Antiphospholipid syndrome (APS) is a immune, thrombotic disorder of unknown etiology, characterized by the association of arterial or venous thrombosis and/or pregnancy morbidity, with the presence of antiphospholipid antibodies (aPL): anticardiolipin antibodies (aCL) and/or lupus anticoagulant (LA) and/or anti-b2-glycoprotein 1 antibodies (anti-b2GP1). In recent years, epigenetic modification has been studied in many autoimmune, immune disorders to elucidate its pathophysiologic correlation, along with genomic, epigenomic profiling. Among them, aberrant DNA methylation (hypo-, or hypermethylation) and its impact on gene expression have been implicated in diverse spectrum of diseases, including immune disease or cancer. The objective of this study is to investigate a global DNA methylation status of antiphospholipid syndrome patients with CpG microarray, and compared it with healthy controls.
Human peripheral whole blood samples were obtained from 6 APS (2 primary APS and 4 secondary APS, which were 3 systemic lupus erythematosus and 1 Behçet's disease) and from 6 age, sex-matched healthy controls. Total genomic DNA extracted using the QIAamp DNA mini and blood kit protocol (Qiagen, Hilden, Germany). To discover aberrantly methylated genes in APS by genome-wide search, we introduce Illumina Infinium Human Methylation 450K BeadChip (Illumina Inc, San Diego, USA) for directly identifying differentially methylated regions of the genomes in each pooled whole blood between APS patients and healthy controls. Immunologic profiles of the patient group including aPL, aCL, LA, anti-b2GP1 were obtained. This study was reviewed by the institutional ethical board and written informed consent was completed.
All patients and the control groups were female and there was no statistical difference in age distribution (age of patients=35. 26 ±10. 34 year). The fluorescent signal intensities were extracted using GenomeStudio Methylation module (1. 8. 5) software. One-hundred and thirty two CpG sites were filtered by criteria of delta mean (difference of average b value from patients and controls)>0. 2, and p value<0. 05 after the quantile normalization to reliably compare data from multiple samples by minimize non-biologic differences. Seventy nine CpG sites (KNDC1, ABHD8, CDK2AP1, MX1, IL16, ROCK2, SNTG2, SORCS2, mi518, mi662, NLRC5, PARP9, etc) were hypermethylated and 53 sites (CYP2E1, BTNL2, DTX2, GCC2, SLC9A3, PLOD1, PXDN, PROZ, ATP4B, etc) were hypomethylated. All differentially methylated CpG sites were located in 5'UTR, TSS1500, TSS200, Exon, intergenic body, 3'UTR. Hierachical clustering (figure 1) and the functional classification analysis using DAVID was done.
Hundreds of candidate genes were screened by BeadChip microarray of peripheral blood in APS patients. High-throuput, next generation sequencing of the individual patients for validation will be underwent and the correlation with immunologic profile and thrombotic features will be investigated.
Kim:National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology: Research Funding.
Asterisk with author names denotes non-ASH members.