Associations of lymphotoxin-a (LTA) rs909253 A/G gene polymorphism, plasma level and risk of ankylosing spondylitis in a Chinese Han population

Lymphotoxin-a (LTA) may be associated with the pathogenesis of inflammatory diseases. To assess the association of the LTA rs909253 A/G polymorphism with plasma level and risk of ankylosing spondylitis (AS) in a Chinese Han population. Genotyping and LTA plasma were tested by mass spectroscopy and enzyme-linked immunosorbent assay (ELISA), respectively. The results showed that the average plasma level of LTA in AS was significantly lower than in the controls (P = 0.000). Our results also indicated that LTA rs909253 A/G was associated with a decreased risk of AS (G vs. A: P = 0.014). Significant differences were also found between the rs909253 A/G genotype and down-regulated plasma level in AS patients, compared with controls. After stratification analysis, a decreased risk of AS was associated with the LTA rs909253 G allele (G vs. A) among female patients, younger patients (Yr. < 30), HLA-B27-positive patients. In addition, In conclusion, LTA rs909253 A/G genotype has a significant relationship with decreased susceptibility to AS.


Results
Characteristics of the study population. The demographic and clinical characteristics of all subjects are summarized in Table 1. Subjects were adequately matched for age and sex (P = 0.345 and 0.815, respectively). The genotype distributions of LTA rs909253 A/G in all subjects are illustrated in Table 2. The observed genotype frequencies for the polymorphism in controls were in HWE for LTA rs909253 A/G (P = 0.109).

Association between LTA rs909253 A/G Polymorphisms and the Risk of AS. Logistic regression
analyses revealed that LTA rs909253 A/G polymorphism was associated with the risk of AS (Table 2). Using genotypes AA as a reference, genotype GG acted as a protection factor for AS patients (GG vs. AA: OR = 0.46, 95%CI = 0.26-0.83; P = 0.010). Using genotypes AG + AA as a reference, genotype GG acted as a protection factor for AS patients (GG vs. AG + AA: OR = 0.74, 95%CI = 0.48-1.15; P = 0.007). Our analysis also revealed that LTA rs909253 G allele was associated with significantly decreased risk of AS (G vs. A: OR = 0.70, 95%CI = 0.52-0.93; P = 0.014) than the rs909253 A allele in the Chinese Han population.

Production of LTA in AS Patients, Controls and Different Genotypes.
The average plasma concentration of LTA was significantly lower in AS patients compared with controls (Table 1). We compared LTA plasma levels on basis of LTA rs909253 A/G genotypes. We found LTA rs909253 A/G genotypes had significant lower levels of LTA in AS patients when compared with control groups except for group genotype GG (Table 1). However, we did not find the significant statistic associations between LTA plasma levels and different LTA rs909253 A/G genotypes in AS patients or control groups (Fig. 1).

Stratification of association between plasma level of LTA and other biomarkers.
Our study indicated the LTA plasma levels of female AS patients were significantly lower than male AS patients. However, no associations were obtained between plasma levels of LTA and sex, age, HLA-B27, C-reactive protein (CRP), or grade of the sacroiliac joint in AS patients (Table 4).

Combined analysis with recent researches of LTA rs909253 A/G polymorphisms in AS.
Combined with recent two other researches found that LTA rs909253 AG increased risk of AS significantly than controls (OR = 1.28; 95% CI = 1.01-1.62; P = 0.038) ( Table 5).

Discussion
In the current case-control association study, our present data suggest that the LTA rs909253 A/G genotype is associated with decreased susceptibility to AS. In addition, we also found LTA rs909253 A/G genotypes had significant lower levels of LTA in AS patients compared to control groups. In stratification analysis, we found a decreased risk of AS was associated with the LTA rs909253 G allele (G vs. A) among female patients, younger patients (Yr. < 30) and HLA-B27-positive patients.
AS can lead to a decrease in the quality of life of patients. However, there is no cure for AS, although treatments and medications can reduce symptoms and pain. Many researchers want to find new ways to prevent and treat AS. Genetic factors may contribute to the development of AS 2 . Approximately 90% of people with AS are the HLA-B27 genotype, and thus, there is a strong genetic association 18 . However, only 1-2% of the persons with the HLA-B27 genotype develop AS. Investigating AS-related genetic factors may be helpful in the prevention and diagnosis of AS. Thus, we explored the associations between the LTA rs909253 A/G polymorphism, plasma level and risk of AS in the Chinese Han population.
LTA is located at the HLA-III region of chromosome 6p, is closely linked to TNF-a. LTA gene consists of four exons and three introns. LTA plays a critical role in inflammatory regulation, anti-virus response and immune activation, similar to TNF-α 19,20 .
There have been studies on rs909253 and gastric cancer, chronic obstructive pulmonary disease (COPD), and coronary heart diseases [21][22][23] . A meta-analysis suggested that rs909253 was correlated with the risk of gastric cancer, and especially in Asians 21 . However, no significantly different genotype frequencies of rs909253 were seen in COPD or coronary heart disease compared with controls 22,23 . In this study, we found that rs909253 was correlated with a decreased risk of AS (G vs. A: OR = 0.70, 95%CI = 0.52-0.93; P = 0.014). Then, we searched PubMed and meta-analyzed our results with the results of two other studies on this locus 15,16 . We found that rs909253 had no risk of AS.
LTA is a soluble protein released by lymphocytes and activated by antigens or mitogens. It can inhibit the activity of tumor cells 24 . Bachmann believed that blocking lymphotoxin might be a promising therapeutic strategy for other autoimmune diseases, such as Hashimoto's thyroiditis and arthritis 25 . As far as we know, there have not been any studies on the level of LTA in AS. Thus, we tested the plasma LTA level in AS and healthy controls. We found that the average plasma level of LTA was significantly lower in AS patients, compared with controls (P = 0.000).  www.nature.com/scientificreports www.nature.com/scientificreports/ We stratified the plasma LTA levels of the AS and control groups according to rs909253 genotype. We found that the LTA levels of the genotypes were significantly lower in the AS group except for group genotype GG (Table 1). Recently, Bolstad AI et al. investigated whether SNPs in the LTA gene clusters were correlated with primary Sjogren 's syndrome, and they found that LTA rs909253 and rs1800629 had significantly association with primary Sjogren's syndrome, and the correlations were mainly due to anti-Ro/SSA and anti-La/SSB antibody-positive primary Sjogren's syndrome 26 . Therefore, we hypothesized that LTA rs909253 may affect mechanistic pathways in AS. We will do functional studies of LTA rs909253 in our future research.
Our study has some limitations. First, because of our hospital-based case-control design, we may have selection bias. Second, we investigated SNPs just based on the functional characteristics, another fine-mapping study is required. Third, we use a medium sample size, so our analytical power is limited, although we also combined the results with other two independent studies. Forth, the control group we recruited was trauma patients, which may have a bias on our study. We will collect healthy patients as control in the future research. Fifth, because of the bias of choice, there is a slight difference in the proportion of HLA-B27 positive patients between the 85 vs. 85 samples and the 190 vs. 190 samples. However, because we use a medium sample size, studies including larger population, more ethnic groups are required.

Materials and Methods
Subjects. We obtained approval of the study protocol from the Ethics Committee of Nanjing Medical University (Nanjing, China). All patients provided written informed consent to be included in the study. We confirmed that all research was performed in accordance with relevant guidelines. One hundred and ninety AS patients were consecutively recruited from the Affiliated Changzhou No.2 People's Hospital of Nanjing Medical   Table 4. Stratification of association between plasma levels of LTA and other biomarkers in ankylosing spondylitis patients. *P value was calculated by non-parametric tests. Bold values are statistically significant (P < 0.05).