1. Candidate Gene Studies in Substance-Dependent Adolescents, their Siblings, and Controls S. E. Young, A. Smolen, M. C. Stallings, R. P. Corley, T. J. Crowley and J. K. Hewitt BACKGROUND Why study the dopamine system? Dopamine is known to mediate motor activity and reward mechanisms. Dopamine imbalance is implicated in psychopathology:  Schizophrenia  Tourette Syndrome  Depression  Substance Abuse  ADHD WHICH CANDIDATE GENES? Dopamine Transporter (DAT1) Mapped to 5p15.3 16 Non-coding region at 3’ end 40 base-pair VNTR (3-11 repeats) DAT1 polymorphisms have been associated with:  Substance Dependence 8,12  ADHD 3,19  Tourette Syndrome 11  Externalizing Behavior 20 Dopamine Receptor (DRD2) Taq I A polymorphism Mapped to 11q22-23 6 RFLP in non-coding region DRD2 Taq1 polymorphism has been associated with:  Substance Dependence 10  Conduct Disorder 9  P300 7 Dopamine Receptor (DRD4) Mapped to 11q15.5 18 48 bp VNTR in exon 3 7 alleles grouped into ‘long’ (7+), ‘short’ (6-) DRD4 has been associated with:  Novelty Seeking 1,5  Substance Dependence 2,17  ADHD 14 CLINICAL SAMPLES Adolescent patients (n = 177) in treatment for substance use problems and antisocial behavior. - Ages: 13-18 years (x = 15.8; s.d. = 1.3) - Gender: 166 males; 13 females Biological siblings (n = 71) of the patients with at least 1 dependence symptom on any substance: - Ages: 12-18 (x = 15.9; s.d. = 1.7) - Gender: 36 males; 35 females CONTROL SAMPLES Adolescents (n = 257) originally ascertained for the Colorado Adoption Project (1 adolescent per family included): - Ages: 16-18 years (x = 17.5; s.d. = 0.3) - Gender: 139 males; 118 females Adolescent twins (n = 184) originally ascertained for the Colorado Longitudinal Twin Study (1 twin per family included). - Ages: 12-13 (x = 12.5; s.d. = 0.3) - Gender: 82 males; 102 females ASSESSMENTS Substance Use and Dependence: Composite International Diagnostic Interview – Substance Abuse Module 4 Average Dependence Symptoms = Sum of symptoms across substances # substances used > 5 times Conduct Disorder Symptoms Diagnostic Interview Schedule for Children 13 SAMPLE CHARACTERISTICS University of Colorado School of Medicine Division of Substance Dependence STUDY I Are there increased frequencies of dopamine risk alleles in the clinical samples compared to the control samples? Allele Frequencies Genotype Frequencies SUMMARY I We found no significant differences in genotypic frequencies for DAT1 among the three groups. Genotypic frequencies for DRD2 were significantly different among the three groups (  2 =12.7; p=.01). There were significant differences in the genotypic frequencies among the three groups for DRD4 (  2 =14.1; p=.01). However, these differences were driven by the sibling sample, and do not remain significant when comparing the patient sample to the control sample. STUDY II Are frequency differences for DRD2 confirmed in family-based analyses? The Transmission/Disequilibrium Test 15 The transmission/disequilibrium test (TDT) assesses deviations from equal transmission of two alleles from heterozygous parents to affected children. TDT = (t 21 – t 12 ) 2 -------------------- t 21 + t 12 -- which is asymptotically distributed as chi-square with one degree of freedom. The TDT requires genotypic data on both parents and an affected child; only heterozygous parents are informative. One advantage of the TDT is that it provides a test of association while avoiding Type I error due to population stratification. Example: (A1 = risk allele) Mother Father FatherMother A1/A2 A1/A1 A1/A1A1/A2 A1/A1 A2/A1 (Transmission) (Non-transmission) Affected ChildAffected Child RESULTS II SUMMARY III Analysis of variance revealed no significant differences in the mean symptom counts for nicotine dependence, alcohol dependence, average dependence or conduct disorder by genotype for DAT1, DRD2 or DRD4. There was a trend towards higher conduct disorder symptoms for the 9/9 genotype for DAT1, which will be followed up as the sample size increases. DISCUSSION We found preliminary evidence for an association between the DRD2 A1 risk allele and substance dependence in an analysis of adolescent patients, their siblings, and controls (Study I). This association was not confirmed when a family- basedTDT was applied (Study II). The sample size was modest (n=107 informative trios); however, the pattern of transmission was not consistent with the predicted association. Risk alleles for DAT1, DRD2 and DRD4 were not associated with symptom severity in the patient (affected) group (Study III). These results obtained in an adolescent sample do not confirm findings previously reported on adult alcohol or nicotine dependent samples. Differences in these findings may also be due, in part, to the fact that our patient sample is a polysubstance abusing sample with extensive comorbid psychopathology. As the patient sample expands, associations between risk alleles and specific substances (e.g., alcohol dependence only) may be possible. ACKNOWLEDGEMENTS This research was supported by the following NIH grants: DA-05131, DA-11015, DA-12845, HD-10333 and MH-43899. We would like to acknowledge Chih-Mae Cheng and Chinatsu McGeary for their technical assistance. We also gratefully acknowledge the study participants for their time and effort. REFERENCES [see handout] Genotype Nic Sx Alc Sx Avg Sx Cond Sx __________________________________________ DAT1 (N) 9/9 (9)1.7 (1.9) 2.3 (2.5) 1.6 (1.0) 8.2 (4.2) 9/10 (65)2.9 (2.3) 2.1 (2.0) 2.5 (1.4) 5.4 (3.0) 10/10 (100)2.8 (2.2) 1.9 (2.0) 2.5 (1.3) 5.8 (2.9) DRD2 A1/A1 (16)2.6 (2.6) 1.8 (2.3) 2.3 (1.6) 4.9 (2.9) A1/A2 (73)2.9 (2.2) 2.2 (2.1) 2.4 (1.3) 5.9 (2.5) A2/A2 (88)2.7 (2.1) 1.9 (1.9) 2.5 (1.2) 5.9 (3.2) DRD4 L/L ( 7)3.6 (2.1) 1.6 (2.4) 2.9 (1.0) 6.5 (3.3) L/S (54)2.6 (2.2) 1.7 (1.5) 2.2 (1.1) 5.9 (3.1) S/S (110)2.8 (2.2) 2.2 (2.1) 2.5 (1.4) 5.7 (3.0) SUMMARY II Of the 104 informative family trios, 32 of the parental genotypes were unambiguously inferred from sibling genotypes. The TDT was non-significant (  2 =.62; p =.43), suggesting that the allelic association shown in Study I (greater frequency of the A1 allele in clinical vs. control samples) may have been due to population stratification. Previous analysis of trios without inferred parental genotypes produced similar results (  2 = 1.03; p =.75). DRD2 DRD2 Non-transmitted Transmitted A1 A2 A1 -- 48 A2 56 -- TDT (  2 ) =(56 – 48) 2 = 0.62; p =.43 (56 + 48) Allele Frequencies Genotype Frequencies STUDY III Are dopamine risk alleles associated with severity of substance dependence or conduct problems? Patient Sample [means(s.d.)]