| Summary |
Given the contribution of central cholinergic circuits to the control of movement and attention, the authors hypothesized that functional CHT gene variants might impact risk for ADHD. They performed a case-control study, followed by family-based association tests on a separate cohort, of two purportedly functional CHT polymorphisms (coding variant Ile89Val (rs1013940) and a genomic SNP 3' of the CHT gene (rs333229), affording both a replication sample and opportunities to reduce potential population stratification biases. Initial genotyping of pediatric ADHD subjects for two purportedly functional CHT alleles revealed a 2-3 fold elevation of the Val89 allele (n=100; P-value=0.02) relative to healthy controls, as well as a significant decrease of the 3' SNP minor allele in Caucasian male subjects (n=60; P-value=0.004). In family based association tests, significant overtransmission of the Val89 variant to children with a Combined subtype diagnosis (OR=3.16; P-value=0.01) was found, with an increased Odds Ratio for a haplotype comprising both minor alleles. These studies show evidence of cholinergic deficits in ADHD, particularly for subjects with the Combined subtype, and, if replicated, may encourage further consideration of cholinergic agonist therapy in the disorder. |
| Total Sample |
For case-control study: the VUMC sample consisted of 50 unrelated and 10 affected sib pairs (n=63 subjects genotyped; 81% male; 51% Cacasian/12% African USAn) ranging from 6-17 years of age; the University of Chicago cohort (n=37 subjects geno- typed; 70% male; 86% Caucasian/14% African USAn) consisted of a subset of unrelated children who had participated in an earlier pharmacogenetic study; The Vanderbilt University control cohort consisted of 290 subjects, between 18-45 years of age that displayed no clinically significant abnormality based upon medical history, physical examination and routine laboratory testing. For family-based study: 403 children from 251 families were included; probands and their siblings between the ages of 4 and 18 (M=10.7, SD=3.9) were recruited. The initial evaluation of the association of CHT SNPs in ADHD was performed as a simple case-control design with affected subjects drawn from prior Vanderbilt and University of Chicago studies. The mean age of the Vanderbilt/Chicago sample was 10.5(SD=0.3) years and was 76% male, 24% female with mostly Caucasian (81%) versus African-USAn (19%) subjects. Controls subjects were drawn from a panel of healthy controls with no medical diagnoses, originally recruited for cardiovascular studies. The mean age of the control sample was 28,SD=7.7 years and was 42% male, 58% female with both Caucasian (44%) and African-USAn (56%) subjects. Owing to significant ethnic variation evident in the CHT Val89 variant, the case-control analyses were restricted to the Caucasian subset of this panel. The study sample used for within-family association analyses comprised 269 boys (67%) and 134 (33%) girls, with an ethnic background of 78% Caucasian, 10% African-USAn, 2% Hispanic, 1% Asian, and 8% of mixed ethnicity.200 of the 251 probands met criteria for a DSM-IV ADHD diagnosis with 70 children (35%) diagnosed with the predominately Inattentive subtype, 12 (6%) with the predominately Hyperactive-Impulsive subtype, and 118 (59%) with the Combined subtype. 36 of the 152 siblings that participated in the study met criteria for ADHD. Thirteen (36%) were diagnosed with the predominately Inattentive subtype, 10 (28%) with the predominately Hyperactive-Impulsive subtype, and 13 (36%) with the Combined subtype. Control subjects ranged in age from 6 to 18 years (M=12.9, SD=2.6), included 138 boys (58%) and 98 girls (42%), and their ethnic background was 92% Caucasian, 2% African-USAn, 2% Asian, and 4% Hispanic. 45 met criteria for the ADHD diagnosis. Twenty-four (53%) were diagnosed with the predominately Inattentive subtype, 8 (18%) with the predominately Hyperactive-Impulsive subtype, and 13 (29%) with the Combined subtype. |
| Sample Collection |
ADHD subjects involved in initial case-control study were consented and enrolled at Vanderbilt University Medical Center (VUMC), Nashville, TN, USA and the University of Chicago Children's Hospital, Chicago, IL, USA. For family-based study, 403 children from 251 families were consented and enrolled from two sites, Atlanta, Georgia and Tucson, Arizona, USA. At the Atlanta site, subjects were recruited through the Center for Learning and Attention Deficit Disorders (CLADD) at the Emory University School of Medicine and the Emory University Psychological Center. At the Tucson site, subjects were recruited through a group psychiatric practice. For family-based study, control subjects were also recruited from sites in Atlanta, Georgia and Tucson, Arizona. Subjects recruited at the Atlanta site represent a subsample of the Georgia Twin Registry, a sample of twins born between 1980 and 1991 recruited via birth records from the general population of Georgia. Twin families were originally recruited by mail to participate in a questionnaire-based study of childhood psychopathology. A subset of these families was contacted by phone to participate in a follow-up lab study of temperament and cognitive development that included DNA collection. Subjects from the Tucson site were drawn from the general population. |
| Diagnosis Description |
Subject ascertainment for samples involved in case-control study was performed under a protocol approved by the Institutional Review Boards of both institutions. The Emory University and University of Arizona Institutional Review Boards reviewed and approved the assessment procedures utilized in family-based study. For case-control study: in the VUMC sample, Kiddie-SADS-Present and Lifetime Version was used to determine DSM-IV criteria for ADHD subtypes; all subjects completed a semi-structured diagnostic interview and met DSM-IV criteria for ADHD; children were excluded if they carried a diagnosis of autism or other brain disorders such as mental retardation. For case-control study: both clinics at the Atlanta site specialize in the assessment of childhood external- izing disorders such as ADHD, Oppositional Defiant Disorder (ODD), Conduct Disorder (CD) and learning disorders; any child previously diagnosed with autism, traumatic brain injury, or neurological conditions (e.g., epilepsy) was excluded from the study, as were children with IQs<75. any other diagnosis previously assigned to a child remained confidential and did not influence inclusion in the study. For more details, please refer to the original publication. |
| Technique |
DNA was collected from either buccal samples or from whole blood and extracted using a commercial DNA isolation kit (Gentra systems, Minneapolis, MN). An allelic discrimination assay was performed in the Vanderbilt Center for Human Genetics Research DNA Resources Core using TaqMan SNP Genotyping Assay reagents (Applied Biosystems, Inc). |
| Analysis Method |
Crosstab analyses using SPSS version 15 (SPSS, Inc., Chicago, IL) were conducted for quality control analyses that included measures of genotype reliability and MZ twin agreement for genotypes. Call rates, Mendelian error rates, and exact Hardy Weinberg Equilibrium (HWE) tests and p-values were also estimated using the program PEDSTATS . For the case-control studies, genotype call rates were found to be within HWE and significance deter- mined using the X2-test. Case-control analyses were then followed-up in an independent sample using family-based tests of association with the associated alleles from the case-control analyses designated as the 'risk' alleles. Extensions of the Transmission Disequilibrium Test (i.e., TDT) that are applicable to both categorical and continuous variables and tests of moderation were used for family-based analyses of association and linkage. Derived from the TDT, the FBAT and PBAT approaches have been extended to incorporate both intact and missing parental genotypes without introducing bias as well as affected and unaffected siblings and extended pedigrees. By specifying an offset equal to the population prevalence of the disorder, the FBAT and PBAT software makes use of genotypic transmissions in case and control subjects by contrasting the number of transmissions of a specific allele in the case population with the number of non-transmissions of that allele in the control population. As such, both case and control subjects can be utilized within a TDT framework. The prevalence of the ADHD diagnosis and the respective subtypes in the control sample as assessed by the EDRS were used as offset values in the current study. A recently developed extension of the TDT was also used to test a priori hypotheses for the contrasts among the diagnostic subtypes. In this method, transmissions of a particular allele from heterozygous parents to children with a subtype of interest are contrasted with transmissions to children who have the other subtypes or who are unaffect-ed. This test was implemented within the FBAT/PBAT analytic framework to test for the association of CHT SNPs with the Inattentive and Combined ADHD subtypes (the Hyperactive-Impulsive subtype was not tested due to small sample size). All of the analyses conducted in FBAT and PBAT yield a Z statistic that was used in hypothesis testing and which was converted into the effect size index R2 using the formula Z2/N, where N=the number of informative families. Odds Ratios (OR) were calculated by first converting the R2 value into the effect size Cohen'sd using the formula d=(2xR)/sqrt (1-R2), and then calculating an OR using the formula described by Chinn. |
| Result Description |
Single marker case-control analysis: In the Vanderbilt/Chicago ADHD panel, the allele frequency for the CHT rs333229 major allele was slightly elevated (79%), though this change did not reach statistical significance. Further examination of the CHT rs333229 allele frequencies in Caucasian and Caucasian male-only groups versus matched controls showed a significant shift in allele distribution that did not differ between the diagnostic subtypes. Similarly, the allele frequency of the hypomorphic rs1013940 were significantly elevated (12%) compared to that (6%) found in healthy controls (P-value=0.023). Further examination of rs1013940 allele frequencies in Caucasian or Caucasian male-only groups versus matched controls revealed an even greater increase in both the Inattentive and Combined. Single marker family-based association analyses: In family-based analyses of association of the overall ADHD diagnosis with the Ile89Val (rs1013940) or 3'SNP (rs333229) using FBAT, no evidence was found for association with either SNP across additive, dominant, or recessive genetic models, regardless of whether only affected cases were analyzed or whether affected cases were contrasted with unaffected controls. However, there was a significant association between the Val89 allele and the Combined subtype, which was strongest under additive and dominant models when the Combined subtype was contrasted with no diagnosis and all other subtypes. They next conducted similar family-based association analyses of ADHD and its diagnostic subtypes with haplotypes comprising both CHT SNPs (Table 4). Linkage disequilibrium between the two markers was virtually absent (D'=.04. R2=.01). For the overall diagnosis of ADHD, as well as the Combined and Inattentive subtypes, Haplotype analysis: In all haplotype tests, transmissions in ADHD cases versus unaffected controls or transmissions in the Combined or Inattentive subtypes versus unaffected controls and all other subtypes were contrasted. All of the haplotype tests for either the ADHD diagnosis or the Inattentive subtype were non-significant. In contrast, the omnibus tests yielded a significant association with the Combined subtype under an additive model and a trend towards an association under a dominant model with the haplotype comprising both Ile89Val and 3'SNP minor alleles showing the strongest associations. |
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