Study of Associations of the GRM8 Gene with Antipsychotic-induced Hyperprolactinemia

: Hyperprolactinemia is one of the common adverse events of antipsychotic therapy. The role of genetic factors in the development of drug-induced side effects is being actively investigated. The present study examined the association of two polymorphisms rs2237748 and rs2299472 in the GRM8 gene encoding the glutamate metabotropic receptor type 8 with antipsychotic-induced hyperprolactinemia in 536 patients with schizophrenia from several regions of Siberia (Russia). The investigated polymorphisms are not associated with drug-induced hyperprolactinemia in patients with schizophrenia. There were no associations of the GRM8 gene polymorphisms with serum prolactin levels in patients taking antipsychotic therapy. Our results did not confirm the involvement of the GRM8 rs2237748 and rs2299472 in the development of antipsychotic-induced hyperprolac-tinemia.


Introduction
Hyperprolactinemia is one of the most common side effects of antipsychotic therapy.With short-term therapy, an increase in prolactin levels is recorded in 80% of patients [1] and in 30-70% of patients receiving long-term maintenance treatment [2,3].
As it is known, a common biochemical property for all antipsychotics is their ability to block postsynaptic dopaminergic receptors.Antipsychotics that cause pronounced neuroendocrine side effects associated, in particular, with an increase in the production of the hormone prolactin, a decrease in the content of growth hormone, functional disorders in the hypothalamic-pituitary-thyroid and hypothalamic-pituitary-gonadal systems, have a strong blocking effect on D2 receptors in the tuberoinfundibular system (hypothalamus, pituitary gland) of the brain [4][5][6][7][8].
Prolactin secretion is under complex neuroendocrine control, which involves agents of various natures: neurotransmitters, biologically active neuropeptides, hormones of peripheral endocrine glands.Factors involved in the regulation of prolactin secretion can be divided into two groups: inhibitory (dopamine, gamma-aminobutyric acid, gastrin, somatostatin, gonadotropin-binding protein) and stimulating (serotonin, thyrotropin-releasing hormone, gonadotropin-releasing hormone, vasointestinal peptide, opiates, neurotensin and substance P, oxytocin, angiotensin 2).
Experiments with rats have also shown that glutamate can directly influence the production of prolactin by the pituitary gland [9].A hypothesis was tested that glutamate would stimulate prolactin release when applied directly to primary cultures of dispersed adult female rat anterior pituitary cells.Glutamate increased the rate of prolactin release within two minutes in a self-limited manner.These experiments demonstrate that anterior pituitary lactotrophs respond to glutamate by increasing the rate of prolactin release.
Abnormal glutamatergic neurotransmission is attracting increasing attention from researchers.This is due to the fact that glutamate is the main excitatory neurotransmitter in the central nervous system (CNS), and the two main groups of glutamate receptors, namely metabotropic and ionotropic (AMPAR, NMDAR and KAR), are closely associated with the etiology of neuropsychiatric disorders [10,11].
Metabotropic glutamate receptors (mGluR 1-8) are divided into 3 groups and belong to a heterogeneous family of G protein-coupled receptors [12].It is assumed that they function as presynaptic regulatory mechanisms that control the release of neurotransmitters and, accordingly, modulate brain excitability through presynaptic, postsynaptic, and glial mechanisms [13].
In the presented study, the associations of the GRM8 gene polymorphisms with hyperprolactinemia after antipsychotic treatment in patients with schizophrenia were studied.

Objective
The aim of the study was to study the association of polymorphic variants rs2237748, rs2299472 of the GRM8 gene with the development of hyperprolactinemia in patients with schizophrenia after treatment with antipsychotic drugs.

Patients
This study was carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki 1975, revised in Fortaleza, Brazil, 2013) for experiments involving humans.We recruited 536 patients with schizophrenia (266 men and 270 women) being treated at clinics of the Mental Health Research Institute, Siberian State Medical University, Tomsk Clinical Psychiatric Hospital (Tomsk), Kemerovo Regional Clinical Psychiatric Hospital (Kemerovo) in the Siberian region.The inclusion criteria were a verified clinical diagnosis of schizophrenia (F20) according to the World Health Organization World Mental Health Composite International Diagnostic Interview (WHO WMH-CIDI) for schizophrenia diagnostics [18], age 18-60 years, and the patient's informed consent.Exclusion criteria for all patients were non-Caucasian physical appearance (e.g., Mongoloid, Buryats or Khakassians), organic mental disorders (e.g., epilepsy, Parkinson's disease) or somatic disorders in the stage of decompensation.

Prolactin Analysis
Measurement of the hormone prolactin concentration in the blood serum was carried out by enzyme immunoassay using the PRL Test System reagent kit.The criterion for neuroleptic hyperprolactinemia was an increase in the concentration of prolactin in the blood: for men -above 20 ng/ml, for women -above 25 ng/ml [19].

Genetic Analysis
Blood samples were taken after 8 h overnight fasting in tubes containing EDTA and stored in several aliquots at −20 •C until DNA isolation using the standard phenol-chloroform method.Inclusion criteria for SNP selection and genotype are described elsewhere [20].Genotyping of 2 single-nucleotide polymorphisms (SNPs) in GRM8 (rs2237748, rs2299472) was carried out with use a QuantStudio™ 3D Digital PCR System Life Technologies amplifier (Applied Biosystems, Waltham, MA, USA) using TaqMan Validated SNP Genotyping Assay kits (Applied Biosystems) based at The Core Facility "Medical Genomics", Tomsk National Research Medical Center of the Russian Academy of Sciences.

Statistical Analysis
Statistical analysis was performed using SPSS software for Windows, version 21.The genotype frequencies were checked for Hardy-Weinberg equilibrium using the chisquare test.Chi-square test and the Fisher's exact test, where necessary, were used for between group comparisons of genotype or allele frequencies at a significance level of p < 0.05.Assessment of the association of genotypes and alleles of the studied polymorphic variants of genes with a pathological phenotype was carried out using the odds ratio (OR) with a 95% confidence interval for the odds ratio (95% CI).

Results
Details about the studied patient population are presented in Table 1.Patients with hyperprolactinemia were characterized by younger age and shorter duration of the disease.Along with this, hyperprolactinemia was observed more often in women (Table 1).Differences in the average value of chlorpromazine equivalent (Daily dose of CPZeq), which characterizes the antipsychotic therapy of patients, in the group with and without hyperprolactinemia did not reach statistical significance (p=0.05).Analysis of the frequency distribution of genotypes and alleles of the studied polymorphisms was carried out in a subgroup of men and in a subgroup of women (Tables 2 and 3).We did not find any significant differences in the distribution of alleles and genotypes of rs2237748 and rs2299472 in groups of patients with schizophrenia with and without hyperprolactinemia.
In the current study, we also analyzed the level of prolactin in a group of men and a group of women with schizophrenia depending on the different genotypes of the studied polymorphic variants of the GRM8 gene (Table 4).We did not find significant associations of the GRM8 gene polymorphisms with serum prolactin levels in the patients with schizophrenia.

Discussion
Use of antipsychotic agents has been associated with hyperprolactinemia, or elevated prolactin levels.Hyperprolactinemia is more than an abnormal laboratory value; elevated prolactin levels can interfere with the functioning of reproductive, endocrine, and metabolic systems.A special role in the pathogenesis of the development of antipsychotic-induced hyperprolactinemia belongs to genetic factors, which may be the basis for the sensitivity of the development of complications in many patients [21][22][23][24][25][26].
Changes in levels of anxiety, exploratory behavior, working memory, and fear extinction identified in deficient mouse models [27] and in various neuropharmacological studies [28,29] have provided evidence Group III metabotropic glutamate receptors (mGluR), including GRM8, should play an important role in the development of pathological processes in the brain at the molecular level.
In our study, we attempted to identify the effect of the GRM8 gene on the level of prolactin in the blood serum and the development of hyperprolactinemia while taking antipsychotic drugs in patients with schizophrenia.We did not find any statistically significant differences for polymorphic variants of the GRM8 gene rs2237748 and rs2299472.

Table 1 .
Demographic and clinical parameters of the patients.

Table 2 .
Distribution of allele and genotype frequencies of the GRM8 gene polymorphisms in male schizophrenia patients with and without hyperprolactinemia.

Table 3 .
Distribution of allele and genotype frequencies of the GRM8 gene polymorphisms in female schizophrenia patients with and without hyperprolactinemia.

Table 4 .
The level of prolactin in the male and female with schizophrenia.