Cytotoxic T-lymphocyte Antigen-4 Polymorphisms and Susceptibility to Osteosarcoma

Abstract

Despite the knowledge of many genetic alterations present in osteosarcoma, the complexity of this disease precludes placing its biology into a simple conceptual framework. Cytotoxic T-lymphocyte antigen-4 (CTLA-4) plays important roles in downregulating T-cell activation, thereby attenuating anti-tumor responses and increasing cancer susceptibility. Polymorphisms in the CTLA-4 gene are associated with different autoimmune diseases and cancers. The current study evaluated the association of four CTLA-4 gene mutations, −1661A/G (rs4553808), −318C/T (rs5742909), +49G/A (rs231775), and CT60A/G (rs3087243), with osteosarcoma in the Chinese population. CTLA-4 polymorphisms were detected by polymerase chain reaction–restriction fragment length polymorphism in 267 osteosarcoma patients and 282 age-matched healthy controls. Results showed that the CTLA-4 gene +49 AA genotype, +49 A allele, and GTAG haplotype were significantly more frequent in osteosarcoma patients than in controls (odds ratio [OR] 2.20, 95% confidence interval [CI] 1.23–2.95, p = 0.007; OR 1.32, 95% CI 1.03–1.69, p = 0.029, and OR = 1.47, 95% CI 1.03–2.09, p = 0.033, respectively). The CTLA-4 +49G/A polymorphism and GTAG haplotype are associated with increased risk of osteosarcoma.

Introduction

O steosarcoma is the most common pediatric bone malignancy in many countries including China and the United States (Ries et al., 1999). Fifty years ago, surgery was the only available treatment, and survival was abysmal at less than 20% (Dahlin and Coventry, 1967). Treatment of patients with osteosarcoma was transformed in the 1980s and 1990s with advances in chemotherapy and orthopedic surgical techniques, leading to long-term survival rates that now approach 70% (Bielack et al., 2002). Unfortunately, further increase in survival has not occurred in the last decade despite multiple attempts at intensification of chemotherapy and trials utilizing new agents (Niswander and Kim, 2010). Therefore, it is necessary to understand the natural history and biology of osteosarcoma to improve our therapeutic approaches.

Several genetic syndromes are associated with osteosarcoma. Hereditary retinoblastoma is caused by germ-line mutations in the tumor suppressor gene RB, which functions by blocking entry of cells into the DNA synthesis (S) phase of the cell cycle (Strahm and Malkin, 2006). Patients with hereditary retinoblastoma have an approximately 100-fold increased risk of developing osteosarcoma, thus strongly implicating RB abnormalities with osteosarcoma (Wadayama et al., 1994; Feugeas et al., 1996; Heinsohn et al., 2007). The Li-Fraumeni syndrome is a hereditary cancer associated with germ-line mutations in the p53 gene, which functions as a sensor of DNA damage or cellular stress (Strahm and Malkin, 2006). Patients with Li-Fraumeni syndrome have an increased risk of osteosarcoma (Strahm and Malkin, 2006).

Cytotoxic T-lymphocyte antigen 4 (CTLA-4, also known as CD152) plays a pivotal role in the negative regulation of T-cell proliferation and activation (Teft et al., 2006). This activation-induced homodimeric glycoprotein receptor on CTLs interacts with B7.1 (CD80)/B7.2 (CD86) ligands on the surface of APCs, resulting in cell cycle arrest and inhibition of cytokine production, which directly eliminates the T-cell proliferation phase (Teft et al., 2006). Furthermore, CTLA-4 may indirectly control effector T cells by its constitutive expression on T-regulatory cells. As an inhibitory mechanism leading to downregulation of T-cell response and peripheral tolerance, besides minimizing harm to normal tissues, CTLA-4 is also able to diminish the generation of effective antitumor responses and thus bring tumor tolerance (Egen et al., 2002). It has been hypothesized that during the early stage of tumorigenesis, CTLA-4 may elevate the T-cell activation threshold, thereby attenuating the anti-tumor response and increasing cancer susceptibility (Egen et al., 2002).

The CTLA-4 gene is located on chromosome 2q33 and 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). Several single-nucleotide polymorphisms (SNPs) in the CTLA-4 gene, −1661A/G (rs4553808), −318C/T (rs5742909), +49G/A (rs231775), and CT60A/G (rs3087243), are associated with different autoimmune diseases and cancers (Bouqbis et al., 2003; Erfani et al., 2006; Wong et al., 2006; Hadinia et al., 2007; Xiao et al., 2010). However, the correlation between CTLA-4 polymorphisms and osteosarcoma remains unclear. The present study investigated the associations of the CTLA-4 −1661A/G, −318C/T, +49G/A, and CT60A/G mutations with osteosarcoma in Chinese population.

Materials and Methods

Study population

The study population consisted of 267 osteosarcoma patients (10–67 years of age) and 282 healthy controls (12–70 years of age), recruited from Changzheng Hospital from July 2006 to July 2010. Patients with familial cancer syndromes were excluded from this study. All subjects were unrelated ethnic Han Chinese. Written informed consent was obtained from each participant. The study was approved by the Review Board of Changzheng Hospital. 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 −1661 A/G, −318 C/T, +49 G/A, and CT60 A/G. These SNPs were 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 2X PCR Mix buffer (Fermentas), and 0.1 mol of each primer (Shenggong). PCR products were digested with restriction enzymes (NEB; Fermentas) according to the manufacturer's protocol and analyzed by 10% polyacrylamide gel electrophoresis or 2%–3% agarose gel electrophoresis. Genotyping primers and restriction enzymes are shown (Table 1). To confirm the genotyping results, 15% of PCR-amplified DNA samples were examined by DNA sequencing. Results between PCR and DNA sequencing analysis were 100% concordant.

Table 1.

Restriction Enzymes and Length of Digested Fragments for CTLA-4 Polymerase Chain Reaction–Restriction Fragment Length Polymorphism

Gene SNP primer	Primer sequence	Restriction enzyme	Length of restriction products (bp)
	F:5′-CTAAGAGCATCCGCTTGCACCT-3′	Mse I	A: 347 + 139
CTLA-4 −1661A/G	R:5′-TTGGTGTGATGCACAGAAGCCTTT-3′		G: 486
	F:5′-AAATGAATTGGACTGGATGGT-3′	Mse I	C: 226 + 21
CTLA-4 −318C/T	R:5′-TTACGAGAAAGGAAGCCGTG-3′		T: 130 + 96+21
	F:5′-AAGGCTCAGCTGAACCTGGT-3′	Eco91I	A:130 + 22
CTLA-4 +49 G/A	R:5′-CTGCTGAAACAAATGAAACCC-3′		G:152
	F:5′-GAGGTGAAGAACCTGTGTGTTAAA-3′	HpyCH4 IV	A: 178
CTLA-4 CT60 A/G	R:5′-ATAATGCTTCATGAGTCAGCTT-3′		G: 107 + 71

Statistical analysis

The SPSS statistical software package ver.13.0 (SPSS) was used for statistical analysis. Demographic data between the study groups were compared by the chi-square test and by Student's t-test. The polymorphisms were 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 (ORs) and 95% confidence intervals (CIs) were calculated using unconditional logistic regression. The linkage disequilibrium (LD) between these polymorphisms and the haplotypes of CTLA-4 (−1661, −318, +49, and CT60) were conducted using the SHEsis software [http://analysis.bio-x.cn/(Bio-X)] (Shi and He, 2005). p-Values less than 0.05 were considered significant.

Results

The CTLA-4 polymorphisms and osteosarcoma susceptibility

A total of 267 osteosarcoma cases and 282 controls were recruited for the present study. All subjects were ethnic Chinese. Demographic and other selected characteristics of the cases and controls are presented (Table 2). Cases and controls did not show statistically significant differences with regard to age and sex (Table 2).

Table 2.

General Characteristics of the Subjects

Characteristics	Osteosarcoma (n = 267) (%)	Control (n = 282) (%)	p-Value
Age
≤20	187 (70.0)	181 (64.2)
>20	80 (30.0)	101 (35.8)	0.145
Mean age	25.1 ± 18.2	29.4 ± 17.7	0.418
Gender
Male	172 (64.4)	156 (55.3)
Female	95 (35.6)	126 (44.7)	0.298
Tumor location
Long tubular bones	213 (79.8)
Axial skeleton	54 (20.2)
Metastasis
With	76 (28.5)
Without	191 (71.5)

The CTLA-4 polymorphisms (−1661 A/G, −318 C/T, +49 G/A, and CT60 A/G) in osteosarcoma patients and healthy controls are summarized in Table 3. All SNPs genotyped were in HWE (p > 0.05). CTLA-4 +49 AA genotype and +49 A allele frequencies were significantly higher in patients than in controls (OR 2.20, 95% CI 1.23–2.95, p = 0.007, and OR 1.32, 95% CI 1.03–1.69, p = 0.029). Analysis of LD between polymorphic sites revealed that the polymoephisms of −1661 and −318 (D′ = 0.922), −318 and +49 (D′ = 0.835), −318 and CT60 (D′ = 0.988), and +49 and CT60 (D′ = 0.871) locus were in strong LD (D′ > 0.80) after Bonferroni adjustment. Also, we analyzed the possible haplotypes including −1661A/G, −318C/T, +49G/A, and CT60 A/G SNPs in all osteosarcoma cases and controls. There were eight observed haplotypes, and the four most common haplotypes (ACGG, ACAA, GTAG, and ACAG) (−1661, −318, +49, and CT60) are shown (Table 3). Among them, a significantly higher frequency of GTAG haplotype was observed in the osteosarcoma group than in controls (OR = 1.47, 95% CI 1.03–2.09, p = 0.033). These data suggest that CTLA-4 +49 AA genotype, +49 A allele, and GTAG haplotype are associated with increased susceptibility to osteosarcoma in the Chinese population.

Table 3.

Genotype, Allele, and Haplotype Frequencies of CTLA-4 Single-Nucleotide Polymorphisms in Osteosarcoma patients and Healthy Controls

Frequencies	Cases (%) (n = 267)	Controls (%) (n = 282)	OR (95% CI)	p-Value
−1661 A/G
AA	177 (66.3)	197 (69.9)	1.00
AG	76 (28.5)	73 (25.9)	1.16 (0.79–1.69)	0.447
GG	14 (5.2)	12 (4.2)	1.30 (0.58–2.88)	0.520
AG+GG	90 (33.7)	85 (30.1)	1.18 (0.82–1.69)	0.370
A	430 (80.5)	467 (82.8)	1.00
G	104 (19.5)	97 (17.2)	1.16 (0.86–1.58)	0.330
−318 C/T
CC	175 (65.5)	195 (69.1)	1.00
CT	77 (28.9)	80 (28.4)	1.07 (0.74–1.56)	0.713
TT	15 (5.6)	7 (2.5)	2.39 (0.95–5.99)	0.057
CT+TT	92 (34.5)	87 (30.9)	1.18 (0.82–1.68)	0.368
C	427 (80.0)	470 (83.3)	1.00
T	107 (20.0)	94 (16.7)	1.25 (0.92–1.70)	0.149
+49 G/A
GG	99 (37.1)	120 (42.6)	1.00
GA	128 (47.9)	140 (49.6)	1.11 (0.77–1.59)	0.574
AA	40 (15.0)	22 (7.8)	2.20 (1.23–3.95)	0.007^a
GA+AA	168 (62.9)	162 (57.4)	1.26 (0.89–1.77)	0.190
G	326 (61.0)	380 (67.4)	1.00
A	208 (39.0)	184 (32.6)	1.32 (1.03–1.69)	0.029^a
CT60 A/G
GG	176 (66.0)	188 (66.7)	1.00
GA	77 (28.8)	83 (29.4)	0.99 (0.68–1.44)	0.962
AA	14 (5.2)	11 (3.9)	1.36 (0.60–3.08)	0.459
GA+AA	91 (34.0)	94 (23.3)	1.03 (0.73–1.47)	0.853
G	429 (80.3)	459 (81.4)	1.00
A	105 (19.7)	105 (18.6)	1.07 (0.79–1.45)	0.660
Haplotype (−1661, −318, +49, CT60)
ACGG	299 (56.0)	338 (59.9)	1.00
ACAA	85 (15.9)	89 (15.8)	1.08 (0.77–1.51)	0.654
GTAG	87 (16.3)	67 (11.9)	1.47 (1.03–2.09)	0.033^a
ACAG	17 (3.2)	22 (3.9)	0.87 (0.46–1.68)	0.684

p < 0.05.

CTLA-4 polymorphisms and clinical parameters of osteosarcoma patients

We further evaluated the association of CTLA-4 −1661 A/G, −318 C/T, +49 G/A, and CT60 A/G polymorphisms with clinicopathological factors in osteosarcoma patients. The stratification analysis including age, gender, tumor location, and metastasis is shown (Table 4). None of the data reached significant difference. These suggest that there is no association between the four mutations and the selected clinical parameters.

Table 4.

Stratification Analysis of CTLA-4 Polymorphisms in Osteosarcoma Patients

Genotype frequency	N (frequency %) Age ≤20 (n = 187)/> 20 (n = 80)	OR (95% CI)	p-Value	N (frequency %) Male (n = 172)/Female (n = 95)	OR (95% CI)	p-Value
−1661 A/G
AA	123 (65.8)/54 (67.5)	1.00		112 (65.1)/65 (68.4)	1.00
AG	54 (28.9)/22 (27.5)	1.08 (0.60–1.94)	0.804	51 (29.7)/25 (26.3)	1.18 (0.67–2.09)	0.560
GG	10 (5.3)/4 (5.0)	1.10 (0.33–3.66)	0.879	9 (5.2)/5 (5.3)	1.10 (0.34–3.25)	0.940
AG+GG	64 (34.2)/26 (32.5)	1.08 (0.62–1.89)	0.785	60 (34.9)/30 (31.6)	1.16 (0.68–1.98)	0.584
−318 C/T
CC	125 (66.8)/50 (62.5)	1.00		108 (62.8)/67 (70.5)	1.00
CT	52 (27.8)/25 (31.3)	0.83 (0.47–1.49)	0.533	54 (31.4)/23 (24.2)	1.46 (0.82–2.59)	0.199
TT	10 (5.4)/5 (6.2)	0.80 (0.26–2.46)	0.696	10 (5.8)/5 (5.3)	1.24 (0.41–3.79)	0.704
CT+TT	62 (33.2)/30 (37.5)	0.83 (0.48–1.43)	0.494	64 (37.2)/28 (29.5)	1.42 (0.83–2.43)	0.203
+49 G/A
GG	68 (36.3)/31 (38.8)	1.00		58 (33.7)/41 (43.2)	1.00
GA	90 (48.1)/38 (47.5)	1.08 (0.61–1.91)	0.792	85 (49.4)/43 (45.3)	1.40 (0.81–2.41)	0.226
AA	29 (15.6)/11 (13.7)	1.20 (0.53–2.71)	0.658	29 (16.9)/11 (11.5)	1.86 (0.84–4.15)	0.125
GA+AA	119 (63.7)/49 (61.2)	1.11 (0.65–1.90)	0.712	114 (66.3)/54 (56.8)	1.49 (0.89–2.50)	0.126
CT60 G/A
GG	119 (63.6)/57 (71.3)	1.00		111 (64.5)/65 (68.4)	1.00
GA	57 (30.5)/20 (25.0)	1.37 (0.75–2.49)	0.308	50 (29.1)/27 (28.4)	1.08 (0.62–1.90)	0.776
AA	11 (5.9)/3 (3.7)	1.76 (0.47–6.54)	0.395	11 (6.4)/3 (3.2)	2.15 (0.58–7.98)	0.244
GA+AA	68 (36.4)/23 (28.7)	1.42 (0.80–2.50)	0.229	61 (35.5)/30 (31.6)	1.19 (0.70–2.03)	0.521

Genotype frequency	N (frequency %) Location: L/A (n = 213)/(n = 54)	OR 95% CI	p-Value	N (frequency %) Metastasis: Yes/No (n = 76)/( n = 191)	OR 95% CI	p-Value
−1661 A/G
AA	142 (66.7)/35 (64.8)	1.00		50 (65.8)/127 (66.5)	1.00
AG	59 (27.7)/17 (31.5)	0.86 (0.44–1.65)	0.640	23 (30.3)/53 (27.7)	1.10 (0.61–1.99)	0.746
GG	12 (5.6)/2 (3.7)	1.48 (0.32–6.92)	0.617	3 (3.9)/11 (5.8)	0.69 (0.19–2.59)	0.583
AG+GG	71 (33.3)/19 (35.2)	0.92 (0.49–1.72)	0.797	26 (34.2)/64 (33.5)	1.03 (0.59–1.81)	0.913
−318 C/T
CC	138 (64.8)/37 (68.5)	1.00		48 (63.2)/127 (66.5)	1.00
CT	62 (29.1)/15 (27.8)	1.11 (0.57–2.17)	0.764	22 (28.9)/55 (28.8)	1.06 (0.58–1.92)	0.852
TT	13 (6.1)/2 (3.7)	1.74 (0.38–8.07)	0.472	6 (7.9)/9 (4.7)	1.76 (0.60–5.22)	0.300
CT+TT	75 (35.2)/17 (31.5)	1.18 (0.62–2.24)	0.607	28 (36.8)/64 (33.5)	1.16 (0.66–2.02)	0.605
+49 G/A
GG	75 (35.2)/24 (44.4)	1.00		24 (31.6)/75 (39.3)	1.00
GA	104 (48.8)/24 (44.4)	1.39 (0.73–2.63)	0.315	36 (47.4)/92 (48.2)	1.22 (0.67–2.23)	0.511
AA	34 (16.0)/6 (11.2)	1.81 (0.68–4.84)	0.231	16 (21.0)/24 (12.5)	2.08 (0.95–4.55)	0.063
GA+AA	138 (64.8)/30 (55.6)	1.47 (0.80–2.70)	0.210	52 (68.4)/116 (60.7)	1.40 (0.80–2.46)	0.241
CT60 G/A
GG	136 (63.8)/40 (74.1)	1.00		47 (61.8)/129 (67.6)	1.00
GA	67 (31.5)/10 (18.5)	1.97 (0.93–4.18)	0.073	24 (31.6)/53 (27.7)	1.24 (0.69–2.24)	0.467
AA	10 (4.7)/4 (7.4)	0.74 (0.22–2.47)	0.618	5 (6.6)/9 (4.7)	1.52 (0.49–4.78)	0.467
GA+AA	77 (36.2)/14 (25.9)	1.62 (0.83–3.16)	0.157	29 (38.2)/62 (32.4)	1.28 (0.74–2.23)	0.786

OR, odds ratio; CI, confidence interval; L, long tubular bones; A, axial skeleton.

Discussion

In the current study, we showed that CTLA-4 polymorphisms were associated with increased susceptibility to osteosarcoma in the Chinese population. CTLA-4 is a potent immunoregulatory molecule that suppresses antitumor responses by downregulating T-cell activation (Egen et al., 2002). Changes in CTLA-4 expression or function may lead to development of tumors; SNPs in CTLA-4 may have functional significance. The −1661A/G and −318C/T mutations are located in the promoter region of the CTLA-4 gene and increase the mRNA level of CTLA-4 (Ligers et al., 2001; Bouqbis et al., 2003). The +49G/A polymorphism, located at position +49 in exon 1, causes an amino acid change (threonine to alanine) in the peptide leader sequence, and adenine at this position is correlated with high expression of the CTLA-4 protein (Ligers et al., 2001). The CT60A/G lies in the 3′-untranslated region and has been reported to be associated with decreasing the soluble CTLA-4 isoform (Ueda et al., 2003). Previous studies have found that polymorphisms in the CTLA-4 gene, especially the +49G/A SNP, are associated with many different autoimmune diseases and cancers such as diabetes, breast cancer, and gastric cancer (Bouqbis et al., 2003; Erfani et al., 2006; Wong et al., 2006; Hadinia et al., 2007; Xiao et al., 2010). We have shown here that CTLA-4 polymorphism and haplotype are correlated with osteosarcoma. The mechanism is not clear yet. Since Nagamori et al. (2002) has reported that the anti-CTLA-4 blocking mAb may prolong the survival time of mice with osteosarcoma, which suggests that changes in CTLA-4 expression may have an impact on osteosarcoma in mice. It is possible that the SNPs causing CTLA-4 changes may also affect human osteosarcoma patients in similar ways. In addition, it has been shown that the CTLA-4 molecule is expressed and functional on human osteosarcoma tumor cells (Contardi et al., 2005), which indicates that CTLA-4 polymorphisms might affect osteosarcoma through multiple pathways.

According to the HapMap Project and Environmental Genome Project, the frequencies of the CTLA-4 +49 A 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.

When compared to patients with or without metastasis, it seemed that the +49 AA genotype was higher in patients with metastasis (p = 0.063) (Table 4). It might be because the increase of CTLA-4 caused by the +49 AA genotype decreased T-cell activation throughout the body. Without the proper function of the T cell response in the whole immune system, the cancer cells might easily spread to other organs. In addition, our data showed that the −318 TT genotype occurred more frequently in patients than in controls, although p-value did not reach significant difference (p = 0.057) (Table 3). It is possible since the −318 TT genotype has been shown to increase the mRNA level of CTLA-4 (Ligers et al., 2001; Bouqbis et al., 2003), it may downregulate T-cell activation. It would be interesting to study these polymorphisms in a larger number of osteosarcoma patients.

In conclusion, this case–control study demonstrated that the CTLA-4 gene +49 AA genotype, +49 A allele, and GTAG haplotype are associated with the development of osteosarcoma in the Chinese population. Our results provide important insights for understanding the genetics of osteosarcoma and would be helpful for the development of CTLA-4 as a possible therapeutic target for this disease.

Footnotes

Acknowledgments

This research was funded by National Nature Science Foundation of China (81071509).

Disclosure Statement

No competing financial interests exist.

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