CTLA-4 polymorphisms associate with breast cancer susceptibility in Asians: a meta-analysis

Introduction

Breast cancer has been the most common type of cancer and the main cause of cancer death among women in the world, which was estimated to have 1.7 million new cases in 2012 (Torre et al., 2015). Breast cancer is an extremely heterogeneous disease in the clinic and the potential molecular mechanism of carcinogenesis has not been clearly understood so far. In recent years, inherited factors were identified to play a critical role in the development of breast cancer (Reeves et al., 2012).

Researches on the field of tumour immunology found that the immune system can influence tumour occurence during the period of elimination, equilibrium and escape (Dunn, Old & Schreiber, 2004). Cytotoxic T-lymphocyte antigen-4 (CTLA-4), which was also designated as CD152, expressed mainly on activated T cells. As an immunosuppressive cytokine, it can inhibit T-lymphocyte proliferation and activation (Sun et al., 2008). Numerous researches have demonstrated that blockage of CTLA-4 function can improve antitumor immunity (Leach, Krummel & Allison, 1996; Ribas et al., 2004; Vandenborre et al., 1999). This indicates CTLA-4 may exert positive effects on carcinogenesis. The human CTLA-4 gene, which is located in human chromosome 2q33, is one of the most important genes involved in immune responses to a variety of antigens (Walunas et al., 2011). CTLA-4 gene comprises four exons and has several important polymorphisms in the entire region, including the +49G>A (rs231775) in exon 1 (Donner et al., 1997), the +6230G>A (rs3087243) in 3^′-untranslated region (Hughes, 2006), the −1661A>G (rs4553808) and −1722 T>C (rs733618) in the promoter region (Johnson et al., 2001), which are the most commonly studied single nucleotide polymorphisms (SNPs). These SNPs are important because they can alter the host immune response by affecting the transcription of CTLA-4 gene, the expression of CTLA-4 protein, and the interaction of CTLA-4 and CD80 ligand (Anjos et al., 2002; Sun et al., 2008; Wang et al., 2002).

Numerous investigations have demonstrated that CTLA-4 genetic polymorphisms may have association with human breast cancer susceptibility (Erfani et al., 2006; Ghaderi et al., 2004; Li et al., 2012; Li et al., 2008; Minhas et al., 2014; Sun et al., 2008; Wang et al., 2007; Zhifu et al., 2015; Kong, 2010). The results showed that some of the polymorphisms such as rs733618 and rs4553808 may increase the breast cancer risk while other polymorphisms such as rs231775 and rs3087243 may reduce the risk of breast cancer. Considering that a single study does not have enough power to detect the overall effects, we conducted a meta-analysis which is a statistical analysis of the data from some collection of studies in order to synthesize the results to obtain a more reliable evaluation of the relationship between the four common SNPs in CTLA-4 gene and breast cancer susceptibility.

Materials and Methods

Our meta-analysis was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines (Moher et al., 2010).

Results

Characteristics of included studies

Finally, nine articles comprising 10 studies investigating CTLA-4 +49A>G (rs231775) and/or +6230G>A (rs3087243) and/or −1722 T>C (rs733618) and/or −1661A/G (rs4553808) polymorphisms were identified for further analysis (Fig. 1). Table 1 presented the characteristics of selected studies. Of the 10 studies, seven were from China, two were from Iran, and one was from India. Additionally, seven studies were based on population and three based on hospitals. Moreover, genotype distributions in the control group of each included studies complied with Hardy-Weinberg equilibriums (HWE) (P > 0.05) except for one study for one SNP (Erfani et al., 2006). The detailed data of the allele frequency and genotype distribution as well as HWE from each study were shown in Table 2.

Table 1:

Characteristics of the studies included in the meta-analysis.

First author	Year	Country	Ethnicity	Genotyping medthod	Source of control	Total sample size (case/control)	SNP
Yu	2015	China	Chinese	PCR-RFLP	PB	376/366	1;2;3;4
Minhas	2014	India	Indian	PCR-RFLP	PB	250/250	1
Li D	2012	China	Chinese	PCR-RFLP	PB	581/566	1;2;3;4
Kong	2010	China	Chinese	PCR-RFLP	HB	315/322	4
Sun	2008a	China	Chinese	PCR-RFLP	PB	1060/1070	1
Sun	2008b	China	Chinese	PCR-RFLP	PB	1037/1070	1
Li H	2008	China	Chinese	PCR-RFLP	HB	328/327	2;3
Wang	2007	China	Chinese	PCR-RFLP	PB	117/148	1;2;4
Erfani	2006	Iran	Iranian	PCR-CTPP	PB	283/245	3;4
Ghaderi	2004	Iran	Iranian	PCR-RFLP	HB	197/151	1

DOI: 10.7717/peerj.2815/table-1

Notes:

PCR

polymerase chain reaction

RFLP

restriction fragment length polymorphism

CTPP

confronting two pairs primers

PB

population based

HB

hospital based

SNP

single-nucleotide polymorphism

SNP No.1, +49A>G (rs231775); 2, +6230G>A (rs3087243); 3, −1722T>C (rs733618); 4, −1661A>G (rs4553808).

Table 2:

Genotype distributions and allele frequencies of CTLA-4 polymorphisms in cases and controls.

Study	Genotype (N)								Allele frequency (N)				P of HWE
	Case				Control				Case		Control
	Total	AA	AB	BB	Total	AA	AB	BB	A	B	A	B
+49A>G (rs231775)
Zhifu et al. (2015)	376	174	175	27	366	174	157	35	523	229	505	227	0.96
Minhas et al. (2014)	250	111	113	26	250	105	121	24	335	165	331	169	0.20
Li et al. (2012)	576	49	281	246	553	54	243	256	379	773	351	755	0.74
Sun et al. (2008)	1,060	101	485	474	1,070	65	446	559	660	1,406	576	1,564	0.15
Sun et al. (2008)	1,037	100	455	482	1,070	73	451	546	655	1,419	597	1,543	0.12
Wang et al. (2007)	117	48	59	10	148	55	70	23	155	79	180	116	0.93
Ghaderi et al. (2004)	197	84	104	9	151	60	72	19	272	122	192	110	0.72
+6230G>A (rs3087243)
Zhifu et al. (2015)	376	257	110	9	366	252	103	11	624	128	607	125	0.90
Li et al. (2012)	581	361	197	23	566	361	182	23	919	243	904	228	0.99
Li et al. (2008)	328	32	124	172	327	20	114	193	188	468	154	500	0.57
Wang et al. (2007)	117	24	47	46	148	18	56	74	95	139	92	204	0.16
−1722T>C (rs733618)
Zhifu et al. (2015)	376	123	186	67	366	137	166	63	432	320	440	292	0.30
Li et al. (2012)	574	184	276	114	551	207	256	88	644	504	670	432	0.55
Li et al. (2008)	328	125	163	40	327	111	168	48	413	243	390	264	0.22
Erfani et al. (2006)	282	225	54	3	245	204	41	0	504	60	449	41	0.15
−1661A>G (rs4553808)
Zhifu et al. (2015)	376	273	91	12	366	281	78	7	637	115	640	92	0.56
Li et al. (2012)	574	405	153	16	551	425	115	11	963	185	965	137	0.33
Kong (2010)	315	204	105	6	322	241	76	5	513	117	558	86	0.72
Wang et al. (2007)	109	62	45	2	148	111	35	2	169	49	257	39	0.68
Erfani et al. (2006)	282	211	65	6	238	184	43	11	487	77	411	65	0.001

DOI: 10.7717/peerj.2815/table-2

Notes:

A

the major allele

B

the minor allele

HWE

Hardy-Weinberg equilibrium

Meta-analysis results

Seven studies containing 3,613 cases and 3,608 controls focused on breast cancer risk with CTLA-4 rs231775 polymorphism. As presented in Table 3, significantly decreased risk was observed in the overall population in all the models except heterozygote (G vs. A: OR = 0.86, 95% CI [0.80–0.92], P = 0.000, Fig. 2A; GG vs. AA: OR = 0.68, 95% CI [0.57–0.81], P = 0.000; GG vs. AA + AG: OR = 0.79, 95% CI [0.71–0.87], P = 0.000; AG + GG vs. AA: OR = 0.85, 95% CI [0.74–0.97], P = 0.02;). In subgroup analyses, rs231775 was also found to significantly reduce the breast cancer risk in Chinese and subgroup based on population under allelic, homozygous and recessive models.

Table 3:

Meta-analysis results of CTLA-4 polymorphisms and BC risk.

SNP	B vs. A		BB vs. AA		AB vs. AA		BB vs. AA + AB		AB + BB vs. AA
	OR (95%CI)	P	OR (95%CI)	P	OR (95%CI)	P	OR (95%CI)	P	OR (95%CI)	P
+49A>G (rs231775)
Overall	0.86 (0.80–0.92)	0.000	0.68 (0.57–0.81)	0.000	0.92 (0.80–1.06)	0.23	0.79 (0.71–0.87)	0.000	0.85 (0.74–0.97)	0.02
Chinese	0.85 (0.79–0.92)	0.000	0.68 (0.56–0.82)	0.000	0.92 (0.73–1.16)	0.49	0.79 (0.71–0.88)	0.000	0.84 (0.65–1.08)	0.17
PB	0.86 (0.80–0.93)	0.000	0.70 (0.59–0.84)	0.000	0.91 (0.78–1.05)	0.19	0.80 (0.72–0.89)	0.000	0.85 (0.69–1.04)	0.12
+6230G>A (rs3087243)
Chinese	0.87 (0.71–1.07)	0.20	0.68 (0.49–0.95)	0.02	0.99 (0.83–1.19)	0.94	0.77 (0.61–0.97)	0.02	0.87 (0.65–1.17)	0.36
PB	0.91 (0.71–1.17)	0.48	0.75 (0.50–1.12)	0.15	1.03 (0.85–1.25)	0.76	0.77 (0.54–1.09)	0.14	1.00 (0.82–1.20)	0.99
−1722T>C (rs733618)
Overall	1.09 (0.93–1.29)	0.29	1.15 (0.79–1.68)	0.47	1.13 (0.96–1.32)	0.15	1.11 (0.90–1.37)	0.32	1.14 (0.98–1.33)	0.09
Chinese	1.07 (0.88–1.29)	0.51	1.12 (0.77–1.63)	0.55	1.12 (0.94–1.33)	0.22	1.10 (0.89–1.35)	0.39	1.11 (0.86–1.43)	0.43
PB	1.19 (1.05–1.34)	0.007	1.37 (1.05–1.78)	0.02	1.22 (1.02–1.47)	0.03	1.21 (0.96–1.54)	0.11	1.26 (1.06–1.50)	0.01
−1661A>G (rs4553808)
Overall	1.34 (1.16–1.53)	0.000	1.22 (0.78–1.92)	0.38	1.45 (1.23–1.70)	0.000	1.12 (0.72–1.76)	0.61	1.43 (1.22–1.67)	0.000
Chinese	1.41 (1.21–1.63)	0.000	1.59 (0.95–2.67)	0.08	1.47 (1.24–1.75)	0.000	1.45 (0.86–2.43)	0.16	1.48 (1.25–1.75)	0.000
PB	1.30 (1.11–1.52)	0.001	1.19 (0.73–1.94)	0.48	1.40 (1.17–1.68)	0.000	1.11 (0.68–1.80)	0.68	1.38 (1.16–1.64)	0.000
HWE	1.41 (1.21–1.63)	0.000	1.59 (0.95–2.67)	0.08	1.47 (1.24–1.75)	0.000	1.45 (0.86–2.43)	0.16	1.48 (1.25–1.75)	0.000

DOI: 10.7717/peerj.2815/table-3

Notes:

A

the major allele

B

the minor allele

CI

confidence interval

OR

odds ratio

PB

population based

HB

hospital based

SNP

single-nucleotide polymorphism

HWE

subgroup excluding the study departing from HWE

There were four studies all of which were from China with 1,402 cases and 1,407 controls investigating the relationship between and breast cancer susceptibility and CTLA-4 rs3087243 polymorphism. The results presented a significantly lower breast cancer risk in homozygous and recessive genetic models in Chinese women (AA vs. GG: OR = 0.68, 95% CI [0.49–0.95], P = 0.02, Fig. 2B; AA vs. GG + GA: OR = 0.77, 95% CI [0.61–0.97], P = 0.02).

For CTLA-4 rs733618 polymorphism, we aseessed 4 studies containing 1,560 cases and 1,489 controls. Overall, our analysis did not suggest any association between rs733618 and breast cancer susceptibility. However, when stratifying by source of control, rs733618 was observed to increase breast cancer risk based on population in three genetic models (C vs. T: OR = 1.19, 95% CI [1.05–1.34], P = 0.007; CC vs. TT: OR = 1.37, 95% CI [1.05–1.78], P = 0.02; CT vs. TT: OR = 1.22, 95% CI [1.02–1.47], P = 0.03).

Five studies involving 1,656 cases and 1,625 controls investigated the breast cancer risk with CTLA-4 rs4553808 polymorphism. We observed a higher risk in overall analysis under three models (G vs. A: OR = 1.34, 95% CI [1.16–1.53], P = 0.000; AG vs. AA: OR = 1.45, 95% CI [1.23–1.70], P = 0.000; AG + GG vs. AA: OR = 1.43, 95% CI [1.22–1.67], P = 0.000, Fig. 2C). The results were similar in Chinese subgroup. When stratifying by source of control, rs4553808 was also noted to increase breast cancer risk in allelic and heterozygous models based on population.

Heterogeneity analysis and sensitivity analysis

As presented in Table 4, no obvious heterogeneity was detected for the four CTLA-4 polymorphisms in most of the genetic models. For the few in which existed significant heterogeneity (I² > 50%), random-effects model was applied.

Table 4:

Heterogeneity-analysis results of CTLA-4 polymorphisms and BC risk.

SNP	B vs. A			BB vs. AA			AB vs. AA			BB vs. AA + AB			AB + BB vs. AA
	I2	P	EM	I2	P	EM	I2	P	EM	I2	P	EM	I2	P	EM
+49A>G (rs231775)
Overall	0%	0.46	F	45%	0.09	F	29%	0.21	F	27%	0.22	F	41%	0.12	F
Chinese	12%	0.34	F	40%	0.15	F	51%	0.09	R	0%	0.59	F	60%	0.04	R
PB	6%	0.38	F	39%	0.14	F	39%	0.15	F	0%	0.56	F	51%	0.07	R
+6230G>A (rs3087243)
Chinese	58%	0.07	R	8%	0.36	F	0%	0.38	F	0%	0.78	F	53%	0.09	R
PB	60%	0.08	R	24%	0.27	F	16%	0.31	F	0%	0.58	F	46%	0.16	F
−1722T>C (rs733618)
Overall	52%	0.10	R	51%	0.10	R	7%	0.36	F	35%	0.22	F	39%	0.18	F
Chinese	64%	0.06	R	59%	0.09	R	37%	0.21	F	31%	0.23	F	57%	0.10	R
PB	0%	0.74	F	0%	0.45	F	0%	0.99	F	0%	0.37	F	0%	0.98	F
−1661A>G (rs4553808)
Overall	27%	0.24	F	9%	0.35	F	14%	0.32	F	6%	0.37	F	24%	0.26	F
Chinese	0%	0.48	F	0%	0.99	F	32%	0.22	F	0%	0.98	F	24%	0.27	F
PB	39%	0.18	F	31%	0.23	F	27%	0.25	F	30%	0.24	F	34%	0.21	F
HWE	0%	0.48	F	0%	0.99	F	32%	0.22	F	0%	0.98	F	24%	0.27	F

DOI: 10.7717/peerj.2815/table-4

Notes:

EM

Effects model

F

fixed effects model

R

random effects model

PB

population based

HB

hospital based

SNP

single-nucleotide polymorphism

HWE

subgroup excluding the study departing from HWE

Each study was sequentially removed to assess the impact of single study on the combined ORs. The result showed that the omission of any study didn’t alter the overall estimations substantially, indicating that our meta-analysis results were robust (Fig. 3).

Publication bias

We implemened Begg’s funnel plot and Egger’s test to asesess the publication bias. As shown in Fig. 4, funnel plot failed to display obvious asymmetry. The Egger’s test result didn’t reveal any publication bias for the four SNPs in CTLA-4 gene and breast cancer risk either (Table 5, P > 0.05).

Discussion

It was reported that mutation in human CTLA-4 gene resulted in quantitative reduction of CTLA-4 expression and led to a severe immunoregulatory disorder (Kuehn et al., 2014). Several investigations have suggested that particular CTLA-4 gene polymorphisms are linked to cancer development or progression (Erfani et al., 2006; Tang et al., 2014; Wang et al., 2007). However, the results from those studies remained conflicting. In one previous study, the author found that rs733618 and rs4553808 polymorphisms in CTLA-4 increased the breast cancer risk whereas rs231775 and rs3087243 polymorphisms did not have significant associations with breast cancer risk (Li et al., 2012). However, in other studies, rs3087243 and rs231775 polymorphisms were found to reduce the risk of breast cancer while rs733618 did not associated with breast cancer risk (Sun et al., 2008; Wang et al., 2007). Since CTLA-4 is important in carcinogenesis and a single study does not have enough statistical power to detect the effects, we carried out this meta-analysis which synthesized the results of the included studies with a statistical analysis of the data from these studies to draw a more reliable conclusion about the association between CTLA-4 SNPs and breast cancer susceptibility.

In the present meta-analysis, we identified that CTLA-4 rs231775 had an association with breast cancer susceptibility. We observed a significantly decreased risk in both overall and subgroup analysis in different genetic models. Some previous meta-analyses have also involved the relationship between rs231775 polymorphism and several tumor sites including breast cancer (Gao et al., 2014; Geng et al., 2014; Wang et al., 2015; Zhang et al., 2011). The results suggested the A allele of rs231775 may contribute to breast cancer susceptibility. Our result confirmed that the A allele of CTLA-4 rs231775 polymorphism has more possibility to increase breast cancer risk than G allele. Nevertheless, our study differs from theirs because we specifically focused on breast cancer and our meta-analysis included more studies than theirs. Therefore, our results are more reliable.

CTLA-4 rs3087243 polymorphism was found to decrease breast cancer risk under homozygous and recessive genetic models in Chinese. Our results implied that individuals carry AA genotype are less susceptible to breast cancer than those carry GG or (GG + GA) genotypes. Previous studies also found that rs3087243 was associated with breast cancer susceptibility as a subgroup of several cancer sites (Yan et al., 2013; Zhao, Duan & Gu, 2014). The results were similar with our research, but our our meta-analysis included one more study and have more statistical power.

We didn’t find any relationship between CTLA-4 rs733618 and breast cancer risk in the overall analysis under any genetic model. However, there was a higher risk in the population-based group under all the genetic models except recessive model. Considering that we selected only four eligible studies and most of them were small-size sample (<500), these results need to be taken with caution. One previous study found a positive signal of rs733618 polymorphism with breast cancer (Li et al., 2012) while other two studies showed negative signal (Erfani et al., 2006; Tang et al., 2014). So further researches with larger sample size should be designed and implemented to validate or refute these conclusions.

In contrast, CTLA-4 rs4553808 polymorphism was realated to an increased breast cancer risk in both overall and Chinese population in allelic, heterozygous and dominant models. This suggested that CTLA-4 −1661G allele is more likely to be a risk factor of breast cancer than A allele. Previous meta-analysis also found rs4553808 may increase cancer risk especially for breast cancer (Geng et al., 2014; Yan et al., 2013; Zhao, Duan & Gu, 2014). But these studies investigated the association of this single SNP with various types of cancer while our study specifically focused on breast cancer and investigated several SNPs. Notably, for this SNP, the P-value of HWE in control of one study was less than 0.05 (Erfani et al., 2006), suggesting that the study population was not representative of a broad population. Nevertheless, we decided to keep this study because deleting it did not affect the pooled ORs significantly.

Several limitations of our research should be noticed. Firstly, the sample size in this meta-analysis was relatively small, especially for rs3087243, rs733618 and rs4553808 polymorphisms. Secondly, our results need to be interpreted with caution since we did not find any studies from Europe, Africa or America and most of the included studies were from China. Therefore, more studies with large population and more ethnic groups are needed to provide sufficient statistical power. Thirdly, other factors such as environmental variants, age, and living habit are generally considered to contribute to cancer susceptibility. Lacking data of these factors for adjustment may impact the estimation of breast cancer risk. Lastly, bias may still exist because we failed to find any studies of other races and we did not have access to gray literature.

Conclusion

In summary, our meta-analysis suggests that rs231775, rs3087243 and rs4553808 polymorphisms in human CTLA-4 gene significantly associated with breast cancer susceptibility in Asians, particularly in the Chinese population. In consideration of the limitations of our work, further large-scale studies including multi-racial populations are required to confirm our findings.

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