Genome wide association studies in presence of misclassified binary responses

Background

Misclassification has been shown to have a high prevalence in binary responses in both livestock and human populations. Leaving these errors uncorrected before analyses will have a negative impact on the overall goal of genome-wide association studies (GWAS) including reducing predictive power. A liability threshold model that contemplates misclassification was developed to assess the effects of mis-diagnostic errors on GWAS. Four simulated scenarios of case–control datasets were generated. Each dataset consisted of 2000 individuals and was analyzed with varying odds ratios of the influential SNPs and misclassification rates of 5% and 10%.

Results

Analyses of binary responses subject to misclassification resulted in underestimation of influential SNPs and failed to estimate the true magnitude and direction of the effects. Once the misclassification algorithm was applied there was a 12% to 29% increase in accuracy, and a substantial reduction in bias. The proposed method was able to capture the majority of the most significant SNPs that were not identified in the analysis of the misclassified data. In fact, in one of the simulation scenarios, 33% of the influential SNPs were not identified using the misclassified data, compared with the analysis using the data without misclassification. However, using the proposed method, only 13% were not identified. Furthermore, the proposed method was able to identify with high probability a large portion of the truly misclassified observations.

Conclusions

The proposed model provides a statistical tool to correct or at least attenuate the negative effects of misclassified binary responses in GWAS. Across different levels of misclassification probability as well as odds ratios of significant SNPs, the model proved to be robust. In fact, SNP effects, and misclassification probability were accurately estimated and the truly misclassified observations were identified with high probabilities compared to non-misclassified responses. This study was limited to situations where the misclassification probability was assumed to be the same in cases and controls which is not always the case based on real human disease data. Thus, it is of interest to evaluate the performance of the proposed model in that situation which is the current focus of our research.

     

Predicting coexistence of plants subject to a tolerance-competition trade-off

Ecological trade-offs between species are often invoked to explain species coexistence in ecological communities. However, few mathematical models have been proposed for which coexistence conditions can be characterized explicitly in terms of a trade-off. Here we present a model of a plant community which allows such a characterization. In the model plant species compete for sites where each site has a fixed stress condition. Species differ both in stress tolerance and competitive ability. Stress tolerance is quantified as the fraction of sites with stress conditions low enough to allow establishment. Competitive ability is quantified as the propensity to win the competition for empty sites. We derive the deterministic, discrete-time dynamical system for the species abundances. We prove the conditions under which plant species can coexist in a stable equilibrium. We show that the coexistence conditions can be characterized graphically, clearly illustrating the trade-off between stress tolerance and competitive ability. We compare our model with a recently proposed, continuous-time dynamical system for a tolerance-fecundity trade-off in plant communities, and we show that this model is a special case of the continuous-time version of our model.     

Genetic inactivation of the pancreatitis-inducible gene Nupr1 impairs PanIN formation by modulating KrasG12D-induced senescence

Nuclear protein 1 (Nupr1), a small chromatin protein, has a critical role in cancer development, progression and resistance to therapy. Previously, we had demonstrated that Nupr1 cooperates with Kras^G12D to induce pancreas intraepithelial neoplasias (PanIN) formation and pancreatic ductal adenocarcinoma development in mice. However, the molecular mechanisms by which Nupr1 influences Kras-mediated preneoplastic growth remain to be fully characterized. In the current study, we report evidence supporting a role for Nupr1 as a gene modifier of Kras^G12D-induced senescence, which must be overcome to promote PanIN formation. We found that genetic inactivation of Nupr1 in mice impairs Kras-induced PanIN, leading to an increase in β-galactosidase-positive cells and an upregulation of surrogate marker genes for senescence. More importantly, both of these cellular and molecular changes are recapitulated by the results of mechanistic experiments using RNAi-based inactivation of Nupr1 in human pancreatic cancer cell models. In addition, the senescent phenotype, which results from Nupr1 inactivation, is accompanied by activation of the FoxO3a-Skp2-p27^Kip1-pRb-E2F pathway in vivo and in vitro. Thus, combined, these results show, for the first time, that Nupr1 aids oncogenic Kras to bypass senescence in a manner that cooperatively promotes PanIN formation. Besides its mechanistic importance, this new knowledge bears medical relevance as it delineates early pathobiological events that may be targeted in the future as a means to interfere with the formation of preneoplastic lesions early during pancreatic carcinogenesis.Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with <5% survival after 5 years and a median survival of <6 months after diagnosis.¹ PDAC progresses from precursor lesions named pancreas intraepithelial neoplasias (PanINs). In this regard, it has been firmly established that oncogenic mutations in KRAS behave as one of the earliest stimuli for the formation of PanINs.^{2, 3} These data are strongly supported by animal models, such as the Pdx1-Cre; LSL-Kras^G12D transgenic mice, in which the pancreas-specific expression of oncogenic Kras promotes PanIN occurrence⁴ and, at a lower frequency, pancreatic cancer. Thus, the role of Kras as an initiating cancer mutation is one of the best-established pathobiological mechanisms required for the development of pancreatic cancer. Noteworthy, however, during the initiation stage, pancreatic cells not only trigger protumoral processes but also cellular events that aim at counteracting transformation. One of these tumor-suppressive processes elicited by Kras activation is cellular senescence (oncogene-induced senescence). In the pancreas, the induction of senescence underlies the resistance of exocrine cells to oncogenic Kras-mediated transformation⁵ so as to prevent tumor promotion, which is often supported by common diseases such as chronic pancreatitis.⁶ Indeed, tissue injury, as it occurs in pancreatitis, weakens the defense mechanism posed by senescence leading to its bypass by exocrine cells, which can then readily form PanINs.⁵ Therefore, the molecular mechanism that supports the development of oncogene-induced senescence (OIS) needs to be fully elucidated, if we want to advance our understanding of pancreatic cancer development.The nuclear protein 1 (Nupr1) is a basic helix–loop–helix molecule that is strongly induced by acute pancreatitis and several other cell stresses.^{7, 8} Nupr1 is also overexpressed in several types of human cancers, including PDAC. In this regard, the expression of genes that are targets for regulation by Nupr1 has been implicated in key protumorigenic pathways, including cell cycle regulation, matrix remodeling, autophagy, cell cannibalism and apoptosis inhibition.^{9, 10, 11, 12, 13, 14, 15, 16} Moreover, the fundamental role that Nupr1 has in pancreatic tumorigenesis is underscorded by recent results, which showed that, in mice, the oncogenic form of Kras^G12D is unable to promote PanINs in the absence of this chromatin protein,¹⁷ although the mechanisms responsible for this effect remain an area of active investigation. Consequently, we designed the current study with the aim of testing the hypothesis that Nupr1 cooperates with oncogenic Kras to induce PanIN formation by modulating the expression of gene networks that are necessary for bypassing senescence. To address this question, we characterized the effects that Nupr1 inactivation has on Kras-induced senescence using genome-wide expression profiling, as well as both cellular and molecular assays for this process. As a result of these experiments, we found that, indeed, the genetic inactivation of Nupr1 induces cellular senescence in exocrine pancreatic cells and reduces Kras-induced PanIN formation. At the molecular level, we demonstrated that this phenomenon is characterized by the upregulation of gene networks, which are known mediators of this phenomenon, by regulating at the G1/S transition. Taken together, these results provide mechanistic insights into how Nupr1 cooperates with Kras to promote the development of pancreatic preneoplastic lesions by discovering and characterizing a role for this pancreatitis-inducible protein in modulating cellular senescence. Thus, the new information emerging from this study has both mechanistic and biomedical implications for a better understanding of the pathobiology of pancreatic cancer.     

TLR2/1 and sphingosine 1-phosphate modulate inflammation,myofibroblast differentiation and cell migration in fibroblasts

Dermal fibroblasts are important regulators of inflammatory and immune responses in the skin. The aim of the present study was to elucidate the interaction between two key players in inflammation, Toll-like receptors (TLRs) and sphingosine 1-phosphate (S1P), in normal human fibroblasts in the context of inflammation, fibrosis and cell migration. We demonstrate that TLR2 ligation strongly enhances the production of the pro-inflammatory cytokines IL-6 and IL-8. S1P significantly induces pro-inflammatory cytokines time- and concentration-dependently via S1P receptor (S1PR)2 and S1PR3. The TLR2/1 agonist Pam₃CSK₄ and S1P (> 1 μM) or TGF-β markedly upregulate IL-6 and IL-8 secretion. Pam₃CSK₄ and S1P alone promote myofibroblast differentiation as assessed by significant increases of α-smooth muscle actin and collagen I expression. Importantly, costimulation with S1P (> 1 μM) induces differentiation into myofibroblasts. In contrast, Pam₃CSK₄ and low S1P concentrations (< 1 μM) accelerate cell migration. These results suggest that TLR2/1 signaling and S1P cooperate in pro-inflammatory cytokine production and myofibroblast differentiation and promote cell migration of skin fibroblasts in a S1P-concentration dependent manner. Our findings provide significant insights into how infectious stimuli or danger signals and sphingolipids contribute to dermal inflammation which may be relevant for skin tissue repair after injury or disease.     

Characterization of Deoxyribonucleic Acids from Streptomycetes and Nocardiae

The relationships among selected streptomycetes, nocardiae, and mycobacteria have been determined, based upon the base composition of their deoxyribonucleic acid (DNA) and upon the ability of their denatured DNA to anneal with single-stranded reference DNA. The streptomycetes constituted a homogeneous group whose DNA contained between 69 and 73 mole% guanine + cytosine (% GC). Moreover, the streptomycetes examined showed 37 to 88% homology with the Streptomyces venezuelae and S. rimosus reference DNA. The nocardial and mycobacterial DNA both contained 62 to 69% GC. The nocardial strains studied fell into either a 62 to 64% GC group or a 68 to 69% GC group, indicating that they should not be assigned to a single species. The nocardiae having 68 to 69% GC showed 24 to 44% homology with S. venezuelae reference DNA. In competition experiments, wherein unlabeled heterologous DNA interfered with binding of labeled homologous DNA, the nocardial DNA with 68 to 69% GC showed a greater degree of homology with the streptomycetes than did the nocardial DNA with 62 to 64% GC. In addition, the DNA from spores of S. venezuelae was cursorily examined, and interactions between S. venezuelae denatured DNA and polyribonucleotides were sought. The buoyant density of the DNA from S. venezuelae spores was distinctly less than that from mycelia. Moreover, denatured S. venezuelae DNA formed a dense complex with polyriboguanylate.     

New insights into the thermostability of bacterial ferredoxins: high-resolution crystal structure of the seven-iron ferredoxin from Thermus thermophilus

The crystal structure of the seven-iron ferredoxin from Thermus thermophilus (FdTt) has been determined at 1.64 A resolution, allowing us to unveil the common mechanisms of thermostabilization within "bacterial-type" ferredoxins. FdTt and other homologous thermophilic seven-iron ferredoxins are smaller than their mesophilic counterparts. Thermostabilizing features are optimized in a minimal structural and functional unit, with an extensive cross-linking of secondary structure elements mediated by improved polar and hydrophobic interactions. Most of the potentially stabilizing features are focused on the vicinity of the functional [3Fe-4S] cluster. The structural [4Fe-4S] cluster is shielded in thermophilic FdTt by an increased number of polar interactions involving the two N-terminal residues. Comparisons with the hyperthermostable ferredoxin from Thermotoga maritima reveal that (1) a reduction in the number of non-glycine residues in strained conformations, (2) improved polar interactions within the common iron-sulfur cluster binding (betaalphabeta)2 motif, and (3) an optimized charge distribution at the protein surface, constitute a common strategy for increasing the thermal stability of these ferredoxins.