Neurochemistry International 124 (2019) 152–161

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Neurochemistry International

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GAD1 polymorphisms are associated with bipolar I disorder and with blood homovanillic acid levels but not with plasma GABA levels T

∗ Aurora Arrúea,b, , Miguel Angel González-Torresb,c,d, Nieves Basterrecheb,d,e, Ainara Arnaizb,f, Olga Olivasb,g, M. Isabel Zamalloaa,b, Leire Erkorekab,d,h, Ana Catalánb,c,d, Mercedes Zumárragaa,b a Departamento de Investigación Neuroquímica, Hospital de Zamudio, Red de Salud Mental de Bizkaia, Zamudio, Spain b BioCruces Health Research Institute, Barakaldo, Spain c Servicio de Psiquiatría, Hospital Universitario Basurto, Bilbao, Spain d Departamento de Neurociencias, University of the Basque Country (UPV/EHU), Leioa, Spain e Unidad de Hospitalización de Corta Estancia, Hospital de Zamudio, Red de Salud Mental de Bizkaia, Zamudio, Spain f Servicio de Rehabilitación, Hospital de Zamudio, Red de Salud Mental de Bizkaia, Zamudio, Spain g Centro de Salud Mental de Gernika, Red de Salud Mental de Bizkaia, Gernika, Spain h Centro de Salud Mental Barakaldo, Red de Salud Mental de Bizkaia, Barakaldo, Spain

ARTICLE INFO

Keywords: 1 (GAD1) Catechol-O-Methyltransferase (COMT) Plasma homovanillic acid Plasma gamma-aminobutyric acid

1. Introduction 1.1. The GABA system

Schizophrenia (SZ) and bipolar disorder (BD) are serious and per- GABA is the main inhibitory of the central nervous sistent mental illnesses affecting 1% and 1–2% of the population, re- system. Postmortem brain studies of individuals with SZ and BD have spectively; both have unknown causes and great clinical heterogeneity. found low levels of numerous mRNAs and proteins, many of them as- Among other neurotransmission systems, brain gamma-aminobutyric sociated with the GABA system (Hashimoto et al., 2003; Torrey et al., acid (GABA) system dysfunction seems to be involved in their physio- 2005; Volk et al., 2016). pathology (Curley et al., 2013; Guidotti et al., 2000; Torrey et al., It is known that GABA is synthetized from glutamate through the 2005). Family genetic studies and genome-wide association studies limiting GAD. There are two isoforms of this enzyme, GAD65 have shown that both conditions are strongly influenced by genetic and and GAD67, encoded by independently regulated (GAD2 and environmental factors (Craddock et al., 2010; van Haren et al., 2012) GAD1), that differ in their binding to the membrane and intracellular but have also suggested that they share some high-risk genes (Gurung distribution. GAD65 is located in synaptic terminals and appears to play and Prata, 2015). In relation to this, both SZ and BD have been asso- a significant role under high rates of synaptic activity (Tian et al., ciated with glutamate decarboxylase (GAD) gene polymorphisms 1999), while GAD67 is found throughout the cell (Martin and Rimvall, (Addington et al. 2005; Chung et al., 2017; Du et al., 2008; Geller et al., 1993) and is responsible for most of the synthesis of GABA (Asada et al., 2008; Lundorf et al., 2005; Ripke, 2011; Straub et al., 2007; Zhao et al., 1997; Chattopadhyaya et al., 2007). Although the two isoenzymes are 2007). coexpressed in all brain regions, a great proportion of with the highest levels of GAD1 expression are found in the cerebellum and

Abbreviations: Schizophrenia, (SZ); bipolar I disorder, (BD I); gamma-aminobutyric acid, (GABA); homovanillic acid, (HVA); plasma, (p); glutamate decarboxylase, (GAD); catechol-O-methyltransferase, (COMT); single nucleotide polymorphisms, (SNP); valine, (Val); methionine, (Met); linkage disequilibrium, (LD) ∗ Corresponding author. Departamento de Investigación Neuroquímica, Hospital de Zamudio, Red de Salud Mental de Bizkaia, Arteaga Auzoa nº45, 48170, Zamudio. Bizkaia, Spain. E-mail address: [email protected] (A. Arrúe). https://doi.org/10.1016/j.neuint.2019.01.004 Received 25 September 2018; Received in revised form 18 December 2018; Accepted 4 January 2019 Available online 06 January 2019 0197-0186/ © 2019 Elsevier Ltd. All rights reserved. A. Arrúe et al. Neurochemistry International 124 (2019) 152–161 cortex (Uhlén et al., 2015). Low levels of GAD1 in the have reported lower pGABA levels in individuals with BD and SZ than prefrontal cortex and hippocampus is one of the findings most com- healthy individuals. Other studies have used pGABA levels as a marker monly reported in the literature on patients with SZ and BD (Guidotti in depression and post-traumatic stress disorder (Pan et al., 2018; Schür et al., 2000; Hashimoto et al., 2003; Torrey et al., 2005; Volk et al., et al., 2016). In relation to dopaminergic neurotransmission, pHVA 2016). levels have been associated with symptom severity and treatment re- Regarding the GAD1 gene, single nucleotide polymorphisms (SNPs) sponse in patients with SZ (Davila et al., 1988) and BD (Basterreche and haplotypes formed by various of these SNPs have been associated et al., 2008; Zumárraga et al., 2009). Further, individuals who were with a higher risk of BD (Chung et al., 2017; Geller et al., 2008; Lundorf homozygous for the ValVal rs4680 SNP of the COMT gene tended to et al., 2005) and SZ, as well as with cognitive phenotypes related to SZ, have higher pHVA levels than MetMet carriers, while ValMet hetero- though various studies have produced different results (Addington zygous individuals had intermediate levels (Zumárraga et al., 2010). et al., 2005; Du et al., 2008; Kirenskaya et al., 2018; Straub et al., 2007; Zhao et al., 2007). The rs3749034 SNP that consists of a G to A base 1.4. Scope of the study change has been the most widely investigated. Straub et al. (2007),inin silico studies, observed that this SNP is localised within the putative Based on evidence of the role of GABA in SZ and BD and given the region for GAD67 transcription and that DNA carrying the G great variability in results obtained from genetic association studies, the allele lacks binding sites for two transcription factors, which are present primary objective of our study was to assess potential associations of in DNA carrying the A allele, this potentially decreasing expression of three GAD1 SNPs and the haplotypes formed by these polymorphisms the GAD1 gene. The G allele of rs3749034 has been associated with an with SZ and BD, comparing with those in healthy controls. Based on the abnormally thin parahippocampal cortex in healthy volunteers (Brauns lower GAD1 mRNA levels found in patients and the potential func- et al., 2013) and low enzyme mRNA levels in the prefrontal cortex and tionality of the genetic variants, the secondary objectives were to assess hippocampus of patients with SZ (Straub et al., 2007). On the other the relationship of pGABA levels with SNPs or GAD1 haplotypes in all hand, assessing the in vivo GABA system activity in the brain cortex of the participants as well any differences by diagnosis. Thirdly, we as- healthy volunteers, it was found that some GAD1 SNPs, rs1978340 and sessed the relationship between the GABAergic and dopaminergic sys- rs769404, with unknown functionality but associated with a high risk tems, by analysing: the relationship of pHVA levels with GAD1 genetic of SZ and BD, modulated GABA levels in this region of the brain variants and haplotypes; the relationship of pGABA levels with COMT (Marenco et al., 2010). rs4680; and lastly, the influence of interaction between the two genes on pGABA and pHVA levels in all three groups. The final goal of this 1.2. Interaction between GABA and dopaminergic systems. Effect of work was to assess whether these biological markers could be helpful catechol-O-methyltransferase (COMT) for characterizing diagnostic groups.

Though not the focus of our work, abnormal dopaminergic function 2. Materials and methods has been linked with the aetiology and physiopathology of SZ and BD in numerous studies. There is a complex interaction between the GABA 2.1. Patients and controls and dopaminergic systems. Animal-based studies have found that GABAergic interneurons have a connection with dopaminergic axons in The sample was composed of 310 healthy controls, 290 patients the prefrontal cortex (Benes et al., 1993; Seo et al., 2016) and that the diagnosed with bipolar I disorder (BD I) and 303 diagnosed with SZ manipulation of GABA at the subcortical level inhibits the activity and according to DSM-IV-TR criteria. All participants, patients and controls, the concentration of in this and other brain regions (Schiffer were given detailed information about the study and signed an in- et al., 2000). Further, in rats, it has been found that the dopaminergic formed consent form prior to participation. The research was approved system activates or inhibits GABAergic activity depending on whether it by the Ethics Committee of the Basque Health Service and conducted in acts on dopaminergic receptors D1 or D2 in the prefrontal cortex line with the principles of the Declaration of Helsinki. At the time of the (Grobin and Deutch, 1998; Tseng and O’Donnell, 2004). On the other interview, data were collected from all participants concerning their hand, COMT is one of the involved in the catabolism of do- place of birth and the place of birth of their parents and grandparents. pamine, acting at the extraneuronal level. Its role is particularly im- The patients were Caucasian, of either sex, and between 18 and 64 portant in the prefrontal cortex where dopamine transporter is virtually years old. They were selected from patients who attended centres of the nonexistent, and hence, dopamine is not inactivated by re-uptake. This Mental Health Network of Biscay and Basurto University Hospital of the enzyme can indirectly regulate GABAergic activity. The functional Basque Health Service. The BD I group was composed of 159 women COMT SNP rs4680 consists of a G to A base change, producing a change and 131 men, with a mean age of 44 (SD 14) years, and the SZ group of of valine (Val) amino acid to a methionine (Met) in the protein, re- 102 women and 201 men with a mean age of 38 (SD 11) years. We sulting in a reduction in the enzyme activity (Lachman et al., 1996). excluded patients with severe organic disease, and any women who Enzyme activity is higher in ValVal carriers than heterozygous in- were pregnant or breastfeeding. dividuals (ValMet), who in turn showed higher activity than MetMet The control group was composed of 141 women and 169 men, of carriers. Caucasian origin, with a mean age of 40 (SD 11) years. These were There is currently growing interest in studies based on gene-gene recruited from healthcare staff at their yearly check-up. Candidate interaction models in SZ and BD. The interaction between GAD1 and participants were interviewed to rule out a personal or family history of COMT polymorphisms has been reported to have an effect on certain serious mental illness. cognitive phenotypes of SZ (Straub et al., 2007), as well as on GABA To study the relationship of GAD1 SNPs and haplotypes with the levels in the cingulate cortex and parahippocampal cortex thickness in baseline pGABA and pHVA levels, we selected 161 inpatients from the healthy volunteers (Brauns et al., 2013; Marenco et al., 2010). initial sample who at the time of admission were under acute decom- pensation and had not received any previous antipsychotic treatment or 1.3. Plasma measurements mood stabilisers for at least 8 days: 74 patients with BD I (63% women with a mean age of 35 ± 10 years) and 87 patients with SZ (40% Plasma levels of GABA (pGABA) and of the main metabolite of women with a mean age of 36 ± 11 years). The mean washout period dopamine, homovanillic acid (pHVA), have been used as a measure of (SD) was 89.6 weeks (96.0) in patients with SZ and 44.9 (75.1) in pa- central activity (Davidson et al., 1987; Konicki et al., 1991; Petty, 1994; tients with BD I. All patients were on a standard hospital diet. For the Petty et al., 1993, 1987). Regarding the GABA system, some studies control group, we randomly selected 164 individuals taking into

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Fig. 1. Schematic representation of 2 and the locations of the GAD1 gene and the SNPs studied. account sex and age, to reach a sample size comparable that of the cases, we used the most likely ones. Haplotypes with frequencies of less patient group (resulting in a group that was 54% women and had a than 3% were removed from the analysis. mean age of 36 ± 11 years). Subsequently, the association between haplotypes and the study groups was assessed with the chi-square test, and odds ratios (ORs) with 2.2. Blood samples corresponding 95% confidence intervals (95% CIs) were calculated with logistic regression using PLINK. To measure the plasma levels of GABA and HVA, a blood sample Analysis of variance was used to assess the effect of the genotypes of was extracted from untreated patients and selected controls at between each GAD1 SNP on pGABA and pHVA levels as well as the relationship 8 and 8:30 a.m., after 12 h of fasting and 30 min of rest. One aliquot of between the rs4680 COMT variants and pGABA levels. Then, the as- blood was used for obtaining the DNA, and the rest was centrifuged and sociations were assessed with Student's t-test considering the least fre- plasma was stored in aliquots under liquid nitrogen until analysis. A quent allele to be dominant. The effect of haplotypes on baseline single blood sample was taken from the other patients and controls for pGABA and pHVA levels was examined with linear regression analysis, obtaining DNA. using PLINK. To study whether the effects differed by diagnosis, ana- lysis of covariance was performed, in which diagnosis was included as a 2.3. Selection and assessment of single nucleotide polymorphisms factor. To assess the effect of the interaction between the two genes on We studied three SNPs of the GAD1 gene, namely, rs1978340, pGABA and pHVA, a general linear model was constructed. For this, rs3749034 and rs769404 (also known as rs11542313 or rs16823181) pHVA and pGABA levels were taken as the dependent variables and and the haplotypes they form. These SNPs are located in the promoter GAD1 and COMT SNPs (considering the least frequent allele to be region, and in the 5′UTR of exon 1 and exon 3 of the gene, respectively dominant), GAD1 haplotypes (presence/absence of thereof) and the (Fig. 1). They are in strong LD: rs1978340 with rs3749034 (LD = 0.95, interactions between them as the independent variables. Diagnosis was ff r2 = 0.122) and with rs769404, (LD = 0.97, r2 = 0.283), and included in the model to assess the e ect of the interaction in each of ff rs3749034 with rs769404 (LD = 0.95, r2 = 0.179). With regards to the three groups. The e ects of sex and age were explored. All the COMT, we assessed the functional rs4680 SNP, located in exon 3. analyses were performed using the Statgraphics Plus program. DNA was extracted from blood using a commercial kit. The SNPs of fi fi the GAD1 gene were identi ed using real-time PCR with speci c 3. Results fluorescent probes for each allele. In the case of the COMT rs4680, we ® used TaqMan Drug Genotyping Assays from Thermo 3.1. Association of the diagnoses with GAD1 SNPs and haplotypes Fisher Scientific. Further details are given in Supplementary Table 1. All the SNPs passed the Hardy-Weinberg test. Table 1 summarises 2.4. Neurochemical methods the allele frequencies and genotypes of GAD1 SNPs together with the analysis of the associations in SZ and BD I patients and the control We measured pGABA levels using high performance liquid chro- group. The distribution of the rs3749034 significantly differed between matography (HPLC) with fluorescence detection with small modifica- BD I and controls. Specifically, the proportions of individuals carrying tions of the method described by Arrúe et al. (2010), while pHVA was the G allele and homozygous (GG) carriers were higher among patients measured using HPLC with coulometric detection following previously with BD I than among healthy controls (G allele: OR = 1.44, 95% reported methods (Zumárraga et al., 1987). CI = 1.09–1.80, p = 0.0109; homozygous GG: OR = 1.43, 95% CI = 1.03–2.00, p = 0.0327). Further, the difference in distribution of 2.5. Statistical analysis rs3749034 alleles between patients with SZ and healthy controls was nearly significant (χ2 = 2.90, p = 0.0887) (Table 1). For the rest of the The association between the genotypes or alleles of GAD1 was as- SNPs (alleles and genotypes), no significant associations were found sessed separately for each condition, with chi-square or Fisher's exact with either BD I or SZ. tests. Haplotype estimation was carried out using the program PLINK In our sample, the A allele of rs3749034 is in phase with G allele of (Purcell, n.d.). When there is more than one heterozygote for SNPs that rs1978340 (LD = 1). Individuals carrying the allele A also carry the GA constitute a haplotype, our approach does not allow us to ascertain with haplotype rs1978340-rs3749034 and the GAT haplotype rs1978340- certainty which haplotypes are present in a given individual; in these rs3749034-rs769404. For this reason, from hereon we will refer to

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Table 1 3.2. Association of pGABA levels with GAD1 SNPs and haplotypes. Description and frequency distribution of the alleles and genotypes of GAD1 in Differences by diagnosis patients with bipolar I disorder, schizophrenia and controls.

SNP BD I Controls SZ Supplementary Table 2 shows the results of assessing the associa- tions of pGABA levels and GAD1 genotypes and whether the associa- N = 290 N = 310 N = 303 tions differed by diagnosis. The pGABA levels were not significantly affected by GAD1 genotypes of rs1978340, rs3749034 or rs769404 and rs1978340 n (%) n (%) n (%) nor were these levels affected by any interactions between the geno- A 201 (35) 210 (34) 208 (34) types and the diagnosis. G 377 (65) 410 (66) 398 (66) Analysing the haplotypes, no association was found between pGABA AA 33 (11) 34 (11) 29 (10) levels and the haplotypes formed by rs1978340-rs3749034 and by AG 135 (47) 142 (46) 150 (49) ff GG 121 (42) 134 (43) 24 (41) rs1978340-rs3749034-rs769404. Further, no e ect of diagnosis was found (data not shown). rs3749034a The levels of pGABA were higher in controls than in patients with A 107 (18) 152 (25) 123 (20) BD I and SZ (F = 18.62, df =2, p < 0.001). G 473 (82) 468 (75) 483 (80) AA 9 (3) 21 (7) 12 (4) AG 89 (31) 110 (35) 99 (33) GG 192 (66) 179 (58) 192 (64) 3.3. Effect of the COMT rs4680 genotype on pGABA level and GAD1 genotype and haplotype on pHVA level rs769404 C 243 (42) 250 (35) 264 (44) fi ff T 335 (58) 370 (65) 340 (55) We did not nd any e ects of the genotypes of the COMT SNP CC 50 (17) 44 (14) 55 (18) rs4680 on pGABA levels or of the GAD1 genotypes on pHVA levels CT 143 (50) 162 (53) 154 (51) (Supplementary Table 2). When we analysed the data, classifying TT 96 (33) 104 (33) 93 (31) genotypes by presence of the least frequent allele (Supplementary Fig. 1), we found that the GAD1 SNP rs3749034 had a nominally sig- BD I: bipolar I disorder; SZ: schizophrenia; SNP: single-nucleotide poly- fi ff morphism, N: number of patients, n: frequency of the alleles and genotypes in ni cant e ect on pHVA levels (Supplementary Fig. 1A). On the other fi ff each diagnostic group. hand, there were no signi cant di erences in pGABA levels between BD I vs Controls. homozygous ValVal and MetMet+ValMet carriers of rs4680 rs1978340: allele: χ2 = 0.07, p = 0.7883. Genotype: χ2 = 0.12, p = 0.9425. (Supplementary Fig. 1B) or interactions with diagnosis (Supplementary rs3749034: allele: χ2 = 6.16, p = 0.013. Genotype: χ2 = 6.81, p = 0.0332. Fig. 2). rs769404: allele: χ2 = 0.30, p = 0.5855. Genotype: χ2 = 1.15, p = 0.5622. Assessing the relationship of pHVA with GAD1 haplotypes by means SZ vs Controls. of linear regression analysis (Table 3), pHVA levels were found to be χ2 χ2 rs1978340: allele: = 0.11, p = 0.9149. Genotype: = 0.09, p = 0.9149. lower in A allele carriers (GA haplotype rs1978340-rs3749034) than χ2 χ2 rs3749034: allele: = 0.90, p = 0.0887. Genotype: = 3.41, p = 0.1818. non-carriers (presence of GA; pHVA = 12.9 ng/ml, 95% rs769404: allele: χ2 = 1.15, p = 0.2534. Genotype: χ2 = 1.93, p = 0.3801. CI = 11.9–13.9, n = 127 and absence of GA: pHVA = 14.3 ng/ml, 95% a Significant association. CI = 13.4–15.1, n = 127; Student's t-test = 2.14, p = 0.0330). No sig- nificant effect on the pHVA levels was found arising from interactions carriers of the A allele of rs3749034. between the rest of the GAD1 haplotypes evaluated and diagnosis (data Table 2 shows the associations between the two GAD1 gene hap- not shown). The effect of the diagnosis on pHVA levels was confirmed, lotypes studied and the three groups. Comparing BD I and healthy levels being higher in patients with SZ than in healthy controls or pa- controls, we found fewer patients with BD I among carriers of the tients with BD I (F = 4.16, df = 2, p = 0.0182). The general linear haplotypes with the A allele of rs3749034 (GA and GAT) and more model did not yield significant results with the inclusion of genotypes, patients with BD I among carriers of GG in rs1978340-rs3749034 and GAD1 haplotypes or diagnosis either for pGABA or pHVA (results not GGT in rs1978340-rs3749034-rs769404. Regarding patients with SZ shown). compared to healthy controls, the association with the haplotypes containing the A allele of rs3749034 nearly reached significance, with the same trend as with BD I.

Table 2 Distribution of the GAD1 haplotypes in the three study groups. Results of analysing the association with BD I or SZ with respect to the control group.

GAD1 BD I Controls SZ BD I SZ

Haplotypes n (%) n (%) n (%) χ2 p OR 95% CI p χ2 p OR 95% CI p

8.36 0.0150a 4.37 0.1155 AG 196 (34) 210 (34) 203 (34) 0.02 0.878 1.01 0.79–1.28 0.9410 0.01 0.9700 0.99 0.78–1.25 0.9260 rs1978340 GA 103 (18) 152 (24) 120 (20) 7.5 0.0062a 0.67 0.51–0.89 0.0055a 3.65 0.0561 0.76 0.58–1.00 0.0516 rs3749034 GG 273 (48) 249 (42) 278 (46) 4.42 0.035a 1.26 1.00–1.58 0.0450a 2.49 0.1041 1.21 0.92–2.97 0.1140

21.29 < 0.001a 8.906 0.0635 AGT 182 (32) 202 (33) 189 (32) 0.12 0.7374 0.95 0.75–1.22 0.7080 0.19 0.6595 0.94 0.74–1.20 0.6180 rs1978340 GAT 103 (18) 149 (24) 118 (20) 6.8 0.0091a 0.69 0.52–0.91 0.0092a 3.55 0.0596 0.77 0.58–1.00 0.0560 rs3749034 GGC 228 (40) 240 (39) 246 (42) 0.12 0.7239 1.04 0.82–1.31 0.7440 0.82 0.3648 1.11 0.88–1.39 0.3750 rs769404 GGT 45 (8) 19 (3) 30 (5) 13.37 0.0003a 2.68 1.55–4.64 0.0009a 2.92 0.4375 1.65 0.92–2.97 0.1080

(1)-Only haplotypes with a frequency above 3% are shown. n is the frequency of each haplotype (with the percentage in brackets). BD I: bipolar I disorder; SZ: schizophrenia; OR: odds ratio, CI: confidence interval. a Results of the association analysis that are statistically significant (p < 0.05).

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Table 3 carried out a haplotype analysis, they observed that rs1978340, Results of the linear regression analysis between the pHVA levels and GAD1 rs872123, rs3749034 and rs769404, which are in strong LD, formed haplotypes. We included all untreated patients and controls. high-risk haplotypes for BD in Danish people, in line with our results. Dependent variable pHVA Haplotype Beta Stat p Later Geller et al. (2008) analysed another GAD1 variant, rs2241165, previously associated with SZ, and reported the transmission of the A rs1978340 AG 18.1 0.13 0.7220 allele as a high-risk allele in families with children with BD. This SNP − a rs3749034 GA 106 3.89 0.0495 was in complete LD with rs3749034, which is consistent with our re- GG 62.4 1.84 0.1760 sults. In contrast, a recent study has found bipolar II disorder to be rs1978340 AGT 32 0.38 0.5390 associated with the A allele of rs3749034, in a group of 280 Thai pa- rs3749034 GAT −102 3.57 0.0596 tients (Chung et al., 2017). The differences in diagnosis, our patients rs769404 GGC 68.2 2.12 0.1470 being BD I, and in rs374034 allele frequencies between ethnic groups GGT −22.8 0.03 0.8560 may explain this discrepancy. fi pHVA: plasma homovanillic acid levels. Genome-wide association studies have not observed a signi cant a pHVA level is significantly associated with the GA haplotype of rs197340- association of the GAD1 gene with BD (Baum et al., 2008; Cichon et al., rs3749034. 2011; Cross-Disorder Group of the Psychiatric Genomics Consortium et al., 2013; Hou et al., 2016; Psychiatric GWAS Consortium Bipolar 3.4. Effect of the interaction between GAD1 and COMT genes on pGABA Disorder Working Group, 2011; Scott et al., 2009; Sklar et al., 2008; and pHVA levels. Differences by diagnosis Strawbridge et al., 2018; Wellcome Trust Case Control Consortium, 2007). Further, although such studies have related SNPs in other genes The results of the analysis of epistasis between GAD1 and COMT with this disorder, the findings have not been reproducible (Baum et al., genotypes and plasma metabolite levels are presented in 2008; Craddock et al., 2010; Psychiatric GWAS Consortium Bipolar Supplementary Table 3. The genotypes were classified as a function of Disorder Working Group, 2011; Scott et al., 2009). This might be at- the presence of the least frequent allele. In the case of pHVA, interac- tributable to differences in the diagnostic criteria used and changes tions of the GAD1 rs1978340 and COMT rs4680 genotypes had a sig- over time in these criteria, as well as bias in the selection of cases and nificant effect on pHVA levels (Fig. 2). Among individuals with the controls. ValVal genotype in rs4680, pHVA levels were higher in carriers of the On the other hand, numerous studies have reported low GAD1 ex- GG genotype in rs1978340 than carriers of AA+AG in this SNP, al- pression in prefrontal cortex and hippocampus of patients with SZ and though we did not find any interaction with diagnosis. No effect on BD (Guidotti et al., 2000; Hashimoto et al., 2003; Torrey et al., 2005; pGABA levels was found arising from interactions of GAD1 genotypes Volk et al., 2016) and an association has been described between low with rs4680 (Supplementary Table 3). Similarly, pHVA and GABA le- levels of GAD1 expression in these areas of the brain and the G allele of vels were not found to be influenced by any interaction between GAD1 rs3749034 in patients with SZ (Straub et al., 2007), which is consistent haplotypes and rs4680 (data not shown). The inclusion of age and sex with the association found in our study between the G allele and BD I. in the aforementioned models did not change the results. We did not find SZ to be significantly associated with the SNPs or haplotypes studied, comparing to controls, although the relationship 4. Discussion between this disease and the G allele of rs3749034 nearly reached significance (p = 0.0887). Some authors have reported that the A, G 4.1. Association of BD I and SZ with GAD1 SNPs and haplotypes and T alleles of rs1978340, rs3749034 and rs769404 respectively are high risk alleles for SZ and that they are related to cognitive phenotypes We found an association between BD I and the G allele of of SZ (Addington et al., 2005; Du et al., 2008; Straub et al., 2007; Zhao rs3749034, related to lower expression of the GAD1 gene (Table 1). We et al., 2007), which we did not analyse in our study. In contrast, a also observed that the G allele in rs3749034 is part of two haplotypes recent meta-analysis revealed a lack of association between SZ and nine associated with a high risk of BD I: GG in rs1978340-rs3749034 and variants of six genes that include the GAD1 polymorphisms rs1978340 GGT in rs1978340-3749034-769404 (Table 2). Few genetic association and rs3749034 (Xu et al., 2014), in agreement with our results. studies have assessed the relationship of BD I with the GAD1 gene and Finally, we found that the GA haplotype in rs1978340-rs3749034 only one study genotyped more than one SNP (Lundorf et al., 2005). In and GAT haplotype in rs1978340-rs3749034-769404 (Table 2) were ff that study, authors analysed 14 genetic variants and found an asso- associated with a protective e ect against BD I and had a close-to-sig- fi ciation between BD and the A allele of rs1978340 in Danish patients but ni cant association with SZ. In general, few protective haplotypes for not in Scottish patients (Lundorf et al., 2005); however, when they these diseases have been described (Fujii and Wylie, 2003; Petryshen

Fig. 2. Effects of the interaction between the rs1978340 of the GAD1 gene and rs4860 of the COMT gene on pHVA levels in untreated patients and controls analysed together. We represent pHVA levels in individuals carrying the allele Met (ValMet+MetMet) and individuals homozygous for the Val allele (ValVal) of rs4680 of COMT, classified as a function of the presence of genotypes with the least frequent allele A in rs1978340 of the GAD1 gene. Results are expressed as mean ± 95% CI (vertical bars) in ng/ml. GAD1: glutamate decarboxylase 1; COMT: catechol-O-methyltransferase; pHVA: plasma homovanillic acid levels. Interaction rs4680 x rs1978340; F = 9.81, p = 0.0019. *ValVal-(AA+AG) car- riers have significantly lower pHVA levels than ValVal-GG carriers (pHVA in ValVal-(AA+AG) carriers = 13.6 ng/ml, 95% CI = 11.9–15.4, n = 40; and pHVA in ValVal-GG car- riers = 16.9 ng/ml, 95% CI = 15.1–18.9, n = 35).

156 A. Arrúe et al. Neurochemistry International 124 (2019) 152–161 et al., 2005). Further research is necessary to confirm that this was not et al., 1988). an incidental finding. We observed that pHVA levels were significantly affected by the Our results are strengthened by the fact that the associations are in interaction between GAD1 rs1978340 SNP and COMT rs4680, regard- the same direction, that is, that BD I is associated with the G allele of less of the diagnosis: individuals with the high COMT activity ValVal rs3749034, related to a decrease in GAD1 expression, and haplotypes genotype and homozygous GG genotype of rs1978340 have higher containing this allele, while the haplotypes containing the A allele in pHVA levels than AA+AG carriers (Fig. 2). If GG in rs1978304 were to rs3749034 may be a protective factor. result in lower GABAergic activity, this could add to the effect of the Val allele of COMT on pHVA levels, previously observed (Zumárraga et al., 4.2. Association of pGABA levels with GAD1 SNPs and haplotypes. 2010) and confirmed in this study (Supplementary Table 3). This is Differences by diagnosis speculative, however, since the functionality of rs1978340 has yet to be confirmed and findings are mixed regarding the association of this SNP Our results were inconsistent with our hypothesis in that pGABA with the diagnosis of SZ or BD, no association between these disorders levels were not related to the SNPs or the haplotypes studied, and nor being observed in our study, unlike in some previous research (Ikeda were the levels affected by interactions between the genotypes and the et al., 2007; Lundorf et al., 2005; Straub et al., 2007). diagnosis (Supplementary Table 2). The enzyme GAD67, encoded by On the other hand, we did not find pGABA levels to be influenced by the GAD1 gene, is responsible for 80–90% of the GABA synthetized in the rs4680 genotype or any interaction with GAD1 SNPs the brain and seems to play a critical role in the regulation of cytosolic (Supplementary Fig. 1). In contrast, Marenco et al. reported an asso- levels and vesicular filling of GABA (Lau and Murthy, 2012). Although ciation of pGABA levels with ValMet (rs4680) heterozygotes, though there is a strong correlation between GAD67 expression and tissue not with the high and low activity genotypes. They also found a weak GABA levels in the brain, only one study has focused on assessing the effect of their interaction with GAD1 rs769404 on pGABA levels, in relationship between GAD1 variants and GABA levels. In that study, accordance with studies suggesting an inhibitory link between dopa- when GABA levels in the cingulate cortex were assessed in healthy mine and GABA in cortical areas that we did not observe. It is possible volunteers by proton magnetic resonance spectroscopy, which mea- that the interaction described by Marenco et al. (Marenco et al., 2010) sures intra-synaptic and intracellular GABA levels, it was found that is detectable in brain tissue but not in plasma, as we have discussed high levels of GABA were associated with the individual being homo- above. zygous for the A allele of rs1978340 and T allele of rs769404 (Marenco We did not detect a differential effect of GAD1 and COMT SNPs on et al., 2010); our results do not concord with that finding (in that we pHVA levels in the three groups. Nevertheless, as indicated in found no association between pGABA and SNPs in GAD1 even analysing Supplementary Fig. 2, a relationship between GAD1 rs3749034 and the controls independently). It may be that the association found by pHVA levels might have been present in controls, but was not statisti- Marenco et al. is brain-region specific, whereas pGABA levels indicate cally significant. A larger study might enable us to confirm this pattern. GABA activity in the entire brain. On the other hand, pGABA levels only High pHVA levels have been associated with SZ and BD (Davila et al., reflect the extracellular levels of GABA, and the relative influence of 1988; van de Kerkhof et al., 2015) and it is plausible that rs3749034 is postsynaptic receptor activity and reuptake may be higher than when involved in this association. measuring total GABA levels in the brain (Cherubini and Conti, 2001; Saliba et al., 2007). 4.4. Potential confounders in plasma measurements Individuals with SZ and BD I having lower pGABA levels than healthy controls is consistent with a previous study (Arrúe et al., 2010) pGABA is of both central and peripheral origin. Changes in con- and with the known lower GAD67 expression in various areas of the centrations of GABA in the plasma and the brain after pharmacological brain in these patients compared to non-affected controls (Torrey et al., manipulation are proportional and correlated (Cai et al., 2010; Löscher, 2005; Volk et al., 2016). These results support the use of pGABA levels 1979; Petty et al., 1996) Peripherally, GABAergic activity has been as an index of brain GABA activity and the view that GAD1 is involved detected in the kidney, liver, spleen, testes, pancreatic B cells and in SZ and BD. mesenteric plexus (Drummond and Phillips, 1974; Erdö et al., 1984; Okada et al., 1976; Párducz et al., 1992), though much lower than that 4.3. Associations of GAD1 SNPs and haplotypes with pHVA levels, and found in the brain where 95% of GABA is synthesized (Petty, 1994; rs4680 with pGABA levels. Interactions and differences by diagnosis Petty et al., 1993) GABA concentrations, GAD1 expression and GAD activity are much higher in the brain than other tissues (Petty, 1994; We found pHVA levels to be associated with r3749034 Petty et al., 1993; Qume and Fowler, 1996; Uhlén et al., 2015). (Supplementary Fig. 1A) and with the haplotype containing the A allele Plasma HVA derives from both peripheral and central sources, of rs3749034 (GA rs1978340-rs3749034) in GAD1 regardless of the however, it seems that under certain conditions plasma levels reflect diagnosis (Table 3). To our knowledge, this is the first study to assess activity in the brain. It has been found that a major fraction of pHVA the link between the GABAergic and dopaminergic systems through the originates from mesenteric organs (Eisenhofer et al., 2004). Never- relationship of pHVA levels with GAD1 genotype. Several lines of re- theless, experiments with central and peripheral dopamine agonists search have shown that a reduction in the GAD67 mRNA level in GA- have indicated that either central and peripheral levels are correlated or BAergic interneurons increases the activity of dopaminergic neurons in that the peripheral contribution remains relatively stable (Bacopoulos various different brain regions (Akbarian and Huang, 2006; Stansfield et al., 1980, 1979; Davidson et al., 1987; Kendler et al., 1981; Konicki et al., 2015). Our results are consistent with this idea, in that low et al., 1991). GAD67 expression and high pHVA levels, which may reflect higher Diet does not seem to have a significant effect on plasma metabo- dopaminergic activity, were associated with individuals being GG lites after night fasting, moderate exercise (Kaminski et al., 1990; Petty, homozygous for rs3749034 (Supplementary Fig. 1A). We also observed 1994); and smoking two cigarettes a day does not seem to influence that pHVA levels were lower in individuals carrying the haplotype pHVA levels in regular smokers (Kaminski et al., 1990). Other data containing the A allele (GA rs1978340-rs3749034) than non-carriers, indicate that pHVA measured on different days is not influenced by probably due to the presence of the A base of rs3749034, related to a treatment with lorazepam, but does significantly increase after a high higher transcription of the enzyme and a greater synthesis of GABA monoamine meal (Donnelly et al., 1996). Acute benzodiazepine ad- (Table 3). These data are, in principle, compatible with findings in ministration strengthens GABA-A function, and hence, could influence studies on rat brain suggesting that GABA may modulate the dopami- pGABA concentrations. A decrease in pGABA levels has been observed a nergic system, causing changes in HVA levels (Lautin et al., 1980; Reid few hours after benzodiazepine administration in individuals with

157 A. Arrúe et al. Neurochemistry International 124 (2019) 152–161 anxiety disorder and healthy controls (Roy-Byrne et al., 1992), though Conflicts of interest this finding was not reproduced in another study by the same research group (Cowley et al., 1996). We have found no data on the long-term The authors declare no commercial or financial relationship that effects of benzodiazepines, and in general, data concerning the role of might be interpreted as potential conflicts of interest in relation to this GABA in anxiety disorders and the effects of benzodiazepines are lim- research. ited and have not been reproduced (Lydiard, 2003). In our study, participants included in the neurochemical analysis were inpatients, all Appendix A. Supplementary data were on a standard hospital diet, and the administration of benzodia- zepines was allowed. Supplementary data to this article can be found online at https:// Another possible confounder is the variation in washout period doi.org/10.1016/j.neuint.2019.01.004. across the sample, this period ranging from 2 weeks to more than a year in both groups. Neither pHVA nor pGABA levels were correlated with Funding the length of washout in either of our groups. In previous studies, pHVA levels have been found to be slightly higher after 3 or 4 weeks of This research did not receive any specific grant from funding washout but not in the first 3 weeks (Davidson et al., 1991); or after 5 agencies in the public, commercial, or not-for-profit sectors. weeks of washout but not in the preceding weeks (Pickar et al., 1986). More recent research has also not found differences in pHVA levels References between patients with different washout periods (van de Kerkhof et al., 2015). We have not found any data in the literature concerning the Addington, A.M., Gornick, M., Duckworth, J., Sporn, A., Gogtay, N., Bobb, A., Greenstein, potential influence of length of washout period on pGABA. D., Lenane, M., Gochman, P., Baker, N., Balkissoon, R., Vakkalanka, R.K., Weinberger, D.R., Rapoport, J.L., Straub, R.E., 2005. GAD1 (2q31.1), which encodes decarboxylase (GAD67), is associated with childhood-onset schizo- 5. Study limitations phrenia and cortical gray matter volume loss. Mol. Psychiatry 10, 581–588. https:// doi.org/10.1038/sj.mp.4001599. ff Akbarian, S., Huang, H.-S., 2006. Molecular and cellular mechanisms of altered GAD1/ Patients had di erent washout periods, though this parameter is not GAD67 expression in schizophrenia and related disorders. Brain Res. Rev. 52, expected to have significantly influenced plasma levels in the condi- 293–304. https://doi.org/10.1016/j.brainresrev.2006.04.001. tions under which this study was conducted. Further, treatment with Arrúe, A., Dávila, R., Zumárraga, M., Basterreche, N., González-Torres, M.A., Goienetxea, benzodiazepines was allowed. Data concerning the possible influence of B., Zamalloa, M.I., Anguiano, J.B., Guimón, J., 2010. GABA and homovanillic acid in the plasma of Schizophrenic and bipolar I patients. Neurochem. Res. 35, 247–253. these drugs on pGABA are mixed (Lydiard, 2003), while they do not https://doi.org/10.1007/s11064-009-0048-z. seem to have a significant impact on pHVA (Donnelly et al., 1996). Asada, H., Kawamura, Y., Maruyama, K., Kume, H., Ding, R.G., Kanbara, N., Kuzume, H., The heterogeneity of SZ and BD I is well known. It is possible that Sanbo, M., Yagi, T., Obata, K., 1997. Cleft palate and decreased brain gamma-ami- ff nobutyric acid in mice lacking the 67-kDa isoform of glutamic acid decarboxylase. the associations and di erences found are present in some subgroups of Proc. Natl. Acad. Sci. 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On the Basterreche, N., Dávila, R., Zumárraga, M., Arrúe, A., González-Torres, M.A., Zamalloa, other hand, we have made multiple comparisons increasing the like- M.I., Anguiano, J.B., Guimón, J., 2008. Biological correlates of the congruence and lihood of type II errors, and hence these results need to be confirmed in incongruence of psychotic symptoms in patients with type 1 bipolar disorder. Neuropsychobiology 58, 111–117. https://doi.org/10.1159/000170391. a larger sample. Baum, A.E., Akula, N., Cabanero, M., Cardona, I., Corona, W., Klemens, B., Schulze, T.G., Extracellular GABA levels are influenced by other molecules asso- Cichon, S., Rietschel, M., Nöthen, M.M., Georgi, A., Schumacher, J., Schwarz, M., ciated with GABAergic activity such as postsynaptic GABA receptors Abou Jamra, R., Höfels, S., Propping, P., Satagopan, J., Detera-Wadleigh, S.D., Hardy, and GABA membrane transporters. Polymorphisms in the genes en- J., McMahon, F.J., 2008. A genome-wide association study implicates diacylglycerol kinase eta (DGKH) and several other genes in the etiology of bipolar disorder. Mol. coding these elements might modify pGABA, and this has not been Psychiatr. 13, 197–207. https://doi.org/10.1038/sj.mp.4002012. analysed in our study. Benes, F.M., Vincent, S.L., Molloy, R., 1993. Dopamine-immunoreactive axon varicosities form nonrandom contacts with GABA-immunoreactive neurons of rat medial pre- Finally, we have not considered the involvement of epigenetic fac- – ff frontal cortex. Synap. N. Y. N 15, 285 295. https://doi.org/10.1002/syn.890150405. tors that a ect GAD1 expression but not the DNA sequence, such as Brauns, S., Gollub, R.L., Walton, E., Hass, J., Smolka, M.N., White, T., Wassink, T.H., histone methylation of CpGs groups (Dong et al., 2015; Huang and Calhoun, V.D., Ehrlich, S., 2013. Genetic variation in GAD1 is associated with cortical Akbarian, 2007) and other GAD1 regulatory network genes (Ruzicka thickness in the parahippocampal gyrus. J. Psychiatr. Res. 47, 872–879. https://doi. fl ff org/10.1016/j.jpsychires.2013.03.010. et al., 2015), which have been shown to in uence di erences in GAD1 Cai, H.-L., Zhu, R.-H., Li, H.-D., Zhang, X.-H., Hu, L., Yang, W., Ye, H.-S., 2010. Elevated expression in the prefrontal cortex and hippocampus in patients with SZ plasma γ-aminobutyrate/glutamate ratio and responses to risperidone antipsychotic and BD compared to healthy volunteers. treatment in schizophrenia. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 34, 1273–1278. https://doi.org/10.1016/j.pnpbp.2010.07.006. Chattopadhyaya, B., Di Cristo, G., Wu, C.Z., Knott, G., Kuhlman, S., Fu, Y., Palmiter, R.D., 6. Conclusions Huang, Z.J., 2007. GAD67-mediated GABA synthesis and signaling regulate in- hibitory synaptic innervation in the visual cortex. 54, 889–903. https://doi. fi org/10.1016/j.neuron.2007.05.015. Our ndings show that GAD1 SNPs are associated with the risk of Cherubini, E., Conti, F., 2001. Generating diversity at GABAergic synapses. Trends BD I. We have also found that haplotypes containing the A allele of Neurosci. 24, 155–162. rs3749034 (GA and GAT) may protect against both disorders. We ob- Chung, Y.-C.E., Chen, S.-C., Chuang, L.-C., Shih, W.-L., Chiu, Y.-H., Lu, M.-L., Chen, H.-C., served that the connection between the GABA and dopaminergic sys- Kuo, P.-H., 2017. Evaluation of the interaction between genetic variants of GAD1 and miRNA in bipolar disorders. J. Affect. Disord 223, 1–7. https://doi.org/10.1016/j. tems may be reflected in the effect on pHVA levels of GAD1 SNPs, and jad.2017.07.024. their interaction with the COMT gene rs4680, regardless of the diag- Cichon, S., Mühleisen, T.W., Degenhardt, F.A., Mattheisen, M., Miró, X., Strohmaier, J., ff nosis. It is be possible that SNPs or haplotypes of the GAD1 gene, alone Ste ens, M., Meesters, C., Herms, S., Weingarten, M., Priebe, L., Haenisch, B., Alexander, M., Vollmer, J., Breuer, R., Schmäl, C., Tessmann, P., Moebus, S., or interacting with the COMT gene, have a role in the genetic control of Wichmann, H.-E., Schreiber, S., Müller-Myhsok, B., Lucae, S., Jamain, S., Leboyer, pHVA levels, a peripheral marker of schizophrenia and bipolar disorder. M., Bellivier, F., Etain, B., Henry, C., Kahn, J.-P., Heath, S., Bipolar Disorder Genome More studies are needed to clarify this relationship and its potential Study (BiGS) Consortium, Hamshere, M., O'Donovan, M.C., Owen, M.J., Craddock, N., Schwarz, M., Vedder, H., Kammerer-Ciernioch, J., Reif, A., Sasse, J., Bauer, M., clinical utility.

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