Polymorphic variants of SLC6A4 and TPH1 genes as predictors of sertraline efficacy and safety in patients with mixed anxiety-depressive disorder: a pilot study

Introduction
Antidepressants are the most commonly prescribed class of drugs [1]. Selective serotonin reuptake inhibitors (SSRIs) are the first-line pharmacotherapy for various mood and anxiety disorders, including mixed anxiety-depressive disorder.
Genetic factors play an important role in modifying the response to antidepressants. The monoaminergic system (including serotonin, dopamine, and norepinephrine) is postulated to be one of the most important neurobiological pathways [2]. The serotonin transporter protein (5-HTT) is involved in the reuptake of serotonin in presynaptic neurons. 5-HTT affects the level of serotonin in the synaptic cleft, which is one of the main targets for antidepressants, especially SSRIs [2, 3]. The enzyme tryptophan hydroxylase TPH1 plays an important role in the synthesis of serotonin and determines the rate of the biosynthesis reaction of this neurotransmitter [4].
A large number of pharmacogenetic studies of the efficacy and tolerability of SSRIs are devoted to the study of functional polymorphic variants of rs4795541 (5-HTTLPR) in the promoter region of the SLC6A4 gene and rs1800532 of the TPH1 gene [1, 4].
A polymorphic variant rs1800532 C>A was found in the TPH1 gene, which is located at the site of potential binding of the transcription factor GATA. This polymorphism can affect gene expression by regulating pre-mRNA processing. [4]. It is known that carriage of the rs1800532*A allele is associated with increased expression of the TPH1 gene [4]. The biallelic polymorphic variant rs4795541 of the SLC6A4 gene is characterized by the presence of two allelic variants: the short allele S, associated with decreased expression of the serotonin transporter protein, and the long allele L, which, on the contrary, is associated with increased expression. It has been established that carriers of the S allele have a lower efficiency of serotonin reuptake, leading to higher levels of serotonin [2]. The results of a number of studies indicate the existence of racial and ethnic differences in the effectiveness of SSRIs when studying the polymorphic variant rs4795541 of the SLC6A4 gene [3, 5]. The aim of this study was to identify pharmacogenetic markers of the effectiveness of sertraline pharmacotherapy for polymorphic variants rs4795541 of the SLC6A4 gene and rs1800532 of the TPH1 gene in patients with mixed anxiety-depressive disorder from the Republic of Bashkortostan.

Materials and methods
As part of a pharmacogenetic study of the efficacy of sertraline, 96 patients diagnosed with mixed anxiety-depressive disorder (F 41.2) from the Republic of Bashkortostan were studied. The average age of the patients was 46.22±1.57 years. Signed informed consent was obtained from each patient.
Exclusion criteria: the presence of other mental disorders; the use of other psychotropic drugs in therapy.
Patients received sertraline at a dose of 50-100 mg per day from the 1st to the 42nd day of inpatient treatment. The effectiveness of sertraline therapy was assessed using psychometric scales [6] (Montgomery-Asberg depression MADRS, Hamilton depression HAM-D (HDRS); Beck depression BDI (self-questionnaire), Beck anxiety BAI (self-questionnaire); hospital anxiety and depression scale (self-questionnaire) HADS, which includes 2 subscales (HADS-D for assessing depression and HADS-A for assessing anxiety) and was carried out the day before the start of therapy, as well as on the 14th, 28th and 42nd days. The results obtained were assessed in points.
Analysis of single nucleotide polymorphic variants (SNP) rs4795541 of the SLC6A4 gene and rs1800532 of the TPH1 gene was carried out by the polymerase chain reaction method in real time on a CFX96 amplifier (Bio-Rad, USA) using commercial kits reagents of the company "Synthol" (Russia).
Statistical processing of the obtained results was carried out using the SPSS v. 13.0 programs. To establish the significance of pharmacogenetic parameters as predictors of the efficacy and safety of pharmacotherapy, nonparametric statistics methods were used. To assess the reliability of differences, the Mann-Whitney criterion and the Benjamini-Hochberg criterion were used. Continuous (quantitative) data are presented as median, interquartile range (IQR), minimum and maximum. Ordinal, categorical and qualitative data are presented as absolute frequencies (number of observations) and relative frequencies (percentages). Comparison of several samples of continuous data was performed using the Kruskal-Wallis H-test (for data that do not obey the law of normal distribution).
The study was approved by the local bioethics committee of the IBG UFRC RAS.

Results
The results of the pharmacogenetic analysis for the studied SNPs rs4795541 of the SLC6A4 gene and rs7997012 of the TPH1 gene are presented in Tables 1, 2.
The frequency distribution of the genotypes of the polymorphic variant rs4795541 of the SLC6A4 gene corresponded to the Hardy–Weinberg equilibrium 2=0.594; p=0.441.
Table 1 shows changes in the number of points on the HADS-D subscale between carriers of different genotypes of rs4795541 of the SLC6A4 gene. Statistically significant differences were found at HADSD0 pfdr <0.05 (table). Statistically significant differences between carriers of different genotypes of rs4795541 of the SLC6A4 gene on the HDRS scale were observed on all days of the study due to higher scores in carriers of the rs4795541*L/L genotype of the SLC6A4 gene: HDRS0, HDRS14, HDRS28, HDRS42 pfdr <0.05 (table).
As a result of comparing scores on the MADRS scale, statistically significant differences in the severity of depressive symptoms were revealed between carriers of different genotypes on MADRS28 pfdr <0.05 due to higher scores in carriers of the rs4795541*L/L genotype (table).
The distribution of genotype frequencies of the polymorphic variant rs1800532 of the TPH1 gene corresponded to the Hardy–Weinberg equilibrium 2=3.539; p=0.094.
Comparison of scores on the HADS-A and HADS-D subscales in carriers of different genotypes for the SNP rs1800532 of the TPH1 gene revealed that from the start of the study to the 28th day, the compared groups were comparable in terms of the studied indicator. On the 42nd day of the study, statistically significant differences were observed between carriers of different genotypes on the HADSA42 pfdr <0.001 and HADSD42 pfdr=0.013 scales. More pronounced anxiety and depressive symptoms were observed in carriers of the rs1800532*A/A genotype (table). Comparison of HDRS scores in carriers of different genotypes of rs1800532 of the TPH1 gene revealed statistically significant differences on all assessment days pfdr <0.05 except HDRS28 pfdr=0.052. More pronounced depressive symptoms were observed in carriers of the rs1800532*A/A genotype (Table).
In addition, comparison of BAI scores demonstrated statistically significant differences between carriers of different genotypes of rs1800532 of the TPH1 gene on BAI28 pfdr=0.040 and BAI42 pfdr <0.001 study days due to more pronounced anxiety symptoms in carriers of the rs1800532*A/A genotype (see Table 1).
As a result of comparing the scores on the MADRS scale, statistically significant differences were revealed in the severity of depressive symptoms between carriers of different genotypes on MADRS42 pfdr <0.001 due to higher scores in carriers of the rs1800532*A/A genotype (table).

Discussion
As part of this study, an attempt was made to conduct a pharmacogenetic study to identify pharmacodynamic genetic markers that affect the efficacy of sertraline in patients with mixed anxiety-depressive disorder from the Republic of Bashkortostan for polymorphic variants rs4795541 of the SLC6A4 gene and rs7997012 of the TPH1 gene. Similar studies are rare and contradictory both in the world and in Russia [3, 4, 7–9, 11–15, 17–19]. In the course of studying the combined effect of SNPs of the SLC6A4 and TPH1 genes on the response to SSRIs in patients with bipolar disorder, it was found that the key factor determining the effectiveness of antidepressants is the SNP rs4795541 of the SLC6A4 gene, while the SNP rs7997012 of the TPH1 gene is not [7].
As a result of this study, it can be assumed that the rs7997012*A/A genotype can be considered as a marker of low effectiveness of sertraline therapy, which is consistent with a number of studies [8, 9]. Thus, our results confirm the findings of Korean colleagues who found a link between the rs7997012 A/A and rs7997012 A/C genotypes and low efficacy of citalopram in the treatment of major depressive disorder (MDD) [8], as well as the results of a number of other studies that have shown a decrease in the efficacy of other SSRIs - paroxetine and fluvoxamine in MDD [7]. In a study aimed at finding the relationship of the SNP rs7997012 of the TPH1 gene with the efficacy of paroxetine in European patients with unipolar depression, together with placebo or pindolol in a double-blind test for 4 weeks, a link was found between the rs7997012*A/A and rs7997012*A/C genotypes with low efficacy of paroxetine, compared with rs7997012*C/C. In contrast, no such difference was observed in the group taking pindolol. The study found that factors such as gender, the presence or absence of mental disorders, the severity of depressive symptoms, and the concentration of paroxetine in the blood plasma did not affect the result. [10].
The results of a number of other studies have shown that the remission rate during treatment with citalopram was worse in patients with MDD with the rs7997012*A/A and rs7997012*A/C genotypes than in patients with the rs7997012*C/C genotype. Scientists suggest that the TPH1 gene is likely to be a modulator of antidepressant activity, especially in cases of remission treatment [8, 11].
As a result of this study, it can be assumed that the rs4795541*L/L genotype of the SLC6A4 gene can be considered as a marker of low efficacy of sertraline therapy. In contrast, some research teams have found an association of the rs4795541*S/S genotype with lower SSRI efficacy and an increased risk of adverse reactions compared to genotypes homozygous or heterozygous for the rs4795541*L allele [3, 4, 12–15]. A meta-analysis that included data from 1,435 Europeans demonstrated lower rates of remission and SSRI efficacy in carriers of the rs4795541*S allele [16]. At the same time, a number of other studies [17, 18], including STAR*D, the largest study to date in terms of sample size [17–19], did not find an association of this SNP rs4795541 of the SLC6A4 gene with response to SSRIs. This may likely be due to ethnic differences in the study groups [3, 5]. It is known that the frequency of the rs4795541*S allele varies in different populations. Thus, if in European populations the rs4795541*S allele occurs with a frequency of 35–50%, then in Asian populations it increases to 72–82% [5]. Thus, studying this polymorphic variant in Asian populations on small samples may be insufficient to detect a more subtle effect.
The inconsistency of the results obtained, in addition to the ethnicity of individuals, may be associated with the class of antidepressants, as well as with the inclusion and exclusion criteria and the main characteristics of the studied patient samples, which require further research. Thus, according to the meta-analysis, an association of the rs4795541*L allele with a better response and longer remission when taking SSRIs was found in Europeans, but not in Asians, which may probably be due to the different frequencies of the rs4795541*L allele in these populations. The frequency of the rs4795541*L allele in Europeans ranged from 50 to 70% in most of the studies included in this meta-analysis, which is similar to previous studies [20]. According to the results of another meta-analysis of the relationship between the rs4795541 SNP of the SLC6A4 gene and the effectiveness of SSRIs, which analyzed 33 studies, it was found that the prognostic significance of this polymorphism for the Asian population is low [21].

Study limitations
It should be noted that this study had a number of limitations. Firstly, the study is a pilot study and was performed on a small number of patients. The second limitation is related to the ethnicity of patients living in the Republic of Bashkortostan. The obtained results cannot be extrapolated directly to all patients, and ethnic characteristics in the frequency of polymorphic variants should be taken into account when implementing personalized programs for pharmacotherapy.

Conclusion
For the first time, in confirmation of the hypothesis that polymorphic variants of the SLC6A4 and TPH1 genes can be predictors of response to SSRIs, the pharmacogenetic effect of polymorphic variants rs4795541 of the SLC6A4 gene and rs7997012 of the TPH1 gene in patients with anxiety-depressive disorder from the Republic of Bashkortostan was shown. The results of the study are preliminary, it is necessary to conduct research on larger samples, as well as on samples from other regions of the Russian Federation.