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.
Highlights
Factor V (FV), which is known as proaccelerin or labile factor, is a 330-kDa multi-domain (A1–A2–B–A3–C1–C2) procofactor of the coagulation cascade showing no intrinsic coagulant activity until its conversion to activated FV (FVa) [1,2,3]
FV deficiency (Online Mendelian Inheritance in Man #227400) is an inherited coagulation disorder that was originally described by Owren in 1947 as a hemorrhagic diathesis due to the absence of a previously unknown coagulation factor [5]
As for the remaining five novel variants, we identified one putative missense substitution, three splicing defects, and an additional missense variant (c.6528G>C/p.K2148N) falling in the last nucleotide of exon 24, again potentially influencing the F5 pre-mRNA splicing process (Table 2)
Summary
Factor V (FV), which is known as proaccelerin or labile factor, is a 330-kDa multi-domain (A1–A2–B–A3–C1–C2) procofactor of the coagulation cascade showing no intrinsic coagulant activity until its conversion to activated FV (FVa) [1,2,3]. FV activation occurs by a limited proteolysis, which is exerted either by thrombin or activated factor X (FXa) at specific arginine residues, i.e., p.Arg709, p.Arg1018, and p.Arg1545; this results in the removal of the large B domain [1,2,3]. FVa, together with FXa, participates in the formation of the prothrombinase complex, which is responsible for the generation of thrombin [1,2,3]. The disease can be classified as either type-I deficiency—which is characterized by FV antigen levels that can be mildly reduced, low, or even unmeasurable (quantitative defect)—or type-II deficiency, showing normal or slightly-decreased antigen levels associated with reduced coagulant activity (qualitative defect) [10]. Altered levels of the coagulation factor are responsible for clinical manifestations going from life-threatening bleeding episodes (mostly in the gastrointestinal tract and the central nervous system) to less severe symptoms, including epistaxis, menorrhagia, easy bruising, hemarthroses, and hematomas [10,11]
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