1、ADHD and DAT1: Affected Family-Based Control Study using TDT,Young Shin Kim, M.D., MPH Dept. of Epidemiology UC Berkeley,Outline,Genetics in Medicine: overview Association Studies ADHD and DAT1: Affected Family-Based Control Study using TDT Data Analysis,Genetics in Medicine; Two Approaches,Genetic
2、EpidemiologyMacroscopic information (role of genetic and environmental factors, the familiarity of a disorder, and the mode of transmission) Molecular GeneticsFundamental information (identification of the normal products of the vulnerability genes, when and where the genes are normally expressed in
3、 the developing CNS, and insights into how specific alleles function to establish disease vulnerability) parametric methods - classical genetic linkage analysisnon-parametric methods - association studies, allele-sharing methods (affected sib-pair or affected relative studies),Genetic Epidemiology (
4、1),Twin study (initial indicator of importance of genetic vs. non-genetic factors)If MZ and DZ twins share their environment to the same extent, a higher concordance rate of disorders in MZ twins than in DZ twins suggests a strong genetic influence on the pathogenesis of the disorder Adoption Study
5、(Useful for distinguishing genetic and environmental influences confounded by the shared environment in family studies) 1) a classic adoption study - compare the rates of disorders in biological relatives of the affected probands with those in the adoptive relatives2) an adoptees study to compare th
6、e rates of disorders in adopted offspring of affected parents with those in adopted offspring of unaffected parents3) a cross-fostering study to compare the rates of disorders in adopted offspring of affected parents who were raised by unaffected parents with those in adopted offspring of unaffected
7、 parents who were raised by affected parents,Genetic Epidemiology (2),Family Genetic Studies- Investigate the rates and patterns of occurrence of disorders in biological relatives of probands with a disorder - Useful tool for exploring the mode of transmission of a disorder - Caution; information bi
8、as, possibility of cultural transmission (phenotypes of interest are transmitted within families by non-genetic mechanism) Segregation Analysis- Infer a best fitting mode of transmission of a disorder by ruling out those modes which do not fit the disorder- Visual inspection of obvious segregation p
9、atterns in pedigrees and performing formal statistical testing,Molecular Genetics (1),Studies of cytogenic abnormalities - Deletion and translocation of chromosomes - Clues to the chromosomal localization of disease vulnerability genesParametric studies - Classical genetic linkage analysisNon-parame
10、tric studies - Association studies - Allele-sharing methods (affected sib-pair or affected relative studies),Molecular Genetics (2),Linkage Analysis (powerful tool due to its ability to locate disease vulnerability genes precisely)Attempt to identify disease vulnerability genes by investigating the
11、association between the transmission pattern of a disorder in the pedigree and the linkage between a known genetic marker and putative genes thought to be responsible for disease, by detecting an RF smaller than 0.5 and estimating the magnitude of the linkage. A limitation of linkage analysis 1) dis
12、ease model dependent (specifically, in psychiatric disorders, depends on correct assumptions regarding the involvement of a single major gene in the disease transmission, genetic homogeneity, and the precise mode of transmission) 2) statistical multiple testing3) limited applicability in disorders w
13、ith complex traits, such as phenotypic variation, phenocopies, incomplete penetrance, genetic heterogeneity, polygenic inheritance and a high frequency of disease causing alleles in the population.,Molecular Genetics (4),Association studies Detect a difference in allele frequencies at a particular l
14、ocus, using a case-control study design. A positive allele association can indicate 1) the marker examined plays a causative role in the disorder 2) the marker is in linkage disequilibrium, and therefore, has no direct effect on the pathogenesis of the disorder but is closely linked to a disease-cau
15、sing gene 3) it is a false positive finding due to either an artifact of population admixture (ethnic difference in allele frequencies) or polymorphism in the marker which leads to consequent multiple testing. Amendment for false positive findings 1) using a homogeneous population or using an intern
16、al comparison group, (affected family-based control) 2) Statistical adjustment for multiple testing The advantages of association studies lack of a requirement for transmission models or assumptions and their potential to detect genes of small effect.,Molecular Genetics (5),A candidate gene approach
17、A special case of marker disease association study with a recombination fraction of 0 between marker and disease locus Candidate genes that are implicated in the pathogenesis of the disorder (e.g.; genes coding for specific NTs or receptors),Molecular Genetics (6),The allele-sharing methodsAffected
18、sib-pair analysis, affected relatives analysis of a pedigree Detect disease vulnerability genes: non-parametric method to detect linkage Investigators examine whether the inheritance pattern of a chromosomal region is inconsistent with random Mendelian segregation by showing that affected relatives
19、or siblings inherit identical copies of the region more often than expected by chance,Association Studies (1),Linkage EquilibriumSpecific alleles at 2 loci; M, D M generation) - linkage disequilibrium can persist for hundreds of generations in tightly linked loci,Association Studies (2),Example; Ank
20、ylosing spondylitis (recessive gene)At least 1 Allele at a marker loci B27(+) B27(-) TotalDisease D(+) 72 3 75 Status D(-) 3 72 75Total 75 75 150RR (relative risk) = Pdevelop disease with risk factor/Pdevelop disease without risk factor= OR (in rare diseases) = 72*72/(3*3) = 576,Association Studies
21、(3),Caution on interpretationAssociation can arise as an artifact due to population admixture (e.g.) association study between trait of the ability to eat with chopsticks and the HLA-A locus in the SF, then allele A1 would be positively associated because both the ability to use chopsticks and allel
22、e A1 is more frequent among Asians than Caucasians,Affected Family-based Control Association Studies (1),Basic Ideas (Rubinstein et al., 1981; Falk & Rubinstein, 1987)To avoid the difficulties in selecting appropriate control group, use parental data in place of non-related controlsSubjects Nuclear
23、family with a single affected child - type at the marker locus - 2 parental alleles not transmitted to the affected child and they serve as a hypothetical control individual,Marker Genotypes in Nuclear Family (concept for AFBAC),M/m m/mM/maffected childhypothetical control; m/m,Affected Family-based
24、 Control Association Studies (2),Comparison with association studiesDisadvantages 1) more genotyping (2 in association studies, 3 in affected family-based control studies)2) Difficulty in sampling of triosAdvantagesovercoming problem of population stratification and false positive results of case-co
25、ntrol studies,Affected Family-based Control Association Studies (3),Case-Control Study Genotype M (+) M (-) TotalCase 23 6 29 Control 14 15 29 Total 37 21 58,AFBAC1 Haplotype Relative Risk (HRR) (1),Genotype-based Analysis (heterozygosity of allele M is not important) ; recessive modelNon-Transmitte
26、d genotype M (+) M (-) M/M, M/m m/m TotalTransmitted M(+) 9 14 23 (W) Genotype M(-) 5 1 6 (X)Total 14 (Y) 15 (Z) 29 (n)each family contribute to one cell (total of 29 families) Assumption; distribution of marker genotypes from NT parental alleles in families is identical to the distribution of marke
27、r genotypes in the population.,AFBAC1 Haplotype Relative Risk (HRR) (2),HRR = W (M (+) affected child frequency) / X (M (-) affected child frequency)Y (M (+) in NT parental alleles) / Z (M (-) in NT parental alleles)= (W*Z)/ (X*Y) = (23*15)/(6*14) = 4.17H0; No associationTest statistics; 2 test (14-
28、5)2/(14+5), df=1, p=0.039 Similarity of HRR with RR in case-control studies = 0, HRR=RR (no recombination = linkage),AFBAC2 Haplotype-Based Haplotype Relative Risk (HHRR) (1),Terlinger unmatched analysis of TDT Parental allele TotalM mTransmitted 52 6 58 Non-transmitted 39 19 58Total 91 25 116,AFBAC
29、2 Haplotype-Based Haplotype Relative Risk (HHRR) (2),Assumption; 1) contribution of the parents are independent2) T and NT genotypes are independent H0; no association Test statistics; 2 test,AFBAC3 Transmission/Disequilibrium Test (TDT) (1),Spielman et al. (1993)Non-Transmitted allele TotalM mTrans
30、mitted M 33 19 52m 6 0 6Total 39 19 58Classify each single parent according to his/her T and NT allele (Terwilliger 58 parents in 29 families,AFBAC3 Transmission/Disequilibrium Test (TDT) (2),H0; no association and linkage (1-2) = 0 (=0; each heterozygous parent transmits its M allele to the affecte
31、d child with probability of - no association=1/2; no linkage)Test statistic; McNemars 2 test = (b-c)2/(b+c) = (19-6)2/(19+6) = 6.76 (p=0.0093)Only heterozygous parents contribute to the analysis Limitation TDT can detect linkage between the marker locus and the disease locus only if association (due
32、 to linkage disequilibrium) is present,DAT1 and ADHD: Affected Family-Based Control Study using TDT,Characteristics of Attention Deficit- Hyperactivity Disorder (ADHD)- Attention problem (inattention, distractibility)- Hyperactivity, impulsivity Common; 3-6% Risk factor for antisocial and drug abuse
33、 in adulthood,Heritability of ADHD,Twin studies 0.8 heritability estimates Family studiesmore ADHD in relatives of probands with ADHD compared to relatives of adoptive parents, normal controls, or psychiatric controls Segregation analysis(1) autosomal dominant transmission with reduced penetrance of
34、 the hypothesized gene(2) single-gene effect vs. polygenic inheritance,DAT1 as a Candidate Gene,DAT1 - VNTR (variable numbers of tandem repeats) of a 40 base-pair repeat sequence on chromosome 15.3- Majority; 10 repeats or 9 repeats DAT1 and ADHD- Dopamine hypothesis; children with ADHD responds wel
35、l to dopamine agonists and has inhibitory effects on the dopamine transporter (DAT1)- Animal studies1) overexpresesion of mutant rat dopamine transporter 2) mucous knockout studies,Aim of the Study,To test the previously found family-based association of ADHD with the dopamine transporter 10-copy (4
36、80 bp) allele in this larger Korean sample,Study Subjects,Challenge; reduce phenocopy (someone plays the behavioral symptoms of ADHD without hypothesized genetics etiology) Inclusion criteria(a) Diagnosis of DSM-IV ADHD, Combined Type, determined by concurrence of two independent diagnosticians afte
37、r review of all clinical information, corroborated by K-SADS and combined parent and teacher reports(b) Age between 6 and 12 (c) WISC-III Full Scale IQ 80(d) Participation of both biological parents(e) Parent informed consent and child assent Exclusion criteria(a) Seizure disorder or neurological di
38、sease, bipolar mood disorder, pervasive developmental disorder, Tourette syndrome or chronic motor tic disorder, or uncorrected sensory impairment.(b) Parental history of bipolar mood disorder,Genetic Lab Procedures,Dopamine transporter genotyping 1) PCR will be carried out in a 10 l volume containi
39、ng 50 ng of genomic template, 0.5 M of each primer, one of which is 5 fluorescently labeled, 200 M of each dNTP (dATP, dCTP, dGTP, dTTP), 1 x PCR buffer, 2 mM MgCl2, and 0.5 units Taq polymerase (Amplitaq Gold). Samples will be amplified on a 9700 thermal cycler with an initial 12 minute step to hea
40、t-activate the enzyme, 40 cycles consisting of a denaturation step of 95 degrees C for 30 sec., an annealing step of 68 degrees C for 30 sec., and an extension step of 72 degrees C for 30 sec. 2) Products will be injected on an ABI 3700 multi-capillary array genetic analyzer with POP6 polymer. Produ
41、cts will be detected by laser-induced fluorescence using sheath flow on the ABI 3700. Electropherograms will be processed with Genescan software and alleles will be called with Genotyper software, blind to all but a number which is consecutively assigned and is not related to whether the subject is
42、a child, father, or mother and without any indication of relationship to adjacent numbers.,Preliminary Analysis,25 complete trios with ADHD combined type. Novel allele found; 365bp (7 repeat allele) Linkage Format,2X2 Table Illustration (1),HRR AnalysisNon-Transmitted genotype 483 (+) 483 (-) TotalT
43、ransmitted 483 (+) 24 1 25 (W) Genotype 483 (-) 0 0 0 (X)Total 24 (Y) 1 (Z) 25 (n)HRR = (25 * 1) / (0 * 1) = 2 test = not calculable due to 0s in cells Accept null hypothesis; no association,2X2 Table Illustration (2),HHRR AnalysisParental allele Total483(+) 483(-)Transmitted 43 7 50 Non-transmitted
44、 46 4 4 50Total 89 11 1002 test = 0.919 (p=0.338) Accept null hypothesis; no association,2X2 Table Illustration (3),TDT AnalysisNon-Transmitted allele Total483 (+) 483 (-)Transmitted 483 (+) 39 4 43 allele 483 (-) 7 0 7Total 46 4 50McNemars 2 test with 1 d.f. = (4-7)2/(4+7) = 0.8182. (p 0.05) Accept
45、 null hypothesis; no association and no linkage,Implications,Possible that there is no association and linkage in Korean children with DAT1; difference with Caucasian children with ADHD Limitations Small sample size and small number of heterozygous allele parents provided limited information on the
46、TDT analysisAnalysis of more samples (aimed at least 120 trios) are underway,Acknowledgment,Child and Adolescent Psychiatry, University of ChicagoLaboratory of Developmental Neurosciences, Center for Developmental Disorders Bennett Leventhal, M.D.Ed Cook, M.D.Soo Jung Kim, M.D.Multi-center CollaborationKen Ah Cheoun, M.D. Yonsei University Medical CollegeBoo Nyun Kim, M.D., Seoul National University Medical CollegeHee Jung Yoo, M.D., Kyungsang National University Medical CollegeThanks to the children and their families participated in this study,
