The Polymorphism of Osteopontin Gene and Receptors are associated with Allergic Rhinitis in a Chinese Han Population

Osteopontin (OPN) is a pro inflammatory cytokine involved in chronic inflammatory diseases. In this study, we evaluated the potential association of OPN polymorphisms with Allergic Rhinitis (AR) in a Chinese Han population. Three single-nucleotide polymorphisms (SNPs) in OPN−rs9138, rs1126616, rs1126772, and eight SNPs of its receptor−rs1448623, rs3770136, rs3738919, rs3911238, rs5918, rs2056131, rs187116, rs16927061 were genotyped in 1020 AR patients and 1173 healthy controls by using a PCR-restriction fragment length polymorphismassay. The allele, genotype, and haplotype frequenciesin the patients and the controls were compared using a χ2 test. Moreover, we performed haplotype analysis by using the online software platform SHE sis. The results revealed a significant association between five SNPs-rs9138, rs1126616, rs1126772, rs1449263, rs187116-and AR in the studied Chinese Han population. Furthermore, the ATG, CCG haplotype were associated with a significantly higher prevalence of AR than were the CTA haplotypes. However, no significant differences were detected in either the genotype or allele frequencies of the other SNPs between the AR and control groups. Overall, this study has identified a strong association between OPN polymorphisms and increased risk of AR in a Chinese Han population.


Ethics statement
This study was approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University. Written informed consent was obtained from all adult participants and from parents or legal guardians of minors.

Patients
From May 2011 to June 2014, 1020 AR patients aged 18 to 60 years were enrolled in this study. All patients were identified by and treated at the outpatient clinic of the Department of Otolaryngology at the First Affiliated Hospital of Chongqing Medical University, Chongqing, China. The diagnosis of AR was based on the patients' medical history, symptoms and the presence of a positive skin pricking test (SPT, Allergopharma, Hamburg, Germany) in response to a panel of common allergens defined by the ARIA 2008 guidelines [18]. The SPT results were diagnosed in accordance with the recommendations of the Subcommittee on Allergen Standardization and Skin Tests of the European Academy of Allergy and Clinical Immunology [19].
A positive SPT result was defined as the formation of a wheal larger than or equal to one half the diameter of the histamine control wheal, and at least 3 mm larger than the diameter of the negative control wheal. A total of 18 inhaled allergens were tested, including house dust, grass, tree, mold, food, and cat and dog dander et al. In contrast, patients with accompanying systemic disease were excluded from the study. A total of 1173 hereditarily-unrelated healthy individuals were enrolled as controls. They did not show clinical features or family history of allergies and had not experienced an upper respiratory tract infection within the 4 weeks prior to the study. All subjects of this study were of Chinese Han ethnic origin and from the Chongqing city of China.
The 11 SNPs were genotyped by restriction fragment length polymorphism (RFLP) analysis. Briefly, peripheral blood samples were collected from each subject and stored at -80°C for further use. Genomic DNA was isolated from blood leukocytes using the Qiagen DNA Blood Mini kit (Qiagen, Valencia, CA, USA). Amplification of target DNA was performed by PCR using the primers listed in Table 2. PCR conditions were as follows: initial denaturation at 95°C for 5 min, 37 cycles of denaturation at 95°C for 30 s, annealing at 58-62°C for 30 s, and extension at 72°C for 30 s, and final extension at 72°C for 5 min. The PCR products were digested with specific restriction enzymes ( Table 2)at 37°C for at least 4 h. Digestion products were visualized on a 4% a garose gel and stained with Goodview (SBS Gene tech, Beijing, China). Genotypes were confirmed by direct sequencing by the Invitrogen Biotechnology Company (Guangzhou, China) using20% of randomly selected PCR products.

Statistical analysis
Call rates for the SNPs studied were compared between AR patients and controls by the χ2 test. The χ2 test was also applied to compare demographic characteristics and the allele and genotype frequencies between the patients and controls. Estimation of genotype frequencies was performed by direct counting. The online software platform SHE sis was used to analyze the haplotype and probabilities. We used non-risk alleles as the reference, and tested all the other haplotypes. Logistic regression analysis was used to analyze the genotype allele, controlling for age, gender, and occupation as the co variables. Odds ratios (OR) and 95% confidence intervals (CI) were calculated to determine the association of SNPs with the risk of AR. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) version 13.0 for Windows (SPSS Inc., Chicago, Ill. USA). P values were corrected (Pc) for the number of alleles tested using the Bonferroni correction method. A Pc value of <0.05 was considered statistically significant.

Subject characteristics
Demographic and clinical characteristics of the participants are presented in Table 1. The patient group consisted of 493 males and 527 females, with a mean age of 31.58 years (SD = 7). The control subjects included 593 males and 580 females and had a mean age of 33.23 years (SD = 8). There was no significant difference between the 2 groups with respect to mean age and gender distribution. About 51.8% of the patients were allergic to house dust mite, 18.1% to pollens, and 30.1% to mixed allergens.

Genetic polymorphisms of OPN and its receptors in AR
The genotype distributions of the 11examined SNPs in the OPN gene and its receptors were in Hardy-Weinberg equilibrium in both the AR and control groups. The call rate for each SNP was 100%. There were no statistically significant differences in the proportions of missing genotype data between cases and controls (P>0.05). The results of genotypic and allelic frequency analysis are shown in Table 3 (Table 4).

Haplotype analysis of OPN allelic variants
A haplo type analysis was performed using the Haplo view V3.32 program and the SHE sis online software platform. The eight possible haplotype frequencies are shown in Table  5. The ATG and CCG haplotypes accounted for 7.0% and 7.6% of the AR patients respectively, and these were significantly higher in the patients than in controls (P = 6.69 × 10 − 5 , OR = 1.700, 95% CI = 1.307-2.213; P = 2.16 × 10 − 4 , OR =1.594, 95% CI = 1.243-2.044 respectively). We also found that the CTA haplotypes were significantly less prevalent in AR patients (P =5.00× 10 −15 , OR = 0.593, 95% CI = 0.523-0.673).

Discussion
OPN, which was originally identified as a bone matrix protein, was subsequently identified as a cytokine (Eta-1) produced by activated T cells and transformed cells [20,21,22]. As a matri cellular protein, Osteopontin (OPN) is expressed in many different cell types, including dendritic cells, T cells, B cells and so on [23]. As a cytokine, OPN can stimulate T cell proliferation, interfere with the differentiation of T cells into Th1 or Th2 and regulate immunoreactions [24,25,26]. Receptors of OPN include certain integrins [27,28,29] and CD44 variants [30,31,32]. With the potential to interact with multiple ubiquitously expressed cell surface receptors, OPN plays active roles in many physiological and pathological processes including wound healing, inflammation, immune response and so on.
So far, OPN has been identified as a biomarker for various types of inflammatory diseases and cancers [33,34] dys regulated and excessive expression of OPN have also been linked to some of the autoimmune disorders such as SLE [35], MS [36], RA [37]. chronic obstructive pulmonary disease [38], and asthma [39]. It has been reported that OPN can stimulate the production of the Th1 cytokine interferon-γ and the Th17 cytokine IL-17 and inhibited the Th2 cytokine IL-10 [23,40]. This was further identified by a recently study, which demonstrated that an increased Th1/Th2 ratio and Th17 cells have been involved in the pathogenesis of Behcet's disease by OPN. So in this study we try to make sure whether OPN play a role in the pathogenesis AR, which is the most popular allergic disease in the upper airway.
We focused on the polymorphisms of the OPN gene and the susceptibility to AR. The selection of the SNPs of OPN was based on earlier reports [13,17,41]. Meanwhile, because OPN exerts its effects through interacting with multiple receptors, the polymorphisms of these receptors were also investigated [11,12].
Our data demonstrated the association of SNPs of rs9138, rs1126616, rs1449623, rs1448623, rs3770136, rs3738919, rs3911238, rs5918, rs2056131, rs187116, rs16927061 in the OPN gene and its receptors ITGA4, ITGAV, ITGB3, CD44 with AR. In the case of rs9138, the AA, AC genotypeand the A allele significantly increased the risk of AR. Meanwhile, the CT, CC genotype and C allele of rs1126616 was also identified as an AR risk factor. For rs1126772, the AG, GG genotype and G allele also significantly increased the risk of AR. When it comes to the receptors of OPN, only the polymorphisms of ITGA4 and CD44 showed relevance. For rs1449623 of ITGAV4, the AR group had an increase in AG, AA genotype and A allele. We alsodetected a risk association between rs187116 of CD44 and AR patients with the CT, TT genotype and T allele.The study has revealed two clinical clues related to AR. First, ourresults identified that three OPN SNPs (rs9138, rs1126616, andrs1126772) and two OPN receptor SNPs (rs1449263 of ITGA4 and rs187116 of CD44) may strongly associated with the risk of AR in Chinese Han patients. Furthermore, the haplo type analysis performed here revealed that the OPN haplotypes ATG and CCGwas significantlymoreprevalent in patientswith AR than in controls. While haplo types of CTA was negatively relevant to AR. Thus we suggest that these SNPs may be chosen as tag SNPs to evaluate thehigh risk of developing AR in the Chinese Han population.
In the present study, to decrease the influenceof confounding factors on the results, we selected the AR patients and controls using strict guidelines and confirmed the genotyping results by direct sequencing. And we observed a novel association between SNPs of OPN and AR in the Chinese Han population. However, it is worth mentioning that there are some limitations to our study. First, we did notdetect the protein level of OPN and receptors in the peripheral blood and perform a functional analysis study, so we could not draw a certain conclusion about the influence of these polymorphisms on the cytokine levels. Second, as it is well known that environmental factors are critical for the development of AR, further studies will be needed to clarify the genetic influence of OPN in AR pathogenesis, including gene-gene and gene-environment interactions.
Madridge J Immunol. ISSN: 2638-2024 In conclusion, this study provides further evidence of the association of OPN and receptors with AR, suggesting the possible involvement of these genes in the susceptibility to AR. This suggests that combining information from common risk polymorphisms could improve disease prediction. Further functional studies of genetic variants could contribute to an increased understanding of the roles of the studied genes in the pathogenesis of AR.