タイトル

1. タイトル Microsatellite mutations of the children of Chernobyl liquidators in Belarus Katsumi Furitsu, Haruko Ryo, Hiroo Nakajima, Taisei Nomura Department of Radiation Biology and Medical Genetics, Graduate School of Medicine, Osaka University, Japan Klaudiya G. Yeliseeva Institute of Genetics and Cytology, National Academy of Sciences of Belarus, Minsk, Republic of Belarus

2. Trans-generational effect of radiation The trans-generational effects of radiation have already been proved in the animal experiments. However, we have not yet had the clear evidence and scientific consensus on this issue in human. In the study of the children of the Atomic-bomb victims in Hiroshima and Nagasaki, they have not yet got the evidence on it, including in the pilot study on the DNA mutation. The scientific assessment of the trans-generational effects of radiation in human is quite critical for the risk estimation of radiation. The numerous future generations might be influenced from the present operation of the nuclear industry and its accidents.

3. Children of Chernobyl liquidators Liquidators worked in the highly contaminated areas after the Chernobyl accident and sustained both external and internal radiation exposures during their duty. The potential health problems in their offspring have been a matter of great concern.

4. Cooperative study with Belarus and Japanon the Trans-generational Effect of Radiation in human We performed a study on Belarusian liquidators, exploring whether increase in the germline mutation rate at microsatellite loci could be found in their progeny.

5. マイクロ／説明 Repeat unit 5’ 3’ T G T G T G T G T G T G T G T G T G T G T G T G 3’ 5’ A C A C A C A C A C A C A C A C A C A C A C A C Microsatellite ＊sequences of1〜６base Repeat units di-, tri-, tetra- nucleotide repeat ＊The number of Repeat unit is under 100 copies. ＊There are about 10５ (or more)microsatellites in the human genome. ＊Most of them are non-coding regions.

6. Mispairing Backward slippage 突然変異生成機構２ Same No. of Repeat units Mispairing Forward slippage Mechanism of the events of microsatellite mutation Normal replication One unit increease One unit decrease 突然変異生成機構 突然変異生成機構

7. マイクロ／理由 The choice of the microsatellite mutations as the marker of this study 1. Ubiquitous in the human genome more than 105 loci 2. Relatively higher spontaneous mutation rate protein coding gene microsatellite10-6 - 10-9 ＜＜ 10-3-10-4 3. Significant increase of the microsatellite mutation rate was reported in the studies on barn swallows and wheat in the Chernobyl contaminated areas. It might be possible to assess the effect of radiation on the mutation rate, even with the limited numbers of subjects.

8. 調査対象ー被曝群／チェルノブイリ事故 Subjects Exposed group: Belarusian families of the Chernobyl liquidatorsEither of the parents worked for several weeks or months in the highly contaminated areas (April 26th, 1986〜July,1987) Non-exposed control group:Belarusian families living in the non-contaminated areas None of the parents were liquidators.

9. 調査対象ー（表） Families analyzed for microsatellite mutations

10. Paternal exposure Pre-conceptional exposure Post-conceptional exposure Post-gonial exposure Gonial exposure Birth date Sperm Meiosis Spermatogonia Fetus Neonate Paternal exposure and stages of spermatogenesis 9 61 34 27 3 months 9 months Conception date Conception

11. Process of the study Culture lymphocytes （up to 〜5×107cells） Belarus Institute of Genetics and Cytology Isolation of lymphocytes Freeze Peripheral blood Japan Osaka University Detection of microsatellite mutations DNA extraction

12. PCR TAC TAC TAC TAC TAC TAC TAC TAC 5’ ATG ATG ATG ATG ATG ATG ATG ATG 5’ Auto-analyzer （ABI PRISM 3100 Genetic analyzer) Analysis of the microsatellite loci Fluorecence-labelled primer 3’ 3’ PCR pimer 3’ 5’ TAC TAC TAC TAC TAC TAC TAC TAC ATG ATG ATG ATG ATG ATG ATG ATG 5’ 3’ denature Strength of fluorecence (amount of PCR products) GeneScan Length of PCR product (base pair)

13. 使用マイクロサテライト Numbers of the marker microsatellites

14. 結果図：正常・父由来／母由 Paternal mutation Maternal mutation Mutation of Y-linked loci Father Father Father Mother Mother Son Child Child 280 260 270 250 bp bp 260 270 280 bp 250 220 230 240 D12S1090 D12S391 Y-GATA-C4 Detection of the mutation in Children using the length of microsatellite loci as the marker Normal Father Mother p(+4) Child p(+4) m(- 4) bp 220 230 240 D12S391

15. マルチ／図 D5S2501 D10S1237 D4S231 D15S657 D18S1270 bp 260 240 280 300 320 340 360 380 400 GeneScan analysis of Multiplex PCR method

16. Merit Detection of the Microsatellite mutations using GeneScan Advantages of this method Systematic and rather simple procedure Detectable for a minor difference of the length of the marker loci (even the difference of one or two base pairs) Several marker loci can be examined together in one course of procedure, using multiplex PCR.

17. 結果表：常染色体変異ー被曝 Autosomal microsatellite mutations in the children of the liquidators (fathers only), exposed before conception

18. 結果表：常染色体変異ー対照 Autosomal microsatellite mutations in the children of the control group

19. 結果表：Y染色体変異 Y-linked microsatellite mutations in the children of liquidators (fathers only) , exposed before conception, and those of un-exposed control

20. 受精前父親／変異数・率 ・ ＝ ・ Numbers and rates of microsatellite mutations

21. Exp.control? Belarusian control might have been unexpectedly exposed to radiation and some chemical contaminants in the environment? The spontaneous mutation rates reported at the same set of loci in several non-Belarusian populations were similar to those in the Belarusian control in our study. Autosomal: mutation rate, ×10-3 (95% CI) Belarusian control: 3.2 (1.9-5.5) Non-Belarusian population: 5.7 (4.6-7.0) Y-linked: mutation rate, ×10-3 (95% CI) Belarusian control: 3.9 (0.6-21.8) Non-Belarusian population: 3.2 (1.9-5.5)

22. Why non positive? No significant increases of mutation rates in the exposed group could be found in this study. This does not mean the absence of radiation effect. Relatively low dose of exposure in Belarusian liquidators might be one of the reasons. The sample size might be too small to detect the significance in such low dose of exposure. Epidemiological concerns: adjustments of parents age, conception rates, marriage satatus, etc. Is microsatellite mutation actually a proper maker to detect the trans-generational effect of radiation in mammals including human?

23. これまでの調査 significance (-) Other reports on the trans-generational effect of radiation using the markers of repeated sequences Microsatellites ＊Families of A-bomb victims (Pilot study) Satoh et al. Slebos et al. (-) ＊Families of Chernobyl liquidators in Ukraine Minisatellites ＊ Families of A-bomb victims (Pilot study) Satoh et al. (-) ＊ Families of Chernobyl liquidators in Ukraine in Estonia Slebos et al. (-) Kiuru et al. (-) (+) ＊Residents of Chernobyl contaminated areas in Belarus (UK control） Dubrova et al. (+) ＊ Residents of Chernobyl contaminated areas in Ukraine (Ukrainian control) Dubrova et al. (+) ＊ Residents near the Semiparatinsk nuclear test site Dubrova et al.

24. Conclusion No significant increase of mutation rate of microsatellite loci was found in this study of the Belarusian liquidators. This does not mean the absence of radiation effect. Trial to apply this detection system of microsatellite mutations to other categoly of the exposed groups, as A-bomb victims and residents of the contaminated areas, might be meaningful. Continuing the international cooperative study on the trans-generational effect of radiation is still critical. This is our duty as scientists for the future generations.

25. Acknowledgements Thanks Le Thi Thanh ThuyHiroaki KawabataDr. Elena V. KrupnovaDr. Valentina D. TrusovaDr. Valery A. RzheutskyDr. Nikolai KartelProf. K. SankaranarayananProf. W. Hoffman Belarusian liquidators and volunteers in Belarus Dr. Reiko WatanukiMs. Yuko Yoshida

26. Спасйбо!

32. 被曝量（表） チェルノブイリ事故処理作業者の被曝量 国連科学委員会報告(2000年)

33. 自然発生変異率ーY 同座位でのＹ染色体マイクロサテライトの 自然突然変異率の比較

34. 自然発生変異率ー常 ベラルーシとその他の国における同座位での マイクロサテライト自然突然変異率の比較（常染色体）

35. Why no evideice Why they cannot get any clear evidence in Hiroshima and Nagasaki? The data during the first 10 years was not abarable, because of the lack of any study project. The sample size was not large enough to detect the significant effect of the epidemiological data. Possible inaccuracy of the dose estimation of the parents. Most of the conception cases might be the exposure at gonial-stage, which might be less sensitive than post-gonial-stage. Using improper markers to detect the mutations caused by radiation.

36. F1 study Hiroshima Nagasaki The study of children of the Atomic-bomb survivors in Hiroshima and Nagasaki by RERF No significant radiation effect has been detected even now. Concluded studybirth defectsmortalitychromosomeabnormalitiesserum proteins Present on-going studycontinued mortality follow-upmolecular studies on DNAclinical multi-factorial adult disease(hypertension, diabetes mellitus, etc., since 2002).

37. H&N vs Cher Different ways of radiation exposure Hiroshima and Nagasaki acutehigh dose ratemainly externalmostly gonial-stage (paternal exposure) Chernobyl chronic or sub-acutelow dose rateboth external and internal exposureall the stages of spermatogenesis and post conception The “Chernobyl Type” of exposure is more common to us, though the usual operation of the nuclear industry.

38. Gene Scan GeneScan PCR産物 母親由来 父親由来 PCR 父親由来 母親由来 240 250 bp 230 父親由来 240 250 230 Y染色体 父親由来 bp リンパ球におけるPCR産物の長さ(塩基数)の解析 常染色体

39. マルチ／図 D5S2501 D10S1237 D4S231 D15S657 D18S1270 bp 260 240 280 300 320 340 360 380 400 Multiplex PCR法による GeneScan解析例

40. マルチプレックス multiplex PCR法で増幅したマイクロサテライト

41. これまでの調査ーマイクロ マイクロサテライト突然変異を指標にした ヒトの放射線の継世代影響調査報告

42. これまでの調査１ーミニ無 ミニサテライト突然変異を指標にしたヒトの放射線の継世代影響調査報告ー１

43. これまでの調査２ーミニ有 ミニサテライト突然変異を指標にしたヒトの放射線の継世代影響調査報告ー２

44. 父親被曝／精子成熟過程２ 授精前被曝 授精後被曝 61人 9人 後精原細胞期被曝 精原細胞期被曝 34人 27人 3ヶ月 9ヶ月 推定受精日 出生日 受精 精子 減数分裂 精原細胞期 胎児 新生児 父親の被曝状況と被曝時の精細胞成熟時期

45. 被曝時期別／変異数・率 授精前父親被曝群での精細胞成熟時期別の変異率

46. On-going study On going study in Japan Pilot study on the children of A-bomb victims using the same microsatellite markers in this study. Study on the microsatellite mutations in the offspring of paternally exposed mice.