High Mutational Heterogeneity, and New Mutations in the Human Coagulation Factor V Gene. Future Perspectives for Factor V Deficiency Using Recombinant and Advanced Therapies

Abstract

Factor V is an essential clotting factor that plays a key role in the blood coagulation cascade on account of its procoagulant and anticoagulant activity. Eighty percent of circulating factor V is produced in the liver and the remaining 20% originates in the α-granules of platelets. In humans, the factor V gene is about 80 kb in size; it is located on chromosome 1q24.2, and its cDNA is 6914 bp in length. Furthermore, nearly 190 mutations have been reported in the gene. Factor V deficiency is an autosomal recessive coagulation disorder associated with mutations in the factor V gene. This hereditary coagulation disorder is clinically characterized by a heterogeneous spectrum of hemorrhagic manifestations ranging from mucosal or soft-tissue bleeds to potentially fatal hemorrhages. Current treatment of this condition consists in the administration of fresh frozen plasma and platelet concentrates. This article describes the cases of two patients with severe factor V deficiency, and of their parents. A high level of mutational heterogeneity of factor V gene was identified, nonsense mutations, frameshift mutations, missense changes, synonymous sequence variants and intronic changes. These findings prompted the identification of a new mutation in the human factor V gene, designated as Jaén-1, which is capable of altering the procoagulant function of factor V. In addition, an update is provided on the prospects for the treatment of factor V deficiency on the basis of yet-to-be-developed recombinant products or advanced gene and cell therapies that could potentially correct this hereditary disorder.

Development and Characterization of a Factor V-Deficient CRISPR Cell Model for the Correction of Mutations

“…As far as the patient is concerned, a familiar segregation analysis indicated that her mother was heterozygous for the already described c.2218C>T, p.Arg740* mutation [ 23 ] with FV plasma levels at 63%, indicative of a mild phenotype, with no noteworthy clinical symptoms. Her father was heterozygous for the c.3279G>A, p.Trp1093* mutation [ 24 ] and presented with FV plasma levels of 21%, also associated with a mild phenotype without significant symptoms.…”

“…This study proposes a new advanced gene editing-based therapy for the correction of a new mutation in the F5 gene (c.3279G>A, p.Trp1093*), previously described by our research group [ 24 ]. A specific CRISPR/Cas9 technology was developed to treat coagulopathies arising from this mutation, which is protected by a Spanish patent (Ref.…”

“…The study was devoted to the development of a custom cell model based on the mutation present in the patient [ 24 ], who had a severe FV deficiency (<1%). A strategy to correct this mutation and restore full production of functional FV was designed.…”

“…It was decided to choose mutation 3279G>A, p.Trp1093* for the therapeutic model proposed because mutation c.2218C>T, p.Arg740* was close to a thrombin binding domain, which made it difficult to perform small deletions without disrupting the production of the KO cell model. Moreover, in heterozygous conditions, the Trp1093* mutation reduces FV plasma levels to 21% [ 24 ].…”

“…This is in line with previous reports arguing for the need to use monoclonal antibodies to target the region of interest, as polyclonal antibodies are not able to differentiate between a full and a truncated protein just on the basis of an ELISA assay [ 41 ]. This means that viscosity-based coagulometric assays should be the method of choice to quantify and evaluate the functionality of FV, and Sanger sequencing to locate the pathological mutations [ 24 , 42 , 43 , 44 ].…”