Rare Variant Association Testing Under Low-Coverage Sequencing

Deep sequencing technologies enable the study of the effects of rare variants in disease risk. While methods have been developed to increase statistical power for detection of such effects, detecting subtle associations requires studies with hundreds or thousands of individuals, which is prohibitively costly. Recently, low-coverage sequencing has been shown to effectively reduce the cost of genome-wide association studies, using current sequencing technologies. However, current methods for disease association testing on rare variants cannot be applied directly to low-coverage sequencing data, as they require individual genotype data, which may not be called correctly due to low-coverage and inherent sequencing errors. In this article, we propose two novel methods for detecting association of rare variants with disease risk, using low coverage, error-prone sequencing. We show by simulation that our methods outperform previous methods under both low- and high-coverage sequencing and under different disease architectures. We use real data and simulation studies to demonstrate that to maximize the power to detect associations for a fixed budget, it is desirable to include more samples while lowering coverage and to perform an analysis using our suggested methods.     

Development and evaluation of a multimedia e-learning resource for electrolyte and acid-base disorders

This article reports on the development and evaluation of a Web-based application that provides instruction and hands-on practice in managing electrolyte and acid-base disorders. Our teaching approach, which focuses on concepts rather than details, encourages quantitative analysis and a logical problem-solving approach. Identifying any dangers to the patient is a vital first step. Concepts such as an "appropriate response" to a given perturbation and the need for electroneutrality in body fluids are used repeatedly. Our Electrolyte Workshop was developed using Flash and followed an iterative design process. Two case-based tutorials were built in this first phase, with one tutorial including an interactive treatment simulation. Users select from a menu of therapies and see the impact of their choices on the patient. Appropriate text messages are displayed, and changes in body compartment sizes, brain size, and plasma sodium concentrations are illustrated via Flash animation. Challenges encountered included a shortage of skilled Flash developers, budgetary constraints, and challenges in communication between the authors and the developers. The application was evaluated via user testing by residents and specialists in internal medicine. Satisfaction was measured with a questionnaire based on the System Usability Scale. The mean System Usability Scale score was 78.4 ± 13.8, indicating a good level of usability. Participants rated the content as being scientifically sound; they liked the teaching approach and felt that concepts were conveyed clearly. They indicated that the application held their interest, that it increased their understanding of hyponatremia, and that they would recommend this learning resource to others.     

A cross-sectional study of barriers to personal health record use among patients attending a safety-net clinic

Background

Personal health records (PHR) may improve patients'' health by providing access to and context for health information. Among patients receiving care at a safety-net HIV/AIDS clinic, we examined the hypothesis that a mental health (MH) or substance use (SU) condition represents a barrier to engagement with web-based health information, as measured by consent to participate in a trial that provided access to personal (PHR) or general (non-PHR) health information portals and by completion of baseline study surveys posted there.

Methods

Participants were individually trained to access and navigate individualized online accounts and to complete study surveys. In response to need, during accrual months 4 to 12 we enhanced participant training to encourage survey completion with the help of staff. Using logistic regression models, we estimated odds ratios for study participation and for survey completion by combined MH/SU status, adjusted for levels of computer competency, on-study training, and demographics.

Results

Among 2,871 clinic patients, 70% had MH/SU conditions, with depression (38%) and methamphetamine use (17%) most commonly documented. Middle-aged patients and those with a MH/SU condition were over-represented among study participants (N = 338). Survey completion was statistically independent of MH/SU status (OR, 1.85 [95% CI, 0.93–3.66]) but tended to be higher among those with MH/SU conditions. Completion rates were low among beginner computer users, regardless of training level (<50%), but adequate among advanced users (>70%).

Conclusions

Among patients attending a safety-net clinic, MH/SU conditions were not barriers to engagement with web-based health information. Instead, level of computer competency was useful for identifying individuals requiring substantial computer training in order to fully participate in the study. Intensive on-study training was insufficient to enable beginner computer users to complete study surveys.     

In vitro inhibition of translation initiation by N,N'-diarylureas--potential anti-cancer agents

Symmetrical N,N'-diarylureas: 1,3-bis(3,4-dichlorophenyl)-, 1,3-bis[4-chloro-3-(trifluoromethyl)phenyl]- and 1,3-bis[3,5-bis(trifluoromethyl)phenyl]urea, were identified as potent activators of the eIF2α kinase heme regulated inhibitor. They reduce the abundance of the eIF2·GTP·tRNA(i)(Met) ternary complex and inhibit cancer cell proliferation. An optimization process was undertaken to improve their solubility while preserving their biological activity. Non-symmetrical hybrid ureas were generated by combining one of the hydrophobic phenyl moieties present in the symmetrical ureas with the polar 3-hydroxy-tolyl moiety. O-alkylation of the later added potentially solubilizing charge bearing groups. The new non-symmetrical N,N'-diarylureas were characterized by ternary complex reporter gene and cell proliferation assays, demonstrating good bioactivities. A representative sample of these compounds potently induced phosphorylation of eIF2α and expression of CHOP at the protein and mRNA levels. These inhibitors of translation initiation may become leads for the development of potent, non-toxic, and target specific anti-cancer agents.     

Physical characterization of the "immunosignaturing effect"

Identifying new, effective biomarkers for diseases is proving to be a challenging problem. We have proposed that antibodies may offer a solution to this problem. The physical features and abundance of antibodies make them ideal biomarkers. Additionally, antibodies are often elicited early in the ontogeny of different chronic and infectious diseases. We previously reported that antibodies from patients with infectious disease and separately those with Alzheimer's disease display a characteristic and reproducible "immunosignature" on a microarray of 10,000 random sequence peptides. Here we investigate the physical and chemical parameters underlying how immunosignaturing works. We first show that a variety of monoclonal and polyclonal antibodies raised against different classes of antigens produce distinct profiles on this microarray and the relative affinities are determined. A proposal for how antibodies bind the random sequences is tested. Sera from vaccinated mice and people suffering from a fugal infection are individually assayed to determine the complexity of signals that can be distinguished. Based on these results, we propose that this simple, general and inexpensive system could be optimized to generate a new class of antibody biomarkers for a wide variety of diseases.     

Expression of Stem Cell Markers in the Human Fetal Kidney

In the human fetal kidney (HFK) self-renewing stem cells residing in the metanephric mesenchyme (MM)/blastema are induced to form all cell types of the nephron till 34^th week of gestation. Definition of useful markers is crucial for the identification of HFK stem cells. Because wilms'' tumor, a pediatric renal cancer, initiates from retention of renal stem cells, we hypothesized that surface antigens previously up-regulated in microarrays of both HFK and blastema-enriched stem-like wilms'' tumor xenografts (NCAM, ACVRIIB, DLK1/PREF, GPR39, FZD7, FZD2, NTRK2) are likely to be relevant markers. Comprehensive profiling of these putative and of additional stem cell markers (CD34, CD133, c-Kit, CD90, CD105, CD24) in mid-gestation HFK was performed using immunostaining and FACS in conjunction with EpCAM, an epithelial surface marker that is absent from the MM and increases along nephron differentiation and hence can be separated into negative, dim or bright fractions. No marker was specifically localized to the MM. Nevertheless, FZD7 and NTRK2 were preferentially localized to the MM and emerging tubules (<10% of HFK cells) and were mostly present within the EpCAM^neg and EpCAM^dim fractions, indicating putative stem/progenitor markers. In contrast, single markers such as CD24 and CD133 as well as double-positive CD24⁺CD133⁺ cells comprise >50% of HFK cells and predominantly co-express EpCAM^bright, indicating they are mostly markers of differentiation. Furthermore, localization of NCAM exclusively in the MM and in its nephron progenitor derivatives but also in stroma and the expression pattern of significantly elevated renal stem/progenitor genes Six2, Wt1, Cited1, and Sall1 in NCAM⁺EpCAM^- and to a lesser extent in NCAM⁺EpCAM⁺ fractions confirmed regional identity of cells and assisted us in pinpointing the presence of subpopulations that are putative MM-derived progenitor cells (NCAM⁺EpCAM⁺FZD7⁺), MM stem cells (NCAM⁺EpCAM^-FZD7⁺) or both (NCAM⁺FZD7⁺). These results and concepts provide a framework for developing cell selection strategies for human renal cell-based therapies.     

Sphingomyelin liposomes with defined fatty acids: metabolism and effects on reverse cholesterol transport

Small unilamellar liposomes prepared from sphingomyelins with defined 14C-labeled fatty acids were studied after injection into rats. The liposomes contained trace amounts of [3H]cholesteryl linoleyl ether (CLE), which served as a nonexchangeable and nonhydrolyzable marker. The liposomes were cleared from the circulation with an initial t1/2 of about 90 min. [14C]18:0- and [14C]18:1-containing sphingomyelins were cleared at a similar rate, but [14C]18:2-sphingomyelin disappeared much faster. The liver accounted for up to 70% of [3H]cholesteryl ether injected with 18:0-sphingomyelin liposomes, and for up to 50% with liposomes prepared from 18:1 or 18:2-sphingomyelin. The initial uptake of the liver appeared to be of the entire particle, and the loss of 14C label with time indicated metabolism of the sphingomyelins. With [14C]18:0-sphingomyelin liposomes, up to 8% of liver radioactivity was recovered in neutral lipids 6 h after injection, and this value was 17 and 22% with [14C]18:2- and [14C]18:1-sphingomyelins, respectively. The recovery in 'carcass' of [3H]cholesteryl ether 3 h after injection of [14C]18:2-sphingomyelin liposomes was 33% and of 14C label, 21%. Injection of 18:1- or 18:2-sphingomyelin liposomes (5.4 mumol/100 g body weight) resulted in a 2-fold increase of plasma unesterified cholesterol; a 30% increase was seen with 18:0 liposomes (2.63 mumol/100 g body weight). In experiments with cultured cells, the unsaturated sphingomyelin liposomes alone enhanced cholesterol efflux more extensively than the saturated ones, but their efficacies became similar when mixed with apoprotein (apo) A-I. At equimolar concentration, apo C-III1 or C-III2 had a smaller effect than apo A-I. It is concluded that 18:1- or 18:2-sphingomyelin tends to form small unilamellar liposomes which may reach also extrahepatic tissues. The liposomes able to enhance cholesterol release in vitro and in vivo. Since they are not a substrate for lecithin-cholesterol acyltransferase, they should be able to deliver the free cholesterol to the liver, where they are also rapidly metabolized.