Cytotoxic T-Lymphocyte Antigen-4 +49G/A Polymorphism Is Associated with Increased Risk of Osteosarcoma

Abstract

Background: Cytotoxic T-lymphocyte antigen-4 (CTLA-4) is a potent immunoregulatory molecule that suppresses anti-tumor response by down-regulating T-cell activation. CTLA-4 gene +49G/A polymorphism (rs231775) has been reported to be associated with various cancers. Objective: The objective of this study is to investigate the association between CTLA-4 gene +49G/A polymorphism and the susceptibility to osteosarcoma in the Chinese population. Methods: The CTLA-4 variant +49G/A was detected by polymerase chain reaction-restriction fragment length polymorphism in 205 osteosarcoma cases and 216 age-matched healthy controls. Data were analyzed using the Chi-square test. Results: Frequencies of the CTLA-4 +49AA genotype and the +49A allele were significantly increased in patients with osteosarcoma compared to healthy controls (OR 2.27, p=0.010, and OR 1.41, p=0.015). Conclusions: Our data suggest that the +49G/A polymorphism of CTLA-4 gene is associated with increased susceptibility to osteosarcoma.

Introduction

Osteosarcoma is the primary malignancy of bone that has peak occurrences in adolescence and after 50 years of age (Dorfman and Czerniak, 1995). Multiple genetic and environmental factors play roles in osteosarcoma pathogenesis. Since the main mechanism of defense against tumor depends on T cell response, polymorphisms in genes coding for T cell response molecules may potentially affect the development of cancers (Ligers et al., 2001).

Cytotoxic T-lymphocyte antigen-4 (CTLA-4 and also known as CD152, Gene ID: NM_005214.3), a member of the immunoglobulin superfamily, is a costimulatory molecule expressed by activated T cells and has the function of down-regulating T-cell activation (Hodi et al., 2003; Phan et al., 2003). Studies have shown that CTLA-4 can affect T cell responses in animal tumor models and cancer immunotherapy trials in humans (Leach et al., 1996; Egen et al., 2002; Hodi et al., 2003). Several mechanisms of CTLA-4 function have been proposed, including ligand competition with the positive T-cell costimulatory CD28 molecule, interference of TCR signaling, and inhibition of cyclin D3 and cyclin-dependent kinases (cdk4/cdk6) production (Masteller et al., 2000; Greenwald et al., 2002; Chikuma et al., 2003). A recent study showing that the CTLA-4 molecule is expressed and functional on human tumor cells such as osteosarcoma and breast cancer indicates that CTLA-4 may affect these cancers through multiple pathways (Contardi et al., 2005).

The CTLA-4 gene, located on chromosome 2q33, consists of four exons that encode separate functional domains: leader sequence, extracellular domain, transmembrane domain, and cytoplasmic domain (Ligers et al., 2001; Ueda et al., 2003). More than 100 single nucleotide polymorphisms (SNPs) have been identified in the CTLA-4 gene region (Ueda et al., 2003), in which the +49G/A mutation (rs231775) has been reported as one of the most important polymorphisms. The +49G/A polymorphism, located at position +49 in exon 1, causes an amino acid exchange (alanine to threonine) in the peptide leader sequence; and the +49A at this position is correlated with high expression of the CTLA-4 protein (Ligers et al., 2001). Previous studies examined the association of this polymorphism with the susceptibility to different autoimmune diseases and malignancy diseases and identified that the +49AA genotype was associated with increased risk of multiple types of cancer such as lung cancer, breast cancer, and cervical cancer (Ghaderi et al., 2004; Erfani et al., 2006; Hadinia et al., 2007; Sun et al., 2008). However, the relationship between this CTLA-4 mutation and osteosarcoma occurrence remains unknown. There have been no reports about the prevalence of CTLA-4 mutation in osteosarcoma. Clarification of the relationship between CTLA-4 gene mutation and osteosarcoma may indicate a role of CTLA-4 function in the etiology of osteosarcoma and provide clues that might help to guide treatment of this disease. To clarify this relationship, we have analyzed the CTLA-4 gene +49G/A polymorphism in clinical cases of osteosarcoma and normal controls.

Materials and Methods

Patients and controls

The study group included 205 newly diagnosed osteosarcoma cases (aged 11-69 years) recruited from the General Hospital of Jinan Military Command and the Shandong Cancer Hospital and Institute from January 2001 to July 2009. All cases were diagnosed without a familial cancer syndrome.

A total number of 215 controls (aged 14-63 years) were recruited from healthy volunteers who visited the three hospitals from July 2001 to July 2009 for general health exams. All the control subjects were matched with the patient population in terms of age, sex, and residence area (urban or rural). All subjects were unrelated ethnic Han Chinese. Written informed consent was obtained from each participant. The study was approved by the Review Boards of the General Hospital of Jinan Military Command and the Shandong Cancer Hospital and Institute. Each study participant provided a peripheral blood sample.

DNA extraction and genotyping

Genomic DNA was extracted from 2 mL frozen whole blood using the DNA Extraction Kit (Fastagen) according to the manufacturer's protocol. A polymerase chain reaction (PCR)-restriction fragment length polymorphism assay was used to detect dimorphism of +49 A/G (rs231775). This SNP was identified from NCBI dbSNP (NCBI Human Genome Build 36.3). The polymorphic region was amplified by PCR with a Thermoblock PCR System (Eppendorf) in a 25 μL reaction solution containing 50 ng genomic DNA, 12.5 μL 2×PCR Mix buffer (Fermentas), and 0.1 mol of each primer (F: 5′-AAGGCTCAGCTGAACCTGGT-3′ and R: 5′-CTGCTGAAACAAATGAAACCC-3′) (Shenggong). PCR products were digested with restriction enzyme Eco9II (NEB, UK; Fermentas) according to the manufacturer's protocol and analyzed by 10% polyacrylamide gel electrophoresis or 2%-3% agarose gel electrophoresis. To confirm the genotyping results, 20% of PCR-amplified DNA samples were examined by DNA sequencing. Results between PCR and DNA sequencing analysis were 100% concordant.

Statistical analysis

The SPSS statistical software package ver.19.0 (SPSS Inc.) was used for statistical analysis. Demographic data between the study groups were compared by the chi-square test and by the Students t-test. The polymorphism was tested for deviation from Hardy-Weinberg equilibrium by comparing the observed and expected genotype frequencies using the chi-square test. For SNP analysis, genotype and allele frequencies of CTLA-4 were compared between groups using the chi-square test, and odds ratios (OR) and 95% confidence intervals (CIs) were calculated using unconditional logistic regression. p-values <0.05 were considered significant.

Results

The CTLA-4 +49G/A polymorphism in osteosarcoma cases and healthy controls

A total of 205 osteosarcoma cases and 216 controls were recruited for the present study. All subjects were ethnic Chinese. Demographic and other selected characteristics of the cases and controls were presented (Table 1). Cases and controls did not show statistically significant differences with regard to age, sex, tumor location, and presence of metastasis.

Table 1.

General Characteristics of the Subjects

	Osteosarcoma (n=205) (%)	Healthy control (n=216) (%)	p
Age
≤20	132 (64.4)	116 (53.7)
>20	73 (35.6)	100 (46.3)
Mean age	27.1±17.7	32.4±18.2	0.406
Gender
Male	119 (58.0)	113 (52.3)	0.237
Female	86 (42.0)	103 (47.7)
Tumor location
Long tubular bones	152 (74.1)
Axial skeleton	53 (25.9)
Metastasis
Yes	39 (19.0)
No	166 (81.0)

We analyzed the association of CTLA-4 +49G/A polymorphism in 205 patients with osteosarcoma and 215 healthy controls (Table 2). The SNP genotyped were in HWE (p>0.05). CTLA-4 +49AA genotype frequency was significantly higher in patients than in controls (OR 2.27, 95% CI 1.21-4.25, p=0.010), and the CTLA-4 +49A allele carrier frequency was also significantly higher in patients than in controls (OR 1.41, 95% CI 1.07-1.87, p=0.015). These data suggest that CTLA-4 +49AA genotype and +49A allele are associated with increased susceptibility to osteosarcoma in the Chinese population.

Table 2.

Genotype and Allele Frequencies of Cytotoxic T-Lymphocyte Antigen-4 +49 Polymorphism in Osteosarcoma Cases and Healthy Controls

Frequencies	Cases (%) n=205	Controls (%) n=216	OR (95% CI)	p
+49 G/A
GG	64 (31.2)	87 (40.3)	1.00
GA	106 (51.7)	108 (50.0)	1.33 (0.88-2.03)	0.178
AA	35 (17.1)	21 (9.7)	2.27 (1.21-4.25)	0.010^a
G	234 (57.1)	282 (65.3)	1.00
A	176 (42.9)	150 (34.7)	1.41 (1.07-1.87)	0.015^a

p<0.05.

CTLA-4 +49G/A polymorphism and clinical-pathological characteristics in patients with osteosarcoma

We further evaluated the association between CTLA-4 +49G/A polymorphism and clinicopathological factors in patients with osteosarcoma. The stratification analysis including age, gender, tumor location, and metastasis are shown (Table 3). None of the data reached significant difference. These indicated that the +49G/A mutation is not associated with the clinicopathological factors such as age, gender, tumor location, and metastasis in patients with osteosarcoma.

Table 3.

Stratification Analysis of Cytotoxic T-Lymphocyte Antigen-4 +49 Polymorphism According to Clinicopathological Characteristics in Patients with Osteosarcoma

	Age				Gender				Location				Metastasis
Frequencies	≤20	>20	OR 95% CI	p	Male	Female	OR 95% CI	p	L	A	OR 95% CI	p	Yes	No	OR 95% CI	p
+49 G/A
GG	40	24	1.00		39	25	1.00		51	13	1.00		11	53	1.00
GA	69	37	1.12 (0.59-2.13)	0.733	63	43	0.94 (0.50-1.77)	0.846	76	30	0.65 (0.31-1.36)	0.246	21	85	1.19 (0.53-2.67)	0.671
AA	23	12	1.15 (0.49-2.72)	0.751	17	18	0.61 (0.26-1.39)	0.235	25	10	0.64 (0.25-1.65)	0.352	7	28	1.12 (0.42-3.45)	0.729
G	149	85	1.00		141	93	1.00		180	56	1.00		43	191	1.00
A	115	61	1.08 (0.71-1.62)	0.727	97	79	0.81 (0.55-1.20)	0.296	124	50	0.77 (0.49-1.20)	0.252	35	141	1.10 (0.67-1.81)	0.700

L, long tubular bones; A, axial skeleton.

Discussion

Antitumor T lymphocytes play a pivotal role in immunosurveillance of malignancy (Pure et al., 2005). CTLA-4 is a potent immunoregulatory molecule that suppresses anti-tumor response by down-regulating T-cell activation (Hodi et al., 2003; Phan et al., 2003). Also, it has been shown that the CTLA-4 molecule is expressed and functional on human tumor cells such as osteosarcoma and breast cancer (Contardi et al., 2005). All these indicate that changes in CTLA-4's expression or function may influence the development of tumors. The current study reports, for the first time, that polymorphisms in CTLA-4 gene could be a risk factor in the development of osteosarcoma.

The immune system is a complex network that has evolved to protect humans against infectious agents and tumor growth. T-cells and natural killer cells are the major anti-tumor factors. Given that the activation of T-cells requires two signals, the CD28 costimulatory pathway has been shown to play a critical role in the induction and regulation of auto-reactive T-cells. Further, the human CTLA4 and CD28 genes are located in the same chromosome region and are closely linked and separated by only 130kb (Buonavista et al., 1992). The gene structure of CTLA4 is very similar to that of CD28, except for 3′ and 5′ flanking sequences. All these data suggest that CTLA-4 and CD28 may be members of the same pathway, but execute different functions. Several groups found that CTLA-4 binds to the same ligands as CD28, that is, CD80 and CD86 molecules, but has at least a 20-fold greater affinity (Linsley and Ledbetter, 1993). In contrast to CD28, CTLA-4 does not provide a positive signal for T-cell activation. Thus, CTLA-4 may contribute to the down-regulation of anti-tumor immune responses via interference with the CD28 costimulatory pathway (Linsley and Ledbetter, 1993). In addition, a study showed that CTLA-4 is expressed on tumor cells such as osteosarcoma and breast cancer, and it can trigger apoptosis on a high dosage of ligand interaction (Contardi et al., 2005). These data indicate that CTLA-4 on these tumor cells is functional and changes in CTLA-4 might affect the development of tumors in a different pathway.

It has been shown that SNPs in CTLA-4 may have functional significance (Ligers et al., 2001; Bouqbis et al., 2003). The +49A allele, which causes an amino acid exchange (from alanine to threonine) in the peptide leader sequence, results in higher mRNA efficiency and increased CTLA-4 production than the 49G allele (Ligers et al., 2001). The +49 G/A SNP is associated with different cancers such as breast cancer, lung cancer, colorectal cancers, and so on (Erfani et al., 2006; Hadinia et al., 2007; Sun et al., 2008). Our study showed that CTLA-4 gene +49AA genotype and +49 allele are associated with higher risk of osteosarcoma in Chinese population. According to the HapMap Project and Environmental Genome Project, the frequencies of the CTLA-4 49A allele are 0.793 and 0.675, respectively, in European Caucasians and Africans (sub-Saharan Africans and African Americans) but 0.331 in Asians. It would be interesting and important to conduct independent studies in other ethnic populations for comparison.

In summary, the current study demonstrated, for the first time, that the CTLA-4 gene +49AA genotype and +49A allele are associated with the development of osteosarcoma in Chinese populations. Our results raise the possibility that a function shift caused by genetic variants in immunity genes could have important consequences for the pathogenesis of osteosarcoma.

Footnotes

Acknowledgments

We thank all patients and normal individuals for participating in this study.

Disclosure Statement

No competing financial interests exist.

References

Bouqbis

, Izaabel

, Akhayat

et al. 2003. Association of the CTLA-4 promoter region (-1661G alleles) with type 1 diabetes in the south Moroccan population. Genes Immun, 4:132-137.

Buonavista

, Balzano

, Pontarotti

et al. 1992. Molecular linkage of the human CTLA4 and CD28 Ig-superfamily genes in yeast artificial chromosomes. Genomics, 13:856-861.

Chikuma

, Imboden

, Bluestone

. 2003. Negative regulation of T cell receptor-lipid raft interaction by cytotoxic T lymphocyte-associated antigen 4. J Exp Med, 197:129-135.

Contardi

, Palmisano

, Tazzari

et al. 2005. CTLA-4 is constitutively expressed on tumor cells and can trigger apoptosis upon ligand interaction. Int J Cancer, 117:538-550.

Dorfman

, Czerniak

. 1995. Bone cancers. Cancer, 75,1 Suppl:203-210.

Egen

, Kuhns

, Allison

. 2002. CTLA-4: new insights into its biological function and use in tumor immunotherapy. Nat Immunol, 3:611-618.

Erfani

, Razmkhah

, Talei

et al. 2006. Cytotoxic T lymphocyte antigen-4 promoter variants in breast cancer. Cancer Genet Cytogenet, 165:114-120.

Ghaderi

, Yeganeh

, Kalantari

et al. 2004. Cytotoxic T lymphocyte antigen-4 gene in breast cancer. Breast Cancer Res Treat, 86:1-7.

Greenwald

, Oosterwegel

, van der

Woude D

et al. 2002. CTLA-4 regulates cell cycle progression during a primary immune response. Eur J Immunol, 32:366-373.

10.

Hadinia

, Hossieni

, Erfani

et al. 2007. CTLA-4 gene promoter and exon 1 polymorphisms in Iranian patients with gastric and colorectal cancers. J Gastroenterol Hepatol, 22:2283-2287.

11.

Hodi

, Mihm

, Soiffer

et al. 2003. Biologic activity of cytotoxic T lymphocyte-associated antigen 4 antibody blockade in previously vaccinated metastatic melanoma and ovarian carcinoma patients. Proc Natl Acad Sci USA, 100:4712-4717.

12.

Leach

, Krummel

, Allison

. 1996. Enhancement of antitumor immunity by CTLA-4 blockade. Science, 271:1734-1736.

13.

Ligers

, Teleshova

, Masterman

et al. 2001. CTLA-4 gene expression is influenced by promoter and exon 1 polymorphisms. Genes Immun, 2:145-152.

14.

Linsley

, Ledbetter

. 1993. The role of the CD28 receptor during T cell responses to antigen. Annu Rev Immunol, 11:191-212.

15.

Masteller

, Chuang

, Mullen

et al. 2000. Structural analysis of CTLA-4 function in vivo. J Immunol, 164:5319-5327.

16.

Phan

, Yang

, Sherry

et al. 2003. Cancer regression and autoimmunity induced by cytotoxic T lymphocyte-associated antigen 4 blockade in patients with metastatic melanoma. Proc Natl Acad Sci USA, 100:8372-8377.

17.

Pure

, Allison

, Schreiber

. 2005. Breaking down the barriers to cancer immunotherapy. Nat Immunol, 6:1207-1210.

18.

Sun

, Zhou

, Yang

et al. 2008. Functional genetic variations in cytotoxic T-lymphocyte antigen 4 and susceptibility to multiple types of cancer. Cancer Res, 68:7025-7034.

19.

Ueda

, Howson

, Esposito

et al. 2003. Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease. Nature, 423:506-511.