Transcriptome signatures from discordant sibling pairs reveal changes in peripheral blood immune cell composition in Autism Spectrum Disorder

Notwithstanding several research efforts in the past years, robust and replicable molecular signatures for autism spectrum disorders from peripheral blood remain elusive. The available literature on blood transcriptome in ASD suggests that through accurate experimental design it is possible to extract important information on the disease pathophysiology at the peripheral level. Here we exploit the availability of a resource for molecular biomarkers in ASD, the Italian Autism Network (ITAN) collection, for the investigation of transcriptomic signatures in ASD based on a discordant sibling pair design. Whole blood samples from 75 discordant sibling pairs selected from the ITAN network where submitted to RNASeq analysis and data analyzed by complementary approaches. Overall, differences in gene expression between affected and unaffected siblings were small. In order to assess the contribution of differences in the relative proportion of blood cells between discordant siblings, we have applied two different cell deconvolution algorithms, showing that the observed molecular signatures mainly reflect changes in peripheral blood immune cell composition, in particular NK cells. The results obtained by the cell deconvolution approach are supported by the analysis performed by WGCNA. Our report describes the largest differential gene expression profiling in peripheral blood of ASD subjects and controls conducted by RNASeq. The observed signatures are consistent with the hypothesis of immune alterations in autism and an increased risk of developing autism in subjects exposed to prenatal infections or stress. Our study also points to a potential role of NMUR1, HMGB3, and PTPRN2 in ASD

Me-Talnetant and Osanetant Interact within Overlapping but Not Identical Binding Pockets in the Human Tachykinin Neurokinin 3 Receptor Transmembrane Domains

ABSTRACT Recent clinical trials have indicated that neurokinin 3 receptor antagonists (S)-(ϩ) - 6.51 F, although not affecting Me-talnetant, led to a significant decrease in affinity and potency of osanetant. The mutation N142 2.61 A, which abolished the potency and affinity of osanetant, led to a significant increase in the affinity and potency of Me-talnetant. The proposed docking mode was further validated using (S)-2-(3,5-bis-trifluoromethyl-phenyl)- N-[4-(4-fluoro-2-methyl-phenyl)-6-((S)-4-methanesulfonyl-3-methyl-piperazin-1-yl)-pyridin-3-yl]-N-methyl-isobutyramide (RO49085940), from another chemical class. It is noteworthy that the mutation F342 7.39 A caused an 80-fold gain of RO4908594 binding affinity, but the same mutation resulted in the complete loss of the affinity of Me-talnetant and partial loss of the affinity of osanetant. These observations show that the binding pocket of Me-talnetant and osanetant are overlapping, but not identical. Taken together, our data are consistent with the proposed docking modes where Me-talnetant reaches deeply into the pocket formed by transmembrane (TM)1, -2, and -7, whereas osanetant fills the pocket TM3, -5, and -6 with its phenyl-piperidine moiety. The tachykinin family comprises the neuropeptides substance P (SP; RPKPQQFFGLM-NH2), neurokinin A (NKA; HKTDSFVGLM-NH2), and neurokinin B (NKB; DMHD-FFVGLM-NH2), which share a common C-terminal sequence, FXGLM-NH2. SP, NKA, and NKB act as neurotransmitters or neuromodulators, and they elicit their effects through three types of neurokinin receptors: NK 1 R, NK 2 R, and NK 3 R, respectively. Neurokinin receptors (NKRs) belong to the superfamily of G protein-coupled receptors (GPCRs) that couple via G q/11 to the activation of phospholipase C, leading to elevation of intracellular Ca 2ϩ levels ABBREVIATIONS: SP, substance P; NK, neurokinin; NKR, neurokinin receptor; GPCR, G protein-coupled receptor; 3D, three-dimensional; Me-talnetant, (S) IP, inositol phosphates; h, human; TM, transmembrane; EC2, extracellular loop 2; I3, intracellular loop 3; HEK, human embryonic kidney; RT, room temperature; PBS, phosphate-buffered saline; CHO, Chinese hamster ovary; WT, wild type; HBSS, Hanks' balanced salt solution; 7TMD, seventransmembrane domain; SAR, structure-activity relationship(s); 3D, three-dimensional; AVP, arginine vasopressin; V 1 R, vasopressin 1 receptor. 0026-895X/08/7306-1736 -1750$20.00 MOLECULAR PHARMACOLOGY Vol. 73, No. 6 Copyright © 2008 The American Society for Pharmacology and Experimental Therapeutics 42754/3336330 Mol Pharmacol 73:1736-1750, 2008 Printed in U.S.A. - 1736 have been implicated in the pathology of psychiatric diseases such as depression, schizophrenia, and anxiety as well as other conditions, including asthma, pain, emesis, and arthritis Although talnetant clearly displayed a reversible and competitive mode of antagonism in the NKB-induced Ca 2ϩ mobilization at cloned hNK 3 R and in the senktide-induced contractions in rabbit isolated iris sphincter muscles 3 H]Me-talnetant and [ 3 H]osanetant bindings, [MePhe 7 ]NKB-stimulated IP formation, and Schild and kinetic analyses to identify the important molecular determinants of NKB, Me-talnetant, and osanetant binding pockets and to elucidate the antagonism mode of both antagonists on wild-type and mutated hNK 3 Rs. Furthermore, these experimental findings allow the construction of a homology model based on the X-ray crystal of bovine rhodopsin Materials and Methods Materials Construction of Point-Mutated hNK 3 Rs. cDNA encoding the human NK 3 R (accession no. P29371) was subcloned into pCI-Neo expression vectors (Promega, Madison, WI). All point-mutants were constructed using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions and using pCI-Neo-hNK 3 R as a DNA template. Complementary oligonucleotide primers (sense and antisense) containing the single site or double sites of mutations were synthesized by Microsynth AG (Balgach, Switzerland). The following polymerase chain reaction conditions were used for repeated extensions of the plasmid template: 95°C for 1 min and 20 cycles of 95°C for 30 s, 55°C for 1 min, and 68°C for 8 min using 50 ng of plasmid DNA, 100 ng of each primer, and 2.5 units of Pfu Turbo DNA polymerase (Stratagene). The entire coding regions of all positive point-mutants were sequenced from both strands using an automated cycle sequencer (Applied Biosystems, Foster City, CA). Cell Culture, Large-Scale Transient Transfection, and Membrane Preparation. Human embryonic kidney (HEK) 293-EBNA cells (Invitrogen, Carlsbad, CA) were adapted to grow in suspension in spinner flasks at 95 to 105 rpm. For transfection experiments, we used a modified medium (DHI special; Invitrogen) and for the gene delivery, we used transfection reagent X-tremeGENEQ 2 (Roche Applied Science, Rotkreuz, Switzerland), which consists of substances A and B. Cells were cultured to a density of 6 to 10 ϫ 10 5 cells/ml, centrifuged for 3 min at 600 rpm, and resuspended in DHI media. The cell density was adjusted to 5 ϫ 10 5 cells/ml, and the culture was incubated for at least 3 h before transfection. The transfection complexes were generated in 1/10 of the culture volume in DHI media at room temperature (RT). For 1 ml of culture, first 0.4 g of DNA was added to 0.1 ml of medium, mixed, after 2 min followed by 0.15 g of X-tremeGENEQ A, mixed, and followed after a further 2 min by 0.5 g of X-tremeGENEQ B. The mixture was incubated for 15 min at room temperature to allow DNA complex formation before it was added to the cells. Forty-eight hours after transfection, the cells were harvested and washed three times with ice-cold PBS, and then they were frozen at Ϫ80°C. The pellet was suspended in ice-cold 50 mM Tris, pH 7.4, buffer containing 10 mM EDTA (10ϫ volume) and homogenized with a Polytron homogenizer (Kinematica AG, Basel, Switzerland) for 30 s at 16,000 rpm. After centrifugation at 48,000g for 30 min at 4°C, the pellet was suspended again in ice-cold 10 mM Tris, pH 7.4, buffer containing 0.1 mM EDTA (10ϫ volume), homogenized, and spun again as described above. The pellet was resuspended in ice-cold 10 mM Tris, pH 7.4, buffer containing 0.1 mM EDTA and 10% sucrose (5ϫ volume). After homogenization for 15 s at 16,000 rpm, the protein content was measured using the bicinchoninic acid method (Pierce, Socochim, Lausanne, Switzerland), with bovine serum albumin as the standard. The membrane homogenate was frozen at Ϫ80°C before use. [ 3 H]Me-talnetant and [ 3 H]Osanetant Bindings. After thawing, the membrane homogenates were centrifuged at 48,000g for 10 min at 4°C, the pellets were resuspended in the binding buffer (50 mM Tris-HCl, 4 mM MnCl 2 , 1 M phosphoramidon, and 0.1% bovine serum albumin at pH 7.4) to a final assay concentration of 5 g of protein/well. Saturation isotherms were determined by addition of various concentrations of [ 3 H]Me-talnetant (0.005-10 nM) or [ 3 H]-osanetant (0.009-3 nM) to these membranes (in a total reaction volume of 500 l) for 75 min at RT. At the end of the incubation, membranes were filtered onto unitfilter (96-well white microplate with bonded GF/C filter preincubated 1 h in 0.3% polyethylenimine ϩ 0.3% bovine serum albumin; PerkinElmer Life and Analytical Sciences, Waltham, MA) with a FilterMate-96 harvester (PerkinElmer Life and Analytical Sciences) and washed four times with ice-cold 50 mM Tris-HCl, pH 7.4, buffer. Nonspecific binding was measured in the presence of 10 M SB222200 for both radioligands. The radioactivity on the filter was counted (5 min) on a TopCount microplate scintillation counter (PerkinElmer Life and Analytical Sciences) with quenching correction after addition of 45 l of MicroScint 40 (PerkinElmer Life and Analytical Sciences) and shaking for 1 h. Saturation experiments were analyzed by Prism 4.0 (GraphPad Software Inc., San Diego, CA) using the rectangular hyperbolic equation derived from the equation of a bimolecular reaction and the law of mass action, of radioligand and 10 concentrations of the inhibitory compound (0.0003-10 M). IC 50 values were derived from the inhibition curve, and the affinity constant (K i ) values were calculated using the Cheng-Prusoff equation is the concentration of radioligand, and K d is its dissociation constant at the receptor, derived from the saturation isotherm 3 H]osanetant. Binding kinetics parameters, K ob and K off values (observed on and off rates, respectively), were derived from association-dissociation curves using the one-phase exponential association and decay equations (Prism 4.0), respectively. K on , half-life and K d were calculated using the K on ϭ (K ob Ϫ K off )/[ligand], t 1/2 ϭ ln2/K, and K d ϭ K off /K on equations, respectively. Statistical significance was determined using a two-tailed t test (Prism 4.0). [ 3 H]Inositol Phosphates Accumulation Assay. [ 3 H]Inositol phosphates (IP) accumulation was measured as described previously Residue Numbering Scheme. The position of each amino acid residue in the seven transmembrane domain (7TMD) was identified both by its sequence number and by its generic numbering system proposed by Alignment and Model Building. The amino acid sequences of the human NK 3 R (accession no. P29371), rat NK 3 R (accession no. P16177), mouse NK 3 R (accession no. P47937), gerbil NK 3 R (accession no. AM157740), human NK 1 R (accession no. P25103), and human NK 2 R (accession no. P21452) were retrieved form the SwissProt database. These amino acid sequences were aligned to the sequence of bovine rhodopsin (accession no. P02699) using the ClustalW multiple alignment program (http://www.ebi.ac.uk/ clustalw/). A slow pairwise alignment using the BLOSUM matrix series Using this alignment and the X-ray structure of bovine rhodopsin Results talnetant concentrations of 0.005-10 nM and [ 3 H]osanetant concentrations of 0.009-3 nM) and best fitted to a one-site model for both radioligands Mapping the Binding Pocket of NK 3 Receptor Ligands 1739 the binding modes of Me-talnetant and osanetant, an alignment of the seven transmembrane helices of the whole NK family toward the transmembrane helices of bovine rhodopsin (Protein Data Bank reference code 1f88) was made. The inverse agonist of rhodopsin, 11-cis-retinal, was used as a template for the locations of Me-talnetant and osanetant. Amino acids, which were found 6.0 Å away from retinal in the X-ray crystal structure of rhodopsin Subsequently, one of our aims was to address the selectivity of Me-talnetant and osanetant for NK 3 R over NK 1 R and NK 2 R. Although it was not possible to generate all possible mutations of interest, we have chosen a subset of 12 mutants to obtain the information to learn which residues influence selectivity. According to the predicted docking poses, the residues at position 3.36, 6.51, and 7.39 that seem to be important factors for the selectivity of Me-talnetant and osanetant were mutated to the respective residues in hNK 1 R or hNK 2 R (V169M, Y315F, and F342M). Phe342 was additionally mutated to alanine, a decision based on the observation that the 4-phenyl substituent of RO4908594 seems to clash into Phe342. Hence, F342A could result in an affinity increase for this compound, an essential test in the validation of the docking poses. NK 1 R is not only characterized by having a methionine in position 7.39 but also the neighboring residue 7.38 differs from NK 3 R (serine in NK 3 R, isoleucine in NK 1 R). Therefore, the double-mutant S342I/F342M was also generated to correctly introduce the NK 1 R region in NK 3 R. The residue at position 7.38 is too far away from the ligands to make a direct interaction with Me-talnetant or osanetant. Nevertheless, although Ser341 is not predicted to be in direct contact with the docked ligands, it could cause a kink of the helix TM7 as a result of its hydrogen-bonding capabilities. Thus, an additional mutant, S341A, was generated. For the case where this mutation would influence Me-talnetant or osanetant binding or the above-mentioned double-mutant would point to an influence of position 7.38 onto binding, single point-mutations S341I (NK 1 R) and S342L(NK 2 R) would have to be tested because they could indirectly influence binding by a change of TM7 helix conformation. But these mutations will be no longer be needed in case that S341A does not change the binding affinities. Comparison ]-NKB, the mutants N138A, L232A, Y315A, and M346A showed 59.4-, 10.0-, 6.2-, and 14.1-fold (statistical significance of P ϭ 0.0009, P ϭ 0.0002, P ϭ 0.02, and P ϭ 0.006), respectively, lower affinity for [MePhe 7 ]NKB than the WT 3 H]osanetant competition binding assay. RO4908594 is a potent dual NK 1 R/NK 3 R antagonist that binds to hNK 1 R, hNK 2 R, and hNK 3 R, with K i values of 0.8 Ϯ 0.1, Ͼ10,000, and 1.6 Ϯ 0.1 nM, respectively Mapping the Binding Pocket of NK 3 Receptor Ligands 1741 TABLE 2 Effects of the mutations on ͓ 3 H͔Me-talnetant displacement by ͓MePhe 7 ͔NKB, osanetant, and SB222200 in the membrane preparations from HEK293-EBNA cells transiently expressing WT and mutated hNK 3 Rs K i and n H values for ͓ 3 H͔Me-talnetant binding inhibition by ͓MePhe 7 ͔NKB, osanetant, or SB222200 were calculated as described under Materials and Methods. Values are mean Ϯ S.E. of the K i calculated from three independent experiments, each performed in duplicate. The mutations that affected the affinity constants of ͓MePhe 7 ͔NKB, osanetant, and SB222200 in comparison with WT are shown in boldface type. Statistical significance was determined using a two-tailed t test. [MePhe 7 ]NKB have been measured in the presence of 0, 10, 30, and 100 nM Me-talnetant or 0, 30, 100, and 300 nM osanetant in CHO cells expressing transiently the WT and mutated hNK 3 Rs. As seen in Mapping the Binding Pocket of NK 3 Receptor Ligands 1743 As observed above The dissociation rates for [ 3 H]Me-talnetant and [ 3 H]osanetant binding to the WT receptor was determined by the addition of an excess amount of SB222200 after equilibrium (30 min and 1 h, respectively) was reached. The reversal of binding for both antagonists was complete, with t 1/2 values of 4.6 and 10 min, respectively ( 4.9 Ϯ 0. Docking of Me-Talnetant, Osanetant, and RO4908594 onto the NK 3 R-7TMD Binding Cavity. To visualize the mutation data, a 3D model of the hNK 3 R-7TMD using the atomic coordinates of bovine rhodopsin (Protein Data Bank reference code 1f88) was constructed. Mapping the Binding Pocket of NK 3 Receptor Ligands 1745 proposed for RO4908594 The proposed docking pose

Reproducibility via coordinated standardization:A multi-center study in a Shank2 genetic rat model for Autism Spectrum Disorders

Inconsistent findings between laboratories are hampering scientific progress and are of increasing public concern. Differences in laboratory environment is a known factor contributing to poor reproducibility of findings between research sites, and well-controlled multisite efforts are an important next step to identify the relevant factors needed to reduce variation in study outcome between laboratories. Through harmonization of apparatus, test protocol, and aligned and non-aligned environmental variables, the present study shows that behavioral pharmacological responses in Shank2 knockout (KO) rats, a model of synaptic dysfunction relevant to autism spectrum disorders, were highly replicable across three research centers. All three sites reliably observed a hyperactive and repetitive behavioral phenotype in KO rats compared to their wild-type littermates as well as a dose-dependent phenotype attenuation following acute injections of a selective mGluR1 antagonist. These results show that reproducibility in preclinical studies can be obtained and emphasizes the need for high quality and rigorous methodologies in scientific research. Considering the observed external validity, the present study also suggests mGluR1 as potential target for the treatment of autism spectrum disorders

Behavioral Characterization of the Novel GABA B Receptor- Positive Modulator GS39783 (N,NЈ-Dicyclopentyl-2- methylsulfanyl-5-nitro-pyrimidine-4,6-diamine): Anxiolytic-Like Activity without Side Effects Associated with Baclofen or Benzodiazepines

ABSTRACT The role of GABA B receptors in various behavioral processes has been largely defined using the prototypical GABA B receptor agonist baclofen. However, baclofen induces sedation, hypothermia and muscle relaxation, which may interfere with its use in behavioral paradigms. Although there is much evidence for a role of the inhibitory neurotransmitter GABA in the pathophysiology of anxiety, the role of GABA B receptors in these disorders is largely unclear. We recently identified GS39783 (N,NЈ-dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine) as a selective allosteric positive modulator at GABA B receptors. The aim of the present study was to broadly characterize the effects of GS39783 in well-validated rodent models for motor activity, cognition, and anxiety. The following tests were included: locomotor activity in rats and mice, rotarod and traction tests (including determinations of core temperature) in mice, passive avoidance in mice and rats, elevated plus maze in rats, elevated zero maze in mice and rats, stress-induced hyperthermia in mice, and pentobarbital-and ethanol-induced sleep in mice. Unlike baclofen and/or the benzodiazepine chlordiazepoxide, GS39783 had no effect in any of the tests for locomotion, cognition, temperature, or narcosis. Most interestingly, GS39783 had anxiolytic-like effects in all the tests used. Overall, the data obtained here suggest that positive modulation of GABA B receptors may serve as a novel therapeutic strategy for the development of anxiolytics, with a superior side effect profile to both baclofen and benzodiazepines

Investigating the factors underlying adaptive functioning in autism in the EU-AIMS Longitudinal European Autism Project.

Individuals with autism spectrum disorder (ASD) exhibit significant impairments in adaptive functioning that impact on their ability to meet the demands of everyday life. A recurrent finding is that there is a pronounced discrepancy between level of cognitive ability and adaptive functioning, and this is particularly prominent among higher-ability individuals. However, the key clinical and demographic associations of these discrepancies remain unclear. This study included a sample of 417 children, adolescents, and adults with ASD as part of the EU-AIMS LEAP cohort. We examined how age, sex, IQ, levels of ASD symptom and autistic trait severity and psychiatric symptomatology are associated with adaptive functioning as measured by the Vineland Adaptive Behavior Scales-Second Edition and IQ-adaptive functioning discrepancies. Older age, lower IQ and higher social-communication symptoms were associated with lower adaptive functioning. Results also demonstrate that older age, higher IQ and higher social-communication symptoms are associated with greater IQ-adaptive functioning discrepancy scores. By contrast, sensory ASD symptoms, repetitive and restricted behaviors, as well as symptoms of attention deficit/hyperactivity disorder (ADHD), anxiety and depression, were not associated with adaptive functioning or IQ-adaptive functioning discrepancy scores. These findings suggest that it is the core social communication problems that define ASD that contribute to adaptive function impairments that people with ASD experience. They show for the first time that sensory symptoms, repetitive behavior and associated psychiatric symptoms do not independently contribute to adaptive function impairments. Individuals with ASD require supportive interventions across the lifespan that take account of social-communicative ASD symptom severity. Autism Res 2019, 12: 645-657. © 2019 The Authors. Autism Research published by International Society for Autism Research published by Wiley Periodicals, Inc. LAY SUMMARY: This study investigated key clinical and demographic associations of adaptive functioning impairments in individuals with autism. We found that older age, lower IQ and more severe social-communicative symptoms, but not sensory or repetitive symptoms or co-occurring psychiatric symptoms, are associated with lower adaptive functioning and greater ability-adaptive function discrepancies. This suggests that interventions targeting adaptive skills acquisition should be flexible in their timing and intensity across developmental periods, levels of cognitive ability and take account of social-communicative ASD symptom severity

Preference for biological motion is reduced in ASD: implications for clinical trials and the search for biomarkers.

BACKGROUND: The neurocognitive mechanisms underlying autism spectrum disorder (ASD) remain unclear. Progress has been largely hampered by small sample sizes, variable age ranges and resulting inconsistent findings. There is a pressing need for large definitive studies to delineate the nature and extent of key case/control differences to direct research towards fruitful areas for future investigation. Here we focus on perception of biological motion, a promising index of social brain function which may be altered in ASD. In a large sample ranging from childhood to adulthood, we assess whether biological motion preference differs in ASD compared to neurotypical participants (NT), how differences are modulated by age and sex and whether they are associated with dimensional variation in concurrent or later symptomatology. METHODS: Eye-tracking data were collected from 486 6-to-30-year-old autistic (N = 282) and non-autistic control (N = 204) participants whilst they viewed 28 trials pairing biological (BM) and control (non-biological, CTRL) motion. Preference for the biological motion stimulus was calculated as (1) proportion looking time difference (BM-CTRL) and (2) peak look duration difference (BM-CTRL). RESULTS: The ASD group showed a present but weaker preference for biological motion than the NT group. The nature of the control stimulus modulated preference for biological motion in both groups. Biological motion preference did not vary with age, gender, or concurrent or prospective social communicative skill within the ASD group, although a lack of clear preference for either stimulus was associated with higher social-communicative symptoms at baseline. LIMITATIONS: The paired visual preference we used may underestimate preference for a stimulus in younger and lower IQ individuals. Our ASD group had a lower average IQ by approximately seven points. 18% of our sample was not analysed for various technical and behavioural reasons. CONCLUSIONS: Biological motion preference elicits small-to-medium-sized case-control effects, but individual differences do not strongly relate to core social autism associated symptomatology. We interpret this as an autistic difference (as opposed to a deficit) likely manifest in social brain regions. The extent to which this is an innate difference present from birth and central to the autistic phenotype, or the consequence of a life lived with ASD, is unclear

The EU-AIMS Longitudinal European Autism Project (LEAP): design and methodologies to identify and validate stratification biomarkers for autism spectrum disorders.

BACKGROUND: The tremendous clinical and aetiological diversity among individuals with autism spectrum disorder (ASD) has been a major obstacle to the development of new treatments, as many may only be effective in particular subgroups. Precision medicine approaches aim to overcome this challenge by combining pathophysiologically based treatments with stratification biomarkers that predict which treatment may be most beneficial for particular individuals. However, so far, we have no single validated stratification biomarker for ASD. This may be due to the fact that most research studies primarily have focused on the identification of mean case-control differences, rather than within-group variability, and included small samples that were underpowered for stratification approaches. The EU-AIMS Longitudinal European Autism Project (LEAP) is to date the largest multi-centre, multi-disciplinary observational study worldwide that aims to identify and validate stratification biomarkers for ASD. METHODS: LEAP includes 437 children and adults with ASD and 300 individuals with typical development or mild intellectual disability. Using an accelerated longitudinal design, each participant is comprehensively characterised in terms of clinical symptoms, comorbidities, functional outcomes, neurocognitive profile, brain structure and function, biochemical markers and genomics. In addition, 51 twin-pairs (of which 36 had one sibling with ASD) are included to identify genetic and environmental factors in phenotypic variability. RESULTS: Here, we describe the demographic characteristics of the cohort, planned analytic stratification approaches, criteria and steps to validate candidate stratification markers, pre-registration procedures to increase transparency, standardisation and data robustness across all analyses, and share some 'lessons learnt'. A clinical characterisation of the cohort is given in the companion paper (Charman et al., accepted). CONCLUSION: We expect that LEAP will enable us to confirm, reject and refine current hypotheses of neurocognitive/neurobiological abnormalities, identify biologically and clinically meaningful ASD subgroups, and help us map phenotypic heterogeneity to different aetiologies