**10. Epigenetic: genomic imprinting, epimutations, histone and DNA methylation**

Epigenetic is a term used to refer to features of organisms, such as DNA and chromatin modifications, that do not involve changes in DNA sequence. The effects of environment on the phenotype are generally mediated through epigenetic mechanisms. These mechanisms, such as DNA methylation, can become programmed (e.g. imprinted). Genomic imprinting is a unique phenomenon wherein genes are expressed in a monoallelic way, and the choice of which allele is expressed is determined by the parental origin of the allele. Disruptions of the epigenome are called epimutations. Some of these appear to be corrected by normal germlinespecific epigenetic reprogramming and are therefore not transmitted transgenerationally, but others are not corrected and are transmitted over multiple subsequent generations [97].

Epigenetic mechanisms act on chromatin accessibility to transcriptional regulation. Then, they regulate DNA structure and gene expression that can be influenced by exposure to environ‐ mental factors. The most studied are the methylation of genes and modification of histones. Interestingly, epigenetic abnormalities are associated with several neurodevelopmental diseases. The connection between ASD and epigenetic comes from the identification of genetic mutations in imprinted regions and genes that control epigenetic processes. As cited before, among the most common chromosomal alterations in ASD are duplications of the imprinted region 15q11–13, which is maternally inheritable [96].

There are several possible explanations for the involvement of imprinted genes in autism. The imprinted brain theory of autism suggests that autism is a disorder of the extreme imprinted brain and would be caused by imbalances that involve increased effects of the 'paternal brain' relative to the 'maternal brain'. Imprinting has been hypothesized to explain the gender difference through the proposed action of unknown paternally imprinted loci on the X chromosome. Also, as mentioned, autism has been strongly associated with chromosomal abnormalities in the imprinted region of chromosome 15q. This includes the Angelman and Prader-Willi syndromes, as well the 15q duplication syndrome, which occurs in up to 5% of individuals with ASD. Imprinted genes may also contribute to autism indirectly as targets of other genes such a regulatory connection between *MECP2* (the gene associated with Rett syndrome) and the imprinted gene *UBE3A* (associated with Angelman syndrome). Besides this, imprinted genes are candidates for association with autism because of their functional haploid state. This feature may make them extremely vulnerable to rare mutations because the gene may be inactivated. Moreover, a single epigenetic change may lead to loss of imprinting, leading to biallelic expression and to gene dysregulation [173]. Both genetic and environmental factors can affect the imprinting process and alter the level of expression of genes. But the full contribution of genomic imprinting to the risk for autism is still unclear.

The imprinted genes are expressed in several types of tissues but are highly expressed in the brain. Human neurons require extensive methyl modifications throughout development and postnatal life. Several important posttranslational modifications of histone core subunits within nucleosomes involve methylation, an epigenetic mechanism. Recent unbiased genomewide analyses have turned up a multitude of novel candidate genes that encode nuclear factors implicated in chromatin remodeling, histone demethylation, histone variants, and the recognition of DNA methylation. Both histone and DNA methylation patterns are highly dynamic processes in the early development phase that correlate with dynamic changes in cell lineage and differentiation events. Interestingly, mutations in autism have been found in several genes encoding proteins involved in demethylase reactions, that is, reactions that remove methyl groups from histones or DNA [81].

The mechanism of action of the *SHANK3* gene (also known as ProSAP2) is an example of this phenomenon in ASD. The three members of the SHANK family, *SHANK1*, *SHANK2* and *SHANK3* are expressed in different regions of the brain. *SHANK3* is strongly reported to be involved in the etiology of autism since several mutations have been identified in a particular phenotypic group of patients. *SHANK3* regulates the structural organization of dendritic spines and is a binding partner of NLS. It codes a synaptic scaffolding protein enriched in PSD of excitatory synapses and plays important roles in the formation, maturation and maintenance of synapses. Haploinsufficiency of this gene is related to the 22q13.3 deletion syndrome (known as Phelan-McDermid syndrome), a developmental disorder which is characterized by severe language and speech delay, hypotonia, global developmental delay and autistic behavior. It is possible that loss of one copy of this gene makes the nervous system more vulnerable to degeneration in the long term and less able to recover after psychiatric and somatic events. Five CpG-islands have been identified in this gene, and tissue-specific expression is epigenetically regulated by DNA methylation. Much evidence in animal models has shown that *SHANK3* variants are expressed in the developing rodent brain with expression being regulated by DNA methylation of intragenic promoters [39, 147].

specific epigenetic reprogramming and are therefore not transmitted transgenerationally, but others are not corrected and are transmitted over multiple subsequent generations [97].

Epigenetic mechanisms act on chromatin accessibility to transcriptional regulation. Then, they regulate DNA structure and gene expression that can be influenced by exposure to environ‐ mental factors. The most studied are the methylation of genes and modification of histones. Interestingly, epigenetic abnormalities are associated with several neurodevelopmental diseases. The connection between ASD and epigenetic comes from the identification of genetic mutations in imprinted regions and genes that control epigenetic processes. As cited before, among the most common chromosomal alterations in ASD are duplications of the imprinted

There are several possible explanations for the involvement of imprinted genes in autism. The imprinted brain theory of autism suggests that autism is a disorder of the extreme imprinted brain and would be caused by imbalances that involve increased effects of the 'paternal brain' relative to the 'maternal brain'. Imprinting has been hypothesized to explain the gender difference through the proposed action of unknown paternally imprinted loci on the X chromosome. Also, as mentioned, autism has been strongly associated with chromosomal abnormalities in the imprinted region of chromosome 15q. This includes the Angelman and Prader-Willi syndromes, as well the 15q duplication syndrome, which occurs in up to 5% of individuals with ASD. Imprinted genes may also contribute to autism indirectly as targets of other genes such a regulatory connection between *MECP2* (the gene associated with Rett syndrome) and the imprinted gene *UBE3A* (associated with Angelman syndrome). Besides this, imprinted genes are candidates for association with autism because of their functional haploid state. This feature may make them extremely vulnerable to rare mutations because the gene may be inactivated. Moreover, a single epigenetic change may lead to loss of imprinting, leading to biallelic expression and to gene dysregulation [173]. Both genetic and environmental factors can affect the imprinting process and alter the level of expression of genes. But the full

The imprinted genes are expressed in several types of tissues but are highly expressed in the brain. Human neurons require extensive methyl modifications throughout development and postnatal life. Several important posttranslational modifications of histone core subunits within nucleosomes involve methylation, an epigenetic mechanism. Recent unbiased genomewide analyses have turned up a multitude of novel candidate genes that encode nuclear factors implicated in chromatin remodeling, histone demethylation, histone variants, and the recognition of DNA methylation. Both histone and DNA methylation patterns are highly dynamic processes in the early development phase that correlate with dynamic changes in cell lineage and differentiation events. Interestingly, mutations in autism have been found in several genes encoding proteins involved in demethylase reactions, that is, reactions that

The mechanism of action of the *SHANK3* gene (also known as ProSAP2) is an example of this phenomenon in ASD. The three members of the SHANK family, *SHANK1*, *SHANK2* and *SHANK3* are expressed in different regions of the brain. *SHANK3* is strongly reported to be involved in the etiology of autism since several mutations have been identified in a particular

region 15q11–13, which is maternally inheritable [96].

326 Autism Spectrum Disorder - Recent Advances

contribution of genomic imprinting to the risk for autism is still unclear.

remove methyl groups from histones or DNA [81].

[75] reported that mothers of autistic children show significantly lower levels of methylfolate and methionine, two essential precursors for methylation in DNA compared to a control group, but methylation-inhibiting protein levels and S-adenosylmethionine (SAM), adenosine, and homocysteine were elevated. SAM has a role in the DNA methyltransferase reaction, which produces S-adenosylhomocysteine (SAH) and methylated DNA. The SAM/SAH ratio is considered to be an indicator of DNA methylation potential.

Additionally, oxidative stress in brain cells caused by environmental and genetic factors leads to decreased activity of the methionine synthase enzyme which participates in DNA methylation processes. When the activity of this enzyme is impaired, affected individuals can exhibit attention deficits and other signs, including autistic behavior symptoms due to defects in the expression of genes controlled by this epigenetic mechanism [42, 104]. Therefore, environmental factors may also activate intracellular pathways during embryon‐ ic development, causing epigenetic changes in neural function that would explain the relationship between environmental signals and the genome in the regulation of individu‐ al differences in behavior [166].

A study in Sweden with 208 autistic children showed an association between advanced paternal age and an increasing risk for ASD in offspring. Autistic-like traits in the normal population are associated to both young and advancing paternal age and the autistic similarity in twins seems to increase with advancing paternal age. Exposure to toxic agents during life, *de novo* mutations in germ lines and epigenetic alterations are correlated factors [72, 90]. Advancing age in mothers has also been reported as a risk factor for ASD [116].

Thus, epigenetic alterations may be the biological targets through which environmental factors can cause autism. Imprinted genes may be associated with autism because they are involved in brain development and also because they may be more vulnerable to genetic or epigenetic mutations. Some features of ASD are highly consistent with epigenetic dysregulation such as the discordance between monozygotic twins, parental origin and the gender-dependent effects of some alterations. The cause of autism is not just by congenital genetic defects but can also be caused by environmental factors via epigenetic factors, with epigenetic modifications being affected by environmental factors including fetal exposure to drugs.
