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Haematologica. 2019 Jul 4. pii: haematol.2019.217539. doi: 10.3324/haematol.2019.217539. [Epub ahead of print]

Next-generation sequencing and recombinant expression characterized aberrant splicing mechanisms and provided correction strategies in factor VII deficiency.

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Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy.
Department of biological haematology, CHU Montpellier, Univ Montpellier, Montpellier, France.
Haemophilia Centre, CHU Brest, Brest, France.
Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy.
Department of Pediatrics, Saint George Hospital University Medical Center, Beirut, Lebanon.
Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy;


Despite the exhaustive screening of F7 gene exons and exon-intron boundaries and promoter region, a significant proportion of mutated alleles remains unidentified in patients with coagulation factor VII deficiency. Here, we applied next-generation sequencing to thirteen FVII-deficient patients displaying genotype-phenotype discrepancies upon conventional sequencing, and identified six rare intronic variants. Computational analysis predicted splicing effects for three of them, which would strengthen (c.571+78G>A; c.806-329G>A) or create (c.572-392C>G) intronic 5' splice sites (5'ss). In F7 minigenes 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 substantial levels of correct transcripts (~70%). However, this variant was associated with the common F7 polymorphic haplotype predicted to further decrease factor VII levels, thus roughly explaining the factor VII levels of 10% in the homozygous patient. Intriguingly, the effect of the c.571+78G>A and c.572-392C>G changes, and particularly of the former, the severest and well-represented in our cohort, was counteracted by antisense U7snRNA variants targeting the intronic 5'ss, thus demonstrating their pathogenic role. In conclusion, the combination of next-generation sequencing of the entire F7 gene with the minigene expression studies elucidated the molecular bases of factor VII deficiency in ten out of thirteen patients, thus improving diagnosis and genetic counselling, and provided a potential therapeutic approach based on antisense molecules, successfully exploited in other disorders.


Deep-intronic Mutation; Disorders of Coagulation and Fibrinolysis; FVII Deficiency; New Generation Sequencing; Splicing Correction

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