RARE DE NOVO AND TRANSMITTED COPY NUMBER VARIATIONS IN AUTISM SPECTRUM DISORDERS: IMPLICATIONS FOR FUNCTIONAL NETWORKS OF GENES INVOLVED IN NEUROGENESIS, NEURONAL METABOLISM, SYNAPTIC FUNCTION, NEUROIMMUNITY, INTRACELLULAR SIGNALING AND CHROMATIN REMODELING

Autistic behaviors were independently identified as recognizable syndromes in the early 20th century by Heller and, subsequently, by Kanner and Asperger. The autism diagnosis spans a broad continuum of what are collectively known as Autism Spectrum Disorders (ASDs) or Pervasive Developmental Disorders (PDDs). ASDs include several conditions, namely full-syndrome autism (Autistic Disorder or Idiopathic Autism), Childhood Disintegrative Disorder, Asperger Syndrome (AS) and Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS). The latest estimates put the population prevalence of ASDs at approximately 1 in 110. Furthermore, ASDs show a 4:1 male to female gender bias, which may rise to 11:1 when considering Asperger disorder. Although heterogeneous, ASDs are united by a combination of three core behavior symptoms: a) impaired language and communication; b) deficiencies in social interaction; c) restricted interest and repetitive stereotypic behavior. Symptoms of ASDs usually begin in early childhood with evidence of delayed development before age 3 years. The etiology of ASDs is complex and encompasses the roles of genes, the mitochondria, the environment, and the immune system. However, there is strong evidence for the importance of complex genetic factors comprised of different form of genetic variations in the etiology of ASDs. Twin and family studies have, indeed, established the preponderant genetic basis of autism and indicate that the heritability of autism is over 90%, which is the highest heritability value so far associated with a neuropsychiatric disorder. The genetic causes of ASDs can be classified as: (1) ASD-related monogenic syndromes; (2) rare chromosomal abnormalities; (3) rare copy number variations (CNVs); (4) rare gene mutations; (5) common genetic variants. Recently, submicroscopic CNVs, de novo and inherited, are emerging as an important category of genetic risk for ASDs with a different impact depending on the type of CNV identified. Screening for CNVs by means of array CGH and SNP array technologies has proven to be one of the more successful strategies for the discovery of ASD candidate loci over the past five years. Furthermore, the same or overlapping CNVs are being identified as risk factors across a few neurodevelopmental disorders, indicating that some ASD loci are likely pleiotropic with variable expressivity. The increased resolution of the array-based approaches suggests that the proportion of ASD cases (both idiopathic and syndromic) that may be ultimately attributed to a rare structural variant is around 10-20%. The de novo CNV rate in ASDs is roughly three to seven times than that in controls and has been reported to be higher in simplex (low-risk) compared to multiplex families (high-risk). Furthermore, CNV screening gave rise to a paradigm shift away from a common variant model of ASD genetic architecture (based on low penetrating variants) to one suggesting a role for multiple rare and distinct genetic risk factors (with a higher penetrance), known as oligogenic heterozygosity model, which does not exclude, however, a modulation of the phenotype by common susceptibility genetic variants. We collected a series of 115 patients (92 males and 23 females) who were diagnosed with ASDs: 41 with idiopathic Autism, 46 with PDD-NOS, 15 with Syndromic Autism, 8 with High-Functioning Autism, and 4 with Asperger syndrome. Genomic DNA from all patients was used to perform array CGH analysis (Agilent Technology) in order to detect CNVs. In particular, 18 patients were analyzed at a lower resolution (44K or 60K Kits) and 97 at a higher resolution (244K Kit). In the event of detection of rare CNVs, if possible, patients\u2019 parents were analyzed to characterize the origin of the unbalanced microrearrangement (de novo or inherited). In 63 of 115 patients (55%), rare CNVs (one or more) were detected that were not already reported in healthy subjects according to the Database of Genomic Variants (DGV). This group comprised 49 patients analyzed by the Agilent 244K Kit (detection rate ~50.5%) and 14 by the 44K or the 60K Kits (detection rate 77.8%); 51 sporadic and 12 familial cases. Overall, 120 rare CNVs were detected, 73 gains (60.8%) and 47 losses (39.2%), ranging from 10 kb to 11 Mb in size. Furthermore, inheritance is unknown for 13 CNVs (10.8%). Twenty of the remaining 107 CNVs were de novo (16.7%), and 87 were inherited (72.5%), 50 from the mother (57.5%) and 37 from the father (42.5%). The ad hoc analysis of the rare CNV gene content using databases revealed a total of 276 genes that were considered good candidates for ASDs. A small percentage of the selected genes (~11%) have been previously reported as causative genes based on mutations and/or CNVs, often de novo, that have been described in autistic patients. Moreover, SNPs in a very few genes (~5%) have been significantly associated with ASDs. However, most of the proposed candidate genes (54%) have not been previously reported in association with ASDs, and a significant percentage (30.5%) have been involved in some microdeletion/microduplication syndromes that are comorbid with ASDs. On the basis of gene expression data, function, and pathway of action, all the selected genes contribute to CNS neurodevelopment and maintenance, acting during embryonic and foetal development as well as in the early postnatal period and, in some cases, in adult life. Sixty-six of the 276 selected genes are implicated in neurogenesis and neurodevelopment (24%), 27 in CNS metabolism (10%), 29 in synaptogenesis and synaptic plasticity (10.5%), 19 in CNS development, homeostasis, and immunosurveillance mediated by the immune system (7%), 81 in intracellular signaling and trafficking (29%), and 51 in transcriptional and translational regulation and chromatin remodeling (18.5%). For 3 of the 276 genes, the function is still unknown (1%). Of note, 89 of 276 selected genes contribute to CNS development and maintenance acting in concert with the immune system. On the basis of the rare CNV gene content, it is possible to confirm in the present cohort the wide genetic heterogeneity associated with ASDs. Indeed, the same affected loci were detected in only a few unrelated patients (LCLAT1 in patients 7 and 33, and MACROD2 in patients 3, 33, and the siblings 40\u201341), whereas most patients demonstrated specific subsets of rare CNVs in combinations characteristic for each patient, thus supporting the genetic complexity of ASDs. Moreover, most of the selected genes have never been reported in association with ASDs, and thus they are suggested as new candidate loci and have been grouped into six functional networks that all contribute to CNS development and maintenance. Although a subgroup of the selected genes contribute to synaptic function, thus confirming the neurological interpretation of ASD as a \u201csynaptopathy\u201d, our findings support the existence of dozens of non-synaptic genes that may be implicated in ASDs, which encompass a wide range of biological function and cellular processes, such as intracellular signaling and chromatin-mediated transcription. In particular, it may be speculated that anomalies in the interplay between the nervous and the immune systems, due to genetic causes, may be responsible for autism development in specific subsets of autistic patients. Finally, the genotype-phenotype correlation in the reported ASD series seems rather complex. First, rare CNVs were found in only 55% of the patients, thus emphasizing the need to analyze large cohorts of autistic patients by means of different high-throughput genome-wide approaches in order to increase the detection rate for these disorders. In addition, most patients positive in the array CGH analysis (~76%) were found to be carriers of more than one CNV, which were present in different combinations, thus supporting the existence of a genetic model characterized by oligogenic heterozygosity. Furthermore, when more rare CNVs are found in a single patient, it is difficult to attribute a \u201cmajor\u201d causative role to one of them, even in case of de novo CNVs, which are generally considered high-penetrance variants. Indeed, the more severe clinical pictures in our cohort (i.e., syndromic autism) do not always correlate with the finding of rare CNVs, neither with the CNV size nor with the presence of de novo CNVs in the subset of patients bearing rare CNVs

A model for predicting the visual complexity of in-vehicle interfaces

This paper describes the development of a model that can be used to estimate the visual complexity of in-vehicle graphical user interfaces (GUI) and to reduce the distraction of in-vehicle interfaces and thus improve the driving performance. The first version of this model was validated using a GUI that was designed for an interactive C2X application. Using the model, the visual complexity for different screens of the GUI was calculated. 22 participants performed a simple ticket reservation task with the GUI while performing a driving task. A significant correlation was found between the visual complexity and the time until action. Although this result indicates the potential of the developed model, the model has to be refined and further validated in future iterations

Effect of task failure on intermuscular coherence measures in synergistic muscles

The term "task failure" describes the point when a person is not able to maintain the level of force required by a task. As task failure approaches, the corticospinal command to the muscles increases to maintain the required level of force in the face of a decreased mechanical efficacy. Nevertheless, most motor tasks require the synergistic recruitment of several muscles. How this recruitment is affected by approaching task failure is still not clear. The increase in the corticospinal drive could be due to an increase in synergistic recruitment or to overlapping commands sent to the muscles individually. Herein, we investigated these possibilities by combining intermuscular coherence and synergy analysis on signals recorded from three muscles of the quadriceps during dynamic leg extension tasks. We employed muscle synergy analysis to investigate changes in the coactivation of the muscles. Three different measures of coherence were used. Pooled coherence was used to estimate the command synchronous to all three muscles, pairwise coherence the command shared across muscle pairs and residual coherence the command peculiar to each couple of muscles. Our analysis highlights an overall decrease in synergistic command at task failure and an intensification of the contribution of the nonsynergistic shared command

Yield of array-CGH analysis in Tunisian children with autism spectrum disorder

Background: Autism spectrum disorder (ASD) is a neurodevelopmental disorder with strong genetic underpinnings. Microarray-based comparative genomic hybridization (aCGH) technology has been proposed as a first-level test in the genetic diagnosis of ASD and of neurodevelopmental disorders in general. Methods: We performed aCGH on 98 Tunisian children (83 boys and 15 girls) diagnosed with ASD according to DSM-IV criteria. Results: “Pathogenic” or “likely pathogenic” copy number variants (CNVs) were detected in 11 (11.2%) patients, CNVs of “uncertain clinical significance” in 26 (26.5%), “likely benign” or “benign” CNVs were found in 37 (37.8%) and 24 (24.5%) patients, respectively. Gene set enrichment analysis involving genes spanning rare “pathogenic,” “likely pathogenic,” or “uncertain clinical significance” CNVs, as well as SFARI database “autism genes” in common CNVs, detected eight neuronal Gene Ontology classes among the top 10 most significant, including synapse, neuron differentiation, synaptic signaling, neurogenesis, and others. Similar results were obtained performing g: Profiler analysis. Neither transcriptional regulation nor immune pathways reached significance. Conclusions: aCGH confirms its sizable diagnostic yield in a novel sample of autistic children from North Africa. Recruitment of additional families is under way, to verify whether genetic contributions to ASD in the Tunisian population, differently from other ethnic groups, may involve primarily neuronal genes, more than transcriptional regulation and immune-related pathways

Two-colour generation in a chirped seeded Free-Electron Laser

We present the experimental demonstration of a method for generating two spectrally and temporally separated pulses by an externally seeded, single-pass free-electron laser operating in the extreme-ultraviolet spectral range. Our results, collected on the FERMI@Elettra facility and confirmed by numerical simulations, demonstrate the possibility of controlling both the spectral and temporal features of the generated pulses. A free-electron laser operated in this mode becomes a suitable light source for jitter-free, two-colour pump-probe experiments

Phenotypic spectrum of NRXN1 mono- and bi-allelic deficiency: A systematic review

Neurexins are presynaptic cell adhesion molecules critically involved in synaptogenesis and vesicular neurotransmitter release. They are encoded by three genes (NRXN1-3), each yielding a longer alpha (α) and a shorter beta (β) transcript. Deletions spanning the promoter and the initial exons of the NRXN1 gene, located in chromosome 2p16.3, are associated with a variety of neurodevelopmental, psychiatric, neurological and neuropsychological phenotypes. We have performed a systematic review to define (a) the clinical phenotypes most associated with mono-allelic exonic NRXN1 deletions, and (b) the phenotypic features of NRXN1 bi-allelic deficiency due to compound heterozygous deletions/mutations. Clinically, three major conclusions can be drawn: (a) incomplete penetrance and pleiotropy do not allow reliable predictions of clinical outcome following prenatal detection of mono-allelic exonic NRXN1 deletions. Newborn carriers should undergo periodic neuro-behavioral observations for the timely detection of warning signs and the prescription of early behavioral intervention; (b) the presence of additional independent genetic risk factors should always be sought, as they may influence prognosis; (c) children with exonic NRXN1 deletions displaying early-onset, severe psychomotor delay in the context of a Pitt-Hopkins-like syndrome 2 phenotype, should undergo DNA sequencing of the spared NRXN1 allele in search for mutations or very small insertions/deletions

Effects of Parvovirus B19 In Vitro Infection on Monocytes from Patients with Systemic Sclerosis: Enhanced Inflammatory Pathways by Caspase-1 Activation and Cytokine Production

Parvovirus B19 (B19V) has been proposed as a triggering agent for some autoimmune diseases including systemic sclerosis (SSc). In this study, we investigated whether B19V infection in vitro differently activates inflammatory pathways, including those dependent on caspase-1 activation, in monocytes from patients with SSc and healthy controls. We showed that B19V can infect both THP-1 cells and primary monocytes but is not able to replicate in these cells. B19V infection increases the production of tumor necrosis factor-\u3b1 and induces NLRP3-mediated caspase-1 activation in both THP-1 cells differentiated with phorbol 12-myristate 13-acetate and in monocytes from patients with SSc but not from healthy controls. B19V infection was sufficient for THP-1 to produce mature IL-1\u3b2. Monocytes from patients with SSc required an additional stimulus, here represented by lipopolysaccharides, to activate cytokine genes. Following B19V infection, however, lipopolysaccharide-activated monocytes from patients with SSc strongly increased the production of IL-1\u3b2 and tumor necrosis factor-\u3b1. Altogether, these data suggest that viral components might potentiate the response to endogenous and/or exogenous toll-like receptor 4 ligands in monocytes from patients with SSc. The B19V-mediated activation of inflammatory pathways in monocytes might contribute to the disease progression and/or development of specific clinical phenotypes