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SUBSTANTIATION OF CLINICAL DIAGNOSIS BY THE APPLICATION OF GENETIC
TRANSFORMATION MARKER
Desyatova M.A.,
Ural State Medical University, Ekaterinburg, Russian Federation
(Junior research scientist) Korotkov A. V.,
Ural State Medical University, Ekaterinburg, Russian Federation
(Junior research scientist) Institute of Medical Cell Technologies, Ekaterinburg, Russian Federation
Kostyukova S.V.,
Ural State Medical University, Ekaterinburg, Russian Federation
(Junior research scientist) Institute of Medical Cell Technologies, Ekaterinburg, Russian Federation
Makeev O.G.
Ural State Medical University, Ekaterinburg, Russian Federation
(Junior research scientist) Institute of Medical Cell Technologies, Ekaterinburg, Russian Federation
Abstract
In this article, the application principle of the polymorphic marker TP53 had been considered and discussed as a universal methodic for assessment of the disruption of the stability of the genetic apparatus in patients with a previously established fact of oncological disease.
Keywords: Genomic instability, DNA, Single nucleotide polymorphism (SNPs), mutation.
Introduction
Recently, there is a need to detect the presence of mutations leading to malignant transformation of the cell. Understanding genetic variations can provide the ability to track cancer diseases, most of which are caused by carcinogenesis, and as a result, obtain information about the mechanisms leading to changes in the internal integrity of the genetic apparatus, as well as identify critical factors affecting the occurrence of cellular transformations. This may be an advantage when
creating a preventive strategy and a preliminary assessment of the predisposition to mutation. At this stage, we are at the introductory stage of DNA diagnostics, as a tool for analyzing a single nucleotide polymorphism. Replacing a single base can lead to some changes in the genome. However, the vast majority of such substitutions may not cause changes in the expression and functioning of the gene, but manifest themselves in the characteristics of the phenotype. In this study, it is relevant
to consider the mutation of the TP53 gene, which encodes a nuclear phosphoprotein that is involved in the regulation of the cell cycle. At the cellular level, the p53 gene protein works as an agent for the elimination of potential tumor cells, prompting programmed cell death, inhibiting the proliferation of damaged cells or causing cell aging [2]. TP53 is the most frequently altered gene in the event of a tumor formation [2]. Earlier studies have shown that when a gene is damaged, the synthesis of an inactive protein that has adopted an abnormal structure is preserved. P53, inactivated by mutations at codons 175, 248, and 273, makes the cell sensitive to DNA-damaging factors and deactivate the corresponding protein [3]. Consequently, the TP53
mutation can serve as an indicator of the presence of a DNA defect - the reparative function and the disruption of the cell death apparatus. However, the question of the significance of TP53 gene mutations for the development of tumors from cells of various germ layers remains unclear.
The goal of the study is to verify the clinically established diagnosis of patients using a marker for the presence of a TP53 gene mutation and to assess the significance of the mutation of the TP53 gene in tumor cells from different germ layers.
Materials and methods
DNA samples were studied from seven patients with an early established cancer (Table 1).
Table 1
Characteristics of patients with a detected mutation in the TP53 gene
Patient Sex Age Diagnosis Germ layer
1 female 39 Liver hemangioma Endoderm
2 male 57 Papillomatous polyp of the gallbladder Endoderm a
3 female 54 Uterine myoma Mesoderm
4 male 71 Prostate cancer Mesoderm
5 female 56 Uterine cancer Ectoderm
6 male 78 Stomach cancer Ectoderm
7 female 35 Tumor of the left hemisphere of the brain Ectoderm
DNA was isolated from the nucleated cells of the peripheral blood of patients by the phenol method. The presence of metastatic cells in the blood is an unfavorable prognostic factor [5]. After isolation, the obtained DNA samples were frozen and stored at - 85 ° C. PCR was performed using the SNP-Express TP53 system (Litekh, Russia) For each sample of the obtained DNA, two polymerase chain reactions were simultaneously performed with two different pairs of specific primers in a programmable thermostat "Terzik" with an exact algorithm in the volume of the reaction mixture of 53 ^l using Lambda Phage Taq polymerase. During the PCR, a three-step cycle was used according to the manufacturer's protocol.
The amplification products were detected by horizontal electrophoresis on 1.5% agarose with the addition of 4 ^l ethidium bromide per 50 ml of the molten
mixture. 10 ^l amplification was placed in the wells of the gel. Electrophoresis was performed for 23 minutes at 150 V.
Results and discussion
Figure 1 shows the results of PCR analysis. High expression is noteworthy when amplified with the TP53 marker Pro47Ser, mutation of the p53 protein in samples 1, 2, 3 and in the positive control sample (K +). In addition, samples 6 and 7 trace tails, which are DNA fragments, indicating the presence of multiple single and double-strand breaks in patient data, which can serve as an indicator of genome instability. A similar pattern is observed with amplification with the TP53 marker Pro72Arg.
Figure 1.
The results of electrophoresis of PCR amplification products of the polymeric marker TP53 Pro47Ser mutation of the p53 protein (A) and TP53 Pro72Arg mutation 2 of the p53 protein (B). K +: positive control.
The results obtained indicate that the development of oncopathology in these patients is due to several genetic defects in the TP53 gene, both as a variant of the Pro47Ser mutation and Pro72Arg TP53.
Despite the fact that DNA samples were obtained from patients with a confirmed diagnosis, the tumors originated from tissues of different origin: endodermal - patients 1 and 2, mesodermal - 3 and 4, ectodermal -5, 6, 7. In each case, we have registered mutations in codons 175, 248, 273. It cannot be ruled out that in this case we are dealing with inherited mutations. The latter assumption is supported by the high fragmentation of genomic DNA in two patients. This suggests that the study of the TP53 gene may be useful in predicting the risk of oncopathology from tissues of different origin. The latter is confirmed in the literature [1]. The significant polymorphism of the TP53 gene identified by us may increase the risk of developing oncopathology, which is also confirmed in the literature [6, 4].
Thus, the study of TP53 mutations can serve as a complex biological marker of oncopathology, since TP53 mutations manifest themselves at different stages of tumor development of various tissue origins and are probably an early indicator of predisposition to tumor development.
Conclusion
1) The study of TP53 mutations can serve as a universal marker of the risk of oncopathology in a particular patient.
2) Mutations in the TP53 gene are accompanied by malignant transformation of cells from tissues originating from different germ layers.
References
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3. Ory, K., Legros, Y., Auguin, C., & Soussi, T. (1994). Analysis of the most representative tumour-derived p53 mutants reveals that changes in protein conformation are not correlated with loss of transactivation or inhibition of cell proliferation. The EMBO Journal, 13(15), 3496-3504.
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