Kim Siegmund

DNA Methylation in the Arginase–Nitric Oxide Synthase Pathway Is Associated with Exhaled Nitric Oxide in Children with Asthma

RATIONALE Genetic variation in arginase (ARG) and nitric oxide synthase (NOS) has been associated with exhaled nitric oxide (FeNO) levels in children. Little is known about whether epigenetic variation in these genes modulates FeNO. OBJECTIVES To evaluate whether DNA methylation in ARG and NOS genes is associated with FeNO. METHODS A subset of 940 participants in the Childrens Health Study were selected for this study. Children were eligible if they had FeNO measurements and buccal cells collected on the same day. CpG loci located in the promoter regions of NOS1, NOS2A, NOS3, ARG1, and ARG2 genes were analyzed. Multiple loci in each gene were evaluated individually and averaged together. DNA methylation was measured using a bisulfite-polymerase chain reaction pyrosequencing assay. Linear regression models were used to investigate the association between DNA methylation and FeNO and whether associations differed by asthma status. MEASUREMENTS AND MAIN RESULTS DNA methylation in ARG2 was significantly associated with FeNO. A 1% increase in average DNA methylation of ARG2 was associated with a 2.3% decrease in FeNO (95% confidence interval, -4 to -0.6). This association was significantly larger in children with asthma (%diff = -8.7%) than in children with no asthma (%diff = -1.6%; p(int) = 0.01). Differences in FeNO by asthma status were also observed for ARG1 (%diff(asthma) = -4.4%; %diff(non-asthma) = 0.3%; p(int) = 0.02). DNA methylation in NOS genes was not associated with FeNO. CONCLUSIONS DNA methylation in ARG1 and ARG2 is associated with FeNO in children with asthma and suggests a possible role for epigenetic regulation of nitric oxide production.

The Association of Tumor Microsatellite Instability Phenotype with Family History of Colorectal Cancer

Family history is a strong predictor of colorectal cancer risk; however, a diagnosis of colorectal cancer among first-degree relatives has not been systematically investigated as a function of the colorectal cancer molecular subtypes related to tumor microsatellite instability (MSI) status. We investigated whether the observable familial colorectal cancer risks differed according to tumor MSI subtypes, stratified as MSI-High (>30% instability), MSI-Low (<30% instability), and MSS (no instability). Data from 3,143 population-based colorectal cancer cases from five institutions were assessed for family history according to the Amsterdam criteria and the Bethesda guidelines, age at diagnosis, sex, tumor location, and MSI status. The distribution of patient characteristics by MSI status was compared using polytomous logistic regression. Overall, 2.8% colorectal cancer cases met the Amsterdam criteria and 37% met the Bethesda guidelines. There were 14% MSI-High, 13% MSI-Low, and 73% MSS colorectal cancers. MSI-High (P < 0.0001) and MSI-Low tumors (P = 0.01) were more proximally located than MSS tumors. MSI-High tumors were more common among females (P < 0.001). The highest proportion of MSI-High tumors occurred in cases <40 years of age whereas the age-dependent distribution of MSI-Low tumors was unchanged. MSI-High tumors showed a statistically significant association with increasing numbers of first-degree relatives with colorectal cancer (P = 0.002); this association disappeared, however, when MSI-High cases meeting Amsterdam criteria were removed from the analysis. MSI-Low tumors did not show a similar association with family history of colorectal cancer. Familial risk associated with MSI-High tumors is primarily driven by the Amsterdam-criteria patients. MSI-Low tumors may represent a distinct subtype of colorectal cancer with respect to certain epidemiologic variables studied here. (Cancer Epidemiol Biomarkers Prev 2009;18(3):967–75)

NOS1 Methylation and Carotid Artery Intima-Media Thickness in Children

Background—Nitric oxide (NO) plays an important role in cardiovascular health by maintaining and regulating vascular tone and blood flow. Epigenetic regulation of NO synthase (NOS), the genes responsible for NO production, may affect cardiovascular disease, including the development of atherosclerosis in children. Methods and Results—We measured percentage DNA methylation using bisulfite conversion and pyrosequencing assays on DNA from buccal cells provided by 377 participants of the Children’s Health Study on whom carotid artery intima-media thickness (CIMT) measurements were also collected. We examined a total of 16 CpG loci located within NOS1, NOS2A, NOS3, ARG1, and ARG2 genes responsible for NO production. CIMT was measured using high-resolution B-mode carotid ultrasound. The association between percentage DNA methylation in ARG and NOS genes with CIMT was evaluated using linear regression adjusted for sex, ethnicity, body mass index, age at CIMT, town of residence, and experimental plate for pyrosequencing reactions. Differences in the association by ethnicity and ancestral group were also evaluated. For a 1% increase in average DNA methylation of NOS1, CIMT increased by 1.2 &mgr;m (P=0.02). This association was greater in Hispanic children of Native American descent (&bgr;=2.3; P=0.004) than in non-Hispanic whites (&bgr;=0.3; P=0.71) or Hispanic whites (&bgr;=1.0; P=0.35). Conclusions—DNA methylation of NOS1 has a plausible role in atherogenesis through regulation of NO production, although ancestry may alter the magnitude of this association.

Should we consider gene x environment interaction in the hunt for quantitative trait loci

We address the question of whether one can obtain increased power for finding a quantitative trait locus (QTL) if a gene×environment (G×E) interaction is incorporated directly into the linkage analysis. We consider both parametric and nonparametric analysis approaches to including G×E interaction. For the former, we utilize joint segregation and linkage analysis to estimate simultaneously the recombination fraction and a G×E interaction effect, as well as the remaining model parameters. The nonparametric approach is based on an extension of the Haseman‐Elston method applied to sib pairs to include a regression of the squared trait difference on marker‐identity‐by‐descent (IBD) probability (π), the sibling covariate sum (z), and π×z. We utilize 50 replicates of the simulated data and compare empirical power of the various approaches to detect MG4, a locus that is involved in a strong interaction with age for Q4 and in a weaker interaction with environmental factor E2 for Q3. Using the parametric approach, including a G×age effect does increase power for detecting linkage between MG4 and Q4 compared with ignoring the interaction (powers 58% and 38%, respectively, to exceed a lod score of 3.0). On the other hand, including a G×E2 interaction has little effect on the power to detect linkage between MG4 and Q3. The nonparametric approach leads to qualitatively similar findings. We conclude that it is beneficial to incorporate G×E interaction into a linkage analysis, provided the interaction effect is of sufficiently strong magnitude.

Copy number variation in the Framingham Heart Study

In this paper we test for association between copy number variation and diabetes in a subset of individuals from the Framingham Heart Study. We used the 500 k SNP data and called copy number variation using two algorithms: the genome alteration detection algorithm of Pique-Regi et al. and the software Golden Helix. We then tested for association between copy number and diabetes using a gene-based analysis. Our results show little evidence of association between copy number and diabetes status. Furthermore, our results indicate a relatively poor level of agreement between copy number calls resulting from the two programs. We then examined potential causes for this difference in results and the implications for future studies.

Impact of chromosomal rearrangement upon DNA methylation patterns in leukemia

Abstract Genomic instability, including genetic mutations and chromosomal rearrangements, can lead to cancer development. Aberrant DNA methylation occurs commonly in cancer cells. The aim of this study is to determine the effects of a specific chromosomal lesion the BCR-ABL translocation t(9:22), in establishing DNA methylation profiles in cancer. Materials and methods We compared DNA methylation of 1,505 selected promoter CpGs in chronic myelogenous leukemia (CML), acute lymphoblastic leukemia (ALL) with and without the Philadelphia chromosome t(9:22), CD34+ hematopoietic stem cells transfected with BCR-ABL, and other tumors without BCR-ABL (acute promyelocytic leukemia (APL) and gastrointestinal stromal tumors (GIST). In this study, the DNA methylation profile of CML was more closely related to APL, another myeloid leukemia, than Ph+ ALL. Although DNA methylation profiles were consistent within a specific tumor type, overall DNA methylation profiles were no influenced by BCR-ABL gene translocation in the cancers and tissues studied. We conclude that DNA methylation profiles may reflect the cell of origin in cancers rather than the chromosomal lesions involved in leukemogenesis.

Abstract B2-23: A Big Bang model of human colorectal tumor growth

What happens in the early and still undetectable human malignancy is unknown because direct observations are impractical. Here we present a “Big Bang” model, whereby a tumor grows predominantly as a single expansion producing numerous intermixed sub-clones, which are not subject to stringent clonal selection. In this model, both public and most detectable private mutations arise during the earliest phase of tumor growth. Multi-scale genomic profiling of 349 individual glands sampled from 15 colorectal tumors revealed the absence of selective sweeps, uniformly high intra-tumor heterogeneity, and sub-clone mixing in distant tumor regions, as postulated by the Big Bang. By integrating the data in a spatial model of tumor growth and statistical inference framework we also verified the most striking prediction of our model, namely that most detectable intra-tumor heterogeneity originates from private alterations acquired early during growth, and not from the later expansion of selected sub-clones. Hence, early sub-clones define the genomic profile of colorectal carcinomas and advanced adenomas, whereas potentially dangerous late-arising sub-clones will go undetected. Moreover, our results suggest that sub-clone mixing may be a biomarker of malignant potential. This new model provides a quantitative framework that explains the origins of intra-tumor heterogeneity and tumor growth dynamics with significant clinical implications. Citation Format: Andrea Sottoriva, Haeyoun Kang, Zhicheng Ma, Trevor A. Graham, Matthew Salomon, Junsong Zhao, Paul Marjoram, Kim Siegmund, Michael F. Press, Darryl Shibata, Christina Curtis. A Big Bang model of human colorectal tumor growth. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B2-23.

Abstract 287: Characterizing the genetic basis of methylome diversity in histologically normal human lung tissue

The genetic regulation of the human epigenome is not fully appreciated. Here we describe the effects of genetic variants on the DNA methylome in human lung based on methylation-quantitative trait loci (meQTL) analyses. We report 34,304 cis- and 585 trans-meQTLs, a genetic-epigenetic interaction of surprising magnitude, including a regulatory hotspot. These findings are replicated in both breast and kidney tissues and show distinct patterns: cis-meQTLs mostly localize to CpG sites outside of genes, promoters, and CpG islands (CGIs) while trans-meQTLs are over-represented in promoter CGIs. meQTL SNPs are enriched in CTCF binding sites, DNaseI hypersensitivity regions and histone marks. Importantly, 4 of the 5 established lung cancer risk loci in European ancestry are cis-meQTLs and, in aggregate, cis-meQTLs are enriched for lung cancer risk in a genome-wide analysis of 11,587 subjects. Thus, inherited genetic variation may affect lung carcinogenesis by regulating the human methylome. Citation Format: Jianxin Shi, Crystal Marconett, Jubao Duan, Paula Hyland, Peng Li, Zhaoming Wang, William Wheeler, Mihaela Campan, Jing Huang, Weiyin Zhou, Tim Triche, Laufey Amundadottir, Amy Hutchinson, Po-Han Chen, Beiyun Zhou, Brian Chung, Pier Alberto Bertazzi, Andrew W. Bergen, Mathew Freedman, Diane Lee, Kim Siegmund, Andrew Clay Warner, Ben Berman, Angela C. Pesatori, Zea Borok, Dario Consonni, Nilanjan Chatterjee, Margaret Tucker, Neil Caporaso, Stephen J. Chanock, Ite A. Laird-Offringa, Maria Teresa Landi. Characterizing the genetic basis of methylome diversity in histologically normal human lung tissue. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 287. doi:10.1158/1538-7445.AM2014-287

Abstract 155: A comparison of DNA methylation in identical twins discordant for Hodgkin lymphoma

DNA methylation is an epigenetic change that may affect gene expression. Environmental determinants are largely unknown but strongly suspected. Because twins are matched on genome and to some extent, early life exposures, a comparison of DNA methylation between twins is of more interest than one between unrelated individuals. To evaluate the possibility of an association between DNA methylation and young adult Hodgkin lymphoma (HL), DNA samples from blood or saliva were collected from both members of 25 identical twin pairs in whom one twin had developed HL prior to age 50 (discordant pairs). The median time since diagnosis was 16 years. DNA methylation status at 27,578 loci in 14,475 genes was measured in each sample using the Illumina Infinium methylation assay. We measured any difference ( 0) in the level of DNA methylation at each locus between the case and the unaffected co-twin and applied a paired t-test. The overall pattern indicated that cases had higher levels of DNA methylation at loci with lower average DNA methylation levels, but the reverse was true at loci with higher overall levels of DNA methylation (e.g., here the cases had lower levels). With respect to specific loci, the HL case consistently had a higher level of DNA methylation compared to his/her unaffected co-twin at locus cg24693053 in the MFSD7 (major facilitator superfamily domain containing 7) gene in 22/25 pairs; this was also the locus with the most significant p-value (p= 0.0000332). Twenty-two out of 25 cases had a lower level of DNA methylation than their unaffected co-twin at loci cg17385448 in the AGMAT (Agmatine ureohydrolase [agmatinase]) gene (p= 0.0001587) and cg09809672 in the EDARADD (EDAR-associated death domain) gene (p= 0.003775846). Seventeen out of 19 cases with the nodular sclerosis subtype had consistently higher DNA methylation levels at 9 loci and lower levels at 7 loci compared to their unaffected co-twin (binomial test p=0.0007). There was no overall effect on the pattern of methylation by time since diagnosis. We observed suggestive differences in DNA methylation levels in normal peripheral blood mononuclear cells collected from young adult HL patients in remission relative to their unaffected identical co-twins. The differences may have resulted from HL disease or treatment, or could have pre-dated the diagnosis. The results are compatible with a role of the cumulative environment on DNA methylation. 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 155.