p53 Codon 72 and MDM2 SNP309 Polymorphisms and Age of ...

Human Cancer Biology

p53 Codon 72 and MDM2 SNP309 Polymorphisms and Age of Colorectal Cancer Onset in Lynch Syndrome Kaisa Sotamaa,1 Sandya Liyanarachchi,1 Jukka-Pekka Mecklin,2 Heikki Ja¨rvinen,3 Lauri A. Aaltonen,4 Pa¨ivi Peltoma¨ki,4 and Albert de la Chapelle1

Abstract

Purpose: The Arg/Pro polymorphism in codon 72 of p53 was recently associated with age of onset of colorectal cancer in Lynch syndrome. A novel polymorphism in the promoter region of MDM2 was associated with age of cancer onset in Li-Fraumeni syndrome.We studied the influence of both polymorphisms on age of onset in Lynch syndrome and of the p53 polymorphism also in sporadic colorectal cancer. Experimental Design: We genotyped p53 codon 72 in 193 individuals with Lynch syndrome mutations, 93 patients with sporadic microsatellite unstable colorectal cancer, and 93 patients with sporadic microsatellite stable colorectal cancer from Finland and 323 Finnish controls. We genotyped 30 colorectal cancer patients with Lynch syndrome mutations from Ohio and 118 U.S. controls.We genotyped SNP309 of MDM2 in the Lynch syndrome groups.We used m2 test, Kaplan-Meier statistics, and Cox regression model to analyze the data. Results: Allele frequencies of both polymorphisms were similar in subjects and controls from both populations and showed Hardy-Weinberg equilibrium. Neither polymorphism was associated with age of colorectal cancer onset in any of the subject groups. Conclusions: This study failed to show any role of the p53 polymorphism on age of colorectal cancer onset in Lynch syndrome and sporadic colorectal cancer.The polymorphism in the MDM2 promoter had no affect on age of onset in Lynch syndrome. Accurate information about age of onset is important in clinical practice, especially in high-risk conditions. As association studies are vulnerable to biologically insignificant variation, both positive and negative findings need to be reported to enable unbiased assessment of the significance of putative risk variants.

Lynch syndrome (hereditary nonpolyposis colorectal cancer) is an autosomal dominant disorder with incomplete penetrance. It causes a predisposition to colorectal cancer, and several other cancers. Germ line mutations in the mismatch repair genes

Authors’ Affiliations: 1Human Cancer Genetics Program, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio; 2Department of Surgery, Jyva«skyla« Central Hospital, Jyva«skyla«, Finland; 3Department of Surgery, Helsinki University Central Hospital; and 4Department of Medical Genetics, University of Helsinki, Helsinki, Finland Received 5/24/05; accepted 6/23/05. Grant support: NIH grants CA67941 and CA16058 (A. de la Chapelle) and the Ohio Biomedical Research and Technology Transfer Commission (A. de la Chapelle); the Satakunta Foundation of the Finnish Cultural Foundation and the Ida Montin Foundation (K. Sotamaa); the Sigrid Juselius Foundation, Finnish Cancer Foundation, and Academy of Finland (P. Peltoma«ki); and European Commission grant QLG2-CT-2001-01861, Finnish Cancer Society, and the Sigrid Juselius Foundation (L. Aaltonen). L. Aaltonen’s work was carried out at the Center of Excellence in Disease Genetics of the Academy of Finland (project no. 44870). The content reflects the views of the grantee and does not necessarily reflect the views of State of Ohio Biomedical Research and TechnologyTransfer Commission. 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: Albert de la Chapelle, Human Cancer Genetics, 646 Tzagournis Medical Research Facility, 420 West 12th Avenue, Columbus, OH 43210. Phone: 614-688-4781; Fax: 1-614-688-4772; E-mail: delachapelle-1@ medctr.osu.edu. F 2005 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-05-1139

Clin Cancer Res 2005;11(19) October 1, 2005

underlie Lynch syndrome. Defective mismatch repair results in replication errors and genetic instability. The penetrance of mismatch repair gene mutations is highly variable. The lifetime risk of colorectal cancer in Lynch syndrome is 70% to 80% in men and lower in women; thus, 30% to 40% of mutation carriers are never diagnosed with colorectal cancer (1). The median age at onset of colorectal cancer is usually quoted as f45 years, ranging between teen age and old age (2, 3). These variations in age of onset are both intra- and interfamilial (4). The mechanisms underlying the variable penetrance are not known. A genetic component has been proposed but only few studies have addressed the question. Genes encoding proteins that influence the metabolism of known carcinogens are obvious modifier candidates. In one study, polymorphisms of the NAT1 and GSTT1 genes were shown to associate with age of onset and location of the tumor in Lynch syndrome (5). Similarly, polymorphisms in the NAT2 gene were implicated in colorectal cancer risk (6). Genes affecting the cell cycle are other obvious candidates. A significant effect on age of onset of colorectal cancer by a polymorphism in the CCND1 gene was reported (7). As far as we know, these findings have not been confirmed or refuted thus far. Very recently, a strong and significant effect on age of onset of colorectal cancer in carriers of Lynch syndrome mutations was reported for the Arg/Pro polymorphism in codon 72 of the p53 gene. It was shown that Arg/Pro heterozygotes developed colorectal cancer 13 years earlier than Arg/Arg homozygotes

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p53 and MDM2 Polymorphisms and Lynch Syndrome

(8). This finding seemed to be not necessarily unexpected in view of the fact that the two alleles are functionally distinct in vitro, the Arg allele being able to induce apoptosis more efficiently than the Pro allele (9). Further, MDM2, a key regulator of p53, is overexpressed in many human tumors (10). A single nucleotide polymorphism in the promoter of MDM2 (SNP309T!G) up-regulates the gene and was associated with a drastically accelerated tumor formation in Li-Fraumeni syndrome as well as in sporadic sarcoma (11). Penetrance (i.e., age at onset) and lifetime risk of cancer are crucial parameters influencing the management of individuals carrying a Lynch syndrome mutation. To be used in clinical practice, penetrance data must be solidly established. We here report studies aimed at further evaluating the putative effects of the codon 72 polymorphism of p53 and the SNP309 polymorphism of MDM2 on the age at onset of cancer in Lynch syndrome, as well as in sporadic colorectal cancer.

Materials and Methods Subjects. We studied 193 confirmed mismatch repair mutation carriers from 62 Lynch syndrome families in Finland, including more than one individual from 32 of the families. At the time of the study, 121 mutation carriers had been diagnosed with colorectal cancer whereas 72 had not. Among these cases, 28 patients from our MDM2 series, but not from our p53 series, were included in a study by Alhopuro et al. (12). All the Lynch syndrome patients and families were diagnosed in part in the traditional way based on family history and early age at onset, and in part by microsatellite instability screening of tumors from consecutive newly diagnosed colorectal cancer patients unselected for family history or age (13). The families emanate from a defined geographic region in Southern and Eastern Finland and, due to the comprehensive nature of the ascertainment, are likely to represent the majority of all Lynch syndrome in this region. For a closer description of the ascertainment of these patients and families, including mutation and microsatellite instability data, please see refs. 13 – 17. In addition, we studied 186 patients with sporadic colorectal cancer (93 with microsatellite unstable tumors; 93 with microsatellite stable tumors) diagnosed as part of the studies quoted above, and from the same geographic region. Mutations in MLH1 and MSH2 had been excluded by sequencing in all sporadic patients with microsatellite unstable tumors. The sole criteria for inclusion in this study were the availability of germ line DNA and updated relevant clinical information. All these patients were of Finnish ethnicity. In addition, we studied 30 mismatch repair gene mutation carriers affected with colorectal cancer and diagnosed in the metropolitan area of Columbus, Ohio. These patients belonged to 29 families; thus, in one case two members of one family were included. The Ohio patients were identified by large-scale molecular screening for Lynch syndrome as described (18). Among the 30 patients, 26 identified themselves as Caucasian, 3 as African American, and 1 as Asian. Inclusion criteria were as described above. All patients gave written consent to genetic studies in compliance with stipulations by the respective institutional review boards. Controls. The 323 control samples from Finland emanated from 99 adult individuals representing a geographically defined weighted sample of the entire Finnish population, and from 224 male blood donors from the same geographic region as the patients. The U.S. control population consisted of 118 samples from self-identified Caucasians purchased from the Coriell Institute (Camden, NJ). p53 genotyping. PCR and single-strand conformational polymorphism analysis were used to genotype the G/C polymorphism in codon 72 p53. The PCR primers used were 5V-CCGGACGATATTGAACAATG-3V and 5V-TCTGGGAAGGGACAGAAGATGAC-3V. A 163 bp PCR fragment


was generated from 25 ng of DNA in 25 AL reaction with a mixture containing 1 dilution of 10 PCR buffer (Applied Biosystems, Foster City, CA), 400 nmol of each primer, 0.4 mmol dATP, dGTP, dTTP, and dCTP, and 1.25 units of Amplitaq Gold DNA Polymerase (Applied Biosystems). The PCR was done at 94jC for 10 minutes, followed by 35 cycles at 94jC for 30 seconds, 56jC for 30 seconds, and 72jC for 1 minute, and a final extension step 72jC for 10 minutes. PCR products were analyzed with single-strand conformational polymorphism analysis as described previously (19). Three distinct patterns were found, and the PCR products were purified with ExoSAP-IT purification kit (U.S. Biochemical Corporation, Cleveland, OH) and identified by sequencing with ABI sequencing system (Perkin-Elmer Applied Biosystems). Subsequently, samples representing each genomic pattern (G/G, G/C, C/C) were used as controls in every single-strand conformational polymorphism gel, but sequencing was not repeated. MDM2 genotyping. PCR and RFLP analysis were used to genotype the T/G MDM2 SNP309 polymorphism. PCR primers used were 5V-CGCGGGAGTTCAGGGTAAAG-3V and 5V-CTGAGTCAACCTGCCCACTG-3V. PCR was done as described above, except that the annealing temperature was 60jC and the number of cycles was 30. The length of the PCR fragment was 158 bp. Digestion was done in 20 AL reaction volume using MspA1I restriction endonuclease (New England Biolabs, Beverly, MA) according to the instructions of the manufacturer. The fragments were visualized in 3% agarose gel electrophoresis. Three distinct patterns were found and identified with sequencing as described above. Subsequently, samples representing each genotypic pattern (T/T, T/G, G/G) were used as controls in every RFLP assay, but sequencing was not repeated. Statistics. Statistical analysis was conducted using R (http://cran. r-project.org/) software with additional packages ‘‘survival’’ and ‘‘genetics’’. m2 test was applied to check the homogeneity in genotype frequency distributions among the control groups, and between patient groups and their controls. If any of the expected counts was below 5, Fisher’s exact test (20) was applied. Genotype frequencies in all groups were checked for the Hardy-Weinberg equilibrium using two tests, m2 test and an exact test (21). Kaplan-Meier survival curves for the three genotypes were used to analyze the age of onset of the first colorectal cancer. The log-rank test was applied to compare the homogeneity of the survival curves between genotype groups. In the combined group of affected and unaffected patients, the median age was defined as the age at which 50% of the subjects were cancer-free. Cox proportional hazard regression analysis with robust variance correction, to adjust for correlation between family members, was applied to estimate any association between the age of onset of colorectal cancer and the genotype.

Results Genotype frequencies at codon 72 of p53. Table 1 summarizes the genotypes and allele frequencies. Considering all genotypes, the frequencies are remarkably similar in all groups of individuals both in Finland and the United States. The frequencies are consistent with Hardy-Weinberg equilibrium. Comparing patient groups with controls showed no significant differences in the distribution of different genotypes. Age of colorectal cancer onset. As shown in Table 2, no significant effects on age of onset were observed in any of the patient groups. The results of the Cox proportional hazards regression analysis with robust variance correction indicated no association between age of onset and genotype (P = 0.417). However, it is notable that in the 193 mutation-positive patients from Finland, the Pro/Pro genotype (present in just five patients) was associated with a higher median age of onset (57 years) than Arg/Pro heterozygotes (46 years) or Arg/Arg

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Table 1. Genotype frequencies at codon 72 of p53 in patient groups and controls Median age* (range) Finland All genotypes Controls Mut+ CRC+ Mut+ CRC Sporadic MSI+ CRC+ Sporadic MSI CRC+ USA All genotypes Coriell controlsc,b Ohio Mut+ CRC+x

n

Female/ male

Arg/Arg

Arg/Pro

Pro/Pro

n (%)

n (%)

n (%)

Pro-allele proportion

45 (6) 26 (8) 3 (2) 2 (3) 7 (8) 7 (8)

0.245 0.274 0.215 0.208 0.220 0.237

0.090 0.215 0.200 0.480

15 (10) 13 (11) 2 (7)

0.264 0.284 0.183

0.321

N/A 43 (19-70) 47.5 (28-93) 72 (26-88) 67 (30-85)

702 323 121 72 93 93

246/456 57/266 46/75 42/30 56/37 45/48

403 (57) 172 (53) 72 (60) 44 (61) 59 (63) 56 (60)

254 (36) 125 (39) 46 (38) 26 (36) 27 (29) 30 (32)

33 (4-98) 46 (23-82)

148 118 30

76/71 65/52 11/19

85 (58) 64 (54) 21 (70)

48 (32) 41 (35) 7 (23)

P, genotype distributions vs controls

Abbreviations: Mut+ CRC+, mismatch repair mutation carriers affected with colorectal cancer; Mut+ CRC, mismatch repair mutation carriers unaffected with colorectal cancer; Sporadic MSI+ CRC+, individuals with microsatellite unstable sporadic colorectal cancer, no known mutations. Sporadic MSI CRC+ = Individuals with microsatellite stable sporadic colorectal cancer. *Median age is the median age of onset for those with colorectal cancer. cAge was not available for 8 of 118 Coriell controls, and gender was not available for 1of 118 Coriell controls. bCaucasian ethnicity. xEthnicity: Caucasian, 26 of 30; African American, 3 of 30; Asian, 1of 30.

homozygotes (49 years). This finding is not statistically significant but resembles that reported by Jones et al. (8). Genotype frequencies at SNP309 of MDM2. As shown in Table 3, genotype frequencies were similar across the control and patient groups. The frequencies were consistent with Hardy-Weinberg equilibrium. Comparing patient groups with controls showed no significant differences in the distribution of different genotypes. Age of colorectal cancer onset. As seen in Table 4, genotype had no significant effect on age of onset, although both in Finland and in the United States the G/G genotype seemed to associate with somewhat higher age than the T/T and T/G genotypes. However, the differences were not even close to being statistically significant, and the numbers of patients were small. Of note, in Li-Fraumeni syndrome, the G allele was the one associated with accelerated tumorigenesis and

earlier onset (11). The results of the Cox proportional hazards regression analysis with robust variance correction indicated no association between age of onset and genotype (P = 0.24).

Discussion We report here findings about the codon 72 polymorphism of p53, which seem to contradict the published evidence (8). This is by no means a sensational outcome. We note that with rare exceptions, association studies of this type often yield different results under different circumstances. Multiple reasons can be advocated to explain divergent findings. Real geographic and ethnic differences may occur. Differences in methodology between studies (both laboratory-based and statistical) are almost the rule. The main source of divergent, even false,

Table 2. Age of onset of colorectal cancer and genotypes at codon 72 of p53 in patient groups n

Median age of onset (y) Arg/Arg

Finland Mut+ CRC+ Mut+ (CRC+ and CRC)* Sporadic MSI+ CRC+ Sporadic MSI CRC+ USA Mut+ CRC+

Arg/Pro

Global P Pro/Pro

121 193 93 93

43 49 72 67

42 46 76 67.5

57 57 67 72

0.662 0.358 0.999 0.516

30

47

46

43

0.688

*Age is defined as the age at which 50% of the subjects were cancer-free.


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p53 and MDM2 Polymorphisms and Lynch Syndrome

Table 3. Genotype frequencies at SNP309 of MDM2 in patient groups and controls n

Finland All genotypes Controls Mut+ CRC+ Mut+ CRC USA All genotypes Coriell controls Ohio Mut+ CRC+

T/T

T/G

G/G

n (%)

n (%)

n (%)

G-allele proportion

P, genotype distributions vs controls

402 209* 121 72

133 (33) 78 (37) 38 (31) 17 (24)

198 (49) 94 (45) 66 (55) 38 (52)

71 (18) 37 (18) 17 (14) 17 (24)

0.423 0.402 0.413 0.500

0.242 0.098

148 118 30

60 (40) 45 (38) 15 (50)

63 (43) 52 (44) 11 (37)

25 (17) 21 (18) 4 (13)

0.382 0.398 0.317

0.492

*A random subset of the 323 controls described in Materials and Methods.

findings is probably various forms of bias of ascertainment or stratification of the study populations. Unfortunately, it is hard to devise perfect experiments in this regard, as has been discussed and reiterated in almost every publication and review of the subject (22). We realize that a detailed comparison between the study of Jones et al. (8) and ours does reveal several putative sources of divergence. Whereas both our series and the series by Jones et al. comprised mutation-positive individuals both with and without cancer, our Finnish series was twice as large (but our U.S. series was much smaller). We used similar statistical methods; however, Jones et al. did not include any control population to verify the comparability of the patients’ allele frequencies with those of the general population. We nevertheless do not attempt to explain the different results by any other factor than, perhaps, relatively small numbers and, therefore, chance. We favor the interpretation suggested by our own results (i.e., that the codon 72 polymorphism does not affect age of onset in Lynch syndrome). Our main rationale for this assertion is as follows. We note that in vitro studies have shown the Pro allele to render the protein less capable of inducing apoptosis than the Arg allele, so intuitively, the Pro allele might be associated with predisposition, more severe disease, or earlier onset. This has already been suggested for several tumor types other than colon, including papillomavirus-induced cervical cancer (23), lung cancer (24), and prostate cancer (25). In such cases, the expectation is that the effect of a deleterious allele such as the Pro allele would show a dosage-dependent association with clinical features. Thus, the earliest onset, and most severe disease, would be expected in Pro/Pro homozygotes. In fact, contrary to this expectation, both our results and those of Jones et al. (8) suggest that Pro/Pro homozygotes have the latest age of onset. Given that the Pro allele has a frequency of 20% to 30% in the population, Pro/Pro homozygotes are relatively rare, and so the result was not significant in either study. Nevertheless, there is a trend with Pro/Pro homozygotes showing the highest median ages of onset of 63 and 57 years, respectively. Whereas there might be molecular scenarios in which heterozygosity per se might confer a deleterious effect (i.e., not seen in homozygosity), we tentatively conclude that the evidence thus far does not speak towards the Pro allele


being associated with more severe penetrance of colorectal cancer in Lynch syndrome, nor in sporadic colorectal cancer with or without mismatch repair deficiency. We note in this context that in 150 patients with sporadic colorectal cancer from Taiwan, the Pro allele was associated with a more advanced Duke’s stage at diagnosis than the Arg allele. In this population, the Pro allele was more common (52%) than the Arg allele; however, only allele frequencies, not genotypes, were shown (26). In a case-control study of 374 patients with sporadic colorectal cancer from Spain, the Pro allele at codon 72 was weakly associated with cancer risk (P = 0.066); however, a polymorphism in the adjacent intron 3 was significantly associated with cancer risk (P = 0.012). The two markers showed strong but incomplete linkage disequilibrium, and when this was taken into account, the authors concluded that the Pro allele was ‘‘clearly unrelated to colorectal cancer’’ (27). Our data on the SNP309 of MDM2 showed no effect on age of onset of colorectal cancer in Lynch syndrome. This polymorphism was recently detected and shown to have a strong effect on tumorigenesis in vitro as well as on age of tumor onset in vivo in Li-Fraumeni syndrome (11, 28). SNP309 has not been explored in many cancers; however, very recently, a study of early-onset uterine leiomyosarcomas, sporadic colorectal cancers, and squamous cell carcinomas of the head

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Table 4. Age of onset of colorectal cancer and MDM2 SNP309 genotypes in patient groups n

Finland Mut+ CRC+ 121 Mut+ 193 (CRC+ and CRC)* USA Mut+ CRC+ 30

Median age of onset (y) Global P T/T

T/G

G/G

42 47

43.5 48

37 61

0.727 0.321

46

43

53.5

0.863

*Age is defined as the age at which 50% of the subjects were cancer-free.



and neck showed no association. In the same study, a trend towards a lower age of onset in females with sporadic colorectal cancer was noted (12). We are not aware of further studies of this type and conclude that in Lynch syndrome, there probably is no association.

Acknowledgments We thank Pia Alhopuro, Heather Hampel, Jennifer LaJeunesse, Jan Lockman, Sini Marttinen, Jim Perko, Saila Saarinen, StephanTanner, and PiaVahteristo for help and advice.

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