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

Abstract

To cite this article: Uitte de Willige S, Rietveld IM, de Visser MCH, Vos HL, Bertina RM. Polymorphism 10034C>T is located in a region regulating polyadenylation of FGG transcripts and influences the fibrinogen c¢/cA mRNA ratio. J Thromb Haemost 2007; 5: 1243-9.Summary. Background: Fibrinogen gamma haplotype 2 (FGG-H2) is associated with reduced fibrinogen c¢ levels and fibrinogen c¢/total fibrinogen ratios and with an increased deepvenous 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 c¢/total fibrinogen ratio and the increased DVT risk.

γ′ Fibrinogen as a novel marker of thrombotic disease

“…This haplotype includes two SNPs in the intron 9/exon 10 region (Figure 1), FGG 9615C > T (rs2066864) and FGG 10,034C > T (rs2066865). The 10,034T allele is located within a GT-rich downstream sequence element in a potential cleavage stimulation factor binding site, and has been shown to decrease the ratio of γ ′ / γ A mRNA in a minigene construct (44) . The 10,034T allele has also been associated with decreased γ ′ fi brinogen levels and a decreased γ ′ fi brinogen/total fi brinogen ratio (34) .…”

The pleiotropic role of the fibrinogen γ′ chain in hemostasis

“…Because FGG-H2 SNPs 10034C3T and 9615C3T [rs2066864] are completely linked, the functionality of these SNPs on ␥A/␥Ј fibrinogen expression was investigated using mini-gene constructs. 98 This study concluded that 10034C3T is responsible for the reduction in ␥A/␥Ј fibrinogen level since this SNP is located in a GT-rich downstream sequence element, which composes a putative cleavage stimulation factor binding site and regulates the usage of the 2 polyadenylation signals in FGG. How FGG-H3 influences ␥A/␥Ј fibrinogen expression is still unknown, but as SNP 9340T3C is situated in intron 9, it may be that this variation somehow also regulates the 2 polyadenylation signals in FGG.…”

Inherited Thrombophilia: Past, Present, and Future Research

“…This notion was confirmed with the finding of APC resistance. Individuals from families with protein S, protein C, or antithrombin deficiency but also APC resistance had a higher risk for VTE when they inherited combined defects rather than only one defect (Koeleman et al, 1994;van Boven et al, 1996). Thus, the penetrance of thrombosis increases in these protein C deficient families after introduction of the factor V Leiden allele in the pedigree (Brenner et al, 1996).…”

“…In particular subtype IIb confers a lower risk than the other subtypes (Finazzi et al, 1987). Risk estimates for developing VTE in the presence of AT deficiency are mainly based on family studies and these show a 10-20 fold increased risk (Lijfering et al, 2009;Mahmoodi et al, 2010;van Boven & Lane, 1997). The risk for developing a recurrent VTE is 10.5% per year without long-term anticoagulant treatment.…”

“…Heritability was also determined for individual coagulation factors involved in clot formation, like prothrombin (49-57%), factor V (44-62%), and von Willebrand Factor (34-75%) (de Lange et al, 2001;Souto et al, 2000b). In addition, 20-30% of consecutive VTE patients report one or more first-degree family members with VTE (Heijboer et al, 1990;van Sluis et al, 2006). T h e s e f i n d i n g s r e a f f i r m t h a t g e n e t i c r i s k f a c t o r s d o p l a y a n i m p o r t a n t r o l e i n t h e development of VTE.…”