Attention-deficit/hyperactivity disorder (ADHD) is a prevalent psychiatric disorder among children, characterized by inappropriate levels of inattention, overactivity and impulsivity. Substantial evidence suggests that, to a large degree, cognitive deficits, particularly deficits in executive control, underlie the etiology of ADHD.3,4 Executive control functions in the preparation, maintenance, delaying and switching of action and thoughts. Although the precise cognitive characteristics of ADHD are debated, the executive control process inhibition has been identified as a potential core deficit in ADHD.5,6 Inhibition is defined as the ability to suddenly and completely stop a planned or ongoing thought and action.7 A second executive control process, working memory, the active maintenance and manipulation of information necessary to guide decision-making and behavioral responses, may also be related to ADHD.3,8
On the basis of animal models, pharmacological interventions and neuroimaging studies, ADHD and its associated cognitive deficits are believed to result from dysregulation of neurotransmitter systems. The dopamine system has been the primary focus in studies of ADHD to date; however, given that the glutamatergic system is involved in both cognitive and motor function, and modulates the dopamine and serotonin systems,9 dysregulation in this system might also underlie the development of ADHD.
The action of glutamate is mediated by several families of receptors, one of which is the N-methyl-D-aspartate receptor (NMDAR) family. NMDARs are heteromeric complexes differentially expressed throughout the central nervous system and development. Several distinct subtypes of NMDAR have been identified. Each NMDAR subtype is composed of at least one obligatory subunit, termed NR1, and one or more of the four NR2 subunits (2A, 2B, 2C, 2D). While the specific biological and pharmacological properties differ for each NMDAR subtype, it has been well established that NMDARs have critical roles in excitatory synaptic transmission, neuronal survival and plasticity, and in cognitive processes and motor functioning.
Numerous studies have investigated the role of NMDARs in cognition. Studies of long-term potentiation, the phenomenon thought to underlie long-term memory, have shown that NMDARs play an essential role in both the summation of cellular responses and induction of neural plasticity. Also, studies in rats have identified hippocampal NMDARs as being involved in multiple aspects of spatial short-term memory.10 Antagonism of NMDARs in non-human primates causes impairments in short-term memory performance.11 Finally, in humans, administration of ketamine, an NMDAR antagonist, decreases declarative memory performance,12 as well as both verbal and nonverbal working memory performance.13,14 These observations demonstrate that NMDARs are involved in multiple cognitive processes, and suggest that NMDAR functioning is related to both short-term and working memory.
Collective evidence from a number of studies also supports a role for the glutamate system in the regulation of motor function. Hyperactivity can be induced by pharmacological disruption of NMDAR glutamatergic transmission.15,16 Also, infusion of NMDA into the ventral hippocampus to stimulate NMDARs was shown to cause dose-dependant short-term hyperactivity.17 Several strains of transgenic mice carrying mutations in, or knockouts of genes encoding NMDAR subunits exhibit changes in motor and cognitive function. Mice lacking the NMDAR 2A subunit gene GluR
1, the mouse homolog of the GRIN2A gene in humans, show increased spontaneous locomotor activity in a novel environment, impaired latent learning associated with selective attention9 and deficits in spatial learning.18
Recently, Fisher et al19 reported their findings from the first genomewide scan for loci linked to ADHD. Although no regions reached genomewide significance in this initial study, regions on 5p12, 10q26, 12q23 and 16p13 had LOD scores greater than 1.5. The significance of the linkage finding in the 16p13 region was increased by genotyping additional families and markers. Results from this analysis yielded a multi-point maximum LOD score of 4.2 near marker D16S3114.1 The GRIN2A gene maps within 2 MB of the 1-LOD support interval surrounding this maximum. Support for GRIN2A as the susceptibility gene in this region was obtained recently in a family-based study in which a significant association between ADHD and the Grin2a-5 polymorphism was observed.2
In the present study, we investigated the relationship of the GRIN2A gene to ADHD and cognitive measures using the transmission disequilibrium test (TDT) to examine the inheritance patterns of four GRIN2A polymorphisms in a sample of 183 small nuclear families ascertained through an ADHD proband. The markers analysed in this study include the Grin2a-5 polymorphism, as well as three additional single-nucleotide polymorphisms (SNPs) dispersed over the length of the gene (Table 1).
The allele frequencies for the Grin2a-5 polymorphism reported for the Turic sample (allele 1=0.69, allele 2=0.31) and this sample (allele 1=0.72, allele 2=0.28) are similar (Table 1). TDT results for the four markers individually are shown in Table 2. No evidence for biased transmission of alleles at any of the markers was observed. Strong linkage disequilibrium (LD) was observed among polymorphisms Grin2a-605, Grin2a-5 and Grin2a-531, located in intron 5, exon 6 and exon 14, respectively. LD was not observed between the intron 3 polymorphism Grin2a-503, and any of the three downstream markers, indicating that LD does not extend across the entire length of the gene. TDT analysis of haplotypes, using the TRANSMIT program, showed no significant evidence for biased transmission of haplotypes (Table 3). There was no significant evidence for a relationship between the transmission of alleles and scores of the three cognitive phenotypes inhibitory control, verbal short-term memory and verbal working memory (Table 4).
Table 4 - FBAT analysis of GRIN2A alleles transmission in relation to inhibitory control, verbal short-term memory and verbal working memory.
Full table The role of NMDARs in cognition and motor activity, and the location of GRIN2A in the 16p13 linkage region identified in an ADHD genome scan, made GRIN2A a strong candidate gene for consideration as an ADHD susceptibility locus. Support for this hypothesis has been shown by the previous association of alleles of the Grin2a-5 polymorphism and ADHD.2 In contrast to those results, we found no significant evidence of linkage in our sample.
It has been observed that strong associations apparent in initial genetic association studies often become less prominent as more data are collected. Three possible reasons could explain this trend: (i) the results of the first study could be a spurious finding (ie false positive), (ii) the results of a subsequent study could be false negative, (iii) the genetic effect of the polymorphism(s) tested is stronger in some samples than in others.20 Turic and colleagues' finding of an association between GRIN2A and ADHD could be spurious. However, their use of a family-based study design would reduce the possibility of population stratification, and thereby reduce the chance of a spurious finding, and their relatively large sample of 238 nuclear families should be fairly robust to false positives. Notably, we also used a family-based control design, and a similar sample size of 183 families with a total of 229 affected children, and therefore should have sufficient power to detect linkage if the polymorphism is of similar effect in both samples.
ADHD is a clinically heterogeneous disorder that has many etiologies, genetic and nongenetic, which may be differentially represented in different samples and in turn influence the strength of a specific genetic effect. Two possible sources could cause this divergence: (i) the population each sample is drawn from differs in these representations and/or (ii) the manner in which the sample is collected leads to different representations. The ethnic composition of the population and/or sample is included in these sources. In contrast to the British Caucasian sample used by Turic et al,21 most of the families in our study are of mixed European Caucasian descent. In addition to issues related to ethnicity, differences between the samples due to participant ascertainment strategies, selection criteria such as age, gender or absence of comorbidities, and/or the source of diagnostic information may have influenced the sample composition and the strength of a specific genetic effect.
To date, a functional consequence of the Grin2a-5 polymorphism has not been detected and, therefore, we must consider that the association finding from Turic et al is likely to be due to LD between the Grin2a-5 polymorphism and a functional DNA variant(s). Although the frequency of the Grin2a-5 polymorphism is similar in the two samples, the frequency of the functional polymorphism may not be. Therefore, an association of GRIN2A and ADHD may be differentially detected, depending on sample composition and the amount of LD between the marker and the functional variant.
In addition to testing for linkage between GRIN2A and ADHD, we also tested for relationships between GRIN2A and three cognitive phenotypes, inhibitory control, verbal short-term memory and verbal working memory. We found that variation in GRIN2A was not related to measures of these cognitive phenotypes. Although NMDARs are thought to play pivotal roles in multiple memory processes including working memory, their involvement in specific aspects and/or tasks remains unclear. Therefore, our analysis of these specific cognitive phenotypes does not rule out the genetic contribution of GRIN2A to other cognitive phenotypes.
Materials and methods
Participants
Participant assessment and diagnostic criteria have been described previously.22 Probands and affected siblings between the ages of 7 and 16 years were recruited from the Child Development and Neuropsychiatry Clinics at the Hospital for Sick Children, and met the Diagnostic and Statistical Manual of Mental Disorder, 4th Edition (DSM-IV) criteria for ADHD. All children were free of medication for a minimum of 24 h before assessment and cognitive testing. This protocol was approved by the Hospital for Sick Children's Research Ethics Board and informed written consent was obtained for all participants. In total, 183 nuclear families including 46 siblings of the probands were genotyped for this study.
Cognitive phenotype measures
Inhibitory control was measured using the stop task paradigm described in greater detail in Schachar et al.6 The stop task is a laboratory paradigm that provides a direct measure of the speed with which one can execute and voluntarily inhibit a speeded motor response. The stop task involves two concurrent tasks. The go task is a simple choice reaction time task that individuals are asked to perform as quickly and as accurately as possible. The stop task involves a tone emitted from the computer, which signals participants to withhold their response on that trial. Tones occur randomly and on 25% of trials. We used a 'tracking' procedure that converges on the delay between the go and stop signals at which individuals are able to stop 50% of the time. We can estimate the latency of the unobserved stop process, stop signal reaction time (SSRT), by subtracting the mean delay at which the subject inhibits 50% of the time from mean go reaction time (see Logan7 and Logan and Cowan23). SSRT was corrected for age based on population-based norms.24
Verbal short-term and working memory was assessed using the digit span subtest of the Wechsler Intelligence Scale for Children – Third Edition (WISC-III). This test provides an overall memory score, which is then divided into two parts, digits forward (DF) and digits backward (DB). DF provides a measure of verbal short-term memory span. The experimenter speaks aloud a sequence of digits (at the rate of one per second) and the child is asked to repeat them in the order in which they were presented. The dependant measure is the number of trials in which the lists are recalled in the correct order. The DB task is a measure of verbal working memory. The presenter reads a series of numbers to the child. The child is then required to repeat the numbers in a reverse order. For both the forwards and the backwards components, age-normed standardized scores were used.25
Genotyping
Four SNPs were genotyped for this study: Grin2a-503, Grin2a-605, Grin2a-5 and Grin2a-531 located in intron 3, intron 5, exon 6 and exon 14, respectively. Previously, the Grin2a-5 and Grin2a-531 polymorphisms were reported to be located in exon 5 and exon 13, respectively2,26; however, the recent finding of a 5' untranslated exon (see NCBI genomic contig NT_010393 and Itokawa et al27) increases the previous numbering of all exons and introns by one.
The SNPs designated Grin2a-503, Grin2a-605 and Grin2a-531 were genotyped with the ABI 7900-HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using the TaqMan 5' nuclease assay for allelic discrimination. Primer and probe sequences, and annealing temperatures used, are listed in Table 1. The PCR reactions (10 
l volume), contained 30 ng of genomic DNA, 10 mol of TaqMan® Universal PCR Master Mix (Applied Biosystems) and 0.25 l of allelic discrimination mix (Applied Biosystems) containing 36 M of each primer and 8 M of each probe. The thermal cycling conditions were 50°C for 2 min, 95°C for 10 min and then 40 cycles of 92°C for 15 s, and the annealing temperature for 1 min. Each 96-well plate contained two negative controls.
Genotypes for the Grin2a-5 polymorphism were determined by restriction enzyme digestion of PCR products. A product of 154 bp was amplified using the primer sequences listed in Table 1. The forward primer is a mutagenic primer which converts the 'C' located three nucleotides upstream of the SNP to a 'G' resulting in an MspA1I site in the PCR product derived from the 'G' variant (Allele 1) of this SNP. The PCR reactions (20 l volume) contained 50 ng of genomic DNA and 1.5 mM of MgCl2. The thermal cycling conditions were as follows: 94°C for 1 min, and then 35 cycles of 94°C for 30 s, 58°C for 30 s and 72°C for 30 s, and then 10 min at 72°C. Amplification product (8 l) was digested with 5 U of MspA1I (New England Biolabs, Beverly, MA, USA) at 37°C for 1.5–2 h. Restriction fragments of 154 bp (site absent: Allele 2) and/or 131 plus 23 bp (site present: Allele 1) were resolved on agarose gels consisting of 2% agarose plus 2% NuSieve 3 : 1 agarose (Mandel Scientific Company, Inc., Guelph, Ontario, Canada) and visualized with ethidium bromide staining.
Statistical analysis
For the categorical analysis of ADHD, the extended TDT program (ETDT) 28 was used to test for biased transmission of individual marker alleles. Transmission of haplotypes was analyzed using the program TRANSMIT.29 Only haplotypes with a frequency above 0.10 were included in the analysis. The two-locus linkage disequilibrium program (2LD; http://www.iop.kcl.ac.uk/loP/Departments/PsychMed/GEpiBSt/software.shtml) was used to calculate the coefficient of LD, D' between marker alleles in the parental chromosomes. The FBAT program30 was used for the analysis of quantitative measures.
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Acknowledgements
This work was supported by grants from the Natural Sciences and Engineering Research Council (JHA), The Hospital for Sick Children Psychiatric Endowment Fund (CLB), the Canadian Institutes of Health Research MT14336 (CLB) and MOP-14336 (CLB). Also, we thank Drs Anita Thapar and Michael O'Donovan for providing information on the Grin2a-5 polymorphism prior to publication.
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