Japanese Journal of Clinical Oncology 31:185-187 (2001)
© 2001 Foundation for Promotion of Cancer Research
Somatic Mutation Rate of the APC Gene
Department of Surgery, Kyoundo Hospital, Sasaki Institute, Tokyo, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Background: Somatic inactivation of the wild-type APC gene is involved in the development of adenoma of familial adenomatous polyposis. This situation is also true in sporadic adenomas. It is of biological interest to know the somatic mutation rate of the APC gene.
Methods: The number of stem cells of the colon (N) and somatic mutation rate of the APC gene in a stem cell in a year (m) can induce age-specific incidence of adenomas. The number of stem cells was estimated as 108 according to previous reports. In the general population, expected adenomas at the end of age n years will be ~Nm2n2/4. In patients with polyposis, the expected number of adenomas will be Nmn/2. By setting several figures for m, the expected incidence of adenomas was compared with the actual occurrences.
Results: If the mutation rate was set between 2/106 and 3/106 mutations/stem cell/year, the calculated numbers were well fitted to the actual data. Expected adenomas in polyposis patients at the age of 20 and 40 years were 2000 and 4000 and these were within actual experiences.
Conclusions: This is the first study to estimate the somatic mutation rate of the APC gene. The estimated somatic mutation rate of the APC gene was between 2/106 and 3/106 mutations/stem cell/year.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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A germline mutation of the APC gene causes familial adenomatous polyposis (FAP). Although the chance of APC gene germline mutations per gamete per generation was estimated as 5/106 (1), the somatic mutation rate of the APC gene has not been estimated. Recent studies (2–4) proved that inactivation of the wild-type APC gene is involved in the development of adenomas of FAP. This complete loss of function on both alleles of the APC gene is also true in sporadic adenomas (2,3). In the general population, the incidence of adenomas in autopsy is around 30% and their incidence and multiplicity increase with increasing age (5,6). In sporadic adenoma cases, the number of adenomas rarely exceeds 30 (5–7). On the other hand, FAP patients usually have hundreds to thousands of adenomas in the colon (7). Based on this knowledge, if we can estimate the stem cells in the colon, we may be able to estimate the somatic mutation rate of the APC gene.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Because mutations in both alleles of the APC gene produce adenoma, if we set two parameters, the number of stem cells of the colon and rectum (N) and the chance of mutation of the APC gene in a stem cell in a year (m), we can calculate age-specific expected number of adenomas in both FAP patients and the general population. Information on the frequency of stem cell divisions in a year is not necessary because the number of stem cells is roughly constant. In a non-FAP person, Nm stem cells will have mutations on one of the two alleles of the APC gene every year. These mutated stem cells would not yet develop adenomas in the general population because the other allele compensates the function of the APC gene. One of these cells must be mutated on the other normal APC gene to develop an adenoma. Consider a stem cell that had an APC gene mutation at the end of the first year (during the period 0 years). In this cell, the chance of the other APC gene being mutated up to the end of n years (interval n – 1 years) will be m(n – 1)/2 because a hit on the damaged allele means nothing and the chance of a hit on the normal allele is half of m(n – 1). Every stem cell that had one APC gene mutation during a period of 1 year would have m(n – 2)/2 mutations on the wild-type APC gene up to the end of n years. Consecutively, at the end of n years, the cumulative stem cells with mutations on both APC genes will be (Nm)(m
In FAP patients, all stem cells have already had one APC gene mutation from birth. Then the chance of mutation on the wild-type APC gene is Nm/2 in every year. Consequently, an FAP patient will have Nmn/2 adenomas at the age of n years.
The number of stem cells was estimated by the method of Mizuno et al. (8) as 108. This figure was based on the data that the total colorectal mucosal surface is around 1000 cm2 (7), 1 cm2 contains 6000–10 000 crypts (9,10) and one crypt contains 10–20 stem cells (9). We estimated age-specific polyp number by changing the mutation rate m from 2/106 to 3/106 mutations/stem cell/year and compared them with the actual polyp numbers that were prospectively counted (5,6,11). We selected studies that were based on autopsy, meticulous endoscopic examinations or almost totally excised specimens and they were prospectively counted without skipping very tiny polyps.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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For the general population, age-specific polyp numbers were estimated by setting several APC gene mutation rates and they were compared with the actual data from Williams et al. (5) and Mitooka et al. (6). If the mutation rate is set as 2/106 mutations/stem cell/year, the adenoma incidences in the general population would be expected to be 0.25, 0.36 and 0.49 at the age of 50, 60 and 70 years, respectively. If it is taken as 2.5/106 mutations/stem cell/year, the incidences will be 0.39, 0.56 and 0.77 at the indicated ages. The actual polyp incidences by Williams et al. (5) were located between these expected numbers at the indicated ages as shown in Table 1. These results mean that if we examine 10 50-year-old persons, we can expect 2.5–3.9 adenomas detected in their colon and rectum. The estimated mutation rate of m = 3/106 was well fitted for the older generation of the data from Mitooka et al. and that of m = 2.5/106 was fitted for the younger group (Table 2).
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Estimated FAP polyp numbers at a mutation rate of 2/106 mutations/stem cell/year were 2000 and 4000 at the ages of 20 and 40 years, respectively. These data were not far from our experiences and others (7,11,12).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Several hypotheses about colorectal cancer development have been proposed and their mutation rates were estimated (8,13). The estimations were equally based on an assumption that colorectal cancer is originated by several hits on different unknown genes, but a hit on an oncogene is essentially different from a hit on a tumor-suppressing gene or a mismatch repair gene. Fortunately, the genetic change of an adenoma is two steps on one gene (2–4). Consequently, the somatic mutation rate of the APC gene can be precisely estimated if proper epidemiological data on adenomas are available. We took the data from Williams et al. (5) and Mitooka et al. (6) for the general population, because the former was prospectively investigated at autopsy without omitting even tiny lesions and the latter represented Japanese data meticulously examined by videoendoscopy. This group had no colorectal symptoms at examination. In their data, hyperplastic polyps accounted for 68% and 62%, respectively. Hyperplastic polyps are usually very tiny and they have little clinical meaning for treatment. Practitioners are apt to omit or miss these small polyps. Consequently, a high proportion of a hyperplastic polyp counting itself means a reliable adenoma counting. The data fitted well to our estimated age-specific adenoma incidences when the somatic mutation rate of the APC gene was set as 2/106–3/106 mutations/stem cell/year. If we are to be strictly scientific, mutation rate is usually expressed as mutations per stem cell division. However, our interest in this study was to estimate how many APC somatic mutations would occur in a year to produce adenoma. In this method, we needed to estimate the number of the stem cells, but did not need to estimate stem cell divisions in a year. In B-cell cultures, the observed spontaneous mutation on the hprt locus ranges from 0.5 to 2.5/107 mutations per stem cell division (13). If the colonic stem cell divisions occur 20–30 times in a year, our data fit to this value, 1/107. When this result is applied to FAP there will be some difficulties because of polyp counting. Polyp counting in FAP is difficult because polyps <0.5 cm comprise 89–100% (7,11) and polyp number depends on how small polyps are included. The number of adenomas reported by Bussey (7) varied from 104 to over 5000. In author’s experiences, the smallest number was 271 and cases with profuse polyps exceeded 10 000 including tiny elevated lesions (11). Nagase et al. (12) divided FAP polyps into sparse and profuse types and the profuse type contained >5000 polyps. In our estimation, polyps of FAP were within these figures when the mutation rate was set as 2/106 mutations/stem cell/year. The estimated polyp number doubled in the 20 year period from 20 to 40 years of age. These estimated figures were within the actual polyp counting; however, the polyp number of FAP did not directly relate to age. Polyp densities were variable among FAP patients with the same germline mutation (14), even if their data were based on photographs that are less accurate than the direct counting. Polyp number does not seem directly related to age within families (11,14). These facts suggest that the somatic mutation rate of the APC gene may be influenced not only by the location of germline mutations, but also by gender, intra-colonic bacterial circumstances, other chemical circumstances such as bile acids and other genetic factors. Non-steroidal anti-inflammatory drugs may be one of these substances, although they are artificial materials (15). In this study we considered that the somatic mutation rate of the APC gene is equal between the first and second mutations, but an allelic mutation rate in heterozygote might be higher (or lower) than that in homozygote. Herrero-Jimenetz et al. (13) estimated the initiation–mutation rate for colorectal cancer as 7/108 mutations per stem cell division and promotion–mutation as 2/107 for females and 8/108 for males. We simplified our method and did not consider the difference between the first and second mutations. There are two other epigenetic factors that would influence the development of adenoma, hypermethylation (16) and penetrance. Because the effects of these two factors in a cell are little known even now, the author included these factors into the estimated mutation rate as a whole; no hypermethylation and 100% penetrance. This is the first study to estimate the somatic mutation rate of the APC gene based on the age-specific preference for adenoma in the colon and rectum. As a conclusion, we estimated the somatic mutation rate of the APC gene to be between 2/106 and 3/106 mutations/stem cell/year.
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FOOTNOTES |
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+ For reprints and all correspondence: Takeo Iwama, Department of Surgery, Kyoundo Hospital, Sasaki Institute 1–8 Kanda Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan. E-mail: iwamata@trio.plala.or.jp
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Received October 25, 2000; accepted February 5, 2001.
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