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In vitro and in sillico Analysis of F9 Gene Missense Mutations at Exons 2 and 3 Causative of Severe Hemophilia B

1Universidad de Guadalajara, Human Genetics, Guadalajara, Mexico, 2Centro de Investigación Biomédica de Occidente, IMSS, Genetics, Guadalajara, Mexico, 3Universidad de Guadalajara, Physiology, Guadalajara, Mexico

Abstract Number: PB0838

Meeting: ISTH 2020 Congress

Theme: Hemophilia and Rare Bleeding Disorders » Hemophilia - Basic

Background: Factor IX (FIX) glycoprotein undergoes numerous post-translational modifications including gamma-carboxylation of 12 glutamic-acid residues in Gla-domain, which is directed by the FIX propeptide and the gamma-glutamyl-carboxylase enzyme. Mutations in propeptide and Gla-domain generate severe deficiency of FIX activity in hemophilia B (HB) patients.

Aims: Analyze the effect of F9 gene missense mutations at exon 2 (p.Glu35Gly) and 3 (p.Lys51Glu, p.Phe55Cys, p.Glu79Gln) on the structure/function of the FIX protein.

Methods: Missense mutations causing severe HB referred in the http://www.factorix.org/database and which did not have a prior functional study, were selected through the correlation of the reported clinical severe phenotype and the output prediction of six in silico tools. F9 cDNA-intron 1 was cloned into the plasmid pcDNA3.1 (+/-) to induce the mutations by site-directed mutagenesis. The plasmids carrying the mutants and wild-type FIX proteins were transfected into HEK-293 cells. After 48 hours of expression, the effect of inhibitors of intracellular transit was evaluated by the production of the secreted proteins to the culture medium and the intracellular proteins obtained from cellular lysates, which were quantified by ELISA assay.

Results: The four mutants decreased their extracellular concentration respect to the wild-type FIX. In order to know the effect of these mutations on intracellular transit, the main degradation pathways were inhibited. In presence of NH4Cl, an inhibitor of lysosomal degradation, mutant proteins p.Glu35Gly and p.Lys51Glu increased their intracellular concentration. In presence of Brefeldin A, an inhibitor of protein transport from the endoplasmic reticulum to Golgi apparatus, mutant proteins p.Phe55Cys and p.Glu79Gln increased their intracellular concentration.

Conclusions: p.Glu35Gly and p.Lys51Glu mutant proteins are degraded mainly by the lysosomal pathway; p.Phe55Cys and p.Glu79Gln mutant proteins are degraded mainly in pre-Golgi compartment. The structural predictions gave an overview of the mutants behavior which was extended by in vitro analysis.

To cite this abstract in AMA style:

Melendez Aranda L, Jaloma Cruz AR, Romero Prado MMdJ. In vitro and in sillico Analysis of F9 Gene Missense Mutations at Exons 2 and 3 Causative of Severe Hemophilia B [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/in-vitro-and-in-sillico-analysis-of-f9-gene-missense-mutations-at-exons-2-and-3-causative-of-severe-hemophilia-b/. Accessed April 16, 2021.

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