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Keywords:

  • CNP;
  • microarray;
  • oligodendrocyte;
  • post-mortem;
  • schizophrenia

Abstract

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Aims:  Although the expression of oligodendrocyte-related genes in post-mortem brains of patients with schizophrenia is consistently reported to be downregulated, the cause of the change remains unclear. The A-allele of rs2070106 within the 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP), an oligodendrocyte-related gene, was reported to show reduced expression compared with the G-allele, and proposed to be associated with schizophrenia.

Methods:  The effect of the rs2070106 genotype on the expression of CNP and other oligodendrocyte-related genes was examined using data previously obtained from DNA microarray studies of post-mortem brains.

Results:  It was found that the effect of rs2070106 genotype on the CNP expression was transcript specific, and that the genotype was not associated with the expression of other oligodendrocyte-related genes.

Conclusions:  The rs207016 genotype is not likely to contribute to the reported coordinated down-regulation of oligodendrococyte-related genes in schizophrenia.

GENE EXPRESSION ANALYSES in post-mortem brains of patients with schizophrenia have indicated a downregulation of oligodendrocyte-related genes.1–9 Multiple lines of evidence obtained from electron micrograph,10,11 immunohistochemistry,12 and in vivo imaging studies,13,14 collectively suggest a certain alteration of oligodendrocytes in brains of patients with schizophrenia. However, the primary cause of the change in oligodendrocytes in schizophrenia patients remains open to question.

In the context of DNA microarray or other molecular biology studies, the primary cause underlying the coordinated downregulation of oligodendrocyte-related genes remains to be elucidated. We previously reported that DNA methylation status in the CpG island of the SRY (sex determining region Y)-box 10 (SOX10) gene correlated with a downregulated expression of SOX10 and other oligodendrocyte-related genes.8 In addition, several oligodendrocyte-related genes have been proposed to be susceptibility genes for schizophrenia, including myelin-oligodendrocyte glycoprotein (MOG),15 myelin-associated glycoprotein (MAG),16,17 proteolipid protein 1 (PLP1),18 gelsolin (GSN),19 oligodendrocyte lineage transcription factor 2 (OLIG2),20 myelin protein zero-like 1 (MPZL1),21 and 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP).22 Although none of them have yet been replicated across different populations,23,24 identifying the primary cause of the change would provide important insights into the pathophysiology of schizophrenia.

Among the proposed susceptibility genes, CNP is an attractive candidate, in that it was identified to have a functional single nucleotide polymorphism (SNP).22 The A-allele of an exonic SNP within the CNP gene (rs2070106) showed reduced expression compared with the G-allele in the brain, and was reported to be associated with schizophrenia.22 The reduced expression of the A-allele of rs207016 was proved by a robust allelic expression analysis, by which effects of complex trans-acting factors such as expression of transcription factors and various confounding issues such as preparation of samples, are expected to be totally cancelled out.25 Allelic expression analysis, although useful to assess the effect of an SNP of interest, does not deal with total expression changes measured in the brain.25 Consequently, the contribution of the rs2070106 genotype to the reported downregulation of the CNP gene is not clear.

If the rs2070106 in the CNP gene is the primary cause for downregulation of oligodendrocyte-related genes in schizophrenia, association between genotype and expression would be detected in the CNP gene and other oligodendrocyte-related genes. The purpose of the present study was to test the effect of the rs2070106 genotype on the expression of the CNP gene and other oligodendrocyte-related genes using data obtained from DNA microarray studies.

METHODS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

We had previously profiled gene expression in two sets of prefrontal cortices using Affymetrix GeneChips (Affymetrix, Santa Clara, CA, USA).26,27 The sets were provided by the Stanley Medical Research Institute (SMRI). Set 1 (34 controls, 33 patients with bipolar disorder, and 35 with schizophrenia) was examined using an HGU133A chip, which contains probe sets for approximately 22 000 genes. Set 2 (15 controls, 11 patients with bipolar disorder, 13 with schizophrenia, and 11 with major depression) was examined using an HGU95Av2 chip, which contains probe sets for 14 000 genes. In the present study DNA microarray data from controls and patients with schizophrenia were used. Each brain set was matched for various demographics such as age, gender, side of brain, post-mortem interval, and so on. Diagnoses were made by two senior psychiatrists based on DSM-IV criteria and medical records. When necessary, telephone interviews with family members were conducted. Diagnoses of controls were based on structured interviews by a senior psychiatrist with family member to rule out Axis I diagnoses. More detailed information about the samples, and a summary of variables can be found elsewhere28 or at the website http://www.stanleyresearch.org/programs/brain_collection.asp The quality control of DNA microarray analysis, and consistency between DNA microarray and quantitative reverse transcriptase–polymerase chain reaction data have been examined and discussed previously.26,27,29 The microarray raw data were processed using MAS5 software (Affymetrix) and imported into GeneSpring 7.3.1 software (SiliconGenetics, Redwood, CA, USA). Data normalization was then performed by dividing each microarray data set by its median value. Normalized expression levels of the probe sets for the CNP gene and other oligodendrocyte-related genes in controls and patients with schizophrenia were used in the present study. Genotyping of rs2070106 in the CNP gene was performed using Taqman assays (Applied Biosystems, Foster City, CA, USA) with an ABI PRISM 7900HT (Applied Biosystems). The probes and Universal PCR Master Mix were obtained from Applied Biosystems. Genomic DNA from post-mortem brain was obtained from the SMRI or extracted from fresh frozen samples as described earlier, and used for genotyping. Statistical analyses were performed using the two-sample Student's t-test or Pearson's correlation using SPSS10.0 J software (SPSS, Tokyo, Japan). P < 0.05 was considered significant. The present study was approved by the ethics committees of RIKEN.

RESULTS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

There were four and one probe sets for the CNP transcripts in the Affymetrix HU95Av2 and HU133A chips, respectively. The four probe sets in the HU95Av2 chip had been designed to detect different transcripts from the CNP gene by the manufacturer. Two of them (39678_at and 39679_at) showed no detectable amounts of transcripts in the brain samples (i.e. the calls calculated by the MAS5 software were absent in all samples). In contrast, the other two probe sets in the HU95Av2 chip (344_s_at and 612_s_at) and one probe set in the HU133A chip (208912_s_at) showed present calls in all samples examined. Close examination of the location of these probe pairs in the CNP gene further indicated that each of them detected different CNP transcripts (Fig. 1). Therefore, we analyzed three probe sets independently. These probe sets for the CNP transcripts did not show sample-pH dependent expression changes30,31 (R = 0.120, 0.085, and 0.010 for 344_s_at, 612_s_at, and 208912_s_at, respectively).

image

Figure 1. RefSeqs and probe sets for 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP). Affymetrix probe sets for CNP correspond to the distinct CNP transcripts. (□) Region where probe pairs were actually located; (▿) short deletions compared with RefSeqs. It should be noted that three probe sets detected different transcripts, due to the short deletions.

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We genotyped an exonic SNP, rs207016, in the same samples used for the DNA microarray analysis. To test the effect of the SNP on the CNP expression, we first compared expression levels between different genotypes using control samples, to cancel out the confounding factors associated with patients. Due to a lack of AA homozygotes among the controls (n = 2 and n = 1 in set 1 and set 2, respectively), we compared expression levels between GG homozygotes and GA heterozygotes within controls (Fig. 2). The expression status of GG and GA genotypes did not differ in 344_s_at (P = 0.341) or 208912_s_at (P = 0.914), but significantly differed in 612_s_at (P = 0.044) on t-test. Confounding factors including age, post-mortem interval, and sample pH were not statistically different between GG and GA genotypes (data not shown). Consistent with a previous report,22 GA heterozygotes had an approximately 1.4-fold reduced expression compared with GG homozygotes in the 612_s_at probe sets. We next compared the CNP expression levels in the combined samples of controls and schizophrenia patients to assess the possible effect of the AA homozygotes. In this analysis, GG genotypes did not show statistical differences compared with AA genotypes (P = 0.485 and 0.827 for 612_s_at and 208912_s_at, respectively). In the 344_s_at probe sets, a tendency for a difference (P = 0.074) was detected on t-test, although the direction of the difference was opposite to a previous report.22 We did not find differences in expression between the GG and GA genotypes in the combined samples (data not shown). Therefore, among the three probe sets for CNP, we detected downregulation of 612_s_at probe sets in controls having GA genotypes compared with those having GG genotypes.

image

Figure 2. Effect of the rs207016 genotype on 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) expression. The expression level of (a) 208912_s_at was measured with the HU133A chip in post-mortem brain set 1. Expression levels of (b) 344_s_at and (c) 612_s_at were measured with the HU95Av2 chip in post-mortem brain set 2. CT, control; SZ, schizophrenia. *P < 0.05.

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We then assessed the effect of the rs207016 genotype on the other oligodendrocyte-related genes for which the expression was reportedly downregulated in brains of patients with schizophrenia (Table 1). In this analysis we used the DNA microarray data of the post-mortem brain set 2, because the significant downregulation of oligodendrocyte-related genes, including the CNP gene, is well-characterized, and has been reported by at least three independent groups.2,4,8 We found that none of the genes showed differential expression, when the expression in GG and GA genotypes was compared within controls or that in GG and AA genotypes was compared in combined samples.

Table 1.  Effect of the rs207016 genotype on oligodendrocyte-related gene expression
Affymetrix IDSymbolProductP
GG vs GAGG vs AA§
  • IDs for the HU95Av2 chip.

  • P were calculated within control subjects (n = 5, n = 8, for the GG and AA genotypes, respectively);

  • §

    P were calculated for combined samples (n = 7, n = 9, for the GG and AA genotypes, respectively).

32538_atTFTransferrin0.3200.258
32612_atGSNGelsolin0.2900.351
35903_atOMGOligodendrocyte myelin glycoprotein0.3780.261
36018_atSOX10SRY (sex determining region Y)-box 100.1450.543
38051_atMALMal, T-cell differentiation protein0.9660.265
38499_s_atMOBPMyelin-associated oligodendrocyte basic protein0.2550.277
38558_atMAGMyelin associated glycoprotein0.3940.381
38653_atPMP22Peripheral myelin protein 220.2680.557
40624_atOLIG2Oligodendrocyte lineage transcription factor 20.2770.878
41158_atPLP1Proteolipid protein 10.4010.421

DISCUSSION

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

We found that the expression of one CNP transcript was associated with the rs207016 genotype in control brains, whereas the expression of the other two transcripts was not influenced by genotype. These results suggest that the reported cis-acting effect of rs20701622 may be different in different transcripts. To date, a number of different types of CNP transcripts have been deposited in public databases. In addition, different CNP protein isoforms have been reported.32,33 The relationship between transcript variants and protein isoforms, however, was not clarified in the present study because all the probe sets for the CNP gene were designed in the 3′-untranslated region (Fig. 1). It should be noted that the small number of AA homozygotes among the controls prevents a direct comparison of the genotype's effect. In addition, when two diagnostic groups were combined, no statistical differences were observed across genotypes. It should also be noted that the effect of various confounding factors such as medication as well as small sample size on the expression analysis could not be ruled out. These results indicate that further tests using larger samples are needed to confirm the transcript-specific cis-acting effect of rs207016.

Considering the complexity of the CNP transcripts, we could not rule out the possibility that the expression of other undetected CNP transcripts was more severely affected by the rs207016 genotype. However, it is unlikely that such transcripts are, directly or indirectly, associated with the reported downregulated expression of oligodendrocyte-related genes, because expression levels of most of the other oligodendrocyte-related genes did not differ between the rs207016 genotypes. Therefore, the rs207016 genotype of the CNP gene is not likely to contribute to the reported decrease in the expression of oligodendrocyte-related genes in schizophrenia.

ACKNOWLEDGMENTS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

This study was supported by a Grant-in-Aid from the Japanese Ministry of Health, Welfare, and Labor. Post-mortem brain samples were donated by the Stanley Medical Research Institute, courtesy of Drs Michael B. Knable, E. Fuller Torrey, Maree J. Webster, and Robert H. Yolken.

REFERENCES

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES
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