DNA Repair Capacity as a Marker of Breast Cancer Susceptibility

Introduction: The wide-ranging prognostic implications of a breast cancer diagnosis highlight the need to better enable women to make informed decisions regarding screening and treatment options. As several cancer susceptibility syndromes have been linked to germline mutations resulting in defective DNA repair, including the predisposition to breast cancer due to BRCA1 and BRCA2 mutations, more subtle defects in DNA repair capacity may contribute to the components driving differential susceptibility within the general population. Hence, understanding the role of DNA repair capacity in breast cancer onset may aid in the development of a more comprehensive risk profile, thereby furthering the effort to target relevant populations for early screening. In the studies undertaken for this dissertation, we employed various methodologies capturing endpoints across different repair pathways detectable in blood to both further elucidate the etiologic basis of breast cancer development and leverage the information into the potential development of a screening biomarker. Methods: For the phenotypic assessment of nucleotide excision repair (NER) capacity, we developed an ELISA-based method to determine benzo(a)pyrene diolepoxide (BPDE)-DNA adduct capacity in lymphoblastoid cell lines. Gene expression levels were assessed with pre-designed Taqman kits in RNA-derived cDNAs from mononuclear cells using a real-time PCR-based platform. Methylation analysis was conducted with in-house designed assays on bisulfite-converted DNA from mononuclear cells using a pyrosequencing platform. Finally, single nucleotide polymorphisms (SNP) genotyping was assessed in DNA derived from white blood cells with pre-designed Taqman SNP genotyping assays using a real-time PCR-based platform. All studies were conducted in sister-sets enrolled in the New York site within the Breast Cancer Family Registry and all statistical analysis was conducted using the R Foundation for Statistical Computing (2011). Results: We did not detect an association between the ELISA-based phenotypic assessment of NER capacity in the lymphoblastoid cells lines of the sister-sets (n=246, 114 sister-sets) and breast cancer risk (OR = 1.0, 95%CI=0.95, 1.04). Furthermore, we did not observe a correlation with previously determined NER capacity in the same population using an immunohistochemical-based method (r= -0.01, p=0.86). In our gene expression study (n=569, 218 sister-sets), women in the lowest tertile of ATM expression had a heightened risk of breast cancer compared to women in the highest tertile of expression, adjusted for age at blood draw and smoking status (OR=2.12, 95%CI=1.09, 4.12). This association was largely restricted to women with an extended family history of breast cancer (pinteraction = 0.06). Additionally, women in the lowest tertile of MSH2 expression also had a heightened risk of breast cancer compared to women in the highest tertile of expression, adjusted for age at blood draw and smoking status (OR=2.75, 95%CI=1.31, 5.79). The association observed between reductions in ATM expression level and breast cancer risk was lost upon incorporating previously determined end-joining capacity of EcoRI-generated sticky end substrates (OR=1.28, 95%CI=0.15, 11.2) and HincII-generated blunt end substrates (OR=1.55, 95%CI=0.15, 15.5) into the model, suggesting that the impact on risk due to reductions in ATM expression maybe partially driven by the reduction in double strand break repair capacity. In our study investigating breast cancer risk due to the impact of epigenetic modulation on DNA repair gene activity (n=569, 218 sister-sets), no association with risk was observed due to differential promoter methylation levels of BRCA1 (OR=1.09, 95%CI=0.98, 1.20), MLH1 (OR=1.19, 95%CI=0.91, 1.55) or MSH2 (OR=0.89, 95%CI=0.48, 1.64). Furthermore, no correlation between BRCA1 and expression (r=-0.05, p=0.39) or MSH2 methylation and expression (r=-0.04, p=0.39) was observed. Finally, our mismatch repair genotyping study (n=714, 313 sister-sets) indicated an association between the variant MutY_rs3219489 (OR=2.23, 95%CI=1.10, 4.52) and breast cancer risk, as well as a borderline association with risk due to the variant MSH2_rs2303428 (OR=1.71, 95%CI=0.99, 2.95). Furthermore, a protective effect was observed due to the variant MLH3_rs175080, restricted to women without an extended family history of breast cancer (pinteraction = 0.03). Conclusion: These studies suggest that the deregulation of targets spanning various DNA repair pathways contribute to the risk of familial breast cancer

Exploring the associations between microRNA expression profiles and environmental pollutants in human placenta from the National Children's Study (NCS)

The placenta is the principal regulator of the in utero environment, and disruptions to this environment can result in adverse offspring health outcomes. To better characterize the impact of in utero perturbations, we assessed the influence of known environmental pollutants on the expression of microRNA (miRNA) in placental samples collected from the National Children's Study (NCS) Vanguard birth cohort. This study analyzed the expression of 654 miRNAs in 110 term placentas. Environmental pollutants measured in these placentas included dichlorodiphenyldichloroethylene (DDE), bisphenol A (BPA), polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs), arsenic (As), mercury (Hg), lead (Pb), and cadmium (Cd). A moderated t-test was used to identify a panel of differentially expressed miRNAs, which were further analyzed using generalized linear models. We observed 112 miRNAs consistently expressed in >70% of the samples. Consistent with the literature, miRNAs located within the imprinted placenta-specific C19MC cluster, specifically mir-517a, mir-517c, mir-522, and mir-23a, are among the top expressed miRNA in our study. We observed a positive association between PBDE 209 and miR-188–5p and an inverse association between PBDE 99 and let-7c. Both PCBs and Cd were positively associated with miR-1537 expression level. In addition, multiple let-7 family members were downregulated with increasing levels of Hg and Pb. We did not observe DDE or BPA levels to be associated with placental miRNA expression. This is the first birth cohort study linking environmental pollutants and placental expression of miRNAs. Our results suggest that placental miRNA profiles may signal in utero exposures to environmental chemicals

Global DNA methylation levels in white blood cell DNA from sisters discordant for breast cancer from the New York site of the breast cancer family registry

Lower global DNA methylation is associated with genomic instability and it is one of the epigenetic mechanisms relevant to carcinogenesis. Emerging evidence for several cancers suggests that lower overall levels of global DNA methylation in blood are associated with different cancer types, although less is known about breast cancer. We examined global DNA methylation levels using a sibling design in 273 sisters affected with breast cancer and 335 unaffected sisters from the New York site of the Breast Cancer Family Registry. We measured global DNA methylation in total white blood cell (WBC) and granulocyte DNA by two different methods, the [H-3]-methyl acceptance assay and the luminometric methylation assay (LUMA). Global methylation levels were only modestly correlated between sisters discordant for breast cancer (Spearman correlation coefficients ranged from -0.08 to 0.24 depending on assay and DNA source). Using conditional logistic regression models, women in the quartile with the lowest DNA methylation levels (as measured by the [3H]methyl acceptance assay) had a 1.8-fold (95% CI = 1.0-3.3) higher relative association with breast cancer than women in the quartile with the highest DNA methylation levels. When we examined the association on a continuous scale, we also observed a positive association (odds ratio, OR = 1.3, 95% CI = 1.0-1.7, for a one unit change in the natural logarithm of the DPM/mu g of DNA). We observed no association between measures by the LUMA assay and breast cancer risk. If replicated in prospective studies, this study suggests that global DNA methylation levels measured in WBC may be a potential biomarker of breast cancer risk even within families at higher risk of cancer

Expression of imprinted genes in placenta is associated with infant neurobehavioral development

<p>Genomic imprinting disorders often exhibit delayed neurobehavioral development, suggesting this unique mechanism of epigenetic regulation plays a role in mental and neurological health. While major errors in imprinting have been linked to adverse health outcomes, there has been little research conducted on how moderate variability in imprinted gene expression within a population contributes to differences in neurobehavioral outcomes, particularly at birth. Here, we profiled the expression of 108 known and putative imprinted genes in human placenta samples from 615 infants assessed by the Neonatal Intensive Care Unit (NICU) Network Neurobehavioral Scales (NNNS). Data reduction identified 10 genes (<i>DLX5, DHCR24, VTRNA2-1, PHLDA2, NPAP1, FAM50B, GNAS-AS1, PAX8-AS1, SHANK2</i>, and <i>COPG2IT1</i>) whose expression could distinguish between newborn neurobehavioral profiles derived from the NNNS. Clustering infants based on the expression pattern of these genes identified 2 groups of infants characterized by reduced quality of movement, increased signs of asymmetrical and non-optimal reflexes, and increased odds of demonstrating increased signs of physiologic stress and abstinence. Overall, these results suggest that common variation in placental imprinted gene expression is linked to suboptimal performance on scales of neurological functioning as well as with increased signs of physiologic stress, highlighting the central importance of the control of expression of these genes in the placenta for neurobehavioral development.</p