Association of BTLA Polymorphisms with Susceptibility to Non-Small-Cell Lung Cancer in the Chinese Population

Studies have reported that B- and T-lymphocyte attenuator (BTLA) polymorphisms may be associated with the risk to different cancers. However, the correlation between those variations and non-small-cell lung cancer (NSCLC) is still unclear. A total of 1,003 NSCLC patients and 901 noncancer controls were recruited in the study, to confirm the association of variations in BTLA gene with the risk of NSCLC. The SNPscan™ genotyping assay was used to obtain the genotypes of the four BTLA polymorphisms (BTLA rs1982809 G>A, rs16859629 T>C, rs2171513 G>A, and rs3112270 A>G). It was found that BTLA rs1982809 polymorphism reduced the risk of NSCLC (GA vs. GG: adjusted odds ratio (OR) = 0.81, 95%confidence interval (CI) = 0.66‐0.99, and P = 0.043). However, the BTLA rs16859629, rs2171513, and rs3112270 polymorphisms showed no significant association between NSCLC patients and controls in overall comparison. In subgroup analyses, we found that BTLA rs1982809 polymorphism reduced the risk of NSCLC (nonsquamous cell carcinoma: GA vs. GG: adjusted OR = 0.79, 95%CI = 0.64‐0.97, and P = 0.026; AA/GA vs. GG: adjusted OR = 0.81, 95%CI = 0.66‐0.99, and P = 0.037; ≥59 years: GA vs. GG: P = 0.036; never alcohol consumption: GA vs. GG: P = 0.013; GA/AA vs. GG: P = 0.016; body mass index (BMI) ≥ 24 kg/m2: GA vs. GG: P = 0.030; GA/AA vs. GG: P = 0.041). The BTLA rs16859629 polymorphism increased the risk of the development of squamous cell carcinoma (CC vs. TT: adjusted OR = 9.85, 95%CI = 1.37‐71.03, and P = 0.023; CC vs. TT/TC: adjusted OR = 9.55, 95%CI = 1.32‐68.66, and P = 0.025). Taken together, the findings of the present suggest that BTLA rs1982809 and rs16859629 polymorphisms may influence the susceptibility to NSCLC in the Chinese population.


Introduction
Non-small-cell lung cancer (NSCLC) accounts for 80 to 85% of all the lung cancer and is the main pathological type of lung cancer (LC). LC has imposed huge diseases burden on human population and accounts for significant number of mortalities across the globe [1][2][3]. As per estimates, LC is currently ranked as first in terms of incidence and mortality [4]. The currently used treatment strategies for LC include surgery combined with adjuvant therapy. Owing to the recent advancements made in the diagnosis and treatment, the clinical outcomes have significantly improved [5]. However, the identification of the potential risk factors for the occurrence of LC is considered essential as it will permit early diagnosis and immediate management.
The B-and T-lymphocyte attenuator (BTLA), an immunosuppressive receptor, was identified after the cytotoxic Tlymphocyte-associated antigen-4 (CTLA-4) and programmed death-1 (PD-1) and is thus the third member of the CD28 immunoglobulin superfamily (IgSF) [6]. The BTLA upon binding with herpesvirus entry mediator (HVEM) inhibits the T cell response. In contrary, blocking of BTLA may in turn activate T cells [7][8][9]. Evidences suggest that BTLA plays a crucial immune regulatory role in human malignancies. Liu et al. [10] have reported that BTLA/HVEM pathway plays an important immunosuppressive role in the regulation of T cells in peripheral blood of hepatocellular carcinoma patients. Quan et al. [11] and Li et al. [12] found that the level of BTLA expression may act as a biomarker for the prognosis of diffuse large B cell lymphoma and NSCLC. In yet another study, Chen et al. [13] concluded that chemotherapy in combination with anti-BTLA antibody improves the prognosis of ovarian cancer in a mouse model. Similarly, Zhang et al. [14] reported that BTLA could directly intervene the effects of miR-32 on cancer cells. Collectively, these studies point towards a correlation between BTLA and the development and progression of human cancers.
Single nucleotide polymorphism (SNP) mutations constitute an important form of genetic variations. Some recent studies have revealed a significant correlation between BTLA SNPs and development of cancer [15][16][17][18][19]. Fu et al. reported that BTLA rs1844089 C>T and rs2705535 A>G variation could increase the risk of breast cancer in human [15]. Partyka et al. and Karabon et al. found that the variation on BTLA rs1982809 G>A gene polymorphism might be a potential risk factor for the development of chronic lymphocytic leukemia (CLL) and renal cell carcinoma [16,17]. In a previous study, we reported that BTLA rs1982809 SNPs increased the susceptibility of esophagogastric junction adenocarcinoma (EGJA) in smoking subgroup analyses [18]. In another study, we found that the BTLA rs3112270 A>G and rs2171513 G>A polymorphisms could modify the risk of esophageal squamous cell carcinoma (ESCC) [19]. Given this background, we hypothesize that some associations may exist between BTLA polymorphisms and NSCLC pathogenesis.
Combined with these previous studies, the BTLA tagging SNPs (rs1982809, rs16859629, rs2171513, and rs3112270) were selected for analysis. Consistently, the present study is    [20]. Each participant was informed about the purpose of the study and was asked to sign a written consent form for participation in the study. The study was approved by the research ethics committee of Fujian Medical University Union Hospital (Approval No. 2018KY023).

Selection of BTLA Tagging
SNPs. The four candidate SNPs were ascertained by applying Genome Variation Server data [18,19,21] (http://gvs.gs.washington.edu/GVS147/). We gave priority to represent the main linkage disequilibrium blocks. The following main criteria were used: (a) minor allele frequency ≥ 0:05, (b) linkage disequilibrium of r 2 < 0:8 between the SNPs, (c) with the 5 kb extent upstream and downstream of the gene regions, and (d) the genotyping value ≥ 95% in the CHB cohort. Four BTLA tagging SNPs (rs1982809, rs16859629, rs2171513, and rs3112270) were finally selected to decipher the association between the BTLA polymorphisms and NSCLC risk. The results are shown in Table 1. 2.3. DNA Extraction and Genotyping. Around 2 mL of the peripheral blood samples of the subjects (early in the morning, empty stomach) was collected and placed in a vacuum ethylene diamine tetra-acetic acid anticoagulant tube. Genomic DNA was carefully extracted from the collected samples with the help of DNA blood mini kit (Promega, Madison, USA) by following manufacturer's guidelines. The BTLA rs1982809, rs16859629, rs2171513, and rs3112270 genotypes were assessed by the SNPscan™ Kit (Genesky Biotechnologies Inc., Shanghai, China) as per manufacturer's guidelines. For the qualitative assessment, 76 samples (4%) were randomly selected and subsequently tested by another laboratory technician. The genotypes of BTLA were well confirmed repeatedly.

PCR-Specific Experimental Procedures
2.4.1. DNA Cleavage. The DNA was diluted to a concentration of 30 to 50 ng/μL. From this 4 μL, DNA was transferred to a 96-well plate followed by the addition of 2.5 μL 4X DNA buffer. Thereafter, the total volume was made up to 10 μL by the addition of sterile ddH 2 O. The contents were mixed well and incubated at 98°C for 5 min. Afterwards, the plates were immediately placed in ice.

Baseline Characteristics.
A total of 1,003 NSCLC cases and 901 controls were recruited in this study. The risk factors that may influence the development of NSCLC are listed in Table 2. Although a good match was observed between the age of the NSCLC patients and the control group (P = 0:139 ), significant differences were observed in sex, smoking, alcohol consumption, and BMI between the two groups (P < 0:001). The genotyping rate of BTLA rs2171513, rs3112270, rs1982809, and rs16859629 was higher than 95% (99.00%, 98.84%, 98.90%, and 97.48%, respectively) as shown in Table 1. Minor allele frequencies (MAFs) in the
Collectively, the results suggest that the genotypes in BTLA rs2171513 and rs16859629 mutations may be associated with the development of NSCLC.
In nonsquamous cell carcinoma (non-SCC), using AA genotype as reference, it was found that GG and GA+GG genotypes of BTLA rs3112270 polymorphisms may decrease the incidence of non-SCC (AG: OR = 0:82, 95%CI = 0:67-  Adjusting the related risk factors, it is concluded that the BTLA rs3112270 polymorphism did not exhibit the tendency to change the risk to non-SCC. However, BTLA rs1982809 polymorphism was a protective factor   The genotype frequencies of BTLA rs1982809 polymorphism in the subgroup analyses are depicted in Table 5. In ≥59-year subgroup, it was found that the variants of BTLA rs1982809 decreased the incidence of NSCLC (GA vs. GG: P = 0:036). In never alcohol and BMI ≥ 24 kg/m 2 subgroup, it was found that similar genotype variants of BTLA rs1982809 might be a protective factor of NSCLC (never smoking subgroup: GA vs. GG: P = 0:013; AA/GA vs. GG: P = 0:016; BMI ≥ 24 kg/m 2 : GA vs. GG: P = 0:030; AA/GA vs. GG: P = 0:041). Additionally, it was found that BTLA rs2171513, rs3112270, and rs16859629 polymorphisms were not associated with the morbidity of NSCLC in subgroups (Tables 6-8). 3.4. SNP Haplotypes. The SHESIS software online (http:// analysis.bio-x.cn/myAnalysis.php) was used to perform the haplotype analysis. If the probability of BTLA haploid in the case-control study is less than 0.03, it is classified as others. In the end, six subgroups were built. The results are shown in Table 9 and Figure 1. No significant linkage disequilibrium (LD) relationship was observed among the BTLA rs16859629, rs1982809, rs2171513, and rs3112270 (r 2 < 0:8). It was found that compared to BTLA T rs16859629 G rs1982809 G rs2171513 A rs3112270 haplotype, the haplotype BTLA T rs16859629 A rs1982809 G rs2171513 G rs3112270 significantly reduced the susceptibility to NSCLC (OR = 0:66, 95%CI = 0:659-0:930, and P = 0:005).

Discussion
The pathogenesis of LC is overly complex. It is believed that LC might be a disease driven by multiple genes [22]. Epide-miological studies have proved the correlation of the etiology of LC and the gene-environment interaction [23,24]. Recently, immune checkpoint inhibitors have attracted remarkable attention in cancer treatment. For instance, PD-1 and CTLA-4 immunity inhibitors have achieved certain efficacy in the treatment of advanced LC [25]. Nonetheless, the cause of LC is still largely unclear. A previous study had reported that overexpression of BTLA could predict a poor prognosis in NSCLC patients [12].
In the present case-control study, the potential relationship between the BTLA rs1982809 G>A, rs16859629 T>C, rs2171513 G>A, and rs3112270 A>G SNPs and susceptibility to NSCLC was explored [23,26]. It was found that BTLA rs1982809 polymorphism might reduce the risk of overall NSCLC. But candidate locus of BTLA rs2171513, rs3112270, and rs16859629 SNPs could not affect the susceptibility to NSCLC. In NSCLC subgroup analysis, BTLA rs16859629 SNPs could increase the risk of SCC. However, BTLA rs1982809 SNPs might reduce susceptibility to non-SCC. In addition, BTLA rs1982809 SNPs could reduce the susceptibility to NSCLC in the BMI ≥ 24 kg/m 2 , ≥59 year, and never drinking subgroups. To the best of our knowledge, the present study for the first time reports the relationship between BTLA SNPs and NSCLC susceptibility in the Chinese population.
BTLA rs1982809, as a locus in the 3 ′ -untranslated region (UTR), has been reported to exhibit an association with the development of some malignancies. The 3 ′ -UTR plays a crucial role in the regulation of mRNA expression [27,28]. Karabon et al. reported that BTLA could exert its effects on Tlymphocytes by affecting mRNA expression levels due to T to C substitutions of rs1982809 [16]. Additionally, it was also reported that BTLA rs1982809 might act as a potential biomarker in predicting the multiple organ dysfunction syndrome. In another study, rs1982809 polymorphism of the BTLA was found to be associated with the risk of kidney cancer [17]. Similarly, in our previous study, we report that the gene variation in BTLA rs1982809 increased the susceptibility to EGJA in ever smoking subjects [18]. In contrary, Cao et al. concluded that the distribution of genotype in BTLA rs1982809 was not different from the ESCC and the control group [19]. In short, the previous results were ambiguous, even contradictory. Considering that genetic variations played different roles in different cancers, 1,904 participants were enrolled to conduct a more precise evaluation. In this study, we showed that the G to A changes of BTLA rs1982809 genotype reduced the overall risk of NSCLC, especially the non-SCC, BMI ≥ 24 kg/m 2 , ≥59 year, and never drinking subgroups. These facts indicated that BTLA rs1982809 polymorphism could play a critical role in the susceptibility to NSCLC. However, our findings should be interpreted with cautions. Studies with larger sample sizes are required to further validate the effects of this locus on NSCLC.
The BTLA rs16859629 SNP, which is located in the intron variant sequence, plays an important role in alternative splicing [29]. It has been reported that SNP located in the intron regions may affect the susceptibility to several human diseases [30][31][32]. When adjusted for including 5