M. Badii (1), O. Gaal (1), V. Kluck (2), G. Cabau (1), L. Peca (1), C. G. Andra (1), R. Popp (1), T. Octavia Crișan (1), L. A. B. Joosten (1,2) and the Hint Consortium
Affiliation(s):
1. Department of Medical Genetics, Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania
2. Department of Internal Medicine and Radboud Institute for Molecular Life Sciences (Rimls), Radboud University Medical Center, Nijmegen, The Netherlands
Introduction: Hyperuricaemia is the background necessary condition for gout and uric acid was traditionally considered to be an inoffensive by-product in other systemic inflammatory diseases. More recently, high uric acid levels are linked with proinflammatory cytokine production in human mononuclear cells. Two SOCS3 intragenic CpG sites were found statistically associated to hyperuricemia in a previous study using whole genome DNA methylation analysis. The SOCS3 gene codes for Suppressor Of Cytokine Signalling 3, being important for the regulation of inflammation. Genetic variants in SOCS3 have also been associated with inflammatory diseases such a Grave’s ophthalmopathy or infantile asthma.
Material and methods: PBMCs from healthy donors were cultured for 24 h with RPMI as control and different concentrations or uric acid solubilised in RPMI with 10% serum. DNA was isolated and treated with bisulfite sodium for conversion and methylation specific PCR was further performed. In parallel, the transcription rate of SOCS3 and IL-1RN was investigated in cells treated with increasing doses of uric acid or in Percoll enriched monocytes of patients with gout, hyperuricemia and normouricemia. The capacity of the cells to be primed with urate was assessed using qPCR and ELISA for IL-1β and IL-1Ra. Gout patients and hyperuricemic controls have been genotyped for SOCS3 variant rs4946170 using TaqMan and the association for this SNP to disease status or markers of inflammation has been tested.
Results: Cells pre-treated with urate showed higher levels of IL-1β and lower levels of IL-1Ra on both transcription and protein secretion. SOCS3 gene expression increased at higher doses of uric acid. In vivo, SOCS3 gene expression presented high variability in gout patients, although in hyperuricemia patients there is a tendency to increased transcription compared to normouricemic controls. Intragenic CG sites showed heterogeneous methylation, while control CpG island in the promoter regions were unmethylated. The SNP was not found to be statistically associated with gout or markers of inflammation.
Conclusions: The observed differential SOCS3 expression indicates that the gene is dynamic in response to uric acid. Several studies associate methylation at intragenic sites with increased gene transcription. However, using current methodology, we were not able to document variation in DNA methylation at the targeted sites for these experimental conditions. SOCS3 seems to be associated with inflammation, however, its causal role in the pathogenesis of gout requires further exploration.
Keywords: hyperuricaemia, DNA methylation, SOCS3