Purpose: Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant syndrome of familial malignancies. Colorectal and endometrial cancers are most frequently observed. The syndrome results mainly from germ-line mutations in DNA mismatch repair genes. A common G-to-C polymorphism at codon 72 in the p53 gene has been associated with increased risk for lung, nasopharyngeal, oral, prostate, and breast cancers and may be a marker for genetic susceptibility to colorectal cancer. We studied the influence of this p53 polymorphism on HNPCC age of onset.
Experimental Design: We determined the p53 genotype of 92 Caucasian mismatch repair mutation carriers, of which, 47 had colorectal cancer. The subjects were genotyped by single-strand conformational polymorphism analysis. We tested the association between age of onset and the p53 genotypes by comparing Kaplan-Meier survival curves, evaluating the homogeneity of the curves using the log-rank test and Wilcoxon’s test, and estimating the association using the Cox proportional hazards regression model to adjust for potential demographic confounding factors.
Results: The HNPCC patients who were heterozygous developed their colorectal cancer 13 years earlier than HNPCC patients who were homozygous for the wild-type allele.
Conclusions: Combining knowledge of an individual’s p53 genotype with information on other genetic and environmental risk factors may improve risk estimates and help to identify individuals who are genetically susceptible to developing HNPCC at an earlier age.
Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant syndrome resulting from germ-line mutations in DNA mismatch repair (MMR) genes. The MMR genes most often mutated are hMLH1 and hMSH2 (1, 2, 3, 4, 5) . HNPCC is characterized by an 80% lifetime risk of developing colorectal and extracolonic malignancies such as endometrial, gastric, ureter, renal pelvis, and ovarian cancers (6, 7, 8, 9) .
Although all of the MMR gene mutation carriers are predisposed to developing the cancers that are characteristic of HNPCC, there is considerable variability in their age of onset. The variability is probably due to a combination of genetic and environmental factors. We investigated the influence of a polymorphism at codon 72 of the p53 gene on HNPCC age of onset in a population of Caucasian MMR gene mutation carriers.
p53 is a tumor suppressor gene that codes for a multifunctional DNA-binding protein that regulates the transcription of hundreds of genes (10) . p53 protein is crucial to maintaining cell-cycle arrest and regulating DNA repair, differentiation, and apoptosis (11) . p53 gene mutations are the most common cancer-related genetic changes, found in ∼50% of human cancers (12) . The roles of p53 mutations differ depending on the type of mutation and on the effect that the mutation has on p53 protein function (13) . p53 mutations can cause expression of abnormal proteins or result in complete absence of p53 expression.
The p53 polymorphism in codon 72 of exon 4 results in a change from arginine (CGC) to proline (CCC; ref. 14 ). The p53Arg72 and p53Pro72 proteins do not differ in their ability to bind to DNA in a sequence-specific manner but do differ in other ways. The p53Arg72 protein induces apoptosis faster and suppresses transformation more efficiently than the p53Pro72 protein (14) . Conversely, the p53Arg72 protein is more susceptible to degradation by the human papillomavirus (HPV) E6 proteins, and degradation of p53 protein by HPV E6 is correlated with increased risk for HPV-associated cancers (15) .
MMR mutation carriers are already at high risk of developing cancer. Previous studies suggest that adverse polymorphic genotypes of the cyclin D1 and N-acetyltransferase 2 genes influence age associated risk for HNPCC (16 , 17) . Likewise, carrying this p53 polymorphism in addition to the MMR mutation may speed the process of carcinogenesis by way of decreasing the efficiency of the normal apoptotic pathway that is mediated by the wild-type p53 protein. MMR proteins have been linked directly or indirectly to DNA damage-induced cell cycle arrest and apoptosis (18, 19, 20) . Also, O6-methylguanine DNA adducts are recognized by MMR proteins, which are necessary for the induction of double-strand breaks during DNA replication. The double-strand breaks, in turn, induce stabilization of p53 protein, triggering apoptosis by up-regulation of Fas/CD95/Apo-1 (21) . Other forms of DNA damage may also induce this MMR/p53 pathway of apoptosis. In this way, MMR mutation carriers who have variant p53 alleles could develop their cancers at an earlier age than MMR mutation carriers with the wild-type p53 gene due to a decreased efficiency in activating apoptosis in tumorigenic cells.
In this study of a population of Caucasian MMR mutation carriers, we found that heterozygotes for the p53 codon 72 polymorphism had a significantly earlier age of onset for HNPCC than homozygotes for the wild-type allele. Sample sizes of other ethnic groups were not adequate for meaningful evaluation.
MATERIALS AND METHODS
We studied 92 confirmed MMR mutation carriers from 47 Caucasian families in our HNPCC registry who were identified and recruited as described previously (16) . Most of the probands were patients seen at the University of Texas M. D. Anderson Cancer Center. Of the 47 families, 16 families were represented in the study by more than one family member (Table 2)⇓ . Of the 92 study participants, 38 (41.3%) were probands and the remaining 54 (58.7%) were recruited relatives of probands. Of the 47 individuals who had colorectal cancer, 33 (70.2%) were probands, thus ascertained because of their cancer history. Of the 45 unaffected MMR mutation carriers, 5 (11.1%) were probands and 40 were recruited relatives. The mean age of individuals with and without colorectal cancer was 44.2 and 43.2 years, respectively.
All of the participants gave informed consent. Age of onset for colorectal cancer was defined as the patient’s age at diagnosis or, for the unaffected carriers, the age at which blood was drawn. The mutation status of all probands was confirmed by a Clinical Laboratory Improvement Act (CLIA)-certified laboratory. Missense mutations of unknown significance were excluded from this study. Each study subject contributed blood and DNA was extracted as described previously (17) . Demographic data are listed in Table 1⇓ .
PCR and Single-Strand Conformational Polymorphism (SSCP) Analysis.
PCR and SSCP analyses were used to genotype the G-to-C p53 polymorphism in codon 72 as described previously (22 , 23) . Briefly, PCR fragments were generated from 100 ng of genomic DNA in a 20-μL reaction mixture containing a 1× dilution of GeneAmp 10× PCR Buffer [500 mmol/L KCl, 100 mmol/L Tris-HCl (pH 8.3), 15 mmol/L MgCl2; Applied Biosystems, Foster City, CA]; 0.2 mmol/L each of dATP, dGTP, dTTP, and dCTP (Invitrogen Corp., Carlsbad, CA); 20 pmol/L each primer (Sigma/Genosys, The Woodlands, TX); 0.1 μL of [32P]dCTP (3000 Ci/mmol; Perkin-Elmer Life Sciences, Inc., Boston, MA); and 0.5 units of AmpliTaq Gold DNA polymerase (Applied Biosystems). The PCR was performed at 94°C for 10 minutes, followed by 28 cycles at 94°C for 30 seconds, 69°C for 30 seconds, and 72°C for 30 seconds and a final extension step at 72°C for 10 minutes. The PCR primers used were 5′-ATGAAGCTCCCAGAATGCCAGAGG-3′ (primer 1) and 5′-ATGCAAGAAGCCCAGACGGAAACC-3′ (primer 2).
For SSCP analysis, PCR product was mixed with an equal volume of loading buffer containing 95% formamide, 20 mmol/L EDTA, 0.05% xylene cyanol, and 0.05% bromphenol blue; heated at 95°C for 5 minutes and quickly chilled on ice for at least 5 minutes. The mixture was separated on a mutation detection enhancement gel (FMC BioProducts, Rockland, ME) that was prepared according to the manufacturer’s instructions, except that the gel solution was diluted 1:4 with 1× Tris-borate-EDTA. The running buffer was also 1× Tris-borate-EDTA. The gels were vacuum dried and autoradiographed.
DNA Sequencing Analysis.
The p53 genotypes of the three different SSCP band patterns were identified by DNA sequencing analysis. The PCR product from a representative sample of each band pattern was analyzed as described previously (17) .
Kaplan-Meier survival analysis estimates the proportion of the population surviving over the ages of the population. Applying this method to our study allows plotting of the proportion of the population that is cancer-free by the participants’ age at the time of evaluation. Such analysis permits visualization and comparison of the dynamics of cancer onset by genotype.
To analyze the data, we defined age of onset for colorectal cancer as the outcome and p53 genotype as an independent variable. We performed all statistical analyses using Stata 8.0 (Stata Corporation, College Station, TX). We tested for Hardy-Weinberg equilibrium using an exact test based on trinomial frequencies (24) . We tested the association between age of onset and the p53 polymorphism by comparing Kaplan-Meier survival curves according to p53 genotype. We evaluated the homogeneity of the survival curves by using the log-rank test and Wilcoxon’s test. We also used the Cox proportional hazard regression model to estimate the association between age-related colorectal cancer risk and p53 genotype, adjusting for potential demographic confounding factors. Hazard ratios and 95% confidence intervals (CIs) were calculated from Cox regression analysis to determine the direction and strength of the association, with and without a robust variance correction (25) , which adjusts for intrafamilial correlations in time to onset for cancer.
Table 1⇓ summarizes the demographics of the study population by p53 genotype, including cancer status, gender, age of onset, and the frequencies of the p53 alleles. The age of onset values were determined from Kaplan-Meier survival-time data, with the 25th, 50th, and 75th percentiles of cancer-free survival time reported. The median age of onset, defined as the age at which 50% of the population is cancer-free, was 40 years for heterozygotes and 53 years for homozygotes for the wild-type p53 allele. Only one age is reported for the CC genotype because only one of the seven homozygotes for the mutant allele had colorectal cancer.
Table 2⇓ describes the structure of the study population in terms of individual family representation. Of the 47 families, 16 (34%) were represented by 2 to 8 members each. Because it is possible that genetic or familial factors in addition to p53 induce a correlation in family members, we applied a robust variance correction in the Cox regression analysis (24) .
There was no significant difference between the age of onset of the subjects with hMLH1 mutations and those with hMSH2 mutations, as assessed by the log-rank test (P = 0.5987). Similarly, we did not observe differences in age of onset between subjects with missense mutations and truncation or deletion mutations when the data were analyzed by the same procedures (P = 0.5386).
A 164-bp PCR fragment was generated, and three different genotypes were distinguished by PCR-SSCP analysis and identified by DNA sequencing analysis (Fig. 1)⇓ . In the study population, the genotypes were in Hardy-Weinberg equilibrium (χ12 = 1.705; exact P = 0.2321; ref. 24 ).
HNPCC Age of Onset and p53 Genotype.
Kaplan-Meier survival analysis showed that the median age of onset in patients who were heterozygous (GC) was 13 years earlier than that of patients who were homozygous for the wild-type (GG) p53 allele (Fig. 2)⇓ .
The Wilcoxon test, which emphasizes observations from early-onset patients, and the log-rank test, which gives equal weight to all failures, were used to examine the homogeneity of the survival curves. Both the Wilcoxon test (P = 0.0297) and the log-rank test (P = 0.0311) demonstrated a significant difference between the curves.
The Cox proportional hazard regression model was used to evaluate the strength of the association between colorectal cancer age of onset and p53 genotype. The analysis showed significant variability in time to onset by genotype (P = 0.025). The hazard ratio indicates that the p53 heterozygotes are 1.75 times more likely (P = 0.07; 95% CI, 0.96–3.21) to get colorectal cancer during any age interval than those with the homozygous wild-type genotype.
Cox analysis with the robust variance correction confirmed the significance of the association with genotypes (P = 0.0044). The robust correction adjusts the variance of the test for homogeneity by genotype by accounting for clustering in time to onset within families. The hazard ratio for p53 heterozygotes with the robust correction is still 1.75 (P = 0.081; 95% CI, 0.93–3.29), indicating that time to onset is not correlated within families.
The number of subjects who were homozygous for the p53 mutation (CC) was too small to provide meaningful results. After dropping the participants with the CC genotype, the Cox analysis with the robust correction was repeated. The strength of the association between colorectal cancer age of onset and p53 genotype remained significant (P = 0.0393). The hazard ratio from this analysis indicates that the p53 heterozygotes are 1.94 times more likely to get colorectal cancer during any age interval than those with the homozygous wild-type genotype (P = 0.04; 95% CI, 1.03–3.63). Dropping the probands from the analysis yielded similar results, with a hazard ratio of 2.62 (P = 0.11; 95% CI, 0.80–8.64).
In this study, we found that subjects who were heterozygous for the p53 codon 72 Arg to Pro polymorphism had a 94% higher risk for colon cancer by year than those who were homozygous for the wild-type allele. Participants with the Arg to Pro polymorphism developed HNPCC 13 years earlier than those who were homozygous for the wild-type allele. Although the possibility exists that the difference in age of onset may be influenced by the fact that probands were recruited on the basis of a family history of colorectal cancer or early-onset age, the bias would be toward the null hypothesis, underestimating the true age difference. However, the estimate of increased relative risk because of the p53 Arg to Pro polymorphism should not be biased. The prospective likelihood approach that we are using for the analysis was shown by simulation studies to yield reliable relative risks when comparing genotypes (26) . Also, the elevated risk in heterozygotes compared with wild-type homozygotes when probands were included in the analysis was nearly identical to the analysis with exclusion of the probands, suggesting these findings are robust to the selection process. Future studies to confirm our results will analyze a larger sample size. In addition, larger studies could evaluate the impact that the Arg/Arg genotype has upon risk for colorectal cancer. Our study, with only seven such subjects could not draw meaningful conclusions.
Our findings are consistent with those of other studies, which report the Pro allele to be an adverse genotype. The Pro allele is associated with a 1.37 to 11.29-fold higher risk for lung cancer (27, 28, 29) , a 3.7-fold higher risk for nasopharyngeal carcinoma (30) , and an 11-year earlier age of onset for oral cancers (31) .
In contrast, a few studies report the wild-type Arg allele to be an adverse genotype. The Arg allele has been associated with a 4.69-fold increased risk for bladder cancer (32) and a 3.1-fold higher risk for gastric cardia cancer (33) . In addition, the mutant Pro allele may protect against HPV-associated cervical cancer. However, this is possibly because the wild-type (Arg) protein is more susceptible to degradation by the HPV E6 protein (15) , which means that both alleles would yield less efficient p53 proteins in the case of this particular cancer.
In conclusion, MMR mutation carriers in HNPCC families have significant variation in their age of cancer onset. The age range for cancer in this study was 21 to 89 years. We showed that the p53 codon 72 polymorphism is associated with a significantly younger age of onset for colorectal cancer, but we know that there are other genetic factors that contribute to this variation such as polymorphisms in cyclin D1 and N-acetyltransferase 2 (16 , 17) . Our long-term goal is to determine how different polymorphisms and/or environmental factors additively or synergistically affect age of onset among MMR mutation carriers. Such knowledge could help to provide more efficient cancer screening, resulting in earlier detection and treatment, better response to treatment, and better chances of survival.
We thank Dr. Maureen Goode for her editorial comments.
Grant support: National Cancer Institute Grant CA 70759, NIH Cancer Center Support Grant CA 16672, and a cancer prevention fellowship from National Cancer Institute Grant R25 CA 57730.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Marsha L. Frazier, Department of Epidemiology, Unit 189, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-3393; Fax: (713) 745-1163; E-mail:
- Received November 14, 2003.
- Revision received April 14, 2004.
- Accepted May 25, 2004.