SUPPLEMENTARY INFORMATION

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1 Supplementary information S1 (table) Examples of genes associated with ASD and their main clinical phenotypes. Genes Observations Chromatin remodelling MECP2 Females with a mutation in the X-linked MECP2 have Rett syndrome, which is characterized by profound cognitive impairment, poor social communication skills and stereotypic hand movements 1. After a period of apparently normal development, a pervasive growth failure begins between 6 and 18 months of age, followed by a major regression of social communication abilities, and motor skills. Although girls have normal head circumference at birth, after the regression period, microcephaly is frequently reported 1,2. Rare cases of MECP2 haploinsufficiency have been reported in males, whereas duplication of MECP2 occurs in both males and females and is associated with early infantile hypotonia, delayed psychomotor development resulting in severe to profound ID, absent to very limited speech, and neurological symptoms, including abnormal gait, epilepsy and spasticity 3. MEF2C Patients with heterozygous de novo MEF2C mutations are mainly non-verbal and have severe ID. Most patients older than 3 years have seizures that are commonly associated with fever 4. Mild to severe hypotonia is often associated with major difficulties in learning to walk. Brain MRI explorations report abnormalities such as delayed myelinization, enlarged ventricles, hypoplastic or thickened corpus callosum 4. HDAC4 Patients with de novo deletions or truncating mutations of HDAC4 on chromosome 2q37.2 present with brachydactyly mental retardation syndrome (BDMR) 5. BDMR is characterized by dysmorphic features, including midface hypoplasia, broad face and nose, brachycephaly, frontal bossing, downturned lower lip and upslanted eyes. Patients also have decreased sensitivity to pain, ADHD, sleep abnormalities, autistic traits and show self injury. CHD8/CTNNB1 Patients carrying a de novo CHD8 mutation present with hypotonia, idiopathic developmental delay, cognitive impairment, lack of social reciprocity and mild dysmorphic features 6. Patients with mutations with beta-catenin (CTNNB1) display ASD, ID and microcephaly 7. CHD8 acts as a negative regulator of Wnt signaling pathway by regulating CTNNB1 activity 6. ADNP/ARID1B Patients carrying heterozygote de novo mutations of the activity dependent neuroprotective protein (ADNP) gene are diagnosed with ASD with mild to severe ID 8,9. Dysmorphic features are present and include a prominent forehead, high hairline, eversion or notch of the eyelid, broad nasal bridge, and thin upper lip. ADNP is part of the BAF complex (also called mswi/snf) that regulates chromatin remodelling. Any mutations of the component of this complex (SMARCB1, SMARCA4, SMARCA2, SMARCE1, ARID1A, and ARID1B) cause distinct, albeit overlapping, syndromic ID disorders. de novo mutations in ARID1B (also known as BAF250B) were found in patients with ASD and in patients with agenesis of the corpus callosum, ID and language defects 5. TBR1 Patients with T-brain-1 (TBR1) are diagnosed with ASD and ID 7,11,12. TBR1 is a critical neuron-specific transcription factor for forebrain development and a

2 binding partner of CASK, a synaptic proteins mutated in ASD and of FOXP2, a transcription factor mutated in language disorder. TBR1 mrna and protein expression levels are induced by neuronal activation (via NMDA and AMPA receptors) 13. The elevation of TBR1 expression is associated with GRIN2B upregulation in both mature neurons and adult brains. Functional experiments have shown that the TBR1 mutations identified in patients with ASD affect the binding of TBR1 to FOXP2 and CASK 14. Protein synthesis and degradation NF1 Patients with mutations in NF1 are diagnosed with neurofibromatosis, a syndrome characterized by neurocutaneous signs, such as cafe -au-lait spots, axillary freckling and cutaneous neurofibromas, and Lisch nodules 15. More than half of these patients are at risk for ASD and more than 25% receive a full diagnosis of ASD, but without ID 15. TSC1/TSC2 Tuberous sclerosis (TS) is a multi-system disorder characterized by widespread tubers in several organs. The majority of children and adolescents with TS have epilepsy, cognitive impairment, disruptive behaviours and ASD. Individuals with mutations in TSC2 have on average more severe symptoms than those with mutations in TSC1 16. FMR1 Males with a CGG expansion upstream of the X-linked FMR1 have a lower level or no FMRP and are diagnosed with fragile X syndrome. They present with mild facial dimorphism, including a long face, large ears, and prominent jaw and macroorchidism. They have mild to severe ID and approximately 30% have ASD 17. UBE3A Maternal duplications of chromosome 15q11-q13, encompassing UBE3A, are strongly associated with ASD. Deletions, mutations and uniparental disomy of the maternal copy of UBE3A cause Angelman syndrome, which is characterized by ID, movement or balance disorder, and severe limitations in speech and language. Patients with 15q11-q13 duplication display mild to profound intellectual disability, ASD or autistic-like behaviour, central hypotonia, minor dysmorphisms and seizures 18. CUL3 Two de novo mutations of the Cullin 3 (CUL3) gene were reported in patients with ASD 19,20. CUL3 mutations also cause pseudohypoaldosteronism type II (PHAII), a rare syndrome featuring hypertension, hyperkalaemia and metabolic acidosis. This protein is a component of the E3 ubiquitin ligase complex involved in the degradation of proteins that regulate electrolyte homeostasis. Synaptic and cytoskeletal proteins NRXN1-3 Patients carrying a heterozygous mutation of NRXN1 have a high risk of developing mild to severe ID (42%), language delay (75%), ASD (42%) and hypotonia (33%) 21. Homozygous mutations in NRXN1 have been involved in the Pitt-Hopkins-like syndrome characterized by severe ID, lack of speech and breathing anomalies, but with no dysmorphic facial features. Inherited microrearrangements affecting NRXN2 and NRXN3 have been reported in patients with ASD and in other psychiatric disorders such as schizophrenia 22,23. NLGNs Patients with mutations in neuroligins NLGN1, NLGN3 and NLGN4X present with Asperger s syndrome or autism without ID, or with ID without ASD 24,25. NLGNs participate to activity-driven synaptogenesis 26. SHANKs SHANK3 haploinsufficiency has been reported in more than 600 patients

3 affected with chromosome 22q13 deletion syndrome, also known as the Phelan McDermid syndrome. Patients with de novo truncating SHANK3 mutations or deletions have neonatal hypotonia, moderate to severe ID, absent to severely delayed speech, normal to accelerated growth, and minor dysmorphic features 27,28. Remarkably, patients carrying a SHANK3 duplication present with Asperger syndrome, ADHD or hyperkinetic disorders The clinical features associated with SHANK1 or SHANK2 mutations appeared less severe than those associated with SHANK3 28. De novo truncating mutation of SHANK3 may account for 2% of individuals with ASD and ID 28. Mutations in SHANK genes have been observed in patients with other psychiatric conditions, such as schizophrenia and bipolar disorder 32. CNTNAP/CNTN Inherited deletions and mutations of contactin (CNTN) genes have been associated with ASD and other syndromes 33, but their role in ASD could be more moderate than originally proposed 34. Deletions of CNTN4 could be responsible for the neurological symptoms of patients with 3p deletion syndrome including ID, ASD, microcephaly, growth retardation and facial dimorphism. Heterozygous CNTNAP2 mutations have been reported in patients with specific language impairment and ASD Homozygous CNTNAP2 mutations cause Pitt-Hopkins-like syndrome and cortical dysplasia focal epilepsy syndrome 38,39. Deletions of CNTNAP4 were reported in patients with ASD (with or without ID) 40. CACNA1C De novo mutations of CACNA1C cause Timothy syndrome, a rare autosomal dominant disorder characterized by physical malformations, as well as neurological and developmental defects, including long QT syndrome, heart arrhythmias, structural heart defects, and syndactyly (webbing of fingers and toes) 41. SCN1A/SCN2A Mutations of SCN1A and SCN2A have been found in patients with ASD 42,43. Dominant mutations in the sodium channel SCN1A cause the Dravet syndrome, a drug-resistant epilepsy that occurs in the first year of life of previously healthy children. The main clinical features are prolonged and repeated febrile and afebrile generalized or unilateral convulsive seizures. GRIN2A / GRIN2B Mutations in GRIN2A or GRIN2B cause variable neurodevelopmental disorders including schizophrenia, bipolar disorder, ASD and ID 44,45. GRIN2A and GRIN2B encode the NR2A and NR2B subunits of the N-methyl-D-aspartate (NMDA) receptor. CASK Male patients with mutations in the X-linked CASK gene suffer from ID and microcephaly with pontine and cerebellar hypoplasia or ID with or with or without nystagmus (involuntary eye movements). CASK is a calcium/calmodulin-dependent serine protein kinase and a member of the membrane-associated guanylate kinase (MAGUK) protein family. MAGUKs are cytoskeletal membrane scaffold that coordinates signal transduction pathways. CASK is also a binding partner of TBR1, a protein associated with ASD. ANK2/ANK3 Patients with ANK2 mutations have ASD but also can suffer from long QT syndrome or cardiac arrhythmia. Patients with ANK3 (also called ankyrin-g) have ASD and ID. ANK2 and ANK3 are localized to the synapse and to the Nodes of Ranvier where they bind to voltage gated ion channels. ANK3 lossof-function increases β-catenin levels in the nucleus, thereby promoting neural

4 progenitor proliferation. DYRK1A Patients with DYRK1A mutations have higher risk for ASD and microcephaly 7. The DYRK1A gene is located in the Down syndrome critical region (DSCR). DYRK1A is a proline-directed serine/threonine kinase that phosphorylates more than 20 substrates involved in the regulation of cytoskeletal proteins and synaptogenesis 46. References 1. Amir, R.E. et al. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl- CpG-binding protein 2. Nature Genetics 23, (1999). 2. Guy, J., Cheval, H., Selfridge, J. & Bird, A. The role of MeCP2 in the brain. Annu Rev Cell Dev Biol 27, (2011). 3. Ramocki, M.B. et al. Autism and other neuropsychiatric symptoms are prevalent in individuals with MeCP2 duplication syndrome. Ann Neurol 66, (2009). 4. Le Meur, N. et al. MEF2C haploinsufficiency caused either by microdeletion of the 5q14.3 region or mutation is responsible for severe mental retardation with stereotypic movements, epilepsy and/or cerebral malformations. J Med Genet (2009). 5. Ronan, J.L., Wu, W. & Crabtree, G.R. From neural development to cognition: unexpected roles for chromatin. Nat Rev Genet 14, (2013). 6. Bernier, R. et al. Disruptive CHD8 Mutations Define a Subtype of Autism Early in Development. Cell (2014). 7. O'Roak, B.J. et al. Multiplex targeted sequencing identifies recurrently mutated genes in autism spectrum disorders. Science 338, (2012). 8. Helsmoortel, C. et al. A SWI/SNF-related autism syndrome caused by de novo mutations in ADNP. Nat Genet 46, (2014). 9. Vandeweyer, G. et al. The transcriptional regulator ADNP links the BAF (SWI/SNF) complexes with autism. Am J Med Genet C Semin Med Genet 166C, (2014). 10. Pinto, D. et al. Convergence of genes and cellular pathways dysregulated in autism spectrum disorders. Am J Hum Genet 94, (2014). 11. Iossifov, I. et al. The contribution of de novo coding mutations to autism spectrum disorder. Nature 515, (2014). 12. De Rubeis, S. et al. Synaptic, transcriptional and chromatin genes disrupted in autism. Nature 515, (2014). 13. Chuang, H.C., Huang, T.N. & Hsueh, Y.P. Neuronal excitation upregulates Tbr1, a highconfidence risk gene of autism, mediating Grin2b expression in the adult brain. Front Cell Neurosci 8, 280 (2014). 14. Deriziotis, P. et al. De novo TBR1 mutations in sporadic autism disrupt protein functions. Nat Commun 5, 4954 (2014). 15. Plasschaert, E. et al. Prevalence of Autism Spectrum Disorder symptoms in children with neurofibromatosis type 1. Am J Med Genet B Neuropsychiatr Genet 168B, (2015). 16. Crino, P.B., Nathanson, K.L. & Henske, E.P. The tuberous sclerosis complex. N Engl J Med 355, (2006). 17. Budimirovic, D.B. & Kaufmann, W.E. What can we learn about autism from studying fragile X syndrome? Dev Neurosci 33, (2011). 18. Hogart, A., Wu, D., LaSalle, J.M. & Schanen, N.C. The comorbidity of autism with the genomic disorders of chromosome 15q11.2-q13. Neurobiol Dis 38, O'Roak, B.J. et al. Sporadic autism exomes reveal a highly interconnected protein network of de novo mutations. Nature 485, (2012). 20. Kong, A. et al. Rate of de novo mutations and the importance of father's age to disease risk. Nature 488, (2012). 21. Dabell, M.P. et al. Investigation of NRXN1 deletions: clinical and molecular characterization. Am J Med Genet A 161A, (2013).

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