**10. Modeling SMS and PTLS in rodents**

**9.** *RAI1***: Retinoic acid induced 1**

262 Recent Advances in Autism Spectrum Disorders - Volume I

thus likely cause loss-of-function alleles [82, 97].

nuclear transcriptional cofactor [101].

ASD phenotypes in non-syndromic patients as well [103].

*Rai1-/-*

Spanning over 120 kb, the *RAI1* gene consists of six exons, of which the third is the largest, containing >90% of the coding region [80, 85, 96]. All of the point mutations identified in SMS patients to date lie within this exon. Most of the mutations are frameshift or nonsense mutations occurring in a specific heptameric poly C tract hotspot region within *RAI1*, and

The *RAI1* transcript is 7.6 kb, encoding a 1906-amino acid, ~200 kDa protein with sever‐ al known domains, including an extended plant homeodomain (PHD) zinc finger in the carboxyl-terminus (residues 1832-1903; [80]), a polymorphic polyglutamine (CAG) tract in the N-terminus that is associated with the severity of the phenotype and medication response in patients with schizophrenia, as well as the age-at-onset of spino-cerebellar ataxia type 2 (SCA2) [96, 98], two polyserine tracts, two transactivation domains [99], and two bipartite nuclear localization signals (NLS). Importantly, the PHD in *Rai1* is highly conserved in the trithorax family of nuclear proteins involved in transcriptional regulation as well as in the formation of a chromatin remodeling complex, suggesting that Rai1 may also function as a transcriptional regulator [100]. Further strengthening this connection, Rai1 is known to be located in the nucleus and have transactivation ac‐ tivity [99], and it shares a similar genomic structure (>50% shared identity and similar zinc finger domains) with another gene, *TCF20*, or stromelysin1 platelet-derived growth factor (PDGF)-responsive element-binding protein (*SPBP*), which is known to act as a

In the human brain, *RAI1* is highly-expressed in the hippocampus and the cerebellar cor‐ tex, and it is globally-upregulated in the occipital, temporal, and parietal lobes according to expression data from the Allen Brain Atlas (Allen Institute for Brain Science). In con‐ trast, it appears to be down-regulated in the cerebellar nuclei, corpus callosum, dorsal thalamus, and frontal lobe, suggesting that its expression is confined to specific brain re‐ gions. Similar to what is seen in humans, *Rai1* is also upregulated in the hippocampus and cerebellum of adult mice [102]. *Rai1* is critical for development, and the majority of

 mouse embryos are resorbed during development by E15.5 [99]. While *Rai1* expres‐ sion is certainly necessary early in fetal development, according to expression data from mouse embryos, peak *Rai1* expression occurs at E18.5 and persists until P4 (Allen Brain Atlas), indicating that it is also required for post-natal development. Although the pre‐ cise function of *RAI1/Rai1* is not currently understood, it is known to be part of a dos‐ age-sensitive pathway that most likely regulates neuronal development and organogensis, that, when perturbed, results in many of the phenotypes observed in both SMS and PTLS. Importantly, RAI1 has been identified in a reconstructed human genenetwork (Prioritizer) as an important candidate gene for involvement in idiopathic au‐ tism, suggesting that this gene may function in a common pathway that may influence Several mouse models interrogating the critical region for SMS and PTLS have been generat‐ ed in the past decade in order to have an appropriate animal model system to evaluate the phenotypes in SMS and PTLS and to further study the molecular mechanism underlying these disorders. The first of these strains was developed in 2003 using a chromosome-engi‐ neering approach described earlier in this chapter [24]. The resulting mouse models harbour either a chromosomal duplication (*Dp(11)17,* modeling PTLS) or deletion (*Df(11)17,* model‐ ing SMS) of ~2 Mb that is syntenic to the SMS/PTLS critical region. Soon after, several small‐ er deletion strains (~590 kb – 1 Mb) were created using retroviral insertion of *loxP* sites in ES cells with one fixed end, with the intent to determine which other genes in the critical region may contribute to the complex phenotypes in SMS [104, 105]. Once SMS patients with point mutations in *RAI1* were identified, a mouse model harbouring a truncated null allele for *Rai1* was generated via gene targeting to further study the function of this dosage-sensitive gene and to compare the phenotype of this model with that of the deletion strains [99, 102]. Likewise, a mouse model harbouring the *Rai1* transgene (*TgRai1*), and globally over-ex‐ pressing *Rai1* at steady-state levels similar to those seen in *Dp(11)17/+* mice, was constructed to further study the function of this gene in PTLS [106].

Initial studies of *Dp(11)17/+* mice determined that they have reduced weight, reduced ab‐ dominal and inguinal fat, and reduced spleen weight [24]. Upon analysis of some of the be‐ havioral traits of these mice, it was determined that they display anxiety-like behaviors, have reduced maximum startle response during the pre-pulse inhibition test, and defects in contextual fear conditioning [107], as well as several other abnormal social behaviors, in‐ cluding decreased nesting, abnormal sociability, and dominant behaviour [108]. Further in‐ vestigation into the neurobehavioral abnormalities in these mice found that they also have decreased preference for social novelty, motor defects, and increased activity levels in the open field [109]. Many of these behavioral phenotypes are reciprocal or opposing to those seen in *Df(11)17/+* mice, underscoring the dosage-sensitive nature of these disorders [109]. For example, a recent study investigating cerebellum-driven licking behavior in *Dp(11)17/+* and *Df(11)17/+* mice found that many of the quantitative licking behavior parameters ana‐ lyzed were altered in a directly-opposing manner [110]. Specifically, the interval between visits to the waterspout, number of licks per visit, and variability in the number of licks per lick-burst were all altered in duplication and deletion animals in opposite directions com‐ pared to wild-type mice (ex: longer versus shorter intervals, etc).

Recently, an extensive battery of behavioral tests were performed and *Dp(11)17/+* mice were observed to display complex social abnormalities, including defects in social recognition, dominant and aggressive behavior, as well as abnormal response to social odors [30]. Fur‐ thermore, these mice were shown to have altered communication, anxiety-like behavior, dis‐ ordered circadian rhythm, learning and memory deficits, motor defects, and stereotypic, repetitive behaviors, confirming that these mice model both the core and associated features of autism. In addition, rearing these mice in an enriched environment mitigated or rescued certain neurobehavioral abnormalities, suggesting a role for gene-environment interactions in the determination of copy number variation-mediated autism severity [30].

Chromosome-engineered mouse models for ASD are ideal for the study of complex disease, as they are mechanistically similar to human patients (targeted duplication/deletion synten‐ ic to human critical interval), they are polygenic (numerous genes are affected), the observed phenotypes equate with common, clinically described features (neurobehavioral pheno‐ types, sleep disorder, etc), and they can be influenced by environmental factors. In addition, autism is known to be highly variable, and it is suspected to be dependent on both genetic and environmental factors, such as low birth weight and gestational age, prenatal exposure to various agents, parental age at birth, diet, infection, xenobiotic and pesticide exposure, among others [113]. Many of these environmental insults are amenable to study using mouse models, as the interaction of these environmental factors with CNVs can be directly

Advances in Autism Research – The Genomic Basis of ASD

http://dx.doi.org/10.5772/53689

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tested in congenic mouse models to control for the effects of genetic background.

logical and genetic mechanisms.

and James R. Lupski1,2

\*Address all correspondence to: jlupski@bcm.edu

**Author details**

Melanie Lacaria1

Houston, TX, USA

**References**

USA

Molecular analysis of these mouse models, as well as patient samples, can also be utilized to dissect the role of specific genes or CNVs responsible for the susceptibility to the influence of environmental factors in these autism-related syndromes. Most importantly, the results of these types of studies can provide useful insights as to how genes/CNVs can interact with environmental factors in the context of complex human diseases; this may lead to strategies to alleviate symptoms of not only rare genomic disorders, but also more common idiopathic forms of autism or ASD. Furthermore, these models represent an important resource for fu‐ ture studies of the pathomechanisms underlying ASD, as well as potential treatments for ASD. They may also foster further investigation into the genomic basis of autism and com‐ plex behavior, as well the underlying genetic mechanisms leading to these pathogenic CNVs. In studying CNV-based models for complex genomic disorders and ASD, we have come to realize that the ideal animal models of ASD should not only phenocopy relevant human symptoms, but the phenotypes should also be based on similar underlying physio‐

1 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX,

2 Department of Pediatrics, Baylor College of Medicine, and Texas Children's Hospital,

[1] Jacquemont ML, Sanlaville D, Redon R, Raoul O, Cormier-Daire V, Lyonnet S, et al. Array-based comparative genomic hybridisation identifies high frequency of cryptic

The phenotypic changes observed in *Dp(11)17/+* and *Df(11017/+* mice are accompanied by changes in gene expression; on average, transcripts in the critical interval are expressed at 138 + 29% of wild-type levels in *Dp(11)17/+* mice, and at 66 + 15% of wild-type levels in *Df(11)17/+* mice [109]. The expression level of these genes can be normalized to roughly that of wild-type mice by crossing *Dp(11)17/+* and *Df(11)17/+* mice to create a double heterozy‐ gote carrying two copies of the genes within the critical region in *cis*, (as opposed to the typi‐ cal *trans* orientation in wild-type mice). However, the presence of the structural variation itself affects expression of genes outside the affected interval, resulting in "genome regula‐ tion" that may ultimately contribute to the phenotype. As a result, these double heterozy‐ gous mice display some abnormal behaviors, including elevated activity levels and decreased preference for social novelty [109].

When *Rai1* is over-expressed in mice (*Rai1-Tg*, modeling PTLS), these mice have growth re‐ tardation, are underweight, display anxiety-like behavior, social dominance, motor abnor‐ malities, and have increased motor activity in juvenile mice. Furthermore, there is a dosagedependent exacerbation of this phenotype [106]. These mice also display abnormal maternal behavior, altered sociability, reduced reproductive fitness, and impaired serotonin metabo‐ lism [111]. Together these results suggest that *Rai1-Tg* mice display a complex neurobeha‐ vioral and metabolic phenotype similar to that of mice harboring the *Dp(11)17* CNV, suggesting that *RAI1* is likely responsible for some, if not many of the phenotypes identified in PTLS. Further support for this hypothesis is indicated by studies of *Dp(11)17/Rai1* doubleheterozygous mice with normalized copy number of *Rai1*, but increased dosage of the sur‐ rounding interval; this study revealed that normalization of *Rai1* copy number was able to correct weight differences, and at least partially rescue phenotypes on behavioral tests for locomotor activity, anxiety, and learning and memory [95].

### **11. Gene-environment interactions**

While genetic defects play a part in the etiology of ASD, environmental effects have long been thought to contribute to these disorders. For example, although the majority of SMS/PTLS patients present with either deletion or duplication of the same ~3.7 Mb genedense region, there is significant variability in the clinical phenotype [112]. Furthermore, while there are some significant differences in the incidence of the abnormalities in pa‐ tients with the common deletion/duplication compared to those patients with smaller or larger-sized CNVs, a clear distinction between these sub-groups of patients cannot be made; many of these phenotypes are therefore likely strongly influenced by genetic back‐ ground as well as environmental effects [83, 94]. While gene-environment interactions may potentially explain the source of the variability seen in these syndromes, investiga‐ tion into the specific environmental factors that may affect outcomes for these genomic disorders has yet to be undertaken.

Chromosome-engineered mouse models for ASD are ideal for the study of complex disease, as they are mechanistically similar to human patients (targeted duplication/deletion synten‐ ic to human critical interval), they are polygenic (numerous genes are affected), the observed phenotypes equate with common, clinically described features (neurobehavioral pheno‐ types, sleep disorder, etc), and they can be influenced by environmental factors. In addition, autism is known to be highly variable, and it is suspected to be dependent on both genetic and environmental factors, such as low birth weight and gestational age, prenatal exposure to various agents, parental age at birth, diet, infection, xenobiotic and pesticide exposure, among others [113]. Many of these environmental insults are amenable to study using mouse models, as the interaction of these environmental factors with CNVs can be directly tested in congenic mouse models to control for the effects of genetic background.

Molecular analysis of these mouse models, as well as patient samples, can also be utilized to dissect the role of specific genes or CNVs responsible for the susceptibility to the influence of environmental factors in these autism-related syndromes. Most importantly, the results of these types of studies can provide useful insights as to how genes/CNVs can interact with environmental factors in the context of complex human diseases; this may lead to strategies to alleviate symptoms of not only rare genomic disorders, but also more common idiopathic forms of autism or ASD. Furthermore, these models represent an important resource for fu‐ ture studies of the pathomechanisms underlying ASD, as well as potential treatments for ASD. They may also foster further investigation into the genomic basis of autism and com‐ plex behavior, as well the underlying genetic mechanisms leading to these pathogenic CNVs. In studying CNV-based models for complex genomic disorders and ASD, we have come to realize that the ideal animal models of ASD should not only phenocopy relevant human symptoms, but the phenotypes should also be based on similar underlying physio‐ logical and genetic mechanisms.
