CEQer: A Graphical Tool for Copy Number and Allelic Imbalance Detection from Whole-Exome Sequencing Data

Copy number alterations (CNA) are common events occurring in leukaemias and solid tumors. Comparative Genome Hybridization (CGH) is actually the gold standard technique to analyze CNAs; however, CGH analysis requires dedicated instruments and is able to perform only low resolution Loss of Heterozygosity (LOH) analyses. Here we present CEQer (Comparative Exome Quantification analyzer), a new graphical, event-driven tool for CNA/allelic-imbalance (AI) coupled analysis of exome sequencing data. By using case-control matched exome data, CEQer performs a comparative digital exonic quantification to generate CNA data and couples this information with exome-wide LOH and allelic imbalance detection. This data is used to build mixed statistical/heuristic models allowing the identification of CNA/AI events. To test our tool, we initially used in silico generated data, then we performed whole-exome sequencing from 20 leukemic specimens and corresponding matched controls and we analyzed the results using CEQer. Taken globally, these analyses showed that the combined use of comparative digital exon quantification and LOH/AI allows generating very accurate CNA data. Therefore, we propose CEQer as an efficient, robust and user-friendly graphical tool for the identification of CNA/AI in the context of whole-exome sequencing data.     

Assaying ATP synthesis in cultured cells: A valuable tool for the diagnosis of patients with mitochondrial disorders

Mitochondrial ATP synthase plays a central role in cell function by synthesising most of the ATP in human tissues. In different cells, active regulation of mitochondrial ATP synthase in response to cellular energy demand has been demonstrated, as well as its alteration under several pathological conditions affecting oxidative phosphorylation (OXPHOS). Traditionally, detection of OXPHOS defects is based on the spectrophotometric measurement of respiratory chain complex activities in muscle biopsies. Considering the broad clinical spectrum of mitochondrial disorders, and the difficulty in arriving at a single diagnostic method, in this study we propose measurement of ATP synthesis in mitochondria from skin fibroblasts as an effective screening tool. In the light of our results this assessment emerges as a useful marker of impaired energy production in primary OXPHOS disorders of childhood and as a tool with the potential to drive further molecular genetic studies.     

CNAReporter: a GenePattern pipeline for the generation of clinical reports of genomic alterations

Background

Genomic copy number alterations are widely associated with a broad range of human tumors and offer the potential to be used as a diagnostic tool. Especially in the emerging era of personalized medicine medical informatics tools that allow the fast visualization and analysis of genomic alterations of a patient's genomic profile for diagnostic and potential treatment purposes increasingly gain importance.

Results

We developed CNAReporter, a software tool that allows users to visualize SNP-specific data obtained from Affymetrix arrays and generate PDF-reports as output. We combined standard algorithms for the analysis of chromosomal alterations, utilizing the widely applied GenePattern framework. As an example, we show genome analyses of two patients with distinctly different CNA profiles using the tool.

Conclusions

Glioma subtypes, characterized by different genomic alterations, are often treated differently but can be difficult to differentiate pathologically. CNAReporter offers a user-friendly way to visualize and analyse genomic changes of any given tumor genomic profile, thereby leading to an accurate diagnosis and patient-specific treatment.     

Enhancing diagnosis, prognosis, and therapeutic outcome prediction of gliomas using genomics

Malignant gliomas are the most frequent type of primary brain tumors. Patients' outcome has not improved despite new therapeutics, thus underscoring the need for a better understanding of their genetics and a fresh approach to treatment. The lack of reproducibility in the classification of many gliomas presents an opportunity where genomics may be paramount for accurate diagnosis and therefore best for therapeutic decisions. The aim of this work is to identify large and focal copy number abnormalities (CNA) and loss of heterozygosity (LOH) events in a malignant glioma population. We hypothesized that these explorations will allow discovery of genetic markers that may improve diagnosis and predict outcome. DNA from glioma specimens were subjected to CNA and LOH analyses. Our studies revealed more than 4000 CNA and several LOH loci. Losses of chromosomes 1p and/or 19q, 10, 13, 14, and 22 and gains of 7, 19, and 20 were found. Several of these alterations correlated significantly with histology and grade. Further, LOH was detected at numerous chromosomes. Interestingly, several of these loci harbor genes with potential or reported tumor suppressor properties. These novel genetic signatures may lead to critical insights into diagnosis, classification, prognosis, and design of individualized therapies.     

Conjugated nonadecadienoic acid is more potent than conjugated linoleic acid on body fat reduction

Conjugated linoleic acid (CLA) has shown a number of health benefits, particularly on controlling body fat while improving lean mass. As one of CLA cognates, conjugated nonadecadienoic acid (CNA, 19-carbon conjugated fatty acid) has been previously reported to have greater efficacy on body fat control. In this report, we compared the efficacy of dietary CLA and CNA on body fat regulation and also compared the mechanism of body fat control using a mouse model. Effects of 0.1% dietary CNA on body fat reduction were comparable to that of 0.5% dietary CLA. The mechanisms of dietary CNA on body fat control were similar to those of CLA: increased energy expenditure and increased fatty acid β-oxidation. Dietary CNA, but not CLA, also improved expression of hormone-sensitive lipase from white adipose tissue, and this may help explain how CNA has better efficacy on body fat control than CLA. Dietary CNA had similar effects as CLA on liver weights; however, unlike CLA, CNA improved glucose tolerance. Thus, CNA has potential to be used as a pharmacological agent to assist current efforts to reduce obesity with less adverse effects than CLA.     

Molecular simulation of cyclohexanyl nucleic acid (CNA) duplexes with CNA,DNA and RNA and CNA triloop and tetraloop hairpin structures

As part of a selection strategy for artificial nucleic acids (XNA) (to be considered as potential new information systems in vivo), we have carried out a modelling study on cyclohexanyl nucleic acids (CNA) duplexes and hairpins. CNA may form a duplex as well as hairpin structures, having the carbocyclic nucleosides in the ⁴C₁ conformation (with equatorial basis). The geometry of ds CNA is close to that of a HNA:RNA duplex. We demonstrated that CNA triphosphates function as a substrate for polymerases. Modelling experiments indicate that the monomers are probably presented to the polymerase in the ¹C₄ conformation.     

A new approach for producing polyclonal antibodies using impure antigens

A new convenient approach has been designed to produce polyclonal antibodies (PcAb). The approach is based on the principle of the immunoglobulin (Ig) class switch in the immune response. We produced six different antibodies (Ab) against calcineurin A subunit (CNA). CNA, His-tagged calcineurin A subunit (His-CNA), single chain calcineurin (CNB-CNA) and single chain calcineurin-calmodulin complex (CaM-CNB-CNA) were expressed in Escherichia coli (E. coli) BL21 strain, and they were used to immunize male BALB/c mice. These Ab were examined by enzyme-linked immunosorbent assay (ELISA) and western blot analysis. The results clearly demonstrated that the specificity of the Ab produced by different protein samples was much higher than that of the Ab produced by a single sample. We used CNA, CaM-CNB-CNA and His-CNA to immunize mice in turn and obtained monospecific PcAb against CNA fortunately by our new approach. Remarkably, our approach not only offered a simple and general alternative to other methods for producing PcAb described previously, but also disclosed a novel process of immunization that could be used to produce monoclonal antibodies (mAb).     

Elevation of plasma β-amyloid level by glucose loading in Alzheimer mouse models

With the emergence of a promising approach to treat Alzheimer disease (AD) targeting the β-amyloid (Aβ) pathway, it is necessary to establish new diagnostic biomarkers that enable the antemortem diagnosis of AD. Although plasma Aβ has been suggested as a non-invasive biomarker, its significance has been inconclusive. Thus, it is important to improve the diagnostic potential of plasma Aβ. One of the potential approaches is to modify plasma Aβ level using various modulators. In this study, we evaluated the influence of glucometabolic status on plasma Aβ level in two lines of AD transgenic mouse. The present study demonstrated that plasma Aβ level rapidly increased after glucose loading. More importantly, the magnitude of the increase in plasma Aβ was significantly larger in AD transgenic mice than in wild-type littermates. These findings might provide a novel diagnostic tool for AD using the elevation of plasma Aβ level after glucose loading.     

The salt bridge of calcineurin is important for transferring the effect of CNB binding to CNA

Calcineurin (CN) is a heterodimer consisting of a catalytic subunit (CNA) and a regulatory subunit (CNB). The crystal structure shows that three residues or regions of CNA are mainly responsible for the interaction with CNB: the CNB binding helix (BBH), the N-terminus, and Glu53 that forms a salt bridge with Lys134 of CNB. In this report, we try to find the role that the salt bridge plays in the interaction between CNA and CNB. We found that mutation of Glu53 greatly reduced its responsiveness to CNB in the phosphatase assay and also that mutation of Lys134 of CNB affected its ability to activate the phosphatase activity of CNA. Structural analysis showed that disruption of the salt bridge affected the compact association of CNA and CNB. Thus, the salt bridge appears to help to stabilize CN and transfer the effects of CNB binding to CNA to activate its phosphatase activity.