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Polymorphism 10034C>T is located in a region regulating polyadenylation of FGG transcripts and influences the fibrinogen γ′/γA mRNA ratio
Author(s) -
UITTE DE WILLIGE S.,
RIETVELD I. M.,
DE VISSER M. C. H.,
VOS H. L.,
BERTINA R. M.
Publication year - 2007
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/j.1538-7836.2007.02566.x
Subject(s) - microbiology and biotechnology , single nucleotide polymorphism , exon , polyadenylation , biology , haplotype , fibrinogen , messenger rna , chemistry , genetics , allele , genotype , gene , biochemistry
Summary. 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.