Association Of Prickle1 Methylation Levels With Ankylosing Spondylitis

BackgroundAnkylosing spondylitis(AS)is a common inflammatory spondylitis,which has complex polygenic etiology and family aggregation.Genome-wide association studies have identified more than 100 loci,including some involved in antigen presentation(HLA-B27,ERAP1,and ERAP2),some involved in Th17 responses(IL6R,IL23R,TYK2,and STAT3).Others are involved in macrophages and T cells(IL7R,CSF2,RUNX3,and GPR65).However,to date,the susceptibility gene association approach has explained only a minority of the heritability of such diseases,and most disease-related variants have been found to be non-coding,suggesting that disease-association mechanisms via transcriptional regulatory effects may play an important role in disease occurrence and progression.Epigenetics is the main interface between genetic and environmental modifiers of disease and strongly influences transcription.DNA methylation is a well-defined epigenetic mechanism and a highly stable epigenetic marker,which is related to the pathogenesis of diseases.In recent years,DNA methylation has become a hot topic in many autoimmune diseases,but less in AS.In a previous study of our research group"The role of DNA methylation in the promoter region of Wnt signaling pathway related genes in the occurrence and development of ankylosing spondylitis and its molecular mechanism",3ml peripheral blood was extracted from 20 AS patients and 20 healthy controls to extract DNA.The high-throughput sequencing method,Methyl Target,was used to detect 2376 Cp G sites in 122 fragments of 57 genes.148 Cp G sites with significantly different methylation levels were found.In this study,one of the genes with differences was selected for further study.The objective of this study is to investigate the role of Prickle1 gene promoter region methylation and m RNA expression in the pathogenesis of ankylosing spondylitis.MethodsThis study is mainly divided into two parts:(1)At first,84 Prickle1 patients and 83healthy controls were enrolled.Epidemiological data,clinical data and 10 ml blood samples were collected for extraction of peripheral blood genomic DNA.The methylation level of methylation sites in the Prickle1 promoter region was determined by sequencing whole-genome DNA modified by heavy sulfite.The relationship between methylation level in Prickle1 gene and 5 Cp G islands and clinical data is statistically analyzed.(2)Subsequently,50 AS patients and 50 healthy controls were applied for extraction of human peripheral blood mononucleocyte specimens for cell RNA extraction.After reverse transcription,GAPDH was used as internal reference,and the Prickle1 expression in PBMC was detected by real-time quantitative PCR.The correlation between Prickle1 expression level and clinical data of AS was statistically analyzed,and the relationship between Prickle1 expression and AS in PBMC was discussed.Continuous data conforming to normal distribution were expressed as mean±standard deviation,and independent sample t test was used for comparison between groups.Median and quartile were used to describe skewness distribution data,and Mann-Whitney U test was used for comparison between groups.Qualitative data were expressed in absolute numbers and percentages and compared by Pearson Chi-square test or exact probability method.Spearman rank correlation coefficient test was used to investigate the relationship between Prickle1 gene and clinical characteristics.The predictive performance of Prickle1 gene for AS-diagnosis was evaluated using subject operating characteristic curve and area under curve.Chart using Graph Pad Prism Version 8.0.2 software.Bilateral P value(?)0.05 was statistically significant.Results(1)There are 49 Prickle1 gene Prickle1 expression sites in AS patients,and the overall methylation level of AS patients Prickle1 gene is lower than that of healthy control group.Four Gp G island,Cp G(Cp G-1,Cp G-3,Cp G-4,Cp G-5)of the average level of methylation in the AS group and HC group differences were statistically significant(P(?)0.001).Subgroup analysis showed a statistically significant difference in Cp G-4methylation between female AS patients and the control group when stratified by sex(P(?)0.05).Male patients with AS,Cp G-1,Cp G-3,Cp G-4,Cp G-5 island and Prickle1methylation level significantly lower than the control group(P(?)0.001);Stratified according to different HLA-B27 status,Cp G-3,Cp G-4,and Prickle1 Prickle1methylation level in HLA-B27-positive patients is significantly higher than that in HLA-B27-negative patients(P(?)0.05);The Cp G-3 methylation level in patients treated with biologics was significantly higher than that in patients treated without biologics(P(?)0.05).Meanwhile,the Prickle1 transcription level of BASFI≥4 groups is significantly higher than that of BASFI<4 groups(P(?)0.05).(2)At the PCR verification stage,the relative expression quantity of Prickle1 in PBMC of AS patients was significantly higher than that in HC group(P(?)0.05).Cp G-1(r_s=-0.217,P(?)0.05),Cp G-4(r_s=-0.291,P(?)0.05),and Cp G-5(r_s=-0.302,P(?)0.05)in AS patients Prickle1 have significant correlation with their relative expression quantity.Prickle1 gene promoter methylation is negatively correlated with HLA-B27,ESR,CRP,ASDAS,PCT,but positively correlated with LYM,MON,Cp G-1 island methylation is negatively correlated with CRP.Cp G-2 was negatively correlated with ASDAS.Cp G-3was negatively correlated with HLA-B27,CRP,PCT,PLT,and positively correlated with LYM.Cp G-4 was positively correlated with LYM.Cp G-5 was negatively correlated with ESR,CRP,ASDAS,PCT,and positively correlated with LYM.There was a significant correlation between Cp G-4 island methylation and Prickle1 m RNA expression in AS patients receiving NSAIDs(r_s=-0.3814,P(?)0.05).(3)The ROC curve analysis shows good diagnostic value in Prickle1 methylation level,with AUC=0.7301,95%CI=0.6746-0.8240,P<0.001,sensitivity 68.67%,specificity71.43%;Its sensitivity and specificity were higher than 50%.The AUC of m RNA ROC curve was 0.6382,95%CI was 0.5290-0.7474,P(?)0.05,sensitivity was 98.00%,specificity was 30.00%.ConclusionsPrickle1 expression in patients with ankylosing spondylitis significantly changes,and the degree of gene methylation decreases significantly,indicating that the Prickle1 gene may participate in the pathogenesis of ankylose spondylitis,which may helpful for the early diagnosis and screening of AS.