Abstract
The DNA repair gene X-ray repair complementing defective repair in Chinese hamster cells 4 (XRCC4) is thought to play a major role in the caretaking of the whole genome via double-strand break repair. However, the association of polymorphic variants of XRCC4 with colorectal cancer susceptibility has never been reported. In this hospital-based case-control study, the association of XRCC4 polymorphisms C-1622T (rs7727691), G-1394T (rs6869366), G-652T (rs2075685), C-571T (rs2075686), intron 3 DIP (rs28360071), S247A (rs3734091) and intron 7 DIP (rs28360317) with colorectal cancer risk in a Taiwanese population was investigated. The genotypes of XRCC4 of 370 patients with colorectal cancer and 370 age- and gender-matched healthy controls were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. We found significant differences in the genetic and allelic frequencies of the XRCC4 G-1394T between the colorectal cancer and control groups (p=0.0003 and 8.32×10−5, respectively). The distributions of other genetic polymorphisms between cases and the control group were not significantly different. We conclude that the G allele of XRCC4 G-1394T may contribute to colorectal carcinogenesis and may be useful for early detection of colorectal cancer.
Colorectal cancer (CRC) is a leading cause of cancer-related morbidity and mortality worldwide. The incidence and age-adjusted mortality of CRC have drastically continued to increase in Taiwan. In recent years, the incidence and mortality of CRC has taken the third place among the common types of cancer. Etiological studies have attributed more than 85% of CRC to environmental factors (1, 2). Meat consumption, cigarette smoking and exposure to carcinogenic aromatic amines are commonly recognized environmental factors for CRC (3-5) and these DNA damage-inducing factors may induce various types of DNA adducts, including double-strand breaks (DSBs) (6). DSBs may lead to severe genome instability, which is closely related to carcinogenesis (7, 8). Two distinct DNA repair pathways are responsible for DSB repair. Homologous recombination (HR) and the non-homologous end-joining (NHEJ) (8). Components involved in NHEJ include the DNA ligase IV–XRCC4 complex, Ku70, Ku80, the catalytic subunit PKcs and artemis (9). Genetic polymorphisms in NHEJ genes influence DNA repair capacity and confer predisposition to several types of cancer, including skin (10), breast (11-13), gastric (14), and oral (15). Among these reports, the study investigating the association of XRCC4 G-1394T polymorphism with oral cancer found that those who had G/T or G/G at G-1394T showed a 3 to 79-fold increased risk of oral cancer compared to those with T/T (15). Since both the stomach and intestine originate from endoderm during embryonic development, we proposed that this genetic polymorphism may also be associated with CRC susceptibility. To the best of our knowledge, there is no report studying the association of XRCC4 with CRC risk. The present study aimed at investigating the potential association of XRCC4 polymorphisms, including C-1622T (rs7727691), G-1394T (rs6869366), G-652T (rs2075685), C-571T (rs2075686), intron 3 DIP (rs28360071), S247A (rs3734091) and intron 7 DIP (rs28360317), with CRC susceptibility.
Patients and Methods
Study population and sample collection. The study population consisted of 370 case patients and 370 cancer-free control volunteers. Three hundred and seventy patients diagnosed with CRC were recruited at the outpatient clinics of general surgery during 2002-2009 at the China Medical University Hospital, Taichung, Taiwan. The clinical characteristics of patients include histological details were all graded and defined by expert surgeons (Dr. Yang's team). All patients voluntarily participated, completed a self-administered questionnaire and provided peripheral blood samples. An equal number of healthy volunteers as controls were selected by matching for age, gender and habits after initial random sampling from the Health Examination Cohort of the hospital. The exclusion criteria for the control group included previous malignancy, metastasized cancer from other or unknown origin, and any familial or genetic diseases. Our study was approved by the Institutional Review Board of the China Medical University Hospital and written-informed consent was obtained from all participants.
Genotyping conditions. Genomic DNA was prepared from peripheral blood leukocytes using a QIAamp Blood Mini Kit (Blossom, Taipei, Taiwan, R.O.C.) and further processed according to our previous papers (15-19). The polymerase chain reaction (PCR) cycling conditions were: one cycle at 94°C for 5 min; 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 10 min. Pairs of PCR primer sequences and restriction enzymes for each DNA product are all listed in Table I.
Statistical analyses. Only those matches with all DNA polymorphism data (case/control=370/370) were selected for final analysis. To ensure that the controls used were representative of the general population and to exclude the possibility of genotyping error, the deviation of the genotype frequencies of XRCC4 single nucleotide polymorphism (SNPs) in the controls from those expected under the Hardy-Weinberg equilibrium was assessed using the goodness-of-fit test. Pearson's chi-square test or Fisher's exact test (when the expected number in any cell was less than five) was used to compare the distribution of the XRCC4 genotypes between cases and controls. Data were recognized as significant when the statistical p-value was less than 0.05.
Results
The frequencies of the genotypes for XRCC4 C-1622T, G-1394T, G-652T, C-571T, intron 3 DIP, S247A and intron 7 DIP for controls and CRC patients are shown in Table II. Genotype distribution of various genetic polymorphisms of G-1394T was significantly different between the CRC and control groups (p=0.0003), while those for C-1622T, G-652T, C-571T, S247A and intron 3 DIP and intron 7 DIP were not different (p>0.05) (Table II).
The frequencies of the alleles for the XRCC4 C-1622T, G-1394T, G-652T, C-571T, intron 3 DIP, S247A and intron 7 DIP between controls and CRC patients is shown in Table III. Allelic frequency distribution of the XRCC4 promoter G-1394 was significantly different between the CRC and control groups (p=8.32×10−5), while those of the others were not (Table III). The conclusion deduced from Table II and III is that XRCC4 promoter -1394 G allele seems to be associated with a higher risk for CRC.
Discussion
To our knowledge, there are no studies investigating the role of XRCC4, which plays an important role in the NHEJ pathway, in CRC. According to our results, the G-1394T polymorphism of the XRCC4 gene was found for the first time to be associated with CRC (Tables II and III). According to previous study, XRCC4 is such an essential protein in taking care of our genome that critical mutations of XRCC4 will lead to cell apoptosis and possibly fatality during the embryonic stage (20). The variant polymorphism of G-1394T of XRCC4 may slightly alter the normal expression level of XRCC4. These changes may be very subtle initially, and cause no obvious pathological change in the human body without alerting the cell via its apoptosis checking system, such as the cell cyclins and their related kinase system. If the cells undergo severe DNA damage, the cell cycle checking points will arrest resulting in either DNA repair or apoptosis. The effects may only lead to a lower capacity of the NHEJ in which the XRCC4 gene is involved. After birth, exposure to environmental carcinogens increases the possibility for genomic instability, and the need for the intracellular repair system to stem this DNA damage will increase too. In those people whose NHEJ repair system cannot perform at its normal efficiency, the raised instability of their genome can be expected. With time, these genetic deficiencies will increase the number of abnormal cells, and thus raise the risk of CRC.
It is known that DSBs are one of the most severe types of DNA damage, and if the DSBs cannot be repaired before the duplication of the genome, they will result in irreversible cellular injuries, which increases the possibility of CRC carcinogenesis, and perhaps of other types of cancer too. Some reports showed that variations of the XRCC4 gene are associated with many other types of cancer (11-15, 21). The finding that the G allele of XRCC4 gene is a risk factor for CRC is consistent with the case for gastric cancer (14). A previous study showed that intestinal-type gastric carcinoma shared common pathways with CRC, indicating that the clinical subtypes of gastric cancer and CRC are both important in genetic study of cancer (22). Moreover, defects in XRCC4 function and failures of the DNA DSB repair pathway may serve as common biomarkers for early carcinogenesis detection. The phenotypes related to DNA DSB repair capacity, and the correlation between genotype and phenotype need further investigation.
In this study, we have screened seven SNPs of the XRCC4 gene and investigated their associations with susceptibility to CRC. We found that the G-1394T polymorphism of the XRCC4 gene appears to be associated with CRC risk and that the G allele is a risk factor. However, the effects of these SNPs of XRCC4 gene need further study, such as promoter assays, to reveal the role of each section or even each nucleotide in the subtle regulation of the transcriptional, translational, and post-translational expression of the XRCC4 gene. In addition, a larger population size for stratification of factors such as gender, anatomic distributions of CRC, and genomic-environmental lifestyle combinatorial studies could provide a better understanding of the complex relationship between XRCC4 and CRC carcinogenesis.
Acknowledgements
This study was supported by research grants from the China Medical University and Hospital (DMR-99-049 and CMU-97-333), Terry Fox Cancer Research Foundation and the National Science Council (NSC 98-2320-B-039-010-MY3).
- Received May 10, 2010.
- Revision received May 21, 2010.
- Accepted May 26, 2010.
- Copyright© 2010 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved