Radiation Effects Research Foundation five year strategic research plan and program management, 1997-2001 Page: 67 of 152

                                
!144         control families) will be selected and screened for mutations at the minisatellite loci during
45           the first two years. It is important to use a larger body of data to confirm our preliminary
46          results, which showed no effect of A-bomb radiation on genetic instability at the
47           minisatellite loci in human germ cells obtained from the original 100 families, including
P48          children derived from 65 exposed gametes with a mean dose of 1.9 Sv. We have
49           assumed that the 65 gametes received, on average, the doubling dose estimated by Neel
150          et al. (1990)), namely, 1.7-2.2 Sv. For a locus with a spontaneous mutation rate of 0.02
51           per gamete, which is the mean mutation rate of the six minisatellite loci examined in the
152          previous study, using standard power function statistics (a Type I error of 0.05 and a Type
53           II error of 0.2), we calculate that we would need to survey two samples (exposed and
54           unexposed) of 1,188 germ cells each to observe a significant difference at the 0.05 level.
55
56           By examining 60 children from an additional 50 exposed families, each one of them having
7           one exposed parent and the mean gonadal dose of the parent being 1.9 Sv, it is anticipated  ,
58          that we can examine the required number of alleles for each sample. Dubrova et al.
59          reported that mutation rates at minisatellite loci in 79 children of parents who lived in
60           heavily polluted areas of Belarus after the Chernobyl accident were twice that of 105
61          control children from the United Kingdom although the estimated individual dose from
62          external and internal chronic exposure to 137Cs of inhabitants of those areas was less than
63          5 mSv per year. By comparing their data with our new data based on the projected
64           additional sampling, it may be possible to determine whether there is a difference in the
65          biological effects of radiation between acute external exposure and chronic internal
exposure.
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8           The pilot study for the screening of D/I/R mutations will be carried out with the
quantitative measurement of intensities of chemiluminescent bands on Southern filters on
0           two hundred families (the original 100 families and the new 100 families). Probes to be
I           used are DNA fragments from the human counterparts of the seven mouse specific loci,
2           other genes located nearby, and genes supposedly related to common chronic diseases
3           such as hypertension, diabetes mellitus and hereditary nonpolyposis colorectal cancer
4           (HNPCC).
5
6           The 2-DE technique will be used in the pilot study. DNA samples from the 200 families
7           will be examined using this technique after digestion with three sets of restriction enzymes,
8           that is, NotI/EcoRV-Hinfi (NotI/EcoRV and Hinfi being used before .and after the first
9           dimensional electrophoresis, respectively), NotI/EcoRV-PvuII and NotI/EcoRV/PvuII-
0           HinfI, products of each set of enzymes being different from those produced with the other
1           two sets of enzymes. These three kinds of DNA digests labeled with 32P from one
2           individual will be electrophoresed separately, and the resulting three gels will be
3           quantitatively analyzed. A total of 2000 spots (fragments) will be suitable for the
detection of the D/I/R type mutations among 6000 spots (fragments) visualized on the
5           three autoradiograms from the three gels. With the current research design (2000 diploid
fragments scored on three gels per individual), 5 mutations would be detected in 120
children from 100 control families, if we assume that the spontaneous mutation rate is 1
8           x 105/fragment/generation.
9
%        Image analysis is an essential part of any 2-DE study of DNA fragments, and this will

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