Polymorphism 10034C>T is located in a region regulating polyadenylation of FGG transcripts and influences the fibrinogen gamma'/gammaA mRNA ratio.
BACKGROUND
Fibrinogen gamma haplotype 2 (FGG-H2) is associated with reduced fibrinogen gamma' levels and fibrinogen gamma'/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.


OBJECTIVE
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 gamma'/total fibrinogen ratio and the increased DVT risk.
Uitte de Willige
S
S
Department of Hematology, Hemostasis and Thrombosis Research Center, Leiden University Medical Center, Leiden, The Netherlands.
Rietveld
I M
IM
De Visser
M C H
MC
Vos
H L
HL
Bertina
R M
RM
D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D005340:Fibrinogen / Q000737:chemistry / Q000235:genetics*; D006239:Haplotypes; D006801:Humans; D066298:In Vitro Techniques; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics; D017384:Sequence Deletion; D014158:Transcription, Genetic; D014162:Transfection; D020246:Venous Thrombosis / Q000097:blood / Q000209:etiology / Q000235:genetics
D017931:DNA Primers; D010446:Peptide Fragments; D012322:RNA Precursors; D012333:RNA, Messenger; D011994:Recombinant Proteins; D005340:Fibrinogen