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1.  Fine-mapping of the 5p15.33, 6p22.1-p21.31 and 15q25.1 regions identifies functional and histology-specific lung cancer susceptibility loci in African-Americans 
Background
Genome-wide association studies of European and East Asian populations have identified lung cancer susceptibility loci on chromosomes 5p15.33, 6p22.1-p21.31 and 15q25.1. We investigated whether these regions contain lung cancer susceptibly loci in African-Americans refined previous association signals by utilizing the reduced linkage disequilibrium observed in African-Americans.
Methods
1308 African-American cases and 1241 African-American controls from three centers were genotyped for 760 single nucleotide polymorphisms spanning three regions, and additional SNP imputation was performed. Associations between polymorphisms and lung cancer risk were estimated using logistic regression, stratified by tumor histology where appropriate.
Results
The strongest associations were observed on 15q25.1 in/near CHRNA5, including a missense substitution (rs16969968: OR = 1.57, 95% CI = 1.25–1.97, P = 1.1 × 10−4) and variants in the 5′-UTR. Associations on 6p22.1-p21.31 were histology-specific and included a missense variant in BAT2 associated with squamous-cell carcinoma (rs2736158: OR = 0.64, 95% CI = 0.48–0.85, P = 1.82 × 10−3). Associations on 5p15.33 were detected near TERT, the strongest of which was rs2735940 (OR = 0.82, 95% CI = 0.73–0.93, P = 1.1 × 10−3). This association was stronger among cases with adenocarcinoma (OR = 0.75, 95% CI = 0.65–0.86, P = 8.1 × 10−5).
Conclusions
Polymorphisms in 5p15.33, 6p22.1-p21.31 and 15q25.1 are associated with lung cancer in African-Americans. Variants on 5p15.33 are stronger risk factors for adenocarcinoma and variants on 6p21.33 associated only with squamous-cell carcinoma.
Impact
Results implicate the BAT2, TERT and CHRNA5 genes in the pathogenesis of specific lung cancer histologies.
doi:10.1158/1055-9965.EPI-12-1007-T
PMCID: PMC3565099  PMID: 23221128
Lung cancer; adenocarcinoma; squamous-cell carcinoma; fine-mapping; African-American; genetic association
2.  Key epigenetic changes associated with lung cancer development 
Epigenetics  2012;7(6):559-566.
Epigenetic alterations are a common event in lung cancer and their identification can serve to inform on the carcinogenic process and provide clinically relevant biomarkers. Using paired tumor and non-tumor lung tissues from 146 individuals from three independent populations we sought to identify common changes in DNA methylation associated with the development of non-small cell lung cancer. Pathologically normal lung tissue taken at the time of cancer resection was matched to tumorous lung tissue and together were probed for methylation using Illumina GoldenGate arrays in the discovery set (n = 47 pairs) followed by bisulfite pyrosequencing for validation sets (n = 99 pairs). For each matched pair the change in methylation at each CpG was calculated (the odds ratio), and these ratios were averaged across individuals and ranked by magnitude to identify the CpGs with the greatest change in methylation associated with tumor development. We identified the top gene-loci representing an increase in methylation (HOXA9, 10.3-fold and SOX1, 5.9-fold) and decrease in methylation (DDR1, 8.1-fold). In replication testing sets, methylation was higher in tumors for HOXA9 (p < 2.2 × 10−16) and SOX1 (p < 2.2 × 10−16) and lower for DDR1 (p < 2.2 × 10−16). The magnitude and strength of these changes were consistent across squamous cell and adenocarcinoma tumors. Our data indicate that the identified genes consistently have altered methylation in lung tumors. Our identified genes should be included in translational studies that aim to develop screening for early disease detection.
doi:10.4161/epi.20219
PMCID: PMC3398985  PMID: 22522909
DNA Methylation; goldengate; lung cancer; molecular epidemiology; pyrosequencing
3.  Smoking and Genetic Risk Variation across Populations of European, Asian, and African-American Ancestry - A Meta-analysis of Chromosome 15q25 
Genetic epidemiology  2012;36(4):340-351.
Recent meta-analyses of European ancestry subjects show strong evidence for association between smoking quantity and multiple genetic variants on chromosome 15q25. This meta-analysis extends the examination of association between distinct genes in the CHRNA5-CHRNA3-CHRNB4 region and smoking quantity to Asian and African American populations to confirm and refine specific reported associations.
Association results for a dichotomized cigarettes smoked per day (CPD) phenotype in 27 datasets (European ancestry (N=14,786), Asian (N=6,889), and African American (N=10,912) for a total of 32,587 smokers) were meta-analyzed by population and results were compared across all three populations.
We demonstrate association between smoking quantity and markers in the chromosome 15q25 region across all three populations, and narrow the region of association. Of the variants tested, only rs16969968 is associated with smoking (p < 0.01) in each of these three populations (OR=1.33, 95%C.I.=1.25–1.42, p=1.1×10−17 in meta-analysis across all population samples). Additional variants displayed a consistent signal in both European ancestry and Asian datasets, but not in African Americans.
The observed consistent association of rs16969968 with heavy smoking across multiple populations, combined with its known biological significance, suggests rs16969968 is most likely a functional variant that alters risk for heavy smoking. We interpret additional association results that differ across populations as providing evidence for additional functional variants, but we are unable to further localize the source of this association. Using the cross-population study paradigm provides valuable insights to narrow regions of interest and inform future biological experiments.
doi:10.1002/gepi.21627
PMCID: PMC3387741  PMID: 22539395
smoking; genetics; meta-analysis; cross-population
4.  Association study of nicotinic acetylcholine receptor genes identifies a novel lung cancer susceptibility locus near CHRNA1 in African-Americans 
Oncotarget  2012;3(11):1428-1438.
Studies in European and East Asian populations have identified lung cancer susceptibility loci in nicotinic acetylcholine receptor (nAChR) genes on chromosome 15q25.1 which also appear to influence smoking behaviors. We sought to determine if genetic variation in nAChR genes influences lung cancer susceptibly in African-Americans, and evaluated the association of these cancer susceptibility loci with smoking behavior. A total of 1308 African-Americans with lung cancer and 1241 African-American controls from three centers were genotyped for 378 single nucleotide polymorphisms (SNPs) spanning the sixteen human nAChR genes. Associations between SNPs and the risk of lung cancer were estimated using logistic regression, adjusted for relevant covariates. Seven SNPs in three nAChR genes were significantly associated with lung cancer at a strict Bonferroni-corrected level, including a novel association on chromosome 2 near the promoter of CHRNA1 (rs3755486: OR = 1.40, 95% CI = 1.18-1.67, P = 1.0 × 10−4). Association analysis of an additional 305 imputed SNPs on 2q31.1 supported this association. Publicly available expression data demonstrated that the rs3755486 risk allele correlates with increased CHRNA1 gene expression. Additional SNP associations were observed on 15q25.1 in genes previously associated with lung cancer, including a missense variant in CHRNA5 (rs16969968: OR = 1.60, 95% CI = 1.27-2.01, P = 5.9 × 10−5). Risk alleles on 15q25.1 also correlated with an increased number of cigarettes smoked per day among the controls. These findings identify a novel lung cancer risk locus on 2q31.1 which correlates with CHRNA1 expression and replicate previous associations on 15q25.1 in African-Americans.
PMCID: PMC3717803  PMID: 23232035
Lung cancer; nicotine dependence; African-Americans; genetic association; smoking
5.  DNA Methylation, Isocitrate Dehydrogenase Mutation, and Survival in Glioma 
Background
Although much is known about molecular and chromosomal characteristics that distinguish glioma histological subtypes, DNA methylation patterns of gliomas and their association with other tumor features such as mutation of isocitrate dehydrogenase (IDH) genes have only recently begun to be investigated.
Methods
DNA methylation of glioblastomas, astrocytomas, oligodendrogliomas, oligoastrocytomas, ependymomas, and pilocytic astrocytomas (n = 131) from the Brain Tumor Research Center at the University of California San Francisco, as well as nontumor brain tissues (n = 7), was assessed with the Illumina GoldenGate methylation array. Methylation data were subjected to recursively partitioned mixture modeling (RPMM) to derive methylation classes. Differential DNA methylation between tumor and nontumor was also assessed. The association between methylation class and IDH mutation (IDH1 and IDH2) was tested using univariate and multivariable analysis for tumors (n = 95) with available substrate for sequencing. Survival of glioma patients carrying mutant IDH (n = 57) was compared with patients carrying wild-type IDH (n = 38) using a multivariable Cox proportional hazards model and Kaplan–Meier analysis. All statistical tests were two-sided.
Results
We observed a statistically significant association between RPMM methylation class and glioma histological subtype (P < 2.2 × 10−16). Compared with nontumor brain tissues, across glioma tumor histological subtypes, the differential methylation ratios of CpG loci were statistically significantly different (permutation P < .0001). Methylation class was strongly associated with IDH mutation in gliomas (P = 3.0 × 10−16). Compared with glioma patients whose tumors harbored wild-type IDH, patients whose tumors harbored mutant IDH showed statistically significantly improved survival (hazard ratio of death = 0.27, 95% confidence interval = 0.10 to 0.72).
Conclusion
The homogeneity of methylation classes for gliomas with IDH mutation, despite their histological diversity, suggests that IDH mutation is associated with a distinct DNA methylation phenotype and an altered metabolic profile in glioma.
doi:10.1093/jnci/djq497
PMCID: PMC3022619  PMID: 21163902
6.  History of parvovirus B19 infection is associated with a DNA methylation signature in childhood acute lymphoblastic leukemia 
Epigenetics  2011;6(12):1436-1443.
Acute lymphoblastic leukemia (ALL) likely has a multistep etiology, with initial genetic aberrations occurring early in life. An abnormal immune response to common infections has emerged as a plausible candidate for triggering the proliferation of pre-leukemic clones and the fixation of secondary genetic mutations and epigenetic alterations. We investigated whether evidence of infection with a specific common myelotropic childhood virus, parvovirus B19 (PVB19), relates to patterns of gene promoter DNA methylation in ALL patients. We serologically tested bone marrow samples at diagnosis of B-cell ALL for PVB19 infection and DNA methylation using a high-throughput bead array and found that 4.2% and 36.7% of samples were seroreactive to PVB19 IgM and IgG, respectively. Leukemia samples were grouped by DNA methylation pattern. Controlling for age and immunophenotype, unsupervised modeling confirmed that the DNA methylation pattern was associated with history of PVB19 (assessed by IgG, p = 0.02), but not recent infection (assessed by IgM). Replication assays on single genes were consistent with the association. The data indicate that a common viral illness may drive specific DNA methylation patterns in susceptible B-precursor cells, contributing to the leukemogenic potential of such cells. Infections may impact childhood leukemia by altering DNA methylation patterns and specific key genes in susceptible cells; these changes may be retained even after the clearance of infection.
doi:10.4161/epi.6.12.18464
PMCID: PMC3256332  PMID: 22139573
childhood leukemia; DNA methylation; parvovirus B19; serology
7.  Identification of Methylated Genes Associated with Aggressive Bladder Cancer 
PLoS ONE  2010;5(8):e12334.
Approximately 500,000 individuals diagnosed with bladder cancer in the U.S. require routine cystoscopic follow-up to monitor for disease recurrences or progression, resulting in over $2 billion in annual expenditures. Identification of new diagnostic and monitoring strategies are clearly needed, and markers related to DNA methylation alterations hold great promise due to their stability, objective measurement, and known associations with the disease and with its clinical features. To identify novel epigenetic markers of aggressive bladder cancer, we utilized a high-throughput DNA methylation bead-array in two distinct population-based series of incident bladder cancer (n = 73 and n = 264, respectively). We then validated the association between methylation of these candidate loci with tumor grade in a third population (n = 245) through bisulfite pyrosequencing of candidate loci. Array based analyses identified 5 loci for further confirmation with bisulfite pyrosequencing. We identified and confirmed that increased promoter methylation of HOXB2 is significantly and independently associated with invasive bladder cancer and methylation of HOXB2, KRT13 and FRZB together significantly predict high-grade non-invasive disease. Methylation of these genes may be useful as clinical markers of the disease and may point to genes and pathways worthy of additional examination as novel targets for therapeutic treatment.
doi:10.1371/journal.pone.0012334
PMCID: PMC2925945  PMID: 20808801
8.  Copy number variation has little impact on bead-array-based measures of DNA methylation 
Bioinformatics  2009;25(16):1999-2005.
Motivation: Integration of various genome-scale measures of molecular alterations is of great interest to researchers aiming to better define disease processes or identify novel targets with clinical utility. Particularly important in cancer are measures of gene copy number DNA methylation. However, copy number variation may bias the measurement of DNA methylation. To investigate possible bias, we analyzed integrated data obtained from 19 head and neck squamous cell carcinoma (HNSCC) tumors and 23 mesothelioma tumors.
Results: Statistical analysis of observational data produced results consistent with those anticipated from theoretical mathematical properties. Average beta value reported by Illumina GoldenGate (a bead-array platform) was significantly smaller than a similar measure constructed from the ratio of average dye intensities. Among CpGs that had only small variations in measured methylation across tumors (filtering out clearly biological methylation signatures), there were no systematic copy number effects on methylation for three and more than four copies; however, one copy led to small systematic negative effects, and no copies led to substantial significant negative effects.
Conclusions: Since mathematical considerations suggest little bias in methylation assayed using bead-arrays, the consistency of observational data with anticipated properties suggests little bias. However, further analysis of systematic copy number effects across CpGs suggest that though there may be little bias when there are copy number gains, small biases may result when one allele is lost, and substantial biases when both alleles are lost. These results suggest that further integration of these measures can be useful for characterizing the biological relationships between these somatic events.
Contact: E_Andres_Houseman@brown.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btp364
PMCID: PMC2723008  PMID: 19542153
9.  Differentiation of lung adenocarcinoma, pleural mesothelioma, and non-malignant pulmonary tissues using DNA methylation profiles 
Cancer research  2009;69(15):6315-6321.
Pathologic differentiation of tissue of origin in tumors found in the lung can be challenging, with differentiation of mesothelioma and lung adenocarcinoma emblematic of this problem. Indeed, proper classification is essential for determination of treatment regimen for these diseases, making accurate and early diagnosis critical. Here we investigate the potential of epigenetic profiles of lung adenocarcinoma, mesothelioma, and non-malignant pulmonary tissues (n=285) as differentiation markers in an analysis of DNA methylation at 1413 autosomal CpG loci associated with 773 cancer-related genes. Using an unsupervised recursively-partitioned mixture modeling technique for all samples, the derived methylation profile classes were significantly associated with sample type (P < 0.0001). In a similar analysis restricted to tumors, methylation profile classes significantly predicted tumor type (P < 0.0001). Random forests classification of CpG methylation of tumors - which splits the data into training and test sets - accurately differentiated MPM from lung adenocarcinoma over 99% of the time (P < 0.0001). In a locus-by-locus comparison of CpG methylation between tumor types, 1266 CpG loci had significantly different methylation between tumors following correction for multiple comparisons (Q < 0.05); 61% had higher methylation in adenocarcinoma. Using the CpG loci with significant differential methylation in a pathways analysis revealed significant enrichment of methylated gene-loci in Cell Cycle Regulation, DNA Damage Response, PTEN Signaling, and Apoptosis Signaling pathways in lung adenocarcinoma when compared to mesothelioma. Methylation-profile-based differentiation of lung adenocarcinoma and mesothelioma is highly accurate, informs on the distinct etiologies of these diseases, and holds promise for clinical application.
doi:10.1158/0008-5472.CAN-09-1073
PMCID: PMC2755616  PMID: 19638575
10.  Breast Cancer DNA Methylation Profiles Are Associated with Tumor Size and Alcohol and Folate Intake 
PLoS Genetics  2010;6(7):e1001043.
Although tumor size and lymph node involvement are the current cornerstones of breast cancer prognosis, they have not been extensively explored in relation to tumor methylation attributes in conjunction with other tumor and patient dietary and hormonal characteristics. Using primary breast tumors from 162 (AJCC stage I–IV) women from the Kaiser Division of Research Pathways Study and the Illumina GoldenGate methylation bead-array platform, we measured 1,413 autosomal CpG loci associated with 773 cancer-related genes and validated select CpG loci with Sequenom EpiTYPER. Tumor grade, size, estrogen and progesterone receptor status, and triple negative status were significantly (Q-values <0.05) associated with altered methylation of 209, 74, 183, 69, and 130 loci, respectively. Unsupervised clustering, using a recursively partitioned mixture model (RPMM), of all autosomal CpG loci revealed eight distinct methylation classes. Methylation class membership was significantly associated with patient race (P<0.02) and tumor size (P<0.001) in univariate tests. Using multinomial logistic regression to adjust for potential confounders, patient age and tumor size, as well as known disease risk factors of alcohol intake and total dietary folate, were all significantly (P<0.0001) associated with methylation class membership. Breast cancer prognostic characteristics and risk-related exposures appear to be associated with gene-specific tumor methylation, as well as overall methylation patterns.
Author Summary
The current standard prognostic indicator for breast cancer is tumor-node-metastasis staging; though, as population-based studies and clinical trials are conducted, molecular characterization of disease is beginning to allow improved markers of prognosis and assist clinicians in choosing the most appropriate therapies. We investigated DNA methylation profiles in over 160 well annotated breast tumor samples and found significant relationships with standard and other known predictors of prognosis, as well as established risk factors for disease: alcohol intake and dietary folate. Recently the United States National Cancer Institute Cancer Biomarkers Research Group articulated a need for a “Strategic Approach to Validating Methylated Genes as Biomarkers for Breast Cancer,” and our work is extremely responsive to this call for a national strategy. Recognizing the increasing use of pre-operative chemotherapy for patients with operable, early-stage disease, there is added complexity in breast cancer staging. Since chemotherapy can considerably decrease tumor size, it is still unclear whether pre-operative or post-operative stage best informs prognosis and treatment decisions for patients electing pre-operative chemotherapy. However, our data clearly illustrate the promise of tumor DNA methylation for augmenting tumor staging and can be attained with minimal tissue in a pre-operative context.
doi:10.1371/journal.pgen.1001043
PMCID: PMC2912395  PMID: 20686660
11.  Epigenetic profiling reveals etiologically distinct patterns of DNA methylation in head and neck squamous cell carcinoma 
Carcinogenesis  2009;30(3):416-422.
Head and neck squamous cell carcinomas (HNSCCs) represent clinically and etiologically heterogeneous tumors affecting >40 000 patients per year in the USA. Previous research has identified individual epigenetic alterations and, in some cases, the relationship of these alterations with carcinogen exposure or patient outcomes, suggesting that specific exposures give rise to specific types of molecular alterations in HNSCCs. Here, we describe how different etiologic factors are reflected in the molecular character and clinical outcome of these tumors. In a case series of primary, incident HNSCC (n = 68), we examined the DNA methylation profile of 1413 autosomal CpG loci in 773 genes, in relation to exposures and etiologic factors. The overall pattern of epigenetic alteration could significantly distinguish tumor from normal head and neck epithelial tissues (P < 0.0001) more effectively than specific gene methylation events. Among tumors, there were significant associations between specific DNA methylation profile classes and tobacco smoking and alcohol exposures. Although there was a significant association between methylation profile and tumor stage (P < 0.01), we did not observe an association between these profiles and overall patient survival after adjustment for stage; although methylation of a number of specific loci falling in different cellular pathways was associated with overall patient survival. We found that the etiologic heterogeneity of HNSCC is reflected in specific patterns of molecular epigenetic alterations within the tumors and that the DNA methylation profiles may hold clinical promise worthy of further study.
doi:10.1093/carcin/bgp006
PMCID: PMC2650795  PMID: 19126652
12.  Epigenetic profiles distinguish pleural mesothelioma from normal pleura and predict lung asbestos burden and clinical outcome 
Cancer research  2009;69(1):227-234.
Mechanisms of action of non-mutagenic carcinogens such as asbestos remain poorly characterized. As pleural mesothelioma is known to have limited numbers of genetic mutations, we aimed to characterize the relationships among gene-locus specific methylation alterations, disease status, asbestos burden, and survival in this rapidly-fatal asbestos-associated tumor. Methylation of 1505 CpG loci associated with 803 cancer-related genes were studied in 158 pleural mesotheliomas and 18 normal pleura. After false-discovery rate correction, 969 CpG loci were independently associated with disease status (Q < 0.05). Classifying samples based upon CpG methylation profile with a mixture model approach, methylation classes discriminated tumor from normal pleura (permutation P < 0.0001). In a random forests classification the overall misclassification error rate was 3.4%, with <1% (n=1) of tumors misclassified as normal (P < 0.0001). Among tumors, methylation class membership was significantly associated with lung tissue asbestos body burden (P < 0.03), and significantly predicted survival (likelihood ratio P < 0.01). Consistent with prior work, asbestos burden was associated with an increased risk of death (HR = 1.4, 95% CI, 1.1 – 1.8). Our results have shown that methylation profiles powerfully differentiate diseased pleura from non-tumor pleura and that asbestos burden and methylation profiles are independent predictors of mesothelioma patient survival. We have added to the growing body of evidence that cellular epigenetic dysregulation is a critical mode of action for asbestos in the induction of pleural mesothelioma. Importantly, these findings hold great promise for using epigenetic profiling in the diagnosis and prognosis of human cancers.
doi:10.1158/0008-5472.CAN-08-2586
PMCID: PMC2744125  PMID: 19118007
Methylation; asbestos; mesothelioma
13.  Aging and Environmental Exposures Alter Tissue-Specific DNA Methylation Dependent upon CpG Island Context 
PLoS Genetics  2009;5(8):e1000602.
Epigenetic control of gene transcription is critical for normal human development and cellular differentiation. While alterations of epigenetic marks such as DNA methylation have been linked to cancers and many other human diseases, interindividual epigenetic variations in normal tissues due to aging, environmental factors, or innate susceptibility are poorly characterized. The plasticity, tissue-specific nature, and variability of gene expression are related to epigenomic states that vary across individuals. Thus, population-based investigations are needed to further our understanding of the fundamental dynamics of normal individual epigenomes. We analyzed 217 non-pathologic human tissues from 10 anatomic sites at 1,413 autosomal CpG loci associated with 773 genes to investigate tissue-specific differences in DNA methylation and to discern how aging and exposures contribute to normal variation in methylation. Methylation profile classes derived from unsupervised modeling were significantly associated with age (P<0.0001) and were significant predictors of tissue origin (P<0.0001). In solid tissues (n = 119) we found striking, highly significant CpG island–dependent correlations between age and methylation; loci in CpG islands gained methylation with age, loci not in CpG islands lost methylation with age (P<0.001), and this pattern was consistent across tissues and in an analysis of blood-derived DNA. Our data clearly demonstrate age- and exposure-related differences in tissue-specific methylation and significant age-associated methylation patterns which are CpG island context-dependent. This work provides novel insight into the role of aging and the environment in susceptibility to diseases such as cancer and critically informs the field of epigenomics by providing evidence of epigenetic dysregulation by age-related methylation alterations. Collectively we reveal key issues to consider both in the construction of reference and disease-related epigenomes and in the interpretation of potentially pathologically important alterations.
Author Summary
The causes and extent of tissue-specific interindividual variation in human epigenomes are underappreciated and, hence, poorly characterized. We surveyed over 200 carefully annotated human tissue samples from ten anatosites at 1,413 CpGs for methylation alterations to appraise the nature of phenotypically, and hence potentially clinically important epigenomic alterations. Within tissue types, across individuals, we found variation in methylation that was significantly related to aging and environmental exposures such as tobacco smoking. Individual variation in age- and exposure-related methylation may significantly contribute to increased susceptibility to several diseases. As the NIH–funded HapMap project is critically contributing to annotating the human reference genome defining normal genetic variability, our work raises key issues to consider in the construction of reference epigenomes. It is well recognized that understanding genetic variation is essential to understanding disease. Our work, and the known interplay of epigenetics and genetics, makes it equally clear that a more complete characterization of epigenetic variation and its sources must be accomplished to reach the goal of a complete understanding of disease. Additional research is absolutely necessary to define the mechanisms controlling epigenomic variation. We have begun to lay the foundations for essential normal tissue controls for comparison to diseased tissue, which will allow the identification of the most crucial disease-related alterations and provide more robust targets for novel treatments.
doi:10.1371/journal.pgen.1000602
PMCID: PMC2718614  PMID: 19680444
14.  Targeted Sequencing in Chromosome 17q Linkage Region Identifies Familial Glioma Candidates in the Gliogene Consortium 
Scientific Reports  2015;5:8278.
Glioma is a rare, but highly fatal, cancer that accounts for the majority of malignant primary brain tumors. Inherited predisposition to glioma has been consistently observed within non-syndromic families. Our previous studies, which involved non-parametric and parametric linkage analyses, both yielded significant linkage peaks on chromosome 17q. Here, we use data from next generation and Sanger sequencing to identify familial glioma candidate genes and variants on chromosome 17q for further investigation. We applied a filtering schema to narrow the original list of 4830 annotated variants down to 21 very rare (<0.1% frequency), non-synonymous variants. Our findings implicate the MYO19 and KIF18B genes and rare variants in SPAG9 and RUNDC1 as candidates worthy of further investigation. Burden testing and functional studies are planned.
doi:10.1038/srep08278
PMCID: PMC4317686  PMID: 25652157
16.  Socioeconomic Status and Lung Cancer: Unraveling the Contribution of Genetic Admixture 
American journal of public health  2013;103(10):e73-e80.
Objectives
We examined the relationship between genetic ancestry, socioeconomic status and lung cancer among African Americans and Latinos.
Methods
We evaluated socioeconomic status and genetic ancestry in a Northern California lung cancer case-control study (1998-2003) of African Americans and Latinos. Lung cancer cases and controls were frequency matched on age, sex and race/ethnicity. Case-control differences in individual admixture proportions were assessed using two-sample t-tests, and analysis of covariance. Logistic regression models examined associations between genetic ancestry, socioeconomic characteristics and lung cancer.
Results
Decreased Amerindian ancestry was associated with higher education among Latino controls and greater African ancestry was associated with decreased education among African lung cancer cases. Education was associated with lung cancer in both Latinos and African Americans, independently of smoking, ancestry, age and sex. Genetic ancestry was not associated with lung cancer in African Americans.
Conclusions
Findings suggest socioeconomic factors may have a greater impact than genetic ancestry for lung cancer among African Americans. The genetic heterogeneity and recent dynamic migration and acculturation of Latinos complicate recruitment, thus epidemiologic analyses and findings should be interpreted cautiously.
doi:10.2105/AJPH.2013.301370
PMCID: PMC3780736  PMID: 23948011
17.  Genetic variants in telomerase-related genes are associated with an older age at diagnosis in glioma patients: evidence for distinct pathways of gliomagenesis 
Neuro-Oncology  2013;15(8):1041-1047.
Background
Genome-wide association studies have implicated single nucleotide polymorphisms (SNPs) in 7 genes as glioma risk factors, including 2 (TERT, RTEL1) involved in telomerase structure/function. We examined associations of these 7 established glioma risk loci with age at diagnosis among patients with glioma.
Methods
SNP genotype data were available for 2286 Caucasian glioma patients from the University of California, San Francisco (n = 1434) and the Mayo Clinic (n = 852). Regression analyses were performed to test for associations between “number of risk alleles” and “age at diagnosis,” adjusted for sex and study site and stratified by tumor grade/histology where appropriate.
Results
Four SNPs were significantly associated with age at diagnosis. Carrying a greater number of risk alleles at rs55705857 (CCDC26) and at rs498872 (PHLDB1) was associated with younger age at diagnosis (P = 1.4 × 10−22 and P = 9.5 × 10−7, respectively). These SNPs are stronger risk factors for oligodendroglial tumors, which tend to occur in younger patients, and their association with age at diagnosis varied across tumor subtypes. In contrast, carrying more risk alleles at rs2736100 (TERT) and at rs6010620 (RTEL1) was associated with older age at diagnosis (P = 6.2 × 10−4 and P = 2.5 × 10−4, respectively). These SNPs are risk factors for all glioma grades/histologies, and their association with age at diagnosis was consistent across tumor subgroups.
Conclusions
Carrying a greater number of risk alleles might be expected to decrease age at diagnosis. However, glioma susceptibility conferred by variation in telomerase-related genes did not follow this pattern. This supports the hypothesis that telomerase-related mechanisms of telomere maintenance are more associated with gliomas that develop later in life than those utilizing telomerase-independent mechanisms (ie, alternative lengthening of telomeres).
doi:10.1093/neuonc/not051
PMCID: PMC3714154  PMID: 23733245
age at diagnosis; glioma; single nucleotide polymorphism; telomerase; telomere
18.  Inherited variant on chromosome 11q23 increases susceptibility to IDH-mutated but not IDH-normal gliomas regardless of grade or histology 
Neuro-Oncology  2013;15(5):535-541.
Introduction
Recent discoveries of inherited glioma risk loci and acquired IDH mutations are providing new insights into glioma etiology. IDH mutations are common in lower grade gliomas and secondary glioblastomas and uncommon in primary glioblastomas. Because the inherited variant in 11q23 has been associated with risk of lower grade glioma and not with glioblastomas, we hypothesized that this variant increases susceptibility to IDH-mutated gliomas, but not to IDH-wild-type gliomas.
Methods
We tested this hypothesis in patients with glioma and controls from the San Francisco Adult Glioma Study, the Mayo Clinic, and Illumina controls (1102 total patients, 5299 total controls). Case-control additive associations of 11q23 risk alleles (rs498872, T allele) were calculated using logistic regression, stratified by tumor IDH status (mutated or wild-type) and by histology and grade. We also adjusted for the recently discovered 8q24 glioma risk locus rs55705857 G allele.
Results
The 11q23 glioma risk locus was associated with increased risk of IDH-mutated gliomas of all histologies and grades (odds ratio [OR] = 1.50; 95% confidence interval [CI] = 1.29–1.74; P = 1.3X10−7) but not with IDH-wild-type gliomas of any histology or grade (OR = 0.91; 95% CI = 0.81–1.03; P = 0.14). The associations were independent of the rs55705857 G allele.
Conclusion
A variant at the 11q23 locus increases risk for IDH-mutated but not IDH-wild-type gliomas, regardless of grade or histology.
doi:10.1093/neuonc/nos324
PMCID: PMC3635511  PMID: 23361564
adult glioma; IDH1 and IDH2 mutation; rs498872; rs55705857; single-nucleotide polymorphism
19.  Analysis of 60 Reported Glioma Risk SNPs Replicates Published GWAS Findings but Fails to Replicate Associations From Published Candidate-Gene Studies 
Genetic epidemiology  2012;37(2):222-228.
Genomewide association studies (GWAS) and candidate-gene studies have implicated single-nucleotide polymorphisms (SNPs) in at least 45 different genes as putative glioma risk factors. Attempts to validate these associations have yielded variable results and few genetic risk factors have been consistently replicated. We conducted a case-control study of Caucasian glioma cases and controls from the University of California San Francisco (810 cases, 512 controls) and the Mayo Clinic (852 cases, 789 controls) in an attempt to replicate previously reported genetic risk factors for glioma. Sixty SNPs selected from the literature (eight from GWAS and 52 from candidate-gene studies) were successfully genotyped on an Illumina custom genotyping panel. Eight SNPs in/near seven different genes (TERT, EGFR, CCDC26, CDKN2A, PHLDB1, RTEL1, TP53) were significantly associated with glioma risk in the combined dataset (P < 0.05), with all associations in the same direction as in previous reports. Several SNP associations showed considerable differences across histologic subtype. All eight successfully replicated associations were first identified by GWAS, although none of the putative risk SNPs from candidate-gene studies was associated in the full case-control sample (all P values > 0.05). Although several confirmed associations are located near genes long known to be involved in gliomagenesis (e.g., EGFR, CDKN2A, TP53), these associations were first discovered by the GWAS approach and are in noncoding regions. These results highlight that the deficiencies of the candidate-gene approach lay in selecting both appropriate genes and relevant SNPs within these genes.
doi:10.1002/gepi.21707
PMCID: PMC3670948  PMID: 23280628
glioma; SNP; GWAS; candidate-gene
20.  SSBP2 variants are associated with survival in glioblastoma patients 
Purpose
Glioblastoma is a devastating, incurable disease with few known prognostic factors. Here we present the first genome-wide survival and validation study for glioblastoma.
Methods
Cox regressions for survival with 314,635 inherited autosomal single nucleotide polymorphisms (SNPs) among 315 San Francisco Adult Glioma Study patients for discovery and three independent validation datasets (87 Mayo Clinic, 232 GliomaSE and 115 The Cancer Genome Atlas patients) were used to identify SNPs associated with overall survival for Caucasian glioblastoma patients treated with the current standard of care, resection, radiation and temozolomide (total n=749). Tumor expression of the gene that contained the identified prognostic SNP was examined in three separate datasets (total n=619). Genotype imputation was used to estimate hazard ratios (HRs) for SNPs that had not been directly genotyped.
Results
From the discovery and validation analyses, we identified a variant in SSBP2 (single-stranded DNA-binding protein 2) on 5q14.1 associated with overall survival in combined analyses (hazard ratio (HR) = 1.64; P = 1.3X10−6). Expression of SSBP2 in tumors from three independent datasets also was significantly related to patient survival (P = 5.3 X 10−4). Using genotype imputation, the SSBP2 SNP rs17296479 had the strongest statistically significant genome-wide association with poorer overall patient survival (HR = 1.79; 95% CI: 1.45–2.22; P = 1.0 X 10−7).
Conclusion
The minor allele of SSBP2 SNP rs17296479 and the increased tumor expression of SSBP2 were statistically significantly associated with poorer overall survival among glioblastoma patients. With further confirmation, previously unrecognized inherited variations influencing survival may warrant inclusion in clinical trials to improve randomization. Unaccounted for genetic influence on survival could produce unwanted bias in such studies.
doi:10.1158/1078-0432.CCR-11-2778
PMCID: PMC3607457  PMID: 22472174
glioma; glioblastoma; GWAS; survival; epidemiology; SSBP2
21.  A low frequency variant at 8q24.21 is strongly associated with risk of oligodendroglial tumors and IDH1 or IDH2 mutated astrocytomas 
Nature genetics  2012;44(10):1122-1125.
SNPs mapped to 8q24.21 have been shown to be associated with glioma development. By means of tag SNP genotyping/imputation, pooled next-generation sequencing (NGS) using long-range PCR, and subsequent validation SNP genotyping we identified seven low-frequency SNPs that were consistently and highly associated with glioma risk (p=10−25 to 10−14). The most associated SNP, rs55705857, remained highly significant after individual adjustment for the other top six and two previously published SNPs. After stratifying by histologic and tumor genetic subtype, the most significant associations were with oligodendroglial tumors and IDH1 or IDH2 mutated gliomas, (ORrs55705857 = 5.1, p=1.1x10−31 and ORrs55705857 = 4.8, p=6.6 x10−22, respectively). Strong associations were observed for IDH1 or IDH2 mutated astrocytomas (grades II–IV) (OR rs55705857=5.16–6.66; p=4.7x10−12 to 2.2x10−8), but not IDH1 or IDH2 wild-type astrocytomas (smallest p=0.26). The conserved sequence block that includes rs55705857 is consistently modeled as a microRNA.
doi:10.1038/ng.2388
PMCID: PMC3600846  PMID: 22922872
Oligodendroglioma; Glioblastoma; IDH1 and IDH2 mutation; single nucleotide polymorphism
22.  A Case-Control Study of Asphalt and Tar Exposure and Lung Cancer in Minorities 
Objectives
Considerable controversy surrounds the carcinogenic potential of asphalt and tar. Since minority individuals may have had relatively high historical exposures, we investigated asphalt and tar exposure and lung cancer risk among African Americans and Latino Americans.
Methods
We conducted a case-control study of lung cancer among African Americans and Latino Americans in the San Francisco Bay area (422 cases, 894 controls). A questionnaire was used to obtain detailed work histories and exposure information. Self-reported exposure to asphalt and tar as well as other factors (eg. smoking, automobile exhaust, and asbestos) were evaluated as predictors of lung cancer risk. Potential effect modification by cytochrome P450 (CYP) 1A1 was also explored.
Results
Self-reported duration of exposure to asphalt and tar was associated with a statistically significant excess risk of lung cancer in the overall population (OR: 1.11, 95%CI: 1.01–1.22), evaluating risk per year of exposure. Years of exposure to automobile exhaust (OR: 1.02, 95%CI: 1.00–1.05) and asbestos (OR: 1.04, 95%CI: 1.02–1.06) were also associated with statistically significant elevations in risk. In Latino Americans, the lung cancer risks associated with polycyclic aromatic hydrocarbon-related exposures were consistently higher in the CYP1A1 wildtype subjects as compared to the variant genotype subjects, and the interaction was statistically significant for smoking and the CYP1A1 M2 polymorphism (p-valueinteraction=0.02).
Conclusions
These data are consistent with the literature suggesting that exposure to asphalt and tar may increase risk of lung cancer. However, it was not possible to separate the effects and asphalt and tar in this study.
doi:10.1002/ajim.21007
PMCID: PMC3196745  PMID: 21882217
asphalt; tar; lung cancer; minorities
23.  Associations of High-Grade Glioma With Glioma Risk Alleles and Histories of Allergy and Smoking 
American Journal of Epidemiology  2011;174(5):574-581.
Glioma risk has consistently been inversely associated with allergy history but not with smoking history despite putative biologic plausibility. Data from 855 high-grade glioma cases and 1,160 controls from 4 geographic regions of the United States during 1997–2008 were analyzed for interactions between allergy and smoking histories and inherited variants in 5 established glioma risk regions: 5p15.3 (TERT), 8q24.21 (CCDC26/MLZE), 9p21.3 (CDKN2B), 11q23.3 (PHLDB1/DDX6), and 20q13.3 (RTEL1). The inverse relation between allergy and glioma was stronger among those who did not (odds ratioallergy-glioma = 0.40, 95% confidence interval: 0.28, 0.58) versus those who did (odds ratioallergy-glioma = 0.76, 95% confidence interval: 0.59, 0.97; Pinteraction = 0.02) carry the 9p21.3 risk allele. However, the inverse association with allergy was stronger among those who carried (odds ratioallergy-glioma = 0.44, 95% confidence interval: 0.29, 0.68) versus those who did not carry (odds ratioallergy-glioma = 0.68, 95% confidence interval: 0.54, 0.86) the 20q13.3 glioma risk allele, but this interaction was not statistically significant (P = 0.14). No relation was observed between glioma risk and smoking (odds ratio = 0.92, 95% confidence interval: 0.77, 1.10; P = 0.37), and there were no interactions for glioma risk of smoking history with any of the risk alleles. The authors’ observations are consistent with a recent report that the inherited glioma risk variants in chromosome regions 9p21.3 and 20q13.3 may modify the inverse association of allergy and glioma.
doi:10.1093/aje/kwr124
PMCID: PMC3202152  PMID: 21742680
allergy and immunology; glioma; hypersensitivity; polymorphism, single nucleotide
24.  Circulating levels of the innate and humoral immune regulators CD14 and CD23 are associated with adult glioma 
Cancer Research  2010;70(19):7534-7542.
Allergy history has been consistently inversely associated with glioma risk. Two serologic markers, soluble CD23 (sCD23) and soluble CD14 (sCD14), are part of the innate and adaptive humoral immune systems and modulate allergic responses in opposite directions, with sCD23 enhancing and sCD14 blunting inflammatory responses. We measured sCD23 and sCD14 in serum from blood that was drawn at a single time point from 1079 glioma patients post diagnosis and 736 healthy controls. Glioma was strongly associated with high sCD14 (highest vs. lowest quartile OR = 3.94 (95% CI: 2.98-5.21) and low sCD23 (lowest vs. highest quartile OR=2.5 (95% CI: 1.89-3.23)). Results were consistent across glioma histologic types and grades, but were strongest for glioblastoma. While temozolomide treatment was not associated with either sCD14 or sCD23 levels among cases, those taking dexamethasone had somewhat lower sCD23 levels than those not taking dexamethasone. However, sCD23 was associated with case status regardless of dexamethasone treatment. These results augment the long observed association between allergies and glioma and support a role for the innate and adaptive humoral functions of the immune system, and in particular immunoregulatory proteins, in gliomagenesis.
doi:10.1158/0008-5472.CAN-10-0815
PMCID: PMC3339633  PMID: 20719886
CD14; CD23; glioma; brain tumor; immune; tumor risk; epidemiology
25.  DNA hypermethylation profiles associated with glioma subtypes and EZH2 and IGFBP2 mRNA expression 
Neuro-Oncology  2011;13(3):280-289.
We explored the associations of aberrant DNA methylation patterns in 12 candidate genes with adult glioma subtype, patient survival, and gene expression of enhancer of zeste human homolog 2 (EZH2) and insulin-like growth factor-binding protein 2 (IGFBP2). We analyzed 154 primary glioma tumors (37 astrocytoma II and III, 52 primary glioblastoma multiforme (GBM), 11 secondary GBM, 54 oligodendroglioma/oligoastrocytoma II and III) and 13 nonmalignant brain tissues for aberrant methylation with quantitative methylation-specific PCR (qMS-PCR) and for EZH2 and IGFBP2 expression with quantitative reverse transcription PCR (qRT-PCR). Global methylation was assessed by measuring long interspersed nuclear element-1 (LINE1) methylation. Unsupervised clustering analyses yielded 3 methylation patterns (classes). Class 1 (MGMT, PTEN, RASSF1A, TMS1, ZNF342, EMP3, SOCS1, RFX1) was highly methylated in 82% (75/91) of lower-grade astrocytic and oligodendroglial tumors, 73% (8/11) of secondary GBMs, and 12% (6/52) of primary GBMs. The primary GBMs in this class were early onset (median age 37 years). Class 2 (HOXA9 and SLIT2) was highly methylated in 37% (19/52) of primary GBMs. None of the 10 genes for class 3 that were differentially methylated in classes 1 and 2 were hypermethylated in 92% (12/13) of nonmalignant brain tissues and 52% (27/52) of primary GBMs. Class 1 tumors had elevated EZH2 expression but not elevated IGFBP2; class 2 tumors had both high IGFBP2 and high EZH2 expressions. The gene-specific hypermethylation class correlated with higher levels of global LINE1 methylation and longer patient survival times. These findings indicate a generalized hypermethylation phenotype in glioma linked to improved survival and low IGFBP2. DNA methylation markers are useful in characterizing distinct glioma subtypes and may hold promise for clinical applications.
doi:10.1093/neuonc/noq190
PMCID: PMC3064601  PMID: 21339190
glioma; DNA methylation; EZH2; Polycomb; PI3K/Akt

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