High-throughput DNA methylation analysis in ITP confirms NOTCH1 hypermethylation through the Th1 and Th2 cell differentiation pathways.

BACKGROUND

Immune thrombocytopenia (ITP) is a prevalent autoimmune disease with a complex aetiology where DNA methylation changes are becoming triggers.

METHOD: To investigate novel abnormally methylated genes in the pathogenesis of ITP, we performed a high-throughput methylation analysis on 21 ITP patients and 9 normal control samples. We analysed the extent of key methylated genes and their downstream cytokines through Luminex assay or qRT-PCR. Then, bone marrow mononuclear cells were extracted from ITP patients, and decitabine (demethylation drug) was added to the culture medium of cultured cells. qRT-PCR and ELISA were used to detect whether decitabine could effectively affect target genes and related cytokines.

RESULTS

Through the STRING and Metascape databases, hypermethylated NOTCH1 can be identified and can influence ITP by regulating many downstream cytokines through Th1 and Th2 cell differentiation pathways. Compared with those in the normal control group, the expression levels of NOTCH1 and its downstream Th2 cytokines (IL-4, IL-10, and GATA3) were significantly decreased and those of Th1 cytokines (IFN-γ, IL-12, and TNF-α) were significantly increased in the ITP group. Decitabine exerts its demethylation effect, so the expression of NOTCH1 and its related cytokines in the ITP group treated with 100 nM decitabine were significantly reversed.

CONCLUSIONS

Our results suggest that the pathogenesis of ITP may exert its influence on epigenetics through alteration of DNA methylation at regulatory regions of the target NOTCH1 gene in the Th1 and Th2 cell differentiation pathways. At the same time, decitabine may achieve a therapeutic effect on ITP by demethylation.