Genetic Polymorphisms of Osteopontin in Association with Ankylosing Spondylitis in a Chinese Population

Purpose: To determine the association of osteopontin (OPN) polymorphisms with ankylosing spondylitis (AS). Methods: A total of 120 cases diagnosed with AS and 106 age- and sex-matched healthy controls were recruited. All the patients were human leukocyte antigen (HLA)-B27 positive. Three single nucleotide polymorphisms were genotyped using direct sequencing Results: The T allele at -443 SNP had significantly higher frequency in AS patients (0.1875) than the controls (0.1085, p < 0.01). The rate of CT+TT genotype in AS patients was significantly higher than those with CC genotype compared with the control (p < 0.01). Conclusion: SNP at -443 of OPN gene can serve as a candidate genetic marker to evaluate the risk of AS, thus indicating that subjects who carry T allele have a significantly higher risk of developing AS. JournalSeek, Journal Citation Reports/Science Edition, Directory of Open Access Journals (DOAJ), African Journal Online, Bioline International, Open-J-Gate and Pharmacy Abstracts


INTRODUCTION
Ankylosing spondylitis (AS) is a chronic inflammatory disorder characterized by inflammation in the spine and sacroiliac joints causing initial bone and joint erosion and subsequent ankylosis [1]. Most patients develop first symptoms of AS younger than 30 years of age [2]. More recent studies have shown that structural damage at initial presentation is the best predictor of further damage [3][4][5].
Osteopontin (OPN) is well known as a major non-collagenous protein related to bone remodeling [6][7].The expression of OPN in different cell types was significantly influenced by transcription factors, and the genetic polymorphisms of the promoter [8]. Previous studies showed that OPN inhibited nucleation, growth, and aggregation of calcium oxalate crystals in vitro [9,10], and was able to directly inhibit the binding of calcium oxalate crystals to cultured renal epithelial cells [11]. Therefore, OPN is an important modulator of stone formation. Mutations in the gene directing the synthesis of OPN may predispose to AS.
Recently, -443T/C polymorphisms on the promoter region of OPN gene have been found to affect gene expression and transcriptional activity [12,13]

Analysis of OPN gene polymorphisms
Three milliliters of fasting venous blood were collected from all subjects. Genomic DNA was extracted from the white blood cells following standard protocols. SNP was determined using direct sequencing of the sense and anti-sense strands following polymerase chain reaction (PCR) amplification of the promoter regulatory region -532 to +94. PCR was performed using 50 ng DNA as a template under the following conditions: 94 °C for 7 min, then 30 cycles of 94 °C for 55 s, an annealing temperature for 50 s and 72 °C for 90 s, with a final extension at 72 °C for 10 min. The PCR products were subjected to cycle sequencing with the respective forward and reverse primer using an automated ABI 3100 DNA sequencer by GeneCore Bio Technologies (Shanghai, China).

Statistical analysis
Statistical analysis was performed using SPSS 11.5 software. One-way ANOVA and t-test were used to compare mean differences for continuous variables. Allele frequency was determined via direct counting. Statistical significance of differences in gene and genotype frequencies between patients and controls was evaluated for each polymorphism using Chisquare test with Yates' correction, and significance was defined as p < 0.05.

OPN genotype and allele frequencies
The OPN genotype was conducted using direct sequencing of DNA fragments from the promoter regulatory region -532 to +94 in AS patients and controls. The frequency of SNP at -443 with CC homozygotes in AS patients was 0.7083 and 0.8396 in the control ( Figure 1).

OPN polymorphism is associated with AS susceptibility
As shown in Table 1, T frequency was significantly increased in AS patients (18.75) compared to the control (10.85, p < 0.05), the rate of the CT+TT genotype in AS patients was significantly higher than in those with CC genotype compared with control (p < 0.05), indicating that subjects who carried T allele have a significantly higher risk of developing AS than in those with C allele.  In this study, we have investigated the difference in OPN gene polymorphisms between AS patients and healthy controls. The T frequency was significantly increased in AS patients compared to the control (p < 0.05), the rate of the CT+TT genotype in AS patients was significantly higher than those with CC genotype compared with the control (p < 0.05), indicating that subjects who carried the T allele had a significantly higher risk of developing AS than those with the C allele. So far, genetic variants in OPN gene have been shown to be involved in susceptibility to other immune-mediated diseases such as SLE [29,30], oligoarticular juvenile idiopathic arthritis [31] and sarcoidosis [32]. Despite promising functional data, previous genotype analyses could not confirm SPP1 as significant disease modifying gene in classical Th17-mediated diseases such as multiple sclerosis [33,34] and rheumatoid arthritis [35]. However, it is first time to report the relationship between OPN polymorphism and AS patients. It is expected that this study should be confirmed in a large and ethnically divergent population in order to make a stronger conclusion about the association between -443 polymorphism with AS.

Limitation of the study
In this study, the sample size of participants was small, thus rendering the results liable to bias. Therefore, a larger-sample study needs to be undertaken.

CONCLUSION
SNP at nt -443 of the OPN gene can serve as a candidate genetic marker to evaluate the risk of AS. Subjects who carry T allele may have a significantly higher risk of developing AS. This relationship is reported here for the first time.