Association of single nucleotide variants of the clock gene (rs1801260) with seizures after alcohol withdrawal

Introduction

Excessive alcohol consumption is one of the most common risk factors for the development of various diseases. Despite the positive dynamics of the 2.5-fold decline in the growth rate of alcoholic beverage sales over the past 10 years, the number of patients recently diagnosed with F10.0-F10.9 (mental and behavioral disorders caused by alcohol use) at the end of 2022 amounted to 37.0 patients per 100 thousand of the population. It is worth noting that among these cases, alcoholic psychoses account for 8.8 patients per 100 thousand of the population¹.

All patients with moderate alcohol dependence may experience alcohol withdrawal syndrome (AWS), which is understood as a symptom complex of somatic manifestations, including increased blood pressure and body temperature, sweating, nausea, vomiting, etc., as well as mental complications such as dysphoria, sleep disturbance, anxiety, etc. [1], which manifest themselves in stages II and III of alcohol dependence syndrome after a sharp significant reduction in the dose or cessation of alcohol consumption [2]. AWS can occur in two forms: uncomplicated form, limited to somatic symptoms with an impairment of the emotional-volitional sphere of mental activity; and complicated one, which includes alcoholic hallucinosis, alcoholic delirium, and alcoholic convulsions [3].

The concept of "alcoholic seizures" was first introduced by the German psychiatrist Emil Kraeplin [4]. Convulsive seizures associated with the use of ethyl alcohol occur predominantly in males [5]; they usually emerge 6–72 hours after the last alcohol use, often without other manifestations of AWS involving generalized tonic-clonic seizures with the absence of a partial character [6]. It should be noted that, in contrast to "classical" epileptic seizures, alcoholic seizures do not have a regular change of phases; they are short-lived and manifest themselves to a lesser extent, while the memory of the seizure retains [2]. The provoking/predictor factors of "alcoholic seizures" include old age; traumatic brain injury; viral and bacterial infections; dysfunction of other organs, especially the presence of neurological diseases; dehydration; hyponatremia or hypokalemia; elevated AST and GGT levels; low platelet counts; a history of previous attacks and other complications of AWS [6][7]. However, not all patients with alcohol dependence syndrome with AWS develop convulsive seizures.

One of the possible causes of the development of convulsive seizures in patients with alcohol dependence syndrome may be circadian rhythm disorders regulated by melatonin, the main hormone of the epiphysis. It has been proven that circadian melatonin along with other systems regulates epigenetic processes in neurons at molecular, cellular, and energetic levels. Melatonin affects the activity of many genes in the central nervous system: PER (Period Circadian Protein), BMAL (Brain and Muscle Arnt-Like protein.), CRY (Cryptochrome) and CLOCK (Circadian Locomotor Output Cycles Kaput) [8][9]. The study by Efremov et al. detected the C allele and CT genotype of the polymorphic CLOCK gene (rs1801260). The obtained data indicated a high risk of sleep disorders in patients suffering from alcohol dependence, associated with CLOCK (rs1801260) [10]. Available data on the melatonin effect on epileptic activity, sleep disorders in patients with alcohol dependence and data on the role of the CLOCK gene (rs1801260) in circadian rhythm impairments suggest their synergistic participation in the development of convulsive seizures, which requires a more detailed investigation of the association between single nucleotide variants of the CLOCK gene (rs1801260) and convulsive seizures in AWS.

The aim of the study was to investigate the association of single nucleotide variants of the CLOCK gene (rs1801260) and convulsive seizures after alcohol withdrawal.

Materials and methods

A longitudinal study of patients with alcohol dependence syndrome was performed with a focus on the presence or absence of convulsive seizures after alcohol consumption. To form the samplings, we developed inclusion, non-inclusion, and exclusion criteria. Inclusion criteria were related to the existence of a verified diagnosis of F10.2 "Alcohol dependence syndrome" corresponding to the International Classification of Diseases 10th revision (ICD-10) criteria; the presence of AWS complicated by convulsive seizures during hospitalization; age from 18 to 55 years; at least 7 days from the date of hospitalization; a period of abstinence from alcohol consumption of at least 7 and no more than 30 days. Non-inclusion criteria were related to the existence of AWS at the time of examination; the existence of dependence on a psychoactive substance other than alcohol and nicotine in the patient; the impossibility of interviewing for other reasons; history of convulsive seizures not associated with AWS, which preceded alcoholism; the existence of comorbid mental pathology including organic one, such as symptomatic and mental disorders (F00-F09); schizophrenia spectrum disorders (F20-F29); affective disorders (F30-39); mental retardation (F70-F79); the existence of somatic pathology in the acute stage; the existence of acute infectious disease; history of craniocerebral trauma; existence of severe cognitive impairments; detection of convulsive seizures not associated with alcohol consumption within a year after inclusion. The criteria for exclusion from the study were refusal to participate in the study and identification of non-inclusion criteria during clinical interviewing.

According to the specified sampling criteria, 399 patients, namely 83 women (21%) and 316 men (79%), were analyzed. The average age of patients was 41.52±8.42 years. Patients were divided into 2 groups. The main group consisted of patients who suffered convulsive seizures in a hospital environment and involved 108 patients, more specifically 12 women (11%) and 96 men (89%); the comparison group consisted of 291 patients, namely 71 (25%) women and 220 (75%) men. The patients were examined from February 2019 to August 2023.

Venous blood samples (10 ml) were collected in all patients using Vacutainer vacuum systems for molecular genetic testing; the samples were frozen at ‒20 °C and transported to the Center for Molecular Medicine of Ufa University of Science and Technology (UUNiT, Ufa), where genotyping was performed. Blood samples were prepared for DNA extraction using the reagent for preliminary treatment of whole peripheral blood "Hemolytic" (AmpliSens®). DNA extraction was performed using the "Ribo-PREP" kit (AmpliSens®). Genotyping of CLOCK genes (rs1801260) was performed using real-time polymerase chain reaction (RT-PCR) on a RotorGene 6000 amplifier (Quigen, Germany) using a reagent kit manufactured by Syntol (Moscow). A comparative genetic examination of two groups of patients was conducted: the first group included patients with alcohol dependence syndrome with convulsive seizures after alcohol withdrawal (n=108); the second group included patients with alcohol dependence syndrome without convulsive seizures after alcohol withdrawal (n=291).

Statistical processing was performed using Microsoft Excel and IBM CSSS Statistics 26. When conducting frequency analysis, the χ² criterion (Pearson Chi-square) was used, and the odds ratio for each genotype was estimated. Differences were considered statistically significant at a significance level of p<0.05.

The study complied with modern ethical standards according to the Helsinki Declaration of the World Medical Association (WMA) and was approved by the local ethics committee of the Federal State Budgetary Educational Institution of Higher Education Bashkir State Medical University of the Ministry of Health of the Russian Federation (Protocol No. 3 dated June 5, 2021).

Results

Frequency analysis of the occurrence of convulsive seizures after alcohol withdrawal in carriers of different genotypes revealed statistically significant differences in the carriage of the CLOCK gene (p= 0.019). However, carriers of the CC genotype had convulsive seizures much less frequently (OR=0.12) than carriers of the TT (OR=1.557) and TC (OR=0.981) genotypes. In addition, the presence of the T allele was associated with convulsive seizures after alcohol withdrawal, which were most pronounced in the homozygous variant. The results of the statistical analysis are presented in Table 1.

Thus, it can be concluded that there is an association between the CLOCK gene and convulsive seizures after alcohol withdrawal in patients with alcohol dependence syndrome.

Discussions

The detection of the relationship between CLOCK gene variants and convulsive seizures after alcohol withdrawal expands the data previously identified in other investigations, since the association of circadian rhythms with seizures of various origins is widely described in studies. In particular, the researchers demonstrated an association between neuronal excitability and circadian rhythms, realized primarily through the activity of gamma-aminobutyric acid (GABA) receptors, which regulates the activity of the suprachiasmatic nucleus of the hypothalamus [6][7]. The circadian rhythm is also associated with convulsive seizures of other genesis not caused by alcohol withdrawal, for instance, epileptic seizures. In addition, Udaya Seneviratne et al. (2017) pointed, for example, to the role of the sleep-wake cycle in the occurrence of psychogenic non-epileptic seizures, the pathogenesis of which has not yet been studied [4]. Thus, we can suspect the involvement of the CLOCK gene in the regulation of convulsive seizure activity without reference to a specific genesis. This makes the study of convulsive seizures after alcohol withdrawal an even more relevant task for obtaining fundamental knowledge on seizures, as well as further personalization of assistance with seizures.

Conclusion

The existence of the T allele of the CLOCK gene in the genotype is associated with convulsive seizures after alcohol withdrawal. Moreover, carriers of the homozygous genotype have a higher chance of experiencing convulsive seizures than carriers of the heterozygous genotype.