Cell cycle arrest and p53 prevent ON-target megabase-scale rearrangements induced by CRISPR-Cas9.

The CRISPR-Cas9 system has revolutionized our ability to precisely modify the genome and has led to gene editing in clinical applications. Comprehensive analysis of gene editing products at the targeted cut-site has revealed a complex spectrum of outcomes. ON-target genotoxicity is underestimated with standard PCR-based methods and necessitates appropriate and more sensitive detection methods. Here, we present two complementary Fluorescence-Assisted Megabase-scale Rearrangements Detection (FAMReD) systems that enable the detection, quantification, and cell sorting of edited cells with megabase-scale loss of heterozygosity (LOH). These tools reveal rare complex chromosomal rearrangements caused by Cas9-nuclease and show that LOH frequency depends on cell division rate during editing and p53 status. Cell cycle arrest during editing suppresses the occurrence of LOH without compromising editing. These data are confirmed in human stem/progenitor cells, suggesting that clinical trials should consider p53 status and cell proliferation rate during editing to limit this risk by designing safer protocols.

CRISPR-Cas9 globin editing can induce megabase-scale copy-neutral losses of heterozygosity in hematopoietic cells.

CRISPR-Cas9 is a promising technology for gene therapy. However, the ON-target genotoxicity of CRISPR-Cas9 nuclease due to DNA double-strand breaks has received little attention and is probably underestimated. Here we report that genome editing targeting globin genes induces megabase-scale losses of heterozygosity (LOH) from the globin CRISPR-Cas9 cut-site to the telomere (5.2 Mb). In established lines, CRISPR-Cas9 nuclease induces frequent terminal chromosome 11p truncations and rare copy-neutral LOH. In primary hematopoietic progenitor/stem cells, we detect 1.1% of clones (7/648) with acquired megabase LOH induced by CRISPR-Cas9. In-depth analysis by SNP-array reveals the presence of copy-neutral LOH. This leads to 11p15.5 partial uniparental disomy, comprising two Chr11p15.5 imprinting centers (H19/IGF2:IG-DMR/IC1 and KCNQ1OT1:TSS-DMR/IC2) and impacting H19 and IGF2 expression. While this genotoxicity is a safety concern for CRISPR clinical trials, it is also an opportunity to model copy-neutral-LOH for genetic diseases and cancers.

Spleen route accelerates engraftment of human hematopoietic stem cells.

Intravenous injections of human hematopoietic stem cells (hHSCs) is routinely used in clinic and for modeling hematopoiesis in mice. However, unspecific dilution in vascular system and non-hematopoietic organs challenges engraftment efficiency. Although spleen is capable of extra medullar hematopoiesis, its ability to support human HSC transplantation has never been evaluated. We demonstrate that intra-splenic injection results in high and sustained engraftment of hHSCs into immune-deficient mice, with higher chimerisms than with intravenous or intra-femoral injections. Our results support that spleen microenvironment provides a niche for HSCs amplification and offers a new route for efficient HSC transplantation.

Online corrective responses following target jump in altered gravitoinertial force field point to nested feedforward and feedback control.

Studies on goal-directed arm movements have shown a close link between feedforward and feedback control in protocols where both planning and online control processes faced a similar type of perturbation, either mechanical or visual. This particular context might have facilitated the use of an adapted internal model by feedforward and feedback control. Here, we considered this link in a context where, after feedforward control was adapted through proprioception-based processes, feedback control was tested under visual perturbation. We analyzed the response of the reaching hand to target displacements following adaptation to an altered force field induced by rotating participants at constant velocity. Reaching corrections were assessed through variables related to the accuracy (lateral and longitudinal end point errors) and kinematics (movement time, peak velocity) of the corrective movements. The electromyographic activity of different arm muscles (pectoralis, posterior deltoid, biceps brachii, and triceps brachii) was analyzed. Statistical analyses revealed that accuracy and kinematics of corrective movements were strikingly alike between normal and altered gravitoinertial force fields. However, pectoralis and biceps muscle activities recorded during corrective movements were significantly modified to counteract the effect of rotation-induced Coriolis and centrifugal forces on the arm. Remarkably, feedback control was functional from the very first time participants encountered a target jump in the altered force field. Overall, the present results demonstrate that feedforward control enables immediate functional feedback control even when applied to distinct sensorimotor processes.NEW & NOTEWORTHY We investigated the link between feedforward and feedback control when applying a double-step perturbation (visual target jump) during reaching movements performed in modified gravitoinertial environments. Altogether, kinematics and EMG analyses showed that movement corrections were highly effective in the different force fields, suggesting that, although feedforward and feedback control were driven by different sensory inputs, feedback control was remarkably functional from the very first time participants encountered a target jump in the altered force field.

Double-Step Paradigm in Microgravity: Preservation of Sensorimotor Flexibility in Altered Gravitational Force Field.

The way we can correct our ongoing movements to sudden and unforeseen perturbations is key to our ability to rapidly adjust our behavior to novel environmental demands. Referred to as sensorimotor flexibility, this ability can be assessed by the double-step paradigm in which participants must correct their ongoing arm movements to reach targets that unexpectedly change location (i.e., target jump). While this type of corrections has been demonstrated in normogravity in the extent of reasonable spatiotemporal constraints underpinning the target jumps, less is known about sensorimotor flexibility in altered gravitational force fields. We thus aimed to assess sensorimotor flexibility by comparing online arm pointing corrections observed during microgravity episodes of parabolic flights with normogravity standards. Seven participants were asked to point as fast and as accurately as possible toward one of two visual targets with their right index finger. The targets were aligned vertically in the mid-sagittal plane and were separated by 10 cm. In 20% of the trials, the initially illuminated lower target was switched off at movement onset while the upper target was concomitantly switched on prompting participants to change the trajectory of their ongoing movements. Results showed that, both in normogravity and microgravity, participants successfully performed the pointing task including when the target jumped unexpectedly (i.e., comparable success rate). Most importantly, no significant difference was found in target jump trials regarding arm kinematics between both gravitational environments, neither in terms of peak velocity, relative deceleration duration, peak acceleration or time to peak acceleration. Using inverse dynamics based on experimental and anthropometrical data, we demonstrated that the shoulder torques for accelerating and decelerating the vertical arm movements substantially differed between microgravity and normogravity. Our data therefore highlight the capacity of the central nervous system to perform very fast neuromuscular adjustments that are adapted to the gravitational constraints. We discuss our findings by considering the contribution of feedforward and feedback mechanisms in the online control of arm pointing movements.

Development of a conversion model between mechanical and electrical vestibular stimuli.

The vestibular end-organs encode for linear and angular head accelerations in space contributing to our internal representation of self-motion. Activation of the vestibular system with transmastoid electrical current has recently grown in popularity; however, a direct relationship between electrically evoked and mechanically evoked vestibular responses remains elusive in humans. We have developed and tested a mechanical-to-electrical vestibular stimulus conversion model incorporating physiological activation of primary vestibular afferents identified in nonhuman primates. We compared ocular torsional responses between mechanical (chair rotation) and model-derived electrical (binaural-bipolar) stimuli in separate experiments for an angular velocity step change (±10 deg/s over 1 s, ±4-mA peak amplitude; n = 10) and multisine angular velocities (±10 deg/s, 9.7 mA peak to peak, 0.05-1 Hz; n = 5), respectively. Perception of whole body rotation (n = 18) to our step-change stimuli was also evaluated. Ocular torsional slow-phase velocity responses between stimulation types were similar (paired two one-sided tests of equivalence: multiple P < 0.002; one-sample t test: P = 0.178) and correlated (Pearson's coefficient: multiple P < 0.001). Bootstrap analysis of perceived angular velocity likewise showed similarity in perceptual decay dynamics. These data suggest that central processing between stimuli was similar, and our vestibular stimulus conversion model with a conversion factor of ∼0.4 mA per deg/s for an angular velocity step change can generate electrical stimuli that replicates dynamic vestibular activation elicited by mechanical whole body rotations. This proposed vestibular conversion model represents an initial framework for using electrical stimuli to generate mechanically equivalent activation of primary vestibular afferents for use in biomedical applications and immersive reality technologies.NEW & NOTEWORTHY With the growing popularity of electrical vestibular stimulation in biomedical and immersive reality applications, a direct conversion model between electrical and mechanical vestibular stimuli is needed. We developed a model to generate electrical stimuli mimicking the physiological activation of vestibular afferents evoked by mechanical rotations. Ocular and perceptual responses evoked by mechanical and model-derived electrical stimuli were similar, thus providing a critical first step toward generation of electrically induced vestibular responses that have a realistic mechanical equivalent.

Congenital Neuronal Ceroid Lipofuscinosis with a Novel CTSD Gene Mutation: A Rare Cause of Neonatal-Onset Neurodegenerative Disorder.

Neuronal ceroid lipofuscinoses represent a heterogeneous group of early onset neurodegenerative disorders that are characterized by progressive cognitive and motor function decline, visual loss, and epilepsy. The age of onset has been historically used for the phenotypic classification of this group of disorders, but their molecular genetic delineation has now enabled a better characterization, demonstrating significant genetic heterogeneity even among individuals with a similar phenotype. The rare Congenital Neuronal Ceroid Lipofuscinosis (CLN10) caused by mutations in the CTSD gene encoding for cathepsin D is associated with a dramatic presentation with onset before or around birth. We report on a female born to consanguineous parents who presented at birth with severe neonatal encephalopathy with massive cerebral and cerebellar shrinking on magnetic resonance imaging. Whole exome sequencing with targeted bioinformatic analysis of a panel of genes associated with prenatal/perinatal onset of neurodegenerative disease was performed and revealed the presence of a novel homozygous in-frame deletion in CTSD. Additional functional studies further confirmed the pathogenic character of this variant and established the diagnosis of CLN10 in the patient.

Experience of a multidisciplinary task force with exome sequencing for Mendelian disorders.

BACKGROUND: In order to optimally integrate the use of high-throughput sequencing (HTS) as a tool in clinical diagnostics of likely monogenic disorders, we have created a multidisciplinary "Genome Clinic Task Force" at the University Hospitals of Geneva, which is composed of clinical and molecular geneticists, bioinformaticians, technicians, bioethicists, and a coordinator. METHODS AND RESULTS: We have implemented whole exome sequencing (WES) with subsequent targeted bioinformatics analysis of gene lists for specific disorders. Clinical cases of heterogeneous Mendelian disorders that could potentially benefit from HTS are presented and discussed during the sessions of the task force. Debate concerning the interpretation of identified variants and the content of the final report constitutes a major part of the task force's work. Furthermore, issues related to bioethics, genetic counseling, quality control, and reimbursement are also addressed. CONCLUSIONS: This multidisciplinary task force has enabled us to create a platform for regular exchanges between all involved experts in order to deal with the multiple complex issues related to HTS in clinical practice and to continuously improve the diagnostic use of HTS. In addition, this task force was instrumental to formally approve the reimbursement of HTS for molecular diagnosis of Mendelian disorders in Switzerland.

[Quantification of ocular dominance for better management of eye disease]. / Vers une quantification de la dominance oculaire pour une meilleure prise en charge des pathologies de l'œil.

INTRODUCTION: The dominant eye is defined as the one we unconsciously choose when we have to perform monocular tasks. In the field of clinical neuro-ophthalmology, it is well-established that ocular dominance plays a key role in several eye diseases. Furthermore, the accurate quantification of ocular dominance is crucial with regard to certain surgical techniques. However, classical preoperative tests cannot determine the amount of ocular dominance. MATERIALS AND METHODS: In order to obtain further insight into the phenomenon of ocular dominance, we study its influence at behavioral and neurophysiological levels (experiments 1 and 2). Based on these new data, we suggest a method to improve quantification of ocular dominance (experiment 3). RESULTS: We demonstrate that ocular dominance has an influence on hand movements and on interhemispheric transfer time. Moreover, we show that an analysis of the dynamics of saccades allows us to sort out participants with strong or weak ocular dominance. CONCLUSION: In conclusion, this better understanding of the phenomenon of ocular dominance, coupled with the analysis of saccadic dynamics, might, in the short or medium term, lead to the establishment of a quick and straightforward battery of tests allowing determination of the amount of ocular dominance for each patient.

[Clinical and biochemical characterization of childhood urolithiasis]. / Caractéristiques cliniques et biochimiques des lithiases urinaires de l'enfant.

OBJECTIVES: Urolithiasis is rare in children, but the incidence has increased over the past few decades. This study aims at describing the clinical and biochemical characteristics, etiology, and treatment of urolithiasis in children. METHODS: This was a retrospective study of all children under 16 years of age seen at the Bordeaux University Children's Hospital with a diagnosis of urolithiasis. The diagnosis was confirmed either radiologically or clinically by the expulsion of the stone. RESULTS: A total of 186 children with a diagnosis of urolithiasis between 1994 and 2012 were included. The median age at diagnosis was 7.4 years. The male-to-female ratio was 1.9. The estimated annual incidence was around 5.5/100,000 children under 15 years of age in the past 5 years. The main presenting feature was nonspecific abdominal pain (71%). Metabolic calculi accounted for 48% of the patients with idiopathic hypercalciuria as the main cause. Genetic diseases accounted for 15% of cases. The proportion of infectious calculi was estimated at 33% and decreased in the past two decades. Stone fragments were sent for analysis in 86 children, and calcium oxalate was the major component (37%), followed by calcium phosphate (33%), purine (9%), and struvite (8%). At least 26% of patients experienced recurrence of stone passage. CONCLUSION: This retrospective study highlighted changes in characteristics of pediatric urolithiasis over time. Childhood-onset urolithiasis requires complete etiological work-up so that a metabolic cause with a high risk of recurrence does not go unrecognized.

Cortical involvement in the StartReact effect.

The rapid release of prepared movements by a loud acoustic stimulus capable of eliciting a startle response has been termed the StartReact effect (Valls-Solé et al., 1999), and premotor reaction times (PMTs) of <70 ms are often observed. Two explanations have been given for these short latency responses. The subcortical storage and triggering hypothesis suggests movements that can be prepared in advance of a "go" signal are stored and triggered from subcortical areas by a startling acoustic stimulus (SAS) without cortical involvement. Alternatively, it has been hypothesized that the SAS can trigger movements from cortical areas through a faster pathway ascending from subcortical structures. Two experiments were designed to examine the possible role of the primary motor cortex in the StartReact effect. In Experiment 1, we used suprathreshold transcranial magnetic stimulation (TMS) during the reaction time (RT) interval to induce a cortical silent period in the contralateral primary motor cortex (M1). Thirteen participants performed 20° wrist extension movements as fast as possible in response to either a control stimulus (82 dB) or SAS (124 dB). PMTs for startle trials were faster than for control trials, while TMS significantly delayed movement onset compared to No TMS or Sham TMS conditions. In Experiment 2, we examined the StartReact effect in a highly cortically represented action involving speech of a consonant-vowel (CV) syllable. Similar to previous work examining limb movements, a robust StartReact effect was found. Collectively, these experiments provide evidence for cortical (M1) involvement in the StartReact effect.

Telesonography: virtual 3D image processing of remotely acquired abdominal, vascular, and fetal sonograms.

PURPOSE: To design and test a new telesonography technique using remote volume acquisition by untrained operators in locations without access to trained sonographers, postprocessing, and interpretation done at expert centers. MATERIALS AND METHODS: The technique was tested with 84 sonograms of organs acquired in pregnant women (n = 8) and patients with various abdominal pathologic conditions (n = 11) located in French Guyana (France), Ceuta (Spain), and Murighiol (Romania). An operator inexperienced in sonography (US) placed the transducer over the predetermined acoustic window for each organ, then swept it from a -45° to a +45° position to scan the targeted organ. The acquired volume dataset was sent to an expert center via the Internet and reconstructed using a proprietary software, which allowed a trained sonographer to navigate through the appropriately reconstructed sonograms. RESULTS: After three-dimensional processing at the expert center, the organs scanned in the obstetrical cases were adequately visualized by the expert in seven of eight (88%) examinations of the fetal head, femur, and umbilical cord and eight of eight (100%) examinations of the fetal abdomen and placenta, whereas in the general abdominal cases, the liver, gallbladder, portal vein, and right kidney were correctly visualized in 10 of 11 (91%) examinations. CONCLUSIONS: Telesonography allowed untrained operators to scan and transfer the US volume datasets over the Internet to an expert center where an expert sonographer could navigate through the reconstructed US volume and visualize sonograms of diagnostic quality.

Multiplex targeted high-throughput sequencing for Mendelian cardiac disorders.

Mendelian cardiomyopathies and arrhythmias are characterized by an important genetic heterogeneity, rendering Sanger sequencing very laborious and expensive. As a proof of concept, we explored multiplex targeted high-throughput sequencing (HTS) as a fast and cost-efficient diagnostic method for individuals suffering from Mendelian cardiac disorders. We designed a DNA capture assay including all exons from 130 genes involved in cardiovascular Mendelian disorders and analysed simultaneously four samples by multiplexing. Two patients had familial hypertrophic cardiomyopathy (HCM) and two patients suffered from long QT syndrome (LQTS). In patient 1 with HCM, we identified two known pathogenic missense variants in the two most frequently mutated sarcomeric genes MYH7 and MYBPC. In patient 2 with HCM, a known acceptor splice site variant in MYBPC3 was found. In patient 3 with LQTS, two missense variants in the genes SCN5A and KCNQ were identified. Finally, in patient 4 with LQTS a known missense variant was found in MYBPC3, which is usually mutated in patients with cardiomyopathy. Our results showed that multiplex targeted HTS works as an efficient and cost-effective tool for molecular diagnosis of heterogeneous disorders in clinical practice and offers new insights in the pathogenesis of these complex diseases.

Contributions to enhanced activity in rectus femoris in response to Lokomat-applied resistance.

The application of resistance during the swing phase of locomotion is a viable approach to enhance activity in the rectus femoris (RF) in patients with neurological damage. Increased muscle activity is also accompanied by changes in joint angle and stride frequency, consequently influencing joint angular velocity, making it difficult to attribute neuromuscular changes in RF to resistance. Thus, the purpose of this study was to evaluate the effects of resistance on RF activity while constraining joint trajectories. Participants walked in three resistance conditions; 0 % (no resistance), 5 and 10 % of their maximum voluntary contraction (MVC). Visual and auditory biofeedback was provided to help participants maintain the same knee joint angle and stride frequency as during baseline walking. Lower limb joint trajectories and RF activity were recorded. Increasing the resistance, while keeping joint trajectories constant with biofeedback, independently enhanced swing phase RF activity. Therefore, the observed effects in RF are related to resistance, independent of any changes in joint angle. Considering resistance also affects stride frequency, a second experiment was conducted to evaluate the independent effects of resistance and stride frequency on RF activity. Participants walked in four combinations of resistance at 0 and 10 %MVC and natural and slow stride frequency conditions. We observed significant increases in RF activity with increased resistance and decreased stride frequency, confirming the independent contribution of resistance on RF activity as well as the independent effect of stride frequency. Resistance and stride frequency may be key parameters in gait rehabilitation strategies where either of these may be manipulated to enhance swing phase flexor muscle activity in order to maximize rehabilitation outcomes.

Effect of gravity-like torque on goal-directed arm movements in microgravity.

Gravitational force level is well-known to influence arm motor control. Specifically, hyper- or microgravity environments drastically change pointing accuracy and kinematics, particularly during initial exposure. These modifications are thought to partly reflect impairment in arm position sense. Here we investigated whether applying normogravitational constraints at joint level during microgravity episodes of parabolic flights could restore movement accuracy equivalent to that observed on Earth. Subjects with eyes closed performed arm reaching movements toward predefined sagittal angular positions in four environment conditions: normogravity, hypergravity, microgravity, and microgravity with elastic bands attached to the arm to mimic gravity-like torque at the shoulder joint. We found that subjects overshot and undershot the target orientations in hypergravity and microgravity, respectively, relative to a normogravity baseline. Strikingly, adding gravity-like torque prior to and during movements performed in microgravity allowed subjects to be as accurate as in normogravity. In the former condition, arm movement kinematics, as notably illustrated by the relative time to peak velocity, were also unchanged relative to normogravity, whereas significant modifications were found in hyper- and microgravity. Overall, these results suggest that arm motor planning and control are tuned with respect to gravitational information issued from joint torque, which presumably enhances arm position sense and activates internal models optimally adapted to the gravitoinertial environment.

Online control of anticipated postural adjustments in step initiation: evidence from behavioral and computational approaches.

Anticipatory postural adjustments (APAs) prior to step execution are thought to be immutable once released. Here we challenge this assumption by testing whether APAs can be modified online if a body perturbation occurs during execution. Two directions of perturbation (resisting and assisting) relative to the body weight transfer were used during the execution of APAs. We found that APAs are modified online (increase in both ground pressure and muscle activity) to compensate for resisting perturbations. The outcomes of a biomechanical model confirmed that the early changes in the APAs resulted from an active control of the APAs and were not merely mechanical consequences of the perturbation. However, no modification of the initial feedforward command was observed for assisting perturbations. The motor command changes for the resisting perturbation may originate from the mismatch between passively originated forces and those actively specified by the central command when acting in the opposite direction. The absence of a mismatch in the assisting perturbation might explain why the central nervous system was not prompted to modify the APAs in this condition.