Natural and engineered carboxy-terminal variants: decreased secretion and gain-of-function result in asymptomatic coagulation factor VII deficiency

Abstract

Articles and Brief Reportshaematologica | 2012; 97 (5) 705 IntroductionThe congenital deficiency of coagulation factor VII (FVII) (OMIM: 227500) is a rare hemorrhagic disorder with an autosomal recessive inheritance pattern.1 Clarification of the relationship between FVII coagulant activity levels in plasma and bleeding diathesis is greatly helped by our understanding of the molecular mechanisms through which F7 gene mutations 2-3 modulate FVII levels. Nonsense mutations, by inducing mRNA degradation 4 and/or premature termination of translation and synthesis of truncated proteins, are often responsible for very severe forms of human diseases. As expected from the crucial role of FVII in coagulation, 5 very few homozygous patients present this type of mutation and these are associated with undetectable FVII levels and severe 6-8 or moderate 9 bleeding. Coagulation serine proteases share extensive homology 10 but are highly variable in their carboxy-terminal region, the functional role of which has still not been established.In this study, we characterized the p.R462X nonsense mutation and provided evidence for its pleiotropic effects, i.e. reduced secretion and increased specific activity, thus explaining the asymptomatic phenotype in patients. Furthermore, a panel of recombinant carboxy-terminal variants contributed to our understanding of the role of this highly variable region in coagulation serine proteases. Design and Methods PatientsThe proposita (PFVII-R462X) is a 12-year old girl who had been diagnosed for FVII deficiency during a routine coagulation screening. The patient presented with a prolonged prothrombin time (8%) and decreased FVII coagulant activity (3 and 5% of normal in 2 samples taken one year apart); all other coagulation factors functioned normally. She had no history of any bleeding and continues to be asymptomatic. F7 gene sequencing 11 identified the c.1384C>T transition (GenBank #NM_000131.3) in a homozygous condition. This Natural and engineered carboxy-terminal variants: decreased secretion and gain-of-function result in asymptomatic coagulation factor VII deficiency 74 -44121 Ferrara, Italy. Phone: international +39.0532974424. Fax: international +39.0532974484. E-mail: pnm@unife.it We report 2 asymptomatic homozygotes for the nonsense p.R462X mutation affecting the carboxy-terminus of coagulation factor VII (FVII, 466 aminoacids). FVII levels of 3-5% and 2.7±0.4% were found in prothrombin time-based and activated factor X (FXa) generation assays with human thromboplastins. Noticeably, FVII antigen levels were barely detectable (0.7±0.2%) which suggested a gain-of-function effect. This effect was more pronounced with bovine thromboplastin (4.8±0.9%) and disappeared with rabbit thromboplastin (0.7±0.2%). This suggests that the mutation influences tissue factor/FVII interactions. Whereas the recombinant rFVII-462X variant confirmed an increase in specific activity (~400%), a panel of nonsense (p.P466X, p.F465X, p.P464X, p.A463X) and missense (p.R462A, p.R462Q, p.R462W) mutations o...

Replacement of the Y450 (c234) phenyl ring in the carboxyl‐terminal region of coagulation factor IX causes pleiotropic effects on secretion and enzyme activity

“…The present findings on FIX differ substantially from our observations in FVII [14], and from those obtained in PC [13]. Indeed, substitutions of R462 (c253) or short deletions at the FVII carboxyl-terminus resulted in reduced secreted levels of proteins with normal specific activity (Fig.…”

An engineered tale-transcription factor rescues transcription of factor VII impaired by promoter mutations and enhances its endogenous expression in hepatocytes

“…2b , red bar), which was consistent with the extent of the increase in the F7 mRNA levels. Most importantly, the expressed FVII was enzymatically active, as measured by a very sensitive functional fluorogenic assay in plasma that evaluates the activation of factor X, the natural substrate of FVII 21 . In this assay, the activity of FVII responsible for triggering the coagulation cascade process inversely correlates with lag time 22 .…”

The carboxyl‐terminal region is NOT essential for secreted and functional levels of coagulation factor X

“…The study rationale stems from previous observations showing that the natural full-length carboxyl-terminus is essential for the secretion of FVII, FIX and PC [6][7][8][9][10][11][12].…”