Differential expression of prostaglandin D2 synthase (PTGDS) in ...

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Feb 26, 2012 - Juan Jesús Marín-Méndez a,1, Ana Patiño-García b,1,⁎, Victor ... a Department of Psychiatry, University Clinic of Navarra, Pamplona, Spain.
Journal of Affective Disorders 138 (2012) 479–484

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Differential expression of prostaglandin D2 synthase (PTGDS) in patients with attention deficit–hyperactivity disorder and bipolar disorder Juan Jesús Marín-Méndez a, 1, Ana Patiño-García b, 1,⁎, Victor Segura c, Felipe Ortuño a, Mª. Dolores Gálvez a, César A. Soutullo a a b c

Department of Psychiatry, University Clinic of Navarra, Pamplona, Spain Department of Pediatrics, University Clinic of Navarra, Pamplona, Spain Proteomics, Genomics and Bioinformatics Unit, Centre for Applied Medical Research, CIMA, Pamplona, Spain

a r t i c l e

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Article history: Received 9 September 2011 Received in revised form 30 January 2012 Accepted 30 January 2012 Available online 26 February 2012 Keywords: ADHD Bipolar disorder Gene expression Prostaglandin D2 synthase

a b s t r a c t Background: As marker genes for bipolar disorder (BP) and attention deficit hyperactivity disorder (ADHD) are not fully identified, we carried out a complete genome analysis to search for genes differentially expressed in ADHD and BP. Materials and methods: We recruited 39 patients (30 ADHD, 9 BP), aged 7 to 23 years. For evaluation of the psychiatric diagnosis, we used a semi-structured interview based on the K-SADS-PL (DSM-IV). RNA was extracted from peripheral blood and analyzed with the GeneChip® Human Genome U133-Plus 2.0 (Affymetrix). For the validation of differentially expressed genes, realtime PCR was used. Results: Hybridization and subsequent statistical analysis found 502 probe-sets with significant differences in expression in ADHD and BP patients. Of these, 82 had highly significant differences. Neuregulin (NRG1), cathepsins B and D (CTSB, CTSD) and prostaglandin-D2-synthase (PTGDS) were chosen for semi-quantitative mRNA determination. The expression of PTGDS was statistically increased in ADHD relative to BP patients (p= 0.01). We found no such differential expression with NRG1, CTSB and CTSD genes (p> 0.05). Conclusions: The gene coding for PTGDS was found to be more expressed in patients with ADHD relative to patients with BP, indicating a possible link with the differential etiology of ADHD. The experimental approach we have used is, at least in part, validated by the detection of proteins directly concerned with brain functions, and shows a possible way forward for studies of the connection between brain function genes and psychiatric disorders. Limitations: Confirmation of our findings requires a larger sample of patients with clearly-defined phenotypes. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Attention deficit hyperactivity disorder (ADHD) is one of the most frequent childhood psychiatric disorders, affecting 2–12% of children and resulting in high utilization of child

⁎ Corresponding author at: Laboratory of Pediatrics, University Clinic of Navarra, University of Navarra, Los Castaños Building, Irunlarrea SN, 31080 Pamplona, Spain. Tel.: + 34 948 425600/6304; fax: + 34 948 425649. E-mail address: [email protected] (A. Patiño-García). 1 JM-M and AP-G contributed equally to this work. 0165-0327/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jad.2012.01.040

mental health services (Soutullo and Diez, 2007). ADHD is frequently comorbid with other psychiatric disorders including bipolar, oppositional and conduct disorders (Biederman et al., 1991; Huh et al., 2011; Jaideep et al., 2006). Bipolar disorder (BP) has an estimated prevalence of 1–10% depending on diagnostic criteria and evaluation tools (Soutullo et al., 2009). BP can be misdiagnosed in children and adolescents due to symptoms that overlap with ADHD, oppositional defiant disorder and conduct disorders, and there is demand for diagnostic tools to distinguish between BP and ADHD (Jaideep et al., 2006; Soutullo and Diez, 2007). Several studies

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suggest that ADHD might be a prodromal presentation of early onset affective disorders, and this would explain the observed high rates of comorbidity (Singh et al., 2006). For ADHD and BP, numerous studies show both an environmental component (Hosang et al., 2010; Thapar et al., 2007) and a strong genetic component with heritability indexes of 80% for BP (Hosang et al., 2010) and 76% for ADHD (Soutullo and Diez, 2007). Incomplete penetrance and genetic heterogeneity, however, complicate association studies. For BP, few genes have been found to be consistently implicated, despite identification, by linkage studies, of about 40 regions of chromosomal susceptibility (Cruceanu et al., 2009). Other meta-analysis studies have not conclusively identified genes associated with increased susceptibility (Segurado et al., 2003). Studies on the genetics of ADHD, include genetic linkage studies (Thapar et al., 2007), genome-wide association studies (GWAS) (Franke et al., 2009), and numerous allelic and susceptibility gene association studies. The latter have found positive associations with genes for dopamine (DA) transporter (DAT1) (Genro et al., 2008), DA D4 receptor (DRD4) (Kustanovich et al., 2004), and catechol-O-methyltransferase (COMT) (Thapar et al., 2005). However, other studies have not replicated these findings (Gizer et al., 2009; Kustanovich et al., 2004). The genetic contribution to phenotypic variation of ADHD and BP is additive (Gillis et al., 1992), which supports the theory that combinations of numerous risk genes, together with environmental factors, are responsible for pathology. Given that marker genes for these pathologies are, at best, equivocal and in view of the hypothesis that ADHD might be a marker for a prodromal or early manifestation of pediatriconset BP, we carried out a complete genome analysis by means of expression arrays in order to search for genes differentially expressed in ADHD and BP. 2. Patients and methods 2.1. Patients and psychiatric evaluation We included patients with ADHD (n = 30) or BP (n = 9) evaluated in the outpatient clinic of the Child and Adolescent Psychiatry Unit of the University Clinic of Navarra between 2005 and 2007. Inclusion criteria were: diagnosis of ADHD or BP according to DMS-IV-TR criteria; Caucasian ethnicity and ability to speak Spanish (to enable communication with staff). Patients with comorbidities were excluded. For clinical evaluation of the psychiatric diagnosis, we used a semi-structured interview based on the K-SADS-PL of the DSM-IV (Kaufman et al., 1997). The study protocol was approved by the Institutional Review Board of our institution. All parents gave their written informed consent, and children also gave their assent. 2.2. Processing of samples White cells were isolated from 10 ml of peripheral blood anticoagulated in EDTA prior of extraction of peripheral blood leukocytes (PBL) by means of Lymphoprep™ (AxisShield PoC AS, Norway). Dry pellets of PBL were obtained and kept at − 80 °C prior to further processing. Samples were homogenized with QIAshredder columns (Qiagen, Germany), and then extraction of total RNA was carried out

with Qiagen's commercial kit RNeasy Mini Kit, following the manufacturer's instructions. Evaluation of the quality of RNA obtained was done by agarose gel electrophoresis; quantity was determined by quantitative capillary electrophoresis. 2.3. Expression arrays: bioinformatics and data analysis Expression arrays were performed with Progenika Biopharma S.A. GeneChip® Human Genome U133 Plus 2.0 (Affymetrix) chips, (54,675 sequences) were used. Background correction and normalization were done using the RMA (Robust Multichip Average) algorithm (Irizarry et al., 2003). A filtering process was performed to eliminate low expression probe-sets. Applying the criterion of an expression value greater than 64 in 6 samples for each experimental condition (BP, ADHD), 18,269 probe-sets were selected for statistical analysis. R and Bioconductor (Gentleman et al., 2005) were used for preprocessing and statistical analysis. LIMMA (Linear Models for Microarray Data) (Smyth, 2004) was used to find the probe-sets that showed significant differential expression between experimental conditions; in view of sample heterogeneity the criterion of significance was p b 0.05. Control samples were from a Gene Expression Omnibus database microarray experiment (GEO, GSE18312). The GSE18312 data-set included samples from BP patients (n= 9) and control individuals (n= 8) hybridized on the Affymetrix Human Exon ST 1.0. To compare expression levels of both experiments, RMA normalization and an additional standardization process (Z-score calculation) were performed for those genes with p b 0.01. After gene level expression calculation, we merged the results to obtain the consensus expression matrix. Functional enrichment analysis of Gene Ontology (GO) categories was carried out using a standard hypergeometric test (Draghici, 2003). Biological knowledge extraction was complemented through the use of Ingenuity Pathway Analysis (Ingenuity Systems, www.ingenuity.com). 2.4. Validation of differential expression findings Semi-quantitative analysis of cathepsin B (CTSB), cathepsin D (CTSD), neuregulin 1 (NRG1) and prostaglandin D2 synthase (PTGDS) mRNA expression was carried out by real-time polymerase chain reaction (RT-PCR) with the ABI PRISM 7300 Sequence Detector and software v1.4 (Applied Biosystems, Foster City, California). Semi-quantitative mRNA levels were expressed as a percentage relative to that of GAPDH mRNA, which was determined with TaqMan assays (Applied Biosystems). Relative levels of expression were determined by the Ct method (2 − Ct). All assays were performed in triplicate. For expression data from real-time PCR, the Mann– Whitney test was used to identify significant differences between ADHD patients and BP patients. 3. Results We recruited 39 patients (30 ADHD, 9 BP) between 7 and 23 years old. The age at diagnosis differed between the groups (p b 0.05): the ADHD group mean (SD) age was 12.21 years (3.77) and that of BP patients was 18.54 years (3.38).

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Samples from 11 ADHD and 8 BP patients were analyzed by means of GeneChip® Human Genome U133 Plus 2.0. These 19 patients were randomly selected to minimize potential statistical differences in recruitment. Hybridization and subsequent statistical analysis identified probe-sets with significant differences in expression between ADHD and BP patients. We found 82 probe-sets with highly-significant differences; and a further 502 with significant differences (data not shown). We ruled out the effect of possible confounding factors: age, sex and treatment received by using an unsupervised clustering analysis to detect groups of samples that might share common characteristics. The combined analysis of our microarray data with the GEO GSE18312 data-set can be used to infer the expression tendency of BP and ADHD samples compared to the expression levels of control individuals. Supplemental Fig. 1 is a representation of the normalized Z-score profiles of PTGDS and CTSD. Of the differentially expressed genes, four were chosen for semi-quantitative mRNA determination: NRG1, CTSB, CTSD and PTGDS. This choice was based on the statistical association previously determined (NRG1, p = 0.005; CTSB, p = 0.051; CTSD, p = 0.003; PTGDS, p = 0.002) and because the four genes have a documented function related either with a CNS pathology or a psychopathology (Bagnoli et al., 2002; Goes et al., 2009; Kikuchi et al., 2003; Taniguchi et al., 2007). CTSB, with p = 0.051, was selected because it belongs to the same family of proteins as CTSD. The protein encoded

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by CTSB is a secretase involved in the processing of amyloid precursor protein; CTSD gene mutations are involved in Alzheimer disease. The PTGDS gene catalyzes synthesis of PGD2, a neuromodulator and trophic factor in the CNS (Pruitt et al., 2007). The NRG1 gene encodes a signalling protein and neuronal growth factor regulating CNS differentiation, synaptogenesis and myelination, with a critical role in schizophrenia and BD. The whole batch of samples (30 ADHD and 9 BD) was used for validation of candidate genes. Determination of gene expression by RT-PCR revealed that expression of the PTGDS gene was significantly increased in ADHD patients (mean (SD): 0.32 (0.4)) relative to BP patients (0.06 (0.04)) (p= 0.01, Mann–Whitney test). We found no such differential expression with NRG1 (0.53 (0.6) in ADHD vs 0.25 (0.06) in BP), CTSB (0.27 (0.15) in ADHD vs 0.21 (0.05) in BP) and CTSD (0.3 (0.2) ADHD vs 0.16 (0.1) in BP) genes (p> 0.05) (Fig. 1). We normalized our expression data to GAPDH expression since the probes for this gene in the array showed no differences between ADHD and BD patients (p-values between 0.690 and 0.813). For the differential probe-sets (p b 0.01), Ingenuity® identified a molecular pathway including the PTGDS gene product (Fig. 2). For the 82 genes with highly significant differential expression, Ingenuity®'s highest scoring network function was “Cellular Function and Maintenance, Small Molecule Biochemistry, Nervous System Development and Function”, which is congruent with the pathologies under analysis.

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Fig. 1. Comparison of the relative expression of the genes CTSB, CTSD, NRG1 and PTGDS in ADHD and BP patients. Data are presented as mean expression values with bars representing standard deviations.

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Fig. 2. Ingenuity output showing the molecular pathway that includes the over-expressed PTGDS gene (2000–2011 Ingenuity Systems, Inc).

4. Discussion There are clinical difficulties in the differential diagnosis of ADHD: especially comorbidity with oppositional defiant disorder, antisocial personality disorder or BP, which produces disparities in the perceived prevalence of the disorders in different studies. To our knowledge, there are no published reports of complete genome scans comparing patients with ADHD and BP without comorbidity. None of numerous searches for marker genes has succeeded in identifying a gene unequivocally associated exclusively with one of the disorders. This, taking into account the typical age at diagnosis for these diseases,

can be explained by the hypothesis that ADHD is a prodrome of pediatric-onset BP (Singh et al., 2006). The study reported here is not a candidate gene analysis, but rather a whole genome scan undertaken to elucidate biochemical or genetic differences between ADHD and BP. Our analysis did not include a control group because our objective was to find genetic markers for the differential diagnosis of ADHD and BP rather than to identify differences in gene expression between patients and controls. The ADHD and BP groups studied were as matched as possible for demographic confounders. Group differences in age, however, were unavoidable and were controlled by unsupervised hierarchical clustering.

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We found the expression of the PTGDS gene to be increased in ADHD relative to BP patients. The protein encoded by the PTGDS gene is a glutathione-independent prostaglandin D synthase preferentially expressed in the brain. It catalyzes the conversion of prostaglandin H2 (the cyclooxygenase-mediated product of arachidonic acid) to prostaglandin D2 (PGD2). PGD2 functions as both a neuromodulator and a trophic factor in the CNS (Pruitt et al., 2007). PTGDS (or L-PTGDS) is a major endogenous amyloid β-chaperone in the brain, with suspected involvement in Alzheimer disease (Kanekiyo et al., 2007). Prostaglandin synthesis plays a pivotal role in metabolic homeostasis, sleep regulation, adipogenesis, allergic response and inflammation. In the search for the basis of ADHD, the emphasis has historically been on neurotransmission per se. However, the functioning of neurotransmitters and their receptors can be profoundly modified by the lipids in their environment, and recent reports support the hypothesis that fatty acid deficiency is a contributory factor to ADHD (Richardson and Puri, 2000). Begemann and co-workers (Begemann et al., 2008), found that genes involved in prostaglandin metabolism, PTGDS and AKR1C3, showed higher expression in bipolar patients with rapid-cycling depressive episodes. Qiu and co-workers identified PTGDS as one of the genes that is repressed in SHR rodents (Qiu et al., 2010). To clarify a role of PTGDS in ADHD children, further genetic and biochemical analysis are needed. It would be useful to analyze the profiles of essential fatty acids, PGD2 and PGH2 in a subset of patients of narrow clinical phenotype. In conclusion, our main finding was that PTGDS gene expression was increased in ADHD relative to BP patients. This result, however, requires confirmation from study of a larger sample of phenotypically clearly-defined patients. In addition, by detecting genes coding for proteins known to be directly concerned with brain function, the study validates to some extent the experimental approach of whole genome scanning as applied to molecular biology research into psychiatric disorders. Supplementary materials related to this article can be found online at doi:10.1016/j.jad.2012.01.040. Role of funding source This work was funded by a grant from the “Fundación Alicia Koplowitz”; this Foundation had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication. Conflict of interest The authors declare that they have no conflicts of interest in connection with the submitted manuscript.

Acknowledgments We thank all patients and families who collaborated in our project. We are indebted to David Burdon for reviewing the English language of this manuscript.

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