Blood Gene Expression Correlated with Tic Severity in Medicated and Unmedicated Patients with Tourette Syndrome

Research Article For reprint orders, please contact: [email protected] Blood gene expression correlated with tic severity in medicated and unmedicated patients with Tourette Syndrome

Background: Tourette Syndrome (TS) has been linked to both genetic and environmental factors. Gene- expression studies provide valuable insight into the causes of TS; however, many studies of gene expression in TS do not account for the effects of medication. Materials & methods: To investigate the effects of medication on gene expression in TS patients, RNA was isolated from the peripheral blood of 20 medicated TS subjects (MED) and 23 unmedicated TS subjects (UNMED), and quantified using whole-genome Affymetrix microarrays. Results: D2 dopamine receptor expression correlated positively with tic severity

in MED but not UNMED. GABAA receptor e subunit expression negatively correlated with tic severity in UNMED but not MED. Phenylethanolamine N-methyltransferase expression positively correlated with tic severity in UNMED but not MED. Conclusion: Modulation of tics by TS medication is associated with changes in dopamine, norepinephrine and GABA pathways.

†1 KEYWORDS: GABRE n gene expression n microarray n NPAS4 n phenylethanolamine Isaac H Liao , N-methyltransferase n PNMT n Tourette Syndrome Blythe A Corbett1, Donald L Gilbert2, Silvia A Bunge3 Tourette Syndrome (TS) is a developmen- into this complex disorder. Ideally, tic severity & Frank R Sharp1 tal neuropsychiatric disorder characterized should be examined in relation to gene expres- 1University of California, Davis, MIND by the presence of patterned movements or sion in brain tissue, but this is not feasible Institute & Department of Neurology, sounds performed involuntarily or in response using the current technology. The advantage 2805 50th St, Sacramento, CA 9581, USA to urges. Clinic-based TS cohorts show of studying blood gene expression is the ability 2Cincinnati Children’s Hospital Medical extremely high rates of obsessive compulsive to characterize the relationship between genes, Center, Department of Pediatric behaviors and attention deficit hyperactivity environmental factors and disease symptoms Neurology Cincinnati, OH, USA 3University of California, Berkeley, disorder (ADHD) symptoms. Currently, there in a noninvasive manner. As such, blood gene Department of Psychology & Helen is no biological marker for this disorder and its expression could provide accessible biomarkers Wills Neuroscience Institute, CA, USA †Author for correspondence: pathophysiology is poorly understood. Despite for TS subgroups, prognosis, pathophysiology Tel.: +1 916 703 0384 high apparent heritability, sometimes in an and treatment efficacy. [email protected] autosomal dominant fashion with increased Our previous study comparing gene expres- penetrance of tics in males and obsessive com- sion of TS subjects to controls found several pulsive behaviors in females, no dominant gene interesting differences [5]. Notably, a subgroup effect has been identified. The current standard of TS subjects overexpressed natural killer cell for diagnosis is based on the observed presence genes. However, one limitation of this patient of tics, codified in Diagnostic and Statistical sample was medication exposure; patients Manual, Fourth Edition, Text Revision (DSM taking medication were grouped with patients IV-TR) [1] as: that were medication-free. Patients with severe tics and comorbidities are often treated Multiple motor and one or more vocal tics pharmacologically using antipsychotics and Tics that occur daily or intermittently but a2-adrenergic agonists, which act upon dopa- must be present for at least 1 year minergic and adrenergic signaling pathways. We hypothesized that these treatments might Onset before 18 years of age influence gene expression and that the degree to The severity and frequency of tics wax and which this occurrs might be associated with tic wane, tend to peak in adolescence and often severity. Therefore, in this study, we evaluated improve by the end of adolescence or early the gene expression of medicated (MED) and adulthood (for reviews, see [2–4]). unmedicated (UNMED) TS subjects in rela- Our research group has used gene-expression tion to medication status and symptom sever- profiling, through the quantification of RNA ity, specifically focusing on the genes involved levels, in peripheral blood to glean insights in catecholamine signaling and metabolism.

Materials & methods using the Agilent 2100 Bioanalyzer (Agilent Subject recruitment & assessment Technologies Inc., Foster City, CA, USA) and Medicated subjects were recruited from the quantified by fiberoptic spectrophotometry Tourette Syndrome Clinic at Cincinnati using the Nanodrop ND-1000 (Nanodrop Inc., Children’s Hospital Medical Center (Ohio, Wilmington, DE, USA). Samples were excluded

USA). UNMED subjects were recruited via if purified RNA did not have both an A260:A280 the Tourette Syndrome Association, clinical absorbance ratio greater than 2.0 and a 28s:18s referrals, local advertisements, physician refer- rRNA ratio equal to or exceeding 1.8. rals and through the University of California at Davis (CA, USA). UNMED subjects were Gene expression measurement excluded if their estimated IQ was less than Biotin-labeled cDNA was synthesized from 75, if they had taken medication to treat tics, 50 ng of RNA from each sample using the or if they had serious neurological, psychiatric Ovation Whole Blood Solution (NuGEN or medical conditions other than TS, comor- Technologies, Inc., San Carlos, CA, USA) bid ADHD or comorbid obsessive compulsive according to the manufacturer’s protocol. cDNA disorder (OCD). Most UNMED subjects was hybridized with probes on Affymetrix also participated in a functional neuroimag- Human Genome U133 Plus 2.0 microarrays ing study of tic severity and cognitive control (Affymetrix Inc., CA, USA). Microarrays were

conducted by Bunge et al. [6]. UNMED sub- washed and stained on a Fluidics Station 450 jects were medication naive as per parental (Affymetrix) and were scanned on a GeneChip® reports, except for two subjects that had previ- Scanner 3000 (Affymetrix). ously taken atomoxetine (Strattera™; Eli Lilly, IN, USA) to treat ADHD symptoms. One of Data analysis these subjects ended medication approximately Scanned microarray images in the form of 1 month before participation in the study; the Affymetrix CEL files were imported into Partek other stopped taking medication 40 h before Genomics Suite 6.4 (Partek Inc., St Louis, participation. Behavioral measures and princi- MO, USA) using Partek default settings (GC pal component analysis of gene-expression data content adjustment, robust multichip average indicate that these two subjects are not outliers. background correction, log base 2 normaliza- Informed consent was obtained from tion). An analysis of variance using the model each participant, parent or legal guardian. ‘expression = YGTSS’ was performed with age, Recruitment, interview and sample collection gender and scan date (i.e., microarray batch) as proceeded according to established institu- covariates of no interest. tional review board protocols. TS diagnosis was Within Partek, the batch remover algorithm based on Diagnostic and Statistical Manual was used to remove the effects of age, gender of Mental Disorders criteria [1]. Tic severity and batch, and the resulting expression data was assessed via direct child and parent inter- were plotted against tic severity for each gene. view using the Yale Global Tic Severity Scale However, because MED and UNMED micro (YGTSS) [7]. arrays were processed separately, it is not possible to completely remove the batch effect. Sample collection Thus, MED and UNMED data cannot be A total of 15 ml of whole blood was collected compared directly. Accordingly, Pearson cor- from each subject via antecubital fossa venipunc- relation coefficients were chosen as a within- ture into six PAXgene Vacutainer tubes (Qiagen, group metric and calculated separately for Hilden, Germany). These tubes contain a solu- MED and UNMED. For each gene, the cor- tion that immediately lyses all of the cells in relation between gene expression and tic sever- whole blood and stabilizes the RNA without ity was measured, and the associated p-value measurable degradation. The RNA represents of each correlation was calculated (with a null genes expressed in all white blood cells, imma- hypothesis of no correlation, or r: 0). ture red blood cells and immature platelets. To test whether gene expression values Blood samples were frozen at -70°C for storage. and YGTSS were normally distributed, a one-sample Kolmogorov–Smirnov test was RNA isolation from whole blood performed within Partek on each probeset Total RNA was isolated using the PAXgene and on the YGTSS. The null hypothesis was Blood RNA Kit (Qiagen) according to the manu that the observed values were from a normal facturer’s protocol. RNA quality was assessed distribution. Of the UNMED data, 47 of

1734 Pharmacogenomics (2010) 11(12) future science group Blood gene expression correlated with tic severity Research Article

54,683 probesets were not normally distrib- MED uted (p < 0.05), and of the MED data, 23 of 6.61 54,679 probesets were not normally distributed (p < 0.05). Given the null hypothesis, the prob- 6.554 ability of obtaining the observed YGTSS values was p = 0.895 for MED subjects and p = 0.98 6.498 for UNMED subjects. Thus, we cannot reject the null hypothesis that the observed values 6.442 are from a normal distribution for almost all probesets and for the YGTSS at the 0.05 6.386 significance level, indicating that the default Partek import setting of log base 2 is an effec- 6.33 tive normalization for this dataset and that the observed YGTSS values are reasonably DRD2 expression 6.274 normally distributed. To focus on the genes that are most likely to 6.218 be related to tics, and to address the problem of multiple comparisons, only genes that were 6.162 most strongly correlated with tic severity (see Supplementary Tables 1 & 2 at www.futuremedicine. com/doi/suppl/10.2217/pgs.10.160) and known 6.106 to be expressed in either catecholamine-related pathways or neurotransmitter pathways were 6.05 -1 2.2 5.4 8.6 11.8 15 18.2 21.4 24.6 27.8 31 further analyzed. Catecholamine-related path- YGTSS ways were chosen because medications that UNMED act on these pathways are known to decrease 3.84 tics in TS patients [8]. To create a list of neuro transmitter genes, we consulted the Kyoto 3.787 Encyclopedia of Genes and Genomes [9] and all genes from the ‘neuroactive ligand–recep- 3.734 tor interaction’ pathway were used. To create a list of catecholamine-related pathways, we con- 3.681 sulted the Gene Ontology (GO) database [10], and all genes associated with GO terms includ- 3.628 ing the keyword ‘catecholamine’ were used. In addition, to maintain gene symbol format com- 3.575 patibility, only genes from GO using Mouse Genome Informatics accession numbers were DRD2 expression 3.522 used. Combining these two lists, and remov- ing 10 genes not represented on the microar- 3.469 rays, yielded a list of 286 genes (see supplementary Table 3 at www.futuremedicine.com/doi/ 3.416 suppl/10.2217/pgs.10.160). 3.363 Results Subject demographics & medications 3.31 Subject demographics are summarized in 7 10.5 14 17.5 21 24.5 28 31.5 35 38.5 42 YGTSS Supplementary Table 4 (www.futuremedicine.com/ doi/suppl/10.2217/pgs.10.160). The mean age Figure 1. DRD2 expression (y-axis) versus Yale Global Tic Severity Scale of MED and UNMED subjects did not differ (x-axis) for medicated and unmedicated Tourette Syndrome subjects. significantly (11.55 vs 10.69, p < 0.20). The MED: r = +0.58; UNMED: r = -0.028. DRD2: D2 dopamine receptor; MED: Medicated; UNMED: Unmedicated; mean tic severity of MED subjects was sig- YGTSS: Yale Global Tic Severity Scale. nificantly less than that of UNMED subjects (13.4 vs 23.6, p < 0.0001). Both MED and taking each type of medication to treat tics UNMED groups were predominantly male (90 and/or comorbid ADHD/OCD are also listed and 78%, respectively). Numbers of subjects in Supplementary Table 4.

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Table 1. Correlation coefficients for gene expression versus tic severity for selected genes. Symbol Probeset ID Gene title MED r UNMED r DRD2 206590_x_at Dopamine receptor D2 0.58† -0.028 † GABRE 204537_s_at GABAA receptor e 0.097 -0.71 PNMT 206793_at Phenylethanolamine N-methyltransferase -0.24 0.52‡ NPAS4 1554299_at Neuronal PAS domain protein 4 0.51‡ 0.45‡ †p ≤ 0.001. ‡p ≤ 0.05. MED r: Correlation coefficient for medicated subjects; UNMED r: Correlation coefficient for unmedicated subjects.

Gene-expression correlations subjects as a single group regardless of medication with tic severity, stratified by status may have overlooked these genes. Thus, it medication status is important for gene-expression studies of TS to Genes that correlated with tic severity (p < 0.05) account for the effects of medication. This may in MED are listed in Supplementary Table 1. be true for other diseases and medications as well. Similarly, genes that correlated with tic severity (p < 0.05) in UNMED are listed in Supplementary Dopaminergic pathways implicated Table 2. 116 genes were shared between the two in this study lists. Of these 116 genes, 53 were correlated in The observed correlation between DRD2 expres- the same direction in MED and UNMED, and sion and tic severity in subjects taking medica- 63 genes were correlated in opposite directions tion (Figure 1), supports the theory that dopamine in MED and UNMED. neurotransmission is involved in tic production. Both typical and atypical antipsychotics block Catecholamine & dopamine receptors, which induce compensa- neurotransmitter-related genes tory increases in DRD2 mRNA and protein correlated with tic severity expression [11]. We postulate that a correlation is Filtering the list of genes correlated with tic observed in MED subjects because this induced severity using the list of catecholamine or neuro expression occurs in varying amounts, result- transmitter-related genes yielded 26 genes in ing in some subjects overcoming their DRD2 UNMED and 22 genes in MED subjects (see blockade to a greater degree than others. Thus, online supplementary Table 5 at www.futuremedicine. in MED subjects, greater DRD2 expression leads com/doi/suppl/10.2217/pgs.10.160). D2 dopa- to greater activation of the direct pathway or mine receptor (DRD2) gene expression had greater inhibition of the indirect pathway, both significant positive correlation with tic severity of which result in greater tic severity. UNMED (YGTSS) in MED but not UNMED subjects subjects, on the other hand, may not exhibit (Figure 1 & Table 1). By contrast, phenylethanol- an association between DRD2 expression and amine-N-methyltransferase (PNMT) expres- tic severity because DRD2 mRNA levels at the sion had significant positive correlation with time of blood draw do not necessarily reflect the tic severity in UNMED but not MED subjects number of active, pre-existing D2 dopamine

(Figure 2 & Table 1) . GABA A receptor e subunit receptors in the substantia nigra. It might be (GABRE) expression had significant negative possible to clarify this issue by combining our correlation with tic severity in UNMED but study methodology with D2 receptor radioli- not MED subjects (Figure 3 & Table 1). Trend lines gand PET studies. were fitted using linear regression and are shown Decreased GABRE expression correlates in Figures 1, 2 & 3. Correlation coefficients and sig- with increased tic severity in UNMED subjects

(Figure 3) nificance levels for these genes are summarized . GABRE is the e subunit of the GABAA Table 1 in . receptor; GABA A receptors are pentamers, with the most common configuration comprising Discussion two a subunits, two b subunits, and one g sub- Expression of PNMT and GABRE both corre- unit [12]. The e subunit can be incorporated into late with tic severity in UNMED but not MED the receptor instead of the g subunit, resulting subjects. These correlations would not have been in increased sensitivity to GABA and increased found if MED and UNMED subjects had been spontaneous channel opening [13,14]. Thus, one analyzed as a single group. Previous studies exam- would expect decreased expression of GABRE to

ining only MED subjects or examining all TS result in decreased GABA A receptor activation

1736 Pharmacogenomics (2010) 11(12) future science group Blood gene expression correlated with tic severity Research Article in areas where GABRE is expressed, such as in MED dopaminergic neurons in the substantia nigra 6.08 pars compacta (SNc) [15]. We speculate that our results may signal a decrease in GABRE expres- 6.037 sion from normal levels in these neurons. The consequent reduction in inhibitory input would 5.994 yield increased dopamine release into striatum, increasing tic severity. 5.951

Adrenergic pathways implicated in 5.908 this study Expression of PNMT correlates with tic sever- 5.865 ity in UNMED but not MED subjects (Figure 2). PNMT is an enzyme that synthesizes epineph-

PNMT expression 5.822 rine from norepinephrine, and is expressed in medullary projections to the locus ceruleus 5.779 (LC) of rats and monkeys [15,16]. By modulating LC activity, PNMT expression could indirectly 5.736 affect SNc activity via excitatory noradrenergic LC projections to the dopaminergic neurons of the SNc. Although LC projections to the SNc 5.693 are relatively sparse compared with LC projec- 5.65 tions to the ventral tegmental area, some still -1 2.2 5.4 8.6 11.8 15 18.2 21.4 24.6 27.8 31 exist [17]. Therefore, higher expression of PNMT, YGTSS which we found to be correlated with greater tic UNMED severity, could result in increased norepineph- 5.8 rine synthesis in projections to the LC, causing greater activation of the LC and, in turn, greater 5.64 activation of the SNc. This would lead to an increase in motor output, such as tics. 5.48 Expression of GABRE also correlates with tic severity in UNMED but not MED subjects. 5.32 GABRE is expressed in the LC of rats and mon- 5.16 keys [18,19]. Decreased expression of GABRE, which correlates with greater tic severity, would decrease GABAergic inhibition of LC and result 5.0 in greater overall LC activation, possibly influenc- ing tic severity. Alternatively, changes in PNMT 4.84 and GABRE expression could also lead to tics PNMT expression without LC involvement, via direct projections 4.68 from the medulla to the SNc [17,20]. Increased PNMT expression in these neurons would lead 4.52 to greater epinephrine synthesis, causing greater activation of SNc and increased dopamine 4.36 release. Decreased expression of GABRE recep- 4.2 tors localized to these PNMT-rich neurons [15] 7 10.5 14 17.5 21 24.5 28 31.5 35 38.5 42 would also cause them to be more active, leading YGTSS to increased SNc activation and tic exacerbation. Furthermore, since the expression of PNMT Figure 2. PNMT expression (y-axis) versus Yale Global Tic Severity Scale and GABRE correlate with tic severity in (x-axis) for medicated and unmedicated Tourette Syndrome subjects. UNMED but not MED subjects, the relation- MED: r = -0.24; UNMED: r = +0.52. MED: Medicated; PNMT: Phenylethanolamine-N-methyltransferase; ship between expression of these two genes and UNMED: Unmedicated; YGTSS: Yale Global Tic Severity Scale. tic severity may be affected by current medication. As such, PNMT and GABRE may be specific pharmaceutical therapy in the treatment partly responsible for the beneficial effects of of TS and tic disorders, particularly to avoid medication, and could represent novel targets for Parkinsonian side effects and tardive dyskinesia

future science group www.futuremedicine.com 1737 Research Article Liao, Corbett, Gilbert, Bunge & Sharp

MED Neuronal PAS domain protein 4 is 5.02 positively correlated with tic severity in both MED & UNMED 4.977 Neuronal PAS domain protein 4 (NPAS4) was positively correlated with tic severity in both 4.934 MED and UNMED subjects, with a correlation coefficient of r: 0.51 (p < 0.02) in MED subjects, 4.891 and r: 0.45 (p < 0.03) in UNMED subjects (Table 1 & Figure 4). Although NPAS4 was not included in 4.848 the list of catecholamine or neurotransmitter- related genes, the fact that its expression has sig- 4.805 nificant positive correlation with tic severity in both MED and UNMED groups is notable. No 4.762 genes from the list of catecholamine or neuro GABRE expression transmitter-related genes were found to be sig- 4.719 nificantly correlated with MED and UNMED subjects in the same direction. 4.676 Neuronal PAS domain protein 4, a transcrip- tion factor, regulates the expression of several 4.633 activity-dependent genes, which determine the number of GABA-releasing synapses that form on excitatory neurons [23]. GABAergic mecha- 4.59 -1 2.2 5.4 8.6 11.8 15 18.2 21.4 24.6 27.8 31 nisms were implicated in TS in a study that YGTSS found decreased numbers of parvalbumin-posi- UNMED tive, GABAergic interneurons in the caudate and 2.80 putamen of subjects with TS but increased numbers of GABAergic neurons in globus pallidus 2.736 pars interna [24]. Our findings point to altered NPAS4 expression as one possible explanation 2.672 for these changes in numbers of GABAergic neurons in TS. In addition, NPAS4 was positively 2.608 correlated with tic severity regardless of whether subjects were taking medications or not, indicat- 2.544 ing that these medications seemed not to affect the relationship between NPAS4 expression and 2.48 tic severity. However, NPAS4 may not be a good candidate for pharmaceutical therapy, given 2.416 that its regulatory effects on the formation of GABA-releasing synapses most likely occur early GABRE expression 2.352 in development, before TS diagnosis is possible.

2.288 Other genes implicated in this study Three catecholamine or neurotransmitter-related 2.224 genes were significantly correlated with tic severity in both MED and UNMED groups: GABRB3, 2.16 GRIN1 and SSTR5. Interestingly, all three of 7 10.5 14 17.5 21 24.5 28 31.5 35 38.5 42 these genes were correlated in opposite directions YGTSS in MED and UNMED groups: GABRB3 and Figure 3. GABRE expression (y-axis) versus Yale Global Tic Severity Scale GRIN1 were positively correlated with tic sever- (x-axis) for medicated and unmedicated Tourette Syndrome subjects. ity in MED subjects and negatively correlated in MED: r = +0.097; UNMED: r = -0.71. UNMED subjects, while SSTR5 was negatively GABRE: GABA receptor e subunit; MED: Medicated; UNMED: Unmedicated; A correlated with tic severity in MED and positively YGTSS: Yale Global Tic Severity Scale. correlated in UNMED subjects. No catechol- associated with typical antipsychotics [21] and amine or neurotransmitter-related genes were sig- abnormalities in glucose metabolism associated nificantly correlated with tic severity in the same with atypical antipsychotics [22]. direction in both MED and UNMED subjects.

1738 Pharmacogenomics (2010) 11(12) future science group Blood gene expression correlated with tic severity Research Article

GABRB3 is linked to Angelman syndrome [25] MED 4.89 and ADHD [26]. GRIN1 is associated with Parkinson’s disease, as well as other brain dis- 4.835 orders [27]. SSTR5 regulates the proliferation of many cell types [28]. The observation that these genes are correlated with tic severity in opposite 4.78 directions in MED and UNMED, indicates that medications affect the relationship between their 4.725 expression and tic severity. However, precisely how this occurs remains to be elucidated. 4.67

Conclusion 4.615 The predominant theory of TS pathophysiology, and other movement disorders, posits an imbal- 4.56 NPAS4 expression NPAS4 ance between the excitatory direct pathway and the inhibitory indirect pathway from the stria- 4.505 tum through the basal ganglia [29]. An overactive direct pathway or an underactive indirect path- 4.45 way would result in increased psychomotor activity. The SNc provides dopaminergic input to the 4.395 striatum, regulating both the direct pathway through DRD1 and the indirect pathway through 4.34 DRD2. Based on the findings in this study, we -1 2.2 5.4 8.6 11.8 15 18.2 21.4 24.6 27.8 31 propose that altered expression of GABRE and UNMED YGTSS PNMT may play a role in increased activation 2.89 of the SNc in TS. Specifically, increasedPNMT expression in adrenergic projections from the 2.831 medulla to the LC would result in greater input to the LC, while decreased expression of GABRE 2.772 in the LC would result in greater sensitivity to this input. As a result, the LC would become 2.713 more activated, providing greater noradrenergic input to the SNc. This would lead to the hypoth- 2.654 esized imbalance between the direct and indirect pathways, manifesting as an increase in motor 2.595 actions, such as tics.

2.536 Future perspective

To further validate the expression values of expression NPAS4 2.477 genes in this study, quantitative real-time PCR (qRT-PCR) can be performed using probes 2.418 for genes of interest. The drawbacks of qRT- PCR are that it is expensive, time-consuming, 2.359 low-throughput and genes of interest must be selected a priori (by contrast, microarray studies 2.30 assess all genes simultaneously). In addition, one 7 10.5 14 17.5 21 24.5 28 31.5 35 38.5 42 recent paper has shown that appropriate preproc- YGTSS essing techniques can increase the correlation between microarray and qRT-PCR results [30]. Figure 4. NPAS4 expression (y-axis) versus Yale Global Tic Severity Scale Gene expression and tic severity were meas- (x-axis) for medicated and unmedicated Tourette Syndrome subjects. ured at only one time point per subject in this MED: r = +0.51; UNMED: r = +0.45. study. Future studies could assess whether tics MED: Medicated; NPAS4: Neuronal PAS domain protein 4; UNMED: Unmedicated; YGTSS: Yale Global Tic Severity Scale. vary in severity in proportion to gene-expression levels over time. Moreover, different types of tics Patients in the MED group were taking might be associated with different changes in medications of different types. However, fur- gene expression. ther dividing the MED group into subgroups

future science group www.futuremedicine.com 1739 Research Article Liao, Corbett, Gilbert, Bunge & Sharp

based on medication type resulted in sample Gregg for processing the microarrays. We also thank Dr Lisa sizes too small for meaningful analysis. Future Lit and Dr Boryana Stamova for help in the early stages of studies interested in the effects of specific medi- this research. cations could restrict participants to those using medications of interest. Financial & competing interests disclosure Similarly, comorbidities commonly associated Support for this study was provided by the Tourette Syndrome with TS – namely ADHD and OCD – were Association; the MIND Institute at UC Davis; a John Merck present in the patient population but were Scholarship in the Biology of Developmental Disabilities (SA not explicitly investigated due to sample size. Bunge); and a gift from Ron and Darin Mittelstaedt via the Future studies could investigate the effects of RDM Positive Impact Foundation. The authors have no other medication on ADHD and OCD in isolation relevant affiliations or financial involvement with any organi to determine whether the gene-expression cor- zation or entity with a financial interest in or financial con relations seen here are unique to TS or if they flict with the subject matter or materials discussed in the also correlate with measures of ADHD or OCD manuscript apart from those disclosed. severity (Conners’ Rating Scale and Children’s No writing assistance was utilized in the production of Yale–Brown Obsessive–Compulsive Scale, this manuscript. respectively). In addition, age and gender were selected as covariates of no interest and further Ethical conduct of research studies could examine these factors explicitly. The authors state that they have obtained appropriate insti tutional review board approval or have followed the princi Acknowledgements ples outlined in the Declaration of Helsinki for all human The authors would like to thank Carol L Baym, Samantha or animal experimental investigations. In addition, for B Wright and Debra Galik for subject recruitment and data investi gations involving human subjects, informed consent collection. The authors thank Ryan R Davis and Jeffrey P has been obtained from the participants involved.

Executive summary Tourette Syndrome (TS) is a movement disorder most likely caused by both genetic and environmental factors. Gene expression measures the regulation of genes in response to the environment. A better understanding of how gene expression is affected by TS medications could identify new drug targets and contribute to our understanding of TS. Materials & methods To assess tic severity, the Yale Global Tic Severity Scale was used in a behavioral assessment of 20 medicated (MED) TS subjects and 23 unmedicated (UNMED) TS subjects. To measure gene expression, RNA from all subjects was purified, reverse transcribed and hybridized to Affymetrix microarrays. Gene-expression data was analyzed using GeneSpring® and Partek® software. Results D2 dopamine receptor expression correlated positively with tic severity in MED but not UNMED subjects.

GABAA receptor e subunit expression negatively correlated with tic severity in UNMED but not MED subjects. Phenylethanolamine N‑methyltransferase expression positively correlated with tic severity in UNMED but not MED subjects. Conclusion Modulation of tics by TS medication is associated with changes in dopamine, norepinephrine and GABA pathways.

4 Singer HS: Tourette’s syndrome: from 7 Leckman JF, Riddle MA, Hardin MT Bibliography behaviour to biology. Lancet Neurol. 4, et al.: The Yale Global Tic Severity Scale: 1 American Psychiatric Association: Diagnostic 149–159 (2005). initial testing of a clinician-rated scale of and Statistical Manual of Mental Disorders: 5 Lit L, Gilbert DL, Walker W, Sharp FRA: tic severity. J. Am. Acad. Child Adolesc. (Revised 4th Edition). Washington, DC, USA, Subgroup of Tourette’s patients overexpress Psychiatry 28, 566–573 (1989). 111–114 (2000). specific natural killer cell genes in blood: a 8 Srour M, Lespérance P, Richer F, 2 Swain JE, Scahill L, Lombroso PJ, King RA, preliminary report. Am. J. Med. Genet. B. Chouinard S: Psychopharmacology of tic Leckman JF: Tourette syndrome and tic Neuropsychiatr. Genet. 144B, 958–963 disorders. J. Can. Acad. Child Adolesc. disorders: a decade of progress. J. Am. Acad. (2007). Psychiatry 17, 150–159 (2008). Child Adolesc. Psychiatry 46, 947–968 (2007). 6 Baym CL, Corbett BA, Wright SB, 9 Ogata H, Susumu G, Kazushige S et al.: 3 Müller N: Tourette’s syndrome: clinical Bunge SA: Neural correlates of tic severity KEGG: Kyoto Encyclopedia of Genes and features, pathophysiology, and therapeutic and cognitive control in children with Genomes. Nucleic. Acids Res. 27, 29–34 approaches. Dialogues Clin. Neurosci. 9, Tourette syndrome. Brain 131, 165–179 (1999). 161–171 (2007). (2008).

1740 Pharmacogenomics (2010) 11(12) future science group Blood gene expression correlated with tic severity Research Article

10 Blake JA, Harris MA: The Gene 17 Mejías-Aponte CA, Drouin C, 24 Kalanithi PSA Zheng W, Kataoka Y et al.: Ontology project: structured vocabularies for Aston-Jones G: Adrenergic and Altered parvalbumin-positive neuron molecular biology and their application to noradrenergic innervation of the distribution in basal ganglia of individuals genome and expression analysis. midbrain ventral tegmental area and with Tourette syndrome. Proc. Natl. Acad. Curr. Protoc. Bioinformatics Chapter 7, retrorubral field: prominent inputs from Sci. USA 102, 13307–13312 (2005). Unit 7.2 (2002). medullary homeostatic centers. 25 Dan B, Boyd SG: Angelman syndrome 11 Sands S, Dickerson D, Morris S, Chronwall B: J. Neurosci. 29, 3613–3626 (2009). reviewed from a neurophysiological Dopamine D2 receptor stimulation alters 18 Moragues N, Ciofi P, Tramu G, Garret M: perspective. The UBE3A-GABRB3 G-protein expression in rat pituitary Localisation of GABA(A) receptor e-subunit in hypothesis. Neuropediatrics 34, 169–176 intermediate lobe melanotropes. Endocrine 6, cholinergic and aminergic neurones and (2003). 325–333 (1997). evidence for codistribution with the q-subunit 26 Comings DE: Clinical and molecular genetics 12 Dunn SM, Bateson AN, Martin IL: Molecular in rat brain. Neuroscience 111, 657–669 (2002). of ADHD and Tourette syndrome. Two neurobiology of the GABAA receptor. Int. 19 Sinkkonen ST, Hanna MC, Kirkness EF, related polygenic disorders. Ann. N. Y. Acad.

Rev. Neurobiol. 36, 51–96 (1994). Korpi ER: GABA A receptor e and q subunits Sci 931, 50–83 (2001). 13 Wagner DA, Goldschen-Ohm MP, display unusual structural variation between 27 Wu S, Wang WF, Shyu HY et al.: Association Hales TG, Jones MV: Kinetics and species and are enriched in the rat locus analysis of GRIN1 and GRIN2B spontaneous open probability conferred by the ceruleus. J. Neurosci. 20, 3588–3595 (2000). polymorphisms and Parkinson’s disease in a

{e} subunit of the GABA A receptor. 20 Nagatsu I, Ikemoto K, Takeuchi T et al.: hospital-based case–control study. Neurosci. J. Neurosci. 25, 10462–10468 (2005). Phenylethanolamine-N-methyltransferase Lett. 478, 61–65 (2010). 14 Ranna M, Sinkkonen ST, Möykkynen T, – immunoreactive nerve terminals afferent to 28 Cordelier P, Estève JP, Najib S et al.: Uusi-Oukari M, Korpi ER: Impact of e and q the mouse substantia nigra. Neuroscience Regulation of neuronal nitric-oxide synthase subunits on pharmacological properties of Letters 245, 41–44 (1998). activity by somatostatin analogs following a3b1 GABAA receptors expressed in Xenopus 21 Keck PE, McElroy SL, Strakowski SM, SST5 somatostatin receptor activation. J. Biol. oocytes. BMC Pharmacol. 6, Soutullo CA: Antipsychotics in the treatment Chem. 281, 19156–19171 (2006). 1 (2006). of mood disorders and risk of tardive 29 Kulisevsky J, Litvan I, Berthier ML et al.: 15 Carlton SM, Honda CN, Denoroy L: dyskinesia. J. Clin. Psychiatry 61(Suppl. 4), Neuropsychiatric assessment of Gilles de la Distribution of phenylethanolamine 33–38 (2000). Tourette patients: comparative study with N-methyltransferase cell bodies, axons, and 22 Elias AN, Hofflich H: Abnormalities in other hyperkinetic and hypokinetic terminals in monkey brainstem: an glucose metabolism in patients with movement disorders. Mov. Disord. 16, immunohistochemical mapping study. schizophrenia treated with atypical 1098–1104 (2001). J. Comp. Neurol. 287, 273–285 (1989). antipsychotic medications. Am. J. Med. 121, 30 Mieczkowski J, Tyburczy M, Dabrowski M, 16 Milner TA, Abate C, Reis DJ, Pickel VM: 98–104 (2008). Pokarowski P: Probe set filtering increases Ultrastructural localization of 23 Lin Y, Bloodgood BL, Hauser JL et al.: correlation between Affymetrix GeneChip phenylethanolamine N-methyltransferase-like Activity-dependent regulation of inhibitory and qRT-PCR expression measurements. immunoreactivity in the rat locus coeruleus. synapse development by NPAS4. Nature 455, BMC Bioinformatics 11, 104 (2010). Brain Res. 478, 1–15 (1989). 1198–1204 (2008).

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