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

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

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

to further study the function of this gene in PTLS [106].

pared to wild-type mice (ex: longer versus shorter intervals, etc).

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

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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‐

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

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 nuclear transcriptional cofactor [101].

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 *Rai1-/-* 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 ASD phenotypes in non-syndromic patients as well [103].
