Further Evidence That MicroRNAs Can Play a Role in Hemophilia A Disease Manifestation: F8 Gene Downregulation by miR-19b-3p and miR-186-5p.

Katarzyna I Jankowska,Chintamani D Atreya,Behnaz Pezeshkpoor,Zuben E Sauna,Joseph Mcgill,Johannes Oldenburg

doi:10.3389/fcell.2020.00669

Abstract

Hemophilia A (HA) is a F8 gene mutational disorder resulting in deficiency or dysfunctional FVIII protein. However, surprisingly, in few cases, HA is manifested even without mutations in F8. To understand this anomaly, we recently sequenced microRNAs (miRNAs) of two patients with mild and moderate HA with no F8 gene mutations and selected two highly expressing miRNAs, miR-374b-5p and miR-30c-5p, from the pool to explain the FVIII deficiency that could be mediated by miRNA-based F8/FVIII suppression. In this report, an established orthogonal in vivo RNA-affinity purification approach was utilized to directly identify a group of F8-interacting miRNAs and we tested them for F8/FVIII suppression. From this pool, two miRNAs, miR-19b-3p and miR-186-5p, were found to be upregulated in a severe HA patient with a mutation in the F8 coding sequence and two HA patients without mutations in the F8 coding sequence were selected to demonstrate their role in F8 gene expression regulation in mammalian cells. Overall, these results provide further evidence for the hypothesis that by targeting the 3′UTR of F8, miRNAs can modulate FVIII protein levels. This mechanism could either be the primary cause of HA in patients who lack F8 mutations or control the severity of the disease in patients with F8 mutations.

Highlights

Hemophilia A (HA) is an X chromosome-linked bleeding disorder that is associated with mutations in the Factor 8 (F8) gene leading to either reduced expression or production of a dysfunctional FVIII protein
Using a reporter system where the F8 3 UTR is fused to the luciferase gene, we demonstrated that two miRNAs, miR-30c-5p and miR-374b-5p modulate F8 gene expression in mammalian cells and, further, in cells that endogenously express FVIII protein, these two miRNAs can reduce the expression of F8 mRNA and FVIII protein (Jankowska et al, 2019)
We have investigated the miRNA mediated control of FVIII gene expression, which involves miRNA:mRNA interactions leading to the dysregulation of mRNA functions

Summary

Introduction

Hemophilia A (HA) is an X chromosome-linked bleeding disorder that is associated with mutations in the Factor 8 (F8) gene leading to either reduced expression or production of a dysfunctional FVIII protein. Several studies indicate that in about 1% of severe and about 3% of mild or moderate HA patients, no mutations were detected in the F8 gene (El-Maarri et al, 2005; Johnsen et al, 2017), suggesting that there are other molecular mechanisms in addition to mutations in F8 that regulate FVIII expression (Rosset et al, 2016; Jankowska et al, 2019). In some extremely rare cases, even intronic mutations are not detected. In a recent study, using Generation Sequencing (NGS), we demonstrated upregulation of eight microRNAs (miRNAs) in two such extremely rare HA patients compared to healthy controls (Jankowska et al, 2019). Using a reporter system where the F8 3 UTR is fused to the luciferase gene, we demonstrated that two miRNAs, miR-30c-5p and miR-374b-5p modulate F8 gene expression in mammalian cells and, further, in cells that endogenously express FVIII protein, these two miRNAs can reduce the expression of F8 mRNA and FVIII protein (Jankowska et al, 2019)

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