Efeito do silenciamento gênico de ACVR1 (r206h) por sirna, sobre a expressão de ACVR1 e BMP4 em células mononucleares de sangue periférico de pacientes portadores de fibrodisplasia ossificante progressiva

Abstract

Introduction: FOP is a complex autosomal dominant genetic rare disease and characterized by congenital skeletal abnormalities being the hallux valgus one pathognomonic feature and heterotopic ossification progressive postnatal. The patients present a heterozygous mutation (c.617G> A, p.R206H) in the ACVR1 gene, on chromosome 2q23-24. Studies of the pathophysiology of FOP involve imbalance in the signaling pathway of BMP and its interaction with multiple molecular pathways and the immune system. ACVR1 mutations are gain of function, activating the signaling pathway of BMP independent of ligand. The substitution of arginine for histidine at mutation creates an allosteric change in the receptor domain, activating the receptor independently of the BMP. Experiments in vitro of primary culture of leukocytes of patients have not been explored and will be important of models in assessing via trigger of the disease interaction with medication used to control acute flare-ups. Objectives: Determination of an experimental model for the silencing of gene expression of ACVR1 (R206H) in cultured peripheral blood mononuclear cells (PBMC) of FOP patients obtained by primary culture and the effect of siRNA on the expression of ACVR1 and BMP4. Methods: Whole blood samples were collected for mononuclear cell cultures (PBMC) of ten volunteers (6 people with FOP and 4 healthy controls). Cells were treated with siRNA and siRNA for mutated ACVR1 and normal ACVR1. Cells were treated with siRNA for ACVR1 mutated and with siRNA for normal ACVR1. Total RNA extracted by Stat60 subjected to RT and PCRtr. Unpaired t test was used to assess gene expression. Results and Conclusion: This work shows that molecular studies of gene silencing using the model of PBMC and carbon nanoparticles is feasible. Since it is a relatively simple model, it will allow future studies in the understanding of the pathophysiology of the FOP. We observed in the present work that there was the silencing of the ACVR1 gene, though the mechanism by which the ACVR1 was superexpressed has not been very clear. But when we observe the expression of the gene ACVR1 and analyze together with the expression of BMP4, we realized that there was a modulation when the cells were treated with RNAi.