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4 Heritable Genetic Effects of Radiation in Human Populations INTRODUCTION AND BRIEF HISTORY search Foundation in 1976. In the late 1940s, the mouse was chosen as the primary surrogate for assessing the genetic Naturally occurring mutations in somatic and germ cells radiosensitivity of humans, and extensive studies were contribute respectively to cancers and heritable genetic dis- initiated in different research centers in the United States, eases (i.e., hereditary diseases). The discoveries by Muller England, and Japan. (1927) of the mutagenic effects of X-rays in fruit flies In the mid-1950s, one major international and several (Drosophila) and by Stadler (1928a, 1928b) of similar ef- national scientific bodies came into existence, including the fects in barley and maize, and the subsequent extension of United Nations Scientific Committee on the Effects of these findings to other types of ionizing radiation (and also Atomic Radiation (UNSCEAR), the Committee on the Bio- to ultraviolet) and other organisms, conclusively established logical Effects of Atomic Radiation (the BEAR committee; the genetic damage-inducing effects of radiation. However, renamed the Committee on the Biological Effects of Ioniz- widespread and serious concern over the possible adverse ing Radiation [BEIR] in 1972) set up by the U.S. National genetic effects of exposure of large numbers of people to Academy of Sciences, and the Committee of the British low levels of radiation first arose in the aftermath of the deto- Medical Research Council. The UNSCEAR and the BEIR nation of atomic bombs over Hiroshima and Nagasaki in committees have continued their work up to the present, pe- World War II, some 20 years after the discoveries of the riodically reviewing the levels of radiation to which human mutagenic effects of X-rays. In June 1947, at the meeting of populations are exposed and improving assessment of the the Conference on Genetics convened by the Committee on somatic and genetic risks of radiation exposure (NRC 1972, Atomic Casualties of the U.S. National Research Council to 1980, 1988, 1990, 1999; UNSCEAR 1993, 2000b, 2001). assess the program of research on the heritable effects of From the beginning of these efforts, it was obvious that in radiation to be undertaken in Japan, the leading geneticists the absence of direct human data on radiation-induced germ voted unanimously to record the following expression of cell mutations, quantitative estimates of genetic risk could their attitude toward the program: “Although there is every be derived only through a knowledge of the prevalence of reason to infer that genetic effects can be produced and have naturally occurring hereditary ill health in the population, been produced in man by atomic radiation, nevertheless the the role of spontaneous mutations in supporting this burden, conference wishes to make it clear that it cannot guarantee and plausible assumptions on the rates of induced germ cell significant results from this or any other study on the Japa- mutations in humans. The methods developed and used by nese material. In contrast to laboratory data, this material is the above committees for risk estimation, therefore, were too much influenced by extraneous variables and too little necessarily indirect. All were geared toward using human adapted to disclosing genetic effects. In spite of these facts, data on genetic diseases as a frame of reference, together the conference feels that this unique possibility for demon- with mouse data on radiation-induced mutations, to predict strating genetic effects caused by atomic radiation should the radiation risk of genetic disease in humans. Both the not be lost . . .” (NRC 1947). Thus came into existence the UNSCEAR and the BEIR committees are cognizant of the genetics program in Hiroshima and Nagasaki under the aus- need to make assumptions given the consequent uncertain- pices of the Atomic Bomb Casualty Commission (ABCC), ties in extrapolating from mouse data on induced mutation the newly formed joint agency of the Japanese Ministry of rates to the risk of genetic disease in humans. Health and Welfare and the U.S. National Academy of Sci- Details of the genetics program that evolved in Japan and ences. The ABCC was renamed the Radiation Effects Re- the vast body of data that emerged from these studies have 91

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92 BEIR VII been published in a series of articles. The most relevant ones Germ Cell Stages and Radiation Conditions of Relevance have now been compiled in a single volume (Neel and Schull From the standpoint of genetic risks, the effects of radia- 1991). The most important finding of these studies is that tion on two germ cell stages are particularly important. In there are no statistically demonstrable adverse genetic ef- the male, these are the stem cell spermatogonia, which con- fects attributable to radiation exposures sustained by the sur- stitute a permanent germ cell population in the testes and vivors. Although cited and discussed in the UNSCEAR and continue to multiply throughout the reproductive life span of BEIR reports over the years, these results did not constitute the individual. In the female, the corresponding cell stages part of the “mainstream thinking” of genetic risk estimators are the oocytes, primarily the immature ones. The latter con- and therefore were not used in risk estimation. stitute the predominant germ cell population in the female. During the past few years, estimates of the baseline fre- Female mammals are born with a finite number of oocytes quencies of Mendelian diseases have been revised and math- formed during fetal development. These primordial oocytes, ematical methods have been developed to estimate the im- as they are called, grow, and a sequence of nuclear changes pact of an increase in mutation rate (as a result of radiation comprising meiosis takes place in them. The latter however exposures) on the frequencies of different classes of genetic are arrested at a particular stage until just before ovulation. diseases in the population. Additionally, there have been sev- Because oocytes are not replenished by mitosis during adult eral advances in our understanding of the molecular basis life and immature oocytes are the predominant germ cell and mechanisms of origin of human genetic diseases and of population in the female, these are clearly the cell stages radiation-induced mutations in experimental systems. As a whose irradiation has great significance for genetic risks. result of these developments, it now is possible to reexamine The radiation exposures sustained by germ cells in hu- the conceptual basis of risk estimation, reformulate some of man populations are generally in the form of low-LET (lin- the critical questions in the field, and address some of the ear energy transfer) irradiation (e.g., X-rays and γ-rays) de- problems that could not be addressed earlier. livered as small doses at high dose rates (e.g., in diagnostic This chapter summarizes the general framework and the radiology) or are greatly protracted (e.g., continuous expo- methods and assumptions used in risk estimation until the sures from natural and man-made sources). In estimating publication of BEIR V (NRC 1990). This is followed by a genetic risks to the population therefore, the relevant radia- discussion of the advances in knowledge since that time, their tion conditions are low or chronic doses of low-LET irradia- impact on the concepts used in risk estimation, and how they tion. As discussed later, most mouse data used for estimating can be employed to revise the risk estimates. Throughout the rates of induced mutations have been collected at high this chapter, the terms “genetic diseases,” “genetic effects,” doses and high dose rates. Consequently, assumptions have and “genetic risks” are used exclusively to mean “heritable to be made to convert the rates of induced mutations at high genetic diseases,” “heritable genetic effects,” and “heritable doses and dose rates into mutation rates for radiation condi- genetic risks,” respectively. tions applicable for risk estimation in humans. GENERAL FRAMEWORK GENETIC DISEASES Goal of Genetic Risk Estimation Since the aim of genetic risk estimation is to predict the The goal of genetic risk estimation, at least as envisioned additional risk of genetic diseases relative to the baseline and pursued by UNSCEAR and the BEIR committees, re- frequency of such diseases in the population, the concept of mains prediction of the additional risk of genetic diseases in genetic diseases and their classification and attributes are human populations exposed to ionizing radiation, over and considered in this section. The term genetic diseases refers above that which occurs naturally as a result of spontaneous to those that arise as a result of spontaneous mutations in mutations. The concept of “radiation-inducible genetic dis- germ cells and are transmitted to the progeny. eases,” which emerged early on in the field, is based on two established facts and an inference. The facts are that (1) he- Mendelian Diseases reditary diseases result from mutations that occur in germ cells and (2) ionizing radiation is capable of inducing simi- Diseases caused by mutations in single genes are known lar changes in all experimental systems adequately investi- as Mendelian diseases and are further divided into autoso- gated. The inference, therefore, has been that radiation expo- mal dominant, autosomal recessive, and X-linked, depend- sure of human germ cells can result in an increase in the ing on the chromosomal location (autosomes or the X chro- frequency of genetic diseases in the population. Worth not- mosome) and transmission patterns of the mutant genes. In ing is the fact that although there is a vast amount of evi- an autosomal dominant disease, a single mutant gene (i.e., in dence for radiation-induced mutations in diverse biological the heterozygous state) is sufficient to cause disease. Ex- systems, there is no evidence for radiation-induced germ cell amples include achondroplasia, neurofibromatosis, Marfan mutations that cause genetic disease in humans. syndrome, and myotonic dystrophy. Autosomal recessive

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HERITABLE GENETIC EFFECTS OF RADIATION IN HUMAN POPULATIONS 93 diseases require homozygosity (i.e., two mutant genes at the of multifactorial diseases and to estimate the recurrence risks same locus, one from each parent) for disease manifestation. in relatives is the multifactorial threshold model (MTM) of Examples include cystic fibrosis, phenylketonuria, hemo- disease liability. The MTM, its properties, and its predic- chromatosis, Bloom’s syndrome, and ataxia-telangietasia. tions are discussed in Annex 4A. The X-linked recessive diseases are due to mutations in genes located on the X chromosome and include Duchenne’s Chromosomal Diseases muscular dystrophy, Fabry’s disease, steroid sulfatase defi- ciency, and ocular albinism. Some X-linked dominant dis- Historically, both UNSCEAR and the BEIR committees eases are known, but for most of them, no data on incidence have always had an additional class of genetic diseases— estimates are currently available. Therefore, these diseases “chromosomal diseases”—in their lists that included those are not considered further in this report. The general point that had long been known to arise as a result of gross (i.e., with respect to Mendelian diseases is that the relationship microscopically detectable), numerical (e.g., Down’s syn- between mutation and disease is simple and predictable. drome, which is due to trisomy of chromosome 21), or struc- tural abnormalities of chromosomes (e.g., cri du chat syn- drome, due to deletion of part or the whole short arm of Multifactorial Diseases chromosome 5 [5p-]). As discussed later, this is really not an The major burden of naturally occurring genetic diseases etiological category, and deletions (microscopically detect- in human populations, however, is not constituted by Men- able or not) are now known to contribute to a number of delian diseases, which are rare, but by those that have a com- constitutional genetic diseases grouped under autosomal plex etiology. The term “multifactorial” is used to designate dominant, autosomal recessive, and X-linked diseases. these diseases to emphasize the fact that there are multiple genetic and environmental determinants in their etiology. RISK ESTIMATION METHODS Their transmission patterns do not fit Mendelian expecta- tions. Examples of multifactorial diseases include the com- In the absence of data on radiation-induced germ cell mon congenital abnormalities such as neural tube defects, mutations that can cause genetic disease in humans, all of cleft lip with or without cleft palate, and congenital heart the methods developed and used for predicting the risk of defects that are present at birth, and chronic diseases of adults genetic disease from the mid-1950s to the present are indi- (i.e., with onset in middle and later years of life) such as rect. Their strengths and weaknesses are reviewed in BEIR V coronary heart disease, essential hypertension, and diabetes (NRC 1990). One such indirect method is the doubling dose mellitus. method, on which attention is focused in this section. It has Evidence for a genetic component in their etiology comes been in use since the early 1970s (NRC 1972, 1990; from family and twin studies. For example, first-degree rela- UNSCEAR 1977, 1982, 1986, 1988) and is used in the re- tives of patients affected with coronary heart disease have a cent UNSCEAR (2001) report. two- to sixfold higher risk of the disease than those of matched controls, and the concordance rates of disease for The Doubling Dose Method monozygotic twins are higher (but never 100%) than those for dizygotic twins (Motulsky and Brunzell 1992; Sankara- The doubling dose method enables expressing of the ex- narayanan and others 1999). pected increase in disease frequency per unit dose of radia- As mentioned earlier, multifactorial diseases are pre- tion in terms of the baseline frequency of the disease class. sumed to originate from the joint action of multiple genetic The doubling dose (DD) is the amount of radiation required and environmental factors; consequently, the presence of a to produce in a generation as many mutations as those that mutant allele is not equivalent to having the disease. For arise spontaneously. Ideally, it is estimated as a ratio of the these diseases, the interrelated concepts of genetic suscepti- average rates of spontaneous and induced mutations in a bility and risk factors are more appropriate. The genetic ba- given set of genes: sis of a common multifactorial disease is the presence of a genetically susceptible individual, who may or may not de- DD = average spontaneous mutation velop the disease depending on the interaction with other rate/average induced mutation rate. (4-1) genetic and environmental factors. These concepts are dis- The reciprocal of the DD (i.e., 1/DD) is the relative muta- cussed further in Annex 4A. The important general point is tion risk (RMR) per unit dose. Since RMR is the reciprocal that unlike the situation with Mendelian diseases, the rela- of DD, the smaller the DD, the higher is the RMR and vice tionships between mutations and disease are complex in the versa. With the doubling dose method, until recently, risk case of multifactorial diseases. For most of them, knowledge was estimated as a product of two quantities—namely, the of the genes involved, the types of mutational alterations, baseline disease frequency, P, and 1/DD: and the nature of environmental factors remains limited. Among the models used to explain the inheritance patterns Risk per unit dose = P × (1/DD). (4-2)

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94 BEIR VII The population genetic theory that underlies the use of later, the estimate of risk will depend on the model used for Equation (4-2) is the equilibrium theory that population ge- their maintenance in the population. neticists use to explain the dynamics of mutant genes in populations. The theory assumes that the stability of mutant The Concept of Mutation Component gene frequencies (and thus disease frequencies) in a popula- tion is the result of the existence of a balance between the The concept of mutation component and the statistic MC, rates at which spontaneous mutations enter the gene pool in which is derived using this concept, help to unify attempts at every generation and the rate at which they are eliminated by predicting how the frequencies of different classes of ge- natural selection (i.e., through failure of survival or repro- netic diseases in the population will change as a result of duction). increases in mutation rate. The mutation component is de- When the mutation rate is increased as a result of radia- fined as the relative increase in disease frequency (i.e., rela- tion in every generation, this balance between mutation and tive to the baseline frequency) per unit relative increase in selection is disturbed by the influx of induced mutations, but mutation rate (i.e., relative to the spontaneous mutation rate). the prediction is that the population will attain a new equilib- First introduced in BEIR I (NRC 1972) to address the prob- rium (over a number of generations) between mutation and lem of the impact of the radiation risk of multifactorial dis- selection. The amount of increase in mutation frequency, the eases in the population, and subsequently elaborated by time it takes for the population to reach the new equilibrium, Crow and Denniston (1981, 1985) and Denniston (1983), and the rate of approach to equilibrium are all dependent on the concept can be used for all classes of genetic disease as induced mutation rates, the intensity of selection, the type of done in BEIR V (NRC 1990). During the past few years, the genetic disease, and whether the radiation exposure occurs concept has been developed further with the necessary alge- in one generation only or generation after generation. It braic formulations, that permit a direct evaluation of the im- should be noted that since the starting population (before pact of an increase in mutation rate for all classes of genetic radiation exposure) is assumed to be in equilibrium between disease in any postradiation generation of interest following mutation and selection, the quantity P in Equation (4-2) rep- exposure to radiation in either one generation only or gen- resents the equilibrium incidence of the disease, and the eration after generation (Chakraborty and others 1998a; product of P and 1/DD is the expected increase in disease Denniston and others 1998). These advances are considered frequency at the new equilibrium. in a later section. Suffice to note here that the inclusion of MC in Equation (4-2) yields the revised equation: Risk Estimation for Different Classes Risk per unit dose = P × (1/DD) × MC, (4-3) of Genetic Disease The application of Equation (4-2) to risk estimation is where MC is the disease class and postradiation generation- straightforward for autosomal dominant diseases since the specific mutation component and the other two quantities relationship between mutation and disease is simple for this are as defined earlier. class of diseases. Population genetic theory predicts that for these diseases, if there is an x% increase in mutation rate in RECENT ADVANCES WITH RESPECT TO THE THREE every generation, at the new equilibrium this increase will QUANTITIES USED WITH THE DD METHOD OF RISK be reflected as an x% increase in the frequency of these dis- ESTIMATION eases. Until recently, estimates of risk for the first, second, or any postradiation generation of interest were obtained The BEIR V report (NRC 1990) reviewed the advances through “back calculation” from the predicted new equilib- that occurred from the mid-1950s to 1990 with respect to P, rium incidence using certain assumptions. If the population the baseline frequency of genetic disease, DD, and MC, the sustains radiation exposure in one generation only, there will three quantities considered relevant for risk estimation with be a transient increase in the mutant frequency in the first the DD method thus far. In the material that follows, atten- postradiation generation, followed by a progressive decline tion is focused on progress made since 1990. to the “old” equilibrium value. The method used to predict the risk of X-linked diseases Baseline Frequencies of Genetic Diseases is approximately similar to that for autosomal dominant dis- eases discussed above. For autosomal recessive diseases, the Mendelian Diseases risk calculation is more involved because when recessive mutations first arise (or are induced), they are present in the Estimates of the baseline frequencies of Mendelian dis- heterozygous state and do not precipitate disease in children eases used by UNSCEAR since its 1977 report and by the of the first few postradiation generations. For multifactorial BEIR III and BEIR V committees (NRC 1980, 1990) have diseases, the situation is complex in that there is no simple been based on the compilations and analysis of Carter relationship between mutation and disease, and as discussed (1976a, 1976b) primarily for Western European and Western

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HERITABLE GENETIC EFFECTS OF RADIATION IN HUMAN POPULATIONS 95 European-derived populations. These are the following (all for all varieties of the above diseases), the total became in live births): autosomal dominants, 0.95%; X-linked, 120%. Footnote f to Table 2-5 of the BEIR V report (NRC 0.05%; and autosomal recessive, 0.25%. Advances in human 1990) offers the following explanation for the 120% figure: genetics during the past two decades now permit an upward “Includes heart disease, cancer, and other selected dis- revision of the above estimates to 1.5% for autosomal domi- orders . . . . Note that the total exceeds 100%. The genetic nant diseases, 0.15% for X-linked diseases, and 0.75% for component in many of these traits is unknown. To the ex- autosomal recessive diseases (Sankaranarayanan 1998). tent that genetic influences are important, the effects are Note that the revised total frequency of Mendelian diseases through genes that have small individual effects but that act is thus 2.4%, which is about twice the earlier figure of 1.25%. cumulatively among themselves and in combination with environment factors to increase susceptibility.” Multifactorial Diseases Estimates of Baseline Frequency of Multifactorial For multifactorial diseases (which include congenital ab- Diseases Used in This Report normalities present at birth and chronic diseases), the esti- mates used by UNSCEAR (1986, 1988, 1993, 2001) derive In examining what would be considered a reasonable es- from data obtained for the population of Hungary (Czeizel timate of baseline frequency of congenital abnormalities for and Sankaranarayanan 1984; Czeizel and others 1988). use in risk estimation, the BEIR VII committee took note of These estimates are 6% of live births for congenital abnor- the vast body of data on their prevalence in different parts malities and 65% of the population affected by chronic dis- of the world, including some large-scale studies carried out eases (excluding cancers). Since most chronic diseases have in North America (Myrianthopoulos and Chung 1974; their onset in middle and late ages (published figures per- Trimble and Doughty 1974; Baird and others 1988). The tain to these age groups), data on the distribution of the estimates vary over a wide range, from about 1% in live population in various age intervals (i.e., ages 0, 1, 2, 3–4, births to a high of about 8.5% in total births (i.e., still- and 5–9, 10–14, . . . 80–84, 85+, etc.; a total of 21 age intervals) live births), depending on, among other things, the defini- for 1977 to 1981 were used to obtain estimates applicable to tion, classification, and diagnostic criteria; entities included; the population as a whole. For example, if the published es- method of ascertainment; duration of follow-up of live-born timate for a given disease pertains to the adult population children; and sample sizes. In one of the largest U.S. studies (i.e., above age 14), the figure was reduced by 21% since (Myrianthopoulos and Chung 1974), the overall frequency of the 0–14 year age group constituted 21% of the total popu- major abnormalities was 8.3% (53,257 deliveries of known lation of 10.7 million (Czeizel and others 1988). outcome), which compares favorably with the estimate of For the BEIR V committee (NRC 1990), the starting about 6% from British Columbia (Baird and others 1988) point for congenital abnormalities was the published data of and of about 6% from Hungary mentioned earlier. This Czeizel and Sankaranarayanan (1984) and Czeizel and documents the premise that under conditions of good ascer- others (1988), which gave an incidence estimate of 6%. tainment, the overall prevalences are similar and are of the This figure was reduced to 2–3% by noting that the 6% fig- order of about 6%. This committee therefore accepts the 6% ure is “. . . so high, in part, because of the unusually high figure as reasonable for use in risk estimation in this report. frequency of congenital dislocation of the hip in Hungary” For chronic multifactorial diseases, the committee pre- (Czeizel and Sankaranaryanan 1984). For chronic diseases, fers to use the estimate of 65% obtained by Czeizel and col- the starting point was the estimate of about 60% based on leagues (1988) in view of the fact that the estimate is based preliminary data of Czeizel and colleagues made available on 26 clear-cut disease entities defined by ICD (Interna- to and used by UNSCEAR in its 1988 report. The BEIR V tional Classification of Diseases) code numbers that were committee reduced the figure of 60% to 30% by (1) sub- studied epidemiologically in a large population. This esti- tracting the estimates for essential hypertension, acute myo- mate was also used by UNSCEAR (1988, 1993, 2001) as cardial infarction, other acute and subacute forms of the best available overall estimate for chronic diseases as a ischemic heart disease, and varicose veins of the lower ex- whole (excluding cancers). Included in the above estimate tremities (together about 25%) and (2) reducing the figure are heart or blood vessel-related diseases, together, about for juvenile osteochondrosis of the spine from 11% (based 25%. For the estimate of 60% mentioned in BEIR V (NRC on radiographic screening) to about 0.5% (on the assump- 1990) under the heading “heart disease” no verifiable source tion that only about 5% of the cases identified by radio- or study is cited. Likewise, for cancers, BEIR V cites an es- graphic screening may be deemed to be of clinical signifi- timate of 30%, again with no citation of the source or the cance). The resulting adjusted figure of about 30% was types of cancers included. As mentioned earlier, both of given as the estimate for the “selected others” subgroup of these numbers represent round number approximations. “other diseases of complex etiology.” Together with the ear- In the view of the BEIR VII committee, the inclusion of lier committee’s figures for heart disease (60%) and cancer cancers in estimating the heritable risks of radiation is not (30%; which were termed “round number approximations” meaningful at the present state of knowledge.

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96 BEIR VII Estimates of Baseline Frequency of Chromosomal Disease Table 4B-1 (see Annex 4B) summarizes the important developments. As evident from that Table, with one excep- The BEIR V report (NRC 1990) and the UNSCEAR tion, most of the DD estimates used in risk estimation by (1993) report assessed the baseline prevalence of chromo- UNSCEAR and the BEIR committees were based on data on somal diseases to be of the order of about 0.4% in live births. both spontaneous and induced mutation rates in mice. The The present committee sees no reason to alter this estimate. one exception was BEIR I (NRC 1972), which used data on spontaneous rate of mutations of human genes and induced Summary of Current Estimates of Baseline Frequencies of rate of mutations in mouse genes. As discussed below, re- Genetic Diseases and Comparison with Those in BEIR V evaluation of the assumptions underlying the use of mouse data on spontaneous mutation rate for DD calculations has Table 4-1 presents these comparisons showing that the shown that these are incorrect and that the use of human data current estimates for Mendelian diseases are higher than on spontaneous mutation rates along with mouse data on in- those used in 1990, while those for the other classes remain duced rates is correct. essentially unchanged. Incorrectness of the Assumption of Similarity of The Doubling Dose Spontaneous Mutation Rates in Mice and Humans—The As discussed earlier DD is one of the important quantities Need to Use Human Spontaneous Mutation Rates for DD used in the equation for the doubling dose method of risk Calculations estimation. Although the DD concept was formulated by Extrapolation of the mouse-based DD to humans for risk Muller (1951, 1954, 1959) in the 1950s and several possible estimation implies the assumption that both the spontaneous estimates and/or ranges of DDs were discussed in the BEAR and the induced rates of mutations are similar in the two report (NRC 1956), in UNSCEAR (1962), and in Lüning species. The assumption of similarity of induced rates of and Searle (1971), actual use of the method to obtain quanti- mutations in both species is defensible on the grounds of tative estimates of risk began only in 1972 (NRC 1972). generally similar gene organization, 70–90% homology in Changes in the conceptual basis and database used for DD DNA sequence of genes, and substantial conservation of estimates from the mid-1950s to the early 1990s have re- synteny for many chromosomal regions between humans and cently been reviewed (Sankaranarayanan and Chakraborty mice. However, the situation is different with respect to 2000a). spontaneous mutations. The reasons spontaneous mutation rates in humans are unlikely to be similar to those in mice have been discussed TABLE 4-1 Estimates of the Baseline Prevalences of (Sankaranarayanan 1998). Briefly, these have to do with the Genetic Diseases Used in BEIR VII and BEIR V differences in the number of cell divisions between the zy- gote and the mature germ cell in the two species. Vogel and Baseline Prevalence Estimates Motulsky (1997) estimate that in human females, the num- per 106 Live Births ber of cell divisions from zygote to the mature egg (Nf) is of the order of about 24. For the mouse female, estimates of Disease Class BEIR VII BEIR V Drost and Lee (1995) suggest that Nf is of the same order. Mendelian So, from the standpoint of Nf, human and mouse females are Autosomal dominant 15,000 10,000 similar. X-linked 1500 400 In human males, however, the comparable number of cell Autosomal recessive 7500 2,500 divisions is much higher; it is about 30 until the age of pu- Chromosomal ~4000 ~4000 berty (taken to be 15 years), ~23 per year thereafter, and 6 Multifactorial Congenital abnormalities 60,000 20,000–30,000 for proliferation and meiosis. Thus, the number of cell divi- Chronic multifactorial 650,000 a sions prior to sperm production (Nm) in a 20-year-old male Other Disorders of Complex Etiology can be estimated to be 30 + (5 × 23) + 6 = 151, increasing to b Heart disease 600,000 381 at age 30 years, 611 at age 40 years, and 841 at age Cancer c 300,000 b 50 years (Crow 1999). The Nm/Nf thus increases with pater- Selected others 300,000 nal age, being 6.3 at age 20, 15.9 at age 30, 25.5 at age 40, aBEIR V included these diseases under “other disorders of complex eti- and 35.0 at age 50. In the male mouse, the number of cell ology.” divisions from zygote to sperm is of the order of about 62 at bIncluded under chronic multifactorial diseases in BEIR VII. age 9 months, assuming a 9-month generation (Chang and cNot specifically considered in this chapter. others 1994; Drost and Lee 1995; Li and others 1996). The SOURCE: Table reproduced with permission from Chakraborty and others Nm/Nf ratio in the mouse is therefore 2.5 (i.e., 62/25), which (1998b). is much lower than in humans. The committee notes that in

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HERITABLE GENETIC EFFECTS OF RADIATION IN HUMAN POPULATIONS 97 most mouse experiments, the parental animals were used at eration), the limited data available on mosaics and clusters at a rather uniform age (usually about 12 weeks), and the ques- present preclude a quantitative assessment of their contribu- tion of paternal age effects has not been specifically ad- tion to spontaneous mutation rates. The main relevance of dressed. germinal mosaicism in the human context is this: the parent Since most spontaneous mutations arise as a result of er- who carries a mosaic mutation for an autosomal dominant or rors in DNA replication, one would expect that the mutation X-linked trait does not have a mutant phenotype and there- rate in human males would be higher than that in females fore would not be considered as having a risk of producing and that there would be an increase in the likelihood of spon- affected children. However, because his or her gonads con- taneous germinal mutations with the age of the male (so- tain mutant and normal cells, he or she may run the risk of called paternal age effect). By and large, these expectations having more than one progeny who carries the mutant gene have been fulfilled. The literature on this subject and the (mutational “clusters”). recent evidence from molecular studies have been reviewed Second, if a substantial proportion of human mutations (Crow and Denniston 1985; Crow 1993, 1997, 1999; Vogel arise as germinal mosaics in one generation and result in and Motulsky 1997; Sankaranarayanan 1998; Green and oth- clusters in the following generation, the frequencies of at ers 1999). least autosomal dominant and X-linked diseases also have to When one considers the large differences in life span be- be corrected upwards to account for this possibility; there is tween humans and mice and the paternal age effect for spon- no reliable way of doing this at present. The published esti- taneous mutations in humans, it is clear that extrapolation mates of human spontaneous mutation rates do not provide from short-lived mice to humans is unlikely to provide a sufficient grounds for assuming that substantial proportions reliable average spontaneous rate in a heterogeneous human of mutations in the germ cells first arose as mosaics and population of all ages. This is one reason to abandon the use subsequently resulted in clusters of mutations; if this had of the mouse data on spontaneous mutation rates in DD cal- been the case, major increases in the frequencies of affected culations and to use human spontaneous mutation rates in- individuals from one generation to the next would have been stead. The following arguments support this: (1) estimates observed, but this does not appear to be true. Further, family of spontaneous mutation rates in humans are unweighted sizes in present-day human populations are limited (in fact, averages of the rates in the two sexes (and therefore auto- they are so small that there is almost never more than one matically incorporate sex differences and paternal age ef- affected offspring from a mating, in contrast to the situation fects), and (2) the sex-averaged rate is relevant in the context in mice where large numbers of progeny are obtained from a of DD calculations (Sankaranarayanan 1998). single male). Both of these arguments support the view that A second reason for not using the mouse spontaneous mutational clusters are much less relevant in humans than in mutation rates for DD calculations is that the whole question mice. of spontaneous mutation rates in mice has now assumed an The advantages of using human spontaneous mutation unexpected complexity due to the noninclusion, until re- rates for DD calculations are (1) they pertain to human dis- cently, of mutations that originated as germinal mosaics (re- ease-causing genes; (2) as mentioned earlier, the mutation sulting in progeny carrying the same mutation [“clusters”] in rate estimates in humans, because they are averaged over the following generation) in estimates of spontaneous muta- both sexes, automatically include sex differences and pater- tion rates in the specific locus experiments (Russell and nal age effects; and (3) in mutation rate calculations, human Russell 1996; Selby 1998a, 1998b; Russell 1999). Accord- geneticists count all mutants that arise anew irrespective of ing to Russell and Russell (1996), if mosaic data are in- whether they were part of a cluster or not; if clusters had cluded, the total spontaneous rate becomes twice that of 6.6 occurred, they would have been included. The committee × 10–6 per gene based on mutations that arose singly. How- therefore accepts the view that the use of human spontane- ever, Selby (1998a, 1998b) has argued that (1) the data on ous rates and mouse induced rates for DD calculations (i.e., clusters should be included in calculating the total spontane- the procedure used in BEIR I; NRC 1972) is more logical, ous mutation rate; (2) his computer simulation studies and it has assessed published data on spontaneous mutation (which incorporate clusters in his model) suggest an increase rate in humans and induced rates of mutations in mice. of the rate by a factor of about 5 compared to that based on mutations that arose singly; (3) the fivefold higher total spon- Doubling Dose Estimation Using Spontaneous Mutation taneous rate is the appropriate numerator in DD calculations; Rates of Human Genes and Induced Rates of Mouse and (4) if paternal age effects are extrapolated from humans Genes to mice, the estimate of spontaneous rate is even higher. In the view of this committee, the above argument cannot be Estimation of the Average Spontaneous Mutation Rate of sustained for humans for the following reasons: Human Genes First, while there is no doubt that a proportion of sponta- neous mutations in human genes arise as germinal mosaics To calculate a representative average spontaneous muta- (and can potentially result in clusters in the following gen- tion rate of human genes, the available estimates for indi-

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98 BEIR VII vidual autosomal dominant diseases published by Childs mated 135 loci) is (2.95 ± 0.64) × 10–6 per locus per genera- (1981) and Vogel and Motulsky (1997) were used, irrespec- tion. This figure is within the range of 0.5 × 10–5 to 0.5 × 10–6 tive of whether these diseases have high or low prevalence per locus used in the 1972 BEIR I report (NRC 1972). or high or low mutation rates. However, the analysis took The list of autosomal dominant diseases used to provide into account the numbers of genes thus far known or esti- the basis for the prevalence estimate (P in Equation (4-3)) mated to underlie each of these disease phenotypes (Vogel encompasses many more than the 26 diseases used in the and Motulsky 1997; Sankaranarayanan 1998; McKusick above calculations (Sankaranarayanan 1998); these other 2000). This represents an important departure from earlier diseases could not be included in the present analysis be- estimates based on disease phenotypes alone, which gener- cause of lack of information on mutation rates. Further, the ally assumed a one-to-one relationship between mutation and mutation rate estimates for X-linked phenotypes have not disease. Details of these diseases, estimates of mutation rates, been included in these calculations; instead, it has been as- and selection coefficients are given in Table 4-2. The sumed that the average spontaneous mutation rate for auto- (unweighted) average mutation rate derived from these data somal dominant genes calculated above can also be used for (for some 26 autosomal dominant phenotypes with an esti- X-linked genes. The justification for this assumption rests on the following lines of reasoning: (1) among Mendelian diseases, autosomal dominants constitute the most impor- tant group from the standpoint of genetic risks, and (2) al- TABLE 4-2 Database for Estimating Average though X-linked recessive diseases are also expected to re- Spontaneous Mutation Rate of Human Autosomal Genes spond directly to an increase in mutation rate, since their Associated with Autosomal Dominant Diseases and Their prevalence is an order of magnitude lower than that of auto- Selection Coefficients(s) somal dominants (i.e., 0.15% versus 1.5%) the assumption Estimated of similar spontaneous rates of mutations for autosomal dominants and X-linked recessives is unlikely to result in No. of Mutation Rate Selection any significant underestimation of the total risk. In fact, for Disease Phenotype Loci (× 106)a Coefficient(s)b this reason, these two classes of diseases are considered to- gether in risk estimation. Achondroplasia 1 11.0 0.8 Amelogenesis imperfecta 1 1.0 0 Aniridia 2 3.8 0.1 The Average Rate of Induced Mutations in Mice Apert’s syndrome 1 3.5 0 Blindness 9 10.0 0.7 To calculate the average rate of induced mutations in Cataracts (early onset) 30 6.0 0.3 mice, the committee used all available data on rates of in- Cleft lip 1 1.0 0.2 duced mutations in defined genes in mice; these relate to Deaf mutism 15 24.0 0.7 recessive specific locus mutations at 12 loci, biochemical Dentinogenesis imperfecta 2 1.0 0 Huntington disease 1 5.0 0.2 mutations (null enzyme mutations, also recessive at a large Hypercholesterolemia 1 20.0 0 number of loci), and autosomal dominant mutations at 4 loci Marfan syndrome 1 5.0 0.3 incidentally detected in the course of the specific locus ex- Multiple exotoses 3 7.7 0.3 periments. The data on these autosomal dominant mutations Myotonic dystrophy 1 18.0 0.3 are all from studies carried out in Harwell; comparable data Neurofibromatosis 2 70.0 0.5 Osteogenesis imperfecta 2 10.0 0.4 from Oak Ridge studies were unavailable. Inclusion of the Osteopetrosis 1 1.0 0.2 data on dominant mutations in mutation rate calculations was Otosclerosis 1 20.0 0 dictated by the consideration that although the underlying Polyposis of intestine 1 10.0 0.2 genes were not well defined at the time these experiments Polycystic kidney disease 2 87.5 0.2 were performed (but mutations were “frequently” observed Porphyria 2 1.0 0.05 Primary basilar impression 1 10.0 0.2 and recorded, indicating that they were among the more ra- Rare diseases (early onset) 50 30.0 0.5 diation-mutable loci), we now know not only their identity Retinoblastoma 1 8.7 0.5 (and the molecular nature of the mutations) but also their Spherocytosis 1 22.0 0.2 human counterparts (the mouse Sl, W, Sp, and T correspond Tuberous sclerosis 2 8.0 0.8 to, respectively, the MGF, KIT, PAX3, and T genes in hu- Total 135 mans; see McKusick 2000). All of the data considered here Average ( 2.95 ± 0.64) 0.294 come from experiments involving stem cell spermatogonia. The data from female mice have not been used because aFor some entries, mutation rate estimates are uncertain (see Childs 1981 there is uncertainty about whether mouse immature oocytes for details). are a good model for assessing the mutational radiosensitiv- bEstimated from reproductive fitness. ity of human immature oocytes (UNSCEAR 1988). The ar- SOURCE: Childs (1981); Vogel and Motulsky (1997). guments rest on (1) the strikingly higher sensitivity of mouse

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HERITABLE GENETIC EFFECTS OF RADIATION IN HUMAN POPULATIONS 99 immature oocytes to radiation-induced killing (the majority mutations is highest at the original seven specific loci (3.03 are destroyed by 0.5 Gy; Oakberg and Clark 1964) in con- × 10–5 per locus per gray) and is about one-third of the above trast to those of human and rhesus monkey immature oo- at the six loci used in the experiments of Lyon and Morris cytes, for which the dose required is at least 100 times higher (1969; i.e., 0.78 × 10–5 per locus per gray; one locus, a, is (Baker 1971) and (2) the observations that no mutations were common to both sets). For various sets of biochemical loci at recovered from oocytes sampled 7 weeks after irradiation in which null mutations have been scored, the estimates vary contrast to the situation with mature and maturing oocytes over a range from 0.24 × 10–5 to 1.64 × 10–5 per locus per (Russell 1965). In view of this uncertainty and in order not gray. The average rate for dominant visible mutations is to underestimate the risk, the committee has used the as- within the above range. The unweighted average of the in- sumption that the rate estimated for males will also be appli- duced mutation rates is 1.09 × 10–5 per locus per gray for cable to females. acute irradiation. The use of this rate for DD calculations, Details of the data used are summarized in Tables 4-3A to however, is somewhat problematic since (1) there is overlap 4-3C and are from experiments involving acute X-irradia- of one or more loci in different data sets; (2) in some studies tion or from high-dose fractionated X-irradiation (usually (see footnote e, Table 4-3A), all of the loci involved could two fractions separated by 24 h) appropriately normalized to not be ascertained; and (3) there is no simple way of taking acute X-irradiation conditions (see Table 4-3A, footnote d; into account the interlocus variation and sampling variance and Table 4-3B, footnotes a and b) to permit easy compar- of induced rates from the derived average estimate of 1.09 × isons. Table 4-3A shows that the average rate of induced 10–5 per locus per gray. TABLE 4-3A Database for Calculating Rates of Induced Mutations in Mice No. of Average Rate/ System Loci Locus/Gy (× 105) Reference 1. The 7-locus system (Lyon and others 1964) (3 and 6 Gy; 7a 3.03 Phillips (1961); acute X- or γ-irradiation or 3 + 3 Gy, 24 h interval) Russell (1965, 1968); Lyon and others (1972); Cattanach and Rasberry (1994); Pretsch and others (1994) 2. The 6-locus system (Lyon and others 1964) 6b 0.78 Lyon and Morris (1969) (6 Gy; acute X-irradiation) 3. Biochemical loci (recessive, null enzyme) 12c 0.70d Charles and Pretsch (1986); (3 + 3 Gy, 24 h interval; X-rays) Pretsch and others (1994) 4. Biochemical loci (recessive, null enzyme) 32e 1.64 Unpublished data of (3 Gy, 3 + 3 Gy, 24 h interval and 6 Gy; X-rays) 32 0.67d S.E. Lewis, cited in 32 0.24 Neel and Lewis (1990) 5. Biochemical loci (recessive, null enzyme) 4f 1.24d Unpublished data of (3 + 3 Gy, 24 h interval; X-rays) J. Peters, cited in Neel and Lewis (1990) 6. Dominant visibles (Sl, W, Sp and T)g (X rays) 4 0.44 See Table 4-3B Unweighted average: 8.74/8 = 1.09 × 10–5 per locus per gray NOTE: Data are from experiments involving irradiation of males (stem cell spermatogonia) and all rates are normalized to single acute X-irradiation condi- tions. aa: non-agouti; b: brown; c: chinchilla; d: dilute; p: pink-eyed dilution; s: piebald; se: short ear; in the work of Pretsch and others (1994), with some strains, mutations at four or five of these loci were scored. ba: non-agouti; bp: brachypodism; fz: fuzzy; ln: leaden; pa: pallid; pe: pearl. cLdh1, Tpi, Gpi1, Pgk, G6pd1, G6pd2, Pk, Gr, Mod1, Pgam, Gapdh, Ldr. dNormalized assuming additivity of the effect of dose fractionation. eAcy1, Car2, G6pd1, Ggc, Es1, Es3, G6pd1, Gpi1, Hba, Hbb, Idh1, Ldh1, Ldh2, Mod1, Mod2, Np1, Pep2, Pep3, Pep7, Pgm1, Pgm2, Pgm3, Pk3, Trf (the identity of the other 8 loci could not be ascertained). fHba, Hbb, Es3, Gpi1. gSl: steel; W: dominant spotting; Sp: splotch; T: brachyury.

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100 BEIR VII TABLE 4-3B Dominant Visible Mutations Recovered in the Course of Mouse Specific Locus Experiments (Spermatogonial Irradiation) Number of mutations at Mutations Expt Number of per Locus No. X-ray Dose (Gy) Progeny Sl W Sp T Total per Gray (× 105) Reference 1 6+6 (8-week interval) 3,612 1 — — — 1 0.58a Lyon and others (1964) 2 6 16,735 — 1 — — 1 0.25 Lyon and Morris (1969) 3 5+5 7,168 1 — — — 1 0.35a Cattanach and Moseley (1974) Cattanach and others (1985) 4 3+3 7,645 2 — — — 2 1.09a Cattanach and Rasberry (1994) Cattanach and others (1985) 5 3+3 15,849 1 1 1 3 6 0.35b Cattanach and Rasberry (1994) Cattanach and others (1985) 6 6 10,897 1 — — — 1 0.38 Cattanach and Rasberry (1994) 7 6 19,285 1 — — — 1 0.22 Cattanach and Rasberry (1994) 8 1+9 10,318 1 — — 1 2 0.24a Cattanach and others (1985) 9 1+9 14,980 — — — 3 3 0.50a Cattanach and others (1985) Unweighted average: 3.96/9 = 0.44 per locus per gray NOTE: Experiments were carried out during 1964–1994 in Harwell, England. All rates are normalized to single acute X-irradiation conditions. aNormalized to single unfractionated irradiation conditions under the assumption of additivity of yields. bNormalized to single unfractionated irradiation (by dividing the rate by 3) on the basis of observations of the enhancement of specific locus mutation frequency (in the same experiment by a factor of 3 [3H1 strain of mice]). The committee therefore used the following approach to account both intra- and interlocus variability) can be given. derive the average induced rate of mutations. All experimen- These data permit an overall average estimate of (1.08 ± tal data were first grouped by loci, so that an unweighted 0.30) × 10–5 per locus per gray (Table 4-3C). With a dose- estimate of the locus-specific induced rates could be derived rate reduction factor of 3 traditionally used1 (Russel 1965; from the average of the estimates from all experiments in- volving each of the loci. Subsequently, these locus-specific 1In the mouse, the dose-rate reduction factor of 3 for spermatogonial rates were averaged across loci to arrive at the average in- irradiations comes not only from the 6 Gy data of Dr. William Russell but duced mutation rate. This procedure permitted calculation of also from the analysis of Dr. Tony Searle published in the Proceedings of the standard error of the estimated rate that incorporated the the Cortina International Radiation Reseach Conference in 1967. Dr. Searle sampling variability across loci as well as the variability of analyzed all of the chronic radiation data in the range from 37.5 to 861 R statistically and showed that the exposure-frequency relationship is linear the rates in individual experiments. In this approach, unpub- and that the straight line of best fit could be described by lished data of Neel and Lewis (1990) were excluded since Y = 8.34 x 10-6 + 6.59 x 10-8X, details of the identity of all the loci and the loci at which where Y is the yield of mutations and X is the exposure in roentgens. The mutations were recovered were unavailable. Although fewer slope is one-third of that for acute X-irradiation (300 and 600 R). data were used (the total number of loci became 34), this Further, the following statement from BEIR V (NRC 1990, p. 110) provides additional substantiation for the dose-rate reduction factor of 3: approach was considered preferable since (1) no locus is “The other important baseline value for spermatogonia is for the response double-counted while averaging over all loci, (2) the loci and to low dose-rate, low-LET irradiations . . . the rate is (7.3 ± 0.8)10–8/locus/ the corresponding mutant phenotypes are clear, and (3) an rad for total doses between 35 and 900 rad (Ru82a). The dose-rate factor is estimate of the standard error of the mean (which takes into 3.0 ± 0.4.”

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HERITABLE GENETIC EFFECTS OF RADIATION IN HUMAN POPULATIONS 101 TABLE 4-3C Locus-Specific Rates for Radiation-Induced experiments involving acute X- or fractionated X-irradiation Mutations in Mice Estimated from Data Tables 4-3A and experiments. In trying to put together all of these data, there 4-3B was no alternative but to use the correction factors suggested by the authors of the respective papers to estimate the rate Locusa Rate per Gray (× 105) SE (× 105) for chronic radiation conditions from the available data. The committee feels that the procedures adopted in estimating an pa 0 0 induced rate of (0.36 ± 0.10) × 10–5 per gray are sound and pe 0 0 G6pd1 0 0 that it is justifiable to use a single estimate for the induced G6pd1 0 0 rate of mutations. Ldh2 0 0 Ldr 0 0 Pgk1 0 0 THE DOUBLING DOSE ESTIMATE Tpi 0 0 Hba2 0 0 With the estimates of (2.95 ± 0.64) × 10–6 per locus for Hbb1 0 0 the rate of origin of spontaneous mutations in humans and Hbb2 0 0 Gapdh 0 0 (0.36 ± 0.10) × 10–5 per locus per gray for induced mutations Pk 0 0 in mice, the DD becomes 0.82 ± 0.29 Gy. This new estimate Mod1 0 0 is not very different from 1 Gy that has been used thus far Sp 0.04 0.04 and was based entirely on mouse data. The conceptual basis W 0.15 0.12 and the database used for estimating the average spontane- Gpi 0.33 0.33 a 0.45 0.24 ous and induced rates of mutations, however, are now differ- T 0.45 0.18 ent. The committee suggests retaining the use of 1 Gy for the ln 0.67 0.67 DD estimate. Ldh1 0.97 0.69 se 0.97 0.33 Sl 1.31 0.51 bp 1.34 0.95 MUTATION COMPONENT OF GENETIC DISEASES Es3 1.67 1.67 Hba1 1.67 1.67 Background c 1.90 0.48 Gr 2.19 1.40 As noted earlier, the MC is one of the quantities in the b 2.35 0.52 equation used to estimate risk of genetic disease using the fz 2.68 1.34 doubling dose method (i.e., risk per unit dose = P × [1/DD] p 2.93 0.56 × MC, where P = baseline disease prevalence, 1/DD = the d 3.14 0.62 Pgam 3.91 1.93 relative mutation risk per unit dose, and MC = the mutation s 7.59 0.89 component). The rationale for including MC in the risk equa- Average rate (acute irradiation) 1.08 0.30b tion is that the relationship between mutation and disease Chronic irradiation 0.36 0.10b varies between different classes of genetic diseases—simple for autosomal dominant and X-linked diseases, slightly com- NOTE: For raw data and their analysis, see Sankaranarayanan and Chakraborty (2000a). plex for autosomal recessive diseases, and very complex for multifactorial diseases—and the use of disease class-specific aIn these calculations, two additional loci (Ldh2 in the experiments of MC makes it possible to predict the impact of an increase in Pretsch and others 1994; Hba2 in the experiments of Peters) have been included based on current evidence (Lewis and Johnson 1986). mutation rate on the frequencies of all classes of genetic dis- bThe standard error of the average rate was calculated taking into ac- eases (Chakraborty and others 1998b; Denniston and others count variation of the rates among loci as well as sampling variation of the 1998; ICRP 1999). experimental data for each locus. General Definition Searle 1967), the rate for chronic low-LET radiation condi- Let P be the disease prevalence before an increase in tions becomes (0.36 ± 0.10) × 10–5 per locus per gray. mutation rate and ∆P its change due to a ∆m change in spon- It is worth reiterating here that this is the first time an taneous mutation rate, m. The mathematical identity attempt has been made to use the mutation data coming not only from the 7 specific loci but also from all loci for which ∆P ∆m ∆P / P = ⋅ (4-4) there are published data (a total of 34 loci; see Table 4-3C) P m ∆m / m taking into account interlaboratory and interexperimental variations in induced rates. Unfortunately, all of the data formalizes the definition of MC. In this equation, since ∆P/P is from biochemical loci and for dominant visibles were from the relative change in disease prevalence and ∆m/m is the

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HERITABLE GENETIC EFFECTS OF RADIATION IN HUMAN POPULATIONS 121 FIGURE 4A-2 Comparisons of the distribution liability in the general population with those in relatives of affected individuals when there are differences in the prevalence of multifactorial disease, according to the multifactorial threshold model with the additional assumption of different thresholds for disease liability in the two sexes. Based on the properties of the normal distribution of li- VG/VP is called “broad-sense heritability of liability,” or “de- ability (made up of both genetic and environmental compo- gree of genetic determination,” and is symbolized by hB2. It nents) that underlies the MTM, methods have been devel- provides a measure of the relative importance of genotype as oped to use data on the population frequency of a given a determinant of phenotypic value (Smith 1975). multifactorial disease to predict the risk to relatives of those The genotypic variance VG can be subdivided into an ad- affected and to estimate, on the basis of correlation in liabili- ditive component (VA) and a component to deviations from ties between relatives, the relative contribution of genetic additivity. Additive genetic variance is the component at- factors to the overall phenotypic variability summarized in tributable to the average effect of genes considered singly, the statistic called “heritability of liability” (h2). as transmitted in the gametes. The ratio VA/VP is called “nar- row-sense heritability,” or hN2, and expresses the extent to which the phenotypes exhibited by parents are transmitted to Concept of Heritability offspring, and it determines the magnitude of correlation In quantitative genetics, the relative contributions of ge- between relatives. The nonadditive genetic variance is due netic and environmental factors to the overall phenotypic to the additional effects of these genes when combined in variation is assessed by analysis of variance (i.e., by estimat- diploid genotypes and arises from dominance (VD), interac- ing the total phenotypic variance, VP, and apportioning it tion (epistasis, VI) between genes at different loci, and assor- into variance due to genetic factors, VG, and variance due to tative mating (VAM). In the absence of these sources of ge- environmental factors, VE). Under the assumption that the netic variance, hN2 = hB2. It is important to note that most of genetic and environmental effects are independent of each the heritability estimates for chronic diseases published in other (i.e., they are not correlated), VP = VG + VE. The ratio

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122 BEIR VII the literature are broad-sense heritability of liability esti- experiments involving irradiation of male mice [spermatogo- mates and are in the range of about 0.3–0.8. nial stem cell irradiations]). They found that for acute X- irradiation of males, although individual estimates varied from 16 to 51 R (with wide confidence limits, except for Other Models of Inheritance of Multifactorial Diseases specific locus mutations), the overall average was about 30 An important assumption of the MTM as discussed above R. For low-dose or chronic low-LET radiation exposure, the is that a large number of factors, each with small effects, suggestion was that it would be between three and four times contributes to liability. However, the assumption of fewer that for acute X-irradiation (i.e., about 100 R). UNSCEAR, contributing factors is also consistent with data from famil- however, did not use the DD method in its 1972 report, but ial aggregation studies, and for this reason, it is not a good in all reports published until 1993, the mouse data-based es- analytical tool for discriminating between different modes timate of 1 Gy has been used. of inheritance. Consequently, attempts to fit the familial data The BEIR I report (NRC 1972) introduced the concept to Mendelian models (with appropriate choice of assump- that DD estimates must be based on the average spontaneous tions on the numbers of loci, penetrance, dominance, etc.) or mutation rate of human genes and the average induced rate to a combination of major locus and polygenic models have of mutations in mouse genes. In that report it was assumed been made, (e.g., Elston and Stewart 1971; Morton and that (1) the spontaneous mutation rate of human genes might MacLean 1974; Kendler and Kidd 1986); although these be in the range of 0.5 × 10–6 to 0.5 × 10–5 per gene and (2) the models are of interest in catalyzing the search for the genes sex-averaged rate of induced recessive mutations in mouse involved, they are now largely superseded by molecular ap- was about 0.25 × 10–7 per locus per rem for low-LET radia- proaches that hold the potential for direct identification of tion conditions. With these estimates, a range of DDs from the genes. 20 to 200 rem was calculated. The induced rate of 0.25 × 10–7 per locus per rem men- tioned above was the unweighted average of the rate of 0.5 × ANNEX 4B: THE DOUBLING DOSE 10–7 per locus per rem for males (at 12 loci, including 7 of Table 4B-1 provides a broad overview of the data used the specific loci have been used in most mouse experiments during the past four decades for estimating doubling doses. and the additional 5 used in the studies of Lyon and Morris It is worth noting that although the present unit for express- 1969) and that of zero assumed for females. It was noted, ing absorbed radiation dose is gray (or sievert when consid- however, that the estimate of 0.25 × 10–7 per locus per rem ering radiations of different qualities), in reviewing the ear- might be too high for at least two reasons: (1) “the gene loci lier estimates in this section the DDs are expressed in the at which these studies were made, were to some extent pre- same units employed in the original publications, namely, selected for mutability” and (2) “the rate of induction of roentgens (R), rads, roentgen-equivalent-man (rem), grays, dominant visible mutations in mice is lower than for reces- and sieverts. Note that for low-LET radiation (e.g., X-rays sives by at least an order of magnitude and dominant muta- and γ-rays), 1 Gy = 100 rads ~ 100 R; 1 rem = 1 rad; and tions constitute a substantial part of the human genetic risk.” 1 Sv = 100 rem. This procedure of using human data on spontaneous muta- Briefly, the notion that the DD for genetic damage in- tion rates was driven by one of the principles stated by the duced in human males at low-dose or chronic low-LET ra- committee—namely, that emphasis should be placed on hu- diation conditions is likely to be of the order of about 100 R man data when feasible—the implicit idea being that if the was already entertained in the early 1960s (UNSCEAR induced rate was extrapolated from mouse to humans, there 1962). This estimate was guided by the findings (from mouse would be one extrapolation uncertainty and if both sponta- studies on recessive specific locus mutations) that chronic neous and induced rates were extrapolated to humans, there X-irradiation would be only about one-third as effective as would be two such uncertainties. acute X-irradiation in males and much less effective in fe- When UNSCEAR (1977) first used the mouse data-based males (Russell and others 1958, 1959). Consequently, it was DD of 100 rads, it did not actually specify the induced rates. suggested that the DD for chronic X-irradiation exposure This was because the estimate of 100 rads was arrived at by conditions was probably at least three times that for acute X- assuming that the DD for low-LET chronic radiation condi- irradiation (i.e., three times that of about 30 R suggested in tions would be three times that of ~30 rads for high-dose- the 1958 UNSCEAR report for acute X-irradiation or about rate acute X-irradiation conditions (for five different end 100 R). points; see Lüning and Searle 1971). In 1971, Lüning and Searle broadened the original con- In BEIR III (NRC 1980), however, the committee aban- cept of the DD to include not only mutations at defined gene doned the method that was used in BEIR I, namely, using loci, but also four other end points of genetic damage human data on spontaneous mutation rates and mouse data (semisterility, dominant visible mutations recovered in the on induced mutation rates in defined genes. The stated ob- course of studies on recessive specific locus mutations, au- jection to the BEIR I method was that it mixed the induced tosomal recessive lethals, and skeletal abnormalities, all from rate of a set of mouse genes preselected for high mutability

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HERITABLE GENETIC EFFECTS OF RADIATION IN HUMAN POPULATIONS 123 TABLE 4B-1 Doubling Dose Estimates Used in Risk Estimation from the 1950s to the Early 1990s Radiation Reference DD Conditions Comments 1956 BEAR report 50–80 R High dose Guided more by general radiation genetic principles (established mostly from Drosophila (NRC 1956) 40 R rate (acute) studies) than by knowledge of mouse or human mutation rates and, therefore, nothing more than educated guesses; among the principles were (1) linear dose-effect relationship for induced mutations and (2) effect independent of dose rate or dose fractionation. The general philosophy and “best” estimates of the Medical Research Council (MRC 1958) and UNSCEAR (1958) were roughly similar UNSCEAR (1962) 100 R Chronic Based on mouse data on the reduced effectiveness of chronic γ-irradiation for the induction of specific locus mutations (Russell and others 1958); assumed that DD for males will be about 3 times that of 30 R assumed in UNSCEAR (1958) for acute X-irradiation conditions; noted that DD for females may be higher Lüning and Searle 16–51 rads Acute Based on mouse data for 5 different end points for males; no DD estimate provided for (1971) ~100 rads Chronic females 1972 BEIR report 20–200 rem Chronic Based on a range of spontaneous rates in humans (0.5 × 10–6 to 0.5 × 10–5) and a sex- (NRC 1972) averaged rate of induction of specific locus mutations of 0.25 × 10–7 per locus per rem in mice Neel and others 46 rem Acute Based on data on mortality of children born to A-bomb survivors through the first 17 years (1974) (Petersen and of life; assumed that for chronic irradiation, the DD for males might be 3 to 4 times 46 rem others 1990) and as much as 1000 rem for females 125 rem (females) Sankaranarayanan (1976); 80–240 rads Chronic Based on mouse data for specific locus mutations induced in spermatogonia and in Searle (1976) mature + maturing oocytes and dominant visibles and translocations induced in spermatogonia UNSCEAR (1977) 100 rads Chronic Rationale stated as follows: “Examination of available evidence in the mouse suggests that the use of a 100-rad DD will not underestimate the risk. The ICRP Task Group has also this figure in its calculations . . .” 1980 BEIR report 50–250 rem Chronic Based on the “best substantiated” estimate of DD of 114 rem for spermatogonial irradiation (NRC 1980) of male mice and approximately halving and doubling the above estimate to arrive at the range of 50–250 rem UNSCEAR (1982) 100 rads Chronic No change from the 1977 report Neel and others (1982); 60 ± 93 rem Acute The first three estimates are based, respectively, on data on UPOs, survival through Schull and others (1982) 135 ± 388 rem childhood, and sex chromosomal aneuploids in the Japanese studies; the authors considered 535 ± 2416 rem that the weighted average of 135 ± 156 rem (last entry) should be multiplied by a factor 135 ± 156 rem of 3 to make it applicable to chronic radiation conditions UNSCEAR (1986) 1 Gy Chronic No change from the 1977 report UNSCEAR (1988) 1 Gy Chronic No change from the 1977 report 1990 BEIR report 100 rads Chronic Overall estimate based on mouse data (both sexes) on several different end points; most (NRC 1990) estimates given as ranges that vary by factors between about 2 and 30 (a reflection of differences in estimated spontaneous and induction rates); multiplication factors between 5 and 10 used when necessary to convert DD estimates for high-dose-rate irradiation to those for chronic irradiation Neel and others (1990) 1.69–2.23 Sv Acute Composite estimates of “minimal DDs” (DDs at 95% lower confidence limits) compatible with Japanese results on UPOs, F1 mortality, F1 cancer, sex chromosomal aneuploids, and mutations altering protein charge or function; on the assumption of a dose-rate reduction factor of 2, the authors suggest that for chronic low-LET, low-level radiation, the figures are likely to be twice those estimated (i.e., about 3.4 to 4.5 Sv) Neel and Lewis (1990) 1.35 Gy Acute Based on an analysis of mouse data on 7 mutational end points (spermatogonial irradiation experiments); the authors suggest that with the use of a dose-rate factor of 3, the DD will be about 3 Gy UNSCEAR (Rabes and others 2000) 1 Gy Chronic No change from the 1977 report SOURCE: Sankaranarayanan and Chakraborty (2000a).

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124 BEIR VII with estimates of human spontaneous rates for more typical tal effect; (4) individuals with liability exceeding the thresh- genes. The BEIR III committee adopted the view that it was old T (i.e., x > T) are affected by the disease, and those for preferable to use a DD estimate obtained from spontaneous whom x < T are unaffected; and (5) unaffected individuals and induced mutations in the same set of loci in the same have a fitness of 1 and unaffected ones of (1 – s). The impact species and used exclusively the data on the seven specific of an increase in total mutation rate as a result of radiation loci obtained in experiments with male mice. The figures exposures—from m to m(1 + k), with k measuring the in- used were 7.5 × 10–6 per locus for spontaneous rates and 6.6 crease relative to the baseline—is assessed in terms of × 10–8 per locus per rem for induced rates from which “the changes in heritability of liability (hx2), and consequent best substantiated” DD estimate of 114 R was calculated. To changes in the MC. This assessment was carried out by as- derive DDs for risk predictions, it approximately halved and suming that the effects of the mutant alleles are either addi- doubled the above estimate of 114 R to obtain a range of 50 tive or synergistic. to 250 rem. Unlike the case of Mendelian diseases, the algebraic for- In BEIR V (NRC 1990), the committee again used prima- mulations of the FLTM do not permit expressing the effects rily mouse data but included several additional end points in in the form of a single equation. However, the predictions of both sexes (dominant lethals, recessive lethals, dominant the model can be evaluated iteratively using the computer visibles, recessive visibles, reciprocal translocations, con- program that was developed for this purpose. The program genital malformations, and aneuploidy). On the basis of all is first run using a specified set of parameter values (muta- these data, it concluded that “considering all endpoints to- tion rate, selection coefficients, threshold, etc.) until the gether, the direct estimates of doubling dose for low dose population reaches equilibrium between mutation and selec- rate radiation have a median value of 70–80 rad, indirect tion. Once this occurs, the mutation rate is increased either estimates based on high dose-rate experiments have a me- once or permanently corresponding to radiation exposure in dian value of 150 rad, and the overall median lies in the one generation only or in every generation, and the com- range of 100 to 114 rad. These estimates support the view puter run is resumed with the new mutation rate while the that the doubling dose for low dose-rate, low-LET radiation other parameters remain the same. The changes in mutation in mice is approximately 100 rad for various genetic end- component and its relationship to heritability of liability are points.” then examined in desired generations and at equilibrium. It Table 4B-1 also shows that the DD estimates made over is worth mentioning that the h2 estimates are not inputs but the years based on genetic data from A-bomb survivors (Neel outputs of the program obtained using different combina- and others 1974, 1982, 1990; Schull and others 1981, 1982; tions of s values, environmental standard deviation, and Otake and others 1990; Neel 1998) were at least some three threshold. to four times that of 1 Gy used by UNSCEAR and the BEIR committee; the so-called Japanese DD estimates, however, ANNEX 4D: DIFFERENCES BETWEEN SPONTANEOUS were never used by the above committees. For the first time, DISEASE-CAUSING MUTATIONS IN HUMANS AND the BEIR V (NRC 1990) report gave a formal “status” to the RADIATION-INDUCED MUTATIONS IN Japanese results by noting that “a doubling dose of 100 rem EXPERIMENTAL SYSTEMS approximates the lower 95% confidence limit for the data from atomic bomb survivors in Japan and it is also consis- The molecular alterations recorded in spontaneous tent with the range of doubling doses in mice.” disease-causing mutations in humans include a wide variety ranging from base-pair changes to whole-gene deletions and some multigene deletions. Radiation-induced mutations ANNEX 4C: ASSUMPTIONS AND SPECIFICATIONS OF studied in experimental systems (including the mouse), how- THE FINITE-LOCUS THRESHOLD MODEL ever, are often multigene deletions, although scored through The assumptions and specifications of the FLTM have the phenotype of the marker loci. The extent of the deletion been discussed in detail by Denniston and colleagues (1998) varies with the locus and the genomic region in which it is and in the ICRP (1999) Task Group report. Briefly, the located. FLTM assumes that (1) the genetic component of liability of Spontaneous mutations arise through a number of differ- a chronic multifactorial disease is discrete and is determined ent mechanisms, and most are dependent on the DNA se- by mutant alleles at a finite number (n) of autosomal gene quence organization of the genes and their genomic context. loci; the total number of mutant alleles at these n loci in a In contrast, radiation-induced mutations originate through given genotype is a random variable g; (2) the environmen- random deposition of energy in the cell. One can, therefore, tal component is continuous and represented by a random assume that the initial probability of radiation inducing a variable e, which has a Gaussian distribution with mean of deletion may not differ between different genomic regions. zero and variance of Ve; (3) the total liability x = f(g) + e, However, their recoverability in live-born offspring seems where f(g) is a function of the number of mutant alleles in the dependent on whether the loss of the gene or genomic region n-locus genotype of the individual and e is the environmen- is compatible with viability in heterozygotes.

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HERITABLE GENETIC EFFECTS OF RADIATION IN HUMAN POPULATIONS 125 Spontaneous mutations can cause either loss or gain of function mutations (e.g., the FGFR3 gene involved in function of the normal gene through different mechanisms. achondroplasia); (2) trinucleotide repeat expansions (e.g., For example, loss-of-function mutations in genes that code Huntington’s disease); (3) dominant negative mutations for structural or regulatory proteins may result in dominant (e.g., the COL1A1 gene involved in osteogenesis im- phenotypes through haploinsufficiency (i.e., a single normal perfecta); and (4) restricted array of point mutations (e.g., gene is not sufficient for normal functioning) or through mutations in the APOB gene involved in one form of famil- dominant negative effects (i.e., the mutant product interferes ial hypercholesterolemia). Also included in this group are with the function of the normal gene in the heterozygote). genes that are relatively small in size and located in puta- While loss of function of a gene can result from a variety of tive gene-rich regions (e.g., the VMD2 gene in Best’s macu- molecular alterations including deletions, gain-of-function lar dystrophy). mutations are likely only when specific changes in the gene The gene is assigned to group 2 (uncertain recoverabil- cause a given disease phenotype. Radiation-induced muta- ity) when (1) it is large, it codes for an essential structural tions, because they are often multigene deletions, cause loss protein, and the known genetic changes are missense or of function through haploinsufficiency. nonsense mutations; (2) whole-gene deletions are rare; Despite the existence of a number of differences be- (3) whole-gene deletions are not rare, but the gene is lo- tween spontaneous and radiation-induced mutations as out- cated in a putative gene-rich region; and (4) information on lined above, radiation mutagenesis studies with a variety of these other genes and their function is insufficient (e.g., experimental systems have been very successful. The pos- BRCA2; VHL [von Hippel-Lindau syndrome]). sible reasons for this are now becoming evident: although Group 3 (potentially recoverable) includes genes that are the choices of marker genes in early studies of induced mu- generally large and constitutional deletions, some extending tations were dictated more by practical considerations (e.g., beyond the confines of genes, and translocations or inver- obtaining sufficient numbers of mutants, unambiguous sions with breakpoints in the gene causing the disease identification through their respective phenotypes) than by phenotype are known despite the putative gene-rich nature their relevance to human genetic diseases, in retrospect it is of the genomic region (e.g., EXT1 [multiple exotoses]; RB1 clear that the “successful” mutation test systems have been [retinoblastoma]). those in which most of these marker genes, and the ge- For X-linked genes, the assessment is based on whether nomic regions in which they are located, are nonessential the induced deletion will be compatible with viability in for the viability of heterozygotes (in vivo) or of the cell car- males and cause disease (since the loss of the whole X chro- rying the induced genetic change (in vitro). Consequently, mosome is compatible with viability but results in 45,X fe- induced mutations—predominantly deletions—could be re- males). covered and studied. Most human genes, however, do not appear to be of this type. ANNEX 4F: RADIATION STUDIES WITH EXPANDED SIMPLE TANDEM REPEAT LOCI IN THE MOUSE AND ANNEX 4E: CRITERIA USED TO ASSIGN HUMAN MINISATELLITE LOCI IN HUMAN GERM CELLS GENES TO ONE OF THREE GROUPS FROM THE STANDPOINT OF THE RECOVERABILITY OF INDUCED Introduction MUTATIONS IN LIVE BIRTHS The mouse and human nuclear genomes, like those of The genes included in the analysis are a subset of those other complex eukaryotes, contain a large amount of highly in which mutations cause autosomal dominant and X-linked repeated DNA sequence families most of which are tran- diseases, which have provided the basis for the overall inci- scriptionally inactive (Singer 1982). Among these are the dence estimates for these diseases discussed earlier (San- simple sequence repeats that are perfect or slightly imperfect karanarayanan 1998). Since not all of them fulfilled the tandem repeats of one or a few base pairs (bp). In the mouse requirements for inclusion (because of insufficient informa- genome, the tandem repeat loci are represented by (1) rela- tion about one or more of the following: gene size, struc- tively short microsatellites (<500 bp) with a repeat size of 1 ture, function, genomic context, etc.), only a subset could to 4 bp; (2) long expanded simple tandem repeats (0.5 to 16 be used. The “gene-richness” or “gene poorness” of given kilobases, repeat size 4 to 6 bp); and (3) true minisatellites genomic regions was assessed using the MIM (Medelian (0.5 to 10 kb) with repeat size of 14 to 47 bp (Gibbs and Inheritance in Man) gene maps that present the cytogenetic others 1993; Bois and others 1998a, 1998b; Blake and others location of “disease genes” and other expressed genes in 2000). given cytogenetic bands (McKusick 2000.). A gene is assigned to group 1 (induced deletions un- Mouse ESTRs likely to be recovered and/or unlikely to cause the pheno- type of the disease under study) when the phenotype of the The ESTRs were originally called minisatellites but have naturally occurring disease is due to specific (1) gain-of- recently been renamed to distinguish them from the much

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126 BEIR VII more stable true minisatellites in the mouse genome (Bois Low-LET Radiation Studies and others 1998a, 1998b). The ESTRs are highly unstable In the studies of Dubrova and colleagues (1993) involv- (i.e., they manifest high spontaneous mutation rates) in both ing irradiation of spermatagonial stem cells (0.5 and 1 Gy of somatic and germ cells. The mutational changes are mani- γ-rays; CBA/H strain), significant increases in the frequen- fest as changes in the number of tandem repeat cores and, cies of mutations at the Ms6-hm and Hm-2 loci were found. hence, allele length. The available data suggest that the Subsequent work with X-irradiation doses of 0.5 and 1 Gy ESTR instability is a replication- or repair-based process in- established that for mutations induced in the above cell stage, volving polymerase slippage similar to mechanisms sug- the dose-effect relationship was consistent with linearity gested for microsatellite instability (Ellegren 2000). (y = 0.111 + 0.338D), where D is the dose in grays (Dubrova and others 1998a, 1998b). From these data, the authors esti- Human Minisatellites mated that the DD for ESTR mutations induced in sper- matogonia was 0.33 Gy for acute X-irradiation, similar to In contrast to mouse ESTRs, the minisatellites in humans that reported for specific locus mutations in mice. consist of longer repeats (10 to 60 bp) that may span from In the above work, spermatids were found to be insensi- about 0.5 kb to several kilobases and show considerable se- tive to mutation induction, a finding at variance with those quence variation along the array (Jeffreys and others 1991; of Sadamoto and colleagues (1994) and Fan and coworkers 1994; May and others 1996; Buard and others 1998; Tamaki (1995) with the C3H/HeN mouse strain. These authors and others 1999; Stead and Jeffreys 2000; Vergnaud and showed that for Ms6-hm locus mutations, all male germ cell Denoeud 2000). The majority of the classical minisatellites stages were sensitive (3 Gy of γ-irradiation). Nonetheless, are GC rich. The fact that some of the human minisatellite both sets of studies demonstrated that increases in mutation loci studied are highly unstable and have very high sponta- frequencies could be detected at radiation doses and sample neous mutation rates of the order of a few percent is now sizes substantially smaller than those used in conventional well documented (Jeffreys and others 1985, 1988, 1995; genetic studies with specific locus mutations. Smith and others 1990; Vergnaud and Denoeud 2000). Mu- tation at these loci is almost completely restricted to the germline and is attributed to complex gene conversion-like High-LET Radiations Studies events involving recombinational exchanges of repeat units Niwa and collegues (1996) found that acute neutrons from between alleles (Jeffreys and others 1994; May and others a 252Cf source (65% neutrons + 35% γ-rays) were 5.9, 2.6, 1996; Jeffreys and Neumann 1997; Tamaki and others 1999; and 6.5 times more effective, respectively, in spermatozoa, Buard and others 2000; Stead and Jeffreys 2000; Vergnaud spermatids, and spermatogonia, than acute γ-irradiation in and Denoeud 2000). inducing mutations at the Ms6-hm locus. In similar studies, Dubrova and colleagues (2000a) noted that in spermatogo- Radiation Studies with Mouse ESTR Loci nial cells, chronic neutrons also from a 252Cf source had a relative biological effectiveness of about 3 relative to chronic The Loci Used γ-irradiation (regression equations: y = 0.136 + 1.135D, neu- trons; doses of 0.125, 0.25, and 0.5 Gy; y = 0.110 + 0.373D, Two ESTR loci have been used thus far in mouse muta- γ-rays; doses of 0.5 and 1 Gy). Additionally (and not unex- tion studies, namely, the Ms6-hm, and Hm-2, both of which pectedly), they found that at the above γ-ray doses of 0.5 and show multiallelism and heterozygosity within inbred strains. 1 Gy, there was no dose-rate effect. It should be remem- The Ms6-hm is <10 kb in size (varying greatly between dif- bered that the lower effectiveness of chronic γ-irradiation ferent mouse strains) and consists of tandem repeats of the recorded in earlier specific locus mutation studies (Russell motif GGGCA. Linkage analysis localized Ms6-hm near the and others 1958) occured at total doses of 3 and 6 Gy. This brown (b) coat color gene on chromosome 4. The germline observation is in contrast to earlier results with specific locus mutation rate is about 2.5% per gamete (Kelly and others mutations (Russell and others 1958) at 3 and 6 Gy showing 1989). The Hm-2 locus is located on chromosome 9 and con- that chronic γ-irradiation was only one-third as effective as sists of GGCA tetranucleotide repeats with alleles contain- acute X-irradiation in inducing specific locus mutations. ing up to 5000 repeat units (i.e., up to 5 kb). The germline mutation rate of this locus is estimated to be of the order of at least 3.6% (Gibbs and others 1993). As discussed below, Mutation Induction at the ESTR Loci—An Untargeted Dubrova and colleagues studied mutation induction at both Process Arising as a Result of Radiation-Induced Genomic of the above loci, whereas the Japanese workers focused their Instability attention only on the Ms6-hm locus. One important conclusion that emerges from these stud- ies is that mutation frequencies in the progeny of irradiated animals are too high to be accounted for by the direct induc-

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HERITABLE GENETIC EFFECTS OF RADIATION IN HUMAN POPULATIONS 127 tion of mutations at the loci studied (i.e., radiation induction germline (i.e., affecting alleles not only from the exposed F0 of germline mutations at ESTR loci is an untargeted pro- male but also from the unexposed F0 female). The latter find- cess). Dubrova and colleagues (1998a, 1998b) concluded ing is similar to that of Niwa and Kominami (2001). that there might be two associated processes: structural dam- In subsequent experiments, Barber and colleagues (2002) age elsewhere in the genome or in other sensor molecules confirmed the transgenerational effects of chronic neutron and, subsequently, indirect mutation at ESTR loci. This irradiation and extended the observations to acute X-irradia- nontargeted origin of radiation-induced mutations at the tion. Additionally, the response of two other inbred mouse ESTR loci is reminiscent of the phenomenon of delayed ra- strains (C57BL/6 and BALB/c) was compared with that of diation-induced genomic instability in somatic cells (dis- the CBA/H strain used in their studies. The rationale for the cussed in Chapters 2 and 3). The experiments of Barber and comparisons rests on earlier findings that BALB/c and CBA/ colleagues (2000) showed that the ESTR mutations in H mice show higher levels of radiation-induced genomic unirradiated or irradiated mice are not associated with a gen- instability in somatic cells than C57BL/6 mice and that this eral genome-wide increase in meiotic recombination rate. difference can be attributed to the strain-specific polymor- Further support for the concept of the nontargeted origin phism at the Cdkn2a (cyclin-dependent kinase inhibitor) and of induced ESTR mutations comes from the work of Niwa Prkdc (DNA-dependent protein kinase catalytic subunit) and Kominami (2001). In their study, male mice received genes (Zhang and others 1998; Yu and others 2001). 6 Gy of γ-irradiation and were mated to unirradiated females In these experiments, (1) spermatogonial neutron to produce F1 progeny from irradiated spermatozoa and stem (0.4 Gy) or X-irradiation (2.0 Gy) of CBA/H mice resulted cell spermatogonia. As in their earlier studies, mutations at in an increase in the mutation rate in both the F1 and the F2 the Ms6-hm locus were studied. The mutant frequencies for generations (derived from unirradiated F1 males and fe- the paternally derived allele increased to 22% and 19% in males); however, although spermatid irradiation did not the F1 progeny from irradiated spermatozoa and spermatogo- cause an increase in mutation rate in the F1 generation nia, respectively (about a twofold increase over the control (which was also the case in their earlier work), there was a rate). The surprising finding was that the mutation frequency clear increase in mutation rate in the F2 progeny, suggesting also was higher (20%) in the maternally derived allele in that destabilization of the F1 germline occurs after fertiliza- progeny descended from irradiated spermatozoa, but not tion, regardless of the stage of spermatogenesis exposed to from spermatogonia. The authors’ interpretation is that the radiation, and that the radiation-induced signal also persists introduction of damage into the egg by irradiated spermato- and destabilizes the F2 germline; (2) transgenerational ef- zoa triggers genomic instability in zygotes and in embryos fects were also observed in neutron-irradiated (0.4 Gy) of subsequent developmental stages, and that this genomic C57BL/6 and X-irradiated (1 Gy) BALB/c mice; and instability induces untargeted mutation in cis (in the pater- (3) there were clear differences in the levels of spontaneous nally derived allele) and in trans (in the unirradiated mater- and transgenerational instability in the order BALB/c > nally derived allele). CBA/H > C57BL/6. In summary, these data permit the con- clusion that the instability associated with radiation-induced germ cell mutations at the ESTR loci persist for at least two Transgenerational Instability generations. Dubrova and colleagues (2000a) and Barber and cowork- ers (2002) provided additional evidence for the involvement Direct Studies of ESTR Mutations in Mouse Sperm of radiation-induced germline genomic instability in the ori- gin of induced ESTR mutations. In these experiments in- In a recent paper, Yauk and colleagues (2002) have re- volving chronic neutron irradiation (0.5 Gy) of spermatogo- ported on mouse experiments involving single molecular nial stem cells, the mutation frequency in the F1 progeny was polymerase chain reaction (PCR) analysis of genomic DNA about sixfold higher than in the control. Breeding from the for studying spontaneous and radiation-induced mutations at unirradiated F1 mice revealed that the mutation rate remained the Ms6-hm locus. These X-irradiated male mice (1 Gy) were high in transmissions from both F1 males (6×) and F1 fe- killed 10 weeks postirradiation, and spermatozoa collected males (3.5×; scored in F2). A part of this increase is due to from caudal epididymis from the mice were screened for germline mosaicism in F1 animals, suggesting that paternal mutations. The findings were that (1) significant increases exposure to radiation results in a destabilization of ESTR in mutation frequency could be detected, with the magnitude loci in the germline of offspring and that some of the muta- being similar to that established by conventional pedigree tions occur sufficiently early in germline development for analysis, and (2) the majority of mutations resulted from significant levels of mosaicism to arise. More importantly, small gains or losses of three to five repeat units. this instability is transmissible through meiosis and mitosis to the F2 generation and appears to operate in trans in the F1

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128 BEIR VII Radiation-Induced Mutations at Human Minisatellite Loci posed (>20 mSv); and (4) the mutation rate in the latter was significantly higher than in the former, and both were higher Studies After the Chernobyl Accident and Around the than in the unexposed UK controls. Semipalatinsk Nuclear Test Site Further evidence showing an increase in minisatellite mutation frequencies has also been obtained from two stud- Dubrova and colleagues (1996) first reported on radia- ies, one in the Kiev and Zhitomir regions of Ukraine that tion-induced minisatellite mutations among children born sustained heavy radioactive contamination after the Cher- between February and September 1994 to parents who were nobyl accident (Dubrova and others 2002b) and another at continuously resident in the heavily polluted rural areas of the Semipalatinsk nuclear test site in Kazakhstan (Dubrova the Mogilev district of Belarus following the Chernobyl ac- and others 2002a). In the Ukraine investigation, the control cident. Blood samples were collected from 79 families (fa- and exposed groups were composed of families containing ther, mother, and child) for DNA analysis. The control children conceived before (n = 98) and after (n = 240) the sample consisted of 105 nonirradiated Caucasian families Chernobyl accident. Eight hypervariable minisatellite from the United Kingdom, sex-matched to the offspring of probes (CEB1, CEB15, CEB25, CEB36, MS1, MS31, the exposed group. DNA fingerprints were produced from MS32, and B6.7) were used. all families by using the multilocus minisatellite probe A statistically significant 1.6-fold increase in mutation 33.15 and two hypervariable single-locus probes, MS1 and rate was found in the germline of exposed fathers, whereas MS31. Additionally, most families were profiled with the the maternal germline mutation rate was not elevated. More minisatellite probes MS32 and CEB1. For the Mogilev than 90% of the children in the exposed cohort came from families, the level of 137Cs contamination was used as a dose the most heavily radioactively contaminated areas of measure, and the families were divided according to the Ukraine, with a level of surface contamination from 137Cs median 137Cs contamination levels into those inhabiting less of >2 Ci/km2. According to gamma spectrometric measure- contaminated areas (<250 kBq m–2) and those inhabiting ments of radionuclide concentration in soil and measure- more contaminated areas (>250 kBq m–2). ments of external exposures (γ-exposure rate in air), the The data showed that the frequency of mutations (1) was whole-body doses from external exposures did not exceed higher by a factor of about 2 in the children of exposed 50 mSv, and similar doses from the ingestion of 137Cs and families relative to control families and (2) showed a corre- 134Cs for the Ukrainian population were also reported. The lation with 137Cs contamination levels as demarcated above. authors note that that all of these doses are well below all The authors suggested that these findings were consistent known estimates of the DD for mammalian germline muta- with radiation induction of germline mutations but also tion of 1 Sv (Sankaranarayanan and Chakraborty 2000b; noted that other nonradioactive contaminants from Cher- UNSCEAR 2001) and, therefore, cannot explain the 1.6- nobyl, such as heavy metals, could be responsible. These fold increase in mutation rate found in exposed families results have been subject to criticism on the grounds that the Between 1949 and 1989, the Semipalatinsk site was the U.K. control population was ethnically and environmentally former Soviet Union’s premier test site for 456 nuclear tests; different and therefore inappropriate for comparisons it was closed in 1991. The surrounding population was ex- (UNSCEAR 2001). Furthermore, from the data presented, it posed mainly to the fresh radioactive fallout from four sur- would seem that the estimated germline doses in the whole face explosions conducted in 1949, 1951, 1953, and 1956, region remain sufficiently uncertain to question the true sig- and the radioactive contamination outside the test zone cur- nificance of an approximately twofold difference in muta- rently is assessed to be low. A total of 40 three-generation tion frequencies. families around the test site (characterized by the highest In a subsequent extension of the above study, Dubrova effective dose >1 Sv) along with 28 three-generation and colleagues (1997) recruited 48 additional families and nonirradiated families from a geographically similar non- used five additional probes and found that the data con- contaminated rural area of Kazakhstan were included in the firmed the approximately twofold higher mutation rate in study (Dubrova and others 2002a). Note that the above dose exposed families compared to nonirradiated families from estimate cited in the paper is from Gusev and colleagues the United Kingdom. In these studies, (1) approximate indi- (1997; based mostly on external radiation), and the World vidual doses for chronic γ-ray exposures were computed for Health Organization (WHO 1998) states that the estimates 126 families in the exposed group using published data on range from <0.5 Sv to 4.5 Sv. All parents and offspring the annual external and internal exposure to 137Cs in soil, were profiled with the eight hypervariable minisatellite milk, and vegetables and family histories after the Cher- probes previously used in the Belarus and Ukraine studies. nobyl accident; (2) the parental dose for each family was The mutation rates in the P0 and F1 generations were estab- taken as the mean value of the paternal and maternal doses lished from the observed frequencies, respectively, in the F1 up to conception of the child; (3) families within the ex- and F2 generations (controls and exposed progeny). posed group could be divided according to the median of The findings were (1) in the controls, the spontaneous the distribution, into less exposed (<20 mSv) and more ex- mutation rates in the P0 and F1 generations were similar;

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HERITABLE GENETIC EFFECTS OF RADIATION IN HUMAN POPULATIONS 129 (2) in the irradiated groups, the P0 rate was significantly ternal and external). Secondly, in the case of A-bomb survi- higher (1.8-fold) and the F1 rate was nonsignificantly (1.5- vors, most of their children were born more than 10 years fold) higher compared to controls; and (3) plotted against after the single, acute parental exposure; in Belarus and the parental year of birth (1950–1960, 1961–1965, and Ukraine, however, the affected areas have been irradiated 1966–1974), the mutation rate in the exposed F1 generation constantly since the Chernobyl accident. Finally, the Japa- showed a negative correlation (i.e., decreased) with the nese data are derived from families in which most of the parental year at birth, with the highest rate in the 1950–1960 children were born to parents of whom only one had sus- cohort (similar to that in the P0 families) and much lower in tained radiation; in the work of Dubrova and colleagues, the the later two time periods (similar to that in the control data pertain to children for whom both parents had been ex- cohorts). posed to chronic irradiation. The authors have interpreted these findings as follows: Livshits and colleagues (2001) found that the children of (1) all P0 parents born between 1926 and 1948 would have Chernobyl cleanup workers (liquidators) did not show an been directly exposed to relatively high levels of radiation elevated rate of minisatellite mutations compared to a Ukrai- from the nuclear tests, and this would explain the 1.8-fold nian control group. The dose estimate for the liquidators was increase in mutation rate; (2) F1 parents born between 1950 <0.25 Gy but is subject to uncertainty (Pitkevich and others and 1956 would be heterogeneous with respect to the doses 1997), and the main exposure was from external γ-irradia- received: some would also have been exposed to high radia- tion (with a relatively minor contribution from the intake of tion doses, while those born later would have received con- radionuclides) received as repeated small daily doses. Inter- siderably lower doses, and this heterogeneity in the parental estingly, children conceived within 2 months of the fathers’ doses could explain the 1.5-fold increase in mutation rate; employment had a higher mutation rate than those conceived and (3) the negative correlation with the year of birth may more than 4 months after the fathers stopped working there. reflect the decreased exposure after the decay of radioiso- This would be consistent with an effect on cells undergoing topes in the late 1950s and after the cessation of surface and spermatogenesis, but not on spermatogonial stem cells. How- atmospheric nuclear tests. ever, none of these differences was statistically significant. More recently, Kiuru and colleagues (2003) compared the frequencies of minisatellite mutations among children of 147 Other Population Studies Estonian Chernobyl cleanup workers. The comparisons were In the mid-1990s, subsequent to publication of the radia- within families (i.e., between children born before and after tion studies with mouse ESTR loci discussed earlier, Kodaira their fathers were exposed to radiation). The post-Chernobyl and colleagues (1995) conducted a pilot feasibility study on children (n = 155) were conceived within 33 months of their germline instability in cell lines established from the chil- fathers’ return from Chernobyl; the “control” children were dren of atomic bomb survivors in Japan. The cell lines were siblings (n = 148) born prior to the accident. Mutations were from 64 children from the 50 most heavily exposed families studied at eight minisatellite loci (CEB1, CEB15, CEB25, (combined gonadal equivalent dose of 1.9 Sv) and 50 chil- CEB36, MS1, MS31, MS32, and B6.7). The estimated mean dren from control families. Mutations at six minisatellite loci dose to the workers was 100 ± 60 mSv, with fewer than were studied using the following six probes: Pc-1, 8TM-18, 1.4% of the cohort receiving more than 250 mSv. ChdTC15, p8g3, 8MS1, and CEB1. A total of 28 mutations A total of 94 mutations (42 in the pre-Chernobyl group were found, but these were at the p8g-3, 8MS-1, and CEB-1 and 52 in the post-Chernobyl group) were found at the eight loci only, and there were no mutations at the other three loci. tested loci. Within-family (i.e., pre- and post-Chernobyl) Twenty-two of these were in the controls (of 1098 alleles comparisons of mutation rates showed that the post- tested; 2%), and six were in children from irradiated parents Chernobyl children had a slightly but not significantly higher (among 390 alleles; 1.5%). Thus, there was no significant mutation rate (0.042 per band) than the pre-Chernobyl chil- difference in mutation frequencies between the control and dren (0.035 per band) with an odds ratio of 1.33 (95% CI: the exposed groups. The use of probes 33.16 and 33.15 in 0.80, 2.20). The available data do not permit an assessment subsequent work did not alter the above conclusion (Satoh of the extent to which differences in paternal age might have and Kodaira 1996; Satoh and others 1996). contributed to this difference. When the cleanup workers The discrepancy between the results of Kodaira and col- were subdivided according to their radiation doses, the mu- leagues, on the one hand, and those of Dubrova and col- tation rate in children born to fathers with recorded doses of leagues (1996, 1998b, 2000a, 2000b) in the Belarus and 200 mSv, showed a nonsignificant increase relative to their other cohorts discussed earlier appears real. To what extent siblings; at lower doses there was no difference. this might be due to differences in type and duration of ra- Weinberg and colleagues (2001) screened children born diation exposure remains unclear. For instance, the A-bomb in families of cleanup workers (currently either in Ukraine survivors were externally exposed to considerable acute or Israel) for new DNA fragments (‘mutations’) using doses of radiation, whereas in the Belarus, Ukraine, and “multisite DNA fingerprinting.” In contrast to the results of Semipalatinsk studies the exposures were chronic (both in- Livshits and colleagues (2001), they reported a sevenfold

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130 BEIR VII increase in mutation rate in these children compared to those 1996). Bleomycin, a radiomimetic agent, selectively targets conceived before the Chernobyl accident and external con- mouse oocytes, but no mutation induction in male germ cells trols. However, the mutants were detected using random has been observed. The only patient treated with pro- amplified polymorphic DNA-PCR, an unreliable technol- carbazine + oncovin + prednisone (for six cycles with 3–4 ogy. These mutants were not validated and had no obvious week intervals between cycles) showed a slight increase in molecular basis (Jeffreys and Dubrova 2001). mutation frequency (1.14% versus 0.79%). Procarbazine is known to be mutagenic to mouse spermatogonia. In the work of May and colleagues (2000), sperm DNA Studies of Cancer Patients samples from three seminoma patients who underwent or- There are some limited data on minisatellite mutations chiectomy and external beam radiotherapy were used to detected directly in sperm sampled from cancer patients who study induction of mutations at the B6.7 and CEB1 loci. have sustained radiotherapy and/or chemotherapy (Armour These men received 15 fractions of acute X-irradiation, with and others 1999; May and others 2000; Zheng and others a total testicular dose (from scattered radiation) ranging be- 2000). All of these studies used the so-called small-pool PCR tween 0.4 and 0.8 Gy. No induced mutations were found. approach (SP-PCR) originally developed for the analysis of spontaneous mutations at human minisatellite loci (Jeffreys ANNEX 4G: DOUBLING DOSES ESTIMATED FROM and others 1994). While this method can overcome the small GENETIC DATA OF CHILDREN OF A-BOMB sample size limitations encountered in pedigree analysis, a SURVIVORS major shortcoming of the SP-PCR approach, compared to the pedigree approach, is the very large variation in sponta- The most recent DD estimates consistent with the Japa- neous mutation rates of individual alleles at a single locus. nese data are those of Neel and colleagues (1990). These Although SP-PCR can be used to evaluate the mutation rate were expressed as “end-point-specific minimal DDs” ex- in the same male before and after mutagenic treatment, it cluded by the data at specified probability levels and “most does not allow amplification of very large minisatellite alle- probable gametic DD” (note that all of these are for the acute les (longer than 5 kb), thus restricting mutation scoring to a radiation conditions obtained during the bombings). For ex- subset of relatively small minisatellite sizes. ample, the minimal DDs at the 95% probability level were In the first of these studies (Armour and others 1999), the following: 0.05 to 0.11 Sv (F1 cancers); 0.18 to 0.29 Sv sperm DNA of two men exposed to the anticancer drugs cy- (UPO); 0.68 to 1.10 Sv (F1 mortality); 1.60 Sv (sex-chromo- clophosphamide, etoposide, and vincristine, plus 2.2 Gy of somal aneuploidy), and 2.27 Sv (electrophoretic mutations). X-rays (scattered radiation from mediastinal radiotherapy), When only UPO, F1 cancers, and F1 mortality were consid- were analyzed for mutations at the MS205 locus known to ered together, the estimated DD at the 95% probability level have a high germline mutation rate (~0.4–0.7% per gamete). was 0.63 to 1.04 Sv. The comparable estimate for sex chro- There were no significant differences in mutation frequen- mosomal aneuploidy and electrophoretic mutations consid- cies in the pretherapy and posttherapy samples (11 and 16 ered together was 2.71 Sv. months, respectively, in the two individuals). Mutation rates The oft-quoted DD range of 1.69 to 2.23 Sv, called the were 0.38% versus 0.47% in the former and 0.10% versus “most probable gametic DD” by Neel and colleagues, was 0.11% in the latter. It should be noted, however, that in obtained by calculating overall spontaneous and induced mouse experiments, cyclophosphamide is mutagenic only in “mutation rates” for the above-mentioned five end-points postmeiotic germ cells, etoposide (a topoisomerase II inhibi- and obtaining a ratio of these two. The former was estimated tor) is mutagenic only in meiotic cells, and vincristine is not by summing the five individual estimates of spontaneous mutagenic, although it is known to prevent the assembly of rates (which yielded 0.00632 to 0.00835 per gamete) and tubulin into spindle fibers (Witt and Bishop 1996; Russell the latter, likewise, by summing the individual rates of in- and others 1998). duction (which yielded 0.00375 per gamete per parental Sv). In the second study (Zheng and others 2000), sperm DNA The ratio 0.00632-0.00835/0.00375 is the DD range which from 10 men treated for Hodgkin’s disease (with different is 1.69 to 2.23 Sv. The overall DDs thus calculated were combinations of chemotherapeutic agents plus 2.5 Gy of found to be between 1.69 Sv (i.e., 0.00632/0.00375) and abdominal X-rays) were analyzed using the MS205 locus. 2.23 Sv (i.e., 0.00835/0.00375) for the acute radiation con- Nine patients treated with either vinblastine or adriamycin ditions during the bombings. In these estimates, the limits and bleomycin did not show any increases in mutation fre- reflect biological uncertainties about the parameters, but do quency. Vinblastine binds to tubulin and, in mice, results in not take into account the additional error inherent in the esti- aneuploidy but not chromosome breakage or mutations. mation process itself, which must be relatively large (Neel Adriamycin is an intercalating agent and an inhibitor of and others 1990). With a dose-rate reduction factor of 2 topoisomerase-II, and in mice, this compound is toxic to (which was used) for chronic low-LET radiation conditions, germ cells but does not cause mutations (Witt and Bishop the relevant DD becomes about 3.4 to 4.5 Sv. Note, how-

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HERITABLE GENETIC EFFECTS OF RADIATION IN HUMAN POPULATIONS 131 ever, that the dose-rate reduction factor traditionally used by two of the above (i.e., F1 mortality, F1 cancers) are multifac- UNSCEAR and the BEIR committees is 3, based on specific torial traits (similar to UPO), and their responsiveness to an locus mutation experiments with male mice. increase in mutation rate will depend on the magnitude of For reasons discussed in the main text, the DDs estimated the mutation-responsive component, which is quite small, as from these data cannot readily be compared with those used Neel and colleagues point out. Consequently, the rates of by UNSCEAR and the BEIR committees. However, the re- induced genetic damage underlying these traits are expected sults with one indicator of damage used in the Japanese stud- to be small, and increases will be undetectable with the avail- ies, namely, untoward pregnancy outcome, which includes able sample sizes at the relatively low radiation doses (about stillbirths, congenital abnormalities, and early neonatal 0.4 Sv) sustained by most of the survivors. deaths, permit a crude comparison with the risk of congeni- The reasons for the lack of significant effects on sex chro- tal abnormalities estimated in this report. The rate of induc- mosomal aneuploidy and electrophoretic mutations are dif- tion defined by the regression coefficient for UPO is (26.4 ± ferent. There is no evidence from mouse studies that radia- 27.7) × 10–4 per parental sievert, compared to the background tion is capable of inducing chromosomal nondisjunction (the risk of 500 × 10–4 assumed in the calculations. The risk of principal basis for the origin of sex chromosomal aneup- congenital abnormalities (estimated from mouse data in this loidy). Since radiation is a poor inducer of point mutations, a document) is 60 × 10–4 per Gy–1 for acute X-irradiation, com- priori one would not expect electrophoretic mutations to be pared to the background risk (human data) of 600 × 10–4. induced by radiation to any great extent as they are known to Considering the uncertainties involved in both of these esti- be due to base-pair changes. Null enzyme mutations would mates, one can conclude that they are of the same order. be expected to be induced, but they are unlikely to be found The other end points—namely, F1 mortality, F1 cancers, at the low dose levels experienced by most survivors. Con- sex chromosomal aneuploidy, and electrophoretic mobility sequently, it is not surprising that the DD estimates of Neel or activity mutations—that have been used in the Japanese and colleagues for these end points (1.60 Sv for sex-chro- studies have not been used in this report and so do not lend mosomal aneuploids and 2.27 Sv for electrophoretic muta- themselves to comparisons. It should be noted that the first tions) are higher than those for the other end points.