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Association between SP‐A rs1965708 gene polymorphism and allergic rhinitis risk in Chinese population

Abstract Background Pulmonary surfactant protein A (SP‐A) in the respiratory tract plays an important role in host. In the present, we assessed the association between SP‐A gene polymorphism and allergic rhinitis. Methods Using a case–control design, we compared the genotype frequencies of SP‐A rs1965708 between allergic rhinitis patients and healthy control group. Genotyping was performed using real‐time quantitative PCR‐based molecular identification methods. Univariate and multivariate logistic regression were performed to quantitatively assess the association between rs1965708 polymorphism and allergic rhinitis, and the odds ratio (OR) and 95% confidence interval (CI) were also calculated. Results 500 patients with allergic rhinitis and 500 healthy controls were included in the study. Compared with the CC genotype, we found that AA genotype of rs1965708 could increase the allergic rhinitis risk in the univariate analysis (OR = 2.63, 95% CI: 1.56–4.54, p = 0.000). For dominant model, we found no significant difference in the dominant model (OR = 1.14, 95% CI: 0.86–1.52, p = 0.367). In the recessive model, the CC genotype could elevate the risk of allergic rhinitis compared with CC + AA genotype (OR = 2.70, 95% CI: 1.61–4.54, p = 0.000). Similar results were also found in the allele model (OR = 1.28, 95% CI: 1.07–1.54, p = 0.008). Interactions between rs1965708 AA or AC and smoking increased the allergic rhinitis risk. Conclusions The rs1965708 variants of SP‐A gene polymorphism are associated with allergic rhinitis, and the A allele could increase the allergic rhinitis risk. The AA SNP variants that interact with smoking may alter the susceptibility to allergic rhinitis.

asthma, sinusitis, nasal polyposis, otitis media, and conjunctivitis and has been identified as one of the top ten reasons for visiting primary care clinics. 3 The incidence of AR has increased recently.
Although the etiology of AR is not well understood, 4 it is established that the occurrence of AR is a consequence of gene-environment interactions.
Surfactant proteins (SPs) are produced and secreted by alveolar type II cells and Clara cells, including hydrophilic SPs A and D (SP-A and SP-D), and the hydrophobic SPs B and C (SP-B and SP-C). The human SP-A and SP-D genes are located on chromosome 10q22-q24. 5 It is suggested that SP-A consists of one SP-A1 molecule and two SP-A2 molecules. Both SP-A1 and SP-A2 transcripts are expressed in adult human alveolar type II cells.
However, SP-A1 and SPA-A2 are differentially expressed in different tissues. 6 Both SP-A1 and SP-A2 transcripts have been expressed in the human small and large intestines, whereas only the SP-A2 genes were predominantly detected in the epithelium of the reproductive system. 7 SP-A was also detected in the human nasal mucosa. Both SP-A and SP-D belong to the C-type lectin superfamily and prevent lung collapse during expiration. It has been demonstrated that these molecules participate in the innate immune response and regulate inflammatory processes. 8 Genetic variations in SP-A1, SP-A2, and SP-D genes are related to susceptibility to several infectious diseases, such as respiratory syncytial virus infection and meningitis. 9,10 SP-A and SP-D directly bind to rough lipopolysaccharides and other components present on the surface of bacteria and other microorganisms. SP-A and SP-D also inhibit the growth of gram-negative bacteria by increasing membrane permeability and the ability to kill bacteria. 11 In addition, SP-A and SP-D bind to different receptors on the surface of several types of immune cells and modulate inflammatory processes and innate immune function by influencing MAPK and other signaling pathways. 12 Recently, Malhotra et al 13 found SP-A-specific binding allergen particles. SP-A can also reduce the proliferation of mononuclear cells in the peripheral blood of asthmatic children caused by house dust mites. The SP-A1 alleles differ at five codons, the amino acids 19, 50, 62, 133, and 219, while the SP-A2 alleles differ at four codons, the amino acids 9, 91, 140, and 223.
Previous studies have reported that several allele variants of SP-A1 and SP-A2 genes are related to respiratory distress syndrome, severe respiratory syncytial virus bronchiolitis, and pulmonary tuberculosis. A study also found that genetic polymorphism of SFTPA and risk of Aspergillus allergy in Caucasian populations, as well as polymorphism of the SFTPA gene, were also strongly associated with susceptibility to asthma in children. 13 In the present study, we assumed that gene polymorphisms of SP-A may be associated with the overall genetic predisposition to allergic airway diseases instead of affecting the occurrence of pulmonary diseases. Previous studies with small sample sizes found a positive association between SP-A rs1965708 and AR. 14 In the present study, we validated the association in different population settings and explored the potential interaction between rs1965708 polymorphism and other factors.

| Data collection and genotyping
The general characteristics and clinical information were collected from the medical records, including age, sex, and body mass index (BMI) calculated using the formula weight (kg)/height (m) 2 ; BMI > 24 was defined as overweight. Smoking was defined as current smoking or smoking previously daily, and drinking was defined as two to four times a month. 16 A history of diseases was also collected. Two 5 ml venous blood samples were extracted in the morning before meals using test tubes containing ethylenediaminetetraacetic acid (EDTA). One sample was used to detect IgE, interleukin-6 (IL-6), IL-8, and IL-10 using enzyme-linked immunosorbent assays. The remaining blood sample was stored at −80°C for genotyping.
Using the DNA Purification Kit, the "salting-out" method was used to extract genomic DNA, which was stored at −80°C. The genotypes of SP-A rs1965708 loci were analyzed from the DNA samples of each participant using the SNPscanTM genotyping method. To determine whether the obtained genotypes of SP-A SNPs could not be influenced by the technology, we randomly selected 110 DNA samples for checking, and no inconformity was found. DNA concentration and purity were measured using a UV spectrophotometer (Pharmacia Biotech). PCR amplification primers and single-base extension primers (forward: 5ʹ-AAGAAAGCAAGTTCTCTGCCTG-3ʹ; reverse: 5ʹ-CTGTGTACATCTCCACACACTT-3ʹ) for the rs1800544 site were designed using Sequenom Genotyping. Amplification was performed in a 5 µL reaction system using multiplex PCR. The PCR assay conditions were as follows: 94°C for 4 min, 45 cycles of 94°C for 20 s, 56°C for 30 s, 72°C for 1 min, and 72°C for 3 min, and the amplicons were maintained at 4°C.

| Statistical analysis
Continuous variables (age, BMI, IgE, IL6, IL8, and IL10) were expressed as the mean ± standard deviation, and Student's t test was used for comparison between the case and control groups.
Categorical variables (sex, smoking, drinking, family history, overweight, and genotype frequency) were expressed using count and percentage, and the chi-square test was used for comparisons between the two groups. The Hardy-Weinberg equilibrium (HWE) was assessed using SP-A genotype frequencies. The following gene models were used to assess the association between gene polymorphism and allergic rhinitis: heterozygote (AC vs. AA), homozygote (AA vs. to select the best genetic model. The smaller the AIC, the better the model. The odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. Subgroup analysis was performed for categorical variables (sex, age, overweight, family history, drinking, and smoking) to compare the relationship between SP-A gene polymorphisms and AR. Furthermore, we also analyzed the association between gene polymorphisms and inflammation factors (IgE, IL6, IL8, and IL10). All the analyses were computed using SPSS 23.0, and twosided p < 0.05 was considered significant.

| General characteristics between the case and control groups
This study included 500 patients with AR and 500 healthy controls. Table 1 summarizes the demographic information and the rs1965708 gene frequency distributions in the study subjects. There were no significant differences in sex ratio (p = 0.746), age (p = 0.291), BMI (p = 0.233), or overweight ratio (p = 0.139) between the case and control groups. However, the case group had a higher smoking ratio than the control group (36.0% vs. 22.8%, p < 0.001). The drinking ratio in the case group was 24.8%, with a rate of 30.0% in the control group, but no significant difference was observed between the two groups (p = 0.076). There was no significant difference in TA B L E 1 Comparisons of general characteristics between case and control group the family history of allergic rhinitis between the case and control groups (p = 0.050). Furthermore, the IgE level was higher in the case group than in the control group (p < 0.0001). The levels of inflammatory factors IL-6, IL-8, and IL-10 were also higher in the case group than in the control group (p < 0.001 for all).

| SP-A gene rs1965708 polymorphism and allergic rhinitis risk
We identified three genotypes (CC, AC, and AA) from the samples. The chi-square test indicated that genotype frequencies were in accordance with the Hardy-Weinberg equilibrium (x 2 = 0.012, p = 0.914).

| Interactions between genes and environment components
To explore the interaction effects between environmental components and genes on the risk of AR, we performed an interaction analysis. Interaction analysis was performed for two factors: smoking and overweight.   Bold values are just in order to emphasize that these P values <0.05

| Association between the rs1965708 variants and clinical parameters
We also analyzed the association between the rs1965708 variants and IgE, IL-6, IL-8, and IL10 in participants with AR. As shown in

| DISCUSS ION
The nasal mucosa is exposed to the outside air for a long time.
Pollutants, microorganisms, and allergens in the external environment can stimulate the nasal mucosa. In this case, innate host defense plays a key role in resisting damage to the nasal mucosa. 17 the AR and nasal polyp groups was higher than that of the control group, and the difference was significant. 22 As the first-line defense molecule of the nasal mucosa, SP-A gene polymorphism may cause structural and functional changes that may lead to the development of allergic diseases. The present study found that (1) there were significant differences in terms of allele and/or genotype frequency in rs1965708, and the A allele increased the risk of AR; (2) overweight weakened the effect of rs1965708 on the risk of AR, while the environmental components, including sex, age, smoking, and drinking, had no effect on the association between the rs1965708 variants and increased risk of AR; (3) interactions between rs1965708 AA or AC and smoking increased the risk of AR; (4) increased AR caused by the rs1965708 variants could be associated with IgE and some inflammatory factors. Our results provide evidence of this assumption.

Surface-active protein A (SP-
In the present study, the frequencies of CC, AC, and AA were  23 In another study on the Chinese population, the reported frequencies of the three genotypes were 96.0%, 4.0%, and 0.0%. 24 These results indicate that the SP-A variant greatly varied in different populations. A previous study also found that SP-A gene polymorphisms were associated with asthma risk, 25  is an important cause of the increasing inflammatory response. 28 The pathogenesis of AR is related to the activation of circulating T lymphocytes and mononuclear macrophages, and its activation is related to cytokines such as IL-1, TNFα, and IL-6. The activation of these cytokines reflects the acute phase of the inflammatory response and has important consequences for the clinical manifestations of AR. 29 Our results also showed that the rs1965708 variants were associated with IgE and inflammation factors. This could be explained as follows: It is known that the human SP-A gene is located on chromosome 10q22-q23, including two highly homologous functional genes (SP-A1 and SP-A2) and one pseudogene. A total of four single-nucleotide polymorphisms (SNPs) mapped to SP-A1 and five SNPs mapped to SP-A2 have been identified. 30 SP-A1 is not associated with the risk of AR, whereas only one SNP of SP-A2 is associated with AR. Allergen inflammatory response is known to play a key role in airway inflammatory disease. Some allergens can be identified and bound to the collagen lectin family. SP-A is a member of the collagen lectin family, which has the same structural characteristics: It is located in a region rich in hemieptidine at the N-terminal of the skin chain. The original area of the Treponema pallidum structure adhesive alphacoiled spiral "bottleneck" area plant lectin area (CRD area) at the C-end of the skin chain. An important biological function of the CRD region is its ability to bind to aspirated allergen particles. 31 The rs1965708 variants belong to the SP-A2 gene and are located in the CRD region. The rs1965708 variant may affect the ability of SP-A2 to allergens. Previous studies also found that SP-A2 has a greater ability to cytophagy, inhibit the secretion of surface-active substances, and induce the release of tumor necrosis factor. 32 This may explain why the rs1965708 variants are associated with allergic rhinitis susceptibility.
The present study had some limitations. First, based on the inherent shortcomings of case-control design, the cause-effect explanation is limited. Second, we only reported one SNP associated with AR because some SNPs have been previously associated with AR, but the interaction with other SNPs should be reported. Third, AR is a disease caused by genes and environments, and more geneenvironmental interactions should be explored for AR risk. Finally, we found that the increased risk of AR caused by these SNP variants could be associated with IgE and some inflammatory factors; however, the specific molecular mechanism needs to be investigated in future studies.
In conclusion, the rs1965708 variants of SP-A gene polymorphisms are associated with AR, and the A allele could increase the risk of AR. The AA SNP variants that interact with smoking may alter the susceptibility to AR. Studies with larger sample sizes are required to validate the present findings.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.

AUTH O R CO NTR I B UTI O N S
ZXH contributed to this research idea and study design. YXH performed data collection. YXH wrote the manuscript. WB, HLL, and YZQ revised the manuscript. ZXH proofread the final version. All authors read and approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
Original data will be provided upon request by correspondence author (zhangxinhaify@foxmail.com).