Functional AGXT2 SNP rs180749 variant and depressive symptoms: Baseline data from the Aidai Cohort Study in Japan

No study has shown the relationship between alanine-glyoxylate aminotransferase 2 (AGXT2) single nucleotide polymorphisms (SNPs) and depressive symptoms. The present case–control study examined this relationship in Japanese adults. Cases and control participants were selected from those who participated in the baseline survey of the Aidai Cohort Study, which is an ongoing cohort study. Cases comprised 280 participants with depressive symptoms based on a Center for Epidemiologic Studies Depression Scale (CES-D) score ≥ 16. Control participants comprised 2034 participants without depressive symptoms based on the CES-D who had not been diagnosed by a physician as having depression or who had not been currently taking medication for depression. Adjustment was made for age, sex, smoking status, alcohol consumption, leisure time physical activity, education, body mass index, hypertension, dyslipidemia, and diabetes mellitus. Compared with the GG genotype of rs180749, both the GA and AA genotypes were significantly positively associated with the risk of depressive symptoms assessed by the CES-D: the adjusted odds ratios for the GA and AA genotypes were 2.83 (95% confidence interval [CI] 1.23–8.24) and 3.10 (95% CI 1.37–8.92), respectively. The TGC haplotype of rs37370, rs180749, and rs16899974 was significantly inversely related to depressive symptoms (crude OR 0.67; 95% CI 0.49–0.90), whereas the TAC haplotype was significantly positively associated with depressive symptoms (crude OR 1.24; 95% CI 1.01–1.52). This is the first study to show significant associations between AGXT2 SNP rs180749, the TGC haplotype, and the TAC haplotype and depressive symptoms.


Introduction
Major depressive disorder (MDD) is a common psychiatric disorder.Environmental and genetic factors are interrelated in the development of MDD.Environmental factors such as emotional abuse and neglect were found to be related to the risk for depressive symptoms (Mandelli et al. 2015;Kwong et al. 2019).As for genetic factors, 102 independent variants and 269 genes were identified in a genome-wide meta-analysis of MDD (Howard et al. 2019).Moreover, recent findings have indicated that epigenetics, including DNA methylation, non-coding RNAs, and histone modifications, are related to the pathogenesis of MDD (Penner-Goeke and Binder 2019; Ménard et al. 2016).
AGXT2 also regulates methylarginines such as asymmetric N G , N G -dimethyl-l-arginine (ADMA) and symmetric N G , N′ G -dimethyl-l-arginine (SDMA) (Ogawa et al. 1987).ADMA is an endogenous inhibitor of nitric oxide synthase (NOS).ADMA dose-dependently inhibits endothelial NOS (eNOS) and neuronal NOS (nNOS) activity in the presence of physiologic cellular l-arginine (Kielstein et al. 2007).AGXT2 impacts the nitric oxide (NO) pathway through the competitive inhibition of NOS by ADMA; as a result, AGXT2 can regulate blood pressure and be related to endothelial dysfunction leading to cardiovascular disease (Yoshino et al. 2021;Rochette et al. 2013).In MDD patients, elevated ADMA and decreased SDMA concentrations were reported (Baranyi et al. 2015).Moreover, decreased peripheral production of NO and plasma concentration of NO were found in MDD patients (Chrapko et al. 2006;Selley 2004).Because NO modulates release of neurotransmitters such as acetylcholine, catecholamines, serotonin, and histamine, reduction of NO in the brain may be related to the pathogenesis of MDD (Prast and Philippu 2001).
The Center for Epidemiologic Studies Depression Scale (CES-D), a short self-report for evaluating depressive symptomatology in the general population, has been widely used to measure depressive symptoms.In a meta-analysis of 28 studies, CES-D had acceptable screening accuracy in the general population (Vilagut et al. 2016) and was useful for adolescents and older adults (Blodgett et al. 2021;Cosco et al. 2020).The present case-control study was conducted to investigate the relationship between four functional AGXT2 SNPs and depressive symptoms based on the CES-D in Japanese adults using baseline data from the Aidai Cohort Study (AICOS).

Study population
The AICOS is an ongoing, population-based, prospective study that was started in 2015 (Fukui et al. 2019;Miyake et al. 2020Miyake et al. , 2021Miyake et al. , 2023;;Hara et al. 2020;Tanaka et al. 2021;Nobori et al. 2022;Kumon et al. 2022).The baseline survey of the AICOS was conducted in Yawatahama City in 2015, Uchiko Town in 2016, Seiyo City in 2017, and Ainan Town in 2017, with total populations of approximately 36,000, 17,000, 38,000, and 22,000, respectively; these four municipalities are among the 20 municipalities in Ehime Prefecture on Shikoku Island, situated south of Japan's main island.Study participants were recruited from individuals who had undergone health checkups conducted by their municipality of residence or through alternative recruitment procedures, and 798, 347, 524, and 755 study participants in Yawatahama City, Uchiko Town, Seiyo City, and Ainan Town, respectively, took part in the baseline survey of the AICOS.In addition, 223 employees of a manufacturing company (PHC Corporation) in Toon City, one of the municipalities in Ehime Prefecture, also participated in the AICOS.In total, 2,647 participants aged 33-89 years (1069 men aged 35-89 years and 1578 women aged 33-85 years) gave written, informed consent and completed a self-administered questionnaire; however, 268 of them had missing or incomplete data for the factors under study and were excluded from the present study.A Japanese version (Shima 1985) of the CES-D, a 20-item questionnaire (Radloff 1977), was incorporated into the questionnaire.Study subjects were asked to score the frequency of the occurrence of specific symptoms over the last week on a 4-point scale (0, "less than 1 day"; 1, "1-2 days"; 2, "3-4 days"; and 3, "5-7 days").All subscores were summed to yield a total score ranging from 0 to 60, and the presence of depressive symptoms was defined as a CES-D score ≥ 16 (Radloff 1977;Shima 1985).In the current case-control study, 280 cases of depressive symptoms based on a CES-D score ≥ 16 were identified.Of the 2099 remaining study participants who were eligible to serve as controls, 65 who were not considered to have depressive symptoms as defined by the CES-D criteria but who had been diagnosed by a physician as having depression or who had been currently taking medication for depression were excluded.The final analyses thus included 280 cases and 2034 control participants.The AICOS was approved by the ethics committee of the Ehime University Graduate School of Medicine (approval No. 1504012).

Measurements
Information on age, sex, smoking habits, alcohol drinking habits, physical activity, education, and current use of antihypertensive, cholesterol-lowering, and diabetic medications was also elicited using the questionnaire at baseline.Research technicians completed missing or illogical data by telephone interview.Never smoking was defined as having smoked fewer than 100 cigarettes in their lifetime.Former smoking was defined as having smoked more than 100 cigarettes in their lifetime but having quit smoking by the time of the survey.Current smoking was defined as having smoked more than 100 cigarettes in their lifetime and continuing to smoke at the time of the baseline survey.Leisure time physical activity was defined as at least 30 min of any type of moderate-to-vigorous physical activity, such as brisk walking, golf, gardening, jogging, or playing tennis, at least once a week.Body height and weight were measured in light clothing without shoes, and the body mass index (BMI) was computed by dividing body weight in kilograms by the square of the height in meters.Blood pressure was measured twice, each time with the study participant in the sitting position after at least 5 min of rest, using an automated sphygmomanometer.The second measurement was used for the present study.Hypertension was defined as current use of antihypertensive medication, systolic blood pressure ≥ 140 mmHg, or diastolic blood pressure ≥ 90 mmHg.After overnight fasting, blood samples were collected from an antecubital vein.Plasma glucose concentrations, hemoglobin A1c levels, serum low-density cholesterol, high-density lipoprotein cholesterol, and triglyceride concentrations were measured at an external laboratory (Shikoku Chuken, Ehime, Japan).Dyslipidemia was defined as current use of cholesterol-lowering medication, serum low-density lipoprotein cholesterol concentration ≥ 140 mg/dL, high-density lipoprotein cholesterol concentration < 40 mg/dL, or triglyceride concentration ≥ 150 mg/dL.DM was defined as current use of diabetic medication, fasting plasma glucose level ≥ 126 mg/dL, or hemoglobin A1c level ≥ 6.5%.

DNA extraction and genotyping
After 7 mL of blood samples treated with EDTA were stored at 2-5 °C within 3 days, blood samples were centrifuged at room temperature and the buffy coat was collected.Genomic DNA was prepared from the buffy coat using a DNA extraction kit (QuickGene-610L; FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) and stored at − 20 °C.According to the manufacturer's instructions, four AGXT2 SNPs, rs37370 (C__1018750_1_), rs37369 (C__11162986_1_), rs180749 (C__1018735_1_), and rs16899974 (C__25742181_10), were genotyped using the prestandardized and experimentally validated TaqMan SNP genotyping assay on the Ste-pOnePlus machine (Applied Biosystems, Foster City, CA, USA).

Statistical analysis
SNPs under study were tested for deviation from Hardy-Weinberg equilibrium using the Chi-squared test.Linkage disequilibrium analysis was performed using Haploview software version 4.2 (Broad Institute, Cambridge, MA) (Barrett et al. 2005).Logistic regression analysis was performed to estimate crude odds ratios (ORs) and 95% confidence intervals (CIs) for depressive symptoms relative to the SNPs under investigation, with the reference category being the homozygote of the ancestral allele based on the National Center for Biotechnology Information SNP database.Multiple logistic regression analysis was performed to adjust for age, sex, smoking status, alcohol consumption, leisure time physical activity, education, BMI, hypertension, dyslipidemia, and DM.Haplotypes and their frequencies were inferred by means of the expectation maximization algorithm using STATA/MP software version 17.0 (Stata-Corp, College Station, TX, USA).For differences in haplotype frequencies between the cases and controls, crude ORs and 95% CIs were estimated based on the frequency of each haplotype relative to all other haplotypes combined.Except for the calculation of linkage disequilibrium and haplotype analysis, all analyses were conducted using the SAS software package version 9.4 (SAS Institute, Inc., Cary, NC, USA).

Comparison of cases with control subjects
Compared with the control participants, cases were more likely to be younger and female and were less likely to report current alcohol consumption and leisure time physical activity (Table 1).No differences were found between cases and controls with respect to smoking status, education, BMI, hypertension, dyslipidemia, and DM.

Relationship between AGXT2 polymorphisms and depressive symptoms
Compared with study participants with the GG genotype of AGXT2 SNP rs180749, those with the GA and AA genotypes had a significantly increased risk of depressive symptoms (Table 3).After adjustment for confounders under study, the positive associations were slightly strengthened: the adjusted ORs for the GA and AA genotypes were 2.83 (95% CI 1.23-8.24)and 3.10 (95% CI 1.37-8.92),respectively, with rs37370 and rs16899974 not materially associated with the risk of depressive symptoms in the multivariate model.

Haplotype analysis of three AGXT2 polymorphisms in relation to depressive symptoms
In both cases and controls, seven haplotypes with a frequency of 1% or more were detected (Table 4).Given that the haplotype order was rs37370, rs180749, and rs16899974, the TGC haplotype was significantly related to a reduced risk of depressive symptoms in comparison with all other haplotypes combined: the crude OR was 0.67 (95% CI 0.49-0.90).In contrast, the TAC haplotype was significantly positively associated with the risk of depressive symptoms compared with all other haplotypes combined: the crude OR was 1.24 (95% CI 1.01-1.52).

Discussion
To the best of our knowledge, the current case-control study is the first to investigate the relationship between AGXT2 SNPs (rs37370, rs180749, and rs16899974) and depressive symptoms.The present study showed that the GA and AA genotypes of rs180749 were significantly positively associated with the risk of depressive symptoms compared with the GG genotype.No relationships  were found between AGXT2 SNPs rs37370 or rs16899974 and depressive symptoms.Haplotype analyses identified a significant inverse association between the TGC haplotype of rs37370, rs180749, and rs16899974, occurring in 13.5% of the controls, and depressive symptoms and a significant positive relationship between the TAC haplotype, occurring in 23.0% of the controls, and depressive symptoms.
The GA and AA genotypes of rs180749 are related to loss of AGXT2 activity (Yoshino et al. 2014a) and can cause ADMA metabolic attenuation.Elevated plasma ADMA levels and decreased circulating NO levels were shown in Agxt2-knockout mice (Caplin et al. 2012).Overexpression of Agxt2 in vitro conversely increased NO production and protected endothelial cells from ADMA-induced inhibition of NO production (Rodionov et al. 2010).In fact, decreased cerebral blood flow in the right anterior cingulate and frontal gyri was shown in persons with MDD (Monkul et al. 2012).Moreover, cerebrovascular disease identified as white matter hyperintensities on T2-weighted MRI was also related to depression, especially late-life depression, and cognitive dysfunction (Taylor et al. 2013).Because ADMA impacts to the regulation of blood pressure, AGXT2 might be related to depressive symptoms through the change of ADMA metabolism that can modify cerebral blood flow (Kielstein et al. 2006).
Although AGXT2 haplotypes also may impact to depressive symptoms via the ADMA metabolism, there is no report on how AGXT2 haplotypes of rs37370, rs180749, and rs16899974 affects the AGXT2 function and the ADMA metabolism.
Two methodological strengths of the current study are that the study participants were homogeneous with respect to their residential area and that adjustment was made for several confounders.
There were several limitations of the present study.The participants were probably not representative of the Japanese general population because the participation rate was low in the baseline survey of the AICOS.For example, the educational level of the present participants was higher than that of the general population.According to a population census conducted in 2010 in Ehime Prefecture (Statistics Bureau Ministry of Internal Affairs and Communications 2012 2010), the proportions of persons aged 60-69 years with low, medium, high, and unknown educational levels were 28.2%, 48.6%, 19.0%, and 4.2%, respectively, in men, and 26.7%, 56.4%, 12.9%, and 4.0%, respectively, in women.The corresponding figures in the present study for persons aged 60-69 years were 11.8%, 55.3%, 32.9%, and 0.0%, respectively, in men, and 16.3%, 52.4%, 31.3%, and 0.0%, respectively, in women.On the other hand, the distributions of all three SNPs under study were within Hardy-Weinberg equilibrium, and any selection bias in relation to genotype distribution was negligible.
A score higher than 15 on the CES-D is considered to indicate suspected MDD (Li and Hicks 2010;Radloff 1977), but not all cases are diagnosed as MDD because an actual diagnosis of MDD should be made by psychiatrists.Moreover, the cutoff score of 16 on the CES-D may be inadequate for certain sex and racial/ethnic groups to evaluate depressive symptoms (Henry et al. 2018).The possibility of nondifferential outcome misclassification would introduce a bias toward the null.The number of study subjects was rather small for a valid genetic association study; however, significant associations were detected.Although adjustment was made for some confounders, residual confounding effects could not be ruled out.

Conclusions
The present case-control study showed that the GA and AA genotypes of AGXT2 SNP rs180749 and the TAC haplotype of rs37370, rs180749, and rs16899974 were significantly associated with an increased risk of depressive symptoms based on the CES-D, whereas the TGC haplotype of these three SNPs was significantly related to a decreased risk of depressive symptoms.These findings should be confirmed by further epidemiological studies with larger numbers of participants.To clarify the relationship between AGXT2 and depressive symptoms, further studies of the molecular biological mechanisms are also desirable.Functional AGXT2 SNP rs180749 variant and depressive symptoms: Baseline data from the Aidai… Medicine (approval No. 1504012).Written, informed consent was obtained from all participants.This study was performed in accordance with the Declaration of Helsinki.
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Table 1
Characteristics of the study populationSD standard deviation a Chi-squared test or Wilcoxon rank-sum test Two-sided P values less than 0.05 were considered statistically significant.

Table 2
Select tagging AGXT2 SNPs and pairwise linkage disequilibrium of AGXT2 polymorphisms MAF minor allele frequency Two-sided P values less than 0.05 were considered statistically significant.(A) Main characteristics of the select tagging AGXT2 SNPs in control subjects

Table 3
b Adjusted for age, sex, smoking status, alcohol consumption, leisure time physical activity, education, body mass index, hypertension, dyslipidemia, and diabetes mellitus.Bold text indicates statistical significanceCES-D Center for Epidemiologic Studies Depression Scale, CI confidence interval, OR odds ratio

Table 4
Haplotype analysis of three AGXT2 polymorphisms in relation to depressive symptoms