Associations Of Methylation Levels Of Gene Promoter Of Interferon Regulatory Factor 8(IRF8)and Ankylosing Spondylitis

Objective 1.The methylation and the m RNA expression levels of interferon regulatory factor 8(IRF8)gene promoter were detected in patients with ankylosing spondylitis(AS)and healthy controls.2.We investigated the associations between methylation of the IRF8 gene promoter and clinical features of AS to explore the relationship between methylation of the IRF8 gene promoter and disease prognosis.Methods 1.During 2016-2019,this study collected 5ml blood samples and epidemiological data in case and control from the Department of Rheumatology and Health Examination Center of the First Affiliated Hospital of Anhui Medical University.Our experiment was divided into two parts.In the first part,99 AS patients and 99 healthy controls were recruited for DNA methylation testing.In the second part,another independent sample of 19 AS patients and 19 healthy controls were enrolled to test m RNA expression levels.2.Firstly,DNA was extracted using the DNA kit(QIAGEN),and the methylation and m RNA expression levels of the IRF8 gene promoter were detected.The extraction method was based on Methyl Target method and quantitative real-time reverse transcription-polymerase chain reaction(q RT-PCR).3.Data analysis was carried out using SPSS software version 23.0(SPSS,Chicago,IL,USA).Appropriate statistical analysis methods were used according to data types and distribution characteristics.If the data meet the normality assumption,the results were described as mean ± standard,otherwise,it would be described as Median(interquartile range,IQR).Qualitative data were described by frequency and percentage.The statistical methods were selected as follows: If the quantitative data meets the normal distribution,t test was used to compare the two groups.Otherwise,the Mann-Whitney U test would be adopted.Qualitative data were compared between the two groups using chi square test;Spearman correlation was used for bivariate correlation analysis.Gender differences,drug treatment and HLA-B27 status may affect the levels of methylation of genes,so the subgroup analysis of gender,the drug and HLA-B27 were performed.The Kruskal Wallis Test was used to compare the statistical differences between the three groups.The Mann-Whitney U test was used to compare the statistical differences between the two groups.To draw the graphs,we used the Graph Pad Prism version 7 for windows(Graph Pad Software,La Jolla,CA USA,www.graphpad.com).P value≤0.05 was considered to be statistically significant.Multiple comparisons using Bonferroni correction.Results 1.The basic characteristics were as follows: The study was divided into two parts.In the first part,the study included 99 AS patients and 99 healthy controls was carried out.The average age of AS patients was 31.14 ± 9.93 years,with the sex ratio 3.5:1.The average age of healthy controls was 31.77 ± 8.69 years,with the sex ratio 5.18:1.There were no statistical differences in age distribution(t=0.475,P=0.635)and gender(χ2=0.861,P=0.353)between AS patients and healthy controls.A total of 19 AS patients and 19 healthy controls were recruited in the second part.There were no significant differences in gender and age between the two groups(age: 32.21 ± 7.58 vs.32.05 ± 7.75 years,P = 0.950;male/female: 13/6 vs.14/5,P = 0.721).2.Association analysis of methylation levels of 91 Cp G sites with AS susceptibility: we selected 91 Cp G sits of IRF8 gene promoter and differential methylation analysis revealed 31 differentially methylated Cp G sites,suggesting significant DNA methylation change in IRF8 gene from patients with AS.3.Methylation levels of four Cp G regions of the IRF8 gene: The median percentage of Cp G-1 methylation was 1.53% in AS patients and 1.40% in healthy controls;The median percentage of Cp G-2 methylation was 3.43% in AS patients and 3.20% in healthy controls;The median percentage of Cp G-3 methylation was 1.23% in AS patients and 1.17% in healthy controls;The median percentage of Cp G-4 methylation was 1.40% in AS patients and 1.35% in healthy controls.The four Cp G regions of the IRF8 gene promoter were hypermethylated in AS patients compared to healthy controls(Cp G-1: 1.53% vs.1.40%,P<0.001;Cp G-2: 3.43% vs.3.20%,P<0.001;Cp G-3: 1.23% vs.1.16%,P<0.001;Cp G-4: 1.40% vs.1.34%,P=0.02).At the overall level,the overall methylation level of IRF8 gene in AS patients was significantly higher than that in healthy controls(IRF8: 1.91% vs.1.78%,P<0.001).4.Subgroup analysis of IRF8 methylation: Subgroup analysis of gender showed that DNA methylation between AS and healthy controls in male and female were statistically significant.Subgroup analysis of the drug showed that AS treated with biological agents had lower methylation compared with AS untreated groups(Z=-2.502,P=0.012).The subgroup analysis of HLA-B27 showed that the methylation levels of IRF8 were higher in the HLA-B27 positive group or the HLA-B27 negative group than that in the healthy control group.There was no statistically significant difference in the methylation levels of IRF8 between the HLA-B27 positive group and the HLA-B27 negative group.5.q RT-PCR validation: The m RNA levels of IRF8 in AS were significantly lower than that in the controls.The median and interquartile range of m RNA levels of IRF8 gene in AS patients was 0.77(0.65,1.00),P = 0.038.6.The correlation between IRF8 gene methylation and clinical manifestations: Correlation analysis suggested a positive correlation between IRF8 gene promoter methylation and disease duration and BASFI.Conclusion 1.Hypermethylation of the IRF8 gene promoter may lead to the development of AS by regulating the expression of its m RNA.2.Biologicals therapy may alter the methylation levels of the IRF8 gene promoter,and the state of HLA-B27 does not alter the methylation levels of the IRF8 gene promoter.3.The methylation levels of IRF8 gene promoter can be used as an indicator of the prognosis of AS disease.