PB0220 - miR-128 in severe hemophilia-A patients with and without inhibitor

Background: miR-128 is a crucial regulator of immune responses and it is implicated in the pathobiology of various autoimmune disorders. Inhibitor development occurs in 1/3 of patients with severe hemophilia A that are treated with FVIII replacement products. Many environmental and genetic risk factors have been investigated, but the real mechanism of inhibitor development remains poorly known.

Aims: To investigate the involvement of miR-128 in inhibitor development.

Methods: Forty-seven severe hemophilia-A patients (n=9 with persistent, n=10 with transient, and n=28 without inhibitor) were investigated. The whole blood was drawn into CPT™ tubes (BD) and the peripheral blood mononuclear cells (PBMCs) were collected by centrifugation. Naïve and Memory CD4+ T cells were negatively selected from PBMCs by using the Human Naive CD4+ T Cell Isolation Kit II and the Human Memory CD4+ T Cell Isolation Kit, respectively (Miltenyi). Total RNA was extracted with the miRNeasy kit (Qiagen) and levels of miR-143 and of the endogenous control RNU6B were evaluated in PBMCs, Naïve, and Memory cells by qPCR using the StepOnePlus Real-Time PCR system (Applied Biosystems). Duplicates of each sample were analyzed and the relative quantities were determined using the 2−∆∆Ct method. For statistical analysis, Student's t-test was applied.

Results: A statistically significant increase in miR-128 was found in PBMCs (p=0.02) and Memory T cells (p=3.50e−03) of patients with (persistent and transient) inhibitor (INH+) compared to patients without inhibitor (INH-) (Figure 1A). A higher expression (from 2- to 4-fold change) in miR-128 was also found in PBMCs (p=0.02) and Memory T cells (p=0.02) of patients with a persistent inhibitor (INH+p) compared to patients without inhibitor (INH-) (Figure 1B).

Conclusion(s): The present study indicates an overexpression of miR-128 in Memory T cells of patients with severe hemophilia A developing inhibitor. Further investigation is required to understand how miR-128 could contribute to pathogenic T-cell differentiation resulting in inhibitor development.