Polymorphism 10034C>T Is Located in a Functional Cleavage Stimulation Factor (CstF) Binding Site of the Fibrinogen Gamma Gene and Influences the γ′/γA mRNA Ratio.
Authors: de Willige Shirley Uitte , Rietveld Inge M. , de Visser Marieke C.H. , Vos Hans L. , Bertina Rogier M. 
Published: October 2019

Previously we found that haplotype 2 of the fibrinogen gamma gene (FGG-H2) is associated with an increased risk of deep venous thrombosis and with reduced fibrinogen γ′ levels, an alternatively spliced form of FGG, and reduced fibrinogen γ′/total fibrinogen ratios (Blood2005; 106(13): 4176–83). Two of the FGG-H2 tagging single nucleotide polymorphisms (SNPs), 9615C>T [rs2066864] and 10034C>T [rs2066865], are located in the region containing the two polyadenylation (pA) signals used in FGG pre-mRNA processing, namely pA1 and pA2, resulting in transcripts coding for γ′-chains and γA-chains, respectively. SNP 10034C>T is located in a Cleavage stimulation Factor (CstF) binding site at nucleotides 10030–10047 of FGG. CstF is a multi-subunit complex required for efficient cleavage and polyadenylation of pre-mRNAs. We hypothesized that 10034T, by strengthening this CstF binding site, favors use of pA2 (synthesis of γA-chains) at the expense of pA1-use (synthesis of γ′-chains). We transiently transfected FGG mini-gene constructs containing exon 9, intron 9 (containing pA1), exon 10 and the 3′ UTR (containing pA2) in liver-derived HepG2 cells and used quantitative real-time PCR to measure the mean relative pA-signal usage (pA1/pA2 ratio) of the different constructs. To study the effects of SNPs 9615C>T and 10034C>T on the relative use of pA1 and pA2, we made constructs carrying the different alleles of these SNPs. Compared to reference construct CC (9615C, 10034C; FGG-H1) of which the pA1/pA2 ratio was set at 100%, the ratio of construct TT (9615T, 10034T; FGG-H2) was 1.4-fold decreased (71.5%, p=0.015). To investigate whether the SNPs individually or collectively caused the reduction in the pA1/pA2 ratio, exchange constructs CT (9615C, 10034T) and TC (9615T, 10034C) were also tested. The ratio of construct CT was almost 1.2-fold decreased (85.3%, p=0.001), while the ratio of construct TC did not differ significantly from the reference construct (101.6%, p=0.890). These results indicate that in our in vitro system SNP 10034C>T, and not SNP 9615C>T, is the major contributor to the reduction of the pA1/pA2 ratio. We further investigated the functionality and importance of the CstF binding site, containing 10034C>T, with a construct in which nucleotides 10013–10056 containing the complete CstF binding site were deleted. Compared to the reference construct, in which the CstF site was still present, the pA1/pA2 ratio of the CstF deletion construct was 2.2-fold increased (222.3%, p<0.001), indicating that this site is involved in the regulation of pA2-usage. The functionality of the CstF site was further confirmed using several mutant constructs in which the CstF consensus was weakened or strengthened by introducing one or two point mutations. Weakening the CstF site resulted in a significant 1.5-fold increase of the pA1/pA2 ratio, while strengthening the site resulted in a significant 1.6-fold decrease of the ratio. From these results we conclude that 10034C>T is located in a functional CstF binding site and is involved in regulating the usage of the pA2-signal of FGG. We propose that the 10034C>T change is the functional variation in the FGG-H2 haplotype and is responsible for the reduction in the fibrinogen γ′/total fibrinogen ratio and the increased risk of deep venous thrombosis.