**1. Introduction**

[156] Zhao, X., Leotta, A., Kustanovich, V., Lajonchere, C., Geschwind, D. H., Law, K.,

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248 Recent Advances in Autism Spectrum Disorders - Volume I

Law, P., Qiu, S., Lord, C., Sebat, J., Ye, K., & Wigler, M. (2007). A unified genetic theory for sporadic and inherited autism. Proc Nati Acad of Sci U S A., , 104(3),

> Many of the recent advances in autism research that have provided fundamental insight into this condition have come from the application of genetic/genomic approaches; these advances have been fomented by the advent of new technologies to interrogate the en‐ tire genome, such as array comparative genomic hybridization (aCGH), single nucleotide polymorphism (SNP) microarrays, transcriptome sequencing, and whole genome or whole exome sequencing (WGS/WES) [1]. With the recent advancement of these technol‐ ogies over more traditional, lower-resolution technologies such as cytogenetic analysis, came the ability to interrogate the entire genome at a high-resolution. With the improve‐ ment of next-generation sequencing technology, as well as the reduction in the cost of this technique, WGS is becoming more commonplace in the search for novel diseasecausing variants in individual patients. Alternatively, many studies have utilized WES, as it is less costly than sequencing the entire genome and coding simple nucleotide var‐ iants (SNVs) can often be more readily interpreted given knowledge provided by the ge‐ netic code. While the reduced cost and more readily interpretable variation have proven to be distinct advantages of this method over whole-genome sequencing, it is well known that many other variants in non-coding or regulatory regions can be pathogenic, and they typically cannot be discerned by whole-exome sequencing, which requires a targeted-capture step to enrich for and focus analysis on the coding sequences of all an‐ notated protein-coding genes [2, 3]. Furthermore, repetitive or G-C rich regions or highly homologous sequences are often excluded by WES, and copy number variations (CNVs) usually cannot be accurately called due to the use of PCR-based sample preparation methods. Nonetheless, the utility of WGS/WES in individual patient diagnosis and man‐ agement has been demonstrated by several recent reports [4-6].

© 2013 Lacaria and Lupski; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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

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

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

Advances in Autism Research – The Genomic Basis of ASD

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Crawley et al, where a full description of these behavioral tests can be found; [23]).

manner [27]. (http://www.sanger.ac.uk/resources/mouse/micer/).

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

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 consortium in the US by WES.

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 phenotypes commonly associated with ASD [22].
