Hong Kong J Psychiatry 2009;19:141-4

ORIGINAL ARTICLE

Dr KX Xue, Mental Health Institute, Guangdong General Hospital, Guangdong Academy of Medical Sciences; Postgraduate Student from Medical College, Shantou University, PR China.
Dr CH Fan, Mental Health Institute, Guangdong General Hospital, Guangdong Academy of Medical Sciences, PR China.
Dr XL Li, Mental Health Institute, Guangdong General Hospital, Guangdong Academy of Medical Sciences, PR China.

Address for correspondence: Dr Chang-he Fan, Mental Health Institute, Guangdong General Hospital, Guangdong Academy of Medical Sciences, PR China.
E-mail: changhefan@yahoo.com

The research is funded by Guangdong Provincial Foundation of Natural Sciences (Grant No. 5002679).

Submitted (Chinese version): 24 Jul 2009; Accepted (Chinese version): 14 Aug 2009

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Introduction

Depression is a common psychiatric disease which may lead to mental disability and suicide. The life-time prevalence of depression is about 5 to 17%.¹ The incidence of depression is highly modulated with environmental factors, however, there is also a strong genetic contribution to the disease pathogenesis with an estimated heritability of 40 to 70%.¹ Family studies, twin studies, and foster children studies show an association between genetic factors and depression.² Until now, there are still no confirmed genes or related DNA sequences associated with the onset of depression. Therefore, studies to identify the genetic associations of depression will be an interesting area of concern.

Results from psychopharmacology and neurobehavioural research show serotonin (5- hydroxytryptamine [5-HT]) dysfunction in the central nervous system is associated with the incidence of depression.² Decreased level of 5-HT is associated with depressed mood, reduced appetite, insomnia, circadian rhythm disturbance, endocrine and sexual dysfunction, anxiety, and reduced activity.³ Furthermore, the central serotonin neural system is the target for most commonly prescribed antidepressants such as tricyclic antidepressants, selective serotonin reuptake inhibitors (SSRIs), and monoamine oxidase inhibitors.⁴ Central serotonin is synthesised at dorsal raphe nuclei of the brainstem.⁵

The biological processes related to serotonin in the body include its biosynthesis, storage, transportation, release, receptor binding, reuptake, and metabolism. Proteins and corresponding related genes which regulate these processes include tryptophan hydroxylase-1 and -2 (TPH1 and TPH2), vesicular monoamine transporter 2 (VMAT2), serotonin transporter (5-HTT), monoamine oxidase A (MAOA), and the serotonin receptors including: 5-HT1A, -2A, -2C, -3, -4,-5, -6, -7.⁶ Therefore, these serotonin-related genes are the candidates for research into the aetiology of depression.

Tryptophan hydroxylase is the rate-limiting enzyme of 5-HT biosynthesis, which includes TPH1 and TPH2. The former (TPH1) is predominately expressed in peripheral organs such as the heart, lungs, duodenum, liver, and adrenal glands, and also in some parts of the brain. It is also the main regulator of peripheral 5-HT synthesis. The latter (TPH2) is neuron-specific and only expressed in the brain 5-HT neuron. In some regions of the brain (such as the hippocampus, frontal lobe, thalamus, hypothalamus, and amygdala), the expression levels of TPH1 and TPH2 are almost equal. However, TPH2 is predominately expressed in the brain stem where 5-HT neurons originate.⁷

Recent studies have shown inconsistent results regarding any association between TPH2 gene polymorphism and unipolar depression.^8-16 In 2005, Zhang et al⁴ found that the human TPH2 gene coding region contains a functional polymorphism, G1463A, leading to the replacement of the 441 arginine position with histidine (R441H). This may cause approximately 80% of TPH2 function loss and significantly reduce 5-HT generation. Zhang et al⁴ also analysed the TPH2 G1463A polymorphism and its relationship with the affective disorder. Results showed that among 87 patients with unipolar depression, 9 carried the 1463A mutant allele, while in 219 control subjects, only 3 carried the mutant allele. This suggested that the R441H mutation may be an important risk factor in unipolar depression. In order to verify this result in the Chinese Han population, this study used an association analysis method to study the TPH2 G1463A polymorphism in the southern Chinese Han population and its relevance to unipolar depression.

Methods

Subjects

All study subjects were of Han nationality in the Guangdong Province of southern China and gave signed informed consent prior to enrolment.

Case Group

A total of 123 patients were included in the study — 42 were male, 81 were female. They were aged 16 to 65 years with a mean of 38 (standard deviation [SD], 14) years. All were recruited from the Guangdong General Hospital Institute of Mental Health outpatient clinics and inpatient services. For all patients, the diagnosis of unipolar depression was made by 2 veteran psychiatrists, according to diagnostic criteria in the Chinese Classification and Diagnostic Criteria of Mental Disorders (3rd edition), and in the Diagnostic and Statistical Manual of Mental Disorders (4th edition). The 24-item version of the Hamilton Depression Scale was used to evaluate the severity of depression and the total scores for all patients were greater than 20. Patients who were diagnosed with neurological disorders, other serious physical illnesses, secondary depression, bipolar disorder, and other functional mental disorders were excluded.

Control Group

Another 122 healthy individuals without severe physical diseases, genetic diseases, and mental illness were recruited as controls. They were hospital staff, internship students, and volunteers from the Guangdong General Hospital Medical Center and without any blood relationships to any of the patients. Among the control were 55 males and 67 females; they aged 18 to 65 years, with a mean of 37 (SD, 14) years. No significant statistical differences were found with regard to gender and age distribution between cases and controls (P>0.05).

Specimen Collection and Genomic DNA Extraction

From each patient suffering from depression and each healthy control, 5 ml of peripheral venous blood was acquired and mixed with sodium citrate anticoagulant. A column-blood genomic DNA extraction kit was used to extract genomic DNA (according to kit instructions).

TPH2 G1463A Genotype Analysis

The amplification refractory mutation system–polymerase chain reaction (ARMS-PCR) method⁴ was used to analyse TPH2 G1463A genotypes. The PCR reaction primers included 1 allele-specific primer (G or A allele) and 2 positive control primers (forward and reverse) flanking the site of the G1463A polymorphism. According to human TPH2 gene coding DNA sequences, a forward positive control primer “F” was designed as: 5'-ATG TGT GAA AGC CTT TGA CCC AAA GAC A- 3 '; a reverse positive control primer “R” as: 5'-TGC GTT ATA TGA CAT TGA CTG AAC TGC T-3 '; G allele-specific primer P^G as: 5’-TAG GGA TTG AAG TAT ACT GAG AAG GCA C; A allele-specific primer P^A as: 5’-TAG GGA TTG AAG TAT ACT GAG AAG GCA T. For each sample, 2 PCR reactions were conducted to detect the possible G / A alleles using primes F + R + P^G for the G allele detection and primers F + R + P^A for the A allele. The 25 μl PCR reaction volume was composed of 12.5 μl GoTaq Green Master Mix, 2 × (reaction buffer, pH > 8.5; 400 μM dNTP; 3 Mmol MgCl²), DNA template 2 μl (≥ 100 μg), each primer 1 μl (10 μmol/l) and ddH²O 8.5 μl. The PCR reaction conditions were: 94ºC predenatured for 5 minutes (94ºC for 30 sec, 63ºC for 30 sec, 72ºC for 30 sec) × 40 cycles, final extension at 72ºC for 5 minutes. For genotyping, 5 μl PCR product samples separated via 2% agarose gel electrophoresis were stained with EB and the results observed. According to electrophoresis banding patterns, TPH2 G1463A genotype was identified: 2 bands of 492-bp and 294-bp indicated for “positive” (G allele or A allele positive, respectively, according to the type of PCR primers used) and one 492-bp band for “negative” (G allele or A allele negative, respectively). Some samples of the PCR products involved DNA sequencing.

DNA Sequencing of Polymerase Chain Reaction Products

In order to verify the accuracy of the ARMS-PCR method in TPH2 G1463A genotyping, 8 samples of PCR products were taken for DNA sequencing. DNA sequencing was conducted following the routine protocol by Shanghai Invitrogen Biotechnology Co., Ltd, Guangzhou Office. It was carried out as follows: (1) recycling of unpurified PCR product in gel; (2) examination of samples’ band brightness to decide the template quantity added to reaction mixture; (3) ddH²O, primes and template were added to the reaction mixture for heat-denaturation at 96ºC; (4) after being cooled on ice and added with 1 µl BDT, the reaction mixture was taken for amplification in a thermocycler for 25 cycles; (5) after being cooled on ice, the mixture was added with EDTA and laid aside for about 5 minutes; (6) centrifugation for 30 minutes at 4ºC after being added with 15 µl anhydrous ethanol; (7) after centrifugation, the upper solution was discarded, and the precipitate added with 50 μl of 70% iced ethanol and centrifuged for another 15 minutes at 4ºC; (8) the precipitate was air dried after discarding the upper solution; (9) 10 µl of deionised formamide added and subjected to denaturation; (10) after being cooled, the sample was taken to the sequencing analyser 3730 to obtain a final result.

Statistical Analysis

The Statistical Package for the Social Sciences (Windows version 13.0; SPSS Inc, Chicago [IL], US) was used to carry out data management and counting of genotype and allele frequency. The allele and genotype frequency distribution differences between the two groups were analysed using the Chi-square (χ²) test. If the theoretical value was less than 5, Fisher’s exact probability analysis would be used.

Results

Agarose gel electrophoresis and DNA sequencing results showed that in all subjects the TPH2 1463A mutant allele was not present (Fig); all 123 cases of unipolar depression and the 122 healthy controls were homozygous for 1463G. According to the report of Zhang et al,⁴ 1463A allele frequency in patients with unipolar depression was 5.17%. In accordance with this frequency, among all patients there should have been 6 subjects carrying the 1463A mutant allele. However, in neither the cases or controls was there a single 1463A allele carrier.

Discussion

Zhang et al⁴ first reported in 2005 that the coding region of human TPH2 gene contains a G1463A functional polymorphism. However, in subsequent studies, many research groups could not replicate Zhang’s results. Garriock et al¹³ analysed TPH2 G1463A polymorphism in a sample of 182 unipolar depression patients which was similar to Zhang’s sample in race and gender distribution, including 83 cases of treatment resistance, 8 bipolar affective disorder patients (treatment resistant), and 186 healthy controls, and found that no subjects were 1463A mutant allele carriers. For the first time, they also studied the 11th intron in the TPH2 gene, searching for the other 2 single-nucleotide deletion mutations (C1487G and T1578G), and the sequence analysis showed no single nucleotide variations. Van Den Bogaert et al¹⁴ carried out TPH2 G1463A genotyping on 2 independent case-control samples. One sample consisted of 135 cases of unipolar depression (mean age, 57 years) and 182 cases of bipolar affective disorder patients (mean age, 56 years) forming the case group, and 364 gender-, age-, and race-matched individuals formed the control group. The other sample consisted of 182 cases of unipolar depression patients (mean age, 47 years) and 182 cases of bipolar affective disorder patients (mean age, 56 years) forming the case group, and 364 cases of gender-, age- and race-matched individuals forming the control group. Within the 2 study samples, there was not a single carrier of 1463A. In addition, they discussed the reason for the discrepancy between Zhang’s and their study — the sample of patients selected in Zhang’s study⁴ had a rather high average age (>60 years), and 7 patients with unipolar depression carrying the 1463A allele were unresponsive to SSRI treatment. Glatt et al¹⁵ carried out G1463A genotype analysis on 1023 cases of unipolar depression from a wide range of ethnic representatives, and found no carriers of the 1463A allele. Zhou et al¹⁶ carried out TPH2 G1463A genotyping on 779 cases of unrelated individuals (403 cases of unipolar depression, with an average age of >60 years) and 1740 patients with major depression, and did not find any carrier of the 1463A allele. They speculated that the TPH2 1463A allele is a very rare mutation, and may not have any relationship with major depression or other behaviours. If there is a relationship with other behaviours, based on their own samples of old-age patients in the study, the 441H allele may be related to late onset of depression. Additionally, Delorme et al¹² used 3 different methods: ARMS-PCR, restriction fragment length polymorphism (RFLP), and direct sequencing of 1071 cases with mental disorders (265 cases of unipolar depression, 297 cases of severe depression, 84 cases of bipolar disorder, 201 cases of obsessive compulsive disorder, 224 cases of autism patients), and 246 healthy volunteers to genotype the TPH2 G1463A polymorphism. They found all subjects were G1463G homozygotes.

This study also found no TPH2 1463A allele carrier within the sample of southern Chinese Han population, and was in accordance with the findings of the above- mentioned authors but inconsistent with Zhang’s earliest results.⁴ The probable underlying reasons may be: (1) with regard to depression itself, there may exist geographic and ethnic differences; (2) compared with Zhang’s study sample (unipolar depression, patients’ age ≥ 60 years), patients in this study had a lower mean age (38; SD, 14 years) and therefore lower onset age of depression. If the age of onset is different, the genetic backgrounds may also be different; (3) the TPH2 1463A allele may be a rare mutant allele and has not been detected in this study due to its relatively small sample size; (4) the 1463A allele may be seen mainly in patients with a poor response to SSRI treatment. Zhang’s study⁴ found that among the 9 cases who carried the A mutant allele, 7 exhibited a poor response to treatment with SSRI, and the other 2 required a very high dose. In this study sample however, no patients had a poor response to SSRI treatment, which may be another reason for inconsistence; (5) since this and other studies did not detect the 1463A allele, the TPH2 gene G1463A polymorphism should not be a major genetic factor in depression. In short, the results of this study do not support a correlation between the TPH2 G1463A gene polymorphism and depression among the southern Chinese Han population.

The present study has some limitations, one of which is its relatively small sample size. Usually, rare mutant alleles are detected only in a large sample. If the TPH2 1463A allele is a rare mutant allele in Chinese Han population, a large sample would be needed to detect it. In this study, although there was no evidence of an association between TPH2 gene G1463A polymorphism and unipolar depression in southern Chinese Han population, our findings cannot exclude a possible correlation between TPH2 gene and unipolar depression, because there are several other polymorphisms affecting this gene.^8-11 To clarify the relationship between 5-HT–related genes like TPH2 and unipolar depression, further studies with a larger sample and consideration of gene-gene and gene-environment interaction are necessary.

Acknowledgements

This paper’s laboratory work was completed in the Guangdong General Hospital Medical Research Center, during which time we received the guidance and support of Prof Zhi-xin Shan and Miss Hong-hong Tan.

References

1. Zill P, Baghai TC, Zwanzger P, Schüle C, Eser D, Rupprecht R, et al. SNP and haplotype analysis of a novel tryptophan hydroxylase isoform (TPH2) gene provide evidence for association with major depression. Mol Psychiatry 2004;9:1030-6.
2. Du L, Bakish D, Hrdina PD. Tryptophan hydroxylase gene 218A/C polymorphism is associated with somatic anxiety in major depressive disorder. J Affect Disord 2001;65:37-44.
3. Oquendo MA, Mann JJ. The biology of impulsivity and suicidality. Psychiatr Clin North Am 2000;23:11-25.
4. Zhang X, Gainetdinov RR, Beaulieu JM, Sotnikova TD, Burch LH, Williams RB, et al. Loss-of-function mutation in tryptophan hydroxylase-2 identified in unipolar major depression. Neuron 2005;45:11-6.
5. Veenstra-VanderWeele J, Cook EH Jr. Knockout mouse points to second form of tryptophan hydroxylase. Mol Interv 2003;3:72-5.
6. Veenstra-VanderWeele J, Anderson GM, Cook EH Jr. Pharmacogenetics and the serotonin system: initial studies and future directions. Eur J Pharmacol 2000;410:165-81.
7. Holden C. Neuroscience. Mutant gene tied to poor serotonin production and depression. Science 2004;306:2023.
8. Tsai SJ, Hong CJ, Liou YJ, Yu YW, Chen TJ, Hou SJ, et al. Tryptophan hydroxylase 2 gene is associated with major depression and antidepressant treatment response. Prog Neuropsychopharmacol Biol Psychiatry 2009;33:637-41.
9. Yoon HK, Kim YK. TPH2 -703G/T SNP may have important effect on susceptibility to suicidal behavior in major depression. Prog Neuropsychopharmacol Biol Psychiatry 2009;33:403-9.
10. Mann JJ, Currier D, Murphy L, Huang YY, Galfalvy H, Brent D, et al. No association between a TPH2 promoter polymorphism and mood disorders or monoamine turnover. J Affect Disord 2008;106:117-21.
11. Lopez de Lara C, Brezo J, Rouleau G, Lesage A, Dumont M, Alda M et al. Effect of tryptophan hydroxylase-2 gene variants on suicide risk in major depression. Biol Psychiatry 2007 1;62:72-80.
12. Delorme R, Durand CM, Betancur C, Wagner M, Ruhrmann S, Grabe HJ, et al. No human tryptophan hydroxylase-2 gene R441H mutation in a large cohort of psychiatric patients and control subjects. Biol Psychiatry 2006;60:202-3.
13. Garriock HA, Allen JJ, Delgado P, Nahaz Z, Kling MA, Carpenter L, et al. Lack of association of TPH2 exon XI polymorphisms with major depression and treatment resistance. Mol Psychiatry 2005;10:976-7.
14. Van Den Bogaert A, De Zutter S, Heyrman L, Mendlewicz J, Adolfsson R, Van Broeckhoven C, et al. Response to Zhang et al (2005): loss-of- function mutation in tryptophan hydroxylase-2 identified in unipolar major depression. Neuron 45, 11-16. Neuron 2005;48:704.
15. Glatt CE, Carlson E, Taylor TR, Risch N, Reus VI, Schaefer CA. Response to Zhang et al. (2005): loss-of-function mutation in tryptophan hydroxylase-2 identified in unipolar major depression. Neuron 45, 11-16. Neuron 2005;48:704-5.
16. Zhou Z, Peters EJ, Hamilton SP, McMahon F, Thomas C, McGrath PJ, et al. Response to Zhang et al. (2005): loss-of-function mutation in tryptophan hydroxylase-2 identified in unipolar major depression. Neuron 45, 11-16. Neuron 2005;48:702-3. 144