. Methods We examined the role of the MnSOD Ala-9Val polymorphism in a population-based case–control study of invasive and in situ breast cancer in North Carolina. Genotypes were evaluated for 2025 cases (760 African Americans and 1265 whites) and for 1812 controls (677 African Americans and 1135 whites). Results The odds ratio for MnSOD Ala/Ala versus any MnSOD Val genotypes was not elevated in African Americans (odds ratio = 0.9, 95% confidence interval = 0.7–1.2) or in whites (odds ratio = 1.0, 95% confidence interval = 0.8–1.2). Greater than additive joint effects were observed for the Ala/Ala genotype and smoking, radiation to the chest, and occupational exposure to ionizing radiation. Antagonism was observed between the Ala/Ala genotype and the use of nonsteroidal anti-inflammatory drugs. Conclusions The MnSOD genotype may contribute to an increased risk of breast cancer in the presence of specific environmental exposures. These results provide further evidence for the importance of reactive oxygen species and of oxidative DNA damage in the etiology of breast cancer.

Background. Cigarette smoking is a well-known risk factor for not only incident events of coronary heart disease, but also preclinical atherosclerosis. Enzymes including glutathione S-transferase mu (GSTM1) and theta (GSTT1) are involved in the metabolism of tobacco smoke chemicals and are expressed in human vessels and smooth muscle cells. Methods. We estimated the effect of interaction of smoking with the deletion polymorphisms of GSTM1 and GSTT1 on the risk of carotid artery atherosclerosis. We analyzed a stratified random sample of 1394 individuals from the Atherosclerosis Risk in Communities (ARIC) study, which is a prospective cohort study of 15,792 white and black men and women randomly selected from four U.S. communities between 1987 and 1989. As an index of generalized atherosclerosis, we used carotid artery intimal-medial thickness, as determined by B-mode ultrasound at the baseline or first follow-up examination in asymptomatic individuals. Results. We found a suggestion of an interaction between the functional GSTT1-1 genotype (GSTT-1) and heavy smoking. For 20 or more pack-years and GSTT1-1, the odds ratio for increased intimal-medial thickness was 4.7 (95% confidence interval = 1.9-11.8); for 20 or more pack-years and GSTT1-0, 1.7(0.5-5.5); and for nonsmokers and GSTT1-1, 0.7(0.3-1.5). We found no interaction between smoking and GSTM1. Conclusions. The results of this analysis of preclinical atherosclerosis extend reports from the ARIC study of an interaction between smoking and GSTT1-1 in relation to the risk of incident coronary heart disease and lower extremity arterial disease.

Tobacco smoke exposure dramatically alters DNA methylation in blood cells and may mediate smoking-associated complex diseases through effects on immune cell function. However, knowledge of smoking effects in specific leukocyte subtypes is limited. To better characterize smoking-associated methylation changes in whole blood and leukocyte subtypes, we used Illumina 450K arrays and Reduced Representation Bisulfite Sequencing (RRBS) to assess genome-wide DNA methylation. Differential methylation analysis in whole blood DNA from 172 smokers and 81 nonsmokers revealed 738 CpGs, including 616 previously unreported CpGs, genome-wide significantly associated with current smoking (p1.2x10-7, Bonferroni correction). Several CpGs (MTSS1, NKX6-2, BTG2) were associated with smoking duration among heavy smokers (22 cigarettes/day, n = 86) which might relate to long-term heavy-smoking pathology. In purified leukocyte subtypes from an independent group of 20 smokers and 14 nonsmokers we further examined methylation and gene expression for selected genes among CD14+ monocytes, CD15+ granulocytes, CD19+ B cells, and CD2+ T cells. In 10 smokers and 10 nonsmokers we used RRBS to fine map differential methylation in CD4+ T cells, CD8+ T cells, CD14+, CD15+, CD19+, and CD56+ natural killer cells. Distinct cell-type differences in smoking-associated methylation and gene expression were identified. AHRR (cg05575921), ALPPL2 (cg21566642), GFI1 (cg09935388), IER3 (cg06126421) and F2RL3 (cg03636183) showed a distinct pattern of significant smoking-associated methylation differences across cell types: granulocytesmonocytesB cells. In contrast GPR15 (cg19859270) was highly significant in T and B cells and ITGAL (cg09099830) significant only in T cells. Numerous other CpGs displayed distinctive cell-type responses to tobacco smoke exposure that were not apparent in whole blood DNA. Assessing the overlap between these CpG sites and differential methylated regions (DMRs) with RRBS in 6 cell types, we confirmed cell-type specificity in the context of DMRs. We identified new CpGs associated with current smoking, pack-years, duration, and revealed unique profiles of smoking-associated DNA methylation and gene expression among immune cell types, providing potential clues to hematopoietic lineage-specific effects in disease etiology.

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. Studies of cytologically normal bronchial epithelial cells from smokers have identified transcription-based biomarkers that may prove useful in early diagnosis of lung cancer, including 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 (p53 response elements, or p53REs) that affect gene expression could influence susceptibility to cancer. To connect p53RE SNP genotype with 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 test and 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 of other studies. 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 SNPs associated with gene expression were identified as cancer risk SNPs by either genome-wide association studies (GWAS) or candidate gene studies. 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.

Cancer studies suggest that the null polymorphisms of glutathione S-transferase M1 or T1 (GSTM1/GSTT1) may affect the ability to detoxify or activate chemicals in cigarette smoke. The potential modification of the association between smoking and coronary heart disease (CHD) by GSTM1 and GSTT1 has not been studied in humans. A case-cohort study was conducted to test the hypotheses that specific genotypes of GSTM1 or GSTT1 affect susceptibility to smoking-related CHD. CHD cases (n=400) accrued during 1987-1993 and a cohort-representative sample (n=924) were selected from a biracial cohort of 15792 middle-aged men and women in four US communities. A significantly higher frequency of GSTM1-0 and a lower frequency of GSTT1-0 were found in whites (GSTM1-0=47.1%, GSTT1-0=16.4%) than in African-Americans (AAs) (GSTM1-0=17.5%, GSTT1-0=25.9%). A smoking-GSTM1-0 interaction for the risk of CHD was statistically significant on an additive scale, with ever-smokers with GSTM1-0 at a approximately 1.5-fold higher risk relative to ever-smokers with GSTM1-1 and a approximately 2-fold higher risk relative to never-smokers with GSTM1-0, after adjustment for other CHD risk factors. The interaction between having smoked >/=20 pack-years and GSTT1-1 was statistically significant on both multiplicative and additive scales. The risk of CHD given both GSTT1-1 and >/=20 pack-years of smoking was approximately three times greater than the risk given exposure to >/=20 pack-years of smoking alone, and approximately four times greater than the risk given exposure to GSTT1-1 alone. The modification of the smoking-CHD association by GSTM1 or GSTT1 suggests that chemicals in cigarette smoke that are substrates for glutathione S-transferases may be involved in the etiology of CHD.

Prior microarray studies of smokers at high risk for lung cancer have demonstrated that heterogeneity in bronchial airway epithelial cell gene expression response to smoking can serve as an early diagnostic biomarker for lung cancer. As a first step in applying functional genomic analysis to population studies, we have examined the relationship between gene expression variation and genetic variation in a central molecular pathway (NRF2-mediated antioxidant response) associated with smoking exposure and lung cancer. We assessed global gene expression in histologically normal airway epithelial cells obtained at bronchoscopy from smokers who developed lung cancer (SC, n = 20), smokers without lung cancer (SNC, n = 24), and never smokers (NS, n = 8). Functional enrichment analysis showed that the NRF2-mediated, antioxidant response element (ARE)-regulated genes, were significantly lower in SC, when compared with expression levels in SNC. Importantly, we found that the expression of MAFG (a binding partner of NRF2) was correlated with the expression of ARE genes, suggesting MAFG levels may limit target gene induction. Bioinformatically we identified single nucleotide polymorphisms (SNPs) in putative ARE genes and to test the impact of genetic variation, we genotyped these putative regulatory SNPs and other tag SNPs in selected NRF2 pathway genes. Sequencing MAFG locus, we identified 30 novel SNPs and two were associated with either gene expression or lung cancer status among smokers. This work demonstrates an analysis approach that integrates bioinformatics pathway and transcription factor binding site analysis with genotype, gene expression and disease status to identify SNPs that may be associated with individual differences in gene expression and/or cancer status in smokers. These polymorphisms might ultimately contribute to lung cancer risk via their effect on the airway gene expression response to tobacco-smoke exposure.

The p53 tumor suppressor regulates its target genes through sequence-specific binding to DNA response elements (REs). Although numerous p53 REs are established, the thousands more identified by bioinformatics are not easily subjected to comparative functional evaluation. To examine the relationship between RE sequence variation—including polymorphisms—and p53 binding, we have developed a multiplex format microsphere assay of protein-DNA binding (MAPD) for p53 in nuclear extracts. Using MAPD we measured sequence-specific p53 binding of doxorubicin-activated or transiently expressed p53 to REs from established p53 target genes and p53 consensus REs. To assess the sensitivity and scalability of the assay, we tested 16 variants of the p21 target sequence and a 62-multiplex set of single nucleotide (nt) variants of the p53 consensus sequence and found many changes in p53 binding that are not captured by current computational binding models. A group of eight single nucleotide polymorphisms (SNPs) was examined and binding profiles closely matched transactivation capability tested in luciferase constructs. The in vitro binding characteristics of p53 in nuclear extracts recapitulated the cellular in vivo transactivation capabilities for eight well-established human REs measured by luciferase assay. Using a set of 26 bona fide REs, we observed distinct binding patterns characteristic of transiently expressed wild type and mutant p53s. This microsphere assay system utilizes biologically meaningful cell extracts in a multiplexed, quantitative, in vitro format that provides a powerful experimental tool for elucidating the functional impact of sequence polymorphism and protein variation on protein/DNA binding in transcriptional networks., Author Summary Characterizing the functional roles of gene regulatory sequences and the impact of genetic polymorphism on protein-DNA binding are rapidly growing areas of genomics. The p53 tumor suppressor regulates its target genes through sequence-specific binding to DNA response elements, and we have developed a multiplex format microsphere assay (MAPD) to probe how sequence variation alters p53 binding. Testing pools of multiplexed oligonucleotides that contain numerous p53 binding targets or different sequence variants, we demonstrate that subtle changes in target sequence can impact p53 binding, and these effects are not captured by commonly used computational binding models. A group of single nucleotide polymorphisms was examined and binding profiles closely matched gene expression measured in a parallel system. We also observed distinct binding patterns that were characteristic of transiently expressed wild type and mutant p53s, suggesting that this method may be useful for probing the impact of protein structural variation on DNA binding. MAPD utilizes biologically meaningful cell extracts in a multiplexed, quantitative format that provides a powerful experimental tool for elucidating the functional impact of sequence polymorphism and protein variation on protein/DNA binding in transcriptional networks.

Both genetic and environmental factors are likely to be important causes of primary brain tumors. The few clues about brain tumor etiology indicate that certain occupations involving exposure to polycyclic aromatic hydrocarbons (PAHs)2 or other aromatic hydrocarbons may be associated with increased risk (Inskip, P.D., et al., 1995), most notably work in the petroleum industry (Carozza et al., 2000; Demers et al., 1991; Preston-Martin, 1989; Thomas et al., 1986, 1987); however, multiple exposures in implicated occupations limit possible conclusions about aromatic hydrocarbons. Smoking, a major source of aromatic hydrocarbon exposure, has been associated with brain tumor incidence in several studies (Burch et al., 1987; Lee et al., 1997), but not consistently so (Inskip, P.D., et al., 1995), and sometimes only among certain subgroups (Efird et al., 2004; Phillips et al., 2005). It is possible that underlying variability in genes responsible for biotransformation and metabolism of aromatic hydrocarbons could hinder the consistency of studies of chemical exposures. For this reason, it may be illuminating to study the association of variants in genes involved in aromatic hydrocarbon metabolism with the risk of brain tumors. The conversion of PAHs to DNA-reactive products depends on a complex series of biotransformations. In the case of benzo[a]pyrene, transformation events include oxidation by cytochrome P-450 enzymes (such as CYP1A1) to create the active benzo[a]pyrene epoxide (Pelkonen and Nebert, 1982; Shimada et al., 1996), hydration by microsomal epoxide hydrolase (EPHX1) to the less toxic benzo[a]pyrene diol, oxidation by P-450 enzymes (such as CYP1B1) to the highly carcinogenic benzo[a]pyrene diol epoxide, detoxification of benzo[a]-pyrene and benzo[a]pyrene diol epoxide by glutathione S-transferases (such as GSTM1, GSTT1, and possibly GSTM3) by addition of reduced glutathione to electrophilic compounds (Omiecinski et al., 2000; Strange et al., 2001), and reduction of oxidative potential of quinones derived from benzo[a]pyrene diol by NAD(P)H:quinone oxidoreductase 1 (NQO1) (Palackal et al., 2002; Pastorelli et al., 1998; Ross et al., 2000). There is evidence from animal experiments that NAD(P)H protects from PAH-induced carcinogenicity; this protection is thought to operate through decreases in quinone-induced DNA adduct formation and DNA mutagenicity, including that induced by benzo[a]pyrene quinine (Joseph and Jaiswal, 1998; Long et al., 2001). In a previous report, we presented case-control study results for some genes known to be involved in metabolism of PAHs or other potential carcinogens, namely, CYP2E1, GSTM1, and GSTT1 (De Roos et al., 2003). For the current investigation, we selected several additional candidate genes related to PAHs or other aromatic hydrocarbons; all of the selected metabolic genes exhibit sequence variation that may relate to function. Substitution of valine with isoleucine in exon 7 of CYP1A1 results in a variant (I462V) with increased arylhydrocarbon hydroxylase activity (Cosma et al., 1993; Crofts et al., 1994; Kiyohara et al., 1996, 1998; Taioli et al., 1995). The functional significance of a CYP1B1 variant, V432L, is not well known; however, some studies suggest that the valine product results in higher catalytic activity toward some PAH dihydrodiols relative to leucine (Shimada et al., 1999), possibly leading to increased levels of reactive intermediates. The GSTM3 gene has a three-base-pair deletion in intron 6, and the two alleles are referred to as GSTM3*A and GSTM3*B (Inskip, A., et al., 1995; Strange et al., 2001). This deletion creates a recognition motif (-aagata-) for the YY1 transcription factor which could potentially affect detoxification activity by GSTM3*B (Strange et al., 2001). The EPHX1 variant Y113H has demonstrated increased activity in vitro but not in vivo (Hassett et al., 1994; Omiecinski et al., 2000). In vitro, EPHX1 activity is increased (about 40%) when associated with the histidine product, probably because of altered protein stability (Hassett et al., 1994). The NQO1 P187S variant resulting from C-to-T substitution leads to reduced enzyme function (Moran et al., 1999; Traver et al., 1997) and thus, presumably, less protection against oxidative damage. We examined the effects of these metabolic gene variants in a parallel comparison of three major categories of malignant and benign brain tumors, namely the gliomas, meningiomas, and acoustic neuromas. Although we selected genes according to their possible relevance to metabolism of PAHs and other aromatic hydrocarbons, the substrate specificity is quite broad, and it is unclear to what extent the selected genes reflect a coherent pathway. Nevertheless, this exploratory approach was considered appropriate, given the dearth of knowledge about causes of brain tumors.

The p53 tumor suppressor protein is a master regulatory transcription factor that coordinates cellular responses to DNA damage and cellular stress. Besides mutations in p53, or in proteins involved in the p53 response pathway, genetic variation in promoter response elements (REs) of p53 target genes is expected to alter biological responses to stress. To identify SNPs in p53 REs that may modify p53-controlled gene expression, we developed an approach that combines a custom bioinformatics search to identify candidate SNPs with functional yeast and mammalian cell assays to assess their effect on p53 transactivation. Among ≈2 million human SNPs, we identified >200 that seem to disrupt functional p53 REs. Eight of these SNPs were evaluated in functional assays to determine both the activity of the putative RE and the impact of the candidate SNPs on transactivation. All eight candidate REs were functional, and in every case the SNP pair exhibited differential transactivation capacities. Additionally, six of the eight genes adjacent to these SNPs are induced by genotoxic stress or are activated directly by transfection with p53 cDNA. Thus, this strategy efficiently identifies SNPs that may differentially affect gene expression responses in the p53 regulatory pathway.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is highly toxic in experimental animals, and is known to induce cytochrome P450 (CYP) gene expression. We investigated the effect of CYP1A1 and CYP1B1 variant genotypes and haplotypes on CYP1A1 and CYP1B1 mRNA expression and ethoxyresorufin-O-deethylase (EROD) activity in lymphocytes from 121 subjects from the Seveso population, Italy, accidentally exposed to TCDD in 1976. The 3′UTR 3801T>C and I462V variants of CYP1A1 were present in 16% and 6% of the subjects, respectively. The frequency of CYP1B1 variants was 85.2% for L432V, 49.6% for R48G and A119S, and 28.7% for N453S. There was complete linkage disequilibrium (LD) among the CYP1B1 variant loci (D′ = −1) and high LD among the CYP1A1 loci (D′ = 0.86). Gene expression measured by RT-PCR did not vary by CYP1B1 genotype in uncultured lymphocytes. However, when lymphocytes were treated in vitro with 10 nM TCDD, CYP1B1 and CYP1A1 mRNA expression was strongly induced and modified by CYP variant alleles. Specifically, the CYP1B1*3 haplotype (L432V) was associated with increased CYP1B1 mRNA expression (P = 0.03), following an additive model; the CYP1A1 I462V polymorphism was positively, although not significantly, associated with CYP1A1 expression. The CYP1B1*3 variant may have affected CYP1B1 expression in subjects highly and acutely exposed to dioxin at the time of the accident. Although based on small number of subjects, a slight increase in eczema (P = 0.05, n = 8) and urticaria (P = 0.02, n = 2) was observed 20 years after the accident in subjects carrying the CYP1B1*3 allele. Genetic variation in cytochrome P450 induction may identify subjects with variable responsiveness to TCDD and potentially increased risk of disease.

Genetic differences that underlie inter-individual variation in the metabolism of common carcinogens are important potential sources of cancer susceptibility. Cytochrome P450 1B1 (CYP1B1), a central enzyme in the activation of the ubiquitous environmental carcinogen benzo[a]pyrene (B[a]P), has several genetic variants. This study investigated six rare mutations and four common polymorphisms for their effects on B[a]P metabolism. Five missense mutations associated with congenital glaucoma (Gly61Glu, Gly365Trp, Asp374Asn, Pro437Leu and Arg469Tryp) dramatically decreased the capacity of CYP1B1 to convert (-)benzo[a]pyrene-7R-trans-7,8-dihyrodiol (B[a]P-7,8-diol) to (+/-)benzo[a]pyrene-r-7,t-8-dihydrodiol-9,10-epoxides. These five mutations resulted in enzymes with 3-12% of normal activity when assayed in vitro using an Saccharomyces cerevisiae microsomal expression system. A 10 bp deletion mutation produced no detectable protein or activity. In contrast, proteins containing all possible combinations of four common single nucleotide polymorphisms (Arg48Gly, Ala199Ser, Val432Leu, Asn453Ser) had modest effects on B[a]P-7,8-diol metabolism. Michaelis-Menten analysis suggested that two alleles, Arg48, Ala119, Val432, Ser453 (RAVS) and Arg48, Ala119, Leu432, Ser453 (RALS), have KM values 2-fold lower than Arg48, Ala119, Val432, Ser453 (RAVN): 1.4+/-0.3 and 1.3+/-0.4 microM, respectively, compared with 2.8+/-0.8 microM (P

GST and CYP2E1 genes are involved in metabolism of several compounds (e.g., solvents) that may play a role in brain cancer etiology. We evaluated associations between polymorphisms in these genes and adult brain tumor incidence. Cases were 782 patients with brain tumors diagnosed from 1994 to 1998 at three United States hospitals. Controls were 799 patients admitted to the same hospitals for nonmalignant conditions. DNA was extracted from blood samples that had been collected from 1277 subjects (80% of all subjects; 604 controls; 422 gliomas, 172 meningiomas, and 79 acoustic neuromas), and genotyping was successfully conducted for GSTM1 null, GSTT1 null, GSTP I105V, GSTP A114V, CYP2E1 RsaI, and CYP2E1 Ins96. The GSTP1 105 Val/Val genotype was associated with increased glioma incidence [odds ratio (OR), 1.8; 95% confidence limits (CLs), 1.2, 2.7], with the estimated effect following a trend of increasing magnitude by number of variant alleles (Ile/Ile: OR, 1.0; Ile/Val: OR, 1.3; Val/Val: OR, 2.1). The CYP2E1 RsaI variant was weakly associated with glioma (OR, 1.4; 95% CL, 0.9, 2.4) and acoustic neuroma (OR, 2.3; 95% CL, 1.0, 5.3), with some indication of stronger associations among younger subjects. Estimated effects of the gene variants differed by glioma subtype. There was evidence of supermultiplicativity of the joint effect of GSTP1 I105V and CYP2E1 RsaI variants on both glioma and acoustic neuroma, even following adjustment of estimates toward a common prior distribution using hierarchical regression models. Previously reported associations between the GSTT1 null genotype and overall glioma incidence were not replicated, but an association with meningioma was observed (OR, 1.5; 95% CL, 1.0, 2.3). These findings may provide clues to both genetic and environmental determinants of brain tumors.

Recent studies suggest that a polymorphism in catechol-O-methyltransferase (COMT) is associated with increased risk of breast cancer. Methylation by COMT is the principal pathway for inactivation of catechol estrogens, which are hypothesized to participate in estrogen-induced carcinogenesis. We examined the association of COMT genotype and breast cancer risk in a population-based, case-control study of invasive breast cancer in North Carolina. The study population consisted of 654 cases and 642 controls, with approximately equal numbers of African-American and white women and women under the age of 50 and aged 50 or over. Contrary to previous reports, we did not observe an association between one or more copies of the low activity COMT allele (COMT-L) and breast cancer risk. Multivariate relative risks (RRs) were 0.8 (95% confidence interval: 0.6-1.1) for COMT-HL and 0.8 (0.6-1.1) for COMT-LL, compared with the COMT-HH genotype. RRs for COMT did not differ among African-American and white women and we did not observe strong modification of RR estimates by menopausal status, body mass index, physical activity or other covariates. Our results suggest that COMT genotype is not related to breast cancer risk.

About 45 million (∼19%) of U.S. adults smoke, 46 million are former smokers and >100 million are exposed to environmental tobacco smoke. Tobacco use is associated with many types of cancer, heart disease, stroke, and respiratory disease. While hundreds of tobacco smoke constituents cause DNA damage, many adverse outcomes are not related to DNA damage and an emerging hypothesis is that exposure-induced epigenetic effects may mediate many of these outcomes. Using epigenome-wide association, Joubert et al (Env Health Perspectives, 2012) observed Bonferroni-corrected statistically significant associations (480,000 tests, p Citation Format: Xuting Wang, Gary S. Pittman, Dan Su, Kelly N. Adamski, Michelle R. Campbell, Bonnie R. Joubert, Zhiqing Y. Huang, Cathrine Hoyo, Susan K. Murphy, Stephanie A. London, Douglas A. Bell. Dose-dependent alteration of CpG methylation in AHRR and GFI1 in mononuclear cell DNA of smokers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3647. doi:10.1158/1538-7445.AM2013-3647

Single nucleotide polymorphisms (SNPs) may influence disease susceptibility by modifying cellular responses to environmental stresses. Regulatory SNPs occur in non-coding gene regions and can modify gene expression in an allele-specific manner by altering transcription factor binding. The tumor suppressor p53, a transcription factor, coordinates the expression of an intricate network of genes in response to DNA damage. Importantly, sequence-specific DNA binding is essential for p53 tumor suppressive activity. Unfortunately, lack of robust computational methods and laborious experimental assays have hampered the discovery and characterization of the effects of SNPs in p53 response elements (p53REs). Therefore, we developed a refined bioinformatics approach to identify SNPs in bona fide and putative p53-DNA binding sites. We also developed the microsphere assay for protein-DNA binding (MAPD) to assess the impact of genetic variation on p53-RE binding following cellular exposure to DNA-damaging agents. Thirty-two SNPs (within 12 bona fide and 20 putative p53REs) identified by our bioinformatics approach were evaluated using the MAPD assay. We monitored p53-DNA binding in nuclear extracts from doxorubicin- or IR-treated human lymphoblastoid and U2OS osteosarcoma cells. Significant increases in p53 binding over the negative control were observed for the majority of the p53REs. p53 bound several candidate elements at levels comparable to the positive control (p21 p53RE). Alleles that were predicted to lower p53 binding indeed displayed reduced binding toward 23 of the 32 sequences. Of these alleles, 10 diminished p53 binding by ≥50%. Base substitutions at conserved C and G bases had the greatest impact on p53-DNA binding. In addition, five p53RE SNPs altered gene expression in a set of genotyped lymphoblastoid cell lines. Cellular binding of p53 to putative REs are currently being validated by ChIP-seq. These findings demonstrate that our bioinformatics approach combined with MAPD and gene expression analysis can identify SNPs that alter p53 activity in response to DNA damage and discover novel genes within the p53 transcriptional network. This strategy also can be employed to identify functional SNPs for future studies in human populations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4744.