Comparison of Sequencing Based CNV Discovery Methods Using Monozygotic Twin Quartets

Background

The advent of high throughput sequencing methods breeds an important amount of technical challenges. Among those is the one raised by the discovery of copy-number variations (CNVs) using whole-genome sequencing data. CNVs are genomic structural variations defined as a variation in the number of copies of a large genomic fragment, usually more than one kilobase. Here, we aim to compare different CNV calling methods in order to assess their ability to consistently identify CNVs by comparison of the calls in 9 quartets of identical twin pairs. The use of monozygotic twins provides a means of estimating the error rate of each algorithm by observing CNVs that are inconsistently called when considering the rules of Mendelian inheritance and the assumption of an identical genome between twins. The similarity between the calls from the different tools and the advantage of combining call sets were also considered.

Results

ERDS and CNVnator obtained the best performance when considering the inherited CNV rate with a mean of 0.74 and 0.70, respectively. Venn diagrams were generated to show the agreement between the different algorithms, before and after filtering out familial inconsistencies. This filtering revealed a high number of false positives for CNVer and Breakdancer. A low overall agreement between the methods suggested a high complementarity of the different tools when calling CNVs. The breakpoint sensitivity analysis indicated that CNVnator and ERDS achieved better resolution of CNV borders than the other tools. The highest inherited CNV rate was achieved through the intersection of these two tools (81%).

Conclusions

This study showed that ERDS and CNVnator provide good performance on whole genome sequencing data with respect to CNV consistency across families, CNV breakpoint resolution and CNV call specificity. The intersection of the calls from the two tools would be valuable for CNV genotyping pipelines.     

IRC-082451, a Novel Multitargeting Molecule,Reduces L-DOPA-Induced Dyskinesias in MPTP Parkinsonian Primates

The development of dyskinesias following chronic L-DOPA replacement therapy remains a major problem in the long-term treatment of Parkinson’s disease. This study aimed at evaluating the effect of IRC-082451 (base of BN82451), a novel multitargeting hybrid molecule, on L-DOPA-induced dyskinesias (LIDs) and hypolocomotor activity in a non-human primate model of PD. IRC-082451 displays multiple properties: it inhibits neuronal excitotoxicity (sodium channel blocker), oxidative stress (antioxidant) and neuroinflammation (cyclooxygenase inhibitor) and is endowed with mitochondrial protective properties. Animals received daily MPTP injections until stably parkinsonian. A daily treatment with increasing doses of L-DOPA was administered to parkinsonian primates until the appearance of dyskinesias. Then, different treatment regimens and doses of IRC-082451 were tested and compared to the benchmark molecule amantadine. Primates were regularly filmed and videos were analyzed with specialized software. A novel approach combining the analysis of dyskinesias and locomotor activity was used to determine efficacy. This analysis yielded the quantification of the total distance travelled and the incidence of dyskinesias in 7 different body parts. A dose-dependent efficacy of IRC-082451 against dyskinesias was observed. The 5 mg/kg dose was best at attenuating the severity of fully established LIDs. Its effect was significantly different from that of amantadine since it increased spontaneous locomotor activity while reducing LIDs. This dose was effective both acutely and in a 5-day sub-chronic treatment. Moreover, positron emission tomography scans using radiolabelled dopamine demonstrated that there was no direct interference between treatment with IRC-082451 and dopamine metabolism in the brain. Finally, post-mortem analysis indicated that this reduction in dyskinesias was associated with changes in cFOS, FosB and ARC mRNA expression levels in the putamen. The data demonstrates the antidyskinetic efficacy of IRC-082451 in a primate model of PD with motor complications and opens the way to the clinical application of this treatment for the management of LIDs.     

Cell death mediated by the N-terminal domains of a unique and highly conserved class of NB-LRR protein

Plant genomes encode large numbers of nucleotide-binding, leucine-rich repeat (NB-LRR) proteins, many of which are active in pathogen detection and defense response induction. NB-LRR proteins fall into two broad classes: those with a Toll and interleukin-1 receptor (TIR) domain at their N-terminus and those with a coiled-coil (CC) domain at the N-terminus. Within CC-NB-LRR-encoding genes, one basal clade is distinguished by having CC domains resembling the Arabidopsis thaliana RPW8 protein, which we refer to as CCR domains. Here, we show that CCR-NB-LRR-encoding genes are present in the genomes of all higher plants surveyed, and that they comprise two distinct subgroups: one typified by the Nicotiana benthamiana N-required gene 1 (NRG1) protein and the other typified by the Arabidopsis activated disease resistance gene 1 (ADR1) protein. We further report that, in contrast to CC-NB-LRR proteins, the CCR domains of both NRG1- and ADR1-like proteins are sufficient for the induction of defense responses, and that this activity appears to be SGT1-independent. Additionally, we report the apparent absence of both NRG1 homologs and TIR-NB-LRR-encoding genes from the dicot Aquilegia coerulea and the dicotyledonous order Lamiales as well as from monocotyledonous species. This strong correlation in occurrence is suggestive of a functional relationship between these two classes of NB-LRR proteins.     

Biological role, protein expression, subcellular localization, and oxidative stress response of paraoxonase 2 in the intestine of humans and rats

Oxidative stress is a cardinal manifestation of various intestinal disorders. However, very little knowledge is available on the intestine's inherent defense mechanisms against free radicals. This study was designed to determine the protein expression, subcellular localization and oxidative stress response of paraoxonase 2 (PON2), a member of a powerful antioxidant family in human and rat intestine. Biochemical and ultrastructural experiments all showed a substantial expression of PON2 in human and rat intestine. Western blot analysis disclosed higher levels of PON2 in the jejunum than in the duodenum, ileum, and colon. Cell fractionation revealed a predominant PON2 association with microsomes and lysosomes in the human jejunum, which differed from that in rats. PON2 was detected in the intestine as early as week 15 of gestation and was significantly increased by week 20. Iron ascorbate-mediated lipid peroxidation induced a marked decrease in PON2 expression in intestinal specimens coincidental to an abundant rise in malondialdehyde (MDA). On the other hand, preincubation with potent antioxidants, such as butylated hydroxytoluene, Trolox, and N-acetylcysteine, prevented iron-ascorbate-generating PON2 reduction in parallel with MDA suppression. Finally, the preincubation of permeabilized Caco-2 cells with purified PON2 led to a protection against iron-ascorbate-induced lipid peroxidation. These observations demonstrate that the human intestine is preferentially endowed with a marked PON2 expression compared with the rat intestine and this expression shows a developmental and intracellular pattern of distribution. Furthermore, our observations suggest PON2 protective effects against prooxidant stimuli in the small intestine.     

Correlation between airway responsiveness and proteoglycan production by bronchial fibroblasts from normal and asthmatic subjects

Asthma is characterized by an airway remodeling process involving altered extracellular matrix deposition such as collagen, fibronectin and proteoglycans. Proteoglycans determine tissue mechanical properties and are involved in many important biological aspects. Not surprisingly, it has been suggested that proteoglycan deposition may alter airway properties in asthma including airway hyperresponsiveness. In chronically inflamed airway tissues, fibroblasts likely represent an activated fibrotic phenotype that contributes to the excessive deposition of different extracellular matrix components. To investigate whether this was the case for proteoglycans, the production of hyaluronan, perlecan, versican, small heparan sulphate proteoglycans (HSPGs), decorin and biglycan was quantified in the culture medium of primary bronchial fibroblast cultures, established from four normal and six asthmatic subjects. Values were further correlated to the airway responsiveness (PC(20) methacholine) of donor subjects. Fibroblasts from subjects with the most hyperresponsive airways produced up to four times more total proteoglycans than cells from subjects with less hyperresponsive or normoresponsive airways. We observed a significant negative correlation between the PC(20) and perlecan, small HSPGs and biglycan, while such correlation was absent for decorin and close to significant for hyaluronan and versican. Altered proteoglycan metabolism by bronchial fibroblasts may contribute to the increased proteoglycan deposition in the bronchial mucosa and to airway hyperresponsiveness characterizing asthma.     

A dual polybasic motif determines phosphoinositide binding and regulation in the P2X channel family

Phosphoinositides modulate the function of several ion channels, including most ATP-gated P2X receptor channels in neurons and glia, but little is known about the underlying molecular mechanism. We identified a phosphoinositide-binding motif formed of two clusters of positively charged amino acids located on the P2X cytosolic C-terminal domain, proximal to the second transmembrane domain. For all known P2X subtypes, the specific arrangement of basic residues in these semi-conserved clusters determines their sensitivity to membrane phospholipids. Neutralization of these positive charges disrupts the functional properties of the prototypical phosphoinositide-binding P2X4 subtype, mimicking wortmannin-induced phosphoinositide depletion, whereas adding basic residues at homologous positions to the natively insensitive P2X5 subtype establishes de novo phosphoinositide-mediated regulation. Moreover, biochemical evidence of in vitro P2X subunit-phospholipid interaction and functional intracellular phosphoinositide-binding assays demonstrate that the dual polybasic cluster is necessary and sufficient for regulation of P2X signaling by phospholipids.     

Structure and pH-induced alterations of recombinant and natural spider silk proteins in solution

The spinning process of spiders can modulate the mechanical properties of their silk fibers. It is therefore of primary importance to understand what are the key elements of the spider spinning process to develop efficient industrial spinning processes. We have exhaustively investigated the native conformation of major ampullate silk (MaS) proteins by comparing the content of the major ampullate gland of Nephila clavipes, solubilized MaS (SolMaS) fibers and the recombinant proteins rMaSpI and rMaSpII using (1) H solution NMR spectroscopy. The results indicate that the protein secondary structure is basically identical for the recombinant protein rMaSpI, SolMaS proteins, and the proteins in the dope, and corresponds to a disordered protein rich in 3(1) -helices. The data also show that glycine proton chemical shifts of rMaSpI and SolMaS are affected by pH, but that this change is not due to a modification of the secondary structure. Using a combination of NMR and dynamic light scattering, we have found that the spectral alteration of glycine is concomitant to a modification of the hydrodynamical diameter of recombinant and solubilized MaS. This led us to suggest new potential roles for the pH acidification in the spinning process of MaS proteins.     

Smelling right: the scent of male lemurs advertises genetic quality and relatedness

Sexual selection theory predicts that competitors or potential mates signal their quality or relatedness to conspecifics. Researchers have focused on visual or auditory modes of signal transmission; however, the importance of olfactory indicators is gaining recognition. Using a primate model and a new integrative analytical approach, we provide the first evidence relating male olfactory cues to individual genome-wide heterozygosity and to the genetic distance between individuals. The relationships between male semiochemical profiles and genetic characteristics are apparent only during the highly competitive and stressful breeding season. As heterozygosity accurately predicts health and survivorship in this population, we identify scrotal olfactory cues as honest indicators of male quality, with relevance possibly to both sexes. Beyond showing that semiochemicals could underlie kin recognition and nepotism, we provide a putative olfactory mechanism to guide male–male competition and female mate choice.