T. Andrushchenko1, S. Goncharov2, L. Dolinchuk3, V. Dosenko2
1State Enterprise “Occupational Medicine Institute named after Yu. I. Kundiev of the National Academy of Medical Sciences of Ukraine”, Kyiv, Ukraine
2O.O. Bogomolets Institute of Physiology of the National Academy of Sciences, Kyiv, Ukraine
3O.O. Bogomolets National Medical University Kyiv, Ukraine
Abstract. Introduction. Polymorphism of DNA repair genes is actively studied in the formation of the individual sensitivity of the genome to damaging mutagenic effects.
Objective of the work. To study the distribution of frequencies of alleles and genotypes of DNA repair genes: XPD (rs13181, rs799793) and ERCC1 (rs11615) in workers of asbestos-cement plants and miners to identify risk markers for bronchopulmonary pathology.
Material and methods. The study included workers of asbestos-cement plants and miners (n=214). Real-time polymerase chain reaction was used to determine genotypes of XPD (rs13181, rs799793) and ERCC1 (rs11615) genes.
Results. The study determined alleles and genotypes associated with the risk of developing bronchopulmonary pathology: - in the population of workers of asbestos-cement plants: XPD*Asn/Asn (rs799793), (p<0.01; χ2=6.62; OR=2,20; 95 %CI: 1,75–2,77); - in the population of miners: XPD*C (rs13181), (p<0.02; χ2=4,99; OR=1,88; 95 %CI: 1,04–3,40); XPD*CC (rs13181), (p<0,003; χ2=8.61; OR=4,29; 95 %CI: 1,41–13,37). The study also detected allele XPD*A (rs13181), which in the population of miners proved to be a marker of resistance to bronchopulmonary pathology (p<0,02; χ2=4.99; OR=0,53; 95 %CI: 0,29–0,96).
Conclusions. The study has identified alleles and genotypes associated with the risk of developing bronchopulmonary pathology in the population of workers in harmful and dangerous production facilities of Ukraine. The study has determined the genotype and allele that can be used as biomarkers of resistance to the pathology of the respiratory system.
Key words: molecular-genetic markers, XPD and ERCC1, bronchopulmonary pathology.
Introduction. Diseases of respiratory organs as a result of exposure to industrial aerosols play a key role in the structure of occupational diseases [1]. The risk of developing a bronchopulmonary disease (BLP) is individual and depends to a large extent on the genetically determined activity of enzymes [2].
To date, the scientific literature has collected sufficient data on the polymorphism of genes in the DNA repair system, which are associated with factors of increased risk of a variety of oncopathologies of various types and localisations [3]. Several dozens of polymorphisms involved in different types of repair systems are known, as well as the fact that polymorphic variants of genes are capable of altering the structure and activity of reparative enzymes [6, 7]. Therefore, the role of polymorphisms in DNA repair genes is actively studied in the formation of the individual sensitivity of the genome to damaging mutagenic effects [8].
In the structure of harmful and dangerous occupational factors that may lead to BLP, there are those that can cause disorders in the DNA repair system: dust of fibrogenic action of various nature, chemical substances, physical factors, which, in turn, may induce mutagenesis. Given the pathogenetic component of DNA damage in the development of BLP, the search for markers of individual sensitivity among polymorphic variants of reparation genes is of high priority.
The purpose of the work is to study the distribution of the frequencies of alleles and genotypes of the genes: XPD (rs13181, rs799793) and ERCC1 (rs11615) in workers of asbestos-cement plants and miners of coal mines to identify risk markers for bronchopulmonary pathology.
Material and methods. The study included two categories of workers in harmful and dangerous industries of Ukraine (n = 214); these are workers of asbestos cement plants (ACP, n = 94) and miners of coal mines of Ukraine (n = 120).
The first category is the workers of the ACP, Balakleiskyi Slate Combine LLC and Kramatorsk Slate LLC (n = 94). Their average age is 42.9 ± 6.7 years, the average harmful experience is 15.8 ± 4.9 years. For the comparative analysis, two groups were formed: study and control. The group included AСP workers with BLP (chronic bronchitis, chronic obstructive pulmonary disease (COPD), pneumoconiosis), the control group included non-BLP workers, but their work experience and working conditions were identical with those of the study group.
The second category of the study respondents was comprised of miners of coal mines in Donetsk, Lugansk and Lviv regions of Ukraine. The mines where they worked were similar in terms of mining and geological conditions for coal mining. The average age of miners in the study was 52.5 ± 7.3 years, the average underground experience is 22.04 ± 5.9 years. The group included miners with BLP (chronic bronchitis, COPD, pneumoconiosis), the control group included miners without BLP, but their age, underground experience and working conditions were identical upon the statistical treatment.
Genetic material (DNA) was isolated from peripheral blood leukocytes. Real-time polymerase chain reaction was used to determine genotypes of XPD (rs13181, rs799793) and ERCC1 (rs11615) genes. The results were statistically processed using Orion 7.0, Statistica, Excel 2000. The probability of differences was determined by χ2 criterion, p-value <0.05 was considered reliable.
Results and discussion. XPD gene products (xeroderma pigmentosum group D) function at the initial stage of the synthesis of all cell proteins as a subunit of the complex protein TFIIH, an accessory factor of RNA polymerase II [6]. This study investigated allelic polymorphisms of XPD (rs13181 and rs799793): replacement of G by A at position 23591 causes a substitution in exon 10 (312 Asp (D) for Asn (N) of amino acid exchange), and substitution of А - С at nucleotide position 35931 causes a substitution in exon 23 of codon 751 Lys (K) for Gln (Q). According to the literature, carriers of СС (Gln/Gln) minor genotype of XPD gene have a reduced ability to repair and this leads to an inferior restoration of DNA damage upon ultraviolet irradiation and action of chemical mutagens [9].
Five polymorphisms are described for ERCC1 gene, this study investigated the allelic polymorphism ERCC1 (rs11615), it is known that ERCC1 118T allele in some studies was associated with a decrease in mRNA quantity and a threefold decrease in the ability to repair DNA damage [4, 10].
The analysis of the study of allelic polymorphisms of XPD (rs13181, rs799793) and ERCC1 (rs11615) genes showed that the frequency of distribution of minor alleles: XPD*С, XPD*Asn, ERCC1*Т in the ACP study group was, respectively: 37 %, 36.9 %, and 35.9 %. And in the control group: XPD*С — 44.8 %, XPD*Asn — 29.2 %, ERCC1*Т — 39.6 %. At the same time, the dominant XPD*А, XPD*Asp, ERCC1*C alleles among the respondents in the ACP study group were as follows: 63 %, 63.1 %, and 64.1 %. Accordingly, in the control group, the frequency of dominant alleles by the studied genes was as follows: XPD*А — 55.2 %, XPD*Asp — 70.8 %, ERCC1*C — 60.4 %. The analysis of frequencies of allele distribution of XPD (rs13181, rs799793) and ERCC1 (rs11615) genes in the population of ACP workers is presented in Table 1.
Table 1. Analysis of frequencies of allele distribution of XPD (rs13181, rs799793) and ERCC1 (rs11615) genes in the population of workers of asbestos-cement plants
When analysing the study of the frequencies of distribution of minor alleles: XPD*С, XPD*Asn, ERCC1*Т in the study group of miners were, respectively: 51.2 %, 35.2 %, and 39.8 %. In the control group of miners: XPD*С — 35.8 %, XPD*Asn — 34.9 %, ERCC1*Т — 38.2 %. Distribution of dominant alleles by the studied genes in the study group of miners: XPD*А – 48.8 %, XPD*Asp – 64.8 %, ERCC1*C – 60.2. Accordingly, in the control group, the frequency of dominant alleles was as follows: XPD*А — 64.2 %, XPD*Asp — 65.1 %, ERCC1*C — 61.8 %. The analysis of frequencies of allele distribution of XPD (rs13181, rs799793) and ERCC1 (rs11615) genes in the population of miners is presented in Table 2.
Table 2. Analysis of frequencies of allele distribution of XPD (rs13181, rs799793) and ERCC1 (rs11615) genes in the population of miners
*XPD*C (rs13181) allele frequency is significantly higher in the study group in comparison with the control (p ≤ 0.02; χ² = 4.99);
*XPD*A (rs13181) allele frequency is significantly higher in the control group in comparison with the study group (p ≤ 0.02; χ² = 4.99);
The study of allele frequencies of the studied genes demonstrated a statistically significant difference in frequencies of XPD*A and XPD*С allele (rs13181) in the population of miners (p ≤ 0.02, χ² = 4.99). In determining OR, a correlation was established for carriers of the minor allele XPD*С (rs13181) in miners with BLP relative to the relative risk of developing BLP (OR = 1.88, 95 % CI: 1.04–3.40), as well as calculated OR of the dominant allele XPD*A (rs13181) (OR = 0.53, 95 % CI: 0.29–0.96) in the population of miners supports a protective role in relation to the risk of development of BLP.
To study the associations of certain genotypes of repair genes with the risk of developing BLP, their frequencies were determined: XPD (rs13181, rs799793) and ERCC1 (rs11615). It should be noted that the genotype frequencies of the polymorphisms studied were close to the population frequencies in the European population, which according to the literature is:
- XPD gene (rs 13181) dominant homozygote — XPD*A/A — 35.4 %, XPD*A/С — 52.4 %, minor XPD*С/С — 12.2 %;
- XPD gene (rs 799793) dominant homozygotes — XPD*Asp/Asp – up to 43 %; heterozygotes XPD*Asp/Asn — 50–53 %, minor homozygotes XPD*Asn/Asn — 17 %;
- ERCC gene (rs11615) dominant homozygotes — ERCC1*С/С — up to 50 %; heterozygotes ERCC1*С/Т — 30 %, minor homozygote ERCC1*Т/Т - 17 % [3, 4, 5, 9, 10].
When analysing the frequencies of XPD gene genotypes (rs799793), the carriers of XPD*Asn/Asn genotype in the population of ACP workers were 13.1 % in the study group, minor homozygotes were not found in the control group (χ² = 6.62, p ≤ 0.01), which indicates an increased risk of developing BLP in the ACP study group (OR = 2.20, 95 % CI: 1.75–2.77). The analysis of the genotype frequencies of XPD (rs13181, rs799793) and ERCC1 (rs11615) in the population of ACP workers is presented in Table 3.
Table 3. Analysis of genotype frequencies of XPD (rs13181, rs799793) and ERCC1 (rs11615) genes in the workers of asbestos-cement plants
XPD*Asn/Asn (rs799793) genotype frequency is significantly higher in the study group in comparison with the control group (p ≤ 0.01; χ² = 6.62);
In the study, the genotype frequencies of the genes studied in the population of miners were also analysed. When studying the genotype distribution of XPD gene (rs13181), the frequency of minor XPD*С/С homozygotes in the study group of miners was 33.4 % and 10.5 % in the control group. In the statistical processing of the results by the χ² method, a statistically significant difference in the frequencies of XPD*С/С (rs13181) genotypes between miners with BLP and the control group (χ² = 8.61, р ≤ 0.003) was established. The analysis of genotype frequencies of XPD (rs13181, rs799793) and ERCC1 (rs11615) genes in the population of miners is presented in Table 4.
Table 4. Analysis of genotype frequencies of XPD (rs13181, rs799793) and ERCC1 (rs11615) genes in miners
XPD*CC (rs13181) genotype frequency is significantly higher in the study group in comparison with the control group (p ≤ 0.003; χ² = 8.61);
Conclusions
1. The study has demonstrated a statistically significant difference in frequencies of the allele XPD*A and XPD*С of XPD gene (rs13181) in the population of miners (p ≤ 0.02, χ² = 4.99), which indicates an association for carriers of the minor XPD*С allele with a risk of bronchopulmonary pathology (OR = 1.88, 95 % CI: 1.04–3.40), while the carriers of the dominant XPD*A allele have a relative resistance to the development of the respiratory system diseases (OR = 0.53, 95 % CI: 0.29–0.96).
2. The genotypes associated with the risk of developing a bronchopulmonary pathology have been identified: XPD*CC (rs13181) in the population of miners, (p ≤ 0.003; χ² = 8.61; OR = 4.29; 95 %CI: 1.41–13.37) and XPD* Asn/Asn (rs13181) in the population of ACP workers, (p ≤ 0.01; χ² = 6.62; OR = 2.20; 95 % CI: 1.75–2.77).
3. Molecular genetic studies are a promising area for developing methods for primary prevention of bronchopulmonary pathology and provide a personalised approach.
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Надійшла до редакції 20.02.2018 р.