DNA Double Strand Break Repair Gene XRCC7 Genotypes were Associated with Hepatocellular Carcinoma Risk in Taiwan Males and Alcohol Drinkers
Yi-Hsien Hsieh1,2*, Wen-Shin Chang3,4*, Chia-Wen Tsai3, Jen-Pi Tsai5, Chin-Mu Hsu3, Long-Bin Jeng3 and
Da-Tian Bau3,4
1Department of Biochemistry, School of Medicine, Chung Shan Medical University, Taichung, Taiwan,
R.O.C.;
2Clinical laboratory, Chung Shan Medical University Hospital, Taichung, Taiwan, R.O.C.;
3Terry Fox Cancer Research Laboratory, China Medical University Hospital, Taichung, Taiwan, R.O.C.; 4Graduate Institute of Clinical Medical Science, China Medical University, Taichung, Taiwan, R.O.C.; 5Department of Nephrology, Buddhist Dalin Tzu Chi General Hospital, Chiayi, Taiwan.
*The two Authors contributed equally to this work
Correspondence to: Da-Tian Bau, Terry Fox Cancer Research Lab, China Medical University Hospital, 2 Yuh-Der Road, Taichung, 404 Taiwan, R.O.C. Tel: +886 422052121 Ext. 7534, e-mail:
Abstract Hepatocellular carcinoma (HCC) is fifth most common cancer worldwide, for which the prevalence and mortality rates are very high in Taiwan. The study aimed at evaluating the contribution of
XRCC7 G6721T, together with cigarette smoking and alcohol drinking lifestyles to the risk of HCC. In this hospital-based case—control study, association of XRCC7 single nucleotide polymorphism G6721T with
HCC risk were examined by PCR-RFLP among 298 HCC patients and 889 age- and gender matched
healthy controls. The results showed that the percentages of TT, GT and GG XRCC7 G6721T were 53.0%,
41.3% and 5.7% in the HCC patient group and 48.9%, 43.1% and 8.0% in non-cancer control group,
respectively. We have further stratified the populations by genders, cigarette smoking and alcohol drinking
status to investigate their combinative contributions with XRCC7 G6721T genotype to HCC risk. The
results showed that the GG genotype of XRCC7 G6721T conducted a protective effect on HCC
susceptibility which was obvious among males and drinkers, but not females, smokers, non-smokers or
non-drinkers (p=0.0058, 0.0069, 0.1564, 0.2469, 0.9354 and 0.3416, respectively). Our results suggested
that the GG and GT genotypes of XRCC7 G6721T had a protective effect on HCC risk in Taiwan,
particularly among males and alcohol drinkers.
Introduction
Liver cancer is the fifth cancer worldwide and the third most common cause of cancer mortality.
Statistically, over 80% of the liver cancer cases were from low income or middle-income countries, and
about 50% were from China population alone [1]. Clinically, hepatocellular carcinoma (HCC) is the main
type of liver cancer, accounting for over 90% of cases. Environmental factors for HCC included chronic
infections with hepatitis B and C viruses, alcohol and alcohol-related cirrhosis, tobacco consumption,
overweight, diabetes and contamination of cereal foodstuff with aflatoxin in selected countries [2-4].
Useful biomarkers for identifying high-risk populations as well as novel early detection and prediction
tools together with preventive care are in urgently need. Low-penetrance susceptibility genes combined
with environmental factors have been believed to play an important role in carcinogenesis, and subtle
polymorphic defects in the DNA repair may ruin the preventive system and initiate and progress the
tumorigenesis.
One of the most detrimental forms of DNA damage is the double strand break (DSB), because DNA
may lose its physical integrity and information content on both strands in this case [5]. Homologous
recombination (HR) and non-homologous end joining (NHEJ) are the two important pathways for
removing the DSBs induced by endogenous and exogenous carcinogens. HR, which acts during the
transition of S to G2 phases of the cell cycle, entails copying the missing information from an undamaged
homologous chromosome. In NHEJ, which acts during all phases of the cell cycle, the broken DNA termini
repair proteins, human X-ray repair cross-complementing group 7 (XRCC7) (MIM: 600899; Genbank
accession no: NM_001469) plays a central role encoding the catalytic subunit of DNA-activated protein
kinase (DNA-PKcs) of NHEJ pathway [6]. It should be noted that NHEJ is the dominant subpathway for
DSB repair in human cells [6]. DNA-PK complex is also known as PRKDC, HYRC, and HYRC1.
Whenever DSB is formed and detected, DNA-PKcs is recruited to the DSBs by the KU70/KU80
heterodimer to form an active DNA-PK complex that is essential for the progression of the NHEJ pathway
[7]. Deficiencies in DNA-PK activity are clinically significant and mice with inactivated components of
DNA-PK show severe combined immunodeficiency as well as ionizing radiation hypersensitivity [8, 9].
There were three some reports investigated the association of XRCC7 polymorphic genotypes with human
diseases such as cancers [10-23], and most of them focused on the three polymorphic variants, rs7003908
(T>G), rs7830743 (A>G), and rs10109984 (T>C). Among them, G6721T of XRCC7 (rs7003908) is located
in the intron 8 of the gene. It is speculated that this polymorphism, may regulate splicing and cause mRNA
instability [7]. Although several studies examined the association of G6721T polymorphism of XRCC7
with several types of cancer have been published [10-14, 16, 20], however, few has been focused on HCC
[21].
As for the overall DSB repair system, some previous reports showed that the genotypes of DSB repair
genes, especially those NHEJ genes, may interact with environmental factors such as smoking or hepatitis
C virus infection in determining the relative risk of HCC [24-26]. In this study, we aimed at revealing the
genotypic frequencies of genotypes of G6721T polymorphism of XRCC7, and focusing on the combined
Materials and methods
Investigated population and sample collection
Two hundred and ninety-eight patients diagnosed with HCC were recruited at the Departments of General
Surgeon at the China Medical University Hospital, Taiwan, in 2004-2010. Each patient and non-cancerous
healthy person completed a self-administered questionnaire and provided their peripheral blood samples.
Originally, three times as many non-cancer healthy volunteers as controls were selected by matching for
age and gender after initial random sampling from the Health Examination Cohort of our hospital. The
exclusion criteria of the controls included previous malignancy, metastasized cancer from other or
unknown origin, and any genetic or familial diseases. The included control population was eight hundred
and ninety-eight. Our study was approved by the Institutional Review Board of the China Medical
University Hospital (DMR103-IRB-094) and written-informed consent was obtained from all participants.
Genotyping conditions
Genomic DNA was extracted from peripheral blood leucocytes using the QIAamp Blood Mini Kit
(Blossom, Taipei, Taiwan) and stored as previously published [27-29]. The paired primers used for
G6721T polymorphism of XRCC7 were: forward TGGTGCTCAGCTTCTGGCTT-3’, and reverse
5’-CATCCCTGCCAGCTCTTCTG-3’. The XRCC7 G6721T polymerase chain reaction (PCR) conditions
extension at 72oC for 10 min.
Restriction fragment length polymorphism (RFLP) conditions
After the PCR process of XRCC7 G6721T, the resultant 301 bp PCR product was mixed with 2 U TaqI
and incubated for 3 h at 65C in CutSmartTM Buffer (New England BioLabs, Taipei, Taiwan). The G form
PCR products could be further digested while the T form could not. Two fragments 235 bp and 66 bp were present if the product was digestible G form. Then, 10 l of product was loaded into a 3% agarose gel containing ethidium bromide for electrophoresis. The genotype analysis was performed by two researchers
independently and blindly. During this process, five of the control samples were excluded since their PCR
products were not sufficient for clear genotyping recognition after PCR-RFLP.
Statistical analyses
Finally, 889 of the controls and 298 cases with genotypic and clinical data were analyzed and the data were
shown in the Tables. Pearson’s Chi-square and Fisher’s exact (when one or more cells were less than five)
tests were used to compare the distribution of the genotypes between case and control groups. Data were
recognized as significant when the statistical p-Value was less than 0.05. The HCC risk associated with the
genotypes was estimated as an odds ratio (ORs) and 95% confidence intervals (CIs) by logistic regression.
Results
controls are summarized in Table 1. Since we have applied frequency matching to select the non-cancer
healthy controls, distributions of age and gender were comparable among the cases and the controls (Table
1). The cases had a significantly higher percentage of smokers (71.5% vs. 65.1%, p=0.0522) and drinkers
(69.1% vs. 58.3%, p=0.0011) than the controls (Table 1).
The distributions of the XRCC7 G6721T genotypes among the controls and the HCC patients are
provided and analyzed in Table 2. The frequencies of TT, GT and TT genotypes at XRCC7 G6721T were
53.0%, 41.3% and 5.7% in the HCC patients and 48.9%, 43.1% and 8.0% in the controls, respectively
(Table 2). Statistically speaking, the XRCC7 G6721T heterozygous GT and homozygous GG genotypes
were not significantly associated with HCC risk.
There were more males than females of HCC patients in Taiwan and we are interested in the
genotypic contribution of XRCC7 G6721T to gender difference of HCC susceptibility. After the
stratification by the gender, it was found that the genotypes of XRCC7 G6721T were differently distributed
among males (p=0.0058) but not females (p=0.1564) (Table 3).
The interaction of the genotype of XRCC7 G6721T and lifestyles such as cigarette smoking and
alcohol drinking of the participants was of great interest since HCC is one of the smoking- and
alcohol-related cancers. The results in Table 4 showed that the genotypic distribution of the variant XRCC7
G6721T genotypes was not different between HCC and control groups among who were ever smokers
(p=0.2469) or those who were non-smokers (p=0.9354) (Table 4). Interestingly, the results in Table 5
showed that the genotypic distribution of the variant XRCC7 G6721T genotypes was different between
non-drinkers (p=0.3416) (Table 5). Overall, it seemed that there was an interaction between XRCC7 G6721T
genotype and drinking lifestyle to the HCC susceptibility.
Discussion
The NHEJ genes such as XRCC4, XRCC5, XRCC6, and XRCC7 take care of the genome integrity and
consequently cell survival. Subtle genetic variations such as single nucleotide polymorphisms in these
genes may escape the cell cycle checking point surveillance, and lead to suboptimal overall DNA repair
allowing DNA damage to accumulate and trigger carcinogenesis [30]. As the catalytic subunit of the
DNA-PK complex, XRCC7 play a role in NHEJ via recognition and repair of the DNA DSBs [31]. Mice with
DNA-PK inactivation were ionizing radiation hypersensitive and immuno-deficient [8, 9]. In addition, cells
defective in DNA-PK components are incapable of repairing DSBs and hypersensitive to ionizing radiation
[32]. The XRCC7 G6721T may regulate alternative splicing and cause the instability of its mRNA, [7], and
further associated with increased cancer risk [10-13, 16, 19]. In this study, we have strengthened our
analyzing power via enrolling a larger population of controls than our previous papers [24, 33, 34]. In
Table 1, the cigarette smoking and alcohol drinking lifestyles were found to be risky environmental factors
for HCC, although the former seemed to be at the bolder line (Table 1). From the results the GT and GG
genotypes of XRCC7 G6721T were not associated with HCC risk in the whole population (Table 2),
however, they seemed to be protective factor of HCC for only the Taiwan males (Table 3). As for the
females, the frequency of wild-type TT genotype was lower in the case group (43.5%) than the control
(TT frequency higher in the cases than in the controls) as in the males (Table 3). As liver cancer is
recognized to be one of the tobacco- and alcohol-related cancers [4, 35], the combined effects of XRCC7
genotypes with smoking and drinking lifestyle on HCC risk in Taiwan were also of interested. As for the
smoking lifestyle, it is found that no association between XRCC7 genotype with HCC risk among either
ever-smokers or non-smokers (Table 4). As for the alcohol drinking lifestyle, the association between
XRCC7 genotypes with HCC risk was obvious among ever drinkers but not non-drinkers (Table 5). The detail explanation of the difference among the subgroups needs further investigation. Very possibly, the
role of XRCC7 in hormone- or alcohol-induced DNA DSBs was somehow different from that in
tobacco-induced DNA DSBs. Overall, although the genetic variation of XRCC7 G6721T was not found to direct
result in an amino acid coding change, it might influenced the expression level or stability of the XRCC7
protein as well as its function in NHEJ and genome maintenance. The low penetration of XRCC7 genetic
variation may contribute to only specific patients’ HCC carcinogenesis so that we could not find a
significant association in the whole HCC population (Table 2) but in special subgroup (Tables 3, 4 and 5).
Overall, male people carrying T allele in XRCC7 G6721T may have lower capacity than those carrying G
allele in DSB removing capacity, especially for alcohol-induced HCC development.
The current study has several limitations and improving directions. First, the hospital-based study
could enhance the representative power by enlarging the sample size of the cases. Second, other
confounding factors such as obesity and virus infection status could not be taken into consideration and
adjusted. In 2011, Long and his colleagues found that XRCC7 G6721T genotype associated with HCC risk
study contained 348 HCC cases and 597 controls, much similar to the level of current study. In vivo studies
could be performed to reveal the contribution of XRCC7 to HCC carcinogenesis such as comparing the
expression alterations at mRNA and protein levels for XRCC7 between the paired tumor- and
non-tumor-sites from the same group of HCC patients with specific XRCC7 genotypes. Also, the role of XRCC7
genotypes among various cancers and populations are still inconclusive in the previous papers and need
further investigations [36].
In conclusion, our findings suggested that G allele of XRCC7 G6721T was associated with lower
Acknowledgements We appreciate Hong-Xue Ji, Chieh-Lun Hsiao, Chia-En Miao and Tissue-bank of
China Medical University Hospital for their excellent technical assistance. This study was supported by
research grants from Terry Fox Cancer Research Foundation and Taiwan Ministry of Health and Welfare
Clinical Trial and Research Center of Excellence (MOHW103-TDU-B-212-113002).
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