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Identification and functional characterization of a novel splicing variant in the F8 coagulation gene causing severe hemophilia A
Author(s) -
Famà Rosella,
Borroni Ester,
Zanolini Diego,
Merlin Simone,
Bruscaggin Valentina,
Walker Gillian E.,
Olgasi Cristina,
Babu Deepak,
Agnelli Giacchello Jacopo,
Valeri Federica,
Giordano Mara,
Borchiellini Alessandra,
Follenzi Antonia
Publication year - 2020
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/jth.14779
Subject(s) - chinese hamster ovary cell , exon , microbiology and biotechnology , rna splicing , exon skipping , biology , alternative splicing , hek 293 cells , mutation , intron , gene isoform , transfection , gene , genetics , rna , cell culture
Background We have identified a synonymous F8 variation in a severe hemophilia A (HA) patient who developed inhibitors following factor VIII (FVIII) prophylaxis. The unreported c.6273 G > A variant targets the consensus splicing site of exon 21. Objectives To determine the impact of c.6273 G > A nucleotide substitution on F8 splicing and its translated protein. Methods Patient peripheral blood mononuclear cells were isolated and differentiated into monocyte‐derived macrophages (MDMs). FVIII distribution in cell compartments was evaluated by immunofluorescence. The splicing of mutated exon 21 was assessed by exon trapping. Identified FVIII splicing variants were generated by site‐directed mutagenesis, inserted into a lentiviral vector (LV) to transduce Chinese hamster ovary (CHO) cells, and inject into B6/129 HA‐mice. FVIII activity was assessed by activated partial thromboplastin time, whereas anti‐FVIII antibodies and FVIII antigen, by ELISA. Results HA‐MDMs demonstrated a predominant retention of FVIII around the endoplasmic reticulum. Exon trapping revealed the production of two isoforms: one retaining part of intron 21 and the other skipping exon 21. These variants, predicted to truncate FVIII in the C1 domain, were detected in the patient. CHO cells transduced with the two FVIII transcripts confirmed protein retention and absence of the C2 domain. HA mice injected with LV carrying FVIII mutants, partially recovered FVIII activity without the appearance of anti‐FVIII antibodies. Conclusions Herein, we demonstrate the aberrant impact of a FVIII synonymous mutation on its transcription, activity, and pathological outcomes. Our data underline the importance of increasing the knowledge regarding the functional consequences of F8 mutations and their link to inhibitor development and an effective replacement therapy.