| Objective:1.To investigate the association between single nucleotide polymorphisms of B7-H3and B7-H4 genes and susceptibility to ankylosing spondylitis;2.Detect the methylation level and m RNA expression level of B7-H3 gene promoter region in AS patients,and explore the role of B7-H3 gene in the pathogenesis of AS at the epigenetic level.Methods:1.The study was divided into two parts.In the first part,649 confirmed AS patients were collected from the Rheumatology Department of the First Affiliated Hospital of Anhui Medical University,and 646 healthy controls with matched gender and age were collected from the Physical Examination Center of the First Affiliated Hospital of Anhui Medical University and blood bank in Hefei City center.Five SNPs of B7-H3 and B7-H4 genes（rs3816661,rs3825859,rs6428679,rs3738414 and rs10801935）were selected.The genotypes of candidate genes were obtained by using SNPscan genotyping technique.Genotype distribution,allele frequency and different genetic models were analyzed to compare the differences between AS patients and healthy controls.The genotypes were stratified by age,sex,HLA-B27 status,BASDAI score,BASFI score and ASDAS score.2.In the second part,84 AS patients and 83 sex-and-age-matched healthy controls were enrolled.Methylation levels of the promoter region of B7-H3 gene in AS patients and healthy controls were measured by bisulfite sequencing.q RT-PCR was used to detect B7-H3 m RNA expression in 20 randomly selected patients and corresponding healthy controls.The differences of methylation levels in different Cp G loci and Cp G regions of B7-H3 gene in AS patients and healthy controls were analyzed.The methylation levels were stratified according to gender and HLA-B27status.3.SPSS 23.0 software was used for all statistical analyses.Continuous data consistent with normal distribution were represented by mean±standard deviation（（?）±SD）,and inter-group comparison was performed by independent sample T test.Median and quartile spacing（M（P25,P75））were used for skewness distribution data,and mann-Whitney U test was used for comparison between groups.The distribution characteristics of qualitative data are expressed in absolute numbers and percentages.The distribution of genotype,allele frequency and genetic model were compared between the case group and the control group using Pearson’s Chi-square test or Fisher’s exact test.Odds ratios（ORs）and corresponding 95%confidence intervals（CIs）were calculated to assess risk.Independent Hardy-Weinberg equilibria（HWE）were tested for polymorphism in the control group using the Chi-square test.Linkage disequilibrium（LD）and haplotype analysis were performed using Haploview 4.2 software.Draw graphs using Graph Pad Prism 7.0.Under multiple comparisons,Bonferroni correction method was used to correct p values,and p values on two-tailed less than 0.05 were considered statistically significant.Results:1.Basic Information:Part I:A total of 649 AS patients were enrolled in the study,including 518 male patients,131 female patients,and 646 healthy controls,including526 male patients and 120 female patients.The mean age of the case group and the control group was 29.30±9.58 years and 28.61±7.85 years,respectively.There were no significant differences in gender（P=0.482）and age（P=0.156）between the case group and the control group.Part II:The study included 84 cases in the case group and 83 cases in the control group.There were 64 male patients,20 female patients,63male controls and 20 female controls.There were no significant differences in age（t=0.157,P=0.876）and gender composition（X~2=0.002,P=0.965）between the case group and the control group.2.There were no statistically significant differences in genotype and allele frequency distribution of all SNPs between the case group and the control group（P<0.05）.The CA+CC dominant genotype of B7-H4 RS10801935 significantly increased the risk of AS compared with wild AA genotype（P=0.013）.Compared with the wild-type GG genotype,GA and GA+AA genotypes at B7-H4 rs37388414 increased the risk of AS,but the difference was not statistically significant after Bonferroni correction（GAvs.GG:P=0.033,GA+AA vs.GG:P=0.045）.SNPs were not significantly associated with clinical features.Stratified analysis showed that GA and GA+AA genotypes at B7-H4 rs3738414 were significantly associated with an increased risk of AS compared with the wild-type GG genotype in male patients and patients with ASDAS≥1.3.After Bonferroni adjustment,the association remained statistically significant in male patients（GA vs.GG:P=0.005,GA+AA vs.GG:P=0.008）,but there was no statistically significant difference in patients with ASDAS≥1.3（GA vs.GG:P=0.028;GG:P=0.034）.The B7-H4 rs3738414 A allele（A vs.G）increased the risk of AS in the male subgroup,but Bonferroni corrected p value was not statistically significant（P=0.027）.Haplotype analysis showed that TAG haplotype was associated with a significantly increased risk of AS（P=0.026,OR=1.190,95%CI=1.020-1.389）.3.Methylation analysis results showed that there were 18 sites of differential expression of methylation between AS patients and healthy controls;The median methylation level of Cp G-1 was 2.81%in patients with AS and 3.47%in healthy controls,with significantly lower methylation levels in patients with AS（Z=-5.121,P<0.001）;The median methylation level of Cp G-2 was 7.17%in patients with AS and7.68%in healthy controls,significantly reduced in patients with AS（Z=-2.579,P=0.01）.The median methylation level of the B7-H3 gene promoter was 4.46%in patients with AS and 5.11%in healthy controls,and the overall methylation level in patients with AS was significantly lower than that in healthy controls.（Z=-4.199,P<0.001）.Stratified analysis showed that there were significant differences in methylation levels between AS patients and healthy controls in both male and female stratified,and methylation levels in AS patients were significantly lower than those in the control group（male:Z=-7.853,P<0.01;female:Z=-5.126,P<0.01）.There were significant differences in methylation levels between healthy control group,HLA-B27positive group and HLA-B27 negative group（H=19.690,P<0.001）.Pair comparison showed significant differences in methylation levels between h LA-B27positive group and healthy control（H=-3.202,P=0.001）and HLA-B27 negative group and healthy control（H=-3.827,P<0.001）.Methylation levels in both HLA-B27 positive and HLA-B27 negative groups were significantly lower than those in healthy controls.There was no significant difference in methylation levels between HLA-B27 positive and HLA-B27 negative groups（Z=-1.667,P=0.096）.Conclusion:1.B7-H4 rs10801935 and RS3738414 polymorphisms were associated with AS susceptibility.The small A allele and GA genotype of B7-H4 rs3738414 increase the risk of AS in male patients.B7-H4 haplotype block TAG can promote the development of AS.2.Changes in the methylation level of B7-H3 gene in AS patients may affect the occurrence and development of AS by changing its m RNA expression level. |
