Autism Spectrum Disorder and Epigenetic Links

Samantha Hamilton Faculty Mentor: Dr. John Sollinger Southern Oregon University

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The of Autism Autism spectrum disorders (ASD) are a group of childhood neurodevelopmental disorders that contribute to problems with social interaction, communication, and repetitive behavior (Grafodatskaya, Chung, & Szatmari 2010). Along with autism, , , and pervasive developmental disorder are all forms of ASD. Among ASD, autism is highly diagnosed. A diagnosis of autism is based on impairments in two domains, reciprocal social communication, and repetitive, stereotyped, and ritualistic verbal and non-verbal behaviors (Toro et al., 2010). The DSM-IV criteria for diagnosis are discussed later. Often the symptoms of autism vary from patient to patient which creates confusion and difficulty in making a diagnosis. The foundation is noted for determining that “If you know one person with autism, you know one person with autism”. The insight of parents is often the first and best tool leading to a diagnosis. Recently research has been conducted linking autism and other ASD to genetic components. Although no single gene has been highlighted as the specific link for autism, many genes appear to play a role in the disorder. Many genetic phenomena involve inherited or sex- linked mutations; however others appear to arise de novo. Both genetic and environmental factors are found to play a role in autism by influencing fetal or early brain development (Grafodatskaya et al., 2010). Many environmental factors have been determined to increase the risk for developing de novo mutations and autism including heavy metals, latitude, precipitation, sun exposure, and vitamin D deficiency (Kinney, Barch, Chayka, Napoleon, & Munir 2010). These de novo mutations are characterized by single-nucleotide polymorphisms (SNPs) and copy number variants that are the result of chromosomal abnormalities, including large deletions and duplications (Guerra, 2011). Researchers have found more than 5,000 copy number variants in people with autism disorder, and usually DNA was missing in more than one section of the genome (Saey, 2010). Recent research also suggests that the genes thought to be contributors for autism are likely to be pleiotropic (Wilkins, 2009). Pleiotrophy is when one gene is responsible for or affecting more than one phenotypic character. The phenomenon of pleiotrophy could explain the high frequency that autism is found within the world.

Symptoms The symptoms of autism vary widely between those diagnosed, as well as the affected areas of communication and social interaction, repetitive behaviors, and physical and mental delay. The symptoms listed below were taken from the National Institute of Mental Health (National Institute of Mental Health (NIMH), 2011) and typically last a lifetime, but early intervention can make a difference in the development achieved and symptoms experienced by the child diagnosed. Children with autism tend to experience difficulty learning how to engage in everyday interactions. Many avoid eye contact and often prefer being alone. Many resist attention or physical contact. Often nonverbal communication is hard to grasp for a child with autism, and they are not able to experience the world from another’s perspective. Empathy is a common character trait not found in autistic children. Often, emotional regulation is a difficult task, and a tendency to become angry and frustrated accompanies physical aggression.

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Some children diagnosed with autism remain mute throughout their lives; however, the majority develop spoken language or learn to communicate through different techniques such as sign language. Often a delay in language or the inability to regulate a conversation accompanies those diagnosed with autism. Normal body language and verbal expression are absent from conversations, making it difficult to perceive what an autistic individual needs. As children, individuals diagnosed with autism often engage in odd, repetitive motions that can sustain through adulthood. Many children need and demand absolute consistency in their daily routines and are extremely stressed by slight changes. Often repetitive behavior is found in the form of an intense preoccupation, sometimes with unusual content such as fans. In as many as 39% of people diagnosed with autism, is also found. It is more commonly found in children who also experience regression in skills. Many children diagnosed with autism also experience gastrointestinal problems, such as gastritis, chronic constipation, colitis, celiac disease, and esophagitis. Although the exact number of autistic children who suffer from these ailments is unknown, it is estimated to be between 46-85% (NIMH, 2011). Another common symptom of autistic children and adults are sleep problems. Research has suggested a link between autism and the synthesis pathway that results in an abnormal circadian rhythm and sleep problems (Fradin et al., 2010). Many children with autism experience unusual responses to certain stimuli, such as vision, hearing, touch, smell, taste, movement, and position. Often“normal” stimuli, for those not affected by autism, can be experienced as painful or unpleasant to those affected. This symptom is Sensory Integration Dysfunction and can involve hypersensitivity or hyposensitivity (NIMH, 2011). The treatment for these symptoms is discussed below and involves treatment for the underlying conditions as well as behavior modification techniques.

Diagnosis Autism characteristics appear during the early stages of development and often are patterned by a period of normal development followed by a period of regression. Typically this happens within the first two to four years of life with a loss of social, cognitive, and language skills as well as stereotypic behaviors (Lopez-Rangel & Lewis, 2006). Studies have found that individuals with ASD have altered neuronal organization, cortical connectivity, pathways, and brain growth (Grafodatskaya et al., 2010). Listed below are the DSM-IV criteria for diagnosis of autism. A total of six or more items from heading (A), (B), or (C), with at least two from (A), and one each from (B) and (C): (A) Qualitative impairment in social interaction, as manifested by at least two of the following:  Marked impairments in the use of multiple nonverbal behaviors such as eye-to-eye gaze, facial expression, body posture, and gestures to regulate social interaction.  Failure to develop peer relationships appropriate to developmental level.  A lack of spontaneous seeking to share enjoyment, interests, or achievements with other people.  A lack of social or emotional reciprocity.

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(B) Qualitative impairments in communication as manifested by at least one of the following:  Delay in or total lack of, the development of spoken language.  In individuals with adequate speech, marked impairment in the ability to initiate or sustain a conversation with others.  Stereotyped and repetitive use of language or idiosyncratic language.  Lack of varied, spontaneous make-believe play or social imitative play appropriate to developmental level.

(C) Restricted repetitive and stereotyped patterns of behavior, interests and activities, as manifested by at least two of the following:  Encompassing preoccupation with one or more stereotyped and restricted patterns of interest that is abnormal either in intensity or focus.  Apparently inflexible adherence to specific, nonfunctional routines or rituals.  Stereotyped and repetitive motor mannerisms.  Persistent preoccupation with parts of objects.

Delays or abnormal functioning in at least one of the following areas, with onset prior to age three years: (A) Social interaction. (B) Language is used in social communication. (C) Symbolic or imaginative play.

The disturbance is not better accounted for by Rett’s Disorder or Childhood Disintegrative Disorder (Diagnostic and Statistical Manual of Mental Disorders; Fourth Edition, 2000). Parents of a child who is exhibiting autism-like signs between 16 and 30 months are often asked to use the Modified Checklist for Autism in Toddlers (M-CHAT; Robins, Fein, & Barton, 1999) to determine if follow up, specialty care is necessary. The M-CHAT is widely used but is a screening tool and should be used in conjunction with proper medical care. See appendix 1 for M-CHAT and scoring guidelines. Often children diagnosed with autism also have other health or mental conditions, with the most common of these being sensory problems, mental retardation, seizures, , or Tuberous Sclerosis (National Institute of Mental Health (NIMH), 2011).

Treatment The treatment for autism is a multifactor process that involves deep commitment from the family and caretakers of the autistic individual. The most frequently applied techniques include: treatment for the core symptoms of autism, applied behavior analysis, pivotal response therapy, verbal behavior therapy, floor time, relationship development intervention, training and education of autistic and related communication handicapped children, social communication/emotional regulation/transactional support, the son-rise program, and treatment for biological and medical conditions associated with autism (Autism Speaks, Inc, 2011). This multifaceted approach to treating a person diagnosed with autism is tailored to each individual need.

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Prevalence The prevalence of autism is estimated to be an average of 1 in 110 children in the United States. The male: female ratio is 4:1 which strongly correlates the idea that a sex-linked mutation may contribute to the disorder (American Academy of Pediatrics, 2011). X-linked loci are being researched to determine the role, if any, they play in males who have an increased vulnerability to autism. In this research it is suggested that the absence of the expression from this loci does not correlate to an autism diagnosis, but rather eliminates a protective factor within the male that makes him more susceptible to genetic mutations leading to autism (Grafodatskaya et al., 2010). The changes in gene expression at these loci have pleiotropic effects and possibly consequences throughout the individual’s . Twin studies have consistently shown that autism has a heritability of 82-90% in monozygotic twins and 1-10% in dizygotic twins with a sibling recurrence of 6% (Toro et al., 2010). However, genetic studies have found similar genes between those affected by autism in only a small percentage of cases contributing to the idea that many genes are responsible for the diagnosis of the disorder (Sanders, 2011). Inheritance A list of over 190 genes thought to contribute to autism or ASD has been developed and can be found at AutDB, a public database for autism research. However, because the association of these genes could not be confirmed by replication, they remain only as possible candidates and the list is continually growing (Toro et al., 2010). Many different genetic syndromes are associated with autism. However, the connection is not clear as to if a disability mimics the symptoms of autism or if a molecular change in an overlapping gene or its expression pattern via epigenetic modification is responsible for the similarities (Grafodatskaya et al., 2010). The most common chromosomal abnormality, seen in 1% of children diagnosed with autism, is the gain of chromosome segment 15q11-q13. This duplication is associated with an autism risk at more than 85% (Grafodatskaya et al., 2010). Many genes are imprinted within the 15q11-13, and the mechanisms that affect the gene expression within this region are only beginning to be uncovered (Lopez-Rangel & Lewis, 2006). One gene strongly linked to autism is DDX53-PTCHD1 and is located on the X chromosome. More often, women who carry a deletion of the gene on one chromosome carry a healthy copy on the other that masks the effects. However, if the X chromosome with the deletion is passed to a son, a diagnosis of autism is common because of hemizygosity (Saey, 2010). The discovery of this gene has lead researchers to continue searching for X-linked mutations as the incidence of autism in boys is four times that of girls. Another gene commonly linked to a higher risk for autism is the CNTNAP2 gene. Researchers have used MRIs to determine that children, who carried the risk gene, showed a difference in connectivity between the communication pathways in the brain (Scott-Van Zeeland et al., 2010). With the risk gene CNTNAP2, the brain was shown to have definite disrupted activation patterns while the diagnosis of autism was not definite. Individuals with ASD have altered neuronal organization, cortical connectivity, neurotransmitter pathways, and brain growth (Grafodatskaya et al., 2010). Several regions of the brain are associated with the criteria for an

Autism Spectrum Disorder and Epigenetic Links Hamilton | 6 autism diagnosis including the frontal and temporal cortex (Guerra, 2010). A carrier of the CNTNAP2 has higher associations of altered brain pathways than someone with normal language. The ongoing research regarding genes responsible for contributing to autism is looking closely at the development and regulation of the brain. The last example of a genetic predisposition to autism is the mutation as a single base insertion in FOXP1. This insertion causes a premature stop codon to introduce a frameshift in the area of the genome affected. The evidence of the severe consequences regarding this mutation is that those affected have language delay, regression, moderate , and nonfebrile seizures, all which are symptoms of autism (O’Roak et al., 2011). Although many genomic studies have been done and a large number of similar regions detected in autism patients, the combination of and large numbers of mutations contribute to the vast complexity of this disorder.

Environment Besides the genetic determinant, the environment has been shown to play a role in autism. Many theories have been explored regarding the relationship between environment (including nutrition, economic status, pollutants, and vaccinations) and ASD (Guerra, 2010). The development and regulation of the brain are affected in autism patients and genetic predispositions, cued by an environmental trigger, are thought to disrupt development of the brain during gestation, early infancy, and childhood causing the classic autism symptoms (Guerra, 2010). The environment has proven to be a contributing factor to autism, not a cause; as one must have a genetic predisposition or epigenetic inheritance to the disorder in order for the environment to elicit a response in brain tissues. Research has consistently shown that autism has a heritability of 82-90% in monozygotic twins (Toro et al., 2010). However, the degree of impairment and range of symptoms varies greatly between the pairs of twins, suggesting that environmental factors do have an association with a diagnosis of autism when combined with genetic predisposition for the disorder (Grafodatskaya et al., 2010). Of these environmental factors the exposure to teratogenic medications, such as thalidomide and valproate, has shown consistent association with autism diagnosis. The risk of autism in children whose mothers took valproate during pregnancy is estimated to be 10.8%, 16 times the general population risk. The mechanisms that cause this increase in autism diagnosis are altered folate metabolism and inhibition of deacetylases, both of which are discussed below (Grafodatskaya et al., 2010). Environmental factors have been found to directly alter genes by influences that lead to improper tagging or detagging of genes, leading to improper translation. Dysregulation of genes that control epigenetic mechanisms leads to changes in genes that regulate epigenetic marks, such as DNA methyltransferases, methyl binding proteins, and enzymes that affect histone modification (Grafodatskaya et al., 2010). Many environmental factors affect the degree of autism, but are also found to directly or indirectly lead to a diagnosis in a person predisposed for the disorder.

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Epigenetics The genome contains information for every different cell function. In contrast, the epigenome varies from tissue to tissue, controls the expression of genes, and gives each cell type a specific identity. Epigenetics refers to heritable changes in gene expression that occur without changes in DNA sequence. The epigenetic effects are related to DNA methylation and histone acetylation (Lopez-Rangel & Lewis, 2006). Acetylation of reduces the affinity between histones and allows RNA polymerase and transcription factors access to gene regions. Deacetylation on histones causes promoter regions to be less accessible because the DNA is too tightly wrapped around the nucleosone (set of eight histones) and turns off gene expression (Lopez-Rangel & Lewis, 2006). Epigenetic modifications affect the regulation of gene expression without affecting primary DNA sequences (Grafodatskaya et al., 2010). Epigenetic modifications are happening both in the germ line and in somatic tissues by genetic, environmental, and stochastic factors. The “epigenotype” of each cell is highly variable within an individual; and, because of this plasticity, the epigenotype is prone to errors. The errors caused by the modifications play a role in autism as the genetic and environmental influences interrupt neurodevelopmental processes (Grafodatskaya et al., 2010). The epigenetic marks define chromatin state by methylation or acetylation. Chromatin is made of histones that control gene expression and DNA replication. Methylation will dissociate histones from the chromatin complex; demethylation tends to favor transcriptionally inactive chromatin; and, deactylation affects the promoter regions of the complex. These play a role in autism as they effect the accessibility of DNA-binding proteins to unwind the DNA double helix and transcribe RNA (Guerra, 2010). DNA methylation or histone acetlyation/phosphorylation can possibly regulate the expression of certain genes in a tissue specific manner. The evidence of this appears in the Reelin (RELN) gene that is epigenetically regulated and implicated in autism by association studies. RELN is necessary for neuronal migration and synaptogenesis in brain nuclei. RELN protein and mRNA were significantly reduced in the frontal and cerebellar areas of brains of autistic individuals (Grafodatskaya et al., 2010). Disturbances of folate metabolism are also associated with autism. The genetic variations do not directly cause autism; however, the interaction of genetic and environmental factors (such as folate and vitamin B intake, amino acid deficiencies, and exposures to toxins) result in a disruption of the expression of epigenetically regulated genes and can cause abnormal brain development (Grafodatskaya et al., 2010). Imprinting (tagging with methyl or acetyl groups) during meiosis is an epigenetic modification that leads to preferential expression of a specific parental allele in somatic cells of the offspring. The role of imprinting is clearly shown by the abnormal development or termination of embryos that inherit two copies of either a maternal or paternal genome as with the fusion of two eggs, rather than the normal one of each (Fradin, et al., 2010). Imprinting changes genetic expression and has consequences for multiple aspects of the phenotype (Wilkins, 2009). Most normally imprinted genes control fetal growth; however, many are also associated with aspects of the phenotype that emerge later in development (Wilkins, 2009). The role of imprinted genes in prenatal growth is primarily in the brain, which correlates to a role in autism. The strongest evidence for the imprinting of a neuraldevelopment gene is the gene

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CLOCK, which codes a protein regulating circadian rhythm. At least five studies have found that an abnormal melatonin synthesis pathway, which results in an abnormal circadian rhythm, is present in autistic individuals (Fradin et al., 2010). This accounts for the common symptom of sleep problems found in autistic individuals. The association between melatonin and vitamin-D levels has been shown to also have a correlation in autism. Vitamin-D deficiency is associated with an increased risk for autism, since vitamin-D influences hormones that activate over 200 target genes, thereby regulating gene expression (Bakare, Munir, & Kinney, 2011). Imprinting via epigenetic marks, such as methylation status, is a potential measure for determining the etiology of autism and other neuropsychiatric disorders. Research Much research is being conducted in order to not only better the understanding of autism, but also treat and possibly, one day, prevent or cure the disorder. One study currently being conducted is considering the role of CD38, a transmembrane antigen that has been studied extensively in leukemia research, in oxytocin (OT) secretion within the brain. OT is a hormone that plays a crucial role in social recognition and memory, as well as bonding and care in rodents (Munesue et al., 2010). A genetic variation of oxytocin receptor gene (OXTR) has been found to be associated with ASD in several ongoing studies (Grafodatskaya et al., 2010). The current research is studying whether CD38 mutations, which alter OT secretion, may contribute to a diagnosis of ASD (Munesue et al., 2010). Disturbances of folate metabolism in individuals with autism have also been found. The interaction of folate with genetic markers is not thought to be a direct correlation to autism, but rather encompass various deficiencies and exposures that may modify the expression of metabolic pathways. Additional, placebo-controlled studies are currently being conducted to determine the role of folate as a supplemental therapy to children with autism (Grafodatskaya et al., 2010). Lead researchers at the University of California, Davis MIND Institute in Sacramento are hopeful as each new genetic clue is discovered regarding autism. However, for any particular gene found to be related to autism, it is only related to about 1-2% of the cases. This demonstrates the complexity of autism, and proves that there is not going to be a single autism gene, but many (MacNeil/Lehrer, 2011).

Conclusion Current research is finding genetic and epigenetic pathways that are common factors in individuals diagnosed with autism; however, the complexity of the potentially modifiable determinants makes developing treatments difficult. Current research is finding links between genes and their expression patterns that leads to a diagnosis of autism. With each new discovery comes further insight to a debilitating disorder that has social and emotional consequences that lie far deeper than the outward symptoms. New technologies allowing whole-genome screening for DNA methylation and chromatin alterations will allow identifying epigenetic markers for autism (Grafodatskaya et al., 2010). By targeting these epigenetic marks such as, DNA methylation and histone acetylation, new therapies are being studied that could possibly, one day, treat or prevent autism.

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Appendix 1: M-CHAT and scoring guidelines

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Appendix 1 continued

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References American Academy of Pediatrics (2011). Children’s health topics: Autism. http://www.aap.org/healthtopics/autism.cfm Autism Speaks Inc. (2011). http://www.autismspeaks.org Bakare, M., Munir, K., & Kinney, D. (2011). Association of hypomelanotic skin disorders with autism: links to possible etiologic role of vitamin-d levels in autism? Hypothesis, 9(1), 1-9. Diagnostic and Statistical Manual of Mental Disorders; Fourth Edition, 2000. American Psychiatric Association. Fradin, D., Cheslack-Postava, K., Ladd-Acosta, C., Newschaffer, C., Chakravarti, A., Arking, D., … Fallin, M. (2010). Parent-of-origin effects in autism identified through genome- wide linkage analysis of 16,000 SNPs. PLoS One, 5(9), doi:10.1371/journal.pone.0012513. Grafodatskaya, D., Chung, B., Szatmari, P., & Weksberg, R. (2010). Autism spectrum disorders and epigenetics. Journal of the American Academy of Child and Adolescent Psychiatry, 49(8), 794-809. Guerra, D. (2011). The molecular genetics of autism spectrum disorders: genomic mechanisms, neuroimmunopathology, and clinical implications. Autism Research and Treatment, 2011, doi:10.1155/2011/398636. Kinney, D., Barch, D., Chayka, B., Napoleon, S., & Munir, K. (2010). Environmental risk factors for autism: do they help cause de novo genetic mutations that contribute to the disorder? Med Hypothesis, 74(1), 102-106. Lopez-Rangel, E, & Lewis, MES. (2006). Loud and clear evidence for gene silencing by epigenetic mechanisms in autism spectrum and related neurodevelopmental disorders. Clinical Genetics, 69, 21-25. MacNeil/Lehrer (Producer). (2011b). Autism’s causes: How close are we to solving the puzzle? [Web]. Available from http://www.pbs.org/newshour/bb/health/jan- june11/autism3causes_04-20.html Munesue, T., Yokoyama, S., Nakamura, K., Anitha, A., Yamada, K., Hayashi, K., … Nakatani, H. (2010). Two genetic variants of CD38 in subjects with autism spectrum disorder and controls. Neuroscience Research 67(2), 181-191. National Institute of Mental Health (2011). What are the Autism Spectrum Disorders? http://www.nimh.nih.gov/health/publications/autism/what-are-the-autism-spectrum- disorders.shtml O'Roak, B. (2011). Exome sequencing in sporadic autism spectrum disorders identifies severe de novo mutations. Nature Genetics, doi:10.1038/ng.835.

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Saey, T.H. (2010, July 3). Some autism cases linked to rare mutations. Science News, 178(1), 12-12. Sanders, Laura. (2011, June 18). Genetic analysis reveals clues to autism's roots. Science News, 179(13), 5-6. Scott-Van Zeeland, A., Abrahams, B., Alvarez-Retuerto, A., Sonnenblick, L., Rudie, J., Ghahremani, D., Mumfor, J., Poldrack, R., Dapretto, M., Geschwind, D., Bookheimer, S. (2010). Altered functional connectivity in frontal lobe circuits is associated with variation in the autism risk gene CNTNAP2. Science Translational Medicine, 3, 56-80. Toro, R., Konyukn, M., Delorme, R., Leblond, C., Chaste, P., Fauchereau, F., ... Bourgeron, T. (2010). Key role for gene dosage and synaptic homeostasis in autism spectrum disorders. Tends in Genetics 26, 363-372. Wilkins, J. (2009). Antagonistic coevolution of two imprinted loci with pleiotropic effects. Evolution, 64(1), 142-151.