Cytotoxic T-Lymphocyte Antigen-4 Genetic Variants and Risk of Ewing's Sarcoma

Abstract

Despite the knowledge of many genetic alterations present in Ewing's sarcoma (ES), the complexity of this disease precludes placing its biology into a simple conceptual framework. Cytotoxic T-lymphocyte antigen-4 (CTLA-4) can decrease T-cell activation and attenuate antitumor responses. Polymorphisms in the CTLA-4 gene have been shown to be associated with different diseases. Here, we investigated the association of four CTLA-4 gene polymorphisms, −1661A/G (rs4553808), −318C/T (rs5742909), +49G/A (rs231775), and CT60A/G (rs3087243), with ES in the Chinese population. A total of 308 ES cases and 362 healthy controls were recruited and CTLA-4 polymorphisms were tested by polymerase chain reaction-restriction fragment length polymorphism. Results showed that frequencies of the CTLA-4 gene +49AA genotype, +49A allele, and GTAG haplotype were significantly increased in ES patients compared to healthy controls (odds ratio [OR]=2.42, 95% confidence interval [CI] 1.43-4.09, p<0.001; OR 1.38, 95% CI 1.11-1.73, p=0.005, and OR=1.46, 95% CI 1.06-2.02, p=0.020, respectively). We further compared CTLA-4 polymorphisms in ES patients based on different clinical parameters and data revealed that ES patients with metastasis had higher numbers of the +49AA genotype than those without metastasis (p=0.004). These results indicated that the CTLA-4 polymorphism could be a risk factor for ES and suggested a potential role of CTLA-4 in the metastasis of this malignancy.

Introduction

After osteosarcoma, Ewing's sarcoma (ES) is the most common bone malignancy in children and adolescents (Dorfman and Czerniak, 1995). Its peak age of occurrence is 15 years, slightly lower than that for osteogenic sarcoma, which has a peak age of 19 years (Ligers et al., 2001). In addition, there is no second peak of occurrence in the eighth decade of life, as is seen with osteogenic sarcoma. ES affects ∼250-400 patients in the United States each year, and 80% of these cases occur before age 18 years (Ligers et al., 2001). There is, however, an increasing number of adults over the age of 30 being diagnosed with this disease (Ligers et al., 2001). Also, studies have shown that the occurrence of this disease has increased rapidly in the Asian population in recent years (Ligers et al., 2001). Currently, most clinical centers performing intensive chemotherapy report long-term survival rates of 50%-70%, suggesting that ES is sensitive to traditional chemotherapy (Ligers et al., 2001). The key prognostic factor in ES is the presence or absence of metastatic disease, and while 80% of patients have clinically localized disease, subclinical metastases are assumed to be present in nearly all patients (Ligers et al., 2001). Recent advances in molecular pathology have greatly enhanced practitioners' ability to classify and differentiate these round-cell tumors (Ligers et al., 2001). The translocation t(11;22)(q24;q12) is the most common translocation diagnostic for ES and is present in ∼85% of ES cases, indicating mutations in human genes may play important roles in this disease (Ligers et al., 2001).

Cytotoxic T-lymphocyte antigen 4 (CTLA-4) plays critical roles in downregulating T-cell proliferation and activation (Hodi et al., 2003; Phan et al., 2003). CTLA-4 is expressed on cytotoxic T-lymphocytes (CTLs) and can interact with B7.1/B7.2 ligands on the surface of antigen-presenting cells, which causes cell cycle arrest and directly eliminates the T-cell proliferation (Leach et al., 1996; Egen et al., 2002; Hodi et al., 2003). Also, studies have shown that CTLA-4 can regulate effector T cells through its role in T-regulatory cells (Leach et al., 1996; Egen et al., 2002; Hodi et al., 2003). CTLA-4 may decrease damage to normal tissues, since it can reduce T-cell response and increase peripheral tolerance (Masteller et al., 2000; Greenwald et al., 2002; Chikuma et al., 2003). CTLA-4 is also known to be able to cause tumor tolerance due to its role in diminishing antitumor responses (Contardi et al., 2005). Interestingly, more and more evidence has shown CTLA may be correlated with the development of bone diseases such as rheumatoid arthritis, osteosarcoma, and ES (Liu et al., 2011; Wang et al., 2011).

Single-nucleotide polymorphisms (SNPs) may have functions and affect the pathogenesis of diseases. The CTLA-4 gene 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 polymorphisms have revealed the potential to affect gene expression; +49G/A (rs231775) has been shown to be associated with different diseases and cancers (Ghaderi et al., 2004; Erfani et al., 2006; Hadinia et al., 2007; Sun et al., 2008). In the current study, we investigated the association of the CTLA-4 −1661A/G (rs4553808), −318C/T (rs5742909), +49G/A (rs231775), and CT60A/G (rs3087243) polymorphisms with ES in the Chinese population.

Materials and Methods

Study population

The study was approved by the Review Boards of the Changzheng Hospital and the Shanghai Corps Hospital. Written informed consent was obtained from each participant or guardians on behalf of the child participants involved in the study. The study population included 308 ES patients (7-38 years of age) and 362 healthy controls (8-42 years of age), recruited from the Changzheng Hospital and the Shanghai Corps Hospital. The diagnosis of ES was established with histological examination in all cases. Patients with melanotic neuroectodermal tumor were excluded from analysis, because this is a tumor distinct from ES. The control population was recruited from people who came for general health exams. People who were relatives were excluded from this study. People with a history of familial cancer syndromes were excluded from this study. 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.

DNA extraction and genotyping

Genomic DNA was extracted from 5 mL frozen whole blood using the DNA Extraction Kit (Qiagen, Inc.) according to the manufacturer's protocol. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was used to detect dimorphism of −318 C/T and +49 G/A. The polymorphic region was amplified by PCR with a Thermoblock PCR System in a 25 μL reaction solution containing 50 ng genomic DNA, 12.5 μL 2xPCR Mix buffer (Qiagen, Inc.), and 0.1 mol of each primer (Shenggong). PCR products were digested with restriction enzymes (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 ensure the reliability of the RFLP results, 20% of the PCR products were randomly selected and sequenced using the fluorescent dideoxy chain termination method with an ABI 3730XL Analyzer (Applied Biosystems) according to the manufacturer's instruction. Results between PCR and DNA sequencing analysis were 100% concordant.

Table 1.

Restriction Enzymes and Length of Digested Fragments for CTLA-4 PCR-RFLP

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

CTLA-4, cytotoxic T-lymphocyte antigen 4; SNP, single-nucleotide polymorphisms; PCR-RFLP, polymerase chain reaction-restriction fragment length polymorphism.

Statistical analysis

The SPSS statistical software package ver.13.0 (SPSS, Inc.) was used for statistical analysis. Demographic data between the study groups were compared by the chi-square test and by the Student's t-test. The polymorphisms were tested for deviation from the Hardy-Weinberg equilibrium by comparing the observed and expected genotype frequencies using the chi-square test. For SNP analysis, the 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 less than 0.05 were considered significant.

Results

The study population included 308 ES patients and 362 healthy controls. Demographic and other selected characteristics of the cases and controls are summarized in Table 2, which included age, gender, tumor origin, tumor location, and metastasis status. We did not detect any statistically significant differences between ES patients and controls with regard to age (p=0.732) and sex (p=0.777). We investigated four CTLA-4 polymorphisms (−1661 A/G, −318 C/T, +49 G/A, and CT60 A/G) in ES patients and healthy controls. The frequencies of the allele and genotype are presented in Table 3. All the four polymorphisms were in HWE (p>0.05). The numbers of CTLA-4 gene +49AA genotype and +49A allele were significantly higher in ES patients than in the controls (OR=2.42, 95% CI 1.43-4.09, p<0.001; and OR 1.38, 95% CI 1.11-1.73, p=0.005). However, we did not find any significance when compared the other three SNPs, CTLA-4 −1661 A/G, −318 C/T, and CT60 A/G, between patients and controls (p>0.05) (Table 3). Further, we analyzed the linkage disequilibrium of these four SNPs. Data showed that the polymorphisms of −1661 and −318 (D′=0.857), −318 and +49 (D′=0.823), −318 and CT60 (D′=0.936), and +49 and CT60 (D′=0.845) locus were in strong LD (D′>0.80) after Bonferroni adjustment. We then constructed haplotypes based on the four CTLA-4 polymorphisms (−1661 A/G, −318 C/T, +49 G/A, and CT60 A/G) and analyzed them in all ES cases and controls. The four most common haplotypes (ACGG, ACAA, GTAG, and ACAG) (−1661, −318, +49, and CT60) are shown (Table 3). We detected that the GTAG haplotype had higher numbers in the ES group than in controls (OR=1.46, 95% CI 1.06-2.02, p=0.020), whereas the other three haplotypes did not show any significance. These results indicated that the CTLA-4 +49AA genotype, +49A allele, and GTAG haplotype were associated with the high risk of ES in the Chinese population.

Table 2.

General Characteristics of the Subjects

Variables	Ewing's sarcoma (n=308) (%)	Control subjects (n=362) (%)	p-Value
Age			0.732
≤20	227 (73.7)	271 (74.9)
>20	81 (26.3)	91 (25.1)
Gender			0.777
Male	189 (61.4)	226 (62.4)
Female	119 (38.6)	136 (37.6)
Origin
Extra-skeletal	41 (13.3)
Skeletal	267 (86.7)
Tumor location
Long tubular bones	122 (39.6)
Axial skeleton	186 (60.4)
Metastasis
With	95 (30.8)
Without	213 (69.2)

Table 3.

Genotype, Allele, and Haplotype Frequencies of CTLA-4 SNPs in Ewing's Sarcoma Cases and Healthy Controls

Variables	Ewing's sarcoma (n=308) (%)	Control subjects (n=362) (%)	OR (95% CI)	p-Value
−1661A/G
AA	209 (67.9)	252 (69.6)	Referent
AG	83 (26.9)	96 (26.5)	1.04 (0.74-1.47)	0.814
GG	16 (5.2)	14 (3.9)	1.38 (0.66-2.89)	0.394
A	501 (81.3)	600 (82.9)	Referent
G	115 (18.7)	124 (17.1)	1.11 (0.84-1.47)	0.463
−318 C/T
CC	213 (69.2)	249 (68.8)	Referent
CT	89 (28.9)	102 (28.2)	1.02 (0.73-1.43)	0.909
TT	6 (1.9)	11 (3.0)	0.64 (0.23-1.75)	0.380
C	515 (83.6)	600 (82.9)	Referent
T	101 (16.4)	124 (17.1)	0.95 (0.71-1.27)	0.721
+49 G/A
GG	110 (35.7)	155 (42.8)	Referent
GA	150 (48.7)	179 (49.4)	1.18 (0.85-1.64)	0.319
AA	48 (15.6)	28 (7.7)	2.42 (1.43-4.09)	<0.001^a
G	370 (60.1)	489 (67.5)	Referent
A	246 (39.9)	235 (32.5)	1.38 (1.11-1.73)	0.005^a
CT60 A/G
GG	210 (68.2)	243 (67.1)	Referent
GA	87 (28.2)	105 (29.0)	0.96 (0.68-1.35)	0.808
AA	11 (3.6)	14 (3.9)	0.91 (0.40-2.05)	0.818
G	507 (82.3)	591 (81.6)	Referent
A	109 (17.7)	133 (18.4)	0.96 (0.72-1.26)	0.749
Haplotype (−1661, −318, +49, CT60)
ACGG	340 (55.2)	437 (60.4)	Referent
ACAA	93 (15.1)	112 (15.5)	1.07 (0.78-1.45)	0.680
GTAG	99 (16.1)	87 (12.0)	1.46 (1.06-2.02)	0.020^a
ACAG	20 (3.2)	27 (3.7)	0.95 (0.52-1.73)	0.872

p<0.05.

OR, odds ratio; CI, 95% confidence interval.

Since polymorphisms may be correlated with certain clinical factors in patients (Ghaderi et al., 2004; Erfani et al., 2006; Hadinia et al., 2007), we divided ES patients based on different clinical parameters and analyzed the CTLA-4 −1661 A/G, −318 C/T, +49 G/A, and CT60 A/G polymorphisms between these subgroups. The criteria to divide ES patients included age, gender, tumor location, and metastasis (Table 4). Data showed that the CTLA-4 gene +49AA genotype and +49A allele had significantly higher numbers in ES patients with metastasis than those without metastasis (OR=2.83, 95% CI 1.39-5.77, p=0.004, and OR=1.61, 95% CI 1.14-2.27, p=0.007, respectively). The −1661A/G, −318C/T, and CT60 A/G SNPs did not reveal any significant differences between patient subgroups. These results indicated that the CTLA-4 +49G/A polymorphism was associated with metastasis of ES in the Chinese population.

Table 4.

Stratification Analysis of CTLA-4 Polymorphisms in Ewing's Sarcoma Patients

	Age (%)				Gender (%)
Genotype frequency	≤20 (227)	>20 (81)	OR (95% CI)	p-Value	Male (189)	Female (119)	OR (95% CI)	p-Value
−1661 A/G
AA	151 (66.5)	58 (71.6)	Referent		126 (66.7)	83 (69.7)	Referent
AG	62 (27.3)	21 (25.9)	1.13 (0.63-2.03)	0.671	51 (27.0)	32 (26.9)	1.05 (0.62-1.77)	0.855
GG	14 (6.2)	2 (2.5)	2.69 (0.59-12.20)	0.184	12 (6.3)	4 (3.4)	1.98 (0.62-6.34)	0.244
A	364 (80.2)	137 (84.6)	Referent		303 (80.2)	198 (83.2)	Referent
G	90 (19.8)	25 (15.4)	1.36 (0.83-2.20)	0.218	75 (19.8)	40 (16.8)	1.23 (0.80-1.87)	0.347
−318 C/T
CC	157 (69.2)	56 (69.1)	Referent		130 (68.8)	83 (69.7)	Referent
CT	65 (28.6)	24 (29.6)	0.97 (0.55-1.69)	0.904	55 (29.1)	34 (28.6)	1.03 (0.62-1.72)	0.901
TT	5 (2.2)	1 (1.2)	1.78 (0.20-15.61)	0.596	4 (2.1)	2 (1.7)	1.28 (0.23-7.13)	0.780
C	379 (83.5)	136 (84.0)	Referent		315 (83.3)	200 (84.0)	Referent
T	75 (16.5)	26 (16.0)	1.04 (0.64-1.69)	0.890	63 (16.7)	38 (16.0)	1.05 (0.68-1.63)	0.819
+49 G/A
GG	79 (34.8)	31 (38.3)	Referent		69 (36.5)	41 (34.4)	Referent
GA	111 (48.9)	39 (48.1)	1.12 (0.64-1.94)	0.695	92 (48.7)	58 (48.7)	0.94 (0.57-1.57)	0.819
AA	37 (16.3)	11 (13.6)	1.32 (0.60-2.91)	0.491	28 (14.8)	20 (16.8)	0.83 (0.42-1.66)	0.602
G	269 (59.3)	101 (62.3)	Referent		230 (60.8)	140 (58.8)	Referent
A	185 (40.7)	61 (37.7)	1.14 (0.79-1.65)	0.490	148 (39.2)	98 (41.2)	0.92 (0.66-1.28)	0.618
CT60 G/A
GG	157 (69.2)	53 (65.5)	Referent		129 (68.3)	81 (68.1)	Referent
GA	63 (27.8)	24 (29.6)	0.89 (0.50-1.56)	0.674	54 (28.6)	33 (27.7)	1.03 (0.61-1.72)	0.918
AA	7 (3.0)	4 (4.9)	0.59 (0.17-2.10)	0.411	6 (3.1)	5 (4.2)	0.75 (0.22-2.55)	0.648
G	377 (83.0)	130 (80.2)	Referent		312 (82.5)	195 (81.9)	Referent
A	77 (17.0)	32 (19.8)	0.83 (0.52-1.31)	0.424	66 (17.5)	43 (18.1)	0.96 (0.63-1.47)	0.848

	Location (%)				Metastasis (%)
Genotype frequency	L (122)	A (186)	OR (95% CI)	p-Value	Yes (95)	No (213)	OR (95% CI)	p-Value
−1661 A/G
AA	82 (67.2)	127 (68.3)	Referent		67 (70.5)	142 (66.7)	Referent
AG	33 (27.0)	50 (26.9)	1.02 (0.61-1.72)	0.934	24 (25.3)	59 (27.7)	0.86 (0.49-1.50)	0.601
GG	7 (5.8)	9 (4.8)	1.21 (0.43-3.36)	0.722	4 (4.2)	12 (5.6)	0.71 (0.22-2.27)	0.558
A	197 (80.7)	304 (81.7)	Referent		158 (83.2)	343 (80.5)	Referent
G	47 (19.3)	68 (18.3)	1.07 (0.71-1.61)	0.760	32 (16.8)	83 (19.5)	0.84 (0.53-1.31)	0.437
−318 C/T
CC	85 (69.7)	128 (68.8)	Referent		67 (70.5)	146 (68.5)	Referent
CT	34 (27.8)	55 (29.6)	0.93 (0.56-1.55)	0.782	26 (27.4)	63 (29.6)	0.90 (0.52-1.55)	0.700
TT	3 (2.5)	3 (1.6)	1.51 (0.30-7.64)	0.619	2 (2.1)	4 (1.9)	1.09 (0.19-6.10)	0.922
C	204 (83.6)	311 (83.6)	Referent		160 (84.2)	355 (83.3)	Referent
T	40 (16.4)	61 (16.4)	0.99 (0.65-1.55)	0.998	30 (15.8)	71 (16.7)	0.94 (0.59-1.49)	0.786
+49 G/A
GG	43 (35.2)	67 (36.0)	Referent		27 (28.4)	83 (39.0)	Referent
GA	58 (47.5)	92 (49.5)	0.98 (0.59-1.63)	0.945	45 (47.4)	105 (49.3)	0.32 (0.75-2.30)	0.332
AA	21 (17.2)	27 (14.5)	1.21 (0.61-2.41)	0.583	23 (24.2)	25 (11.7)	2.83 (1.39-5.77)	0.004^a
G	144 (59.0)	226 (60.8)	Referent		99 (52.1)	271 (63.6)	Referent
A	100 (41.0)	146 (39.2)	1.08 (0.77-1.49)	0.667	91 (47.9)	155 (36.4)	1.61 (1.14-2.27)	0.007^a
CT60 G/A
GG	85 (69.7)	125 (67.2)	Referent		66 (69.5)	144 (67.6)	Referent
GA	33 (27.0)	54 (29.0)	0.90 (0.54-1.50)	0.683	26 (27.4)	61 (28.6)	0.93 (0.54-1.60)	0.794
AA	4 (3.3)	7 (3.8)	0.84 (0.24-2.96)	0.786	3 (3.1)	8 (3.8)	0.82 (0.21-3.18)	0.772
G	203 (83.2)	304 (81.7)	Referent		158 (83.2)	349 (81.9)	Referent
A	41 (16.8)	68 (18.3)	0.90 (0.59-1.38)	0.639	32 (16.8)	77 (18.1)	0.92 (0.58-1.44)	0.711

p<0.05.

L, long tubular bones; A, axial skeleton.

Discussion

CTLA-4 plays important roles in decreasing T-cell activation and attenuating antitumor responses (Pure et al., 2005). Polymorphisms in the CTLA-4 gene have been shown to be associated with different diseases (Ghaderi et al., 2004; Erfani et al., 2006; Hadinia et al., 2007). In this study, we identified that CTLA-4 polymorphisms are associated with increased susceptibility to ES in the Chinese population. This research demonstrated that polymorphisms in the CTLA-4 gene could act as risk factors for ES. Further, we observed that the +49G/A SNP was correlated with ES metastasis, suggesting that polymorphisms in CTLA-4 may affect the prognosis of this disease.

CTLA-4 binds to CD80/CD86 to modulate the immune response (Hodi et al., 2003; Phan et al., 2003; Contardi et al., 2005). Recent studies have revealed that CTLA-4 +49 G/A and CD86 +1057G/A polymorphisms are associated with osteosarcoma, which is the most common pediatric bone malignancy in the world (Wang et al., 2011; Liu et al., 2011). Also, study has revealed that the CD86 +1057G/A polymorphism is correlated with ES (Wang et al., 2012). In the current research, we found that CTLA-4 +49G/A polymorphism was associated with increased susceptibility to ES in the Chinese population. All these results indicate that the CTLA-4/CD86 pathway may play important roles in the development of bone diseases. It has been reported that SNPs in CTLA-4 can affect the function of this gene. The −1661A/G and −318C/T SNPs are located in the promoter region of the CTLA-4 gene and have been shown to increase the mRNA level of CTLA-4 (Buonavista et al., 1992). The +49G/A polymorphism, located at position +49 in exon 1, causes an amino acid exchange (threonine to alanine) in the peptide leader sequence and adenine at this position is correlated with high expression of the CTLA-4 protein (Linsley and Ledbetter, 1993). The CT60A/G lies in the 3′-untranslated region and has been reported to be associated with decreasing the soluble CTLA-4 isoform (Contardi et al., 2005). 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 (Erfani et al., 2006; Hadinia et al., 2007; Sun et al., 2008). In addition, many studies have revealed that the +49G/A can affect the prognosis of many diseases (Erfani et al., 2006; Hadinia et al., 2007; Sun et al., 2008). Here, we identified that CTLA-4 +49G/A polymorphism is correlated with increased risk of ES, especially metastatic ES, which suggests CTLA-4 may have a broad function in regulating human cancers. Since the current research was performed in the Chinese population, it would be interesting and important to conduct independent studies in other ethnic populations for comparison.

In conclusion, this study demonstrates that the CTLA-4 gene +49G/A SNP can be a new risk factor for ES, especially metastatic ES in the Chinese population. It raises the possibility that a function shift caused by genetic variants in immunity genes could have important consequences for the pathogenesis of ES.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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