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THU0484 Serum Levels of Extracellular DNA (EXDNA) and EXDNA-Complexed Proteins at Ankylosing Spondylitis (AS), Correlation with Biomarkers – Crp, Esr, Methylation Level and Leukocyte Count
  1. N. Shubaeva1,
  2. V. Kuzmin2,
  3. O. Kupavtzeva1,
  4. V. Kurova2,
  5. E. Aleksandrova1,
  6. S. Erdes1,
  7. J. Krasnaya1,
  8. A. Speransky1
  1. 1Nasonova Research Institute of Rheumatology
  2. 2State Institute of Biochemical Physics of Russian Academy of Sciences, Moscow, Russian Federation

Abstract

Background We suggest that differential gene expression in patients with AS includes the immune system, intracellular or extracellular signaling pathway, and bone matrix biosynthesis pathway, which might play important roles in hip joint ligament ossification. The development of inflammation in the AS pathogenesis (sacroileitis, spondylodiscitis, arthritides, etc.) is associated with an increase of C-reacting protein, CRP and BASDAI rates and ESR, often complexed with HLA-B27.

Objectives We set a task to study the biomarkers indicative of the cell death processes and their possible patterns (apoptosis, necrosis), as well as methylation levels, through an investigation of an associations with inflammation intensity (ESR, CRP) biomarkers, exDNA level and the proteins complexed with exDNA.

Methods Phenol-based technique was used to extract exDNA from blood serum of 37 patients with AS and 18 healthy donors. 85% patients were positive for HLA-B27, BASDAI 5.31±3.28. The exDNA concentration was determined using fluorescent technique with Hoechst 33258. The protein component bound to exDNA, was determined using fluorescence of the protein-dye complex (coumarin-benzotiametincyanine, 580 nm). The level of methylation by exDNA restriction with methyl-specific nucleases HpaII and MspI. The statistical processing - Statistica 8.0.

Results 71% of AS patients showed an increase in exDNA concentration, and 50% demonstrated high methylation levels (1.56±0.34 a.u.), which correlated with the exDNA concentrations and increased leukocyte count (p=0.008, t=0.7467). Raised concentration of monomethine proteins, complexed with DNA, as compared with the normal, was found in 74% of AS patients. A direct correlation was detected between the levels of monomethine proteins and the level of DNA methylation (p=0.03, t=0.867). 84% of patients with CRP concentration >5 mg/l, demonstrated a rise in exDNA concentration (501.5±238.5 ng/ml). Direct correlation between ESR and CRP (p=0.04, t=3.85) was observed, and an increased leukocyte count (10.26±4.8) was found. In a group where CRP level were <5 mg/l, only 50% of patients had elevated exDNA (314.3±254.6 ng/ml) and methylation levels. One can suppose that hypermethylated DNA is released in the process of apoptosis. The statistically significant association between methylation level and elevated leukocyte count suggests a role of hypermethylated DNA in the increased leukocyte resistance to death, pathogenesis, and inflammation at AS.

Conclusions Correlation between elevated monomethine protein and leukocyte count in the association with high DNA methylation levels can be considered as a marker of neutrophil resistance to apoptosis and participation of neutrophils in the processes of destruction and inflammation. Hence, the data we obtained helps us to develop a concept of AS pathogenesis as cause-effect relationships of the inflammation processes, destruction, and proliferation with an increase in cell death rate and in hypermethylated DNA. Presence of hypermethylated DNA is associated with an elevated level of monomethine proteins and higher leukocyte count that possibly involves chromatin remodeling a mechanism regulating protein and genome function.

Disclosure of Interest : None declared

DOI 10.1136/annrheumdis-2014-eular.3187

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