Polymorphism 10034C>T is located in a region regulating polyadenylation of FGG transcripts and influences the fibrinogen γ′/γA mRNA ratio

Abstract

Background: Fibrinogen gamma haplotype 2 (FGG-H2) is associated with reduced fibrinogen γ′ levels and fibrinogen γ′/ total fibrinogen ratios and with an increased deep-venous thrombosis (DVT) risk. Two FGG-H2 tagging single nucleotide polymorphisms (SNPs), 9615C>T and 10034C>T, are located in the region of alternative FGG pre-mRNA processing. 10034C>T is located in a GT-rich downstream sequence element (DSE) that comprises a putative cleavage stimulation factor (CstF) binding site. Objectives: To investigate the functionality of SNPs 9615C>T and 10034C>T, and the importance of the DSE containing 10034C>T. Methods: Different minigene constructs containing FGG exon 9, intron 9, exon 10 and the 3′ region were transiently transfected into HepG2 cells and quantitative real-time polymerase chain reaction was used to measure relative polyadenylation (pA) signal usage (pA1/pA2 ratio). Results: Compared with the reference construct CC (9615C-10034C; FGG-H1; pA1/pA2 ratio set at 100%), the pA1/pA2 ratio of construct TT (9615T-10034T; FGG-H2) was 1.4-fold decreased (71.5%, P = 0.015). The pA1/pA2 ratio of construct CT (9615C-10034T) was almost 1.2-fold decreased (85.3%, P = 0.001), whereas the pA1/pA2 ratio of construct TC (9615T-10034C) did not differ significantly from the reference construct (101.6%, P = 0.890). Functionality of the putative CstF binding site was confirmed using constructs in which this site was deleted or its sequence altered by point mutations. Conclusions: SNP 10034C>T is located in a GT-rich DSE involved in regulating the usage of the pA2 signal of FGG, which may represent a CstF binding site. We propose that the 10034C>T change is the functional variation in FGG-H2 that is responsible for the reduction in the fibrinogen γ′/total fibrinogen ratio and the increased DVT risk. © 2007 International Society on Thrombosis and Haemostasis.