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The chaperone‐like sodium phenylbutyrate improves factor IX intracellular trafficking and activity impaired by the frequent p.R294Q mutation
Author(s): ,
S. Pignani
Affiliations:
Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy
,
A. Todaro
Affiliations:
Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy
,
M. Ferrarese
Affiliations:
Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy
,
S. Marchi
Affiliations:
Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, University of Ferrara, Ferrara, Italy
,
S. Lombardi
Affiliations:
Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy
,
D. Balestra
Affiliations:
Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy
,
P. Pinton
Affiliations:
Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, University of Ferrara, Ferrara, Italy
,
F. Bernardi
Affiliations:
Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy
,
M. Pinotti
Affiliations:
Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy
A. Branchini
Affiliations:
Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy
Correspondence: Alessio Branchini, Department of Life Sciences and Biotechnology, University of Ferrara, Via Fossato di Mortara 74, 44121 Ferrara, Italy|Tel.: +39 053 297 4485|E‐mail: brnlss@unife.it
ISTH Academy. Branchini A. Oct 4, 2018; 234167
Dr. Alessio Branchini
Dr. Alessio Branchini

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Background
Missense mutations often impair protein folding and intracellular processing, which can be improved by small compounds with chaperone‐like activity. However, little has been done in coagulopathies, where even modest increases of functional levels could have therapeutic implications.
Objectives
To rescue the expression of factor IX (FIX) variants affected by missense mutations associated with type I hemophilia B (HB) through chaperone‐like compounds.
Methods
Expression studies of recombinant (r)FIX variants and evaluation of secreted levels (ELISA), intracellular trafficking (immunofluorescence) and activity (coagulant assays) before and after treatment of cells with chaperone‐like compounds.
Results
As a model we chose the most frequent HB mutation (p.R294Q, ~100 patients), compared with other recurrent mutations associated with severe/moderate type I HB. Immunofluorescence studies revealed retention of rFIX variants in the endoplasmic reticulum and negligible localization in the Golgi, thus indicating impaired intracellular trafficking. Consistently, and in agreement with coagulation phenotypes in patients, all missense mutations resulted in impaired secretion (< 1% wild‐type rFIX). Sodium phenylbutyrate (NaPBA) quantitatively improved trafficking to the Golgi and dose dependently promoted secretion (from 0.3 ± 0.1% to 1.5 ± 0.3%) only of the rFIX‐294Q variant. Noticeably, this variant displayed a specific coagulant activity that was higher (~2.0 fold) than that of wild‐type rFIX in all treatment conditions. Importantly, coagulant activity was concurrently increased to levels (3.0 ± 0.9%) that, if achieved in patients, would ameliorate the bleeding phenotype.
Conclusions
Altogether, our data detail molecular mechanisms underlying type I HB and candidate NaPBA as affordable ‘personalized’ therapeutics for patients affected by the highly frequent p.R294Q mutation, and with reduced access to substitutive therapy.
Keyword(s)
blood coagulation factor deficiencies, factor IX, hemophilia B, molecular medicine, missense mutation
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