Evaluation of PCR-based preimplantation genetic diagnosis applied to monogenic diseases: a collaborative ESHRE PGD consortium study

Preimplantation genetic diagnosis (PGD) for monogenic disorders currently involves polymerase chain reaction (PCR)-based methods, which must be robust, sensitive and highly accurate, precluding misdiagnosis. Twelve adverse misdiagnoses reported to the ESHRE PGD-Consortium are likely an underestimate. This retrospective study, involving six PGD centres, assessed the validity of PCR-based PGD through reanalysis of untransferred embryos from monogenic-PGD cycles. Data were collected on the genotype concordance at PGD and follow-up from 940 untransferred embryos, including details on the parameters of PGD cycles: category of monogenic disease, embryo morphology, embryo biopsy and genotype assay strategy. To determine the validity of PCR-based PGD, the sensitivity (Se), specificity (Sp) and diagnostic accuracy were calculated. Stratified analyses were also conducted to assess the influence of the parameters above on the validity of PCR-based PGD. The analysis of overall data showed that 93.7% of embryos had been correctly classified at the time of PGD, with Se of 99.2% and Sp of 80.9%. The stratified analyses found that diagnostic accuracy is statistically significantly higher when PGD is performed on two cells versus one cell (P=0.001). Se was significantly higher when multiplex protocols versus singleplex protocols were applied (P=0.005), as well as for PGD applied on cells from good compared with poor morphology embryos (P=0.032). Morphology, however, did not affect diagnostic accuracy. Multiplex PCR-based methods on one cell, are as robust as those on two cells regarding false negative rate, which is the most important criteria for clinical PGD applications. Overall, this study demonstrates the validity, robustness and high diagnostic value of PCR-based PGD.     

A New Workflow for Whole‐Genome Sequencing of Single Human Cells

Unbiased amplification of the whole‐genome amplification (WGA) of single cells is crucial to study cancer evolution and genetic heterogeneity, but is challenging due to the high complexity of the human genome. Here, we present a new workflow combining an efficient adapter‐linker PCR‐based WGA method with second‐generation sequencing. This approach allows comparison of single cells at base pair resolution. Amplification recovered up to 74% of the human genome. Copy‐number variants and loss of heterozygosity detected in single cell genomes showed concordance of up to 99% to pooled genomic DNA. Allele frequencies of mutations could be determined accurately due to an allele dropout rate of only 2%, clearly demonstrating the low bias of our PCR‐based WGA approach. Sequencing with paired‐end reads allowed genome‐wide analysis of structural variants. By direct comparison to other WGA methods, we further endorse its suitability to analyze genetic heterogeneity.     

Subcortical substrates of TMS induced modulation of the cortico-cortical connectivity

BackgroundTranscranial magnetic stimulation (TMS) can modulate transiently the physiological brain oscillations, e.g. the alpha rhythm. It has been hypothesized that this effect is not limited to the stimulated region but involves subcortical and distant cortical areas.MethodsWe applied single pulse TMS to the primary motor cortex (M1) of healthy subjects to interfere the cortical oscillatory activity recorded by simultaneous EEG and calculated the cortico-cortical coherence and power in the alpha and beta band. To study the structural substrate of the functional connectivity we performed diffusion tensor imaging and fractional anisotropy analysis (FA). To capture the pathways involved we applied probabilistic tractography to reconstruct the entire network.ResultsSuprathreshold TMS of M1 induced a consistent enhancement of interhemispheric cortico-cortical alpha band coherence that lasted ca. 175 ms. after the pulse has been applied. The changes were confined to the interhemispheric central EEG electrodes (i.e. C3-C4). There were no consistent changes in the beta band. Power analysis revealed a longer lasting increase in the beta band after TMS pulses. A cluster in the contralateral thalamus showed a linear relationship between regional FA and TMS induced change in alpha band coherence. Probabilistic tractography presents the transcallosal and the contralateral thalamocortical pathways as essential for the observed oscillatory synchronisation.ConclusionTMS induces an enhancement of oscillatory interaction between corresponding central regions of both hemispheres in the alpha band. The contralateral thalamus, transcallosal fibres and the contralateral thalamocortical pathways may constitute critical brain structures mediating the TMS induced change in oscillatory coupling.     

Oxytocin Reduces Reward-Driven Food Intake in Humans

Experiments in animals suggest that the neuropeptide oxytocin acts as an anorexigenic signal in the central nervous control of food intake. In humans, however, research has almost exclusively focused on the involvement of oxytocin in the regulation of social behavior. We investigated the effect of intranasal oxytocin on ingestion and metabolic function in healthy men. Food intake in the fasted state was examined 45 min after neuropeptide administration, followed by the assessment of olfaction and reward-driven snack intake in the absence of hunger. Energy expenditure was registered by indirect calorimetry, and blood was repeatedly sampled to determine concentrations of blood glucose and hormones. Oxytocin markedly reduced snack consumption, restraining, in particular, the intake of chocolate cookies by 25%. Oxytocin, moreover, attenuated basal and postprandial levels of adrenocorticotropic hormone and cortisol and curbed the meal-related rise in plasma glucose. Energy expenditure and hunger-driven food intake as well as olfactory function were not affected. Our results indicate that oxytocin, beyond its role in social bonding, regulates nonhomeostatic, reward-related energy intake, hypothalamic-pituitary-adrenal axis activity, and the glucoregulatory response to food intake in humans. These effects can be assumed to converge with the psychosocial function of oxytocin and imply possible applications in the treatment of metabolic disorders.The hypothalamic nonapeptide oxytocin is released into the circulation by axonal terminals in the posterior pituitary and, moreover, acts directly on central nervous receptors. Oxytocin, which has been highly preserved during mammalian evolution, regulates physiological functions related to reproduction and mother-infant interaction, such as lactation, and in recent years, has been shown to modulate affiliative behavior (1). Research in humans has almost exclusively focused on the role of oxytocin in the regulation of prosocial behavior, including trust, attachment, and sexual behavior (2–5), largely ignoring potential effects of the neuropeptide on ingestive behavior and metabolism. In fact, evidence from rodent studies indicates that the neuropeptide acts as a strong inhibitor of food intake and affects energy expenditure and glucose homeostasis (6–9). Oxytocinergic neurons in the hypothalamic paraventricular nucleus are assumed to mediate the food intake–limiting effect of leptin, an adipokine that provides the brain with negative feedback on body fat stores and sensitizes caudal brainstem nuclei to satiety factors such as cholecystokinin (10). Hypothalamic oxytocin signaling, moreover, mediates anorexigenic effects of the satiety factor nesfatin-1 in a leptin-independent manner (11). Importantly, oxytocin reduces food intake not only in normal-weight rodents but also in animals with diet-induced obesity (8,12,13), so oxytocinergic pathways might be a promising target of clinical interventions in obese patients.The direct manipulation of neuropeptidergic central nervous signaling pathways can be achieved via the intranasal administration of peptides, which is known to bypass the blood–brain barrier and result in significant cerebrospinal fluid elevations in substance levels within 40 min, without the need for systemic infusion (14,15). This approach has been validated, among others, for vasopressin, a close homolog of oxytocin (14), and intranasal oxytocin administration has been shown to reliably modulate neuropsychological functions in a series of studies (2–5) in the absence of relevant side effects (16). Surprisingly, however, the effect of intranasal oxytocin on energy metabolism, including ingestive behavior, has not been investigated in humans so far. The assessment of respective effects of intravenous oxytocin (17) is hampered because peripheral oxytocin is not readily transported across the blood–brain barrier (18).In the present experiments, we studied the contribution of oxytocin signaling to the control of ingestive behavior and energy expenditure in normal-weight, healthy men, with a particular view to endocrine regulators of metabolism, such as ghrelin and insulin, as well as hypothalamic-pituitary-adrenal (HPA) axis secretory activity. Ingestive behavior is not only regulated homeostatically (i.e., by central nervous pathways that respond to energy depletion) but also by nonhomeostatic brain circuits that process the reward-related, “hedonic” qualities of food intake (19). Therefore, we applied a twofold assessment of food intake that relied, on the one hand, on a large breakfast buffet after an overnight fast to investigate homeostatic, primarily hunger-driven energy intake (20–22), and on the other hand, on a collection of snacks of varying palatability offered after breakfast intake for the measurement of reward-driven food intake (22–24).     

A retrospective analysis of the therapeutic effects of 0.01% atropine on axial length growth in children in a real-life clinical setting

Graefe's Archive for Clinical and Experimental Ophthalmology - Several randomized controlled studies have demonstrated the beneficial effects of 0.01% atropine eye drops on myopia progression...     

P50 Suppression and Its Neural Generators in Antipsychotic-Na?ve First-Episode Schizophrenia Before and After 6 Months of Quetiapine Treatment

Background: Numerous studies have demonstrated sensory gating deficits in schizophrenia. However, only a few longitudinal studies report on the effects of antipsychotic treatment on sensory gating deficits and their results are inconsistent. In the present study, P50 suppression and its neural generators were investigated in antipsychotic-naïve first-episode patients with schizophrenia before and after 6 months of treatment with quetiapine. Methods: Thirty-four antipsychotic-naïve first-episode schizophrenia patients and age and gender matched healthy controls were tested in an auditory sensory gating paradigm at baseline and after 6 months. During this period, the patients were treated with quetiapine, while controls received no treatment. Sixteen patients completed the study. Results: Patients showed significant reduced P50 suppression compared with controls at baseline but not at follow-up. Furthermore, a significant positive correlation between baseline P50 suppression and dose of quetiapine at follow-up was found. P50 suppression in patients receiving above median dosages of quetiapine increased significantly from baseline to follow-up. At baseline, a frontocentral source was significantly more active in patients than in controls at the time of the testing stimulus. Conclusions: The present findings suggest that P50 suppression deficits are already present at an early stage of schizophrenia. Furthermore, particularly those patients with more severe gating deficits appeared to need higher dosages of quetiapine, although their clinical symptoms did not seem to indicate this. Quetiapine treatment significantly improved these gating deficits. Furthermore, a frontocentral source in the brain appeared to be involved in the deficient P50 gating of the patients.