**7. Modeling 15q11-13 CNVs in mice**

AS is a neurodevelopmental disorder that is characterized by severe developmental de‐ lay, intellectual disability, microcephaly, seizures, lack of speech, ataxia, and dysmorphic facial features [11, 59]. Patients with AS are often described as having happy demeanors, however hyperactivity, attention deficits, aggression, and repetitive or stereotypic behav‐ iors have also been described [59]. AS has been associated with ASD in several studies, however, the severity of the cognitive impairments in most patients with AS may pre‐

It has been estimated that up to ~5% of cases of ASD can be attributed to maternal duplica‐ tion of the genomic region reciprocal to the PWS-AS critical region on chromosome 15q11-13, making it one of the most common chromosomal abnormalities observed in pa‐ tients with ASD [10, 62]. Due to the presence of imprinting at this locus (discussed above), parent-of-origin effects are seen, and, for interstitial duplications, maternal origin confers an increased risk for clinical phenotypes. Paternal duplications are much less common, and do not appear to lead to ASD, as familial cases have been described where a seemingly normal carrier mother transmits a paternally-derived duplication to their child [63]. However, a small number of subjects with paternal duplication of 15q11-13 and various clinical pheno‐ types have been described [64]. Phenotypes are dosage-sensitive at this locus; one extra ma‐ ternal copy of 15q11-13 results in partial autism penetrance, while two extra copies (caused by idic15 or interstitial triplication) result in a much higher penetrance of autism as well as additional phenotypes that are typically more severe than those seen in patients with dupli‐ cations [62]. In the case of triplications, parent-of-origin effects are no longer observed, and both paternal and maternal duplications are associated with poor clinical outcomes [65]. This loss of parent-of-origin effects is interesting, and it may be an indication of the underly‐ ing mechanism that may give rise to a predisposition for these phenotypes. Many heteroge‐ neous and complex phenotypes can be associated with increased copy number of this region including intellectual disability, apraxia, dyslexia, seizures, hypotonia, developmental de‐ lay, gait abnormalities, hyperactivity, schizophrenia, and ASD [66]. Patients with ASD due to duplication of 15q11-13 also display several stereotypic and repetitive behaviors, includ‐ ing rocking, licking, and hand-flapping, among others, that are often directed towards sen‐ sory stimulation, suggesting that the underlying cause of these phenotypes may be due to a

Interestingly, recent post-mortem evaluation of the brains of patients harboring maternal duplication of 15q11-13 suggested that accumulation and deposition of abnormal intracel‐ lular and extracellular amyloid β protein (Aβ) in the specific regions and neuron types in the brains of patients with maternal duplication of 15q11-13 may underlie or contrib‐ ute to some of the neurobehavioral phenotypes associated with ASD [67]. However, fur‐ ther studies are needed to confirm this hypothesis. Several animal models for the CNVbased syndromes associated with chromosome 15q11-13 have been developed to facilitate

clude an accurate diagnosis [11, 59].

258 Recent Advances in Autism Spectrum Disorders - Volume I

disregulation of sensory inputs or signaling [63, 64].

research into these disorders.

**6. Duplication of 15q11-13**

The first mouse model for PWS was generated by targeted deletion of part of the imprinting center on chromosome 15q11-13 [68]. While these mice model several aspects of PWS, in‐ cluding hypersensitivity to sensory input in the form of increased acoustic startle response and decreased prepulse inhibition, it is not known whether these mice exhibit other autisticlike behaviors, such as impaired social interactions or altered communication [69]. Further behavioral characterization is needed to determine whether these mice accurately recapitu‐ late the neurobehavioral phenotypes seen in patients with PWS and/or ASD.

Mouse models for Angelman syndrome were initially generated by disrupting maternal ex‐ pression of *Ube3a*; these mice exhibit increased anxiety-like behavior that may be due to the disruption of a glucocorticoid receptor transactivation in the brain [70]. They also display various motor defects [71], abnormal cerebellum-driven licking behavior [72], sleep disturb‐ ance [73], abnormal EEG patterns, and cognitive defects in the conditioned fear and Morris water maze tests [74]. However, these mice displayed hypoactivity and normal social seek‐ ing behavior, in contrast to what is seen in human patients with AS and/or ASD [75]. Impor‐ tantly, human patients with mutations in *UBE3A* typically have a milder phenotype than those patients harboring interstitial deletions, so this mouse model may not accurately re‐ flect the majority of AS patients, who have a deletion containing this gene, as well as many other genes at this locus. Indeed, when a larger 1.6 Mb deletion model was generated by chromosome engineering that encompasses the genomic region from *Ube3a* to *Gabrb3*, the phenotype of these mice was more severe than that of mice with deletion of *Ube3a* alone [76]. Similar to *Ube3a* deficient mice, large deletion mice had significant motor impairment, anxiety-like behavior, and abnormal EEG, but they also had learning and memory defects and abnormal communication [76]. These larger deletion mice may be an appropriate model for CNV-associated ASD, however further investigation into the social neurobehavioral phe‐ notypes is necessary.

Chromosome-engineered mouse models harboring a duplication of 6.3 Mb on mouse chro‐ mosome 7 syntenic to the duplication of human chromosome 15q11-13 associated with ASD were developed to study the underlying mechanism behind the phenotypes associated with this CNV in humans [28]. The core features of autism, including abnormal social interac‐ tions, stereotypic or ritualistic behavior, and impaired communication were all evaluated in this mouse model, and patDp/+, but not matDp/+ mice were determined to have reduced so‐ ciability compared to wild-type mice. Ultrasonic vocalizations were evaluated in pups sepa‐ rated from the dam, and an abnormal USV pattern was observed in patDp/+ but not matDp/+ mice. Specifically, patDp/+ mice appeared to have delayed development of com‐ munication, they emitted a greater number of USVs, and some pups emitted vocalizations at abnormally high frequencies (>70 kHz). In order to evaluate communication between older mice, pairs of mice 7-8 weeks of age were observed in a resident intruder paradigm and the pattern of USVs was measured during this interaction. Vocal communication between pairs of pat/Dp/+ mice was significantly reduced compared to the vocalization recorded between WT pairs, giving further support to the notion that patDp/+ mice have a defect in social communication. However, no defects in olfactory communication or function were observed in these mice. Restricted behaviors and inflexibility were evaluated with a battery of behav‐ ioral tests, including the Morris water maze and the Barnes maze. These tests revealed that patDp/+ mice do not respond as flexibly to a change in situation as wild-type or matDp/+ mice. No overt defects in learning and memory were observed in either the patDp/+ or matDp/+ mice by the Morris water maze or conditioned fear test, although patDp/+ mice did display generalized fear and elevated anxiety-like behavior. The abnormal neurobehavioral phenotypes observed in this CNV-based model of ASD could not be attributed to gross mor‐ phological or histological changes in the olfactory bulb, cerebral cortex, hippocampus, amygdala, corpus callosum, or cerebellum. Nor were any abnormalities in the number of Purkinje cells detected in the cerebellum, suggesting that the underlying pathomechanism responsible for these phenotypes is likely due to aberrations in molecular pathways that re‐ main to be determined.

Both SMS and PTLS manifest a broad range of opposing or overlapping phenotypes. SMS is characterized by multiple congenital anomalies, including otolaryngologic, ophthalmologic, brain, cardiac, craniofacial, and renal abnormalities, as well as intellectual disability (ID), brachydactyly, sleep disturbance, hearing impairment, obesity, scoliosis, and other neurobe‐ havioral abnormalities [84, 85]. Specifically, SMS patients display aggressive and self-injuri‐ ous behaviour, including polyembolokoilamania [84], as well as characteristic repetitive behaviors, including autoamppexation or "self-hugging," which is an identifying feature of the disorder [86, 87]. More recently, SMS patients have also been described as meeting the

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The PTLS duplication was the first predicted reciprocal duplication to be described [89]. PTLS was identified and initially defined much later than SMS, ([89] versus Smith et al. 1986); as a result, fewer PTLS patients have been medically examined and fewer studies of the clinical phenotypes are available in the literature. The clinical features that have been ob‐ served in patients with PTLS are distinct from those seen in SMS [15], although cognitive and neurobehavioral abnormalities are present in both disorders. PTLS patients lack the self-injurious behaviors, abnormal facies, and sleep disturbance, as well as some of the con‐ genital anomalies found in most individuals with SMS. The features observed in greater than 90% of PTLS patients are developmental delay, neurobehavioral abnormalities, lan‐ guage impairment, cognitive impairment, poor feeding, hypotonia, and oropharyngeal dys‐ phasia [15, 90]. When evaluated by objective clinical assessment, the majority of PTLS patients have autistic features such as decreased eye contact, atypicality, withdrawal, anxi‐ ety, and inattention, meeting criteria for a diagnosis of autistic spectrum disorder (ASD) or pervasive developmental disorder not otherwise specified, and making ASD the most com‐

Most PTLS patients have no distinctive facial abnormalities but they can have a triangularshaped face. The other clinical features present in over half of patients include sleep apnea, abnormal EEG, attention deficit, hypermetropia, and cardiovascular abnormalities [15]. These cardiovascular abnormalities can typically include both structural and conduction de‐ fects, such as atrial or ventricular septal defects, bicuspid aortic valve, dilated aortic root, di‐ lation of the pulmonary annulus, patent foramen ovale, or hypoplastic left heart [15, 91-93].

Upon molecular analysis, most [22 of 35] PTLS patients included in the first multidisci‐ plinary study were determined to carry a common recurrent 3.7 Mb duplication in 17p11.2 mediated by the same proximal and distal SMS-REPs which also mediate the re‐ ciprocal common recurrent SMS deletion [15]. Others have uncommon and sometimes complex genomic rearrangements, all of which involve duplication in 17p11.2 [94]. The smallest PTLS duplication identified to date occurred in a single patient and is 1.3 Mb in size. This duplicated segment contains 14 genes, including both *RAI1,* the major contribu‐ ting gene for the reciprocal deletion causing SMS, as well as the steroid-metabolism reg‐ ulating gene, *SREBP1*. This patient demonstrates all typical PTLS phenotypes [94]. Whether, or to what extent, PTLS results from *RAI1* gene over-dosage still remains to be elucidated, although mouse studies (described below) have shown that it is likely re‐

criteria for autism spectrum disorder (ASD) [88].

mon and consistent feature observed in PTLS. [14].

sponsible for at least some of the symptoms [95].

The gene *Ube3a* (also known as E6-AP) codes for E3 ubiquitin-protein ligase, which belongs to a family of E3 ligase genes that are involved in synaptogenesis and have recently been linked to the pathogenesis of ASD [62]. Given the known association of the gene *UBE3A* with AS and its maternal-specific expression pattern in neurons, a mouse model that overex‐ presses this gene was generated to test the hypothesis that *Ube3a* is responsible for many of the phenotypes associated with duplication of 15q11-13 [62]. Transgenic overexpression of *Ube3a* via bacterial artificial chromosome (BAC) recombineering in mice resulted in autisticlike neurobehavioral phenotypes, including defects in communication, abnormal social be‐ havior, and increased repetitive or stereotypic behavior [62]. Similar to the phenomenon observed in human patients, these effects were also determined to be dosage-sensitive, as the phenotypes were more penetrant in mice with three-fold overexpression of *Ube3a* than in those observed in mice with two-fold overexpression of this gene. Furthermore, it was de‐ termined that glutamatergic synaptic transmission was suppressed, providing a potential mechanism underlying the neurobehavioral phenotypes.
