Method for determining individual sensitivity of human genome to radon exposure

FIELD: medicine.

SUBSTANCE: determining individual genome sensitivity to radon exposure is ensured by genetic blood test to identify predisposing and protective genotypes: marker Arg280His of gene XRCC1 - predisposing genotype Arg/Arg, protective genotype Arg/His; marker Argl94Trp of gene XRCC1 - predisposing genotype Arg/Arg, protective genotype Arg/Trp; marker Asnl48Glu of gene APE1 - predisposing genotype Glu/Glu, protective genotypes Asn/Asn, Asn/Glu; marker A2455G of gene CYP1A1 - predisposing genotype A/G, protective genotypes A/A and G/G; a deletion marker in gene GSTM1 - predisposing genotype o/o, protective +. High individual sensitivity to high radon dose is stated by observing the quantitative prevalence of predisposing genotypes or the equal quantity of predisposing and protective genotypes. High individual resistance to high radon dose is determined by the quantitative prevalence of protective genotypes.

EFFECT: use of the method allows estimating genetically determined predisposition to formation of high level of chromosomal aberrations even before exposing to a radiating factor.

5 tbl, 2 ex

 

The invention relates to the field of medicine, human genetics and can be used to assess individual radiosensitivity of the human genome (where live or work in conditions of exposure to high doses of radon).

There is a method of determining the genotoxic sensitivity to the complex chemical mutagens, manifested in the form of a certain level of chromosomal aberrations (CA), applicable for the assessment of professional suitability of individuals to work in environments with increased hazard for genetic markers (A.S. SU 1621718 A1, CL G01N 33/48, publ. 22.04.1988). The essence of the method lies in the fact that the Respondent take the blood from the cubital vein, cultivate her 48 hours in the presence of phytohemagglutinin, accumulation of chromosomes in metaphase use treatment by colchicine. This is followed by hypotonic treatment and fixation of the cells. The resulting suspension dig on a chilled glass slides and dried in air. With the help of the method of differential staining of chromosomes reveal extreme variants of polymorphism. For this purpose, preparations stained by the dye from the Institute after the preliminary denaturation in a saturated solution of Ba(OH)2and incubation in standard saline solution of 2×SSC at 60°C. the Evaluation segments performed semiquantitative method on a 5-point system. For ekström the global options accept options: 1 or 5 points, percentagesa inversion. The presence of the genotype of these options is associated with a high frequency of chromosomal abnormalities. Thus, in the presence of the genotype extreme variations of heterochromatin judged unsuitable for persons working in enterprises with high toxic hazard.

There is also known a method of determining individual sensitivity and suitability of individuals to work in the conditions of increased coal dust (patent RU 2316764 C1, CL G01N 33/48, publ. 10.02.2008). The essence of the method lies in the fact that the subjects for the study take 0.5-1.0 ml of blood from the cubital vein. Spend the cultivation of lymphocytes and receive chromosome by a standard method. Drugs sostarivayut" in thermostat at 37°C for 24 hours. The staining perform drawing on the preparation of silver nitrate and define the following cytogenetic indicators: total activity Adair (Ag-positive agricoevrazia regions of chromosomes), the number Adair, argentopyrite D-chromosomes, argentopyrite G-chromosomes, associative index, the number of associations per cell with associations, the number of associate of acrocentrics in a cage with associations. Based on the totality of the resulting figures calculate the confidence level high level HA and when it is 50% or less define the procedure the regional suitability of the test to work in the conditions of increased coal dust.

It should be noted that these methods imply different approaches to predict the level of CA when exposed to chemical compounds. Known methods of biological indication of radiation effects, allows to confirm the fact of oblojennosti and its degree of severity, based on the determination in peripheral blood lymphocytes of the subject HA dicentrics type and E-37-rozetka-forming lymphocytes (patent RU 2126156 C1, CL G01N 33/53, publ. 10.02.1999).

The closest way to determine individual radiosensitivity can serve as a method of rapid detection of irradiated patients with elevated frequencies of HA (patent RU 2141 658 C1, CL. G01N 33/48, publ. 20.11.1999). As a marker to identify patients with elevated frequencies of HA after radiation treatment using anomalies of interphase nuclei of lymphocytes type "tails" in the blood smears. With the increased frequency of occurrence of such cells (0.8% and above) identify patients with a high incidence of HA. The disadvantages of this method include the lack of prognostic significance, because in this way it is not about the risk of accumulation of chromosomal abnormalities, not about the definition of individual genome sensitivity to radiation exposure, but only about the display - identifying individuals with elevated levels of chromosomal mutations after radiation the frame exposure. This significantly narrows the scope and informative way.

Due to the successes of molecular medicine and development of an adequate molecular-genetic methods in recent years has made possible the identification of the relationship of genetic polymorphisms of DNA repair enzymes and biotransformation of xenobiotics with the response to the influence of environmental factors. Currently found potential genes-candidates, which may affect the frequency of spontaneous and induced chromosomal damage in humans. It is established that the null genotypes for glutathione-S-transferase (GSTM1 and GSTT1) is associated with higher levels of HA induced by mitomycin C (Grigoriev S.A. Study of genetically determined sensitivity to the effects of environmental mutagens in induced mutagenesis in humans. Abstract. Diss. M, 2007). Shown an increase in the frequency of the ring and dicentrics chromosomes in carriers of XRCC1 280 His/His in conditions of exposure to indoor radon (Kiuru, A., Lindholm C., Heilimo I. et al., 2005. Inflluence of DNA repair gene polymorphisms on the yield of chromosomal aberrations // Environmental and molecular mutagenesis. V.46 1.3. P.198-205). In vitro experiments have shown that individuals with the wild type XRCC1 194 Arg/Arg demonstrate a greater number of chromosome breaks per cell in the processing of cell cultures by bleomycin and tiolovymi the epoxides of benzo[a]pyrene (Wang y, Spitz M.R., Zhu Y. et al., 203. From genotype to phenotype: correlating XRCC1 polymorphisms with mutagen sensitivity // DNA Repair (Amst). 2 (8). P.901-908). However, these studies only recognise the contribution of individual genotypes in the formation of chromosomal aberrations, not offered specific ways of using the results to determine the individual sensitivity of the person to the action of mutagens.

The main objectives of the proposed invention are: to provide a method for determining individual sensitivity of the human genome to the effects of natural radiation (radon) and increase the prognostic value by engaging in the forecast system molecular genetic markers (genes, DNA repair enzymes and biotransformation of xenobiotics).

This is due to the fact that in the proposed method of determining individual sensitivity of the human genome to the effects of radon, including the exploration of human blood and the determination of individual level HA, identify predisposing and protective genotypes of candidate genes: marker Arg280His gene XRCC1 - predisposing genotype Arg/Arg, protective genotype Arg/His; marker Arg194Trp gene XRCC1 - predisposing genotype Arg/Arg, protective genotype Arg/Trp; marker Asn148Glu gene ARE - predisposing genotype Glu/Glu, protective genotypes Asn/Asn, Asn/Glu; marker A2455G gene CYP1A1 - predisposing genotype A/G, protective the genotypes a/a and G/G; marker deletion in the gene GSTM1 - predisposing genotype o/o protective +, and make a conclusion about the high individual sensitivity to the action of high doses of radon in the quantitative prevalence of predisposing genotypes or equal to the number of predisposing and protective genotypes, and the high individual resistance to the effects of radon in the quantitative predominance of protective genotypes.

The novelty of this approach lies in the development of complex molecular-genetic characteristics, assessment of the totality which is necessary and sufficient to determine the individual sensitivity of the human genome to the effects of radon (in doses above 200 Bq/m3).

First established the role of the polymorphism of some genes biotransformation of xenobiotics (GSTM1 and CYP1A1) in the formation of chromosome aberrations in conditions of chronic exposure to high doses of radon. Among a wide range of known polymorphic genes reparations selected markers associated with an increased risk of chromosomal abnormalities in terms of the natural radiation - Arg280His Arg194Trp and XRCC1 gene, Asn148Glu gene ARE.

Assessment of the totality of gene polymorphisms of DNA repair enzymes and biotransformation of xenobiotics used in the proposed method, has a high efficiency and accuracy determined by the ü potential risk of formation of chromosome aberrations in persons, exposed to natural radiation. This can be used to assess individual sensitivity to the action of radon in residential buildings, and industrial conditions, such as miners, working in conditions of exposure to very high doses of radon.

The method is performed in the following sequence:

1. Form groups of observations.

For this form two groups of donors with high (above average) and low levels of HA formed under conditions of chronic high exposure to radon. In our study, were surveyed Teens (211 people)living in the boarding school. It was experimentally found that the concentration of radon in indoor air boarding school exceeds the permissible level for operating buildings (up to 200 Bq/m3and reaches 583 Bq/m3in the winter, and 334 Bq/m3during the spring period.

The choice of teenagers is justified by the fact that in this case, minimizing the influence of factors such as bad habits, chronic diseases and the impact of professional contact with the production of harm. In addition, compact when all of the sample (living together in a boarding school for at least 3 months prior to study) provides maximum commonality in power settings and conditions of accommodation is found. All donors had no chronic diseases, were non-smokers. In the survey did not include children receiving medical treatment, and also held x-ray examination within 3 months. to collect the material. For each subject was decorated Protocol informed consent signed by the parents or persons performing the guardianship of minors. Age and sex characteristics of the groups are presented in table 1.

Preparations preparations of metaphase chromosomes was performed using standard polymicrobial cultivation of lymphocytes [Hungerford R.A., 1965. Leukocytes cultured from small inocula of whole blood and the preparation of metaphase chromosomes by treatment with hypotonic KC1 // Stain Techn. Vol.40. P.333-338]. Cultured whole venous blood, which was taken from the cubital vein. In the culture vial was placed 0.5 ml of blood, 0.1 ml of phytohemagglutinin (Paneco), 6 ml of medium RPMI 1640 (Paneco), 1.5 ml of fetal calf serum. Duration of cultivation was 48 h For 2 h before fixation in culture was added colchicine at a final concentration of 0.5 μg/ml At the end of cultivation, the cells were treated with hypotonic solution of 0.55% KCl for 10-15 min at 37°C. Fixation was performed in 3 shifts chilled ethanol-acetic latch (3:1). The cell suspension was excavated on a clean, chilled, moistened with water slides. PR is parathas encrypted and were stained with 2%dye solution from the Institute (Merk). Accounting HA conducted without karyotyping. The selection of metaphases to be included in the analysis, and criteria for the registration of cytogenetic damage was consistent with generally accepted recommendations [Bochkov N.P., Kuleshov, NP, Zhurkov B.C., 1972. The analysis of spontaneous chromosomal aberrations in cultures of human leukocytes // Cytology. T. No. 10. Pp.1267-1273]. Viewed 100-300 metaphases. Evaluated the frequency of cells with HA.

The average frequency of HA in the examined donors entire sample was and 5.30±0,16%. It significantly significantly exceeds the value of the base background levels for the region (2,86%), which confirms the fact genotoxic effects of radon. It is notable that, despite the similarity factors: the intensity of radon exposure, age, nutrition, health, 43% of children demonstrate particularly high levels of chromosomal aberrations. The 91 persons frequency of cells with HA was above average and was 7,42±0,18%. Obviously, the reasons for this increased sensitivity is genetically determined. Of these donors, and was formed by experienced group. In the comparison group included 120 people. The average frequency of cells with HA in this group was below average and was 3,48±0,12%. The difference between these groups was statistically significant (p<0,01).

2. Form a table of the relative risk.

Conduct frequent comparison the t allele genotypes and combinations of genotypes of polymorphic markers of candidate genes in the experimental and control groups. The comparison of the frequency is performed according to the criterion χ2with the amendment of Yates with SPT Statistica 6.0. The relative risk and confidence interval are calculated according to the formula:

wherea+0.5 to the number of donors with high levels of HA of native speakers of a given gene/genotype; b+0.5 to the number of speakers of a given gene/genotype with low levels of HA; c+0.5 to the number of donors with high levels of HA without this gene/genotype; d+0.5 to the number of donors with low levels of HA without this gene/genotype (adjusted for the small number of observations).

To construct a confidence interval, we take the value L=lnOP, which approximately can be considered normally distributed with a mean of:

or

and standard deviation:

The lower and upper confidence bounds of the index, between which the rates are with probability 1-a, respectively values:

where- value of student's criterion, the corresponding error probability α; in particular, when constructing a 95% confidence interval, α=0.05 and; when 99% on the credentials of the interval α=0.01 and ; and at the 99,9% confidence interval, α=0.001 and.

As the lower and upper confidence limits take antilogarithm values fromand:

where e=2,718.

If the confidence interval does not include the value of 1.0, then the feedback effect of the disease is considered statistically significant.

The value of the relative risk for all cause genotypes are shown in table 2. With a relative risk of more than 1.5 conclude about predisposition, with a relative risk of less than 0.7 is about genetic resistance to the formation of high levels of chromosomal disorders.

3. Form the table protective and predisposing genotypes (table 3).

4. Detailed description of the method.

Example 1.

The examined So 15 years in a sterile tube type "vacutainer" (with EDTA) was taken by venous blood in a volume of 1.5 ml DNA was isolated using phenol-chloroform extraction. For genotyping of nucleotide substitutions A2455G gene CYP1A1, Arg280His Arg194Trp and XRCC1 gene, Asn148Glu gene ARE used PCR and a set of reagents developed NPF Liteh" (Moscow). Detection of mutations in the genes CYP1A1 (TS), GSTM1 (deletion) was performed in accordance with the instructions of the reagent kits manufacturer (LLC Siring", Novosibirsk, Russia). Amplification wire is whether using amplifier "Terzic". Electrophoretic separation of the amplified fragments was performed in 3% agarose and subsequent color bromide by ethidium. Obtained values of the genotypes and alleles are presented in table 4. Established 6 predisposing genotypes. Based on the obtained results the conclusion about the existence of high-risk genetically determined chromosomal abnormalities when exposed to radon. This conclusion was confirmed by the fact that when pathogenetic analysis of the subject So the level of CA aberrations was 6.5%.

Example 2.

The examined So 11 years typing of polymorphic markers was performed as described in example 1. The results are presented in table 5. Identified the prevalence of protective genotypes (4 protective and 2 predisposing genotype). The conclusion of genetically determined resistance to the formation of chromosomal aberrations in terms of radon exposure. This conclusion was confirmed by the fact that the cytogenetic analysis of surveyed So the real level of HA was 3.5%.

5. The effectiveness of the method.

Determined polymorphic genes-candidates in the comparison group in adolescents exposed to chronic exposure to radon. Found that out of 120 people with a relatively small frequency HA (3,48±0,12%)) in 22 adolescents had dominated retrypolicy genotypes or equal to the number of predisposing and protective genotypes. This suggests that their individual sensitivity to radon exposure is increased. The average frequency of HA from them (3,84%±0,35%) was higher than the other 98 of 120 adolescents with prevalence of protective genotypes and with individual resistance to radon (3,38±0,78%). It is obvious that with increasing time of exposure to radon frequency HA 98 adolescents with genetically determined individual resistance will increase to a lesser extent than in 22 adolescents with genetically determined individual sensitivity. Thus, the group with a heightened sensitivity to the action of radon (91 people), formed using a known method of analysis HA, increased by 22 people through the use of the proposed method, which includes the determination of polymorphic candidate genes. That is, the efficiency of the method was increased by 24.2%.

6. Output.

The proposed method has greater prognostic significance, because it allows the assessment of a genetically determined predisposition to the formation of elevated levels of chromosomal aberrations before exposure to the radiation factor.

Table 1
Age and sex characteristics is istica groups
IndexExperimental group (donors with a high level of chromosomal aberrations)The comparison group (donors with a low level of chromosomal aberrations)
Age, years13,3013,24
Boys/girls52,7%/47,3%51,6%/48.3%of
Just man91120

Table 2
The risk of high levels of chromosomal aberrations associated with the genotypes of the major candidate genes
Polymorphic markerGenotypeThe relative risk (RR and 95% CI)
Arg/Argof 11.26 [6,92-18,31]
Arg280His gene XRCC1Arg/His0, 09 [0,05-0,15]
His/His
rg/Arg 3,75 [2,24-6,28]
Arg194Trp gene XRCC1Arg/Trp0,27 [0,16-0,45]
Trp/Trp
Asn/Asn0,43 [0,17-1,14]
Asn148Glu gene AREAsn/Gluof 0.37 [0.14 to 0,94]
Glu/Glu2,30 [0,88-6,06]
A/A0,32 [0,12-0,86]
A2455G gene CYP1A1A/G3,11 [1,16-8,30]
G/G0,41 [0,21-0,82]
T/T1,76 [0,67-4,62]
TS gene CYP1A1T/C0,57 [0,22-1,49]
C/Cto 2.29 [1,51-3,49]
a deletion in the gene GSTM10/0 (deletion)3,00 [1,56-5,73]
0/+ and +/+0,33 [0,17-0,64]

Table 3
Protective and predisposing genotypes high-risk chromosomal abnormalities from donors exposed to indoor radon
Polymorphic markersPredisposing genotypesProtective genotypes
Arg280His gene XRCC1Arg/ArgArg/His
Arg194Trp gene XRCC1Arg/ArgArg/Trp
Asn148Glu gene APE1Glu/GluAsn/Asn, Asn/Glu
A2455G gene CYP1A1A/GA/A, G/G
TS gene CYP1A1T/T, C/CT/C
a deletion in the gene GSTM10/0 (deletion)+

Table 4
Results genotyping 1
MarkerGenotypeRR
Polymorphic marker Arg280His gene XRCC1Arg/Argof 11.26
Polymorphic marker Arg194Trp gene XRCC1Arg/Arg3,75
Polymorphic marker Asn148Glu gene AREGlu/Glu2,30
Polymorphic marker A2455G gene CYP1A1A/G3,11
Polymorphic marker TS gene CYP1A1T/T1,76
Polymorphic marker deletion in the gene GSTM10/03,00

Table 5
Results genotyping 2
GenotypeRR
Polymorphic marker Arg280His gene XRCC1Arg/Argof 11.26
Polymorphic marker Arg194Trp gene XRCC1Arg/Arg3,75
Polymorphic marker Asn148Glu gene AREAsn /Glu0,37
Polymorphic marker A2455G gene CYP1A1A/A0,32
Polymorphic marker TS gene CYP1A1T/C0,57
Polymorphic marker deletion in the gene GSTM1+0,33

The method of determining individual sensitivity of the human genome to the effects of radon, including the identification of polymorphic markers of candidate genes by genetic studies of human blood, characterized in that determine predisposing and FR is active genotypes of candidate genes: marker Arg280His gene XRCC1 - predisposing genotype Arg/Arg, protective genotype Arg/His; marker Arg194Trp gene XRCC1 - predisposing genotype Arg/Arg, protective genotype Arg/Trp; marker Asnl48Glu gene ARE-predisposing genotype Glu/Glu, protective genotypes Asn/Asn, Asn/Glu; marker A2455G gene CYP1A1 - predisposing genotype A/G, the protective genotypes a/a and G/G; marker deletion in the gene GSTM1 - predisposing genotype o/o protective +, and make a conclusion about the high individual sensitivity to the action of high doses of radon in the quantitative the prevalence of predisposing genotypes or equal to the number of predisposing and protective genotypes, and the high individual resistance to the effects of high doses of radon - in the quantitative predominance of protective genotypes.



 

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2 ex

FIELD: medicine, biology.

SUBSTANCE: invention relates to nutrient medium used for accumulation of cells for the following cytological and/or immunocytochemical analysis carrying out. Invention relates to medium containing salts NaCl, KCl, anhydrous CaCl2, MgSO4 x 6 H2O, MgCl2 x 6 H2O, Na2HPO4 x 2 H2O, KHPO4, NaHCO3, and also glucose and Henx's solution, 10% albumin solution and polyglucin taken in the ratio 1:1:1. Invention provides enhancing the preservation of cells.

EFFECT: improved an valuable properties of nutrient medium.

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