Background
Gliomas are the most common type of primary intracerebral neoplasm in China as well as in the West, and comprise more than 40% of primary brain tumors in humans [
1‐
3]. Although the etiology of gliomas remains unclear, exposure to ionizing radiation (IR) and genetic alterations are unequivocally associated with an increased risk of gliomas [
4].
DNA double-strand breaks (DSBs) can be generated during V(D)J recombination, class-switch recombination at the immunoglobulin heavy chain (IgH) locus or meiosis and result from a variety of factors including ionizing radiation and reactive oxygen species [
5]. Inadequacy or defects in DSB repair can lead to large-scale loss of genetic information and can have disastrous consequences such as genomic instability, immunodeficiency, radiosensitivity, cell death and oncogenic transformation [
6,
7]. DSBs are sensed by the MRN (MRE11, RAD50, and NBS1) complex, which catalyzes activation of ATM [
8,
9]. Two major pathways have evolved in mammalian cells to repair DSBs: non-homologous end-joining (NHEJ) and homologous recombination (HR). The central components of the NHEJ pathway are Ku70 (XRCC6), Ku80 (XRCC5), DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4 and DNA ligase IV (LIG4) proteins [
10]. RAD51 interacts with other important repair proteins, including BRCA1, BRCA2, XRCC2, and XRCC3 and plays a central role in the HR activation through the use of sister-chromatid sequences as a template for precise repair [
11].
Recent evidence suggests that several single nucleotide polymorphisms (SNPs) in the DSB repair pathway genes may be prognostic biomarkers for GBM survival and modulate gamma-radiation-induced mutagen sensitivity in glioma patients [
12,
13]. Genetic variants in DSB repair pathway genes have been extensively studied in multiple cancers. However, few studies have specifically identified any association between genetic variations in the DSB repair pathway genes and the risk of gliomas. Here we investigate the role of 10 potential SNPs in
XRCC3,
BRCA2,
RAG1,
XRCC5,
LIG4,
XRCC4 and
ATM in the development of gliomas, and further evaluate their gene-gene and gene-environment interactions in the development of glioma.
Discussion
Accumulating evidence demonstrates that the DSB repair pathway plays a critical role in repairing double-strand breaks caused by a variety of exposures. Although genetic variants in DSB repair pathway genes are considered as potential risk factors for various cancers, less evidence exists as to the potential role of the DSB repair pathway genes polymorphisms on glioma susceptibility. To our knowledge, this study is the first to provide a comprehensive evaluation of the relationship between polymorphisms in both NHEJ and HR pathway genes and susceptibility to gliomas. On the basis of our analysis of 384 controls and 384 glioma patients, we observed that one splice-site SNP in XRCC4 (rs1805377, IVS7-1A>G, splice-site) and one non-synonymous SNP in LIG4 (rs1805388, Ex2 +54C>T, Thr9Ile) are associated with the increased susceptibility to gliomas in a Chinese population.
Previous researches on the function of the
XRCC4 rs1805377 and
LIG4 rs1805388 polymorphisms have been informative in understanding the potential roles of these two polymorphisms in the development of gliomas. The
LIG4 rs1805388 polymorphism results in a nonsynonymous amino acid change from threonine to isoleucine at the N-terminal of the LIG4 protein that is essential for its activity [
15]. Two linked polymorphisms rs1806389 (T9I) and rs1805388 (A3V) in the N-terminal of LIG4 mildly but reproducibly reduce adenylation and ligation activities (2-3fold) [
16] and increase the hydrophobic nature of this region of the protein [
17]. The
XRCC4 rs1805377 polymorphism in intron 7 may have functional significance since the nucleotide change potentially abolishes an acceptor splice site at exon 8 [
18,
19].
Gene-gene interaction was also studied since XRCC4 and LIG4 proteins form a tight and specific complex that catalyzes ligation of processed DNA ends. Although LIG4 interacts with the coiled-coil region of human XRCC4 via the region that lies between the two C-terminal BRCT domains [
15,
20,
21], the combined analysis of multiple SNPs revealed that
LIG4 rs1805388 which causes a nonsynonymous amino acid change at the N-terminal of the LIG4 protein and
XRCC4 rs1805377 interacted to modulate the risk of gliomas as a joint effect. Tseng et al. [
22] performed a gene-gene interaction analysis which revealed that polymorphisms in the
XRCC4 (rs1805377) and
LIG4 (rs1805388) genes interacted to modulate the risk of lung cancer (adjusted OR, 8.75) and demonstrated that
LIG4 rs1805388 and
XRCC4 rs1805377 polymorphisms are linked significantly with high fractional allelic loss (FAL), an indicator of genomic instability. Taken together, considering the functional relevance of these two proteins, an individual SNP or combinations of these two SNPs may change the activity of the LIG4-XRCC4 complex and pose a substantial influence on the development of gliomas.
Overwhelming evidence indicates that our findings are biologically plausible. NHEJ is a multistep process initiated by the XRCC5/XRCC6 dimer (also known as Ku80/Ku70) which immediately binds to both broken ends of DNA and recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) forming the trimeric DNA-PK holoenzyme [
23,
24]. Finally, the LIG4–XRCC4 complex in vivo carries out the ligation step to complete repair [
10]. XRCC4 serves as a multipurpose partner for the LIG4 protein, facilitating LIG4 stability and stimulating LIG4 adenylation [
21]. Consistent with the need for effective repair of DSBs by NHEJ,
XRCC4- or
LIG4-deficient mouse fibroblasts exhibit marked sensitivity to ionizing radiation, growth defects and premature senescence [
25,
26]. The deficiency of DSB repair has led to significant improvements in radiation sensitization of gliomas [
27]. Furthermore,
XRCC4 or
LIG4 null mice die in late embryogenesis accompanied by defective lymphogenesis and massive apoptotic cell death of newly generated postmitotic neurons [
28,
29]. Many studies in the past have shown that the deficiency of
LIG4 or
XRCC4 in animals can lead to increased rates of neoplastic transformation. Although loss of p53 expression rescues neuronal death and embryonic lethality,
XRCC4 or
LIG4/p53 double-null mice routinely succumbed to RAG-dependent pro-B lymphomas with translocations/amplifications of c-myc and IgH loci [
28,
29]. Nijnik et al. found that LIG4
Y288C mice (a mouse model for human LIG4 syndrome) exhibit multiple defects in lymphocyte development and a hypomorphic
LIG4 mutation can confer strong predisposition to lymphoid malignancies [
30]. In addition to tumors of the immune system, Sharpless et al. demonstrated that
LIG4 haploinsufficiency with decreased NHEJ activity contributes to development of soft tissue sarcomas that possess clonal amplifications, deletions and translocations [
31]. A defective DNA double-strand break repair pathway in the nervous system can also lead to brain tumors. Lee et al. demonstrated that
LIG4/p53 double-null mice can develop medulloblastoma [
32]. Consistent with this notion,
XRCC4/p53 doubly deficient in nestin-expressing neuronal progenitor cells can lead to early onset of neuronally differentiated medulloblastomas [
33]. Significant down-regulation of
XRCC4 was found in grade II, III, IV of astrocytoma compared to normal brain tissues and decreased expression of
XRCC4 was significantly associated with a poor prognosis (
P < 0.05) [
34]. These studies raise the possibility that decreased
LIG4 or
XRCC4 activity plays a role in human carcinogenesis.
Since tobacco is a well-confirmed inducer of DNA damage, in particular DSBs [
35], we performed stratified analysis to estimate the interaction between the genotypes and smoking status. As shown in Table
4,
LIG4 rs1805388 were associated with an increased risk of gliomas among smokers under a dominant model. Our data indicated the presence of an interaction between the NHEJ pathway genes and smoking status. In addition, smokers with less efficient DSB repair capacity may be more likely to develop gliomas.
Currently, the number of genome-wide association studies (GWAS) has been growing rapidly, leading to the discovery of many new variants associated with complex diseases. Two recent genome-wide association studies (GWAS) of risk of glioma in European populations did not identify an association between the
XRCC4 rs1805377 and
LIG4 rs1805388 polymorphisms and glioma risk [
36,
37]. There are several possible reasons for the contradictory findings between GWA studies and our present study. First, it might be due to genetic heterogeneity (both allelic and locus heterogeneity) in different ethnic populations or the different reporting criteria for a
P value. Second, the frequencies of XRCC4 rs1805377 and LIG4 rs1805388 polymorphisms and patterns of linkage disequilibrium (LD) are very different in two HapMap populations (CEU and CHB). Thirdly, it could be that the association of this variant may be population-specific and the interaction between genes and environmental factors vary in different human populations. Our results require confirmation in further GWA studies of gliomas in Chinese population.
Our study has several strengths. First, all tested SNPs were in Hardy-Weinberg equilibrium in controls. Second, in this study, a standardized genotyping approach was performed and quality control samples indicated a high degree of reproducibility of the genotyping results. Third, we were able to examine the association between the 2 SNPs and the risk of gliomas in a well-described and racially homogeneous population of the same ethnicity. Moreover, we use a pathway-based approach to estimate the combined effect of LIG4 and XRCC4 genes, which may provide enhanced risk assessment. Finally, we used a relatively comprehensive analysis of 10 polymorphisms in 7 candidate genes involved in DNA double-strand break repair pathways. However, our current study has some limitations. First, we were not able to explore the exact biological mechanism of how XRCC4 rs1805377 and LIG4 rs1805388 polymorphisms affect the development of gliomas. Second, this study was a hospital-based case–control study; thus, selection bias in the present study may have led to spurious findings. However, the controls and cases were matched on age and sex, which may have minimized selection bias. Third, our findings need to be replicated in other independent studies. Fourth, we did not perform the stratified analysis by glioma grade due to the limited sample size. Therefore, large-scale studies and functional evaluation are warranted to replicate these findings in an independent population that is well-powered to performed stratified analysis by glioma grade.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
PZhao and PZou participated in collection of data and manuscript preparation. PZou and LZ performed the statistical analysis. WY, CK, YY and TJ provided the samples. PZhao participated in study design and critically revised the manuscript. PZhao and TJ participated in study design and manuscript preparation. All authors read and approved the final manuscript.