Progress in Neuro-Psychopharmacology & Biological Psychiatry 32 (2008) 18841888

Contents lists available at ScienceDirect

Progress in Neuro-Psychopharmacology & Biological Psychiatry

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / p n p b p

P2RX7 Gln460Arg polymorphism is associated with depression among diabetic patients

Geza Nagy a, Zsolt Ronai b, Aniko Somogyi a, Maria Sasvari-Szekely b, Omar Abdul Rahman b, Attila Mate a, Timea Varga a, Zsoa Nemoda b,
a b

Semmelweis University, 2nd Department of Internal Medicine, Budapest, 1088, Szentkiralyi u. 46, Hungary Semmelweis University, Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Budapest, 1088, Puskin u. 9, Hungary

a r t i c l e

i n f o

a b s t r a c t
Aims: Both diabetes mellitus and major depression are public health concerns, and the co-occurrence of these illnesses is highly frequent. Acting as a potential risk factor, hyperglycemia might facilitate the manifestation of depression in patients genetically predisposed to affective disorders. In the present study, candidate polymorphisms of the serotonin transporter, the tryptophan hydroxylase 2 (TPH2) genes, as well as of the brain-derived neurotrophic factor BDNF, and the P2RX7 purinergic receptor genes were analyzed in Hungarian diabetic population. We assumed that genetic inuence would be stronger on depressive symptoms in the poor glycemic control group (PC: HbA1C N 7%) compared to the good glycemic control group (GC: HbA1C 7%). Methods: After excluding patients with current antidepressant medication, 218 diabetic patients' Hospital Anxiety and Depression Scale (HADS) scores were used in multivariate analysis of variance. Based on the HbA1C levels, 81 patients were in the GC group, and 137 belonged to the PC group. Results: After correcting for multiple testing, only the association of the P2RX7 Gln460Arg (rs2230912) polymorphism with depressive symptoms remained signicant. Patients with the G-allele (Arg-variant) had higher scores on the HADS depression scales (p = 0.007). A gene x glycemic control interaction (p = 0.032) was observed on the anxiety scale at the TPH2 promoter polymorphism: the -703T-allele decreased anxiety scores only in the GC group (p = 0.008). Conclusions: Our results support the role of the P2RX7 rs2230912 G-allele in the development of depression and emphasize the importance of good glycemic control, acting as a potential protective factor in diabetic patients. 2008 Elsevier Inc. All rights reserved.

Article history: Received 20 June 2008 Received in revised form 27 August 2008 Accepted 27 August 2008 Available online 4 September 2008 Keywords: Hospital Anxiety and Depression Scale P2RX7 Serotonin transporter TPH2

1. Introduction The prevalence of major depression is two times higher among diabetic patients compared to controls, affecting 1132% of the diabetic population depending on the method of assessment (Anderson et al., 2001). During depressive episodes, the poor adherence to antidiabetic treatments can cause problems in the management of diabetes mellitus (DM). In turn, poor glycemic control increases the risk for microvascular and macrovascular complications resulting in lower quality of life, as well as higher morbidity and mortality in diabetic patients (Ciechanowski et al., 2000). Even though the relationship between DM and depression has been investigated from many points of view, the explanation of the comorbidity is still
Abbreviations: 5-HTTLPR, serotonin transporter gene-linked polymorphic region; BDNF, brain-derived neurotrophic factor; P2RX7, purinergic receptor P2X, ligand-gated ion channel, 7; TPH2, tryptophan hydroxylase 2; DM, diabetes mellitus; GC, good glycemic control group (HbA1C 7%); PC, poor glycemic control group (HbA1C N 7%). Corresponding author. Institute of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, POB 260, H-1444, Hungary. Tel.: +36 1 4591500x4017; fax: +36 1 2662615. E-mail address: nemzso@puskin.sote.hu (Z. Nemoda). 0278-5846/$ see front matter 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2008.08.021

unclear. One hypothesis suggests that poor glycemic control may adversely affect mood. In an early study, Lustman and his colleagues demonstrated that hyperglycemia can provoke anxiety symptoms in type 1 diabetic patients using hyperglycemic clamp (Lustman et al., 1981). Another group has shown that depressive symptoms get better by improving metabolic control in type 2 diabetic patients (Testa and Simonson, 1998). Higher glycated hemoglobin (HbA1C) levels predicted reduced responsiveness to antidepressant interventions and worse course of depression in a study conducted by Lustman and coworkers over 5 years (Lustman et al., 1997). As hyperglycemia is responsible for the development of micro- and macrovascular complications, poor glycemic control may be a key factor in the development of depression among diabetic patients (Lustman et al., 2000). In the development of depression both genetic susceptibility and environmental factors (e.g. childhood abuse and exposure to stressful life events) play an important role, probably via gene x environment interaction (Caspi et al., 2003). It is not known whether the development of depressive symptoms is augmented by metabolic factors, such as poor glycemic control. To test this possibility, we investigated four depression-related candidate genes in a Hungarian diabetic population. In

G. Nagy et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 32 (2008) 18841888

1885

addition to the well-known VNTR (Variable Number of Tandem Repeats) in the serotonin transporter gene, the newly indicated SNPs (Single Nucleotide Polymorphisms) in the tryptophan hydroxylase 2 (TPH2) gene, the P2RX7 purinergic receptor gene, and the brain-derived neurotrophic factor BDNF gene were analyzed. Disruption in the serotonin neurotransmitter system has been linked to diverse psychopathology, including depression (Jans et al., 2007). The most frequently prescribed antidepressant drugs target the serotonin transporter, hence the most widely studied polymorphism in depression-related genetic association analyses is located in the serotonin transporter gene (5-HTT, SLC6A4). This VNTR was found in the promoter region, therefore referred to as 5-HTT gene-linked polymorphic region (5-HTTLPR). The polymorphic region consists of a 44-bp insertion/ deletion resulting in a long (L) or short (S) variant, respectively. Lower 5HTT expression levels were associated with the short variant (Lesch et al., 1996). Meta-analyses suggested a small but detectable effect of the short allele on affective disorders and depression-related personality traits (Lotrich and Pollock, 2004; Levinson, 2006). Later studies indicated a functional A/G SNP (rs25531) within the 5-HTTLPR, therefore the distinction between LA and LG alleles became important (Parsey et al., 2006; Hu et al., 2006). Using the reclassied genotype grouping system (where LG is grouped to S, and designated as S'), Zalsman et al. (2006) reported an association between the S' allele and more severe depression. Patients with S' allele scored differentially on the Hamilton Depression Scale in terms of high vs. low life events scores, conrming the original interaction effect reported by Caspi et al. (2003). The brainspecic enzyme of tryptophan hydroxylase (TPH, the rate-limiting enzyme in serotonin synthesis) was discovered a few years ago and named as TPH2 (Walther et al., 2003). We have chosen to investigate the promoter polymorphism at the -703 position (rs4570625), as it was associated with altered amygdala reactivity, a key-regulator of emotional behaviors (Brown et al., 2005). The brain-derived neurotrophic factor is produced and secreted by cortical neurons as an inactive precursor and then activated by different proteases to perform its role in neurogenesis and neuroprotection. The Val66Met polymorphism alters the pro-region of the protein; the Metform showed lower depolarization-induced secretion in in vitro and in vivo settings (Egan et al., 2003). This functional SNP has been associated with cognitive decits and mood disorders; also, four studies reported low serum BDNF levels in depressed patients (reviewed by Post, 2007). In addition, the role of BDNF in glucose metabolism has been studied in type 2 diabetic patients: plasma BDNF levels were decreased in DM patients compared to controls, but there was no association between diabetes and the Val66Met polymorphism (Krabbe et al., 2007). The P2RX7 gene has been introduced as a candidate gene for affective disorders only recently (Barden et al., 2006; Lucae et al., 2006), after scanning the 12q24.31 chromosome region indicated by linkage studies. The purinergic receptor P2X7 is an ATP-binding calcium channel expressed on neurons as well as on microglial cells in various brain regions (Sperlgh et al., 2006). The non-synonymous SNP Gln460Arg is located in the C-terminal domain of the P2X7 ion channel, suggesting a role in receptor trafcking (Denlinger et al., 2006). Here we report a genetic association study of depressive symptoms (assessed by self-report questionnaire) in a diabetic population. We hypothesized that poor glycemic control (acting as an environmental/ metabolic risk factor) could facilitate the development of depressive symptoms and/or good glycemic control could protect from the progression of mood disorders in patients with predisposing genetic risk factor(s). Therefore, we conducted multivariate analysis of variance analyses testing the effect of genetic risk factors and glycemic control (assessed by HbA1C level). 2. Methods Diabetic patients (Caucasians, of Hungarian origin, N = 236) were recruited from the inpatient and outpatient services of the 2nd

Department of Internal Medicine at the Semmelweis University. The diagnosis of DM was based on fasting plasma glucose levels or 75 g oral glucose tolerance test according to the criteria of the WHO. Exclusion criteria were any severe or acute medical illnesses that may affect the presence of depressive symptoms, e.g. liver cirrhosis, end stage renal disease, or cancer. The study design was in accordance with the principles of the Helsinki Declaration and was approved by the Local Ethics Committee (TUKEB). Every patient provided written informed consent for their participation. Among the 236 patients 24 had been previously diagnosed for depression (thus the prevalence of clinically signicant depression was 10.2% in this population). Eighteen diabetic patients had antidepressant medication at the time of study and were excluded from the analyses, since antidepressant treatment inuences the severity of depressive symptoms. Therefore, altogether 218 diabetic patients were included in the genetic analyses (for demographic variables and clinical data of the patients see Supplementary Table). Mood disorder symptoms were assessed by the Hospital Anxiety and Depression Scale (HADS, Zigmond and Snaith, 1983). It is a selfreport questionnaire, consisting of 14 items (7 for anxiety and 7 for depression) invented to screen for anxiety disorders and depression in non-psychiatric medical patients. The Hungarian version of this test is a reliable self-assessment in medical practice (Muszbek et al., 2006). Internal consistency of the subscales was assessed by calculating Cronbach alpha values and was found satisfactory (HADS-anxiety: 0.79, HADS-depression: 0.78). According to the results of Snaith (2003), a score of 11 or higher indicates probable presence of mood disorder, whereas points 810 are considered as borderline. Among the 218 patients 21.1% had borderline and 12.4% had clinical score on the HADS-anxiety scale, whereas 20.6% had borderline and 6.4% had clinical score on the HADS-depression scale. The glycated hemoglobin HbA1C level was used to assess the glycemic status. Participants were divided into two groups: Patients with HbA1C levels equal or below 7% were considered to have good glycemic control (GC group, N = 81, HbA1C 6.32 0.50), those who had HbA1C over 7% were put in the poor glycemic control group (PC group, N = 137, HbA1C 9.01 1.62). Neither the demographic variables, nor the average anxiety and depression scores of the two patient groups differed signicantly (Supplementary Table). Although more patients from the PC group had high anxiety score compared to the GC group patients (16.1% vs. 6.2% had the clinical value of 11 or more points), there was no signicant difference in the categorical distribution of the HADS anxiety and depression scores (HADS-anxiety: 2(2) = 4.60, p = 0.10, HADS-depression: 2(2) = 0.47, p = 0.79). Non-invasive DNA sampling was applied, DNA was isolated from buccal cells using the DNA-purication kit obtained from Gentra (Minneapolis, US). Genotyping of the 5-HTTLPR and the rs25531 was performed by the method of Wendland et al. (2006). Biallelic genotype classication using the designated functional allele S' for S and LG alleles, was carried out as described previously (Parsey et al., 2006). The TPH2-703 G/T SNP (rs4570625), BDNF Val66Met SNP (rs6265), and P2RX7 Gln460Arg SNP (rs2230912) were genotyped by pre-designed TaqMan kits C_226207_10, C_15853715_20, and C_11592758_10, respectively (Applied BioSystem, Foster City, USA). No signicant deviations from HardyWeinberg equilibrium were detected for any of the polymorphisms (for the triallelic 5-HTTLPR: 2(3) = 1.78, p = 0.62; for TPH2-703 G/T: 2(1) = 0.49, p = 0.48; for BDNF Val66Met: 2(1) b 0.001, p N 0.99; for P2RX7 Gln460Arg 2(1) = 0.83, p = 0.36). Statistical analyses were performed using 13.0 version of SPSS for Windows. Because of the HADS subscales' non-normal distribution, square-root-transformation was applied; in all subsequent analysis the transformed HADS-anxiety and HADS-depression scores were used. Independent samples t-test and Pearson correlation were carried out to asses the sex and age effect on the two subscales. Multivariate analyses of variance were used to test the effects of genetic factors and glycemic control on the HADS subscales. Since four genetic polymorphisms were

1886

G. Nagy et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 32 (2008) 18841888

Table 1 Effect of the serotonergic polymorphisms on anxiety and depressive scores 5-HTTLPR Genotype S'S' S'L' L'L' MANOVA Glyc. Contr. Genotype Genotype Others LALA MANOVA Glyc. Contr. Genotype N 42 110 66 df 1 2 N 152 66 df 1 1 HADS-a 6.45 4.30 6.23 3.80 5.35 3.61 F 1.08 0.40 HADS-a 6.29 3.93 5.35 3.61 F 0.58 0.77 HADS-d 5.10 3.81 5.06 3.55 4.23 3.20 F 0.04 0.33 HADS-d 5.07 3.61 4.23 3.20 F 0.002 0.73 TPH2-703 G/T SNP Genotype G/G G/T T/T MANOVA Glyc. contr. Genotype Genotype G/G G/T + T/T MANOVA glyc. Contr. Genotype N 130 79 9 df 1 1 N 130 88 df 1 1 HADS-a 6.27 3.74 5.41 3.91 7.56 4.48 F 2.17 4.11 HADS-a 6.27 3.74 5.63 4.00 F 1.90 2.84 HADS-d 4.98 3.51 4.49 3.43 5.53 4.24 F 1.92 1.83 HADS-d 4.98 3.51 4.58 3.50 F 1.02 1.29

p 0.30 0.68

p 0.84 0.72

p 0.14 0.04

p 0.17 0.18

p 0.45 0.38

p 0.96 0.40

p 0.17 0.09

p 0.31 0.26

The average scores for anxiety (HADS-a) and depression (HADS-d) subscale of the Hospital Anxiety and Depression Scale are presented in mean SD for the three genotype groups of the serotonin transporter gene-linked polymorphic region (5-HTTLPR) and the tryptophan hydroxylase 2 gene promoter polymorphism (TPH2-703 G/T SNP). Based on the triallelic (S-LG-LA) 5HTTLPR system, three functional genotype groups were constructed: the S'S' genotype group consisted of SS, SLG, LGLG; the S'L' group combined the SLA and LALG genotype groups; whereas L'L' stands for the LALA genotype. The MANOVA main effects of glycemic control and genotype on both subscales are presented in separate columns. In the lower part of the table the functionally distinct LALA group was compared to all the other genotypes, whereas at the -703 G/T SNP the rare homozygote genotype was grouped to the heterozygotes.

analyzed, the p-value threshold for multiple comparison was calculated by the False Discovery Rate adjustment (Benjamini and Hochberg,1995). 3. Results Association analyses of polymorphisms in the serotonin transporter, the TPH2, the BDNF, and the P2RX7 genes on anxiety and depressive symptoms were performed using the Hospital Anxiety and Depression Scale (7 questions, 021 points for each subscale). Average HADS anxiety and depression scores were in the normal range (below 7 points) in the overall sample, as well as in the GC and the PC groups. Gender and age effects were assessed on the two subscales: Only a modest correlation of age was observed (HADS-anxiety: r = 0.129, p = 0.058; HADS-depression: r = 0.127, p = 0.061), while sex affected both subscales signi cantly (HADS-anxiety: males = 4.72 3.34, females = 7.20 3.92, t(1,216) = 5.13, p b 0.001; HADS-depression: males = 4.29 3.34, females = 5.31 3.59, t(1,216) = 2.31, p = 0.022). The two subscales correlated with each other signicantly (r = 0.589, p b 0.001). Since gender might be an important factor in anxiety and depression related symptoms, sex was also entered as betweensubject factor in the multivariate analysis of variance testing the effects of genetic factors and glycemic control on the two subscales (age was included as a covariate). The rare homozygote genotype groups at the SNPs were either left out from the analyses (upper part of the Tables), or grouped to the heterozygote group (lower part of the Tables).

Analyzing the promoter polymorphism of the serotonin transporter gene, neither genetic, nor glycemic control effect was observed (Table 1). Although patients carrying the S' allele of the 5-HTTLPR achieved higher points on both subscales, signicant impact of this polymorphism could not be detected. The TPH2 promoter polymorphism affected the anxiety scale signicantly when the rare T/T homozygote group was left out from the analysis: The G/T heterozygote group had lower scores compared to the G/G group (F(1,200) = 4.11, p =0.044, 2 =0.02, power = 0.52). But this effect became only a tendency when the T/T homozygotes were grouped to the heterozygotes (Table 1 lower part). The BDNF Val66Met (C/T) SNP did not have any effect on either subscales in our diabetic population (Table 2). On the other hand, the Gln460Arg (A/G) polymorphism of the P2RX7 gene yielded signicant results: Both anxiety and depressive scores increased signicantly in the G-allele present group compared to the A/A group (leaving out the rare G/G homozygote group: HADSanxiety: F(1,204) = 4.86, p = 0.029, 2 =0.023, power = 0.59; HADS-depression: F(1,204)= 6.71, p =0.010, 2 = 0.032, power =0.73; grouping together A/G and G/G genotypes: HADS-anxiety: F(1,209) = 5.16, p = 0.024, 2 = 0.024, power = 0.62; HADS-depression: F(1,209) = 7.47, p = 0.007, 2 = 0.034, power =0.78). After correcting for multiple testing, only the association between P2RX7 polymorphism and depressive symptoms remained signicant (p b 0.012). Regarding the interaction effects of the MANOVAs, one genotype glycemic control interaction effect was observed at the TPH2-703 G/T SNP: the T-allele decreased the anxiety scores only in the good

Table 2 Effect of the BDNF Val66Met (C/T) SNP and P2RX7 Gln460Arg (A/G) SNP on anxiety and depressive scores BDNF Genotype C/C C/T T/T MANOVA Glyc. Contr. genotype Genotype C/C C/T + T/T MANOVA Glyc. Contr. genotype N 147 64 7 df 1 1 N 147 71 df 1 1 HADS-a 6.13 3.85 5.83 3.91 5.14 3.72 F 0.49 0.19 HADS-a 6.13 3.85 5.76 3.87 F 0.95 0.78 HADS-d 4.59 3.53 5.25 3.40 5.57 3.91 F 0.29 1.70 HADS-d 4.59 3.53 5.28 3.43 F 0.53 1.52 P2RX7 Genotype A/A A/G G/G MANOVA glyc. contr. genotype Genotype A/A A/G + G/G MANOVA glyc. contr. Genotype N 145 68 5 df 1 1 N 145 73 df 1 1 HADS-a 5.53 .3.71 7.00 4.07 6.40 2.61 F 0.95 4.86 HADS-a 5.53 .3.71 6.96 3.98 F 1.12 5.16 HADS-d 4.31 3.22 5.74 3.83 7.00 4.00 F 0.38 6.71 HADS-d 4.31 3.22 5.82 3.83 F 0.89 7.47

p 0.48 0.66

p 0.59 0.19

p 0.33 0.03

p 0.54 0.01

p 0.33 0.38

p 0.47 0.22

p 0.29 0.02

p 0.35 0.01

The average scores for anxiety (HADS-a) and depression (HADS-d) subscale of the Hospital Anxiety and Depression Scale are presented in mean SD for the three genotype groups in the upper part of the table. The MANOVA main effects of glycemic control and genotype on both subscales are presented in separate columns. In the lower part of the table the rare homozygote genotype was grouped to the heterozygotes.

G. Nagy et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 32 (2008) 18841888

1887

glycemic control group (GC group: G/G 6.31 3.56 vs. G/T 3.88 2.55, PC group: G/G 6.24 3.88 vs. G/T 6.15 4.25, interaction effect F(1,200) = 4.64, p = 0.032, 2 = 0.023, power = 0.57). This effect could be seen as a tendency at the depression scale (GC group: G/G 5.06 3.68 vs. G/T 3.38 2.50, PC group: G/G 4.92 3.42 vs. G/T 5.04 3.70, interaction effect F(1,200) = 3.09, p = 0.080, 2 = 0.015, power = 0.42). Within the GC group (N = 81) the TPH2 genotype effect on anxiety scores remained signicant when the rare T/T group was left out from the analysis (F (1,73) = 9.4, p = 0.003, 2 = 0.114, power = 0.86) or when it was grouped together with the G/T group (F(1,76) = 7.47, p = 0.008, 2 = 0.089, power = 0.77). 4. Discussion In the present genetic association analyses the Hospital Anxiety and Depression Scale (HADS) was used to assess anxiety and depressive symptoms in patients with diabetes mellitus. The questions concentrate on general symptoms of emotional disturbances without asking about somatic symptoms (e.g. appetite, sleep problems) which could be due to other clinical conditions. As it was pointed out by earlier studies, it is highly relevant to screen for depressive symptoms among diabetic patients (Anderson et al., 2001). Based on the pioneering work of Lustman and co-workers showing the correlation of depression and glycemic control (Lustman et al., 1997), we had hypothesized that good glycemic control (and sufcient plasma level of insulin) might prevent from developing depressive symptoms in diabetic patients even when carrying susceptibility gene variants. It is still a question if glycemic control can specically affect the development of psychiatric disorders, or it is only the general negative effect of chronic diseases which leads to mood-imbalances in diabetic patients. Hitherto, only one study reported association between a genetic risk factor and depressive symptoms in patients with chronic disease: Among patients with bromyalgia the 5HTTLPR S/S genotype group had the highest depressive score (Offenbaecher et al., 1999). Our results did not show signicant difference between the 5-HTTLPR genotype groups (although patients carrying the S' allele tended to have higher depressive scores than those without this risk allele). Investigation of other patient populations might clarify the importance of negative environmental factors experienced during the clinical course: The psychological burden of a chronic disease (independently of the exact diagnosis) might play a major role in the development of depressive symptoms. On the other hand, specic risk factors, such as poor glycemic control in DM, might have additional negative effects. The tryptophan hydroxylase 2 (TPH2) gene is in the limelight of research associated with serotonin-related disorders, such as major depression, bipolar disorder, or other emotional disturbances. Two independent studies indicated higher Harm Avoidance and Neuroticism in subjects without the -703T-allele in healthy Caucasian populations (Gutknecht et al., 2007; Reuter et al., 2007). In accordance with these ndings, our results showed that the T-allele decreased the anxiety scores of the HADS questionnaire (p = 0.044), and this effect was true only in the good glycemic control group (p = 0.003). It might be speculated that the good glycemic control group represents a moreor-less healthy group where the genetic effect of this TPH2 promoter polymorphism can be observed, while in the poor glycemic control group this genetic effect disappears probably due to the more pronounced effect of environmental risk factors. Recently, a few genetic variants have been implicated in depression beside the serotonergic polymorphisms. The most promising candidate gene has been the brain-derived neurotrophic factor (BDNF) gene, suggesting an important role of neurogenesis and/or neuroprotection in the development of mood disorders. However, the latest literature review does not support the consistency of the association studies concerning the BDNF Val66Met polymorphism in depression (Groves, 2007). In line with the contradictory results, we could not detect any difference between the genotype groups in depression scores.

Regarding the purinergic receptor P2RX7 gene, three studies reported positive association of the Gln460Arg (A/G) polymorphism with mood disorders. Barden and his colleagues observed an over-transmission of the G-allele to patients with bipolar disorder in a family-based study (Barden et al., 2006). Lucae and her co-workers reported an association between the G-allele and major depressive disorder in a case-control setting (Lucae et al., 2006). The most recent large-scale case-control study conrmed the association between the P2RX7 rs2230912 G-allele and bipolar disorder (McQuillin et al., 2008). We have detected signicant association between the P2RX7 G-allele and depressive symptoms: those with at least one G-allele had higher depressive scores compared to the AA homozygotes. Similar genetic effect was observed among patients with major depression or bipolar disorder in a Hungarian population (Hejjas et al., 2008). The P2X7 purinergic receptors are transmitter-gated cation channels and mediate fast ATP signaling at synaptic sites where ATP has been released from vesicles. They are proposed to promote excitatory neurotransmitter release at presynaptic sites (probably through direct effect of calcium) (Sperlgh et al., 2007). The Arg-variant resulted in enhanced P2X7 pore activity on human monocytes (Denlinger et al., 2006), therefore, the G-allele might have a gain-of-function in the central nervous system as well. The role of P2X7 receptor in the development of depression is presently unclear; however, acting as modulator of glutamatergic neurotransmission, this receptor may be a possible candidate in psychiatric disorders. There are a few limitations in our study: First, patients were not screened for neurological and psychiatric disorders with conventional tests and interviews, therefore, vascular depression can not be excluded from our population. In order to leave out participants with probable mental illnesses, the medical documentations were looked through, and patients with a history of psychiatric disorder other than depression, as well as patients currently being treated with antidepressants were excluded from the study. Second, our cross-sectional study design precluded the determination of cause-effect relationship. Prospective studies have demonstrated that depression can act as a risk factor for incident diabetes (Golden et al., 2008). In pre-existing diabetes, however, a reciprocal relationship is likely to exist between depression and glycemic control: depression may lead to poor self care and increased risk for complications, in turn, the declination of physical condition may be demoralizing and depressogenic. In the absence of detailed emotional state progression, the nature of relationship between glycemic control and the development of depressive symptoms could not be characterized in our population. Third, we did not discriminate between different types of DM in the association analyses, therefore specic factors of type 1 and type 2 DM could not be measured. On the other hand, the distribution of the two types of DM in our patient sample represents their prevalence in the Hungarian diabetic population, and gives us a general picture about primary care diabetic patients. 5. Conclusion The present study describes association analyses of depressionlinked genetic polymorphisms in diabetic patients. We have conrmed the association between P2RX7 Gln460Arg polymorphism and depression, measuring anxiety and depressive symptoms among patients 2times more prone to develop mood disorders compared to the general population. Subsequent studies are needed to clarify the role of glycemic control on the development of depressive symptoms by comparing the genetic effects in patients with diabetes mellitus and other chronic medical condition. Acknowledgement This work was supported by Hungarian funds ETT 448/2006 (Somogyi A) and KPI HEF_06_1-SZER_POL (Nemoda Z). Zsoa Nemoda acknowledges the nancial support of Janos Bolyai Research

1888

G. Nagy et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 32 (2008) 18841888 Lesch KP, Bengel D, Heils A, Sabol SZ, Greenberg BD, Petri S, et al. Association of anxietyrelated traits with a polymorphism in the serotonin transporter gene regulatory region. Science 1996;274:152731. Levinson DF. The genetics of depression: a review. Biol Psychiatry 2006;60:8492. Lotrich FE, Pollock BG. Meta-analysis of serotonin transporter polymorphisms and affective disorders. Psychiatr Genet 2004;14:1219. Lucae S, Salyakina D, Barden N, Harvey M, Gagne B, Labbe M, et al. P2RX7, a gene coding for a purinergic ligand-gated ion channel, is associated with major depressive disorder. Hum Mol Genet 2006;15:243845. Lustman P, Carney R, Amado H. Acute stress and metabolism in diabetes. Diabetes Care 1981;4:6589. Lustman PJ, Grifth LS, Freedland KE, Clouse RE. The course of major depression in diabetes. Gen Hosp Psychiatry 1997;19:13843. Lustman PJ, Anderson RJ, Freedland KE, de Groot M, Carney RM, Clouse RE. Depression and poor glycemic control: a meta-analytic review of the literature. Diabetes Care 2000;23:93442. McQuillin A, Bass NJ, Choudhury K, Puri V, Kosmin M, Lawrence J, et al. Case-control studies show that a non-conservative amino-acid change from a glutamine to arginine in the P2RX7 purinergic receptor protein is associated with both bipolarand unipolar-affective disorders. Mol Psychiatry 2008 Electronic publication. Muszbek K, Szekely A, Balogh EM, Molnar M, Rohanszky M, Ruzsa A, et al. Validation of the hungarian translation of hospital anxiety and depression scale. Qual Life Res 2006;15:7616. Offenbaecher M, Bondy B, de Jonge S, Glatzeder K, Kruger M, Schoeps P, et al. Possible association of bromyalgia with a polymorphism in the serotonin transporter gene regulatory region. Arthritis Rheum 1999;42:24828. Parsey RV, Hastings RS, Oquendo MA, Hu X, Goldman D, Huang YY, et al. Effect of a triallelic functional polymorphism of the serotonin-transporter-linked promoter region on expression of serotonin transporter in the human brain. Am J Psychiatry 2006;163:4851. Post RM. Role of BDNF in bipolar and unipolar disorder: clinical and theoretical implications. J Psychiatr Res 2007;41:97990. Reuter M, Kuepper Y, Hennig J. Association between a polymorphism in the promoter region of the TPH2 gene and the personality trait of harm avoidance. Int J Neuropsychopharmacol 2007;10:4014. Snaith RP. The Hospital Anxiety And Depression Scale. Health Qual Life Outcomes 2003;1:29. Sperlgh B, Vizi ES, Wirkner K, Illes P. P2X7 receptors in the nervous system. Prog Neurobiol 2006;78:32746. Sperlgh B, Heinrich A, Cslle C. P2 receptor-mediated modulation of neurotransmitter release an update. Purinergic Signal 2007;3:26984. Testa MA, Simonson DC. Health economic benets and quality of life during improved glycemic control in patients with type 2 diabetes mellitus: a randomized, controlled, double-blind trial. JAMA 1998;280:14906. Walther DJ, Peter JU, Bashammakh S, Hortnagl H, Voits M, Fink H, et al. Synthesis of serotonin by a second tryptophan hydroxylase isoform. Science 2003;299:76. Wendland JR, Martin BJ, Kruse MR, Lesch KP, Murphy DL. Simultaneous genotyping of four functional loci of human SLC6A4, with a reappraisal of 5-HTTLPR and rs25531. Mol Psychiatry 2006;11:2246. Zalsman G, Huang YY, Oquendo MA, Burke AK, Hu XZ, Brent DA, et al. Association of a triallelic serotonin transporter gene promoter region (5-HTTLPR) polymorphism with stressful life events and severity of depression. Am J Psychiatry 2006;163: 158893. Zigmond AS, Snaith RP. The hospital anxiety and depression scale. Acta Psychiatr Scand 1983;67:36170.

Fellowship of Hungarian Academy of Sciences. We thank Krisztina Lakatos (Institute for Psychology, Hungarian Academy of Sciences) for valuable discussions regarding statistical analyses, and all the patients who made this research possible. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.pnpbp.2008.08.021. References
Anderson RJ, Freedland KE, Clouse RE, Lustman PJ. The prevalence of comorbid depression in adults with diabetes: a meta-analysis. Diabetes Care 2001;24:106978. Barden N, Harvey M, Gagne B, Shink E, Tremblay M, Raymond C, et al. Analysis of single nucleotide polymorphisms in genes in the chromosome 12Q24.31 region points to P2RX7 as a susceptibility gene to bipolar affective disorder. Am J Med Genet (Neuropsychiatr Genet) 2006;141:37482. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Statist Soc B 1995;57:289300. Brown SM, Peet E, Manuck SB, Williamson DE, Dahl RE, Ferrell RE, et al. A regulatory variant of the human tryptophan hydroxylase-2 gene biases amygdala reactivity. Mol Psychiatry 2005;10:8848. Caspi A, Sugden K, Moftt TE, Taylor A, Craig IW, Harrington H, et al. Inuence of life stress on depression: moderation by a polymorphism in the 5-HTT gene. Science 2003;301:3869. Ciechanowski PS, Katon WJ, Russo JE. Depression and diabetes: impact of depressive symptoms on adherence, function, and costs. Arch Intern Med 2000;160:327885. Denlinger LC, Coursin DB, Schell K, Angelini G, Green DN, Guadarrama AG, et al. Human P2X7 pore function predicts allele linkage disequilibrium. Clin Chem 2006;52:9951004. Egan MF, Kojima M, Callicott JH, Goldberg TE, Kolachana BS, Bertolino A, et al. The BDNF val66met polymorphism affects activity-dependent secretion of BDNF and human memory and hippocampal function. Cell 2003;112:25769. Golden SH, Lazo M, Carnethon M, Bertoni AG, Schreiner PJ, Roux AV, et al. Examining a bidirectional association between depressive symptoms and diabetes. JAMA 2008;299:27519. Groves JO. Is it time to reassess the BDNF hypothesis of depression? Mol Psychiatry 2007;12:107988. Gutknecht L, Jacob C, Strobel A, Kriegebaum C, Muller J, Zeng Y, et al. Tryptophan hydroxylase-2 gene variation inuences personality traits and disorders related to emotional dysregulation. Int J Neuropsychopharmacol 2007;10:30920. Hejjas K, Szekely A, Domotor E, Halmai Z, Balogh G, Schilling B, et al. Association between depression and the Gln460Argpolymorphism of P2RX7 gene: a dimensional approach. Am J Med Genet (Neuropsychiatr Genet) 2008 Electronic publication. Hu XZ, Lipsky RH, Zhu G, Akhtar LA, Taubman J, Greenberg BD, et al. Serotonin transporter promoter gain-of-function genotypes are linked to obsessivecompulsive disorder. Am J Hum Genet 2006;78:81526. Jans LA, Riedel WJ, Markus CR, Blokland A. Serotonergic vulnerability and depression: assumptions, experimental evidence and implications. Mol Psychiatry 2007;12:52243. Krabbe K, Nielsen A, Krogh-Madsen R, Plomgaard P, Rasmussen P, Erikstrup C, et al. Brainderived neurotrophic factor (BDNF) and type 2 diabetes. Diabetologia 2007;50:4318.