The document discusses epigenetics and its connection to autism spectrum disorder (ASD). Some key points: - Epigenetics refers to modifications that alter gene expression without changing DNA sequence, and can be transmitted to daughter cells. Epigenetic processes like DNA methylation and histone modifications regulate gene expression. - Studies have found epigenetic involvement in ASD, as the disorder is genetically complex with no single gene cause. Disorders like Rett syndrome and Fragile X syndrome linked to ASD also have epigenetic components. - A study found that mice lacking the Mbd1 gene, involved in epigenetic regulation, exhibited autism-like behaviors including reduced social interaction and

1. Epigenetics of Autism Spectrum Disorder Chinyelu Mozie University of Texas at Dallas March 25, 2009

2. What is Epigenetics Weinhold: “ any process that alters gene activity without changing the DNA sequence, and leads to modifications that can be transmitted to daughter cells” (p. 163).

3. What is epigenetics Means above the genome Born with epigenome All cells have same genes Tells our cells what sort of cells they should be-cell differentiation skin, heart, hair, liver, etc Regulating gene function Affect the way we produce proteins Can be transgenerational Contains heritable epigenetic information Interaction with gene and environment

4. What is epigenetics Does not only occur in utero but throughout life span Conrad Waddington credited for term epigenetics in 1942 Increase of usage of word in 1990’s

5. Epigenetic Processes Gene silencing X-chromosome inactivation Imprinting

6. Mechanisms DNA Methylation addition of methyl groups to DNA at CpG sites Chromatin/Histone modifications Addition and removal of acetyl groups from DNA http://www.universe-review.ca/F11-monocell.htm Epigenetic Mechanisms

7. Some Background Information Deoxyribonucleic acid Watson and Crick in 1953 Hold genetic instructions on we look and behave Four nucleotides bases Guanine Adenine Cytosine Thymine http://instruct.westvalley.edu/svensson/CellsandGenes/ DNA-structure

8. Some Background Information (cont.) DNA organized into chromosome chromatin Genes located on chromosome http://images1.clinicaltools.com/images/gene Chromosome Structure

9. Background Information Central Dogma DNA RNA protein Transcription DNA to RNA Translation RNA to protein Translation/transcription process

10. DNA Methylation Addition of methyl groups to CpG sites remains unmethylated at CpG islands Most common method in gene silencing Methylated gene Inactive Prevents transcription factors from binding to promoter Non methylated gene active Transcription factors able to bind to promoter

11. Chromatin/Histone modifications Addition and removal of acetyl groups from histones. Addition of acetyl groups (acetylation) decondense chromatin, uncoiling the DNA strand which causes : gene to become accessible to transcription factors, exposes promoter of gene. transcribed gene-expression of gene Removal of acetyl groups (deacetylation) cause chromatin to condense, tight coiling of DNA. gene becomes inaccessible for transcription factors. silent gene

12. Epigenetic Mechanisms for Repressing Transcription Environ Health Perspect. 2006 March; 114(3): A160–A167

13. Role in Environment Driven by multiple factors in environment: Single nutrients ex: maternal diet in rats, exposure to parents, grandparents during famine Toxins- pesticides, smoking, drinking ex: cancer Behavior ex: grooming of rat pups

14. Single nutrients Study by Dr. Jirtle (2006) concerning epigenetic changes by dietary supplements Epigenetic regulation of agouti gene pregnant mothers diet Vitamin B12, folic acid, choline and betaine. brown coat color in offspring inherited by next generation pregnant mother diet BPA yellow coat color in offspring A pup of a different color . Supplementation of maternal diet with genistein and other compounds induced alterations in DNA methylation that were reflected in offspring coat color changes. Environ Health Perspect. 2006 March; 114(3): A160–A167 .

15. Famine First proof of environmental effect being inherited in humans Research by Marcus Pembrey and Olov Bygren(2008) concerning famine exposure Effect of famine was different in grandmother and grandfather Grandmother susceptible while still in the womb Grandfather affected in late childhood

16. Famine Women who experienced famine earlier in pregnancy had paternal granddaughters die earlier in life Famine affected genetic makeup within X chromosome IGF2 gene different in siblings not exposed Men who experienced famine at age 10 had paternal grandsons that lived longer than those whose grandfathers didn’t experience famine diabetes You are what your parents and grandparents ate

17. Cancer P-53: tumor suppressor gene Excessive deacetylation/methylation silences gene Results in uncontrollable cell growth Epigenetic Therapy Reactivating gene by removing methyl tags

18. Behavior Study by Dr. Moshe Szyf (2004) showed that grooming, nursing, and licking of a rat pup can affect long-term behavior of offspring (p. 164). Rats pups receiving high nurturing methyl groups removed by nurturing signals, activated GR receptor gene-calm adults Rats pups receiving low nurturing methyl groups remain attached to DNA, inactivated (gene silenced) GR receptor gene-anxious adults Hippocampus in pups Production of GR protein (glucocorticoid receptor) Cortisol (stress hormone) binds to these receptors Affects more than just GR gene

19. Behavior Epigenetic difference in high and low nurtured rats. Can be reversed Place pups with licking mother Drugs that add and remove methyl tags

20. Twin Studies Provide evidence of epigenetic influences in humans Good way in determining heritability of disorders MZ - same DNA, same sex DZ - different DNA, different sex Muhle et al. (2004) concordance rate High concordance in MZ-60-90% Low concordance in DZ- 0-10% Fraga et al. (2005) showed epigenetic differences arise in older twins Ex: cancer

21. Connection between Epigenetics and Autism ASD comprises of a complex group of behaviorally related disorders that are genetic in origin No single gene found Multiple genes contribute to autism Risch et al. (1999) found 2-15 genes contributing to susceptibility Cannot be traced to single mutation or gene Involvement of epigenetic regulatory mechanisms

22. Connection between Epigenetics and Autism (cont.) Can use other disorders that share same clinical features to map out genetic and epigenetic components Overlapping phenotypes Rett Syndrome Angelman’s syndrome and Prader-Willi syndrome Fragile X syndrome

23. Chromosomes/Genes 46 chromosomes (23 pairs) 45-46: sex chromosomes XX-woman XY-man Phenotype Observable manifestation of trait Genotype Genetic constitution

24. Rett Syndrome Neurological disorder classified as a PDD by the DSM-IV Symptoms cognitive impairments problems with socialization no verbal skills X-linked neurodevelopmental disorder Caused by mutations in the MECP2 gene on Xq25 n-Rett Syndrome Mother Father XX XⁿY Children XXⁿ XY

25. Fragile X syndrome Genetic disorder caused by mutation of the FMR1 gene on the X chromosome Some symptoms meet the diagnostic criteria for autism X-linked dominant condition -girls: mild MR -boys: severe MR Mother Father X X ª XY X X ª X ª Y

26. Angelman Syndrome/Prader-Willi Syndrome Both produced by same genetic mutation AS: deletion of maternal copy, maternal mutation of UBE3A gene or UPD of chromosome 15q11-q13 Severe mental retardation, happy demeanor, non-verbal PWS: deletion of paternal copy or UPD of chromosome 15q11-q13 Language, motor and developmental delays, excessive weight gain Result from imprinting http://www.ucl.ac.uk/~ucbhjow/bmsi/bmsi_4.html AS/PWS Diagram

27. Methyl-CpG binding domain (MBD). MBD1-gene Mbd1: protein MECP2-gene MeCP2: protein Multiple rules in regulating gene expression in neurons Capable of binding specifically to methylated DNA

28. Fig. 1. The overlapping disorders, phenotypes and genotypes regulated by MECP2/MeCP2 through epigenetic mechanisms. AS, Angelman's syndrome; RTT, Rett syndrome.

29. Study Study by Allan et al. (2008) Mice lacking key regulatory protein Mbd1 show autism-like symptoms Exhibit core deficits reduced social interaction learning deficits anxiety defective sensory motor gating depression abnormal serotonin activity

30. Study (cont.) Methyl-CpG binding proteins (MBDs) are central components of DNA methylation-mediated epigenetic gene regulation. Role in transcriptional repression Mutation found in MBD1 gene in autistic patient and direct relatives Analyzing Mbd1-/- Understand role of Mbd1 in adult neurogenesis and learning Understand importance of epigenetic regulation in mammalian brain development and cognitive functions.

31. Subjects Mbd1-/- knockout (KO) mice Mutant No gross abnormality/ mild reduction of forebrain weight Nearly normal lifespan Mbd1 (WT) Most common phenotype population Sex male and female Age 2-3 months

32. Methods Social interaction Place novel toy in tent for first 5min then mouse in for last 5min Sensorimotor gating PPI(Prepulse inhibition) prepulse stimulus (auditory tone) startle stimulus (air puff) 120 and 300ms interstimulus interval access how well they are able to inhibit stimulus Learning deficits Cued for fear conditioning and contextual fear conditioning foot shocks to train mice tested 24hrs later using auditory tone (cued test) and spatial context (context test) learning and memory accessed by how long they freeze t

33. Methods (cont.) Anxiety Light-dark preference test Elevated plus-maze Open arms Susceptibility to depression Learned helplessness test foot shocks Forced swim test Serotonin receptor Wet dog shake observed in behavioral experiments indicator of increased serotonin activity

34. Results Social Interaction Mbd1-/- Have normal motor activity and exploratory behavior Reduced interest in social interaction No gender differences Figure 1: Mbd1-/- mice have impaired social interaction Allan, A. M. et al. Hum. Mol. Genet. 2008 17:2047-2057; doi:10.1093/hmg/ddn102 Copyright restrictions may apply.

35. Results (cont.) Sensorimotor Gating Mbd1-/- Impaired sensorimotor gating Allan, A. M. et al. Hum. Mol. Genet. 2008 17:2047-2057; doi:10.1093/hmg/ddn102 Figure 2: Mbd1-/- mice have impaired sensorimotor gating as assessed by prepulse inhibition (PPI) Copyright restrictions may apply.

36. Results (cont.) Learning Deficits Mbd1-/- Exhibited deficits in fear-conditioning test-assessed amygdala-mediated learning. Deficits in contextual fear conditioning test-assessed both hippocampus and amygdala mediated learning. Figure 3: Mbd1-/-mice exhibited deficits in fear-conditioning learning tests Allan, A. M. et al. Hum. Mol. Genet. 2008 17:2047-2057; doi:10.1093/hmg/ddn102 Copyright restrictions may apply.

37. Results (cont.) Anxiety Mbd1-/- Increased anxiety Spent less time in well-lit room Spent less time in open arms and more in open arms General locomotion same between genotypes (data not shown) Allan, A. M. et al. Hum. Mol. Genet. 2008 17:2047-2057; doi:10.1093/hmg/ddn102 Copyright restrictions may apply. Figure 4: Mbd1-/- mice exhibited increased anxiety

38. Results (cont.) Susceptibility to depression Mbd1-/- Enhanced susceptibility to depressive behaviors Longer latency in escaping shocks Greater number of failed escapes Exhibited more non-escape-directed behaviors and fewer escape-directed behaviors Figure 5: Mbd1-/- mice exhibited enhanced susceptibility to depressive behaviors Allan, A. M. et al. Hum. Mol. Genet. 2008 17:2047-2057; doi:10.1093/hmg/ddn102 Copyright restrictions may apply.

39. Other Findings Loss of Mbd1 leads to elevated expression of Htr2c receptor. Increased binding of serotonin to receptor Receptors in hippocampus and medial frontal cortex Htr2c does not function properly in Mbd1-/- Low affinity of serotonin to receptor Dysregulation of Htr2c Abnormal serotonin system linked to autism

40. Stahl SM. Mechanism of action of serotonin selective reuptake inhibitors: serotonin receptors and pathways mediate therapeutic effects and side effects. J Affect Disord. 1998;51:215-235

41. Copyright restrictions may apply. Allan, A. M. et al. Hum. Mol. Genet. 2008 17:2047-2057; doi:10.1093/hmg/ddn102 Mbd1 directly regulates the expression of serotonin receptor Htr2c

42. Current Research Dr. Walter Kaufmann at Kennedy Krieger Institute and Dr. Andy Feinberg at Johns Hopkins University compared brain scans of identical twins discordant for autism found that hippocampus smaller in twin with severe autism Hypothesis: since genome the same, one has epigenetic change Searching for methyl marks in DNA

43. Future Studies Recent attention of epigenetics in ASD Autism Speaks: $3.6 million to investigate environmental risk factors for autism Dr. Yong-hui Jiang at Baylor College of Medicine how folic acid supplementation affects epigenetic modulation of SHANK3 protein expression Dr. Robert Plomin at Institute of Psychiatry in London Looking at epigenetic markers in twins who do not share dianosis of autism.

44. Future Studies (cont.) Dr. Emile Rissman at University of Virginia BPA (Bisphenol A) Dr. Bruce Hammock at University of California at Davis Vitamin D levels lack of Vitamin D could influence brain development and function It’s too early to speculate on treatment for epigenetic factors, but mouse models should help evaluate treatment

45. References Allan, A.M., Liang, W., Luo,Y., Pak, C., Li, X., Szulwach, K.E., Chen, D., Jin, P., Zhao, (2008). The loss of methyl-CpG binding protein 1 leads to autism-like behavioral deficits. Human Molecular Genetics, 17(13) , 2047-2057. Fraga, M.F., Ballestar, E., Paz, M.F., Ropero, S., Setien, F., Ballestar, M.L.., et al. (2005). Epigenetic differences arise during the lifetime of monozygotic twins. PNAS, 102(30) , 10604- 10609. Jirtle, R.L., Dolinoy, D.C., Weidman, J.R., Waterland, R.A. (2006). Maternal genistein alters coat color and protects mouse offspring from obesity by modifying fetal epigenome. Environmental Health Perspectives, 114(4), 567-572. Lewis, M., Lopez-Rangel, E. (2006). HotSpots: Loud and clear evidence for gene silencing by epigenetic mechanisms in autism spectrum and related neurodevelopmental disorders. Clinical Genetics, 69 , 21-25. Muhle, R., Trentacoste, S.V., Rapin, I. (2004). The genetics of autism, Pediatrics, 113(5), 472-486. Pembrey, M.E, Bygren, L.O., Kaati, G. (2008). Sex-specific male-line transgenerational responses in humans. Human Genetics, 14, 159-166. Risch, N., Spiker, D., Lotspeich, L., Nouri, N., Hinds, D., Hallmayer, J., et al. (1999). A geomic screen of autism: evidence for a multilocus etiology. Human Genetics, 65, 493-507. Szyf, M., Weaver, I.C.G., Cervoni, N., Champagne, F.A., D’Alessio, A.C., Sharma, S., et al. (2004). Epigenetic programming by maternal behavior. Nature Neuroscience, 7(8), 847-854. Weinhold, B. (2006). Epigenetics: The science of change. Environmental Health Perspective, 114(3), 160-167.