1. Genetic Validation in Alcoholism Ru-Band Lu, MD Professor Department of Psychiatry National Cheng Kung University, Taiwan

2. Genetic Component and Alcoholism  Family studies (Merigangas 1990)  Twin studies (Pickens et al. 1991)  Adoption studies (Cadoret et al. 1985)  Genetic component in alcoholism –More than 50 % (Reich et al. 1999)

3. The Importance of Subtypes Alcoholism  Heterogenesis of mental illnesses  Understanding different pathogenesis  Different treatments and prognosis

4. Alcoholism Subtypes  Jellineck (1960) : 2 subtypes  Morey and Skinner (1986) : 3 subtypes  Lesch (1988) : 4 subtypes  Zucker (1987) : 4 subtypes  Cloninger (1987) : 2 subtypes  Babor (1992) : 2 subtypes

5. Limitations  Lack of cutting point  Different risk factors in different cultures  Inconvenience for clinical use  Lack of genetic validation

6. Clinical Subtyping of Alcoholism (Huang et al., 2004)  Alcoholic patient often meeting most of the diagnostic criteria (DSM-IV-TR, 2000)  More homogeneous mental illness from the diagnostic criteria perspective.  Higher frequent comorbidity with other mental illnesses

7. Clinical Subtyping of Alcoholism (Huang et al., 2004)  Pure Alcoholism  Anxiety-Depression Alcoholism  Antisocial Alcoholism  Mixed Alcoholism

8. Clinical Subtypes of Alcoholism  Pure Alcoholism (Pure ALC) – late onset – without comorbidity with mental illnesses – less alcohol related problems  Anxiety- Depression Alcoholism (ANX/DEP ALC) – early adult onset – comorbid anxiety or depression – heavy alcohol consumption

9. Clinical Subtypes of Alcoholism  Antisocial Alcoholism (Antisocial ALC) – adolescent onset (very early onset) – comorbid antisocial personality disorder – more alcohol related problems  social consequence  Mixed Alcoholism (Mixed ALC) – comorbid major mental illnesses – comorbid multiple substance disorder – Alcoholism with bipolar disorders among Han Chinese

10. DRD2 Gene and Alcoholism (Blum et al. 1990)  An important candidate gene of alcoholism  Controversial results or borderline significance (Reich et al., 1999)  No association with non-subtyped alcoholism among Han Chinese population in Taiwan (Lee et al., 1999)

11. A: Alcoholics; C: Controls; P: Percentage; F: Allele Frequency Author (A : C) Alcoholism, N (%) TaqI-ANon-alcoholism (n,%) A1A1A1A2A2A2A1 (P) A1 (F)A1A1A1A2A2A2A1 (P)A1 (F) Association Blum et al. 1990 (22:24) Neiswanger et al.1995(52:30) Noble et al.2000 (92:85) Kono et al. 1997,(21: 93) Ishiguro et al.1998(209:152) 1 (4.5)13 (59.1)8 (36.4)63.634.10 (0)4 (16.7)20 (83.3)16.78.3 1 (2.0)28 (53.8)23 (44.2)55.828.80 (0)4 (13.3)26 (86.7)13.36.7 6 (6.5)44 (47.8)42 (45.7)54.330.43 (3.5)13 (15.3)69 (81.2)18.811.2 6 (28.6)7 (33.3)8 (38.1)61.945.210 (10.8)49 (52.7)34 (36.5)63.537.1 34 (16)111 (53)64 (31)694321 (14)64 (42)67 (44)5635 No Association Bolos et al. 1990(40:127) Heinz et al.1996 (97:113) Anghelescu et.2001(243:98) Goldman et al.1998 (276:161) Lu et al. 1996, (20:25) Lee et al. 1999, (128:85) 2 (5.0)13 (32.5)25 (62.5)37.521.38 (6.3)30 (23.6)89 (70.1)29.918.1 3 (3.1)31 (32)63 (64.9)35.119.84 (3.5)35 (31.0)74 (65.5)34.519.0 13 (5.3)75 (30.9)155 (63.8)36.220.83 (3.1)32 (32.7)63 (64.3)35.819.4 96 (35)130 (47)50 (18)8258.363 (39.1)70 (43.5)28 (17.4)82.660.3 2 (10)10 (50)8 (40)6060353 (13)3 (13)14 (58)7 (29 )717142 28 (21.9)26 (52)33 (25.8)73.94811 (12.9)44 (51.8)30 (35.5)64.738.8 DRD2 Gene and Alcoholism (Huang et al.,2004)

12. Genes and Clinical Subtypes of Alcoholism

13. DRD2 Gene and Subtypes of Alcoholism  Association – Anxiety-Depression Alcoholism (Huang et al., 2004)  No Association – Pure Alcoholism (Huang et al., 2004) – Antisocial Alcoholism and Non-Alcoholism (Lu et al., 2002) – Mixed Alcoholism (data not shown)

14. DRD2 TaqI A and TaqI B Haplotypes Chinese Han Alcohol Dependence in ANX/DEP, and Controls Group Haplotype Number (2n) Haplotype frequency(%) p-value a Linkage disequilibrium p-value b A1/B1A1/B2A2/B1A2/B2DD′ Controls 3040.3380.0140.0330.6150.044 c 0.22111.000<0.001 Pure ALC d 1420.3870.0000.0140.5990.366 e 0.23180.999<0.001 ANX/DEP ALC d 2260.4730.0000.0310.4960.005 f 0.24011.000<0.001 ANX/DEP d 2580.3530.0040.0270.6160.711 g 0.22111.000<0.001 a p value of Fisher’s Exact test b p value of linkage disequilibrium in each of the four study groups c Controls vs. Pure ALC vs. ANX/DEP ALC χ 2 value=15.788,df=9 d ANX/DEP ALC, anxiety-depressive alcohol dependence ; Pure ALC, pure alcohol dependence; ANX/DEP, anxiety-depressive disorders; p value of the haplotype difference between ANX/DEP (p=0.023, χ 2 =8.324,df=3); ANX/DEP ALC vs. pure ALC (p=0.122, χ 2 =4.057,df=2) e Controls vs. Pure ALC ; χ 2 value=3.155,df=3 f Controls vs. ANX/DEP ALC ; χ 2 value=11.834, df=3 g Controls vs. ANX/DEP ; χ 2 value=1.444, df=3 (Huang et al., 2004)

15. Haplotype Frequency of the DRD2 TaqI A and TaqI B polymorphisms Group Haplotype Number (2n) Haplotype frequency (%) P Value † Linkage disequilibrium P Value ‡ A1/B1A1/B2A2/B1A2/B2DD’ Normal Controls2300.320.010.050.620.10 a 0.200.94<0.0001 Antisocial ALC § 1440.450.010.040.500.12 b 0.230.97<0.0001 Antisocial Non-ALC § 3160.420.010.050.530.14 c 0.210.95<0.0001 † p value of Fisher’s Exact test ‡ p value of linkage disequilibrium in each of two groups a Antisocial ALC vs. Antisocial Non-ALC vs. Normal Controls, χ 2 value= 10.65, df = 6 b Antisocial ALC vs. Normal Controls, χ 2 value = 5.74 df = 3 c Antisocial Non-ALC vs. Normal Controls, χ 2 value = 5.54 df = 3 § Antisocial ALC, subjects with antisocial personality disorder and alcohol dependence; Antisocial Non-ALC, subjects with antisocial personality disorder but without alcohol dependence (Lu et al., 2002)

16. MAOA Gene and Subtype Alcoholism  Association – Pure Alcoholism (Huang et al., 2007) – Mixed Alcoholism (Hu et al., 2013)  No Association – Anxiety-Depression Alcoholism (Huang et al., 2007) – Antisocial ALC or Non-Alcoholism (Lu et al., 2003; Wang et al., 2007)

17. Frequencies of MAOA polymorphisms in Han Chinese men with alcoholism and control subjects Groups/genotypes Sample size Promoter-uNTR; no (%) χ2χ2 P value Sample size EcoRV; no.(%) χ2χ2 P value 3-repeat4-repeat+- Control subjects 197123 (62.4)74 (37.6)5.1600.160201165 (62.2)76 (37.8)5.1740.159 Pure ALC 10753 (49.5)54 (50.5)4.7360.03010854 (50.0)54 (50.8)4.2830.039 ANX/DEP ALC 11872 (61.0)46 (39.0)0.0630.80211845 (38.1) 0.0030.954 MAO A= monoamine oxidase-A; VNTR= variable number of tandem repeats; EcoRV=restriction enzyme rs 1137070; pure ALC= pure alcohol dependence; ANX/DEP ALC= alcohol dependence and anxiety or depression or both. *Control subjects v.s. pure ALC v.s. ANX/DEP ALC; df=3 +Control subjects v.s. pure ALC Control subjects v.s. ANX/DEP ALC (Huang et al., 2007)

18. MAOA-uVNTR and DRD2 gene interaction for the risk of BP with ALC Model ALC+BP BOR95% CIp-Value MAOA-uVNTR 3-repeat−0.8390.4320.198–0.9420.035* DRD2 TaqI A1/A1−0.1230.8840.323–2.4190.810 DRD2 TaqI A1/A2−1.1820.8330.392–1.7700.635 MAOA-uVNTR 3-repeat ∗ DRD2 TaqI A1/A1 1.1863.2730.810–13.2240.096 MAOA-uVNTR 3-repeat ∗ DRD2 TaqI A1/A2 1.2393.4511.240–9.6080.018** ALC+BP: alcoholism with bipolar disorder. B, coefficients; CI, confidence interval; OR, odds ratio. Reference groups are: MAOA-uVNTR 4-repeat, DRD2 TaqI A2/A2 genotypes, and controls;covarying for age. ** pb0.05. (Hu et al., 2013)

19. MAOA Haplotypes for the uVNTR and EcoRV RFLP Polymorphisms in Different Groups Sample (N) Haplotype Number (Frequency x100%) χ2χ2 p-value (2R +)(3R -)(3R +)(4R -)(4R +) Community control 771 (1.3)2 (2.6)40 (51.9)34 (44.2)0 (0.0)11.580.480 Jail control (ASB) 381 (2.6) 20 (52.6)14 (36.8)2 (5.3) Antisocial Non- ALC 501 (2.0) 27 (54.0)19 (38.0)2 (4.0) Antisocial ALC410 (0.0)1 (2.4)26 (63.4)11 (26.8)3 (7.3) ASPD: Antisocial Personality Disorder without meeting the diagnosis of Alcohol Dependence Antisocial ALC: Antisocial Personality Disorder with meeting the diagnosis of Alcohol Dependence ASB: Antisocial Behavior without meeting the diagnosis of ASPD-S-ALC or ASPD-C-ALC MAO A-uVNTR : Variable number of tandem repeat locating at upstream of the MAO A gene 2R, 3R and 4R= 2 repeats, 3 repeats and 4 repeats (+) and (-) = Presence and absence of restriction sites for EcoRV (Wang et al., 2007)

20. ADH1B, ALDH2 Gene and Subtypes of Alcoholism ADH1B*2 and/or ALDH2*2 Allele  Protection  Pure, Anxiety-Depression, Mixed (data not shown) and Antisocial NON-Alcoholism (Lu et al., 2005; 2012)  No protection  Antisocial Alcoholism (Lu et al., 2005; 2012)

21. ADH1B for the Risk of Antisocial Alcoholics Community controls vs. antisocial alcoholics Variable Regression coefficient Standard error Odds ratio 95% confidence interval P-value ADH1B a ADH1B * 2/ * 2-0.3790.3150.6840.369-1.2690.229 Age0.0820.0211.0861.043-1.131<0.001 a Coding of ADH1B genotypes are * 1/ * 1 and * 1/ * 2=0, * 2/ * 2=1;reference group is ADH1B * 1/ * 1 and * 1/ * 2. (Lu et al., 2005;2012)

22. Dependent variable in regression model Total ASPD (N= 297) a (Antisocial ALC and Antisocial Non-ALC) Antisocial ALC (N=133) b Antisocial Non-ALC (N=164) c BSig. Odds ratio 95%CIBSig. Odds ratio 95%CIBSig. Odds ratio 95%CI Lower Bound Upper Bound Lower Bound Upper Bound Lower Bound Upper Bound DRD2 A1-.10.78.91.451.81.36.451.43.573.62-.35.38.70.321.55 DRD2 A2.24.381.27.752.13.43.261.53.733.22.15.601.16.662.05 ALDH2 *1*1-.24.40.79.451.38.64.091.89.903.96-1.01.01.36.18.74 DRD2*A1 + X ALDH2*1*1 1.10.043.011.058.62.42.521.53.425.521.69.015.391.5318.98 DRD2*A2 X ALDH2*1*1.10.811.10.512.36-.37.46.69.261.85.57.241.76.694.50 Logistic regression of ALDH2 and DRD2 genes for ASPD, antisocial ALC and non ALC a Antisocial Participants (Antisocial ALC and Antisocial Non-ALC) vs. Healthy Controls. b Antisocial ALC vs. Healthy Controls. c Antisocial Non-ALC vs. Healthy Controls; A1 + (*A1/*A1 or*A1/*A2) Reference category: normal control, DRD2 A2A2 genotype, ALDH2*1*2+*2*2 genotype. (Lu et al., 2005;2012)

23. Specific Meanings  A candidate gene reported for many times, still controversial; this gene still the pathogenesis of the subtype mental illness  The commonly reason (Parsian et al., 1991)  phenotypic heterogeneity  different definitions of control groups  different racially or ethnically mixed study populations  DRD2 gene a pathogenesis of ANX/DEP alcoholism

24. Specific Meanings  The ALDH2*1 risk drinking and un-subtype alcoholism (Chen et al. 1999)  50% of Han Chinese belonging ALDH2*1/*1 but nearly 100% in Western populations (Agarwal and Goedde, 1992)  ASPD only 0.1 - 0.3% in Han Chinese (Hwu et al. 1988), but 3.3% among Caucasian Americans (Goodwin & Hamilton 2003)

25. Specific Meanings  70% ASPD with alcoholism higher than 15-20% alcoholism from community in Western population (Goodwin & Hamilton 2003)  6% ASPD with alcoholism (from jail) higher than1.5-3% alcoholism from community in Han Chinese (Hwu et al. 1988; Lu et al., 2013)  ASPD demonstration higher prevalence rate of alcoholism than general population in Han Chinese and Western populations  Alcoholism one of the symptoms of ASPD?

26. Specific Meanings  A Dutch family with a MAOA deficiency in exon 8, male demonstration severe aggressive behavior and borderline mental retardation (Brunner et al., 1993)  Difference genotype frequencies in exons 8 and 14 of MAOA genes Higher ASPD with alcoholism than controls (Parsian., 1999)  MAOA 3-repeat & early childhood maltreatment development higher antisocial behavior in adult male (Capri., 2002)

27. Specific Meanings  Aggressive and/or antisocial behavior, the major symptoms of ASPD, but not the diagnosis of ASPD  Probable reasons for the positive or negative finding of ASPD lack of antisocial non-alcoholism as a controls (Lu et al., 2005; 2012)

28. The Roles of MAO, ADH and ALDH in Dopamine Catabolism

29. Dopamine Metabolism AR s ADH DOPET DOPET: 3,4-dihydroxyphenylethanol ARs : aldose (aldehyde) reductase DOPAL:3,4-dihydroxyphenylacetaldehyde COMT:catechol-O-methyltransferase DOPAL (Feldman et al., 1997)

30. Gene to Gene and Clinical Subtypes of Alcoholism

31. ALDH2 and MAOA Gene  Association – MAOA-uVNTR 3-repeat and ALDH2 gene  Antisocial Alcoholism  Antisocial Non-Alcoholism – ALDH2 x MAOA in ANX/DEP, Antisocial & Mixed alcoholism (Lee et al., 2009; 2010; Hu et al., 2013)

32. Antisocial ALC and Non-ALC after Stratification for MAOA-uVNTR polymorphisms Gene and GroupSample Size (n) Genotype number (Frequency, %) χ2p-valueOdds ratio 95% Confidence Interval ALDH2*1/* 1 ALDH2*1/*2 +*2/*2 MAOA 3-repeat Antisocial ALC 8149(60.5)32(39.5)18.7871.46E- 05 3.9132.085- 7.344 Antisocial Non-ALC9627(28.1)69(71.9) MAOA 4-repeat Antisocial ALC5126(51)25(49)1.1780.2781.5020.719- 3.137 Antisocial Non-ALC6627(40.9)39(59.1) Breslow-Day Homogeneity test of odds ratio: χ2=3.772, df=1, p=0.052 (Lee et al., 2009)

33. Logistic regression of interaction for MAOA and ALDH2 gene and the risk of ANX/DEP ALC BSEp-valueOdds Ratio 95% Confidence Interval for Odds ratio Lower Bound Upper Bound MAOA a -.519.426..233.595.2581.372 ALDH2 b -2.142.4122.0E-7.117.052.263 ALDH2 *MAOA 1.017.512.0362.9181.0707.962 B, coefficients; SE, standard errors; p<0.05 is considered significant Reference group: normal control, MAOA-uVNTR 3 allele, ALDH2 *1*1 genotype a MAOA-uVNTR 4-repeat to 3-repeat allele b ALDH2 *1*2+*2*2 to *1*1 genotype DRD2 x MAOA x ALDH2 gene in ANX/DEP Alcoholism (Lee et al., 2010)

34. Logistic regression analysis of MAOA gene and ALDH2 gene and their interaction for the risk of Antisocial ALC a. The reference category is: Antisocial Non ALC, MAOA 4-repeat, ALDH2*1/*2+*2/*2 Antisocial ALC vs Antisocial non ALC a Bdf.Sig.Exp (B) 95% Confidence Interval for Exp (B) Lower BoundUpper Bound ALDH2.3831.3131.466.6973.084 MAOA-.4101.227.663.3411.291 ALDH2 * MAOA1.0741.0322.9271.0997.795 (Lee et al., 2009)

35. MAOA-uVNTR and DRD2 gene interaction for the risk of (bipolar disorder) BP with ALC Model ALC+BP BOR95% CIp-Value MAOA-uVNTR 3-repeat−0.8390.4320.198–0.9420.035 DRD2 TaqI A1/A1−0.1230.8840.323–2.4190.810 DRD2 TaqI A1/A2−1.1820.8330.392–1.7700.635 MAOA 3-repeat ∗ DRD2 TaqI A1/A1 1.1863.2730.810–13.2240.096 MAOA 3-repeat ∗ DRD2 TaqI A1/A2 1.2393.4511.240–9.6080.018 ALC+BP: alcoholism with bipolar disorder. B, coefficients; CI, confidence interval; OR, odds ratio. Reference groups are: MAOA-uVNTR 4-repeat, DRD2 TaqI A2/A2 genotypes, and controls;covarying for age.. (Hu et al., 2013)

36. ALDH2 and DRD2 Gene Interaction  Association – ALDH2*1/*1, DRD2 A1/A1 genotypes and ANX/DEP Antisocial Non-Alcoholism and ASPD (Huang et al.,2004; Lu et el.,2012) – ALDH2 * DRD2 gene in ASPD and Antisocial Non-Alcoholism (Lu et el., 2012)  No Association – ALDH2 & DRD2 gene and Pure (Huang et el., 2004) – Mixed Alcoholism (data not shown)

37. Variable ANX/DEP ALCPure ALC Odds ratio 95% CIP-value Odds ratio 95% CIP-value ALDH2 * 1/ * 1 DRD2A1/A13.581.36-9.480.0101.5650.47-5.220.466 DRD2A1/A22.101.02-4.320.0432.2780.99-5.230.052 ALDH2 * 1/ * 2& * 2/ * 2 DRD2A1/A11.600.43-6.040.4850.8570.15-5.010.864 DRD2A1/A22.450.85-7.0600.0961.5720.46-5.390.472 Multiple Logistic Regression Analysis of the DRD2 TaqI A for Risk of Alcohol Dependence with Stratification of ALDH2 genotypes The genotype of ALDH2 * 2/ * 2 was not found in the alcohol-dependence subjects. Reference group is DRD2 A2/A2. Model 1:Neither controlling for age, gender nor stratification of ADH1B and Model2:Stratification of ADH1B and controlling for gender and age. Model 3:Stratification of ALDH2 and controlling for gender and age. (Huang et al., 2004)

38. GenotypeSubgroup DRD2 genotype distribution (and %)Χ2Χ2 p value A1A1A1A2A2A2 Antisocial ALC vs. Controls ALDH2*1/*1Antisocial ALC 16(21.0)30(39.5) 4.2110.122 Normal control 12(10.6)46(40.7)55(48.7) ALDH2*1/*2+*2/*2Antisocial ALC 12(21.0)29(50.9)16(28.1)1.5300.465 Normal control 24(18.3)58(44.3)49(37.4) Antisocial Non ALC vs. Controls ALDH2*1/*1Antisocial Non ALC 13(23.2)28(50.0)15(26.8)9.0840.011 Normal control 12(10.6)46(40.7)55(48.7) ALDH2*1/*2+*2/*2Antisocial Non ALC 14(13.0)55(50.9)39(36.1)1.6490.483 Normal control 24(18.3)58(44.3)49(37.4) Antisocial personality disorder vs. Controls ALDH2*1/*1Antisocial PD 29(22.0)58(43.9)45(34.1)8.0080.018 Normal control 12(10.6)46(40.7)55(48.7) ALDH2*1/*2+*2/*2Antisocial PD 26(15.8)84(50.9)55(33.3)1.2980.522 Normal control 24(18.3)58(44.3)49(37.4) DRD2TaqI A in Han Chinese Subjects after Stratification for ALDH2 polymorphisms (Lu et al., 2010; 2011)

39. Dependent variable in regression model Total ASPD (N= 297) a (Antisocial ALC and Antisocial Non- ALC) Antisocial ALC (N=133) b Antisocial Non-ALC (N=164) c BSig. Odds ratio 95%CIBSig. Odds ratio 95%CIBSig. Odds ratio 95%CI Lower Bound Upper Bound Lower Bound Upper Bound Lower Bound Upper Bound DRD2 A1-.10.78.91.451.81.36.451.43.573.62-.35.38.70.321.55 DRD2 A2.24.381.27.752.13.43.261.53.733.22.15.601.16.662.05 ALDH2 *1/*1-.24.40.79.451.38.64.091.89.903.96-1.01.01.36.18.74 DRD2*A1/ + A1 * ALDH2*1/*1 1.10.043.011.058.62.42.521.53.425.521.69.015.391.5318.98 DRD2*A2 /*A2 * ALDH2*1/*1.10.811.10.512.36-.37.46.69.261.85.57.241.76.694.50 Logistic regression of ALDH2 and DRD2 genes for ASPD, antisocial ALC and non ALC a Antisocial Participants (Antisocial ALC and Antisocial Non-ALC) vs. Healthy Controls. b Antisocial ALC vs. Healthy Controls. c Antisocial Non-ALC vs. Healthy Controls; A1 + (*A1/*A1 or*A1/*A2) Reference category: normal control, DRD2 A2A2 genotype, ALDH2*1*2+*2*2 genotype. (Lu et al., 2010; 2011)

40. Specific Meanings  Stratification of ALDH2*1/*1, DRD2 gene ASPD and ANX/DEP ALC (Lu et al., 2012).  54.3% ASPD with anxiety disorder in western population  Positive correlation with ASPD and anxiety disorder. (Goodwin & Hamilton., 2003)  Anxiety disorders and ASPD associated with higher odds of alcohol use disorder (Goodwin and Hamilton, 2003)

41. Specific Meanings  ALDH2 * DRD2 gene and BP (Lee et al., 2010)  70-90% BP with anxiety disorder in Western, 20-40% in Han Chinese populations (Chang et al., 2012).  50% of Han Chinese belonging ALDH2*1/*1 but nearly 100% in Western populations (Agarwal and Goedde, 1992)  ALDH2 gene relation to alcoholism, ASPD, anxiety disorder & bipolar disorder.

42. In Summary  DRD2 Association ─ ANX/DEP Alcoholism  MAOA Association ─ Pure and Mixed Alcoholism  ALDH2 Association ─ Pure, ANX/DEP Alcoholism and Antisocial non-alcoholism  ADH1B and ALDH2 Association ─ Pure, ANX/DEP and Mixed Alcoholism

43. In Summary  ALDH2*1/*1, DRD2 A1 /A1 − Anxiety-Depression Alcoholism − ASPD and Antisocial Non-ALC  MAOA 3-repeat,ALDH2 Association – Antisocial Alcoholism  MAOA X ALDH2 Association – Anxiety-Depression Alcoholism – Antisocial Alcoholism – Mixed Alcoholism  ALDH2 X DRD2 Association – ASPD and Antisocial Non-ALC

44. Ongoing Studies  Preliminary studies of low dose (5mg) of memantine : (6 M )  Pure and anxiety-depression alcoholism 1/3 completing discontinue drinking; 1/3 markedly reduced drinking; 1/3 poor response  Antisocial and Mixed Alcoholism (bipolar alcoholism) Poorer response than pure and anxiety-depression alcoholism

45. Comments  Possible mechanism of mematine effect  Inhibition NADPH in microglial and stimulation neurotrophic factors (BDNF etc.) in astroglial cells  Plasma level of memantine (5mg/day): 10-50 ng/ml (mean 16 ng/ml). No effect in NMDA receptor (required > 86.3 – 431.5 ng/ml) (Parsons et al., 1999)

46. Conclusion  More studies about MAO, ADH1B, ALDH2 and dopamine/5-HT related genes in different ethnics of major mental illnesses  The lower prevalence of a disease, the higher statistic meaning for a fixed number of trait loci (Rice et al., 2001)  Han Chinese populations playing a specific contribution to the molecular genetic studies of alcoholism (Lu et al., 1999)  Inflammatory press & neurodegenoration involving alcoholism & substance use disorders.  Alcoholism: An important role in substance use disorder and addiction behavior

47. Acknowledgements  Professor Kenneth K. Kidd, Ph. D.  Professor Ting-Kai Li, Ph. D.  Research Scientist Judy R. Kidd, Ph.D  Professor Jean-Shih Chen, Ph. D  Research Scientist Andrew J. Pakstis, Ph. D.  Professor Shin-Jiun Yin, Ph. D.  Professor Huei-Chen Ko, Ph. D  Associate Professor San-Yuan Huang, M.D., Ph. D.  Associate Professor Yaun-Hwa Chou, M.D., Ph. D.  Assistant Professor Wei-Wen Lin, M.D., Ph. D.  Assistant Professor Jia-Fu Lee, M.D., Ph. D.

48. Acknowledgements  Assistant Professor Yi-Chyan Chen, M.D., Ph. D.  Assistant Professor Michael V. Osier, Ph. D.  Assistant Professor Tso-Jen Wang, M.D., Ph. D.  Hsin-Yi, Lo M.D., Ph. D. student  Cheng-Yi Hahn, M.D., M.S., Ph. D. student  Sheng-Yu Lee, M.D., M.S.  Ms. Ju-Ping Weng, M.S.  Ms. Yi-Syuan Wu, M.S.  Ms. Pei-Ling Wu, M.S.  Mr. Tsun-En Lu, M.S.  Ms. Yu-Shan Wang.

49. THANK YOU FOR YOUR ATTENTION

51. Strategies for developing therapy for drug addiction Pre-inflammatory Factors (ROS, NO., TNF , ILs, PGs) Reactive Microgliosis (Self-propelling) Drugs or toxin (Extro-neurotoxin) Activation Genetic factors (Endo-neurotoxin) Microglia (Working Model) Dopaminergic Neuronal Damage

52. NADPH oxidase (PHOX) Activated O2O2 O2 –O2 – NADPH NADP + + H + Resting gp91 p22 rap1a p40 p47 rac2 p67  MAC-1 LPS out in PKC P P Naloxone, Dextromethorphan Gly-Gly-Phe

53. O2O2 O 2 – Dual Functions of Superoxide Radicals gp91 Microglia MAC1 TLR4 LBP CD14 PHOX ROS H2O2H2O2 O 2 – TNF-  IL-1 NO ONOO - 2) Gene Expression 1) Neurotoxicity NF-kB,AP-1 PKC LPS DA neurons

54. Transfection of gp91 PHOX, a membrane subunit of PHOX, increases [ 3 H]-naloxone binding 0 100 200 300 [ 3 H]-(-)- Nal Binding Capacity (% WT) WT [ 3 H]-(+)-Nal * Gp91- transfected 400 COS-7 Cells (WT) Ligand Binding Assay Stable Transfection COS-7 gp91 ( gp91 ) gp91 *

55. Serotonin Metabolism (Svensson et al. 1999) The metabolism pattern of serotonin (5-HT). MAO = monoamine oxidase; ADH = alcohol dehydrogenase; ALR = aldehyde/aldose reductase; ALDH = aldehyde dehydrogenase; 5-HIAL = 5-hydroxyindole-3-acetaldehyde; 5-HTOL = 5-hydroxytryptophol; 5-HIAA = 5-hydroxyindole-3-acetic acid

56.  ADH1B*2 allele: protective factor  ALDH 2 *2 allele: protective factor (Thomasson et al. 1991) Ethanol Metabolic Genes and Alcoholism

57.  ALDH2 deficiency, which slows the elimination of acetaldehyde (Thomasson et al. 1991)  Higher acetaldehyde levels generated by the more active ADH isozymes should deter heavy drinking (Thomasson et al. 1991) Ethanol Metabolic Genes and Alcoholism

58. (Thomasson et al. 1991) a Number of individuals in group. Note that some exons did not amplify well or gave ambiguous results; thus some individuals were excluded. b Fraction of group with each genotype; because of rounding errors, some groups’ frequencies do not sum to 1.00. c Alcoholics are significantly different from nonalcoholics (P<.002). The ADH2 genotype distribution among alcoholics did not fit the Hardy-Weinberg equilibrium; all other genotype distribution did. d Alcoholics are significantly different from nonalcoholics (P<.005). ADH and ALDH Genotype Frequencies and Allele Frequencies Genotype Frequency b Allele Frequency Group (N a )ADH1B*1/*1ADH1B*1/*2ADH1B*2/*2ADH1B*1ADH1B*2 Nonalcoholics (47)…….0.060.400.530.270.73 Alcoholics (49)…………0.37 c 0.31 c 0.33 c 0.52 d 0.48 d Genotype Frequency b Allele Frequency ALDH2*1/*1ALDH2*1/*2ALDH2*2/*2ALDH2*1ALDH2*2 Nonalcoholics (50)…….0.520.360.120.700.30 Alcoholics (50)…………0.88 d 0.12 d 0.00 d 0.94 d 0.06 d

59. Contradictory Findings of ADH  ADH1B*1 among most of world population (~90%) (Bosron et al. 1993)  ADH1B*2 among Han Chinese (~70%) ( Osier et al. 2002)  ADH1B*2 among Taiwanese aborigines (70-86%) (Chen et al. 1996)  Alcohol prevalence (30-50%) among Taiwanese aborigines >15 times Han Chinese in Taiwan  No support the variance of ADH1B related to rice (Kidd et al., 2010)  Ethnic genetic heterogeneity of ADH1B*2

60. Figure 1. Blood ethanol concentrations after administration of a low-to-moderate dose of ethanol (0.3g/kg) to men with different ADH2 and ALDH2 allelotypes during 4-h period. All subjects were homozygous ADH3*1. The genotypic groups are denoted as ADH2*1/*1, ALDH2*1/*1 ( , n=12); ADH2*1/*2, ALDH2*1/*1 ( , n=10); ADH2*2/*2, ALDH2*1/*1 ( , n=11); ADH2*1/*1, ALDH2*1/*2 (, n=10); ADH2*1/*2, ALDH2*1/*2 (, n=12); ADH2*2/*2, ALDH2*1/*2 ( , n=11). Vertical bars (only the upper or lower portion shown) represent standard errors of the means. Statistically significant differences between the groups at corresponding times by ANOVA: a p<0.001 vs. ADH2*1/*1, ALDH2*1/*1 b p<0.01 vs. ADH2*1/*2, ALDH2*1/*1 c p<0.001 vs. ADH2*2/*2, ALDH2*1/*1 d p<0.05 vs. ADH2*2/*2, ALDH2*1/*2 (Peng et al. 1999)

61. Figure 2. Alterations of blood ethanol in men with homozygous ALDH2*1/*1 and heterozygous ALDH2*1/*2 genotypes, both included three different ADH2 allelotypes (see Fig. 1), following a low-to- moderate dose of ethanol (0.3g/kg) during 4-h period. All subjects were homozygous ADH3*1. The genotypic groups are denoted as ALDH2*1/*1 ( , n=33) and ALDH2*1/*2 (, n=33). Vertical bars (both the upper or lower portion shown) represent standard errors of the means. Statistically significant differences between the groups at corresponding times by Student’s t-test: a p<0.05 vs. ALDH2*1/*1 b p<0.01 vs. ALDH2*1/*1 c p<0.001 vs. ALDH2*1/*1Fig. 1 (Peng et al. 1999)

62. Figure 3. Blood acetaldehyde concentrations after administration of a low-to-moderate dose of ethanol (0.3g/kg) to men with different ADH2 and ALDH2 allelotypes during 4-h period. All subjects were homozygous ADH3*1. The genotypic groups are denoted as ADH2*1/*1, ALDH2*1/*1 ( , n=12); ADH2*1/*2, ALDH2*1/*1 ( , n=10); ADH2*2/*2, ALDH2*1/*1 ( , n=11); ADH2*1/*1, ALDH2*1/*2 (, n=10); ADH2*1/*2, ALDH2*1/*2 (, n=12); ADH2*2/*2, ALDH2*1/*2 ( , n=11). Vertical bars (only the upper or lower portion shown) represent standard errors of the means. Statistically significant differences between the groups at corresponding times by ANOVA: a p<0.05 vs. ADH2*1/*1, ALDH2*1/*1 b p<0.01 vs. ADH2*1/*2, ALDH2*1/*1 c p<0.05 vs. ADH2*1/*2, ALDH2*1/*1 d p<0.01 vs. ADH2*1/*2, ALDH2*1/*1 e p<0.05 vs. ADH2*2/*2, ALDH2*1/*1 f p<0.01 vs. ADH2*2/*2, ALDH2*1/*1 (Peng et al. 1999)

63. Process of DSM Diagnosis  DSM-III : reliability of mental illnesses  DSM-III-R → DSM-IV-TR – developed the validity for diagnostic criteria