Page 315 - Haematologica March 2020

Coagulation & its Disorders
Next-generation sequencing and recombinant expression characterized aberrant splicing mechanisms and provided correction strategies in factor VII deficiency
Ferrata Storti Foundation
Haematologica 2020 Volume 105(3):829-837
Paolo Ferraresi,1 Dario Balestra,1 Caroline Guittard,2 Delphine Buthiau,2 Brigitte Pan-Petesh,3 Iva Maestri,4 Roula Farah,5 Mirko Pinotti1 and Muriel Giansily-Blaizot2
1Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy; 2Department of Biological Haematology, CHU Montpellier, Université Montpellier, Montpellier, France; 3Haemophilia Centre, CHU Brest, Brest, France; 4Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy and 5Department of Pediatrics, Saint George Hospital University Medical Center, Beirut, Lebanon
ABSTRACT
Despite the exhaustive screening of F7 gene exons and exon-intron boundaries and promoter region, a significant proportion of mutat- ed alleles remains unidentified in patients with coagulation factor VII deficiency. Here, we applied next-generation sequencing to 13 FVII-defi- cient patients displaying genotype-phenotype discrepancies upon conven- tional sequencing, and identified six rare intronic variants. Computational analysis predicted splicing effects for three of them, which would strength- en (c.571+78G>A; c.806-329G>A) or create (c.572-392C>G) intronic 5’ splice sites (5’ss). In F7 minigene assays, the c.806-329G>A was ineffective while the c.571+78G>A change led to usage of the +79 cryptic 5’ss with only trace levels of correct transcripts (3% of wild-type), in accordance with factor VII activity levels in homozygotes (1-3% of normal). The c.572- 392C>G change led to pseudo-exonization and frame-shift, but also sub- stantial levels of correct transcripts (approx. 70%). However, this variant was associated with the common F7 polymorphic haplotype, predicted to further decrease factor VII levels; this provided some kind of explanation for the 10% factor VII levels in the homozygous patient. Intriguingly, the effect of the c.571+78G>A and c.572-392C>G changes, and particularly of the for- mer (the most severe and well-represented in our cohort), was counteracted by antisense U7snRNA variants targeting the intronic 5’ss, thus demonstrat- ing their pathogenic role. In conclusion, the combination of next-generation sequencing of the entire F7 gene with the minigene expression studies elu- cidated the molecular bases of factor VII deficiency in 10 of 13 patients, thus improving diagnosis and genetic counseling. It also provided a potential therapeutic approach based on antisense molecules that has been success- fully exploited in other disorders.
Introduction
The inherited deficiency of factor VII (FVII), the crucial enzyme triggering blood coagulation,1 is the most common of the rare coagulation disorders transmitted in an autosomal recessive manner. The clinical features are highly variable, ranging from severe (i.e. intracranial or gastro-intestinal hemorrhages) to milder (i.e. epis- taxis) or asymptomatic forms,2 and the relationship with plasma FVII activity (FVII:C) levels is often elusive. On the other hand, molecular genetic studies com- bined with functional assays and recombinant expression investigations, detailing the residual FVII levels associated with F7 gene mutations, have clearly helped define genotype and coagulation and clinical phenotype relationships,3-5 with implications for diagnosis, prognosis and counseling.
Over 220 point-mutations,6 a few large genomic rearrangements, and six com-
Correspondence:
MIRKO PINOTTI
pnm@unife.it
MURIEL GIANSILY-BLAIZOT
m-giansily@chu-montpellier.fr
Received: March 5, 2019. Accepted: July 2, 2019. Pre-published: July 4, 2019.
doi:10.3324/haematol.2019.217539
Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/105/3/829
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