1. Alex AM, Saradalekshmi KR, Shilen N, Suresh PA, Banerjee M. {{Genetic association of DNMT variants can play a critical role in defining the methylation patterns in autism}}. {IUBMB life}. 2019.
Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder with impairments in social communication, restricted, repetitive and stereotyped behaviors. Both genetic and environmental factors are known to contribute toward pathophysiology of Autism. Environmental influences on gene expression can be mediated by methylation patterns which are established and maintained by DNA methyltransferases. Several studies in the past have investigated the role of global methylations in Autism. The present study is aimed to investigate the role of genetic variations in the DNA methyltransferase which might be critical in defining the threshold for environmental factors toward susceptibility to autism. Polymorphisms in DNA methyltransferases, DNMT1, DNMT3A, DNMT3B, and DNMT3L were screened for association with ASD in 180 autistic patients and 260 healthy controls from a south Indian population. DNMT1 rs10418707 and rs10423341, and DNMT3A rs2289195 were found to be significantly associated at genotypic and allelic level with ASD. Functional prediction indicates that these SNPs have a role in transcriptional regulation and increased expression, indicating that hypermethylation might be induced by its genotype status. The study might reflect the role of genetics variants in DNMTs in defining the threshold of environmental impact in influencing the disease or phenotype variations in ASD. (c) 2019 IUBMB Life, 2019.
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2. Angelakos CC, Tudor JC, Ferri SL, Jongens TA, Abel T. {{Home-cage hypoactivity in mouse genetic models of autism spectrum disorder}}. {Neurobiology of learning and memory}. 2019.
Genome-wide association and whole exome sequencing studies from Autism Spectrum Disorder (ASD) patient populations have implicated numerous risk factor genes whose mutation or deletion results in significantly increased incidence of ASD. Behavioral studies of monogenic mutant mouse models of ASD-associated genes have been useful for identifying aberrant neural circuitry. However, behavioral results often differ from lab to lab, and studies incorporating both males and females are often not performed despite the significant sex-bias of ASD. In this study, we sought to investigate the simple, passive behavior of home-cage activity monitoring across multiple 24-hour days in four different monogenic mouse models of ASD: Shank3b(-/-), Cntnap2(-/-), Pcdh10(+/-), and Fmr1 KO mice. Relative to sex-matched wildtype (WT) littermates, we discovered significant home-cage hypoactivity, particularly in the dark (active) phase of the light/dark cycle, in male mice of all four ASD-associated transgenic models. For Cntnap2(-/-) and Pcdh10(+/-) mice, these activity alterations were sex-specific, as female mice did not exhibit home-cage activity differences relative to sex-matched WT controls. These home-cage hypoactivity alterations differ from activity findings previously reported using short-term activity measurements in a novel open field. Despite circadian problems reported in human ASD patients, none of the mouse models studied had alterations in free-running circadian period. Together, these findings highlight a shared phenotype across several monogenic mouse models of ASD, outline the importance of methodology on behavioral interpretation, and in some genetic lines parallel the male-enhanced phenotypic presentation observed in human ASDs.
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3. Antoine MW, Langberg T, Schnepel P, Feldman DE. {{Increased Excitation-Inhibition Ratio Stabilizes Synapse and Circuit Excitability in Four Autism Mouse Models}}. {Neuron}. 2019; 101(4): 648-61.e4.
Distinct genetic forms of autism are hypothesized to share a common increase in excitation-inhibition (E-I) ratio in cerebral cortex, causing hyperexcitability and excess spiking. We provide a systematic test of this hypothesis across 4 mouse models (Fmr1(-/y), Cntnap2(-/-), 16p11.2(del/+), Tsc2(+/-)), focusing on somatosensory cortex. All autism mutants showed reduced feedforward inhibition in layer 2/3 coupled with more modest, variable reduction in feedforward excitation, driving a common increase in E-I conductance ratio. Despite this, feedforward spiking, synaptic depolarization, and spontaneous spiking were largely normal. Modeling revealed that E and I conductance changes in each mutant were quantitatively matched to yield stable, not increased, synaptic depolarization for cells near spike threshold. Correspondingly, whisker-evoked spiking was not increased in vivo despite detectably reduced inhibition. Thus, elevated E-I ratio is a common circuit phenotype but appears to reflect homeostatic stabilization of synaptic drive rather than driving network hyperexcitability in autism.
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4. Bacrot S, Monnot S, Haddad G, Barcia G, Rachid M, Boisson M, Pasquier N, Rondeau S, Munnich A, Steffann J, Bonnefont JP, Raynaud M. {{Prenatal diagnosis of Fragile X syndrome: small meiotic recombination events at the FMR1 locus}}. {Prenatal diagnosis}. 2019.
OBJECTIVE: Fragile X syndrome (FXS), the most common inherited cause of intellectual disability, is caused by an expansion over 200 CGG repeats (full mutation) in the FMR1 gene. Intergenerational instability of an expanded FMR1 allele is linked to the carrier’s gender (female), the CGG repeat size and the number of AGG interspersions within the CGG repeat, making genetic counseling a complex task. The objective of our work was to emphasize the importance of combining haplotype analysis with FMR1 linked markers and CGG repeat sizing for prenatal diagnosis (PND) of FXS. METHODS: Two PND of FXS were performed using haplotype analyzes and sizing of the FMR1 allele. RESULTS: We detected two cases of meiotic recombination at the FMR1 locus, i.e., reciprocal double crossover or non-crossover, resulting in coexistence of the mutant maternal haplotype and the normal-size maternal CGG repeat. CONCLUSION: These rare and unexpected cases (1/120 frequency in our experience) have to be kept in mind in PND of FXS since they prohibit using polymorphic marker haplotyping as the only tool to predict the fetus status.
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5. Brusov OS, Simashkova NV, Karpova NS, Faktor MI, Nikitina SG. {{[Thrombodynamic parameters of hypercoagulation of blood in children with childhood autism and schizophrenia]}}. {Zh Nevrol Psikhiatr Im S S Korsakova}. 2019; 119(1): 59-63.
AIM: To detect blood plasma coagulability in children with mental diseases using a thrombodynamics test. MATERIAL AND METHODS: The study included two groups of children. Group 1 included 11 patients with infantile psychosis in autism (F84.02) (4 girls and 7 boys). Group 2 included 8 patients with childhood schizophrenia (F20.8xx3) (4 girls and 14 boys). A test was performed with T-2 Thrombodynamics analyzer (LLC Hemacore, Moscow, Russia). RESULTS: Thrombodynamic parameters, such as initial, steady-state velocity and spontaneous clots adjusted velocity (Vi, Vst and V, mum/min, respectively) and clot size at 30 minute of thrombodynamics test (CS mum) were significantly increased in the total group of patients (n=29). The time of appearance of spontaneous clots (Tsp), the time of clot lag time (Tlag) and clot density (D) did not differ significantly from the normal values (p=0.98; p=0.27 and p=0.21, respectively). In the autism group (n=11), Vi, Vst and V were significantly higher than normal values, while CS, Tsp, Tlag and D did not differ from norm. In the schizophrenia group (n=18) V, Vst and CS, and Vi were significantly increased. Tsp, Tlag and D did not differ from normal values. Differences between the parameters of thrombodynamics in 1 and 2 groups were not statistical significant. CONCLUSION: It was shown for the first time that clotting (hypercoagulability) of the blood plasma in patients with autism and childhood schizophrenia was increased. This can cause thrombosis in small vessels of the brain. Early spontaneous clots appear in many patients that indicating the presence of systemic inflammation, possibly associated with an exacerbation of neuroinflammation. The thrombodynamics test allows detection of predisposition to hypercoagulability in the early stages when other methods are not sensitive enough.
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6. Calhoun SL, Pearl AM, Fernandez-Mendoza J, Durica KC, Mayes SD, Murray MJ. {{Sleep Disturbances Increase the Impact of Working Memory Deficits on Learning Problems in Adolescents with High-Functioning Autism Spectrum Disorder}}. {J Autism Dev Disord}. 2019.
Sleep disturbances (SD) are prevalent in individuals diagnosed with Autism Spectrum Disorder (ASD). Less is known about the effects of SD on cognition and learning in adolescents with high-functioning ASD (HF-ASD). Adolescents with HF-ASD (N = 96) were evaluated for the relationships of SD to working memory and learning problems. Results found SD to modify the relationship between working memory and learning problems. Working memory deficits were associated with learning problems among those with SD, while not among those without SD. SD and working memory deficits should be targeted in interventions for these adolescents with HF-ASD (e.g., cognitive behavior therapy for insomnia, pharmacological treatments). Future studies should examine if improvement in SD reduces the impact of working memory deficits on learning problems.
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7. Cordone V, Pecorelli A, Benedusi M, Santini S, Jr., Falone S, Hayek J, Amicarelli F, Valacchi G. {{Antiglycative Activity and RAGE Expression in Rett Syndrome}}. {Cells}. 2019; 8(2).
Rett syndrome (RTT) is a human neurodevelopmental disorder, whose pathogenesis has been linked to both oxidative stress and subclinical inflammatory status (OxInflammation). Methylglyoxal (MG), a glycolytic by-product with cytotoxic and pro-oxidant power, is the major precursor in vivo of advanced glycation end products (AGEs), which are known to exert their detrimental effect via receptor- (e.g., RAGE) or non-receptor-mediated mechanisms in several neurological diseases. On this basis, we aimed to compare fibroblasts from healthy subjects (CTR) with fibroblasts from RTT patients (N = 6 per group), by evaluating gene/protein expression patterns, and enzymatic activities of glyoxalases (GLOs), along with the levels of MG-dependent damage in both basal and MG-challenged conditions. Our results revealed that RTT is linked to an alteration of the GLOs system (specifically, increased GLO2 activity), that ensures unchanged MG-dependent damage levels. However, RTT cells underwent more pronounced cell death upon exogenous MG-treatment, as compared to CTR, and displayed lower RAGE levels than CTR, with no alterations following MG-treatment, thus suggesting that an adaptive response to dicarbonyl stress may occur. In conclusion, besides OxInflammation, RTT is associated with reshaping of the major defense systems against dicarbonyl stress, along with an altered cellular stress response towards pro-glycating insults.
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8. Ford T, Kenchington R, Norman S, Hancock J, Smalley A, Henley W, Russell G, Hayes J, Logan S. {{The agreement between the referrer, practitioner and research diagnosis of autistic spectrum conditions among children attending child and adolescent mental health services}}. {Eur Child Adolesc Psychiatry}. 2019.
We aimed to explore the levels of agreement about the diagnoses of Autistic Spectrum Conditions between the referrer, CAMHS practitioner and a research diagnosis, as well as the stability of the practitioner’s diagnosis over time in a secondary analysis of data from 302 children attending two Child and Adolescent Mental Health Services over two years. Kappa coefficient was used to assess the agreement between the referrer and research diagnosis. Kendall’s tau b coefficient was used to assess the agreement between the practitioner and the research diagnosis assigned using the Development and Well-Being Assessment, as well as the agreement between the referrer’s indication of presenting problems and the practitioner diagnosis. Diagnostic stability was explored in children with and without a research diagnosis of Autistic Spectrum Condition. There was a moderate level of agreement between the referrer and research diagnosis (Kappa = 0.51) and between practitioner’s and research diagnosis (Kendall’s tau = 0.60) at baseline, which reduced over the subsequent two years. Agreement between the referrer and practitioner’s diagnosis at baseline was fair (Kendall’s tau = 0.36).The greatest diagnostic instability occurred among children who practitioners considered to have possible Autistic Spectrum Conditions but who did not meet research diagnostic criteria. Further studies could explore the approaches used by practitioners to reach diagnoses and the impact these may have on diagnostic stability in Autistic Spectrum Conditions. Standardised assessment using a clinically rated diagnostic framework has a potential role as an adjunct to standard clinical care and might be particularly useful where practitioners are uncertain.
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9. Foss-Feig JH, Velthorst E, Smith L, Reichenberg A, Addington J, Cadenhead KS, Cornblatt BA, Mathalon DH, McGlashan TH, Perkins DO, Seidman LJ, Stone WS, Keshavan M, Tsuang MT, Walker EF, Woods SW, Cannon TD, Bearden CE. {{Clinical Profiles and Conversion Rates Among Young Individuals With Autism Spectrum Disorder Who Present to Clinical High Risk For Psychosis Services}}. {J Am Acad Child Adolesc Psychiatry}. 2019.
OBJECTIVE: The overlap versus independence of autism spectrum disorder (ASD) and schizophrenia is a topic that has garnered the attention of generations of clinicians and scientists. Although high rates of psychotic symptoms have been identified in individuals with ASD, the nature, prevalence, and prognostic significance of subclinical psychotic experiences in ASD remain poorly understood. METHOD: This study sought to compare baseline characteristics, clinical profiles, and conversion outcomes between young individuals at clinical high risk for psychosis (CHR) who presented with or without a prior ASD diagnosis during the second phase of the North American Prodrome Longitudinal Study (NAPLS, N=764). RESULTS: Patients with CHR and ASD (CHR/ASD+, n=26) tended to exhibit greater social and social cognitive difficulties, but expressed relatively similar levels of core psychosis symptoms to patients with CHR but no ASD (CHR/ASD-). Risk for conversion to co-occurring psychosis (18.2% CHR/ASD+ versus 16.8% CHR/ASD-) was equivalent between CHR/ASD+ and CHR/ASD- groups, and the NAPLS2 Psychosis Risk Calculator predicted conversion to psychosis equally well across groups. CONCLUSION: These results suggest that baseline psychosis symptoms, predictors of risk for conversion, and ultimate conversion rates are similar in patients with CHR with and without ASD. They further suggest that ASD must not be considered a mutually exclusive diagnosis when such youth present in CHR settings. Future research is needed to better track trajectories in larger cohorts of individuals with CHR and comorbid ASD and to understand whether treatment recommendations effective in the broader CHR population are useful for this particular population as well.
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10. Goel A, Portera-Cailliau C. {{Autism in the Balance: Elevated E-I Ratio as a Homeostatic Stabilization of Synaptic Drive}}. {Neuron}. 2019; 101(4): 543-5.
In this issue of Neuron, Antoine et al. (2019) find reduced feedforward inhibition in cortical neurons in four genetic mouse models of autism but without evidence of increased spontaneous or sensory-evoked activity.
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11. Guerra S, Spoto A, Castiello U, Parma V. {{Sex Differences in Body Ownership in Adults With Autism Spectrum Disorder}}. {Front Psychol}. 2019; 10: 168.
A strong male prevalence has been observed in autism spectrum disorder (ASD) since its definition, but the behavioral manifestations of sex disparity have yet to be clarified. Here, we investigate sex differences in the perception of the Numbness Illusion (NI), a procedure based on a tactile conflict, in adults with ASD and with typical development. We aim to assess if women and men with ASD perceive NI-dependent body ownership differently and whether sex differences emerge in individuals with typical development. To elicit the NI, participants pressed their right-hand palm against the confederate’s hand and stroked with the thumb and the index finger of their left hand the joined index fingers in a synchronous or asynchronous way. Results reveal that women with ASD present a reversed and atypical pattern for the NI compared to men with ASD and a group of matched controls. In particular, women with ASD report a stronger illusion than men with ASD, that is more evident in the asynchronous conditions. In the asynchronous condition, women in the ASD group report stronger NI as compared to women and men in the Control group, whereas men with ASD only to men in the Control group. In the typical sample, the NI emerges only in the synchronous condition and no sex difference is observed. We discuss our results in terms of potential advantage of women in sociality and sensory information processing that might lead women with ASD to use different modalities to solve the illusion compared to men with ASD. In sum, these outcomes describe sex differences in individuals with ASD in the domain of illusory perception. This may be used in the future to support the characterization of the female phenotype of autism.
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12. Kyriakopoulos M. {{Editorial: Autism Spectrum Disorders in Individuals at Clinical High Risk for Psychosis: Possible Association With Social Deficits but Not Conversion Rates}}. {J Am Acad Child Adolesc Psychiatry}. 2019.
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13. Lopez Perez D, Kennedy DP, Tomalski P, Bolte S, D’Onofrio B, Falck-Ytter T. {{Visual Search Performance Does Not Relate to Autistic Traits in the General Population}}. {J Autism Dev Disord}. 2019.
Autism spectrum disorder (ASD) is commonly conceived as the extreme end of a continuum. Research suggests that autistic individuals outperform typically developing controls in visual search. Thus, enhanced visual search may represent an adaptive trait associated with ASD. Here, using a large general population sample (N = 608, aged 9-14 years), we tested if higher levels of autistic traits are associated with enhanced visual search. Visual search was evaluated using both manual responses and eye movements, and autistic traits were measured using the Social Responsiveness Scale. Contrary to our hypothesis, no significant relation between autistic traits and visual search were observed. The theoretical implications of these results are discussed.
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14. Montazeri F, de Bildt A, Dekker V, Anderson GM. {{Network Analysis of Behaviors in the Depression and Autism Realms: Inter-Relationships and Clinical Implications}}. {J Autism Dev Disord}. 2019.
Depression-, anxiety-, OCD- and autism-related behaviors were assessed in 118 high-functioning individuals with autism spectrum disorders (ASD) and in 2016 controls. The ASD group had a higher rate of clinical depression and markedly higher « insomnia » and « restlessness » scores. Network analysis and hierarchical cluster analysis in the ASD group revealed that depression and anxiety items clustered together, but separately from autism-related items. Compared to controls, « insomnia » and « restlessness » items in the ASD network of depression items were much more central (higher closeness, and betweenness centrality). Combined networks of depression-, anxiety-, and OCD-related items revealed that the control group depression item module was not preserved in ASD. The results indicate that depression is atypical in autism and suggest specific intervention targets.
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15. Rabiee A, Samadi SA, Vasaghi-Gharamaleki B, Hosseini S, Seyedin S, Keyhani M, Mahmoodizadeh A, Ranjbar Kermani F. {{The Cognitive Profile of People with High-Functioning Autism Spectrum Disorders}}. {Behav Sci (Basel)}. 2019; 9(2).
Several studies have examined the cognitive profile of people with high-functioning autism spectrum disorders (ASD) (IQ > 70), and its relationship with the symptoms of ASD and attention deficit hyperactivity disorder (ADHD), using the Wechsler Intelligence Scale for Children-IV (WISC-IV). However, no data exist on the similarities or differences in this profile in less affluent countries. The present study examined the cognitive profile and its relationship with the symptoms of ASD and ADHD in 30 subjects aged 6(-)16 years with high-functioning ASD and compared the results with those of 30 typically developing (TD) subjects. In line with previous research findings, the WISC-IV cognitive profile analysis of subjects with high-functioning ASD showed a good competence in Matrix Reasoning and weaknesses in Comprehension, but the main distinguishing point was the competence in processing speed in both groups. In the present study, the Verbal Comprehension Index correlated negatively with the communication symptoms, and the Working Memory Index correlated positively with the social symptoms in the ASD group. Given the similarities that exist between the results of the present research and previous studies, it may be concluded that there are similarities in the cognitive profile of individuals with ASD.
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16. Scahill L, Lecavalier L, Schultz RT, Evans AN, Maddox B, Pritchett J, Herrington J, Gillespie S, Miller J, Amoss RT, Aman MG, Bearss K, Gadow K, Edwards MC. {{Development of the Parent-Rated Anxiety Scale for Youth with Autism Spectrum Disorder}}. {J Am Acad Child Adolesc Psychiatry}. 2019.
OBJECTIVE: Anxiety is common in youth with autism spectrum disorder (ASD). There is no accepted outcome measure for anxiety in this population. METHOD: Following a series of focus groups with parents of youth with ASD, we generated 72 items (scored 0-3). Parents of 990 youth with ASD (age 5-17; 80.8% male youth) completed an online survey. Factor analysis and item response theory (IRT) analyses reduced the content to a single factor with 25 items. Children with a least mild anxiety (N=116; age 5-17; 79.3% male youth) participated in a comprehensive clinical assessment to evaluate validity and reliability of the 25-item Parent-rated Anxiety Scale for ASD (PRAS-ASD). RESULTS: In the online sample, the mean PRAS-ASD score was 29.04 +/- 14.9 (range 0 to 75). Coefficient alpha was 0.93. The IRT results indicated excellent reliability across a wide range of scores with low standard errors. In the clinical sample (N=116), the PRAS-ASD mean was 31.0 +15.6 (range 1-65). Pearson correlations with parent ratings of ASD symptom severity, repetitive behavior and disruptive behavior ranged 0.33 to 0.66 supporting divergent validity of the PRAS-ASD. Pearson correlation with a parent-rated measure of anxiety used in the general pediatric population of 0.83 supported convergent validity. Forty participants (32 boys, 8 girls; mean age of 11.9 + 3.4) returned at Time 2 (mean =12.2 days) and Time 3 (mean = 24.2 days). Intraclass correlation showed test-retest reliabilities of 0.88 and 0.86 at Time 2 and Time 3, respectively. CONCLUSION: The 25-item PRAS-ASD is a reliable and valid scale for measuring anxiety in youth with ASD.
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17. Stefano V, Eleonora N, Viviana C, Deny M, Viola A, Sara L, Antonio N, Marco T. {{Copy number variants in autism spectrum disorders}}. {Prog Neuropsychopharmacol Biol Psychiatry}. 2019.
In recent years, there has been an explosive increase in genetic studies related to autism spectrum disorder (ASD). This implicated the accumulation of a large amount of molecular data that may be used to verify various hypotheses and models developed to explore the complex genetic component of ASD. Several lines of evidence support the view that structural genomic variation contributes to the pathogenesis of ASD. The introduction of more sophisticated techniques for whole-genome screening, including array comparative genome hybridization and high-resolution single nucleotide polymorphism analysis, has allowed to identify an increasing number of ASD susceptibility loci. Copy number variants (CNVs) are the most common type of structural variation in the human genome and are considered important contributors to the pathogenesis of neurodevelopmental disorders, including ASD. In this review, we describe the accumulated evidence concerning the genetic events associated with ASD, and summarize current knowledge about the clinical relevance of CNVs in these disorders.
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18. Vivanti G, Volkmar FR. {{Review: National Guideline for the Assessment and Diagnosis of Autism Spectrum Disorders in Australia (Whitehouse, Evans et al. 2018)}}. {J Autism Dev Disord}. 2019.
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19. Yerys BE, Tunc B, Satterthwaite TD, Antezana L, Mosner MG, Bertollo JR, Guy L, Schultz RT, Herrington JD. {{Functional Connectivity of Frontoparietal and Salience/Ventral Attention Networks Have Independent Associations With Co-occurring Attention-Deficit/Hyperactivity Disorder Symptoms in Children With Autism}}. {Biol Psychiatry Cogn Neurosci Neuroimaging}. 2019.
BACKGROUND: Children with autism spectrum disorder (ASD) and co-occurring attention-deficit/hyperactivity disorder (ADHD) symptoms have worse functional outcomes and treatment response than those without ADHD symptoms. There is limited knowledge of the neurobiology of ADHD symptoms in ASD. Here, we test the hypothesis that aberrant functional connectivity of two large-scale executive brain networks implicated in ADHD-the frontoparietal and salience/ventral attention networks-also play a role in ADHD symptoms in ASD. METHODS: We compared resting-state functional connectivity of the two executive brain networks in children with ASD (n = 77) and typically developing control children (n = 82). These two executive brain networks comprise five subnetworks (three frontoparietal, two salience/ventral attention). After identifying aberrant functional connections among subnetworks, we examined dimensional associations with parent-reported ADHD symptoms. RESULTS: Weaker functional connectivity in ASD was present within and between the frontoparietal and salience/ventral attention subnetworks. Decreased functional connectivity within a single salience/ventral attention subnetwork, as well as between two frontoparietal subnetworks, significantly correlated with ADHD symptoms. Furthermore, follow-up linear regressions demonstrated that the salience/ventral attention and frontoparietal subnetworks explain unique variance in ADHD symptoms. These executive brain network-ADHD symptom relationships remained significant after controlling for ASD symptoms. Finally, specificity was also demonstrated through the use of a control brain network (visual) and a control co-occurring symptom domain (anxiety). CONCLUSIONS: The present findings provide novel evidence that both frontoparietal and salience/ventral attention networks’ weaker connectivities are linked to ADHD symptoms in ASD. Moreover, co-occurring ADHD in the context of ASD is a source of meaningful neural heterogeneity in ASD.
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20. Zhang Q, Yang X, Wang J, Li J, Wu Q, Wen Y, Zhao Y, Zhang X, Yao H, Wu X, Yu S, Wei L, Bao X. {{Correction: Genomic mosaicism in the pathogenesis and inheritance of a Rett syndrome cohort}}. {Genet Med}. 2019.
The second author Jiarui Li is now listed as a co-first author according to her contribution to this paper. The list of authors who contributed equally now reads: Qingping Zhang, Xiaoxu Yang, Jiaping Wang, and Jiarui Li. This has now been corrected in both the PDF and HTML versions of the Article.
