Affiliations of authors:J. Cloos, E. J .C. Nieuwenhuis,M. L .T. van der Sterre, G. B. Snow, B. J. M. Braakhuis, Department of Otolaryngology/Head and Neck Surgery, University Hospital Vrije Universiteit, Amsterdam, The Netherlands; D. I. Boomsma (Department of Biological Psychology), D. J. Kuik (Department of Epidemiology and Biostatistics), F. Arwert (Department of Human Genetics), University Vrije Universiteit, Amsterdam, The Netherlands.
Correspondence to: Boudewijn J. M. Braakhuis, Ph.D., Department of Otolaryngology/Head and Neck Surgery, University Hospital Vrije Universiteit, P. O. Box 7057, 1007 MB, Amsterdam, The Netherlands (e-mail: BJM.Braakhuis{at}AZVU.nl).
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ABSTRACT |
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INTRODUCTION |
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A functional approach to determine individual susceptibility to carcinogenic assaults is to screen for chromatid breaks after in vitro G2-phase bleomycin treatment of cultured peripheral blood lymphocytes. For environmentally related cancers, such as colon cancer, lung cancer, and head and neck squamous cell carcinoma (HNSCC), the biologic relevance of this marker has been well established (8,9). We and others (8-10) showed an increase in the mean level of chromatid breaks per cell in cancer patients compared with healthy control persons. This high level was especially found in those patients with multiple primary tumors (10). The mean number of breaks per cell score was not influenced by smoking or alcohol use by the subjects. It was shown in a meta-analysis that a high susceptibility (defined as 1.0 or more breaks per cell) itself slightly increased cancer risk but did not reach statistical significance. Of interest, however, in combination with exposure to carcinogens, a large increase (up to an odds ratio of 57.5) of risk for HNSCC was found (11).
For persons at high risk of cancer (particularly, members of families with a high frequency of common cancers and HNSCC patients who have been successfully treated for their primary tumor and are at risk for a second tumor), it is important to ascertain whether this susceptibility phenotype has a genetic basis. Such knowledge will increase the value of this susceptibility marker and encourage further studies of the (genetic) mechanisms underlying this susceptibility. We investigated the heritability of the susceptibility to chromatid breaks in pedigrees from HNSCC patients who have been successfully treated and have no evidence of cancer recurrence for at least 1 year. Because familial resemblance can be due to shared environment as well as to shared genes, the number of chromatid breaks was also assessed in 25 pairs of monozygotic twins (unrelated to the HNSCC patients), who are genetically identical. Any larger resemblance for monozygotic twins compared with the first-degree relatives (who share, on average, 50% of their genes) could suggest the importance of genetic factors. In this study, we employed the powerful tool of pedigree analysis to estimate the heritability of mutagen sensitivity.
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METHODS |
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All subjects (n = 135) whose data were used for the estimation of the heritability of the susceptibility marker were healthy volunteers from 53 different pedigrees and consisted of dizygotes (n = 85; stratified in three groups on the basis of their relation to the 14 HNSCC patients who were enrolled in this study; I = siblings of the HNSCC patients [n = 32]; II = offsprings of the HNSCC patients [n = 25]; III = dizygotes, volunteers unrelated to HNSCC patients [n = 28]), and monozygotes, volunteers unrelated to HNSCC patients (group IV: 25 pairs of identical twins, n = 50). Heparinized blood samples were collected from the first-degree relatives (groups I and II) of 14 HNSCC patients previously treated at our department and had no more evidence of any residual or recurrence disease for at least 1 year (n = 57). Heparinized blood samples were also obtained from volunteers not related to HNSCC patients (group III, consisting of 14 dizygotic pairs: eight dizygotic twins and six sibling pairs with only a small age difference [mean age difference ± standard deviation = 3.5 years ± 1.4 years] among the members of the group III).
All 14 probands (individuals through whom family pedigrees were ascertained) had been
successfully treated for HNSCC at our department. Patients were selected on the basis of their
having several first-degree relatives. When the patients were referred to our hospital for
follow-up, they were asked permission to contact their first-degree relatives to participate in this
study. Patient and tumor characteristics and the details of how many relatives of each patient
volunteered are summarized in Table 1. For staging of the HNSCC, the
criteria of the International Union Against Cancer (12) were used. In line
with our earlier studies (10,11), eight (57%) of 14 patients were
determined not to be sensitive to bleomycin-induced chromatid breaks (breaks per cell <1.0).
Blood was drawn from all of the participants after they signed an informed consent form. For all
twin pairs, the names and addresses were obtained from the National Netherlands Twin Registry
(13). Zygosity of the twins was assessed by genotype analysis of six
unlinked microsatellite loci (with heterozygosities >90%) in two different multiplex
polymerase chain reactions (14). Zygosity analysis was performed at the
TNO Prevention and Health, Gaubius Laboratory, Division of Vascular and Connective Tissue
Research, Leiden, The Netherlands. The geographic distribution of both the twins and the family
members of patients was throughout the whole of The Netherlands. All subjects had given
written informed consent and the study design was approved by the local ethical committee.
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Chromatid Breakage Assay
Duplicate cultures were set up for each subject. Whole blood (0.5 mL) was diluted 10 times in RPMI-1640 medium (BioWhittaker, Inc., Walkersville, MD) with 2 mM L-glutamine (Life Technologies, Paisley, U.K.) supplemented with 15% fetal calf serum (Hyclone Laboratories, Inc., Logan, UT), 1.5% phytohemagglutinin (Life Technologies), 100 U/mL penicillin, and 100 µg/mL streptomycin (BioWhittaker, Inc.). After the cells were cultured for 3 days at 37 °C and 5% CO2, they were incubated for 5 hours with bleomycin (30 mU/mL) (Lundbeck, Amsterdam, The Netherlands). To arrest the cells at metaphase, 0.04 µg/mL Colcemid (Sigma Chemical Co., St. Louis, MO) was added to the cultures 1 hour before harvesting. This yields cells in metaphase that were damaged by the bleomycin in the late S-G2 phase of the cell cycle. The cells were swollen in hypotonic solution (0.06 M KCl) and fixed in Carnoy's fixative (3 : 1 [vol/vol] methanol : acetic acid). After the cells were dropped on wet slides, the metaphase spreads were air-dried and stained with Giemsa (Merck, Darmstadt, Germany). Before 50 metaphase spreads were scored on each slide for the presence of chromatid breaks, the slides (two slides per person) were coded to ensure objective "blinded" screening. The mean number of breaks per cell of 100 metaphases was used as a measure for the individual susceptibility. As has been published previously (15), the scoring of gaps did not influence the outcome of the assay and was omitted in further investigations. Since DNA damage was introduced in late S-G2 phase of the cell cycle, chromosome aberrations such as translocations were not present in the metaphases. Background levels (spontaneous) of chromatid breaks without damage induction by bleomycin that have been determined in previous studies were very low (breaks per cell values of approximately 0.06) and did not differ between patients and control subjects. Therefore, data representing spontaneous breaks were not included.
Descriptive Statistics
Differences between groups with respect to mutagen sensitivity were assessed by use of Student's t test. The influences of age and sex on mutagen sensitivity were determined by use of regression and likelihood methods. Intraclass correlations were calculated by use of analysis of variance.
Heritability Estimation
A pedigree-based maximum likelihood method developed by Lange et al. (16) was used to analyze resemblances among family members for chromatid breaks
(used as a continuous phenotype). Data from twins and other family members were analyzed
simultaneously as so that the pedigree data consisted of a total of 53 pedigrees (twins and
dizygote pairs were also put into the file as pedigrees without a proband), a sample size of 135
subjects (including 50 monozygotic twins), and 14 probands (whose data were evaluated
separately and used only for ascertainment correction by conditioning on probands). This
ascertainment correction was needed because the pedigrees of the cancer patients were not a
random selection of the general population (16). Different models of
familial resemblance were fitted to the data. These models specified the variation in phenotype to
be due to the genotype and/or the environment. Sources of variation considered were additive:
genetic influences (i.e., the sum of the effects of the individual alleles across all loci that
contribute to variation), common environmental influences shared by family members who are
living or have lived in the same household, and a random environmental deviation that is not
shared by family members. These sources of variation can be considered as unobserved, or latent
factors, which affect the (continuous) phenotype, and can be estimated from the observed
patterns of resemblance between relatives. For each pedigree of n individuals, a vector
of observations (x) is defined, and a vector of expected values (E[x]) can be calculated. E(x) can depend on fixed variables, such as sex
or age. The covariances among family members for that part of the dependent variable that is not
accounted for by the fixed variables depend on the relationships between the pedigree members
and on the genetic model assumed for dependent variables. We have modeled the variance not
accounted for by the fixed effects as due to additive genetic influences, shared family
environment, and random environmental factors. For a given E(x) and
expected covariances matrix , the ln-likelihood of obtaining the observation vector x is:
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where | | denotes the determinant of the matrix and ' denotes transpose.
The joint likelihood of obtaining all pedigrees is the sum of the likelihood of the separate
pedigrees. Estimation involves selection of parameter values under a specific model that
maximizes the joint likelihood of all pedigrees. The FISHER package of pedigree analysis (16) was used for genetic modeling. The likelihoods obtained for the
different models were compared with chi-squared difference tests where 2
= 2(L1 - L0). L1
and L0 denote the ln-likelihoods of the general (H1)
hypothesis and a constrained (H0) hypothesis. The degrees of freedom (df) for this test are equal to the number of constrained parameters between H1 and H0 (17). The following parameters were
estimated in the general model: a sex effect and an age regression on means and three variance
components (genetic, shared, and unique environmental variances). First, it was tested if additive
genetic influences and common environmental influences could be constrained at zero
(hypothesis of no familial resemblance in chromatid breaks) and next if either additive genetic
influences or common environmental influences could be set to zero. The fixed effects on the
means included a sex effect and an age regression. The variance components part of the analysis
applies to the covariances of family members (while simultaneously modeling the age and sex
effect).
The conditional likelihood approach as implemented in the FISHER package of pedigree analysis was used to correct for ascertainment. The heritability estimate was calculated as the contribution of the genetic variance in the total variance (genetic and environmental).
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RESULTS |
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Plot of the data of the mean number of bleomycin-induced chromatid
breaks per cell of all of the 135 healthy study subjects without cancer
indicated a good approximation of the normal distribution (Fig.
1). The effect of sex on the mean number of breaks
per cell was not statistically significant (
2 = 0.70
with 1 df; P = .40). The regression of age was
positive (slope: b = 0.007) and reached significance
(
2 = 4.84 with 1 df; P = .027).
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The actual heritability estimate was determined by use of a
pedigree-based maximum likelihood method in which the (continuous)
breaks per cell data from twins and other family members were analyzed
simultaneously. The model that was fitted to the data in the FISHER
package of pedigree analysis specified sex differences and age
regression on the mean phenotype and considered genetic, unique, and
shared environmental influences on variances and covariances between
family members. The largest likelihood should be for the full model. As
shown in Table 4, there was no evidence for the
influence of a shared family environment (
2 = 1.08 with
1 df; P = .29). The reduced model with the fixed shared
environment does not fit the data significantly worse than the full
model. Our interpretation is that shared environment does not
contribute to familial resemblances. Genetic influences were
statistically significantly shown in the reduced model with fixed
genetic variances that fits the data significantly worse
(
2 = 4.26 with 1 df; P = .036). Particularly
for the reduced model that excludes any familial resemblance (genetic
and shared environment), the likelihood is statistically significantly
reduced (
2 = 25.46 with 1 df; P<.001). The
heritability estimate of 75% was calculated (from the most optimal
reduced model that excludes the influence of a shared environment) as
the genetic variance (0.04775) divided by the total variance (0.04775 + 0.01613 =
0.06388).
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DISCUSSION |
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It is tempting to speculate that persons with a high susceptibility phenotype in our study may,
in fact, be heterozygotes of the chromosomal instability syndromes. However, this hypothesis is
not supported by what is known about obligate heterozygotes (e.g., parents of AT homozygotic
patients). AT heterozygotes, for instance, are estimated to represent 0.5%-2.8% of
the general population (25), while about 16% of the control
persons are hypersensitive to bleomycin-induced chromatid breaks (defined as breaks per cell
1.0) (15). Moreover, although obligate AT heterozygotes do have a
higher susceptibility to radiation-induced chromatid breaks compared with control persons, they
have increased risk mainly for breast cancer (25) and not for HNSCC.
This may indicate that the levels of bleomycin-induced chromatid breaks that we report are
related to other genes predisposing to HNSCC, although it cannot be excluded that genes of other
chromosomal instability syndromes are involved.
The relationship between a high susceptibility for chromatid breaks and the development of environmentally related cancer has been established in retrospective (8,10,11) and a limited number of prospective (26,27) studies. The major conclusion from these former studies is that the intrinsic susceptibility and exposure to carcinogens act in concert to modulate cancer risk. The great refinement of cancer risk assessment by use of the susceptibility to bleomycin-induced chromatid breaks indicates the importance of this biomarker. The fact that this factor has a high heritability estimate underscores the relevance of genetic factors for cancer development. This will probably be important not only for HNSCC but also for all cancers in tissues that are in direct contact with the environment, such as the colon and the lung. The fact that a similar genetic factor does play a role in the development of various types of cancers may explain cancer proneness in families in which several types of cancers occur. It has previously been noted that familial clustering of environmentally related cancers does exist (28,29). It will be very interesting to screen the DNA of these families to link a high susceptibility phenotype to mutations (or polymorphisms) in the known susceptibility genes, such as p53 (also known as TP53), BRCA1, and BRCA2 (30). The high heritability estimate that we describe in this study for susceptibility to bleomycin-induced chromatid breaks may also facilitate the discovery of new-cancer predisposing genes. The phenotype, mean chromatid breaks per cell, can be a valuable determinant because it may be indicative of the cancer-prone phenotype and can be used as an end point in these studies. It has the advantage that it can be assessed before the cancer has occurred. This is very important because the study of cancer-prone families is hampered by the fact that the affected individuals often have already died.
When a common genetic defect is traced that is prevalent among a relatively large part of the
population (e.g., breaks per cell level 1.0), it may account for cancer predisposition in a
large proportion of the cancer cases in general compared with the fraction that is due to inherited
cancer syndromes (probably <5%) (31). It is, therefore,
important to recognize that the susceptibility to DNA damage varies in different individuals.
Avoidance of exposure to (environmental and occupational) carcinogens, especially in sensitive
persons, may then become an important factor in the prevention of cancer (32). The heritability estimate of 75% challenges us to focus further research into
finding the gene(s) involved in the susceptibility to bleomycin-induced chromatid breaks.
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NOTES |
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We thank Dr. P. Slagboom, TNO Prevention and Health, Gaubius Laboratory of Vascular and Connective Tissue Research, Leiden, The Netherlands, for the monozygosity test of the DNA samples of twins. We also thank Professor Dr. J. P. Vandenbroucke, Department of Clinical Epidemiology, University Hospital, Leiden, for his critical review of our manuscript.
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Manuscript received August 3, 1998; revised April 20, 1999; accepted May 10, 1999.
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