Attention-deficit hyperactivity disorder and the adrenergic receptors α1C and α2C

Abstract

The adrenergic system has been hypothesized to be involved in the etiology of attention-deficit hyperactivity disorder (ADHD) based on pharmacological interventions and animal models. Noradrenergic neurons are implicated in the modulation of vigilance, improvement of visual attention, initiation of adaptive response, learning and memory. In this study we tested the genes for two adrenergic receptors, α1C (ADRA1C) located on chromosome 8p11.2, and α2C (ADRA2C) located on chromosome 4p16, as genetic susceptibility factors in ADHD. For the adrenergic receptor α1C we used a C to T polymorphism that results in a change of Cys to Arg at codon 492 for the linkage study. For the adrenergic receptor α2C gene we examined a dinucleotide repeat polymorphism located approximately 6 kb from the gene. We examined these polymorphisms in a sample of 103 families ascertained through an ADHD proband. Using the transmission disequilibrium test, we did not observe biased transmission of any of the alleles of these polymorphisms. We conclude that the alleles at the polymorphisms tested in these two genes are not linked to the ADHD phenotype in this sample of families.

While the recent investigation of the molecular genetics of ADHD has focused on the dopaminergic system, an extensive body of literature has implicated the involvement of the adrenergic system in ADHD.^1,2 The notion that the adrenergic system is involved in ADHD derives from several lines of research including the manipulation of the catecholamine system in animals and the pharmacological response to adrenergic agonists in humans. In rodents, norepinephrine depletion results in increased distractibility and motor hyperactivity and in non-human primates, stimulation of the noradrenergic system has been shown to improve cognitive function and distractibility.³ The improvement in ADHD symptoms with tricyclic antidepressants has been attributed to the actions of these drugs in the reuptake of norepinephrine.¹

Noradrenergic receptors are divided into three major categories: alpha1, alpha2, and beta. Activation of alpha 1 receptors in rats promotes vigilance (sustained attention), decreases impulsivity, and influences working memory and behavioral activation while having only a minor role in the modulation of long-term memory.⁴ The alpha adrenergic receptors have been classified as α1A, α1B, and α1D corresponding to their ligand binding properties. Four α adrenoreceptors, α1a, α1b, α1c, and α1d, have been cloned⁵ and the cloned α1c receptor⁶ has been identified as the receptor corresponding to the alpha 1 A-ligand binding site.⁵

The gene for the α1C receptor has been localized by somatic cell hybridization⁷ and by multi-point linkage analysis to 8p11.2.⁸ Thus far only a single polymorphism has been identified for this gene, a C to T change located in codon 492 resulting in a change of cysteine to arginine.⁹ This C to T change results in the change in the restriction enzyme recognition site for PstI.^8,9 This polymorphism alters the amino acid sequence potentially changing the function of this protein. However, no difference in receptor function has been observed between the two protein variants.⁹ The pharmacological characteristics and the receptor-mediated [Ca²⁺]_i response can be desensitized in a similar manner for either of these receptor variants.

The α2 adrenergic receptors have been implicated in cognitive function and improved performance in working memory tasks under distracting conditions.³ These receptors have been divided into three subtypes; α2A, α2B, and α2C. The gene for the α2C subtype has been localized to chromosome 4p16, close to the gene responsible for Huntingtons disease,¹⁰ and two polymorphic dinucleotide repeats have been localized in close proximity to the ADRA2C gene.¹⁰ For this linkage study, we used the (GT)_n repeat (marker adra2c1) that was localized closest to the ADRA2C gene as a genetic marker for the gene. Researchers have used this polymorphism to investigate this gene as a susceptibility factor in ADHD, employing a quantitative approach and ADHD symptom scores in 274 individuals diagnosed with Tourette syndrome and 62 controls.¹¹ Of the individuals with Tourette syndrome, 144 also met DSM-IV criteria for ADHD. Using regression analysis the authors showed significant correlation (P = 0.0046) between scores for ADHD and the sizes of the alleles of this polymorphism segregated by allele size ≤ 183 bp and > 183 bp. A significant correlation was also found between these alleles and school academic performance as determined by subject self report. However, the results for these two analyzes were not significant when the results were corrected for multiple testing.

In this study we tested for linkage to these two genes by looking at biased transmission of alleles of the Cys4-92Arg polymorphism in the ADRA1C gene and the marker adra2c1 located near the ADRA2C gene. We used the transmission disequilibrium test (TDT) to test for the presence of biased transmission of the alleles of these polymorphisms from the parents to the affected children in families identified through an ADHD proband.

We calculated the allele frequencies for the alleles at each polymorphism using the sample of parental chromosomes. For the ADRA1C Cys492 allele (allele 1, C) the frequency was 0.438 and the frequency of the Arg492 allele was 0.562. The allele frequencies for the ADRA2C polymorphism in the parental chromosomes are shown in Table 1.

Table 1.

Allele frequencies of the ADRA2C polymorphism in the parental chromosomes

Size (bp)	Frequency
181	0.009
183	0.396
185	0.544
187	0.037
189	0.000
191	0.014

We tested for evidence for linkage in the presence of linkage disequilibrium by means of the TDT.¹² No evidence was found for biased transmission of either allele of the ADRA1C polymorphism to the probands in this sample of ADHD families (Table 2). The Cys492 allele was transmitted 51 times compared to 64 transmissions of the Arg492 allele (TDT χ² = 1.470, 1 df, P = 0.225). There was also no evidence for biased transmission of any of the alleles of the ADRA2C polymorphism (Table 3). In our sample only the two common alleles for the adra2c1 marker provided enough informative transmissions to calculate the TDT—the 183-bp and 185-bp alleles. For the 183-bp allele there were 88 informative transmissions: it was transmitted 47 times and not transmitted 41 times (TDT χ² = 0.409, 1 df, P = 0.523). For the 185-bp allele, there were 94 informative transmissions: it was transmitted 45 times vs 49 times not transmitted (TDT χ² = 0.170, 1 df, P = 0.680).

Table 2.

Results of the transmission disequilibrium test for ADRA1C

Allele	Cys492	Arg492
Transmitted	51	64
Not transmitted	64	51
Chi-square	1.470	1.470
P values	0.225	0.225

Table 3.

Results of the transmission disequilibrium test for ADRA2C

Alleles	181 bp	183 bp	185 bp	187 bp	191 bp
Transmitted	1	47	45	2	5
Not transmitted	2	41	49	6	2
Chi-square		0.409	0.170
P values		0.523	0.680

Although there is strong evidence for the involvement of the adrenergic system in ADHD, the search for the genes responsible for the susceptibility in this pathway may not be straightforward. It is unknown how many genes contribute to the overall regulation of the adrenergic system and thus might contribute to the genetic susceptibility to ADHD. This study investigated two of the adrenergic receptor genes but found no evidence for linkage. Other adrenergic receptor genes can be tested to determine their potential genetic contribution to ADHD. As well as adrenergic receptors, other genes involved in regulating the adrenergic system might well contribute to the genetic susceptibility to ADHD. Although the search for susceptibility genes in the adrenergic pathway seems daunting, an informed choice of candidate genes will enable researchers to determine the correct genes. For the dopamine system, two genes have been implicated in the etiology—the dopamine receptor D4 and the dopamine transporter. In both these cases, the evidence for these genes being susceptibility factors in ADHD appears convincing.

A previous study using a quantitative approach has reported a linkage finding for the ADRA2C gene and ADHD symptoms in subjects with Tourette syndrome and controls.¹¹ In our study we saw no evidence for linkage with ADRA2C and the phenotype of DSM-IV ADHD in families ascertained through an ADHD proband. The methodological differences between the two studies prevent a direct comparison of the results. One major difference is that our study excluded probands and siblings with TS or chronic multiple tics and subjects with an IQ less than 80 on both the Performance and Verbal Scales of the Wechsler Intelligence Scale for Children III.

It is possible that these two genes contribute to the ADHD phenotype but are not genes of major effect. Our sample may not have sufficient power to detect linkage in this case. Also these two genes may contribute to a particular part or subtype of the phenotype. As previously summarized,¹³ some studies have reported children with ADHD to have higher levels in the serum or urine of the noradrenergic metabolite 3-methoxy-4-hydroxyphenyl glycol (MHPG), while others have reported lower MHPG levels compared to controls, suggesting that there may be subgroups of ADHD based on adrenergic function. One study suggested that this distinction may be related to comorbid reading disabilities in the ADHD subjects.¹³ This finding suggests that perhaps adrenergic dysfunction is a factor in only a subset of ADHD children. Currently we have less than 20 subjects in our sample with comorbid reading disabilities and thus we lack sufficient power to subdivide our sample by comorbid disorders. A larger sample size is required to identify a susceptibility factor relating to a particular subtype.

Another consideration in interpreting our results is that for our linkage studies, we have been able to use only a single polymorphism for each gene. The alleles of these polymorphisms may not be in linkage disequilibrium with any potential phenotype-causing allele. For example, in our study of the dopamine transporter gene we examined three polymorphisms and did not see significant evidence for linkage with any of the polymorphisms when examined alone, however, one haplotype created by the combination of these three polymorphisms was significantly linked to the phenotype.¹⁴ While the PstI polymorphism is located within the coding region of this gene, which is relatively small, it is still possible that these alleles are not in linkage disequilibrium with a potential susceptibility allele, and linkage could be missed. For the ADRA2C gene, the polymorphism we used is located approximately 6 kb away from the ADRA2C gene, thus increasing the possibility that linkage could be missed. Ideally, additional polymorphisms in the coding region of both genes should be genotyped to strengthen the possibility of detecting linkage. At this time we are currently screening both these genes in a search for other common polymorphisms, which so far have eluded detection.

Methods

Diagnostic assessment

The assessment and characteristics of the subjects for this study have been previously described.^15–18 Information for the diagnosis of ADHD and comorbid conditions was gathered through a semi-structured interview for parents (Parent Interview for Child Symptoms, PICS-IV; Schachar and Ickowicz, unpublished) and teachers (Teacher Telephone Interview-IV, TTI; Tannock and Schachar, unpublished). This information was supplemented with the following questionnaires and child assessments: Conners Parent and Teacher Rating Scales–Revised,¹⁹ the Ontario Child Health Survey Scales–Revised,²⁰ Wide Range Achievement Test–III,²¹ Clinical Evaluation of Language Fundamentals 3rd Edition,²² Children’s Depression Inventory,²³ and Children’s Manifest Anxiety Scale.²⁴ Children were free of medication for a minimum of 24 h before their assessment. This protocol was approved by The Hospital for Sick Children’s Research Ethics Board, and informed written consent was obtained for all participants.

Subjects were included if they met DSM-IV criteria for ADHD: six symptoms of inattention and/or hyperactivity-impulsiveness either in the home or school setting as determined by clinical interview, evidence of pervasiveness, defined as a minimum of four symptoms in the non-criterion setting, onset before 7 years of age. Subjects were excluded if they scored below 80 on both the Performance and Verbal Scales of the Wechsler Intelligence Scale for Children III,²⁵ or showed evidence of neurological or chronic medical illness, bipolar affective disorder, psychotic symptoms, Tourette syndrome, or chronic multiple tics.

Families

We used a sample of 103 nuclear families identified through an ADHD proband. Both parents were genotyped in 86 of the families and a single parent was genotyped for 17. The sample also included 28 siblings meeting the same criteria for participation in the study as the probands.

Isolation of DNA and marker typing

DNA was extracted from blood lymphocytes using a high salt extraction method.²⁶ The ADRA1C PstI polymorphism was genotyped according to Shibata et al.⁹ The 502-bp fragment of ADRA1C was amplified using a reaction mixture of 100 ng of genomic DNA, the primers P1 5′ atg ctc cag cca aga gtt ca 3′ and P2 5′ tcc aag aag agc tgg cct tc 3′, 1.5 mM MgCl₂, and 1 unit of Taq polymerase. Amplification of the 502-bp fragment by PCR was done as follows: an initial denaturation stage of 4 min at 94°C followed by 35 cycles of denaturing at 94°C for 30 s, annealing at 58°C for 30 s, and an extension of 72°C for 30 s. A final extension step of 72°C was added for 10 min after the last cycle. Ten microliters (μl) of the PCR product were digested with 8 units of PstI restriction enzyme (New England Biolabs, Beverly, MA, USA) for approximately 2 h at 37°C. The alleles were detected after separation on a 2% aga-rose gel. Allele 1 (C, Cys) is not cut with the restriction enzyme and is seen as a 502-bp band. Allele 2 (T, Arg) is cut into two bands of 477 and 25 bp.

The ADRA2C polymorphism was genotyped as previously described²⁷ with minor modifications. The 179–193 bp fragment of ADRA2C was amplified using a reaction mixture of 100 ng of genomic DNA, the primers adra2c-1 5′ cgc tgc ctc cct tcc acc tgt tg 3′ and adra2c-2 5′ agt ggg cag ggc ggg gca ggt 3′, the PCRx Enhancer System (1× PCR Amplification Buffer, 1× PCRx Enhancer Solution, 1.5 mM MgSO₄ from Gibco BRL (Life Technologies, Gaithersburgh, MD, USA), and 1 unit of Taq polymerase. The adra2c-1 primer was labeled with 6-FAM. Amplification of this fragment was as follows: an initial denaturation stage of 4 min at 94°C followed by 35 cycles of denaturing at 94°C for 30 s, annealing at 58°C for 30 s, and an extension of 72°C for 30 s. A final extension step of 72°C was added for 10 min after the last cycle. The PCR products were run on an ABI 310.

Statistical analysis

The transmission disequilibrium test (TDT) was calculated using the ETDT program.²⁸