ISSN 10227954, Russian Journal of Genetics, 2010, Vol. 46, No. 6, pp. 740–749. © Pleiades Publishing, Inc., 2010. Original Russian Text © A.V. Aghajanyan, I.I. Suskov, 2010, published in Genetika, 2010, Vol. 46, No. 6, pp. 834–843.
HUMAN GENETICS
Genomic Instability in Chidren Born after the Chernobyl Nuclear Accident (in vivo and in vitro Studies) A. V. Aghajanyana and I. I. Suskovb, † a
Federal State Institution Russian Scientific Center of Roentgenology and Radiology, Moscow, 117997 Russia; email: ann
[email protected] bVavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, 119991 Russia Received April 7, 2009
Abstract—Analysis of peripheral blood lymphocytes in children born after the accident at the Chernobyl Nuclear Power Plant in the period from 1987 to 2004 (permanent residents of territories contaminated with radionuclides, n = 92; and children of irradiated fathers–liquidators, n = 88) revealed increased levels of aberrant cells (ACs) and aberrations of the chromosomal type as compared to the control (P < 0.05). In three subgroups of children with different initial AC frequencies (children with high AC frequencies, ≥3%; children with medium AC frequencies, 2%; and children with low AC frequencies, ≤1%), the levels of aberrations of the chromosomal type are increased as compared to the control (P < 0.05). The levels of aberrant cells and chromosome aberrations (CAs) in the subgroup of children with ≥3% frequencies significantly differ from those in the subgroup of children with ≤1% AC frequencies. No dependence of the AC and CA frequencies on the year of birth after the Chernobyl accident was revealed. After fractional and single γirradiation (137Cs) of blood in vitro in the 10–30 cGy dose range, the average CA frequencies in the first and second mitoses increased in a similar way depending on the initial AC frequencies in the children and parents. All these results suggest an individual character of genomic instability induced by low radiation doses and its transgen erational phenomenon in the organisms of children. DOI: 10.1134/S1022795410060153 †
INTRODUCTION
The monitoring carried out after the nuclear acci dent in 1986 at the Chernobyl Nuclear Power Plant showed an increase in the incidence of diseases (mainly somatic) in the first generation of children born and permanently living on radionuclidecon taminated territories and in children of fathers–liqui dators [1–3]. Also, increased levels of chromosome aberrations (CAs) are observed in them [4–8]. A recent trend is to attribute increased CA levels in the human organism not only to direct action of radi ation, but also to the phenomenon of radiation induced genomic instability, which was discovered at the end of the 20th century, first in experimental stud ies and then confirmed in people [9–15]. Genomic instability is destabilization of the genome (spontane ous and induced by external factors) that can manifest itself as chromosome aberrations, gene mutations, etc. It occurs in the progeny of divided cells and disturbes the functions of cells, tissues, organs, and systems of the human organism. The basis of this phenomenon can be primary potential DNA changes realized in subsequent cell divisions [16]. After multiple redupli cations, spontaneous or radiationinduced genomic instability of cells of one or both irradiated parents can † Deceased.
be transgenerationally expressed in somatic cells of their offsprings as well [17–23]. Genomic instability induced by low doses of radia tion and its transgenerational phenomen were earlier demonstrated in children of parents irradiated as a result of the Chernobyl accident (children from radio nuclidecontaminated territories born to irradiated fathers and mothers and children of fathers–liquida tors) [13–15, 18, 19]. The purpose of this work was to study genomic instability and its transgenerational phenomenon in the organisms of the abovemen tioned children born after the Chernobyl nuclear acci dent, both in intact lymphocytes and after in vitro test ing irradiation of blood at low doses. MATERIALS AND METHODS Subjects examined. Two groups of children born after the Chernobyl accident in 1987–2004 and a con trol group of children were examined. The children with various somatic pathologies were examined in the Center of Radiation Protection of the Institute of Pediatrics and Children’s Surgery, Moscow (Head of the Center Professor L.S. Baleva). Questioning of the children and their parents was made according to the protocol. Families exposed only to ionizing radiation, without other additional mutagenic exposures, were selected.
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One group included 92 children born to irradiated fathers and mothers and constantly living on radioac tively contaminated territories (the town of Novozy bkov and the Novozybkovskii raion of the Bryanskaya oblast, Russian Federation) with the level of 137Cs soil contamination 135–688 kBq/m2. The age of the chil dren at the time of examination was from 1 to 15 years. During the Chernobyl accident their parents were exposed to radiation in different periods of their onto genetic development (8–17 years) and then continued living under conditions of chronic exposure to low radiation doses. The average age of the fathers and mothers at the time of conception was 32 ± 1.5 and 26 ± 2.2 years, respectively. The second group included 88 children born to irradiated fathers (liquidators of the accident at the ChNPP) and nonirradiated mothers and constantly living on territories of the Russian Federation without radionuclide contaminations. The age of the children at the time of examination was from 2 to 16 years. Their fathers (liquidators) participated in the cleanup works within the 30km zone in 1986–1987. The duration of the works varied from two weeks to six months. The average effective dose was 231 mSv. The individual doses varied from 50 to 480 mSv. The period since the end of irradiation of the fathers till the con ception of the children was from 1 month to 14 years. The average age of the fathers and mothers at the time of conception was 29 ± 2.1 and 24 ± 2.4 years, respec tively. The control group included nonirradiated children similar in age from noncontaminated territories (n = 12). Lymphocyte cultures and preparation of slides. Venous blood samples (0.8 ml) were cultured for 48 and 72 h at 37°С in 6.5 ml of RPMI1640 medium with 15% of fetal calf serum, 2.5% of phytohaemag glutinin, 10 mM 5bromodeoxyuridine (5BrdU), and antibiotics. 3 h before the end of cultivation, 0.2 μg/ml colcemid was added, and then centrifugation fol lowed. After that the blood samples were incubated in hypotonic solution (0.075 M KCl) at 37°С for 20 min and fixed three times in methanol–glacial acetic acid mixture (3 : 1). Preparation of slides was performed according to standard procedures. To differentiate metaphases of the first and subse quent mitoses, the chromosomes were stained by the FPG method [24]. After storage of the slides in the dark for 14 days, they were kept for 12 min in 50 μg/ml Hoechst 33258 solution and then washed three times with distilled water. Then the slides were placed in phosphate buffer (pH 7.0) preliminarily warmed up to 60°С and exposed to UV light for 18 min. Thereafter they were stained according to Giemsa. Conditions of experiment. Experiments with 137Cs testing γirradiation in vitro were carried out using peripheral blood samples from nine children and six parents. Three children were born and permanently lived in contaminated areas. Three other children were born to fathers–liquidators and nonirradiated moth RUSSIAN JOURNAL OF GENETICS
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ers. The remaining three children were from the con trol group with a nonspecific somatic pathology and lived on noncontaminated territories. The parents were irradiated fathers (two liquidators and one resi dent from a radioactively contaminated territory) and nonirradiated mothers (three). Single doses were 10, 20, and 30 cGy. Fractional doses were 10 + 10 cGy and 10 + 10 + 10 cGy (at 24h intervals). During fractional irradiation all blood sam ples were stored at 37°С. Scoring of chromosome aberrations. Chromosome aberrations were scored by examining monochromi cally stained metaphase plates under a light micro scope (Carl Zeiss, Germany). The search for metaphases was carried out at a low magnification (10 × 10). Their analysis was performed at a large mag nification (100 × 10) with the use of immersion [25, 26]. For each blood sample 250–350 metaphase plates with 44–46 chromosomes were analyzed. Partial kary otyping with identificatin of homologous chromo somes was made. Chromosomes 1, 2, 3 of group A, 4, 5 of group B, 6, 7–12 of group C and X chromosome, 13–15 of group D, 16, 17, 18 of group E, 19, 20 of group F, 21, 22 of group G and Y chromosome were identified. Aberrations of the chromatid type (single fragments, isochromatid fragments, and chromatid exchanges) and aberrations of the chromosomal type (singlebreak aberrations: acentric fragments, centro meric breaks, and deletions; doublebreak aberra tions: dicentrics, centric and acentric rings, atypical monocentrics) were scored. Statistical analysis. The significance of differences was determined by the Student’s t test. MS EXCEL and STATISTICA 6 software packages were used. RESULTS In vivo Table 1 presents the results of cytogenetic examina tions of children–offsprings of irradiated parents and children of the control group. The average AC fre quencies in peripheral blood lymphocytes of the chil dren living in contaminated areas were 2.22 ± 0.15 (P < 0.05). All types of chromosome aberrations were observed. Aberrations of the chromatid types (mainly single fragments) were detected in 98% of the chil dren. Singlebreak aberrations of the chromosomal type were revealed in 83% of the children. Acentric fragments prevailed among them, and their average frequencies significantly exceeded the control (P < 0.01). Among doublebreak aberrations of the chro mosomal type, dicentrics and rings were found in 20% of the children and atypical monocentrics in 25% of the children. The average AC frequencies in peripheral blood lymphocytes of the children born to fathers–liquida tors were 2.28 ± 0.17 (P < 0.05). Aberrations of both chromatid (in 99% of the children) and chromosomal 2010
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Table 1. Types and frequencies of chromosome aberrations in lymphocytes of children Children of irradiated parents Control group of children from contami children of fathersliqui children (n = 12) nated territories (n = 92) dators (n = 88)
Parameters examined Number of analyzed metaphases Frequency of aberrant cells (%) ± SEM (the range of individual frequencies of aberrant cells) Frequency of chro Chromatid type mosome aberra tions per 100 cells ± SEM (the range of individual frequen cies of chromosome aberrations) Chromosomal type
single fragments chromatid exchanges isochromatid fragments acentric fragments deletions centromeric breaks dicentrics rings atypical monocentrics
21620 2.22 ± 0.15* (0.33÷6.0) 1.57 ± 0.12 (0.33÷4.0) 0.02 ± 0.01 (0÷0.66) 0.04 ± 0.01 (0÷0.40) 0.38 ± 0.04* (0÷1.82) 0.13 ± 0.03 (0÷1.20) 0.03 ± 0.02 (0÷0.50) 0.08 ± 0.01 (0÷0.50) 0.02 ± 0.02 (0÷0.50) 0.12 ± 0.02 (0÷0.80)
20257 2.28 ± 0.17* (0.33÷8.0) 1.54 ± 0.13 (0.33÷6.50) 0.03 ± 0.02 (0÷1.0) 0.05 ± 0.04 (0÷0.50) 0.39 ± 0.02* (0÷1.67) 0.06 ± 0.01 (0÷0.83) 0.02 ± 0.01 (0÷0.50) 0.07 ± 0.03 (0÷0.10) 0.02 ± 0.01 (0÷0.67) 0.08 ± 0.03 (0÷1.67)
3350 1.13 ± 0.12 (0.33–3.0) 1.05 ± 0.12 (0.33–2.67) 0 0 0.08 ± 0.02 (0–0.67) 0 0 0 0 0
Note: n, the number of examined children. * Significant differences from the control (P < 0.05).
types were detected. Single fragments prevailed. Sin glebreak aberrations of the chromosomal type were revealed in 72% of the children. Acentric fragments prevailed among them, and their average frequencies significantly exceeded the control (P < 0.01). Among doublebreak aberrations of the chromosomal type, dicentrics and rings were found in 19% of the children and atypical monocentrics in 18% of the children. The average AC frequency in lymphocytes of the children from the control group is 1.13 ± 0.12. Chro mosome aberrations are represented mainly by single chromatid fragments in 100% of the children and by very rare acentric fragments in 16% of the children. The distribution of all examined groups of children according to individual AC frequencies is presented in Fig. 1. Individual AC frequencies ≥3% were observed in 31% (n = 29) of the children living on contaminated territories, in 35% (n = 31) of the children of fathers– liquidators, and in 9% (n = 1) of the children from the control group. Individual 2% AC frequencies were observed in 36% (n = 33) of the children from contam inated areas, in 40% (n = 35) of the children of fathers–liquidators, and in 18% (n = 2) of the children from the control group. Individual AC frequencies ≤1% were observed in 33% (n = 30) of the children
from contaminated areas, in 25% (n = 22) of the chil dren of fathers–liquidators, and in 73% (n = 9) of the children from the control group. But what types of chromosome aberrations occur in the children of irra diated parents with different individual AC frequen cies? For a more thorough analysis of the CA spectrum, the examined children (residents of contaminated areas and children born to fathers–liquidators) were subdivided into three subgroups according to the ini tial AC frequencies (children with high AC frequencies ≥3%, children with medium 2% AC frequencies, and children with low AC frequencies ≤1%) (Table 2). The highest levels of aberrations of the chromatid and chromosomal types were observed in lymphocytes of the children with the initial AC frequencies ≥3%. Sta tistically significant differences from the control (P < 0.05) were revealed by the levels of aberrant cells, aber rations of the chromatid type and singlebreak aberra tions of the chromosomal type. The children of this subgroup (initial AC frequency ≥3%) displayed signif icant differences (P < 0.05) in the frequencies of aber rant cells and aberrations of the chromatid and chro mosomal types from the subgroup of children with the initial AC frequencies ≤1%. The average AC levels in
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lymphocytes of the children with the initial AC fre quencies of 2% were 2.01 ± 0.26 and 1.77 ± 0.04, respectively. Increased frequencies of aberrations of the chromosomal type were observed. Statistically sig nificant differences (P < 0.05) from the control were revealed in the level of acentric fragments. In lympho cytes of the children with the initial AC frequencies ≤1%, the average levels of aberrant cells and aberra tions of the chromatid type were practically similar to those in the control group of children. However, there were significant differences (P < 0.05) from the control in the levels of acentric fragments. Doublebreak aber rations of the chromosomal type were also observed. Figure 2 shows individual frequencies of aberrant cells and aberrations of the chromatid and chromo somal types for children of irradiated parents (children living on contaminated territories and children born to fathersliquidators) depending on the year of birth. A wide individual variation of the AC and CA frequen cies is observed. Increased individual AC and CA fre quencies are displayed by children born soon after the Chernobyl accident. Reduced individual AC and CA frequencies are characteristic of children born 14 and more years after the accident. No dependence of the individual AC and CA frequencies (time–effect rela tionship) on the year of birth was revealed among the children of the main two groups. In vitro After testing single and fractional in vitro irradia tion (20 and 30 cGy) of blood lymphocytes of six chil dren of exposed parents, significantly increased AC frequencies were revealed in them in mitoses 1 and 2 as compared to those in the children of nonirradiated parents (Fig. 3). Statistically significant differences were detected in the AC frequencies between irradi ated fathers and nonirradiated mothers in mitosis 1 after a single exposure to 20 cGy and in mitosis 2 after a fractional exposure to 20 and 30 cGy. The spectrum and frequencies of induced chromo some aberrations in mitoses 1 and 2 after single and fractional in vitro γirradiation of peripheral blood lymphocytes at 10, 20, and 30 cGy did not practically differ in the group of children living on teritories with radionuclide contaminations and in the group of chil dren born to liquidators, but they were higher than in the control group of children, although no statitically significant differences were observed [15]. Therefore these children were subdivided into subgroups accord ing to the initial AC frequencies. Significant differ ences in the AC level in mitosis 1 were revealed between children with a high initial AC frequency (3.0–4.0%) (two children born to liquidators and one child from a contaminated territory) and children of the control group both in intact lymphocytes and after single irradiation at 10 cGy and 20 cGy and fractional irradiation at 30 cGy (Fig. 4) as well as in the total fre quencies of all aberrations of the chromosomal type in RUSSIAN JOURNAL OF GENETICS
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Fig. 1. The distribution of groups of children according to individual frequencies of aberrant cells: (a) children from contaminated territories; (b) children of fathers–liquida tors; (c) control group of children.
intact lymphocytes and at the fractional doses of 20 and 30 cGy. Significant differences were observed in the AC level in mitosis 2 in intact lymphocytes and at the fractional doses of 20 and 30 cGy as well as in the level of all aberrations of the chromosomal type in intact lymphocytes and at the fractional dose of 30 cGy. After testing irradiations of lymphocytes of children with a low initial AC frequency (1.5–2.0%) (two children from contaminated territories and one child born to a liquidator), an increase in the CA fre quency is observed as compared to the control, but it is insignificant. Figure 5 presents individual AC frequencies after fractional and single irradiations in mitoses 1 and 2 in the children and their parents. A wide individual vari ation of the AC frequencies is observed in intact lym phocytes and at all doses of single and fractional irra diation. The dose–response curves for the AC fre quencies in the children with a high initial AC 2010
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Table 2. Types and frequencies of chromosome aberrations in lymphocytes of children of irradiated parents with different initial frequencies of aberrant cells Frequency of aberrations per 100 cells ± SEM Examined groups of children
Children from con taminated territories
Subgroups of chil dren (number, %)
AC frequencies ≥3% (n = 29, 31%) AC frequencies 2% (n = 33, 36%) AC frequencies ≤1% (n = 30, 33%)
Children of AC frequencies ≥3% fathersliq (n = 31, 35%) uidators AC frequencies 2% (n = 35, 40%) AC frequencies ≤1% (n = 22, 25%)
aberrations of the chro aberrations of the chromosomal Frequency Number of matid type type analyzed of aberrant cells % ± SEM metaphases single fragments, chro acentric fragments, dicentrics, matid exchanges, isoch deletions, centro rings, atypical monocentrics meric breaks romatid fragments 7646
3.90 ± 0.18*, **
2.68 ± 0.20*, **
0.80 ± 0.08*, **
0.36 ± 0.07**
8901
2.01 ± 0.26
1.48 ± 0.07
0.40 ± 0.05*
0.13 ± 0.04
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1.04 ± 0.06
0.48 ± 0.06
0.34 ± 0.04*
0.10 ± 0.03
7646
3.94 ± 0.23*, **
2.80 ± 0.21*, **
0.79 ± 0.01*, **
0.39 ± 0.07**
8901
1.77 ± 0.04
1.14 ± 0.07
0.44 ± 0.05*
0.16 ± 0.05
6200
0.94 ± 0.07
0.64 ± 0.08
0.27 ± 0.07*
0.03 ± 0.02
Notes: * Significant differences from the control (P < 0.05). ** Significant differences between the subgroups of children with AC frequencies ≥3% and children with AC frequencies ≤1% (P < 0.05).
frequency (3.0–4.0%) and in their irradiated fathers are similar in mitoses 1 and 2. In the children with a low initial AC frequency (1.5–2.0%), the dose– response curve for the average AC frequencies is inter mediate between the curves for the control group of children and nonirradiated mothers. This experiment demonstrates that after fractional and single irradia tion in vitro the CA frequencies increased in mitoses 1 and 2 in the dose range from 10 to 30 cGy depending on the initial AC frequencies both in the children and parents. DISCUSSION Cytogenetic changes revealed in children living on radioactively contaminated territories and irradiated in the intrauterine and postnatal periods as a result of longterm exposure to low doses of ionizing radiation point to radiationinduced genomic instability in their organisms. This phenomenon was revealed in succes sive cell divisions in children from the Bryansk oblast, Ukraine [12, 14]. The fathers and mothers of these children were maximally irradiated during the Cher nobyl accident and soon after it at early stages of their ontogenetic development, when the formation and growth of different organs and systems of the organ ism, including the generative system, occurs [27]. Therefore, radiationinduced genomic instability in the organisms of the examined children is determined not only by the chronic exposure to longlived isotopes (137Cs, 90Sr, etc.) in the intrauterine and postnatal
periods, but probably is transgenerational in character [18, 19]. Cytogenetic changes observed in lymphocytes in the group of children born to fathers–liquidators and nonirradiated mothers are analogous to those in the group of children constantly living in contaminated areas. It should be noted that these children were not directly exposed to radiation. Thus, an assumption can be made about radiationinduced transgenera tional genomic instability in their organisms. The mechanisms of this phenomenon are poorly under stood. Nevertheless, it can be assumed that in the course of spermatogenesis stem spermatogonia with radiationinduced potential DNA changes or with chromosome aberrations undergo reproduction, growth, maturation, transformation into mature sper matozoa and then participate in fertilization. Being in a zygote, the unstable paternal genome is also involved in different processes of embryogenesis (histogenesis, organogenesis, systemogenesis) [28], and this very likely leads to varous manifestations of transgenera tional (gametic) genomic instability induced by low dose radiation in somatic cells of children of fathers– liquidators. This phenomenon was revealed in 144h cultures of lymphocytes of children whose fathers paticipated in the liquidation of the Chernobyl acci dent in Ukraine [12]. Increased levels of induced chromosome aberra tions observed in mitoses 1 and 2 in children of irradi ated parents after testing lowdose irradiations of blood samples in vitro confirm the expression of
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Fig. 2. Individual frequencies of aberrant cells and chromosome aberrations in children of irradiated parents depending on the year of birth. (a), (b), (c) children from contaminated territories; (d), (e), (f) children of fathers–liquidators; (a), (d) aberrant cells; (b), (e) aberrations of the chromatid type; (c), (f) aberrations of the chromosomal type. RUSSIAN JOURNAL OF GENETICS
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genomic instability and its transgenerational phenom enon in their organisms (Fig. 2) [33]. An increased radiosensitivity of lymphocytes was observed in chil dren born to irradiated parents after testing exposure of blood samples to a medium [17]. The action of lowdose ionizing radiation is known to be characterized by the absence of a dose threshold, by biological amplification of induced genomic abnor malities, and by increased sensitivity to endoge nous/exogenous factors [29–34]. Therefore, it is espe
cially important to study individual genomic instabil ity induced by low radiation doses and its transgenerational effect in somatic cells of the chil dren’s organism. The average AC frequencies in the children of the main two groups significantly differ from the control. As regards the individual AC frequencies, their range is rather wide. One third part of the children with a high initial AC frequency (≥3%) belongs to a special risk group. Increased levels of aberrations of the chromatid
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and chromosomal types revealed in lymphocytes of these children may be a result of both the action of var ious mutagenic factors and the expression of radia tioninduced genomic instability in their organisms. However, genomic instability induced by low radiation doses and its transgenerational effect are also observed RUSSIAN JOURNAL OF GENETICS
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in the children with low and medium initial AC fre quencies, which is confirmed by increased levels of aberrations of the chromosomal type. The differential character of increase in the levels of chromosome aberrations after in vitro exposure to 10–30 cGy depending on the initial AC frequencies is likely to be 2010
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indicative of genotypical peculiarities of genomic insta bility and its transgenerational phenomenon (Fig. 3). This can be associated with individual radiosensitivity and activation of the repair systems. One should also take into account the health condition and pathophys iological processes in these children [1–3, 18]. Exper iments with adapting irradiation of blood samples at low doses showed that the proportion of children with an increased level of radiosensitivity increases in groups of sick children [32]. The absence of dependence of the AC and CA fre quencies on the year of birth of children after the acci dent at the Chernobyl Nuclear Power Plant suggests a nonspecific character of the action of lowdose ioniz ing radiation on somatic and sex cells of the parents, zygotes and somatic cells of the children (Fig. 2) [33]. It is most likely that low doses promote the accumula tion of cells with real/potential DNA changes, thus increasing the risk of transformation of inherited pathological recessive alleles/loci from the hetero to the hemi or homozygous state [34]. Thus, the results presented above (in vivo and in vitro) demonstrate the individual expression of genomic instability induced by low radiation doses and its transgenerational phenomenon in the organ isms of children born to parents irradiated as a result of the Chernobyl nuclear accident. This may be a prereq uisite for increased morbidity among these children, and further studies are thus required.
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ACKNOWLEDGMENTS Our special thanks to the personnel of the Labora tory of Ecological Genetics (head Dr. A.V. Rubanov ich), Vavilov Institute of General Genetics, Russian Academy of Sciences, for the help in the work. The work was supported by the Russian Founda tion for Basic Research (grant no. 050449463a) and by the Programs of the Presidium of the Russian Academy of Sciences “Fundamental Sciences to Medicine” and “Gene Pool Dynamics”.
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