CC BY
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UDC 616.24
Mazlumyan A.A.
Undergraduate of Faculty of General Medicine State Medical University Kursk
FUNCTIONAL ACTIVITY OF RIBOSOMAL GENES IN PATIENTS WITH MULTIFACTORIAL DISEASES AND PULMONARY PATHOLOGIES
Abstract
Functioning and regulation processes of ribosomal genes (RG) transcription in humans have been studied in order to understand mechanisms through which polymorphic variants of transcription genes factors of RG and detoxification enzymes are involved in the development and pathogenesis of lung pathology and multifactorial diseases. Statistical material shows that allelic features of these genes, combined with allelic variants of genes of transcription factors of RGs affected the expression of the latter, thus determining the functional basis of the human phenotype, including the pathological phenotype. Study of genes polymorphisms transcription factors of RG and xenobiotics biotransformation genes among patients of various nosological forms and controls was made for the first time.
Key words
Preventive genetic research, bronchial asthma, allergic rhinitis, lung pathology, professional chronic bronchitis,
chronic obstructive pulmonary disease.
Introduction
Studying the influence of genetic changes on the development of pathological reactions from the lungs, the consequence of which is a fixed reaction of the body is quite acute [1-6]. Absence of timely genetic control and screening for further examination, initially manifested allergic symptoms turn into dangerous diseases: bronchial asthma, allergic rhinitis, occupational chronic bronchitis, chronic obstructive pulmonary disease [7,8]. These diseases significantly impair the life quality at risk groups [9]. Preventive genetic research will allow further development of treatment methods, including the prevention of these diseases and their detection at early stages, and provide the opportunity to carry out health-improving and medical processes more efficiently [10-12]. Preclinical diagnostics, which is based on genetic methods, makes it possible to identify a range of possible environmental factors that can provoke the occurrence or exacerbation of a particular pulmonary pathology but also identify a range of environmental factors that could trigger the emergence or exacerbation of a particular pulmonary pathology [13,14]. The subject of study was the functioning and regulation processes of the transcriptional complex of human ribosomal genes.
Materials and methods
The material is presented by three statistical samples: people with bronchial asthma (n=147), with allergic rhinitis (n=120), and people with lung pathology (n=351): patients (work experience from 7 to 38 years) with professional chronic bronchitis (n=164), and patients with chronic obstructive pulmonary disease (n=187), where n is the sample size. The absence of relevant diseases in an individual was taken into account when forming the control group (n=918). The groups of patients did not differ from the control group, both by sex and age (p<0.05; level of significance).
In the experimental part of the work, chromosomes and DNA analysis were carried out, obtained from the one-time biological material of the study groups. The study used three groups of methods: cytogenetic, molecular-genetic and genetic-statistical methods.
Cytogenetic methods
Preparations of metaphasic chromosomes are obtained by standard methods using a semi-micro method from human peripheral blood lymphocytes [15]. Cellular cultures are fixed for 72 hours after stimulation with phyto gemaglutenin according to standard methods [16]. The resulting suspension was laid out on chemically clean chilled glasses, dried, marked and stored at room temperature until staining [17]. The method of differential chromosome coloration proposed by [18] with some modifications was used to identify kernel organizers. Analysis of a set of indicators of functional activity of ribosomal genes (FARG) was carried out on 20 metaphasic plates for each individual without karyotyping. Indicators of FARG were evaluated visually with the help of the microscope "ZEISS" (increase: eyepiece 10x, lens 90x) on a scale of five [19]. In addition to estimating the total activity of ribosomal genes (RG) on 10 acrocentric chromosomes, the activity of RG by chromosome D group and G group was separately considered. A total of 24.400 metaphasic plates have been analyzed: 9.740 are in a sample of children and 14.660 are in an adult sample; of these, 2.140 are children with cerebral palsy, 10.960 are children with cerebral palsy, 1.740 are children with allergic responses, 5.860 metaphases with bronchial asthma, and atopic dermatitis - 1860, with allergic rhinitis - 1720, and patients with lung pathology - 3700 metaphases: patients with professional CB (chronic bronchitis)- 2200, patients with COPD (chronic obstructive pulmonary disease) - 1500.
Molecular genetic methods
Genomic DNA was isolated from frozen (T= -20°C) venous blood by a standard two-stage phenolchloroform extraction method [20]. Genetic statistical methods
The database was developed as part of the medical and genetic counseling of patients and the accumulation of one-time biological material. The resulting database was processed using the Statistica 6.0 program. Data analysis methods variation statistics [21-23]. The results obtained were checked for normal distribution and assessed by the Shapiro-Wilk test [24] and Nonparametric Mann-Whitney and x2 tests were used to compare two independent groups on a single basis [25]. The appropriateness of the genotype distributions to the expected Hardy-Weinberg equilibrium values in the sample and comparison with the frequencies of the alleles and genotypes of the different groups were assessed using the x2 Pearson criterion, provided the sample size did not exceed 10 cases, used the criterion x2 Yate's correction on continuity and precise Fisher's test [26, 27]. About the association of alleles or genotypes with a predisposition to diseases were judged by the size of the odds ratios (OR) - an indicator reflecting how many times the probability to be in the group «case» (patients) differs from the probability to be in the group «control» (healthy) for the carrier of the studied genotype [28,29]. The relationship between cytogenetic traits and molecular genetic markers was studied with the help of one-factor dispersion analysis, in which the distribution of the quantitative traits values in groups with different genotypes and was compared with the subsequent pairwise variance comparison (Wald-Wolfowitz test) [30]. To analyze the influence of genotypes paired combinations on the indicators of the FARG, two-factor dispersion analysis was used with the calculation of explained phenotypic dispersion. In all cases, the level of statistical significance was assumed to be 95% (p < 0.05) [31-33].
Results and discussion
For the first time, features of complex FARG at several independent pathologies of the pulmonary system were studied. Statistically significant differences in the level of FARG in the sample compared to control were found in the group with bronchial asthma and patients with allergic rhinitis, as well as for COPD patients. For each sample examined, characteristic interactions within a set of different indicators of FARG were identified, allowing some compensation for potential "population norm" deviations. The most variable were the FARG level on D chromosomes, the number of active ribosomal cystrones and the number of acrocentric chromosome associations, and the most stable was the FARG level on G chromosomes.
The statistics show that polymorphism of genes transcription factors (GTF) of RG and detoxification
enzymes in Russian residents of the Central Black Earth region is characterized by a wide allele diversity. The level of heterozygosity observed ranged from No=0.12-0.16 for polymorphism 462 I>V of the CYP1A1 gene of different samples to No=0.55-0.57 for polymorphism 6 A>C of the TAF 1B gene. High enough heterozygosity values are also observed for polymorphisms 72 R>P Tp53 and 149 C>T of the DNMT3B gene. The average level of heterozygosity was observed for polymorphisms: 139 H>R of the EPHX 1 gene and 105 IV of the GSTP gene. In general, according to the frequencies of polymorphic genes alleles studied ribosome gene transcription factors and detoxification enzymes, studied in population sample of Russian inhabitants of the Central Black Earth region.
Conclusions
The processes GTF of RG functioning and regulation in humans have been studied. Statistical material shows that allelic features of these genes, combined with allelic variants GTF, affect the expression of the latter, thus determining the functional basis of the human phenotype, including the pathological phenotype. The dependence was established and the degree of influence of polymorphisms of various genes on the formation of phenotypic traits specific complex. FARG indicators in the studied pathogenetically independent multifactorial diseases showed characteristic features: statistically significant differences in FARG levels compared with controls were found in group with bronchial asthma and allergic rhinitis, as well as in the group with chronic lung disease. Of the FARG complex among pathogenetically independent MDs, the most stable cytogenetic indicator was the level of FARG for group G chromosomes, the most variable was the FARG indicator for D chromosomes, the number of active ribosomal cistrons, and the number of associations of acrocentric chromosomes. Study of polymorphisms transcription factors ribosomal and xenobiotics biotransformation genes among patients of various nosological forms and controls was made for the first time. Acknowledgements
The author expresses his gratitude to Pr. E.V. Trubnikova, scientific supervisor, and As. Pr. S.A. Avakova, scientific editor, for valuable advice during the work on the research. References
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© Mazlumyan A.A., 2023
