Association Between a G-Protein Β3 Subunit Gene Polymorphism and The

Association between a G-protein b3 subunit gene polymorphism and the symptomatology and treatment responses of major depressive disorders

H-J Lee1 ABSTRACT 1 The genes involved in signal transduction are major candidates in association J-H Cha studies on affective disorders and responses to antidepressants. We 1 B-J Ham investigated whether the C825T polymorphism of the b3 subunit of G C-S Han1 protein (GNB3) gene is associated with the symptom severity or treatment Y-K Kim1 response of major depressive disorders (MDDs) in a Korean sample of 106 S-H Lee2 MDD patients; our study also included 133 healthy controls. Hypertensive 3 subjects were excluded from the study because association between GNB3 S-H Ryu variants and hypertension has been reported in previous studies. We found R-H Kang1 significantly more carriers of the 825T allele in MDD patients than in normal M-J Choi1 controls (w2 ¼ 6.37, P ¼ 0.012; OR ¼ 2.19, 95% CI 1.18–4.05). The T-allele M-S Lee1 carriers showed higher scores than those with the CC genotype in the baseline total and in some subcategories of the Hamilton Depression Rating 1Department of Psychiatry, Korea University Scale (Po0.05). We also found a statistically significant association between College of Medicine, Seoul, Korea; 2Department T-allele carriers and antidepressant treatment response (Po0.05). These of Psychiatry, Inje University College of Medicine, results suggest that the T allele of the C825T polymorphism in the GNB3 3 Seoul, Korea; Department of Psychiatry, Konkuk gene is associated with MDD. It was also demonstrated that MDD patients University College of Medicine, Seoul, Korea bearing the T allele had a severe symptomatology and a better response to Correspondence: antidepressant treatment than patients without the T allele. Dr M-S Lee, Department of Psychiatry, The Pharmacogenomics Journal (2004) 4, 29–33. doi:10.1038/sj.tpj.6500217 Anam Hospital, Korea University College of Published online 2 December 2003 Medicine, 126-1, 5-Ka, Anam-Dong, Sungbuk-Ku, Seoul 136-705, Korea. Tel: þ 82-2-9205815 Keywords: G protein; GNB3; polymorphism; major depressive disorder; antidepres- Fax: þ 82-2-9272836 sants; treatment response E-mail: [email protected]

INTRODUCTION Hundreds of cell-surface receptors for neurotransmitters and other ligands use G proteins to transduce intracellular signaling pathways. G proteins play important roles in determining the specificity and temporal characteristics of cellular responses to signals. Gb proteins form dimeric complexes with Gg subunits that interact with Ga subunits to generate a heterotrimeric G protein. Upon receptor activation, G proteins dissociate into free Ga and Gbg subunits that can activate various effectors. Effector proteins of the Gbg complex include phospholipases, adenylyl cyclases, ion channels, G-protein-coupled receptor kinases, and Received: 19 June 2003 phosphoinositide-3 kinases.1,2 Revised: 05 October 2003 3 Accepted: 14 October 2003 Recently, Siffert et al detected a single-nucleotide polymorphism (SNP) of Published online 2 December 2003 C825T in exon 10 of the gene encoding the b3 subunit of G protein (GNB3). The Association between GNB3 gene and depression H-J Lee et al 30

T allele of this SNP is associated with the occurrence of a df ¼ 1, P ¼ 0.041; OR ¼ 1.46, 95% CI 1.02–2.10). The geno- splice variant (Gb3s) that leads to the deletion of 41 amino types of C825T SNP were classified into two groups: T þ (TT acids. The Gb3s variant has been reported to be not only and CT genotypes) and TÀ (CC genotype), because the associated with increased signal transduction and ion expression of the more active splice variant of GNB3 transport, but also with pathophysiological conditions such depends on the presence of at least one T allele.3 At least as hypertension3–7 and obesity.8,9 one copy of the T allele (T þ ) was carried by 82.1 and 67.7% More recently, Zill et al10 found an association between of those in the MDD and control groups, respectively, and the C825T SNP of GNB3 and depression. They reported that the presence of T allele (T þ ) was associated with an the frequency of the T allele was significantly higher in increased likelihood of MDD (w2 ¼ 6.37, df ¼ 1, P ¼ 0.012; depressive patients than in healthy controls and schizo- OR ¼ 2.19, 95% CI 1.18–4.05) (Table 1). phrenic patients. They also found a significant association Table 2 presents a comparison of characteristics between between the TT genotype and responses to antidepressant genotype groups of the MDD patients, including sex, age, treatment. Most recently, Serretti et al11 reported the same baseline symptom clusters, and therapeutic response. Sig- results on treatment response to selective serotonin reuptake nificant differences between the two subject groups were inhibitors in a large sample of depressive Italian patients. present in the baseline total, core, psychic anxiety, and Bondy et al12 also reported a significant increase in the delusion HAM-D scores, and in the baseline CGI-S scores GNB3T allele in depressive disorder, and that the TT (Table 2). A total of 106 of the MDD patients completed 8 genotype in the presence of the D allele of the angioten- weeks of antidepressant treatment and underwent evalua- sin-I-converting-enzyme gene is associated with a more than tion using the HAM-D and CGI-S scores. There were no five-fold increase in the risk of major depression. However, significant differences in end-point (8-week) total and no association between depression and the GNB3T allele was subcategory HAM-D scores (total HAM-D scores; found in an Asian population.13,14 TT ¼ 7.376.4, TC ¼ 6.574.9, CC ¼ 8.776.2; F ¼ 0.97, In the present study, the relationships between the C825T P ¼ 0.38; the other data were not presented). However, the SNP of the GNB3 gene and the clinical symptoms of major percentage changes in the HAM-D (total and psychic depressive disorders (MDDs) in a Korean sample of 106 MDD anxiety) score and CGI-S score after 8-week treatment were patients are compared with 133 healthy controls. In higher for the T þ genotype group than for the TÀ group addition, the association between the C825T SNP and (Table 2). Further, the responder percentage was higher for antidepressant therapeutic response was tested. the T þ group than for the TÀ group. Interestingly, however, frequencies of remitters were not significantly different RESULTS between the subject groups (Table 2). The genotypes and allele distributions for the GNB3 C825T SNP for the MDD patients and controls are given in Table 1. DISCUSSION The sex distributions of the MDD patients and the control The results of our study suggest that the T allele of a population were not different (male : female, MDD 28 : 78, functional polymorphism (C825T) in the GNB3 gene is controls 44 : 89; w2 ¼ 1.25, P ¼ 0.26), but their age distribu- associated with MDD. It was also demonstrated that MDD tions were different (MDD, 47.1713.3 years; controls, patients bearing the T allele had a more severe symptoma- 43.4710.2 years; t ¼ 2.32, P ¼ 0.021). The distributions of tology and a better response to antidepressant treatment the C825T SNP genotypes for these two groups were in than patients without it. Although this finding is consistent Hardy–Weinberg equilibrium (MDD: w2 ¼ 2.04, df ¼ 1, with previous studies in Caucasians,4,10,12 it differs from that P ¼ 0.15; controls: w2 ¼ 0.41, df ¼ 1, P ¼ 0.52). in a Japanese population.13 There was a significant difference in the genotype The frequencies of the T allele in Asian populations (42– distribution between MDD patients and the controls 53%) are different from those in Caucasians (27–42%). In (w2 ¼ 6.37, df ¼ 2, P ¼ 0.041). Comparison of allele frequen- the Japanese sample of the study of Kunugi et al,13 the cies revealed an excess of T allele in MDD patients (w2 ¼ 4.19, frequency of the T allele was 0.53 in both depressive and

Table 1 Genotypes, allele, and allele carrier frequencies of C825T polymorphism of the G-protein b3 subunit among patients with major depressive disorder and normal controls

Genotypes Allele frequencies Allele carrier frequencies

TT CT CC T C T+ TÀ

Major depressive disorder (n ¼ 106) 27 (25.5%) 60 (56.6%) 19 (17.9%) 0.54 0.46 0.82 0.18 Controls (n ¼ 133) 28 (21.1%) 62 (46.6%) 43 (32.3%) 0.44 0.56 0.68 0.32

T+, allele subjects include TT or CT genotypes; TÀ, allele subjects include CC genotype only. Genotypes: w2 ¼ 6.37, df ¼ 2, P ¼ 0.041; alleles: w2 ¼ 4.19, df ¼ 1, P ¼ 0.041 (OR ¼ 1.46, 95% CI 1.02–2.10); allele carriers: w2 ¼ 6.37, df ¼ 1, P ¼ 0.012 (OR ¼ 2.19, 95% CI 1.18–4.05).

The Pharmacogenomics Journal Association between GNB3 gene and depression H-J Lee et al 31

Table 2 Demographic data and antidepressant treatment response among C825T SNP genotypes and allele carrier status in patients with major depressive disorder

TT TC CC ANOVA T+ vs TÀ

FPP

Sex (F/M) 6/21 16/44 6/13 w2 ¼ 0.51 0.78 0.57 Age 46.3 (11.7) 47.5 (12.9) 46.9 (17.1) 0.82 0.92 0.94 Baseline HAM-D Core 12.9 (3.9)a 11.6 (3.1)b 9.5 (2.6)a,b 4.98 0.009 0.007 Sleep 3.5 (1.7) 3.7 (2.5) 3.1 (1.7) 0.62 0.54 0.325 Activity 4.0 (1.8) 3.8 (1.6) 3.9 (1.1) 0.16 0.85 0.887 Psychic anxiety 4.1 (1.5)a 3.5 (1.5) 2.6 (1.1)a 4.26 0.017 0.016 Somatic anxiety 4.1 (1.4) 4.2 (1.4) 4.2 (1.4) 0.024 0.98 0.991 Delusion 3.0 (1.4) 3.1 (1.4) 2.1 (1.6) 2.95 0.058 0.017 Total 26.8 (7.0)a 25.5 (6.7)b 21.4 (4.9)a,b 3.41 0.038 0.014 Baseline CGI-S 4.5 (1.0)a 4.5 (1.0)b 3.8 (0.8)a,b 3.12 0.049 0.014 Score change of HAM-D (%)c Core 71.7 (27.5) 69.2 (25.3) 56.2 (30.1) 1.80 0.17 0.065 Sleep 73.3 (37.4) 85.2 (28.6) 71.0 (38.0) 1.48 0.24 0.276 Activity 68.9 (35.9) 69.9 (31.6) 64.2 (36.7) 0.18 0.84 0.559 Psychic anxiety 71.6 (32.0)a 69.4 (32.8)b 27.9 (72.1)a,b 6.45 0.002 0.036 Somatic anxiety 71.1 (34.1) 63.4 (32.4) 55.4 (35.7) 1.00 0.37 0.279 Delusion 79.4 (26.7) 80.6 (26.3) 73.1 (43.9) 0.33 0.72 0.424 Total 70.8 (27.0) 73.0 (20.4) 57.1 (30.4) 2.71 0.072 0.023 Score change of CGI-S (%)c 53.2 (24.2) 51.8 (19.6) 40.3 (23.4) 2.06 0.13 0.046 Responders/nonresponders 17/10 42/18 8/11 w2 ¼ 4.83 0.089 0.035 Remitters/nonremitters 14/13 37/23 9/10 w2 ¼ 1.53 0.46 0.37

Data are reported as mean (SD); T+, allele subjects include TT or CT genotypes; TÀ, allele subjects include CC genotype only. Responders were defined as at least 50% decrease in the HAM-D total score after 8 weeks of medication. Remitters were defined as subjects having a HAM-D total score of seven or less points after 8 weeks of medication. Bold represents Po0.05. a,bMeans significantly different 2 Â 2, according to LSD post hoc test. cScore change (%) ¼ (baseline scoreÀ8-week score) Â 100/baseline score. control groups. However, in our Korean sample, the symptoms, especially in core depressive symptom, psychic frequencies of the T allele were significantly different anxiety, and delusion. We also suggest that the Gb3s variant between depressive and control groups, at 0.54 and 0.44, is related to better and faster treatment responses due to respectively. Unfortunately, GNB3T allele frequency has enhanced signal transduction. It should be pointed out that never been reported in Korean population previously, so the GNB3 825T allele carrier status was not related to the we could not compare our data with other Korean data. T- remission rate after the 8-week antidepressant treatment. allele frequency of 0.44 in our controls is similar to those in This result indicates that the 825T allele of GNB3 is controls of a Taiwanese (43.5%)15 and a Chinese (47.7%)8 associated with the severity of depressive disorder and its population, although it is somewhat different from another improvement by antidepressant treatment, but not with Taiwanese study (50.0%).14 Thus, we cautiously suggest that end-point outcome after 8 weeks of antidepressant it is unlikely that the allele frequencies obtained in this treatment. study were due to sampling problems or genotyping errors. It is also interesting that significant effects of T-allele The T allele was associated with Gb3s, a splice variant of carriers on depression and therapeutic response in our study GNB3 in which nucleotides 498–620 of exon 9 were deleted. might support the dominant effect of the Gb3s variant, as This deletion causes the loss of 41 amino acids, producing a has been reported previously.3 On the other hand, some shortened, more active, splice variant of GNB3. Evidence studies have suggested a recessive effect concerning the from transient-expression experiments suggests that Gb3s is therapeutic response of antidepressants.10,11 a biologically active protein that enhances signal transduc- There are several limitations in this study. Firstly, the age tion.3,16,17 Recently, Rosskopf et al18 reported that Gb3s- distribution differed between MDD patients and controls, in containing G protein results in an increased number of that the mean age of the controls was substantially lower activated Gas and free Gbg dimers, which subsequently than that of patients. It is possible that some individuals in activate the downstream effector systems. the control group will develop depressive disorder in later From the results of the present study, we hypothesize that life, which may have lessened the difference between two the Gb3s variant related with the 825T allele is associated groups. Secondly, we did not control the use of medication, with the vulnerability to depression and severe depressive and hence we could not completely exclude the effects of

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the different medication; however, there was no significant subjects were excluded because they showed a past history difference in the classes of medications used. Furthermore, or family history of substance abuse/dependence or major because all antidepressants have direct or indirect effects on psychiatric disorders (eg, schizophrenia or mood disorders). G proteins, our results are valuable, irrespective of the Venous blood was drawn from each individual after antidepressant administered. Thirdly, another limitation is obtaining written informed consent using a protocol that hypertensive patients were excluded in our samples. approved by the Ethics Committee of the Korea University Although the association data on hypertension and GNB3 Medical Center. All of the sample population was Korean. are divergent, the possibility remains that GNB3 825T alleles The 106 MDD patients received various classes of are enriched in hypertensive individuals. Since hyperten- pharmacological treatment: 42.5% received selective sero- sion prevalence increases with age, excluding hypertensives tonin reuptake inhibitors, 27.4% received serotonin and in control and MDD, patient groups which differ by 4 years noradrenaline reuptake inhibitors, 18.9% received mirtaza- could confound the outcome of this study. Fourthly, the pine, 9.4% received nefazodone, and 1.9% received tricyclic relatively small sample size limits the generalizability of our antidepressants. findings. Future studies should use a larger-sized sample, The therapeutic response was evaluated by calculating and should control the drugs as well as other confounding percentage reductions in total and subcategory HAM-D factors. scores ((baseline scoreÀ8-week score) Â 100/(baseline In conclusion, our results might suggest that the investi- score)). Responders were defined as those with a minimum gated GNB3 C825T polymorphism is a predisposing factor 50% decrease in the HAM-D total score after 8 weeks of for MDD, and is associated with the depressive symptom medication, while remitters were defined by a HAM-D total severity. We also found that the 825T allele was statistically score of seven points or less after 8 weeks of medication. significantly associated with a better clinical response to Ratings were assigned blind to genotyping. antidepressant treatment. These results can be explained by more rapidly occurring adaptive changes in 825T allele Genotyping carrier with increased signal transduction. Genomic DNA was extracted from leukocytes using a QIAamp Blood Kit (Qiagen, Germany). A polymerase- chain-reaction-based method was used for genotyping of MATERIALS AND METHODS the C825T polymorphism in GNB3 gene, as described Subjects previously.3,10 A total of 106 MDD patients were included in this study (age, 47.1713.3 (mean7SD) years; age at onset, 43.3714.6 years; male : female, 28 : 78). It was the first episode of MDD Statistical analyses in 16% of the patients, while the other 84% had several The presence of Hardy–Weinberg equilibrium was tested 2 episodes before (mean 3.4173.53). The proportion of out- with the w -test for goodness of fit. To evaluate specific patients vs inpatients was 79 and 21%, respectively. depressive-cluster symptoms, the HAM-D items were All subjects were examined by trained psychiatrists using grouped according to the following factors, as described by 26 the Structured Clinical Interview of the Korean version of Serretti et al: core (items 1, 2, 7, 8, 10, 13), sleep (items 4– the DSM-IV,19 leading to diagnoses according to DSM-IV 6), activity (items 7, 8), psychic anxiety (items 9, 10), criteria.20 The severity of depression was assessed using the somatic anxiety (items 11, 12, 13), and delusion (items 2, 15, 2 21-item Hamilton Depression Rating Scale (HAM-D)21 and 20). Categorical data were analyzed using the w -test. the Clinical Global Impressions of Severity (CGI-S) scale.22 Differences for continuous variables were evaluated using Only subjects with a minimum score of 18 on the HAM-D Student’s t-test or one-way analysis of variance, followed by entered the study. Patients with severe medical illness were the LSD multiple-range test for comparison among groups. excluded in order to avoid cases with secondary depression. The cutoff P-value was set at 0.05. A correction for multiple Patients with hypertension were also excluded, based on the testing was not performed because of the explorative findings that 825T allele of GNB3 is associated with approach used for a genetically complex trait in which the hypertension,3–7 although contradictive negative results relationship between genotype and phenotype has not been have also been reported.23–25 Subjects with a history of established, and thus such corrections might inappropri- alcohol or drug dependence, neurological disorders, or any ately increase the likelihood that real effects will be missed 27 concomitant DSM-IV axis-I psychiatric disorders were also (ie, increased type-II error rates). Statistical analyses were excluded. The control group of normals included 133 carried out using SPSS for Microsoft Windows. randomly selected individuals, who visited the hospital for regular health screening (a free biannual government- ACKNOWLEDGEMENTS funded service provided to all adult Koreans). This work was supported by a grant of the Korea Health 21 R&D After an initial examination of 185 case records, 30 Project, Ministry of Health & Welfare, Republic of Korea (KPGRN- R-04). subjects with a past history of major medical illness including hypertension or abnormal laboratory results were excluded. In all, 155 physically healthy subjects were DUALITY OF INTEREST interviewed by well-trained psychiatrists, of whom 22 None declared.

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ABBREVIATIONS 12 Bondy B, Baghai TC, Zill P, Bottlender R, Jaeger M, Minov C et al. Combined action of the ACE D- and the G-protein beta3 T-allele in CGI-S clinical global impression of severity major depression: a possible link to cardiovascular disease? Mol DNA deoxyribonucleic acid Psychiatry 2002; 7: 1120–1126. GNB3 b3 subunit of G protein 13 Kunugi H, Kato T, Fukuda R, Tatsumi M, Sakai T, Nanko S. Association HAM-D Hamilton Depression Rating Scale study of C825T polymorphism of the G-protein beta3 subunit gene MDD major depressive disorder with schizophrenia and mood disorders. J Neural Transm 2002; 109: SNP single-nucleotide polymorphism 213–218. 14 Lin CN, Tsai SJ, Hong CJ. Association analysis of a functional G protein beta3 subunit gene polymorphism (C825T) in mood disorders. Neuropsychobiology 2001; 44: 118–121. 15 Chang HW, Yen CY, Liu SY, Singer G, Shih IeM. Genotype analysis using REFERENCES human hair shaft. 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