A Genetic Variant of BTLA Gene is Related with Increased Risk and Clinical Manifestations of Breast Cancer in Chinese Women

Background: B and T lymphocyte attenuator (BTLA), an immunoinhibitory receptor, is shown to suppress the lymphocyte activation. Several studies addressed the relationship between BTLA rs1982809 polymorphism and cancer's risk. Methods: To identify the effects of this polymorphism on the risk of breast cancer (BC), this study was designed in Chinese women from China, Jiangsu Province. This study involved 324 BC patients and 412 controls. Results: We observed that BTLA rs1982809 polymorphism elevated the risk of BC. Similar nding was also shown in the subgroup of premenopausal women and those aged < 55 years old. In addition, this polymorphism correlated with the estrogen receptor (ER) status, C-erbB-2 status, Ki-67 status, tumor node metastasis (TNM) stage and tumor size of BC patients. Conclusions: Collectively, BTLA rs1982809 polymorphism shows a signicant association with elevated risk and clinical features of BC in Chinese women. Further studies involving other races are urgently needed to replicate these ndings.


Introduction
Breast cancer (BC) is the second frequent cancer, and it is also the most common malignancy in women worldwide [1]. Nearly 2.1 million new diagnosed BC patients and approximately 627,000 BC-related deaths occurred in 2018 [2]. BC is a heterogeneous disorder clinically, which has diverse pathology subtypes and molecular subtypes with varied aetiologies, risk factors, and responses to prognoses and treatments [3,4].
Due to the implementation of mammographic screening and improvement of treatment methods for BC, the diagnosis and prognosis of BC improved increasingly. Endogenous hormone levels, exogenous hormone intake, high alcohol consumption, smoking, less physical activity, some breast diseases, and genetic susceptibility are all risk factors for BC [5][6][7]. Up to date, several GWAS studies have demonstrated important loci associated with BC susceptibility [8][9][10][11][12].
B and T lymphocyte attenuator (BTLA) is a suppressor molecule, which belongs to the immunoglobulin superfamily. BTLA is mainly expressed in B lymphocytes, T lymphocytes, macrophages and dendritic cells.
BTLA is an immunoinhibitory receptor which could suppress lymphocyte activation by delivering inhibitory signals, with similarities to programmed cell death-1 (PD-1) or cytotoxic T lymphocyte antigen-4 (CTLA-4) [13,14]. BTLA could negatively regulate immune responses, and BTLA-de cient T cells led to the elevated proliferation of T cells [15]. Herpes virus entry mediator (HVEM), the ligand of BTLA, could interact with BTLA, thereby resulting in the inhibition of T-cell activation and cytokine production [13,16,17]. Liu et al. indicated that BTLA/HVEM pathway was associated with peripheral T cell suppression in hepatocellular carcinoma patients, which may serve as a promising target for hepatocellular carcinoma immunotherapy [18]. Sekar et al. observed the effects of BTLA on type I Natural Killer T cells in limiting anti-tumor immunity [19].
Considering the important role of BTLA in cancer immunology, we assumed that genetic variations in BTLA gene may be associated with cancer susceptibility.
The BTLA gene is located on the chromosome 3q13.2. Previous studies have investigated whether BTLA rs1982809 polymorphism was linked with the risk of cancer, including renal cell carcinoma [20], chronic lymphocytic leukemia [21], esophagogastric junction adenocarcinoma [22], esophageal squamous cell carcinoma [23] and lung cancer [24]. However, no studies explored the association of this polymorphism and BC risk. Thus, we aimed to determine whether BTLA rs1982809 polymorphism conferred susceptibility to BC in Chinese women.

Subjects
This research enrolled a total of 324 BC patients and 412 age-matched controls from Huaian No.1 People's Hospital. BC was diagnosed according to imaging and pathological manifestations. Clinical information of all individuals was recorded from electronic medical records and questionnaires. In addition, 412 healthy volunteers, receiving health examination in the same hospital, were selected as controls. Controls with other cancers or family history of cancer were not recruited in this study. The Institutional Review Board of our hospital approved this study. Informed consents were obtained from all individuals. This research was also in compliance with the standards of Declaration of Helsinki.

Genotyping
Venous samples were got from all subjects. Peripheral venous blood samples from all subjects were used to extract DNA. BTLA rs1982809 polymorphism was genotyped using the PCR method. The primers for this polymorphism were 5'-GCTCTCGAGTCCCGTCCCAGTAGTTGAAGGGTCCTGCTA-3'(F) and 5'-TGCCAGCTGCTTCGAGCGAAGCCATTGCTGC-3'(R). We genotyped 10% chosen samples again to evaluate the genotyping error; and the concordance rate was 100%.
Statistical analysis χ 2 test and t test were utilized to analyze the categorical variables and continuous variables, respectively. A goodness-of-t χ 2 test was intended to estimate the deviation from HWE in controls. The odds ratios (ORs), as well as 95% con dence intervals (95% CIs), were determined with logistic regression analysis with adjustment of age and Body Mass Index (BMI), and menopausal status or not. P value < 0.05 was regarded as signi cant. Statistical analysis was performed by SPSS 22.0.

Characteristics of all enrolled individuals
The characteristic information and clinical features of all subjects are listed in Table 1. This study recruited 324 cases and 412 controls aged 50.48 ± 10.16 and 49.62 ± 9.54 years old, respectively. No obvious Page 4/14 differences were obtained among age, BMI and menopausal status (P > 0.05). Other clinical features of BC are also summarized in Table 1.  In addition, we interpreted the effects of the BTLA rs1982809 polymorphism on the risk of BC in the subgroups by BMI, age, and menopausal status (Table 3). Data detected that the rs1982809 polymorphism was more likely to intensify the risk of BC among premenopausal women and those aged < 55 years old.

Discussion
In this presented study, we observed that BTLA rs1982809 polymorphism intensi ed the risk of BC in Chinese women. Further subgroup analyses obtained that this increased risk effect was more evident in premenopausal women and those aged < 55 years old. Additionally, we found that rs1982809 polymorphism was linked to ER status, C-erbB-2 status, Ki-67 status, TNM stage and tumor size of BC patients.
Recently, several studies paid attention to the link between BTLA rs1982809 polymorphism and cancer risk.
Partyka et al. showed that this SNP strengthened the risk of renal cell carcinoma in a Polish population [20]. A similar nding was shown in another study by their research team, and they found that BTLA rs1982809 polymorphism elevated the risk of chronic lymphocytic leukemia [21]. A Caucasian study from Tunisia also replicated this positive result; they observed that rs1982809 polymorphism was shown to elevate the risk of lung cancer in Tunisian patients [24]. As for Asian populations, no positive relationship was observed between this SNP and esophageal squamous cell carcinoma [23] or esophagogastric junction adenocarcinoma risk in Chinese Han population [22]. From the ndings of abovementioned studies, we assumed that BTLA rs1982809 polymorphism might be speci c for Caucasians, but not for Asians. However, we observed that BTLA rs1982809 polymorphism elevated the risk of BC in Chinese women in this study. Fu et al. revealed that BTLA gene polymorphisms were connected to the intensi ed risk of BC in Chinese women [24]. Nevertheless, they did not investigate rs1982809 polymorphism [24]. Honestly, this was the rst Chinese study to observe this nding. Further studies in other Chinese populations should be conducted to verify the ndings of this study.
In addition, we performed subgroup analyses, and revealed that BTLA rs1982809 polymorphism enhanced the risk of BC in premenopausal women and those aged < 55 years old. Thus, we should pay more attention to those populations who had mutation of this SNP, because these individuals were more prone to the occurrence of BC. Last, we observed that BTLA rs1982809 polymorphism was associated with the ER status, Ki-67 status, C-erbB-2 status, TNM stage and tumor size of BC patients, suggesting that this SNP was signi cantly associated with the clinical manifestations of BC. It is worth noting that other SNPs of BTLA gene were also shown to be related with some clinical features of BC in Chinese women from Heilongjiang Province [33].
This study had several limitations. One, the sample size was not large enough. Two, the ndings of this study might not be not applicable to other races, because all enrolled individuals were Chinese. Three, we only investigated one SNP of BTLA gene. Four, whether BTLA rs1982809 polymorphism was associated with the gene and protein levels is unknown. Five, whether this SNP affected the prognosis of BC needs further investigation. Six, the biological functions of rs1982809 polymorphism remained to be explored. Last, the interaction between this polymorphism and environmental factors should be considered.
Collectively, this case-control study observes that BTLA rs1982809 polymorphism is remarkably associated with higher risk and clinical manifestations of BC in Chinese females. Further retrospective studies are needed to validate these ndings.

Declarations
Ethics approval and consent to participate The Institutional Review Board of The A liated Huaian No.1 People's Hospital of Nanjing Medical University approved this study. Informed consents were obtained from all individuals.

Consent for publication
Not applicable.

Data availability statement
The data that support the ndings of this study are available from the corresponding author upon reasonable request.