School-age children with attention deficit hyperactivity disorder (ADHD) have difficulties in interpersonal relationships, in addition to impaired facial expression perception and recognition. For successful social interactions, the ability to discriminate between familiar and unfamiliar faces is critical. However, there are no published reports on the recognition of familiar and unfamiliar faces by children with ADHD.We evaluated the neural correlates of familiar and unfamiliar facial recognition in children with ADHD compared to typically developing (TD) children. We used functional near-infrared spectroscopy (fNIRS) to measure hemodynamic responses on the bilateral temporal regions while participants looked at photographs of familiar and unfamiliar faces. Nine boys with ADHD and 14 age-matched TD boys participated in the study. fNIRS data were Z-scored prior to analysis.During familiar face processing, TD children only showed significant activity in the late phase, while ADHD children showed significant activity in both the early and late phases. Additionally, the boys with ADHD did not show right hemispheric lateralization to familiar faces.This study is the first to assess brain activity during familiar face processing in boys with ADHD using fNIRS. These findings of atypical patterns of brain activity in boys with ADHD may be related to social cognitive impairments from ADHD. Attention deficit hyperactivity disorder (ADHD) is characterized by major symptoms of hyperactivity, impulsivity, and inattention, which result in problems in the individual’s quality of life , 2. It iThe early development of facial processing has been considered fundamental to the development of social abilities and theory of mind . SeveralHowever, there is limited knowledge regarding basic facial processing in ADHD. Tye et al. (2013) were the first to explore the face inversion effects and gaze processing in children with ADHD using event related potentials (ERPs) . They fofNIRS is a neuroimaging device which provides an index of brain function by measuring changes in the concentration of oxygenated hemoglobin (oxy-Hb), deoxygenated hemoglobin (deoxy-Hb), and total-hemoglobin  using observation of the diffuse transmittance of near-infrared light at an appropriate range of wavelengths , 13. fNIresolution, and provides a non-intrusive environment requiring less restriction of the body and head , 20. fNITo our knowledge, there are no published reports on the recognition of familiar and unfamiliar faces in children with ADHD. Thus, the purpose of this study is to compare the neural correlates involved in familiar (mother) and unfamiliar facial recognition in children with and without ADHD using a fNIRS system, which was placed over the bilateral temporal regions.Seventeen ADHD boys and 16 TD boys were recruited. All diagnoses were guided by the DSM-5  and weret = 3.68, p = 0.048) than those of the ADHD participants .After excluding subjects whose fNIRS data failed the motion criteria (frequent sharp changes in the time series of the raw fNIRS data), 23 subjects remained: 9 boys with ADHD , and 14 TD boys  , which can record 24 channels simultaneously with 12 channels over the right temporal area and 12 over the left. This instrument generates two wavelengths of NIR (695 nm and 830 nm) and measures the time courses of oxyhemoglobin (oxy-Hb) and deoxyhemoglobin (deoxy-Hb) levels and their sum  with a 0.1 s time resolution. The fNIRS probes contained nine optical fibers (3 × 3 arrays) composed of five emitters and four detectors. The distance between the emitters and the detectors was set at 3 cm. Each pair of adjacent emitting and detecting fibers defined a single measurement channel.The placement of the probes was set on the bilateral temporal cortices centered at T5 and T6 according to the International 10–20 system  The analysis of fNIRS data in the present study was similar to that reported by Nakato et al. . Before The raw data on oxy-Hb, deoxy-Hb, and total-Hb concentrations from each channel were digitally band-pass-filtered at 0.02–1.0 Hz to remove noise caused by heartbeat pulsations or any longitudinal signal drifts. The mean concentration of each channel within a subject was then calculated by averaging data across the trials in a time-series with a 0.1 s resolution.Based on mean concentrations in the time-series, we calculated the Z-scores for oxy-, deoxy-, and total-Hb in the familiar face and the unfamiliar face conditions for each channel within a subject , 24–27. The Z-scores obtained from the 12 channels within the left and right temporal areas were averaged across trials for every subject in order to increase the signal-to-noise ratio. Although raw NIRS data consist of relative values that cannot be averaged directly across subjects or channels, normalized data including Z-scores can be averaged regardless of the unit. This calculation of the Z-score is a reliable analysis for changes in Hb concentration in the brain regions under the sensors since the analysis is independent of the differential path length factor (DPF). On the basis of these methodological advantages for fNIRS data, we applied the same analysis of the Z-score in the present study as in previous fNIRS studies with children , 24–27.t-test against a chance level of 0 (baseline) was conducted for the mean Z-score during the early phase and late phase in both temporal areas.Consistent with our previous studies using fNIRS , 24–27, Data from the 23 boys who looked at faces over more than three trials in both the familiar face and unfamiliar face conditions were obtained. Available trials were averaged across trials for each condition . The mean number of removed trials for the familiar face condition was 0.33 (SD 0.74) while that for the unfamiliar face condition was 0.30 (SD 0.64). There was no significant difference between the numbers of available trials under each condition.On the basis of the wavelengths of the Hitachi ETG-4000 system (695 nm and 830 nm), estimations of oxy-Hb concentration have been reported to better reflect changes in regional cerebral blood flow than deoxy- or total-Hb concentrations .t-test with the mean Z-scores for oxy- deoxy-, and total-Hb concentrations for the 3–10 s (the early phase) and 10–17 s (the late phase) durations of the test period in each familiar and unfamiliar face condition  = 3.80, p = 0.005; left temporal cortex; t (8) = 2.79, p = 0.023. They also showed a significant increase in both temporal cortices in the late phase: right temporal cortex; t (8) = 5.27, p = 0.0007; left temporal cortex; t (8) = 2.53, p = 0.035. However, in the unfamiliar face condition, there was no significant change in the concentration of oxy-Hb in the ADHD boys in either the early or late phase. TD boys showed no significant change in the early phase; however, in the late phase, they showed significant increases in the familiar face condition in the right temporal cortex: t (13) = 3.12, p = 0.008. Three-way ANOVA (2 × 2 × 2: group x condition x hemisphere) showed the only main effect in the condition .In the familiar face condition, the Z-scores for oxy-Hb concentrations in the ADHD boys were significantly increased in both temporal cortices in the early phase: right temporal cortex; t (8) = 2.47, p = 0.039; left temporal cortex; t (8) = 4.97, p = 0.001. In the late phase, they showed significant decreases in the left temporal cortex: t (8) = 2.42, p = 0.041. TD boys showed no significant change in the concentration of deoxy-Hb in either condition in either the early or late phase.The Z-scores for deoxy-Hb concentrations of the ADHD boys showed no significant change in the familiar face condition. However, in the unfamiliar face condition, the Z-scores of the ADHD boys were significantly decreased in both the right and left temporal cortices in the early phase: right temporal cortex; t [p = 0.021), but only in the familiar face condition. TD boys showed no significant change in the concentration of the total-Hb in either condition in both the early and late phases.The Z-scores for the total-Hb concentrations in the ADHD boys showed no significant change in either condition in the early phase. However, in the late phase, they showed significant increases in the right temporal cortex (t  = 2.67, The current study compared the neural correlates of viewing familiar and unfamiliar facial images in ADHD and TD boys using fNIRS. To measure the neural activities of facial recognition, the fNIRS probes were set on the bilateral temporal regions, including on the STS, which plays an essential role in processing faces. These measuring procedures were the same as in previous studies , 23–27. fNIRS monitors blood volume and oxygenation in the brain. In this study, the hemodynamic response, as indicated by oxy-Hb, increased gradually during the test period in both groups and was found to occur predominantly in the right hemisphere in TD boys. Neuroimaging studies have reported that the major aspects of facial processing arise predominantly in the right hemisphere , 31. TheHemodynamic responses to familiar faces were found to occur predominantly in the right temporal area in TD boys. In contrast, ADHD boys showed significantly increased concentration of oxy-Hb to familiar faces in both the right and left temporal areas compared with the baseline. The TD boys showed right hemispheric dominance in processing faces, which is consistent with many other neuroimaging studies , 31. WheAside from the major symptoms of ADHD, boys with ADHD have been documented to have social cognitive impairments with impaired facial recognition and expression perception , 6, 7. AIn conclusion, we have demonstrated that differential brain activity occurs between ADHD and TD boys in response to the sight of a familiar face. During familiar face processing, ADHD children showed significant activity in both the early and late phase, while TD children showed significant activity only in the late phase. Additionally, the dominance of the right temporal areas for facial processing was found only in TD children during the familiar face presentation. These findings of atypical patterns of brain activity in ADHD boys may be related to social cognitive impairments in ADHD. We believe that these insights make a number of important additions to the literature and would be of considerable interest to researchers of biopsychosocial medicine.


Attention-deficit/hyperactivity disorder (ADHD) is a common neurodevelopmental disorder characterized by impaired attention, impulsivity and hyperactivity. The “neonatal 6-hydroxydopamine” (6-OHDA) lesion is a commonly used model of ADHD in rat. However, a comprehensive assessment of ADHD‐like symptoms is still missing, and data in mouse remain largely unavailable. Our aim was to analyse symptoms of ADHD in the mouse neonatal 6‐OHDA model. 6‐OHDA mice exhibited the major ADHD‐like symptoms, i.e. hyperactivity (open field), attention deficit and impulsivity . Further, the model revealed discrete co‐existing symptoms, i.e. anxiety‐like (elevated plus maze test) and antisocial  behaviours and decreased cognitive functioning (novel object recognition). The efficacy of methylphenidate, a classical psychostimulant used in the treatment of ADHD, was also evaluated. A histological analysis further supports the model validity by indicating dopamine depletion, changes in cortical thickness and abnormalities in anterior cingulate cortex neurons. A principal component analysis of the behaviour profile confirms that the 6‐OHDA mouse model displayed good face and predictive validity. We conclude that neonatal dopamine depletion results in behavioural and morphological changes similar to those seen in patients and therefore could be used as a model for studying ADHD pathophysiological mechanisms and identifying therapeutic targets. Three core clinical symptoms define ADHD: hyperactivity, inattention and impulsivity3. ADHD patients also often coexisting complains such as poor academic performance, social disabilities and emotional deficits5.Attention-deficit/hyperactivity disorder (ADHD) is a heterogeneous neurodevelopmental psychiatric disorder that affects 5–8% of children and can persist at adulthood in 50% of cases6, neuropsychological studies consistently demonstrated impaired prefrontal cortex (PFC) functions in ADHD patients correlated with reduced PFC size, blood flow and glucose metabolism7. Moreover, ADHD patients have impaired dopamine (DA) and norepinephrine (NE) transmission that affects PFC function8. Consequently, the primary drugs used for treating ADHD enhance DA and NE transmission. Psychostimulants  are the most commonly used pharmacological treatments and target dopaminergic system. ADHD treatments remain however poorly efficient and are accompanied with side effects9. Thus, a better understanding of ADHD neurochemical mechanisms is key to improve treatments and valid animal models are required for this purpose.In line with possible dysfunctions in the networks involved in controlling and regulating attention and/or impulsivity10. Several animal models of ADHD currently exist but none were developed specifically for modeling neurodevelopmental alterations that occur during the disease onset and progression, neither to model several aspects of the behavioral and executive functional symptoms. The most widely used model of ADHD is the inbred spontaneously hypertensive rat (SHR). Besides, the most classical neurodevelopmental model of ADHD created by lesioning brain systems is obtained by neonatal 6-hydroxydopamine (6-OHDA) injection. In this model, studies mainly focused on hyperactivity symptoms12. However, data regarding impulsive behavior or attention deficits remain unclear13.Ideally a reliable animal model should display all the symptoms present in ADHD patients, and respond similarly to the same pharmacological interventionsThe aim of the present study is to evaluate the repertoire of major and comorbid symptoms of ADHD in a 6-OHDA lesioned mouse model. We evaluated face validity through assessing spontaneous activity during juvenile period, coexisting complains in adolescence and the persistence of ADHD-like symptoms at the adulthood. The efficacy of methylphenidate, a classical psychostimulant acting as a catecholamine transporter inhibitor and used in the treatment of ADHD, was also used to test predictive validity of the model. We further analyzed specific morphological characteristics of ADHD in our model, focusing on PFC, particularly anterior cingulate cortex (ACC) neurons. Finally, a principal component analysis of the behavior profile demonstrates the importance of individual symptoms in the pathology.To examine whether 6-OHDA mice display dopamine depletion, we examined TH-immunoreactivity (IR) in the striatum and midbrain of sham and 6-OHDA adult mice. We found a high density of TH-IR fibers in the striatum of sham mice Fig. . In cont). No significant difference was seen in body weight between the two groups .We then evaluated body and brain weight in 6-OHDA mice and sham Fig. 1–2. No s2ups Fig. . Howeverp < 0.05). In 6-OHDA mice, the area of pyramidal neurons is smaller than in sham . Moreover, the diameter of apical dendrites of the layer II-III pyramidal neurons at 10 μm  and 100 μm  from the soma showed a significant reduction in 6-OHDA mice at both distance .Since the dorsal anterior cingulate cortex (ACd) is one of the main targets involved in the modulation of attention and executive functions, we investigated the morphology of layer II-III pyramidal neurons of the ACd using Golgi staining Fig. . QualitaCollectively, these data indicate that 6-OHDA mice exhibited the morphological characteristics of ADHD, including dopamine depletion, changes in cortical thickness and abnormalities in ACd neurons.In order to characterize the ADHD symptoms, we first investigated the spontaneous locomotor activity of 6-OHDA and sham mice in an open field at p24. = 411.40; F = 71.95 and F = 205.57, p < 0.001; respectively), the treatment  = 40.11; F = 32.66 and F = 57.59, p < 0.05; respectively) and the interaction between lesion and treatment  = 11.67; F = 11.67 and F = 12.57, p < 0.001; respectively). At P24, Student-Newman-Keuls post-hoc analysis showed that 6-OHDA mice presented higher locomotor activity than shams . = 20.40; F = 33.55; F = 67.24; F = 132.23, p < 0.001; respectively), whereas the treatment  = 1.73; F = 0.52; F = 0.40; F = 0.61, p > 0.05; respectively) and the interaction treatment/lesion  = 0.021; F = 0.014; F = 0.03; F = 0.007, p > 0.05; respectively) did not affect the parameters. In the elevated plus-maze (EPM) test, the time spent in the open arms  and the number of entries  were reduced in the 6-OHDA group as compared to the sham group   = 915.87; F = 644.06, p < 0.001; respectively), treatment  = 12.71; F = 30.58, p < 0.05; respectively) and the interaction treatment/lesion  = 14.22; F = 11.67, p < 0.001; respectively). The post hoc analysis showed that the sham animals approached the control animals, whereas the 6-OHDA animals avoided them , whereas the same doses increased the avoidance behavior in sham mice  as compared to vehicle-injected 6-OHDA mice, while the opposite was seen in sham mice  was significantly affected by the lesion  = 2209.82; F = 1726.65, p < 0.001; respectively), treatment  = 91.50; F = 88.51; p < 0.05; respectively) and the interaction treatment/lesion  = 12.25; F. 6-OHDA mice displayed a significant increase of the agonistic behavior (number of biting attacks) against a stimulus mouse as compared to sham mice . The same treatment has an opposite effect in sham mice , and a discrimination ratio (Dr) above 0.5 (see suppl. methods) Fig. . = 200.74 and F = 131.70, p < 0.001), the treatment  = 25.21 and F = 19.20, p < 0.05) and the interaction treatment/lesion  = 14.53 and F = 28.11; p < 0.001). In the novel object recognition test, the exploration time of a new object was significantly reduced in 6-OHDA mice , whereas reduced in sham mice , while the opposite was seen in sham mice  = 156.62; F = 360.00; F = 228.60; F = 305.01, p < 0.001; respectively), while the session  = 0.034; F = 0.11; F = 1.21; F = 4.42, p > 0.05; respectively) and the interaction lesion/session  = 0.017; F = 0.017; F = 2.39; F = 6.94, p > 0.05; respectively) had no effect. During the acquisition phases of the last 4 sessions of training at 1 s of SD, 6-OHDA mice were less accurate than sham mice  and the stimulus duration (SD) to evaluate impulsivity and attention, respectively, when mouse performances remained stable. = 45.64, p < 0.001), while the ITI and the interaction lesion/ITI had no effect on accuracy parameter  = 0.015, p > 0.05; F = 3.51, p > 0.05; respectively). Furthermore, the statistical analysis indicated a significant effect of lesion  = 183.84; F = 85.65 and F = 205.96, p < 0.001; respectively) and ITI  = 5.13, p < 0.01; F = 5.28, p < 0.01; F = 4.34, p < 0.05; respectively) on the omission, premature and preservative responses. In contrast, the interaction lesion/ITI had no effect on those parameters  = 1.27; F = 1.42; F = 1.39, p > 0.05; respectively). When the ITI was lengthened from 5 to 7 or 10 sec, a significant increase in premature , perseverative , and omissions  responses was observed in 6-OHDA mice . This suggests that 6-OHDA mice impulsivity was disclosed by ITI increase. Meanwhile, we did not observe difference on accuracy in 6-OHDA mice when the ITI increased  as main factors, showed a significant effect of lesion  = 185.52; omission: F = 172.02; premature responding:F = 743.99 and perseverative response: F = 37.62, p < 0.001) and the SD  = 3.11; F = 3.46; F = 6.82 and F = 4.74, p < 0.05; respectively), whereas, the interaction lesion/SD had no effect  = 1.51; F = 2.00; F = 1.03 and F = 1.23, p > 0.05; respectively). In fact, when SD decreased from 1 to 0.8 or 0.5 s, the post-hoc analysis indicated a significant decrease in accuracy  and an increase in omissions  in 6-OHDA mice  and perseverative responses   Fig. . No effe = 116.99; F = 50.04; F = 126.28; F = 58.36, p < 0.001; respectively), treatment  = 23.15; F = 35.40; F = 50.25; F = 74.25, p < 0.01; respectively) and the interaction treatment/lesion  = 6.95, p < 0.01; F = 6.97, p < 0.01; F = 5.46, p < 0.05; F = 6.76, p < 0.01; respectively). In 6-OHDA mice, Mph at 5.0 mg/kg produced a significant increase in accuracy , a decrease in omissions , premature , and perseverative responses , an increase in omissions , premature , and perseverative responses , there is a significant effect of lesion  based on the 20 variables measured in the different behavioral tests (see methods). The PCA enabled the representation of the variables Fig.  and expe14, but also other comorbid affections.The diagnostic of ADHD, like other psychiatric disorders, relies on behavioural assessment. Animal models of ADHD must mimic clinical symptomatology and in particular the three core symptoms of hyperactivity, impulsivity, and impaired attentionHere we provide evidence that the 6-OHDA mouse model exhibits known major symptoms of the human pathology, namely hyperactivity in a novel environment at a juvenile stage, inattention and impulsive-like behavior at adulthood. Moreover, the 6-OHDA adolescent mouse exhibits co-morbid symptoms including increased anxiety, antisocial and aggressive behaviors, and deficits in learning and memory. We also discuss below the mechanisms underlying dopamine depletion-induced pathophysiology and we point to the interest of the 6-OHDA model in mimicking the effects of known treatments.15. Selective removal of DA projections to forebrain in neonatal rats leads to age-limited spontaneous motor hyperactivity18 at an age corresponding to human periadolescence19. Only one study used such a model in mouse and reproduced similar locomotor impairment12. However, data regarding impulsive behavior or attention deficits remain unavailable in mouse13, or contradictory in rat20. Moreover, existing studies do not describe discrete co-morbid symptoms.Disrupting brain systems through neonatal 6-OHDA lesion is a classical neurodevelopmental model of ADHD in rat21 that can be illustrated by ADHD patient performance of the continuous performance task23. ADHD subjects have slower and more variable reaction times, and make more errors of omission indicative of poor attentional ability21. In addition, they make more errors of commission, demonstrating reduced behavioral inhibition and impulsivity. In our study, hyperactivity is determined in 6-OHDA mice with the open field test. One of our main findings was that 6-OHDA adult mice displayed deficits in inhibitory control in the 5-CSRTT, a task used in rodents that requires behavioral inhibition24. 6-OHDA mice exhibit an increased number of perseverative responding under baseline conditions and increased premature responding during the inter-trial interval challenge. Impulsive choice reflects, to a greater degree, decision-making processes rather than motoric inhibition25. This is generally considered to reflect a failure of the «executive system» represented by frontal cortical areas exerting a top-down control to limbic and paralimbic areas26.ADHD adult patients show inattention and elevated impulsivityLikewise, 6-OHDA mice displayed a greater loss of accuracy when attention was challenged. Interestingly, this effect was present all along the session, indicative of a deficit in selective attention and difficulty to maintain sustained attention. Taken together, these data demonstrate that 6-OHDA adult mice exhibit attention deficit and impulsivity.28. Access to novelty (e.g. object or environment) can elicit approach behaviors in rodents. Starting from this observation29, a new behavioral test was developed in the late 1980s: the so-called object recognition test30. The test is based on the rodent tendency to interact more with a novel than a familiar object. The exploration time of a new object during the test trial was not significantly increased in 6-OHDA mice, suggesting that cognitive abilities (e.g. learning) and/or recognition memory were impaired in 6-OHDA adolescent mice. These findings are in agreement with previous studies showing cognitive impairments in spatial discrimination task in rats10 and in mice12. Anxiety disorder is also a common comorbidity of ADHD27 that we explored with the EPM test. 6-OHDA adolescent mice exhibited anxiety-like behavior in agreement with previous results12. Another set of discrete symptoms of ADHD is characterized by aggression and disruptive behavior34. We found that 6-OHDA adolescent mouse showed an antisocial behavior, including reduced social interaction and aggressive attitude.Beside the major symptoms, ADHD children exhibit cognitive impairments and short-term memory deficits35. SHR shows several major ADHD-like symptoms such as hyperactivity36, impulsivity and poor attention37. However, SHR rats also show hypertension that is not reported in patients with ADHD14, thus making difficult to disassociate the effects of the two disorders. Indeed, hypertension is a potential confounding factor for SHR as a model for ADHD, suggesting that altered norepinephrine (NE) transmission39 may contribute to hypertension rather than hyperactivity10. Moreover, the WKY control often shows low activity levels, and has even been suggested as a model of depression42.Very few animal models of ADHD have been able to mimic multiple deficits at the same time. In the early 1960s, the spontaneously hypertensive rat (SHR) was developed by inbreeding Wistar-Kyoto (WKY) rats43. DAT KO mice show hyperlocomotion in novel environment43 and impaired learning and memory44. However, DAT KO mice display extremely elevated dopamine levels in the striatum and nucleus accumbens45 unlike ADHD patients47. Moreover, testing this model predictive validity with psychostimulants is impossible because of the absence of the DAT protein, the primary target of these drugs48. Other mouse genetic models have been proposed but lack face and/or predictive validity, e.g. the Coloboma Mutant Mouse49.Another genetic model is the mouse strain lacking the dopamine transporter (DAT KO)51. Furthermore, multiple lines of evidence recently supported the view that neuroanatomical alterations exist in ADHD patients. Studies have reported decreased brain volume in patients with ADHD, slowed maturation and reduced connectivity in the prefrontal cortex, anterior cingulate cortex, basal ganglia, and cerebellum13. In particular, the dorsal cingulate cortex, which plays a key role in the modulation of attention and executive functions52, appears to be dysfunctional in patients with ADHD4. Moreover, lateral prefrontal development in children with ADHD is delayed by several years53, and the anterior cingulate cortex is thinner in adults with ADHD54. Studies using functional magnetic resonance imaging in ADHD children sitting still or performing a continuous task also showed smaller sizes of DA target areas, including the prefrontal cortex and striatum and deficits in the basal ganglia55. Our data demonstrate that 6-OHDA mouse model of ADHD also exhibits such anatomical characteristics of ADHD, including decreased in brain weight and volume brain, smaller sizes of DA target areas, cortical thickness and abnormalities in dorsal anterior cingulate neurons.Hyperactivity in human subjects with ADHD is accompanied by decreased dopamine in striatum, prefrontal cortex, septum, midbrain and amygdala56, attention57, and working memory58 in ADHD patients. Comparable findings have been obtained in rats59. We show here that therapeutic-equivalent doses of Mph effectively attenuate the ADHD-like symptoms in 6-OHDA mouse model. Mph reduces hyperactivity of 6-OHDA mice. Moreover, pre-treatment with Mph 30 min before testing 6-OHDA mice in 5-CSRTT resulted in increased attention. The PCA also confirms that Mph is most effective on both the major and discrete symptoms. In contrast, excessive DA stimulation might cause PFC dysfunction leading to impaired inhibition of undesirable behavior, and a deficit in sustaining attention60. This can explain the opposite effects of Mph observed in sham group.Animal models of ADHD should be capable of predicting therapeutic effects in patients. Human studies have shown that Mph increases impulse controlMice are nocturnal animals and displayed their main activity peak at the beginning of the dark phase. They have more locomotor activity during night time than they would during the day. However, in the literature, most protocols use a normal and non-inverse light–dark cycle. All behavioral tasks were performed here at the same period (morning or afternoon) for all animals used to avert any circadian related fluctuation in the performance of the animals.61. However, ADHD symptoms are the result of several dysfunctional loci interacting within a neural network to produce observable behaviours. Nevertheless, 6-OHDA lesion, although artificial, has comparable behavioural consequences and hence has important implications for research on the specific hypothetical construct61. The fact that so many of the ADHD symptoms can be simulated highlights the possibility that the syndrome may have multiple aetiologies, which however may impinge on common neural systems. These systems respond to a common pharmacological treatment, e.g. psychomotor stimulants. Hence, it is possible that the 6-OHDA mouse model reproduces the symptoms of the human disease through the dysfunction of this common target system of the pharmacological treatment.Neurochemical lesion studies have been able to assign specific roles in the regulation of behaviour to discrete brain areas, circuits and neurotransmitter systems, and to mirror the effects of dysfunctional anatomical loci as seen in humans with ADHD65. This reveals a profound compensation and attempts to maintain homeostasis in the residual dopamine terminals in adulthood.Because psychostimulants, which increase catecholamine neurotransmission, have been the primary ADHD treatment for decades, clinicians and researchers conjectured that hypodopaminergic function is the neurobiological mechanism underlying ADHD. However, a feature of the 6-OHDA rat model of ADHD is the hyperactivity of the remaining dopaminergic system, and changes in dopamine receptor expression and function, even if the overal effect is a decrease of dopaminergic transmission66. Behavioral studies using noradrenergic drugs on animal models indicate that norepinephrine transmission does, indeed, affect ADHD symptoms, but the outcomes are mixed. Enhancing norepinephrine transmission by blocking the NET improves hyperactivity in neonatal 6-OHDA-lesioned rats15. From these studies, one cannot infer a causal relationship between increasing/decreasing norepinephrine neurotransmission and severity of ADHD symptoms. Instead, these studies suggest that norepinephrine has dual effects on ADHD-like behaviors61.While both dopamine and norepinephrine are known to regulate motor activity, attention, learning, and cognition, dopamine has been the focus of ADHD research. In the neonatal 6-OHDA-lesioned rat model, selective dopamine depletion is achieved by pretreatment with desipramine, which protects noradrenergic nerves. This model illustrates that dopamine depletion alone is sufficient to produce ADHD-like behaviors63. Conversely, an increase in serotonergic transmission via serotonin agonist m-CPP or SERT blocker, fluoxetine, greatly reduces hyperactivity68.Currently no serotonergic medications are prescribed in the treatment of ADHD. most studies using animal models of ADHD suggest that serotonin acts to compensate for aberrant dopamine and/or norepinephrine signaling. 6-OHDA-lesioned rats exhibit serotonergic hyperinnervation and the elimination of serotonergic hyperinnervation by administration of the selective serotonergic toxin 5,7-DHT greatly potentiates hyperactivity69. The use of 6-OHDA animal models of ADHD can inform on the cognitive functions sub-served by the lesioned area. It aids to a better understanding of this disorder by parceling the ADHD syndrome into distinct comorbid deficits and provides a valid and suitable animal model for pharmacological tests. We demonstrated in the present study that neonatal 6-OHDA mouse model of ADHD is a valid and reliable model for pre-clinical studies on this neurodevelopmental disorder.Although animal models created by the use of neurotoxins do not inform about the causes of ADHD, they are useful tools for studying the contribution of specific brain areas or circuits to cognitive processes that are affected by this pathologySwiss male mice, bred in the central animal facility of Cadi Ayyad University, Marrakech, Morocco. After birth, mice (weighing 4 g) were housed with their mothers in litters and kept under constant temperature conditions (22 °C ± 2), using a 12 h light/12 h dark cycle (lights on at 7 am), with water and food ad libitum. The study received approval of the Council Committee of research laboratories of the Faculty of Sciences, Cadi Ayyad University. All procedures were conducted in accordance with the approved institutional protocols and within the provisions for animal care and use prescribed in the scientific procedures on living animals, European Council Directive (EU2010/63). All efforts were made to minimize any animal suffering.We used 60 12. Pups received 6-OHDA hydrobromide  or vehicle in one of the lateral ventricles after desipramine hydrochloride pretreatment , under hypothermal anesthesia. The site of injection was set at 0.6 mm lateral to the medial sagittal suture, 2 mm rostral to the lambda and 1.3 mm in depth from the skull surface. 60 mice (P5) were divided on Sham and 6-OHDA groups (n = 30/each group). Each experimental animals (sham and 6-OHDA) were divided into three sub-groups , Mph 3.0 mg/kg and 5.0 mg/kg). After injection, 20% of the lesioned mice died before weaning, whereas 60–80% developed hyperactivity together with dopamine (DA) depletion . Mice that did not meet these 2 criteria were excluded from the data analysis.Intracerebroventricular injection of 6-OHDA was performed at P5 following published protocolion Fig. 1–2. Mice72. The injection was given once before open field test, elevated plus maze test, social interaction test and then before the training day (2nd day) of the novel object recognition test  45 min prior to test sessions for all the behavioral tests. The Mph doses were chosen based on previous studiesest Fig. .Figure 675. The slides were visualized with an Olympus microscope and images were acquired with an Olympus D72 camera.At the end of the Mph test, in the 5-CSRTT, the saline mice of both groups  were used for histology. Tissue preparation, Nissl staining, Golgi Cox staining and TH Immunohistochemistry were assessed as described previously78. For the operant test, we selected only saline mice from sham and 6-OHDA groups  to evaluate attention and impulsivity (5-CSRTT training from P50)79. As used in various studies83, and reviewed by Robbins84, the percentage of correct responses, also termed response accuracy, reflects errors of commission without including errors of omission and is one of the two variables best accounting for attentional performance. The percentage of omissions (no response after stimulus presentation) is the second variable accounting for attention; it reflects detection failures. The number of premature responses is an index of impulsivity. The number of perseverative responses corresponds to another form of inhibitory deficit related to impulsive/compulsive behavior.Spontaneous activity (open field test) was assessed in all groups at P24. From P40, behavioral and cognitive deficits were tested in all groups see Fig. : anxietyst and 2nd weeks, ITI = 7 s; 3rd and 4th weeks, ITI = 10 s; 5th and 6th weeks, SD = 0.8 s; and 7th and 8th weeks, SD = 0.5 s. After the behavioral challenge, mice were habituated to saline injections for 1 week. During the pharmacological challenge, Mph (3.0 and 5.0 mg/kg) were injected twice a week before the testing session for five weeks , once the animals showed a stable performance in the task, the inter-trial interval (ITI) was increased (7–10 s) and the stimulus duration (SD) was decreased (0.8–0.5 s) to challenge impulsivity and attention, respectively Fig. . Each paeks Fig. . 0.9% sa85.Statistical analyses were conducted using SigmaPlot 11.0 software . Results were presented as mean ± standard error of the mean (SEM). The Student’s t-test was used for two-sample comparisons. For multiple sample comparisons, Two-Way ANOVA was performed, followed by Student-Newman-Keuls post-tests for behavioral analysis. Two-Way Repeated measure ANOVA followed by Holm-sidak Method post-tests was used to analyze the performance in the 5-CSRTT during baseline, ITI and SD manipulations. For the pharmacological challenges, the mean of all sessions of each 5-CSRTT parameters taken in two different sessions (vehicle and each drug dose) was used as within-subjects factor and the lesion as the between-subjects factor. A Holm-sidak Method post-tests was used to follow-up significant main effects and interactions. Principal component analysis was performed using R software (Ade4 package) based on the 20 behavioral variables that have been measured in this studysupplementary data


Reduced executive functions (EF) are commonly associated with developmental conditions , although EF seems to be typical in children with callous unemotional (CU) traits. Regulatory function (RF) is a proposed infant precursor that maps on onto factors driving later EF. Here, we first test whether RF is specifically and negatively associated with ASD and ADHD traits, but not CU traits. Second, we test whether RF can act as a protective factor, by moderating the association between infant markers and subsequent ASD and ADHD traits.N = 42) and low (N = 37) familial risk for ASD. Data come from the 14-month infant visit  and the 7-year visit .Participants were 79 infants at high  and ADHD inattention  but not hyperactivity  or CU traits . RF moderated the association between infant AOSI score and ASD traits, with a significant effect in those with low RF , not high RF . Similarly, for ADHD, infant activity level was associated with later ADHD inattention in those with low  but not high RF . For ADHD hyperactivity symptoms, activity level was predictive at both high and low levels of RF.Infant RF was negatively associated with later traits of ASD  contains supplementary material, which is available to authorized users. Executive functions (EF) refer to a set of skills including planning, online monitoring, inhibition and working memory, which support the ability to set and achieve goals . EF diffChildren with autism spectrum disorder (ASD) show high levels of co-occurring conditions including ADHD  and callDifficulties in EF have been extensively observed amongst children and adults with autism spectrum disorder (ASD) , particusuperior conflict monitoring ).In contrast to ASD and ADHD inattention, no association was found between infant RF and later CU traits. The difference in the association between RF and later CU traits compared to both ASD and ADHD inattention traits approached significance. This is consistent with previous findings that, unlike in other developmental disorders , EFs do Our second aim, following the hypothesis put forward by Johnson , was to For ADHD traits, infant activity level was associated with later ADHD inattention in those with low but not high RF. Children showing high infant activity levels but also strong EFs may have a greater ability to choose when and what to pay attention to, resulting in lower inattentive traits later in development. For ADHD hyperactivity, on the other hand, there was no evidence of moderation by RF, with activity level predicting later hyperactivity irrespective of RF. This may be because whilst children with high RF are able to control their behaviour at times, for example when concentrating on a task, they continue to manifest hyperactivity at other times, which results in parents forming a global opinion of activity levels being high. To test this hypothesis, it would be important for future studies to use objective measures of activity, such as accelerometer data.Whilst the current study set out to test a moderating role of EF, based on the hypothesis by Johnson , it is iThe current sample includes both high- and low-risk infants. Whilst the main effect of risk group is included in the analysis, the relatively modest sample size precludes testing more complex models with interactions between group and RF, as well as 3-way group × RF × infant marker interactions. It seems likely that the protective role of RF may work differently in those with and without familial ASD risk, and future studies should test this explicitly. Future more highly powered studies should also investigate the potential moderating role of sex. Bedford et al. (2016)  demonstrAnother limitation is the use of parent report measures for disorder traits, which likely share measurement error and could have been subject to rater bias. However, such ‘halo effects’, where parents form a global impression of a child and consequently rate them in a similar way across multiple domains, would be more likely to result in similar associations across all disorder traits, which we did not find. Future studies, with larger sample sizes, should use structural equation modelling approaches, such as bifactor models, to attempt to separate out the shared and distinct variance across disorder traits. Whilst an experimenter-led observational measure was used for early infant autism behaviours (AOSI), activity level was also parent rated, from the IBQ-R. It was chosen because it is the only available infant marker  that has been shown to specifically predict later ADHD traits, but as noted above, future studies should replicate this with objective measures such as accelerometer data.These results have potentially significant implications when thinking about targeting EF in interventions. Executive function difficulties are related to lower adaptive functioning in both ASD , 75 and In conclusion, our findings showed an association between infants’ regulation ability and both ASD and ADHD inattention traits in mid-childhood. We also found suggestive evidence in support of the hypothesis that strong regulation abilities  may have a protective effect across developmental disorders . Early mAdditional file 1:Supplementary Materials. (DOCX 24 kb)


Here we show that upon EGFR activation, 6PGD is phosphorylated at tyrosine (Y) 481 by Src family kinase Fyn. This phosphorylation enhances 6PGD activity by increasing its binding affinity to NADP+ and therefore activates the PPP for NADPH and ribose-5-phosphate, which consequently detoxifies intracellular reactive oxygen species (ROS) and accelerates DNA synthesis. Abrogating 6PGD Y481 phosphorylation (pY481) dramatically attenuates EGF-promoted glioma cell proliferation, tumor growth and resistance to ionizing radiation. In addition, 6PGD pY481 is associated with Fyn expression, the malignancy and prognosis of human glioblastoma. These findings establish a critical role of Fyn-dependent 6PGD phosphorylation in EGF-promoted tumor growth and radiation resistance.6-Phosphogluconate dehydrogenase (6PGD) is a key enzyme that converts 6-phosphogluconate into ribulose-5-phosphate with NADP 6-phosphogluconate dehydrogenase is commonly upregulated in cancers. Here, the authors show that activation of EGFR induces phosphorylation of this enzyme at Y481 to activate the pentose phosphate pathway, which consequently reduces ROS and accelerates DNA synthesis to promote tumor growth and radioresistance. These aberrant alterations in metabolism provide both excessive energy and metabolic intermediates that are necessary for the rapid growth of cancer cells2. Aerobic glycolysis, also known as the Warburg effect, is a typical example: even in the presence of ample oxygen, rather than taking advantage of mitochondrial oxidative phosphorylation, most cancer cells rely more on glycolysis to produce adenosine 5’-triphosphate (ATP) and metabolic intermediates for biosynthesis of macromolecules and subsequent cell proliferation3.The reprogramming of cellular metabolism commonly exists in many types of cancer cells4. For instance, glucose-6-phoshate (G-6-P), derived from glycolysis, enters the pentose phosphate pathway (PPP), which generates nicotinamide adenine dinucleotide phosphate (NADPH) and ribose-5-phosphate (R-5-P)4. In normal conditions, >80% of total cytosolic NADPH is used for biosynthesis, with most of these NADPH consumed by fatty acid synthesis5. NADPH is also a crucial antioxidant. In contrast, it can also be used to produce glutathione (GSH), which in turn eliminates reactive oxygen species (ROS) that is produced during cell proliferation and generated by other stimuli, such as ionizing radiation (IR) and radical-generating compounds7. Another product R-5-P is a precursor for de novo, as well as salvage pathway of nucleic acid biogenesis that is important for mitosis and DNA repair8.Enhanced aerobic glycolysis in transformed cells provides more intermediates to be utilized in glycolytic shunts+ to NADPH. This protein often functions as a homodimer9. Accumulating data suggest that 6PGD is hyperactive in different types of cancer cells and plays a fundamental role in tumor growth13. In lung cancer cells, depletion of 6PGD leads to accumulation of p53 and subsequent cell senescence13. 6PGD can also be acetylated in lung cancer cells, which activates 6PGD to produce NADPH and Ru-5-P, thereby promoting lipids and RNA synthesis and reducing ROS levels14. Moreover, Ru-5-P, generated by 6PGD, inhibits 5' adenosine monophosphate-activated protein kinase (AMPK) activity to promote fatty acid synthesis by disrupting upstream LKB1 complex15. However, whether 6PGD can be phosphorylated and how this phosphorylation contributes to cancer progression remains unknown.6-Phosphogluconate dehydrogenase (6PGD) is the third enzyme of the PPP that catalyzes the oxidative decarboxylation of 6-phosphogluconate (6-PG) to ribulose-5-phosphate (Ru-5-P) with concomitant reduction of NADP16. Substantial evidence suggests that EGFR plays a causal role in GBM pathogenesis and resistance to treatment17. However, how EGFR signaling reprograms cell metabolism to support GBM progression, especially the resistance to treatment, remains unclear.The epidermal growth factor receptor (EGFR) is frequently overexpressed in approximately 40% of glioblastoma (GBM). In approximately 50% of tumors with EGFR amplification, a specific EGFR mutant (EGFRvIII) can be detected. This mutant, which is generated from a deletion of exons 2–7 of the receptor, is constitutively active and highly oncogenicIn this study, we investigate the role of 6PGD phosphorylation in EGFR-promoted tumor growth and radiation resistance, highlighting the fundamental role of Fyn-dependent 6PGD phosphorylation in brain tumor development.+ , and infected these cells and human primary GSC11 GBM cells with the lentivirus expressing Flag-tagged 6PGD (Flag-6PGD). Immunoblotting analyses of immunoprecipitated Flag-6PGD with anti-phospho-serine, anti-phospho-threonine, or anti-phospho-tyrosine antibodies showed that 6PGD was phosphorylated at tyrosine, but not at serine or threonine, upon EGFR activation Fig. . Mass sp+ Fig. . So, we + Fig. . Collect18. So, we overexpressed HA-Src or HA-Fyn in HEK293T/EGFR cells stably expressing Flag-6PGD. Immunoblotting analyses of immunoprecipitated Flag-6PGD with anti-phospho-tyrosine and anti-6PGD pY481 antibodies showed that Fyn, but not Src, phosphorylated 6PGD Y481 , and transiently transfected HEK293T/EGFR or stably infected GSC11 cells with Flag-6PGD in the absence or presence of tyrosine kinase inhibitors, such as Afatinib (EGFR inhibitor), Ruxolitinib (JAK family inhibitor), Saracatinib (Src family inhibitor), and Amuvatinib (c-Kit inhibitor), respectively . We thus hypothesized that Y481 phosphorylation likely activates 6PGD by either fostering its dimerization or increasing its NADP+ affinity. We first tested whether EGF stimulation regulates 6PGD dimer formation in U87/EGFR cells co-transfected with Flag-6PGD and HA-6PGD in the absence or presence of EGF treatment. Coimmunoprecipitation analyses showed that EGF treatment did not change the amount of HA-6PGD precipitated by Flag-6PGD, suggesting that EGF does not regulate 6PGD dimerization  and the turnover rate (kcat) of purified recombinant 6PGD with or without Y481 phosphorylation. The data showed that Fyn greatly decreased Km value of 6PGD WT while Y481F mutation abrogated such function of Fyn  or GSC11 cells and rescued these cells with shRNA-resistant (r) Flag-6PGD WT or Y481F mutant Fig. . As show81F Fig. . In cont81F Fig. . R-5-P, 81F Fig. . FurtherPPP Fig. . In addiPPP Fig. .Fig. 46-We next investigated the biological function of 6PGD Y481 phosphorylation. Cell proliferation assays showed that 6PGD knockdown greatly inhibited cell proliferation of U87/EGFRvIII, which were completely restored by the rescued expression of r6PGD WT, but only partially (~50%) restored by that of r6PGD Y481F Fig. . SimilarTo determine the role of 6PGD Y481 phosphorylation in tumorigenesis, we intracranially injected endogenous 6PGD-depleted U87/EGFRvIII cells with rescued expression of r6PGD WT or Y481F into randomized athymic nude mice. Bioluminescence imaging of mice showed that tumor cells rescued with r6PGD WT elicited a rapid tumor growth, whereas the cells rescued with the expression of r6PGD Y481F had much slower tumor growth Fig. . Consist20. The PPP is central for the production of the reducing equivalents, NADPH that reduces glutathione via glutathione reductase, which converts reactive H2O2 to H2O by glutathione peroxidase6. So, we wondered whether EGF-induced 6PGD Y481 phosphorylation is involved in the resistance of tumor cells to IR. To test this hypothesis, we first examined ROS levels in 6PGD-depleted U87/EGFRvIII, U251/EGFRvIII, or GSC11 cells with rescued expression of r6PGD WT or Y481F in the absence or presence of IR treatment. The experiments showed that, before IR treatment, tumor cells rescued with r6PGD Y481F had similar ROS levels with the cells rescued with r6PGD WT. In contrast, after IR treatment, higher ROS levels were detected in the cells rescued with r6PGD Y481F than in the cells rescued with r6PGD WT .To define the clinical relevance of our finding that Fyn phosphorylates 6PGD Y481 upon EGFR activation, we performed immunohistochemistry (IHC) analyses in 117 glioma specimens using anti-6PGD pY481 and anti-Fyn antibodies. The antibody specificities were validated using IHC analyses of GBM specimens with specific blocking peptides or proteins , were compared between the patients with low 6PGD Y481 phosphorylation (0–170 staining) and the patients with high 6PGD Y481 phosphorylation (175–300 staining). Patients whose tumors had low levels of 6PGD Y481 phosphorylation had a median survival of 19 months, and patients whose tumors had high levels of 6PGD Y481 phosphorylation had a significantly lower median survival of 13 months Fig. . IDH mutTo further explore this relationship, we examined whether the levels of 6PGD Y481 phosphorylation correlates with the grades of glioma malignancy. Levels of 6PGD Y481 phosphorylation in samples from patients (40 cases) with low-grade diffuse astrocytoma  were compared with those in samples from patients (40 cases) with high-grade GBM. IHC analyses showed that lower levels of 6PGD Y481 phosphorylation were present in low-grade tumors than in GBM specimens Fig. . Most lo+-binding affinity to activate the PPP for NADPH production and nucleotides synthesis, which subsequently promotes tumor cell proliferation and radiation resistance  and dihydrolipoamide S-acetyltransferase (DLAT) acetylates 6PGD K76 and K294 to enhance 6PGD activity in lung cancer cells14. Altogether, these studies suggest that, in response to complex tumor microenvironment, 6PGD Y481 phosphorylation along with other PTMs regulates 6PGD activity and subsequently activate the PPP to maintain redox homeostasis and accelerate DNA synthesis, which ultimately enhances tumor cell proliferation and resistance to treatment.Metabolic enzymes are frequently regulated by various types of posttranslational modifications (PTMs), such as phosphorylation, acetylation, and ubiquitination. Among the PTMs, phosphorylation is especially important for protein function as this modification activates (or deactivates) almost half of the enzymes, thereby regulating their functionIn addition, IHC analyses of tumors from GBM patients indicate that 6PGD Y481 phosphorylation correlates with the malignancy and prognosis of GBM. This result implicates the prognostic potential of 6PGD phosphorylation in GBM.24. It has been recently reported that EGFR signaling regulates tumor cell metabolism to promote tumor growth. For example, EGFR activation increases pyruvate kinase isozymes M2 (PKM2) expression by activating protein kinase C epsilon type/nuclear factor kappa-light-chain-enhancer of activated B cells (PKCε/NF-κB) signaling pathway, which therefore promotes glycolysis and subsequent tumor cell proliferation25. EGFRvIII expression upregulates heterogeneous nuclear ribonucleoprotein A1 splicing factor to increase glycolytic gene expression26. EGFR/phosphoinositide 3-kinases (PI3K)/Akt signaling also activates SREBP-1, which upregulates ACC, FAS, and SCD1 to promote de novo fatty acid synthesis27. We found here that EGFR activation can enhance 6PGD activity to activate the PPP, which produces NADPH for detoxification of intracellular ROS and R-5-P for nucleotides synthesis.The EGFR and the EGF family of peptide growth factor have a central role in the pathogenesis and progression of various carcinoma types20. Radicals react with nearby DNA in a very short time, resulting in breakage of chemical bonds or oxidation of the affected DNA. EGFR-activated PPP produces a large amount of NADPH that provides the reducing equivalents for oxidation reduction involved in protecting against ROS-caused DNA damage, allowing GSH generation. In addition, de novo nucleotide biosynthesis is often upregulated in cancer cells28, as sufficient nucleotide pools are required to maintain DNA fidelity during replication29 and to bypass oncogene-induced senescence30. Multiple levels of interaction between purine biosynthesis and DNA damage response have been discovered31. Upon EGFR activation, increased rate of PPP yields elevated levels of R-5-P and consequently 5-phosphoribosyl-1-pyrophosphate (PRPP). The increases in PRPP then result in excess purine biosynthesis that provides sufficient nucleotide pools to support DNA damage repair. Collectively, these results show that EGFR signaling activates PPP through 6PGD to enhance the radiation resistance of tumor cells and provide molecular basis to overcome GBM radioresistance by inhibiting 6PGD phosphorylation.Although the activation of pro-proliferative and anti-apoptotic pathways by EGFR is an established concept, recent reports reveal a novel link between EGFR signaling and the repair of radiation-induced DNA double-strand breaks (DSBs). However, how EGFR signaling contributes to the radiation resistance of tumors remains elusive. In our study, we found that EGFR activation promotes Fyn-dependent 6PGD Y481 phosphorylation, which enhances 6PGD activity and activates pentose phosphate pathway to provide NADPH for ROS detoxification and nucleotides pool for DNA damage repair. Radiation-induced ionizations may act directly on the cellular component molecules (such as DNA) or indirectly on water molecules, causing water-derived radicals, including ROS or NOS32. Fyn localizes in the lipid raft micro-domains of the plasma membrane where it is associated with lipid raft proteins CD36 that facilitates long-chain fatty acid uptake in skeletal muscle and adipose tissue33. In addition, the loss of Fyn improves peripheral tissue insulin sensitivity by relieving a selective negative modulation of AMP kinase activity in adipose tissue and skeletal muscle34. However, whether or how Fyn directly regulates tumor cell metabolism remains unknown. Here we show that, upon EGFR activation, Fyn directly phosphorylates 6PGD and enhances 6PGD activity, which consequently activates the PPP.Fyn is a member of the large Src family of non-receptor tyrosine kinases. In GBM, Src and Fyn have been found to be effectors of oncogenic EGFR signaling, which has led to tumor invasion and cancer cell survival. Several studies have implicated Fyn in the regulation of insulin signaling through lipid raft-dependent signalingAntibodies: mouse monoclonal antibodies against Flag  and anti-Flag M2 affinity gel (A2220) were purchased from Sigma . Rabbit monoclonal antibodies against HA , β-actin , phospho-STAT3 Y705  and phospho-tyrosine (pTyr) , and rabbit polyclonal antibodies against Fyn  and phospho-EGFR Y1068  were obtained from Cell Signaling Technology . Rabbit polyclonal antibodies against 6PGD , GST , and Flag , and mouse monoclonal antibody against His  were purchased from Proteintech . Rabbit polyclonal antibody against phospho-serine (pSer)  was purchased from Millipore . Mouse monoclonal antibody against human IDH1-R132H  was purchased from Medical & Biological Laboratories . Mouse monoclonal antibody against phospho-threonine (pThr)  was purchased from Santa Cruz Biotechnology . The custom-designed rabbit polyclonal antibody against phospho-6PGD Y481 [CTNWTGHGGTVSSSS(pY)NA]  were obtained from Abclonal Technology .+(N0505), 6-phospho-d-gluconate (P7877), thiamine pyrophosphate (C8754-1G), and sodium arsenate dibasic heptahydrate (A6756-50G) were purchased from Sigma . d-Erythrose 4-phosphate sodium salt (G3117) and Cibacron blue agarose (sc-294028) were from Santa Cruz Biotechnology . 32P-labeled adenosine triphosphate (ATP) (NEG002250UC) was purchased from PerkinElmer . ATP (tlrl-atp) were purchased from InvivoGen .Reagents: puromycin (540411-100MGCN) and hygromycin (400052-20MLCN) were bought from Merck/Millipore . DNA transfection reagent PolyJet (100688) was purchased from Signagen Laboratories . GelCode Blue Stain Reagent (24590) was obtained from Pierce . Recombinant human Fyn (active) (F15-10G-10) and EGFR (active) (E10-11G-10) were purchased from SignalChem . Recombinant human Src (active) (ab51424) was obtained from Abcam . NADPKits: NADP/NADPH assay kit (MAK312-1KT) was obtained from Sigma. ROS assay kit (S0033) was obtained from Beyotime . CellTiter-Glo ATP quantification kit (G7570) was obtained from Promega.-1), and bFGF (10 ng ml-1)35. All cell lines tested negative for mycoplasma.U87 (TCHu138), U251 (TCHu58), and HEK293T (GNHu17) cells were obtained from the cell library of the Chinese Academy of Sciences and maintained in high glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Human primary GSC11 GBM cells were maintained in DMEM/F-12 50/50 supplemented with B27, EGF . PCR-amplified EGFR or EGFRvIII was cloned into pCDH-Blastcidin. 6PGD and Fyn mutations were generated using the QuickChange site-directed mutagenesis kit .The pGIPZ control was generated with the control oligonucleotide 5ʹ-CTCGCTTGGGCGAGAGTAA-3ʹ. pGIPZ 6PGD shRNA was generated with 5ʹ-AGGACTGTCTCCAAAGTTG-3ʹ oligonucleotide targeting the coding region of the 6PGD transcript. pGIPZ Fyn shRNA was generated with 5ʹ-TGAGTTTTCACAAAGGAGA-3ʹ oligonucleotide or 5ʹ-AGACAATGAGTACACAGCA-3ʹ oligonucleotide targeting the coding region of the Fyn transcript.r6PGD contains same sense mutations of c108a, t109a, c110g, and c111t in the coding region.Cells were seeded in 35 mm, 60 mm, or 100 mm plates and then transfected with indicated plasmids using PolyJet according to the manufacturer’s instructions.2, 0.1 mM NADP+. The change in absorbance at 340 nm owing to increase of NADPH was measured using BioTek Synergy Neo Multi-Mode Plate Reader .6PGD activity was measured at room temperature. 6PGD was immunoprecipitated from the lysates of glioma cells and subjected to 6PGD enzymatic activity assays in the reaction buffer containing 100 mM Tris (PH 7.4), 2 mM 6-phosphogluconate, 10 mM MgCl2. The final concentration of NADP+ was from 2/15 to 1/300 mM. The velocity was measured using BioTek Synergy Neo Multi-Mod Plate Reader . The Km and Vmax was calculated by nonlinear regression/Michaelis–Menten using GraphPad Prism software. The kcat was calculated using Vmax/(6PGD enzyme concentration).Kinetics of 6PGD was measured at room temperature. His-6PGD WT and His-6PGD Y481F were purified from bacteria and subjected to kinetics measurements in the reaction buffer containing 100 mM Tris (PH 7.4), 2 mM 6-PG, 10 mM MgCld-Glucose-1,2-13C2 and d-( + )-glucose were purchased from Sigma . Fructose-6-phosphate (F-6-P), G-6-P, R-5-P, and Ru-5-P were the products of Santa Cruz Biotechnology . Sedoheptulose-7-phosphate (S-7-P) was purchased from Cayman Chemical . Ammonium acetate and ammonium hydroxide were of liquid chromatography mass spectrometry (LC-MS) grade and purchased from Anpel Laboratory Technologies Inc .High performance liquid chromatography-mass spectrometry (HPLC-MS) grade acetonitrile and methanol are the product of Fisher Scientific. g and 4 °C for 15 min, 800 μl of supernatant was evaporated to dryness under nitrogen gas. The residue was reconstituted in 100 μl of 50% aqueous acetonitrile, and the supernatant was performed in UPLC-MS/MS analysis.The cell mixture was added 80% volume of methanol and processed by five cycles of 1-min ultra-sonication and 1-min interval in ice-water bath. After centrifugation at 16,000 -1.Ultra performance liquid chromatography-mass spectrometry/mass spectrometry (UPLC-MS/MS) analysis was performed on a Waters Acquity UPLC system  coupled to a Triple Quad™ 5500 tandem mass spectrometer . The samples were separated using a Waters UPLC BEH Amide column  with a linear gradient elution system of mobile-phase (A) 10 mM ammonium acetate and 0.1% ammonium hydroxide in water and (B) 10 mM ammonium acetate and 0.1% ammonium hydroxide in acetonitrile/water . The gradient elution conditions were 0–1 min, 95% B; 2 min, 70% B; 6 min, 60% B; 6.1 min, 50% B; 7 min, 50% B; 7.1 min, 95% B; 12 min, 95% B. The column temperature was 30 °C. The injection volume was 4 μl. The flow rate was maintained at 250 μl minThe analytes separated from column were ionized in an electro spray ionization source in negative mode (ESI–). Source temperature: 500 °C, curtain gas (CUR): 25 psi, ion source gas 1 (GAS1): 50 psi, ion source gas 2 (GAS2): 50 psi, collision gas (CAD): 8 psi, ion spray voltage (IS): –4500 V, entrance potential (EP): –10 V, collision cell exit potential (CXP1): –10 V. The dwelling time was set at 20 ms. The multiple reaction monitoring (MRM) was used to acquire data in optimized MRM transition (precursor > product), declustering potential (DP), and collision energy (CE) as Supplementary Table Analyst  was used to analyze data. Using the default parameters and assisting manual inspection to ensure the qualitative and quantitative accuracy of each compound, extract and output chromatographic retention time and peak area.-1) for 30 min. The precipitated complexes were boiled at 95 ℃ for 10 min. Flag-6PGD were separated from the complexes using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel and trypsinized36. In brief, peptides digested with a modified in-gel trypsin procedure were extracted by removing the ammonium bicarbonate solution, followed by washes with a solution containing 50% (v/v) acetonitrile and 5% (v/v) formic acid. The samples were then dried and reconstituted in 5 μl of solvent A . The peptides were analyzed by LC-MS/MS on a Q Exactive mass spectrometer .Immunoprecipitated Flag-6PGD using anti-Flag antibody from U87/EGFR cells treated with or without EGF . In al, 5 μCi 32P-labeled ATP or 20 µM ATP was added in the kinase buffer to start the reaction. The reactions were performed in a total volume of 50 μl at 37 °C for 30 min and then terminated by adding SDS-PAGE loading buffer.In brief, bacterially purified recombinant His-6PGD (2 μg) was incubated with HEK293T-purified WT or KD HB-Fyn (0.5 μg) or commercial GST-Fyn, GST-Src, or GST-EGFR  in kinase buffer  and loaded with 10 mM carboxy-H2DCFDA for 30 min. The cells were harvested, resuspended in PBS and analyzed using fluorescence-activated cell sorter (FACS).Intracellular ROS levels were measured using a ROS measuring kit. The amount of intracellular ROS was measured by detecting dichlorodihydrofluorescein, which is the cleavage product of carboxy-H+, 6 mM MgCl2, 5 mM sodium arsenate, 0.08 mM d-Erythrose 4-phosphate and 0.01% thiamine pyrophosphate, 2 μg glyceraldehyde-3-phosphate dehydrogenase, 1 μg transketolase. The reaction was carried out at room temperature for 30 min. Then 2 μg ribulose-phosphate 3-epimerase was added and the increase absorption of OD340 was measured.To determine cellular concentration of Ru-5-P, the cell lysates were centrifuged in a cold room at 4 °C for 10 min at maximum speed, and the supernatants were applied to Amicon Ultra tubes with 5 kDa cut off filter (Millipore). The final reaction buffer (100 μl) contains: 50 mM imidazole pH = 7.6, 10 mM NADIntracellular ATP concentration was measured using a CellTiter-Glo ATP quantification kit according to the instruction of manufacturer.+ binding ability of 6PGD was determined by the affinity of 6PGD to Cibacron blue agarose, which mimics NADP+. U87/EGFR cells transfected with Flag-6PGD in 60 mm dish were harvested and incubated with 10 μl Cibacron blue agarose at 4 °C for 30 min. After washing with cell lysis buffer for three times, Cibacron blue agarose was boiled and subjected to western blotting.The NADPpCold I-His-6PGD WT or Y481F was transformed into BL21-CodonPlus competent cells. His-6PGD proteins were extracted by sonication of these BL21 cells, which were subjected to isopropyl β-D-1-thiogalactopyranoside (IPTG) (0.5 mM) induction for 24 h at 15 ℃, followed by the purification using HisTrip HP column by The AKTA FPLC System . The column was washed using wash buffer  until the absorbance reached the baseline. The proteins were eluted by an imidazole gradient elution buffer . The proteins were further purified using Superdex 200 Increase 10/300 GL column by the AKTA FPLC System. The purified proteins were concentrated using Amicon® Ultra-4 centrifugal filter concentrator (Millipore) and quantitated by BCA protein assay kit (Pierce).HEK293T/EGFR cells were transfected with pCMV-Flag-6PGD WT or Y481F. Flag-6PGD proteins were immunoprecipitated from these cells by anti-Flag M2 affinity gel (Sigma). The immunoprecipitated complex were washed three times using hash wash buffer containing 0.1% SDS followed by elution with Flag peptides. The Flag peptides were removed and proteins were concentrated using Amicon® Ultra-0.5 centrifugal filter concentrator (Millipore) and quantitated by BCA protein assay kit (Pierce).In all, 1 μg His-6PGD WT and Y481F, or 2 μg Flag-6PGD WT and Y481F proteins were subjected to SDS-PAGE. The protein purity was verified by GelCode Coomassie blue stain reagent (Pierce) following the instructions of the manufacturer and western blotting. For Coomassie blue staining, the gel was washed with deionized water for 15 min and then incubated with GelCode stain reagent for 1 h. The reagent was next replaced with ultrapure water for 1 h. The gel image was obtained by gel imaging systems (Tanon).In vitro kinase assays were performed by incubating bacterial purified recombinant His-6PGD WT or Y481F with or without recombinant active Fyn. After the reaction, His-6PGD variants were pulldown using nickel NTA agarose beads. The beads were washed using wash buffer containing 20 mM imidazole for three times, and the proteins were eluted by an elution buffer containing 250 mM imidazole. Amicon® Ultra-0.5 centrifugal filter concentrator (Millipore) were used to concentrate and desalt the proteins.2). His and Flag peptides were used as the negative control. NADP+ was diluted to a final concentration of 1 mM . Temperature equilibration was allowed for 1–2 h till to 25 °C prior to the experiment. The calorimetric cell was washed three times using dialysis buffer . All solutions were thoroughly degassed by centrifugation before use.ITC assays were performed by the ITC200 Micro-calorimeter . His peptides, Flag peptides, purified His-6PGD variants, or purified Flag-6PGD variants were diluted to a final concentration of 50 μM . The titration enthalpy data were corrected for the small heat changes in titrations of small molecular solution into the dialysis buffer.His peptides, Flag peptides, or 6PGD variants were slowly injected into the calorimetric cell to avoid air bubbles. The experiment was conducted by consecutively injecting NADPU87/EGFR cells transfected with Flag-6PGD in 35 mm dish was harvested using 200 μl cell lysis buffer. Glutaraldehyde was added in 50 μl cell lysates and mixed to a final concentration of 0.025%. The cross-linking assay was carried out in room temperature for 15 min, and then 10 μl 1 M Tris-HCl (pH = 7.5) was added to stop the reaction.Cells were plated on glasses for 24 h and were treated with BrdU (20 μM) for 20 min. Then cells were washed and fixed in 4% paraformaldehyde (PFA) for immunofluorescence assays.4 cells in DMEM with 10% fetal calf serum were seeded in six-well plates. Cells were counted with Thermo countess II FL.In all, 5 × 10For colony formation assays, 5000 cells per well were plated in six-well plate in triplicates and cultured for 14 days before staining viable colonies with nitro blue tetrazolium (Sigma).6 cells were seeded in six-well plates. Twenty-four hours after X-ray radiation (10 Gy) treatment, cells were harvested with trypsin using 600 g centrifugation. Cells was stained with Trypan blue and counted using Thermo countess II FL.In total, 1 × 10+ ratios were determined by measuring NADPH/NADP+ concentrations according to the protocol of NADP/NADPH assay (Sigma). In brief, NADP+/NADPH assay is based on a glucose dehydrogenase cycling reaction, in which the formed NADPH reduces a probe into a highly fluorescent product. The fluorescence intensity of this product, measured at ex = 530 nm/em = 585 nm.NADPH/NADP5 (in 5 μl of DMEM per mouse) luciferase-expressing 6PGD-depleted U87/EGFRvIII cells with reconstituted expression of 6PGD WT or Y481F were intracranially injected into randomized 8-week-old female athymic nude mice. Briefly, a small hand-controlled twist drill that is 1 mm in diameter is used to make a hole in the animal’s skull. The cell suspension is drawn up into the cuffed Hamilton syringe. The needle of the Hamilton syringe is slowly lowered into the central hole of the guide screw until the cuff rests on the screw surface. The cell suspension is slowly injected into the mouse’s brain37. Five mice per group in each experiment were included.In Figs. -1d-luciferin (Xenogen) and subsequently anesthetized with isoflurane inhalation. Bioluminescence imaging with a CCD camera  was initiated 10 min after injection. Bioluminescence from the region of interest was defined manually. Background was defined using a region of interest from a mouse that was not given an intraperitoneal injection of d-luciferin. All bioluminescent data were collected and analyzed using IVIS software.Seven, 9, or 11 days after inoculation, the mice were intraperitoneally injected with 100 μl of 7.5 mg ml2/2. Data represent the mean ± SD of five mice.Eleven days after inoculation and bioluminescence imaging, animals were sacrificed, and the brain of each mouse was harvested, fixed in 4% formaldehyde, and embedded in paraffin. Tumor formation and phenotype were determined by histological analysis of H&E-stained sections. Calculation to formulate tumor volume was V = ab5 (in 5 μl of DMEM per mouse) luciferase-expressing 6PGD-depleted U87/EGFRvIII cells with reconstituted expression of 6PGD WT or Y481F were intracranially injected into randomized 8-week-old female athymic nude mice and then subjected to IR (γ) radiation (4 Gy) in 5 and 10 days after inoculation. Five mice in each group were included.In Figs. Four, 9, 12, 15, 17, 19 days after inoculation, bioluminescence imaging of mice was carried out as described above. Survival durations of the implanted mice were compared.The use of mice was in compliance with ethical regulations and was approved by the institutional review board at Institute of Biochemistry and Cell Biology.The tissue sections from paraffin-embedded human GBM and astrocytoma specimens were stained with antibodies as indicated. We quantitatively scored the tissue sections according to the percentage of positive cells and staining intensity. We rated the intensity of staining on a scale of 0–3: 0, negative; 1, weak; 2, moderate; and 3, strong. We assigned the following proportion scores: X means X% of the tumor cells were stained (0 ≤ X ≤ 100). The score (H-score) was obtained by the formula: 3 × percentage of strongly staining area + 2 × percentage of moderately staining area + 1 × percentage of weakly staining area, giving a range of 0–300. Scores were compared with overall survival, defined as the time from the date of diagnosis to death or last known date of follow-up. All patients received standard adjuvant radiotherapy after surgery, followed by treatment with an alkylating agent (TMZ in most cases). The use of human brain tumor specimens and the database was approved by the Institutional Review Board at XinHua Hospital School of Medicine and the institutional review board at First Affiliated Hospital of Wenzhou Medical College. Informed consent was obtained from all patients.t-test (two tailed). p < 0.05 was considered to be significant.We determined the significance of differences in the human glioma data using Pearson’s correlation test and Student’s Further information on experimental design is available in the Nature Research Reporting Summary linked to this article.Supplementary InformationSource DataReporting Summary


Sotos syndrome is a congenital overgrowth syndrome associated with intellectual disability. This study investigated communicative abilities of children with Sotos syndrome (n = 31), using the Children’s Communication Checklist, second edition. A cross-syndrome approach was used to establish the specificity of these abilities. Children with Williams syndrome (n = 34) were used as a comparison group. In both groups, the majority of participants had communicative impairment. Children with Sotos syndrome had an uneven pragmatic language profile and greater impairment with social relations, compared with restricted interests. Overall, children with Sotos syndrome had difficulties with both language structure and pragmatic language and a specific profile of relative difficulty with using nonverbal communication, using context-appropriate language and understanding peer relationships. Sotos syndrome is a congenital overgrowth syndrome with an estimated incidence of approximately 1 in 14,000 (Tatton-Brown and Rahman Cross-syndrome comparisons are useful for assessing similarities and differences between phenotypes associated with distinct syndromes and can lead to identification of unique characteristic features of syndromes. These comparisons enable the specificity of strengths and difficulties associated with a particular syndrome to be established and can inform understanding of specific factors to target for intervention. Williams syndrome (WS) is a congenital syndrome with a similar incidence to that of Sotos syndrome . In addition, effective communication requires the ability to understand how to use language appropriately, or pragmatic language (e.g. using context-appropriate language and understanding the communicative partners’ experience of the interaction). Language structure ability is not necessarily related to pragmatic language ability. For example, developmental language disorder (DLD) is associated with relative difficulty with structural aspects of language, compared with functional pragmatic language, whereas ASD is associated with relative difficulty with pragmatic aspects of language, compared with language structure  Bishop  is desigTo date, language structure skills and pragmatic language ability have not been explored within the Sotos syndrome population and understanding of the communicative abilities of individuals within this population is limited. It is important to identify the prevalence and profile of language and communication difficulties within the Sotos syndrome population as impairments may have social and educational implications. For example, language skills are critical for social interaction so may impact peer relationships and identification of language and communication difficulties will enable appropriate services and provisions to be identified for individuals with Sotos syndrome. In addition, comparison of the strengths and difficulties of children with Sotos syndrome with those of children with WS will enable the specificity of the language and communicative profile associated with Sotos syndrome to be established. Once specific impairments have been established, these can be explored further in order to identify factors which may underlie these impairments. Ultimately, this approach will enable optimal strategies and interventions to be devised to address specific impairments.The primary aim of this study was to investigate the communication abilities of children with Sotos syndrome and to identify relative communicative strengths and difficulties associated with this syndrome, as assessed by the CCC-2. It was hypothesised that children with Sotos syndrome would have significant communicative difficulties. A secondary aim of this study was to compare the communicative abilities of children with Sotos syndrome with those of a matched group of children with WS in order to determine whether the communicative profiles are syndrome-specific. This is the first cross-syndrome comparison of Sotos syndrome and WS.The sample comprised 31 children with Sotos syndrome  and 34 children with WS , matched on chronological age and sex. Mean age of the Sotos syndrome participants was 9.12 years  and mean age of the WS participants was 9.4 years . The parent/caregiver of each child completed the CCC-2. Families of children with Sotos syndrome were recruited via the Child Growth Foundation  and advertisements on a Sotos syndrome support group on social media. Families of children with WS were recruited via the Williams Syndrome Foundation .The CCC-2 is a standardised 70-item questionnaire designed to assess communicative abilities in children (4–16 years 11 months) and can be used to identify children with significant communicative problems. Age-appropriate norms are provided. Items are coded on a Likert scale to determine the frequency of communicative difficulties and communicative strengths . The CCC-2 has 10 subscales which assess: (A) speech; (B) syntax; (C) semantics; (D) coherence; (E) inappropriate initiation; (F) stereotyped language; (G) use of context; (H) nonverbal communication; (I) social relations; (J) interests. Each of the subscales has 7 items; 5 relate to communicative difficulties and 2 relate to communicative strengths. A General Communication Composite (GCC) score provides an indication of the communicative ability of a child and is calculated as the sum of scaled scores for subscales A–H. Based on the standardisation sample, a GCC score of 54 is equivalent to the 10th percentile and GCC scores < 28 are equivalent to < 1st percentile. In addition, a language structure score  and a pragmatic language score  can be calculated in order to directly compare language structure skills and pragmatic language skills. For both language structure and pragmatic language, scores > 24 indicate typical functioning, scores of 17–24 indicate borderline functioning and scores < 17 indicate impaired functioning. The CCC-2 has been found to be a valid measure for differentiating between individuals with typical communicative ability and those with significant communicative impairments and within the CCC-2 standardisation sample, internal consistency of the subscales ranged from 0.65 to 0.80 Bishop .t-test was used to compare overall communicative ability of children with Sotos syndrome and children with WS. The analysis revealed no significant difference between GCC scores for the Sotos syndrome participants  and GCC scores for the WS participants ; t(63) = 0.23, p = .820, d = 0.06, indicating that both syndromes are associated with a similar level of overall communicative ability.Communication ability was assessed on the basis of GCC scores, with GCC scores < 55 (≤ 10th percentile) indicating that a child has significant communicative difficulties. For participants with Sotos syndrome, GCC scores ranged from 1 to 57 (< 1st percentile–13th percentile), demonstrating that the majority of children with Sotos syndrome had communicative difficulties of varying severity, with only one participant scoring in the normal range. GCC scores for participants with WS ranged from 4 to 60 (< 1st percentile–15th percentile), indicating that the majority of children with WS had communicative difficulties and once again, only one participant scored in the normal range. An independent samples F = 0.34, p = .564, ηρ² = .005, indicating no discrepancy between language structure skills and pragmatic language skills overall within this cohort. There was no effect of group; F = 0.05, p = .820, ηρ² = .001 and no skill x group interaction; F = 3.18, p = .079, ηρ² = .048. This suggests that both children with Sotos syndrome and children with WS had a similar degree of impairment with language structure skills and pragmatic language skills.In order to determine whether children with Sotos syndrome had an uneven profile of greater difficulty with either language structure or pragmatic language, scores for these abilities were compared. The Sotos syndrome profile was also compared to the WS profile to establish any syndrome-specific differences. Language structure scores were calculated as the sum of scaled scores for subscales A, B, C and D for participants with Sotos syndrome  and participants with WS . Pragmatic language scores were calculated as the sum of scaled scores for subscales E, F, G and H for participants with Sotos syndrome  and participants with WS . A 2 × 2 (Group: Sotos/WS x Language skill: structure/pragmatic) mixed measures ANOVA revealed no main effect of skill; X2 = 0.44, p = .801, and no significant association between group and pragmatic language ability category, X2 = 3.40, p = .183. This indicates that, for both Sotos syndrome and WS, a similar proportion of individuals had impaired, borderline and typical language structure and pragmatic language abilities (see Table A Chi square test of independence was used to assess the association between group (Sotos/WS) and category of language ability . There was no significant association between group and language structure ability category, R2 = 0.64, F = 16.27, p < .001, and inspection of the beta weights revealed that both language structure skills  and age  were significant predictors of pragmatic language ability. The same analysis was used to assess variance in pragmatic language ability for participants with WS. The regression equation was significant; R2 = 0.25, F = 3.33, p = .033, and inspection of the beta weights revealed that both language structure skills  and age  were significant predictors of pragmatic language ability. This suggests that language structure skills and age are significant predictors of pragmatic language ability for both children with Sotos syndrome and children with WS. Although the predictors were the same in both groups, language structure skills and age explained more variance in pragmatic language ability for children with Sotos syndrome (64%) than children with WS (25%). This indicates that other factors may account for variability in pragmatic language ability within the WS population.Although children with Sotos syndrome had similar overall difficulty with both language structure and pragmatic language abilities, there was significant variability in level of functioning of these abilities within each group. In order to investigate this variability, multiple regression was used to establish the extent to which variance in pragmatic language ability is explained by language structure skills, age and sex. The regression equation was significant; F = 2.16, p = .099, ηρ² = .067, indicating that children with Sotos syndrome had similar ability in all four language structure skills assessed by the CCC-2.In order to determine whether children with Sotos syndrome had particular difficulty with specific aspects of language structure, a repeated measures ANOVA was used to compare scaled scores for the four language structure subscales: (A) speech (e.g. pronunciation), (B) syntax (e.g. use of pronouns), (C) semantics (e.g. differentiation of words which sound similar) and (D) coherence (e.g. description of a sequence of events). A scaled subscale score ≤ 5 is indicative of significant communicative problems. The analysis identified no effect of subscale; F = 3.61, p = .014, ηρ² = .054 but no significant subscale x group interaction; F = 1.23, p = .302, ηρ² = .019 and no significant effect of group; F = 0.45, p = .504, ηρ² = .007. As there was no significant subscale x group interaction, this suggests that children with these syndromes had similar difficulty with the language structure skills assessed by the CCC-2. Figure The language structure skills of children with Sotos syndrome were compared with those of children with WS in order to investigate syndrome-specific differences in these abilities. A 2 × 4  mixed measures ANOVA was used to compare scaled scores for the four language structure subscales. The analysis identified a significant main effect of subscale; F3, 89 = 3.61F = 11.32, p < .001, ηρ² = .274, indicating that children with Sotos syndrome had an uneven profile of pragmatic language skills. Post-hoc paired samples t-tests  were used to compare scaled scores for the four pragmatic language subscales. The significant comparisons were stereotyped language > use of context, t(30) = 5.67, p < .001, d = 1.02; stereotyped language > nonverbal communication, t(30) = 4.68, p < .001, d = 0.84; inappropriate initiation > use of context, t(30) = 4.60, p < .001, d = 0.83. There was a trend for inappropriate initiation > nonverbal communication, t(30) = 2.76, p = .01, d = 0.49. This suggests that, in terms of the pragmatic language profile, children with Sotos syndrome have greater difficulty with use of context and nonverbal communication, whereas inappropriate initiation and stereotyped language are less problematic.In order to determine whether children with Sotos syndrome had particular difficulty with specific aspects of pragmatic language, a repeated measures ANOVA was used to compare scaled scores for the four pragmatic language subscales: (E) inappropriate initiation , (F) stereotyped language (e.g. repeating back things other people have said), (G) use of context (e.g. consistency of communication skills across different situations) and (H) nonverbal communication . The analysis identified a significant effect of subscale; F = 1.33, p = .253, ηρ² = .021. There was a significant main effect of subscale; F = 17.58, p < .001, ηρ² = .218, and a significant subscale x group interaction; F = 4.86, p = .003, ηρ² = .072, indicating that both groups had uneven pragmatic language profiles but the nature of these profiles differed.The pragmatic language abilities of children with Sotos syndrome were compared with those of children with WS in order to determine the specificity of the Sotos syndrome pragmatic language profile. A 2 × 4  mixed measures ANOVA was used to compare scores for the four pragmatic language subscales. The analysis identified no significant effect of group; p < .013 required for significance) were used to compare scores for the four pragmatic language subscales between the groups. The comparisons revealed a trend for the Sotos syndrome participants to have better stereotyped language scores, compared with the WS participants, t(63) = 2.19, p = .032, d = 0.55. There were no significant differences between groups for inappropriate initiation scores, t(63) = 1.27, p = .210, d = 0.32; use of context scores, t(63) = 1.46, p = .148, d = 0.37; or nonverbal communication scores, t(63) = -1.05, p = .298, d = 0.26. This suggests that the Sotos syndrome pragmatic language profile is characterised by less difficulty with stereotyped language. Figure As there was a significant subscale x group interaction, post-hoc comparisons  = − 5.99, p < .001, d = 1.08, indicating that children with Sotos syndrome had greater difficulty with social relations, compared with restricted interests. In order to establish whether this profile of behaviours was syndrome-specific, a 2 × 2  mixed measures ANOVA was used to compare scaled scores for the ASD subscales between participants with Sotos syndrome and participants with WS. The analysis revealed a significant main effect of subscale; F = 27.74, p < .001, ηρ² = .306 and a significant subscale x group interaction; F = 8.79, p = .004, ηρ² = .122, indicating that both groups had uneven ASD subscale profiles but the nature of these profiles differed. There was no effect of group; F = 0.04, p = .835, ηρ² = .001.The CCC-2 has two subscales which assess behaviours typically associated with ASD; social relations (e.g. interaction with other children) and interests (e.g. preference for the same favourite activity). In order to determine whether children with Sotos syndrome had relative differences in these behaviours, a paired samples p < .025 required for significance) were used to compare scores for the two ASD subscales within the Sotos syndrome and WS participants. As reported above, participants with Sotos syndrome had significantly greater difficulty with social relations, compared with restricted interests. However, the same analysis with participants with WS revealed no significant difference between the social relations subscale and the interests subscale, t(33) = − 1.60, p = .120, d = 0.27. Overall, this suggests that children with Sotos syndrome tend to have less difficulty with restricted interests compared with social relations, and this profile was specific to the Sotos syndrome participants. Figure As there was a significant subscale x group interaction, post-hoc comparisons (using a Bonferroni correction bscale, t3 = − 1.6The primary aim of this study was to investigate communicative abilities of children with Sotos syndrome, using the CCC-2. A cross-syndrome approach was used to compare communicative abilities of children with Sotos syndrome with a matched group of children with WS in order to investigate the specificity of the communicative profile associated with Sotos syndrome. The findings indicate that both syndromes are associated with communicative impairment and a similar level of overall communicative ability. There were no relative differences between overall language structure skills and pragmatic language skills, indicating that children with Sotos syndrome have similar difficulty with both of these linguistic abilities. This was also observed for children with WS. Furthermore, language structure skills and age were significant predictors of pragmatic language ability for both syndromes but accounted for greater variation in pragmatic language ability for the Sotos syndrome participants. Within the Sotos syndrome group, children had an uneven pragmatic language profile and ASD profile but similar difficulty with all language structure abilities assessed by the CCC-2. Specifically, the pragmatic language profile was characterised by greater difficulty with use of context and nonverbal communication but relatively less difficulty with inappropriate initiation and stereotyped language. The ASD profile was characterised by greater difficulty with social relations, compared with restricted interests. Both the pragmatic language profile and ASD profile were specific to the Sotos syndrome participants.The present study included a sample of 31 children with Sotos syndrome and this is the largest study to date to explore communicative abilities within this population. Overall, children with Sotos syndrome had communicative difficulties, compared with typically developing peers of the same age, as evidenced by low GCC scores. Only one participant with Sotos syndrome had a GCC score > 10th percentile. Furthermore, there was no significant difference between GCC scores for children with Sotos syndrome and children with WS, indicating that these populations are associated with a similar degree of overall communicative ability. However, it is important to note that there was considerable variability in communicative ability within each group. Previous research has established that both Sotos syndrome and WS are associated with relative strength in verbal ability, compared with nonverbal reasoning ability, as assessed by standardised cognitive assessments (Lane et al. Previous research indicates that different conditions are associated with uneven profiles of language structure and pragmatic language abilities (Geurts and Embrechts Although previous research has reported that children with Sotos syndrome have communication impairment and language delays, the communicative profile had not been explored in detail (Finegan et al. Although children with Sotos syndrome had difficulty with both ASD subscales, participants had significantly greater impairment with social relations, compared with restricted interests. Difficulty with these behaviours is consistent with previous research indicating that Sotos syndrome is associated with increased prevalence of behaviours typically associated with ASD (Lane et al. Due to the rarity of the syndrome, the present study used a parental questionnaire to assess communicative abilities, in order to obtain a relatively large sample of children with Sotos syndrome. The advantage of this methodology is that parents spend a lot of time with their children so are familiar with abnormal communicative abilities which may not be observed during a clinical test or observation. However, it will be important for future research to further understanding of the social and communicative abilities associated with Sotos syndrome by using paradigms during live social interactions, in order to directly assess these skills in children with the syndrome. For example, this approach has been used to investigate social attention in children with autism (Hanley et al. The present study focused on children with Sotos syndrome but it will be important for future research to investigate language and communication in adults with Sotos syndrome. Previous research indicates that difficulties with social interaction, restricted interests and repetitive behaviours may be less severe in adulthood, compared with childhood, within the Sotos syndrome population (Lane et al. In summary, the findings from the present study demonstrate that the majority of children with Sotos syndrome have communicative impairment, compared with typically developing peers and that children within this population struggle with both language structure and pragmatic language. Children with Sotos syndrome have an uneven pragmatic language profile characterised by relative difficulty with use of context and nonverbal communication, compared with inappropriate initiation and stereotyped language. This pragmatic language profile was specific to Sotos syndrome. Overall, the findings inform understanding of specific communicative difficulties that are common in children with Sotos syndrome and demonstrate the need for support with language and communication development within this population. In particular, children with Sotos syndrome may require additional support with understanding and using nonverbal communication and using context-appropriate language and may have difficulty with peer relationships.