Abstract
Purpose
The repair enzyme RAD18 plays a key role in the post-replication repair process in various organisms from yeast to human, and the molecular function of the RAD18 protein has been elucidated. Single nucleotide polymorphism (SNP) of arginine (Arg, CGA) or glutamine (Gln, CAA) at codon 302 is known on RAD18; however, the association between the SNP and the risk of any human cancers including non-small-cell lung cancer (NSCLC) has not been reported. We therefore investigated the relationship between the polymorphism and the development and progression of human NSCLC.
Methods
The study population included 159 patients with NSCLC and 200 healthy controls. The SNP was genotyped by polymerase chain reaction with the confronting two-pair primer (PCR-CTPP) assay. Genotype frequencies were compared between patients and controls, and the association of genotypes with clinicopathological parameters was also studied.
Results
The Gln/Gln genotype was significantly more frequent in NSCLC patients (20.7%) than in healthy controls (11.5%)(P = 0.003). The increased risk was detected in NSCLC patients with the Gln/Gln genotype [Odds ratio (OR) = 2.63, 95% confidence interval (CI)=1.38–4.98]. As to the relationship of the SNP with clinicopathological parameters of NSCLC, significantly higher risks were detected in lung squamous cell carcinoma (LSC) (OR = 4.40, 95% CI = 1.60–12.1).
Conclusions
Our results suggested that Gln/Gln genotype of the RAD18 SNP has the increased risk of NSCLC, especially of LSC. This is the first report to provide evidence for an association between the RAD18 Arg302Gln polymorphism and human NSCLC risk.
Keywords: SNPs, RAD18, Non-small-cell lung cancer (NSCLC), Cancer predisposition
Introduction
DNA in living cells is damaged by environmental damaging agents and mutagens, such as UV light and mutagenic chemicals (Hoeijmakers 2001). DNA damage must be repaired by DNA repair systems. However, when the DNA repair systems are stalled or saturated, and such DNA damages are thus not removed before the onset of DNA replication, single-stranded gaps are generated. These gaps will be filled by the postreplication repair (PRR) system. The RAD6 pathway is known to be central to PRR (Lawrence 1994) and RAD6 epistasis group proteins, such as RAD5, RAD18, RAD30, MMS2 and UBC13, are all involved in the pathway. In this pathway, RAD18 and RAD6 are two of the most important proteins and play a key role. RAD18 is a single-strand DNA binding protein with a RING finger domain, and has ubiquitin-ligating enzymes (E3) activity (Joazeiro and Weissman 2000). RAD6 is an ubiquitin-conjugating enzyme (E2) in the proteasome protein degradation system (Sung et al. 1990, 1991b; Wood et al. 2003). RAD18 forms a tight complex with RAD6 (Bailly et al. 1994, 1997a; b). Although RAD6 interacts with several ubiquitin-ligating enzymes (E3), the interaction with RAD18 is essential for carrying out PRR (Wood et al. 2003; Bailly et al. 1994; Dohmen et al. 1991; Sung et al. 1991a).
RAD18 knockout cells of mouse embryonic stem cells (Tateishi et al. 2003) and of chicken DT40 cells (Yamashita et al. 2002) were hypersensitive to various DNA-damaging agents and showed defective PRR. Genomic instability of these cells was demonstrated by increased rates of the sister chromatid exchange and integration of exogenous DNA (Tateishi et al. 2003; Yamashita et al. 2002). RAD18 contributes to the maintenance of genomic stability through PRR and dysfunction of RAD18 increases the frequency of homologous recombination as well as illegitimate recombination (Shekhar et al. 2002). Furthermore, dysfunction of RAD18 is thought to lead to the development of cancer (Friedberg 2003).
The genetic polymorphisms of DNA repair genes have been analyzed to determine susceptibility to several cancers, including lung (Ito et al. 2004; Ryk et al. 2006), colorectal (Yamamoto et al. 2005), breast (Costa et al. 2006), head and neck (Huang et al. 2005), bladder cancer (Zhu et al. 2007) and leukemia (Bolufer et al. 2006). The RAD18 gene is known to have a single nucleotide polymorphism (SNP) at codon 302, encoding either arginine (Arg, CGA) or glutamine (Gln, CAA), as known as rs#373572 in the dbSNP; NCBI Reference SNP (refSNP) Cluster Report. In the present study, we found a significant correlation of the SNP with NSCLC. This is, to our knowledge, the first report providing evidence for an association between the RAD18 Arg302Gln polymorphism and human NSCLC risk.
Materials and methods
Subjects
We studied frozen specimens of 159 cases stored at −80°C obtained from Japanese patients with primary NSCLC treated by curative intent surgical resection in Okayama University Hospital (Okayama, Japan), after acquiring informed consent from each patient, between 1994 and 2003. The case groups consisted of 105 lung adenocarcinomas (LAD), 48 lung squamous-cell carcinomas (LSC), 3 adeno/squamous-cell carcinomas and 3 large cell carcinomas (107 men, 52 women; mean age 66.2 years). The clinical stage and pathological grade in most patients were confirmed by operation and pathology. The clinical staging and histological classification of cancers were defined according to the criteria of UICC Tumor-Node-Metastasis Classification of Malignant Tumors (TNM), sixth edition, 2002, (ICD-O C34 for lung). For the controls, each of the 200 healthy controls we analyzed was selected by computer-aided randomization among five individuals matched in smoking habit, gender and age (within 5 years) for each lung cancer patient, all of which were from the subjects of cohort studies on a Japanese general population older than 40 years of age in a town near the Saitama Cancer Center. A population of this town has increased because of a population influx from other areas, with a social increase rate of about 5% every year for 15 years. Informed consent was obtained from all cases and controls concerned. This study was approved by The Bioethics Committee of Okayama University Medical School.
DNA extraction
Genomic DNA of 159 patients was isolated from the non-cancerous region of the resected specimens or from the mononuclear cells of the peripheral blood using SDS/proteinase K treatment, phenol-chloroform extraction and ethanol precipitation. Genomic DNA of 200 healthy controls was extracted from peripheral lymphocytes.
Genetic analysis
Genotyping of the RAD18 Arg302Gln polymorphism was carried out by polymerase chain reaction using the confronting two-pair primer (PCR-CTPP) technique (Hamajima et al. 2000; Hamajima 2001). According to the sequence of the human RAD18 gene shown in database, we designed two sets of paired primers. The first set of primers was as follows: forward primer 1, 5′-ATA CCC ATC ACC CAT CTT C-3′ and reverse primer 1, 5′-GTC TTC TCT ATA TTT TCG ATT TCT T-3′ for the A (Gln) allele amplifying a 146 bp band. The second set of primers was as follows: forward primer 2, 5′-TTA ACA GCT GCT GAA ATA GTT CG-3′ and reverse primer 2, 5′-CTG AAA TAG CCC ATT AAC ATA CA-3′ for the G (Arg) allele amplifying a 106 bp band. A 206 bp band was designed between the forward primer 1 and the reverse primer 2. Genomic DNA (20 ng) was assessed in 20 μl of reaction mixture containing 40 μM of each dNTP, 1X PCR buffer, 8 pmol of the forward primer 1 and reverse primer 2, 24 pmol of the forward primer 2 and reverse primer 1 and 0.5 unit of the Taq DNA polymerase (Takara, Kyoto, Japan). The PCR amplification was initiated by a denaturing step at 94°C for 3 min, followed by 35 cycles at 94°C for 30 s, 64°C for 1 min, 72°C for 1 min, and a final extension step at 72°C for 7 min. For genotyping, the PCR products were subjected to electrophoresis in 3% agarose gel with ethidium bromide staining and then visualized on a UV transilluminator. The allele types were determined as follows; 205 and 106 bp for the G/G (Arg/Arg) genotype, 205 and 146 bp for the A/A (Gln/Gln) genotype and 205, 146 and 106 bp for the G/A (Arg/Gln) genotype. In order to confirm the allele types, some PCR products were processed with the Big Dye terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA), then analyzed and confirmed on an ABI 3100 sequencer (Applied Biosystems).
Statistical analysis
We compared the allele frequencies of the polymorphism in the RAD18 gene between NSCLC patient group and healthy control group. The distribution of the RAD18 genotype (Arg/Arg, Arg/Gln, Gln/Gln) in all of the patients and the controls was tested for adherence to the Hardy–Weinberg equilibrium. The Chi-square test was used to compare the genotype distribution between patients and controls. The odds ratio (OR) and 95% confidence interval (95% CI) were used to estimate the risk of association with genotype. The OR and 95% CI was adjusted for age, gender and smoking habit by an unconditional logistic regression model using the SPSS software Ver.12.0 (SPSS Inc., Tokyo, Japan).
Results
Assessment of cancer risk by RAD18 genotyping
The characteristics of the 159 NSCLC patients and the 200 healthy controls are shown in Table 1. There were no significant differences in gender, age or smoking status between these two groups. Pack-year equivalents were used for smoking status (however, we could not obtain the smoking status for 5 of 159 NSCLC patients).
Table 1.
Characteristics of NSCLC patients and healthy controls
| Patients | Controls | P-value | |
|---|---|---|---|
| n (%) (n = 159) | n (%) (n = 200) | ||
| Gender | 0.874b | ||
| Male | 107 (67.3) | 133 (66.5) | |
| Female | 52 (32.7) | 67 (33.5) | |
| Age (years ± SD)a | 66.2 ± 9.94 | 65.6 ± 9.42 | |
| Smoking habit | 0.909c | ||
| No-smoker | 50 (31.4) | 63 (31.5) | |
| Smoker | 104 (65.4) | 137 (68.5) | |
| <20 pack-years | 5 (4.8) | 17 (12.4) | |
| ≥20 pack-years | 97 (92.3) | 87 (63.5) | |
| Unknown | 2 (2.9) | 33 (24.1) | |
| Unknown | 5 (3.2) | 0 (0.0) |
aAge shows the mean age of each group with standard deviation
b P-values were for the differences in the number of males and females between patients and controls and were calculated by Chi-square test
c P-values were for the differences in the number of smokers and non-smokers between patients and controls and were calculated by Chi-square test
The representative PCR-CTPP patterns and sequence patterns were shown in Fig. 1a, b, respectively. Significant differences in the genotype frequency were evident between NSCLC patients and controls (Table 2). The frequencies of Arg/Arg, Arg/Gln and Gln/Gln genotype were found to be 29.6, 49.7 and 20.7% in the NSCLC patients and 43.0, 45.5 and 11.5% in the controls, respectively. All of the results fitted the Hardy–Weinberg equilibrium. In comparison to Arg/Arg genotype, the most significantly increased risk was found in NSCLC patients with Gln/Gln genotype with an adjusted OR of 2.57 (95% CI, 1.35–4.89). Thus, this result suggested that the homozygous Gln/Gln genotype has an increased risk of NSCLC.
Fig. 1.
The single nucleotide polymorphism at codon 302 of the RAD18 gene. a The PCR-CTPP patterns of the RAD18 SNP. The PCR product was electrophoresed in 3% agarose gel. Two fragments of 205 and 106 bp show the G/G (Arg/Arg) genotype, two fragments of 205 and 146 bp show the A/A (Gln/Gln) genotype, and three fragments of 205-, 146 and 106 bp show the G/A (Arg/Gln) genotype. The case number and genotypes are shown at the top and bottom, respectively. b The direct sequence patterns of the RAD18 SNP. The SNP, Arg (CGA) or Gln (CAA), is indicated by an arrow above the sequence
Table 2.
The RAD18 genotypes in patients and controls
| RAD18 | Patients | Controls | P-value | OR (95% CI) | |
|---|---|---|---|---|---|
| Genotype | N (%) | N (%) | Crude | Adjustedb | |
| Arg/Arg | 47 (29.6) | 86 (43.0) | 1 (Reference) | 1 (Reference) | |
| Arg/Gln | 79 (49.7) | 91 (45.5) | 0.051a | 1.59 (1.00–2.53) | 1.60 (1.00–2.56) |
| Gln/Gln | 33 (20.7) | 23 (11.5) | 0.003a | 2.63 (1.38–4.98) | 2.57 (1.35–4.89) |
| Total | 159 | 200 | |||
| Allele frequencies | 0.002 | ||||
| Arg | 173 (54.4) | 263 (67.8) | |||
| Gln | 145 (45.6) | 137 (34.2) | |||
a P-values were calculated for the difference in genotype frequencies against Arg/Arg by Chi-square test
bORs were adjusted for age, gender and smoking status. Patients whose smoking status was not known were excluded when ORs were calculated
The association between the RAD18 genotype and clinicopathological features
We next analyzed the relationship between the genotype distribution and the clinicopathological parameters. Strong association between the risk of lung squamous-cell carcinoma (LSC) and genotype distribution was shown in Table 3. The adjusted OR of LSC patients with Gln/Gln genotype was 4.40 (95% CI, 1.60–12.1), whereas the same genotype exhibited a marginal risk for lung adenocarcinoma (LAD) with a borderline significance (adjusted OR = 1.97, 95% CI, 0.94–4.12). Differentiated grade, TNM classification, gender and smoking habit were not associated with the frequency of genotype or allele (Table 4).
Table 3.
Association between the RAD18 genotype distribution and histological cell type of patients
| Characteristics | Genotype (%) | ORa (95% CI) | ||||
|---|---|---|---|---|---|---|
| Arg/Arg | Arg/Gln | Gln/Gln | Total | Arg/Gln | Gln/Gln | |
| Controls | 86 (43.0) | 91 (45.5) | 23 (11.5) | 200 | ||
| All patients | 47 (29.6) | 79 (49.7) | 33 (20.7) | 159 | 1.60 (1.00–2.56) | 2.57 (1.35–4.89) |
| LAD | 34 (32.4) | 53 (50.5) | 18 (17.1) | 105 | 1.51 (0.89–2.56) | 1.97 (0.94–4.12) |
| LSC | 11 (22.9) | 25 (52.1) | 12 (25.0) | 48 | 2.40 (1.09–5.29) | 4.40 (1.60–12.1) |
| Others | 2 (33.3) | 1 (16.7) | 3(50.0) | 6 | ||
LAD lung adenocarcinoma, LSC lung squamous-cell carcinoma
aORs were adjusted for age, gender and smoking status. The Arg/Arg genotype of healthy controls was defined as the reference
Table 4.
Association between the RAD18 genotype and clinicopathological parameters of patients
| Characteristics | Genotype (%) | Allele (%) | ||||||
|---|---|---|---|---|---|---|---|---|
| Arg/Arg | Arg/Gln | Gln/Gln | Total | p-value | Arg | Gln | P-value | |
| Differentiated grade | ||||||||
| Well | 18 (34.6) | 23 (44.2) | 11 (21.2) | 52 | 59 (56.7) | 45 (43.3) | ||
| Moderate | 16 (25.8) | 35 (56.5) | 11 (17.7) | 62 | 0.420a | 67 (54.0) | 57 (46.0) | 0.683a |
| Poor | 9(25.0) | 19(52.8) | 8(22.2) | 36 | 0.613a | 37 (51.4) | 35 (48.6) | 0.484a |
| Unknown | 3 | 3 | 3 | 9 | ||||
| TNM classification | ||||||||
| I | 31 (33.0) | 43 (45.7) | 20 (21.3) | 94 | 105 (55.9) | 83 (44.1) | ||
| II, III, IV | 16 (26.7) | 33 (55.0) | 11 (18.3) | 60 | 0.530 | 65 (54.2) | 55 (45.8) | 0.772 |
| Unknown | 0 | 3 | 2 | 5 | ||||
| Gender | ||||||||
| Male | 28 (26.2) | 57 (53.3) | 22 (20.5) | 107 | 113 (52.8) | 101 (47.2) | ||
| Female | 19 (36.5) | 22 (42.3) | 11 (21.2) | 52 | 0.254 | 60 (57.7) | 44 (42.3) | 0.133 |
| Unknown | 0 | 0 | 0 | 0 | ||||
| Smoking habit | ||||||||
| Smoker | 25 (25.3) | 53 (53.5) | 21 (21.2) | 99 | 103 (52.0) | 95 (48.0) | ||
| No-smoker | 22 (40.0) | 22 (40.0) | 11 (20.0) | 55 | 0.558 | 66 (60.0) | 44 (40.0) | 0.351 |
| Unknown | 0 | 4 | 1 | 5 | ||||
a P-values were calculated against Well-differentiated grade by Chi-square test
Discussion
In the present study, we examined whether the SNP at codon 302 in the RAD18 gene is associated with the risk for development of NSCLC, and found significant differences in the genotype distribution between the NSCLC patients and the healthy controls. Our findings suggest that this SNP is associated with the development of the NSCLC, and the susceptibility to the NSCLC is enhanced by the Gln/Gln genotype. However, this SNP does not appear to be associated with progression or metastasis of the NSCLC, as the RAD18 genotype showed no correlation with the clinicopathological characteristics, except histological types. The Gln/Gln genotype was detected more frequently in the NSCLC patients, and the individuals with the Gln/Gln genotype showed a 2.6-fold higher risk of NSCLC. Furthermore, as for the LSC patients, a strong association between the Gln/Gln genotype and the development risk was detected (OR = 4.40, 95% CI = 1.60–12.1). Notably, the heterozygotes (Arg/Gln) exhibited an intermediate risk, still with statistic significance, for both whole NSCLC (OR = 1.60, 95% CI = 1.00–2.56) and LSC (OR = 2.40, 95% CI = 1.09–5.29), indicating a dose-response effect of the Gln allele. This shows that the Gln allele may be defined as the responsive risk-allele. It would be of great interest to see the effects of the SNP on incidence of NSCLC in Europeans and Africans, since the frequency of the individuals with the Gln/Gln genotype is much higher (60%) in these races than in Asian people (8–18%)(rs#373572 in the dbSNP). Giving the high risk of the Gln/Gln genotype for LSC among NSCLC, the ethnic difference may well explain, at least in part, the higher proportion of LSC among NSCLC in Caucasians than in Asians.
We recognize that this specific population of cancer patients does not seriously deviate from the general Japanese population because Japan is an almost racially homogeneous nation and Okayama has experienced population influxes from other areas, such as Tokyo and Osaka (the urban city representing Japan) and the Chugoku and Shikoku Districts (surrounding Okayama).
RAD18 is one of the most important proteins involved in the PRR pathway. In the PRR pathway, an interaction between RAD18 and RAD6 is essential for carrying out PRR (Wood et al. 2003; Bailly et al. 1994; Dohmen et al. 1991; Sung et al. 1991b). Since RAD6, which has no DNA binding activity, interacts with RAD18, it has been proposed that RAD18 recruits RAD6 to the site of DNA damage via its physical interaction where RAD6 and its complex then modulate stalled DNA replication through their ubiquitin-conjugating activity (Haracska et al. 2004; Watanabe et al. 2004). There have been reports that the proliferating cell nuclear antigen (PCNA), a DNA polymerase sliding clamp that is involved in DNA synthesis and repair, is a substrate of the ubiquitin conjugating enzyme, and it is ubiquitinated in a RAD18- and RAD6-dependent manner (Hoege et al. 2002; Stelter and Ulrich 2003; Kannouche et al. 2004). Therefore, the monoubiquitination of PCNA through RAD18 and RAD6 is necessary for carrying out DNA PRR. RAD18 interacts with RAD6 through the RAD6-binding domain in the C-terminal region (AA371-410) (Fig. 2). Considering that Gln/Gln genotype was detected more frequently in NSCLC patients, substitution of Arg by Gln may reduce the RAD6-binding activity. Furthermore, RAD18 has several other functional domains as well, such as the RING-finger motif (Costa et al. 2006), zinc-finger motif (Mackay and Crossley 1998; Akhtar and Becker 2001) and E3 ubiquitin-ligase domain (Marchler-Bauer et al. 2005). The RING-finger motif, residing in the N-terminal region, and the E3 ubiquitin-ligase domain together confer an ubiquitin ligase activity on RAD18. The RAD18 Arg302Gln polymorphism is located in the E3 ubiquitin-ligase domain (Fig. 2). Therefore, this SNP may affect the E3 ubiquitin-ligase activity of RAD18. It is also possible that this SNP may affect the interaction between RAD18 and other proteins involved in PRR through its structural change, which is generated by the substitution of one amino acid residue, a basic amino acid residue (Arg) to a neutral residue (Gln).
Fig. 2.
The location of the polymorphism and the functional motifs of RAD18 protein. The SNP (Arg302Gln) is indicated by an arrowhead above the motif. The motifs of the RAD18 protein are depicted in dark gray and/or by arrows. RING, RING-finger motif (AA25-63); ZINC, zinc-finger motif (AA201-223); Ubiquitin ligase, E3 ubiquitin ligase domain (AA16-304); RAD6 BD, RAD6 binding domain (AA371-410). AA #, amino acid number
Our data provide evidence for an association between the RAD18 Arg302Gln polymorphism and the risk of NSCLC. It is possible that this polymorphism may influence susceptibility to a variety of human cancers through incomplete PRR. The sample size we analyzed was small; however, we recognized that our findings were true because the findings of this study are statistically significant. Analysis with threefold or more of normal control population against our patient population will define more precise values for statistical analysis. Further study with sufficiently larger populations and functional analysis of this polymorphism will be required in order to clarify this issue.
Acknowledgments
We gratefully thank the members of our University Hospital for cooperation in specimen sampling. This work was supported by a Grant-in-Aid for Scientific Research on Priority Area (No. 12213084) to K. S from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Abbreviations
- LAD
Lung adenocarcinoma
- LSC
Lung squamous cell carcinoma
- NSCLC
Non-small-cell lung cancer
- OR
Odds ratio
- PCNA
Proliferating cell nuclear antigen
- PCR-CTPP
Polymerase chain reaction with the confronting two-pair primers
- PRR
Postreplication repair
- SNP
Single nucleotide polymorphism
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