Single nucleotide polymorphisms in the EXO1 gene and risk of colorectal cancer in a Japanese population
Hiromasa Yamamoto1,2,
Hiroko Hanafusa2,
Mamoru Ouchida2,
Masaaki Yano1,2,
Hiromitsu Suzuki1,2,
Masakazu Murakami1,
Motoi Aoe1,
Nobuyoshi Shimizu1,
Kei Nakachi3 and
Kenji Shimizu2,*
1 Department of Cancer and Thoracic Surgery and 2 Department of Molecular Genetics, Graduate School of Medicine and Dentistry, Okayama University, 2-5-1 Shikata-cho, Okayama 700-8558, Japan and 3 Department of Radiobiology/Molecular Epidemiology, Radiation Effects Research Foundation, 5-2 Hijiyama Park, Minami-ku, Hiroshima 732-0815, Japan
* To whom correspondence should be addressed. Tel: +81 86 235 7378; Fax: +81 86 235 7383; Email: shimke47{at}md.okayama-u.ac.jp
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Abstract
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EXO1 is a member of the RAD2 nuclease family and functions in DNA replication, repair and recombination. We investigated the relationship of single nucleotide polymorphisms (SNPs) at exon 10 (T439M) and exon 13 (P757L) of the EXO1 gene with development, progression and metastasis of colorectal cancer. For T439M, the Thr/Met genotype [odds ratio (OR) = 2.03, 95% confidence interval (CI) 1.043.98] and Thr/Met and Met/Met genotypes combined (OR = 2.37, 95% CI 1.234.56) demonstrated significant association with the development of colorectal cancer after adjusting for age, gender and smoking status. For P757L, patients with the Leu/Leu genotype showed a reduced risk of colorectal cancer (adjusted OR = 0.398, 95% CI 0.1830.866) when the Pro/Leu and Pro/Pro genotypes were combined and used as the reference. The Leu/Leu genotype also had a reduced risk (adjusted OR = 0.373, 95% CI 0.1640.850) when the Pro/Leu genotype was used as the reference. Individuals who carried both putative risk genotypes (Thr/Met and Met/Met for T439M and Pro/Leu for P757L) showed an adjusted OR of 4.95 (95% CI 1.5615.7) compared with those who carried both low risk genotypes. Analysis of microsatellite instability (MSI) revealed that tumors from individuals who carried both putative risk genotypes tended to have a higher frequency of MSI positives than those from patients who carried both low risk genotypes, although a significant correlation was not found between EXO1 genotype and MSI status. This is the first report to provide evidence for an association of EXO1 gene polymorphisms with colorectal cancer risk. The EXO1 genotypes were not associated with any clinicopathological characteristics in colorectal cancer patients.
Abbreviations: CI, confidence interval; EXO1, exonuclease 1; HNPCC, hereditary non-polyposis colorectal cancer; MMR, mismatch repair; MSI, microsatellite instability; MSI-H, MSI high; MSI-L, MSI low; MSS, microsatellite stable; OR, odds ratio; RFLP, restriction fragment length polymorphism; SNP, single nucleotide polymorphism; TNM, UICC TumorNodeMetastasis classification
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Introduction
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The exonuclease 1 (EXO1) gene, located at chromosome 1q42q43, contains one untranslated exon followed by 13 coding exons and encodes an 846 amino acid protein (1,2). The gene product is a member of the RAD2 nuclease family and functions in DNA replication, repair and recombination (35). Recently it has been shown that it plays a role in both 5'
3' and 3'
5' mispair-dependent excision repair in vitro (6). Wei et al. (7) found that mammalian EXO1 functions in mutation avoidance and is essential for male and female meiosis. They also reported that EXO1 inactivation predisposes mice to the development of tumors late in life and specifically increases the risk of lymphoma. EXO1 can interact physically with the DNA mismatch repair (MMR) proteins MSH2 and MLH1 in both yeast and human cells and with MSH3 in human cells (2,812). Tishkoff et al. (13) reported that the expression of a 3.0 kb EXO1 transcript was detected in significantly higher level in testis, thymus, colon and placenta. Elevated expression of the EXO1 gene in the colon is intriguing because hereditary non-polyposis colorectal cancer (HNPCC), which is characterized primarily by the development of early onset colorectal cancer and a number of other epithelial malignancies, can be attributed to inherited defects in MSH2/MLH1-dependent mismatch repair (14). Wu et al. (15) proposed that the EXO1 gene could be associated with HNPCC predisposition because EXO1 protein strongly interacts with MSH2 protein. More recently, the EXO1 gene showed negative association with HNPCC, although it is involved in DNA MMR (16,17). However, we cannot exclude a role of EXO1 as a low penetrance cancer susceptibility or modifying gene, because the studies performed to date have only focused on suspected HNPCC cases.
Genetic polymorphisms of DNA repair genes have been reported to determine susceptibility to several cancers, including lung, esophageal, bladder, nasopharyngeal and non-melanoma skin cancers (1822). Although polymorphisms in some genes have been studied in relation to colorectal cancer (2325), no study has been conducted on the association between polymorphisms in the EXO1 gene and colorectal cancer risk. Wu et al. (15) identified 12 missense single nucleotide polymorphisms (SNPs) in the EXO1 gene in exon 6 (D249N), exon 7 (G274R and N279S), exon 9 (R354H), exon 10 (T439M, V458M and V460L), exon 11 (K589E and G670E), exon 12 (C723R and S725S) and exon 13 (P757L), while the function of these polymorphisms remains unclear.
Microsatellite instability (MSI) is caused by a failure of the MMR system. Such MMR defects may be caused either by a germline MMR gene mutation, affecting mainly MLH1 or MSH2, or by somatic MMR gene inactivation, most commonly through epigenetic silencing via methylation of the MLH1 promoter. EXO1 genotype may be associated with cancer incidence and with MSI status because EXO1 protein strongly interacts with the MSH2 and MLH1 proteins. In our present study we have investigated whether SNPs at T439M and P757L of the EXO1 gene are associated with the risk of development, progression and metastasis of colorectal cancer. We also evaluated an association between EXO1 SNPs and MSI status using five microsatellite markers (BAT25, BAT26, D5S346, D2S123 and D17S250) that the National Cancer Institute Workshop on Hereditary Nonpolyposis Colorectal Cancer Syndrome proposed for MSI assessment in HNPCC, collectively known as the NCI panel (26).
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Materials and methods
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Patients and control groups
We analyzed 102 Japanese patients chosen from those who were histologically diagnosed as having primary colorectal cancer and underwent surgical operation at Okayama University Hospital in 19942003, which gathers patients from not only Okayama but also various regions, mainly the Chugoku and Shikoku Districts (around Okayama), in Japan. We confirmed microscopically that all colorectal cancer patients have primary colorectal carcinomas. Clinical stage and pathological grade in all colorectal cancer patients were confirmed by operation and pathology. The clinicopathological staging and histological classification were according to the criteria of the UICC TumorNodeMetastasis Classification of Malignant Tumours (TNM), 6th edition, 2002, colon and rectum (ICD-O C 18-C 20). The 110 controls were randomly selected from a prospective cohort study among a general Japanese population. Written informed consent was obtained from all colorectal cancer patients and controls. The characteristics of the 102 colorectal cancer patients and 110 controls are shown in Table I. There were no significant differences in gender and age at recruitment between the colorectal cancer patients and controls (P > 0.05). Pack-year equivalents ([cigarettes/day ÷ 20] x [smoking years]) were used for smoking status (we could not obtain smoking status for 3 of 102 patients). Patients who smoked for
20 years were less frequent than controls compared with never smokers (P = 0.001).
DNA extraction
Genomic DNA of 102 patients was isolated from freshly frozen non-neoplastic colorectal mucosae using SDS/proteinase K treatment, phenolchloroform extraction and ethanol precipitation. Tumor DNA was also isolated from freshly frozen neoplastic colorectal mucosae. Genomic DNA of 110 controls was extracted from peripheral lymphocytes.
Genotyping at T439M
Genotype at T439M was analyzed by PCRrestriction fragment length polymorphism (RFLP). In brief, PCR was performed in a 20 µl reaction mixture with 20 ng genomic DNA, 2.5 mM each dNTP, 1x PCR buffer, 8 pmol each primer (forward primer, 5'-TCT CTA AGT ACA GGT GAA ACA AAG; reverse primer, 5'-GAG CTA TTT TTC TTG GTC TTC TAC) and 0.5 U rTaq DNA polymerase (Takara, Kyoto, Japan). Amplification conditions were 3 min of initial denaturation at 94°C, followed by 35 cycles of 30 s at 94°C, 30 s at 58°C and 30 s at 72°C, with a final extension of 7 min at 72°C. The 125 bp PCR products were digested overnight at 37°C with BsaAI (New England Biolabs, Beverly, MA). BsaAI digestion of the PCR product gives rise to 100 and 25 bp fragments for the Thr (ACG) allele and a single 125 bp fragment for the Met (ATG) allele. Digested fragments were subjected to electrophoresis on 3% agarose gel and visualized under UV light.
Genotyping at P757L
Genotyping at P757L was also conducted by PCRRFLP. PCR was performed with forward primer 5'-CAG AAT GGT CTT AAA ATG GGT GT and reverse primer 5'-TTC AGA ATA AGA AAC AAG GCA AC. Amplification conditions were 3 min initial denaturation at 94°C, followed by 30 cycles of 30 s at 94°C, 30 s at 56°C and 30 s at 72°C, with a final extension of 7 min at 72°C. The 255 bp PCR products were digested overnight at 37°C with MnlI (New England Biolabs). MnlI digestion gives 153 and 102 bp fragments for the Pro (CCT) allele and a single 255 bp fragment for the Leu (CTT) allele.
Analysis of MSI
Tumor and normal DNA was analyzed for MSI using the NCI panel of five microsatellite markers: BAT25, BAT26, D5S346, D2S123 and D17S250. Oligonucleotide forward primers were fluorescently 5'-labeled. PCR was performed in a 20 µl reaction mixture with 20 ng genomic DNA, 2.5 mM each dNTP, 1x PCR buffer, 4 pmol each primer and 0.5 U rTaq DNA polymerase (Takara, Kyoto, Japan). Each primer sequence was as follows; BAT25, forward primer 5'-TCG CCT CCA AGA ATG TAA GT, reverse primer 5'-TCT GCA TTT TAA CTA TGG CTC; BAT26, forward primer 5'-TGA CTA CTT TTG ACT TCA GCC, reverse primer 5'-AAC CAT TCA ACA TTT TTA ACC C; D5S346, forward primer 5'-ACT CAC TCT AGT GAT AAA TCG G, reverse primer 5'-GTT TCC ATT GTA GCA TCT TGA C; D2S123, forward primer 5'-ACA TTG CTG GAA GTT CTG GC, reverse primer 5'-CCT TTC TGA CTT GGA TAC CA; D17S250, forward primer 5'-GCT GGC CAT ATA TAT ATT TAA ACC, reverse primer 5'-GGA AGA ATC AAA TAG ACA AT. Each amplification condition was as follows: BAT25, BAT26 and D2S123, 3 min initial denaturation at 94°C, followed by 28 cycles of 30 s at 94°C, 1 min at 55°C and 1 min at 72°C, with a final extension of 7 min at 72°C; D5S346 and D17S250, 3 min initial denaturation at 94°C, followed by 28 cycles of 30 s at 94°C, 1 min at 58°C and 1 min at 72°C, with a final extension of 7 min at 72°C. Then MSI was analyzed in an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA) using GeneScan Analysis 3.7 software (Applied Biosystems). MSI was indicated by the presence of novel peaks in the tumor tissue that were not seen in normal control tissue from the same patient or by a difference in microsatellite lengths between the two samples. Tumors exhibiting MSI at two or more markers were defined as MSI high (MSI-H). Tumors showing instability at only one marker were defined as MSI low (MSI-L). Tumors in which no markers exhibited MSI were considered to be microsatellite stable (MSS).
Statistical analysis
All statistical analyses in this study were performed using SPSS software version 12.0 (SPSS Inc., Japan). Odds ratio (OR) and 95% confidence interval (CI) were adjusted for age, gender and smoking status using an unconditional logistic regression model. Accordance with the HardyWeinberg equilibrium was examined for colorectal cancer patients and controls using the
2 test. In colorectal cancer patients the correlation between the genotype and clinicopathological characteristics was examined by
2 and Fisher's exact probability tests. Values of P < 0.05 were considered significant.
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Results
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Risk of colorectal cancer by genotype at T439M and P757L in the EXO1 gene
Figure 1 shows the representative PCRRFLP patterns of the T439M and P757L genotypes in the EXO1 gene. Digestion of the PCR product (125 bp) with BsaAI at T439M resulted in a single fragment of 125 bp for the Met allele and two fragments of 100 and 25 bp for the Thr allele. Digestion of the PCR product (255 bp) with MnlI at P757L resulted in a single fragment of 255 bp for the Leu allele and two fragments of 153 and 102 bp for the Pro allele.

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Fig. 1. Polymorphisms of EXO1 exon 10 (T439M) and exon 13 (P757L). (A) T439M locus. (B) P757L locus. Numbers above the panel are case numbers. Genotypes are shown below each panel.
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EXO1 genotypes in colorectal cancer patients and healthy controls are shown in Table II. For T439M, the Thr/Thr, Thr/Met and Met/Met genotypes were found in 63 (63.6%), 30 (30.3%) and 6 (6.1%) of 102 colorectal cancer patients (the genotypes of three patients could not be identified because of unsuccessful PCR) and in 88 (80.0%), 22 (20.0%) and 0 (0%) of 110 controls, neither of which deviated from those expected from the HardyWeinberg equilibrium (P = 0.649 and 0.507, respectively). Met allele frequencies were 21.2 and 10.0% in patients and controls, respectively (P = 0.00148). For P757L, the genotypes Pro/Pro, Pro/Leu and Leu/Leu were found in 35 (34.3%), 53 (52.0%) and 14 (13.7%) of 102 colorectal cancer patients and in 36 (32.7%), 47 (42.7%) and 27 (24.5%) of 110 controls. The distributions of genotypes at P757L in patients and controls also fitted the HardyWeinberg equilibrium (P = 0.690 and 0.342, respectively). Leu allele frequencies did not show a significant difference (P = 0.197) between patients and controls (39.7 and 45.9%, respectively). For T439M, the Thr/Met genotype (OR = 2.03, 95% CI 1.043.98) and Thr/Met and Met/Met genotypes combined (OR = 2.37, 95% CI 1.234.56) demonstrated a significant OR after adjusting for age, gender and smoking status when the Thr/Thr genotype (wild-type) was defined as the reference. An OR for the Met/Met genotype could not be calculated. For P757L, none of the Pro/Leu genotypes (OR = 1.17, 95% CI 0.6102.24), Leu/Leu genotypes (OR = 0.436, 95% CI 0.1831.04) or Pro/Leu and Leu/Leu genotypes combined (OR = 0.893, 95% CI 0.4851.65) demonstrated a significant OR when the Pro/Pro genotype (wild-type) was defined as the reference. However, when the Pro/Leu and Pro/Pro genotypes were combined and used as the reference, the patients with the Leu/Leu genotype showed a reduced risk of colorectal cancer (adjusted OR = 0.398, 95% CI 0.1830.866). The Leu/Leu genotype also had a reduced risk (adjusted OR = 0.373, 95% CI 0.1640.850) when the Pro/Leu genotype was defined as the reference. Therefore, the relative risk of developing colorectal cancer did not show an alleledose relationship for P757L.
We also analyzed the joint effect of polymorphisms at T439M and P757L (Table III). There was no linkage disequilibrium between T439M and P757L polymorphisms. We considered carriers of the genotypes found to be at the lowest risk of disease (Thr/Thr for T439M and Leu/Leu for P757L) as the reference for this analysis. As shown in Table III, individuals who carried only one of the two polymorphisms associated with colorectal cancer risk (i.e. Thr/Met and Met/Met for T439M or Pro/Leu for P757L) were not at significant risk for development of colorectal cancer, whereas individuals who carried both putative risk genotypes showed an adjusted OR of 4.95 (95% CI 1.5615.7). Adjustment for age, gender and smoking status was not significantly effective in all these estimates.
Association between EXO1 genotypes and MSI status
Among tumors from the 102 colorectal cancer patients analyzed, 6 (5.9%) were MSI-H, 11 (10.8%) were MSI-L and the remaining 85 were MSS. We evaluated association between EXO1 SNPs and MSI status (Table III). As shown in Table III, individuals who carried both putative risk genotypes tended to have a higher frequency of MSI-positive (MSI-H and MSI-L) tumors than those who carried both low risk genotypes, although no significant correlation was found between EXO1 genotype and MSI status (P = 0.422).
Association between EXO1 genotype and clinicopathological characteristics
We analyzed the association between the EXO1 genotype and clinicopathological characteristics in colorectal cancer patients (Table IV). We compared the patients with Thr/Met and Met/Met for T439M and Pro/Leu for P757L with the other patients. However, there were no significant differences between these two groups.
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Discussion
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The EXO1 gene has been investigated in association with HNPCC (1417,27). Wu et al. (15) found several EXO1 mutants in HNPCC patients who were shown to be negative for germline mutations in the MLH1, MSH2 and MSH6 genes. Among a total of 30 alterations in the EXO1 exons, 14 were unique to HNPCC patients. However, the other 16 were thought to be polymorphisms because they occurred at similar frequencies in both patients and controls. Thompson et al. (17) reported that the EXO1 gene does not appear to be associated with HNPCC, but did not refer to these polymorphisms. Therefore, we investigated T439M and P757L among these polymorphisms.
In the present study we have examined whether SNPs at T439M and P757L in the EXO1 gene are associated with the risk for development, progression and metastasis of colorectal cancer. We found significant differences in genotype distribution of the EXO1 gene between colorectal cancer patients and controls. We recognize that this population of colorectal cancer patients does not seriously deviate from the general Japanese population because Japan is an almost racially homogeneous nation and Okayama has had population influxes from other areas such as Tokyo and Osaka (urban cities representing Japan) and the Chugoku and Shikoku Districts (around Okayama). For T439M of EXO1, an OR of 2.03 was observed among individuals with the Thr/Met genotype and an OR of 2.37 was observed among individuals with the Thr/Met and Met/Met genotypes combined. For P757L of EXO1, patients with the Leu/Leu genotype had a reduced risk of colorectal cancer when the Pro/Leu and Pro/Pro genotypes combined or Pro/Leu genotype was defined as the reference (OR = 0.398 and 0.373, respectively). Interestingly, individuals with putative risk genotypes for both T439M (Thr/Met and Met/Met) and P757L (Pro/Leu) were at a 5-fold higher risk of colorectal cancer. As for an association between EXO1 SNPs and MSI, individuals who carried both putative risk genotypes tended to have higher frequency of MSI-positive tumors than those who carried both low risk genotypes, although no significant correlation was found. This fact may support the proposal that high risk genotypes of the EXO1 gene cause colorectal cancer through incomplete MMR. Our findings are the first to suggest an association between polymorphisms in the EXO1 gene and risk of colorectal cancer. No other reports have found an association between other cancers and EXO1 SNPs. Although intriguing, however, these joint effects should be interpreted with caution, given the modest size of the present study to evaluate joint effects. The EXO1 genotype was not significantly associated with clinicopathological characteristics. Therefore, the EXO1 gene does not appear to be associated with progression or metastasis of colorectal cancer.
As functional motifs of EXO1 protein, an EXO1 nuclease domain was identified in the N-terminal 391 amino acid residues (27) and a proteinprotein interaction domain with MMR proteins was identified on the basis of sequence alignment and functional domain analysis (2,912,28) (Figure 2). T439M is located in the MLH1 interaction domain and P757L is located within the region required for interaction with MSH2. It is known that mutations of MSH2 and MLH1 are associated with typical HNPCC, resulting in a total loss of MMR function (29). In the case of T439M, a polar amino acid (Thr) is replaced by a non-polar amino acid (Met). Considering that the genotype Met/Met were not found in the control subjects in T439M, it is possible that substitution of Thr by Met may strongly affect interaction with other MMR proteins. As for P757L, this substitution may influence proteinprotein interaction because prolines tend to destabilize (kink)
-helices due to the lack of a backbone hydrogen bond and steric constraints (3032). It is notable that the MMR proteins MSH2, MLH1 and MSH6 are constituents of BASC, the BRCA1-associated genome surveillance complex (33). If EXO1 is also involved in this complex through MMR proteins, the protein may play a more general role(s) in the protection of DNA in addition to MMR.

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Fig. 2. Functional motifs of EXO1 and the locations of polymorphisms. Nuclease domains and proteinprotein interaction regions are shown by arrows. SNPs examined in the present study (T439M and P757L) are indicated above the motif by inverted triangles.
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In conclusion, our data provide evidence for an association between the EXO1 gene polymorphisms at T439M and P757L and the risk of development of colorectal cancer. It is possible that these polymorphisms may influence susceptibility to colorectal cancer through incomplete DNA repair. The association was more prominent for individuals who carried both of the two putative risk genotypes. Further study with sufficiently larger populations and functional analysis of these polymorphisms will be needed to clarify the unsolved issues.
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Acknowledgments
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This work was supported by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (grant no. 12213084) to K.S.
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Received July 13, 2004;
revised October 15, 2004;
accepted November 7, 2004.