# Key role for gene dosage and synaptic homeostasis in autism spectrum disorders

Roberto Toro, Marina Konyukh, Richard Delorme, Claire Leblond, Pauline

Chaste, Fabien Fauchereau, Mary Coleman, Marion Leboyer, Christopher

Gillberg, Thomas Bourgeron

<figure><img src="image_1.png" /></figure>

► To cite this version:

Roberto Toro, Marina Konyukh, Richard Delorme, Claire Leblond, Pauline Chaste, et al. Key role for gene dosage and synaptic homeostasis in autism spectrum disorders. Trends in Genetics, 2010, 26 (8), pp.363-372. 10.1016/j.tig.2010.05.007. pasteur-01967134

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<figure><img src="image_2.png" /></figure>
Key role for gene dosage and synaptic homeostasis
in autism spectrum disorders

Roberto Toro<fnref n="1" />,<fnref n="2" />, Marina Konyukh<fnref n="1" />,<fnref n="2" />, Richard Delorme<fnref n="1" />,<fnref n="2" />,<fnref n="3" />,<fnref n="6" />, Claire Leblond<fnref n="1" />,<fnref n="2" />, Pauline Chaste<fnref n="1" />,<fnref n="2" />,<fnref n="3" />, Fabien Fauchereau<fnref n="1" />,<fnref n="2" />,<fnref n="4" />, Mary Coleman<fnref n="5" />, Marion Leboyer<fnref n="6" />,<fnref n="7" />,<fnref n="10" />, Christopher Gillberg<fnref n="8" />,<fnref n="9" /> and Thomas Bourgeron<fnref n="1" />,<fnref n="2" />,<fnref n="4" />,<fnref n="6" />*

<footnote n="1">¹ Human Genetics and Cognitive Functions, Institut Pasteur, 75015 Paris, France</footnote>

<footnote n="2">² CNRS URA 2182 “Genes, synapses and cognition”, Institut Pasteur, Paris, France</footnote>

<footnote n="3">³Department of Child and Adolescent Psychiatry, Hôpital Robert Debré, Assistance Publique-
Hôpitaux de Paris, 75019 Paris, France</footnote>

<footnote n="4">⁴University Denis Diderot Paris 7, 75005 Paris, France</footnote>

<footnote n="5">⁵Foundation for Autism Research</footnote>

<footnote n="6">⁶Foundation FondaMental French National Science Foundation</footnote>

<footnote n="7">⁷INSERM U955, IMRB, Psychiatry Genetic team, Faculty of Medicine, Université Paris Est-Créteil I, Créteil, France</footnote>

<footnote n="8">⁸Department of Child and Adolescent Psychiatry, Göteborg University, 41119 Göteborg, Sweden</footnote>

<footnote n="9">⁹Institute of Child Health, London, United Kingdom</footnote>

<footnote n="10">¹⁰, AP-HP, Henri Mondor-Albert Chenevier Hospitals, Department of Psychiatry, Creteil, F-94000, France</footnote>

<footnote>* Correspondence should be addressed to Thomas Bourgeron, PhD, Human Genetics and Cognitive Functions, Institut Pasteur, 25 rue du Docteur Roux, 75015 Paris, France. Tel: 33-1-40-61-32-16. Fax: 33-1-40-61-39-53. E-mail: thomasb@pasteur.fr</footnote>
Abstract

Autism spectrum disorders (ASD) are characterized by impairments in reciprocal social communication, and repetitive, stereotyped verbal and non-verbal behaviors. Genetic studies have provided a relatively large number of genes that constitute a comprehensive framework to better understand this complex and heterogeneous syndrome. Based on the most robust findings, three observations can be made: first, genetic contributions to ASD are highly heterogeneous with most likely a combination of alleles with low and high penetrance. Second, the majority of the mutations apparently affect a single allele, suggesting a key role for gene dosage in the susceptibility to ASD. Finally, the broad expression and function of the causative genes suggest that alteration of synaptic homeostasis could be a common biological process associated with ASD. Understanding the mechanisms that regulate synaptic homeostasis should shed new light on the causes of ASD and may provide means to modulate the severity of the symptoms.
## Introduction

The diagnosis of autism is based on impairments in two major domains – reciprocal social communication, and repetitive, stereotyped and ritualistic verbal and non-verbal behaviors. The term “autism spectrum disorders” (ASD) is used as a shorthand to refer to any patient that meets these diagnostic criteria. But beyond this unifying definition lies an extreme degree of clinical heterogeneity, ranging from profound to moderate impairments, but always with functional disability. Indeed, autism is not a single entity, but rather a complex phenotype thought to be caused by different types of defects in common pathways, producing similar behavioral phenotypes. The prevalence of ASD overall is about 1/100, but closer to 1/300 for typical autism¹. ASD are more common in males than females with a 4:1 ratio²,³. Twin and family studies have conclusively described ASD as the most “genetic” of neuropsychiatric disorders, with concordance rates of 82-92% in monozygotic twins versus 1-10% in dizygotic twins; sibling recurrence risk is 6%²,³.

From 15 to 70% of children diagnosed as suffering from ASD have intellectual disabilities⁴, and it is now understood that, like intellectual disability, autism symptoms can be caused either by gene mutations or by chromosomal aberrations (Box 1). In approximately 10-25% of the affected individuals, autism is “syndromic”, ie. occurring in a child with a known genetic or environmental toxin disorder, such as fragile X, tuberous sclerosis, neurofibromatosis, valproic syndrome, or autism caused by brain herpes simplex infection²,⁴.

In the last years, various independent studies and large scale international efforts have identified a growing number of candidate genes for ASD and suggest a set of mechanisms that could underlie the ASD phenotype.

## Genetic variations and the modes of inheritance of ASD

Due to the absence of classical Mendelian inheritance, ASD were first thought to be a polygenic trait involving many loci. Therefore, model free linkage studies, such as affected sib-pair analyses, were performed to identify susceptibility genes. Many genomic regions were detected, but only a restricted number of loci were replicated in independent scans (e.g., chromosome 7q31 and 17q11). To homogenize the genetic and phenotypic data and to gain higher statistical power, collaborative efforts
were initiated, such as the autism genome project (AGP), that genotyped 1496 sib-pair families using
the Affymetrix 10K single nucleotide polymorphisms (SNP) array <sup>5</sup>. Nevertheless, no genome-wide
significant loci could be detected, and the signals on chromosome 7q31 and 17q11 were lost. The
absence of relevant targets identified by linkage studies prompted geneticists to use an alternative
method: association studies with dense SNP arrays. In theory, association studies are sensitive to
allelic heterogeneity whereas linkage studies are not. Nevertheless, association studies provide major
advantages compared with linkage studies. First, studies can include large sample of patients since
they are not restricted to multiplex families with two or more affected children. Second, the genomic
regions associated with the trait are much narrower than in linkage studies, due to loss of strong
linkage disequilibrium between relatively close genomic regions. In addition, SNP arrays can be used
to detect structural variants such as copy number variations (CNVs) <sup>6</sup>.

By using these approaches, several genes were associated with ASD. A list of 190 genes is
available at AutDB, a public, curated, web-based, database for autism research
([http://www.mindspec.org/autdb.html](http://www.mindspec.org/autdb.html)). However, it should be noted that most of these genes remain
only candidates since their association was not always confirmed by replication and/or functional
validation. In Tables 1 and 2, we present a list of some of these genes, and we consider the
mechanisms that might be affected. Depending on the penetrance of the mutation on the risk for ASD,
we derive two main categories of genes or loci. In the first (Table 1), genes or loci appear to have a
high penetrance, but are mutated in a limited number of individuals (sometime a single individual). In
this category, variations are mostly composed of *de novo* or rare point mutations, CNVs and
cytogenetically detected deletions/duplications (Box 1). The second category of genes includes the so-
called susceptibility genes to ASD (Table 2). Here, the variations are mostly composed of SNPs or
inherited CNVs observed in the general population and associated with low risk for ASD. Especially,
in this category of genes, the association with ASD should be taken with great care, since the three
largest genome-wide association studies (GWAS) performed on more than 1000 patients in each study
could not detect the same genes associated with ASD <sup>7-9</sup>.

Two opposite models have been proposed to explain the inheritance of ASD. Some
researchers have considered autism as a polygenic trait (i.e., a group of patients with a relatively
homogeneous phenotype produced by multiple genes, each of low effect). Others have considered autism as a very phenotypically heterogeneous group of different disorders (autisms), thought to be caused by different genes in common pathways (in this model, eventually a single highly penetrant mutation would be sufficient to produce autism). The heterogeneous model was recently supported by both the absence of strong linkage and/or association loci replicated by whole genome scans and by the identification of apparently monogenic forms of autism, each time affecting a limited number of patients, 1-2% for the most replicated genes. Nevertheless, the polygenic model of autism cannot be excluded for several reasons. First, it is now well established that the clinical outcome of most of the monogenic disorders are actually modulated by additional genetic variations. Second, in polygenic traits such as blood pressure and height for example, the effect of a single common variant on the phenotype seems to be very low (OR<2) and requires large sample size (>15000) to be detected <sup>10</sup>. In ASD, the current genome scans – the largest were performed on cohorts of less than 2000 cases – might lack the statistical power necessary to detect such common alleles with low effect. Finally, the number of deleterious mutations within the human genome of one individual remains difficult to establish, but it is most likely that, in a large proportion of patients, the combination of multiple rare variants such as CNVs, coding and regulatory variations could affect specific biological pathways and therefore increase the risk for autism.

## Abnormal gene dosage in ASD

In recent years, enormous progress has been achieved in detecting rare and frequent structural variants of the human genome, such as deletions, duplications and inversions <sup>11</sup>. At least eight studies have searched for such genomic imbalances in patients with ASD <sup>5, 12-19</sup>. Differences in patient inclusion criteria, genotyping methodologies and algorithms to detect deletions and duplications make the comparison of these results difficult. Nevertheless, it seems that there is a significant increase in rare inherited and *de novo* CNVs in the ASD population compared with the general population. The overall rate of *de novo* CNVs in ASD could range from 5-10% compared with 1% in the general population <sup>15</sup>. Not surprisingly, the frequency of rare and *de novo* CNVs increases for simplex families with one affected child (7-10%) compared with multiplex families with at least two affected children (2-3%) <sup>15</sup>.
The presence of dysmorphic features in the patient also increases the odds of detecting a rare or *de novo* CNV in up to 27.5% ¹². Based on the current findings, the majority of the CNVs apparently affect only one copy of the gene (which can be either deleted or duplicated), suggesting that abnormal gene dosage or expression might play a key role in the susceptibility to ASD.

Several mechanisms could explain why abnormal gene dosage would increase the risk for ASD. First, deletions may reveal a mutation on the second allele (Fig. 1A), as it has been recently shown for *NRXN1* and *CNTNAP2*, two genes associated with ASD. Patients with Pitt-Hopkins-like syndrome (severe mental retardation, autistic behavior, epilepsy, and breathing anomalies) have been reported to carry one CNV affecting either *NRXN1* or *CNTNAP2*, and a deleterious point mutation affecting the same genes on the second allele ²⁰. Therefore, this apparently dominant trait was actually a recessive trait when a mutation screening of the remaining allele was performed. In theory, the second mutation could be present only in some tissues, maybe in specific parts of the brain (Fig. 1B). This situation has not been observed in ASD yet, but is well known in familial forms of cancer where both an inherited and a somatic mutation are required to develop the disease (the “two hit” or Knudson model). Gene dosage problems may also appear even if the second copy does not present a mutation, but is silenced by epigenetic hallmarks, as it has been observed to be the case in disorders affecting imprinted loci, such as Angelman or Prader-Willi syndromes ²¹. Similarly, allelic exclusion (i.e., the expression of a single allele in one cell) could turn off the unaffected allele in all cells or in specific cell types (Fig. 1C). Allelic exclusion was well characterized for immunoglobulins and olfactory receptors and is one of the mechanisms ensuring that a single antibody or receptor is expressed per cell ²². This mechanism, first considered as rare, could be in fact more frequent than originally thought ²³. Especially in the brain, allelic exclusion has been observed for cell adhesion molecules such as protocadherins ²⁴. If the cadherins and protocadherins associated with ASD ⁷, ¹⁴, ²⁵ were subject to allelic exclusion, then it would be possible that turning off the expression of the unaffected copy could totally deplete the cell of the protein product.

Second, abnormal gene dosage might by itself represent a risk factor for ASD. It is known from work in yeast that genes with high sensitivity to dosage are enriched in regulatory and structural proteins ²⁶, that need to interact with precise stoichiometry. Thus, lower or higher levels of a single
component of these complexes might disorganize the assembly of the machinery and alter its
biological function. In particular, scaffolding synaptic proteins such as SHANK involved in specific
protein-protein interactions are expected to be especially sensitive to dosage (Figure 1D). Finally,
some mutation associated with ASD could act as gain of function. Indeed, the knock-in mice carrying
the Nlgn3 R451C mutation – previously identified in one patient with autism and his brother with
Asperger syndrome <sup>27</sup> – displayed different synaptic and behavioral phenotypes compared to the
complete Nlgn3 knock-out <sup>28</sup>.

Abnormal level of synaptic proteins

Several lines of evidence indicate that mutations in genes regulating various aspects of synaptogenesis and neuronal circuit formation (Fig. 2) are associated with an increased risk for ASD. Among these, several genes seem to regulate the level of proteins at the synapse. Two X-linked genes, *MeCP2* and *FMR1*, are involved in autism “secondary” to Rett and fragile X syndromes, respectively. MeCP2 (Fig 2B) is a protein that directly and/or indirectly regulates neurotrophic factors, such as Brain Derived Neurotrophic factor (BDNF), by binding to methylated DNA <sup>29</sup>. Deletions or mutations of *MECP2* are associated with Rett syndrome in females, whereas duplications of *MeCP2* are associated with mental retardation and ASD in males and psychiatric symptoms, including generalized anxiety, depression, and compulsions in females <sup>30</sup>. FMRP (Fig 2A, B) is a selective RNA-binding protein that transports mRNA into dendrites and regulates the local translation of some of these mRNAs at synapses in response to activation of metabotropic glutamate receptors (mGluRs). In the absence of FMRP, there is an excess and a dysregulation of mRNA translation leading to altered protein synthesis dependent plasticity <sup>31</sup>.

Mutations of other genes associated with ASD seem to affect the level of synaptic proteins by
dysregulating overall cellular translation <sup>31</sup>. Patients with neurofibromatosis, tuberous sclerosis, or
Cowden/Lhermitte Duclos syndromes have higher risk than the general population to have ASD.
These disorders are caused by dominant mutations in the tumor suppressor genes *NF1*, *TSC1/TSC2*
and *PTEN* (Fig. 2). These proteins act in a common pathway as negative effectors of the rapamycin-
sensitive mTOR-raptor complex, a major regulator of mRNA translation and cellular growth in mitotic
cells³¹. The mutations observed in ASD have been predicted to enhance the mTORC1 complex, which
could lead to abnormal synaptic function due to an excess of protein synthesis. Interestingly loss of
*Tsc1/Tsc2* or *Pten* in mice results in neuronal hypertrophy ³², and patients presenting mutations in
*NF1*, *TSC1/TSC2* and *PTEN* have a higher risk for macrocephaly. Further modulation of the PTEN
and MTOR pathways is exerted by serotonin and the proto-ocongene cMET, two pathways that were
associated with ASD ³³, ³⁴.

Consistent with the hypothesis of a relationship between abnormal levels of synaptic proteins
in ASD, many studies have reported mutations in genes involved in synaptic protein ubiquitination,
including *UBE3A*, *PARK2*, *RFWD2* and *FBXO40* ¹³ (Fig. 2A). Protein degradation through
ubiquitination proceeds through the ligation of ubiquitin to the protein to be degraded. This post-
translational modification directs the ubiquitinylated proteins to cellular compartments or to
degradation into the proteasome. The ligation of ubiquitin is reversible and could be used to regulate
specific protein levels at the synapse. In mice, many proteins of the post-synaptic density, including
the mouse orthologs of the ASD-associated SHANK proteins, have been demonstrated to be targeted
by ubiquitination in an activity-dependent homeostatic manner ³⁵. Ubiquitination involves activating
enzymes (E1), conjugating enzymes (E2) and ligases (E3). Substrate specificity is usually provided by
the E3 ligases, which typically have substrate-binding sites. UBE3A (also called E6-AP) is an E3
ligase encoded by an imprinted gene (only expressed from the maternal copy) and is responsible for
Angelman syndrome ²¹. In ASD, *de novo* maternal duplications of chromosome 15q11-q13 including
*UBE3A* have been observed in 1-3 % of the patients ²¹. It is still not clear whether *UBE3A* alone
contributes to the risk of ASD, since other candidate genes are also duplicated on chromosome 15q11-
q13, however, its role at the synapse has been recently demonstrated in mice ³⁶, ³⁷. In cultured
hippocampal neurones *Ube3a* is localized at the pre- and post-synaptic compartments, but also at the
nucleus. Experience-driven neuronal activity induces *Ube3A* transcription, and then, Ube3A regulates
excitatory synapse development by controlling the degradation of Arc, a synaptic protein that
promotes the internalization of the AMPA subtype of glutamate receptors ³⁷. This might have many
consequences for synaptic structure, as suggested by Ube3a maternal-deficient mice, which exhibit
abnormal dendritic spine development, including spine morphology, number and length ³⁶, and a
reduced number of AMPA receptors at excitatory synapses<sup>37</sup>.

Finally, the transcription factor *MEF2C* (Fig. 2B), involved in the regulation of the number of synapses appears to be a risk factor for intellectual disability<sup>38</sup>, and could therefore also be associated with ASD. Taken together, the genetic results obtained in humans and the functional studies mostly obtained in mice suggest that different independent mechanisms could alter the level of synaptic proteins; however, the actual nature of the impaired synaptic function(s), and its association with ASD phenotype remains to be characterized.

## Abnormal formation of neuronal circuits in ASD

The main category of genes associated with ASD is related to the development and the function of neuronal circuits<sup>39</sup>. At the synaptic membranes, cell adhesion molecules, such as NLGNs and NRXNs (Fig. 2) are major organizers of excitatory glutamatergic and inhibitory GABAergic synapses, and contribute to the activity-dependent formation of neuronal circuits in mice<sup>40</sup>. Mutations identified in patients with ASD were found to alter the ability of NLGNs to trigger synapse formation in cultured neuronal cells<sup>41, 42</sup>. The disorders associated with NLGN-NRXN mutations can greatly vary among individuals, and this appears to be the case even for subjects of the same family, carrying the same mutation. Mutations of the X-linked *NLGN4X* have been associated with mental retardation<sup>43</sup>, typical autism<sup>42, 44</sup>, Asperger syndrome<sup>27</sup> and more recently with Tourette syndrome<sup>45</sup>. In one case, a *NLGN4X* deletion was observed in a male with normal intelligence and apparently no autistic features<sup>46</sup>. *NRXN1*, by contrast, has been implicated in disorders such as schizophrenia and Pitt-Hopkins-like syndrome, but has been also found in asymptomatic carriers<sup>20</sup>.

Interestingly, NLGN and NRXN might also play a role in social interaction in other species than humans without affecting overall cognitive functions. Mutant mice carrying a R451C *Nlgn3* mutation displayed an increased number of GABAergic synapses and inhibitory currents<sup>28</sup>, normal<sup>47</sup> to reduced social interaction<sup>28</sup> and a reduction of ultrasonic vocalization in pups<sup>47</sup>. The knockout mice for *Nlgn4* displayed reduced social interactions and ultrasonic vocalizations at the adult stage<sup>48</sup>. By contrast, mutant knockin *Nlgn3* and knockout *Nlgn4* displayed enhanced to normal learning compared with wild-type mice<sup>28, 48</sup>. Furthermore, in the mouse model for fragile X, an enhanced
Nlgn1 expression improved social behavior, whereas no effect on learning and memory was observed
⁴⁹. Finally, in the honeybee, sensory deprived animals had a lower level of Nlgn1 expression, but a
generally increased level of the Nlgn2-5 and Nrxn1 expression compared with hive bees ⁵⁰.

The postsynaptic density plays a major role in the organization and plasticity of the synapse,
and mutations affecting scaffolding proteins, such as SHANK2, SHANK3 and DLGAP2, are
recurrently found in ASD ¹⁹, ⁵¹, ⁵². Deletions at 22q13 and mutations of *SHANK3* could be present in
more than 1-2% of ASD patients (Box 1) ⁵¹⁻⁵³. Shank proteins are a family of three members, which
are crucial components of the postsynaptic density. Together with their binding partners, they have
been shown, *in vitro*, to regulate the size and shape of dendritic spines ⁵⁴. They also link glutamate
receptors to the cytoskeleton and variations in genes regulating cytoskeletal dynamics were associated
with mental retardation and ASD ¹⁹, ⁵⁵.

The role of neurotransmitter transporters and receptors in the susceptibility to ASD is still
unclear. Because of the abnormally high levels of serotonin in ASD patients ³³, the serotonin
transporter *SLC6A4* was extensively analyzed, and the results pointed toward dimensional rather than
categorical roles for *SLC6A4* in stereotypic behaviors ⁵⁶. For glutamate, only weak associations for
*GRIK2* were detected ⁵⁷, and a duplication of the X-linked *GRIA3* receptor gene was observed in a
patient presenting typical autism ¹². Concerning GABA, the most robust findings concern the
duplication of the GABA receptor subunit gene-cluster on chromosome 15q11-13 and the observation
of maternal over-transmission of a rare variant of the GABA(A) receptor beta3 subunit gene
(GABRB3) ⁵⁸.

Finally, proteins, related to axonal growth and synaptic identity, are now also suspected to
play a role in ASD. Semaphorins are membrane or secreted proteins (Fig. 2) that influence axon
outgrowth and pruning, synaptogenesis and the density and maturation of dendritic spines. SNPs
located close to the semaphorin *SEMA5A* were associated with ASD in a large cohort ⁸. Independently,
the level of *SEMA5A* mRNA was found to be lower in brain-tissue and B-lymphoblastoid cell lines
from patients with ASD compared with controls ⁵⁹. The contactin family of proteins is involved in
axonal guidance as well as in the connection between axons and glial cells, and ASD patients have
been found to have deletions of the contactin genes *CNTN3* and *CNTN4* and the contactin associated
protein CNTNAP2<sup>25, 60-63</sup>. In addition, inherited CNVs or SNPs have been found in other cell-adhesion
proteins – cadherins (CDH9, CDH10, CDH18) and protocadherins PCDH9 and PCDH10<sup>7, 14, 25</sup> –
which might contribute to the susceptibility to ASD by altering neuronal identity.

Abnormal synaptic homeostasis in ASD

Different homeostatic mechanisms allow neuronal cells to maintain an optimal level of neuronal activity despite global changes in the overall activity of the network <sup>64-66</sup>. Recent evidence suggests that homeostasis plays a role in the adaptation of synaptic plasticity by changing levels of activity <sup>35, 64</sup>, and might be also associated with the downscaling of synaptic weights during sleep <sup>65</sup>. During development and the first years of life, activity plays an important role in the refinement of brain connections, and many results suggest that these processes are under homeostatic control at the synapse <sup>67</sup>. The genes and the mechanisms that we have surveyed in this article might disrupt synaptic homeostasis at various levels <sup>68</sup>. The synthesis and degradation of different postsynaptic density proteins has been shown to vary as a function of activity <sup>35</sup>. Mutations in ubiquitin-dependent degradation could directly interfere with this process, as would also be the case if mutations are present in scaffolding genes such as the Shank family. Synaptic homeostasis has been shown to depend on local protein synthesis, on Ca<sup>2+</sup> concentration and on a tight regulation between the pre- and the post synaptic sides of the synaptic contact mediated by cell adhesion molecules such as NLGN and NRXN <sup>69</sup>. Finally, synaptic homeostasis is not independent from cellular homeostasis and therefore should be affected by mutations altering gene expression level as well as neuronal numbers and shape such as mutations related to the mTOR pathway.

If synaptic homeostasis is altered in ASD, environmental factors that influence this regulatory
process could also modulate its severity. As reviewed elsewhere <sup>33, 70</sup>, abnormal serotonin and/or
melatonin levels and altered sleep or circadian rhythms might constitute risk factors for ASD <sup>71</sup>. Sleep
has been proposed as an important mechanism to regulate synaptic homeostasis. During wakefulness
there appears to be a global increase in the strength of excitatory synapses, scaled down during sleep
to a baseline level <sup>65, 72</sup>, a mechanism that can play an important role in learning and memory <sup>73</sup>. In
addition to mutations of genes directly involved in synaptic processes, we have recently proposed that,
in some cases, ASD could result from the interplay between abnormalities in synaptic and clock genes,
and that restoring circadian rhythms might therefore be beneficial for the patients and their families ⁷⁰.

Most of the genes considered in this review are thought to be expressed throughout the brain,
however, neuroimaging studies seem to converge into a stereotypical network of brain regions where
differences between ASD and control populations can be detected. These two results would not need
to be in contradiction, if different brain networks were differently resilient to variations in synaptic
homeostasis (Fig. 3). From an evolutionary standpoint, brain networks involved in more recently
acquired cognitive skills, such as language or complex social behavior, might have less compensatory
mechanisms compared with more ancient biological functions that have been shaped by a much
stronger selective pressure.

Concluding remarks and perspectives

It is a matter of time for geneticists to be able to obtain whole genome sequences of ASD patients.
Exploring epigenetic alterations should be also more feasible in the near future, thanks to the
availability of brain tissue samples and stem cells from patients. Animal models based on genetic
results are now under scrutiny in many laboratories and the consequence of the mutations and their
reversibility is analyzed from cell to behavior. However, more than ever we need to recognize the
inherent heterogeneity of the genetic correlates of ASD. A true understanding of the relationship
between genetic mutations and ASD phenotype will not be possible if we persist in considering autism
as a binary value in our analyses. Advancement in the research on ASD requires the expertise from
different fields, but only clinicians and psychiatrists will be able to determine what we are actually
looking at (i.e. the autism phenotype or, rather, the different autism phenotypes). If to date common
variants seem to be difficult to identify in heterogeneous samples of less than 1000 families, future
studies should tell if increasing sample size or meta-analyses, phenotypic stratification, pathway
analyses and SNP x SNP interactions can identify common variants associated with sub groups of
patients with ASD.
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Box 1. Matched syndromes of reciprocal imprinting or deletions and duplications

A series of matched syndromes have been found which have aberrations in identical or very similar genomic regions. These syndromes usually contain subgroups with autistic features, but each matched pair of syndromes might have different symptoms in other clinical categories.

On chromosome 7q11, a microdeletion causes the Williams-Beuren syndrome, which is the opposite of autism: if anything, they are overly friendly and have strengths in language, face processing, and sociability. Strikingly, the same genomic region was found duplicated in several patients with severely impaired articulation and expressive speech abilities<sup>74</sup>.

On chromosome 15q11-13, maternal deletions cause Angelman syndrome (40-80% autism), which has mental retardation, handflapping and mild spasticity or ataxia<sup>13, 21</sup>. Paternal deletions or maternal UPD cause the Prader-Willi syndrome of hypotonia, obesity, short stature with small hands and feet and hypogonadotropic hypogonadism. Regarding duplications, maternal duplications are a relatively common cause of ASD with autistic features found in 40% of patients with the duplications<sup>21</sup>.

On chromosome 16p11.2, deletions and duplications of ~600kb are inherited or *de novo* and were identified in 0.5-1% of the patients with ASD <sup>14, 75, 76</sup>. The duplication of the same region is also associated with schizophrenia <sup>14, 75-77</sup>.

On chromosome 17p11.2, deletions cause Smith-Magenis syndrome characterized by infant hypotonia, short stature, a dysmorphic face, mental retardation, congenital cardiac defects, inverted melatonin cycles and often sleep disorders and self-abusive behaviors<sup>78</sup>. Duplication of the same genomic region causes the Potocki-Lupski syndrome where about two-thirds of the children meet ASD criteria<sup>79</sup>.

On chromosome 22q11.2, deletions are mostly *de novo* and encompass 1.5 to 3 Mb of genomic DNA including 24-30 genes. The patients present with the DiGeorge 1 syndrome, where 50% meet autism criteria<sup>80</sup>. Other individuals might have learning disabilities or schizophrenia<sup>80</sup>. Reciprocal duplications are mostly inherited from healthy parents and these children also have a higher risk for ASD with hypotonia and both mental and growth retardation<sup>81</sup>.
On chromosome 22q13.3, the microdeletion syndrome is characterized by normal to accelerated growth combined with neonatal hypotonia, minor dysmorphic features, absent to severely delayed speech and global developmental delay <sup>51</sup>. Up to 44% of these children meet autistic criteria and a duplication of the same region, including the *SHANK3* gene, was observed in a boy with Asperger syndrome who learned language preciously, in a girl with ADHD and in several patients with schizophrenia <sup>51, 52, 82</sup>.
## Figure legends

**Figure 1. Figure 1. Possible consequences of abnormal gene dosage.** Abnormal gene dosage seems to play a key role in the susceptibility to ASD. Several mechanisms can explain how abnormal gene dosage can alter brain development. In panel A, a gene has two alleles, A1 and A2 (in yellow and blue respectively, left column). The deletion of allele A1 could completely devoid the cell of the gene product if the other allele A2 is mutated (middle column) as it has been shown for patients with NRXN1 and CNTNAP2 deletions; or silenced by epigenetic hallmarks as in Angelman or Prader-Willi syndromes (right column). The second mutation could devoid the brain from the gene product only in specific regions (panel B) if, as in the "two-hits" or Knudson model, the first allele presented a germinal mutation while the mutation in the second allele (somatic mutation) occurred later during development. Another mechanism involves allelic exclusion, which has been already observed for cell adhesion molecules (panel C). For example, if the protocadherins involved in ASD were subject to allelic exclusion, brain cells would produce either allele A1 or A2 in the normal case (left side of panel C). If allele A1 was deleted, then no protein would be produced in the cells where A2 is silent, and only A2 would be present in the brain, produced by the remaining cells. Finally, an imbalance of gene product due to a deletion or a duplication could lead to disorganized protein complexes. Scaffold proteins (such as the SHANK family) at the post-synaptic density assemble with other proteins in a precise stoichiometry (panel D, left). Both the absence (middle) or excess (right) of protein could affect this equilibrium.

**Figure 2. Biological processes involved in ASD.** The genes associated with ASD appear to participate into three main biological processes. First (panel A), at the synapse cell adhesion proteins such as cadherins (CDH), protocadherins (PCDH), neuroligins (NLGN) and neurexins (NRXN) are involved in synaptic recognition and assembly. Within the postsynaptic density, scaffold proteins such as SHANK3 and DLGAP2 assemble the synaptic components and provide a link between membrane proteins and the actin skeleton. FMRP transports mRNA at the dendrites and regulates local translation of synaptic proteins. In the cytoplasm, the mTOR pathway regulates translation and is influenced by proteins such as PTEN, NF1, TSC1/TS2 and c-MET. The E3 ligase UBE3A is involved in the
targeting of synaptic proteins to the proteasome. Receptors for glutamate (GLUR) and GABA (GABAR) are playing a central role in producing excitatory and inhibitory currents, respectively. IMPP2L is a peptidase within the inner membrane of the mitochondria. Second (panel B), in the nucleus the methyl binding protein MECP2 and transcription factors such as MEF2C regulate the expression of neuronal genes involved in the formation of neuronal circuits and synaptic functions. The FMRP protein transports and regulates the translation of mRNA at the synapse. Finally (panel C), at the nodes of Ranvier proteins such as CNTN and CNTNAP2 organize the tight junctions between the axon and the myelinating glia. At the membrane or in the intercellular space, cell adhesion molecules and secreted proteins such as NRCAM or SEMA5A act as guidance cues for axonal outgrowth.

**Figure 3. Different resilience of brain networks to gene dosage.** Depending on their different resilience, the functional effects of abnormal gene-dosage could seem localized, even if the genetic abnormalities are widespread. Brain networks involved in evolutionary older biological processes might have developed more compensatory mechanisms than those supporting more recent cognitive functions. In the illustration we distinguish four possibilities, networks insensitive to gene-dosage (dark blue), networks only sensitive to duplications (light blue) or deletions (orange), and finally, those unable to compensate for gene-dosage abnormalities (red). Nodes represent brain regions, and edges between nodes represent their functional link. Wavy edges represent sub-optimal functional links.

**Databases used in this review**

DECIPHER v4.3 https://decipher.sanger.ac.uk/application/

Autism Genetic Database (AGD) http://wren.bcf.ku.edu/

Autism CNV Database http://projects.tcag.ca/autism_500k/

AutDB http://www.mindspec.org/autdb.html

BioGPS http://biogps.gnf.org/#goto=welcome

UCSC Genome browser http://genome.ucsc.edu
Acknowledgments

This work was supported by the Institut Pasteur, Université Denis Diderot Paris 7, INSERM, CNRS,
Fondation Orange, Fondation de FranceFondation FondaMentale.
<table><caption>Table 1. Genes associated with high risk for ASD</caption><thead><tr><th>Gene</th><th>Chromosome</th><th>Function</th><th>Evidence</th><th>Inheritance</th><th>Diagnosis</th><th>Reference</th></tr></thead><tbody><tr><td>FMR1</td><td>Xq27</td><td>Synaptic translation</td><td>Mutations</td><td>de novo (permutations)</td><td>ASD, Fragile X syndrome</td><td>83</td></tr><tr><td>MECP2</td><td>Xq26</td><td>Chromatin remodeling</td><td>CNV, mutations</td><td>de novo (rarely inherited)</td><td>ASD, Rett syndrome</td><td>84</td></tr><tr><td>TSC1</td><td>9q34.13</td><td>mTOR / PI3K pathway</td><td>CNV, mutations</td><td>de novo, inherited</td><td>ASD, Tuberous sclerosis</td><td>85</td></tr><tr><td>TSC2</td><td>16p13.3</td><td>mTOR / PI3K pathway</td><td>CNV, mutations</td><td>de novo, inherited</td><td>ASD, Tuberous sclerosis</td><td>85</td></tr><tr><td>NF1</td><td>17q11.2</td><td>mTOR / PI3K pathway</td><td>CNV, mutation</td><td>de novo, inherited</td><td>ASD, Neurofibromatosis</td><td>86</td></tr><tr><td>PTEN</td><td>10q23.31</td><td>mTOR / PI3K pathway</td><td>CNV, mutations</td><td>de novo, inherited</td><td>ASD, Cowden syndrome</td><td>87</td></tr><tr><td>CACNA1C</td><td>12p13.33</td><td>Calcium channel</td><td>Mutation</td><td>de novo</td><td>ASD, Timothy syndrome</td><td>88</td></tr><tr><td>DPYD</td><td>1p21.3</td><td>Pyrimidine base biosynthesis</td><td>CNV</td><td>de novo</td><td>ASD</td><td>14</td></tr><tr><td>RFWD2</td><td>1q25.1-q25.2</td><td>Ubiquitination</td><td>CNV</td><td>de novo, inherited</td><td>ASD</td><td>13</td></tr><tr><td>NRXN1</td><td>2p16.3</td><td>Synaptic CAM</td><td>CNV, mutations, SNP</td><td>de novo, inherited</td><td>ASD, SCZ</td><td>5, 89</td></tr><tr><td>CNTN4</td><td>3p26.3</td><td>Synaptic CAM</td><td>CNV</td><td>Inherited</td><td>ASD, MR</td><td>13, 25, 63, 90</td></tr><tr><td>MEF2C</td><td>5q14.3</td><td>Transcription factor</td><td>CNV, mutations</td><td>de novo</td><td>MR, Seizures</td><td>38</td></tr><tr><td>SYNGAP1</td><td>6p21.3</td><td>Synaptic Ras GAP</td><td>CNV</td><td>de novo</td><td>ASD, MR</td><td>19</td></tr><tr><td>CNTNAP2</td><td>7q35-7q36.1</td><td>Synaptic CAM</td><td>CNV, rare variants *</td><td>Inherited</td><td>ASD, MR, SCZ, TS,</td><td>20, 60-62, 91, 92</td></tr><tr><td>DPP6</td><td>7q36.2</td><td>dipeptidyl-peptidase activity</td><td>CNV</td><td>de novo, inherited</td><td>ASD</td><td>14</td></tr><tr><td>DLGAP2</td><td>8p23.3</td><td>Synaptic scaffold</td><td>CNV</td><td>de novo</td><td>ASD</td><td>14</td></tr><tr><td>ASTN2</td><td>9q33.1</td><td>Neuron-Glial Interaction</td><td>CNV</td><td>Inherited</td><td>ASD, SCZ, ADHD</td><td>13</td></tr><tr><td>SHANK2</td><td>11q13</td><td>Synaptic scaffold</td><td>CNV</td><td>de novo</td><td>ASD</td><td>19</td></tr><tr><td>NBEA</td><td>13q13.2</td><td>Synaptic protein</td><td>Translocation</td><td>de novo</td><td>ASD</td><td>93</td></tr><tr><td>UBE3A</td><td>15q11-q13</td><td>Ubiquitination</td><td>CNV</td><td>de novo, inherited</td><td>ASD</td><td>13</td></tr><tr><td>SHANK3 (del 22q13)</td><td>22q13</td><td>Synaptic scaffold</td><td>CNV, mutations</td><td>de novo, inherited</td><td>ASD, MR, SCZ</td><td>51, 52, 94</td></tr><tr><td>NLGN3</td><td>Xq13.1</td><td>Synaptic CAM</td><td>Mutation</td><td>Inherited</td><td>ASD</td><td>95</td></tr><tr><td>IL1RAPL1</td><td>Xp21.3-p21.2</td><td>Synaptic receptor</td><td>CNV, mutations</td><td>de novo, inherited</td><td>ASD, MR</td><td>96</td></tr><tr><td>NLGN4</td><td>Xp22</td><td>Synaptic CAM</td><td>CNV, mutations</td><td>de novo, inherited</td><td>ASD, MR, TS</td><td>95</td></tr><tr><td>PTCHD1</td><td>Xp22.11</td><td>Hedgehog receptor activity</td><td>CNV</td><td>Inherited</td><td>ASD</td><td>14</td></tr><tr><td>GRIA3</td><td>Xp25</td><td>Synaptic receptor</td><td>CNV</td><td>Inherited</td><td>ASD</td><td>12</td></tr></tbody></table>

ASD Autism Spectrum Disorder; SCZ Schizophrenia; MR Mental Retardation; ADHD Attention-Deficit Hyperactivity Disorder; MDC1D congenital muscular dystrophy; BP Bipolar, TS Tourette syndrome; * in contrast to mutations, the functional role of the rare variants was not confirmed
<table><caption>Table 2. Proposed susceptibility genes for ASD</caption><thead><tr><td>Gene</td><td>Chromosome</td><td>Function</td><td>Evidence</td><td>Diagnosis</td><td>References</td></tr></thead><tbody><tr><td>ASMT</td><td>PAR1</td><td>Melatonin pathway</td><td>Inherited CNV, SNPs, mutations</td><td>ASD</td><td>81, 97, 98</td></tr><tr><td>DISC1/DISC2</td><td>1q42.2</td><td>Axonal growth</td><td>Inherited CNV</td><td>ASD, SCZ</td><td>99</td></tr><tr><td>TSNAX</td><td>1q42.2</td><td>Cell differentiation</td><td>Inherited CNV</td><td>ASD, SCZ</td><td>99</td></tr><tr><td>DPP10</td><td>2q14.1</td><td>Dipeptidyl-peptidase activity</td><td>Inherited CNV</td><td>ASD</td><td>14</td></tr><tr><td>CNTN3</td><td>3p12.3</td><td>Synaptic CAM</td><td>Inherited CNV</td><td>ASD</td><td>25</td></tr><tr><td>FBXO40</td><td>3q13.3</td><td>Unknown function</td><td>Inherited CNV P = 3.3 x 10<sup>-3</sup></td><td>ASD</td><td>13</td></tr><tr><td>SLC9A9</td><td>3q24</td><td>Transporter</td><td>Inherited CNV, mutations</td><td>ASD, ADHD, MR</td><td>25</td></tr><tr><td>PCDH10</td><td>4q28</td><td>Synaptic CAM</td><td>Inherited CNV</td><td>ASD</td><td>25</td></tr><tr><td>PARK2</td><td>6q26</td><td>Ubiquitination</td><td>Inherited CNV P = 3.3 x 10<sup>-3</sup></td><td>ASD, PD</td><td>13</td></tr><tr><td>IMMP2L</td><td>7q31.1</td><td>Mitochondrial protease</td><td>Inherited CNV</td><td>ASD, TS, ADHD</td><td>100</td></tr><tr><td>PCDH9</td><td>13q21</td><td>Synaptic CAM</td><td>Inherited CNV</td><td>ASD</td><td>14, 25</td></tr><tr><td>MDGA2</td><td>14q21.3</td><td>GPI anchor protein</td><td>Inherited CNV P = 1.3 x 10<sup>-4</sup></td><td>ASD</td><td>18</td></tr><tr><td>BZRAP1</td><td>17q22</td><td>Benzodiazepine receptor binding</td><td>Inherited CNV P = 2.3 x 10<sup>-5</sup></td><td>ASD</td><td>18</td></tr><tr><td>PLD5</td><td>1q43</td><td>Phospholipase D</td><td>SNP rs2196826 P = 1.1 x 10<sup>-8</sup></td><td>ASD</td><td>9</td></tr><tr><td>SLC25A12</td><td>2q31.1</td><td>Synaptic receptor</td><td>SNP rs2056202 P = 1 x 10<sup>-3</sup></td><td>ASD</td><td>101, 102</td></tr><tr><td>CDH9/CDH10</td><td>5p14.2</td><td>Synaptic CAM</td><td>SNP rs4307059 P = 3.4 x 10<sup>-8</sup></td><td>ASD</td><td>7</td></tr><tr><td>SEMA5A</td><td>5p15.2</td><td>Axonal guidance</td><td>SNP rs10513025 P = 2 x 10<sup>-7</sup></td><td>ASD</td><td>8</td></tr><tr><td>TAS2R1</td><td>5p15.2</td><td>Receptor</td><td>SNP rs10513025 P = 2 x 10<sup>-7</sup></td><td>ASD</td><td>8</td></tr><tr><td>GRIK2</td><td>6q16.3</td><td>Synaptic receptor</td><td>SNP rs3213607 P = 0.02</td><td>ASD, SCZ, OCD, MR</td><td>57</td></tr><tr><td>POU6F2</td><td>7p14.1</td><td>Transcription factor</td><td>SNP rs10258862 P = 4.4 x 10<sup>-7</sup></td><td>ASD</td><td>9</td></tr><tr><td>RELN</td><td>7q22.1</td><td>Axonal guidance</td><td>GGC repeat in the 5' UTR P < 0.05</td><td>ASD, BP</td><td>103</td></tr><tr><td>NRCAM</td><td>7q31.1</td><td>Synaptic receptor</td><td>SNP rs2300045 P = 0.017</td><td>ASD</td><td>104</td></tr><tr><td>MET</td><td>7q31.2</td><td>Tyrosine kinase</td><td>SNP rs1858830 P = 2 x 10<sup>-3</sup></td><td>ASD</td><td>34</td></tr><tr><td>EN2</td><td>7q36.3</td><td>Transcription factor</td><td>SNP rs1861972 P = 9 x 10<sup>-3</sup></td><td>ASD</td><td>105</td></tr><tr><td>ST8SIA2</td><td>15q26.1</td><td>N-glycan processing</td><td>SNP rs3784730 P = 4 x 10<sup>-7</sup></td><td>ASD</td><td>9</td></tr><tr><td>GRIN2A</td><td>16p13.2</td><td>Synaptic receptor</td><td>SNP rs1014531 P = 2.9 x 10<sup>-7</sup></td><td>ASD, SCZ</td><td>106</td></tr><tr><td>ABAT</td><td>16p13.2</td><td>Enzyme</td><td>SNP rs1731017 P = 1 x 10<sup>-3</sup></td><td>ASD, GABA-AT Deficiency</td><td>106</td></tr><tr><td>SLC6A4</td><td>17q11.2</td><td>Serotonin Transporter</td><td>Meta analysis P > 0.05</td><td>ASD, OCD</td><td>107</td></tr><tr><td>ITGB3</td><td>17q21.3</td><td>Cell-matrix adhesion</td><td>SNP Leu33Pro P = 8.2 x 10<sup>-4</sup></td><td>ASD</td><td>108</td></tr><tr><td>TLE2 / TL6</td><td>19p13</td><td>Wnt receptor signaling pathway</td><td>SNP rs4806893 P = 7.8 x 10<sup>-5</sup></td><td>ASD, FHM2, AHC</td><td>109</td></tr><tr><td>MACROD2</td><td>20p12</td><td>Unknown function</td><td>SNP rs4141463 P = 2 x 10<sup>-8</sup></td><td>ASD</td><td>9</td></tr></tbody></table>

ASD Autism Spectrum Disorder; SCZ Schizophrenia; PD Parkinson Disease; TS Tourette syndrome; ADHD Attention-Deficit Hyperactivity Disorder; MR Mental Retardation; FHM2 Familial Hemiplegic Migraine 2; AHC Alternating Hemiplegia of Childhood; OCD Obsessive-Compulsive Disorder; BP Bipolar Disorder.
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<figure><img src="image_9.png" /><figcaption>Figure 3</figcaption></figure>