| Summary |
The purpose of this study was to compare genetic association studies of three polymorphisms of the alpha-2A adrenergic receptor gene (ADRA2A) with radioligand binding studies of the alpha-2A adrenergic receptor protein in platelets from a sample of children without or with ADHD. The pediatric subjects ranged from 6 to 18 years of age. A thorough clinical assessment of each child resulted in one of the following DSM-IV ADHD diagnoses: inattentive, hyperactive/impulsive, combined, or no ADHD. No significant linkage was found between the ADRA2A polymorphisms (MspI, HhaI, and DraI) and any of the phenotypes tested. Association analysis, however, did detect significant linkage disequilibrium for the DraI polymorphism. Association was also evaluated considering the three ADRA2A single nucleotide polymorphisms as haplotypes. The HhaI-DraI and the MspI-HhaI-DraI haplotypes were significantly associated with ADHD. The platelet alpha-2 adrenergic receptor density did not differ between children without or with ADHD.The affinity of the receptor for the radioligand however, differed significantly between those without and with ADHD. In addition, there were some significant correlations between binding parameters and severity of ADHD in this well-characterized clinical population, and significant association was found between these measures of receptor function and MspI and DraI polymorphisms. Thus, both the genetic and the binding studies indicate that the alpha-2 adrenergic receptor may play a role in ADHD. |
| Total Sample |
Eighty families participated in the genotyping. Of these, 55 had both parents, and 25 had only one parent. A total of 159 children were ascertained, and 155 children (92 boys and 63 girls) assented to blood or buccal swab samples for genotyping. Sixty-five of the families had two children, and 5 had three children in the study. |
| Diagnosis Description |
All probands and siblings were assessed for the diagnosis and severity of ADHD and for comorbid psychiatric disorders using the following assessment measures: Schedule for Affective Disorders and Schizophrenia for School-Age Children (K-SADS) ADHD Module [Ambrosini, 2000]; ADHD-IV: Parent-Investigator Version [DuPaul et al., 1998]; Conners' Parent Rating Scale-Revised: Short Form (CPRS-R:S) [Conners, 1997]; and a clinical psychiatric interview and/or complete K-SADS. An investigator with clinical training in psychometric assessment administered the KSADS and the ADHD-IV: Parent-Investigator Version. Parents were asked to complete the ADHD assessments based on the subject's behavior when the child was not on medication. Demographic information and a medical history were also collected on each subject. |
| Technique |
DNA was obtained using the PureGene (Gentra) extraction method. Three single nucleotide polymorphisms (SNPs) were typed for the ADRA2A gene, rs1800544, rs180045, rs583668. The MspI polymorphism was typed using amplification by the primers, followed by restriction digest and agarose electrophoresis with staining by ethidium bromide. This produced either one fragment 174 bp in size or two fragments of 212 and 53 bp. The HhaI and DraI SNPs were typed according to the procedure of Park et al. [2004]. As with the MspI polymorphism, the amplification products were digested with the appropriate enzyme and detected by electrophoresis. The HhaI region was amplified by primers 5'-CCAAGTTATCAGGCCACCGA- 3' and 5'-TGCTCCTGGCGGAACATGAA-3' producing either a fragment 160 bp in size or two of 138 and 22 bp, with allele frequencies of 0.138 and 0.862, respectively. The DraI region was amplified by 5'-TACAAGGGCATGGCTCACAA- 3' and 5'-CCAAGGCCAGGATTTCAACA-3' and produced either one 221 bp fragment or two fragments of 130 and 91 bp. |
| Analysis Method |
Association analyses with the quantitative phenotype scores were done using the QTDT procedure [Abecasis et al., 2000] with empirical significance values calculated through 1,000 permutations, while the FBAT procedure [Horvath et al., 2001] was used for the dichotomous affected trait. Sib-pair linkage analysis was done using the SIBPAL program in the S.A.G.E. 5.0 package [S.A.G.E., 2004]. Single-point association analysis was done utilizing two different approaches, both of which are family-based transmission disequilibrium tests. Since the three marker loci are tightly linked to each other, they also tested association of the loci as haplotypes using the GENEHUNTER 2.0 TDT procedure. |
| Result Description |
No significant linkage was found between the ADRA2A polymorphisms (MspI, HhaI, and DraI) and any of the phenotypes tested. Association analysis, however, did detect significant linkage disequilibrium for the DraI polymorphism. The HhaI-DraI and the MspI-HhaI-DraI haplotypes were significantly associated with ADHD. Both the genetic and the binding studies indicate that the alpha-2 adrenergic receptor may play a role in ADHD. |
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