Т. Andrushchenko¹, S. Honcharov², V. Dosenko²

¹State Institution “Kundiiev Institute of Occupational Health of the National Academy of Medical Sciences of Ukraine ”, Kyiv, Ukraine
²Bohomolets Institute of Physiology of the National Academy of Sciences of Ukraine, Kyiv, Ukraine

Abstract. Introduction. Distribution of the following allelic variants of DNA repair genes: АТМ (rs664677), XRCC7 (rs7003908), and MLH1 (rs1799977) in the population of personnel of harmful and hazardous occupation has been studied. The studied polymorphisms are recognized as cancer-specific markers of various types and localization of malignant neoplasms, as well as markers of radiosensitivity/resistance to radiation exposure.
Objectives of the work: to find out the significance of polymorphisms of repair genes of double-strand DNA breaks: XRCC7 (rs7003908), АТМ (rs664677), and mismatch repair: MLH1 (rs1799977) in the formation of an individual predisposition to the development of chronic diseases of the bronchopulmonary system in miners and personnel ofasbestos-cement plants.
Materials and methods. Respondents of the study group was the personnel of asbestos-cement plants and miners with chronic bronchopulmonary disease; the control group was made up of personnel without diseases of the respiratory system. The genotypes of the following genes were determined by real-time polymerase chain reaction: АТМ (rs664677), XRCC7 (rs7003908), and MLH1 (rs1799977).
Results. It was established that the minor alleles of ATM•T and MLH1•G, minor homozygote ATM•TT and heterozygote MLH1•AG are associated with the risk of developing chronic diseases of the bronchopulmonary system. It has been revealed that the dominant alleles of ATM•A, MLH1•A; dominant homozygotes ATM•AA; MLH1•AA and heterozygote ATM•AT contribute to resistance to the development of the respiratory system conditions.
Conclusion. The following alleles: ATM•T (Р<=0,06, χ²=3,44; OR=1,44; 95 % Cl: 0,96–2,17); MLH1•G (Р<=0,002, χ²=5,06; OR=1,61; 95 % Cl: 1,04–2,49) and genotype: ATM•TT (Р<=0,01, χ²=6,61; OR=2,48; 95 % Cl: 1,16–5,31); MLH1•AG (Р<=0,002, χ²=9,00; OR=2,32; 95 % CI: 1,29–4,21) associated with the risk of bronchopulmonary conditions development have been established. Also alleles: ATM•A (Р<=0,06, χ²=3,44; OR=0,69; 95 % CI: 0,46–1,04); MLH1•A (Р<=0,002, χ²=5,06; OR=0,62; 95 % CI: 0,40–0,96) and genotype: MLH1•A/A (Р<=0,003, χ²=8,73; OR=0,43; 95 % CI: 0,24–0,79) that form resistance to the development of pulmonary system conditions in certain occupational groups have been established.
Key words: SNP, ATM, XRCC7, MLH1, bronchopulmonary pathology.

Introduction. Bronchopulmonary conditions (BPCs) dominate in the structure of occupational conditions, its prevalence stipulates the need for the development of new methods of primary prevention and early diagnostics [1]. Currently, data on single nucleotide polymorphisms (SNP) in DNA repair genes associated with high-risk factors for a variety of cancers of various types and localization have been accumulated. It was established that the violation in the system of control over DNA repair and apoptosis processes is caused not only by genetic and epigenetic lesions but also by the variability of the functioning of genes due to SNP [2].

Double-strand break repair (DSBR) is very important for the survival of highly differentiated cells. Two DSBR mechanisms exist non-homologous end joining (NHEJ) and homologous recombination (HR) [4, 6]. Double-strand DNA breaks may occur during the normal replication process and as a result of DNA damaging agents which are considered to be the most severe forms of genotoxic damages. Different variants of DSBR errors lead to various types of mutations and chromosome rearrangements that induce genome instability and carcinogenesis [3, 6].  Among the well-known genetic factors, NHEJ genes play an important role in the repair of double-strand DNA breaks under the action of exogenous factors [12]. The gene XRCC7, known as Protein kinase, DNA-activated, catalytic polypeptide (PRKDC), located on the 8th chromosome (8q11), consisted of 85 exons and 86 introns, is an important DNA regenerator involved in the NHEJ [8]. XRCC7 encodes a protein that is a large catalytic subunit of the DNA-PKcs complex that forms an active protein kinase with the Ku heterodimer and initiates repair by NHEJ [13, 14]. Currently, over 100 SNPs have been known in XRCC7 gene, some of which correlate with malignant tumours, in particular, with lung carcinoma [3, 7, 11, 12].

Ataxia-telangiectasia mutated (ATM) gene is localised on the 11th chromosome (11q22-23), has a length of 150 thousand nucleotide sequences and consists of 66 exons. ATM encodes DNA-dependent protein kinase, localised mainly in the nucleus. This enzyme is involved in the mitogenic signalling of meiotic recombination and in the regulation of the cell cycle. There are ATM 88 polymorphisms, the carriers of mutant alleles are characterized by sensitivity to radiation, multiple developmental defects, predisposition to malignancies [10].

DNA mismatch repair (MMR) takes a special place among the cellular systems. Due to MMR, it is possible to store genetic information, when organisms are exposed to conditions under which the rate of mutations sharply increases [9]. As the result of errors of DNA polymerase and recombination, synthesised DNA strands show noncomplementary residues of nucleosides: instead of canonical pairs G-C and А-T, DNA has the following pairs as G-G, А-А, А-С, G-T that locally bend double strand of macromolecule [4]. MLH1 (mutL (E.coli) homolog 1) is located on the 3rd chromosome and consists of 19 exons and 757 codons [5]. MLH1 encodes a protein that regulates the replacement of improperly coupled DNA bases and is inactivated by methylation. 126 SNPs of MLH1 and MSH2 were registered, most of which are on the intronal surface [10].

The structure of occupational factors that determine the development of BPCs has those that can reduce the effectiveness of DSBR and MMR. Therefore, the search for markers of individual predisposition to BPCs in personnel of harmful and hazardous industries among SNP DSBR and MMR is relevant.

Objective of work is to find out the significance of polymorphisms of repair genes of double-strand DNA breaks: XRCC7 (rs7003908), АТМ (rs664677), and DNA mismatch repair: MLH1 (rs1799977) in the formation of an individual predisposition to the development of chronic diseases of the bronchopulmonary system in miners and personnel of asbestos-cement plants.

PROCEDURE. The study included two categories of personnel of harmful and hazardous industries in Ukraine (n = 215): personnel of asbestos-cement plants (ACPs) (n = 95 and miners in coal mines of Ukraine (n = 120). Study and control groups were formed for comparative analysis. The study group included personnel of ACPs and coal miners with BPCs (chronic bronchitis, chronic obstructive pulmonary disease, pneumoconiosis), control group included respondents, who had no history of BPCs, however, their work experience and working conditions were comparable with data from respondents of the test group. General characteristics of groups is provided in Table 1.

Table 1. General characteristics of study respondents.

The material for the study was venous blood that has been collected under sterile conditions into 2.7 mL Monovette tubes with anticoagulant (ethylenediaminetetraacetic acid potassium salt) (11.7 mmol/L) (Sarstedt, Germany). A unified bank of genetic material was created for persons who contacted with genotoxic agents (dust of fibrogenic action, chemicals and physical factors) to assess the long-term consequences of the effect of man-made factors using modern molecular genetic technologies. DNA was isolated from peripheral blood white blood cells using NeoPrep100DNA, NEOGENE, Ukraine. Using 7500 Fast Real-Time PCR System (Applied Biosystems, USA) and TaqMan SNP, genotypes were identified for genes: АТМ (rs664677), XRCC7 (rs7003908), MLH1 (rs1799977), and analysis by allele discrimination was performed (Fig. 1).

Fig. 1. Results of discrimination of alleles of АТМ (rs664677) gene using Real-time PCR in the respondents of control group (a) and in the respondents of study group (b).

Note: I — AA homozygotes, II — AT heterozygotes, III — TT homozygotes, VI — DNA-free test.

The obtained results were statistically processed using Orion 7.0, Statistica 10, Excel 2000. At the same time, the probability of differences was determined by χ2-criterion, p-value <0.05 was considered significant.

RESULTS AND DISCUSSION. Analysis of the study of allelic polymorphisms of ATM (rs664677), XRCC7 (rs7003908) and MLH1 (rs1799977) showed that the frequency of minor alleles: ATM • T and MLH1 • G in the study group was significantly higher in comparison with the control group and was the following: АТМ • Т — 47. 8%; MLH1 • G — 36.1, respectively in the control group:  АТМ • Т — 38.8 %; MLH1 • G — 26.0. At the same time, the rate of dominant alleles in АТМ (rs664677) and MLH1 (rs1799977) was significantly higher among respondents of the control group:  АТМ • А — 61.2 %; MLH1•А — 74.0, respectively in the study group: АТМ•А — 52.2 %; MLH1 • А — 63.9.  In the study of the distribution of alleles in XRCC7 (rs7003908), no significant differences were found. Analysis of the rate of distribution of alleles of ATM (rs664677), XRCC7 (rs7003908) and MLH1 (rs1799977) in the population of miners and ACPs personnel is presented in Table 2.

Table 2. Analysis of the rate of distribution of alleles of ATM (rs664677), XRCC7 (rs7003908) and MLH1 (rs1799977) in the population of miners and personnel of asbestos-cement plants.

The next stage of our study included investigation of the rate of genotypes of АТМ (rs664677), XRCC7 (rs7003908) and MLH1 (rs1799977). It should be noted that the obtained values of rate of genotypes were close to the population frequencies of the European population, which according to the literature data are the following:

-  dominant homozygotes: АТМ • AA — 30–35 %; XRCC7 • СС — 33 %; MLH1 • АА — 25–45 %;
-  heterozygotes: АТМ  • AТ — 50 %; XRCC7 • СТ — 50 %; MLH1 • АG — 35–45 %;
-  minor homozygotes: АТМ • ТТ — 13 %; XRCC7 • ТТ — 17 %; MLH1 • GG — up to 10 % [5, 11].

During the study of the rate of genotypes  of ATM (rs664677), it was found that dominant AA homozygotes in the control group were 35.2 %; heterozygotes AT — 52 %; minor TT homozygotes — 12.8 %, and respectively in the study group: АТМ • AA — 31.1 %; АТМ • AТ — 42.2 %; АТМ • ТТ — 26.7 % (р ≤ 0.03). The obtained results indicate that the distribution of the rate of genotypes of ATM (rs664677) significantly differs from the rate of the minor homozygotes in the control group and in the group of personnel with BPCs (Table 3).

During the study of the rate of genotypes of XRCC7 (rs7003908), it was found that dominant AA homozygotes in the control group were 44.8 %; AT heterozygotes — 40.8 %; minor TT homozygotes — 14.4 %, and respectively in the study group: XRCC7 • СС — 46.7 %, XRCC7 • СТ — 42.2 %, XRCC7 • ТT — 11.1 % (р ≤ 0.7). The obtained results indicate that the distribution of the rate of genotypes of XRCC7 (rs7003908) is not significantly different in the control group and in the study group (Table 2).

The rate of allele variant of MMR gene — MLH1 (rs1799977) in our study was the following: MLH1 • АА — 56 %; MLH1 • АG — 36.0 %, MLH1 • GG — 8 % in the control group. In the study group, respectively: dominant AA homozygotes — 35.6 %, AG heterozygotes — 56.7 %, minor GG homozygotes — 7.7 % (P ≤ 0.008). Analysis of the rate of distribution of genotypes of ATM (rs664677), XRCC7 (rs7003908) and MLH1 (rs1799977) in the population of miners and ACPs personnel is presented in Table 3.

Table 3. The rate of distribution (%) of genotypes of ATM (rs664677), XRCC7 (rs7003908) and MLH1 (rs1799977) in the population of miners and personnel of asbestos-cement plants.

Using statistical data processing: OR (odds ratio), χ², alleles and genotypes associated with the risk of BPCs development have been established, and they were the following: minor alleles ATM • T and MLH1 • G, minor homozygote ATM • TT and heterozygote MLH1 • AG. And also, alleles and genotypes that contribute to the resistance to the development of the respiratory system conditions have been established. These are dominant alleles of ATM • A, MLH1 • A; dominant homozygotes ATM • AA; MLH1 • AA and heterozygote ATM • AT.

Thus, the results indicate the association between the altered alleles of ATM (rs664677) and MLH1 (rs1799977) and the probability of the risk of developing BPCs.

CONCLUSION

1. The following alleles: АТМ • Т (Р ≤ 0.06, χ² = 3.44; OR = 1.44; 95 %CI: 0.96–2.17); MLH1 • G (Р ≤ 0.002, χ² = 5.06; OR = 1.61; 95 %CI: 1.04–2.49) and genotype: АТМ • ТТ (Р ≤ 0.01, χ² = 6.61; OR = 2.48; 95 % CI: 1.16–5.31); MLH1 • АG (Р ≤ 0.002, χ² = 9.00; OR = 2.32; 95 %CI: 1.29–4.21) associated with the risk of bronchopulmonary conditions development have been established.

2. Alleles: АТМ • A (Р ≤ 0.06, χ² = 3.44; OR = 0.69; 95 %CI: 0.46–1.04); MLH1 • A (Р ≤ 0.002, χ² = 5.06; OR = 0.62; 95 %CI: 0.40–0.96) and genotype: MLH1 • АА (Р ≤ 0.003, χ² = 8.73; OR = 0.43; 95 %CI: 0.24–0.79) that form resistance to the development of pulmonary system conditions in certain occupational groups have been established.

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Надійшла до редакції 29.06.2018 р.