Comunicación presentada en ISTH Congress

21 de junio de 2025

de Pablo-Moreno JA., Miguel-Batuecas A., Bermejo-Alvarez P., Porras N., Gonzalez-Brusi L., de Pablo-Moreno J., Rodriguez-Bertos A., Revuelta L. y Liras A.

Factor V deficiency is an ultra-rare autosomal recessive disorder characterized by the absence or dysfunction of coagulation factor V (FV) and variability in bleeding symptoms. Over 250 mutations have been identified, with missense mutations being the most common, usually resulting in milder phenotypes. In contrast, deletion mutations cause frameshift changes, leading to premature stop codons and more severe disease phenotypes. Animal models are crucial for treatment development, nevertheless, their scarcity has limited progress, with fresh frozen plasma the only available alternative.
The aim is to describe a spontaneous knockout model of FV deficiency in mice, generated through CRISPR/Cas9 with a Frame Disrupting mutation (FVFD).
The FVFD mutation was unexpectedly discovered while generating the missense Thr1857Met mutation in mice via CRISPR/Cas9. A heterozygous F1 generation was obtained and crossed to achieve a homozygous F2 generation for coagulation studies, including FV levels, prothrombin time (PT), and activated partial thromboplastin time (aPTT). Histological studies were performed.
A spontaneous knockout mouse model was generated with a four-base pair deletion (c.5554_5557del Val1852 fs Ter1862 [FVFD]), causing a previously undescribed stop codon in humans. Crosses yielded fewer heterozygous (Hz) and homozygous (Hm) individuals than expected, with Hm mice being nonviable. Hz mice exhibited prolonged coagulation times and reduced FV levels compared to wild-type (WT) mice (Figure 1). Hm mice displayed severe diffuse hemorrhages, including internal bleeding in subcutaneous tissue and encephalus (intraparenchymal and submeningeal hemorrhages - Figure 2), and reduced FV immunoexpression in tissues.
The FVFD mutation is lethal in homozygous mice, causing severe hemorrhagic phenotypes and rendering individuals nonviable. CRISPR/Cas9, as seen in this case, can generate unintended mutations. This study is the first to use immunohistochemistry to assess FV tissue expression in an animal model, offering insights into molecular mechanisms and enabling parallels with severe human cases. This mutation may aid future diagnostics if identified in humans

	Departamento de Genética, Fisiología y Microbiología. Facultad de Ciencias Biológicas. Universidad Complutense (UCM).
	Departamento de Reproducción Animal. Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA).
	Servicio de Patología y Veterinaria Forense (SAP). Centro de Vigilancia Sanitaria Veterinaria (VISAVET). Universidad Complutense (UCM).
	Departamento de Medicina y Cirugía Animal. Departamento de Fisiología (Fisiología Animal). Facultad de Veterinaria. Universidad Complutense (UCM).

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- Un nuevo modelo en ratón con deficiencia grave de factor V revela claves sobre esta enfermedad. Premio a la mejor comunicación en forma de poster en el Congreso Internacional ISTH 2025 - unaesperanzaparacelia.org