Expression of the α1A-adrenergic receptor in schizophrenia

Neuroscience Letters 401 (2006) 248–251

Expression of the ␣1A-adrenergic receptor in schizophrenia Dan A. Clark ∗ , Dalu Mancama, Robert W. Kerwin, Maria J. Arranz Clinical Neuropharmacology (P051), Institute of Psychiatry, De Crespigny Park, Denmark Hill, London SE5 8AF, United Kingdom Received 31 October 2005; received in revised form 10 February 2006; accepted 9 March 2006

Abstract The ␣1 -adrenergic receptors may contribute to cognitive functions relevant to schizophrenia. Following the discovery of an association between polymorphisms in the regulatory region of the ␣1A -adrenergic receptor and schizophrenia we investigated the expression of mRNA for this receptor between schizophrenics (n = 19) and controls (n = 19) using a TaqMan approach in post-mortem brains. No differences were found suggesting that mRNA levels are not altered in schizophrenia. Genotypic data for the subjects found that possession of the previously associated genotypes did not convey a difference in mRNA expression suggesting that these polymorphisms do not affect the level of transcription. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Adrenergic alpha-1 receptors; mRNA expression; Schizophrenia; ADRA1A

The ␣1 -adrenergic receptors have been implicated in cognitive functions relevant to schizophrenia [4,9,11,16]. Despite this, attempts to determine the expression of central ␣1 -adrenergic receptors in psychiatric patients show no consensus as to whether binding is elevated, reduced or unchanged [2,8]. Furthermore, these studies have not been specific enough to distinguish between the three ␣1 -adrenergic subtypes (␣1A , ␣1B , ␣1D ) which appear to have distinct roles [7,17]. A recent case-control association study from our group focusing on just one of these subtypes, revealed a strong association with schizophrenia from two polymorphisms and a haplotype in the ␣1A -adrenergic receptor gene (ADRA1A) [5]. As these polymorphisms are located in the regulatory/promoter region of the gene [12,15], we hypothesised that these polymorphisms influenced receptor expression levels and that this conferred susceptibility to schizophrenia. To investigate this we measured the levels of mRNA for the ADRA1A gene in post-mortem tissue derived from the brains of schizophrenic and unaffected controls and genotyped these samples for the previously investigated single nucleotide polymorphisms (SNPs) in the regulatory region of this gene. Post-mortem brain samples were supplied by the Institute of Psychiatry Brain Bank (London, United Kingdom). Patient samples were a randomly chosen total of 19 individuals previously diagnosed with schizophrenia according to ICD-10 criteria [18].

∗

Corresponding author. Tel.: +44 207 848 0343; fax: +44 207 848 0051. E-mail address: [email protected] (D.A. Clark).

0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2006.03.025

All were chronic schizophrenics who had been undergoing longterm typical antipsychotic treatment but with no information on treatment regime available. The control samples comprised 19 individuals without any history of psychiatric or neurobiological illness. All subjects were of European Caucasian descent and tissue was biopsied from the prefrontal cortex (Broadmann areas 9 and 10) of the brain using standardised dissection protocols. This brain region was selected based on evidence implicating noradrenaline in the prefrontal cortex in cognitive functions relevant to schizophrenia [3,4,6,9]. Data for the pH level of each sample was not available, however, before snap freezing the samples were observed to lie within the range of pH 6.2–6.7. Experiments were conducted in accordance with the declaration of Helsinki. In all instances tissue had been donated with informed consent and under the approval of the Institute of Psychiatry’s ethics committee. Total RNA and DNA were isolated from 100 mg of brain tissue using a Sigma TRI-Reagent RNA isolation kit (SigmaAldrich, Gillingham, UK) and Nucleon BACC3 DNA extraction kit (Nucleon Biosciences, Coatbridge, UK) in accordance with the manufacturer’s guidelines. For TaqMan analysis first strand complementary DNA (cDNA) synthesis of RNA was performed using the Qiagen Omni-script Reverse Transcriptase polymerase chain reaction (RT-PCR) kit with oligo-dT primers (10 ␮M) (Qiagen, Crawley, UK). cDNA quantification was performed using the TaqMan polymerase chain reaction (PCR) technique on an Applied Biosystems 7900HT sequence detection system (Applied Biosystems,

D.A. Clark et al. / Neuroscience Letters 401 (2006) 248–251

Warrington, UK). We eliminated the need for an absolute measurement using a standard curve by opting for a relative quantification method, reporting (for each subject) the level of ADRA1A cDNA as a ratio of the level of cDNA from the ubiquitously expressed ␤-Actin gene. The primers and probe for ␤-Actin were provided in a premix from Applied Biosystems (p/n: 4333763T, Applied Biosystems, Warrington, UK). Those for the ADRA1A gene were supplied by Applied Biosystem’s Assay-on-demand service (assay ID: Hs00169124 m1, Applied Biosystems, Warrington, UK). The probe for this assay was designed to span the boundaries of the adjoining exons 1 and 5 to avoid quantifying any remaining genomic DNA. This design meant the probe quantified all full-length receptor isoforms (ADRA1A v1 to ADRA1A v5) but not the truncated isoforms (ADRA1A v6 to ADRA1A v16). More detail on the construction of these isoforms can be found in [10]. Quantification of the target and control was carried out in duplicate in separate reactions on ABI prism 384-well optical reaction plates (Applied Biosystems, Warrington, UK). For each reaction, 1.5 ␮L of cDNA was used with 0.5 ␮L of the probe and primer premix (for either the target or control), 5 ␮L of Taqman Universal PCR master mix (Applied Biosystems, Warrington, UK) and 3 ␮L of water. The Applied Biosystems 7900HT was configured for absolute quantification, with thermal cycling conditions comprising 50 ◦ C for 2 min and 95 ◦ C for 10 min, followed by 45 cycles of 95 ◦ C for 15 s and 60 ◦ C for 1 min. For both the ␤-Actin and ADRA1A assays, two types of negative controls were run simultaneously in duplicate. One had no cDNA while the other no TaqMan Universal PCR Master Mix. In both cases the reaction volume was made up to 10 ␮L with water. Using the genomic DNA extracted from the brain tissue the subjects were genotyped for the eight previously studied single nucleotide polymorphisms (−9625-G/A, −7255-G/A, −6274C/T, −4884-A/G, −4155-G/C, −2760-A/C, −1873-G/A and −563-C/T) in the regulatory region of the ADRA1A gene using the same assays described previously [5]. Data analysis was carried out using the same protocol as explained in a previous study [13] using the Applied Biosystems 7900HT sequence detection software (SDS version 2.1, Applied Biosystems, Warrington, UK). Threshold cycle values were used to define the initial amount of target cDNA for each of the subjects. These were derived from the change in the reporter fluorescence (
Rn) which was normalised against the passive reference (contained in the TaqMan Universal PCR master mix) by dividing the emission intensity of the reporter fluorescence by the emission intensity of the passive reference. The threshold cycle was determined as the first PCR or thermal cycle at which a statistically significant increase in
Rn was first detected. Further details can be found in the TaqMan gene expression product guide [1]. By using the ubiquitously expressed ␤-actin as the endogenous control or calibrator, we normalised levels of ADRA1A cDNA by determining the ratio of ADRA1A cDNA relative to that of the ␤-Actin cDNA. These ratios were examined for normality of distribution and homogeneity of variance using a combination of box plots and histograms (not shown). In the control group a positive skew was apparent that became more normally distributed after log10 transformation of the ratios.

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Neither group indicated departure from homogeneity of variance following logarithmic transformation so the data for both groups was analysed on this scale. As a precaution all group comparisons were adjusted for age and PMI by including these variables as covariates in an ANCOVA model. To determine whether the investigated polymorphisms influence levels of the ADRA1A receptor cDNA the model was adjusted for each polymorphism in turn to include genotype as a fixed factor. Statistical testing was performed at a significance level of 5% and all analyses were performed in SPSS (Release 11, Chicago, Illinois). The sample was calculated using nQuery Advisor (Release 6, Statistical Solutions, Ireland) to have an 85% power of detecting a 10% difference in threshold cycle value with a 95% confidence. The patient and control groups were identically matched for gender distribution with 11 males and 8 females in each. The groups were similarly matched for age of death and PMI (see Table 1). Ratios of ADRA1A cDNA showed no difference between patients [1.08 (1.05–1.11 95% confidence interval (CI))] and controls [1.08 (1.05–1.10 95% CI) (F = 0.048, d.f. = 1, p = 0.83]. The subjects were genotyped for eight previously investigated ADRA1A polymorphisms (−9625 G/A, −7255 G/A, −6274 C/T, −4884 A/G, −4155 G/C, −2760 A/C, −1873 G/A and −563 C/T). No significant differences were found in the distribution of the genotypes between patients and controls. To determine whether genotype status may influence mRNA expression we grouped the subjects according to genotype and compared mean ratios of ADRA1A cDNA between groups. Table 2 shows how no evidence was found to support a role of these polymorphisms in influencing ADRA1A mRNA levels. In the light of evidence from a recent association study [5] we proposed alterations to normal ␣1A -adrenergic receptor gene expression in schizophrenia. To investigate this hypothesis, we chose to measure the levels of ADRA1A mRNA in the prefrontal cortex. This region was selected given the evidence implicating the noradrenergic innervations of the prefrontal cortex in cognitive functions relevant to schizophrenia [3,4,6,9]. mRNA was extracted from the brain tissue of 19 schizophrenics and 19 unaffected controls and cDNA was formed in a first-strand reverse transcriptase PCR. The ADRA1A cDNA was quantified in relation to cDNA levels of the ubiquitously expressed ␤-Actin gene using the Taqman approach. With no difference between patients and controls, levels of ADRA1A mRNA in the prefrontal cortex do not appear to change in schizophrenia. Given that the rate and amount of protein synthesised is related to the quantity of mRNA [14], it is possible that levels of the ␣1A -adrenergic receptor are not changed in schizophrenia either. Of course there are recognised limitations to such an extrapolation, such as the influences of mRNA degradation, non-coding RNA and post translational events. Genotypic data from the subjects on regulatory region ADRA1A polymorphisms was used to determine if possession of any of these genetic variants conveyed a significant influence on the level of ADRA1A mRNA by comparing ratios between the groups of individuals possessing the various genotypes. No evidence was found to support an influence of these polymorphisms in the levels of ADRA1A receptor mRNA.

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D.A. Clark et al. / Neuroscience Letters 401 (2006) 248–251

Table 1 Summary statistics for ADRA1A mRNA expression analysis

Demography Mean age ± S.D. Mean PMI ± S.D. Gender distribution Mean ratio of ADRA1A mRNA expression after adjustment for age and PMI (95% CI) Distribution of ADRA1A polymorphism genotypes

Patients (n = 19)

Controls (n = 19)

Tests for group differences

69.9 ± 20.2 48.6 ± 25.0 8 females, 11 males

63.8 ± 17.5 48.7 ± 21.9 8 females, 11 males

2-tailed t-test, p = 0.43 2-tailed t-test, p = 0.59

1.08 (1.05–1.11)

1.08 (1.05–1.10)

F = 0.048(1), p = 0.83

−9625 G/A

G/G G/A A/A

100% 0% 0%

G/G G/A A/A

100% 0% 0%

n/a

−7255 G/A

G/G G/A A/A

32% 42% 26%

G/G G/A A/A

16% 68% 16%

χ2 (2) = 2.69, p = 0.26

−6274 C/T

C/C C/T T/T

79% 21% 0%

C/C C/T T/T

90% 10% 0%

χ2 (1) = 0.79* , p = 0.33

−4884 A/G

A/A A/G G/G

74% 16% 11%

A/A A/G G/G

58% 42% 0%

χ2 (2) = 4.63, p = 0.10

−4155 G/C

G/G G/C C/C

26% 32% 42%

G/G G/C C/C

16% 68% 16%

χ2 (2) = 5.35, p = 0.07

−2760 A/C

A/A A/C C/C

26% 47% 26%

A/A A/C C/C

32% 53% 16%

χ2 (2) = 0.64, p = 0.73

−1873 G/A

G/G G/A A/A

63% 26% 11%

G/G G/A A/A

53% 42% 5%

χ2 (2) = 1.21, p = 0.55

−563 C/T

C/C C/T T/T

26% 32% 42%

C/C C/T T/T

32% 42% 26%

χ2 (2) = 1.07, p = 0.59

The statistical test for differences in mean mRNA ratios was carried out on the log10 adjusted ratios but the ratios quoted in this table are those prior to logarithmic adjustment. PMI: post-mortem interval. S.D.: standard deviation. (*) denotes Fisher exact test used: The distribution of the test statistics under the null hypothesis of no association of genotype with group membership has been derived by permuation.

As our previous study found association between these polymorphisms and schizophrenia [5] these results can be interpreted in several ways. A simple comparison of statistical powers between this study and the original association shows that this study has less power and therefore a lesser ability to detect a genetic association. This interpretation lends itself to the possibility that the original finding is correct and that the study presented here is a false negative. Alternatively, as the original study utilised a population isolate, it is possible that the original findings were due to the extensive linkage disequilibrium that may exist in isolate populations rather than the SNPs under investigation. Therefore, the non-isolate population studied here with different and probably less extensive LD relationships would not show association between the investigated SNPs and disease nor altered gene function. Finally, there is always the possibility that our initial finding of association was a false positive. Although this study does not provide evidence for a role of the ␣1A -adrenergic receptor in schizophrenia or an influence

in transcription for the investigated ADRA1A polymorphisms, it is important that these results are considered taking into account the limitations of this study. Firstly, this study only detected mRNA for the full-length isoforms (ADRA1A v1 to ADRA1A v5) and not the truncated isoforms (ADRA1A v6 to ADRA1A v16). It is possible that schizophrenics differ from controls only in the number of truncated isoforms that are expressed and that the polymorphisms of the ADRA1A regulatory region influence this. Further work requiring additional primers and probe sets would be required to confirm this. Secondly, only one brain region (the prefrontal cortex) was investigated, and any possible effect may be regional. And in addition to these limitations, there are also those inherent to investigations using post-mortem brain samples such as the unknown influence of prior medication and agonal state. In summary, the results of this study do not support a functional role for the investigated polymorphisms in influencing levels of ADRA1A mRNA expression. Furthermore, these results

D.A. Clark et al. / Neuroscience Letters 401 (2006) 248–251

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Table 2 Mean ratios of ADRA1A mRNA levels between the investigated polymorphisms Polymorphism

Mean ratios of ADRA1A expression

F-test statistic

−9625 A/G

n = 37 1.08 (1.06–1.10) A/A

n=0 n/a A/G

n=0 n/a G/G

n/a

−7255-G/A

n=8 1.08 (1.05–1.13) G/G

n = 21 1.07 (1.05–1.10) G/A

n=8 1.09 (1.05–1.13) A/A

F = 0.38, d.f. = 2, p = 0.69

−6274-C/T

n = 31 1.07 (1.05–1.09) C/C

n=6 1.09 (1.05–1.14) C/T

n=0 n/a T/T

F = 0.46, d.f. = 1, p = 0.50

−4884-A/G

n = 24 1.07 (1.05–1.09) A/A

n = 11 1.09 (1.05–1.14) A/G

n=2 1.03 (0.96–1.11) G/G

F = 0.78, d.f. = 2, p = 0.47

−4155-G/C

n=8 1.06 (1.05–1.13) G/G

n = 19 1.07 (1.04–1.09) G/C

n = 10 1.09 (1.06–1.13) C/C

F = 1.16, d.f. = 2, p = 0.33

−2760-C/A

n = 11 1.08 (1.04–1.11) C/C

n = 18 1.07 (1.04–1.10) C/A

n=8 1.09 (1.05–1.13) A/A

F = 0.10, d.f. = 2, p = 0.90

−1873-G/A

n = 21 1.08 (1.05–1.10) G/G

n = 13 1.08 (1.05–1.11) G/A

n=3 1.06 (1.00–1.13) A/A

F = 0.13, d.f. = 2, p = 0.88

−563-C/T

n = 13 1.07 (1.04–1.11) C/C

n = 13 1.08 (1.05–1.11) C/T

n = 11 1.08 (1.05–1.11) T/T

F = 0.02, d.f. = 2, p = 0.98

The mean ratios of ADRA1A expression are the pre-logarithmic adjustment values and are reported with 95% confidence interval in brackets.

find no evidence for change of ADRA1A mRNA expression in schizophrenia. [10]

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