Genetic simulation tools for post-genome wide association studies of complex diseases.

Genetic simulation programs are used to model data under specified assumptions to facilitate the understanding and study of complex genetic systems. Standardized data sets generated using genetic simulation are essential for the development and application of novel analytical tools in genetic epidemiology studies. With continuing advances in high-throughput genomic technologies and generation and analysis of larger, more complex data sets, there is a need for updating current approaches in genetic simulation modeling. To provide a forum to address current and emerging challenges in this area, the National Cancer Institute (NCI) sponsored a workshop, entitled "Genetic Simulation Tools for Post-Genome Wide Association Studies of Complex Diseases" at the National Institutes of Health (NIH) in Bethesda, Maryland on March 11-12, 2014. The goals of the workshop were to (1) identify opportunities, challenges, and resource needs for the development and application of genetic simulation models; (2) improve the integration of tools for modeling and analysis of simulated data; and (3) foster collaborations to facilitate development and applications of genetic simulation. During the course of the meeting, the group identified challenges and opportunities for the science of simulation, software and methods development, and collaboration. This paper summarizes key discussions at the meeting, and highlights important challenges and opportunities to advance the field of genetic simulation.

Small heat shock proteins Hspb7 and Hspb12 regulate early steps of cardiac morphogenesis.

Cardiac morphogenesis is a complex multi-stage process, and the molecular basis for controlling distinct steps remains poorly understood. Because gata4 encodes a key transcriptional regulator of morphogenesis, we profiled transcript changes in cardiomyocytes when Gata4 protein is depleted from developing zebrafish embryos. We discovered that gata4 regulates expression of two small heat shock genes, hspb7 and hspb12, both of which are expressed in the embryonic heart. We show that depletion of Hspb7 or Hspb12 disrupts normal cardiac morphogenesis, at least in part due to defects in ventricular size and shape. We confirmed that gata4 interacts genetically with the hspb7/12 pathway, but surprisingly, we found that hspb7 also has an earlier, gata4-independent function. Depletion perturbs Kupffer's vesicle (KV) morphology leading to a failure in establishing the left-right axis of asymmetry. Targeted depletion of Hspb7 in the yolk syncytial layer is sufficient to disrupt KV morphology and also causes an even earlier block to heart tube formation and a bifid phenotype. Recently, several genome-wide association studies found that HSPB7 SNPs are highly associated with idiopathic cardiomyopathies and heart failure. Therefore, GATA4 and HSPB7 may act alone or together to regulate morphogenesis with relevance to congenital and acquired human heart disease.

Percent of lung involved in disease on chest X-ray predicts unfavorable treatment outcome in pulmonary tuberculosis.

Radiology may better define tuberculosis (TB) severity and guide duration of treatment. We aimed to systematically study baseline chest X-rays (CXR) and their association with TB treatment outcome using real-world data. We used logistic regression to associate TB treatment outcomes with CXR findings, including percent of lung involved in disease (PLI), cavitation, and Timika score, alone or in combination with other clinical characteristics, stratifying by drug resistance status and HIV (n = 2,809). We fine-tuned convolutional neural nets (CNN) to automate PLI measurement from the CXR DICOM images (n = 5,261). PLI is the only CXR finding associated with unfavorable outcome across drug resistance and HIV subgroups [Rifampicin-susceptible disease without HIV, adjusted odds ratio (aOR) 1·11 (1·01, 1·22), P-value 0·025]. The most informed model of baseline characteristics tested predicts outcome with a validation mean area under the curve (AUC) of 0·769. PLI and Timika (AUC 0·656 and 0·655 respectively) predict unfavorable outcomes better than cavitary information (best AUC 0·591). The addition of PLI improves prediction compared to sex and age alone (AUC 0·680 and 0·627, respectively).PLI>25% provides a better separation of favorable and unfavorable outcomes compared to PLI>50%. The best performing ensemble of CNNs has an AUC 0·850 for PLI>25% and mean absolute error of 11·7% for the PLI value. PLI is better than cavitation for predicting unfavorable treatment outcome in pulmonary TB in non-clinical trial settings and it can be accurately and automatically predicted with CNNs. One Sentence Summary: The percent of lung involved in disease improves prediction of unfavorable outcomes in pulmonary tuberculosis when added to clinical characteristics.

Predictive capabilities of baseline radiological findings for early and late disease outcomes within sensitive and multi-drug resistant tuberculosis cases.

Purpose: This study compares performance of Timika Score to standardized, detailed radiologist observations of Chest X rays (CXR) for predicting early infectiousness and subsequent treatment outcome in drug sensitive (DS) or multi-drug resistant (MDR) tuberculosis cases. It seeks improvement in prediction of these clinical events through these additional observations. Method: This is a retrospective study analyzing cases from the NIH/NIAID supported TB Portals database, a large, trans-national, multi-site cohort of primarily drug-resistant tuberculosis patients. We analyzed patient records with sputum microscopy readings, radiologist annotated CXR, and treatment outcome including a matching step on important covariates of age, gender, HIV status, case definition, Body Mass Index (BMI), smoking, drug use, and Timika Score across resistance type for comparison. Results: 2142 patients with tuberculosis infection (374 with poor outcome and 1768 with good treatment outcome) were retrospectively reviewed. Bayesian ANOVA demonstrates radiologist observations did not show greater predictive ability for baseline infectiousness (0.77 and 0.74 probability in DS and MDR respectively); however, the observations provided superior prediction of treatment outcome (0.84 and 0.63 probability in DS and MDR respectively). Estimated lung abnormal area and cavity were identified as important predictors underlying the Timika Score's performance. Conclusions: Timika Score simplifies the usage of baseline CXR for prediction of early infectiousness of the case and shows comparable performance to using detailed, standardized radiologist observations. The score's utility diminishes for treatment outcome prediction and is exceeded by the usage of the detailed observations although prediction performance on treatment outcome decreases especially in MDR TB cases.

Gata4 directs development of cardiac-inducing endoderm from ES cells.

The transcription factor Gata4 is essential for normal heart morphogenesis and regulates the survival, growth, and proliferation of cardiomyocytes. We tested if Gata4 can specify cardiomyocyte fate from an uncommitted stem or progenitor cell population, by developing a system for conditional expression of Gata4 in embryonic stem cells. We find that in embryoid body cultures containing even a low ratio of these cells, expression of Gata4 is sufficient to enhance significantly the generation of cardiomyocytes, via a non-cell-autonomous mechanism. The Gata4-expressing cells do not generate cardiac or other mesoderm derivatives. Rather, Gata4 expression directs the development of two types of Sox17+ endoderm. This includes an epCam+Dpp4+ subtype of visceral endoderm. In addition, Gata4 generates similar amounts of epCam+Dpp4- definitive endoderm enriched for Cxcr4, FoxA2, FoxA3, Dlx5 and other characteristic transcripts. Both types of endoderm express cardiac-inducing factors, including WNT antagonists Dkk1 and Sfrp5, although the visceral endoderm subtype has much higher cardiac-inducing activity correlating with relatively enhanced levels of transcripts encoding BMPs. The Gata4-expressing cells eventually express differentiation markers showing commitment to liver development, even under conditions that normally support mesoderm development. The results suggest that Gata4 is capable of specifying endoderm fates that facilitate, with temporal and spatial specificity, the generation of cardiomyocyte progenitors from associated mesoderm.

Radiologist observations of computed tomography (CT) images predict treatment outcome in TB Portals, a real-world database of tuberculosis (TB) cases.

The TB Portals program provides a publicly accessible repository of TB case data containing multi-modal information such as case clinical characteristics, pathogen genomics, and radiomics. The real-world resource contains over 3400 TB cases, primarily drug resistant cases, and CT images with radiologist annotations are available for many of these cases. The breadth of data collected offers a patient-centric view into the etiology of the disease including the temporal context of the available imaging information. Here, we analyze a cohort of new TB cases with available radiologist observations of CTs taken around the time of initial registration of the case into the database and with available follow up to treatment outcome of cured or died. Follow up ranged from 5 weeks to a little over 2 years consistent with the longest treatment regimens for drug resistant TB and cases were registered within the years 2008 to 2019. The radiologist observations were incorporated into machine learning pipelines to test various class balancing strategies on the performance of predictive models. The modeling results support that the radiologist observations are predictive of treatment outcome. Moreover, inferential statistical analysis identifies markers of TB disease spread as having an association with poor treatment outcome including presence of radiologist observations in both lungs, swollen lymph nodes, multiple cavities, and large cavities. While the initial results are promising, further data collection is needed to incorporate methods to mitigate potential confounding such as including additional model covariates or matching cohorts on covariates of interest (e.g. demographics, BMI, comorbidity, TB subtype, etc.). Nonetheless, the preliminary results highlight the utility of the resource for hypothesis generation and exploration of potential biomarkers of TB disease severity and support these additional data collection efforts.

SEER cancer registry biospecimen research: yesterday and tomorrow.

The National Cancer Institute's (NCI) Surveillance, Epidemiology, and End Results (SEER) registries have been a source of biospecimens for cancer research for decades. Recently, registry-based biospecimen studies have become more practical, with the expansion of electronic networks for pathology and medical record reporting. Formalin-fixed paraffin-embedded specimens are now used for next-generation sequencing and other molecular techniques. These developments create new opportunities for SEER biospecimen research. We evaluated 31 research articles published during 2005 to 2013 based on authors' confirmation that these studies involved linkage of SEER data to biospecimens. Rather than providing an exhaustive review of all possible articles, our intent was to indicate the breadth of research made possible by such a resource. We also summarize responses to a 2012 questionnaire that was broadly distributed to the NCI intra- and extramural biospecimen research community. This included responses from 30 investigators who had used SEER biospecimens in their research. The survey was not intended to be a systematic sample, but instead to provide anecdotal insight on strengths, limitations, and the future of SEER biospecimen research. Identified strengths of this research resource include biospecimen availability, cost, and annotation of data, including demographic information, stage, and survival. Shortcomings include limited annotation of clinical attributes such as detailed chemotherapy history and recurrence, and timeliness of turnaround following biospecimen requests. A review of selected SEER biospecimen articles, investigator feedback, and technological advances reinforced our view that SEER biospecimen resources should be developed. This would advance cancer biology, etiology, and personalized therapy research. See all the articles in this CEBP Focus section, "Biomarkers, Biospecimens, and New Technologies in Molecular Epidemiology." Cancer Epidemiol Biomarkers Prev; 23(12); 2681-7. ©2014 AACR.

An injury-responsive gata4 program shapes the zebrafish cardiac ventricle.

A common principle of tissue regeneration is the reactivation of previously employed developmental programs. During zebrafish heart regeneration, cardiomyocytes in the cortical layer of the ventricle induce the transcription factor gene gata4 and proliferate to restore lost muscle. A dynamic cellular mechanism initially creates this cortical muscle in juvenile zebrafish, where a small number of internal cardiomyocytes breach the ventricular wall and expand upon its surface. Here, we find that emergent juvenile cortical cardiomyocytes induce expression of gata4 in a manner similar to during regeneration. Clonal analysis indicates that these cardiomyocytes make biased contributions to build the ventricular wall, whereas gata4(+) cardiomyocytes have little or no proliferation hierarchy during regeneration. Experimental microinjuries or conditions of rapid organismal growth stimulate production of ectopic gata4(+) cortical muscle, implicating biomechanical stress in morphogenesis of this tissue and revealing clonal plasticity. Induced transgenic inhibition defined an essential role for Gata4 activity in morphogenesis of the cortical layer and the preservation of normal cardiac function in growing juveniles, and again in adults during heart regeneration. Our experiments uncover an injury-responsive program that prevents heart failure in juveniles by fortifying the ventricular wall, one that is reiterated in adults to promote regeneration after cardiac damage.

A reverse genetic approach to test functional redundancy during embryogenesis.

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Raloxifene modulates estrogen-mediated B cell autoreactivity in NZB/W F1 mice.

OBJECTIVE: Estrogen has been found to exacerbate disease activity in murine lupus and to induce a lupus-like syndrome in nonspontaneously autoimmune mice. This has led to the consideration that estrogen may be a risk factor for the development of systemic lupus erythematosus (SLE), and selective estrogen receptor modulators (SERM) may serve to ameliorate lupus activity. We evaluated the effects and mechanism of action of the SERM raloxifene in murine lupus. METHODS: Effects of raloxifene on the development of lupus in NZB/W F1 mice were evaluated in the presence and absence of estrogen by assessing the serum DNA reactivity, glomerular IgG deposition and kidney damage, B cell maturation and selection, and activation status of marginal zone and follicular B cells. RESULTS: Compared to estradiol-treated mice, mice treated with estradiol and raloxifene had significantly lower serum anti-DNA antibody levels and less kidney damage. These effects of raloxifene were due, at least in part, to antagonism of the influence of estrogen on DNA-reactive B cells. Raloxifene was found to prevent estrogen-mediated suppression of autoreactive B cell elimination at the T1/T2 selection checkpoint, to reduce estrogen-induced CD40 overexpression on follicular B cells, making them less responsive to T cell costimulation, and to ameliorate estrogen-mediated CD22 downregulation on marginal zone B cells, thereby decreasing their responsiveness to B cell antigen receptor-mediated stimuli. CONCLUSION: Raloxifene suppressed estrogen-mediated effects on the survival, maturation, and activation of autoreactive B cells in NZB/W F1 mice.

Prolactin alters the mechanisms of B cell tolerance induction.

OBJECTIVE: Autoimmune diseases predominantly affect women, suggesting that female sex hormones may play a role in the pathogenesis of such diseases. We have previously shown that persistent mild-to-moderate elevations in serum prolactin levels induce a break in self tolerance in mice with a BALB/c genetic background. The aim of this study was to evaluate the effects of hyperprolactinemia on the mechanisms of B cell tolerance induction. METHODS: Effects of prolactin on splenic B cell subsets were studied in female BALB/c mice. B cell receptor (BCR)-mediated apoptosis and proliferation of transitional B cells were analyzed by flow cytometry. Expression of apoptotic genes was examined by microarrays and real-time polymerase chain reaction analysis. B cells coexpressing kappa/lambda light chains were assessed by flow cytometry and immunohistochemistry. Activation status of transitional type 3 (T3) B cells was evaluated by BCR-induced calcium influx studies. RESULTS: BCR-mediated apoptosis of the T1 B cell subset, a major checkpoint for negative selection of autoreactive specificities, was decreased in prolactin-treated mice. Microarray studies indicated that this event may be mediated by the prolactin-induced up-regulation of the antiapoptotic gene interferon-gamma receptor type II and down-regulation of the proapoptotic gene Trp63. Prolactin treatment also altered the amount of receptor editing, as indicated by the increased number of transitional B cells coexpressing kappa/lambda light chains. Additionally, hyperprolactinemia modified the level of B cell anergy by increasing the degree of BCR-induced calcium influx in the T3 B cells. CONCLUSION: Persistently elevated serum prolactin levels interfere with B cell tolerance induction by impairing BCR-mediated clonal deletion, deregulating receptor editing, and decreasing the threshold for activation of anergic B cells, thereby promoting autoreactivity.