| Summary |
They tested for linkage of DRD1 to ADHD by examining the inheritance of four biallelic DRD1 polymorphisms [D1P.5 (-1251HaeIII), D1P.6 (-800HaeIII), D1.1 (-48DdeI) and D1.7 (+1403Bsp1286I)] in a sample of 156 ADHD families. Owing to linkage disequilibrium between alleles at the four markers, only three haplotypes are common in current sample. Using the transmission/disequilibrium test (TDT), they observed a strong bias for transmission of Haplotype 3 (1.1.1.2) from heterozygous parents to their affected children (P=0.008). Furthermore, using quantitative trait TDT analyses, they found significant and positive relationships between Haplotype 3 transmission and the inattentive symptoms, but not the hyperactive/impulsive symptoms, of ADHD. These findings support the proposed involvement of DRD1 in ADHD, and implicate Haplotype 3, in particular, as containing a potential risk factor for the inattentive symptom dimension of the disorder. Since none of the four marker alleles comprising Haplotype 3 is predicted to alter DRD1 function, they hypothesize that a functional DRD1 variant, conferring susceptibility to ADHD, is on this haplotype. To search for such a variant they screened the DRD1 coding region, by sequencing, focusing on the children who showed preferential transmission of Haplotype 3. DNA from 41 children was analysed, and no sequence variations were identified, indicating that the putative DRD1 risk variant for ADHD resides outside of the coding region of the gene. |
| Total Sample |
In this study, 156 nuclear families ascertained through an ADHD proband were analysed. The sample consists of 123 families in which both parents were genotyped and 33 families in which a single parent was genotyped. Genotypes of affected siblings of probands, that is, siblings who met the diagnostic criteria for ADHD, were also included in the analysis. In the sample of 156 families, 36 affected siblings were identified, giving a total of 192 affected children in the study. Among the children included in this study, the distribution of ADHD subtypes is as follows: 25% Predominantly Inattentive Type, 18% Predominantly Hyperactive/Impulsive Type, 57% Combined Type. |
| Diagnosis Description |
Subject assessment and diagnostic criteria for inclusion in this study have been described previously. Briefly, all of the children were between 7 and 16 years of age and met the DSM-IV criteria for ADHD. Children who scored below 80 on both the Performance and Verbal Scales of the WISC-III were excluded from the study, as were children who exhibited neurological or chronic medical illness, Tourette syndrome, chronic multiple tics, bipolar affective disorder, psychotic symptoms or other anxiety, depressive or developmental disorder that might better account for their behaviour. Children with a family history of bipolar disorder or schizophrenia were also excluded from the analysis. Diagnoses of ADHD and comorbid conditions were based on information obtained from semistructured interviews of parents (Parent Interview for Child Symptoms) and teachers (Teacher Telephone Interview for Children's Academic Performance, Attention, Behaviour and Learning: DSM-IV Version), and from the following standardized questionnaires and assessments: Conners Parent and Teacher Rating Scales-Revised, Ontario Child Health Survey Scales-Revised, Wide Range Achievement Test- Revision 3, Clinical Evaluation of Language Fundamentals-3rd Edition, Children's Depression Inventory and Children's Manifest Anxiety Scale. Children were free of medication for at least 24 h prior to assessment. The assignment of subtypes, as recognized by the DSM-IV , was based on scores for the inattentive and hyperactive/impulsive symptom dimensions of ADHD, as reported in the interviews with parents and teachers. This study protocol was approved by the Hospital for Sick Children Research Ethics Board, with written informed consent obtained for all participants. |
| Technique |
DNA was extracted from peripheral blood using a high-salt extraction method. Genotypes for each of four DRD1 markers were determined by PCR amplification of 60-100 ng DNA, followed by restriction enzyme digestion of the PCR products. Sequence analysis of the DRD1 gene was carried out by direct sequencing of four PCR products, designated D1Seq1, D1Seq2, D1Seq3 and D1Seq4. Each was amplified from 60-100 ng of genomic DNA. PCR products were treated with Exonuclease I and Shrimp Alkaline Phosphatase according to the manufacturer's instructions, and sequenced directly using either of the two primers that was used for PCR amplification. Fluorescent automated DNA sequencing was carried out using the ABI PRISM BigDye Terminator v3.0 Cycle Sequencing System and Sequencher analysis software. |
| Analysis Method |
The degree of linkage disequilibrium (pairwise association) between marker alleles in this study was evaluated according to Lewontin, expressing the coefficient of linkage disequilibrium as D'. Parental allele frequencies and 2-marker haplotype frequencies, used in the calculation of D', were determined using the Estimate Haplotypes (EH) program. TDT analyses, considering ADHD diagnosis as a categorical trait, were carried out using the extended TDT (ETDT) program to examine the transmission of individual marker alleles, and the TRANSMIT program to examine the transmission of haplotypes. For biallelic markers (as in this study), the ETDT program excludes families for which only one parental genotype is available, given the potential for erroneously biased results if such families are included. Quantitative trait TDT analyses, examining transmission of Haplotype 3 in relation to DSM-IV symptom scores, were carried out using the FBAT program (v1.4.1) and a logistic regression-based extension of the TDT. As with ETDT, families with a missing parent were excluded from the logistic regression-based analysis. As this method does not support the analysis of haplotypes, Allele 1 of the D1P.6 marker was used as a proxy for Haplotype 3. Transmissions from heterozygous parents were assigned a binary variable of either '1' (for transmission of D1P.6 Allele 1) or '0' (for nontransmission of the allele) and logistic regression analysis, using SPSS software, was carried out to test the relationship between this variable and the offspring phenotype scores. For both the FBAT and logistic regression analyses, the quantitative phenotypes of inattentive and hyperactive/impulsive symptom counts were tallied for parents and teachers separately. All P-values in this study are reported without correction for multiple testing. |
| Result Description |
Four polymorphic DRD1 markers were analysed: D1P.5, D1P.6, D1.1 and D1.7. Consistent with the strong degree of linkage disequilibrium observed in each case, among the 16 haplotypes that are theoretically possible for these markers, only three (ie 1.2.2.1, 2.2.2.1 and 1.1.1.2) are common in current sample. Trends for biased allele transmission for each of the individual markers analysed were found (ie biased towards transmission of D1P.5 Allele 1, P=0.180; D1P.6 Allele 1, P=0.086; D1.1 Allele 1, P=0.084; D1.7 Allele 2, P=0.089). Significant evidence for linkage was obtained when the transmission of DRD1 haplotypes was analysed. First, a global test of association on three degrees of freedom yielded P=0.030. In addition, considering the three common haplotypes individually, strong evidence for biased transmission of Haplotype 3 was observed (P=0.008), together with marginally significant evidence for nontransmission of Haplotype 2 (P=0.045). Using the FBAT program63,64 we found significant and positive relationships between transmission of Haplotype 3 and inattentive symptom scores reported by both parents (P=0.024) and teachers (P=0.030). These relationships were confirmed using a logistic regression-based method (P=0.008 and 0.045, respectively), with odds ratios showing that for every increment of 1 in the parent- or teacher-reported symptom score, the odds of Haplotype 3 transmission increased ~1.2-fold. In contrast to these results, no significant relationships between hyperactive/impulsive symptom scores and Haplotype 3 transmission were evident using either method of analysis. |
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