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Brief Report   |    
Effect of Metabotropic Glutamate Receptor 3 Genotype on N-Acetylaspartate Measures in the Dorsolateral Prefrontal Cortex
Stefano Marenco, M.D.; Sonya U. Steele, B.Sc.; Michael F. Egan, M.D.; Terry E. Goldberg, Ph.D.; Richard E. Straub, Ph.D.; Anjail Z. Sharrief, B.Sc.; Daniel R. Weinberger, M.D.
Am J Psychiatry 2006;163:740-742. doi:10.1176/appi.ajp.163.4.740

Abstract

Objective: This study was carried out to confirm prior evidence of an effect of a single nucleotide polymorphism (SNP) in the metabotropic glutamate receptor 3 (GRM3) gene (a putative risk factor for schizophrenia) on measures of N-acetylaspartate in healthy comparison subjects. Method: Fifty-four carefully screened healthy volunteers genotyped at SNP rs6465084 underwent magnetic resonance spectroscopic imaging (MRSI) at 3 T and selected neuropsychological testing. Results: The A/A genotype group exhibited a significant reduction of N-acetylaspartate/creatine levels in the right dorsolateral prefrontal cortex compared to the G carriers. A tendency in the same direction was seen in the left dorsolateral prefrontal cortex and in the white matter adjacent to the prefrontal cortex. Conclusions: These findings provide further evidence that GRM3 affects prefrontal function and that variation in GRM3, monitored by SNP rs6465084, affects GRM3 function.

Abstract Teaser
Figures in this Article

The metabotropic glutamate receptor 3 (GRM3) gene is a key molecule in regulating synaptic glutamate concentrations. Single nucleotide polymorphisms (SNPs) in the GRM3 gene have been associated with schizophrenia in several independent reports (1–3). Recently, Egan et al. (4) showed that the A allele of SNP rs6465084 in intron 2 of the GRM3 gene was associated with an increased risk of schizophrenia, reduced verbal fluency (an index of prefrontal function that has been consistently found to be impaired in patients with schizophrenia and in their healthy relatives), and reduced prefrontal cortical levels of N-acetylaspartate/creatine, measured in vivo with nuclear magnetic resonance spectroscopy. N-Acetylaspartate/creatine levels reflect the abundance of N-acetylaspartate, an amino acid believed to be an index of neuronal function. Reductions of N-acetylaspartate/creatine levels in the prefrontal cortex of patients with schizophrenia have been demonstrated in a number of studies. The purpose of the present study was to examine the effect of the A allele of SNP rs6465084 of GRM3 on N-acetylaspartate/creatine levels in a new cohort of healthy comparison subjects studied with higher spatial resolution on a 3 T scanner. We hypothesized that N-acetylaspartate/creatine levels in the prefrontal cortex of A allele homozygotes would be reduced compared to G allele carriers.

The authors studied 54 carefully screened healthy Caucasian American comparison subjects of European ancestry. Demographic information is presented in a table available online (Data Supplement 1 at http://ajp.psychiatryonline.org). All of the participants underwent a structured diagnostic interview and a neurological evaluation, and subjects with any current or past psychiatric or neurological diagnosis, current medical illness, or family history of psychosis were excluded. Written informed consent was obtained from all subjects. The subjects were genotyped at rs6465084 in intron 2 of the GRM3 gene, as described by Egan et al. (4). The subjects completed a battery of tests, including verbal fluency (letters and categories), and an IQ test (WAIS–Revised; see reference 4 for more details). Proton magnetic resonance spectroscopic imaging (MRSI) was performed with a 3 T GE magnet (General Electric Medical Systems, Milwaukee), with a multislice imaging technique similar to that in previous publications by our group (four slices [7.5 mm cubic voxel dimensions]; spin echo slice selection; TR=2300 msec, TE=280 msec, no water suppression, and lipid signal from the scalp suppressed with outer volume saturation pulses) (5). The MRSI acquisition was conducted on a plane parallel to the main axis of the hippocampi. Regions of interest were drawn on the left and right dorsolateral prefrontal cortex, the cingulate cortex, the white matter of the centrum semiovale, the left and right hippocampal formation, and the occipital cortex on structural magnetic resonance imaging (MRI) scans registered to the MRSI slices. Metabolite signals were calculated as the integral of the magnitude spectrum in a range of 0.25 ppm surrounding peaks for N-acetylaspartate, creatine, and choline and were reported as metabolite ratios averaged over the voxels in the regions of interest. Extensive quality control procedures were undertaken to eliminate voxels with obvious artifacts. A low number of voxels in the regions of interest (less than three voxels in slice one and less than seven voxels in slice four) resulted in rejection of that particular region of interest for further statistical analysis. All quality control procedures were performed blind to genotype status. We collapsed the A/G (N=21) and G/G (N=4) genotype subjects into a G carrier group in the analysis. The differences in metabolite ratios and letter and category fluency task scores between the two genotype groups were analyzed by unpaired t tests. One-tailed statistics with a significance level set at p<0.05 were used for the dorsolateral prefrontal cortex because of the directional hypothesis based on prior findings by our group (4) (no correction for multiple comparisons). Potential confounding factors, such as age, sex, handedness, education, and IQ, were also examined.

The main results are presented in Data Supplement 1 at http://ajp.psychiatry.org. The A/A genotype group exhibited a significant reduction of N-acetylaspartate/creatine levels in the right dorsolateral prefrontal cortex compared to the G carriers, a finding that would survive corrections for six multiple comparisons. There were tendencies in the same direction in the left dorsolateral prefrontal cortex and the adjacent white matter of the centrum semiovale. No other metabolite ratios or regions of interest were significantly different between the groups. The genotype groups were well matched for age, handedness, years of education, and IQ (see table); however, there tended to be more women in the A/A genotype group. When the authors used gender as a covariate in an analysis of variance, the effect of genotype remained significant in the right dorsolateral prefrontal cortex (effect of gender: F=0.33, df=1, 51, p=0.28; effect of genotype: F=5.80, df=1, 51, p=0.01). There were no significant differences in verbal fluency performance between the A/A carriers and the G carriers.

The authors found that the GRM3 genotype at SNP rs6465084 predicted N-acetylaspartate/creatine levels in the dorsolateral prefrontal cortex in a new group of normal subjects, thus confirming an earlier report from our group (4) that had used a less highly resolved and sensitive MRI technique (the current scans were performed with a 1.5 T scanner at half the resolution of the original report). The A/A homozygotes (i.e., those with a genotype associated with an increased risk for schizophrenia) had lower N-acetylaspartate/creatine levels in the dorsolateral prefrontal cortex, possibly indicating decreased neuronal function. It is remarkable that a genotype effect was detected on this phenotype with such a small group. Based on evidence that GRM3 modulates synaptic glutamate, that it is a receptor for N-acetyl-aspartyl-glutamate, and that N-acetylaspartate is related to mitochondrial activity and to glutamate levels (6, 7), the prefrontal N-acetylaspartate reduction may reflect an alteration in genetically regulated glutamate neurotransmission or innervation patterns. Our data represent further evidence that glutamate system dysfunction may play a role in the prefrontal functional abnormalities seen in schizophrenia. Our in vivo data also add support to evidence that SNP rs6465084 monitors a functional variation in GRM3. Our group has shown that the same genotype is associated with reduced expression of presynaptic vesicular markers (8) and of the glial glutamate transporter in postmortem human brain tissue (4), but conclusive effects on GRM3 mRNA or protein levels have not been found. Although it is possible that this intronic SNP may cause altered splicing, we cannot infer from the currently available data that this is a functional locus. We did not observe an expected difference in verbal fluency between the genotypes. This may be due to a lack of power in this rather small group or possibly to a covariate that was not identified in this study (e.g., undetected ethnic stratification). The limitations of our study were that we did not correct for the respective contribution of gray and white matter to our regions of interest and that we did not obtain “absolute” measures of N-acetylaspartate levels, which are made significantly more imprecise at 3 T for long times of echo because of the effects of T 2 relaxation and B 1 inhomogeneities.

+Presented in part at the 59th meeting of the Society for Biological Psychiatry, New York, April 28–31, 2004, and at the 43rd meeting of the American College of Neuropsychopharmacology, San Juan, Puerto Rico, Dec. 12–16, 2004. Accepted Jan. 31, 2005; revision received April 8, 2005; accepted April 21, 2005. From the Genes and Cognition Program, Clinical Brain Disorders Branch, Intramural Research Program, NIMH, NIH. Address correspondence and reprint requests to Dr. Marenco, Genes and Cognition Program, Clinical Brain Disorders Branch, Intramural Research Program, Bldg. 10, Rm. 4S235, 10 Center Dr., NIMH, NIH, Bethesda, MD 20892; marencos@mail.nih.gov (e-mail).Funded by the NIMH Intramural Research Program.The authors thank Alan S. Barnett, Ph.D., and Jan Willem Van Der Veen, Ph.D., for their contribution to the MRI aspects of this article and Brad Zoltick, M.A., for writing programs that allowed more efficient processing of the data.

1. Marti SB, Cichon S, Propping P, Nothen M: Metabotropic glutamate receptor 3 (GRM3) gene variation is not associated with schizophrenia or bipolar affective disorder in the German population. Am J Med Genet 2002; 114:46–50
 
2.Fujii Y, Shibata H, Kikuta R, Makino C, Tani A, Hirata N, Shibata A, Ninomiya H, Tashiro N, Fukumaki Y: Positive associations of polymorphisms in the metabotropic glutamate receptor type 3 gene (GRM3) with schizophrenia. Psychiatr Genet 2003; 13:71–76
 
3. Chen Q, He G, Wu S, Xu Y, Feng G, Li Y, Wang L, He L: A case-control study of the relationship between the metabotropic glutamate receptor 3 gene and schizophrenia in the Chinese population. Schizophr Res 2005; 73:21–26
 
4. Egan MF, Straub RE, Goldberg TE, Yakub I, Callicott JH, Hariri AR, Mattay VS, Bertolino A, Hyde TM, Shannon-Weickert C, Akil M, Crook J, Vakkalanka RK, Balkissoon R, Gibbs RA, Kleinman JE, Weinberger DR: Variation in GRM3 affects cognition, prefrontal glutamate, and risk for schizophrenia. Proc Natl Acad Sci USA 2004; 101:12604–12609
 
5. Van Der Veen JW, Weinberger DR, Tedeschi G, Frank JA, Duyn JH: Proton MR spectroscopic imaging without water suppression. Radiology 2000; 217:296–300
 
6. Bates TE, Strangward M, Keelan J, Davey GP, Munro PM, Clark JB: Inhibition of N-acetylaspartate production: implications for 1H MRS studies in vivo. Neuroreport 1996; 7:1397–1400
 
7. Petroff OA, Errante LD, Rothman DL, Kim JH, Spencer DD: Neuronal and glial metabolite content of the epileptogenic human hippocampus. Ann Neurol 2002; 52:635–642
 
8. Kleinman JE, Hyde TM, Halim ND, Horowitz C, Straub R, Egan MF, Weinberger DR, Lipska BKB: Expression of GRM3 in schizophrenia, in 34th Annual Meeting of the Society for Neuroscience. San Diego, Society for Neuroscience, 2004, p 909
 
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References

1. Marti SB, Cichon S, Propping P, Nothen M: Metabotropic glutamate receptor 3 (GRM3) gene variation is not associated with schizophrenia or bipolar affective disorder in the German population. Am J Med Genet 2002; 114:46–50
 
2.Fujii Y, Shibata H, Kikuta R, Makino C, Tani A, Hirata N, Shibata A, Ninomiya H, Tashiro N, Fukumaki Y: Positive associations of polymorphisms in the metabotropic glutamate receptor type 3 gene (GRM3) with schizophrenia. Psychiatr Genet 2003; 13:71–76
 
3. Chen Q, He G, Wu S, Xu Y, Feng G, Li Y, Wang L, He L: A case-control study of the relationship between the metabotropic glutamate receptor 3 gene and schizophrenia in the Chinese population. Schizophr Res 2005; 73:21–26
 
4. Egan MF, Straub RE, Goldberg TE, Yakub I, Callicott JH, Hariri AR, Mattay VS, Bertolino A, Hyde TM, Shannon-Weickert C, Akil M, Crook J, Vakkalanka RK, Balkissoon R, Gibbs RA, Kleinman JE, Weinberger DR: Variation in GRM3 affects cognition, prefrontal glutamate, and risk for schizophrenia. Proc Natl Acad Sci USA 2004; 101:12604–12609
 
5. Van Der Veen JW, Weinberger DR, Tedeschi G, Frank JA, Duyn JH: Proton MR spectroscopic imaging without water suppression. Radiology 2000; 217:296–300
 
6. Bates TE, Strangward M, Keelan J, Davey GP, Munro PM, Clark JB: Inhibition of N-acetylaspartate production: implications for 1H MRS studies in vivo. Neuroreport 1996; 7:1397–1400
 
7. Petroff OA, Errante LD, Rothman DL, Kim JH, Spencer DD: Neuronal and glial metabolite content of the epileptogenic human hippocampus. Ann Neurol 2002; 52:635–642
 
8. Kleinman JE, Hyde TM, Halim ND, Horowitz C, Straub R, Egan MF, Weinberger DR, Lipska BKB: Expression of GRM3 in schizophrenia, in 34th Annual Meeting of the Society for Neuroscience. San Diego, Society for Neuroscience, 2004, p 909
 
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