Mutation Research, 284 (1992) 25-36
25
© 1992 Elsevier Science Publishers B.V. All rights reserved 0027-5107/92/$05.00
MUT 0374
Heterozygous manifestations in four autosomal recessive human cancer-prone syndromes: ataxia telangiectasia, xeroderma pigmentosum, Fanconi anemia, and Bloom syndrome * Ruth A. Heim, Nicholas J. Lench and Michael Swift Biological Sciences Research Center, Unit,ersity of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7250, USA
(Accepted 16 October 1991)
Keywords: Heterozygotes; Ataxia telangiectasia; Xeroderma pigmentosum; Bloom syndrome; Fanconi anemia
Four rare autosomal recessive syndromes associated with a high cancer risk in homozygotes ataxia telangiectasia (A-T), Bloom syndrome (BS), Fanconi anemia (FA), and xeroderma pigmentosum (XP) (Table 1) - offer insight into the genetics of cancer, diabetes mellitus, congenital malformations, and mental retardation. Heterozygous carriers of genes for the respective syndromes constitute a substantial proportion of the general population and may also have an increased risk of developing cancer and other health problems associated with the homozygotes. By studying heterozygotes for genes that predispose an individual to a common disorder, it may be possible to dissect the contribution that single genes make to the pathogenesis of complex disorders. Incidence and gene frequency G e n e frequencies can be measured directly by testing individuals in a population for allelespecific D N A mutations or protein variants. When this is not possible, three indirect methods can be used to estimate the allelic frequencies of autoso-
Correspondence: Dr. Michael Swift (present address:) New York Medical College, Valhalla, NY 10595, USA.
mal recessive syndromes. The most familiar approach uses the H a r d y - W e i n b e r g formula in which p is the frequency of the wild-type allele and q the frequency of the disease allele. If the incidence of the homozygotes (q2) can be measured by counting individuals with the distinctive phenotype, then the frequency of the disease allele is calculated simply as the square root of q. The observed incidence of a syndrome may be much smaller than the true birth or conception incidence, however, leading to a substantial underestimate of the disease allele frequency. The allele frequency q may also be estimated by the Dahlberg formula ( R o m e o et al., 1983) if both the frequency of first cousin marriages among the parents of homozygotes for a given autosomal recessive syndrome and the frequency of first cousin marriages in the general population are known. The latter is usually unknown or difficult to estimate. Even so, the frequency of one autosomal recessive syndrome relative to another can be estimated from the rates of parental consanguinity in each. The allele frequency can also be estimated from the proportion of families in which affected homozygotes have cousins, aunts or uncles, or nieces or nephews who are also homozygotes. When an allele is relatively common, as is the case for cystic fibrosis, it is simply more likely for a random person marrying into a cystic fibrosis
Acute lymphocytic leukemias and lymphomas in childhood, Chronic lymphocytic leukemias in adulthood, Also breast, pancreas, stomach, bladder, ovary, oral cavity.
Ataxia telangiectasia a
Bloom Mainly solid syndrome h tumors, including skin, breast, large intestine, oral cavity and lung. Also leukemia, lymphoma, Hodgkin disease.
Cancer predisposition
Syndrome
Sun sensitivity (ultraviolet),
No evidence,
Photophobia
Erythema. Facial telangiectasia, Small hyperand hypopigmented areas. Cafe-au-lait spots.
Oculocutaneous telangiectasia, Freckling. Progeric changes of hair and skin. Cafe-au-lait spots. Hyperpigmented macules,
Dermatological features
Possible mild mental deficiency.
Infant/ childhood onset of progressive cerebellar ataxia. Choreoathetosis. Oculomotor defects. Dysarthic speech. Dystonic posturing of fingers.
Neurological function and mental retardation
MAJOR CLINICAL FEATURES ASSOCIATED WITH HOMOZYGOTES
TABLE 1
Diabetes mellitus, usually insulinindependent.
Mild diabetes mellitus.
Diabetes mellitus
Growth retardation.
missing. Growth retardation. Ovarian dysgenesis.
or
Thymus abnormal
Structural abnormalities
Impaired. IgA, IgG and IgM.
T- and B-cellmediated immunodeficiency. IgA, IgE, and IgG 2.
Immune function
Normal.
Normal.
Hematological function
Chronic lung disease. Infertility. Gastrointestinal infections.
Alphafetoprotein. Radiosensitivity. Progressive pulmonary disease.
Other features
Mainly basal and squamous cell skin cancers, malignant melanomas,
Xeroderma pigmentosum d
Erythema with edema and blistering, Freckles. Pigmented and achromic macules, Telangiectasias. Actinic keratoses.
Cafe-au-lait spots. Fine reticular melanosis,
Progressive neurological degeneration and mental retardation in certain families.
Mental retardation, microcephaly in some patients,
No evidence.
Diabetes mellitus, insulinindependent,
None.
Growth retardation. Wide spectrum congenital malformations, including renal and ocular anomalies, skeletal malformations. Reduced natural killer cell activity.
Abnormal. Low natural killer cell function.
Normal.
Variable progressive pancytopenia. Increased fetal hemoglobin. Lymphopenia.
References: a Gatti et al. (1991); Swift (1990); b German and Passarge (1989); Cohen and Levy (1989); c Schroeder et al. (1976); Cohen and Levy (1989); ~ Kraemer and Slor (1984); Cohen and Levy (1989); c Okuyama and Mishina (1987); Jacobs and Karabus (1983).
Extreme sensitivity to sunlight (ultraviolet).
Mainly acute None. myelogenous leukemia, Also solid tumors, including liver, stomach, skin, esophagus, female genitalia, and breast, e
Fanconi anemia c
28 family to carry the cystic fibrosis allele, than it is for a rare recessive syndrome such as XP. With sufficient data, q can be estimated by pedigree analysis (Swift et al., 1986). Heterozygous carriers of a gene for a rare autosomal recessive syndrome are common in the general population. For example, if a syndrome has a t r u e incidence (q2) of 1/40,000 then the heterozygote frequency is 1//100; for a true incidence of 1/360,000 the frequency is 1/300; and even if the incidence is 1/I(Y' the frequency is still 1/500. The heterozygote frequency will be even higher if a disorder is genetically heterogeneous, because q must be calculated separately for each independent gene. For example, if a disorder with a clinical incidence of 1/40,000 is actually due to the expression of four independent genes with equal incidence (1/160,000), then the total incidence of heterozygotes is 1/50 instead of the 1/100 estimated for a single-gene disorder. Patients with A-T, FA, or XP have been reported from all parts of the world (Swift, 1990; German and Passarge, 1989; Kraemer and Slot, 1984). In the United States patients with A-T and FA are seen at about the same frequency approximately twice as often as patients with XP (M. Swift, personal observation). BS is perhaps the rarest of the four syndromes, except among Ashkenazi Jews, where there may be a founder effect. 46 of 132 cases of BS reported to the BS Registry by 1987 (35%) were of Ashkenazi Jewish descent (German and Takebe, 1989). For A-T, the overall incidence observed in the United States was approximately 1/300,000 and the highest observed incidence within the United States was 1/90,000 (Swift et al., 1986). In Britain, the observed incidence of A-T was 1/100,000 (Pippard et al., 1988). The overall incidence of XP, without taking genetic heterogeneity into account, was estimated as approximately 1/250,000 in Europe and the United States (Robbins et al., 1974). German et al. (1977) estimated the incidence of BS among Ashkenazi Jews to be approximately 1/60,000. An overall incidence value for BS in the United States, derived from data in the BS Registry, was estimated as 1/6,331,000 (German and Takebe, 1989). Similarly, the observed incidence of FA in the United States and Ger-
many could be estimated from data already available in the FA Registry (Auerbach et al., 1989), but has not yet been published. The point prevalence of FA among white Afrikaners was found to be 1/22,000 in South Africa (Rosendorff et al., 1987), a high incidence undoubtedly due to the founder effect evident in other genetic disorders of Afrikaners. Gene frequencies estimated from all these incidence values are likely to be underestimates because of difficulties in diagnosis (especially of FA and BS) and in case-finding. T h e proportion of consanguineous marriages among parents of homozygotes was found to be 1.8% in A-T families in the United States (Swift et al., 1986); 7.7-23.9% in FA families worldwide (Schroeder et al., 1976); 12-100% in XP families worldwide (Kraemer and Slor, 1984; Takebe et al., 1987); and 39% in non-Jewish BS families worldwide (German and Takebe, 1989). The proportion of homozygotes among the non-sib blood relatives of probands has been reported only for A-T in the United States, giving a maximum likelihood estimate of gene frequency for A-T of 0.007 (Swift et al., 1986). Clinical and laboratory manifestations in heterozygotes Since heterozygotes for A-T, FA, BS, and XP have population frequencies in the range of 0.22% or more, it is important to know whether they, like the homozygotes (Table 1), are predisposed to cancer, diabetes mellitus, or other health problems. No obvious clinical signs distinguish these heterozygotes. However, clinical manifestations in heterozygotes have been identified by comparing obligate heterozygotes and blood relatives of homozygotes With population or intrafamilial spouse controls. Distinctive cellular abnormalities have been described in A-T, FA, BS, and XF homozygotes (Table 2). It is important to know which of these, if any, are also found in heterozygous cells; evidence of abnormalities in such cells would support clinical evidence that a single copy of a mutant allele produces a phenotypic effect. In addition, cellular anomalies in heterozygous cells might form the basis for tests that identify heterozygotes.
Possibly 4 or more.
No evidence for more than one.
Possibly 2 or more.
Nine: A - H and Variant.
Ataxia telangiectasia a
Bloom syndrome b
Fanconi anemia c
Xeroderma pigmentosum d
Ultraviolet light, Alkylating agents, including mitomycin C, aflatoxin, Oxygen.
Cross-linking agents including diepoxybutane, mitomycin C, nitrogen mustard, Oxygen.
Ultraviolet. X-Rays. BrdU. Oxygen radicals,
X-Rays, drugs including bleomycin, streptonigrin, neocarzinostatin, Oxygen.
Toxic agents that induce an abnormal cellular response
Evidence for some s p o n t a n e o u s chromosome r e a r r a n g e m e n t in cells from unaffected skin. Induced c h r o m o s o m e aberrations include chromatid deletions and dicentrics, and sisterchromatid exchanges.
Variable excess of s p o n t a n e o u s and induced c h r o m o s o m e aberrations especially gaps and breaks, micronuclei, sisterchromatid exchanges, G 2 chromatid aberrations.
Excess s p o n t a n e o u s and BrdU-induced sisterchromatid exchanges. Also c h r o m o s o m e breaks and aberrations, especially quadriradials.
Excess spontaneous and induced chromosome breaks and r e a r r a n g e m e n t s in G o and G 1. N o n - r a n d o m c h r o m o s o m e 7 and 14 translocations. Induced G 2 aberrations.
Chromosome aberrations
"~ cell killing by UV.
"r cell killing by toxic agents.
Not studied.
1' cell killing by toxic agents,
Cell survival
Not studied.
G 2 arrest.
Prolonged G o . G 2 arrest.
Induced and s p o n t a n e o u s cell cycle anomalies: G 2 arrest, radioresistant D N A synthesis.
Cell cycle anomalies
? frequency of somatic cell mutations, e
? levels spontaneous recombination and somatic mutation. Infrequent spontaneous mutation. $ transformation by SV40.
Hypothesized.
Hypothesized.
Hypothesized.
Defective $ mutation nucleotide induced by UV. excision (groups A - H ) or post-replication (XP variant) repair of induced D N A damage.
Mutation
D N A repair anomalies
References: a Gatti et al. (1991); b Nicotera (1991); G e r m a n and Passarge (1989); c C o h e n and Levy (1989); d K r a e m e r and Slor (1984); C o h e n and Levy (1989); e Bigbee et aL (1989).
Complementation groups
Syndrome
MAJOR LABORATORY PHENOTYPES ASSOCIATED WITH HOMOZYGOTES
TABLE 2
30
For each of the syndromes, the biological mechanisms responsible for their multiple phenotypes are consistent with DNA repair a n d / o r DNA processing defects. XP homozygous cells are defective in various steps of the excision repair pathway for the repair of induced DNA damage (Cleaver, 1990). Two genes associated with XP have been cloned and are thought to be DNA helicases (Tanaka et al., 1990; Weeda et al., 1990). In BS, a defective ligase activity may well be the biological basis of the phenotype (Barnes et al., 1991; Nicotera, 1991), but specific enzymatic errors have not yet been shown to be responsible for the A-T or FA phenotypes. The molecular basis for clinical or cellular manifestations in heterozygotes is, therefore, not known.
Ataxia telangiectasia There is good evidence that heterozygous carriers of the A-T gene are predisposed to cancer (Swift et al., 1976, 1987, 1991; Morrell et al., 1990; Pippard et al., 1989; Borresen et al., 1990). Female breast cancer with onset before 65 years of age is clearly associated with A-T heterozygosity. Diagnostic X-ray exposure may be a risk factor (Swift et al., 1987, 1991). Other cancers possibly associated with A-T heterozygosity are those of the pancreas, stomach, bladder, ovary, lung, prostate, and oral cavity, and chronic lymphocytic leukemia, malignant melanoma, and lymphoma. If A-T heterozygotes constitute 1.4% of the United States population and have a 6.8fold breast cancer risk (Swift et al., 1987), then 9% of all breast cancer cases in the United States could be attributed to A-T heterozygosity. Female A-T heterozygotes may also be at increased risk for late-onset diabetes mellitus. These carriers are three times more likely than non-carriers to develop a late-onset diabetes mellitus clinically similar to the mild diabetes seen in the homozygotes (Morrell et al., 1992). Each possible association between A-T heterozygosity and specific cancers or diabetes could be tested using the index-test method described below. Ceils from A-T homozygotes show reduced survival after X-irradiation or treatment with spe-
cific cytotoxic compounds under a wide variety of experimental conditions (Taylor et al., 1975; Shiloh et al., 1983). In contrast, ceils from obligate A-T heterozygotes have shown differential survival only under specific exposure conditions (Chen et al., 1978; Paterson et al., 1985; Shiloh et al., 1982; Cole et al., 1988; Weeks et al., 1991). However, survival of cultured fibroblasts after y-irradiation does not reliably discriminate indit~idual A-T heterozygotes from non-carriers (Paterson et al., 1985; Weeks et al., 1991). ~Similarly, various attempts to reliably identify individual A-T heterozygotes by diverse assays have not been successful (Rosin et al., 1989; Rudolph et al., 1989; Pawlak et al., 1990). Numerous attempts to find a clastogenic response to X-irradiation or radiomimetic drugs in A-T heterozygous cells have identified differences between cells in some studies but not others (Natarajan et al., 1982; Nagasawa et al., 1985; Bender et al., 1985; Parshad et al., 1985; Waghray et al., 1990; Sanford et al., 1990; Wiencke et al., 1992). Methodological differences or genetic heterogeneity may account for the variation between studies, but it is clear that individual A-T heterozygous cells are heterogeneous in their cytogenetic response to X-rays. Wiencke et al. (1992) concluded from a large sample that the increase in chromatid aberrations seen in many A-T heterozygous cells after X-irradiation is not a reliable method of discriminating A-T heterozygotes from non-carriers.
Fanconi anemia Petridou and Barrett (1990) found several clinical differences between 16 obligate FA heterozygotes and 40 unaffected controls. In heterozygotes fetal hemoglobin was significantly increased, the number of natural killer cells was significantly decreased, and mitogenic responses to phytohemagglutinin and interleukin-2 were reduced. Three heterozygotes had neutropenia and there were also significant differences in skeletal proportions. These results support those of Welsheimer and Swift (1982), who found that heterozygotes are predisposed to the genitouri-
31 nary and distal limb malformations frequently observed in FA homozygotes. Female FA heterozygotes are predisposed to diabetes mellitus, being approximately six times more likely to develop the disease than non-carriers (Swift et al., 1972; Morrell et ai., 1986). No systematic surveys have been reported, but there is certainly an excess of diabetes in FA homozygotes (Woodard et al., 1981). FA was the cancer-prone autosomal recessive syndrome that generated the idea that heterozygous carriers may also be predisposed to cancer (Swift, 1971). A study of 25 families suggested an association between bladder, stomach and breast cancer and FA heterozygosity, although there was no overall excess of cancer among the blood relatives (Swift et al., 1980). This study had a modest sample size compared, for example, to the more recent A-T family studies (Swift et al., 1987; 1991). Possible associations between specific cancers and diabetes and FA heterozygosity could be tested using the index-test method described below. Elevated spontaneous chromosome breaks and rearrangements are seen in almost all FA patients (Auerbach et al., 1989); the diagnosis can be made unequivocally by combining clinical data with cytogenetic evaluation of the frequency of aberrations induced by D N A cross-linking agents such as diepoxybutane or mitomycin C (Auerbach and Wolman, 1976; Auerbach et al., 1989). Rosendorff and Bernstein (1988) found that the mean chromosome aberration frequency for FA heterozygotes was significantly elevated above controls when induced by diepoxybutane but not by mitomycin C. The degree of overlap between the groups, however, precludes the use of this assay as a reliable diagnostic test for FA heterozygosity. This conclusion is supported by earlier, less definitive studies (Auerbach and Wolman, 1978; Berger et al., 1980; Cervenka and Hirsch, 1981; Auerbach et al., 1981; Cohen et al., 1982). Miglierina et al. (1991) found that there is a cell cycle disturbance following exposure to nitrogen mustard in FA heterozygous cells that can be detected by flow cytometry. The value of this finding in testing for FA heterozygotes is not yet known. Similarly, Petridou and Barrett (1991)
found significant differences between FA heterozygotes and controls that may well form the basis for a test for FA heterozygotes.
Xeroderma pigmentosum XP is not a single genetic disorder: a total of nine complementation groups and a variant have been identified (Cleaver, 1990). The hypothesis that XP heterozygotes are predisposed to nonmelanoma skin cancer was supported by finding significantly more of these cancers in blood relatives of XP patients than in their spouse controls, in 31 North American XP families of unknown complementation group (Swift and Chase, 1979). The excess cancers appeared to be concentrated in four XP families living in the Southern United States. Other cancers apparently in excess in the 31 families were those of the lung, stomach, and prostate. Predisposition to cancer was also studied in an unspecified number of British XP families representing 48 patients. Only two skin cancers were found among the parents and grandparents of the probands (Pippard et al., 1988). There were, however, three stomach and two lung cancers in the grandparents, and a single prostate cancer in a father. Microcephaly and progressive mental retardation are seen in XP homozygotes, particularly those of complementation groups A and D. In 31 XP families four cases of microcephaly and 11 of mental retardation were observed, concentrated in the families known to belong to groups A and D (Welshimer and Swift, 1982). This excess was significant and suggests that some XP mutations predispose the heterozygote to mental retardation and microcephaly. It will be possible to test the association of XP heterozygosity with specific cancers and with neurological dysfunction using the index-test method, described below. A limited number of investigations have suggested that spontaneous chromosome aberrations are present in XP homozygous cells (Kraemer and Slor, 1984; Aledo et al., 1989). Certainly, an increase in aberrations is seen after such cells are exposed to UV and certain cytotoxic agents (reviewed in Kraemer and Slor, 1984). In UV-irradiated XP heterozygous cells, Bielfield et al. (1989)
32 found an increase in sister-chromatid exchanges and micronuclei; with combined data from these two endpoints, they were able to distinguish individual XP heterozygous and control cells approximately 90% of the time. The study was exceptional because 19 parents and sibs of seven XP heterozygotes of known complementation groups were compared to 24 controls. An increase in spontaneous chromosome aberrations has been observed in two obligate XP heterozygotes (Casati et al., 1990). Quantitative differences between DNA repair in XP heterozygous and wild-type cells have been observed with several different assays (Giannelli and Pawsey, 1974; Squires and Johnson, 1988) but not in others (Kleijer et al., 1973; Day, 1974; Selsky and Greer, 1978). Each of these assays was technically arduous, and only between two and ten XP heterozygous or control cell lines were used in any single study. Differences in complementation group may account for some of the variation between reported studies of cellular phenotypes in XP heterozygous cells, but no assay for an XP heterozygous phenotype has yet been shown to reliably detect XP heterozygotes in families or in populations. Perhaps a DNA repair assay directed at a single genetic form of XP would be sensitive and specific for that mutant allele in heterozygotes.
Bloom syndrome There are anecdotal reports of cancers in obligate BS heterozygotes (German, 1974), but no systematic studies of clinical findings in BS heterozygotes have been reported. A BS Registry has collected data for all reported cases since the early 1960s (German and Passarge, 1989). The predisposition of the BS heterozygote to cancer or diabetes could be assessed, at modest cost and effort, by methodical follow-up of the families identified through this Registry. It is regrettable that this has not yet been done, since 1/125 Ashkenazi Jews are BS heterozygotes. BS heterozygous cells have been studied very little, perhaps because nothing is known about the disease predisposition of BS heterozygotes. Limited studies have shown no evidence for a significant excess of chromosome aberrations or
sister-chromatid exchanges in BS heterozygous cells (Chaganti et al., 1974; Bartram et al., 1976; Sperling et al., 1976; Hustinx et al., 1977; Kuhn and Therman, 1979), although these are typical findings in BS homozygous cells (Nicotera, 1991). It would be logical and cost-effective to assess the clinical effects of BS heterozygosity from the BS Registry data before attempting to develop a reliable test for the BS heterozygote.
Overview of heterozygote tests There is no question that groups of A-T, FA, or XP heterozygous cells can be distinguished from controls under several, but not all, experimental conditions. These differences may reflect abnormalities that provide the cellular basis for observed clinical differences between carriers of an A-T, FA, or XP gene and non-carriers. An accurate test for identifying heterozygotes in the general population is of obvious clinical value. For example, there is very good evidence for an association between A-T heterozygosity and cancer, especially female breast cancer. Cancer incidence in the 1% of the population who are A-T heterozygotes could be minimized by reducing exposure to all sources of ionizing radiation, including diagnostic X-rays. These heterozygotes should be identified and observed for the earliest signs of cancer, since early treatment is associated with improved survival. Eventually, it may be possible to protect A-T heterozygotes from developing cancers by rational therapy deduced from an understanding of the metabolic action of the A-T gene. To be useful, tests for identifying A-T, FA, BS, or XP heterozygotes in the general population must be fully sensitive and specific. No assay yet developed is reliable enough for heterozygote identification for clinical or research purposes. Proposed heterozygote tests have been technically demanding, have had varying outcomes depending on the laboratory performing the technique, or were not tested blindly on an adequate number of obligate heterozygotes and controls. Reliable heterozygote tests could be based on DNA analysis once the gene locus (or loci) for each syndrome is identified and all the mutants characterized. At present, the genes for A-T, FA,
33 and BS are not known, and only the genes for XP complementation groups A and B have been cloned (Tanaka et al., 1990; Weeda et al., 1990). Substantial progress has been made in mapping the A-T locus on chromosome 11q22-23 (Gatti et al., 1991). Preliminary linkage data have assigned an FA gene in a subset of FA families to chromosome 20q (Mann et al., 1991). Proposed associations between being heterozygous for an A-T, BS, FA, or XP mutant allele and a common disease must be confirmed before attempting to identify heterozygotes for clinical purposes. Each hypothesized association can be tested most efficiently and reliably by the indextest method, which relies on precise identification of heterozygotes within the families of homozygous probands. In these families, heterozygotes can be identified even before the gene is cloned if the mutant allele can be traced by polymorphic D N A markers tightly linked to the gene locus.
Establishing heterozygote risk by the 'index-test method' The index-test method is a recently described genetic method for establishing an association between any candidate allele and any common chronic disease such as cancer or diabetes mellitus (Swift et al., 1990). Its statistical power is good, so that the sample size required is practical, and it is not affected by variations in population gene frequencies, by ascertainment bias, or by genetic heterogeneity of the common disease. For example: the index-test method could be used to test the possible associations of nonmelanoma skin, or gastric cancer with heterozygosity for XP group A mutant alleles. Group A is the predominant type of XP in Japan (Takebe et al., 1987). The following is an outline of such an application. (1) Identify the 'index cases' - those Japanese XP group A patients who carry mutations in the group A gene. They are called index cases because they identify (i.e., index) the families in which the alleles of interest are segregating. (2) Find as many families as possible in which these alleles are segregating. Depending on how great a risk the heterozygote has for a specific
cancer, 20 families might provide adequate statistical power (see Table 2 in Swift et al., 1990). (3) Identify as many 'test individuals' as possible in each family. To test the association with skin cancer, the test individuals would be blood relatives of the index case who have skin cancer (to test the association with gastric cancer a different group of test individuals is required: blood relatives with gastric cancer). In each group of test individuals exclude homozygotes, obligate heterozygotes, and any individual whose status as an XP heterozygote depends on the status of another test individual. (4) Obtain DNA from all test individuals using bloodl saliva, tissue, or paraffin-embedded tissue blocks. Use DNA analysis to determine which test individuals are heterozygous for the specific XP mutation segregating in their family. (5) Count the total number of XP heterozygotes in the selected group of test individuals. This is the 'observed number' of XP heterozygotes in this group. (6) Determine the 'expected number' of XP heterozygotes in the group of test individuals. Table 1 in Swift et al. (1990) gives the probability that a blood relative of the index case is heterozygous, based on the allele frequency and the blood relative's relationship to the index case. (7) Compare the observed number of XP heterozygotes to the expected number. If the observed number is greater than expected, test the significance of the difference. Estimate the relative risk of the cancer in question for XP group A heterozygotes from the odds ratio. (8) A significant excess of XP group A heterozygotes among the test individuals with a cancer is strong evidence that the XP group A allele predisposes heterozygotes to that cancer.
Conclusion There is cumulative evidence that the heterozygote phenotypes for the autosomal recessive syndromes A-T, XP and FA are distinct from wild-type at both the cellular and clinical levels. Little is known about BS heterozygotes, but perhaps this review will encourage new investigations. If current efforts to clone the respective disease genes are successful, analyzing each gene's
34 m o l e c u l a r e f f e c t s will l e a d t o a n u n d e r s t a n d i n g the complex phenotypes
of
and to measures for im-
proving the health of the general population.
Note added in proof c D N A s t h a t c o r r e c t t h e d e f e c t in F A c e l l s designated complementation group C have rec e n t l y b e e n c l o n e d ( S t r a t h d e e e t al., 1992, N a t u r e , 356, 7 6 3 - 7 6 7 ) .
References Aledo, R., G. Renault, M. Prieur, M.F. Avril, B. Chretien, B. Dutrillaux and A. Aurias (1989) Increase of sister chromatid exchanges in excision repair deficient xeroderma pigmentosum, Hum. Genet., 81, 221-225. Auerbach, A.D., and S.R. Wolman (1976) Susceptibility of Fanconi's anaemia fibroblasts to chromosome damage by carcinogens, Nature, 261,494-496. Auerbach, A.D., and S.R. Wolman (1978) Carcinogen-induced chromosome breakage in Fanconi's anemia, Nature, 271, 69-71. Auerbach, A.D., B. Adler and R.S.K. Chaganti (1981) Prenatal and postnatal diagnosis and carrier detection of Fanconi anemia by a cytogenetic method, Pediatrics, 67, 128135. Auerbach, A.D., A. Rogatko and T.M. Schroeder-Kurth (1989) International Fanconi Anemia Registry: Relation of clinical symptoms to diepoxybutane sensitivity, Blood, 73, 391396. Barnes, D.F., A.F. Tomkinson, K. Kodama and T. Lindahl (1991) DNA ligase defect in Bloom's syndrome, Am. J. Hum. Genet., 49 (Suppl.), 295 (Abstract). Bartram, C.R., T. Koske-Westphal and E. Passarge (1976) Chromatid exchanges in ataxia telangiectasia, Bloom syndrome, Werner syndrome, and xeroderma pigmentosum, Ann. Hum. Genet., 40, 79-86. Bender, M.A, J.M. Rary and R.P. Kale (1985) G2 chromosomal radiosensitivity in ataxia telangiectasia lymphocytes, Mutation Res., 152, 39-47. Berger, R., A. Bernheim, M. Le Coniat, D. Vecchione and G. Schaison (1980) Sister chromatid exchanges induced by nitrogen mustard in Fanconi's anemia. Application to the detection of heterozygotes and interpretation of the results, Cancer Genet. Cytogenet., 2, 259-267. Bielfeld, V., M. Wiechenthal, M. Roser, E. Breitbart, J. Berger, E. Seemanova and H.W. Rudiger (1989) Ultraviolet-induced chromosomal instability in cultured fibroblasts of heterozygote carriers for xeroderma pigmentosum, Cancer Genet. Cytogenet., 43, 219-226. Bigbee, W.L., R.G. Langlois, M. Swift and R.H. Jensen (1989) Evidence for an elevated frequency of in vivo somatic cell mutations in ataxia telangiectasia, Am. J. Hum. Genet., 44, 402-408.
Borresen, A.-L., T.I. Andersen, S. Tretli, A. Heiberg and P. Moiler (1990) Breast cancer and other cancers in Norwegian families with ataxia telangiectasia, Genes Chromosomes Cancer, 2, 339-340. Casati, A., M. Stefanini, R. Giorgi, P. Ghetti and F. Nuzzo (1991) Chromosome rearrangements in normal fibroblasts from xeroderma pigmentosum homozygotes and heterozygotes, Cancer Genet. Cytogenet., 51, 89-101. Cervenka, J., and B.A. Hirsch (1983) Cytogenetic differentiation of Fanconi anemia, 'idiopathic' aplastic anemia, and Fanconi anemia heterozygotes, Am. J. Med. Genet., 15, 211-223. Chaganti, R.S.K., S. Schonberg and J. German (1974) A manyfold increase in sister chromatid exchanges in Bloom's syndrome lymphocytes, Proc. Natl. Acad. Sci. USA, 71, 4508-4512. Chen, P.C., M.F. Lavin and C. Kidson (1978) Identification of ataxia telangiectasia heterozygotes, a cancer prone population, Nature, 274, 484-486. Cleaver, J.E. (1990) Do we know the cause of xeroderma pigmentosum? Carcinogenesis, 11,875-882. Cohen, M.M., and H.P. Levy (1989) Chromosome instability syndromes, Adv. Hum. Genet., 18, 43-149, 365-367. Cohen, M.M., S.J. Simpson, G.R. Honig, H.S. Maurer, J.W. Nicklas and A.O. Martin (1982) The identification of Fanconi anemia genotypes by clastogenic stress, Am. J. Hum. Genet., 34, 794-810. Cole, J., C.F. Arlett, M.H. Green, S.A. Harcourt, A. Priestley, L. Henderson, H. Cole, S.E. James and F. Richmond (1988) Comparative human cellular radiosensitivity: II. The survival following gamma-irradiation of unstimulated (G~) T-lymphocytes, T-lymphocyte lines, lymphoblastoid cell lines and fibroblasts from normal donors, from ataxia telangiectasia patients and from ataxia telangiectasia heterozygotes, Int. J. Radiat. Biol., 54, 929-943. Day III, R.S. (1974) Studies on repair of adenovirus 2 by human fibroblasts using normal, xeroderma pigmentosum, and xeroderma pigmentosum heterozygous strains, Cancer Res., 34, 1965-1970. Gatti, R.A., E. Boder, H.V. Vintners, R.S. Sparkes, A. Norman and K. Lange (1991) Ataxia telangiectasia: an interdisciplinary approach to pathogenesis, Medicine, 70, 99117. German, J. (1974) Bloom's syndrome, lI. The prototype of human genetic disorders predisposing to chromosome instability and cancer, in: J. German (Ed.), Chromosomes and Cancer, John Wiley, New York, pp. 601-617. German, J., and E. Passarge (1989) Bloom's syndrome. XII. Report from the Registry for 1987, Clin. Genet., 35, 57-69. German, J., and H. Takebe (1989) Bloom's syndrome. XIV. The disorder in Japan, Clin. Genet., 35, 93-110. German, J., D. Bloom, E. Passarge, K. Fried, R.M. Goodman, I. Katzenellenbogen, Z. Laron, C. Legum, S. Levin and J. Wahrman (1977) Bloom's syndrome. VI. The disorder in Israel and an estimation of the gene frequency in the Ashkenazim, Am. J. Hum. Genet., 29, 553-562. Giannelli, F., and S.A. Pawsey (1974) DNA repair synthesis in human heterokaryons. II. A test for heterozygosity in
35 xeroderma pigmentosum and some insight into the structure of the defective enzyme, J. Cell. Sci., 15, 163-176. Hustinx, T.W.J., B.G.A. Ter Haar, J.M.J.C. Scheres, F.J. Rutten, C.M.R. Weemaes, R.L.E. Hoppe and A.H Hanssen, (1977) Bloom's syndrome in two Dutch families, Clin. Genet., 12, 85-96. Jacobs, P., and C. Karabus (1984) Fanconi's anemia. A family study with 20-year follow-up including assiciated breast pathology, Cancer, 54, 1850-1853. Kleijer, W.J., E.A. de Weerd-Kastelein, M.L. Sluyter, W. Keijzer, J. de Wit and D. Bootsma (1973) UV-induced DNA repair synthesis in cells of patients with different forms of xeroderma pigmentosum and of heterozygotes, Mutation Res., 20, 417-428. Kraemer, K.H., and H. Slor (1984~ Xeroderma pigmentosum, Clin. Dermatol., 2, 33-69. Kuhn, E.M., and E. Therman (1979) No increased chromosome breakage in three Bloom's syndrome heterozygotes, J. Med. Genet., 16, 219-222. Mann, W.R., V.S. Venkatraj, R.G. Allen,, Q. Liu, D.A. Olsen, B. Adler-Brecher, J.-I. Mao, 13. Weiffenbach, S.L. Sherman and A.D. Auerbach (1991) Fanconi anemia: evidence for linkage heterogeneity on chromosome 20q, Genomics, 9, 329-337. Miglierina, R., M. Le Coniat and R. Berger (1991) A simple diagnostic test for Fanconi anemia by flow cytometry, Anal. Cell. Pathol., 3, 111-118. Morrell, D., C.L. Chase, L.L. Kupper and M. Swift (1986) Diabetes mellitus in ataxia telangiectasia, Fanconi anemia, xeroderma pigmentosum, common variable immune deficiency and severe combined immune deficiency families, Diabetes, 35, 143-147. Morrell, D., C.L. Chase and M. Swift (1990) Cancers in 44 families with ataxia telangiectasia, Cancer Genet. Cytogenet., 50, 119-123. Morrell, D., C.L. Chase and M. Swift (1992) Diabetes mellitus in relatives of ataxia telangiectasia patients in 146 families, submitted for publication. Nagasawa, H., S.A. Latt, M.E. Lalande and J.B. Little (1985) Effects of X-irradiation on cell-cycle progression, induction of chromosomal aberrations and cell killing in ataxia telangiectasia (AT) fibroblasts, Mutation Res., 148, 71-82. Natarajan, A.T., M. Meijers, A.A. van Zeeland and J.W.I.M. Simons (1982) Attempts to detect ataxia telangiectasia (AT) heterozygotes by cytogenetical techniques, Cytogenet. Cell Genet., 33, 145-151. Nicotera, T.M. (1991) Molecular and biochemical aspects of Bloom's syndrome, Cancer Genet. Cytogenet., 53, 1-13. Okayuma, S., and H. Mishina (1987) Fanconi's anemia as a nature's evolutionary experiment on carcinogenesis, Tohoku J. Exp. Med., 153, 87-102. Parshad, R., K.K. Sanford, G.M. Jones and R.E. Tarone (1985) G{2} chromosomal radiosensitivity of ataxia telangiectasia heterozygotes, Cancer Genet. Cytogenet., 14, 163-168. Paterson, M.C., S.J. MacFarlane, N.E. Gentner and B.P. Smith (1985) Cellular hypersensitivity to chronic gammaradiation in cultured fibroblasts from ataxia telangiectasia
heterozygotes, in: R.A. Gatti and M. Swift (Eds.), AtaxiaTelangiectasia: Genetics, Neuropathology, and Immunology of a Degenerative Disease of Childhood, Alan R. Liss, Inc., New York. Pawlak, A.L., M. Kotecki and R. Ignatowicz (1990) Increased frequency of chromatid breaks in lymphocytes of heterozygotes of ataxia telangiectasia after in vitro treatment with caffeine, Mutation Res., 230, 197-204. Petridou, M., and A.J. Barrett (1990) Physical and laboratory characteristics of heterozygote carriers of the Fanconi aplasia gene, Acta Paediat. Scand., 79, 1069-1074. Pippard, E.C., A.J. Hall, D.J. Barker and B.A. Bridges (1988) Cancer in homozygotes and heterozygotes of ataxia telangiectasia and xeroderma pigmentosum in Britain, Cancer Res., 48, 2929-2932. Polani, P.E. (1979) DNA repair defects and chromosome instability disorders, in: Human Genetics: Possibilities and Realities, Ciba Foundation Symposium 66, Excerpta Medica, Amsterdam. Robbins, J.H., K.H. Kraemer, M.A. Lutzner, B.W. Festoff and H.G. Coon (1974) Xeroderma pigmentosum. An inherited disease with sun sensitivity, multiple cutaneous neoplasms, and abnormal DNA repair, Ann. Intern. Med., 80, 221-248. Romeo, C., P. Menozzi, A. Ferlini, S. Fadda, S. Di Donato, G. Uziel, B. Lucci and L. Capodaglio (1983) Incidence of Friedreich ataxia in Italy estimated from consanguineous marriages, Am. J. Hum. Genet., 35, 523-529. Rosendorff, J., and R. Bernstein (1988) Fanconi's anemia Chromosome breakage studies in homozygotes and heterozygotes, Cancer Genet. Cytogenet., 33, 175-183. Rosendorff, J., R. Bernstein, L. Macdougall and T. Jenkins (1987) Fanconi anemia: Another disease of unusually high prevalence in the Afrikaans population of South Africa, Am. J. Med. Genet., 27, 793-797. Rosin, M.P., H.D. Ochs, R.A. Gatti and E. Boder (1989) Heterogeneity of chromosomal breakage levels in epithelial tissue of ataxia telangiectasia homozygotes and heterozygotes, Hum. Genet., 83, 133-138. Rudolph, N.S., H. Nagasawa, J.B. Little and S.A. Latt (1989) Identification of ataxia telangiectasia heterozygotes by flow cytometric analysis of X-ray damage, Mutation Res., 211, 19-29. Sanford, K.K., R. Parshad, F.M. Price, G.M. Jones, R.E. Tarone, L. Eierman, P. Hale and R.A. Waldmann (1990) Enhanced chromatid damage in blood lymphocytes after G 2 phase X irradiation, a marker of the ataxia telangiectasia gene, J. Natl. Cancer Inst., 82, 50-54. Schroeder, T.M., D. Tilgen, J. Kruger and F. Vogel (1976) Formal genetics of Fanconi's anemia, Hum. Genet., 32, 257-288. Selsky, C.A., and S. Greer (1978) Host-cell reactivation of UV-irradiated and chemically-treated herpes simplex virus-1 by xeroderma pigmentosum, XP heterozygotes and normal skin fibroblasts, Mutation Res., 50, 395-405. Shiloh, Y., E. Tabor and Y. Becker (1982) The response o f ataxia telangiectasia homozygous and heterozygous skin fibroblasts to neocarzinostatin, Carcinogenesis, 3, 815-820.
36 Shiloh, Y., E. Tabor and Y. Becker (1983) Abnormal response of ataxia telangiectasia cells to agents that break the deoxyribose moiety of DNA via a targeted free radical mechanism, Carcinogenesis, 4, 1317-1322. Sperling, K., U. Goll, J. Kunze, E.-K. Ludtke, M. Tolksdorf and G. Obe (1976) Cytogenetic investigations in a new case of Bloom's syndrome, Hum. Genet., 31, 47-52. Squires, S., and R.T. Johnson (1988) Kinetic analysis of UVinduced incision discriminates between fibroblasts from different xeroderman pigmentosum complementation groups, XPA heterozygotes and normal individuals, Mutation Res., 193, 181-192. Swift, M. (1971) Fanconi's anemia in the genetics of neoplasia, Nature, 230, 370-373. Swift, M. (1990) Genetic aspects of ataxia telangiectasia, lmmunodefic. Rev., 2, 67-81. Swift, M., and C. Chase (1979) Cancer in families with xeroderma pigmentosum, J. Natl. Cancer Inst., 62, 1415-1421. Swift, M., L. Sholman and D. Gilmour (1972) Diabetes mellitus and the gene for Fanconi's anemia, Science, 178, 308-310. Swift, M., L. Sholman, M. Perry and C. Chase (1976) Malignant neoplasms in the families of patients with ataxia telangiectasia, Cancer Res., 36, 209-215. Swift, M., R.J. Caldwell and C. Chase (1980) Reassessment of cancer predisposition of Fanconi anemia heterozygotes, J. Natl. Cancer Inst., 65, 863-867. Swift, M., D. Morrell, E. Cromartie, A.R. Chamberlin, M.H. Skolnick and D.T. Bishop (1986) The incidence and gene frequency of ataxia telangiectasia in the USA, Am. J. Hum. Genet., 39, 573-583. Swift, M., P.J. Reitnauer, D. Morrell and C.L. Chase (1987) Breast and other cancers in families with ataxia telangiectasia, New Engl. J. Med., 316, 1289-1294. Swift, M., L.L. Kupper and C.L. Chase (1990) Effective testing of gene-disease associations, Am. J. Hum. Genet., 47, 266-274. Swift, M., D. Morrell, R. Massey and C.L. Chase (1991) Cancer incidence in 161 ataxia telangiectasia families studied prospectively, New Engl. J. Med., 326, 1831-1836.
Takebe, H., C. Nishigori and Y. Satoh (1987) Genetics and skin cancer of xeroderma pigmentosum in Japan, Jpn. J. Cancer Res. (Gann), 78, 1135-1143. Tanaka, K., N. Miura, I. Satokata, I. Miyamoto, M.C. Yoshida, Y. Satoh, S. Kondo, A. Yasui, H. Okayama and Y. Okada (1990) Analysis of a human DNA excision repair gene involved in group A xeroderma pigmentosum and containing a zinc-finger domain, Nature, 348, 73-76. Taylor, A.M.R., D.G. Harnden, C.F. Arlett, S.A. Harcourt, A.R. Lehmann, S. Stevens and B.A. Bridges (1975) Ataxia-telangiectasia: a human mutation with abnormal radiation sensitivity, Nature, 258, 427-429. Waghray, M., S. AI-Sedairy, P.T. Ozand and M.A. Hannan (1990) Cytogenetic characterization of ataxia telangiectasia (AT) heterozygotes using lymphoblastoid cell lines and chronic gamma-irradiation, Hum. Genet., 84, 532-534. Weeda, G., R.C.A. van Ham, W. Vermeulen, D. Bootsma, A.J. Van Der Eb and J.H.J. Hoeijmakers (1990) A presumed DNA helicase encoded by ERCC-3 is involved in the human repair disorders xeroderma pigmentosum and Cockayne's syndrome, Cell, 62, 777-791. Weeks, D.E., M.C. Paterson, K. Lange, B. Andrais, R.C. Davis, F. Yoder and R.A. Gatti (1991) Assessement of chronic gamma radiosensitivity as an in vitro assay for heterozygote identification of ataxia telangiectasia, Radiat. Res., 128, 90-99. Welshimer, K., and M. Swift (1982) Congenital malformations and developmental disabilities in ataxia telangiectasia, Fanconi anemia, and xeroderma pigmentosum families, Am. J. Hum. Genet., 34, 781-793. Wiencke, J.K., D.W. Wara, J.B. Little and K.T. Kelsey (1992) Heterogeneity in the clastogenic response to X-rays in lymphocytes from ataxia telangiectasia heterozygotes and controls, Cancer Causes and Control, 3, 237-245. Woodard, T.L., G.A. Burghen, A.E. Kitabchi and J.A. Wilimas (1981) Glucose intolerance and insulin resistance in aplastic anemia treated with oxymetholone, J. Clin. Endocrinol. Metab., 53, 905-908.
25
© 1992 Elsevier Science Publishers B.V. All rights reserved 0027-5107/92/$05.00
MUT 0374
Heterozygous manifestations in four autosomal recessive human cancer-prone syndromes: ataxia telangiectasia, xeroderma pigmentosum, Fanconi anemia, and Bloom syndrome * Ruth A. Heim, Nicholas J. Lench and Michael Swift Biological Sciences Research Center, Unit,ersity of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7250, USA
(Accepted 16 October 1991)
Keywords: Heterozygotes; Ataxia telangiectasia; Xeroderma pigmentosum; Bloom syndrome; Fanconi anemia
Four rare autosomal recessive syndromes associated with a high cancer risk in homozygotes ataxia telangiectasia (A-T), Bloom syndrome (BS), Fanconi anemia (FA), and xeroderma pigmentosum (XP) (Table 1) - offer insight into the genetics of cancer, diabetes mellitus, congenital malformations, and mental retardation. Heterozygous carriers of genes for the respective syndromes constitute a substantial proportion of the general population and may also have an increased risk of developing cancer and other health problems associated with the homozygotes. By studying heterozygotes for genes that predispose an individual to a common disorder, it may be possible to dissect the contribution that single genes make to the pathogenesis of complex disorders. Incidence and gene frequency G e n e frequencies can be measured directly by testing individuals in a population for allelespecific D N A mutations or protein variants. When this is not possible, three indirect methods can be used to estimate the allelic frequencies of autoso-
Correspondence: Dr. Michael Swift (present address:) New York Medical College, Valhalla, NY 10595, USA.
mal recessive syndromes. The most familiar approach uses the H a r d y - W e i n b e r g formula in which p is the frequency of the wild-type allele and q the frequency of the disease allele. If the incidence of the homozygotes (q2) can be measured by counting individuals with the distinctive phenotype, then the frequency of the disease allele is calculated simply as the square root of q. The observed incidence of a syndrome may be much smaller than the true birth or conception incidence, however, leading to a substantial underestimate of the disease allele frequency. The allele frequency q may also be estimated by the Dahlberg formula ( R o m e o et al., 1983) if both the frequency of first cousin marriages among the parents of homozygotes for a given autosomal recessive syndrome and the frequency of first cousin marriages in the general population are known. The latter is usually unknown or difficult to estimate. Even so, the frequency of one autosomal recessive syndrome relative to another can be estimated from the rates of parental consanguinity in each. The allele frequency can also be estimated from the proportion of families in which affected homozygotes have cousins, aunts or uncles, or nieces or nephews who are also homozygotes. When an allele is relatively common, as is the case for cystic fibrosis, it is simply more likely for a random person marrying into a cystic fibrosis
Acute lymphocytic leukemias and lymphomas in childhood, Chronic lymphocytic leukemias in adulthood, Also breast, pancreas, stomach, bladder, ovary, oral cavity.
Ataxia telangiectasia a
Bloom Mainly solid syndrome h tumors, including skin, breast, large intestine, oral cavity and lung. Also leukemia, lymphoma, Hodgkin disease.
Cancer predisposition
Syndrome
Sun sensitivity (ultraviolet),
No evidence,
Photophobia
Erythema. Facial telangiectasia, Small hyperand hypopigmented areas. Cafe-au-lait spots.
Oculocutaneous telangiectasia, Freckling. Progeric changes of hair and skin. Cafe-au-lait spots. Hyperpigmented macules,
Dermatological features
Possible mild mental deficiency.
Infant/ childhood onset of progressive cerebellar ataxia. Choreoathetosis. Oculomotor defects. Dysarthic speech. Dystonic posturing of fingers.
Neurological function and mental retardation
MAJOR CLINICAL FEATURES ASSOCIATED WITH HOMOZYGOTES
TABLE 1
Diabetes mellitus, usually insulinindependent.
Mild diabetes mellitus.
Diabetes mellitus
Growth retardation.
missing. Growth retardation. Ovarian dysgenesis.
or
Thymus abnormal
Structural abnormalities
Impaired. IgA, IgG and IgM.
T- and B-cellmediated immunodeficiency. IgA, IgE, and IgG 2.
Immune function
Normal.
Normal.
Hematological function
Chronic lung disease. Infertility. Gastrointestinal infections.
Alphafetoprotein. Radiosensitivity. Progressive pulmonary disease.
Other features
Mainly basal and squamous cell skin cancers, malignant melanomas,
Xeroderma pigmentosum d
Erythema with edema and blistering, Freckles. Pigmented and achromic macules, Telangiectasias. Actinic keratoses.
Cafe-au-lait spots. Fine reticular melanosis,
Progressive neurological degeneration and mental retardation in certain families.
Mental retardation, microcephaly in some patients,
No evidence.
Diabetes mellitus, insulinindependent,
None.
Growth retardation. Wide spectrum congenital malformations, including renal and ocular anomalies, skeletal malformations. Reduced natural killer cell activity.
Abnormal. Low natural killer cell function.
Normal.
Variable progressive pancytopenia. Increased fetal hemoglobin. Lymphopenia.
References: a Gatti et al. (1991); Swift (1990); b German and Passarge (1989); Cohen and Levy (1989); c Schroeder et al. (1976); Cohen and Levy (1989); ~ Kraemer and Slor (1984); Cohen and Levy (1989); c Okuyama and Mishina (1987); Jacobs and Karabus (1983).
Extreme sensitivity to sunlight (ultraviolet).
Mainly acute None. myelogenous leukemia, Also solid tumors, including liver, stomach, skin, esophagus, female genitalia, and breast, e
Fanconi anemia c
28 family to carry the cystic fibrosis allele, than it is for a rare recessive syndrome such as XP. With sufficient data, q can be estimated by pedigree analysis (Swift et al., 1986). Heterozygous carriers of a gene for a rare autosomal recessive syndrome are common in the general population. For example, if a syndrome has a t r u e incidence (q2) of 1/40,000 then the heterozygote frequency is 1//100; for a true incidence of 1/360,000 the frequency is 1/300; and even if the incidence is 1/I(Y' the frequency is still 1/500. The heterozygote frequency will be even higher if a disorder is genetically heterogeneous, because q must be calculated separately for each independent gene. For example, if a disorder with a clinical incidence of 1/40,000 is actually due to the expression of four independent genes with equal incidence (1/160,000), then the total incidence of heterozygotes is 1/50 instead of the 1/100 estimated for a single-gene disorder. Patients with A-T, FA, or XP have been reported from all parts of the world (Swift, 1990; German and Passarge, 1989; Kraemer and Slot, 1984). In the United States patients with A-T and FA are seen at about the same frequency approximately twice as often as patients with XP (M. Swift, personal observation). BS is perhaps the rarest of the four syndromes, except among Ashkenazi Jews, where there may be a founder effect. 46 of 132 cases of BS reported to the BS Registry by 1987 (35%) were of Ashkenazi Jewish descent (German and Takebe, 1989). For A-T, the overall incidence observed in the United States was approximately 1/300,000 and the highest observed incidence within the United States was 1/90,000 (Swift et al., 1986). In Britain, the observed incidence of A-T was 1/100,000 (Pippard et al., 1988). The overall incidence of XP, without taking genetic heterogeneity into account, was estimated as approximately 1/250,000 in Europe and the United States (Robbins et al., 1974). German et al. (1977) estimated the incidence of BS among Ashkenazi Jews to be approximately 1/60,000. An overall incidence value for BS in the United States, derived from data in the BS Registry, was estimated as 1/6,331,000 (German and Takebe, 1989). Similarly, the observed incidence of FA in the United States and Ger-
many could be estimated from data already available in the FA Registry (Auerbach et al., 1989), but has not yet been published. The point prevalence of FA among white Afrikaners was found to be 1/22,000 in South Africa (Rosendorff et al., 1987), a high incidence undoubtedly due to the founder effect evident in other genetic disorders of Afrikaners. Gene frequencies estimated from all these incidence values are likely to be underestimates because of difficulties in diagnosis (especially of FA and BS) and in case-finding. T h e proportion of consanguineous marriages among parents of homozygotes was found to be 1.8% in A-T families in the United States (Swift et al., 1986); 7.7-23.9% in FA families worldwide (Schroeder et al., 1976); 12-100% in XP families worldwide (Kraemer and Slor, 1984; Takebe et al., 1987); and 39% in non-Jewish BS families worldwide (German and Takebe, 1989). The proportion of homozygotes among the non-sib blood relatives of probands has been reported only for A-T in the United States, giving a maximum likelihood estimate of gene frequency for A-T of 0.007 (Swift et al., 1986). Clinical and laboratory manifestations in heterozygotes Since heterozygotes for A-T, FA, BS, and XP have population frequencies in the range of 0.22% or more, it is important to know whether they, like the homozygotes (Table 1), are predisposed to cancer, diabetes mellitus, or other health problems. No obvious clinical signs distinguish these heterozygotes. However, clinical manifestations in heterozygotes have been identified by comparing obligate heterozygotes and blood relatives of homozygotes With population or intrafamilial spouse controls. Distinctive cellular abnormalities have been described in A-T, FA, BS, and XF homozygotes (Table 2). It is important to know which of these, if any, are also found in heterozygous cells; evidence of abnormalities in such cells would support clinical evidence that a single copy of a mutant allele produces a phenotypic effect. In addition, cellular anomalies in heterozygous cells might form the basis for tests that identify heterozygotes.
Possibly 4 or more.
No evidence for more than one.
Possibly 2 or more.
Nine: A - H and Variant.
Ataxia telangiectasia a
Bloom syndrome b
Fanconi anemia c
Xeroderma pigmentosum d
Ultraviolet light, Alkylating agents, including mitomycin C, aflatoxin, Oxygen.
Cross-linking agents including diepoxybutane, mitomycin C, nitrogen mustard, Oxygen.
Ultraviolet. X-Rays. BrdU. Oxygen radicals,
X-Rays, drugs including bleomycin, streptonigrin, neocarzinostatin, Oxygen.
Toxic agents that induce an abnormal cellular response
Evidence for some s p o n t a n e o u s chromosome r e a r r a n g e m e n t in cells from unaffected skin. Induced c h r o m o s o m e aberrations include chromatid deletions and dicentrics, and sisterchromatid exchanges.
Variable excess of s p o n t a n e o u s and induced c h r o m o s o m e aberrations especially gaps and breaks, micronuclei, sisterchromatid exchanges, G 2 chromatid aberrations.
Excess s p o n t a n e o u s and BrdU-induced sisterchromatid exchanges. Also c h r o m o s o m e breaks and aberrations, especially quadriradials.
Excess spontaneous and induced chromosome breaks and r e a r r a n g e m e n t s in G o and G 1. N o n - r a n d o m c h r o m o s o m e 7 and 14 translocations. Induced G 2 aberrations.
Chromosome aberrations
"~ cell killing by UV.
"r cell killing by toxic agents.
Not studied.
1' cell killing by toxic agents,
Cell survival
Not studied.
G 2 arrest.
Prolonged G o . G 2 arrest.
Induced and s p o n t a n e o u s cell cycle anomalies: G 2 arrest, radioresistant D N A synthesis.
Cell cycle anomalies
? frequency of somatic cell mutations, e
? levels spontaneous recombination and somatic mutation. Infrequent spontaneous mutation. $ transformation by SV40.
Hypothesized.
Hypothesized.
Hypothesized.
Defective $ mutation nucleotide induced by UV. excision (groups A - H ) or post-replication (XP variant) repair of induced D N A damage.
Mutation
D N A repair anomalies
References: a Gatti et al. (1991); b Nicotera (1991); G e r m a n and Passarge (1989); c C o h e n and Levy (1989); d K r a e m e r and Slor (1984); C o h e n and Levy (1989); e Bigbee et aL (1989).
Complementation groups
Syndrome
MAJOR LABORATORY PHENOTYPES ASSOCIATED WITH HOMOZYGOTES
TABLE 2
30
For each of the syndromes, the biological mechanisms responsible for their multiple phenotypes are consistent with DNA repair a n d / o r DNA processing defects. XP homozygous cells are defective in various steps of the excision repair pathway for the repair of induced DNA damage (Cleaver, 1990). Two genes associated with XP have been cloned and are thought to be DNA helicases (Tanaka et al., 1990; Weeda et al., 1990). In BS, a defective ligase activity may well be the biological basis of the phenotype (Barnes et al., 1991; Nicotera, 1991), but specific enzymatic errors have not yet been shown to be responsible for the A-T or FA phenotypes. The molecular basis for clinical or cellular manifestations in heterozygotes is, therefore, not known.
Ataxia telangiectasia There is good evidence that heterozygous carriers of the A-T gene are predisposed to cancer (Swift et al., 1976, 1987, 1991; Morrell et al., 1990; Pippard et al., 1989; Borresen et al., 1990). Female breast cancer with onset before 65 years of age is clearly associated with A-T heterozygosity. Diagnostic X-ray exposure may be a risk factor (Swift et al., 1987, 1991). Other cancers possibly associated with A-T heterozygosity are those of the pancreas, stomach, bladder, ovary, lung, prostate, and oral cavity, and chronic lymphocytic leukemia, malignant melanoma, and lymphoma. If A-T heterozygotes constitute 1.4% of the United States population and have a 6.8fold breast cancer risk (Swift et al., 1987), then 9% of all breast cancer cases in the United States could be attributed to A-T heterozygosity. Female A-T heterozygotes may also be at increased risk for late-onset diabetes mellitus. These carriers are three times more likely than non-carriers to develop a late-onset diabetes mellitus clinically similar to the mild diabetes seen in the homozygotes (Morrell et al., 1992). Each possible association between A-T heterozygosity and specific cancers or diabetes could be tested using the index-test method described below. Ceils from A-T homozygotes show reduced survival after X-irradiation or treatment with spe-
cific cytotoxic compounds under a wide variety of experimental conditions (Taylor et al., 1975; Shiloh et al., 1983). In contrast, ceils from obligate A-T heterozygotes have shown differential survival only under specific exposure conditions (Chen et al., 1978; Paterson et al., 1985; Shiloh et al., 1982; Cole et al., 1988; Weeks et al., 1991). However, survival of cultured fibroblasts after y-irradiation does not reliably discriminate indit~idual A-T heterozygotes from non-carriers (Paterson et al., 1985; Weeks et al., 1991). ~Similarly, various attempts to reliably identify individual A-T heterozygotes by diverse assays have not been successful (Rosin et al., 1989; Rudolph et al., 1989; Pawlak et al., 1990). Numerous attempts to find a clastogenic response to X-irradiation or radiomimetic drugs in A-T heterozygous cells have identified differences between cells in some studies but not others (Natarajan et al., 1982; Nagasawa et al., 1985; Bender et al., 1985; Parshad et al., 1985; Waghray et al., 1990; Sanford et al., 1990; Wiencke et al., 1992). Methodological differences or genetic heterogeneity may account for the variation between studies, but it is clear that individual A-T heterozygous cells are heterogeneous in their cytogenetic response to X-rays. Wiencke et al. (1992) concluded from a large sample that the increase in chromatid aberrations seen in many A-T heterozygous cells after X-irradiation is not a reliable method of discriminating A-T heterozygotes from non-carriers.
Fanconi anemia Petridou and Barrett (1990) found several clinical differences between 16 obligate FA heterozygotes and 40 unaffected controls. In heterozygotes fetal hemoglobin was significantly increased, the number of natural killer cells was significantly decreased, and mitogenic responses to phytohemagglutinin and interleukin-2 were reduced. Three heterozygotes had neutropenia and there were also significant differences in skeletal proportions. These results support those of Welsheimer and Swift (1982), who found that heterozygotes are predisposed to the genitouri-
31 nary and distal limb malformations frequently observed in FA homozygotes. Female FA heterozygotes are predisposed to diabetes mellitus, being approximately six times more likely to develop the disease than non-carriers (Swift et al., 1972; Morrell et ai., 1986). No systematic surveys have been reported, but there is certainly an excess of diabetes in FA homozygotes (Woodard et al., 1981). FA was the cancer-prone autosomal recessive syndrome that generated the idea that heterozygous carriers may also be predisposed to cancer (Swift, 1971). A study of 25 families suggested an association between bladder, stomach and breast cancer and FA heterozygosity, although there was no overall excess of cancer among the blood relatives (Swift et al., 1980). This study had a modest sample size compared, for example, to the more recent A-T family studies (Swift et al., 1987; 1991). Possible associations between specific cancers and diabetes and FA heterozygosity could be tested using the index-test method described below. Elevated spontaneous chromosome breaks and rearrangements are seen in almost all FA patients (Auerbach et al., 1989); the diagnosis can be made unequivocally by combining clinical data with cytogenetic evaluation of the frequency of aberrations induced by D N A cross-linking agents such as diepoxybutane or mitomycin C (Auerbach and Wolman, 1976; Auerbach et al., 1989). Rosendorff and Bernstein (1988) found that the mean chromosome aberration frequency for FA heterozygotes was significantly elevated above controls when induced by diepoxybutane but not by mitomycin C. The degree of overlap between the groups, however, precludes the use of this assay as a reliable diagnostic test for FA heterozygosity. This conclusion is supported by earlier, less definitive studies (Auerbach and Wolman, 1978; Berger et al., 1980; Cervenka and Hirsch, 1981; Auerbach et al., 1981; Cohen et al., 1982). Miglierina et al. (1991) found that there is a cell cycle disturbance following exposure to nitrogen mustard in FA heterozygous cells that can be detected by flow cytometry. The value of this finding in testing for FA heterozygotes is not yet known. Similarly, Petridou and Barrett (1991)
found significant differences between FA heterozygotes and controls that may well form the basis for a test for FA heterozygotes.
Xeroderma pigmentosum XP is not a single genetic disorder: a total of nine complementation groups and a variant have been identified (Cleaver, 1990). The hypothesis that XP heterozygotes are predisposed to nonmelanoma skin cancer was supported by finding significantly more of these cancers in blood relatives of XP patients than in their spouse controls, in 31 North American XP families of unknown complementation group (Swift and Chase, 1979). The excess cancers appeared to be concentrated in four XP families living in the Southern United States. Other cancers apparently in excess in the 31 families were those of the lung, stomach, and prostate. Predisposition to cancer was also studied in an unspecified number of British XP families representing 48 patients. Only two skin cancers were found among the parents and grandparents of the probands (Pippard et al., 1988). There were, however, three stomach and two lung cancers in the grandparents, and a single prostate cancer in a father. Microcephaly and progressive mental retardation are seen in XP homozygotes, particularly those of complementation groups A and D. In 31 XP families four cases of microcephaly and 11 of mental retardation were observed, concentrated in the families known to belong to groups A and D (Welshimer and Swift, 1982). This excess was significant and suggests that some XP mutations predispose the heterozygote to mental retardation and microcephaly. It will be possible to test the association of XP heterozygosity with specific cancers and with neurological dysfunction using the index-test method, described below. A limited number of investigations have suggested that spontaneous chromosome aberrations are present in XP homozygous cells (Kraemer and Slor, 1984; Aledo et al., 1989). Certainly, an increase in aberrations is seen after such cells are exposed to UV and certain cytotoxic agents (reviewed in Kraemer and Slor, 1984). In UV-irradiated XP heterozygous cells, Bielfield et al. (1989)
32 found an increase in sister-chromatid exchanges and micronuclei; with combined data from these two endpoints, they were able to distinguish individual XP heterozygous and control cells approximately 90% of the time. The study was exceptional because 19 parents and sibs of seven XP heterozygotes of known complementation groups were compared to 24 controls. An increase in spontaneous chromosome aberrations has been observed in two obligate XP heterozygotes (Casati et al., 1990). Quantitative differences between DNA repair in XP heterozygous and wild-type cells have been observed with several different assays (Giannelli and Pawsey, 1974; Squires and Johnson, 1988) but not in others (Kleijer et al., 1973; Day, 1974; Selsky and Greer, 1978). Each of these assays was technically arduous, and only between two and ten XP heterozygous or control cell lines were used in any single study. Differences in complementation group may account for some of the variation between reported studies of cellular phenotypes in XP heterozygous cells, but no assay for an XP heterozygous phenotype has yet been shown to reliably detect XP heterozygotes in families or in populations. Perhaps a DNA repair assay directed at a single genetic form of XP would be sensitive and specific for that mutant allele in heterozygotes.
Bloom syndrome There are anecdotal reports of cancers in obligate BS heterozygotes (German, 1974), but no systematic studies of clinical findings in BS heterozygotes have been reported. A BS Registry has collected data for all reported cases since the early 1960s (German and Passarge, 1989). The predisposition of the BS heterozygote to cancer or diabetes could be assessed, at modest cost and effort, by methodical follow-up of the families identified through this Registry. It is regrettable that this has not yet been done, since 1/125 Ashkenazi Jews are BS heterozygotes. BS heterozygous cells have been studied very little, perhaps because nothing is known about the disease predisposition of BS heterozygotes. Limited studies have shown no evidence for a significant excess of chromosome aberrations or
sister-chromatid exchanges in BS heterozygous cells (Chaganti et al., 1974; Bartram et al., 1976; Sperling et al., 1976; Hustinx et al., 1977; Kuhn and Therman, 1979), although these are typical findings in BS homozygous cells (Nicotera, 1991). It would be logical and cost-effective to assess the clinical effects of BS heterozygosity from the BS Registry data before attempting to develop a reliable test for the BS heterozygote.
Overview of heterozygote tests There is no question that groups of A-T, FA, or XP heterozygous cells can be distinguished from controls under several, but not all, experimental conditions. These differences may reflect abnormalities that provide the cellular basis for observed clinical differences between carriers of an A-T, FA, or XP gene and non-carriers. An accurate test for identifying heterozygotes in the general population is of obvious clinical value. For example, there is very good evidence for an association between A-T heterozygosity and cancer, especially female breast cancer. Cancer incidence in the 1% of the population who are A-T heterozygotes could be minimized by reducing exposure to all sources of ionizing radiation, including diagnostic X-rays. These heterozygotes should be identified and observed for the earliest signs of cancer, since early treatment is associated with improved survival. Eventually, it may be possible to protect A-T heterozygotes from developing cancers by rational therapy deduced from an understanding of the metabolic action of the A-T gene. To be useful, tests for identifying A-T, FA, BS, or XP heterozygotes in the general population must be fully sensitive and specific. No assay yet developed is reliable enough for heterozygote identification for clinical or research purposes. Proposed heterozygote tests have been technically demanding, have had varying outcomes depending on the laboratory performing the technique, or were not tested blindly on an adequate number of obligate heterozygotes and controls. Reliable heterozygote tests could be based on DNA analysis once the gene locus (or loci) for each syndrome is identified and all the mutants characterized. At present, the genes for A-T, FA,
33 and BS are not known, and only the genes for XP complementation groups A and B have been cloned (Tanaka et al., 1990; Weeda et al., 1990). Substantial progress has been made in mapping the A-T locus on chromosome 11q22-23 (Gatti et al., 1991). Preliminary linkage data have assigned an FA gene in a subset of FA families to chromosome 20q (Mann et al., 1991). Proposed associations between being heterozygous for an A-T, BS, FA, or XP mutant allele and a common disease must be confirmed before attempting to identify heterozygotes for clinical purposes. Each hypothesized association can be tested most efficiently and reliably by the indextest method, which relies on precise identification of heterozygotes within the families of homozygous probands. In these families, heterozygotes can be identified even before the gene is cloned if the mutant allele can be traced by polymorphic D N A markers tightly linked to the gene locus.
Establishing heterozygote risk by the 'index-test method' The index-test method is a recently described genetic method for establishing an association between any candidate allele and any common chronic disease such as cancer or diabetes mellitus (Swift et al., 1990). Its statistical power is good, so that the sample size required is practical, and it is not affected by variations in population gene frequencies, by ascertainment bias, or by genetic heterogeneity of the common disease. For example: the index-test method could be used to test the possible associations of nonmelanoma skin, or gastric cancer with heterozygosity for XP group A mutant alleles. Group A is the predominant type of XP in Japan (Takebe et al., 1987). The following is an outline of such an application. (1) Identify the 'index cases' - those Japanese XP group A patients who carry mutations in the group A gene. They are called index cases because they identify (i.e., index) the families in which the alleles of interest are segregating. (2) Find as many families as possible in which these alleles are segregating. Depending on how great a risk the heterozygote has for a specific
cancer, 20 families might provide adequate statistical power (see Table 2 in Swift et al., 1990). (3) Identify as many 'test individuals' as possible in each family. To test the association with skin cancer, the test individuals would be blood relatives of the index case who have skin cancer (to test the association with gastric cancer a different group of test individuals is required: blood relatives with gastric cancer). In each group of test individuals exclude homozygotes, obligate heterozygotes, and any individual whose status as an XP heterozygote depends on the status of another test individual. (4) Obtain DNA from all test individuals using bloodl saliva, tissue, or paraffin-embedded tissue blocks. Use DNA analysis to determine which test individuals are heterozygous for the specific XP mutation segregating in their family. (5) Count the total number of XP heterozygotes in the selected group of test individuals. This is the 'observed number' of XP heterozygotes in this group. (6) Determine the 'expected number' of XP heterozygotes in the group of test individuals. Table 1 in Swift et al. (1990) gives the probability that a blood relative of the index case is heterozygous, based on the allele frequency and the blood relative's relationship to the index case. (7) Compare the observed number of XP heterozygotes to the expected number. If the observed number is greater than expected, test the significance of the difference. Estimate the relative risk of the cancer in question for XP group A heterozygotes from the odds ratio. (8) A significant excess of XP group A heterozygotes among the test individuals with a cancer is strong evidence that the XP group A allele predisposes heterozygotes to that cancer.
Conclusion There is cumulative evidence that the heterozygote phenotypes for the autosomal recessive syndromes A-T, XP and FA are distinct from wild-type at both the cellular and clinical levels. Little is known about BS heterozygotes, but perhaps this review will encourage new investigations. If current efforts to clone the respective disease genes are successful, analyzing each gene's
34 m o l e c u l a r e f f e c t s will l e a d t o a n u n d e r s t a n d i n g the complex phenotypes
of
and to measures for im-
proving the health of the general population.
Note added in proof c D N A s t h a t c o r r e c t t h e d e f e c t in F A c e l l s designated complementation group C have rec e n t l y b e e n c l o n e d ( S t r a t h d e e e t al., 1992, N a t u r e , 356, 7 6 3 - 7 6 7 ) .
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