**2. Chromosome engineering mouse models for ASD**

Some of the first studies providing a high-resolution view of the entire genome have re‐ vealed that a large number of CNVs are present in the genomes of healthy individuals, and that CNVs account for a greater proportion of the nucleotide variation between two given individual genomes than can be attributed to SNVs [7-9]. These structural alterations can reach up to several megabases in length, but a much higher frequency is observed for small‐ er (<1 kb) CNVs [2]. And, as one would expect, the likelihood of CNVs becoming pathogenic rises when they have an increased size and/or occur in gene-dense regions of the genome [8]. Traditionally, structural variation (CNV) was not considered to play a causative role in autism or ASD. However, recent studies have revealed that not only single-gene alterations, but also CNVs can lead to autism or ASD. In fact, it is now becoming increasingly evident that CNVs account for a larger proportion of new autism diagnoses than single-gene disor‐ ders. Recurrent CNVs at specific genomic loci have been associated with autism, including 15q11-q13, 16p11.2, 17p11.2, 22q13.3, 7q11.23, and 2q37, among others [1, 10-16]. While sev‐ eral of these loci are associated with known Centers for Mendelian Genomics, numerous CNVs have also been observed in idiopathic autism, underscoring the importance of these

The application of next-generation sequencing technology to evaluate CNVs has also re‐ cently been described in a report that utilized whole-transcriptome sequencing analysis of the genomes of a cohort of patients with autism spectrum disorder (ASD) [18]. This approach allows for the evaluation of CNVs and overcomes some of the problems asso‐ ciated with CNV-calling in WES. With several large-scale projects currently underway, the future of next-generation sequencing and whole-genome analysis in the study of au‐ tism will most definitely provide many new insights into the etiology of this disease. Currently, Autism Speaks is working in collaboration with the company BGI to gener‐ ate the largest database of sequenced genomes of individuals with ASD, a project known as the "Autism Genome 10K." Similarly, the National Institute of Mental Health in the US has funded another large-scale "Autism Genome Project." Mendelian/ syndromic forms of autism are also currently being studied by the Genomic Disorders

Among the variants identified in the large-scale studies of patients with autism report‐ ed to date, many gene networks/pathways have been implicated, including genes for neuronal adhesion [18, 19], ubiquitin degradation [19], chromatin remodelling [5, 20], sodium channels [13], proteolysis [21], cytoskeletal organization [21], signal transduction [18], neuropeptide signalling [18], neurogenesis/synaptogenesis [18], neuronal migration [22], basic metabolism, and RNA splicing [22], among others. While these pathways may seem diverse, repeated "hits" in these networks support the "many genes, com‐ mon pathway" hypothesis [22]. Importantly, although the biological function of ASD susceptibility genes identified via these whole-genome studies do not appear to lie within the same network, they likely converge to disrupt neuronal function in brain re‐ gions that support language, social cognition, and behavioral flexibility, resulting in the

structural variations in the future of all types of autism research [17].

consortium in the US by WES.

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phenotypes commonly associated with ASD [22].

Since ASD is known to be a highly heterogeneous disorder with both genetic and environmen‐ tal components, modeling the disease in rodents, where environmental and background ef‐ fects can be largely controlled and systematically manipulated and studied, is of great advantage in the study of the pathomechanisms underpinning autism. Furthermore, numer‐ ous tools for genetic manipulation and for behavioral analysis that are currently available and developed for genetic studies in rodents can be leveraged to facilitate this avenue of research. Importantly, behavioral assays have been developed and validated to objectively quantify the phenotypes relative to autism, including both core and associated autistic-like phenotypes, such as abnormal social behavior, communication deficits, and repetitive behaviors, as well as autism-associated anxiety-like behaviors, motor defects, learning and memory deficits, sleep disorders, sensory hypersensitivity, and seizures, among others [23] (Table 1; adapted from Crawley et al, where a full description of these behavioral tests can be found; [23]).

With the importance of CNVs in the etiology of ASD established, chromosome-engineered mouse models were then generated for the study of autism or ASD. The first such mouse strains were developed over a decade ago using a chromosome-engineering approach [24]. This technique allows for the creation of a targeted duplication or deletion in the desired lo‐ cation by first generating the rearrangement in mouse embryonic stem (ES) cells which can then be established as mouse strains via *Cre/loxP* site-specific recombination [25, 26]. To gen‐ erate the desired rearrangement, two gene-targeting steps are required to prepare each end point for a selectable recombination event (Figure 1). Importantly, the type of rearrangement (deletion, duplication, inversion) depends on the relative orientation of the *loxP* sites; if the sites are in the same orientation, the region between them can be deleted or duplicated, but if they are in opposite orientation, an inversion results [25]. The *cis* or *trans* configuration is also relevant; *trans* insertion (insertion in each chromosome homologue) of loxP sites ena‐ bles generation of both deletion and duplication in the same ES cells. Transient transfection of the ES cells with a vector expressing *Cre* recombinase facilitates the recombination be‐ tween the targeted *loxP* sites, and cells containing the event can be selected for using hypo‐ xanthine aminopterin thymidine (HAT)-containing media due to the reconstitution of a functional Hprt cassette as a result of the recombination [25]. The resulting mouse models harbor either a chromosomal duplication or deletion of a defined region that is syntenic to the copy number variable region in humans. Importantly, chromosome-engineered mouse models are distinct from monogenic animal models in that they harbor structural chromoso‐ mal rearrangements resulting in specific, targeted CNVs with genomic intervals that may span several megabases and contain numerous genes, many of which may be of unknown function. In contrast, monogenic animal models primarily utilize a reverse-genetics ap‐ proach to knock-out or transgenically-overexpress the specific single gene of interest, limit‐ ing the study to that one particular gene. A publicly-available resource can facilitate chromosome engineering for the targeted manipulation of the mouse genome; the mutagen‐ ic insertion and chromosome engineering resource (MICER) can be utilized to access vectors to create chromosomal rearrangements or to study gene disruptions in a high-throughput manner [27]. (http://www.sanger.ac.uk/resources/mouse/micer/).


**Figure 1. Generating defined chromosome rearrangements.** Defined endpoints of the desired interval for rear‐ rangement (A and B) are modified by gene targeting in embryonic stem (ES) cells to allow for introduction of a *loxP* site (blue triangle), a non-functional portion of the HPRT cassette [5' or 3'), and a positive selectable marker (Neo or Puro). The targeted ES cells are then transiently transfected with a vector expressing *Cre* recombinase, which facilitates recombination between the loxP sites, resulting in either deletion (Df) or duplication (Dp) of the intervening region. In this example, reconstitution of the Hprt cassette via recombination between *loxP* sites lying in a shared intron be‐ tween the two halves confers resistance to hypoxanthine aminopterin thymidine (HAT), which can be used to select for the deletion event. Two positive selectable markers (Puro and Neo) identify the duplication event. A full explana‐

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We will discuss specific examples of chromosome-engineered mouse models of ASD for which the neurobehavioral phenotypes have been described using established rodent be‐ havioral assays, including mice harboring duplication or deletion of the genomic interval syntenic to human chromosome 16p11.2, 15q11-q13, and 17p11.2, which all model CNVs that have been identified in patient populations with autism [28-30]. Most of the CNV-based animal models of ASD described to date have focused on syndromic forms of autism, as the

By definition, genomic disorders result from structural changes in the genome, wherein the genomic instability often reflects a susceptible genome architecture, that leads to disease traits [31]. These structural rearrangements or CNVs commonly cause the disruption or complete loss or gain of dosage-sensitive gene(s). Alternatively, CNVs can cause gene fu‐ sion, position effects, transvection effects, or the unmasking of a recessive allele or function‐ al polymorphism [32]. Genomic disorders are therefore distinct from traditional genetic syndromes, which are typically caused by DNA sequence-based changes [33]. Within the past decade, several technologies, including array comparative genomic hybridization (aCGH), next-generation sequencing, and single nucleotide polymorphism (SNP) genotyp‐

tion of this technology and more examples of chromosome recombineering are outlined by Mills et al [25].

underlying genetic cause of these genomic disorders is often well-described.

**Table 1.** Behavioral tests for the evaluation of autistic-like features in mice

**Core features of autism Analogous feature/assay in mice** Abnormal social interactions Preference for social novelty

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Impaired social communication Vocalizations during direct social interaction

Repetitive or restrictive behavior Perseverative holeboard exploration

ASD- Associated features Analogous feature/assay in mice

Theory of mind deficits Social transmission of food preference

Motor defects Rotarod test for motor learning

Aggression Resident intruder attack

Sleep disturbances Circadian running wheel

**Table 1.** Behavioral tests for the evaluation of autistic-like features in mice

Hypersensitivity to sensory stimuli Pre-pulse inhibition

T-maze

Wire hang Dowel walking Footprint analysis

Acoustic startle

Seizures Sensitivity to audiogenic or drug-induced seizures

Anxiety Elevated plus maze

Intellectual disability Morris water maze

Social recognition

Home cage monitoring - nesting patterns Direct social interactions / juvenile play Social approach to a stranger mouse

Parental retrieval of separated pups Ultrasonic vocalizations by separated pups

Behavioral stereotypies/ home cage observation Reversal of a position habit in maze tasks

Response to olfactory cues

Marble burying

Novel object exploration

Light/dark exploration Open field analysis

Avoidance of aggressive encounters

Contextual and cued fear conditioning

Tube test for social dominance

Hot plate test for nociception

Home cage monitoring - activity levels

**Figure 1. Generating defined chromosome rearrangements.** Defined endpoints of the desired interval for rear‐ rangement (A and B) are modified by gene targeting in embryonic stem (ES) cells to allow for introduction of a *loxP* site (blue triangle), a non-functional portion of the HPRT cassette [5' or 3'), and a positive selectable marker (Neo or Puro). The targeted ES cells are then transiently transfected with a vector expressing *Cre* recombinase, which facilitates recombination between the loxP sites, resulting in either deletion (Df) or duplication (Dp) of the intervening region. In this example, reconstitution of the Hprt cassette via recombination between *loxP* sites lying in a shared intron be‐ tween the two halves confers resistance to hypoxanthine aminopterin thymidine (HAT), which can be used to select for the deletion event. Two positive selectable markers (Puro and Neo) identify the duplication event. A full explana‐ tion of this technology and more examples of chromosome recombineering are outlined by Mills et al [25].

We will discuss specific examples of chromosome-engineered mouse models of ASD for which the neurobehavioral phenotypes have been described using established rodent be‐ havioral assays, including mice harboring duplication or deletion of the genomic interval syntenic to human chromosome 16p11.2, 15q11-q13, and 17p11.2, which all model CNVs that have been identified in patient populations with autism [28-30]. Most of the CNV-based animal models of ASD described to date have focused on syndromic forms of autism, as the underlying genetic cause of these genomic disorders is often well-described.

By definition, genomic disorders result from structural changes in the genome, wherein the genomic instability often reflects a susceptible genome architecture, that leads to disease traits [31]. These structural rearrangements or CNVs commonly cause the disruption or complete loss or gain of dosage-sensitive gene(s). Alternatively, CNVs can cause gene fu‐ sion, position effects, transvection effects, or the unmasking of a recessive allele or function‐ al polymorphism [32]. Genomic disorders are therefore distinct from traditional genetic syndromes, which are typically caused by DNA sequence-based changes [33]. Within the past decade, several technologies, including array comparative genomic hybridization (aCGH), next-generation sequencing, and single nucleotide polymorphism (SNP) genotyp‐ ing platforms, have been utilized to detect and analyze CNVs in the genome and to investi‐ gate the mechanism by which these CNVs are generated [34]. CNVs can be formed by several mechanisms, such as non-allelic homologous recombination (NAHR), non-homolo‐ gous end joining (NHEJ), or fork stalling and template switching (FoSTeS) [35]. NAHR, which is often mediated by low copy repeats (LCRs) with high (~95%) sequence similarity flanking the rearranged region, is the most common mechanism by which recurrent CNVs are created. Often this mechanism can result in recurrent genomic rearrangements that are observed in multiple patients with the same disorder, as in Charcot-Marie-Tooth disease type 1A, Prader-Willi syndrome, and Smith-Magenis Syndrome, among many others [32, 33]. The genomic architecture rendering genomic instability at three loci that are enriched for LCRs are shown in Figure 2.

[13]. The 16p11.2 locus is flanked by two directly repeated segmental duplications of ~145 kb, which mediate the NAHR that results in the loss or gain of ~600 kb intermediate region

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Interestingly, the microduplication of this region has also been linked to schizophrenia, sug‐ gesting the presence of an underlying biological link between these two disorders [41, 42]. This phenomenon also gives a potential genetic basis for the hypothesis of Crespi et al, which states that autism and schizophrenia represent diametric disorders of the social brain [43]. Thus, schizophrenia and autism might reflect mirror traits of the opposing extremes of behavioral phenotypes reflecting evolution of the social brain [43]. The phenotypes caused by CNV at the 16p11.2 locus are extremely heterogeneous, and, in addition to ASD, they have been reported to include metabolic disorders [44-47], cardiac anomalies [40, 48], de‐ pressive disorder [49], speech delay [50], mental retardation [40, 51, 52], vertebral anomalies [52], syringomyelia [53], abnormal head size [36], and epilepsy [36, 40], as well as other vari‐ ous congenital anomalies and behavioral abnormalities [44]. As the phenotypes of many more patients harboring CNVs in this genomic region are delineated, the full phenotypic spectrum associated with this locus will likely become more well-defined, and the critical genomic interval and dosage-sensitive genes responsible for the phenotypes will be deter‐ mined. Indeed, a more recent study described a patient pedigree for a family with multiple generations of autism or ASD that also carry a smaller-sized deletion within the common deletion of 16p11.2, thereby reducing the "critical" interval for ASD to a 118 kb region con‐ taining only 5 genes: *MVP*, *CDIPT1*, *SEZ6L2*, *ASPHD1*, and *KCTD13 [54]*. To date, none of these genes have been significantly associated with an elevated risk for ASD, which indi‐ cates that the situation is likely much more complex [37, 55]. Furthermore, correlation be‐ tween the phenotypes of patients harboring different- or similar-sized CNVs is confounded by extreme heterogeneity and variability of symptoms. For example, a family with three af‐ fected members harboring identical 16p11.2 deletions was recently described to have mini‐ mal symptom overlap between family members [56]. Subsequent studies have aimed at using model organisms to identify the key dosage-sensitive genes within this region that give rise to the abnormal phenotypes [29, 57, 58]. Among these, chromosome-engineered mouse models harboring reciprocal deletion or duplication of the mouse chromosome syn‐ tenic to human chromosome 16p11.2 have been generated to study the physiological and be‐

havioral phenotypes associated with these chromosome abnormalities [29].

Mouse models were generated through a chromosome engineering approach for the study of human 16p11.2 deletion and duplication CNVs [29]. It was observed that ~50% of mice harboring the deletion CNV die shortly after birth, while duplication mice sur‐ vive to adulthood, suggesting that the deletion CNV results in a more severe phenotype than the duplication [29]. A similar phenomenon has been observed in other genomic disorders caused by reciprocal CNVs, including Smith-Magenis and Potocki-Lupski syn‐ dromes [15]. Expression of the genes within the 16p11.2 region corresponds to gene dos‐

**4. Animal models for human 16p11.2 CNVs**

containing ~27 protein-coding genes [9, 12, 40].

**Figure 2. The genomic structure of loci associated with CNV-based ASD. (A)** Chromosomes 15q11-13, **(B)** 16p11.2, and **(C)** 17p11.2, are enriched for LCRs, or segmental duplications (indicated by red arrows), which facilitate non-allelic homologous recombination (NAHR), resulting in the generation of CNVs (blue and red bars). This figure was generated using the genome browser provided by UCSC (http://genome.ucsc.edu/index.html?org=Hu‐ man&db=hg18&hgsid=289381925].
