The subjects of the present study consisted of 125 unrelated patients with MDD, who were recruited from the Han population of Jiangsu (China) between 2010 and 2013. All patients (58 males, 67 females) were hospitalized in the Department of Geriatric Psychiatry, Wuxi Psychiatric Hospital (Wuxi, China). Diagnosis of MDD was confirmed using the Mini International Neuropsychiatric Interview, and by a minimum score of Hamilton Depression Rating Scale (HDRS) (25,26). All cases met the DSM-IV diagnostic criteria for MDD and were severe enough to require follow-up in a specialized psychiatric outpatient clinic at the Department of Geriatric Psychiatry, Wuxi Psychiatric Hospital (27,28). The HAMA scale was used to assess the severity of symptoms (29) by means of structured questionnaires, information on specific demographic and clinical variables, including family history of suicide was obtained.

A total of 50 MDD + suicide patients (defined as the MDD + suicide attempts group; 23 males, and 27 females) who were consecutively admitted to our psychiatric departments following a suicide attempt, were included in the present study. Suicide was defined as intentional self-harm, in order to end one’s life. The remaining 75 MDD control patients (defined as the MDD group; 35 males, and 40 females) who had not attempted suicide were selected for the present study. Written informed consent was obtained from each patient, and the protocol of the study was approved by the local ethics committee of Nanjing Medical University (Nanjing, China).

Venous blood was collected from the patients and immediately frozen in aliquots at −80°C or below, prior to analysis. For genotyping, genomic DNA was extracted from EDTA-supplemented blood samples using a commercial DNA extract kit (Wizard^® Genomic DNA Purification kit; Promega Corp., Madison, WI, USA).

Genomic DNA (3 μg) from the cells was denatured with 0.3 M NaOH (Sigma-Aldrich, Shanghai, China) at 37°C for 10 min. Bisulfite treatment (Sigma-Aldrich) was performed as described previously (30). Primers specific for TPH2 MSP were designed using MethPrimer (31) and synthesized by Sangon Biotech Co. (Shanghai, China). Bisulfite-treated DNA was used for amplification of the TPH2 promoter. Primers specific for TPH2 were as follows: Unmethylated (U) TPH2 sense, 5′-TTTGTAATTTGATTGTGGTTATTGG-3′ and anti-sense, 5′-ACAATCAACTACCTACTTAAAACACT-3′; and methylated (M) TPH2 sense, 5′-GGTTTGTAATTTGATTGTGGTTATC-3′ and anti-sense, 5′-CGATCAACTACCTACTTAAAACGCT-3′, which amplify a 164 bp product and a 165 bp product, respectively. MSP consisted of 38 cycles at 95° C for 45 sec, 60°C for 50 sec and 72°C for 30 sec, followed by a 10-min extension at 72°C in a DNA Thermocycler (Agilent Technologies, Inc., Santa Clara, CA, USA). The amplification products were separated by 2% agarose gel electrophoresis (Sangon Biotech Co.) and visualized by ethidium bromide staining (Sangon Biotech Co.) and ultraviolet transillumination (FR 2000 UV transillumination; Shanghai Furi Science & Technology Co., Ltd., Shanghai, China). Methylation was defined as M/(M+U)≥0.5; and unmethylation was defined as M/(M+U)<0.5.

RT-qPCR was performed using SYBR Green I chemistry (Invitrogen Life Technologies, Carlsbad, CA, USA). Specific primers were designed to target human TPH2 (PrimerBank ID: 169234956c2; 130 bp). Total RNA was isolated from the blood samples. The first-strand cDNA was synthesized from a moloney murine leukemia virus-reverse transcriptase kit using 2 μg total RNA according to the manufacturer’s instructions (Takara Bio, Inc., Dalian, China). Primer sequences of TPH2 for the RT-qPCR reaction were as follows: Forward, 5′-CAAAAATGACGACAAAGGCAACA-3′ and reverse, 5′-CCTCAGTGCTTTTACCAATCCA-3′. Primer sequences of β-actin for the RT-qPCR reaction were as follows: Forward, 5′-CTGGGACGAATGGAGAAA-3′ and reverse, 5′-AAGGAAGGCTGGAAGAGTGC-3′. The qPCR was performed using the Mx3000P qPCR system (Stratagene, La Jolla, CA, USA) and the PCR reaction mixture consisted of cDNA in 20 μl SYBR Premix Ex Taq. The qPCR was performed under the following conditions: 5 min at 95°C, followed by 40 cycles of 30 sec at 95°C, 30 sec at 58°C and 50 sec at 72°C. As an internal control for qPCR, β-actin mRNA expression was amplified from the same cDNA samples. All of the results were normalized to β-actin. Cycle threshold (Ct) values for triplicate reactions were averaged and relative TPH2 expression levels were determined using the comparative CT method (32), using the average Ct values for TPH2 and β-actin.

All data were generated without knowledge of the clinical status of the samples, and were analyzed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). Differences between the groups were calculated using the unpaired t-test. Associations between categorical variables were examined using Pearson’s χ² and Fisher’s exact tests. Data are expressed as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

Demographic and clinical characteristics of the subjects of the present study are presented in Table I. The study population consisted of 50 MDD + suicide patients [mean age ± standard deviation (SD), 36.8±10.2 years] and 75 matched MDD patients (mean age ± SD, 35.3±11.0 years). For the MDD + suicide group, 22 patients attempted suicide by drug overdose, hanging and drowning, and 28 patients attempted suicide by violent methods, such as through several deep cuts. No statistically significant differences were observed between the MDD + suicide and MDD groups for age, age at onset, course of disease or HDRS scores.

To accurately quantify the relative mRNA expression levels of TPH2, an RT-qPCR assay was conducted on samples from the MDD + suicide and MDD groups. The overall results are summarized in Fig. 1. TPH2 mRNA expression levels were lower in the MDD + suicide group [mean−∆Ct ± standard error (SE),−9.07±0.24], as compared with the MDD group (mean−∆Ct ± SE,−7.91±0.21). The difference between TPH2 expression levels in the MDD + suicide and MDD groups was significant (P=0.0005; Fig. 1). These results suggested that the downregulation of TPH2 gene expression may have an important role in the development of suicidal behaviors.

The methylation status of the TPH2 promoter region was analyzed in the MDD + suicide and MDD groups. The TPH2 promoter was methylated in 36.0% (18/50) of MDD + suicide patients, as compared with in 13.0% (10/75) of MDD patients. Furthermore, the TPH2 promoter was unmethylated in 64.0% (32/50) of MDD + suicide patients, as compared with 87.0% (65/75) of MDD patients. The differences in TPH2 methylation status between the MDD + suicide and MDD groups were statistically significant (P=0.0029; Fig. 2).

To determine whether TPH2 promoter methylation was correlated with the suppression of TPH2 mRNA expression in MDD, qPCR was used to detect the mRNA expression levels of TPH2 in all patients (Fig. 3). The mRNA expression levels of TPH2 were significantly decreased in the patients with TPH2 promoter methylation, as compared with those possessing an unmethylated TPH2 promoter (mean−∆Ct ± SE, −9.76±0.25 and −8.02±0.17, respectively; P<0.0001; Fig. 3A).

In the MDD + suicide group, the mRNA expression levels of TPH2 were −9.93±0.38 in patients possessing a methylated TPH2 promoter and −8.59±0.30 in those with an unmethylated TPH2 promoter (P=0.0084; Fig. 3B).

In the MDD group, the mRNA expression levels of TPH2 were −9.46±0.48 in the patients with a methylated TPH2 promoter, and −7.74±0.23 in those possessing an unmethylated TPH2 promoter (P=0.0058; Fig. 3C). These results suggested that in patients with MDD exhibiting methylation of the TPH2 promoter, TPH2 mRNA expression is likely to be downregulated.

The methylation of specific gene promoters is considered a marker for various types of psychiatric disorder (33). The correlation of TPH2 methylation with the clinicopathological parameters of the MDD + suicide and MDD groups is shown in Table II. The methylation status of the TPH2 promoter was associated with depression, hopelessness and cognitive impairment (P=0.011, 0.022 and 0.015, respectively) in the MDD + suicide group. In addition, methylation of TPH2 was only associated with depression (P=0.014) in the MDD group. Methylation of TPH2 was not associated with the remaining clinicopathological parameters evaluated, including gender, diurnal change, sleep disorders, anxiety and slow movement.