**12. Exome sequencing**

**11. Epigenetic in autism**

228 Recent Advances in Autism Spectrum Disorders - Volume I

mental factors via an epigenetic mechanism.

intragenic promoters (Uchino et al., 2006).

Particularly in light of recent findings on mutations in the genes that encode synaptic mole‐ cules associated with the communication between neurons, genetic factors are considered to be the most important contributors to the pathogenesis of autism. Epigenetic mechanisms, such as DNA methylation and modifications to histone proteins, regulate DNA structure and gene expression, but without changing DNA sequence. Epigenetic abnormalities are as‐ sociated with several neurodevelopmental diseases. Many features of autism are consistent with an epigenetic dysregulation, such as discordance of monozygotic twins, parental origin and the gender-dependent effects of some alterations. Since epigenetic modifications are known to be affected by environmental factors such as nutrition, drugs and mental stress, ASD are not only caused by congenital genetic defects, but may also be caused by environ‐

An example of this phenomenon in ASD is the mechanism of action of the *SHANK3* gene. *SHANK3* is strongly suspected of being involved in the etiology of ASD since several muta‐ tions have been identified in a particular phenotypic group of patients with ASD. SHANK3 (also known as *ProSAP2*) regulates the structural organization of dendritic spines and is a binding partner of neuroligins; genes encoding neuroligins are mutated in autism and As‐ perger's syndrome (Durand et al., 2007). It codes a synaptic scaffolding protein enriched in the postsynaptic density of excitatory synapses and plays important roles in the formation, maturation and maintenance of synapses. Haploinsufficiency of the *SHANK3* gene causes a developmental disorder, 22q13.3 deletion syndrome (known as Phelan-McDermid syn‐ drome), which is characterized by severe language and speech delay, hypotonia, global de‐ velopmental delay and autistic behavior. Five CpG-islands have been identified in the gene, and tissue-specific expression is epigenetically regulated by DNA methylation. Cumulative evidence in animal models has shown that several *SHANK3* variants are expressed in the developing rodent brain with their expression being regulated by the DNA methylation of

Additionally, oxidative stress in brain cells occurs due to environmental and genetic causes and leads to decreased activity of the methionine synthase enzyme, which participates in DNA methylation processes. So, when the activity of this enzyme is impaired, affected indi‐ viduals can exhibit attention deficits and other signs, including autistic symptoms, due to defects in the expression of genes that are controlled by this epigenetic mechanism (Na‐ viaux, 2008; Dhillon et al., 2011).Therefore, environmental factors may activate intracellular pathways during embryonic development thereby causing epigenetic changes in neural function that would explain the relationship between environmental signals and genome in

Studies carried out in Sweden involving 208 autistic children with Pervasive Developmental Disorder-Not Otherwise Specified (PDD-NOS) and Asperger's syndrome (AS) obtained in‐ teresting results. The authors observed that advancing paternal age is associated with an in‐ creasing risk for ASD in offspring, that autistic-like traits in the normal population are affected by both young and advancing paternal age and that autistic similarity within pairs

the regulation of individual differences in behavior (Zhang & Meaney, 2010).

The development of the next generation sequencing has radically modified the scientific landscape, making it possible to sequence all exomes of any person. The power of this ap‐ proach has been demonstrated by a number of studies which have identified pathogenic mutations in diseases that have been difficult to make by traditional genetic mapping.

Exons are coding regions of the genome responsible for the development of functional ele‐ ments of the body, such as proteins. It has become clear that exome sequencing has great potential with respect to sporadic diseases and the identification of *de novo* mutations. Whole-exome sequencing (WES) of patient-parent trios has proven to be a successful screen‐ ing tool for the identification of candidate genes causing complex phenotypes such as in ASD, learning disability, and schizophrenia (Choi et al., 2009; Bilguvar et al., 2010; Vissers et al., 2010; Gilman et al., 2011; Pagnamenta et al., 2012; Sanders et al., 2012).

O´Roak et al. (2012) studied all coding regions of the genome for parent-child trios exhibit‐ ing sporadic ASD, under the hypothesis that *de novo* mutations underlie a substantial frac‐ tion of the risk for developing ASD in families with no previous history of ASD or related phenotypes (simplex families). They observed that these mutations are overwhelmingly pa‐ ternal in origin and positively correlated with paternal age, consistent with the modest in‐ creased risk for children of older fathers. It was also possible to estimate 384-821 loci which could be considered pathogenic.

The analysis the homozygosity is used to define loci that may be involved in recessive ho‐ mozygous mutations that cause diseases characterized by genetic heterogeneity (Lencz et al., 2007; Nalls et al., 2009). Starting from this concept, Chahrour et al. (2012) used homo‐ zygosity analysis to identify probands from non-consanguineous families that showed evi‐ dence of distant shared ancestry, suggesting potentially recessive mutations. The WES of 16 probands revealed validated homozygous, potentially pathogenic, recessive mutations that segregated perfectly the disease in 4 of 16 families. The candidate genes that were found, *UBE3B, CLTCL1, NCKAP5L,* and *ZNF18,* encode proteins involved in neuronal activities.

Neale et al. (2012) studied 175 trios to assess *de novo* mutations. They found strong evidence that *CHD8* and *KATNAL2* are genuine autism risk factors. However, the small increase in the rate of *de novo* events, when taken together with the protein interaction results, were con‐ sistent with an important but limited role for these mutations in ASD, similar to that docu‐ mented for *de novo* CNVs. According the authors, the data indicated that most of the observed *de novo* events are unconnected to ASD; those that do confer risk are distributed across many genes and are incompletely penetrant. The results support polygenic models in which spontaneous coding mutations in any of a large number of genes increases risk by 5 to 20-fold.

**13. Conclusion**

**Acknowledgements**

**Author details**

Agnes Cristina Fett-Conte1

Knowledge about the biological mechanisms involved in the etiology of ASD has increased significantly over the past three years. A genetic etiology of these disorders is certain, as cer‐ tain as is their complexity. An understanding of the genetic factors involved is crucial to es‐ tablish future intervention strategies. Although the current emphasis on deciphering ASD has demonstrated the necessity of multidisciplinary approaches, clinical geneticists have an important role in diagnosis and research of autism. The interpretation of this new genetic data requires a set of skills. It is important to know how to get and to interpret genetic tests, family pedigrees, to analyze dysmorphic, neurologic, and medical phenotypes, to interpret

Despite the numerous known or, at least, allegedly involved causes of predisposition for ASD, the etiology is identified in a few cases (~ 10%) thereby highlighting the importance of genetic testing in affected individuals. The discovery of an etiological agent in a given case will, very probably, not interfere in treatment. However, this will reduce the distress of pa‐ rents by explaining the cause of the problem and clarify about the possibility of familial re‐ currence. On identifying the etiologic agent, genetic counseling can be better targeted. Thus, a clinical-genetic evaluation of the patient is important as are the karyotypic analysis, molec‐ ular test for FRAXA, the investigation of inborn errors of metabolism, performing imaging tests and multiplex ligation-dependent probe amplification (MLPA) for at least three hot spots in ASD (15q11-13, 16p11.2 and 22q11.2). These are strategies available to better assess the etiology ASD. Certainly, in the not too distant future, other more sophisticated genetic research tools will be commercially available. The question that remains is whether the in‐

heterogeneity, develop rational genetic models, and to design researchs.

terpretation of results will accompany the speed of technical advances.

Financial support: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP).

, Ana Luiza Bossolani-Martins2

1 Medical School – FAMERP/FUNFARME, São José do Rio Preto, São Paulo, Brazil

2 IBILCE/UNESP, São José do Rio Preto, São Paulo, Brazil

and Patrícia Pereira-Nascimento2

Genetic Etiology of Autism http://dx.doi.org/10.5772/53106 231

Iossifov et al. (2012) did not find significantly greater numbers of *de novo* missense muta‐ tions in ASD children versus unaffected, but gene-disrupting mutations (nonsense, splice site and frameshifts) were twice as frequent in the first group. Based on this differential and the number of recurrent and total targets of gene disruption they estimated between 350 and 400 autism susceptibility genes. Many of the genes are associated to the FMRP protein, rein‐ forcing links between autism and synaptic plasticity. They suggested that genes associated to *FMRP* are especially targets of cognitive disorders that are dosage-sensitive.

Another aspect of exomes should also be considered. Mitochondria are cellular organelles that function to control energy production necessary for brain development and activity. Al‐ though each individual is typically characterized by a single mitochondrial DNA type, the fact is that each individual is a population of mitocondrial DNA genomes, and the presence of multiple types within an individual is termed heteroplasmy. Although each individual is typically characterized by a single mitocondrial DNA type, in fact to date, more than 400 mi‐ tochondrial mutations have been associated with human disease and most were observed in heteroplasmic states, with pathogenic mutations coexisting with normal mitochondrial ge‐ nomes. This suggests that the heteroplasmic level is of particular interest, as the disease phe‐ notype becomes evident only when the percentage of mutant molecules exceeds a critical threshold value. Although this value differs for different mutations and in different tissues, it is usually in the range of 70%~90%. However, all the various techniques that have been employed to detect heteroplasmy have disadvantages. WES allows rapid detection of not only nuclear mutations but also mitochondrial mutations that also seem to be involved in the etiology of ASD. In this context, Li et al. (2012) sequenced the mitochondrial genome of 131 healthy individuals of European ancestry. In 32 individuals they identified 37 hetero‐ plasmies at frequencies of 10% or higher at 34 different sites in the mitochondrial DNA indi‐ cating that variations commonly occur in mitochondrial DNA. These variations may impact on energy levels and influence brain development and function. Next generation sequencing should provide novel insights into genome-wide aspects of variation or heteroplasmy useful in the study of human disorders including autism.

All these results show that there are a lot of regions/genes being identified by very advanced methods, but no common etiology can be proposed. It is clear that whatever the proposed model to explain ASD, all aspects such as environmental, oligogenic, *de novo* mutations, polygenic, multifactorial, pleiotropic effects, combination of locus heterogeneity, heteroplas‐ my, among others, do not apply to all cases. Perhaps ASD emerge due to highly specific and individual biological patterns. The possibility of distinguishing primary and secondary ef‐ fects will require a better understanding of the underlying biology and identification of the association between genetic and environmental factors within the phenotypic context of each family. The bottom line is that you must have a systemic view of the problem.
