Genetic association between selected cytokine genes and glioblastoma in the Han Chinese population

Glioblastoma (GBM) is one of the most malignant and deadly brain tumors, and occurs more commonly in adults, especially in males. According to the classification of World Health Organization, it is classified as the highest grade of IV. Although GBM has been researched for many years, the etiology of it remains unclear. It possibly arises from genetic and epigenetic alterations in normal astroglial cells [1], implying that the genetic factors play the mainly role in GBM genesis.

Cytokines play a significant role in cancer diagnosis, prognosis and therapy. Current studies suggest that the occurrence and development of tumors such as glioma, gastric cancer and breast cancer are associated with cytokine genes [2‐5]. Many abnormal secretion and expression of cytokines have been found in GBM. To examine whether cytokine genes also contribute to risk of GBM, we selected seven tag single nucleotide polymorphisms (tSNPs) in six cytokine genes, which previously reported to be associated with glioma susceptibility [2, 4, 6‐10], to perform the study in the Han Chinese population using a case–control study.

A multiplexed SNP MassEXTEND assay was designed with the Sequenom MassARRAY Design 3.0 Software. Seven tSNPs in the cytokine genes were included in GBM cases and controls. A total of 374 participants, including 72 GBM cases (44 males, 28 females; mean age at diagnosis 44 ± 15) and 302 controls (119 males, 183 females; mean age 46 ± 18) were successfully genotyped for further analysis. All of the tested tSNPs are in Hardy-Weinberg equilibrium (HWE) in the controls of this study (Table 1). The average tSNPs call rate was 99.12% in both cases and controls. χ² test revealed one tSNP was significantly associated with GBM risk at a 5% level (rs1801275, IL4R, OR = 1.71, 95% CI, 1.00 - 2.92, p = 0.047).

We assumed that the minor allele of each tSNP was a risk factor compared to the wild-type allele. MAF of cases and controls are listed in Table 1. Genetic models were applied for analyzing the association between tSNPs and GBM risk by unconditional logistic regression analysis, which adjusted for age and gender. Our results showed that the genotype “TC” of rs20541 in IL13 gene was associated with an increased risk of GBM in over-dominant model (OR = 2.00, 95% CI, 1.13- 3.54, p = 0.015). The genotype “CT” of rs1800871 in IL10 gene showed a decrease risk in the over-dominant model (OR = 0.57, 95% CI, 0.33 – 0.97, p = 0.037). The genotype “AG” of rs1801275 in the IL4R gene showed an increase risk in the over-dominant model (OR = 2.29, 95% CI, 1.20 – 4.35, p = 0.0081), (Tables 2, 3 and 4).

We further analyzed whether the seven tSNPs have a different effect on GBM risk in gender specific population, and found that the allele “G” of rs2243248 in the IL-4 gene showed a decrease risk in female (OR = 0.35, 95% CI,0.13 - 0.94, p = 0.0032), but the allele “T” showed a decrease risk in male (OR = 0.30, 95% CI, 0.17 - 0.53, p = 0.0032) (Table 5).

We genotyped seven tSNPs in this case–control study in the Han Chinese population, and found two risk tSNPs and one protective tSNP using genetic model analysis. In addition, we also found one tSNP have different risk effect on GBM in gender specific population. All the results suggested that the polymorphisms of these cytokine genes may play an important role in the risk of GBM in the Han Chinese population.

Interleukins are a part of cytokine, encoded by interleukin genes and produced by a variety of cells. They can deliver information, activate and regulate immune cells, mediate T and B cells activation, proliferation and differentiation. Cytokines play a significant role in cancer diagnosis, prognosis and therapy. The immune system’s failure to recognize the malignant tumor cells and perform an effective response may be the result of tumor-associated cytokine deregulation [15].

IL10 (interleukin-10) has pleiotropic effect in immunoregulation and inflammation, which plays a key role in immunosuppressive and antiangiogenic process, suggesting its possible involvement in carcinogenesis [16]. It has been demonstrated that polymorphisms of the IL-10 gene are associated with multiple cancer, such as gastric cancer, non-small cell lung cancer and breast cancer [3, 17‐20], and our results indicating that the polymorphisms of IL-10 are associated with GBM.

IL13 encodes IL-13, an immunoregulatory cytokine produced primarily by activated Th2 cells. IL-13 is thought to the pathogenesis of allergen-induced asthma [21]. Besides, the polymorphisms of IL-13 involved in some other diseases, such as eczema, allergic rhinitis [22, 23]. GBM etiology remains unclear, but IL-13 has been shown to be over expressed in a majority of glioma cell lines and GBM tumor tissues [15]. There are consistent reports of inverse association between risk of adult glioma and personal history of allergy and autoimmune disease, but the molecular mechanism still unclear, there still need further investigate.

IL-4 is a ligand for interleukin-4 receptor, inducing macrophage activation and synergizing with colony-stimulating factors in promoting the growth of hematopoietic cells. Previously researches have suggested the IL-4 polymorphisms were significantly associated with the risk of adult glioma [4]. Another report showed that IL-4 induced an aberrant activation of Stat3 in GBM cells but not in normal human astrocytes, and speculated that IL-4 induce aberrant activation of Stat3 may contribute to the pathogenesis of GBM cells [8].

IL4R encodes the alpha chain of the interleukin-4 receptor that can bind interleukin 4 and interleukin 13 to regulate IgE production [24]. A soluble form of the encoded protein can inhibit IL-4 mediated cell proliferation. In our study rs1801275 in the IL4R gene can predict 2.29-fold GBM susceptibility by the over-dominant model. In addition, another article also reported rs1801275 could increase the risk of GBM (OR = 1.61, 95% CI, 1.05 – 2.47) in a population-based case–control study [25], it is consistent with our results that IL-4 gene are associated with GBM.

Helper T (Th) cell can secret multiple cytokines. Th1 cell mainly produced IL-2, IFN-γ and TNF, mediating cellular immune response and involving in delayed type hypersensitivity [26]. Th2 cell secreted IL-4, IL-6, IL-10 and IL-13, mainly mediating humoral immune response [26]. Under normal circumstance, Th1 and Th2 cytokines are in dynamic equilibrium. In the anti-tumor immunity Th1 cytokines should have a more important role. But in glioma tissues, there is obviously predominant expression of Th2 type cytokines, it may result tumor cells escape from immune response, so the abnormal secretion of IL-4, IL-10 and IL-13 may play an important role in the occurring and developing of human glioma [27, 28]. Besides, previously results showed that IL4, IL4R and IL13 genes may play an important role in glioma survival [29]. All of these suggest multiple cytokines are associated with tumor development and progression survival.

In addition, gender difference should be considered in the association analysis, because many genes have been demonstrated function differently in male and female. Such as 5-HTTLPR gene, females with the l/s genotype showed higher anxiety than those with the s/s genotype in both state and trait anxiety. Oppositely, males with the s/s genotype showed high anxiety than those with the l/s genotype [30]. The human gene BDNF genotyping 196G/G carriers can increase the risk of multiple sclerosis only in females, but not in males [31]. However, few researches take gender difference into consideration in association analysis of susceptibility gene. We analyzed whether cytokine genes have different effect on GBM in gender specific population, and found that the allele “G” of rs2243248 in the IL-4 gene showed a decrease risk of GBM in female (OR = 0.35, 95% CI, 0.13 – 0.94, p = 0.0032), but the allele “T” showed a decrease risk in male (OR = 0.30, 95% CI, 0.17 – 0.53, p = 0.0032). We speculated that the expression of IL-4 polymorphisms maybe regulated by sex hormone.

In conclusion, our findings combined with previously results, suggest that the polymorphisms of cytokine genes have potential role in GBM development, and we advocate that gender difference should be taken into consideration in research of susceptible gene. However, the exact function of the polymorphisms of the genes and the regulatory mechanism for gene expression have not been researched clearly, the molecular mechanisms still need to be further investigated.