**9. Copy Number Variations (CNVs) in ASD**

Copy Number Variations (CNVs), submicroscopic variations that are less than 500kb in size in DNA, are widespread in normal human genomes. The most common CNVs tend to be large (> 500Kb) and contain several genes, while rare or *de novo* CNVs are smaller (< 100Kb), sometimes interrupting a single gene. On average, there are more than 1000 CNVs in the ge‐ nome, accounting for ∼4 million base pairs of genomic difference. Although SNPs outnum‐ ber CNVs in the genome by three orders of magnitude, their relative contributions to genomic variation (as measured in nucleotides) are similar. Thus, in addition to 0.1% of ge‐ netic difference at the nucleotide sequence level, another 0.1% of genetic difference is appa‐ rent at the structural level (Malhotra & Sebat, 2012).

vestigated both the total level of ASD symptoms as well as scores of three ASD symptom domains, thus taking into account potential differential genetic origins of different ASD symptom domains. QTL linkage analyses for the different ASD domains were carried out using 5407 SNPs spanning the entire genome. Findings suggest that some QTLs are ASD specific, although the 15q QTL potentially has pleiotropic effects for ADHD and ASD (Nij‐ meijer et al., 2011). The genetic analysis of quantitative traits that are phenotypically linked, such as in ASD and ADHD, can reduce the heterogeneity of diagnosis and indicate loci re‐

There is a QTL related to language delay located close to the 7q35-36 region. Interestingly this region is mapped in the *CNTNAP2* gene, a strong candidate for predisposition to autis‐ tic behavior; it is well known that communication abilities are qualitatively impaired in au‐ tistic individuals. The significant delay in language ("age of first word") is observed in about half of affected children (Alárcon et al., 2008). The relationship of SNPs in the *CNTNAP2* gene has already been described, as has the association of the gene and its SNPs to language development delay in autistic and non-autistic individuals (Alárcon et al., 2008;

Additionally, the transcription factor encoded by the *FOXP2* gene has already been linked to the development of language. This factor binds to the promoter of the *CNTNAP2* gene regu‐ lating its expression during development. There are reports of changes in the *FOXP2* bind‐ ing site in patients with ASD, which suggests that a reduced expression of the *CNTNAP2* gene may be the underlying etiology of one of the phenotypic characteristics of ASD (Vernes et al., 2008; Poot, et al., 2009). In addition to these, it has been suggested that *WNT2* and *EN2*

Promising results on the influence of genetic bases in neurobehavioral disorders have also been obtained through studies on CNVs. All these emerging genetic technologies have brought more valuable approaches to improve the understanding of the etiology of ASD. Advances in the use of molecular biology tools have provided a promising manner to study gene-gene and gene-environment relationships in disorders (Gurrieri, 2012; Li et al., 2012). This combination of tools in the search for the etiology will reflect in the possibility of target‐ ing the diagnosis, prognosis, early interventions and genetic counseling. However, more da‐ ta and the reproducibility of findings are necessary to establish the genetic components of

Copy Number Variations (CNVs), submicroscopic variations that are less than 500kb in size in DNA, are widespread in normal human genomes. The most common CNVs tend to be large (> 500Kb) and contain several genes, while rare or *de novo* CNVs are smaller (< 100Kb), sometimes interrupting a single gene. On average, there are more than 1000 CNVs in the ge‐ nome, accounting for ∼4 million base pairs of genomic difference. Although SNPs outnum‐ ber CNVs in the genome by three orders of magnitude, their relative contributions to

Arking et al., 2008; Tan et al., 2010; Stein et al., 2011; Whalley et al., 2011).

are related to language development in autism (Lin et al., 2012).

**9. Copy Number Variations (CNVs) in ASD**

lated to susceptibility (Lu et al., 2011).

224 Recent Advances in Autism Spectrum Disorders - Volume I

these diseases.

The rate of genome-wide nucleotide substitutions is estimated at 30–100 per generation and ∼1 per exome. In contrast, the global rate of structural mutation events is lower: CNVs > 10 Kb in size occur at a rate of ∼0.01–0.02 per generation (Marshall et al., 2008; Conrad et al., 2011; Levy et al., 2011; Sanders et al., 2011). Nucleotide substitutions probably account for the majority of disease risk alleles, but based on sheer size and potential to impact genes (or multiple genes), structural mutations are, on average, more pathogenic. Thus, CNVs, *de novo* CNVs in particular, seem to be a class of variants that have large effect on disease risk (Mal‐ hotra & Sebat).

CNVs are gaining importance in the scenario of ASD. They represent a significant source of genetic diversity and seem to significantly contribute to changed behavioral phenotypes (Se‐ bat et al., 2007; Rees et al., 2011). To have an idea, *de novo* CNVs have already been reported to be three to five times more common in families of individuals with ASD than in controls, and more often presenting the syndromic form of autism, that is, with the most severe phe‐ notypes (Miller et al., 2010; Pinto et al., 2010; Shen et al., 2010; Sanders et al., 2011). In fact, CNVs, in particular *de novo* CNVs involving many genes, confer risk for ASD. However, al‐ though they are important in this respect, they rarely interrupt a single gene or have com‐ plete penetrance and many give a wide-ranging risk including risk for other problems such as intellectual deficiency, epilepsy and schizophrenia (Geschwind, 2011; O'Roak et al., 2011).

Up to 40% of CNVs in autism are inherited from apparently normal parents, consistent with the suggestion of incomplete penetrance. Both *de novo* (non- inherited) or inherited CNVs occur at the same locus in unrelated individuals, and some of them coincide with those seen in other gene-related diseases associated with ASD, including developmental delay and in‐ tellectual deficiency (Cook & Scherer, 2008; Lee & Scherer, 2010). Thus, some apparently have a pleiotropic effect.

*De novo* CNVs have been observed in from 7-10% of cases in simplex families, in 2-3% in multiplex families and approximately 1% of normal controls. Rare *de novo* CNVs have al‐ ready been observed in 5.8-7.9% of carriers and in 1.7-1.9% of unaffected siblings in simplex families (Levy et al., 2011; Sanders et al., 2011), while *de novo* mutations in coding regions participate in < 20% of cases of ASD (Malhotra & Sebat, 2012). In addition, about 10% of ASD cases with *de novo* CNVs have two or more CNVs (Sebat et al., 2007; Christian et al., 2008; Marshall et al., 2008).

Many of the variations occur in gene regions that contain synaptic genes, and it seems that some involve haploinsufficient regions or dominant inheritance. Others seem to express re‐ cessive forms as in the cases of the *NHE926*, *PCDH10* and *DLA1* genes identified in studies of individuals with consanguineous parents. Other rare variations were found deleted in ho‐ mozygous (Bourgeron, 2009; Ramocki & Zoghbi, 2008; Morrow et al., 2010).

There are descriptions of *de novo* and inherited CNVs, sometimes in combination in a given family, implicating many novel ASD genes such as *SHANK2, SYNGAP1, DLGAP2* and the X-linked *DDX53-PTCHD1* locus, of an enrichment of CNVs disrupting functional gene sets involved in cellular proliferation, projection and motility, and GTPase/Ras signaling, (Pinto et al., 2010). Another pathway with many of these variations is that related to the develop‐ ment of synapses, and maintenance and motility of neurons, the NLGN-NRXN-SHANK pathway (Bourgeron, 2009).

established before glutamatergic transmission suggesting that GABA is the principal excita‐ tory transmitter during early development (Takayama & Inoue, 2004; Ben-Ari et al., 2007).

Genetic Etiology of Autism http://dx.doi.org/10.5772/53106 227

In multiplex families, i.e. those with more than one affected individual, different individuals have similar microdeletions or microduplications and exhibit autistic, broad or normal phe‐ notypes (Fernandez et al., 2010; Kumar et al., 2008). The microduplication of 15q11-13, which seems to have a major impact on the expression of ASD, is a good example of differ‐ ent effects in carriers. For example, Veltman et al. (2005) reported a female proband referred for evaluation of a possible ASD. Genetic analyses indicated that the proband, her father and one of her sisters, carried a paternally derived interstitial duplication involving 15q11-13. The proband showed evidence of ASD (PDD-NOS), borderline mental retardation, mild hypotonia and joint laxity. Her father and her sister were of normal intelligence and neither was thought to have an ASD, although speech/language difficulties and some autis‐ tic type behaviors were reported to have been present early in the development of the sister.

In studies of idiopathic cases, the most common change is the 16p11.2 microduplication/ microdeletion, comprising a region of around 600 Kb. Microduplication of 16p11.2 is present in autistic and schizophrenic individuals but is also seen in normal individuals. This varia‐ tion is also observed in cases of ASD with and without dysmorphic signs (Fernandez et al., 2010; Shinawi et al., 2010). Microdeletions of 16p11.2 seem to be more penetrating (around 100%) and are associated with the presence of major dysmorphic signs, while microduplica‐ tions have reduced penetrance (~50%) and are associated with minor dysmorphic signs (Ra‐

One possible explanation for these cases, as already mentioned in previous sections, is that probably there are other genetic components that are contributing to the disease in these families that, depending on how they are associated in individuals, result in different phe‐ notypes (Griswold et al., 2012). But it is also very likely that different CNVs display different penetrance depending on the sensitivity of the affected gene to dosage (number of copies),

Endophenotypes (or intermediate phenotypes) are defined as heritable traits that form a causal link between genes and observable symptoms. Brain based endophenotypes offer several important advantages over clinical phenotypes in the search for pleiotropic genes in ASD. They provide insight in the causal chains of action leading from gene to symptom ex‐ pression, they aid in forming etiologically more homogeneous subgroups of patients (Gould

In addition to the variability of simple and more complex genetic mechanisms proposed in the expression of the ASD phenotype, environmental factors via epigenetic mechanisms

mocki & Zoghbi, 2008; Bourgeron, 2009; Fernandez et al., 2010).

the function of the gene and the affected region.

& Gottesman, 2006).

seem to be very important.

But, CNVs have been regarded as "rare variants" within the panorama of the etiology of ASD and it is important to emphasize the care that should be taken in associating them in the etiology, especially those not previously described. Accuracy is required in the study of these changes during the formation of control groups. On the other hand, when it comes to autism, on finding the same CNVs in carriers and controls is not a reason to dismiss their participation in the phenotype, given the complexity of factors that may be acting jointly and in combination in different cases.
