Introduction

Glutamatergic neurotransmission is the major excitatory system in human brain. It is involved in many basic neuronal functions including fast synaptic transmission, neuronal migration, proliferation and excitability, synapse formation, stability and plasticity, and long-term potentiation. Altered glutamatergic neurotransmission has been implicated in many different CNS processes, physiological and pathological,¹ because of these wide-ranging functions. Given this, genes encoding glutamate receptors represent candidate genes of interest for neuropsychiatric disorders.² There are two classes of glutamate receptor, ionotropic and metabotropic. Ionotropic receptors are further classified based on their binding to a range of ligands into N-methyl D-aspartate receptors (NMDA), alpha-amino-3 hydroxy-5-methyl-4-isoxazole propionate receptors (AMPA), kainate (KA) and delta receptors.

Attention deficit hyperactivity disorder (ADHD) is a childhood onset disorder with estimated prevalence rates of between 1.4 and 6%.^3,4 There is consistent evidence demonstrating the important contribution of genetic factors to this disorder and the search for susceptibility genes has so far been based on candidate gene association studies⁵ although genome scans are now beginning to be published.⁶ The pathophysiology of ADHD is unknown although there has been much interest in the role of a variety of neurotransmitters particularly dopamine.⁴ There is also increasing interest in the involvement of glutamatergic pathways in ADHD.⁷ In particular, animal studies^8,9,10,11 have demonstrated that glutamatergic transmission and NMDA receptors play a role in motor activity (and overactivity) as well as learning and memory. We are therefore at present undertaking a systematic analysis of glutamate receptors, transporters and other glutamate-related genes in ADHD. To date there has been one published genome-wide linkage scan of ADHD.⁶ This was based on 126 affected sib pairs and yielded multipoint maximum LOD scores of >1.5 in three regions, of which one was chromosome 16p13. More recently, sib-pair analysis of 277 affected sib pairs, by the same group,¹² suggested evidence of a major susceptibility locus on chromosome 16p13 (maximum LOD score of 4.2, P=5 10^-6). This region has also been implicated in the aetiology of autism by three genome-wide scans.¹² GRIN2A, which encodes the glutamate receptor NMDA2A subunit, maps within 2 Mb of the most significant linkage marker (D16S3114). GRIN2A is thus a functional as well as positional candidate gene. We therefore targeted this gene as a priority for family-based association analysis.

Methods and materials

The sample in this study consisted of 238 families in which the children met full diagnostic criteria for DSM-III-R/ DSM-IV ADHD or ICD-10 hyperkinetic disorder after comprehensive assessment using standard research diagnostic interviews (full details of assessment and diagnostic procedures are described in Holmes et al.).¹³ A total of 157 of these families were complete trios (child and both parents). Subjects, of whom 90% were male and 10% female, were aged between 5 and 15 years (mean age 9.6 years) and of Caucasian origin. Those with IQ test scores below 70, autism (screened using the Autism Screening Questionnaire),¹⁴ Tourette's syndrome and any neurological condition were excluded. The study was originally based on 161 families but sample collection is ongoing and given the positive findings, genotyping was later extended to include more recently collected families to expand the sample size.

We previously identified six sequence variants (two synonymous, three in UTRs, and one intronic) in exons 1, 5, 10 and 13 and intron 9 of GRIN2A after systematic DHPLC analysis of 14 unrelated Caucasian schizophrenics.² This sample has power of 0.8 to detect alleles with a frequency of 0.05 in the sampled population. As we have previously shown² that the allele frequencies at GRIN2A are the same in schizophrenics and the general population, our power estimate of 0.8 to detect alleles with a frequency of 0.05 in the general population applies. In the previous study, only three of the SNPs, CTG(L)325CTA(L) in exon 5, CGG (R)695 CGA (R) in exon 10 and #5765C/T in the 3' UTR (exon 13), had a minor allele frequency >2% in controls. To confirm these markers were not appreciably enriched in the ADHD sample, we initially genotyped all six polymorphisms in pooled samples of 100 UK Caucasian ADHD cases in DNA pools.¹⁵ All fragments were amplified using primers and conditions described by Williams et al.² This analysis confirmed that the minor alleles for three of the variants were at frequencies below the resolution of the pooling method, which empirically, we have found to be <2%. The other three SNPs were genotyped in the original association sample (n=161 families) by single nucleotide primer extension using a fluorescence polarisation (FP) assay¹⁶ based upon AcycloPrime™ reagents (Perkin Elmer Life Science Products, Boston, MA, USA) according to the manufacturer's recommendations. Analyses were performed using a LJL Biosystems Analyst™ platform.

The genotypic data were analysed for complete parent/child trios and mother/child duos using the statistical package TRANSMIT.¹⁷ Data from all available families were analysed and we also used the option of specifying a maximum allowable ambiguity for parental haplotypes of two. However, as the use of incomplete families may lead to spurious association, we also undertook TDT analysis for complete trios only. Evidence for association between haplotypes constructed from the three markers and ADHD was sought using TRANSMIT. Linkage disequilibrium (LD) between markers (D' and r²) was calculated from the estimated haplotype probabilities produced by TRANSMIT.

Results

Initially, the study was based on a sample of 161 families. The exon 5 synonymous SNP (Grin2a_5) yielded evidence for association (P=0.01). No significant evidence was obtained for the exon 13 SNP #5765C/T (Grin2a_13; P=0.13) or the exon 10 SNP (Grin2a_10; P=0.08). Construction of the haplotypes from the three SNPs in the initial sample yielded weaker evidence for association (²=13.67; 7df, P=0. 34) than did the exon 5 SNP alone in the same sample. Results are presented in Table 1. Similarly, haplotypes of all combinations of pairs of markers also yielded no extra association evidence beyond that achieved with the exon 5 SNP alone. Each of the markers showed moderate to strong LD (Table 2). In particular, the exon 5 and exon 10 SNPs were in strong LD, which presumably explains the trend for association detected with the exon 10 SNP.

Table 1 - Results of TDT and haplotype analyses for exons 5, 10 and 13 (3'UTR) SNPs using TRANSMIT.

Full table

Table 2 - Linkage disequilibrium of exons 5, 10 and 13 (3'UTR) SNPs.

Full table

Given that the exon 13 SNP #5765C/T and the exon 10 SNP add no further information to the analysis, these were not followed up further in the newly recruited sample. However, we further genotyped 77 additional, newly recruited families for the exon 5 SNP CTG(L)325CTA(L). The enlarged sample continued to provide evidence for association (P=0.01; Table 1) with this SNP. More conservatively, when the analysis was restricted to complete trios (n=157 families), the results remained significant (G allele; 78 transmissions, 55 nontransmissions; P=0.04; 10 000 simulations). The estimate for the odds ratio was 1.4.

Discussion

Our data suggest that genetic variation in GRIN2A may confer increased risk of ADHD and that this, in part, might be responsible for the linkage result on 16p reported by Smalley et al. ¹² The prior probability for association between ADHD and GRIN2A is enhanced by evidence that glutamatergic pathways may be involved in the pathophysiology of ADHD. First, there is indirect evidence from neuroimaging, functional neuroanatomy and neuropsychology studies. Glutamatergic innervation is most dense in areas of the CNS that have been strongly implicated as being affected in ADHD. Specifically, functional brain imaging and neuropsychological studies have implicated the involvement of the prefrontal cortex (PFC) and frontostriatal pathways in the pathophysiology of ADHD^4,18,19 as well as the cerebellum.²⁰ The main afferent and efferent pathways in the PFC are glutamatergic, with important glutamatergic innervation from the prefrontal and frontal cortex to the striatum.^21,22 Glutamate receptors are densely distributed in the basal ganglia, hippocampus, cerebellum and amygdala.^21,22 Animal studies also implicate the involvement of glutamate and NMDA receptors in ADHD. First, the hyperactivity of Slc6a3 knockout mice lacking the dopamine transporter (Dat1) is more severe when NMDA neurotransmission is blocked and decreases when glutamate transmission is enhanced.⁸ Furthermore, NMDA blockade appears to prevent inhibition of the hyperactivity by stimulant drugs, the main pharmacological agents used to treat ADHD.⁸ Findings from mice expressing low levels of NMDA receptors also suggest that glutamate, specifically via frontostriatal glutamatergic pathways, is involved in motor activity.⁹ Other work examining the consequences of disruption of NMDA receptor genes in mice has implicated the involvement of NMDA receptors in learning and memory ¹¹ as well as activity.¹⁰ Individuals affected by ADHD show cognitive deficits as well as motor overactivity with deficits characteristically affecting working memory, learning and executive functioning.⁴ Thus, there are good reasons for expanding candidate gene analysis of ADHD to embrace genes encoding glutamatergic system receptor subunits as candidate genes for ADHD.

The exon 5 SNP showing evidence for association is synonymous, that is, it does not change the predicted amino-acid sequence of NMDAR2A. Changes that alter amino-acid sequences may not be the rule in complex diseases^23,24 and if the association is a true positive, despite the fact that it is synonymous, it is possible that this variant per se confers susceptibility because synonymous polymorphisms may have functional effects on transcription, mRNA stability, splicing, editing or translational efficiency. An alternative explanation is that the association has arisen by virtue of linkage disequilibrium. If the latter explanation is correct, the true susceptibility variant is unlikely to be represented within exons as we have screened all the exons directly. Thus far, we have found no evidence that this SNP alters gene expression,²⁵ but have not investigated the other possibilities. Prior to further detailed functional examination of this SNP and analysis of intronic and distal regulatory elements, the first priority is for urgent independent replication given that the statistical evidence for association is modest and therefore we cannot rule out that our findings have arisen by chance. Others using unselected samples should note that a sample size of 325 complete trios will be required to provide 80% power to detect our estimated effect size (estimated equivalent odds ratio=1.4).

In summary, following on from recent linkage study findings that suggested GRIN2A as a positional as well as functional candidate gene for ADHD, our association findings suggest that further examination of GRIN2A polymorphisms in subjects with ADHD is warranted.

References

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Acknowledgements

This work is supported by the Wellcome Trust. Jane Holmes, Tracey Hever and Helen Pay were involved in the first wave of data collection. We thank all the clinicians and especially all the families who participated in this study.