Alyssa Cwanger

DNA Methylation Is Associated with Altered Gene Expression in AMD

PURPOSE Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Evidence suggests oxidative stress plays a role in the disease. To assess the potential contribution of epigenetic regulation of antioxidant genes relevant to AMD pathogenesis, we evaluated DNA methylation, a tissue-specific genetic modulation that affects gene expression. METHODS Using the Infinium HumanMethylation27 Illumina platform, we performed DNA bisulfite sequencing to compare the methylation status in postmortem retina pigment epithelium (RPE)/choroid between patients with AMD and age-matched controls. Gene expression was assessed with the Affymetrix Exon Array. TaqMan gene expression assays were used for relative quantification (RT-PCR) confirmation of the expression array results: Glutathione S-transferase isoform mu1 (GSTM1) and mu5 (GSTM5) promoter methylation was confirmed by CpG island bisulfite pyrosequencing. To assess protein levels and localization, we used Western analysis, immunohistochemistry, and immunofluorescence with murine and human samples. RESULTS The mRNA levels of GSTM1 and GSTM5 were significantly reduced in AMD versus age-matched controls in RPE/choroid and neurosensory retina (NSR), which corresponded to hypermethylation of the GSTM1 promoter. mRNA and protein levels were decreased (RPE to a greater extent than NSR) in AMD postmortem samples, irrespective of age. Immunohistochemistry and immunofluorescence confirm the presence of the enzymes in the NSR and RPE. CONCLUSIONS Comparison of DNA methylation, together with mRNA levels, revealed significant differences between AMD versus normal retinas. The evidence presented suggests that GSTM1 and GSTM5 undergo epigenetic repression in AMD RPE/choroid, which may increase susceptibility to oxidative stress in AMD retinas.

Dexras1, a small GTPase, is required for glutamate-NMDA neurotoxicity

Dexras1, a small G-protein localized predominantly to the brain, is transcriptionally upregulated by the synthetic glucocorticoid dexamethasone. It has close homology to the Ras subfamily but differs in that Dexras1 contains an extended 7 kDa C-terminal tail. Previous studies in our laboratory showed that NMDA receptor activation, via NO and Dexras1, physiologically stimulates DMT1, the major iron importer. A membrane-permeable iron chelator substantially reduces NMDA excitotoxicity, suggesting that Dexras1-mediated iron influx plays a crucial role in NMDA/NO-mediated cell death. We here report that iron influx is elicited by nitric oxide but not by other proapoptotic stimuli, such as H2O2 or staurosporine. Deletion of Dexras1 in mice attenuates NO-mediated cell death in dissociated primary cortical neurons and retinal ganglion cells in vivo. Thus, Dexras1 appears to mediate NMDA-elicited neurotoxicity via NO and iron influx.

A High Serum Iron Level Causes Mouse Retinal Iron Accumulation Despite an Intact Blood-Retinal Barrier

The retina can be shielded by the blood-retinal barrier. Because photoreceptors are damaged by excess iron, it is important to understand whether the blood-retinal barrier protects against high serum iron levels. Bone morphogenic protein 6 (Bmp6) knockout mice have serum iron overload. Herein, we tested whether the previously documented retinal iron accumulation in Bmp6 knockout mice might result from the high serum iron levels or, alternatively, low levels of retinal hepcidin, an iron regulatory hormone whose transcription can be up-regulated by Bmp6. Furthermore, to determine whether increases in serum iron can elevate retinal iron levels, we i.v. injected iron into wild-type mice. Retinas were analyzed by real-time quantitative PCR and immunofluorescence to assess the levels of iron-regulated genes/proteins and oxidative stress. Retinal hepcidin mRNA levels in Bmp6 knockout retinas were the same as, or greater than, those in age-matched wild-type retinas, indicating that Bmp6 knockout does not cause retinal hepcidin deficiency. Changes in mRNA levels of L ferritin and transferrin receptor indicated increased retinal iron levels in i.v. iron-injected wild-type mice. Oxidative stress markers were elevated in photoreceptors of mice receiving i.v. iron. These findings suggest that elevated serum iron levels can overwhelm local retinal iron regulatory mechanisms.

Bayesian probability of malignancy with BI-RADS sonographic features.

The purpose of this study was to develop a quantitative approach for combining individual American College of Radiology Breast Imaging Reporting and Data System (BI‐RADS) sonographic features of breast masses for assessing the overall probability of malignancy.

Comparison of naive Bayes and logistic regression for computer-aided diagnosis of breast masses using ultrasound imaging

This study compares the performance of two proven but very different machine learners, Naïve Bayes and logistic regression, for differentiating malignant and benign breast masses using ultrasound imaging. Ultrasound images of 266 masses were analyzed quantitatively for shape, echogenicity, margin characteristics, and texture features. These features along with patient age, race, and mammographic BI-RADS category were used to train Naïve Bayes and logistic regression classifiers to diagnose lesions as malignant or benign. ROC analysis was performed using all of the features and using only a subset that maximized information gain. Performance was determined by the area under the ROC curve, Az, obtained from leave-one-out cross validation. Naïve Bayes showed significant variation (Az 0.733 ± 0.035 to 0.840 ± 0.029, P < 0.002) with the choice of features, but the performance of logistic regression was relatively unchanged under feature selection (Az 0.839 ± 0.029 to 0.859 ± 0.028, P = 0.605). Out of 34 features, a subset of 6 gave the highest information gain: brightness difference, margin sharpness, depth-to-width, mammographic BI-RADs, age, and race. The probabilities of malignancy determined by Naïve Bayes and logistic regression after feature selection showed significant correlation (R2= 0.87, P < 0.0001). The diagnostic performance of Naïve Bayes and logistic regression can be comparable, but logistic regression is more robust. Since probability of malignancy cannot be measured directly, high correlation between the probabilities derived from two basic but dissimilar models increases confidence in the predictive power of machine learning models for characterizing solid breast masses on ultrasound.

Combined Naïve Bayes and logistic regression for quantitative breast sonography

Sonography is commonly used as an adjunct to mammography for early detection of breast cancer. We are developing methods to classify solid breast masses in sonograms as malignant or benign. The goal of this study was to combine two independent probabilistic classifiers to improve computer-aided diagnosis of breast masses. Naïve Bayes and logistic regression were used for supervised classification of masses from extracted morphological sonographic features, in combination with mammographic BI-RADS (categories 1 to 5) and patient age. Solid masses with biopsy-proven diagnoses were analyzed. Training and testing were performed using leave-one-out cross validation. Diagnostic performance was evaluated by the area under the curve (AUC) of the receiver operating characteristic (ROC). Agreement between predictions from the two classifiers was used to differentiate benign and malignant masses. The results show that logistic regression and Naïve Bayes performed with ROC area of 0.902 ± 0.023 and 0.865 ± 0.027, respectively. The combined use of logistic regression and Naïve Bayes demonstrated reduction in biopsies by 48%, with malignancy missed in 2% of cases (false negative rate of 6.4%).

Iron-Induced Retinal Damage

Iron is a vital element needed for daily functioning. Excess iron, however, can damage the retina by producing the highly toxic hydroxyl radical. For this reason, it is important to understand how iron is absorbed from the gut, transported to the eye, and regulated locally within the retina. This chapter reviews irons potential role in age-related macular degeneration and other retinal diseases as well as mechanisms of retinal iron homeostasis. Then, retinal protection through promising iron chelators and lifestyle modification, including reduction in red meat consumption, is discussed.