Venetta Thomas

Genome-wide association study of prostate cancer in men of African ancestry identifies a susceptibility locus at 17q21

In search of common risk alleles for prostate cancer that could contribute to high rates of the disease in men of African ancestry, we conducted a genome-wide association study, with 1,047,986 SNP markers examined in 3,425 African-Americans with prostate cancer (cases) and 3,290 African-American male controls. We followed up the most significant 17 new associations from stage 1 in 1,844 cases and 3,269 controls of African ancestry. We identified a new risk variant on chromosome 17q21 (rs7210100, odds ratio per allele = 1.51, P = 3.4 × 10−13). The frequency of the risk allele is ∼5% in men of African descent, whereas it is rare in other populations (<1%). Further studies are needed to investigate the biological contribution of this allele to prostate cancer risk. These findings emphasize the importance of conducting genome-wide association studies in diverse populations.

Identification of UreR binding sites in the Enterobacteriaceae plasmid-encoded and Proteus mirabilis urease gene operons.

The closely related Proteus mirabilis and Enterobacteriaceae plasmid‐encoded urease genes are positively regulated by the AraC‐like transcriptional activator UreR. In the presence of the effector molecule urea, UreR promotes transcription of ureD, the initial gene in the urease operon, and increases transcription of the divergently transcribed ureR. Here, we identify UreR‐specific binding sites in the ureRp–ureDp intergenic regions. Recombinant UreR (rUreR) was expressed and purified, and gel shift and DNase I protection assays were performed with this protein. These analyses indicated that there are two distinct rUreR binding sites in both the plasmid‐encoded and P. mirabilis ureRp–ureDp intergenic regions. A consensus binding site of TA/GT/CA/TT/GC/TTA/TT/AATTG was predicted from the DNase I protection assays. Although rUreR bound to the specific DNA binding site in both the presence and the absence of urea, the dissociation rate constant k–1 of the rUreR–DNA complex interaction was measurably different when urea was present. In the absence of urea, the dissociation of the protein–DNA complexes, for both ureRp and ureDp, was complete at the earliest time point, and it was not possible to determine a rate. In the presence of urea, dissociation was measurable with a k–1 for the rUreR–ureRp interaction of 1.2 ± 0.2 × 10−2 s−1 and a k–1 for the rUreR–ureDp interaction of 2.6 ± 0.1 × 10−3 s−1. This corresponds to a half‐life of the ureRp–rUreR interaction of 58 s, and a half‐life of the ureDp–rUreR interaction of 4 min 26 s. A model describing a potential role for urea in the activation of these promoters is proposed.

Activation of transcription at divergent urea‐dependent promoters by the urease gene regulator UreR

The Proteus mirabilis and plasmid‐encoded urease loci contain seven contiguous structural and accessory genes (ureDABCEFG) and the divergently transcribed ureR, which codes for an AraC‐like transcriptional activator. Previously, it was shown that the plasmid‐encoded ureR to ureD intergenic region contained divergent promoters (ureRp and ureDp). Transcription from these promoters required both the effector molecule urea and the activator protein UreR. In this report, we demonstrate that the P. mirabilis urease gene cluster contains similar divergent urea‐ and UreR‐dependent promoters. The ureR gene products from either urease locus were able to activate transcription at both the plasmid‐encoded and P. mirabilis promoters. The minimal concentration of urea required to activate transcription at ureRp or ureDp from either gene cluster was approximately 4 mM. The transcriptional start sites for the plasmid‐encoded and P. mirabilis divergent promoters were similar in an Escherichia coli DH5α background, as determined by primer‐extension analysis. However, in P. mirabilis HI4320, transcription of ureR initiated predominately at an alternative site. Physical mapping and inhibition studies were used to localize the UreR‐binding sites within the plasmid‐encoded ureRp and ureDp intergenic sequences to regions of 68 bp and 86 bp, respectively. Gel shift analysis demonstrated that UreR bound to a 135 bp fragment in the approximate centre of the plasmid‐encoded ureR to ureD intergenic region. The results presented here suggest that the P. mirabilis and plasmid‐encoded urease gene clusters utilize similar mechanisms of transcriptional activation in response to urea.

Urea-dependent Signal Transduction by the Virulence Regulator UreR

Identification of the environmental triggers involved in the expression of virulence genes is a fundamental objective in studies of bacterial pathogens. For uropathogens, urea, found in the urinary tract at concentrations of up to 500 mm, functions as an environmental signal. Urea freely diffuses into the bacterium Providencia stuartii and activates UreR, a member of the AraC family of transcriptional activators. Active UreR promotes transcription of virulence-associated urease genes and alerts the organisms of its immediate milieu. Thus, the UreR·urea complex has a dual role, acting as both a transcriptional activator as well as an environmental sensor. Here, we describe the molecular events associated with activation of gene expression by urea-bound UreR. The K d of the urea·UreR binding reaction was measured as 0.2 mm by fluorescence quenching assays, and the shape of the binding curve indicated a single specific urea-binding site on UreR. Histidine residues are critical for urea binding in urease, and therefore to identify the urea-binding site in UreR, five mutant UreR forms were generated with histidine to alanine substitutions. Two of the mutants (UreRc) exhibited a constitutive phenotype by both activating transcription and binding to DNA with an increased affinity in the absence of urea. The UreRc bound urea with an affinity similar to that of wild-type UreR. We concluded, therefore, that the mutations resulting in constitutive activity were not involved in the UreR·urea interaction. UreR was activated, then, either by binding urea or by histidine to alanine substitutions at one of two positions. Circular dichroism indicated little change in the structure of UreR when activated, and size-exclusion chromatography demonstrated that both rUreR and rUreRc were dimers in both the presence and absence of urea. Thus, the structural changes associated with activation are subtle.

Intracellular signaling of tumor necrosis factor-α in brain microvascular endothelial cells is mediated by a protein tyrosine kinase and protein kinase C-dependent pathway

The intracellular signaling pathways responsible for tumor necrosis factor (TNF)-alpha stimulation of lymphocyte adhesion to brain microvascular endothelial cells (BMEC) were studied using inhibitors of protein kinase C (bisindolylmaleimide HCl, H-7, or staurosporine), or protein tyrosine kinase (genistein). Each of these blocked the ability of BMEC to respond to TNF-alpha. In contrast, BMEC treated with H-89, an inhibitor of protein kinase A, or the adenylate cyclase inhibitor, dideoxyadenosine, responded normally to TNF-alpha. Forskolin, an adenylate cyclase agonist, significantly increased lymphocyte adhesion to BMEC. These data indicate that intracellular signaling by TNF-alpha in BMEC is mediated through a protein kinase C and tyrosine kinase dependent pathway.

Methodological Considerations in Estimation of Phenotype Heritability Using Genome- Wide SNP Data, Illustrated by an Analysis of the Heritability of Height in a Large Sample of African Ancestry Adults

Height has an extremely polygenic pattern of inheritance. Genome-wide association studies (GWAS) have revealed hundreds of common variants that are associated with human height at genome-wide levels of significance. However, only a small fraction of phenotypic variation can be explained by the aggregate of these common variants. In a large study of African-American men and women (n = 14,419), we genotyped and analyzed 966,578 autosomal SNPs across the entire genome using a linear mixed model variance components approach implemented in the program GCTA (Yang et al Nat Genet 2010), and estimated an additive heritability of 44.7% (se: 3.7%) for this phenotype in a sample of evidently unrelated individuals. While this estimated value is similar to that given by Yang et al in their analyses, we remain concerned about two related issues: (1) whether in the complete absence of hidden relatedness, variance components methods have adequate power to estimate heritability when a very large number of SNPs are used in the analysis; and (2) whether estimation of heritability may be biased, in real studies, by low levels of residual hidden relatedness. We addressed the first question in a semi-analytic fashion by directly simulating the distribution of the score statistic for a test of zero heritability with and without low levels of relatedness. The second question was addressed by a very careful comparison of the behavior of estimated heritability for both observed (self-reported) height and simulated phenotypes compared to imputation R2 as a function of the number of SNPs used in the analysis. These simulations help to address the important question about whether todays GWAS SNPs will remain useful for imputing causal variants that are discovered using very large sample sizes in future studies of height, or whether the causal variants themselves will need to be genotyped de novo in order to build a prediction model that ultimately captures a large fraction of the variability of height, and by implication other complex phenotypes. Our overall conclusions are that when study sizes are quite large (5,000 or so) the additive heritability estimate for height is not apparently biased upwards using the linear mixed model; however there is evidence in our simulation that a very large number of causal variants (many thousands) each with very small effect on phenotypic variance will need to be discovered to fill the gap between the heritability explained by known versus unknown causal variants. We conclude that todays GWAS data will remain useful in the future for causal variant prediction, but that finding the causal variants that need to be predicted may be extremely laborious.

Abstract 4704: Racial/ethnic differences in double-strand-break repair signaling in breast cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC To evaluate whether deficient DNA repair contributes to elevated DNA damage and breast carcinogenesis, we used a newly developed flow-cytometry assay for rapid assessment of H2AX phosphorylation (gamma-H2AX) in response to double strand breaks (DSBs) in peripheral lymphocytes from 64 breast cancer cases and 71 controls. Basal or ionizing radiation (IR)-induced gamma-H2AX was not influenced by age, smoking status, family history (FH), age at menarche, age at first birth or parity and body mass index (BMI). With limited sample size, basal or IR-induced gamma-H2AX level did not differ between breast cancer cases (mean+SD, 6.65+1.18; 29.23+6.88) and controls (6.48+0.95; 27.19+7.26). In subgroup analysis of women with younger age at first live birth ( 25 kg/m2). In younger cases, a higher gamma-H2AX was observed in AA (p=0.042) compared to Caucasians (32.71+5.43 vs. 27.32+5.33). In never smokers, AA cases had a significantly higher level of IR-induced gamma-H2AX than Caucasians (32.70+5.77 vs. 27.58+6.41, p=0.03). In cases without FH, AA had a significantly higher level of IR-induced gamma-H2AX than Caucasians (32.38+6.90 vs. 28.02+7.09, p=0.044). In cases with a higher BMI, AA had a significantly higher level of IR-induced gamma-H2AX than Caucasians (33.36+5.94 vs. 26.93+6.00, p=0.001). Our results, although based on a relatively small group of subjects, indicate that DSBs response is significantly higher in AA breast cancer cases. Future larger studies are warranted to test whether this racial/ethnic difference in IR-induced gamma-H2AX levels contribute to breast cancer risk, tumor phenotypes, treatment response, and/or clinical outcome. 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 4704.

Abstract A45: Inflammatory biomarker c-reactive protein in radiotherapy-induced skin toxicities of breast cancer patients.

Purpose: Post-surgery adjuvant radiotherapy (RT) significantly reduces recurrence and metastasis of breast cancer. However, many patients experience early adverse skin reactions (EASRs) that impact quality of life. This study evaluated an inflammatory biomarker C-reactive protein (CRP) in RT-induced EASRs in breast cancer patients undergoing RT. Methods: We recruited 159 breast cancer patients undergoing RT after breast conserving surgery. The pre- and post-RT plasma CRP levels were measured using a highly-sensitive ELISA CRP assay. RT-induced EASRs were assessed at weeks 3 and 6 using the National Cancer Institute Common Toxicity Criteria (v3.0). Results: RT-induced grade 2+ skin toxicities were observed in 8 (5%) and 79 (50%) patients at weeks 3 and 6, respectively. Significantly higher proportions of African Americans developed grade 3 skin toxicities (13.8% vs. 2.3% in Whites) at week 6. In multivariate analysis, there was a suggestive association between grade 2+ skin toxicities at week 3 and >2 mg/L pre-RT CRP (OR=4.68, 95%CI=0.49, 44.94). However, grade 2+ skin toxicities at week 6 were significantly associated with elevated: CRP > 1 mg/L (OR=3.26; 95%CI=1.35, 7.85, p=0.01), obesity (OR=2.39; 95%CI=1.19, 4.83, p=0.02), or combined both factors (OR=7.80; 95%CI=2.59, 23.49, p<0.001). Conclusion: This is the first study to suggest that CRP has value as an inflammatory biomarker in RT-induced EASRs, particularly combined with obesity. Future larger studies are warranted to validate our findings and facilitate the discovery and development of anti-inflammatory agents to protect normal tissue from RT-induced adverse effects and improve quality of life in breast cancer patients undergoing RT. Citation Format: Jorge L. Rodriguez-Gil, Anne Cooley, Venetta Thomas, Cristiane Takita, Jean Wright, Martine Poitevien, Glenn O. Allen, Isildinha M. Reis, Wei Zhao, Jennifer J. Hu. Inflammatory biomarker c-reactive protein in radiotherapy-induced skin toxicities of breast cancer patients. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A45.

Abstract 2613: Racial/Ethnic specific polygenic models of breast cancer risk

Breast cancer is the most frequently diagnosed cancer and the second leading cause of cancer death in American women. Earlier family linkage studies have identified high-penetrance breast cancer genes, such as BRCA1, BRCA2, and TP53. Moreover, genes involved in DNA repair, such as CHEK2, ATM, BRIP and PALB2, have been associated with moderate breast cancer risk. Recent Genome-wide Association Studies (GWAS) have identified multiple single nucleotide polymorphisms (SNPs) associated with breast cancer risk. We evaluated whether breast cancer risk is associated with multiple SNPs in DNA damage signaling and repair and immune and inflammatory responses in addition to SNPs identified from GWAS. We hypothesize that targeting critical candidate pathways may yield important information regarding SNP-SNP interactions in breast cancer risk. We selected SNPs from DNA damage signaling and cell cycle control, base excision repair, nucleotide excision repair, double-strand break repair, mismatch repair, and immune and inflammatory responses. We have genotype results of 3,754 SNPs in 910 controls (763 Whites and 147 African Americans) and 793 breast cancer cases (667 Whites and 116 African Americans). 16.2% and 20.6% of controls and cases (respectively) had a first degree relative with breast cancer. 48% and 55.7% of the controls and cases (respectively) had their first child before or at the age of 24. 40.5% and 42.2% of the controls and cases (respectively) had a history of smoking. Our study results suggest that 6 SNPs were associated with breast cancer risk in Whites: (1) 2 SNPs from GWAS - rs3817198 in LSP1 (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2613. doi:1538-7445.AM2012-2613

Abstract 3204: Association between oxidative damage and early adverse skin reactions following postoperative adjuvant radiotherapy in breast cancer patients

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Breast cancer is the most frequently diagnosed cancer and the second leading cause of cancer death in American women. Lumpectomy followed by radiotherapy (RT) has significantly improved survival. However, about 30% of patients develop a grade 2 or worse early or late skin reaction, pain, breast edema and poor cosmetic results that impact quality of life. Inter-individual variability in the development of RT-induced adverse reactions in normal tissue is well-documented for both acute and late effects. Therefore, identifying individuals which may have severe reactions could assist the radiation oncologist in tailoring a treatment regimen which provides adequate care with minimal undesirable effects. One of the hallmarks of radiation induced DNA damage is the oxidation of the nucleotide guanine. Repair and excretion of this modified base known as 8-hydroxydeoxyguanosine (8-OHdG) serves as a biomarker of oxidative stress and DNA damage. In an ongoing study, we investigated the urinary level of 8-OHdG (presented as ng/mg creatinine) in urine of the first 103 breast cancer patients undergoing adjuvant RT following breast conservation surgery. The normal skin toxicity at 3 weeks and at the end of treatment was assessed and associated with 8-OHdG levels. There was a significant correlation between pre- (55.8+35.2) and post-RT (55.5+37.4) urinary 8-OHdG levels (Pearson correlation coefficient=0.44; p<0.001). Within our patient population, Non-Hispanic Whites had a significantly higher 8-OHdG level (80.9+52.6) compared to Hispanic Whites (48.2+25.2, p=0.001) and Black or African Americans (43.7+20.9, p=0.008). With limited sample size, at 3 weeks of post-RT, patients with no apparent skin reaction showed a non-significantly lower pre-RT 8-OHdG (46.4+23.5) compared to patients with a grade 1 or 2 skin toxicity (57.8+37.5, p=0.18). Similar trend was also observed for post-RT 8-OHdG (45.6+20.7 vs. 57.7+40.3; p=.20). At the end of RT, patients with detectable skin toxicity had a baseline 8-OHdG level of 56.0+35.6 while patients without a skin reaction had a baseline level of 39.6+17.6. In summary, our preliminary data with limited sample size showed significant racial/ethnic differences in urinary 8-OHdG levels and promising but not significant association between 8-OHdG levels and skin toxicity grade. Larger studies are warranted to validate these interesting findings. The outcome of the ongoing study (n=1000) will target effective intervention and treatment strategies, and ultimately improve quality of life and progression-free survival in high-risk breast cancer patient populations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3204. doi:10.1158/1538-7445.AM2011-3204