Gene mutations in the PI3K/Akt signaling pathway were related to immune thrombocytopenia pathogenesis.

BACKGROUND: Immune thrombocytopenic (ITP) is an autoimmune bleeding disease with genetic susceptibility. Twenty newly diagnosed active primary ITP patients who had not been treated with glucocorticosteroids, immune globulin or immunosuppressants prior to sampling were enrolled in this study. Bone marrow blood mononuclear cells were used for whole exome sequencing to further elucidation the variant genes of ITP. METHODS: High-molecular-weight genomic DNA was extracted from freshly frozen bone marrow blood mononuclear cells from 20 active ITP patients. Next, the samples were subjected to molecular genetic analysis by whole-exome sequencing, and the results were confirmed by Sanger sequencing. The signaling pathways and cellular processes associated with the mutated genes were identified with gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses. RESULTS: The results showed that there were 3998 missense mutations involving 2269 genes in more than 10 individuals. Unique genetic variants including phosphatase and tensin homolog, insulin receptor, and coagulation factor C homology were the most associated with the pathogenesis of ITP. Functional analysis revealed these mutation genes mainly affect Phosphatidylinositol 3 kinase/serine/threonine kinase B signaling pathways (signal transduction) and platelet activation (immune system). CONCLUSION: Our finding further demonstrates the functional connections between these variant genes and ITP. Although the substantial mechanism and the impact of genetic variation are required further investigation, the application of next generation sequencing in ITP in this paper is a valuable method to reveal the genetic susceptibility.

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.

Characterization of Th1- and Th2-associated chemokine receptor expression in spleens of patients with immune thrombocytopenia.

PURPOSE: In view of the numerous clinical observations and laboratory studies that suggest a critical role for the spleen in immune thrombocytopenia (ITP) pathophysiology, we aimed to characterize Th1-associated chemokine receptors CXCR3 and CCR5 and Th2-associated chemokine receptor CCR3 in spleens of ITP patients and assess the significance of their differential expression in the clinical setting. METHODS: The histopathology of spleens was observed using hematoxylin-eosin staining (HE), and the positive rate of CXCR3, CCR5 and CCR3 expression in spleens of 24 ITP patients and 12 patients with traumatic splenic rupture as normal controls was detected by immunohistochemistry using the SP method. CXCR3, CCR5 and CCR3 protein expression was analyzed by Western blot and mRNA levels were investigated by real-time polymerase chain reaction (RT-PCR). RESULTS: Reactive hyperplasia could be seen in follicles of the white pulp, the germinal central zone was enlarged and the marginal zone was thickened, the central arteries were thickened and fibrotic, and the density of the capillary vessel was increased in ITP patients. ITP group displayed a higher rate of expression of Th1-associated chemokine receptors CXCR3 and CCR5 (83.3% vs. 75%, 100% vs. 83.3%) but lower rate of expression of Th2-associated chemokine receptor CCR3 (50% vs. 66.7%) compared with the controls (P < 0.05, respectively). Western blot analysis revealed that CXCR3 and CCR5 protein expression was significantly increased in ITP patients while CCR3 was significantly reduced (P < 0.05, respectively). Meanwhile, ITP patients displayed increased mRNA levels of CXCR3 and CCR5 but decreased gene expression of CCR3 (P < 0.05, respectively). CONCLUSION: The data suggested that the abnormal expression of Th1/Th2 chemokine receptors may participate in splenic immune disorder in patients with ITP. Using corresponding inhibitors may inhibit Th1-dominant expression and mitigate the progress of the disease.

[Expression of CXCR3 and CCR5 chemokine receptor in spleens of patients with primary immune thrombocytopenia].

OBJECTIVE: To study CXCR3 and CCR5 chemokine receptor expression in spleens of patients with primary immune thrombocytopenia (ITP) and its clinical significance. METHODS: The splenectomy specimens from 10 ITP patients (ITP group) and 8 patients with traumatic splenic rupture (normal control group) were studied. Immunohistochemistry (IHC) was used to study the positive rate of CXCR3 and CCR5. Western blot was performed to detect CXCR3 and CCR5 protein expression, while real-time polymerase chain reaction (RT-PCR) was conducted to analyze their mRNA expression. RESULTS: The positive rate of CXCR3 and CCR5 were both higher in ITP group (90% and 100%, respectively) than those in control group (75% and 87.5%, respectively)(P < 0.05). The differences were statistically significant (P < 0.05). Protein and mRNA level of CXCR3 in ITP group were 3.0 and 3.5 times as high as those in control group, respectively. Those of CCR5 in ITP group were 1.2 and 1.7 times as high as those in control group, respectively. CONCLUSION: High expression of CXCR3 and CCR5 may play a part in the splenic immune disorders in patients with ITP.