ABSTRACT
High coagulation factor VIII (FVIII) levels comprise a common risk factor for venous thromboembolism (VTE), but the underlying genetic determinants are largely unknown. We investigated the molecular bases of high FVIII levels in 2 Italian families with severe thrombophilia. The proband of the first family had a history of recurrent VTE before age 50 years, with extremely and persistently elevated FVIII antigen and activity levels (>400%) as the only thrombophilic defects. Genetic analysis revealed a 23.4-kb tandem duplication of the proximal portion of the F8 gene (promoter, exon 1, and a large part of intron 1), which cosegregated with high FVIII levels in the family and was absent in 103 normal controls. Targeted screening of 50 unrelated VTE patients with FVIII levels ≥250% identified a second thrombophilic family with the same F8 rearrangement on the same genetic background, suggesting a founder effect. Carriers of the duplication from both families showed a twofold or greater upregulation of F8 messenger RNA, consistent with the presence of open chromatin signatures and enhancer elements within the duplicated region. Testing of these sequences in a luciferase reporter assay pinpointed a 927-bp region of F8 intron 1 associated with >45-fold increased reporter activity in endothelial cells, potentially mediating the F8 transcriptional enhancement observed in carriers of the duplication. In summary, we report the first thrombophilic defect in the F8 gene (designated FVIII Padua) associated with markedly elevated FVIII levels and severe thrombophilia in 2 Italian families.
Subject(s)
Biomarkers/analysis , Factor VIII/genetics , Gene Duplication , Genetic Predisposition to Disease , Thrombophilia/pathology , Adult , Aged , Case-Control Studies , Female , Follow-Up Studies , Humans , Male , Middle Aged , Pedigree , Prognosis , Thrombophilia/genetics , Whole Genome Sequencing , Young AdultSubject(s)
Factor V Deficiency/complications , Factor V Deficiency/genetics , Factor V/genetics , Intracranial Hemorrhages/epidemiology , Intracranial Hemorrhages/etiology , Mutation , RNA Splicing , DNA Mutational Analysis , Factor V Deficiency/diagnosis , Female , Homozygote , Humans , Incidence , Infant, Newborn , Intracranial Hemorrhages/diagnosis , Intracranial Hemorrhages/prevention & control , Pedigree , RNA Splice Sites , Severity of Illness IndexABSTRACT
Antisense molecules are emerging as a powerful tool to correct splicing defects. Recently, we identified a homozygous deep-intronic mutation (F5 c.1296+268A>G) activating a cryptic donor splice site in a patient with severe coagulation factor V (FV) deficiency and life-threatening bleeding episodes. Here, we assessed the ability of 2 mutation-specific antisense molecules (a morpholino oligonucleotide [MO] and an engineered U7 small nuclear RNA [snRNA]) to correct this splicing defect. COS-1 and HepG2 cells transfected with a F5 minigene construct containing the patient's mutation expressed aberrant messenger RNA (mRNA) in excess of normal mRNA. Treatment with mutation-specific antisense MO (1-5 µM) or a construct expressing antisense U7snRNA (0.25-2 µg) dose-dependently increased the relative amount of correctly spliced mRNA by 1 to 2 orders of magnitude, whereas control MO and U7snRNA were ineffective. Patient-derived megakaryocytes obtained by differentiation of circulating progenitor cells did not express FV, but became positive for FV at immunofluorescence staining after administration of antisense MO or U7snRNA. However, treatment adversely affected cell viability, mainly because of the transfection reagents used to deliver the antisense molecules. Our data provide in vitro and ex vivo proof of principle for the efficacy of RNA therapy in severe FV deficiency, but additional cytotoxicity studies are warranted.
