1. Genes and behavioral disorders A-Min Huang, Ph.D. Associate Professor, Department of Physiology, NCKU, College of Medicine 2010

2. Objective To learn the development of psychiatric genetic approaches –The gene-to-disorder approach –The endophenotype approach –The gene-environment interaction approach –Integrating experimental neuroscience and the gene-environment interaction approach Main reference: Nat. Rev. Neurosci. 7:583-590, 2006

3. Approaches to psychiatric genetics research Nat. Rev Neurosci 7:583-590, 2006

4. The gene-to-disorder approach

5. Assumption –Direct linear relations between genes and behavior Goal –To correlate psychiatric disorders with individual differences in DNA sequence Methodology –Linkage analysis –Association analysis –Microarray

6. Summary Susceptibility loci for schizophrenia and bipolar disorder Susceptibility loci for schizophrenia: Chromosome 1, 4, 5, 6, 8, 9, 10, 13, 15, 17, 22, X Susceptibility loci for bipolar: Chromosome 1, 3, 4, 12, 13, 18, 21, 22, X 13q32; 22q11-13

7. The Journal of Clinical Investigation 115:1440-1448, 2005 DTNBP1: dystrobrevin-binding protein 1, ch6 NRG1: Neuregulin 1, ch8 DAO: D-amino acid oxidase, ch12 DAOA: D-amino acid oxidase activator, ch13 RGS4: Regulator of G protein signaling 4, ch1 CAPON: C terminal PDZ domain ligand of neuronal nitric oxide synthase, ch1 PPP3CC: protein phosphatase 3, catalytic subunit, ch8 COMT, DRD2, DRD3, HTR2A Finding Schizophrenia genes

8. RGS4 gene and schizophrenia -- found by microarray studies Strategy High-density cDNA microarray analysis PFC area 9 from 6 patients and 6 controls Verification by in situ hybridization Comparison in a second cohort of schizophrenic and control subjects (5 pairs) Mirnics K, et al. (2000) Neuron 28, 53–67

9. Prefrontal cortex as a major locus of dysfunction Reduction in gray matter volume Increase in cell packing density Reduced neuropil hypothesis

10. Dual color labeling Control experiment I Cortical mRNA from a control subject Cy3 labeling Cy5 labeling Incyte UniGEM-V microarray 7000 cDNA spots (250 gene groups) Signal ratio of each cDNA (Cy5/Cy3)

11. Six matched pairs compared Cortical mRNA from C1 Cy3 labeling Cy5 labeling Cortical mRNA from S1 Cortical mRNA from C6 Cy3 labeling Cy5 labeling Cortical mRNA from S6 Cortical mRNA from C2 Cy3 labeling Cy5 labeling Cortical mRNA from S2 … … …

12. 250 gene groups were analyzed. More than 98% of the gene groups were not different. 3735 genes are detectable; 4.8 % of the genes having  |1.9| fold; 2.6% were up-regulated; 2.2 % were down-regulated. Cy5/Cy3 intensity across six pairwise comparisons Cy3

13. RGS expression in PFC - microarray RGS : Regulator of G protein signaling, GTPase activating protein that will reduce response duration of postsynaptic neuron 5HT2 & D2R are G- protein coupled receptor Molecular Psychiatry (2001) 6, 293–301

14. Confirmation by in situ hybridization RGS4 mRNA was decreased in the cortex of the schizophrenia patients Molecular Psychiatry (2001) 6, 293–301

15. Schizophrenia Major depression Molecular Psychiatry (2001) 6, 293–301

16. Genomic organization of RGS4 and flanking regions Established single nucleotide polymorphism, SNP Human Molecular Genetics (2002) 11: 1373–1380

17. Support for RGS4 as a Susceptibility Gene for Schizophrenia (G/A) (T/G) (G/A) (A/G) BIOL PSYCHIATRY (2004) 55:192–195 Control: 675; Case: 683 Control: 673; Case: 773

18. BIOL PSYCHIATRY (2004) 55:192–195 SNP1: A/G SNP4: T/G SNP1: A/G SNP18: A/G

19. Confirming RGS4 as a Susceptibility Gene for Schizophrenia 249 cases and 231 controls from Ireland Not schizoaffective American Journal of Medical Genetics (2004) 125B:50–53

21. Genetics of mood disorder Biol Psychiatry 60:84-92, 2006

22. (continued) Biol Psychiatry 60:84-92, 2006

23. Problems with the gene-to- disorder approach Replication failures are routine Overall progress has been slow

24. The endophenotype approach

25. Endophenotypes (I) Specificity: –The endophenotype is more strongly associated with the disease of interest than with other psychiatric conditions. State-independence: –The endophenotype is stable over time and not an epiphenomenon of the illness or its treatment. Heritability: –Variance in the endophenotype is associated with genetic variance. Neuropsychopharmacology (2004) 29, 1765–1781

26. Endophenotypes (II) Familial association: –The endophenotype is more prevalent among the relatives of ill probands compared with an appropriate control group. Cosegregation: –The endophenotype is more prevalent among the ill relatives of ill probands compared with the well relatives of the ill probands. Biological and clinical plausibility: –The endophenotype bears some conceptual relationship to the disease.

27. Rationale for the endophenotype approach

28. Endophenotypes

30. The above two approaches are main-effect approaches Assumption: genes cause disorder

31. The gene-environment interaction approach

32. Assumption Environmental pathogens cause disorder Genes influence susceptibility to pathogens Two observations Mental disorders have environmental causes People show heterogeneity in their response to those causes

33. Childhood –genetic risk, disturbed family environment, childhood sexual abuse, and childhood parental loss Early adolescence –neuroticism, self-esteem, and early-onset anxiety and conduct disorder Late adolescence –educational attainment, lifetime traumas, social support, and substance misuse Adulthood –History of divorce and past history of major depression The last year –Marital problems, difficulties, and stressful life events Environmental risk factors for major depression Am J Psychiatry 159:1133-1145 (2002)

34. Science 297, 851-854, 2002 Role of MAOA genotype in the cycle of violence in maltreated children

35. VNTR polymorphism of MAOA Hum Genet 103:273–279, 1998 (30 bases)

36. Science 297, 851-854, 2002 行為異常 暴力犯罪 性情傾向暴力 反社會人格異常症狀

37. Influence of Life Stress on Depression: Moderation by a Polymorphism in the 5-HTT Gene Science 301, 386-389, 2003

38. Map of the human 5-HTT promoter

39. Serotonin transporter gene polymorphism: s and l forms J. Neurochem. 66 : 2621-2624, 1996 17q1 1.2

40. l allele and 5-HTT expression Homozygous for the l allele have higher concentrations of 5-HTT mRNA and express nearly twofold greater 5-HT reuptake in cultured human lymphoblast cell lines

41. Promoter activity

42. 5-HT uptake, binding, and mRNA concentration

43. Distribution for neuroticism scores and percentage

44. Science 301, 386-389, 2003

46. Biol Psychiatry 57,1117-1127 (2005) Cannabis use x psychosis x COMT genotype polymorphism

47. Biol Psychiatry 57,1117-1127 (2005)

49. How does genotype moderate the psychological effects of cannabis use? Nat. Rev. Neurosci. 7:583-590, 2006

50. Are gene-environment interaction studies replicated? Most gene-environment interaction findings have emerged too recently to be evaluated Two of these findings are promising –MAOA genotypes and maltreatment –5-HTT long and short genotypes and life stress

51. 5-HTT long and short genotypes and life stress-replicated studies Mol. Psychiatry 9, 908-915 (2004) Mol. Psychiatry 10, 220-224 (2004) Arch. Gen. Psychiatry (in the press) PNAS 101, 17316-17321 (2004) Bio. Psychiatry 59, 673-680 (2006) Arch. Gen. Psychiatry 62, 529-535 (2005) Br. J. Psychiatry 188, 210-215 (2006) Am. J. Psychiatry (in the press) Int. J. Neuropsychopharmacol 7 Jun 2006

52. PNAS 101:17317-17321, 2004

53. Biol Psychiatry 59:673-680, 2006

55. 5-HTT long and short genotypes and life stress- failures to replicate Psychol. Med. 35, 101-111 (2005) Bio. Psychiatry 59, 224-229 (2006) Females or males? Younger or older adults? First onset or recurrent depression?

56. Limitation of the G-E approach Not able to understand the biological mechanisms involved in an interaction

57. Integration of experimental neuroscience with the gene- environment interaction approach

58. Phase a Phase bPhase c Nat. Rev. Neurosci. 7:583-590, 2006

59. MAOA knock-out mice MAOA gene polymorphism and the cycle of violence Imaging study of the MAOA gene polymorphism and emotional arousal Neuroscience evidence base Epidemiological G-E interaction research Experimental neuroscience

60. BOLD fMRI Science. (2002) 297:400-403

62. DRD4 VNTR polymorphism and substance-use disorders Candidate genesAddiction  ˇ Subjects’ reactions Experimental stimuli Genotype Craving Alcohol or Cigarette DRD4 VNTR

63. DRD4 VNTR polymorphism Am. J. Hum. Genet. 74:931–944, 2004 Associations of the seven-repeat (7R) allele of the human dopamine receptor D4 (DRD4) gene with both the personality trait of novelty seeking and attention deficit/hyperactivity disorder have been reported. The 7R variant exhibits a blunted ability to reduce cAMP levels, in comparison with that of the common 4R variant (Asghari et al. 1995).

64. Nat. Rev. Neurosci. 7:583-590, 2006

65. Towards a nomological network A nomological network refers to the interlocking system of laws — the predicted pattern of theoretical relationships — which define a construct.

66. 1. Animal models of environmental pathogen exposure are needed 2. Studies that compare human genotype groups on their responses to experimentally administered environmental stimuli are needed

67. 3. more epidemiological cohort studies should collect neuroscience measurements –DNA –Neuropsychological tests –Heart rate reactivity –Immune system markers –Imaging paradigms

68. 4. move beyond SNP to a set of gene polymorphisms 5. Genome-wide scans for new disease genes 6. to explain demographic patterns of disorder –Males or females –Younger or older

69. Conclusion: Neuroscience and gene-environment interaction research are joining forces to look for answers Why do some people who are exposed to an environmental pathogen develop mental disorders, while others do not? Why do some disorders excessively afflict one sex or one age group? How can tow people experiencing the same environmental pathogen later develop very different disorders? How does an environmental pathogen, especially one that is psycho-social in its nature, get under the skin to alter the nervous system and generate mental disorders?

70. Mid-term paper Please design a study that compare human genotype groups on their responses to experimentally administered environmental stimuli By October 29, 2010.