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Understanding the Impact of Aberrant Splicing in Coagulation Factor V Deficiency

Authors
  • Paraboschi, Elvezia Maria1, 2
  • Menegatti, Marzia3
  • Peyvandi, Flora3, 4
  • Duga, Stefano1, 2
  • Asselta, Rosanna1, 2
  • 1 (S.D.)
  • 2 Humanitas Clinical and Research Center, IRCCS, Via Manzoni 56, Rozzano, 20089 Milan, Italy
  • 3 (F.P.)
  • 4 Department of Pathophysiology and Transplantation, Università degli Studi di Milano, 20100 Milan, Italy
Type
Published Article
Journal
International Journal of Molecular Sciences
Publisher
MDPI AG
Publication Date
Feb 20, 2019
Volume
20
Issue
4
Identifiers
DOI: 10.3390/ijms20040910
PMID: 30791524
PMCID: PMC6412230
Source
PubMed Central
Keywords
License
Green

Abstract

Rare inherited coagulation disorders (RICDs) are congenital deficiencies of the plasma proteins that are involved in blood coagulation, which generally lead to lifelong bleeding manifestations. These diseases are generally qualitative and/or quantitative defects that are associated with monoallelic or biallelic mutations in the relevant gene. Among RICDs, factor V (FV) deficiency is one of the least characterized at the molecular level. Here, we investigated four unrelated patients with reduced plasma FV levels (three severe, one mild), which were associated with a moderately severe bleeding tendency. Sequence analysis of the FV gene identified seven different variants, five hitherto unknown (p.D1669G, c.5789-11C>A, c.5789-12C>A, c.5789-5T>G, and c.6528G>C), and two previously reported (c.158+1G>A and c.5789G>A). The possible pathogenic role of the newly identified missense variant was studied by in silico approaches. The remaining six genetic defects (all putative splicing mutations) were investigated for their possible effects on pre-mRNA splicing by transient transfection experiments in HeLa cells with plasmids expressing appropriate hybrid minigenes. The preparation of minigene constructs was instrumental to demonstrate that the two adjacent variants c.5789-11C>A and c.5789-12C>A are indeed present in cis in the analyzed FV-deficient patient (thus leading to the c.5789-11_12CC>AA mutation). Ex vivo experiments demonstrated that each variant causes either a skipping of the relevant exon or the activation of cryptic splice sites (exonic or intronic), eventually leading to the introduction of a premature termination codon.

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